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BACTERIOLOGY 
 
 IN 
 
 ABSTRACT 
 
 BY 
 
 A. B. WALLGREN. B. S.. M. D. 
 
 Assistant Professor of Biology, University of 
 Pittsburgh; Pathologist to the Pittsburgh 
 Hospital, St. Margaret's Hospital and 
 Columbia Hospital; Author of 
 Histology in Abstract and 
 Pathology in Absti ict 
 
 PITTSBURGH, PENNA. 
 
 Published by 
 Medical Abstract Publishing Company 
 Jenkins Arcade Building 
 Pittsburgh, Pa. 
 
PREFACE TO SECOND EDITION 
 
 The demand created by the first 
 Abstract in Bacteriology necessitated the 
 production of a second edition, which, while 
 considerably enlarged, contains only the 
 very elements of Bacteriology. Although 
 containing only the first principles of 
 Bacteriology, it is hoped that this book 
 will be of use to the student wishing a 
 small work for a ready guide. 
 
 For a detailed work the student must 
 necessarily refer to the works of Jordan, 
 Mallory and Wright, Chester, Frost, Frost 
 and McCampbell, Marshall, Park and 
 Williams, Hiss and Zinsser, Besson, 
 Gorham, Muir and Ritchie, Simon, etc., 
 all of which have been freely consulted in 
 the preparation of this Abstract. 
 
 The Author is very much indebted to 
 Eleanor C. Doty for valuable aid m the 
 preparation of manuscript and the reading 
 of proofs. 
 
5 ^9, 9 6^ 
 
 \A/ /6' b 
 history" OF BACTERIOLOGY. 
 
 ^ With the introduction of the micro- 
 J scope some order was brought into 
 S understanding- that, group of organisms 
 ^ which Linnaeus had termed "chaos." 
 ^ That disease and decay were due to 
 \ minute organisms had been the theory 
 S for centuries. The conception of con- 
 \, tagion, or the transmission of disease 
 ^ from one human being to another, was 
 ' centuries old. This fact had been rec- 
 ognized by Aristotle and had been re- 
 iterated by medieval philosophers, and 
 ^ had led to the division of contagious 
 ^ diseases, by Pracastor (1546), into those 
 diseases transmitted "per contactum" 
 - and those conveyed indirectly "per 
 i fomitem." It was on account of these 
 k facts of transmissibility of disease that 
 ^ physicians of the eighteenth century 
 saw an explanation in the microorgan- 
 \ isms discovered in the latter half of the 
 ; seventeenth century by the Jesuit, 
 Kircher (1659), and the Dutch linen- 
 draper, van Leeuwenhoek (1675). These 
 two men were able by the improved 
 microscope to demonstrate microorgan- 
 isms in water, intestinal contents, etc. 
 They made out short, straight, and 
 curved rods, and described their motil- 
 ity. There can be no doubt that the 
 bodies seen by these two men were, at 
 least in part, bacteria. During the cen- 
 tury following the work of these two 
 investigators, a more exact description 
 ^of these forms of life led Muller to 
 attempt a systematic classification. A 
 ^more extensive study and classification 
 ^was later made by Ehrenburg. 
 
 Needham, in 1749, published an 
 ^^rticle in which he favored the opinion, 
 ^held by many, namely, that the 
 ^minute organisms described by Leeuw- 
 enhoek and others were produced by 
 t^tepontaneous generation. He held to 
 .this opinion from the fact that he had 
 placed putrefying material and vege- 
 \table infusion in sealed flasks, exposing 
 Pthem for a short time to heat, and later 
 y^ound this infusion to be filled with 
 -^^microorganisms. BufCon supported him 
 "^in his views. Abbe' Spallanazanni re- 
 peated the experiments of Needham, 
 employing greater care in sealing his 
 flasks, and subjecting them to a greater 
 exposure of heat. His results were un- 
 
4 BACTERIOLOGY. 
 
 like those of Needham and consequently 
 did not support the theory of spon- 
 taneous generation. Schulze's (1836) 
 failure to find a living organism in in- 
 fusions which had been boiled and to 
 which air had been admitted only after 
 passage through acid solutions, did not 
 convince many that spontaneous gener- 
 ation was impossible, and that the life 
 that appeared in the infusions was due 
 to the presence of minute organisms 
 having entered the infusion by reason 
 of faulty technique. The question of 
 spontaneous generation was not definite- 
 ly settled until Pasteur (1860) conduct- 
 ed a series of experiments, the results 
 of which absolutely refuted the doctrine 
 of spontaneous generation. 
 
 Plenciz of Vienna (1762), expressed 
 his belief in the direct etiological con- 
 nection between microorganisms and 
 certain diseases, and also suggested 
 specific treatment for these diseases. 
 
 Rayer, in 1815, found rod shaped or- 
 ganisms in the blood of animals sick 
 with splenic fever. The real advance 
 in the development of bacteriology 
 came in 1837 when Schwann showed 
 that yeasts were living organisms and 
 had to do with the process of fermen- 
 tation. The same view was held by the 
 Frenchman Cagniard-Latour. 
 
 Pasteur paralleled his researches up- 
 oti spontaneous generation with experi- 
 ments upon fermentation along the 
 lines suggested by Cagniard-Latour, 
 publishing his classical studies upon 
 the fermentation that occurred in beer 
 and wine, due to yeasts, and was also 
 able to show that a number of other 
 fermentations, such as those of lactic 
 and butyric acid, as well as the decom- 
 position of organic matter by putrefac- 
 tion, were directly due to the action of 
 microorganisms. The doctrine of spon- 
 taneous generation had received its final 
 refutation in all but one particular. It 
 was not understood why sterility could 
 not always be obtained from the appli- 
 cation of definite degrees of heat. This 
 was finally explained by Cohn (1871), 
 who demonstrated the presence of bac- 
 terial spores, also demonstrating their 
 resistance toward heat and other influ- 
 ences. 
 
 Pollender, in 1855, reported the pres- 
 ence of rod shaped bodies in the blood 
 
BACTERIOLOGY. 5 
 
 and spleen of animals dead of anthrax, 
 previously reported by Rayer. Convinc- 
 ing proof of the observations of Rayer 
 and Pollender was . brought out by 
 Davaine (1863), who also succeeded in 
 demonstrating that anthrax could be 
 transmitted by means of blood contain- 
 ing the rods described by Rayer and 
 Davaine, and could never be transmitted 
 by blood from which the rods were ab- 
 sent. Anthrax, therefore, was the first 
 disease in which bacterial causation 
 was demonstrated. 
 
 Obermeier (1868) demonstrated the 
 presence of a spirillum in the blood ot 
 patients suffering from relapsing fever. 
 
 Rindfleisch, von Recklinghausen, Wal- 
 deyer described microorganisms in tis- 
 sues containing abscesses. 
 
 Klebs (1870) described similar mi- 
 croorganisms found in pus. 
 
 Koch (1880) introduced nutrient me- 
 dia upon which bacteria could be cul- 
 tured, thus laying the foundation for 
 an exact science. 
 
 Weigert, Koch, and Ehrlich intro- 
 duced the use of aniline dyes which 
 facilitated the morphological study of 
 bacteria. 
 
 Koch published the discovery of the 
 typhoid bacillus, fowl cholera bacillus 
 and pneumococcus in 1880 and the 
 tubercle bacillus in 1882. 
 
 THE BIOLOGY OF BACTERIA. 
 
 ORIGIN, DISTRIBUTION, ETC. 
 
 Bacteria cannot arise denovo. They 
 must develop from pre-existing bacteria 
 or their spores. One kind of organism 
 will not produce another kind. 
 
 DISTRIBUTION. 
 
 Air. In the air there are bacteria 
 throughout, except at great altitudes. 
 
 Soil. In soil, bacteria are very abund- 
 ant except at great depths. Garden 
 soil will contain from a hundred 
 thousand to as many millions per gm. 
 Bacteria are less numerous in the 
 peat or acid moor soils, in the soils 
 containing an excess of alkali and in 
 sandy soils. Were it not for bac- 
 teria, plant life could not exist 
 as they perform functions in the soil, 
 
6 BACTERIOLOGY. 
 
 fitting it physically and chemically 
 for the growth of higher plants. 
 They decompose organic matter di- 
 rectly, and minerals of the soil in- 
 directly, converting them into com- 
 pounds that are taken up and assim- 
 ilated by plants, etc. 
 "Water, as a rule, is the home of bac- 
 teria. 
 
 Wells contain from a very few to 
 several hundred bacteria per c. c, 
 though in deep wells, such as arte- 
 sian, and in the springs of some dis- 
 tricts, there may be no bacteria. 
 There are about the same number of 
 bacteria in lakes and ponds as in 
 wells, except where there is sewage 
 contamination. 
 
 Streams usually contain more bac- 
 teria than any of the above by rea- 
 son of sewage contamination or wash- 
 ings from water sheds, etc. 
 Foods. Bacteria are most abundant in 
 most foods. Some healthful foods 
 contain great numbers of bacteria, as 
 is illustrated by the lactic acid bac- 
 teria of sour milk. 
 
 Other bacteria present in food, in 
 great numbers, may be harmless. 
 
 The bacteria of foods may be di- 
 vided into three groups: — 
 
 (1) Those beneficial — which bring # 
 
 about the fermentations, as in 
 the preparation of pickles, 
 sauerkraut, etc. 
 
 (2) Those producing fermentations 
 
 and decay. 
 
 (3) Those producing disease. 
 Fermentation bacteria act upon 
 
 carbohydrates, as starch and cellu- 
 lose, breaking them down. Decay- 
 bacteria decompose proteins. 
 Body. The normal living tissues of the 
 body are free from bacteria. 
 
 Bacteria are found in great num- 
 bers on the skin, in the alimentary 
 tract, and can be considered as a 
 flora peculiar to this situation as they 
 ordinarily do no harm. Certain bac- 
 teria (pathogenic) may enter the body 
 and produce disease. 
 
 MICROORGANISM NORMAL TO THE 
 HUMAN BODY. 
 
 A great number of species of bacteria 
 develop on the skin of the body and 
 
BACTERIOLOGY. 
 
 7 
 
 in the various body cavities which 
 open to the surface. Normally the tis- 
 sues of the body are sterile. Bacteria 
 are not found in the blood stream, in 
 the muscles, nor in the glands oi nor- 
 mal individuals although the lymph 
 fluids lying- near the intestines may 
 occasionally be infected by the intest- 
 inal bacteria. Organisms may or may 
 not be present in the tissues of the 
 individuals. Some diseases are classi- 
 fied as septicemias and bacteriemias be- 
 cause of the general distribution of the 
 bacteria in the blood stream. In other 
 cases, the organisms are localized and 
 do not get far from the site of infec- 
 tion. 
 
 The exposed surfaces of the body and 
 the cavities of the body that communi- 
 cate with the surface possess a bac- 
 terial flora that may be considered 
 normal to the part. 
 The Flora of the Skin. 
 
 The organisms that are constantly 
 present and evidently multiply upon 
 the surface: — 
 
 Micrococcus (staphylococcus) epider- 
 midosis albus, an organism which 
 is closely related to the pus pro- 
 ducing cocci. This organism fre- 
 quently penetrates into the lower 
 layers of the epidermis. 
 
 Micrococcus aureus and albus are fre- 
 quently present, but are usually of 
 a low virulence. These are the or- 
 ganisms that produce infection of 
 wounds, abscesses, and boils. 
 
 Streptococci may frequently be iso- 
 lated from the skin. 
 
 Bacterium smegmatis is nearly con- 
 stantly present in places where the 
 skin is moist and greasy. It does 
 not seem to penetrate into the skin 
 but lives upon the skin excreta. 
 
 In the wax secreted in the external 
 auditory canal, the micrococcus 
 cereus flavus is generally found. 
 
 Very few bacteria are found normal- 
 ly on the conjunctiva. The tears 
 seem to remove or destroy any or- 
 ganism that may gain entrance. 
 
 The organisms that accidentally form 
 a skin flora are the bacteria that 
 contaminate the skin when it comes 
 into contact with dust or dirt. The 
 soil bacteria are quite common to 
 the skin, particularly under the 
 
8 BACTERIOLOGY. 
 
 finger nails and in the hair. These 
 include the soil aerobic forms, as 
 the bacillus subtilis group and cer- 
 tain anaerobic bacteria. 
 
 The intestinal organisms, as the ba- 
 cillus coli, are commonly present 
 by reason of contact with diseased 
 individuals. 
 The Flora of the Mouth. 
 
 A great number of tne organisms that 
 have been isolated from the mouth 
 are probably due to accidental con- 
 tamination. Mueller has described 
 a number of species found present 
 in the normal mouth. Of these, he 
 decided that six were constantly 
 present; i. e., leptothrix and the 
 spirocheta dentium. These organ- 
 isms are obligative anaerobes and 
 find favorable conditions for growth 
 between the teeth and in teeth un- 
 dergoing decay. 
 
 Anaerobic bacilli related to B. putri- 
 ficus. 
 
 Non virulent varieties of streptococ- 
 cus pyogenes. 
 
 Non virulent varieties of micrococcus 
 aureus and albus. 
 
 Avirulent pneumococcus. 
 
 The decay of teeth is said to be due 
 to the development of acids by cer- 
 tain lactic acid bacteria present 
 upon the surface, causing a more or 
 less complete removal of the lime 
 
 * present; while the decomposition of 
 the remaining substance of the 
 teeth is probably brought about by 
 common anaerobic bacteria. Occa- 
 sionally the mouth of a healthy in- 
 dividual may contain pathogenic 
 organisms, as the bacterium diph- 
 theria and bacterium infiuenza. 
 The Flora of the Stomach. 
 
 Ordinarily bacteria do not develop in 
 the stomach by reason of its acid- 
 ity. If for any reason the acidity 
 is diminished, bacteria belonging to 
 the butyric acid group and the bac- 
 terium lactis aerogenes may de- 
 velop. Under certain conditions, 
 the lactic acid bacteria of the Bul- 
 garian type may develop. 
 Flora of Intestine. 
 
 The first portion of the intestine is 
 relatively free from bacteria by 
 reason of the fact that the bile is 
 antiseptic to certain microorganisms 
 
BACTERIOLOGY. 
 
 9 
 
 and will, more or less, completely 
 inhibit their growth. In the lower 
 part of the small intestine bacteria 
 become more numerous. The bac- 
 terium lactis aerogenes and bacil- 
 lus coli are present in small num- 
 bers. In the colon, bacteria develop 
 freely. The bacterium lactis aero- 
 geneus and the bacillus coli are 
 nearly always present. These to- 
 gether with the bacillus bifRdus and 
 bacillus acidophilus inhibit the 
 growth of any organism of the 
 putrefactive type by the formation 
 of an acid, and possibly by the for- 
 mation of metabolic products. An- 
 aerobic forms, as the bacillus putrif- 
 icus and bacillus aerogenes capsul- 
 atus, are also present. 
 
 It is quite possible that in some of 
 the herbivorous animals the organ- 
 isms are of some assistance in the 
 digestion of food. In the human 
 intestine it is believed that products 
 of decomposition brought about by 
 the putrefactive bacteria are ab- 
 sorbed through the intestinal wall 
 and appear in the same form or as 
 related compounds in the urine, and 
 cause some of the changes charac- 
 teristic of old age, particularly the 
 hardening of the arterial wall 
 (arterial sclerosis). 
 
 Metchnikofe believed that the putre- 
 factive bacteria might be elimin- 
 ated by using foods containing 
 great numbers of lactic acid bac- 
 teria, which would prevent the de- 
 velopment of the putrefactive form 
 by the presence of the lactic acid 
 bacteria and also the lactic acid. 
 The Plora of the Respiratory Tract. 
 
 The flora of the upper respiratory 
 passages, as the nose, can not be 
 said to be characteristic. The bac- 
 teria isolated from the nasal cav- 
 ities are generally the bacteria of 
 the air. The air of the bronchi, the 
 bronchioles and alveoli of the lungs 
 are usually entirely free from mi- 
 croorganisms by reason of the filtra- 
 tion of air through the nasal pass- 
 ages, and the passage of the air 
 over the nasal and oral mucous 
 membrane, which is covered with 
 mucous and also with ciliated cells, 
 which serve to some extent to wash 
 
10 BACTERIOLOGY. 
 
 the mucous containing the bacteria 
 from the surface. Infections of 
 the nasal mucous membrane are, 
 however, not uncommon. The bac- 
 terium influenza, the streptococcus 
 pyogenes, the micrococcus pyogenes, 
 aureus and albus, bacterium diph- 
 theria, the micrococcus intracel- 
 lularis, meningitidis and occasion- 
 ally the bacterium mallein produce 
 infection through the membrane of 
 the nasal cavities. 
 The Flora of the Genito-Urinary Tract. 
 
 The exposed mucous surfaces of the 
 genito-urinary tract usually harbor 
 a number of harmless bacteria. 
 Organisms closely related to the pus 
 producing cocci are commonly pres- 
 ent. The secretions of the vagina 
 are somewhat bactericidal, but cer- 
 tain bacteria are constantly present 
 and the properties of the vaginal 
 secretion is supposed to be in part 
 due to the presence of these organ- 
 isms, e. g. Doederlein's bacillus. 
 
 Normally the uterus does not contain 
 bacteria, although their presence is 
 not uncommon in the later stages 
 of pregnancy. The urinary bladder 
 is normally free from bacteria. 
 
 In the secretions about the external 
 genitals, acid-fast bacteria, partic- 
 ularly the bacterium smegmatis, are 
 constantly present. 
 
 THE CATABOLIC ACTIVITIES OF BACTERIA 
 
 Study of sulistances that result from 
 the action of the life of bacteria and 
 the changreb that they produce in the 
 various media of growth, is really a 
 branch of organic chemistry. So we 
 can only make mention of them here. 
 The function of bacteria is essentially 
 a destructive one. They split up the 
 higher nitrogenous and non-nitro- 
 genous compounds into simple sub- 
 stances. 
 
 The various substances that are found 
 in the media of bacterial growth 
 comprise : — 
 
 1. The components of the bacterial 
 
 cell proper, as the proteins. 
 
 2. The secretions of the cell, as the 
 
 ferments and toxins. 
 
 3. The substances that are the re- 
 
 sult of the action of the organ- 
 
BACTERIOLOGY. 
 
 11 
 
 isms upon the medium of 
 growth: — 
 The toxic substances in bacterial 
 cultures may be classified as 
 (a). Intracellular and (b) Ex- 
 tracellular, according- as they 
 are contained within the bac- 
 terial cell or free in the culture 
 medium. The extracellular sub- 
 stances may be purely products 
 of bacterial secretion which 
 have separated from the cell or 
 they may be decomposition 
 products derived from the cul- 
 ture meuium. 
 
 (1) The proteins may produce sup- 
 purations or fever, or may 
 cause inflammatory processes. 
 They are comparatively resist- 
 ant to heat and are thus sharply 
 distinguished from the ferments 
 and toxins. The best known 
 examples are mallein, derived 
 from the bacillus of glanders, 
 and tuberculin from that of 
 tuberculosis. These substances 
 are pyogrenlc (fever) when in- 
 jected into animals sick with 
 glanders or tuberculosis but 
 have very slight effect upon 
 healthy subjects. 
 
 (2) Second group are ferments and 
 possibly toxins. 
 
 Ferments are complex bodies 
 about which very little is 
 known, except the effects they 
 produce. By their presence and 
 probably without entering into 
 intimate chemical combination, 
 they possess the power of 
 breaking up more highly organ- 
 ized nitrogenous and non-nitro- 
 genous compounds into simple 
 and more diffusible molecules. 
 They are termed enzymes or un- 
 formed ferments in contradis- 
 tinction to the bacteria them- 
 selves. That the action of fer- 
 ments is not due directly to the 
 organism is shown by the fact 
 that bactericidal substances, 
 such as phenol 5%, chloroform, 
 ether, etc., have no effect on 
 them and that cultures freed 
 from bacteria by filtration still 
 possess fermentative power. 
 
BACTERIOLOGY. 
 
 The action of ferments is termed 
 fermentation, but this term is 
 more especially limited to the 
 effect of certain ferments upon 
 non-nitrogenous compounds, par- 
 ticularly the carbohydrates. The 
 result of fermentation upon ni- 
 trogenous material is called 
 putrefaction, which generally 
 occurs with, though often with- 
 out, the formation of odorous 
 gases and other substances. 
 
 The intracellular origin of certain 
 ferments has been demon- 
 strated by their experimental 
 separation from the bacteria 
 when placed under high pres- 
 sures. The resulting bacteria- 
 free liquid possesses the same 
 fermenting qualities as the cul- 
 ture itself. 
 
 Ferments like toxins are of un- 
 known composition, are highly 
 destructible by chemical agents 
 and heat, and cause effects out 
 of all proportion to their bulk 
 or amount. They are frequent- 
 ly mechanically precipitated 
 with various indifferent bodies. 
 When injected into animals 
 both are capable of exciting the 
 formation of antibodies (anti- 
 ferments and antitoxins). 
 The principal ferments are: — 
 
 Proteolytic ferments. Transform- 
 ing albumins into more soluble 
 and diffusible substances. 
 
 These ferments find their analogy 
 in the ferments of the stomach 
 and pancreas. 
 
 They digest the various albumins 
 with the formation of album- 
 oses and those end products of 
 hydrolysis which are collective- 
 ly spoken of as peptones. 
 
 One form very often met with 
 liquefies gelatin. It acts in 
 an alkaline medium and is 
 therefore akin to the animal 
 ferment trypsin. This lique- 
 faction of gelatin affords a 
 means of distinguishing many 
 species of organisms. 
 
 Amylolytic ferments (amy las is 
 diastases) transform starches 
 into sugar and are found in 
 many bacterial cultures, e. g., 
 
BACTERIOLOGY. 13 
 
 B. mallei, B. pneumonia, etc. 
 They find their analogy in the 
 secretions of the pancreas and 
 the ptyalin of saliva. 
 InvertlniT ferments (Invertases.) 
 They are apparently related to 
 the amylolytic ferments and 
 are to a certain extent iden- 
 tical with them. They invert 
 disaccharides to monosacchar- 
 ides and according- to their spe- 
 cific action upon cane sugar, 
 maltose and lactose, they are 
 termed — invertases, maltases 
 and lactases, respectively. They 
 have their analogies in the sa- 
 liva and pancreatic juice. Such 
 ferments are found in cultures 
 of spirillum cholera and Metch- 
 nikovi. 
 
 Emulsifying' Ferments are formed 
 by but few bacteria. One ex- 
 ample is micrococcus pyogenes 
 tenuis. 
 
 Coag'ulating' Ferments. In these 
 we have a means of differenti- 
 ating bacteria by their co- 
 agulation of milk. This coagu- 
 lation is not due to acids pro- 
 duced in the medium but to the 
 action of a ferment. Some 
 varieties of bacteria produce a 
 ferment that has the power of 
 dissolving this coagulum when 
 formed (casease). Other bac- 
 teria produce both ferments — 
 the coagulating and the dis- 
 solving. 
 
 Jjipolytic Ferments. Fat splitting 
 ferments (lipases). 
 
 They cause hydrolysis of fats into 
 fatty acids and glycerin. They 
 have their analogues in the 
 pancreatic and gastric juice. 
 
 Ureases (hydrolytic) break up 
 urea into ammonium carbonate 
 and hippuric acid into glycocol 
 and benzoic acid. They are in 
 such bacteria as the micrococ- 
 cus urea, bacterium urea, bacil- 
 lus fluorescens, etc. 
 
 Oxidases. Produced by several 
 bacteria, especially the chrom- 
 ogens. They act upon complex 
 organic compounds changing 
 them to colored bodies. 
 
14 BACTERIOLOGY. 
 
 Beductases. This ferment is con- 
 tained by all but a few bacteria, 
 among- the exceptions being the 
 Bact. acidi lactici. They will 
 reduce nitrates to nitrites, sul- 
 phur to hydrog-en sulphide, etc. 
 
 Effects of Perments. The single 
 or combined action of these 
 various ferments cause cer- 
 tain kinds of fermentation 
 distinguished by the principal 
 substance produced. Alcoholic, 
 lactic acid, and butyric-acid 
 fermentation of the sugars, 
 acetic acid fermentation of al- 
 cohol (B. acidi lactice. B. buty- 
 ricus, B. acidi butyrici, B. 
 aceticus, etc.); cellulose fer- 
 mentation with the production 
 of carbonic acid gas and am- 
 monia; nitrification, in which 
 oxidation of ammonium leads to 
 the production of nitrites and 
 secondarily, conversion of ni- 
 trites into nitrates; mucoid fer- 
 mentation of grlucose and invert 
 sugar are examples. 
 
 Toxins will be taJien up later. 
 
 The study of those substances result- 
 ing from the actions of the life of 
 bacteria in the media of growth, is 
 accomplished by the so-called 
 
 Biochexuical Methods, 1. e.. 
 
 Test the cultivations of the or- 
 ganism for the presence of — 
 
 1. Soluble enzymes — protiolytic, di- 
 
 astic, invertase. 
 
 2. Org'anic acids — (a). Quantitative- 
 
 ly — i. e. Estimate the total 
 acid production. (b). Qual- 
 itatively, for formic, acetic, 
 propionic, butyric and lactic. 
 
 3. Ammonia. 
 
 4. Neutral Volatile substances — 
 
 ethyl, alcohol, aldehyde, acetone. 
 
 5. Aromatic products — Indol — 
 
 phenol. 
 
 6. Soluble pigrments. 
 
 7. Beducingr Powers (a) coloring 
 
 matters, (b) nitrates to nitrites. 
 
 8. Gas production — H2 S., CO2 H. 
 
 Estimate the ratio between the 
 last two ^ases. 
 Prepare all cultivations for these 
 methods of examination under 
 optimum conditions, previously de- 
 
BACTERIOLOGY. 15 
 
 termined for each of the organisms 
 it is intended to investigate as to — 
 
 (a) Reaction of medium. 
 
 (b) Incubation temperature. 
 
 (c) Atmospheric environment. 
 Keep careful records of these points, 
 
 also the age of the cultivation used 
 in the final examination. 
 
 Examine the cultivations for the var- 
 ious products of bacterial metabol- 
 ism after 48 hours growth, and 
 never omit to examine "control" 
 (uninoculated) tulbe or flask of 
 medium from the same Ibatch, kept 
 for a similar period under identical 
 conditions. 
 
 If the results are negative, test fur- 
 ther cultivations at 3, 5 and 10 days. 
 
 CHEMICAL CONSTITUENTS OF 
 THE BACTERIAL CELL. 
 
 The chemical composition of bacteria 
 varies with their food supply. 
 
 About 85% of the bacterial body is water. 
 The remainder is chiefly proteids 
 which constitutes from 50 to 85% of 
 the dry substance. After the proteid 
 material has been extracted there are 
 fats and in some instances true wax 
 (fatty acid combinations with higher 
 alcohols. In traces of cellulose and 
 ash the ash constitutes about 1 to 2% 
 of the dry substance, and is made up 
 largely of phosphates and chlorides 
 of potassium, sodium, calcium and 
 magnesium. 
 
 NUTRITION OF BACTERIA. 
 
 In order that bacteria may develop and 
 multiply, they must be supplied with 
 the substances of which they consist, 
 in proper quantity and in forms suit- 
 able for assimilation, namely, carbon, 
 oxygen, hydrogen, nitrogen, inorganic 
 salts and varying quantities of phos- 
 phorus and sulphur. 
 
 Carbon is necessary for their nourish- 
 ment, and may be obtained from 
 proteids, carbohydrates and fats, or 
 from their derivatives. 
 
 Ozygren. Free oxygen is necessary for 
 the growth of many bacteria 
 (obligatory aerobes). For these it is 
 obtained directly from the atmosphere 
 in the form of free O. Another class 
 of bacteria is unable to develop in the 
 
16 BACTERIOLOGY. 
 
 presence of free oxygen (obligatory- 
 anaerobes), and they obtain their 
 oxygen indirectly by the enzymatic 
 processes of fermentative and proteo- 
 lytic cleavage, from carbohydrates 
 and proteids, or by reduction from 
 reducible bodies. 
 
 There is still another large group of bac- 
 teria which develops well under both 
 aerobic and anaerobic conditions. 
 Some of these have a preference for 
 free oxygen, but will thrive without 
 it (facultative anaerobes). In others 
 the reverse is true (facultative 
 aerobes.) 
 
 Nitrosren is in most cases obtained from 
 proteids. The diffusible proteids are 
 the most important, but many of the 
 non-diffusible albumins may be rend- 
 ered assimilable by the proteolyzing 
 enzymes possessed by many bacteria. 
 
 A large number of bacteria may develop 
 on media containing no proteid, in 
 which case the organism produces a 
 synthetic proteid. 
 
 Many bacteria may obtain their nitrogen 
 from creatin, creatinin, urea and 
 urates and ammonia compounds and ni- 
 trates. 
 
 A few bacteria, as the bacilli in the root 
 tubercles of legumins and the nitro- 
 gen-fixing bacteria of the soil, obtain 
 their supply of nitrogen directly from 
 the free N of the air. 
 
 Hydrogren is obtained in combination as 
 water and together with the carbon 
 and nitrogen containing substances. 
 
 Salts. The phosphates are necessary 
 constituents of culture media and are 
 taken in as phosphates of magnesium, 
 calcium, sodium or potassium. 
 
 The chlorides are not absolutely essen- 
 tial (Proskener and Beck). 
 
 The sodium salts seem better for pur- 
 poses of cultivation than potassium 
 salts. 
 
 The sulphur is usually taken in as 
 soluble sulphates. The thiobacteria 
 of Winogradsky demand free H2 S. 
 
 The iron in the higher bacteria is taken 
 in as ferrous compounds and is oxid- 
 ized in the bacterial body into ferric 
 compounds. 
 
BACTERIOLOGY. 17 
 
 BIOLOGIC ACTIVITIES OF BACTERIA. 
 
 Without the bacterial processes, which 
 are constantly active in the reduction 
 of complex organic substances to their 
 simpler compounds, the chemical in- 
 terchange between the animal and 
 vegetable kingdom would fail and all 
 life would cease. 
 
 They are paramount factors in the cycle 
 of living matter supplying the links 
 in the constant circulation of nitrogen 
 and carbon compounds necessary be- 
 tween the plant and the animal king- 
 doms, through their anabolic or con- 
 structive process in the one and their 
 catabolic or destructive process in the 
 other. 
 
 The catabolic activities of bacteria con- 
 sist in the fermentation of carbohy- 
 drates and in the cleavage of proteids 
 and fats (see ferments or enzymes; 
 also denitrifying bacteria in nitrogen 
 cycle). 
 
 THE ANABOLIC OR SYNTHETIC 
 ACTIVITIES OF BACTERIA. 
 
 The depletion of the soil by constant 
 withdrawal of nitrogenous substances 
 by plants would soon be complete, 
 were it not for certain forces con- 
 stantly at work replenishing the sup- 
 ply from the free nitrogen of the air. 
 
 Bacteria, to a large extent, return nitro- 
 gen to the soil (see "Nitrogen fix- 
 ation," also "Nitrification" in nitrogen 
 cycle). 
 
 ^isrht Production by bacteria, is seen in 
 certain salt water forms. Much of 
 the phosphorescence observed at sea 
 is caused by bacteria. They are close- 
 ly allied to the putrefactive bacteria 
 and in the sea are usually found 
 upon rotting animal matter. 
 
 The light production is dependent upon 
 free access of oxygen and their lum- 
 inous quality is not a true phospho- 
 rescence in that it does not depend 
 upon previous illumination. 
 
 The formation of pigment by bacteria is 
 also a result of anabolic activity. 
 
 ANALYTIC CHANGES PRODUCED BY 
 MICRO-ORGANISMS. 
 
 It is the purpose to briefly outline the 
 changes induced, with particular 
 
18 BACTERIOLOGY. 
 
 reference to the so-called cycles of 
 certain of the elements and the part 
 played by microorganisms in inducing 
 these changes. The most important 
 elements to be considered are nitrogen, 
 carbon, sulphur and phosphorus. 
 1. The Cycle of Nitrogren in Nature. 
 Nitrogen is found in nature in 3 prin- 
 cipal forms: — Free in the air, as a 
 gas; in inorganic compounds such 
 as ammonia, nitrites, and nitrates; 
 and in organic compounds. 
 Microorganisms are important in 
 changing nitrogen from one form or 
 combination to another; in fact, 
 without their activity, it would be 
 impossible for higher animals and 
 plants to exist on the earth. It will 
 probably be most convenient to 
 start with complex organic nitro- 
 genous compounds in the study of 
 the nitrogen cycle and the changes 
 to be considered are: 
 (a) Ammonification, which is the 
 conversion of complex nitrogen- 
 ous compounds into simpler 
 forms, and ultimately into am- 
 monia. 
 
 This occurs in several distinct 
 stages. The complex protein 
 molecule is broken up into some- 
 what simpler compounds — the 
 » proteoses, and then into pep- 
 tones; (termed peptonization). 
 These peptones are broken down 
 by various organisms with the 
 formation pf polypeptids and 
 amino acid's primarily, and a 
 considerable number of second- 
 ry products. 
 
 The amino acids are still further 
 decomposed with the production 
 of ammonia. 
 
 The whole process of successive 
 cleavages of proteins is some- 
 times termed proteolysis. The 
 principal nitrogenous waste pro- 
 duct of the decomposition of 
 proteins in the body is urea. 
 This is actively transformed by 
 certain bacteria into ammonium 
 carbonate. It is evident that 
 all organic nitrogenous com- 
 pounds are ultimately reduced 
 to ammonia by the process of 
 Ammonification. 
 
BACTERIOLOGY. 
 
 19 
 
 This is of very great economic 
 importance in agriculture, as 
 nitrogen in this form is readily- 
 changed so as to become avail- 
 able to higher plants. 
 
 The various steps in ammonifica- 
 tion may be brought about by 
 different organisms. A few spe- 
 cies can attack native proteins; 
 many, however, can utilize and 
 change only the peptones, the 
 peptids and amino acids. The 
 organisms changing urea to am- 
 monium carbonate constitute a 
 very distinct group. 
 
 (b) Nitrification. (Nitrifying Bac- 
 teria). Certain microorganisms, 
 common in the soil, particularly 
 a coccus (nitrosococcus), are 
 able to oxidize ammonia to 
 nitrous acid. They secure their 
 energy for growth by this 
 change. The process is termed 
 nitrosation. Normally, the ni- 
 trous acid is neutralized, at 
 once, by the bases of the soil 
 thus forming nitrites. These 
 nitrites soon undergo the next 
 change, nitration, or oxidation 
 to nitrates by other species of 
 soil bacteria. The nitrates, and 
 to a less degree the ammonia, 
 constitute the source of nitro- 
 gen for higher plants. 
 
 No nitrogenous manure is effect- 
 ive in increasing crop yield that 
 is not capable of being ammoni- 
 fied and nitrified. 
 
 (c) Nitrog-en Assimilation is in 
 large part a function of the 
 higher plants. 
 
 The nitrates, and to a less degree 
 the ammonia, produced by bac- 
 terial activity in the soil are 
 taken up through the roots and 
 built up into protoplasm and 
 complex proteins. 
 
 These may decaj^ or they may be 
 eaten by animals, but ultimate- 
 ly they are decomposed by mi- 
 croorganisms. 
 
 This alternate synthesis of pro- 
 teins by higher plants and dis- 
 integration by microorganisms 
 constitutes the principal part of 
 the nitrogen cycle. 
 
BACTERIOLOGY. 
 
 (d) Denltrlfication. Nitrates, in the 
 absence of oxygen and in the 
 presence of organic matter, may- 
 reduce to nitrites by bacterial 
 activity, and these nitrites fur- 
 ther decompose with liberation 
 of free nitrogen. Under these 
 conditions, microorganisms take 
 the oxygen from the molecule of 
 nitrate or nitrite. Some species, 
 for example, will live under 
 anaerobic conditions if nitrates 
 are present, otherwise they are 
 aerobic. 
 
 This fact is of some significance 
 in agriculture in explaining loss 
 of fertility in water-logged soils. 
 
 (e) Kitrog'eii Fixation. If gaseous 
 nitrogen is lost from the cycle 
 as a result of denitrification, 
 there must be some method 
 whereby it can be again fixed or 
 combined. 
 
 A certain number of species 
 among the bacteria and moulds 
 are known to possess this 
 power. 
 
 Certain of the higher plants have 
 mould-like fungi which live up- 
 on their roots and take up the 
 atmospheric N (e. g. Alders, 
 Russian olives, and certain 
 other trees, the orchids and 
 many plants living in peat bogs 
 and swamps). 
 
 These organisms are termed my- 
 corrhizas. 
 
 The B. radicicoliiB, a minute bac- 
 terium, produces nodules or tu- 
 bercles on roots of many legum- 
 inous plants as the bean, pea, 
 olive and alfalfa. These swell- 
 ings are found to be made up of 
 cells tightly packed with bac- 
 teria. 
 
 These organisms take N from the 
 air and directly or indirectly 
 transfer it in part to the host 
 plant. Legumens, unlike most 
 plants, therefore, can grow in 
 soil devoid of N., provided the 
 roots are supplied with nodules. 
 The efficiency of legumens in in- 
 creasing the fertility of the soil 
 is due to this fixation of nitro- 
 gen. 
 
BACTERIOLOGY. 21 
 
 There are also a few living soil 
 bacteria which can take up 
 nitrogen from the air. These 
 belong to two groups, anaerobes 
 and aerobes; some spore bearing 
 soil bacilli (Clostridium), in the 
 presence of proper food, such as 
 certain carbohydrates, can fix 
 some nitrogen under anaerobic 
 conditions. These forms are not 
 very important in the soil. 
 
 Much more important are the 
 aerobic Azotobacter. These se- 
 cure energy for the fixation of 
 nitrogen by the oxidation of 
 carbohydrates. They are prob- 
 ably very important in soil fer- 
 tility. 
 
 2. Carbon Cycle. This cycle in 
 nature as affected by microorg- 
 anisms is more simple than that 
 of N. 
 
 It is well here to emphasize two 
 facts: — (1) All plants and an- 
 imals alike are continuously 
 developing CO2; (2) Some plants 
 can synthesize organic com- 
 pounds, principally carbohy- 
 drates and fats, from CO2. 
 
 All active cells are constantly 
 breaking down carbon com- 
 pounds; some can also build 
 them up. All plants containing 
 chlorophyll or leaf-green use 
 CO2 and water to produce starch 
 and sugars, gaining the energy 
 necessary by means of , the ab- 
 ■ sorption of sunlight. A few 
 bacteria containing bacterio- 
 purpurin are also capable of 
 using light for this purpose. 
 Some forms oxidize ammonia i/) 
 nitrites, nitrites to nitrates, 
 H2 S to sulphur or sulphur to 
 H2 SO4, and utilize the energy 
 thus secured in building up food 
 materials. 
 
 The carbon cycle, then, consists 
 of the alternate building up of 
 carbon into organic compounds 
 and their subsequent disintegra- 
 tion with ultimate oxidation of 
 the Carbon to CO2. 
 
 3. Stilphizr Cycle. The decomposi- 
 tion of organic compounds con- 
 taining sulphur usually results 
 in the evolution of H2 S. This 
 
22 BACTERIOLOGY. 
 
 is readily oxidized by many 
 aerobic bacteria with the pro- 
 duction of free sulphur and sul- 
 phuric acid. These organisms 
 are abundant in sewage and in 
 water of sulphur springs. In 
 these springs, they may form 
 masses of considerable size. The 
 sulphur granules may be seen 
 within the cells of the organism. 
 
 Reduction of Sulphur Compounds 
 with formation of H2 S occurs 
 when sulphates in the presence 
 of organic matter are subjected 
 to anaerobic conditions. 
 
 The sewage of some cities is very 
 offensive because the city water 
 contains sulphates in consider- 
 able quantity. Bacteria cause 
 decomposition of organic mat- 
 ter of the sewage reducing the 
 sulphate and the sulphite is 
 formed. 
 
 Phosphorus and Calcium Cycles 
 in nature show a change which 
 is influenced by microorganisms. 
 
 CLASSIFICATION OF BACTERIA. 
 
 Involution, Structure, Reproduction, 
 Biologrical Classification. 
 
 Bacteria are minute unicellular or- 
 ganisms which may occur free and 
 singular, or in larger or smaller ag- 
 gregations, thus forming multicellular 
 groups or colonies, the individuals of 
 which are, however, physiologically 
 independent. They occupy the lowest 
 plane of plant life. The position which 
 they occupy in plant life is shown 
 l>elow: — 
 
 Plants. 
 
 A. Cryptogramia (flowerless plants 
 forming spores). 
 
 1. Fteridophyta. e. g. ferns, horse- 
 
 tails and club mosses. 
 
 2. Bryophyta. e. g. liverworts and 
 
 mosses. 
 
 3. Thallophyta. e. g. 
 
 Myxomycetes (slime-fungi). 
 
 4. Scliizophyta (fission plants) 
 
 Schizophyceae (fission algae) 
 Schizomycetes (fission fungi 
 or Bacteria. 
 
BACTERIOLOGY. 23 
 
 Diatomea (diatomis), Chloro- 
 phyceae (green algae). 
 
 Rhodophycear (red algae), 
 Phaeophyceae (brown algae). 
 
 Characeae (stone worts), Hy- 
 pomycetes (fungi). 
 
 Lichens. 
 
 B. Fhanerog-amia^ (flowering plants, 
 forming seeds). 
 
 Bacteria, Schizomycetes, are classi- 
 fied by Migula into: — 
 1. Hubacteria, cells contain no 
 
 sulphur granules or bacterio- 
 
 purpurin. 
 
 1. Family Coccaceae, spherical 
 
 forms 
 Genus: 
 
 (a) Streptococcus, non-motile; 
 
 cells divide in one plane. 
 
 (b) Micrococcus, non-motile, 
 
 cells divide in two planes. 
 
 (c) Sarcina, non-motile; cells 
 
 divide in three planes. 
 
 (d) Planococcus, motile; cells 
 
 divide in two planes. 
 
 (e) Planosarcina, motile; cells 
 
 divide in three planes. 
 
 2. Family Bacteriaceae, straight, 
 
 rod-shaped forms without 
 envelope. 
 Genus: 
 
 (a) Bacterium, non-motile. 
 
 (b) Bacillus, motile; flagella 
 
 over whole surface. 
 
 (c) Pseudomonas, motile; flagel- 
 
 la polar. 
 
 3. Family Spirillaceae, curved 
 
 rod-shaped froms without 
 envelope. 
 Genus: 
 
 (a) Spirosoma, non-motile; cells 
 
 rigid. 
 
 (b) . Microspira, motile; one,' 
 
 rarely two or three polar 
 flagella. 
 
 (c) Spirillum, motile; polar 
 
 tufts of flagella. 
 
 (d) Spirochaeta, cells flexible. 
 
 4. Family Chalamydobacteriaceae, 
 
 cells with envelopes. 
 Genus: 
 
 (a) Chlamydothrix, unbranched 
 threads; cell-division in 
 one plane; (b) crenothrix 
 unbranched threads; cell 
 
24 BACTERIOLOGY. 
 
 division in three planes; 
 sheath visible. 
 
 (c) Phrag-midothrix, unbranch- 
 
 ed threads; cell-division 
 in three planes; sheath 
 scarcely visible. 
 
 (d) Sphoerotilus, branched 
 
 threads. 
 
 II. Thiobacteria, cells contain sulphur 
 granules or bacterio-purpurin; red 
 or violet color, never green. 
 
 1. Family Beggiatoaceae, thread- 
 
 forming, without bacterio- 
 purpurin. 
 Genus: 
 
 (a) Thiotrix, attached threads; 
 
 non-motile. 
 
 (b) Beggiatoa, unattached 
 
 threads; motile. 
 
 2. Family Rhodobacteriaceae, cells 
 
 contain bacterio-purpurin 
 and sulphur granules; red or 
 violet. 
 
 A. Subfamily Thiocapsaceae, cells di- 
 
 vide in three planes. 
 Genus: Triocystis. Thiocapsa. 
 Thiosarcina. 
 
 B. Subfamily Lamprocystaceae, cells 
 
 divide first in three, then in two 
 planes. 
 
 Genus: Lamprocystis. 
 Q. Subfamily Thiopediaceae, cells di- 
 vide in two planes. 
 
 Genus: Thiopedia. 
 
 D. Subfamily Amebobacteriaceae, cells ^ 
 
 divide in one plane. 
 Genus: Amebactor. Thiothece. 
 Thiodictyon. Thiopolycoccus. 
 
 E. Subfamily Chromatiaceae. 
 Genus: Chromatium. Rhabdochro- 
 
 matium. Thiospirillum. 
 
 A commonly used classification subdi- 
 vides bacteria into:* — 
 
 I. Xiower Bacteria, which are mi- 
 croscopic in size, multiply by 
 fission and contain no chloro- 
 phyll. 
 
 1. Cocci, are globular in form. 
 
 (a) Single coccus. 
 
 (b) Diplococcus. 
 
 (c) Staphylococcus. 
 
 (d) Streptococcus. 
 
 (e) Tetrads. 
 
 (f) Sarcina. 
 
 2. Bacilli, are straight rods, 
 (a) Long. 
 
BACTERIOLOGY. 
 
 25 
 
 (b) Short. 
 
 (c) Diplobacillus. 
 
 (d) Irregular. 
 
 3. Spirillae are curved or spiral 
 rods. 
 
 (a) Comma. 
 
 (b) Spiral. 
 
 II. Kig'lier Bacterlar, have a more com- 
 plex organization. They consist 
 of filaments built up of separate 
 individuals, some of which seem 
 related to physiologic labor and 
 some seem for the purpose of re- 
 production. 
 
 They possess the following charac- 
 teristics: They are attached, 
 unbranched, filamentous forms, 
 showing a differentiation between 
 base and apex; growth apparently 
 apical; exaggerated pleomorphism; 
 pseudo branching from opposition 
 of cells and are classified into — 
 Beggiota and thiothrix; free swim- 
 ming forms, which contain sul- 
 phur granules. 
 
 Crenothrix, cladothrix and lepto- 
 thrix do not contain sulphur gran- 
 ules. 
 
 Streptothrix; a group which exhibits 
 true but not dichtomous branch- 
 ing and contains some pathogenic 
 species. 
 
 Branched forms (normal though un- 
 usual) must not be confused with 
 involution forms. They are di- 
 vided into: — 
 
 1. True branching' — a bud springs 
 out from the bacteria, e. g. Bacil- 
 lus tuberculosis, and the bacillus 
 of diphtheria. 
 
 2. Dichotomous, which is often 
 confounded with true branching. 
 It is, however, a misnomer, as it 
 means a branching in two equal 
 parts. 
 
 3. Fseudodichotomous, or false 
 branching is due to the opposi- 
 tion of seperate organism. The 
 streptococci may produce false 
 branching by one cocci dividing at 
 right angles to chain and in this 
 way producing a new chain of 
 cocci which branches from the 
 original chain. 
 
26 BACTERIOLOGY. 
 
 INVOLUTION FORMS OF BACTERIA. 
 
 Degreneratiou Forms or 
 Fleomorphism. 
 
 Bacteria grown on artificial media, or 
 having- grown in the same media for 
 some time; i. e. under conditions not 
 favorable for their growth may show 
 abnormal or unusual shapes (pleo- 
 morphism). 
 
 Involution forms characterized by alter- 
 ations of shape are not necessarily 
 dead, but those forms characterized by 
 a loss of staining power are always 
 dead. 
 
 STRUCTURE. 
 
 Cell membrane or Capsule is a dense, 
 highly refractile, gelatinous outer 
 portion or covering of the cell wall 
 of some bacteria. It will absorb 
 moisture and swell. Organisms hav- 
 ing a capsule, when in suitable 
 solution, make the solution gelatin- 
 ous or slimy. This condition gives 
 rise to slimy bread and ropy milk. 
 The composition of the capsule may 
 be nitrogenous or non-nitrogenous. 
 Substances such as mucin, mannans, 
 . galactans and dextrins have been 
 identified. 
 
 An organism may produce a capsule 
 • under certain conditions only, as in 
 the blood, urine or milk, but not in 
 most culture media. 
 
 The membrane prevents certain bac- 
 teria, such as the streptococcus and 
 the staphylococcus, from becoming 
 separated, forming them into chains 
 or bunches. 
 
 Bacteria growing in gelatinous masses, 
 secreted by the cell, is known as 
 Zoogrloea. It can be seen in sewage 
 and on filter beds. 
 
 The capsule is not easily demonstrated 
 by the ordinary staining methods. 
 Cell wall lies between the capsule and 
 the cell protoplasm, from which it 
 is modified. Its chemical composi- 
 tion differs in different bacteria. 
 
 In some bacteria it is of a cellulose 
 reaction in others an albumin, and 
 in chemical composition it resembles 
 the chitin of the lower invertebrates. 
 
 All the food passes through it by 
 
BACTERIOLOGY. 
 
 27 
 
 diffusion, it having no selective 
 power. It can be easily demon- 
 strated. 
 
 Cell content is mainly protoplasm, com- 
 posed of mycoprotein. As a rule it 
 is homogeneous, but may contain 
 granules, fluid spaces, fat droplets, 
 pigment, sulphur and chlorophyll. 
 That portion next to the cell wall, 
 called ectoplast, is an important 
 structure as it has to do with nutri- 
 tion. In many bacterial cells it is 
 semipermeable, allowing some sub- 
 stances to pass through, inhibiting 
 others. A demonstration of its ac- 
 tion as an osmotic membrane may 
 be had by placing certain bacteria in 
 strong sugar solutions, causing the 
 protoplasm to shrink (plasmolysis). 
 A definite nucleus has not been demon- 
 strated, though the granules pres- 
 ent are probably nuclear in nature. 
 Their behavior during cell division 
 would probably indicate them to be 
 a primitive nucleus. 
 
 Metachromatic graniaes (e. g. diphthe- 
 ria) derive their name from their 
 ability to take up basic aniline dyes, 
 as does chromatin. 
 
 Sheath. Certain bacteria growing in a 
 chain secrete a firm membrane 
 (sheath) in such a way as to form 
 a tube in which the organism lives, 
 e. g. Chlamydothrix, Crenothrix and 
 Cladothrix substances, such as iron 
 or calcium compounds, may be de- 
 posited in this sheath. 
 
 MOBILITY. 
 
 1. Mobility by Plaffella, delicate hair- 
 like appendages which according to 
 some investigators are out-growths 
 of the cell membrane, or of the cell 
 wall itself. A greater majority, 
 however, believe them to be out- 
 growths through the cell membrane 
 from the protoplasm. They are 
 called: — 
 
 (a) Monotrichous, when situated 
 singly at one pole. 
 
 (b) Amphitrichous, when situated 
 singly at each pole. 
 
 (c) Lophotrichous, when situated 
 plurally at each pole. 
 
 (d) Peritrichous, when scattered 
 around the entire cell. They 
 
28 BACTERIOLOGY. 
 
 move the organism fast or slow 
 in any direction away from its 
 original position when first ob- 
 served. 
 
 They are very difficult to demon- 
 strate as they are very delicate, 
 easily break off and disintegrate. 
 Dark-field illumination and spe- 
 cial staining methods are re- 
 quired for their demonstration. 
 
 2. :Locomotion by undulatinsr mem- 
 branes has been observed in some 
 bacteria. 
 
 3. Amoeboid locomotion has been found 
 in rare instances. 
 
 4. Brownlan, vibratory or molecular 
 movement. The bacteria vibrate, 
 but do not change their position. 
 The movement is due to transmis- 
 sion by external physical causes. 
 
 REPRODUCTION. 
 
 Under this head is considered the:* — 
 
 1. Active Stagre (Vegetative), i. e. by 
 fission or simple cell division. When 
 conditions such as heat, moisture 
 and nutrition are favorable, to- 
 gether with the absence of the dele- 
 terious effects of other bacteria, or 
 
 , their products 
 
 (a) The cell becomes elongated and 
 the protoplasm aggregates at 
 opposite poles. 
 
 (b) The cell-wall constricts, usual- 
 ly midway between the proto- 
 plasmic aggregations, gradually 
 forming a septum in the interior 
 of the cell. 
 
 (c) The septum divides the cell into 
 two equal parts. 
 
 (d) The daughter cells may remain 
 united by the gelatinous en- 
 velope for a variable time. 
 Eventually they separate and 
 they themselves subdivide. 
 
 This division may take place in 
 one, two or three planes, de- 
 pending upon the nature of the 
 organism. 
 
 Division may be completed in less 
 than 30 minutes. 
 
 2. Resting Stagre (Spomlation). 
 Spore formation, is endogenous 
 (Endosporous) or Arthrogenous 
 (Arthrosporous). 
 
BACTERIOLOGY. 29 
 
 Tike requistes for spore formation were 
 once supposed to be: — 
 
 (a) An exhaustion of nutriment. 
 
 (b) The generation within the me- 
 dium of toxic material from the 
 accumulation of metabolic pro- 
 ducts. 
 
 (c) The environment becomes un- 
 favorable, e. g., temperature. 
 
 In other words, when conditions 
 became such that the cell could 
 no longer maintain life, the 
 organism turned itself into a 
 spore in order that it might 
 escape annihilation. 
 
 This is not altogether correct, as 
 sporulation takes place only 
 when conditions present are 
 most favorable to the well — 
 being of the cell. The tempera- 
 ture at which spores are best 
 formed is constant for each 
 org^anism, but varies with the 
 different species, aerobes re- 
 quire oxygen for sporulation but 
 anaerrobes will not spore in its 
 presence. 
 
 Endogenous spore formation. The pro- 
 toplasm of the cell becomes differ- 
 entiated and concentrates into a 
 a small granule which increases in 
 size, or several granules are formed, 
 which coalesce and grow to form 
 an oval or rarely cylindrical mass. 
 Further contraction takes place, 
 the outer layers become still more 
 differentiated and form a distinct 
 spore membrane. Some authorities 
 maintain that the spore membrane 
 consists of two layers, the ex- 
 osporium and the endosporium. 
 The spore is now a clearly defined 
 
 highly refractile body. 
 The cell contains but one spore, situ- 
 ated usually in the middle, occasion- 
 ally at one end (four exceptions 
 have been recorded, e. g. B. inflatus). 
 It is of the same diameter, or a lit- 
 tle less, as that of the cell itself. 
 
 The shape of the parent cell may be 
 unaltered (e. g. B. anthrax) or al- 
 tered (e. g. B. tetanns), and this 
 serves for a classification of spore- 
 bearing bacilli: viz: — 
 (a) Cell body unaltered in shape. 
 
30 
 
 BACTERIOLOGY. 
 
 (b) Cell body altered in shape. The 
 terms applied to each are: — 
 
 (1) Clostridium. (Spindle shape). 
 Swollen at the center and thin 
 at the poles. 
 
 (2) Cuneate. (Wedge-shape). 
 
 (3) Clavate. (Key-hole shaped). 
 Swollen at one pole and unal- 
 tered at the other. 
 
 (4) Capitate. (Drum-stick shaped). 
 The endospores remain within the cell 
 
 for a variable time, but are eventu- 
 ally set free by the swelling up and 
 the solution of the cell membrane of 
 the parent by the surrounding 
 liquid or by the rupture of the mem- 
 brane. The spore now presents the 
 following characteristics: — 
 
 (a) Well formed, dense cell mem- 
 branes, rendering their staining 
 difficult; and when stained, 
 equally difficult to decolorize. 
 
 (b) Highly refractile, which differ- 
 entiates it from vacuoles. 
 
 (c) Higher resistance (spore re- 
 sistance) than the parent or- 
 ganism on account of the low 
 water content of plasma, low 
 heat conducting power and the 
 low permeability of the spore 
 membrane to such lethal agents 
 as chemicals, light, heat, desic- 
 cation, starvation, time, etc., 
 this resistance varying some- 
 what with the particular spe- 
 cies. 
 
 Bacteria grown on media poor in 
 nutrient material are likely to be- 
 come asporogrenOTis ; i. e., they be- 
 come sterile and do not produce 
 spores. This condition may be 
 temporary or permanent. 
 
 Arthrogrenous spore formation is seen 
 only in the micrococci. One com- 
 plete element resulting from fission 
 becomes differentiated for this pur- 
 pose, enlarges, and developes a 
 dense cell wall. This process is 
 probably not real spore formation 
 but a relative increase in resistance. 
 They have never been seen to germ- 
 inate, nor is their resistance very 
 marked, as they fail at culture after 
 having been exposed to 80° C tem- 
 perature for 10 minutes. 
 
 Spore Germination. When placed under 
 favorable conditions of heat, mois- 
 
BACTERIOLOGY. 51 
 
 ture, nutrition, etc., the spores 
 germinate, usually within 24 to 36 
 hours and successively undergo the 
 following- changes: — 
 
 (1) Swell up slowly and enlarge, 
 through absorption of water. 
 
 (2) Lose their refrangibility (grow 
 dull). 
 
 (3) One of the following processes 
 is observed (a particular pro- 
 cess is constant for the same 
 species) : — 
 
 (a) The spore grows out into the 
 new organism without throwing 
 off its membrane. 
 
 (b) It loses its spore membrane by 
 solution. 
 
 (c) It loses its spore membrane by 
 rupture. 
 
 (d) Endo-germination. The spores 
 germinate within the parent 
 body. The germinal rod be- 
 comes detached, leaving the 
 empty capsule within the 
 parent. 
 
 The rupture may be polar or 
 
 equatorial. 
 The polar rupture may take place 
 
 at one pole only or at both 
 
 poles. 
 
 In the cases where the spore 
 membrane is discarded, the cell 
 membrane of the new bacillus 
 may be formed from: — 
 
 (a) The inner layer of spore mem- 
 brane, which has been split up 
 into a parietal and visceral 
 layer. 
 
 (b) The outer layers of the cell 
 protoplasm, which has become 
 differentiated for that purpose. 
 
 The new organism now increases 
 in size, elongates and takes on 
 vegetative growth. 
 Foxrmatioxis of Gonidia. In the higher 
 bacteria (filamentous bacteria), as 
 in Mycobacteriaceae, a number of 
 specialized cells or spores are form- 
 ed (short rods or coccoid forms) by 
 multiple segmentation or differen- 
 tiation, usually at the free tip of 
 the filament, and are termed gonidia 
 (conidia). 
 
 They may be termed resting bodies, 
 as they remain dormant for a vari- 
 able period until favorable condi- 
 tions are brought about, when they 
 
32 BACTERIOLOGY. 
 
 elongate and produce the vegetative 
 form from which they arose. 
 
 Many of these gonidia have been con- 
 sidered as degenerative forms, but 
 this is unlikely as degenerative 
 elements would not produce new 
 vegetative cells. According to A. 
 Coffen Jones, tubercle bacilli pro- 
 duce gonidia. 
 
 The resistance of the diphtheria or- 
 ganism to unfavorable conditions 
 would make it likely that the gran- 
 ular segments so often produced are 
 of the nature of gonidia. 
 
 BIOLOGICAL CLASSIFICATION. 
 
 1. Bacteria are classified according to 
 their life functions into: — 
 
 (a) Saprog-enic. (Saprophytes), or 
 putrefactive bacteria, are those 
 that live only on dead organic 
 matter. 
 
 (b) Zymog'enlci, f>r fermentative 
 bacteria, are those which pro- 
 duce soluble ferments or en- 
 zymes during the course of their 
 growth. The ferments possess 
 the power of breaking up more 
 highly organized nitrogenous 
 and non nitrogenous compounds 
 
 » into simple and more diffusible 
 
 substances. The action of fer- 
 ments upon non-nitrogenous 
 compounds is called fermenta- 
 tion. The action of ferments 
 upon nitrogenous compounds is 
 called putrefaction, often pro- 
 ducing odorous gases and 
 ptomaines, which are complex 
 alkaloids resembling those 
 found in plants. 
 The principal bacterial ferments 
 are: — 
 
 Proteolytic (Converts proteins 
 into proteose, peptone and 
 further products of hydrolysis). 
 
 Diastase (Converts starches into 
 sugar). 
 
 Znvertase (Converts saccharose 
 into a mixture of dextrose and 
 levulose). 
 
 Sennin or coagroIatiniT (Coagu- 
 lates milk independent of the 
 action of acids). 
 
 (c) Pathofifenic, or disease produc- 
 ing bacteria, are those causing 
 
BACTERIOLOGY. 
 
 33 
 
 various pathologrical conditions 
 and producing- the diseases 
 known as (infectious diseases). 
 Bacteria are classified according to 
 their food requirements into: — 
 
 (a) PrototropMc, (e. g. nitrifying 
 bacteria) are those which re- 
 quire no organic food. 
 
 They change albuminoids into 
 skatol, indol, leucin, and these 
 into nitrites and nitrites into 
 nitrates. 
 
 (b) Met atrophic, (e. g. saprophytes 
 and facultative parasites), are 
 those which require organic 
 food. 
 
 The saprophytes are easily culti- 
 vated; some will grow in al- 
 most pure distilled water and 
 some will grow in pure solu- 
 tions of carbohydrates. 
 
 The facultative parasites need 
 highly organized foods as pro- 
 teids or other sources of nitro- 
 gen and carbon and salts. 
 
 (c) Paratrophlc, (e. g. obligate 
 parasites) are those which re- 
 quire living food. They will 
 not live outside the living body. 
 
 Bacteria are classified according to 
 their metabolic products into: — 
 (a) Chromogrenic, or pigrment-pro- 
 ducing bacteria, are those which 
 produce vivid pigments (yellow, 
 orange, red, violet, fluorescent, 
 etc.,) during the course of their 
 life and growth. The coloring 
 matter is usually an intercell- 
 ular excrementitious substance; 
 though it occassionally appears 
 to be stored within the body of 
 the organism. They are there- 
 fore classified into: — 
 Chromoparous bacteria, when the 
 pigment is diffused out upon 
 and into the surrounding 
 medium. 
 
 Chromophorous bacteria, when the 
 pigment is stored in the cell 
 protoplasm of the organism. 
 
 Parachromophorous bacteria, when 
 the pigment is stored in the cell 
 wall of the organism. 
 
 Different species of chromogenic 
 bacteria differ in their require- 
 ments as to environment for the 
 production of their character- 
 
34 
 
 BACTERIOLOGY. 
 
 istic pigrments; some need oxy- 
 gren, light or high temperatures; 
 others favor the opposite condi- 
 tions. 
 
 (b) Photogrenlc, or light-producing 
 bacteria, are those which exhibit 
 phosphorescence when culti- 
 vated under suitable conditions. 
 
 (c) Aerog'enlc, or gas producing 
 bacteria, are those which pro- 
 duce hydrogen, carbon dioxide 
 and sulphuretted hydrogen, etc. 
 
 Toxins. Many bacteria, especially the 
 pathogenic, elaborate or secrete 
 poisonous substances, concerning 
 which little exact knowledge is 
 available, though many appear to 
 be enzymic in their action. They 
 seem to be akin to the venom of 
 serpents and other animals and to 
 certain poisonous principles of 
 plants. 
 
 It has been estimated that 1-1000 gm. 
 of tetanus toxin will kill a horse 
 weighing 1,200 pounds. They were 
 first called ptomaines or cadaveric 
 alkaloids, but this term is now ap- 
 plied to poisons which form in de- 
 composing meat, cheese, etc., as a 
 result of chemical change caused by 
 bacteria; they have also been termed 
 > toxalbumlns, as they give all the 
 reactions of albumin. It is probable, 
 however, that a toxalbumin is but 
 a combination of the toxin and the 
 substances derived from the me- 
 dium of growth. 
 
 A certain group of toxins are retained 
 within the organism and are only 
 set free after its death. 
 
 Toxins are usually divided into: — 
 
 Xntracellnlar (inseparate) are those 
 which are bound up with the pro- 
 toplasm of the organism. 
 
 No means has as yet been devised for 
 their separation of extraction. Anti- 
 bacterial seram is used to combat 
 this type. 
 
 Sxtracellnlar (soluble) are excreted 
 by the organism and are diffused 
 into and held in solution by the sur- 
 rounding medium. 
 
 Anti-toxin serum is used to combat 
 this type. 
 
 End-products of metabolism are or- 
 ganic acids (lactic, butyric, pro- 
 pionic, benzoic, formic, acetic, oxalic. 
 
BACTERIOLOGY. 
 
 35 
 
 succinic, salicylic, gallic and tan- 
 nic), alkalies (ammonia), aromatic 
 compounds (indol, phenol), reducing 
 substances (nitrates to nitrites), 
 and gases (sulphuretted hydrogen, 
 carbon dioxide, etc.) 
 Growth. Certain conditions are neces- 
 sary to the life and growth of 
 bacteria; any marked change in 
 these conditions will inhibit the 
 growth or destroy them. Water is 
 absolutely essential for their growth. 
 
 1. Influence of atmosphere. Certain 
 bacteria require oxygen for their 
 growth and death will follow if this 
 is not available. They are termed 
 olillg'ate aerobes. 
 
 A certain group of bacteria will thrive 
 equally well with or without oxygen. 
 They are termed facultative anae- 
 robes. 
 
 Certain bacteria live and multiply 
 only when there is complete exclu- 
 sion of free oxygen. They are 
 termed obllgrate anaerobes. 
 
 2. Influence of heat. A temperature 
 of from 10'' to 40° C is necessary 
 to the life and growth of bacteria. 
 Practically no growth occurs below 
 ^° C, and very little above 40° C. 
 The most favorable temperature for 
 the majority of microorganisms is 
 from 30° C to 37° C. Saprophytes 
 grow between 0° and 30° C, the 
 optimum being 15° to 20° C. 
 
 Parasites, flourish between 10° and 45° 
 but best at body temperature, 37° C. 
 
 The maximum and minimum temper- 
 atures at which growth takes place, 
 as well as the optimum, are fairly 
 constant for each bacterium. They 
 may be classified, according to their 
 optimum temperature, into: — 
 
 MIN. OPT. MAX. 
 
 (a) Psychrophilic 
 (chiefly water 
 
 organisms) . . 0° C. 15° C. 30° C. 
 
 (b) Mesophi 1 1 c 
 (includes 
 pathogenic 
 
 forms) 15° C. 27° C. 45° C. 
 
 (c) Thermophylic 
 
 bacteria 45° C. 55° C. 70° C. 
 
 Each bacterium has its own Thermal 
 death point. 
 
36 BACTERIOLOGY. 
 
 The "thermal death point" of an or- 
 ganism is that temperature which 
 causes the death of the vegetative 
 forms when the exposure is con- 
 tinued for a period of 10 minutes. 
 
 It is between 50° and 60° C in the 
 most pathogenic, while below the 
 lower limit their growth is only 
 inhibited. An exposure to 250° C. 
 has been made without preventing 
 the organisms future development. 
 
 Spores are extremely resistant; some 
 are killed only after an hour's ex- 
 posure to 115° C. 
 
 3. Influence of ligrlit. Many organisms 
 are indifferent to the presence of 
 light. On the other hand 
 
 Daylight frequently inhibits the 
 growth and alters to a greater or 
 lesser extent the biochemical char- 
 acters of the organisms; e. g., 
 chromogenicity or power of lique- 
 faction. Pathogenic bacteria under- 
 go a progressive loss of virulence 
 when cultivated in the presence of 
 daylight. 
 
 Direct sunlight destroys them as does 
 also electric light, but to a less ex- 
 tent. Violets rays are very effective 
 in the destruction of bacteria. 
 .4. Influence of electricity. Electrical 
 currents inhibit or destroy the 
 growth of bacteria, not directly, 
 but probably by the products of 
 electrolysis. 
 
 Roentgen rays are bactericidal to 
 bacteria in living tissues, but have 
 little effect on cultures. 
 
 5. Influence of movements. Movements, 
 if slight and of a flowing character, 
 do not seem to affect the growth of 
 bacteria, but violent shaking kills 
 them. 
 
 CULTIVATION OF BACTERIA. 
 
 Culture Media, Tubing* Media, 
 Sterilization. 
 
 As it is difficult and sometimes impos- 
 sible to study the growth of bacteria 
 m their natural habitat, it becomes 
 necessary to isolate individual mem- 
 bers of microorganisms, to observe 
 their growth, morphology, phenomena, 
 etc., by their cultivation on artificial 
 nutrient naedia. 
 
BACTERIOLOGY. 
 
 37 
 
 APPARATUS BEQUIBEB. 
 
 Test tubes. Several sizes should be 
 kept in stock. The ordinary tubes 
 in most use are the %"x5" Board 
 of Health tubes. Small tubes 5x0.9 
 cm. for use in inverted position in- 
 side tubes containing- carbohydrate 
 media as gas collecting tubes. 
 
 Plorence Plasks of 250,500 and 1000 
 cc. capacity will be found very con- 
 venient. 
 
 Erlenmeyer Plasks, with narrow neck 
 of 75, 100, 150 and 250 cc. capacity. 
 
 Petri Dishes or Plates, 1.5 cm. high X 
 10 cm. diameter put up in bundles 
 of 6, wrapped in paper or cloth, 
 sterilized and put aside for use. 
 They can also be put up in specially 
 prepared metallic boxes. 
 Pipettes of 1 cc. plain, 1 cc. gradu- 
 ated in 0. 1 cc, and .01 cc. capacity; 
 also pipettes of 10 cc. capacity 
 g-raduated in .1 cc. Each variety 
 should be stored in large test tubes 
 or in special metallic boxes, steril- 
 ized and put aside for use. 
 
 Capillary pipettes (Pasteur's) are pre- 
 pared from small bore soft glass 
 tubing, heated and pulled out to a 
 fine capillary tube at one end. 
 
 Blood pipettes (Pakes) are made, from 
 1 cm. bore soft glass tubing-, in a 
 manner similar to Pasteurs, except 
 that they are pulled out at both 
 ends. Wright's tubes are similar 
 to Pakes' except that one end is 
 turned at an angle. They are stored 
 in test tubes, sterilized and put 
 aside for use. 
 
 Permentation tubes, used for the col- 
 lection and analysis of gases liber- 
 ated from media during the growth 
 of some bacteria. They may be 
 plain or graduated. They are 
 plugged with cotton and sterilized. 
 
 Platinum wire, fitted into a glass or 
 aluminum handle, to be used for 
 inoculations. 
 
 CULTURE MEDIA. 
 
 The greater number of these media are 
 primarily fluid, but in order to bet- 
 ter study the characteristics of in- 
 dividual organisms, through their 
 colonies many media are therefore 
 
BACTERIOLOGY. 
 
 rendered solid by the addition of 
 substances like grelatin or agar in 
 varying proportions. Gelatin is 
 employed for the solidification of 
 those media on which it is intended 
 to cultivate bacteria at room tem- 
 perature or in the "cold" incubator. 
 Gelatin, in the precentage usually 
 employed, becomes liquid at 25° C. 
 
 Agar, in the precentage usually em- 
 ployed, only becomes liquid when 
 exposed to 90° C for a considerable 
 period and again solidifies at 40° C. 
 
 Such media is spoken of a liquefiable 
 media. Other media as potato, co- 
 agulated blood serum, etc., can not 
 be again liquefied and are therefore 
 spoken of as solid media. 
 
 Meat Extract forms the basis of sev- 
 eral of the nutrient media and is 
 prepared as follows: — 
 
 1. Add to 1000 cc. distilled water, in 
 
 an enameled pot, 500 gms. of 
 finely minced fresh lean meat. 
 
 2. Heat gently in a water bath, at a 
 
 temperature that at no time ex- 
 ceeds 40° C, for 20 minutes; this 
 will dissolve out the soluble pro- 
 teids, extractives, salts, etc. 
 
 3. Raise the temperature of the mix- 
 
 ture to boiling and maintain for 
 10 minutes; this precipitates 
 some of the albumins, haemog- 
 lobin, etc., from the solution. 
 
 4. Strain through muslin (sterile) 
 
 or a perforated porcelain fun- 
 nel, then filter through paper 
 into a sterile flask and when 
 cool make up loss by evapora- 
 tion to 1000 cc. with distilled 
 water. 
 
 5. If not needed at once, sterilize 
 
 for 20 minutes on 3 consecutive 
 days. 
 
 Wyeth's beef-juice, or Liebig's 
 extract of meat, 3 gms. to 1000 
 cc. of distilled water heated and 
 filtered as above, may be sub- 
 stituted, except where the more 
 highly parasitic bacteria are to 
 be cultivated. 
 The Reaction of Meat Extract as pre- 
 pared above is always acid, due to 
 acid phosphates of potassium and 
 sodium, acids of the glycollic series, 
 and acid organic compounds. 
 
BACTERIOLOGY. 
 
 39 
 
 Prolonged boiling causes the extract 
 to undergo hydrolytic changes 
 which increase its acidity. It 
 should therefore be boiled for at 
 least 45 minutes, when it will be- 
 come stable, and the total acidity is 
 to be estimated when the solution 
 >v is at the boiling point. 
 
 The meat extract reacts acid to 
 phenolphthalein, though it may re- 
 act neutral or alkaline to litmus, 
 due to 
 
 (1) The insensitiveness to some or- 
 
 ganic acids. 
 
 (2) The formation of dibasic sodium 
 
 phosphate, formed during the 
 process of neutralization. 
 STANDARDIZING THE REACTION. 
 
 1. Fill a burette with standardized 
 
 n/20 NaOH. 
 
 2. Measure out 5 cc. of media and 
 
 45 cc. distilled water into a 
 beaker (should be at a temper- 
 ature of 100° C). 
 
 3. Add to the contents of the beaker, 
 
 5 drops of a 0.5% (50% alcohol) 
 solution of phenolphthalein. 
 
 4. From the burette, run the n/20 
 
 NaOH solution carefully into 
 the test media, constantly stir- 
 ring until the end-point is 
 reached, as indicated by a deep- 
 rose color. 
 
 5. Read ofC the amount of NaOH 
 
 solution required to neutralize 
 the 5 cc. of media. 
 
 6. Verify the reaction by another 
 
 titration. 
 
 7. Calculate the amount of standard- 
 
 ized normal NaOH it will take 
 to neutralize the remaining 990 
 cc. of media. 
 (For all practical purposes it can 
 be estimated as still having 
 1000 cc. of media and adding the 
 normal NaOH, viz: — if it re- 
 quires 5 cc. of the n/20 NaOH 
 to neutralize 5 cc. of the media, 
 then 50 cc. of the normal NaOH 
 will be required to neutralize 
 the 1000 cc. of media. In other 
 words, move the decimal point 
 one to the right, e. g., burette 
 reading is 5.3 cc, then 53. cc. 
 will neutralize the 1000 cc. of 
 media). The sign + (plus) is 
 prefixed to the media if it is 
 
40 
 
 BACTERIOLOGY. 
 
 acid and the sign — (minus) 
 if it is alkaline, e. g., media + 
 10 indicates that it reacts acid 
 to phenolphthalein and would 
 require the addition of 10 cc. 
 normal NaOH per 100 cc. for 
 neutralization. 
 8. Titrate again the neutralized 
 media to insure results. In as 
 much as the titration for a last 
 control is often wanted, it may 
 be well to use a deka-normal 
 NaOH for the neutralization of 
 the bulk, so as not to bring the 
 total quantity of media greatly 
 above the original 1000 cc, as 
 might be the case if the N/20 
 or normal NaOH were used. 
 Nearly all bacteria have a well 
 marked "optimum reaction" 
 which happily approximates 
 close to + 10, therefore this 
 standard may be used for all 
 media unless otherwise indi- 
 cated. 
 
 The standardizing 1000 cc. of 
 media to + 10 is accomplished 
 by merely subtracting 10 of the 
 NaOH from the initial calcu- 
 lation. This renders the reac- 
 tion -f 10. 
 
 FILTRATION OF MEDIA. 
 
 Flnld Media are filtered through filter 
 paper folded in the "physiological- 
 filter form so as to accelerate the 
 rate of filtration. 
 
 ^Iquefiable Media are filtered through 
 "paper Chardin," which is a special- 
 ly made filter paper. 
 Gelatin if made properly will filter 
 
 through this paper readily. 
 Agar, likewise, if properly made will 
 filter readily, but not so rapidly as 
 gelatin. 
 
 A special hot-water jacket has been 
 constructed to surround the glass 
 funnel; the temperature of the 
 water in the jacket is maintained 
 at 90° C. and facilitates the filtra- 
 tion. If care is taken the liquefiable 
 media can be filtered through ab- 
 sorbent cotton efficiently. 
 
BACTERIOLOGY. 
 
 41 
 
 STOCK MEDIA. 
 
 Bouillon. Put 500 cc. double strength 
 meat extract into a litre flask and 
 add 300 cc. distilled water. 
 Mix 10 gms. peptone and 5 gms, salt 
 into a smooth paste with 200 cc. of 
 distilled water previously heated to 
 60° C. Add the emulsion to the 
 meat extract and heat in the Arnold 
 for 45 minutes to dissolve the pep- 
 tone and to render the acidity of 
 the meat extract stable. Estimate 
 the reaction and control the results. 
 Heat agrain in Arnold for 30 minutes 
 to completely precipitate the phos- 
 phates. Filter through paper into 
 flask. Sterilize, or tube and ster- 
 ilize. 
 
 Ag'ar=Ag'ar. (Agar is derived from sea 
 plants along the coast of Japan. It 
 has some of the properties of 
 gelatin, but is less affected by heat). 
 Weigh a 2 litre double Agate ware 
 boiler and note it. Put 500 cc. dou- 
 ble strength meat extract into the 
 boiler. Mix 10 gms. of peptone, 
 5 gms. of salt into a paste with 
 150 cc. distilled water. Add the 
 paste and 15 to 20 gms. of Agar 
 (powdered if available) to the meat 
 extract. Heat over flame to 100° C. 
 for 25 minutes (stirring constantly) 
 or more for complete solution of 
 Agar. Weigh the pan and to Its 
 contents add enough water to make 
 up the bulk of the medium to 1 
 litre. 
 
 Titrate, control the result, calculate 
 the amount of soda solution re- 
 quired to make the medium + 10 
 and add it to the medium. Place in 
 the Arnold or over the flame for 20 
 minutes to complete the precipita- 
 tion of the phosphates, etc. 
 
 Cool the medium to 60° C, add the 
 whipped white of two eggs, place 
 it over the gas burner or in Arnold 
 until the egg-albumin has formed 
 into a firm floating mass. Filter 
 through paper, tube and sterilize. 
 Gelatiji is used for determining the pro- 
 teolytic ferments of bacteria by its 
 liquefaction. Other distinctions ar# 
 also met with. 
 
42 
 
 BACTERIOLOGY. 
 
 Weigrh a 2 litre double Agate boiler 
 and note it. Put 500 cc. double 
 strength meat extract into the 
 boiler. Mix 10 gms. of peptone, 5 
 gms. of salt into a paste with 150 
 cc. distilled water. Add the paste 
 and 100 to 150 gms. sheet gelatin 
 (cut into small pieces) to the meat 
 extract. Heat over flame to 100° C. 
 for 10 minutes (stirring constantly 
 till there is complete solution of the 
 gelatine. Weigh the pan and its 
 contents and add enough water to 
 make up the bulk of the medium to 
 1 litre. Titrate, control the result, 
 calculate the amount soda solution 
 required to make the medium + 10 
 and add it to the medium. Place 
 in the Arnold or over the flame for 
 20 minutes to complete the precipi- 
 tation of the phosphates, etc. Cool 
 the medium to 60° C, add the 
 whipped white of two eggs, place 
 it over the flame or in the Arnold 
 until the egg albumin has formed 
 into a flrm floating mass. Filter 
 through paper, tube and sterilize. 
 
 Blood Serum. The blood is collected 
 at the slaughter house in sterile 
 glass cylinders and allowed to stand 
 for 15 minutes to form clot to pre- 
 vent the serum from being stained 
 with haemoglobin. When removed 
 to the laboratory the clot is separ- 
 ated from the sides of the cylinder 
 by a sterile glass rod and placed in 
 the ice chest for 24 hours. The 
 serum is then drawn out with 
 sterile pipettes and placed in sterile 
 test tubes (5 cc. in each). The 
 tubed serum is heated on two suc- 
 cessive days. The third day, 
 heat the tubes in a slanting posi- 
 tion in a serum inspissator to 
 about 72° C. which coagulates the 
 serum. Place the tubes in the in- 
 cubator at 37° C for 48 hours to 
 eliminate the tubes that have been 
 contaminated. Store in a cool place. 
 The serum can be sterilized by the 
 fractional method by exposure in a 
 water bath to a temperature of 56* 
 C. for 30 minutes on each of 6 con- 
 secutive days. Store in the fluid 
 condition and coagulate in the in- 
 spissator when needed. 
 
BACTERIOLOGY. 43 
 
 Guy's Citrated Blood Agrar. A small 
 rabbit is killed by chloroform, 
 nailed out on a board, hair moisten- 
 ed thoroug-hly with 2% solution of 
 lysol. skin is reflected (with sterile 
 instruments) over the thorax, thorax 
 opened (sterile), pericardium opened 
 (sterile), surface of left ventricle 
 seared with hot iron, the point of a 
 sterile capillary pipette is thrust 
 through the wall of the ventricle, 
 pipette filled with blood by suction, 
 transfer the blood to a small 
 Erlenmeyer flask containing a num- 
 ber of glass beads and 5 cc. con- 
 centrated sodium citrate solution, 
 agitate, set aside for 2 hours, with 
 a sterile 10 cc. graduated pipette, 
 transfer 1 cc. citrated blood to a 
 tube of liquefied agar, mix, allow 
 agar to cool in slanting position, 
 place tubes in incubator for 48 
 hours, after which time store the 
 uncontaminated tubes for future 
 use. 
 
 Potato. Cylinders are cut out of a well 
 washed peeled potato. The cyl- 
 inders are cut obliquely from end to 
 end, forming them into wedges. 
 The fresh potato is strongly acid 
 and in order to approximate + 10 
 the cylinders are placed in 1% solu- 
 tion of sodium carbonate for 30 
 minutes. Each wedge is placed in 
 a test tube into which has been pre- 
 viously inserted a piece of ab- 
 sorbent cotton moistened with 
 sterile water, with its base resting 
 upon the cotton. The tubes are 
 then replugged and sterilized in 
 Arnold on each of 3 consecutive 
 days. 
 
 The acid of the potato can also be 
 abstracted by placing the wedges in 
 running water for 24 hours. 
 
 Dorset's Effgr. Sterilize in the autoclave 
 for 20 minutes 1 litre of a .85% 
 solution of sodium chloride and cool 
 to 20^ C. Wash 12 fresh eggs with 
 water, then with pure formaline and 
 allow them to dry. Break the eggs 
 into a sterile graduate, noting their 
 total volume. Add the salt solution 
 to the eggs in proportion of 1 to 3. 
 Whip the mixture with an egg- 
 whisk thoroughly and filter through 
 
44 
 
 BACTERIOLOGY. 
 
 coarse muslin into a sterile flask. 
 (A few drops of alcoholic solution 
 of basic fuchsin to Rive a definite 
 pink color, or a few drops of water 
 proof Chinese ink added to the 
 medium at this stag-e will facilitate 
 the subsequent "fishing of col- 
 onies"). 
 
 Tube and solidify at a slant in the 
 inspissator at 75° C. for one hour. 
 Incubate for 48 hours and eliminate 
 the contaminated tubes. The sterile 
 tubes are capped with rubber caps 
 and stored for future use, 
 
 Hgg. A number of eggs are broken into 
 a vessel and thoroughly mixed with 
 a little water, tubed and sterilized 
 at a slant in the Arnold on each of 
 3 consecutive days. 
 
 Dunham's Peptone. 10 gms. of peptone 
 and 5 gms. of salt are emulsified 
 with 250 cc. of distilled water pre- 
 viously heated to 60° C. The 
 emulsion is placed in a flask and 
 made up to 1 litre with distilled 
 water. Heat in Arnold for 30 
 minutes, filter through paper, tube 
 and sterilize in Arnold. 
 
 Dextrose Bouillon. Make bouillon in the 
 manner outlined above and add to 
 • it 1% of dextrose. Tube and ster- 
 ilize as for bouillon. This media 
 is generally used in the fermenta- 
 tion tubes. The ordinary glucose 
 will answer as well except that 
 during its sterilization it will 
 deepen greatly in color. 
 
 Milk. 1 litre of fresh milk is put into- 
 a large separating funnel and heat- 
 ed in the Arnold for 1 hour. Esti- 
 mate the reaction. (If it is higher 
 than + 20 or lower than -f 10 re- 
 ject it.) Cool to separate the fat. 
 Draw off the fat-free milk into 
 sterile tubes and sterilize in the 
 Arnold for 20 minutes on each of 5 
 successive days. Incubate for 48 
 hours and eliminate any contam- 
 inated tubes. 
 
 Xiitmus Milk. The milk is prepared as 
 described above, and fat-free is 
 drawn off into a sterile flask. Suf- 
 ficient sterile litmus solution is 
 added to give it a deep lavender 
 color. Tube and sterilize as above. 
 
BACTERIOLOGY. 45 
 
 SPECIAL MEDIA. 
 
 ANAEROBIC CUXiTUBES. 
 
 Kltasato's Glucose Formate Bonlllon. 
 
 Dissolve 20 gms. of glucose and 4 
 g-ms. of sodium formate in 1 litre 
 bouillon. Tube and sterilize in 
 Arnold. 
 
 Weyle's Sulphlndlsrotate Bouillon. Dis- 
 solve 20 gms. glucose and 1 gm. of 
 sodium sulphindigotate in 1 litre 
 bouillon. Tube and sterilize in 
 Arnold. 
 
 Kitasato's Glucose Formate Gelatine. 
 
 Dissolve 20 gms. of glucose and 4 
 gms. of sodium formate in 1 litre 
 of hot gelatin. Filter through 
 paper, tube and sterilize in Arnold. 
 
 Weyl's Sulphlndlgrotate Gelatin. Dis- 
 solve 20 gms. glucose and 1 gm. of 
 sodium sulphindigotate in 1 litre 
 hot gelatine. Filter through paper, 
 tube and sterilize in Arnold. 
 
 Xltasato's Glucose Formate Agrar. 
 Dissolve 29 gms. glucose and 4 gms. 
 sodium formate in 1 litre hot agar. 
 Tube and sterilize in Arnold. 
 
 Sulphlndlgrotate Agar. Dissolve 20 
 gms. glucose and 1 gm. sodium 
 sulphindigotate in 1 litre hot agar. 
 Tube and sterilize in Arnold. 
 
 All the sulphindigotate media are of 
 a blue color. During the growth of 
 the anaerobes, the media is oxidized 
 and changed in color to a light 
 yellow. 
 
 MacConkey's Bile Salt Broth. Emul- 
 sify 20 gms. of peptone in 200 cc. 
 distilled water previously warmed 
 to 60° C. Dissolve 5 gms. sodium 
 taurocholate and 5 gms. of glucose 
 in the emulsion. Wash the emul- 
 sion into a flask with 800 cc. of dis- 
 tilled water and place in Arnold 
 for 20 minutes at lOO** C. Filter 
 through paper and add sterile 
 litmus solution until the medium 
 is of a deep purple color. Tube into 
 gas tubes and sterilize in Arnold 
 for 20 minutes on 3 consecutive 
 days. 
 
 FOB THE STUDY OF THB ORGAN- 
 ISM'S CKEMICAi; COMPOSITION'. 
 Uschlnsky's Asparagine. Dissolve 8.4 
 gms. asparagine, 10 gms. ammonium 
 lactate, 5 gms. sodium chloride, 0.2 
 
46 BACTERIOLOGY. 
 
 gms. magnesium sulphate, 0.1 gm. 
 calcium chloride and 1 gm. acid 
 potassium phosphate, in 1 litre of 
 distilled water. Add 40 cc. glycerine, 
 tube and sterilize in Arnold. 
 
 This media can be made up into 
 gelatine or agar. 
 
 Usclxiiuiky's Proteid Free Broth. Dis- 
 solve 0.1 gm. calcium chloride, 0.2 
 gms. magnesium sulphate, 2 gms. 
 acid potassium phosphate, 3 gms. 
 potassium aspartate, 5 gms. sodium 
 chloride and 6 gms. ammonium lac- 
 tate, in 1 litre of distilled water. 
 Add 30 cc. glycerine, tube and steril- 
 ize. 
 
 FOB THE STUDY OF THE ORGAN- 
 ISM'S BIO-CHEMICAl^ REACTION. 
 Bnnhatn'g Inosite-free Bouillon. In- 
 oculate 1 litre of bouillon with the 
 B. lactis aerogenes and incubate for 
 48 hours. Heat in Arnold for 20 
 minutes. Estimate the reaction and 
 make it + 10. Inoculate with the 
 B. coli communis and incubate for 
 48 hours. Heat in Arnold for 20 
 minutes. 
 
 Fill 2 fermentation tubes, tint with 
 litmus solution and sterilize; in- 
 oculate with the B. lactis aerogenes. 
 If no acid or gas is formed the 
 medium is sugar free; but if acid or 
 gas is present, again make the 
 bouillon to + 10 reinoculate with 
 either of the above bacteria and in- 
 cubate; make another test. Repeat 
 above procedure till neither acid or 
 gas appears. 
 
 Stand the medium in a cool place to 
 
 allow the growth to sediment. 
 Filter the top medium through 
 paper till clear. Tube and sterilize 
 in the Arnold. 
 Nitrate Bouillon. Dissolve 5 gms. of 
 potassium nitrate in 1 litre 
 bouillon. Tube and sterilize in 
 Arnold. 
 
 Iiitmus Bouillon. Add enough sterile 
 litmus solution to 1 litre of bouillon, 
 to give it a dark lavender color. 
 Tube and sterilize in Arnold, (+10 
 media will usually react faintly 
 alkaline or occasionally neutral to 
 litmus). 
 
BACTERIOLOGY. 47 
 
 Iron Bouillon. Dissolve 1 gm. of 
 ferric tartrate in 1 litre bouillon. 
 Tube and sterilize in Arnold. 
 
 £ead Bouillon. Dissolve 1 grm. of 
 lead acetate , in 1 litre bouillon. 
 Tube and sterilize in Arnold. 
 
 Fake's Nitrate Peptone. Emulsify 10 
 gms. peptone with 200 cc. ammonia 
 — free distilled water previously 
 heated to 60* C. Wash emulsion 
 into a flask and make up to 1 litre 
 with ammonia-free distilled water. 
 Heat in Arnold for 20 minutes. 
 Dissolve 1 gm. of sodium nitrate in 
 the above. Filter through paper, 
 tube and sterilize. 
 
 Rosalie Acid Peptone. Make a .5%, 
 80% alcoholic, solution, of rosalic 
 acid (corallin) for a stock solution. 
 Add to 100 cc. Dunham's peptone, 
 2 cc. of the corallin stock solution. 
 Heat in Arnold for 30 minutes. 
 Filter through paper, tube and 
 sterilize. 
 
 Pakes' Iron Peptone. Emulsify 30 
 gms. of peptone with 200 cc. tap 
 water (heated to about 60" C.) 
 Wash it into a flask with 800 cc. of 
 tap water. Dissolve in it 5 gms. 
 of salt and 3 gms. of sodium phos- 
 phate. Heat in Arnold for 30 min- 
 utes. Filter and tube. Add to each 
 tube 0.1 cc. of a 2% neutral solu- 
 tion of ferric tartrate. Sterilize. 
 
 l^ead Peptone. Prepare as for iron 
 peptone except to substitute 0.1 cc. 
 of a 1% neutral aqueous solution 
 of lead acetate for the ferric tar- 
 trate. 
 
 Capaldi-Proskauer No. 1. Dissolve 
 2 gms. sodium chloride, 0.1 gm. 
 magnesium sulphate, 0.2 gms. cal- 
 cium chloride and 2 gms. mono- 
 potassium phosphate in 1 litre of 
 distilled water. Add to the mixture, 
 2 gms. of asparagin and 2 gms. of 
 mannite. Take 25 cc. of mixture 
 and titrate it against n/10 sodium 
 hydrate using litmus as an indi- 
 cator. Calculate amount of sodium 
 hydrate necessary to make the so- 
 lution neutral to litmus and add 
 it. Filter and add to it 5% of 
 neutral litmus solution. Tube and 
 sterilize in Arnold. 
 
 Capaldi-Proskauer No. 2. Dissolve 20 
 gms. of peptone and 1 gm. of man- 
 
48 BACTERIOLOGY. 
 
 nite to 1 litre of distilled water. 
 Neutralize as In No. 1, filter and 
 add litmus solution as above. Tube 
 and sterilize in Arnold. 
 
 Glucose Gelatine. Dissolve 20 gms. 
 of glucose in 1 litre of hot gelatin, 
 filter through paper, tube and steril- 
 ize in Arnold. 
 
 Glucose Asrar. Dissolve 20 gms. of 
 glucose in 1 litre of hot agar, filter, 
 tube and sterilize in Arnold. 
 
 Urine Gelatine. Fresh urine with a 
 sp. gr., of 1010 (if above 1010, it is 
 diluted with sterile water until that 
 sp. gr., is reached) is collected in a 
 sterile fiask, heated to the boiling 
 point and the reaction estimated 
 and noted. 10% of gelatine is added 
 and the mixture heated in the 
 Arnold for 1 hour. Estimate the 
 reaction to that of the original 
 urine. Cool to 60" C. and clear with 
 egg. Filter through paper, tube and 
 sterilize in Arnold. 
 
 Heller's Urine Gelatin. Same as above 
 with addition of 1% of peptone and 
 0.5% of salt. 
 
 Urine Agfar. To fresh urine with a 
 specific gravity of 1010 is added 1.5 
 , to 2% of powdered agar. Heat in 
 the Arnold for 1 and % hours. Cool 
 to 60** C. and clear with eggs. 
 Filter, tube and sterilize in Arnold. 
 
 Eiss' Serum Dextrose. Blood is col- 
 lected and allowed to clot. It is 
 then expressed in a graduated cyl- 
 inder and for every 100 cc. of serum 
 300 cc. of distilled water is added. 
 Heat in the Arnold for 30 minutes. 
 Filter if turbid. (If not needed at 
 once it can be sterilized for 3 days 
 and stored). 
 
 Dissolve 10 gms. of dextrose in 1 
 litre of the above serum. Filter 
 through paper and add 50 cc. of the 
 "neutral litmus solution." Tube and 
 sterilize in Arnold. 
 
 FOR THE STUDY OF CHBOMOGENZC 
 ORGANISMS. 
 
 Eisenbergr's MUk Rice. Mix 70 cc. 
 
 bouillon and 210 cc. milk. In a 
 mortar rub up 100 gms. of rice 
 powder and the mixture into a 
 paste. Spread the paste out in 0.5 
 cm. thick layer over the bottom of 
 
BACTERIOLOGY. 
 
 49 
 
 petri-dishes. With the lids removed, 
 heat over a water bath at 100* C. 
 until the mixture solidifies. 
 Replace the lids and sterilize in the 
 Arnold. 
 
 FOB THE STUDY OF FKOSPKOBES- 
 CENT AND FKOTOGESriC OR- 
 GANISMS. 
 Fish Bouillon. Dissolve 26.5 gms. 
 sodium chloride, 0.75 gm. potassium 
 chloride and 3.25 gms. magnesium 
 chloride in 500 cc. distilled water. 
 Add the solution to 500 gms. of 
 herring, cod or mackerel (in an 
 enameled pot) and heat over a 
 water bath, gently at 40^* C. for 20 
 minutes, then rapidly raise to 100" 
 C, maintaining it at this tempera- 
 ture for 10 minutes. Strain through 
 muslin. Emulsify 5 gms. of pep- 
 tone in 200 cc. of fish water, then 
 mix it thoroughly with the rest. 
 Heat in the Arnold for 20 minutes. 
 Filter through paper and when cold 
 make up to 1 litre with distilled 
 water. Tube and sterilze in Arnold. 
 If it is to be used as a basis for 
 gelatin or agar it should be made 
 up double strength. The gelatine 
 or agar is then prepared in the 
 ordinary manner. 
 
 FOB THB STUDY OF BABTH BAC- 
 
 TBBIA. 
 
 Idpnian and Brown's Bairthy Salts 
 Agfar. (Enumeration of soil or- 
 ganism). Emulsify 20 gms. agar 
 with 200 cc. distilled water and 
 wash it by means of 400 cc. of dis- 
 tilled water into a double boiler. 
 Add to it an emulsion made with 
 .5 gm. of peptone and 50 cc. of dis- 
 tilled water and heat at 100° C. for 
 20 minutes. Add to the mixture, 
 10 gms. of dextrose, 0.5 gm. potas- 
 sium phosphate, 0-2 gm. magnesium 
 sulphate and 0.06 gm. of potassium 
 nitrate. Adjust the weight of the 
 mass to the calculated figure for 
 1 litre (1025 gms.) by adding dis- 
 tilled water at 100° C. Titrate and 
 adjust the reactif)n to -|- 5. Cool to 
 60° C, clear with egg, filter, tube 
 and sterilize, in Arnold. 
 
 Beyrinck's No. 1. (Cultivation of ni- 
 trogen fixing organisms). Dissolve 
 1 gm. potassium hydrogen phos- 
 
50 BACTERIOLOGY. 
 
 phate, 0.2 gm. magrnesium sulphate 
 and 0.02 gm. sodium chloride in 1 
 litre of water. Add 1 cc. of a 1% 
 solution of manganese sulphate. 
 Add 20 gms. dextrose and heat in 
 Arnold for 20 minutes. Filter, tube 
 and sterilize. 
 
 Beyrinck's No. 2. (For growth of 
 Azobacter). It is the same as 
 No. 1, except that mannite is sub- 
 stituted for dextrose. 
 
 Winogradsky's for Nitric Orgranisms. 
 Dissolve 1 gm. potassium phosphate, 
 0.5 gm. magnesium sulphate, 0.01 
 gm. calcium chloride and 2 gms. 
 sodium chloride in 1 litre of dis- 
 tilled water. Fill into flasks in 
 quantities of 20 cc. and add to each 
 a small amount of freshly washed 
 magnesium carbonate. Sterilize in 
 Arnold as usual. Add to each flask 
 2 cc. of a sterile 2% solution of 
 Ammonium sulphate. Incubate and 
 eliminate any flask not sterile. 
 
 Winogradsky's for Nitrous Organisms. 
 Dissolve 1 gm. ammonium sulphate 
 and 1 gm. of potassium sulphate in 
 1 litre of distilled water. Add 5 to 
 10 gms. basic magnesium carbonate 
 , previously sterilized by boiling. 
 Fill into flasks, etc., as for the nitric 
 organisms. 
 
 rOB THE STUBY OF WATER OB- 
 OANISMS. 
 
 Hesse and Keyden Naehrstoff Agar. 
 
 (For enumeration of organisms) 
 Emulsify 12.5 gms. of agar in 250 
 cc. of distilled water. Wash the 
 emulsion into a double boiler with 
 250 cc. of distilled water. Heat in 
 water bath till agar is dissolved 
 and add to it an emulsion made 
 from 7.5 gms. NaehrstofC-Heyden 
 (albumose) with 200 cc. cold dis- 
 tilled water. 
 Adjust weight of mass (1020 gms.) 
 to 1 litre by adding distilled water 
 at 100° C. Clear with eggs. Tube 
 and sterilize in Arnold. 
 
 FOB THE STUDir OF FI^ANT OR- 
 GANISMS. 
 
 Haricot Bouillon, (For bacteria from 
 tubercles of legumens). Add to 1 
 litre of distilled water, 250 gms. 
 of haricot beans, 10 gms. nodium 
 
BACTERIOLOGY. 
 
 51 
 
 chloride and 1 cc. of a 1% solution 
 of sodium bicarbonate. Heat in 
 Arnold for 30 minutes. Filter and 
 add 20 gms. saccharose. Tube and 
 sterilize. 
 
 Haricot Affar. In the usual way add 
 15 gms. of agar to haricot bouillon, 
 adjust the weight and reaction, cool 
 to 60° C, clear with eggs. Filter, 
 add the 20 gms. saccharose, tube 
 and sterilize. 
 
 Hay Infusion. Macerate 10 gms. of 
 chopped hay with 1 litre of distilled 
 water that has been heated to 70° C. 
 in a flask; close flask with rubber 
 stopper and place in a water bath 
 at 60° for 3 hours. Replace the 
 stopper with a cotton plug and heat 
 in Arnold for 1 hour. Filter through 
 paper, tube and sterilize. 
 
 Beets, Carrots, Turnips and Parsnips 
 are prepared in the manner de- 
 scribed for potato. 
 FOR STUDY COIiI-TYFHOID GROUP 
 OP OSaANISMS. 
 
 Carbolized Bouillon. Dissolve 1 gm. 
 of carbolic acid in 1 litre of 
 bouillon. (2.5 to 5 gms. are also 
 used.) Tube and sterilize in Arnold. 
 
 Carbolized Gelatin. Dissolve 5 gms. 
 of carbolic acid in 1 litre of hot 
 nutrient gelatin. Tube and sterilize 
 in Arnold. 
 
 Carbolized Agrar. Dissolve 1 gm. of 
 carbolic acid in 1 litre of hot nu- 
 trient agar. Tube and sterilize in 
 Arnold. 
 
 Parietti's Bouillon. Mix 4 cc. of pure 
 hydrochloric acid with 100 cc. of a 
 5% carbolic acid solution and allow 
 to stand for a few days. Prepare 
 several nutrient bouillon tubes, each 
 containing 10 cc, sterilize, add to 
 each the above solution by means 
 of a sterile pipette in quantities of 
 0.1, 0.2 and 0.3 cc. Incubate for 
 48 hours to eliminate the contam- 
 inated tubes. 
 
 Litmus Gelatin. Add to nutrient 
 gelatin enough sterile neutral litmus 
 to give it a deep lavender color. 
 Tube and sterilize in Arnold. 
 
 Litmus Lactose Bouillon. Emulsify 4 
 gms. of peptone with 200 cc. of meat 
 extract at 60° C. Mix 2 gms. of 
 salt and 20 gms. of lactose with 
 the emulsion. 
 
52 BACTERIOLOGY. 
 
 Add to the mixture 200 cc. of meat 
 extract and 600 cc. of distilled water 
 and heat in Arnold for 30 minutes. 
 
 Neutralize carefully to litmus paper. 
 Heat in Arnold for 20 minutes. 
 Filter through paper and add sterile 
 litmus solution to color medium a 
 deep purple. Tube and sterilize in 
 Arnold. 
 
 Wurtz's ZiltmuB ]&actose Gelatin. 
 
 Render 1 litre nutrient gelatin — 5. 
 Heat in Arnold for 20 minutes. 
 Clear with egg. Dissolve 20 gms. 
 lactose in the medium. Filter and 
 add enough litmus solution to color 
 medium a pale lavender. Tube and 
 sterilize in Arnold. 
 
 Wurte's liltmus Iiactose Agfar. Render 
 1 litre nutrient agar — 5. Heat in 
 Arnold for 20 minutes. Cool to 60° 
 C. and clear with egg. Dissolve 20 
 gms. lactose solution to color 
 medium. Filter and add enough 
 litmus solution to color medium a 
 pale lavender. Tube and sterilize 
 in Arnold. 
 
 MacConkey's BUe Salt Agrar. Emul- 
 sify 15 gms. powdered agar with 
 200 cc. tap water at 60" C. Mix the 
 emulsions. Dissolve 5 gms. sodium 
 » taurocholate in 300 cc. tap water 
 and with it wash the emulsions 
 into a double boiler. Heat in water 
 bath or Arnold for 20 minutes. Ad- 
 just the weight of the medium mass 
 for 1 litre (1040 gms.) Cool to 
 60° C. and clear with eggs. Filter 
 and add 10 gms. lactose. Tube and 
 sterilize in Arnold. 
 
 Fawcus' Bile Salt Agrar. Emulsify 
 20 gms. of agar with 100 cc. of dis- 
 tilled water. Wash the emulsion 
 into a double boiler with 500 cc. of 
 distilled water. Heat until the agar 
 Is dissolved. Cool to 60"* C. and 
 clear with eggs. Filter and add 5 
 gms. of sodium taurocholate, 20 
 gms, of peptone and 5 gms. of 
 lactose. Adjust reaction to + 15. 
 Filter, add 20 cc. of a 1% aqueous 
 solution of picric acid. Tube and 
 sterilize In Arnold. 
 
 Glycerine Potato Bonillon. Grate 1 
 kilo of potatoes previously well 
 washed and peeled. Weigh and add 
 distilled water in proportion of 1 cc. 
 for every gm. of potato. Place in 
 
BACTERIOLOGY. 
 
 61 
 
 ice chest for 12 hours. Strain and 
 filter through paper. Note amount. 
 Add an equal quantity of distilled 
 water. Heat in Arnold for 60 
 minutes. Add 4% glycerine. Mix 
 and filter. Tube and sterilize. 
 
 XSlsuer's Potato Gelatine. Grate 1 kilo 
 of potatoes previously well washed 
 and peeled. Weigh and add dis- 
 tilled water in proportion of 1 cc. 
 for every gm. of potato. Place in 
 ice chest for 12 hours. Strain and 
 filter through paper. Note the 
 amount. Add 15% of gelatin and 
 place in Arnold till dissolved. Esti- 
 mate the reaction and adjust it to 
 4- 25. Cool to 60° C. and clear with 
 eggs. Add 1% powdered potassium 
 iodide. Filter through paper, tube 
 and sterilize in Arnold. 
 
 Braiim's Puchsin Ag'ar. Prepare a 
 fuchsin solution as follows: — Dis- 
 solve 3 gms. basic fuchsin in 60 cc. 
 absolute alcohol. Put aside for 24 
 hours, then centrifugalize thorough- 
 ly and decant the top fluid and place 
 in a well-stoppered bottle. 
 
 Dissolve 10 gms. lactose in 1 litre 
 nutrient agar. Adjust the reaction 
 to — 5. Filter and thoroughly mix 
 with 5 cc. of fuchsin solution. Add 
 to the mixture 25 cc. of a freshly 
 prepared 10% aqueous solution 
 sodium sulphite. Tube and sterilize. 
 Store in a dark place. 
 POB THE STUDY OP MIIiK OBGAK- 
 ISMS. 
 
 Iiitmus Whey. Curdle fresh milk by 
 adding rennet and warming to 60" 
 C. Filter off the whey and neu- 
 tralize to litmus by adding a 40% 
 solution of citric acid. Heat in 
 Arnold for one hour to coagulate 
 all the proteids. If the whey is 
 cloudy, put it aside in ice-chest for 
 48 hours, decant and filter into a 
 sterile flask. Add litmus solution 
 till the whey is of a deep purple 
 red color. Tube and sterilize. 
 
 Petruschky's Litmus Whey. Add 1.5 
 cc. of hydrochloric or glacial acetic 
 acid to 1 litre fresh milk. Filter 
 off the casein and neutralize to 
 litmus by adding normal sodium hy- 
 drate solution. Boil and neutralize 
 to litmus by adding n/10 sodium 
 hydrate solution. Filter and add 
 
54 BACTERIOLOGY. 
 
 litmus solution till the whey is of 
 a deep purple color. Tube and 
 sterilize. 
 
 Whey Gelatin. Curdle fresh milk by 
 adding rennet and warming to 60** 
 C. Filter and estimate the reaction. 
 Add 10% gelatine and place in 
 Arnold till dissolved. Weigh and 
 estimate the reaction of the mass. 
 Restore the mass to the original re- 
 action of the whey by sodium hy- 
 drate solution. Cool to 60° C. and 
 clear with eggs. Filter, tube and 
 sterilize in Arnold. 
 
 iitmus Whey Agar or Gelatin. Add 
 1.5 cc. of hydrochloric or glacial 
 acetic acid to 1 litre fresh milk and 
 boil for 5 minutes. Filter and 
 render whey faintly alkiline to 
 litmus. Prepare an emulsion from 
 a few cc. of the whey and 10 gms. 
 of peptone. Add the emulsion to 
 the whey. Mix in 50 gms. gelatin 
 (or 15 gms. agar) and heat in the 
 Arnold till dissolved. Clear with 
 eggs, filter and adjust the weight 
 of the medium mass for 1 litre. Add 
 15 gms. dextrose. Color with 
 sterile litmus solution. Tube and 
 sterilize in Arnold on each of 5 
 .consecutive days. 
 
 Gelatin Agfar. Emulsify 5 gms. powd- 
 ered agar with 100 cc. of distilled 
 water. Add to it 400 cc. of double 
 strength meat extract and 100 gms. 
 of gelatin. Heat in the Arnold till 
 dissolved. Emulsify 10 gms. of 
 peptone and 5 gms. salt with 100 cc. 
 double strength meat extract heated 
 to 60° C. and add it to the medium 
 mixture. Heat in the Arnold for 
 15 minutes, adjust the weight of 
 the medium mass for 1 litre by 
 adding distilled water at 100° C, 
 estimate the reaction and adjust it 
 to + 10; heat in Arnold for 20 
 minutes, cool to 60° C, clear with 
 eggs, filter through paper, tube and 
 sterilize in Arnold. (This medium 
 will allow incubation at a tempera- 
 ture above 22 C. If incubation at 
 30° C. is to be employed, use 10% 
 of gelatin and 0.5% of agar in the 
 medium. If incubation at 37° C. is 
 used, make the medium with 12% 
 gelatin and 0.75% agar. Avoid the 
 addition of too much agar, as the 
 
BACTERIOLOGY. 
 
 55 
 
 liquefying ferment may be retard- 
 ed or absent). 
 rOR THE STUDY OF DIPI.OCOCCUS 
 PNEUMONIA. 
 
 Washbouru's Blood Agfar. Incubate 
 ag-ar slants for 48 hours to evapor- 
 ate ofC some of the water of con- 
 densation; under aseptic conditions 
 open the thorax of a small rabbit 
 and with sterile pipettes deliver 
 from the heart a small quantity of 
 blood over the surface of each of 
 the agar slants; allow the blood to 
 coagulate in a slanted position; in- 
 cubate the blood agar for 48 hours 
 and eliminate any contaminated 
 tube. 
 
 FOB THE STUDY OF DIFI^OCOCCUS 
 MENINGITIDIS INTRACEI^IiU- 
 I.ARIS. 
 
 Wassenuann Ascitic Agrar. Add to 
 
 210 cc. of distilled water 90 cc of 
 ascitic fluid and 6 gms. of nutrose. 
 Heat over a flame, with constant 
 -shaking, until the fluid boils and the 
 nutrose is dissolved. Add the mix- 
 ture to 600 cc. of melted nutrient 
 agar, heat in the Arnold for 30 
 minutes, filter, tube and sterilize. 
 
 FOR THE STUDY OF QONOCOCCUS. 
 
 I^ipschuetz's Egrg* Albumin Broth. In 
 
 a flask containing some sterile glass 
 beads place 4 gms. of powdered egg 
 albumin and 200 cc. of distilled 
 water previously warmed to 37** C. 
 Dissolve the egg-albumin by shak- 
 ing. Add 40cc. of n/10 NaOH. 
 Allow to stand for 30 minutes with 
 frequent shaking. Filter, sterilize 
 by boiling two or three times at 
 intervals of two hours; add 600 cc. 
 nutrient bouillon, fill in quantities 
 of 50 cc. into small fiasks, incubate 
 for 48 hours and eliminate any 
 contaminated flask. 
 Egrgr Albumin Agfar. This is prepared 
 in the same manner as the above 
 except that nutrient agar is sub- 
 stituted for the 600 cc. of nutrient 
 bouillon. 
 
 Serum Bouillon. Under aseptic pre- 
 cautions, hydrocele, pleuritic or 
 ascitic fluid is collected in sterile 
 flasks; add to it twice its bulk of 
 sterile nutrient bouillon; if neces- 
 sary filter; tube, sterilize in water 
 
56 BACTERIOLOGY. 
 
 bath at 56° C. for 30 minutes on 
 each of 5 consecutive days, incubate 
 for 48 hours and eliminate any con- 
 taminated tubes. 
 Wertlieimer's Serum Agar. Prepare 
 nutrient agar using 2% of agar, 2% 
 peptone, 5% salt and q. s. meat ex- 
 tract. Adjust the reaction to + 10, 
 filter, tube in quantities of 5 cc. and 
 sterilize. After last sterilization 
 cool to 42° C. and add 5 cc. sterile 
 blood serum from human placenta 
 to each tube, slope the tubes; in- 
 cubate, when solid, for 48 hours 
 and eliminate any contaminated 
 tubes. 
 
 Heiman's Semm Affar. Prepare nu- 
 trient agar using 2% of agar, 1.5% 
 peptone, 0.5% salt and q. s. meat 
 extract. Adjust the reaction to + 
 10, filter, tube in quantities of cc. 
 and sterilize. After the last sterili- 
 zation cool to 42° C. and add 3 cc. 
 sterile hydrocele fluid, ascitic fluid 
 or pleuritic effusion, to each tube; 
 slope the tubes; incubate, when 
 solid, for 48 hours and eliminate 
 any contaminated tubes. 
 
 FOB THE STUDY OF B. DIPHTHERIA. 
 
 I^oeffler's Blood Serum. Prepare nu- 
 trient bouillon using veal meat ex- 
 tract instead of beef. Add to the 
 bouillon 1% of glucose and when 
 dissolved add 300 cc. of clear blood 
 serum to every 100 cc. of bouillon. 
 Fill into a sterile tube and complete 
 as for blood serum. 
 
 Councilman and Mallory's Blood 
 Serum. Collect blood in slaughter- 
 house, coagulate, remove the serum 
 and tube (avoid air bubbles). Heat 
 the tubes at a slant in the hot air 
 sterilizer at 90° C. till coagulated 
 (% hour), then sterilize in Arnold 
 for 20 minutes on each of 3 succes- 
 sive days. 
 
 FOR THE STUDY OF B. TUBERCU- 
 I.OSIS. 
 
 Glycerine Bouillon. Add 60 cc. of 
 
 glycerine to 1 litre of nutrient 
 bouillon. Tube and sterilize in 
 Arnold. 
 
 Glycerine Agrar. Add 60 cc. of glyc- 
 erine to 1 litre of nutrient agar. 
 Tube and sterilize in Arnold. 
 
 Glycerine Blood Senuu. Add 5% of 
 
BACTERIOLOGY. 
 
 57 
 
 glycerine to blood serum before 
 tubing", then proceed as described 
 under Blood Serum. 
 Glycerinated Potato. Prepare the 
 ordinary potato slants and soak 
 them in 25% solution of glycerine 
 for 15 minutes. Moisten the cotton 
 pads at the bottom of the tube with 
 a 25% solution of glycerine. Tube 
 and sterilize in Arnold for 20 
 minutes on each of 5 consecutive 
 days. 
 
 NEUTBAZi UTMUS SOXiUTION. 
 
 Place 50 gm. of litmus in 300 cc. 95% 
 alcohol and put it aside until al- 
 cohol acquires a green color (com- 
 pleted in about 7 days with daily 
 shaking). Decant oft the green 
 alcohol and again treat with 300 cc. 
 95% alcohol and repeat shaking. 
 
 Repeat process until on adding fresh 
 alcohol, the fluid only becomes 
 tinged violet. 
 
 Pour ofC alcohol, leaving litmus as 
 dry as possible, connect up bottle 
 to an air pump and evaporate off 
 the last trace of alcohol. 
 
 Transfer the dry litmus to a liter 
 flask, measure in 600 cc. distilled 
 water and allow to remain in con- 
 tact for 24 hours, with frequent 
 shaking. 
 
 Filter the solution and add to it one 
 or two drops of pure sulphuric acid 
 until litmus solution is distinctly 
 wine-red in color. 
 
 Add excess of pure solid baryta and 
 allow to stand until the reaction is 
 again alkaline. 
 
 Filter. 
 
 Bubble CO2 through the solution until 
 reaction is acid. 
 
 Sterilize at 100 C. for 30 minutes for 
 3 days. This also drives ofC C02, 
 leaving the solution neutral. 
 
 TUBING OF NUTRIENT MEDIA. 
 
 After filtration the media is placed in 
 flasks or tubes. The flasks and tubes 
 must first have been washed, dried 
 and their mouths firmly closed with 
 a cotton plug about 1 and i/^ inches in 
 length (allowing about V2 inch or 
 more to extend beyond the mouth). 
 The flasks and tubes are then placed 
 
58 BACTERIOLOGY. 
 
 in the hot-air oven for about one 
 hour at 150° C. which bakes and 
 molds the plugs and sterilizes the ap- 
 paratus. 
 
 The liquefiable media (liquefied) and the 
 liquid media may be tubed by means 
 of a pipette attached to a funnel by 
 a short rubber tube on which is fitted 
 a pinch-cock to regulate the flow of 
 media into the tube. The tube should 
 contain about 1 and inches or 10 cc. 
 of media. 
 
 A special apparatus for tubing media 
 has been constructed that allows the 
 media to be measured into each tube. 
 
 Fluid media containing carbohydrates 
 may be filled into Smith's fermenta- 
 tion tubes or into the Durham gas 
 tubes; the latter tubes are ordinary 
 media tubes, having smaller tubes 
 inverted inside them. When first 
 filled the small tubes will float on the 
 surface, but after sterilization all the 
 contained air will be replaced by 
 media. 
 
 The media must now be sterilized in 
 the Arnold for 3 consecutive days. 
 
 STERILIZATION. 
 
 Dr;jr Heat produced by means of a "hot- 
 air oven" heated by a gas burner. 
 Hot air at 150** C. will destroy all 
 bacteria and their spores in about 
 30 minutes. An exposure at about 
 180° C. for a few minutes only will 
 do the same. This method of 
 sterilization can be used with glass, 
 metal or small bulk fabric only. 
 Large masses of fabric are not 
 readily sterilized by dry heat on ac- 
 count of its poor penetrative power. 
 
 MOIST HEAT. 
 
 Fractional Sterilization. A water bath 
 with a temperature of 56° C, if 
 maintained for 30 minutes, will de- 
 stroy vegetative bacteria. It will, 
 however, have no effect on spores. 
 
 It is used for sterilizing albuminous 
 fluid media that would coagulate at 
 a higher temperature. 
 
 Method. The water bath is heated by 
 a Bunsen flame to 56° C. If the 
 bath is not controlled by a thermo 
 regulator it must be watched care- 
 fully. The material to be sterilized 
 
BACTERIOLOGY. 
 
 59 
 
 is now placed in the bath so that 
 it will be at least 2 cm. below the 
 lev'"^ of the water. The temperature 
 of t' i bath will probably fall some- 
 whtd', but will again in a few 
 minutes rise to 56'' C. The material 
 is removed and subjected to the 
 rapid cooling effect of running" 
 water. The vegetative forms are 
 killed and it is now put for 24 
 hours in a cool, dark place, at the 
 end of which time some of the 
 spores will have germinated and 
 assumed the vegetative form, these 
 are killed by a similar exposure to 
 56° C. on the second, third, fourth, 
 fifth and sixth day successively. 
 
 The water li)atli at a temperature of 
 60°C. for 60 minutes is used in the 
 sterilization of "bacterins," or so- 
 called "vaccines." 
 
 One exposure is all that is necessary 
 for reasons to be explained later 
 (see "preparation of bacterins"). 
 
 Water bath at 100"* C. (Water sterili- 
 zation) destroys vegetative bacteria 
 almost instantly. Spores are de- 
 stroyed in from 5 to 15 minutes. It 
 is used for metal instruments, rub- 
 ber stoppers, rubber and glass tub- 
 ing, etc. 
 
 STEAM. 
 
 Steam at 100* C will destroy vegeta- 
 tive bacteria in from 15 to 20 
 minutes and the spores in from 1 
 to 2 hours. The various culture 
 media are sterilized by this method. 
 
 Koch's Steam Chest was constructed 
 for this form of sterilization. It is 
 a tall, cylindrical vessel divided by 
 a perforated diaphragm into an up- 
 per steam chamber and a lower 
 water chamber. The chest is heat- 
 ed by gas-burners. 
 
 Arnold's Steam Sterilizer is a modifi- 
 cation of Koch's chest. It is very 
 efficient and much used. 
 
 Method: 
 
 When live steam issues steadily from 
 the sterilizer, the material to be 
 sterilized is placed within the steam 
 compartment and allowed to remain 
 for 20 minutes, if the media is 
 liquid, and for 30 minutes if the 
 
 * media is liquefiable or solid. 
 
60 
 
 BACTERIOLOGY. 
 
 This will kill all vegetative bacteria. 
 During the hours of cooling the 
 spores will germinate and can then 
 be destroyed by repeating the pro- 
 cess in 24 hours. ♦ At the end of 
 another 24 hours the media is sub- 
 jected to another sterilization. 
 
 The method is spoke]«i of as a discon- 
 tinuons or Intermittent sterilization. 
 
 Continuous Sterilization. An exposure 
 to steam at 100° C. for 1 to 2 hours 
 is sometimes practiced, but is not 
 to be recommended. 
 
 SuperlieatecL Steam. Chamberland's 
 Autoclave consists of a metallic 
 cylinder fitted with a movable lid 
 which seals the cylinder by means 
 of bolts. It also has a manometer, 
 vent cock and safety valve. It per- 
 mits the heating of steam, under 
 pressure to 115° C., ^^nd will destroy 
 both vegetative bacV^r^i^ia and spores 
 within 15 minutes. If the pressure is 
 increased so as to raise the temper- 
 ature to 120° C, the vegetative bac- 
 teria and spores will be killed in 
 10 minutes. 
 
 Although it is a short, effective meth- 
 od of sterilization and was formerly 
 employed to a great extent for 
 media, on account of hydrolytic 
 •changes in media subjected to high 
 temperatures, which renders it un- 
 fit for the cultivation of the more 
 delicate organisms, its use has been 
 restricted to disinfecting old cul- 
 tures, contaminated articles, etc. 
 
 FIZkTEBS FOB STEBII^IZATION OP 
 AIB AND I^IQUIDS. 
 
 Cotton wool is used in the laboratory 
 for sterilizing air or gases. It is 
 put as a loose plug in a glass tube 
 or a modified tube (air filter) and 
 sterilized in the hot air oven. If 
 the cotton plug is prevented from 
 becoming moist (from air or 
 liquids) it will prevent organisms 
 from entering. 
 
 Porcelain Filters are used for steril- 
 izing liquids. 
 
 The liquids are passed through a 
 cylindrical vessel, closed at one end 
 like a test tube, made of either 
 porous "biscuit" porcelain, hard- 
 burnt and unglazed (Chamberland) 
 or of Kleselguhr, a fine diatoma- 
 
BACTERIOLOGY. 61 
 
 ceous earth (Berkfield) and are 
 called "candle" or "bougies." In 
 passing the liquids through these 
 candles, the bacteria are retained in 
 the pores of the filter which renders 
 the liquid germ-free. 
 
 BACTERIAL CULTIVATION. 
 
 Zdentlflcatlon of Bacteria. Culture 
 Characteristics. Staining*. 
 
 Aerobic Bacteria. 
 
 1. Tube Cultures, accomplished by means 
 
 of a straight or a looped end plat- 
 inum wire fastened to the end of a 
 glass or aluminum rod. The pro- 
 cedure is as follows: 
 
 (a) Sterilize the wire by heating in 
 
 a flame. 
 
 (b) Remove cotton plug from tube 
 
 and hold the end of plug that 
 has not been within the tube 
 between the fingers. 
 
 (c) Touch wire to the material to be 
 
 transferred. 
 
 (d) Stroke or smear (stroke, or 
 
 "streak" culture, is made by 
 drawing the wire as lightly as 
 possible along the center of the 
 surface of the medium. "Smear" 
 culture is made by rubbing the 
 loop all over the surface of the 
 m e d i u m) the contaminated 
 straight wire over the surface 
 of the slanted media; or if the 
 media is not slanted, "stab" 
 culture the solidified media with 
 the wire. This is also employed 
 for the so-called "shake cul- 
 tures." 
 
 (e) Replace the cotton plug. 
 
 (f) Sterilize the wire in the flame. 
 
 Label for identification and with 
 the date of inoculation. 
 
 (g) Place all inoculated tubes, ex- 
 
 cept that containing gelatin, in 
 the incubator. 
 
 2. Plate Cultures. Petri dishes are used. 
 
 These are two shallow glass dishes, 
 so made that one will cover the 
 other. These cultures are made in 
 order that the appearances of the 
 separate colonies may be studied. 
 Having first sterilized the Petri dishes 
 in a hot air oven, the procedure for 
 plating the culture is as follows: 
 
62 
 
 BACTERIOLOGY. 
 
 (a) Several tubes of agar or gelatin 
 
 are melted, then cooled to a 
 temperature not destructive to 
 the bacteria; i2° C. for agar and 
 lower for the gelatin. A water 
 bath with a constant tempera- 
 ture of about 43" C. is very con- 
 venient. 
 
 (b) Place 3 sterile Petri dishes in a 
 
 row and number them 1, 2 and 3. 
 
 (c) Looped-end wire is sterilized over 
 
 flame. 
 
 (d) A loopful of culture is then 
 
 shaken in the tube of melted 
 media. This tube is marked 
 No. 1, or first dilution. Shake 
 with an even circular move- 
 ment so as to diffuse the in- 
 oculum throughout the medium. 
 
 (e) Sterilize the loop and transfer 2 
 
 loopfuls of No. 1 to another 
 tube of melted media. This is 
 marked No. 2, or second dilu- 
 tion. Mix as before. 
 
 (f ) In like manner transfer 3 loopfuls 
 
 of No. 2 to another tube of 
 melted media and this is marked 
 No. 3, or third dilution. Mix as 
 before. 
 
 (g) Sterilize the wire. 
 
 (h) Flame the plug of tube No. 1, 
 
 remove it, flame the lips of the 
 tube, raise the cover of Petri 
 dish No. 1 and pour the inocu- 
 lated liquefied medium into it 
 so as to form a thin layer over 
 the bottom of the plate. 
 No. 2 and 3 are poured in a sim- 
 ilar manner. 
 Place agar dishes in the incu- 
 bator. The gelatin dishes are 
 to remain at room temperature. 
 The dilutions are made in order that 
 the colonies may be thinned out, 
 thus allowing their accurate study 
 and sometimes their separate re- 
 covery, when under various condi- 
 tions there may be more than one 
 kind of bacterial colonies present. 
 In the pouring of the plates, No. 1 
 (1st dilution) rarely gives a plate 
 of any value, therefore it is re- 
 placed by a tube of bouillon or salt 
 solution; the plate (No. 1) is not 
 poured. 
 
BACTERIOLOGY. 
 
 63 
 
 When the main object of the dilutions 
 is to obtain subcultivations from a 
 number of individual bacteria, "sur- 
 face plates" are prepared. 
 
 Method. Liquefy three tubes of lique- 
 fiable media and pour each tube into 
 a separate Petri dish and allow to 
 solidify. When cold place a drop 
 of the inoculum on the surface of 
 the media close to one side of the 
 plate and with a platinum wire, 
 glass rod or aluminum wire (bend 
 about 4 cm. of one end at a right 
 angle, sterilized) smear the drop 
 over the surface with the short arm 
 of the spreader (holding the plate 
 vertical). Rub the infected spreader 
 over the surface of No. 2 plate then 
 over No. 3 plate. Sterilize the 
 spreader, label and incubate the 
 the plates. 
 
 ANAEROBIC BACTERIA. 
 
 Anaerobic cultures are made by grow- 
 ing the organisms, by means of cul- 
 ture tubes or plates, in the absence 
 of oxygen. This is accomplished 
 by: — Exclusion of the air from the 
 cu4tivation; exhaustion of the air 
 from the vessel containing the culti- 
 vation by an air pump; displacement 
 of air by an indifferent gas, e. g. 
 hydrogen; absorption of oxygen by 
 means of pyrogallic acid rendered al- 
 kaline with caustic soda (nitrogen 
 atmosphere) ;. a combine of two or 
 more of the above. 
 
 METHODS. 
 
 Hesse's. A deep stab in agar or 
 gelatine is made with the needle 
 containing the organism, and the 
 tube is then nearly filled with 
 melted sterile media, or a layer of 
 sterilized oil is poured upon the sur- 
 face 1 to 2 cm. deep. 
 
 Another method, when dealing with 
 pure cultivation is to make a plate 
 of agar or gelatin, inoculate one 
 spot of the surface and place over 
 the spot a sterile cover slip or 
 piece of mica, well pressed down to 
 exclude air bubbles. 
 
 Roux's. Aspirate inoculated media 
 into capillary pipettes and seal each 
 end of pipette in a blow flame. 
 
BACTERIOLOGY. 
 
 Another method, sometimes spoken of 
 as the "biological method," is to 
 make a deep stab into gelatin or 
 agar and then pour a layer of a 
 broth cultivation of a vigorous 
 aerobe over it. 
 
 Buchner's. An inoculated culture tube 
 is placed within a larger tube, the 
 lower part of which contains an 
 alkaline solution of pyrogallic acid. 
 The tube is closed with a rubber 
 stopper. Use 1 gm. of pyrogallic 
 acid for every 100 cc. of air capac- 
 ity of the larger tube. 
 
 Wrigrht's. Make a tube cultivation, 
 
 • cut off the projecting part of cot- 
 ton plug, push the plug into the 
 tube (2 to 3 cm. distance); with a 
 pipette run about 1 cc. of a 10% 
 solution of pyrogallic acid onto the 
 plug; with another pipette an equal 
 amount of soda and close the tube 
 quickly with a rubber stopper. 
 
 Exhaustion of Air. Make a tube cul- 
 tivation, replace the cotton plug 
 with a perforated rubber stopper, fit 
 in a glass tube bent at a right 
 angle with a construction of about 
 3 cm. above the stopper, connect 
 glass tube with a water or air 
 pump (interposing a wash-bottle 
 containing sulphuric acid), exhaust 
 the air and seal the glass tube at 
 the construction, using a blow-pipe 
 flame, before disconnecting the 
 pump. 
 
 Kovy's. Place cultivations inside 
 Novy's jar, connect up delivery tube 
 with hydrogen apparatus, attach 
 rubber tubing to exit tube, collect 
 sample of issuing gas (over water) 
 for testing; when air is completely 
 displaced turn the stopper to close 
 entry and exit tubes and disconnect 
 the gas apparatus. 
 
 Bnlloch's. Place cultivations in a 
 glass dish resting in the center of a 
 glass slab; put pyrogallic acid at 
 one side of the dish; put sodium 
 hydroxide near the pyrogallic acid; 
 smear flange of Bulloch's jar with 
 resin ointment; smear stop-cocks 
 with resin ointment; connect short 
 tube with gas supply: open both 
 stop-cocks; connect a piece of glass 
 tubing by means of a piece of rub- 
 
BACTERIOLOGY. 
 
 65 
 
 ber tubing (with a screw-clamp) to 
 the long tube; collect issuing gas 
 and test; when air is displaced shut 
 off stop-cock of entry; shut off stop 
 cock of exit, screw down clamp; re- 
 move glass tube from rubber con- 
 nection; connect up short tube to 
 air pump; open stop-cock of short 
 tube; aspirate small quantity of 
 gas; shut off stop cock; disconnect 
 air pump; fill 10 cc. bulb pipette 
 with water and insert it into rub- 
 ber tubing on long tube as far as 
 screw clamp; open screw clamp; 
 run in water till stopped by in- 
 ternal pressure and shut off stop 
 cock. Incubate. 
 Botkins. Place a leader cross in a 
 glass dish (20 cm. in diameter, 8 
 cm. deep) put tube cultivation in a 
 glass jar or plate cultivations in a 
 wire frame resting them on the 
 cross, adjust U-shaped pieces of 
 glass tubing in a vertical position 
 on opposite sides of the dish, place 
 a bell jar over the cultures enclos- 
 ing one arm of each U-tube (resting 
 it on the cross), fill the dish with 
 glycerine or mercury to a depth of 
 about 5 cm. and connect one U-tube 
 with a gas apparatus. 
 
 IDENTIFICATION OF BACTERIA. 
 
 In order to identify an organism after 
 isolation it must be studied as to 
 cultural characters, morphology, chem- 
 ical products of growth, biology and 
 pathogenicity, as no microorganism 
 can be identified by any one character 
 or property. 
 
 STUDY OP GROWTH CHARACTER- 
 ISTICS. (MICROSCOPIC METHOD). 
 
 Plate Cultures. In gelatin note the 
 presence or absence of liquefaction 
 in the surrounding medium. Note 
 the shape and character of the 
 liquefaction, if present. 
 
 In the agar, no liquefaction takes 
 place. The liquid found on the sur- 
 face is merely the water of con- 
 densation. 
 
 The colonies are to be examined at 
 intervals of 24 hours, — with the 
 naked eye, with a hand lens, and 
 under low power microscope or dis- 
 
BACTERIOLOGY. 
 
 secting. microscope. Distinguish the 
 superficial from the deep colonies 
 and note the characters of the 
 .colonies, as to: — 
 
 1. Size. Diameter at various ages. 
 
 2. Shape. Functifonu (minute, hemi- 
 
 spherical) ; round; elliptical (oval) ; 
 irreg-ular (no recognized shape); 
 fusiform (spindle-shaped); coch- 
 leate (like snail shell); amoeboid 
 (streaming, irregular); mycelioid 
 (mold-like); filamentous (irreg- 
 ular mass of filaments) ; floccose 
 (dense, wooly structure); rhizoid 
 (root-like) ; conglomerate (aggre- 
 gate of colonies of similar size 
 and form); toruloid (aggregate of 
 of colonies like budding of yeast) ; 
 and rosulate (rosette-like). 
 
 3. Surface Elevation. 
 
 (a) Creneral character of surface as 
 
 a whole. Flat (thin, leafy, 
 spread over the surface) ; ef- 
 fused (spread over the sur- 
 face as a thin, veilly layer, 
 more delicate than the "flat") ; 
 raised (growth thick, with 
 abrupt, terraced edges); con- 
 vex (surface the segment of 
 a circle but very flatly con- 
 vex); pulvinate (surface the 
 segment of a circle, but de- 
 decidedly • convex); capitate 
 (surface hemispherical); um- 
 bilicate, (having a central pit 
 or depression); conical (cone 
 with rounded apex) ; and um- 
 bonate (having a central con- 
 vex, nipple-like elevation). 
 
 (b) Detailed characters of Surface. 
 
 Smooth (surface even); al- 
 veolate (marked by depres- 
 sion separated by thin walls 
 so as to resemble a honey- 
 comb); punctate (dotted with 
 punctures like pin-pricks) ; 
 bullate (like a blistered sur- 
 face rising in convex prom- 
 inences, rather coarse) ; vesic- 
 ular (more or less covered 
 with minute vesicles due to 
 gas formation, more minute 
 than the "bullate"); verrucose 
 (wart-like, bearing wart-like 
 prominences) ; squamose 
 (scaly) ; echinate (beset with 
 
BACTERIOLOGY. 
 
 67 
 
 pointed prominences) ; papil- 
 late (beset with nipple or 
 mamma-like processes); ru- 
 gose (short, irregular folds, 
 due to shrinkage of surface 
 growth); corrugated (in long 
 folds, due to shrinkage) ; con- 
 toured (an irregular but 
 smoothly undulating surface, 
 resembling the surface of a 
 relief map); and rimose 
 (abounding in chinks, clefts 
 cracks). 
 
 4. Internal Structure of Colony. (Mi- 
 croscopic). 
 
 Refraction weak: Outline and sur- 
 face of relief not strongly defined. 
 
 Refraction strong: Outline and 
 surface of relief strongly defined; 
 dense, not filamentous colonies. 
 
 (a) General. Amorphous (without 
 
 any definite structure) ; hy- 
 aline (clear and colorless); 
 homogeneous (structure uni- 
 form) ; and homochromous 
 (color uniform). 
 
 (b) Granulations or Blotching's. 
 
 Finely granular; coarsely 
 granular; grumose (coarser 
 than the preceding, with a 
 clotted appearance, and part- 
 icles in clustered grains) ; 
 moruloid (having the char- 
 acter of a mulberry, segment- 
 ed, by which the colony is di- 
 vided in more or less regular 
 segments); and clouded (hav-. 
 ing a pale ground, with ill- 
 defined patches of a deeper 
 tint). 
 
 (c) Colony Marking" or Stripingf. 
 
 Reticulate (in the form of a 
 network, like the veins of a 
 leaf); areolate (divided into 
 rather irregular, or angular 
 spaces by more or less definite 
 boundaries); gyrose (marked 
 by wavy lines, indefinitely 
 placed) ; marmorated (show- 
 ing faint, irregular stripes, or 
 traversed by vein-like mark- 
 ings, as in marble) ; rivulose, 
 (marked by lines like the 
 rivers of a map) and rimose 
 (showing chinks, cracks, or 
 clefts). 
 
68 
 
 BACTERIOLOGY. 
 
 (d) Pilamentous Colonies. Fila- 
 mentous; floccose (composed 
 of filaments, densely placed); 
 and curled (filaments in 
 parallel strands, like locks or 
 ringlets). 
 
 5. Hdges of Colonies. Entire (without 
 
 toothing or division); undulate 
 (wavy); repand (like the border 
 of an open umbrella) ; erose (as if 
 gnawed); irregular (toothed); lob- 
 ate; lobulate (minutely lobate); 
 auriculate (with ear-like lobes) ; 
 lacerate (irregularly cleft, as if 
 torn) ; fimbriate (fringed) ; ciliate 
 (hair-like extensions, radiately 
 placed); tufted; filamentous and 
 curled. 
 
 6. Optical Characters (after Shuttle- 
 
 worth). 
 
 (a) General Characters. Trans- 
 
 parent (transmitting light); 
 vitreous (transparent and col- 
 orless); oleaginous (trans- 
 parent and yellow, olive to 
 linseed-oil colored) ; resinous 
 transparent and brown, varn- 
 ish or resin-colored) ; trans- 
 lucent (faintly transparent) ; 
 porcellaneous (translucent and 
 white); opalescent (trans- 
 lucent, greyish-white by re- 
 flected light) ; nacreous (trans- 
 lucent, greyish-white, with 
 pearly lustre); sebaceous 
 (translucent,' yellowish or 
 greyish-white); butyrous 
 (translucent and yellow); 
 ceraceous (opaque and white, 
 chalky); dull (without lust- 
 er); glistening (shining); 
 fluorescent and iridescent. 
 
 (b) Chromog'enicity. Color of pig- 
 
 ment, pigment restricted to 
 colonies, pigment resticted to 
 medium surrounding colonies 
 and pigment present in col- 
 onies and in medium. 
 Streak or Smear Cultures. In gelatin 
 and agar note the points as indi- 
 cated under plate cultures. In 
 blood serum note the presence or 
 absence of liquefaction. 
 Gelatin Stab Cultures. Note as to 
 1. Surface Growth. Same as in plate 
 cultures. 
 
BACTERIOLOGY. 
 
 69 
 
 2. l^ine of Puncture. Filiform (uni- 
 
 form growth) ; nodose (closely 
 ag-g^regated colonies); beaded 
 (loosely placed or disjointed 
 colonies); papillate (beset with 
 papillate extensions); echinate 
 (beset with acicular extensions); 
 villous (beset with short, undi- 
 vided, hair-like extensions); 
 pulmose (a delicate, feathery 
 growth) ; arborescent .(branched 
 or tree-like, beset with branched 
 hair-like extensions). 
 
 3. Area of Iiiquef action (if present). 
 
 Crateriform (a saucer-shaped 
 liquefaction); saccate (shape of 
 an elongated sack, tubular, cyl- 
 indrical); infundibuliform (shape 
 of a funnel, conical); napiform 
 (shape of a turnip); fusiform 
 (outline of a parsnip, narrow at 
 either end, broadest below the 
 surface) ; and stratiform (lique- 
 faction extending to the walls of 
 the tube and downward horizon- 
 tally). 
 
 4. Character of tlie I^iquefied Gelatin. 
 
 Pellicle on surface; uniformly 
 turbid; granular; mainly clear, 
 but containing flocculi; deposit at 
 apex of liquefied portion. 
 
 5. Production of Gas Bubbles. 
 SHAKE CUXiTUBBS. Presence or ab- 
 sence of liquefaction; production of 
 gas bubbles; bulk of growth at the 
 surface (aerobic) ; bulk of growth in 
 depths (anaerobic). 
 
 FI^UID MEDIA. 
 
 1. Surface of the Liquid. Presence 
 
 or absence of froth due to gas 
 bubbles; presence or absence of 
 pellicle formation; character of 
 pellicle. 
 
 2. Body of the LiCLuid. Uniformly 
 
 turbid; flocculi in suspension; 
 granules in suspension; clear, 
 with precipitate at the bottom of 
 tube; coloration of fluid, presence 
 or absence. 
 
 3. Precipitate. Character; amount; 
 
 color. 
 
 CARBOHYDRATE MEDIA. Note: 
 Growth; reaction; gas formation. 
 
 lilTMUS MII^K CULTIVATIONS. Note: 
 Reaction (unaltered, acid or alkaline) ; 
 odor; gas formation; consistency (un- 
 
70 
 
 BACTERIOLOGY. 
 
 altered, peptonized or coagulated) ; 
 clot (solid, flocculent or rag-ged and 
 broken up by gas bubbles; coagulum 
 undissolved; coagulum finally pepton- 
 ized, completely or incompletely; re- 
 sulting solution, clear or turbid) ; 
 whey (abundant or scanty, clear or 
 turbid, coagulated by boiling or not). 
 
 STUDY .OF BACTERIA BY MICRO- 
 SCOPIC METHODS. 
 I.IVING BACTERIA: Note motility or 
 non-motility. If the organism is one 
 which forms spores observe — spore 
 formation and spore germination. 
 METHODS OF EXAMINATION. 
 1. Ordinary Method. 
 
 If specimen from solid media is 
 used, a drop of water is placed 
 on a clean slide. 
 If specimen from liquid media is 
 used, a drop of the media con- 
 taining the bacteria is used. 
 
 (a) Flame the cotton plug of 
 tube containing the culture; ex- 
 tinguish the burning cotton. 
 
 (b) Hold test tube containing cul- 
 ture between thumb and finger of 
 left hand. 
 
 (c) Hold platinum needle between 
 thumb and forefinger of right 
 hand, and sterilize by heating red 
 hot. Allow to cool. 
 
 (d) Remove cotton plug with the 
 third and fourth finger; insert 
 needle, and transfer minute por- 
 tion of the bacterial culture to the 
 slide. 
 
 (e) Return plug to tube and ster- 
 ilize needle. 
 
 (f) Place a clean cover glass over 
 specimens and examine first with 
 1-6 objective, then with 1-12 of 
 oil immersion pushed gently into 
 a drop of cedar oil placed on the 
 cover glass. Use the fine adjust- 
 ment. Examination should be 
 made by dim light. 
 
 During the examination, stains and 
 other regents may be run in 
 under the coverslip. The non- 
 toxic basic dyes for **intra-vitam" 
 staining of bacteria are neutfal, 
 red, quinoline blue, methylene 
 green and vesuvian in 0.5% 
 aqueous solutions. 
 
BACTERIOLOGY. 
 
 71 
 
 2. Burris' Negrative Stain is some- 
 
 times employed to simulate dark 
 g^round illumination. It is pre- 
 pared by mixing 25 cc. of liquid 
 black ink (any liquid waterproof 
 black drawing inks) and 1 cc. of 
 tincture of iodine. Allow the mix- 
 ture to stand for 24 hours, cen- 
 trifugalize, pipette off the super- 
 natant liquid to a clean bottle, 
 and then a crystal of thymol or 
 1 drop of formalin as a preserva- 
 tive. 
 
 'With a sterilized loop place one 
 drop of the liquid ink close to one 
 end of a slide; sterilize the loop 
 and place a drop of the fluid cul- 
 ture (or emulsion of solid cul- 
 ture) on the slide by the side of 
 the ink; mix thoroughly; sterilize 
 the loop; with another slide 
 spread the mixture across the 
 slide (by placing the end of the 
 slide used as a spreader trans- 
 versely and at an angle of about 
 60° on the mixture and allow the 
 fluid to spread across the slide 
 and fill the angle of incidence; 
 draw it toward the end) ; dry in 
 the air and examine with 1/12 oil 
 objective. 
 
 3. "Hanging" drop" method. 
 
 (a) Paint a ring of vaseline around 
 the hollow in a "culture slide." 
 
 (b) Place bacterial culture in a 
 small drop on a clean cover glass. 
 
 (c) Invert slide over the cover 
 glass, the drop to be within the 
 vaseline ring, but not to touch its 
 sides, and press down so as to 
 seal tight. 
 
 (d) Invert carefully and examine. 
 This method is for demonstration 
 of bacterial motility. It may be 
 kept for examination from day to 
 day s6 that spore formation and 
 germination can also be studied. 
 
 To Study Spore F6miation. Prepare the 
 hanging drop from vegetative forms, 
 add a trace of 0.5% magenta solution 
 to render bacilli more distinct, place 
 slide under microscope (using a warm 
 stage if necessary) ; with the 1/6 lens 
 select a bacillus for observation, then 
 substitute the 1/12 oil immersion and 
 observe the formation of the spore. 
 
72 
 
 BACTERIOLOGY. 
 
 To Study Spore Germination. Prepare 
 the hanging drop from old cultiva- 
 tions in which no living vegetative 
 forms are present and observe the 
 process of germination as in "spore 
 formation." 
 
 FIXED AND STAINED BACTERIA. 
 Bacteria are rendered more prominent 
 by the use of dyes and by their aid, 
 note — 
 
 1. The points in morphologfy, as: — 
 
 Shape, size and pleomorphism 
 if present, record — predominant 
 character of the variant forms, 
 the media on which they are ob- 
 served, at what period of develop- 
 ment). 
 
 2. The details of structure, as: — 
 
 Flagella (if present, record — 
 method of staining, position, ar- 
 rangement and number) ; spores 
 (if present, record — method of 
 staining, shape, size, position 
 within cell, condition as to shape 
 of parent cell, optimum medium 
 and temperature, age of cultiva- 
 tion, condition of environment as 
 to temperature and atmosphere, 
 methods of germinations); invo- 
 lution forms (if present, record — 
 method of staining, character 
 e. g. living or dead), shape, on 
 what medium observed, age of 
 medium and environment) ; me- 
 tachromatic granules (if present, 
 record — method of staining, char- 
 acter of granules, number of 
 granules and color of granules). 
 REACTION OF STAINS. 
 
 1. Gram's Method. Positive (not de- 
 
 colorized) or negative (decolor- 
 ized.) 
 
 2. Neisser's Method. If granules are 
 
 present, record their position and 
 number. 
 
 3. Ziehl-Neelsen's Method. Acid-fast 
 
 or decolorized. 
 
 4. Simple Aniline Dyes. Record those 
 
 giving best results. 
 
 STRAINING METHODS. 
 
 Most bacteria stain easily and are there- 
 fore easily decolorized. 
 
 Some bacteria can withstand alcohol 
 and some withstand strong solutions 
 of mineral acids without decolorizing. 
 
BACTERIOLOGY. 
 
 73 
 
 It is often necessary to use heat or a 
 mordant in order that the stain may 
 penetrate the cell. 
 
 Potassium hydrate, aniline oil, alcohol, 
 carbolic acid (1-5%) and acetic acid 
 (1-5%) are the common mordants 
 used. 
 
 Foxrmtaas of Staining' Solution. 
 
 1. Simple aniline stains are prepared 
 
 by saturating" alcohol with methy- 
 lene blue, dahlia, fuchsin, Vesu- 
 vian, gentian violet or thionine. 
 
 To these stock solutions alcohol may 
 be added from time to time, al- 
 ways allowing an excess of un- 
 dissolved stain to remain on the 
 bottom of the vessel. 
 
 When required for use add 5 cc. of 
 the saturated alcohol solution to 
 95 cc. of distilled water and filter. 
 
 The methylene blue stain is the 
 only one that is permanent. The 
 others must be made fresh as 
 required for use. 
 
 All stains should be filtered before 
 using, unless otherwise specified. 
 
 2. Aniline-Gentian Violet. 
 
 Anilene Water 10 parts. 
 
 (Aniline oil 5 cc. and distilled 
 water 100 cc. are well shaken to- 
 gether and filtered. Make fresh 
 every time). 
 Saturated alcoholic solution of g-en- 
 tian violet 1 part. 
 
 3. Aniline-fuchsin. 
 
 Aniline water (see Aniline Gentian 
 V.) 10 parts 
 
 Saturated alcoholic solution of 
 fuchsin 1 part. 
 
 4. Alkaline Methylene Blue. 
 
 (a) IiOe£B.er's. 
 
 Sat. ale. sol. methylene blue 
 
 30 parts. 
 
 Sol. potass, hydrate (1-10,000) 
 100 parts. 
 
 (b) Kochs. 
 
 Sol. potass, hydrate (10 
 
 per cent) 0.2 part. 
 
 Sat. ale. sol. methyl. 
 
 blue 1.0 part. 
 
 Water (distilled) 200.0 parts. 
 
 5. Carbolic acid solutions. 
 
 (a) Kuhne's Methylene-blue. 
 
 Methylene blue 1.5 gm. 
 
 Abs. alcohol 10.0 cc. 
 
74 BACTERIOLOGY. 
 
 Carbolic acid solution 
 
 (1-20) 100 parts. 
 
 Stain for 5 minutes, 
 (b) Ziehl's carbol fuclLsln. 
 
 Basic fuchsin 1 part. 
 
 Abs. alcohol 10 parts. 
 
 Carbolic acid solution 
 
 (1-20) 100 parts. 
 
 Filter. 
 
 6. Gram's Iodine Solutioxi. 
 
 Iodine 1 part. 
 
 Potass, iodid 2 parts. 
 
 Distilled water 300 parts. 
 
 7. Gab'bet's Acid Blue (a rapid stain). 
 
 25 per cent solution of 
 
 sulphuric acid 100 parts. 
 
 Methylene blue 2 parts. 
 
 Allow dilute acid to stand 24 hours 
 before adding the methylene blue. 
 
 8. Unna's Borax Methyl Blue. 
 
 Borax 1 part. 
 
 Methyl blue 1 part. 
 
 Water 100 parts. 
 
 9. Unna's Polyclirome Methylene Blue. 
 
 Potassium carbonate ... 1 piart. 
 
 Methylene blue 1 part. 
 
 Water 100 parts. 
 
 Must be ripened for months. 
 
 10. NicoUe's Carhol-thionine. 
 
 Sat. sol. thionine in ale. (90 
 
 per cent) 10 cc. 
 
 Aqueous sol. ac. carbol. (1 
 
 per cent) 100 cc. 
 
 Stain sections one-half to one min- 
 ute. 
 
 Contrast Stains. 
 
 11. Eosin Aqueous Solution. 
 
 Bosin (aqueous) 1 gm. 
 
 Water (distilled) 100 cc. 
 
 Absolute alcohol 5 cc. 
 
 12. Bosin Alcoholic Solution. 
 
 Eosin (alcoholic) 0.5 gm. 
 
 Alcohol (70%) 100 cc. 
 
 13. Safranine Aqueous Solution. 
 
 Safranine 0.5 g. 
 
 Water (distilled) 100 cc. 
 
 14. Neutral Bed Aqueous Solution. 
 
 Neutral red 1.0 gm. 
 
 Water (distilled) 100 cc. 
 
 15. Vesuvin (or Bismarck Brown). 
 
 Vesuvin 0.5 gm. 
 
 Water (distilled) 100 cc. 
 
 Special Stains. 
 
 16. MacConkey's Stain (for capsules). 
 
 Dahlia 0.5 gm. 
 
BACTERIOLOGY. 
 
 75 
 
 Methyl green (crystals) . . 1.5 gm. 
 
 Water (distilled) 100 cc. 
 
 Mix well in mortar, then add 
 
 Fuchsin (sat. alcohol sol.). 10 cc. 
 Water (distilled) to make.. 200 cc. 
 
 17. Muir's Mordant (for capsules). 
 
 Mercuric bichloride (sat. aq. 
 
 sol.) 2 cc. 
 
 Tannic acid (20% aq. sol.). . .2 cc. 
 Potash alum. (sat. aq. sol.)..5cc. 
 
 18. Biblsert's Stain (for capsules). 
 
 Axjetic acid (glacial) 12.5 cc. 
 
 Alcohol (absolute) 50.0 cc. 
 
 Water (distilled) 100.0 cc. 
 
 Warm to 36° C. and saturate with 
 dahlia. 
 
 19. Muir's Mordant (for flaffella). 
 
 Tannic acid (10% aq. sol.)..10cc. 
 
 Mercuric bichloride (sat. aq. 
 
 sol.) 5 cc. 
 
 Alum. (sat. aq. sol.) 5 cc. 
 
 Carbol fuchsin (Ziehl) 5 cc. 
 
 Allow to settle for a few hours, 
 
 decant off the clean fluid into 
 
 tubes and centrifugalize. 
 It will keep for a couple of weeks, 
 
 but is at its best 4 or 5 days after 
 
 its manufacture. Must be cen- 
 
 trifugalized each time before use. 
 
 20. l^oeffler's Mordant (for flagrella). 
 Tannin (20% aq. sol.) .... 10 parts. 
 Ferrous Sulphate (sat. aq. 
 
 sol.) 5 parts. 
 
 Decoc. of logwood (1 to 8 
 
 aq. sol.) 3 parts. 
 
 Carbolic acid (1% aq. sol.) 4 parts. 
 Must be freshly prepared. 
 
 21. IiOe£B.er's Stain (for flagrella). 
 
 Methylene-blue 4 gms. 
 
 Aniline water (freshly satu- 
 rated and filtered) 100 cc. 
 
 or — 
 
 Methyl ene-violet 4 gms. 
 
 Freshly saturated and filtered 
 Aniline water (freshly satu- 
 rated and filtered) 100 cc. 
 
 or — 
 
 Fuchsin 4 gms. 
 
 Aniline water (freshly satu- 
 rated and filtered) 100 cc. 
 
 22. Fitfield's Mordant (for flagrella). 
 
 Tannic acid 1 gm. 
 
 Water 10 cc. 
 
 23. Pitfield Stain, 
 
 Alum. (sat. aq. sol.) 10 cc. 
 
76 
 
 BACTERIOLOGY. 
 
 Gentian violet (sat. ale. sol.) . 1 cc. 
 Water (distilled) 5 ce. 
 
 24. Van Ermengrem's Fixing- Plnid (for 
 flagella). 
 
 Osmic acid (2% aq. sol.) . . . . 10 cc. 
 Tannic acid (20% aq. sol.) . . .20 cc. 
 
 Acetic acid (glacial) 1 cc. 
 
 Prepare a few days before using-. 
 Filter when needed. It should be 
 violet in color. 
 
 25. Van Ermengrem's sensitising" Solu- 
 tion (for flagella). 
 
 Silver nitrate (0.5% aq. sol?) 
 Keep in the dark and filter just 
 before using. 
 
 26. Van Ermengem's Reducing solu- 
 tion (for flagella). 
 
 Gallic acid 5 gms. 
 
 Tannic acid 3 gms. 
 
 Potassium acetate (fused)*10 gms. 
 
 Water (distilled) ...350 cc. 
 
 Prepare fresh and filter. 
 
 27. Bung-e's Mordant (for flagella). 
 Tannic acid (20% aq. sol.)..10cc. 
 Ferrous sulphate (sat. aq. 
 
 sol.) 5 cc. 
 
 Fuchsin (sat. ale. sol.) 1 cc. 
 
 28. Pappenheini's Corallin, Methylene- 
 . blue Solution (for B. tuberculosis). 
 
 Corallin 1 gm. 
 
 Methylene-blue (sat. alco. 
 
 sol.) 100 cc. 
 
 Glycerine 30 cc. 
 
 29. Spengler's picric acid alcohol. 
 
 Alcohol (absolute) 20 cc. 
 
 Picric acid (sat. aq. sol.) . . . .10 cc. 
 Water (distilled) 10 cc. 
 
 30. Neisser's Stain (for diphtheria) 
 
 SOLUTION I. 
 
 Methylene-blue 1 gm. 
 
 Alcohol (96%) 20 cc. 
 
 Dissolve and add 
 
 Water (distilled) 950 cc. 
 
 Acetic acid (glacial) 50 cc. 
 
 SOLUTION II. 
 
 Vesuvian 2 gms. 
 
 Water (distilled) 1000 cc. 
 
 31. Modified Niesser's Stain (for diph- 
 theria). 
 
 SOLUTION L 
 Methylene-blue (sat. ale. 
 
 sol.) 4 cc. 
 
 Acetic acid (5% aq. sol.).. 96 cc. 
 
 SOLUTION II. 
 
 Neutral red 2.5 gms. 
 
 Water (distilled) 1000 cc. 
 
BACTERIOLOGY. 
 
 77 
 
 m.{ 
 
 32. Elirlich's Haematoxylin (tissue 
 staining). 
 
 J ( Haematoxylin 2 gms. 
 
 ^' Alcohol (absolute) 100 cc. 
 
 Ammonium alum 2 gms. 
 
 Water (distilled) 100 cc. 
 
 Mix I. and II. stand for 48 hours, 
 then filter and add 
 
 Glycerine 85 cc. 
 
 Acetic acid (glacial) 10 cc. 
 
 Expose to the light for ong month 
 then filter. 
 
 33. Mayer's Haematin (tissue stain- 
 ing) 
 
 -,- / Haematin 1 gm. 
 
 ^- \ Alcohol (909c warmed to 
 
 (37° C.) 50 cc. 
 
 11.^ Potash alum. 50 gms. 
 
 I Water (distilled) 100 cc. 
 
 Pour the two solutions slowly and 
 
 simultaneously into a flask by 
 
 means of a large funnel to insure 
 
 thorough mixing. 
 
 34. Mayer's Alum Carmine (tissue 
 
 staining). 
 
 Alum 2.5 gms. 
 
 Carmine 1 gm. 
 
 Place in a beaker on a sand bath 
 and add successive small quanti- 
 ties of distilled water; keep mix- 
 ture boiling for 20 minutes. The 
 solution should make up to 100 cc. 
 Filter. 
 
 35. Picrocarmine. (tissue staining). 
 
 Picrocarmine 2 gms. 
 
 Water (distilled) 100 cc. 
 
 TECHNIQUE FOR ORDINABY 
 STRAINING. 
 
 (a) Prepare clean cover glass and 
 slide. 
 
 (b) Place drop of water on glass or 
 slide. 
 
 (c) Transfer with sterile needle a 
 minute portion of culture to the 
 drop of water and spread evenly 
 over surface of glass. 
 
 (d) Allow film to dry. 
 
 (e) Fix by passing the glass 3 times 
 through a Bunsen flame. 
 
 (f) Cover specimen with a stain. Al- 
 low it to stain from 2 to 10 
 minutes. 
 
 (g) Wash in water. 
 
 (h) Dry and mount in balsam, 
 
 (i) Examine with 1-12 oil immersion 
 
78 
 
 BACTERIOLOGY. 
 
 — use Abbe condenser. If speci- 
 men is good, label and preserve. 
 TECHNIQUX: FOB DIFFEBEXTTIAI^ 
 STAINING. 
 
 1. Gram's Method. (Gram's stain). 
 This is a differential stain the value 
 depending upon the mycoprotein of 
 \ certain bacteria forming with ^ini- 
 line dyes and an iodid, a compound 
 insoluble in alcohol. Such organ- 
 isms are said to **stain by Gram" 
 or to be "Gram positive." 
 
 (a) Stain specimen for 5 minutes in 
 aniline gentian-violet. 
 
 (b) Wash in water. 
 
 (c) Stain with Gram's iodine solution 
 for 1 minute, or until the film is 
 black or dark brown. 
 
 (d) Wash in 95 per cent alcohol until 
 no more color comes away. 
 
 (e) Dry. and contract-stain in Bis- 
 marck-brown (2-3 minutes) or 
 eosin (1 minute). 
 
 This step may be omitted when or- 
 ganisms are in pure culture. 
 
 (f) Wash, dry and mount. 
 
 The important bacteria retaining the 
 
 stain are (gram positive). 
 
 Smegna bacillus 
 
 Anthrax bacillus 
 
 Tubercle bacillus 
 
 Tentani bacillus 
 
 Leprae bacillus 
 
 Diphtheria bacillus 
 
 Rhinoscleromatis bacillus 
 
 B. Aerogenes capsulatus 
 
 B. Botulinus 
 
 B. Subtilis 
 
 Staphylococcus 
 
 Streptococcus 
 
 Pneumococcus 
 
 Micrococcus tetragenus 
 
 Urethra-coccus. 
 The important bacteria that de- 
 colorize are (gram negative). 
 
 Gonococcus 
 
 Displococcus intracellularis 
 
 B. Mucosus capsulatus 
 
 Bacillus typhoid 
 
 Bacillus coli 
 
 B. Enteritidis 
 
 Bacillus mallei 
 
 Bacillus influenza 
 
 B. Proteus 
 
 B. Morax-Axenfeld 
 
 B. Malignant oedema 
 
BACTERIOLOGY. 
 
 79 
 
 B. Pyocyaneus 
 Bubonic plague bacillus 
 Koch-Weeks's bacillus 
 Cholera-asiatica spirillum 
 Micrococcus catarrhalis 
 Paratyphoid bacillus 
 Dysenteric bacillus 
 Fecal alkalligenes bacillus 
 
 2. Gram-Claudius Method. 
 
 (a) Stain in methyl violet (1% aq. 
 sol.) for 3 to 5 minutes. 
 
 (b) Treat twice with picric acid (sat. 
 aq. sol.) 
 
 (c) Wash in water and dry. 
 
 (d) Decolorize with clove oil. 
 
 (e) Wash in xylol. 
 
 (f) Mount in xylol balsam. 
 
 3. Gram-Weig'ert Method. 
 
 (a) Stain for 5 minutes with aniline 
 gentian violet. 
 
 (b) Wash in water. 
 
 (c) Stain with Gram's iodine solution 
 for 1 minute or until the film is 
 black or dark brown. 
 
 (d) Wash in water and dry in air. 
 
 (e) Wash in aniline oil (1 part) and 
 xylol (2 parts) until no more 
 color come away. 
 
 (f) Wash in xylol. 
 
 (g) Mount in xylol balsam. 
 
 4. Ziehl-Neelsou Method (for B Tuber- 
 
 culosis and other acid-fast bacilli). 
 
 (a) Prepare films as usual. 
 
 (b) Stain in carbol-fuchsin, steaming, 
 but not boiling, for 5 minutes; 
 cool for 25 minutes. 
 
 (c) Wash in 25% sulphuric acid for 
 3 to 5 seconds. 
 
 (d) Wash in water (Faint red color 
 returns). 
 
 (e) Wash in alcohol till no more 
 color comes away. 
 
 (f) Wash in water. 
 
 (g) Counterstain in weak methylene 
 blue. 
 
 (h) Wash in water, dry, and mount. 
 The lepra bacillus and the smegma 
 
 bacillus also stain by this meth- 
 od. The B. lepra stains quickly; 
 the B. Smegmatis is decolorized 
 by the alcohol. 
 
 5. Pappenheim's Method. (Supposed to 
 
 differentiate between B. tubercu- 
 losis and other acid-fast micro- 
 organisms), 
 (a) Prepare films as usual. 
 
BACTERIOLOGY. 
 
 (b) Stain in carbol-fuchsin without 
 heat for 3 minutes. 
 
 (c) Without washing in water treat 
 the film with 3 or 4 successive 
 applications of Pappenheim's 
 (corallin) stain. 
 
 (d) Wash in water. 
 
 (e) Dry and mount. 
 
 Neisser's Method (for B. diphtheria). 
 
 (a) Prepare films as usual. 
 
 (b) Treat with solution I, (Neisser's 
 stain) for 1 to 3 seconds. 
 
 (c) Wash in water. 
 
 (d) Treat with solution II, (Neis- 
 ser's stain) for 3 to 5 seconds. 
 
 (e) Wash, dry and mount. 
 
 By this method the body of the or- 
 ganism is stained brown and the 
 oval polar granules are blue. 
 Modified Neisser's Method (for B. 
 diphtheria). 
 
 (a) Prepare films as usual. 
 
 (b) Treat with solution I, (Modified 
 Neisser's stain) for 2 minutes. 
 
 (c) Wash in water. 
 
 (d) Treat with Gram's iodine solu- 
 tion for 10 seconds. 
 
 (e) Wash in water. 
 
 (f) Treat with solution II, (Modified 
 Neisser's stain) for 30 seconds. 
 
 (g) Wash, dry and mount. 
 
 This must be used on cultivations 
 grown upon blood serum, incu- 
 bated at 37° C. for from 9 to 1« 
 hours. 
 
 The body of the organism is stained 
 a light red and the granules are 
 black. 
 
 Hunt's Method (for diphtheria). 
 
 (a) Prepare films as usual. 
 
 (b) Treat with aqueous methylene- 
 blue for 1 minute. 
 
 (c) Wash in water and dry. 
 
 (d) Treat with tannic acid (10% so- 
 lution) for 1 minute. 
 
 (e) Wash in water and dry. 
 
 (f) Treat with an aqueous solution 
 of methyl-orange for 1 minute. 
 
 (g) Wash, dry and mount. 
 
 Grain's Method with addition of Bis- 
 marck-brown (for gonococcus). 
 
 (a) Prepare film with the urethral 
 pus and fix. 
 
 (b) Treat with aniline gentian violet; 
 stain for 15 seconds. 
 
 (c) Wash in water. 
 
BACTERIOLOGY. 
 
 81 
 
 (d) Treat with Gram's iodin solution 
 and permit to remain for from 
 1 to 2 minutes. 
 
 (e) Wash specimen in 70 per cent 
 alcohol until but a faint violet 
 color remains. 
 
 (f) Stain for 2 minutes with sta. ale. 
 sol. of Bismarck-brown. 
 
 (g") Wash in water, dry and mount 
 in balsam. 
 By this method the gentian violet 
 stains all bacteria present, but 
 the treatment with the iodine 
 solution and alcohol decolorizes 
 the gonococcus, while the other 
 bacteria in the urethra remain 
 violet. 
 
 The addition of the Bismarck- 
 brown stains the previously de- 
 colorized gonococcus a light 
 brown. Nuclei of pus and 
 epithelial cells are stained a ma- 
 hogany color, while the bodies 
 of cells are somewhat lighter in 
 color. 
 
 10. Wheal and Chown (Oxford) Method 
 
 (for actinomyces). 
 
 (a) Prepare films as usual. 
 
 (b) Stain with Ehrlich's haema- 
 toxylin till nuclei are a faint 
 blue after washing with tap 
 water (examine with micro- 
 scope). 
 
 (c) Stain in hot carbol-fuchsin for 
 5 to 10 minutes. 
 
 (d) Wash in tap water. 
 
 (e) Decolorize in alcohol. 
 
 (f) Dehydrate in alcohol. 
 
 (g) Clear in xylol. 
 
 (h) Mount in xylol balsam. 
 
 Can also be used for sections. 
 TECHNIQUE FOB CAFSUXiE STAIZT- 
 ING. 
 
 1. John's Method. 
 
 (a) Prepare films as usual. 
 
 (b) Warm in a 2% solution of gen- 
 tian violet till steam arises. 
 
 (c) Wash, dry and mount. 
 
 2. Welch's Method. 
 
 (a) Prepare films as usual. 
 
 (b) Flood with acetic acid (2%) for 
 2 minutes. 
 
 (c) Remove acetic acid by means of 
 filter paper or blow it oft with 
 a pipette. 
 
82 BACTERIOLOGY. 
 
 (d) Treat with aniline gentian violet 
 for 5 to 30 seconds. 
 
 (e) Wash, dry and mount. 
 
 3. Hiss' Method. 
 
 (a> Prepare films as usual. 
 
 (b) Treat with a mixture of gentian 
 violet or fuchsin (5 cc.) and dis- 
 tilled water (95 cc.) heated until 
 it steams. 
 
 (c) Wash in a solution (20%) of 
 cupric sulphate crystals. 
 
 (d). Wash, dry and mount. 
 
 4. Ritotoert's Method. 
 
 (a) Prepare film as usual. 
 
 (b) Treat with Ribbert's stain for 1 
 to 2 seconds. 
 
 (c) Wash, dry and mount. 
 
 5. MacConkey's Method. 
 
 (a) Prepare film as usual. 
 
 (b) Treat with MacConkey's stain for 
 5 to 10 minutes. 
 
 (c) Wash thoroughly, dry and mount. 
 
 6. Muir's Method. 
 
 (a) Prepare film as usual. 
 
 (b) Treat with carbol-fuchsin, warm 
 until steam begins to rise and 
 allow stain to act for 30 seconds. 
 
 (c) Wash ^ quickly with methylated 
 spirit. 
 
 (d) Wash in water. 
 
 (e) Treat with Muir's mordant for 
 5 seconds. 
 
 (f) Wash in water. 
 
 (g) Treat with methylated spirit for 
 30 seconds (film should now be 
 a pale red). 
 
 (h) Wash in water. 
 
 (i) Stain with aqueous solution of 
 methylene-blue for 30 seconds. 
 
 (j) Wash in water. 
 
 (k) Dehydrate in alcohol. 
 
 (1) Clear in xylol and mount. 
 
 TECHNIQUE FOB SPORE STAINING. 
 
 1. Sing'le Stain. 
 
 (a) Prepare film as usual, except that 
 you pass film through the flame 
 15 or 30 times instead of the 
 usual three. This will destroy 
 resisting power of spore mem- 
 brane and permits the stain to 
 reach the interior. 
 
 (b) Stain with methylene-blue or 
 fuchsin. 
 
 (c) Wash, dry and mount. 
 
 2. Double Stain. 
 
BACTERIOLOGY. 
 
 83 
 
 (a) Prepare film as usual (flame 3 
 times). 
 
 (b) Treat with carbol-fuchsin, steam- 
 ing for 20 minutes. 
 
 (c) Wash in water. 
 
 (a) Decolorize in acid alcohol (97 cc, 
 70% alcohol and 3 cc. hydro- 
 chloric acid) for a few seconds, 
 in 2 part alcohol and 1 part of 
 1% acetic acid, or in 1% sulphuric 
 acid. 
 
 (e) Wash in water. 
 
 (f) Examine under the 1/6 objective, 
 (film mounted in water). 
 
 The spores should be red and the 
 rd^s unstained or faintly pink. 
 
 (g) Counter stain with weak methy- 
 lene-blue for 3 to 4 minutes or 
 g-entian violet for 1 minute. 
 
 (h) Wash, dry and mount. 
 
 3. Abbott's Method. 
 
 (a) Prepare film as usual. 
 
 (b) Treat with Loeffler's alkaline 
 methylene-blue, heat carefully till 
 steam arises and allow hot ^ stain 
 to act for 1 to 5 minutes. 
 
 (c) Wash in water. 
 
 (d) Decolorize in a solution made up 
 of 1 part of 2% nitric acid and 
 98 parts of 80% alcohol. 
 
 (e) Wash in water. 
 
 (f) Counter stain in eosin (1% aq. * 
 sol.) 
 
 (g) Wash, dry and mount. 
 
 4. Mueller's Method. 
 
 (a) Prepare film as usual. 
 
 (b) Treat with absolute alcohol for 
 2 minutes, then in chloroform for 
 2 minutes. (This dissolves out 
 any fat or crystals that might 
 retain the spore stain). 
 
 (c) Wash in water. 
 
 (d) Treat with a 5% aqueous solu- 
 tion of chromic acid for 1 minute. 
 
 (e) Wash in water. 
 
 (f) Decolorize in 5% aqueous solution 
 of sulphuric acid for 5 seconds. 
 
 (g) Wash in water. 
 
 (h) Counter stain with Kuehne's car- 
 bolic methylene-blue for 1 to 2 
 minutes. 
 
 (i) Wash, dry and mount. 
 TECHNIQUE FOR FZiAGEUA STAIN- 
 ING. 
 
 Bacteria shouM be from smear agar 
 cultures, 12 to 18 hours old if incu- 
 
84 BACTERIOLOGY. 
 
 bated at 37° C, 24 to 30 hours if 
 incubated at 22° C. 
 
 In preparing the films a small quantity 
 of the growth is removed by means 
 of the platinum loop and transferred 
 to a few cc. of distilled water in a 
 watch glass. Gently mix the bacteria 
 with the water by moving the loop to 
 and fro, without touching the side 
 
 . of the watch-glass. Flame a cover 
 slip and spread a thin film, using no 
 force. Dry in air, protect the film 
 from dust. Hold the cover slip be- 
 tween finger and thumb, and fix by 
 passing 3 times through the flame. 
 
 1. Xioefder's Metliod. 
 
 (a) Prepare film as described above. 
 
 (b) Treat with Loeflfler's mordant, 
 hold it high above the flame and 
 heat, steaming for 1 minute. 
 
 (c) Wash in water (distilled) and dip 
 carefully in absolute alcohol. 
 Wash again in water. 
 
 (d) Filter on to the film a few drops 
 ' of Loefiler's "flagella stain" and 
 
 warm as before for 1 minute. 
 
 (e) Wash, dry and mount. 
 
 2. Bungfe's Method. 
 
 Same as Loeffier's, except, that 
 Bunge's mordant is substituted 
 for Loeflaer's. 
 
 3. Pitfield's Method. 
 
 (a) Prepare film as described above. 
 
 (b) Mix equal parts of Pitfield's 
 mordant and stain, boil the mix- 
 ture, and while still, hot immerse 
 the film in it for 1 minute. 
 
 (c) Wash in water. 
 
 (d) Examine in water; if satisfactory, 
 dry and mount. 
 
 4. Muir's Modined Fitiield Method. 
 
 (a) Prepare film as described above. 
 
 (b) Treat with Muir's mordant, hold 
 it high above the fiame and heat, 
 steaming for 2 minutes. 
 
 (c) Wash in water and dry carefully. 
 
 (d) Treat with Muir's stain (for 
 flagella) and warm as before for 
 2 minutes. 
 
 (e) Wash carefully, dry and mount. 
 
 5. Van Ermengrem^s Method. 
 
 (a) Prepare film as described above. 
 
 (b) Treat with Van Ermengem's fix- 
 ing solution, heat carefully, 
 steaming for 5 minutes, 
 
 (c) Wash in water. 
 
BACTERIOLOGY. 
 
 85 
 
 (d) Wash 0 in absolute alcohol. 
 
 (e) Wash in distilled water. 
 
 (f) Treat with Van Ermengem's 
 "sensitizing solution" for to 1 
 minute; remove excess of fluid 
 with filter paper. 
 
 (g) Transfer film to a watch-glass 
 containing Van Ermengem's "re- 
 ducing solution" for % to 1 min- 
 ute; remove excess of fluid with 
 filter paper. 
 
 (h) Treat again with the "sensitizing 
 solution" until film commences to 
 turn black. 
 
 (i) Wash in distilled water, dry and 
 mount. 
 
 TECHNIQUE FOB STAINING BAC- 
 TERIA IN TISSUES. 
 
 This is ' practically the same as prepar- 
 ing tissue for histological study. 
 Small pieces of tissue are selected 
 and 
 
 Fixed in alcohol (most used; formalin, 
 Zenker's fluid or Mueller's fluid 
 are also used but are not so good 
 as the alcohol fixative). 
 
 Hardened, unless alcohol is used as 
 fixative; if not, then the tissues 
 must be kept for 24 hours in 50%, 
 75%, 90% and absolute alcohols. 
 
 Dehydrated, by transferring the tis- 
 sues to fresh absolute alcohol. 
 
 Cleared by xylol or chloroform. 
 
 Embedded in paraflan, (Ceiloidin Is 
 also used but is not pref eralple). 
 
 iSectioued. Sections are fioated on 
 slide which has been lightly 
 smeared with a mixture of equal 
 parts egg albumin and glycerine i 
 to which is added a crystal of 
 camphor or a drop or two of 
 carbolic acid. It is now put 
 ;aside in the incubator (or warm- 
 ing chamber) for 4 or 5 hours. 
 
 Stained Iby 
 
 XoeiHer's Method. 
 
 1. Dissolve out paraffin with xylol. 
 
 2. Remove xylol by flushing section 
 
 with absolute alcohol. 
 
 3. Stain in alcoholic methylene-blue 
 
 solution for 5 to 15 minutes, or 
 in Loeffier's alkaline methylene 
 blue for from 1 to 24 hours. 
 
 4. Wash in 1-1000 solution of acetic 
 
 acid for about 10 seconds. 
 
86 
 
 BACTERIOLOGY. 
 
 5. Treat with absolute alcohol for 
 
 10-20 seconds. 
 
 6. Clear in xylol. 
 
 7. Mount In balsam. 
 Gram-Weig-ert Metliod. (To stain Gram 
 
 positive bacterial). 
 
 1. Dissolve out paraffin with xylol. 
 
 2. Remove xylol by flushing section 
 
 with absolute alcohol. 
 
 3. Stain in alum carmin for about 15 
 
 minutes. 
 
 4. Wash thoroughly in water. 
 
 5. Filter aniline gentian violet solu- 
 
 tion on to the section and allow 
 to stain for about 25 minutes. 
 
 6. Wash thoroughly in water. 
 
 7. Treat with Lingol's iodine until 
 
 section ceases to become any 
 blacker. 
 
 8. Wash thoroughly in water. 
 
 9. Treat with a mixture of equal parts 
 
 of aniline oil and xylol until no 
 more color comes away. 
 
 10. Wash thoroughly with-xylol. 
 
 11. Decolorize and dehydrate with ab- 
 
 solute alcohol until there remains 
 only a very faint bluish tint. 
 
 12. Clear with xylol. 
 1^. Mount in balsam. 
 
 The fibrin and hyaline tissue are 
 stained deep blue while Gram 
 positive bacteria appear a deep 
 blue violet color. 
 To Stain Acid fast Bacteria. 
 
 1. Prepare sections for staining as 
 
 above. 
 
 2. Stain with haematin solution 10 to 
 
 20 seconds, to obtain a pure 
 nuclear stain. 
 
 3. Wash in water. 
 
 4. Stain with carbol fuchsin for from 
 
 20 to 30 minutes at 47° C. 
 
 5. Wash in water. 
 
 6. Treat with aniline hydrochlorate, 
 
 2% watery solution, for from 2 to 
 5 seconds. 
 
 7. Decolorize i'n 75% alcohol till sec- 
 
 tion appears free from stain (15 
 to 30 minutes). 
 
 8. Dehydrate with absolute alcohol. 
 
 9. Clear with xylol. 
 10. Mount in balsam. 
 
 To Stain Actinomyces. 
 Mallory's Method. 
 
 1. Prepare sections for staining as 
 above. 
 
BACTERIOLOGY. 
 
 87 
 
 2. Stain with a saturated watery solu- 
 
 tion of eosin for 10 minutes. 
 
 3. Wash in water. 
 
 4. Apply aniline gentian violet for 
 
 from 2 to 5 minutes. 
 
 5. Wash in normal saline solution'. 
 
 6. Apply Weig^ert's iodine solution 
 
 (Iodine 1 part, K. I. 2 parts and 
 water 100 parts) for 1 minute. 
 
 7. Wash in water and blot. 
 
 8. Clear in aniline oil. 
 
 9. Wash in several changes of xylol. 
 10. Mount in balsam. 
 
 STUDY OF CHEMICAL PRODUCTS OF 
 GROWTH (BIOCHEMICAL METHODS). 
 
 Effect of Physical Ag-ents on Growth 
 Study of Disinfectants. 
 
 1. TEST POB THE PRESENCE OP 
 ENZYME PRODUCTION. 
 
 (a) Proteolytic by 
 
 Preparing cultivations in flask 
 bulk (50 cc), using blood serum 
 and milk serum filtered through 
 porcelain; incubate; after which 
 the liquid is made faintly acid 
 (acetic acid 1%) and boiled; a 
 precipitate of unaltered proteins 
 is thrown down. Filter. Mix 
 10 cc, of the filtrate and 1 cc. 
 of caustic soda (30%) in a test 
 tube; add, drop by drop, of 
 copper sulphate solution (0.5%). 
 A pink color which becomes 
 violet as copper sulphate is 
 added = proteose and peptone. 
 Saturate the rest of filtrate with 
 ammonium sulphate. The pre- 
 cipitate = proteose. 
 
 Filter and divide the filtrate into 
 3 parts. 
 
 (1) In one part use excess of 
 caustic soda (30% aq. sol.) to 
 displace the ammonia from the 
 ammonium sulphate, then add 
 drop by drop of the copper sul- 
 phate solution (0.5%), — a pink 
 color = peptone. 
 
 (2) Boil second part with Millon's 
 reagent (a solution of mer- 
 curic nitrate in water contain- 
 ing free nitrous acid, — a pink 
 color = peptone. 
 
BACTERIOLOGY. 
 
 (3) Add to the 3rd part some 
 glyoxylic acid solution, then run 
 in sulphuric acid (cong), — 
 
 A violet ring at upper level of 
 acid =: tryptophane. 
 
 (b) Diastase, by preparing inosite- 
 
 free bouillon tube cultivations 
 and incubate. Add equal parts 
 of the cultivations and a thin 
 starch paste (made with water 
 and starch to which is added 
 2% of thymol); incubate the 
 mixture and incubate at 37° C. 
 for 6 to 8 hours. Filter. Test 
 the filltrate for sugar, using 
 "Fehlings test" — a yellow or 
 orange precipitate — sugar. 
 
 (c) Invertase, by preparing inosite- 
 
 free bouillon tube cultivations 
 and incubate. Mix equal parts 
 of the cultivation and carbolized 
 sugar solution (carbolic acid 2 
 parts, cane sugar 2 parts and 
 water 96 parts) in a test tube; 
 allow to stand for several 
 hours. Filter. Test the filtrate 
 as in the Diastase. 
 
 (d) Reunin, by preparing inosite-free 
 
 bouillon tube cultivations and 
 incubate. Heat the cultivation 
 to 55° C. for 30 minutes (to 
 sterilize). With a sterile pi- 
 pette run 5 cc. of the cultivation 
 into each of 3 tubes of litmus 
 milk. Incubate at 22° C, and 
 examine each day for 10 days. 
 
 Absence of coagulation = absence 
 of rennin ferment. 
 
 Fermentation Reactions are made 
 upon peptone water containing 
 2% respectively of each of the 
 following: — a monosaccharide 
 (dextrose), disaccharide (lac- 
 tose), trisaccharide (mellitose), 
 polysaccharide (dextrin) and 
 glucocide (amygdalin) ; also 
 1% respectively of each of the 
 following organic salts: — so- 
 dium citrate, formate, lactate, 
 maltate and tartrate. Make tube 
 cultivations in each of the 
 above, observe from day to day 
 for 10 days and note growth, 
 reaction, gas production. 
 
BACTERIOLOGY. 89 
 
 2. TEST FOB THE PRESENCE OF 
 ACID PRODUCTION. 
 
 (a) Quantitative.. Prepare glucose 
 
 bouillon cultivation in bulk 
 (100 cc.) in a flask; also "con- 
 trol" flask of medium. Incubate 
 both flasks. Heat in Arnold for 
 30 minutes to sterilize. De- 
 termine the titre of the "inoc- 
 ulated'.' and "control" medium; 
 the difference between the titre 
 gives the total acid production 
 of the bacterium in terms of 
 normal NaOH. 
 
 (b) Qualitative. Prepare glucose or 
 
 lactose bouillon cultivation, in 
 bulk (500 cc), in a litre flask 
 and add 10 gms. of sterilized 
 precipitated chalk. Incubate. 
 Put a cube (about 1 cc.) of 
 paraffin into the cultivation and 
 connect it up with a condenser. 
 Distill over 200 to 300 cc. This 
 distillate (1st distillate "A") 
 will contain alcohol, etc., (see 
 Alcohol production). The first 
 residue "a," will contain the vol- 
 atile and fixed acids. Filter 
 the first residue ("a") and make 
 up the filtrate (first filtrate "a") 
 with distilled water to 500 cc. 
 and divide into 2 parts. Treat 
 250 cc. (1st portion of filtrate 
 "a") with 20 cc. phosphoric acid 
 (cong.) to liberate the volatile 
 acids and distill (second dis- 
 tillate "B") to small bulk. The 
 second distillate ("B"), may- 
 contain formic, acetic, propionic, 
 butyric and benzoic acids. Add 
 baryta water till alkaline and 
 evaporate to dryness. Add 50 cc. 
 absolute alcohol, allow to stand, 
 stirring frequently, for 2 or 3 
 hours. 
 
 Filter and wash with alcohol. 
 
 Filtrate (2nd "b") may contain 
 barium propionate, barium bu- 
 tyrate. Evaporate to dryness 
 and dissolve (2nd filtrate "b") 
 with 150 cc. water. Acidify 
 with phosphoric acid and dis- 
 till (2nd "b"). Saturate the 
 distillate with calcium chloride 
 and distill over a few cc. — 
 (third distillate "c"). Test dis- 
 
BACTERIOLOGY. 
 
 tillate for butyric acid (add 
 3 cc. alcohol and 4 drops sul- 
 phuric acid cong. Smell of pine- 
 apple — butyric acid). 
 Besidue (3rd "C") may contain 
 barium acetate, barium formate, 
 barium benzoate. Evaporate off 
 alcohol and dissolve up residue 
 on filter in hot water and neu- 
 tralize. 
 
 Divide the solution into 4 por- 
 tions. 
 
 (1) Add ferric chloride solution 
 (4% aq.). Brown color = acetic 
 or formic acids; buff ppt. — 
 benzoic acid. (2) Add silver ni- 
 trate solution (1% aq.), then 1 
 drop ammonia water and boil; 
 black ppt. of metallic silver = 
 formic acid. (3) Evaporate to 
 dryness; mix with equal quanti- 
 ties of arsenius oxide and heat 
 on platinum foil; unpleasant 
 smell of cacodyl = acetic acid. 
 (4) Add a few drops of mer- 
 curic chloride solution in a test 
 tube, and heat to 70° C, pre- 
 cipitate of mercurous chloride 
 which is slowly reduced to 
 mecury = formic acid. 
 
 Second residue ("b"). Wash from 
 filter paper, dissolve by heating 
 with dilute hydrochloric acid 
 (25%) and add calcium chloride 
 solution and ammonia until al- 
 kaline. 
 
 White precipitate insoluble in 
 
 acetic acid — oxalic acid. 
 2iLd Portion of first filtrate C<a") 
 
 (2nd 250 cc.) should be ex- 
 amined for (the ether soluble 
 acids) lactic, oxalic, succinic, 
 benzoic; salicylic, gallic and 
 tannic acids by evaporating the 
 filtrate to a thin syrup, acidify 
 strongly with phosphoric acid. 
 Extract by agitation in a sepa- 
 ratory funnel with 5 times its 
 volume of ether. Evaporate the 
 ethereal extract to a thin syrup. 
 Add 100 cc. of water and mix. 
 Add a slight excess of sodium 
 carbonate to a small portion of 
 the mixture and evaporate to 
 dryness on a water-bath, dis- 
 solve in 5 to 10 cc. of pure sul- 
 
BACTERIOLOGY. 
 
 91 
 
 phuric acid, add 2 drops of 
 copper sulphate (sat. sol.) place 
 in a test tube and heat in boil- 
 ing water-bath for 2 minutes; 
 cool, add 2 or 3 drops of thio- 
 phene solution (0.15 cc. in 100 cc. 
 alcohol) and warm gently. 
 Cherry red color = lactic acid. 
 If, on the addition of the sul- 
 phuric acid, a brown color is 
 produced, another sample should 
 
 . be taken and boiled with animal 
 charcoal before evaporating. 
 
 If lactic acid is present, prepare 
 zinc lactate by boiling part of 
 the solution of the ether extract 
 with excess of zinc carbonate, 
 filtering and evaporating to 
 crystalize. The crystals ob- 
 tained have a characteristic 
 form, and it dried at 110° C, 
 should contain 26.87% of zinc. 
 
 Test a part of the rest of the so- 
 lution of the ether extract for 
 oxalic acid by heating with di- 
 lute hydrochloric acid and add- 
 ing calcium chloride solution 
 and ammonia until alkaline. 
 White precipitate insoluble in 
 acid =: oxalic acid. Neutralize 
 the remainder and add ferric 
 chloride solution (4% aq. sol.). 
 Red brown gelatinous precipi- 
 tate = succinic acid. Buff pre- 
 cipitate = benzoic acid and 
 other acids related to benzoic 
 acid. Violet color = salicylic 
 acid. Inky black color precipi- 
 tate =: gallic or tannic acid. 
 
 TEST FOB THE PRESENCE OF 
 AMMONIA PBODT7CTION. 
 
 Prepare cultivation in bulk (100 
 cc.) in a 250 cc. flask and in- 
 cubate together with a control 
 flask. 
 
 After incubation add 2 gms. of 
 calcined magnesia to each flask, 
 then connect up with con- 
 densors and distill. Place 50 cc. 
 of the distillate from each in a 
 Nessler tube and add 1 cc. 16 
 gms. Hgclz in 500 cc. pure water, 
 35 gm. K. I. in 200 cc. pure 
 water. Pour Hgclg solution into 
 K. I. solution until faint show 
 of excess is indicated. Add 160 
 
92 
 
 BACTERIOLOGY. 
 
 gms. KOH. Dilute to 1000 cc 
 and add a strong solution of 
 Hgcls until red mercuric iodide 
 just begins to be prominent. 
 Let excess of mercuric iodide 
 settle to bottom. Reagent should 
 have a pale straw color to each 
 tube. Yellow color = ammonia. 
 The depth of color is propor- 
 tionate to the amount present. 
 
 4. TEST FOB THE FBESENCi: OF A3j- 
 COHOIklC FBODUCTION. 
 
 Prepare glucose or lactose bouil- 
 lon cultivation in bulk (500 cc.) 
 in a litre flask and add 10 gms. 
 of sterilized precipitated chalk. 
 Incubate. Put a cube (about 
 1 cc.) of paraffin into the culti- 
 vation and connect it up with 
 a condenser. Distill over 200 
 or 300 cc. 'Divide the distillate 
 into 4 portions and test for the 
 production of alcohol, acetalde- 
 hyde, acetone. Add Lugol's 
 Iodine, then a little NaOH so- 
 lution and stir till the color of 
 the iodine disappears. Pale- 
 yellow crystalline precipitate of 
 iodoform (with characteristic 
 smell) appearing in the cold, = 
 acetaldehyde, or acetone; ap- 
 pearing only on warming = al- 
 cohol. The precipitate may be 
 absent, . though odor is pro- 
 nounced' Add Schiff's reagent 
 (sulphuric acid 3 parts and a 
 10% solution of ferricchloride, 
 1 part). Violet or red color = 
 aldehyde. 
 
 Add to 10 cc. of the solution, 2.5 
 cc. of a 25% sulphuric acid, and 
 a crystal or two of potassium 
 bichromate and distill. Reduc- 
 tion of the bichromate to a 
 green color and a distillate, 
 which smells of acetaldehyde 
 and reacts with Schilf's reagent 
 = presence of alcohol in the 
 original liquid. Add a few drops 
 of sodium nitroprusside liquid 
 solution (5%), add ammonia to 
 make alkaline, then saturate 
 with ammonium sulphate crys- 
 tals. Acetone gives little color 
 on the addition of ammonia, but 
 after the addition of ammonium 
 
BACTERIOLOGY. 
 
 93 
 
 sulphate a deep permanganate 
 color is found, which takes 10 
 minutes to reach its full depth. 
 Aldehyde gives a carmine red not 
 changed by the ammonium sul- 
 phate. 
 
 5. TEST rOR THIS PRODUCTION OF 
 INDOIi, (a product of putrefaction). 
 Make several peptone water test 
 tube cultivations and incubate. 
 Allow the culture to cool to 
 room temperature and remove 
 2 cc. of the cultivation by means 
 of a sterile pipette, transfer to 
 clean tubes then add 2 cc. para- 
 dimethylamino-benzaldehyde so- 
 lution (paradimethylamino-ben- 
 zaldehyde 4 gms., absolute alco- 
 hol 380 cc.) add 2 cc. potassium 
 persulphate solution (sat. aq. 
 sol.). A delicate rose-pink color 
 appearing throughout the mix- 
 ture which deepens slightly on 
 standing indicates indol. 
 A method used for the test in 
 several laboratories is by the 
 ordinary nitrosoindol reaction. 
 Inoculate several tubes, each 
 containing 10 cc. of glucose-free 
 bouillon or peptone water, allow 
 the culture to grow for 5 to 10 
 days. To each tube of the cul- 
 ture add 10 drops of pure con- 
 centrated sulphuric acid, and 
 then 1 cc. of a sodium nitrate 
 solution (.2%). If a pink color 
 develops within 10 minutes, in- 
 dol is present. In recording the 
 production of indol it is neces- 
 sary to state the age of the cul- 
 ture, since indol may be pro- 
 duced in 10 days and not in five 
 days. 
 
 The reaction may appear immedi- 
 ately, or a faint reaction may 
 appear after a long standing. 
 
 The test can also be performed in 
 one stage by making a mixture 
 of concentrated commercial sul- 
 phuric, hydrochloric or nitric 
 acid, all of which hold a trace 
 of nitrite in solution. A red 
 color within 20 minutes pres- 
 ence of indol. 
 
94 BACTERIOLOGY. 
 
 6. TEST FOR THE PBODUCTION OF 
 FHENOIi (a product of putrefac- 
 tion). 
 
 Prepare a 50 cc. nutrient bouillon 
 cultivation in a 100 cc. Erlen- 
 meyer flask and incubate. After 
 incubation add 5 cc. of sul- 
 phuric acid solution (25%), 
 connect up with a condenser, 
 distil over 15 to 20 cc. Divide 
 the distillate into 3 portions. 
 To one portion add 0.5 cc. of 
 Millon's reagent. Digest one 
 part by weight, of mercuric 
 chloride with 2 partso by weight, 
 of nitric acid sp. gr. 1.42 and 
 dilute the resulting solution 
 with 2 volumns of water and 
 heat to boiling. Red color 
 phenol. 
 
 To another portion add 0.5 cc. of 
 a ferric chloride solution (1% 
 aq. sol.). Violet color = phenol. 
 (If the distillate is acid the re- 
 sult will be negative). 
 
 To another portion add strong 
 bromine water. 
 
 A crystalline white ppt. of tri- 
 bromo-phenol or a turbidity = 
 phenol. 
 
 In recording the presence or ab- 
 sence of phenol in cultures, the 
 age of the culture and tempera- 
 ture of growth should be stated. 
 If indol and phenol appear to- 
 gether in the same culture, it is 
 well to separate them before 
 making the tests, by the Hoppe- 
 Deyler method, in the following 
 manner: Make a 200 to 300 cc. 
 mosite-free bouillon flask culti- 
 vation and incubate, after which, 
 render deflnitely acid with 
 acetic acid and connect up with 
 a condenser. Distill over 50 to 
 70 cc. The distillate will con- 
 tain both indol and phenol. 
 Render the distillate strongly 
 alkaline with caustic potash and 
 redistill. The distillate will 
 contain indol (make test for 
 same) while the residue will 
 contain phenol. When the resi- 
 due is cold, saturate it with 
 carbon dioxide and redistill. 
 Test this distillate for phenol. 
 
BACTERIOLOGY. 95 
 
 7. TEST FOR THE PRODUCTION OF 
 
 REDUCING AGENTS. 
 
 Color destruction. Prepare tube 
 cultivations in nutrient bouil- 
 lon faintly colored with litmus, 
 rosolic acid, neutral red, and in- 
 cubate. Examine the cultures 
 from day to day and note 
 whether any color change oc- 
 curs. 
 
 Reduction of Nitrates to Nitrites. 
 
 Prepare tube cultivations of ni- 
 trate bouillon on nitrate peptone 
 and incubate together with un- 
 inoculated controle tubes. This 
 is necessary as the medium 
 may take up nitrates from the 
 atmosphere and an opinion as 
 to the absence of nitrates in 
 the cultivation is based upon an 
 equal coloration of the medium 
 in the controle tube. Test both 
 the culture and the control for 
 the presence of nitrates by add- 
 ing drops of sulphuric acid 
 (25%) to the tubes, then run in 
 2 or 3 cc. metaphenylenediamine 
 (5% aq. sol.) into each tube. 
 Brownish-red color = nitrites. 
 The depth of color is in pro- 
 portion to the amount present. 
 
 8. TEST FOR THE PRODUCTION OF 
 
 PIGMENTS. 
 
 Make tube inoculations upon the 
 various media and incubate at 
 37° and 20** C. temperatures; 
 grow them also under aerobic 
 and anaerobic atmospheres; 
 also, exposed to and protected 
 from light. Note the conditions 
 most favorable to the formation 
 pigments. Note the solubility 
 of the pigment in hot and cold 
 water, alcohol, ether, chloro- 
 form, benzol and carbonbisulph- 
 ide. Note the effect of acids and 
 alkalies upon the cultivations or 
 upon solutions of the pigment. 
 
 9. TEST FOR THE PRODUCTION OF 
 
 GAS. 
 
 Inoculate fermentation tubes filled 
 with sugar bouillons and incu- 
 bate. Examine at intervals of 
 24 hours and mark the levels 
 of the fluid when the evolution 
 of gas has ceased, measure the 
 
96 
 
 BACTERIOLOGY. 
 
 length of the column of gas 
 with a millimeter scale. Ex- 
 press this column as a percent- 
 age of the entire length of the 
 closed branch. 
 
 To roughly determine the relative 
 proportions of CO2 and H — 
 fill the bulb of the fermenta- 
 tion tube with caustic soda so- 
 lution; close the mouth of the 
 bulb with a rubber stopper; in- 
 vert the tube 10 times, that the 
 soda may be brought into in- 
 timate contact with the gas; 
 return the gas to the end of 
 the closed tube, and measure. 
 The loss in volume of gas = 
 carbon dioxide. 
 
 The residual gas = hydrogen. 
 Transfer the gas to the bulb, 
 apply a light and cause it to 
 explode. 
 Sulplmretted Hydrog'ezi. 
 
 Inoculate tubes of iron peptone 
 or lead peptone media and in- 
 cubate together with control 
 tubes. Examine every 24 hours 
 for several days. The libera- 
 tion of H2 S will cause the 
 yellowish-white precipitate to 
 darken to a brownish-black or 
 jet black, the depth of color 
 being in proportion to the 
 amount of H2 S present. 
 STUBY OF THE BIOI.OGY OF CUl^- 
 TUBES BY FKYSICAI^ METHODS. 
 
 The growth and development of 
 cultures are to be examined 
 under conditions of 
 1. Atmosphere. Prepare 4 sets of cul- 
 tivation in: — 
 (a) Slanted glucose formate agar and 
 incubate aerobically at 37° C, 
 slanted glucose formate gela- 
 tine and incubate aerobically at 
 20° C. Seal the cultures in 
 Buchner's tubes according to 
 Buchner's method. 
 
 (c) Slanted glucose formate agar; 
 
 glucose formate bouillon. Grow 
 anaerobically by placing them 
 in Bulloch's apparatus accord- 
 ing to Bulloch's method and in- 
 cubate at 37° C. 
 
 (d) Slanted glucose formate gelatin; 
 
 glucose formate bouillon. Grow 
 
BACTERIOLOGY. 
 
 97 
 
 anaerobically by placing: them 
 in Bulloch's apparatus accord- 
 ing to Bulloch's method and 
 incubate at 20° C. 
 
 Make observations upon the culti- 
 vations both microscopically and 
 * microscopically at intervals of 
 
 24 hours, until the completion 
 of 7 days incubation. By this 
 method it can be determined 
 as to whether an organism is 
 an obligate aerobe, a faculta- 
 tive anaerobe or an obligate 
 anaerobe. 
 
 A rough estimate of the above 
 may be made by an observa- 
 tion of the growth in fermenta- 
 tion tubes. If there is a growth 
 in the closed arm as well as in 
 the bulb of the tube, it indi- 
 cates that the organism is a 
 facultative anaerobe; while a 
 growth odburing only in the 
 bulb or in the closed arm shows 
 that it is an obligate aerobe or 
 anaerobe respectively. 
 
 In addition to the observation of 
 growth in the presence or ab- 
 sence of oxygen it is often 
 necessary to observe the growth 
 in — Gases other th.au Ozysrezi 
 (SO2, N2O, NO, CO2, etc.) 
 
 Prepare tube cultivations upon 
 solid media and place them in 
 Bulloch's apparatus. Replace 
 the contained air with the 
 selected gas and incubate under 
 optimum temiperature. 
 
 Examine the cultivations at 24 
 hour intervals for 7 days, by 
 removing a tube from the ap- 
 paratus each day. 
 
 If no growth is visible, incubate 
 the tube under optimum condi- 
 tions of temperature and at- 
 mosphere, by which the length 
 of exposure to the gas neces- 
 sary to kill the organism is de- 
 termined. 
 Temperature. 
 
 (a) Test the ran§re of temperature 
 (minimum and maximum). 
 
 Prepare 10 tube cultivation in 
 fluid media of optimum reac- 
 action. Arrange a series of in- 
 cubators with fixed tempera- 
 
BACTERIOLOGY. 
 
 tures varying from ^° C. to 
 50° C. (Water baths can be 
 used if incubators are not 
 available). Incubate one tube 
 aerobically and one anaerobic- 
 ally in each incubator. Ex- 
 amine at % hour intervals for 
 from 5 to 18 hours.' Note the 
 temperature (optimum) at 
 which the growth is first ob- 
 served. 
 
 Continue the incubation for 7 
 days. Note the extremes of 
 temperature at which growth 
 takes place (minimum and 
 maximum temperatures) 
 
 (b) Test for tlie (absolute) optimnm. 
 
 temperature. 
 Prepare 10 tube cultivations in 
 fluid media of optimum reac- 
 tion. Arrange a series of in- 
 cubators w|th fixed tempera- 
 tures varying 1° C. for 5 de- 
 grees on either side of the 
 optimum observed in testing 
 for the range of temperature, 
 and incubate a tube in each 
 incubator. Examine at l^ hour 
 intervals and note the tempera- 
 ture ("absolute optimum") at 
 which growth is first observed. 
 
 (c) Test for the Thermal Death-point. 
 Veg-etative Forms. 
 
 Moist t. d. p. is the temperature 
 which kills a watery suspen- 
 , sion of the organism after an 
 exposure for 10 minutes. 
 
 Make the test by preparing tube 
 cultivation on solid media of 
 optimum reaction, and incubate 
 for 48 hours. Examine cultiva- 
 tions microscopically to deter- 
 mine the absence of spore; 
 place 3 loopfuls of the surface 
 growth in each of 12 test tubes 
 containing 5 cc. salt solution; 
 mix the organisms through the 
 salt solution; transfer the mix- 
 ture from each tube into a 
 sterile 250 cc. flask and mix; 
 place 5 cc. of this mixture into 
 each of 12 sterile test tubes 
 and number them consecutive- 
 ly; regulate a water bath to 
 40° C. and suspend one tube in 
 it so that the upper level of 
 
BACTERIOLOGY-. 
 
 99 
 
 the bacterial suspension in the 
 tube is about 4 cm. below the 
 surface of the water in the 
 bath, and the bottom of the 
 tube is about the same distance 
 ''rom the bottom of the bath; 
 suspend and adjust a "control 
 tube" containing 5 cc. of salt 
 solution, under similar condi- 
 tions, plug the tube with cot- 
 tion and pass a thermometer 
 through the plug so that its 
 bulb is immersed; watch the 
 themometer in the test until it 
 records 40" C. Note the time 
 and at the end of 10 minutes 
 at this temperature, remove the 
 bacterial suspension and cool it 
 by placing the lower end of the 
 tube in running water. Make 
 
 3 gelatin or agar plates, con- 
 taining respectively 0.2, 0.3 and 
 0.5 cc. of the suspension, and 
 incubate; place the remaining 
 
 4 cc. of the suspension into a 
 flask containing 250 cc. of 
 bouillon and incubate. Observe 
 the cultivations from day to 
 day for 7 days, at the end of 
 which time "no growth" can 
 be recorded. 
 
 The same technique is to be car- 
 ried out in the remaining 11 
 tubes, varying the conditions 
 so that each tube will be ex- 
 posed to a temperature 2° C. 
 higher than the immediately 
 preceding one (i. e., 42* C. 44° C. 
 4°6 C. 48" C, etc.) 
 
 Note the lowest temperature at 
 which no growth takes place 
 up to the end of seven days' in- 
 cubation, = the thermal death- 
 point. 
 
 Dry t. d. p. is that temperature 
 which kills a thin film of the 
 organism after an exposure for 
 10 minutes. 
 
 Make, under sterile conditions, an 
 emulsion with three loopfuls 
 from an optimum cultivation in 
 
 5 cc. normal salt, and with the 
 microscope determine the ab- 
 sence of spores. If spores are ab- 
 sent, make 12 cover-slip sterile 
 films and place each in a sterile 
 
100 BACTERIOLOGY. 
 
 Petri dish to dry. Expose each 
 dish in the hot-air oven for 
 ten minutes at temperatures 
 varying- 5° C. between 60° C. 
 and 120'' C, with a sterile 
 forcep removing- each ^Im from 
 the oven as soon as the, ten 
 minutes are completed, placing 
 it in a flask containing 200 cc. 
 bouillon; incubate and prepare 
 subcultivations from the flasks 
 showing evidence of growth, to 
 determine that no contamin- 
 ation has taken place. 
 Spores. 
 
 Moist t. d. p. is that time ex- 
 posure to a temperature of 100 
 C. necessary to kill all spores 
 present in a suspension. 
 
 Make 12 agar slant cultivations 
 and - incubate under optimum 
 conditions. Examine micro- 
 scopically to determine the 
 presence of spores. Under 
 sterile conditions place about 
 5 cc. normal saline into each 
 tube and by means of a plat- 
 inum wire emulsify the surface 
 g-rowth; add the 60 cc. emulsion 
 to 140 cc. of normal saline con- 
 tained in an - Erlenmeyer flask; 
 place the flask in a water-bath 
 of' boiling water. When the 
 temperature of the flask reaches 
 100° C. remove by means of a 
 sterile pipette 5 cc. of the sus- 
 pension from which plate and 
 flask cultivations are made. Re- 
 peat process intervals of 25 
 minutes steaming. Control the 
 experiments by removing the 
 suspension at intervals of or 
 1 minute during the 5 or 10 
 minutes preceding the previous- 
 ly determined t. d. p. 
 
 Dry t. d. p. Make an ag-ar slant 
 cultivation and incubate under 
 optimum conditions for spore 
 formation. Under sterile con- 
 ditions place about 5 cc. nor- 
 mal saline into the tube and 
 emulsify the growth. Deter- 
 mine the presence of spores by 
 microscopic examination. Make 
 12 cover-slip films and place 
 each in a separate Petri dish. 
 
BACTERIOLOGY. 101 
 
 Expose each dish in turn for 
 10 minutes to a different fixed 
 temperature, varying 5° C. be- 
 tween 100° C. and 160° C. 
 
 Complete examination as in the 
 vegetation forms. 
 . REACTION OF MEDIUM. 
 
 Baug-e. Make a 24 hour bouillon 
 culture of the organism. Pi- 
 pette 0.1 cc. of the cultivation 
 into a tube containing 9.9 cc. 
 of sterile bouillon and mix. 
 Inoculate a series of tubes of 
 nutrient bouillon of varying 
 reactions, from + 25 to — 30, 
 viz.: + 25, + 20, + 15, + 10. 
 + 5, neutral — 5, — 10, — 15, 
 — 20, — 25, — 30, with 0.1 cc. 
 of the diluted cultivation and 
 incubat,e. 
 
 Make half hour observations 
 from the third to the twelfth 
 hours and note the tube or 
 tubes in which the growth first 
 appears. (Probably the opti- 
 mum reaction). At the end of 
 a 48 hour incubation, note the 
 extremes of acidity and akalin- 
 ity in which growth has taken 
 place; this indicates the range 
 ^ of reaction." 
 
 Optimum Reaction. The steps are 
 indicated in "range reaction," 
 but must be fixed within nar- 
 rower limits by inoculating a 
 series of tubes which have a 
 variation in reaction of 1 in- 
 stead of 5 for five points on 
 either side of the tube or tubes 
 in which the growth first ap- 
 pears in the "range reaction." 
 RESISTANCE TO I^ETKA]^ AGENTS. 
 Desiccation. Make an agar-slant 
 cultivation and incubate for 48 
 hours, after which examine to 
 determine the absence of spores. 
 Pipette about 5 cc. sterile nor- 
 mal saline into the tube and 
 suspend the growth in it. Make 
 thin spreads of the suspension 
 on sterile cover slips and place 
 them inside of sterile Petri- 
 dishes to dry. When dry, ele- 
 vate the lids of the Petri-dish 
 in such a way as to allow ven- 
 tilation and place the dish in a 
 
BACTERIOLOGY. 
 
 MuUer's desiccator, the upper 
 chamber of which is filled with 
 pure H2 SO., cover with a bell 
 jar, and exhaust the air. At 5 
 hour intervals admit air, re- 
 move a cover slip, and under 
 sterile conditions tr^insfer it to 
 a culture flask. Reseal the 
 desiccator and exhaust the air. 
 Incubate the culture flasks for 
 7 days if necessary and pour 
 plates from those flasks which 
 show growth to determine the 
 absence of contamination. 
 Hourly observations should now 
 be made for 5 hours preceding 
 and succeeding the death-time 
 determined above. 
 
 iiigriit. 
 
 (a) Diffuse Dayliffht. Make a tube 
 cultivation in nutrient bouillon 
 and incubate for 48 hours. 
 Pour 10 gelatin and 10 agar 
 plates, each containing 0.1 cc. 
 of the bouillon culture. Place 
 one agar and one gelatin plate 
 into the hot and cold incu- 
 bators, respectively, as controls. 
 On the center of the cover of 
 the remaining plates fasten a 
 black paper, cut in the shape 
 of a cross or some other figure, 
 and expose the plates to the 
 action of diffuse daylight, for 
 1, 2, 3, 4, 5, 6, 7, 8, 10, 12 hours, 
 after which incubate and ex- 
 amine after 24 and 48 hours 
 and compare with the two con- 
 trols. If the growth is absent 
 from that portion of the plate 
 not covered by the paper, con- 
 tinue the incubation and daily 
 observations for 7 days. 
 
 (b) Direct Stinligrlit. Prepare as 
 
 above, except that plates ar^ 
 placed in the direct rays of tht 
 sun. Stand a small glass disl 
 (14 cm. X 5 cm.) on top of eacl: 
 plate and fill with a 2% watery 
 solution of potash alum (to ab 
 sorb the heat of sun's rays) 
 Make exposure and incubate a: 
 in experiment above. 
 
 (c) Colours. Test separately witl 
 
 violet, blue, green, red, orang- 
 and yellow. Prepare plate cul 
 
BACTERIOLOGY. 103 
 
 tivations as in "light" experi- 
 ments. Fasten a strip of black 
 paper (3 cm. wide) across the 
 cover of each plate; then paint 
 the colors over the remaining 
 portions of the cover. The 
 colors are prepared by placing 
 2% of the following dyes: — 
 
 Chrysoidine (red), aurantia (or- 
 ange), naples yellow (yellow), 
 malachite green (green), bluish 
 eosin (blue), and methylviolet 
 (violet), in pure photographic 
 collodion. 
 
 Expose the plates to bright day- 
 light for varying periods as in 
 preceding experiments. 
 
 Heat. (See thermal d. p.) 
 
 Antiseptic and Disinfectants. Bi- 
 chloride of mercury, formalde- 
 hyde, carbolic acid are general- 
 ly selected for the test, noting 
 the strength of solution and 
 duration of exposure necessary 
 to produce death. 
 
 They are examined with refer- 
 ence to 
 
 (a) Inhibition Coefficient, — that % of 
 disinfectant present in the me- 
 dium which is sufficient to pre- 
 vent the growth and multiplica- 
 tion of bacteria therein. 
 Prepare a series of 6 bouillon 
 cultivations of an organism in 
 tubes containing 10 cc. of me- 
 dium. Mark the tubes from 
 
 1 to 6; place in No. 1 tube — 
 
 2 cc. of a 5% carbolic acid so- 
 lution (1:100); in No. 2 — 1 cc. 
 (1:200); in No. 3 — 0.6 cc. 
 (1:300); in No. 4 — 0.5 cc. 
 (1:400); in No. 5 — 0.4 cc. 
 (1-500); and in No. 6 — 0.6 cc. 
 (1:1000). 
 
 Prepare a series of 6 bouillon cul- 
 tiva^tions, using 
 
 Prepare a series of 6 bouillon cul- 
 tivations, using 
 
104 BACTERIOLOGY. 
 
 Incubate the three sets and ex- 
 amine them from day to day 
 for 7 days and note those tubes, 
 if any, in which growth takes 
 place. 
 
 (b) Inferior letlial coefficient — the 
 
 time exposure necessary to kill 
 vegetative forms suspended in 
 water at 20° to 25° C. in which 
 the disinfectant is present in 
 concentration insufficient to 
 cause plasmolysis. 
 Prepare 48 hour agar slant culti- 
 vations of each of the "test" 
 organisms and examine to de- 
 termine the absence of spores. 
 Prepare solutions of different 
 percentages of each disinfect- 
 ant and make a series of hang- 
 ing-drop preparations from the 
 culture, using the different per- 
 centage to prepare the emulsion 
 on each cover slip. Examine 
 these under the microscope to 
 determine the strongest solu- 
 tion which does plasmolyze the 
 organism. Make control pre- 
 parations of these two solli- 
 tions and determine the per- 
 centage to be tested. Transfer 
 10 cc. of sterile water into the 
 agar cultures and suspend the 
 growth in it, after which it is 
 transferred to a flask contain- 
 ing 90 cc. of sterile water and 
 well shaken. 10 cc. of this dilu- 
 tion is placed into each of 10 
 sterile test tubes; one is placed 
 in the 20° C. incubator as a 
 control; to each of the other 
 tubes a sufficient quantity of 
 a concentrated solution of the 
 disinfectant is added to pro- 
 duce the percentage previously 
 determined. Incubate at 20° C. 
 Remove the control tube and 
 one of the other tubes contain- 
 ing the disinfectant at hourly 
 intervals; make subcultivations 
 upon agar and incubate. Ex- 
 amine these cultures from day 
 to day for 7 days and determine 
 the shortest exposure necessary 
 to cause the death of vegeta- 
 tive forms. 
 
BACTERIOLOGY^ 105 
 
 (c) Superior lethal coefficient, that 
 time exposure necessary to kill 
 spores suspended in water at 
 20° to 25° C. in which the dis- 
 infectant is present in concen- 
 tration insufficient to cause 
 plasmolysis. 
 Make agar-slant cultivations of 
 the "test" organism and incu- 
 bate under conditions previous- 
 ly determined for the formation 
 of spores. 
 Employ that percentage solution 
 of the disinfectant determined 
 in the inferior lethal coefficient 
 and complete the investigation 
 as detailed therein, increasing 
 the interval between planting 
 the subcultivations to two, 
 three or five hours if advisable. 
 Where it is necessary to leave 
 the organism in contact with a 
 strong solution of the disin- 
 fectant for a long period of 
 time, all traces of the disin- 
 fectant must be removed by 
 several centrif ugations and 
 washing of the bacteria with 
 sterile water. 
 
 5TUDY OP THE FATKOGENICITY OP 
 AN ORGANISM. 
 
 •"or the study of the pathogenicity, the 
 use of living animals has become a 
 necessity in order that the — 
 
 Pathogrenic properties of bacteria 
 already isolated in pure culture 
 may be determined. 
 
 The conditions influencing the vi- 
 rulence of an organism and the 
 pathogenic effect produced by its 
 entrance into and multiplication 
 within the tissues of the body, 
 etc., can only be carried out by 
 animal inoculation. 
 
 Baising* or '^exalting"' the Virulence 
 of an Organism. 
 
 When it is desired to raise the vi- 
 rulence of a feebly pathogenic 
 organism, one of the following 
 methods of inoculation is neces- 
 sary — 
 
 (a) A highly susceptible animal is 
 inoculated with a pure culture of 
 the organism and then passed 
 , from animal to animal as rapidly 
 
106 BACTERIOLOGY. 
 
 as possible, always selecting the 
 method of inoculation (e. g. 
 peritoneal) which places the or- 
 ganism under the most favorable 
 conditions for growth and multi- 
 plication. 
 
 (b) An animal is inoculated with a 
 pure culture of the organism to- 
 gether with a pure culture of 
 some other organism, which is in 
 itself of a virulency sufficient to 
 produce the death of the animal. 
 The inoculation of the two organ- 
 isms may be made into the same 
 situation or in different parts of 
 the body. 
 
 By the association of these two 
 organisms, the one of low viru- 
 lence will often acquire a high 
 virulence, which may be still 
 further raised by the passage 
 from animal to animal. 
 
 (c) An animal is inoculated with a 
 pure culture of an organism to- 
 gether with an injection of a 
 toxin (e. g., elaborated by the 
 proteins group, etc.) The na- 
 tural resistance of 'the animal is 
 lowered, the organism allowed to 
 multiply, its virulence is then 
 raised by passage from animal to 
 animal. 
 
 Attenuating- or 3^owering- the Viru- 
 lence Qf an Orgranism. 
 
 This is usually brought about by 
 
 (a) Cultivating the organism in a 
 media unsuitable by reason of its 
 composition or reaction. 
 
 (b) Cultivating the organism in 
 suitable media at an unsuitable 
 temperature. 
 
 (c) Cultivating the organism in 
 suitable media, in an unsuitable 
 atmosphere. 
 
 (d) Cultivating the organism in 
 suitable media, under unfavorable 
 conditions as to light, motion, etc. 
 
 (e) Passing the organism through 
 naturally resistant animals. 
 
 (f) Exposing the organism tc 
 desiccation. 
 
 (g) Exposing the organism to gas- 
 eous disinfections. 
 
 (h) By a combination of two or 
 more of the above methods. 
 
BACTERIOLOGY. 107 
 
 II. Isolation of Fathogrenlc Bacteria. 
 
 Certain parasitic bacteria are with 
 great difficulty isolated from as- 
 sociated saprophytic bacteria by 
 the ordinary culture methods by 
 reason of the difficulty with 
 which they grow upon artificial 
 culture media. 
 
 If the parasite and its associated 
 saprophyte are injected into an 
 animal susceptible to the paras- 
 ite, the pathogenic organism can 
 readily be Isolated from the tis- 
 sues of the infected animal. 
 
 Special media may be excellent for 
 the growth of these certain 
 parasites, but the associated 
 saprophytes will also grow so 
 abundantly as to overgrow the 
 parasite; therefore, if the ma- 
 terial containing the parasitic or- 
 ganism is inoculated under the 
 skin of a susceptible animal, the 
 pathogenic organism, in a few 
 days, will have entered the blood 
 stream and killed the animal, 
 leaving the saprophytes at the 
 seat of inoculation. 
 
 Cultivations made at post mortem 
 from the animal's heart blood 
 will produce a pure growth of the 
 pathogenic organism. 
 
 In obtaining the culture from the 
 infected animal, complete asepsis 
 must be the rule. The fur or 
 feathers are drenched with a 2% 
 lysol solution, to prevent the 
 hairs from flying about and enter- 
 ing the body cavities during the 
 autopsy, and also to render in- 
 ocuous any vermin that may be 
 present. With sterile forceps 
 and scalpel incise and reflect the 
 skin. Sear the whole exposed 
 surface with heated searing irons; 
 remove a part of the body wall 
 with a new set of sterile instru- 
 ments and proceed to make the 
 culture. 
 
 III. Identification of Pathog-enic Bac- 
 
 teria. 
 
 The morphological and cultural re- 
 semblances of certain pathogenic 
 bacteria are in some cases so 
 great as to make identification 
 impossible; the same organism 
 
108 BACTERIOLOGY. 
 
 may under carying- condition take 
 on an appearance so different 
 from the typical that results are 
 very confusing". A simple inocu- 
 lation may decide the point at 
 once. 
 
 IV. Study of the Problems of Im- 
 munity. 
 
 It is only by careful study of the 
 behavior of the animal cell and 
 the body fluids together with the 
 infecting organism that we may 
 understand the complex problem 
 whereby the cell successfully re- 
 sists the invading organism. 
 During the inoculation studies, 
 instances of both racial and indi- 
 vidual natural immunity will be 
 met with. Natural immunity, 
 however, is relative only and 
 never absolute and an organism 
 must not be put down as non- 
 pathogenic until many different 
 methods of inoculation have been 
 accomplished upon animals of 
 different species. (See immunity.) 
 
 METHODS USED POR THE STUDY 
 OP THE PATHOGENICITY OP 
 . AIT ORGANISM: 
 
 1. The livings Orgfanism. 
 
 (a) The Psychrophilic Bacteria. 
 
 (Grow only at or below 18° — 
 20° C.) 
 
 Cultivations are prepared under 
 optimum conditions in nutrient 
 broth, and after seven days' in- 
 cubation, an amount of the cul- 
 ture corresponding to 1% of the 
 body weight of a frog is injected 
 into the frogs dorsal lymph sac. 
 
 Observe, if necessary, for 28 days. 
 (See Animal Inoculation). 
 
 (b) Mesophilic Bacteria (grow at 
 35° — 37'' C.) 
 
 Cultivations are prepared under 
 • optimum conditions in nutrient 
 broth, and after 48 hours incu- 
 bation, inoculate a white mouse 
 subcutaneously at the root of the 
 tail, , with an amount of the cul- 
 ture corresponding to 1% of its 
 body weight. Inoculate a second 
 mouse of the same weight, or 
 nearly so, ultraperitoneally with 
 a similar dose. 
 
BACTERIOLOGY. 109 
 
 Observe carefully until death oc- 
 curs, or for 28 days. (See Animal 
 Inoculation). 
 
 If death takes place a post-mortem 
 examination is to be made. If 
 death takes place shortly after 
 the injection, the inoculation ex- 
 periment should be repeated two 
 or three times; and if the or- 
 ganism invariably exhibits patho- 
 genic effects, ascertain, if possi- 
 ble, its minimal lethal dose (from 
 the g-rowth upon solid media) for 
 frogs or white mice, respectively. 
 Now make tests on white rats, 
 guinea pigs and rabbits. 
 The Toxins. 
 
 Prepare bulk cultivations of the 
 organism in glucose * formate 
 broth and incubate for 14 days 
 under optimum conditions. For 
 the determination of: 
 
 (a) Intracellular or Insolul>le Tox- 
 ins. 
 
 Heat the fluid culture in a water 
 bath at 60° C. for 30 minutes. 
 Inoculate a tube of sterile bouil- 
 lon with an equal amount of the 
 heated culture and incubate under 
 optimum conditions to demon- 
 strate the absence of the living 
 organism. 
 
 Inject intravenuously that amount 
 of the cultivation corresponding 
 to 1% of the body weight of the 
 selected animal. 
 
 Observe during life, or until the 
 28 th day, and in the event of 
 death make a complete post mor- 
 tem examination. 
 
 The experiment should be repeated, 
 and if a positive result is ob- 
 tained, the minimal lethal does 
 of "killed culture" is estimated. 
 
 (b) Extracellular or Soluble Toxins. 
 Filter the cultivation through a 
 
 porcelain filter into a sterile filter 
 f flask. Inoculate the various an- 
 imals subcutaneously with a 
 quantity corresponding to 1% of 
 the body weight, and observe, if 
 necessary, for 28 days. 
 Inoculate a control tube of bouillon 
 and incubate to demonstrate the 
 absence of living organisms. 
 
110 BACTERIOLOGY. 
 
 Repeat the experiment, and if a 
 positive result is obtained .de- 
 termine the minimal lethal dose 
 of the toxin. 
 
 ANIMAI. INOCUI^ATION. 
 
 Animals employed in the study of 
 pathogeneses are the cold blooded 
 frogr; toad and lizard; the warm 
 blooded mouse, rat, guinea pig, rabbit 
 and monkey; the hot blooded fowl 
 and pigeon. 
 
 Before animals are inoculated they 
 should be carefully examined so as 
 to avoid the employment of any al- 
 ready diseased. This examination 
 should take in the observation of the 
 animal at rest and in motion; the ap- 
 pearance of the fur, feathers or 
 scales, inspection of the eyes, and of 
 the external orifices of the body; 
 tactile examination of the body and 
 limbs, palpation of the groins and 
 abdomen and in many cases micro- 
 scopical examination of fresh and 
 stained blood-films. 
 
 The mouse and rat may suffer from 
 septicemia, the cysticercus form of 
 taenia murina.; the cystic form of 
 .taenia crassicollis has its habitat in 
 their lives: scabies; favus and try- 
 panosoma Lewisi. 
 
 The guinea pig may suffer from scabies, 
 coccidiosis and tuberculosis. It is 
 well to test the animal by injecting 
 0.5 cc. of Koch's old tuberculin, which 
 will cause death in those diseased 
 within 48 hours. 
 
 The rabbit may suffer from psoric acari. 
 One form (sarcoptes minor) first 
 shows itself as yellowish scales and 
 crusts around the nose, mouth and 
 eyes, spreads to the bases and outer 
 surfaces of the ears, to the fore and 
 hind limbs and into the groins and 
 around the genitals. Another form 
 (psoroptes communis cuniculi) com- 
 mences at the bottom of the concha in 
 the form of white, yellowish masses 
 of crusts, scales, feces and dead 
 acari. The coccidium oviforme is a 
 frequent infection. Infection with 
 ordinary pyogenic bacteria frequently 
 occurs in the rabbit. 
 The monkey is very prone to tuber- 
 culosis and should be injected with 
 1 cc. Old tuberculin. 
 
BACTERIOLOGY. Ill 
 
 Anematode (oesophagostoma inflatum) 
 resembling the anchylostomum, par- 
 asitic in cattle, Is frequently present 
 in the tissues of the monkey, may 
 bore through the intestinal wall and 
 produce small cysts in the mesentery. 
 
 The pigeon may be infected by haemos- 
 poridia and pigeon diphtheria. 
 The fowl may suffer from scabies and 
 ringworm, fowl cholera or fowl sep- 
 ticaemia. 
 
 Animal inoculation is purely surgical 
 operation, therefore, in its perform- 
 ance strict attention must be paid to 
 asepsis and precautions adopted to 
 guard against contamination of the 
 material to be introduced into the 
 animal. 
 
 The Material used for Inoculation may 
 
 be 
 
 1. Cultures of bacteria grown — 
 
 (a) In fluid media; a definite meas- 
 ured quantity injected by means 
 of a syringe or (if a large bulk 
 is to be introduced) by means of 
 a gravity apparatus consisting of 
 a funnel, rubber tubing and an 
 injection needle. 
 
 (b) On solid medi^.; a fluid sus- 
 pension is made by washing the 
 culture with a little bouillon or 
 normal saline, and then injected i 
 as above. 
 
 2. Metabolic products of bacteria (Tox- 
 
 ins). Prepared as previously de- 
 scribed and injected as described 
 under cultures of bacteria. 
 
 3. Pathological products (fluid secre- 
 
 tions and excretions, solid tis- 
 sues) are treated as fluid cultiva- 
 tions. If the material is very 
 thick a small portion of bouillon 
 or normal saline solution may be 
 used to dilute it. Solid tissues 
 are rubbed up in a sterile mortar 
 with a small portion of bouillon. 
 The Methods of Inoculation. 
 Tne animal is held firmly by an assist- 
 ant or secured to an operating table, 
 liquid soap applied to the area select- 
 ed for inoculation with a small pad 
 and lathered freely by the aid of 
 warm water; shave thoroughly; wash 
 with 1% lysol solution; wash off lysol 
 with ether and allow the ether to 
 
112 BACTERIOLOGY. 
 
 evaporate; then inoculate by method 
 selected from the following: 
 
 1. Cutaneous MetlLOd. (No anaesthetic). 
 
 Make numerous short parallel 
 superficial incisions with the 
 point of a sterile scalpel and 
 when the oozing has ceased, rub 
 the inoculum into the scarifica- 
 tions. Cover the area with a pad 
 of sterile gause secured by ad- 
 hesive or collodion. 
 
 2. Subcutaneous Method. (No anaes- 
 
 thetic if inoculum is solid ethyl 
 chloride spray). 
 
 If the inoculum is fluid pinch up a 
 fold of skin between finger and 
 thumb and inject with a hypor- 
 dermic syringe. 
 
 If the inoculum is solid, raise a 
 fold of the skin in a pair of 
 forceps and make a small inci- 
 sion. By means of a probe make 
 a small pocket in the subcu- 
 taneous tissue and introduce the 
 tissue inoculum into it. Close 
 the wound in the skin with a clip 
 (Michel's) or a suture and cover 
 the area as in cutaneous method. 
 
 3. * Intramuscular. No anaesthetic if 
 
 the inoculum is fluid but if solid 
 use A. C. E. anaesthetic. The 
 method is practically the same as 
 in the subcutaneous, except that 
 the injection is made deep into 
 the muscle. 
 
 4. Intraperitoneal. (No anaesthetic). 
 
 For liquid inoculum the method is 
 essentially the same as in the 
 subcutaneous, except that the en- 
 tire thickness of the. abdominal 
 walls is pinched up into a tri- 
 angular fold. Ascertain that there 
 are no coils of intestine included 
 by slipping the peritoneal sur- 
 faces one over the other. 
 For the solid inoculum, an A. C. E. 
 anaesthetic is , used and the 
 aponeuroses between the recti 
 muscle are divided upon a direc- 
 tor, the peritoneum likewise, the 
 inoculum introduced: the peri- 
 toneum closed with Lembert's 
 sutures; the aponeuroses and skin 
 incision are closed together with 
 interrupted suture. 
 
BACTERIOLOGY. 113 
 
 Intracranial. (A. C. E. anaesthetic). 
 
 (a) Subdural. By the use of a 
 
 trephine open the skull in the 
 parietal seg^ment at the point of 
 intersection of the medium line 
 and a line joining the posterior 
 canthi, perforate the dura and 
 with a syringe deposit the ma- 
 terial immediately below this 
 membrane carefully so as to pro- 
 duce no injury. 
 
 (b) Intracerebral. Same as in in- 
 tracranial except that the needle 
 is pushed into the substance of 
 one or the other cerebral hemis- 
 pheres. 
 
 Intraocular. (Cocaine anaesthetic).* 
 Two needles are fitted to a 
 syringe. One is attached to the 
 syringe and the required dose of 
 inoculum is taken into it; the 
 needle is then removed. The other 
 needle is used to pierce the cornea, 
 allowing the aqueous to escape 
 through it, then without removal 
 the syringe is attached and the 
 inoculation is made into the an- 
 terior chamber. 
 
 Intrapulmonary. (No anaesthetic). 
 The fluid inoculum is injected 
 through the 5th and 6th inter- 
 costal space into the lung tissue. 
 
 Intravenous. (No anaesthetic). The 
 The fluid inoculum must be pre- 
 pared with care in order that 
 when injected a fatal embolism 
 may be obviated. If possible, the 
 fluid should be. filtered through 
 sterile filter paper to do away 
 with small fragments of tissue. 
 Eliminate the possibility of air 
 bubbles. After the usual prepar- 
 ation of the skin, plunge the 
 needle of the syringe through the 
 skin into the lumen of the vein 
 and slowly inject the inoculum. 
 Withdraw the needle and press a 
 pledget of cotton over the punc- 
 ture. 
 
 The jugular vein may be utilized in 
 the guinea pig; the posterior 
 auricular vein in the rabbit; the 
 internal saphenous vein in the 
 dog or monkey. 
 
 Inhalation (No anaesthetic). The 
 animal is placed in a closed 
 
114 BACTERIOLOGY. 
 
 metal box and through a hole in 
 one side of it the nozzle of the 
 spraying apparatus (ordinary 
 nasal spray will do) containing 
 the fluid inoculum is introduced 
 and sprayed into the interior of 
 the box. On completion of the 
 spraying, the animal is sprayed 
 thoroughly with a 10% solution 
 of formaldehyde and the animal 
 returned to its cage. The inhala- 
 tion chamber is thoroughly disin- 
 fected. In another method, for 
 both fluid and powdered inoculum, 
 frequently used, a wooden gag 
 provided with a square orifice 
 through which a tracheal or 
 oesophageal tube may be passed 
 down through the larynx into the 
 trachea. Connect the straight 
 portion of a Y-shaped tube to the 
 laryngeal tube; couple one branch 
 of this to a separatory funnel 
 containing the fluid inoculum or 
 insufflator containing the powder- 
 ed inoculum and the other to a 
 hand bellows. Allow the fluid 
 inoculum to run down into the 
 lungs by gravity, or below the 
 powdered inoculum into the lungs 
 by means of a bellows. 
 
 10. Intrag'astric. (No anaesthetic). By 
 
 use of a gag similar to the one 
 mentioned above, insert a soft 
 rubber catheter into the stomach 
 and allow a measured quantity of 
 the inoculum to run down into the 
 stomach. With some sterile salt 
 solution wash out the last traces 
 of the inoculum in the catheter 
 and then withdraw it. 
 
 11. Feeding'. Pieces of sterilized bread 
 
 are soaked in the fluid inoculum, 
 or small pieces of tissue inoculum 
 are placed in sterile dishes and 
 offered to the animal. 
 The possession of pathogenic prop- 
 erties by an organism is indicated 
 by the infection of the experi- 
 mental animal. Infection is con- 
 sidered to have taken place 
 
 (a) When the death of the animal 
 is produced by the inoculum. 
 
 (b) When, without producing death, 
 the inoculum causes local or gen- 
 
BACTERIOLOGY. 115 
 
 eral changes of a pathological 
 character, 
 (c) When either with or without 
 death, or the production of local 
 or general changes, certain sub- 
 stances make their appearance in 
 the body fluids which can be 
 shown to exert some specific ef- 
 fect when brought into contact 
 with cultivations of the organism 
 originally inoculated. 
 
 The observation upon the animals 
 inoculated must begin immediate- 
 ly and only terminate with the 
 death of the animal. If the an- 
 imal appears to be unaffected it 
 should be killed at the end of 
 2 or 3 months and a complete 
 post-mortem carried out. 
 
 The examination of the animal 
 should consist of 
 
 (a) General Observation daily as to 
 general appearance, weight, and 
 temperature. 
 
 (b) Special Observations. 
 
 1. As weekly examination of the 
 site of inoculation and the neigh- 
 boring glands palpated. 
 
 2. As to any local reaction (sup- 
 puration carefully examined both 
 microscopically and culturally). 
 
 3. Frequent examination of the 
 blood histologically. 
 
 4. Examination of the blood bac- 
 teriologically for the presence of 
 the organism previously injected 
 into the animal. 
 
 Method: 
 
 Sterilize a glass syringe and moisten 
 its interior with a sterile solution 
 of sodium citrate (sodium citrate 
 10 gm., sodium chloride 0.75 gm., 
 distilled water, 100 cc.) If more 
 than 5 cc. of blood is required, re- 
 tain about V2 cc. of the sodium 
 citrate solution in the syringe to 
 prevent coagulation of the blood. 
 Prepare the animal and introduce 
 the syringe needle into the lumen 
 of the selected vein; collect suf- 
 ficient blood; withdraw the needle; 
 deliver the citrated blood into a 
 flask containing 250 cc. of nu- 
 trient broth and incubate until 
 growth occurs or until the expi- 
 ration of 10 days. 
 
116 BACTERIOLOGY. 
 
 5. Examination of the blood sero- 
 logfically to demonstrate the pres- 
 ence of antiV3odies as antitoxin, 
 agg'lutinin, precipitin, opsonin, 
 and immune body or bacteriolysin. 
 (See under immunization). 
 Conditions Necessary to Infection are 
 1. The micro-org-anism must "be patho- 
 g*enic. It must be a parasite. 
 Organisms that are parasitic are 
 not necessarily pathogenic; how- 
 ever, certain requirements must 
 be met in order that an organism 
 may be in/ectious for any given 
 animal, and by this is meant, the 
 ability of an organism to live and 
 multiply in the animal fluids and 
 tissues. 
 
 Organisms which do not grow at 
 body temperature are not patho- 
 genic, neither are the strictly 
 aerobic organisms as they are 
 not able to obtain oxygen in 
 available combination from carbo- 
 hydrates. Aerobic organisms are 
 practically unable to multiply in 
 the blood stream and produce 
 general infection. 
 2., The org'anism must "be virulent. 
 
 Pathogenic organisms differ very 
 much in their power to incite 
 disease. This variation in viru- 
 lence occurs not only among dif- 
 ferent species of pathogenic or- 
 ganisms, but may occur within 
 the same species. Certain organ- 
 isms when kept upon artificial 
 media or in unfavorable environ- 
 ment for some time, are much 
 less virulent than those isolated 
 from the bodies of man or an- 
 imal. 
 
 3. The number of ors^anisms which 
 
 g'ain entrance to the animal tissue 
 must be of sufficient number. 
 
 A small number of organisms, even 
 though of the proper species and 
 of sufficient virulence, may be 
 overcome by the defenses of the 
 body. The more virulent the or- 
 ganism, the smaller the number 
 necessary to produce disease. 
 
 4. An Infection Path by which bacteria 
 
 gain entrance is of importance in 
 determining whether or not dis- 
 ease will occur. Streptocpcci 
 
BACTERIOLOGY. 117 
 
 when swallowed may cause no ef- 
 fect, while if rubbed into the 
 abraded skin will give rise to a 
 severe reaction. Typhoid rubbed 
 into the skin may not give rise 
 to any reaction of moment, while 
 if swallowed may .cause fatal in- 
 fection. Animals are protected 
 from bacterial invasion by the 
 skin and mucous membrane, and 
 when these are healthy and unin- 
 jured, micro-organisms are usual- 
 ly kept out, though they may oc- 
 casionally pass through uninjured 
 skin and mucosa. There can not 
 be much doubt that the tubercle 
 bacilli may pass through the in- 
 testinal mucosa into the lymph- 
 atics without causing local lesion. 
 5. Animal must be susceptilble. 
 
 Susceptibility is relative and not 
 absolute. It may be natural to 
 a certain race; it may be acquired 
 by the presence of conditions 
 which lower vitality; it may be 
 inherited, by reason of an in- 
 herited tendency. 
 Even though virulent pathogenic 
 organisms may pass through an 
 injured portion of the skin or 
 mucosa, it does not necessarily 
 follow that infection will take 
 place, as animals have an im- 
 munity (see "Immunity") which, 
 if normally vigorous and active, 
 will usually overcome a certain 
 number of the invading organ- 
 isms. If this immunity is weak 
 by reason of depression, or the 
 invading microorganisms are very 
 virulent or plentiful, infection 
 takes place. 
 
 INFECTIONS. 
 
 When microorganisms have gained an 
 entrance into the animal body and 
 give rise to disease, the process is 
 spoken of as infection. 
 
 In contact with the body of animals is 
 a vast flora of microorganisnis, some 
 constant parasites, some transient in- 
 vaders, some harmless saprophytes 
 and some capable of becoming patho- 
 genic. 
 
118 BACTERIOLOGY. 
 
 The phenomena of infection are reac- 
 tions between the microorganism and 
 the body defense. 
 
 In order to cause infection bacteria 
 must gain entrance to the body by 
 paths adapted to their own cultural 
 requirements and must be permitted 
 to multiply. 
 
 They may then give rise merely to 
 local inflammation, necrosis and ab- 
 scess formation; they may remain at 
 the point of entrance and elaborate 
 toxins which are absorbed and cir- 
 culated by the blood; they may, from 
 tne local lesion, gain entrance into 
 the lymphatics and blood vessels and 
 be carried freely into the circulation, 
 where, if they survive, bacteriaemia 
 or septicaemia follows; they may be 
 carried by the blood to other parts of 
 the body and find lodgment in any 
 of the organs and give rise to sec- 
 ondary foci of inflammation, necrosis, 
 and abscess formation (pyemia). 
 
 The disease arising as the result of the 
 infection may depend wholly or in 
 part upon the mechanical injury pro- 
 duced by the inflammatory process, 
 
 . the disturbed function caused by the 
 presence of the bacteria in capillaries 
 and tissues, and by the absorption of 
 the products resulting from the reac- 
 tion between the body cells and the 
 bacteria. 
 
 The symptoms characteristic of in- 
 fectious diseases, to a large extent, 
 result from the abosrption of: — 
 
 Bacterial Poisons, produced by the or- 
 ganisms themselves. 
 
 (a) Ptomaines were discovered by 
 
 Brieger during his investigations 
 into the* nature of the poisons 
 evident in bacterial infections. 
 These bodies isolated by him 
 from decomposing beef, fish and 
 human cadavers, although pro- 
 duced from proteid material by 
 bacterial action, and the cleavage 
 products derived from the culture 
 medium, they are not true bac- 
 terial poisons in the sense in 
 which the term is now employed. 
 
 (b) Toxins. The poisons produced by 
 
 all pathogenic microorganisms are 
 soluble, secretory products of the 
 bacterial cell, passing from the 
 
BACTERIOLOGY. 119 
 
 cell into the culture medium dur- 
 ing their life. 
 
 They may be obtained free from the 
 bacteria by filtration and in a 
 purer state from the filtrate by 
 chemical precipitation, etc. 
 
 The bacillus of diphtheria and the 
 bacillus of tetanus are examples. 
 If a several day bouillon growth 
 of these organisms is passed 
 through porcelain filters, the fil- 
 trate will often be extremely 
 toxic, while the residue will be 
 inactive or very weak. If the 
 residue possesses any toxicity at 
 all, the symptoms appearing will 
 be quite different from those pro- 
 duced by the filtrate. Other or- 
 ganisms act in an opposed man- 
 ner, e. g., spirillum cholera and 
 bacfillus typhosus. If these are 
 cultivated and filtered, the filtrate 
 will be toxic only to a slight de- 
 gree, while their residue may be 
 very toxic. This is evidently due 
 to poisons not secreted into the 
 medium but rather attached to 
 the bacterial body. They are 
 termed endotoxins, and the 
 greater number of pathogenic 
 bacteria seem to act under this 
 class. 
 
 Mode of action of bacterial poisons 
 
 is much the same as the ability 
 of the various narcotics and al- 
 kaloids to select special tissues 
 or organs and enter into a com- 
 bination with them, either chem- 
 ically or physically, or both. 
 
 Soluble toxins like the bacillus of 
 tetanus and the botulinus bacil- 
 lus attack specifically the nervous 
 system. Certain poisons elabor- 
 ated by certain organisms as the 
 staphylococci, streptococci, etc., 
 attack the red blood cell (haemo- 
 lysin) while others attack the 
 white blood cell (leukocidin"). 
 
 These toxins when in solution can 
 be removed by the acjdition of 
 their specific tissue, e. g., solution 
 of tetanus toxin, if treated with 
 brain substance and centrifuged 
 leaves the solution free from 
 toxin; likewise haemoly tic poisons 
 can be removed from solutions by 
 
120 BACTERIOLOGY. 
 
 contact with red blood cells, but 
 only when the red blood cells of 
 a susceptible species are em- 
 ployed. 
 
 THEORY OF IMMUNITY. 
 
 Several theories have been advanced to 
 account for the various phenomena 
 of immunity. Pasteur advanced the 
 
 "Ezliaustion theory," in which bacteria 
 by their growth in the body used up 
 or exhausted something vitally neces- 
 sary to their subsequent growth. 
 
 detention theory in which certain 
 noxious agents are retained by the 
 body, which prevent further growth 
 of bacteria. 
 
 Cellular or hiologric theory of Metchni- 
 "koSf or "Phagocytosis." 
 
 Humoral or chemical theory of ZShrlich, 
 or "Side Chain." 
 
 The theory accepted by most bacteri- 
 ologists is a combination of the 
 theories of Metchnikoff and Ehrlich, 
 and is called the Cellulo -humoral. 
 The theories of immunity, accept- 
 able at the present time, are 
 based upon two branches of 
 . study: — 
 
 (1) A conception formulated by the 
 German school under the leader- 
 ship of Ehrlich, Pfeiffer, Kruse; 
 deal entirely with the phenomena 
 occurring in reaction between bac- 
 teria or bacterial products and 
 body fluids. 
 
 (2) The participation of the cellular 
 elements of the body in its re- 
 sistance to infectious organisms. 
 Phagocytosis was formulated by 
 studies at the Pasteur Institute 
 in Paris, under the leadership of 
 Metchnikoff. 
 
 IMMUNITY. 
 
 It is plain that the mere entrance of 
 pathogenic organism into the animal 
 body through the skin or mucosa does 
 not necessarily lead to the develop- 
 ment of an infection. The body must 
 therefore possess certain means of 
 defense in order that the pathogenic 
 grerms after they have gained en- 
 trance into the tissue and fluids will 
 
BACTERIOLOGY. 
 
 121 
 
 be disposed of, or, at least, be pre- 
 vented proliferating and elaborating 
 their poisons. The condition which 
 enables the body to accomplish this 
 is spoken of as resistance, and when 
 this resistance is especially marked, 
 it is spoken of as ''immunity." 
 
 Immunity, therefore, denotes that con- 
 dition of an organism which enables 
 it to resist an attack of the particular 
 bacteria and their toxic secretion 
 against which they are said to be 
 immune. The varieties of immunity 
 are: — 
 
 1. Natural Immunity, as an inheritance 
 
 from immune ancestors. 
 
 (a) Species Immunity. — Many infec- 
 tious diseases common to man 
 do not occur in animals; e. g. 
 gonorrhea and syphilis do not oc- 
 cur in animals except when pro- 
 duced experimentally and this 
 with the greatest difficulty. Lep- 
 rosy, influenza, etc., have not been 
 transmitted to animals, likewise 
 human beings are immune to dis- 
 eases which attack animals. 
 
 (b) Racial Immunity. Separate races, 
 or varieties within the same spe- 
 cies, often display differences in 
 their immunities towards patho- ' 
 genie organisms; e. g., Algerian 
 sheep show a much higher resist- 
 ance to anthrax than do our 
 domestic sheep. The difference in 
 resistance towards tuberculosis 
 between the Caucasian and the 
 American Indian, the Eskimo and 
 Negro is very striking. Converse- 
 ly, the comparative immunity of 
 the negro from yellow fever, 
 which shows very great virulence 
 toward the Caucasian. 
 
 (c) Individual Immunity Is noticed to 
 some extent in man and may 
 probably be attributed to indi- 
 vidual variation in the body 
 metabolism; e. g., depressions in 
 gastric acidity predispose to in- 
 fection of gastrointestinal origin; 
 anatomical differences may act as 
 predisposing factors towards in- 
 fection. 
 
 2. Acquired Immunity. 
 
 (a) Active Immunity is^naturally ac- 
 quired by having had a previous 
 
122 BACTERIOLOGY. 
 
 infection. This is illustrated by 
 an infection with typhoid fever, 
 yellow fever, and many of the 
 exanthemata. A single attack ot 
 any of these diseases protects 
 the individual for a limited period 
 and frequently for life. 
 It may be artificially acquired by: 
 
 (1) Inoculations with weakened, at- 
 tenuated cultures of bacteria. 
 
 (2) Inoculation with sublethal doses 
 of fully virulent microorganisms. 
 Successive inoculations with 
 gradually increased doses of the 
 virus creates an immunity suffi- 
 cient to resist ten times the toxic 
 dose. 
 
 (3) Injecting with gradually in- 
 creased doses of dead microorgan- 
 ism. Used especially in that 
 class of bacteria in which the 
 cell bodies (endo toxins) had been 
 found to be more poisonous than 
 their extra-cellular products 
 (toxins). 
 
 (4) Injecting gradually increased 
 doses of the bacterial product 
 (toxins). 
 
 (b) Passive Imnmnity is acquired by 
 injection of the serum of animals 
 that have been rendered immune 
 by artificial methods, into the in- 
 dividual infected or to be pro- 
 tected. 
 
 This type of immunity is used 
 chiefly against diseases caused by 
 bacteria which produce powerful 
 toxins, and the sera of animals 
 immunized against such toxins 
 are called antitoxic sera. 
 
 Passive immunity against micro- 
 organisms that do not have mark- 
 ed toxin formation has not been 
 successful. The microorganisms 
 which are injurious by reason oi 
 the content of the bacteria cell, 
 rather than by the secreted 
 soluble toxins, probably do not 
 produce antitoxins in the sera oi 
 immunized animals. . 
 
 The substances produced by their 
 immunization seem directed 
 against the invading organisms in 
 that they have the power of de- 
 stroying the specific germ used m 
 the production of immunity. 
 
BACTERIOLOGY. 123 
 
 The anti bacterial sera are used in 
 laboratory animals to immunize 
 them against a large number of 
 ^^erms, and if used just before, 
 at the same time or soon after 
 infection, they seem fairly ef- 
 fective. 
 
 In human disease their use has been 
 disappointing, except when the 
 anti bacterial sera could be 
 brought in direct contact with the 
 germs, as in closed cavities or 
 localized lesions; e. g., Plexner's 
 sera for meningitis. 
 
 ANTIBODIES. 
 
 The treatment of the animal body with 
 bacteria or their products gives rise 
 to a variety of reactions which result 
 in the presence of "antibodies." These 
 bodies are not produced by bacteria 
 or their products only. They may be 
 produced by a variety of poisons of 
 plant and animal origin. 
 
 Nuttall, Fodor and Pluggs (1886), noted 
 the bacterial properties of normal 
 blood. A study of the blood sera of 
 immunized animals by Beljarff showed 
 no change from normal as to index 
 of refraction, specific gravity, and al- 
 kalinity. 
 
 Joachim, Moll, Hiss and Atkinson found 
 immunized blood sera richer in glob- 
 ulin than normal serum. 
 
 Very little light was thrown upon the 
 phenomenon of immunity until Nut- 
 tall, Fodor and Buchner demonstrated 
 the power of normal blood serum to 
 destroy bacteria. This property of 
 the blood diminished with age and 
 was destroyed by heating to 56° C. 
 Buchner called this themolabile sub- 
 stance alexin. 
 
 Behring, Kitasato and Wernicke, in 1890 
 and 1892, showed that the blood sera 
 of actively immunized animals against 
 the toxins of diphtheria and tetanus 
 would protect normal animals against 
 the poisons of these diseases. Behring 
 called this substance, contained in the 
 blood sera of immunized animals, 
 antitoxins. 
 
 Soon after this Ehrlich produced anti- 
 toxin against some of the higher 
 plants. Calmette produced antitoxin 
 
124 BACTERIOLOGY. 
 
 against snake and scorpion poisons; 
 Kempner against the poison of bacil- 
 lus botulinus, etc. 
 
 The formation of antitoxins directed 
 against the soluble toxins did not ex- 
 plain the immunity acquired against 
 bacteria which produced no soluble 
 toxin. Pfeifeer (1894), threw light 
 upon this when he injected into the 
 peritoneal cavity of cholera-immune 
 guinea-pigs, cholera spirilla. 
 
 The microorganism often underwent 
 complete solution, determined by 
 hanging-drop preparations. 
 
 MetchnikofC and Bordet showed that 
 this lytic process would also take 
 place in vitro. The constituents of 
 the blood serum which gave rise to 
 this destructive phenomenon were 
 called 'bacteriolysins. 
 
 Gruber and Durham then discovered 
 another specific property of immune 
 serum to which the name agrgrlutiniu 
 was applied. Certain bacteria, when 
 brought into contact with the serum 
 of animals immunized against them, 
 became clumped, lost their motility 
 and firmly agglutinated. 
 
 In 1897, Kraus demonstrated that pre- 
 cipitates were formed when the fil- 
 trates of cultures of typhoid, cholera, 
 etc., ^ere mixed with their specific 
 immune sera. These substances he 
 called precipitins. 
 
 The large variety of substances, some 
 poisonous, some inocuous, that pos- 
 sess the power of stimulating anti- 
 body formation in the sera of animals 
 are termed autigrens or antibody- 
 producers. 
 
 Ehrlich proposes three forms of recep- 
 tors in explanation of all varieties of 
 antibodies. (See Side Chain Theory). 
 1st order liaptines or receptors, 
 when free in the circulation, con- 
 stitute the antitoxins and anti- 
 ferments. 
 2nd order haptines or receptors, 
 when free in the circulation, serve 
 as anchorage and for the further 
 digestion of antigens. They are 
 precipitins and agglutinins. 
 3rd order haptines or receptors 
 merely anchor suitable substances 
 and exert no action till combined 
 with the complement. When free 
 
BACTERIOLOGY. 125 
 
 in the circulation with a chemical 
 group having" affinity for the 
 antig-en and a complementophile 
 group are the amboceptors of 
 bacteriolytic, cytolytic and hemo- 
 lytic sera. 
 
 The second group of receptors which 
 give rise to agglutinins and pre- 
 cipitins Ehrlich believes to be made 
 up of a single hatophore group for 
 the anchorage of the ingested ma- 
 terial, and an attached zymophore 
 group, or ferment, which changes the 
 anchored substance preparatory to its 
 absorption by the cell protoplasm. 
 
 Bordet has shown that it is not the 
 agglutining itself which agglutinates, 
 but the agglutinin in combination 
 with its antigen is agglutinated by 
 the salt solution. He therefore dis- 
 agrees with Ehrlich and concludes 
 that the phenomenon of agglutination 
 consists of the union of the antibody 
 with its antigen in a celloidal solu- 
 tion, and that the actual agglutina- 
 tion is a secondary phenomenon de- 
 pending possibly upon a change in the 
 physical properties of the emulsion. 
 The feame he holds to be true of pre- 
 cipitins. 
 
 The antibodies which can be demon- 
 strated are agglutinin, precipitin, op- 
 sonin and bacteriolysin. 
 
 These substances cannot»be isolated in 
 purity apart from the blood serum, 
 consequently, methods have been 
 elaborated to permit of their recog- 
 nition. 
 
 The serum from the experimental an- 
 imal (specific serum) is studied and 
 compared with the serum from an 
 uninoculated animal of the same spe- 
 cies (normal serum). In order that 
 the differences existing in the serum 
 of various individuals may be elim- 
 inated, a mixture of sera obtained 
 from several normal animals (pooled 
 serum) is usually used. 
 
 Collection of Serum. 
 
 Shave the dorsal surface of the ear, 
 wash with lysol, remove lysol by 
 dropping ether over it and allowing 
 the ether to evaporate, puncture the 
 vein and collect the blood by means 
 of a small blood-collecting pipette, 
 touch the issuing drop of blood with 
 
126 BACTERIOLOGY. 
 
 one end of the pipette, which is held 
 at an angle so that the blood will 
 flow down into it. When the tube is 
 about two thirds full, hold it by the 
 end containing the blood, the clean 
 end pointing obliquely upward, warm 
 this end with the bunsen flame to 
 expel some of the contained air; then 
 seal it in the flame. Shake the blood 
 into the closed end and seal the other 
 end in the flame. 
 
 When the blood has clotted place the 
 pipette in the centrifuze, the end first 
 closed pointing downward, and cen- 
 trifugalize thoroughly. The blood 
 cells will then be found collected in 
 a firm mass at one end, and above 
 them wil appear the clear serum. 
 
 Mark the pipette above the serum with 
 a file and break it off at this point; 
 the serum is now accesible for test- 
 ing. 
 
 IMMUNIZATION. 
 
 In order to study the pathogenic pow- 
 ers of any particular bacterium, the 
 active immuniation of one or more 
 normal animals becomes necessary. 
 This is done by various methods; 
 seldom by one method only, but usual- 
 ly by a combination of methods 
 adapted to suit each particular case. 
 The ordinary methods used are as 
 follows: 
 
 1. Active Immunity. (See "Immunity"). 
 An illustration of how the general 
 methods of immunization are carried 
 out is as follows: — 
 
 A full grown rabbit weighing from 1200 
 to 1500 gms. or over is most suitable 
 for immunization. 
 
 A small rabbit is inoculated intraperi- 
 toneally with one or two loopfuls of 
 2i hour optimum culture of the viru- 
 lent organism selected. Death should 
 follow within 24 hours, or at most, 
 in 48 hours. Under aseptic precau- 
 tions the rabbit is "posted," and a 
 loopful of heart blood is transferred 
 to 50 cc. of sterile broth. This is 
 incubated at 37° C. for 24 hours. Also 
 prepare several cultures on optimum 
 media from the heart blood of the 
 rabbit; label them all O. C. (original 
 culture). Incubate at 37* C. for 24 
 
BACTERIOLOGY. 127 
 
 hours, after which seal the mouth of 
 the plug- tube of all but one culture 
 with an Indian rubber cap painted 
 with shellac or paraffin, and replace 
 in incubator. (Prevents evaporation 
 and culture will remain virulent for 
 a considerable period of time). 
 Suspend the 24 hour broth culture in 
 a water bath at 60° C. for one hour 
 in order to kill the culture. Cool 
 immediately. Now determine the ster- 
 ility of the cultivation by transferr- 
 ing 1 cc. to each of several tubes of 
 broth; incubate at 37° C. for 24 hours. 
 If a growth occurs, heat the culture 
 in the water bath again at 60° C. for 
 
 1 hour. Test again for sterility. If 
 sterile, inject the suitable animal 
 mentioned above intravenously with 
 
 2 cc. of the killed culture; also inject 
 10 cc. into the peritoneal cavity. 
 Watch the animal the next few days; 
 it will lose some weight and may 
 show pyrexia. 
 
 When the temperature and weight have 
 become normal again, inject a killed 
 culture in a mount of 5 cc. intraven- 
 ously and 20 cc. intraperitoneally. 
 Weight and pyrexia reaction like but 
 less marked than that following the 
 first inoculation will probably follow. 
 
 Subcultivate on optimum medium, the 
 uncapped O. C. ; incubate for 24 hours 
 at 37° C. ; determine the minimal 
 lethal dose upon a number of mice. 
 
 One week after the last killed culture 
 injection, prepare a fresh optimum 
 subculture from another O. C. tube 
 and incubate for 24 hours. Prepare 
 the minimal lethal dose and inject 
 subcutaneously into the abdominal 
 wall. A local reaction, a pyrexia and 
 loss in weight will probably be ob- 
 served. In about ten days, inject a 
 similar minimal lethal dose into the 
 peritoneal cavity. 
 
 Note weight and temperature of animal 
 carefully, regulating the time of the 
 animal's inoculation by its general 
 condition and continue to inject liv- 
 ing cultivations into the peritoneal 
 cavity in gradually increased doses by 
 multiple of ten. 
 
 At intervals of 2 months the animal's 
 serum is tested for its specific anti- 
 bodies. 
 
128 BACTERIOLOGY. 
 
 Under favorable conditions, after 6 
 months' work, the animal may be in- 
 jected intra peritoneally with an 
 entire optimum cultivation of the 
 organism without any ill effect. 
 
 The animal serum, if withdrawn in 
 about a week following the injection, 
 will, if injected in doses of 0.01 cc. 
 into a mouse, protect it against ten 
 times the minimal lethal dose of the 
 organism. 
 
 Immunity has been created by reason 
 of the formation of an antibody spe- 
 cific to the bacterium in question and 
 was sufficient in amount to destroy 
 enormous doses of the living organ- 
 ism, the antigen in this case being 
 a bacterial protoplasm of the organ- 
 ism with its endotoxin. 
 
 If death did not immediately follow the 
 injection of the antigen, specific anti- 
 bodies are always formed in greater 
 or lesser extent; and in experimental 
 work a sufficient amount of any re- 
 quired antibody may be obtained 
 without carrying the process of im- 
 munization to completion. * 
 
 If the immunization of a rabbit toward 
 bacillus typhosus be carried out along 
 the lines indicated above, it will be 
 noticed that after a few injections of 
 the killed cultivations that the blood 
 serum of the animal contains specific 
 agglutins for the bacillus typhosus, 
 
 ,and if the object of the experiment has 
 been directed toward the preparation 
 of agglutinin, the inoculation may be 
 stopped, even though the animal is 
 not yet strictly immune. 
 
 Antibodies may also be formed in re- 
 sponse to antigens other than micro- 
 organisms, as can be demonstrated 
 by the injection into animals of for- 
 eign proteins, such as egg albumin, 
 blood sera or the red blood cells from 
 an animal of different species. This 
 will lead to the formation of specific 
 antibodies possessing affinities for 
 their specific antigen. Hemolysin is 
 a common antibody of this type and 
 is found in the blood serum of an 
 animal that has previously been in- 
 jected with the washed red blood cells 
 from an animal of a different species. 
 This serum will possess the power 
 of disintegrating the blood cells of 
 
BACTERIOLOGY. 129 
 
 the variety employed as antigen, and 
 cause these red blood cells of any 
 other species of animal. 
 The action of this serum is due to the 
 presence of two distinct bodies; the 
 one, hemolysin, and the other, com- 
 plement. 
 
 Hemolysin (immune body, copula, sen- 
 sitizingr "body, and amboceptor) is a 
 
 thermostable antibody which is form- 
 ed by the repeated injection of foreign 
 red blood cells into an animal. Al- 
 though it is itself inert, it will link 
 up the complement present in normal 
 serum to the red blood cells of the 
 variety used as antigen. A combina- 
 tion of the two results is Hemolysis. 
 
 Hemolysin is obtained by collecting 
 fresh blood serum from an animal 
 that has been inoculated with ma- 
 terial in question and then exposed 
 to a temperature of 56° C. for 15 to 
 30 minutes to destroy the comple- 
 ment. It is now spoken of as in- 
 activated serum. It is reactivated by 
 the addition of fresh normal serum 
 which contains the complement. 
 
 Although hemolysin is of importance in 
 making clear many problems of im- 
 munity, its main and practical im- 
 portance is in its application of the 
 hemolytic system to certain laboratory 
 methods having for their object the 
 identification of infected entity or the 
 diagnosis of existing infection. 
 
 For its use directed towards the meth- 
 ods of laboratory diagnosis, it is very 
 convenient to prepare hemolytic serum 
 specific for human blood. Ox blood, 
 sheep blood, or goat blood may, how- 
 ever, be used instead. 
 
 Complement (or alexin) is a thermo- 
 labile oxidizable body present in the 
 normal serum of every animal in a 
 variable but unalterable amount. It 
 is a substance which exerts a lytic 
 effect upon all foreign matter intro- 
 duced into the blood or tissues. It 
 is, in itself, comparatively inert and 
 is capable of exerting its greatest 
 lytic effect only when in the presence 
 of and in combination with a specific 
 antibody or immune body. 
 
 It is obtained by collecting fresh blood 
 serum from any healthy normal an- 
 imal. Guinea pig serum is most fre- 
 quently employed. 
 
130 BACTERIOLOGY. 
 
 Preparation of hemolytic sernm. 
 
 Take 2 cc. of citrated human blood col- 
 lected from a vein aseptically, place 
 it in the centrifuge and centrifugalize 
 it thoroughly. Wash it with normal 
 saline and centrifugalize again. Re- 
 peat this procedure twice, after which, 
 by means of a sterile pipette, transfer 
 the washed blood cells into a sterile 
 capsule. Add 5 cc. of normal saline 
 and mix thoroughly. By means of a 
 sterile glass syringe, inject the blood 
 suspension into the peritoneal cavity 
 of a healthy rabbit weighing at least 
 2500 gms. At the end of seven days, 
 inject the washed blood cells from 
 10 cc. of human blood mixed with 
 5 cc. of normal saline. After another 
 interval of 7 days, repeat the injection 
 of washed blood cells from 10 cc. of 
 human blood mixed with 5 cc. of 
 normal saline. 
 
 After 5 days, collect about 2 cc. of the 
 rabbit's blood, allow it to clot, sepa- 
 rate the serum and transfer it to a 
 sterile test tube. Place the test tube 
 in a water bath of 56'' C. for 15 
 minutes to inactivate the serum, then 
 the serum quantitively for hemolytic 
 
 . properties as follows: — 
 
 Titration of Hemolytic Serum. 
 
 1. Two test tubes marked A and B 
 
 each containing 9 cc. of normal 
 saline. 
 
 2. Add 1 cc. of rabbit serum to tube 
 
 A; mix thorouhgly. 
 1 cc. of this mixture is added to 
 tube B; mix thoroughly. 
 
 3. Place ten small test tubes in a 
 
 rack and number them from 1 
 to 10. 
 
 4. By means of a pipette place into 
 Tube No. 1. 0.5 cc. of hemolytic 
 
 serum = 0.5 cc. hemolytic serum. 
 
 Tube No. 2. 0.2 cc. of hemolytic 
 serum = 0.1 cc. hemolytic serum. 
 
 Tube No. 3. 0.5 cc. of tube A mix- 
 ture =1 0.05 cc. hemolytic serum. 
 
 Tube No. 4. 0.3 cc. of tube A mix- 
 ture = 0.03 cc. hemolytic serum. 
 
 Tube No. 5. 0.2 cc. of tube A mix- 
 ture = 0.02 cc. hemolytic serum. 
 
 Tube No. 6. 0.1 cc. of tube A mix- 
 ture = 0.01 cc. hemolytic serum. 
 
 Tube No. 7. 0.5 cc. of tube B mix- 
 ture = 0.005 cc. hemolytic serum. 
 
BACTERIOLOGY. 
 
 131 
 
 Tube No. 8. 0.03 cc. of tube B mix- 
 ture = 0.003 cc. hemolytic serum. 
 
 Tube No. 9. 0.02 cc. of tube B mix- 
 ture — 0.002 cc. hemolytic serum. 
 
 Tube No. 10. 0.01 cc. of tube B mix- 
 ture = 0.001 cc. hemolytic serum. 
 
 5. To each of the above ten tubes add 
 
 1 cc. of the red blood cells sus- 
 pension. 
 
 6. Add sufficient normal saline to the 
 
 tubes containing a small amount 
 of material, to bring the columns 
 of fluid to the same level. 
 
 7. Shake each tube so as to thoroughly 
 
 mix its contents. Plug the mouth 
 of the tube with cotton and place 
 all in the incubator at 37° C. for 
 1 hour. 
 
 8. Remove the tubes from the incu- 
 
 bator and into each tube, by 
 means of a pipette, place 0.1 cc. 
 complement (guinea pig serum); 
 replace tubes in incubator for 1 
 hour. 
 
 9. Remove the tubes from the incu- 
 
 bator and if all the tubes are not 
 completely hemolized, stand on 
 one side, in ice-chest if possible, 
 for 1 hour. 
 
 10. Examine all tubes for — 
 Complete hemolysis — clear red so- 
 lution, no deposit of red cells at 
 the bottom of the tube. 
 
 Absence of hemolysis — clear or 
 turbid colorless fluid, with a de- 
 posit of red cells at the bottom of 
 the tube. 
 
 The smallest amount of hemolytic 
 serum causing complete hemolysis is 
 known as the minimal hemolytic dose 
 (M.H.D.) and, if hemolysis has oc- 
 curred in all of the tubes down to 
 No. 7, the M.H.D. of this particular 
 serum is 0.005 cc. 200 minimal hemo- 
 lytic doses per cc. This serum is 
 strong enough for experimental work. 
 As a matter of fact, complete hemo- 
 lysis down to tube No. 6 will indicate 
 a serum sufficiently strong (= 100 
 M.H.D. per cc.) If the first one or 
 
 ^ two tubes show complete hemolysis 
 only, the rabbit should receive further 
 injections in order to raise the hemo- 
 lytic power to the proper high level. 
 
132 BACTERIOLOGY. 
 
 THE STORAGE OF HEMOLYSIN. 
 
 If the rabbit serum hemolytic contents 
 is found to be sufficient, chloroform 
 the rabbit and remove aseptically as 
 much blood as possible from the heart 
 and place it in sterile centrifuge tubes. 
 Place the tubes in the incubator at 
 37° C. for 2 hours, after which times 
 centrifugalize thoroughly. Pipette off 
 the clear serum and fill in quantities 
 of 1 cc. into small pipettes, sealed 
 hermatically, in the blow pipe flame, 
 avoid scorching the serum. Place the 
 small pipette containing the serum, 
 after having been sealed in the water 
 bath at 56° C. for 30 minutes (de- 
 stroying the complement) ; i. e., in- 
 activating the serum and at the same 
 time insuring sterility. A longer ex- 
 posure reduces the hemolytic power. 
 Place the pipette in a metal box and 
 store in the ice-chest. 
 
 IjYSINS are substances occurring in 
 normal and immune sera which have 
 the power of destroying and dissolv- 
 ing bacteria and dissolving or liberat- 
 ing the hemoglobin of the red blood 
 cells and also have a lytic action on 
 the various body cells. 
 
 When acting on bacteria, they are called 
 bacterolysins; on the red blood cells, 
 hemolysins; on the body cells, cyto- 
 lysins. The piechanism of the process 
 is complex. Certain substances which 
 kill bacteria and the body cells but 
 do not actually dissolve them are 
 spoken of respectively as bactericidal 
 substances and cytotoxins. 
 
 Blood cells of one animal, injected into 
 another animal of another species, 
 gives rise to a hemolytic substance in 
 the blood serum of the animal in- 
 jected, which is specific for the 
 variety of cells injected. These hemo- 
 lysins are termed heterolysins. 
 
 Ehrlich and Morgenroth injected the 
 washed red blood cells of one goat 
 into another and found that the serum 
 of the injected goat would, after a 
 time, develop hemolytic power against 
 the blood cells of the goat whose 
 blood cells had been used but did not 
 possess hemolytic power toward the 
 red blood cells of all goats. Such 
 substances producing hemolysins in 
 
BACTERIOLOGY. 
 
 133 
 
 members of its own species are called 
 isolysius. 
 
 The injection of isolysins produced anti- 
 isolysins which were again specific. 
 They were not able to produce sub- 
 stances that would hemolyze the an- 
 imals own red blood cells (autolysin). 
 
 Lytic substances can be prepared for a 
 large number of bacteria and for 
 many body cells. These bodies may 
 be increased markedly during the pro- 
 cess of immunization. The substances 
 having the power to produce lysins 
 are called lysinogen and are distinct 
 antigens as the lysins are antibodies. 
 The lysins may be prepared by in- 
 jecting the live cells, the dead cells, 
 the disintegration products of cells 
 and in some cases the metabolic pro- 
 ducts of cells. 
 
 From the fact that the bacteriolytic 
 digestive power of immune serum 
 after being destroyed by heating or 
 attenuated by time can be restored — 
 "reactivated" — by the addition of 
 small quantities of normal blood 
 serum, Borded concluded that the 
 bactericidal or bacteriolytic action of 
 the serum depended upon two sub- 
 stances. One present in normal serum 
 and thermolabile, he identified as 
 Buchner's alexin. The other, more 
 stable, produced or increased in serum 
 
 - by immunization, he called the "sensi- 
 tizing substance," which he believed 
 
 : acted upon the bacterial cells and 
 rendered them susceptible to the ac- 
 tion of alexin. 
 
 Ehrlich called the thermolable substance 
 or alexin "compliment" and showed 
 that it was always present in normal 
 serum and was very little, if at all, 
 increased during the process of im- 
 munization. 
 
 The "sensitizing substance" he called 
 the "immune body," and this he 
 showed to be increased during im- 
 munization. Ehrlich argued that when 
 bacteria or blood cells were injected 
 into the animal, certain chemical 
 components of the injected substances 
 were united to side chains of proto- 
 plasm of the tissue cells. The ex- 
 cessive production of these receptors 
 caused their detachment and subse- 
 quent invasion into the circulation as 
 
134 
 
 BACTERIOLOGY. 
 
 "immune bodies.'* These immune 
 bodies must therefore possess atom 
 complexis, which endow it with chem- 
 ical affinity for the bacteria or red 
 blood cells used in its production. 
 
 The complement does not combine di- 
 rectly with the blood cell or bacteria, 
 but does so through the intervention 
 of the immune body which possesses 
 two atom groups or haptophores; one 
 the cytophile haptophore group, pos- 
 sessing strong chemical affinity for 
 the blood cell or bacteria; the other 
 complementophile haptophore group- 
 possessing a weaker affinity for the 
 •complement. 
 
 Blood cell ,<rvc^ rmiTTTN ^ 
 
 The Structure of I^ysins. 
 
 Ijysins and bactericidal substances are 
 composed of a thermolabile part — 
 "complement" — which is destroyed at 
 a temperature of 56° to 60° for 30 
 minutes and a thermolstable part — 
 "amboceptor" — having a double com- 
 bining power. The amboceptor with- 
 stands a 60° C. temperature for 24 
 hours, but will be destroyed at a tem- 
 perature of 70° C. Ehrlich believes 
 these amboceptors to be free chemical 
 receptors of the body cells produced 
 toy the same method as are the anti- 
 toxins, but differing from them in 
 that they have two combining groups; 
 one called the citophyle group, with 
 which the amboceptor combines with 
 bacteria or other cells; and the other, 
 the complementaphyle group, with 
 which it combines with the comple- 
 ment. 
 
 J The complement seems to be a normal 
 constituent of the blood sera and 
 other body fluids and is undoubtedly 
 produced by the various body cells, 
 leucocytes, etc. During the immuni- 
 zation of animals, it increases but 
 
 or , 
 
 t . t 
 
 ^^Complement 
 
 Cy fopb\\C Complemenlophilc 
 
 Haptophore 
 
BACTERIOLOGY. 
 
 135 
 
 slightly, if at all. It is supposed to 
 be made up of two groups, one a hap- 
 tophore group, which combines with 
 the amboceptor, and another a zyma- 
 phore group which really produces the 
 lytic action after the haptophore has 
 combined with the amboceptor. On 
 heating the complement, the zymo- 
 phore group is destroyed and a com- 
 plementoid is produced. This sub- 
 stance is similar to a toxid and will 
 combine with amboceptor but no lysis 
 will result. (See antitoxins). 
 
 It is the amboceptor that is increased 
 during process of immunization. The 
 complement will not combine with the 
 cells unless the amboceptor is present 
 and has first combined with the cells. 
 It is probable that in the body fluids 
 there are many complements which 
 may activate a variety of ambocep- 
 tors. Various sera have been noted 
 which possess amboceptors for cer- 
 tain cells, but they are not lytic be- 
 cause they do not possess the neces- 
 sary complement. For example, dog 
 serum contains amboceptors for the 
 anthrax bacterium, but no comple- 
 ment. If, in such cases, a foreign 
 complement, such as guinea pig or 
 rabbit serum is added, there will be 
 lysis of the bacterial cells. 
 
 Occassionally the absence of a comple- 
 ment may benefit an animal, and this 
 may account for the seeming natural 
 immunity as illustrated when the 
 venom (this is nothing more than am- 
 boceptors) of a poisonous snake is 
 injected into an animal, such as the 
 hog, which possesses no complement, 
 no lysis of the cells takes place. On 
 the other hand, should the animal, 
 such as rabbit or man, possess, as 
 they do, the necessary complement, 
 lysis will take place. 
 
 Substances are sometimes normally 
 present in sera which have the power 
 of combining with amboceptors which 
 may be present and in turn, prevent 
 the amboceptors from combining with 
 the cells so that when the comple- 
 ment is added there will be no lysis. 
 These substances are spoken of as 
 anti amboceptors and they may be de- 
 veloped in an animal by immuniza- 
 tion with amboceptors of definite kind 
 
136 BACTERIOLOGY. 
 
 (anti antibodies). Certain other sub- 
 stances which may also engage the 
 amboceptors but cannot be called anti- 
 amboceptors in the true sense, ac- 
 complish the same purpose and are 
 therefore classed with these bodies. 
 
 Deviation of the Complement. The com- 
 plement may be deviated in several 
 ways, and as a result lysis may be 
 prevented. Sometimes there is noted 
 in sera normal substances which com- 
 bine with the complement and prevent 
 it from combining with the ambo- 
 ceptors. These substances are spoken 
 of as anti complements and may be 
 produced by the immuniation of an- 
 imals with complements. 
 
 The complement may occasionally be 
 absorbed by tissue cells and in this 
 way prevented from combining with 
 the amboseptor. In certain cases, 
 where the serum contains an excess 
 of amboceptors and only a small 
 amount of complement, it may be 
 deviated by reason of the fact that the 
 cells will then have taken up all the 
 possible amboceptors, leaving an 
 abundance of free amboceptors in the 
 serum to combine with the comple- 
 ment, so that no lysis will take place. 
 (This theory advanced by Neisser and 
 Wechsberg has created wide discus- 
 sion but is believed to be erroneous). 
 
 Fixation of Complement (as a test for 
 antibodies). The demonstration of 
 this was first worked out by Neisser 
 and Wechsberg and it is now used for 
 testing the sera for unknown anti- 
 bodies similar to bactericidal sub- 
 stances and lysins. The reaction is 
 made use of in the recently devised 
 test for syphilis, (See Wasserman 
 reaction). 
 
 Filtration of Immune Body and Comple- 
 ment. 
 
 Filtration of the serum will allow the 
 amboceptor to pass through while the 
 complement is held back. The am- 
 boceptor filters equally well, whether 
 or not mixed with the complement. 
 
 COMPLFMENT FIXATION. 
 Test for Immune Body (Amboceptor). 
 
 The term "complement fixation" is ap- 
 plied to a method of investigation by 
 which even the small quantities of 
 
BACTERIOLOGY. 
 
 137 
 
 any given amboceptor can be demon- 
 strated in the serum. The method 
 was devised by Bordet and Gengrou 
 and prepared as follows: 
 
 Tube No. 1 
 
 Bacterial Ambo- 
 cepto 
 
 (Plague immune 
 serum, heated). 
 
 Plague Emulsion. 
 
 Complement 
 
 (Fresh Normal 
 serum). 
 
 — Allowed to act for 
 
 Hemolytic ambo- 
 ceptor 
 
 (Heated hemo- 
 lytic serum). 
 
 Red blood cells. 
 
 Tube No. 2 
 
 Normal serum, 
 heated. 
 
 Plague Emulsion. 
 
 Complement 
 
 (Fresh Normal 
 serum). 
 
 5 hours, then add — 
 
 Hemolytic ambo- 
 ceptor. 
 
 (Heated hemo- 
 lytic serum). 
 
 Red blood cells. 
 
 = Results = 
 
 No hemolysis. Hemolysis. 
 
 (Fluid turbid or (Fluid clear and 
 with red ppt). red). 
 
 The conclusion to be drawn from this, 
 would be, that in No. 1, the presence 
 of amboceptor had led to the absorp- 
 tion of all the complement and that 
 in No. 2, there being no bacteriolytic 
 immune body to sensitize the bacteria 
 and enable them absorb complement, 
 the complement was therefore left 
 free to activate the added hemolytic 
 amboceptors. 
 
 This principle of complement fixation, 
 discovered by Boedet and Gengou in 
 1901, has been utilized in bacteriolog- 
 ical investigations and in the prac- 
 tical diagnosis for the determination 
 of specific antibodies in serum. 
 
 The reaction depends upon the fact that 
 when an antigen is mixed with its 
 inactivated antiserum, in the presence 
 of complement, the complement Is 
 firmly fixed by the combined ambo- 
 ceptor and antigen in such a way that 
 it can no longer be found free in the 
 
138 BACTERIOLOGY. 
 
 mixture. If this mixture is now al- 
 lowed to stand at a suitable tempera- 
 ture for an hour or more, then an 
 emulsion of red blood cells and in- 
 activated hemolytic serum are added, 
 no hemolysis will take place as there 
 is no free complement present to com- 
 plete the hemolytic system. (See e. g. 
 below). 
 
 -Complement 
 
 ^Hemolytic ) Hemolytic System 
 Amboceptor [ (Hemolysis) 
 
 =Red blood celr 
 
 N/1 
 
 Immune Body 
 (Antibody) 
 
 ZZ. Antigen 
 
 Eg. = Test tube containing—^ 
 — Complement. 
 
 — Patient's serum containing 
 immune body (antibody). 
 
 — Antigen. 
 
 Placed in incubator at 37.5° for 
 
 one hour, then add — 
 
 — Hemolytic ambocepto 
 — Red blood cells. 
 = Results = 
 
BACTERIOLOGY. 139 
 
 No hemolysis occurs by reason of the 
 
 immune body combining with the 
 complement and antigen — 
 
 (I) (+) (5) 
 
 Allowing the red blood cell and the 
 hemolytic amboceptor to remain free — 
 
 CI) (2) (3) 
 
 No completion of hemolytic system. 
 
 If the original mixture contains no anti- 
 body for the antigen used, the com- 
 plement present is not fixed and Willi 
 be available for the activation of the 
 hemolytic serum. (See (1) (2) (3> 
 above). 
 
 The reaction is therefore seen to de- 
 pend upon the fact that neither 
 antigen alone nor amboceptor alone 
 can fix the complement, but that this 
 fixation is carried out only by the 
 combination of antigen plus ambo- 
 ceptors. Any specific amboceptor can 
 be determined by this method, pro- 
 vided the hemologous or stimulating 
 antigen is used and vice versa. 
 
 DETERMINATION OF ANTIBODIES 
 BY COMPLEMENT FIXATION. 
 
 When testing immune sera for certain^ 
 amboceptors in man or animals by 
 microorganisms which can be culti- 
 vated, either the whole organism or- 
 its extracts may be used as antigen.. 
 
 Bordet and Gengou use a thick 6alt so- 
 lution emulsion of a 24 hour agar 
 slant culture of the organism. In the 
 use of tubercle bacilli, 80 mg. ot 
 bacilli are emulsified in 1 cc. of salt 
 solution. 
 
 Wasserman and Bruck prepare antigen 
 by emulsifying 10 agar slant cul- 
 tures in 10 cc. of sterile distilled 
 water, after which it is placed in a 
 
140 BACTERIOLOGY. 
 
 shaking apparatus and shaken for 24 
 hours. 0; 5% of carbolic acid is added 
 and the fluid cleared by centrifuga- 
 tion. The old or the new tuberculins 
 or "Bacillary Emulsion" are used. 
 Method. 
 
 H. S. = Hemolytic serum (heated for 
 15 min. at 56° C, i. e. inactivated). 
 
 Comp. = Complement (Fresh guinea 
 pig serum). 
 
 H. R. B. C. = Human red blood cells. 
 
 S. S. — Specific serum from inocu- 
 lated animals, — inactivated. 
 
 P. S. = Control "pooled serum" from 
 normal animals of the same species, 
 — inactivated. 
 
 Ant. = Antigen (organism grown on 
 solid media and previously having 
 served as antigen in the inoculated 
 animals). 
 
 Place in test tubes 
 
 12 3 
 
 0.1 cc. Comp. — 0.1 cc. Comp. 
 
 0.2 cc. S. S. 0.2 cc. S S. 
 
 1.0 cc. Ant. 1.0 cc. Ant. 1.0 cc. Ant. 
 
 4 5 
 
 0.1 cc. Comp. 0.1 cc. Comp. 
 
 0.2 cc. P. S. 
 
 0.1 cc. Ant. 
 
 Incubate at 37° C. for one hour 
 
 Add to each tube 1 cc. of H. R. B. C. 
 
 and 4 minimal hemolytic doses (see 
 titration of hemolytic serum) of H. S. 
 
 Incubate at 37° C. for one hour 
 
 Results 
 
 No. 1 — No hemolysis = indicates the 
 presence in the serum of the in- 
 oculated animal of a specific anti- 
 body to the organism used in the 
 inoculations; since it shows that 
 the complement has been bound by 
 the immune body to the bacterial 
 antigen and none has been left free 
 to enter into the hemolytic system. 
 
 Hemolysis indicated that no appreci- 
 able amount of antibody has yet 
 been formed in response to the in- 
 
BACTERIOLOGY. 
 
 141 
 
 oculations; i. e., there is no infec- 
 tion, since the complement remained 
 unfixed at the time of the addition 
 of the H. R. B. C. solution and the 
 H S 
 
 No. 2 = No hemolysis ( 
 
 No. 3, 4, 5 = Hemolysis V''°lt° No. 1. 
 
 It may sometimes be convenient to 
 sensitize the H. R. B. C. just be- 
 fore they are needed. This is done 
 before the completion of the first 
 period of incubation. 
 Method. 
 
 Place 5 cc. of H. R. B. Q. and 20 min- 
 imal doses of H. S. in a sterile test 
 tube and allow them to remain at 
 room temperature for 15 minutes. 
 The red cells are then sensitized 
 and ready for use. 
 
 When the tubes are removed from 
 the incubator at the end of the first 
 hour, 1 cc. of the sensitized red 
 cells are added to each tube; mixed 
 thoroughly and the tubes returned 
 to the incubator for the second 
 period. 
 
 Complement Fixation for the Determ- 
 ination of Immune Body of Syphilis. 
 
 The so called "Wasserman Reaction" 
 
 (although not strictly belonging to 
 the domain of bacteriology) has re- 
 cently become so prominent as a 
 diagnostic agent in syphilis that an 
 outline of it will be given. 
 1. Antigfen. 
 
 Wasserman first made use of salt 
 solution extracts of the organs 
 (spleen, etc.) of a syphilitic fetus, 
 in which the uncombined products 
 (free syphilitic antigens) of spiro- 
 chaete pallida were assumed to be 
 present. He cut the tissue into 
 small pieces and added 4 parts by 
 weight of a normal salt solution 
 containing 0.5% of carbolic acid to 
 1 part of the tissue. This was 
 placed in the shaking apparatus for 
 24 hours and then centrifugalized. 
 The supernatent liquid was used as 
 the antigen. Porges and Meyer pre- 
 pared antigen by extracting syphi- 
 litic organs with alcohol. The 
 syphilitic liver was chopped up and 
 extracted with 5 volumes of abso- 
 lute alcohol for 24 hours, filtered 
 
1^2 BACTERIOLOGY. 
 
 through paper and then the alcohol 
 was evaporated in vacuo at a tem- 
 perature not exceeding 40° C. About 
 1 gm. of the greenish residue is 
 emulsified in 100 cc. of salt solu- 
 tion containing 0.5% of carbolic 
 acid and filtered through thin paper. 
 The filtrate is used as the antigen. 
 
 Nogruchi prepared antigen by- thor- 
 oughly macerating normal liver or 
 spleen in five times its volume of 
 absolute alcohol. Place it in the 
 incubator and allow it to extract 
 for 6 to 8 days with thorough stir- 
 ring daily. It is then passed 
 through cheese cloth and filtered 
 through paper. The extract is now 
 evaporated to dryness at room tem- 
 perature and the sticky, brownish 
 residue is dissolved in a small 
 quantity of ether and 4 times its 
 volume C. P. acetone is added. A 
 heavy, sticky, brown precipitate 
 settles to the bottom. This mass 
 is used as the antigen and may be 
 preserved under acetone. The ace- 
 tone soluble fraction is discarded. 
 When wanted for use, about 0.2 
 gm. of the mass is dissolved in 
 about 5 cc. of ether, 100 cc. of 
 normal salt solution added and 
 shaken till the ether has evap- 
 orated. The antigen is now titrated, 
 and when this is accomplished it 
 is ready for use. 
 
 The use of syphilitic organs for the 
 preparation of antigen is not neces- 
 sary in order to obtain a subtsance 
 which will combine with the syphi- 
 litic immune body. 
 
 Many nonspecific antigens will give 
 reasonably reliable results. Porges 
 and Meier found that a 1% com- 
 mercial lecithin in carbolized salt 
 solution furnished a suitable anti- 
 gen. This has, however, not been 
 universally accepted. 
 
 They also found that normal foetal 
 liver would give good results; like- 
 wise others have successfully used 
 an alcoholic extract of guinea pig 
 heart. 
 
 The ingredient furnishing the immune 
 body binding power is unknown as 
 yet, although it is claimed to be 
 due to the lipoids. 
 
BACTERIOLOGY. 
 
 143 
 
 Antigen must be standard before It 
 can be used for the actual test. 
 The substances used as antigens 
 often have the power, if used in 
 too large quantities of binding the 
 complement. It is therefore neces- 
 sary to determine the largest quan- 
 tity of antigen which may be used 
 without binding complement. This 
 may be done by mixing graded 
 quantities of antigen with a con- 
 stant quantity of complement, in 
 duplicate sets, and adding to each 
 tube of one set 0.2 cc. of a normal 
 serum and to the other 0.2 cc. of a 
 known syphilitis serum. These 
 substances are allowed to remain 
 together for one hour and then red 
 blood cells and inactivated hemo- 
 lytic serum are added. The quan- 
 tity which has caused complete in- 
 hibition with the syphilitic serum, 
 but none with normal serum, is the 
 one to be used in the subsequent 
 tests. 
 
 A dilution of the antigen should be 
 made with salt solution in such 
 a way that 1 cc. shall contain the 
 required amount of antigen. (E. g. 
 if 0.05 cc is wanted, mix 0.5 cc. with 
 9.5 cc. of salt solution. 1 cc. of 
 this can be used in each tube in the 
 test). 
 
 The Hemolytic Semm, Amboceptor. 
 
 Prepared as outlined on page 130, 
 using sheep's blood. 
 By reason of the fact that small 
 amounts of precipitins for sheep's 
 serum may be present in the serum, 
 due to insufficient washing of the 
 corpuscles employed in the immuni- 
 zation, and cause the formation of 
 precititates which have a tendency 
 to carry down the complement from 
 a mixture, it is therefore necessary 
 that the serum is of high potency 
 in order that the quantities used 
 for the reaction may be as small as 
 possible. 
 
 The smallest amount of hemolytic 
 serum that has caused complete 
 hemolysis in 1 cc. of a 5% emul- 
 sion of washed blood corpuscles is 
 spoken of as the hemoljrtic nnlt. 
 
 Many make use of two hemolytic 
 units for the actual reaction. 
 
144 BACTERIOLOGY. 
 
 Complement (fresh guinea pig serum) 
 is obtained by anesthetizing a 
 guinea pig, incise the carotid artery 
 and allowing the blood to flow into 
 a large Petri dish. The dish is put 
 away in the ice chest until the 
 serum has separated, which is then 
 carefully removed. 
 
 The serum may be centrifugized to 
 insure complete separation from 
 blood cells. The complement in 
 G. P. S. is for practical purposes, 
 constant in quantity. It should be 
 kept, except when in actual use, or 
 a low temperature, and should not 
 be used after 3 days from the time 
 of preparation. 
 
 Sheep's Corpuscles are obtained by 
 receiving the blood of a sheep in — 
 
 (1) a flask containing glass beads, 
 and shaking thoroughly for about 
 10 minutes to completely defibrin- 
 ate the blood. The corpuscles are 
 washed free from the serum by 
 centrifugalization in salt solution 
 
 (2) a flask containing a sterile solu- 
 or 
 
 tion of 0.5 % sodium citrate and 
 0.85% sodium chloride, which will 
 prevent clotting and the corpuscles 
 may be washed free from the 
 citrate solution by centrifugaliza- 
 tion in salt solution. Thorough 
 washing with salt solution is essen- 
 tial in order to preclude the oc- 
 curence of precipitates and to re- 
 move traces of complement. The 
 bulk of the centrifugalized cor- 
 puscles is measured and 19 parts of 
 sterile salt solution is added. This 
 forms a 5% enlulsion of corpuscles 
 which is the solution employed for 
 the test. 
 
 Serum to toe tested for Syphilitic anti- 
 body. 
 
 3 to 5 cc. of blood is removed from a 
 vein, or if conditions will not per- 
 mit such procedure, the blood may 
 be obtained from the finger or ear. 
 The quantity of blood must always 
 be of suflJicient quantity to furnish 
 1 cc. of clear serum. The serum is 
 now inactivated by heating at 56° 
 C. for 20 to 30 minutes. 
 
 Noguchi advises inactivation at 54° C. 
 as the 56° C. destroys the syphilitic 
 antibody in part. 
 
BACTERIOLOGY. 
 
 145 
 
 <1? o 
 
 § \ p.^ 
 
 c3 C O d 
 
 ft ^ o w 
 
 d 2 o 
 o w o o 
 
 iH o 
 O O* CO 
 
 <V rj 
 
 m M C q! 
 
 ft g o<; 
 
 ci 2 d 
 o S o 
 
 ft <u d !3 
 
 ^.2ft"^ 
 g:;^ o d 
 d c3 c5 
 
 (M^ tH O 
 O O CO 
 
 bflS 
 
 01 13 <D 4-> 
 
 2 ^ 
 
 g S O cj 
 
 d 2 d d 
 
 o o a 
 
 C<l tH O 
 
 O O eo 
 
 ft (V « 
 
 ?3 (13 
 
 ■ ^.2ft.^ 
 
 <D ^ <V 
 
 ft.^ 
 
 <=> 55 S 1:^ 
 
146 BACTERIOLOGY. 
 
 Place in water bath at 40* C. for 
 
 1 hour 
 
 Add to each tube 1 cc. of a 5% solution 
 of sheeps' corpuscles and two units 
 of amboceptor. 
 
 Place in water bath at 40** 'C. for 
 
 1 to 2 hours 
 
 Xtesnlts. 
 
 If test is positive, tubes 3A and 4A 
 will show no liemolysis while all 
 other tubes show complete hemo- 
 lysis. 
 
 Tube No. 1. "A" shows active comple- 
 ment. "B" shows antigen alone is 
 not sufficient to deviate comple- 
 ment. 
 
 Tube No. 2. "A" shows no deviation of 
 complement in presence of normal 
 serum alone. "B" shows that the 
 particular serum alone will not 
 deviate the complement. 
 
 Tube No. 3. "A" shows that antigen 
 is specific in that it deviates com- 
 plement in the presence of syphi- 
 litic antibody. "B" shows syphi- 
 litic antibody will not alone deviate 
 the complement. 
 
 Tube No. 4. "A" shows the presence 
 
 . of an antibody specific to the anti- 
 gen employed in that the comple- 
 ment was deviated by antigen in 
 presence of test serum. 
 Nogruchi's Modification of the Wasser- 
 man Test. 
 
 Anti-human hemolytic amboceptor is 
 used instead of an anti-sheep am- 
 boceptor. It is obtained by 4 or 5 
 injections of washed human cor- 
 puscles into rabbits. The ambo- 
 ceptor unit is obtained as in the 
 original Wasserman. Two units are 
 used. 
 
 The fact that human serum contains 
 normally no amboceptor active 
 against the human red corpuscle is 
 important and has an advantage 
 over the original Wasserman. 
 
 Human serum, normally, may contain 
 a variable quantity of amboceptor 
 for sheep's corpuscles, consequent- 
 ly the actual amount of hemolytic 
 amboceptor used in the original 
 Wasserman is uncertain. This is 
 not so in the Noguchi, as the actual 
 quantity of amboceptor is known 
 exactly by titration. 
 
BACTERIOLOGY. 
 
 147 
 
 Antig'en. Prepared as in Wasserman. 
 
 Complement. A 40% fresh guinea pig 
 serum is made by mixing 1 part of 
 serum with 1.5 parts of salt solu- 
 tion. 0.1 cc. of this solution is used 
 for the test. 
 
 Siiman Corpuscles. Normal corpus- 
 cles, or those of the patient himself, 
 may be employed. The patient's 
 corpuscles should not be used for 
 other tests than that on the 
 patient's own serum. 1 cc. of a 1% 
 emulsion of washed corpuscles is 
 used for the test. 
 Patient's Serum to be tested for the 
 Syphilitic antibody. 
 
 Obtained as in Wasserman or in 
 Wright's tube* 
 
 About 2 cc. should be taken. 
 
148 
 
 BACTERIOLOGY. 
 
 1 
 
 o O 
 
 E S ""SI 
 
 ^ ;3 S 
 O ^ 
 
 ^ c 
 
 o <r) <P 
 
 .5 o 
 o 
 
 eo < 
 
 o 
 
 o S £ 
 _, o o 5-1 *^ 
 
 ^ S 0^ • 
 > g ^ o . 
 
 .-^ o ^ 
 
 5 ^ » 
 
 go 
 
 w 5 ft 
 
 S S <i> c^^.S ' 
 " ft g d ^ 
 
 -M 2 ^ w ^ 
 
 o 
 
 s s § ^ 
 
BACTERIOLOGY. 149 
 
 Shake, and place in water bath at 
 
 38°-40° for one hour 
 
 Add to each tube two units of ambocep- 
 tor and the human red blood cell 
 emulsion. 
 
 — ^Shake, and replace in water bath for 
 one hour or more till controls are 
 hemolized 
 
 BesTilts. 
 
 If test is positive there will be no 
 hemolysis in tubes lA and 2A while 
 all others are hemolized. 
 Determinatiou of Autig'en by Comple- 
 ment Fixation. 
 
 In testing for suspected antigen, the 
 procedure is reverse to that of test- 
 ing for suspected antibodies. The 
 serum or bacterial extract to be 
 tested for antigen is brought into 
 contact with an antibody specific 
 for the antigen in the presence of 
 complement; and at the end of an 
 hour at suitable temperature, free 
 complement is again determined by 
 hemolytic reaction, as in the anti- 
 body tests. 
 
 Hemolytic Amboceptor. Prepared in 
 the rabbit for sheep corpuscles. 
 
 Inactivated and titrated as for Was- 
 serman test. 
 
 Two units are used. 
 
 Bacterial Antiserum. Prepared by 
 immunizing a rabbit. It must be 
 highly potent. The smallest quan- 
 tity of the immune serum which 
 will fix the complement in the 
 presence of an emulsion or extract 
 of the microorganism in question is 
 determined by experiment. 
 
 The bacterial emulsion is prepared by 
 scraping the growth from 24 hour 
 agar slant cultures, drying it, and 
 macerating in a mortar with salt 
 solution until a slight opalescent 
 emulsion is formed. 
 
 Prepare a series of tubes, each con- 
 taining 0.1 cc. of the bacterial emul- 
 sion, 0.1 cc. complement and gradu- 
 ally diminishing quantities of in- 
 activated specific immune serum, 
 ranging from 0.1 cc. downward. 
 
 Incubate the tubes at 38°-40° C. for 
 1 hour. 
 
 Add the required quantities of red 
 blood cells and hemolytic immune 
 serum. The smallest quantity of 
 
150 BACTERIOLOGY. 
 
 immune serum which has complete- 
 ly inhibited hemolysis is the unit. 
 A quantity slig-htly in excess of the 
 unit is used in the test. 
 
 Complement. Fresh guinea pigr serum 
 (0.1 cc. used in routine work). 'It 
 should however be titrated if pos- 
 sible and used in double the quan- 
 tity necessary to produce hemolysis 
 of 1 cc. of a 5% emulsion of blood 
 cells, in the presence of two units 
 of amboceptor. 
 
 Sheep Corpuscles. Prepared as in 
 Wasserman test. 
 
 Patient's Serum. Obtained by the 
 usual method and inactivated at 
 56" C. for 20 minutes. 
 Test. 
 
 Prepare a series of tubes, each con- 
 taining: 
 
 1. Complement, 0.1 cc. or the deter- 
 
 mined quantity. 
 
 2. Antiserum, the determined quan- 
 
 tity. 
 
 3. Serum to be tested for antigen in 
 
 diminishing quantities from 1 cc. 
 downward. 
 
 4. Salt solution for dilution to 3 cc. 
 
 5. Control tubes containing the same 
 
 ingredients without the antiserum. 
 
 6. Incubate for 1 hour at 40° C. 
 
 7. Add required quantities of ambo- 
 
 ceptor and red cells. 
 
 8. Incubate again. 
 
 Results = A positive reaction if there 
 is no hemolysis in the tubes con- 
 taining the patient's serum. 
 
 Proteid Differentiation by Complement 
 Fixation, such as human or animal 
 blood was shown by Gengou in 1902. 
 The test is said to be more delicate 
 and reliable than the precipitation 
 tests. 
 
 Hemolytic amboceptor. Prepared as 
 for Wasserman test. 
 
 Complement. Prepared as for Wasser- 
 man test. 
 
 Sheep Corpuscles. Prepared as for 
 Wasserman test. 
 
 Specific Antiserum. Prepared by im- 
 munizing a rabbit with the proteid 
 material for which the test is to 
 be made. .Filtrate by using dim- 
 inishing quantities of the antiserum 
 in a series of test tubes containg 
 the determined quantity of com- 
 
BACTERIOLOGY. 151 
 
 plement, and the antigen which is 
 
 to be tested for, i. e., the holologrous 
 
 serum with which the antiserum 
 
 has been produced. 
 The test should be so delicate as to 
 
 determine 0.0001 cc. of the antigen, 
 
 consequently this quantity is added 
 
 to each tube. 
 The tubes are incubated for 1 hour. 
 
 The hemolytic amboceptor and red 
 
 cells are then added. 
 The unit represents the smallest 
 
 quantity of antiserum which has 
 
 completely inhibited hemolysis. 
 One and one-half to two units are 
 
 used for the test. 
 Solution of th.e proteid material to be 
 
 tested. Prepare as for precipitin 
 
 test. 
 Test. 
 
 Prepare a series of tubes containing: 
 
 1. Complement, quantity determined 
 
 by titration. 
 
 2. Antiserum, quantity determined by 
 
 titration. 
 
 3. Diminishing quantities of the sub- 
 
 stance in which the antigen is 
 suspected, ranging from 0.1 cc. 
 downward to 0.0001 cc. 
 
 4. Salt solution to make dilution to 
 
 3 cc. 
 
 5. Control tubes containing the same 
 
 ingredients without the antiserum. 
 
 6. Incubate for 1 hour at 40° C. 
 
 7. Add j^equired quantities of ambo- 
 
 ceptor and red cells. 
 
 8. Incubate again. 
 
 Results =: A positive reaction if there 
 is no hemolysis in the tubes con- 
 taining the suspected antigen. 
 
 EHBI^ICH'S SIDE-CHAIN THEORY de- 
 rives its name from its analogy to 
 what happens in the Benzol ring when 
 its replaceable hydrogen atoms are 
 substituted by "side-chains." 
 
 The theory itself is based upon the 
 mechanism of cell nutrition in its re- 
 lation to the mode of production of 
 specific antitoxins. 
 
 In order that a cell may be nourished, 
 the nutritive substance must enter 
 directly into chemical combination 
 with some elements of the cell pro- 
 toplasm. 
 
 The highly complex protoplasmic mole- 
 cules of cells are made up of a central 
 
152 BACTERIOLOGY. 
 
 atom-group, upon which the special- 
 ized activities of the cell depend, and 
 several outer atom-groups (side- 
 chains) by which the cell entered into 
 chemical relation with food and other 
 substances brought to it by the cir- 
 culation. 
 
 In just the same way the nutritious sub- 
 stances are brought into relation with 
 the cell by means of the side atom- 
 groups, so will also isomeric toxins. 
 
 These side-chains are called "receptors," 
 and if they have an affinity, by reason 
 of isomerism or chance for a given 
 toxin they unite with the toxin and 
 are tltferefore rendered useless for 
 their normal physiological function of 
 nutrition. 
 
 These receptors are probably cast off 
 and regenerated by the normal re- 
 parative mechanism of the body. 
 
 The regenerative process does not stop 
 at simple replacement of the cast off 
 elements, but goes on to over compen- 
 sation, so that they are reproduced 
 in excess of the physiologic needs of 
 the cell, and therefore cast of£ to cir- 
 culate in the blood as antitoxins. 
 These receptors (outer-atom-.groups, 
 antitoxins) retain their specific affin- 
 ity for the toxins used in their pro- 
 duction and will unite with the poison 
 before it can reach the sensitive cells, 
 and in this way protect the cell from 
 the poison. ^ 
 
 Toxin analysis (Ehrlich). 
 
 Toxin solutions deteriorate with time; 
 i. e. a toxin bouillon which contained 
 80 toxin units per 1 cc. was found to 
 contain but 40 units after 4 or 5 
 months. 
 
 Ehrlich found that such bouillon re- 
 tained its full original power ot 
 neutralizing antitoxin. 
 
BACTERIOLOGY. 
 
 153 
 
 The toxin molecule must therefore con- 
 tain two separate atom-groups. One 
 stable-group, possessing- the power of 
 binding antitoxin, he called the "hap- 
 tophore" or "anchoring" group. The 
 other, the one by which the toxin 
 molecule exerts its deleterious action, 
 is more easily changed or destroyed, 
 he calls the "toxophore," or poison 
 group. 
 
 In the toxin-bouillon in which a part of 
 its poison has been lost while the 
 neutralizing antitoxin power still re- 
 mains, it is quite evident that the 
 toxophore group, or some of the toxin, 
 must have been changed or destroyed. 
 Altered in this way he calls it 
 "toxoid." 
 
 Substances found in fresh bouillon, 
 which have a weaker affinity for anti- 
 toxin than toxin itself, called "toxins," 
 are primary secretory products of the 
 bacteria. 
 
 The "toxoids" are of two kinds — namely, 
 those which have a stronger affinity 
 for antitoxin than toxin itself (pro- 
 toxoids), and those whose affinity for 
 antitoxin is equal to that of toxin 
 (syntoxoids). 
 
 The toxon has a haptophore group sim- 
 ilar to that of toxin, but a different 
 toxophore group. It differs from 
 toxin in that it lacks the power to 
 produce acute symptoms; it causes 
 gradual emaciation and paresis in an- 
 imals. 
 
 The toxophore group, producing the 
 harmful results, is divided into toxin, 
 toxoid and toxon. 
 
154 BACTERIOLOGY. 
 
 The haptophore group of the toxin, 
 then, possesses the affinity for the re- 
 ceptor or antitoxin. 
 
 The haptophore groups of all three of 
 these substances are alike. In toxid, 
 the toxophore group has been de- 
 stroyed or altered; in toxon it is 
 qualitatively different from that of 
 toxin. It should therefore produce 
 antitoxins. 
 
 AGG-IiUTIITINS. 
 
 While investigating the Pfeiffer reac- 
 tion with B. coli, Gruber and Durham 
 noticed that if the immune serum 
 was added to bouillon cultures of B. 
 coli, the cultures would loose their 
 turbidity and flake-like clumps would 
 sink to the bottom of the tube. 
 
 Widal applied this agglutination reac- 
 tion to the practical diagnosis of 
 typhoid. 
 
 Gruber and Durham believed the ag- 
 glutinins to be identical with the im- 
 mune body concerned in the Pfeiffer 
 reaction, which injured the bacteria, 
 thereby rendering them susceptible to 
 alexins. It has since been shown that 
 agglutinins and bactericidal sub- 
 stances are in no way parallel. 
 Strongly agglutinating sera may be 
 very weak in bactericidal power and 
 strongly agglutinating sera may be 
 very weak in agglutinating power; the 
 relative quantity of these substances 
 depends upon the method of immuni- 
 zation. 
 
 Agglutinated bacteria are not killed by 
 the algglutination and are often as 
 virulent as non-agglutinated cultures. 
 Agglutinins remain active after ex- 
 posure to over 55° C. temperature. 
 Some will withstand 65° to 70* C. 
 and can not be reactivated by the ad- 
 dition of normal sera. This excludes 
 the participation of complement in 
 this reaction. 
 
 The agglutinins do not dialyze. 
 
 Normal sera contain small amounts of 
 agglutinin — "normal agglutinins" — 
 probably due to the various micro- 
 organisms parasitic upon the animal 
 body. 
 
 Agglutinins can be produced by intro- 
 ducing microorganisms subcutaneous- 
 ly, intravenously or intraperitoneally. 
 Almost all the known bacteria will 
 
BACTERIOLOGY. 
 
 155 
 
 produce agglutinin, and it will, as a 
 rule, appear in the blood of animals 
 three to six days after the micro- 
 organisms' introduction; increase to 
 a maxim at the 7th to the 13th day, 
 they then fall oft till they reach a 
 level, at which they remain for a long 
 time. 
 
 Agglutination is not limited to bacteria; 
 just as hemolysins are produced by 
 the injection of red blood cells, so 
 hemagglutins are similarly formed. 
 
 Ag-grltitiuating' Test. 
 Microscopic Method. 
 
 1. Collect a small amount (5-10 drops) 
 
 of blood in a small glass tube. 
 
 2. Separate the serum by centrifu- 
 
 gation. 
 
 3. By means of Wright's capillary 
 
 pipette or the white mixing pi- 
 pette accompanying the hemocy- 
 tometric counting chamber, dilute 
 the serum with normal salt solu- 
 tion in proportion of 1 to 20. 
 From this, subdilute in propor- 
 tion of 1-40, 1-80, 1-160, etc., and 
 place each dilution in a separate 
 sterile watch glass. 
 (Dilutions can also be made by the 
 drop method, using a capillary 
 pipette from which a drop of 
 serum is placed in a watch glass 
 and then normal saline dropped 
 into it till proper dilution is ob- 
 tained). 
 
 4. With the platinum loop, place a 
 
 drop of the serum from each of 
 the dilutions of serum on a cover 
 glass, and inoculate each with a 
 loopful of a 24 hour old bouillon 
 growth of the organism. 
 
 5. Press the cover slips carefully over 
 
 the chamber of culture slides, the 
 margins of v/hich have been 
 singed with vaseline (hanging 
 drop method). 
 See that the various dilutions are 
 properly indicated on the slides. 
 6. Examine immediately under the 1/6 
 objective of the microscope, and 
 discard the slides if any clumps 
 are observed. If the mount is 
 satisfactory. 
 7. Set the mount aside and re-examine 
 at the end of y2 hour. If reaction 
 is positive, all the microorgan- 
 
156 BACTERIOLOGY. 
 
 isms will be found motionless and 
 gathered in clumps of variable 
 size. 
 
 This will be the case at least in the 
 lowest dilutions, while in the 
 hig-her ones it may be necessary 
 to wait until another half hour 
 has expired. 
 
 The hig-her the dilution in which 
 complete clumping is obtained, 
 the greater is the diagnostic 
 value. 
 Macroscopic method. 
 
 This test is made in series of small test 
 tubes of 0.5x5 cm. size. In these test 
 tubes 1 cc. of the different serum dilu- 
 tions and the bacterial emulsion are 
 mixed. They are now placed in the 
 incubator for a few hours and then 
 kept at room temperature. 
 
 Hiss has observed that agglutination 
 will be hastened in some instances if 
 after their removal from the in- 
 cubator, they are placed in the ice 
 chest. ^ 
 
 Vv^hen agglutination takes place, clumps 
 of bacteria are seen to form, which 
 settle to the bottom, much like snow 
 tfakes on the tube. The surface of 
 the sediment is heaped up and irreg- 
 ular. The supernatant fluid becomes 
 entirely clear. 
 
 When the reaction is negative, the sedi- 
 ment is an even, granular one with 
 flat surface, and the emulsion remains 
 turbid. 
 
 PRECIPITINS. 
 
 Kraus ,(1897) demonstrated that sera of 
 animals immunized against a partic- 
 ular microorganism when mixed with 
 the clear filtrate of bouillon culture 
 of the particular organism, would give 
 rise to visible precipitate. In that the 
 precipitate occurred only when the 
 filtrate of a bouillon culture of an 
 organism and the sera of animals 
 immunized with same organism led 
 Kraus to name them "specific precipi- 
 tates," or precipitin. 
 
 Precipitin formation is not limited to 
 bacterial immunization, but is also 
 found like the phenomena of agglut- 
 ination and lysis. Substances produc- 
 
BACTERIOLOGY. 
 
 157 
 
 ing- the phenomena in sera are called 
 precipitinogens. 
 
 Precipitins like agglutinins may be in- 
 activated by heating to from 60° to 
 70° C. and cannot be reactivated by 
 adding normal sera, etc. Inactivated 
 precipiptin, while unable to produce 
 precipitates, will bind the precipi- 
 tinogen. This is shown when inacti- 
 vated precipitin is mixed with pre- 
 cipitinogen no reaction occurs if fresh 
 precipitin is added. 
 
 Precipitin is, therefore, like toxin, made 
 up of two atom-g-roups, a stable hap- 
 tophore and a labile precipitophore 
 group. It is the opinion of many that 
 precipitins are identical in structure 
 with amboceptors. Just as in ag- 
 glutins there is in precipitin a certain 
 degree of "group reaction"; that is, 
 the precipitin obtained with a colon 
 bacillus will cause a precipitation 
 with culture filtrates of allied organ- 
 isms. This may be easily adjusted, 
 however, by the use of proper dilu- 
 tion similar to that used in ag-g-lutina- 
 tion tests. 
 
 Wasserman and others found other lise 
 for this reaction as a means of dis- 
 tinguishing the blood of one species 
 from that of another. Precipitins 
 have not been demonstrated in normal 
 sera. 
 
 PRECIPITIN TESTS. 
 
 These tests jnay not only be applied to 
 bacteria, but also to the various 
 proteid substances. 
 Bacterial Antisera. 
 
 1. The "bacterial antisera are produced 
 by injecting- rabbits by intraperi- 
 toneally or intravenously with 
 emulsions of organisms (either 
 broth cultures or salt solution 
 emulsions of agar cultures) in 
 gradually in^jreasing quantities on 
 5 or 6 occasions, at intervals of 
 from 5 to 6 days. In 7 to 12 days 
 after the last injection, the an- 
 imal is bled and a preliminary 
 test made as to the precipitating 
 value of the serum. 
 If this is insufficient, more injec- 
 tions may be made. In 5 to 12 
 days after the last injection the 
 animal is bled and the sera pre- 
 served by sealing- in glass bulbs 
 
158 BACTERIOLOGY. 
 
 and kept in the dark at a low 
 temperature. A preservative as 
 chloroform may be added. 
 The antisera should be absolutely- 
 clear. If turbid, it may be filtered 
 through porcelain candles, 
 
 2. The bacterial filtrates for test are 
 
 produced by growing- the organ- 
 ism in broth composed of 0.5% 
 Liebig's extract of beef, peptone 
 1%, salts 5% with a reaction 
 of -f 5. 
 
 The cultures are incubated from a 
 week to several months, and then 
 filtered through porcelain or 
 Berfefeld candles until perfectly 
 clear. 
 
 The extracts may also be made by 
 emulsifying agar cultures in salt 
 solution and incubating them at 
 37° C. for a week or more, then 
 filtering. 
 
 When the two reagents have been 
 completed, the test is made as 
 follows: — 
 
 Mix in a series of narrow test 
 tubes. 
 
 (a) Tube No. 1. — 0.5 cc. antibac- 
 terial serum and 1 cc. bacterial 
 filtrate. 
 
 Tube No. 2 — 0.5 cc. normal 
 serum and 1 cc. bacterial filtrate. 
 Tube No. 3. — 0.5 cc. anti bac- 
 teria serum and 1 cc. salt solu- 
 tion. 
 
 Tube No. 4. — 0.5 cc. salt solu- 
 tion serum and 1 cc. bacterial fil- 
 trate. 
 
 (b) Place tubes in incubator at 37* C. 
 
 (c) If test is positive, tube No. 1 
 shows a haziness, which develops 
 Into a distinct cloudiness or even 
 a flocculent ppt. within one hour. 
 Tubes 2, 3, 4 remain clear. 
 
 The precipitating antisera against 
 proteid solutions are prepared by 
 methods analogous to those em- 
 ployed for the production of anti- 
 bacterial sera. If tests are to be 
 made upon proteid material, as 
 (blood stains, meat (as detection 
 of horse meat substitution for 
 beef), etc., they should be ex- 
 tracted with salt solution, in an 
 approximate dilution of 1-50Q. 
 
BACTERIOLOGY. 
 
 159 
 
 The solutions are filtered to insure 
 clearness. 
 
 To test the unknown proteid with 
 serum of an animal immunized 
 with the proteid sought, mix in 
 a series of narrow test tubes: — 
 
 Tube No. 1. — 0.1 cc. immune serum 
 and 2 cc. unknown proteid solu- 
 tion. 
 
 Tube No. 2. — 0.1 cc. immune serum 
 and 2 cc. known proteid solution 
 of variety suspected (similarly 
 diluted). 
 
 Tube No. 3. — 0.1 cc. Immune serum 
 and 2 cc. proteid solution of dif- 
 ferent nature (similarly diluted). 
 
 Tube No. 4. — 0.1 cc. immune serum 
 and 2 cc. salt solution. 
 
 Tube No. 5. — 2 cc. unknown pro- 
 teid solution. 
 
 Test is positive when a precipitate 
 appears in tubes No. 1 and No. 2, 
 but not in any of the others. 
 
 ANTITOXINS. 
 Semm Vaccine Antltozln. 
 
 Antitoxins are produced for all bacteria 
 producing- soluble toxin, and for the 
 toxic substances of a large number of 
 other plant and animal cells. They 
 are called antitoxins because they 
 combine with and render inert the 
 soluble toxins. (See side-chain theory). 
 They are labile chemical substances 
 which resist analysis or probably sim- 
 ilar to euglobulins, and are composed 
 of molecules of large size. It was at 
 one time supposed that antitoxin was 
 but a toxin in a different form. This, 
 of course, has been disproved. The 
 amount of antitoxin produced is much 
 greater than the amount of toxin 
 which is injected or produced during 
 an infection. The union between a 
 toxin and an antitoxin is of a chem- 
 ical nature. The union of these two 
 substances forms a compound that is 
 harmless and differs from the toxin 
 and the antitoxin in that it is much 
 more stable. Toxins have a greater 
 affinity for the three haptophile re- 
 ceptors of cells (free antitoxin) than 
 for those still associated with the 
 cells. The toxin and antitoxin always 
 
160 BACTERIOLOGY. 
 
 combine, if possible, before the toxin 
 and the body cells enter into chemical 
 union. In certain cases when the 
 toxin has been bound by the body 
 cells and the antitoxin is produced in 
 sufficient amount or injected, the toxin 
 cell chemical union will be broken up 
 and the toxin and antitoxin will com- 
 bine. This is illustrated in diphtheria, 
 and antitoxins of this kind effect 
 cures because the union between the 
 toxin and the cell is comparatively 
 unstable. This is not true in cases 
 such as tetanus, in which the toxin 
 is so strongly combined with the cells 
 of the nervous system and other body 
 cells that it is with difficulty that 
 their union is broken by the addition 
 of antitoxin. The union here between 
 the toxin and the body cells is so 
 stable that exceedingly large doses of 
 the antitoxin are required, and these 
 rarely act with any degree of success. 
 
 This explains why tetanus antitoxin is 
 of so little use therapeutically. It is, 
 however, of great use as a prophy- 
 lactic when the toxin is free and be- 
 ing produced in the body. 
 
 Bhrlich, in an accurate stud> of the 
 neutralization of the toxin by the 
 antitoxin, noted that the addition of 
 fractional amounts of the antitoxin 
 to the Lj° of the toxin (complete neu- 
 tralization of one antitoxic unit) and 
 the injection of the resulting mix- 
 tures into guinea pigs, there was not 
 a corresponding decrease in the de- 
 gree of toxicity. The toxin, there- 
 fore, seems to be made up of various 
 parts; a part seeming to have great 
 affinity for the antitoxins is not real- 
 h ly toxin and is called Protoxoids. 
 These compose about % of the amount 
 of toxins necessary to saturate one 
 immunity unit. After ^ of the anti- 
 toxin is added, the mixture of anti- 
 toxin becomes less toxic for the ex- 
 perimental animals, down to the point 
 where % of the amount of toxin 
 necessary to saturate one unit of anti- 
 toxin is used. This fraction is con- 
 sidered the true toxin. Here, again, 
 in as much as the toxicity of the mix- 
 ture does not decline, it has been 
 demonstrated that it is due to another 
 part of the toxic molecule which has 
 
BACTERIOLOGY. 
 
 161 
 
 less avidity for the antitoxin than the 
 toxin itself and the protoxoid. This 
 part of the molecule is called epi- 
 toxoid, true toxoid or toxon. The 
 toxon molecule necessary to saturate 
 one unit of antitoxin is, therefore, 
 made up of 1^4 protoxoid, i/4 true toxin 
 and % epitoxoid, toxoid or toxon. 
 
 ANTITOXINS. 
 
 Antitoxins are employed in the form 
 of sera which may be either liquid, 
 dry or specificated. The immunity that 
 is produced^ by the use of antitoxins 
 is passive and lasts for a period of 
 a few weeks only. The reason for 
 the Passive Immunity is due to the 
 fact that the individual receives no 
 substances which stimulate the pro- 
 duction of protected bodies, but the 
 individual receives, however, those 
 protective antibodies which have been 
 produced in the blood of some other 
 species. When antitoxin is injected 
 and becomes absorbed, the neutraliza- 
 tion of these specific toxins takes 
 place; therefore, it may be used both 
 as a prophylactic and therapeutic 
 agent. . 
 
 The most important antitoxins used at 
 the present time are those of diph- 
 theria and tetanus. Some other anti- 
 sera have been extensively used with 
 good results, while others are still in 
 the experimental stage; i. e., anti 
 streptococci serum, anti dysentery 
 serum, anti hog cholera serum, anti 
 pneumococci serum and anti tubercle 
 serum. 
 
 Biplitherltic antitoxin. 
 
 The organism is grown on Loeflfler's 
 blood serum in the incubator at 37° C, 
 care being taken that the culture is 
 pure. 
 
 The pure diphtheria culture is now 
 transferred to large flasks of beef 
 broth and incubated at 37° C. for a 
 period of about two weeks, during 
 which time, the rapid growth of the 
 organism has elaborated its specific 
 toxin and thrown it off into the broth. 
 The culture is now examined micro- 
 scopically in order to determine the 
 absence of contamination. A pre- 
 servative, such as carbolic acid or 
 
162 
 
 BACTERIOLOGY. 
 
 trikresol, is added and the culture 
 then passed through a Burkef eld filter. 
 The diphtheritic toxin (filtrate) Is 
 then placed in the refrigerator until 
 wanted for use. 
 
 The horses used in the manufacture ot 
 anti diphtheritic serum are removed 
 from the detention stable where they 
 have been confined for several weeks, 
 during which time they are subjected 
 to a thorough physical examination 
 and tested for the presence of gland- 
 ers by the Mallein test. They are now 
 admitted to the antitoxin stable and 
 injected subcutaneously with the 
 diphtheria toxin. The first dose in- 
 jected is but a fraction of a cc, then 
 increasingly larger doses are injected 
 until the animal is able to receive 
 300 cc. or more at a single injection. 
 The intervals between the injections 
 and the rate of increase in the doses 
 at any time depends upon the condi- 
 tion of the animal. In order that a 
 constant process of antitoxin forma- 
 tion may take place in the body of 
 the horse, and that a potent serum 
 may be produced, the Injection of the 
 
 » toxin should be made as rapidly as 
 the reactions, which follow each in- 
 jection, will allow. 
 
 The toxin treatment usually occupies a 
 period of about six weeks, after which 
 the horse is allowed to rest for about 
 two weeks in order that all of the 
 toxin injected may be absorbed. 
 
 By means of a sterile canula, rubber 
 tube and glass cylinders, the animal 
 is now bled from the jugular vein 
 under the proper aseptic and anti- 
 septic conditions by securing as much 
 blood as the horse can conveniently 
 yield. After the serum separates, 
 usually at the end of 24 to 48 hours, 
 the clear fiuid is poured into large, 
 serile, glass containers. A preserva- 
 tive is added and the material is 
 transferred to the laboratory, where 
 it is filtered through a Burkefeld 
 filter. 
 
 The serum is now submitted to tests as 
 to potency, safety and microbial con- 
 tamination. 
 
 Potency Test. Varying amounts of 
 serum are mixed with the L + dose 
 of diphtheria toxin ancl injected into 
 
BACTERIOLOGY. 
 
 163 
 
 a series of guinea pigs, each weigh- 
 ing 250 gms. 
 
 (The L + dose of toxin is the least 
 amount of toxin which, when mixed 
 with one unit of standard antitoxin 
 and injected into a guinea pig of 250 
 gm. weight, is sufficient to kill the 
 animal in four days. 
 
 By this test it is possible to determine 
 the smallest amount of antitoxin 
 which will protect the guinea pig of 
 250 gm. weight when the animal has 
 received simultaneously the L -f dose 
 of toxin. This minimum amount of 
 antitoxin represents one unit. If one 
 five hundredth cc. of the antitoxin 
 represents the smallest amount which 
 is capable of neutralizing the L + 
 dose of toxin, the antitoxin then pos- 
 sesses a potency of 500 units per cc.) 
 
 Safety Test. Several guinea pigs are 
 each injected subcutaneously with 
 2 cc. of the serum and held under 
 close observation until satisfied that 
 the serum contains no injurious 
 properties. 
 
 Microbial contamination. Inoculate cul- 
 ture media with large amounts of 
 antitoxin under aerobic and anaerobic 
 conditions. Place in the incubator, 
 and if after a period of 72 hours no 
 growth occurs, the serum is read^ for 
 use. If, however, a growth appears, 
 the serum is refiltered and re- 
 examined for microbial contamination. 
 
 The serum is now put up in sterile glass 
 cylinders so constructed that steril- 
 ized needles and pistons may be ap- 
 plied and the antitoxin injected di- 
 rectly from the containers. Each 
 container bears a label indicating the 
 number of antitoxin units enclosed 
 and the date of preparation. A num- 
 ber of these packages are now opened 
 and examined for contamination, and, 
 if free from this, the serum is ready 
 for distribution. 
 
 Tetanus Antitoxin. 
 
 The preparation of Tetanus Antitoxin 
 differs but little from that of the 
 diphtheritic antitoxin. A pure culture 
 of bacillus tetani is inoculated into 
 large flasks of glucose bouillon and 
 placed under anaerobic conditions 
 • (see anaerobic cultivations), or, be^ 
 fore inoculation, drive oft the free 
 
164 BACTERIOLOGY. 
 
 oxyg-en by boiling^ the glucose bouil- 
 lon and then covering the liquid me- 
 dium by a layer of oil. Incubate 
 these cultures at 37° C. for several 
 weeks, after which examine micro-, 
 scopically; add a preservative and* 
 pass the culture through a Burkefeld 
 filter and then through Pasteur filter. 
 
 The filtraton process had better be car- 
 ried out in an isolated room used only 
 for the preparation of tetanus toxin 
 on account of the danger of contam- 
 inating any other material or biolog- 
 ical products with the tetanus bacillus^^ 
 
 The tetanus antitoxin is obtained by 
 injecting horses with the toxin along 
 the lines laid down in the preparation 
 of diphtheritic antitoxin. The serum 
 is tested relative to potency, safety 
 and absence from microbial contam- 
 ination. 
 
 The unit of tentanus antitoxin is ten 
 times the least quantity of antitetanic 
 serum necessary to save the life of a 
 300 gm. guinea pig for 96 hours, 
 against the official dose of a standard 
 toxin furnished by the hygienic lab- 
 oratory of . the Public Health and 
 Marine Hospital Service. 
 
 Anti Streptococci Serum. Bouillon cul- 
 tures of the streptococcus pyogenes 
 ar?? killed by heating and injected into 
 horses in increasingly larger doses. 
 Generally but one strain of the or- 
 ganism is used and the serum is 
 called "Monovalent." Frequently, how- 
 ever, several strains of the organism 
 are used and the serum is then desig- 
 nated "Polyvalent." The procuring 
 of the serum, etc., is carried out along 
 the lines laid down in the preparation 
 of the anti diphtheritic serum. The 
 obtained antitoxin is tested in regard 
 to safety and freedom from microbial 
 contamination, but not as to potency, 
 in as much as there are no known 
 methods of standardizing the product. 
 
 Anti Gonococcus Serum. Killed cultures 
 of M. Gonorrhoea are injected in- 
 traperitoneally into large, healthy 
 rams in increasingly larger doses; 
 finally live cultures are injected. The 
 degree of acquired immunity is de- 
 termined by frequent agglutination 
 tests. The serum is tested as to 
 safety and freedom from microbial 
 
BACTERIOLOGY. 
 
 165 
 
 contamination but not as to potency. 
 Anti Dysentery Serum. Both Monova- 
 lent and Polyvalent antitoxic sera for 
 epidemic dysentery have been pre- 
 pared b5^ Shig-a by injecting horses 
 with the filtrate from bouillon cul- 
 tures of the bacillus. It is still in 
 the experimental stage. 
 
 VACCINES. 
 
 Preventative medicine depends to a con- 
 siderable extent upon the use of vac- 
 cines, antitoxins and certain other 
 specific biological preparations, such as 
 diphtheritic antitoxin, small-pox vac- 
 cines, tuberculins, etc. As stated in 
 other parts of the book, the infection 
 of the animal organism is due to the 
 absence of natural or acquired re- 
 sistance. 
 
 An acquired resistance or immunity 
 may, therefore, be brought about by 
 the application of a vaccine or an 
 antitoxin. The application of small- 
 pox vaccine (although believed not 
 to be bacterial in origin but will 
 illustrate the point in question) 
 causes a reaction in the body, or a 
 mild form of the disease, and brings 
 about an active immunity which is 
 relatively permanent in duration. The 
 use of diphtheritic antitoxins causes 
 a passive immunity and affords temp- 
 orary protection by neutralizing- the 
 diphtheritic toxin molecules. 
 
 Vaccines are weakened or modified ^ 
 viruses. Small-pox, black-leg- and 
 anthrax vaccines may be used with 
 safety only on individuals free from 
 the specific disease in question, be- 
 cause if given to an individual suf- 
 fering from the specific disease, the 
 introduction of the attenuated or- 
 ganisms or virus, would tend to in- 
 crease the infection. The g-eneral ac- 
 tion of these vaccines is, therefore, 
 preventative, or prophylactic, and not 
 curative. 
 
 There are several methods employed in 
 the preparation of vaccines. The gen- 
 eral plan is to attenuate or modify 
 the viruses so that they may be in- 
 jected into the normal animal body 
 without danger of producing serious 
 diseased lesions. (See active immuni- 
 zation). 
 
166 BACTERIOLOGY. 
 
 The following methods are usually 
 used: — 
 
 (1) Attenuation by growth at a temp- 
 erature above the optimum (see 
 anthrax vaccine). 
 
 (2) Attenuation by passage of virus 
 through some species other than 
 the animals for which the virus 
 is specific (see small-pox vac- 
 cine). 
 
 <3) Attenuation of virus by drying 
 at constant temperature (see 
 Rabbi's treatment). 
 
 (4) Attenuation by chemical. Patho- 
 genic bacteria are grown in the 
 presence of weak antiseptics, 
 which weakens their disease-pro- 
 ducing powers. 
 
 (5) By the simultaneous injection of 
 a virus together with its pro- 
 tective serum (hog cholera). 
 
 (6) By the combination of pathogenic 
 bacteria with bacteria of other 
 species antagonistic to them, as 
 illustrated by the restraining ac- 
 tion of yeast upon pyogenic bac- 
 teria and antagonism of the Ps. 
 pyocyanea toward the bacterium 
 anthracis. 
 
 . (7) The filtration of liquid cultures 
 of pathogenic organism and the 
 separation of the organism from 
 the toxin (the toxin is used to 
 immunize animals in the produc- 
 tion of antitoxin). 
 (8) The liestruction of young living 
 cultures of specific bacteria by 
 moist heat at a temperature 
 slightly above their thermol death 
 point. 
 
 Anthrax Vaccine. (Pasteur's Method). 
 
 Cultivate the bacterium from the 
 
 blood of a typical case of anthrax or 
 
 agar broth. 
 Prepare two vaccines as follows: — 
 
 Vaccine No. 1. (less active). Grow 
 the anthrax organisms at a temper- 
 ature of 42° C. for a period of 15 
 to 20 days (this produces an as- 
 porogenous race). At the end of 
 this time, suspend cultures in a 
 sterile physiological salt solution. 
 
 Vaccine No. 2. Same as No. 1, except 
 that it is grown for 10 to 15 days. 
 Both vaccines must now be tested 
 for activity and safety by animal 
 
BACTERIOLOGY. 167 
 
 inoculation. No. 1 should kill white 
 mice, but should not cause death in 
 guinea pigs or rabbits. No. 2 
 should kill white mice and guinea 
 pigs but not rabbits. 
 Healthy animals are injected sub- 
 cutaneously with 1 cc. of No. 1. 
 From several days to a few weeks 
 after the injection of No. 1, the 
 second vaccine is injected. A severe 
 reaction with occasional death fol- 
 lows the use of the vaccine. These 
 accidents can be attributed to care- 
 less methods in standardizing and 
 administering the vaccine. The ob- 
 jection to this method lies in the 
 danger of using a living organism. 
 Good results have been obtained 
 from the use of killed and dried 
 anthrax organisms. 
 
 Small-pox Vaccine. The first method 
 employed in small-pox vaccinations 
 was inoculating healthy individuals 
 with the virus from a mild case of 
 ' the disease. Since 1796 small-pox 
 vaccination has been carried out by 
 vaccinating with small-pox virus. As 
 yet, it has not been conclusively de- 
 termined that the cow-pox of cattle 
 and the small-pox of man possess in- 
 timately related causative factors, but 
 abundant evidence proves the efficacy 
 of cow-pox virus as a specific prophy- 
 lactic against small-pox in man. 
 Under sterile conditions, the virus, or 
 seed, is secured by removing the ex- 
 tradite from the vesicles on -infected 
 heifers. This virus is now inoculated 
 into calves or yearlings that have 
 been placed in detention stables where 
 they are inspected and carefully 
 tested for tuberculosis. Before their 
 admittance to the vaccine laboratory, 
 they have passed as healthy in every 
 way and have had their bodies 
 scrubbed with soap and water and a 
 weak antiseptic solution. 
 The virus is inoculated as follows: — 
 The ventral surface of the body is 
 shaved and cleansed, and under 
 sterile conditions, the skin is scari- 
 fied in parallel lines over the greater 
 portion of the abdomen. The stock 
 virus is inoculated clear through 
 the scarified area. The animal is 
 then placed in the propagating room 
 
168 BACTERIOLOGY. 
 
 and all possible precautions taken 
 to avoid contamination by bacteria. 
 In from 5 to 7 days, characteristic 
 vesicles appear on the inoculated 
 area, filled with a thick, heavy ex- 
 tradite. Animal removed to the 
 operating- table, the field is washed 
 with sterile water and the contents 
 of the vesicles are removed with 
 ^ a sterile pipette. (By recent order 
 of the Federal Government, animals 
 used in this work must be slaught- 
 ered before the vaccine is obtained 
 and then carefully autopsyed). The 
 cow-pox extradite is mixed with 
 about 50% glycerine, which adds as 
 a preservative. Safety tests are 
 made by inoculation of small por- 
 tions into guinea pigs. It is then 
 placed in the refrigerator. The 
 glycerine and low temperature 
 gradually destroy extraneous con- 
 tamination. Potency tests are made 
 on calves and rabbits or guinea 
 pigs. Inoculations are made in the • 
 culture media to exclude both 
 aerobic and anaerobic bacteria. 
 For the presence of the tetanus ba- 
 cillus 1 cc. of the product is trans- 
 ferred to glucose beef bouillon and 
 placed under anaerobic conditions at 
 a temperature of 3 71/^*" C. for about 
 10 days. Any growth is removed 
 by filtration and the filtrate is in- 
 jected in the guinea pig. Absence 
 of symptoms in the injected animals 
 shows the absence of tetanus toxin 
 in the cultures. At the completion 
 of these tests, the product is placed 
 in small capillary tubes or upon 
 ivory points, sealed in glass con- 
 tainers. 
 
 If kept in a cool, dark place, it will 
 retain its activity for a period of 
 about 8 months. 
 
 Babies Vaccine. Originated by Pasteur 
 in 1885, with slight modifications it 
 continues to be the only specific treat- 
 ment for rabies. 
 
 Method. A dog suffering from rabies is . 
 killed, a small portion of the brain 
 removed, emulsified in sterile water 
 or salt solution; inject a few drops 
 of the emulsion subdurally into a 
 rabbit. This inoculation should pro- 
 duce symptoms of "dumb rabies" and 
 
BACTERIOLOGY. 
 
 169 
 
 causer the death of the rabbit in 14 
 to 18 days. The virulence of the 
 strain of rabbit material is increased 
 by making- subdural inoculations until 
 the incubation is shortened to about 
 six days. When the rabbit shows 
 symptoms on the sixth or seventh day 
 after the inoculation, the virulence of 
 the virus is called fixed virus and is 
 now used for the preparation of the 
 vaccine. 
 
 The spinal cord of the rabbit dying 
 within seven days is removed anti- 
 septically and suspended over caustic 
 potash and dried at a temperature of 
 23° C. for a period of from 1 to 15 
 days. The patient is now vaccinated 
 with a suspension of a spinal cord 
 which has been attenuated by drying" 
 for 14 or 15 days. On the succeeding 
 days of the treatment, the injection is 
 made of spinal cord which has been 
 less and less attenuated. 
 
 Treatment usually lasts about 21 days, 
 or until the patient has received an 
 injection of the least attenuated virus. 
 It is very important that the treat- 
 ment be begun as early as possible, 
 ment be begun as early as possible, 
 when bitten by a rabid animal, in 
 secured before the expiration of the 
 incubation period. 
 
 Hog" Cholera Vaccine. (Dorset-Niles 
 Serum). Obtain the hog cholera virus 
 by withdrawing the blood from the 
 carotid artery suffering from the dis- 
 ease. Test it for activity, as a given 
 strain of virus may not produce the 
 acute form of hog cholera. Raise the 
 virulence, if necessary, by passing 
 through a series of young pigs until it 
 uniformly produces symptoms in 4 
 to 6 days and death is less than 15 
 days. This degree of virulence is 
 necessary in manufacturing the serum. 
 
 The blood used in the process of hyper- 
 immunization should be obtained from 
 susceptible pigs, weighing from 50 to 
 100 pounds each. The animals used 
 as hyperimmunes should be healthy 
 and weigh from 100 to 300 pounds 
 and possess either natural or acquired 
 immunity to the disease. The blood is 
 secured from the diseased pig by al- 
 lowing the blood to flow from the 
 jugular vein into a sterile pan, or by 
 
170 BACTERIOLOGY. 
 
 drawing: it under aseptic conditions 
 from the carotid artery. The blood 
 obtained is defibrinated and the serum 
 is secured. 
 
 The immune hog"s are hyperimmunized 
 by the slow or by the quick method. 
 
 The slow method. The animals receive 
 several injections at intervals of 
 every few days, each succeeding dose 
 of the virus being increased in pro- 
 portion to the weight of the animal. 
 
 The quick method. Animal is injected 
 with one large dose of the virus, the 
 amount determined by the weight of 
 the aninlal. 
 
 In from one to two weeks after the 
 hyperimmune animal has received the 
 last injection of the virus, the end of 
 the tail is severed with a sharp in- 
 strument and several hundred cc. of 
 blood collected defibrinated, a pre- 
 servative added and placed in the re- 
 frigerator. Repeat this process sev- 
 eral times at 7 to 10 day intervals, 
 when the animal is ready for rehyper- 
 immunizing. 
 
 In rehyperimmunizing the animal about 
 y2 of the quantity of the virus used 
 in the first process is injected. Re- 
 
 * hyperimmunize 2 or 3 times, then 
 relieve the animal of all its blood. 
 Mix the different lots of serum repre- 
 senting the different bleedings and 
 test the potency of the product by 
 injecting subcutaneously four sus- 
 ceptible pigs weighing about 50 
 pounds with 2 cc. of the virus. Two 
 of these pigs are simultaneously in- 
 jected with about 20 cc. of the serum. 
 If the serum possesses the necessary 
 activity, the two test pigs will re- 
 main normal, except for a thermol 
 reaction and slight* clinical symp- 
 toms, while the two controlled pigs 
 should show symptoms in 5 or 6 days 
 and die in less than 15 days. 
 
 The treatment now consists in simul- 
 taneously injecting hyperimmune se- 
 rum and virus intramuscularly into 
 healthy hogs. The amount of hyper- 
 immune serum injected varies from 
 10 to 70 cc, depending upon the 
 weight of the hog. 
 
 Other Vaccines in General. 
 
 After Pasteur had shown that It was 
 possible to produce active immunity 
 
BACTERIOLOGY. 171 
 
 in animals by substituting: chicken 
 cholera, anthrax and swine plague, the 
 thought naturally suggested itself 
 that the same should be possible in 
 the case of some of the organisms 
 which are pathogenic to man. At- 
 tempts in this direction showed that 
 it was not only impossible to protect 
 laboratory animals against infections 
 like typhoid fever and cholera, but 
 man could also be protected, not only 
 by the use of living cultures, but 
 even with the killed organisms. The; 
 great question naturally has been how 
 large a dose of bacterial should be 
 injected and how frequently the in- 
 jection should be made in order that 
 one might secure sufficient protection. 
 Pfeiffer and Kolle were the first to 
 attempt this in a human being through 
 a bacterialytic content of the serum. 
 Wright then introduced a method in 
 which he thought that by the opsonic 
 contents of the blood the degree of 
 protection might be indicated. It has 
 been shown, however, that a parallel 
 between the size of the dose and the 
 serum content of protective sub- 
 stances and the degree of immunity 
 does not exist, and the method now 
 employed are the outcome of actual 
 triumph. 
 
 Freparation of Vaccines. 
 
 Typhoid Pever. The culture from 
 which the vaccine is to be made is 
 brought to a certain degree of viru- 
 lence by passage through animals 
 and that when grown in bouillon it 
 should yield from one thousand to 
 two thousand million bacilli per cc. 
 (This procedure is not absolutely 
 necessary). The vaccine should,, 
 however, be polyvalent; i. e., it 
 should be made from a number or 
 different strains. The medium for 
 the growth is generally a 1% pep- 
 tone broth. Each of the strains is 
 inoculated into separate fiasks and 
 grown at 37 C. for 24 to 48 hours, 
 after which they are carefully 
 mixed and sterilized in a water 
 bath at 60° C. (Some authorities 
 advocate 52*' C.) The flask is to 
 remain in the hot water for 10 to 
 15 minutes and then removed. 
 (Some authorities indicate an ex- 
 
172 BACTERIOLOGY. 
 
 posure for 1 hour. Necessity of this 
 questioned). The sterility of the 
 contents is now tested by placing 
 .1 to 10 cc, according to the amount 
 of the material, in agar or liy in- 
 oculating broth. 
 
 Determine the niimber of bacteria per 
 cc. by Wright's method. Mark a 
 capillary pipette v/ith a glass pencil 
 about % of an inch from the end, 
 puncture the thumb and charge the 
 pipette with a volume of blood as 
 indicated by the mark on the pi- 
 pette. Now charge the pipette with 
 a like volume of the bacterial emul- 
 sion and then with three volumes 
 of a 9/10% salt solution (keeping 
 the individual portions separate 
 from one another by little air bub- 
 bles). The blood and bacterid are 
 now thoroughly mixed by repeated- 
 ly blowing the contents of the capil- 
 lary pipette upon the slide. Small 
 drops of this emulsion are now 
 mounted on a clean slide and spread 
 out like a blood film, dried and 
 stained with Jenner's or Hastiiig's 
 stain. A small square diaphragm 
 of paper is placed in the ocular of 
 the microscope, the "red cells and 
 bacteria are counted in successive 
 filed until 1000 of, the red cells have 
 been counted. The number of red 
 cells in one cc. of blood is ap- 
 proximately 5,000,000, and as the 
 red cells a.nd the bacteria must be 
 present in the same ratio to one 
 another as in the original units of- 
 volume, the number of bacteria per 
 cc. of the vaccine is ascertained ac- 
 cording to equation: Number of 
 red cells counted : number of bac- 
 bacteria counted : : 5,000,000 : X. 
 
 A more accurate method has been 
 suggested by Hopkin based upon the 
 concentration of bacterial culture 
 by centrifugation and the prepara- 
 tion of standard emulsions from the 
 sediment. This requires an espe- 
 cially constructed centrifugation 
 tube, which is prepared by the 
 International Instrument Company, 
 Cambridge, Mass. 
 
 Wright recommends a first injection 
 of 750.000.000 to 1,000.000,000 organ- 
 
BACTERIOLOGY. 
 
 173 
 
 isms and double this amount for the 
 second injection. 
 Fearing that the injection of a large 
 dose of organisms may be followed 
 by a diminution in the protective 
 substances of the body (negative 
 phase), owing to an interaction be- 
 tween the normal antibacterial sub' 
 stances and the bacterial antigen, 
 the individual may be temporarily 
 less resistant to the corresponding 
 infection. Wright, therefore, sug- 
 gests that in persons who are likely 
 ^to be exposed to typhoid fever soon 
 ^ after the first injection, this should 
 be smaller than usual, and that its 
 effect is to be supplemented later 
 by a correspondingly stronger injec- 
 tion. 
 
 Anti-typhoid vaccination, as indicated 
 above, is for prophylaxis only. 
 Various attempts, however, have 
 been made to use it as a curative 
 agent. Some writers have expressed 
 themselves favorably upon this 
 point; others condemn it strongly. 
 At any rate, it will require a great 
 deal of investigation before definite 
 conclusions can be reached. It is 
 impossible to tell when and how 
 much we inject, or as to whether 
 it is beneficial or harmless, unless 
 Wright's opsonic index will prove 
 of some value as determined by 
 future study. 
 Asiatic Cholera. 
 
 Two methods of vaccination against 
 this disease have been carried out 
 with positive results in both meth- 
 ods. 
 
 Haffkine's method depends upon the 
 use of the cholera spirillum after it 
 has been attenuated by the growth 
 at temperature above the optimum. 
 Vaccines of different strengths are ' 
 used. 
 
 Kolle's method depends upon the use 
 of heated (killed) cultures of the 
 organisms. 
 Bubonic PlagTie. 
 
 The same methods employed in the 
 vaccination against Asiatic cholera 
 are used in the vaccination against 
 Bubonic Plague. Cultures of the , 
 plague bacillus, killed by heating at 
 a temperature of 60® C. for 1 hour. 
 
174 BACTERIOLOGY. 
 
 Bacterial Vaccines or Bacterins for 
 Therapeutic Purposes. 
 
 Recent studies would tend to show 
 that bacterins (killed bacteria) may- 
 be employed as curative agents in 
 those infections which tend to 
 chronicity, and in which toxins play 
 little or nor part. Wright and 
 Doug-las first advanced the theory 
 of opsonins including the suggestion 
 that the subcutaneous injection of 
 a given species of bacteria, killed 
 by heating, conferred to the blood 
 when injected a greater opsonic 
 activity towards the species of or- 
 ganisms in question. 
 
 In preparing bacterial vaccines, (based 
 upon the opsonic theory), the spe- 
 cific organism is isolated, grown for 
 24 hours at 37° C, emulsified in 
 sterile physiological salt solution, 
 heated in a water bath at 60° C. for 
 ^ hour, standardized as to number 
 of bacteria in 1 cc, and a preserva- 
 tive added (making the emulsion 
 correspond to V2% lysol and tested 
 for its sterility as in preparation 
 of typhoid bacteria). 
 
 The use of these bacterial vaccines 
 has brought splendid results in the 
 treatment of furunculosis, acne, 
 sycosis and other infections caused 
 t>y pyogenic organisms. 
 
 Two kinds of vaccine are used: — the 
 so-called autogenous vaccines; i. e., 
 vaccines that are derived from the 
 individual organism which is re- 
 sponsible for the particular infec- 
 tion, and the so-called stock vac- 
 cines, which are prepared from 
 stock cultures of the specific or- 
 ganisms responsible for the infec- 
 tion. 
 
 The question as to whether or not 
 autogenous vaccines are imperative 
 has created a great deal of discus- 
 sion. It would seem theoretically, 
 at least, that the probable existence 
 of many strains of a given type of 
 organism would make the autogen- 
 ous vaccines preferable to the stock 
 vaccines. 
 
 TUBERCULINS. 
 
 Koch's Tuherculln. (Old). An inocu- 
 lation of the bovine or liuman bact, 
 
BACTERIOLOGY. 
 
 175 
 
 tuberculosis is made into several 
 flasks of beef bouillon to which 5% 
 glycerine has been added. The cul- 
 tures are carefully, placed on the sur- 
 face of the medium. After an incu- 
 bation at 37° to 38° C. for a period of 
 6 to 10 weeks or longer, the growth 
 that slowly spreads over the surface 
 finally falls to the bottom (it is neces- 
 sary that during the incubation, the 
 cultures remain undisturbed and have 
 access to plenty of air without temp- 
 erature fluctuations in order that they 
 may complete the elaboration of ac- 
 tive tuberculinic substance). The cul- 
 tures are removed from the incubator 
 and sterilized in streaming steam. 
 Evaporate the cultures over a water 
 bath to 0.1 its original volume; re- 
 move the bacteria by passing the 
 cultures through filter paper and a 
 Burkefeld filter; add a preservative. 
 
 The active substance of a tuberculin is 
 apparently an albuminous derivative 
 insoluble in alcohol and is elaborated 
 during the organism's multiplication. 
 The product used is harmless for 
 healthy animals, but exerts a toxic 
 action upon those affected with tuber- 
 culosis. This tuberculin is used as a 
 diagnostic agent, not as a prophylactic 
 agent. Its injection into individuals 
 affected with tuberculosis is followed 
 in from 2 to 10 hours by a rise of 
 temperature, which continues for a 
 few hours then subsides. 
 
 The dose of this tuberculin for cattle 
 is 0.25 cc. 20.7 cc. By reason of its 
 syrupy consistency and small dose, it 
 is usually diluted with seven parts of 
 weak carbolic acid solution; 2 cc. of 
 the diluted tuberculin is used as the 
 dose for cattle. The product is tested 
 for activity by injecting known tuber- 
 culosus animals and the activity of 
 the product is indicated by the typical 
 reaction which follows. 
 
 Other methods than the one described 
 above of applying tuberculin as a di- 
 agnostic agent have been instituted by 
 Kalmette, von Pirquet and Morrow. 
 
 Kalmette's Method consists in the instil- 
 lation in the eye of one drop of a 
 1% solution Koch's purified or refined 
 tuberculin (prepared by treating the 
 original tuberculin r with absolute al- 
 
176 BACTERIOLOGY. 
 
 cohol, washing and drying the pre- 
 cipitate). A positive reaction is in- 
 dicated by a congestion of the pal- 
 pebral and ocular conjunctiva a few 
 hours after its application. 
 
 Von Pirquet's Method. The patient's 
 arm is cleansed; one drop of tubercu- 
 lin (old) is placed on the skin of the 
 cleansed area and the skin underneath 
 the drop is scarified. Two or more 
 areas are treated in this way. It is 
 well to scarify another small area 
 as a control, this area to be treated 
 with a drop of sterile salt solution, 
 or a solution of glycerijae and dilute 
 carbolic acid in substitution for the 
 tuberculin. 
 
 The appearance of a reddish zone in 
 from 12 to 24 hours under the tuber- 
 culin areas indicates a positive reac- 
 tion. 
 
 Morrow's Method. An ointment is pre- 
 pared from equal parts of tuberculin 
 (old) and hydrous lanolin and vigor- 
 ously rubbed on a small portion of the 
 skin of the abdomen. A distinct 
 granular or papular eruption at the 
 point of application after about 24 
 • hours indicated a positive reaction. 
 
 Kocli's Tulberculin in "T. R." (tuber- 
 culin residum) is prepared by repeat- 
 ed centrifugation of a suspension in 
 water of the dried and ground organ- 
 isms. The supernatent fluid "t.O." 
 after the first centrifugalization is 
 discarded, and the final product, con- 
 sisting of the constituents of the bac- 
 teria which are insoluble in water, 
 contains the T. R. 1 cc. of the tuber- 
 culin T. R. should contain the equiva- 
 lent of 1 mg. of the dry tubercle 
 solids. 
 
 Koch's Tuberculin "B. E." (bacillary 
 emulsion) is composed of a suspen- 
 sion of crushed or thoroughly ground 
 tubercle bacilli in 5% glycerine solu- 
 tion. Each cc. should contain the 
 equivalent of 1 mg. of tubercle solids. 
 
 Koch's T. R. and B. E. are used as 
 therapeutic agents, the B. E. being 
 regarded most favorably b3^ clinicians. 
 They are administered by subcutane- 
 ous injections. The initial dose 
 recommended by Wright is one four 
 hundredth to six hundreth mg. The 
 Intervals between the successive treat- 
 
BACTERIOLOGY. 177 
 
 ments varies from three to ten days. 
 
 MALLEIN. 
 
 Mallein is used for the diagnosis of 
 glanders. It is prepared from cul- 
 tures of the bact. mallei by partic- 
 ally the same method as those em- 
 ployed in the preparation* of tuber- 
 culin. The organism used in the pre- 
 paration of Mallein should be viru- 
 lent. It is inoculated into flasks of 
 glycerine bouillon having reaction of 
 3 and incubated at a temperature of 
 37° C. for several weeks. The cul- 
 tures are removed from the incubator, 
 heated in streaming steam, passed 
 through a Burkefeld filter, the filtrate 
 concentrated, preserved and put up in 
 vials ready for use. 
 
 A few hours after the injection of 
 mallein into a horse affected with 
 glanders, a severe local reaction and 
 a rise of temperature usually follows. 
 The local swelling caused by the mal- 
 lein is considered by some to be as 
 diagnostic as the rising temperature. 
 
 Metcliniioif^s Ph.ag'ocytic Theory. 
 
 The term "phagocyete" is given to any 
 cell capable of incorporating bac- 
 teria and of destroying them by a 
 process of digestion. 
 Phagocytic cells comprise: — 
 
 1. Microphages, polymorphonuclear 
 
 leucocytes. , 
 
 2. Macrophages are all other leuco- 
 
 cytes, endothelial cells and con- 
 nective tissue corpuscles having 
 phagocytic power. 
 
 When animals are subjected to an 
 irritant, phagocytosis occurs. The 
 leucocytes are attracted by chemo- 
 taxis to the zone of irritation and 
 envelop the irritating substance. 
 
 The organisms that escape from one 
 cell are seized by others, but if 
 their multiplication is excessive 
 they overpower the phagocytic 
 leucocytes and invade the blood 
 serum. The blood serum and the 
 body fluids are likewise bac- 
 tericidal, due to the disintegra- 
 tion of phagocytes — phag'olysis, — 
 the properties of these cells be- 
 ing imparted to the serum. This 
 property is due to two constitu- 
 
178 
 
 BACTERIOLOGY. 
 
 ents of the plasma. The one (the 
 specific immune body) circulates 
 In the plasma and resists a temp- 
 erature of 100° C. The other, or 
 "cytase" (digestive ferment), de- 
 rived from the disintegrated pha- 
 gocytes, corresponds to Buchner's 
 "alexins" and Ehrlich's "lysins." 
 It ie destroyed at 60° C. 
 
 AGGRESSINS. 
 
 Certain bacteria may be injected into 
 an animal in considerable quantities 
 without producing any effect other 
 than the temporary local disturbance 
 following the subcutaneous admini- 
 stration of the material. 
 
 Certain other bacteria, on the other 
 hand, as the bacillus of anthrax or 
 chicken cholera, may, if injected even 
 in the most minute dose, give rise to 
 a rapid fatal septicemia. Within the 
 same species fluctuations in virulence 
 may take place, depending upon a 
 variety of influence, but variations in 
 the susceptibility of the inoculated 
 subject do not furnish a sufficient ex- 
 planation for the reaction so that the 
 explanation must be put down to the 
 activities of the bacteria themselves. 
 
 Pathogenic bacteria differ from non 
 pathogenic bacteria in their power to 
 overcome the protective mechanism of 
 the animal body, and to proliferate 
 within it. They do this by reason of a 
 certain definite substance which they 
 give ofC in the nature of the secre- 
 tion, which protects them against 
 phagocytosis. These substances were 
 named by Bail "aggressins." The ag- 
 gressins are probably absent in test 
 tune cultures, but can be found in the 
 animal body in the exudates occur- 
 ring about the sight of inoculation in 
 rapidly fatal infections. Bail was 
 able to show that fatal infections 
 could be produced in animals by the 
 Injection of sub lethal doses of bac- 
 teria if a small quantity of aggressin 
 was administered at the same time. 
 He believed that the aggressin para- 
 lyzed the phagocytic and other pro- 
 tective agencies which made it pos- 
 sible for the bacteria to proliferate. 
 He further showed that animals were 
 
BACTERIOLOGY. 
 
 179 
 
 successfully immunized with a§:gres- 
 sins. These animals were not only 
 immune themselves, but contained a 
 substance in their serum which per- 
 mitted the passive immunization of 
 other untreated animals. 
 
 Bail's theory has been attacked by Was- 
 serman. Citren. Wolfe and others. 
 These men claim that much of the 
 aggressive character of Ball's ex- 
 udates is due to their containing lib- 
 erated bacterial poisons (endotoxin). 
 
 Opsonins. Wright and Douglas demon- 
 strated that there were present in 
 blood serum and the body fluids cer^ 
 tain substances that had the power 
 of rendering bacteria susceptible to 
 phagocytosis. These substances were 
 termed opsonia^s (I prepare food for). 
 The opsonins act chemically upon cer- 
 tain substances within the bacteria 
 and sensitize them. Phagocytosis de- 
 pends almost wholly on these specific 
 opsonins which are present in many 
 normal sera for the various bacteria. 
 Its presence was demonstrated by 
 washing leucocytes free from all se- 
 rum, when they refused, except in 
 rare cases, to take up bacteria. Bac- 
 teria which have been placed in con- 
 tact with blood serum or body fluids 
 and thoroughly washed, will, when 
 placed in contact with leucocytes, be 
 taken up by them. Opsonins may be 
 produced in animals not containing 
 them by the process of immunization. 
 Opsonins are destroyed at about 60° C. 
 for thirty minutes. They will remain 
 active for several days at 0° but will 
 deteriorate rapidly in the withdrawn 
 blood if stored at a temperature of 
 37° C. Many opsonins have the fea- 
 tures of agglutinins and precipitins, 
 although they bear some points of re- 
 semblance to antitoxins and comple- 
 ments. They possess a haptophore 
 group with which they combine with 
 the bacteria, and a functional group, 
 which sensitizes the microorganisms 
 for phagocytosis. 
 
 Opsonins may be increased in the serum 
 of normal or infected individuals by 
 the injection of heated (60°) cultures 
 of these speciflc etiological micro- 
 organisms. These substances are 
 called opsonogrens or vaccines (seo 
 
180 BACTERIOLOGY. 
 
 Bacterins) and are extensively used 
 in the treatment of various pus in- 
 fections,-due to the staphylococci, and 
 also in tuberculosis and to a less ex- 
 tent 'in pneumonia. 
 Wright, in his work, makes use of the 
 so called opsonic index in order to 
 estimate the changes in the resist- 
 ance of a patient against the given 
 infection. 
 
 The Determination of the Opsonic Index 
 
 (in order that the concentration of 
 opsonins in an individual may be 
 recorded). 
 
 1. By means of Wright's capsule col- 
 
 lect blood from the finger. Seal 
 the capsule at both ends; allow 
 the blood to clot; and hasten the 
 separation of th^ serum by a few 
 revolutions in the centrifuge. 
 
 2. Make a bacterial emulsion by rub- 
 
 bing up a few loopfuls of a 24 
 hour slant agar culture with a 
 little physiological salt solution; 
 this emulsion must be even. 
 
 3. Bleed 10 to 15 drops from the ear 
 
 or finger, directly into 5 or 6 cc. 
 of a normal saline solution con- 
 taining 1V2% sodium citrate. Cen- 
 trifugalize for 5 or 6 minutes, at 
 the end of which time the cor- 
 puscles at the bottom of the tube 
 will be covered by a thin, greyish 
 pellicle consisting chiefly of leu- 
 cocyte. Pipette these off with a 
 capillary pipette (by careful, 
 superficial, scratching movements 
 over the surface of the buffy coat 
 forming the greyish pellicle). 
 
 The serum, the bacterial emulsion 
 and leucocytes having thus been 
 prepared, the test is carried out 
 as follows: 
 
 With a greased pencil make a mark 
 upon a six or seven inch capillary 
 pipette, about 2 to 3 cm. from the 
 end, and successively draw into 
 the pipette up to the mark, cor- 
 puscles, bacteria and serum, 
 separating them from one another 
 by small air bubbles. Equal quan- 
 tities of each having thus been 
 secured,^ they are thoroughly 
 mixed by repeatedly drawing 
 them in and out of the pipette 
 upon a slide. The mixture is then 
 
BACTERIOLOGY. 
 
 181 
 
 drawn into the pipette; the end 
 is pealed; incubated at 37i^° for 
 about 15 to 30 minutes. 
 
 A control, or normal serum, is pre- 
 pared and treated in exactly the 
 same way. (The normal or con- 
 trol serum is obtained by a •'Pool" 
 or mixture of the sera of 5 or 6 
 supposedly normal individuals). 
 
 After incubation, the end of the 
 pipette is broken oft, the con- 
 tents are again mixed, and smears 
 are made upon glass slides in the 
 ordinary way, and stained with 
 Wright's or Jenner's stain, and 
 the number of bacteria contained 
 in each leucocyte is counted. The 
 contents of about 80 to 100 cells 
 are usually counted and averages 
 taken. This average number of 
 bacteria in such leucocytes is 
 spoken of as the phagocytic in- 
 dex. The phagocytic index of the 
 iested serum divided by that of 
 the normal "pool" serum, gives 
 the opsonic index. (Suppose the 
 leucocytes of the infected indi- 
 vidual take up an average of five 
 bacteria. In this case, the pha- 
 gocytic index is said to be five. 
 Again, suppose the leucocytes of 
 the normal individual take up 15 
 bacteria. The phagocytic indejc 
 in this case would be 15. The 
 opsonic index of the infected in- 
 dividual would therefore be 0.33 
 +, as the normal individual pha- 
 gocytic index is taken as the de- 
 nominator of a fraction and the 
 phagocytic index of the infected 
 individual as enumerator, there- 
 fore, it would be 5/15 or 1/3). 
 
 The opsonic index would therefore 
 seem a fair indication as to the 
 resistance of the particular indi- 
 vidual to the infecting micro- 
 organism. By the judicious use 
 of vaccines, the opsonic index 
 may be raised to at least 1.0 or 
 even more, showing that the leu- 
 cocytes are actively phagocytic 
 and opsonins increased in concen- 
 tration of the blood serum. In 
 such cases, recovery will be indi- 
 cated. 
 
182 BACTERIOLOGY. 
 
 The opsonic index gives a fair idea 
 as to the resistance of an indi- 
 vidual to an infecting micro- 
 organism. 
 
 Virulent bacteria are not phago- 
 cytized. Virulent streptococci 
 and pneumococci are not as easily 
 taken up as the non virulent 
 forms. It would seem from this 
 that some toxic or poisonous sub- 
 stance produced by the bacteria is 
 antagonistic to opsonins, or it 
 may be that an anti opsonin is 
 formed. 
 
 The presence of opsonins in the body 
 fluids of an animal is not absolute 
 proof that such animal is highly 
 resistant to infection. The re- 
 sistance depends upon the activ- 
 ity of the phagocytes, and in 
 certain cases where the opsonins 
 are high in concentration the pha- 
 gocytes are not active. In certain 
 cases the reverse is true, and here 
 the opsonins and phagocytosins 
 are of the utmost importance dur- 
 ing the immunity of the in- 
 dividual. 
 
 LEUCOCYTIC EXTRACT. 
 
 Hiss conceived the plan of injecting 
 into infected subjects the substances 
 jthat compose the chief cells, or all 
 the cells usually found in extradites, 
 in the most diffusible forms and as 
 little changed by manipulation as pos- 
 sible, in order that the living leuco- 
 cytes which exert the protective ac- 
 tion against bacterial infection might 
 be considerably reinforced as directly 
 as possible with a further supply of 
 the weapons that they use against 
 the microorganisms. 
 
 Hiss also assumed that extracts from 
 the leucocytes would be more effica- 
 cious than the living leucocytes them- 
 selves, in that if they were diffusible 
 they would be distributed impartially 
 to all parts of the body by the cir- 
 culatory system. In this way, quick 
 absorption would relieve the tired out 
 leucocytes and would also protect by 
 any toxin-neutralizing or other power 
 they might possess, the cells of high- 
 Iv specialized functions. 
 
BACTERIOLOGY. 
 
 183 
 
 Method of obtaining' tliese substances 
 
 (for animal experiments and treat- 
 ment of human subjects). Rabbits of 
 1500 gms. weight or over are injected 
 intraplurally with aleuronat (prepared 
 by making a 3% solution of starch 
 and meat extract broth, without heat- 
 ing; after the starch has gone into a 
 thorough emulsion, 5% of powdered 
 aleuronat is added; after thorough 
 mixing, boil for 5 minutes; fill into 
 sterile potato tubes in quantities of 
 20 cc. in each tube; sterilize in the 
 autoclave). 
 10 cc. of the mixture is injected into 
 each plural cavity, in the intercostal 
 spaces at the level of the end of the 
 sterum, in the anterior axillary line, 
 taking care that the lungs are not 
 punctured. At the end of 24 hours a 
 copious cellular exudate will have ac- 
 cumulated in the plural cavities. Kill 
 the animal with chloroform and under 
 rigid sterility open the anterior chest 
 wall and pipette the exudate into 
 sterile centrifuge tubes. Centrifugal- 
 ize immediately before clotting can 
 take place; decant the supernatent 
 fluid. Add to the leucocytic sediment 
 about 2 cc. of sterile, distilled water, 
 and make into an emulsion by means 
 of a platinum spatula. Make smears, 
 stain with Jenner's blood stain and 
 examine for possible bacterial con- 
 taminations. It is well to test for 
 contamination by the culture method. 
 Now add to each tube about 20 vol- 
 umes of sterile, distilled water to one 
 volume of the sediment; set aside in 
 the incubator for 8 hours. Test again 
 for sterility. Store in the refriger- 
 ator, where further extraction takes 
 place, until the extract is used. Hiss 
 and Zinsser have injected the extract 
 subcutaneously as treatment in cases 
 of epidemic cerebro-spinal meningitis, 
 in lobar pneumonia, in staphylococci 
 infections and in erysipelas with 
 beneficial results. 
 
 ANAPHYLAXIS OR HYPER 
 
 SUSCEPTIBILITY. 
 
 When a foreign proteid is introduced 
 into the body, after' a time there will 
 appear a specific hypersusceptibility 
 
184 BACTERIOLOGY. 
 
 of the animal for this proteid. If, 
 after a definite interval, a second in- 
 jection of the same substance is given, 
 violent symptoms of illness will be 
 produced, and often death. 
 
 As early as 1893, Behring noticed that 
 animals highly immunized against 
 diphtheric toxin would occasionally 
 show marked susceptibility to in- 
 jections of small doses of the toxin. 
 
 Wolfe and Esiner believe that all cells 
 and proteid material contain a toxic 
 substance which is characterized by 
 its inability to produce a neutralizing 
 antibody when injected into animals. 
 The first injection produces a lysin 
 for the proteid injected, which pos- 
 sesses the power of liberating such 
 poisons from the complex molecules; 
 consequently, when a second injection 
 is given there is a rapid liberation of 
 the toxic fraction, and an injury to the 
 animal results. This view has been 
 supported experimentally by Vaughn 
 and Wheeler, who have been able to 
 extract from various proteids toxic 
 substances which give rise in animals 
 to symptoms not unlike those of typ- 
 ical anaphylaxis. 
 
 PATHOGENIC MICRO-ORGANISIMS, 
 THE STAPHYLOCOCCI 
 (MICROCOCCI). 
 
 The Staphylococcus Pyogrenes, Anretis 
 and Alhus. These organisms are found 
 to cause infections, such as boils, ab- 
 scesses, osteomyelitis, pyemia, etc., 
 throughout the world. These organ- 
 isms stain readily in pus with aniline 
 dyes, and the simple sowing upon or-- 
 dinary media is usually suflflcient for 
 cultures, but, if pure cultures are 
 wished, plating should be resorted to. 
 
 Man seems to be considerably more sus- 
 ceptible to staphylococci infections 
 than other animals. The virulence of 
 the organisms varies and is increased 
 by successive passage through an- 
 imals of the same species, but re- 
 mains unaltered for animals of other 
 species. Virulent cultures, injected 
 into the peritoneal cavity of animals, 
 may kill in from 48 hours to a week, 
 or longer, with abscess formation, 
 
BACTERIOLOGY. 
 
 185 
 
 especially in the kidney. Malignant 
 or ulcerative endocarditis has been 
 experimentally produced; likewise, 
 osteomyelitis. Simply rubbing- the 
 virulent cultures in the skin of man 
 often produces furuncles. 
 Immunization can be secured by in- 
 jections of the dead or live cocci in 
 graduated doses. The serum posses- 
 ses slight bactericidal and agglutin- 
 ated properties, also a high degree of 
 opsonic power. The serum is protec- 
 tive only when used slightly before 
 or along with the injection of the 
 organism, hence, of little value. Active 
 immunization is extensively practiced 
 with autogenous strains of the organ- 
 isms. 
 
 Tlie variety aureus is spherical in shape. 
 On solid media it is found singly, in 
 pairs or in rows of three or four, but 
 generally in irregular groups, like 
 bunches of grapes. In liquid media, 
 the single and paired forms are most 
 frequent. It is .gram positive. Temp- 
 erature range of growth is 10° to 43°. 
 Optimum about 30°. Grows readily on 
 all culture media of a slightly alka- 
 line reaction. On agar, after 24 hours, 
 small, round, greyish white or yellow 
 colonies appear. The characteristic 
 orange yellow pigment may not ap- 
 pear until later. In broth, growth is 
 rapid with diffused clouding, with a 
 thin pellicle and a heavy sediment 
 after several days. In gelatine, col- 
 onies sink into cups of liquefaction. 
 Liquefaction is due to a thermol label 
 ferment substance called. Gelatinis. 
 Milk is coagulated in three or four 
 days time. Potato, abundant growth 
 not as moist or smooth as on agar. 
 Acid but no gas is produced in dex- 
 trose, lactose and saccharose media. 
 Presence of fatty acids produces char- 
 acteristic odor of cultures. Pigment 
 appears in the aerobic but not in the 
 anaerobic culture. The pigment is in- 
 soluble in water but soluble in alcohol, 
 chloroform, ether and benzol. The 
 toxins are largely intracellular. In 
 , the more virulent strains grown in 
 moderately alkaline broth, a thermol 
 label hemolytic substance can be ob- 
 tianed by filtration through porcelain 
 filters. Another toxic substance is 
 
186 BACTERIOLOGY. 
 
 found that causes the death of leu- 
 cocytes (leucocidia). This toxin is 
 less stable than the one mentioned 
 above (staphylo-haemolysin). 
 
 Staphylococci are more resistant than 
 are the other non spore-bearing bac- 
 teria. An hour or more at 60* is 
 necessary to kill watery suspensions; 
 70° is necessary to kill in 10 minutes. 
 Resistance is much greater if organic 
 material is present. Low tempera- 
 tures have little effect; thirty per 
 cent have survived 30 minute ex- 
 posure to liquid air. They resist dry- 
 ing and direct sunlight to a marked 
 degree. They may be found in pulp 
 that has been dried for several 
 months. To the germicides, especial- 
 ly in the presence of organic matter, 
 they are more resistant than other 
 vegetative bacteria. 
 
 The variety albus. This organism dif- 
 fers from the pyogenes aureus simply 
 In the absence of the golden color. 
 Morphologically, culturally and path- 
 ogenically, it is identical. Its toxin 
 and enzyme producing power is gen- 
 erally less than the aureus, otherwise, 
 its biological relationship is so close 
 RS' to be demonstrated by its agglu- 
 tinations in the aureus immune. 
 
 The Staphylococcns, Epidermidis Alhus 
 was described by Welch and may give 
 rise to stitch abscesses. It is merely 
 one of the non pathogenic forms of 
 the staphylococcus pyogenes albus. 
 
 The Staphylococcus Fyogfenefl Citrens 
 differs from the staphylococcus pyo- 
 genes aureus in its bright yellow or 
 lemon colored pigment. It may be as 
 pyogenic, biit is less often found in 
 connection with pathological lesion. 
 
 A large number of staphylococci differ- 
 ing from those mentioned above have 
 been observed. Few of these have any 
 pathological significance and so far as 
 known they have no toxin producing 
 properties. They are frequently met 
 with as contaminations in the course 
 of bacteriological work. 
 
 Micrococcus Tetragfemis was discovered 
 in 1881 by Gaffney in the pus of 
 tubercular patients. Stained smears 
 of the pus containing the micrococcus 
 show them in the form of tetrads 
 larger than the staphylococci. They 
 
BACTERIOLOGY. 
 
 187 
 
 are flattened along their adjacent sur- 
 faces and are surrounded by a thick, 
 halo like capsule. It stains easily 
 with the usual aniline dyes, also by 
 Gram's. It grows on all the ordinary 
 media. On agar, the colonies first ap- 
 pear as transparent spots which later 
 become greyish white, but are al- 
 ways more transparent that the other 
 staphylococci cultures. 
 On gelatine, growth is slow; no lique- 
 faction. Broth, is clouded. Potato, 
 growth is whrte and moist showing 
 a tendency to confluence. Milk, co- 
 agulated. Litmus milk, acid forma- 
 tion. 
 
 In man, the organism is probably with- 
 out any pathogenic significance when 
 found in the sputum or saliva. In a 
 few isolated cases, it may, however, 
 be the cause of abscess formation. 
 Bezancon isolated the organism from 
 a case of meningitis and Forneaca re- 
 ported a case of tetragenus septi- 
 cemia. 
 
 The organism Is seldom found in con- 
 nection with disease, but it is often 
 found in considerable numbers in 
 sputum examined for pneumococci or 
 tubercle bacilli. 
 
 Experimentally, the organism is especi- 
 ally (pathogenic for Japanese mice. If 
 injected subcutaneously, death occurs 
 in three or four days. Grey mice, 
 rats, guinea pigs and rabbits are less 
 susceptible and show only a localized 
 reaction at the point of inoculation. 
 
 THE STREPTOCOCCI. 
 
 The Streptococci Pyogrenes grow in long 
 chains and. ferment lactose, saccha- 
 rose and salicin but do not coagulate 
 milk. This group comprises most of 
 the streptococci which cause suppur- 
 ative lesions or severe systemic in- 
 fections. 
 
 Streptococcic infections are endemic 
 among all races and under all social 
 conditions. They are more frequently 
 found in the human being, however, 
 than in horses, cattle and laboratory 
 animals. The period of incubation is 
 probably about one to three days. 
 
 Streptococci seem to be always present 
 on the exposed surface of the body 
 
188 BACTERIOLOGY. 
 
 and are capable of causing infection 
 should any local lowered resistance 
 occur. The symptoms of septicemia 
 are a rapid rise in temperature to a 
 105" P. or over, chills, rapid, irregular 
 and weak pulse, respiration labored, 
 may be vomiting, and constipation or 
 diarrhea. Headache more or less se- 
 vere, sometimes delirium. Death may 
 occur in two or three days or within 
 a week. Mild cases may recover. 
 
 Death from septicemia causes the body 
 to putrefy rapidly. The glandular 
 organs, especially the spleen, tend to 
 be swollen and soft, and parenchy- 
 matous degenerations are found to a 
 greater or less extent. The endo- 
 thelium of the heart and vessels is 
 blood stained, which is a character- 
 istic feature of streptococcic septi- 
 cemia. Bronchitis and broncho pheu- 
 monia are usually found. 
 
 Erysipelas is an inflammation of the 
 skin and sometimes of the mucous 
 membrane brought about by the strep- 
 tococci. The area involved is definite- 
 ly outlined. Oedema ^nay be white 
 marked where the skin covers loose 
 pigment. Fever with its accompani- 
 ment is present. There may be vomit- 
 ing, constipation or diarrhea, severe 
 headaches or delirium. Death may 
 occur without any apparent compli- 
 cation or death may follow meningitis, 
 pericarditis or nephritis. 
 
 Superficial cutaneous infections are met 
 with and, if mild, may be similar to 
 the localized abscesses caused by 
 
 ' staphylococci. But if severe the in- 
 fection is followed by rapid spreading 
 oedemia, lymphangitis, severe sys- 
 temic manifestations, a grave cellu- 
 litis, often threatening life and re- 
 quiring energetic surgical interference. 
 
 The respiratory organs may be invaded 
 leading to bronchitis, pneumonia and 
 empyema. It may be present as a 
 secondary infection in tuberculosis. 
 The infection of the lung and plura 
 frequently leads to pericardial in- 
 volvement. 
 
 Streptococci may invade bones and pro- 
 duce a severe form of osteomyelitis. 
 If occurring in the mastoid bone, it 
 may lead to meningitis. 
 
BACTERIOLOGY. 
 
 189 
 
 In the throat and mouth, pharyngitis 
 may be produced together with the 
 tonsillitis that may be easily mistaken 
 for the diphtheria. The inflamma.tory 
 throat present in scarlatina is almost 
 always due to the streptococcus. A 
 secondary infection of this organism 
 following diphtheria is a frequent and 
 serious complication, 
 
 Str»eptococcic throat infections have re- 
 cently appeared as epidemics. Sev- 
 eral small epidemics took place in 
 England. Severe epidemics have ap- 
 peared in this country; one in Boston, 
 one in Chicago and another in Balti- 
 more. Investigation traced the infec- 
 tion in the majority of these cases 
 to a single milk supply. Those sec- 
 ondary cases occurred by contact. 
 Complications, such as suppurative 
 adenitis, otitis, erysipelas, peritonitis 
 and septicemia, were frequent. A' 
 capsulated hemolytic streptococcus 
 was found in each epidemic. 
 
 Prom any local process, streptococci 
 may pass into the circulation, causing 
 sepsis. The septicemia occurring dur- 
 ing the puerperium is often caused by 
 this organism. Streptococci have been 
 found in appendiceal abscesses. 
 
 Secondary foci in the viscera may take 
 place and lead to pyemia, if localized 
 upon the valves of the heart, septic 
 endocarditis results. All forms of 
 streptococcic infection, whether acute 
 or chronic, is followed by a high 
 mortality. The diagnosis in these 
 cases is usually made by means of 
 blood cultures in plain broth or other 
 suitable media. 
 
 The streptococci vary somewhat in size. 
 In shape they may be rounded or oval, 
 or with one aspect flattened when they 
 occur in pairs. The chains formed 
 may be long or short, and a grouping 
 into pairs is quite frequent, even 
 when the organism is formed into 
 chains. The organism is non-motile 
 and without spores. It stains with 
 the ordinary aniline dyes, gram posi- 
 tive, temperature range from 15 to 45, 
 the optimum about 37°. The organ- 
 ism is aerobic and facultative anae- 
 robic. A strict anaerobic species has 
 been said to have been isolated from 
 feces. Culture media should be 
 
190 BACTERIOLOGY. 
 
 slightly alkaline in reaction. Acid 
 is produce(J which has a inhibitory 
 action upon its growth. Acids are 
 formed from the monosaccharides 
 lactose, saccharose and salicin. Gas 
 production is negative. Nitrates are 
 reduced to nitrites in some cases. 
 Hydrogen sulphide is produced by a 
 group called streptococcus faecalis. 
 No pigment except a slight brownish 
 tinge in some gelatin cultures. It is 
 actively hemolytic which, however, is 
 lost on cultivation. The toxic pro- 
 ducts of the organisms have been 
 deeply investigated without any defin- 
 ite facts discovered. On agar, a visi- 
 ble growth appears in 18 to 24 hours, 
 as small, round, translucent colonies, 
 with even or notched borders, center 
 thick and margins thin. The colonies 
 show a tendency to remain discreet. 
 In nutrient broth, the long chain 
 varieties produce granular deposits 
 or small flocculi or large flakes at the 
 bottom and along the sides of the 
 tube, leaving the remainder of the 
 broth clear. Certain few long chain va- 
 rieties produce a uniform cloudiness. 
 The short chain varieties generally 
 produce a cloudiness in the medium 
 which remains so for a number of 
 days even though a fine, granular de- 
 posit accumulates at the bottom of 
 the tube. The gelatin colony is the 
 same as that of the agar. Stab cul- 
 tures are at first finely granular fili- 
 form which later become beaded and 
 may assume a brownish color. The 
 gelatin is not liquefied. Milk becomes 
 strongly acid and coagulation may 
 take place. On potato, no growth re- 
 sults except in some cases when there 
 seems to be an invisible growth. 
 LoeflEier's blood serum is a favorable 
 medium. 
 
 The streptococci will die out rapidly in 
 cultures due to the accumulation of 
 their own products. The organism 
 may be found alive after several 
 weeks or months at room temperature 
 in pus, blood or sputum. The thermal 
 death point is 45°. Direct sunlight 
 Will kill within a few hours. 
 
 Immunity following the recovery from 
 natural streptococcic infections is 
 very slight, if any, and never of a 
 
BACTERIOLOGY. 
 
 191 
 
 permanent nature. Septicemias once 
 established are generally fatal and 
 erysipelas can recur frequently. Ac- 
 tive immunity may be produced in 
 rabbits, goats, horses and other 
 domestic animals by treatment with 
 gradually increased doses of the cul- 
 tures. The bacterial substances, op- 
 sonins, agglutinins and precipitins 
 have been demonstrated in the im- 
 mune serum, which, however, shows 
 no therapeutic success. 
 The Streptococcus Mitis is a saprophytic 
 type of the mouth, showing the same 
 culture characteristics as the strep- 
 tococcic pyogenes, but grows in short 
 chains. 
 
 The Streptococcus Anglnosns is a type 
 found frequently in scarlet fever 
 throats and differs only from the 
 streptococcus pyogenes in coagulating 
 milk. 
 
 The Streptococcus Salivarius is a short 
 chain type frequently found in the 
 mouth, rarely pathogenic, which fer- 
 ments lactose, saccharose and rafR- 
 nose. It coagulates milk. 
 
 The Streptococcus Fecalis is a short 
 chain type found normally in the in- 
 testine and is occasionally pathogenic, 
 which ferments lactose, saccharose 
 and mannlte. 
 
 The Streptococcus Eg.uinus is a short 
 chain type found normally in horse 
 dung and is never pathogenic. It 
 ferments lactose. 
 
 The Streptococcus Mucosus. This or- 
 ganism was described by Howard and 
 Perkins in 1901. It was isolated by 
 Schottmuller from cases of para- 
 metritis, peritonitis, meningitis and 
 phlebetis. Some have claimed it to 
 be the cause of a variety of lesions; 
 others describe it as a harmless or- 
 ganism of the normal mouth. Morph- 
 ologically, it shows a tendency to pro- 
 duce chains. On solid media, it often 
 appears as a diplococcus. It is cap- 
 sulated and is therefore similar to 
 the pneumococcus but does not have 
 the typical lancet shape. The fact 
 that it ferments inulin media and on 
 account of its agglutinating proper- 
 ties, it might more accurately be 
 placed, in the group of pneumococci 
 than in the group of streptococci. 
 
192 BACTERIOLOGY. 
 
 The Poyntou and Paine Streptococcus 
 
 (Rheumaticus). A diplococcus iso- 
 lated from eight cases of acute rheu- 
 matic fever and with which Poynton 
 and Paine produce lesions in rabbits 
 which they considered typical of rheu- 
 matism. The organism was recovered 
 from the blood from the pericardial 
 fluid or the tonsil of the patients. It 
 was described as a minute, gram 
 negative diplococcus growing in acid 
 media under anaerobic conditions, but 
 would grow under aerobic conditions. 
 Attemps to confirm their work have 
 met with negative results. Rosenow 
 » has, however, reported a streptococcus 
 isolated from the joints of articular 
 rheumatic patients and has been able 
 to produce non-suppurative arthritis, 
 endocarditis and pericarditis in rab- 
 bits. He describes the organism as 
 intermediate in character between the 
 streptococcus viridans and the strep- 
 tococcus hemolytica. 
 
 DIFI^OCOCCnS PNEUMONIA. 
 
 (Pneumococcus, Diplococcus lanceolatus. 
 Micrococcus Pneumonia, Streptococ- 
 . cus Pneumonia). 
 
 The occurence of a diplococcus in a 
 large majority of cases, especially of 
 the lobar type of pneumonia, has 
 caused this coccus to be regarded as 
 practically specific. About 90% of all 
 cases of acute, lobar pneumonia is 
 caused by the pneumococcus, the re- 
 mainder being due to streptococci in- 
 fluenza, bacillus. Friedlander's bacilli 
 and exceptionally to other micro- 
 organism's. Lobular pneumonia is also 
 caused by the pneumococcus with al- 
 most equal regularity. The incuba- 
 tion period of this organism is two 
 or three days. The onset of the dis- 
 ease is marked by a chill, pain and a 
 rise in temperature. The respirations 
 become frequent. The fever runs be- 
 tween 102° and 105° F. for five to ten 
 days and then in favorable cases 
 terminates by a sudden drop to normal 
 within a few hours. 
 
 The pathological findings are (first 
 stage) congestion and oedema of the 
 lungs, followed by (second stage) the 
 lung becoming solid, airless and ot 
 a dark red color, the alveoli showing 
 
BACTERIOLOGY. 193 
 
 microscopically, a fibrous exudate 
 with large numbers of red cells, some 
 leucocytes and desquamated epithe- 
 lium; (third stage) the lung becomes 
 slightly softer and of a grey color, 
 microscopically the red cells degen- 
 erate and leucocytes are increased in 
 number. (fourth stage) Resolution 
 takes place by liquefaction and ab- 
 sorption of the contents of the alveoli) 
 and the entrance of air. 
 I Death occurs from toxemia or compli- 
 cations, such as endocarditis, menin- 
 gitis, etc. 
 
 For animals, the pathogenic properties 
 of the pneumococcus varies. Natural 
 infection is not common. Mice and 
 rabbits are most susceptible to arti- 
 ficial infection; while guinea pigs, 
 dogs, rats and cats are more resistant. 
 Birds are nearly immune by reason 
 of their high temperatures. Subcu- 
 taneous or intraperitoneal injections 
 of the virulent organism from cul- 
 tures or sputum kill mice and rabbits 
 by the development of septicemia and 
 peritonitis. The virulence of the 
 pneumococcus may be increased by 
 passage through susceptible animals 
 until an extremely small dose would 
 kill a mouse. The virulence of the 
 cultures obtained from man may vary 
 considerably in their virulence for 
 animals. 
 
 The organism appears to be a common 
 inhabitant of the respiratory tract, 
 acquiring virulence only under some 
 special condition that lowers the gen- 
 eral vitality, and gaining entrance 
 through the respiratory mucosa, and 
 during the disease, it is frequent to 
 find positive blood cultures; a fact 
 which accounts for the development 
 of complications, as meningitis and 
 endocarditis. The toxemia results 
 probably from lysis of the organism 
 and it has been shown that autolysis 
 of cultures in salt solution gives rise 
 to a soluble toxic portion and an in- 
 soluble toxic portion. 
 
 Immunity can be shown to exist after 
 an attack for a short time only. 
 
 Specific therapeutic agents, such as anti 
 pneumococci sera, vaccines of dead 
 cultures and autolysates and leuco- 
 cyte extracts have been tried with 
 some promise of results. No one 
 
194 BACTERIOLOGY. 
 
 method, however, has been applied 
 sufficiently with success enough to 
 warrant general adoption. In the 
 Ksputum, a Gram stained specimen is 
 sufficient to detect the diplococcus, 
 but positive identification mi^st be 
 made by culture. Culture medium 
 made rich by the addition of blood 
 serum from man or animals is used. 
 
 Inoculations are made from the blood 
 organs or sputum. Sputum injected 
 into white mice or rabbits will often 
 cause a fatal septicemia, and the or- 
 ganisms may then be obtained in pure 
 culture from the heart's blood. The or- 
 ganisms appear in pairs, as oval or 
 lancet shape cocci, with their con- 
 tiguous surface flattened and the 
 distal ends pointed. The organism 
 may vary from this type to spherical 
 or short bacillary form. The organ- 
 ism may also appear singly or in 
 chains of a length usually not more 
 than about six or eight individuals. 
 Well developed capsules envelope the 
 single organism, the pairs or the 
 chains. There are no spores or 
 flagella. The organism stains with 
 
 » the ordinary aniline dyes and is Gram 
 positive. The temperature range is 
 from 25** to 37*'. It is both aerobic 
 and anaerobic and grows in a slightly 
 alkaline media. Glycerine, nutrose 
 and dextrose media are favorable to 
 their growth. On agar, small, trans- 
 parent, finally granular colonies ap- 
 pear. On a serum or ascitic fluid 
 agar, the colonies are slightly larger 
 and more opaque. Broth is slightly 
 but uniformly clouded. Milk is acidi- 
 fied and coagulated. On potato, a 
 growth occurs but is invisible. Fer- 
 mentation with acid production takes 
 place in the majority of carbohydrates, 
 even inulin. On blood agar, a green- 
 ish zone appears about the growth, 
 but no clear zone of hemolysis as ap- 
 pears in the growth of streptococci. 
 The differentiation from other strepto- 
 cocci is sometimes difficult but the fol- 
 lowing characters are important dis- 
 tinguishing features: — the lanceolate 
 shape; the capsule; fermentation of 
 inulin; absence of hemolytic power; 
 agglutination in anti pneumococcic 
 serum; susceptibility to lysis by the 
 
BACTERIOLOGY. 
 
 195 
 
 action of bile salts. Acid is a char- 
 acteristic product and if allowed to 
 accummulate, rapidly kills the organ- 
 ism. The toxic products are closely 
 united with the cell bodies and only 
 released when the cells are broken up. 
 The thermal death point is 52°. Light 
 is a very efficient germicide unless 
 protected in thick masses of sputum. 
 Desiccation is resisted well in the 
 sputum or in blood of infected an- 
 imals. The ordinary germicides, if 
 used in their usual strength, will kill 
 the organism in a few minutes. 
 
 THE MICROCOCCUS INTBACEZiIkU- 
 IkABIS MENINGITIBIS. 
 
 In 1887, Weichselbaum discovered a 
 micrococcus in the exudate of cerebro- 
 spinal meningitis and called it Diplo- 
 coccus Intracellularis Meningitidis 
 after obtaining it in pure culture and 
 studying its characteristics. He suc- 
 ceeded also in obtaining the diplo- 
 coccus from the nasal secretion of 
 the individual sick from the disease. 
 Albreth and Ghon (1901) demon- 
 strated the organisms in healthy in- 
 dividuals. It is now believed that the 
 organism is not infrequently present 
 in the nasal cavities. The respiratory 
 tract through winter and spring pre- 
 sents a place of infection and where 
 an increase in the virulence of the 
 organisms take place. Meningitis, 
 in some cases, will follow an infection 
 of the nasal mucous membrane but 
 not in others. Why this is so is not 
 yet known. Infected persons, together 
 with the material recently soiled by 
 the nasal secretions, are dangerous. 
 
 The organism does not show marked 
 pathogenicity for adult animals. It is 
 most pathogenic for mice and guinea 
 pigs, less so for rabbits and dogs. 
 Large, subcutaneous injections in 
 animals cause death. Mice injected 
 into the plural or peritoneal cavity 
 usually become sick and die within 
 36 to 48 hours. In man, the most 
 marked lesions occur at the base of 
 the brain. The cord is also infected. 
 The exudate formed varies from a 
 slightly turbid, serous fluid to a thick, 
 fibrinous consistency. In chronic 
 cases, encephalitis and dilatation of 
 the ventricles may take place. Oc- 
 
196 BACTERIOLOGY. 
 
 casionally, secondary inflammation of 
 the nasal cavities and their accessory 
 sinuses, catarrhal inflammations of 
 the middle ear, acute bronchitis and 
 pneumonia, may take place. Eisner 
 examined the blood during the early 
 days of the disease in forty cases and 
 found the organism present in ten. 
 The diagnosis of the cerebrospinal 
 meningitis may be made by means 
 of lumbar puncture, allowing the 
 spinal fluid to settle, making smears 
 of the sediment and staining by 
 means of a blood stain, when the or- 
 ganism may be demonstrated, usually 
 inside of the leucocyte, in the form 
 of a diplococcus of a coffee bean 
 shape or as a tertracoccus. It bears 
 a close resemblance to the gonococcus. 
 It never appears within the nucleus 
 of the polynuclear leucocyte and rare- 
 ly within other cells. It may be dis- 
 tinguished from the other organisms 
 frequently met with in meningitis 
 (pneumococcus, streptococcus and 
 staphylococcus) by its rapid decolor- 
 ation by the Gram solution. In many 
 cases there are very few diplococci 
 
 . present in the spinal fluid, so that a 
 failure to find them - by microscopic 
 examination should not be taken to 
 prove that the disease did not exist, 
 therefore, cultures from the fluid 
 should be made immediately upon its 
 withdrawal. The organisms tend to 
 diminish as the disease advances. A 
 considerable amount of fluid should 
 be used for culture. 
 
 Immunization of animals by repeated 
 inoculation results in the formation 
 of agglutinins. A considerable per- 
 centage of cultures are relatively in- 
 agglutinable. Strains that do agglu- 
 tinate respond to the agglutinins de- 
 veloped in an animal immunized with 
 a true strain. During recent years 
 attempts have been made to treat the 
 disease by injections subcutaneous 
 and intraspinious of a meningococcus 
 immune serum. Wasserman in 1907, 
 obtained recoveries of 32.7% in 102 
 patients treated by the serum obtained 
 from horses immunized with pure 
 cultures of the meningoccus and toxic 
 meningococcic extracts. Flexner and 
 Jobling have more recently treated 
 
BACTERIOLOGY. 197 
 
 the disease by the use of a similar 
 serum injected intraspinously, after 
 some of the spinal fluid had been 
 withdrawn, with excellent results. 
 Hiss and Zinsser claim to have favor- 
 ably influenced the course of the dis- 
 ease by the use of subcutaneous in- 
 jections of leucocytic extract. 
 
 Culturally, the organism grows between 
 25^* and 40° C, best at 37%°. Some- 
 times it may grow at 23°C. in artifi- 
 cial media. While it often lives for 
 weeks, it may die within a few days 
 and must therefore be transplanted 
 to fresh material at least every two 
 days. It grows scai*cely at all in 
 bouillon. On agar, usually a scanty 
 growth appears. Sometimes a few 
 colonies may grow vigorously. Com- 
 paratively good growth takes place 
 on Loeffier's blood serum, blood serum 
 or ascitic fluid agar. Glucose added 
 to the media in proportion of \% 
 favors the growth. If the organism 
 has been grown successfully for some 
 time, it will produce a good growth 
 at the end of 48 hours on nutrient 
 agar or glucose agar. The colonies 
 appear as a flat layer about one-eighth 
 of an inch in diameter. They are 
 greyish white in color, flnely granular 
 and nonconfluent unless very close to- 
 gether. On Loeffier's blood serum, the ' 
 colonies are round, whitish, shining, 
 viscid, with smooth and sharply de- 
 fined outline. They tend to become 
 confluent but do not liquefy the serum. 
 
 The organisms are readily killed by 
 heat, disinfectant, sunlight and dry- 
 ing. In the dried state, a few cocci 
 may live for one to three days. After 
 the cultures have been maintained for 
 several weeks, by daily replanting, 
 transplantation once a month will 
 suffice. 
 
 GONOCOCCUS (BIFIkOCOCCVS 
 GONORBKOEA.) 
 
 The Gonococcus was discovered by 
 Neisser (1879) in the purulent secre- 
 tion of acute urethritis and vaginitis, 
 also in the acute conjunctivitis of the 
 new born. Bumm succeeded, in 1885, 
 in cultivating this organism upon 
 human blood serum. He isolated the 
 organism in pure culture and sue- 
 
198 BACTERIOLOGY. 
 
 ceeded in producing a disease by in- 
 oculating- these cultures upon the 
 healthy urethra. 
 
 The organism is usually seen in diplo- 
 cocci form, flattened along the sur- 
 faces, facing each other, which gives 
 it a coffee bean shape. Stained in 
 gonorrheal pus from acute cases, the 
 organisms are found both intra and 
 extracellularly; a great number of 
 them are characteristically crowded 
 within the leucocyte. They are never 
 found within the nucleus. The in- 
 tracellular position, which is consid- 
 ered diagnostic, is not found to any 
 great extent in the secretions from 
 chronic cases. It stains easily with 
 the usual aniline dyes. It is decolor- 
 ized by Gram, which is of differential 
 value if applied to the pus from the 
 male urethra. In exudates from the 
 vagina or from the eye, the morph- 
 ological characteristics are not so re- 
 liable on account of the presence of 
 other Gram negative organisms. In 
 the examination of a chronic discharge 
 for the presence of the organism, it is 
 necessary to attempt cultures by rea- 
 son of the fact that a negative morph- 
 ological examination cannot be re- 
 garded as conclusive. 
 
 A true gonnorheal urethritis has not 
 been produced experimentally in an- 
 imals. The infection occurs spon- 
 taneously In man. The common seat 
 of infection is in the male and female 
 genital tracts, in the conjunctiva, and 
 it may also produce a cystitis, a pros- 
 tatitis and stomatitis. Sometimes it 
 enters the blood stream, giving rise to 
 septicemia and secondarily produces 
 endocarditis and arthritis. The or- 
 ganism has been found in a few cases 
 of periostitis and osteomyelitis. 
 
 Acute infections of the genito-urinary 
 passages in man may be followed by 
 a prolonged* chronic infection, which 
 may remain quiescent for years and 
 be a source of social danger. 
 
 In female children particularly, the in- 
 fection is not rare, and in institu- 
 tions it may travel from bed to bed 
 assuming epidemic characters. 
 
 Subcutaneous and intraperitoneal injec- 
 tions of the organism into animals 
 would produce local necrosis and sup- 
 puration, probably due to the organ- 
 
BACTERIOLOGY. 
 
 199 
 
 ism's endotoxin. This toxin has been 
 isolated by Nikolaysen from the bac- 
 teria by extracting with distilled , 
 water or a solution of sodum hydrate. 
 The toxin will resist a temperature of 
 a 120° and is fully as toxic for an- 
 imals as the living cultures. 
 
 Christmas asserts to have demon- 
 strated a true, soluble toxin which 
 
 , is denied by Wassermann and Niko- 
 laysen, who do not believe that a 
 general immunity is developed in in- 
 dividuals infected wit'h the organism. 
 Christmas and Torey report success- 
 ful immunisation of animals, and 
 Torey has successfully treated human 
 cases by injections of human serum 
 from immunized animals. Bacterins 
 have been used with apparently real 
 benefit in inflammations of joints and 
 in very localized chronic infections of 
 the urethra and bladder. 
 
 The gonococcus grows best at blood 
 temperature. Its temperature range 
 is from 25° to 40°. It may be grown 
 upon nutrient agar that has been 
 streaked with human blood. It may 
 be grown on a nutrient agar contain- 
 ing 5% of glycerine. (See also media 
 for the study of the gonococcus). 
 After protracted cultivation, the or- 
 ganism will frequently grow on media 
 containing no serum. Some strains 
 will even grow on plain nutrient agar. 
 
 The cultures frequently die if kept at 
 room temperature for from 48 to 72 
 hours. In the ice-box they may live 
 for several weeks, and on plain nu- 
 trient agar they frequently live for 
 one week at a temperature of 36° C. 
 
 At the end of 24 hour cultivations, a 
 delicate growth appears, the colony 
 is translucent, finely granular with a 
 scalloped margin, which is sometimes 
 scarcely to be differentiated from tne 
 culture media. The color is usually 
 greyish white with a tinge of yellow. 
 A streaked culture appears as a grey 
 white translucent growth with rather 
 thick edges. 
 
 The organism has but little resisting 
 powers against outside influences. 
 Weak disinfecting solutions kill it 
 readily. It does not survive exposure 
 to a temperature of 45° C. for six 
 hours or a temperature of 60° for . 
 thirty minutes. Gonorrhea pus is not 
 
200 BACTERIOLOGY. 
 
 very resistant to desiccation if in thin 
 layers, but if smeared in thick layers, 
 as on linen, it has lived for 49 days 
 and it has also lived when dried on 
 glass for 29 days. 
 
 MICROCOCCUS CATABBHAX.IS. J 
 
 This organism is occasionally found iiP 
 the secretions of normal mucous 
 membrane, generally of the respira- 
 tory tract, and may be very abundant 
 in certain diseased conditions of the 
 mucous membrane. At times they 
 may produce catarrhal inflammations, 
 also pneumonia. They occur in pairs, 
 sometimes in fours, never in chains. 
 They are coffee bean in shape, slight- 
 ly larger than the gonococcus and 
 negative to Gram stain. According to 
 Ghon and PfeifCer they are of slight 
 pathogenic significance and are. of im- 
 port, aside from their production of 
 catarrhal inflammation, only in sim- 
 ilarity to the meningococcus and the 
 gonococcus. Certain cultures of the 
 micrococcus catarrhalis may prove as 
 pathogenic for white mice, guinea 
 pigs and rabbits as the meningococ- 
 cus, while other cultures are less 
 pathogenic. The range of temper- 
 ature for growth is from 20* to 40°; 
 optimum, 37^*. On nutrient agar, the 
 growth appears as greyish white or 
 yellowish white circular colonies of 
 about the same size as the meningo- 
 cocci. The borders of the colony are 
 irregular and abrupt. On serum agar, 
 the growth is luxuriant. Gelatin is 
 not liquefied. Bouillon is clouded with 
 a frequent development of a pellicle. 
 Milk, not changed. No gas production. 
 It is differentiated from the gonococ- 
 cus in that it grows easily on simple 
 culture media which is not true of the 
 gonococcus. 
 
 It is differentiated from the meningococ- 
 cus by cultural characteristics and 
 agglutination reaction. The micro- 
 coccus catarrhalis develops at tem- 
 peratures below 20** C. while the 
 meningococcus does not develop at a 
 temperature below 25" C. 
 
 Infections due to the micrococcus catar- 
 rhalis has been successfully treated 
 by bacterins. 
 
BACTERIOLOGY. 201 
 Pseudp Meiiingrococcus. 
 
 This organism was described by Elser 
 and Huntoon as a diplocoGcus very 
 similar to the meningococcus, and 
 cannot be differentiated from it ex- 
 cept by serum reaction. It is gram 
 negative. 
 
 Micrococcus Fharyngls Siccus. 
 
 This organism was described by von 
 Lingelsheim as an organism similar 
 to the micrococcus catarrhalis from 
 which it may be differentiated by fer- 
 mentation tests, and from the men- 
 ingococcus and other gram negative 
 cocci by the firm adherence and dry- 
 ness of its colonies. 
 
 Diplococcus Mucosus. 
 
 This organism was described by von 
 Lingelsheim. It is similar to the 
 meningococcus in its colony formation 
 but more sticky and mucoid. It pos- 
 sesses a distinct capsule, which can 
 be demonstrated by a capsule stain. 
 Chromog'eiiic Gram-ueg'ative Cocci. 
 
 A study of these organisms has been 
 made by Elser and Huntoon. They 
 produce a greenish yellow pigment on 
 all the ordinary culture media. , At 
 times the pigment is absent, partic- 
 ularly when grown upon sugar free 
 culture miedia, and are to be dis- 
 tinguished from the meningococcus 
 only by sugar fermentations and 
 serum reaction. 
 
 MICBOCOCCUS MEXiZTENSIS. 
 Malta Fever. 
 
 The Micrococcus Melitensis was dis- 
 covered by Bruce in 1887 in the spleen 
 in a case of Malta fever, and subse- 
 quent investigation proved it to be the 
 causative agent of Malta fever. This 
 disease is endemic along the shores 
 of the Mediteranean, in South Africa, 
 India, China, Japan, the West Indies 
 and the Philippines. The disease does 
 not seem to be transmitted from per- 
 son to person. The period of incuba- 
 tion is usually about 6 to 10 days. 
 The ordinary variety of the fever is 
 intermittent in character and lasts 
 for a period of from one to three 
 weeks with intermissions and remis- 
 sions, and may occur from time to 
 time during a period of many months, 
 
202 BACTERIOLOGY. 
 
 accompanied by constipation and gen- 
 eral debilities, with various compli- 
 cations, such as neuralgia, arthritis, 
 orchitis, etc, • Malignant cases have 
 been described, which may be fatal in 
 a week or ten days. -The mortality is 
 2% and at autopsy the spleen is found 
 to be large and very soft. The liver 
 is large and congested, both organs 
 having undergone parenchymatous de- 
 generation. The organisms are abund- 
 ant in the blood and all the organs. 
 Animals eliminate the organism in 
 the urine, and milk of goats has been 
 found to be a prolific source of in- 
 fection in that the organisms are 
 passed with the feces and so contam- 
 inate the milk. Safeguarding the milk 
 will largely eliminate the disease. 
 
 The organism is an oval coccus, some- 
 times described as a bacillus, occur- 
 Ing in pairs, in irregular groups and 
 in short chains. It is generally con- 
 sidered non motile but recently it has 
 been described as motile and possess- 
 es a single flagellum at the ex- 
 tremity . of the long diameter of the 
 oval coccus. It stains by ordinary 
 aniline dyes and iis Gram negative. It 
 
 » grows at room temperature, best at 
 body temperature, either in an acid or 
 alkaline medium. The most favorable 
 media for blood cultures is peptone 
 broth to which bile has been added. 
 On agar, after 48 hours, small, whitish 
 or yellowish colonies appear. In 
 broth, a slight cloudiness appears 
 after 46 days. The culure remains 
 alive for" several weeks or months. 
 The gelatine growth is very slow with 
 no liquefaction. 
 
 Injected animals produce specific agglu- 
 tinins, which are of practical aid in 
 diagnosis. 
 
 Micrococcus Zymogreues. 
 
 The Micrococcus Zymogenes was ob- 
 tained by McCallum and Hastings 
 from a case of acute endocarditis. It 
 has been found in a few other path- 
 ological processes. The organisms 
 occur in pairs and sometimes in short 
 chains. It grows on agar and fer- 
 ments lactose and glucose. Gelatine 
 is slowly liquefied. 
 
BACTERIOLOGY. 203 
 
 PATHOGENIC MICBO-OBOANISMS. 
 
 Bacillus Pyocyaneue. 
 (Bacillus of Green azid of Bine Pus.) 
 
 The blue and green pus frequently 
 found in many suppurating wounds is 
 due to the action of the bacillus py- 
 ocyaneus. Gessard, in 1882, demon- 
 strated this chromogenic microorg- 
 anism as a causative factor in this 
 peculiar type of suppuration. 
 
 The organism is usually found as a short 
 straight rod, occasionally slightly 
 curved. The size is subject to con- 
 siderable variation. They are fre- 
 quently united in pairs or in chains of 
 4 to 6 elements, occasionally growing 
 into long filaments and twisted spirals. 
 Spores are not found. The bacillus 
 is actively motile and each possesses 
 a single flagellum placed at one end. 
 It stains with the ordinary aniline 
 dyes but not with Gram. 
 
 The bacillus is widely distributed in 
 nature; it is found on the healthy 
 skin of man, in the feces of many 
 animals, in water contaminated by 
 animal or human material, in pur- 
 ulent discharges and in serous wound 
 infections. 
 
 It is most pathogenic for guinea pigs 
 and rabbits. Subcutaneous or in- 
 traperitoneal injections of 1 cc. of the 
 bouillon culture usually cause death 
 in from 24 to 36 hours. When smaller 
 quantities are injected into subcu- 
 taneous tissues, the animal usually 
 recovers, producing only a local ab- 
 scess, and it is subsequently immune 
 against a second inoculation with a 
 dose which would prove fatal to an 
 unprotected animal. 
 
 In man, it is found occasionally in con- 
 nection with suppurative lesions of 
 various parts of the body; frequently 
 as a secondary infection; sometimes 
 as the primary cause of the infection 
 which does not usually take place un- 
 less the individual's general condition 
 and resistance are abnormally low. 
 Under such conditions, it may be the 
 cause of chronic otitis media. It has 
 been cultivated from the stools of 
 children suffering from diarrhea and 
 has been found at autopsy distributed 
 throughout the organs of children 
 dead of gastro-enteritis. It has been 
 cultivated from the spleen at autopsy 
 
204 BACTERIOLOGY. 
 
 from a case of general sepsis follow- 
 ing mastoid operations, Wassermann 
 demonstrated the bacillus to be an 
 etiological factor in an epidemic of 
 umbilical infections in new born chil- 
 dren. 
 
 The organism is anaerobic motile ba- 
 cillus capable of growing anaerobical- 
 ly but under this condition produces 
 no pigment. Grows on all artificial 
 media at room temperature, but best 
 at 37° C. It transmits to some of the 
 culture media a bright green color in 
 the presence of oxygen. On agar, a 
 wrinkled, moist, greenish white layer 
 is developed. The surrounding me- 
 dium is bright green, which subse- 
 quently becomes darker, changing to 
 a blue green or almost black. In 
 bouillon, the growth appears as a 
 delicate, fluorescent sediment, chang- 
 ing the color of the bouillon to green. 
 The milk is coagulated and changed 
 to a yellowish green color. Gelatine 
 is liquefied. The liquefaction in stab 
 cultures occurs first near the surface, 
 in the form of a small funnel, and 
 extends downward and becomes strati- 
 form, imparting a greenish yellow 
 
 . color to that portion of the medium 
 which is in contact with the air. 
 
 Two pigments are produced by this or- 
 ganism; the one, a fluorescent green 
 common to many bacteria, is soluble 
 in water but not in chloroform; the 
 other, of a blue color (pyocynin) is 
 soluble in chloroform and may be ob- 
 tained from pure solution in long 
 blue needles. This pigment dis- 
 tinguishes the bacillus pyocyaneus 
 from the other fluorescing bacteria. 
 
 Besides the ferment that causes the 
 liquefaction of gelatine, there is one 
 which acts upon albumen. It is called 
 Pyocyanase, has the power to dissolve 
 bacteria and it is believed to have 
 some protective power when injected 
 Into animals. By reason of this fact, 
 it has been used as local treatment in 
 a number of cases of diphtheria. An- 
 imal infection is followed by the pro- 
 duction of antitoxin and antibacteri- 
 cidal substances. Wassermann found 
 agglutinins present in the immune 
 sera and Eisenberg claims that such 
 agglutinins are active also against 
 some of the other fluorescent bacteria. 
 
BACTERIOLOGY. 205 
 
 BACIXil^US PROTEUS (VUI^GABIS) 
 
 The Bacillus Proteus Vulgaris was dis- 
 covered along with other species of 
 proteus by Houser (1885), in putrefy- 
 ing substances. It is one of the most 
 widely distributed putrefactive bac- 
 teria, and is usually a harmless para- 
 site when located in the mucous mem- 
 brane of the nasal cavities where it 
 only decomposes the secretions with 
 the production of putrefactive odor. 
 Its pathogenic powers are usually 
 slight. It is found occasionally in the 
 discharge from cases of otitis media 
 in combination with other bacteria. 
 Houser isolated the organism from a 
 case of purulent peritonitis, from 
 purulent puerperal endometritis, and 
 from a phlegmonous inflammation of 
 the hand. Next to bacillus coli com- 
 munis the proteus vulgaris appears 
 most frequently concerned in the 
 etiology of pyelonephritis. In this 
 condition, together with that of 
 cystitis, the bacillus is frequently 
 found in pure culture or associated 
 with other bacteria. Krogius* uroba- 
 cillus liquefaciens septicus was prob- 
 ably a variety of the proteus bacillus. 
 Many epidemics of meat poisoning 
 have also been attributed to members 
 of the proteus family. Buker, from 
 extended researches, concluded that 
 the proteus plays an important 
 part in the morbid symptoms which 
 characterized cholera infantum. Levi 
 obtained a pure culture from the 
 vomited material and the stools in the 
 case of a man who shortly after died 
 of cholera morbus. The blood col- 
 lected at the autopsy was sterile. 
 Weinsbers cultivated a proteus ba- 
 cillus from the putrid meat which 
 had caused acute gastroenteritis in 63 
 individuals. Similar epidemics have 
 been reported by others. In some of 
 these the bacilli were very toxic when 
 injected into animals but could not 
 be recovered from the organs after 
 death. The organism grows best at 
 temperature at or above 25® C. on 
 all media. It is a facultative an- 
 aerobe. The bacillus appears or oc- 
 curs commonly as a broad, long rod 
 but varies greatly in size. Flexible 
 
206 BACTERIOLOGY. 
 
 filaments may be formed, which are 
 sometimes more or less wavy or 
 twisted like braids of hair. It does 
 not form spores and stains readily 
 with fuchsin or gentian violet. In 
 broth, it produces a rapid clouding 
 with a pellicle and mucoid sediment 
 formation. In gelatin, the colonies 
 are characteristically irregular with 
 rapid liquefaction, which is, however, 
 diminished or even inhibited under 
 anaerobic conditions. On agar, and 
 other solid media, the characteristic 
 irregular colonies are produced. From 
 a central flat, greyish white poly- 
 nucleus irregular streamers grow out 
 over the surrounding medium, giving 
 it a stellate appearance. On potato, 
 a dirty yellowish growth appears. 
 Milk is coagulated with an acid reac- 
 tion at first, later the casein is redis- 
 solved. In peptone solutions, endol 
 and phenol is produced. It grows well 
 in urine and decomposes urea into 
 carbonate of ammonia. 
 
 BACIIiZknS MAI^IiEZ. 
 
 Glander Bacillus. 
 
 TJie Bacillus of Glanders was first ob 
 tained in pure culture by Loeffler and 
 Schutz in 1882. It causes an infec- 
 tious disease called glanders, which is 
 prevalent chiefly among horses, but is 
 occasionally transmitted to other 
 domestic animals and man. 
 
 The organism is a rather small rod with 
 rounded ends, usually straight, but 
 may be slightly curved. Separate in- 
 dividuals in the same culture vary 
 greatly in size and this is a charac- 
 teristic of the organism. In old cul- 
 tures, involutions appear as short, 
 vacuolated almost coccoid individuals. 
 It is stained easily with methylene 
 blue, showing irregularity in its 
 staining qualities; the deeply stained 
 areas alternate with areas that are 
 faintly stained or entirely unstained. 
 This staining irregularity is charac- 
 teristic. The organism is non motile 
 and does not form spores. It is decol- 
 orized by Gram's. 
 
 Infection with the glander bacillus oc- 
 curs spontaneously most frequently in 
 horses. It may occur in asses, cats 
 and more rarely in dogs. The disease, 
 in some cases, infects man, if in 
 
BACTERIOLOGY': 207 
 
 habitual, contact with horses. Cattle, 
 dogs, rats and birds are immune. Ex- 
 perimental inoculations have been suc- 
 cessful in guinea pigs and rabbits. 
 
 The infection takes place through the 
 mucosa of the mouth and nasal pass- 
 ages and occasionally through the di- 
 gestive tract in horses. It is believed 
 that injury to the skin or mucosa is 
 necessary for the entrance and the 
 development of the bacilli. 
 
 In horses, the disease occurs in an 
 acute or chronic form, depending upon 
 the susceptibility of the subject or the 
 relative virulence of the organism. 
 The acute form of the disease is 
 usually limited to the nasal mucosa 
 and the upper respiratory passages. 
 It begins with fever and prostration 
 after 2 or 3 days; there is at first a 
 ^serous nasal discharge whiph later be- 
 comes seropurulent; coincident with 
 this, there is ulceration of the nasal 
 mucosa and swelling of the neighbor- 
 ing lymphatic glands, which may 
 break down and form pus discharging 
 sinuses and ulcers. The lungs now 
 become involved and death follows 
 within 4 to 6 weeks. The chronic type 
 is accompanied by multiple swellings 
 of the skin and general lymphatic en- 
 largement, and in this form is spoken 
 of as "farcy." In this type, the onset 
 is more gradual, together with the 
 nasal inflammation. The swelling of 
 the skin in some cases shows a tend- 
 ency to break down and ulcerate. The 
 disease may last for several years 
 and may occasionally end in a cure. 
 This is by far the most frequent type 
 of the disease in horses. When the 
 disease occurs in man, it is quite like 
 that of the horse except that the 
 point of origin is more frequently in 
 the skin than in the nasal mucosa. 
 The onset of the disease is violent, 
 with fever and systemic symptoms. A 
 nodule appears at the point of infec- 
 tion surrounded by lymphangitis and 
 swelling. Occasionally a papular 
 eruption occurs, which may become 
 pustular and clinically may simulates 
 small pox. Death usually follows in 
 8 to 10 days. The chronic form in 
 man is much like that in the horse, 
 ^but is more often fatal. 
 
208 B ACTERIOLOGY. 
 
 The diagnosis of glanders may be made 
 by isolating and indentifying the or- 
 ganism from the center of the glander 
 Tiodule, the nasal secretions and occa- 
 sionally from the blood. In the ma- 
 jority of cases, however, isolation is 
 difficult and animal inoculation be- 
 comes a necessity. Intraperitoneal 
 inoculation with material containing 
 the bacillus is made into male guinea 
 pigs, which leads within two or three 
 days to tumefaction and inflammation 
 of the testicles. This method is spoken 
 of as "Strauss Test," which should be 
 reinforced by cultures of the testic- 
 ular pus, the spleen and the peritoneal 
 exudate of the animal on potato. 
 
 The organism is aerobic but growths 
 may also take place under an- 
 aerobic conditions. The temperature 
 range is 22° to 43' C, optimum 37 
 C. It grows easily on all culture 
 media, whether neutral or slightly 
 alkaline or slightly acid in reaction. 
 Glycerine or small quantities of glu- 
 cose added to the media favors its 
 cultivation. On agar, the colonies ap- 
 pear after 24 hours as yellowish 
 white, first transparent, later opaque 
 •spots, with even border. Old cultures 
 become more yellow. On gelatin the 
 the growth is slow, of a greyish white 
 color, with no liquefaction. In broth, 
 there is at first (clouding later) a 
 heavy, slimy sediment with pellicle 
 formation. Broth later assumes a 
 brown color. Milk is coagulated. 
 Litmus milk indicates acid. On potato 
 media, which is not too acid, an 
 abundant growth appears within 48 
 hours, completely covering the surface 
 as a yellowish, transparent, slimy 
 mass, which grows darker until it be- 
 comes a deep reddish brown. This 
 growth is considered diagnostic, al- 
 though the potato growth of the ba- 
 cillus pyocyaneous is very similar. 
 
 The culture will, if kept cool and in the 
 dark in sealed tubes, live for months 
 and years. Sunlight, if strong, will 
 kill it within 24 hours. Heat will 
 kill it if exposed for 2 hours to 60" C; 
 one hour if 75° C. Its resistance to 
 chemical disinfections as well as dry- 
 ing is not very high. 
 
 The toxin (Mallein) belongs to the class 
 of endotoxins and is obtained by ex- 
 
BACTERIOLOGY. 209 
 
 traction of dead bacilli (see Mallein 
 under vaccines). It differs from other 
 bacterial poisons in its extreme re- 
 sistance to temperatures of a 120** C. 
 and prolonged storage. It is not a 
 powerful poison to healthy animals, as 
 , considerable doses can be given with- 
 out producing death. It is used for 
 diagnostic purposes. The injection of 
 the Malein may cause reactions in the 
 presence of other diseases than gland- 
 ers, such as bronchitis, periostitis, 
 etc., and is therefore not so valuable 
 specifically as tuberculin for diag- 
 nosis. 
 
 Recovery from glanders will not pro- 
 duce immunity. Agglutinins are form- 
 ed in the serum of subjects suffering 
 from the disease, and may be used 
 for diagnostic purposes if used in 
 dilutions of at least on6 to five 
 hundred. 
 
 BACIXiXinS AITTKBACZS. 
 
 The bacillus of anthrax was first ob- 
 served in the blood of infected an- 
 imals by Pollender in 1849. Experi- 
 mental infection in animals with the 
 blood containing the bacilli was made 
 by Davaine in 1863. 
 
 The bacillus causes an acute infectious 
 disease. Is very prevalent among an- 
 imals, particularly, sheep and cattle. 
 The infection not infrequently occurs 
 in horses, hogs and goats. It is the 
 most wide spread of all infectious 
 diseases. It is more common in 
 Europe and Asia than in America. Its 
 ravages among the cattle in Russia 
 and Siberia, the sheep in France, 
 Hungary, Germany, Persia and India 
 have been more severe than those of 
 any other animal plague. Local epi- 
 demics have occured in England and 
 it is there called Splenic Fever. 
 
 The disease also occurs in man, particu- 
 larly among stablemen, shepherds, 
 tanners, butchers and those who work 
 in wool and hair. Two forms of the 
 disease have' been described; the ex- 
 ternal anthrax, or malignant pustules, 
 and the internal anthrax of which 
 there are intestinal and pulmonary 
 forms, the so-called wool-sorter's dis- 
 ease. The bacillus is a *non motile 
 straight rod. In the blood of animals, 
 they are usually single or in pairs. 
 Grown on artificial media, they form 
 
210 BACTERIOLOGY. 
 
 long threads. The end of the indi- 
 vidual bacillus is square. In the 
 threads, the corners of the bacillus are 
 so sharp that the ends in contact in 
 a chain often touch each other only 
 at the corner, leaving an oval chink 
 between the ends of the organism. On. 
 artificial media, the organism forms 
 oval spores, centrally located, which 
 are not found in the blood of animals. *. 
 The organisms are easily stained by 
 the usual aniline dyes and are gram 
 positive. Especially stained organisms 
 from the animal tissues or the blood 
 occasionally shows a capsule. This 
 has never been demonstrated in cul- 
 tures on ordinary media. 
 
 The anthrax bacillus is pathogenic for 
 cattle, sheep, guinea pigs rats and 
 mice, their degree of susceptibility 
 varying greatly, even among different 
 members of the same species, as 
 shown by the high resistance of 
 Algerian sheep and the high sus- 
 ceptibility of the European variety. 
 Dogs, hogs, cats, birds and cold 
 blooded animals are relatively im- 
 mune. The organism is definitely 
 .pathogenic to man though less so than 
 for cattle, etc. 
 
 Separate races of the organism may 
 vary much in their virulence. A single 
 strain may remain constant as to viru- 
 lence, if preserved, dried upon threads 
 or kept in sealed tubes in dark places. 
 The virulence of the organism may 
 be reduced by the various attenuat- 
 ing procedures, which is of import- 
 ance in prophylactic immunization. 
 Experimental inoculation subcutane- 
 ously is followed at first by no symp- 
 toms, and some animals appear per- 
 fectly well until a few hours or less^ 
 before death. The duration of the ^ 
 disease depends upon the resistance 
 of the infected subject. The quantity 
 of the infectious material injected 
 has little bearing on the outcome, as 
 a single bacillus is frequently suffi- 
 cient to bring about a fatal result. 
 The bacilli are not found in the blood 
 until immediately before death. They 
 are, however, conveyed from the point 
 of inoculation by the blood and 
 lymph streams to all the organs, as 
 has been demonstrated when the tail 
 or ear of animals was inoculated, 1 
 
BACTERIOLOGY. 211 
 
 without preventing a fatal infection. 
 The bacilli do not, as a rule, multiply 
 in the blood, at least, not at first. 
 They may proliferate at the point of 
 inoculation and probably in the or- 
 g-ans and when the resistance of the 
 animal is overcome. They invade the 
 circulation and multiply within it. 
 Autopsy upon suoh animals shows at 
 the point of inoculation, edematous, 
 hemorrhagic infiltration. The spleen 
 is congested and enlarged. The kid- 
 neys are congested and there may be 
 hemorrhagic spots upon the mucous 
 membrane. Death brought about by 
 the anthrax bacillus is probably due 
 in a large extent to obstruction of 
 the capillaries, although a true toxin 
 has never been demonstrated, the 
 toxic clinical picture of the disease 
 presented in some animals and in 
 man precludes the possibility that 
 such poisons do not exist, though 
 neither the culture filtrates nor the 
 dead bacilli have any noticeable toXic 
 effect upon test animals and exert no 
 immunizing action. 
 Infection of animals takes place by 
 way of the alimentary canal. The 
 sjpores of the bacteria resist the g^r^ 
 trie juice and develop into the vegeta- 
 tive form in the intestines, where 
 they increase and invade the system. 
 Subcutaneous infections may occur 
 when there are small punctures and 
 abrasions about the mouth. When 
 Infection takes place upon a visible 
 part, there is formed a diffused local 
 swelling, somewhat like a large car- 
 buncle, the center of which is marked 
 by a black, necrotic slough, or may 
 contain a pustular depression. In- 
 fection by way of inhalation is rare 
 among animals. The disease in in- 
 fected cattle and sheep is very acute 
 and kills within one or two days. 
 There is about 80% mortality. In 
 man infection generally takes place 
 through a small cutaneous abrasion. 
 It may also occur by inhalation and 
 through the alimentary tract. The 
 cutaneous infection occurs usually 
 through an abrasion of the skin in 
 men who handle live stock, in 
 butchers and tanners of hides. The 
 primary lesion, appearing at the sight 
 of inoculation within 12 to 24 hours, 
 
212 BACTERIOLOGY. 
 
 resembles an ordinary small furuncle, 
 with a central vesicle filled with a 
 sero sanguineous and later a sero 
 purulent fluid. This changes in the 
 center to a black, necrotic mass 
 which is surrounded by an edematous 
 areola. If early and prompt excision 
 Is made of the mass, the patient re- 
 covers; if not, local gangrene and 
 general systemic infection may occur 
 and lead to death within 5 or 6 days. 
 The pulmonary Infection (wool-sorter's 
 disease), rare In this country, occurs 
 in persons who handle raw wool, 
 hides or horse hair, by their inhaling 
 or by their swallowing the spores. 
 The disease has manifested itself as 
 a violent, irregular pneumonia, which, 
 In the majority of cases, leads to 
 death. 
 
 Infection through the alimentary canal, 
 rare in man, usually takes place 
 through the ingestion of uncooked, 
 infected meat, the initial lesion locat- 
 ing in the small intestine, producing 
 violent enteritis, with bloody stools 
 and great prostration, which results 
 in death. 
 
 The anthrax bacillus grows very lux- 
 uriantly under aerobic conditions, 
 while it develops slowly and tersely 
 under anaerobic conditions. Its op- 
 timum temperature is 37^' C. but 
 will grow at temperature as low 
 as 15° and as high at 45*. It may be 
 cultivated upon all the ordinary 
 media; a slightly alkaline or neutral 
 media seems to be the optimum reac- 
 tion. On agar plates, vigorous colonies 
 appear in 12 to 24 hours. They are 
 irregular in outline, wrinkled, and if 
 examined under the microscope, they 
 seem to be made up of hair-like 
 tangle of thread spreading in wavy 
 layers from a more compact central 
 knot. On gelatin plates, they appear 
 within 24 to 48 hours as opaque pin 
 head size white disks. As the colony 
 increases in size, their outline be- 
 comes less regular, and under the 
 microscope Is similar to that of the 
 agar plates. Liquefaction takes place 
 in about three or four days. In the 
 gelatin stab there is at first a thin, 
 white line along the puncture. This 
 growth continues into the formation 
 of thin filaments, which diverge from 
 
BACTERIOLOGY. 213 
 
 the stab and take on an appearance 
 not unlike a small, inverted "Christ- 
 mas tree." Liquefaction begins at 
 the top. In broth there is at first a 
 rapid growth with uneven clouding 
 and a pellicle formation, later a 
 slimy mass somewhat like a cotton 
 fluff appears. On potato, there is a 
 rapid, white, dry growth. Milk is 
 slowly acidified and coagulated. 
 
 On account of the spores, the anthrax 
 is extremely resistant to chemical 
 and physical agents. The vegetative 
 form is killed by an exposure to a 
 temperature of 54 C. for ten minutes. 
 The spores in a dry state will live 
 for many years. The exposure of the 
 spore to dry heat at 140° C, for 
 three hours, is necessary to kill. Live 
 steam at 100° C.kills them in from 5 to 
 10 minutes. Low temperature does 
 not seem to have a great deal of ef- 
 fect on them. The spore's resistance 
 to chemicals varies with different 
 strains. Direct sunlight will k411 the 
 spore within 6 to 12 hours. 
 
 Active immunization of small labora- 
 tory animals is very difficult but can 
 be accomplished wih extremely at- 
 tenuated cultures. 
 
 Passive immunity by means of serum, 
 of actively immunized sheep has been 
 produced and practically applied by 
 Sobernheim. The injection of such 
 serum has been found to protect an- 
 imals from anthrax and to confer an 
 immunity which lasts often as long 
 as two months. 
 
 No specific nor bactericidal nor bac- 
 terialitic properties have been dem- 
 onstrated in the immune serum. Ag- 
 glutinins have not been satisfactorily 
 demonstrated. 
 
 ANTHBAX-IiIKE BACIIiU. 
 
 In nearly all laboratories there are 
 strains of true anthrax bacilli which 
 have become so attentuated that they 
 are practically non pathogenic. They 
 do not, however, differ from the viru- 
 lent organisms in their culture or 
 morphological characteristics. There 
 are likewise in the laboratory certain 
 non virulent bacteria which do not 
 resemble the anthrax bacillus cultur- 
 ally but do so morphologically (see 
 below). 
 
214 BACTERIOLOGY. 
 
 Bacillus Anthracoides. This is a non- 
 pathogenic, gram positive organism, 
 indistinguishable from the bacillus 
 anthracis, except morphologically the 
 ends are more rounded and culturally 
 the growth is more rapid, together 
 with a more rapid liquefaction of 
 gelatin. 
 
 Bacillus Badicosus. This is a non- 
 pathogenic organism cultivated from 
 city water supplies. Morphologically, 
 it is somewhat larger than the an- 
 thrax bacillus and the individual ba- 
 cilli are more irregular in size. Cul- 
 turally, the growth is most active at 
 room temperature, with very rapid 
 liquefaction of gelatin. 
 
 Bacillus SufetUis. (Hay Bacillus). This 
 is a practically non-pathogenic Gram 
 positive organism found in brackish 
 waters and infusions of vegetable 
 matter, and occasionally as a saphro- 
 phyte or secondary invader in chronic 
 suppurative lesions, as in old sinuses 
 an^ infected wounds. It is not very 
 closely related to the anthrax bacillus. 
 It occurs as straight rods and is 
 actively motile in young cultures in 
 which the bacilli appear singly or in 
 
 » pairs. In older cultures, chains are 
 formed and the bacilli become mo- 
 tionless. Spores are formed, usually 
 slightly nearer one pole than the 
 other. On gelatin and agar the ba- 
 cilla grow as a dry, corrugated pelli- 
 cle. Gelatin is liquefied. Micro- 
 scopically the colonies are irregularly 
 round with fringed edges and made 
 up of interlacing threads. 
 
 BACIZiI^US DIPHTHERIA. 
 (Xlebs-Zioeffler Bacillus) 
 
 The - Bacillus of Diphtheria was dis- 
 covered by Klebs in 1883, having ob- 
 served the bacillus morphologically 
 from the pseudo membranes of diph- 
 theritic throats. An organism was 
 isolated and cultivated by Loeffler in 
 1884, which corresponded to the 
 morphological characters of the ba- 
 cillus discovered by Klebs. He in- 
 oculated the organisms upon the in- 
 jured mucous membrane of animals 
 and produced lesions which resembled 
 the false membranes of the disease in 
 human individuals. Loeffler was, 
 however, very conservative in nis de- 
 
BACTERIOLOGY. 215 
 
 scription of this organism as a 
 causative agent of diphtheria by rea- 
 son of his failure to find the organ- 
 ism in all cases examined, and his 
 finding the organism in the throats 
 of a liealthy individual, together with 
 his inability to explain the systemic 
 manifestations of the infection. All 
 existing doubt as to the etiology of 
 the disease was overcome by Loeffler's 
 further studies together with the 
 publication of articles, on the nature 
 of the toxin produced by the diph- 
 theria bacillus, by Roux and Yersin 
 m 1888. 
 
 The bacillus of diphtheria is subject to 
 a number of morphological variations 
 which depend to a certain extent upon 
 the age of the culture and upon the con- 
 stitution of the medium of which it is 
 grown. These factors do not, however, 
 control the appearance of the organism 
 with any degree of regularity, in as 
 much as all of the variations may be 
 observed in the same growth. The 
 difference in its morphology probably 
 represents stages in the growth and 
 degeneration of the individual organ- 
 ism. Certain characteristics in the 
 morphology of the organism facili- 
 tate its recognition. They appear as 
 slender, straight or slightly curved 
 rods. Their thickness throughout 
 their length Is rarely uniform. They 
 may show club shaped thickenings at 
 one or both ends. Sometimes they 
 are more thick at the center and 
 taper towards the ends. If they are 
 thickened at one end, they take on a 
 slender wedge-shaped form, and are 
 usually straight, of smaller size than 
 the others mentioned and stain uni- 
 formly. This type has been re#Brred 
 to by Beck as "ground type" and be- 
 lieved by him to be young individual. 
 Branched forms have also been noted, 
 which are probably abnormal or in- 
 volution forms. The organisms stain 
 readily with watery aniline dyes. An 
 irregularity of staining is a charac- 
 teristic of diagnostic value and is 
 best obtained by the use of Loefller's 
 alkaline methylene blue, which, if ap- 
 plied for from 5 to 10 minutes, will 
 cause the bacilli to be traversed by 
 stained and unstained bands, which 
 give to the organisms a striped or 
 
216 BACTERIOLOGY. 
 
 beaded appearance. If the organisms 
 are long, they may take on the ap- 
 pearance of short streptococci; others 
 may appear granular. The bacilli of 
 abput 18 hours' culture may show 
 stained oval bodies, most frequently 
 situated at the end, and are spoken 
 of as polar or Babes-Ernst bodies. 
 Special stains for these bodies have 
 been brought out by Neisser and 
 others, who claim for them differen- 
 tial value in distinguishing the diph- 
 theria bacillus from other morph- 
 ologically like organisms. These polar 
 bodies are probably chromatic gran- 
 ules. The organism is stained by 
 Gram, but care must be taken in 
 timing the stain. Carelessness may 
 lead to irregular results. 
 
 The bacillus of diphtheria causes a 
 specific local action upon mucous 
 membranes, the so-called pseudo 
 membrane. The disease depends in 
 part uiibn the mechanical surface of 
 this membrane, and in part upon the 
 toxins which are produced by the 
 organism. The most frequent sites 
 of diphtheria are the mucous mem- 
 branes of the throat, larynx and nose. 
 » Occasionally they have been found in 
 the ear, upon thfe mucous membrane 
 of the stomach and vulva and upon 
 the conjunctiva and skin. The or- 
 ganism may extend from the larynx 
 and cause a diphtheritic broncho- 
 pneumonia. Although the organism 
 has been isolated after death from the 
 spleen and liver, a true diphtheritic 
 speticemia is not probable. 
 
 The organism is very pathogenic to 
 dogs, cats, fowls, rabbits and guinea 
 pigs. Rats and mice will resist it, 
 if* administered in extremely large 
 doses. Membranes analogous to those 
 found in man, have been prod?uced in 
 susceptible animals, but only when 
 mechanical injury to the mucosa has 
 preceded the inoculation with the 
 bacillus. If small quantities (one- 
 half to 1 cc.) of a broth culture are 
 Injected subcutaneously into a guinea 
 pig, symptoms appear within 6 to 8 
 hours which are followed by death 
 within 36 to 72 hours. At autopsy, 
 a serosanguineous exudate will be 
 found at the point of inoculation. 
 The lymph glands are edematous. 
 
BACTERIOLOGY. 217 
 
 The kidney, liver, speen and lungs 
 are congested. There may be exudates 
 in the pleural and peritoneal cavities. 
 A severe congestion of the suprarenal 
 bodies is characteristic and almost 
 pathognomonic. 
 
 Agglutinins for the diphtheria bacillus 
 have been developed in an amount to 
 act in one to five thousand dilutions 
 of the serum by the injections of the 
 bodies of the organism into animals. 
 The serum of convalescent patients 
 has ordinarily but little agglutinating 
 power. The test is not used in diag- 
 nosis. Antitoxins have been prepared 
 and used in treatment with very 
 beneficial results (see diphtheria anti- 
 toxin). Active immunization has been 
 recommended by Theobald Smith by 
 the use of mixtures of toxin and anti- 
 toxin, in this way producing an im- 
 munity. There are a great number 
 of objections to this method of im- 
 munization. 
 
 The organism is an aerobe, but will 
 grow under anaerobic conditions in 
 the presence of carbohydrate. The 
 temperature range is 19" to 42° C, 
 optimum 37%** C. Any temperature 
 above this impedes the development 
 of the toxin. The organism is iso- 
 lated from mixed cultures very read- 
 ily. Cultures are taken from throats 
 and placed upon Loeffler's blood se- 
 rum, upon which they are permitted 
 to grow at 371/^* C. for from 18 to 24 
 hours. An emulsion is now made from 
 the growth with about 5 cc. of bouil- 
 lon; two or three loopfuls of this emul- 
 sion is streaked over the surface of 
 a sugared agar, incubated for 24 
 hours and the characteristic colonies 
 transferred to Loeffler's media. 
 
 The diphtheria bacillus grows readily 
 on most of the rich laboratory media. 
 The most favorably reaction for its 
 growth is probably about alka- 
 linity. Loeffler's media is the most 
 widely used media for the cultivation 
 of the organism. Swabs from sus- 
 pected throats are smeared over the 
 surface of Loeffier media and incu- 
 bated at 37%** C. At the end of 12 
 to 24 hours minute, greyish white, 
 bristling colonies of the diphtheria 
 bacillus are developed. These enlarge 
 and grow to such an extent that they 
 
218 BACTERIOLOGY. 
 
 outstrijp the accompanying micro- 
 org-anisms. This method is of value 
 for diagnostic purposes. On agar, the 
 colonies appear within 24 to 36 hours 
 as small, translucent, greyish spots, 
 quite characteristic and easily recog- 
 nized. The colonies become irreg- 
 ularly round or oval, with a dark 
 nucleus like center, fringed by a loose, 
 coarse, granular disk. The edges of 
 the colony are irregular. The addi- 
 tion of 1% dextrose, 2% nutrose, 
 6% glycerine renders the agar favor- 
 able for rapid growth but makes it 
 a very poor media for the preserva- 
 tion of the culture. A meat infusion 
 gelatin is a favorable media but be- 
 cause of the low temperature at which 
 the media must be kept, the growth is 
 very slow. Gelatin is not liquefied. 
 The organism grows readily in milk 
 with no coagulation. Endol is not pro- 
 duced in peptone solutions. 
 The organism has a thermal death point 
 of 58° C. It is killed if exposed to a 
 boiling heat for one minute. Low 
 temperatures are borne readily. Desic- 
 cation and exposure to light are not 
 as fatal to it as to most other patho- 
 , genie organisms. Chemical disin- 
 fectants will kill the organisms 
 readily. 
 
 PSEUDO DIPHTHERIA BACIZiZkUS. 
 
 (Bacillus Hoffmaimi). 
 
 The Pseudo diphtheria Bacillus was 
 described by Hoffmann in 1888, who 
 cultivated the organism from the 
 throats of normal individuals, and in 
 several instances from the throats 
 of diphtheritic persons. The organ- 
 ism resembles the diphtheria bacillus 
 but differs from it in its non-patho- 
 genicity to guinea pigs. It was at 
 first regarded as an attentuated form 
 of diphtheria bacillus, but further 
 study showed it to be. unquestionably a 
 separate species. It is a non-motile 
 bacillus, shorter and broader than the 
 bacillus of diphtheria. It is usually 
 straight and may be slightly clubbed 
 at one end. Stained with Loeffler's 
 methylene blue, it may show un- 
 stained bands, but unlike the diph- 
 theria bacillus, these bands rarely 
 number more than one, and never 
 
BACTERIOLOGY. 
 
 219 
 
 more than two. No polar bodies have 
 been demonstrated by special stains. 
 Distinguished culturally from the diph- 
 theria bacillus, it grows more lux- 
 uriantly upon simple media. On agar 
 plates, the colonies are larger, less 
 transparent and more white. In liquid 
 media, there is clouding and less 
 tendency to pellicle formation. It 
 does not form acid upon the various 
 sugared media. Animals immunized 
 with it do not possess increased re- 
 sistance to the diphtheria bacillus. It 
 is entirely inocuous to ordinary 
 laboratory animals. 
 
 BACZI.I.US XEROSIS. 
 
 The Bacillus Xerosis, almost indentical 
 with the diphtheria bacillus, was dis- 
 covered by 'Kutschert and Neisser 
 from the eyes of patients suffering 
 from a , chronic conjunctivitis called 
 Xerosis. They believed it to be the 
 etiological factor in this disease, but 
 the fact that it has been frequently 
 isolated from normal eyes, precluded 
 it as a causative factor. It is prob- 
 ably a harmless parasite, found more 
 often in the slightly inflamed than 
 in the normal conjunctiva, 
 
 No absolute differentiation morpholog- 
 ically can be made between the diph- 
 theria bacillus and the bacillus 
 xerosis. Polar bodies have occasion- 
 ally been seen. Its growth on Loef- 
 fler's blood serum agar, glycerine agar 
 and in broth is probably more delicate 
 but very similar to that of bacillus 
 diphtheria. It is not easily cultivated 
 upon ordinary meat extract media. 
 It will not grow on gelatin at room 
 temperature and on glycerine or glu- 
 cose agar; the colonies are micro- 
 scopically identical with those of 
 diphtheria. It differs, however, from 
 the bacillus of diphtheria in produc- 
 ing acid from saccharose but not from 
 dextrin. It is non pathogenic to an- 
 imals and does not form a toxin. 
 
 BACIZiI^US INFI^UENZA. 
 (Pfelffer Bacillus). 
 
 Epidemics of influenza can be traced 
 back to the fifteenth century. The last 
 serious epidemic occurred in the years 
 1889 to 1890. Beginning in the East, 
 traveled through Russia, became pan- 
 
220 BACTERIOLOGY. 
 
 demic in Europe, invaded America and 
 became prevalent in China, Japan, Aus- 
 tralia and Africa. Hundreds of thou- 
 sands of people were infected and the 
 mortality was high. Since then more 
 or less of it has been present, especi- 
 ally during the winter months. Many 
 acute^ inflammations of the respira- 
 tory mucous membrane due to pneu- 
 mococci and streptococci give symp- 
 toms similar to those produced by the 
 bacillus of influenza, which was final- 
 ly isolated by Pfeiffer In 1892 from 
 the purulent bronchial secretions of 
 a patient suffering from the disease 
 and grown upon blood agar. The 
 Bacillus of Influenza is an extremely 
 small, non motile organism of irreg- 
 ular length, with rounded ends, rarely 
 forming chains. The organisms do 
 not take the ordinary aniline dyes 
 well and are best demonstrated by 
 staining with a 10% aqueous fuchsin 
 for 5 to 10 minutes, or with Loeffler's 
 alkaline methylene blue for 5 minutes. 
 They are Gram negative. Occasional- 
 ly a slight polar stain may be ob- 
 served. In the smears from the 
 bronchial secretions, the bacilli lie 
 close together in thick, irregular 
 clusters without definite parallelism. 
 The fact that they very rarely 
 form chains is considered character- 
 istic. The organisms are found 
 in the nasal passages and bronchial 
 secretions of those sick from the dis- 
 ease. The organs affected most fre- 
 quently in man are the upper respira- 
 tory passages and lungs. The disease 
 takes the form of a broncho or lobular 
 pneumonia. The broncho pneumonias 
 produced by the organism do not 
 differ essentially from those produced 
 from other microorganisms, conse- 
 quently, a bacteriological diagnosis is 
 imperative. The infection is not in- 
 frequently followed by abscess or 
 gangrene of the lung, and occasional- 
 ly a chronic interstitial process is de- 
 veloped. The organisms have been 
 found in the middle ear, the meninges, 
 the brain and spinal cord. Although 
 the general character of the symp- 
 toms suggests septicemia, the organ- 
 ism has not been found in the circu- 
 lating blood. The incubation period 
 is short, having been shown to develop 
 
BACTERIOLOGY. 221 
 
 In 24 hours. The organism may re- 
 main in the bronchial secretions of 
 convalescents or even in the secre-, 
 tions of normal individuals for many 
 years. Animals are not susceptible 
 to the infection, excepting the monkey, 
 in which influenza-like symptoms 
 have been produced by rubbing pure 
 cultures upon the unbroken nasal 
 mucosa. Rabbits inoculated intravt^n- 
 ously suffer from severe symptoms 
 which are probably purely toxic. The 
 Immunity produced by an attack of 
 influenza, if any, is of very short 
 duration. 
 
 The organism is aerobic. The isolation 
 is not easy. PfeifCer succeeded in 
 growing the bacillus upon serum agar 
 plates which had been smeared with 
 the pus from the bronchial secretions 
 of patients. Agar smeared with blood 
 is a favorable media for its growth. 
 The organism grows well symbiotic- 
 ally with staphylococci which seem 
 to create a favorable environment for 
 their development. The organism 
 does not grow at room temperature 
 at Z1V2° C, and on favorable media, 
 the colonies appear in from 18 to 24 
 hours as minute, colorless, transpar- 
 ent, discrete droplets. In order to 
 keep the cultures alive they should 
 be stored at room temperature and 
 transplanted every four or five days. 
 The organism is very sensitive to 
 heat, desiccation and disinfectants. 
 
 ^CNFIiUENZA-I^IKE BACIUI. 
 
 Fseudo Influenza Bacillus. This organ- 
 ism was found by Pfeiffer in the 
 broncho pneumonic process of chil- 
 dren. It is slightly larger than the 
 bacillus of influenza, non motile and 
 Gram negative, with tendency to form 
 threads and involution forms. It is 
 strictly aerobic. Woolstein believes 
 the organism to be the same as the 
 bacillus of influenza, after having 
 studied it both culturally and by ag- 
 glutination tests. Strains of similar 
 bacilli have been isolated from cases 
 of pertussis. 
 
 Koch-Weeks Bacillus. This bacillus was 
 described by Koch in 1883 and Weeks 
 in 1887 in connection with an acute 
 conjunctival inflammation. Morph- 
 
222 BACTERIOLOGY. 
 
 ologically it resembles the bacillus of 
 influenza, though more slender and of 
 greater length. It grows at a tem- 
 perature of 371/^° C. only, and can be 
 cultivated upon serum or ascitic fluid 
 without hemoglobin, in which respect 
 it differs from the bacillus of in- 
 fluenza. It is Gram negative. 
 The Bacillus of Fleuro Fneimioiila of 
 Babbits is a small Gram negative ba- 
 cillus slightly larger than the bacillus 
 of influenza and grows upon ordinary 
 media. The organism was described 
 by Beck. 
 
 The Bacillus Murisepticus and Bacillus 
 Bhusiopathiae are organisms morph- 
 ologically similar to the influenza 
 group but can be easily separated 
 from them because of their luxuriant 
 growth on ordinary media. They are 
 more closely related to the hemor- 
 rhagic septicemic group of organisms. 
 
 BACZIil^US BOBDET-GBNGOir. 
 
 (Bacillus of Whoopiugr Coug'h). 
 
 This organism was discovered by Bordet 
 and Gengou in the sputum of a child 
 ill with pertussis. It is as yet not 
 positive as to the specificty of this 
 organism for whooping cough. Cul- 
 'tivation was not successfully carried 
 out until 1906. Since then almost 
 pure cultures of the organism have 
 been obtained during the early par- 
 oxysm of the disease, and for this 
 reason it is thought likely to be a 
 speciflc cause. In early cases, true 
 influenza bacilli have often been 
 found, and these seem to remain in 
 the sputum of such patients for a 
 longer period and in larger numbers 
 than the bacillus of Bordet-Gengou. 
 The organism though slightly larger 
 than influenza bacillus resembles it 
 greatly, but shows some morpholog- 
 ical differences and less tendensy to 
 pleomorphism. It is a small ovoid 
 bacillus found scattered in enormous 
 numbers among the pus cells, some- 
 times within the cell, in the sputum 
 early in the disease. Occasionally it 
 resembles a micrococcus, though gen- 
 erally the form is constant, slightly 
 enlarged individuals may be encount- 
 ered. The poles of the cell may stain 
 more deeply than the center. It. is 
 stained with alkaline methylene blue, 
 dilute carbofuchsin or aqueous fuchsin. 
 
BACTERIOLOGY. 
 
 223 
 
 It is decolorized by Gram. The group- 
 ing is separated, though sometimes in 
 end to end pairs. 
 
 Inoculated into the respiratory tract of 
 monkeys it has failed to produce the 
 disease. One to two cc. of an extract 
 made by emulsifying the agar growths 
 with a little salt solution, dried in 
 vacuo and ground in the mortar, di- 
 luted with salt solution and centri- 
 fugalized and decanted, and injected 
 intravenously into rabbits, will kill 
 within 24 hours. A subcutaneous in- 
 oculation will produce necrosis and 
 ulceration without marked constitu- 
 tional symptoms. Specific agglutinins 
 obtained from immunized animals 
 distinguishes the organism from that 
 of the bacillus of influenza. 
 
 It is cultivated from the sputum on a 
 medium made by adding 100 gms. ol 
 sliced potato to 200 cc. of 4% watery 
 solution of glycerine, steamed in an 
 autoclave; 50 cc. of this extract is 
 mixed with 150 cc. of 6% salt solu- 
 tion, then 5 gms. of agar are added. 
 It is now melted in an autoclave and 
 filled in quantities of 2 to 3 cc. into 
 test tubes and sterilized. To each 
 tube is now added an equal volume 
 of sterile defibrinated rabbit's or hu- 
 man blood. The substance is mixed 
 and the tube slanted. On such a 
 media, growth appears after 24 to 48 
 hours as small greyish rather thick 
 colonies. The second generation on 
 this media becomes rapid and lux- 
 uriant and after several generations, 
 they resemble the growth of the 
 typhoid bacillus. Later, it seems less 
 dependent upon the presence of 
 hemoglobin than does the bacillus of 
 influenza. It will develop at as low 
 a temperature as 5° C. but grows 
 best at 371/2° C. 
 
 BACIl^IiUS MORAX-AXENFEI^D. 
 
 The Bacillus of Morax-Axenfeld was 
 discovered by Morax in 1896 from a 
 type of chronic catarrhal conjuncti- 
 vitis, which attacked both eyes, espe- 
 cially in the angles of the eye and 
 most severe at or about the caruncle. 
 The swelling produced is not great 
 and there is hardly ever ulceration. 
 The condition becomes subacute and 
 
224 BACTERIOLOGY. 
 
 chronic and may be diagnosed by- 
 smear preparations made trom the 
 pus, which is especially abundant 
 during the night. The organism is a 
 short thick diplobacillus. It may, 
 however, appear singly or in short 
 chains. The ends are rounded, the 
 center slightly bulged. They are 
 stained by the aniline dyes and de- 
 colorized by grams. Inoculation with 
 pure cultures has produced subacute 
 conjunctivitis in man. The produc- 
 tion of lesions in lower animals has 
 been unsuccessful. 
 The organism is cultivated only upon 
 alkaline media containing blood or 
 blood serum. At the end of 24 hours 
 on LoefHer's blood serum, small areas 
 of liquefaction are noticed, later the 
 entire media is liquefied. Upon serum 
 agar, delicate, greyish, droplike col- 
 onies, not unlike those of the gono- 
 coccus, are formed. 
 
 BACIUUS OF ZUB NEEDEN. 
 
 Zur Needen described a small, slightly 
 curved, non motile diplobacillus in 
 ulcerative conditions of the cornea, 
 to which he attributed etiological im- 
 » portance. The organism is stained 
 by the ordinary aniline dyes, though 
 poorly at the ends and decolorized by 
 Gram's. 
 
 Corneal ulcers are produced by inocula- 
 tion of guinea pigs. 
 
 Upon agar within 24 hours, trans- 
 parent, slightly fluorescent, round, 
 raised, rather coarsely granular col- 
 onies are formed which show a tend- 
 ency to confluence. Gelatin is not 
 liquefied. Milk is coagulated. Upon 
 potato, there appears a thick, yellow- 
 ish growth. Upon dextrose media, 
 there is acid formation but no gas. 
 Indol is not produced in peptone. 
 Bacillus of DxLcrey. 
 (Bacillus of Soft Chancre). 
 
 This bacillus was first described and 
 obtained in pure culture by Ducrey in 
 1889. It produces a lesion, which oc- 
 curs usually upon the genitals or 
 the skin surrounding the genitals 
 of an acute. Inflammatory, destructive 
 nature and called "soft chancre" or 
 "chancroid." The lesion begins usual- 
 ly, as a small pustule, which soon 
 ruptures and forms a small, round. 
 
BACTERIOLOGY. 225 
 
 depressed, irregular ulcer, with under- 
 mined edges and a necrotic floor, dis- 
 charging seropurulent fluid which is 
 extremely infectious. This ulcer 
 spreads rapidly and leads usually to 
 lymphatic swellings in the groin, 
 which later give rise to abscesses 
 spoken of as "buboes." The lesion 
 differs from the syphiltic chancre in 
 that there is no induration. 
 It is an extremely small, non-motile 
 bacillus, generally appearing in short 
 chains and in parallel rows, though 
 it may be found irregularly grouped. 
 It stains easily though irregularly 
 with aniline dyes. More stained at 
 the poles, decolorized by Gram's. The 
 bacilli are found in the pus, often 
 within the leucocyte. Various in- 
 vestigators have succeeded in pro- 
 ducing lesions in man by inoculating 
 with pure cultures. Attempts to in- 
 oculate animals have beeja unsuccess- 
 ful. 
 
 The organism grows on agar medium to 
 which blood has been added. Coag- 
 ulated blood kept for several days 
 in sterile tubes is a very favorable 
 medium. The organism is isolated 
 by aspirating an unruptured bubo 
 with a sterile hypordermic syringe 
 and transferring the pus in quantities 
 directly to the agar. If no buboes 
 are present, the primary lesion may 
 be cleansed with water or salt solu- 
 tion, material scraped from the bot- 
 tom of the ulcer by means of a stiff 
 platinum loop and smeared over the 
 the surface of blood agar plates. 
 After a period of 48 hours, small, 
 transparent, grey, finely granular, 
 isolated colonies appear upon the 
 agar plate. These rarely grow larger 
 than pin head size. The cultures die 
 readily at room temperature but may 
 be kept alive in the incubator for a 
 week or more. 
 
 SFIBIIiI^UM CHOLERAS ASIATICAE. 
 (The Comma Bacillus of Koch). 
 
 This organism was discovered by Kocn 
 in 1883 in the dejecta of patients suf- 
 fering from Asiatic cholera. 
 
 Asiatic Cholera, a disease occurring s 
 spontaneously only in man, is en- 
 demic in eastern countries, partic- 
 ularly India. From time to time it 
 
226 BACTERIOLOGY. 
 
 has become epidemic in Europe and 
 Asia, not infrequently sweeping over 
 almost the entire earth. The last 
 great epidemic began about 1883, dur- 
 ing which time there were 800,000 
 victims in Russia alone, and reached 
 Germany in 1892. Prom there it en- 
 tered America and Africa. 
 The vibrio or spirillum of cholera is a 
 small, curved rod. The curvature may 
 vary from the comma like form to a 
 distinct corkscrew like spiral, with 
 one or two turns. It is actively 
 motile by reason of a single polar 
 flagellum. The comma form predom- 
 inates in young cultures, while the 
 longer forms are more numerous in 
 old cultures. Prolonged artificial cul- 
 tivations without passage through 
 the animal body tend to change the 
 form of the organism into a straight 
 type. They take the ordinary watery 
 aniline dy^s well and are decolorized 
 by gram. The infection is essentially 
 of the intestine and contracted by 
 the injections of the organisms with 
 water, food or contaminated material. 
 A few organisms entering into the 
 stomach may be checked by the nor- 
 mal gastric secretions by reason of 
 the organism's sensitiveness to an 
 acid reaction. Entering the intestine, 
 however, they proliferate and rapidly 
 outgrow the normal intestinal flora. 
 Autopsies show extreme congestion 
 of the intestinal walls, with occa- 
 sional ecchymosis and localized ne- 
 crosis of the mucosa, with swelling 
 of the solitary lymph-follicles and 
 Peyer's patches. The organisms 
 penetrate the mucosa and lie within 
 it in the layers next to the submucosa. 
 The intestines are filled with watery, 
 slightly bloody, or "rice water" stools, 
 which is characteristic and from 
 which pure cultures of the organism 
 may be isolated. They are, in fact, 
 found only in the intestines and their 
 contents. The parenchymatous de- 
 generation in other organs is of toxic 
 origin. In animals the disease never 
 appears spontaneously so that special 
 methods were necessary in order to 
 produce the disease experimentally. 
 Subcutaneous inoculations, unless in 
 large quantities of the organism, in 
 rabbits and guinea pigs very seldom 
 
BACTERIOLOGY. 227 
 
 produce more than a slight illness. 
 Intraperitoneal inoculation, if in 
 proper quantities, generally leads to 
 death. Different strains of the cholera 
 spirillum vary greatly in their viru- 
 lence and this may be enchanced by 
 repeated passage through animals. 
 
 The poisonous action of the cholera or- 
 ganism depends upon the formation 
 of true secretory toxins and upon 
 endotoxins. Which of these is para- 
 mount in producing the disease can- 
 not be stated definitely. 
 
 Active immuniation is accomplished by 
 inoculation of dead cultures or small 
 doses of living bacteria. One attack 
 confers protection against subsequent 
 infections. Specific bacteriolysins and 
 agglutinins are found in the serum of 
 immunized animals, which are of 
 great importance in making a bac- 
 teriological diagnosis of the true or- 
 ganism. For protective inoculation 
 of man, see ''Cholera Vaccine." 
 
 The organism is aerobic and facultative 
 anaerobic. It grows between 22° C. 
 and 40° C, with an optimum of ZTY2° 
 C. The method of isolation is ac- 
 complished by inoculating a set of 
 gelatin plates, a set of agar plates 
 and a set of Dunham's peptone tubes 
 with the suspected material. If pres- 
 ent in great quantities, they may be 
 picked up from the plate colonies. 
 When less numerous, they may be 
 found in the topmost layers of the 
 peptone broth after 8 or 10 days at 
 371/^° C, from which plate dilutions 
 can be prepared and the colonies 
 picked up and identified by means of 
 cultural and agglutinative tests. 
 
 The organism grows on all the ordinary 
 culture media of moderate alkalinity. 
 Slight acidity will not, however, pre- 
 vent growth. In gelatin plates at 
 room temperature, yellowish grey pin 
 head colonies appear within 24 hours. 
 The colonies increase in size; the 
 gelatin becomes liquefied. Magnified 
 the colonies appear coarsely granular 
 with irregular margins. In the 
 gelatin stab, the liquefaction is fun- 
 nel shaped. Upon agar plates, grey- 
 ish opalescent colonies appear within 
 18 to 24 hours. These are easily 
 ""differentiated, from other bacteria of 
 the feces, by reason of their trans- 
 
228 BACTERIOLOGY. 
 
 parency. Coagulated blood serum is 
 liquefied. On potato, the growth is 
 heavy and of a brownish color. Milk, 
 the growth is heavy without coagu- 
 lation. In broth, there is a general 
 clouding with pellicle formations. In 
 Dunham's peptone, indol is produced. 
 The organism is not very resistant to 
 drying. Boiling destroys them im- 
 mediately. They are killed after an 
 hour's exposure to a temperature of 
 60° C. The common disinfectants, in 
 very weak solution, will destroy them 
 after a short exposure. The organism 
 frozen in ice may live for three or 
 four days. 
 
 ORGANISMS AZiIiIED TO CHOZkERA 
 SFIBH^IiVM. 
 
 The examination of the stools of per- 
 sons suffering from cholera have re- 
 vealed, in a small percentage of cases, 
 spirilla that somewhat closely re- 
 semble the true cholera organisms, 
 and they are of bacteriological im- 
 portance by reason of the difficulty 
 which they add to the work of dif- 
 
 . ferentiation. Some bear only morph- 
 ological resemblance, while others 
 can be distinguished from the true 
 cholera organism only by the serum 
 reactions and the pathogenicity to 
 animals. 
 
 The Spirillum Metchnikovl was dis- 
 covered by Gamaleia in 1888, in the 
 intestinal contents and the blood of 
 fowls dying of an infectious disease, 
 which prevailed in certain parts of 
 Russia during the summer months, 
 and which presents symptoms resem- 
 bling fowl cholera. It is identical 
 with the spirillum of cholera in its 
 morphological and staining reactions. 
 It possesses a single polar flagella 
 and is actively motile. Culturally, it 
 is similar to the cholera spirillum, ex- 
 cept for more luxuriant growth and 
 more rapid liquefaction of gelatin. It 
 also gives the indol reaction in pep- 
 tone media. Differentiation from the 
 cholera spirillum is made by inocu- 
 lating minute quantities subcutane- 
 ously into pigeons, producing there- 
 by a rapidly fatal septicemia. It is 
 more pathogenic for guinea pigs than 
 is the cholera spirillum. There is no 
 
BACTERIOLOGY. 229 
 
 lysis or agglutination by cholera im- 
 mune serum. 
 
 The Spirillum of Pinkler-Prior was Iso- 
 lated by Pinkler and Prior in 1884 
 from the feces of patients having 
 cholera nostras. It is like the true 
 cholera spirillum, though somewhat 
 longer and thicker and less uniformly 
 curved and not so uniform in dia- 
 meter, the central portion being 
 usually wider than the pointed ends. 
 Culturally, it resembles the cholera 
 spirillum except that its growth is 
 more rapid and thick upon the or- 
 dinary culture media. It does not 
 form indol in peptone solutions nor 
 does it give specific serum reaction 
 with cholera immune serum. 
 
 Tlie Spirillum Massauali was isolated 
 by Pasquale in 1891 at Massauah 
 from a doubtful case of cholera. In 
 its pathogenicity, it closely resem- 
 bles the spirillum Metchnikovi in that 
 it is able to produce septicemia in 
 pigeons. Culturally and morpholog- 
 ically it resembles the cholera spirul- 
 lum. It possesses four flagella and 
 does not give specific serum reaction 
 with cholera immune serum. 
 
 The Spirillum Deneke was isolated from 
 butter by Deneke. It greatly resem- 
 bles the spirillum of Finkler-Prior. 
 It does not produce indol in peptone 
 media. 
 
 ACID FAST GROUP OF ORGANISMS. 
 
 This so-called "acid fast" group of or- 
 ganisms derives their name by the 
 non-permeability of the ordinary 
 stains unless exposed to them for a 
 long time or by heating the solutions. 
 The stain having entered the organ- 
 ism will retain it even when treated 
 with alcohol and strong acids. The 
 acid fast nature seems to depend 
 upon the fatty substances contained 
 within the organisms. 
 
 Tlie Bacillus of Tuberculosis. 
 
 This bacillus was isolated by Koch in 
 1882 and established the etiological 
 relationship of the bacillus to the 
 disease by infecting guinea pigs and 
 other animals with pure cultures of 
 the bacillus, and producing the charac- 
 teristic lesions. Previous to this tne 
 transmission of tuberculous material 
 was accomplished by Klenkce in 1843 
 
230 BACTERIOLOGY. 
 
 and Willeman in 1865 and the tubercle 
 bacillus had been demonstrated in 
 tissue sections by Baumgarten early 
 in 1882. 
 
 The tubercle bacillus is a slender, 
 straight, or slightly curved rod usual- 
 ly rounded at the ends. The diameter 
 may be uniform throughout, thougn 
 more often they appear beaded and 
 irregularly stained. This irregular- 
 ity in staining generally appears in 
 old cultures and may be regarded as 
 vacuola. The bodies of the bacilli 
 may bulge slightly in three or four 
 places, presenting oval or round 
 knobs which take the stain deeply 
 and are very resistant to decolorization. 
 A cell membrane has been described 
 which confers resistance to the or- 
 ganism against drying, etc. It gives 
 a cellulose reaction and the waxy 
 material obtained from the culture by 
 extraction is believed to be contained 
 within it. Branched forms of the or- 
 ganism have been demonstrated by 
 various observers, and by reason of 
 this fact, it is probable that the 
 tubercle bacillus is not a member of 
 the family schizomycetes but belongs 
 to the higher bacteria. 
 
 The bacilli do not stain easily with the 
 ordinary aniline dyes. They must be 
 exposed to the stains for a long time 
 or the stain solution must be heated. 
 After having been stained, however, 
 they are extremely resistant to acids 
 and decolorizations. For methods of 
 staining see section on Staining 
 Formulas. 
 
 Very young cultures are often not acid 
 fast and it is not always possible to 
 demonstrate acid fast bacilli in pus 
 from cold abscesses, in sputum, in 
 serous exudates and in lesions of the 
 lymph nodes which can be shown by 
 animal inoculation to be tuberculant. 
 In this material. Much calls attention 
 to Gram positive granules arranged 
 singly in short chains or irregular 
 clumps, which he believed to be non 
 acid fast tubercle bacilli. There is no 
 doubt as to the truth of his belief in 
 that his work has repeatedly been 
 confirmed. These Gram positive 
 bodies, however, are not of great di- 
 agnostic value as other bacilli form 
 granules of the same appearance. 
 
BACTERIOLOGY. 231 
 
 Small rods and splinters are also 
 found which are Gram positive and do 
 not stain by the carbofuchsin method. 
 Other organisms of the acid fast 
 group, which may be difficult to dif- 
 ferentiate from the tubercle bacillus, 
 are the bacillus of leprosy and the 
 smegma bacillus. By reason of the 
 distribution of the smegma bacillus 
 in feces, urine or even sputum, it be- 
 comes necessary to apply to sus- 
 pected specimens the other stains 
 which are devised for the differenti- 
 ation of this bacillus from that of 
 tuberculosis. Pappenheim's stain is 
 the one most frequently employed for 
 thiis purpose. Stained by Pappen- 
 heim's method the tubercle bacilli re- 
 main red; the smegma bacilli appears 
 blue. Tubercle bacilli are Gram posi- 
 tive. When tubercle bacilli are pres- 
 ent in extremely small numbers in 
 the sputum and other material, it 
 may be impossible to find them by 
 direct examination, and often the 
 only method of demonstrating them 
 will be by animal inoculation. Meth- 
 ods of concentration have been de- 
 vised by which the bacilli may be 
 found when only a few are present. 
 One method is to add hydrogen per- 
 oxide to the sputum. This dissolves 
 the mucous and allows the solid 
 particles to settle by centrifugation. 
 Another method, much used today, is 
 by the use of "antiformin," which ia 
 made up of equal parts of liquor 
 soda chlorinated (sodium carbonate 
 
 ^ 600 parts, chlorinated lime 400 parts 
 and distilled water 4,000 parts) and 
 a 15% solution of caustic soda. The 
 sputum is pored into a 10 to 15% so- 
 lution of antiformin and allowed to 
 stand for several hours. The other 
 elements of the sputum, as the cells 
 and bacteria, will be dissolved out, 
 leaving only the acid fast bacteria in 
 the residue. The tubercle bacilli are 
 not killed by this process and after 
 sufficient washing, they may be cul- 
 tivated or can produce lesions in 
 guinea pigs. 
 
 The organism produces in man and 
 susceptible animals, a specific phe- 
 nomenon of inflammatory foci, known 
 as tubercles. Tuberculosis is the 
 most common disease in man. ISTagel 
 
232 BACTERIOLOGY. 
 
 in a large series of autopsies found 
 lesions of healed or active tubercu- 
 losis in a large percentage of cases. 
 The disease is less common in the 
 rural districts than in large towns. 
 The pulmonary infection is the most 
 common type in man, though tuber- 
 culosous process may be found in the 
 skin, the bones, the joints, the or- 
 gans of special sense, the abdominal 
 viscera, and peritoneum. Infection 
 takes place by inhalation or through 
 the skin, or through the digestive ap- 
 paratus. Behring has caused a great 
 deal of discussion by stating that he 
 believed a large percentage of all 
 cases of tuberculosis originated in 
 childhood by way of the intestinal 
 tract. He therefore brought to notice 
 the problem of the virulence of bovine 
 tubercle bacilli for human beings, as 
 he assumed that the infection of 
 children is due to the use of infected 
 milk. From the contributions of 
 Parke and Krumweide, it would seem 
 that human adults are relatively In- 
 susceptible to bovine infection which 
 may take place, but is unusual. 
 
 A more relative susceptibility is found 
 under 16 years of age, and the danger 
 of milk infection is without doubt 
 great; in fact, one third of the cases 
 arising from this source. The danger 
 of bovine tuberculosis is greatest 
 under 5 years of age. 
 
 The above statement would indicate 
 that Behring's original statement 
 cannot be upheld, though it does, 
 without doubt, point to the great 
 dangers of milk infections. 
 
 The tubercle bacillus (human) is patho- 
 genic for guinea pigs; less so for 
 rabbits and still less so for dogs. It 
 is slightly pathogenic for cattle. 
 
 The work connected with the isolation 
 of specific toxins has led to a chem- 
 ical analysis of the organism, which 
 shows it to consist of about 8Sy2% 
 of water; 20 to 26% of the residue 
 can be extracted with ether and al- 
 cohol. This material consists of fat- 
 ty acids and waxy substance (fatty 
 acids in combination with higher al- 
 cohol). The residue, after the al- 
 cohol ether extraction, is made up 
 chiefly of proteids, which can be ex- 
 tracted with dilute alkaline solutions 
 
BACTERIOLOGY. 233 
 
 and consist principally of nucleo al- 
 bumens, a fraction of which Is sus- 
 pected of being the pathogenic prin- 
 cipal of the bacillus in that it shows 
 high toxicity. The remainder con- 
 tains cellulose, representing probably 
 the frame work of the cell membrane 
 and the ash which is rich in calcium 
 and magnesia. 
 
 (For the toxins and their method of 
 preparation see Tuberculins). Num- 
 erous attempts have been made to 
 passively immunize tubercular sub- 
 jects with the sera of immune an- 
 imals. The methods most used for 
 the production of such serum is that 
 of Maragliano, who believes that 
 there is a toxic albumin in the cul- 
 tures of the tubercle bacilli, which is 
 destroyed by the heating employed in 
 the usual production of tuberculins. 
 He therefore prepares his substance 
 by filtering unheated cultures and 
 precititates the filtrates with alcohol. 
 He now makes a watery extract of 
 the bacillary bodies and with these 
 two substances he immunizes horses. 
 After 4 to 6 months of treatment, he 
 withdraws blood from the horse and 
 separates the serum. This serum 
 called Maragliano's serum is exten- 
 sively used in Italy. Its value is 
 very doubtful. 
 
 Marmorex claims that the toxin pro- 
 duced by the bacillus of tuberculosis 
 is dependent to a great extent upon 
 the medium on which It Is grown. 
 He believed that the substance ob- 
 tained in tuberculin was not true 
 toxins of the bacillus and that the 
 true toxins could only be elaborated 
 by a younger phase of the bacillus, 
 as it occurs within the animal body 
 or on media composed of animal tis- 
 sue. He therefore grows his cultures 
 on a medium of leucotoxic serum and 
 liver tissue. Such cultures he be- 
 lieves to contain no tuberculant. The 
 sera produced by immunization with 
 these cultures is supposed by him to 
 have high curative powers. 
 
 The organism is aerobic with a temper- 
 ature range of 30° to 42° C. with an 
 optimum of 37%. The organism is 
 not easily cultivated. Isolation from 
 tuberculous material is materially 
 aided by inoculation into guinea pigs. 
 
234 BACTERIOLOGY. 
 
 The animals often withstand the acute 
 infection produced by contaminating 
 organisms and in 4 to 6 weeks die 
 from the tuberculous infection. The 
 bacilli may then be cultivated from 
 the lymphnodes or other foci which 
 contain only tubercle bacilli. Koch 
 isolated the organism from the 
 sputum as follows: — 
 The sputum is thoroughly washed in 
 running water to free it from mucus. 
 It is then washed in 8 or 10 changes 
 of sterile water. The material for 
 cultivation is taken from the center 
 of the washed mass, if possible. 
 Select the caseous material, which is 
 often present in such sputum, and 
 either inoculate culture media direct- 
 ly or inoculate animals as indicated 
 above. On blood serum, at the end 
 of 8 to 14 days, small, dry, greyish 
 white, scaly colonies with corrugated 
 surfaces appear. At the end of 3 to 
 4 weeks' cultivation these colonies 
 joined together cover the surface of 
 the medium as a dry, whitish, wrin- 
 kled membrane. The organism will 
 grow well on agar slants to which 
 
 1 to 2 cc. of rabbit's blood has been 
 •added. Glycerine agar is a favorable 
 
 media for its growth. Glycerine 
 bouillon of a slightly alkaline reac- 
 tion is an excellent culture medium. 
 This medium is placed in thin layers 
 into wide mouthed flasks and then in- 
 oculated by carefully floating flakes 
 of the culture upon the surface. The 
 organism will appear as a thin, 
 opaque, floating film, which rapidly 
 thickens into a white, wrinkled, or 
 granular layer, spreading out in all 
 directions and covering the entire sur- 
 face of the bouillon in from 4 to 6 
 weeks. Later, portions of the mem- 
 brane will sink to the bottom. The 
 organism will also grow freely upon 
 glycerine potato. 
 The life of cultures kept in a favorable 
 environment (access of oxygen) is 
 from 2 to 8 months. In sputum, they 
 remain alive and virulent for 6 weeks, 
 and in dried sputum for more than 
 
 2 months. 
 
 In fluid media, the organism is killed 
 at a temperature of 60° C. in from 
 15 to 20 minutes, and at 80° C. in ^ 
 minutes; at 90" C. in from 1 to 
 
BACTERIOLOGY. 235 
 
 minutes. They withstand dry heat at 
 a 100° C. for 1 hour. They are re- 
 sistant to cold; 5% carbolic acid will 
 kill the organism in a few minutes. 
 If used as a disinfectant for sputum 
 by reason of the fact that the mucous 
 coat will protect the bacilli, the dis- 
 infectant should be allowed to act for 
 5 to 6 hours. Direct sunlight will 
 kill the organism in a few hours. 
 
 The Bacillus of Bovine Tuberculosis. 
 The difference between the reaction to 
 the infection in the bovine type of the 
 disease and that of man was studied 
 by Koch in his early work. He 
 thought this difference to be due to 
 the nature of the infected subjects. 
 Theobal Smith, in 1898, pointed out 
 the difference between the bacilli iso- 
 lated from man and those isolated 
 from cattle. He determined that the 
 bovi^p bacilli were usually shorter 
 than the human bacilli and they did 
 not grow as well upon artificial media. 
 Grown upon slightly acid glycerine 
 bouillon, the bovine organism will re- 
 duce the acidity until a neutral or 
 slightly alkaline reaction is reached. 
 It differs in this respect from the 
 human bacillus, which produces but 
 slight reduction of the acidity during 
 the first week of growth. After this, 
 the acidity increases and never reaches 
 neutrality. Marked differences have 
 also been shown to extgt in the patho- 
 genicity of these org'anisms towards 
 various animal species. Guinea pigs 
 die more quickly and show more ex- 
 tensive lesions when inoculated with 
 the bovine type than when inocu- 
 lated with the human bacillus. The 
 bovine bacillus will kill a rabbit with- 
 in 2 to 5 weeks. The human bacillus 
 produces a mild disease, which lasts 
 frequently for 6 months, and at times 
 fails to kill a rabbit at all. 
 
 Many attempts, with little or no suc- 
 cess, have been made to infect cattle 
 with the human organism. Infection 
 of the human individual with the 
 bacillus of the bovine type has been 
 reported and proven by Smith, Parke, 
 Krumweide and others. 
 
 Bacillus of Avian. Tuberculosis. Koch 
 discovered bacilli in the lesions of dis- 
 eased fowl, which closely resembled 
 the bacillus of tviberculosis. Nocard 
 
236 BACTERIOLOGY. 
 
 and Roux differentiated these bacilli 
 as a definite species. In its staining 
 characteristics and in its morphology, 
 it is almost identical with the human 
 bacillus of human tuberculosis. Cul- 
 turally, it differs, however, in that 
 growth is more rapid and takes place 
 at a temperature of 41*' to 45" C. 
 Guinea pigs are very resistant to this 
 type, while rabbits die quickly from 
 the avian tuberculosis. Prolonged 
 cultivation and passage through the 
 mammalian bodies will cause the or- 
 ganism to approach the mammalian 
 type. Nocard has, on the other hand, 
 succeeded in rendering the mam- 
 malian tubercule bacilli pathogenic 
 for fowl by keeping them enclosed in 
 celloidln sacs within the periton- 
 eum of chickens for 6 months. 
 TulberciQosis of Cold Blooded Animals. 
 A bacillus resembling the bacillus of 
 tuberculosis in its morphology and 
 acid fastness was isolated by Dubarre 
 and Terre from cold blooded animals, 
 as fish, frogs, turtles and snakes. It 
 is non-pathogenic to warm blooded 
 animals but will kill a frog within 4 
 weeks. It grows at a temperature 
 'from 15° to 30° C. Attempts have 
 been made to show a close relation- 
 ship between the tubercle bacillus of 
 cold blooded animals and that of 
 warm blooded animals. Kuster, re- 
 viewing the ^work of many investi- 
 gators, states that spontaneous tuber- 
 culosis may occur in fish, snakes, 
 turtles and frogs, and that the organ- 
 ism which causes these diseases is 
 specific for cold blooded animals and 
 similar in many respects to the 
 tubercle bacillus of warm blooded an- 
 imals, but in them they do not pro- 
 duce progressive disease. The human, 
 bovine and avian tubercle bacilli 
 when inoculated into cold blooded an- 
 imals can produce lesions which sim- 
 ulate tuberculosis and the bacilli may 
 remain in these lesions for a long 
 period of time without losing their 
 pathogenicity for guinea pigs. 
 
 BACII^Iil RESEMBIVING TUBEBd^E 
 BACII^IiZ. 
 
 Bacllltis of Timothy. This organism 
 was isolated by Muller from timothy 
 grass and dust in haylofts. Will grow 
 
BACTERIOLOGY. 237 
 
 rapidly on ag"ar and is of a deep red 
 or dark yellow color. 
 Baclllxis Butyricus (Butter Bacillus): 
 This organism was isolated by Petri, 
 Korn and others from milk and but- 
 ter. It resembles the bacillus of tu- 
 berculosis in that it is slightly acid 
 fast but can be differentiated cultur- 
 ally. It is slightly pathogenic for 
 guinea pigs but not for man. 
 
 ZkUSTGABTEN'S BACIIiZ.US. 
 
 Bacillus Smegmatis. 
 
 Lustgarten, in 1884, discovered an acid 
 fast bacillus in syphilitic lesions 
 which he believed to be the cause of 
 this disease. It has since been shown 
 that a bacillus which corresponds in 
 its morphology but differing slightly 
 in certain staining peculiarities to the 
 bacillus described by Lustgarten, oc- 
 curs as a harmless saprophyte in the 
 normal smegma from the prepuce or 
 vulva. The bacillus is a straight or 
 ciirved organism resembling the tu- 
 bercle bacillus in its morphology. It 
 is found lying singly or sometimes in 
 groups within the interior of cells 
 having a round, oval or polygonal 
 form and apparently somewhat 
 swollen. It stains with almost as 
 much difficulty as the tubercle bacillus, 
 but is more easily decolorized. A 
 method of differentiation was devised 
 by Pappenheim (see stain) which de- 
 pends upon the fact that prolonged 
 treatment with alcohol and rosolic 
 acid will decolorize the smegma bacil- 
 lus but not the tubercle bacillus. 
 
 Attempts have been made, without suc- 
 cess, to cultivate Lustgarten's bacillus 
 on artificial media. The smegma 
 bacillus has no pathogenic signi- 
 ficance. Attempts to infect animals 
 have been unsuccessful. It might be 
 well to state here «that although the 
 so called smegma bacillus and the 
 bacillus of Lustgarten are almost 
 Identical, the identity of the two 
 bacilli has not been definitely estab- 
 lished. 
 
 I^EFBOSY BACZZ^l^US. 
 
 Bacillus ILeprae, 
 
 The Bacillus of Leprosy was discovered 
 by G. A. Hansen, in 1879, in the tis- 
 sues of the nodular lesions of indi- 
 
238 BACTERIOLOGY. 
 
 viduals sufferingr from leprosy. The 
 organisms were found lying in small 
 clumps intra and extracellularly, as 
 well as in the serum that oozed from 
 the tissue during its removal. The 
 disease was present and widely dis- 
 tributed long before the Christian 
 era, extending down through the 
 Middle Ages to the present time. It 
 is most common in India and China. 
 It is found in Norway, Russia and 
 Iceland; and in the United States in 
 Louisianna, California and Minnesota. 
 The bacillus of leprosy is a small, 
 slender, acid fast rod, resembling the 
 tubercle bacillus in form, though 
 somewhat shorter and not so fre- 
 quently curved. The rods have point- 
 ed ends, and when stained have the 
 unstained spaces frequently seen in 
 the tubercle bacilli. The organism 
 stains easily with the ordinary aninine 
 dyes, also by Gram's. 
 Attempts to inoculate animals with 
 leprosy have been unsuccessful. Sub- 
 cutaneous inoculations of cultures 
 into guinea t>ig"S have produced local 
 lesions which resemble the leprous 
 'lesions in man. Duval states that he 
 was able to continue the growth on 
 later transfers. The bacilli are usual- 
 ly found in large numbers especially 
 in the tubercles of the skin, in the 
 conjunctiva and cornea, the mucous 
 membrane of the mouth, gums and 
 larynx, and in the interstitial pro- 
 cesses of the nerves, testicles, liver, 
 spleen and kidneys. The bacilli 
 nearly always lie within the cells in 
 the old ceilters of infection, are 
 larger and often polynuclear. Some 
 observers have claimed to have found 
 giant cells similar to those of tuber- 
 culosis in the nodules. The hair fol- 
 licles, sebaceous sweat glands, are 
 often attached. A true caseation does 
 not occur, but ulceration results. 
 In the anaesthetic form of leprosy, the 
 bacilli are most comonly found in the 
 nerves and less frequently in the skin. 
 The organism may also occur in the 
 blood, partly free and partly within 
 the leucocyte, particularly during the 
 febrile stage, which precedes the 
 reaking out of the tubercles. The 
 acilli have also been found in the 
 
BACTERIOLOGY. 239 
 
 intestines, in the lungs and in the 
 sputum, but not in the urine. ' 
 
 The contagiousness of leprosy is far 
 less than that of most other bacterial 
 diseases. The question of direct in- 
 heritance of the disease from the 
 mother to the unborn child has 
 brought out a considerable difference 
 of opinion. Many attempts have been 
 made to infect healthy individuals 
 with the material containing the ba- 
 cilli without conclusive results. Arn- 
 ing, although he successfully infected 
 a condemned criminal in the Sanj^- 
 wich Islands with fresh leprous tu- 
 bercles, did not produce positive evi- 
 dence of the transmissibility of the 
 disease in that way, as Swift pointed 
 out that the man had other oppor- 
 tunities of becoming infected. A wide- 
 spread idea before the discovery of 
 the organism that the disease was 
 associated with the constant eating of 
 dried fish, or certain kind of food, has 
 been abandoned. 
 
 The relation of leprosy to tuberculosis 
 is evidenced by their similarity in 
 many respects, and still more so by 
 the fact that leprosy will react both 
 locally and generally to injections of 
 tuberculin in the same manner as 
 tuberculosis, but to somewhat less 
 extent. 
 
 Cultivation of the leprosy bacillus has 
 not met with success. 
 
 Clegg, in 1908, reported that he had been 
 able to cultivate an acid fast bacillus, 
 from cases of leprosy, in symbiosis 
 with ameba and cholera vibrio. By 
 heating a symbiotic culture of the 
 leprosy bacillus to 60° C. it was ob- 
 tained in pure culture. From the first 
 culture, different media were success- 
 fully inoculated. On agar, the surface 
 colonies are small and brownish. 
 Blood serum is liquefied after ten 
 days; lactose is not fermented. Duval 
 succeeded in obtaining cultures by the 
 Clegg method, but in spite of ex- 
 tensive work along this line, the 
 opinions are still divided as to the 
 specific nature of the organisms cul- 
 tivated by these two investigators. 
 
240 BACTERIOLOGY. 
 
 BACII.Z.US OF BAT I^EFBOSY. 
 
 A disease occurring- spontaneously among 
 house rats in Odessa was first ob- 
 served by Stefansky, characterized by 
 subcutaneous induration, swelling of 
 lymphnodes, falling out of the hair, 
 emaciation and sometimes ulceration. 
 In the diseased rats, under the skin 
 of the abdomen or flank, a thickened 
 area of adipose-like tissue, though 
 more nodular and grey and less shiny 
 than fat, may be found, within which 
 are acid fast bacilli resembling the 
 bacillus leprae. These organisms may 
 also be found in the lymphnodes and 
 sometimes in small nodules of the 
 liver and lung. 
 
 The disease can be transmitted from rat 
 to rat experimentally. The disease is 
 not exactly like human leprosy clin- 
 ically. 
 
 PATHOGEKIC ANAEROBIC GBOUF 
 OF OBGANISMS. 
 
 BACII^I^US TETANI. 
 
 The infectious nature of lockjaw, or 
 tetanus, was demonstrated by Carlo 
 and Rattone, in 1884. Kitasato by 
 'anaerobic methods demonstrated the 
 tetanus bacillus in 1889, and definitely 
 solved the etiology of ' the disease. 
 The bacillus of tetanus is found to 
 occur in the superficial layers of the 
 soil. The soil of cultivated and ma- 
 nured fields is thickly sown with this 
 organism, probably because of its 
 presence in the dejecta of some 
 doniestic animals. 
 
 The organism is a slender bacillus, the 
 vegetative forms of which are slight- 
 ly motile by reason of the numerous 
 peritrichial flagella. The organism 
 develops spores which are character- 
 istically located at one end and give 
 to it the diagnostic drum stick ap- 
 pearance. The vegetative forms oc- 
 cur chiefiy in young cultures, which, 
 after 24 hours' incubation, develop 
 into the spore forms, and as the cul- 
 tures grow old, the spores will super- 
 sede the vegetative form. In very old 
 cultures only spore forms and spores 
 are found. The organism stains easily 
 with aniline dyes and also by Gram. 
 The organism is extremely patho- 
 genic, though viewed from the stand- 
 
BACTERIOLOGY. 241 
 
 point of universal distribution of the 
 bacilli in nature, the infection is in- 
 frequent. The spores of the organism 
 introduced into the animal body, free 
 from toxins, the disease may not be 
 produced, by reason of their suscep- 
 tibility to destruction by phagocytosis 
 and to other protective agents before 
 vegetative forms can develop and the 
 toxin formed. Tetanus will, however, 
 develop in animals if introduced into 
 deep, lacerated wounds in which there 
 has been considerable tissue destruc- 
 tion. Common pus cocci or other 
 more harmless parasites may aid in 
 furnishing a suitable media for the 
 growth of the tetanus bacillus. There 
 is an incubation period of from 5 to 
 7 days in acute cases to from 4 to 5 
 weeks in chronic ones. Guinea pigs 
 inoculated with the organism sufCer 
 in from 1 to 3 days from a rigidity 
 of the muscles nearest the point of 
 infection, which rapidly spreads to 
 other parts of the body and is 
 followed by death in 4 or 5 days after 
 the time of injection. Autopsies ot 
 animals or human beings, dead of 
 tetanus, do not present marked lesions. 
 The point of infection may be triffling 
 in appearance. The organs show no 
 pathological changes except a general 
 moderate congestion. The bacilli are 
 infrequently found at the point of in- 
 fection and have rarely been demon- 
 strated in the blood or viscera. Tiz- 
 zoni and Creite have succeeded in cul- 
 tivating the tetanus bacillus from the 
 spleen and heart's blood of infected 
 human beings. The pathogenicity of 
 the organism is dependent upon the 
 soluble toxin which it produces. It 
 is one of the most powerful poisons 
 known. Filtrates of broth cultures in 
 quantities of 0.000.005 cc. will often 
 prove fatal to mice of 10 gm. weight. 
 Different species of animals show 
 great variation in susceptibility. 
 Human beings and horses are prob- 
 ably the most susceptible species in 
 proportion to their body weight. The 
 susceptibilty of the horse calculated 
 for grams of body weight is twelve 
 times that of a mouse. The hen Is 
 extremely resistant, in fact 200,000 
 times more resistant than the mouse. 
 The toxin injected subcutaneously 
 
242 
 
 BACTERIOLOGY. 
 
 first produces spasms in the muscles 
 nearest the point of inoculation. In- 
 travenous inoculation usually results 
 in a general spasm of all the muscles. 
 The action of the tetanus toxin is 
 upon the central nervous system. The 
 manner in which the toxin reaches the 
 central nervous system is by way of 
 the motor nerve. The toxin Injected 
 into the circulation will reach all the 
 motor nerve endings simultaneously, 
 producing a general tetanus. The 
 poison cannot pass directly into the 
 central nervous system but must 
 follow the route of nerve tracts (see 
 also antitetanic serum). 
 The bacillus of tetanus is an obligative 
 anaerobe and if cultivated strictly 
 under these conditions, it will grow 
 readily upon meat infusion broth 
 which becomes clouded in 24 to 36 
 hours. Upon meat infusion gelatin, 
 there is slow liquefaction. On agar, 
 at the end of 48 hours, compact col- 
 onies not unlike subtilis colonies make 
 their appearance. In agar stabs, the 
 growth appears as fine, radiating pro- 
 cesses grown from the central stab. 
 Milk is a favorable media and is not 
 * changed. On potato, a hardly visible 
 growth appears. The media may be 
 rendered more favorable for growth 
 by the addition of a 1 or 2% of 
 glucose, maltose or sodium formate. 
 The vegetative forms of the bacillus 
 are not more resistant against heat or 
 chemical agents than the vegetative 
 forms of other microorganisms. The 
 spores, however, will resist dry heat 
 at 80° C. for 1 hour, live steam for 
 about 5 minutes. They are killed by 
 a 5% carbolic acid solution in 12 to 
 15 hours; 1% of bichloride of mer- 
 cury in 2 to 3 hours. Direct sunlight 
 will diminish their virulence and 
 ultimately destroy them. 
 
 BACIIiI^nS OF SYMPTOMATIC 
 ANTHRAX. 
 
 An infectious disease occurring among 
 sheep, cattle and goats, spoken of as 
 "Quarter-evil" or "Blackleg" is often 
 confused with true anthrax by reason 
 of a slight similarity in clinical symp- 
 toms. It is, however, caused by a 
 very different microorganism found 
 widely distributed in the soil, from 
 
BACTERIOLOGY. 
 
 243 
 
 which the infection is generally ob- 
 tained. The bacillus of symptomatic 
 anthrax is a spore bearing bacillus of 
 drum stick shape, or spindle shape, 
 and is anaerobic. It was first ob- 
 tained in pure cultures by Kitasato. 
 It is usually seen singly and never 
 forms chains. In the vegetative form 
 the organism is motile, but soon loses 
 this power on account of the oxygen 
 to which it is exposed. It shows well 
 defined flagella and develops spores. 
 The organism may be demonstrated 
 by the aid of microscope in the blood 
 without staining, if done soon after 
 death. It stains easily by the sim- 
 ple aniline dyes or by Gram's. 
 
 The bacilli are pathogenic for cattle, 
 sheep and goats. Most cases appear 
 among cattle. Guinea pigs are very 
 susceptible. Horses, little suscepti- 
 ble; dogs, cats, rabbits and birds are 
 immune. Man appears to be abso- 
 lutely immune. Infection takes place 
 through abrasions or wounds, and de- 
 pends to some extent upon the degree 
 of virulence, which varies greatly in 
 this organism. A soft, puffy, swelling 
 appears at the point of entrance at 
 from -Z to 24 hours after the inocu- 
 lation. This area is found to be 
 emphysematous, which spreads rap- 
 idly, often reaching the abdomen and 
 chest in a day. It is accompanied 
 with high fever and extreme general 
 prostration. Death usually results 
 within 3 to 4 days. At autopsy, the 
 swollen area is found infiltrated with 
 a thick blood tinged and foamy exu- 
 date. The subcutaneous tissues and 
 muscles are edematous and filled with 
 gas. The organs show parenchy- 
 matous degeneration and hemorraghic 
 areas. The organisms are found in 
 enormous numbers in the area sur- 
 rounding a central focus, immediately 
 after death, but very few are found 
 in the blood and internal organs. The 
 unburied carcasses become bloated 
 with gas and the organs filled with 
 bubbles, by reason of the general 
 distribution of the bacilli. 
 
 A soluble toxin is produced by the or- 
 ganism in considerable quantities in 
 broth containing blood or albuminous 
 animal fiuids, but not to any extent 
 in ordinary broth. The toxin is quite 
 
244 BACTERIOLOGY. 
 
 resistant to heat, but deteriorates on 
 exposure to air. 
 
 Arloing actively immunized cattle by 
 subcutaneous inoculation with vac- 
 cines prepared from the tissue of in- 
 fected animals. Two vaccines are 
 prepared; No. 1 is made from the 
 juice of infected meat, which is dried 
 and heated to 100" C. for six hours. 
 No. 2 is also made from the juice of 
 infected meat heated to 90° C. for 
 six hours. No. 1 is injected in quan- 
 tities of 0.01 to 0.02 cc, emulsified in 
 sterile salt solution, near the end of 
 the animal to be protected. The same 
 quantity of No. 2 is injected in the 
 same way after 14 days. 
 
 Kitt prepared a vaccine from the powd- 
 ered meat which was heated to 94° C. 
 for six hours, which has been largely 
 used in America. A passive immuni- 
 zation with the serum of actively im- 
 munized sheep has been used in com- 
 bination with the methods of active 
 immunization. 
 
 The organism grows readily under an- 
 aerobic conditions upon the ordinary 
 media, which may be rendered more 
 favorable by the addition of glucose 
 glycerine or nutrose. The organism 
 will grow equally well on slightly 
 alkaline or slightly acid medium and 
 is an active gas producer. On agar 
 plates the colonies appear round with 
 a compact, slightly granular center 
 from which a thin zone is given off. 
 This zone will under the microscope 
 appear as a tangle of fine threads. In 
 agar stabs, the growth appears with- 
 in 18 hours and spreads into the 
 media as a diffused fine cloud. Gas 
 bubbles are formed which later in- 
 crease to such extent as to cause ex- 
 tensive splitting of the medium. On 
 gelatin plates, round or oval colonies 
 with a compact center, about which 
 are fine, radiating filaments, make 
 their appearance in about 24 hours. 
 The gelatin is liquefied. In gelatin 
 stab cultures the growth Is less rapid, 
 but is similar to the growth in agar 
 stabs. 
 
BACTERIOLOGY. 245 
 
 BACni^US OF MAI^IGKANT EDEMA. 
 (Bacillus Oedamatis Maligul). 
 
 Pasteur in 1877 described an organism 
 which he isolated under anaerobic 
 conditions in impure cultures from 
 the tissues of guinea pigs and rabbits 
 which he had inoculated with putrefy- 
 ing animal tissues. He named it 
 **Vibrion septique." Koch, in 1881, sug- 
 gested the term "bacillus of malignant 
 edema," by reason of the fact that the 
 organism did not produce a true sep- 
 ticemia. The organism was later 
 found to occur in garden soil and in 
 dust by Gaffky. The bacillus of ma- 
 lignant edema is a long, slender rod, 
 resembling somewhat the bacillus of 
 anthrax. It occurs singly, but fre- 
 quently appears in long threads with 
 irregular subdivisions; or without 
 subdivision as long homogeneous fila- 
 ment. The organism is motile, pos- 
 sessing .numerous laterally placed 
 flagella. Motility is often absent. 
 Oval spores are found irregularly 
 placed in the center or slightly nearer 
 the end and cause a bulging of the 
 body. It stains readily by the usual 
 aniline dyes but is decolorized by 
 Gram. 
 
 The organism seems to be pathogenic 
 for all animals. Subcutaneous inocu- 
 lation of pure cultures produces an 
 acute edematous inflammation at the 
 point of inoculation within 24 to 36 
 hours. This edema spreads through- 
 out the subcuticular and deeper layers 
 and consists of a thin fluid which is 
 slightly bloody. The lymphnodes be- 
 come enlarged and hemorrhagic. Gas 
 is formed causing subcutaneous em- 
 physema. The toxemia is general, and 
 in small animals the disease is usual- 
 ly fatal. At autopsy the organisms 
 are found in the edema about the 
 local lesion. The organism is not 
 found in the blood or internal organs 
 shortly after death, though later they 
 may become distributed throughout 
 the body. The internal organs show 
 parenchymatous degeneration with oc- 
 casional hemorrhages. 
 
 Infection with malignant edema Is rare. 
 It has been seen in horses, in cattle 
 and in sheep. Infection generally 
 takes place after tramua, and in man 
 
246 BACTERIOLOGY. 
 
 secondarily, after compound frac- 
 tures, or upon the site of suppurating 
 wounds. 
 
 A recovery from the infection will pro- 
 duce immunity. Immunity may also 
 be produced by the injection of solu- 
 ble toxin in bacteria-free filtrates of 
 the organism. Very small amount of 
 this toxin is capable of killing guinea 
 pigs. Immunity can also be produced 
 by injection with the toxic filtered 
 sera of animals dead from the disease. 
 
 The bacillus of malignant edema may 
 be cultivated under strict anaerobic 
 conditions upon any of the ordinary 
 media. The addition of .glucose to the 
 media will favor its growth. Gases 
 are produced through proteid cleav- 
 age. On gelatin plates, small, grey, 
 spherical colonies, with microscopic 
 radial filaments, appear in about 3 
 days. The gelatin is liquefied. In the 
 gelatin stab, growth appears along 
 the entire stab to within a short dis- 
 tance of the surface. Processes de- 
 velop laterally with a formation of 
 gas. In agar, the growth appears as 
 a white line along the entire length 
 of the stab, which takes on a lateral 
 * cloud-like extension, and in the pres- 
 ence of sugar, bubbles will be formed 
 throughout the medium. In broth, 
 there is general clouding and a gran- 
 ular precipitate. Milk is slowly co- 
 agulated. 
 
 BACIXiI^irS AEBOGENES 
 CAFSUIiATUS. 
 
 The Bacillus Aerogrenes CapsulatTiB is 
 
 found in soil, dust and brackish water 
 and in the intestine of human beings 
 and animals. They were first ob- 
 served by Welch in 1892, having ob- 
 tained it during autopsy from the 
 blood of a case of ruptured aortic 
 aneurysm. His attention was called 
 to the blood by reason of air bubbles 
 appearing throughout the vessels. 
 The organism appears as a straight rod 
 of varying length, and somewhat 
 thicker but not unlike the anthrax 
 bacillus. On rare occasions the or- 
 ganism may be slightly curved, and 
 still rarer it may be so short as to 
 appear almost coccoid. The organism 
 generally appears singly with rounded 
 ends, but may appear as short chains 
 
BACTERIOLOGY. 247 
 
 when the ends are almost square. The 
 chain formation takes place in the 
 blood. Long chains are never seen in 
 artificial culture media which dis- 
 tinguishes the organism from the 
 bacillus of anthrax. Spores are seen 
 in blood serum cultures, rarely upon 
 plain agar and never in the animal 
 body. They are oval in form and 
 may be centrally or polarly located. 
 It is non motile and enclosed by a 
 capsule which cannot always be 
 demonstrated. The capsules are best 
 seen when preparations are made 
 from animal fluid, though they may 
 be seen in specimens grown upon arti- 
 ficial culture media. It is stained 
 readily by the ordinary aniline dyes 
 and by Gram, if taken from tissues. 
 If taken from artificial culture media, 
 they are partially decolorized by Gram 
 by reason of involution forms. 
 The bacillus is highly pathogenic for 
 guinea pigs but very slightly so for 
 rabbits. The virulence varies with 
 the strain. In general, its patho- 
 genicity for laboratory animals is 
 slight. The bacillus has been isolated 
 from numerous cases of emphy- 
 sematous gangrene in man, which is 
 characterized by a rapidly necrotizing 
 inflammation accompanied by subcu- 
 tanecrus emphysema. Infection, when 
 it takes place, follows trauma, especi- 
 ally compound fractures. The organ- 
 ism has also been found in the uterus 
 in puerperal infection. It has also 
 been found in infectious processes of 
 gastrointestinal and biliary tracts, the 
 lungs, the pleura and the meninges. 
 *The organism may be isolated from 
 mixed cultures by animal inoculation. 
 About 1 cc. of the suspected material 
 is emulsified with about 5 cc. of sterile 
 salt solution and filtered through 
 sterile paper. 1 to 2 cc. of this sus- 
 pension is injected into the ear vein 
 of a rabbit. The rabbit is then killed 
 and placed in the incubator for five 
 to eight hours, at the end of which 
 times the carcass will be found to 
 be distended with gas. Autopsy will 
 show gas to be distributed as bubbles 
 ' throughout the organs. Prom these 
 bubbles, the organism may be taken 
 for indentification or culture. The or- 
 ganism is grown under obligatory an- 
 
248 BACTERIOLOGY. 
 
 aerobic conditions upon any of the 
 usual media of a neutral or slightly 
 alkaline reaction. The addition of glu- 
 cose or lactose will favor the growth. 
 Upon plate cultures a flat grayish, 
 translucent, round colony with slight- 
 ly irregular and fringed margin ap- 
 pears within 24 hours. Gelatin is 
 slowly liquefied, but occasionally 
 liquefaction does not occur. In sugar- 
 ed agar stabs or slants, there is a 
 rapid formation of gas which is con- 
 sidered of diagnostic value. In broth, 
 there is general clouding, and with- 
 in 40 hours a heavy, white flocculent 
 sediment is formed. Froth appears 
 upon the surface of the broth tubes, 
 if undisturbed, due to the formation 
 of gas. On potato, the growth is 
 scanty. On coagulated blood serum, 
 the growth is heavy with slight pep- 
 tonization of the medium. Milk is 
 rapidly coagulated and rapidly acidi- 
 fied. Gas is formed. 
 
 THE BACIUUS ENTEBITIDIS 
 SFOROGENES. 
 
 This organism closely resembles the 
 bacillus aerogenes capsulatus. It is 
 • a spore bearing organism usually 
 present in the intestinal tract of man. 
 It is found in sewage, milk, dust and 
 food stuffs, such as wheat, oatmeal, 
 rice, etc. Cline believed that it pro- 
 duced diarrhea when taken in milk. 
 This fact is disputed by many. 
 
 BACII^I^US BOTUIiINUS. 
 
 The Bacillus Botulinus was discov- 
 ered by van Ermengen, in 1896, who 
 isolated the organism from a ham, 
 the eating of which had caused dis- 
 ease in a number of persons. He 
 found the bacilli within the muscle 
 fibers of the ham in great numbers 
 and was also able to cultivate the 
 same microorganism from the stomach 
 and spleen of one of the individuals 
 who ^ied from the infection. The 
 bacillus is a large, straight rod with 
 rounded ends, appearing singly or 
 grouped in very short chains. It is 
 slightly motile and develops oval 
 spores which are situated near the 
 end of the bacillus, on rare occasions 
 at the middle. It is stained readily 
 by the ordinary aniline dyes also by 
 
BACTERIOLOGY. 249 
 
 Gram, if care be taken that there is 
 not over decolorization with the al- 
 cohol. 
 
 Ingestion of meat infected with this 
 org-anism produces the botulism or 
 allantiasis in man. After a period of 
 incubation of from 24 to 48 hours, the 
 symptoms are: chilliness, trembling, 
 giddiness, followed by headache, occa- 
 sionally by vomiting. The chief diag- 
 nostic symptoms are due to the tox:ic 
 interference with the cranial nerves, 
 producing loss of accommodation, 
 dilated pupils, ptosis, aphonia and 
 dysphagia, etc. Fever is usually ab- 
 sent. Consciousness is rarely lost. 
 These symptoms difCer from the meat 
 poisonings caused by other micro- 
 organisms. 
 
 Animals inoculated with living cultures 
 or toxins will show the symptoms in- 
 dicated above. Guinea pigs seem to 
 be the most susceptible. They are 
 killed by injections of minute quan- 
 tities of the toxin. Before death, 
 which occurs within 24 to 36 hours, 
 general motor paralysis, dyspnea, and 
 hypersecretion of mucus from nose 
 and mouth may occur. 
 
 Autopsies show a general hyperemia of 
 the organs with much parenchyma- 
 tous degeneration and many minute 
 hemorrhages. 
 
 The poisoning occuring in man is due 
 to the toxins that have been formed 
 by the bacillus in the ingested meat. 
 It has been shown that little or no 
 toxin is produced by the bacillus after 
 it has been introduced into the ani- 
 mal body. It produces the disease by 
 the absorption of the toxin that is 
 secreted by the organisms. This toxin 
 Is active not only when injected sub- 
 cutaneously, but also when intro- 
 duced through the gastrointestinal 
 canal. A specific antitoxin has been 
 produced by Kempner. 
 
 The organism is easily cultivated under 
 strict anaerobic conditions in the or- 
 dinary culture media of a neutral or 
 moderately alkaline reaction and at 
 a temperature not exceeding 35" C. 
 On gelatin, the growth is rapid and 
 abundant with the formation of gas 
 and rapid liquefaction. On glucose 
 gelatin plates, the colonies appear as 
 round, yellowish, transparent spots 
 
250 BACTERIOLOGY. 
 
 surrounded by a zone of liquefaction. 
 On agar plates, the colonies are yel- 
 lowish, opalescent and round in shape, 
 with a finely fringed border. Stab 
 cultures in glucose agar produce a 
 thin white growth along the line of 
 stab which does not reach the surface 
 of the medium. The medium is split 
 by the formation of gas. Milk is not 
 coagulated. Disaccharides and poly- 
 saccharides are not fermented. The 
 gas that is formed in cultures, chiefly 
 hydrogen and methane. 
 
 KEMOBBKAGIC SEPTICEMIA GROUP 
 OP ORGANISMS. 
 BACTERIUM PESTIS. 
 (Bacillus of Bubonic Plargnie). 
 
 The bacterium of bubonic plague was 
 described by Yerdin and Kitasato in 
 1893, independently of each other, and 
 is now recognized as the etiological 
 factor in bubonic plague, epidemics 
 of which have been recognized since 
 the second century. About half the 
 population of the Roman Empire died 
 in the sixth century, and during the 
 fpurteenth century an epidemic (The 
 Black Death) swept over Europe and 
 killed about twenty-five million peo- 
 ple. About two million died of this 
 disease in India during 1901 to 1904. 
 Smaller epidemics have appeared in 
 numerous parts of the world, as 
 China, Egypt and South Africa. 
 
 The organism is a short, thick bacillus 
 with well-rounded ends. It is non- 
 motile, and appears singly, in pairs 
 and occasionally in short chains. The 
 organism shows distinct polar stain. 
 The size of the organism varies, and 
 in old cultures involution forms may 
 appear either as club-shaped diph- 
 theria-like bacilli or as swollen 
 coccoid forms. The involution forms 
 are of diagnostic importance by rea- 
 son of their irregularity, and seem to 
 be more numerous in artificial media. 
 There are no spores present, and cer- 
 tain observers have demonstrated a 
 erelatinous capsule. Occasionally, 
 branched forms may be observed. It 
 stains easily with aniline dyes, par- 
 ticularly, at ^he poles. It is Gram 
 negative. 
 
BACTERIOLOGY. 251 
 
 The organism is extremely pathogenic 
 for rats, mice, guinea pigs, rabbits 
 and monkeys. Even insects die from 
 the infection. Among animals, the 
 disease has been found chiefly in rats 
 and squirrels. Dogs may occasionally 
 become infected. Two distinct types 
 are observed clinically, depending 
 upon the mode of infection, which 
 takes place by the entrance of organ- 
 ism through the skin or by the res- 
 piratory tract. In the cutaneous 
 infection, which may take place 
 through the most minute lesion, the 
 disease is first localized in the nearest 
 lymphnodes, and from these primary 
 dwellings the bacilli may enter the 
 blood and produce secondary foci. If 
 entrance has taken place through the 
 respiratory tract, a pneumonia is pro- 
 duced which usually proves fatal 
 within four or five days. Cardiac de- 
 pression is a very characteristic 
 symptom of systemic infection. The 
 diagnosis may be made during life by 
 finding the bacilli in the aspirated 
 fluid from a bubo, or from the 
 sputum. Identification is made mor- 
 phologically, culturally, animal inocu- 
 lation and agglutination tests. 
 
 At autopsy, in man, the bacilli are 
 found in the primary lesion in the 
 blood and in the spleen, the liver and 
 lymphatics. Hemorrhages into the 
 serous cavities may be found. The 
 pneumonic type is usually of the 
 bronchopneumonic variety, with ex- 
 tensive swelling of the bronchial 
 lymphnodes. When the disease has 
 been prolonged, tubercle-like foci may 
 be found in the spleen, liver and lung. 
 
 The typical lesions found in rats, who ' 
 play an important role in the spread 
 of the disease, becoming infected 
 from the cadavers of plague victims 
 or the eating of the bodies of rats, 
 dead from the disease, are engorge- 
 ment of the subcutanous vessels and 
 a pink colorization of the muscles. 
 The bubo when present is suflacient 
 for diagnosis. The area siirrounding 
 the buboes are markedly injected and 
 sometimes hemorrhagic. A pleural in- 
 fusion is present. The liver has 
 undergone a fatty change. The spleen 
 is large, friable and often presents 
 pinpoint granules on the surface. 
 
252 BACTERIOLOGY. 
 
 The systemic symptoms are due to the 
 absorption of the toxin, which are of 
 the endotoxin and also of a true 
 soluble toxin variety. 
 
 A single attack will immunize. The 
 antibodies developed are agglutinins, 
 bacteriolytic and possibly antitoxin. 
 The agglutinins are of importance in 
 diagnosis. Active immunization is 
 accomplished by the inoculation of 
 the whole dead bacteria. The serum 
 of immunized animals has been tried 
 as a therapeutic agent and gives en- 
 couraging results when administered 
 early. 
 
 The bacillus is isolated in pure culture 
 from the lesion during life or at 
 autopsy. The organism grows read- 
 ily, best upon meat infusion of a 
 neutral or moderate alkaline reaction 
 and at a temperature of 30* C. It 
 will grow at temperatures ranging 
 from 20*' to 38° C. On agar, minute 
 colonies with a compact center sur- 
 rounded by an irregular, indented, 
 granular margin appears within 24 
 hours. On gelatin, colonies like those 
 upon agar appear after two or three 
 days. It is not. liquefied. In bouillon, 
 t^he organism grows slowly, sinking 
 to the bottom or adhering to the 
 sides of the tube as granular deposit. 
 Occasionally a delicate pellicle is 
 formed. Milk is not coagulated. In 
 litmus milk, there is slight acid 
 formation. No indol is formed upon 
 peptone. 
 
 The organisms are eliminated in the 
 exudates from surrounding buboes 
 and from the sputum in the pneu- 
 monic type and are present through- 
 out the body after death; therefore, 
 the dead bodies of human beings and 
 of rats are sources of infection for 
 other rats, which become chronic car- 
 riers of the disease; and even though 
 showing no symptoms themselves, 
 they must necessarily be important 
 factors in the maintenance and 
 spread of the disease. 
 
 If in a dark and moist environment, the 
 organism may live outside of the 
 body for months and even years. In 
 cadavers, they may live for weeks 
 and months, if protected from dry- 
 ness. Complete drying will kill the 
 organism in two or three days, and 
 
BACTERIOLOGY. 253 
 
 dried artificially, they may be killed 
 within four or five hours. A dry heat 
 of 100° C will kill the organism in one 
 hour, boiling the organism will kill it 
 in a few minutes. They are very re- 
 sistant to cold. Direct sunlight 
 destroys them within four or five 
 hours. They are not very resistant 
 to antiseptics. 
 Prophylaxis consists in isolating in- 
 fected animals, followed by thorough 
 disinfection, involving even the kill- 
 ing of fleas and the destruction of 
 rats, squirrels or other animals which 
 may serve as carriers. Haffkine's 
 vaccination method has also been 
 shown to be a valuable prophylactic 
 measure. 
 
 BACTEBIUM TUI^ABEINSZ!. 
 
 McOoy has described a disease which 
 occurs in the California ground squir- 
 rel which closely resembles, so far as 
 the lesions are concerned, the plague 
 infection of these animals. McCoy 
 was able to transmit plague-like 
 lesions in most animals inoculated 
 with infected material, and later, in 
 1912, McCoy and Chapman isolated 
 the bacterium on an egg medium. 
 The organism is small and often cap- 
 sulated, staining poorly with methy- 
 lene blue, better with carbo fuchsin 
 or gentian violet. 
 
 BACTERIUM AVISEFTICUS. 
 (Bacillus of Chicken Cholera). 
 
 The bacillus of chicken cholera was 
 discovered in the blood of infected 
 animals by Pasteur in 1880. This 
 • disease is widely prevalent in chick- 
 ens, ducks, geese and a large variety 
 of smaller birds. The organism is a 
 short non-motile rod, staining easily 
 with analine dyes. It is decoLorized 
 by Gram. It often appears as a 
 diplococcus by reason of marked polar 
 staining qualities. Spores are not 
 formed. Occasionally vacuolated 
 forms, not unlike those of tl^e bacillus 
 pestis, occur. The infection of birds 
 is extremely acute, accompanied by 
 diarrhea, often bloody stools, great 
 exhaustion, drowsiness, and ending 
 fatally within a few days. Autopsy 
 shows hemorrhagic infiltration of the 
 
254 BACTERIOLOGY. 
 
 intestinal mucosa, enlarg-ement of the 
 liver and spleen and frequently a 
 bronchopneumonia. 
 
 The bacilli may be found in the blood, 
 in the organs and in the exudates, if 
 present, and in large numbers in the 
 dejecta. It grows well upon the ordi- 
 nary culture media at a temperature 
 of 25° to 40° C. Broth is clouded uni- 
 formly with a later formation of a 
 pellicle. Upon agar, minute white or 
 yellowish, first transparent, later, 
 opaque colonies appear within 24 to 
 48 hours. No liquefaction of gelatin. 
 No coagulation of milk. No gas is 
 formed in sugar broth, which, how- 
 ever, becomes acid, and in peptone, 
 indol is formed. 
 
 Infection takes place probably through 
 food and water contaminated by dis- 
 charges of diseased birds. The feed- 
 ing or subcutaneous inoculation with 
 cultures, even in the most minute 
 dose, will produce a quickly develop- 
 ing septicemia which is uniformly 
 f3.tal. 
 
 The bacillus is extremely pathogenic 
 for rabbits, if subcutaneous inocula- 
 tions are made; less so for hogs, 
 sheep and horses. The disease does 
 not follow the ingestion of infected 
 material by these animals. 
 
 BACTERIUM SUIS£FTICUS. 
 (Bacillus of Swine Plague). 
 
 The bacillus of swine plague causes 
 an epidemic disease among hogs char- 
 acterized by a bronchopneumonia, 
 which is followed by a general sep- 
 ticemia. A pleural exudate of a sero- 
 sanguineous nature, together with 
 enlargements of the bronchial lymph 
 glands, the liver and the spleen, is 
 frequently found. The gastrointesti- 
 nal . tract is rarely involved. At 
 autopsy a nonmotile Gram negative 
 bacillus, almost identical with the 
 bacillus of chicken cholera, may be 
 found in the lungs, in the exudates, 
 in the liver, spleen and in the blood. 
 The disease is seldom acijLte, but is 
 almost uniformly fatal in young pigs. 
 
 Spontaneous infection usually occurs 
 by inhalation. Experimentally the 
 disease has been produced by sub- 
 cutaneous inoculation. Mice, guinea 
 
BA<5tERIOLOGY. 255 
 
 pigs and rabbits inoculated subcu- 
 taneously with small doses of the 
 organism are killed within four or 
 five days. Active and passive im- 
 munization has been successful. 
 Kitt and Mayr have shown that the 
 serum of horses immunized with 
 chicken cholera would sometimes pro- 
 tect against bacillus suisepticus. 
 
 BACTERIUM BOVISZSFTIUM. 
 
 The bacterium boviseptium produces a 
 disease and affects a wide variety of 
 domestic and wild animals. It has 
 been reported from many portions of 
 North America, some sections of 
 South America and many European 
 countries, and is known as Corn Stalk 
 disease, Buffalo disease and pneu- 
 monenteritis, etc. The domestic ani- 
 mals most commonly affected are 
 cattle, sheep, horses and goats. The 
 onset of the disease is sudden and 
 the case acute. It does not spread 
 from herd to herd, but appears in 
 isolated outbreaks. Body infection 
 probably occurs by inoculation. The 
 characteristic lesions found at au- 
 topsy are hemorrhages, which occur 
 subcutaneously under the mucus 
 membrane or under the serous mem- 
 brane and also in the lymph glands. 
 The lesions produced by this bac- 
 terium indicate a general distribution 
 through the body. 
 
 The mortality ranges from 50 to 80 per 
 cent. The acute and rapidly fatal 
 cases, where the autopsy shows only 
 trifling lesions, would indicate the 
 formation of active toxins. 
 
 Very little is known concerning the 
 elimination of this organism from the 
 diseased body, but isolation and dis- 
 infection are to be recommended on 
 general principles. 
 
 The disease resembles anthrax and 
 symptomatic anthrax in some of its 
 characteristics. It may be readily 
 differentiated from either of these 
 diseases by microscopic examination. 
 
 The organism resembles the bacterium 
 of chicken cholera, of rabbit septi- 
 cemia and the bacillus of cholera 
 suisepticus so closely that laboratory 
 differentiations are extremely difficult. 
 The bacterium is small, with rounded 
 ends, closely resembling a diplococ- 
 
256 
 
 BACTERIOLOGY. 
 
 cus. Involution forms may appear. 
 Shows by polar staining, decolorizes 
 by Gram, produces no spores, has no 
 flagella and is nonmotile. 
 
 THE COl^ON-TYFHOID DYSENTERY 
 GBOUF OF BACII^I^I. 
 
 The organisms belonging to this group 
 present great differences in their 
 pathogenic characters, but possess so 
 many points of similarity in their 
 morphological and biological charac- 
 teristics that their differentiation be- 
 comes extremely difficult. The group 
 considered will be the Coll group, the 
 Enteritidis group, the Dysentery 
 group and the Typhosus group. 
 
 The differentiation of these organisms 
 is important in that certain of the 
 bacilli are specifically pathogenic, 
 while others are essentially saphro- 
 phytic and become pathogenic only 
 under exceptional conditions. 
 
 BACIUUS COI^I GBOUF. 
 
 These organisms, sometimes called 
 "Lactose Fermenters," are frequently 
 nonpathogenic, for man but may be- 
 ,come distinctly pathogenic under cer- 
 tain conditions. Their degree of 
 virulence upon inoculation of lower 
 animals varies greatly. 
 
 Motility is not marked, or none. Dex- 
 trose and lactose are fermented with 
 acid production. Milk is quickly 
 coagulated with acid production. 
 Indol is not produced by most va- 
 rieties. 
 
 THE BACIIiI^TJS COZiI. 
 
 Under the name of Colon Bacilli are 
 grouped a number of varieties which 
 differ from one another in minor 
 characteristics, but correspond in cer- 
 tain cardinal points, which warrants 
 their consideration under one heading. 
 
 The Bacillus Coll Commtmls is the most 
 prominent type of the group, and 
 therefore will be chiefly considered. 
 This organism was discovered by 
 Buchner in 1885. It is a constant 
 inhabitant of the intestinal canal of 
 human beings and animals. It is 
 occasionally found in the soil, air, 
 water and in milk. It is, in fact, 
 found in all thickly populated neigh- 
 
BACTERIOLOGY. 257 
 
 borhoods. In man, the bacillus coll 
 appears normally in the intestine, 
 being found in greatest numbers at 
 or about the ileocecal valve diminish- 
 ing from here upward to the duo- 
 denum and downward as far as the 
 rectum. The organism is frequently 
 found in the tissues and in blood after 
 death without visible lesions of the 
 intestinal mucosa. It is probable that 
 it may enter the circulation a few 
 hours before death. Extensive inves- 
 tigations have been carried out to de- 
 termine whether or not the presence 
 of the organism in the intestines 
 possesses a definite physiological 
 function of advantage to its post. 
 This question has not been definitely 
 settled though it might be stated that 
 the function of the organism in the 
 intestine is not inconsiderable if only 
 because of its possible antagonism to 
 certain putrefactive bacteria, as has 
 been demonstrated by Bienstock. The 
 bacillus coli communis is a short 
 rod, varying in thickness from one- 
 third to one-fifth its length. Under 
 certain conditions of cultivation, it 
 may appear more slender or shorter 
 and even coccoid in form. It usually 
 appears singly, but occasionally in 
 short chains. There are no spores. 
 When first isolated from the body, it 
 may be extremely motile, while old 
 laboratory strains of the organism 
 may show almost no motility. Ordi- 
 narily the motility of the colon 
 bacillus is intermediate between these 
 two extremes. Stains readily with 
 the ordinary aniline dyes and is 
 decolorized by Gram. 
 The pathogenicity of the organism for 
 animals is slight and varies with 
 the different strains. If 1 cc. of a 
 bouillon culture is injected intraperi- 
 toneally into guinea pigs, death will 
 often ensue. If large doses are in- 
 jected intravenously into rabbits, 
 symptoms of violent intoxication are 
 presented, followed by death in from 
 24 to 48 hours. Moderate doses 
 inoculated subcutaneously usually 
 produce nothing more than a local 
 abscess, from which the animal re- 
 covers. It is probable that death 
 results from the action of poisons lib- 
 erated from the disintegrating bac- 
 
258 BACTERIOLOGY. 
 
 teria, and not from multiplication of 
 the bacilli themselves. 
 
 In man a largre variety of lesions pro- 
 duced by the organisms have been 
 described. The manner in which the 
 organism becomes pathogenic is not 
 clear. A number of explanations have 
 been advanced. First, that whenever 
 an infection is produced by the 
 bacillus coli, it is produced by one 
 that has been recently acquired from 
 another host; second, that the viru- 
 lence of the organism may be en- 
 chanced by inflammatory processes 
 brought about by other organisms; 
 third, infection may possibly take 
 place by reason of a reduction in the 
 resistance of the host. Whatever the 
 cause for the infection, it is doubtful 
 whether septicemia produced by the 
 colon bacillus is due to an actual pri- 
 mary invasion of circulation by the 
 bacillus, although a few unquestion- 
 able cases have been reported. 
 Diseases, such as cholera nostras and 
 cholera infantum, have been attrib- 
 uted to these organisms, but without 
 being supported with satisfactory evi- 
 dence. It is likely that in most of 
 the intestinal diseases formerly attrib- 
 . uted to these organisms the organism 
 plays but a secondary part. In peri- 
 tonitis following perforation in the 
 intestine, the organism is always 
 present, but can never be found in 
 pure culture, being usually accom- 
 panied by stpahylococci and strepto- 
 cocci and other micro-organisms. It 
 is therefore hard to determine 
 whether or not these bacilli could be 
 considered as a primary cause of peri- 
 tonitis. The organism may give rise 
 to a mild suppurative process, in as 
 much as it is able to proliferate 
 within the peritoneum. Welch re- 
 ports a case of peritonitis in which 
 the bacillus coli was isolated in pure 
 culture. The organism has also been 
 isolated from liver abscesses from 
 the bile and from the center of gall 
 stones, consequently inflammatory 
 conditions have been attributed to it 
 in these situations. 
 
 The organism is found more frequently 
 in the urine than any other organism. 
 It may be present in normal indi- 
 viduals. It is frequently seen during 
 
BACTERIOLOGY. 259 
 
 convalescence from typhoid. It may 
 disappear spontaneously or cystitis 
 may supervene and occasionally an 
 ascending pyonephrosis. The organ- 
 ism may cause localized suppurations 
 in all parts of the body, most fre- 
 quently seen, however, about the anus 
 and the genitals. 
 
 The toxic action of the colon bacillus 
 is due to endotoxins. The injection 
 of gradually increased doses of living 
 or dead colon bacilli will produce 
 specific bacteriolytic agglutinating 
 and precipitating substances. The 
 injection of any specific race of colon 
 bacilli produces in the immunized ani- 
 mal high agglutination value only for 
 the individual cuUure used for im- 
 munization. 
 
 The large number of varieties of colon 
 bacilli described during the early 
 days of bacteriology were, in many 
 cases, based upon a temporary de- 
 pression of one or another cultural 
 characteristic, although some Were 
 undoubtedly closely related. They 
 were, however, distinct groups. 
 
 The constant and distinct varieties of 
 the bacillus coli do not occur. The 
 most common is the 'bacillus coli com- 
 munior (Dunham), and is believed to 
 be more abundant in the human and 
 animal intestines than the coli com- 
 munis itself. It possesses all the 
 cardinal characteristics of the colon 
 group. It differs, however, from the 
 bacillus coli communis, in that it 
 produces acid from the saccahrose as 
 well as from dextrose and lactose; 
 whereas the coli communis does not 
 form acid or gas from saccharose. 
 The Bacillus Coli Communis is an 
 aerobe and facultative anaerobe. It 
 will grow upon all of the ordinary 
 media at a temperature ranging 
 from 20° to 40° C, optimum 37%** C. 
 In broth, general clouding with later 
 pellicle and a light, slimy sediment is 
 formed. Upon agar, gray colonies 
 appear within 12 to 18 hours, which 
 gradually become more and more 
 opaque. The surface colonies often 
 show a characteristic grape leaf 
 structure, or may be round and fiat 
 and show a definitely raised, glisten- 
 ing surface. Upon agar slant, the 
 growth occurs in a uniform layer. On 
 
260 BACTERIOLOGY. 
 
 gelatin, the growth is rapid with no 
 liquefaction. On potato, the growth 
 is abundant and of a gray white, 
 glistening layer which later turns to 
 a yellowish brown, and in old cultures 
 often to a dirty greenish brown. 
 Indol is formed in peptone solution. 
 Milk is acidified and coagulated. In 
 lactose litmus agar, acid is formed 
 and the medium becomes red. Gas 
 bubbles appear along the line of the 
 stab inoculation. Gas is formed in 
 dextrose, lactose and mannite, but not 
 in saccharose. Acid and gas are 
 formed in levulose, lactose and mal- 
 tose. 
 
 The isolation of the colon bacillus from 
 mixed cultures''^ is accomplished by 
 plating upon lactose litmus agar. 
 Cultures of the colon bacillus are 
 characterized by an odor not unlike 
 that of diluted feces. The acids 
 formed by the organism from sugar 
 are lactic, acetic and formic acids. 
 The gas produced is chiefly carbon 
 dioxide and hydrogen. 
 
 BACTERIUM IkACTUS AEBOGENES. 
 (Bacillus Aerog'enes). 
 
 This organism was first isolated by 
 Escherich (1885) from the feces of 
 infants. It is almost constantly pres- 
 ent In milk, and together with one or 
 two other microorganisms, is the 
 chief cause of the ordinary souring 
 of milk. It is also widely distributed 
 in nature in feces, in water and in 
 sewers. It is anaerobe and facultative 
 anaerobe. Distinguished from the 
 colon bacillus in that it is non-motile, 
 very seldom forms chains, and when 
 cultivated in milk it possesses a dis- 
 tinct capsule; also in that it will fer- 
 ment polysaccharides as starch and 
 does not produce indol. 
 
 The organism is but slightly pathogenic 
 to man. It is almost constantly in 
 the human intestines. In infants it 
 may give rise to flatulence and has 
 been known to produce cystitis. In 
 rare instances it has formed gas in 
 the blood. The different strains of 
 the organism vary in their patho- 
 genicity for animals. 
 
 It grows abundantly at a temperature 
 between 25" to 30° C on all the ordi- 
 
BACTERIOLOGY. 261 
 
 nary culture media. Upon agar and 
 gelatin, heavy, white, mucoid colonies 
 appear which have a tendency to con- 
 fluence. In broth, there is general 
 clouding and pellicle formations; a 
 sour or cheesy odor. Upon potato, 
 growth is heavy and gas is formed. 
 Milk, coagulated and acidified. The 
 organism's chief characteristic is that 
 it is. capable of producing a large 
 amount of acid, chiefly lactic, and of 
 being able to withstand this quan- 
 tity of acid without being in- 
 jured. All carbohydrates, except sac- 
 charose, are fermented with the 
 formation of gas. 
 
 BACII^XiUS MUCOSUS CAFSUl^ATXTS. 
 (Friedlauder's Bacillus. Bacterium 
 Pneumonia, Fneumobacillus ) . 
 
 Friedlander's Bacillus, the Bacillus of 
 Rhinoscleroma, the Bacillus of Ozena, 
 etc., together with a number of bac- 
 teria reported as allied to Friedland- 
 er's, mainly upon morphological 
 grounds, are classified together as the 
 "Friedlander group" of organisms. 
 
 This organism was discovered by Fried- 
 lander in 1882, and believed by him to 
 be the cause of lobar pneumonia. 
 He described it as a micrococcus. 
 Researches by Frankel and Weichsel- 
 baum proved this to be a short, in- 
 capsulated bacillus, which occurred 
 in lobar pneumonia on rare occa- 
 sions only. The organism is a short, 
 plump bacillus with rounded ends, 
 varying greatly in size, even in the 
 same culture. In animal and human 
 lesions the organism is almost coccoid 
 in form. It appears singly in diplo- 
 form or in short chains. They are 
 nonmotile. It is surrounded by a cap- 
 sule in animals when taken from ani- 
 mal fluid and sometimes in the smears 
 from agar or gelatin. The capsule is 
 two or three times the size of the 
 bacillus, and when seen in chains or 
 in groups, several bacilli may be en- 
 closed within one capsule. The cap- 
 sule will disappear in prolonged cul- 
 tivation on agar or gelatin. It stains 
 easily with the ordinary stains, but is 
 decolorized by Gram. 
 
 It causes pneumonia probably in about 
 7 or 8 per cent, of all cases in this 
 
262 BACTERIOLOGY. 
 
 country. When causing the disease, 
 the pneumonia is extremely severe 
 and usually fatal. It has been found 
 in ulcerative stomatitis and nasal 
 catarrh. It has been reported as oc- 
 curring- in severe tonsilitis in children, 
 in the pus from suppurations in the 
 antrum of Highmore and nasal 
 sinuses and in cases of ozena, believed 
 by Abel to be the specific cause. It 
 has been found in empyemic fluid, in 
 pericardial exudate and in spinal fluid. 
 Cases of septicemia have been de- 
 scribed, caused by Friedlander's 
 bacillus. It has been believed to be 
 associated with some forms of 
 diarrheal enteritis in that it is an 
 occasional inhabitant of the normal 
 intestine. 
 
 It is pathogenic for mice and guinea 
 pigs, also for rabbits. Inoculated into 
 susceptible animals, it will produce 
 inflammation and death by septicemia. 
 Intraperitoneal inoculatipn, a mucoid 
 stringy exudate is found which is 
 characteristic. 
 
 Immunization with graded doses of 
 dead bacillus has been produced in 
 isolated cases. Specific agglutinins 
 ,in the immune serum have occasion- 
 ally been found, potent only against 
 the particular strains used. The 
 organism is an aerobe and facultative 
 anaerobe and grows easily on all cul- 
 ture media. Temperature ranges 
 from 18° to 40° C, optimum 37 V^'' C. 
 On agar, grayish white mucous light 
 colonies of a slimy, semifluid consis- 
 tency appear. After three or four 
 days a tendency to confluence causes 
 a large part of the surface to be cov- 
 ered with a film of glistening, sticky 
 exudate, which, if fished, comes ofC 
 in a tenacious, stringy matter. In 
 broth, there is rapid abundant growth 
 with pellicle formation. General 
 clouding and later a stringy sediment. 
 Stab cultures in gelatin show at first 
 a white line of growth along the 
 course of the puncture; later, there 
 appears a grayish glucose droplet on 
 the surface, which enlarges and gives 
 the growth a nail-like appearance, 
 which is regarded as diagnostic. The 
 gelatin is not liquefied. On potato, 
 an abundant growth appears of a 
 slightly brown color. There is no 
 
BACTERIOLOGY. 263 
 
 indol formed in peptone solution. In 
 milk, the growth is rapid, with irreg- 
 ular coagulation. All carbohydrates 
 (except lactose) are fermented with 
 formation of gas. 
 
 BACIZil^US OF HHINOSCIiEBOMA. 
 
 The bacillus was discovered by • von 
 Prisch in 1882. It produces a disease 
 called in rhinoscleroma in man, which 
 consists of a slowly grown granu- 
 lomatous inflammation, located at the 
 external larynx, or the mucosa of 
 nose, mouth, pharynx or larynx. 
 Within the interior of the lesions are 
 many large, swollen cells, within 
 which the bacilli lie; also in the in- 
 tracellular spaces. The disease is 
 rare in America. It is slowly pro- 
 gressive and comparatively intract- 
 able to surgical treatment, seldom 
 affecting the general health unless by 
 obstruction to the air passages. The 
 bacillus may be isolated from the 
 lesions. Morphologically and cultur- 
 ally, it is almost identical with Fried- 
 lander's bacillus. It differs from 
 Priedlander's bacillus in forming no 
 gas in dextrose bouillon, and pro- 
 ducing no acid in lactose bouillon and 
 never coagulating milk. 
 
 BACIIkLnS OZENA. 
 
 Abel and others have shown that an 
 organism morphologically and cultur- 
 ally almost identical with the bacillus 
 mucosus capsulatus is nearly always 
 present in ozena. While he states 
 that it does not form gas in dextrose 
 bouillon and is less pathogenic for 
 mice than is the bacillus of Fried- 
 lander, it cannot be definitely stated 
 as to whether it is a separate species 
 or merely an atypical form of the 
 bacillus of Friedlander. 
 
 BACII^IiUS ENTERITIDIS GROUP. 
 
 Most members of this group of organ- 
 isms are under certain conditions dis- 
 tinctly pathogenic for many of the 
 lower animals and for man. The 
 motility is usually marked. Dextrose 
 is fermented with gas formation. 
 Lactose is not fermented. Milk is not 
 coagulated. No indol or only a slight 
 amount is produced (bacillus alka- 
 ligenese does not ferment sugars). 
 
264 BACTERIOLOGY. 
 
 Gartner's discovery in 1888 of the 
 bacillus enteritidis in association 
 with epidemics of meat poisoning 
 gave impetus to the study of a num- 
 ber of bacteria resembling in many 
 characteristics the colon or typhoid 
 bacilli. 
 
 They are often spoken of as "group of 
 intermediates" and classified as inter- 
 mediate between the colon and the 
 typhoid types. 
 
 By reason of the pathological conditions 
 with which they have been associated, 
 the terms "hog-cholera group," "en- 
 teritidis group," "paracolon group" 
 or "paratyphoid group," were applied 
 to the chief members under investi- 
 gation. 
 
 The microorganisms are morpholog- 
 ically indistinguishable from the 
 colon and typhoid bacilli. Patho- 
 genically they have attracted atten- 
 tion in their connection with meat 
 poisoning and with protracted fevers 
 that are indistinguishable from mild 
 typhoidal infections. 
 
 Bacillus Enteritidis (Gartner) was dis- 
 covered and isolated from the meat 
 of a cow, the ingestion of which had 
 • produced the symptoms of acute 
 gastrointestinal catarrh in fifty-seven 
 people. The organism was demon- 
 strated in the spleen and in the blood 
 of one patient who died from the dis- 
 ease. The organism is actively mo- 
 tile, forms no indol and produces gas 
 in dextrose media. If fed to mice, 
 guinea pigs, rabbits and sheep, it will 
 induce acute gastrointestinal symp- 
 toms. The bodies of the organisms 
 contain an extremely toxic substance 
 which differs from the endotoxins of 
 other bacteria in that it is extremely 
 resistant to heat. Sterilized cultures 
 have the same pathogenic effect as 
 the living cultures. 
 
 Bacillus Morselle, described by Van 
 Ermengem in 1891 in an epidemic of 
 meat poisoning at Morselle, differing 
 slightly in minor characteristics, is 
 almost identical with Gartner's bacil- 
 lus. 
 
 Bacillus Psittacosis, isolated by Nocard 
 in 1892 from infections in parrots, 
 showed a close resemblance to Gart- 
 ner's bacillus. 
 
« BACTERIOLOGY. 
 
 265 
 
 Bacillus Typhimurium, isolated by 
 Loeffler, was in 1893 shown to be sim- 
 ilar to Gartner's bacillus and also to 
 the so-called "hog-cholera bacillus," 
 by T. Smith and Moore from their 
 studies of the disease of swine. They 
 first used the term of "hog cholera" 
 group. 
 
 Paracolon and Paratyphoid Group were 
 introduced by Gilbert in 1893 to des- 
 ignate the organisms of this group 
 resembling more nearly the biolog- 
 ical characters, the colon bacillus on 
 the one hand and typhoid bacillus on 
 the other. 
 
 Bacillus bovls morbificans was isolated 
 by Basenan in 1894 in an epidemic 
 of meat poisoning; differing slightly 
 in minor characteristics, it is almost 
 identical with Gartner's bacillus. 
 
 Paracolon Bacillus was isolated by 
 Widal and Nobecourt in 1897 from 
 an esophageal abscess following 
 typhoid fever. This organism showed 
 a close resemblance to Gartner's 
 bacillus, and following the Gilberts' 
 suggestion they named it "B. para- 
 colon." 
 
 Gwyn, in 1898, isolated an organism 
 from the blood of a patient who pre- 
 sented all the symptoms of typhoid 
 fever, but the patient's serum did not 
 have any agglutinating power for the 
 bacillus typhosus. Its culture char- 
 acteristics were similar to those of 
 Gartner's bacillus which was agglu- 
 tinated by the patient's serum. He 
 called it "paracolon bacillus." The 
 paracolon and paratyphoid can be dis- 
 tinguished without difficulty from the 
 typhoid bacillus. They produce gas 
 in gl\icose media, and in this respect 
 they differ from typhoid, but, unlike 
 B. coli, they do not produce gas from 
 lactose, coagulated milk, or, as a rule, 
 from indol. 
 
 Agglutination tests applied to the in- 
 termediates show that the members 
 of the paracolon group do not all 
 show mutual reactions, and the 
 group, like the B. coli, is therefore 
 composed of a number of distinct 
 races. The paratyphoids, most of 
 which have been isolated from cases 
 simulating typhoid fever, belong 
 chiefly to two strains. An active 
 serum prepared from either strain of 
 
266 BACTERIOLOGY. 
 
 the bacilli will agglutinate all the 
 others of that strain. They are des- 
 ignated as type A and type B. 
 
 A similar organism was isolated by 
 Gushing, in 1900, from a costo- 
 chondral abscess during convalescence 
 from typhoid fever. 
 
 En,cillus Icteroides, associated by Sana- 
 relli with yellow fever, was shown by 
 Reed and VarroU, in 1899, to be cul- 
 turally similar to the bacillus of hog- 
 cholera. 
 
 Bacillus Paratyphoid, Schottmuller, in 
 1900, isolated bacilli from five cases 
 which corresponded to bacilli pre- 
 viously described. 
 
 Cultural and agglutination studies of 
 the organisms obtained showed that 
 they could be divided into two sim- 
 ilar yet distinctly different types; one 
 of them very close to the typhoid 
 type (B. Paratyphoid); the other, 
 closer to the Gartner bacillus. 
 
 Type A has been isolated from the nor- 
 mal intestines of animals by Morgan 
 and is not considered very important 
 as a causative agent of human dis- 
 ease. Kutscher therefore suggests 
 that, except in rare instances, this 
 •organism is a nonsaphrophtic para- 
 site. Type B not infrequently pro- 
 duces an infection. 
 
 Clinically, the diseases caused by this 
 class of bacteria may be divided into: 
 
 Group 1. Those which fall into the 
 category of meat poisoning (Para- 
 colon) more like those due to B. 
 Gartner, having sudden, violent onset 
 of gastrointestinal symptoms directly 
 following the ingestion of meat, and 
 characterized by profound toxemia. 
 
 Group 2. Those in which the disease 
 simulates a mild form of typhoid 
 fever, lasting from twelve to 
 eighteen days, and differing only 
 by the absence of the specific agglu- 
 tination reaction for typhoid bacilli. 
 
 Bacillus Alkalig'enes. This bacillus re- 
 sembles somewhat a colon bacillus 
 which has lost its power to ferment 
 sugars. Morphologically and cultur- 
 ally it, is more like the typhoid 
 bacillus. It ferments no sugars. It 
 is frequently present in the intestines 
 and may have pathogenic properties. 
 
 Bacillus Cliolerae Suis (Bacillus of Hogr- 
 Cholera). This organism is an ac- 
 
BACTERIOLOGY. 267 
 
 tively motile bacillus; grows readily 
 in bouillon; renders milk at first 
 slightly acid, then strongly alkaline; 
 dissolves casein and ferments dex- 
 trose with acid production. It is 
 found almost regularly present in 
 hogs, sick of cholera, but is not the 
 essential cause of the disease. Al- 
 though it is not an essential factor 
 in exciting hog-cholera, it is believed 
 to be of importance as an added in- 
 fection. It is pathogenic for hogs, 
 causing, when fed, a fatal enteritis. 
 Theobald Smith and Moore, in 1893, 
 studied this disease and noted a great 
 similarity between the organism, the 
 bacillus of the Gartner group and the 
 bacillus typhi murium isolated by 
 Loeffler. 
 
 Bacillus of Swine Plague. This is a 
 nonmotile bacillus which grows 
 feebly in bouillon. Does not coagu- 
 late milk and ferments glucose with- 
 out production of gas. When fed to 
 pigs it does not usually cause illness. 
 
 It is closely related to the hemorrhagic 
 septicemic group. 
 
 THE BACUZiUS DYSENTERY 
 GROUP. 
 
 This group of organisms, often 
 grouped with bacillus typhosus are 
 pathogenic for man and by inocula- 
 tion less pathogenic for animals. 
 The organisms are nonmotile, fer- 
 ment dextrose without formation of 
 gas, do not ferment lactose, do not 
 coagulate milk nor produce indol. 
 The bacillus dysenteria (Shiga) does 
 not ferment mannite. The bacillus 
 paradysenteria (Park) ferments mal- 
 tose and mannite. The paradysen- 
 teria (Plexner) ferments mannite 
 only. 
 
 Bacillus Dysenteria (Shigra). The etiol- 
 ogy of dysentery was obscure until 
 Shiga (1898) found a bacillus in the 
 stools of patients suffering with 
 dysentery which had not before been 
 identified. It was present in all 
 the cases of epidemic dysentery ex- 
 amined but was not found in the stools 
 of healthy persons. 
 
 The blood of dysenteric patients agglu- 
 tinated the bacilli which were iso- 
 lated, but the organisms were not 
 agglutinated » to any such degree by 
 
268 BACTERIOLOGY. 
 
 serum from healthy individuals. The 
 org-anism is a short rod similar to the 
 colon group of org^anisms. Stains easily 
 with the aniline dyes with the ends 
 showing a tendency to deeper stain- 
 ing than the center. 
 
 It decolorizes by Gram's. No spores 
 or flagella have been demonstrated. 
 On gelatin the colonies appear more 
 like the typhoid than the colon 
 bacilli. It is not liquefied. On agar 
 the colonies resemble those of the 
 typhoid bacilli. 
 
 On potato, a delicate, scarcely visible, 
 brownish growth is formed. In bouil- 
 lon, a diffuse cloudiness is formed 
 with a slight deposit appearing 
 after some days. Occasionally a 
 pellicle is formed. 
 
 Litmus Milk becomes a pale lilac after 
 24 hours, which returns to the orig- 
 inal color after three to eight days. 
 Neutral red agar is not changed. 
 
 It does not form indol, except, perhaps, 
 in intestine, or ferment mannite, mal- 
 tose or saccharose. Animals injected 
 with this type of org-anism produce 
 specific ag-glutinins which only in a 
 small way combine ' with the other 
 type of the group. 
 
 In man, the organisms produce an acute 
 dysentery with symptoms of cramps, 
 diarrhoea and tenesmus. The stools, 
 at first feculent, then seromucous, 
 become bloody or composed of coffee- 
 ground sediment. At the height of 
 the disease there are ten to fifty 
 stools in the twenty-four hours. The 
 blood usually disappears after from 
 two to seven days. The disease is 
 especially limited to the mucous mem- 
 brane of the large intestines. The 
 vessels of the surfaces appear con- 
 gested and prominent. The mucous 
 membrane is covered with a yellowish 
 mucous and seems to be absent in 
 places. The solitary follicles are en- 
 larged, especially in the sigmoid 
 flexure, and in some instances are 
 depressed and appear to be necrotic 
 in their center. Microscopically, the 
 mucous glands are normal except 
 over the solitary follicles, where they 
 are slightly broken down and contain 
 polynuclear leukocytes. The capil- 
 laries of the follicles are extremely 
 congested. The submiicosa is thick- 
 
BACTERIOLOGY. 269 
 
 ened and slightly edematous. The 
 connective tissue cells have under- 
 gone a slight hyaline degeneration. 
 The deeper coats of the intestine are 
 not involved. 
 
 The small intestine seems to be slightly 
 distended. The mesenteric glands are 
 large and red. Peyer's patches are 
 swollen slightly, but without ulcera- 
 tion. Microscopically, the mucous 
 membrane appears normal. 
 
 In the severe cases the entire lumen 
 of the intestines may be filled with a 
 pseudomembrane of a diphtheretic 
 character. In young children the 
 lesions appear to be more superficial 
 even in fatal cases. 
 
 Animals injected intravenously with 
 the organism show symptoms of 
 diarrhoea and paralysis, which is fol- 
 lowed by death. Animals are likewise 
 very sensitive to killed cultures. 
 Autopsies on animals killed from in- 
 jections into the peritoneum of living 
 or dead bacilli show a hyperemic peri- 
 toneum, the cavity of which is more 
 or less filled with serous or bloody 
 serous exudate. The liver may be cov- 
 ered with a fibrinous mass, the small 
 intestine filled with fluid, the large 
 intestine usually empty, and the 
 mucous membrane of both hyperemic 
 and may sometimes be hemorrhagic. 
 Subcutaneous injections of the dead 
 or living organisms produces infiltra- 
 tions of tissue and frequently abscess 
 formation. The organism produces 
 both an extracellular and a cellular 
 toxin. 
 
 Bacillus Paradysentery (Farke). "A." 
 
 In 1902, Parke and Dunham described 
 an orgariism which they found in a 
 small epidemic of dysentery occuring 
 in men which differed somewhat from 
 the organisms previously described, 
 in that it produces endol. The organ- 
 isms ferment mannite with the pro- 
 duction of acid, but does not act 
 upon maltose or saccharose. Animals 
 injected with this organism develop 
 immune bodies and agglutinins that 
 are specific for this type. 
 Bacillus Paradysentery (Plexner). "B." 
 Flexner in 1899, while investigating 
 the dysentery in the Philippines, iso- 
 lated an organism which corresponds 
 to Shiga's organism, but differs from 
 the other in that it produces endol. 
 
270 BACTERIOLOGY. 
 
 ferments mannite and acts strongly 
 upon maltose and feebly upon saccha- 
 rose. This type of organism is near- 
 est to the Colon group. 
 
 The two mannite fermenting types are 
 widely scattered over the world and 
 caused epidemics of dysentery of a 
 milder type than that produced by 
 the Shiga organism and have been 
 described by many investigators. 
 These two types have also been 
 described at times in mixed infec- 
 tions where dysentery symptoms are 
 almost or entirely absent. 
 
 Passive immunization of animals and 
 human beings with the serum of im- 
 munized horses has been attempted 
 by Shiga, Kruse and others, who have 
 reported a reduction of mortality by 
 the use of such sera. Todd has dem- 
 onstrated the neutralization of the 
 solutions of toxin by an immune 
 serum. 
 
 By reason of the different varieties of 
 dysentery bacilli, polyvalent sera is 
 of considerable value. It is given 
 subcutaneously in 20 cc. doses once 
 or twice a day for several days, or 
 until convalescence is established. 
 
 BACUI^US TYPHOSUS GROUP. 
 
 The Bacillus Typhosus is an actively 
 motile organism pathogenic for man 
 and less pathogenic by inoculation for 
 lower animals. Dextrose is fermented 
 without gas formation; lactose not 
 fermented; milk not coagulated and 
 no formation of indol. 
 
 The organism was discovered by Ebert 
 (1880) in the spleen apd diseased 
 areas of the intestine of typhoid 
 cadavers. 
 
 It was obtained in pure culture by 
 Gaffky in 1884. 
 
 The organism is a short plump rod 
 having rounded ends. Under favor- 
 able conditions it is actively motile. 
 The degree of motility varies in dif- 
 ferent cultures. The flagella are 
 preipherally arranged of twelve or 
 more in number. Many of the shorter 
 forms have but a single terminal 
 flagellum. 
 
 The bacillus stains a little less in- 
 tensely with the ordinary aniline dyes 
 than do most other bacteria. Bipolar 
 
BACTERIOLOGY. 271 * 
 
 staining is sometimes marked and 
 decolorized by Gram's. 
 
 The organism is aerobic and facultative 
 anaerobic bacillus developing best at 
 37° C. Its growth is retarded above 
 40° C and below 30° C. Below 10° C 
 its growth almost ceases. It does not 
 form spores. Some of the bacilli are 
 killed within a few hours when dried, 
 though a few will remain alive for 
 months under the same conditions. 
 Usually they are killed by an ex- 
 posure to 60° C for one minute. 
 
 In natural water it remain^ alive for 
 36 days (Klein). 
 
 In ice it may remain alive for three 
 months (Prudden). 
 
 It is killed by 1-500 bichloride or 5% 
 carbonic acid within five minutes. 
 
 Upon agar plates, small grayish col- 
 onies appear within 18 to 24 hours. 
 
 These colonies are first transparent, 
 later they become opaque. Upon 
 agar slants, the transparent, filiform 
 grayish streak is formed. 
 
 Upon gelatin plates, characteristic irreg- 
 ular (grape-leaf) transparent, bluish- 
 white colonies appear. Magnified, they 
 are of homogenous structure marked 
 by a delicate network of furrows. 
 
 As the colonies grow older they grow 
 heavier, become more opaque and lose 
 their early differential value. 
 
 In gelatin stab cultures the growth is 
 mostly on the surface as a thin scal- 
 loped extension which gradually 
 reaches the sides of the tube. In the 
 stab proper there is but a limited 
 growth of yellowish brown color. 
 Gelatin is not liquefied. 
 
 Bouillon is uniformly clouded. When 
 the medium is slightly filkaline a 
 delicate pellicle may be formed after 
 18 to 24 hours' growth. 
 . On potato, a characteristic almost in- 
 visible growth appears after 24 to 48 
 hours which usually covers the sur- 
 face of the medium, though it may be 
 restricted to the point of inoculation. 
 The growth may also, however, be 
 quite heavy and of a yellowish-brown 
 color with a greenish halo like that 
 of B. Coli. Milk is not coagulated. 
 The neutral violet color In litmus 
 whey becomes more red during the 
 first 48 hours; the fluid remaining 
 clear. 
 
272 BACTERIOLOGY. 
 
 In Dunham's peptome solution there is 
 no production of indol. 
 
 The organism does not produce gas 
 when grown in dextrose, mannite, lac- 
 tose and saccharose Droth, but it does 
 produce acid in dextrose, levulose, gel- 
 actose, mannite, maltose and dextrin 
 broth. 
 
 In shake or stab cultures of Rothberg- 
 er's neutral red no change is pro- 
 duced while the colon group reduce 
 the red, decolorize the media and pro- 
 duce gas. 
 
 When inje\3ted into animals, no typicai 
 pathological changes are ' produced. 
 The sickness or fatal results after 
 such injections can be attributed to 
 the toxemia produced by the endo- 
 toxins liberated from the dead bac- 
 teria. 
 
 Animals inoculated subcutaneously by 
 bacilli, freshly obtained from typhoid 
 cases, rapidly die. 
 
 In the peritoneal cavity they may in- 
 crease with the production of a fatal, 
 peritonitis. If the bacilli are accus- 
 tomed to the animal body the viru- 
 lence may be so increased as to prove 
 fatal to the animal when injected 
 .with very small cultures. 
 
 The organism produces in man an in- 
 fectious disease in which the organ- 
 isms pass into the blood, and by this 
 channel they pass to all parts of the 
 body and become localized in the tis- 
 sue such as the bone marrow, lym- 
 phatic tissues and spleen, liver and 
 kidneys. 
 
 The lesions of the intestine consist of 
 an inflammatory enlargement of the 
 solitaryjs and agminated lymph 
 nodules. In the more severe cases 
 the hyperplasia is frequently followed 
 by ulceration and necrosis. 
 
 Ulceration and sloughing may involve 
 the muscular and peritoneal coats 
 with the production of perforation. 
 Peritonitis and death usually follows, 
 though in rare instances adhesions 
 may close the perforation. 
 
 The mesenteric lymph nodes undergo 
 changes like those of the ilium. The 
 spleen is enlarged by reason of con- 
 gestion and hyperplasia. The liver 
 and to less extent the kidney are apt 
 to show foci of cell proliferation. 
 
BACTERIOLOGY. 273 
 
 The typhoid bacillus may in rare cases 
 act as "pus producer." 
 
 The complications occuring in typhoid 
 fever are usually due to secondary 
 or mixed infections with the sta- 
 phylococcus, pneumococcus, strepto- 
 coccus, pyocyanens and colon bacillus. 
 
 The organism is present in the urine 
 of typhoids in about 20% of cases 
 during the third or fourth week. 
 When pneumonia is caused by B. 
 typhosus it can be found in the spu- 
 tum. 
 
 During typhoid fever the organism is 
 always found in the gall bladder. The 
 organism usually disappears from the 
 body in the fourth or fifth Week, but 
 may remain for months or years in 
 the urine and throughout life in the 
 gall bladder. 
 
 Abscesses have been found one year 
 after recovery from typhoid fever. 
 
 One to five per cent, of individuals hav- 
 ing had typhoid continue to pass 
 typhoid bacilli for years, maybe for 
 life. 
 
 A number of so-called typhoid carriers 
 have been reported which if not de- 
 tected are very dangerous * as con- 
 stant spreaders of typhoid fever. The 
 treatment of these cases has not been 
 satisfactory. Medicinal treatment 
 and immunization have yielded slight 
 results. 
 
 Recovery from typhoid fever produces 
 an immunity which may last for 
 years, except in about 2% of cases. 
 The second attack in these, however, 
 is usually of a mild type. 
 
 Serum of animals immunized possesses 
 bactericidal and feeble antitoxic prop- 
 erties against B. typhosus, and an 
 attempt has been made to treat 
 typhoid by this method, and although 
 good results have been reported by a 
 number of men the majority have 
 found little or no value in its use. 
 
 Where danger of typhoid infection 
 exists, the use of protective vaccines 
 advocated by Wright are indicated. 
 (See typhoid vaccine.) 
 
 Vaccination durin-g the course of the 
 fever has been advised by certain 
 individuals, but the results obtained 
 by Richardson does not show any 
 effect except that relapses seem to 
 be less. 
 
274 BACTERIOLOGY. 
 
 For the diagnosis of typhoid see the 
 Gruber-Widal reaction (Widal test). 
 
 DISEASES DUE TO THE HIGHER 
 BACTERIA. 
 
 ^eptothrlz. Forms which appear as 
 simple threads without branching. 
 Members of this group have been 
 found associated with certain inflam- 
 mation of the mouth and pharynx. 
 The organisms were identified by 
 morphology. 
 
 None of the inflammations were accom- 
 panied by severe systemic symptoms 
 and the organisms may be regarded 
 as comparatively harmless sapro- 
 phyteg appearing in connection with 
 some other specific inflammation. 
 
 Cladothrix. Thread-like, forms with 
 false branching, due to the fragmen- 
 tation of the threads. 
 
 By reason of the difficulty in difCeren- 
 tiation of this form from the strepto- 
 thrix, it is likely that most cases of 
 infection ascribed to these organisms 
 have really been due to streptothrix 
 infection. *A rigid difCerention of 
 true and false branching only can 
 determine whether or not cladothrix 
 infection may occur. 
 
 Streptothrix. (Nocardia.) Forms with 
 numerous true branches and spores 
 which usually appear in chains. 
 
 Numerous cases of infection of various 
 parts of the body of man and animal 
 have been reported. A member of 
 this group has been described by 
 Nocard as the etiological factor in. a 
 disease "farcies du boeuf," occuring 
 among cattle in Guadeloupe. 
 
 Trevisan proposed the name "Nocardia" 
 for this organism, and Wright calls 
 attention to the misuses of the term 
 "streptothrix" and points out +hat the 
 term Nocardia should be used in its 
 place. 
 
 Members of this group have also been 
 reported in the pus from a cerebral 
 abscess,in pulmonary disease simu- 
 lating tuberculosis, suppuration of 
 bone and of the skin and the intes- 
 tinal canal. Streptothrices vary con- 
 siderably in morphology. In infec- 
 tious lesions they most aften appear 
 as rods and filaments with branches. 
 Sometimes the filaments may be long 
 and intertwined, and branch may 
 
BACTERIOLOGY. 275 
 
 show club-shaped ends. Young" cul- 
 tures may consist of rod-shaped forms 
 not unlike bacilli of the diphtheria 
 group. 
 
 They stain easily with Loeffler's methy- 
 lene blue or aqueous fuchsin. Culti- 
 vation upon agar and gelatin plates 
 has been made. Grayish-white, glis- 
 tening, flat colonies appear at the end 
 of four or five days. In bouillon, a 
 floculent precipitate and surface pel- 
 licle is formed of the thread, without 
 clouding. 
 
 The organisms will grow readily upon 
 fresh, sterile kidney-tissue of rabbits. 
 When cultures are inoculated into 
 rabbits and guinea pigs, subcutaneous 
 abscesses, bronchopneumonia, and 
 suppuration, according to the mode 
 of infection may be produced. 
 
 Actinomyces (Ray fungus) is charac- 
 terized by the formation of club- 
 shaped ends and the stellate arrange- 
 ment of its threads. 
 
 The organism was first observed by 
 Bollinger (1877) in diseased cattle 
 and by Israel (1878) in man. 
 
 The organisms appear in the pus from 
 discharging lesions as small granular 
 bodies, resembling sulphur granules, 
 visible microscopically. 
 
 They are ordinarily soft and can be 
 easily crushed under the cover slip, 
 but occasionally, in old l,esions, they 
 may be hard, owing to calcification. 
 
 They may be recognized easily by 
 crushing the granules under the cover 
 glass, and examining them, unstained, 
 with the microscope. Fresh speci- 
 mens may also be stained by Gram's. 
 
 The colony as it appears in tissue sec- 
 tions or pus smear consists of a 
 rosette arrangement. The central 
 portion of the colony is a dense mass 
 of mycelium and spherical bodies. 
 From this felted central mass there 
 extends ray or club-like bodies. Club- 
 shaped enlargements at the end of 
 filaments frequently appear and are 
 regarded as a distinguishing charac- 
 teristic of actinomyces. 
 
 The organism grows on a variety of 
 media. On glycerine agar the colonies 
 develop into transparent drop-like 
 bodies in four or five days at 37°. Old 
 colonies become white or yellowish 
 with a powdery surface. Some va- 
 
276 BACTERIOLOGY. 
 
 rieties appear distinctly aerobic and 
 others anaerobic. Gelatin is nearly 
 always liquefied. 
 In artificial culture filaments appear 
 which are very long and slender. 
 They show true branching, but have 
 no septa. 
 
 The young- colony is a loose mass of 
 filaments; older colonies become dense 
 and fertile. Rod-shaped and spherical 
 forms may also appear and some fila- 
 ments develop conidia. 
 
 Tissue sections, stained with carmine, 
 followed by Gram's or Wigert's, give 
 good results. 
 
 Actinomycosis - (lumpy jaw, wooden 
 tongue) is an infectious disease which 
 spreads rapidly. Cattle are most com- 
 monly affected, but humans, horses, 
 sheep and dogs are susceptible. 
 
 The disease usually runs a chronic 
 course and is distinguished especially 
 by enlargement of affected parts, by 
 hardening of the tongue and by 
 suppuration. 
 
 Head parts, including the facial bones, 
 are commonly affected; lungs and va- 
 rious other internal organs and even 
 . the vertibrae may be involved. The 
 extent of injury depends upon the 
 location and the size of the involved 
 area. 
 
 There are several varieties of acti- 
 nomyces, and it is probably specific 
 in its relation to the disease, but it 
 is frequently aided by pus-producing 
 bacteria. 
 
 It is, vegetative on various grasses 
 especially wild barley, and infection 
 occurs by inoculation with the awns 
 and barbs of such grasses through 
 the mucous membrane of the mouth 
 or alimentary tract. Infection by in- 
 halation may occur. 
 
 It is probable that some special stage 
 of development is necessary either 
 within the diseased body or upon 
 some plants in order that it may irf- 
 f^ct animals' bodies, as direct inocu- 
 lation with pus usually giver 
 negative results. Inoculation with 
 pieces of diseased tissue occasionally 
 gives positive results. 
 
 The pus scattered over fodder, mangers 
 and feed racks probably serves in- 
 directly as a source of dissemination. 
 
BACTERIOLOGY. 277 
 
 An active toxin is evidently not pro- 
 duced. The disturbance caused by 
 the disease is apparently due to harm- 
 ful growths in the tissue and to 
 secondary infection. 
 
 Suppuration is one of the conspicuous 
 features, as is also the development 
 of much new granulation tissue which 
 tends to degenerate at the center. 
 Soft organs affected show a tendency 
 to multiple abscesses. 
 
 Actino'bacillosis is probably to be dis- 
 tinguished from actinomycosis. It is 
 very similar in history and clinical 
 evidence but apparently different as 
 .to specific cause. The cause of acti- 
 nobacillosis seems to be a bactei*ium 
 found also in rosette-like masses re- 
 sembling those of actinomycosis. 
 
 MYCETOMA. 
 (Madura Foat). 
 
 This disease is very much like acti- 
 nomycosis. It is more or less lim- 
 ited to warmer climates; India, espe- 
 cially Madura. It consists of a 
 chronic productive inflammation, most 
 frequently attacking the foot, less 
 often the hand, very rarely other 
 parts of the body. 
 
 Nodular swellings occur, which break 
 down and lead to abscess formation 
 and later to sinuses which discharge 
 purulent fluid containing the charac- 
 teristic hard, brittle, black, granular 
 bodies resembling grains of gun- 
 powder. The granules may be gray- 
 ish white or yellow and soft and 
 grumous. 
 
 The appearance of these granules gives 
 rise to two varieties of the disease: 
 
 The melanokL variety is caused by a 
 member of the hyphomycetes group. 
 
 The ochroid variety is believed by many 
 to be actinomycosis. 
 
 The bones are often involved and a 
 rarefying osteitis results. 
 
 In broth, the growth is rapid, composed 
 of long hyphae, which form a struc- 
 ture of a powder puff appearance. 
 
 On agar, at the end of a week, a thick 
 grayish meshwork of hyphae spread 
 over the surface. In old cultures, 
 black granules appear among the 
 mycelial meshes. 
 
278 BACTERIOLOGY. 
 
 On potato a dense, velvety membrane 
 appears with a pale brown center and 
 a white periphery. Brown drops ap- 
 pear in old cultures. 
 
 FATKOGENIC MOUZkDS. 
 (Hyphomycetes, Eumycetes). 
 
 The relation of moulds to bacteria 
 shows them to occupy a place higrher 
 than the higher bacteria which they 
 resemble, ii> that they grow in fila- 
 ments, but show in majority of cases 
 a more complicated structure in pos- 
 sessing a more distinct wall and a 
 definite nucleus and in their repro- 
 ductive organs. The hyphae branch 
 and grow into a network called 
 mycelium. 
 
 In the lower forms each hypha is a 
 single cell, septa only occuring when 
 fructification begins. 
 
 In the higher forms the filaments are 
 made up of rows of cells. 
 
 Most forms produce endospores in a 
 spore sac (sporangia) situated at the 
 end of a hypha. 
 
 Certain varieties have a primitive sex- 
 ual process, a conjugation of two 
 cells with the formation of a zygo- 
 spore, from which a sporangium car- 
 rier may arise and develop a spor- 
 angium. 
 
 Spores may also be* produced in so- 
 called gummae (chlamydospores), 
 which are swollen portions, segmented 
 in the course of a hypha. 
 
 Spores may be formed as conidia. The 
 common molds grow easily on arti- 
 ficial media and therefore are very apt 
 to infect the media during bacteriol- 
 igical cultivations. They grow espe- 
 cially well in acid medium and are 
 therefore very often found on fruit. 
 The majority of moulds are not path- 
 ogenic, but some are however true 
 parasites and produce a number of 
 very common diseases. Their arti- 
 ficial cultivation is more diflacult than 
 the ordinary varieties. 
 
 Certain varieties of the common mucor 
 has been reported pathogenic for man 
 in that they have been found to pro- 
 duce eye and ear infections, also in 
 a case of enteritis with secondary 
 perttonitls. Autopsy of the latter 
 showed also multiple abscesses of 
 brain and lungs. 
 
BACTERIOLOGY. 279 
 
 The aspergillus (aspergillus fumigatus, 
 more frequent) is found more often 
 pathogenic to birds, producing a 
 pseudo tuberculosis. Such cases have 
 also been reported in man. 
 
 Many varieties are found in plant dis- 
 eases and indirectly may be of danger 
 to man as when they form poisonous 
 substances as in the infection of grain 
 by claviceps purpurea (ergot poison- 
 ing), etc. 
 
 The more common pathogenic forms for 
 man are: 
 
 Trlchopliyton (Blngrwonn Funsrus). 
 
 1. Tinea circlnata produces ringworm of 
 
 the body. 
 
 2. Tinea tonsurans (produces ring- 
 
 3. Tinea barbae or worm of the 
 
 tinea sycosis t hairy parts. 
 According to Sabourand, there are two 
 distinct types of the fungus, tri- 
 chophyton causing ringworm in man. 
 
 (a) Tinea microsporon, with small 
 
 spores, is the common fungus of 
 T. Tonsurans of children, and its 
 special seat of growth is in the 
 substance of the hair. The 
 spores are contained in a mycel- 
 ium which is not visible, and 
 appear piled up like zoogloea 
 massess forming a dense sheath 
 around the hair. 
 
 (b) Tinea megralosporon, with large 
 
 spores, is essentially the fungus 
 of ringworm of the beard and the 
 smooth parts of the skin. The 
 spores are always contained in 
 distinct mycelium filaments, 
 which may either be resistant 
 when the hair is broken up or 
 fragile and easily breaking up 
 into spores. One-third of the 
 cases of T. Tonsurans of children 
 are due to trichophyton megalo- 
 sporon. Cultivation is simple on 
 acid glucose agar or gelatine. 
 Acliorion Schoenleinii (Favus). This 
 fungus was discovered by Schoen- 
 leinii in 1839. It attacks chiefly the 
 hairy portion of the body of man and 
 some domestic animals. It is com- 
 municated by contagion-. Want of 
 cleanliness is a predisposing factor. 
 The disease Is extremely chronic and 
 very resistant to treatment. 
 
280 BACTERIOLOGY. 
 
 In man, it is found most frequently en 
 the scalp of persons in weak health, 
 especially from phthisis, and in 
 undernourished children upon the 
 scalp. Other portions of the skin may 
 also be involved. Pathologically, the 
 disease represents the reaction of the 
 tissues to the irritation caused by 
 the growth of the fungus. The 
 spores invade the hair follicles. The 
 fungus grows in the epidermis, the 
 density of the growth causes pres- 
 sure on the parts below, lowering the 
 vitality of the hair. 
 
 The disease first appears as a small 
 sulphur-yellow disc (scutulum), 
 pierced by a hair, which lesion is 
 characteristic. It is readily culti- 
 vated on artificial media. 
 
 Kaposi has reported a case of confluent 
 favus in which patients died with 
 symptoms of severe gastrointestinal 
 irritation. The presence of the fungus 
 in the stomach and intestines was 
 demonstrated at autopsy. 
 
 Microsporou furfur (Fityriasles Versi- 
 color). This organism was discov- 
 ered by Eichstedt in 1846, invades 
 only the most superficial layers of 
 the skin and produces the disease 
 (Chiefly in those living under condi- 
 tions of uncleanliness, or among 
 those who combine a tendency to pro- 
 fuse perspiration and uncleanliness. 
 The organism does not give rise to 
 any considerable pathological changes 
 in the skin or hair. 
 
 The organism shows preference for 
 locations such as the chest, abdomen, 
 back, and axillae, less frequently neck 
 and arms, exceptionally it attacks the 
 face. It appears as scattered spots 
 of a color which varies from cream- 
 coffee to reddish-brown. 
 
 Soor Fimgrus: Oidium Albicans (Thrush). 
 This organism was described by 
 Langenbeck in 1839. It produces a 
 disease of the oral mucous membrane 
 of infants during the early weeks of 
 life. It occurs most frequently in 
 children suffering from malnutrition. 
 It has been found as a slight mycosis 
 in the vagina of women and in rare 
 cases attacks adults, especially those 
 whose health has been undermined 
 by diseases, such as diabetes, typhoid, 
 etc. A few cases have been reported 
 
BACTERIOLOGY. 281 
 
 in which the fungus was isolated 
 from abscesses of the lung, spleen, 
 kidney and brain. It can readily be 
 cultivated in the ordinary media of 
 either acid or alkaline reaction. 
 
 The oidium albicans appears both as a 
 yeast and a mycelium, and therefore 
 seems to occupy a position between 
 the true moulds and the yeasts. It 
 grows at times to long threads, and 
 under certain conditions, almost ex- 
 clusively, it will multiply by budding. 
 THX: PATHOGENIC YEASTS. 
 (Blastomy cetes) . 
 
 These organisms have been of great 
 importance in brewing and baking 
 and recently have been reported to 
 have caused infections in man and 
 animals. 
 
 The position which the yeast occupies 
 in systematic biology has as yet not 
 been accurately determined. Their 
 chief characteristic is in their 
 method of reproduction by budding. 
 Yeasts can at times develop short 
 hyphae and in rare cases reproduce 
 by segmentation. 
 
 The most important property of yeasts 
 is that of producing alcoholic fer- 
 m.entation, and has been studied ex- 
 tensively along this line. The work 
 of Pasteur and Hansen along these 
 lines developed the fact that differ- 
 ences in the flavors and other qual- 
 ities of beer, wine, etc., were de- 
 pendent upon the particular species 
 of yeast employed for the fermenta- 
 tion. The fermentative property is 
 produced by an enzyme known as 
 "zymase," which transforms sugar 
 into ethyl alcohol. Various yeasts also 
 produce other ferments which split 
 higher carbohydrates (saccharose, 
 maltose, starch) and prepare them 
 for action of the zymase. 
 
 The yeasts employed in practice are 
 spoken of as "culture yeasts" and 
 those which act as disturbing factors 
 in fermentation are called "wild 
 yeasts"; the latter usually producing 
 only a slight degree of fermentation. 
 The culture yeast cell is oval or ellip- 
 tical in shape, while the wild species 
 are more often round or globular and 
 known as "torula" forms. Sausage- 
 shaped and thread forms are also 
 found. 
 
282 BACTERIOLOGY. 
 
 The individual cell is highly refractive 
 and varies greatly in size even in 
 those of the same species or the same 
 culture. The cell contains a nucleus, 
 which can be demonstrated by stain- 
 ing. During the process of budding 
 the nucleus moves toward the peri- 
 phery and divides, the limiting mem- 
 brane of the cell ruptures or a pro- 
 trusion develops (daughter cell), this 
 rapidly increases in^ size and assumes 
 the shape of the mother cell. 
 
 Spore formation takes place in the 
 yeast, which is of importance in th'e 
 continuation of the species and also 
 for propagation. The nucleus divides 
 into several fragments, each of which 
 becomes the center of a new cell. 
 These new cells (within the original 
 cell) possess a firm membrane, a cell 
 nucleus and a little dense protoplasm. 
 As a rule one cell does not produce 
 more than four spores, called "astro- 
 spores." The number of spores 
 formed varies, however, but is con- 
 stant for a species. 
 
 The Pathogenic forms are: 
 
 Saccharomyces Busse, isolated by Busse 
 in 1894 from a woman's tibia. Au- 
 topsy showed broken down nodules 
 an several bones, in the lungs and 
 kidney. 
 
 Saccharomyces tumefaciens, isolated by 
 Curtis in 1895 from multiple tumors 
 on the hips and neck which resembled 
 microscopically softened myxosar- 
 coma. 
 
 This yeast is pathogenic for mice, rats 
 and dogs. In generalized blastomyco- 
 sis, the lung seems to be the seat of 
 primary infection. 
 
 Cases described by Rixford and Gil- 
 christ as coccidiosis (thought to be 
 a protozoan disease) were unquestion- 
 ably due to blastomycetes. Fontaine, 
 Hasse and Mitchell reported a typical 
 case of systematic blastomycosis. 
 Lundogaard reported a case of optlial- 
 mia due to a yeast. Tokishige re- 
 ported an epidemic of ulcerous skin 
 diseases among horses in Japan to be 
 due to one of the saccharomyces. 
 Kartulis described 100 cases of a skin 
 affection occuring in the gluteal re- 
 gions of men from which he isolated 
 the ordinarj'- saccharomyces cere- 
 visiae. Kessler reported a skin 
 
BACTERIOLOGY. 283 
 
 lesion in an infant as due to a blas- 
 tomycete. 
 
 Attempts have been made to connect 
 the development of cancerous growth 
 with blastomycetes by reason of the 
 similarity between the yeasts and the 
 inclusions or so-called parasites of 
 cancer and also by the fact that 
 yeasts will, when injected into the 
 animal body, produce tumor-like 
 nodules. These masses are not 
 tumors in a patholog-lc sense, but 
 merely masses of yeast cells mixed 
 with inflammatory tissue prolifera- 
 tion. 
 
 DISEASES OF UNKNOWN ETIOZ^OGY. 
 
 Measles. Many bacteria as well as sup- 
 posed protozoan bodies have been de- 
 scribed by various investigators as 
 occuring on the mucous membrane or 
 in the blood of those sick with the 
 disease. 
 
 ■ Home, in 1759, claimed to have pro- 
 duced measles of a modified and 
 milder type by rubbing- cotton swabs 
 saturated with the blood of patients 
 affected with measles on wounds 
 made on the arms of other individ- 
 uals. It is not certain that he pro- 
 duced the disease at all. 
 
 Positive results in experimental inocu- 
 lation have been reported by Stewart 
 (1799), Speranza (1822), Katowa 
 (1842), and Nigirr (1850). These re- 
 sults are, however, not satisfactory. 
 
 Heckton, in 1905, produced the disease 
 experimentally by injecting the blood 
 of a measle patient, during the fourth 
 day of the disease, into two students. 
 
 Attempts at cultivation were negative, 
 but the virus of measles will live 
 for at least 24 hours when mixed with 
 acitic broth. 
 
 Scarlet Pever is an acute, febrile, highly 
 infectious disease characterized by a 
 diffuse punctate erythematous skin 
 eruption accompanied by catarrhal, 
 croupous or gangrenous inflammation 
 of the upper respiratory tract and by 
 manifestations of general systemic 
 infection. Both streptococci and pro- 
 tozoa have been described as the 
 etiological factor in the disease. 
 
 Crooke in 1885 demonstrated strepto- 
 cocci in the cadavers of scarlet fever 
 victims. 
 
284 
 
 BACTERIOLOGY. 
 
 Babinsky and Sommerfield in 1900 re- 
 ported the presence of streptococci in 
 the heart's blood Of eight rapidly fatal 
 cases of scarlet fever. 
 
 Mallory in 1904 described protozoan 
 bodies found in the skin of four 
 scarletina cases. The bodies described 
 and found by him between the epithe- 
 lial cells were small and not unlike 
 the Plasmodium of malaria. They 
 stain easily by methylene blue. 
 
 The bodies are still under investigation. 
 Field and others have failed to dem- 
 onstrate. The streptococci are, how- 
 ever, certainly present, but are con- 
 sidered secondary invaders, and by 
 reason of this fact Moser adopted the 
 use of antistreptococcic serum and 
 claims exceptional results. 
 
 Typhus Pever. This is an infectious 
 disease of a five-day or more incuba- 
 tion period and characterized by high 
 temperature and a petechial rash. 
 The etiologrical factor has not been 
 definitely determined. 
 
 Nicolle in 1909 transmitted the disease 
 to the chimpanzee, and from this to 
 the macacus with typical eruption in 
 each case. He was not able to trans- 
 mit the disease from monkey to 
 monkey. 
 
 Anderson and Goldberger in 1909 trans- 
 mitted the typhus fever of Mexico 
 (kabardillo) directly from the human 
 being to the macacus and capuchin. 
 
 Ricketts and Walker in 1910 from re- 
 searches came to the following con- 
 clusions: 
 
 1. M. rhesus can be infected by 
 
 injecting the blood of man dur- 
 ing the 8 to 10-day period of the 
 fever. 
 
 2. It could not be transmitted from 
 
 monkey to monkey. 
 
 3. The disease may produce so mild 
 
 symptoms in the monkey as to 
 be unrecognized clinically, vac- 
 cination results. 
 
 4. Immunity test is proof of pre- 
 
 vious occurrence or nonoccur- 
 rence of the disease within a 
 period of one month. 
 
 5. It is transmitted to the monkey 
 
 by the bite of the louse. 
 
BACTERIOLOGY. 285 
 
 6. A monkey was infected with 
 
 typhus by introducing feces 
 and abdominal contents of in- 
 fected lice into small incisions. 
 
 7. The blood of patients taken from 
 
 the 7th to 12th day and stained 
 (Giemsa) will show bacilli of 
 the hemorrhagic septicaemic 
 group morphology. 
 
 8. No cultures could be obtained, 
 
 but fresh preparations showed 
 forms like those above with- 
 out motility. 
 
 9. Dejecta of lice were examined 
 
 and the organism found in the 
 infected lice and occasionally 
 in noninfected ones. 
 
 Plotz has recently reported the cultiva- 
 tion of a gram positive pleomorphic 
 anaerobic organism from the blood of 
 cases of Brillo disease and also from 
 typhus cases. Complement fixation 
 was obtained when this organism was 
 used as antigen. 
 
 Smallpox, or variola, is an acute infec- 
 tious disease characterized by an epi- 
 dermic eruption of macules, vesicles 
 and pustules, which upon healing pro- 
 duce cicatrices of varying extent and 
 depth. 
 
 The disease was first described by 
 Phozes in the tenth century. It may 
 have developed first in certain re- 
 gions of Asia and central Africa. 
 
 Severe epidemics have swept China and 
 Eastern countries many centuries 
 before Christ, also Europe, especially 
 at the time of the Crusades. 
 
 The disease was widespread when 
 Jenner (1798) showed conclusively 
 that vaccination with cowpox afforded 
 protection. 
 
 The etiological factor is as yet not 
 determined. Streptococci, often found 
 in the vesicles and pustules and con- 
 tribute materially to the fatal out- 
 •come of the disease, are secondary 
 invaders. 
 
 Guarneiri (1892) named certain inclu- 
 sions present in the epithelial cells of 
 smallpox lesions in a rabbit's cornea, 
 
 "Cytorrhyctes variola," and believed 
 them to be protozoa. 
 
 Councilman believed the bodies (vaccine 
 bodies) to be protozoa, and describes 
 two cycles in its development, one 
 intracellular and the other intranu- 
 
286 BACTERIOLOGY, 
 
 clear, and that intranuclear infection 
 occurs only in smallpox. 
 
 Calkins, working with Councilman, also 
 believes them to be protozoa of the 
 class rhizopoda. 
 
 Ewing admits that the vaccine bodies 
 are probably specific for variola, but 
 calls attention to inclusions found in 
 diphtheria, measles, g-landers and 
 other infectious processes which can- 
 not be considered as etiological fac- 
 tors in these diseases. He believes 
 that all forms so far described are 
 degeneration products, some specific, 
 others not. The similarity of cow- 
 pox (vaccinia) and small pox has 
 been the subject of controversy. Many 
 observers claim that although related 
 to each other they are essentially 
 different. 
 
 Others maintain, and this seems to be 
 the prevailing opinion, that cowpox 
 or vaccinia, when inoculated into man, 
 represents the altered and attenuated 
 variety of variola. It has been claim- 
 ed that cowpox was originally trans- 
 mitted to cattle by human beings 
 affected with smallpox. The immun- 
 ity caused by successful vaccination 
 is not permanent and varies in its 
 juration in different individuals. Al- 
 though immunity may last for 10 to 
 15 years it is well to be vaccinated 
 every year, if exposed to the disease. 
 If the vaccination is unnecessary it 
 will not be successful. (See small- 
 pox vaccine.) 
 
 Babies (Hydrophobia) is an acute in- 
 fectious disease of mammals, depend- 
 ent upon its specific virus and com- 
 municated to susceptible animals by 
 the saliva of an infected animal com- 
 ing in contact with a broken surface, 
 usually through a bite. No bacteria 
 have been discovered that are con- 
 sidered as factors in the disease. In 
 1903, Negri described peculiar svriic- 
 tures which he observed in the cell of 
 the central nervous system of rabid 
 animals, which he claims are not only 
 specific for hydrophobia but are prob- 
 ably animal parasites and cause the 
 disease. His later studies confirm 
 his previous work and, so far as the 
 diagnostic value of these bodies is 
 concerned, he has been corroborated 
 by numerous investigators. 
 
BACTERIOLOGY. 287 
 
 Williams in 1906, was convinced that 
 these cell inclusions were animal or- 
 ganisms and called attention to the 
 similarity between their structures 
 and that of the rhizopoda. He gave 
 them the name Neurorycetes Hydro- 
 phobia. 
 
 It is not possible, at the present time, 
 to decide absolutely whether or not 
 the Negri bodies should be regarded 
 as parasites or specific degeneration 
 products. 
 
 The virus of rabies has been shown to 
 be partially filterable through course 
 Berkefeld filters. The retained por- 
 tion is always more virulent than the 
 filtrate. This would seem to indi- 
 cate that there are some forms just 
 
 ^ within the limit of visibility and 
 others larger which correspond with 
 what "we know of the variation in size 
 of the Negri bodies. 
 
 The largest forms of Negri bodies are 
 about 18 microns and the smallest 
 about 0.5 micron. They are round, 
 oval, oblong, triangular or ameboid. 
 They show a hyalene-like cytoplasm 
 with an entire margin containing one 
 or more chromatin bodies which have 
 a more or less complicated and reg- 
 ular arrangement. 
 
 The demonstration of Negri bodies in 
 tissues is carried out by procuring a 
 small piece of tissue from the cere- 
 bellum or from the center of the hip- 
 pocampus major and fixing it for 12 
 hours in Zenker's fluid. It is then 
 washed in water and dehydrated in 
 graded alcohol, embedded in paraffin 
 and sectioned. The sections are at- 
 tached to slides and placed in the fol- 
 lowing solution from 12 to 24 hours: 
 Methylene-blue (Grueber 00) one 
 
 per cent 35 cc. 
 
 Eosin (Gruebler BA) one per cent 
 
 35 cc 
 
 Distilled water 100 cc. 
 
 Then differentiate in: 
 
 Absolute alcohol 30 cc. 
 
 Sodium hydrate, 1 per cent in ab- 
 solute alcohol 5 cc. 
 
 Allow them to remain for about five 
 minutes, wash in absolute alcohol 
 then place in water, then in water 
 slightly acidified with acetic acid. 
 Dehydrate in absolute alcohol, clear 
 in xylol and examine. 
 
288 BACTERIOLOGY. 
 
 The nerve cells are stained pale blue 
 and in their cytoplasm close to the 
 nucleus or near the axis-cylinder pro- 
 cess are seen oval bodies stained a 
 deep pink. They show a more deeply 
 stained periphery than the interior 
 which often contains small vacuole- 
 like bodies. There may be one, three 
 of four in a single cell. 
 
 The Negri bodies may be rapidly dem- 
 onstrated in smears of brain tissues 
 for diagnostic purposes as follows: 
 
 A small pin head sized piece of brain 
 tissue taken from the cerebellum or 
 the center of the hippocampus major 
 is placed on one end of the slide 
 under a cover glass and then gently 
 squeezed until the tissue is flattened 
 out into a thin layer. The glass cover 
 is then gently slipped across the slide 
 so as to smear the brain tissue along 
 the entire surface. These smears are 
 fixed in methyl alcohol and stained 
 by the Giemsa method. The bodies 
 are stained light blue in contrast to 
 the darker and more violet cell bodies. 
 
 The smears may also be stained by 
 van Gieson's stain as follows: 
 
 Fix the smears in methyl alcohol, wash 
 
 . in water, cover with the fresh pre- 
 pared stain, steam, rinse in water and 
 dry. 
 
 Distilled water 10 cc. 
 
 Saturated alcoholic solution of 
 
 rosanalin violet 2 drops. 
 
 Saturated a^queous solution of 
 methylene-blue diluted one-half 
 
 2 drops. 
 
 The Negri bodies stain magenta; their 
 contained granules, blue; the air cells, 
 blue; and the red blood cells, yellow. 
 Or by the Williams and Lowden modi- 
 fication: 
 
 Distilled water 10 cc. 
 
 Saturated alcoholic solution of 
 
 basic-fuchsin 3 drops. 
 
 Loefl^er's alkaline methylene-blue 
 
 2 cc. 
 
 The bodies assume a brilliant hue and 
 contain in their interior darkly stained 
 irregular particles, probably chro- 
 matim bodies. 
 All of the work should be controlled by 
 careful animal inoculation. The work 
 of the smear method in diagnosis has 
 
BACTERIOLOGY. 
 
 289 
 
 been summarized by Parke and Wil- 
 liams as follws: 
 
 1. Negri bodies demonstrated, diag- 
 
 nosis rabies. 
 
 2. Negri bodies not demonstrated in 
 
 fresh brains, very probably not 
 rabies. 
 
 3. Negri bodies not demonstrated in 
 
 decomposing brains, uncertain. 
 .4. Suspicious bodies in fresh brains, 
 probably rabies. (See rabic vac- 
 cine.) 
 
 Whooping' Cougrli. (See Bordet and 
 Gengou Bacillus.) 
 
 Bordet and Gengou in 1906, described a 
 bacillus which they consider the • 
 specific organism because they obtain 
 with it the complement fixation re- 
 action. 
 
 Woolstein, 1909, was not able to cor- 
 roborate their work. This bacillus 
 differs slightly from a bacillus which 
 differed only slightly from the ba- 
 cillus of influenza that had been de- 
 tected by Jockman, Krause and Wool- 
 stein in practically all cases of 
 whooping cough during the acute 
 stages. 
 
 Mumps. Diplococci had been considered 
 as possibly being the exciting or- 
 ganism. 
 
 Noma. A streptothrix pseudo diph- 
 theria bacillus, diphtheria bacilli are 
 the organisms most usually present 
 in cases of noma but it is, as yet, 
 undecided whether the disease is due 
 to one or to several microorganisms. 
 A special predisposition of the tissues 
 is necessary. 
 
 Articular rheumatism. (See Poynton 
 and Paine diplococcus). Most bac- 
 teriologists believe the exciting factor 
 has not yet been identified. Strepto- 
 cocci have been of all bacteria most 
 frequently found in the search for the 
 etiological factor in the synovial 
 fluid, blood vegetation of heart valves, 
 and in exudates on tonsils, etc. 
 
 The streptococci and the other coci 
 found are probably important second- 
 ary infections. 
 
 Beriberi. Both bacteria and protozoon 
 microorganisms have been considered 
 as exciting factors but nothing def- 
 inite has been proven. 
 
290 BACTERIOLOGY. 
 
 Pellagrra. Some investigators believe 
 this to be due to a microorganism, 
 while others believe it to be an in- 
 toxication similar to that of ergot 
 poisoning. 
 
 THE ui;tra microscopic 
 
 ORGANISMS. 
 
 Infective material from a number of in- 
 fectious diseases may with certain 
 precautions be passed through stone 
 filters of varying degrees of porosity 
 and still reproduce the disease with 
 all its characteristic symptoms when 
 inoculated into susceptible animals. 
 Microscopic examination of the fil- 
 trate, except in one or two diseases, 
 does not show the faintest sign of 
 particulate matter. 
 
 The precautions necessary in such fil- 
 tration are: 
 
 1. A perfect filter, which will abso- 
 
 lutely retain all known bacteria, 
 allowing none to pass into the 
 filtrate. 
 
 2. The filtration must be completed 
 
 within a moderate time by reason 
 of the fact that bacteria may, in 
 a media which contains a certain 
 amount of albuminous material, 
 grow through the filter. 
 
 3. The material to be filtered should 
 
 be greatly diluted and first passed 
 through filter paper so as to 
 avoid the clogging action of ex- 
 traneous material. 
 If. with these precautions, the filtrate 
 is pathogenic, ascertain whether the 
 symptoms are due to the microorgan- 
 isms and not to a toxin. This may 
 be determined with almost absolute 
 certainty by inoculating a series of 
 animals successively with the filtrate 
 obtained from a previously so inocu- 
 lated animal. 
 Anterior Poliomyelitis. Landsteiner and 
 Popper thought the virus belonged 
 to the class of invisible protozoa. 
 They were able to transmit the dis- 
 ease to apes. They made intraperi- 
 toneal inoculations with spinal cord 
 and produced typical symptoms and 
 lesions. They were unable to trans- 
 mit the disease from ape to ape. 
 Flexner transmitted the disease from 
 monkey to monkey by intracerebral 
 Inoculations. Landsteiner and Le- 
 
BACTERIOLOGY. 291 
 
 vaditi transmitted the disease from 
 monkey to monkey and found that the 
 virus lived four days outside of the 
 body. They found the virus in the 
 salivary glands and suggested the 
 moist or dry saliva as a source of 
 contagion. Plexner transmitted the 
 disease by means of inoculations into 
 the blood or peritoneal cavity, also by 
 subcutaneous inoculation, and found 
 the virus to be filterable. 
 
 The virus has been shown to be pre- 
 servable under glycerin for ten days 
 and retains its virulence to from 7 to 
 11 days, when dried. The virus will 
 remain active when frozen for a 
 period of 40 days, but is extremely 
 sensitive to heat, being destroyed by 
 a temperature of 45° to 50° C. main- 
 tained for 30 minutes. Flexner and 
 Noguchi placed small bits of an emul- 
 sion of the brain of monkeys dead of 
 poliomyelitis under conditions similar 
 to the cultivation of the Treponema 
 pallidum. Noguchi found after five 
 days that an opalescence appeared, 
 which increased until the tenth day 
 when sedimentation began. Micro- 
 scopical examination by Giemsa's re- 
 vealed small ovoid bodies arranged in 
 pairs, short chains and masses. Sim- 
 ilar bodies were later found in poli- 
 omyelitis tissue. Cultures were ob- 
 tained from glycerinated virus, fresh 
 virus and from filtered and infiltered 
 material. When these cultures were 
 injected into monkeys, typical lesions 
 and death were produced up to the 
 eighteenth generation of cultivation 
 on artificial media. 
 
 Foot and Mouth Disease. This is a 
 highly infectious disease occurring 
 chiefly in cattle, sheep and goats, 
 more rarely in other domestic an- 
 imals. It appears as a vesicular 
 eruption upon the mucus membrane 
 of the mouth and upon the delicate 
 skin between the hoofs. Usually the 
 disease is mild. The vesicles form 
 small ulcers' and pustules which 
 gradually heal with a disappearance 
 of systemic symptoms. The disease 
 may be complicated by a gastro- 
 intestinal or pulmonary infection and 
 may end in death. The disease is 
 generally transmitted from animal to 
 animal by means of the virus con- 
 
292 BACTERIOLOGY. 
 
 tained in the vesicles. On rare oc- 
 casions, the disease is transmitted to 
 man, usually by direct contact or by 
 drinking the milk of animals suffer- 
 ing from the disease. The course of 
 the disease in man is usually very 
 mild. 
 
 Loeffler and Frosch in 1898, diluted the 
 contents of an unbroken vesicle with 
 20 to 40 times its volume of water, 
 passed the solution through a Berke- 
 feld filter and found that the filtrate 
 remains infectious for some time. 
 The virus of the disease is readily 
 destroyed by heating to 60° G. Loef- 
 fler has actively immunized horses 
 and cattle with greater doses of the 
 virus obtained from vesicles and with 
 the sera of such animals has produced 
 passive immunity. One attack of foT)t 
 and mouth disease protects against 
 subsequent attacks. This immunity 
 may last for years, but a case of re- 
 currence within a single year has 
 been reported. 
 
 Yellow Pever is an acute infectious dis- 
 ease which prevails endemically in 
 the tropical countries with no char- 
 acteristic lesion except jaundice and 
 hemorrhage. Other lesions that ex- 
 ist are those common to toxemia. 
 
 Sanarelli, 1897, found the bacillus 
 icteroid circulating in the blood and 
 in the tissues of most yellow fever 
 patients and it was thought by many 
 to be the causative organism, but has 
 been rejected. The U. S. Army Com- 
 mission (Reed, Carroll, Agramonte 
 and Lazear) established the fact that 
 the disease was carried from one in- 
 fected person to another through the 
 agency to the mosquito. This Com- 
 mission established the following 
 facts: 
 
 1. Yellow fever is transmitted under 
 
 natural conditions only by the 
 bite of a mosquito (Stegomyia 
 calopus) that at least 12 days 
 before had fed upon the blood 
 of the patient, sick with this dis- 
 ease during the first three days 
 of his illness. 
 
 2. Yellow fever can be produced arti- 
 
 ficially by subcutaneous injection 
 of blood of a person sick with 
 this disease during the first three 
 days of his illness. 
 
BACTERIOLOGY. 293 
 
 3. Yellow fever is not conveyed by 
 
 fomites. 
 
 4. The bacillus icteroid has no causa- 
 
 tive relation to yelow fever. 
 Although the specific parasite has not 
 yet been discovered, the following 
 facts have been brought out: 
 
 1. The causative agent requires two 
 
 hosts for the completion of its 
 cycle. (A mammal and arthro- 
 \ pod.) 
 
 2. There is a definite time between 
 
 the bite of a mosquito and the 
 infectivity of the blood (average 
 5^ days) and a definite time that 
 the blood remainl^ infective (three 
 days.) 
 
 3. The blood after passing through 
 
 the finest porcelain filters remains 
 infective during these three days. 
 
 4. The blood will lose its virulence in 
 
 48 hours when exposed to the air 
 and at a temperature of 24° to 
 30^ C. If protected from the air 
 by oil, at the same temperature, 
 it remains virulent for from 5 to 
 8 days. It becomes non-virulent 
 if heated at 55** C. for five 
 minutes. 
 
 5. The bite of an infected mosquito 
 
 does not become infectious until 
 12 days (at a temperature of 
 31° C.) after it has bitten the 
 first patient. 
 The infective blood filtrates show noth- 
 ing with dark field illumination, ex- 
 cept small motile granules similar to 
 those found in healthy persons. 
 The necessity for a second host and the 
 long incubation time required before 
 the host becomes infected, after biting 
 a yellow fever patient, seem to point 
 to a protozoan organism as a causa- 
 tive factor. 
 Dengue, Ashburn and Craig claim to 
 have reproduced the disease in sus- 
 ceptible individuals along the lines of 
 procedure employed in yellow fever. 
 The intermediate host in natural in- 
 fection they claim to be culex 
 fatigans. 
 
 South African Horse Sickness occurs in 
 warm weather, as a rule, and seems 
 to be more common in animals which 
 pass the night outside. The disease 
 manifests itself by uneasiness, diflfi- 
 culty in breathing, and the appearance 
 
294 BACTERIOLOGY. 
 
 of reddish froth from the mouth. 
 The temperature rises in the daytime, 
 but drops at night. Edematous swell- 
 ing of the head and neck may appear 
 in severe cases. 
 Contagrious FlenropneTunonia of Cattle. 
 This disease does not affect other 
 species. It appears as inflammation 
 of the lung's and pleura with necrosis. 
 Nocard and Roux have cultivated an 
 organism in collodion sacs placed in 
 the peritoneal cavity of a rabbit, 
 using a mixture of serum and bouil- 
 lon. After two weeks a very faint 
 turbidity appears in the sacs, coin- 
 cidently the fluid becomes infected. 
 The causative factor in this disease 
 has been made to grow and produce 
 disease in new animals and, as at the 
 highest limit or present magnifica- 
 tion, it is seen to consist of minute 
 granules. 
 
 Rinderpest. A fatal European and 
 African disease of cattle is character- 
 ized by inflammation of the intestinal 
 mucous membrane. No organism can 
 be seen. 
 
 Trachoma is a disease of the eye which 
 is characterized by a progressive fol- 
 
 » licular inflammation of the conjunc- 
 tiva followed by cicatrization. 
 
 Prowazek (1907) announced the dis- 
 covery of small organisms the cause 
 of the disease and named them 
 Chalamydozoa and believes they oc- 
 cupy a place between bacteria and 
 protozoa. 
 
 The organism is found only in the 
 early acute cases. 
 
 Prowazek states that the organism 
 grows in a characteristic manner in 
 the conjunctival epithelial cells. It is 
 so small that it cannot at first be 
 seen, only the mantle can be demon- 
 strated, which stains blue with 
 Giemsa. The organism appears as a 
 small red granule within the blue 
 body. As the organisms increase in 
 size and numbers the blue mantles 
 disappear, leaving a mass of small, 
 round, or slightly elongated red 
 bodies. 
 
 Lipschutz points out the fact that 
 dhalamydozoa, although visible, pass 
 through filters, and with Borrel 
 claims to have discovered a similar 
 organism in Molluscum contagiosum 
 
BACTERIOLOGY. 295 
 
 of man and birds. He also believes 
 that the Volpino's motile granules 
 discovered in vaccinia by the ultrami- 
 croscpoe and his own bodies of rabies 
 belong to the same class. 
 By reason of their round form he sug- 
 gested the name "Strongyloplasmen." 
 
 SFIBbCHETAE AND Al^I^IES. 
 
 The microorganism known as spirocheta 
 (name introduced by Ehrenberg in 
 1838, who differentiated it from 
 spirillum by its flexibility) are slender, 
 undulating, cork-screw like threads 
 which vary both structurally and cul- 
 turally from bacteria. . The organisms 
 were formerly regarded as bacteria, 
 belonging to the general group of the 
 spirillum. Schaudinn, by a careful 
 morphological study, claims that 
 many of these forms are protozoa. 
 Other observers have not agreed with 
 him. 
 
 The reason for considering these or- 
 ganisms as protozoa are: 
 
 1. Their flexibility and the indication 
 
 in many of longtitudinal division 
 and of undulating membrane. 
 
 2. The demonstration of forms inter- 
 
 mediate betwefen trypanosomes 
 and the spirochetes (SP. bal- 
 bianii). 
 
 3. The spirochetal forms of certain 
 
 trypanosomes (TR. Noctuae.) 
 The reasons for favoring the bacterial 
 nature of spirochetes are; 
 
 1. The rigidity of some forms, the 
 
 lack, of undulating membrane in 
 most and of definite nuclei ap- 
 paratus in all, the evidence of 
 transverse division of all and of 
 flagella arising from the blepharo- 
 plast in some. 
 
 2. The cultivation of certain forms 
 
 are the SP. refringens and the 
 SP. Obermeieri for many gener- 
 ations '-without development of 
 trypanosome forms. 
 It is probable that the spirochetes 
 and their allies occupy a posi- 
 tion intermediate between the 
 protozoa and bacteria. 
 Spirochetes of the Month. 
 I Non-pathogenic forms commonly found 
 in normal mouths are: 
 
296 BACTERIOLOGY. 
 
 1. Spirocheta buccalis has three to 
 
 ten irregular flat coils. No true 
 cilia have been demonstrated. 
 Some authorities claim for it an 
 undulating- membrane. It stains 
 violet with Giemsa. 
 
 2. Spiroclieta Dentium is much smaller 
 
 than the buccalis and somewhat 
 similar to the pallidum in stain- 
 ing qualities and fixity of its 
 coils when in motion. It stains 
 with Loeffier's flagella stain. A 
 flagellum is present, but no un- 
 dulating membrane or nuclear 
 material has been demonstrated. 
 Spirals are numbered from four 
 to twenty. This organism has 
 been cultivated. 
 
 3. A form which seems to occupy a 
 
 position between the two men- 
 tioned above has been found in 
 the mouth, but has less regular 
 spirals. 
 
 Spiroclieta refrlngrens is an organism 
 that is found in the mouth. It is also 
 frequently associated with triponema 
 pallidum in the various lesions of 
 syphilis, with which it is probably a 
 secondary invader. The irregular, 
 
 » wide, flat spirals number from three 
 to fifteen and change their shape dur- 
 ing motion. Stains easily and quick- 
 ly with Giemsa. 
 
 Schaudinn stated that it possesses an 
 undulating membrane. A terminal 
 cilia has been demonstrated by 
 Levaditi, who also cultivated the or- 
 ganism in collodion sacs in the peri- 
 toneal cavity of a rabbit. 
 
 Spiroclieta Vincentl (Vincent's angfina). 
 Vincent's angina consists of an in- 
 flammatory lesion in the mouth, phar- 
 ynx or throat, situated most fre- 
 quently on the tonsils, beginning as 
 an acute stomatitis, pharyngitis, or 
 tonsilitis, which soon leads to the 
 formation of the pseudo-membrane 
 closely resembling that caused by the 
 bacillus of diphtheria. This may be 
 followed by distinct ulcers with a 
 well defined margin and punched out 
 appearance. The disease is usually 
 mild, but occasionally moderate fever 
 and systemic disturbances are noted. 
 Vincent described the presence of two 
 organism as causative agents of the 
 disease; the one a large spindle- 
 
BACTERIOLOGY. 297 
 
 shaped, or fusiform bacillus; the 
 other, a spirocheta similar to the 
 "middle form" found in the mouth. 
 By reason of the fact that th^e two 
 organisms were always found to- 
 gether, they were at first believed to 
 represent two forms which lived in 
 symbiosis. Tunnicliff believes that 
 these two forms merely represent dif- 
 ferent stages of development of the 
 same organism. 
 
 The bacilli vary in length, thick at the 
 center, from which they taper gradual- 
 ly towards the ends, and end in blunt 
 or sharp points. The organism is 
 usually straight, though sometimes it 
 may be slightly curved. They stain 
 with the stronger aniline dyes and 
 usually decolorize by Gram's. They 
 stain more deeply near the end and 
 show a banded or striped alternation 
 of stain and unstained areas in the 
 central bodies. 
 
 The spirilla are usually somewhat 
 longer than the fusiform bacilli. 
 Microorganisms, as staphylococci, 
 streptococci and not infrequently 
 diphtheria bacilli, usually accompany 
 the microorganisms of Vincent's 
 angina, and by reason of this fact it 
 is impossible to decide that the fusi- 
 form bacilli and spirilla are primary 
 etioligical factors. Animal inocula- 
 tion with these microorganisms has 
 led to little results. 
 
 Various fusiform bacilli which morph- 
 ologically are indistinguishable from 
 those found in Vincent's angina may 
 frequently be found from smears 
 from the gums, from carious teeth, 
 and occasionally mixed with micro- 
 organisms in the pus from old sinuses. 
 Weaver and Tunnicliff have found 
 spirilla and fusiform bacilli in great 
 numbers present in a gangrenous dis- 
 ease of the gums and cheeks, called 
 noma. Here again, it is uncertain 
 whether the organisms are primarily 
 the etiological factor in the disease 
 or merely secondary invaders. 
 
 Spiroch.eta Cbermeieri (relapsing fever). 
 This organism was discovered by 
 Ob«rmeier in 1873 in the l)lood of 
 patients suffering from relapsing 
 fever. The organisms are long, 
 slender, flexible, spiral or wavy fila- 
 ments with pointed ends, with from 
 
298 BACTERIOLOGY. 
 
 four to ten or more undulations. 
 Compared with the red blood cells 
 among which they are seen, the or- 
 ganiam may vary from one-half to 
 ten times the diameter of a corpuscle. 
 They stain somewhat faintly with 
 watery solution of basic aniline dyes, 
 and stain best by the Romanowsky 
 method or its modification. They are 
 negative to Gram. A terminal flagel- 
 lum has been demonstrated by Novy. 
 
 The organisms are found in the blood 
 or blood organs and never in the 
 secretions, and only during the fever 
 and not in intermission. In the fresh 
 preparation from the blood, they ex- 
 hibit active movements accompanied 
 by very rapid rotation in the long 
 axis of the spiral filaments, or un- 
 dulating movement. Their movements 
 will be active for a considerable time 
 if kept in blood serum in 0.6 per cent 
 sodium chloride solution. They are 
 killed quickly at 60° C. but will re- 
 main alive some time at 0° C. Many 
 unsuccessful attempts at cultivation 
 have been made. Novy finally suc- 
 ceeded in cultivating them in celloidin 
 capsules placed in the peritoneal cav- 
 
 * ity of rats. 
 
 The disease has been produced in 
 monkeys, rats and mice by inoculat- 
 ing them with the blood containing 
 the spirochetes. In man the organism 
 produces the following symptoms: 
 
 The microorganism was found in a 
 large percentage of the cases exam- 
 ined, both in the cutaneous papules 
 and in ulceration. He determined that 
 no monkeys are susceptible to inocu- 
 lation. The monkeys susceptible to 
 inoculation with yaws do not become 
 immune to syphilis, neither do those 
 having syphilis become immune for 
 
 yaws. Further specific differences be- 
 tween the two disease have been 
 shown by the Bordet-Gengou reaction. 
 By reason of the morphological simil- 
 arity to the tryponema pallidum, it 
 should probably be called treponema 
 pertenus. 
 
 Spirocheta GalUnamm is an acute in- 
 fectious disease occuring among 
 chickens, chiefly in South America. 
 It is caused by a spirocheta which, 
 morphologically, is very similar to 
 the spirocheta of Obermeirei.' It Is 
 
BACTERIOLOGY. 299 
 
 easily demonstrated in the circulating 
 blood by staining the blood with 
 Giemsa's stain or by dilute carbo 
 fuschin. The organism has lately 
 been successfully cultivated by 
 Noguchi* under anaerobic conditions. 
 The disease has been transmitted 
 from animal to animal by subcu- 
 taneous injection of blood. Other 
 birds are susceptible as well. Mam- 
 mals have not been successfully in- 
 oculated. The disease is generally 
 transmitted to the chicken by a spe- 
 cies of tick which acts as an inter- 
 mediate host and causes the infection 
 by a bite. Active immunization may 
 be carried out by the injection of in- 
 fected blood in which the spirochetes 
 have been killed. The serum of im- 
 mune animals has a protective action 
 upon birds. Sacharoff had previously 
 reported an organism named spi- 
 rochete anserina, which caused a dis- 
 ease in geese, principally in Russia 
 and Northern Africa, which clinically 
 and pathologically corresponds to the 
 disease caused by the spirocheta gal- 
 linarum, and it is not impossible that 
 these two organisms may be identical. 
 
 A "rapid rise of temperature which re- 
 mains high for five to seven days 
 then drops by crisis. At the end of 
 seven days, another rise of temper- 
 ature is followed by an earlier crisis. 
 There may be a second or third re- 
 lapse. The organisms multiply rapid- 
 ly in the blood from the beginning 
 of the fever. They begin to disappear 
 a short time before the crisis, and 
 after the crisis it is nearly impossible 
 to find them in the circulating blood. 
 The disease is not often fatal. The 
 mortality in different epidemics varies 
 from ten to two per cent. 
 
 The mode of transmission of the disease 
 is not clear, though infection prob- 
 ably occurs through the bite of blood 
 sucking insects. In the African dis- 
 ease the transmission occurs through 
 the intermediation of a tick (Orni- 
 thodoros moubata), which infects 
 itself when sucking blood from an 
 infected human being. 
 
 Recovery from an attack usually results 
 in a more or less definite immunity. 
 The individuals who have recovered 
 have hyper immunized blood. Both 
 
300 BACTERIOLOGY. 
 
 the active and passive immunity may 
 last for months. 
 Spirocheta Duttonl. Button in 1905 
 showed the cause for African tick 
 fever to be due to an organism that 
 was morphologically very similar to 
 the SP. Obermeieri. Novy and Frankel 
 believed that this organism is another 
 variety if not another species of the 
 group. 
 
 The organism can be transferred to 
 monkeys by the bites of young ticks 
 at their first feeding, after hatching 
 from infected parents. Button acci- 
 dentally inoculated himself through a 
 break in the skin while performing an 
 autopsy upon an infected subject, and 
 died from the disease. 
 
 Spirocheta Carter! was described by 
 Carter in 1877 as causing relapsing 
 fever in Bombay. 
 
 Spirocheta Pallida (Treponema Palli- 
 dum). Schaudinn working with Hoff- 
 man, in 1905, while investigating a 
 number of primary syphilitic indura- 
 tions and secondary large lymphnodes, 
 discovered a spirochete and named it 
 spirocheta pallida. He thought that 
 the organism was the cause of the 
 » disease. Further study by him re- 
 vealed a delicate flagellum at each 
 end, but left the existence of the 
 undulating membrane which he at 
 first thought present in doubt, so he 
 called it tryponema pallidum. Ex- 
 tensive studies of human syphilis and 
 experimental syphilis of lower an- 
 imals has since corroborated the 
 work of Schaudinn and Hoffman. The 
 organism is a very delicate structure, 
 closely resembling the spirocheta 
 dentium in morphology and staining 
 reaction. The spirals number from 
 four to twenty and are quite deep. 
 The angle of the spiral turn is very 
 short. There are anterior and flagella- 
 like prolongations. On rare occasions 
 double flagella appear at one end, 
 which Schaudinn interprets as be- 
 ginning longitudinal division. Alive 
 the organism is not very refractive, 
 hence seen with difficulty. Its char- 
 acteristic movements are rotation on 
 its long axis, quivering movem,ents up 
 and down the spirals, slight forward 
 and backward motion and bending of 
 the entire body. The organism stains 
 
BACTERIOLOaY. 301 
 
 red by Giemsa's method, as does also 
 the spirocheta dentium. Mo®t other 
 spirochetes stain blue. The organisms 
 have been found constantly present in 
 the primary and secondary lesions of 
 all carefully investigated cases. The 
 presence of the spirochetes in the 
 blood has been demonstrated by van 
 Bandi and Simanelli. In the tertiary 
 lesions the organism is found less 
 regularly than in the primary and 
 secondary lesions. In congenital 
 syphilis the organism has been found 
 in the lungs, liver, spleen, pancreas, 
 kidneys, and in isolated cased in the 
 heart muscle. 
 The organism may be demonstrated in 
 the living state by the hanging drop 
 method, which is, however, difficult 
 and uncertain. A better method is 
 by means of dark field illumination. 
 The material taken for examination 
 should be straight from syphilitic 
 lesions, and if not dilute enough for 
 examination, it should be emulsified 
 in a drop or two of human syphilitic 
 fluid. 
 
 The organism cannot be stained with 
 the weaker aniline dyes, therefore the 
 special method recommended by 
 Schaudinn and Hoffman is generally 
 used. 
 
 1. Make smears, if possible, from the 
 
 depth of the lesion and free as 
 possible from blood. 
 
 2. .Fix in methyl alcohol for ten to 
 
 twenty minutes and dry. 
 
 3. Cover with a fresh solution of: 
 
 Distilled water .10 cc. 
 
 V Potassium carbonate, 1 to 1000 
 
 5 to 10 drops. 
 
 Giemsa's solution. 10 to 12 drops. 
 Allowing the mixture to act for 
 one to four hours, preferably in 
 a moist chamber. 
 
 4. Wash in running water. 
 
 5. Blof and examine. 
 
 The organism is stained with a 
 
 violet or reddish tint. 
 The organism may be demonstrated 
 
 in tissues by the method of 
 
 Levaditi. 
 
 1. Fresh tissue is cut into small 
 
 pieces of two to four m.m. thick- 
 ness. 
 
 2. Fix in ten per cent formula for 24 
 
 hours. 
 
302 BACTERIOLOGY. 
 
 3. Wash in water. 
 
 4. DeiJnydrate in 96% alcohol for 24 
 
 hours. 
 
 5. Wash in water. 
 
 6. Place in a three per cent silver ni- 
 
 trate solution at a temperature of 
 37%° C. and in the dark for three 
 to five days. 
 
 7. Wash in water. 
 
 8. Place in a freshly prepared solu- 
 
 tion of: 
 
 Pyrogallic acid 2 to 4 g-rams 
 
 Formalin 5 cc 
 
 Distilled water 100 cc. 
 
 Allow to remain in this solution for 
 24 to 48 hours at room temper- 
 ature. 
 
 9. Wash in water. 
 
 10. Dehydrate in graded alcohol. 
 
 11. Embed in parafiine and cut thin 
 
 sections. 
 
 12. Examine without further staining 
 
 or counter stain with Giemsa's 
 solution or Hemotoxin. 
 
 Attempts at cultivation were at first 
 unsuccessful. Later cultivations have 
 been reported by Schereschewsky and 
 Muehlens but did not succeed in car- 
 rying out Koch's postulates with the 
 c^iltures they obtained. 
 
 Noguchi has successfully cultivated the 
 spirochete as follows: 
 
 Into tubes (20 cm. high and 1.5 cm. 
 wide) he placed 10 cc. of a serum 
 water made of three parts of distilled 
 water and one part of coarse sheep 
 or rabbit serum. These were steril- 
 ized by the fractional method, after 
 which a small piece of sterile rabbit 
 kidney or testicle and a bit of testicle 
 of syphilitic rabbit were placed in 
 each tube. 
 
 The serum in the tubes was now cov- 
 ered with sterile parafl^in oil and 
 placed in an anaerobic jar at 33%° C. 
 for ten days, at which time, the 
 spirocheta had developed greatly to- 
 gether with bacteria. He obtained 
 pure cultures from these cultivations 
 by allowing the spirochetes to grow 
 through Berkefeld filters; also by 
 what he considers a better method, 
 preparing high tubes of three parts of 
 very slightly alkaline or natural agar 
 to which a piece of sterile tissue had 
 been added. These tubes are inocu- 
 lated from the impure cultures with a 
 
BACTERIOLOGY. 303 
 
 long- pipette. The spirocheta and bac- 
 teria grow close to the tissue and 
 along- the stab. At the end of ten 
 days to two weeks, the spirocheta 
 wander from the stab and appear as 
 hazy colonies. The tubes are cut and 
 the colonies are directly transplanted 
 to other serum agar tissue tubes. 
 Noguchi carried out Koch's postulates 
 with syphilis. 
 So far as is known, syphilis in nature 
 appears only in man. All experi- 
 mental inoculations of animals were 
 unsuccessful until MetchnikoflC and 
 Roux (1903) succeeded in transmit- 
 ting the disease to a female chim- 
 panzee. Klebs stated, 1879, that he 
 had produced syphilis in monkeys by 
 the inoculation of human virus. Since 
 then Lazear has also successfully in- 
 oculated monkeys. Nicolle succeeded 
 in inoculating the lower monkeys 
 (Macacus) with syphilis. Attempts 
 to transmit syphilis from the tertiary 
 lesions have been unsuccessful. The 
 org^anisms can be demonstrated both 
 in primary lesion and in the second- 
 ary enlarged gland of the inoculated 
 animal. Bertarelli produced an ulcer- 
 ative lesion of syphilis by inoculating 
 upon the cornea and into the anterior 
 chamber of the eye, and later found 
 the spirocheta within this situation. 
 Syphilis generally remains localized 
 in rabbits as well as in the lower 
 monkeys. Parodin, in 1907, inoculated 
 syphilis into the testicles of rabbits, 
 and this method has proven to be the 
 most simple in obtaining the spi- 
 rocheta from lesions in man and in^ 
 definitely carried along by continuous 
 transinoculation from one rabbit to 
 another. 
 
 After the development of the primary 
 lesion man is usually insusceptible 
 to reinoculation during- the active 
 stage of the disease, but in some cases 
 both man and monkey can be reinocu- 
 lated. Reinoculation in the tertiary 
 state produced precocious lesions of 
 tertiary type, gumma and tubercles. 
 Injections of large quantities of 
 syphilitic serum into chimpanzee has 
 failed to produce definite immunity. 
 
 For the Bordet-Gengou phenomena see 
 Wasserman reaction under Comple- 
 ment Fixation. 
 
304 BACTERIOLOGY. 
 
 Spiroclieta Pertenuis (Framboesia trop- 
 ica, yaws). Yaws, a disease resem- 
 bling- syphilis, occurs in tropical 
 and subtropical countries, and Castel- 
 lan! in 1906 announced that he had 
 found a spiroorganism which bore a 
 close resemblance to the spirocheta 
 pallida. He named it spirocheta 
 pertenuis. 
 
 Spiroolieta Fhasredenls is an organism 
 of probably a new species cultivated 
 by Noguchi from the phagedemic 
 lesions on human external genitals. 
 
 Spiroclieta Macro dentium is believed by 
 Nog-uchi to be identical with Vincent's 
 spirocheta. 
 
 Spirocheta Microdentium cultivated by 
 Noguchi from the tooth deposits in 
 children. 
 
 Spirocheta Calligrymm cultivated by 
 Noguchi from condylomata is prob- 
 ably a new species. 
 
 THE BACTERIOLOGY OF I^IILK. 
 
 The use of cow's milk as a food, espe- 
 cially for infants, has caused it to be 
 closely studied. 
 
 Milk usually contains about 87% of 
 water and about 13% of solids. Of the 
 solids there is approximately 4% of fat; 
 the remaining- 9 7o is composed of about 
 5% lactose, about 3.3% protein (casei- 
 nogen 4 parts, albumen 1 part) and 
 about .7% of ash (salts). There are in 
 addition hydrolitic enzymes as galac- 
 tase, a proleolytic enzyme and oxidase. 
 
 Milk is a favorable culture medium 
 for the development of bacteria and 
 "therefore very well fitted to convey the 
 germs of infectious diseases. - 
 
 It is ordinarily impracticable to se- 
 cure milk entirely free from bacteria. 
 In the milk ducts and in the teats of 
 even healthy cows a certain number of 
 bacteria may be found, although within 
 the udder milk is sterile. 
 
 If pyogenic or systemic disease of 
 bacterial origin exists, the milk may be 
 infected. 
 
 Certain forms of bacteria seem to de- 
 velop within the milk cistern and within 
 the larger milk ducts. The first milk 
 drawn from the teats is generally loaded 
 with bacteria, in the later milk they are 
 comparatively few in number in com- 
 parison. 
 
BACTERIOLOGY. 305 
 
 Usually milk drawn from the udder 
 contains less than 100 bacteria per 1 
 C.C., although in some cows, seemingly 
 normal, there may be large numbers. 
 
 There are changes taking place In 
 milk, due to micro-organisms, which in 
 a sense may be considered normal and 
 may be divided into a stage of bacterial 
 action, a development of lactic acid, a 
 neutralization of lactic acid and a de- 
 composition or putrefaction. 
 
 Incidental changes, brought about by 
 bacteria, such as sweei curdling, ropy, 
 soapy or color formation may infre- 
 quently be met with. 
 
 The most important source of bacteria 
 in milk is probaly due to contamination 
 of the milk from dust particles contain- 
 ing bacteria, which are dislodged from 
 the hair and skin of the udder and sides 
 of the cow during the process of milk- 
 ing. It is therefore necessary to have 
 the animal carefully groomed and adja- 
 cent body surfaces thoroughly moisten- 
 ed in order that this source of contami- 
 nation may be eliminated while milking. 
 The organisms from this origin are 
 largely of fecal origin. 
 
 Dust in the building in which cows 
 are milked,. from dusty fodder or bedding, 
 is also a source of contamination, and 
 are usually the B. subtilis and putrefac- 
 tive types of bacteria. 
 
 The hands of milkers, unless carefully 
 cleaned, will also afford an opportunity 
 for milk infection. This contamination 
 may more readily carry infection from 
 the milkers to other individuals, than an 
 infection from the cow itself to man. 
 
 Milking utensils may also prove a 
 dangerous source of infection, in that 
 imperfectly soldered joints may harbor 
 innumerable bacteria. Utensils should 
 be thoroughly scalded, or the entire ves- 
 sel heated to the boiling point of water 
 to destroy the organisms present. 
 
 Careless handling, such as allowing 
 milk to stand in open cans or the use of 
 unclean dippers, etc., or contaminated 
 water used for rinsing milk vessels, is 
 frequently a cause of contaminating 
 clean milk. 
 
 The number of bacteria in fresh milk 
 will decrease for a time, which indicates 
 a germicidal action. The length of time 
 
306 BACTERIOLOGY. 
 
 of bactericidal action differs with the 
 number and kinds of bacteria and with 
 the conditions under which the milk is 
 kept. Arguments as to the reduction of 
 bacteria are many. Some hold that re- 
 duction is due to agglutinating power of 
 milk, so that in reality there is no 
 actual reduction in bacterial number. 
 Others argue that all bacteria gaining 
 entrance to milk do not find favorable 
 environment and die more rapidly than 
 those which find environment suitable. 
 
 It would seem, however, that milk 
 must contain a certain amount of bac- 
 tericidal action by reason of germicidal 
 substances contained in blood, which 
 must be given off. at least in part, with 
 the milk. 
 
 The number of leucocytes present in 
 milk would also be a factor, as their 
 phagocytic power would not be lost im- 
 mediately with the milking. 
 
 The bactericidal property, however, 
 can in no case completely sterilize milk, 
 as the bactericidal action is specific, 
 that is, certain bacteria are destroyed 
 by it, while others are not affected. 
 
 The lactic acid organisms, present in 
 m'ilk develop rapidly, particularly if 
 milk is kept in a warm place. When these 
 organisms develop 0.4% of lactic acid 
 the milk will be decidedly sour in taste. 
 When .75% to .80% acidity is reached, 
 curdling of milk takes place. 
 
 The ordinary lactic acid bacteria will 
 rarely produce more than a 1.25% acid- 
 ity. The Bact. bulgaricum group of 
 bacteria will, however, produce a much 
 higher percentage of acidity. 
 
 Sour milk may be kept under anaero- 
 bic conditions for a long time without 
 producing any change in its composition. 
 If exposed to the air, however, certain 
 mords (e.g.. Oidium lactis) develop on 
 the surface* of the milk, using the lactic 
 acid as food, oxidizing it to CO2 and 
 water. By reason of this the acidity of 
 the milk is neutralized. Some of the 
 acid may also be neutralized by the milk 
 caseinogen. 
 
 When the excess milk acidity has been 
 neutralized, the various putrefactive 
 bacteria develop and the milk, particu- 
 larly the caseinogen, rapidly decom- 
 poses. 
 
BACTERIOLOGY. 307 
 
 Milk heavily inoculated with the B. 
 subtilis group of organisms may not 
 sour, but undergo sweet curdling in- 
 stead. This is due to the overgrowth of 
 the lactic acid organisms and the pro- 
 duction of a rennet-like enzyme by the 
 subtilis group. The curd is later more 
 or less completely digested. 
 
 Certain organisms produce the so- 
 called ropy milk by the formation of 
 gums from carbohydrates and mucin- 
 like substances from the proteins, while 
 certain other organisms may produce 
 red, yellow, blue and even black milk. 
 
 The undesirable flavors, sometimes 
 produced in milk^ characterized as soapy 
 and bitter milks, are produced by 
 bacteria. 
 
 The number of bacteria present in a 
 given sample of milk depends upon the 
 contamination taking place during the 
 milking process, the time which elapses 
 after the milking, the temperature at 
 which the milk is held, the care in 
 handling and the matter of non-pasteur- 
 ization or pasteurization. 
 
 The temperature at which milk Is 
 held is very important. The acid-pro- 
 ducing organisms and most other forms 
 grow slowly if at all at low tempera- 
 tures. Milk should therefore be cooled 
 as soon as possible after drawing and 
 kept at a low temperature so as to pre- 
 vent the multiplication of bacteria. If 
 this is carried out milk may be kept 
 from souring several days. 
 
 If it is not quickly cooled and is kept 
 at room temperature it may sour In less 
 than 24 hours. 
 
 The number of bacteria may be great- 
 ly reduced by pasteurization. 
 
 Infectious transmitted "by mlllc. 
 
 The most important infections trans- 
 mitted by milk are the diarrhoeas and 
 dysenteries of infants. The intestinal 
 tracts of infants seem particularly sus- 
 ceptible to infection of micro-oranisms 
 belonging to the enteriditis, paratyphoid 
 and dysentery groups. The summer 
 complaints of infants are, in large part, 
 due to the use of milk containing these 
 organisms. Wherever it is impossible to 
 obtain an infection-free milk for infant 
 feeding, pasteurization becomes neces- 
 sary. 
 
308 BACTERIOLOGY. 
 
 Typhoid fever epidemics have fre- 
 quently been traced to an infection 
 through the milk supply. This is also 
 true for scarlet fever and diphtheria. 
 
 The use of tuberculous milk is the 
 common cause of a number of cases of 
 tuberculosis in children, the milk hav- 
 ing been contaminated by the organism 
 entering the milk within the udders of 
 cows or, what is more likely, by con- 
 tamination through the feces of animals 
 sick with the disease. 
 
 Milk should come from herds that 
 have been tested by tuberculin, and from 
 which all of the tuberculous animals 
 have ^ been removed. 
 
 Anthrax, foot and mouth disease, and 
 malta fever have infrequently been 
 transmitted through milk. 
 Milk Analyses. 
 
 1. Plate various dilutions of milk on 
 nutrient agar (+10 reaction). 
 
 2. Incubate at 37° C. for 48 hours, or 
 at 22° C. for 5 days. 
 
 Milk properly drawn will not contain 
 more than 500 to 1000 bacteria per 1 c.c. 
 Milk as sold in cities is from 36 to 48 
 hours or over old before use, contains 
 many times the above number. 
 
 Good milk may contain about 1,000,000 
 bacteria per 1 c.c, or even more when it 
 begins to sour, so that it is evident that 
 numbers alone are of little moment ex- 
 cept that they indicate the care used in 
 milking and delivery to the consumers. 
 
 Certain cities have classified milk into 
 uninspected milk, inspected milk, pas- 
 teurized milk and certified milk. 
 
 Uninspected milk has no sanitary con- 
 trol. 
 
 Inspected milk is a milk which comes 
 from cows tuberculin tested, and 
 which is drawn and cared for under 
 sanitary conditions. 
 
 Pasteurized milk is a milk which has 
 been heated for a short period of 
 time at a temperature considerably 
 below the boiling point, and then 
 followed by a rapid chilling. Its 
 object is the destruction of harmful 
 bacteria and their products. 
 
 The two methods of pasteurization 
 are: 
 
 (1) The "holder process" in which 
 the milk is heated to 60-65° C. and 
 
BACTERIOLOGY. 309 
 
 held at this temperature for about 
 one-half hour. It is then cooled 
 rapidly and bottled. 
 (2) The "flash or continuous pro- 
 cess" in which the milk is heated 
 to 80-85° C. and held at this tem- 
 perature for 30 seconds to one 
 minute. It is then cooled and 
 kept at a low temperature until 
 distributed. 
 The holder method is held to be the 
 most efficient, as it destroys tiie larger 
 percentage of bacteria. 
 
 Certified milk is now a milk obtained 
 from animals free from contagious 
 or infectious disease; ^ attendants 
 must be in good health; stables 
 must be sanitary, well lighted, and 
 free from dust; milking vessels 
 must be sterile, and every precau- 
 tion must be used to prevent the en- 
 trance of bacteria to the milk. After 
 milking it must be quickly cooled, 
 sealed in bottles, and kept cold until 
 delivery. In most cities where cer- 
 tified milk is inspected it must not 
 contain more than 10,000 bacteria 
 per 1 c.c. 
 
 THE BACTERIOLOGY OF WATER. 
 
 All natural waters contain micro-or- 
 ganisms, which gain entrance from 
 many sources. The vapors arising from 
 the sea or land contain no organisms, 
 but as soon as precipitation takes place, 
 the organisms enter the water from the 
 air and soil. 
 
 Certain organisms, because of their 
 ability to find sufficient nutriment for 
 life and growth in water, may be 
 spoken of as belonging to the "water 
 flora." Some bacteria, found in water, 
 flourish only during rain and flood sea- 
 sons, while other bacteria, such as in- 
 testinal organisms survive for a short 
 period only. 
 
 The organisms found in water may be 
 divided into: 
 
 1. The Natural Water Bacteritt, which 
 are frequently numerous and gener- 
 ally harmless to man. Certain species 
 will predominate at one season and 
 disappear at another. Some bacteria 
 contained in water have, by reason of 
 their biochemical properties, been di- 
 vided into: 
 
810 BACTERIOLOGY. 
 
 (a) A bacillus fluorescence llquefa- 
 ciens group, recognized by the green 
 fluorescence of the colonies and liq- 
 uefaction of gelatine, is more fre- 
 quently found in water than in any 
 other form. 
 
 (b) A bacillus fluorescence non-llque- 
 faciens group, produce the charac- 
 teristic fluorescence, but do not 
 liquefy gelatine, are very abundant 
 in river water "and are represented 
 by the B. f . longus, B. f . tennis, B. f. 
 aureus and B. f. crassus. 
 
 (c) A groxLj? of liquefy ing- and milk 
 acidifying bacilli. These are com- 
 n^on to certain seasons. Some are 
 soil organisms, some are related to 
 
 , the proteus group, others are the B. 
 liquefaciens, B. punctatus and B. 
 circulans. 
 
 (d) A chromog'enic bacilli group such 
 as B. prodigiosus, B. ruber, B. indi- 
 cus B. rubescens, B. rubefaciens, B. 
 aquatilis, B. ochracens, B. aurantia- 
 cus, B. fulous, etc., are often pres- 
 ent in water. 
 
 At certain times, in river and brook 
 waters, violet organisms as B. violacius 
 or B. janthinus, B. lividus, B, amie- 
 thy'stinus and B. coerulens are found. 
 
 (e) A chromogenic cocci group. Sar- 
 cina lutea is the most common spe- 
 cies, though this group is not nu- 
 merous in water. 
 
 (f) A non-chromog'enic cocci group, 
 such as the non-liquefying M. can- 
 dicans, M. nivalis, M. aquatilis, and 
 a liquefying' type as the M. coro- 
 natus. 
 
 2. Soil bacteria from surface wash- 
 ing-. Numerous soil organisms are 
 found in natural waters during 
 floods and after rains. Certain spe- 
 cies of these organisms may remain 
 in the water for a long time. 
 The most common organisms of this 
 group are the B. mycoides, B. subtilis, 
 B. megatherium, B. mesentericus (vul- 
 gatus, fuscus and ruber). 
 
 The colonies of these organisms are 
 characteristic rhizoid, liquefying -gela- 
 tine, and produce spores. One of the 
 thread bacteria (cladothrix dichotoma) 
 may also be present. It is frequently 
 found in both fresh and stagnant water 
 and in most soils. 
 
BACTERIOLOGY. 311 
 
 For the isolation of these organisms, 
 beef peptone gelatine is used. "When 
 other media are used a different flora, 
 such as the nitrofying organisms, yellow 
 chromogens, etc., appear. 
 
 3. Intestinal Bacteria, usually of sew- 
 agre origin. 
 
 (a) Protein group. The B. vulgaris, 
 B. zenkeri, B. mirabilis, B. zopfii, B. 
 
 cloacae and the sewage proteins of 
 Houston. 
 
 These organisms are very abundant in 
 sewage, but are not present in very large 
 numbers in contaminated water. This 
 group of organisms are mobile, liquefy 
 gelatine, produce gas in dextrose and 
 saccharose broth (sometimes a little in 
 lactose), reduce nitrates, coagulate 
 milk, produce indol and impart a fecal 
 odor to the media. 
 
 (b) Sewag'e streptococci. The strepto- 
 cocci in water is indicative of re- 
 cent sewage contamination. They 
 die quickly outside of the body. By 
 their action on the various sugars 
 an equine, human and bovine type 
 may be differentiated, which may be 
 used as indicative of recent contam- 
 ination from street washings, hu- 
 man excreta or cultivated fields. 
 
 (c) B. enteritidis sporog'enes, though 
 usually present in the intestinal 
 tract of man, cannot be considered 
 as an indicator of excretal pollution 
 by reason of its presence in dust, 
 food stuffs, etc., and the resistance 
 of its spores. 
 
 (d) B. Coli. The bacillus coli is ac- 
 cepted as the bacterial indicator of 
 sewage pollution of water. 
 
 (e) Bact. lactis serog'enes, next to B. 
 coli, may be regarded as an indicator 
 of sewage pollution of water. 
 
 (f ) B,i typhosus, reported to have been 
 isolated from water in a very few 
 instances, will live in pure water 
 from 8 to 10 days. When exposed to 
 the action of sewage bacteria, it will 
 live for from 5 to 6 days. 
 
 (g) Msp. comma. The spirillum of 
 Asiatic cholera is an intestinal or- 
 ganism and spreads the disease 
 largely through water. It has been 
 frequently isolated from infected 
 waters. 
 
312 BACTERIOLOGY. 
 
 The number of "bacteria in water. 
 
 The bacterial purity of natural waters 
 depends upon the source from which the 
 waters are derived, together with the 
 special and local condition in relation to 
 contamination. 
 
 Bain. The number of bacteria present 
 in rain water depends upon the 
 month of the year and the dryness 
 of the air. When there is consider- 
 able dust in the air, the first rain 
 will be very rich in bacteria, but 
 during the latter hours of prolonged 
 rain the water may be comparative- 
 ly sterile. 
 
 The rain in densely inhabited cities 
 always contains more bacteria (av- 
 eraging about 19 per cc.) than the 
 rain falling on open farm land or 
 upland pastures, in which the num- 
 ber of bacteria will average about 
 4.3 per cc. 
 Snow. The results from snow fall are 
 similar to those from rain, except 
 that the number of bacteria present 
 per cc. is larger; 334 to 463 bacteria 
 per 1 cc. of snow water has been 
 recorded, while Binot did not find a 
 •single mocro-organism present in 8 
 cc. of water from mountain-top 
 snow. 
 
 Hail Stones. The number of bacteria 
 obtained from hail stones varies 
 from 628 to 21,000 per cc. by reason 
 of surface water being carried by 
 storms. 
 
 Well Water. Deep well water ordinarily 
 contains but few organisms. Usu- 
 ally less than 50 per cc. on gelatin 
 at 20° C. and about 5 per cc. on agar 
 plates. 
 
 Shallow well water's bacterial content 
 varies with the amount of rain fall, 
 even though they are well located 
 and constructed. The water in pol- 
 luted wells may contain enormous 
 numbers of organisms; 20,000 bac- 
 teria per cc. on gelatin has been 
 reported. 
 
 Spring" Water corresponds in bacterial 
 content to that of deep wells. 
 
 Upland Surface Water contains but few 
 bacteria if draining from barren 
 land. Cultivation and habitation 
 may change this considerably. 
 
BACTERIOLOGY. 313 
 
 Pure waters contain from 50 to 300 
 bacteria per 1 cc. when grown on 
 g-elatin and from 1 to 10 on agar. 
 River Water. The bacterial content of 
 river water is influenced by sewage 
 contamination temperature, rain 
 fall, vegetable debris, etc. 
 Iiake Water is generally much purer 
 than the waters of rivers. The bac- 
 terial content near the shore is 
 greater than further out by reason 
 of the influence of habitation. 
 Sea Water near the shore and in the 
 neighborhood of seaports may con- 
 ^ tain a large number of bacteria. In 
 the water remote from the coast 
 there are few bacteria. 
 The number of bacteria in natural 
 waters is influenced by: 
 1. Temperature. A low temperature 
 decreases the parasitic types, but 
 the number of other bacteria pres- 
 ent during the hot summer months 
 is generally somewhat less than 
 during the cooler months. 
 Water should be examined for its 
 bacterial content immediately after 
 collection, as there is usually a re- 
 duction in the number during the 
 first few hours, to be followed later 
 by a large increase. 
 The samples collected for analysis 
 should be kept cool, although very 
 polluted waters snow a marked de- 
 crease of intestinal types if the 
 sample is kept cool. 
 Iiigrht, although germicidal, does not in- 
 fluence the number of bacteria in 
 water, probably "Dy reason of the 
 water's turbidity and the speed of 
 the current. The greatest germi- 
 cidal effect of sunlight is produced 
 in shallow, clear and slowly moving 
 water. Direct light is not efficient 
 as a purifier of water.- 
 Pood Supply. In water containing a 
 large amount of organic matter the 
 number of bacteria is greatly in ex- 
 cess of that in which there is but 
 little of such material. Sewage 
 water is rich in organic matter and 
 therefore contains great numbers of 
 bacteria. The number of bacteria 
 present in a given water is therefore 
 proportionate to the diminution of 
 organic material. Self purification 
 
814 BACTERIOLOGY. 
 
 of streams is dependent mainly 
 upon the causes producing- insuffi- 
 ciency or unsuitability of the bac- 
 teria's food supply. 
 
 Oxidation. The oxygen absorbed on the 
 surface of waters, in rapids, falls 
 and tidal rivers can be considered 
 as a very minor agent in the purifi- 
 cation of water. 
 
 Iiow Plants and Animals as algse, river 
 plants and numerous protozoa re- 
 duce the organic matter of water 
 and thereby reduce the food supply 
 of bacteria. The chemical products 
 of hig-her forms are injurious to 
 bacteria and many bacteria are in- 
 gested by the unicellular animals. 
 
 Dilution. Polluted water flowing into 
 larger bodies of pure water, as into 
 a river or lake, is at once diluted, 
 thus diminishing the bacteria's food 
 supply, likewise also diffusing the 
 bacteria through a greater volume 
 of water; the greater the dilution 
 the fewer sewage bacteria will .be 
 found. 
 
 Sedimentation. .The suspended matter 
 of still water tends to sediment, and 
 this in itself brings about its puri- 
 'fication. 
 
 Water Analyses. 
 
 The improbability of getting typhoid 
 bacilli from suspected water, except 
 under unusually favorable condi- 
 tions, caused a return to the esti- 
 mation of the number of intestinal 
 bacteria. 
 
 It is known that the group of colon 
 bacilli have a somewhat longer exist- 
 ence than the typhoid bacilli, and as the 
 colon bacilli come chiefiy or wholly from 
 the intestinal passages of men and ani- 
 mals, it is fair to assume that typhoid 
 bacilli could not occur without the pres- 
 ence of the colon bacillus, except in 
 rare cases, as, for example, pollution 
 with urine only. The latter could of 
 course occur abundantly without the ty- 
 phoid bacillus. 
 
 During the past few years the atten- 
 tion of sanitarians has been seriously 
 devoted to the interpretation of the 
 presence of smaller or larger numbers 
 of colon bacilli in water, until at pres- 
 ent upon the quantitative analyses 
 (measuring within certain limits, de- 
 
BACTERIOLOGY. 315 
 
 composing org-anic matter) and the 
 colon test (indicating more specifically 
 that pollution derived from intestinal 
 discharges of man or animals) the bac- 
 teriological analyses of water is based. 
 
 The determination of the number of 
 bacteria is also of value. 
 
 TechxiicLue. Utmost care is necessary 
 to get reliable results. A speck of 
 dust, a contaminated dish, a delay 
 of a few hours, an improperly ster- 
 ilized agar or gelatin, a too high or 
 too low temperature, may introduce 
 an error or variation in results 
 which would make a reliable test 
 impossible. 
 In the collection of sample: — 
 
 1. Utmost carefulness in collection is 
 necessary. 
 
 2. An immediate test is essential as 
 bacteria readily increase or de- 
 crease in number after collection. 
 
 Prankland records a case (well water 
 sample) of water sample, kept for 3 
 days at a moderate temperature, in 
 which the bacteria increased from 7 to 
 495,000. 
 
 Jordan reports a case of sample water 
 in which the bacteria decreased in 4'8 
 hours from 535,000 to 54,000. 
 
 Park and Williams, of New York City, 
 record a case in a sample from Croton 
 river irt*^" which B. Colon present in- 
 creased from 10 to 100 per c.c, during 
 24 hours. 
 
 , 3. It is better to make cultures in the 
 open field or in a house, rather 
 than to wait 12 hours for the con- 
 veniences and advantage of a lab- 
 oratory. 
 
 4. If sent to the laboratory, water 
 should be kept at about 5°C. 
 (41° P.) during transit. 
 
 Quantity of water to "be used in tests. 
 
 1. It is of great importance to add 
 proper amounts of water to the 
 broth in the fermentation tubes 
 and in the media for planting. 
 Usually 1 c.c. and 0.01 c.c. are 
 added to the fermentation tubes 
 and 10 c.c, of melted nutrient agar 
 or gelatin.' 
 
 2. If possible always make duplicate 
 tests. 
 
 3. When necessary to know whether 
 colon is present in larger amounts 
 than 1 c.c, quantities as large as 
 
316 BACTERIOLOGY. 
 
 10 c.c. or 100 c.c. can be added to 
 bouillon, and then after a few 
 hours 1 c.c. are added to fermen- 
 tation tubes. 
 
 4. Less than 20 colonies and more 
 than 200 on a plate give inaccurate 
 counts, the smaller number being 
 too few to judge an average and 
 the larger number interfere with 
 each others growth. 
 
 When as many as 10,000 colonies 
 develop in the agar contained in one 
 plate, it will be found that there 
 will develop in a second plate con- 
 taining but 1-10 the amount of 
 water from 20 to 50% as many 
 colonies. This shows that crowd- 
 ing of the colonies had prevented 
 the growth of all but 1-5 to % of 
 them. 
 
 5. The chemical composition of the 
 medium affects the results of the 
 analyses. 
 
 Nutrient agar of a 1.5% acid re- 
 action gives slightly lower counts 
 than gelatin, but on account of its 
 convenience in summer and its 
 greater uniformity, it is more gen- 
 erally used for routine work. 
 6.. The American Public Health Asso- 
 ciation has adopted a standard re- 
 action of 1% acidity which is the 
 average optimum for water bacte- 
 ria. 
 
 Only a certain proportion of bac- 
 teria develop and all we can ask is 
 that our count represents fairly* 
 the quick growing sewage forms. 
 7. The temperature is very important. 
 Plate cultures, as a rule, are grown 
 at 20°C.-21°C. for days, and at in- 
 cubator temperature (37.5°C.) for 
 from 24 to 48 hours. 
 Some bacteria do not develop in 4 
 days, but these are neglected. 
 
 The number ' of bacteria growing 
 at room temperature is usually 
 much greater than those growing 
 at 37.5°C. 
 
 As all the intestinal groups of 
 bacteria grow at body temperature, 
 while many of the water types do 
 not, some investigators believe it 
 important to develop the bacteria 
 at both temperatures so as to com- 
 pare the results. (Advantage in 
 coli tests). 
 
BACTERIOLOGY. 317 
 
 8. In making litmus lactose agar 
 plates, the colon, if present, will 
 take on a red color in the blue 
 field. If many coli are present 
 the whole medium becomes red by 
 reason of the acidity. Later, at 
 48 hours or so, by reason of an 
 alkali being produced by the form- 
 ation of NH3, the blue color may 
 return. 
 
 Sigrnlficance of Coli Bacilli in Water. 
 
 The colon test has been applied with 
 satisfaction and confidence in the exam- 
 ination of water, shell fish, and other 
 articles of food by many authorities, 
 while other authorities have denied its 
 value. 
 
 Bacteriologists have found bacilli re- 
 sembling certain members of the colon 
 group in apparently unpolluted well 
 water. 
 
 The discovery that animals have colon 
 bacilli identical, in the usual character- 
 istics studied, with those of man has 
 complicated matters. 
 
 A fresh hill side stream may be 
 loaded with colon bacilli from the wash- 
 ings of horse or cow manure used as 
 fertilizer in the soil of the field through 
 which the stream runs, or the stream 
 may be polluted by a stray cow or horse. 
 Swine, hens, birds etc., may contaminate 
 in unsuspected ways. 
 
 The number of colon bacilli, rather 
 than their presence, in any body of sur- 
 face water is therefore of importance. 
 
 In well and spring water the presence 
 of the colon bacilli indicates contami- 
 nation: The absence of the colon bacil- 
 lus in water proves it harmless so far 
 as bacteriology can prove it. 
 
 When the colon is present in numbers 
 that may enable one to isolate it from 
 1 c.c. quantities in a series of tests, 
 it is reasonable proof of animal or hu- 
 man contamination ana the conditions 
 should be investigated. 10 colon in 1 c.c. 
 indicates serious contamination. 
 
 Surface water from inhabited regions 
 will always contain numerous colon 
 bacilli after a heavy rainstorm or 
 shower. 
 
 The washing from roads and culti- 
 vated fields contain necessarily large 
 numbers of colon bacilli. 
 
 Wilson reports that in only two out 
 of 58 samples of presumably non-pol- 
 
318 BACTERIOLOGY. 
 
 lute waters did he recover colon bacilli 
 in 1 c.c. samples; even in 21 stag-nant 
 pools he only found colon bacilli in 5 
 of the Ic.c. samples. 
 
 The experience of all those who have 
 studied the subject practically, is that in 
 delicacy the colon test surpasses chemi- 
 cal analysis; in constancy and definite- 
 ness it also excells the quantitative bac- 
 terial count. All tests must, however, 
 be supplemented by inspection. 
 
 Analyses 
 
 1st Method. 
 
 1. Plate 1 c.c. of water in each of 2 
 or 3 Petri dishes containing Hess's 
 agar. Incubate for from 24 to 48 
 hours at 37.5°c., at end of which 
 time, count the colonies appearing 
 on the plate. 
 
 2. Plate 1 c.c. of water in each of 2 
 or 3 Petri dishes containing gel- 
 atin. Incubate for from 48 to 96 
 hours at 20-21°c., at end of which 
 time, count the colonies appearing 
 on the plate; make note of the 
 number of liquefiers. 
 
 3. Place 1 c.c. of water in each of 
 10 fermentation tubes containing 
 dextrose bouillon. Incubate at 
 37.5''c. for 48 nours. Note the 
 
 » quantity of gas produced, if any, 
 at' the end of 24 hours, also at the 
 end of 48 hours. Determine the 
 ratio of CO2 to H. 
 
 Fermentation with the proper 
 gas ratio in but one tube would 
 suggest that 1 colon bacillus is 
 present in 10 c.c. of water, etc. 
 Make report on number of bacteria 
 
 present in 1 c.c. of water grown on 
 
 agar. 
 
 Make report on number of bacteria 
 present in 1 c.c. of water grown on 
 agar. 
 
 Make report on number of bacteria 
 present in l.c.c. of water grown on 
 gelatin. 
 
 Make report on number of liquefiers 
 present in 1 c.c. of water grown on 
 gelatiu. 
 
 Make report on number of colon bacilli 
 present in 1 c.c. of water grown in 
 dextrose bouillon. 
 2iid Method. 
 1. Plate 1 CO. of water in each of 2 
 or 3 Petri dishes containing litmus 
 lactose agar and Incubate at 37.5 "c. 
 
BACTERIOLOGY. 319 
 
 for from 24 to 48 hours, at end of 
 which time note number of red 
 colonies, and trasfer these to each 
 of the necessary number of fermen- 
 tation tubes containing 1% dex- 
 trose bouillon. Incubate the tubes 
 for 24 hours. If gas is not pres- 
 ent, the red colonies are not colon 
 toacUli. 
 
 If g"as is present, test gas ratio, then 
 apply Riva's test for colon 1, 2 
 and 3. 
 
 Blva Test No. 1. 
 
 Boil in a test-tube about 5 c.c. of 
 media from the fermentation tubes, 
 with about 3 c.c. of a 10% solution 
 of Na OH. 
 No change in color=colon bacillus. 
 Change in color to a pink=not colon, 
 
 but ordinary saccharolyte. 
 Biva Test No. 2. 
 
 Depends upon the ability of colon to 
 exhaust all sugar in a 1% dextrose 
 bullion in 24 hours. Sugar change 
 as a matter of fact ceases at the 
 end of the 18th hour. 
 Determine the presence or absence 
 of sugar in the media of fermentation 
 tube culture by boiling a small quantity 
 in a test-tube containing about 5 c.c. of 
 Pehling's solution. A reduction of the 
 copper by the sugar present, changing 
 blue color to deep yellow to red ppt., 
 indicates colon Ibacillus. 
 
 No reduction of the copper, if no sug- 
 ar is present, consequently no change 
 in blue color=:not colon. 
 Biva Test No. 3. 
 
 Add to about 5 c.c. of fermentation 
 tube culture, contained in a test- 
 tube*, about 3 c.c. of a 50% solution 
 of H2 SO4 then add 2 or 3 c.c. of a 
 10% solution of NaOH. 
 
 A pink to red contact ring— Indol re- 
 action=colon. 
 
 3rd Metliod. 
 
 Vide — 1, 2 and 3 of 1st method. Then 
 apply Riva's 1, 2 and 3 tests to 
 the fermentation tube cultures 
 showing gas formation. 
 
 4th Method. 
 
 Quantitative Examination. 
 
 A. 1. Plate 1 c.c, 0.5 c.c, 0.3 c.c and 0.2 
 c.c. of water in agar. 
 
 2. Plate 0.5 c.c. and 0.1 c.c. of water 
 In agar. 
 
 3. Plate a controls agar. 
 
320 BACTERIOLOGY. 1 
 
 4. Label each plate with the num-1 
 ber of the sample, the quantity 
 of water contained and the date. 
 
 5. Incubate at 37.5° C. 
 
 B. 1. Place 9.9 c.c. sterile distilled 
 
 water in a sterile capsule. 
 
 2. Add 0.1 c.c. of the water sample 
 to 9.9 c.c. of water in the capsule. 
 This will give a dilution of 1 in 
 100. 
 
 3. Plate 0.5 c.c, 0.3 c.c. and 0.2 c.c. 
 of diluted water in gelatin. 
 
 4. Label each plate with the quan- 
 tity of water it contains — that 
 is, 0.005 c.c, 0.003 c.c, and 0.002 
 c.c. 
 
 5. Plate 0.5 c.c, 0.3 cc. and 0.2 c.c. 
 of water sample in gelatin. 
 
 6. Plate a controle. 
 
 7. Label each plate with the quan- 
 tity of water it contains. 
 
 8. Incubate at 20° C. 
 
 C. 1. Plate 0.5 c.c, 0.3 c.c. and 0.2 c.c. 
 
 of water sample in wort gelatin. 
 2. Label the plates and incubate at 
 20° C. 
 
 D. 1. After 48 hours incubation, count 
 ^ and record the number of colonies 
 
 that developed upon the various 
 plates. 
 
 2. Replace the gelatin and the wort 
 plates in the incubator; observe 
 a gain at 3, 4 and 5 days. 
 
 3. Calculate and record the number 
 of organisms present per cc. of 
 the original water from the av- 
 erage of thC' SIX gelatine plates 
 at the latest date possible up to 
 seven days. The presence of 
 liquefying bacteria may render 
 the calculation necessary at an 
 earlier date, hence the importance 
 of daily observations. 
 
 Qualitative Examination. 
 
 In routine examination of water, the 
 qualitative examination of water is usu- 
 ally limited to a search for B. Colli and 
 its allies, streptococci and some ob- 
 servers insist on a search for the B. 
 enteritidis sporogenes. "The last organ- 
 ism is relatively scarce in water, there- 
 fore, the collection of a large quantity 
 of water is usually necessary. 
 
 During epidemics or tne examination 
 of new and unknown waters, the coli- 
 typhoid group are to be searched for 
 and on occasion the presence or ab- 
 
BACTERIOLOGY. 321 
 
 sence of vibriocholera, B. anthracis or 
 B. tetani may need to be determined. 
 
 When pathogenic or excremental bac- 
 teria are present in water, their 
 number are few and it is, therefore, nec- 
 essary to adopt either the enrichment or 
 the concentration method of examina- 
 tion. 
 
 A. Snrichment Metbod. The harmless 
 non-pathogenic bacteria are de- 
 stroyed or their growth inhibited, 
 while the growth of the parasitic 
 bacteria are encouraged by ar- 
 ranging the environment a& to re- 
 action of media, incubation tem- 
 perature and atmosphere so as to 
 favor the growth of the patho- 
 genic forms at the expense of the 
 harmless saprophytes. 
 Metbod. 
 
 1. Number a set of Bile salt broth 
 tubes 1-5. 
 
 2. Number a set of Bile salt broth 
 tubes la-5a. 
 
 3. Number one flask 6 and another 7. 
 
 4. To tubes No. 1 and la add 0.1 c.c. 
 water sample. 
 
 To tubes No. 2 and 2a add l.c.c. 
 water sample. 
 
 To tubes No. 3 and 3a add 2. c.c. 
 water sample. 
 
 To tubes No. 4 and 4a add 5c.c. 
 water sample. 
 
 To tubes No. 5 and 5a add lO.c.c. 
 water sample. 
 
 5. Put up all the tubes in Buchner's 
 tubes and incubate aerobically 
 at 42» C. 
 
 6. Pipette 25 c.c. of double strength 
 bile salt broth into flask 6 and 50 
 c.c. double strength bile salt broth 
 into flask 7. 
 
 7. Pipette 25 c.c. water sample into 
 flask 6 and 50 c.c. water sample 
 into flask 7. 
 
 8. Incubate the two flasks aerobically 
 at 42" C. 
 
 9. After the end of 24 hours incuba- 
 tion, note each culture: 
 
 (a) The presence or absence of vis- 
 ible growth. 
 
 (b) The reaction of the medium as 
 indicated by the colour change, 
 if any, the litmus has undergone. 
 
 (c) The presence or absence of gas 
 formation as indicated by a froth 
 on the surface of the medium and 
 
322 BACTERIOLOGY. 
 
 the collection of g-as in the inner 
 "gras" tube. 
 
 10. Replace those tubes which show 
 no signs of growth in the incuba- 
 tor. 
 
 Examine after another period of 
 24 hours with reference to points 
 indicated above. 
 
 11. Remove culture tubes which show 
 visible growth from the Buchner's 
 tubes, whether acid production and 
 gas formation are present or not. 
 
 12. Examine all tubes showing growth 
 by hanging-drop preparations. 
 Note such as show the presence 
 of chains of cocci. 
 
 13. Prepare surface plate cultivations 
 upon nutrose agar from each tube 
 that shows growth either macro- 
 scopically or microscopically and 
 incubate for 24 hours aerobically 
 at 37.5 C. 
 
 14. Examine the growth on the plate 
 either with the naked eye or with 
 hand lens. Pick off for subculti- 
 vation of the coll group, typhoid 
 group, paratyphoid group and the 
 streptococci. (Practice will facili- 
 tate the recognition of the groups). 
 
 ;15. The colifonn or tsrphiromi colonies 
 are streak or smear subcultivated 
 upon nutrient agar and incubated 
 aerobically for 24 hours at 37.5" C. 
 
 (a) Examine growth of each tube 
 macroscopically and microscopic- 
 ally. 
 
 If growth is Impure, replate on 
 nutrose agar, pick olf colonies and 
 resubcultivate till pure, then add 
 5 c.c. sterile normal saline or ster- 
 ile broth and emulsify the entire 
 surface growth with it. 
 
 (b) From the emulsion prepare a 
 series of subcultivations by loop 
 smears on slanted gelatin, slant- 
 ed agar potato, and by adding 0.1 
 c.c. of emulsion to nutrient broth, 
 litmus milk, dextrose peptone, 
 levulose peptone, galactose pep- 
 tone, maltose peptone, saccharose 
 peptone, raffinose peptone, dulcite 
 peptone, marmite peptone, glycerin 
 peptone, inulin peptone and dex- 
 trin peptone. 
 
 (c) Differentiate the bacilli by 
 means of the cultural and biologi- 
 cal characters into: 
 
BACTERIOLOGY. 323 
 
 1. SBcherlcli gronp. B. Coli commu- 
 nis, B. Coli communior, B. lactis 
 aerogenes and B. Cloacae. 
 
 2. Gaertner group: B, enteritidis (of 
 Gaertner), B. paratyphosus A. B. 
 parathyphosus B. and B. cholerae 
 suum. 
 
 3. Ebert group: B. typhosus, B. dy- 
 sentariae (Shigra), B. dysentariae 
 (Flexner), and B. fercalis alcali- 
 grines. 
 
 (d) Confirm results by specific ag- 
 glutinating- sera obtained from ex- 
 perimentally inoculated animals. 
 If a positive result is obtained by 
 this method, it needs only a sim- 
 ple calculation to determine the 
 smallest quantity of the sample 
 that contains at least one of the 
 organisms of indication, e.g. if 
 growth due to B. Coli in tubes 
 from 4 to 10, it follows that at 
 least one colon bacillus is pres- 
 ent in every 10 c.c. of the water 
 sample, but not in every 5 c.c. 
 
324 
 
 BACTERIOLOGY. 
 
BACTERIOLOGY. 325 
 
 16. Pick off streptococcus colonies and 
 
 subcultivate upon nutrient agar as 
 directed in steps a and b of 15. 
 Differentiate the streptococci iso- 
 lated into members of the (a) 
 Saprophytic group — short-chained 
 cocci. 
 
 (b) Parasitic (pathogenic) group 
 — long-chained cocci by their cul- 
 tural characters and record nu- 
 merical frequency as indicated af- 
 ter — of 15. 
 
 Determine the pathogenicity for 
 mice and rabbits of the strepto- 
 cocci isolated. 
 B. Concentration Metbod. Organisms 
 in water that are few in number 
 are best sought for by the concen- 
 tration method. The quantity of 
 water required for this examina- 
 tion is about 2000 c.c. 
 
326 BACTERIOLOGY. 
 
BACTERIOLOGY. 327 
 
 Metbod. 
 
 1. Fit up filtering: apparatus as in the 
 accompanying diag-ram (after 
 Eyre). 
 
 2. Filter the entire 2000 c.c. of water 
 through the filter candle. 
 
 3. When filtration is completed, screw 
 up the clamp so as to occlude the 
 two pieces of pressure tubing. 
 
 4. Reverse the position of the glass 
 tubes in Wolff's bottle, so that the 
 one nearest the air pump now dips 
 into the H2SO4. 
 
 5. Slowly open clamps and allow air 
 to gradually pass through the acid, 
 and enter flask, and so restore 
 pressure. 
 
 6. Unship the apparatus, remove the 
 cork from mouth of candle. 
 
 7. Pipette 10 c.c. of sterile broth into 
 the interior of the candle, and by 
 means of a sterile test-tube brush 
 emulsify the slimy residue which 
 lines the candle, with the broth. 
 
 Practically all of the bacteria 
 contained in the original 2000 c.c. 
 of water will now be contained in 
 the 10 c.c. emulsion of broth, so 
 that 1 c.c. of emulsion is equiv- 
 alent, so far as the contained or- 
 ganisms are concerned, to 200 c.c. 
 of the original water. 
 Coll-Typlioid Group. 
 
 1. Number 9 tubes of bile salt broth 
 from 1-9. 
 
 2. To No. 1 add 1 c.c. of the original 
 water sample before filtration is 
 commenced. 
 
 To No. 2 add 2 c.c. of the original 
 water sample before filtration is 
 commenced. 
 
 To No. 3 add 5 c.c. of the original 
 water sample before filtration is 
 commenced. 
 
 3. To No. 4 add 0.05 c.c. (equivalent 
 to 10 c.c. of original water sample). 
 To No. 5 add 0.125 c.c. (equivalent 
 to 25 c.c. of original water sample). 
 To No. 6 add 0.25 c.c. (equivalent 
 to 50 c.c. of original water sample). 
 To No. 7 add 0.5 c.c. (equivalent to 
 100 c.c. of original water sample). 
 To No. 8 add 1.0 c.c. (equivalent to 
 200 c.c. of original water sample). 
 To No. 9 add 2.5 c.c. (equivalent to 
 500 c.c. of original water sample). 
 
328 BACTERIOLOGY. 
 
 4. Put up each tube anaerobically in 
 a Buckner's tube and incubate at 
 42" C. 
 
 5. Subsequent steps are same as those » 
 described under enrichment meth- 
 od. 
 
 B. Enteritidis Sporogrenes. 
 
 1. Transfer 5 c.c. of emulsion from 
 the filter to a sterile test-tube and 
 plug carefully. 
 
 2. Place test-tube into the interior of 
 a benzole bath, and expose to a 
 temperature of 80° C. for 20 min- 
 utes. 
 
 3. Number 10 tubes of litmus milk 
 from 1-10. 
 
 4. Remove test-tube from benzole 
 bath and shake well to distribute 
 spores through fluid. 
 
 5. Add to each tube of litmus milk a 
 measured quantity of suspension 
 vide coli group. 
 
 6 Incubate anaerobically at 37.5** C. 
 (Put up in Buchner's tubes or in 
 Bulloch's apparatus, or pour layer 
 of sterile vaseline on surface of 
 fluid). 
 
 7. Examine after 24 hours, 
 (a) Acid reaction. 
 
 » (b) Presence of clotting and sep- 
 aration of clear whey, 
 (c) Presence of gas. 
 
 8. Replace tubes showing no signs of 
 growth in incubator for another 
 24 hours and again examine vide 7. 
 
 9. Remove tubes showing growth, 
 carefully pipette off whey, and ex- 
 amine microscopically. 
 
 10. Inoculate 2 guinea-pigs subcuta- 
 neously with 0.5 c.c. of whey and 
 observe result. 
 
 ▼IMo Cbolera. 
 
 1. Number ten tubes of peptose water 
 from 1-10. 
 
 2. To each tube add a measured quan- 
 tity of emulsion, vide coli group. 
 
 3. Incubate auaerobically at 37.5" C. 
 for 24 hours and examine for deli- 
 cate pellicle formation, which if 
 present, examine microscopically. 
 
 4. Inoculate fresh tubes of peptone 
 water from tubes showing pellicle 
 formation and incubate for 24 
 hours. 
 
 5. Test peptone water for nidol and 
 nitrite. 
 
BACTERIOLOGY. 329 
 
 6. Pick off colonies resembling chol- 
 era colonies and subcultivate on all 
 ordinary media. 
 
 7. Test vibrio isolated against serum 
 of an animal immunized to cholera 
 for agglutination. 
 
 B. Anthrax. 
 
 1 and 2 vide B. enteritidis sporagenes. 
 
 3. Inoculate a young white rat sub- 
 cutaneously with Ic.c. of emulsion. 
 Observe during life, if animal dies, 
 make post mortem examination. 
 
 4. Make nutrient agar plates with 
 0.2 c.c, 0.3 c.c, and 0.5 c.c. quan- 
 tities of suspension and incubate 
 at 37.5'' C, for 24 or 48 hours. 
 
 5. Pick off anthrax-like colonies and 
 subcultivate on all ordinary media. 
 
 6. Inoculate another white rat as in 3, 
 using 2 loopfuls of agar subculti- 
 vation emulsified with 1 c.c. steri^^ 
 bouillon. Observe as in 3. 
 
 B. Tetani. 
 
 1. Vide 1 and 2 of B Anthrax. 
 
 2. Add 1 c.c. of suspension to each 
 of 3 tubes of glucose formate 
 broth, incubate anaerobically in 
 Buchner's tubes at 37.5** C. 
 
 3. From such tubes showing visible 
 growth after end of 24 hours in- 
 cubation, inoculate guinea-pigs 
 subcutaneously, using 0.1 c.c. of 
 bouillon cultivation. Observe vide 
 3 B Anthrax. 
 
 4. From the same tubes pour agar 
 plates and incubate anaerobically in 
 Bulloch's apparatus at 37.5" C. 
 
 5. Subcultivate suspicious colonies on 
 various media, incubate anaerobi- 
 cally, making controle cultivation 
 on glucose formate agar, stab and 
 streak, to incubate anaerobically 
 and carry out further inoculation 
 experiments with resulting 
 growths. 
 
 Interpretation of 1>acteriolosrical water 
 analysis. 
 
 In the analysis of water, data, such as 
 the kind of water, the method of collec- 
 tion, the sampling, rain fall, transmis- 
 sion, etc., miist be recorded in order that 
 the results may be properly interpreted. 
 Several analyses are necessary and 
 should be made^egularly and systemat- 
 ically. 
 
330 BACTERIOLOGY. 
 
 The number of micro-organisms per- 
 missible in potable water depends to a 
 g-reat extent upon the kind of micro- 
 organisms present. Great numbers of 
 bacteria indicate a large amount of or- 
 ganic matter. The number of bacteria 
 in deep wells and springs should not ex- 
 ceed 50 per cc. on gelatin at 20° to 22** C. 
 Organisms in excess of the above fig- 
 ures would indicate pollution, except 
 after rains or floods. 
 
 The number of organisms grown on 
 agar at a temperature of 37%'* C. is 
 probably more important than the num- 
 ber present in the "gelatine count" in as 
 much as many water bacteria do not 
 grow at this heat, whereas the sewage 
 and soil organisms grow very rapidly at 
 371/^" C. The agar count would there- 
 fore eliminate the water flora, but 
 ^ould obscure the sanitary results by 
 reason of the presence of soil organisms. 
 
 The agar count of deep waters should 
 not exceed 10 per cc. and for surface 
 water it should not exceed 100 per cc. 
 
 Isolation and identification of specific 
 disease organisms from water would 
 condemn such a water as unfit for use; 
 bifct by reason of the difficulty of such 
 an examination their isolation is not 
 often attempted. 
 
 The isolation of the colon bacillus 
 from water is easily carried out and its 
 presence is generally looked upon as 
 significant and indicative of sewage pol- 
 lution. The number of bacilli coli in a 
 certain amount of water sufficient to 
 condemn it varies in the opinion of dif- 
 ferent authorities. Prescott and Win- 
 slow hold that if present in 1 cc. of 
 water it is reasonable proof of serious 
 pollution. Savage suggests that the 
 bacillus coli should be absent from 10 
 cc. in surface waters, such as rivers 
 used for drinking purposes, shallow 
 wells and upland surface waters. 
 
 The streptococcus is also an indication 
 of sewage contamination, and should be 
 absent from the amounts of water men- 
 tioned above for the bacillus coli. 
 
 The bacillus enteritidis sporogenes 
 should not be present in 1,000 cc. water 
 from deep wells nor in 400 cc. from sur- 
 face waters. 
 
BACTERIOLOGY. 331 
 
 THE BACTERIOLOGY OF SEWAGE. 
 
 Sewage is a menace to the public 
 health because of the frequent presence 
 of pathogenic bacteria. It is made up of 
 the products of man and animal waste. 
 A constant or characteristic bacterial 
 flora cannot be established. A classifi- 
 cation based upon bacterial activity 
 rather than upon the species, the genus, 
 the group or type has been adopted by 
 reason of the organism's activity in 
 sewage purification. 
 
 Certain exceptions to these general 
 principles are taken in case of such or- 
 ganisms as the bacillus coli, the sewage 
 streptococcus and the bacillus enteri- 
 ditis. These are to a certain extent 
 characteristic sewage bacteria and their 
 interest in them as individuals has to do 
 with water bacteriology. 
 
 According to the general character of 
 changes which the sewage organisms 
 bring about they are divided into two 
 large groups as follows: 
 
 (1) The anserobic or putrefactive 
 bacteria which bring about the 
 withdrawal of oxygen from one 
 molecule or part of a molecule 
 and the subsequent oxidation of 
 another molecule or part of the 
 same molecule. The energy re- 
 leased in this process is utilized 
 in the vital functions of the or- 
 ganisms. It involves the reduc- 
 tion of urea, the hydrllis of pro- 
 tein, and of cellulose, the emul- 
 slfication of fats, the reduction of 
 nitrates and sulphates and pos- 
 sibly phosphates. 
 
 (2) The Ozidizingr Bacteria. They 
 are distinguished by the fact 
 that oxygen is added to the 
 molecule, the product containing 
 more oxygen than the initial 
 substance. Carbon dioxide, water 
 and nitrates are produced in 
 distinction from methane hy- 
 drogen and ammonia which 
 characterizes the anserobic re- 
 actions. 
 
 Fathogfenic Bacteria. It is assumed 
 that they are always present in sewage. 
 Sewage can be made harmless by being 
 sterilized but can be freed from offense 
 only by the destruction of organic mat- 
 ter, and this is obtained almost wholly 
 
332 BACTERIOLOGY. 
 
 through bacterial processes, except when 
 chemical precipitants are used. 
 
 There are two general methods em- 
 ployed for the cultivation of those bac- 
 teria which are of assistance in sewage 
 purification. They may be cultivated in 
 niters of sand or coarser material, or in 
 specially constructed tanks called "sep- 
 tic tanks." 
 
 Septic Tank Furification. 
 
 Cameron, 1895, introduced the "septic 
 tank." In this tank, the sewage was ad- 
 mitted at the bottom and flowed out at 
 the top after about 24 hours' subjection 
 to anaerobic conditions acting upon the 
 organic matter as indicated above. Soil 
 and sand filters act not only mechan- 
 ically, but also bacteriologically and 
 ofCer one of the best means of purifying 
 sewage bacteriologically. Sewage is 
 conducted to beds, allowed to pass 
 through and then after a few hours 
 again poured on. This purification is 
 produced by the action of aerobic bac- 
 teria. The best results are obtained by 
 combining the two processes; first, the 
 anaerobic treatment to break down the 
 solid material, then the sand filtration to 
 oxidize the compounds and render these 
 products harmless. 
 
 The biological processes remove bac- 
 teria not by any specific antagonistic 
 action but by delaying their passage and 
 permitting the natural decrease that oc- 
 curs when multiplication is prevented. 
 
 Sewagre Analysis. 
 
 On account of the great numbers of 
 bacteria in sewage it becomes neces- 
 sary to make dilutions ranging from 
 1-1000 to 1-10000 and then plating, 
 etc., as in water analyses. 
 In sewage chemistry, putrefaction Is 
 that change which takes place naturally 
 in sewage after anaerobic conditions 
 have become established. It involves 
 the reduction of urea, the hydrolysis of 
 protein and of cellulose, the emulsifica- 
 tion of fats, the reduction of nitrates 
 and sulphates and possibly of phos- 
 phates and those other changes which 
 are characterized by the withdrawal of 
 O and the hydrolysis of complex mole- 
 cules. These changes are always noted 
 in sewage under anaerobic conditions, 
 and the terms putrefactive and anaerobic 
 change are for the present purposes 
 practically synonymous. 
 
BACTERIOLOGY. 383 ^ 
 
 BACTERIOLOGY OF SOIL 
 
 Many varieties of bacteria are pres- 
 ent in the soil. Some by reason of con- 
 tamination through animal feces and 
 other waste products, but the majority 
 are real soil bacteria in that they live 
 and multiply chiefly in the soil. They 
 have important functions* to preform in 
 continuing- the earth's supply. Some of 
 the bacteria make carbon, nitrogen, hy- 
 drogen and other compounds locked up 
 in the dead bodies of animals and 
 plants available for plants. Other bac- 
 teria manufacture food for plants from 
 the gases of the air and the inorganic 
 elements of the earth, which in their 
 simple forms were not available. They, 
 therefore, form an important link in the 
 earth's life cycle. Food, moisture and 
 proper temperature are necessary for 
 their activities. 
 
 In a grain of rich loam there may 
 be many millions, while an equal quan- 
 tity of sand may be almost free from 
 bacteria. 
 
 Various species of soil bacteria have 
 an influence upon each other. The an- 
 aerobic bacteria are enabled to develop 
 by the aerobic species utilization of the 
 free oxygen, while still other species 
 make assimilable substances which can- 
 not be used by others. 
 
 Carbon compounds are broken up by 
 the soil bacteria. Starch is manufac- 
 tured by plants then converted into cel- 
 lulose, wood fats and sugar, which when 
 formed, cannot be utilized by other 
 plants. 
 
 The largest part of these substances 
 are broken up by micro-organisms; a 
 smaller portion are transformed within 
 the animal body. Alcohol is fermented 
 from sugars and starch by the yeasts 
 and molds with the production of carbon 
 dioxide, or acid fermentation by the 
 action of bacteria takes place, with the 
 production of acids and often of carbon 
 dioxide. 
 
 Cellulose is attacked by certain vari- 
 eties of bacteria, acting both In the 
 presence and absence of free oxygen. 
 Moulds also act on cellulose with pro- 
 duction of carbon dioxide, gas and other 
 products. Wood is first attacked by 
 fungi, then by bacteria. 
 
 Animals utilize plant proteids and re- 
 duce them to simpler compounds as 
 
384 BACTERIOLOGY. 
 
 urea, etc., but these compounds are not 
 suitable for plant use, so that micro- 
 organisms must break tnese compounds 
 into more simple form. Yeasts, molds 
 and fungi decompose these substances 
 (plant proteids) to a certain extent, but 
 the chief decomposition is carried out by- 
 bacteria. 
 
 In the absence of oxygen, the process 
 is incomplete with the production of 
 H2S, NH3 and CH4, and is termed putre- 
 faction. The presence of oxygen gives 
 rise to more complete decomposition 
 with the production of CO2, N and H2O. 
 
 The variety of organisms producing 
 these changes are many. Some are 
 found in decaying vegetable matter, 
 others in animal tissue. 
 
 There Is a process of oxidation pro- 
 duced by bacteria, in which ammonia 
 compounds are changed to nitrates and 
 thus utilized by plants (nitrification). 
 The ammonia is first oxidized to nitrite, 
 then into a nitrate, which is taken up 
 by the plant roots from the soil. The 
 two organisms isolated causing a change 
 of ammonia to nitrites, are the nitroso- 
 menas and the nitrosococcus. One vari- 
 ety of organism changing nitrites to 
 nitrates is called nitrobacter. These 
 organisms appear to depend upon 
 mineral substances for their food. 
 A small amount of organic matter in 
 the media acts as antiseptics. Plants 
 take up most of their nitrogen in the 
 form of nitrates; hence these bacteria 
 are important. When the soil becomes 
 acid, the growth ceases. Air is neces- 
 sary for their action as the process is 
 one of oxidation. 
 
 There is also a reduction process 
 called dentrification. Nitrates yield a 
 part or all of its oxygen and becomes 
 changed to nitrites, ammonia, and free 
 nitrogen. 
 
 In the partial change, the soil does 
 not lose its available nitrogen which 
 takes place in the total change, by 
 changing nitrites and ammonia by the 
 nitrifying bacteria to nitrates. 
 
 The types of nitrogen reduction are: 
 
 1. The reduction of nitrates to nitrites 
 and ammonia. 
 
 2. The reduction of nitrates and ni- 
 trites to gaseous oxides of nitrogen. 
 
 3. The reduction of nitrites with the 
 development of free nitrogen. 
 
BACTERIOLOGY. 386 
 
 Certain plants are able to use the ni- 
 trogen of the air through the aid of 
 bacteria growing in and producing en- 
 largments (tubercles), on the roots. The 
 root bacteria are called B. radicicola and 
 may remain active in the soil for long 
 periods even though there is no legu- 
 minous vegetation. 
 
 The organism diffuses rapidly in soils 
 that are in proper conaition, so that if 
 a soil lacks the organism, it cannot be 
 introduced to it until the soil has been 
 made fit for the organisms development. 
 Buchanan concludes that: 
 
 1. The B. radicicola varies considera- 
 bly in its morphology when appro- 
 nutrients, as the salts of organic 
 acids, are induced into the artifi- 
 cial media. Sodium succinate pro- 
 duces a most luxuriant growth to- 
 gether with the greatest variety of 
 bacteroids. 
 
 2. The B. radicicola, in the roots of 
 legumens, inay show the same type 
 of bacteroids as seen in suitable 
 artificial media, and again the same 
 type may not be the same as pro- 
 duced in culture-media and that 
 produced in the nodule by the same 
 form. 
 
 3. The B. radicicola probably includes 
 a group of closely related varie- 
 ties or species which differ from 
 each other and morphological char- 
 acters. 
 
 4. The organism of the nodule re- 
 sembles morphologically both the 
 yeasts and the bacteria. The dif- 
 ference between this form and the 
 forms included ander bacillus and 
 pseudomonas justify the generic 
 use of a separate generic name of 
 Rhizobium. 
 
 Winogradsky states that certain an- 
 aerobic, spore-bearing, bacilli (Clostri- 
 dium Pasteurianum) outside of the roots 
 perform the same function as those 
 within the roots. Their power of nitro- 
 gen fixation is increased in the presence 
 of sugar and decreased in the presence 
 of nitrogenous substances. 
 
 Beyerinck and Bailey have described 
 aerobic species of nitrogen fixing bac- 
 teria, to which the name of Azotobacter 
 has been given. 
 
 The inoculation of soils and an in- 
 vestigation of soils and crops best fitted 
 
38« BACTERIOLOGY. 
 
 for the growth of these bacteria has 
 been carried out with the result of 
 greatly Improving inpoverished soils. 
 
 The use of seeds inoculated with a 
 special variety of bacteria suitable for 
 the plant and soil has been largely 
 practiced with marked results. Exces- 
 sive bacterial development may at times 
 be harmful to the soil. 
 
 The exhaustion of the soil following 
 the constant raising of the same crop is 
 now thought to be due partly to the 
 inability of a few restricted species of 
 bacteria, continued in the soil, to pro- 
 duce the substances necessary for the 
 nutrition of the special crop, or that the 
 bacteria use up the substances in the 
 soil necessary to crop growth. j 
 
 The greatest number of bacteria are 
 found a little below the surface of the 
 soil. 
 
 Some of the bacterial products act 
 upon the inorganic constituents of the 
 soil. The CO2 and organic acids act up- 
 on the compounds of lime and magnesia, 
 and convert them into more soluble sub- 
 stances. The same is true of the rock 
 phosphates, the silicate of potassium, 
 sulphates, etc. 
 
 Quantitative Analysis of soil for bacteria 
 
 Include 4 distinct investigations: 
 
 1. Enumeration of Aerobic organism. 
 
 2. Enumeration of Spores of Aerobes. 
 
 3. Enumeration of Anaerobic organ- 
 isms (also facul. Anaerobes). 
 
 4. Enumeration of Spores of organ. 
 Further by a combination of results , 
 
 of 1-2 and 3-4, the ratio of spores 
 to vegetative forms is obtained. 
 
 1. Obtain soil under sterile conditions. 
 
 2. Weigh and make proper dilutions 
 for counting. 
 
 3. (A) Aerobe— 
 
 Pour set of gelatin plates. 
 Incubate at 20° C. 
 Pour set of agar plates. 
 Incubate at 371/2" C. 
 
 4. Count plates for 3, 4 or 5 days. 
 
 (a) The number of aerobic micro 
 organisms per 1 gm. soil. 
 
 (b) The number of yeast and 
 moulds per 1 gm. soil. 
 
 (c) The number of aerobe growing 
 at 37" C, per 1 gm. soil. 
 
BACTERIOLOGY. 837 
 
 (B) Anaerobes spores and Vegr. 
 Pour set of plates in glucose. 
 Formate gelatin and agar. 
 Incubate in Bulloch's apparatus. 
 
 (C) Aerohes and Anaerobes (spores 
 only). 
 
 (1) 5 c.c. of soil dilution in sterile 
 tube. 
 
 (2) Differential sterilize at 80 for 10 
 minutes. 
 
 ( 3 ) Pour plates and incubate anaerobi- 
 cally. 
 
 (4) After long incubate, count. 
 Qualitative — f or 
 
 Coli group — Typhoid group. 
 
 B. Anthrax, B. Tetanus, B. Malig 
 
 Oedema. 
 
 Nitrous Organism, Nitric Organisms. 
 Nitrous Orgran. 
 
 10 tubes of Winogradsky's Sol. No. 1. 
 
 Label from 1-10. 
 
 Inoculate each tube with varying 
 dilutions and incubate at 30** C. 
 Nitric Organisms. 
 
 10 tubes of Winogradsky's Sol. No. 
 11, and incubate as in 1. 
 Incubate at 30" C. 
 Examine after 24-48 and from those 
 tubes that show signs of growth make 
 subcultivations in fresh tubes of same 
 media and incubate at 30" C. Make 
 further subcultivation from these and 
 again incubate. 
 
 If growth occurs in these sub-cul- 
 tures, make surface smears on plates of 
 Winogradsky's silicate Jelly. Pick off 
 colonies as make appearance and sub- 
 cultivate in each of these two media. 
 W. Sol. for Nitric. 
 
 1. K. Phosphate, 1 gm. 
 Mg. Sulph., .5 gm. 
 
 Ca. Chloride, .01 gm. 
 Na Chlor, 2 gm. 
 Dissolve in A.D., 1000 c.c. 
 
 2. Fill into flasks in quantities of 20 
 c.c, and add to each a small quan- 
 tity of freshly washed mg. Carb. 
 
 3. Sterilize in steamer at 100 for 3 
 days. 
 
 4. Add to each flask, 2 c.c. of sterile 
 2% sol Ammonia Sulph. 
 
 5. Incubate at 37° for 48 hours and 
 eliminate any contaming a growth. 
 
 Tlie Media for the growth of Nitros 
 
 Organisms. 
 1. Ammon. Sulph., 1 gm. 
 
 K. Sulph., 1 gm. 
 
 A.D., 1000 c.c. 
 
838 BACTERIOLOGY. 
 
 2. Add 5-10 gm. basic mg". Garb (ster- 
 ilize by boiling:). 
 
 3. Pill flasks and sterilize as in No. 1. 
 W. SUicate JeUy. 
 
 Sol. A. 
 
 Ammon Sulph., 40 gm. Mgr. 
 
 Sulph., 0.05 gm. 
 Calcin Chloride, .01 gm., A.D. 
 
 50 c.c. 
 
 Sol. B. K. Phosphate .10 gm. Na Carb 
 .60 gm. A.D. 50 c.c. 
 Silica acid 3.4 gm., A.D. 100 c.c. 
 Pour them into a large dish, (Por- 
 celain). 
 
 5. Mix of Sol. A & B then add suc- 
 
 cessive small quantities of mixed 
 salts to silicic acid sol. (stir const.) 
 . with glass rod till a Jelly of right 
 consistency is found. 
 
 6. Spread layer of Jelly over several 
 Petri dishes. 
 
 Sterilize for 30 minutes on 3 days. 
 
 THE BACTERIOLOGY OF AIR. 
 
 The atmosphere is not the normal 
 habitat of bacteria. Their growth and 
 multiplication can not take place in it 
 under ordinary conditions. The air is 
 kept in motion by the wind, so that fine 
 pa»rticles are constantly being carried 
 into it from the ground, especially so in 
 inhabited areas. The bacteria in the 
 dust of the field and street are carried 
 along with the dust particles of the air 
 and are usually of the harmless soil 
 variety ©r the almost harmless intes- 
 tinal bacteria of animals. Pathogenic 
 human bacteria are rarely carried in 
 harmful numbers except under excep- 
 tional circumstances, and are usually in 
 form of spores, e. g., anthrax bacillus, 
 tetanus bacillus. On a dry, windy day 
 the air contains many thousands of bac- 
 teria per cubic meter. In warm weather 
 the rain carries down the bacteria of the 
 air. After a storm there are very few 
 bacteria present in the air. The bac- 
 teria in the air of the country are much 
 less than the bacteria in the air of the 
 cities. Forests decrease the number of 
 bacteria. Bacteria are very few on high 
 mountains; also on the high seas. The 
 bacteria that multiply in the streets and 
 in the soil are almost always sapro- 
 phytic. The bacteria present in the air 
 of dwellings depend upon factors such 
 
BACTERIOLOGY. 339 
 
 as the opening of windows to the out- 
 side, the cleanliness of the dwellingr and 
 the stirring up of dust t)y sweeping. It 
 is nearly impossible to separate the ef- 
 fects of the bacteria which we inhale 
 from that of the dust particles which 
 they accompany. Both probably act as 
 slight irritants and so predispose to 
 definite infections. 
 
 It is problematical as to the impor- 
 tance of air as a means of conveying dis- 
 ease, though there can be no doubt that 
 smallpox, measles, scarlet fever, etc., 
 are transmitted readily, and pulmonary 
 anthrax and tuberculosis, pneumonia, 
 influenza, diphtheria and meningitis 
 may result from inhalation of the or- 
 ganisms. 
 
 The distance through which the air 
 may carry the causative agents of dis- 
 ease requries further study and must 
 necessarily depend upon a variety of 
 conditions, as time, degree of moisture, 
 air currents, factors producing desicca- 
 tion, effects of sunlight, etc. 
 Bacterial Air Examination. 
 
 Gelatin or agar plates may be exposed 
 to the air for a definite length of 
 time and then incubated at both 
 25° and 37%° C. temperatures. 
 
 The number of colonies appearing on 
 the plates will indicate in a general way 
 the bacterial content of the air. The 
 results must necessarily vary according 
 to the degree of moisture in the atmos- 
 phere, air currents, etc., and therefore 
 furnishes no standard for comparative 
 results. 
 
 A method in use at the present time, 
 from which more accurate results may 
 be obtained, follows, vide: 
 
 1. Pill 10 litres of water into an aspi- 
 rating bottle. 
 
 2. Construct a sand filter from a 
 small glass tube qontaining about 4 
 cm. depth of quartz sand held in 
 place by means of a wire screen. 
 Sterilize in hot air oven. 
 
 3. Insert sand filter in a perforated 
 rubber stopper which fits mouth of 
 aspirating bottle. 
 
 4. By allowing water to flow from 
 lower opening of aspirating bottle, 
 
340 BACTERIOLOGY. 
 
 10 litres of air is now drawn 
 through the sand niter. (Refill as- 
 pirating: bottle and aspirate as 
 many times as necessary to give the 
 quantity of air required for the 
 test.) 
 
 5. Remove the sand filter and care- 
 fully pour the sand into a known 
 quantity of sterile bouillon or water. 
 
 6. From the bouillon-sand mixture 
 (which contains in suspension the 
 total number of bacteria contained 
 in the quantity of air aspirated 
 through the sand) plate 1 c.c. in gel- 
 atin or agar and from the colonies 
 appearing thereon estimate the 
 number of bacteria contained in a 
 given quantity of air. 
 
 By the use of the aspirating bottle the 
 air may be drawn directly through a 
 measured quantity of sterile bouillon or 
 water in an Erlenmeyer flask as follows: 
 
 (1) Erlenmeyer flask of 250 c.c. ca- 
 pacity containing 50 c.c. Bouillon 
 or water. 
 
 (2) Rubber stopper to fit mouth of 
 flask perforated with 2 holes and 
 fitted as follows: Take a 9 cm. 
 length of glass tubing and bend up 
 3 cm. at one end at right angles 
 to main length. Pass long arm of 
 the angle through one of the per- 
 forations in the stopper. It must not 
 come in contact with the bouillon. 
 
 Take a glass funnel 5 or 6 cm. in 
 diameter, with a stem 12 cm. in length, 
 and bend stem close up to the apex of 
 the funnel, in a gentle curve through a 
 quarter of a circle; pass the long stem 
 through the other perforation in the 
 rubber stopper. Make sure the end of 
 the stem of the funnel is immersed in 
 the bouillon. 
 
 (3) Sterilize flask, contents and fit- 
 tings. 
 
 (4) Attach aspirating bottle to small 
 glass tube and operate as in "4" of 
 sand filter. 
 
 (5) Maae plates from bouillon as in 
 "6" of sand filter. 
 
INDEX 
 
 Adiorion Schoen- 
 
 leinii, 279. 
 Acid fast bacteria, 
 86. 
 
 group of organ- 
 isms, 229. 
 production test 
 for, 89, 90. 
 
 Actinobacillosis, 
 277. 
 
 Actinomyces, 
 86, 275. 
 
 Active stage of 
 bacteria, 28. 
 
 Aerobes, 16. 
 
 Aerogenio bacteria 
 34. 
 
 Agglutinin, 
 
 124, 154, 155. 
 
 Agressin, 178. 
 
 Agar agar, 41. 
 sulphindigo- 
 tate, 45. 
 
 Alcoholic produc- 
 tion, test for, 92. 
 
 Alexin, 123, 129. 
 
 Amboceptor, 
 129, 143. 
 test for, 136. 
 
 Ammonia produc- 
 tion, test fon# 
 91. ^ 
 
 Ammonification, 
 18. 
 
 Amylases, 12. 
 
 Amylolytic fer- 
 ments, 12. 
 
 Anaphylaxis, 183. 
 
 Anabolic 
 
 activities, 17. 
 
 Anaerobes, 16. 
 
 Anaerobic cultiva- 
 tion, 63. 
 cultivation 
 methods, 63. 
 
 Analyses of soil, 
 336. 
 
 Anthrax, 209. 
 vaccine, 166. 
 
 Anthrax-like 
 bacilli, 213. 
 
 Animal inocula- 
 tion, 110. 
 
 Anterior poliomy- 
 litis, 290. 
 
 Antiamboceptor, 
 135. 
 
 Antibacterial 
 serum, 34. 
 
 Antibodies, 12, 123. 
 determination 
 of, 138. 
 
 Anticomplements, 
 136. 
 
 Antidysenteric 
 
 serum, 165. 
 Antiferments, 12. 
 Antigens, 124, 141. 
 Antigonococci 
 
 serum, 164. 
 Antiseptics, test 
 
 for, 103. 
 Antistreptoccic 
 
 serum, 164. 
 Antitoxins, 12, 123, 
 
 159. 
 
 Antitoxic serum, 
 34. 
 
 Articular rheuma- 
 tism, 289. 
 
 Aromatic producits, 
 14. 
 
 Anthrogenous 
 
 spore forma- 
 tion, 30. 
 
 Asparagin Uschin- 
 sky's, 45. 
 
 Asporogenousi bac- 
 teria, 30. 
 
 Asiatic cholera, 
 225. 
 
 cholera vaccine, 
 173. 
 
 Atmospheric opti- 
 mum, test for, 
 96. 
 
 Autolysin, 133. 
 Azotobacter, 21. 
 
BaoUluB aerosrenes, 
 
 260. 
 
 aerogenes capsu- 
 latus, 246. 
 
 alkaligenes, 
 263, 266. 
 
 amethystinus, 
 310. 
 
 auranticus, 310. 
 anthracis, 209. 
 anthracoides, 
 
 214. 
 blue pus, 203. 
 Bordet-&eng^on, 
 
 222. 
 
 Botulinus, 248. 
 Bovis morbifi- 
 
 cans, 265. 
 bubonic plague, 
 
 250. 
 
 butyricus, 237. 
 chicken cholera, 
 253. 
 
 cholera sins, 266. 
 circulans, 310. 
 cloacae, 311. 
 coerulens, 310. 
 coli, 256. 
 coli communior, 
 ' 259. 
 
 ©oli communis, 
 256. 
 
 coli groi^p of or- 
 ganisms, 256. 
 
 diphtheria, 214. 
 
 Ducrey, 224. 
 
 dysenteria 
 Shiga, 267. 
 
 group of organ-^ 
 isms, 267. 
 
 enteritidis, 263,. 
 264. 
 
 enteritidis 
 sporagenes, 
 248, 328. 
 
 fluorescence 
 aurens, 310. 
 
 fluorescence 
 crassus, 310, 
 
 fluorescence 
 longus, 310. 
 
 fluorescence 
 liquefacieus 
 group of or- 
 ganisms, 310. 
 
 fluorescence non 
 liquefacieus 
 group of or- 
 ganisms, 310. 
 
 fluorescense ten- 
 nis, 310. 
 
 fulous, 310. 
 
 green pus, 203. 
 
 glanders, 206. 
 
 hog cholera, 266. 
 
 Hoffmanni, 218. 
 
 mdicus, 310. 
 
 influenza, 219. 
 
 ioteroides, 266. 
 
 janthinus, 310u 
 
 leprosy, 237. 
 
 leprosy, rat, 237. 
 
 liquefacieus, 310. 
 
 lividus, 310. 
 
 Lustgarten, 237. 
 
 malignant ode- 
 ma, 245. 
 
 mallei, 206. 
 
 megathesium, 
 310. 
 
 mesentericus, 
 310. 
 
 mirabilis, 310. 
 
 Morax-Axenfeld, 
 223. 
 
 mucosus capsu- 
 
 latus, 261. 
 murisepticus, 
 222. 
 
 mycoides, 310. 
 
 ochracens, 310. 
 
 ozena, 261, 263. 
 jparadysenteria 
 ^. "A" Parke, 
 269. 
 
 paradysenteria, 
 **B" Flexner, 
 269. 
 
 paratyphoid, 266. 
 
 pleuro-pneu- 
 monia of rab- 
 bits, 222. 
 
 prodigiosus, 310. 
 
 proteus vulgaris, 
 205. 
 
 psittacosis, 
 264. 
 
 radicicolus, 20. 
 radicosus, 214., 
 rat leprosy, 240. 
 
rhinoscleroma, 
 
 261, 263. 
 rhusiopathlar, 
 
 222. 
 
 rubefacens, 310. 
 
 ruber, 310. 
 
 rubescens, 310. 
 
 smegmatio, 237. 
 
 soft chancre, 224. 
 
 subtilis, 310, 314. 
 
 swine plague, 
 254, 267. 
 
 symptomatic an- 
 thrax, 242. 
 
 tetani, 240. 
 
 timothy, 236. 
 
 tuberculosis, 229. 
 
 tuberculosisv av- 
 ian, 235. 
 
 tuberculosis, bo- 
 vine, 235. 
 
 typhimurium, 
 265. 
 
 typhosus, 270. 
 typhosus g-roup 
 
 of organisms, 
 
 270. 
 
 violaceus, 310. . 
 
 vulgaris, 311. 
 
 whooping cough, 
 222. 
 
 xeroses, 219. 
 
 Zenkeri, 311. 
 
 Zopfii, 311. 
 
 Zurneeden, 224. 
 Bacilli resembling 
 
 bacillus of tub- 
 erculosis, 236. 
 Bacteria in air, 5. 
 
 in body, 6. 
 
 in foods, 5. 
 
 in soil, 5. 
 
 in water, 5. 
 
 normal to human 
 
 body, 6. 
 Bacteria reaction 
 
 to stains, 72. 
 Bacteriaceae, 23. 
 Bacterial cultiva- 
 tion, 61. 
 
 enzymes, 11. 
 
 ferments, 11. 
 
 growth charac- 
 teristics, 67. 
 
 identification, 65. 
 
 mobility, 27. 
 
 nutritian, 15. 
 
 proteins, 11. 
 
 sheath, 27. 
 Bacteraemia, 118. 
 Bacterins, 174, 
 Bacteriology of air 
 338. 
 
 of milk, 304. 
 
 of sewage, 331. 
 
 of soil, 333. 
 
 of water, 309. 
 Bacteriolysins, 
 124. 
 
 Bacterium avisep- 
 ticus, 253. 
 boviseptium, 
 255. 
 
 diphtheria, 214. 
 Bacterium lactus 
 aerogenes, 260. 
 pneumonia, 261. 
 suisepticus, 254. 
 tularense, 253. 
 Beri-beri, 289. 
 Beyrinck's media, 
 
 49, 50. 
 Bile salt broth, 45. 
 Biochemical mejth- 
 
 ods, 14. 
 Biology of bac- 
 teria, 5. 
 Biologic activitiea) 
 of bacteria, 17. 
 classification of 
 bacteria, 32. 
 Black death, 250. 
 Black leg, 242. 
 Blastomycetes, 
 281. 
 
 Blood agar, 43. 
 Blood serum, 42. 
 Bouillon, 41. 
 Branched baciteria, 
 25. 
 
 Bubonic plague, 
 250. 
 
 Bubonic plagn© 
 
 vaccine, 173. 
 Buffon, 3. 
 
 Calcium cycle, 22. 
 Capsule of bac- 
 teria, 26. 
 Carbon cycle, 21. 
 Casease, 13. 
 Cell membrane, 26. 
 
 wall, 26. 
 
 content, 27. 
 
Chalamydobax;- 
 teriaceae, 28. 
 
 Chalamydozoa, 
 294. 
 
 Chemical constitu- 
 ents of bacterial 
 
 cell, 15. 
 Chicken cholera, 
 
 253. 
 
 Chromogenic bac- 
 teria, 33. 
 
 Chromparous bac- 
 teria, 33. 
 gram negative 
 cocci, 201. 
 
 Cladothrix, 274. 
 
 Classification of 
 bacteria, 22. 
 
 Clostridium, 21. 
 
 Coagulating fer- 
 ments, 13, 32. 
 
 Coccaceae, 28. 
 
 Cohn, 4. 
 
 Colon-typhoid dys- 
 entery, group 
 of bacilli, 256, 
 
 Coma bacillus of 
 Koch, 225. 
 
 Complement, 129, 
 144, 150. 
 fleviation, 136. 
 fixation of, 136, 
 
 139, 140. 
 method of fixa^ 
 tion, 140. 
 
 Contagious 
 
 pleuro-pneu- 
 monia of cat- 
 tle, 294. 
 
 Cornstalk disease, 
 255. 
 
 Copula, 129. 
 
 Cultivation of bac- 
 teria, 36, 61. 
 
 Culture media, 36, 
 37. 
 
 Davaine, 5. 
 
 Degeneration 
 
 forms of bac- 
 teria, 26. 
 
 Denitrification, 20. 
 
 Dextrose bouillon, 
 44. 
 
 Diastases, 12, 32. 
 differential 
 staining, 78. 
 
 Dilutions in culti- 
 vation, 62. 
 
 Diplococcus gonor- 
 rhoea, 197. 
 lanceolatus, 192. 
 mucosus, 201. 
 pneumonia, 192. 
 
 Diphtheria, 214. 
 
 Diptheretic anti- 
 toxins, 161. 
 
 Diseases produced 
 by higher bac- 
 teria, 274. 
 of unknown eti- 
 ology, 283. 
 
 Disinfectants, test 
 of, 103. 
 
 Dissolving fer- 
 ments, 13. 
 
 Distribution of 
 bacteria, 5. 
 
 Dorlset's egg me- 
 dia, 43. 
 
 Dunham's inosite- 
 free bouillon, 
 46. 
 
 Dysentery, 267. 
 
 Ebert group of 
 organisms, ' 
 323. 
 
 Ectoplast, 27. 
 
 Egg media, 44. 
 
 Ehrlich, 5. 
 
 Eisenberg's rice- 
 milk media, 48. 
 
 Endogenous spore 
 formation, 29. 
 
 Endotoxins, 119. 
 
 Enzymes, 11. 
 
 Enzyme produc- 
 tion, 87, 88. 
 
 Erhenberg, 3. 
 
 Eschrich group of 
 organisms, 323. 
 
 Elubacteria, 23. 
 
 Eumycetes, 278. 
 
 Extracellular tox- 
 ins, 34, 109. 
 
 Facnltative anaer- 
 obes, 16. 35. 
 
 Pat splitting fer- 
 ments, 13. 
 
 Pavus, 279. 
 
Ferment at ion, 12, 
 32. 
 
 Ferments, 12. 
 
 amylolytic, 12. 
 
 coagulating", 13. 
 
 effects of, 14. 
 
 emulsifying, 13. 
 
 inverting, 13. 
 
 lypolytic, 13. 
 
 oxidases, 13. 
 
 proteolytic, 12. 
 
 reducing, 14. 
 
 ureases, 13. 
 Filtering media, 
 
 40, 60. 
 Flagellar motility, 
 27. 
 
 Flexner's bacillus, 
 269. 
 
 Foot and mouthi 
 disease, 291. 
 
 Formation of gon- 
 idia, 31. 
 
 Fracas t or, 3. 
 
 Friedlander's bac- 
 illus, 261. 
 group of organ- 
 isms, 261. 
 
 Frombresia trop- 
 ica, 304. 
 
 Fusiform bacillus, 
 297. 
 
 G-artner's bacillus, 
 264. 
 
 group of organ- 
 isms, 323. 
 Gas production, 
 
 test for, 95. 
 Gelatin, 41. 
 
 stab culures, 68. 
 Genito-urinary 
 
 tract flora, 10. 
 Glanders, 206. 
 Gonidia, 31. 
 Gonococcus, 197. 
 Growth conditions 
 for bacteria, 
 35. 
 
 Haemorliaglc sep- 
 ticemia group 
 of organisms, 
 250. 
 
 Haemolytic serum, 
 130, 143. 
 unit, 143. 
 
 Haemolysin, 129, 
 132. 
 
 Hanging drop, 71. 
 
 Haptins, 124. 
 
 Hay bacillus, 214. 
 
 Heller's urine 
 agar, 48. 
 
 Hesse's method of 
 anaerobic cul- 
 tivation, 63. 
 
 Heterolysis, 132. 
 
 Higher bacteria, 
 25. 
 
 History of bacter- 
 iology, 3. 
 
 Hog cholera, 266. 
 vaccine, 169. 
 
 Hydrophobia, 286. 
 
 Hydrolytic fer- 
 ments, 13. 
 
 Hypersusceptibil- 
 ity, 183. 
 
 Hyphomycetes, 
 278. 
 
 Immtinity, study 
 
 of, 108, 120. 
 Immune body, 120. 
 
 test for, 136. 
 Immunization, 
 
 study of, 126, 
 
 127, 128. 
 Indol production, 
 
 test, 93. 
 Infection, 114, 116, 
 
 118. 
 
 blood examina- 
 tion methods 
 in, 115. 
 conditions ne- 
 cessary 'to, 
 116, 117. 
 general observa- 
 tion of, 115. 
 special observa- 
 tion of, 115. 
 Infectious dis- 
 eases, 33. 
 Infection trans- 
 mitted by milk, 
 307. 
 Influenza, 219. 
 Influenza-like bAr 
 
 cilli, 221. 
 Inoculation, llOu 
 materials used, 
 111. 
 
methods of, 111, 
 112, 113. 
 Inseperate toxins, 
 34. 
 
 Intestinal flora, 8. 
 
 Intracellular tox- 
 ins, 34, 109. 
 
 Invertases, 13, 32. 
 
 Inverting" fer- 
 ments, 13. 
 
 Involution forms 
 of bacteria, 26. 
 
 Isolation of bac- 
 teria, 107. 
 
 Isolysins, 133. 
 
 Kata.l>olic activi- 
 ties of bac- 
 teria, 10, 17. 
 
 Kirch er, 3. 
 
 Ritasato glucose 
 formate agar, 
 45. 
 
 bouillon, 45. 
 gelatin, 45. 
 Klebs, 5. 
 
 Klebs-Lroffler ba- 
 cillus, 214. 
 Koch, 5. 
 
 Kock-Week'si ba- 
 ' cillus, 221. 
 
 Jjactases, 13. 
 
 Latour, 4. 
 Leprosy, 237. 
 Lepra bacillus, 
 237. 
 
 Leptothrix, 274. 
 
 Leucocytic ex- 
 tract, 182. 
 
 Ligrht production 
 by bacteria, 17. 
 
 Linnaeus, 3. 
 
 Lipolytic, 13. 
 
 Lipases, 13. 
 
 Litmus gelatin, 57. 
 milk, 44. 
 neutral solution, 
 57. 
 
 Lower bacteria, 24. 
 
 Leeuwenhoek, 3. 
 
 Lumpy jaw, 276. 
 
 Lustgarten's ba- 
 cillus, 237. 
 
 Lysins, structure 
 of, 134. 
 
 Madura foot, 277. 
 
 Maltases, 13. 
 
 Malta fever micro- 
 coccus, 201. 
 
 Maliign*ant oede- 
 ma, 245. 
 
 Mallein, 117. 
 
 McConkey's bile 
 isalt broth, 45. 
 
 Meat extract, 38. 
 
 Media, 36, 37. 
 agar ag^ar, 41. 
 agar ascitic,Was- 
 sermann's, ^55. 
 agar bile salt, 
 McConkey's, 
 52. 
 
 agrar blood, 
 Washboum's, 
 55. 
 
 agar, Braun's 
 fuchsin, 53. 
 
 agar carbolized, 
 57. 
 
 agar glucose, 49. 
 
 agar earthy- 
 salts, Lipman 
 and Brown, 49. 
 
 ag-ar glucose 
 formate, Kit- 
 sat o, 45. 
 
 agrar glycerine, 
 56. 
 
 ag'ar Haricot, 50. 
 ag-ar Hesse-Hay- 
 
 den-Naehrtoff, 
 
 50. 
 
 agar litmus lac- 
 tose, Wurtz's, 
 52. 
 
 agar serum, Hei- 
 
 man's, 56. 
 agar serum, 
 
 'Wertheimer's 
 
 56. 
 
 agar sulphindi- 
 g-otate, Weyl's, 
 45. 
 
 asparagin, Us- 
 chinsky's, 45. 
 
 Beyrinck's me- 
 dia, 49, 50. 
 
 bile salt broth, 
 McConky's, 
 45. 
 
 bouillon, 41. 
 
bouillon carbol- 
 Lzed, 51. 
 
 bouillon iglucose; 
 
 formate, Kitas- 
 ato, 45. 
 
 bouillon glycer- 
 ine, 56. 
 
 bouillon glycer- 
 ine, potato, 52. 
 
 bouillon. Hari- 
 cot, 50. 
 
 bouillon inoslte- 
 free, Dun- 
 ham's, 46. 
 
 bouillon litmus 
 lactose, 57. 
 
 bouillon litmus 
 lactose, 
 Wurtz's, 52. 
 
 bouillon, Pari- 
 ett's, 57. 
 
 bouillon serum, 
 55. 
 
 bouillon sulphin- 
 
 digotate, 
 
 Weyl's, 45. 
 blood agar, Guy's 
 
 citrated, 42. 
 blood serum, 42. 
 blood serum, 
 
 Councilman 
 and Mallory, 56. 
 blood serum, 
 
 glycerine, 56. 
 blood serum, me- 
 dia, Loeffler's 
 
 56. 
 
 Capaldi-Pros- 
 kauer No. 1, 
 47. 
 
 Capaldi-Pros- 
 kauer No. 2, 
 47. 
 
 dextrose bouil- 
 lon, 44. 
 Dorset's egg, 43* 
 egg, 43. 
 
 egg albumin 
 agar, 55. 
 
 egg albumin 
 broth, Lip- 
 schutz's, 55. 
 
 filtration, 40, 60. 
 
 fish bouillon, 49. 
 
 fiuid, 37. 
 
 for chromogenlo 
 group of or- 
 ganisms, 48. 
 
 for coli-typhoid 
 group of or- 
 ganisms, 51. 
 
 for diptheria 
 bacillus, 56. 
 
 for diplococcus 
 meningitidis, 
 55. 
 
 for diplococcus 
 pneumonia, 55« 
 
 for earth organ- 
 isms, 49. 
 
 for gonococcus, 
 55. 
 
 for milk organ- 
 isms, 53. 
 
 for nitrous or- 
 ganisms, Win- 
 ogradsky's, 50. 
 
 for nitric organ- 
 isms, Wino- 
 gradsky's, 50. 
 
 for nitrogen fix- 
 ing organisms, 
 49, 50. 
 
 for phosphores- 
 cent organ- 
 isms, 49. 
 
 for photogenit 
 organisms, 49. 
 
 for plant organ- 
 isms, 50. 
 
 for tuberculosis 
 bacillus, 56. 
 
 for water organ- 
 isms, 50. 
 
 gelatin, 41. 
 
 gelatin carbol- 
 ized, 51. 
 
 gelatin Eisner's 
 potato, 53. 
 
 gelatin glucose 
 formate, Kit- 
 sato, 45. 
 
 gelatin litmus 
 lactose, 51 
 
 gelatin litmus 
 lactose, 
 
 Wurtz's, 52. 
 gelatin 'sulphin- 
 
 digotate, 
 
 Weyl's, 45. 
 hay infusion, 51. 
 iron bouillon, 47. 
 
iron peptone, 
 Pake's, 47. 
 
 lead bouillon, 47. 
 
 lead peptone, 47. 
 
 liquefiable, 38. 
 
 litmus bouillon, 
 46. 
 
 litmus milk, 44. 
 milk, 44. 
 nitrate bouillon, 
 46. 
 
 niitrate peptone, 
 47. 
 
 peptone, Dun- 
 ham's, 44. 
 
 potato, 43. 
 
 potato ;glycerin- 
 ized, 57. 
 
 proteid-free 
 broth, 46. 
 
 rice-milk, Eisen- 
 berg, 48. 
 
 rosalic acid pep- 
 tone, 47. 
 
 serum dextrose. 
 Hiss, 48. 
 
 special, 45. 
 
 sterilization of, 
 58. 
 
 tubing of, 57. 
 T^hey agar, 54. 
 whey gelatin, 54. 
 whey litmus, 53. 
 whey agar, 54. 
 whey gelatin, 54. 
 whey litmus, Pe- 
 truschky's, 53. 
 Measles, 281. 
 Metachromic 
 
 granules, 27. 
 Metatropic bac- 
 teria, 33. 
 MetchnikofC's pha- 
 gocytic the- 
 ory, 177. 
 Method of fixing 
 bacteria, 72. 
 of staining bac- 
 teria, 72. 
 Mesophilic bac- 
 teria, 33. 
 Micrococcus aqua- 
 tilis, 310. 
 candicans, 310. 
 catarrhalis, 200. 
 coronatus, 310. 
 
 intracellularis 
 meningitidis, 
 195. 
 
 melintensis, 201. 
 
 pharyngis, 201. 
 
 pneumonia, 192. 
 
 tetragenes, 186. 
 
 Zymogenes, 202. 
 Microscopic meth- 
 od for study 
 of bacteria, 
 70, 71. 
 Microspiro coma, 
 311. 
 
 Microsporon fur- 
 fur, 280. 
 Milk, 504. 
 
 analyses, 308. 
 Mobility of bac- 
 teria, 27. 
 
 amoeboid, 28. 
 
 Brownian, 28. 
 
 by indulating 
 membrane, 28. 
 
 by flagella. 
 Mouth flora, 8. 
 Muller, 3. 
 Mumps, 289. 
 Mycetoma, 277. 
 Mycorrhizas, 20. 
 
 Needham, 3. 
 
 Negri bodies, 287. 
 
 Neutral volatile 
 substances, 14. 
 
 Nitration, 19. 
 
 Nitrification, 19. 
 
 Nitrifying bac- 
 teria, 19. 
 
 Nitrogen assimila- 
 tion, 19. 
 
 Nitrogen cycle, 18. 
 fixation, 20. 
 
 Nitrosation, 19. 
 
 Nitrosococcus, 19. 
 
 Nocardia, 274. 
 
 Noguchi modifica- 
 tion of Was- 
 serman, 146, 
 148. 
 
 Noma, 289. 
 
 Nutrition of bac- 
 teria, 15. 
 
 Obenuier, 5. 
 
 Obligative aero- 
 bes, 15, 85. 
 
anaerobes, 16, 35. 
 Odium albicans, 
 280. 
 
 Opsonins, 179. 
 
 Opsonic index, 180. 
 
 Organic acids, 14^ 
 
 Organisms decol- 
 orized by- 
 Gram's, 78. 
 stained by 
 
 Gram's, 78. 
 allied to cholera 
 spirillum, 228. 
 
 Origin of bacteria, 
 5. 
 
 Oxidizing fer- 
 ments, 13. 
 
 FaJce's iron pep- 
 tone, 47. 
 nitrate peptone, 
 47. 
 
 Paracolon bacilli, 
 » 265. 
 group of organ- 
 isms, 265. 
 Paracliromophor- 
 ousf bacteria, 
 33. 
 
 Paratrophic bac- 
 teria, 33. 
 
 Paratyphoid bacil- 
 lus, 266. 
 group of organ- 
 isms, 265. 
 
 Parke bacillus, 
 269. 
 
 Pasteur, 4. 
 
 Pathogenic 'anaer- 
 obic organ- 
 isms, 240. 
 
 Pathogenic bacilli, 
 203. 
 
 bacteria, 32, 184. 
 moulds, 278. 
 properties of 
 
 bacteria, 105. 
 yeasts, 281. 
 Pathogenicity, 
 method of 
 \ study, 108. 
 study of, 105. 
 Pellagra, 290. 
 Peptone, Dun- 
 ham's, 44. 
 Pake, 47. 
 
 PfeifCer bacillus, 
 219. 
 
 Phagocytic the- 
 ory, 117. 
 
 Phagolysis, 177. 
 
 Phenol production, 
 test for, 94.' 
 
 Phosphorous cy- 
 cle, 22. 
 
 Photogenic bac- 
 teria, 33. 
 
 Pigment produc- 
 tion of bac- 
 teria, 17. 
 production, test 
 for, 95. 
 
 Pi'tyriasies versi- 
 color, 280. 
 
 Plasmolysis, 27. 
 
 Plate cultures, 60. 
 study of, 65, 68. 
 
 Plenciz, 4. 
 
 Plemorphism, 26. 
 
 Pneumonia, 261. 
 
 Pneumobacillus, 
 261. 
 
 Pneumococcus, 
 192. 
 
 Pneumoenteritis, 
 255. 
 
 Pollender, 4. 
 
 Potatoe media, 43. 
 
 Precipitins, 124, 
 156, 157. 
 
 Proteid differenci- 
 ation, 150. 
 
 Proteins differen- 
 ciation, 150. 
 
 Protein group in- 
 testinal bac- 
 teria, 310. 
 
 Proteolytic fer- 
 ments, 12, 32. 
 
 Prototropic bac- 
 teria, 33. 
 
 Pseudodiph- 
 
 theria bacillus, 
 218. 
 
 Pseudo influenza, 
 221. 
 
 Pseudo meningo- 
 coccus, 201. 
 
 Psychrophilic bac- 
 teria, 35. 
 
 Ptomains, 118. 
 
 Putrefaction, 12, 
 32. 
 
Quarter evil, 242. 
 
 Babies, 286. 
 
 vaccine, 168. 
 Ray fungus, 275. 
 Rayer, 4. 
 
 Reaction of media 
 optimum, 101. 
 
 Receptors, 124. 
 
 Reducing agents, 
 test for, 95. 
 ferments, 14, 
 
 Reckinghausen, 5. 
 
 Relapsing fever, 
 297. 
 
 Rennin, 32. 
 
 Reproduction of 
 bacteria, 28. 
 
 Resistance of leth- 
 al agents, test 
 for, 101, 02. 
 
 Respiratory flora, 
 9. 
 
 Resting stage of 
 
 bacteria, 28. 
 Rinderpest, 294. 
 Rindfleisch, 5. 
 Ring worm, 279. 
 
 fungus, 279. 
 Riva test, 319. 
 
 SaprbfTenic bac- 
 teria, 32. 
 
 Saprophytes, 32. 
 
 Scarlet fever, 283. 
 
 Scharomyces, 
 Busse, 282. 
 
 Scharomyces tum- 
 efaciens, 282. 
 
 .Schizomycetes, 23. 
 
 Schultz, 4. 
 
 Schwaun, 4. 
 
 Sensitizing body, 
 129. 
 
 Septicaemia, 118. 
 Septic tanks 332. 
 Sewage analyses, 
 332. 
 bacteria, 311. 
 streptococci, 311. 
 Shake cultures, 69. 
 Sheath of bacteria, 
 27. 
 
 Shiga bacillus,267. 
 Skin flora, 7. 
 Smallpox, 285. 
 vaccine, 166. 
 
 Smear culture, 68. 
 Smegma bacillus, 
 237. 
 
 Soft chancre, 224. 
 Soluble engymes, 
 14. 
 
 Soor fungus, 280, 
 South African 
 
 horse sickness, 
 
 293. 
 
 Spallanzanni, 3. 
 Special media, 45. 
 Spirillaceae, 23. 
 Spirillum cholera 
 asiaticae, 225. 
 Deneke, 229. 
 Finklei - Pry or, 
 229. 
 
 Massanah, 229. 
 Metchnikovi, 
 228. 
 
 Spirochetae and 
 allies, 295. 
 
 buccalis, 296. 
 
 calligyrum, 304. 
 
 Carter!, 300. 
 
 dentium, 296. 
 
 Duttoni, 300. 
 
 gallinarum, 298. 
 
 macrodentium, 
 304. 
 
 mouth of, 295. 
 
 Obermeiri, 297. 
 
 pallada, 300. 
 
 pertenuis, 304. 
 
 phagedens, 304. 
 
 refrigens, 296. 
 
 Vincenti, 296. 
 Spore, 36. 
 
 formation 28. 
 
 study of, 71. 
 
 germination, 30. 
 
 study of, 72. 
 
 resistance, 30. 
 Sporulation, 28. 
 Staining of bac- 
 teria, 72. 
 
 bacteria, acid 
 fast, 72. 
 
 bacteria, Mallory 
 method, 86. 
 
 bacteria, in tis- 
 sues, 86. 
 
 bacteria, in tis- 
 sues, Gram- 
 Wiogert meth- 
 od, 86. 
 
bacteria, in, tis- 
 sues, Loefller 
 method, 85. 
 Staining- of cafp- 
 sule, 81. 
 
 capsule, Hiss' 
 method, 82. 
 
 capsule, John's 
 method, 81. 
 
 capsule, McCon- 
 key's method, 
 82. 
 
 capsule, Muir's 
 method, 82. 
 
 capsule, Rilb- 
 bert's method, 
 82. 
 
 capsule, Welch's 
 
 method, 81. 
 Staining of flagel- 
 
 la, 83. 
 flag-ella, Bung-e's 
 
 method^ 84. 
 flagella, Loef- 
 
 fler's method, 
 
 84. 
 
 flag-ella, Muir's 
 method modi- 
 fied, Pitfeld, 
 84. 
 
 flagella, Pit- 
 feld's (method, 
 84. 
 
 flagella, Van Em- 
 engen's meth- 
 od, 84. 
 Staining: of spores. 
 82. 
 
 spores, Abbot's 
 method, 83. 
 
 spores, double 
 s^tain method, 
 82. 
 
 spores, Muller'is 
 method, 83. 
 
 spores, sing-le 
 stain method, 
 82. 
 
 Staining- techni- 
 que, 77. 
 technique, dif- 
 ferential, 78. 
 technique, dif- 
 ferential, 
 Gram's meth- 
 od, 78. 
 
 technique, dif- 
 ferential, 
 Gram's Claud- 
 ius* method, 
 79. 
 
 techniquev dif- 
 ferential. 
 Gram's negra- 
 tive method, 
 78. 
 
 technique, dif- 
 ferential, Nels- 
 ser's method, 
 80. 
 
 technique, dif- 
 ferential, 
 Pappenheim's 
 method, 79. 
 
 technique, dif- 
 ferential, Zi- 
 ehl - Nielson 
 method, 79. 
 
 technique, ordi- 
 nary, 77. 
 Stains, 72. 
 
 aniline fuchsin 
 violet, 73, 
 
 aniline gentian, 
 73. 
 
 alkaline methy- 
 lene blue, 73. 
 
 alkalinei methy- 
 lene blue, Lio- 
 efller's, 73. 
 
 alkaline methy- 
 lene blue^ 
 Koch's, 73. 
 
 Bismark brown, 
 74. 
 
 Stains, capsule, 81. 
 
 capsule, McCon- 
 key's, 74. 
 
 capsule, Muir''s 
 mordant, 75. 
 
 capsule, Rib- 
 bert's, 75. 
 
 carbolic acid so- 
 lution, 73. 
 
 contrast, 74. 
 
 contrast. eosin 
 solutions, 74. 
 
 contrast, neutral 
 aqueous solu- 
 tion, 74. 
 
contrast, saif- 
 franim aque- 
 ous solution, 
 74. 
 
 contrast, Vesuv- 
 ian solution, 
 74. 
 
 differential, 80. 
 differential. 
 Gram with 
 Bismark 
 brown, 80. 
 differential, 
 Hunt's modi- 
 fied, 80. 
 differential, 
 
 Neisser's mod- 
 ified. 80. 
 differential, 
 Wheal and 
 Chown, 81. 
 Bhrlich's haema- 
 
 toxylin, 77. 
 eosin alcoholic 
 
 solution, 74. 
 eosin aqueous 
 
 solution, 74. 
 flagella, 75. 
 flageUa, Bunge's 
 
 mordant, 76. 
 flagella, Loef- 
 
 fler's, 75. 
 flagella, Loef- 
 fler's mordant, 
 75. 
 
 flagella, Muir's 
 
 mordant, 75. 
 flagella, Pit- 
 
 feld's, 75. 
 flagella, Pit- 
 
 f eld's mordant, 
 
 75. 
 
 flagella. Van Em- 
 engen's flxing 
 solutioni 76. 
 
 flagella. Van Em- 
 engen's sensit- 
 izing solution, 
 76. 
 
 flagella, Van Em- 
 engen's reduc- 
 ing solution, 
 
 Gabtoett's acid 
 
 blue, 74. 
 Gram's iodine 
 
 solution, 74. 
 
 Kuhne's meth- 
 lene blue> 73. 
 
 Mayer's alum 
 carmine, 77. 
 
 Mayer's Haema- 
 tin, 77. 
 
 Papenheim's, 76. 
 
 Niesser's, 76. 
 
 Nicolles carbol- 
 thionin, 76. 
 Stain, simple ani- 
 line, 73. 
 
 special, 76. 
 
 Spengler's, 76. 
 
 stock solutions, 
 
 73. 
 
 Unna's borax 
 methylene 
 blue, 74. 
 
 Unna's poly- 
 chrome methy- 
 lene blue, 74. 
 
 picro - carmine, 
 77. 
 
 Ziehl's carbol- 
 fuchsin, 74. 
 
 Standardization of 
 media, 39. 
 
 Staphylococcus, 
 184. 
 
 pyogenes, 184. 
 pyogenes, albus, 
 186. 
 
 pyogenes, au- 
 
 rens, 185. 
 epidermidosis, 
 albus, 186. 
 Sterilization of 
 
 media, 58. 
 Stomach flora, 8. 
 Streptococcus, 187. 
 anginosus, 191. 
 equinus, 191. 
 fecalis, 191. 
 mitis, 191. 
 pneumonia, 191. 
 pyogenes, 187. 
 rheumaticus of 
 Poynton and 
 Paine, 192. 
 Structure of bac- 
 teria, 26. 
 Sulphur cycle, 21. 
 Synthetic activi- 
 ties of bac- 
 teria, 17. 
 
Temperature opti- 
 mum, test for, 
 97. 
 
 Tetanus, 240. 
 
 Tetanus antitox- 
 ine, 163. 
 
 Thermal death 
 point, 35, 98, 
 99, 100. 
 
 Thermophilic bac- 
 teria, 35. 
 
 Thiohaoteria, 24. 
 Winogradsky, 16. 
 
 Thrush, 280. 
 
 Tinea barbae, 279. 
 cirinata, 279. 
 megalosporon, 
 279. 
 
 microsporon, 279. 
 syncosis, 279. 
 tonsmans, 279. 
 
 Tox albumins, 34. 
 
 Toxins, 34, 109, 
 108, 109. 
 
 Toxin analysis, 
 152. 
 
 Toxoids, 153. 
 
 Toxons, 153. 
 
 Trachoma, 294. 
 
 Treponema palli- 
 da, 300. 
 
 Trichophyton, 279. 
 
 Trypanosome, 295. 
 
 Tube cultures, 61. 
 
 Tuberculins, 174. 
 
 Tuberculosis, 229. 
 avian, 235. 
 bovine, 235. 
 
 Tuberculosis of 
 cold iblooded 
 animals, 236, 
 
 Typhoid fever, 270. 
 
 Typhoid grroup of 
 organisms, 
 270. 
 
 Typhoid vaccine, 
 171. 
 
 Typhus fever, 284. 
 
 Ultra microscopic 
 organisms,290. 
 
 Uschinsky's aspar- 
 agin, 45. 
 proteid free 
 t)roth, 46. 
 
 Vaccines, 165. 
 
 in general, 170. 
 preparation of, 
 171. 
 
 Vegetative stage 
 of bacteria, 28. 
 
 Vibro cholera, 328. 
 
 Vibro septique,245. 
 
 Vincent's angina, 
 . .296. 
 
 Virulency of path- 
 ogenic organ- 
 isms, 105. 
 of attenuating 
 or lowering, 
 106. 
 
 of raising or ex- 
 alting, 105. 
 
 Waldeyer, 5. 
 
 Wasserman reac- 
 tion, 141, 145. 
 Water, 309. 
 analyses, 314. 
 bacteria, 309. 
 flora, 309. 
 Weigert, 5. 
 Weyle's sulphindi- 
 gotate bouil- 
 lon, 45. 
 gelatine, 45. 
 Whooping cough, 
 289. 
 bacillus, 222. 
 Winogradsky 's ni- 
 tric organism, 
 media, 50, 377. 
 nitrous organ- 
 ism, media, 50, 
 377. 
 
 silicate jelly,338. 
 Wooden tongue, 
 276. 
 
 Yaws, 304. 
 Yellow fever, 292. 
 
 Zoo^loea, 26. 
 
 Zymogenic (bacte- 
 ria, 32. 
 
UNIVERSmr OF ILLINOIS-URBANA 
 
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