UC-NRLF **0 A^ ^^^. n B M aiD 331 FATE OF TUBERCLE BACILLI OUTSIDE THE ANIMAL BODY BY CHARLES FRANCIS BRISCOE A. B. Indiana University, 1899 A. M. University of Illinois, 1905 THESIS Submitted in Partial Fulfillment of the Requirements for the Degree of DOCTOR OF PHILOSOPHY IN BACTERIOLOGY THE GRADUATE SCHOOL of the UNIVERSITY OF ILLINOIS 1912 FATE OF TUBERCLE BACILLI OUTSIDE THE ANIMAL BODY BY CHARLES FRANCIS BKISCOE ^ ' A. B. Indiana University, 1899 A. M. University of Illinois, 1905 THESIS Submittea in Partial Fulfillment of the Requirements for the Degree of DOCTOR OF PHILOSOPHY IN Bacteriology IN THE GRADUATE SCHOOL of the UNIVERSITY OF ILLINOIS 1912 T • • a* • • • UNIVERSITY OF ILLINOIS Agricultural Experiment Station BULLETIN NO. 161 FATE OF TUBERCLE BACILLI OUTSIDE THE ANIMAL BODY By CHAS. F. BRISCOE URBANA, ILLINOIS, NOVEMBER, 1912 3i^77 Letter oe Transmittal University of Illinois, June, 1912. Dr. Eugene Davenport, Director of the Agricultural Experiment Station. Sir : I have the honor to transmit and recommend for publication the accompanying paper prepared under my direction by Chas. F. Briscoe, Ph.D., upon the Fate of Tubercle Bacilli Outside the Animal Body. This is a companion bulletin with No. 149 of the . University of Illinois Agricultural Experiment Station issued under date of February, 191 1, entitled Tuberculosis of Farm Animals, by Chas. F. Briscoe and W. J. MacNeal. That bulletin was based upon a general study of the disease in bovine ani- mals — its characteristics, modes of dissemination and infection, methods of recognition and prevention, and the relation to tuberculosis in man. The import of the present paper is well expressed in the title, with the understanding that the studies were made principally upon the bovine type of the causative organism, Much previous work had been done upon the same subject, as is indicated in the literature herein cited, but important contribu- tions to existent knowledge are made in the results of the experiments em- bodied in this publication, while earlier announcements have been confirmed or disproved. The whole matter is of such tremendous significance that any additions to knowledge and any further dissemination of knowledge upon the subject is of wide and vital importance. The information here presented will be welcomed by specialists, sanitarians, and the general public. T. J. Burrill, Chief in Botany {including Bacteriology). Summary of BullKTin No. 161 1. There are four recognized types of tubercle bacilli; human, bovine, avian, and a type that infects cold-blooded animals. Only the first two types have any important part in the infection of man. Page 284 2. The beaded appearance of these germs led the' early inves- tigators to a belief in spore-formation. This is nov^ known not to occur. The fatty content of the bacilli varies from 10 to 42 percent, which is five times as much as found in any other micro- organism. It appears that this fatty material has little or nothing to do with the duration of their viability. Page 284 3. The tubercle bacillus does not secrete a soluble toxin, but that poisons are formed is well known. It has been shown by various investigators that tubercles can be produced in test animals by the injection of dead cultures. Tubercles thus produced may be mistaken for those produced by living germs. Page 284 4. A clear criterion of death is necessary in reporting results on the duration of life of the tubercle bacillus. ^This fact many investigators have disregarded. Page 285 5. The tubercle bacillus may be classed with the nonspore- bearing organisms as to viability ; but in this class it is one of the most resistant, especially as to drying and to the antagonism of decay organisms in water and foul matter. Page 286 6. The action of sunlight upon bacteria was first studied by Downes and Blunt in 1877. Numerous investigators have con- tinued this study to the present day. One of the most important facts brought out is that l^acteria when directly exposed to the sun are killed in a few minutes. This is due to the effect of the ultra violet light. The ultra violet rays are now cheaply produced artificially by the mercury vapor lamp. This lamp is destined to play an important part in sterilization and disinfection. Pages 287-291 7. It is shown by the results of all the investigators given in Table 2 that tubercle bacilli when exposed directly to the sun are killed in a few minutes to a few hours. The time of killing is less at higher altitudes; but it is ten to fifteen times longer in diffuse light. Pages 292-295 8. Tuberculous sputum reduced to dust and inhaled by test animals causes tuberculosis. A much less amount is necessary to produce tuberculosis by inhalation than by ingestion. This, how- 279 ever, must not be taken to indicate that inhalation plays a more important part than ingestion as a cause of tul>erculosis. The more important method depends upon the opportunity of infection from each. Investigators do not agree on this question. At pres- ent both inhalation and ingestion should be considered dangerous sources of tuberculous infection. Pages 295-306 9. The Mills-Reincke phenomenon, which has been given a mathematical ecjuivalent by Hazen's theorem, viz., ''Where one death from typhoid fever Jias been avoided by the use of better water, a certain number of deaths, probably two or three, from other causes have been avoided," has been found by Sedgwick and MacNutt to be sound and conservative. Their studies show that tuberculosis has decreased in certain cities of Massachusetts, which decrease is evidently due, in part, to the improvement of their water supply. Pages 307-308 10. It is reported in the literature that tubercle bacilli live for a very long time, several months to more than a year, in water and other material. Pages 309-310 11. In experiments to determine the time that tubercle bacilli live in various conditions the chief difficulty is the ''index of death" for these germs. This is true since cultivation of the tubercle bacilli from contaminated material is not feasible, and since the dead germs produce, in test animals, tubercles indistin- guishable by microscopic appearance from those produced by live tubercle bacilli. Pages 31 1-3 13 12. Pure cultures of nonspore-bearing organisms and the vege- tative cells of spore-bearing germs when exposed to direct sun- light in thin smears are killed in ^ to 6 minutes ; the human, bovine, and avian types of tubercle bacilli exposed in the same way were killed in i to 4 minutes. Pages 314-317 13. When exposed to desiccation in a dark, well-ventilated place, the nonspore-bearing organisms and the vegetative cells of spore-bearing organisms died in i to 4 days; spores of B. subtilis and B. vulgatiis' used as controls were not killed in 35 days ; the human and bovine type of tubercle bacilli exposed at the same time and under the same conditions were dead within 4 and 8 days re- spectively. Pages 317-318 14. Pure cultures of bovine tubercle bacilli mixed in cow manure and exposed in a two-inch layer in a pasture field in the sunshine remained alive and virulent for two months. Pages 323-3^4 280 15- As would be expected, these germs exposed in cow manure retained their virulence longer in the shade than in the sunshine, as shown both by the greater severity of the disease produced in the guinea pigs inoculated with the germs exposed in the shade, than that produced in the guinea pigs inoculated on the same day with the germs exposed in the sunshine, and by the greater length of time that the guinea pigs which were inoculated with the germs exposed in the sunshine remained alive. Pages 325-326 16. Tubercle bacilli in the manure of a naturally infected cow exposed in the same manner as the artificially infected manure were dead within two weeks after exposure. Pages 327-334 17. Tubercle bacilli in garden soil and in a dead tuberculous guinea pig buried in garden soil were alive on the 213th and the 71st days, respectively, and dead on the 230th and 99th days, after first exposed. Pages 334-339 18. Tubercle bacilli live for more than a year in running water. A watering trough harboring these germs may be a dangerous source of infection to cattle. The better disposition of dead tuber- culous animals is to destroy by burning. Tubercle bacilli in drink- ing water is one of the possible sources of infection for man. In- fection is not prevented by dilution, since clumps containing a great number of these organisms may be inclosed in mucoid ma- terial w^hich prevents their separation and destruction. Pages 340-359 19. Tubercle bacilli in market butter placed in cold storage live for more than ten months, which is a longer time than such butter is usually kept in storage. Pages 359-363 20. General discussion. Pages 364-365 21. References. Page 366 281 FATE OF TUBERCLE BACILLI OUTSIDE THE ANIMAL BODY By CHAS. F. BRISCOE, Instructor in Botany' INTRODUCTION The fact that one-tenth of all deaths in the human family are due to tuberculosis, and that millions of dollars worth of farm animals are lost annually from its ravages, makes any knowledge concerning the fate of tubercle bacilli outside of the animal body of great value. These questions have often come from stock owners : How long is it necessary to keep healthy stock from a field where tuber- culous cattle have been previously allowed to run? How long do tubercle bacilli live in manure, in a watering trough, and in a dead tuberculous animal ? And again, some authors have questioned the reports in the literature that tubercle bacilli remain alive and viru- lent for periods of a year and more outside of the animal body. They have suspected that in such cases the tubercles, found in the test animals after inoculation with such tuberculous material, kept for these long periods, had been produced by dead tubercle bacilli. It has been, in part, the purpose of this bulletin to answer these questions. The first section is devoted to brief notes on the biology of the tubercle bacillus; then a tabular review of the literature is given vvith brief discussions; and lastly an account of the experimental work, followed by a general discussion. The experiments deal with pure cultures of tubercle bacilli, and with those of some other bacteria for purposes of comparison as to their duration of life in sunlight and under desiccation; and with the time limit of life of tubercle bacilli in cow manure, garden soil, water, butter, and dead tuberculous animals. BIOLOGY OF THE TUBERCLE BACILLUS MORPHOI OGY "^^^^ tubercle bacillus varies in form according to type, method of growth, and age of the individual culture. It is a slightly curved, rod-shaped organism measuring from 0.3 to 0.5 microns in diameter and from 2 to 5 microns in ^Now Professor of Bacteriolog-y in the Mississippi Agricultural Experiment Station, Agricultural College, Mississippi. 283 284 ' . Bulletin No. i6i [November: length. There are two well-recognized types that infect mammals, the human and the bovine. The latter is shorter and thicker and stains more uniformly. The avian type, found in domestic fowls and birds, and a type that infects the cold-blooded animals, espe- cially fishes and frogs, play almost no part in the infection of man. The tubercle bacillus often presents a beaded appearance. This is more common in the specimens found in old pus and sputum. It is due to a fragmentation of the protoplasm. This peculiar struc- ture of the organism led the earlier scientists to the belief in spore formation. Koch^^, in his first paper, Die Aetiology der Tuhercii- los, held to this belief, and this idea of spore formation has found place in many publications even to the present day. It is now known, from the relation of these organisms to the action of heat, sun- shine and chemical disinfectants, that they do not form spores They are killed very readily, in thin layers, by the direct rays of the sun, and also at a temperature of 60° C. in fifteen to twenty minutes. In these respects they are like other nonspore-bearing organisms. Their power of resistance to drying and to the SUBSTANCE antagonism of decay organisms appears to be greater than that of other nonspore-bearing or- ganisms, tho less than that of the spore organisms. This power of resistance is no doubt due, in part at least, to the content of waxy or fatty substance found largely in the outer layer of the tubercle bacillus. The presence of this waxy material gives them their well-known character of "acid proof" power when stained. (When this waxy or fatty substance is extracted with ether, stains are no longer held on treatment with an acid or alcohol.) The bacillus has the largest amount of fatty substance of any known micro-organism. The fat content varies, according to different in- vestigators, from 10 to 42 percent; while in other micro-organisms an alcohol ether-extractive has been found to vary only from 1.7 to 10. 1 percent. The tubercle bacillus does not secrete a soluble Th'e ^^ERCLE ^^^^'^^ ^^ ^o ^' diphtheria and B. tetani. It has not BACILLUS l^eei"i demonstrated that the tubercle bacillus forms a true toxin. Levene^"^ proved the absence of tox- albumins from extracts of the bacillus. That poisons are formed is well known, tho their character is not understood. Baldwin^^ thinks that the symptoms and toxemia of tuberculosis are accounted for by the presence of the nucleic acid products in the blood. Koch'^-'^, Von Prudden and Hodenpyle^ ''"'•'', Vissman^''^'*, Straus and Gamaleia^**, Sternberg^ '*•''•, Krompecher^^, Miller^', and Rosenau^^^ /p/i?] Fate of Tubercle Bacilli Outside the Animal Body 28S have produced Uiberculosis in lest animals (guinea pigs and rab- bits) by injecting dead cultures. These results have been confirmed in the laboratory here. (See experiments page 312). The injection of dead cultures killed by heat or DEADCULT'uRES^^y chemical disinfectants will produce necrosis, alDscesses, caseation, emaciation, and death. So closely do these lesions resemble those produced by the living germs that it is difficult to know whether we are dealing with dead or with living cultures. Rosenau^^^ suggests that much of the work done upon the duration of life of tubercle bacilli has little value for lack of a clear criterion of death. Lesions from dead cultures, as from live ones, may be characterized by giant cells. Tuberculin cannot be depended upon to distinguish between the lesions pro- duced by dead tubercle bacilli and those produced by live germs. The reactions caused by the injection of 2 cc. of tulDerculin under the skin of guinea pigs with lesions produced by dead cultures are similar to those caused by a like injection into guinea pigs with lesions produced, from living cultures, even to the death of the guinea pig. Most investigators have not taken into account the fact that dead tubercle bacilli produce lesions, and in reporting their results have taken for granted, when at autopsy even local lesions have been found, that the tubercle bacilli have been alive and virulent. The only safe method of distinguishing is by inoculating a second- ary healthy guinea pig with a small amount of the tuberculous material from the lesions of the original test animal. If the bac- teria are alive, there develops a generalized tuberculosis, usually severe; if dead, either no lesions at all or only a slight localized effect will occur. Cultivations from this tuberculous material from both the original and the secondary test animal will give further evidence. If the organisms are dead, no growth occurs; if alive, growth is usually evident. This method, tho time consuming, is ecessary to obtain the most trustworthy information. That the tubercle bacillus does not possess the re- '^^'■ppj?'^ ^^ sistance to external agents as do the spore-bearing piLLi TO OTHER •^^S^^^sms IS Unquestioned. The spores of Bact. ORGANISMS anthracis have been known to hold their virulence from ten to twelve years (Aiello and Drago^). We have found that the spores of B. subtilis dried on an agar slant and remaining in this state for eight years, gave growth when seeded into broth. It might be expected, however, that tubercle bacilli, from their waxy-fatty content and from their analogy to spores in respect to staining qualities, would be more resistajit to 286 Bulletin No. i6i [November, external injurious agencies such as heat, drying, Hght, chemicals, and putrefaction. This, in general, is found not to be the case. Rosenau^-'^ says, "The tubercle bacillus may be classed with the nonspore-bearing organisms so far as its viability is concerned." This statement is surely correct with respect to heat and light. The thermal death point of the tubercle bacillus, as determined by the more careful investigators (Herr^^, 1901 ; Hess^^, 1901 ; Rus- sell and Hastings 127^ 1904; and Rosenau ^^^ 1908), is given at 60° C, with an exposure of from fifteen to twenty minutes. This is practically the same as the thermal death point determined for most other nonspore-bearing organisms. (Sternberg^^^, Smith^*'^^). As regards desiccation, and more especially the antagonism offered by decay and foul matter, tubercle bacilli appear rather to take an intermediate position between spore- and nonspore-bearers. Rickards, Slack and Arms^^^ found that tubercle bacilli in sputum resisted drying for 88 days; LeNoir and Camus^^ found these organisms alive after 33 days ; Kuss"^^ found them alive from 20 to 30 days, and killed in 40 to 60 days; NoeteP'^^ found them yet living after 35 days. Maffucci^^ shows that pure cultures of avian tubercle bacilli dried on silk threads live for 14 days. Kirstein^'^ finds pure cultures dried in dust living from 3 to 8 days. In my own experiments they lived 8 days. Olher nonspore-bearing organ- isms die in a very short time, as B. violaceus, i day; B. typhoid, 3 days; and B. coli, 3 days. There are reported in the litera- ture cases where nonspore-bearing organisms have resisted drying, under special conditions, for a very long time. Rosenau^ ^-^ finds B. pestis to live over four months on a piece of dry sponge. Sirena and Alessi^^^ report that the pneumonia diplococcus, when dried on silk threads kept in a moist room, did not die until after 192 days. More comparative work must be done with cultures of different nonspore-bearers and with tubercle bacilli exposed under the same conditions before definite conclusions can be drawn. In the presence of foul material tubercle bacilli live from a month to a year and more (see Table 7). In my own experi- ments they were always found to live in water for 202 days and as long as 441 days. In this work much care was taken to deter- mine that the tuberculosis produced in the test animals was by living germs. Careful workers like Jordan, Russell and Zeit^^ have found B. typhosus living in sewage from 3 to 4 days only ; Russell and Fuller^ 2^ report from 8 to to days. It would be expected that tubercle bacilli protected by the mucoid material, as found in sputum and diseased tissues, in which these germs more frequently occur, and also by their abundance of naturally waxy constituents, Kjij] Fate of Tubercle Bacilli Outside the Anmmal Body _ 287 would be protected against drying and injuries from the presence of foul material. This conclusion is well borne out by the litera- ture and by the experimental data given in this bulletin. REVIEW AND DISCUSSION OF THE LITERATURE The Action of Sunlight on Bacteria Historical The killing power of sunlight on bacteria was first noted by Downes and Blunt^^ in 1877. They worked with mixed cultures suspended in culture solutions and found that the organisms were killed in a short time, as shown by the solutions remaining clear. Tyndall^^'^ questioned the results of Downes and Blunt but later published results'""^^ confirming them. Duclaux^" (1879) was the first to test the action of sunlight upon pure cultures and thus place the work upon a scientific basis. Arloing^ (1885) was the first to test the action of light upon pathogenic bacteria {Bad. anthracis). He was also the first to use the electric light in su)cji experimental work. Feltz^^ (1890) was the first to test the action of light upon tubercle bacilli. The classic works of Buchner^^ and Ward^^'^ (1892-3) deserve special mention. Buchner was the first to ex- hibit the killing power of light in reproducing characters by the growth of the organisms in that part of the Petri dish protected from the sunlight. The organisms are thickly seeded in a solid medium like agar, and the desired characters cut from black paper are fastened over this Petri dish. The organisms exposed to the sunlight are killed ; where protected by the black paper, they grow, forming the letters. Ward called especial attention to the effect of different rays of the sun, which he separated by the use of screens of colored glass and colored solutions. There early arose the questions : To what is the killing of bac- teria due? and. Are spores more easily killed than the vegetative cells? Arloing^ (1885) showed that spores of Bacf. anthracis in broth were killed in two hours, while to kill the vegetative cells required from twenty-six to thirty hours. Nocard^^^ (1885) sug- gested that during the exposure the spores developed into bacilli and the light acted upon the more sensitive vegetative cells. Straus^ ^^ (1886) apparently confirmed this suggestion of Nocard by exposing anthrax spores in broth and in distilled water. He found that the spores in the broth were killed in nine hours while those in distilled water were not killed in this time. In 1886 Ar- loing*^ repeated his experiments, exposing the cultures on ice so that the spores could not develop, and confirmed his earlier results. Roux^-''"' (1887) finds the explanation in the fact that spores are more readily killed by the action of sunlight in the presence of 288 Bulletin No. i6i [November, oxygen than when the oxygen is excluded. He thinks that from the nature of the spore constituents more oxidation products are formed in the spore than in the vegetative cell, and that for this reason the spore is more readily killed. Dieudonne^^ (1894) con- cludes that the formation of hydrogen peroxid by the action of sunlight on the presence of oxygen forms an important part in the killing of bacteria. Kruse"^^ (1895) and Richardson^^* (1893) also came to this same conclusion. Thiele and Wolf^*^ (1906) carried out carefully planned experiments in which their cultures of ^. prodigiosiiSj B. pyocyaneus and B. coli were exposed in the presence of air, oxygen, or hydrogen. They took special precau- tions to confine the gases used, with mercury joints, so as not to allow the slightest diffusion. They found that the bacteria were killed as readily anaerobically as aerobically, and conclude, there- fore, that the killing of the bacteria by light is not assignable to the indirect influence of oxidation of water. They exposed their cultures in broth diluted i- 1000, physiological salt solution and Elbe river water. It can readily be shown that by the exposure of physiological salt solution to sunlight for the length of time the investigators ex- posed their cultures there is formed some condition sufficient to kill bacteria. The question whether the bactericidal action is due to an injurious chemical formed by the action of the sunlight, or whether it is a direct bactericidal action of the light, is still unset- tled. The facts thus far established indicate that the action is due to both a chemical and a direct bactericidal action, sometimes one and sometimes the other being predominant, depending upon the condition of the experiment. A more important fact, which has been especially emphasized in a recent publication (see Weinzirl, Table 2) is that bacteria when directly exposed to sunlight are killed in a few minutes, in contrast to most of the published results of a few hours to a few days. This is due to the well-known germicidal effect of the ultra violet rays. Since glass is an excel- lent screen for these rays, an exposure of bacteria in glass contain- ers lengthens very greatly the time necessary to kill. Powerful beams of ultra violet rays, are now artificially pro- duced with the c|uartz mercury vapor lamp. A number of these machines are upon the market. The rays from them are so effec- tive that bacteria directly exposed in thin layers are killed almost instantly. Indeed, so strong are the most powerful of these lamps that the skin accidently exposed is killed and later sloughs off. It is stated that the sterilization of a city water supply can be done economically with these lamps. A test^ at Marseilles, France, shows that the cost per million gallons of water is only ten dollars, 191^] Fate of Tubercle Bacilli Outside the Animal Body 289 i. e., one cent per looo gallons. This indicates a feasible system of supplying a city with a good, potable water from a contaminated source. Years ago it was suggested by Marshall Ward that the dis- infection of cow sheds might well be done with electric lights. Tho this was thought fanatical at the time, it may be feasible with such light as the present powerful mercury vapor lamp produces. Why cannot the "toning up" effect that is to be felt in a room which has been exposed to the sunlight thruout the day be in a way brought about by exposing to one of these powerful mercury vapor lamps, for a few hours in the evening, such rooms as have no access to the sun, at the same time securing the great benefit that comes from disinfection by its powerful beams? For further discussion of this most interesting subject we refer our readers to the recent numbers of the periodicals, more espe- cially the chemical and engineering journals. The following table gives a brief review of the results found in the literature upon the action of sunlight on bacteria. Table 1 Action of Sunlight Upon Bacteria Author Date Conditions Not killed Killed Downes and Blunt^' Duclaux^^ Duclaux^^ Duclaux''^ Arloing*' Straus' ^^ Arloing-* Downes^=* Roux^^' 1877 1879 1885 1885 1885 1886 18S6 1886 1887 Cultures in Pasteur solutions Tyrothrix filiformis spores Tyrothrix scaber from milk: A drop dried in an empty flask jl4 days In broth culture vegetative cells. . . Kept in the dark 3 yrs. Micrococci, young- cultures veal broth: j In dark jover 1 yr. In sunshine, May to June | In sunshine, July. ..... In dried condition Bact. anthracis spores in broth 1 to IK hrs. Bact. anthracis vegetative cells in broth 2 hrs. Bact. anthracis spores: In broth In distilled water 9 hrs. Bact. anthracis spores in broth on ice 4° C Bacteria in mixed cultures, diffuse light Bact. anthracis spores: Without air 83 hrs. With air 5 to 6 hrs. 35 days 60 days IS days 40 days 15 days 8, 3 and even 2 days after 2 hrs. 26 to 30 hrs. 9 hrs. 5 hrs. 5 days 29 to 54 hrs. 290 Bulletin No. i6i Table 1.^— Continued [November, Author Date Conditions Not killed Killed Pansini'«8 1889 B. violaceus, B. prodig-iosus, B. pyo, cyaneus, Bact. anthracis, Msp comma, Murisepticus, and Staph* pyogenes aureus: Exposed to diffuse daylight, devel- opment hindered 24to48hrs. E^xposed to sunshine in tube cultures 1 day. In hang-ing drop on needle >^ to 2 J^ hrs. I^aurent**'' 1890 Bacillus of Kiel on potato 1 to 3 hrs. 5 hrs. Janowsky«2 1890 B. typhosus in culture media 4 to 10 hrs. Buchner*® 1892 B. typhosus on agar plates 1 to iy2 hrs. Buchner^*^ 1893 B. typhosus, B. pyocananeus, and Msp. comma on plates 1.6 meters under water A% hrs. Geisler^" 1892 B. typhosus (almost all killed) 3 to 6 hrs. Momont^oo 1892 " Asporegene anthrax" in phenol broth: Dried and exposed in air ■. . 5 to Sy2 hrs. 6}^ hrs. Dried and exposed in vacuo In moist condition in the air 2>^ hrs. In moist condition, no air 50 hrs. Dried anthrax spores in the air 100 hrs. Dried anthrax spores in vacuo 100 hrs. Moist anthrax spores in the air 44 hrs. Moist anthrax spores in vacuo . 100 hrs. Ward^" 1893 Bact. anthracis spores on agar 4 to 10 hrs. Frankland and 1893 Bact. anthracis spores in Thames Appleyard'** . river water: In diffuse daylight ^ In direct sunshine Bact. anthracis vegetative cells: 6 mo. 151 hrs. In unsterilized water. 56 hrs. 84 hrs. L CLl.±\^ M.OV^XXCl.^C^J. ..«..•••.....•• Gaillard*» 1894 B. typhosus in water 3 to 4 hrs. Dieudonne'^ 1894 B. coli, B. typhosus, Bact. anthracis, B. prodigiosus, and B. fluorescens By electric arc light 900 candle- % to 2,1^ hrs. power 5 hrs. 8 hrs. Bact. anthracis spores: In the air. 3'A hrs. Air excluded 3K hrs. B. tetani spores, air excluded 9 hrs. Kruse^« 1895 Bact. anthracis spores in hanging drop 1^ hrs. 2 to 5 hrs. B. typhosus: ' In broth, air present 2}4 to 7 hrs. In broth, air replaced with hydrogen 7 hrs. Bact. anthracis, diffuse light (13 days 80 days) ^ Tho the spores were not killed, they were so weakened that one cubic centi- meter did not kill mice. A broth culture of the same in much smaller quantity killed mice. /9/-'l Fate of Tubercle Bacilli Outside the Animal Body TABiyB \.— Continued. 291 Author Date Beck and Schultz' " Gehrke^** Kedzior^'' Jones" ^ Mettler^-'"' Huber«^ Thielei*' and Wolf Weinzirl'«« 1896 1899 1899 1900 1904 1905 1906 1906 Neumark^ Orsi'*'« McNaug-htand Korich*'-* 1907 1907 1910 Conditions Not killed Killed Bacteria Bacteria: In clear water Ag^ar cultures B. pyocyaneus, B. diphtheria, Msp. metchnikovy, and .spores of Bact. anthracis, exposed on g-elatin B. cartovorus, directly exposed. ...... Msp. comma, B. typhosus, and B. coli in broth culture Streptococci and B. diphtheria in broth B. coli, and B. typhosus in diluted broth, Elbe river water, or physi- ological salt solution Eixposed directly to the sun in thin smears: B. cholera suis, B. prodigiosus, B. ty- phosus B. typhosus (repeated), B. dysenteria, B. prodig-iosus (repeated), B. pyocyaneus Msp. comma B. diphtheria Pus cocci B. coli B. friedlander B. phosphorescens Sar. aurantiaca. Pink air micrococci Eixposed to direct sunlight on agar plate: B. anthracis spores B. anthracis veg"etative cells B. coli - *'Schweinpest bacilli" Chicken cholera Schwein erysipelas B. typhosus and Msp. comma in sun- lififht B. coli in distilled water B. coli: In distilled water in air at 0° to 1S°C. in diffuse daylight 25 days In strong sunlight, numbers much decreased. 6 hrs. In strong sunlig-ht ... j Dried in a desiccator Ill days 5 min. 6 hrs. short time 2 to 8 hrs. 6 hrs. 1^ to "hyi \\x 10 min. few hours 2 to 3 hrs. 9 hrs. 5 to 10 min. 2 to 5 min. 2 to 6 min. 5 min. 5 to 15 min. }4 to 2 min to 1 min. 1 to 2 min. 90 min. 120 min. 25 to 30 min. 20 to 25 min. 60 min. 110 to 120 min. 8 to 10 min. 240 min. 8 to 10 hrs. 12 hrs. 12 hrs. 292 Bulletin No. i6i [November, The Duration of Lii^E of Tubkrcle Baciuj in Sunsiiink and IN Diffuse Dayught INDEX OF Every author consulted, except Weinzirl, used the DEATH animal test to determine when tubercle bacilli were dead. Weinzirl, using only pure cultures, em- ployed the method of cultivation. No investigator whose work is recorded in Tables 2 and 3 has called attention to the fact that dead cultures produce tuberculosis (for discxission see page 112). Just what error a neglect of attention to this point has introduced into the results given in the following table cannot be definitely known. Tho it is certain that dead tubercle bacilli do produce tuberculosis, yet in no case was it found in my own experimental work that localized tuberculosis was produced by dead organisms. In every case the secondary guinea pigs inoculated from cases of localized tuberculosis became tuberculous and usually severely infected. Cul- tures from diseased tissues produced growth, either from the origi- nal or from the secondary inoculated guinea pigs, or from both. It appears, therefore, that the error arising from not taking into account tuberculosis produced by dead cultures is probably slight. TIME REQUIRED P^^^ cultures of tubercle bacilli when exposed in TO KILL thin layers to the direct sunlight are killed in from a few minutes to a few hours. This has been the result obtained by all investigators. To kill these germs in sputum requires only a slightly longer time. The mucoid mass is a slight protection which increases with the thickness and opacity of the material, but even here tubercle bacilli are killed in a very short time. With two exceptions, that of Feltz^^ and of Mitchell and Crouch^^ (see Table 2), no investigator reports these bacilli surely living after twenty-four hours' exposure to the sun. In these two cases the sputum was exposed on soil, and a small layer of soil would afford good protection from the action of the sunlight. EFFECT OF Experiments definitely planned to test the killing HIGH ALTITUDE power of the sun on tubercle bacilli at different altitudes have been carried out by a Russian, Tres- kinskaja^^^. In our country Mitchell and Crouch^"^ have determined the effect of the sun on tuberculous sputum at a height of 1356 meters. Treskinskaja used pure cultures emulsified in one-percent peptone solution, spread in thin layers and exposed directly to the sun. These were killed as follows: At a height of 1560 meters, in three hours ; at 903 meters, in four hours ; at sea level, in four hours. The peptone solution when dried gave a thin protective layer covering these organisms. Treskinskaja thought that this protection would be about equal to that of a thin layer of sputum. IQI2] Fate of Tubercle Bacilli Outside the Animal Body 293> The difference in killing power is due to the difference in ultra violet light. These rays are largely absorbed by the atmosphere, especially when laden with moisture. According to Langley, only 39 percent of the ultra violet light reaches the sea level. The higher the altitude, the dryer the atmosphere, the more intense is the light and the greater its killing power. Mitchell and Crouch found that tubercle bacilli in sputum were not killed in 35 hours, but were killed in 45 hours. These experi- ments are not comparable with the experiments of Treskinskaja, done at nearly the same altitude, since the soil and sputum give far more protection than exposure in a thin layer on glass from a one-percent peptone solution. DISCREPANCIES ^^ ^^ ^^^^ ^'^ ^^^ expected that tubercle bacilli under IN RESULTS different conditions will be killed in the same length of time. It is indeed difficult to expose different pure cultures under the same conditions. There may be a difference in the age of the culture, in the uniformity of the emulsion, in the manner of exposing, and in the means of deter- mining the time of killing the germs. When exposed on threads of linen or silk some of the germs may be well protected and live for a much longer time than when exposed in a thin layer on a glass slip or on sterilized glazed paper. Then if the material in which they are exposed, as sputum, is of such a nature as to give protection from drying as well as from the rays of the sun, the difference will be still greater. This accounts, partly at least, for the diff'erence in the results of Sawitsky^^^, which showed that tubercle bacilli in sputum on linen threads lived two and one-half months, and the fact reported by WeinzirP^^, that a pure culture in thin layers exposed directly to the sun was killed in a few min- utes. It is not possible to give definite reasons for all the discrep- ancies found in the results shown in Table 2 ; but excepting those of Feltz^^, w^hose material mixed in the soil might well give com- plete protection from the sun, it will be seen that tubercle bacilli exposed to the sunlight are killed within a few minutes to a few hours. DIFFUSE LIGHT It takes ten to fifteen times as long for tubercle bacilli to be killed in diffuse light as in direct sun- light; yet these germs are killed much sooner in diffuse daylight than in the dark, when under the same conditions otherwise, as may be seen by comparing the results of Rickards, Slack and Arms^^^, TwichelP^^, and Ransome and Delepine^^*. Tables 2 and 3, respectively, give in brief the literature upon these subjects. 294 Bulletin No. i6i [November. Tabi^E 2.— EjFifECT OK Sunshine; on Tuberci.!? BACiTyi.i Author Date Conditions Not killed Killed Feltz»« 1890 Sputum and soil mixed and exposed to direct sunlight, tested by gui- nea pig inoculation 137 da3's after 137 Exposed to changing climatic condi- days flQJJS ., ,.,,« 2K mo. after2>^mo. few min. to few hrs. Koch''' 1890 In pure culture Sawitskyi2 8 Ransome and 1891 Soutum on stretched linen 2^ mo. Delepine^i^ 1894 Soutum exoosed to lisrht 45 days 4 days (12 >4 hrs. sun) Pure culture on glazed paper Renzi' 1 ' 1894 Sputa mixed with 10 parts water, ex- nosed at 28° C . . . 6 hrs Migneco»« 1895 Sputum on stretched linen and woolen 10 to 15 hrs. 24 to 30 hrs. 2 hrs. Straus' ■'=' 1895 Culture in a glass container, in broth In a thick layer of dried sputum 1 to 12 hrs. G-ardiner^'^ 1898 Tri ^hrs. Jousset«« 1900 Sputum exposed to direct sun Another similar experiment Ihr. 5 to 7 hrs. 1 hr. Mitchell and 1900 Tuberculous sputum placed upon Crouch^* sterilized soil 35 hrs.* 45 hrs. Abba and Barelli^ 1901 6 days Annett* 1902 Tested 105 specimens of sputa taken at random; 5, or 4.76 percent con- 4.76 percent 2 to 24 hrs. 1903 Tuberculous sputum size of spitting. 48 hrs. Cadeac^® 1905 Tuberculous sputum on a board 24 hrs. Tuberculous sputum on glass 24 hrs. 48 hrs. Bang-i^ 1905 Tubercle bacilli on agar, exposed to a ~ 30-ampere lamp at 30 cm. distance 6 min. TwitchelP^" 1905 Tuberculous sputum, direct rays 1 hr. 7 hrs. Didonna^** 19U6 Exposed culture to sun 2 to 8 hours; then inoculated guinea pigs in order to immunize them. Pro- duced only a local abcess 2 to 8 hrs. 2 WeinzirP«" 1906 Pure culture on paper slips or glass covers 2 to 10 min. Rickards, 1909 Sputum exposed in sunshine 6 hrs. Slack and Arms*i» 148 Treskinskaja 1910 Summer months, direct sun: Pure culture tubercle bacilli, height 1560 m Pure culture tubercle bacilli. 903 m Pure culture tubercle bacilli, sea level ... • 3 hrs. 4 hrs. 4 hrs. * Virulence diminished after 20 hours. ^It is not certain whether the organisms were alive or dead, since dead organ- isms can produce tubercles. j(?i^] Fate of Tubercle Bacilli Outside the Animal Body 295 TABI.E 3 Action of Diffuse I^ight Upon Tuberci^e Bacii,i.i Author Date Galtier^« 1889 Sawitskyi28 Ransome and Delepine^'* Ivucibelli»« 1892 1894 1899 Jousset®*' 1900 1902 Hill«» 1903 Cadeaci« TwitchelP^" 1905 1905 Sormanii3« Rickards, Slack and Arms^i* Weinzirli^» 1906 1908 1908 Conditions Not killed jKiUed Tuberculous org-ans: Dried at 30° Dried at room temperature Sputum on stretched linen Tubercle bacilli on dried paper Sputum dried on glass Fluid sputum in reagent glass Sputum, Series 1 Series 2 Sputum in fine drops on glass: Mixed with dust On threads On coarse cloth Sputum in reagent glass in glass cup- board Sputum on glass • Tuberculous sputum in paraffined bot- tles Dry sputum in a room Dry sputum in a room • • Fine emulsion of tubercle bacilli dried on paper slips (average of ten trials) 38 days (some) 30 days 4 to 7 hrs. 5 days 20 days 2 to 6 days 124 days 1 mo. 1 mo. 2.8 days 30 days (usually)af- ter 30 days 2J4 mo. 2 days 18 days 4 mo. 4 to 7 hrs. 8 to 14 days 8 days 10 days 16 days 4 to 10 days 175 days 4.4 days Effect of Desiccation upon Tubercle Bacilu in Sputum AND Other Material The difference in the length of time that different bacteria withstand desiccation is very great. B. cartovorus is killed in a few minutes to a few hours (Jones^^) ; B. tuberculosis may be killed in a few days ( 14 days, Maffucci^^) to several days (88 days, Rickards^ ^^) ; and spores of Bact. anthracis live twelve years (reported above). The difference depends upon the kind of substance on which they are exposed and upon the difference in the kind and form of organism exposed. Harding and Prucha^^ have shown that Bact. campestrc remains alive much longer when dried on cabbag^e seed than when dried on glass covers; on glass it was dead at the end of ten days ; on the seed it remained alive for thirteen months. This difference is no doubt largely due to the difference in the hygroscopic moisture retained by these substances. The kind and form of the organism exposed to drying has even more to do with its capability of living. The spore form lives very much longer than the vegetative form. 296 Bulletin No. i6i [November, It appears that tubercle bacilli, especially in sputum and other mucoid material, withstand desiccation better than other nonspore- formers. The difference is not great and there are many apparent exceptions where other nonspore-bearers live for a very long time, like the one given above after Harding and Prucha^^. Diplococcus pneumonia has been reported as living for 192 days when dried on silk (Sirena and Alessi^^^). The important fact which has been so thoroly established in recent years is that tuberculous sputum reduced to dust causes tuberculosis when experimental animals (guinea pigs, rabbits, cats, dogs and calves) are made to breathe such dust-laden air. How great the danger is to man and cattle to breathe dried tuberculous material is yet a disputed question. In 1882 Koch, in his work on ''Die Aetiologie der Tuberculose", pointed out that dried tuber- culous sputum is one of the most important factors in causing tuberculosis. It was not until 1905 when the work of Calmette and Guerin^^ was published that ingestion was brought forward as a very important factor in producing tuberculosis. It was sug- gested by the advocates of this theory that practically all tubercu- losis is produced by ingestion — that even inspired material is swal- lowed and what apparently is inhalation tuberculosis is actually tuberculosis by ingestion. This conclusion will not hold in the light of the most recent experiments, especially those carried out at Breslau and collectively published by Fliigge^^ (1908), ''Ver- breitungsweise and Bekampfung der Tuberkulose". It is shown here that in all animals with which experiments have, been made it required a hundred to a thousand times more tubercle bacilli to produce tuberculosis by feeding than it does by inhalation. So the thought that the swallowing of a part of the few tubercle bacilli necessary to produce tuberculosis by inhalation is the cause of in- gestion tuberculosis is wholly precluded. A complete discussion of this most interesting subject cannot be given here. The data in the two following tables show the fact mentioned above, that it requires many times more germs to pro- duce tuberculosis by feeding than by inhalation ; also another most important fact, that severe tuberculosis may be produced by either method of infection. It must not be understood that the facts exhibited in these two tables in any way indicate which is the more frequent method of infection. For what matter if It does require one thousand times more tubercle bacilli to produce tuber- culosis, by feeding than by inhalation, who knows whether we are, on the average, taking in one thousand times more of these germs in the food than in the breath? On one hand, Calmette^^ is found declaring that ''In the ordi- nary daily life, the infection of the digestive^ organs is predomi- n;i. Fate of Tubekcle Bacilli Outside the Animal Body 297 to S . 5 o rt c o tfl "-• OJ ra 'd ^ tJ (U O 4) tc 3 t« s s g 1> n jL "^ A> ?i 3 »: o * X ? O lis ^■^ u CI rt 3 -O 'd fi a CO o u o :3 CO a to 'd > 'd c ri 3 o QJ ■-J (U ri J3 -4-> ■M M ^ c U-) a o (1) -♦-• ii! 'd 'S'rt ■Ij" a •d s 'u s 3 S.8 SO CO 'd art o s ri "d c ri CO ■M ho a "> o CO e c o c c a a a *> ■> '> '> o o o o PQfQWW a fi a ri ri rt BBB 3 3 3 WWW s s a a a 3 03 a (U a ^3 ri a B 4) ^ (U W < 298 Bulletin No. i6i [November, a .t! <^ o ri 1^ . ■ cti (U JI tQ •-1 fl'^^;:.^ o 00 Cfl 'O c/5 O ^ o t/3 O }h in ;- ; t/3 r1 .1-1 be o O -M B->^ rt ^ ^ o 4> OJ o '-' LJ G r- oj (1) (u r" O t>. rt -d fO rt c . fl^ be •J bci:.S « u O h£ +++ + + e+ a; o 9 '^ S'd "" ^ ^ ^ rt > 3 o ^ 0) > o o W W be be be bo_ be be a o o be en o O be be u o cd c^ cd o o o be be bo bo be be C! c fl o s 3 o o o bo o « pq M 1^ a ^ o -d c rt © (1> c T^ '0 -^^ o ;x, o 19 1 2 Fatk ov Trr.ERci'.E Bacilli Outside the Anjmal Body 299 iiant" ; aiul on the other hand Chausse-'' says, "I'nherenlosis in adult bovine is due to inhalation in 98 percent of the cases, if not more; in calves in about 90 percent, ingestion and congenital in- fection being responsible for the remainder." Dr. RaveneP^^, a strong believer in ingestion as one of the important causes of tuberculosis says, "One must take an impartial view of the v^hole problem, and be willing to agree that both channels of infection are open. In animals, however, the alimentary tract seems to be a more common port of entry". At present, at least, both ways of infection should be consid- ered very dangerous and every precaution taken to guard against them. A brief summary of the literature concerning the length of time that tubercle bacilli live in dried tuberculous material and concerning the infectiousness of such material is given in Table 6. TABI.K 6. -Effect of Desiccation of Tuberci^e Bacii,i.i in Sputum and Other Materiai^ Author Date Conditions Not killed Killed Villemin^^^ 1869 1882 1883 1883 1884 1886 1886 1887 1888 • 1888 1888 Dried tuberculous sputum several hrs. 8 wks. 3 wks 12 days 126 days 186 days 2 mo. 6 mo. 20 to 38 days 43 days 76 days 80 days 150 days 15, 30 and 38 days 30 days Koch^-* Dried tuberculous sputum. Cochez24 Malassez and VignaP^ Schill and Fischer^ **♦ Tubercle bacilli incased in sputum Dried sputum killed a dog-, by inhala- tion Alternate drying and moistening of tuberculous sputum 8 times Tuberculous sputum with spores, dried on glass 179 days 226 days 10 mo. Tuberculous sputum without spores, dried on glass DeThoma^^ Dried tuberculous sputum Sormani' ^^ Dried tuberculous sputum virulence Galtier'*® Tuberculous sputum dried on linen. . . Dried tuberculous material deceased thereafter no virulence after 4 mo. Cadeac and Malet^^ Pieces of dried tuberculous lung ex- posed on paper in laboratory Dried tuberculous lung allowed to de- compose in outer air 102 days Same repeated. Same repeated after 150 De Souza=« Inhalation of dried tuberculous mate- rial caused tuberculosis in 12 of 14 guinea pigs. Time of drying not stated days Galtier^« Inoculation of tuberculous material dried at 30° C, or by breathing dried tuberculous material Same material dried at room temper- ature after 30 days 300 Bulletin No. i6] 'November, Tabi,k 6 Continued Author Date Conditions Not killed Killed Cornet27 Feltz3« Sawitskj^2 8 Stone^''" Koch^2 Maffucci^o Marpmann^^ Ransome and Delepine^*^ Hance»3 1889 1890 1891 1891 1892 1892 1893 1894 1895 Kirchner*'* Dust samples examined for tubercle bacilli were found positive: In hospitals In insane asylums In dwelling" houses Dried tuberculous sputum in road dust: Kxposed to weather Kxposed to sun 1895 Dried sputum, in rooms Tuberculous sputum dried under or- dinary conditions and exposed in the dark in living" rooms Same exposed to sunlight Dried tuberculous sputum, only de- creased in virulence Guinea pigs were inoculated with tuberculous sputum dried for 2 weeks, for 4 weeks, and for 8 weeks. In each case tuberculosis developed as with fresh material. Pure culture of avian tubercle bacill dried on silk threads, inoculated into a hen abdominally Same repeated. Hen not tuberculous abdominally; one tubercle in the lung Tubercle bacilli frequently found in street dust by microscopic test . . . Tuberculous sputum exposed in watch glasses: To air and light 4 days; then 15 days in the dark To air and light 8 days; then 11 days in the dark In closed cupboard In dark air shaft, produced slight tuberculosis in a rabbit The author, under the direction of Dr. Trudeau of the Adirondack Cottage Sanitarium, examined 81 samples of dust from the differ- cottages. Four guinea pigs died too early to make the test for tu bercle bacilli; of those remaining 5, or 4.9 percent, became tubercu lous. These were from a group of 10 guinea pigs inoculated with the dust from one small cottage. The author later inoculated 9 guinea pigs with dust from a New York hospital, 5 of which died in 3 days, and 1 of the 4 remaining became tuberculous Fourteen guinea pigs were inoculated with 7 samples of dust from rooms in homes of consumptives; 9 guinea pigs died early and 5 re- mained healthy. In a second ser- 47.6 percent 17.6 43.6 over 7 mo. about 140 days 7 to 9 mo. IV2 mo. 2^ mo. 3 yrs. 2 to 8 wks. 14 days 2 mo. (?) (?) 19 days 19 days 45 days 2 mo. (?) 19 days 4.9 percent 25 percent all killed i(jij\ Fate of Tuisekcle Bacilli Outside the Animal Body 301 Table 6 Continued Author Date Conditions Not killed Killed Kirchner ^'^ (continued) Cornet2« Laschtsch- enko^^ Neisser^**-' Beninde^' Cornet^e Sticker^39 Jones** Peterson' Heymann®® 1893 1898 1898 1899 1899 1899 1900 1900 1901 ies three rooms were investig-ated, from which 8 guinea pigs were in- oculated; one died early and one of the other 7 became tuberculous. Tuberculous sputum placed on a car- pet and allowed to dry naturally produced tuberculosis in 35 out of 36 guinea pigs which were placed in a room at a distance of 134 to 300 cm. from tbis carpet Tuberculous sputum dried in thick Jayers on paper and cloth was not virulent after 9 months Tuberculous sputum rubbed up with dust and aspirated at a velocity of 3 to 5 cm. per second will pro- duce tuberculosis, when inocu- lated into guinea pigs, in a large majority of cases Handkerchiefs that were used by con- sumptives from 2 to 12 hours were dried for 24 hours, and then rubbed up in a box. The dust from these was aspirated and injected into two guinea pigs for each test. In 12 tests 8 were positive and 4 nega- tive Dried sputum in one case lost viru- lence in 3 months; another time remained virulent from 6 to 8 months Tested 29 samples of sputum dried from 2 to 21 days, by inoculating guinea pigs with dried sputum dust and by causing them to inhale the dried sputum dust. Tuberculosis was produced in 27 out of 29 tests. The author inoculated guinea pigs interperitoneally with the mucus from the nostrils of 131 healthy persons. Three of these guinea pigs died of tuberciilosis on the 8th, 14th and 59th days after in- oculation. No tubercle bacilli could be found by stains of the nasal mucus ^ One of 8 guinea pigs became tuber- culous after inhaling sputum, dried for 2 months; while sputum dried for 3 months gave one posi- tive in 6 The aspirated air from sputum, dried 6 "days and injected into guinea pigs, produced tuberculosis 14 percent 8 positive 8 mo. Positive 27 of 29 tests 9 mo. 4 negative 3 mo. 2 mo. 3 mo. 6 days Ut is probable that these three guinea pigs, or at least the first two, died of spontaneous tuberculosis. 302 Bulletin No. i6i Tabi^E ^—Continued. [November, Author Date Conditions Not killed killed Heymann^-' (continued) Gotschlich Hill^ 1903 1903 NoetePo^ 1904 Cadeac*^* Kirstein«» 1905 1905 Dust from rooms of consumptives: Samples taken on a brush No. of No. of Percent samples positive positive Private rooms 59 5 8.5 Hospitals 61 5 8.2 Samples taken on a moist sponge Private rooms 57 9 15.78 Hospitals 62 25 40.3 Total sa'ples 239 44 18.4 Hospitals 123 30 24.3 Pri'te homes 116 14 12.0 No tubercle bacilli were found in 119 samples of dust taken from 15 places where consumptives were in the habit of visiting. Samples of tuberculous sputum from 10 rooms, using" 496 swabs, were , taken by rubbing them on carpet, ' furniture, etc. In 8 of the 10 cases the sputum was proven to contain tubercle bacilli from stained preparations; the other two were not tested. Tubercle bacilli dried on glass rods were dead after 16 days. Clothing that had been and that was being worn daily by consumptives was enclosed in a large box of three cubic meters content. The dust was beaten and shaken out and allowed to settle; then it was collected and thoroly rubbed up in 5 cc. of broth. The original material, and dilutions of .1 and .01 were respectively injected into guinea pigs: Coat and vest worn daily Jacket and hose worn daily New jacket, hose and old vest with no evidence of contaminated sputum. Old coat Coat, hose and plush vest, none worn for three weeks Wool jacket and old hose, not worn for five weeks Tuberculous sputum exposed on glass to air and light Tuberculous sputum, in thick layers dried on a marble slab Tubercle bacilli from pure culture or from tuberculous sputum were sprayed upon collected dust, and then after different lengths of time tested for virulence by inoc- ulation of guinea pigs. Dust from: Positive Positive Negative Positive Positive Positive 2, 4and6dys 4,6andlOdy3. 14 days 191-'] Fatf-: of Ti'UKRCLE Bacilli Oi'Tsini-: the Animal Body 303 TABiyR 6. — Continued Author Date Conditions Not killed 1 JCilled Kirstein^" (continued) Park and Williams' »" Tvvitchell»^« Sormani^*^ lycNoir and Camus'*® Rodetand^^* Delanoe Kohlisch' 1905 1905 1906 1907 1907 1908 Books and papers Sputum dust Cloth ravelings Street dust Dried tuberculous sputum gradually loses its virulence, but still infective Tuberculous sputum: In paraffined bottles in dark moist box In paraffined bottles in diffuse light In paraffined bottles in thermostat. In cotton-stoppered bottles in dark moist box In cotton-stoppered bottles in dark closet On ice Tuberculous sputum: On handkerchiefs. On towels On carpets In sand in moist light place In sand in dry light place. In open bottles, out doors, winter . . Dried tuberculous excretion at 35° C. L/arge quantities of air were aspirated from tuberculous hospitals and the dust injected under the skin of guinea pigs. I^rom 4 tests aspi- rating respectively 270, 2000, 20000 and 53000 liters, in no case were tubercle bacilli found Eleven samples of dust from local offices and private dwellings were inoculated into guinea pigs, with wholly negative results Fresh tuberculous sputum or a pure culture of tubercle bacilli was inti- mately mixed with dust obtained from dwellings and factories, and the number of tubercle bacilli that a guinea pig inhales in a given time was determined. It was found that tuberculosis was pro- duced in the test animals when as low as 50 tubercle bacilli were in- haled, and that the inhalation of 270 or more of these germs al- ways produced tuberculosis. An- other series of experiments were made with dust from 15 dwellings where consumptives were living. The rooms were small and poorly kept. E^ighteen guinea pigs breathed for some time in dense clouds of this dust. None of these guinea pigs became tuberculous. 8 days 4 days 5 days 3 days 2 to 3 mo. 14 days 7 daj^s 10 days 8 days 170 days 124 days 33 days 188 days 175 days 100 days 157 days 172 days 100 days 102 days 141 days 153 days 70 days 70 " 110 days 110 " 39 " 70 " 123 " 148 " 30 " 70 " 110 " 132 *♦ 15 days all 11 sampl»^s 304 Bulletin No. i6i Table 6 . —Continued [November, Author Date Conditions Not killed Killed Kuss^ 1908 Tuberculous sputum dried in thin layers in the dark, free to the air: Virulence wholly preserved Virulence diminished Virulence much decreased, yet a larg-e dose produced a tuberculous g-uinea pig- Virulence entirely lost Tuberculous sputum, dried in thin layer, in diffuse light of a room rapidly decreased in virulence: Noticeable decrease Marked decrease Very noticeable decrease Not entirely lost Entirely lost Two g-uinea pig's were exposed to the dust made by shaking- of hand- kerchiefs on which tuberculous sputum had been dried for 25 days. Neither became tuberculous Six guinea pigs were exposed to the dust from a plank containing sputum that had been drying for 17 days. Remained healthy Experiments in a box of 125-liter con- tent: Tuberculous sputum dried for 6 days in the dark was powdered and 1 to 2 grams suspended in the air of this box. Guinea pigs with their heads projecting into this box were made to breathe this dust from 30 to 60 minutes. In all cases the guinea pigs became tubercu- lous Experiments in a room of 30 cubic meters: Series i — 125 cc. of tuberculous sputum were dried on a carpet from 11 to 13 days. Nine guinea pigs were swung 60 to 90 inches above this carpet. Then the car- pet was beaten and swept for 20 minutes. E^our of the 9 guinea pigs became tuberculous. Also the dust obtained by the aspira- tion of 60 liters of air, at a point 60 inches above this carpet, was inoculated into a guinea pig, which became severely tubercu- lous Series 2. — 20 cc. of tuberculous spu- tum were dried on another carpet for 6 days. Six guinea pigs were suspended above this carpet, which was beaten and swept for 12 minutes. Two of the 6 guinea pigs became tuberculous. Also 12 to 14 days 18 days 20 to 30 days 3 days 7 days, 10 days 15 days 40 to 50 days 20 days 25 days 15 days 6 days 11 to 1: days J0T2] Fate of Tubercle Bacili.i Outside the Animal Ropy 305 Tabi,!? 6 Continued Author Kuss'^ (continued) lyeNoir and Camus" ^ Date Conditions IveNoir and Camus" ^ 1908 1908 1908 dust obtained by the aspiration of 71 liters of air produced a tuber- culous guinea pig" Series j. — Six of the 9 guinea pigs exposed to the beatings and sweepings of the two carpets of Series 1 and 2, described above, became tuberculous Tuberculous sputum rich in tubercle bacilli was mixed with dust a part of which had been sterilized, another part not sterilized, and each mixed sample was dried in a large open flask and kept in the laboratory for 33 days. Each sam- ple was inoculated into a series of 5 guinea pigs: Series J.— Bust not sterilized. Three of the 5 guinea pigs became tu- berculous only in the lymphatic glands near the point of inocula- tion* Series 2 Dust sterilized. Three of the 5 guinea pigs died of acute in- fection; the other 2 had tubercu- lous lymph glands near the point of inoculation, and one of the two had a tubercle in one lung* Again, the author examined dust from a tuberculous hospital: Series j. — The dust was collected from the cornice near the ceiling, wash-board, window-sill, window- seat, bed railings and floor. All was thoroly mixed, divided into two lots of .6 gram each, and placed in two flasks. One flask was protected from the light; the other was exposed to diffused light for 5 days and to sunlight 3 days. Each sample was inocu- ulated into a series of 5 guinea pigs. In each case only one of the 5 test animals became tuber- culous. Series ercle bacilli a part of which showed signs of disintegra- tion. Glycerine egg media and a guinea pig were inoculated with pus material from the point of inoculation and a liver abcess. Neither of these revealed living tubercle bacilli. A third experiment was made using the same EXPERIMENT Ml strain of tubercle bacilli. An emulsion was made in sterile 0.8 percent salt solution. The emul- sion showed a faintly milky appearance. Three tubes of glycerine egg were seeded with this emulsion of tubercle bacilli and Guinea Pig 1666 was injected interperitoneally with i cc. The remaining portion of the emulsion was divided into two parts : one was heated at 85° C. for ten minutes, the other was heated in the autoclave at 115° C. for ten minutes. With each of these portions three glycerine egg slants were seeded and a guinea pig was in- oculated interperitoneally with i cc. The unheated portion gave excellent growth and produced severe generalized tuberculosis in Guinea Pig 1666. The part heated to 85° C. for ten minutes gave no growth on either of the three glycerine egg tubes after six weeks incubation at 38° C. Guinea Pig 1167, killed 6y days after inoculation, showed local tuberculosis in the right superior inguinal near the point of inoculation and a few small lesions in the liver. All other organs were apparently healthy. A stained preparation from pusl material of these lesions showed tubercle bacilli. Cul- tures on glycerine egg and the inoculation of a second healthy guinea pig from this material gave negative results. The part heated to 115** C. for ten minutes neither gave growth on the glycerine egg media nor produced any effect when i cc. was in- jected interperitoneally into Guinea Pig 1168. I. Dead cultures, when not killed at too high a CONCLUSIONS temperature, produce tuberculous lesions in guinea pigs. 2. Secondary guinea pigs inoculated from tuberculous material from lesions produced by dead cultures always remain healthy. 3. In determining the length of time tubercle bacilli live when exposed to various conditions outside the animal body, it is nec- essary to inoculate a second healthy guinea pig, especially when only local lesions are produced in the guinea pig inoculated with the original material, in order to be sure the tubercles are not produced by dead tubercle bacilli. 314 Bulletin No. i6i [November, Ei'p'KCT OF Light Upon Si^ori;- and Nonspokk-Bkaring Organisms A few spore-bearing and nonspore-bearing or- CULTURE organisms were tested by exposing them to the direct rays of the sun. The spore-bearing organ- isms, B. subtilis and B. mesentericiis viilgatiis, were grown npon peptoneless agar (made with three grams of beef extract and fif- teen grams of agar per liter) for six days at 32° C. An examina- tion of the cultures showed an abundance of well-developed s'pores. The vegetative cells of these two organisms were obtained by re- peated growths in beef broth. The broth cultures were kept at ^y° C. and repeated every twenty-four hours for six days. A microscopic examination at the end of this time showed no spores. B. diphtheria cultures were obtained from twenty- four hours' growth upon Loeffler's blood serum. All other organisms used were from fresh broth cultures grown at the optimum temperature for the organism tested. MANNER OF The cultures grown on the solid media were sus- EXPOSURE pended in 0.8 percent salt solution. A concen- tration of slightly milky appearing emulsion was formed. Then a loop of this emulsion was spread in a very thin smear on either a small slip of sterile glazed paper or a small sterile glass cover. These were then exposed to the direct rays of the sun for definite periods of time between the hours of ten in the morning and three in the afternoon. The exposed slips, with the exception of those containing B. diphtheria, were aseptically drop- ped into sterile broth and incubated at a temperature of 30° C. for one week. The exposed smears of B. diphtheria were seeded on IvOeffler's blood serum and incubated at 37° C. for one week. The cultures were carefully examined for growth, and if growth was evident further tests were made to see if it was the same as the original culture or an accidental contamination. P_Q,,,_e * A summary of the results is given in Table 8. It will be noted that the nonspore-bearing organ- isms were all killed in a few minutes. ( J^ to 6 minutes). While the spore-bearing organisms were not killed in the time exposed (180 minutes). 79/^] Fate of Tubercle Bacilli Outside the Animal Body 315 TabIvE 8. — Organisms Exposed to Direct Sunl,ight Name of organism Not killed Killed B. subtilis spores^ B. subtilis, spores^ , B. mesentericus vulg-atus, spores B. subtilis, vegetative cells B. mesentericus, vegetative cells B. prodigiosus B. diphtheria B. coli B. typhosus B. violaceus 120 min. 180 min. 180 min. Yi min. 1 min. 6 min. 2 min. 1 min. 2 min. 1 min. 2 min. Yz mini after 6 min. 3 min. 2 min. 3 min. ^Time of exposure was not continued longer; no end point was reached. Effect of Direct Sunlight Upon Tubercle Bacilli CULTURE AND "^^^ effect of direct sunlight was tested several EMULSION times upon cultures from three types of tubercle bacilli. The organisms used for exposure were always from active cultures grown upon glycerine ^gg from two to three weeks. A heavy emulsion was made by rubbing up some of the culture on the inside of the neck of a sterile glass-stoppered graduate flask with a sterile glass rod. From time to time a few drops of sterile o.8 percent salt solution were added. At first the culture was rubbed into a fine paste with the addition of only a few drops of the salt solution; then about 6 cc. of o.8 percent salt solution were added and, the contents shaken thoroly. The emulsion then showed a decidedly milky appearance and was filtered thru sterile glass wool to remove the larger clumps. The examination of a stained preparation, made in a similar way as the smears that were used for exposure to sunlight,, showed the in- dividual organisms usually well separated. There were, however, clumps of twenty-five to thirty organisms still to be found. PREPARATION Smears from the prepared emulsions were made of^smearI^'^^ upon small slips of sterile glazed paper. Pins pushed thru the corners of these small paper slips were then stuck into the bottom of a pasteboard box having a snug fitting lid. The lx)x and the slips were sterilized at 150° C. for one hour. A small loopful of the emulsion was smeared in a thin layer upon each of the sterile glazed paper slips. These were ex- posed at once to the sun for the desired length of time. Triplicate smeared slips for each period of exposure were seeded upon culture media suitable for the growth of tubercle bacilli. The media used was 5 percent glycerine beef -juice agar or glycerine tgg. 316 Bulletin No. i6i [November, The glycerine tgg was prepared by opening, aseptically, ten good fresh eggs and pouring the whole contents into a sterile flask. To the well-beaten eggs there were added 200 cc. of 5 percent gly- cerine beef broth. This emulsion was placed in sterile test tubes by means of a sterile Pasteur's bulb pipette. It was solidified, in a slanting position, at y^° C. for two hours. After placing the exposed slip on the surface of the media, it was rubbed and scraped by means of a small sterile platinum spatula so as to dislodge some of the dried tubercle bacilli, and a small amount of the glycerine egg or glycerine agar was smeared over this slip until it was thoroly moistened. The cotton stoppers were flamed, dipped into parafline and pushed into the tubes, which were then stopped with paraffined stoppers. The cultures were incubated at 38° C. for one month and then carefully examined for growth. Table 9 indicates the method of recording the re- RESULTS suits for the separate tests. A summary of the results of the various tests from the three types of cultures is given in Table 10. It is seen that these germs are killed in a very short time ( i to 4 minutes) . These results agree with those of Weinzirl (see Table 2). Tabi,e 9.— BacilIvUS Tubercui^osis, Human Type Exposed to Sunshine (Between 11 and 12 A. M., Nov. 2, 1911) Time of exposure, minutes Conditions Result of g-rowth Remarks Controls, not exposed + Eixcellent errowth (( (( (( + (( (( (( H- (( (( Yz Sun bright, no clouds. . . + (( 4< i (< (( i( (( + a (( 11 ( ( ( ( (( +? Contamination 1 <( n a (< + Excellent growth 1 " *' '* " + Moderate growth 1 (( (< (< (( + . btiri' 2| rO «r> ro 00 lO rH „ I> l> t>. ^O VO vo lO 0) a 'oj a s J3 '-M S r^ ri ri «-i a^ t^ ,H "S lO O ^O VO lO -"t -"t S a he s 3 .§ 00 00 00 00 00 vO vo be ^ 0) > ^0 'S 7i P "S 2 0) S VO 00 vo CO rO M ■* VO lO -^ tJ- T}- T-( s s H 00 00 00 C?\ 00 no lO ^s -^ c« J^ O O «.9^.^.oi^i:^. rH rO VO t> X 1-H S.^.S.^.^ t>.* I> VO 00 rO o a\ lO iH tH •^- 0^ O tH tH M cOiO 00 c> >% 'O ;3 o O rH n CO 'O lO O tH ro 6 >^>^ 5 lO CO O CO vo tH 00 c ; ,H C^ CO UJ !>. a\ v^ rt ^ ri CO a\ CO o t^ CO u rH CO CO Tl- TJ tc a> 1> O r-( a^ rH CO .-( T-( CO '<1- 00 c<) r-. iH tH 0) o VO '^ ?i iS 00 gj o a; tn . r-t r-t 15 iJ bcbcfcio-^^- > • Q 1 3 S 3 D O O ^ -< < -< 'X O ^ hS igi2] Fate of Tubercle Bacilli Outside the Animal Body 323 of the test; the number of clear days durmgthis time and also the number of partly and of wholly cloudy days. There is also given the number of hours of sunshine, the amount of rainfall, the highest maximum daily temperature and the lowest minimum daily temperature occurring between each test ; and an average daily maximum, and an average daily minimum temperature during the time of exposure up to the time the test was made. There is also given the average monthly temperature occurring during the time of exposure. ResuivTs of the Tests FROM ARTIFI- Bxposed in the Sunshine. — ^The results of testing FECTED * ^^^^ samples of cow manure infected with a pure MANURE culture of tubercle bacilli exposed in the sunshine are given in Table 13. As previously mentioned (page 320), the test of the sample on July 29, 19 10, the day it was first exposed, produced two severely tuberculous guinea pigs. Since exposure seven tests have been made. On the 7th and i6th days the four guinea pigs inoculated contracted severe generalized tuber- culosis. Only miscroscopic and cultural tests were made from the two guinea pigs inoculated on the 7th day of exposure, since the infection in both guinea pigs was so severe it appeared certain to be from living tubercle bacilli. This was later shown to be true from the culture tests. Microscopic and culture tests from the diseased tissues of both of the tuberculous guinea pigs infected with the sample taken on the i6th day of exposure, as well as an inoculation test from the tissues of one of them, showed charac- teristic tubercle bacilli. The test of the sample made 31 days since first exposed produced in both guinea pigs inoculated only slight tuberculosis of the right superior inguinal lymphatics. Microscopic, cultural, and inoculation tests of the diseased tissues from these guinea pigs showed typical tubercle bacilli. On the 49th day of exposure (September 16, 19 10) the test showed that the virulence of the tubercle bacilli had considerably decreased, but was sufificient to produce slight tuberculosis in one of the two guinea pigs in- oculated. The guinea pigs were inoculated subcutaneously with the centrifuge sediment of 40 cc. of an emulsion made by thoroly grinding three grams of the dried sample of infected manure in 150 cc. of 0.8 percent salt solution. The pus from the right supe- rior inguinal of the guinea pig that became tuberculous was shown by miscroscopic, cultural, and inoculation tests to contain charac- teristic tubercle bacilli. The other guinea pig, when killed 53 davs after inoculation, was found to be healthv. Tests made on 324 Bulletin No. t6i [November, u5 •- 8 p y o5 Ox :^ ^^ b.ii p cfl C rt O 3 fl TJ t« S flj cfl rt Ui + 1 > > o o Ui u ^^ 0) 0) c a 00 a\ On OO + ++ + + ++ + 00 ++ ++ + ++ ++ ++- ++ + > > •J lU 'T^ be .tlj 3 ;3 o o ;- u ^^ 'd'd N N 00 3 ;:( o o u u (V I> VO VO rH fl "C . > . > o o 1-1 S tfl C 0) fl a; 4j Cfl 4j !« • bod 00 00 00 ^ -i ?^ bo 'a 55 + 1 ++ ++ + + ++ rt is Ssg- ++ ++ ++ + + ++ bo gs u ■>-> o > > £.2 t« t« 03 '/I 03 tn o o P4 m tfl tfl Cfl cfl t« ^ 1 Efl tC .O O O o o ^ % BQ I/l .^-a S ^ ;3 3 fl o 22 SS ea o o o ^ v o o o -4-> -4-* O O O U u u Wl tH o o II 3 ;3 1 sS* 0) J3 X!^ o o o 0) dJ ^; TO'd a a-s Td'd •2^ o o ^ 0) D (U (u a; O O 1-1 ft iH tH yi »H r-t rH rH rH fH r- rt tH tH rH ii ^^n J>ul, c!^ ^ vAvi J^^L Jsck vi^ fH iH Cq C;! r-( rH tH r- n N r^r< tili otol) otol) oi, ol) J>cl> »H rH riri. rH rH rl^ri. oil l>QO ^i2 ^1 11 00 oc IS 00 00 tgIS o o i> t> vo vo 1—1 fH a\ c^ r-i r- ro fO r-i rH rH rH ro fO rt Ti- 00 OC ri M t^t^ c3 o^ y-i rH 1-i rH c /} p. 1 o 326 Bulletin No. i6i [November, the 8 1 St, 123d and 171st days of the exposure to sunshine showed the tubercle baciUi to be dead or at least not sufficiently virulent to produce tuberculosis when the centrifuge sediment was injected sub- cutaneously into guinea pigs. It thus appears from these tests that pure cultures of bovine tubercle bacilli, when mixed with cow manure and exposed in an open place in a pasture field, remained alive in this instance for ap- proximately two months. Exposed in the Shade. — The part of the artificially infected manure exposed in a place protected from sunshine was tested on the same days and in the same manner as was the part exposed in the sunshine. These results are given in Table 14. The parts tested on the 7th, i6th, 31st and 49th days were shown at each testing to contain virulent tubercle bacilli. The infection of the guinea pigs in the first two tests was severe. The guinea pigs in- oculated with samples taken on the 31st and 49th days of the exposure were infected with tuberculosis but not so severely as the guinea pigs inoculated on the 7th and i6th days. However, they were much more severely infected than the guinea pigs in- oculated with the sample taken from the part exposed in the sun- shine and tested upon the same days. The three samples taken on the 8 1st, 123d and 171st days were shown not to contain viru- lent tubercle bacilli. Three of the guinea pigs inoculated with the first two samples remained healthy until killed on the 49th and 55th days respectively after inoculation of the sample. One of the two guinea pigs inoculated with the sample taken on the 8ist day died five days later with an acute infection. The two guinea pigs inoculated with the sample taken on the 171st day died of acute infection with no evidence of tuberculosis. From these tests it appears that tubercle bacilli mixed with cow manure remain virulent to guinea pigs for 49 days after ex- posure in a place protected from the sun. All tests made later than this date showed these organisms to have lost their virulence. Virulence was retained longer in the shade than in the sunshine, as shown by the production of more severe tuberculosis in the guinea pigs inoculated from samples exposed in the shade than that produced in the guinea pigs inoculated on the same days with sam- ples exposed in the sunshine. jp/ (U • rt o . > ss 1) M ci 3 § • hflo l-H 0-a^ 00 ■M he 55 + ' 1 U . ct3 6.H • • he W, Q, + ; ; 1 , ^ o c « rj +-> U O ^ rA2 rt ^ ^o .J he c o o 'a 8 p .S5 -1 >^b o >% t->t>> ^t>> >^ >^ ^:5 0) J 55 55 X ja '^'5 <+-l -M 'ri 13 oj oJ -M -M ^ fl '-' a; OJ «, <1^ OJ OJ OJ a; o OtO K^ iJW WW WK WW S gg o !3 o rl .—1 ct O '2 s OJ he ^ X3 6 H M-t Ox •d TJ ^- 'd'd 'd'd TJ 'O 0) t: ^ •iH .tH 3 2 WW M^ WW •ays Ince ocu- tion o CO Tj- 00 M M ON ON 0^ ON 00 ■^ \o lO lO -* "* Tf -rj- p w^c^ T-( •<-• 1 o o O O o o OO oo tH tH tH 1-t r-i ,H rH rH tH ,h Datec inocu lation ^ ^ J^J^ J.^ ol)ol) ^^ tH fH M CJ r-( tH rH rH r^ n ol) o!. ol^oi) C^^ n rO l^ l^ tH i-H ri ri lO lO l^ l^ t- 1^ 00 00 00 00 00 00 D ampl ex- posed days o o rO rO "* ^ rO rO lOiO T-( tH t<) rO VO o o o r-( rH 1 a Q p. 1 J9J2] Fate of Tubercle Bacilli Outside the Animal Body 32'J 3 -3^ + : ; [ Cfl o ^ ' ' .22 ' 'd p- '-' a, .2 o + : ] :^^ ■ ''0 - o 2-s ^ 0) • o S a -M (1> • rt o . > • s^ ^ ;:2 o o . bx)6 rH -M 1— i '^•^^ S : o he o5 + : *a nJ 6.2 0) .S *3 be + *. 1 ^ ^ o C tfl fl "3 C/] rt ^ o u 'a fl t^ >%b t^ >^ t-. t>> K>^ o .-^^ J3.C X .c :S'5 55 o >.Si:^ -t-l -M t/3 O -^s d ?^. 'rt 13 rt rt rt d V. c-^! oj Si (U 0) 0) 0) o^a-^ WW Wrt WW S 5 ^ «M o^ Id 'd TCd •Cd 'O'd fl) -M ol) <^c}^ clcL J.J. • 1 R"^-^ iH r-t tH tH 00 c^ vr>i> O rH -rtlfi l> t^ »H •-» fO ro ID lO t>. l^ 00 00 00 00 00 00 0) e 55 00 rt o o ^^ ss CC p< 1 330 Bulletin No. i6i [November, Series 2 PREPARATION A second series of experiments exposing cow ma- '^p°c;a'mplf9^^^ nure artificially infected with a pure culture of bovine tubercle bacilli was made. The prepara- tion and exposure of these samples w^ere made in the same man- ner as in Series I (see pages 319 and 320). The places of exposure were within a few feet from the places where the samples were ex- posed in the first series. WEATHER Table 17 gives the weather conditions during the CONDITIONS time of exposure of this series. The same plan is followed as in Table 12 giving the weather conditions in the first series. These conditions were just the re- verse of those in the first series, the first series beginning in the hot month of August, the second in the cooler and more moist month of March. No especially unusual weather conditions oc- curred during this period. Manure Exposed in the Sunshine. — The results of RESULTS exposing this infected manure in the sunshine are given in Table 18. The tubercle bacilli were still alive at the third test after 45 days of exposure. Tho the tuber- culosis produced in each of the two guinea pigs from this test was only slight, as shown when these guinea pigs were killed and ex- amined 53 days later, Guinea Pig 973, inoculated with the dis- eased tissues from these two, produced severe generalized tuber- culosis. Typical cultures were obtained from the diseased tissues of both the original and the secondary inoculated guinea pigs. The samples taken after this date produced no tuberculosis in the six guinea pigs inoculated. Manure Exposed in the Shade. — The results of exposing the sample in the shade are given in Table 19. Here we find that the tubercle bacilli were alive for 73 days, while in the sample ex- posed in the sun they were dead at this testing. Also the test made on the 45th day shows more severe tuberculosis in the guinea pigs inoculated .from the sample kept in the shade than from the sample in the sun. I. As shown by the results in both series of ex- CONCLUSIONS periments, a pure culture of bovine tubercle bacilli mixed with cow manure and exposed in the sun- shine in a pasture, remains alive and virulent for approximately two months. 2. The virulence of the tul^ercle bacilli in cow manure was re- tained in the samples protected from the sunshine longer than in those exposed in the sun, as shown both by the increased length /p/i-I Fate of TriiERCLE Bacilli Outside the Animal Body 331 < S be ^3 a S C^l CO -* to ^ O Ti- oi> a\ t> 00 rt iO VO t>. C» 00 § o c o to in t>- CO d On ON c^ o oi> >o CO LO t>l> 00 Cfl ;3 «+H JiS o o ^ C^ VO O CO On CI t^ Lo ri t^ o >o .9 J C<1 c^ rH r-t 0\ o^ OO + + + + ^ v ^ H 3 »; 0) -l-> D t> . > c 0) c C^ ON + +! + + !+++++ > > Cfl cc 4: o Vh U 01 0) N NI Vh Vh C fl dj (y 00 dJ OJ W E HH MH hM HH ^1 53 a -0 • T-l 5w TO T^ i53 1 1) i 55 ^ 10 10 r-t S2 !8 % »H ^ r-( r-( tH tH tH »H r-^ ?H r-l tH tH r-( fO CO 4 : I I :iz ^^ ON cn 10 vo VO 10 l> t> 0\ Q\ 0\ 0\ 0\ o 88 I9I2] Fate of Tubercle Bacilli Outside the Animal Body 333 a -M < G CQ '^ =.s i ++ : + 1 o5 ; '. •^ '0 2- 1 0) S 03 • ++ ; + '-O .2 §1 • 11 : -^ . p . -4-1 • d :o ' s o , ^ CQ n • a\ , o . h£>o . t>i^ • M t— 1 o-a^ ; • CNa\ :S ■ o5 : + + I -h+ : + ' ■p. ri o.i^ I '. ^ S. 1 o 5^ 1 + ++ ++ : + ' *3 ho S CO I O V IV > > 03 tfl (fl 0) 0) (U ■g %t>> {>, >^ 2 '■ y p o o BB ^ ^ ^^ ^ ^ o «+ -( Vh u u -»-> tn -*-» ■4-> -M -M 3 :3 ^3 JSJ M a> o i>i> ' lO t^ rO CO tH 00 lO V) vo roco ^>a^ VO VO t> CO Q mGji .. . T" Tm~ tH tH r-i tH iH tH tH iH iH iH «4-4 , y. H T-i r-( T-1 tH r-t tH tH tH tH tH tH O ;3 S3 fli o O ^ ^.i r^A cJ^cJ, J.J. t^tl ri. ol) "S^- (^.S^ q N tH r-t ^ ^ tH tH M u J^cJ) 44 ^^ vivi vA 0^ cill ? rt- COr^ to rf (0 1> vo h- ^ t> -t T-t N tS •^ ■* lO vo I>1> o\ tH T\ a\ a>a\ 0\ o\ 0\Q\ a\(y\ OS o lH ample ex- osed, days . 1 :30 T^ ss S ^ tH tH tH tH 11 ^ p, 1 334 Bulletin No. i6i [November, of time that they remained alive and by the severity of the disease produced in the guinea pigs inoculated upon the same days from each of ,these samples. This difference, however, was doubtless due mainly to the difference in drying. 3. Tubercle bacilli in the manure of a naturally infected cow were dead within two weeks after exposure. More experimental re- sults are necessary before trustworthy conclusions can be drawn, 4. Danger of infecting farm animals from tuberculous ma- nure is indicated. Duration of Life of Bovine Bacilu in Garden SoiIv PURPOSE Some investigators state that tubercle bacilli will OF TEST live in soil and in dead carcasses buried in the soil from two to three years. If this be true, ma- nure from tuberculous cattle when put upon cultivated fields which are later used to pasture stock may be a source of infection for farm animals, especially hogs. For this and other reasons it was thought advisable to test the length of time a pure culture of bovine tubercle bacilli will live in garden soil. The culture of bovine tubercle bacilli was of the CULTURE same strain as that used in the experiment forarti- fically infecting the cow manure. About four milli- grams of pure culture were obtained from a four- weeks' growth on the surface of two large tubes of glycerine agar. The organisms were carefully removed from the glycerine agar and emulsified as described under ''Cultures and Emulsions," page 315. SAMPLE "^^^ sample of soil with which the tubercle bacilli OF SOIL emulsion was mixed was obtained from a garden plot that had been in cultivation only two years. Previous to that time, this plot of ground had been in sod for at least fifteen years. The part from which the soil sample was ob- tained had been well manured with horse manure the first year it was under cultivation. The second year no manure was added but the ground was well stirred and made into a lettuce bed. A suf- ficient amount of this soil was obtained to fill a }i-'mch. mesh wire basket having the dimensions of 4x5x6 inches. This required 1700 grams. This pulverized soil was placed in a large pan and the emulsion of tubercle bacilli sprinkled over it. These were thoroly mixed by being constantly stirred for some time. The amount of emulsion was sufficient to make the soil quite wet and sticky. iQi^] Fate of Tubercle Bacilli Outside the Animal Body 335 PLACE OF '^'^'^^ ^i^n'.'ims of this infected soil were removed to EXPOSURE be tested for tubercle bacilli as a control. The re- maining soil was put into the wire basket and covered with a wire gauze. It was buried in another garden that had been under cultivation for one year and had received no ma- nure or fertilizer of any kind. The place where it was buried had been previously well pulverized. The basket with the infected soil was buried six inches under the surface of the ground. TESTS OF Samples of this infected soil were tested for tuber- SAMPLES cle bacilli on the first day of exposure and on the 7th, 1 6th, 34th, 55th, and thereafter about once a month for 352 days. Ten grams of soil were removed by dig- ging down beside the mouth of the basket and with a sterile pota- to knife making an opening to the center of the basket. After the removal of the sample, the opening was filled by pressing the soil in around this opening with a potato knife. The wire gauze was placed over the mouth of the basket and the garderi earth filled in over it to a depth of six inches. The soil sample taken to the lab- oratory was thoroly shaken with 200 cc. of 0.8 percent salt solu- tion in a 300 cc. flask. After standing ten minutes until the coarser sediment had fallen to the bottom, 40 cc. were removed and placed in two sterile centrifuge tubes and centrifuged for five minutes. The supernatant liquid was drawn ofY and put into two other centrifuge tubes and centrifuged for thirty minutes at 2000 revolutions per minute. The supernatant liquid was drawn ofif and discarded. The last 5 cc. of the liquor and sediment were thoroly mixed and in- jected subcutaneously into guinea pigs, in graded doses, giving one i^ cc, another i cc, and for the first test a third received ^ cc. RESULTS OF ^^^ results considering the length of time that THE TEST tubercle bacilli live in the soil are recorded in Table 20. The guinea pigs inoculated from samples taken on the day of exposure, and on the 7th, i6th and 34th day after exposure showed in each case, when killed and ex- amined, severe generalized tuberculosis. Microscopical and cul- tural tests showed the germs from the diseased tissues to be char- acteristic of active, living tubercle bacilli. Tests after this time indi- cated a weakening in virulence, but slight tuberculosis was produced in the test animals from material taken on the 213th day of expos- ure. Macroscopic, cultural, and guinea-pig tests from the diseased tis- sues of the original guinea pig showed the tubercle bacilli in the soil sample to be active and virulent. Five tests were made after this date. In every case the guinea pigs, when killed and examined, were found to be healthy. The two testings after the last one in which tubercle bacilli were found, viz., the ones made on the 336 Bulletin No. i6i [November, 3 5^" : : + • : + + + ;5 O ^ • ■ ■-^2 •d 6.^ ; ! ! ; '. '. % ^§^ : ;+ ■ ' + + + -0 1^^ : : . r r •- V. 6 o : :ji o '. '. ' XI (1) ^ fl, ^ D ' o 2-s • • S V. • • ;:! j: ?i jX 3 Ih • .2 o a . --Ma) • -M 0) -*J 4^ 4-> V ' d o • • . t> • . > . > > • S CO : : c a^ * G 0) G o c ' H- O X s: V a > > > a; > > «H -4-> r- CO to Ifl ^ a; -i -t-> ^ o mmZ ^^ -^ a3 ^X5 .^ --H 0) ^d a; 1^ fl cd • .-1 .tH (U J3 S3 rC'd 5-^ 'S'd IK U 'd'd <3J ^>^-^ ;S;3 N 0) N N O G a; a; o2 ';3 =1 C fl {ti 22 ^'-S el Sg (U c> 0) V. CJ 0) i;| ^ A fl c fl fl a; C fl -s-s ^ (U a; oJ fl OJ a; •2 c X 5^^ 00 ^5 00 ;3 (u H ? ^ 'd "d '0 'd'd ns-d 'd'd Td -d 'd •d a> -*-! ^^ (U ^ .i> lO lO CO CO P tfl^C^ O O O o o o o o o o o o o f 1 rH r-t tH y-< y-{ r-< r-i rH rH tH tH »H r-( >atec nocu ation ^^^ 6 c^/ R'^^ rH iH -^ V) vo r-i CQ l:^ CO VO t^ 00 Ov n CO lOii^l vo vo \o vo vo 00 CO rH tH CO CO t- t^ t-- t>. l>. t-t- l^l^ 00 00 00 X mple ex- sed, ays ooo I> 1> VO vo CO CO lO lO VO 'vo r-t iH 00 00 rt onj 1 i /} p^ igi. Fate of Tubercle Bacilli Outside the Animal Body 337 ; : + >l- • ; '; : + : + : . ; • 1 .s • • 3 vi ;ls : : • . > • P ■n : c 4; : c *+3 *^ "^ "d ' u o o o o 2 I) .5 k,t>> >^t>, >^tx, t>^>^ >^ ^ JZ A X Si C "•" ■4-> j3 C V/, 0) OJ KM " Ct j3 o o o - .^ <1 << -<« > 55 e J U3 •0 X , - "O v.v. 'O'd -d-d 'd'O 0) a> 'd'd 0) 0) 1 0) c > <—( O) V -< 'PC a s q'5 Sg 22 S3 22 2 Mf to fO lO >o rH 00 ro Tt lO lO VOVO u)iO t>t^ ^ 00 ■^ S^ o o r^ r-t rH r-t r-( r-i iH iH »H rH r-i r-t rH 00 tOVO ?^^ 00 00 o o •^ -^ o *o g > 00 00 ^ o\ o^ as c^ o\ 0^ G\ o\ o\ rH ^?? 28 s ^5^ ^^ ss ss ^^ °2S T-t r- tH T-( tH M s ti ;^^ : G (u ; s ,5 *3 ciJ • p w o .bed S ^ G e-a^ • • o 4-> > > 3 V 0) .^ -^ u o Cfl Cfl cfl o a O O O § o 'B 99 (>>o> > H-< V. o o o ^ x: X X.P *l «H Ui v^ -4-> HH cA fl^ aj aj »-H rt M 2 o 03 rj oj WW X oj .tJ (J :^ »« 'O'd ■« (U OJ D < ■M N N N .»-( -iH frt ^ rt 'c3 73 H in «H Ih q ri, 1 1 rH r^ H > 1 d G rt cJ. >i>i 2Z 1, Tf fOrh Ti Q «r> rf lO N ^^ ^^ 8 o o W rH r-i T-t M + 13 ^.2 • ^ 2 -M -M : fl n o pj o • td 1^ O 4) o > 0) vu 0) a; t5 .t: 't« '5 'S o fl C o o o .*-< •'-' -4-1 <4H H-< ;3 3 3 o o o O O o Wi u Vh -M 0) fl c -M -4-» !3 a o 'O'd •d ?3 O Cfl cfl Cfl te « NJ NJ ■<1 O o • fH 15 (^ 00 H ^ w i^ >yl k^ W Pm HH W lo a\ o\ C^ tH CO ^^ t^ l> cr\ Q-, t^ I> 00 CO 00 00 00 c» igiJ] Fate of Tubercle Bacilli Outside the Animal Body 345 : + + + : : + + + + ." pi -2 . > . > c C OJ c > > T— 1 ^ 0) *I2 «; _t2_tr .i2 .2 ^ a> C C! '(/) '5 'tfl '5 be Jj ^ .2.2 o c « 1 o 3 c 9 c >^ >% t>> « ^~ t—t >— 1 x^ ^ u ^ ^ • S nj cd g OJ S 3 3 3 -*-> J3 OJ .2 0) a; {3 (V -d N 1 0) MH o :3 o < o 0) ■4-) a ti c fl P 0) u^ ^ y-{ LO •* Th T»- rH rH rH rH rH rH rH rH 1-t rH 4^ u^u^ cJ. 1 rH J^ ^ 1 vH r-< ti 4 1 M«^ rH r-{ rH y~i rH rH rH r^ r-< c^c^ rh^ 4 J> ^ ti oi, c)^ 1 rH rH rH r-l M 0^ 0^ is 5? en i 1 rH 1 y-\ y{ s tH r< M »H T-t « rH o y^ i> i ] gg ?J?^ § k 5 * ^ ^ i 2 he o fl -d a; o ho a'd S^ t; a; rt <« u he 3 a o D CO o + + + + + + + + J3^ h£hc to CO ^ o o o ^^ dj a; 00 4) t> 0) (U CO CO CO cfl" n ;3 3 U m U U 00 o o rH rH 00 00 +++++++++ 'd '0 'd "d 'd'O 'd -d a; (U (U a ) 0) (U (/} ^ ^ > > > > > ^ o o a, d) .!2 a ; d) a; u u ui m *cfl u 1 cfl w P S, ^ C 5 O ^ HJ o o 93 p i 9 9 t>^ >, t>> >% o >, S 8 o o t J o O j: X X X c J3 u u V 4 V-l V^ -4-1 -*-' -4-) o o o o a ^ 3 P p a; (U 'd'O »— ' X i ns 'O a; «4H SH a> 0) ra a ; 0^ OJ 13 rt fl J ri 1^ V ■4-' -M u u V ■1 Wi U 3 13 XT) rt rO VO ^ -^ "«}■ •* .5 rH »H tH r-« tH ^ i iH rH rH rH tH ,^ 1-^ rH a; T-( rH i-» tH iH T- i r-t i-{ rH rH rH rH r-i iH -+-• ^^ ^>^ C^) . J^ cA 1 ^ 1 rH 1 Tj- rh 1-1 rH 1-H f- i 1 r-) T-t (H rH 1 Ar^ cJjcJ) 1 „ O 2 .i oi) oi ^ i rH ho p li as 1 ^ 1 1 tH 8 rH rH rH rH rH § -4-» O Z 1 »H n 00 a 5 l^ ^^ o On ,^ t^ uo VO M M rf x> O ■^ t^ 00 o «r) *o 00 ^ C1M c^ r 1 CO ■* •^ rt lo lO ^ ^ 348 Bulletin No. i6i [November, 3 J, 1> : : : : ; + \ \ + ' ;^ o5 ; ■ ■^ . . ■ '0 p- ■ . 0) .3^ o ■ ■ : + + • ^^ ; ; ; ''0 fi 6 o •:|^ ; ; 3 : w g G a • • -^ <^ -M , < O 58 j •" '■■n d '. :0 : 9 o ^ o . bed ' r^ <7\ : ' fH t^ Q a 0-a^ \ ^ 'eg • ;^ \ \ « : Iz; ^ J. tu 0} •a 52 + + + + ++ ++ ■ • M . M »4 (D a ii ^ + + + + ++ ++ *3 be Ml ss • ; w O (U > > > > > <^ D tfj (U 4-> _bc^bc ^ o -*-> 4-" o o ■4-1 izi ^ P 3 9 9 ». .s p9 S^ H o o o o o CA tf) o o > o Vh ^1 Vh Vh V- u •^ ^ 1:j«4H o tfl a; (U 'S'S a> 0) 53 fl 2.S go; fi9 o < pq 2 o ^ ^ ^ ^ t2 »2 ^^ o o P p P p 9 9 3 P 'J* .»H fe ci "♦-' "*^ "*-• +-» o o -*-> ^ o o o :^ 'd '0 ^3 'd "d'd g N N N N 3 'rt "ri 'c3 'rt H^l rt rt ^ii }h Vh )-l u Ui u J" ft o ^ 00 o M 1, 0j2 'd '0 ^3 'd 'd'd "d 'd 'd a) -*-• S ^ TJ 'd a;'d 2 3 g i 33 33 oj a; SS 35 -4^ till vo OS n CO ri M rH rH ^^ 00 I> Tl- ^ uo lO CO CO vo >0 l^ 5 O 3 fl 0) o o o~ o o o oo r^ rH rH rH •< 1-{ T^ r-( r-( rH rH tH rH rH rH T-i r-i H vA ^ J. C^ cLc!> 1 1 rH rH c!,c!, 44 -M o*+3 rH <^ M rH rH CO CO 1 1 C^ M rt fl nj ol a!, 3 rl rH rH r-{ M M fici g g o 00 ro 00 ON O 00 00 OrH « 00 %t a; o\ ro rj- IT) vo vo t>r^ o o •H a£p M "«»■ X> C) M rj -rl- 00 00 rH rH r-i i~{ 1-^ r-{ C^ N rt O-d a 2 p. 1 iQij] Fate of Tubercle Bacilli Outside the Animal Body 349 The tuberculosis produced in the two guinea pigs inoculated at the fifth testing from the sample of standing water contained in the cotton-stoppered bottle, was neither extensive nor severe. Those from the running water had a higher virulence than the tubercle bacilli in the standing water, but the tuberculosis produced was not so severe as that from the former samples. It should be noted that the time of killing these guinea pigs was much earlier, and it may be for this reason alone that the virulence appeared to be weakening. This fact was noted at the time of killing the four guinea pigs inoculated with the bovine tubercle-bacilli-in- fected waters, and so the two guinea pigs inoculated with water infected with the tubercle bacilli of the human type were not killed at this time. Thirteen days later one of these two guinea pigs died with generalized tuberculosis. Unfortunately the death of this guinea pig was not noted until a short time afterwards, and it could not be determined whether tuberculosis was the only cause of death. The other of the two guinea pigs inoculated with the sample of water containing the human type was killed 72 days later, and the examination showed severe generalized tuberculosis. Later, on the 147th day, w^e find severe generalized tuberculosis produced from each of the three samples. After this date only two more tests were made from the sample in the cotton-stoppered l3ottle, one on the i8oth and the last one on the 2026. day of ex- posure. These two tests show^ the lessening of the virulence of the germs : for from the former no tubercles could be noted 24 days later, when the two inoculated guinea pigs died of acute infection; from the latter, one guinea pig died too early to make the test; the other, killed 78 days after inoculation, showed only a doubtful sign of tuberculosis. However, an inoculation of an emulsion of the tissues from the point of inoculation and the right superior inguinal glands produced, in a secondary guinea pig, gen- eralized tuberculosis. From the diseased tissues of this guinea pig an active culture was obtained. Thus no end point was reached from the sample of standing water with the bovine tubercle bacilli in the cotton-stoppered bottle. The other two samples, kept in porous flower pots which were transferred to the pool in the court- yard, remained alive for 441 days, but were dead on the 470th day. From the emulsion of the bovine type severe tuberculosis was produced in the guinea pigs even in the last test in which these germs were found alive. From the emulsion of the human type only localized tuberculosis w^as produced at the last test. The sec- ondary guinea pig inoculated from the diseased tissues of this last test animal showed severe generalized tuberculosis, and cultures from the diseased tissues of both the original and the secondar}^ test animal showed these germs to be active. No test made later than this showed any indication of live tubercle bacilli. Five such ncirativc tests were made. 350 Bulletin No. i6i [November, Series 2. Pure Cultures Before the close of the first series it was recognized that these experiments were of such importance that a repetition of the same was advisable. PREPARATION ^^^^ cultures used and the preparation of the OF EMULSIONS emulsions were similar to that in Series I. Bmulsion of the Human typt. — The sample of water used for the emulsion of the human type was obtained from the pool June 15, 1911- With the sample was obtained also a considerable amount of floating green and yellow sediment scraped from the inside of the large tiling enclosing this pool of water. A microscopic examination showed very abundant algae — anabena, diatoms, desmids, scenedesmus, and confervoideae ; also several kinds of bacteria. Among the animalcule there were vorticellae, amoeba, a few paramoecium, water-eels and rotifers. There were thoroly shaken 450 cc. of this algal water to which was added an emulsion of approximately 5 mg. of human tubercle bacilli. This sample was divided into two equal portions : one portion was placed in an unglazed earthen, cylindrical jar eight inches tall and three inches in diameter; the other portion was placed in a small, unglazed vase which was stopped tightly with a one-hole rubber stopper in which was inserted a small glass tube that reached above the surface of the water and allowed the escape of any gases that might accumulate in the vase. This vase was placed on the bottom of the courtyard pool twenty inches below the surface of the water. The other sample in the 8-inch jar was placed on a slate shelf four inches below the surface of the water. This jar was open to the sunlight but the direct sun could not reach the surface of the emulsion inside. During the winter the part of this jar projecting above the surface of the waterj was crumbled by freezing. (See Fig. i.) Emulsion of the Bovine type. — The emulsion of the bovine type was made in a way similar to that of the human type. Five milligrams of these organisms from a young culture on glycerine tgg were removed and rubbed up thoroly in a sample of 225 cc. of water obtained from the drinking trough at the dairy cattle barns, and thoroly mixed. A microscopical examination showed the following: (a) vegetation — spirogyra, . oscillaria (large and small species, the small species being very abundant), a few dia- toms, abundant protococcus, desmids, scenedesmus and anabena ; (b) animal enle—SimoQha J paramoecia, stylontia and vinegar eels. This sample was placed along with the emulsion of tlie human type in a similar cylindrical jar on the slate shelf below the sur- face of the water in the pool. 79/. Fate of Tubercle Bacilli Outside the Animal Body ^51 RESULTS OF ^^^^ results of the tests of the bovine and the TEsSrs human types exposed in the open, unglazed, cylin- drical jars are given in Tables 25 and 26. The last test in each, made 259 days since first exposed, showed only local tuberculosis in each of the four guinea pigs inoculated. Tho there was not time to determine by cultures and by secondary in- oculations whether these organisms were living, it is likely that they were. The end point in this series was not reached. The tuberculosis produced in the test animals from all other samples, except those dying with acute infection, was severe and general- ized. A typical autopsy is shown in the case of Guinea Pig 11 24 (Fig. 3), which was inoculated with tubercle bacilli of the human type after exposure for 212 days in running water. The results of the tests from the other part of the emulsion of human tubercle bacilli placed in the submerged vase are reported in Table 27. The last test, made on the 259th day, gave gener- alized tuberculosis in the two guinea pigs inoculated. Fig. 3. Generai^ized Tubercui^osis in Guinea Pig 1124 Inocui^ated with Human Tubercle Bacii.i.i after being Exposed in Running Water eor 212 Days. 352 Bulletin No. i6i [November, 3 ;3J + + + + cfi O-M .2 v> 'd P- 4> S CO + + + + + '-d 1 M-i 1 . ^ ^* x5 3 o .2 §1 -4-' ;3 ;3 pi a; 3 ^ bi o . hc6 »H CO o M ^ H o ro O 00 ri ro 1— 1 ^•p.^ O 1-H tH T-l r-( tH rH ^ 4J 12; he 5s + c^. + + + + + : M [4 2-E " o + + + + + + + + + + PQ *3 he ■gs ; a O u 1 o u 0) 0) fl o cn ID > r/i .2 0) tfl > a; M he •1-1 he 121 w tA o O c«^ ^ Cfl" tl H o O o O 13 J_4 .r-( a 'i «i c« w 1 o a o o 9 ;3 "3 ^ 9 o CO a> ■4-> fl rt -♦-> 2^ ^ 5:^ c 'S3 'S s 2 o ID N C .2 o a ^d N "d '^ 0) rt 'd 0, 9 (D o o u <1 'Si (U rt CI -3 5 3 4> 0) 13 to 3 ^ fl c o o o S o 0) (U (U (U ^ p p 1 3 s s s 3 a s •I-) M s a 3 rf rt ^ o -M ^ ■«1- ro rO »H fs O 00 C^ ON (N lO lO rt vrj Tj- vc 00 00 w p 'tc _G ri ,4 O i fl 0) o o rt fl rt J) T-t rH 1 4 S rH rH rH 3 CI 1 rH ri ri rH tH rH >i, ^ ti J. =1, 5 1 o rO rO 00 tH '^ ri CO M a^ On ^ 00 ON c lO >o >o ri rH rH c1 n C) 1 c « p< 1 I9I2_ Fate of Tubercle Bacilli Outside the Animal Body 353 3 + + + j ; 1 O^ • • '"M T? 2-s ■ '. 0) .§§^ + + + \ ' + + + :^^ I • *'d r. ^ M-l 6 o \ ^ ^ U3 OP ' ^ ^ a; o 1 ga s -M ;3 • • Jl ^ w li 0) tfl \ ; P H S H 0) CO < ^ 3 5^ oil ^ M '^ fO UO o\ o 7^ o M »H 8 r-i rH rH rH rH rH tn he o5 \ 0- + + 1 : + + + g ■p. • M rt 6.H ', I i4 .s '3 be + + + + ; + + + + + pq «H o D D w 0) ffl > tfl M .22 > 0) w o 0) t« 1 to '55 o 'S CA to ^1 K '£ Cfl o c fl u o 9 '3 to" o o 1 'tfi O o ^o o s o u 1 o ^ D V« -4-' V ^ r ;3 o ^ d o «+H § J2 3 9 o J-l .2 •f P o CA dJ •4-> i 1 t> C^ 6 ci 1 rH ! 1 rH to r-{ rH rH r-\ t^ M ri I> U5 rH ^ fO s "^ Tt 'O 00 0\ tH li^ l> QO Q^ o n VO yi 0\ o\ s s O S rH y-\ rH rH rH r-> 0) S oi « d o m rO 00 •^ 00 CI <^l CO M O^ a\ »H '^ c» M M uo o t^ r-{ lo lO rH rH rH rH rH n M ri C/ 2 p. 1 354 Bulletin No. i6i [November, 55 'd S-2 1 '0 c o o 2-1 o a. pi o O o M • »-l oil 1H c P4 tt o5 + s be t3 U 1 02 1 2U -f - 4 - + + + > '3 be s^ < <4-l s o 4-> 2: •5 0) M !3 > c ' > hT «) t; "d B O ^ a ^c 4 -4- 0) B S a 3 1 > r^ ID t. Ct oj a ct! < U w a > a > V-i 1 (U s c a c 3 t Z e fe c < o w «« p:. O^ X. -o •c TJ a V "ti 0, 'C 'C CJ n: 1 IL> 'Si a 0. i r- t cJ- cJ) S «ll 00 oc iH i> c r-( T- H 'd 6S • .§& + + + + + + . + + SS 1 ■'0 - - - - .- - .. O 6 o ^ 0) -M o H-> 0) -M (D . -M D ■*■' D -M OJ o nJ O • , > . > . t> ^ > • . > . ► • ^ . > §^ 53 OJ S3 Oj ] ] fl 03 be 'a HH rH rH • • rH rH rH rH rH r-i rH -+ - + + • ; + + -U -T- 4- + .+ — d 6a -f - + + + + H- -r + + + *3 be ^^ 1 c ^-1 V. u U V-i Vh S-i u U U .2 1 Cfl V p > > > > > > > > 0) >• Vj tw a, o d) V (U (U aT *c? "m "«j "m fl *tfl 'tn 'S *cfl ^ -4-* C C c o C o o o o O o O o ■*-> o o o .2 a. r- "72 "^ r^ i-^ 3 o 3 '3 '5 9 o 9 o o o o o o ^ o ^ 55 o ^ o o o <4H «4H a; C/3 o o S 'c3 13 s c 0) cu X3 1 I" -d c ctJ .S.5 3 ;3 0) OJ OJ ;3 ^ -*-> _2 o ;3 ^ ^ N 'd a; .2 3 <1 -d N -d .2 'd 'd N 0) <4-l c O !3 S ;3 o .»-( • a lU (U (U a; >o >o f^ 00 rO ro i^ i> ^ M "* 00 -* rO CO VO tJ- I-- i^ l^ 00 '^ o VO lO rl- rH ^ -^ CO Q oj^C rt •^^ 1 r- T-H T-t y-> rH rH rH rH rH rH y-i rH rH M ri ri ri 3 o i fl OJ o o S 1 1 rH jlS Z rH ? S rH »• 4 1 1 1 ■j-i o *r1 tH rH rH r-t <^ rH rH rH r-t rH rt C rt ci) w^ u^ vo VO l^ ^ oi ^ ^ S 1 rH rH rH i-< CO ro cJ) CO CO C\ rOTi- ON M ^ lO o ^ 8 M 00 00 0^ vo 0\ 0\ ^^ ^ O rH s rH i rH rH rH r-t rH r-i r^ rH rH ample ex- osed, days o o o §^S^ ^ CA cr\ C^ ro a\ t^ 00 y-\ ■* -rj- rH t^ !>. rO 'O i^ t^ On n lO l^ n VO o 00 r-i rH rH r-t rH M f^ CS ro rO ro CO 11 ^ Q a jgi-'] Fate of Tubercle Bacilli Outside the Animal Body 357 tuberculosis, save those that died too early with acute infection. The two guinea pigs used in the third test, made 99 days af- ter exposure, remained healthy. Probably only non-tuberculous tissue was obtained for this test. Later tests taken from broken- down tissues showed virulent tubercle bacilli. The tests were positive up to the 321st day; after this date the test animals all died with acute infection, the last one, however, not too early to have shown tubercle bacilli. On the 229th day and later no structure of tissue could be determined except the hair and bones, and the bones were almost as fragile as garden earth. Sediment from the bottom of the flower pot was obtained for the samples. Tuberculous Sputum To determine the length of time that tubercle bacilli from tuberculous sputum will live in run- ning water, a sample was obtained from an ad- vanced case of tuberculosis. A stained smear of this sputum showed numerous bacilli. A very small sample was inoculated into a guinea pig the first day of exposure. This guinea pig died in three days of acute infection. RESULTS The results are given in Table 29. Tuberculosis has been produced in the test animals up to the last test, 232 days since exposure. However, only local tubercu- losis was produced at this last test and there has not been sufficient time to determine whether the organisms are alive or dead. The test made just previous to the last one, 187 days after exposing the sputum m, water, produced severe generalized tuberculosis in Guinea Pig No. 11 26 (see Fig. 4). SOURCE OF SAMPLE TabIvE 29. Tubercle Bacii.i,i in Water Kind and source of the organisms Human tubercle bacilli: a. Pure culture in flower pot. Series 1 b. Pure culture in 8-inch cylinder. Series 2 c. Pure culture in submerged vase d. In tuberculous guinea pig e. In sputum Bovine tubercle bacilli: a. Pure culture in flower pot, Series 1 b. Pure culture in 8-inch cylinder, Series 2 c. Pure culture in cotton-stoppered bottle. . Not killed 441 days 259 days 259 days 321 days 232 days 441 days 259 days 202 days Killed 470 days 381 days 470 days 358 Bulletin No. i6i [November, o p + + 1 o^ • v» 'd 2-^ . ! .Ho + + + (H Cfl S!^ g ''5 - - , • > > > -< cfl Cfl Vlfl Cfl cfl Cfl « P:^ ■tfl" o Cfl" CO Cfl" 13 ^ c 3 c • iH a " ^ o o '« ^ o in 1 _o Cfl *3 o J S • ^ u '-^-t W Oh o 'B ^ 9 o '3 9 o ,X m o 4) o o D o o 0) (U c c c c a; (U 0) n § 'd 'd -d 'd v •d 'd t: -s ^ (U (U d) (U 1 r-H I'd s 3 M s 3 s P 2 4j pii " S S g p ^1 §-5 fO 00 VO M I> ■^• rH i> ON p 1 CO U5 -t rH ^ O -* 00 Q cfl^C jti 'S ' r^ »H rH tH r-t ri rH w "~C ^ 00 O VO o r^ ^ 1 rH o rH rH r-t r- r- rH o 0\ to 0\ l^ ^ I> ri ri CO VO o\ ri 00 ro ro c3 *^ Q^ r-\ r-{ rH M C^ c /} Ph jQTj] Fate of Tubercle Bacilli Oi'tside the Animal Body 359 General Sitiiiinary A suniniary of the results showing the length of time that tubercle bacilli from various materials live in water is given in Table 29. Conclusions 1. Tubercle bacilli live for more than a year (441 days) in running water. 2. The length of time that human and bovine tubercle bacilli live in water is practically the same. 3. These organisms live in sputum exposed in water for more than 232 days. 4. They live in the tissues of a dead guinea pig exposed in water for more than 321 days but are dead in 381 days. 5. A watering trough harboring tubercle bacilli may become a dangerous source of infection to cattle. 6. A dead tuberculous animal in a stream on your neighbor's farm may be a means of infecting your stock. 7. The better disposition of dead tuberculous animals is to destroy by burning. 8. Tubercle bacilli in drinking water is one of the possible sources of infection for man. N 9. Infection is not prevented by dilution, since clumps contain- ing a great number of these organisms may be inclosed in mucoid material which prevents their separation and destruction. Duration of Lii^E of Bovine Tube:rci.e Bacilli in Butter INTRODUCTORY Considerable attention is at present being directed STATEMENT to the presence of tubercle bacilli in foods, more especially milk, butter and cheese. It has been determined by Sedgwick and Winslow and by Park that typhoid l)acilli frozen in water die very rapidly. After an hour's freezing 30 to 60 percent were destroyed, and in two weeks 99 percent were killed. The remaining one percent lived for a number of weeks. Tubercle bacilli in butter kept at a temperature below freezing are not killed in this, way, as determined by Mohler, Washburn and Rogers in 1909. In order to obtain further information upon this subject the following experiments were planned. Butter was mixed with an emulsion of a pure culture of bovine tubercle bacilli and placed in small vials which were stored in the three following places: 360 Bulletin No. i6i [Norember, 1. The cold storage of the Monarch Refrigerating C()ni[)any, Chicago, 111., at io° C. below freezing. 2. The University of Illinois Dairy storeroom at 4° C. (above freezing). 3. The basement of a dwelling in Urbana, Illinois, kept at an average of approximately 20° C. PREPARATION "^ pound of fresh butter was obtained from the OF SAMPLES creamery of the University of Illinois directly from the moulding board. It was salted as usual for the market, one ounce of dry salt to one pound of butter. After the butter is mixed, pressed and drained it has a salt content of two to three percent. It was not chilled, but at once taken to the bacterioligocal laboratory and mixed with the emulsion of tubercle bacilli. An emulsion of 3 mg. of bovine tubercle bacilli in 100 cc. of 0.8 percent salt solution was made, and the butter melted at 35° C. was thoroly shaken with this emulsion. From about 10 to 15 cc. of this emulsion were put into small sterile glass vials and stopped with sterile cork stoppers. Thirty such samples were prepared, ten of which were stored in each of the three places mentioned above. TESTING THE '^^^ samples were tested when prepared and at SAMPLES varying internals afterward on the same day from each of the places stored. They were brought to the laboratory, melted at a temperature of about 38° C, and two guinea pigs were each injected subcutaneously with i cc. of the melted butter from each sample. The samples kept at Chicago at 10° C. below freezing were always in excellent condition ; those kept in the basement of the dwelling became very rancid and slightly mouldy; and those kept in the University Dairy storage showed slight moulding in part of the bottles. RESULTS The results of the tests are given in Tables 31, 32 33. No end point was reached. Generalized tu- berculosis was produced in the test animals from each of the three samples taken on the 274th day. It was noted that the tuberculo- sis produced by the samples kept at the lower temperature was more severe. This was probably due to the killing out of other organisms that at higher temperatures acted antagonistically to the tubercle bacilli. jplj] Fate of Tuhkrcle Bacilij Ol'tsidk the Animal Body 361 ■ : + 1 U +- -d P- :+ ''0 ^^ 1 d 1 o .2 6o rt o ; ; ; ; : ^ % . bod ■ % . . ^u^ ■ rH * • • ' « ■4-» t/i -4-> be o5 ++ + : ++ : : : + :+ ++ M 'a M d 6.H ^ .§^ ++ ++ - + ++ ++ ++ ++ ++ ++ V '5 be ^^ M *fH w > > > > > > > > > •S > t1 > > > 01 0) o Wi Ui u u u, v^ Ui l-i u u ^s u u 3 3 o 2S (U 0? -M s o ^X! .Q^ ^^ XJX! .Q X3 -d^ c-^ ^Xl 'd "d :3 :3 ;:« ;3 3 :3 s ;3 ^ S fl ^ S5 s ;3 4) 4> to rt ■*-• ■*-> -M ■+-• -M 4-> d -^ -♦-> -*-> N Nl ^ 'd'd 'd'd 'd'd 'd'd 'd 'd n'O ^-d 'd'd '^ 'r^ w Oj 0) OJ 0) D a> Oj (U OP a; OJ 0) be ^ ? 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