LIBRARY OF THE UNIVERSITY OF CALIFORNIA. BIOLOGY LIBRARY G Ocular or Eye piece Draw-Tube Rack and Pinion for coarse adjustment Micrometer Screw for fine adjustment Abbe Condenser Iris Diaphragm DIRECTIONS FOR LABORATORY WORK IN BACTERIOLOGY FOR THE USE OF THE MEDICAL CLASSES IN THE UNIVERSITY OF MICHIGAN. FREDERICK G. NOVY, Se.D., M.D., JUNIOR PROFESSOR OF HYGIENE AND PHYSIOLOGICAL CHEMISTRY. GEORGE WAHR: PUBLISHER AND BOOKSELLER, ANN ARBOR, MICH. : / 7 Li!-' - Copyrighted 1894. GEORGE \VAHK. ANN ARBOR COURIER, PRINTERS AND BINDERS. PREFACE. No attempt has been made in the following pages at a formal, systematic presentation of Bacteriology. The subject-matter has been arranged entirely with reference to progressive work in the laboratory and, more especially, corresponds with the work as carried on in the Hygienic Laboratory of the University of Michigan. The course covers a period of twelve weeks of daily afternoon work. Illustrations of the various bacteria and of their cultural character- istics have been expressly omitted, as the student is expected to sketch from observation the form of .each organism and its peculiar- ities of growth in the colony, and in tube culture. Blank pages are provided for this purpose and for such additional notes as may bo desirable. The works that have been drawn upon freely in the preparation of these pages are Fraeukel's Grundriss der Bakterienkunde, Eisenberg's Bakteriologische Diagnostik, and Fliigge's Die Mikro- organisnien. The larger works of Baumgarten and of Sternberg were likewise frequently consulted, and in many instances recourse was had to the original sources. For the frontispiece plate, which is intended to show the com- ponent parts of the microscope, I am indebted to the firm of E. Leitz, of Wetzlar. F. G. NOVY. ANN ARBOR, April, 1894. 132699 LABORATORY Y/ORK IN BACTERIOLOGY. i. FORM AND CLASSIFICATION. Bacteria as single-celled microscopic plants. .Relation to algae to fungi to yeasts. Classified according to external form into Micrococcus spherical. Bacillus rod-shaped. Spirillum screw-shaped. The term bacterium still used occasionally to desig- nate a very short bacillus. Vibrio is the term applied to organisms which may form spirals, but commonly grow in seg- ments of a spiral giving rise to comma or ^ N shaped forms. Lack of a natural classification of bacteria. Variation in form as a result of natural conditions environment. Young and fully developed cells. Nature of the medium on whhh the growth occurs. Solid and liquid media. Temperature. Unfavorable media give rise to involution forms degenerations. Variations resulting from artificial conditions methods of examination. Deposition of aniline dyes. Simple and double stains, as of tubercle and of leprosy bacillus. Contraction of protoplasm by alcohol by iodine. Action of heat. Constancy of form and of species. Demonstration of, Micrococcup, Bacillus, Spirillum. PREPARATION OF NUTRIENT GELATIN. LABORATORY WORK. Place 500 g. of chopped lean beef into a clean 1^-2 litre flask; add 1000 c. c. of tap-water and insert a cotton plug into the mouth of the flask. Shake the flask repeatedly during the next 15-30 rain., and then immerse in boiling water in a water-bath and heat for 15-30 min. Filter through muslin and to the filtrate or meat extract thus obtained add, 100 g. of gelatin 10 g. of dry peptone 5 g. of common salt. Warm in the water-bath until the gelatin melts, then neutralize or render slightly alkaline by cautious addi- tions of a saturated sodium carbonate solution (avoid excess of alkali). Continue heating in a water-bath, with occasional shaking, for f to 1 hr. Filter through a plaited filter. The filtrate should be, (1) perfectly clear, (2) should be neutral or slightly alkaline in reaction, (3) should not become cloudy or coagulate when boiled in a test tube for 1 to 2 minutes, (4) should solidify when cooled. If the filtrate is cloudy and strongly alkaline correct the reaction by the addition of dilute acetic acid. If it becomes cloudy or coagulates when warmed, continue heating in the water-bath. If the filtered gelatin answers the above requirements then fill, with the aid of a small funnel, into test-tubes to a depth of 1 to 1^ inches. Avoid touching the neck of the tube with gelatin. These test-tubes are first thoroughly cleansed, allowed to dry, then plugged with cotton, placed upright in a wire basket and sterilized in a dry heat oven at 150 to 175 C. for 1 hour. After the tubes have been filled with gelatin they are again sterilized by heating in the steam sterilizer, (100 C.) for 15 minutes, each day, for the next consecutive three days. MEMORANDA. MEMORANDA. II. STRUCTURE OF THE BACTERIAL CELL. The cell- wall possibly composed of cellulose or woody- fibre Difficultly seen its demonstration by icdine. Capsulated bacteria. ZoOgloea the result of fusion of the cell-walls of many bacteria. The cell contents protoplasm existence of a nucleus. Homogeneous granular. As a rule the cell is colorless. A few are slightly colored. Chlorophyll, the green coloring matter of plants, is present in a siunll number. The granulose reaction given by some blue color with iodine, same as with boiled starch. Motion Brownian, or molecular movement. Real motion. Actual motion observed in many bacilli, in spi- rilla and in two micrococci. Whips or flagella. Their arrangement on the bacillus, spirillum, vibrios, micrococciis. Giant whip-. Demonstration of, Action of iodine on protoplasm, Capsules, Whips. PREPARATION OF POTATO CULTURES. Select three sound potatoes and then clean them thoroughly, under the tap, with the aid of a brush. By means of a knife remove any bad spots or depressions that 8 may exist, since these frequently harbor bacteria which are highly resistant to destruction. Place the potatoes, thus prepared, in a solution of mercuric chloride (1-1000) for | to 1 hour; transfer to a tin pail with a perforated bottom, and then place tin's in a steam sterilizer. Heat for f to 1 hour, the potatoes should be well cooked. Remove the pail and allow the potatoes to partially cool. A "moist chamber" is prepared by placing around filter paper on the bottom of the lower dish and moisten- ing it with mercuric chloride, allowing any excess of the solution to drain off. Three potato knives are sterilized by heating in the flame till the edge begins to redden. They are then set aside to cool, with the edge up, on a block or over the edge of the table so that the blade does not touch anything. The partially cooled potato is now picked up with the left hand, which previously has been dipped in mercuric chloride, and cut into halves by a horizontal section with a sterilized knife. Each half of the potato is carefully placed in the moist chamber with the cut surface turned upwards. Contact with the cut surface must be avoided. To inoculate, transfer a small portion of the bacterial growth by means of a sterilized and cooled wire or knife to the potato, and then thoroughly spread this material over the surface avoiding the outer inch. The potato is held in the fingers of the left hand, which has been dipped in mercuric chloride. The number of bacteria which is thus transferred to the surface of the potato is usually so great that when they develop the entire surface is covered with a continuous growth. To isolate bac- teria, therefore, it is necessary to resort to dilution cul- ture. For this purpose a small amount of material is taken from the surface of the inoculated potato, or No. 1, by means of a sterilized knife, and transferred in the same manner as before to the surface of potato No. 2, where it is likewise spread thoroughly, and evenly, over the surface. The number of bacteria transplanted to the second potato MEMORANDA. MEMORANDA. -9- is still, as a rule, too great. Hence, with a sterilized knife a minute amount of material is taken from the surface of potato No. 2 and transferred to that of potato No. 3, where it is spread as before. If the inoculation is properly carried out the third potato will have-but a few bn.cteria scattered over the sur- face, and separated by inch or more. Each germ thus isolated from its neighbor soon multiplies so that in 24 to 36 hours a small growth of about the size of a pin head becomes visible. This isolated growth is known as a colony, and inasmuch as it is derived from a single cell it is &pure culture of that organism. In this and all subsequent work successful results and freedom from danger depend upon the rigid sterilization of all articles used. Hence, sterilize all instruments, wires, etc., immediately before use, and immediately after use, before placing them back on the table or in the tumbler. LABORATORY WORK. Make dilution culture (on three potatoes) of the Micrococcus prodigiosus. Mass culture (single potato) of Orange sarcine. Red bacillus of water. Violet bacillus of water. 10 III. LIFE HISTORY OF A BACTERIAL CELL. Young cells grow, attain full size, multiply. Spore formation, analogy of spores to seeds of higher plants. Observed in many bacilli, few spirals, but not in micrococci. Sporogenic granules their coalescence the spore. Spore germination observed in but few instances. Bacillus snbtilis, Cohn ; Bacillus butyricus, Praz- movski. Bacillus anthraci?, Koch ; Bacillus megaterium, DeBary. Each spore gives rise to but one bacterial cell, and a cell develops but one spore. Spore formation, therefore, a means of reproduction, not of multiplication. Structure of a spore dense, highly resistant cell- wall the contents. Behavior of aniline dyes. Action of heat, cold, desiccation, chemicals. Their importance as resting or permanent forms. Position of the spore in the cell median or termi- nal with or without enlargment. Clostridium form Drumstick or u Kopchen v form. Attempts at classification Endospore and arthro- spore bacteria. Spore formation not the result of exhaustion of soil, but like the flower and fruit of plants represents the high- est stage of development. It occurs only under favorable conditions medium, temperature, oxygen. Asporogenic bacteria, result of unfavorable environ- ment influence of calcium. MEMORANDA. MEMORANDA. 11 Multiplication of bacteria always takes place by division one cell forms two and only two new cells. Threads result from division of bacilli which remain adherent end to end by the undivided cell membrane. Diplocoocus Streptococcus Staphylococcus. Division in two directions results in Tetrads. Division in three directions results in sarcines. Demonstration of, Sporogenic granules, threads, diplococci, Staphyl- ococci, streptococci, tetrads, sarcines. Spores, median and terminal. 12 IV. THE MICROSCOPE AND ITS COMPONENT PARTS. The stand Coarse adjustment by rack and pinion. Fine adjustment by micrometer screw. The optical part mirror, Abbe condenser with iris diaphragm, objectives, eye-pieces. (Structure of the Abbe condenser its object. In examining unstained specimens contract the diaphragm. In examining stained specimens open the diaphragm. Requirements of good objectives : Magnification, defining power, resolving power. Achromatic and apochromatic objectives. Homogeneous oil-immersion objectives advant- ages. The cedar oil must be removed at the close of the day's work by touching the lens carefully with soft filter paper. Slides and cover-glasses for microscopic work must be rigidly clean. The cover-glasses should be kept in alco- hol and wiped dry when needed. If surface is at all greasy, heat the cover-glasses with sulphuric acid and potassium bichromate, wash well with water and transfer to alcohol. EXAMINATION OF BACTERIA IN HANGING DROPS. This is intended to show the bacteria in the living condition their form and arrangement, presence or absence of motion, the appearance of the protoplasm, division of the cells, sporogenic granules, spores, etc. MEMORANDA. MEMORANDA. 13 Place a small drop of water in the center of a clean cover glass. With a sterilized and cooled platinum wire, which is fused into the end of a glass rod, 6 or 7 inches long, transfer a minute amount of the bacterial growth from the surface of one of the potato cultures, to this drop of water. Place the cover-glass on a table or on a block. Select a clean " concave slide " and apply a ring of vaseline with a brush or stick to the edge of the cavity. Invert the slide thus prepared over the cover- glass with the drop and gently press down till the little chamber is sealed air tight. The slide is now ready for microscopic examination, and if properly made the drop of water will be flat, and will not run when the slide is placed on its edge. Place the slide, with the cover-glass uppermost, on the stage of the microscope, .and first find the edge of the drop with a low power No. 3 objective. If too much light is present, constrict the diaphragm. The edge of the drop should be seen as a sharp line passing through the field of the microscope. Holding the slide between the thumb and forefinger of the left hand, slowly move it so that the edge of the drop constantly remains in the field. In this way the entire edge or circumference of the drop should be examined, chiefly for the purpose of practice in moving a slide under the microscope. Owing to the min- ute size of bacteria they cannot be seen under this mag- nification. To observe the individual cells, therefore, recourse must be had to the higher powers No. 7 objec- tive, or the -iV inch homogeneous oil-immersion objective. Examination with No. 7 objective. Having found the edge of the drop with No. 3 objective, replace this by No. 7, by rotating the nose-piece. Then lower the tube of the microscope by the coarse adjustment till the objective almost touches the cover-glass. The field of the micro- scope now is usually very dark, hence open the diaphragm a trifle to admit enough light to see distinctly. With the fine adjustment now raise the microscope tube till the edge of the drop is brought out distinctly. By focussing 14 the edge carefully the bacteria will be readily detected. Now move the slide as mentioned above, so as to examine the entire edge of the drop, and also the center. Study the characteristics of the microorganisms present. In working with high powers, while focussing, it is desirable to constantly hold the slide between the thumb and forefinger of the left hand, imparting to it a slight motion. If this motion is arrested it is due to pressure of the objective, which has been lowered too far, and unless the pressure is promptly relieved, damage may result. Examination with A- homogeneous oil immersion objective. Having studied the bacteria in the hanging drop with the No. 7 objective, replace No. 3 objective and again find the edge of the drop. Now raise the tube of the microscope, bring the iV objective into position. Place a drop of cedar oil on the center of the cover-glass, and lower the tube till the objective touches the oil. As the field is now very dark open the diaphragm slightly. Focus the edge of the drop with the fine adjustment, holding the slide between the fingers of the left hand. Examine carefully the bacteria present, their motion, structure, etc., and also different parts of the drop in the manner already indicated. LABORATORY WORK. Make hanging drops of the four kinds of bacteria growing on the potatoes, and examine as above. Too much time cannot be devoted to the work at this point, as the practice thus obtained is indispensable to the easy and successful manipulation of the microscope. MEMORANDA. MEMORANDA. V. REQUIREMENTS OF BACTERIA. Bacteria, like all living cells, require certain nourish- ing substances. Absence of chlorophyll inability to acquire carbon from carbonic acid. Carbon, therefore, derived from pre- formed carbon compounds sugar, proteids, etc. Nitrogen derived from organic and inorganic sources --proteids, nitrates, nitrites, ammonia. Presence of moisture necessary for growth. Suitable reaction of medium neutral or slightly alka- line. These conditions solutions of organic nitrogenous substances widely distributed in nature. Hence the occurrence of bacteria almost everywhere on the surface of the ear tli. Almost total absence of bacteria at high altitudes, in air of mid ocean, and in deeper layers of the earth. Absence of bacteria in organs and circulating fluids of the healthy normal body. Theory of spontaneous generation, the outcome of the wide distribution of bacteria and lack of knowledge con- cerning the resistance of bacteria and their spores. Its overthrow. Classification of bacteria according to habitat sapro- phytic and parasitic. No sharp line of distinction to be drawn facultative and obligative. Temperature requirements. Each organism has its minimum, optimum and maximum temperature for growth. In general the minimum temperature is about 15 C.; the maximum about 40. 16 Optimum temperature of saprophytic bacteria, that of the room, or summer, 25 to 30 C. Optimum temperature of parasitic bacteria, that of the body, about 37.5 0. Influence of cold of heat, 70 and above. Growth of some bacteria at 0. At 50, 60, 70 C. Injurious action of diffuse light, and especially of sunlight. LABORATORY WORK. Continuation of that of preceding day. MEMORANDA. MEMORANDA. VI. CHEMISTRY OF BACTERIA. All living cells take in food, elaborate certain pro- ducts and give off waste or metabolic products. Production of, Gases Carbonic acid, hydrogen sulphide, nitro- gen, etc. Acids Acetic, lactic, butyric, phenyl-propionic, etc. Amido acids. Nitrous and nitric acids. Alkalies Ammonia, and its substitution com- pounds, the amines. Ptomaines Alkaloidal compounds like the vege- table alkaloids. May be poisonous (toxines) and non-poisonous. Proteids Bacterial proteids may be highly poisonous. Compare with the phytalbumoses of plants, and the proteids in venom of ser- pents. Soluble ferments or enzymes Analogy to the soluble ferments of the animal body and of certain plants. Alcohols Ethyl, propyl, butyl; phenol, etc. Reducing powers oxidizing powers. Classification of bacteria according to function. Zymogenic fermentation bacteria. Saprogenic putrefaction bacteria. Chromogenic pigment producing bacteria. A erogenic gas producing bacteria. Photogenic light producing or phosphorescing bacteria. Fermentations as vital phenomena, the result of activity of microorganisms, so-called organized ferments, 18 bacteria, yeasts, moulds, etc. ; unorganized or soluble fer- ments (enzymes) produced by bacteria. Alcoholic, acetic, butyric acid, etc., fermentations. Ferment changes in the mouth dental caries. Abnormal fermentation in the stomach in the intes- tines, Summer diarrhoea of infants. Ammoniacal fermentation of urine Hydrothionuria. Putrefaction is putrid fermentation Proteids acted upon whereas in true fermentations, starches, sugars, and cellulose are acted upon. Poisonous foods. Liquefaction of albumin, gelatin, etc., by bacteria due to soluble peptonizing ferments. Liquefying and non-liquefying bacteria. Inversion of starch by bacteria coagulation of milk. Nitrification in the soil. Production of pigment, as a rule, is a secondary process, taking place outside of the cell. The oxygen of the air acts on a colorless or leuco-product. Sometimes the pigment may be formed directly by the cell primary product. Phosphorescence the result of intracellular activities. In fermentation and putrefaction more or less com- plex, dead animal and vegetable substances are acted upon by microorganisms; transformed into relatively simp- ler compounds, and eventually into inorganic forms, as carbonic acid, ammonia, nitrates, nitrites, etc., which are now utilizable by living plants. Thus, lifeless remains become indispensable to new life, and bacteria, in their role of scavengers of nature, prove beneficial. Certain bacteria may live on living matter in the ani- mal body, and in plants, not only at their expense, but even to their injury, producing changes which may result in disease and death. Division of bacteria into pathogenic and non-patho- genic, toxicogenic and non-toxicogenic. MEMORANDA. MEMORANDA, l/U 19 STAINING OF BACTERIA. Aniline dyes are commonly employed for this purpose, and those which are used most frequently are fuchsine, gentian violet, methyl violet, methylene blue, and vesuvin or Bismarck brown. The first two are quite permanent, stain rapidly and deeply and as a rule are to be preferred. Methylene blue stains slowly and is excellent for special purposes. Saturated solutions in absolute or strong alcohol are first prepared and serve as stock. These concentrated solutions are only very rarely used as such. Ordinarily they are diluted with water. For this purpose pour into the small staining bottle (1 oz.), which is provided with a pipette, some of the concentrated solution to a depth of about i inch, then fill the bottle with water. This dilute stain must be perfectly clear from cloud or precipitate, and should not be transparent. The dilute stains do not keep for any length of time owing to the deposit of the dye. In such cases a fresh solution should be made. SIMPLE STAINING OF COYER-GLASS PREPARATIONS. Place a small drop of water on a cover-glass which is held between the thumb and forefinger of the left hand. The cover-glass must be perfectly clean so that when the drop is subsequently spread over the surface with a plat- inum wire, it will spread in a thin film and not gather into minute globules. The drop of water taken should prefer- ably be so small that when spread in a thin film over the cover-glass, it will dry almost immediately, and thus evenly cover the whole surface. Large drops of water by drying slowly, tend to leave unsightly " shore-lines." With a sterilized platinum wire pick up a minute amount of the growth from one of the potato cultures and touch the drop of water cautiously once or twice. Then sterilize the wire, and when cool, spread the drop of water evenly over the whole surface of the cover glass, avoiding, however, contact with the fingers. In a few minutes the water evaporates, and if desired, this may be hastened by 3 20 waving the cover glass, to and fro, over a flame, at a height of 6 or 8 inches. Care must be taken not to transfer too much material to the drop of water, for in such cases the cover-glass on subsequent staining will be found to be one mass of bacteria. The cover-glass prepartion which has now been dried in the air or over a flame, must be u fixed" before apply- ing the staining solution. For this purpose, take hold of the cover-glass with a narrow-pointed pair of forceps, and pass it rapidly through the flame, from above down- ward, keeping the specimen side up, away from direct contact with the flame. If the exposure to the flame is too long the bacteria may be so altered as to refuse to take the stain subsequently, or at most poorly. On the other hand, if not heated enough, the bacteria will readily wash away on treatment with the dye, or with water. As soon as the fixed cover-glass is cool, and while still holding it in the forceps, cover the specimens with the dilute aniline stain. Allow this to act for the necessary length of time (i ^ 1 min.) and then wash off com- pletely by means of a syphon or bulb wash-bottle. Place the cover-glass now on a piece of filter-paper, resting on the right index finger, and rotate carefully till the lower side is perfectly clean and dry. Now invert the specimen, with the moist specimen side downward, onto a clean glass slide. Sufficient water should be present to fill in the space between the cover-glass and slide. Specimens should never be examined dry. Place the slide thus prepared on the stage of the microscope and first examine with the No. 7 objective and subsequently with the ^ homogeneous oil-immersion objective, in the same way as was done in examining hanging drops. A good cover-glass should show the bacteria well stained, not in masses, but separated from each other, and evenly distributed over the entire cover-glass. If the stained bacteria are seen to move about it is due to insuf- ficient fixing in the flame. MEMORANDA. 21 LABORATORY WORK. Practice staining cover-glass preparations made from the different potato cultures, em- ploying the five aniline dyes mentioned above. Also stain preparations made from the white matter on the teeth, near the edge of the gums, and look for comma bacilli, spirilla, and leptothrix threads. To make permanent stained preparations, the speci- men which has proven satisfactory on preliminary exam- ination in water as above, can be floated off the slide by first bringing a drop or two of water near the edge of the cover-glass. If any oil is on the upper side it should be carefully removed by rotating the cover-glass on a piece of filter-paper. The specimen is then allowed to dry in the air, or by gently waving over aflame. A clean glass slide is then selected and a suitable drop of Canada bal- sam placed in the center. The dry cover-glass is then inverted, specimen side down, and carefully lowered until it touches the balsam. If necessary, gentle pressure is applied so as to cause the balsam to spread out under the cover-glass. The following synopsis will be of service : Simple Stain. Cover-glass preparation. Air- dried. 3 x through flame. Dilute stain (^ 1 min.). Water (and examine). Air-dried. Canada balsam. GELATIN PLATE CULTURE. Tha- object of this method, as with the dilution potato culture already made, is to isolate the several kinds of bacteria that may be present. The isolated organisms developing in a solid, transparent medium, form colonies which are easily perceived and from which transplan- tations can be readily made. Pure cultures of the differ- ent kinds of bacteria 'are thus obtained. 22 First, sterilize six glass plates by placing them in an iron box and heating this in the dry heat sterilizer, at a temperature of 150-175 0., for one hour. Then remove the box and allow it to cool. PJace three of the sterilized gelatin tubes in a water- bath which has been warmed to about 30-35 0. When the gelatin melts the tubes are ready for inoculation. With a sterilized, cooled platinum wire pick up a minute amount of the growth of the potato culture of Micrococcus prodigiosus. Place one of the liquefied gelatin tubes between the thumb and index finger of the left hand, so that it is almost horizontal. The neck of the tube with its plug, as well as the palm of the left hand, is turned to the right. While still holding the platinum wire in the right hand, grasp the cotton plug with the little finger of that hand, and remove it by slight rotation. Now pass the inoculated wire into the tube and thoroughly mix the bacteria, thus introduced, with the gelatin. Then with- draw the wire, replace the cotton plug, and sterilize the platinum wire in a flame. With a colored wax pencil mark the tube thus inocu- lated with J. Likewise mark another liquefied gelatin tube with 2. Place tube 1 in the left hand in the same position as before, and then next to it, tube 2. Remove the cotton plug of tube 2 and place it between the adjoin- ing index and middle fingers. Then remove the cotton plug of tube 1 and place it between the ring and little finger. Now, with a sterilized cooled platinum wire, the end of which is provided with a small loop, transfer a loopful of gelatin from tube 1 to tube 2 and mix well. Return the platinum wire to tube 1 and again transfer a loopful of gelatin to tube 2. Repeat this once more, so that all told, three transfers of inoculated gelatin have been made. Replace the cotton plugs into their respect- ive tubes, sterilize the platinum wire and set the tubes in a tumbler having a layer of cotton on the bottom. Mark a new liquefied gelatin tube with 3. Then place 2 in the same position in which No. 1 was just held, and MEMORANDA. MEMORANDA, 23 next to it place tube 3. Remove the cotton plugs and place in their respective places as before. With the ster- ilized cool platinum wire make three successive transfers of gelatin from tube 2 to tube 3. Return the cotton plugs to their tubes, sterilize the wire, and set the tubes aside in the tumbler. Each of the three gelatin tubes has now been inocu- lated. Tube 1 usually has a very large number of bac- teria, while tube 2 has less and tube 3 should have but a small number, so that subsequently when colonies develop these should be separated from one another by an appre- ciable distance. It is necessary therefore, to take a very minute amount of material for the inoculation of tube 1 in order to obtain good dilutions. In transferring gelatin from one tube to another care must be taken to prevent the wire from coming in contact with the neck or walls of the tubes. Prepare the ice plating apparatus for use and then level it. Remove a sterilized glass plate from the iron box by grasping the edges with two fingers ; place it upon the ground plate of the ice apparatus and cover with the bell jar. As soon as the plate is cool it is ready to receive the gelatin. Before pouring the contents of the tubes upon the plates it is necessary, as a matter of precaution, to sterilize the neckof the tube. To accomplish this, cut off the cotton which projects from the tube, push in the plug a trifle, and then rotate the neck of the tube in a flame till the cotton begins to turn yellow. As soon as the neck oi the tube cools the gelatin can be poured. To do this remove the cotton plug with a pair of forceps, ster- ilized in the flame, and place it between the fingers of the left hand. Transfer the tube to the right hand, raise the bell jar somewhat and pour the gelatin onto the centre of the plate. With the lip of the tube spread out the gela- tin as rapidly and as fully as possible, avoiding, however, the edges of the plate. Allow the gelatin to solidify under cover of the bell-jar. 24 Prepare a " moist chamber" in the same way as for potato cultures except that tap water may be used for moistening instead of mercuric chloride. On three small pieces of paper write the name of the germ or material, the number of the plate and date. Now place a glass bench on the bottom of the moist chamber and on it the label for plate 1. Transfer the gelatin plate from the ice appa- ratus to the bench. Pour the remaining gelatin tubes on plates in the same manner as described and when cool transfer to the benches which are arranged one above the other, in the moist chamber. Each chamber can hold a stack of six plates. The moist chamber containing the plates is now set aside for 1-2-3 days, during which time colonies will develop and be ready for further examination. LABORATORY WORK. Make plate cultures of Micrococ- cus prodigiosus and of Bacillus Indicus. MEMORANDA. MEMORANDA. 25 YJII. EXAMINATION OF COLONIES. In a greater or less period of time, varying usually from 1 to 3 days, colonies develop on the plates, and as soon as they become of suitable size they are ready for examination. A careful study of the colonies on a plate should first be made with the unaided eye. Several char- acteristics of growth can often thus be recognized quite early. Especial attention should be given to the form and appearance of the colonies; the presence or absence of pigment; liquefaction or non-liquefaction of gelatin, etc. It should be remembered, however, that a given organism may give rise to at least two kinds of colonies which sometimes are quite different in appearance. Thus, we may have surface colonies, and also deep colonies. The former developing on the surface of the gelatin are unhindered in their development, and may, therefore, spread out and thus acquire peculiar characteristics, more- over, having ready access to oxygen, pigment formation and liquefaction will be first seen in connection with these surface colonies. The deep colonies, on the other hand, are surrounded on all sides by solid gelatin, and hence, much the same resistance to growth will exist in all direc- tions. The result is that deep colonies, as a rule, are much alike in appearance. Thus, the form is usually round or oval, with sharp edges, and the contents are slightly granular and yellowish. The plate should now be placed upon the stage of the microscope and the colonies carefully examined with a low-power No. 3 objective. Further characteristics can thus be brought out which have escaped the eye. The study of the micro-organisms which compose the 26 colonies should now be made. This is done by making hanging drop examinations and stained preparation accord- ing to the directions already given. The object in making plate cultures is to obtain colo- nies which, since they are derived from a single cell, are pure cultures of that organism. To perpetuate and keep up the pure culture thus obtained, it is necessary to resort to transplantation. For this purpose the colony to be transplanted is touched with a sterilized and cooled, straight platinum wire. A portion of the colony will adhere to the end of the wire and can be transferred to a tube of sterilized gelatin. The wire is usually pushed down the centre of the tube, in which case we have what is known as a stich or stab culture. The operation of touching the colony is one that requires the greatest care to prevent contamination with foreign colonies, or other material, thus vitiating the pure culture. For this reason it is always carried out under a microscope, and so far as patience is concerned it certainly is not inaptly called "fishing." The gelatin plate is placed on the stage of the micro- scope and, with the No. 3 objective, a suitable colony for transplantation is selected. It is desirable to have but one colony in the field of the microscope. A straight platinum wire, previously sterilized and cooled, is held in the right hand in the pen position. The hand is supported by rest- ing the little finger on the right corner of the stage. The platinum wire is then inserted about midway between the front lens of the objective and the surface of the gelatin. It is held steadily in this position, and on looking into the microscope an indistinct shadow is seen. The wire is slowly drawn back till the end of the shadow or indistinct wire is directly over the colony. Should the wire in doing this touch the objective or the gelatin it must be sterilized at once and the operation repeated. When the end of the wire has been brought over the colony, gradually lower the point till it touches the colony or cuts it into two. MEMORANDA. MEMORANDA. 27 Now carefully remove the wire without touching the microscope or some other portion of the gelatin. A tube of solid gelatin is held in the left hand in an almost hori- zontal position, the plug is then removed by grasping it with the little finger of the right hand, and the platinum wire, which has a small portion of the colony attached to it, is slowly forced down the centre of the gelatin to the bottom of the tube. The cotton plug is at once replaced, the wire sterilized and the tube set aside. The stich culture thus made is a pure culture and is now labelled with the name of the organism and date and set aside to develop. In a few days development takes place along the line of inoculation and more or less char- acteristic growth results. The manner of growth should be daily observed. Hanging-drop and stained preparations can, of course, be made if desired, from tube cultures. When the gelatin is very old, and hence too solid, it tends to split as soon as the platinum wire is forced into it. This is remedied by melting the gelatin and allowing it to re-solidify. LABORATORY WORK. Examine carefully the colonies with the eye and under the microscope. Make hanging- drop and stained preparations from the colonies. Prac- tice "fishing" and make stich cultures from each of the- different colonies. The line of study of the Micrococcus prodigiosus and Bacillus Indicus and of the various bacteria to be pres- ently taken up, consists first, in making plate cultures. Colonies are thus obtained, the characteristics of which are to be thoroughly studied. Hanging-drop examination! and stained preparation are next made, thus becoming; familiar with the organism itself. Stich cultures in gela tin are then made and also streak cultures on potato or agar which will be presently described. Finally drawings should be made showing the form of the colony, the form of the organism, the appearance of the stich culture, etc. 4 28 Summarized then, each organism, subsequently de- scribed, is to be studied by making Plates, Colonies, Hanging-drop examination. Stained preparation, Stich culture in gelatin, Streak culture on potato, or agar, or both. Drawings. MEMORANDA. MEMORANDA. 29 IX. MODIFIED GELATIN PLATE CULTURES. Several modifications of the Koch plate method as just described have been introduced whereby the same, if not better results are obtained with less apparatus. In the plate method contamination not infrequently results from exposure to the air while on the ice apparatus, or subsequently when kept in the large, moist chamber. An examination of one plate necessitates the exposure of the remaining plates to contamination with the organisms in the air. Furthermore, it not infrequently happens that the gelatin on an upper plate undergoes liquefaction and then drips over the edges of the plates on those below it. To overcome these difficulties Petri introduced the use of shallow dishes which are about 10 cm. in diameter. Petri Dish Culture. Place the Petri dishes in a wire basket and sterilize in the diy-heat oven by heating I hour at a temperature of 150-175 C., then allow to cool. Inoculate three gelatin tubes with the organism to be plated in the same manner as for ordinary plates. Cut off the projecting cotton, sterilize the lip of the tube as before, then pour the contents of each tube into one of the cool, sterilized Petri dishes, properly labeled. Replace the cover and gently tilt the dish from side to side so as to cause the gelatin to spread evenly over the bottom. Allow the gelatin to solidify, then set the dishes aside for colo- nies to develop. When the colonies develop examine on the stage of the microscope and transplant as with ordi- nary plates. In this method each dish constitutes a plate by itself. It can be readily examined and the risk of contamination is reduced to a minimum. In addition to that the use of 30 the ice-machine, plates, benches, plate boxes, etc., is done away with. Esmarck Roll- Tube Cultures. In this method the advantages of the plate method are secured without the use of any extra apparatus, as plates or dishes. The inoculated gelatin instead of being poured out onto steril- ized plates or into dishes is solidified in a thin film on the inside wall of the test-tube. Another advantage of this method is that it is well adapted for those organisms which grow very slowly, and require a week or two to form distinct colonies. Desiccation of the gelatin can be readily prevented in the roll- tube, whereas it is much more difficult in plate or dish cultures. Inoculate three gelatin tubes with the material to be plated, in the usual manner. Then cut off the cotton plug on each tube and cover the end with a rubber cap. Place the tube in a horizontal position, or nearly so, in a dish of cold, or ice-water and rotate it carefully until the gelatin solidifies in an even thin layer over the inside wall of the tube. Avoid contact of the gelatin with the cotton plug. Now set aside in a cool place to develop and then examine the colonies under a microscope and make trans- plantations. The operation of fishing in this case will, of course, require special care. LABORATORY WORK. Make Esmarch roll-tubes of saliva (1 loopful); and of the Violet bacillus of water. Make Petri dishes of the Red bacillus of water. MODIFIED POTATO CULTURES. The method of making potato cultures of bacteria is open to much the same objections as the ordinary gelatin plate method. Two modifications, analogous to the two modified gelatin plate cultures, are commonly employed and are excellently adapted for their purpose. . . Esmarch Potato Cultures. The Esmarch dishes, which are about 6 cm. in diameter and 2cm. high, are ster- ilized in the dry heat oven in the usual manner, and then allowed to cool. A small, sound potato is selected and MEMORANDA. MEMORANDA. rcKSIl 31 held with the thumb and forefinger of the left hand. With a potato knife held vertically, the outer edge of the potato is pared circularly. Two horizontal sections, one upper and a lower one, are now made and the clean, sound core of the potato thus obtained is slipped into a sterilized Esmarch dish. Each dish is thus supplied with a clean potato section. The dishes are then placed in a steam sterilizer and steamed for f to 1 hour. The pota- toes will then be sterilized and cooked. The inoculation of the cold sterilized potato is made in essentially the same way as in the ordinary potato culture?. As each potato is in a small sterilized dish by itself the risk of contamination is very small under care- ful and rapid manipulation. LABORATORY WORK. Make dilution cultures of Micro- coccus prodigiosus, using three of the Esmarch potato dishes. Test-Tube Potato Cultures. These were introduced independently and almost simultaneously by Bolton of this country, Globig, of Germany and Roux of France. In convenience and reliability of cultures, the method leaves nothing to be desired. Clean, plug and sterilize about 6 or 8 large test-tubes (| x (> inches). Also clean several large potatoes and place them in boiling water or in steam sterilizer for about j of an hour. By means of a cork-borer, having nearly the diameter of the test tubes, punch out a number of cylinders from the cooled potatoes. Place these cylinders on a clean piece of paper, trim off the ends and then cut each cylinder diagonally into two pieces. Into each of the sterilized test-tubes place one of these pieces of pota- toes with the circular end lowermost. Each tube then contains a piece of potato having an inclined surface. Now sterilize again in a steam sterilizer for about f to 1 hour. Instead of a cork borer an excellent substitute may be obtained by cutting a test tube in two. By making cylinders from previously boiled potatoes, the pieces in 32 the tubes remain perfectly white, whereas cylinders made direct from the raw potato will frequently become discol- ored by the subsequent heating. The inoculation of the sterilized potato tubes is easily done. If it is desired to obtain dilution cultures, that is, colonies, this can best be accomplished by making several parallel streaks on the surface of the potato with the end of the platinum wire, if necessary repeating the inocula- tion in two or three tubes, using the same wire. When transplanting a pure culture, as a portion of a colony, a single streak should be made along the middle of the inclined potato. LABORATORY WORK. Make^streak cultures from the colonies of the bacteria which have been studiedthus far. MEMORANDA. 34 BACILLUS PRODIGIOSUS. MONAS PRODIGIOSA OF EHRENBERG. MICROCOCCUS PRODIGIOSUS OF OLDER WRITERS. Origin. Found on starchy substances, rice, potatoes, moist-bread ; also on meat, albumin, milk, etc. May form at times local epidemics, infecting foods as bread, meat, sausages, with production of pink or red color. " Bleed- ing" bread or wafers. Form. Short rod, slightly longer than its width. May form short threads, especially in slightly acid media. Usually sinirle or in pairs. Motility. Ordinarily shows no motion, except a marked Brownian movement. In acid or very dilute media appears to have slight motion. Sporulation. Has not been observed. Possesses marked resistance to desiccation. Anilin Dyes. Stain readily. Growth. Very rapid. Gelatin Plates. Deep colonies, round or oval, with sharp border and light brown color. Surface colonies irregular, rough border, granular, with reddish centre, and surrounded by clear, liquefied gelatin. Stich Cultures. Rapid, funnel-shaped liquefaction, extending along entire line of inoculation. Red scum forms on surface of liquid, eventually settles and entire contents of tube colored bright red. Streak Cultures. On agar, forms abundant, moist, spreading growth, having an intense red color which is non-diffusible. On potatoes, especially rapid, slimy growth, with marked pigment production. The pigment, when old, has a metallic fuchsine-like lustre. Odor of trimethylamine. On blood serum, growth as on agar, with liquefaction. Milk, Growth takes place and the pigment is held in solution by the fat globules. Oxygen requirements. Is a facultative anaerobe. Pigment is formed only in presence of oxygen. Temperature. Grows best at ordinary room tem- perature. In incubator ceases to form pigment, and may temporarily lose this property, i. e., becomes attenuated. Behavior to Gelatin. Rapidly liqueties as result of formation of soluble ferment. This liquefying property may be diminished or temporarily lost by growth in acid media. Aerogenesis. Strong odor of trimethylamine on potatoes. Pathogenesis. No pathogenic power. Its soluble products in large amounts may have a toxic action. The cellular proteids may induce suppuration. Animals in- susceptible to malignant oedema are rendered susceptible by injection of B. prodigiosus. Rabbits inoculated with anthrax are saved by injection of B. prodigiosus. MEMORANDA. 36 BACILLUS INDICUS. Koch. Origin. Isolated in India from the contents of the stomach of a monkey. Form. Small, narrow, very short rod with rounded ends. Motility. Actively motile. Sporulation. Not definitely observed. Anilin Dyes. Readily stain. Growth. Is rapid. Gelatin Plates. Deep colonies are yellowish, with wavy contour. Sur- face colonies grayish yellow, finely granular, with fibrillated borders. Show movement of contents, rapidly liquefy and may show a light pink color. Stich Cultures. Rapid liquefaction along line of inoculation. Dense flocculent growth settles on the bottom, and is grayish or light pink in color. A delicate scum forms on the surface and is colored from a light pink to brick red. Streak Cultures. On agar, forms a low, moist, spreading growth, which usually is faint pink in color. On potatoes, the growth is low, not slimy as M. prodigiosus,and the color is more marked than on other media. On blood serum, liquefaction results with or without pigment production. Oxygen requirements. Grows best in the pres- ence of air, but is a facultative anaerobe. Pigment pro- duction depends upon the presence of oxygen. Temperature. The optimum is about 35 C. Pig- ment absent in cultures that develop in the incubator. Behavior to Gelatin. Liquefies very rapidly. Pigment production. Varies greatly. May be grayish to bright brick red. Usually is light pink, so that present cultures may be considered to be attenuated. Pathogenesis. Has marked toxic action, and when injected in large amounts into the abdominal cavity, or into the veins of rabbits and guinea-pigs, proves fatal. Rabbits develop marked diarrhoea and die in from 3 to 20 hours. On post-mortem the intestines show a severe inflammatory condition of the mucous membrane and at times ulcerations. MEMORANDA. 38 BACILLUS RUBER OF KIEL. J. BREUNIG. BACTERIOLOGISCHE UNTERSUCHUXG DES TRIXKVVAS- SERS DER STADT KIEL (iNAUG. -DISSERTATION) KIEL, 1888. EM. LAURENT. ANNALES DE L'INSTITUT PASTEUR IV, 464, 1890. Origin. Drinking waler of Kiel. Form. Rods about three to five or seven times as long as wide. Motility. Somewhat motile, and the motion de- pends on presence of oxygen. Sporulation.--Not observed. Anilin Dyes. Stain readily. Growth. Rapid and abundant. Gelatin Plates. Deep colonies are oval, pale yellow, with wavy or even border. The surface colonies are blood red in color, spread rapidly and have a sinuous border; are surrounded by a clear zone and liquefy gelatin. Stich Cultures. Develop along the line of inoculation and liquefaction takes place. The fluid becomes strongly colored and gas may form in the deeper layers. Streak Cultures. On agar, at 30-35, the growth is at first a pale rose, and later becomes a brick red. On potatoes, at 30-35, develop rapidly, forming a purple red growth. At lower temperatures the color is less intense, and at first is orange, later carmine red. Milk. At 35, coagulation takes place in 24 honrs, without a trace of coloration, due to rapid growth and production of acidity. At ordinary tem- perature the coagulation takes place slowly and the fluid gradually colors. Oxygen requirements. Is a facultative anaerobe, but requires oxygen to form the pigment Temperature. Grows from 10 to 42C. The opti- mum is 30-35, and above this the growth ceases to be colored. Direct insolation kills it in 5 hours. By exposure of 3 hours is not killed but no longer produces the pig- ment, i. e , becomes attenuated. Behavior to Gelatin. Liquefies gelatin quite rapidly. Aerogenesis. Some gas bubbles may form in gela- tin tubes. Pathogenesis. No action observed. MEMORANDA, 40 BACILLUS RUBIDUS. RED BACILLUS OF WATER. Origin. Water. Form. Long-, narrow rod; forms threads. Motility. Ac-lively motile. Sporulation. No spores observed. Anilin Dyes. Stain readily. Growth. Fairly rapid. Gelatin Plates. Small, yellow, finely granular colonies, with irregular border. Liquefies gelatin. Stick Cultures. Liquefies gelatin slowly along line of inoculation. The mass of bacteria settles to the bottom, colored yellowish brown, and a thin folded scum forms on the surface. Streak Cultures. On ogar, thin, irregular bordered, slightly folded and colored growth. On potatoes, the most characteristic growth is developed, which spreads and lias a bright brick red color. On blood serum, liquefaction takes place and red pigment forms. Oxygen requirements. Is aerobic. Temperature. Does not grow at the temperature of body. Behavior to Gelatin. Liquefies. Pathogenesis. No effect observed. MEMORANDA. 42 BACILLUS VIOLACEUS. VIOLET BACILLUS OF AVATEIJ. Origin. Water of the river Spree at Berlin, and of the Thames at London ; also in well water. Form. Long, narrow rods, about three times as long as wide ; forms threads. Motility. Actively motile. Sporulation. Forms median spores. Anilin Dyes. React readily. Growth. Is moderately rapid. Gelatin Plates. Irregular colonies, with loose fibrillaled borders. The center shows quite early a violet color. Liquefies. Stich Cultures. Funnel-shaped liquefaction along entire line of inocu- lation. A violet sediment collects on the bottom, while the liquefied gekxtin above is perfectly clear. Streak Cultures. On agar, forms a thin, moist, bright violet covering. On potatoes, the growth is somewhat slow but very characteristic, forming a bright violet, eventually dark covering On blood serum, the violet color is produced and liquefaction takes place. Oxygen requirements. Is a facultative anaerobe. Oxygen is necessary to pigment formation. Temperature. Does not grow at higher tem- peratures. Behavior to Gelatin. Liquefies. Pathogenesis. Has no effect. MEMORANDA. 44 BACILLUS FLUORESCENS PUTIDTJS. Fliigge. FLUORESCING BACILLUS OF WATER. Origin. Putrid media, water. Form. Short, small rods, with rounded ends. Motility. Very actively motile. Speculation. No spores observed. Anilin Dyes. Stain readily. Growth. Rapid . Gelatin Plates. Deep colonies are small, round, finely granular. Surface colonies spread rapidly and form at first a very thin plaque, with irregular, wavy border, which shows markings. Later a bluish green color diffuses through the surrounding gelatin. Odor of trimethylamine. No liquefaction. Stich Cultures. No growth in lower part of tube. Surface of gelatin, covered with grayish white growth, while the fluorescing pigment gradually diffuses downward into the gelatin. Streak Cultures. On agar, a moist, spreading growth. The agar becomes colored, but later the color fades. On potatoes, a thin grayish or brownish, moist growth lorms. Oxygen requirements. Aerobic. Temperature. Ordinary room temperature is best. Behavior to Gelatin. Does not liquefy. Pathogenesis. Without action. MEMORANDA, 46 BACTERIUM PHOSPHORESCENS. Fischer. THIS IS BUT ONE OF A NUMBER OF BACTERIA FOUND IN SEA-WATER WHICH POSSESSES THE PROPERTY OF PHOSPHORESCING IN THE DARK. PHOTOBACTEBIUM. Origin. In water of the harbor of Kiel, also on sea fish. Form. Short, thick bacillus, with rounded ends; sometimes almost a coccus. Usually in pairs, may form threads. Involution forms soon develop. Motility. No motion. Sporulation. Not observed. Anilin Dyes. Slain readily. Growth. Moderately rapid, and the cultures show a greenish phosphorescence in the dark. Gelatin Plates. Show small, white, glistening colonies, which do not liquefy gelatin. The border is sharp, irregular, and contents are granular, and show several concentric rings. Stich Cultures.- -Granular growth along the line of inoculation, but is most abundant on the surface, forming a thin grayish white covering. Even- tually the gelatin is colored a yellowish brown. Streak Cultures. On agar, potatoes, etc., growth is limited to the line of inoculation. Grows also well on fish, beef, bread, fats, etc. Oxygen requirements. Is a facultative anaerobe. The production of light depends upon the presence of oxygen, and is therefore most marked on the surface growths. The intensity of the iight may diminish and eventually become lost attenuation. May be restored by growth on suitable media, as salt fish, etc. Temperature. Does not grow in incubator. grow at C. Behavior to Gelatin. Does not liquefy. Pathogenesis. No effect on animals. MEMORANDA. 48 SARCINA AURANTIACA. ORANGE SAKCIXE. Origin. From air, weiss-beer. Form. Small, spherical cocoi, grouped in 2 and 4, and also forming package-shaped masses. Motility. None. Sporulation. None. Anilin Dyes. Slain very easily and are likely to over stain. Growth. Rather rapid. Gelatin Plates. Show round, sharp-edged colonies, which are granular and of an orange-yellow color. Liquefy. Rtich Cultures. Liquefies gelatin along entire line of inoculation. Even- tually an orange-colored deposit of bacteria forms on the bottom and the liquid above becomes clear. Streak Culture^ ')n (if/or, forms a thick, orange-colored growth. On potatoes, the pigment i.s excellently developed. Oxygen requirements. Is aerobic. Temperature. Higher temperatures unfavorable. Behavior to Gelatin. Liquefies rapidly. Aerogenesis. Not observed. Pathogenesis. Has no effect. MEMORANDA. 50 SARCINA LUTEA. Schroter. YELLOW SARCIXE. Origin. Air. Form. Larger cocci than the orange sarcine and forms more perfect package-shaped masses. Motility. None. Sporulation. None. Anilin Dyes. React readily and are likely to over- stain. Growth. Very slow. Gelatin Plates. Colonies develop very slowly as minute yellowish spots, which show an irregular outline and are markedly granular. The colonies do not liquefy gelatin. SticJt Cultures. Gi'owth is especially developed on the surface and ex- tends but slightly down the line of inoculation. Lower half of tube is usually free from growth. The color is bright yellow and in very old tubes liquefaction slowly shows itself, so that eventually a bright yellow deposit forms on the bottom while the liquefied gelatin above is perfectly cjear. Streak Cultures. On a/< Cultures. Growth occurs along the entire line of inoculation, but liquefaction is more energetic in the upper part. The liquefied gelatin remains turbi'd for some time and a thin, grayish, folded scum forms on the top. .S7/v/r CtiUii'rt'N.On ac/(ir, forms a dull white or grayish, folded growth. On potato!*, the most characteristic growth develops. The surface is rapidly covered with a thick, white, strongly folded, coherent growth. Later the color become s ;i dirty brown or red. Milk. 1 coagulated. Oxygen requirements. A e robe. Temperature. ({rows at ordinary as well as higher temperatures. Behavior to Gelatin. Liquefies rapidly. Pathogenesis. No effect observed. MEMORANDA. 56 BACILLUS MEGATERITJM. De Bary. Origin. From boiled cabbage leaves. Form. Cylindrical rods, with granular contents, 3 to 6 times as long as broad, with rounded ends. Are usually slightly bent and may form threads. Involution forms common. Motility. Slow, amseboid motion. Lateral flagella. Sporulation. Forms median spores. Anilin Dyes. Stain readily, though irregularities due to granular protoplasm may be seen. Growth. Rapid. Gelatin Plates. Colonies are at first irregular, small, yellowish masses, but subsequently show marked radiating or branching forms, which soon Mqnef y the gelatin . Stick Cu Itures. Rapid growth and liquefaction along the line of inocu- iution. May show threads of bacteria penetrating outward into the solid gel- atin. Eventually the gelatin is wholly liquefied and a flocculent mass accu- mulates on the bottom; the supernatant liquid clears up without formation of scum on top. Streak Cultures. On agar, forms a dull white or grayish covering. On potatoes, grows rapidly as a thick, slimy, grayish white mass, which is rich in spores and involution forms. Oxygen requirements. Is aerobic. Temperature. Optimum about 20C. May grow in incubator. Behavior to Gelatin. Liquefies. Pathogenesis. No effect observed. MEMORANDA. 58 BACILLUS RAMOSUS. ROOT OR WURZEL BACILLUS. Origin. Very common in earth ; occurs also in river and spring water. Form. Rather large rod?, thicker than the Hay bacillus; with slightly rounded ends. Threads common. Motility. Slowly motile. Sporulation. Large median spores occur. Anilin Dyes. Stains well. Growth. Rapid. Gelatin Plates. The colonies present a characteristic appearance, resem- bling somewhat fine branching rootlets, hence the name. At first the colo- nies are round, dark and with bristly borders. Subsequently the colonies branch and ramify throughout the gelatin which is liquefied. Stick Cultures. Are also characteristic. Growth develops along the line of inoculation and from this threads penetrate or radiate into the surround- ing gelatin. The growth is more rapid at the top than in the lower parts of the tube so that the appearance of an "inverted pine tree" results. Later the gelatin is liquefied completely. The bacterial growth accumulates on the bottom while the Ijquid above becomes clear and has a thin scum on the surface. Streak Cultures. On agar, forms a grayish growth, spreading outward from the streak so that the appearance often is not unlike thatof a centipede. On potatoes, a slimy, whitish growth which develop spores. Oxygen requirements. Is aerobic. Temperature. Grows at ordinary temperature and also in incubator. Behavior to Gelatin. Liquefies. Pathogenesis. Without effect, even in very large doses. MEMORANDA. 60 PROTEUS VULGARIS. Hauser. INCLUDED IN THE BACTERIUM TERMO OF OLDER WRITERS. Origin. Very widely distributed. Is commonly present in the putrefaction of animal proteids; has also been met with in water, in meconium, in purulent ab- scesses, and in blood and tissues of two cases of fatal putrid infection of intestines. Form. Rods, of varying length, from short oval forms to those which are 2 to 6 times as long as wide. It is usually bent and grows in pairs; may also form twisted, interwoven threads. Roundish involution forms are common. Motility. Actively motile. Flagella very numerous and lateral. Sporulation. Not observed, though cultures are re- sistant 1o desiccation and retain vitality for many months. Anilin Dyes. Stain readily. Growth. Very rapid. Gelatin Plates. Rapid and extensive liquefaction of the gelatin. The colonies are yellowish brown, with bristly borders, and in soft gelatin tend to spread over the surface and assume peculiar figures. Detached portions of colonies can be seen to move about "swarming islets." Disagreeable odor and alkaline reaction. Stich Cultures. Rapid liquefaction along entire line of inoculation, so that in a few days the entire contents are liquefied. The fluid is at first dif- fusely cloudy, but later clears up and a flocculent sediment settles on the bot- tom, while on ihe top a grayish white layer is formed. Streak Cultures. On agar, forms a grayish, slimy, rapidly spreading growth. On potatoes, it forms a dirty colored, sticky covering. Oxygen requirements. Facultative anaerobe. Temperature. Optimum lies between 20 and 24. Grows excellently in the incubator. Behavior to Gelatin. Rapidly liquefied. Aerogenesis. Forms hydrogen sulphide. Pathogenesis. Small doses have no effect. Injec- tion of large quantities of cultures, or filtrates from these, produces in rabbits and guinea-pigs toxic effects, and even death may result. It is therefore toxicogenic, but not pathogenic. NOTE. When surface colonies as those above present special character- istic they can be reprinted on cover-glasses. To make such an impression or "Klatsch" preparation, select a suitable spreading colony, wilh the aid of No 3 objective, then raise the lube of the microscope and carefully drop a clean- cover-glass on top of the colony. Apply gentle pressure with a pair of forceps, then grasp the edge of the cover-glass and carefully remove: allow to dry in the air; fix and stain in the usual manner. In making the reprint only the growth should adhere to the cover-glass. Considerable gelatin, solid or liquid, on the cover-glass, is undesirable and interferes. MEMORANDA. 62 BACTERIUM ZOPFII. Kurth. Origin. From the intestines of chicken. Form. Rods, 2 to 5 times as long as wide. Forms threads, which in gelatin are often peculiarly bent or twisted. Motility. Actively motile. Sporulation. Spore-like bodies are formed, which resist desiccation, but are readily destroyed by heat, and are readily stained by anilin dyes. Anilin Dyes. Stain easily. Growth. Rapid. Gelatin Plates. The colonies form delicate cloudy patches of radiating threads, and under the microscope show, in addition to the network of threads, numerous rounded little masses or bunches of cells. Stick Cultures. Marked growth in the upper part of the tube and almost absent in the lower part. Shows fine radiating lines which, at or near the surface, penetrate deepest into the surrounding gelatin. Streak Cultures. On agar, forms a very thin, dry, grayish growth. Oxygen requirements. Is aerobic. Temperature. Grows best at ordinary temperature. Can grow at 37-40, but tends to develop involution forms and to die out. Behavior to Gelatin. Is not liquefied. Pathogenesis. No effect on animals. NOTE. Make "Klatsch" or impression preparations of the colonies. MEMORANDA. 64 SPIRILLUM RTJBRTJM. Von Esmarch. Origin. Isolated from the putrefied cadaver of a mouse. Form. Clear, transparent, thick cells, which com- monly are single, appearing as large bent rods or comma bacilli (vibrio). May form spirals of 3 or 4 or even 40 windings. Involution forms are common in old cultures. Motility. Actively motile. Each end of a spiral has one wavy flagellum. Sporulation. True spores not observed. Anilin Dyes. Stain slowly but well. Growth. Extremely s?o\v. Gelatin Plate 8 Owing to the very slow development of colonies ordinary plates cannot be used. In roll tubes, colonies develop in from 7 to 10 days, and at first are minute and grayish; later the center becomes tinged with pink and eventually becomes red. The edge is smooth and contents finely granular. Stick Cu//?nvx. Are the most characteristic. Growth takes place along the entire line of inocukvtion, forming a row of colonies. The growth spreads slightly on the surface and is colored a light pink. The pigment formation is most marked along the stich where oxygen is absent. It passes through a light pink to a beautiful dark wine-red color. Ordinary bacterial pigments are formed only in the presence of air and are secondary products, whereas this pigment is formed in the absence of air and is primary. Streak Cultures. On agar, forms moist, thick, non-spreading patches, which, when old, possess a light pink or red color, especially near the center. On potatoes, develops slowly, forming minute deep red colonies. On blood serum, the growth is much the same as on agar. Milk. In fluid.media, milk, bouillon, etc., forms long spirals. Oxygen requirements. Is a facultative anaerobe. Temperature. Grows between 16 and 40. Opti- mum about 370. Behavior to Gelatin. Not liquefied. Pathogenesis. Has no effect. NOTE. Make Esmarch Roll-tubes of the Spirillum rubrum. MEMORANDA, 66 BACILLUS ACIDI LACTICI. Hueppe. BACILLUS OF LACTIC ACID FERMENTATION. IS ONLY ONE OF A LARGE NUMBER OF BACTERIA GIVING RISE TO LACTIC ACID. Origin. Sour milk. Form. Short, thick rods, about one-half as long as long as wide; usually in pairs, rarely in chains. Motility. Has no motion. Brownian movement, however, is marked. Sporulation. Hound, terminal spores observed. Anilin Dyes. Stain readily. Growth. Abundant and fairly rapid. Gelatin Plates. The deep colonies are round or oval, yellow, sharp bor- dered, finely granular. The surface colonies spread, forming thin plaques, with irregular, wavy borders. The outer zone of the colony is at first almost transparent and shows markings resembling the venation of leaves. Stich Cultures. Slight growth along the stich, but on the surface it is considerable and spreads rapMly as in thin, dry, pearly-white covering. In old cultures bundles of crystals form along the stich at or near the surface. Streak Cultures. On agar, forms a grayish white, moist, spreading growth, which offers no. special characteristics. On potatoes, it forms a brownish yellow, slimy covering. 31Wc. In sterilized milk converts the lactose or milk-sugar into lactic acid and carbonic acid. The acid reaction thus produced causes a precipita- tion of the casein or curd. This change occurs only in presence of air. Oxygen requirements. Is a facultative anaerobe. Temperature. Grows between 10 and 45. Opti- mum about 350. Behavior to Gelatin. Not liquefied. Aerogenesis. Gas is produced in milk. Pathogenesis. No effect. 0.75 per cent, lactic acid stops the growth. Production of lactic acid in the mouth and dental caries ; abnormal fermentations in the stomach, in the intestines. Lactic acid bacteria favor the growth of anaerobic bacteria. MEMORANDA. 68 BACILLUS BUTYRICUS. Hueppe. BACILLUS OF BUTYRIC ACID FERMENTATION. IS ONLY ONE OF A LARGE NUMBER OF AEROBIC AND ANAEROBIC BACTERIA WHICH GIVE RISE TO BUTYRIC ACID. THE VIBRION BUTYRIQUE OF PASTEUR WAS THE FIRST ANAEROBE DISCOVERED (1861). Origin. Milk. Form. Lon ;, narrow rods, with rounded ends , fre- quently in pairs, may form threads. Motility. Actively motile. Sporulation. At about 30 forms bright, oval, me- dian spores. Anilin Dyes. React well. Growth. Rapid. Gelatin Plates. The deep colonies form yellowish masses, whereas the surface ones liquefy rapidly and then form grayish-brown, granular patches with fibri Hated borders. Mich Cultures. Rapid liquefaction along entire line of inoculation. The gelatin becomes colored yellowish and on the surface a thin, folded, grayish white scum forms. The liquid remains cloudy for some time but later the growth settles to the bottom. Streak Cultures. On agar, forms a light, yellow, sticky covering. On potatoes, forms a light brown, transparent growth which sometimes becomes folded. Milk. Without change in the amphoteric reaction the casein gradually coagulates, as with rennet. Subsequently after about 8 days the casein is redissolved or peptonized with formation of pepton, leucin,tyrosin, ammonia and bitter products. From hydrated milk sugar and lactates it forms butyric acid. Oxygen requirements. Is aerobic. Temperature. Can grow at ordinary temperature, but its optimum is 35 to 40 C. Behavior to Gelatin. Liquefies. Aerogenesis. Butyric acid formed. Fathogenesis. No effect. MEMOEANDA, 70 BACILLUS CYANOGENUS. Fuchs, (1841). BACILLUS OF BLUE MILK. Origin. In blue milk. Form. Small, rather narrow rod. 5 , with slightly rounded ends, 2 to 3 times as long as wide. Frequently grows in pairs, very rarely in threads. Motility. Very actively rnolile. Sporulation. Small terminal spores observed in gelatin, milk, etc., at ordinary temperature. Anilin Dyes. Stain easily. Growth. Rapid. Gelatin Plates, The deep colonies are round with sharp, smooth border, and yellowish granular contents. The surface colonies are moist, elevated, convex masses, which are round, finely granular and dark colored. Stich Cultures Little or no growth in the lower part of the stich. Spreads over the surface as a thick, moist, dark gray covering. A dark steel- blue color diffuses downward into the gelatin. The shade of color varies with the reaction of the medium. In neutral or acid media it is quite blue, whereas in very alkaline media it is dark or even black. The cultures when old become dark colored. Streak Cultures. On agar, forms a dirty gray, thick, moist covering, and the medium becomes diffusely colored. On potatoes, it likewise forms a thick, raised, slimy growth, which rapidly spreads and becomes colored. On blood scrum, no color is formed. Milk. In sterilized milk it produces no acid or coagulation, but the liquid becomes colored a slate gray which with acids turns blue, In unsteril- ized milk, that is in presence of lactic acid bacteria, the color is sky-blue. The color is developed from casein, not from lactose. Oxygen requirements. Aerobic. Temperature. Can grow at ordinary temperature, or in incubator. The pigment is best developed at low temperatures, 15-18C. Behavior to Gelatin. Not liquefied. Pathogenesis. No effect on animals. NOTE. Make " Kiatsch " or impression preparations of the colonies. MEMORANDA. 72 OIDIUM LACTIS. DOES NOT BELONG TO THE BACTERIA, BUT IS A SIMPLE MOULD. Origin. Almost invariably present in milk and in butler. Form. A delicate white mycelium of wavy threads. No special fruit organ. Large oblong spores. Anilin Dyes. Eeact readily. Growth. Rapid. Gelatin Plates. Delicate white stars form, which rapidly enlarge, and on the surface spread as flat, whitish, dry masses. Under the microscope the colonies show radiating branched hyphse. Stich Cultures. Growth takes place along the entire line of inoculation, but most abundantly at or near the surface. A branching network of threads extends outward into the solid gelatin. On the surface a grayish white, dry, low growth forms. In old cultures only the upper layer of gelatin shows the radiating lines. Streak Cultures. On agar, it forms a grayish white, thin growth. Milk. Growth occurs without any change in its composition. Temperature. Grows best at ordinary temperature. Can grow in inc.ubator. Behavior to Gelatin. Does not liquefy. Pathogenesis. No effect on animals. MEMORANDA. MEMORANDA. 73 BACTERIOLOGICAL, EXAMINATION OF WATER. The water to be examined must be received in a sterilized bottle or flask, thoroughly protected against subsequent contamination. Furthermore, in view of the rapid multiplication of bacteria, a given sample of water should be examined as soon as possible after collection. The method commonly employed consists in the deter- mination of the number of bacteria present in a given volume, 1 c. c., and the recognition of the several species or kinds of microorganisms present. This process, as carried out, is as follows : Place several 1 c. c. pipettes, graduated in 1-10 c. c., in a pipette box and sterilize in the dry heat oven in the usual way. Liquefy 3 gelatin tubes and with a sterilized cooled pipette transfer into tube No. 1 one c.c. of the water ; into tube No. 2 place one-half c.c., and into tube No. 3 one drop of the water. Gently agitate the contents of the tubes, to secure complete mixture, then pour the gelatin onto sterilized glass plates, observing the usual precau- tions in making plate cultures. Set aside the gelatin plates thus obtained for two or three days and then count the colonies when sufficiently developed. When only a small number of colonies are present the counting can be done with the unaided eye, but when, as it frequently happens, the number is very large, it is desirable to make use of a counting apparatus that of Wolffhiigel is usually employed. The gelatin plate on which the colonies are to be counted is placed on the black glass base and covered with a glass plate ruled into squares. The number of colonies under six or more squares is thus easily determined, and in this way the 74 average number of colonies per square readily ascertained. By determining the number of squares which the gelatin on the plate covers, and multiplying this figure by the average number of colonies per square, the total number of colonies on the plate is found. Since each colony is derived from a single cell this number then represents the number of bacteria present in 1 c. c. or ^ c. c. or 1 drop of the water. The number of bacteria found should always be expressed as so many per c. c. To ascertain the kind of bacteria present, the colonies are examined under the microscope in the usual way. A seen from the preceding work the form of the colony and its behavior to gelatin may sometimes assist in its identification. Hanging-drop examinations, stained prepa- rations and stich cultures will still further assist the recognition. The chief object of the bacteriological examination of water is to determine the presence or absence of patho- genic or toxicogenic bacteria. In the above method this is done by recognizing the colony of the specific organism sought for. When the pathogenic bacteria, as the cholera or typhoid fever bacillus for example, are present in large numbers, and this is very rarely the case, the identification can perhaps be easily d*one. On the other hand a few pathogenic bacteria in the presence of a large number of saprophytic organisms can be easily overlooked, and in such cases their recognition becomes well-nigh impossible. In view of these facts the following method has been devised and used in this laboratory since 1888. It is based upon the fact that the majority of bacteria present in water are common saprophytes which grow at the ordi- nary temperature, cannot grow at the temperature of the body, and cannot, therefore, produce toxic or pathogenic effects. Further, that those bacteria which can develop at the temperature of the body may or may not be patho- genic, and this is ascertained by animal experiment. The process as used is as follows : MEMORANDA. MEMORANDA. /o - To sterilized beef tea or bouillon tubes add 1 c. c., ^ c. c., and one drop of the water by means of a sterilized pipette. Set aside in the incubator at 37 to 39 C. for 24 hours. If no growth occurs at this temperature it is at once sufficient evidence that the water is free from disease- producing organisms. On the other hand, if growth develops, injections of 1 c. c. of the culture are made intra- peritoneally into white rats by- means of a sterilized Koch syringe. The recovery of the animal indicates the absence of pathogenic bacteria. If death occurs the toxic or pathogenic form can be found arid isolated from the organs and tissues of the animal (see anthrax). When it is desired to examine snow or ice this should be melted in a sterilized flask and the water thus obtained is examined as above. The number of bacteria present in water from various sources is subject to the greatest variation. Thus spring water may be sometimes wholly free of microorganisms, but as a rule the number is less than 50 per c. c., and may, in exceptional case, contain 3,000 per c. c. In well waters considerable variation has been observed, but usually the number is less than 500 per c. c. The water of deep wells may be said to be free of microorganisms. The same may be said to be true of the water of lakes. The number of bacteria present in river water varies from a few hundred to as many thousand, but in the neighbor- hood of large cities it may reach hundreds of thousands per c. c. In Paris the river water in the wool-washing stations has been shown to contain from 12 to 40 millions per c. c. LABORATORY WORK. Make plate cultures of two samples of water tap-water and well-water. Also Petri dishes of milk. The gelatin tubes are inoculated with milk in the same manner as with water. 76 BACTERIOLOGICAL EXAMINATION OF SOIL. The collection of samples of earth from various depths can be readily accomplished by means of Fraenkel's earth - borer. For each culture experiment a definite quantity of the soil should be weighed out, or a measured volume taken. The latter is the simpler procedure, and can be done with a Loffler platinum spoon (1-50 c. c.) which serves the purpose of a standard volume. With a sterilized Loffler spoon transfer one spoonful of the earth to a tube of liquid gelatin. Mix thoroughly with a sterilized platinum wire and then make an Esmarch roll-tube. The soil and the organisms present are thus brought into perfect contact with gelatin, and after a lapse of a few days colonies develop. These can be readily counted, and, if necessary, with the aid of an Esmarch roll-tube counter. The kind of organisms bacteria, mould?, etc. can be determined by the study of the colonies, and by further culture and examination. In this wav it is easy to determine approximately the number and kind of organisms present. Unfortunately this method is not adapted for the detection of anaerobic bacteria which are apparently widely distributed in the earth, and are represented by the well-known bacilli of tetanus, malignant oedema and symptomatic anthrax. These have thus far been obtained only by indirect methods from the soil. Thus animals are inoculated with earth, and from the tissues and organs after death the bacteria are isolated. The surface layers of soil, to a depth of about two feet, are exceedingly rich in bacteria. The number has been found to vary from 100,000 to 350,000, and may even reach several million, per c. c. The number rapidly decreases with the depth, and at 9 to 12 feet the soil is practically sterile. LABORATORY WORK. Examine three samples of soil by the above method. MEMORANDA. MEMORANDA. 77 BACTERIOLOGICAL EXAMINATION OF AIR. To determine the number and kind of bacteria present in the air is a problem of considerable importance, and can be accomplished quite satisfactorily with Hesse's appara- tus. The large, wide tube is sterilized, nutrient gelatin introduced, and a large Esmarch roll-tube then made. The tube thus prepared is connected with an aspirating bottle of known volume. In this way a definite volume of air can be drawn through the apparatus. The bacteria pres- ent in the air are deposited on the moist gelatin walls of the tube, and subsequently develop, forming colonies- These are counted and the number of colonies per liter of air is thus ascertained. The kind of bacteria present can be determined in the usual way. The method of Petri, though somewhat more compli- cated, requires less time and gives excellent results. The air is filtered by means of an aspirator or air pump through a tube filled with sterilized sand. The sand, which then contains the bacteria originally in the air, is transferred to a Petri dish containing gelatin, thoroughly mixed, and set aside to develop. The number of bacteria present in the open air is very small and rarely exceeds 3-4 per liter. Usually the num- ber is much less than this. Spores of moulds are more abundant in the air than are bacteria. Air of mid- ocean and of high altitudes is practically free of microorganisms. 78 PREPARATION OF BREAD FLASKS. Moist bread, like potatoes, owing to its slightly acid acid reaction, is an excellent medium for the growth of some organisms, especially moulds. Prepare some dry powdered bread, which can be read- ily done by over-toasting it in the dry-heat oven, and then crushing or pulverizing the dry mass. Keep in a stoppered bottle. Clean, plug, and sterilize in the dry-heat oven six small Erlenmeyer flasks. When cool cover the bottom of the flasks to a depth of about ^ inch with the dry powdered bread, then add water till the mass becomes thoroughly moist and soft. Sterilize in steam sterilizer for three con- secutive days, \ hour each day. Inoculate the bread flasks with the following moulds : Penicillium glaucum. .Mucor corymbifer. " rhizopodiformis. Aspergillus niger. " flavescens. " fumigatus. All these flasks, except the first, should be placed in the incubator at about 37 C., for 24 to 36 hours. They should then be examined for the characteristic fruit organs and spores. Transfer a portion of the growth to a watch- glass containing about 50 per cent, alcohol, to which a drop or two of ammonium hydrate has been added. When the growth becomes moist, transfer a portion to a drop of glycerine on a slide. Tease out the specimen thoroughly and carefully with needles or pins. Cover with a cover- glass and examine with No. 7 objective the fruit organs and the structure. If the specimen is satisfactory it may MEMORANDA. MEMORANDA. 79 be made permanent by placing a ring of asphalt, with the aid of a turn-table, around the edge of the cover-glass. Make Petri dishes of white yeast; Esmarch roll tubes of red yeast and of black yeast. Examine baker's or brewer's yeast (Saccharomyces cerevisise) in hanging drop and in stained preparations. Observe the form and structure of the yeast cell and the method of multiplication by budding. MEMORANDA. MEMORANDA. 82 PENICILLITJM GLAUCUM. ONE OF THE MOST COMMON GREEN MOULDS. Origin. Widely distributed in the air, water, soil. Color. Green. Mycelium. Consists of horizontally arranged, straight or slightly wavy, jointed mycelial threads from which the fruit hyphge rise vertically. Fruit-organs. --The ends of the fruit hyphae are forked, and on the ends are the intermediate spore bear- ers, or "sterigmae, also sometimes called basidia. Each of these in turn bears a row of spores or conidia, so that the appearance of the whole is that of a brush. Gelatin plates. The colonies form whitish floccules which rapidly increase in size, and at the same time the center colors green. The gelatin is liquified quite early. A low objective will show the above chai'acteristics of growth. Bread flasks. Show a low, finely flocculent covering, which at first is white but soon changes to a distinct green. Temperature. Optimum temperature is from 22 to 26 C. Does not grow at the temperature of the body. Behavior to Gelatin. Liquefies. Pathogenesis. Has no effect on animals. Mi ]V:CIMM?A. 84 MTJCOR, CORYMBIFER. Lichtheim. THE MOST COMMON AND WIDELY DISTRIBUTED MUCOR IS MUCOR MUCEDO, OCCURRING ON EXCRETA, ETC. Origin. Is of rare occurrence, and was found as a contamination on bread-gelatin plates. Is present in white bread, and has been found in the ear-passages of man. Color. Forms a snowy, cotton-like growth. Mycelium. Loose, wavy, branching, slender mycel- ial threads. Fruit-organs. The fruit hyphae branch forming clusters or corymbs which terminated with spherical or pear-shaped sporangia. Within these are the oval or elongated spores. Growth. Rapid and extensive. Bread flasks. In the incubator forms a white, elevated, cotton-like growth which soon fills the flask. Temperature. Grows slow at ordinary tempera- ture; best at 37 C. Pathogenesis. Intravenous injection of the spores into rabbits produces death in 3 to 4 days. The kidneys, mesenteric glands, Peyer's patches contain mycelial masses. The Peyer's patches are swollen and ulcerated. Intraperitoneal injections produce the same results. Dogs are immune. MEMORANDA. 86 MUCOR RHIZOPODIFORMIS. Lichtheim. Origin. White bread kept at 37 0. Color. At first white, but later becomes grayish. Mycelium. The mycelial threads are colorless and thicker than in the preceding mucor, and are not jointed or divided. Fruit-organs. The fruit hyphae occur in groups or bunches, which adhere to the nutrient medium by means of special root tufts. The large sporangia on the ends of the hyphae contain rounded spores which are larger than those of the preceding organism. Growth. Rapid. Gelatin plates Development is best when the gelatin is made with bread infusion. It forms a coarse grayish-black mass which liquefies the gelatin. Bread flasks. The growth is lower than that of M. corymbifer, and is grayish, owing to the dark colored sporangia. An ethereal or aromatic odor is present. Temperature. Slow growth at 12 to 15, but develops best at 37 0. Behavior to Gelatin. Liquefies. Pathogenesis. Has a similar effect as M. corymbi- fer, but is more pathogenic. MEMORANDA. ASPERGILLUS NIGER. Van Tieghem. Origin. In putrid organic substances ; in lungs of birds. Color. Black or dark brown. Mycelium. The arrangement is much the same as in penicillium. Fruit-organs. The fruit hyphse are swollen or flask or club-shaped at the end, and this enlargement is covered willi radially arranged minute bottle-shaped bodies the intermediate spore bearers or sterigmse from which rows of spores extend. Sterigmee divided. Growth. Slow. Bread flasks Forms a slow growth which becomes very black. Temperature. Its optimum is about 35 C. Pathogenesis. Intravenous injection of spores in rabbits is not followed by as malignant results as with the next two forms. ASPERGILLUS FLAVESCENS. Wreden. Origin. White bread. Color. At first whitish, eventually pale yellow. Mycelium. The mycelial threads and spores are smaller than those of A. niger. Fruit-organs. The club shaped ends of the fruit hyphge are covered with sterigmae, from which extend rows of spore?, as in A. niger. Growth. Rapid. Bread flasks. Grows best on bread. Forms a yellowish, low growth. Temperature. Optimum about 28 C. Grows well in incubator. Pathogenesis. Is more pathogenic than A. niger, and less than A. fumigatus. MEMORANDA, 90 ASPERGIKLUS FUMIGATUS. Lichtheim. Origin. White bread. In the air passages of a bird. Color. Greenish or bluish green growth, resembling very much that of penicillium. Mycelium. About same as preceding. Fruit-organs. About same as preceding, but spores only about one-half as large. Growth. Is best on bread and is rapid. Bread flasks. The growth is low and at first is bluish green, but when old is grayish green. Temperature. The optimum is 39-40 C. Can grow at the ordinary temperature. Pathogenesis. Intravenous injections of spores in rabbits and dogs produced death in a few days. Mycelia are found in the kidneys, heart muscle and other muscles, and occasionally in the liver. MEMORANDA. 92 RED YEAST. SEVERAL RED YEASTS ARE KNOWN. THE RED, WHITE AND BLACK YEASTS ARE NOT TRUE YEAST-PLANTS. Origin. Very common in air. Color. Bed or pink. Form. Round [or oval cells with granular proto- plasm which stains irregularly. Multiplies by budding- distinction from bacteria. Motility. None. Sporulation. None. Anilin Dyes. Stain readily. Growth. Abundant, though somewhat slow. Gelatin Plates. Colonies are small, round, elevated, moist and pink- colored. Stich Cultures. Growth absent from the lower part of the tube. Spreads slowly over the surface, forming a thick, moist, bright red covering. Streak Cultures. On agar, develops in a few days as a thick, slimy, spreadftig, pink-colored growth. On potatoes, forms the same pigment. Temperature. Grows best at ordinary temperature. Behavior to Gelatin. Does not liquefy. Aerogenesis. Does not produce alcohol. Pathogenesis. No effect on animals. MEMORANDA. 94 r o> 8 ^ 1 % qa a VI 'zoinyee 1 ^ S o 1 i ~ I | s 5 J =i S CQ fc c^ IB p. wi. ^ ' S /. fefl 4 X i; O S S ^ g <2 << " fa * % y heating in steam sterilizer, 4- hour each day, for three consecutive days. At the end of the third heat place the agar tubes, while still liquid, in a slightly inclined position, so that the agar reaches to within 1 to H inches of the plug, and allow it to solidify. Instead of preparing an extract of fresh meat it is sometimes more convenient to employ commercial meat extract, such as Liebig's. In that case 2.5 g. of Liebig's extract with the usual amount of peptone and common salt is added to 100U c. c. of water. To this solution or bouillon the ordinary proportion of agar or gelatin is added, and the nutrient media are otherwise prepared in exactly the same manner as already given. When a perfectly transparent agar is desired it is, as a rule, necessary to filter though paper. Thiscan be accom- plished most rapidly by placing a filter-stand with funnel and plaited filter, slightly moistened, in a steam sterilizer. When the funnel is thoroughly heated the boiling agar solution is transferred to the filter. MEMORANDA. MEMORANDA. 97 PATHOGENIC BACTERIA. By the application of the gelatin plate method it is possible to readily separate a given organism from other forms which may be present and thus obtain a. pure cul- ture. The isolated colony as it develops on a plate fur- nishes the first pure cultivation since it is derived from a single micro-organism. Transplantations made from a colony, if made with proper piecautions, in turn yield pure cultures or growths containing but a single species. Tube cultures can thus be made in gelatin, bouillon, agar, blood serum, potato, etc., and where it is desired, as in the study of chemical products of bacteria, flask cultures can be made. It is evident that in order to demonstrate that a given bacterium is the cause of a certain fermentation, or of the production of some pigment or of phosphorescence, etc., it is necessary that it should, first, be isolated and obtained in pure cultures, and that, second, pure cultures of the organism grown under the same or similar conditions, should give rise to the original phenomena the produc- tion of the same fermentation, pigment, phosphorescence, etc. Having thus demonstrated that a given organism is the cause of certain changes it does not follow that this organism has the exclusive power to do so. Thus, in alco- holic fermentation the yeast plant is commonly said to be the cause, but a large number of different species of yeasts are known which have this power, and not only the yeasts but many bacteria possess similar properties. Again a considerable number of bacteria have been shown to be capable of inducing acetic, lactic, butyric acid fermen- tations, the ammoniacal and hydrogen sulphide fermen- tations of urine, the phosphorescence of sea-water, etc. The most that can be said of a given organism which 98 induces a certain change, therefore, is that it is the cause in that particular instance. The possibility of other organisms giving rise to the same changes, or effect, or chemical products, must be conceded, and the demonstra- tion of the relations of an organism to such a change rests with the proof that it is a cause. Just as there are organisms which induce changes in dead animal or vegetable matter, there are others which are capable of inducing similar changes in living animals and plants, thus living at the expense and frequently to the detriment of the host. The infectious diseases in man, animals and plants, possess as an essential characteristic the property of transmissibility. They are the result, first, of infection that is the entrance of a specific micro organ- ism, and second, of intoxication due to the poisonous pro- ducts elaborated by the microorganism. Poisonous chem- ical compounds may produce the symptoms and the changes observed in an infectious disease. They are the cause of those symptoms and changes, but they are not the cause of disease, since the symptoms and changes thus obtained are not transmissible from one individual to another. Chemical substances have no power of multiplication and the effect observed is, there- fore, directly proportional to the amount of the chemical compound introduced. Microorganisms, however, have the power of multiplication, and the introduction of a minute amount, even a single cell, may bring about entirely dis- proportionate results. The invading organism is therefore the cause of the disease since it imparts the characteristic property of transmissibility, and, through the action of its chemical products, produces the symptoms and effects of that disease. In order to positively demonstrate the causal relation of a microorganism to a given disease, it is necessary to meet the following requirements, commonly known as the four rules of Koch : (1.) The organism must be present in all cases of that disease. MEMORANDA. MEMORANDA. 99 (2.) The organism must be isolated and obtained as an absolutely pure culture. (3.) The pure culture of the organism when intro- duced into susceptible animals must produce the disease. (4.) In the disease thus produced the organism must be found distributed the same as in the natural disease. To these four requirements, a fifth may be added, namely: That the chemical products of the organism must produce the characteristic symptoms and effects of that disease. The demonstration of the constant presence of an organism in a disease is accomplished by hanging-drop examination, stained cover-glass preparation, or by stain- ing sections of tissues and organs. Frequently the direct detection of the organism is difficult owing either to its scarcity or to the absence of definite characteristics. In such cases artificial, culture or animal experiment will prove the presence of the organism. The mere fact that an organism is constantly present in a given disease does not prove that it is the cause of that disease. It certainly is strong presumptive evidence that the organism does bear a causal relation to that dis- ease, but at the same time the possibility must be admitted that it may be an accompaniment, or even a consequent of ihat disease. To complete the chain of evidence it is necessary, therefore, to obtain the organism in a pure culture, and, inoculation of animals with such cultures must reproduce the disease. The isolation of the organism and the preparation of pure cultures is accomplished by the gelatin plate method or its modifications. The isolated colony which develops on a plate is derived from a single cell, and is, therefore, a pure culture. Transplantations from the colony, when properly made, into tubes of gelatin, agar, or bouillon, in turn are pure cultures. Subsequent transplantations from tube to tube can be made as often as may be desired, or as may be necesssary. Each growth thus obtained is called 100 a generation. In many cases, as in tuberculosis, anthrax, and in hog cholera, the organisms have thus. been carried through several hundred consecutive generations without impairment of pathogenic properties. In other instances, as in glanders, the organism does not find in our artificial media the conditions favorable for its growth and as a result it undergoes a physiological alteration so that the cultures become less and less active till finally they cease to have any effect on animals. This change in the physi- ological properties of an organism known as attenuation is frequently accompanied by a corresponding decrease in the vitality of the growth so that, when the virulence is wholly lost, the culture soon dies out. Sometimes, how- ever, the organism adapts itself to the artificial media and continues to grow although with diminished pathogenic properties. The above four rules have been fully complied with in a large number of infectious diseases. In others the first two rules ;ire s;itij-fied bill the third is nor, owing to the difficulty of obtaining a susceptible animal. Ay:ain, the first rule maybe ihoonly one complied with, as in leprosy, where the isolaiio'i of the organism has not, thus far, been unquestionably successful. And again a large number of infectious diseases remain, in which even the presence of a specific organism has not been definitely shown. Although many of the infectious diseases have been shown to be due to bacteria, it must not be forgotten that other low forms of plant and animal life possess sim- ilar properties. Thus there are infectious diseases due to fungi and also such as are due to animal parasites sporo- zoa, etc. MEMORANDA. MEMORANDA. 101 METHODS OF INFECTION. 1. Cutaneous application. 2. Subcutaneous application. In mice and rats this can best be done on the back, over the root of the tail. 3. Subcutaneous injection. The Koch syringe is commonly used. Pravaz syringe. 4. Intravenous injection. The large veins in the ears of rabbits are frequently used ; also the jugular and femoral veins. 5. Intraperitoneal injection. 6. Intrapleural injection. 7. Injection into the anterior chamber of the eye. 8. Infection along respiratory tract. (a) Inhalation, (b) Injection into the trachea. 9. Infection of alimentary canal. (a) With food or drink. (b) Through a stomach-tube the contents of the stomach are previously rendered alkaline. (c) Intraduodenal injection. The inoculation of animals with pure cultures of microorganisms must be made with rigid precautions to prevent the introduction of foreign organisms. At the site of inoculation the hair must be carefully cut away ; the exposed skin is then well washed with alcohol, and finally is thoroughly moistened with mercuric chloride (1-1000). The instruments employed in making the inoc illations, as knives, scissors, forceps, lance, wire, etc., must be sterilized in a flarne shortly before use. The Koch syringe is sterilized in the dry oven. After having used the instruments they are at once sterilized. 102 In working with the pathogenic microorganisms the student must specially observe the utmost precaution against personal infection. The rule to sterilize every instrument shortly before, and immediately after use, before it has left the hands, must be strictly attended to. Direct contact of the hands with infectious matter must be carefully avoided, and when such contact has taken place prompt disinfection must be resorted to. On no account must lead pencils or glass rods be held in the mouth, or labels moistened on the tongue. Gelatin or agar plates must be set aside for some hours in mercuric chlo- ride (1-1000). Old tube cultures are best sterilized by heating in the steam sterilizer for about one-half hour. If by accident infectious matter is dropped on the table or floor it must at once be covered with mercuric chloride. At the close of the day's work the table must be well washed with the mercury solution, and the hands thoroughly disinfected. MEMORANDA. MEMORANDA. 103 POST-MORTEM EXAMINATION. Demonstration of post-mortem on guinea-pig that died aftor subcutaneous inoculation with the anthrax bacillus. The animal is placed on a board ; the feet are extended and tacked or nailed down. The hair over the abdomen and thorax is moistened thoroughly with a cloth soaked in mercuric chloride. With a pair of sterilized forceps the skin over the lower part of the abdomen is raised and a slight transverse nick is made with sterilized scissors. Into the opening thus made the lower blade of the scis- sors is introduced and an incision is made along the median line to the neck. While making the incision the skin is kept raised by means of the forceps to. avoid cutting through the abdominal or thoracic walls. At each end of this incision lateral cuts are made in the direction of the extremities, and the two flaps of skin, thus prepared, are carefully reflected, thus exposing the entire abdominal and thoracic walls. The condition of the subcutaneous tissue, of the abdominal walls, of the blood-vessels and the presence or absence of oedema, gas, etc., should be noted. The scissors and forceps are sterilized, and when cool a similar incision is made into the abdominal wall and extended through the cartilages of the ribs to the neck. Special care must be taken to prevent cutting into the intestines or internal organs. Lateral cuts are made as before, and after nicking the. ribs on the inside of the thorax close to the vertebral column, the entire abdominal and thoracic walls can be reflected, thus exposing to view all the internal organs. The condition of the abdominal and thoracic cavities should be observed ; also the appearance of the peritoneum, liver, spleen, kidneys, heart, lungs, etc. 10 104 With re-sterilized forceps and scissors (lie spleen, kidneys, liver, etc., should be removed to sterilized Petri or Esmarch dishes and can be used for subsequent exam- ination. In making post-mortem examinations the utmost care must be taken to prevent the introduction of foreign microorganisms, and at the same time to prevent- scatter- ing any infectious matter, from the animal. For that reason the hair on the skin is thoroughly moistened to prevent it from flying about or entering the opening in the body. The forceps, knives and scissors must be sterilized in a flame for each separate incision. When blood or pieces of tissue adhere to the instruments, they should not be placed at once into the flame, otherwise the sudden heating will cause the material to spurt and scatter about. To avoid this the material should first be dried by holding I he instruments close to the flame. This precaution should also be observed when sterilizing wires which are covered with gelatin. LABORATORY WORK, WITH ASTHKAX TISSUE. Isolation of the bacillus in pure culture. The bacil- lus of anthrax which is present in the blood, tissues and organs of the guinea pig, must be isolated and obtained in pure culture. This can be readily accomplished by the gelatin plate method. For this purpose a small piece of liver, about half the size of a grain of wheat, is cut off with a sterilized pair of scissors. The piece of tissue is placed on the loop of a sterilized platinum wire and transferred to a tube of liquefied gelatin. By rubbing the piece against the walls of the tube with the wire the blood can be squezed out and the oigani-m piesent is thus spread throughout the gelatin. From this tube, which is No. 1, transfers are made in the usual manner to tube No. 2, and from this to tube No. 3. Gelatin plates are then made in the usual manner, and set aside for two or three days to develop. MEMORANDA. MEMORANDA. 105 When the colonies develop their form should be care- fully studied as it is very characteristic, and, if possible, impression preparations should b6 made from the surface colonies and stained with methylene blue. As the colony is a pure culture of the anthrax bacillus, transplantations to tubes in turn yield pure cultures. Make a stich culture in gelatin and a streak culture on inclined agar. This latter is made by simply drawing the end of the platinum wire along the middle on the surface of the agar. The agar tube is placed in the incubator at 37 to 39 G. for one or two days, then removed and examined. Another agar tube is liquefied and -J to 1 drop of cal- cium hydrate is added, thoroughly mixed, and the tube is then set aside in an inclined position till the agar solidifies. Then make a streak culture on this Ca (OH) 2 -agar, and set it aside to develop in the incubator. With the pure cultures of the anthrax bacillus thus obtained the student can inoculate a number of white mice, white rats and rabbits, and in these, after death, the organism can in turn be detected and isolated. In this way each one has an opportunity to demonstrate all four rules of Koch with reference to anthrax, thus proving that the anthrax bacillus is the cause of the disease. MICROSCOPICAL EXAMINATION. Hanging-drop. Take a clean f -inch cover-glass and pass it once through the flame. Transfer a small drop of sterile bouillon to the cover-glass and then add to it, with a sterilized wire, a minute amount of the heart-blood. Apply the concave slide, ringed with vaseline, and examine the hanging-drop, thus prepared, with the No. 7 objective. Study the characteristics of the anthrax bacillus as it exists in the blood, and compare its size with that of the blood-cell. Then label the slide and set aside in the incubator for 24 hours. Examine the slide on the following day and observe the formation of threads, of sporogenic granules and possibly of spores. 106 Finally make permanent stained mounts of these threads by transferring a small portion of this drop culture to a minute drop of water on a clean cover-glass ; spread, dry, fix and stain the preparation in the usual manner. Stained preparations. Place about two dozen clean cover-glasses on the lid of a slide box. Pick up a piece of the spleen, kidney or liver in a pair of forceps, and while holding the cover-glass down with another pair of forceps, lightly streak the cut surface of the organ over the cover- glass. A very thin and even film is desirable. In this way streak all the cover-glasses, then allow them to dry in the air and fix cautiously by passing once or twice through the flame. These cover- glasses are commonly known as streak preparations. Stain some of the fixed cover-glasses will) simple ani- lin dyes, as gentian violet or i'uchsine ; examine and study the specimens carefully and make permanent prep- arations. The remainder of the cover-glasses will serve for double-staining by Gram's method. Gramas method. This excellent method for demon- strating the presence of certain bacterin, as anthrax, in the fluids and tissues of the body is based upon the fact that the protoplasm of the bacterial cell when stained with anilin water-gentian violet, and then treated with iodine forms a difficultly soluble compound. By proper exposure to a solvent the dye can now be removed from the entire cover-glass, but not from the bacterial cell. The deeply stained violet rods lie on a colorless back-ground, which on treatment with a contrast color, as eosine or picro car- mine, becomes stained a light pink. The method is as follows : A solution of anilin water-gentian violet is first pre- pared. Anilin oil is placed in a test-tube to a depth of about half an inch. The tube is then filled with water, closed with the thumb and thoroughly shaken in order to obtain a saturated aqueous solution of anilin. The liquid is then passed through a small filter and collected in MEMORANDA. MEMORANDA. 107 another test'- tube. The filtrate should be perfectly clear, not cloudy. To the anilin water thus obtained a satu- rated alcoholic solution of gentian violet is added till the iluid is deeply colored. Some of the anilin water-gentian violet thus pre- pared is poured out into a watch-glass. A streak cover - glass preparation of anthrax is now carefully fixed in tlie flame. Care must be taken not t.o over-heat the specimen, as the anthrax bacillus when over-heated does not stain satisfactorily. The fixed cover-glass is then placed between the thumb and forefinger, with the specimen side down, and carefully dropped upon the surface of the stain in the watch-glass. It is then allowed to float on the dye for 10 to 15 minutes. Sometimes it is necessary to warm the dye on the radiator or on an iron plate in order to obtain a rapid and intense stain. The cover-glass is then picked up with the forceps, thoroughly washed with water, and immersed in a solution of iodine in potassium iodide. This is made by dissolving 2 g. of potassium iodide and 1 g. of iodine in 300 c. c. of distilled water. The specimen is allowed to remain in the iodine for ^ to 1 minute, or even several minutes. Care must be taken not to expose too long to the action of iodine, as it tends to contract the protoplasm into granules. The cover-glass is then removed from the iodine, washed with water, and moved about in a watch glass of strong alcohol, to which, if necessary, a drop of acetic acid may be added, to assist the decoloration. From time to time the cover-glass should be washed with water and examined with No. 7 objective to ascertain the progress in decoloration. When finally a colorless back-ground is obtained for the deeply stained violet bacilli the washing in alcohol is discontin- ued. The cover-glass is then washed with water and stained with dilute eosine for ^ to ^ minute. The eosine is an acid anilin dye, and therefore stains the protoplasm of cells, nuclei, etc., but not bacteria. Care must be taken not to overstain the preparation with eosine, as it would 108 tend to diminish the sharp contrast that is desired. The specimens after staining with the eosine is thoroughly washed with water and examined under the microscope. It should show the deeply stained violet bacilli on a light pink back-ground. Weigert's picro-carmine solution, or Bismarck brown, can be also used for contrast colors. The Gram's method is applicable to many pathogenic bacilli and to most micrococci. A notable exception among the latter is the gonococcus. The other important organisms that do not stain with this method are the bacillus of typhoid fever, of Asiatic cholera, of glanders, of chicken cholera, of rabbit septicaemia; also Friedlaender's pneumo-bacillus, and the spirillum of recurrent fever. The following synopsis of the staining methods for streak preparations will be of service: Cover glass preparation. Air-dried. 3 x through tlame. Simple slain : Gram?s stain : Dilute anilin dye Anilin water gentian violet (i to | min.). (10 10 15 min.; if hot, 2 to Water (and examine). 5 inin.). Air-dried. Water. Canada balsam. Iodine in potassium iodide (| to 1 to 3 min.). Water. Alcohol. Water (and examine). Contrast color (eosine or picrocarmine, few sec.). Water (and examine). Air dried. Canada balsam. The hanging-drop examination and the streak prepa- rations stained by the simple and double method as MEMORANDA, MEMORANDA. 109 described, serve the purpose of demonstrating 1 the pres- ence of the anthrax bacillus in the different organs and tissues of the body. The form, size, etc., of the bacillus found under these conditions should be compared with the growth of the organism in pure cultures in different media. For this purpose make hanging-drop examina- tions and permanent simple stains of the bacillus grown in the stich culture in gelatin, on ordinary agar, and on calcium hydrate agar. The preparation of impression cover-glasses of the anthrax colonies and simple stains of bouillon hanging-drop culture have been mentioned. Double stain for spores. The growth of the anthrax bacillus on calcium hydrate agar when examined, as mentioned above, in hanging-drop will show the presence of an abundance of bright, highly refracting oval bodies, or spores, which may be observed free and also within the parent cell. Simple stains of this growth with fuchsine, etc., will show the bacilli deeply stained, whereas the spores remain colorless. This is undoubtedly due to the dense impenetrable wall which surrounds the spores and prevents the dye from passing into the spore, as well as to a special composition of the spore contents. By proper treatment with strong anilin dyes it is possible to force the stain into the spore. Once within the spore it is as difficult to remove the dye as it was to cause it to enter. By suitable decoloration it is, therefore, possible to remove the stain from everything on the cover-glass, except from the spores. Then, on the application of a contrast color the specimens will show a bright red spore within a blue bacillus. The method of double staining of spores is as follows : The cover-glass preparation from the calcium hydrate agar is dried in the air and fixed in the usual manner. The cover-glass is held in the forceps, in the left hand, with the specimen side up and covered with a solution of carbolic fuchsine. This is held over a Bunsen flame, so that vapors are given off from the liquid. Active ebullition 110 should be avoided. From time to time the liquid which is lost by evaporation is replaced by a fresh addition of the carbolic-fuehsine, and under no condition should the dye be allowed to dry down on the cover-glass. Best results in heating are obtained with the flame turned low, so that it is not over two inches high. After heating the speci- men in this manner for two or three minutes the stain is thoroughly washed off with water and the cover-glass examined wiih the No. 7 objective. Colorless spores should no longer be visible, but everything should be stained a deep red. If the spores are not colored the heat- ing with carbolic-fuchsine is repeated until they become stained. The cover-glass may be floated on hot carbolic- fuchsine in an Esmarch dish for ^ to 1 hour. The cover glass having deeply stained spores is then moved about in dilute alcohol, and, from time to time, washed with water and examined with the No. 7 objective. As soon as the bacilli are decolored the washing in alco- hol is discontinued. The specimen then shows bright red spores within cells that are almost or wholly colorless. The cover-glass is then stained for a short time with methylene blue, washed with water and examined. The spores should be stained deep red while the bacillus itself should be light* blue. Spores may be readily simple stained by passing the cover-glass, after it has been fixed, 8 to 10 times through the flame. Then the specimen is heated for 1 to 2 min- utes with carbolic-fuchsine. The carbolic-fuchsine solution known also as ZiehTs solution, is prepared by adding 1 g. of fuchsine and 13 c. c. of absolute alcohol to 100 c. c. of 5% carbolic acid. The solution is heated on the water-bath until everything dissolves and the solution has a clear bright red color. MEMORANDA. MEMORANDA. Ill SPORE STAINS. Cover-glass preparations. Air dried. Simple: 12 x through flame. Carbolic-fuchsine (hot inin.). Water (and examine). Air dried. Canada balsam. Double: 3 x through flame. Carbolic-fuchsine (hot 2 to 5 min ). Water (and examine). Dilute alcohol. Water (and examine). Contrast color (metbylene blue, ]- to min.j. Water (and examine). Air- dried. Canada balsam. 2; to Metchnikoff's cellular theory of immunity, the white blood cell is endowed with the power of taking into itself, and ultimately destroying, the invading organism. Phagocytic action can be readily demonstrated in frog-* inoculated with anthrax. For this purpose a pure culture of the amiirax baciilus is intro-. duced into the dorsal lymph sac of a frog, and at the end of 12 or 18 hours it is killed with chloroform. Make cover glass preparations with the. fluid in the dorsal lymph sac and stain some with simple anilin dyes and others after Gram's method. Pliagocytes. Accordin; i i 112 Summary of laboratory work with anthrax: From guinea-pig : Gelatine plates, Colonies Impression preparations. Stich cultures Hanging-drop and permanent mounts. Agar streak cultures Hanging-drop and perma- nent mounts. Ca (OH)2-agar streak cultures Hanging-drop and permanent mounts. Bouillon hanging-drop of blood threads perma- nent mounts. Streak preparations. Simple stain. Gram's stain. From frog phagocytes, simple and Gram's stain. Spores simple and double stain. MEMORANDA. 114 BACILLUS ANTHRACIS. Davaine. Pollender. (1849). SYNONYMS OF ANTHRAX. SPLENIC FEVER (IN CATTLE) : WOOL-SORTER* S DISEASE, MALIGNANT PUSTULE (IN MAN) ; MILZBRAND (Germ.} ; CHARBON., SANG DE KATE (/*>.). Origin. In the blood and tissues in anthrax. Form. Large, clear, homogeneous rods, with slightly rounded ends ; si/e varies witli different media, but the length is less than the diameter of a blood cell. Occurs in blood in short threads of 2-4-0 cells, which may show slightly swollen ends. In bouillon and on agar forms long threads. Involution forms. Motility. lias no motion. Sporulation Forms median, oval spores, without enlarge- ment of cell. Alter long cultivation it may lose the property of forming spores asporogenic variety. I; such cases the addition of fo-1 drop of Cu(OH)2 to an agar tube favors spore formation. Optimum temperature, 30 C. Not formed below 18 C. Spores possess variable resistance. Spores not formed within the body. Anilin Dyes. Stu.n readily, also by Gram's method. Growth. Is rapid. <;<'lii/iti !'!(cs. Deep colonies form round, granular, yellowish-brown masses, with irregular borders. Surface colonies are very characteristic, and according to the consistency of the gelatin the border is fibril I tiled, or shows very wavy strands of threads Medusa head. Liquefy. Mich Cultures. .Short threads radiate from the line of inoculation into the surrounding" gelatin, imparting a brush-like appearance. Cup-shaped liquefaction forms on top and gradually extends till the contents are wholly liquefied. The mass of bacteria settles to the bottom and leaves a perfectly clear solution above, without scum. Streak Cultures. On agar, forms a dry, grayish-white growth On potatoes, the growth is abundant, white, cream-like and rather dry; spores. Oxygen requirements. Is aerobic, but can grow in the body as a facultative amierobe. Temperature. Grows between 12 and 45 C. Optimum :>7 . Behavior to Gelatin. Liquefies. Attenuation By heating for ten minutes at 35 C. ; ^-1 minute at 100. By growing at 42.5 for four weeks. By action of chemicals, mercuric chloride, carbolic acid, etc. By insolation. By growth under pressure. In the body of immune animals, as frogs. Immunity. Obtained with attenuated cultures, first and second vaccine of Pasteur; with sterilized cultures; with extract of thymus gland and of testes. Pathogenesis White mice, guinea-pigs, rabbits, sheep, cat- tle, horses and man are susceptible. Dogs, old white rats, birds and frogs are insusceptible. Subcutanec us application kills in 24- 48 hours. Post-mortem shows subcutaneous oedema and enlarged spleen. Bacilli everywhere. Infection. (1) Through the food, presence of spores, Intes- tinal anthrax in sheep and '.attle. (2) Through wounds, Inocu- lation anthrax in man (malignant pustule), (3) Through the air, Lung anthrax in man, the wool-sorter's disease and possibly rag- picker's disease. MEMORANDA. 116 BACILLUS OF SYMPTOMATIC ANTHRAX. Feser and Bellinger (1878). SYNONYMS OF SYMPTOMATIC ANTHRAX. BLACK LEG, QUARTER EVIL; CHARBON SYMPTOMATIQUE (Fr.) ', RAUSCHBRAND (Germ.}. Origin. In the subcutaneous tissue, muscles, serous exudate, etc., of symptomatic anthrax. Form. Rather large, narrow rods, with distinctly rounded ends ; almost invariably single, may form in twos. About three times as long as wide. Involution forms appear in old cultures swollen in the middle or at the ends. Motility. Actively motile. Spore bearing rods eventually lose their motion. Shows lateral flagella, also giant whips. Sporulation. Spores develop readily in all media as bright oval bodies, situated near one end which is somewhat enlarged. Anilin Dyes. Stain readily. Not by Gram's method. Spores readily double stained. Growth. Rapid, and gives off a strong butyric acid odor. Acid or alkaline. glucose media are best. Requires anaerobic con- tions. Plates. On gelatin, forms irregular masses surrounded by a dense whorl of threads. Liquefies. On ayar, the form of colonies varies. Usually appears as a dense mass of threads. Stick Cultures. In glucose gelatin development takes place in the lower part of the tube; the contents are liquefied and gas is produced. Energetic growth and gas production in glucose agar. The contents of the tube are torn into several parts. Giant whips common. (NovY.) Streak Cultures. On glucose cigar, in hydrogen forms a whitish spread- ing film. On blood serum good growths; giant whips (Loftier). Bouillon. Becomes cloudy; gas bubbles accumulate on the surface; -after several days the growth settles to the bottom, forming a compact, adherent sediment. Liquid above remains cloudy for several days. Glucose gelatin, colored with litmus, develops growth in incubator under ordinary conditions. The color of the litmus changes to a wine-red, showing formation of acids. Heavy flocculent sediment on the bottom. Milk. The casein is coagulated. Starch is not inverted. Oxygen requirements. Is an obligative anaerobe. Grows in vacuum, hydrogen, carbonic acid, etc. Temperature. Grows slowly at ordinary temperature. Best at 37-38 C. Behavior to Gelatin. Liquefies. Aerogenesis. Energetic production of gas, having a disagree- able odor; is inflammable and consists of marsh gas, etc. Attenuation. Bouillon cultures soon lose virulence but main- tain their vitality. Attenuation takes place at 42-43. Spore bear- ing material heated to 80 and 100 becomes attenuated. Virulence restored by inoculating animals, and at same time injecting some lactic acid. Virulence maintained in solid media. Immunity. Can be obtained (1) by inoculating small amounts of virulent organism ; (2) by intravenous injections; (3) by injecting heated cultures, 100 and 80 C. ; (4) with inactive old cultures; (5) with filtered cultures. Pathogenesis. Young cattle, sheep, goats, guinea-pigs are highly susceptible. Horse, ass, white rat are less so; while hogs, dogs, cats, ordinary rats, rabbits, doves, ducks, chickens are wholly immune. Subcutaneous injection in guinea-pigs produces death in 24-48 hours. An extensive subcutaneous oedema is present. The muscles are dark and infiltrated. Infection. Takes place naturally by inoculation through wounds ; not through the food or air. Poisoned arrows used in fishing in Norway. MEMORANDA. 118 BACILLUS CEDEMATIS MALIGNI. Pasteur. (1877). VIBRION SEPTIQUE OF PASTEUR. SYNONYMS OF MALIGNANT (EDEMA. SEPTICEMIE (Fr.); MALIGNES ocDEM (Germ.). Origin. From animals inoculated with garden soil; from horse and from man (septicemie gangretieuse). Form. Rods about three times as long as wide, with rounded ends; usually single, but may form threads, especially in the body. In size, etc., resembles the bacillus of S. anthrax ; is narrower than anthrax bacillus. Motility. Actively motile. Show lateral flagella; also giant whips (NovY). Sporulation. In bouillon and agar, spores appear in 24 hrs. The best temperature is about 37 C. The spores are median or nearly so, with corresponding enlargement of the parent cell. Anilin Dyes. React readily. Is stained by Gram's method. Spores stain double. Growth Is very rapid, especially on glucose media. Requires anaerobic conditions. Plates. On gelatin, colonies develop in 2-3 days, and under the micro- scope resemble those of the Hay bacillus. As they become larger gas bubbles form. On agar plates at 37 the colonies appear as an irregular, dense net- work of threads. &lich Cultures. In gelatin, growth occurs in the lower part of the tube; the gelatin is liquefied, gas given off and the growth settles on the bottom. Agar cultures are torn into several parts by the gas which is formed. In the liquid on the bottom of the tube, giant whips can be found by staining. Streak Cultures. On agar, offer no special characteristics. Grows on potatoes without forming a scum. Bouillon. Becomes cloudy, and in 1-2 days the growth settles on the bottom as a low, adherent sediment, and in a few days the liquid becomes clear. Glucose gelatin, colored with litmus. In air at 37 C. is liquefied and litmus first reduced, then in presence of oxygen Becomes red acid production. Milk. Develops a good growth; a part of the casein is precipitated. Starch is not changed to sugar. Oxygen requirements. Is an obligative anaerobe. Grows in vacuum, hydrogen, carbonic acid, etc. Temperature. Growth is best at the temperature of the body. Can grow at ordinary temperature. Behavior to Gelatin Liquefies. Aerogenesis. On glucose media, especially when distinctly alkaline, it gives rise to the production of gas. Attenuation. Bouillon cultures retain virulence for months. Immunity. One attack of malignant (edema does not protect against a second. 100 c. c. of heated or filtered cultures injected into guinea-pigs in three portions confers immunity ; 6-8 c. c. of the serous exudate accomplish the same result. Pathogenesis, Rabbit susceptible distinction from sympto- matic anthrax. The horse, hog, dog, cat, chicken, dove, guinea- pig and mice are susceptible. Cattle are immune. Subcutaneous inoculation in guinea-pigs of % c - c - or more of bouillon culture produces death in about 24 hours. Marked subcutaneous, spread- ing, reddish oedema. Bacilli present, single or in threads, in subcu- taneous tissue, serous surfaces as peritoneum, etc. ; scarce in the blood. 25-30 c. c. of the filtered bouillon culture, injected subcu- taneously, kills guinea-pigs. Infection. Takes place exclusively by inoculation through wounds. Poisoned arrows of the New Hebrides. Rag-picker's disease. MEMORANDA, _120 BACILLUS CEDEMATIS MALIGNI, NO. II. Novy. (1893). Origin. From guinea-pigs inoculated with milk nuclein obtained from casein by digestion with artificial gastric juice. Form. In the animal body it occurs usually in single rods, 4-5 times as long as wide ; may also' occur in short threads. On arti- ficial media it develops as straight or bent rods, sometimes forming peculiarly twisted threads. The contents are often granular, and show a bright body at one end. Motility. Possesses a slight swaying motion, which is often absent. Has lateral flagella, and in pure culture, as well as in the animal, it gives rise to giant whips which may attain a length of 40-50-72 microns. Speculation. Spore formation not observed. Aniliii Dyes. Stain readily. Gram's method applicable. Growth. Depends upon the vitality of the organism. When taken from an animal it grows rapidly. Plates: On glucose agar good colonies develop in 2-3 days at 37 C. Show a very irregular, fibril ated border, and often give rise tongas bubbles. May contain giant whips. Stick cultures. Develop only in the lower part of the tube. In glucose agar having proper alkalinity, it develops rapidly, forming a plainly visible growth along the line of inoculation; the agar is soon torn into several parts by the gas that is produced. Cultures soon die out. Streak cultures. Develop on glucose agar only when oxygen is com- pletely excluded-. It forms a white film which spreads over the surface. On acid agar involution forms develop. Bouillon. An excellent growth develops which in 24 hours settles to the bottom as a loose, flocculent sediment; the liquid above becomes clear. Glucose f/elatin, colored with litmus. Is liquified and acid is produced the litmus is turned red. Oxygen requirements. Is an obligative anaerobe. Grows in vacuum, hydrogen, nitrogen, carbonic acid, illuminating gas. Temperature. Does not grow below 25 C. Optimum temper- ature about 39 C. Can withstand freezing for 24 hours. Behavior to Gelatin. Liquefies. Aerogenesis. In alkaline media gives rise to gases. Volatile acids, as butyric acids, etc., are formed in artificial culture and also in the body (of rabbits). Attenuation. Cultures left in hydrogen, or exposed to light, lose their virulence. Is not attenuated when left in the dark or when frequently passed through animals. Lost virulence can be reconstituted by inoculation with a "mixed" culture containing Proteus vulgaris. Immunity. Not conferred by a non- fatal inoculation, or by old, weakened cultures, or by the serous exudate of the pleural cavity. Pathogenesis. Subcutaneous injection of % c. c. of hydrogen bouillon cultures kills guinea-pigs, rabbits, white rats, white mice, doves, in 12-24 hours. Marked subcutaneous cedema present; serous exudates in thoracic and abdominal cavities. Cover-glass preparations made from the subcutaneous tissue, or serous surfaces, as peritoneum, shows usually enormous numbers of bacilli, and frequently giant w r hips are also present. MEMORANDA. 122 BACILLUS TETANI. Nicolaier (188-4J. SYNONYMS OF TETANUS. LOCK JAW ; \\TNDSTARRKRAMPF (Germ.}. TETANOS (Fr.). Origin. Found in animals that diedof tetanus after inoculation with earth ; from traumatic tetanus of man and animals ; from head-tetanus. Form. Large, narrow rods with rounded ends ; frequently forms threads. Motility. Is motile. Simple stains with gentian violet of old agar culture may show long spirals. Sporulaticn. Occurs rapidly, in 24-48hours at 37 C. Forms terminal spores, with enlargement drum-sticks. Anilin Dyes. Stain rapidly. Gram's method is applicable. Spores can be double-stained. Growth Pure cultures obtained by heating the spore-bearing material to 80 C., to destroy the ordinary bacteria. Growth slow. Plates. At ordinary temperature colonies develop in gelatin in 4-7 days, and resemble those of the Hay bacillus or Proteus. The gelatin is slowly liquefied and gas produced. On agar plates the colonies appear as fain't clouds which, under the microscope, are seen to be made up of a whorl of threads which are finer than those of other anaerobes. Stich cultures. Development restricted to the lower part of the tube. Cultures of glucose gelatin lubes show along the line of inoculation a cloudy growth, radiating outward into the surrounding gelatin; resembles that of the Root, bacillus. Eventually the gelatin is liquefied. Gas bubbles present. In glucose agar at 37 C. the growth is sometimes indistinct and shows radi- ations. Streak cultures.- -On glucose (tf/nr develop rapidly. Houillnn. At :>7 becomes diffusely cloudy and remains so for several days; eventually the growth settles to the bottom, forming a scarcely visible sediment distinction from preceding anaerobes. Glucose geldfin colored with litmus. At "7 C. becomes liquefied; a very small sediment forms, and the culture remains blue, showing absence of acid formation distinction from preceding. Milk Grows well in milk without inducing any change. Does not invert starch. Oxygen requirements. Is an obligative anaerobe Grows in vacuum, hydrogen, nitrogen, and carbonic acid. Temperature. Does not grow below 16 C. The optimum is about 38 C. Behavior to Gelatin. Liquefies. Aerogenesis. Gives rise to gaseous products, also disagree- able penetrating odor. Hydrogen sulphide. Attenuation. Partial loss of virulence by culture. Thymus bouillon attenuates. Immunity. Iodine trichloride; thymus bouillon cultures; blood serum of artificially immunized rabbits, horse, sheep, dog ; milk of immunized goat. Pathogenesis. Man, horse, sheep, guinea-pigs, young cattle, goats, white mice and white rats, are susceptible. Rabbits and dogs are less susceptible. Ducks and chickens are immune. The bacillus is present at the point of inoculation, although in small numbers. Intensely poisonous products. The filtered bouillon culture in a dose of 0.0002 c. c. kills mice, and 0.002 c. c. kills guinea-pigs. Infection. Occurs through wounds, Poisoned arrows of the New Hebrides. MEMORANDA. MEMORANDA. 123 CULTURES OF ANAEROBIC BACTERIA. Obligative anaerobic bacteria, those which grow only in the absence of oxygen, require special conditions for cul- tivation. Their growth is favored by the addition of 1 to 2 per cent, of glucose to the nutrient, medium, whether gel- atin, bouillon or agar. Freshly prepared media are, as a rule, best adapted for culture purposes. The numerous methods which have been proposed for obtaining growths of anaerobic bacteria can be classified under the following heads: (1.) Exclusion of oxygen. (2.) Exhaustion of air. (3.) Absorption of oxygen. (4.) Displacement of air. (5.) Cultures apparently in the presence of air. The well-known method of Liborius, of culture in deep layers of gelatin or agar, depends upon the exclusion of air. The method is .simple and very convenient. The culture-tubes contains glucose agar or gelatin, l|-2 inches high. Stich cultures are made in the usual manner. Growth develops in the lower two-thirds of the medium, while the upper layer of J to inch serves to exclude the air. In order to insure complete exclusion of oxygen, the contents of an ordinary agar orgelatin tube can be lique- fied and then, with proper precautions against contamina- tion, poured on top of the inoculated medium and quickly cooled. This extra layer is, as a rule, unnecessary. Colonies of anaerobic bacteria can be obtained by making ordinary gelatin or agar plates, and then placing* a sterilized glass plate on top to exclude oxygen. Vacuum cultures are frequently resorted to. Gruber's 124 tubes with constricted necks are commonly employed for this purpose. The air is pumped out, after the medium is inoculated, and the tube is then sealed in a llame. The absorption of oxygen can be accomplished by means of an alkaline solution of pyrogallic acid. In Buclmer's method the inoculated tube is placed within a larger tube, which is closed with a rubber stopper, and which contains on the bottom the pyrogallate solution. The displacement of air by some inert gas, as hydro- gen, is frequently made use of in cultivating anaerobic bacleria. Special tubes, as those of Li bonus, have been introduced for this purpose. A current of hydrogen is passed through the inoculated tube until all the air has been displaced, after which it is sealed in a ilame. Plate cultures in hydrogen can be readily obtained with Botkin's apparatus a bell jar inverted over liquid paraffin, or mercury. Cultures of anaerobic bacteria can be readily obtained by inoculating glucose gelatin tubes, colored with litmus, and placing them in the incubator. Although the con- tents of the tubes are liquid, and apparently the air has free access, yet energetic growth takes place. These cul- tures preserve their vitality for a considerable period of time and have the additional advantage of being readily accessible. The most convenient method for obtaining cultures in a vacuum, or in an atmosphere of any desirable gas, is to use some form of bottle in which the ordinary culture tubes can be placed, and the exhaustion or displacement of air carried out. Fig. 1 shows such a bottle, which is provided with a special stopper through which a current of gas, as hydrogen, carbonic acid, etc., can be passed. The bottle is sealed air-tight by merely turning the stopper through ninety degrees. Fig. 2 shows a simple and eilicient form of bottle in which the same result is obtained by means of two glass stop-cocks. Ordinary test-tubes containing glucose gelatin, bouil- Fig. 1. 12 Fig. MEMORANDA. 125 Ion, agar, etc., are inoculated in the usual manner. The projecting part of the cotton plug is cut off close to the mouth of the tube, and the plug slightly raised, with sterilized forceps, to facilitate diffusion of the gas. The tube is then placed in the bottle by means of a pair of long forceps and the apparatus connected with a Kipp's hydrogen generator. The current of hydrogen should be passed first through an alkaline solution of lead acetate, and then through a six per cent, solution of potassium permanganate. After passing through the apparatus the gas passes through a small wash-bottle containing water, which serves as a valve. If carbonic acid is used it should be passed through a saturated solution of sodium carbonate. A rapid current of gas is passed through the bottle for 1 to 2 hours, it is then sealed by turning the stopper, and set aside in the incubator to develop. The apparatus can also be used for vacuum cultures, in which case it is connected with a Chapman aspirator and the air pumped out. The alkaline pyrogallate method can be employed with excellent results. (NovY, Central- Uatt fur Bakteriologie, 14, 581, 1893). By far the easiest and simplest method for obtaining plate cultures of anaerobic bacteria is that devised also in this laboratary. The apparatus, which has the form of a desiccator, is provided with the special stopper seen in the bottle, Fig. 1. Petri plates are placed in the appara- tus, hydrogen is then passed through for 1 to 2 hours, and finally it is sealed by turning the stopper. LABORATORY WORK. Liquefy four glucose agar tubes by heating in the water bath ; then allow to solidify in an upright position. When cool make deep stich cultures of Bacillus of symptomatic anthrax. Bacillus of malignant oedema. Bacillus of malignant oadema, No. II. Bacillus of tetanus. 126 If the agar in the tube is less than one inch high, it will be necessary to pour on top, after inoculation, the con- tents of another agar tube, taking care to sterilize the mouths of both tubes. Set aside the inoculated tubes in the incubator for 24 to 48 hours. The cultures are then examined in hanging-drop. Simple stains are made, also double stains of spores. The drop or two of liquid which sometimes accumulates on the surface of the agar, and invariably on the bottom of the tubes can be stained for ordinary nagella and for giant-whips. Make the following cultures at the same time as the preceding : (1) Streak culture on inclined glycerine agar of the tubercle bacillus, using either a pure culture, or a tubercle from a guinea-pig inoculated with tuberculosis. Spread the material thoroughly over the surface of the medium. (2) Streak culture on ordinary inclined agar of the Achorion Schonleinii the fungus of favus. (3) Streak culture on ordinary inclined agar of Actin- omyces the fungus of lumpy-jaw. After these three inoculations have been made the cotton plug of each tube is cut off close to the mouth of the tube, and this is then sealed either with a rubber cap, or with sealing wax, or with paraffin of a high melting point, 56 C. The sealed tubes are then placed in the incubator for several weeks. STAINING OF FLAGELLA. In order to obtain good stains of nagella special care must be given to the preparation of the cover-glasses. These must contain as little organic matter as possible in order to prevent the formation of a dirty precipitate on the cover-glass. Excellent cover-glasses can be made by dilution. A small loopful of the turbid fluid from the bottom of the agar tube culture of (Edema bacillus No. II, MEMORANDA. MEMORANDA. 127 or from that of the Bacillus of malignant oedema, is trans- ferred to a large drop of distilled water in the center of a wide cover-glass. By means of a straight platinum wire, three transfers are made from this drop to another drop of distilled water on a second cover-glass. This second cover glass will now contain only a small number of bacteria and very little foreign matter. By means of a platinum wire, with a very small loop, not much larger than a pin-head, transfers can now be made to t> or 8 clean wide cover glasses. Each small loopful is spread at once over as much of the surface of the cover-glass as possible. The thin film of liquid evaporates almost immediately, and the cover-glass can then be fixed by passing it once through the flame. Overheating the cover-glass is very likely to destroy the slender flagella. The cover-glass, with the specimen side up, is held in a pair of forceps and covered, by the aid of a pipette, with Loffler's mordant solution. The cover-glass is then held over the flame, which should be turned low, for about a minute. The liquid should be warmed so as to give off vapors, but should not be actually boiled. As fast as evaporation takes place fresh mordant solution should be added, and at no time should it be allowed to dry down on the cover-glass. The mordant must then be thoroughly and completely washed off the cover-glass by a jet of water. If the edge has dried down, it should be loosened with a pin or knife and then washed off. To still further clean the cover- glass, it should be dipped for a few seconds in absolute alcohol and again washed with water. The mordanted cover-glass is then covered with a satu- rated solution of anilin water fuchsine, or with carbolic fuchsine, and heated over the flame for 1 to 2 minutes, observing the same precaution as before. The specimen is then thoroughly washed with water and examined with the -i 1 / inch oil-immersion objective. 128 Summary for staining flagella : Dilution cover-glass preparation. Air-dried. 1 x through flame. Mordant, hot (1 to 2 min.). Water. Alcohol (few seconds). Anilin-water fuchsine, hot (1 to 2 min.). Water (and examine). Air-dried. Canada balsam. The mordant employed is prepared by dissolving 20 g. of tannic acid in 80 c. c. of distilled water. To 10 c. c. of this tannic acid solution, add 5 c. c. of ferrous sulphate solution (1-2), and 1 c. c. of saturated alcoholic solution of fuchsine. The stain employed is made by adding 4 to 5 g. of fuchsine to 100 c. c. of anilin water (p. 106). Both mor- dant and stain should be kept warm while in use. MEMORANDA. MEMORANDA. MEMORANDA. 130 EXAMINATION OF SPUTUM FOR THE TUBERCLE BACILLUS. Ziehl-Neelsen Method. A ]oopful of the sputum is transferred to a wide cover-glass and thoroughly spread over the surface. It is then allowed to dry in the air, or by moving it to and fro over the flame, and then fixed in the usual way. The cover-glass is held in the forceps, specimen side up, and covered with carbolic fuchsine solu- tion. It is warmed over the flame for 1 to 2 minutes, avoiding actual ebullition, and then washed with water. The specimen is now dipped in dilute nitric acid (a watch- glass is filled with water and 3 or 4 drops of nitric acid added), for a few seconds. Then transferred to dilute alcohol (60 to 70X), where it is moved about till it is almost decolored. After this it is washed with water and stained for a few seconds with methylene blue. The lat- ter is washed off with water and the specimen examined under the microscope. It should show the tubercle bacil- lus stained bright red, on a light blue back-ground. Heavily stained preparations can be obtained by floating the prepared cover-glass on the carbolic-fuchsine solution for 15 or 30 minutes, then decoloring, as before. Cover-glass preparation. Air-dried. 3 x through flame. Carbolic-fuchsine, hot, (1 to 2 min.). Water. Dilute nitric acid (few seconds). Dilute alcohol. Water. Methylene blue (i to % min.). Water (and examine). Air-dried. Canada balsam. A 2 per cent, aqueous solution of anilin hydrochloride can be used to excellent advantage instead of the dilute nitric acid. MEMORANDA. 132 BACILLUS TUBERCULOSIS. Koch. (1882). TUBERCLE BACILLUS. Origin. In tuberculosis of mammals. Lupus vulgaris. The bacillus, present in chicken tuberculosis is distinct from that in mammals. Form. Very narrow, rather long rods which are smaller than the diameter of a red blood cell. The ends are distinctly rounded and the bacillus itself may be straight or more frequently is slightly bent or nicked. Occurs usually single but may form short threads of 3-6 cells. In the sputum, tissues, etc., is" frequently found in small bunches. Motility. Has no motion. Sporulation. Frequently shows a number of bright bodies within the cell, but these cannot be considered as true spores. .The bacillus itself possesses a relatively high power of resistance to heat, desiccation, acids, putref action, etc. Anilm Dyes Stains very slowly and difficultly with simple anilin dyes; readily with hot carbolic-fuchsine, or anilin-water fuchsiue or gentian violet. When once stained it is difficult to decolor, whereas ordinary bacteria do so readily. Specimens from sputum and tissues can therefore be readily double stained dis- tinction from all known bacteria, except the leprosy bacillus. Can be stained by Gram's method. Growth. Takes place very slowly, requiring usually several weeks to become clearly visible. Furthermore, a special tempera- ture, at or near that of the body, and special media as blood-serum or glycerine-agar, etc., are necessary. Plntes .No growth has been obtained on plates. Colonies can be readily obtained by making successive streaks on glycerine-agar or blood-serum. Colonies obtained direct from the sputum are round, white, opaque, and raised, resembling colonies of white yeast. On subsequent culture the colonies are dry, grayish scales. Under the microscope they appear as interwoven, twisted strands of threads. Stick Cultures. Can be obtained on glycerine-agav. Growth restricted to the upper part of the tube. It spreads over the surface as a thick, raised plaque which at first is white, but later becomes yellowish. Streak Cultures.- On glycerine agar or blood-serum eventually develops an abundant dry, granular, raised growth, which at first is grayish, but later takes on a light yellow tinge. Similar growths develop on potatoes. Jiouill on. Grows well, especia'ly on the surface in bouillon which con- tains the usual amount of glycerine, 5-6 per cent. Such bouillon cultures, filtered and concentrated, constitute the so-called tuberculin. Oxygen requirements. Free access of oxygen necessary for growth. Is a facultative anaerobe (FHAENKEL). Temperature. The optimum is about 37-39C. Slight varia- tions above or below this stop the growth. It cannot, therefore, grow at ordinary temperatures. Behavior to Gelatin. No growth. Attenuation. Slight attenuation probably does result with age, but otherwise it has not been positively demonstrated. Pathogeneais. Man, monkeys, cattle', guinea-pigs, field mice, rabbits, and cats are susceptible. White mice, rats, and dogs are somewhat insusceptible. Chickens are immune. Inoculation of pure cultures produces in susceptible animals tuberculosis. The formation of tubercles and of giant cells. The bacilli may be very abundant, at other times are scarce and difficult to find. Infection Takes place most frequently along the respiratory tract Inhalation tuberculosis. May occur tlirough wounds Inocu- lation tuberculosis, and also through food Intestinal tuberculosis. Placental infection. MEMORANDA. 134 BACILLUS LEPRJE. Hansen. (1879). LEPROSY BACILLUS. Origin. Found in the leprous nodules of the skin and mucous membrane, lymphatic glands, liver, spleen, marrow, etc. Not in the blood. Form. Rather large, narrow rods, which resemble the tubercle bacillus. Motility . Non- motile. Sporulation. Bright bodies frequently observed within the cell, as is the case of the tubercle bacillus. Doubtful if these are spores. Anilin Dyes. Stain readily. Can be stained by Gram's method, also by the method for the tubercle bacillus. Sections kept in alcohol lose the property of double staining. Growth. Has not been obtained under artificial conditions with certainty. The cultures of Bordoni- UfFreduzzi on glycerine blood-serum inoculated with the marrow of long bones. Apparently is an obligative para- sitic organism. Pathogenesis. While the constant presence of the leprosy bacillus in leprous tissue leads to the prevailing view that it is the cause of that disease, it should never- theless be remembered that as yet unquestioned pure cultures have not been obtained and hence successful in- oculations are impossible. Direct infection with leprosy tissues has given but few positive results. Infection. The mode in which this occurs in man is entirely unknown. MEMORANDA. 136 AGAR PLATE CULTURES. The ordinary gelatin plates are applicable for the isolation of colonies of only those organisms which can grow at ordinary room temperature. Above 25 C. the nutrient gelatin melts and cannot therefore be employed as a solid medium for the growth of those organisms which develop only at a higher temperature. In such cases plates can be made with ordinary, or glycerine or glucose agar. The method of making agar plate cultures is briefly as follows: Three agar tubes are immersed in boiling water in a water-bath until the contents are liquefied. The burner is then removed from under the water-bath and the water with the immersed tubes is allowed to cool slowly until a temperature of 45 C. is reached. Tube 1., is inoculated with the material to be plated and the usual dilutions to tubes 2 and 3 are made as rapidly as possible. The cotton plugs are then cut off short, pushed in slightly, and the lips of the tubes sterilized in the flame. The inoculated contents are then poured into sterilized Petri dishes, or on ordinary sterilized plates. Inasmuch as the agar solidifies at about 40 it will require rapid work to inoculate the tubes and pour the contents before solidification takes place. Ice-water must not be used to congeal the agar. The Petri dishes or plates are then placed in the incubator. Esmarch roll-tube cultures can also be made with agar in the same manner as described for gelatin cultures. The tubes should be rotated in ordinary tap water. LABORATORY WORK. Make glycerine agnr Petri dishes from the spleen of a guinea-pig inoculated with glanders. When the colonies develop, make streak cul- tures on inclined glycerine ngar. Examine the spleen and also the pure cultures in the usual manner by making hanging-drops and cover-glass preparations. MEMORANDA. 138 BACILLUS MALLEI. Loftier and Schtitz. (1882). BACILLUS OF GLANDERS. MORVE (Fr.) | ROTZ (Germ.} } MALLEUS (.Ld.). Origin. Found in the nodules, ulcers, discharges, etc., of glanders or farcy. Form. Rods with rounded ends, straight or slightly curved, shorter and thicker than the tubercle bacillus. May grow in pairs or in short threads. Motility. Has no motion. Sporulatipn. Bright bodies are frequently found in the cells, as in the tubercle bacillus; are considered by Loffler as the first indication of degeneration. Keal spores are said to have been double stained. The bacillus itself is highly .resistant to desiccation. Anilin Dyes. Is readily stained and also decolors rapidly. Carbolic-fuchsine, or alkaline anilin gentian violet, or anilin fuchsine stain well, especially when warmed. Not stained by Gram's method. Growth. Occurs only at relatively high tempera- tures. Growth is rapid. Glycerine agaris the best medium. Plates. Cannot be obtained with gelatin. On glycerine agar at 37 Q C. forms excellent colonies in a day or two. These are round, grayish, and glis- tening in appearance, with granular contents and smooth sharp borders. Stich Cultures. Can be made in glycerine agar, not in gelatin. Streak Cultures. --O\\ glycerine agar forms a thick, moist, slimy, semi- transparent growth. On potatoes the growth is very characteristic. At first it forms a thin, transparent, honey or amber-colored growth which later be- comes reddish-brown. On blood-serum forms yellowish, transparent spots which eventually fuse together and yield a slimy, whitish growth. Bouillon. Grows readily and abundantly. Oxygen requirements. Is a facultative anaerobe. Temperature. Does not grow below 25 or above 42 C. The optimum is about 37 C. Behavior to Gelatin. Scarcely any growth. Attenuation. Takes place rapidly when grown on artificial media; must therefore be frequently passed through an animal, otherwise the virulence is lost and the organism dies out. Mallein the filtered cultures of the glanders bacillus analogous to tuberculin. Immunity. Sm all amounts of bouillon cultures in- jected intravenously into dogs confer immunity. Pathogenesis. Man, horse, ass, guinea-pigs, field mice, cats, and goats are highly susceptible. Ordinary and white mice, cattle, and hogs are immune, while dogs, rabbits, and sheep are slightly susceptible. White mice become susceptible when fed with phloridzin. Susceptible animals on inoculation develop typical glanders. In guinea-pigs death results in 4-6-8 weeks. Field mice die in a few days. Enlarged lymphatics, nodules in liver, spleen, etc. Bacilli present. Infection. Through wounds inoculation glanders. One instance in man with pure culture. Along the re- spiratory tract probably the usual source of infection in horses. MEMORANDA. 140 BACILLUS DIPHTHEBIJE. Klebs, Loffler (1883). BACILLUS OF DIPHTHERIA. Origin. Found in diphtheritic pseudo-membranes, and in very small numbers in the spleen, liver, etc., of diphtheria. Form. Rather large thick rods which are straight or slightly bent and have rounded ends. The form is subject to considerable variation, and rods with swollen, club-shaped ends are frequently met with involution forms. Motility. Has no motion. Sporulation. Spores have not been observed. Tne bacillus is very susceptible to desiccation, or to heat of 50 and above. Anilin Dyes. Dimple anilin dyes react poorly. Can be best stained with carbolic-fuchsine, or with Loffler's alkaline methylene blue (30 c. c. of cone, alcoholic solution of methylene blue + 100 c.c. of a 0.01 per cent, solution of potassium hydrate). Is also stained by Gram's method. Growth. Is very rapid at higher temperatures and on special media as glycerine agar and blood serum. Plates. On gelatin plates left at about 24 C. forms very small, round, white colonies wh'ich have granular contents and irregular borders; do not liquefy gelatin. Cn glycerine agar plates, kept in the incubator, excellent colonies form in 24-48 'hours The deep colonies are round, or oval, coarsely granular. The surface colonies are flat, grayish white, glistening, with irregu- lar borders and coarsely granular contents. Rl.ich Cultures. In gelatin a very limited, scarcely preceptible growth of small, round, white dots marked involution forms present. Streak Cultures. On glycerine agar show a thin, grayish, spreading, adherent film, which is quite characteristic. On potato the growth is invisible or forms a dry, thin glaze irregular forms of the bacillus are numerous. On blood-serum it forms a thick white, opaque growth. Bouillon. Becomes diffusely clouded and the growth eventually sub- sides on the sides of the tube and on the bottom. A pellicle may form on the surface. Oxygen requirements. Is a facultative anaerobe, but grows best in presence of oxygen. Temperature. Very slight growth at 20-25 C. The maxi- mum is about 42 and the optimum 35-37 C. Behavior to Gelatin. Does not liquefy. Attenuation. Cultures directly isolated from membranes show frequently marked variation in virulence. By artificial cul- ture the virulence is still further diminished. Cultures can be at- tenuated by growth at 40 in a current of air. Immunity. Is produced by filtered bouillon cultures heated to 60-70 C. Also by injections of thymus bouillon cultures prev- iously heated to 65-70. Partial results with iodine trichloride. Pathogeiiesis. Mice and rats are wholly immune. Finches, sparrows, doves, chickens, rabbits, guinea-pigs and cats are suscep- tible. Subcutaneous inoculation in guinea-pigs produces death in 24-48 hours. Pseudo-membranous masses form at point of inocula- tion ; an extensive hemorrhagic oedema forms under the skin and exudates occur in the pleural cavity. Inoculation in the trachea of cats, chickens, doves, rabbits, etc., is followed by pseudo-membrane formation, and by death. In some animals as rabbits typical diph- theritic paralysis of the extremities can be observed. The highly poisonous toxalbumin. Infection. Exact mode of infection is not known, but un- doubtedly occurs through the air. NOTE. Make glycerine agar Petri dishes of the diphtheria bacillus. MEMORANDA. 142 MICROCOCCUS PNEUMONIA CROUPOS.2E. Sternberg (1880). Frankel (1883). SYNONYMS: MICROBE OF SPUTUM SEPTIC^MIA, DIPLOCOCCUS PNEU- MONIA, FKANKEL'S DIPLOCOCCUS. Origin. Occasionally in saliva of healthy persons; especially in " rusty " sputum of pneumonia. The same organism or scarcely distinguishable varieties, are present in cerebro-spinal meningitis, pleuritis, peritonitis, pericarditis, etc. Form. Oval or lance-shaped diplococci, may form chains of 4-6 cells and resemble a streptococcus. Owing to its oval form it is sometimes regarded as a bacillus. In the animal body it is sur- rounded by large capsules. Motility. Has no motion. Sporulation. Unknown. Anilin Dyes. Stains readily, also by Grain's method. The capsules remain colorless. Growth. Takes place somewhat slowly and only at higher temperatures, and on alkaline media. Plates. On gelatin plates kept at 24 C. small, round, sharply denned, tly granular, whitish colonies develop slowly. On agar plates in the in- -cubator, in 48 hours, delicate, glistening, transparent drops form which under slightly granular, whitish colonies develop slowly. On agar plates in the in- -cubator, in 48 hours, delicate, glistening, transparent drops form the microscope are round, sharply bordered and finely granular. Stich Cultures. In. gelatin a row of small, white granules develop along the line of inoculation. Does not liquefy. Streak Cultures. On agar, in the incubator, the growth develops as a thin layer of delicate, glistening, almost transparent drops. Dies out rapidly. On blood-serum forms a transparent film-like dew drops. No growth on potato. Bouillon. Excellent growth occurs; vitality preserved for some time. Milk. Is a favorable culture medium, becomes coagulated. Oxygen requirements. Is a facultative anaerobe. Temperature. Growth occurs only between 24 and 42. Its optimum is about 37 C. Behavior to Gelatin, Does not liquefy. Attenuation. Cultures from different sources show marked difference in virulence. When grown on artificial media it rap- idly attenuates and soon dies out, unless it is passed through a sus- ceptible animal, as a rabbit, every few weeks. Rapidly attenuates *it42O. Immunity. Intravenous injection of very small amount of virulent culture ; injections of filtered cultures, especially when heated to 00 C. ; blood serum of immune animals. Blood-serum from pneumonic patients immunizes rabbits against the pure cul- ture. Pathogenesis. Subcutaneous injection of 0.1 0.2 c. c. of bouillon cultures in rabbits produces death in 24-48 hours. The diplococcus is found in the blood and internal organs and is sur- rounded by a capsule. Tracheal injections in rabbits produce true pneumonia. Mice and rabbits are highly susceptible ; guinea-pigs, sheep, dogs are less susceptible. Is the" recognized cause of croup- ous pneumonia. NOTE. Make agar Petri dishes from the peritoneum exudate in a rabbit. Make cover-glass preparations from the peritoneum, surface of intestines and tieart-blood, and stain by simple and by Gram's method. MEMORANDA. 144 PNEUMOBACILLUS OF FEIEDLAENDER. (1883). ALSO KNOWN AS FRIEDLAENDER's PNEUMOCOCCUS. Origin. Is frequently found in normal saliva ; also in lungs and " rusty " sputum of pneumonia. Form. May appear as an oval coccus, but in reality is a short, thick rod, which may grow in pairs and even in short threads. In the animal body it is enveloped by a capsule. Motility. Has no motion. Sporulation. No spores observed. Cultures retain vitality for many months. Anilin Dyes. The cell is stained readily but the capsule remains colorless. Is not stained by Gram's method distinction from Rhinoscleroma bacillus and Frankel's fiiplococcus. Growth. Is rapid and abundant. Platen. On gelatin plales it develops rapidly. The deep colonies are round or oval, sharply bordered, finely granular and yellowish. The surface colonies are quite characteristic and appear as thick, moist, glistening, white masses which do not lend to spread but rather to become convex and raised. No liquefaction. Stich Cultures.- (Irmvth takes place along 1he entire line of inoculation and is especially developed on the surface forming a " nail-shaped " culture. As the culture becomes old the gelatin near the surface becomes brownish in color and small gas bubbles may form. Streak Cultures. On ac/ar forms a thick, white, moist, shiny growth On blood-serum develops as a grayish, shinv mass. On potatoes forms a thick, yellowish, sticky growth, showing gas bubbles. Oxygen requirements. Is a facultative anaerobe. Temperature. Grows rapidly at low temperatures, 16-20; also in the incubator. Behavior to Gelatin. Does not liquefy. Aerogenesis. Abundant production of gas in 4 per cent, gelatin ; potato cultures grown in the incubator also give rise to gas. Pathogenesis. Is pathogenic for mice and young rats; guinea-pigs and dogs are less susceptible, while rab- bits are immune. While not the cause of pneumonia, its frequent presence in that disease may serve to bring about a " mixed infection." NOTE Make gelatin plates and cover-glass preparations from the lungs and blood of a young rat which has received an intrapleural injection. MEMORANDA. 146 BACILLUS OF RHINOSCLEROMA. Frisch (1882). Origin. In the tumors of rhinoscleroma, a rather rare disease occurring in Austria and Italy. Form. Short, thick rods with rounded ends resem- bling the Friedlaender's pneumobacillus, may form short threads. Are likewise surrounded by a colorless capsule. Cells of Mickulicz. Motility. Has no motion. Sporulation. Not observed. Anilin Dyes. Stain readily, and show colorless capsule; also stained by Gram's method. Growth. Growth resembles in almost every respect that of the Friedlaender bacillus, The colonies, stich and streak cultures, agree so closely as to be scarcely distin- guishable. Oxygen requirements. Is a facultative anaerobe. Temperature. Grows rapidly at ordinary tempera- ture. The optimum is about 36-38 entire line of inoculation, and especially so on i he surface wh^re it spreads as a thin, grayish white covering. Gelatin eventually becomes cloudy, due to the production of acids. Streak Cnttui'm. Jn ut/nr and on hi >')n potatoes at ordinary temperatures, but in the in- cubator in u few tla\ s it gives rise to a yi llowi^h-gray transparent covering. Oxygen requirements. Is a facultative anaerobe. Temperature. Grows at ordinary temperature and also in the incubator. Behavior to Gelatin. Does not liquefy. Attenuation. Artificial cultures soon lose their virulence. It was in connection with this organism that attenuation was first ob- served by Pasteur (1880). Influence of oxygen, of heat. Immunity. Is produced in chickens and pigeons by inocula- tion with first and second vaccines. Pathogenesis. Chickens, geese, pigeons, sparrows, mice and rabbits are susceptible. Guinea-pigs, sheep, horses are less suscep- tible and only local abscesses form. After death the bacilli are found distributed throughout the body a true septicaemia. Infection. Usually results in chicken through the food and along the alimentary canal. May possibly also occur through scratches and wounds. MEMORANDA. BACILLUS OF HOG CHOLERA. Detmers (1880). BILLING'S SWINE PLAGUE BACILLUS. BACTERIUM OF HOG CHOLERA (SALMON AND SMITH). Origin. In the blood, organs and intestinal contents of swine that died of hog cholera. Form. Short, small rods, resembling those of chicken cholera. On some media, as gelatin, it may form long rods. Occurs single or in pairs. Motility. Is actively motile. Has long, wavy flag- ella. Shows no motion in serum or in blood. Sporulation. Not observed. Anilin Dyes. At first impart a bi-polar stain, but on sufficient exposure the entire rod is colored. Is not stained by Gram's method. Growth. Is fairly rapid. Plates. In a couple of days colonies develop on gelatin plates. The deep colonies are very small, yellowish-brown and spherical. The surface colonies spread slightly. No liquefaction. Stich Culture*. &\\o\v along the line of inoculation a white line or row of colonies, while on the surface of the gelatin a thin, very slowly spreading growth forms. Streak Cultures Qn agar forms a moist grayish-white growth without any special characteristics. On potatoes a straw yellow growth develops, re- sembling somewhat that of glanders. Oxygen requirements. Is a facultative anaerobe. Temperature. Grows well at ordinary temperature. Best at about 37 0. Behavior to Gelatin. Does not liquefy. Attenuation. Artificial cultures retain their viru- lence apparently indefinitely, same as the anthrax bacillus. Immunity. Can be produced experimentally by inoculation with filtered cultures ; with repeated small doses of blood, previously heated to 54-58 C , from infec- ted rabbits. Pathogenesis. Hog, mice, rabbits and guinea-pigs are highly susceptible ; pigeons are less susceptible, while chickens, sheep and calves are immune. -\ c.c. of bouillon culture injected subcutaneously into rabbits kills in about four days. Bacilli distributed everywhere. Infection. May result through the food, also by in- oculation through wounds. MEMORANDA. ISO BACILLUS OF HOG ERYSIPELAS. Pasteur (1883). SYNONYMS. SCIIWEINEKOTHLAUF (Germ.}', ROUGET (Fr.). Origin. In the blood, internal organs, etc., of swine infected with the disease. Form. Very small, narrow rods resembling needle-shaped crystals. Are usually single, but may occur in pairs and even in threads. Motility. Has motion. Sporulation. Spore formation is not known. Anilin Dyes. Stain readily. Gram's method gives excellent results. Growth Is rather slow. Growth. On gelatin plates the colonies are very characteristic and ap- pear as diffuse cloudy patches which are sometimes difficult to see. Little or no surf are growth. No liquefaction Stick Cultures. In gelatin are likewise very characteristic. The growth develops along the line of inoculation as a delicate, cloud-like radiating col- umn. As the culture becomes old a depression forms at the top, due to slow liquefaction and corresponding evaporation. .Sometimes liquefaction can be observed. Streak Cultures. On agar and on blood- serum it forms a scarcely visible thin film or group of colonies. No growth on potatoes. Bouillon. A very delicate diffuse cloudiness forms which can best be seen on slight agitation. Resembles the bouillon culture of the Tetanus bacillus. Oxygen requirements. Is a facultative aerobe. Best growth under anaerobic conditions. Temperature. Grows slowly at ordinary temperature. Best at 36 C. Behavior to Gelatin. Does not perceptibly liquefy gelatin. Aerogenesis Produces hydrogen sulphide in pure cultures, and in the body. This gas is also produced by the anaerobic bac- teria and to a less extent by nearly all pathogenic bacteria. Attenuation Old cultures become attenuated and this re- sult can also be obtained by growing the virulent germ at high tem- peratures, about 42 C.,for*some time (Pasteur). Immunity. By inoculation with attenuated cultures first and second vaccine of Pasteur perfect immunity can be produced. One attack of the disease confers immunity. Pathogenesis. Swine, rabbits, pigeons, white mice, house mice 'are susceptible, while guinea-pigs and chickens are insusceptible. Bacilli distributed throughout the organism ; are single or in pairs, and very often can be seen to be enclosed in cells. Infection. Probably occurs naturally in swine through the food. MEMORANDA. 182 ~ BACILLUS OF MOUSE SEPTIC^MIA. Koch (18T8J. SYNONYMS. BACILLUS MURISEPTICUS. MAUSESEPTIKAMIE (Germ.). Origin. From mice after inoculation with putrid blood. Form. The rods are narrower and thinner than those of the rouget bacillus, but otherwise resemble the latter very much. Motility. Appears to possess motion. Said to be non-motile by some. Sporulaticn. Round, glistening bodies, or spores form within the cells. Anilin Dyes. Stain rapidly. Gram's method is applicable. Growth. Is rather slow and resembles very closely that of the rouget bacillus. Plates. The colonies on the gelatin plate resemble those of the rouget bacillus, except that they spread somewhat more rapidly and are especially delicate and transparent in appearance. Stich Cultures. Show this distinction in growth quite sharply. While the cloudy growth of the rouget bacillus is dense and somewhat limited to the line of inoculation, that of the mouse septicaemia bacillus spreads readily throughout the entire gelatin. This difference is clearly seen in young cultures. Streak Cultures. On agar the growth is scarcely to be distinguished from that of the rouget bacillus. Bouillon The bacillus develops a growth similar to that of the bacillus of rouget. Oxygen requirements. Is a facultative aerobe. Grows better when air is excluded. Temperature. Grows well at ordinary temperature, also in the incubator. Behavior to Gelatin. Ordinarily no liquefaction can be ob- served. Sometimes, however, it is present. Aerogenesis. Produces less hydrogen sulphide than the rou- get bacillus. Attenuation Old cultures possess diminished virulence. Immunity. Rabbits that recover after one inoculation with the pure culture are rendered immune against subsequent inocula- tion. Fathogenesis. White mice, house mice, pigeons, sparrows and rabbits are susceptible. Chickens, guinea-pigs and field mice are wholly immune. After death the bacilli are distributed throughout the body, single or in pairs, and frequently inclosed in cells. MEMORANDA. 16 184 ACTINOMYCES. Bellinger (1877). SYNONYMS. RAY-FUNGUS. STRAIILENPILZ (Germ.}. Origin. From actinomycosis or lumpy-jaw in cattle, hogs, and in man. Form. The exact position of this organism is uncer- tain, but it is closely related to the fungi or moulds. It forms nodules which consist of a whorl of mycelial-like multiple branched threads. These radiate outward from a central point and become club-shaped. In pure cultures only slender, wavy threads are formed, and the club-shaped or swollen ends, so commonly present in tissues are lacking. Anilin Dyes. Stain readily with carbolic fuchsine; also by Gram's method. Growth. Develops somewhat slowly, requiring sev- eral days in the incubator. Streak Cultures. On agar, the growth begins as minute, isolated colo- nies, which slowly enlarge, forming thick, convex, glistening, yellowish masses. These colonies are exceedingly hard and for examination should be crushed between two glass slides previously sterilized by passing several times through the flame. Cover-glass preparations are then made and stained in the usual manner. Oxygen requirements. Said to grow best in the absence of air, but grows very well on the surface of agar. Temperature. Grows only at or near the body temperature. Pathogenesis. In rabbits, intraperitoneal injection of the pure culture produces typical actinomycotic nodules on the peritoneum, mesentery, intestinal walls, etc. MEMORANDA. 186 ACHORION SCHONLEINII. Schonlein (1839). THE FUNGUS OP FAVUS. Origin. Found in the scaly accumulations on the skin of persons afflicted with favus. Form. Apparently belongs to the moulds. It shows on microscopical examination peculiarly twisted threads, which show divisions and give off branches at right angles. Fruit-organs. No true fruit organs observed, but on special media as on blood-serum at 30 0. conidia or spores form. Anilin Dyes. Stains well, also by Gram's method. Growth. Is rather slow. Plates. On gelatin plates, the colonies grow slowly and form whitish, stellate masses, which rapidly liquefy the gelatin. No conidia present. Slich Cultures. Growth is very poor in the lower part of the gelatin tube. On the surface it forms a white covering, the lower side of which is light yellow. Liquefies. Streak Cultures. On agar it forms a closely adherent, whitish, dry mass. Temperature. Dies out at the ordinary tempera- ture. The optimum is about 30 C. Behavior to Gelatin. Liquefies. Pathogenesis. Inoculation with pure culture pro- duces typical favus in man. The favus fungus is closely related to that of Herpes tonsurans the Tricophyton tonsurans (1845); to that of Pityriasis versicolor the Microsporon furfur (1846); and also to the Oidium lactis. MEMORANDA. 188 MONILIA CANDIDA. Robin (1847). SYNONYMS. THRUSH FUNGUS; OIDIUM ALBICANS, SACCHAROMYCES ALBICAXS. SOORPILZ (Germ.}. Origin. Found in the mouths of infants ; in thrush. Form. Occupies an intermediate position between the moulds and yeasts. On gelatin plates and on sugar media it forms yeast-like cells, whereas in the deeper part of the stich culture it forms mycelial threads. Anilin Dyes. Stain readily. Growth. Is rapid and abundant. Plates. Snow-white colonies form on gelatin plates and no liquefaction takes place. Stich Cultures. In gelatin show growth along the line of inoculation, while on the surface a milk-white, thick mass forms. Streak Cultures. On agar, forms a glistening, moist, thick, white growth. On potatoes, it grows rapidly as a thick, white, yeast-like mass. Temperature. Grows at ordinary temperature, also in incubator. Behavior to Gelatin. Does not liquefy. Pathogenesis. Intravenous injection in rabbits produces death in 1-2 days. The internal organs are per- meated with a growth of long mycelial threads. MEMORANDA. MEMORANDA. MEMORANDA. MEMORANDA. 189 SPECIAL WORK. Direct microscopical examination of streak prepara- tions made from the organs and tissues of infected ani- mals, as well as cultural experiments will reveal the pres- ence of microorganisms. In order to ascertain the presence and especially the distribution of organisms within tissues and organs it is necessary to harden these, then to cut sections and finally to stain the sections by suitable meth- ods. Hardening. For this purpose alcohol is usually employed and gives excellent, results. The tissue is cut into small pieces which are either transferred direct to a wide-mouthed bottle containing 92-96 per cent, alcohol, or are first placed on pieces of filter paper and then in the alcohol. The pieces of tissue should remain in this alcohol for at least 3 or 4 days, or until it is desired to make sec- tion?, when they are transferred to absolute alcohol for one or two days. The tissue is then hardened and ready for cutting sections. To do tljis a piece of the tissue is at- tached to a small cork by means ot a glycerine gelatin mixture made by warming 1 part of gelatin, 2 parts of water and 4 parts of glycerine. The cork is then securely clamped to the microtome and sections made. The tissue and knife must be kept moist with alcohol and the sec- tions are at once transferred to alcohol by means of a camel hair brush. Very satisfactory and rapid hardening can be obtained with a solution of mercuric chloride made by saturating an aqueous five per cent, glacial acetic acid solution with mercuric chloride. The tissue can be fixed in this solution in 4 to 12 hours. It is then passed through a series of 190 alcohols of different strength, 60, 80, 96X and absolute, in each of which the tissue remains for 24 hours. Cutting sections. As already stated the tissue which has been hardened in alcohol may be cut directly and sections can thus be obtained which are fairly good. Another method for obtaining sections is to employ the freezing microtome, in which case the alcohol is first re- moved from the tissue by placing the pieces in water. This is accomplished in cold water in 4-8 hours, depend- ing: on the size of the pieces. Warm water about 38 C. will remove the alcohol more rapidly, in 1 to 2 hours. The tissues can then be frozen and sections cut. The knife should be kept moistened with water and the sec- tions are transferred at once to water. Undoubtedly the most satisfactory method of prepar- ing sections is to first imbed the hardened tissue either in celloidin, or in paraffin. Either method gives excellent results. The method for imbedding in paraffin which is usually employed in the laboratory is briefly as follows : The hardened tissue is placed in absolute alcohol for 24 hours; then for 4 to 5 hours in chloroform, then in a chloroform-paraffin solution over night. The pieces are then placed in paraffin which melts at about 42 C. and kept in a water air-bath at a temperature of about 50 for 24 hours. From this the tissue is transferred to hard paraffin which melts at about 48 C. This may be obtained by taking equal parts of 42 and 56 paraffin. After 24 hours the piece of tissue is "blocked.-' . Two glass L's are fitted together so as to make a trough of suitable size, and this then filled with melted paraffin (48 C.) The piece of tissue is transferred by means of a pair of forceps, slightly warmed, to the center of the block and the whole allowed to cool. The solid block, if necessary, is then trimmed, fastened to the microtome and sections cut with a dry knife. From absolute alcohol the pieces of tissue can be placed first in toluene for 24 hour,s; then in a mixture of MEMORANDA. MEMORANDA. 191 equal parts of toluene and paraffin for 24 hours and then in paraffin for 24- hours. The paraffin can be dissolved from the sections by means of xylol or turpentine. The sections are then placed in absolute alcohol and finally transferred to 10% alcohol, in which they may be kept for any length of time and are now ready for staining, Paraffin sections sometimes tend to curl or become folded. This difficulty can be readiiy overcome by plac- ing the sections in a Petri dish containing tepid water. This must not be so warm as to melt the paraffin. The dish may be kept on an iron plate which is heated gently at one end. The sections spread out on the surface of the warm water. They can be received on strips of paper and transferred to a glass-slide or cover-glass which is covered with a film of albumin. The section is then dried with a piece of paper and caused to adhere by slightly warming the glass slide. It can then be stained in the usual man- ner. The method is very convenient in working with very thin sections or with very delicate tissues. (BORREL). Staining of Sections. The presence of organisms in sections of tissues is sometimes very difficult to demon- strate although they may be easily shown to be present in ordinary streak cover-glass preparations. This is fre- quently due to the absence of any sharp means of differ- entiating the organism from the surrounding tissue. In many cases, however, a sharp differentiation can be ob- tained by double staining either by Gram's method, or, as in the case of leprosy and tuberculosis, by the Triplication of the usual process for staining these bacilli. Simple stain. The student should begin with c ec- lions of the spleen, kidney and liver of a guinea-pig which died of anthrax. The sections are transferred by means of a needle to the dilute anilin .dye, as fuchsine or gentian violet and are allowed to remain there for 10 to 30 minutes. They are then transferred to water slightly acid- ulated with acetic acid, or to very dilute alcohol. This is 192 done to remove the excess of dye and to differentiate the bacteria in the tissue. From the decolorizing solution the section is transferred to water then, by means of a spa- tula, to a glass slide and examined with a No. 7 objective. If the section is still too intensely stained it should be re- turned to the decolorizing agent and after a while again examined. When satisfactorily stained the sections should be placed in absolute alcohol, for a few seconds, till thoroughly dehydrated, they are then cleared up in oil of cloves, cedar or anise; placed in xylol and examined on a slide. If satisfactory the cover-glass is carefully lifted off and the xylol removed from the section by means of a piece of filter paper. A drop of Canada balsam is then applied to the section and the whole covered with a clean cover-glass. The exposure to absolute alcohol and to oil of cloves should be carefully watched as both tend to re- move the stain. The oil of cloves can indeed be relied upon to remove any excess of dye that may be present. Instead of the ordinary dilute anilin stain, Lo (Tier's methylene blue (page 140) or ZiehFs carbolic fuchsine (page 110) can be employed in special cases to excellent advantage. A dilute ZiehTs solution gives particularly good results. The sections remain in this for about half an. hour and are then transferred to absolute alcohol which is very slightly acidulated with acetic acid. As soon as the color changes to a peculiar reddish violet tint the section is removed, cleared up in xylol, examined and mounted in balsam. (Pfeiffer's method.) Double stain Gramas method. Those microorgan- isms which can be stained by this method can be readily detected in sections, as the preparations when properly made show the heavily stained violet bacilli on a light pink background. The student should begin with sections of the kidney of the anthrax guinea-pig. A strong solution of anilin water gentian violet is prepared according to the directions given on page 106. It should be warmed slightly on the radiator, or on an iron plate. The sections are placed MEMORANDA. MEMORANDA. 193 in this slain for 15 to 30 minutes. They are then washed in anilin water to remove excess of the dye, and thus to prevent the formation of unsightly deposits on subsequent contact with iodine. The sections are then placed in the solution of iodine in potassium iodide (p. 107) for 2 or 3 minutes. From this they are transferred to absolute alco- hol and gently moved about till most of the stain is re- moved. The sections should be still slightly stained, not completely decolored. They are then placed in Weigert's picrocarmine solution or in eosine, for 1-2-3 minutes, de- hydrated in absolute alcohol for a few seconds and then transferred to oil of cloves in which the sections are al- lowed to remain till all the gentian violet lias been re- moved. Oil i/f cloves, especially when dark colored, has strong decolorizing properties and will remove all traces of gentian violet from the tissues without affecting the bacilli to any extent. The sections are then placed jn xylol and examined and if satisfactory mounted in Can- ada balsam. ANTHRAX SECTIONS. Simple Stain: Gra'tns Method: Dilute anilin stain Anilin water-gentian violet (10 to 30 min.). (warirr 15 to 30 min.). Acetic water. Iodine in potassium iodide Water (and exam- (2-3 min.). ine). Absolute alcohol. Absolute alcohol Picrocarmine (1-2-3 min.). (few seconds). Absolute alcohol (few Oil of cloves. seconds). Xylol (and exam- Oil of cloves (till violet ine). ceases to be given off). Canada Balsam. Xylol (and examine). Canada balsam. Malignant oedema. Prepare sections from the ab- dominal wall, kidney and liver of a guinea-pig which died after inoculation with the bacillus of malignant oedema. 194 The tissues and organs should be removed 48 hours after the death. Stain with dilute carbolic fuchsine according to Pfeiffer's method as given on page 192. Symptomatic anthrax. Prepare sections from the same tissues as above from a guinea-pig which was not ex- amined till 48 hours after the death. Stain by the same method and compare the two organisms. Bacillus cedematis maligni, No. II. Section the thickened abdominal wall and stain after Gram's method. Tubercle bacillus. Prepare sections of tubercular human lung, also of the spleen, liver, and mesenteric tu- bercles of a guinea-pig inoculated with tubercular spu- tum. Stain the sections according to the following method which is a modification of the Ziehl-Neelsen method. The sections are placed in Ziehl's carbolic fuchsine, slightly warmed, for 15-30 minutes. They are then trans- ferred to Ebner's solution where they are moved about till the color ceases to be given off. The sections should still possess a slight pink color. They are then placed in dilute methylene blue for ^-1 minute. From this they are trans- ferred by means of a spatula to absolute alcohol for -J-l minute. The sections must not remain in the alcohol till all the blue disappears. They are then placed in oil of anise, transferred to xylol and examined. If satisfactory the section is mounfed in Canada balsam. Ebner's decalcifying solution is prepared according to the formula: Sodium chloride 0.5, hydrochloric acid 0.5, alcohol 100, distilled water 30. Instead of using Ebner's solution for decoloring the tis- sues a 2 per cent, aqueous solution of anilin hydrochloride can be employed with excellent results as it has little or no tendency to decolor the tubercle bacilli. (KiiHNE, BORREL). The sections are stained in Ziehl's solution as above, then placed for a few seconds in the 2 per cent, aqueous solution of anilin hydrochloride, then washed in alcohol and counter-stained as above. Leprosy bacillus. Sections of the skin of a leper MEMORANDA. MEMORANDA. 195 can be double stained according to the method described for the tubercle bacillus. It can be summarized as fol- lows : Sections. Carbolic fuchsine (warm 15-30 min.). Ebner's solution (till it turns a light pink). Dilute methylene blue (-J 1 min.), Absolute alcohol (-J-1 min.). Oil of anise. Xylol (and examine). Canada balsam. Leprosy sections left in dilute alcohol soon lose their capacity for double staining. They can be simple stained with methylene blue, or with carbolic fuchsine by Pfeif- fer's method. Glanders bacillus. The detection of this bacillus in tissue i? rather difficult owing to its marked peculiarity of readily becoming decolorized. Sactions from the spleen, of a guinea pig should be simple stained with Lo filer's alkaline meihylene blue (p. 140) or with carbolic fuchsine. Typhoid fever bacillus. The E berth bacillus al- though it is siained readily and intensely is very likely to become decolorized in the ordinary method of staining. Sections of human spleen are stained in Loffler's alkaline methylene blue for 24 hours. Then washed and decolored in water, dehydrated in anilin oil, allowed to dry on a slide and finally cleared up with xylol. Simple stains can be made with carbolic fuchsine, decolorizing carefully in acid- water and alcohol. Frankel's diplococcus. Sections from the lung, spleen, liver etc., of a rabbit can be stained by Gram's method, Loffler's diphtheria bacillus. Sections of diph thentic membranes, or of muscles from the neighborhood of the point of inoculation should be stained with Loffler's alkaline methylene blue, or by Gram's method. 196 Staphylococcus pyogenes aureus.- This organism also the Streptococcus pyogenes can be detected in sections of the kidney, suprarenal body etc. in pyaemia of man, or in the organs of rabbits inoculated with pure Cultures. The sections should be stained by Gram's method, or with carbolic fuchsine. Chicken cholera bacillus. This can best be de- monstrated in sections of the liver, spleen and pectoral muscles of a pigeon. Simple staining with carbolic fuch- sine or anilin water gentian violet will give fair results. Micrococcus tetragenus. The kidneys, lungs etc. of white mice and guinea-pigs give excellent preparations when stained by Gram's method. MEMORANDA. OF THE ( UNIVERSITY ) OF MEMORANDA. 197 TESTING OF DISINFECTANTS. In studying the action of physical and chemical agents on bacteria it is necessary to rigidly adhere to certain re- quirements without which the results would be of little value, if not wholly contradictory. The conditions which underly.the testing of disinfectants may be summed up as follows. (1 ). Variable resistance of spores and of the vegetat- ing forms of one and the same organism. It has been shown in recent years that considerable variation may exist in the resistance which an organism possesses to destruc- tion. Thus, while there are some spores of anthrax which are readily destroyed by steam-heat, 100 C., others have been known to withstand this temperature for 10-12 min- utes. Again it was formerly stated that anthrax spores were destroyed by 5 per cent, carbolic acid in two days but the researches of Fraenkel have shown that spores of anthrax may be had which are not destroyed by an ex- posure of 30 to 40 days. In view of these facts several standards have been proposed. Thus Fraenkel designates anthrax spores which are destroyed by 5 per cent, carbolic in less then 10 days as feebly resistant; in 10 to 20 days as of average resistance; in 20 to 30 days as very resistant; in 30 to 40 days as extremely resistant. Geppert's stand- ard an fchiMX sp)t35rir e those which are infectious after boiling for one minute 1 c. c. of a spore suspension which is added to 30 c. c. of boiling water. Esmarch has sug- gested as a standard anthrax spores which when fixed on silk threads resist steam-heat of 100 G. for 10 minutes. (2). The influence of the medium in which the organ- ism is tested. Thus it has been shown that to destroy an- thrax spores in bouillon it requires'20 tin)3s as much mer- 198 curie chloride (1-1000) than when they are suspended in water, and 250 times as much when they are distributed in blood serum. (3). The temperature at which the disinfection is made. The higher the temperature at which the experi- ments are made the more rapid and energetic will be the action of the disinfectant. Cholera bacteria are not de- stroyed bymercuric chloride (1-1000) in one hour at 3, whereas at 36 C. they are killed in a few minutes. (4). Immediate and thorough contact of all the or- ganisms present with the disinfectant. This can be done perfectly only with bacterial suspensions in which each or- ganism is entirely free and separate from others. To ob- tain such a suspension it is necessary first to filter through glass wool and then to agitate the liquid thoroughly at a temperature of about 37 until microscopical examination shows no aggregations of bacteria. Silk threads which have been soaked in bacterial suspensions and then dried are open to the objection that on exposure to the disinfectant organisms are unequally exposed and some even protected by their position and hence when transplanted soon de- velop. The same objection, to a less degree, applies to cover-glasses on which a thin film of the suspension has been deposited. (5). The number of bacteria in a given experiment. It can readily be shown that the greater the number of bacteria present the more slowly does the disinfection take place. In order therefore that results may be com- parable, approximately the same number of organisms should be present in each experiment. This is readily as- certained by diluting a small portion of the bacterial sus- pension witli 1-2000 parts of sterilized water and then making a gelatin plate with one drop of this dilution. (6). The amount of the disinfectant which is carried over in each trial inoculation. Thus, when the disinfect- ant is applied to the bacterial suspension and at the end of stated intervals transfers of 1-3 loopfuls of the mixture MEMORANDA. MEMORANDA. 199 are made to sterilized nutrient media, a sufficient amount of the disinfectant may be carried over to prevent the growth of the organism which mny still possess vitality. This has been a most serious source of error in the past. The error is more marked, the greater the antiseptic power of the disinfectant. It is of course less marked where the substance has weak antiseptic properties and where the transplantation occurs into relatively large amounts of the nutrient medium (10 to 15 c. c.). It must be remembered that probably in all cases the first action of a disinfectant is to attenuate the organism and that when the latter is in this condition a much smaller amount of the disinfectant will act as an antiseptic and prevent growth. This has been especially shown to be the case with reference to the action of mercuric chlo- ride on anthrax spores. Formerly it was disposed that these were killed by this substance in a strength of 1 to 1000 in one minute but if the mercury which is held fast by the silk thread, and which cannot be removed by mere washing, is removed by the action of hydrogen sulphide it can be shown that the organism is alive and infectious even alter an exposure of four hours. It may even possess vitality alter an exposure of 24 hours. The first action of the disinfectant in this instance is to attenuate the organ- ism the growth of which is then prevented by mere traces of the mercury. One part in two million according to Geppert suffices to produce this result. (7). Observation of the trial inoculation tubes over a considerable length of time. The failure of tubes to grow within 24 hours is not a positive indication that the organ- ism has been destroyed by the disinfectant. In the at- tenuated condition the organism will grow much more slowly then it would if normal and in possession of full vi- tality. Moreover, as stated already, traces of the disin- fectant which are carried over in the experiment will still further tend to retard the growth. For these reasons the 200 tubes should be kept under observation for 1 2 3 weeks before definite conclusions can be drawn. (8). Temperature at which the trial inoculation tubes are kept. The organism which has been exposed to the action of the disinfectant should be placed under con- ditions which are most favorable to its growth. That is, the best nutrient medium and the most suitable tempera- ture should be furnished. Transplantations made into gel- atin and kept at ordinary room temperature frequently fail to grow while parallel bouillon and agar cultures, kept in the incubator,develop. It is therefore desirable to make the transplantation onto the surface of inclined agar tubes or into bouillon and to keep the tubes under observation at a temperature of about 37.5 C. for two or three weeks. (9). Negative experiments with animals inoculated with organisms exposed to heat, or to the action of chemi- cals prove nothing. The organism may be dead or it may have become attenuated and is therefore without action, although it may still grow on artificial media. Thus, an- thrax spores exposed to the boiling temperature for 2 min- utes no longer kill guinea-pigs, but nevertheless can grow in tubes, even after 5 minutes exposure. Again, positive experiments may be obtained by inoculating white mice or guinea-pigs with the mixture of bacteria and disinfectant at a time where transplantation of a corresponding amount on a nutrient medium fails to grow owing to the antiseptic power of the disinfectant carried over. METHODS FOR TESTING DISINFECTANTS. (1). . Silk threads. This method was introduced by Koch and has been extensively used. Threads of silk, linen, or cotton are cut up into lengths of about 1 cm. They are placed in a sterilized plugged test-tube and sterilized in the dry-heat oven. A cloudy suspension of the spores or bacteria to be tested is made in sterilized water. The pieces of sterilized threads are immersed in this suspen- sion for some minutes, then transferred with sterilized for- MEMORANDA. 18 MEMORANDA. 201 ceps to a sterilized Petri dish and allowed to dry. They can then be placed in a test-tube and kept for future use. To ascertain the disinfecting action of a solution a thread impregnated with the bacteria to be tested is im- mersed in it fora given length of time, as for instance 2 minutes. It is then removed with sterilized forceps and gently washed in sterilized water or alcohol. Finally it is transferred to a tube of nutrient bouillon (10-15 c. c.) and then set aside in the incubator for a week or more. Simi- lar tests with exposures of 2, 5, 10, 3.0, and 60 minutes should be made. The objections to this method are twofold and have already been incidentally mentioned. In the first place the bacteria on the thread may not be evenly exposed to the action of the disinfectant and secondly the disinfec- tant itself may be transferred to the nutrient medium. The attempt is made to obviate the latter objection by washing the threads and while this may be successful in some cases, in others it fails. Thus mercuric chloride is apparently held fast by the fibre and can only be re- moved by the action of hydrogen sulphide (GEPPERT). (2). Cover glasses. This method was introduced by Geppert and has been used bySpirig and others. Ordinary microscopic cover-glasses are cut in two, cleaned and rendered free from far, and finally sterilized. They are then immersed in the bacterial suspension, or in bouillon cultures, transferred to a sterilized wire gauze, under a bell jar, and allowed to dry. To test a disinfectant a dry cover-glasses is immersed in it for a given length of time as in the case of the silk threads. It is then removed with sterilized forceps and washed in about 400 c. c. of sterilized water for about -|-| hour. Then placed in sterilized bouil- lon and set aside in the incubator. The advantages of this method are (1) that a thin film of evenly spread bacteria is employed, and (2) that the cover-glass does not unite with the disinfectant, as is the case with the silk threads. It is open to the objection, 202 which holds true also for the silk threads, that the process of desiccation tends to lower the vitality of the organism. Furthermore it may be urged that the disinfectant has not free access to all sides of the bacteria. (3.) Bacterial suspensions. This method in some of its modifications is the one which is commonly employed and, if used with proper precautions, yields perfectly reli- able results. The first essential is to secure a suitable sus- pension of the organism to be tested. For this purpose the fresh growth on the surface of 3 or 4 agar tubes is care- fully removed and thoroughly rubbed up in about 10 c. c. of sterilized distilled water. In order to remove the coarse floccules the suspension is filtered through glass wool, and the filtrate immersed in a water-bath at 37.5 C. and fre- quently agitated till a microscopic examination shows no longer the presence of groups or masses of bacteria. In this suspension now the number of bacteria present can determined as already stated. By means of a sterilized pipette, graduated in 1-10 c. c., an exact volume, 3 c. c., is transferred into each of several sterilized test-tubes. To the suspension in one of these tubes an equal volume of the disinfectant, of double the strength to be tested, is added. At intervals of 2, 5, 10. 20, 30, 60 minutes etc. transfers are made to sterilized bouillon or agar tubes and these are then set aside in the incubator for at least one week. The inoculations should be made in duplicate and 2 or 3 loopfuls used for each tube. The method as given is open to the objection that an appreciable amount of the disinfectant is transferred each time to the culture tubes and that it may prevent growth. This is specially true with substances which possess marked antiseptic properties, as mercuric chloride. Where possible, the disinfectant should be rendered inert. Thus, traces of mercuric chloride can be removed by precipita- tion with hydrogen sulphide. With other substances the error is not so marked and is partly counterbalanced by MEMORANDA. MEMORANDA. 203 growing the tubes in the incubator for many days. (SCHAFFER.) LABORATORY WORK. The student should test, by the methods given, the disinfecting action of mercuric chloride (1-1000), carbolic acid (5 per cent), hydrochloric acid (0.4 per cent.). Anthrax bacillus, anthrax spores, staphy- lococcus pyogenes aureus, cholera, typhoid fever and dipththeria bacilli may be used, suspended in distilled water, bouillon and blood-serum. The blood-serum required in this work may be readily prepared as follows: The flowing blood from an ox or calf is received into a large sterilized Erlenmeyer flask and when firmly clotted is carried to the laboratory and placed in the ice-chest. After 24 48 hours the clear yel- low serum separates out. A portion of this may be placed in a beaker, diluted with 510 parts of distilled water, then filled into tubes and sterilized as in the case of or- dinary bouillon. The dilution with water prevents coagu- lation of the blood-serum. Another portion of the blood- serum may be filled direct in tubes which are then placed in an inclined position in an air-bath and the temperature slowly raised to about 80 C. which is maintained for about one hour. The serum coagulates in the inclined position. On the following day the tubes should be steri- lized in the steam sterilizer for ^ 1 hour. To obtain undiluted fluid blood-serum the blood is re- ceived directly from the artery or vein into sterilized jars or flasks which are protected against contamination from the air. As soon as the serum separates it is transferred by means of a sterilized pipette to the sterilized tubes. When this is properly done no organisms are introduced into the serum and hence it requires no subsequent steri- lization. >i, OF THE UNIVERSITY of MEMORANDA. MEMORANDA. MEMORANDA. 205 List of Apparatus and Aecessorie: 1 1 1 6 1 1 2 12 12 6 H 1 100 100 3 12 12 8 200 50 100 3 100 4 1 Flask, 2 litre. I " " y* " " 50 c.c. Erlenmeyer. Funnel, 15 cm. diameter. 6 " Moist Chain hers. Esma'ch Dishes. Petri Dishes. Staining Di>hes, with covers. Watch Glasses, 5 cm. diarn. Test Glass. 18cm. high. Test Tubes, 150x14 mm. 125x12 " Tumblers. Glass Plates. Glass Benches. Glass Rods, 18 cm. Cover Glasses, No. 1, % in. diarn. Glass Slides. Concave Slides. Labels for Slides. Slide Boxes. Slide Disinfecting Jar, with top, Six 10 cm. Disinfecting Jar, with top, 15x20 cm. Stain bottles, 1 oz. with pipettes, in stand. Glass Pipettes, 1 c.c. with iron box. Woltfhii^el Colony Counter. Novy Bottle for Anaerobic Tube Culture. Novy Anaerobic Plate Apparatus. Cylinders, graduated, 25, 100, 1000 c.c. Bottles, 2 oz. Waste Dish. Wire Gauze. Wire Basket, large 18x18x24 cm. " " small 10x12x18 cm. Iron Sterilizing Box,5xl4Uxl7V cm Bunsen Burner, with tubing. Test tube Stand, for 48 tubes. Support Board, large, 25x25 cm. " small, 8x20 cm. Platinum Wires, No. 23, 5 cm, Pair Pincers, narrow pointed, 10 cm. Pair Scissors, 14 cm. Scalpel. Potato Knives. Colored Wax Pencil. Potato brush. Iron Water-bath, with tripod, 18 cm. diam. Wash-bottle, siphon or bulb. Ice apparatus for plates. Battery Jars, 11x11 cm. Rat Jars, with leaded top. 1 Crucible Forceps. 1 Chapman Aspirator. 1 Kipp's hydrogen generator. 1 Koch steam Sterilizer, with crown ourner. 1 Dry Heat Sterilizer, with crown burner. 1 Incubator, with safety lamp. 2 Thermoregulatorss. 1 Thermometer, 200 C. 1 " 00 C. 1 Microtome. 1 Microscope, with 3 objectives, %, 1-0, and 1-12; 2 eyepieces; Abbe condenser and iris diaphragm. 1 Roll of Cotton. 12 Sheets of Filter Paper. Rubber caps. Fnchsine. Gentian Violet. Methylene Blue. Methyl Violet. Bismarck Brown. Kpsine. Picrocarrnine, Weigert's. Anilin Oil. Anilin Hydrochloride. Iodine. Potassium Iodide Mercuric Chloride. Carbolic Acid. Sulphuric Acid. Nitric Acid. Hydrochloric Acid. Acetic Acid. Pyrogallic Acid. Tannic Acid. Ferrous Sulphate. Sodium Carbonate. Sodium Hydrate. Ammonium Hydrate. Oil of Cloves. Oil of Cedar, Oil of Anise. Xylol Alcohol. Ether Chloroform. Paraffin, 40, 46, 52, 56 C. Collodium. Celloidin Sealing Wax Tube of Canada Balsam, Gelatin, silver. Agar-Agar. Peptone^ sice. Witte. Glucose. Glycerine. Litmus. Vaseline. Extract of Meat. ERRATA. Page 15. Above the fifth line from the bottom insert: Classification according to oxygen requirements aerobic and anaerobic. Gradations in requirements facultative and obligative. Page 122. On the 23rd. line read "Cultures in" Page 123. First line, read "Culture" instead of "Cultures." MEMORANDA. INDEX. Abbe condenser. 12. Achorion. 126, 186. Aetinomyces, 126. 184. Aerobic bacteria, 204. Aerogenic bacteria, 17. Agar, nutrient, 95. plates, 136. " roll-tubes. 136. " streak cultures. 126. Air, 77. Alkaline methylene blue, 140. Amoeba coli, 174. Anaerobic apparatus for tubes, 124. " *' " plates, 125. Anaerobic bacteria, 116-122, 204. * culture of, 123. Anilin water, 106. " fuchPine, 128. " gentian violet, 106. " hydrochloride, 130, 194. Animal inoculations, 101, 200. Animal parasites, 100. Anthrax bacillus, 114. " sections, 191. " work with, 104. Arthrospore, 10. Asiatic cholera, 148, 158. Aspergillus flavescens, 88 " furnigatus, 90. " niger, 88. Asporogenic bacteria, 10. Attenuation, 100, 176, 199, 200. Bacillus, 5 " acidi lactici, 66. " anthracis, 114. " butyrieus, 68. " of chicken cholera, 176. ' coli communis, 158. " cyanogenus, 70. " diphtheria?, 140. " fluorescens putidus, 44. of Friedlaehder, 144. ' of hog cholera, 178. " of hog erysipelas, 180. Indicus, 36. " leprae, 134. mallei, 138. " mega teri urn, 56. " mesentericus vulgatus, 54. " murisepticus, 182. " Neapolitanus, 156. " oedernatis rnaligni, 118. Bacterium, 5. coli commune, 158. " of hog cholera, 178. phosphorescens, 46. " terrno, 60. " Zopfli, 62. Black-leg, 116. Blue milk, 70. Blue pus, 168. Blood-serum, 203. Botkin apparatus, 124. Bouillon, 95. Bread flasks, 78. Buchner's method, 124. Butyric acid, 17, 68, 120. Calcium hydrate agar, 103. Capsule, 7, 142, 144, 172. Carbolic fuchslne, 110. Caries, dental, 18, 66. Cedar oil, 12. Cell wall, 7. Charbon, 114. Cheese spirillum, 152. Chemistry of bacteria, 17. Chicken cholera, 154, 176. 196. " tuberculosis, 132. Chlorophyll, 7, 15. Cholera, Asiatic, 148, 158. " of chicken, 154, 176. " nostras, 150. Chromogenic bacteria, 17. Classification, 5, 15, 17, 18, 94, 204. " of plants, 94. Clostridium, 10. Colony, 9, 21,97. " examination of 25. Comma bacillus, 148. Concave slide, 13. Condenser, Abbe, 12. Cover-glasses, 12. " " preparation, 19, 106. " " for disinfection, 201. Croupous pneumonia, 142. Cutting sections, 190. Deep layer cultures. 123, 125. Deneke's bacillus, 152. Diaphragm, Iris, 12. Diphtheria, 140. sections, 195. Diplococcus, 11. of pneumonia, 142. Disinfection, methods of, 197, 200. No. II, 120.Drum-stick forms, 10. prodigiosus, 34. Eberth's bacillus, 160. pyocyaneus, 168. Ebner's solution, 194. ramosus, 58. Emmerich's bacillus, 156. of rhinoscleroma, 146. Endocarditis, 164, 166. rnber of Kiel, 38. Endospore, 10. rubidus, 40. Enzyme, 17. subtilis, 52. Erysipelas, 162. 164. of symptomatic anthrax, 116. Escherlch's bacillus, 158. tetani, 122. Esmarch potato culture, 30. " tuberculosis, 132. Esmarch roll culture, 30. " typhi abdominalis, 160. Facultative, 15, 204. " violaceus, 42. Farcy, 138. Bacteria, 5, 94. Favus, 126, 186. 208 Fermentation, 17. Kinkier-Prior's bacillus, 150. Fhigellu, 7. ' staining of. 126. Flnoresfriuw bacillus. 44. Frankel'sdiplococrus. 142, l'.5. FnetJleenUers baeiliu.-, 144. Fungi, 5, bjectives, 12. (Edema, malignant. 118, 120. Oidium albicans, 88. lactis, 72. Orange sarcine. 48. Osteomyelitis, HJ ,. Paraffin sections, 190 Parasitic bacteria, 15. Pathogenic bacteria, 18. 97. Penicillium glaucum, 82. Pericarditis, 142. Peritonitis, 142. Petri dish cultures, 29. Pfeiffer's method, 192. Phagocytes, li.l. l > hosphore^cence, IS, 46. Photobacterium, 46 Phoiogenic bacteria, 17. Pigment, 18,04. Pityriasis, 180. Plasmodium, 174. Plants. 94. Plates, sterilization of, 22, 29. Plates, culture on, 21, 29, 130. Pleuritis, 142. Pneumococcus, 144. Pneumonia, 142, 144, 195. Poisoned arrows, 110, 1 18, 122. Post-mortem examination, 103. Potato bacillus, 54. cultures. 7, 30. " Esmarch cultures, 30. " tube culture, 31. Precautions. !, 102, 101. Proteids, 17. Proteus vulgaris, 00. Ptomaines, 17. Puerperal fever, 161. Pure culture, 9, 21,26, 97. Putrefaction, 18. Pya>miu, 104 sections. 196. Pyrogallate met hod, 124, 125. Quarter-evil, 1 10. Kag-picker'-s disease. 114, 118. Kay-fungus, 184. Recurrent lever, 171 Red bacillus of Kiel, 38. ". \vater, 40. Ked yeast, 92. Relapsing lever, 174. Reproduction, of bacteria, 10. Khinoscleroma, 146. Roll culture, agar, 136. gelatin, 30. Root bacillus, 53. Rouget, J80. 209 Rules of Koch, 98. Saccharomyces, 79, 188. Sjiliva. 21, 30, 142, 144, 172. Saprogenic bacteria, 17. Saprophytic bacteria, 15. Sarcine, 11. " orange, 48. " yellow, 50. Sections, "89. " Gram's stain, 192. " simple stain, 191. " tubercle, 194. Septicemie, 118. Silk threads, 198,200. Soil, 7(i. Spirillum, 5. " Oberrneieri, 174. rubrurn, 64. " tyrogenum, 152. Spirocheete, 174. Splenic fever, 114. Spontaneous generation, 15. Spores, 10, 109, 197. Spores, double stain, 109. Sporogenic granules, 10, 105. Sporozoa, 100. Sputum, 130, 142, 144,172. Sputum septica3mia, 142. Siaining coverglasses, simple, 19, 106. Gram's, 106. " tubercle, 130. Staining sections, 191. Stains, 19. Staphylococcus. 11. " pyog. albus, 166. " aureus, 166, 196. " citreus. 166. Sterilization of media, 6, 8, 78, 96, 203. " plates, 22, 29. " " tubes, 6. Stich cultures, 27. Streak cultures, 32, 126. Streak preparations, 106. Streptococcus, 11. erysipelatis, 162. pyogenes, 164, 196. Summer diarrhoea, 18. Suppuration, 162-172. Suspensions for disinfection, 202. Swine plague, 178. Symptomatic anthrax, 116, 194. Temperature. 15, 198. Testing disinfectants, 200. Tetanus, 122. Tetrads. H. Threads, 11. Threads for disinfection, 198. Thrush, 1 88 Toxicogenic bacteria, 18. Tricophyton, 186. Tubercle bacillus, 126, 130,132. sections, 194. ; sputum, 130. Tuberculin, 1&2. Tuberculosis of chicken, 132. Typhoid fever, 160, 195. TTrine, fermentation of, 18. Vficuum cultures, 123, 125. Vibrio, 5. of Asiatic cholera, 148. of Deneke, 152. of Finkler-Prior, 150. Metchnikovi, 154, 176. proteus, 150. Vibrion butyrique, 68. Vibrion septique, 118. Violet bacillus of water, 42. Water, 73. Whips, 7. Wool-sorter's disease, 114. Wurzel bacillus, 58. Yeast, 5, 94. " baker's, 79. " black, 79. " red, 79, 92. " white, 79. Yellow sarcine, 50. Ziehl-Neelsen method, 130. Ziehl's solution, 110. Zoogloea, 7. Zymogenic bacteria, 17. MEMORANDA. BOOK T ,. ^ 132699 JUBRARY G i THE I r " * fI70IJ TBRARY