COLUMBIA DKlYERSlfl. COLLEGE OF FHYSICIANS ffi& SURGEONS, DEPARTLiENT OF BACTERIOLOGY. ~'~ rt «" "}v Jt" ,c ",~ />• R -^ Laboratory Manual in Medical Bacteriology /M-&K63 __ Columbia (Bntoetsitp intljeCttpoflfttigmrk College of iPjjpgtctang anb burgeon* Htfcrarp Digitized by the Internet Archive in 2010 with funding from Open Knowledge Commons http://www.archive.org/details/laboratorymanual01colu Laboratory Manual IN Medical Bacteriology Edited by the Staff of the Department of Bacteriology College of Physicians and Surgeons Columbia University New York 1929 Kfc$R&5 ill mi Columbia Stotoetsitp College of $fjp£irian* anb gmrseons library LABORATORY MANUAL IN MEDICAL BACTERIOLOGY by the Staff of the Department of Bacteriology Third Edition College of Physicians and Surgeons Columbia University New York Copyright 1929, by Frederick P. Gay Published by JAMES T. DOUGHERTY 427 W. 59th Street N. Y. ^2*5£^ /9£f c,/ INTRODUCTION Bacteriology as a general science is a subdivision of Botany. Bacteria may be studied taxonomically as plant forms, or practically in their relation to various industrial processes. The overwhelming importance of certain bacteria as the agents of diseases in plants, animals, and man have set aside the pathogenic bacteria as a spe- cialized group around which has been created one of the most im- portant branches of medicine. Medical bacteriology, viewed from another angle, is the logical introduction to Pathology which under- lies the whole practice of medicine. Pathology is the science of disease. In its larger sense the term may be used to designate a group of closely correlated medical sciences and also the applied aspects of these sciences in the recog- nition and treatment of disease. The function of instruction in Pathology is to serve as an introduction to the study of clinical medicine, to outline the abstract conceptions of disease processes, to point out the variations from normal function and structure which occur in the animal body as the result of disease. The complete consideration of any particular disease would logically consider, first, the cause of the disease, secondly, its course, and thirdly, its effect on the normal body. We find that dis- eases are due to three general causes : ( 1 ) external inanimate agents, (2) external animate agents, and (3) a set of causes as yet unknown. The diseases due to external animate agents, oi the infectious diseases, as they are called, comprise about one-half of all disease entities, and are the cause of approximately one- half of the deaths. The infectious diseases, therefore, are nu- merically important and possess the additional advantage of afford- ing facilities for study m their three successive phases, as already outlined. The microorganismal causes of disease can be, for the most part, isolated outside the animal body and studied separately (bacteriology and protozoology). Their effects can be watched in the animal body and the changes induced therein studied (in- fection and immunity) and finally the resultant effects or lesions of the diseases they produce can be examined (morbid anatomy and histopathology). In the diseases due to external inanimate agents and in those due to unknown causes, the origin in specific instances is not always recognizable and the sequence of events is frequently obscured. The final results as measured by metabolic and struc- tural changes are, however, rather more striking than in the in- fectious diseases. Many of the effects produced can be simulated by the methods of experimental pathology. These latter studies in the experimental animal serve, moreover, as an introduction to the more extensive investigations of diseased function and structure that will be taken up in hospital work. This syllabus outlines a laboratory course in: (1) The methods of general bacteriology. The principles of asepsis, sterilization, preparation of culture media. The isolation in pure culture and identification of various saprophytic bacteria with some conception as to the ubiquity of these micro-organisms in life. The biochemistry of bacterial growth. (2) An intensive study of the commoner and more important bacteria that cause disease in man and animals. (3) The reproduction experimentally in animals of certain in- fectious diseases by inoculation of their respective bacterial agents (Infection). (4) A study of the defense mechanism in the animal body against bacteria (Immunity). The laboratory work is presented in the form (1) of exercises which state the more important technical details and the well recognized facts which should be studied by all those who learn bacteriology and (2) problems of a more difficult and experimental nature to be assigned to and worked on by individual students who will present their results to the whole class. References to current literature are given in footnotes ; a con- sultation of some of them is recommended, in addition to text- book reading, in connection with the class exercises, and will be essential in order to understand and perform the individual problems. GENERAL REFERENCES Textbooks, Manuals and Compilations Am. Pub. Standard Methods for the Examination of Water Health Assn., and Sewage, ed. 5, 1925. Standard Methods of New York, N. Y Milk Analysis, ed. 5, 1927. Benecke Bau und Leben der Bakterien. Leipzig and Berlin, B. G. Teubner, 1912. Bergey Manual of Determinative Bacteriology. Baltimore, Williams & Wilkins, 1923." Besson Practical Bacteriology, Microbiology and Serum Therapy. Trans, from the French by Hutchins. New York, Longmans, Green & Co., 1913. Paris, J. B. Bailliere et Fils, 1911. Bordet & Studies in Immunity. New York, Wiley, 1909. Gay Bouchard Nouveau Traite de Pathologie Generale, vol. 1. Paris, Masson & Cie, 1912. Brumpt Precis de Parasitologic Paris, Masson & Cie, 4th Ed., 1928. Buchanan General Systematic Bacteriology. Baltimore, Williams & Wilkins. Buchanan Physiology and Biochemistry of Bacteria. Wil- & Fulmer liams & Wilkins, Baltimore, 1928. Calkins Protozoology. New York & Philadelphia, Lea & Febiger. 1909. Castellani & Manual of Tropical Medicine (3rd edition). New Chalmers York, William Wood, 1919. Chandler Animal Parasites and Human Disease. New York, William Wood, 1919. Chester A Manual of Determinative Bacteriology. New York, MacMillan, 1909. Clark The Determination of Hydrogen Ions. (3rd edi- tion). Baltimore, W r illiams & Wilkins, 1928. Craig Parasitic Amoebae of Man. Philadelphia and London, Lippincott, 1911. Delater & Nouveau Precis de Bacteriologie. Paris, Gauttier & Grandclaude Villars, 1928. Dobell Clifford & O'Connor Doflein Duclaux Ehrlich & BcMuan Fanthan, Stephens & Theobald Fitzgerald Ford Garrison Greaves Guyer Haden Hegner & Taliaferro Zinsser Jordan Jordan & Falk Karsner & Ecker Kendall Kolle & Hetsch Kolle, Kraus & Uhlenhuth Kolmer The Intestinal Protozoa of Man. London, Bale, 1921. Lehrbuch der Protozoenkunde. Jena, Gustav Fischer, 1911. Traite de Microbiologic Paris, Masson & Cie, 1900. Studies in Immunity. New York, Wiley, 1910. The Animal Parasites of Man. London, Bale, 1916. The Practice of Preventive Medicine. St. Louis, Mosby, 1922. Text book of Bacteriologv, Philadelphia & London. W. B. Saunders, 1927.' History of Medicine. (4th edition) Philadelphia & London. Saunders, 1929. Elementary Bacteriology. Philadelphia. Saunders & Co., 1928. Animal Micrology. Chicago University Press, 1917. Dental Infection and Systemic Disease. Philadel- phia & New York. Lea & Febiger, 1928. Human Protozoology. New York, MacMil- lan, 1925. Textbook of Bacteriology, (6th edition). New York & Chicago, Appleton, 1927. A Textbook of General Bacteriology. Philadel- phia & London, W. B. Saunders & Company (9th edition) 1928. Newer Knowledge of Bacteriology and Immun- ology. University of Chicago Press, 1928. Principles of Immunology. Philadelphia & Lon- don, Lippincott, 1921. Bacteriology, General, Pathological, Intestinal. Philadelphia & New York, Lea & Febiger, 1921. Die experimentelle Bakteriologie, etc., (ed. 7) 2 vols. Berlin, Urban & Schwarzenberg, 1929. Handbuch der Pathogenen Mikroorganismen. Jena, Gustav Fischer, (edition 3), 1928. Infection, Immunity and Biologic Therapy, (3rd ed.) Philadelphia, Saunders, 1925. Kolmer Kolmer Lafar Leitz Long MacCallum Mallory & Wright Marshall McClung Michaelis Minchin Nichols Park, Williams & Krumweide Philibert Prowazek Stitt Tanner Wadsworth Wells Wells Wenyon Wood Laboratory Diagnostic Methods. New York, D. Appleton & Co., 1925. Serum Diagnosis by Complement Fixation, Phila- delphia & New York, Lea & Febiger, 1928. Technical Mycology, trans, by Salber. London, C. Griffin, 1911. Enzyme Actions & Properties. New York, Wiley, 1929. London, Chapman & Hall. Short History of Pathology. Baltimore, Williams & Wilkins, 1928. A Textbook of Pathology (4th edition). Phila- delphia, Saunders, 1928. Pathological Technic, (7th ed.) Philadelphia, Saunders, 1918. Microbiology. Philadelphia, Blakiston, 1917. Handbook of Miscroscopical Technique, New York, Hoeber, 1929. Hydrogen ion Concentration. Baltimore, Williams & Wilkins, 1926. Introduction to the study of Protozoa. London, Arnold, 1917. Carriers in Infectious Diseases. Baltimore, Wil- liams & Wilkins, 1922. Pathogenic Microorganisms. (8th ed.) Philadelphia & New York, Lea & Febiger, 1924. Manuel de Bacteriologie Medicale. Paris, Masson & Cie. 1928. Handbuch der Pathogenen Protozoen. Leipzig, Johann Ambrosius Barth, 1912. Practical Bacteriology (8th edition). Philadelphia, Blakiston, 1928. Mycology of Foods, Examination of Milk. New York, Wiley, 1919. Standard Methods of the Division of Labatories and Research of the New York State Depart- ment of Health, New York, D. Appleton & Co., 1927. Chemical Pathology, (ed. 5), Philadelphia, Saun- ders, 1925. Chemical Aspects of Immunity New York, Chemi- cal Catalog Co., 1925. Protozoology. London, Balliere, 1926. Chemical and Microscopical Diagnosis, (3rd edi- 10 tion), New York, iVppleton, 1917. Zinsser Infection and Resistance (3rd edition). New York, MacMillan, 1923. Zinsser A Laboratory Course in Serum Study, (2nd edi- Hopkins& tion). New York, MacMillan, 1921. Ottenberg Principal Periodicals and Current Literature Abstracts of Bacteriology (Prior to 1925) American Journal of Hygiene American Journal of Pathology American Journal of Tropical Medicine Annales de lTnstitut Pasteur Archives of Pathology Biological Abstracts (Beginning 1926) British Journal of Experimental Pathology Bulletin de Tlnstitut Pasteur Bulletin U. S. Hygienic Laboratory Bulletins of the U. S. Public Health and Marine Hospital Service Centralblatt fur Bakteriologie, Parasi- tenkunde und Inf ektionskrankheiten ; Originate; Referate Comptes Rendus de la Societe de Biologic Ergebnisse der allgemeinen Pathologie Index Medicus (Prior to 1927) Jahresbericht iiber pathogenen Micro- organismen Jahresbericht iiber Immunitatsforschung Journal of the American Medical Asso- ciation Journal American Public Llealth Associa- tion Journal of Bacteriology Journal of General Physiology Journal of Laboratory & Clinical Medicine Journal of Experimental Medicine Journal of Hygiene Journal of Immunology Journal of Infectious Diseases Journal of Medical Research (Prior to Sept. 1924) Baltimore. Baltimore. Boston. Baltimore. Paris. Chicago. Philadelphia. London. Paris. Washington. Washington. Jena. Paris. Wiesbaden. Chicago. Leipzig. Stuttgart. Chicago. New York. Baltimore. Baltimore. St. Louis. Baltimore. London. Baltimore. Chicago. Boston. 12 Journal of Pathology and Bacteriology Edinburgh. Quarterly Cumulative Index Medicus Chicago. Tropical Disease Bulletin London. Zeitschrift fur Chemotherapie und ver- Leipzig. wandte Gebiete Zeitschrift fur Hygiene und Infektions- Berlin. krankheiten Zeitschrift fur Immunitatsforschung und Jena. experimentale Therapie ; Originate, Referate GENERAL INSTRUCTIONS Desk and Locker Space : Each student should be assigned a desk with locker, a separate locker for tray of stains and reagents, and a tray in the incubator room. Supplies and Apparatus : Students will be supplied with a tray of stains and reagents, Bunsen burner and tubing, ring stand, granite cup and thermometer. Each student should provide the following individual equipment: One textbook. One microscope. One apron, waiter's coat or laboratory smock. Two towels. One handkerchief for cleaning slides One set of dissecting instruments. One pair of slide forceps. Two platinum or nichrome needles with holder. Two rubber nipples. One box of microscope slides. One box cover slips, No. 1, 22mm, (circles or squares.) One package of lens paper. 2 padlocks and ke}'s for lockers. One laboratory notebook. One wax pencil (glass or skin-marking). One small triangular file. 4 hollow-ground slides. General Rules: The wire used in making cultures should be sterilized by heating to redness in the Bunsen flame before and after every use made of it. If it is covered with viscous material, dry at the side of the flame before sterilizing to avoid scattering infectious material. Sterilize hollow-ground slides and slide cover-slip preparations by placing in the jar of disinfectant provided. Displace the cover- slip with forceps or needle, which should then be flamed. 14 Instruments that have been used on infected animals should be boiled before putting away. Cultures and infected tubes that are to be discarded should be left on the desk; they will be collected and sterilized after the class session. Put non-infectious solid waste into the buckets provided. Turn Bunsen burners low when not in use, to avoid over-heating the laboratory and wasting the gas. Watch for gas leaks. At the close of the day's work, put all cultures to be studied further, in the incubator, or when directed, in the desk locker ; put away all apparatus, and wash the hands with soap and water. Accidents: In case a living culture is spilled accidentally, notify one of the instructors and cover immediately with disinfectant. If the fingers come in contact with living culture material, rinse the hands in disinfectant and scrub with soap and water. In case of personal accidents, such as cutting or pricking the fingers, or splashing in- fective material or culture in the eye, report at once to the instruc- tor in charge. Use every precaution to avoid such accidents. Smoking is not permitted during class hours. Keep fingers and objects in use away from the mouth. Use and Care of the Microscope : Good illumination is essential in the examination of bacteria. Use the plane mirror for daylight and the concave mirror when using electric light for illumination. The sub-stage condenser should always be employed; it should be raised or low- ered slightly with slides of varying thickness to focus the rays of light sharply upon the field to be examined. The usual Abbe Con- denser is satisfactory but the aplanatic condenser gives more bril- liant illumination. When the low-power objective is used the iris diaphragm should be closed to a very small diameter; with the high-power dry objec- tive it should be opened slightly and with the oil immersion lens it should always be wide open. Oil immersion objectives containing a fluorite system yield tht most perfect definition. In the use of the oil immersion lens, place a drop of cedar oil on the portion of the slide which is to be ex- amined and lower the objective with the coarse adjustment until the front lens touches the drop of oil. Observe the lens from the side and continue to lower the lens until it almost touches the slide. Then apply the eye to the ocular and focus up slowly with the coarse adjustment until the field comes into view. Subsequent focusing is done with the fine adjustment. 16 Before putting the microscope away carefully wipe off the cedar oil from the immersion objective with lens paper, using xylol to soften and remove gummed oil. 18 Exercise 1 Handling of Bacterial Cultures Bacteria are cultivated most commonly in tubes, Petri plates, and flasks, which are used to contain liquid media such as nutrient broth, and similar media which have been brought to a semi-solid or jelly-like consistency by the addition of agar-agar or other sub- stances. The composition of the media commonly used and their methods of preparation are given in a separate section in the back of the manual. In opening tube and flask cultures, the cotton stopper is removed with a rotary motion, while the tube or flask is held inclined to- ward the horizontal plane; the mouth of the tube or flask is then passed through the Bunsen flame to destroy bacteria which cling to shreds of cotton or particles of dust that might fall into the culture. Samplings of the cultures are made with a wire fixed in a holder. The wire should be of nichrome or platinum, of 26 to 28 B. & S. gauge, and about 7 cm. in length. It may be used in a special holder or may be welded in "a very hot Bunsen flame or blast-lamp to a rod of aluminum or glass 3 to 5 mm. in diameter. Before and after every use of the wire it should be sterilized by holding in the Bunsen flame until it is heated to redness. One wire should be straight, the other should have a loop 2 mm. in diameter made in the free end by means of round-nosed pliers. After the culture is sampled the mouth of the tube should be flamed again and the stopper returned. While the stopper is out of the tube it should not be allowed to come into contact with any object. The micro Bunsen burner held by a burette clamp in the hori- zontal plane gives the most satisfactory flame but is not generally employed. Semi-solid or solid media only are used in Petri plates. Cultures in Petri plates are protected against contamination by the cover. In sampling, the cover should be raised just enough to permit the wire to be used freely. Petri plate cultures are inverted before being stored or placed in the incubator, in order to prevent the liquid expressed from the medium, the so-called water of condensa- tion, from flowing over the medium and carrying contamination from the edges of the plate. 20 Exercise 2 Fresh Preparations Material needed: 24 hour broth cultures of Staphylococcus albus Streptococcus viridans Bacillus pyocyaneus Bacillus subtilis The hanging drop is often used in the examination of fresh preparations. Spread a thin ring of vaseline with a tooth-pick about the concavity of a hollow ground slide. With the flamed loop, place a drop of the fluid to be examined in the center of a clean cover slip resting on a flat place on the desk. Then invert the hollow ground slide over the cover slip and press gently down to make the ring of vaseline adhere to the cover slip, with the drop in the center of the concavity. Invert the slide again and it is ready for examination. A slide cover-slip preparation answers almost every purpose of the hanging drop, and has the advantage of distributing the bacteria more nearly in a single plane. This facilitates focussing and there is less danger of crushing the cover slip with the lens. Such a preparation is made by placing with the flamed loop a small drop of the fluid to be examined on a slide, and placing a cover slip over the drop so that it spreads out in a thin film between cover slip and slide. Examination of either preparation should be made first with the low, than the high power dry lens, which usually gives sufficient magnification. In exceptional cases the oil-immersion lens is used; it is especially valuable with dark-field illumination in examination of the spiral organisms. Make a fresh preparation from each of the bouillon cultures. Observe the morphology of each organism. Examine for motility: distinguish between (1) true motility, shown by movement across the field of vision, (2) Brownian motion, which is a rapid vibratory or dancing movement due to molecular forces, and (3) uniform movement of many individuals due to currents in the fluid in which they are suspended. 22 Exercise 3 Stained Preparations Material needed: Agar cultures of Staphylococcus albus Bacillus coli Bacillus subtilis Killed suspension of Bacillus tuberculosis While en masse some bacteria are pigmented, microscopically the individuals are nearly colorless. Their morphology is therefore best studied in stained preparations. There are many stains for special purposes; only the principal bacterial stains are mentioned here. They are best demonstrated with 24 hour cultures on plain agar. Staining solutions are supplied to students ready to use. The formulas are to be found on page 178. Preparation of Films : Take a clean glass slide and mark and number three divisions with a wax pencil. With sterilized loop, place a very small drop of water in the center of each division. The slide must be clean and free from greasy material, as otherwise the water will not adhere to the glass and it will be impossible to spread the drop out. Flame the loop. Open a culture tube and flame its mouth, barely touch the culture growth with the loop, and emulsify the material taken up in one of the drops of water on the slide. Flame the mouth of the tube and replace the plug after withdrawing the loop. Spread the drop in a thin film over an area of about one square cm. Make similar films of other organisms in the other drops. Allow the film to dry spontaneously, or in damp weather accel- erate drying by holding the slide high over the flame at a tempera- ture which does not burn the hand. Fix the film, when thoroughly dry, by passing the slide three times through the flame, with the film side uppermost. Cool before staining. 1. Simple stain. Cover the fixed film with Loeffler's methylene blue. Allow to stand 1 minute. Wash, drain, and blot ; dry thoroughly by holding high above flame. Safranin or dilute carbol fuchsin may be used in a similar way. 2. Gram stain. A. Cover fixed film with Sterling's gentian violet, 15 sec. B. Wash off gentian violet with water. C. Cover with Gram's iodine solution and allow to stand 15 seconds or until the film has assumed throughout a grayish brown color. 24 D. Wash off iodine with 95% alcohol, changing alcohol two times, and allow final alcohol to remain on slide 1 minute. E. Wash with water. F. Cover with safranin one minute. Bismarck brown two minutes or dilute fuchsin y 2 minute, also make good counter stains. G. Wash with water, blot and dry. 3. Ziehl-Neelson stain (acid-fast stain). A. Prepare film from suspension of B. tuberculosis, and on another portion of the same slide one of B. subtilis. Fix by heat, avoiding overheating. B. Place slide on ring stand, cover slide with carbol fuchsin and heat with Bunsen flame until the stain begins to steam. Heat occasionally, and keep the slide steaming for three minutes. Do not let the stain boil; add more stain if necessary before the film dries. C. Wash with water. D. Decolorize with several changes of acid alcohol, until no more dye is removed. E. Wash with water. F. Counterstain with Loefrler's methylene blue one minute. G. Wash, blot and dry. The student should make stained preparations from each of the agar cultures, using simple and Gram stains, and an acid fast stain of B. tuberculosis and another organism. Examine the stained films with the oil immersion lens. In the methylene blue stains, look for metachromatic granules or areas of irregular stain- ing in the bacteria. With the Gram stain, observe the difference in final color. Organisms retaining the gentian violet color are spoken of as Gram positive while those which lose the purple color and take the counterstain are called Gram negative. The technic of the Gram stain should be repeated until the student is certain of his results. In the acid-fast stain, if the staining has ben properly carried out, the background takes the counterstain, and only certain or- ganisms that possess a waxy material preserve the red color of the carbol fuchsin after the treatment with acid alcohol. Such organisms are called acid-resistant, or acid-fast. Make a careful study of each organism with the different stains. Sketch and write brief description in note-book. 26 Exercise 4 Inoculation of Culture Media A. Material needed: 24 hr. broth culture of staphylococcus albus 1 plain agar slant 1 bouillon tube 1 gelatin tube 2 deep agar tubes 1 Petri plate 1. The technic of inoculation of different culture media will be demonstrated by the instructor. Students will make inocu- lations, from stock cultures supplied, upon: (a) Plain agar slant (b) Bouillon tube (c) Gelatin tube (Stab culture) 2. Place two deep agar tubes in agate cup filled with water and heat to boiling; at this temperature the agar melts. Cool water to 45 °C. 3. Inoculate one tube of agar, distributing the culture material throughout the agar by elevating the tube quickly from an inclined position, with a rotary motion. 4. Flame the mouth of the second tube and pour the agar aseptically into a sterile Petri plate, raising the cover on one side only. Allow the agar to solidify before handling plate. When solid, inoculate the surface of the agar by drawing the loop filled with culture material gently over the surface in parallel streaks. Cover and invert plate. 5. Store the gelatin tube at room temperature, in the desk locker. Place other cultures in tray in incubator room. B. Examine the cultures after incubation. 1. Make fresh preparation from bouillon tube, and stain film made from one of the cultures on solid media. 2. Note character of growth on agar slant for (a) amount (b) consistency (c) pigmentation. 3. Examine bouillon tube for (a) turbidity (b) pellicle (c) sediment. 4. Note appearance of gelatin stab culture and form of liqui- fied portion. 5. Note size, appearance and distribution of colonies in agar tube culture. 6. Study isolated colonies on agar plate with the unaided eye and with the low power of the microscope. 7. Make drawings in note book to illustrate each culture. 28 Exercise 5 The Isolation of Bacteria: Aerobic Bacteria from the Air Material Needed: A. 3 Deep agar tubes 3 Petri Plates B. 2 agar slants One purpose of this exercise is to examine the possibilities of aerial contamination and the necessity for proper care in avoiding and detecting this source of error in bacteriological technic. A. 1. Pour three Petri plates of plain agar as in previous exercise. Allow the agar to solidify. 2. Remove the cover from one plate and expose the agar to the air for 5 minutes by the watch. 3. Expose the second plate to the air for 20 minutes. 4. Keep third plate unexposed as a control. 5. Students at odd desks keep plates in desk lockers, those at even desks place in tray in incubator room. B. 1. Study plates exposed to air and observe the different types of colony. It is one of the marks of the experienced bac- teriologist to be able to judge the probable relationships of a bacterial culture by the microscopic appearance of well separated isolated colonies. In addition to the size of the isolate colonies, consistency, pigments, elevation, character of the edge, and other points may frequently be noted with value. See in appendix Chester's Terminology for descrip- tion of Colonies p. 92. 2. With wax pencil make a circle on bottom of Petri plate about well isolated colonies, and place an identifying num- ber beside each circle. Fish from these colonies to slides, make film and stain by Gram. The technic of fishing from colonies will be demonstrated by the instructor. 3. Inoculate agar slants from two colonies showing different types of organism, preferably one coccus and one bacillus. 4. Enumerate colonies on each of the plates. Tabulate obser- vations. 5. Incubate subcultures at 37° C. G. 1. Study cultures fished from plates; make fresh and stained preparations to check the purity of culture. 30 Exercise 6 Isolation from Mixed Culture by Plating Material Needed: A. Mixed culture in broth of Micrococcus catarrhalis Staphylococcus aureus Bacterium coli Five deep agar tubes Five Petri plates B. Five plain agar slants A. 1. Pour plate method. Melt five tubes of agar in agate cup and cool to 45 °C. Letter tubes a, b, c, d, and e. Inocu- late (a) with one loop of mixed culture supplied. Mix thoroughly as in Ex. 4. Inoculate (b) from (a) with three loops. Mix. Inoculate (c) from (b) with five loops. Mix. The agar in all the tubes should be kept melted by standing between manipulations in water in the cup at 45 °C. The contents of each tube, including the uninoculated control, (d) should be poured aseptically into a separate sterile Petri plate. Arrange the pour plates in a stack and place the agate cup on top of the stack. This will warm the cover of each plate and prevent condensation of water on it. 2. Streak plate method. Pour tube (e) of melted agar into a sterile Petri plate, allow the agar to harden. Take a loop of the mixed culture and make three closely parallel streaks along one side of the plate. Flame the loop and cover the remaining surface of the agar with parallel streaks one cm. apart at right angles to the previous ones, touching the inoculated portion and passing from it to the opposite side of the plate. Any similar method for spreading the mixture out in diminishing concentration will serve. The last portion streaked should show well separated colonies. 3. Invert all plates, label with wax pencil, and incubate at 37°C. B. 1. Study plates made at previous hour. The uninoculated control^ (d) should be sterile. If it is not, it indicates contamination due to faulty technic. One or more of the inoculated plates should show well separated colonies. On pour plates distinguish between deep and surface colonies. Sketch the different types of colony in each situation. 2. Mark with wax pencil on bottom of plate surface colonies sufficiently separated to permit of fishing. Make films and stain by Gram. Attempt to find each of the species present in the original mixed culture. 3. Fish under the low power of the microscope from one colony of each type to a plain agar slant. Incubate sub- cultures at 37° C. 32 4. Make blue-print of pour plate showing fewest colonies. See Broadhurst, Blue Printing Directly from Agar Plates Jour. Bact. 3, 1918, p. 187. C. Examine cultures fished from plates. Test for purity of culture by fresh and stained preparations. Describe charac- teristic^ of each culture, and confirm previous findings as to staining reaction and morphology. 3-1 Biochemical Activities or Bacteria Exercise 7 Oxygen — Carbon Dioxide Exchange (Respiration) Material Needed: 24 Hr. culture of Eact. coli Bacillus subtilis Million tubes (5cc.) Sterile vaseline 2 bouillon tubes with CO- trap Phenol red indicator M XaOH (carbonate free; 100 Paraffined corks Oxygen indicator (Indigo carmine 0.1%, KsCOa l c /c ; Dextrose 1%. Allow to stand over night to permit reduction of the indigo.") A. 1. Inoculate tubes of bouillon with B. coli and B. subtilis. Cover surface of medium in these tubes and in uninoculated control tube with melted vaseline. M 2. With capillary pipettes, add 5 drops to XaOH and 2 100 drops of phenol red indicator to the distilled water contained in the small CO- trap within the larger tube. Then inoculate bouillon in tube with B. subtilis. Plug the tube tightly with paraffined cork. Add XaOH and indicator in a similar way to the small tube of the uninoculated control, and plug tightly with cork. Do not flame mouths of these tubes or hold them near Bunsen flame at any time, as this would permit entrance of a large amount of CO2. B. 1. Test for presence of oxygen in tubes treated according to direction- in par. 1 of previous lesson. Take up a small amount of oxygen indicator (reduced indigo carmine; with a capillary pipette. Protect the indicator from air by draw- ing up first some vaseline., then the indicator, and again a -mall amount of vaseline to seal the tip of the capillary. The vaseline may be melted by placing the tube in a vessel of water at 40°C. Push the tip of the pipette through the vaseline covering the uninoculated control tube and inject half the indicator solution into the bouillon. "With fresh pipettes inject indicator solution into the inoculated tubes. Avoid forcing even the smallest amount of air beneath the vaseline seal of the tubes to be tested. In the presence of molecular oxygen the indicator solution immediately develop- a bluish-green color. 2. Examine tubes containing C0 2 trap. COo absorbed by the liquid in the small tube increases its acidity, as indicated 36 by change in color of the phenol red. What is the source of the volatile acid produced in the inoculated tubes? Reference Efimov, V. V., Colorimetric Method for Oxvgen. Biochem, Z, 155, 1925, p. 371. Novy, F. G., Roehm, H. R., Soule, M. H., Novy, F. G.. Jr., Microbic Respiration. J. Inf. Dis., 36, 1925, pp. 109, 168, 245, 343. Coulter, C. B., and Isaacs, M. H. Reduction Potentials of the Tvphoid Bacillus. J. Exp. Med. 49, 1929, p. 711. 38 Exercise 8. Fermentation A. Material needed: 24 hr. culture of B. subtilis B. lactis aerogenss Yeast 2 dextrose agar slants 3 dextrose bouillon tubes (15cc.) 1 dextrose fermentation tube (Smith type) 1. Inoculate the dextrose agar slants with B. subtilis. Plug one tube tightly with parafined cork. 2. Inoculate two tubes of bouillon and the Smith fermentation tube with B. lactis aerogenes and one tube of bouillon with yeast. Incubate at 37°C. B. Material needed: pH indicators Phenol Red Brom-thymol-blue Methyl Red M/50 NaOH Ether Concentrated H^SO* 5% H2SO4 Saturated solution CuSOi Thiophene, 0.5% alcoholic solution 10% Na 2 C0 3 Solution I 2 K1 1% solution K-Ci-.Ot 10% NaOH 1. Observe relation between oxygen supply and fermentation in agar cultures of B. subtilis. Make slide cover slip preparation from both tubes ; allow a drop or two of Grams iodine to flow under cover slip. Examine with high-power dry lens and oil immersion for presence of glycogen granules in the bacilli. Are they present in preparations from both cultures? 2. Test cultures in bouillon for products of fermentation of dextrose. a. Organic Acids. Determine the pH of the culture, by adding 3 drops of each of the indicators to 3 cc. portions of culture and examining in the com- parator block, b. Titrate a 5cc. portion to pH 7.6 with phenol red and M/50 NaOH Does the value found represent all the acid which has been produced in growth ? See Exercise 7. 40 c. Test for lactic acid. The first part of this test should be done at one of the large tables, away from Bunsen flames. Shake out a lOcc. portion with 5 cc. ether. Place the tube in the refrigerator while the ether layer is separating. Pipette the ether layer into a dry test tube and evaporate on the water bath. Dissolve the residue in 2cc. of distilled water. Add 5 cc. cone. H 2 S0 4 and 1 drop of saturated CuS0 4 and mix. Heat in boiling water 1 minute, cool under tap, add 2 drops of 0.5% alcoholic solution of Thiophene. Shake and replace tube in boiling water. A cherry-red color develops with lactic acid. 3. Test yeast culture for production of alcohol. a. To \y 2 cc. portion of broth culture add a few drops of 10% Na 2 C03 and 5 to 20 drops of I 2 KI adding enough to render the mixture bright yellow for 3^ -minute (Grams iodine solution may be used). Warm slightly, note odor of iodoform, and set aside. Examine the separated yellow iodoform crystals (rosettes) under the micro- scope. Acetone and other substances also give this reaction. b. Distill off from a 10 cc. portion, using a bent glass tube and a rubber stopper, 3 cc. of distillate. Add 2-4 drops of Potassium dichromate, acidify with dilute H 2 S0 4 and boil. Note odor of alde- hyde, changing to that of acetic acid. 4. Test culture of B. lactis aerogenes for acetyl-methyl carbinol. To a 5 cc. portion add an equal amount of 10% NaOH and a few drops of IT 2 2 . Incubate for 24 to 72 hours. The development of a red color indicates the presence of acetyl methyl carbinol. The test is known as the Voges-Proskauer reaction. The significance is discussed by Levine, M., Tour. Bact., 1. 1916, p. 153. 5. Observe gas accumulation in closed arm of Smith fermenta- tion tube. Mark the level of the liquid medium in this arm and with a scale measure the length of the column of gas. A^dd 10% NaOH to the open arm of the tube, filling it completely ; place the thumb over the mouth of the tube and invert several times. Allow the remaining gas to collect in the closed arm and carefully remove thumb. Mark the level of the medium, measure the column of hydrogen and calcu- late the ratio between H 2 and C0 2 produced by the culture. 42 Exercise 9. Protein Metabolism The chemical changes considered in this exercise are not brought about by bacteria universally, and, on the other hand, represent only a few of the many chemical transformations which bacteria may produce in protein. The substances which are responsible for immune reactions, as well as the bacterial toxins, are products of protein or nitrogen metabolism but are too complex and of too uncertain nature to be studied here. A. Material needed: 24. h. culture of B. pyocyaneus B. sporogenes B. coli 1 gelatin tube 1 Loeffler's serum slant 2 milk serum tubes (5 c. c.) 1 Dunham's peptone (10 c. c.) 1. Inoculate Loeffler's serum and gelatin with B. pyocyaneus. Make stab culture in gelatin and incubate at room temperature. 2. Heat milk serum tubes in boiling water for 15 minutes, cool rapidly and inoculate heavily with B. sporogenes. 3. Inoculate Dunham's peptone with B. coli. B. Material needed: Ammonium sulfate (crystals) NaOH 10% CuS0 4 0.1% Thymol blue indicator Lead acetate solution Ehrlich Indol reagents. 1. Examine cultures in gelatin and Loeffler's serum for liquefac- tion of the medium. Make observations over a period of 5 to 7 days. If the gelatin becomes liquified by melting, place in incubator for 24 to 48 hours, then test for ability of the medium to harden by placing in refrigerator. 2. Test culture in milk serum for proteolysis. Precipitate the proteins from a 5 c.c. portion by the addition of ammonium sulfate to saturation, and heat to 50° to 60° C. for thirty minutes. Filter through paper; to filtrate add strong NaOH and a few drops of dilute copper sulfate. A violet color indicates the presence of pep- tone, resulting from the proteolysis of the protein originally present. 3. Test culture in milk serum for ammonia liberated by deamin- ization of amino acids. Moisten a plug of absorbent cotton with distilled water and 2 or 3 drops of thymol blue indicator, and push the plug into the culture tube to within 2 cm. of the surface of the medium. Place the tube in boiling water. Liberation of ammonia is indicated by development of the alkaline color in the indicator. 44 4. Add lead acetate to a culture in milk serum and test for libera- tion of S from sulphur-containing ammo-acids. ' 5. Test the cultures in Dunham's peptone for the production of indol. Moisten a small plug of absorbent cotton with the Ehrlich reagent and insert into tube containing 5 c.c. of culture ; push the plug down to within 2 cm. of the surface of the liquid. Place tube in boiling- water. Indol present is volatilized along with water and condenses in the cotton plug where it reacts with the reagent to give a reddish color. 46 Exercise 10 Oxidation-Reduction Activity A. Material needed: 24 hr. cultures of B. coli. B. phosphorescens Streptococcus viridans 2 tubes of nitrate broth 1 tube of bouillon with indigo carmine 1 tube of infusiott bouillon 1 tube of sea water agar. 1. Inoculate one tube of nitrate broth and the indigo carmine bouillon with B. coli. 2. Inoculate sea water agar with B. phosphorescens. 3. Inoculate infusion bouillon with Strept. viridans. B. Material needed: Nitrate reagents (sulfanilic acid, napthylamine) Dilute acetic acid Saturated ammonium sulfate solution 5% solution of sodium nitroprusside Concentrated ammonium hydroxide 1% solution a-naphthol in 95 % alcohol 1% solution paraphenylenediamine — HC1 in water 1% sodium carbonate solution 3% suspension of washed sheep cells 1. Test the nitrate broth culture and the uninoculated control tube for the reduction of nitrate to nitrite as follows : Transfer 4 c.c. of culture to a clean test tube. Add gradually 2 c.c. of freshly mixed nitrate reagent solutions. Place tube in warm water for 20 minutes. A pink color develops in the presence of nitrites. Observe promptly. 2. Observe reduction of indigo carmine in culture containing the dye. Agitate tube vigorously and note restoration of color on aeration. 3. Test for reduced glutathione in culture of B. phosphorescens. Transfer a quantity of culture to the concavity of a hollow-ground slide. Add dilute acetic acid and heat slide over flame until it begins to steam. Carefully decant acetic acid and add saturated ammonium sulfate. Change the latter solution twice. To the culture material in ammonium sulfate add 5 to 10 drops of sodium nitroprusside and allow to stand 3 to 5 minutes ; then add concentrated ammonium hydroxide. A reddish color develops at once and fades in a few seconds, in the presence of sulfhydryl. Acetone as well as other substances also give the reaction. 4. Observe spectrum of reduced cytochrome in culture of B. phos- phorescens. Transfer culture material from agar slant to a cell 3 mm. deep, illuminate with ribbon-filament bulb, using the substage condenser and examine through the microspectroscope. -is 5. Oxidation of Dextrose. Refer to Exercises 7 and 8. 6. Indophenol oxidase. Emulsify a quantity of B. phosphorescent culture in 5 c.c. bouillon in a test tube. Add 5 drops of 1% alpha naphthol, 5 drops of 1% paraphenylene diamine, and 5 drops of 10% sodium carbonate solution. Allow to stand 30 minutes. A reddish color, turning to purple develops on oxidation of the reagents by the oxidase. 7. Observe oxidation of oxyhemoglobin to methemoglobfci by Streptococcus viridans. With capillary pipette place 1 c.c. of red cell suspension and 1 c.c. of bouillon culture of streptococcus into a small test tube. Allow tube to stand in water bath at 37° C. for one half to one hour. A greenish brown color develops in the erythro- cytes as the oxyhemoglobin becomes oxidized to methemoglobin. 50 Exercise 11 Differential Hydrolysis of Carbohydrates Material needed : 24 hr. cultures of B. coli B. paratyphosus A. B. alkaligenes B. subtihs 1 tube of dextrose agar 1 " " lactose " 1 " " saccharose " 1 " " mannit " 15 c.c. per tube, containing andrade indicator 1 tube of starch agar. Solution of iodine in 50% ethyl alcohol A. Melt the agar containing the different carbohydrates and pour Petri plates. When cool, inoculate each plate with each of the organisms supplied, making a single streak across the plate. Number or otherwise identify each streak on the bottom of the plate. Incubate at 37°. B. 1. Examine cultures for acid produced by the fermentation of the dextrose resulting from hydrolysis of the sugars. Record the results in a table. 2. With a pipette, place 4-5 drops of solution of iodine upon the starch-agar plate, and allow the iodine to flow at right angles across the culture streaks. Observe reaction, and note clearing of medium about portions of growth not treated with iodine. 52 Exercise 12 Production of Pigment and Light Material needed: 24 hr. cultures of Staphylococcus aureus B. prodigiosus B. pyocyaneus B. phosphorescens 3 agar slants 1 tube of broth 1 tube of sea-water broth A. 1. Inoculate Staphylococcus aureus, B. prodigiosus and B. pyo- cyaneus on dextrose agar slants. 2. Inoculate B. pyocyaneus into broth. 3. Inoculate the sea-water broth with B. phosphorescens. 4. Incubate all cultures at room temperature in the dark. B. 1. Observe presence, color and distribution of pigment in cul- tures. 2. Examine broth cultures of B. procyaneus. Agitate tube vigor- ously and observe appearance or deepening of color. Allow the tube to stand, and note any change in appearance of pigment. Offer an explanation for the change. 3. Shake out culture of B. pyocyaneus with 3 c.c. of chloroform. Note solubility of pigment in this solvent. Pipette off the aqueous layer and evaporate the chloroform solution to dry- ness on the water bath. 4. Test solubility of the dry pigment in water, alcohol and ether. 5. Make similar preparations of pigment from cultures of other bacteria, and apply the lipocyan test to the dry pigment, as follows : Add 1 drop of concentrated H 2 S0 4 and observe for production of intense blue color. This reaction is given only by the pigments known as carotinoids from their resemb- lance to plant carotin. 6. Carry the culture of B. phosphorescens into a dark room. Allow the eyes to become accustomed to the darkness and examine the culture for luminescence. Agitate the tube and note any change in the production of light. 7. Add a few drops of KsFe (CN) 6 solution to the luminescent culture. Observe the tube in the dark. Does it luminesce when agitated ? References : Buchanan and Fulmer, Physio'logv & Biochemistry of Bacteria. Vol. 1, Chapter III. Harvey. The Nature of Animal Light. Lippincott, Philadelphia, 1920. Morrison, L. F., Studies in Luminous Bacteria. Jour. Gen. Physiol., 7, 1925, p. 741. 54 Pathogenic Micro-Organisms Throughout the rest of the course we concern ourselves largely with microorganisms in their relation to disease. No attempt is made to study all pathogenic microorganisms in this course. Only the most representative and important species are con- sidered as illustrative of general principles or of particularly im- portant details. Some of the most important diseases of known animate causation cannot readily be studied owing to technical difficulties that would require too much time or skill for the student to master. The ingenuity of each individual may be indulged in connection with the assigned problems which now assume significance. It is desirable that the student should learn to think of the patho- genic bacteria in their position in natural taxonomic groups as well as in their relation to specific diseases. Practical utility in bacteriology has out-stripped strictly scientific classification ; interest has centered in the pathogenic members of a given genus rather than in an attempt to collect all possible related members of it. Efforts are now under way, however, to classify bacteria in a more exact fashion. On page 190 will be found a summary of Bergey's Manual of Determinative Bacteriology, simplified to cover the medical aspects of bacteriology. The ideal source of material for the study of pathogenic bacteria is diseased tissue from which they are originally isolated. Whenever possible such material in the form of pus, secretions, blood, urine, feces, or tissues from human beings or animals will be furnished. Stock cultures are used in other instances. Exercises in infection and immunity are presented in connection with the exercises on pathogenic bacteria. These exercises will be assigned to small groups of students, and will be undertaken as original problems and reported to the class at large. Knowledge of the scientific background of the subject illustrated by the experi- ment is expected, but only the student's experimental observations should be reported, leaving to general discussion the formulation of appropriate theory and the development of ideas regarding the signi- ficance of the experiment. Brief statement only of the object and method of each exercise is given. The student should seek more complete information in the references to the literature of the subject which are given. In every instance a plan of experimentation should be submitted to the in- structor for approval after references have been read and before the experimental work is started. 56 Exercise 13 Genus Staphylococcus Usually parasitic. Cells in irregular groups, rarely in packets. Usually Gram-positive. Growth good as a rule on artificial media. Gelatin may be liquified, nitrates may be reduced. Pigment pro- duced : white, lemon yellow or orange. S. aureus is the principal pathogenic member. It is the most common organism causing suppuration. A. Material needed: 24 hr. culture of S. aureus Material from infected animal .2 agar plates 1 tube gelatin 1 tube bouillon 1. Majce fresh and stained preparations of culture and of material from infected animal. 2. With stock culture of S. aureus streak one agar plate and inoculate gelatin and bouillon tubes. Incubate gelatin tube in desk locker. 3. Streak out material from infected animal on the other agar plate. B. Material needed: 2 agar slants 3 bouillon tubes 1 blood agar plate 1. Examine plate made from stock culture of S. aureus; mark isolated colonies. Fish from these and examine stained films using methylene blue and Gram stains, and make subcultures to agar slant and bouillon tube. 2. Carry 2 loops of bouillon culture of S. aureus over to sterile tube of bouillon, and carry over 3 loops from this to tube of melted agar at 45° C. Add 1 c.c. of sterile blood, mix and pour plate. 3. Examine agar plate inoculated with material from infected animal for typical colonies. Fish from isolated colonies to agar slant and bouillon tube. 1. Compare cultures made from stock strain of S. aureus and from animal material. Make fresh and stained preparations. Note character of growth and grouping of organisms in bouillon culture and examine agar slants for pigment formation. 2. Examine gelatin tube from A-2 for liquefaction. 3. Examine blood agar pour plate for hemolysis. Reference : 1. Neisser Die Staphylokokken. Kolle, Kraus and Uhlenhut. 2. Buchanan Studies in the Nomenclature and Classification of the Bacteria. Jour. Bact., 2, 1917, 603. 3. Julianelle Studies of Hemolytic Staphylococci. Tour. Inf. Dis., 31, 1922, 256. 58 Infection and Immunity 1. Methods of obtaining whole blood, red corpuscles, leucocytes, and blood serum. A variety of methods are in use. Students will learn under the guidance of the instructor and will demonstrate to the class the methods of obtaining blood from laboratory animals and separating cells and serum. As general references consult Kolmer Zinsser, Hopkins and Ottenberg 2. Formation of sterile exudates. a. Serous exudate. Place ear of rabbit in water at 56° C. for 3 minutes. Follow course of reaction for 48 hours. b. Purulent exudate. Rub drop of croton oil into ear of rabbit. Follow course of reaction for 48 hours. Reference : Maccallum. 3. Local abscess. Inoculate a rabbit intradermally with culture of S. aureus. Examine inoculated area daily for production of lesions and for presence of staphylococcus. Reference : Maccallum. 4. Pyemia. Inoculate a rabbit intravenously with a culture of S. aureus. Make daily observations on temperature and weight of the animal and make cultures of blood. Autopsy. Examine internal organs in the gross and in microscopic section for lesions and make cultures to determine presence of S. aureus. Reference : Kolmer. 5. Preparation of a Bacterial Vaccine. Secure a pure culture of S. aureus by isolation. Prepare vaccine by the method given in Zinsser. Kill by minimum exposure to heat, with consideration of the thermal death point of the microorganism. Test for sterility of the preparation. Standardize in two different ways. Zinsser, Hopkins and Ottenberg, p. 176. Wadsworth, p. 457 Jordan and Falk, p. 285. 6. Leucocytosis. Inject intravenously into rabbits sterile milk and bacterial vaccine. Make leucocyte counts hourly and observe the stages of leucopenia and hyperleucocytosis. Reference : Stitt Zinsser, Infection and Resistance. 60 TRIBE STREPTOCOCCUS Cells spherical or hemispherical. Planes of fisson parallel, re- sulting in pairs or chains of cells. Generally Gram positive. Es- sentially parasitic, except Leuconostoc, and growth not abundant as a rule on artificial media. Exercise 14 GENUS STREPTOCOCCUS Chiefly parasites. Fresh cultures not dissolved by bile. Char- acteristic reactions on red blood cells. The streptococci produce a wide variety of infections. The most common locations in which the streptococcus is found are external wound infections, erysipelas, severe acute infections of the naso- pharynx and neighboring structures, scarlet fever, the lung and pleura in certain forms of pneumonia and the blood in streptococcus bacteriemia. A. Material needed: 24 hr. culture of Strept. hemolyticus Strept. viriclans Material from experimental animal infected with Strept. hemolyticus 2 blood agar plates 1. Make fresh and stained preparations of stock cultures and of material from infected animal. 2. Divide one blood agar plate into two portions by marking on the bottom with a wax pencil. On one portion of the plate streak out the culture of streptococcus hemolyticus, on the other streak out streptococcus viridans. 3. Streak out material from infected animal on the second blood agar plate. Incubate cultures at 37° C. B. M-aterial needed: 3 bouillon tubes 1 dextrose agar plate 1. Study plate culture of streptococcus hemolyticus and strepto- coccus viridans. Observe characteristics of isolated colony, and examine for hemolysis or methemoglobin formation about colonies. 2. Fish from isolated colonies of each form of streptococcus; inoculate plain and dextrose bouillon tubes and streak out on a portion of dextrose agar plate. 3. Examine plate culture of material from infected animal for colonies typical of streptoccccus hemolyticus. Fish from isolated colonies to bouillon tube and to a portion of dextrose agar plate. C. Material needed: 3% washed red blood cell suspension pH indicators Sterile bile or bile salt solution Wasserman tubes Water bath at 37° C. 62 1. Observe character of growth in bouillon cultures of streptococci. Examine by fresh and stained preparations, making- preparations from sediment in tubes and from supernatant fluid. 2. Study agar plate cultures with hand lens and low power micro- scope and observe colony characteristics. 3. Test for fermentation of dextrose by pH change in dextrose bouillon cultures. 4. From plain bouillon cultures of each type of streptococcus, pipette 15 drops into each of two small test tubes. To the first add 10 drops of 3% red blood cell suspension, to the second tube 5 drops of sterile bile or bile salt solution. Prepare control tubes of blood plus sterile bouillon, and culture plus sterile bouillon. Place in water bath one hour. Examine for hemolysis, methemoglobin formation and solution by bile. Carry out appropriate tests to identify organism isolated from infected animal as streptococcus hemolyticus. References : Gay — Recent Aspects of Streptococcus Infections. Jour. Lab. and Clin. Med., vol. 3 (1918), p. 3. Methods for the Isolation and Identification of S. hemolyticus. Adopted by the Medical Department, U. S. Army, 1918. Holman — The Classification of Streptococci. Jour. Med. Res., vol 34 (1916), p. 377. Foster — The Biochemistry of Streptococcus hemolyticus. Jour. Bact., vol. 16 (1921), p. 211. Dochez, Avery and Lancefield — Studies on the Biology of Streptococcus. I. Antigenic Relationships between Strains of Streptococcus hemolyticus. Jour. Exp. Med, vol. 30 (1919), p. 179. Lancefield, R. — Antigenic Complex of Streptococcus Hemolyticus. J. Exp. Med., 48, 1928, 91, 469, 481. Dochez, A. R. — Etiology of Scarlet Fever. Medicine, 4, 1925, 251. Dick & Dick — Results with Skin Test for Susceptibility to Scarlet Fever. J. A. M. A, 84, 1925, 1477. 7. Septicaemia. Inoculate rabbits intravenously with pathogenic strain of strepto- coccus hemolyticus. Follow course of infection by observations of temperature, weight, and blood cultures. Autopsy. Make culture from hearts blood and from organs. Hopkins and Parker, Jour. Exp. Med, 27, 1918, p. 1. Zinsser, Infection and Resistance. 8. Sero-Fibrino-Purulent Exudate. Produce streptococcus empyema in a rabbit by intrapleural inocu- lation. Examine exudate daily by thoracentesis. Follow course of temperature and weight, and take blood cultures. Autopsy. Examine pleurae in the gross and in microscopic section, and make cultures. Gay and Stone, J. Inf. Dis, 26, 1920, p. 268. Gay and Morrison, J. Inf. Dis, 33, 1923, P 337. 64 Exercise 15 Genus Neisseria (Gram Negative Diplococcus Group) Strict parasites, failing to grow or growing very poorly on usual artificial media. Cells normally in pairs, occasionally in tetrads. Gram negative. The pathogenic members of this group are : (1) Neisseria intracel- lular or meningococcus, the organism causing epidemic meningitis and found in the inflamed meninges and blood stream of patients, and in the naso-pharynx of patients and healthy carriers: (2) the Neisseria gonorrhoeae, or gonococcus, causing urethritis and less commonly conjunctivitis and arthritis. True infection in the ordi- nary laboratory animals is not possible and stock cultures are the source of class material. It should be borne in mind in connection especially with this and the following group of bacteria, that in be- coming adapted to culture media the bacteria have lost many of the characteristics of freshly isolated strains. A. Material needed: 24 hr. cultures of meningococcus gonococcus micrococcus catarrhalis Films of pus from urethritis 3 deep dextrose agar tubes 2 c.c. sterile ascitic fluid 2 c.c. sterile laked blood 3 Petri plates 1. Melt agar and cool to 45°. Pour plates of simple dextrose agar, ascitic fluid agar and laked blood agar, mixing the ascitic fluid and the blood thoroughly with the agar as directed in a previous exercise. 2. Divide each plate into three compartments with the wax pencil. Make streak inoculations in one compartment of each plate with each of the cultures supplied. Incubate at 37°. 3. Make fresh and stained preparations of the stock cultures, using both methylene blue and Gram stains, and stain the films of urethritis pus supplied. Note characteristics of morphology and staining reaction, and grouping of cocci in pus films. B. Material needed: Saline suspension of meningococcus (killed) Anti-meningococcus serum 1/20 Normal horse serum 1/20 3 Wassermann tubes Water bath at 55° 2 dextrose ascitic agar slants, with Andrade or litmus indicator 2 maltose ascitic agar slants, with Andrade or litmus indicator 66 1. Study plate cultures. Note characteristic differences in growth of the different organisms. Examine isolated colonies with the hand lens, as well as with the low power microscope. Attempt to identify the meningococcus by its colony characteristics, especially on the laked blood medium. 2. Inoculate the meningococcus and gonococcus upon dextrose and maltose fermentation agar slants. Incubate at 37°. 3. Examine stained films of growth on plates. Repeat Gram stain if necessary to show characteristic staining reaction. 4. With capillary pipette place 15 drops of saline emulsion of meningococcus into each of three small tubes. To the first tube add 15 drops of saline, to the second tube 15 drops of normal horse serum diluted 1/20 and to the third 15 drops of antimeningococcus serum diluted 1/20. Place tubes in waterbath at 55° for thirty minutes. Agitate tubes by flicking with the finger after the first five minutes incubation. Examine tubes for agglutination, using the hand lens if gross agglutination is not apparent. References : Meningococcus Elser and Huntoon — "Studies on Meningitis." Jour. Med. Res. (1909), vol. XX, p. 371. Flexner — Mode of Infection, Means of Prevention and Specific Treatment of Epidemic Meningitis. Jour. A. M. A., vol. 69 (1917). Foster and Gaskell — Cerebro-Spinal Fever. Cambridge University Press, England. 1916. Gonococcus: Cole and Lloyd — Preparation of Solid and Liquid Media for the Cultivation of the Gonococcus. Jour. Path, and Bact., vol. 21 (1917), p. 267. Cooke and Stafford — A studv of the Gonococcus and Gonococcal Infection. Jour. Infect. Dis., vol. 29 "(1921), p. 561. Torrey and Buckell — A Serological Studv of the Gonococcus Group. Jour. Immunology, vol. 7 (1922), p. 305. Infection and Immunity 9. Phagocytosis. The action of leucocytes on bacteria. Prepare the peritoneal cavity of a guinea pig by injecting sterile broth or aleuronat-starch mixture into the cavity. On the following day inject intraperitoneally a small quantity of a broth culture of a coccus. Withdraw small amounts of fluid from the peritoneum at 15 minute intervals. Prepare films, fix with methyl alcohol and stain by Wright stain. 10. Action of macrophages on nucleated red blood cells. Inject suspension of washed pigeon or hen erythrocytes into the peritoneal cavity of a guinea pig which has been prepared by the injection of gum-arabic beef extract broth 48 hours previously. Withdraw fluid 68 at 15 minute intervals. Stain fresh preparations woth neutral red; Prepare films, fix with methyl alcohol and stain with Wright stain. References : Kolmer, p. 1121 Gay and Morrison— J. Inf. Dis., 33, 1923, 338 Gay and Clark— Arch. Path. & Lab. Med., 1, 1928, 847 Zinsser — Infection and Resistance 11. Mechanism of Action of Opsonin. Obtain pleural exudate from a rabbit, suspension of cocci (Meningococcus or Pneumo- coccus), and normal and immune horse or rabbit sera. (a) Demonstrate difference in phagocytosis due to presence of normal and immune opsonin. (b) Determine whether the action of opsonin is on the bacteria or on the leucocytes. References : Zinsser et al, p. 168 Kolmer, p. 1171 70 Exercise 16 Genus Vibrio Short slightly curved rods, single or united into spirals. Motile, 1 to 3 polar flagella. Gram negative. Vibrio cholerae is the principal member and probably the only one of the group that is pathogenic for man. The organism is found in the intestinal canal of patients and carriers. A. Material needed: 24 hr. cultures of V. cholerae V. metschnikovi 2 deep agar tubes 2 Petri plates 1. Pour agar plates. Make streak inoculations on the surface of the medium from the cultures of two types of Vibrio. Care should be taken to make the inoculation sufficiently light. 2. Make stained and fresh preparations from stock cultures. Examine for motility. B. Material needed: 2 gelatin tubes 2 alkaline agar slants 2 Dunham's peptone solution Study agar plates. From isolated colonies of each organism make inoculation on the test media. 1. Examine gelatin tube for liquefaction. 2. Test Dunham's peptone solution for indol production. 3. Examine growth on alkaline agar medium. Compare with growth of B. coli on the same medium. In practical work the two sources from which the isolation of the cholera vibrio is usually attempted are water, and the stools of patients and convalescents. With either of these materials pre- liminary enrichment by growth in peptone water is of advantage as, during the first five to eight hours of incubation, the cholera vibrio outgrows other forms in this medium and accumulates in a pellicle on the surface. This pellicle is then plated out on gelatin or Dieu- donne's alkaline agar. Reference : Teague and Travis— Tour. Inf. Dis. 18, 1916, p. 601. Infection and Immunity 12. Bactericidal Action of Normal Serum. Demonstrate the bactericidal action of normal serum. Fol- low the method described in Zinsser, Hopkins and Otten- berg, p. 20, but use V. metschnikovi instead of B. typhosus. Reference : Zinsser et al., p. 20. Kolmer, p. 357. 13. Bacteriolysis. The Pfeiffer phenomenon in vitro. Immunize a rabbit against V. cholerae. Assist instructor in trial bleeding and titration. Read Bordet and Gay, p. 56, and Kolmer, pp. 385 and 1151, on the Pfeiffer phenomenon. Out- line a method for demonstrating this phenomenon in vitro, with rabbit anticholera serum and guinea pig alexin. Demon- strate bacteriolysis by the use of dilution plates and by films stained with dilute carbol fuchsin. 14. Normal Hemolysin. Demonstrate the hemolytic power of normal blood serum for foreign cells, using serum and cells of laboratory animals. Reference : Zinsser et al., p. 22. Kolmer, p. 394. Kolmer & Casselman, J. Inf. Dis., 16, 1915, 441. 15. Experimental Hemolysin. Immunize a rabbit against sheep ery- throcytes. Make a trial bleeding and test the hemolytic power of the serum. Make final bleeding when rabbit is sufficiently immunized. Demonstrate (a) Dual nature of hemolytic action. (b) Specificity of hemolytic serum. (c) Union of sensitizer with cells. References : Bordet & Gay, p. 134. Zinsser et al., pp. 26, 34, 46. Zinsser. Infection and Resistance. 74 Exercise 17 Genus Proteus Pleomorphic rods. Motile, flagella peritrichous. Gram negative. Dextrose and saccharose fermented, not lactose. Actively proteo- lytic and occasionally hemolytic. The proteus group is widely distributed in nature, and represents the common organisms of aerobic putrefaction. Its chief import- ance in medical bacteriology is its frequent occurrence as a con- taminant in cultures, especially from organs at autopsy. It appears infrequently as a pathogen of considerable virulence in deep-seated abscesses. It is as a rule easily identified by the amoeba-like rapidity spreading colonies which it develops on moist solid media. Genus Pseudomonas (Fluorescent Group) Slender rods, actively motile. Gram negative. Carbohydrates utilized. Proteolytic. Characteristic pigment produced. B. pyocyaneus is the only member of the chromobacteriae of pathogenic significance. It is encountered essentially as a sapro- phyte in suppurative processes due to other organisms. Its inde- pendent pathogenicity is apparently very low. Its persistence in wounds and hospital wards is remarkable. A. Material needed: 3 gelatin tubes 24 hr. cultures of B. proteus 3 Petri plates B. pyoc}% H CI, rinsed in clear water, dried, and polished with old linen. Used slides should be treated with xylol to soften the dried cedar-oil or balsam, then boiled in a solution of soap or washing-soda. If the slide is perfectly clean, a drop of water may be spread over its sur- face in a thin even film ; if it is greasy, a drop of water collects into small droplets and a film cannot be spread. Cover Slips: The most useful sizes are the 18 mm. squares or circles for ordi- nary preparations. Larger rectangular slips are better for covering blood-films and serial sections. Cover slips, which have been used, or are greasy, should be treated as used microscopic slides by boiling in soap or soda solution and then cleaned by dropping into alcohol in an evaporating dish. Concentrated HXO s is then added. This should be done under a hood. After a few minutes the acid reacts with the alcohol and fumes of N0 2 are given off. Warming may be necessary to start the process. After thorough rinsing, the slips may be dried and polished, or kept in alcohol and dried as needed. Cover-slips should be handled only by the edges or with forceps. They may be kept ready for use in glass jars intended for the pur- pose, or in a clean tumbler covered with a Petri plate. Sterilization of Glassware: Sterilization by hot air is employed for dry glassware. It is also used for metals subject to rust. It cannot be used for culture media, for rubber or other organic materials. The dry-air sterilizer should be full each time it is run. for the sake of economy. It is heated by gas. The temperature should be raised gradually to 160° C, timed for one hour at 160° to 180°, and the gas turned off. It may be run at 180 for 30 minutes, but this temperature makes paper and cotton fibers very brittle. Never open the door until the chamber has cooled below SOX for fear of breakage due to draughts. All glassware should be dry before fitting plugs or placing in the sterilizing oven. Flasks and culture tubes should be plugged with non-absorbent cotton. Two types of plugs are used, rolled and pushed. Rolled plugs are best, but take longer to make. Pushed plugs are adequate for ordinary purposes. Both types will be demonstrated. A push plug for a standard tube, 150 x 18 mm. should extend 25 mm. inside the tube and 20 mm. outside. Large plugs for flasks 152 preserve the same proportions, and should always be of the rolled type. They are most satisfactory if wrapped in gauze or cheese- cloth to prevent fraying. Durham tube vials are placed open end downward in plugged culture tubes. Petri plates are fitted together and stacked cover side uppermost in the metal containers or wrapped in paper in groups of 2 or more plates. Pipettes are plugged loosely at the mouth end only and placed tip inward in the metal containers. The protruding threads of cotton should be singed off. Capillary pipettes for qualitative purposes are prepared as fol- lows : A length of glass tubing, 8 mm. diam. x 25 cm. long is plugged with cotton at each end. After sterilization, the tube is softened at the middle in the Bunsen flame and the two ends drawn apart slowly. The capillary portion should be about 40 cm. long. It is softened in the flame, and the two halves separated and sealed. Steam Sterilization at 100° C: This method is extensively employed in bacteriology for the steri- lization of culture-media, especially gelatin and those containing sugars, and for articles of rubber. Material to be sterilized is ex- posed at 100° C for 20 minutes on each of three successive days. The accepted principle of this intermittent or "fractional" method of sterilization is that one exposure is sufficient to kill all vegetative forms of bacteria; between the heatings the spores may pass into vegetative forms, which are destroyed during the subsequent heat- ings. The door should always be opened when time is up and the ma- terial being sterilized removed at once to avoid wetting the stoppers with water of condensation. 154 CULTURE MEDIA Culture media provide the artificial chemical and physical envi- ronment necessary for bacterial growth. They supply water, inor- ganic salts, and carbon, nitrogen and other elements in available form. Different species of bacteria vary greatly in their require- ments. The basis for the media ordinarily employed in the study of the common bacteria is nutrient broth or bouillon. Agar or gelatin is added merely to solidify the medium. To an aqueous extract of meat or beef extract, proteoses and other necessary nitrogenous compounds are added in the form of commercial peptone. The salt content and buffer value of the medium may be increased by the addition of sodium chloride and sodium phosphates. Media may be enriched by the addition of sugars, serum, blood or other substances. Hydrogen Ion Concentration: No requirement is more important than the proper hydrogen ion concentration. For the precise determination of the cH of a solu- tion, the electrometric method is employed. Sufficiently accurate results in the measurement of bacteriological culture media may be obtained by colorimetric methods. A comprehensive discussion of the methods may be found in : Clark, W. M. : The Determination of Hydrogen Ions. 3rd ed., Balti- more, Williams & Wilkins, 1928. Clark, W. M., & Lubs, H. A. : The Colorimetric Determination of Hydrogen Ion Concentration and Its Applications in Bacteriology. Jour. Bacteriology 1917, 2, 1, 109, 191. A summary will he found in most of the text books. In applying the colorimetric method, an indicator is selected which has its maximum color change in the neighborhood of the cH to be determined. As standards for comparison one may use a series of solutions of graded pH, with an indicator added, in test tubes; or a printed color chart made to correspond to such tubes may be used, with perhaps more convenience but less accuracy. For comparison of colored or turbid solutions a special device known as the comparator is useful. This is arranged so that through two apertures in a block of wood one may view light trans- mitted through the standard tube plus a tube of medium without indicator, and beside, the light through a tube of distilled water plus media with indicator. In this way the alteration of the standard 156 color by the turbidity and color of the medium is the same in both fields observed. In well buffered solutions such as culture media, dilution with distilled water does not appreciably alter the pH and is of advan- tage in reducing the turbidity, lessening the error from the color solution itself and delaying the solidification of agar solutions. In testing pure solutions of strong acids or alkalies, dilution introduces a large error. To determine the pH of an unknown solution a drop or two of the following indicators may be added to separate small portions of the solution in test tubes. Amount of Strength Indicator of added per Solution 10 c.c. of ph Range Color Range Used Solution Thymol blue (acid range) 1.2-2.8 Red-Yellow .04% l.Oc.c. Bromo-Phenol blue 3.0-4.6 Yellow-Blue .04% O.Sc.c. .Methyl red 4.4-6.0 Red-Yellow .02% 0.3c.c. Brom-cresol purple • 5.4-7.0 Yellow-Purple .04% 0.5c. c. Brom-thvmol blue 6.0-7.6 Yellow-Blue .04% 0.5c.c. Phenol red • 6.6-8.2 Yellow-Red 0.2% 0.5c.c. Cresol red 7.2-8.8 Yellow-Red .02% 0.5c.c. Thymol blue (alkaline range) . . 8.2-9.8 Yellow-Blue .04% 0.5c.c. Indicator solutions are prepared by treating the powder with normal alkali before dissolving. For details see Clark, loc-cit. If the solution is acid to one indicator and alkaline to another, its pH lies between the ranges of these two indicators. An indicator is then selected covering this range and the standard amount indi- cated in the table is added to 10 c.c. of the solution to be tested. This tube is then compared with a series of tubes of graded hydro- gen ion concentration containing the same indicator or with the color chart. The pH of the unknown solution is that of the tube which it most closely matches. The range of reaction of culture media ordinarily used in the study of pathogenic bacteria is satisfactorily covered by phenol red as an indicator. Take a clear tube containing 8 c.c. of distilled water and add 2 c.c. of the medium to be tested and 0.50 c.c. of phenol red solution (.02 c /c aqueous). To this add very cautiously X/20 XaOH solution from a burette until the color matches that of the standard tube of the reaction desired (for class work pH7.6). Multiply the burette reading by 25 and add this amount of N/1 XaOH to each liter of medium to be corrected. Mix well and check the correction by determining the pH of the corrected medium. 158 Titration of Total Acidity: The determination of total acidity, by titration to an arbitrary end-point (color change of phenolphthalein) was formerly the only means employed for the adjustment of the cH. This method gives a measure of the reserve acidity, and after adjustment to the proper cH, of the reserve alkalinity of a culture medium, and as such is still occasionally of value in determining the buffer value of a medi- um. It is used according to the directions for the preparation of "standard" media as follows : To 45 c.c. distilled water in a white enameled dish or beaker, add 1 c.c. phenolphthalein solution (0.5 per cent in 50 per cent ethyl alcohol). Boil 1 minute. Test for neutrality by adding 1 drop N/20 NaOH, which should give a dis- tinct purple color in the originally clear fluid. Add 5 c.c. of the media to be tested. Again boil 1 minute. Read the burette. Run into the hot mixture enough X/20 XaOH to produce a faint but distinct and permanent pink color. Standard methods of water analysis prescribe a color produced by a combination of 25 per cent red (wave length approximately 658) with 75 per cent white on the color top for comparison. Read from the burette the amount of N/20 XaOH used to neutralize the total quantity. Three or more trials should be made until the student can check his previous results within 0.1 c.c. burette read- ing. Record each result obtaining original data and select those which agree for computation. The burette reading gives the per- centage normality directly, since as much N/1 XaOH will be re- quired to neutralize 100 c.c. of media as X/20 to neutralize one- twentieth of 100 c.c. or 5 c.c. The burette reading therefore shows the number of cubic centimeters of N/1 XaOH required to reduce the reaction of 100 c.c. of medium to neutrality to phenolphthalein; but while phenolphthalein is most suitable for the titration of or- ganic acids, its turning point is slightly alkaline to the true neutral point, therefore the reaction of standard media is adjusted, not to neutrality to phenolphthalein. but to + 1 or N/100 acid. The num- ber of cubic centimeters of N/1 XaOH to be added to every 100 c.c. of media is therefore not that indicated by the burette reading, but that number minus 1. Check the result by repeated titration. 160 Liouid Media 1. Milk. Secure freshly separated milk. Test for freedom from exces- sive acid by heating a tubeful in a bath of boiling water for a few minutes ; if it coagulates on heating, it is necessary to add barely sufficient sodium carbonate as determined by titration to prevent coagulation. (If too much alkali is added, subsequent coagulation by bacterial growth is prevented and the usefulness of the medium destroyed; the practice is therefore not generally recommended). When it is shown that the milk does not coagulate on heating it may be tubed and sterilized in the Arnold sterilizer on three suc- cessive days for 30 minutes each day. Sterilization in the auto- clave renders milk unfit for culture media. If indication of acid production by bacteria is desired other than that shown by coagulation, sufficient concentrated blue litmus solu- tion, Andrade's indicator, or bromcresol purple may be added previ- ous to sterilization. 2. Nitrate Solution. (1) Mix the following: 0.1 per cent peptone. 0.02 per cent nitrite-free potassium (or sodium) nitrate. (2) Sterilize in the autoclave. 3. Dunham's Peptone Solution. Peptone (must contain tryptophane for indol test) 10 gms. NaCl C. P 5 gms. Distilled water 1000 c.c. Dissolve by boiling, filter through paper, and sterilize 30 minutes at 20 pounds pressure in the autoclave. 4. Meat Extract Bouillon. ( 1 ) Mix 3 gms. Liebig's Beef Extract. 10 gms. peptone. 5 gms. NaCl. 1000 c.c. distilled water. (2) Boil over free flame to dissolve. (3) Restore water lost by evaporation with distilled water to 1000 c.c. (4) Titrate and if necessary readjust to +1 (N/100 acid) to phenolphthalein. (5) Sterilize in the autoclave under 15 pounds pressure for 30 minutes. (6) Filter through paper; distribute as required. (7) Sterilize as in step 5. 5a. Meat Infusion Bouillon. 162 (1) Stir 1 lb. of very fresh lean chopped beef heart into 1 liter of water. (Distilled preferred.) (2) Boil vigorously for 8-10 minutes. (3) Strain through cheese-cloth and restore to its original volume with distilled water. (4) Add (Bacto) Peptone — 1% NaCl — 0.5% and stir until fully dissolved (5) Titrate and adjust to pH 7.8-8.0. (6) Heat in autoclave at IS pounds pressure for 10 minutes. (7) Filter through one paper until clear. (8) Sterilize in autoclave at 15 pounds pressure for 15-20 minutes. b. Carbohydrate Broth. The above prepared meat infusion after the addition of sugars or other carbohydrates may be used for differential and diagnostic purposes. (1) Inoculate a sterile flask of broth with culture of B. coli communis and incubate for 24 hours. The bacteria will ferment any monosaccharids which may be present in the broth — thus rendering it sugar free and acid. (2) Autoclave for half -hour to kill B. coli. (3) Add a small amount of technical Kieselguhr to clog the filter for clearing the medium. (4) Filter through paper. (5) Add 1% of the carbohydrate desired. (6) Titrate and adjust to pFI 7.8. (7) Tube and sterilize in the Arnold for one-half hour on three successive days. The autoclave should not be used in this case because the high temperature will tend to split up the more complex sugars. c. Indicators. To facilitate the determination of acidity in culture media due to bacterial growth indicators are added, either before sterilization or after growth has occurred. Litmus Solution is prepared by adding 5% of Merck's purified, or Kahlbaum's litmus to distilled water. To dissolve, the solution must be heated in the Arnold for 2-3 hours, with occasional shaking. It is then filtered through paper and sterilized. It should be kept sterile as molds will grow m it otherwise. It is added to culture medium to a concentration of 3% to 5%. Andrade Indicator is made up in the following manner : Acid f uchsin 0.5 ( ", 164 Distilled water - 100 c.c. N/1 NaOH - 16 c.c. Filter through paper — add to the medium to a concentration of 1%. Upon the addition of Andrade, the medium takes on a red color. This color should fade within one to two hours. If it remains red or slightly reddish, the medium should be readjusted to a higher alkalinity. Acid production turns Andrade Indicator red. Phenolphthalein Indicator. The medium should be alkaline to this indicator. Sufficient 1-2% alcoholic solution must be added to produce a faint pink color. This color is discharged when acid is produced. Other Indicators such as Phenol Red, Brom-cresol-purple and Cresol Red are used. See p. 158. These above indicators may be reduced by bacterial action, so a very small amount should be added after growth has occurred. 6. Buffered Broth. The addition of alkaline phosphates or other salts to liquid medi- um renders it especially favorable for many delicately growing or- ganisms. 0.5% Xa 2 HP04 or MgC0 3 may be added to meat in- fusion broth. This obviates the necessity of titration, and if left in excess automatically prevents the accumulation of a high acidity. This medium is eminently suitable for cultivating such organisms as streptococcus, pneumococcus and obligative anaerobes. Sugars should be added aseptically as excessive heating in the presence of a slightly alkaline reaction caramelizes the carbohydrate, thus giving the medium a dark color, and to a degree inhibiting growth. 7. Gelatin. Meat extract bouillon is used as a base for this medium. (1) Add 18-22% gelatin. (2) Heat in the Arnold occasionally stirring until fully dis- solved. ( 3) Titrate and readjust to pH 7.8-8.0. (4) Cool to 50°C. (5) Add well beaten white of an egg (1 egg for each liter of medium). (6) Coagulate by heating in the Arnold for 45 minutes. (7) Filter through cotton which has been placed in a steaming hot funnel. (8) Tube 12 c.c. each. 166 (9) Sterilize in Arnold sterilizer 30 minutes on three successive days. 8. Meat Infusion Agar. This medium is prepared with meat infusion bouillon serving as a base. (1) Add to the already prepared broth 2% of agar-agar. (2) Heat in autoclave for 40 minutes at 15 pounds pressure to dissolve the agar. (3) Cool to 80°C. Titrate and adjust to pH 7.8-8.0. (4) Cool to 60° C. and then add well beaten white of one tgg to every liter of media. (5) Autoclave for 40 minutes at 15 pounds pressure to coagu- late the tgg albumin. (6) Filter through a non-absorbent cotton filter; the funnel should be kept hot by a steam jacket or heating coil. (7) Sterilize in autoclave. 9. Blood and Serum Agar. Beside the use of carbohydrates, serum, blood, or albuminous fluids may be added to enrich media for producing growth. One part of the enriching substance is usually added to three parts of the medium under aseptic conditions. In adding enriching substances (blood, serum) to melted agar it must be first cooled to 45 °C. Then, in case of a slant, the tube must be placed at the desired angle and left to harden. Blood may be added in the form of whole blood, defibrinated, or citrated blood. 10. Chocolate Agar, especially used for influenza bacillus, is made by adding blood to melted agar already cooled to 50° C. and then reheated to 85 °C. for 1-2 minutes. It is then allowed to harden. 11. Endo Medium. For the isolation of typhoid and allied bacilli. The basis is ordi- nary beef extract agar, slightly alkaline to litmus or about pH 8.0. This should be sterilized in bottles in 250 c.c. amounts. When needed, 1% lactose is added and the agar is autoclaved for 20-30 minutes. This serves the double purpose of melting the agar and sterilizing the sugar. Decolorize with 1%-1.5% of a freshly pre- pared 10% watery solution of sodium sulphite. The color of the agar while fluid should be pinkish and almost colorless when con- gealed. Plates are poured and allowed to harden. The medium must be mixed each time it is needed and the plates used fresh. Kliger and Defandorfer recommend a reaction of pH 7.6 to 7.8 for the basic agar, the substitution of the bisulphite salt and the addi- tion of 0.5 c.c. of a fuchsin-sulphite mixture (10 c.c. of 10% 168 NaHS0 3 with 1 c.c. of saturated alcoholic fuchsin), for the B. dysenteriae group. Plates should not be poured too hot as the con- sequent water of condensation will interfere with the isolation of individual colonies. 10. Eosin Ad ethylene Blue Agar. (1) Prepare meat extract agar adjusted to pH 8.0. (2) Add 0.5% each of lactose and saccharose. (3) Heat in the Arnold for 10 minutes. (4) To 1 liter of agar add 20 c.c. of 2% yellow eosin and 20 c.c. of 2% methylene blue. (5) Tube 12 c.c. to a tube and sterilize in autoclave for 20 minutes at 15 pounds pressure. (6) When needed melt and pour into sterile Petri dish. 11. Eosin Brilliant Green Media. For use in isolating typhoid organisms from urine. (1) To 50 c.c. of already prepared melted meat extract agar add 0.5 grams lactose, 1 c.c. of 3% eosin solution and 1 c.c. of brilliant green solution. (2) Tube 15 c.c. to a tube and sterilize in autoclave for 20 minutes, at 15 pounds pressure. (3) When needed melt and pour into sterile Petri dish. 12. Russell's Sugar Media. (1) Prepare one liter of meat extract agar adjusted to pH 7.8. (2) Add 1% lactose. 0.1% glucose. (3) Add either sufficient litmus to give it the proper color or 1% Andrade Indicator. (4) Tube and slant, leaving a generous "Butt" at bottom of the tube for stab inoculation. It has been found of great help in isolating para-typhoid bacilli to add 1% saccharose. The fermentation of this sugar distinguishes para-typhoid-like bacilli which are frequently found in stools. 13. Hiss Serum Water Medium. (1) To fresh beef blood serum add three volumes of distilled water. (2) Heat in Arnold for 15 minutes. (3) Add enough concentrated litmus solution to obtain a deep blue color. (4) While hot, add 1% of the carbohydrate desired. (5) Test reaction by addition of 1 c.c. N/20 HC1 to 5 c.c. of media. A distinct pink should result. If necessary, 170 adjust with a few drops of N/1 NaOH or HO, as the case may be. (6) Sterilize in Arnold for 25 minutes on three successive days. 14. Dorset Egg Medium. The eggs are thoroughly cleaned with water of any adherent dirt, and then washed with 5% carbolic solution and allowed to partially dry. The ends are then gently dried in the flame and pierced with sharp forceps, which have been flamed. The hole at one end should be about }i inch in diameter and the membrane broken ; at the other end, which is to be blown into, the hole should be smaller and the membrane left unbroken, if possible. The eggs are then blown into a sterile Erlenmeyer flask, the blowing being done from the cheeks. To the egg is then added water (10 per cent by volume of the weight of the eggs). This is mixed by twirling the flask or by gently stirring with a glass rod. Bubbling must be avoided. The mixture is then filtered through cheesecloth by gravity and tubed. The tubes are then slanted and coagulated by heating to 70° C. for two or two-and-a-quarter hours on two successive days. No further sterilization is employed. The medium is incubated to test its sterility. 15. Pctrojf's Egg Medium. Five hundred grams of beef or veal are infused in 500 c.c. of a 15 per cent solution of glycerin in water, in a cool place. After twenty-four hours the meat is squeezed in a sterile press and the infusion collected in a sterile beaker. The shells of eggs are steri- lized by immersion for ten minutes in 70 per cent alcohol. They are broken into a sterile beaker, well mixed and filtered through sterile gauze. One part of meat juice is added to two parts of egg by volume. One per cent alcoholic solution of gentian violet is added to make a final proportion of 1 : 10,000. The three ingredients are well mixed. The medium is tubed and inspissated as with the Dorset medium. 16. Lead Acetate Agar. (1) To meat extract agar (1 per cent), adjusted to pH. 7.0 to 7.2 add 1 per cent glucose. (2) Tube 5 c.c. to the tube for stabs and sterilize in the auto- clave at 15 pounds for 15 minutes. (3) While still hot add aseptically to each tube x / 2 c.c. of a 5 per cent sterile solution of lead acetate and roll. (4) This will give a concentration of .05 per cent lead acetate to the resulting media. 17. Sabourand's Maltose Agar Medium for the cultivation of fungi. 172 This medium to be satisfactory must be prepared from crude maltose and to be accurate should be made from ingredients which are obtained at E. Cogit et Cie., Blvd. St. Michel, Paris, France. Maltose Brute de chanut 40 grams Peptone de Chassaing 10 grams Agar 1 5 grams Distilled water 15 c.c The reaction is defined only by the reaction of the special in- gredients. It is about pH 6.5. 19. Casein Digest Medium. Medium for the Cultivation of Lactobacillus Group. (1) Boil solution containing H 2 1 liter Peptone 1 5 grams Beef extract 3 grams (2) Filter and cool to 60°C. (3) Add Casein 1 5 grams Trypsin 3 grams (4) Titrate and adjust to pH 7.4. (5) Add 5% chloroform to prevent growth. (6) Incubate at 37° for 48 hours. (7) Boil for several minutes to evaporate the chloroform. (8) Add 15 grams of agar and heat to dissolve. (9) Titrate and adjust to pH. 7.0. (10) Add the whites of eggs; autoclave to coagulate. (11) Filter. (12) Add 15 grams of lactose. (13) 50 c.c. of 2% Sodium Oleate Solution. (14) Sterilize. 20. Sea Water Medium. (1) Obtain sea water. (2) Autoclave and filter. (3) Add. Peptone 1 % NaCl 0.5% Beef extract 0.3% Agar 2% (if desired) (4) Autoclave. (5) Titrate and adjust to pH 8.0. (6) Filter through same filter until clear. (7) Tube and sterilize in autoclave for 25 minutes at 15 pounds pressure. 174 COMPARATIVE TABLES OF WEIGHTS AND MEASURES 1 inch = 2.54 centimeters. 1 foot =0.3048 meters. 1 yard = 0.9144 meters. 1 mile =1.61 kilometers. 1 micromillimeter (micron) =0.000001 meter = 0.00003237 inch. 1 millimeter = 0.001 meter = 0.3937 inch. 1 centimeter = 0.01 meter = 0.3937 inch. 1 decimeter = 0.1 meter = 3.937 inch. 1 meter =1 meter = 39.37 inches = 3.28 feet. .1 kilometer = 1000 meters = 3281 feet. 1 grain = 0.0648 grams. 1 ounce, avoirdupois = 28.35 grams. 1 ounce, troy = 31.10 grams. 1 pound, avoirdupois = 453.6 grams. 1 pound, troy = 373.2 grams. 1 gram =15.43 grams. 1 kilogram = 2.205 pounds, avoirdupois. 1 kilogram = 2.679 pounds, troy. 1 gallon, U. S. Liquid=3785 cubic centimeters. 1 quart, U. S. Liquid =946 cubic centimeters. 1 pint, U. S. Liquid = 473 cubic centimeters. 1 ounce, U. S. Liquid= 29.57 cubic centimeters. 1000 cubic centimeters =1.057 U. S. Liquid quarts. CENTIGRADE AND FAHRENHEIT THERMOMETER SCALES Centigrade Fahrenheit —0.4° Freezing Blood heat Temperature of inactivation of sera Thermal death point of most non-sporulat- ing bacteria Water boils at sea level, 760 mm. Hg. 5 pounds steam pressure 10 pounds steam pressure 15 pounds steam pressure 20 pounds steam pressure 176 -18° —0.4' 0° 32° 37 P 98.6 C 56° 132.8 C 60° 140° 100° 212° 107.8° 226° 115.5° 240° 121.1° 250° 126.7° 260° Medical Bacteriology STAINS AND REAGENTS The primary step in the preparation of many stains is the making of saturated solutions, which for the following stains contain the designated number of grams of solid stain per 100 cubic centi- meters : Aqueous Alcoholic Methylene blue 1 6.68 0.66 Gentian violet 1 1.75 4.42 Basic fuchsin 1 0.66 2.94 In practice it is customary to add about 20 per cent in excess of these figures, shaking thoroughly and allowing to stand ; there should be a slight deposit of dye in the bottom of the bottle, indicating really a supersaturated solution. The supernatant fluid must be filtered for use and any insoluble residue may be restored to the bottle. 1. Loe flier's Methylene Blue. 2 Saturated alcoholic solution methylene blue 30 c.c. Aqueous KOH 1-10,000 100 c.c. 2. Sterling's Modification of Gram's Method. 3 Anilin oil 2 c.c. Alcohol, 95 per cent 10 c.c. Distilled water 88 c.c. Gentian violet 5 gms. Shake anilin oil and alcohol and add 88 c.c. distilled water. Grind gentian violet in a mortar and add anilin solution slowly while grind- ing. Filter. This solution keeps and stains in one-half to 1 minute. 3. Gram's Iodine Solution. Iodine crystals ., 1 gm. Potassium iodide 2 gms. Distilled water q. s 300 c.c. Grind in mortar with 5 c.c. distilled water. 1 Stitt, Practical Bacteriology, Blood Work, Parasitology. New York, Blakiston. 2 Loeffler, Mit. a. d. k. Gesundh., Bd. 2 (1884), S. 421. 3 Gram, Fortschr. d. Med., Bd. 2 (1884), S. 185; U. S. Army Medical War Manual No. 6, p. 27. 178 Medical Bacteriology 4. Bismarck Brown Counterstain. Bismarck brown r 10 gms. Distilled water 1000 c.c. Boil to dissolve, cool and filter. 5. Safranvn, Counterstain. Safranin 5 gms. Distilled water 1000 c.c. Boil to dissolve, cool and niter. 6. Ziehl-Nielson Carbol Fuchsin* Basic fuchsin 10 gms. Ethyl alcohol, 95 per cent 100 c.c. Phenol, 5 per cent aqueous 1000 c.c. Dissolve and filter. 7. Acid Alcohol for Testing Acid-fast Organisms. Ethyl alcohol, 95 per cent 800 c.c. HC1 C. P. Cone. : 30 c.c. 8. Neisser Stain for Diphtheria Bacilli. (1) Methylene blue 1 gram Alcohol, 96% 20 c.c. Glacial acetic acid 50 c.c. Water 950 c.c. (2) Bismarck brown 0.2% solution. 9. Toluidin Blue Stain for Diphtheria. Toluidin blue 0.25 gms. Acetic acid 2.0 c.c. Absolute alcohol 5.0 c.c. Distilled water 100.0 c.c. 10. Giemsas Stain (old). Mallory and Wright, Pathological Technique, p. 427. Azur II eosin 0.3 gin. Azur II 0.8 gm. Glycerine 250. gms. Methyl alcohol 250. gms. 180 \ 11. Nitrite Reagents 1. Sulphanilic Acid. Dissolve 0.5 gm. in 150 c.c. of acetic acid of Sp. Gr. 1.04. (Acetic acid of 1.04 prepared by diluting 400 c.c. of cone, of sp. gr. 1.75 with 700 c.c. of water.) 11. A-Napthylamin. Dissolve 0.1 gr. in 20 c.c. of water, boil, filter (if necessary) and to clear filtrate add 180 c.c. of acetic acid, Sp. Gr. 1.04. 12. Ehrlich's Indol. Reagent (A) Paradimethylamidobenzaldehyde 4 parts Absoltue alcohol 380 parts Concentrated HO 80 parts 13. Andrade's Indicator. Acid fuchsin 0.5 gms. Distilled water 100. c.c. N/1 NaOH 16. c.c. Add 1 c.c. per 100 c.c. of media. 14. Liquor Creosolis Compositus. Linseed oil 300 gms. Cresylic acid 500 gms. NaOH 54 gms. Alcohol 30 c.c. Water to make 1000 c.c Heat the oil in a vessel over water bath at 70° C. Dissolve NaOH in 50 c.c. water, warm to 70°C. and add to the oil. Mix thoroughly, add the alcohol, and continue heating till a small portion tested is found soluble in boiling water without the separation of an oily layer. While yet warm add the cresylic acid and mix thoroughly. Keep at 70°C. until a clear solution is produced. Finally add the remainder of the water to make a total volume of 1000 c.c. This solution can be used in 1 to 2 per cent as a hand wash, and slightly stronger for general disinfection purposes. 182 CHESTER'S TERMINOLOGY FOR DESCRIPTION OF COLONIES 1. Form of Colonies. Plate Culture Puntiform : dimensions too slight for defining form by naked eye, minute, raised, semispherical. Round : of a more or less circular outline. Elliptical : shape of an ellipse. Fusiform : spindle-shaped, tapering at each end. Cochleate: spiral or twisted like a snail shell. Amoeboid : very irregular, streaming. Myceloid: a filamentous colony with the radiate character of a mold. Filamentous : an irregular mass of loosely woven filaments. Floccose : of a densely wooly structure. Rhizoid : of an irregular branched, rootlike character, as in Bad. mycoides. Conglomerate : an aggregate of colonies of similar size and form. Toruloid: an aggregate of colonies like the budding of the yeast plant. Rosulate : shaped like a rosette. 2. Detailed Character of Surface Smooth : surface even, without any of the following distinctive characters. Alveolate : marked by depressions separated by thin walls, so as to resemble a honeycomb. Punctuate : dotted with punctures like pin pricks. Bullate: like a blistered surface, rising in convex prominences, rather coarse. Vesicular : more or less covered with minute vesicles, due to gas formation; more minute than bullate. Verrucose : wartlike, bearing wartlike prominences. Squamose : scaly, covered with scales. Echinate : beset with pointed prominences. Papillate : beset with nipple-or mamma-like processes. Rugose : short, irregular folds, due to shrinkage of surface growth. Corrugated: in long folds, due to shrinkage. Contoured : an irregular but smoothly undulating surface, like the surface of a relief map. Rimose : abounding in chinks, clefts, or cracks. 184 3. Internal Structure of Colony (Microscopic) Amorphous: without definite structure as below specified. Hyaline : clear and colorless. Homogeneous: structure uniform throughout all parts of the colony. Homochromous : color uniform throughout. Granulations or blotchings. Finely granular. Coarsely granular. Grumose: coarser than the preceding, a clotted appearance, par- ticles in clustered grains. Moruloid : having the character of a morula, segmented, by which the colony is divided into more or less regular segments. Clouded: having a pale ground, with ill-defined patches of a deeper tint. 4. Colony Marking or Striping (Surface) Reticulate : in the form of a network like the veins of a leaf. Areolate : divided into rather irregular or angular spaces by more or less definite boundaries. Gyrose : marked by wavy lines indefinitely placed. Marmorated: showing faint, irregular stripes, or transversed by veinlike markings as in marble. Rivulose : marked by lines, like the rivers of maps. Rimose: showing chinks, cracks or clefts. Filamentous : as already defined. Floccose : composed of filaments densely placed. Curled: filaments in parallel strands, like locks or ringlets, as in agar colonies of B. authracis. 5. Edges of Colonies Entire: without toothing or division. Undulate: wavy. Repand : like the border of an open umbrella. Erose : as if gnawed, irregularly toothed. Lobate: border broadly rounded with equally broad sinuses. Lobulate : minutely lobate. Auriculate: with earlike lobes. Lacerate : irregularly cleft, as if torn. Fimbriate: fringed. Ciliate : hairlike extensions, radiately placed. Filamentous : as already defined. Curled: as already defined. 186 6. Optical Characters (After Shuttlewokth) Transparent : transmitting light Vitreous : transparent and colorless. Oleaginous: transparent and yellow :.live tc linseed oil tolored. Resinous: transparent and brown, varnish tc resin colored. Translucent: fairly transparent. Porcelaneous : transparent and white. Opalescent: translucent, grayish white by rehtitei light smoky brown by transmitted light Nacreous : translucent, grayish white, with pearly luster. Sebateous: transiu:ent. yellowish or grayish white. Butyrous : translucent and yellow. Ceraceous : translucent and wax colored. Dpaque: not transmitting light. Cretaceous: opaque and white thalky: dull without luster. Dull : without luster. Glistening : shining. Iridescent: rainbow-like colors. :-i CLASSIFICATION AND IDENTIFICATION OF MICRO-ORGANISMS Artificial Key to the Classes of Protozoa KINGDOM. Animals. Phylum I. Protozoa — one-celled animals. Lacking permanent structure for locomotion and nourish- ment, these functions being performed by means of pseu- dopodia. Class 1. Sarcodina Locomotion by flagella Class 2. Mastigphora Locomotion by cilia Class 3. Infusoria Always parasitic in metazoa, without specialized means for ingestion of food, which is obtained by osmosis — reproduc- tion by sporogony Class 4. Sporozoa For details of further classification consult: Kingsley, Hertwig's Manual of Zoology (New York, Holt). Calkins, Protozoology (New York), Besson, Practical Bacteriology, Microbiology and Serum Therapy; trans. H. J. Hutchins (London, Longmans, 1913). Artificial Key to the Classes of Non-Chlorophyllaceous Thallophyta KINGDOM. Plants. With seeds Subkingdom Phanerogamia Without seeds „ _ Subkingdom Cryptogamia Roots, stems and leaves clearly differented. Phylum Pteridophyta (ferns) Roots, stems and leaves imperfectly differentiated, archegonia present. Phylum Briophyta (mosses) Roots, stems and leaves not differentiated. Phylum Thallophyta (algae, fungi, and bacteria) With chlorophyll (algae). Without chlorophyll. Spores reproductive in function (fungi).* Reproduced sexually by zygospores or oospores, asexually by conidia, hypae aerial. Class Hemiascomycetes 190 Reproduction by spores, borne in basidia in definite number, eight, in asci, which are always surrounded by a perithe- cium _ Class Ascomycetes Reproduction by spores, borne in variable number, in asci of which the perithecium is absent or rudimentary. Class Hemiascomyetes Reproduction by spores, borne in basidia in definite numbers, mostly four, basidia being either one-celled or cruciately septate Class Basidiomycetes Reproduction by spores borne on promycelium which may be cruciately septate Class Hemibasidiomycetes Reproduction by conidia only, borne either in perithecia or on conidiophores. Life histories in some cases incompletely known and therefore sometimes referred to "fungi imper- fecti" .._ Class Deuteromycetes Spores when present resistant in function (bacteria). Repro- duction by fission only Class Schizomycetes *For detailed study and references consult : Massee, Text book of Fungi (Duckworth & Co., 1906). Brumpt, Precis de Parisitologie (Paris, Masson, 1921). Buchanan, Household Bacteriology (New York, Macmillan, 1915). Anderson, Yeast-like Fungi of the Human Intestinal Tract., Tour. Inf. Dis., vol. 21 (1917), p. 341. CLASS SCHIZOMYCETES (NAEGELI) MIGULA* Typically unicellular plants, cells usually small and relatively primitive in organization. The cells are of many shapes, spherical, cylindrical, spiral or filamentous : cells often united into groups, families or filaments; occasionally in the latter showing some dif- ferentiation among the cells, simulating the organization seen in some of the blue-green, filamentous alga. Multiplication typically by cell fission. Endospores are formed by some species of the Eubacteriales, conidia by some of the filamentous forms. Chloro- phyll is produced by none of the bacteria (with the possible excep- tion of a single genus). Many forms produce pigments of other types. The cells may be motile by means of rlagella ; some of the forms intergrading with the protozoa are flexuous, a few fila- mentous forms (as Beggiatoa) show oscillatory movement similar to that of certain blue-green algae (as Oscillatoria). 192 KEY TO THE ORDERS OF THE CLASS SCHIZOMYCETES (BUCHANAN) 1. Simple and undifferentiated forms, the true bacteria. Order I. Eubacteriales 2. Specialized or differentiated forms. a. Plant like b. Mold like. Order II. Actinomycetales bb. Not mold like. c. Sheathed. Order III. Chlamydobacteriales cc. Not sheathed. d. Sulphur bacteria. *Lohnis and Smith, Life Cycles of the Bacteria, Jour. Agric. Res., vol. 6 (1916), p. 675. * This attempted classification of the Schizomycetes or bacteria is based on the most recent attempt that has been made to deal with this important and difficult subject. Bergey's Manual of Determinative Bacteriology (Williams & Wilkins, Baltimore, 1923) presents an elaborate classification covering all varieties of bacteria both saprophytic and pathogenic. In the following pages we have attempted to schematize this classification for the pathogens. It is thoroughly realized that any such classification is subject to revision and will undoubted!}' be modified, but it seems of advantage to the student to give this outline as indicating modern progress in classification and indicating relationships which should at least be understood in their gen- eral aspects. Order IV. Thiobacteriales dd Slime-mold like. Order V. Myxomycetales aa. Protozoan like. Order VI. Spirochaetales ORDER I EUBACTERIALES Key to families, tribes and genera of the order Eubacteriales. I. Organisms obligate aerobes, using oxygen for direct oxida- tion of carbon, hydrogen or nitrogen or compounds of these. Cells 194 usually rod shaped, occasionally spherical. Motile or non-motile. Branched involution forms are produced. Endospores never formed. Usually water or earth forms. Family I. Nitrobacteriacae (Includes 2 Tribes; 9 Genera; and 30 species). II. Cells in their free form spherical; during division somewhat elliptical. Division in one, two or three planes. If the cells remain in contact after division they are usually flattened in the plane of division, and occur singly, in pairs, tetrade, packets, chains or in ir- regular masses. Motility rare. Endospores absent. Metabolism, unlike that of nitrobacteraceae (Family I) is here complex, usually involving the utilization of amino acids or carbohydrates. Family II. Coccaceae. Tribe I. Neissereae. Strict parasites, failing to grow or growing very poorly on usual artificial media. Cells normally in pairs, occasionally in tetrads. Gram negative. Colonies have distinct crumbs scattered on surface. Genus I. Neisseria Trevisan (7 species). Species. (1) Neisseria gonorrheae (2) " intracellularis (3) catarrhalis (4) " sicca (S) " perflava (6) " flava (7) " subflava Tribe II. Streptococcae. Parasites (thriving only or best on or in the animal body) ex- cept genus leuconostoc ; grow well under anaerobic conditions. Many forms grow with difficulty on serum-free media, none very abundantly. Planes of fission usually parallel, producing pairs, or short or long chains, never packets. Pigment, if any, orange or white. Genus II Diplo coccus (1 species) Parasites growing poorly or not at all on artificial media. Cells usually in pairs. Type Species. Diplococcus pneumoniae. Genus III Leuconostoc (3 species). Genus IV Streptococcus (24 species). 196 Chiefly parasites. Normally forming short or long chains. Never in packets. Generally Gram-positive. Inulin rarely at- tacked. Variable activity in blood. Species. I Parasitic or hemiparasitic. A. Hemolytic group (1) Streptococcus pyogenes (2) scarlatinae (3) mixtos (4) equi (5) mastitidis (6) cuniculi (7) felini (8) stenos B. Viridians group. (9) Streptococcus mitior (10) fecalis (11) equinus (12) bovis (13) ignavus C. Colonies gray. No hemolysis or green coloration. (14) Streptococcus anhemolyticus (13) saprophytics II. Saprophytic (9 species) Genus V. Staphylococcus (6 species). Usually parasitic. Cells as a rule in irregular groups, rarely in packets. Usually Gram-positive. Growth fair to good on arti- ficial media. Gelatin may be liquified. Nitrates may be reduced. (Produce hemolysis on blood agar.) Pigment white, orange or lemon yellow. Species. I. Orange pigment. (1) Staphylococcus aureus II. Lemon yellow pigment. (2) Staphylococcus citreus III. White or colorless growth on solid media. (3) Staphylococcus epidermidis (4) " albus (5) " pharyngis (6) 198 Tribe III. Micrococceae. (3 genera; 42 species). Facultative parasites or saprophytes, as a rule, aerobic. Grow- well on artificial media. Planes of fission often at right angles. Cell aggregates in groups, packets or masses. Generally Gram- positive. Many species form red or yellow pigment. Family III. Spirillaceae. Cells elongated, more or less spirally curved. Cell division al- ways transverse, never longitudinal. Cell non-flexuous, usually without endospores. As a rule, motile by means of polar flagella, sometimes non-motile. Typically water forms, though some species are intestinal parasites. Genus I. Vibrio. (12 species). (a) Cells short, bent rods, rigid, single or united into spirals. Motile. Polar flagella (1 to 3) or flagellum. Aerobic, facultative anaerobic. No endospores. Usually Gram-negative. Species. A. Pathogenic for man or laboratory animals (9 species). Type: Vibrio comma Koch. B. Non-pathogenic (3 species). Genus II. Spirillum (4 species). Family IV. Bacteriaceae. Rod-shaped cells without endospores. Motile or non-motile. Metabolism complex, amino-acids being utilized and generally carbohydrates. Usually Gram-positive. Tribe I. Chrombactereae. Produce pigment on solid media. The pigment may be red, yel- low, violet, blue or green. Genus I Serratia (23 species). Genus II Flavobacterium (46 species). Genus III Chromobacterium (9 species). Genus IV Pseudomonas (20 species). Tribe II. AchromobaUereae. Non-pigment forming rods, occurring in water and soil. Motile or non-motile. Gram-negative. Rods small to medium in size. Produce a brownish growth on potato. Genus V Achromobacter (51 species). Tribe III. Cellulomonadeae. Short rods occurring in soil, having the property of digesting cellulose. Motile or non-motile. Chromogenic or non-chromo- 200 genie. Growth in ordinary culture media often not vigorous. Gram- negative. One genus. Genus VI Celhdomonas (31 species). Tribe IV. Erwiniae. Plant pathogens. Growth usually whitish, often slimy. Indol generally not produced. Usually ferment carbohydrates with acid or acid and gas. Motile or non-motile. Gram-negative. Genus VII Erwina (11 species). Genus VIII Phytomonas (36 species). Tribe V. Zopfeae. Gram-positive rods, growing freely on artificial media. Do not attack carbohydrates. One genus. Genus IX Zopfius (2 species). Species. 1. Zopfius zopfiii 2. Zopiius zenkeri Tribe VI. Bactereae. Gram-negative rods generally growing well on artificial media. Generally attack carbohydrates, forming acid and often gas com- posed of C0 2 and H 2 . When motile the flagella are peritrichous. Genus X. Escherichia (22 species). Motile or non-motile rods commonly occurring in the intestinal canal of normal animals. Attack numerous carbohydrates forming acid and frequently acid and gas. Do not produce acetyl-methyl- carbinol. Type Species. Escherichia coli Genus XI Aerobacter (6 species). Genus XII Proteus (6 species). Highly pleomorphic rods. Filamentous and curved rods are common as involution forms. Gram-negative. Actively motile possessing peritrichous flagella. Produce characteristic amoeboid colonies on moist media and decompose proteins. Ferment dextrose and sucrose but not lactose. Do not produce acetyl-methyl-carbinol. Type Species. Proteus vulgaris Genus XIII Salmonella (17 species). Motile forms occurring in the intestinal canal of animals in vari- ous inflammatory conditions. Attack numerous carbohydrates with 202 the formation of acid and gas. In general do not form acetyl- thyl-carbinol. Species. (1) Salmonella schotmulleri (2) aertrycke (3) typhi-murium (4) veboda (5) columbensis (6) enteritidis (7) psittacosis (8) abortivo-equina (9) suipestifer (10) icteroides (11) paratyphi (12) pullora (13) wolinae (14) watareka (15) morgani (16) guimai (17) foetida Genus XIV Eberthella (25 species). Motile or non-motile rods, occurring in the intestinal canal of man, usually in different forms of enteric inflammation. Attack a number of carbohydrates with the formation of acid but no gas. Do not form acetyl-methyl-carbinol. Species. A. Motile (10 species). Type : Eberthella typhi B. Non-motile (15 species). Type : Eberthella dysenteriae para-dysenteriae (Flexner para-dysenteriae (Hiss) para-dysenteriae (Strong) Genus XV Alcaligines (9 species). Motile or non-motile rods, generally occurring in the intestinal canal of normal animals. Do not form acetyl-methyl-carbinol. Do not ferment any of the carbohydrates. Type Species. Alcaligines fecalis Tribe VII. Encapsulated^. Short rods, somewhat plump with rounded ends, mostly occur- 204 ring singly. Encapsulated. Xon-motile. Gram-negative. Ferment a number of carbohydrates with the formation of acid and gas. Encountered principally in the intestinal tract of man. Aerobic, growing well on ordinary culture media. Genus XVI Encapsulates (6 species). Type Species. Encapsulatus pneumoniae Tribe VIII. Laetobacillae. Rods, often long and slender. Gram-positive. Xon-motile. Without endospores. Usually produce acid from carbohydrates, as a rule lactic. When gas is formed it is C0 2 without Ho. The organisms are usually somewhat thermophilic. As a rule micro- aerophilic. Surface growth on media is poor. Genus XVII Lactobacillus {26 species). Tribe IX. Bacteroideae. Motile or non-motile rods, without endospores. Show good growth on ordinary culture media ; without pigment formation. Obligatory anaerobes. Corns XVIII Bacteroides (17 species). Tribe X. Pasteurelleae. Gram-negative rods showing bipolar staining. Parasitic forms with slight fermentative powers. Aerobic, facultative. Powers of carbohydrate fermentation slight ; no gas production. Gelatin not liquified. Parasitic., frequently pathogenic., producing plague in man and hemorrhagic septicemia in the lower animals. Genus XIX Pasteur ella (7 species). pcacs. { 1 ) Pasteu rella avicida (2) muriseptica (3) cuniculicida (4) suiseptica (5) boviseptica (6) tularensis (7) pestis Tribe XI. Hemophileae. Minute rod-shaped cells, sometimes thread forming and pleomor- phic. Xon-motile. Strict parasites growing best or only in the presence of hemoglobin and in general requiring blood serum, ascitic fluid, or certain growth accessory substances. Gram-negative. 206 "lenus XX Hemophili i 7 species). Species (1) Hemophilus influenzae 2 hemolyticus (3) - pertussis 4 " ^njunctivitidis 5 ■ lacunatus 6 i iurrevii (7) canis' Genus XXI D ter ( 10 species ) . FciKu-. '.' . Baclllacac. Rods producing endospores, usually Gram-positive. Flagella when present, peritrichous. Often iecompose protein medium. Genus I. Bacillus (75 species^. _ Aerobic forms. Mostly saprophytes. Generally liquify gelatin. " ft en occur in long chains and form rhizoid colonies. Form of rod usually not greatly : hanged at sporulation. 7- : : Sresies. "(1) Motile Bacillus subtilis 2 Non-motile Bacillus anthracis Genus II. Clcstridiur-: i'41 species-. Anaerobes or microaerophiles. often parasiti:. Rods frequently eni^r^-: i it sooruiatiom producing tlostridium or plettrichum forms. Pathogenic sue ties. (1) Clos trioiu an welchii Type I (2) " Type II (3) tt •• Type III ■+.' tt " Type Im- 5 It ogens (6) tt fall ax "~ tt oedentatis-maligni i tt chauvei (9) tt oedemati : ; ic tt novyi n " Eotulinum Type A 12: (I Type B (13) 11 history ticum (H it tyrosinogenes : v (15) tt sporogenes (16) SI tetani (17) a lucilae ORDER II. ACTINOMYCETALES. Cells usually elongated., frequently filamentous and with a de- cided tendency to the development of branches, in some genera giving rise to the formation of a definite branched mycelium. Cells frequently show swellings, clubbed or irregular shapes. Xo pseudoplasmodium. Xo deposits of free sulphur or iron. Xo bac- terio-purpurin. Endopores not produced but conidia are developed in some genera. Usually Gram-positive. Xon-motile. Some species parasitic in animals or plants. Some strongly aerobic and oxida- tive. Complex proteins frequently required. Growth on culture media often slow; some genera showing mold-like colonies. Xo water forms. Key to the Families of the Order Actixomycetales. A. Filamentous forms, often branched, sometimes forming mycelia. Conidia sometimes present. Some species parasitic. Family I. Actixomycetacea. B. Parasitic forms. Rod-shaped, rarely filamentous, and with only slight and occasional branching. Xo conidia. Family II. aIycobacteriaceae. Family I. AcHnomycetaceae. Filamentous forms, often branched and sometimes forming my- celia. Conidia sometimes present. Some species are parasitic. Genus I. Actinobacillus (10 species). Genus II. Leptotrichia (1 species). Thick, long, straight or curved filaments, unbranched, frequently clubbed at one end and tapering to the other. Gram-positive when young. Filaments fragment into short, thick rods. Anaerobic or facultative. Xo aerial hyphae or conidia. Parasites or facultative parasites. Type Speeies. Leptotrichia buccalis. Genus III. Actinomyces (64 species) Organisms growing in form of much-branched mycelium, which 210 may break up into segments which function as conidia. Some- times parasitic, with clubbed ends or radiating threads conspicu- ous in lesions in the animal body. Some species are microaerophilic or anaerobic. Non-motile. Type Species. Actinomyces bovis. Genus IV. Erysipelothrix (1 species). Rod-shaped organisms with a tendency to the formation of long filaments which may show branching. The filaments may also thicken and show characteristic granules. No spores. Non-motile. Gram-positive. Do not produce acid. Microaerophilic. Usually parasitic. Type Species. Erysipelothrix rhusiopathae Family II. Mycobacteriaceae. Parasitic forms. Rod-shaped, frequently irregular in form but rarely filamentous and with only slight and occasional branching. Often stains unevenly (showing variations in staining reaction within the cell). No conidia formed. Genus I. Mycobacterium (17 species). Slender rods which are stained with difficulty, but when once stained are acid-fast. Cells sometimes show swollen, clavate or cuneate forms and occasionally even branched forms. Growth on media slow. Aerobic. Type Species (Parasitic in man). Mycobacterium tuberculosis leprae Genus II. Corynebacterium (16 species). Slender, often slightly curved rods with a tendency to club and pointed forms, with branching forms in old cultures. Barred, uneven staining. Not acid-fast. Gram-positive. Non-motile. Aerobic. No endo-spores. Some pathogenic species produce a powerful exo- toxin. Characteristic snapping motion is exhibited when cells divide. Type Species. Corynebacterium diphtheriae Genus III. Fusiformis (2 species). Obligate parasites. Anaerobic or microaerophilic. Cells frequently elongate and fusiform, staining somewhat unevenly. Filaments 212 sometimes formed : non-branching. Non-motile. No spores formed Growth in laboratory media feeble. Type Species. Fusiformis dentium Genus IV. Pfeifferella (1 species.) Xon-motile rods, slender. Gram-negative. Staining poorly, some- times forming threads and showing a tendency toward branching. Gelatin maybe slowly liquefied. Do not ferment carbohydrates. Growth on potato characteristically honey-like. Type Species. Pfeifferella mallei ORDER III. CHLAMYDOBACTERIALES. Filamentous bacteria, algae-like typically water forms, frequently sheathed, without true branching although false branching may be present. The sheath is frequently impregnated with iron. Conidia may be developed but never endospores. Sulphur granules or bac- teriopurpurin never present. Mature cells or filaments not motile or protozoan like. Family I. Chlamydobacteriaceae. Genus I. Leptothrix (4 species). Four other non-pathogenic Genera; 5 species ORDER IV. THIOBACTERIALES. Cells various, typically containing either granules of free sulphur or bacteriopurpurin, or both, usually growing best in the presence of hydrogen sulphide. The cells are plant-like, not protozoan-like, not producing a pseudoplasmodium or a highly developed resting stage. Spores are rarely or never formed. There are listed in Bergey under this Order. 3 families, 2 sub-families, 5 tribes, 30 genera and 62 species. ORDER V. MYXOBACTERIALES. Motile, rod-like organisms multiplying by fission, secreting a gelatinous base and forming a pseudoplasmodium-like aggregation before passing into a more or less highly developed cyst-producing, resting stage in which the rods may become encysted in groups without modification, or may be connected into spore masses. (1 family; 3 genera: 21 species.) ORDER VI. SPIROCHAETALES. Protozoan-like in many characteristics. Cells usually relatively 214 COLUMBIA UNIVERSITY LIBRARY This book is due on the date indicated below, or at the expiration of a definite period after the date of borrowing, slend^ as P rov ided by the rules of the Library or by special ar- gitudi ran g emen t with the Librarian in charge. both. Fanui- C c- c G A s Pan spirilli flagellr G Pan taperin. ■ DATE BORROWED DATE DUE DATE BORROWED DATE DUE * r I L : 1 C2B(239)M100 lon- rs or rane. rigid hout orm 216 COLUMBIA UNIVERSITY LIBRARY This book is due on the date indicated below, or at the expiration of a definite period after the date of borrowing, as provided by the rules of the Library or by special ar- rangement with the Librarian in charge. DATE BORROWED , DATE DUE DATE BORROWED _ DATE DUE c.2.e £.3= w: :: vert -ci clans e C72 1929