Main Lib. BlOtOGY UBRAfW G A LABORATORY GUIDE IN ELEMENTARY BACTERIOLOGY BY WILLIAM DODGE FROST INSTRUCTOR IN BACTERIOLOGY, UNIVERSITY OF WISCONSIN ILLUSTRATED 1901 PUBLISHED BY THE AUTHOR MADISON, WIS. THE LIBRARY OF CONGRESS, Two COfifs RECEIVED MAR, 20 1901 COPYWOMT ENTRY , SLABS O. XXc. No. Afv?o COPY A. F.S Main Lib. Agria. Uept, COPYRIGHT, 1901, BY WILLIAM DODGE FROST. OUPLI1..1 TRACY, GIBBS & CO., PRINTERS, MADISON, WIS. 55 S PREFACE. The. following pages constitute, substantially, the material which has been furnished the stu- dents in Bacteriology at the University of Wisconsin, in mimeograph form, for several years. They contain directions for the performance of certain fundamental exercises in Bacteriology. In a rapidly developing subject it is important that the various exercises be worded so as to lend themselves readily to changes which become desirable from time to time. With this end in view the exercises have been divided, where possible, into a general and a special part. The general directions contain the essential part of the exercise which does not permit of any considerable variation, while the special directions embrace such features as are most subject to modification, as for instance, the particular organism to be used, the kind of medium, the incubation temperature, etc. Desirable changes here are easily indicated when the exercise is assigned. Some of the exercises can be performed in a few minutes, while others require several days for their completion. No attempt has been made to group them according to their length, nor to divide the text into lessons, but as far as possible they are arranged in the order in which they would be logically used in the laboratory. The right hand pages have been left for notes and drawings with the idea that notes in perma- nent form are the only ones of value to the student in subsequent years. The charts of the various organisms furnish a most satisfactory means for recording the observa- tions made during the study of a germ and are especially convenient for reference. Part I. is the work required of students taking the General Course in which special emphasis is placed on the biology of bacteria. It is completed in the first semester. Part II. which is given during the second semester includes the more specialized phases of the work, particularly as applied to the student preparing for medicine. References have been made to all of the leading English text-books and occasionally to original sources. It is expected that the student will make constant use of these references. My thanks are due Prof. H. L. Russell under whose general direction the work outlined here is given, for valuable help in the selection and arrangement of the material and for generous council. I am also indebted to Mr. E. G. Hastings, Assistant Bacteriologist to the Wisconsin Experiment Station, for critical reading of manuscript and proof. WILLIAM DODGE FROST. MADISON, Wis., January, 1901. (iii) 263429 CONTENTS. List of References vi List of Apparatus vii Laboratory Rules viii PART I.— GENERAL BACTERIOLOGY. CHAPTER I. MORPHOLOGY AND ELEMENTARY TECHNIQUE. EXERCISE. PAGE. I. Cleaning Glassware 2 II. Plugging Flasks and Tube? 2 III. Sterilization of Glassware 2 IV. Preparation of Bouillon 4 V. Filling Test-tubes and Flasks with Culture Media 6 VI. Sterilization of Culture Media 8 VII. Preparation of Gelatin 10 VIII. Preparation of Agar 10 IX Preparation of Potatoes 12 X. Preparation of Water-blanks 12 XI. Care of Culture Media 14 XII. Platinum Needles 14 XIII Test-tube Cultures 14 XIV. Incubation of Cultures 16 XV. Cleaning Slides and Cover-glasses. 18 XVI. Preparation of Staining Solutions. 18 XVII. Simple Cover-glass Preparation. .. 20 XVIII. Use of Microscope 22 XIX. Hanging-drop Preparation 24 EXERCISE. PAGE. XX. Test-tube Cultures Illustrating Form Types 26 XXI. Study of Test-tube Cultures 26 XXII. Microscopical Study of Form Types 26 XXIII. Drawing Bacteria 28 XXIV Study of Cell Grouping 30 XXV. Study of Involution Forms 30 XXVI. Gelatin Plate Cultures 32 XXVII. Agar Plate Cultures 34 XXVIII. Roll Cultures 34 XXIX. Study of Plate Cultures 36 XXX. Use of Decolorizing Agents 36 XXXI. Gram's Stain 36 XXXII. Tubercle Stain (Gabbett) 38 XXXIII. Staining Endospores 38 XXXIV. Study of Endospores 40 XXXV. Flagella Stain 40 XXXVI. Capsule Stain 42 CHAPTER II. PHYSIOLOGY OF BACTERIA. EXERCISE. PAGE. XXXVII Preparation of Special Media 44 XXXVIII. Effect of Reaction of Media on Growth 44 XXXIX. Effect of Concentration of Media on Growth 46 XL. Effect of Temperature Variations on Rate of Growth 46 XLI. Determination of Thermal Death Point 46 XLII. Comparative Efficiency of Dry and Moist Heat 48 XLIII. Effect of Desiccation 48 XLIV. Effect of Chemicals on Bacteria . . 48 XLV. Relation to Oxygen 50 XLVI. Effect of Direct Sunlight 50 EXERCISE. PAGE. XLVII. Detection of Gas 50 XLVIII. Quantitative Analysis of Gas 50 XLIX. Detection of Acids 52 L. Quantitative Determination of Acids 52 LI. Detection of Nitrites in Cultures. . 52 LU. Detection of Ammonia 52 LI 1 1. Detection of Sulphuretted Hy- drogen 54 LI V. Detection of Indol 54 LV. Determination of Chemical En- zymes in Cultures 54 LVI. Variation in Enzyme Production. . 54 LVII. Variation in Color Production .... 56 CHAPTER III. TAXONOMY. PAGE. I Points to be observed in the study of Bacteria. . 57 I Classification of Bacteria (Migula). PAGE. 60 CHAPTER IV. SYSTEMATIC STUDY OF REPRESENTATIVE NON-PATHOGENIC BACTERIA. EXERCISE. PAGE. LVIII. Preparation of Special Media 63 LIX. Bacillus prodigiosus 64 LX. Variety of Pigments 66 LXI. Separation of Bacterial Coloring Matter 67 EXERCISE. PAGE. LXII. Bacterium phosphorescens 68 LXIII. Bacillus acidi lactici 70 LXIV. Bacillus vulgaris 72 (iv) CHAPTER V. BACTERIOLOGICAL ANALYSIS. EXERCISE. PAGE. LXV. Comparative Analysis of Air 80 LXVT. Quantitative Determination of Number of Bacteria in Air 80 LXVII. Relation of Bacteria in Air to Dust Particles 82 LXVIII. Estimation of Number of Bacteria in Soil . . 82 EXERCISE. PAGE. LXIX. Water Analysis 82 LXX. Quantitative Analysis of Milk. ... 84 LXXI. Efficiency of Pasteurization 84 LXXII. Testing Antiseptic Action of Chem- icals 84 LXXIII. Testing Disinfecting Action of Chemicals 86 PART II. MEDICAL BACTERIOLOGY. CHAPTER VI. PATHOGENIC AEROBES. EXERCISE. PAGE. LXXIV. Preparation of Culture Media 88 LXXV. Streptococcus pyogenes 90 LXXV1. Micrococcus pyogenes *92 LXXVII. Micrococcus melitensis 94 LXXVIII. Micrococcus aureus 96 LXX IX. Micrococcus gonorrhoeae 98 LXXX. Micrococcus intracellularis 100 LXXXI. Sarcina tetragena 102 LXXXII. Bacterium anthracis 104 LXXXIII. Bacterium pneumoniae 106 LXXXI V. Bacterium pneumonicum 108 LXXXV. Bacterium cuniculicida no LXXXVI. Bacterium rhusiopathiae 112 EXERCISE. PAGE. LXXX VII. Bacterium tuberculosis 114 LXXXVIII. Bacterium mallei 116 LXXXIX. Bacterium diphtheriae 1 18 XC. Bacterium influenzae 120 XCI. Bacillus typhosus 122 XCII. Bacillus pestis 124 XCIII. Bacillus suipestifer 126 XCIV. Bacillus icteroides 128 XCV. Pseudomonas aeruginosa 130 XCVI Microspira comma 132 XCVII. Microspira metschnikovi 134 XCVIII. Microspira finkleri 136 CHAPTER VII. PATHOGENIC ANAEROBES. EXERCISE. XCIX. Bacterium welchii 150 C. Bacillus chauvaei 153 EXERCISE. CI. Bacillus oedematis .... 154 CII. Bacillus tetani 156 CHAPTER VIII. ANIMAL INOCULATION AND STAINING OF BACTERIA IN TISSUE. EXERCISE. PAGE. CIII. Animal Inoculation 162 EXERCISE. PAGE. CIV. Preparation of Tissue for Exami- nation 170 CV. Staining Sections IJ2 CHAPTER IX. BACTERIOLOGICAL DIAGNOSIS. EXERCISE. PAGE. CVI. Examination of Buccal Secretion. . 178 CVII. Examination of Sputum 180 CVIII. Examination of Blood 184 CIX. Examination of Faeces 188 CX. Examination of Urine 192 EXERCISE. PAGE. CXI. Examination of Transudates and Exudates 194 CXII. Diagnosis of Rabies 198 CXIII. Examination of Material from Hu- man Autopsies 198 CHAPTER X. DETECTION OF PATHOGENIC BACTERIA IN WATER AND MILK SUPPLIES. EXERCISE. PAGE. CXIV. Examination of Water for Patho- genic Bacteria 200 EXERCISE. PAGE. CXV. Examination of Milk for Patho- genic Bacteria 200 [v] LIST OF TEXTS AND REFERENCE WORKS WITH ABBREVIA- TIONS USED. A.— Abbott: Principles of Bicteriology. Lea Bros. '& Co. Philadelphia, 5th Edit.. 1899. B. — Bowhill: Manual of Bacteriological Technique. Oliver & Boyd, London. 1899. F. — Fischer: Structure and Functions of Bacteria. Clarendon Press, New York, 1900. Fr. — Frankland: Micro-organisms of Water. Longmans, Green & Co , 1894. G. - Gage: The Microscope. Comstock Pub. Co., Ithaca, N. Y., 7th Edit., 1899. H.— Hewlett: Manual of Bacteriology. Blakiston, Son & Co., Philadelphia, 1898. J. H. — Jordan's Translation of Hueppe: Principles of Bacteriology. Opan Court Pub. Co., Chicago, 1899. v. J. — v. Jaksch: Clinical Diagnosis. Charles Griffin & Co., London, 4th Edit., 1899. K. &D.— Kanthack & Drysdale: Practical Bacteriology. MacMillan Co., New York, 1895. L. — Lafar: Technical Mycology. Vol. i. Lippencott Co , Philadelphia, 1898. L.& K. — Levy & Klemperer: Clinical Bacteriology. Saunders & Co., Philadelphia, 1900. L. & N. — Lehmann & Neumann: Atlas and Essentials of Bacteriology. Wood & Co., New York, 1897. M. — Moore: Laboratory Directions for Beginners in Bacteriology. Ginn & Co , New York, 1000. M. & R. — Muir & Ritchie: Manual of Bacteriology. MacMillan Co , New York, 2nd Edit., 1899. M. & W.— Mallory & Wright: Pathological Technique. Saunders & Co , Philadelphia, 1897. McF. — McFarland: Text-Book of Pathogenic Bacteria. Saunders & Co., Philadelphia, 2nd Edit., 1898. N. — Novy: Laboratory Work in Bacteriology. Geo. Wahr, Ann Arbor, Mich., 2nd Edit., 1899 Ne. — Newman: Bacteria. Putnam, New York, 1899. P. — Park: Bacteriology in Medicine and Surgery. Lea Bros. & Co. , Philadelphia, 1899. P. B. C — Proceedings of the Bacteriological Committee from Jour. Amer. Pub. Health Assn. Vol. XXII. P. & M. — Peamain & Moor: Applied Bacteriology. Bailliere, Tindall & Cox, London, 1897. S. — Sternberg: Manual of Bacteriology. Wood & Co. , New York, 1893. Si. — Simon: Clinical Diagnosis. Lea Bros. & Co., Philadelphia, 2d Edit., 1897. W.— Woodhead: Bacteria and Their Products. Charles Scribner & Sons, New York, 1892. Wm. — Williams: Manual of Bacteriology. Blakiston, Son & Co., Philadelphia, 1898. [vi] LIST OF APPARATUS. This list comprises the apparatus which is to be under the exclusive control of the student and does not include the general laboratory outfit, such as sterilizers, incubators, microscopes, general chemical supplies, etc. FOR INDIVIDUAL USE. A. 50 (| oz.) cover-glasses, 18mm. (Jin.) square and 0.17 mm. thick (No. 2). 50 glass slides. 100 labels, 2 cm. square. 13 cm. platinum wire (No. 27). 1 pair cover-glass forceps (Cornet or Stew- art). 1 pair fine pointed forceps. 2 slide boxes for 50 slfdes. 1 hanging-drop slide. 1 towel. B. 1 flask, lOOOcc. 1 flask, 400 cc. 3 flasks, 250 cc. 1 flask, 100 cc. 200 test-tubes (15 X 120 mm.). 15 Petri dishes (10 cm). 2 fermentation tubes. 2 glass tumblers. 4 tin cans. 2 glass rods for platinum needles. 3 pipettes, 1 cc. 1 brass tube to hold pipettes (25 X 250 mm.). 8 stain bottles with pipettes, in block. 1 waste dish. 1 yard of muslin. 3 sheets of filter paper. 3 sheets of lens paper. FOR GROUP USE (About Four Students). 1 glass funnel, 12 cm. 1 glass funnel, 5 cm. 1 filtering flask with rubber stopper. 2 stirring rods. 1 pipette, 5 cc. 1 thermometer, 0-100" C. 10cm. rubber tubing. 1 cm. dia See Fig. 1. 1 Mohr stopcock. 1 anaerobic jar for plates. 1 anaerobic jar for tubes. 1 potato knife. 1 Bunsen burner witli tubing. 1 piece of wire gauze. 1 tripod with reducing rings 1 rice cooker. 3 small wire baskets. 1 enamel pan. 1 roll of cotton wool. YT, lb. absorbent cotton. 1 piece of Russia iron, 12 cm. square. 1 graduated cylinder, 300 cc. 1 graduated cylinder, 100 cc. 1 graduated cylinder, 25 cc. 1 evaporating dish, 10 cm. 1 disinfecting jar. 1 copper cup. 1 ring stand with clamp. 1 test-tube brush. [vii] LABORATORY RULES. I. Food should not be eaten in the laboratory and lead pencils or labels should not be moistened with the tongue. II. All possible cleanliness should be observed in the care of apparatus, desk, etc. III. The platinum needles used in making- cultures should be sterilized shortly before and immediately after use and before they are laid down. When the needles are covered with infectious material they should be held at the side of the flame until dry before being- sterilized; this will avoid the danger of scatter- ing this material about the laboratory. IV. If infectious matter should by accident come in contact with the hands or be dropped on the table or floor, corrosive sublimate (1:1000) should be imme- diately applied. V. Solid material, culture media and corrosive sublimate should not be put in the sink but in crocks provided for the purpose. Burnt matches, pieces of paper, etc., should also be put in the crocks and not on the floor. VI. All cultures of bacteria should be labeled with the name of the organism, the name of the student and the date. VII. Discarded cultures shouldbe covered with corrosive sublimate and placed in a proper receptacle and under no condition should they be left lying about the laboratory. Pipettes which have been used to handle infectious material should be placed in a glass cylinder containing a disinfectant or potassium bi- chromate and sulphuric acid. VIII. Whan using the steam sterilizer see that there is enough water present before lighting the gas and do not leave the laboratory until the gas has been turned off. IX. Before beginning an exercise read over the directions and look up some of the references. Keep notes of everything done and the conclusions reached on the right hand pages in this Guide. Make drawings wherever they will be of value. Outline with pencil and fill in with India ink. The laboratory Guide should be kept in the laboratory. X. At the close of the day's work the tables should be washed with corrosive sublimate and the hands disinfected by washing in the sublimate solution (or a germicidal soap) and then in soap and water. [viii] PART I. GENERAL BACTERIOLOGY PART I.— GENERAL BACTERIOLOGY. CHAPTER I. MORPHOLOGY AND ELEMENTARY TECHNIQUE. EXERCISE I. CLEANING GLASSWARE. GENERAL DIRECTIONS. All glassware to contain culture media must be thoroughly clean. New glassware should be washed in hot soap-suds (a test-tube brush will be needed for the test-tubes) , rinsed in tap water and then placed for a few minutes in water to which about 1% of hydrochloric acid has been added to remove free alkali frequently present on new glass, and then thoroughly rinsed in tap water. It is then allowed to drain. Test-tubes and flasks are best dried by placing them on a drain board especially prepared, or standing them mouth down in a box with a cloth bottom or on filter paper. Glassware containing media (discarded cultures, etc.), is best cleaned by first stand- ing in water for some hours, or by being steamed and pouring out the material while in a liquid condition and then cleaning as above with the exception of the use of the hydro- chloric acid. REFERENCES. A. 120; H. 39; K. & D. 81; M. & W. 74; N. 158; P. 223. SPECIAL DIRECTIONS. Clean as directed above, all flasks, test-tubes, fermentation tubes and Petri dishes in your possession. EXERCISE II. PLUGGING FLASKS AND TUBES. GENERAL DIRECTIONS. When the flasks, test-tubes and fermentation tubes are thoroughly dry they are to be plugged with cotton. The cotton for this purpose should be of the best non-absorbent quality, i. e., as free from foreign matter as possible. The plugs should be sufficiently loose to permit the interchange of gases and at the same time tight enough to support the weight of the vessel and its contents, to prevent their being pulled out in handling the vessel. The cotton should be rolled into a cylinder of the proper diameter and long enough to extend into the mouth about 2? cm. (1 in.) and pro- ject sufficiently to protect the lips from dust. The plug should be pushed in straight and not twisted; the surface next to the glass must be perfectly smooth, presenting no creases for the entrance of dust. REFERENCES. A. 121; H. 39; M. & W. 74; M. & R. 56; McF.107; P. 223. SPECIAL DIRECTIONS. Plug all test-tubes, flasks and fermentation tubes in your possession. EXERCISE III. STERILIZATION OF GLASSWARE. GENERAL DIRECTIONS. The glassware thus prepared is ready for sterilization, which process is accomplished in an apparatus called the hot air sterilizer. This is a sheet iron or copper box with a double wall which permits of rapid heating. The apparatus should General Bacteriology. be so arranged that a temperature of 150° C. can be quickly reached and readily main- tained. In such a sterilizer all glassware to be used for the reception of culture media, such as flasks, test-tubes, Petri dishes, etc., is submitted to a temperature of 140-150° C. for 1 hour, or until the cotton plugs are slightly browned; this change being due to the incipient charring of the cotton. The test-tubes are placed erect in square baskets made of galvanized iron wire. When the air in the sterilizer has cooled to about 40° C. the glassware can be taken out and stored ready for use. The Petri dishes are not to be opened until used for culture purposes. REFERENCES. A. 71 and 121; H. 32; L. & K. 74; M. & R. 36; N. 159; McF. 106; P. 223; S. 51. SPECIAL DIRECTIONS. All glassware prepared in I. is to be sterilized for one hour at 150° C. The small pipettes should be placed in brass tubes, provided for the purpose, and also sterilized. EXERCISE IV. PREPARATION OF BOUILLON. GENERAL DIRECTIONS. Any one of the three methods (A B or C) may be used. They are arranged in order of preference, but method C is the most convenient, and hence most used. A. B. Secure meat as under A a, add 1 liter of distilled water, weigh (see e below), cook for ^ hour at about 70° C. , and pro- ceed as directed under e below. Weigh out three grams of beef extract (such as Liebig's), add 1 liter of water, and then proceed as directed under e be- low. a. From 500 grams (1^-lb.) of lean beef, remove the fat and connective tis- sue and mince (Hamburg steak). 6 . Add 1 liter of distilled water and after thoroughly shaking set in ice chest for 12 to 24 hours. c. Squeeze through a cloth and add enough distilled water to make 1 liter and place in vessel to cook. This may be done either in a flask which is heated in a water-bath or a sterilizer, or in a rice cooker. In this case use a 50 % solution of calcium chloride in outer vessel instead of water as by this means the contents of the inner vessel can be brought to a rapid ebullition, something impossible by the use of water alone. d. Boil \ hour and make up loss of water. e. Add to any of the above solutions: 1% (10 gms.) peptone (Witte) and -5-% (5 gms.) common salt (NaCl), then weigh solution, with vessel, so that the water which is subsequently driven off in cooking can be accurately replaced. /. Heat until ingredients are in solution, then restore the water lost by evaporation. g. Neutralize or render slightly alkaline. This is a very important step and calls for great care. Method A is more accurate and should be employed for special or research work. For ordinary routine work B may be employed. C. General Bacteriology. B. Use a normal solution of so- dium hydroxide (yNaOH). Add to the hot solution a few cc. at a time, at first, later a few drops, stirring thoroughly with a glass rod. After each addition, test by placing a drop of the solu- tion by means of the glass rod on a strip of red litmus paper, and then moisten the paper with distilled water. The addition should continue until the red litmus paper is turned blue, but no change occurs on blue litmus paper. A. 1 . ) Titrate as follows : Pipette off 5 cc: of the fluid into a 4-inch evaporating dish, add 45 cc. of distilled water, boil for three minutes, add 1 cc. of phenol- phthalein (0.5% substance in 50% alcohol), and then carefully run in, drop by drop, from a burette a twen- tieth normal * solution of sodium hydroxide (^VNa OH) until the solution turns a faint pink color. Treat two other samples in the same way. If the amount of Na OH required is approximately the same in each case the average can be taken as the amount necessary to neutralize 5 cc. Calculate the amount necessary to neutralize the whole (1000-15 cc.). Since this amount would dilute the medium too much, a stronger solution (normal) is used, hence, 2.) Neutralize by adding ^Vth of the volume cal- culated above of a normal solution of sodium hydrox- ide. Test the accuracy of the work at this point by the addition of a few drops of phenolphthalein to a cc. or so of the medium. If a faint pinkish tint is not obtained, titration and neutralization must be re- peated. If by mistake more alkali is added than is required, the reaction can be corrected by the use of a normal solution of hydrochloric acid. h. Boil for 5 minutes and restore weight. i. Test reaction and adjust if necessary. j. Add 0.5 to 1.5% of a normal hydrochloric acid if neutralized by method A, oth- erwise omit. The amount of acid to be added varies with the purpose for which the medium is to be used, e. g., in water analyses +1.5 (acid) is preferable, with the path- ogenic bacteria a smaller amount of acid (+ 0.5) more nearly meets requirements. k. Filter through moistened filter paper (Abbott p. 96), or absorbent cotton, (VII. m). If the filtrate is not perfectly clear, cool to 60° C., add the white of an egg, thoroughly mix and boil for 5 minutes without stirring. The filtrate (bouillon) should be of a light straw color, perfectly clear, and should not give a precipitate on boiling. REFERENCES. A. 90; M. & R. 43; McF. 124; N. 234; P. 212; P. B. C. 18-24. SPECIAL DIRECTIONS. Prepare 1 liter of bouillon according to method C. Secure and put to soak meat for VII. EXERCISE V. FILLING TEST-TUBES AND FLASKS WITH CULTURE MEDIA. GENERAL DIRECTIONS. In filling tubes be careful not to allow the media to touch the neck of the vessels as this will cause the cotton to stick to the glass when the plugs are removed. Place the culture fluid to be tubed in a funnel arranged with a delivery •Normal solutions are prepared so that one liter at 16° C. shall contain the hydrogen equivalent of the active reagent weighed in grams (Sutton). For present purposes a 4 % solution of sodium hydrate is sufficiently accurate. 8 General Bacteriology. tube and stopcock (fig. 1), from which it can be run into sterile vessels. Test-tubes should contain 6-10 cc. of medium (about 3 cm. deep). Flasks are to be filled about three-fourths full. SPECIAL DIRECTIONS. Fill 15 test-tubes . and preserve remainder of bouillon in larger flasks. EXERCISE VI. STERILIZATION OF CULTURE MEDIA. O f FIG. 1. Appara- tus for filling test- tubes. EXPLANATORY. To accomplish this steam is used almost exclusively either as streaming steam or under pressure. The unconfined steam is applied in an apparatus known as a steam sterilizer. Of the various patterns the Arnold is perhaps the most satisfactory. It is effective, economical in the use of gas, and does not allow the escape of large quantities of steam into the room, as a large part is condensed to be re- converted into steam. For student use the form shown in fig. 2 is very convenient. The method of using these different forms is identical. Always have plenty of water present before heating. The discontinuous method is most frequently employed. Exposure is made on three consecutive days for 20 minutes, be- ginning to count time when the material reaches the temperature of the steam, which will vary with different substances and the volume treated. Between successive steamings culture media should be kept under conditions favorable to bacterial development (room or incubator temperature). For the employment of steam under pressure the autoclave is essential. The lid should contain a thermom- eter as well as a steam gauge, safety and outlet valve. A thermo-regulator is also desirable. The following table gives the temperature corresponding to atmospheres of pressure : Atmospheres. Degrees C. 1 100 1.5 112.2 2 121.4 2.5 128-.8 3 135.1 This table is only true when all of the air in the apparatus is replaced by steam, and hence the steam must be allowed to escape freely before the outlet valve is closed. A sin- gle exposure of 20 minutes at a temperature of 120° C. (one additional atmosphere) is sufficient to kill all. germ life. After the proper exposure, care must be taken not to allow the steam to escape too rapidly, otherwise the culture media may be forced against the plugs owing to the unequal pressure. GENERAL DIRECTIONS. Ordinary media may be sterilized by either method. Sugar media cannot be sterilized in the autoclave as it must not be heated above 100° C. The solidifying property of gelatin is impaired if submitted to a temperature of 120° C. longer than 15 minutes, and at a temperature above 120° C. momentarily. REFERENCES. A. 55-73; M. & R. 37; McF. 109; N. 161; P. 213. SPECIAL DIRECTIONS. Sterilize bouillon prepared in IV. for 20 miuutes in a steam sterilizer oa three consecutive days. FIG. 2. Simple sterilizer consisting of a galvanized iron pail with a cover a and a false bottom b. 10 General Bacteriology. N. B. Some time is required to raise the temperature of the media to that of the steam, especially if the vessels are large. All media should be carefully examined every day for a week or more, and if "specks" or the least cloudiness appears, the medium is not sterile and the process of sterilization must be repeated. All receptacles containing media should be labeled after sterilization. For this pur- pose labels can be purchased, the size used for glass slides, or gummed paper in sheets can be cut into squares (2 cm.). The labels are to be attached to each vessel 1 cm. from the lip. The kind of medium and the date of preparation should be written across the top, as io"is-L>99 leaving the rest of the label to be filled in when the medium is inoc- ulated. EXERCISE VII. PREPARATION OF GELATIN. GENERAL DIRECTIONS. a to d. Same as bouillon. (IV.) e. Add 1% peptone; 0.5% salt and 10-15% * of best white gelatin, and weigh. /. Heat until ingredients are dissolved. g. Neutralize. h. Boil 5 minutes and restore weight. i. Test reaction. j. If neutralized by method A add 5 cc. of a normal hydro- chloric acid. In method B omit acid. k. Cool and add egg and boil 5 minutes. I. Filter. Arrange the apparatus shown in fig. 3. Use absorbent cotton. The funnel and flask should first be heated with warm water. Start the filter pump before pouring in the culture medium. This prevents the unfiltered gelatin from TiTsTT^aratus for filtering passing between the COtton and the glass. media through absorbent cotton; "' layer of cotton: *• tubes for Tnhp (V } "*• J making connection with air W. Sterilize. pump; ct Bunsen valve to prevent -r •, i entrance of water into flasks. o. Label. REFERENCES. A. 95; H. 42; M. & R. 46; McF. 127; N. 153; P. B. C. 26. SPECIAL DIRECTIONS. Make 1 liter, using method A. Fill 30 test-tubes. Put the remainder in flasks, sterilize in steam sterilizer or autoclave. Remember long exposure to high heat injures the solidifying properties of gelatin. EXERCISE VIII. PREPARATION OF AQAR. GENERAL DIRECTIONS. a. Add 15 grams of agar-agar threads (finely chopped) to 500 cc. of water and either (l)boil until the agar-agar is dissolved (about^hour) and make up loss of water by evap- oration, or (2) dissolve in autoclave by heatingup to 120° C., closing off gas and allow- ing to cool. * The amount to be varied according to the season of the year, 10 per cent in winter, 12-15 per cent in summer, but it should be remembered that different quantities affect the appearance of the culture. 12 General Bacteriology. b. l)-4) Same as a-d in the preparation of bouillon (IV.), except, that only one- half (500 cc.) of the amount of water is added to the beef or extract. 5) Add 1% peptone and 0.5% salt. 6) Heat until peptone is dissolved. 7) Neutralize. 8) Mix a. and b. (in case beef is used it will be necessary to cool a. to about 60° C. before mixing). 9) Boil 5 minutes and restore weight. 10) Test reaction. 11) Addition of egg will be necessary only where extract is used. 12) Filter as incase of gelatin, (IV. m.) 13) Tube. 14) Sterilize in steam for 15 minutes on three suc- cessive days or in autoclave for 20 minutes at 120° C. After the last sterilization place most of the tubes in a sloping position to harden (fig. 4), these are known as agar slopes. Those solidified horizontally can be used for plate cultures. 15) Label. REFERENCES. A. 100; H. 43; M. & R. 48; McF. 129; N. 235; P. B. C. 27; S. 43; Journal of Applied Microscopy, 1898, 1; 106. SPECIAL DIRECTIONS. Use meat extract, make 1 liter, fill 25 tubes and after last sterilization incline 20 of them. Place the remainder in flasks and sterilize. FIG. 4. Method of sloping agar. (23 EXERCISE IX. PREPARATION OF POTATOES. (BOLTON.) GENERAL DIRECTIONS. a. Select a number of rather large test-tubes (150x20 mm.) place a small wad of absorbent cotton in the bottom of each (fig. 5 a), plug and sterilize as usual. b. Wash a large potato, then with a cork borer slightly smaller than the test-tubes punch out cylinders about 5-6 cm. long. c. Divide these diagonally and trim to shape indicated in fig. 5 b. d. Add a few drops of distilled water to each test-tube and place pieces of potato in position. e. Sterilize on three consecutive days for 30 to 45 minutes. Unless the tubes are to be used immediately, they should be sealed. (XI.) The dark color can be prevented by immersing the pieces between c and d in running water for from 12-18 hours. REFERENCES. A. 104; M. & R. 54; McF. 134; N. 183; P. 216; P. B. C. 28; S. 47. SPECIAL DIRECTIONS. Prepare 15 test-tubes of potato, sterilize, label, and seal with paraffin. (XI. 2.) EXERCISE X. PREPARATION OF WATER-BLANKS. GENERAL DIRECTIONS. Water-blanks are prepared by placing exactly 10 cc. of a physiological salt solution (6 gms. per 1,000 cc. of water) in test-tubes and sterilizing in autoclave 15 minutes at 120 ° C., or in steamer 15 minutes on three successive days. SPECIAL DIRECTIONS. Prepare and sterilize 10 water-blanks. 0. FIG. 5. Bolton's potato tube. 14 General Bacteriology. EXERCISE XI. CARE OF CULTURE HEDIA. When sterile culture media (or test-tube cultures) are to be kept for some time they must be protected from evaporation and stored in a dark, cool place. Evaporation may be checked to a considerable extent, (1) by storing them iu tin cans, e. g. quinine cans. Care must be taken, however, that these do not become too damp, in which case the mould fungi frequently grow through the cotton plugs; (2) flasks and test-tubes may be sealed by removing the plugs, dipping same in melted paraffin (melting point about 50° C.) and then replacing them; (3) by cutting off the projecting cotton and drawing over the mouth of the vessel a rubber cap (made for the purpose) which has been sterilized in a solution of mercuric bichloride (1: 1,000, spoken of in the lab- oratory as "sublimate solution") ; or (4) By use of a cap of tin-foil. In this case the foil should be put on as soon as the tubes are filled, and sterilized with the medium. All media should be carefully examined every day for a week or more, and if spots or the least cloudiness appears, the medium is not sterile and the process of sterilization must be repeated. EXERCISE XII. PLATINUM NEEDLES. GENERAL DIRECTIONS. These are made by — C \ fusing a piece of No. 27 platinum wire (5 cm. long) into a glass rod or tube (18 cm. long) . (Fig. 6.) Each student should have two such needles; FlG'8- platinum needles- in one the wire should be straight (designated "needle") and the other bent to form a "loop". This loop should be formed around a No. 10 wire. These instruments must be sterilized shortly before and immediately after use by heating the wire to a glow in the gas flame. The handle should also be passed through the flame two or three times. Cool before using. If the habit of sterilizing is thoroughly acquired much trouble will be avoided and possible danger prevented. These needles will be in constant use. REFERENCES. A. 125; M. & B. 58; N. 172; P. B. C. 33, foot note. EXERCISE XIII. TEST-TUBE CULTURES. EXPLANATORY. The extreme minuteness and slight variation in the form of dif- ferent bacteria render a thorough study of them by direct microscopic observation a dif: ftcult and well nigh impossible task. In their study, therefore, it is necessary to depart from the usually accepted rules that govern the determination of the life history of other forms of life and resort to special methods. The most successful of these are those known as culture methods. According to these methods the bacteria are sown on vari- ous food substances and upon these they develop forming masses easily visible to the naked eye. The manner of their growth and the changes which they produce in these media make it possible to detect differences which would otherwise escape attention. The most common culture media, bouillon, gelatin, agar and potato have already been prepared, and others will be described as needed. Cultures maybe made either in test-tubes (streak or stab cultures), or on glass plates, as plate cultures. The plate culture is especially important and is used (a) to obtain pure cultures; and (6) for ascertaining the character of the colonies as an aid to 16 General Bacteriolv;/ >/ . FIG. 7. Method of holding test-tubes. their diagnosis. The tube-cultures are serviceable in giving opportunity for a further study of the characters as well as to furnish the most convenient method of maintain- ing the cultures. GENERAL DIRECTIONS. Bacteria when ob- tained in "pure culture" are usually grown in test-tube cultures. To make these a small portion of a previous culture is transferred to fresh cul- ture media by means of the platinum needles. a. Stab Cultures are made in test-tubes con- taining solid, transparent media, such as gelatin and agar. The end of a sterile needle is infected with the material to be transferred. The needle is then thrust into the medium to the bottom of the test-tube and withdrawn. In this way the bacteria are left along the entire length of the needle track. For method of holding tubes see fig. 7. They are held in an inclined position to prevent the possi- bility of infection. b. Streak Cultures are cultures made by drawing the needle or loop over the surface of the medium (test-tubes with media having sloped surfaces or plate cultures). Agar, potato and blood serum are frequently used in this way, and occasionally gelatin. c. Fluid Cultures (bouillon, milk, etc.), are inoculated by transferring the desired material to them on either the needle or loop. REFERENCES. A. 146; H. 51; M. & E. 60; McF. 146. SPECIAL DIRECTIONS. a. Make a gelatin stab, an agar streak, a potato streak, and a bouillon culture of Bacillus subtilis (EHRENB.) COHN (hay bacillus) and Bacillus coli (EsCH.) MIG. (colon bacillus) from agar cultures supplied. b. Label each tube, writing the name of the organism, the date of inoculation and your own name. c. Place the gelatin in the cool chamber, and the other cultures in the incubator at 28° C. (XIV). EXERCISE XIV. INCUBATION OF CULTURES. EXPLANATORY. Most bacteria grow at ordinary temperatures (22° C.), but their growth is usually hastened by a higher temperature (e. g. 28°-30° C.). The pathogenic, or disease-producing bacteria grow best at the temperature of the human body (38° 0.). All bacteriological laboratories are, therefore, supplied with apparatus arranged for maintaining constant temperatures, known as thermostats or incubators. The non-pathogenic cultures are usually kept at 28° C., while the pathogenic ones are kept at 38° C. All gelatin cultures, however, must be kept at a temperature several degrees below the melting point of gelatin, i. e., not above 22° C. Ordinarily the temper- ature of the locker, especially near the floor, will be found satisfactory. In a very warm room, particularly in the summer, an artificially cooled chamber will be necessary. Test-tube cultures are stored in the various incubators in tin cans or glass tumblers with a layer of cotton in the bottom, while the Petri dishes are stacked in low piles. REFERENCES. A. 136; H. 48; M. & R. 88; N. 178 & 243; P. 231; P. & M. 37. 18 General Bacteriology. SPECIAL DIRECTIONS. a. Incubate all cultures of the non- pathogenic bacteria at 28° C., except the gelatin. Keep these in the cool chamber. After growth has taken place, the cultures can be taken from the incubator and kept at the room temperature. b. Study and make diagrams of an incubator, a Reichert thermo-regulator, a Roux thermo-regulator and Koch's safety burner. EXERCISE XV. CLEANING SLIDES AND COVER-GLASSES. GENERAL DIRECTIONS. Slides can be sufficiently cleaned by washing in water or alcohol and drying with a towel. The cover-glasses for bacteriological work, however, must not only be freed from visible dirt but must be rendered free from fat. One of the best methods is the following: New cover-glasses are cleaned by washing in water and drying from alcohol between driers (two blocks 20xlOx2J- mm. covered with several layers of cotton cloth or chamois skin) , and then heating them on a piece of sheet iron or in hot air sterilizer for one hour at about 200° C. They are best kept in a clean Petri dish and handled with forceps. (Novy). Old slides and covers having balsam on them should first be dropped one by one into a cleaning solution (potassium bichromate 60, sulphuric acid 60, water 1000) , and boiled for one-half hour and then treated as above. SPECIAL DIRECTIONS. Clean £ oz. of cover-glasses and place them in a clean Petri dish. EXERCISE XVI. PREPARATION OF STAINING SOLUTIONS. GENERAL DIRECTIONS. The dyes most useful for staining bacteria are the basic anilin dyes which come in powdered or crystalline form. (Gruebler's dyes are standard.) Those in most common use are Fuchsin, Methylen blue, Gentian violet and Bismark brown. They keep well in powdered form, with perhaps the exception of Methylen blue, but because of greater convenience and equally good keeping qualities, saturated alcoholic solutions are kept in stock. These are made by adding the dry dye to 95% alcohol to saturation and filtering. This form can not be used for staining bacteria. The following solutions are required to begin work with: 1. Aqueous solution of Gentian violet. Saturated alcoholic solution of Gentian violet, - 2.5 cc. Distilled water, 47.5 cc. 2. Saturated aqueous solution of Bismark brown. 3. Ziehl's carbol-fuchsin. Saturated alcoholic solution of Puchsin. 5 cc. Solution of carbolic acid (5%)- - 45 cc. 4. Loeffler's Methylen blue. Saturated alcoholic solution of Methylen blue, - 15 cc. Potassium hydrate ( 1 : 10 , 000 ) , 50 cc . 5. Ehrlich's Anilin Oil Gentian violet. Saturated alcoholic solution of Gentian violet, 6 cc. Absolute alcohol, - 5 cc. Anilin water, - - 50 cc. Anilin water is prepared by adding 2-3 cc. of anilin oil drop by drop to 50 cc. of water, thoroughly shaking and then filtering through moistened filter paper until per- fectly clear. 20 General Bacteriology . This stain should stand 24 hours and then be filtered. It does not keep well and must not be used when more than 14 days old. 6. Grain's Iodine solution. Iodine, Potassium iodide, Distilled water, 7. Gabbett's Methylen blue solution. Methylen blue (dry), Sulphuric acid, Distilled water, 8. Alcohol, 96%. REFERENCES. A. 156; H. 75; M. & W. 245; M. & R. 103; McF. 90; P. 200. SPECIAL DIRECTIONS. Prepare the solutions of dyes from the saturated alcoholic solutions (furnished) and place them in 2 oz. bottles arranged with pipettes and neatly labeled. The bottles are conveniently kept in a block. Fig. 8. EXERCISE XVII. SIMPLE COVER-GLASS PREPARATION. 1 gm. 2 gm. 300 cc. 2 gms. 25 cc. 75 cc. Block for stain bottles. GENERAL DIRECTIONS. Bacteria may be studied under the microscope in a living condition in a hanging drop preparation (XIX) ; but on account of their hyaline charac- ter, which makes the examination difficult, the student should first learn to stain them and later make the hanging drop preparation. With a few exceptions all bacteria can be stained by the following process : A small drop of distilled water is placed on a clean cover-glass by means of the platinum loop. With a sterile needle a portion of the material to be examined is secured and while the cover-glass is held in the fingers of the left hand the bacteria on the needle are introduced into the water, thoroughly mixed and then spread in a thin film over as much of the surface of the cover-glass as possible. When the bacteria are taken from fluid media a drop of water will not be necessary. In this case use a loop. The film is now allowed to dry. If the drop is sufficiently small this will be a short process. It may be hastened by holding the cover-glass high over the flame, but it should always be held in the hand to prevent over- heating, which spoils the preparation. When the film is thoroughly dry place the cover-glass in a pair of Cornet or Stewart forceps and "fix'' the bacteria in the flame. This is done by passing the preparation through the upper portion of a gas flame, film side up. Three passages should be made, each consuming about one second of time. The forceps are now placed on the table and the film flooded with one of the anilin dyes. After the stain has acted for five or ten minutes it is washed off into a waste dish with a stream of distilled water, and while the cover-glass is still wet it is placed, bacteria side down, on a clean glass slide, being careful to avoid air bubbles. The surplus water is then taken up by means of a small piece of blotting or filter paper. The preparation is now ready for microscopical examination. (For directions see XVIII). 22 General Bacteriology. The preparation can be made permanent either by allowing the water under the cover-glass to dry before it is removed, or by floating it off with water and afterwards drying. When dry a drop of Canada balsam, dissolved in xylene, is placed on the cover- glass and this is then lowered on to the slide again. Resume . a. Spread film, b. Air dry, c. Fix, (I. Stain, e. Mount, /. Examine, g. Mount in balsam, or, /. Mount in balsam, g. Examine. "The great mistake made by beginners is to take too much growth." (M. & R.) REFERENCES. A. 151; H. 71; L. & K. 104; M. & W. 89; M. & R. 95; McF. 91; N. 147; P. 198; P. B. C. 11; S. 25. SPECIAL DIRECTIONS. Make cover-glass preparation from agar streak of B. subtilis (XIII) staining with au aqueous solution of gentian violet for 5 minutes. EXERCISE XVIII. USE OF MICROSCOPE. GENERAL DIRECTIONS. For bacteriological purposes a microscope with a magnifying power of at least 500 diameters is needed. There should be a coarse adjustment (rack and pinion) as well as a fine micrometer screw; and the following accessories: two eye pieces, one 1 in. (25 mm.) and one 2 in. (50 mm.); three objectives, one 1 in. (16 mm.), one i in. (4mm.), ory in. (3.5 mm.) and one oil immersion yV in. or rV in- (2 mm.); a triple nose-piece, and an Abbe substage condenser with iris diaphragm mounting. In the use of the microscope the following points should be noted: a. LIGHT. The proper angle at which the mirror should be placed is best determined by removing the eye-piece and so arranging the mirror that the unobstructed light from the window covers the whole field. The ideal light is that from a white cloud. Direct sunlight should never be used. b. ABBE CONDENSER. The purpose of the condenser is to furnish a large cone of light, and as it is corrected for parallel rays the plane side of the mirror should always be used, except when artificial light is employed. When highly stained objects are to be exam- ined, the open diaphragm should be used, but when the, structural rather than the color picture is desired, it will be necessary to diminish the light by closing the diaphragm. When the high powers are employed, raise the condenser as high as possible; for low powers a lower position will give better definition. c. FOCUSING. Turn the proper objective in place and rack down until the objective nearly touches the cover- glass. This should be done while the eye is held at one side and directs the movement. Then with the eye at the tube slowly move up with the micrometer screw- Never rack down with the eye at the tube. d. USE OP OIL- IMMERSION. The oil-immersion objective is indispensable to the proper study of bacteria. It is constructed upon the principle that a drop of fluid having the same refractive index as the objective, prevents the dispersion of light, thus permitting the use of lenses having a greater numerical aperture and longer working distance for 24 General Bacteriology. the same degree of amplification than is possible with the dry system. In using an immersion lens, place a small drop of oil on the preparation, then carefully lower the objective until it touches the oil drop and nearly touches the cover-glass. Apply eye to the ocular and focus upward very slowly with fine adjustment until the definition is clear. At the close of the day's work the oil must be removed from the objective and cover- glass. This is best accomplished by wiping them with apiece of Japanese paper made for the purpose. In case the oil should accidentally dry on the objective, it can be removed by adding a little more oil and allowing it to stand for a few minutes; it can then be wiped off with paper. If this method does not succeed, the objective should be taken to the instructor. Great care must be observed since solvents of the oil are also sol- vents for the lens mountings. REFERENCES. See Gage; A. 190; H. 104; M. & R. 93; McF. 86; N. 123; P. 206. SPECIAL DIRECTIONS. a. Examine cover-glass preparations made in (XVII) first with \ in. objective, and then with the oil-immersion objective. If the specimen is satisfactory wipe off the oil and mount in Canada balsam. b. Practice making cover-glass preparations by staining specimens from each of your cultures. Use Loeffler's methylen blue for the gelatin and bouillon; aqueous solution of gentian violet for agar, and carbol-fuchsin for potato. Examine, mount permanently and hand to instructor for inspection. EXERCISE XIX. HANaiNQ-DROP PREPARATIONS. GENERAL DIRECTIONS. These are made by adding a small portion of bacterial cul- ture from solid media to a drop of water on a clean cover-glass, or in case of fluid media by placing a loop of the culture medium on the cover- glass. A hollow ground glass slide having the rim of the cavity previously coated with vaseline, is inverted and lowered over the cover-glass enclosing the drop. With a careful, quick movement the prepara- tion is now brought right side up. Instead of the hollow ground glass-slide an ordinary glass-slide to which a small section of a glass or rubber tube has been cemented can be used, and in some cases is preferable. In examining the preparation under a microscope focusing is a somewhat difficult process and must be carried out with great care. Use a narrow diaphragm. Find the edge of the drop with the low power (f in. objective) adjusting slide so that edge of drop passes through the center of the field; then turn on the high power (^ in. objective) and focus without moving the slide. The edge of the drop is selected because the bacteria are here nearest the cover-glass and hence more easily focused upon than where they are deeper in the drop. REFERENCES. A. 195; H. 101; L. & K. 102; M. & W. Ill; M. & R. 94; McF. 88; N. 142; P. 209. SPECIAL DIRECTIONS. a. Make hanging-drop preparation of B. subtilis from agar or bouillon. (XIII) 6. Make same preparation of B. coli. (XIII) c. Make same preparation of organism supplied. (Micrococcus) d. Make same preparation of water containing particles of india ink or carmine in suspensiqn. Study character of movement in all cases. Distinguish between vital and molecular movement. 26 General Bacteriology. In cases where vital movement is questionable, remove the cover-glass and place a drop of formalin or chloroform in the bottom of the cell; replace the cover-glass, ex- amine and note change in character of movement, if any. EXERCISE XX. TEST-TUBE CULTURES ILLUSTRATING FORM TYPES. a. Make test-tube cultures in bouillon, gelatin, agar and potato of the following organisms : Micrococcus (any species). Sarcina lutea SCHROETEB. Pseudomonas fluoresce ns (FLUEGGE) MIG. Bacillus mycoides FLUEGGE. Microspira Metschnikovi MIG. (or any vibrio). Spirillum rubrum v. ESMARCH. 6. Incubate all cultures, except gelatin, at 28° C. EXERCISE XXI. STUDY OF TEST-TUBE CULTURES. GENERAL DIRECTIONS. As soon as growth becomes visible a systematic and careful study of the cultures should be made. A detailed list of the points to be noted will be found in Chapter III, and should be consulted in writing up the descriptions. The sum- mary below will, however, be found useful. For bouillon cultures note : 1) condition of fluid, 2) character of sediment, 3) pres- ence or absence of membrane, and 4) characteristic odor. For solid cultures (agar and potato slopes), note: 1) Form of growth, 2) size, 3) surface elevation, 4) consistency, 5) color, 6) effect on media, and 7) characteristic odor. For gelatin stab cultures, note: 1) Effect on media, a. non-liquefying, i) line of puncture, ii) surface, b. liquefying, i) shape of liquefied area, ii) condition of fluid, iii) character of sediment, 2) characteristic odor. The study should be continued from day to day as long as changes are noted. Make drawings wherever they will be of service in elucidating the descriptions. REFERENCES. P. B. C. (Charts by Cheesman.) SPECIAL DIRECTIONS. Study and write careful descriptions and make necessary drawings of all cultures made. EXERCISE XXII. MICROSCOPICAL STUDY OF FORH TYPES. a. Make cover-glass preparations from the agar streaks (XX) and stain with an aqueous solution of gentian violet or with Loeffler's methylen blue. b. Examine with the oil-immersion objective, write the names of the organisms in their proper place in the table below. Name. Sketch. ( medium . Coccaceae (spherical) (. small . . ( large . Bacteriaceae (elongated) ( small . ( curved- . Spirillaceae (spiral) (. twisted . 28 General Bacteriology. c. Make similar preparations from the gelatin and potato and note any variations in form, size. etc. d. All these preparations are to be mounted in balsam, sketched (XXIII) and handed to instructor for inspection. EXERCISE XXIII. DRAWING BACTERIA. GENERAL DIRECTIONS. In drawing bacteria only a few organisms occuring in the microscopic field should be sketched, but these should be made of considerable size so that the exact outline maybe indicated. Furthermore they should be drawn to scale and individuals selected to give range in form and size. To measure microscopic objects an ocular micrometer is used, and the first step will be to determine its value. Place the ocular micrometer on the diaphragm in the ocular, use a stage micrometer as an object and focus. The image of the scale on the stage mi- crometer will appear imposed on that of the ocular micrometer. Make the lines of the two micrometers parallel and then make any two lines of the stage micrometer coincide with any two on the ocular micrometer, pulling out the draw-tube if necessary. Divide the value of the included space or spaces on the stage micrometer by the number of divisions on the ocular micrometer required to include them, and the quotient so obtained will give the valuation of the ocular micrometer in fractions of the units of measure of the stage micrometer (Gage). If result is not in terms of micron (//) it should be con- verted to such, as this is the unit in micrometry. In making drawings represent a micron by two millimeters on paper. This will give a magnification of 2,000 ( X 2,000) . REFERENCES. G. 100-108. SPECIAL DIRECTIONS. a. Determine the value of the ocular micrometer and fill out blanks in following table: No of Microscope Make . . . Ocular in ., or mm. Objective. Tube length. Value of single di- vision on scale in /*. | in. (16 mm.) ^ in. (4 mm.) Oil-immersion. 6. Make drawings of cover- glass preparations made in XXII in place provided in table. 30 General Bacteriology. EXERCISE XXIV. STUDY OF CELL GROUPING. HANGING DROP PREPARATIONS. a. Make hanging-drop preparations from bouillon cultures prepared above and also from those supplied. b. Examine with oil-immersion objective and assign organisms to their proper place, as determined by cell grouping, in the following scheme : Name. Sketch. Isolated ( Bacilli Filaments • (. Cocci Plane surface, Tetrads C Regular Masses -j Irregular I I Zoogloea IMPRESSION PREPARATIONS. The exact relation of cell to cell as they develop in the colony can frequently be determined with greater accuracy by studying a "contact prep- aration" which is prepared as follows: a. Melt a gelatin tube and slope it, when solid make a streak culture of B. mycoides FLUEGG.E and when growth has taken place dip the tube in hot water to loosen gelatin which is then slipped out of the tube. b. Lower gently a clean cover-glass over the surface. Apply a slight pressure by tapping glass. Raise coyer-glass by one edge taking care that natural arrangement of adherent bacteria is not disturbed. c. Thoroughly air dry the same, then fix and stain in the ordinary manner. d. Examine the thinner layers noticing the arrangement of cells with reference to each other and draw a sufficient number to illustrate this relationship. AGAR HANGING-DROP CULTURES. a. Melt a tube of agar and cool to 43° C. b. Sterilize a cover- glass by passing it two or three times through the flame quickly. c. With the needle make a streak on the cover-glass about 3 mm. long of B. subtilis. d. With the loop place a drop of liquid agar so as to cover up streak. e. Seal cover-glass to hollow ground slide. Incubate and later examine and sketch. EXERCISE XXV. STUDY OP INVOLUTION FORMS. a. Grow Bacillus subtilis (EHRENB.) MIG. in bouillon and also in a solution con- taining 0.1% asparagin, 10% sugar, and by means of stained cover- glass preparations compare the individual organisms in each casein regard to their form and size. The de- generated or involution forms are more apparent by staining. Draw several cells illus- trating a variety of involution forms. b. Examine a culture of Bacterium diphtheriae (LOEFFLER) MIG. on Loeffler's blood serum. Read M. & R. 5. 32 General Bacteriology. EXERCrSE XXVI. GELATIN PLATE CULTURES. EXPLANATORY. Plate cultures are only possible with the liqueflable solid media, gelatin and agar. In making them the bacteria are mixed with the medium while it is in a fluid state in such quantities that the individuals are separated from each other by several millimeters when it is spread out on a horizontal surface to cool. As the medium solidifies, the organisms become fixed and their growth results in the formation of "colo- nies." These vary in size and appearance according to the peculiarities of the organism and the age of the culture, but are of the greatest service in the study and identification of the various species. These cultures are prepared as follows: GENERAL DIRECTIONS. Three gelatin tubes are marked Nos. 1, 2 and 3 and melted by placing them in a water bath at a temperature of 42° C. For this purpose a small cup of water placed on a tripod can be used (Fig. 9). They are inoculated by introducing the material to be studied into tube No. 1. The quantity of this material varies. The amount cling- ing to the platinum needle will be sufficient if a pure culture is used, while in other cases several loops or even drops are neces- sary. The inoculated material is thoroughly mixed with the gelatin in No. 1. This is done by rolling the tube gently be- tween the palms of the hands, instead of shaking, so as to pre- vent the introduction of air bubbles. With a sterile loop three loopfuls of fluid gelatin are now transferred from No. 1 to No. 2, and mixed. For method of handling tubes see Fig. 7. In like manner three or more loops from No. 2 are carried over to No. 3, which in turn is well mixed. The cpntents of the tubes Nos. 1-3 are now poured into separate sterile Petri dishes. The process of pouring is performed as follows: The Petri dish is placed on the desk; the gelatin tube is taken in the right hand, the cotton plug removed with the left hand; the mouth of the tube sterilized by flaming it once or twice, and when the glass is cool FIG. 10. Method of pouring plates, the gelatin is poured into the lower half of the dish while the cover is slightly raised (Fig. 10), but not inverted or laid on the table. The cover of the dish is then replaced, the test-tube filled with a solution of corrosive sublimate, and the cotton plug returned. The gelatin is spread over the entire bottom of the dish by tipping it from side to side. It is then allowed to harden by placing the dish on the cooling apparatus or leaving it on horizontal surface at room temperature. A simple, inexpensive and effective cooling apparatus is a piece of soapstone, such as is sold at hardware stores (Fig. 11). In winter this can be cooled by hanging it out of doors, at other seasons by im- mersing it in cold water. The three Petri dishes thus prepared should be properly labeled and placed un- der conditions where the gelatin will remain solid and yet growth takes place. The temperature of the laboratory should not be allowed to exceed 23° C. or gelatin cultures are in danger of melting while under examination. Within a few days colonies will make their appearance, in varying numbers, depending upon the dilution used. FIG. 9. Method of melting gela- tin. FIG. 11. Soapstone used for solidifying gela- tin in Petri dishes. 34 General Bacteriology. X Inasmuch as the first plate is invariably too thickly seeded to be of much service, this gelatin tube is often replaced by a water blank, which is treated exactly as the gela- tin tube No. 1, but is not of course "plated" but simply serves to dilute the material. REFERENCES. A. 124; H. 57; L. & K. 88; M. & W. 108; M. & R. 61; McF. 140; 171; P. 224; S. 72. SPECIAL DIRECTIONS. a. Make three gelatin plate cultures, as directed above, and inoculate with B. sub- introducing a minute portion of agar culture (XIII) into tube No. 1, two loops of No. 1 into No. 2, and three of No. 2 into No. 3. Label, and when the gelatin has solidi- fied, place plates in cool chamber (XV). b. Also make a "blank" plate from an uninoculated gelatin tube, observing all pre- cautions to prevent contamination. This will serve as a control or check on your other plates. If any colonies develop on this it indicates carelessness. EXERCISE XXVII. AQAR PLATE CULTURES. GENERAL DIRECTIONS. These are made in the same way as the gelatin plates ex- cept that the high meltingpoint (96° C.) of agar makes it necessary to use boiling water to melt it. Inasmuch as the vitality of vegetative bacteria is destroyed at a temperature much above 42° C., it must be cooled down before inoculating, but as agar solidifies at 39-40° C. it must not, therefore, be cooled below that point. It is best to keep the melted agar at about 42° C. for 10 minutes before it is inoculated. For this purpose a water- bath should be so arranged that the temperature can be controlled by means of a thermo-regulator. A cheap and yet satisfactory 03 W M ITl ^ arrangement is represented in Fig. 11. Inoculate, make dilu- V\ 1 tions and pour as in case of gelatin, except that before the agar is poured, it is well to slightly warm the Petri dishes by placing them iu the incubator at 38° C. for a few minutes, other- wise the agar may solidify in lumps in the plate. In cooling, agar shrinks somewhat, and in doing so water is expressed from the solid jelly. In the incubator this condenses on the under side of the cover of the Petri dish to such an extent that drops run down on to the culture surface thus causing the developing superficial colonies to "run." To obviate this the Petri dishes, FIG. 11. Water-bath for cooling agar. when placed in the incubator, should be inverted. REFERENCES. H. 61; L. & K. 94; M. & R. 66; N. 285; P. 225; P. B. C. 28. SPECIAL DIRECTIONS, a. Make three agar plates of B. coli; use one loop of bou- illon culture (XIII) for tube No. 1 and proceed as in XXVI. b. Place in incubator at EXERCISE XXV11I. ROLL-CULTURES (Esmarch). GENERAL DIRECTIONS. These are essentially plate cultures in which the medium instead of being poured out into dishes is solidified in a thin, even layer on the inner surface of the test-tubes. This is best accomplished by means of a piece of ice placed in a dish on a piece of cloth by which it can be kept in the desired position (Fig. 12). A horizontal groove is melted in the ice by means of a test-tube filled with hot water. In this groove the test-tubes, inoculated as in case cuUu '""of plate cultures, are rapidly whirled until the medium is thor- oughly set. Both agar and gelatin can be used, although gelatin cannot be used sue- FIG. 12. 36 General Bacteriology. eessfully with those species which liquefy this medium. In the case of agar the tubes should be placed in a horizontal position a few hours (over night) until the medium has become attached to the tube ; afterwards they can be stored in the usual receptacles for tube cultures. REFERENCES. A. 131; M. & R. 65; McF. 143. SPECIAL DIRECTIONS. «. Melt a tube of gelatin and without inoculating it practice making a roll-culture as described above. Avoid tipping the tube enough to get medium on cotton plug. Remelt and roll again until the knack is acquired. b. Make two roll-cultures in gelatin of B. coli (XIII), using a water-blank instead of gelatin tube No. 1. c. Make two agar cultures of B. subtilis in same way. d. Incubate b. in cool chamber, andc. at 28° C. EXERCISE XXIX. STUDY OF PLATE CULTURES. MACROSCOPIC. As the colonies appear, note: «. form, b. size, c. surface elevation, d. consistency, t. color. Both the surface and deep colonies should be described as they are frequently very different. Drawings should always be made wherever they will be of value; study should be continued as long as changes are noticed. (See Chapter III, I. A. a.-f.) MICROSCOPIC. The colonies appearing on the plates are to be studied under a low power of the microscope. Use a f in. (16 mm.) objective. The Petri dishes can be inverted, and thus avoid the danger of exposing the culture to contamination from the air except with gelatin where liquefying organisms are present. Observe, «. structure of colony as a whole ; 6. character of margin. (See Chapter III. I. A./ifcgr.) REFERENCES. P. B. C. (Cheesman's Charts.) SPECIAL DIRECTIONS. Study, write descriptions and make drawings of all plate cultures. Use blank pages for description and sketch of cultures. EXERCISE XXX. USE OF DECOLORIZING AGENTS. Make three cover-glass preparations from a 24 hour old culture of B. subtilis, stain- ing them with an aqueous solution of gentian violet. Mount in water and examine. While they are still under the microscope, place at one side of the cover-glass a few drops of one of the following solutions, and by means of a strip of filter paper at the opposite side draw the liquid under the cov er-glass until all the color is removed. In this, way determine the relative value of alcohol (95%), acetic acid (5%), and nitric acid (30%) as decoloring agents. EXERCISE XXXI. GRAM'S STAIN. EXPLANATORY. This is a differential stain and one of the most useful. Some bac- teria when stained by this method exhibit a dark violet color, others remain perfectly colorless, thus rendering possible the differentiation of bacteria which are morphologically nearly or quite identical, and also greatly facilitating the demonstration of certain bac- teria in animal tissue. Most of the pathogenic micrococci retain the violet stain although there are important exceptions. The bacilli and spirilla may or may not remain colored. GENERAL DIRECTIONS. a. Spread film. 6. Air dry and fix. 38 General Bacteriology. c. Stain with anilin-oil gentian violet 5 minutes. d. Pour off stain and without washing: e. Apply iodine solution 2 minutes (use several changes). /. Decolorize with 96% alcohol until drippings do not stain white filter paper. g. Wash in water and counter-stain with Bismarck brown. h. Mount in water and examine. i. Dry and mount in balsam. REFERENCES. A. 162; H. 78; L. & K. 106; M. & W. 91; M. & R. 110; McF. 99; N. 287; P. 203. SPECIAL DIRECTIONS. Stain films of young cultures of B. coli and B. subtilis. Also a film of an organism supplied. EXERCISE XXXII. TUBERCLE STAIN (Qabbett). EXPLANATORY. All of the differential methods of staining the tubercle bacterium depend upon the fact that this germ is very resistant towards the ordinary stains and in order to be stained at all must be treated with a dye containing a mordant and this either allowed to remain in contact with the micro-organism several hours or be applied hot. The latter method is the quicker and is usually employed, although it does not give as good results. When once stained this germ withstands the effect of decoloriz- ing agents to such an extent that it is possible to remove the dye from all other objects on the cover-glass preparation (as in sputum) while it retains its own color. The appli- cation of a second dye, of a complementary color, readily distinguishes this germ from all others in the field. A few other bacteria have similar staining properties. (See Part II.) Red is the usual stain and blue the counter stain. Gabbett's method is one of the simplest. GENERAL DIRECTIONS. a. Spread film (sputum from tuberculous patient) . b. Air dry and fix. c. Stain with hot carbol-fuchsin 2 minutes. d. Wash in water. e. Treat with Gabbett's solution | to 1 minute. /. Wash in water and examine. g. Dry and mount in balsam. REFERENCES. A. 162; M. & W. 92; McF. 214; P. 304. SPECIAL DIRECTIONS. Stain three samples of sputa which contain varying numbers of the tubercle bacteria. EXERCISE XXXIII. STAINING ENDOSPORES. GENERAL DIRECTIONS. A. Simple stain. a. Prepare film as usual. 6. Fix by passing through flame 10 or 12 times instead of 3 times. (This prevents the vegetative portion from taking the stain) . c. Stain 2-5 minutes in hot carbol-fuchsin. d. Mount and examine. 40 General Bacteriology. B. Differential stain (Hauser's method). a. Make cover-glass preparation of a spore-bearing culture, fix and stain with hot carbol-fuchsin until spores are thoroughly colored. This must be determined by mount- ing in water and examining under microscope. b. Cautiously decolorize with acetic acid, 5%, until stain is removed from the vege- tative portion only. This to be determined as above. c. Wash in water and counter- stain with methylen blue. d. Examine. Crimson spores will be seen in blue bacilli. REFERENCES. Other methods, see A. 164-167. SPECIAL DIRECTIONS. Stain by each method spores in cultures of B. subtilis or other spore-bearing organisms. EXERCISE XXXIV. STUDY OF ENDOSPORES. a. Make cultures on peptoneless agar, or agar to which a few drops of calcium hydrate has been added, of the following organisms and incubate at 28° or 38° C. depend- ing upon the organisms : Bacillus subtilis (EHRENB.) COHN. Bacterium anthracis (KOCH) MIG. Bacillus amylobacter VAN TIEGHEM (or any clostridium form). Bacillus tetani NICOLAIER (or any "drumstick" bacillus). Pseudomonas erythrosporus (CORN) MIG. b. When the cultures are 48 hours old mount films without staining, examine and note: 1) Form. 2) Size. 3) Color. 4) Power to refract light. 5) Relation to mother-cell. (1) Median or central. (2) Intermediate. (3) Terminal or polar. (4) Clostridium form. (5) Drumstick form. c. Make drawings. READ: J. H. 26; L. 60; L. & N. 76; M. & R. 6; N. 46; P. 46; P. B. C. 15; S. 114. EXERCISE XXXV. FLAQELLA STAIN (Bunge). GENERAL DIRECTIONS. a. Make an agar streak of the organism to be stained. b. After 18 to 24 hours, by means of the platinum needle, remove a portion of the growth (being careful to avoid the culture medium) to a large drop of tap water on a perfectly clean cover-glass (XV.) and allow to stand 5 minutes rather than spread, as there is less danger of breaking off the flagella. c. Spread carefully 2 or 3 loopfulsof this drop on each of several clean cover-glasses and dry at room temperature. d. Fix by passing the cover- glass through the top of the flame while it is held in the hand, not in the forceps, as over heating will injure the preparation. 42 General Bacteriology. e. Flood the cover- glasses thus prepared with the following solution (Mordant) : Liquor ferri sesquichlorfdi diluted with distilled water 1:20, 1 part; saturated aqueous solution of taunic acid, 3 parts. This mixture improves with age but should be filtered before using. Allow to act 1 minute. /. Wash in water and dry between filter paper. g. Stain with hot carbol-fuchsin for about one minute. h. Wash in water, dry and mount in balsam. REFERENCES. M. & W. 103; McF. 104; P. 205. Other methods M. & R. 115; McF. 101; A. 167. SPECIAL, DIRECTIONS. Stain B. typhi from cultures furnished, also try B. coli and B. subtilis. EXERCISE XXXVI. CAPSULE STAIN (Welch). GENERAL DIRECTIONS. a. Spread film without the use of water. b. Air dry. c. Fix. d. Apply glacial acetic acid, and drain it off immediately. Do not wash in water. e. Stain with anilin-oil gentian violet (Ehrlieh) which is to be renewed several times to remove acid. /. Wash in 1 to 2% salt solution. g. Examine in salt solution. (Balsam causes capsule to shrink.) REFERENCES. A. 163; P. 203; P. B. C. 13. SPECIAL DIRECTIONS. Use pneumonic ("rusty") sputum, or blood of rabbit in- fected with the pneumococcus. CHAPTER II. PHYSIOLOGY OF BACTERIA. EXERCISE XXXVII. PREPARATION OF SPECIAL MEDIA. The following media will be necessary for the work outlined in this chapter: a. GLUCOSE BOUILLON. To ordinary bouillon add 1% glucose (c. P.), tube and sterilize in steamer, not in autoclave, 2 test-tubes and 2 fermentation tubes. b. GLUCOSE GELATIN. 1% glucose (c. P.), tube and sterilize in steamer, 6 tubes. c. GLUCOSE AGAR. 1% glucose (c. P.), 5 tubes. d. LACTOSE AGAR. 1% lactose (c. P.), 2 tubes. e. LITMUS SOLUTION. To 10 gms. of the dried material add 500 cc. of distilled water, digest in a warm place, decant clear liquid and add a few drops of nitric acid to produce a violet color. (Button). Place in flasks or test-tubes and sterilize in steamer three times, 1 tube. /. DUNHAM'S SOLUTION. Sodium chloride 0.5 gm. | fi u u n . dissolved fllt tube and steril. Peptone (Witte) 1. gms. \ . . . , Water 100. ) ze' 4 g. NITRATE 'SOLUTION. Sodium chloride 0.5 gm. Peptone (Merk) 1 gms. ) Potassium nitrate 0.2 [-Filter, tube and sterilize, 3 tubes. Water 1,000 j k. LITMUS MILK. 1) Freshly separated milk (or if this is not available, new milk is placed in a sep- aratory funnel in an ice chest over night to allow the separation of the cream and milk then drawn off) is titrated with /jNaOH and rendered slightly alkaline to phenolphtha- lein by the addition of rNaOH. 2) Litmus solution is then added until medium is faintly blue. 3) Tube and sterilize in the steamer for 30-45 minutes on 3 or 4 consecutive days. During the summer months particularly very resistant bacterial forms abound in the milk so that it is necessary to increase the number of applications or length of exposure. The efficiency of the sterilizing process should be tested by placing the flasks in the in- cubator for several days to see if anv change occurs, 2 tubes. In addition to the above have 15 tubes of bouillon (9 to contain exactly 10 cc. XLI & XLIV), 10 tubes of gelatin, 15 tubes of agar, 6 water-blanks and 5 potato tubes. EXERCISE XXXVIII. EFFECT OF REACTION OF HED1A ON GROWTH. GENERAL DIRECTIONS. a. Melt 6 tubes of gelatin and add, under aseptic precautions, to three of them, re- spectively, 0.1 cc., 0.3 cc., and 0.5 cc. of a normal solution of hydrochloric acid, and to the other three the same amounts of a normal sodium hydrate. 46 General Bacteriology. b. Thoroughly mix, solidify gelatin in ice water and then inoculate (stab) each tube with the organism to be studied, making a control culture in a tube of neutral gelatin. c. Incubate at 18° C. and note the effect of the chemicals on the rate, amount and character of the growth. REFERENCES. L. & N. 87; McF. 46. SPECIAL DIRECTIONS. Use B. subtilis and B. coli. EXERCISE XXXIX. EFFECT OF CONCENTRATION OF MEDIA ON GROWTH. a. Pour about 2 cc. of "condensed milk" into each of two sterile test-tubes, dilute one with five times the volume of sterile water. b. Inoculate both with a pure culture of B. subtilis and incubate at 28° C. Explain changes which occur. c. Test extract of beef or syrup in the same way. EXERCISE XL. EFFECT OF TEMPERATURE VARIATIONS ON RATE OF GROWTH. GENERAL DIRECTIONS. a. Make four agar streak cultures of organism to be studied. b. Incubate them at the following temperatures: Ice chest (7° C.), room (20° C.), low incubator (28° C.), blood heat (38° C.). c. By frequent observations as to luxuriance of growth, determine the optimum temperature of growth for each. REFERENCES. F. 73; L. & N. 98. SPECIAL DIRECTIONS. Use B. campestris and B. coli. EXERCISE XLI. DETERMINATION OF THERMAL DEATH POINT. GENERAL DIRECTIONS. a. Make a bouillon culture of the organism to be tested. b. 48 hours later heat a large water-bath to 45° C. Place in this, in close proximity to a thermometer, a test-tube (16 mtn. in diam.) containing exactly 10 cc. of standard bouillon. (Reaction +1.5.) c. After 15 minutes exposure at this temperature remove the cotton plug from the tube, inoculate the broth with three loopfuls (standard size, XII) of the culture prepared above (a.), and carefully mix by slightly agitating the tube, without removing it from the bath. d. After a further exposure of 10 minutes remove the tube from the bath and place it in a vessel of ice cold water to cool. Then incubate at a temperature favorable to the development of the organism under observation. e. In the same manner expose the organism to the following temperatures: 50°, 55°, 60°, and 65° C. /. In all cases incubate at least a week and take as the thermal death point the low- est temperature at which growth fails to appear. (In more accurate work the tempera- ture should be determined within 2° C.). REFERENCES. P. B. C. 32. SPECIAL DIRECTIONS. Use B. coli or B. typhosus. 48 General Bacteriology. EXERCISE XLH. COHPARATIVE EFFICIENCY OF DRY AND MOIST HEAT. GENERAL DIRECTIONS. a. Charge a water blank with culture of a spore-bearing bacillus, shaking it well to break up the clumps. b. Sterilize eight cover-glasses by passing them several times through the flame, and place four in each of two sterile Petri dishes. c. With a sterile loop place an equal quantity of the bacterial suspension (a.) on each cover-glass, and dry by placing Petri dishes in the incubator with the covers slightly raised. d. When dry place one Petri dish in the dry sterilizer (near the thermometer), and the other in the steamer. e. Keep both sterilizers at a temperature of 100° C., and at the end of 5, 10, 20 and 40 minutes respectively, remove one cover-glass from each Petri, place it in a sterile Petri dish and pour a tube of liquefied gelatin or agar over it. Tip the dish from side to side to dislodge as many of the bacteria as possible from the cover-glass, solidify the medium and incubate. REFERENCES. L. 101; S. 146. SPECIAL DIRECTIONS. Use an old (spore-bearing) culture of B. subtilis. Arrange data in the form of a table. EXERCISE XLIII. EFFECT OF DESICCATION. GENERAL DIRECTIONS. a. Prepare five cover-glasses each of a spore-bearing and a non-spore-bearing cul- ture, as directed in XLII. b. Place them in a sterile Petri dish, and dry in the incubator. c. Next morning and every twenty-four hours later plate one of the cover-glasses. d. In this way determine the length of time the organism in question can withstand desiccation. * REFERENCES. F. 77; L. & N. 93; McF. 46; S. 151. SPECIAL DIRECTIONS. Use a young culture of B. coli and an old (spore-bearing) cul- ture of B. subtilis. Tabulate results. EXERCISE XLIV. EFFECT OF CHEMICALS ON BACTERIA. GENERAL DIRECTIONS. a. Inoculate three tubes containing 10 cc. of sterile bouillon, with three loopfuls of a 24-hour old broth culture of organism to be studied. b. Add 0.1 cc. of a 5% solution of carbolic acid to one tube (No. 1); 0.6 cc. to an- other (No. 2) ; and 2 cc. to the third (No. 3) . c. Two hours later transfer three loopfuls from each tube to sterile bouillon and in- cubate all of the tubes at 38° C. d. The carbolic acid in No. 1 and its sub-culture does not prevent growth. In No. 2 no growth, but abundant in its sub-culture (acts as an antiseptic). In both No. 3 and its sub-culture no growth (acts as a disinfectant) . REFERENCES. F. 81; L. & N. 90; L. 107; McF. 46. SPECIAL DIRECTIONS. Use B. coli. 50 General Bacteriology. EXERCISE XLV. RELATION TO OXYGEN. GENERAL DIRECTIONS. a. Pour a tube of melted agar into a sterile Petri dish, and when the medium has hardened make several parallel streaks with a platinum loop charged with an aerobic or- ganism. b. Sterilize a piece of mica or a cover- glass, by passing it several times through the flame and place this over several of the streaks. This is to shut out the air and should therefore be in perfect contact with the medium. c. Make another plate in the same way using an anaerobe. REFERENCES. F. 60; L. & N. 95; L. 180; McP. Chap. VIII. SPECIAL DIRECTIONS. Use B. siibtilis and an anaerobe. EXERCISE XLVI. EFFECT OF DIRECT SUNLIGHT. GENERAL DIRECTIONS. a. Make an agar plate of the organism to be studied (seeding rather thickly). b. When agar has thoroughly set, invert the Petri and paste on under side a piece of black paper from which has been cut out a number of letters, e. g., student's initials. c. Expose this dish, paper side up, to the direct sunlight for a number of hours (4-6). d. Remove the paper and incubate. REFERENCES. F. 71; L. &N. 101; L. 77; McF. 46; S. 151. SPECIAL DIRECTIONS. Use B. prodigiostis (Ehrenb.) Fluegge or B. typhosus. EXERCISE XLVII. DETECTION OF GAS (Shake Culture). GENERAL DIRECTIONS. a. Melt a tube of glucose agar (or gelatin) and inoculate with a gas-producing organism. b. Thoroughly mix and solidify quickly by placing in ice water. c. Incubate over night. REFERENCES. L. & N. 153; M. & R., 85. SPECIAL DIRECTIONS. Use B. coli; incubate. Make sketch. EXERCISE XLVIII. QUANTITATIVE ANALYSIS OF GAS (Fermentation Tube). GENERAL DIRECTIONS. a. Inoculate the open arm of a fermentation tube with a gas-producing organism. b. Incubate at 38° C. c. By frequent observations determine: 1. Whether growth takes place in the open or closed arm, i. e., whether it is aero- bic or anaerobic. ^2. The rapidity and total amount of gas formation. Use gasometer. (Plate I. B.) 3. Kinds of gas. When the culture has ceased producing gas, completely fill the open arm with a 2 % solution of sodium hydrate ; place the thumb over the mouth of the tube and thoroughly mix the Na OH with the gas in the closed arm, then without remov- ing the thumb return the gas to the closed arm, remove the thumb, when the medium will rise in the closed arm to take the place of the absorbed CO2. Measure. The re- 52 General Bacteriology. maining gas is considered as hydrogen; bring this into the open arm, remove the thumb and introduce alighted match. Air mixred with the hydrogen present causes a slight ex- TT plosion. Express the amount of CO2 and H. in the form of a proportion. ^— = . CU2 REFERENCES. A. 203; McF. 54; M. & R. 86. SPECIAL DIRECTIONS. Use B. coli, also try B.-subtilis. EXERCISE XLIX. DETECTION OF ACIDS (Wurtz). GENERAL DIRECTIONS. a. Melt a tube of lactose agar (gelatin can be used) and add enough of a sterile, blue litmus solution to give it a distinct color, cool to 42° C., inoculate it with an acid- pro- ducing organism and pour in the usual manner. b. When the agar has solidified invert the dish and place it in the incubator. REFERENCES. McF. 54. SPECIAL DIRECTIONS. Use B. coli and incubate at 38° C. EXERCISE L. QUANTITATIVE DETERHINATION OP ACIDS. GENERAL DIRECTIONS. a. Inoculate 5 test-tubes of glucose bouillon (or milk) with an acid-producing organism. b. At periods 24 hours apart remove, with a sterile pipette, 5 cc. of the medium from each and titrate with a twentieth normal potassium (or sodium) hydrate solution, using phenolphthalien as an indicator. c. Plot the results, expressing the number of cc. of hydrate solution as abscissae and the daily intervals as ordinates. SPECIAL DIRECTIONS. Use B. coli and incubate at 38° C. EXERCISE LI. DETECTION OF NITRITES IN CULTURES. GENERAL DIRECTIONS. a. Make a culture of a reducing organism in a test-tube of the nitrate solution (XXXVII. g.). 6. Incubate at 28° C. for 1 week, add 1 cc. of each of following solutions: 1) Sulphanilic acid (para-amido benzenesulphonic acid) 0.5 gm. Acetic acid (sp. gr. 1.04) 150 cc. 2) «-amido-naphthalene acetate. Boil 0.1 gram of solid a-amido-naphthalene in 20 cc. of water, filter the solution through a plug of washed absorbent cotton, and mix the nitrate with 180 cc. of diluted acetic acid. All water and vessels used must be free from nitrites. (Leffman and Beam.) The presence of a nitrite is indicated by a pink color. c. A tube of the original medium should be incubated and tested as a control. REFERENCES. A. 215; McF. 56. SPECIAL DIRECTIONS. Use Bacillus vulgaris. (Hauser.) Mig. EXERCISE LII. DETECTION OF AMMONIA. GENERAL DIRECTIONS. a. Make bouillon culture and incubate. 54 General Bacteriology. b. Place in neck of tube a piece of filter paper which has been dipped in Nessler's reagent (for formula see works on water analysis). A yellow to reddish brown color indicates the presence of ammonia. REFERENCES. L. & N. 141. SPECIAL DIRECTIONS. Use sewage to inoculate medium. EXERCISE LIII. DETECTION OP SULPHURETTED HYDROGEN. GENERAL DIRECTIONS. a. Make a culture in a test-tube, or better, a flask of bouillon and incubate at 38° C. b. Twenty-four hours later fasten in the flask, by means of the cotton plug, a strip of filter paper moistened with lead acetate. c. The presence of sulphuretted hydrogen is indicated by change of color from brownish to blue. The color change is often slight and can be best detected by frequent observations. REFERENCES. L. & N. 138. SPECIAL DIRECTIONS. Use B. coli or sewage. EXERCISE LIV. DETECTION OF INDOL. GENERAL DIRECTIONS. a. Make a culture in a tube of glucose-free broth* (or Dunham's solution). b, 24 hours to 1 week later add a few drops of concentrated sulphuric acid and 1 cc. of sodium nitrite solution. (Sodium nitrite, 0.02 gms. Distilled water, 100 gms.) The presence of iudol is indicated by the production of a deep red color. REFERENCES. L. & N. 142; McF. 56; M. & R. 87. SPECIAL DIRECTIONS. Use B. coli. EXERCISE LV. DETERMINATION OF CHEMICAL ENZYHES IN CULTURES. GENERAL DIRECTIONS. a. Make two gelatin stab cultures of a rapidly liquefying organism and incubate several days or until the gelatin has all been liquefied. b. Pour one into a tube of gelatin to which carbolic acid (yV cc. of a 5% sol. per cc. of medium) has previously been added. Mark the line which separates the liquid and solid gelatin. c. Add the other tube of liquefied gelatin to a tube of carbolized milk. d. Make control cultures in the carbolic media with a pure culture of the organism used above to show that the acid inhibits the growth and that the changes are not due to the living organism. REFERENCES. McF. 53. SPECIAL DIRECTIONS. Use B. subtilis. EXERCISE LVI. VARIATION IN ENZYME PRODUCTION. Make stab cultures of Pseudomonas aeruginosa (SCHROETER) MIG. (B. pyocyaneus), or any slow liquefier, in ordinary neutral gelatin and also glucose gelatin. Compare rate of liquefaction in each. *This is prepared from beef by inosulating the meat infusion with an organism capable of fer- menting sugar, such as B. coli, and allowing it to stand several hours at !)8° C. The meat is then strained and the bouillon prepared in the usual manner. This is recommended for testing for indol. 56 General Bacteriology. EXERCISE LV11. VARIATION IN COLOR PRODUCTION. Make an agar streak of B. prodigiosus. Incubate at 38° C. 24 hours later transfer to fresh media. Continue the process of daily transplanting from cultures of previous day until chromogenic property is lost, even at the room temperature. CHAPTER III. TAXONOMY. POINTS TO BE OBSERVED IN THE STUDY OF BACTERIA. The following scheme gives an idea of the points to be noted in the description of an organism together with some of the more common descriptive terms. CULTURE CHARACTERS. 1. GELATIN PLATE: A. Surface colonies. a. Form: Punctiform, too small to be defined by naked eye; circular; oval; irregular; fusiform; cochlate, twisted like a snail shell; amoeboid, very irregular like changing forms of amoebae; conglomerate, an aggregation of colonies. b. Size, expressed in millimeters. c. Surface Elevation: flat; spreading; thin; raised, growth thick with abrupt, terraced edges; convex, surface segment of a circle but very flatly convex; pulvinate, sur- face the segment of a circle but decidedly convex; capitate, hemispherical; rough, irregular elevations and depressions; contoured, like the undulating surface of a relief map; papil- late, horn like projections; rugose, wrinkled; alveolate, depressions separated by thin walls; pitted; sulcate, ridged or furrowed. d. Consistency: thin; membraneous, thin, dry, separating from medium; coria- ceous, thick like leather or parchment; viscous, ropy; slimy ; gelatinous; brittle. e. Color: transparent; vitreous, transparent and colorless; oleaginous, trans- parent and yellow, olive to linseed oil colored; resinous, transparent and brown, varnish or resin colored; translucent; paraffinous, translucent and white, porcelaneous ; opales- cent, translucent, grayish-white by reflected light, smoky-brown by transmitted light; nacreous, translucent, grayish- white with pearly lustre; sebaceous, trans- lucent, yellowish or grayish- white, tallowy; butijrous, translucent or yellow; ceraceous, translucent and wax colored; opaque; cretaceous, opaque and white; chalky, dull without lustre; glossy, shining; fluorescent; iridescent. f. Margin (To be determined by low power of microscope): entire; undulate; repand; erase, finely eroded as if gnawed; lobed; articulate; laciniate, cut jaggedly into deep narrow lobes; lacerate, cut variously into irregular segments' fimbricate, edge bordered by slender processes thicker than hairs; ciliate, tufted; floccose, wooly, filaments in fleecy masses; curled, filaments in locks or ringlets: filamentous, consisting of loosely placed, interwoven filaments, not so dense as floccose. g. Internal structure (To be determined by microscope) : homogeneous, uniform throughout; concentrically zoned; marmorated, traversed by veins as in some kinds of marble, marbled; finely punctate; areolate, marked out with small spaces, reticulate; moruloid, having the character of a morula, resembling a mulberry; segmented; finely granular; coarsely granular; grained, as in lumber; curled, composed of twisted bundles of parallel filaments as in locks or ringlets; floccose; filamentous. h. Change in Medium: consistency; color; odor. 58 General Bacteriology. B. Deep colonies: a. Form. 6. Size. c. Color. d. Internal structure. 2. AGAB PLATES: A. Surface colonies. ) „ . , . ,. ,.-, > Same points as in gelatin plate, (I). B. Deep Colonies. j 3. GELATIN STAB CULTURES. A. Non-liquefying. a. Line of puncture: filiform, uniform growth without any special characters; tuberculate; papillate, covered with papilla?; echinulate, minutely prickly; villous, beset with long or short undivided hair-like extensions; arborescent, beset with branched hair- like extensions; beaded, composed of small round more or less conjointed colonies; banded longitudinally- b. Surface: (Same as surface colonies gelatin plates 1 c.) B. Liquefying. a. Shape of liquefied area: crateriform, saucer shaped liquefaction of gelatin; saccate, shape of an elongated sack, tubular; cylindrical; funnel formed; napiform, out- line of a turnip; fusiform, outline of a parsnip; stratiform, liquefaction extending to the walls of the tube and then downward horizontally. b- Fluid: clear; turbid; flocculenf. c. Sediment: flocculent; stringy ; granular. d. Membrane: character; color. 4. STREAK CULTURES: a. Form. b. Size. c. Surface elevation. d. Consistency. P i . f Same as for colonies on gelatin plates (1;. /. Margin. g. Internal structure. h. Change in medium- 5. POTATO. A. Growth apparent. (Same as plate cultures). B. Growth not apparent. 6. BOUILLON: o. Character of fluid : clear; turbid; etc. b. Sediment. c. Membrane. 7. MILK. A. No visible change, even after boiling. B. Curd formed: a. Time required. b. Character of curd: hard; soft. c. Digestion- General Bacteriology. 59 d. Character of whey: clear; turbid; flocculent. e. Reaction. /. Gas. g. Odor. 8. BLOOD SERUM: (Same as streak cultures) . MORPHOLOGICAL CHARACTERS. a. Form. 6. Cell grouping. c. Size. 1. In terms of the inicromillimeter; breadth, average and extreme length- 2. In terms of human blood cell. d. Stain. 1. Aqueous solutions; stains easily or with difficulty; uniformly or irreg- ularly. 2. Special stain ; Gram; tubercle; etc. e. Motility. 1- Brownian movement- 2. Vital movement; sluggish or active; rotary or direct; most favorable temperature; age; media; etc. 3- Flagella; stained by Loeffler, Bunge or Van Ermengem's method; dis- tribution, monotrichal, lophotrichal or peritrichal- /. Capsule; stained by Ziehl, Gram or Welch's method; most favorable con- ditions; broad or narrow; present in serum, milk or on agar streaks. g. Spores; time required for formation; media; position in cell, center or end; effect on shape of cell, clostridium, or drumstick; germination, time, temperature; stain, Hauser or Moeller's method; temperature limits- h. Vacuoles (plasmoloysis). i. Crystals, j- Involution forms- k. Pleomorphism- 1. Effect of various media. 2. Effect of reaction of media- PHYSIOLOGICAL CHARACTERS- a. Effect of desiccation. b. Relation to temperature; minimum; optimum; maximum; thermal death point. c. Relation to oxygen; under mica plate; in hydrogen. d. Relation to light; (Buchner's Experiment XL VI.). e. Relation to antiseptics and disinfectants • /- Pigment production; relation of development to oxygen; relation of de- velopment to character of medium; changes produced by alkali and acid; solubility; spec- trum analysis. g- Gas production; rate, quantity and formula produced on glucose, lactose, and saccharose media. 60 General Bacteriology. h. Acid and alkali production; carbohydrates present ; carbohydrates absent, t. Relation of growth to acidity and alkalinity of medium; growth in 1.5, 3 and 4 % alkali; growth in 1.5, 3, 4 and 5 % acid. j. Reduction of nitrates; to nitrites; to ammonia. k. Production of sulphuretted hydrogen. I- Production of indol. m. Enzyme production; proteolytic; diastatic. n. Characteristic odor. o. Pathogenesis : 1. Modes of inoculation by which its pathogenic properties are demonstrated. 2. Quantity of material required. 3. Duration of the disease and its symptoms- 4. Lesions produced and the distribution of the bacteria in the inoculated animals. 5. Which animals are susceptible and which are immune. 6. Variations in virulence and the probable causes to which they are due- 7. Detection of toxic or immunizing products of growth. 8- Widal test. 9. Pfeiffer's phenomenon- REFERENCES: Chester, Report Delaware Experiment Station, 1897; A. 216; P. B. C. (Cheesman's Charts). CLASSIFICATION OF BACTERIA. (MIQULA.) I. Cells globose in a free state, not elongated in any direction before divisions in 1, 2, or 3 planes. COCCACEAE ZOPH emend. MIG. A. Cells without organs of motion. a. Division in one plane, 1. Streptococcus BILLROTH. b. Division in two planes, 2. Micrococcus (HALLIER) COHN. c . Division in three planes, • 3. Sarcina Goodsir. B. Cells with organs of motion. «. Division in two planes, - 4. Planococcus MIGULA. b. Division in three planes, • 5. Planosarcina MIGULA. II. Cells cylindrical, longer or shorter, and only di- vided in one plane, and elongated to twice the normal length before the division. (1) Cells straight, rod-shaped without sheath, non-motile by means of flagella. BACTERIACEAE MIGULA. A. Cells without organs of motion, - 6. Bacterium EURENB. B. Cells with organs of motion (flagella). a. Flagella distributed over the whole body, 7, Bacillus COHN. b- Flagella polar, 8. Pseudomonas MIGULA. (2) Cells crooked, without sheath. SPIRILLACEAE MIGULA. A. Cells rigid, not snake-like or flexuous. General Bacteriology. 61 «. Cells without organs of motion (flag- ella), - 9. Spirosoma MIGULA. b. Cells with organs of motion (flagella) 1. Cells with 1, very rarely 2-3 polar flagella,. - - 10. Microspira SCHEOETER. 2. Cells with polar flagella- tufts, 11. Spirillum EHRENB. B. Cells flexuous, - 12. Spirochaeta EHRENB. (3) Cells enclosed in a sheath. CHLAMYDOBACTERIACEAE MIGULA. A. Cell contents without granules of sulphur. a. Cell threads unbranched. 1) . Cells division always only in one plane, - 13. Streptothrix COHN. 2). Cell division in three planes pre- vious to the formation of condia- i). Cells surrounded by very delicate scarcely visible sheath (marine), - - 14. Phragmidiothrix ENGLER. ii). Sheath clearly visible (fresh water), - - 15. Crenothrix COHN. b. Cell threads branched, - 16. Cladothrix COHN. B. Cell contents containing sulphur granules. 17. Thiothrix WINOGRADSKY (4). Cells destitute of a sheath, united into threads motile by means of an undulating membrane. BEGGIATOACEAE. Only one genus. (The single species is scarcely separable from Oscillaria) - 18. Beggiatoa TRAVISAN. CHAPTER IV. SYSTEMATIC STUDY OF REPRESENTATIVE NON-PATHOGENIC BACTERIA. EXERCISE LVIII. PREPARATION OF SPECIAL MEDIA. Tube and sterilize the following media for work in Chapters IV and V: 80 tubes of plain agar. 2 tubes of lactose agar. 20 tubes of gelatin. 8 tubes of bouillon. 10 fermentation tubes of glucose bouillon. 8 tubes of potato. 8 tubes of milk. 8 tubes of Dunham's solution. 10 water-blanks. EXERCISE LIX. BACILLUS PRODIQIOSUS (Ehrenb.) Fluegge. EXPLANATORY. This organism was first described by Ehrenberg (Erhandlunger der Berliner Akademie) in 1839 and named Monas prodigiosa. It is the oldest known chromogenic bacterium. It is commonly found in the air of Europe and has a very interesting his- tory on account of its casual relation to bread epidemics — "bloody bread," "bleeding host," etc. It is questionable if it occurs sponta- neously in this country. It is slightly pathogenic. Introduced intraperitoneally into guinea pigs in large quantities it produces death. Inoculated into animals naturally immune to malignant oedema it renders them susceptible. Rabbits inoculated with anthrax are protected by a subsequent inoculation with this organism. It is grown with the streptococcus of erysipelas to produce Coley's Fluid for treatment of inoperable malignant tumors. REFERENCES. Lafar, 137-138. MORPHOLOGICAL CHARACTERS. Age of cultures. Incubatioa temp. (°C.) SKETCHES. i. Form: c Gelatin 2 Size .. 5 Motility * PHYSIOLOGICAL CHARACTERS. i. Relation to temperature ; 2. Relation to free oxygen: 3. Relation to other agents, such as desiccation, light, disinfectants, etc. : 4. Pigment production: 5. Gas produ ction in glucose media: a. Shake culture b. Fermentation tube, growth in: (i) open arm: (3) rate of development: 24 hours per cent., 48 hours (4) reaction in open arm: 6. Acid or alkali production, litmus milk 7. Reduction of nitrates; to nitrites 8. Indol production; 24 hours , 48 hours fecal odor; 24 hou rs ,48 hours 9. Enzyme production : proteolytic ....(s) closed arm: percent., 72 hours per cent., hours . ...(5) gas formula : H : CO2 : : : per cent. , to ammonia. . days, days. . diastatic. 10. Characteristic odor. 11. Hathogenesis [64] CULTURE CHARACTERS. Reaction of Medium, Incubation Temp.(°C) 34 HOURS. 48 . HOURS 0 Dvrs SKETCHES. (1) Gelatin plate : (a) Surface Colonies. (b) Deep Colonies. (2) Agar plate: (a) Surface Colonies. (b) Deep Colonies. (3) Gelatin Stab. A r A (4) Agar streak. ^_ S A V / A (5) Potato. Bouillon. (7) Special Media. [65] EXERCISE LX. VARIETY OF PIGMENTS. Make agar or potato streak cultures of the following organisms, incubate at 38° C.. study, describe and sketch. AGAR STREAK. 24 HOURS. 48 HOURS. SKETCHES. Bacillus indicus or Sarcina aurantiaca or Sarcina lutea or Pseudomonas fluorescens (B. fluorescens) Pseudomonas aeruginosa (B. pyocyaneus) Pseudomonas violacea or [66] General Bacteriology. 67 EXERCISE LXI. SEPARATION OF BACTERIAL COLORING HATTER. a. Make four agar streaks of Bacillus prodigiosus, which are to be kept in the dark until the coloring matter is well formed. b. Add about 10 cc. of ether to each tube and shake vigorously until the red pig- ment has all been dissolved out. c. Pour into a large test-tube and allow to stand over night in the dark, then pipette off the colored portion. d. Divide this into four parts and treat them as follows: 1. Evaporate on glass slide and examine crystals formed under microscope. 2. Add a few drops of hydrochloric acid, drop by drop. 3. Add a few drops of sodium hydroxide. 4. Stand in 'direct sunlight. EXERCISE LXII. BACTERlUn PHOSPHORESCENS Fischer. GENERAL CONSIDERATIONS. Described by Fischer in 1887 (Zeitschrift fttr Hygiene, Baud II, p. 92). Found in Kiel harbor, dead sea fish, oysters and occasionally on meat in shops. The production of light is shown in the dark, especially when the organism is grown on a medium made by boiling two salt herrings in a liter of water, adding 100 gins, of gelatin to the filtrate without neutraliza- tion, tubing and then sterilizing (Lehmann). Phosphorescence can even be restored to attenuated cultures by growth on this medium. Inasmuch as oxygen is necessary to light production surface growths are best. REFERENCES. Lafar 160-164. MORPHOLOGICAL CHARACTERS. •I V 3* Incubation temp. (°C.) SKETCHES. i. Form: c Gelatin . .... 2 Size in growths 4. Staining powers: 5. Motility * a. Character of movement b. Flagella stain PHYSIOLOGICAL CHARACTERS. i. Relation to temperature : 2. Relation to free oxygen: 5. Relation to other agents, such as: desiccation, light, disinfectants, etc.: . 4. Pigment production: 5. Gas production in glucose media: a. Shake culture A. Fermentation tube, growth in: (i) open arm: (3) rate of development: 24 hours per cent., 48 hours . (4) reaction in open arm: 6. Acid or alkali production, litmus milk 7. Reduction of nitrates; to nitrites 8. Indol production; 24 hours 48 hours fecal odor; 24 hours 48 hours 9. Enzyme production : proteoly tic . . . . (2) closed arm : per cent., 72 hours per cent., • -.(5) gas formula, H : CO2 : : hours per cent. . to ammonia. . days, days. . diastatic — 10. Characteristic odor. 11. Pathogenesis [68] CULTURE CHARACTERS. Kc'art'um of Medium. Incubation Temp. (°C) 24 HOURS. 48. . . HOURS. 6.. . DAYS. SKETCHES. [69] A EXERCISE I. Mil. BACILLUS ACIDI LACTIC! Hueppe. GENERAL CONSIDERATIONS. First described in 1884 by Hueppe in Mitteil. aus dem Kaiserl. Gesundlieitsamte, Bd. II, p. 1837. This organism may be taken as a type of the bacteria causing sour milk. REFERENCES. Lafar, 223-244. MORPHOLOGICAL CHARACTERS. • v ~°s o'a *u Incubation temp. (°C.) SKETCHES. i. Form: c Gelatin 2 Size 5 Motility b Flagella stain . PHYSIOLOGICAL CHARACTERS. i. Relation to temperature : 2. Relation to free oxygen: '. 3. Relation to other agents, such as: desiccation, light, disinfectants, etc.: 4. Pigment production: 5. Gas production in glucose media: a. Shake culture 4. Fermentation tube, growth in: (i) open arm: (3) rate of development: 24 hours percent., 48 hours. (4) reaction in open arm: 6. Acid or alkali production, litmus milk 7. Reduction of nitrates; to nitrites 8. Indol production; 24 hours 48 hours fecal odor; 24 hours 48 hours 9. Enzyme production : proteolytic ...(2) closed arm: . per cent., 72 hours per cent., ...(5) gasformula, H : CO, : : — hours.. percent. to ammonia.. days, days. .diastatic. 10. Characteristic odor., it. Pathogenesis [70] CULTURE CHARACTERS. Reaction of Medium. Incubation Temp. CC) 24.. ..HOURS. 48 HOURS. 6 DAYS. [71] SKETCHES. , A i A EXERCISE LX1V. BACILLUS VULQARIS (Hauser) Migula. PROTEUS VULGARIS. GENERAL CONSIDERATIONS. Described by Hauser in 1885 as Proteus vulgaris (Ueber Faulnis Bakterien). It is widely distributed and is commonly found in putrefactive substances. It is one of several related species included under the old name of "Bacterium termo." While in small doses and under ordinary conditions it is harmless, at times and in large doses it may be pathogenic. REFERENCES. Lafar 194-199. MORPHOLOGICAL CHARACTERS . i ~2 V 3 t" Incubation temp. («C.) SKETCHES. i. Form: b. Agar c. Gelatin d Other media . 2 Size. rf. Special stains 5. Motility : a. Character of movement b. Flagella stain 6. Spores 7. Special characters, such as: deposits, vacuoles pleomorphic and involution forms, capsules, etc PHYSIOLOGICAL CHARACTERS. i. Relation to temperature: 2. Relation to free oxygen: 3. Relation to other agents, such as : desiccation, light, disinfectants, etc: 4. Pigment production: 5. Gas production in glucose media: a. Shake culture *. Fermentation tube, growth in: (r) open arm: (3) rate of development: 24 hours per cent., 48 hours . (4) reaction in open arm: 6. Acid or alkali production, litmus milk 7. Reduction of nitrates; to nitrites 8. Indol production; 24 hours ,48 hours fecal odor; 24 hours 48 hours 9. Enzyme production : proteolytic . ..(2) closed arm: per cent., 72 hours per cent., hours. ...(5) gas formula, H : CO, : : : per cent. , to ammonia. . days . days . diastatic 10. Characteristic odor. 11, Pathogenesis [72] CULTURE CHARACTERS. Reaction of Medium, Incubation Temp.C'C) 24 HOURS 48. .. . HOURS 0 Dws SKETCHES. (1) Gelatin plate : (a) Surface Colonies. (b) Deep Colonies. • (2) Agar plate: (a) Surface Colonies. (b) Deep Colonies. (3) Gelatin Stab. A A (4) Agar Streak. x._'/ A A v / A (5) Potato. Bouillon. (7) - Special Media. [73] Name of organism Source, habitat, etc. MORPHOLOGICAL CHARACTERS. Age of Cultures. Incubation temp. (°C.) SKETCHES. i. Form: t b Loeffler's methylen-blue 5 Motility: PHYSIOLOGICAL CHARACTERS. i. Relation to temperature: . 2. Relation to free oxygen: 3. Relation to other agents, such as: desiccation, light, disinfectants, etc.: 4. Pigment production: 5. Gas production in glucose media: a. Shake culture b. Fermentation tube, growth in: (i) open arm: (3) rate of development: 24 hours per cent., 48 hours. (4) reaction in open arm: 6. Acid or alkali production, litmus milk 7. Reduction of nitrates; to nitrites » 8. Indol production; 24 hours 48 hours fecal odor; 24 hours ,48 hours 9. Enzyme production: proteolytic (2) closed arm : .per cent., 72 hours per cent., (5) gas formula, H : CO« : : . hou rs per cent. , to ammonia .days, .days. .diastatic. 10. Characteristic odor. 11. Pathogenesis [74] CULTURE CHARACTERS. Reaction of Medium, Incubation Temp. ("Q 24 HOURS. 48 HOURS. 6 DAYS. SKETCHES. [75] A A Name of organism Source, habitat, etc. . . MORPHOLOGICAL CHARACTERS. en u «- n-. s o~ 85 Incubation temp. (°C.) SKETCHES. i. Form: • c Gelatin . .... d Other media . ... . 2 Size . .. X 5. Motility. .... b. Flagella stain . ... PHYSIOLOGICAL CHARACTERS. i. Relation to temperature : 2. Relation to free oxygen: 5. Relation to other agents, such as: desiccation, light, disinfectants, etc.: 4. Pigment production: 5. Gas production in glucose media: a. Shake culture b. Fermentation tube, growth in: (i) open arm: (3) rate of development: 24 hours per cent., 48 hours (4) reaction in open arm: 6. Acid or alkali production, litmus milk 7. Reduction of nitrates; to nitrites 8. Indol production; 24 hours ,48 hours fecal odor; 24 hours , 48 hours 9. Enzyme production : proteolytic ....(2) closed arm: percent., 72 hours per cent hours . ..(5) gas formula, H : CO2 : : per cent. , to ammonia . . days, days. . diastatic... 10. Characteristic odor. 11. Hathogenesis [70] CULTURE CHARACTERS. Reaction of Medium, Incubation 24 ... . HOURS. 48 ... HOUKS. 6 DAYS SKETCHES. Temp. («C) (1) Gelatin plate: (a) Surface Colonies. (b) Deep Colonies. (2) Agar plate: (a) Surface Colonies. (b) Deep t Colonies. (3) . Gelatin Stab. - ^ o (4) Agar A /\ Streak. i 'N ,X ^ (5) A A A Potato. y & (6) Bouillon. (7) Special Media. [77] Name of organism . . Source, habitat, etc. MORPHOLOGICAL CHARACTERS. i "°2 £ Incubation temp. (oC.) SKETCHES. i. Form: c Gelatin 2 Size : 5. Motility . PHYSIOLOGICAL CHARACTERS. i. Relation to temperature: 2. Relation to free oxygen: 3. Relation to other agents, such as: desiccation, light, disinfectants, etc.: 4. Pigment production: , 5. Gas production in glucose media: a. Shake culture b. Fermentation tube, growth in: (i) open arm: (3) rate of development: 24 hours per cent., 48 hours. (4) reaction in open arm: 6. Acid or alkali production, litmus milk 7. Reduction of nitrates; to nitrites 8. Indol production; 24 hours 48 hours fecal odor; 24 hours 48 hours 9. Enzyme production : proteolytic ...(2) closed arm: .per cent., 72 hours per cent., • ••(5) gas formula, H : COs : : hours., percent. to ammonia., days, days. .diastatic. 10. Characteristic odor. u. Pathogenesis [78] CULTURE CHARACTERS. Reaction of Medium, 24 HOURS 48 HOURS. 6 DAYS. SKET 01 ES Incubation Temp. CC) (1) Gelatin plate: (a) Surface Colonies. (b) Deep Colonies. (2) Agar plate : ' (a) Surface Colonies. (b) Deep Colonies. (3) Gelatin Stab. l> u (4) Agar Streak. • • ' A A /• \ 1 \ y V / (5) Potato. A A (6) . '• Bouillon. ^^ ^^ (7) Special Media. - [79] CHAPTER V. BACTERIOLOGICAL ANALYSIS. EXERCISE LXV. COMPARATIVE ANALYSIS OF AIR (Koch's Hethod). a. Plate three tubes of gelatin and expose by removing lid for 20 minutes in the following places: 1. Laboratory, 2. Cellar, 3. Out of doors. b. Replace the lids and keep plates at 22° C. for several days. c. Count the colonies; if the number of colonies is greater than 100, use the counting plate figured in Plate I. A. and count a portion and estimate the whole number. d. Calculate the area of the Petri dish by multiplying the square of the diameter by 0.785. e. Express the results in terms of the number of organisms which fall per square foot per minute. This method enables one to make a rough comparison of the number of organisms occurring in the localities examined, but to determine the number per volume the fol- lowing method must be employed. REFERENCES. H. 390. EXERCISE LXVI. QUANTITATIVE DETERMINATION OF NUHBER OF BACTERIA IN AIR (Petri-Sedgwick Method). GENERAL DIRECTIONS. a. A piece of glass tubing 6 mm. (fin.) in diameter by 15 cm. (6 in.) long is drawn out at one end in a gas flame and sealed. b. Fill this tube about one-third full with granulated sugar, insert a cotton plug next . d. Fasten the tube, pointed end up, in a clamp, remove the first cotton plug and con- nect with an aspirator. (Fig. 14). FIG. u. Aspirator for e. Break off the pointed end of the tube filtering air. an(j m h «-• 3 3£ 4> ~ So < Incubation temp. (°C.) SKETCHES. i. Form: 2. Size 5. Motility. . PHYSIOLOGICAL CHARACTERS. i. Relation to temperature : t. Relation to free oxygen: 5. Relation to other agents, such as: desiccation, light, disinfectants, etc.: 4. Pigment production: }. Gas production in glucose media: a. Shake culture b. Fermentation tube, growth in: (i) open arm: (3) rate of development: 24 hours percent., 48 hours (4) reaction in open arm: 6. Acid or alkali production, litmus milk 7. Reduction of nitrates: to nitrites 8. Indol production; 24 hours 48 hours fecal odor; 24 hours 48 hours g. Enzyme production : proteolytic — (a) closed arm: per cent., 72 hours • •(5) gas formula, H per cent ................ hours per cent. , to ammonia. . days, days. . diastatic — 10. Characteristic odor. 11. Pathogenesis [92] CULTURE CHARACTERS. Reaction of Medium, Incubation Temp. (°C) 24 HOURS. 48... . HOURS. 0.. . DAYS. SKETCHES. ,-"• /\ A. [93] EXERCISE LXXVII. MICROCOCCUS HELTINESIS Bruce. This organism is the cause of Malta (ever and is found especially in the spleen of the diseased. REFERENCES. Bruce: Ann. de 1' Inst. Pasteur, 1899, 8; 239. Durham: Jour. Path, and Bact., 1898, 5; 377. H. 301 ; M. & E. 449. MORPHOLOGICAL CHARACTERS. vi H 'oB 8? <° Incubation temp. <<>C.) SKETCHES. i. Form: e. Gelatin ... 2 Size d Special stains 5 Motility b Flagella stain . . • PHYSIOLOGICAL CHARACTERS. r. Relation to temperature :. . 2. Relation to free oxygen: , 3. Relation to other agents, such as: desiccation, light, disinfectants, etc.: 4. Pigment production: 5. Gas production in glucose media: a. Shake culture b. Fermentation tube, growth in: (i) open arm: (3) rate of development: 24 hours per cent., 48 hours. (4) reaction in open arm; 6. Acid or alkali production, litmus milk y. Reduction of nitrates; to nitrites 8. Indol production; 24 hours 48 hours fecal odor; 24 hours 48 hours , 9. Enzyme production : proteolytic ...(2) closed arm: .per cent., 72 hours per cent., ...(5) gasformula, H : CO* : : hours., per cent. to ammonia.. days days .diastatic. 10. Characteristic odor.. 11. Pathogenesis [94] CULTURE CHARACTERS. Kc:u lion of Medium. Incubation Temp. («C) ''4 HOURS 48 HOURS. 0 DAYS. SKETCHES. (1) Gelatin plate : (a) Surface Colonies. (b) Deep Colonies. (2) Agar plate : (a) Surface Colonies. (b) Deep Colonies. (3) Gelatin Stab. . A A / \ (4) Agar Streak. • 'v X A A A (5) Potato. (6) Bouillon. - - Special Media. ^ [95] EXERCISE LXXVIII. MICROCOCCUS AUREUS (Rosenbach) Mig. STAPHYLOCOCCUS PYOGENES AURKUS ; GOLDEN PUS ooocus. First described in 1884 by Eosenbach. It is the most common organism in pus.— 80^. REFERENCES. Eosenbach: Mikroorganismen bei dem Wundinfektionskrankheiten des Mensohen. A. 260; H. 130; L. & K. 115; M. & E. 160; M. & W. 121 ; McF. 184; P. 401 ; S. 265. MORPHOLOGICAL CHARACTERS. • E "32 V 3 1° Incubation temp. (°C.) SKETCHES. i. Form: c Gelatin 2 Size b. Loeffler's methylen-blue b. Flagella stain 6, Spores 7. Special characters, such as: .. . deposits, vacuoles pleimorphic and involution PHYSIOLOGICAL CHARACTERS. i. Relation to temperature: 2. Relation to free oxygen: 3. Relation to other agents, such as: desiccation, light, disinfectants, etc: 4. Pigment production:. 5. Gas production in glucose media: a. Shake culture b. Fermentation tube, growth in: (i) open arm: (3) rate of development: 24 hours per cent., 48 hours. (4) reaction in open arm : 6. Acid or alkali production, litmus milk 7. Reduction of nitrates; to nitrites 8. Indol production; 24 hours 48 hours fecal odor; 24 hours ,48 hours 9. Enzyme production : proteolytic . . ..(2) closed arm: per cent., 72 hours per cent hours ...(5) gas formula, H : CO, : : : per cent. , to ammonia. . days days . diastatic 10. Characteristic odor 11. Pathogenesis [96] CULTURE CHARACTERS. Reaction of Medium Incubation Temp. («C) 24 HOURS 48 . . HOURS 6 . . DAYS SKETCHES. (1) Gelatin plate: (a) Surface Colonies (b) Deep Colonies (2) Agar plate : (a) Surface Colonies. (b) Deep Colonies. t (3) Gelatin Stab. A A ' \ (4) Agar Streak. X A / \ k y A (5) Potato. \ (6) Bouillon. • - - V (7) Special Media. V / [97] EXERCISE LXX1X. MICROCOCCUS GONORRHOEA (Neisser) Fluegge GONOCOCOUS ; DlPLOCOCCUS OF GONORRHOEA. First described in 1879 by Neisser. It is constantly found in gonorrhoeal discharges and may produce disease on any mucous mem- brane; urethra, bladder, rectum, conjunctiva (causing ophthalmia neonatorum), and even cause arthritis (gonorrhoea! rheumatism), endocarditis, salpingitis and general septicaemia. L. & K. 311 ;M. & R. 189; M. & W. 130; McF. 201 ; P. 522; S. 288. MORPHOLOGICAL CHARACTERS. Age of Cultures. Incubation temp. (°C.) SKETCHES. i. Form: 2 Size b. Loeffler's methylen-blue c. Gram's stain d. Special stains 5. Motility: a. Character of movement b. Flagella stain ; 6. Spores 7. Special characters, such as: ..". . deposits, vacuoles : pleomorphic and involution forms, capsules, etc PHYSIOLOGICAL CHARACTERS. :. Relation to temperature: a. Relation to free oxygen: 3. Relation to other agents, such as: desiccation, light, disinfectants, etc.: . 4. Pigment production: 5. Gas production in glucose media: a. Shake culture A. Fermentation tube, growth in: (i) open arm: (3) rate of development: 24 hours per cent., 48 hours. (4) reaction in open arm: 6. Acid or alkali production, litmus milk 7. Reduction of nitrates; to nitrites 8. Indol production; 24 hours 48 hours fecal odor; 24 hours 48 hours 9. Enzyme production: proteolytic (2) closed arm: . per cent., 72 hou rs per cent., hou rs . (5) gas formula, H : COi : : : per cent. , to ammonia .days, .days. .diastatic. 10. Characteristic odor, n. Pathogenesis [98] Medical Bacteriology. 99 The Micrococcus gonorrhoeae does not grow on the ordinary artificial media but may be cultivated on the following: a. Blood agar. Blood drawn from the finger, under aseptic precautions, in a cap- illary pipette is placed on the surface of agar either in tube or Petri dish. This blood is then inoculated with the material containing the organism (pus or pure culture) and smeared over the surface of the agar either with the loop or better by means of a sterile camel's hair brush. 6. Wertheim's method. Human blood-serum (from placenta or pleuritic or other effusion may be used) in a fluid and sterile condition is placed in two or three test-tubes. These are heated to 40° C. and inoculated with the material containing the organism, making dilutions from one to another if necessary. To each tube is then added an equal quantity of nutrient (ordinary or 2%) agar thoroughly liquefied and cooled to 40° C. The two are then thoroughly mixed and quickly poured into Petri dishes and placed in the incubator at 38° C. Colonies appear in 24 hours. c. Rabbit blood-serum may be used either in a fluid or solid condition. EXERCISE LXXX. MICROCOCCUS INTRACELLULARIS (Weichselbaum) Mig. DIPLOCOOCUS OF CEREBRO-SPINAL MENINGITIS. First described in 1887 by Weichselbaum. It is found in the meningeal exudate of certain cases of epidemic cerebro-spinal menin- gitis and in nasal secretions in a number of cases. REFERENCES. Weichselbaum: Fortschritte der Medicine, 1887; Councilman : Rept. Mass. State B. of H. 1898; A. 285; H. 138; M. & R. 172; M. & W. 135; P. 516; S. 310. MORPHOLOGICAL CHARACTERS. • V 3 o- O = BU Incubation temp. (°C.) SKETCHES. i. Form: « c Gelatin . . .' 2 Size PHYSIOLOGICAL CHARACTERS. i. Relation to temperature : a. Relation to free oxygen: 3. Relation to other agents, such as: desiccation, light, disinfectants, etc.: 4. Pigment production: 5. Gas production in glucose media: a. Shake culture *. Fermentation tube, growth in: (i) open arm: (3) rate of development: 14 hours percent.. 48 hours (4) reaction in open arm: 6. Acid or alkali production, litmus milk ~. Reduction of nitrates; to nitrites 8. Indol production; 24 hours 48 hours fecal odor; 24 hours 48 hours 9. Enzyme production : proteolytic ....(21 closed arm: percent., 72 hours per cent hours per cent. ..(5) gas formula, H : COZ :: : , to ammonia . . days. . days. . diastatic. 10. Characteristic odor. 11. Pathogenesis [100] CULTURE CHARACTERS. Reaction of Medium, Incubation Temp. (°C) 24 HOURS 48 HOURS 0 DAYS SKETCHES. (1) Gelatin plate: (a) Surface Colonies. (b) Deep Colonies. • (2) Agar plate: (a) Surface Colonies. (b) Deep Colonies. (3) Gelatin Stab. A A /\ A (4) Agar Streak. (5) Potato. (6) Bouillon. • - - (7) Special Media. [101] EXERCISE LXXXI. SARCINA TETRAQENA (Qaffky) Mig. MlCROCOCCTJS TETRAGENUS. First described in 1883 by Gaffky. It is found in phthisical cavities and sputum and it occasionally occurs in mixed infections as abscesses connected with carious teeth, about the neck, jaws, and middle ear, rarely elsawhere. REFERENCES. Gaffky: Langenbeck's Archev, 1883, 28:500. A. 309; H. 130; M. & R. 171 ; M. & W. 133;McF. 443 ; P. 472 ; S. 314. MORPHOLOGICAL CHARACTERS. 8 "33 $ Incubation temp. (°C.) SKETCHES. i. Form: c Gelatlii z Size a. Aqueous gentian-violet * b. Loeffler's methylen-blue c. Gram's stain 5. Motility: PHYSIOLOGICAL CHARACTERS. i. Relation to temperature : 2. Relation to free oxygen: 3. Relation to other agents, such as: desiccation, light, disinfectants, etc.; 4. Pigment production: 5- Gas production in glucose media: a. Shake culture *. Fermentation tube, growth in: (i) open arm: (3) rate of development: 24 hours percent., 48 hours. (4) reaction in open arm: 6. Acid or alkali production, litmus milk 7. Reduction ol nitrates; to nitrites 8. Indol production; 24 hours 48 hours fecal odor; 24 hours 48 hours 9. Enzyme production : proteolytic ... (2) closed arm: .percent., 72 hours percent.. hours per cent. • ••(5) gas formula, H : COj : : : to ammonia., days. days. .diastatic. 10. Characteristic odor., 1 1 . Pathogenesis [102] CULTURE CHARACTERS. (1) (2) (3) Reaction of Medium Incubation Temp. ("C (4) (5) Potato. (6) Bouillon. Special Media. 24. . . . HOURS. 48 HOURS. (5 . . . DAYS. SKETCHES. A (\ [103] EXERCISE LXXXII. BACTERlUn ANTHRACIS (Koch) nig. BACILLUS OF ANTHRAX. First described by Robert Koch in 1876. Found in the blood and tissue in cases of anthrax or splenic fever. REFERENCES. Koch: Cohn's Beitraege zur Biologie der Pflanzen, 1870, 2; 277. Chester: Dept. Delaware Exp. Station, July, 1895. A. 448; H. 151 ; L. & K. 287; M. & R. 295; M. & W. 156; McF. 356; P. 547; S. 328. MORPHOLOGICAL CHARACTERS. i °s 41 3 *5 Incubation temp. (°C.) SKETCHES. i. Form: c Gelatin . d Other media . 2 Size. _ d. Special stains 5. Motility : b. Flagella stain 6. Spores 7. Special characters, such as: deposits, vacuoles pleomorphic and involution forms, capsules, etc PHYSIOLOGICAL CHARACTERS. i. Relation to temperature:. 2. Relation to free oxygen: 3. Relation to other agents, such as : desiccation, light, disinfectants, etc: 4. Pigment production: 5. Gas production in glucose media: a. Shake culture ft. Fermentation tube, growth in: (i) open arm: (3) rate of development: 24 hours per cent., 48 hours. (4) reaction in open arm: 6. Acid or alkali production, litmus milk 7. Reduction of nitrates; to nitrites 8. Indol production; 24 hours 48 hours fecal odor; 24 hours 48 hours 9. Enzyme production : proteoly tic ...(2) closed arm: per cent., 72 hours per cent ...(5) gas formula, H : COj : : hours. per cent. , to ammonia . . days days . . diastatic 10. Characteristic odor. . 1 1 . Pathogenesis [104] CULTURE CHARACTERS. Reaction of Medium Incubation 24 HOURS 48 HOURS. 6 DAYS. SKETCHES. Temp. (°C (1) Gelatin plate : (a) Surface Colonies (b) Deep Colonies (2) . i Agar plate : (a) Surface Colonies. (b) Deep Colonies. (3) Gelatin Stab. o ^ (4) Agar - A /\ J 1 Streak. i i •X«, ^^ (5) A A / 4 / \ / 1 i \ Potato. & (6) Bouillon. & sJ (7) Special Media. ^ [105] EXERCISE LXXXIII. BACTERIUM PNEUMON1AE (Welchselbaum) Mlg. PNEUMOCOCCUS ; DIPLOOOCOUS OP PNEUMONIA; MICROOOCOOS LANCEOLATTJS. First described by Sternberg in 1880. Found in saliva and nasal secretion of healthy persons — about 20 per cent. Usually present in "rusty sputum" of pneumonia. EEPEEENOES. Weichselbaum : Am. Jour. Med. Sci., July, 1886: Welch: Johns Hop. Hosp. Bulletin, 1892, 8; 125; A. 303. H. 273; L. & K. 118; M.& R. 204; M. & W. 128; McF. 345; P. 498; S. 298. MORPHOLOGICAL CHARACTERS. Age of Cultures. Incubation temp. (°C.) SKETCHES. i. Form: c Gelatin 2 Size in growths a. Aqueous gentian-violet S. b. Loeffler's methylen-blue d. Special stains 5. Motility: - a. Character of movement b. Flagella stain 6. Spores 7. Special characters, such as: deposits, vacuoles pleomorphic and involution forms, capsules, etc PHYSIOLOGICAL CHARACTERS. i. Relation to temperature: 2. Relation to free oxygen: 3. Relation to other agents, such as: desiccation, light, disinfectants, etc.: 4. Pigment production: 5. Gas production in glucose media: a. Shake culture b. Fermentation tube, growth in: (i) open arm: (3) rate of development: 24 hours per cent., 48 hours. (4) reaction in open arm: 6. Acid or alkali production, litmus milk , 7. Reduction of nitrates; to nitrites 8. Indol production; 24 hours ,48 hours fecal odor; 24 hours ,48 hours 9. Enzyme production: proteolytic (2) closed arm: .per cent., 72 hours percent., hours. (5) gas formula, H : CO« : : : per cent. . to ammonia . days . . days . .diastatic. 10. Characteristic odor. 11. Pathogenesis [100] CULTURE CHARACTERS. Reaction of Medium, Incubation Temp. (°C) 24 HOURS 48 HOURS 6 . DAYS SKETCHES. (1) Gelatin plate : (a) Surface Colonies. (b) Deep Colonies. (2) Agar plate: (a) Surface Colonies. (b) Deep Colonies. (3) Gelatin - Stab. A A l\ /\ A (4) Agar Streak. (5) Potato. (0) Bouillon. Special Media. ^ ^^ [107] EXERCISE LXXXIV. BACTERIUM PNEUHONICUn (Friedlander) nig. FRIEDLANDER'S BACILLUS. First described by Friedlander in 1882. Found frequently in normal saliva, lungs, "rusty sputum" of pneumonia, and has been found in air and water. REFERENCES. Friedlander: Virchow's Archiv, 32; 319; H. 278; L. & K. 119; M. & R. 211; McF. 353; P. 458; S. 296. MORPHOLOGICAL CHARACTERS. en |B Incubation temp. (°C.) SKETCHES. x. Form: b. Loeffler's methylen-blue 5 Motility 7, Special characters, such as: PHYSIOLOGICAL CHARACTERS. i. Relation to temperature :. 2. Relation to free oxygen: j. Relation to other agents, such as: desiccation, light, disinfectants, etc.: . 4. Pigment production:.. 5. Gas production in glucose media: a. Shake culture b. Fermentation tube, growth in: (i) open arm: (3) rate of development: 24 hours per cent.. 48 hours (4) reaction in open arm: 6. Acid or alkali production, litmus milk 7. Reduction of nitrates; to nitrites 8. Indol production; 24 hours 48 hours fecal odor; 24 hours 48 hours 9. Enzyme production : proteolytic .-. ....(2) closed arm: per cent., 72 hours per cent ..(5) gas formula. H : COi : : — hours... per cent. , to ammonia. . days. . days. . diastatic. 10. Characteristic odor. n. Pathogenesis [108] CULTURE CHARACTERS. Reaction of Medium Incubation Temp. (°C; 24 ... HOUES. 48 HOURS. C DAYS SKETCHES. (1) Gelatin plate: (a) Surface Colonies. (b) Deep Colonies. (2) Agar plate: (a) Surface Colonies. (b) Deep Colonies. . (3) Gelatin Stab. A A !\ Agar Streak. 'N / A f \ >• X A (5) Potato. (6) Bouillon. ^ - Special Media. ^ ) [109] EXERCISE LXXXV. BACTERIUH CUNICULICIDA Koch. BACILLUS OP CHICKEN CHOLERA; BACILLUS OF SWINE PLAGUE; BACILLUS SEPTICAEMIAS HEMORRHAGICAE. First described by Koch in 1878. Found in blood, organs and excreta of chickens suffering with fowl cholera, and swine suffering from swine plague. REFERENCES. Koch: Wundiiifektionskrankheiten. Septikaemie bei Kaninchen, 1878; Smith: Report on Swine Plague, Bureau of Animal Industry, U. S. Dept. Agri., 1891 ; Smith & Moore: Bull. 6, B. A. I., 1894; H. 208; McF. 409; S. 408. MORPHOLOGICAL CHARACTERS. tn s ~^2 w"a & Incubation temp. (oC.) SKETCHES. i. Form: * c Gelatin 2 Size .... 5 Motility b Flagella stain . . .". PHYSIOLOGICAL CHARACTERS. i. Relation to temperature:. 2. Relation to free oxygen: 3. Relation to other agents, such as: desiccation, light , disinfectants, etc.: 4. Pigment production: 5. Gas production in glucose media: a. Shake culture A. Fermentation tube, growth in: (t) open arm: (3) rate of development: 24 hours percent., 48 hours. (4) reaction in open arm: 6. Acid or alkali production, litmus milk 7. Reduction of nitrates; to nitrites 8. Indol production; 24 hours , 48 hours fecal odor; 24 hours 48 hours 9. Knzyme production : protcolytic ...(2) closed arm: .percent., 72 hours per cent ...(5) K4 HOURS 48 HOURS 6 DAYS SKEI I '1 tES Incubation Temp. (°C (1) Gelatin plate : (a) Surface - Colonies (b) Deep Colonies. (2) Agar plate: (a) Surface Colonies. (b) Deep Colonies. (3) Gelatin Stab. o u (4) Agar Streak. A A A ^N ,S v / (5) Potato. • A t \ A (6) Bouillon. ^, ^^ (7) Special Media. • ^ J [113] EXERCISE LXXXVII. BACTERIUH TUBERCULOSIS (Koch) dig. BACILLUS OF TUBERCULOSIS. First described by Koch in 1882. Found in diseased tissue of man and animals and phthisical sputum. REFERENCES. Koch: Berlin. Klin. Wochenschr., 1882, 15; 221; Smith: Jour. Exp. Med., 1898, 3: 451; A. 312; H. 189; L. & K. 251; M. & R., 224; M. & W. 148; McF. 208; P. 263; S. 375. MORPHOLOGICAL CHARACTERS. Age of Cultures. Incubation temp. (°C.) SKETCHES. i. Form: c Gelatin a. Aqueous gentian-violet .' b. Loeffler's methylen-blue c. Gram's stain d Special stains - 5. Motility: a Character of movement b Flagella stain pleomorphic and involution forms, capsules, etc • PHYSIOLOGICAL CHARACTERS. i. Relation to temperature: . j. Relation to tree oxygen: 3. Relation to other agents, such as: desiccation, light, disinfectants, etc.: 4. Pigment production: 5. Gas production in glucose media: a. Shake culture b. Fermentation tube, growth in: (i) open arm: (3) rate of development: 24 hours per cent., 48 hours. (4) reaction in open arm: 6. Acid or alkali production, litmus milk T. Reduction of nitrates; to nitrites 8. Indol production; 14 hours 48 hours fecal odor; 24 hours 48 hours 9. Enzyme production: proteolytic (2) closed arm: .per cent., 73 hours per cent., hours... (5) gas formula, H : COj : : : percent. , to ammonia . .days, .days. .diastatic. 10. Characteristic odor. 11. Pathogenesis [114] Medical Bacteriology. 115 B. tuberculosis does not grow upon the ordinary artificial media, but may be grown upon blood serum [see p. 89 (1) ] and bouillon, agar and potato to which 5% of glyc- erine has been added. The tubercle bacterium is very sensitive to temperature varia- tions and should therefore be kept at a temperature varying at most only a degree or two from 38° C. It is also extremely sensitive towards desiccation and for this reason the cotton plug should be well paraffined or replaced by a cork through which a small cotton plugged glass tube passes and the incubator kept saturated with moisture. For methods of culture and isolation see Smith: Jour. Exp. Med., 1898, 3; 456. EXERCISE LXXXVIH. BACTERIUM MALLEI (Loeffler) Mig. BACILLUS OP GLANDERS. First described by Loeffler in 1886. Found in the nodules, ulcers, discharges, etc., of glanders or farcy. REFERENCES. Loeffler: Arbeit, aus dem Kais. Gesundheitsamte, 1886, 1 ; 141 : A. 339; H. 217; L. & K. 300; M. & R. 268; M. & W. 164; McF. 248; P. 598 ;S. 39C. MORPHOLOGICAL CHARACTERS. • v 3 o±: &3 < Incubation temp. (°C.) SKETCHES. i. Form: c Gelatin 2 Size 5 Motility PHYSIOLOGICAL CHARACTERS. i. Relation to temperature : 2. Relation to free oxygen: . 3. Relation to other agents, such as: desiccation, light, disinfectants, etc.: 4. Pigment production: 5. Gas production in glucose media: a. Shake culture *. Fermentation tube, growth in: (i) open arm: (3) rate of development: 24 hours per cent., 48 hours (4) reaction in open arm: 6. Acid or alkali production, litmus milk j. Reduction of nitrates; to nitrites 8. Indol production; 24 hours 48 hours fecal odor; 24 hours 48 hours 9. Enzyme production : proteolytic ....(2) closed arm: per cent., 72 hours per cent., ..(5) gas formula, H : COZ : : hours per cent. , to ammonia. . days, days. . diastatic. 10. Characteristic odor. 11. Pathogenesis [110] CULTURE CHARACTERS. Reaction ol Medium. Incubation Temp. (°C) 24 HOURS. 48 . . HOURS C DAYS. SKETCHES. (1) Gelatin plate: (a) Surface Colonies. (b) Deep Colonies. B (2) Agar plate: (a) Surface Colonies. (b) Deep Colonies. (3) Gelatin Stab. A A A (4) Agar Streak. ' • 'v / A i\ v / A (5) Potato. - ^J v J (6) Bouillon. - - (7) Special Media. V ) [117] EXERCISE LXXXIX. BACTERIUM DIPHTHERIAS (Loeffler) Mig. BACILLUS OF DIPHTHERIA.; KLEBS-LOEFFLER BACILLUS. First described in 1883 by Klebs. First cultivated in 1884 by Loeffler. Found in the false membrane in cases of diphtheria and in small numbers in spleen, liver, etc. ; occasionally in healthy throats. REFERENCES. Klebs: Verhandl. d. Kongressfuer innere Medizin, 1883, II. Loeffler: Mitth. aus dem Kais. Gesundheitsamte, 1884, 2; 421. A. 349; H. 162; L. & K. 207; M. & R. !i53; M. & W. 137; McF. 284; P. 329; S. 356. MORPHOLOGICAL CHARACTERS. • V "ol '"'3 #3 Incubation temp. (°C.) SKETCHES. i. Form: c Gelatin . . . . ... d Other media . 2 Size . - * 5^ Motility PHYSIOLOGICAL CHARACTERS. i. Relation to temperature : 2. Relation to free oxygen: 3. Relation to other agents, such as: desiccation, light , disinfectants, etc.:. 4. Pigment production: 5. Gas production in glucose media: a. Shake culture b. Fermentation tube, growth in: (i) open arm: (3) rate of development: 24 hours percent., 48 hours. (4) reaction in open arm: 6. Acid or alkali production, litmus milk y. Reduction of nitrates: to nitrites 8. Indol production: 24 hours 48 hours fecal odor; 24 hours 48 hours 9. Knzyme production : proteolytic ...(2) closed arm: .percent., 72 hours percent hours. ...(i) gas formula, H :CO, : : : per cent. to ammonia.. days. days. .diastatic. 10. Characteristic odor. 11. Pathogenesis [118] CULTURE CHARACTERS. Reaction of Medium Incubation Temp. (°C) 24 HOURS 48 HOURS. 6 DAYS. SKETCHES. (1) Gelatin plate: (a) Surface Colonies. (b) Deep Colonies. (2) Agar plate : (a) Surface Colonies. (b) Deep Colonies. (3) Gelatin Stab. A A A ' \ A (4) Agar Streak. (5) Potato. (6) • Bouillon. ^ - (7) Special Media. • [119] EXERCISE XC. BACTERIUCl INFLUENZAE (R. Pfeiffer) Lehm. & Neum. BACILLUS OF INFLUENZA; LA GRIPPE. First described in 1892 by R. Pfeiffer. Found in the sputum and nasal secretions of the diseased. REFERENCES. Pfeiffer: Z. f. H. 1893, 13; 357; A. 334; H. 280; L. & K. 281 ;M. & R. 431; M. & W. 162; McF. 440; P. 320; S. 370. MORPHOLOGICAL CHARACTERS. | •32 4) 3 & Incubation temp. (°C.) SKETCHES. x. Form: c Gelatin . d. Other media . 2. Size. 5. Motility: a. Character^ movement If. Flagella stain 6. Spores r 7. Special characters, such as: deposits, vacuoles pleomorphic and involution forms, capsules, etc PHYSIOLOGICAL CHARACTERS. i. Relation to temperature:. i. Relation to free oxygen:, 3. Relation to other agents, such as: desiccation, light, disinfectants, etc:. . . 4. Pigment production: 5. Gas production in glucose media: a. Shake culture A. Fermentation tube, growth in: (j) open arm: (2) closed arm: (3) rate of development: 24 hours per cent., 48 hours percent., 72 hours percent hours per cent. (4) reaction in open arm: (5) gas formula, H : COj : : : 6. Acid or alkali production, litmus milk 7. Reduction of nitrates; to nitrites , to ammonia 8. Indol production; 24 hours ,48 hours days fecal odor; 24 hou rs 48 hou rs days g. Enzyme production : proteolytic diastatic 10. Characteristic odor. 11. Pathogenesis [120] Medical Bacteriology. 121 B. influenzae does not grow on the ordinary artificial culture media but may be cul- tivated on agar slopes upon the surface of which blood has been smeared. The blood from man, rabbits, guinea-pigs and frogs can be used, but that from pigeons is best. The blood may be obtained from a needle prick and spread over the medium with a loop. The skin should first be washed with alcohol and then ether and the first drops should not be used. The sterility of these tubes should be tested by placing them in an incu- bator for 24 hours previous to inoculation. EXERCISE XCI. BACILLUS TYPHOSUS Oaffky. BACILLUS OF TYPHOID FEVER; EBERTH'S BACILLUS. First described by Eberth in 1880, first cultivated by Gaffky, 1884. It is found in the faeces and urine of typhoid patients. REFERENCES. Eberth: Virchow's Archiv. 1880, 81; 58 and 1881, 83; 480. Gaffky: Mitth. aus dem Kais. Gesundheitsamte, 1884, 2; 372; A. 309; H. 223; L. & K. 16(i; M. & R. 317; M. & W. 141; McF. 306; P. 402; S. 337. MORPHOLOGICAL CHARACTERS. Age of Cultures. Incubation temp. (°C.) SKETCHES. i. Form: ,- b Loeffler's methylen-blue 5 Motility 7 Special characters, such as: PHYSIOLOGICAL CHARACTERS. i. Relation to temperature: . 2. Relation to free oxygen: 3. Relation to other agents, such as: desiccation, light, disinfectants, etc.: 4. Pigment production: 5. Gas production in glucose media: a. Shake culture b. Fermentation tube, growth in: (i) open arm: (3) rate of development: 24 hours — per cent., 48 hours. (4) reaction in open arm: 6. Acid or alkali production, litmus milk 7. Reduction of nitrates: to nitrites 8. Indol production; 24 hours 48 hours fecal odor; 24 hours 48 hours 9. Enzyme production: proteolytic (2) closed arm: .per cent., 72 hours percent hours. (5) gas formula, H : CO. : : per cent. , to ammonia .days. . days . .diastatic. 10. Characteristic odor. 11. Pathogenesis [122] CULTURE CHARACTERS. Reaction of Medium. Incubation 24 . HOURS 48 HOURS. 6 DAYS. SKETCHES. Temp. (UC) (1) Gelatin plate : • (a) Surface Colonies. (b) Deep Colonies. (2) Agar plate : (a) Surface Colonies. •- (b) Deep Colonies. (3) Gelatin Stab. ^ ^ (4) Agar A A / \ f \ Streak. i i ^/ \Tx i i (5) A / \ A Potato. « & ^ (6) Bouillon. A ^ w (7) Special Media. [123] EXERCISE XCII. BACILLUS PESTIS Kitasato and Yersin. BACILLUS OF BUBONIC PLAGUE. Described at about the same time independently by Kitasato and Yersin in 1894. Found in the buboes, and occasionally in the faeces, urine and blood and, in the pneumonic form, in the blood. EEFERENCES. Kitasato: Lancet, 1894, 3; 428; Yersin: Ann. de 1' Inst. Past., 1894, 8; 663; A. 393; H. 359; L. & K. 300; M. & R. 437; McF. 483; P. 606. MORPHOLOGICAL CHARACTERS. V **- s Incubation temp. (°C.J SKETCHES. i. Form: ' c Gelatin ". d Other media 2 Size 5 Motility . .... PHYSIOLOGICAL CHARACTERS. i. Relation to temperature : 2. Relation to free oxygen: 3. Relation to other agents, such as: desiccation, light, disinfectants, etc.: 4. Figment production: 5. Gas production in glucose media: a. Shake culture b. Fermentation tube, growth in: (i) open arm: (3) rate of development: 24 hours per cent., 48 hours (4) reaction in open arm: 6. Acid or alkali production, litmus milk 7 . Reduction of nitrates; to nitrites 8. Indol production; 24 hours 48 hours fecal odor; 24 hours 48 hours 9. Enzyme production : proteoly tic ....(2) closed arm: per cent., 72 hours percent., hours. ..(5) gas formula, H : CO2 : : : per cent. , to ammonia. . days, days. . diastatic. 10. Characteristic odor. 11. Hathogenesis [134] CULTURE CHARACTERS. • Reaction of Medium, Incubation Temp. (°C) 24 HOUKS 48 HOURS G DAYS SKETCHES. (1) Gelatin plate: (a) Surface Colonies. (b) Deep Colonies. (2) Agar plate : (a) Surface Colonies. (b) Deep Colonies. (8) Gelatin Stab. A /\ (4) Agar Streak. i ^ / A / \ A (5) Potato. ^^ ^^ Bouillon. (7) Special Media. 0 [125] EXERCISE XC1II. BACILLUS SUIPESTIFER Kruse. BACILLUS OF HOG CHOLERA. First described by Klein, 1884, first cultivated by Salmon and Smith in 1885. Occurs in blood, organs and intestinal contents of hogs suffering from hog cholera. REFERENCES. Salmon & Smith : Kept Bureau Anim. Ind., 1885-91; H. 269; McF. 413; S. 413. MORPHOLOGICAL CHARACTERS. • £ ~°S — y A A A A (4) Agar Streak. (5) Potato. *& X x' (6) Bouillon. - - (7) Special Media. [129] EXERCISE XCV. PSEUDOMONAS AERUQINOSA (Schroeter) nig. BACILLUS PYOCYANEUB OR BACILLUS OF BLUE-GREEN Pus. First described in 1872 by Schroeter. Found in green pus, and is widely distributed in nature. REFERENCES. Schroster: Cohn's Baitraege zur Biologie, 1872, 1; 126. Barker: Jour. Am. Med. Asso., 1897, July 31. Jordan: Jour. Exp. Med. 1899, 627. Lartigan, Ibid., 1898; 595; A. 287; H. 138; L. & K. 120; M. & R. 170; M. & W. 160; McF. 197; P. 535; S. 454. MORPHOLOGICAL CHARACTERS. Age of Cultures. Incubation temp. (°C.) SKETCHES. i. Form: b Loeffler's methylen-blue 5 Motility 7 Special characters, such as: PHYSIOLOGICAL CHARACTERS. i. Relation to temperature: . 2. Relation to free oxygen: 3. Relation to other agents, such as: desiccation, light, disinfectants, etc.:.. . . 4. Pigment production: 5. Gas production in glucose media: a. Shake culture b. Fermentation tube, growth in: (i) open arm: (3) rate of development: 24 hours per cent., 48 hours. (4) reaction in open arm: 6. Acid or alkali production, litmus milk 7. Reduction of nitrates; to nitrites 8. Indol production; 24 hours 48 hours fecal odor; 24 hours 48 hours 9. Enzyme production: proteolytic (2) closed arm: .per cent., 72 hours percent., hours. (5) gas formula, H : COS : : : per cent. . to ammonia . .days, .days. .diastatic. 10. Characteristic odor. 11. Pathogenesis [130] CULTURE CHARACTERS. Reaction of Medium, Incubation Temp. (°C) 24 HOURS 48 HOURS 6 DAYS SKETCHES. (1) Gelatin plate : (a) Surface Colonies. (b) Deep Colonies. (2) Agar plate : (a) Surface Colonies. * (b) Deep Colonies. (3) Gelatin Stab. A 1 /\ / \ I \ (4) Agar Streak. - ^ A / \ / \ A (5) Potato. (6) Bouillon. - ^ (7) Special Media. ^ [131] EXERCISE XCVI. MICROSPIRA COMMA (Koch) Schroetei-. COMMA BACILLUS; CHOLERA VIBRIO. First described by Koch in 1884. Found in the intestinal contents of cholera patients and has also been isolated several times from a water supply. REFERENCES. Koch: Berl. Klin. Wochenschr. 1884, no. 31 u. 32: A. 401; H. 244; L. & K. 181; M. & R 402; M. & W. 152; McF. 311; P. 508; S 500. MORPHOLOGICAL CHARACTERS. en B VM = 0~ V 3 BU Incubation temp. (°C.) SKETCHES. i; Form: c Gelatin 5 Motility PHYSIOLOGICAL CHARACTERS. i. Relation to temperature :. .. z. Relation to free oxygen: 3. Relation to other agents, such as: desiccation, light, disinfectants, etc.: . 4. Figment production: 5. Gas production in glucose media: a. Shake culture 6. Fermentation tube, growth in: (i) open arm: (3) rate of development: 24 hours per cent., 48 hours (4) reaction in open arm: 6. Acid or alkali production, litmus milk 7. Reduction of nitrates; to nitrites 8. Indol production; 24 hours 48 hours fecal odor; 24 hours ,48 hours 9. Enzyme production : proteoly tic ....(2) closed arm: per cent., 72 hours percent., hours. ..(5) gas formula, H : CO2 : : : per cent. , to ammonia. . days. days. . diastatic. 10. Characteristic odor. 11. Pathogenesis [132] CULTURE CHARACTERS. Reaction of Medium. Incubation Temp. (°C) 24 . HOCES. 48 HOURS. 0 .... DAYS. SKETCHES. (1) Gelatin plate : (a) Surface Colonies. • (b) Deep Colonies. (2) Agar plate : * (a) Surface Colonies. (b) Deep Colonies. (3) Gelatin Stab. A /\ i\ (4) Agar Streak. A f \ v / A, (5) Potato. (6) \ Bouillon. ^ - (7) Special Media. [133] EXERCISE XCVII. MICROSPIRA flETSCHNlKOVI nig. VIBRIO METSCHNIKOVI. First described in 1888 by Gamaleia. Found in intestinal contents, blood and organs of chickens suffering from a disease resem- bling chicken cholera. REFERENCES. Gamaleia: Ann. d 1' Inst, Past. 1888, 2; 482. A. 441 ; H. 256; M. & R. 436; McF. 332; P. 593; S. 511. MORPHOLOGICAL CHARACTERS. E 'oS w'a & Incubation temp. (°C.) SKETCHES. i. Form: a. Bouillon r b. Agar ,. .. c Gelatiu d Other media 2 Size b. Loeffler's methylen-blue • 5. Motility: PHYSIOLOGICAL CHARACTERS. i. Relation to temperature : 2. Relation to free oxygen: 3. Relation to other agents, such as: desiccation, light , disinfectants, etc.: 4. Pigment production: 5. Gas production in glucose media: a. Shake culture b. Fermentation tube, growth in: (i) open arm: (3) rate of development: 24 hours per cent., 48 hours. (4) reaction in open arm: 6. Acid or alkali production, litmus milk 7. Reduction of nitrates: to nitrites 8. Indol production; 24 hours , 48 hours fecal odor; 24 hours 48 hours 9. Enzyme production: protcolytic ...(2) closed arm: , .percent., 72 hours percent., hours percent. ...(5) gas formula, H : CO« : : : to ammonia., days. days. . diastatic. 10. Characteristic odor., 11. Pathogenesis [134] CULTURE CHARACTERS. Reaction of Medium. Incubation Temp. (°C) 24 HOURS 48 . . HOURS C DAYS. SKETCHES. (1) Gelatin plate : (a) Surface Colonies. (b) Deep Colonies. (2) Agar plate: (a) Surface Colonies. (b) Deep Colonies. • (3) Gelatin Stab. A i A A (4) Agar Streak. A /\ ^ / A (5) Potato. (6) Bouillon. - - (7) Special Media. V } [135] EXERCISE XCVIII. fUCROSPIRA FINKLERI Schroeter. SPEILLUM OF FINKLER AND PRIOR. First described in 1884 by Finkler & Prior. Deutsche Med. Wochenschr., 1884, 632. REFERENCES. A. 429; H. 257; M. & R. 428; McF. 326; P. 589; S 509. MORPHOLOGICAL CHARACTERS. B oj 0»"p 1" Incubation temp. (°C.) SKETCHES. i. Form: c Gelatin . d Other media 2 Size 5. Motility : b. Flagella stain 6. Spores 7. Special characters, such as: ." deposits, vacuoles pleomorphic and involution forms, capsules, etc PHYSIOLOGICAL CHARACTERS. i. Relation to temperature:. 2. Relation to free oxygen: , 3. Relation to other agents, such as: desiccation, light, disinfectants, etc:. 4. Pigment production: 5. Gas production in glucose media: a. Shake culture *. Fermentation tube, growth in: (:) open arm: (3) rate of development: 24 hours (4) reaction in open arm: 6. Acid or alkali production, litmus milk 7. Reduction of nitrates; to nitrites 8. Indol production ; 24 hou rs fecal odor; 24 hours 9. Enzyme production : proteoly tic per cent., 48 hours. . ..(2) closed arm: per cent., 72 hours percent hours per cent. ..•(5) gas formula, H : COj : : : , to ammonia. , 48 hours. . .. , 48 hours . days . days . diastatic 10. Characteristic odor. 11. Pathogenesis [186] CULTURE CHARACTERS. Reaction of Medium, Incubation 24 HOURS 48 HOURS (i DAYS. SKETCHES. Temp. ("C) (1) Gelatin plate : (a) Surface Colonies. (b) Deep Colonies. (2) Agar > plate: (a) Surface Colonies. (b) Deep Colonies. (3) Gelatin Stab. L u (4) Agar A A Streak. 1 1 ! ^ V J (5) A / \ ,A / \ / i 1 \ Potato. J (6) Bouillon. ^ ^ (7) Special Media. ^ ^^ [137] Name of organism Source, habitat, etc. MORPHOLOGICAL CHARACTERS. Age of Cultures. Incubation temp. (°C.) SKETCHES. i. Form: 2 Size a. Aqueous gentian-violet 1 b. Loeffler's methylen-blue 5 Motility • pleomorphic and involution forms, capsules, etc PHYSIOLOGICAL CHARACTERS. i. Relation to temperature: . 2. Relation to free oxygen: 3. Relation to other agents, such as: desiccation, light, disinfectants, etc.:.. 4. Pigment production: 5. Gas production in glucose media: a. Shake culture b. Fermentation tube, growth in: (i) open arm: (3) rate of development: 24 hours per cent., 48 hours. (4) reaction in open arm: 6. Acid or alkali production, litmus milk 7. Reduction of nitrates; to nitrites 8. Indol production; 24 hours ,48 hours , fecal odor; 24 hours ,48 hours , g. Enzyme production: proteolytic (2) closed arm: .per cent., 72 hours per cent., , (5) gas formula, H : COS : : . hou rs per cent . , to ammonia .days. .days. .diastatic . 10. Characteristic odor 11. Pathogenesis '. . [188] CULTURE CHARACTERS. Reaction of Medium. 24 HOURS 48 HOURS. 6 ... DAYS SKET ' '! ES Incubation Temp. (°C) (1) Gelatin plate : (a) Surface Colonies. (b) Deep Colonies. (2) Agar plate : (a) Surface Colonies. (b) Deep Colonies. • (3) Gelatin Stab. L U (4) Agar A A / \ Streak. u ^ / (5) A ,/\ A Potato. U U (6) Bouillon. Special Media. - [139] Name of organism . . Source, habitat, etc. References MORPHOLOGICAL CHARACTERS. SKETCHES. Form: a. Bouillon. *. Agar . . . . c. Gelatin . . d. Other media 2. Size 3. Cell groupings and arrangements in growths 4. Staining powers: a. Aqueous gentian-violet.. *. Lot-filer's methylen-blue. c. Gram's stain d. Special stains 5. Motility .' a. Character of movement . b. Flagella stain 6. Spores 7. Special characters, such as: deposits, vacuoles pleomorphic and involution forms, capsules, etc. PHYSIOLOGICAL CHARACTERS. i. Relation to temperature :.. 2. Relation to free oxygen: 3. Relation to other agents, such as: desiccation, light, disinfectants, etc.: 4. Figment production: 5. Gas production in glucose media: a. Shake culture b. Fermentation tube, growth in: (i) open arm: (3) rate of development: 24 hours percent., 48 hours (4) reaction in open arm: 6. Acid or alkali production, litmus milk 7. Reduction of nitrates; to nitrites 8. Indol production; 24 hours 48 hours fecal odor; 24 hours 48 hours 9. Enzyme production : proteoly tic ....(2) closed arm: per cent., 72 hours per cent ..(5) gas formula, H : CO2 : : , hours per cent. , to ammonia . . days, days. . diastatic. 10. Characteristic odor it. Pathogenesis '. ... [140] CULTURE CHARACTERS. Reaction of Medium, Incubation Temp. (°C) 24 HOURS 48 .... HOURS C . DAYS. SKETCHES. (1) Gelatin plate: (a) Surface Colonies. (b) Deep Colonies. (2) Agar plate : (a) Surface Colonies. (b) Deep Colonies. (3)- • Gelatin Stab. J A /\ (4) Agar Streak. A ,' \ >> y A (5) Potato. (6) Bouillon. - - (7) Special • Media. [141] Name of organism . . Source, habitat, etc. MORPHOLOGICAL CHARACTERS. M _£ Incubation temp. (°C.) SKETCHES. i. Form: c Gelatin - • PHYSIOLOGICAL CHARACTERS. i. Relation to temperature : 2. Relation to free oxygen: 3. Relation to other agents, such as: desiccation, light, disinfectants, etc.:. 4. Pigment production: 5. Gas production in glucose media: a. Shake culture b. Fermentation tube, growth in: (i) open arm: (3) rate of development: 24 hours per cent., 48 hours. (4) reaction in open arm: 6. Acid or alkali production, litmus milk 7. Reduction of nitrates: to nitrites 8. Indol production: 24 hours ,48 hours fecal odor; 24 hours 48 hours 9. Enzyme production : proteolytic ...(2) closed arm: .percent., 72 hours per cent., ...(5) gas formula, H : COa : : .hours percent. to ammonia. days. days. . diastatic. 10. Characteristic odor. 11. Pathogenesis [142] CULTURE CHARACTERS. Reaction of Medium Incubation Temp. (°C 24 HOURS 48 HOURS. G DAYS. SKETCHES. (1) Gelatin plate: (a) Surface Colonies. (b) Deep Colonies. (2) Agar plate : (a) Surface Colonies. (b) Deep Colonies. (3) Gelatin Stab. A A (4) Agar Streak. * A 1 \ A (5) Potato. (6) Bouillon. - - (7) Special Media. [143] Same of organism . . Source, habitat, etc. References MORPHOLOGICAL CHARACTERS. m D k. 4)*a 1" Incubation temp. ("C.) SKETCHES. i. Form: d. Special stains 5. Motility: . b. Flagella stain - 6. Spores 7. Special characters, such as: deposits, vacuoles pleomorphic and involution forms, capsules, etc PHYSIOLOGICAL CHARACTERS. i. Relation to temperature:. 2. Relation to free oxygen: 3. Relation to other agents, such as: desiccation, light, disinfectants, etc: 4. Pigment production: 5. Gas production in glucose media: a. Shake culture b. Fermentation tube, growth in: (i) open arm: (3) rate of development: 24 hours (4) reaction in open arm: 6. Acid or alkali production, litmus milk 7. Reduction of nitrates; to nitrites 8. Indol production; 24 hours fecal odor; 24 hours 9. Enzyme production : proteolytic (2) closed arm: per cent., 48 hours per cent., 72 hours per cent., hours per cent. (5) gasformula, H : CO, : : : , to ammonia. ., 48 hours. . . , 48 hours. . . days . days . diastatic 10. Characteristic odor. 11. Pathogenesis [144] CULTURE CHARACTERS. Reaction of Medium, Incubation Temp. (°C) 24 HOURS 48 HOURS (i . . DAYS SKETCHES. (1) Gelatin plate : (a) Surface Colonies. • (b) Deep Colonies. (2) Agar plate: (a) Surface Colonies. (b) Deep Colonies. (3) Gelatin Stab. A A (4) Agar Streak. *v X A A ^ / A (5) Potato. (6) Bouillon. - - (7) Special Media. ^ ^ [145] Name of organism Source, habitat, etc. MORPHOLOGICAL CHARACTERS. Age of Cultures. Incubation temp. (°C.) SKETCHES. i. Form: c Gelatin . d Other media 4. Staining powers: • a. Aqueous gentian-violet b, Loeffler's methylen-blue - c. Gram's stain d. Special stains 5. Motility: a. Character of movement * b. Flagella stain 6. Spores • PHYSIOLOGICAL CHARACTERS. i. Relation to temperature: 2. Relation to free oxygen: 3. Relation to other agents, such as: desiccation, light, disinfectants, etc.: 4. Pigment production: 5. Gas production in glucose media: a. Shake culture b. Fermentation tube, growth in: (i) open arm: (3) rate of development: 24 hours per cent., 48 hours. (4) reaction in open arm: 6. Acid or alkali production, litmus milk 7. Reduction of nitrates; to nitrites 8. Indol production; 24 hours 48 hours fecal odor; 24 hours ,48 hours 9. Enzyme production: proteolytic (2) closed arm: .per cent., 72 hours per cent (5) gas formula, H : COS : : .hours percent. , to ammonia . .days. . days . .diastatic. 10. Characteristic odor. 11. Pathogenesis [14C] CULTURE CHARACTERS. Reaction of Medium. Incubation Temp. (°C) 24 . , . HOURS. 48. . . HOURS. DAYS. SKETCHES. (1) Gelatin plate : (a) Surface Colonies (b) Deep Colonies. (2) Agar plate: (a) Surface Colonies. (b) Deep Colonies. (3) Gelatin Stab. (4) Agar Streak. (5) Potato. (6) Bouillon. (?) Special Media. A A A [147] CHAPTER VII. PATHOGENIC ANAEROBES. Anaerobic bacteria may be furnished conditions, which permit of their development, in a variety of ways and a very considerable number of pieces of apparatus have been devised to secure this end. In a general way all of the methods may be grouped under the following heads: 1. Displacement of air. 2. Absorption of oxygen. 3. Exhaustion of air. 4. Exclusion of air. 5. Miscellaneous methods, in the presence of reducing substances as litmus, or a strongly aerobic germ, etc. The first two methods are the most reliable. In the displacement method, hydro- gen, carbon dioxide or illuminating gas may be used; hydrogen is best. This gas is readily prepared by the action of sulphuric acid (1:8) on zinc. Either a Kipp generator may be used or one of a simpler construction. The gas should be washed, 1st. in lead nitrate to absorb the sulphuretted hydrogen, 2nd. in silver sulphate to absorb any arseniuretted or phosphuretted hydrogen, and 3rd. in potassium hydrate to remove sul- phur and carbon dioxide. The cultures are made in media containing glucose (which should preferably be freshly prepared and always boiled immediately before being inoculated), either as test- tube or plate cultures. Novy's anaerobic jars are perhaps the most satisfactory recep- tacles for the cultures. (For careful description of same, see N. 306.) In the second method (Buchner's method) an alkaline solution of pyrogallic acid is used to absorb the oxygen. The cultures may be placed in Novy jars or similar re- ceptacles ; for tube cultures a large wide mouthed bottle fitted with a rubber cork does very well. The dry pyrogallic acid is placed in the bottom of the receptacles, about 1 gram to every 100 cc- of air space, the tubes are put in place, then about 10 cc. of a normal sodium hydroxide is added to each gram of pyrogallic acid, and the apparatus immediately and hemetically sealed. REFERENCES. A. 206; L- & K. 98; M. & R. 68; M. & W. 117; McF. 153; P. 233; S. 78. EXERCISE XCIX. BACTERIUM WELCHII Mig. BACILLUS AEROGENES CAPSULATUS. First described by Welch in 1892. Occurs at autopsies in which gas bubbles are present in the larger vessels, accompanied by the formation of numerous small cavities in the liver containing gas. It has been found also in emphysematous phlegmons, in puerperal sepsis, in peritonitis and in other conditions (M. & W.). Widely distributed in nature. (Welch.) REFERENCES. Welch and Nuttall: Bull. Johns Hopkins Hospital, 1892, 8; 81; Welch & Flexner: Jour. Exp. Med., 1896, 1; 5; H. 140; M. & W. 173; McF. 463; P. 545; S. 731. MORPHOLOGICAL CHARACTERS. i "32 IB Incubation temp. (°C.) SKETCHES. i. Form: c Gelatin 5 Motility . . .... PHYSIOLOGICAL CHARACTERS. i. Relation to temperature:.. 2. Relation to free oxygen: 3. Relation to other agents, such as: 'desiccation, light, disinfectants, etc.:. 4. Pigment production: 5. Gas production in glucose media: a. Shake culture *. Fermentation tube, growth in: (i) open arm: (3) rate of development: 24 hours percent., 48 hours. (4) reaction in open arm: 6. Acid or alkali production, litmus milk 7. Reduction of nitrates: to nitrites 8. Indol production; 24 hours 48 hours fecal odor; 24 hou rs , 48 hours 9. Enzyme production : proteolytic ...(2) closed arm: .per cent., 72 hours per cent., ...(5) gas formula, H : CO« : : — hours.. percent. to ammonia.. days, days. .diastatic. 10. Characteristic odor. 11. Pathogenesis [150] CULTURE CHARACTERS. Reaction of Medium, Incubation Temp. (»C) 24 HOURS 48 HOUES. G DAYS. SKETCHES. (1) Gelatin plate: (a) Surface Colonies. (b) Deep Colonies. (2) Agar plate: (a) Surface Colonies. (b) Deep Colonies. (3) Gelatin Stab. A A A (4) Agar Streak. A f \ V / A (5) Potato. • (6) Bouillon. ' - - (7) Special Media. [151] EXERCISE C. BACILLUS CHAUVAEI Arloing, Cornevin and Thomas. BACILLUS OF SYMPTOMATIC ANTHRAX. First described by Arloing, Cornevin and Thomas in 1887. It occurs in the subcutaneous tissue, muscles and serous exudate of ani- mals suffering from sj'mptomatic anthrax. REFERENCES. Arloing, Cornevin and Thomas ; Le Charbon symptomatique du baeuf , 2nd edit. Paris, 1887 ; A. 482 ; H. 304 ; McF. 453; P. 503 ; S. 403. MORPHOLOGICAL CHARACTERS. i •32 f Incubation temp. (°C.) SKETCHES. i. Form: b. Agar d Other media . 2 Size. 4. Staining powers: . . . T 5. Motility: a. Character of movement b. Flagella stain 6. Spores 7. Special characters, such as: deposits, vacuoles pleomorphic and involution forms, capsules, etc PHYSIOLOGICAL CHARACTERS. i. Relation to temperature:. 2. Relation to free oxygen: 3. Relation to other agents, such as : desiccation, light, disinfectants, etc: 4. Pigment production: 5. Gas production in glucose medte: a. Shake culture b. Fermentation tube, growth in: (i) open arm: (3) rate of development: 24 hours (4) reaction in open arm: 6. Acid or alkali production, litmus milk 7. Reduction of nitrates; to nitrites 8. Indol production; 24 hours fecal odor; 24 hours 9. Enzyme production : proteolytic per cent., 48 hours. . ..(2) closed arm: per cent., 72 hours per cent., • ••(5) gas formula, H : COj : : hours. per cent. , to ammonia . , 48 hours. , 48 hours.. . days days . . diastatic . 10. Characteristic odor. 11. Pathogenesis [152] CULTURE CHARACTERS. Reaction of Medium, 24 HOURS 48 ... HOURS. fl . . . DAYS SKEI ITU JES Incubation Temp.("C) (1) Gelatin plate : (a) Surface Colonies. (b) Deep Colonies. (2) Agar plate: (a) Surface Colonies. (b) Deep Colonies. (3) Gelatin Stab. L/ u (4) Agar Streak. A i /\ / \ / \ - ^ ^ y (5) Potato. A / \ / t A (6) Bouillon. ^ ^_^ (7) Special Media. ^ ^^ [153] EXERCISE Cl. BACILLUS OEDEMATIS Liborius. BACILLUS OF MALIGNANT OEDEMA. First described by Pasteur in 1877. Widely distributed in soil and putrefying material. Few cases on record of infection of man. REFERENCES. Z. f. H., 1886; 1 : 158; A. 476; H. 302; L. & K. 305; M. & R. :!94; M. & W. 175; M. & W. 459; P. 543; S. MORPHOLOGICAL CHARACTERS. • £ "32 $ Incubation temp. (°C.) SKETCHES. i. Form: c Gelatin . . ... a. Aqueous gentian-violet b, Loeffler's methylen-blue 5. Motility: a. Character of movement pleomorphic and involution forms, capsules, etc PHYSIOLOGICAL CHARACTERS. i. Relation to temperature: . 2. Relation to free oxygen: 3. Relation to other agents, such as: desiccation, light, disinfectants, etc.: 4- Pigment production: i. Gas production in glucose media: a. Shake culture A. Fermentation tube, growth in: (i) open arm: (3) rate of development: 24 hours per cent., 48 hours. (4 ) reaction in open arm : 6. Acid or alkali production, litmus milk 7. Reduction of nitrates; to nitrites 8. Indol production; 24 hours 48 hours fecal odor; 24 hours 48 hours 9. Enzyme production: proteolytic (2) closed arm: .per cent., 72 hours per cent., (5) gas formula, H : COS : : . . ..hours. per cent. . , to ammonia .days, .days. .diastatic. 10. Characteristic odor. 11. Pathogenesis [154] CULTURE CHARACTERS. Reaction of Medium, Incubation 24 HOURS 48 HOURS 6 . . DAYS SKETCHES. Temp. (°C) (1) Gelatin plate : (a) Surface • t Colonies. (b) Deep Colonies. (2) Agar plate: (a) Surface Colonies. (b) Deep Colonies. (3) Gelatin , Stab. o 0 (4) Agar A A Streak. i • * . V^ ^^ (5) A A Potato. b; ^ (6) Bouillon. ' o ^ (7) Special Media. ^ ' v y [155] EXERCISE Cll. BACILLUS TETANI Nicolaier. Discovered by Nicolaier, 1884. First cultivated by Kitasato, 1889. Occurs in man and animals suffering from the disease and widely distributed in nature, especially in soil. REFERENCES. Nicolaier : Deutszche Med. Wochenschrift, 1884; Kitasato: Deutsche Med. Wochenschrift, 1889; A. 469; H. 290; L. & K. 230; M. & R. 376;M.&W. 171; McF. 274;P. 385; S. 482. MORPHOLOGICAL CHARACTERS. tfi . Ordinarily the organisms are very sparse and large quantities must be used, 100- 1000 cc. are placed in flasks aud 1% of peptone and 0.5% salt are added, the fluid made alkaline and incubated at 38° C. for 6-24 hours. Then gelatin plate cultures are made from the upper layers and the suspicious colonies worked up as above. EXERCISE CXV. EXAHINATION OF HILK FOR PATHOGENIC BACTERIA. B. DIPHTHERIAE. Where B. diphtherias is suspected in milk, make a considerable number of streak cultures on Loefflers's blood serum and incubate at 38° C. for 8-12 hours and examine growth microscopically very carefully for B. diphtheria*-. BACTERIUM TUBERCULOSIS (Koch) Mig. Hammond's method of examining milk for B. Tuberculosis. See Sputum, CVII. Animal Inoculation. Concerning the transmission of material containing Bacteria in Mails, see Postal Guide, 1898 Ruling No. 82, p. 901. Part of which is as follows: "That the order of the Postmaster General of June 1, 18!)li, forbidding the use of mails for the transmission of specimens of germs of cholera or other diseased tissues, is hereby modified to this extent: "Specimens of diseased tissue may be admitted to the mails for transmission to United States, State or municipal laboratories only when inclosed in mailing packages constructed in accordance with the specifications hereinafter enumer- ated. Upon the outside of every package shall be written or printed the words: 'Specimen for Bacteriological examination.' No package containing diseased tissue shall be delivered to any rep- resentative until a permit shall have first been issued by the Postmaster General, certifying that said institution has been found to l>e entitled, in accordance with the requirements of this regula- tion, to receive such specimens." [200] INDEX. ABBE condenser. 22. Acids, detection of in cultures, 52; quantitative determination of, 52. Agar, glucose, 44; glycerine, 115; hanging-drop culture in, 30; lac- tose, 44. Agar plate cultures, 34; character of colonies on, 58. Agar slopes, 12. Air, analysis, comparative, 80; quan- titative, 80. Amoeba coli, in faeces, 192; in exu- dates, 198. Ammonia, detection of in cultures, 52. Anaerobic cultures, 149. Animal inoculation. 162. Anilin dyes, 18. Anilin oil gentian violet, 18. Antiseptic action, 48. Antiseptics, method of testing, 84. Aspirator, 80. Autoclave, 8. Autopsies, examination of material from, 198. BACILLUS acidi laclici, 70. aerogenes capsulatus, 150, amylobacter, 40. of anthrax, 104. campestris, 46. of blue-green pus, 130. of bubonic plague, 124. ckauvaei, 152; 172; 176. of chicken cholera, 110. coli, 16; 24; 84; 36; 46; 48; 52; 54; in the blood, 186; in transu- dates and exudates. 196. of diphtheria, 118. of Friedlander, 108. of glanders, 116. of hog cholera, 128. icteroides, 128; 188. of influenza, 120. of malignant oedema, 154. mycoidts, 26 oedematis, 154; 172; 176; 198. pestis, 124; 164; 184; 186; 196. prodigiosus, 50; 56; 64; 67. pyocyaneus, 130. rouget du pore, 112. of septicaemia haemorrhagica, 110. subtilis, 16; 22; 24; 30; 84; SB; 40; 46; 48; 50; 52; 54. suiptstifcr, 128; 184; 186. of swine erysipelas, 112. of swine plague, 110. Bacillus of symptomatic anthrax, 152. tetani, 40; 156; 172; 198. tuberculosis, 114. of typhoid fever. 122. typhosus, 42; 46; 50; 122: 188; 192; 200. vulgaris, 52: 72. Bacteria, in air. 80; transmission thiough the mail, 200. Bacteriological analysis, 80; diagnosis, 178. Bacterium anthracis, 40; 104; 162; 184. cuniculicida, 110; 174. dipHtheriae. 30; 118; 164; 172; 178; 200. injluenzae, 120; 182; 186. leprae, 172; 174; 198. mallei, 116; 164; 174; 186; 196. phosphorescens, 68. pneumoniae, 106; 162; 172: 174; 184; 186; 196. pneumonicum, 108; 162. pseudo-diphtheriae, 180. rhusiopalhiae, 112; 172. tuberculosis. 114; 162; 172; 174: 182; 186; 192; 196; 200. welchii, 150; 196. Bismarck brown, 18. Blank for animal experiments, 168. Blood, examination of, 184. Blood serum, character of growth on, 59; collection of, 184; Loeffler's mixture, 88. Bouillon, character of growth in, 58; glucose, 44; preparation of, 4. Brownian movement, 24. Buccal secretions, examination of, 178. Bunge's flagella stain, 40. CANADA balsam, 22. Capsule stain, 42. Carbol-fuchsin, 18. Cell grouping, study of, SO. Chemicals, effect on bacteria, 48. Cholera red, 192. Cholera vibrio, 132. Classification of bacteria, 60. Cleaning glassware, 2. Colon bacillus, 16. See B. coli. Color production, variation in, 58. Coloring matter, separation of, 67. Comma bacillus, 132. Concentration of media, effect on bac- terial growth, 46. Cover-glass preparation, 20. Cover-glass, cleaning of, 18. Culture characters, description of, 57. Cultures, fluid, 16; incubation of, 16; stab, 16; streak, 16, test-tube, 14. Culture media, care of, 14; preparation of 4; 10; 12; 44; 63; 88; steriliza- tion, 8. DECOLORIZING agents, use, 36. Desiccation, effect, 48. Diplococcus of cerebro-spinal men- ingitis, 100. of gonorrhoea, 98. of pneumonia, 106. [203] Disinfectant, 48. Disinfectants, testing, 88. Drawing bacteria, 28. Dunham's solution, 44, Dust, relation of bacteria to, 82. CBERTH'S bacillus, 122. " Ehrlich's anilin oil gentian violet 18. Eisner's medium, 190. Embedding tissue, 170. Endospores, staining, 38; study of, 40 Enzymes, 54. Esmarch rolls, 34. CAECES, examination of, 188. Fermentation tube, 50. Filter for gelatin, 10. Flagella stain, 40. Fluid cultures, 16. Form types, study of, 26. Fraenkel's soil borer, 82. Frost's gasometer, 50. Fuchsin, carbol, 18; Ziehl's, 18, pABBETT'S methylen blue. 20; tu- *J bercle stain, S8. Gas analysis, 50; detection, 50. Gasometer, 50. Gelatin glucose, 44; preparation, 10; sterilization, 10. Gelatin plate cultures, character of colonies on 57; preparation, 32. Gelatin stab culture, character of growth in, 58; inoculation, 16. Gentian violet, 18. Glassware, cleaning and steriliza- tion, 2. Glucose media, 44. Golden pus coccus, 98. Gonococcus, 98. Gram's, iodine solution. 20; stain, 38. H^MATOXYLIN and eosin stain. 172. Hanging-drop preparation, 24. Mauser's spore stain, 40. Hay bacillus, 16. j Heat, effect on bacteria, 48. [ Hiss' media, preparation, 190; use, 190- 200. IMPRESSION preparation, 30. * Incubators, 16. Indol, 54. Involution forms, 30. Iodine solution, Gram's 20; Weigert's 172. JT-LEBS-LOEFFLER bacillus, 118. !»- Koch's method of air analysis. 80. Index. 205 LABELS, 10. Lactose agar, 44. Litmus, lactose agar-plate, 52; milk. 44: solution, 44; 52. Loeffler's blood serum, 88; tissue stain, 172. MAILING bacteria, 200. Methylen blue, Gabbett's, 20; Loeffler's, 20. Micrococcus aureus, 96; 172. gonorrhotae, 98; 174; 192; 194. inlracellularis, 100; 196. lanceolatus, 106. melilensis, 94. pyogmes, 92; 172. tetragenus, 102. Micrometer, ocular and stage, 28. Microscope, use, 2*2. Micros/lira comma, 132; 190; 200. finkleri, 136. metschnitovi, 26; 134 Milk, character of growth in, 58; ex- amination for pathogenic bacteria, 200; litmus. 44; pasteurization, 84; quantitative analysis, 84. Monilia Candida, 180. Morphological characters, 59. Movement, study of, 24. NEISSER'S diphtheria stain, 178. Nitrites, detection, 52. Nitrate solution, 44. Non-pathogenic bacteria, 63. OBSERVATION of inoculated 'ani- mals, 164. Oil-immersion objective, 22. Oxygen, effect on bacteria, 50. nARIETTrS method, 188; 200. * Pasteurization of milk, 84. Pathogenic aerobes, 88; anaerobes, 149; bacteria in water and food sup- plies, 200. Petri dishes, 32. Petri-Sedgwick's air analysis, 80. Phenolphthalein, 6. Physiological characters, 59. Pigment, production, 56; varieties, 66. Pipettes, sterilization, 4. Plasmodium malariae, 186. Plate cultures, gelatin, 32; agar, 34; study, 36. Platinum needles, 14. Plugging flasks and tubes, 2. Pneumococcus, 106. Post-mortem, examination, 164. Potato, character of grown on, 58; in- oculation, 16; preparation, 12. Proteus vulgaris, 72. Pseudomonas aeruginosa, 54; 130; 196. erythro sporus, 40. fluorescens, 26. Pyogenic micrococci. 180; 186; 192; 194. RABIES, diagnosis, 198. Reaction of media, 6; effect on growth of bacteria, 44. Roll cultures, 34. Russell's water sampler, 82. SARCINA lulea, 26. tetragena, 102; 162; 172; 174. Sections, cutting, 170; staining, 172. Shake culture, 50. Slides, cleaning, 18. Soap stone for cooling plate cultures, 32. Soil, analysis, 82. Spirillum of Finkler and Prior. 138. otermeitri, 186; 192. rubrum, 26. Sputum, 180. Stab culture, 16, Stain bottles, 20. Staining solutions, 18, Staphylococcus epidermidis albus, 92. pyogenes albus, 92; aureus, 96. Steam sterilizers, 8. Sterilization, culture media, 8; discon- tinuous, 8; gelatin, 10; glassware, 2; instruments. Irt4. Sterilizers, Arnold, 8; hot air, 2; sim- ple form, 8. Streak cultures, character of growth on, 58; inoculation, 16. Streptococcus pyogencs, yd; 162; 194. Streptothrix actinomyces, 172; 184; 198. Study of bacteria, 57. Sugar media, preparation, 44; sterili- zation, 8; 44. Sulphuretted hydrogen in cultures, 54. Sunlight, effect on bacteria, 50. TAXONOMY, 56. * Temperature variations, effect on bacteria, 46. Test-tube cultures, inoculation, 14; 26; study, 26. Test-tubes, cleaning, 2; filling, 6. Thermal death point determinations, 46. Thermostats, 16. Tissue, embedding, 170; hardening, 170; staining, 172. Transudates and exudates, examina- tion of, 194. Tubercle stain, 38. Typhoid blood, Widal reaction, 188. I TRINE, examination of, 192. V I BRIO metschnikovi. 184. Vital movement, 24. WATER analysis. 82; examination for pathogenic bacteria, 200. Water blanks, 12. Weigert's stain, 172. Welch's capsule stain, 42. Wertheim's medium for gonococcus, 99; 194. Widal reaction, 188. Wurtz's lactose litmus agar plate, 52 ZIEHL'S carbol fuchsin, 18. Ziehl-Neelsen stain, 182. , MAR 20 1901 143 1 4 268429 £T UNIVERSITY OF CALIFORNIA LIBRARY