i a il i eat ‘ i i int LIBRARY NEW YORK STATE VETERINARY COLLEGE ITHACA, N. Y. This Volume is the Gift of Dr, V. A. Moore Cornell University The original of this book is in the Cornell University Library. There are no known copyright restrictions in the United States on the use of the text. http://www.archive.org/details/cu31924000870109 A ae LABORATORY (ZUIDE IN ELEMENTARY BACTERIOLOGY BY WittiamM DODGE FROST INSTRUCTOR IN BACTERIOLOGY, UNIVERSITY OF WISCONSIN ILLUSTRATED 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. Ina 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. MapIson, WiIs., January, 1901. (iii) CONTENTS. List of References...........-. 83 Sit cecusded 8 8-0 E SA MENOWE RE 400 Oi Swi eae e he spree A fds aeaiaon dash aupe Ree ise vi List of Apparatus. ........0. cece cece eee ee eet e ne terete erent eters f nee nee tense tence enenes vii Laboratory Rules........ cc eee cece ene ee een eee eee ene teen esaaea cert sree ttesanees . Vili PART I.—GENERAL BACTERIOLOGY. Cuarter I. MoreHoLtoGy AND ELEMENTARY TECHNIQUE. EXERCISE. PAGE. | EXERCISE. _ PAGE. I. Cleaning Glassware.............. 2 XX. Test-tube Cultures Illustrating II. Plugging Flasks and Tubes....... 2 . Form Types s4 2 ssieseuws s+ oss 26 III. Sterilization of Glassware........ 2 XXI. Study of Test-tube Cultures 26 IV. Preparation of Bouillon ......... 4 XXII. Microscopical Study of - Forn V. Filling Test-tubes and Flasks with TY POS ec asssecieeny cetera! wos eee ace 26 Culture Media ................ 6 XXIII. Drawing Bacteria...............- 28 VI. Sterilization of Culture Media.... 8 XXIV. Study of Cell Grouping .......... 30 VII. Preparation of Gelatin. ........ 10 XXV. Study of Involution Forms....... 30 VIII. Preparation of Agar............. Io XXVI. Gelatin Plate Cultures........... 32 IX. Preparation of Potatoes. ....... 12 XXVIL. Agar Plate Cultures ............. 34 X. Preparation of Water- blanks nigh as 12 XXVIII. Roll Cultures ......... .... 202008 34 XI. Care of Culture Media..>........ 14 |. XXIX. Study of Plate Cultures.......... 36 XII. Platinum Needles ............... 14 XXX. Use of Decolorizing Agents ...... 36 XIII ' Test-tube Cultures. ...5.,........ 14 XXXKI. Gram's Stain ..........0.02 sees 36 XIV. Incubation of Cultures. % . 16 XXXII. Tubercle Stain (Gabbett)......... 38 XV. Cleaning Slides and Cover- glasses. 18 XXXIII. Staining Endospores............. 38 XVI. Preparation of Staining Solutions. 18 XXXIV. Study of Endospores .... ....... 40 XVII. Simple Cover-glass Preparation... 20 XXXV. Flagella Stain...........---0..0. 40 XVIII. Use of Microscope .. .......... 22 XXXVI. Capsule Stain.... .............. 42 XIX. Hanging-drop Breparehion: ocr 24 CuarTer II. PxHystoLoGy oF BACTERIA. EXERCISE. PAGE. | EXERCISE, PAGE. XXXVIL. Preparation of Special Media..... 44 XLVII. Detection of Gas............... 50 XXXVIII. Effect of Reaction of Media on XLVIII. Quantitative Analysis of Gas . 50 GROWN onic cis oa Wnaewon nies 44 XLIX. Detection of Acids ............ 52 XXXIX. Effect of Concentration of Media L. Quantitative Determination :of Acids 52 on. Growth 2 66. ..608 senenes 46 LI. Detection of Nitrites in Cultures... 52 XL. Effect of Temperature Variations LII. Detection of Ammonia....... .. 52 on Rate of Growth............. 46 LILI. Detection of Sulphuretted Hy- XLI. Determination of Thermal Death TORBEN cris. teas ss aipails va sistas 54 FOUR, coal veenmens bok eend omeee 46 LIV. Detection of Indol............... 54 XLII. Comparative Efficiency of Dry and LV. Determination of Chemical En- Moist’Heéat 0.2 2 ss cesinee eg ee 48 zymes in Cultures ...........6. 54 XLIIL. Effect of Desiccation ............ 48 LVI. Variation in Enzyme Production... 54 XLIV. Effect of Chemicals on Bacteria... 48 LVII. Variation in Color Production.... 56 XLV. Relation to Oxygen............ +. 50 : XLVI. Effect of Direct Sunlight ........ 50 CuapTerR III, Taxonomy. PAGE, Points to be observed in the study of Bacteria.. 57 | Classification of Bacteria (Migula)............. mao CuapTeR IV. SysTEMATIc Stupy oF REPRESENTATIVE Non-PATHOGENIC BACTERIA. EXERCISE. ; : PAGE. | EXERCISE. PAGE LVIII. Preparation of Special Media..... 63 LXII. Bacterium phosphorescens ....... 68 ~ LIX. Bacillus prodigiosus ore eee 64 LXIII. Bacillus acidi lactici:........... , 70 LX. Variety of Pigments ............. 66 LXIV. Bacillus vulgaris ...........0... 72 LXI. Separation of Bacterial Coloring Mattef wa ieanas cetncamaaaa dick ce 67 (iv) CHAPTER V. BACTERIOLOGICAL ANALYSIS. EXERCISE. PAGE. | EXERCISE. PAGE. LXV. Comparative Analysis of Air...... 80 LXIX. Water Analysis... .......-.0eeeeee 82 LXVI. Quantitative Determination of LXX. Quantitative Analysis of Milk. 84 Number of Bacteria in Air..... 80 LXXI. Efficiency of Pasteurization...... 84 LXVII. Relation of Bacteria in Air to Dust LXXII, Testing Antiseptic Action of Chem- PartiGlesic: o. See fusing a piece of No. 27 platinum wire (5 cm. long) into a glass rod or tube (18cm. long). (Fig. ei , RENEE 6.) Hach student should have two such needles; ee 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. Thehandle 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. & R. 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- ficult 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 may be 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 (b) for ascertaining the character of the colonies as an aid to 16 General Bacteriology. 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 tobe 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- Fic. 7. Method of holding test-tubes. 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, ete.), are inoculated by transferring the desired material to them on either the needle or loop. REFERENCES. A. 146; H. 51; M. & R. 60; McF. 146. SprEcIAL 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 (Escu.) Mic. (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. ExeLaNnatory. 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° C.). 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 avery 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 & 248; 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 DirREcTIONS. 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 20x10x2} 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 DrrEcTIoNS. Clean } oz. of cover-glasses and place them in a clean Petri dish. j 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 ee. Distilled water, - - - - - - 47.5 ee. 2. Saturated aqueous solution of Bismark brown. 8. Ziehl’s carbol-fuchsin. , Saturated alcoholic solution of Fuchsin. - - ‘ - 5 ce. Solution of carbolic acid (5%)- - = E ‘ 45 ec. 4. Loeffier’s Methylen blue. Saturated alcoholic solution of Methylen blue, S(t - 15 cee. Potassium hydrate (1:10,000), —- - @ Z 50 ec. 5. Ehrlich’s Anilin Oil-Gentian violet. Saturated alcoholic solution of Gentian violet, - - - 6ee. Absolute alcohol, - : - - - - 5 5 ee. Anilin water, - - - - - ? 7 - 50cee. Anilin water is prepared by adding 2~8 ec. of anilin oil drop by drop to 50 ce. 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. Gram’s Iodine solution. Todine, - - - - - - - - 1 gm. Potassium iodide, - - - - - - 2 gm. Distilled water, - - - - - - - 300 ec 7. Gabbett’s Methylen blue solution. Methylen blue (dry), - - - - - 2 gms. Sulphuric acid, - - - - - - - 25 ec. Distilled water, - - - - - - 75 ee. 8. Alcohol, 96%. REFERENCES. A. 156; H.75; M. & W. 245; M.&R. 103; McF. 90; P. 200. SpEecIaL DrrEcTIONS. 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. Fic. 8. Block for stain bottles. GENERAL Directions. Bacteria may be studied under the microscope in a living condition in a hanging drop preparation (XIX); buton 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: ? Filter, tube and sterilize, 3 tubes. Water 1,000 7 h. Lirmus Minx. 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 ¢y>Na0H and rendered slightly alkaline to phenolphtha- lein by the addition of {NaOH. eK 2) Litmus solution is then added until medium is faintly blue. 3) Tube and sterilize in the steamer for 30-45 minutes on 38 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 any 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 XXXVIN. EFFECT OF REACTION OF MEDIA 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 ce. of a normal solution of hydrochloric acid, and to the other three the same arnounts 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 ec. 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. ; -¢. 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 mm. in diam.) containing exactly 10 cc. of standard bouillon. (Reaction +1.5.) i 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. f. 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. Specian Directions. Use B. coli or B. typhosus. 48 General Bacteriology. EXERCISE XLII. COPMPARATIVE 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 mee 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 XLII. 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. 938; McF. 46; S. 151. Specisu 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 ce. of sterile bouillon, with three loopfuls of a 24-hour old broth culture of organism to be studied. b. Add 0.1 ec. of a5% solution of carbolic acid to one tube (No. 1); 0.6 ee. 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 acover-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. Rererences. F. 60; L. & N. 95; L. 180; McF. Chap. VIII. SpeciaL Directions. Use B. subtilis 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 Petriand 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; 8. 151. SPECIAL DirEcTIONS. Use B. prodigiosus (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. & RB., 85. SpecraL Directions. Use B. coli; incubate. Make sketch. EXERCISE XLVI. 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: P 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 CO,. Measure. The re- B. GASOMETER FOR FERMENTATION TUBE ora e ne \ coca IEn gan Ze= Zen > TORRE 1RQarananaenl RSIS 7 MA ti- meters. The numbers indicate the area o e various A sc LT = = ss ot oH Hye cross-lines divide the figure into square cen A. PLATE COUNTER. See p log Het rH HH Re aa {J a ee) Ch, 1 ap O) . oe Ao if. | . , el LeU /o a of, See p. 50. Ly ae Wh L] a eet 52 General Bacteriology. maining gas is considered as hydrogen; bring this into the open arm, remove the thumb and introduce alighted match. Air mixed with the hydrogen present causes a slight ex- plosion. Express the amount of CO, and H. in the form of a proportion. _H _ as, CO. Rererences. A. 203; McF. 54; M. & R. 86. SprciaL 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. SprcraL DirEcTIONS. Use B. coli and incubate at 38° C. EXERCISE L. QUANTITATIVE DETERIIUNATION OF 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 ce. 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. SprciaL 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.). b. Incubate at 28° C. for 1 week, add 1 ce. of each of following solutions: 1) Sulphanilic acid (para-amido benzenesulphonic acid) 0.5 gm. Acetic acid (sp. gr. 1.04) 150 ce. 2) @-amido-naphthalene acetate. Boil 0.1 gram of solid a-amido-naphthalene in 20 ce. of water, filter the solution through a plug of washed absorbent cotton, and mix the filtrate 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 LI, 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 OF 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. 188. SpeciaL Directions. Use B. colt 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 ec. of sodinm nitrite solution. (Sodium nitrite, 0.02 gms. Distilled water, 100 gms.) The presence of indol is indicated by the production of a deep red color. Rererences. L. & N. 142; McF. 56; M. & R. 87. SpeciaL Directions. Use B. colt. EXERCISE LY. DETERMINATION OF CHEMICAL ENZYMES 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 (zy ce. of a5% sol. per ec. 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. ’ SpectaL Directions. Use B. subtilis. EXERCISE LVI. VARIATION IN ENZYME PRODUCTION. Make stab cultures of Pseudomonas aeruginosa (SCHROETER) Mia. (B. pyocyaneus), or any slow liquefier, in ordinary neutral gelatin and also glucose ais Compare rate of liquefaction in each. *This is prepared from beef by inoculating the meat infusion with an organism capable of fer- menting sugar, such as B. coli, and allowing it to stand several hours at 38° 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 LVIIl. 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 IIL. 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 ofa 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; macreous, translucent, grayish-white with pearly lustre; sebaceous, trans- lucent, yellowish or grayish-white, tallowy; butyrous, 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; erose, finely eroded as if gnawed; lobed; articulate; laciniate, cut jaggedly into deep narrow lobes; lacerate, cut variously into irregular segments; jfimbricate, 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. b. Size. ce. Color. d. Internal structure. 2, AGAR PLATES: A. Surface colonies. B. Deep Colonies. 38. GELATIN Stas CULTURES. A. Non-liquefying. a. Line of puncture: filiform, uniform growth without any special characters; tuberculate; papillate, covered with papille; 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; flocculent. c. Sediment: flocculent; stringy ; granular. d. Membrane: character; color. 4. STREAK CULTURES: . Form. ) Size. Surface elevation. . Consistency. . ; : : ; Gctor: ( Same as for colonies on gelatin plates (1). } Same points as in gelatin plate, (1). Margin. Internal structure. . Change in medium. / 5. PoTaTto. A. Growth apparent. (Same as plate cultures). B. Growth not apparent. 6. BovmLLoN: a. Character of fluid: clear; turbid; ete. bv. 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. FR mMeasecs General Bacteriology. 59 d. Character of whey: clear; turbid; flocculent. e. Reaction. f. Gas. g. Odor. 8. Buoop Serum: (Same as streak cultures). MORPHOLOGICAL CHARACTERS. a. Form. b. Cell grouping. c. Size. 1. In terms of the micromillimeter; 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. Speéial stain; Gram; tubercle; ete. e. Motility. 1. Brownian movement. 2. Vital movement; sluggish or active; rotary or direct; most favorable temperature ; age; media; ete. 3. Flagella; stained by howtos, Bunge or Van Ermengem’s method; dis- tribution, monotrichal, lophotrichal or peritrichal. f. Capsule; stained by Ziehl, Gram or Welch’s method; most favoriile con- ditions; broad or narrow; present inserum, 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). 1. Crystals. j. Involution forms. k. Pleomorphism. 1. Effect of various media. 2. Effect of reaction of media. PHYSIOLOGICAL CHARACTERS. . Effect of desiccation. . Relation to temperature; minimum; optimum; maximum; thermal death oe point. c. Relation tooxygen; under mica plate; in hydrogen. d. Relation to light; (Buchner’s Experiment XLVI.). e. Relation to antiseptics and disinfectants. f. 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. i. Relation of growth to acidity and alkalinity of medium; growth in 1.5, 3 and 4 % alkali; growth in 1.5, 8, 4 and 5 % acid. . Reduction of nitrates; to nitrites; to ammonia. . Production of sulphuretted hydrogen. - Production of indol. . Enzyme production; proteolytic; diastatic. . Characteristic odor. 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. eo 8 Bare. REFERENCES: Chester, Report Delaware Experiment Station, 1897; A. 216; P. B. C. (Cheesman’s Charts). CLASSIFICATION OF BACTERIA. (MIGULA.) I. Cells globose in a free state, not elongated in any direction before divisions in 1, 2,or 3 planes. CoccacEkaAE ZOPH emend. Mia. 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. a. Division in two planes, - - 4. Planococcus Miguua. b. Division in three planes, - - . Planosarcina Miqauua. 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 ENRENB. B- Cells with organs of motion (flagella). a. Flagella distributed over the whole oO body, - - - - 7. Bacillus Coun. b. Flagella polar, - 2 - 8. Pseudomonas Miguua. (2) Cells crooked, without sheath. SPIRILLACEAE MIGULA. A. Cells rigid, not snake-like or flexuous. General Bacteriology. a. Cells without organs of motion (flag- ella), - - =~ = = 9. Spirosoma Miquua. b. Cells with organs of motion (flagella) 1. Cells with 1, very rarely 2-3 polar flagella, - - - 10. Microspira SCHROETER. 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, - - - - 18. Streptothrix Coun. 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 Coun. b. Cell threads branched, - - 16. Oladothric Conn. 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. 61 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 PRODIGIOSUS (Ehrenb.) Fluegge. EXPLANATORY. This organism was first described by Ehrenberg (Erhandlunger der Berliner Akademie) in 1839 and named Mong prodigiosa. It is the oldest known chromogenic bacterium. It is commonly found in the air of Europe and has a very interestinghi, tory on account of its casual relation to bread epidemics—‘“‘bloody bread,” ‘bleeding host,” etc. It is questionable if it occurs Spontg neously in this country. Itis slightly pathogenic. Introduced intraperitoneally into guinea pigs in large quantities it produces deat, Inoculated into animals naturally immune to malignant oedema it renders them susceptible. Rabbits inoculated with anthrax are protecta by a subsequent inoculation with this organism. It is grown with the streptococcus of erysipelas to produce Coley’s Fluid for — of inoperable malignant tumors. a REFERENCES. Lafar, 137-138. : SKETCHES, cultures. Incubation temp. (°C.) MoRPHOLOGICAL CHARACTERS. Age of 1, Form: @, Bouillon: sce vececc vere dichnes cbid nase Gawcarsiew bo Ad cia hd Eee weinte Miaqes seid GEN VERA TH A ami Re RIE wale wie Semana Tis Ra Sabie [95 ie anise eit ad, Other media BAS ZO ie da teray ee we 3. Cell groupings... atid ‘ATrANGEMEN tS! ai.c se iciencises vigeRaswa) aie wis tide ay apie ailtiondis ais nye Sueisinws wiaiRia ouassie: Wlars Subeahd SG cals ate avetaye e/Rdia SadceraCoee dub, Qddgonc ese gagvecd| B56 fF inuiare | ean Goinisles | ESM RMON a, Aqueous gentian-violet.......... 0... cece ee cece eee ee eee eee eee 1 aiesanesalivd eaCSoaeione sls 9% late SERS GSS AERTS i ce ateactiret || tee sacellanese eee 6;..Loeffier’s: meth ylens blues 2 sccsacw a ssecncraine sews damint tie Aacd 20 a6 aoa canadian oe Denaeeewaoas ubie sedans daapa cede Ren Bid Baud spent ees @: Grany’S Stain + wrceacaaw im peieg eaters $e jiica las Yel weoshateeatadiaa ye. gigas iene eMae us Se ewaw aarti maine: GR Gee ies dion ice’ cae mdall ela ator 4, Flagella stain 6. Spores:.. is Special Characters; SuGh AS .22..0dcnd id dee cseudeade 5 bea scedeesd ze gee, ae Sb sev drend nud ccrenngaren aya td lode attivhton sah agerdcanboad seajsopene cted dhe vanenstenbccrenewbil, des ucleedae and [reswinte edvaal ded aeaee deposits, VaCuolesy sires ay se iain weakaincarse.ag Wis winulins de Wakiwteninn Geen ata ae SaeemlO es Fu ieee ae Renamer aIY Baines Sa SinatiA ad em wind tunel] alae atecete Posse ass She pleomorphiciand:involution forms, capsules, tevicsciics scl clsjegie oldnriua te ag snes da done im aatens endaules yx av apace nenad waaeayed | Ay oi gaeall aeew ooze: PHYSIOLOGICAL CHARACTERS. i ‘Relationto temperature r:iictesnnarsraicanastiy se ks einen shad whadaw te aeremaaaniNamoaie maine aMaet, wLentie sk meine AUS Hage AR mtidatea Bans OA Mae Soaaeenie a ae aoe hme teas diate Ce a: Relation to:free: Ox ye etl os ned seats cova ine sracate tatters sa aus BE ay Relays Sh SUR Wo NE BERG prsnsltA e Ae wieisicna 4-4 eayeidd. doa sesdsorepany gearelign be wyend bobs AG MAMebea Sa echo alma RM aaeaea Mamaia 3. Relation to other agents, such aS......... 0... cee cece eee eee eee eee een 8 Seid ienaaara ivggte seajesSele Denspupn end -Take ha ahaa. saliia tages Vee wae sncdajeritiptavar Wie Wed aahoteumbdann Sok -ave eS tara ats eat eee ete desiccation, light, disinfectants, etc.:.......... 2... 5. eolecus aicenee Scclan ssa Renanarn east ne ase en aah yaalelove haan eecatpesiae 4. Pigment production: 5. Gas production in glucose media: @: Shake Cultures ov xsi: as vheee- se se tees east agi ge os ye Shui Ad eee ee RIRe BERR G Rete ROE SEE i doar isda tengiauele en aie Syste Sia adhe toje teas aR yada zamemeEee ET 6, Fermentation tube, growth in: (1) opemarm:............ 0. cece eee ee eee waisisenegitney (2) closed arm: ..... (3) rate of development: 24 hours ............... percent., 48 hours ......... per cent., 72 hours ............. percent., .... .. hours (4) reaction in open armi..... 2... ee eee cee eee cet eee ener sees eeee (5) gas formula: H:COg:: 6. Acid or alkali production, litmus milk. .... 0... 6c. cece cece eee ee eee ene EEE ERLE RE en Ene EE EE Ooo cE ed Ee EE EEE Ebb bed bbb bebe cece cece cece cued 4 7. Reduction of nitrates; to nitrites eat ncansde tI rats a foe ee be 8. Indo] production; 24 hours ........ 62. .c cece cece ce eee ‘ fecal Sders 24, WOUTS soa pacus so be ccuswuwns coronene vane g. Enzyme production : proteolytic ..... ar PathO@emesis: << 9-22rq i iesaus id me geeteeceaatees selseeg! Easinre mime it me seaRepietes 1a ie ne cnubdeudeh yee eae cocks [64] CULTURE CHARACTERS. Reaction. of Medium, Incubation Temp. (°C) 24.. .» Hours. SKETCHES. (1) latin late: ~ Surface Colonies, x Deep Colonies. ~ (2) ar late : Surface Colonies. Deep Colonies. (3) atin. sab. (5) Sy [65] EXERCISE LX. VARIETY OF PIGMENTS. Make agar or potato streak cultures of the following organisms, incubate at 28°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) or Pseudomonas aeruginosa {B. pyocyaneus) or Pseudomonas violacea or a [66] General Bacteriology. 67 EXERCISE LXI. SEPARATION OF BACTERIAL COLORING FATTER. 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. BACTERIUM PHOSPHORESCENS Fischer. GENERAL CONSIDERATIONS. Described by Fischer in 1887 (Zeitschrift fir Hygiene, Band II, p. 92). Found in Kiel harbor, dead sea fish, oysters and occasionally on meat in shops. The production of light isshown in the dark, especially when the organism jy grown on a medium made by boiling two salt herrings in a liter of water, adding 100 gms. 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. REFERENOES. Lafar 160-164. MORPHOLOGICAL CHARACTERS. Sultures. Age of Incubation temp. (°C.) SKETCHES, 1, Form: a. Bouillon... d. Other media 6 POs cnc navasns . Cell groupings and arrangements :2 39% ctstanaseme iene ve ce seteeinee memeuuies teed bai iasaQieineaebbchnetate aeacage)S ane Aoeged eC ues Ga chance elveraneein Macrae ees a, Aqueous gentian-violet..... 5. Loeffler’s methylen-blue.... . c. Gram’s stain d. Special stains OMA GAIA 92 cee cM ON ea NN Oh ae A A Sa set DOO Sass ese ae AT Ae NA Teer thE ed se teria as a. Character of movement 6. Flagella stain SOR a ceeded 5 oie vance ded a biSiuahges ay 1s Se Satara wie Sides acd aE athe @aentd etched ne DRA e RIAN ey aaa Rien ante mss Ege desiccation, light, disinfectants, etestii. sis iwns cnisisdarsaeeneiae neley epinsiaasinweaverd’s Gs £6 tetas nedvse meee gout 4. Pigment production:...... 0... .c0. cee cette eee EEE eee nee ca ee cone EE EE Stuy ctbE bebe dEE bEbE bbeL bebe bebe babe babe bebbibee cbebuansat " g Gapedueeaminemneeda?, ==CU~C~C~‘“‘C‘“‘C“‘(Sé~*C*‘“(‘ a SO @, Shake culture .. 20.0... cee eee cee eee ee EEE EEE EERE EEE EEE DEE EEE be bbe bel 5. Fermentation tube, growth in: (x) open arm... ..eccceceeecesecceeceee cesveeee(@) 10800 AIM! ceecsoerecee (3) rate of development: 24 hours ......,........ per cent., 48 hours .......... percent., 72 hours ........, seee POL Cent.) . consis cad fa) weacilot It OPE APMG, .0.icincensi ater oxen nasrunwed nus goeenien aa v++00(5) gas formula, H :CO,:: 6. Acid or alkali production, litmus milk. .... 1. ..1e see ee cece eee eee eee eet e cede ceae tenet end crite dabei bebe bbe ce 7. Reduction of iti Ps tea atta aay ane cdcrgcercnad ah eh dnaain ae daa tah en Noein mae natn. ae cree tees 8. Indo] production; 24 hours ........-.60. cee eee eee te eee 6 ABR OUES scsars davsins winded vase Mae weboeenpee ina a fecal odor; 24 HOUTS .......66 06 ce eee eee econ een eens AB NOUTS (5:5 seers alsin? sdayownn al Seu hoy odcees 9. Enzyme production : proteolytic ........ 0... cece cece eee eee ener nsec ners lets veceucens 10, Characteristic Odor... .... 2.2.6 e cece cee eee ee ee ee ee eects neE tebe cite sien bee eens 11, Pathogenesis .........-6..scceeeee eens cere eee eee nee een tnee tbl bte ibe cbae eee. [68] CuLTURE CHARACTERS. Reaction es 24.....5 Hours. ABs aise Hours. Cra vrais 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. a A. i /N /\| 47 \| | Agar . i AM | Streak. \ N a (5) - / / \ / a {7 lL |/ / \ Potato. | NN, (6) Bouillon. (7) Special Media. [69] EXERCISE LXIH. BACILLUS ACIDI LACTICI Hueppe. ‘GENERAL CONSIDERATIONS. First described in 1884 by Hueppe in Mitteil. aus dem Kaiserl. Gesundheitsamte, Bd. II, p. 1837, This organism may be taken as a type of the bacteria causing sour milk. REFERENCES. Lafar, 222-244. ; SZ | Sa SKETCHES; os = =I MORPHOLOGICAL CHARACTERS. BO | gs ; 1. Form: @. Bouillon ............ 2, Size ........ 3. Cell groupings....... and arrangements .. in growths.... 4. Staining powers: a, Aqueous gentian-violet .. 8. Loeffler’s methylen-blue. .. ah GRA eee eaiaish Seta ASE Baas Re RIACRIASH ae Moe NAGA Aevetads TAR Sek deal do eamaye erates lemeinaierats.| denied c. Gram’s stain... da. Special stains . 5. Motility:...... 00... eee a. Lineaeter a! MmoveMien tices de iv ingens sarees 01 peed een eraninnea i icamen renee ing. Ra th Remon areTEs sh oP wake saved mean vor eet xpos Be PICO ASA ons eecavazas v4 490s Guaes wr baues SAK Ba eer) Cuan ERY 9-H CRRA A PRE 4 HE 05 Ua SRG SRR WH WR wed ceeds bewne vere 6. Spores..... diguibitabaes atanbighd Holand ane dha. secaiaraus asypauesialancland © Shatss voesialan aed rueeanen teal erara aa 7s DPOcial Characters, SUCHIAS! counter cs cen cantrauciWonn Sa ne van Juste Uvicw dake da pe pad duauyd nepmeMe ta sa deNeaa Te pulaeweey eee aeadenanlte-04 mprilloctes wane deposits, vacuoles............. ag Fa de lawton args erblnctie wh oe eawtantne yee arcaanre akgh desks tamleeasaese Bead Gemiee tates de Avon nace ayers Pleomorphic and involution fornis, capsules. Gidecisc <4. asmaaaneiwadaude ts dosadeamde i eloweues ee camer 2 ° > Bp a 2 ° > @ bet iP wu Ss ° im ~ a GE POSITS; VACUO] ES! re toresa acess Hates (ais vis: wataien wiacia He Testateale va ai MMNCEIRRIDIS a Aatalclaedbida ie ny ovis bhatemaneaNabniniale aa gUingla wuglamnnyynde se ait lewis estarceell eiepens Salers pleomorphic and involution forms, capsules, etC.. 1.0.0... cece cee eee eee eee rete tere rete eben ene tae eeen eres pacale Nae PHYSIOLOGICAL CHARACTERS. i, Relation. to temperature: ss sasvinscrc as de ction eee s sw Hawin sctiaisne He wis tein cinyans Mawhsniala Siewince dave Bahasa YeneamAiTe oe AP Amieeee ne Ree die VE MODS Wadee UG iaes Slneeue doe oda aeR Se 5. Gas production in glucose media: @; Sha kere dl (ure ae aawanscnnrmnmias sana laeeven ve tains Boaters eh ae wrest a. ta telly aa ea ae reine nce aig ad ccs Dealt ddan ace oll BASae hte Aaa cheba aldose ibs 6. Fermentation tube, growth in: (1) open arm:........ 0 eee cee eee eee +++es.+-(2) closed arm: .. (3) rate of development: 24 hours 8. Indo) production; 24 HOUWIS .2.~ 02.5 casas neracenneey © te o0e fecal odor; 24 hours ............. sii ais enecen Votes se a Bagyme production 2 BreteOlytie acy 14 ce ocwn scien vavuioany os 19 pew elewens ba EAE wae in ad wenws Pathageneeis «a o.ceess heewes i a See oeS Ae an BNE os ce con aceene CULTURE CHARACTERS. Reaction of Medium, Incubation Temp. (°C) SKETCHES. (1) Gelatin plate: (a) Surface Colonies. (b) Deep Colonies. (2) Agar plate: (a) Surface Colonies. | (b) Deep Colonies. (3) Gelatiu Stab. (4) Agar Streak. Potato. (6) Bouillon. (7) ‘Special Media. [77] Name Of Organism 0.0.0.0... c ee cece cen eek eee en en ne EEE TREE E TEER EERE EEE FEET EEE EES CRETE EEE Ree eee eee ayy, Source, habitat, etc. ........... ccc cece eee piney Ses vete PRN ge ee ayer ets as pibsetees neem: doh a aceeaieds ee eee References.... ........cc ccc eees cece eees asa Saree Smee aes baie talent a ROG ELIE ed ates PEAS ARR ROS cbecaailad eed. a Incubation temp. (°C.) MorPHOLOGICAL CHARACTERS. BO 1. Form: Wi TIOMUNON « svciivsdavatcwaiee yoda cated obs Gdeksd Wi WAASSRIE AE RARS 1A. Senee DEE ges Fs PCa ende sd veneers eae be caaeieh | 08 CoE eR eN aN te d. Other media @ SIZE: s; sdea nas emacenenk tet: seve aueosaw weddeuN SER deed Se oe Seah Meee a Palgeead aa pee GaGa ae GATE A inaetOM deen da pndeMNIE OEE 3. Cell groupings andiarrangements) i ssciiccrandis swrea es ndieaieeeenveaven ones Ginsane dda atagdvonse-edane aegonie marae atom AG Tk BAST TY BYOWLNE. gesy coe vide bean 2s cs chow us eens naam’ va Ras Wena ss Hew ReeN RTA EARREG ER PET EeMSe Hed era RRR ERE KOM) Bed Ts Ene DRIFT Bed EP & Staining POWers! cove crap sces cous cowed vat awen news a a gnawed eg nett we nmin He coke wdawny Rou MER REE RE RE ARE Rh Oae ob sa eae dea eR ahenIe Rinne modi wade @, Aqueous gentian-violet 0.0.00. ccc cece cee ce eee cette tee ete bebe seed nee e eee n een en rete este ee nese eres cee tne cseenees laren ces fall eerie ete $e b, Loeffler’s meth Vlen=blue. ...jcc-sa ia cece: ace sak one ne.ng ob enue deeeis te enuineniobe 14 Se cage Ree AE aS MORE cane ecm hee Pod rr | Cre ier Oy Grails Stain sss ve ss eeaerocancrmaini taste se aetna awe eis Mmm GrALeMnY we cid deme da dha h/aeide Saait Seimes Hamers SAN Saga | ea aeoligaeee ae d. Special stains .......... 00: cece cece cee ete ete tee cent tere tee een een ne ee eee tees ene eee ene eere rete rere tater refer tences [tere cers 7. Special characters, such as: deposits, vacuoles. pleomorphic and involution forms, capsules, tC. ........ 06.0 ccc cece cece cee cene cess cute cet ceteteteettetsteettisteseses[esseeeea[eeee eens PHYSIOLOGICAL CHARACTERS. 5. Gas production in glucose media: a. Shake culture 8, Tndol prodwetions 24 HoUTSs excuse 14 1a case coun ove cnye eats fecal Odor? 24 HOUTS =. vs cccinie ce es seedaege Hagin oy ee 9. Enzyme production: proteolytic............. 0... cece cece cee eee eee siavssaeere Sialeretine an oy oudea CULTURE CHARACTERS. Reaction of Medium, Incubation Temp. (°C) SKETOHES. (1) telatin 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) 3ouillon. (7) ‘pecial Media. [79] CHAPTER V. BACTERIOLOGICAL ANALYSIS. EXERCISE LXV. COMPARATIVE ANALYSIS OF AIR (Koch’s [ethod). 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. e. 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 NUMBER OF BACTERIA IN AIR (Petri-Sedgwick Method). GENERAL DIRECTIONS. g a. A piece of glass tubing 6 mm. (2 in.) Hf in diameter by 15 em. (6 in.) long is drawn out at one end ina gas flame and sealed. b. Fill this tube about one-third full : with granulated sugar, insert a cotton plug next a;:-, to the sugar and one at the end of the tube (Fig.13). c. Sterilize in the hot air sterilizer for A 1 and % hours at 130° C. (sugar melts at a higher temperature) . . » ey d. Fasten the tube, pointed end up, in ~ a clamp, remove the first cotton plug and con- -nect with an aspirator. (Fig. 14). Fic.14. Aspirator for é. Break off the pointed end of the tube filtering air. and draw a measured quantity of air through Fic.13. Apparatus for fil- the sugar. tering air through sugar. A, ready for sterilization. SPECIAL DIRECTIONS. B, point broken off and at- a. Filter 50 liters of air. taghed ty aspirator, b. Dissolve sugar in 10 ce. of sterile water and make plates, using 1 ec. of the mixture. : c. Incubate, count colonies as above and estimate the number of organisms per liter of air. ; REFERENCES. A. 551; H. 393; L. & K. 392; McF. 164; N. 449; §. 541. 82 General Bacteriology. EXERCISE LXVII. RELATION OF BACTERIA IN THE AIR TO DUST PARTICLES. a. Pour a tube of gelatin into a Petri dish and solidify. b. Remove the lid and shake a dust-brush or cloth over it. c. 18-24 hours later, examine under low power of microscope to determine the rela- tion of the developing colonies to the dust particles. EXERCISE LXVIII. ESTIMATION OF NUMBER OF BACTERIA IN SOIL. a. With a sterile knife collect a sample of soil in a sterile test-tube or Petri dish. Samples at various depths can be secured by means of an earth.borer. (Fig. 15). b. Weigh out 1 gram and dilute 1000 times with sterile water. c. Make three gelatin plate cultures using 1 cc., $cc. and zy ce. of this suspension. Incubate. d. Count the colonies as they develop and estimate the number of bacteria per gram of soil. e. Many of the bacteria of the soil are anaerobic and can only be grown in the absence of free oxygen. See Part II. Chap. VII. for methods of cultivation. REFERENCES. A. 556; H. 394; L. & K. 389; McF. 174; N. 444; S. 567. Fig. 15. Fraenkel’s Soil Borer. EXERCISE LXIX. WATER ANALYSIS. ; QUANTITATIVE ANALYSIS. a. Collect a sample of water in a sterile test-tube or bottle. Fig. 16 shows a form of apparatus used in taking samples of water at vari- ous depths. b. Make two gelatin plates using $cc. and 7y cc. of the water. e. Count the colonies as they appear, and estimate the number per ce. ‘d. Make agar plates and compare results with those obtained above. : e. Analyze asurface water (lake or river), a deep well and a spring water. QUALITATIVE ANALYSIS. a. Detection of putrefactive organisms. Examine gelatin plates, made above and (1) determine number of liquefying organisms per ce. (2) search for the presence of proteus forms. (B. vulgaris.) b. Detection of Faecal Bacteria. Pi Giatr Leip 1) Inoculate a fermentation-tube containing glucose bouillon (1%) with 1 ce. of water. 2) Make litmus lactose agar plate using 1 ce. water. 3) Ineubate both at 38° C. 4) Compare growth obtained with that of B. coli. REFERENCES. A. 526; H. 878; L. & K. 396; McF.169; M. & R. 79: N P. 245; S. 553. For the determination of the various Species present ace Bratt Micro-organisms of Water; Fuller: Report Am. Public Health Assoe., 1899, 580 . 422; kland’s 84 General Bacteriology. EXERCISE LXX. QUANTITATIVE ANALYSIS OF MILK. . Obtain a sample of milk in a sterile vessel. . Dilute milk 1000 times with sterile water. . Make plates as under soil (LX VIII). . Count colonies and estimate number of bacteria per cc. a we a EXERCISE LXXI. EFFICIENCY OF PASTEURIZATION, a. Place same milk as used in previous experiment in the bottles of a pasteurizing apparatus, such as Freeman’s, and pasteurize as per printed directions, or place the milk in ordinary milk bottles or fruit jars, filling to a uniform level; these are then to be placed in a flat bottomed pail which is to be filled with water and heated to71° C.(160° F.), remove source of heat, cover and allow to stand 30 minutes. Remove bottles and cool as quickly as possible without danger to glass. b. Determine bacterial content of pas- teurized product by making plates. A dilu- tion of 100 will probably be sufficient. Ex- press results so as to indicate per cent. of or- ganisms destroyed by the process. Compare the keeping qualities of the pasteurized pro- duct with that of the raw milk by keeping samples of both under similar conditions, . e. g. in locker or ice chest, making frequent Fic. 17. A home-made pasteurizer (Russell.) observations. Pasteurized milk should not have a permanently cooked taste. REFERENCES. Bull. Wis. Exp. Station No. 44. Russell, Outlines of Dairy Bacte- riology, 95 (4th Edit.). EXERCISE LXXH. TESTING ANTISEPTIC ACTION OF CHEMICALS. GENERAL DIRECTIONS. a. Fill a number of test-tubes with a measured quantity of agar (5 cc.). b. Add to the agar varying but measured amounts of the substance to be tested. If the antiseptic is not volatile, or affected by heat, sterilize. ce. Inoculate the tubes thus prepared, together with a control, with B. colt and make rolls. : d. Keep these cultures under observation in the 28° C. incubator. e. If no growth appears within 96 hours repeat the experiment, using smaller amounts of the antiseptic. In this way determine the amount of chemical (in %) which just pre- vents growth. SpeciaL Directions. Test in this way carbolic acid (5 %), aleohol (95 %). REFERENCES. A. 566; H. 411; N. 527; 8, 156. 86 General Bacteriology. EXERCISE LXXHI. TESTING DISINFECTING ACTION OF CHEMICALS, SUSPENSION METHOD. a. Make a culture of the organism to be studied in tubes of bouillon containing 5 ce. b. Incubate at 38° C. for 24 hours. c. Add to this an equal amount (5 cc.) of the disinfectant to be tested, of double the required strength. : ‘ _d, At the end of 5, 10, 20, 40, and 60 minutes make agar rolls, using two or three loopfuls of the mixture for each roll. e. In this way determine the time of exposure necessary to kill the organism used. f. Test in this way the value of corrosive sublimate (1:1000) and Lysol (5 %), using B. coli. COVER-GLASS METHOD. a. Make a bouillon culture of the organism to be studied and incubate at 38° C. for 24 hours. b. By means of a burette, pipette, or loop, place the same sized drop on each of several sterile cover-glasses and dry as directed in the experiment on desiccation (XLII. c. When the cover-glasses are dry, they are to be immersed in the disinfectant for the desired time, then removed and transferred to tubes of melted agar which are then made into rolls. d. Test by this method carbolic acid (5%), aleohol (95%) and formaldehyde (10%), using B. coli. REFERENCES. A. 558; N. 518; P. 152; 8. 158. PART II. MEDICAL BACTERIOLOGY. | PART II.—MEDICAL BACTERIOLOGY. CHAPTER VI. PATHOGENIC AEROBES. EXERCISE LXXIV. PREPARATION OF CULTURE MEDIA. The following media will be necessary for the work outlined in the following chap- ters. This is exclusive of a few special media which are described under special heads and are to be performed as a part of the exercise in which they are used. 100 tubes of agar. - 12 tubes of glucose agar. 100 tubes of gelatin. 12 tubes of glucose gelatin. 80 tubes of bouillon. 30 fermentation tubes of glucose bouillon. 30 tubes of potato. 30 tubes of milk. 30 tubes of glucose free broth or Dunham’s solution. 380 water blanks. 30 tubes of blood serum: a. Collection of the blood. Sterilize Mason fruit jars, by successive washings in corrosive sublimate, distilled water, alcohol and ether (or sterile Erlenmeyer flasks may be used). These are to be carried to the slaughter house and the blood from a beef caught directly into them. They are then allowed to stand undisturbed for 15-30 minutes, or until the clot has firmly attached itself to the sides of the vessel, when they may be removed to the laboratory. b. Separation of the serum from the blood clot. The clot is separated from the sides of the vessel by means of a sterile knife or glass rod, and the vessel placed in the ice chest. After standing 48 hours the clot will have shrunken away from the walls of . the vessel leaving the clear serum on the top and at the sides. This can now be pipetted or siphoned off. If the serum contains a large number of red blood corpuscles it can be placed in rather tall cylinders (graduates) and allowed to stand 24 hours longer, when the clear straw colored serum can be readily. separated. This may be preserved for a long time by the addition of + % chloroform and kept in a tightly corked bottle in a cool place. c. Loeffler’s mixture. This consists of 3 parts of blood serum and 1 part of glu- cose bouillon (1 %). d. Sterilization. Fill sterile test-tubes (about 3 em. deep) with the serum and ster- ilize either: Medical Bacteriology. 89 (1) By heating to 60-65° C. for 1 hour on 5 successive days, and finally plac- ing the tubes in a sloped position in inspissator (or sloping tray ina high temperature in- cubator or steamer) and heated above the coagulating point of the serum (70° C.) In this method the clear serum is used and not Loeffler’s mixture and a transparent medium ob- tained. This method is not usually employed, but the following: (2) Loeffler’s mixture is used and the tubes are immediately placed in a sloping position in an inspissator, or steamer and heated up to 95° ©. for 1 hour on three con- secutive days. If a higher temperature is employed bubbles are formed which rupture the surface of the medium in their escape. When sterile the tubes should be sealed with paraffin or otherwise. REFERENCES. A. 106; H. 45; L. & K. 88; M. & R. 50; M. & W. 81; McF. 181; N. 463; P. 219; S. 87 & 35. EXERCISE LXXV. STREPTOCOCCUS PYOGENES Rosenbach. First described by Rosenbach in 1884. It is found in abscesses, pyemia, puerperal fever, and erysipelas. It is frequently present jn mixed or secondary infections and occurs in the mouth and sputum and on the mucous membranes of the nose, urethra, vagina, ate, REFERENCES. Rosenbach: Mikroorganismen bei den Wundinfectionskrankheiten des Menschen, 1884. A. 268; H. 133; L. & K. 117; M. & R. 168; M. & W. 124; McF. 190; P. 476; 8. 274. ltures. e of u MoRPHOLOGICAL CHARACTERS. Incubation temp. (°C.) *e 1. Form: @ Bouilotigs os acess as tiscahecug vase eepanreieane xe ais Medhats SSAA Sh Ge ace Shine 2a de MESS SAE RE Se thee FEMME Rome eauree eR ERA cols | pete eee 35 Cell Prot PINES ceca seve caves Maes Beem oe Ne SS aad brated ave addadiaued poke aan dead ead eenn b Dash tea on iesoe wecuieen ede Aaa Bec Re eR es RE AUTRE GS ic 3:2 sca Se oh aevdedaskd aedeoihes oa Sdoi uAemh Vaan diiada Wade auulis vale Gawd emaRRe ag URI e ag SeadeNas en tale Lae anual nad asa @s Aqueous Pentian-Viol etic: cs ys ulsins Ye Big septeidisen cis wiitiens oie aadrermacdiasne died aqraesd odusionjoe sade Akelounlsmndanus rb aa ceneeomen eens ib; Loetier's methylenebl tess isa csaantsd ssacerdsiecyaisasad i-oaeevarafuat amavoa de ee Aaenceve amd taveanin oe he ausian var hades ad alee ea Scenes aba €. Grain’ S\S tain egies eens siesina iad aa wees Gans Stee saw Se ae aan Nesige suis! tie saa Sagl ded ae F eadeeate ait an wpen cake a, Character of movement.................00065 5. Sons dad ls scaoncgSed sata divcxGobah 0 Sea wus, Riel tceedeseh Gi emeHN Sex recaueaane oe NUTT Vina eRage BOE ala, ade Py. PRA RIN SIAID scare astied bu 1g Haan sintieka anaes sane wmalore eee balybbcial wa ou neces maelans euaeeee wae de: puhew Wada Rens Bs Gs wubBee es Gate Ree an if SPOLES! sisisirsiy sewwicawnanin rasan fas ifs mltnat a sbaisroied aaah oie ies aveaieiarainsaiped'svohygaliig a7 ste AASy Saws Tie OEE TS RAGE Retaiege ebiclar ae dakaeabeoee a8 Souder N uw ho] ® °. 2. p tat i) oa > iat » a o o a we a c is) = ry 2 deposits, vacuoles pleomorphic and involution forms, capsules, ete. 0. ... ec ece cece cece cece sete sees ueneenee ees PHYSIOLOGICAL CHARACTERS. 5. Gas production in glucose media: as Shakeiulturess oe ni poncccisiianstiaynie dd amen endaundwnmaos so ae Meeaxomsios valeurs ox &. Fermentation tube, growthin: (1) opemarm:..................... (3) rate of development: 24 hours................ per cent., 48 hours............ per cent., 72 hours......... per cent. hours (5) gas formula, H : COg:: $. Indo] production: 44 houre a .w0s pecas erreves vies ceews vie fecal odor; 24 hours .......... 2c. cee cece eee e ee eens Enzyme production: proteolytic CULTURE CHARACTERS. Reaction gt Medians 24...... Hours. 48...... Hours. Pee Days. SKETCHES. Temp. (°C) (1) Gelatin plate : (a) Surface Colonies. (b) Deep Colonies. (2) Agar plate: (a) Surface Colonies. (b) Deep Colonies. (3) Gelatin Stab. (4) ON N \ Agar \ H Streak. Potato. (8) ouillon. (7) i _pecial ~ Media. [91] EXERCISE LXXVI. MICROCOCCUS PYOGENES (Rosenbach) Mig. : STAPHYLOCOCOUS PYOGENES ALBUS; STAPHYLOCOCCUS EPIDERMIDIS ALBUS. First described by Rosenbach, in 1884. One of the common organisms found in pus. Occurs on the skin, in sputum, air, water, dust and soil. ; = REFERENCES. Rosenbach: Mikroorganismen bei dem Wundinfectionskrankheiten des Menschen. 1884. McF. 184; P. 470; §, 9% a , = at we | S$ é SKETCHES, , = a MORPHOLOGICAL CHARACTERS. 85 g2 < - 1. Form: > Bouillon sepscacione’satsdaitie voy sleicclgsaiglg gia buainigitig Seb ge nique ysis See eee asta hasan Salas ialcstcara odes inane: whe be eactesSace ana ha arial crate oa ae | das aelanmies Di PRA sss sis iti ap St a aaldehal bbe Sdisishad dea sEiTn Noa dla aad bed dioetsdcAd 4 gh oectavadbed ad Gaatvee be Bs UA La GaNE PaNieioiae sea UR TE TERRE tela Ski [leamaaessa | Meateeeate 6s Greatness ras ay gels delaras ae’ slaetds yaireiges ds a amen vanoeuemm ds ov ellaomes avaealg oo ug aeelas Mobdaan soaheualad arene a | oavdarenl bewneeae @s OMe Medias escent air. _adjunsciie: uecl Gece eA ebAAn Oe be dectasiaain ecmuduerke Go) kd waveiegd Secyacans wea etyedaas Nod ieee an aieas LE Ga bisa) even Seal Se areas POUL G25 cd Ae trudebp eeaca fatal nbeed ha esas wrstecirih eats oe aparece ee Gian Matec «Sab eccat aaittidedai giacth Austin fasts wncaheay Se oa heme ee | eee ea ariCellsgroupings aici sachet ances ao aten Aas haetgahn al son etatseee ai ted corte ae prea nag anni d oa ebaceie E daeyatatatgs De tetian a exe ol earn el Race cet drt and ‘arrangementsiwe: av aandssamgnenessg wees eae niSemaeadN BT Gh ceded ue RURG Mew els obaats Meuee a ay Saeacan anes heat sete deel aechs Guaed Gabe onde BRE REIN ete ca vices Sa se Saas aoecn ww RUS G4 ee BIE Hed RI RAGIna 6 HA Ad paein cresdiel whedbiehs pd by Ged GAL GR GSAX HE GARI od edie Seid AMA NAHE mane anes 4, Staining DOWEIS 3 cox nasasnanivhan cata saveteaeten aut iach sae MN Ann Rhea mieemeae ae 5 aistaibavslplina ni ieainioa aie ata ininaticla Si Ro-araueAe Nie Bela wisibteciaiein| ga a Aelia Becks : a. Aqueous gentian-violet.!....... 0.0... cece cece eee cece ee id eibtanane werd Gleenalen Ge soUMENER SGOT ote RiIERIA Hate Me ae GiamMAd ear few BF Gavelleelad HERE Be Teeter’ & wie lyg OR UG ses ia er ner emavneutamen v4 we eas WRU eee tam Ee COmER Raa mun ees han MOY eee A Geto | a Sa eon ada aoa Cp GYAN 'S:Btalm ascecnams san eae th Hy atee Gabteneecematy wh de Wed aiaaurbiehigisiddauad da opr ammeuphestierandseiesititee wo ae rasaylahh dio qayiesis Aut acaba onda ek Wop cial eehave tires dz SpecialiStainsycie crue ieuieesavaetskeaae wat, “aapiiwunetan: aieauandseatasaded wrea aia sat nie awed ober onae [leeleaerealls chaise se Ha IMM OUR DE Yio as ee ssvacea eal ib ve devzcastna Fab tahetedet ckcnre ta cevovdeevn tarcteeconant ee ce eodenel avait eeeannh ud ty ss stars Worle cats sods eat a nataaata tated tne labenaynsh Gas Mlacealee”wnaee! IES Bare eae @.. Character of movement enue scisareaigusaicne ns sanenige aparuelis ia-sip goa aumian ad Mesite S4-sa awobarnemne vary Saye nen GHaihty dese ides saad yu lle otemenae B. PNAS CNA SEA sce ca vweraging Sy das ealmey wae ealnl aaen 48.4 BOMB Nuay ta HeKALIN TA Ed EA HORAK Wie KREG be Cae cmdins hyena oa de souaasnyl nianss oe laercance 6), SPOLeSnemenmeremen ae teineate ta ao Meret as Se mek hn sens eens Po octal se cick Ekta dasscrl aie ake Ryluost Alou arora wa Sa aaa ute near odlgeae ain aaa ced alas el Sts taneecs 7 ISPCClAL Characters; SUCH AS 5.52 srac.tieissesses inva om aon raise est nee tl atcee ans dada wala biain diate Adal te Reg ueeenst Mianoetn valde emeonmerntedetesny vent Bice alllicedhec ou laces DS POSIES) VACUOLES raissiasciciais 3 sis sing gueccimc nt a tre sapsthansvenanstcearaccie! ie bal Sain gamegsha aiataueenanenteca na ge asoetateetieg Gecteetanle be Sy aewdindersciBananone alacnte aeOoave Wescegencerweel ecacen sess pleomorphic and involution forms, capsules, etc.. 2... 0.6.6 ccc ccc eee eee cee ec ea eee neta eae sete cctetensnteceratsesscraefeccevessbereeces PHYSIOLOGICAL CHARACTERS. ut. (Relation €o temperature 'sevsjcis < c0 cuteacre ve Haves enemas aiaidiaise Mcseisi be Ye: vale uculglele waalaed uekseiatnie o9-Db HOM gal Wie widielsoaed Ws 4.4 baum aachoubawiee ad 2. Relationtortree Oxy Pen tenants sates oe pe geste ott eae ce a dieiaeaGuiont a deiceam ad's 5 bau aneth ers sh OUaW ae eulbela dvine waa Mn mec e §.. RElBHOD:tG CtHEP AGENTS; SUCH AS ss cecics cciscins. ada dictermvteue Daarmnan hon wigh) BAaveruund Lee aelg Acne omesAMMAEee eetoash acme desiccation, light, disinfectants, et0.isacaaus sas casie apse svesnuscnn Weeacinn node ws x2 dammed abbceimnawme ns. dvajewd cadinawer ang 4c. Pigment production tas ciaearis svwssaysvdmwianiens sa seie genie nsaeelay de se gtas oetlen ds mie Soaudiaedng ho ede euannneaueeae seleranatdne 5. Gas production in glucose media: ai Shake: culture: ig xcdousdsds wecdaseaeee doo wtnas nereblnerautees jutactalh ea shed vane amin eee tac ag alin wen eae 6. Fermentation tube, growth in: (1) open arm:.... 0.0.0... cee eee cece eee cece eae (2) closed arm: ... (3) rate of development: 24 hours ..............4 per cent., 48hours ......... percent., 72 hours ....... evade per cent., ............., hours .... 0.000 per cent. (4) reaction in open arm:...... 2.0... eee eee eee eee tence ee eee eeee sees eeeeeeee(5) gas formula, H: CO, 33 6. Acid or alkali production, litmus milk. 0.0.2.0... cece cee cee cee cee cee ee eu cous eeeseteavunnenanees | 9, Reduction of nitrates; to nitrites..c ee cee adiny cea wenwes ex: G SE Ratt din Saas sonse aseapvbeeth atu aie A ty Westen moeneh do 8. Indo] production; 24 hours ............ 00 cece ee eee cee ee feGal Odors 24 NOUTS! sic ia ss aaootmamidnedeendisenene g. Enzyme production : proteolytic to; Characteristic odoniiassascasnseareoie onsaningaivine ste aiaiyaa wn tganlngas 9 Ti jehe doesen Rance 11. Pathogenesis CULTURE CHARACTERS. Reaction of Medium, 94 Incubation ' Temp. (°C) Pasenscs 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. (5) Potato. (6) Bouillon. (7) Special Media. [93] EXERCISE LXXVII. MICROCOCCUS IMELTINESIS Bruce. This organism is the cause of Malta fever and is found especially in the spleen of the diseased. REFERENOES. Bruce: Ann. de!’ Inst. Pasteur, 1899, 8; 289. Durham: Jour. Path. and Bact., 1898, 5; 377. H. 361; M. & R, 449, i le) 6B | Ba SKETCHRS, os pa : MORPHOLOGICAL CHARACTERS. 20 ceo t. Form: @s BOUIN ONS secs casnteis acnuaceewnnds af ts ceed Meaiaws veoh kiana 0; AG ar: cs ce drnaeaieive oepaeviie tealnanwaniad nde Reus wnedicig 35 ou Spalaemeeeeee s 6} Gelatin sowseieasnsae vee amen rveniaees xh Ua eae Heads Osea GR sa Ss Theda Heal amees He at, ee DCA aaa wen ddd vars dates eiemaae ere pasd meni Wd Kens 1A SRAM AS Big SABE ahi ic agers ssw eelntare Mover SAE Hiroe ORAS N A we cernlnn Ae aely Bed aed ae 3, Cell groupings: : ssicces ss se suis walsdaciod v5 oe Shou aisle nest dg ame genase ware setinnmain Hane MERRIER he He ot and arrangements Ue BROW ETS ve veidd Oi Goad eeadd cater eds V4 das Hes acre TORs wine sc gderoe Oe RARE BMA ReteereRA ess dx Slate DOMES? ccs vows wanes nesanen wees dies cues Weedead Kise Hae Reer Tesi mawe LEE BETeSE RRR TURNER Rees CARR ND te gee 3 end tien a, Aqueous gentian-violet ........ ... cece cee cece eee eee eee eens 6. Loeffler’s methylen-blue.............. cece cess cree ee te es 2 Grams Star tigr is sie e shade siv sie Sessa dpaeeeeas od ST HeisE Ae IOS DRA BERS @z, Special Sains) ss .aciivs ciweiewice ne dee waiemessauueneeas. ng sawed vnwespinens ne ae Ssihog SE Puente 6G GEMM ae Ee OPEN Fee %, Moa iiecaecaviengnus aces swung ide sanw iawa Soe Akane Res SHH 05 AF MER EE RE RRR RUSE NRO ve TEe SENSE RE Lh Haile pode GER AONE AAS OO CFKE ORNa Res Baa @. Character of MOVEMENL.... 2.6... e eee cee cee tee cree eee rene cee nee tenet etn eeee ene eeteeetennereeer cere sete ceealaceceeeeleeee eens 6. Plagella stain. sisi .caicccah ae ows ea ee eeeenacas Ye, tabi Syste eaets ie tial ssasnarey ae sie a ooo: Nay evades wares cola ale Wace an's use’ dvdcel econ Se Rashida Ga 6, SPOreSiscassae nea cinend peewee wasie dies ania dees qiass eos voit sieialacein nop dip weve hioie se AURGM Gls Ue aD AHEAD ARATE BG HE Sat Wielein seein ee eeenaiie Gea Apieg dea [Gree nee 7. Special characters, such as: .......... 0.60. e00 ee Galop ie ieeshaicncacseh a matte eteyenecaitead Sa dee yi deli died ear: a did anegcbe le! cn SHEAR a? yb ac aa hcinn MSc doiealSeaaciaaes let liaayea oeuass deposits, vacuoles............. -. oss tapsa ste eyunsate sep tope“s ‘sen coetergita patspe astndcvasines ais Sisk Ge ci oaTeI Re eaaTEIa win aes: MISS Reese ala eT cavthoaa eoaiataceel ancrap areata lee cpa pleomorphic and involution forms, capsules, etc......... 0.6. ccee cece ence eee teen ee een enter tte tene rr te ceteceteneen lees tees lore eees PHYSIOLOGICAL CHARACTERS. 1 Relation to Gem pe rats Oi Blk [cA I a1 => cat 01s bf: eNaea tam everett Resear Per meen a ean rem oP rere it fre erere Tera tty ert eran RMR EAT crc circ Sarr ot ey an eA aot arc De SIZE gaia vi no Wlalteecva ms tmchae Sak siore Bea siesaeinanierm bia ae Campa aia Beate Te slo waea (acer e a aia ce eeANG Gated asya fale a Sub I Aoeeels Reon aoe Die ele Gand Pee dre tinvets [a eagle 3. Cell groupings; 214s esnsuse sins soca contr oe saak gaasedade srummed exe tegen cig ties BOTS Mbeae mapa Ee Re bEN Jai ae Rae Ie HE Teas and‘arrangéments: gos. ncy ede wey ace neta ee oh aaeen iG ye eee eee Se MERE BROS eG ap Fe SEE es Baigine Cee ee ee eRe BES IMs GLOW ENS Nines ass dois Soda aiee SeaisaauSnle ykedk ale aasuuind oechetaldiearaate Ae Ab Sacahd samba deeatin aise Seated Mees ang emNN seh Rae aia ees aala ee aie 4. Staining powers: @, Aqueous gentian-violet ssn cwcidyessu tate os va ani anyone asaiaarng 24 +a rgpite mba sien aesteiaes Sh eeias eisleba aac Tok Rea reeels gone 6, Loefiler’s meth ylen-bluéew 2:23 asic oe disinaene be ee Game teens a eae Ne BA EE aS Ra ei iG Kisienlg 2g lead on oat eee ON ad. Special! stains «sa gs se-seeseye sess iernccen sx siea yas aitcnwis ai a aw aceaeiers tin sie -asapar a nrersites PERRO ee NG alg Siig HE Ha NTE NNEE bee nde I Alo oe POS UNE TFE A er noemern ERT Tay air Ps ER? ersnten rnc sqeaca is iran tis rine Ratio Ne Ba UMN ae EN ret? @: ‘Character of Movement cegecs sesee ye rs ead acta assem ac Ye ie Gamat ad cei ee aeun eases eatnaag He sau Tem amemee sa 58 Oss Plage) a Star igs s oi Socyiasessai ues tnaceeecs assis ia wees uctaeetep spein ss looal te ate eso ee wether Seco tp aac aceon eh Satis oee wave Map Seemaae eee Sk aFs Ge AS POr eSiees Soa chara ahh sean esederes atest osc Apo ENR au ate Rais heh hearer A Nacht ate, hac den Va. Seas Moonee aa BGI nia tease Sala ro mee de 7: Special:characters, SUChias? .ivss suacun ee he va mone eivaahe Aavean we veers oils naubianlaiinga ye uy aeGieminaag Aaaited di lp elising Ae BREA as Se Ge posits; WacCuolesics cae 5c si esouss cussccielsreie yes cease ley Ay ay a RNS ae gee Sa Ss cana rennet cemeipeee Ra aes, heed Desa oe pleomorphic.and ‘involution forms, capsules, Gt@. 0... sc sccrd sp. oan veacned ad nd same eb ak swaiele aueted ow wtcnene 85 aig Seeloule ee Gaeta ess Fb PHYSIOLOGICAL CHARACTERS. Tf; CRelation-tortempera ture ts aos. wi cas charts eerie ey i IS vas Os naa sao tin: ahsiars ves tid dated. ed pa Mao eR Gen weed stares sewn é on 2; Reélation-to: free Oxy Pens cance cede sw dasewesats ox tease ame ssod wens sae Aa AaMaW Vawe apie uaa SEAS EOD RS ee Moo boiee §; Relation to other agents; SUCH JaSte2. cis v6 say canta 24 aaemeredaiee awed Dade E MEATY Yas Gee abuekeeudu bb ea duban kee anions ” desiccation; light disinfectants; €tCsi-xeecy ties vadidewe Bees Gales gop ad aasedde Ac oe AS edad beet ened ean ae “Pigment produ ction tii eoiyesntens no aye cisnis oeeet tue. 30d b dechecteyet ene vs Ged ence hi Seeslue AN ete ee avs ch hese ea 5. Gas production in glucose media: a, Shake culture é. Fermentation tube, growth in: (tr) (3) (4) rate of development: 24 hours................ per cent., 48 hours..... 6,. Acid or alkali production, litmus Mi)Ks 02.06. cc. esa eee chee ve ne Sees Dena eae nea oe o 7. Reduction of nitrates; to nitrites........ 0.6... eee eee eee eee eee 8. Indol production) 24 hoursie.s css seas ede ceewr yee ss ee ames fecal odori2d: hourSsscisackeaeceahusues aeeawe oe aes 9: Enzyme production: proteolyti¢.. co.cc: crse seco reendan an yeoman weer s semana rete ds Tos: Charactenisti@: 0d Otis ssecinancrnsceearere ne anes Reaver os eee ters Il. OPEN: ATM Sas ci eseweok ag 14 9S aE se Sie aes ++.....per cent., 72 hours....... FEAChiOn 1M OPeMarMlivccscens soso ns Kaede mess He daceteR gine o ois diawio be ps4 DIN OUTS epheue date cc ioc ds se aii pes tease teres ee bey 24 48 HOULS pore eye oe os Patho@enesisweccy cise ay co nagesdsacimntnds Se slctacys sed mm tanaren Heeaee Guile Ninel deca ome enaan eo\(2)) CLOS@CAIRIM) csek ae avers sino aoe wig cage DEDCEN yaa hi geno eeemes hours...........per cent, -++.+.:(5) gasformula, H:CO, :: Seeeveialy COAMMIMO MANS 2 aca neg wataa's ae aasemeincwine sas seen eeeenee ......diastatic.... 2... [110] CULTURE CHARACTERS. Reaction, of Medium, Incubation Temp. (°C) 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. [111] EXERCISE LXXXVI. BACTERIUM RHUSIOPATHIAE (Kitt) lig. BACILLUS OF SWINE ERYSIPELAS: ROUGET. First described by Loeffler in 1882. Found in blood, internal organs, etc., of swine infected with the disease. REFERENCES. Loeftler: Arb. aus dem Kaiserl. Gesundheitsamte, 1885, 1; 46; McF. 426; S. 420. SKETCHEs, Cultures. Incubation temp. (°C.) Age of MoRPHOLOGICAL CHARACTERS. 1. Form: GEE RGA OT ve eee eras ae ne oak fe Specs cheese es Lala hevaten sR HE ae aa one 2s Gate HAS eam TENET EEE aoe RNR earn ye Te Py ype an Bec Ae oct das Seas nse a celiac ats os AGE Be NE Sa ae en see 2 ns RTS SIR ecg reas Ment r a Se aR BIRR PANE DRI TESA 1G IE SY ESV ARTI ice aoe as Se ee Ra BE tina of Sd RAEI SSPE SERTER a eo SeeE eset Rs bette Te fst Beas > hoot TRA BL USES ES SBCA RR erate NSE TIE RSIS SG be CEG Te IVE CL IEA na, onesies cle ese ees ee ted ore cpe dete dns Tad Sea CGY Ca eth RNAI SRS es a aha ers enhta) Se ata Suere re sbtea tn Se lee SIRE SS Sa Psat Ba SU sore ae setae ae erase ee hoe detects MA ence nent aueTaCgUe spl aeRNaaE Abad aller (esi eaans Seeissee are ie MOR OLE LED TENE TS ARR Nara Tee A auton esate) Sie SE | EES SY SCO SOUP INES’ darsrea vo hc os, weadarsemnseus ees ay Ae hia Riad a oat ih ds) dele niet ethe nFSaheralvayarers eae lreresce pee wee AG SF CUREIR In Sy AE Nee Sse Nati Oat Sera and arrangements icc ties dated eshahensgeeda ak wediciwmncehadae Sith Rea Sate sents SD La SAE eee eee Rae S cane etree nae ae ne pies in growths............. 5 ese esl se steed stoke beg ESAs Gee Bae acd Bas A SA ERTE: TG RG CHAI REUNITE Gee eS TE Ase oA fapnralay ota 9G naesaie = eeie | BE Le RS Syamndeeles 4. Staining POWEIS2s sink esas aneneanerode ae sraaieiaiee en. shee pie be ame nemosnon RSM eeraiea sce Dicinieh wes Meee BERIT Marrs crest RANE Selah diate | ASAE SRS a. Naeous: Pen tian-Viol Otis se. s sh yee vate ee Mites te aa alas ato trays Afalcbanta meting ats aeteieet noveeclcgemeia eS he LG Retest, Se Sats heigige Paeia istritl mie a &. Loetler’s imetivleme blu Gscscc5c.c sive enc as 36 nds Sakeweags 14 AREER TE NG BONG CRD eon a nmeNRRY Fai TINS a REED WE MAE HNN Hee | PF SRA RRO TS Gy GAIA SSE ATI dois seessmee scenere secs ave stcretecevshe peasadey dee BB vee? Gebenea vive de Yaad egsdfs ise atoaccek imme ae aaa Mare ween ea ag ise NREL Bi Sienna seis ae ga eee Besishd fitic eta az ae de! SpeGiall SCAMS ans scene ws re Apeenlis aslo alg oom auc asaeae ctelosy nat mae Tae ADS. £i0.S Cisaclisa it ed Seem RSD ie Reem a wats Hy SP rs aoe aerate heheh eg gM GHANM EH esate yscds a saat ntcbense BS Racneltoay Gens EARNS OU Se SUISSE rae oe seeetecae dal te aps ae eas tata ose AN kA ct ie Slee nents BE Se IMRAN HE AER AAG ia area eS wn a. Chawacteriof movement 4 .sda sete cs.cc dec Qanvas ness HAS RERE SY HE SESE CRM W LA OE Rae RaR Th ae Gane e TEES Tee GA ee eet tennis dae damnit Peiear|pineecntay! & Mlageélla-stait’ <2 cccesa: savsxectiwsatdeed otanecer ett Ot Spots. cecsenn ts gure ey eee H.°S peclalicharac bens, UCM AS pac. bcay oe decunterg nea wesc eM Osee Ma MAB NN Oops a yitan neved ok sence a Seats aya aathaite:iubath Seaeanslag ee deposits, vacuoleS...............ee cece eens pleomorphic and involution forms, capsules, etc .......-. 6.05.00. cesses PHYSIOLOGICAL CHARACTERS. fz Relation: to:femiperature? ccc. as ug adackous oeceme der St (eee waaa se aie Beets Gear era eae vanes as gree adamemuldn Ay ante pes omawe we salam a Ronit: a. Welatiow to free ORY PEI cc ve ov aece cess cance ya come anes ope ae bu wa ie veda HPIT RG ede ee PEE PERE ARE Dewey Ad EX ANA ER RENNER SEE Oa FT ROE TE AIA ARR ERAT Le RS eee RENEE COORD 3. Relation to other agents, such aS:........ 0.6.0. eeceee eee ees desiccation, light, disinfectants, etc:.. 4. Pigment production: 5. Gas production in glucose media: i Slralces Gill Cues ei teaiaa Nek ee ah cease eke cuca eee I ieeeciar eS Bd Gales Mona gaa ie Saat ae 4. Fermentation tube, growthin: (1) open arm:..............0.0 -2e eee eee sees... (2) closed arm: at (3) rate of development: 24 hours .....2...0...0. per cent., hours... 0.0505 percent, 72 hours... oe cece Per CONt., oo... sees eee MOUNTS peer cee . per cent, (Qn action in Open nme: a teen eer func weil ese te Siok es eve cna nets (5) gas formula, H:COe:: 6: Acid oralkaltproduction, Htmusanilke: acs. o3gasucnd Ub vaeielas ca de Lacege ee Miieasaoeviton vale ; 9, Reduction of mitrates; to nitrites’: cogs. x sa neense sansa sane gess pemes oveaianemsys oideesy CO AMMONIA. cc 4 bsy sedan se tae teeta 8: Indolproductiows 24 Hours witud secsien a vane sty ee wet hi sy 48 NOUNS nts Ve tadisc Ld So dad ne eaeeatecp Reeve Weve edemnione S NOS feca] odor; 24 hours ...........- Soy Wada ens Saag dos ated OSNOUT Sista tyne day : 6: Enzyme production ® proteolytics.n:cna01 sranten ates oaiauhein mores ames opel: ... diastatic i 16,0 Characteristi@odor css 5 acxany eo fe oe eR bees 11, Pathogenesis, .... 2.24 2.6 cone eeee ee beeen seus CULTURE CHARACTERS. Reaction of Medium, Incubation Temp. (°C) eee SKETCHES. (1) relatin plate: a) Surface Colonies. b) Deep Colonies. (2) Agar plate: a) Surface Colonies. b) Deep Colonies. (3) Felatin Stab. (4) Agar Streak. (5) otato. (6) 3ouillon. (7) pecial Media. A : \} h \ er [118] EXERCISE LXXXVII. BACTERIUM TUBERCULOSIS (Koch) lig. 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 M. & R., 224; M. & W. 148; McF. 208; P. 263; 8. 375. 21; Smith: Jour. Exp. Med., 1898, 3: 451; A. 312; H. 189; L. & x 95). MoRPHOLOGICAL CHARACTERS. e of lentes: A Incubation temp. (°C.) SKETCHEs, 1. Form: (GMOS TONND A Voy 9 Pees he De CnUAR COL ir Beran arc tre We ate io tM REY Mee Pn eo POTN ee Ee Se OME TS eS ic eet are rer? ec INGE circ aoe St cise oe ae eaves aes tae Se GY des a wig ge aaa UaER Hg SR SGM be SUED OE Mee EERO ONE RG ie RAE SO OCR RRS Salat eee 6) (Gelatittive aciyhitin i sanenke te Ae Te Wad @\ 1 Vcd ce V0 bE: eer eset lee ira eee eae a one Mes TiCe ee aE EAA er aRT, 8 PENORI Rts ated ny Gehint ar Me Te HAG clamor RC ONE ee ree ie 7 BAS ZE ays Se sctleesier tee lewis ede WES RIAD RTI TRAE EE DENSE Ht 1G BE WOIe HSU RRA Tes PEAT SURE SETAE DAN ERD: OS Nie Soe es DEST 3. Cell: BrOuUpINGSsatenscaekecistns secnssiomeias +a ddan nate oe ‘and arrange CMments as at cheecrnam ae sean Aaah es Sacer eta tH ea eee Late eee ee MEL Na Bering heap ta cae tue eee Wide [Wy aye TED HO WENS oe b.ck coh sys 5G ab Hb cGesnite GH garaged BO Hee dank Canmenr ge take 4. Staining powers: isis: ecasa teen ac cgi wean iad cis eee ee a. Aqueous gentian-violet ................0... bs Teo Mer’ ssnethy len-bl WG ae eae eect eee ery deena eee oe eh ee hee Nola alg pee a tag oh eee Se nt cao ease nr aradatuaeie Gis GATTI S SEAM os ssid nese ene ae see pian RAPS Ce Entei EAA oh Mea CMDR ek Sol Steer RAE oe eV EA Rh Ge Sesten be aie t= tae oe Bokeh ea | ad. Special/stains: .uweasececuseeieon Aiden Rote os ieee & Motility ie ees dense wens sy +s see wn a@. Character of movements a csac0 ine tea: aeeenrgs peta hs da ba aw dee ay haa de ba Seawind Seated Oz srlagellasStalny ssa ecen aes cto et canes Gas a ares foe ea oe eS at as aces Poo iach ate eran aaa datos ras eases re RT ANS cite (6: SPORES: Oa cihsn es deca e teat unto oa ska ge Mile nnanal Woon RN iain aa ketonne mame mitienlatrtatmeccctin ola AY FE ete 9. (Special Characters, Suchias3 isco curses anshe we ae sean AeA y oevaaan ce Ue ke a3 OUR Sea dete was oma aoaedee aheas ME PaSitS Vv ac uOles cn sak crete see ei manr ee enatene aicWie as eoenge oa octet ings, Bt. ys ala EY Syne oe aupybeSoanyeoubey dee he a nwchol ae pleomorphic and involution forms, capsules, etc... 06-2... 2 cee eee cece eee eee PHYSIOLOGICAL CHARACTERS. 1. Relation to temperature:............. 2, Relation to freeoxy gen? anc cumnanisais ve dqieiatecea sees paces gauwa oe 3. Relation to:other agents, Such asin is capa seceescen nena sa ae wr one aA8 SEES Ne we de ai wielgucd Hb ducal Bede cig desiccation, light, disinfectants ete .c..5 60 ceseaie on an Sanders snags ambenee dewewe 4. = Bigaient production ders see 5 voters arcane sae cheba ncato ha Gite tieaigeiaa core asap dauslita gy meamher eae he 5. Gas production in glucose media: Ga ShralkeevCull ture. aie ze) os as ices oa asa eae ON Done anesns taaye oor OMe Secs asiee 6. Fermentation tube, growthin: (1) openarm:................. (3) rate of development: 24 hours....... (4): TEACHION. In! OPEN Arnie c.u eaves i ie Ae ee reve va weiss sess cobs wa Sucuteen Sos 6; GH and arrangements ins weve eee enizaoria teh nig oot sinac amended Ha melameals ates ITIP EOW ENS scacecetnde ce sehes ao hare AE AUER OE BAR RR Na a bed Sed te aaa SU nee ae adele zaaLeacin nent seme meee UE ea 4. Staining powers: a. Aquéous gentian-violeti sess ecm came canes ue aia aeeN nS HORS ALAS TE SEDI AE Gd wan oald ee OVO aR NY Soe EES b,.leoemer’s nrethylen=zblucin sx tas /cncssviaas fas alata aalomiann nah eoualsiae teas Paola he UMMA ce FEAT RE aan Betactn as ne eae eeraS 6 Grams Stainwenesdse- was eis as acne id ae Seok oeea ant een In ee Pdi ee eee eR RAG Adltn ails ates who saaieise nthe saan meres] ahs aenatinallla aman GSS P ECVAIS PALMS 56.52. Sy Aensse esd, J sce cisosch ete ch 2 Sey aca byes depts ese ato ial tude Gh aceasta eeeseiet he ve nN SARIN ARP 4 cave wee AME ah Bis MLO UGY ste riecc. ataxia ca rsurpais cays ASSIS Vena Te eG NE AVE WEIN Sena des tales main ACR iA ei etal 5 Culestare aut Wyiealiten sdk ae fechas eens meee NE a @. Character-of movement cess sens emecantace ea atena cium 4 a8 dainael sh dann nda Bo SND We my eas GU GS Tura REE Ane Mane aaa 6. SS PORES, 5s. Fos.5, se seeasshs as ese 3 SS OS Na aiSd GS GS BES Ca a ENE STG Ur Bee RES EB a DEED. Stir erev at dina ye aie ape] ate tes 5: Special characters, Such Ase sac eewate sce cpacere sictane eater tai ates ae cues arora eee ca ah arm ap tess eee ere NSU HE RG IO Ea bE Eerdae se rain | Resa eaa ibe ee een deposits, vacuoles............. pleomorphicand! involationfornis; capsules; €tC vies aa hecee ake we eeng Ayes fa inate sa 3 RA re ee ee ng ed Ae eA We PHYSIOLOGICAL CHARACTERS. Tt, Relation to: temperatures: . 2. scuce sone as sestisaserenag geass Ziv TREAT OMCOMTEE ONY REM: Ssh scege si ures a arabe ots Pe Ges hd Ws wi Ad cee eutia se te ancl pian eben ee eT Hek seanvchotaauseee ale beimeeese acletid ates pc Toreaeaee dete eae ed eee clube re ome openssl stove alemiorma eet 3. drelation: tooth emarents Such, asians cans sae Gani Zagato anyedta iy anahkas anise a athe scl uel antag ne eunlome wap ge Beate yeh dia cs unt gotnaute aerial eh ale 8 hace anil dag Mee desiccation, light, disinfectants, etc.:......... 4e- PIP MEN tp Od UGLO IAs ook wind te edione Avot AneR My RE AatLAAOR SAE ENA @ MRL ces Ue else Soles i dan od Sued aps a shawrerme rang hhc ne Geucane 5. Gas production in glucose media: @: Shake cultures. a2 Gacscey sense debieaes audi ae ge cule &. Fermentation tube, growth in: (1) (3) (4) open arm:. rate of development: 24 hours................ Faction M-OpeNalMgwacieioces tate se caus shveeeas anes dzae wee guess abs sposiien (2) Closed ATMA uae avs ge aly von Mowe ona set dundied Huds amma rioetae amen ane HMR per cent., 48 hours............percent., 72 hours.............. DEF CONtiy cs ssess seas ......(5) gasformula, H: CO, :: 6. Acid of alkali productiom, [itimusiinilkey vo. naawee wae geek heen ate de Fee Mena a4 Vatyea iva adiwernaes 7. Reduction of nitrates; to nitrites........ 8, Indel productions 24 hourtsij.2c secede ase aa ea ieee naan feCal“ddor: Se NOUTSsuswed cas cae Hea dcwmmiey g. Enzyme production: proteolytic.................0...... To 0 ChiapactenStic- Od Omscsnweve tase orate fey peg P Sede scar dwar tes Bac ee se Methane wen deere ew AB SNOUTS UCAS Ge hese enim eee aay puis CAS LAN CHicarstraiaky da, caste hene conte ri,. Pathogenesisi..9; adistet Gate Mchat eat inereancz a [118] ages cae ROULS ace . se... per cent. Foam Ona. 2 3: Noss atestnnst. ne eaten Ss Beenie emlau wemsmatia a pan aanamanmeina putes eee theadee AAYS wasyrasnmedays ye ca ix eeancwneiel aM CULTURE CHARACTERS. Reaction of Medium, Incubation Temp. (°C) 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. (7) Special Media. [119] EXERCISE XC. BACTERIUM 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, 18; 357; A. 334; H. 280; L. & K. 281; M. & R. 431; M. & W. 162; McF. 446; P. 320; 8. 370, MoRPHOLOGICAL CHARACTERS. Age of Cultures. Incubation temp. (°C.) SKETCHEs, 1. Form: N 10. TI. @3 Bouillon: cnweaads qekcordencneauoncseesesmet aes By BOE otek pce ta dakaabaacnccais aawe cine hax awed 65.54 ca nue dau be bambre da 1s OURS WORE ERA: isla hE aR Qawe eR DERE et eee 6; Gelatiny se varuasenagieteca oeseiasee Gangs oy lees Ae ae Same esian oaategle @. Other medias. cc samc neta wigs sa eastern sais Peed dace Vanes eastbound des BADIZEs s veMened ged oY REREG TEBE RA Te eee a ae eRe Ghia sass seme « Cell Groupings... .5 occ cnnsice wa cees ss ANG ATFANGEMENtS wives shton ak are te else bh aMA DOS te sa hen bce eavaiomensias Gea Dahle eee Sh aahesli dats ch aiaG nema soins Tee in GOW thSsisise gies se acice ids eal edawtne va ve atea able ga SAimay eres Fs eg aie ge aie Die hee etonent ae Wienninn APRN BS A ANI ae Vase a alte ee @. Aqueous Gentian-Vidlet. occ iooccdcce ns so saiesa gene neways 06 9G GBS ROHS La igang ATT Sh een Se Renee RE Re ee IS) eng Rn 5. Loeffler’s methylen-blueioi. sic cs di cate cee ce cre neee ence ca ee nears se emibion ea aie galsed ea o Eb Sale be de 89Re a wens SERN oe aa ee eR €) Gram’ s'stai nie: sc ac tn ies c aloes aan seeeenea a Sanveasen 4a HemaiAT en ehes be ohecinee nb aa Si aang UEaas Pasay yee nies @: Spéciallistains: suse os gene sagecnea os a teins Weak Sy LAME Ne Melon Gada Fee a nsla na ane meme Bieri NA sealnee doe bie cam lsahendtn are gaa wie ie Gi as, Character: of move m entre acces vachitaceeeeenvraean ch stindene Gavin Atm Sos eed eee ma Saes swe ee ealee . Plagellastaine ¢acceneierasiasnade someacia dee owaira ee WS DOMES 1g gciscssnecd neste caging aaa Cen aG tie tea lenis 3s Yr ain apa aie rad ee dice teaitctbe tes camenans ecient Wk ata eo das Special characters,Such/ ase asics secure: nt cessed bad selina de Moiyeni ag Remade ed Seueaieavnseseieanaacnn ad eposits, VacuoleSicsovis senkons save gave wees Hae ak, calendar oeae Raa A pleomorphie and involution fornis; capsules, Cte: cs <5 spare cesses dese ong mriona nb eytiede + pmonseoedad Gumgen dene gma iy MEEVEE Yi spac aca oee ru: eaceed avn in. Sens te ehegee bE Sven GG c Fi bcd, a a ROU AS oe leisln Sb Soa E EIN eR ace oeet is REN gee ERNE Rata acted ark eens eto hota es as Aes Ras PHYSIOLOGICAL CHARACTERS. Relation: -tontelmpe natures ssciss. aeinusesun tip. dyna vk facectcintustthrashentgs rusiesols Be gous: da lidlevateya) Daluliae aS castin’ uageen ede aera encase na eie Septevaue eAalowe es sewertey avant Jak dvadunt ddeuiye. stances ieuysieesnecmaratian ae Relationitd freeiox yeens ace zak ve eeenes See ada Gia va nara gs We iyns dena Now lee oa eal daielneany ue ES ae aa emmiRauia 26 a8 Satine es Lig ae seen Gee Ae ? Sette onnans Relationstoothermagents,.Suchvas®: jog) .g.z aces cee av euetea oar ae monk aateteraiad aaa arate Sat nnc ds aieys ra Leer aad Per pS Me uae INe Daten eager : Garant ‘desiceation; lights disinfectants;; €tC sxicuhe tay set aie Ae Uo as ess te osetia seg aANa a ainpene eee a ciubatyc wis azsaevagen nd Soehear avcimienNeeoiore hae a beeen Pigiient production x. save snd syd aim on valida caine wlecewels pape tate ones aaeakiRa le Aoteadia ya nal hhey ea he neulie aba e nee Ae Ye ge gee tenets Gas production in glucose media: Gs Shialcerewl EU] spose ets cstes cnerecs eaters nh daa Ee Se a pie de hee, cia eeeaileindl elena 8. Fermentation tube, growthin: (1) opemarm:..... ......... (3) (4) rate of development: 24 hours ............... per cent., 48 hours..... FEAaction An OPeMAannss suiansd snes odenh wehraGavat eons UAahA Whe de nena een Se) EVISE AMIN Gil wos w be Opag a be baoRe eb mnd ae Saad oe Oy cad enad aoe meneame ...... per cent., 72 hours......... .......(5) gas formula, H:COe:: ....per cent. sia tees HOULSiss 36-6 vess seer cent. Avid or alkali production, [ities MANU sacica ce ox a: eatrnn wasn we on once aus sndneGe Hs Oh 4a Hi Bedi causes daakave % Reduction ofnitratess tomitites: cose se ee apoio eee te eat wad eaew Sede cat Tndol productions 24 hours... voc. ceni veneers fecal odor; 24 hours Enzyme production? proteolyti¢cscwies.: vest veces ec sueenan pees Gene Ree fees dae laens 25 ABOUTS fcc vetat aca tee amas ....., to ammonia... ... Characteristic od otisc seston es heck sas bees Gigs Woe ahne ar aredan bla yeas oa da beteue tte oe PathogenesisSins ics cess: th eonoestuedae 12 Seek Verge ee ee Siemens arenes iduning ee GAYS seacia tintianieasens eee ein eae ee rueya 8 MOUNS stein tineag eet hae her danse la se ee AA loan gee eave trie ended rains Gomme LAYS wear Terai waives athena satel OLAS tALiCs je: sisemieanetnise siombctanes nt wdc wh ke nea nan soaNOS tanto na Sa male one ’ 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 Gaffky. 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; 486. Gaffky: Mitth. aus dem Kais. Gesundheitsamte, 1ggq 9. B72; A. 369; H. 223; L. & K. 166; M. & R. 317; M. & W. 141; McF. 366; P. 402; 8. 337. MoRPHOLOGICAL CHARACTERS. Age of Cultures. Incubation temp. (°C.) SKETCHES, 1. Porm: Ms BOUIN GMs nize bse ables Garner 6. Agar CAG CLAW ecnctecamaratetheke uk Sanwa sone enon ant da, Other media.... De ULC sashes Okey et sae nies 3. Cel] groupings........ and arrangements . In GROWtHS \c.uies can ieee Bow Ane Sader ere atee & aa ehs Ge Stain POW ETS} de soie yooh cieomno ia gies ae RAGS Ee Ce a, Aqueous gentian-violet b. Loeffler’s methylen-blue..... @, Grain’s Stalin seg cnes nevi wees wees as d. Special stains .. 5a. Motilitytirs cgi ccaees a. Character of movement 6. Flagella stain ie SOLES 2nd a ennnracn cate eno ce RIGS We See Wate ete Nea SoA Sa onian eae soo seni aide ge tones 7, Special characters, suchas: «021.0... -48: He BOSIOS: VICUICIES 3.08 ous awneis sea venwomwy as yee nen es eenieeris% wees pleomorphic and involution forms, capsules, etc. © ......-.. 0.00025. PHYSIOLOGICAL CHARACTERS. 1. Relation to temperature: ......... 2. Relation to free oxygen: .............-. 3. Relation to other agents, such as: desiecdtion; light, disintectants; 206%. .0000je wi arti ceeds tensenep maser acids oe ewan 24 aedeloleagy batee be bee 4. Pigment production:............. _ Ree res - 5. Gas production in glucose media: a. Shake culture...... b. Fermentation tubé,.growth in: (1) Opemsarm? oc. in coonce ny 25.0 ona Beek ncad ee cae (2) (3) rate of development: 24 hours........ veeveee per cent., 48 hours............per cent., 72 hours............ per cent., (4) reaction in open arm: ...(5) gas formula, H: COo:: 6. Acid or alkali production, litmus milk 7. Reduction of nitrates; to nitrites ...... cose. ese enseiein eee 8, Indol production; 24 NOWTS «20.25.4024 o2 sees eo a eis oh , 48 hours fecal Odore2a HOUTS sc. wa ccnmorcnseeresd een ne ries (onal Gina Seer NN css ie 9. Enzyme production: proteolytic... neterline tates to, Characteristic odor....... 11. Pathogenesis .... GIOSEG a Pini acerca: Vode aenteoign Ha anise : & Sep ibO- ANIM ONIA wens le ee A Be per cent. CULTURE CHARACTERS. Reaction of Medium, Incubation Temp. (°C) a6 3S Hours. 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. Potato. (8) Bouillon. (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 feaces urine and blood and, in the pneumonic form, in the blood. REFERENCES. Kitasato: Lancet, 1894, 2;428; Yersin: Ann. del’ Inst. Past., 1894, 8; 662; A. 292; H. 259; L. & K. 200; M, & R, 437. McF. 433; P. 606. MORPHOLOGICAL CHARACTERS. Cultures. Incubation temp. (°C.) Age of 1, Form: Be OWT ca oe Sur sa vencnlene Wpeied ceded 24 Ma eG Geen ew te a we ety Kr oe Ga as red Kewanee ¥ DU INGAT si ahcnee es SNOUT ae ay cs darrian a ic ee es to ae ain ace Seo GAR Men ee a Hs PONS GHEY AE GE GUT 4S GOOSEN TE HATE d, Other medias. sc cciccccaccs: Bie SUZ Ow son ereocpenin ne Annie a Ah cree late NAA a tap rolep Nk tale Aten Ree ae lcci ta Thane Lanier had eal ater 3 Cell BLOUPINES ashes cecke rs hs whe taming tren ae aes ear asaya GA ae utah AIR aEe a MAR tinraedun ik WA ered ANCVATHANIGEMMENS 3) 37.5 e asain den, G aaah mayne ELIAS LULU fv DGG etapa ease lvotepepeciaa alae cates gs orate - seca os toes ee eR eee AME TO WENS see ctcreeeciseigsiins veg Sims wrrcrican wie iehasie arnada ne ako etme eRleela sea SG Ad Heloa as wimenlg Ge ymecealove at etd esata suai ah BE Meme en qo Staining POWE Sins sn sacks auton cu uy memes og ends a. Aqueous gentian-violet..................... B -OCMEer Suet nh Vle d= DIU Cie ce 3 uiadarctiee ato Se ree uss actvale igen ak Siecaeltes eredsicd aed pacpauaes Saree mae ewe mele EMGTATI S| STAIR a aon «knots ane nace mene eenetenes oan ieee aneeer name d. Special stains .............. 5: Motility: vss ex cneseia nes @:, Chara Cher Ol MOVE Mie ME se arash carers Moa aaa soe har acAl Cog ca ae es dtc ih GPs tetha Hit sg ma ete Pah weer vocals creche hems ans testes etalon he OI SPOLESS assis skaie ohrsrnaenns ono 7. Special characters, such as:...........-... cece eeee GEPOSITS WACUOlES mache AM agiias om ARAL Se Toei Set ead nec eee eel pte day pleomorphic and involution forms, capsules, etc............... es | SKETCHES, PHYSIOLOGICAL CHARACTERS. t.~ Relation fo températuresiccas os nce an Ghat saaatanoatan 2; Relation to:free oxy Pens c6 oscar can czinoans sees exec 5. Relation to otheragents, such ast svc ov ye nd bites sae ea ssn Gare Se ean Svea desiccation, light, disinfectants, etc.:..........0.... 0. 4. Pigment productioniza. ccc. usceaeseis seas cada Hee awe 5. Gas production in glucose media: a. Shake culture ...... é. Fermentation tube, growth in: (1) open arm:.... (3) rate of development: 24 hours ............... (4) reaction in open arm:................. 6. Acid or alkali production, litmus milk. 2... ......... 7. Reduction of nitrates; to mtrites........ 0............. 8. Indo] production; 24 hours fecal odor; 24 hours ............... meee ta ae g. Enzyme production : proteolytic .................... tO; ‘Characteristic: odotws< a: ix cacneaimotenese aan ir. Pathogenesis ........ per cent., 48 hours .... Jip 4B NOUNS sors Sa hs ares oe ee ireeeae oo lhok onsen eek WO WNOULB acerd pricey esd bps) Mid ee ans +++. per cent., 72 hours ........ sees... (5) gas formula, ++...) to ammonia............. occ hours... 00.06. per cemt, CULTURE CHARACTERS. Reaction at Meginins OA. she: Hours ABs ceed Hours. | 6...... 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. “~ aN ri \ 7 N . , ALi 2 Agar | \ \ Streak. \ 1 y ~ (5) A f / \ / ‘ \ 4 \ Potato. ; ‘ =f \ y, (6) Bouillon. (7) Special Media. [125] Macy EXERCISE XCIII. BaCILLus BACILLUS SUIPESTIFER Kruse. 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: Rept. Bureau Anim. Ind, 1885-91; H. 269; McF. 413; 8. 413. MORPHOLOGICAL CHARACTERS. e of ultures. SKETCHES, Incubation temp. (0C.) t. Form: Bs (BOMMIN 0 Nsasssae aes wee seers tociolstiets ota avore OS NLD oo ssc yc scr saveo ks Dhar BS UE Foes eevee dig ONL TS De AOS a aio Sp aN Ns Sh iiSS GUNS Shae ISV ica atch BE TRIB ae ed GI GSTS TLE STE Tt te eo caesar ae G5 Gelatin. a sists casa) crane haan cutee de sacaucgame @even Semeeie ween Semin Lem he Keene ve ee enon inige Gu umbend ys SA aRlenia Asani amas eeu GQ OE OE We AIA oi 45.5 Asean 2 a eat ot cecal naire veh 0 ats YO a CONE eA? Sd AE AY SHRI BAUS eR OR AO Neda Nal airilon i Qan Se ceaine Ba sxeeiains aves ANG AT TAN SEMEN US = rccahae dees canine J suo te ate ONG REQ HUUAES Sen ued (adr nd LE HEGRE: OS uae Estee aeG LORD abl SONGS SOUNDER EES Es SUZ © rcicyz ng Sib pastries pea evecare bes ncn re eho Remeclore isl padentenege rau cheney seat Geligrouping seahorse rake heey kale Anne ey DRCA AE durable Re eh aed atea A en EE Red eT. andeteeims Ti SLOW UNG seated 4.5 oenpuiet atest ganna heen aatue nk uiitea Aids chaos uh as . Staining powers: @, Aqueous sentian=violett cc tee. we aaa acane ws Ws Be eed SOUS CE eee es > Teoether’semethiylen; DIU GC xvkatic. oats cctanaaiie aia SecA esed Vie mwa ae moog HH URE RE ra DAS YER Home Me ME Be Eee enone d; Special stains. cic vsaasenys sts ataatnos pauiorrme ms aayates w @ Chara GteriOi Mm Ovienie nitive sceg sie ele a 7a eee ae Se Ara sO oa el ee ae a turn Rane Roe Usa od rd SSeS ete ae ee 6. Flagella stain D 7. Special characters, such as: ...... deposits, vacuoles............ pleomorphic and involution forms, capsules, ete. :. 02563 ce% vdecs eeereiw 25 08 vere ad Keene sis a eee Re dar be o PHYSIOLOGICAL CHARACTERS. desiccation, light, disinfectants, etc.:......... Gas production in glucose media: SME OUIIIE Veteran Sy-cca aon Beda dart tvaaslor aca apienene ene WA a nrctac re yeaah ia taeda Roo neue, Del aee wc heerte iy Wha te ee paar 9S Cn alae Und Decne mid | Mea Ne Pigment produc Onin sate ioaGacnie dios women eink ae CATAS os eae: GSMA KEIGU EU GE Ss a ead ah tua tie en ee EES. cae Soy ETE Ree () 6. Fermentation tube, growth in: (3) rate of development: 24 hours (4) reaction in open arm:..........-. 6, Reduction of nitrates; to nitrites........ NI Indol production; 24 hours fecal odor; 24 hours.... Enzyme production: proteolytic............. Characteristic odor.............. Pathogenesis........... bg © open aie. Acid or alkali production, litmus milk. .................. Relation: to: temperature ices conse acta ttsno maa aoe eaeinnedt wheat yuan wae AM ast nine) Beate sg te G2) ClOS Od) arin 4 eee eaves dbne bhe per cent., 72 hours........., ..(5) gasformula, H:CO.:: eoery tO AMMONIA. 0. e505 pAB TOUTS A622 .28 pone. .... 48 hours AE PETICEN C5 a5 03 Anta ede pi hOUTS...52c0.0.. percent CULTURE CHARACTERS. Reaction of Medium, Incubation ‘Temp. (°C) SKETOHES. (1) Gelatin plate: (a) Surface Colonies. (b) Deep Colonies. (2) Agar plate: (a) Surface Colonies. (b) Deep Colonies. (3) Gelatin Stab. (4) Agar Streak. (6) Bouillon. . (%) Special Media, [127]. EXERCISE XCIV. BACILLUS ICTEROIDES Sanarelli. First described in 1897 by Sanarelli, and claimed by him to be the cause of yellow fever. REFERENCES. H. 369; M. & R. 453; McF. 400; P. 609. MORPHOLOGICAL CHARACTERS. Cultures. Incubation temp. (°C.) Age of SKETCHEs. 1. Form: BC BOUIOT ew aniadage pes macmansenolsaee yelctans iach a OG NPAT aoc cee sink oF Sa Sete Din weened gram Daiolagae Dt oui e dina a ie MaAPA Oe AEE Pa Ne ee 6) Gelatin: 2 scevadiss cea sn Gav dae are as he pana nee ee d.. Other media’ ciuwsseie sevens conncaei ehh 4 dated poset see ese Geis 23 ESS Ba SIZ EH nse Nysins Bt yr OR AMN Be SAID Coseselvsrele cet clus le ns OS ubiess BMG RSMO RR: AE EAS tala Se ch ciadle siSaseiees ese MORE TG RANTS AS fe aeabites A STRUT INS ie MARR ima a A 9. Cell SP FOUPINES sos sie de as seg hoa teal eaian mia degein sy BASE Da GRAN EG ea Rae ae et ome eee REA Merelniad Ge dante aie tens ine th RATER Sea and arrangements..... 0.2.0... eee nce tee ee eee ete renee rete teen ttt teen esas UH GOO WE ss conde noes, Sadat ecsvautense or ta donee aie ie Dees Fs LEE Be eae aaa Aes ERM RENE Oy FFs BERT POTTER ES Keene ets 4: Staining POWESrStins 1g earn by ek chin aa een en se seas a. Aqueous gentian-violet ..................5. 6 Tooeer's methylene blue ice cy os oes cccsad db ayo ng a4 po bak bAGW ORT HRS ENE ES £4 OP REHAY Bs Lids eRe RHE PN ROR I we Be mE ¢; ‘Grami’s' Stainless ecu saccuaranwrn ares id. Special stains: sys os weiniv as ood iey sa newer sony sam aide ne sa dhewenbiiies eiteeehant as aan Sipe a auereadlane it aac Ginn Se BSNS Bee 08 Gate wie wn a. Character of movement........ 6. Flagellastaineja. co. 2 secre ne acne ssneey a o SOLES Hs. sp hackaisaryoee Sasa ay N MEPORITS; VACWOIEG:s .1xeaye us ains a5 waar whancdiqude se ce nemsweare peeAntee eee aaws pleomorphic and involution forms, capsules; €t@) veer ieee ove ners cane oaciea eo Cho) Fee aE hen aed, Sakae se eee ve MOREY Fay. i6 5 dock coi d dadeeode vs a weg oan tek Rei US Gio aes A ee Se eE TS ta ee eH een sea ne ted RINE AE RESET REE HE De etek ee Sead eR ARLEN S, GUO HE: an ene 7i40 thay dole sneebasphred Lede seme sae e dnd ebat hd af deded Lap euhtne whe as RANTS PHYSIOLOGICAL CHARACTERS. 1. Relation to temperature:..... B. “Relationstortree OxVRe na eri eons ornate sae Saami rea chen ish Me ot aalataade welatdedascrinee srday: sun ieaadngd «ttle atin es Pa ntaRMerag aa ]nay aavbIa Bedale al Gmomrdle dip Asie weebuaiae Se Mea madnve nine seeneles 3. Relation to other agents, such as:........-....-.5 dawcation, Ndt, disintectante: Bhetentac 52s eantn ned wher sine abasted aidanmue smi ed toate ol end Mee elas phe domme we Rates Sead ere teas ‘i Saat Rs HET ale TON 4. Pigment production icc: scagercesess cow 5. Gas production in glucose media: Phe ESTA IGENC AI IIE LDL G ictadare eat re onc cia ec tne Shae 62) SRG DY HIG ROSS NE RAE TRIB REA te i aiaitls eV eo pst maa ms ae nee nee ot eters oe Gita cee b. Fermentation tube, growthin: (1) open arm (3) rate of development: 24 hours .......... (4) reactionin open arm:.............-.. Hakka em ania, deManatiee its selina amen C2) CIOSCUUATINE oer area aaectac ot ewline ieeaaacemnies Weaken es Wineineae dye Danan ad anaemia eta outs PET CONC; 48 NOUTS, cece ec s PEP CONE. 72 NOULSs 2. cadeacess se DEr Camby, aance ones ..(5) gas formula, H : COe:: 6. Acid or alkali production, litmus milk.... 2.0.0... 066. c cece eee e te eee eee 7. Reduction of nitrates; to nitrites .... 8. Indol productions 24 HOUTS singe. ceeer sees sph bee eet ee fecal odor; 24 hours ............ 9. Enzyme production: proteolytic.........-.. 2.6.06. c eee eee eee ee Toy. (CharacteristicOdOre. aang cessed Sater wie ans ae Oh Bs Ge Ae oe Sey Ya eae ela Gouna Ts PathoOpeMesisss ras ose.ccnen ss mateacee pdson winced hain Ho Lasid tke ebasninlce demmalnsahen Ayes s deer OFAMMDIN OD La siete. aati seasrccesitic.c aonaaeate xi rlonuae sam euscercns osama eet te ate meahons Bun Cento mag FpitdORILOU TSA Re nenery as aes e a uagohet rine deel ink ht Aaah teen sheng .., 48 hours AGN AS FAL GE Sens ewan enciamine eae [128] vee eewshOUrS.... 006.05. per cent. tele eds AVS! on cigaruansa lca ssanemaien telahye saiera SAVES a Saath fone Soa as Se 2 ars Sh ayS ues esee de auasehet ie) er Sas a, martin eae LAV S A cgeciean is enwmiegeete rae Oa CULTURE CHARACTERS. Reaction of Medium, Incubation Temp. (°C) 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. (6) Bouillon. (7) Special Media. [129] EXERCISE XCV. PSEUDOMONAS AERUGINOSA (Schroeter) Mig. BACILLUS PYOCYANEUS OR BACILLUS OF BLUE-GREEN PUS. First described in 1872 by Schroeter. Found in green pus, and is widely distributed in nature. REFERENCES. Schroeter: Cohn’s Beitraege 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; 8, 454, SKETCHES, Cultures. Incubation temp. (°C.) Age of MORPHOLOGICAL CHARACTERS. 1. Form: LEV OMEN Te pcs foe eS euSeGe ay Sicsles os See e te op n S EBL AS Coy ea SUSERN ioe oe eee as a do In MONEE: Nw AE eg gONaioa cada RES SI RAIS, AS ed Mer | ite det cree Oy AGAT wate sabe eee eaden 35 He a ae Pate Me ae aes Ga teehee Lae a Aearan Reais be te Waar en Mare tare wu makes os REM eREN lees Tale ee desea es Gre] Bt Rieae sc tecesee: armeatieted obdk GRUsnG ge seeibitis, we Weep seereaasatit racers Sia aamcdeen vera metndent, Sanh .G Mee ve ore east cra mals oe wai vdageees alos ad ncoereed dees ety bbec bi ceesah RE NS aaa @ OUMER INEGI ia aco hee el ale ats eee weit Nee a ae lanl a RE le ae eld ete Ces as del doe pe Mie ide cig (aetna esha nce [rae setae BEGG OF SA San SSID Bette cis GeO ty en gk ia aa Secs Bk Bee AAs as Cos eee SUES GIES ine OR OI GRE GS USA a act Ae eeh aad ate Nacesh inate g gstiissne e/a schall anereaes ke ses ee 3: Celligrou ping icc axes cree weiner ae cack tae hag eke ae GeReEe PaO RE An emkle ae PRA an sem ee Anbiee wien ries ie GWE | Toe a Dowel ee de be TOWNS cs Se ee eee cert, Se es oe acaiiin Ne Maton ase had wh) ge nie ee a ee ei a Cae trae oe ah 2 gies co er Avra eee Nant ha a walled ae Be Ae Staiain Ge POW ELSE A oa sistke Seale wekepeEncy nee ee SSC agg LAGU Sena vecded oe Sheietoaul ada yav oe Oe tee inde ne eB Marcos Ae ve in pan cE eal aed oad enced | ceeectonsaqall wees eee ae: Aqueous pemtlanviclet.. cc: 1414 sewn c2e ween nce ws nae aA SweRte a He wn ee BH CHE MRRUR Bs a oadew ROEDER PEIN Sb9E Kee enwaeeee [Eis dade 0: Woether sine thiylemeb] 0 Ore este ve tunes vec an eich pean od He oh ate nm ende an wn achat Sie ne we AAI S ae en ee ene nee eens | GAIGRANUS S ba ls sn eedeto ween uk ee hee ene a eae ee, einai ae aethiops aie ape wang ai gumitee adie ce ote nmaathscnds ga teatgelg Auto a udebek Seb [ors ae ersrceall eyua oer Me SPECLALSbAln Sis porecn tae eomeemine eR eibay Mee cecas crise eum aN nee ease iceashegs Stacia asa a ae Ale Sa nt ua Rg Oe Bou AthA Ma Bee. Sater est IMT Ga UE AM ye 5 crit Aces tees nb acs a aot ed eat y0d eeepc Sy arb vend eicnealo oa Ne caaeee dah ADVE IA Stargate A axe Meee ORE wn @. Character oLimovements +. scnucacieaninet yess as auans poner the aenmaadiennets bs FlagellaStainie. sedis ocwamavecs tetas eck wet wattn< ste sennieae oe wee Maes wa meee se a ae os Saeed TH BRAD ap doeewod Sowaean bt aba be a pS DOMES a5 i 8 Sees toee acca cae SA Sa ne etn eh a ys ca NT ular ad She nt ea oA Gao GG, Siceatan teh yaa Oa eevee eee 7,, Special characters, Such aS? .aisiaze cd aaa a gs Riad Re ves Mate We aioe tp oe Aaae Hawes eee Males oy Babee es GE POSTIS . VACUOLES Hau tts camancresss cece) a cathein a edetanirante Sas meninle Jk innaeteheyae law Beek Gave Wemaie as aoe kms pleomorphic and involution forms, capsules, et . 0.0.6... ee eee eee eee neces eee ee ees PHYSIOLOGICAL CHARACTERS. nr, “Relation to temperatures cn cie: cs ecerae caise-wads cine a Semana einen mieten as a heemin ae aie ay ais Rina en Adele we ed 2, Relatiom tor tree Oxy. Gels o3s ress soos eh auras as HEME ee a ane tne ela et ia Rh bee GB ee Tue dk 3. Relation'tovotheragents; SUCKS: giv daves ica pers eitele sd demhe Bod saad bee TH Teena tue en desiccation, light, disintectants, etc.:.......... 0.0.0.0. cee ae 4. “PISIM ENE PTOAUELIONS:: seta nerg ov akan gated ean saa ace sh on vemes mn epTAhe ead vip eal Ze dan ad ge matnnee 5. Gas production in glucose media: : SAK Cultures, cc tasiGs cna cnetic ne me maniie aeteeortema wae Ha Waiay saa aude arom lng RE AMY Wee eed oe od als 6. Fermentation tube, growthin: (1) opemarm: 0.2... 0... cece ee cece ce ee (2) closed arm: (3) rate of development: 24 hours........ ....... percent., 48 hours..........., per cent., 72 hours............ per cent., (4) reaction in open arM:.... 1... ieee eect et eee teeters eee eee ee (5) gas formula, H : COs. #2 6. Acid or alkali production, litmus milk ....................-2... cure earn hours.... . " io nitrates; to nitri Lilche Rasa kas eee bees A 7. Reduction of nitrates; to nitrites Peete cee eee eee eeeeeee vey CO AMMONIA ........... 8. Indo] production; 24 hours...... At ae Se pemsea ues ae ea OG HOU ES skeen tees cE Bed fecal od Greed NOUTS sss a: -cia serene rauiy wrale-eesacnay MO NOUNS) «cx udesie sae gece 5» Enzyme priduction: protec Pits cic coer eiony es neeee ra eenees¥eende Gey ade ce Sava AS CALLE space ws anes nichancetien et ti 10s.) Characteristic: Odor acscenaied tended caede as ve letlondacurd calemoboe nous BSE RUGS eacuimauode moe al ndeaccen 1%, Pt CSS sete sesh tote thes pu aoe PaO RP eon tees Seat at aaes = CULTURE CHARACTERS. Reaction of Medium, Incubation Temp. (°C) SKETCHES. (1) Gelatin plate: (a) Surface Colonies. (b) Deep Colonies. (2) Agar plate: (a) Surface Colonies. (b) Deep Colonies. (3) Gelatin Stab. Potato. (8) Bouillon. (7) Special Media. [131] EXERCISE XCVI. MICROSPIRA COMMA (Koch) Schroeter. 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. 82: A. 401; H. 244; L. & K. 181; M. & R. 402; M. & W. 152; McF, 311: P. 568; S500, SKETCHES, e of Cultures. Incubation temp. (°C.) MorpPHOLOGICAL CHARACTERS. Age 1. Form: iG BOWilOiy was cg cc soe yeas OS See ae he ee Pa Be Oa Ba a eed B eSina een eee dd gt CaS Glee Bee Ga eenin ease ametinnl couse. | eee tate Oi INE 4 cacteteeieie, os aisha! seatey tao anbeana ee igh dlab aA Gaba RRIpRG NE Eetetie Andante tex SNERSAIN aa apendanaeht ohana ies: paslchalleA sas dentine alee la dai gos Be Rll area tnten PRONE TY Gra Gy CUAILING, Se oi haart Renteare i ea tenc Bossy ue eants Seo eee Magis De kara eee se ae sate aman Se ATE Oi ahene He Shiva raae ee Sethu en oe aoe RS ene BEY ainte | ebalgtaa tell oases, teers d, Other media........ sogcad ae ssyclaanacheg dete awensasie/elalabrornict ens nad akotarata hedslederaustewa otne. ev nah «i PeaveNslanvie Sbeuesayes vy ara ceamcorei@ cneoulntbaane aah hie rh deere lg eave wets | ones peer Be SIPC wea ath Bg fG aA ge eae Bo Send aw Gaede be bee eteN whereie De aecaicermie se Re ey eth ee tees om Bi etic Sodiart eee anaes ae bach ae ANG LAT PAMMEMENES sy5:3 ce wes ca ssralind Mee eA PEELE eR RAD AG BA Hees Mamet eae SNe ae Hoos amNnS 7AeR RAS Wa wean |anoen as Hallsen cen THER OWENS Art ar clo elae es Rie laa, aie sloshed on iereneevnstels La iseuclaiae emits s SLAM. POWETS! a4. as Ghee sraneet se wa xen ese oay = a. Aqueous gentian-violet......... Ox Thoefilerscnmveth yle ac DIl es seve hic aon ae ett et acs ersutee aaatayannabarsyeieptadia laa tebeabie im a apn weaieaoea rebates oe asad ae esa aisle Ieee @; Special’stains|: 2s cecissa de oeebess oped) Caviar pins Agree eee ae is rari CONC Ue erent CHE ce ee nerds eri tures Tee Oe nC ani err ey at Mean ae eeeargre ee ee AE ACRE Oe ee ae ere wa a, Character of movement ....0.0.. 066.60. cee bs PlaeellasSta nna sams cas tacen ar aura celeines emacs ake ras Peaasereheacnee 6; SPOreSies cerns . Special characters, such as:........... NI deposits, vacuoles .... pleomorphic and involution forms, capsules, etc.. PHYSIOLOGICAL CHARACTERS. i: Relahionecto, temperature se... sesccss opens se neneeta) asieewiendase vaca ee 2. Relation to free oxyrens, vs j...e op ee kann s. Relation to other agents, such as:............ desiccation; light, disinfectants, etev.c cocci vesagaeccie vase cans 4. Pigment production:.............. 5. Gas production in glucose media: a. Shake culture ...... 6. Fermentation tube, growth in: (1) openarm:............0........0000... ses... (2) Closed arm: (3) rate of development: 24 hours ............... per cent., 48 hours RAE per cent., 72 hours ............. percent., .............. hours per cent, Qh) TEACHOMUM OPE ADM... nis ss tenses cca were ee tengigle de aacieaokamonnea(§) ‘gasfornula, Hi + COg:: : i 6. Acid or alkali production, Jitmus milk. .... ..0........ ao RedwetroiGl rintratess toaitritesi in wasce cha rcesg ewns area ean eo oats emumet aw clases iste, OO BAMIIG NLA a7 ah 45 ie ok od meaivee A amaaye nooale 8. Indo] production; 24 hours ......... ; Bis aol eerie GH OM MIOMIT Sea 4 <3 pads cichs Se urahe Me AINE anes catwikr Sashes Sita days vad RECA OH OTS SH TOUTS oc pada rtd wt oe obey AON we ete ae GO OUTS ucinman ee meta ued desan cnc BaeretosAt eee ts days oe | QO: Enzyire: production 2 prove ov ytie: wees sc acesveteneate ea ee ae ies aise dees eybri a tuectierareantCl TAS CALI Cigna wend renee Ue aetna ee 10. Characteristic odor..... ee Teese antes cues satan ily 11. Pathogenesis CULTURE CHARACTERS. Reaction of Medium, Incubation Temp. (°C) 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. (7) Special Media. [133] EXERCISE XCVII. First described in 1888 by Gamaleia. bling chicken cholera. REFERENCES. Gamaleia: Ann. dl’ Inst, Past. 1888, 2; 482. MICROSPIRA [ETSCHNIKOVI Ilig. VIBRIO METSCHNIKOVI. A. 441; H. 256; M. & R. 426; McF. 332; P. Found in intestinal contents, blood andorgans of chickens suffering from a disease regem. 593; 8. 511, MORPHOLOGICAL CHARACTERS. Age of Cultures. SKETCHES, Incubation temp. (eC.) 1, Form: Gr IOUUNOR acdsee his Raate aiiyerselor Miseen ears terelercion: Maney beta nee Dep E Se ese ie eae ae Ct Net f20 ACES (Rs VO SBE Cpab ge A Senco cSdencps car LeUI yt Aen na aan ae ep OLS TA van MeN ACE EG Bi AGG AIAN aces ie enti dal a clea ipa sam ced sa 1a Salepluninns AEN He yiwd Honed eemea ee ey aie YA AY ANIERS oA RNR TAs ERAN DEE VA Ge Es MOTMG reAIVE GEA sec sects st Oth as eOe ce teased ys Sok ee ctcoee oh saan eae REND cyanea ane ooeraiyee? 2 SUZ Oh bach 4 Sua ha heehee eo ih Ratuar th Oee art AS gets nade enrane aie ate eeemtiae ere once 3. Cell groupings............... andvarrangem Cmts: pay jecatar eke yale satis wwe taclaan seid ciatchudaed hid (aiaute AE diesud Gaihardaga inva musik tives DING De Heh ae Oe PA ES cass cesar oor arcoreivas od Oa dud Sh aC SaG wh oe hekananea needs Gialamas oa wameua ane aees Wau eaunoakmnrrad aus ya waeeue le canoetaslomue ass 4. Staining powers: @: “Aqueous Pentianzviol@ts scacerecg. 4 Adee oq deaunee ie bee aed em tame erates eee ic EAOIAT Se AV ler ieee: aye ay sek sacha acvans ah bake Sabie Spaced Ge GP tama mtGh eee Robe Aa Ee. Due eae Aa naan elas (Gh Garten’ SIS CARMA Zeek cecisecatss saat bliss’ cgetchd SauceesessUs %/e lence gd RAINE sects cpcnaenytcidesoe ard Mb die a rupee izes lesene ete Sums A SOPC ClAll’S CAINS says seiavasteaceesi aes naas dam taneatrea relies 53: M Ott y sepesmaniis vrldeacuie we apaniamncelos a muamee rey tse ae Guay rama gs dann ee eaten nt acre intial eh de els ad hs Bs AAS AOE OS Ti VS IS Ug ales crea acai case oS ih Gach A BO Dank Cava Ad ty OO oer Baa eee ren bs Plagellastainy erg ieacansy vhnoae ab ranks cdoeraaok 7 soPeClaliCHatacters Suc hv Ass wuseieavaceacvee uses Se untan deep acca ol Lee Gacy dendis Sa cae ea uaa SR Mee Rhee eke aR Bara MA OeeNT EE GEDOSIES WacWOlesies creat sie. neta ete lott ot on a cmt n yaaa atari senior eaegees teeta tadaeds aioe mie eae pleomorphic and involution forms, capsules, etc................ PHYSIOLOGICAL CHARACTERS. 1. Relation to HOHE MA URE S12 5.5 3 leaaydiach rose were. 72 Sracla ftv elton Lenser hein nes nvacais oe eeeeeded cata aitayeeaul ay WS ete Bate nme aoe leroy en i LT Oey i oa nate iaveaoee cyan gs jaye elo agle 2: Relation tontnee: Oxy oe tis inamaron ee dete eaves sa aeetonnre Sx tages Giga heveeats Stuse ates sseete ant Getcha wees Ad Divan aiee tip eccnae 3. Relation to other agents, such as:................. desiccation, light, disinfectants, etc.:......... 4. Pigment: production tis sc crweiids 2 seoctoneis aoe anaiy on adeve ¢ 5. Gas production in glucose media: i SNAKE CUI TCL aio se ytd atow Peta ea Aw tis Mysto Guest vais vate Melaka eee wn by daul eae RE open ca ek OSS 6. Fermentation tube, growth in: (t) openarm:......... (3) (4) 6. Acid or alkali production, litmus milk. ...... 7. Reduction of nitrates; to nitrites.... 8. Indol production; 24 hours................ fecal odor; 24 hours... g. Enzyme production: proteolytic................. to. Characteristic odor... scckca2ns2 Din PAthOgemesis; ocreenscinne uncer eau vamos: rate of development: 24 hours................ per cent., 48 hours............percent., 72 hours......... Be Set (GO MOUTSH Ain sendadae Renton eter cee ene Con eae eye Se aE tk geese Ov UMOUIS HW ssster ih tes Desde aycaeed weeine deaeeuae Sie tadetay seeks 2) closed) “animes ctasnsca tins Aske seg iioty tid ) aia el selec ap momenta wees ee ea aan ee vieeo..-.(5) gasformula, H:CO.:: ecceaigs TOMATO Marea netics cy con aya agate marcia wid a Seared dose ciauaatee aioli om ace aaa oA emne oTe ener ACTASCALICS sm ease akon a eseis DER COM si: atratinvrs sa ween hours. .i¢ccce0 per cent. : ayaa Mee ciel eae eee CULTURE CHARACTERS. Reaction of Medium, Incubation -|Temp. (°C) 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. (7) Special Media, oe [185] EXERCISE XCVIII. MICROSPIRA FINKLERI Schroeter. SPRILLUM 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. SKETCHES, Cultures. Incubation temp. (°C.) Age of MORPHOLOGICAL CHARACTERS. 1. Form: Gs Bouillon awausisevameide do ae see swine eeaaaieR. Sak Baws SEARING ee apaieetn de aa” Sean Mueke Wey Roane aeamas Enodis pene eaemaaeal seat Mes sautececces SING ainsi sds cy EA eRe teats Hise see gee yal mete SE ea eae onscieae Ata eae ota oe centcler te ates eo shctctae STO RAC PSTN STEM arcana Ne tee eo eeaehoc eas FOIE AG ae eas ae ries Bae aes ve sco eS a GS eee ae REL. ALY ELAN ad ER bo Eivneion Aaeoaie nis Bagh vba htaears. woe [BEER eee Meus ae Me UNCP WOU, wees aos cedn va se meuds un pacien te aa seas Wunee a ORME RG oN Ed EERE NERA » RAGA eRe Whe anane LARIEE wa Te pe Os Pee el eetA Hone wae Bet Z Ci sceecta nicer cone oe cin Seeds maya yas eA AED we Pa PED Wie ORNL ES dec aga aS os gE REIEIG 5 aa Stan AUD CIEE S Sy DEUS AL Ue Gibal ene Te wl Esbestet 3. \Cell roupine sis Wnts ei ace tecacc an uty suse F sce ae bie uate weve uated otek oe A tiecion alec eaih bt, Aeiey ces amelie metaun a aetetia datcaierve hap val avdaa bebe at eet ATG ATFANSEMENES 3 ye5 ios he taka ek vaca deey deve ee ones omusdod ate ptensteset vets ecard nl ot timeefeay tes -o fale Apalnldla vio urbe ma Ala boe arale ot iaie sel pene cese Seqleue Lae in growths............. pr naan miniidsaih WRG AIA St rg Analy eee SNA MOU MRL ee aU ROR eats ang wis 38 mn aleynen aah nu Gen rd aie weeeneays gene dd 20 eesti le Rete eer ayn [[aaiem wate ie SLAIN POW ELS Ss alee array vse cernrn FA sa atone ee ety ca aesases nee che a SG Ess Wo GER tae LamalEED Deuba thc Pualiash Leg bo bialh potbin 2 et ada tg eon eeneell Sia | I es 2 AQUEOUS: SENANAVIOLSE cada ciose Se oi etateyencesc causa oa co cstawc aia Sax css pots hee te ote avec eretorn ee omeedtiece minccsen lea arasteds sent cebe rsh cabocu on Shel uot meneesee bs Woefler!s:meth ylen=b] ie cases cisscsaratcr aries oss ex densa htse/e cle orvdels Se age ctae vce uaeomc tare el aeons ar ang ate anna aD es ee iva apace So | esomee arate GM GTAIN!S Stal; trois hctaucienlecos aalaectomisds awe cH reet as Be Sears ste Ruet ipsa Dacia antigenic ay Sloss cod OOSIEIRGG Du ceostaeeiouaTG oS SNORT DE AOE AH aR deat EE ROA teen ed Els SSP ECVal Steam S oie sere ecco id asters eb cates ns, zac eSapahod Bs RSU RES Aen SeceUNU SPEIER Te RS a PE zeus crear ene spease vent lleeebsle nella enelstees Cag) Koy a Ul Unenetena en a etesat See et ae AND RN dr Re eerie ne i eet orn oe Ree EET Ab A NAAT AAA SVN Aan eR Bre WRT AW rt] MIRREN EN Fen /AUA a. Character of movement .................... Rib eseishe Parnes 1 ta Saletan Ue PA ga han Ouledz ae pw NP NOR A Min Ra Nua RS ws, UAT AT ta ie neetotU rated leat b-Rlagellastaintcoiiila snegnen char games sigpmogs Marat aerate ganceanaes is, Nehyead 4 teiaitdim Gale ladieedethe eae sewed Meeieiote cee: 6, SPOTS! 4 asucesawaet eacdewe ay ak 7;.Specialscharacters :Suchyas? a. 2:c:mapiaisnaeina saetern ne ab Sh tie Gene Mi ae eT: gas eo ndenh ec oeinu aac ohekech Geposits;, VACUOlES Hs a0 Aue auew dueuerviag das yaw sane add sige asc cone ea ek orn arena pleomorphic and involution forms, capsules, etc ................. 00. PHYSIOLOGICAL CHARACTERS. 1. Relation to temperature:..... ...... 2:2 Relation to free Ox yeenthes 2g hoes Iwan okra Aue saeco te catia cher oats atsctt ia Rata alee ume eMac eer antec SANE Pe sg ne eas oe pe la ell Be Ca MEN adhe sul pasa 3. Relation to other agents, such as:............0.00.0.. 0.0.0... desiccation, light, disinfectants, etc:........ de JRIBRIENE prod UGtIOT ein nae gts te Drees 25, clans ajehAl oul helena lc esa ae plore aacseenctOlaeoe nip ma ants 5. Gas production in glucose media: Gs Shalkcevcul Cure ian wlan aia dare apatite Wes oe a sa uy mea WP A See Naya: Seo els Se aol ged aeiauce aie es eel : Sicon ah dasesa SACRE &. Fermentation tube, growthin: (1) openarm:................ Hntni diame Serge yee na(2) CIOSEM) AMM x ciaia-sn comes vsure (3) rate of development: 24 hours ......... .... per cent., 48 hours........... per cent., JANONTS. 152 ccaeee er wePEL COME. «cnc caseate saat hours............per cent. Ca TOROTIOE I BPCN MEN on cu tea ah yieelng oy ov ears he wace waa an Gk patel mmc eho (5) gas formula, H:COe:: 6. Acid or alkali production, litmus milk...... 7. Reduction of nitrates; to nitrites .... SCRE AE ean Ae dat a STB AS Te ah ee LE Bane tus meets: GEO AIM ON Latins ied fos wants wa hoes Acute oa anes 8. Indol production; 24 hours................0... ae Smeets ndol p 4 to AS ROUT Se csisseyirys sat cuca ede inewecng alent e staan ap iin can. 4 Hanis ceca tee AVS sppdatna drvalus. vaten nowt wvnces ies a feca] odor; 24 hours ............ Asses BA Sees 4 s cto HOMNOUESH? sem sptcete ea giainnc das tan oue sciniyniee cau ndte, deych, Si a beinele ae dx GAYS che 44 nes osaneenekataues eee 9. Enzyme production: proteolytic............... Meeesecr asin Memb ea mS se couse mite. 22 CUASTAIC) inv awueg unis wasn aunartak wae ia dayaa tne wmean naventrimstan wah newest nate 10,. Characteristic odor: sc.0ci00 04 si gewaeeaaead ex 11. Pathogenesis.................. [136] CULTURE CHARACTERS. Reaction er bes la Q4....45 Hours. AB cexpecines Hours. Bid.oaes 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. kod , (4) / Agar H Streak. (5) / a ox Potato. (6) Bouillon. (7) Special Media. [137] Naine of organism SOurCE, “habitat ete? cacy cena: wae eels cates es RR ee ea Rare cata PE nee eet Sphan Lee oR RELETON GCG s e mrcemntnytiiag tice ae once a. i evs afew a aS Se Ran eee ge Jed humdi Re caesar ter se euioneue beeen a | & = sate On 5a SKETCHES, tr os A zs ~ me 3 3 a MORPHOLOGICAL CHARACTERS. &5 | gs A na 1. Form: a. Bouillon........ BSD eb eee bad het esutee ek eth eee Seed ae A Se GAN ng eye Sacer cto Na le [avs nce esr aoe ae ate Obs ANGE ABS Aoconsse a0 fa hanahestiat eshte anc pon theca Bache AVP Screed ped as ey PIA I alder one saleke Aha bakes ge extend cha needle et eects ae Cie GLAND hd AAtynee Woh Rah WTAE LR Dea At hE GO ER eae ONE Syne NO eA bE Saget N a keke SEEN area eta e assem SG ADCS Ge AR re IEE | GE AE SS a SOE IVT COLT Ase, reals aetna oe if MPSA Desehiea a ares tc estas aarp Pascoe errant batole leloyes AA Fas ade eenctts a/b: ap ua ees Sacynaaperaintagm ones sk aeeh aaa raas ctor | wee easy sad Ne S's | Be | SV A Cr ssay sie Sigsu Wargo eee Oe Eat RON TRO EAR SG eR Li Seuss hse ead eect a GaN P RI a SUN CECA NOE Sete pePR. BV tee a rales een Mit seat dna Seni ts Se orale | 32 Celli PLOUPINGSa sy pacce ah any at waael Al wit dar ah UEd em seeamNe ree cleo sues Marin ant apa Rte PUTED Gane ee i ewe sepanaaatyi el Eee neato Oecd Mass ooo ANNA SA PHAM PCTS MES a aacteacscaniriawetee KAM Gar caetsoesta con eberae as clecedzes neha ets Guna onthe aes Waseetc hata aon See been Seis hed es see ater aye sda et scaoa neha ana | eee seats | ea gusto TNS BTOWENS Soaenntcii eaten la sts. ashen las mast ROhtas see seralin Senge Wk). Aaa PE Sea ernde encanta wei ed Syan ee Monriaean oA atr-capmd se nme Melle uve: @ oe | eeeenente ee FAMINE OWE TS ites gas caeoe ie ae eaves eva teams se BEG Arata PREIS ae ees es Sah & cote Hy Wig Sapeenes I Ra CAA Setar ese llevan din ll cearten wal ayih il Sata Bs | Gi AGU COUS © ENILIAN IGE bert ah SNS toe ae RS Eee SileinG tenia Mc Ratt Geiger eA Nita Matte seks ELL AMEE aA ANAM Sieg 4 salar th tll lated hecnnecte | neetiaiats G6; soe Mer s:me tyler abies nera-s.caye terres ase Se seed serves seeyecsaecee aa a weaves tae eta pete iA Si IS gata ate anak chgtkivee coe | Mea derencte ase | feoestenctone G5 HG WAT SESUA Diet tee. aa searhetnceee tat Races ee pees ate baa fad Winsiinsal Pi abewoel te, as vcedestoe ad aba nbn Pee ellaimitte aa as Utne eee aed imal de dis eed cetshs appre ist ace a ans waco lbs weg eee a | id. Special StalnStseveasa sass pee evah ee ed. Kio Ree NEAR ES as uae oe eee a eahia sy wis alas eet tae soe ees eee ee | BVO ERLE Y 22 icici metoene es ota Sa cia Ee Eada ak ak amie nel aas De RERUNS SE SDR TT OG Sah Some hadoe ALAIN Lis MA nis anata bmellts | aaiGharactemofmovemient:cansiccime ant vaciath ob tarioty eae iid &. Flagella stain ........... Os SDOLES isg ceria te sarin oa a Mlertioae Ac etiam lntienie sit emai iantalm nor cianehaimsiehg ots odaas Teoma aan aoe Seman ex pee ener as SES EGE ab a ene ee BER EE Ghee RA URE TSG Hie Sahat Siete oa calmed ioeomieon secon fides [Geert : 7. Special characters, such as:............ EPOSIES VACUOIES oa Nine 2 suice S60 A cele ebes pant gomenh Brag OE Ae taG as 6 pleomorphic and involution forms, capsules.ete . .. ...scsce eves seas cada ben sede even geek ouUydubyueeouteacas PHYSIOLOGICAL CHARACTERS. t. Relation to temperature:............. 2. Relation to free oxygen: 3. Relation to other agents, such as:..............-0000. 000s desiccation, light, disintectants, etc.:..........0....00.. 4.. Pigment: productiontiicoc2s7e20s tae Reehei ne cre cans 5. Gas production in glucose media: CoS Wwallcere ail bn tetas tera is cent detent sas eens MG Meade Se trates yh eae eten Beer ne ee thee Ca ae ad A &. Fermentation tube, growthin: (1) openarm:........ -seeeeeeee. (2) closed arm: (3) rate of development: 24 hours........ Lo ae PETEeN EY AB ar Be err NG Sirs SL 3 p ent., 48 hours........ vas pemeent., 72 WOUTS!: ¥. 4.6400 Per Cents, osc-sccas oy HOWTS os ¥y vere wee per cea. 6. NI (4) reaction in open arm: Acid or alkali production, litmus milk .... Reduction of nitrates; to nitrites .......... i Indol productions -2¢ NOUS «: sass ns eases veeearanties ox ve feca] odor; 24 hours .... Enzyme production: proteolytic. Characteristic odor. Pathogenesis . -....-..(5) gas formula, H : COo:: .., to ammonia ., Agden, a CHaS PatiCh Geddy ae ee os aan eicind ae CULTURE CHARACTERS. Reaction of Medium, Incubation Temp. (°C) 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) (6) 3ouillon. (7) “special - Media. [139] Name of organism.............00.00 006: est bennernics Sources habitat. ievGs ss x cle vaeeta teavs coe de eho BE gaan TR tu ae eam He ees Renee ee ne ar ir yt ny MorPHOLOGICAL CHARACTERS. 7) o Q 62 o> ie) x Incubation temp. (°C.) SKETCHES. 1. Form: i, Boul VOM soi. bct cave ose oe ee eee gsr Sy USF ADR GP ERE NOE ren TREE tS, SSRIGPA GTA OSS 6. Agar . 6 Gelatin: cesar a: Other wmediar. vi: cascutaiewnes a's Bo SEl MSO UuplnpeSixs sso Gaeageoia tue are educa ud wi eanstseionans aude helen One and arrangements................5- in growths...... 4. Staining powers: . a, Aqueous gentian-violet......... See re eens 6. Loeffler’s methylen-blue..................... Gs, GUAM’ S Sta nai. mcs re ees da. Special stains me MOLLY, cards eect w a, Character of movement ...............- O63 Plag@ella: Stain inc-cxs yeas secs fate Sone) a » SPOTeS: 6.00 55 ed BUNGE Hie Special characters, such as:.. N deposits, vacuoless ic neces cee eae pleomorphic and involution forms, capsules, etc. PHYSIOLOGICAL CHARACTERS. 1, Relation to temperature t............. 06.0055 desiccation, light, disinfectants, etc.:.............. 4. Pigment-prodiiction tics yeesics anes wee 5. Gas production in glucose media: a. Shake culture . 6. Fermentation tube, growth in: (1) open arm: (3) rate of development: 24 hours .... (,) reaction in open arm:..... 6. Acid or alkali production, litmus milk. Reduction of nitrates; to nitrites........ BOUIOMES ss seate -saotslcbainds vet was its paken Gia aed cae lacke te cecion mete oe Nan ted nena ah dens Indl ai. cad Warnes aula hit rare idea ora amanda satel seipencaes | seared Dr EAA Seats ie COM a Nh ae says Hee Sad eit eteccek det a ctety lasts ade hee A nase Sete ake ae en at nto sneer tae | Aes [see e ae Gs Gellatk tay: areata vidas ena te sate ne Sse nes aes eel ia Hata es ats oR RAL ohaserate EES oe UE RU ee He A PEs oe Ma iste led artista ell oediiet ato d. Other media . Dy SN Cress specriesserisat a nackte 3. Cell groupings and arrangements............. in growths. .........0.. 4. Staining powerst.iiccccccws se cewen ees a. b. Aqueous gentian-violet c. d. Special stains . 5. Motility:. a. Character of movement..................... 6. Flagella stain. ........ 6,, SPOTS os ase aes 7 = SPCC lal CHATACLERS) SUC) ASH teri iuclven Ria inckhivan ee 3a Haoceertcntwesie ae sactinns os ope ia aman erenag ot vet cabcok duvtaeat Geeta deposits, vacuoles pleomorphic and involution forms, capsules, ete . TsoettlerShime thy len=blue.acnas. deen eaves syste ci omceted comove spel oaersenal ame eden GeraMS 3S Calle eye ae arcseeasgteenietctearomacen die SKETCHES, PHYSIOLOGICAL CHARACTERS. 1. Relation to temperature:..... a 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.... 6. Fermentation tube, growthin: (1) open arm:..... .....(2) closed arm: ( (4) 3) 6, Reduction of nitrates; to nitrites .... N Indo] production; 24 hours. oo fecal odor; 24 hours . Enzyme production: proteolytic... 10, Characteristic odor.. 11. Pathogenesis rate of development: 24 hours reaction IM OPEN ALME...c ie ceneeee t +. percent., 48 hours........... percent., 72 hours......... ...percent., ................,hours .-(5) gas formula, H:COe:: Acid or alkali production, litmus milk SAS MOMTSYsc2 cues ox tiee ones sei MB OUTS srves thks At ein’ weresece nelaunclon aeteth minis cece eeveee Per cent CULTURE CHARACTERS. Reaction of Medium, Incubation Temp. (°C) 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, (7) Special. Media. [145] NaING OL OTPANISM 245525) nos Adee aaeeceenn Bae ae ee Ag eee ean ae SG ER ane piers Get nln oie gn Greener ca Ee nla Aaa : Souree; habitat et@scc. dete iad eS ale ae ac eda tise oR a hae a IR ae vee eget caren BP SSS ag le tae onal Cae enters eee oes Ref6reniGess a scniile ead 8 aeee eee Sea ORGS a Se REAGAN SY Ene ew GiWAS eee Gina ade poets Be Faye REN RRR AY fe APO TE rt pore LER Ea TEs Incubation temp. (°C.) SKETCHES, 1. Form: Gx, BOUIN OM gate ra vet eatatntoc.y ta Paha é; Gelatin ison v5 ys esas vi GAOT ET MVEA ag 22 2A hee nie desis be Fadel Bede BE SUZOe acres ate sysvahesmiess i rinter sake oie 3. Cell: rOUPiNgS; escese weayeseaerecddad oa na neeatncne: orem anne meme Bese we and arrangements ........ TIO TOWENS yue-3 peg Batigenanuerena mat Aoeneenl 4. Staining powers: . a. Aqueous gentian-violet b,. Loefiler’s methylén- blues. i454 cee se ve es os Qaedine ee eedd es aw inamuaye ge eeen ge oe ses c. Gram’s Stain............ id; Special StainSss.cs vs seremcas Ha Anant aot tea aie 65 MOC Nyy cee dake ys ne fonds vavwieds e+ es natinwapaccie ania ta vera a. Character ohinovemen teased: ui teers kine ve ph eenalg Me anette fa the at sieutla lens Hela asiviere pW ala Rais ahs ometa aes aye eats a Aiayepa saan Apasts Be We ella Stay 5 daca xi Vesey a Soave eam 9S a Ro a CE ay ve a ad a on ge Gate sa aes SE KS Se GAS POLES: warnctaoacraeetisien as wien esta Soe Saertacnan Le a nome Abas telnet Loe lear ot mietais paateaita aera G seem e ee eae 4, Special chatacters, SUCH aS? «<2... 2.c crass esa mene es oe sais GOpORe, VACROIES «jc cesnv ss wae Kull g a5 SaeeR Ye Hea wh Pade ts De Ae M4 ved genes eae peL ee eg La Ye weed Seu es oN 14 pleomorphic and involution forms, capsules, etc............ PHYSIOLOGICAL CHARACTERS. 1. Relation to temperature:............. 2. Relation to free oxygen: ........... 3. Relation to other agents, such a$?.... 0.0. 0s00se0% desiccation, light, disinfectants, etc.:...... 4. Pigment production:...............-. 5. Gas production in glucose media: a; Shakerculturesatisc heshecces toed (1) 24 HOUTS:s sie eces 6. Fermentation tube, growth in: (3) (4) rate of development: 6. Acid or alkali production, litmus milk ............ 60... .00 2 eee eee eee 7. Reduction of nitrates; to nitrites .............. 8. Indol production; 24 hours.......... 0... ..00 sees fecal odow:24: HOURS an.4 522. ceaeet eae wee ee ale 2 9. Enzyme production: proteolytic........ 2.65.02. - te eee ete eee Characteristic odor....... Pathogenesis .... OPENGAMUE cme maeeGee Loans .., 48 hours........ .... per cent., 48 hours............percent., 72 hours reaction im Open ALN: os vewrovsion Hot ectenins a eemygriee deca chem eRe eE He nap SAR OMITS Sieaa nainctie me ge as atte RRMA and ose oe ne hale Rata (2), | ChOSE GI AMUN hn dnivenicmn sehen sc aie eea deutch per cent., tise veneS) gas formula, H : COs: : per cent. a sys tOMATMIMMI OMIA a ee nhs os ae ye ewe wheres seen PsA Si ine neds We sads saneweGees Male ORS osc elocask vosexs dees eee vow o'Glagtatic:«.. CULTURE CHARACTERS. | Reaction of Medium, 24 Incubation = Temp. (°C) saaieys Hoors. 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. (4) PA NN \ \ Agar Streak. q 5) . : * / a eN Potato. (6) Bouillon. (7) Special Media. [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 zine. 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 8rd. 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 ce. of air space, the tubes are put in place, then about 10 ce. 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 mii a ii 1892, 3; 81; Welch & Flexner: Jour. Exp. Med., 1896, 1;5; A. 140; M. & W. 173; McF. 463; P. 545; S. 781. SKETCHES. Incubation temp. (@C.) MoRPHOLOGICAL CHARACTERS. BO 1. Form: @. Bowillon Juccsscxvsese vac Repent biuret sh ea cays deren antida wate ea Seandnared ontatoteen he Semen eee aInnaaihs eek temiaisiaies | smc] deleWeeter Be COU POU B IBS isis a eadinisiind sob asatsind Maio wissser a te wielens@u ds 4 dhadra Sein radioisaalbGe yanis gega He OEMS a woe Meeeb ta Bik oa kines BH APPANBOMEDIE cc picn see gaeens dacs Meena rawdied eres uel shed rendoda ee ee tke aparewad anaes ei epde PERE ED MORE WON ATO WES Sc cree sid aassassians Vasvsuisenelns acre ceereidraaghtnacta Hie by ameeaesion mendes MERE REG eins WARTS OE NG Aaah alayeaeaananl HE dewenee e erausreners @ Staining Powers: 2s eccers dacmecin cs oe Seas cane do LISkSag be Hewes aot sasbs de MacwleMilad se eaun Heine Menard Tee mia ie Se dateieptle meio i @, AQUEOUS FON EAT =VIOLEE ij cc nie a eb aigniaes ealeiaitiasaininie oo ie leis cde nae seldn inde heele As TR MANES 86 be Ree eet pu getgtne rly sem &. Loeffler’s methylen-blue ) Gran’ s Stains sieisigitzetacioetyavisie Se wa ccliie ders anctapss usdig is easrsiais otsisied ve ible a aise wie RE iets SE Re wvemMae aaded ele Miasie wreeteuesiaten] sie! Geese ffeateleyens ee @: Special Stains: ve gisns ponacoe vs te ores ceva vaeean bene FAIRE RDO RR ET Gren errr rere eee ee ete ib der aed secant lb Wantawece Bie MGCL ty Sas sisaccsinc ue ub side wosded ganacnd Ue BE ht eine aes ow ie DERRAS Sakae amas Neke wote Heoies are ee oS A eR eRe Ed eeR ve eames [ae oe chee seeds Gi Character of MNOVEMENE souls Nes2 Gi dh dane neGaawdyad Anaedee Suva syy waves wealnetora WE8G: bdfahe Ghesadubscicuduelaey dendpersaltienasert haces Sie Mek Gee a [SOLER So Mesa b-. Plagella'stainsescac wacicir axa deaiesiaias peeians ie aida camer oa reine Ge ENA OG TL EAN Betex FORCE CRU IT, | Dee Sl Net er Ox SPOreSiccein cansmes sa teeter wan prelialenas vig careaieeiid Mesiemad ae sel nels@g hase aE Daletesld oh Hpae wlNNes 9.9 vaeretha Ne Se SideareetREe avdalesie ats SaaeR eal alee Agia Sse RAMS as Spectal CHATACTETS, SUCGHCAS! 4.41 Hadacaucdd te teditaaneseddenguuad es ii ei GM BRS ila, Ge ea eb a AG HER ALD DAES ALB DETR O eae bebe HAE Sasa déposits, vacnolesiseciccsncsac: — wins eswtiia erin tates Sa ea Sean heed Seb neonate a4 Mate ae maecde bh uboh eadwen GA a ambainel ke aeames lesaceune ‘pleomorphic and dnvolution forms; capsulesy, eteisuiinss sic ciize xiarcisie dare aivisiesustais viaiate wicca cia semiereiaie BHC aiclige wictatha atin wiaickgiasied eaten ages [ls08 Pplenite PHYSIOLOGICAL CHARACTERS. 1. Relation to temperature : Sees joka Sen tha hdd els slop a tae py “a ana “a i dear aphdd Tua PddUASAE (Sha Sve GiRNEin Cea Se avoia raean ose Maca AatS EINE <45 GAG BlecaLR 4H HAGE RARE REINS ahaa DIR REIS. ci-Re Ta Menmaeee SoeaiAO 5. Gas production in glucose media: @. Shakesculture ci sisise se wets aeawsetitad sed wiped sadaietensitins ve nwueeSiarein dimen aitiby va ae Geena NEUTUle Rha wine se. aitlad He Ga sed edna gS ean’ visas by Wale banSard naan asain eH . b, Fermentation tube, growth in: (1) open arm: (3) rate of development: 24 hours.............-.. (4) reaction I OPE AP. coc sne nice coed erinwe tops besos on oe me Nena meReuMBE ae nee, 6. Acid or alkali production, litmus milk. 7, Réduction of nitrates: to nitrites .:ccs.2 2x2 sexy ca ve eamep Ree eee easy Ss de cnet eenaonaed vee . &. Indel production; ap hourSicsansee ca oc vows senraee se oe tent fecal Odor 24. hOUTS 3:23 sscsiecce ve aoven aieays eae aes g. Enzyme production: proteolytic.............. Side auletaa 24 Migeae BY te ve Sut hesien nod oe aicdde i CULTURE CHARACTERS. Reaction ‘ of Medium, : Incubation 24 Moe Hours, 48...... Hours. 6...... 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. (4) AN AN / Agar / _ Streak, ( (5) Potato. (6) Bouillon. (7) Special Media, , bh [151] EXERCISE C. BACILLUS CHAUVAEI Arloing, Cornevin and Thomas. “BACILLUS OF SYMPTOMATIO 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 symptomatic anthrax. REFERENCES. Arloing, Cornevin and Thomas; Le Charbon symptomatique du baeuf, 2nd edit. Paris, 1887; A. 482; H. 304; McF 453; P. 563; S. 493. : | ary Sf | 64 SKETCHES, MorpHoLocicaL CHARACTERS. 85 | 38 i. Form: a. BO ti gcanse ni actos swiocan vees eadee en aled, Gwe Weed RevadGRiGR OUT) txaRwn SAgdslad Mer weed eur weNs TA RANSY CLAN IG WER SOON eee eet eres Ds BAG sili sitiallin Maks CE ewidin a Leip an RRA RA TD Gm in ERNE SEAR HAA MERRY VO ROENS FG A EER ANS RRR SR ES ENE REM REE a ss ateaie ne HIER BIN [8 eS 6 GOVAN 6 oak ey cesin de ceine id Sau ois Sunaina doawRa ene U MAG DER blew Seas na APTA Riad HD. 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Special: stains ¢ sox sa sdaics aisles Ysictesiawsisiwis sseratsns w stevciaivaate via ila ecensinsicdate arasaial aed BN Awa ata UAROLE ne Nea leimay bd domsiole: abies inioneadstIADe |i La 4 eleledes[ niereitrels oe §> Motility tssc.ues ie v5 cies ogee raneanies wis ik dae AGs Sey ata pe nh See eG wins cee Mee 55-45 cieeatehaR ion acuedion are Ne NaIelN BiRlanaialeai Bia Sietwakemcinaiehen [Os Wes team] aieiaiolns a, Character of movement ..............0..00...e0 eee 4 Wad neenyeue wasn eS omo asi schdetwen nie MDS RENN ay emMommi nade oy ay geen (nine gsis b: Wlagella stain: cocesnsccatenaaei aedeacae eae eeuh aide Ger a Addie head Sean Senne whe aame Re iy shag raid ints ayoats vk aecbae attoonenerenvel| coy gate I meade nt Bs G: SSPOTEs is: coveesce see yeree guiemie ds &. sts) he ace Sargstwnsiainss geietene is’ o aChva aubtardatasayshartoslsvavetal iarbye a alae clemstarw: webualga wee Sey saat otrtoeg eranaih aie obit ceeetibatetlt [ve aly aayeilla dardone tod a yf, Betas KH APARI ATS, BUR WANE ye casonaes 14: bs ween txuadadawes He aL Reua ae Wee He badd Nom wees Waed ois Maa aeewublig Gang aedyseids | yy ey ue. Mebamtely ee Meposits, VACUOLES PAathOgetesis .iiisiecanasicrcrsawsancaane wick wes tcae mcahtelanials og aneieharentanh ceenaeN ee Maat CULTURE CHARACTERS. Reaction of Medium, Incubation Temp. (°C) SKETCHES. (1) ~ Gelatin ~ plate: (a) Surface R Colonies. (b) Deep ~ Colonies. (2) Agar plate: * (a) Surface Colonies. (b) Deep Colonies. (3) Gelatin Stab. (4) . ie Streak. A Bouillon. 4 a j \ A \ | SS oo [161] . CHAPTER VIII. ANIMAL INOCULATION AND STAINING OF BACTERIA IN TISSUE. EXERCISE Cill. ANISLAL INOCULATION. MeEtTHops oF INOCULATION. Animal inoculation is practiced to determine the path- ogenic properties of an organism and also the character of the tissue changes produced. The animals commonly used are white mice and rats, rabbits, guinea pigs and pigeons. Inoculations are usually made intraperitoneally, intravenously or subcutaneously, and in special cases into the pleural cavity, brain, eye, etc., etc. Mice require a holder, the inoculation being made at the root of the tail. Other animals can usually be held by an assistant. : Subcutaneous. The place selected is usually the abdominal wall. Pigeons are inoculated in the pectoral muscles; the hair or feathers should be removed and the skin washed with a disinfectant, e. g., 5% carbolic acid. a. For liquids a sterilized hypodermic syringe is used. A fold of the skin is raised, the needle of the syringe inserted and the requisite amount of culture injected. b. For solid material a pocket is made which is stitched, or sealed with contractile eollodion, after the material is introduced. Intraperitoneal. Prepare seat of inoculation as above, then plunge needle directly into the peritoneal cavity. Intravenous. A rabbit is generally chosen for this purpose and the inoculation made into the dorsal vein of the ear. Slight pressure at the base of the ear will render the vein more prominent. Avoid the introduction of air, which causes immediate death, and keep the animals under close observation for one hour. Inoculation into Lymphatic system. Fluid cultures or suspensions of bacteria can be injected into the lymphatics by way of the testicles, by plunging the point of the needle into the substance of the testicle and injecting the desired amount of fluid. Inoculation into the Pleural Cavity. Where necessary the needle is introduced into the pleural cavity between the ribs. It is very difficult to perform this experiment with- out injuring the lung. Inoculation into the Anterior Chamber of the eye. Rarely practiced. The eye is treated with a few drops of cocaine (2% solution) and then the needle is inserted through the cornea just in front of its junction with the sclerotic, the needle passing into the anterior chamber in a plane parallel to the plane of the iris. . The following inoculations are those most frequently made: Streptococcus pyogenes. Mice or rabbits, intravenous. Sarcina tetragena. Guinea pigs and white mice, subcutaneous. Bacterium anthracis. Guinea pigs or rabbits, subcutaneous. —— pneumoniae. Rabbits and mice, subcutaneous. —— pneumonicum. Mice and young rats, intraperitoneal. tuberculosis. Guinea pigs, rabbits and field mice, any method of inoculation will produce the disease. 164 Medical Bacteriology. B. mallei. Male guinea pigs, infection of lymphatics. —— diphtheriae. Guinea pigs, rabbits and fowl, subcutaneous and intratracheal. Bacillus pestis. Rats, mice, guinea pigs and rabbits, subcutaneous. — suipestifer. Rabbits and mice,- subcutaneous. STERILIZATION OF INSTRUMENTS. These are best sterilized by boiling in a solution of soda or borax for 15 minutes. This is accomplished in an especially designed ap- paratus or in an ordinary enamel stew pan. In case of emergencies the instruments may be dipped in benzene or alcohol and burned. This is less injuriousto the instru- ment than heating in the direct flame. : Use blank, p. 168, for preservation of data. OBSERVATION OF INOCULATED ANIMALS. After inoculation the animals should be placed in separate cages, or if placed together they must be described or marked so as to be easily identified. They must also be kept under constant observation and the following conditions noted: Temperature. Loss of Weight. Peculiar position in cage. Loss of appetite. Condition of the coat or hair. Condition of the secretion of the air passages, conjunctiva and kidneys; diarrhea or hemorrhage from the bowels. 7. The condition of the seat of inoculation. ‘ The animals should be fed regularly, weighed at the same hour each day and the temperature taken at the rectum. Post Mortem EXAMINATION. Perform the autopsy as soon as possible after death. When delay cannot be avoided, place the animal in the ice-chest until such time as is convenient. OaRowe A. 1. Inspect externally and note presence and character of any lesion. 2. Sterilize a suitable post-mortem board with corrosive sublimate solution, 1 to 1000, place the animal belly upwards and tack the four legs fast to the board. 3. Wash the surface of the thorax and abdomen with corrosive sublimate solu- tion, make an incision through the skin at the pubis, introducing one blade of the scis- sors, and extend the incision as far as the chin. 4. Carefully dissect the skin away from the abdomen, thorax, axillary, inguinal, and cervical regions, and fore and hind legs, and pin it to the board as far as possible from the thorax and abdomen. It is from the skin that the chances of contamination are greatest. . B. All incisions from now on are made with sterilized instruments. 1. Take an ordinary potato-knife, heat it quite hot, and place it on the abdomen in the region of the linea alba until the fascia begins to burn; the knife is then held trans- versely to this line over the center of the abdomen, making two sterilized tracks through which the abdomen may be opened by crucial incisions: two burned lines are also made along the sides of the thorax. 166 Medical Bacteriology. 2. Make a central longitudinal incision from the sternum to the genitalia with sterile scissors, the abdominal wall being held up with sterilized forceps, or a hook to prevent the viscera being injured. A transverse incision is made in a similar man- ner. Cut through the ribs with strong sterilized scissors along the sterilized tracks on the sides of the thorax, when the whole anterior wall of the thorax is easily lifted and entirely removed by severing the diaphragm connections. 3. When the thoracic and abdominal cavities are fully exposed, a careful examina- tion of the organs and surroundings is made without disturbing them. Culture plates (Petri-dish) or roll cultures are prepared from the blood, liver, spleen, . kidneys, and any exudates present. The method is as follows: (1) Heat a scalpel and scorch a small surface of the pen from which the cultures are to be made. (2) Heat the scalpel again and penetrate the capsule of the organ with the point, and through the opening insert a stout sterilized platinum loop, push it into the tissues, twist around, and obtain enough material from the center of the organ to make the culture. Cultures from blood are usually made from one of the dart cavities, the surface being seared with a hot knife before opening. As soon as the culture material is ob- tained, cover-glass specimens are prepared from each organ and existing exudates. Small pieces of each organ are also preserved for future examination. When the autopsy is finished the remainder of the animal should be burned and the instruments should be sterilized. Wash the post-mortem board with sublimate solution. The cover-glasses and other material likely to contain infectious matter must also be sterilized when of no further use. Cultures are to be incubated at 38° C., growth examined microscopically, and by means of sub-cultures. REFERENCES. The above is } taken largely from Bowhill, 74; see also A. 219; N. 260; and other texts. 168 Medical Bacteriology. BLANK FOR ANIMAL EXPERIMENTS. ATVI 555 cs pains ioe Socio NOK a eal ee ees ABE oo eececcetecees Weight... Specimens received .o...0... occ cceccceccevecceececececscuesecteceeueevseetsesttgeteseenss o’elock.......... MM. Organs Experiment: STV eA 5 5 pa cya eed aca casas de a le en Sin esc mane NIE easy M. ARES cca a apn Dameaee Gs We esd aaeo sea iad bees dawie eda oa Sry gO ONTO, oa IML Findings: Bacteriological Examination: Histological Examination: neo adiaatedabapubaben new 170 Medical Bacteriology. EXERCISE CIV. PREPARATION OF TISSUE FOR EXAMINATION. Portions of the diseased tissue, removed at autopsy, should be cut into cubes hav- ing edges about 5 mm. long and treated as follows: 1). Frxme. Use 15 or 20 times their volume of 95% alcohol for 24 hrs. The speci- mens should be placed on cotton to keep them near the top and the alcohol changed atter 3 or 4 hours, if they are not to be sectioned immediately carry to 80% alcohol. Where larger sections are desired they should be left a longer time in the alcohol. 2). PREPARATION FOR SECTIONING. A. Paraffin Method. | a. Absolute Alcohol 6-24 hours. | _b. Xylene 6-24 hours. | c. Paraffin melting at 50°C. and kept in anoven or water- bath at a temperature a few de- grees above the melting point of the paraffin. | d. Embed. Pour’ melted paraffin into a paper box or other suitable receptacle and with warm forceps, arrange block of tissue in proper position and coolrapidly by plunging into cold water. | B. Celloidin Method. | a. Mixture of ether and absolute alcohol (equal parts) 24 hours. | b. Thin celloidin (about 6%) 24 hours to several weeks. | c. Thick celloidin (about 12%) 24 hours to several weeks. | d. Remove block of | tissue to a piece of wood fiber covered with ‘‘thick’’ celloidin, orient, dry a few minutes in air then place in 80% alcohol for 6-24 hours. | Cc. Freezing Method. a. Place in 1% Formalin 2 hours. | b. Place tissue on plate of freezing microtome in water or better first soak tissue- in a syrupy solution of gum-arabic and moisten plate with same before freezing. 8). SECTIONING. Cut sections from 10-12 » thick. 4). MANIPULATION OF SECTIONS. a. Celloidin sections can be preserved in 80 % alcohol and are best stained by placing the sections first in water and then in the stain. The various reagents are best used in watch glasses and the sections transferred from one to the other by means of a section lifter. b. Paraffin sections should be fixed to the slide or cover-glass as follows: A water- bath is heated up to a few degrees below the melting point of the paraffin, the sections are placed on the water where they will straighten out and are then transferred to the slide or more conveniently to the cover-glass by simply dipping the same into the water and drawing up the section by means of the fine point of a pair of forceps or a needle, draining off the water and drying the section in an incubator for a few hours. The sec- tions are more secure if the cover-glasses are first smeared with a thin coat of egg albumin. When the sections are once fixed to the cover the staining can be carried on in the forceps as with ordinary cover-glass preparations. Before staining, however, the paraffin must he removed; this is done with xylene and this in turn removed with absolute alcohol. REFERENCES. A. 173; M. & W. 204-239; N. 581. 172 Medical Bacteriology. EXERCISE CV. STAINING SECTIONS. GENERAL HISTOLOGICAL METHOD. Hematoxylin and Eosin. a. Transfer sections from alcohol to distilled water. ; db. Stain in alum-hematoxylin 2, 5 to 30 minutes. The stain may be prepared as follows (Boehmer): 1. Hematoxylon erystals, - - - - - - 1 gram. Absolute alcohol, - - - - - - - 10ce. 2. Alum, - - - - - - - - 20 grams. Distilled water, - - - - - - - 200 ec. Cover the solutions and allow them to stand over night. The next day mix them and allow the mixture to stand for one week in a wide-mouthed bottle lightly plugged with cotton. Then filter into a bottle provided with a good cork. The solution is now ready for use but its staining powers improve with age. c. Wash the sectionsin several changes of water until they have lost all traces of a red tint. d. Counter-stain with eosin (jy to + % in 60 % alcohol) 1 to 5 minutes. e. Alcohol, 95 %, two or three changes to dehydrate and remove excess of counter: stain. f. Clear in oil of origanum or Dunham’s mixture, white oil of thyme 4 parts, oil of cloves 1 part. GENERAL BACTERIOLOGICAL METHODS. A. Loeffler’s Universal Method. a. Take sections out of alcohol into Loeffler’s methvlen blue for 5-30 minutes. b. Decolorize in acetic acid (0. 1%) 10 to 20 seconds. c. Dehydrate in absolute alcohol, two or three changes, a few seconds. d. Clear in xylene. e. Mount in balsam. B. Weigert’s Method. a. From aleohol to Ehrlich’s waitin water gentian violet 5-15 minutes. b. Wash in 0. 6% salt solution. c. Dry with filter paper. d, Place in potassium iodide and iodine solution (iodine 1 part, potassium iodide 2 parts, water 100 parts). e. Dry with filter paper. J. Decolorize in a mixture of anilin oil 2 parts and xylene 1 part, 2-5 minutes. g. Clear in xylene. h. Mount in balsam. This stain can only be used with those organisms which take the Gram stain, namely: S. pyogenes, M. pyogenes, M. aureus, Sar. tetragena, B. anthracis, B. pneumoniae, B. rhusiopathiae, B. tuberculosis, B. leprae, B. diphtheriae, P. aeruginosa, B. Welchii, B. chauvaet, B. oedematis, B. tetani and Strevtothrix actinomyces. 174 Medical Bacteriology. SPECIAL BACTERIOLOGICAL MEtTHopS. Particular organisms may be stained as follows: Pyogenic micrococci. Loeffier’s or Weigert’s method. Micrococcus gonorrhoeae. Loeffler’s method gives the best results. Sarcina tetragena. Loeffler’s or Weigert’s method. Bacterium anthracis. Loeffler’s or Weigert’s method. Bacterium pneumoniae. Weigert’s method. Bacterium pneumonicum. The following method is recommended for staining the cap- sules in sections (M. & W.): a. Stain for 24 hours in the incubator in the following solution: Saturated alcoholic solution of gentian violet - - - - 50cc. Distilled water - - - a - . - - 100cc. Glacial acetic acid - - - - - - - 10ec. b. Wash out in 1% solution of acetic acid. c. Alcohol. d. Xylene. e. Canada balsam. Bacterium cuniculicida. Loeffler’s Method. Bacterium tuberculosis. a. Weigert’s method (staining with anilin oil gentian vale 24 hours at room temperature, or 2-8 hours at 40° C.). bp. Ziehl-Neelsen’s Method. 1. Stain with carbol-fuchsin (12-24 hrs. room temperature, 1-3 hrs. 40° C.) 2. Decolorize with nitric acid (10%) a few seconds and then with alcohol (60-90%) until color is nearly all extracted. 8. Counter-stain with methylen blue. 4. Dehydrate with absolute alcohol (a few seconds). 5. Clear with clove oil. 6. Xylene (and examine). 7. Mount in balsam. Bacterium leprae. This organism is stained with the tubercle stain, unless the sec- tions have been kept in alcohol for some time, in which case Weigert’s method can be employed. To differentiate this organism from B. tuberculosis, stain as follows: . An aqueous solution of fuchsin 6-7 minutes. Acid alcohol (nitric acid 1, aleohol 10) + minute. Wash in water. Counter-stain in a saturated aqueous solution of methylen blue. Alcohol. Xylene. . Balsam. The bacteria of leprosy stain readily by this method, tubercle bacteria do not. Bacterium mallei. Slow Method. a. Stain in Loeffler’s methylen blue 6-8 hours. b. Wash in distilled water. c. Tannic acid solution (10 %) 4-5 hours. a Ms aces 176 ‘Medical Bacteriology. d. Wash thoroughly in water. e. Dehydrate in absolute alcohol. jf. Clear in xylene and mount. Quick Method. . Stain in carbol-methylen blue 10-30 seconds. . Wash in distilled water. . Tannic acid solution (10 %) 4-1 minute. . Counter-stain with a weak solution of eosin until sections are red. Wash in water until pink. : Dehydrate in absolute alcohol. . Clear in xylene and mount. Bacterium diphtheriae. Loeffler’s or better Weigert’s method. Bacillus typhosus. . Loeffler’s methylen blue or carbol-fuchsin 15 min.—24 hrs. . Wash slightly in distilled water. Place in 10% solution of tannic acid for 10-60 min. . Dehydrate rapidly in alcohol. Clear in xylene. Examine. . Mount in balsam. Such sections examined under a low power will be found to contain heavily stained masses, which under a high power prove to be clumps of bacilli. Not infrequently the bacilli are difficult to detect in tissue from typhoid cadavers. Bacillus suipestifer. Loeffler’s method. Bacterium Welchii. Weigert’s and Loeffler’s methods. Bacillus chauvaei. Use Pfeiffler’s stain: a. Dilute carbol-fuchsin % hour. b. Absolute alcohol slightly acidutated with acetic acid until section is a reddish violet tint. : c. Xylene and examine. d. Mount in balsam. Bacillus oedematis. Pfeiffer’s stain. Streptothria actinomyces. . Ziehl’s carbol-fuchsin, 10 minutes. . Wash in distilled water. Picrie acid (cons. ale. solution). . Wash in distilled water. Wash in alcohol (50%). Dehydrate in absolute alcohol. . Clear in xylene. . Balsam. Tissue stained yellow, rays red. REFERENCES. M. & W. 2389-286; N. 537. ea MPrasae aeme aAaows FeMeracsws CHAPTER IX. BACTERIOLOGICAL DIAGNOSIS. EXERCISE CVI. EXAMINATION OF BUCCAL SECRETION. DEFINITION. The secretion of the mouth, or saliva, is a mixed product derived in part from the mucous glands within the mouth and also from the parotid, submax- illary, and sublingual glands. In disease the normal character of the different parts may vary or there may be various exudates and growths present. COLLECTION. Material for bacteriological examination is best obtained by means of a sterile probang or forceps. This material may be examined directly by means of cover-glass preparations or by means of cultures. 1. Method of Preparing Outfit. Wind a small piece of absorbent cotton on the end of a wire (about 1 mm. in diameter and 14 em. long). Thrust the other end of the wire through the cotton plug of a test-tube or fasten in a cork and sterilize at 150° C. for lhour. This with a tube of nutrient medium (usually Leoffler’s Blood serum) is placed in a box for transportation. 2. Method of Using Outfit. The patient is placed in a good light and the probang gently but firmly rubbed over the suspected area of the throat and then drawn gently over the surface of the medium, both tubes securely stoppered and the outfit sent to the laboratory. The organisms to be sought for are B. diphtheriae, the pyogenic cocci and Monilia candida. BacCTERIUM DIPHTHERIAE.. The presence of this germ in the mouth usually results in a formation of a pseudo- membrane a portion of which is to be removed with a pair of forceps or by means of the outfit described above. It should, 1) be examined directly for the ae bacillus by smearing on a cover-glass and staining by following methods: a. Loeffier’s methylen blue. b. Gram’s stain. ce. Neisser’s stain: a. 1 gram methylen blue dissolved in 20 ce. of alcohol (96%), is added to 950 cc. of distilled water and 50 ec. of glacial acetic acid; b. 2 grams of bismark brown dissolved in a liter of distilled water. Films are stained in a. 2 to 3 seconds, washed in water, stained in 0. 3 to 5 seconds, dried and mounted. 2) Usually, however, mere microscopical examination is not sufficient, and culture methods must be employed. In fact this method ought always to be used. In this case make smears on Loeffler’s blood serum and incubate them at 36-38° C. for 12-24 hours and then examine the growth in cover-glass preparations. The diphthe- ria organism if present should show: a. Characteristic appearance with Loeffler’s methylen blue. b. Positive Neisser stain. c. Positive Gram stain. 180 Medical Bacteriology. 3) Occasionally micro-organisms (pseudo-diphtheria bacilli among others) are met with that very closely resemble the Klebs—Loeffler bacillus and render a positive diag- nosis doubtful. In such eases attention to following table will be helpful: B. Diphtheriae B. pseudo-diphtheriae 1) Form 2) Size 3) Threads 4) Grouping 5) Involution forms 6) Motility 7) Stains a. Loeffler’s methylen blue b. Gram c. Neisser 8) Spores 9) Alkaline potato 10) Sugar agar and gelatin stab cultures 11) Neutral litmus milk 12) Anaerobic cultures in H 13) Nitroso-indol reaction 14) Inoculation experiments (Guinea pig subcutaneous) Slender and of same diameter throughout Average 1.2-2 Not formed Parallel grouping more or less characteristic but do not touch Common Immotile Stains readily giving banded or polar stain Positive Characteristic stain with very young cultures, six hours. Absent Growth almost invisible Full length of stab Acid reaction Grows well After 7 days Death 36-48 hours. Thicker at center than ends, plumper and shorter and less variable than B. diphtheriae Averaging 1-1.6 @ Not formed Parallel but lie closer together Rare Immotile Stains more regularly Polar stain rare Positive Not under 24 hours Absent Visible and cream colored in 2 days Only at upper part Alkaline reaction No growth After 21 days Non-pathogenic PyoGcEenic Micrococct. 1) Stained cover-glass preparations are examined and if micrococci are found make: | 2) Smear cultures, or better agar plate cultures and work up the colonies as they appear. Monta CANDIDA (Organism of Thrush). The material is collected by removing a portion of the patches or membrane and ex- amining it: 1) Under the microscope in a drop of glycerine. 2) Cover-glass preparations stained with carbol-fuchsin or Gram’s method. 3) By means of smear cultures on agar or blood serum, the resulting growth being examined either in glycerine mounts or stained cover-glass preparations. REFERENCES. EXERCISE CVII. v. J. 95; 8. 101. See also various texts under special organism. EXAMINATION OF SPUTUM. Definition. By this term is meant all of the material derived from the air passages by the act of coughing or hawking. For diagnostic purposes it is best collected in a salt- mouthed bottle (about 2 oz. capacity) which has been sterilized. The morning sputum is best and before being collected the mouth should be rinsed out with water. METHOD OF COLLECTION. 182 Medical Bacteriology. BACTERIUM TUBERCULOSIS. Place the sputum in a Petri dish over a black surface and select one of the little cheesy masses, if these are present, and smear it on a cover- glass. Where these particles are not present a loop or two of the thick portion is used. The cover-glass preparations are to be stained by one of the following methods: 1) Gabbett, see Part 1, p. 38. 2) Ziehl-Neelson: a. Carbol-fuchsin ten times through the flame. b. Nitric acid (30%) momentarily. c. Water. d. Aleohol (60%) until red color disappears. It may be necessary to immerse preparation in acid a second time, but the greatest care must be exercised to prevent extraction of dye from tubercle bacterium. é. Loeffler’s methylen blue, 1 minute. f. Mount and examine: ‘While the tubercle bacteria may be detected when present in considerable numbers with a } in. objective when there are few present a 7s in. oil immersion will be neces- sary, and this ought to be used to search all slides where the tubercle germ has not been _ found with a lower power. A mechanical stage is a great convenience in a systematic search. At least two preparations should be stained and thoroughly examined before a meg ative result is pronounced. The viscosity of sputa may be overcome and the bacteria concentrated where the num- ber is very small by 1) Ribbert’s method which consists in the addition of a 2% solution of caustic potash and boiling. This dissolves the mucus and the bacteria are then depos- ited with the sediment. This sediment can be obtained by allowing the mixture to stand in a conical glass vessel or more quickly by the use of a centrifuge. 2) Ham- mond’s method: 1. Add 5% of crystallized carbolie acid (in the case of sputum add 5 times its bulk of a 5% solution of carbolic acid). 2. Place 15 ec. in the tubes of a centrifuge and whirl for 15 minutes. 3. Pour off supernatant fluid and treat precipitate with 8 cc. of a 5% KOH solution. Mix thoroughly and allow to stand 2 minutes. 4. Fill to 15 ce. mark with distilled water and whirl 20 minutes. 5. Make cover-glass preparation of sediment (or purify same by repeated washings and centrifugalizations with distilled water). A centrifugal machine should be able to make at least 2,500 revolutions per minute. This speed ought to be maintained for 15 minutes. Sputum may be preserved by ad- dition of small quantity of carbolic acid (5%). Negative results are of positive diagnostic value only when repeated examinations are made of different samples taken at different times. BACTERIUM INFLUENZAE. This micro-organism is frequently present in enormous numbers (100 or more) and sometimes in almost pure cultures in the greenish purulent masses in the sputum. It stains readily with the ordinary dyes, and when lightly stained presents the bipolar stain. Carbol-fuchsin diluted 10 times is one of the best stains. Gram’s stain is negative. Sputum from suspected cases should be collected either by means of a probang or in a bottle and examined: 184 Medical Bacteriology. 1) Microscopically by staining, with a weak carbol-fuchsin, smears from the puru- lent masses. If a very small bacillus is in large clumps, which fails to retain stain by Gram’s method, the evidence is strong that it is the influenza bacillus; the diagnosis should be confirmed, however, by 2) Cultures on blood agar. Animal inoculations are without effect. BaCTERIUM PNEUMONIAE. The sputum of patients suffering from pneumonia is usually of a rusty color due to presence of blood. The ‘‘pneumococcus’’ is readily seen in such material when stained by Gram’s method, or with carbol-fuchsin and momentarily washed with alcohol, as lancet-shaped organisms with outer ends pointed and surrounded by a clear area—the capsule. The capsule can be easily stained by Welch’s method. (See XXXVI.) This organism is also frequently found in the sputum of healthy persons and small numbers may be detected by means of animal inoculation. The rabbit or mouse are most susceptible and should be inoculated intraperitoneally. As a result of infection with this organism the animal quickly dies with a typical septicaemia, the micro-organ- isms being found in great numbers in the blood current. BACILLUS PESTIS. This micro-organism is frequently found in the sputum especially in the pneumonic form of the disease—for methods of detection see CX. STREPTOTHRIX ACTINOMYCOSES. This organism has been occasionally found in sputum and in such eases the peculiar morphology of the colonies is well brought out by Gram’s method. See CX. REFERENCES. v. J. 114; S. 245. See also various texts under particular organisms. EXERCISE CVII. EXAMINATION OF BLOOD. For serum test (Widal reaction) the blood may be collected and dried (see below), but in other cases where cultures are to be made the blood must be collected aseptically in sterile receptacles and hermetically sealed. For this purpose Sternberg’s bulb is ex- cellent. The skin should first be sterilized by use of corrosive sublimate or carbolic acid followed with alcohol. It is usually well in any case to make cover-glass smears at the bed-side for micro- copical examination. These are best made as follows: Place a drop of blood about the size of a pin-head on a perfectly clean cover-glass and then a second cover-glass on this; this flattens the drop of blood out into a thin film. Immediately and before coag- sulation can take place the two are drawn apart horizontally and the films allowed to dry. (Cabot.) BacTERIUM ANTHRACIS. In case of animals dead of suspected anthrax, blood or portion of spleen should be removed with least possible danger from infection or distri- bution of bacilli and studied as follows: 1. Microscopical examinations of blood or the spleen pulp of animals show (when stained with Loeffler’s methylen blue) large bacteria in chains (5 or 6 segments) pre- senting the bamboo appearance. 2. In hanging drop preparation large, homogeneous, immotile bacilli. 3. Agar plate cultures should also be made and from the separate colonies subcul- tures; the gelatin stab being especially characteristic. 186 Medical Bacteriology. 4, In important cases (as in man) guinea pigs, or white mice, should be inoculated, and in case of death organism isolated and identified. SPIRILLUM OBERMEIERI. This organism is found in the blood only during a par- oxysm. It is a long slender organism 6 or 7 times the diameter of a red blood corpuscle. (45) They have a brisk vibratile movement in the direction of their long axis. They are very sensitive to reagents of all kinds. Even the addition of distilled water will cause them to disappear. Fresh blood is best, but dried smears may be used and stained with fuchsin or by Gunther’s method: a. Dried films are treated with acetic acid (5%) 10 seconds, this is removed by blowing and holding film over flask of strong ammonia previously shaken. b. Stained in Ehrlich’s gentian violet. c. Washed with water. d. Dried. e. Mounted in balsam or xylene. jf. Examined. Pyvocenic Micrococcr. These are occasionally found and for method of detection see CX. : - BacTERIUM MALLEI. Sometimes found in the blood of those suffering with Gland- ers. It may be detected in the blood-smears. For special methods see CX. B. PNEUMONIAE. This germ is frequently present in fatal cases 24 to 48 hours be- fore death. The blood should be drawn with a sterile hypodermic syringe and about 1 ce. of blood mixed with a tube of melted agar at 43°C. and poured into a Petri dish. Characteristic colonies appear in 24 to 48 hours. B. TuBERcULOSIS. In ease of miliary tuberculosis they may be very rarely found in sufficient numbers to be detected by staining methods, see sputum CVII. B. INFLUENZAE. Canon claims to have stained and cultivated this organism in blood, but this needs confirmation. B. cout. This organism may be found in the blood, for methods of isolation and identification see Faeces CIX. BacILLuS PESTIS. This germ occurs in the blood in certain cases at least but ap- pears to require considerable skill in detecting it due to its variable appearance. Broth tubes should be infected and animals inoculated. BACILLUS SUIPESTIFER. a. Make agar plate and streak cultures from spleen of dead animal, and work up the colonies as they appear. ‘ b. Widal Reaction (for technique see below under B. typhosus). PLASMODIUM MALARIAE. a. Examination of fresh blood. A droplet of blood from finger or lobe of ear is placed on a glass slide, covered with a cover-glass and then the cover-glass is ringed with vaselin. Examination should be made with a 75 in. oil immersion. b. Stained. Prepare films as directed above and stain with methylen blue and eosin or treat films with a very weak acetic acid 2 or 3 drops to 30 cc. of water; to remove haemoglobin wash with water and stain with following solution for } y minute: Borax - - : 7 : : : - - 5.0 parts. Methylen blue - - - : - : - - 0.5 Bes Water - - - eee - - = - '- 100.0 parts.. Wash, dry and mount in balsam (Manson). 188 Medical Bacteriology. BACILLUS ICTEROIDES. Make agar streaks from blood or fragment of liver (where liver is obtained it is best wrapped in cloth and kept in incubator at 88°C. for 12 hours before cultures are made to encourage development of the micro-organisms, which are usually only sparingly present in tissue). Keep the cultures at 38°C. for 12-16 hours and then at 22°C. for same time; the characteristic appearance is a transparent, bluish growth surrounded by an opaque zone. If this is not obtained other culver must be prepared and a thorough study of the organisms isolated made. REFERENCES. v.J. 45; 8. 79. See also texts under particular organisms. Wipau Reaction. Directions for collecting samples of blood. ‘‘ Wash with boiled water the part from which the blood is to be obtained (lobe of ear, end of finger, or toe in infant). Prick deeply the skin with a clean needle.’’ Remove two or three large drops of blood on a clean glass slide, alluminum foil, piece of isinglass or letter paper. Allow the blood to dry. Then place in an envelope and send to laboratory and test as follows: a. Make a hanging drop preparation from a 24-72 hour old agar, or bouillon, cul- ture of Bacillus typhosus. b. If the bacilli are actively motile, remove the cover-glass, add to the culture a small drop of a solution of typhoid blood (diluted from 10-50 times), return the cover- glass to the slide and seal well with vaselin. ¢. Examine with a high dry power (% in. obj.) rather than with the oli immersion. In a typical reaction the motility is almost immediately affected and soon motion ceases altogether while the bacilli collect in clumps, i. e. become ‘‘agglutinated.’’ REFERENCES. v.J. 49; 8.79. See also texts under particular organism. EXERCISE CIX. EXAMINATION OF FAECES, The material expelled from the rectum and comprising the substances from the food and the secretions of the alimentary tract come under this head. The number of micro-organisms occuring here is enormous, and comprise a large number of species and among them several pathogenic forms particularly B. typhosus. M. comma, B. tubercu- losis and Amoeba coli. BacILLUS TYPHOSUS. This organism occurs in the faeces in the case of typhoid pa- tients, but on account of the large number of other organisms its detection is very diffi- eult The following methods are the most serviceable: Parietti’s Method. This method consists in adding Parietti’ s solution (carbolie acid 5 grams: hydrochloric acid 4 grams, and distilled water 100 ec.) to bouillon in the following manner: A number of tubes of bouillon have a varying quantity of the above solution added, e. g, 1 drop to one tube, 2 to another, 3 to another, and so on. These tubes are inoculated with a small quantity, (one or two loops), of the faeces and then placed in the 38° C. incubator. Twenty-four hours later the tube containing the largest amount of Parietti’s solution which shows growth probably contains B. coli and B. typhosus if it is present. The organisms may be separated most quickly and easily by the use of the lactose litmus agar plate. The blue colonies should be worked up, and especially tested for its agglutinating power on typhoid blood. Instead of the use of the lactose litmus agar plate, either Elsner’s or Hiss’ methods may be used. 190 Medical Bacteriology. Esner’s Medium. Method of preparation: Peel and cut up 500 gms. of old potatoes of medium size, add 1000 ce. of water and boil 1 and 4 hours. Mash potatoes thoroughly; strain through a cloth and add water to filtrate to make a liter. Add 15 % gelatin and boil 10 minutes. Cool to 60° C. and add white of one egg and boil 15 minutes. Filter through cotton, then paper. Titrate and make gelatin 2-3 % acid. Just before tubing add 1 % potassium iodide (10 ce. of a solution in which 1 ce. contains 1 gram of potassium iodide). Tube and sterilize three times. Plates of this medium are made in the usual way and kept at 15-18°C. On this me- dium the typhoid germ forms very finely granular, small, bright droplets resembling condensed moisture, while the colon bacillus gives rise to larger, brown colonies, which are more granular and spread more. Hiss’ Plate Medium. This contains: 10 grams of agar. 25 grams of gelatin. 5 grams of beef extract (Leibig). 5 grams of sodium chloride. 10 grams of glucose. 1000 grams of water. _ It is made by first dissolving the agar, salt and extract in the water, then the gelatin is added and dissolved, the reaction changed by use of NaOH and phenolphthalein so that it will contain not less than 2% normal acid, cleared with two eggs and filtered, glu- cose added and the medium tubed and sterilized. Make plate cultures in ordinary way and incubate at 38° C. for 18 hours, then ex- amine the colonies microscopically. The colonies of B. typhosus have irregular out- growths and fringing threads. The colonies of B. coli, on the other hand, are much large and as a rule are darker in color and do not form ehyaada, The colonies may be further examined by the use of Hiss’ Tube Medium. 5 grams of agar-agar. 80 grams of gelatin. 5 grams beef extract (Leibig). 5 grams sodium chloride. 10 grams glucose. 1000 grams water. Made as plate medium except that is is to contain 1.5% normal acid. Within 18 hours at 38° C. the typhoid bacilli produce a uniform clouding. The colon bacilli do not produce uniform clouding and do produce gas. All suspected cultures should be tested with typhoid blood (Widal reaction). The typhoid organism may be isolated from the stools during the first two weeks of” the disease. MICROSPIRA COMMA. 1. Microscopical examination of ‘‘rice-water’’ discharges for spirilla lying parallel. 2. Culture methods. Gelatin or agar-plates should be made from the rice-like flakes; other flakes should be inoculated into flasks of peptone water (Dunham’s solution) and inoculated at 38° C. The surface growth 6-12 hours later is to be examined microscop- 192 Medical Bacteriology. ically and by means of plates. Then test the peptone cultures for nitroso-indol (cholera red reaction) by the addition of a few drops of sulphuric acid. BACTERIUM TUBERCULOSIS. This organism has been found in the stools in cases of intestinal ulcerations, and may come, in cases of phthisis, from ingested sputa. AMOEBA COLI. 1. A drop of the mucous portions of stool is placed on a glass slide, covered with a cover-glass and examined with a magnification of about 500 diameters (4 in. objective). Examination should be conducted on a warm stage in order to get amoeboid movements. 2. Preparations may be stained with methylen blue and carmine. The nucleus is stained with the carmine. 3. Discharge may be hardened and stained by Mallory’s method as follows: a. Fix tissues in alcohol. b. Stain (paraffin) sections in a saturated aqueous solution, of thionin for 5-20 minutes. : c. Wash in water. d. Differentiate in a 2% aqueous solution of oxalic acid 4-1 minute. e. Wash in water. f. Dehydrate in aleohol (95%). g- Clear in oil of bergamot. h. Wash with xylene and mount in balsam. Nuclei of Amoebae brownish red, other nuclei blue. REFERENCES. v. J. 199; Si. 228. See also texts under various organisms. EXERCISE CX. EXAMINATION OF URINE. For bacterial examination urine should be drawn with a sterile catheter into a sterile bottle. BACTERIUM TUBERCULOSIS. For method of staining see under Sputum, CVII. It is best to centrifuge the product and care must be taken to differentiate from the Smegma bacterium. or this purpose stain cover-glass smears as follows (Bunge & Franteroth.): 1) Absolute aleohol, 3 hours. 2) Chromic acid, 15 minutes. 3) Stain in hot carbol-fuchsin. 4) Decolorize in sulphuric acid (25%) 2-3 minutes. 5) Counter-stain with a saturated alcoholic solution of methylen blue. The smegma bacillus is decolorized by this method. Tubercle bacterium in urine is frequently present in clusters while the smegma bacterium occurs singly. Injection of guinea pigs, smegma bacillus is non-pathogenic. The following organisms have also been found in the urine. For methods of isolation see references. PYROGENIC MIcROcoccI.. CXI. M. GONORRHOEAE. CXI. B. TypHosus. CIX. S. OBERMEIERI. CVIII. REFERENCES. v. J. 273; Si. 504. and texts under the various organisms. 194 Medical Bacteriology. EXERCISE CXI. EXAMINATION OF TRANSUDATES AND EXUDATES. The material should be collected in sterile vessels under aseptic precautions. Make several cover-glass preparations and stain one with Loeffler’s methylen blue and the others with gentian violet or carbol-fuchsin. Mount and examine. a. If staphylococci alone are present search for the pyogenic micrococct. b. If streptococci suspect S. pyogenes. c. If diplococci.or tetracocci. 1. Within the pus-cells test for M. gonorrhoeae or M. intracellularis. 2. Free. 8. tetragena: d. If bacilli any of the following may be searched for: 1. B. coli. This organism is likely to be found especially in suppurative peritonitis and diseases of the urinary organs. 2. B. anthracis. 8. B. pneumoniae. 4, B. tuberculosis. 5. B. leprae. 6: B. mallet. 7. B. pestis. 8. P. aeruginosa. 9. B. welchti. 10. B. oedematis. 11. B. tetani. e. Streptothrix actinomyces. f. Amoeba coli. PYOGENIC MICROCOCCI. These organisms are frequently present in pus and should be isolated and identified in pure cultures as microscopical examinations alone will not suffice. STREPTOCOCCUS PYOGENES. This organism is not infrequently present and can be readily identified by culture methods. MicROcoccUS GONORRHOEAE. Pus should be collected in a sterile receptacle or spread on cover-glasses and allowed to dry, but should not be allowed to dry and then wet up again to spread, as this destroys the pus-cells, and hence the valueof the material for diagnosis. Stain: 1. a. Loeffler’s methylen blue 3-5 minutes. b. Wash in water. e. Dry, mount. in balsam and examine with #5 in. oil immersion. d. Look for a biscuit-shaped diplococcus within the pus-cells. 2. By Gram’s method. a. Anilin oil gentian violet 15 minutes. bd. Wash in water. c. Treat with iodine solution 2 minutes. . d. Decolorize with alcohol. e. Counter-stain with Bismark brown, $ minutes. f. Wash, dry and mount in balsam. g- Examine with oil immersion. If the gonococci are present they will be stained brown. If diagnosis is of great importance make cultures as follows: — 1) Make 6 or more streak cultures on blood agar or better make plates on Wertheim’s medium (p. 99). Grow at 38° C. 2) Make a set of ordinary agar plates or streak cultures and keep at 38° C. The gonococcus grows on the first two media but not on the plain agar. The gonococcus is the only organism that: 196 Medical Bacteriology. 1) Occurs in groups (cell-colonies) in pus-cells. 2) Is decolorized by Gram’s method. 3) Does not grow on agar at room or blood heat. (Foulerton). MICROCOCCUS INTRACELLULARIS. Pus may be obtained by lumbar puncture which is performed as follows. The back — of the patient and the operator’s hands should be made sterile. Theneedle (4cm. X 1 mm. for children) should be boiled 10 minutes. The patient should lie on the right side, with the knees drawn up and the uppermost shoulder so depressed as to present the spinal column to the operator. The puncture is generally made between the third and fourth lumbar vertebrae. The thumb of the left hand is pressed between the spinous processes and the point of the needle is entered about 1 em. to the right of the median line, and on a level with the thumb nail and directed slightly upwards and inward toward the median line. Ata depth of 3 or4em. in children and 7 or 8 in adults the needle enters the subarachnoid space and the fluid flows usually by drops. This is allowed to drop into an absolutely clean test-tube, which has previously been plugged and sterilized. From 5 to 15 ec. of the fluid is a sufficient quantity for examination. Cultures should be made at once on blood agar and plain agar (M. & W. 371.). After standing some hours, the sediment should be examined in cover-glass preparations, stained with Loeffler’s methylen blue and by Gram’s method. Micrococcus intracellularis stains by Loeffler’s method and appears as a diplococcus in groups in the pus cells, is decolorized by Gram’s method, and grows on blood-agar and feebly on ordinary agar at 38° C. The following organisms are also found occasionally. For methods of diagnosis see exercises indicated. cour. COIX. PNEUMONIAE. Stain for capsule. Cultivate on blood-agar. CVII. TUBERCULOSIS. OVII. LEPRAE-. For method of staining, see CV. MALLET. a, Widal reaction (If in man typhoid and diphtheria must be excluded in case of a positive reaction). b. Examination of discharge. 1. Microscopical examination usually without result. 2. Cultures, glycerine agar and potato from pus. ec. Animal inoculation, Straus method. B. PESTIS. a. Make plate cultures from blood and buboes and work up colonies. b. Make subcutaneous inoculation into guinea pigs from bubo, and if death ensues search for B. pestis. P. AERUGINOSA. Hasily recognized by its culture characters. B. WELCHII. This germ is non-pathogenic for rabbits but Welch and Flexner have shown that if a rabbit is inoculated intravenously with 0.5 to 1 cc. of a bouillon culture and killed after a lapse of 5 or 10 minutes and the animal kept at 18°-20° C. for 24 hours or at 30°-35° C. for 4 to 6 hours, the organism will multiply in the blood and produce large quantities of gas in the vessels and organs. This effect is characteristic. wo Oo ho 198 Medical Bacteriology. B. OEDEMATIS. a. Make cover-glass ee paratows from fluid of affected parts. b. Also make anaerobic cultures. If material contains spores it should be heated to 80° ©. for 10 minutes before it is seeded. B. TETANI. a. Make cover-glass preparation from pus and search for drumstick bacillus. b. Make cultures in glucose bouillon and agar-plates and develop in hydrogen. c. Inoculate animals with the discharge, and also with the bouillon culture, and - watch for characteristic symptoms. S. ACTINOMYCES. a. Place one of the minute sulphur yellow nodules in a drop of glycerine on a glass slide and then apply gentle pressure. b. Even the low powers of a compound microscope will then show something of the clustered arrangement which can be more carefully studied under a higher power. ce. Intraperitoneal inoculation of guinea pig. One month later nodules on peritoneum. AMOEBA COLI. CIX. REFERENCES. v. J. 405; Si. 514 and 518. See also texts under the various or- ganisms. EXERCISE CXII. DIAGNOSIS OF RABIES. a. The medulla of the suspected animal is removed under aseptic precautions, as soon as possible after death. In case the animal is some distance from the laboratory it is best to cut off the head, pack it in ice and ship by express. b. Place a piece of the medulla about the size of a pea, in 4 or 5cc. of sterile bouillon and thoroughly grind up the same. c. Anaesthetize a rabbit with ether, clip the hair from between the eyes and ears and disinfect with a carbolic acid solution. d. Make a longitudinal incision through the skin and subcutaneous tissue along the median line, while a crucial incision is made through the periosteum on one side of median line thus avoiding haemorrhage from the longitudinal sinus. The periosteum is then pushed back and a disc of the skull (3 inch in diameter) removed with a trephine and the dura mater exposed. e. With a sterile hypodermic syringe introduce 2 or 3 drops of the suspension of medulla beneath the dura mater, stitch the skin, disinfect, dry and seal the wound with collodion. ‘The rabbits apparently experience no inconvenience; the wound heals rapidly and the rabid symptoms appear in from 15 to 30 days, although sometimes they may occur earlier or much later. EXERCISE CXIII. EXAMINATION OF MATERIAL FROM HUSAN AUTOPSIES. At human autopsies smears from the organs should be made on cover-glasses and afterwards stained and examined. Plate-cultures should also be made from the various organs or instead parallel streaks over blood serum, agar-slopes or agar-plates. In all cases the surface from which the material is to be obtained should first be burned to avoid infection of cultures with extraneous germs. Portions of the various organs should also be preserved and hardened in alcohol. CHAPTER X. DETECTION OF PATHOGENIC BACTERIA IN WATER AND MILK SUPPLIES. EXERCISE CXIV. EXAMINATION OF WATER FOR PATHOGENIC BACTERIA. Bacittus Typuosus. In the examination of water it is best to concentrate the bacteria by filtering a large amount of the water through a Berkefeld filter and use the slime on the filter to make the plates. a. Parietti’s method, see CIX. b. Hiss’ method. Make plate cultures and incubate at 38° C. for 18 hours. Inocu- late suspicious colonies into Hiss’ tube medium, fermentation tube, milk and make indol test. Also try Widal reaction. : c. Animal Inoculation. (Michigan method). 1) Inoculate suspected water into bouillon tubes or flasks, and incubate at 38° C. 2) Twenty-four to forty-eight hours later inoculate one ec. into the peritoneal cavity of a white rat. 3) If animal recovers B. typhosus is not present. If animal dies hold autopsy and isolate and study organism causing death. MrcrosPira CoMMA. a. If there is reason to believe that the spirilla are very numerous gelatin plate cul- tures can be made directly from the water, and the suspicious colonies worked up. b. Ordinarily the organisms are very sparse and large quantities must be used, 100- 1000 ec. are placed in flasks and 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. EXAMINATION OF [SILK FOR PATHOGENIC BACTERIA. B. DIPHTHERIAE. Where B. diphtheriae 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. diphtheriae. 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, 1893, 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 be entitled, in accordance with the requirements of this regula- tion, to receive such specimens.” [200] BBE condenser, 22. Acids, detectlon of in cultures, 52; quantitative determination of, 52. Agar, glucose, 44; glycerine, 115; hanging-drop culture in, 380; lac- tose, 4. Agar plate cultures, 34; character of colonies on, 58. C Agar slopes, 12. Air, analysis, comparative, 80; quan- titative, 80. Amoeba colt, 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. ACILLUS acidi lactici, 10. aerogenes capsulatus, 150, amylobacter, 40. of anthrax, 104. campestris, 46. of blue-green pus, 130. of bubonic plague, 124. chauvaet, 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. ot diphtheria, 118. of Friedlander, 108. of glanders, 116. of hog cholera, 126. icterotdes, 128; 188. of influenza, 120. of malignant oedema, 154. mycoides, 26. oedematis, 154; 172; 176; 198. pestis, 124; 164; 184; 186; 196. prodigtosus, 50; 56; 64; 67. pyocyaneus, 130. rouget du porc, 112, of septicaemia haemorrhagica, 110. subtilis, 16; 22; 24; 380; 34; 36; 40; 46; 48; 50; 52; 54. suipestifer, 126; 164; 186. of swine erysipelas, 112. of swine plague, 110. Bacillus of symptomatic anthrax, 152. tetant, 40; 156; 172; 198. tuberculosis, 114. of typhoid fever. 122. typhosus, 42; 46; 50; 122; 188; 192; 200. . vulgaris, 52; 72. INDEX. 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; 1785 200. influenzae, 120; 182; 186. leprae, 172; 174; 196. mallet, 116; 164; 174; 186; 196. phosphorescens, 68. pneumoniae, 106; 162; 172; 174; 184; 186; 196. pneumonicum, 108; 162. pseudo-diphtheriae, 180. rhustopathiae, 112; 172. tuberculosis, 114; 162; 172; 174: 182; 186; 192; 196; 200. welchit, 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. Weapoiane balsam, 22. Capsule stain, 42. Carbol-fuchsin, 18. Cell grouping, study of, 30. Chemicals, effect on bacteria, 48. Cholera red, 192. Cholera vibrio, 182. Classification of bacteria, 60. Cleaning glassware, 2. Colon bacillus, 16. See B. colz. Color production, variation in, 56. Coloring matter, separation of, 67. Comma bacillus, 182. 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. ECOLORIZING agents, use, 36. Desiccation, effect, 48. Diplococcus of cerebro-spinal men- ingitis, 100. of gonorrhoea, 98. of pneumonia, 106. [203] Disinfectant, 48. Disinfectants, testing, 86. Drawing bacteria, 28. Dunham’s solution, 44, Dust, relation of bacteria to, 82. BERTH’S bacillus, 122. Ehrlich’s anilin oil gentian violet, 18, Elsner’s medium, 190, Embedding tissue, 170. Endospores, staining, 38; study of, 40 Enzymes, 54. Esmarch rolls, 34. AECES, 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, ABBETT’S methylen blue, 20; tu- bercle stain, 38. Gas analysis, 50; detection, 50. Gasometer, 50. Gelatin glucose, 44; preparation, 10; sterilization, 10. Gelatin plate cultures, character of colonies on 57; preparation, 82. 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, 96. Gonococcus, 98. Gram’s, iodine solution, 20; stain, 36. FEMATOXYLIN and eosin stain, 172. Hanging-drop preparation, 24. Hauser’s spore stain, 40. Hay bacillus, 16. Heat, effect on bacteria, 48. Hiss’ media, preparation, 190; use, 190; 200. MPRESSION preparation, 30. Incubators, 16. Indo, 54. Involution forms, 30. lodine solution, Gram’s 20; Weigert’s, 172. LEBS-LOEFFLER bacillus, 118. Koch’s method of air analysis, 80. ABELS, 10. Lactose agar, 44. Litmus, lactose agar-plate, 52; milk, 44; solution, 44; 52. Loeffler’s blood serum, 88; tissue stain, 172. AILING bacteria, 200. Methylen blue, Gabbett’s, 20; Loeffler’s, 20. Micrococcus aureus, 96; 172. gonorrhoeae, 98; 174; 192; 194. tntracellularis, 100; 196. lanceolatus, 106. melttensts, 94. pyogenes, 92; 172. tetragenus, 102. Micrometer, ocular and stage, 28. Microscope, use, 22. Microspira comma, 132; 190; 200. Jfinkleri, 136. metschnikovt, 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. EISSER’S diphtheria stain, 178. Nitrites, detection, 52. Nitrate solution, 44. Non-pathogenic bacteria, 63. BSERVATION of inoculated ani- mals, 164. Oil-immersion objective, 22. Oxygen, effect on bacteria, 50. ARIETTI'S 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. Index. Phenolphthalein, 6. Physiological characters, 59. Pigment, production, 56; varieties, 66. Pipettes, sterilization, 4. Plasmodium malariae, 186. Plate cultures, gelatin, 82; 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. ABIES, diagnosis, 198. Reaction of media, 6; effect on growth of bacteria, 44. Roll cultures, 34. Russell’s water sampler, 82. ARCINA lutea, 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, 136. obermetert, 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, 164. 205 Sterilizers, Arnold, 8; hot air, 2; sim- ple form, 8. Streak cultures, character of growth on, 58; inoculation, 16. Streptococcus pyogenes, 90; 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. AXONOMY, 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, (a examination of, 192. IBRIO metschnikovi, 134. Vital movement, 24. ATER 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 IEHL’S carbol fuchsin, 18. Ziehl-Neelsen stain, 182.