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http://www.archive.org/details/cu31924003265943
A LABORATORY GUIDE IN ELEMENTARY BACTERIOLOGY
A LABORATORY GUIDE
IN
Elementary Bacteriology
BY
WILLIAM DODGE FROST, PuH.D.
Associate Professor of Bacteriology, University of Wisconsin
FIFTH REVISED EDITION
spew Bork THE MACMILLAN COMPANY
LONDON: MACMILLAN & CO., LTp.
1913
All Rights Reserved
Eve
De a al COPYRIGHT, 1901, 1902 anD 1904 BY
WILLIAM DODGE FROST
Set up and electrotyped. Published October, 1904 Reprinted September, 1907 Reprinted July, 1909 Reprinted April, 1911 Reprinted July, 1912 Reprinted July, 1913
fue MaAson-HEnry Press Syracuse, New York.
PREFACE TO THE FOURTH EDITION
In this edition minor changes have been made. <A few of the experiments have been rewritten; some of the old methods have been replaced by later, and it is hoped better, methods.
The general plan of the book remains the same. Its object, as heretofore, is to give adequate directions for the performance of certain fundamental exercises in bacteriology. In attempting this two considerations have been kept in mind, first, that in a rapidly developing subject it is important that the directions for 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 directions 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 con- siderable 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 incuba- tion temperature, ete. Desirable changes here are easily indi- cated when the exercise is assigned; second, that each experiment should be complete in itself. Thus some of the experiments can be performed in a few moments, while others require several days for their completion., No attempt has been made to group them into lessons. The order of the experiments is believed to be a logical one, but may be readily adapted to meet the needs under varying conditions.
The various bacteria are studied in groups. This arrangement is in keeping with recent tendencies, and it is hoped that it will impress the student with the similarity between closely related forms, and also emphasize certain minute but important differ- ences.
The system of classification adopted is that suggested by Migula and is the one most widely accepted.
The nomenclature used is determined by rules generally adopt- ed by systematists. To those. who still prefer the old names, the synonyms will be found useful.
(v)
vi PREFACE
The charts of the various organisms furnish a most satisfactory means for recording the observations made during the study of a germ and are especially convenient for reference.
Blank pages have been left for notes and drawing with the idea that notes in permanent form are the only ones of value to the student in subsequent years.
References have been made to the leading text-books and ocea- sionally to original sources. It is expected that the student will make constant use of these references.
My acknowledgments are due to my colleagues, Professor C. A. Fuller and Miss Vermillion Armstrong.
W. D. FROST.
Madison, Wis., January, 1911.
TABLE OF CONTENTS.
List of Apparatus........... bcd canroh beter CAD ioe pewaN HGH elolhna ew aiiuialice een alee Laboratory Rules cei. ¢. pees eeied 4 sucsig ae me Ree ee eather oenarewe wenden s DOL List of References........ sige s Bente SEEM WER Sass veeee RIT
PART I.—GENERAL BACTERIOLOGY CHAPTER I. MORPHOLOGY AND ELEMENTARY TECHNIQUE.
EXERCISE PAGE 19. Use of Microscope........ 34 20. Drawing Bacteria........ 36 21. Hanging-drop Prepara-
TOM aude cntonsniine intebauie? « 38 22. Microscopical Study of Form Types........+++. 40
23. Study of Cell Grouping... 42 24. Study of Involution Forms 46
25. Study of Endospores..... 46 26. Flagella Stain............ 48 27. Capsule Stain............ 50 28. Stain for Metachromatic Grantlesesi 50 sean ess 50
29. Morphology of Yeasts and Moulds Compared with
Bacteria ............4. 50 30. Gelatin Plate Cultures.... 52 31. Agar Plate Cultures..... 56 32. Roll Cultures............ 56
33. Study of Plate Cultures... 58 34. Use of Decolorizing
35. Gram’s Stain............ 60 36. Tubercle Stain (Gabbett) 62
CuHapTeR II. PuHystoLoGgy oF BACTERIA.
EXERCISE PAGE 1. Cleaning Glassware....... 2 2. Plugging Flasks and TUbC8i3¢:5 cance eeac Seon: | 3. Sterilization of Glassware 4 4, Preparation of Bouillon... 6
5. Filling Test-tubes and
Flasks with Culture Me- CLUES ‘spanstanaweie 6 aoauniov aueroandee 10
6. Sterilization of Culture IMG Tain autnl crsihiatscurseuilots 10 7. Preparation of Gelatin... 14 8. Preparation of Agar...... 16 9. Preparation of Potatoes.. 18
10. Preparation of Water- DIN KS hc wadeaes esienss 18 11. Care of Culture Media.... 18 12. Platinum Needles........ 20 13. Test-tube Cultures........ 22 14, Incubation of Cultures.... 24
15. Study of Test-tube Cul- TUTCSis cameos eevee sexes 26
16. Cleaning Slides and Cover- BIASSES, ccvase ca cunwiins icoen 26
17. Preparation of Staining Solutiotiass.cccsacenwewne 28
18. Simple Cover-glass Prep- BPATIONS, jcaccasiacnea wa atic 30 EXERCISE PAGE
37. Preparation of Special Medias ss esenucedene see 64
38. Effect of Reaction of Me- dia on Growth......... 66
39. Effect-of Concentration of Media on Growth...... 66
EXERCISE PAGE
40. Effect of Temperature Variations on Rate of GIOWED. so. scek sictsiand eee 68
41. Determination of Thermal Death Point........... 68
Vili TABLE OF CONTENTS CHaPTER II. PHYSIOLOGY OF BacTERIA.— Continued EXERCISE PAGE | EXERCISE PAGE 42. Comparative Efficiency of 51. Detection of Nitrites in Dry and Moist Heat.... 70 CUltMPES coe nines 76 43, Effect of Desiccation..... 70 52. Detection of Ammonia... 78 44. Effect of Chemicals on 53. Detection of Sulphuretted Bacteridsss ss <s06 ss 04 os 72 Hydrogen........... oo 45. Relation to Oxygen...... ie 54. Detection of Indol....... 78 46. Effect of Direct Sunlight.. 72 55. Determination of Chemical 47. Detection cf Gas......... 74 Enzyms in Cultures.... 80 48. Quantitative Analysis of 56. Variation in Enzym Pro- Gass Ss nciune hasan padi T4 CMiPCEIO Ns: ois eaah aie aes 80 49. Detection of Acids and Al- 57. Variation in Color Produc- Kal eS ss: sa eistaszee pieeneietce 76 CLO Uyevopese dS des ol tnawaie peeleene 80 50. Quantitative Determina- tion of Acids.......... 76 CHAPTER JII. Taxonomy. PAGE PAGE Points to be Observed in the Bacteria Arranged in Classes Study of Bacteria...... 82 and Groups...........- 91 Classification of Bacteria (Mi- CULA ie date euthe ivetemee As 89 CHAPTER IV. Systematic STuDY oF REPRESENTATIVE Non PaTHOGENIC BACTERIA. EXERCISE PAGE | EXERCISE PAGE 58. Preparation of Special 62. Separation of Bacterial Medias ses gti 04 peace a Of Coloring Matter........ 108 59. Saprophilic Class......... 95 63. Zymogenic Class......... 109 60. Chromogenic Class....... 103 64. Saprogenice Class......... 113 61. Variety of Pigments..... 107 65. Phosphorescent Class..... 121 CHAPTER V. BACTERIOLOGICAL ANALYSIS. EXERCISE PAGE | EXEKCISE PAGE 66. Comparative Analysis of 70. Quantitative Analysis of MAP io oe eee eee seers « 126 Milltss iti snc tiene wwe 2 132 67. Quantitative Determina- 71. Efficiency of Pasteuriza- tion of Number of Bac- HOD io net ae ta et oe 132 $OPIA. In. AIT esas ness 126 72. Testing Antiseptic Action 68. Water Analysis.......... 128 of Chemicals........... 134 69. Estimation of Number of 73. Testing Disinfecting Ac- . Bacteria in Soil....... 132 tion of Chemicals..... . 186 PART II.—MEDICAL BACTERIOLOGY. CHAPTER VI. PaTHOGENIC AEROBES. EXERCISE PAGE | EXERCISE PAGE 74. Preparation of Culture 76. Pus Coceus Group........ 145 Mediass: 23 sans ase: >. 138 77. Malta Fever Group...... 153 75. Erysipelas Group......... 141 78. Diplocoeccus Group....... 157
TABLE OF CONTENTS ix
CHAPTER VI. PATHOGENIC AEROBES.— Continued.
EXERCISE PAGE | EXERCISE PAGE 79. Sarcina Group........... 163 87. Tubercle Group.......... 213 80. Anthrax Group.......... 167 88. Colon Group............ . 219 81. Friedlander Group....... 171 89. Hog Cholera Group....... 227 82. Swine Plague Group...... 183 90. Typhoid Group.......... 235 83. Glanders Group.......... 191 91. Pseudomonas Group...... 247 84. Diphtheria Group........ 199 92. Cholera Group........... 251 85. Pneumonia Group........ 207 93. Streptothrix Group....... 263 86. Influenza Group.......... 211
CHAPTER VII. PaTHOGENIC ANAEROBES
EXERCISE PAGE | EXERCISE PAGE 94. Emphysema Group....... 273 96. Tetanus Group........... 289 95. Oedema Group........... 277
CHaprTerR VIII. ANIMAL INOCULATION AND STAINING OF BACTERIA IN TISSUE.
EXERCISE PAGE | EXERCISE PAGE 97. Animal Inoculation....... 294 99. Staining Sections..... so. 312 98. Preparation of Tissue for
Examination............ , 310
CuapTeR IX. BAcTERIOLOGICAL DIAGNOSIS.
EXCRCISE PAGE | EXERCISE PAGE
100. Examination of Buccal 105. Examination of Transu- Secretion............... 320 dates and Exudates.... 346
101. Examination of Sputum.. 326 | 106. Diagnosis of Rabies...... 354
102. Examination of Blood.... 330 | 107. Examination of Material
103. Examination of Feces.... 338 from Human Autopsies 356
104. Examination of Urine.... 346
CHAPTER X. DETECTION OF PATHOCENIC BACTERIA IN WATER AND MILK
SUPPLIES.
EXERCISE PAGE | EXERCISE PAGE
108. Examination of Water for 109. Examination of Milk for Pathogenic Bacteria.... 358 Pathogenic Bacteria.... 360
APPENDIX A. A Key to the Indentification of the Common Pathogenic and a few of the
Well Known Saprophytic Bacteria.......... 0. cece cece cece eens 361 APPENDIX B.
Charte for the study of additional bacteria .......... ee eee ee eee ee eee 365 APPENDIX C.
MOVES cx cosaxciee npriia. Deke a dlids Aig snndln on iauninee aud iauetale GCE cued Badstecens BEVIS HES CRREAD SS 389
LIST OF APPARATUS
This list comprises the apparatus which is to be under the
exclusive control of the student and does not include the general laboratory outfit, such as sterilizers, incubators, microscopes, gen- eral chemical supplies, etc.
50
A.
(4% 02.) cover-glasses, 18 mm. (34 in.) square and 0.17 mm. thick (No. 2).
glass slides.
labels, 2 cm. square.
em. platinum wire (No. 27). pair cover-glass forceps (Cornet or Stewart).
pair fine pointed forceps.
slide boxes for 50 slides. hanging-drop slide.
towel.
yard of muslin.
B.
flask, 1000 ec.
flasks, 400 ee.
flask, 250 ee.
flask, 100 ee.
test-tubes (15 x 120 mm.). Petri dishes (10 em.). fermentation tubes.
glass tumblers or tin eups. small wire baskets.
glass rods for platinum needles.
(x)
3 pipettes, 1 ee.
1 brass tube to hold pipettes (25 < 250 mm.).
8 stain bottles with pipettes, in
block.
sheets of filter paper.
sheets of lens paper.
test-tube brush.
glass funnel, 12 em.
glass funnel, 5 em.
stirring rods.
pipette, 5 ee.
thermometer, 0-100° C.
em. rubber tubing, 1 em. dia.
See Fig. 1.
Mohr stopcock.
potato knife.
Bunsen burner with tubing.
piece of wire gauze.
rice cooker.
graduated cylinder, 300 ee.
graduated cylinder, 100 ee.
graduated cylinder, 25 ce.
evaporating dish, 10 em.
disinfecting jar.
copper cup.
ring stand with clamp.
a CREP NH HH ww
Bee ee
Bee eee
LABORATORY RULES
1. Before beginning an exercise read over the directions and look up some of the references. Keep notes of everything done and the conclusions reached on the right hand pages in this Guide. Make drawings wherever they will be of value. Outline with pencil and fill in with India ink. The Laboratory Guide should be kept in the laboratory.
II. All possible cleanliness should be observed in the care of apparatus, desk, etc.
III. After working with the pathogenic bacteria the tables should be washed with corrosive sublimate and the hands disinfected by washing in the sublimate solution (or a germicidal soap) and then in soap and water.
IV. Solid material, culture media and corrosive sublimate should not be put in the sink but in crocks provided for the purpose. Burnt matches, pieces of paper, etc., should also be put in the crocks and not on the floor.
V. When using the steam sterilizer see that there is enough water pres- ent before lighting the gas and do not leave the laboratory until the gas has peen turned off.
VI. Food should not be eaten in the laboratory and lead pencils or labels should not be moistened with the tongue.
VII. All cultures of bacteria should be labeled with the name of the organism, the name of the student and the date. ;
VIII. The platinum needles used in making cultures should be sterilized shortly before and immediately after use, and before they are laid down. When the needles are covered with infectious material they should be held at the side of the flame until dry before being sterilized; this will avoid the danger of scattering this material about the laboratory.
IX. Discarded cultures should be covered with corrosive sublimate and placed in a proper receptacle, and under no condition should they be left lying about the laboratory. Pipettes which have been used to handle infec- tious material should be placed in a glass cylinder containing a disinfectant, or potassium bichromate and sulphuric acid.
X. If infectious matter should by accident come in contact with the hands, or be dropped on the table or floor, corrosive sublimate (1:1000) should
be immediately applied.
(XI)
LIST OF TEXTS AND REFERENCE WORKS WITH ABBREVIATIONS USED
A— Abbott: Principles of Bacteriology. Lea Bros. & Co., Philadelphia, 5th Edit., 1899. A. 2.— Abbott: Hygiene of Transmissuble Diseases. Saunders & Co., Philadelphia, 2nd Edit., 1903. B.— Bowhill: Manual of Bacteriological Technique. Oliver & Boyd, London, 2nd Edit., 1902. Cc.— Chester: A Manual of Determinative Bacteriology. The Macmillan Co., New York, 1901. Cn.— Conn: Agricultural Bacteriology. Blakiston’s Son & Co., Philadelphia, 1901." Cn. 2— Conn: Bacteria in Milk. Blakiston’s Son & Co., 1903. Co.— Connell: A Laboratory Guide in Practical Bacteriology. Author, WKing- ston, Ontario, 1899. Cu.— Curtis: Essentials of Practical Bacteriology. Longmans, Green & Co., New York, 1900. B.— Emery: Handbook of Bacteriological Diagnosis. Blakiston’s Son & Co., Philadelphia, 1902. Ey.— Eyre: Bacteriological Technique. Saunders & Co., Philadelphia, 1903. F.— Fischer: Structure and Functions of Bacteria. Clarendon Press, New York, 1900. Fl.— Fluegge: Die Mikro-organismen. IF. C. W. Vogel, Leipzig, 1896. Fr.— Frankland: Micro-organisms of Water. Longmans, Green & Co., New York, 1894. G.— Gage: The Microscope. Comstock Pub. Co., Ithaca, N. Y., 8th Edit., 1901. Go.— Gorham: Laboratory Course in Bacteriology. W. B. Saunders & Co., Philadelphia, 1901. H.— Hewlett: Manual of Bacteriology. Blakiston’s Son & Co., Philadelphia, 2nd Edit., 1902. Ho.— Horrocks: Introduction to the Bacteriological Examination of. Water. Blakiston’s Son & Co., Philadelphia, 1902. J. H— Jordan's Translation of Hueppe: Principles of Bacteriology. Open Court Pub. Co., Chicago, 1899. v. J— vy. Jaksch: Clinical Diagnosis. Charles Griffin & Co., London, 4th Edit., 1899. K.— Kloecker: Fermentation Micro-organisms. Longmans, Green & Co., New York, 1903. Ik. & D.—Kanthack & Drysdale: Practical Bacteriology. ‘he Macmillan Co., New York, 1895.
Kk. & W.—Kolle & Wassermann: Handbuch der Pathogenen Mikro-organismen, I., I1. and IJI., and atlas. Gustav Fischer, Jena, 1903.
L.— Lafar: Technical Mycology, Vol. I. Lippincott Co., Philadelphia, 1898. Vol. II., Part I., 1903. ‘
L. & K.—Levy & Klemperer: Clinical Bacteriology. Saunders & Co., Philadelphia, 1900.
L. & N.— Lehmann & Neumann: Atlas and Essentials of Bacteriology. W. B. Saun- ders & Co., Philadelphia, 1901.
M.— Moore: Laboratory Directions for Beginners in Bacteriology. Ginn & Co., New York, 1900.
(xm)
TEXTS AND REFERENCE WORKS xili
Mig.—- Migula: System der Bakterien. Gustav Fischer, Jena, 1900. M. & R.—Muir & Ritchie: Manual of Bacteriology. The Macmillan Co., New York, 8rd Edit., edited by Harris, 1903. M.& W.—Mallory & Wright: Pathological Technique. W. B. Saunders & Co., Phila- delphia, 2nd Edit., 1903.
McF.— McFarland: Text-Book of Pathogenic Bacteria. W. B. Saunders & Co., Philadelphia, 4th Edit., 1903.
N.— Novy: Laboratory Work in Bacteriology. Geo. Wahr, Ann Arbor, Mich., 2nd Edit., 1899.
Ne.— Newman: Bacteria. Putnam, New York, 2nd Edit., 1903.
P.— Park: Bacteriology in Medicine and Surgery. Lea Bros. & Co., Philadel- phia, 1899.
P. B. C.—Proceedings of the Bacteriological Committee from Jour. Amer. Pub. Health Assn., Vol. XXII. P. & M.—Peamain & Moor: Applied Bacteriology. Baillére, Tindall & Cox, London,
2nd Edit. P. & W.—Prescott & Winslow : Elements of Water Bacteriology. Wiley & Sons, 1904. R.— Roger: Infectious Diseases. Lea Bros. & Co., Philadelphia, 1903. S.— Sternberg: Manual of Bacteriology. Wood & Co., New York, 1893. Ss. 2— Sternberg: Immunity. Putnam & Sons, New York, 1903. si.— Simon: Clinical Diagnosis. Lea Bros. & Co., Philadelphia, 3rd Edit., 1897. w.— Woodhead: Bactcria and their Products. Charles Scribner & Sons, New York, 1892. Wmo.— Williams: Manual of Bacteriology. Blakiston'’s Son & Co., Philadelphia,
38rd Edit., 1904.
PART I
GENERAL BACTERIOLOGY
PART I—GENERAL BACTERIOLOGY
CHAPTER I
MORPHOLOGY AND ELEMENTARY TECHNIQUE
EXERCISE 1. CLEANING GLASSWARE.
GENERAL Directions. All glassware to contain culture media must be thoroughly cleaned. New glassware should be washed in hot soap-suds (a test-tube brush will be needed for the test-tubes), rinsed in tap water and then placed for a few minutes in water to which about 1% of hydrochloric acid has been added to remove free alkali frequently present on new glass, and then thoroughly rinsed in running water. It is then allowed to drain. Test-tubes and flasks are best dried by placing them on a drain board spe- cially prepared, or standing them mouth down in a box with a cloth bottom or in a wire basket.
Glassware containing media (discarded cultures, ete.,) is best cleaned by first standing in water for some hours, or by being steamed and pouring out the material while in a liquid condition and then cleaning as above with the exception of the use of the hydrochloric acid.
Rererences. A. 126; H. 44; P. 223.
SpecisL Directions. Read Rule I. Clean as directed above, all flasks, test-tubes, fermentation tubes and Petri dishes in your
possession. 2
4 GENERAL BACTERIOLOGY
EXERCISE 2. PLUGGING FLASKS AND TUBES.
GENERAL Directions. When the flasks, test-tubes and fer- mentation tubes are thoroughly dry they are to be plugged with cotton. The cotton for this purpose should be non-absorbent and of the best quality, 7. ¢., as free from foreign matter as possible. The plugs should be sufficiently loose to permit the interchange of gases and at the same time tight enough to support the weight of the vessel and its contents, otherwise they are apt to be pulled out in handling the vessels. The cotton should be rolled into a eylin- der of the proper diameter and long enough to extend into the mouth about 214 em. (1 in.) and project sufficiently to protect the lips from dust. The plug should be pushed in straight and not twisted; the surface next to the glass must be pérfectly smooth, presenting no creases for the entrance of dust.
Rererences. A. 127; H. 44; M. & R. 49; MecF. 164; P. 223.
Specran Directions. Plug all test-tubes, flasks and fermenta- tion tubes in your possession.
EXERCISE 3. STERILIZATION OF GLASSWARE.
GENERAL Directions. The glassware thus-prepared is ready for sterilization, which process is accomplished in an apparatus called the hot air sterilizer. This is a sheet iron or copper box with
a double wall which permits of rapid
| heating. The apparatus should be so ar-
OQ ranged that a temperature of 150° C. can
be quickly reached and readily main-
tained. In such a sterilizer all glassware
to be used for the reception of culture
Pate. such as flasks, test-tubes, Petri
dishes, etc., is submitted to a temperature
of 140-150° C. for 1 hour, or until the
cotton plugs are slightly browned; this
change being due to the incipient char-
ring of the cotton. The test-tubes are
Ke placed erect in square baskets made of
Ried. HabMleBlertiiecy, galvanized iron wire. When the air in
(Muir & Ritchie). the sterilizer has cooled to about 40° C.
the glassware can be taken out and stored ready for use. The Petri dishes are not to be opened until used for culture purposes.
Rererences. A. 75 and 127; H. 36; M. & R. 29; McF. 164; Py 228:
6 GENERAL BACTERIOLOGY
SpeciaL Directions. All glassware prepared in 1. is to be sterilized for one hour at 150° C. The small pipettes should be placed in brass tubes, provided for the purpose, and also sterilized.
EXERCISE 4. PREPARATION OF BOUILLON..
GENERAL Directions. Any one of the three methods (A, B or C) may be used. They are arranged in order of preference, but method C is the most convenient, and hence most used.
A. B. C.
a. From 500 grams (1% lbs.) of lean} a. Ditto. a. Weigh out beef remove the fat and connective tis- three grams of sue and mince (or use Hamburg steak). beef extract (such as Lie- big’s).
b. Add 1 liter of distilled water and} 6. Add 1literof| 6. Add1 liter after shaking thoroughly set in ice/distilled water. of distilled wa- chest for 12 to 24 hours. ter.
ce, Squeeze through a cloth and add| c. Place in ves-| ¢. Place in enough distilled water to filtrate to|sel for cooking,| vessel for cook- make 1 liter and place in vessel to cook./then cook for 14| ing. hour at about 70° C., filter through paper and make up to 1 liter.
d. Add to any of the above solutions: 1% (10 gms.) peptone (Witte) and 44% (5 gms.) common salt (NaCl), then weigh solu- tion, with vessel, so that the water which is subsequently driven off in cooking can be accurately replaced.
Cooking may be done either in a flask which is heated in a water- bath or sterilizer, double-walled boiler, or rice-cooker. In case a rice- cooker is used a 50% solution of calcium chloride should be placed in the outer vessel instead of water as by this means the contents of the inner vessel can be brought to a rapid ebullition, something impossible by the use of water alone.
e. Heat, not above 60° C., until ingredients are in solution, then restore the water lost by evaporation.
f. Neutralize. This is a very important step and calls for great care. Of the following methods, A is more accurate and should be employed for special or research work. For ordinary routine work B may be employed.
8 GENERAL BACTERIOLOGY
A.
1.) Titrate as follows: Pipette off 5 ee. of the fluid into a 10.cm. evaporating dish, add 45 ce. of distilled water, boil for three minutes, add 1 cc. of phen- olphthalein (0.5% substance in 50% al- eohol), and then run in carefully, drop by drop, from a burette a twentieth normal’ solution of sodium hydroxide (#,NaOH) until the solution turns a faint pink color. Treat two other sam- ples in the same way. If the amount of NaOH required is approximately the same in each case the average can be taken as the amount necessary to neu- tralize 5 ce. Calculate the amount nec- essary to neutralize the whole (1000—15 ee.). Since this amount would dilute the medium too much, a stronger solu- tion (normal) is used, hence,
B.
Use a normal’ solution of so- dium hydroxide (2 NaOH). Add to the hot solution a few cc. at a time at first, later a few drops, stirring thoroughly with a glass rod. After each addition, test by placing a drop of the solution by means of the glass rod on a strip of phenolphthalein paper. (Prepared by dipping filter pa- per in a solution.) The addition
- should continue until the test pa-
per is turned a faint pink color.
2.) Neutralize by adding z5th of the volume calculated above
of a normal solution of sodium hydroxide.
Test the accuracy of
the work at this point by the addition of a few drops of phenol- phthalein to a ce. or so of the medium. If a faint pinkish tint is not obtained, titration and neutralization must be repeated.
g. Boil for 5 minutes and restore weight.
h. Test reaction and adjust if necessary.
1. Add 0.5 to 1.5% of a normal hydrochloric acid. The amount of acid to be added varies with the purpose for which the medium is to be used, e. g., in water analysis + 1.0 (acid) is preferable, with the pathogenic bacteria a smaller amount of acid (-L 0.5) more nearly meets requirements.
j. Heat until precipitate appears flaky and then filter through moistened filter paper. (For method of folding see Abbott p. 96).
The filtrate (bouillon) should be of a light straw color, per- fectly clear, and should not give a precipitate on boiling.
Rererences. A. 94; H. 45; M. & R. 35; McF. 180; P. 212;
P. B. C. 18-24.
Specrau Directions. Prepare 1 liter of bouillon according to method C. Secure and put to soak meat for 7. See Rule IV.
*Normal solutions are prepared so that one liter at 16° C. shall contain the hydrogen equivalent of the active reagent weighed in grams (Sutton). For present purposes a 4% solution of sodium hydrate is sufficiently accurate.
10 GENERAL BACTERIOLOGY
EXERCISE 5. FILLING TEST-TUBES AND FLASKS WITH CULTURE MEDIA.
GENERAL Directions. In filling tubes be careful not to allow the media to touch the neck of the vessel as this will cause the cotton to stick to the glass when the plugs are removed. Place the culture fluid to be tubed in a funnel arranged with a delivery tube and stopcock (Fig. 2), from which it can be run into sterile vessels. Test-tubes should contain 6-10 cc. of medium (about 3 em. deep). Flasks are to be filled about three-fourths full.
SpeciaAL Directions. Fill 15 test-tubes and preserve remainder of bouillon in flasks.
EXERCISE 6. STERILIZATION OF CULTURE MEDIA.
EXPLANATORY. To sterilize culture media steam is used almost exclusively either as streaming fia.2. Appara- steam or under pressure. The unconfined steam {Up for fling test- is applied in an apparatus known as a steam ster- ilizer. Of the various patterns the Arnold is perhaps the most satisfactory. It is effective, economical in the use of gas, and does not allow the escape of large quantities of steam into the room as a large part is condensed to be reconverted into steam. A simple steam sterilizer is shown in Fig. 3, and for student use is very convenient. The method of using either form is identical. Always have plenty of water present before heating. Exposure is made on three consecutive days.for 20 minutes, be- ginning to count time when the ma- terial reaches the temperature of the
Fig. 3. Simple sterilizer consisting steam, which will vary with different
Seseel pina Aan ga withacover substances and the volume treated.
Between successive steamings culture
media should be kept under conditions favorable to spore germi-
nation, 7.e, at the room temperature. This method of steriliza- tion is known as the discontinuous method or Tyndalization.
For the employment of steam under pressure the autoclave is essential. The lid should contain a thermometer as well as a
12 GENERAL BACTERIOLOGY
steam gauge, safety and outlet valve. A thermo-regulator is also desirable. The following table gives the temperature correspond- ing to atmospheres of pressure indicated on the gauge:
TABLE OF TEMPERATURES CORRESPONDING TO STEAM-
PRESSURES. Temperatures Steam-Pressure Temperatures Steam-Pressure F. Cc, Lbs. F. C. Lbs. 212° 100° 0 251° 121.5° 15 228° 109° 5 260° 126.5° 20 240° 115.5° 10 287° 141.5° 40
This table is true only when all of the air in the apparatus is replaced by steam, and hence the steam must be allowed to escape freely before the outlet valve is closed. A single exposure of 20 minutes at a temperature of 120° C. (one additional atmos- phere) is sufficient to kill all germ life. After the proper exposure, care must be taken not to allow the steam to escape too rapidly, otherwise the culture media may be forced against the plugs owing to the unequal pressure. ,
GENERAL DirEcTIOoNS. Ordinary media may be sterilized by either method. In case of gelatin and sugar media the temperature should not exceed 110° C. for 15 minutes.
Fig. 4. Autoclave;
Rererences. A. 59-77; H.37;M. & R. 29; McF. a, safety valve; b, 166; P. 218. blow-off pipe: ¢
gauge; (Muir & Ritchie).
SpeciaAL Directions. Sterilize bouillon pre- pared in 4 for 20 minutes in a steam sterilizer on three consecutive days, or in the autoclave at 120° C. for 20 minutes. Rule V.
N. B. Some time is required to raise the temperature of the media to that of the steam, especially if the vessels are large.
All media should be carefully examined every day for a week or more, and if ‘specks’? or the least cloudiness appears, the medium is not sterile and the process of sterilization must be repeated.
14 GENERAL BACTERIOLOGY
EXERCISE 7. PREPARATION OF GELATIN.
GENERAL DIRECTIONS.
a.
b. $ Same as bouillon (4). C. d. Add 1% peptone, 0.5% salt and 10-15%' of the best gold label, sheet gelatin, and weigh.
e. Heat until ingredients are dissolved. f. Neutralize.
g. Boil 5 minutes and restore weight. h. Test reaction.
4. Boil until albumin coagulates and floats in the clear fluid. If beef extract is used it will be necessary to first cool below 60° C. and thoroughly stir in an egg.
j. Filter. Arrange the apparatus shown in Fig. 5. Use absorbent cotton. The funnel and flask should first be heated with warm water. Usually the hot gelatin will filter without the use of the pump. If the pump is needed it should be started be- fore pouring in the culture medium. This prevents the unfiltered gelatin from pass- ing between the cotton and glass.
k. Add 5.0 ee. (0.5 %) of a normal hy-
LTO iy
o °o oc cam
Fic. 5. Apparatus for filter-
drochloric acid solution. ing media through absorbent cotton; a, coil of wire over l, Tube. (5). laid with layer of absorbent
*
Bate ? cotton; b, tubes for making m. Sterilize in the steamer for 20 connection with air pump: c,
j : . Bunsen valve to prevent en- minutes on three consecutive days or in the trance of water into flasks,
autoclave at 110° C. for 15 minutes.
Rererences. A. 99; H. 47; M. & R. 40; McF. 184; F. 215; P. B. C. 26.
SpeciaL Directions. Make 1 liter, using method A. Fill 30 test-tubes. Put the remainder in flasks, sterilize in steam sterilizer or autoclave. Remember long exposure to high heat injures the solidifying properties of gelatin.
1The amount to be varied according to the season of the year, 10 per cent. in winter, 12-15 per cent. in summer, but it should be remembered that differ- ent quantities affect the appearance of the cultures.
16 GENERAL BACTERIOLOGY
EXERCISE 8. PREPARATION OF AGAR (RaveEnet).
GENERAL DIRECTIONS.
Add 15 grams of agar-agar threads (finely chopped) to 500 ce. of water and either (1) dissolve in autoclave by heating up to 120° C., closing off gas and allowing to cool, or (2) boil until the agar-agar is dissolved (about 14 hour) and make up loss of water by evapora- tion. While the agar is being dissolved proceed as follows:
a. Same as in the preparation of bouillon (4 a).
b. Add 500 ee. of distilled water.
c. Same as bouillon (4 c).
d. Add 10 gms. of peptone and 5 gms. of salt.
e. Heat until peptone is dissolved.
T. Neutralize.
g. Cool to 60° C., add agar solution and mix (in ease extract is used it will be necessary to add an egg at this point).
h. Boil until albumin is coagulated and floats in the clear liquid and restore weight.
i. Test reaction.
j. Add 0.5% normal hydrochloric acid. k, Filter as in case of gelatin. (7).)
l. Tube.
m. Sterilize in steam for 15 minutes on three successive days or in autoclave for 20 minutes at 120° C. After the last sterilization place most of the tubes in a sloping position to harden (Fig. 6), these are known as agar slopes. Those solidified in an upright position, frequently called ‘‘deep stick agar,’’ Fie.6 Method of sloping agar. are used to make plate cultures. 2
-”
Rererences. A. 104; H. 47; M. & R. 88; McF. 185; P. 215; P. B. C. 27; Journal of Applied Microscopy, 1898, 1; 106.
SPECIAL Directions. Use meat extract, make 1 liter, fill 25 tubes and after last sterilization incline 20 of them. Place the remainder in flasks and sterilize.
18 GENERAL BACTERIOLOGY
EXERCISE 9. PREPARATION OF POTATOES (Borroy). GENERAL DIRECTIONS.
a. Select a number of rather large test-tubes (150x20 mm.), place a small wad of absorbent cotton’ in the bottom of each (Fig. 7 a), plug and sterilize as usual. ==
b. Wash a large potato, then with a cork borer ee slightly smaller than the test-tubes punch out cylinders about 5-6 em. long.
c. Divide these diagonally and trim to shape indi- eated in Fig. 7 0.
d. Add a few drops of distilled water to each test- tube and place pieces of potato in position.
e. Sterilize on three consecutive days for 30 to 45 minutes. Fia.7. Bolton’s
Unless the tubes are to be used immediately, they Potato tube: should be sealed. (11.) The dark color can be prevented by im- mersing the pieces between c and d in running water for 12-18 hours.
Rererences. A. 107; H. 49; M. & . 47; McF. 190; P. 216; P. B. C. 28; 8. 47.
SpeciaL Directions. Prepare 15 test-tubes of potato, sterilize and seal with paraffin. (11.2.)
EXERCISE 10. PREPARATION OF WATER-BLANKS.
GENERAL Directions. Water-blanks are prepared by placing exactly 10 ce. of a physiological salt solution (6 gms. per 1,000 ec. water) in test-tubes and sterilizing in autoclave 15 minutes at 120° C., or in steamer 15 minutes on three successive days.
Speciau Directions. Prepare and sterilize 10 water-blanks.
EXERCISE 11. CARE OF CULTURE MEDIA.
When sterile culture media (or test-tube cultures) are to be kept for some time they must be protected from evaporation and stored in a dark, cool place. Evaporation may be checked to a con- siderable extent, (1) by storing them in tin cans, e. g. quinine cans. Care must be taken, however, that these do not become too damp in which case the mould fungi frequently grow through the cotton plugs; (2) flasks and test-tubes may be sealed by removing the
1 Gage recommends glass beads. A smaller cylinder of potato may also be used instead of the cotton; in this case the tubes would be sterilized empty.
20 GENERAL BACTERIOLOGY
plugs, dipping same in melted paraffin (melting point about 50° C.) and then replacing them; (3) by cutting off the projecting cotton and drawing over the mouth of the vessel a rubber cap (made for the purpose) which has been sterilized in a solution of mercuric bi- chloride, or rubber dam, easily obtained from dentists, fastened on with a rubber band, may also be used; or (4) by use of a cap of tin- foil. In this case the foil should be put on as soon as the tubes are filled, and sterilized. with the medium.
All media should be carefully examined every day for a week or more, and if spots or the least cloudiness appears, the medium ts not sterile and the process of sterilization must be repeated.
All receptacles containing media should be labeled after steriliza- tion. For this purpose labels can be purchased, the size used for glass slides, or gummed paper in sheets can be cut into squares (2em.). The labels are to be attached to each ves- nes setae sel 1 em. from the lip. The name of the student, Date the kind of medium and the date of preparation should be written across the top, leaving the rest of the label to be filled in when the medium is inocu-
lated. Rule VII.
EXERCISE 12. PLATINUM NEEDLES.
GenrraL Directions. These are made by fusing a piece of No. 27 platinum wire (5 em. long) into a glass rod or tube (18 em.
— Ss long). (Fig. 8.) The dan-
ger of having the wire crack O- C } off when the needle is heated Fig. 8. Platinum Needles. is lessened if a little piece (4%4 em.) of fusible glass is soldered on the glass rod before the wire is melted in. Each student should have two such needles; in one the wire should be straight (designated ‘‘needle’’) and the other bent to form a ‘‘loop.’’ This loop should be formed around a No. 10 wire. These instruments must be sterilized shortly before and im- mediately after use by heating the wire to a glow in the gas flame. The handle should also be passed through the flame two or three times. Cool before using. If the habit of sterilizing is thoroughly acquired much trouble will be avoided and possible danger prevented. These needles will be in constant use.
Rererences. A. 131; H.42;M.&R. 51; McF. 196; P. B. C. 33, foot note.
22 GENERAL BACTERIOLOGY
EXERCISE 13. TEST-TUBE CULTURES.
EXxpLaNaTory. The extreme minuteness and slight variation in the form of different bacteria render a thorough study of them by direct microscopic observation a difficult 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. <Ac- cording to these methods the bacteria are sown on various food sub- stances 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 differ- ences 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 cult- ures), 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 their diagnosis. The tube-cultures are serviceable in giving oppor- tunity for a further study of the characters as well as to furnish the most convenient method of maintaining the cultures.
GENERAL Directions. Bacteria when obtained in ‘‘pure eulture’’ are usually grown in test-tube cultures. To make these a small portion of a previous culture is transferred to fresh culture OF by means of the platinum needles.
. Stab Cultures are made in test: tubes containing solid, trans- eae media, such as gelatin \ and agar. The end of a ster- | ile needle is infected with the material to be transferred. The needle is then thrust into the medium to the bottom of the test-tube and withdrawn. In this way the bacteria are left along the entire length of the needle track. For method of holding tubes see Fig. 9. They are held in an inclined position to prevent the possibility of infection
: Fig. 9. Method of holding test-tubes. from the air. a, cotton plugs.
24 GENERAL BACTERIOLOGY
b. Streak Cultures are cultures made by drawing the needle, or better, the 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. Liquid Cultures (bouillon, milk, ete.) are inoculated by trans- ferring the desired material to them on either the needle or loop.
RereRENcES. A. 152; H.58;M. & R. 51; McF. 198.
SPECIAL DIRECTIONS.
a. Make a gelatin stab, an agar streak, a potato streak, and a bouillon culture of Bacillus subtilis (HHRENB.) CoHN (hay bacillus) and Bacillus coli (Escu.) Mia. (colon bacillus) from agar cultures supplied. Rule VIII.
b. Label cach tube, writing the name of the organism, the date of inoculation and your own name. Rule VII.
c. Place the gelatin in the cool chamber, and the other cultures in the incubator at 28° C. See next Hixercise.
EXERCISE 14. INCUBATION OF CULTURES.
ExpLaNaTory. Most bacteria grow at ordinary temperatures (22° C.), but their growth is usually hastened by a higher tempera- ture (e. g. 28°-30° C.) The pathogenic, or disease-producing bac- teria grow best at the temperature of the human body (38° C.). All bacteriological laboratories are, therefore, supplied with appara- tus 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, how- ever, must be kept at a temperature several degrees below the melt- ing point of gelatin, 2. €., not above 22° C. Ordinarily the tempera- ture of the locker, especially near the floor, will be found satisfac- tory. In avery warm room, particularly in the summer, an artifi- cially 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.
Rererences. <A. Ch. VIII; H.55;M. & R. 82; P. 231.
SPECIAL DIRECTIONS.
a. Incubate all cultures of the non-pathogenic bacteria at 28° C.,
BaAcILLUS SUBTILIS (EHRENB.’ CoHN
25
Gelatin Stab: Grown 24 hours at....... aitarstaraitin Os
Ss) .o) Be a 3 c| n wn ny & 3 2 } 6 4 4 te 2 G8: WOWES Ubinas. accceiwian: daigicwe oC CORYS Abiascissncnrasgauomeedwes 6s, Agar Streak: Grown 24 hours at.........--- asa. Cs 3 oO ° ° a 3 4 4 AB OUTS Abas svscnrernewesseweedion aCe 6 days at...... silastic? Os Potato: Grown 24 hours at...... siacvenguemnensiyy °C. ro) | o | o 3 S) Sy mn 7) 3 5 to) ts) 4 4 oe 45 ROUTE RE viens neseenwxsenvewns °C. Sdays lis vacasevinencasasennes Oy Bouillon: Grown 24 hours at.......---eseeeeee eee ee 3 3 | 3 é 8 8 | : g 3 =] 4 i Ei 24 : Aa ee ee,
@ hours at....... a eho eeasoa pia Aras OF | 6 GAYE Ab... sc ccseveesereeveee oe Gy
26 GENERAL BACTERIOLOGY
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 15. STUDY OF TEST-TUBE CULTURES.
GENERAL Directions. As soon as growth becomes visible a sys- tematic and careful study of the cultures should be made. A de- tailed list of the points to be noted will be found in Chapter III, and should be consulted in writing up the descriptions. The sum- mary below will, however, be found useful.
For bouillon cultures note: 1) condition of fluid, 2) character of sediment, 3) presence or absence of membrane, and 4) charac- teristic odor.
For solid cultures (agar and potato slopes), note: 1) Form of growth, 2) size, 3) surface elevation, 4) consistency, 5) color, 6) effect on media, and 7) characteristic odor.
For gelatin stab cultures, note: 1) Effect on media, a. non-lique- fying, i) line of puncture, ii) surface, b. liquefying, i) shape of liquefied area, ii) condition of fiuid, iii) character of sediment, 2) characteristic odor.
The study should be continued from day to day as long as changes are noted. Make drawings wherever they will be of service in elucidating the descriptions.
SpeciaAL Directions. Study, write careful descriptions and make necessary drawings of all the cultures made. For recording results use the table on pages 25 and 27.
EXERCISE 16. CLEANING SLIDES AND COVER GLASSES.
GENERAL Directions. Slides can be sufficiently cleaned by washing in water or aleohol 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 by rubbing them between driers (two wooden blocks 20x10x214 mm. covered with several layers of cotton cloth or chamois skin), and then heating them on
Baciuuus cour (Escu.) Mic. av
Gelatin Stab: Grown 24 hours at...
8 °C
°.
hours at ».-hours at........°C
48 HOUFS At... seeccee seco neers °C. COATS Avice convimamoascens oC. Agar Streak: Grown 24 hours at......ccseeeeeee ss 3 Oo ° ° HN n mn : : 4 A
4S OWES Athy cay vases veces nvene *C. GAYS Bhs aeveensxies cessewones Ay
Potato: Grown 24 hours at
} 8G) c
. hours at.......°
seaeeshours at.....
SB HOUTS Ateses aceaesews weed: °C. 6 GAYS Btiiccciesemsigt Seaway e
Bouillon: Grown 24 hours at.......-......00s0e °C.
°C,
Fad Gt
hours at . hours at......
48 hours at... 1+.
GOA S Bhs es wissiscs sscemaineesigion °C,
28 GENERAL BACTERIOLOGY
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 1,000), and boiled for one-half hour and then treated as above.
SpectaL Directions. Clean 1% oz. of cover-glasses and place them in a clean Petri dish.
EXERCISE 17. PREPARATION OF STAINING SOLUTIONS.
GENERAL Directions. The dyes most useful for staining bac- teria are the basic anilin dyes which come in powdered or erystal- line form. (Gruebler’s dyes are standard). Those in most com- mon use are Fuchsin, Methylen blue, Gentian violet and-Bismarck brown. They keep 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 alcoholie solution of Gentian violet.......... 2.5 ee. Distilled: water’ -. a caniecesegecs eae ea Mees pada Re ewetee 47.5 cee. 2. Saturated aqueous solution of Bismarck brown.
3. Ziehl’s carbol-fuchsin.
Saturated alcoholic solution of Fuchsin............... 5 ec. Solution of carbolic acid (5%)... ... cece cece c ee ee ene 45 ce. 4, Loeffler’s Methylen blue. Saturated alcoholic solution of Methylen blue............ 15 ee. Potassium hydrate (1:10,000)'........ ccc. eee cee ee 50 ee. 5. Ehrlich’s Anilin Oil Gentian violet.? Saturated alcoholi¢ solution of Gentian violet......... 6 ec. Absolute aleobok 6s access gine 4 eG 6a Roald cea woes borkceuee 5 ee AULD Wabel: etaoivek aio iatd ae ecided e4uutedemaadhawas 50 ee.
*This dilution can be readily made by taking 1 ec. of a 10% potassium hydrate solution, making this up to 100 ce., then taking 5 ce. of this and making it up to 50 ce.
*Some prefer anilin oil made as follows: Solution A., 2 ee. of anilin oil and 100 ec. of distilled water; Solution B., 25 ce. of filtered saturated aleo- holic solution of gentian violet and 75 ce. of Solution A. Mix and filter. This stain remains good for a long time.
30 GENERAL BACTERIOLOGY
Anilin water is prepared by adding 2-3 cc. of anilin oil, drop by drop, to 50 ec. of water, thoroughly shaking and then filtering through moistened paper until perfectly clear.
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.
Todime: was ase pets dee eee nev s wed ¥ veg shee y sine She sa elge s 1 gm.
Potdesltint 4Odtde cctv Aie veces eis eats ds sa eames 2 gm.
Drstillédiswater «scan oischgccak wtinetana mines dcuay atest ayeraue a 300 ce 7. Gabbett’s Methylen blue solution.
Methylen, blue (Cdry ae soe ese eee cae win ea nee e eg ares 2 gms.
Sulphuri¢ Ged. wcsssew ses vedere eee see seas eeeees aare's s 25 ee.
DISHES WATET fois a. scaqusisue dr suade dcereeas Fane rnaralen te howe eaecwe. 75 ce.
8. Alcohol, 96%. REFERENCES. A. 163; H. 85; M. & R. 97; P. 200.
SpeciaL DirEcTIONS. Prepare the solutions of dyes from the saturated alcoholic solutions (furnished) and place them in 2 oz. bottles arranged with pipettes and neatly labeled. The bottles are conveniently kept in a block. Fig. 10.
EXERCISE 18. SIMPLE COVER-GLASS PREPARATION Fic. 10. Block for stain bottles.
GENERAL Directions. Bacteria may be studied under the micro- scope in a living condition in a hanging drop preparation (21); but on account of their hyaline character, which makes the exami- nation difficult, the student should first learn to stain them and later make the hanging drop preparation. With a few exceptions all bacteria can be stained by the following process: A small drop (about the size of a pinhead) of distilled water is placed on a clean cover-glass by means of the platinum loop. With a sterile needle a portion of the material te be examined is secured and while the cover-glass is held in the fingers of the left hand the bacteria on the needle are introduced into the water, thoroughly mixed and then spread in a thin film over as much of the surface of the cover-glass as possible. When the bacteria are taken from fluid media a drop of water will not be necessary. In this case use a loop. The film is now allowed to dry. If the drop is sufficiently small this will be a short process. It may be hastened by holding the cover-glass high
32 GENERAL BACTERIOLOGY
over the flame, but it should always be held in fingers to prevent over- heating, which spoils the preparation.
When the film is thoroughly dry place the cover-glass in a pair of Cornet or Stewart foreeps and ‘‘fix’’ the bacteria in the flame. This is done by passing the preparation through the upper portion of a gas flame, film side up. Three passages should be made, each consuming about one second forceps. (Muir & Ritchie). of time. The forceps are now placed on the table and the film flooded with one of the anilin dyes. After the stain has acted for five to ten minutes it is washed off into a waste dish with a stream of dis- tilled water, and while the cover-glass is still wet it is placed, bac- teria side down, on a clean glass slide, being careful to avoid air bubbles. The surplus water is then taken up by means of a small piece of blotting or filter paper.
The preparation is now ready for microscopical examination. (For directions see next exercise).
The preparation can be made permanent either by allowing the water under the cover-glass to dry before it is removed, or by floating it off with water and afterwards drying. When dry a drop of Canada balsam, dissolved in xylene, is placed on the cover-glass and this is then lowered on to the slide again.
Résumé.
a. Spread film,
b. Air dry,
ce, Fix,
d. Stain,
e. Mount in water, e. Dry,
f. Examine, } or hi: Mount in balsam, g. Dry and mount in balsam. g. Examine.
“‘The great mistake made by beginners is to take too much growth,’’ (M. & R.) and too large a drop.
Rererences. A. 159; H. 80; M. & R. 98; McF. 145; P. 198; P. B.C. 11.
SPECIAL DIRECTIONS.
a. Make cover-glass preparation from agar streak of B. subtilis (13) staining with an aqueous solution of gentian violet for five minutes.
oa) Hee
GENERAL BACTERIOLOGY
b. Practice making cover-glass preparations by staining speci- mens from each of your cultures. Use Loeffler’s methylen blue for the gelatin and bouillon; aqueous solution of gentian violet for agar, and carbol-fuchsin for potato. Examine, mount permanently and hand to instructor for inspection.
EXERCISE 19. USH OF MICROSCOPE.
GENERAL Directions. For bacteriological purposes a microscope with a magnifying power of at least 500 diameters is needed. There should be a coarse adjustment (rack and pinion) as well as a fine micrometer screw ; and the following accessories: Two oculars, one 1 in. (25 mm.) and one 2 in. (50 mm.) ; three objectives, one ? in. (16 mm.), one 4 in. (4 mm.), or $ in. (3.5 mm.) and one oil immersion +; in. or 7g in. (2 mm.); a triple nose-piece, and an Abbe substage condenser with iris diaphragm mounting.
In the use of the microscope the following points should be noted:
a. Licut. The proper angle at which the mirror should be placed is best determined by removing the ocular and so arranging the mirror that the unobstructed light from the window covers the whole field. The ideal light is that from a white cloud. Dzérect sunlight should never be used.
b. Aspe ConDENSER. The purpose of the condenser is to furnish a large cone of light, and as it is corrected for parallel rays the plane side of the nurror should always be used, except when artificial light is employed. When highly stained objects are to be examined, the open diaphragm should be used, but when the structural rather than the color picture is desired, it will be necessary to diminish the light by closing the diaphragm. When the high powers are employed, raise the condenser as high as possible; for low powers a lower posi- tion will give better definition.
c. Focusine. Turn the proper objective in place and rack down until the objective nearly touches the cover-glass. This should be dene while the eye ws held at one side and directs the movement. Then with the eye at the tube slowly move up with the micrometer screw. Never rack down with the eye at the tube.
d. Use or O1n-Immersion. The oil-immersion objective is indis- pensable to the proper study of bacteria. It is constructed upon the principle that a drop of fluid having the same refractive index as the
36 GENERAL BACTERIOLOGY
objective, prevents the dispersion of light, thus permitting the use of lenses having a greater numerical aperture and longer working distance for the same degree of amplification than is possible with the dry system. In using an immersion lens, place a small drop of immersion oil on the preparation, then carefully lower the objective until it touches the oil drop and nearly touches the cover-glass. Apply eye to the ocular and focus upward very slowly with fine ad- justment until the definition is clear. At the close of the day’s work the oil must be removed from the objective and cover-glass. This is best accomplished by wiping them with a piece of Japanese paper made for the purpose. In case the oil should accidentally dry on the objective, it can be removed by adding a little more oil and allowing it to stand for a few minutes; it can then be wiped off with paper. If this method does not succeed, the objective should be taken to the instructor. Great care must be observed since solvents of the oil are also solvents for the lens mountings.
REFERENCES. See Gage; A.199;H.118;M. & R. 87; P. 206.
SpeciAL Directions. Examine cover-glass preparations made in previous exercise, first with 4 in. objective, and then with the oil- immersion objective. If the specimen be satisfactory, sketch as directed in next exercise.
EXERCISE 20. DRAWING BACTERIA
GENERAL Directions. In drawing bacteria only a few organisms occurring in the microscopic field should be sketched, but these should be made of considerable size so that the exact outline may be indi- eated. Furthermore they should be drawn to scale and individuals selected to give range in form and size.
To measure microscopic objects an ocular micrometer is used, and the first step will be to determine its value. Place the ocular micrometer on the diaphragm in the ocular, use a stage micrometer as an object and focus. The image of the scale on the stage micro- meter will appear imposed on that of the ocular micrometer. Make the lines of the two micrometers parallel and then make any two lines of the stage micrometer coincide with any two on the ocular micrometer, pulling out the draw-tube if necessary. Divide the value of the included space or spaces on the stage micrometer by the number of divisions on the ocular micrometer required to in- elude them, and the quotient so obtained will give the valuation of the ocular micrometer in fractions of the units of measure of the stage micrometer (Gage). If the result be not in terms of the micron
38 GENERAL BACTERIOLOGY
(#) it should be converted to such, as this is the unit in micrometry. REFERENCES. G. 100-108. SPECIAL DIRECTIONS. a. Determine the value of the ocular micrometer and fill out blanks in following table:
No. of Microscope.............. MBG ici xia aaceiicirsiend APSRMENS Ocular........ Mey, OF vice ccna mm. Value of single di- Objective. Tube length. vision on scale in p. 2% in. (16 mm.)
1% in. (4 mm.)
Oil-immersion.
b. Measure the bacteria on the preparations made in Exercise 18 and sketch a few individuals from each.
In making drawings, represent a micron by two and one-half millimeters on paper. This will give a magnification of 2,500 diame- ters, represented thus: > 2,500.
EXERCISE 21. HANGING-DROP PREPARATION.
GENERAL DrrEcTIons. These are made by adding a small portion of bacterial culture from solid media to a drop of water on a clean cover-glass, or, in case of fluid media, by placing a small loopful of the culture medium on the cover-glass. A hollow ground glass slide having the rim of the cavity previously coated with vaseline, is inverted and lowered over the cover-glass enclosing the drop. With a careful, quick movement the preparation is now brought right side up.
WLLLZZZ. TOOT TI TITLE
Fig. 12. Hanging-drop preparation. a, Hanging drop; b, Vaselin.
40 GENERAL BACTERIOLOGY
Instead of the hollow ground glass-slide an ordinary glass-slide to which a small section of a glass or rubber tube has been cemented can be used, and in some eases is preferable.
In examining the preparation under a microscope, focusing is a somewhat difficult process and must be carried out with great care. Use a narrow diaphragm. Find the edge of the drop with the low power (2 in. objective), adjusting slide so that edge of drop passes through the center of the field; then turn on the high power (4 in. objective) and focus without moving the slide. The edge of the drop is selected because the bacteria are here nearest the cover-glass and hence more easily focused upon than where they are deeper in the drop.
RerFereNces. <A. 204; H.114;M.& BR. 87; McF. 141; P. 209.
SPECIAL DIRECTIONS.
a. Make a hanging-drop preparation of water containing parti- eles of India ink or carmine in suspension. This illustrates molec- ular or Brownian movement.
b. Make a preparation using straw infusion or tartar from teeth to note variations in rate and character of vital movement.
c. Make hanging-drop preparation of B. subtilis from agar or bouillon (13).
d. Make same preparation of B. coli (18).
In cases where vital movement is questionable, remove the cover- glass and place a drop of formalin or chloroform in the bottom of the cell; replace the cover-glass, examine and note change in character of movement, if any.
EXERCISE 22. MICROSCOPICAL STUDY OF FORM TYPES.
a. Make bouillon and agar streak cultures of the following or- ganisms :
Micrococcus (any species).
Sarcina lutea SCHROETER.
Pseudomonas fluorescens (FLUEGGE) Mic.
Bacillus mycoides FLUEGGE.
Microspira Metschnikovi Mia. (or any vibrio).
Spirdlim rubrum v. ESMARCH.
b. Incubate cultvres at 28° C. for 24 hours.
42 GENERAL BACTERIOLOGY
c. Make cover-glass preparations from the agar streaks and stain with an aqueous solution of gentian violet or with Loeffler’s methylen blue.
d. Examine with the oil-immersion objective, and write the names of the organisms in their proper places in the table below:
Shape of Relative : : Name. Sketch. organism. size. Medium. Spherical, Small. Large. Elongated. Small. Short. Spiral. Long.
e. Make sketches of each organism.
f. Mount all preparations in balsam and hand them to instructor for inspection.
EXERCISE 23. STUDY OF CELL GROUPING.
IMPRESSION PREPARATIONS. The exact relation of cell to cell ag they develop in the colony can frequently be determined best by studying a ‘‘contact preparation’’ which is prepared as follows:
a. Melt a gelatin tube and slope it, when solid make a streak culture of B. mycoides and when growth has taken place dip the tube in hot water to loosen gelatin, which is then slipped out of the tube.
b. Lower gently a clean cover-glass over the surface. Apply a slight pressure by tapping glass. Raise cover-glass by one edge, taking care that natural arrangements of adherent bacteria are not disturbed.
c. Thoroughly air dry the same, then fix and stain in the ordinary manner.
44 GENERAL BACTERIOLOGY
d. Examine the thinner layers, noticing the arrangement of cells with reference to each other, and draw a sufficient number to illus- trate this relationship.
Hanainc-Drop PREPARATIONS.
a. Make hanging-drop preparations from bouillon cultures pre-
pared above (22) and also from those supplied.
b. Examine with oil-immersion objective and assign organisms to their proper places, as determined by cell grouping, in the follow-
Ing scheme: Arrangement. Form. Name. Sketch. Spheres. Isolated. Rods. Spirals. Spheres. Filaments. Rods. Spirals. Plane surfaces. Spheres. Regular masses. Spheres. Spheres. Irregular masses. Rods.
Acar Hancinc-Drop CULTURES (Wesbrook).
a. Melt a tube of agar and cool to 43° C. b. Sterilize a cover-glass by passing it two or three times through
the flame quickly.
c. With the needle make a streak on the cover glass about 3 mm.
long of B. subtiles.
46 GENERAL BACTERIOLOGY
d. With the loop place a drop of liquid agar so as to cover up streak.
e. Flame a hollow-ground slide and seal the cover glass to it. In- cubate and later examine and sketch.
RerereNnces. Hill, Hanging Block, Jour. Med. Research, 1902, 2; 202.
EXERCISE 24. STUDY OF INVOLUTION FORMS.
a. Grow Bacillus subtilis (HHRENB.) Mia. in bouillon, and also in water containing 0.1% asparagin, 10% sugar, and by means of stained cover-glass preparations compare the individual organ- isms in each case in regard to their form and size. The degenerated or involution forms are more apparent by staining. Draw several cells illustrating a variety of involution forms.
b. Examine a culture of Bacterium diphtheriae (LOEFFLER) Mia. on Loeffler’s blood serum. Read M. & R. 5.
EXERCISE 25. STUDY OF ENDOSPORES.
a. Make cultures on peptoneless agar, or on an agar tube to which a few drops of calcium hydrate have been added, of the following organisms and incubate at 28° or 38° C.:
Bacillus subtilis (HHRENB.) COHN.
Bacterium anthracis (KocH) Mic. (or Bacillus mycoides FLUEGGE).
Bacillus amylobacter Van TreGHEM (or any clostridium form).
Bacillus tetani Nicouater (or any ‘‘drumstick”’ bacillus).
b. When the cultures are 48 hours old mount films without stain- ing, examine and fill out following table:
Size of Spore. Position. Name of organism. Sketch.
Smaller than di- Median.
ameter of mother-
si Polar. Median.
Larger than diam- eter of mother-
cell. Polar.
48 GENERAL BACTERIOLOGY
c. Simple stain for spores.
1. Prepare film of B. subtilis.
2. Fix by passing through flame 10 or 12 times instead of 3 times. (This prevents the vegetative portion from taking the stain).
3. Stain 2-5 minutes in hot earbol-fuchsin.
4. Mount and examine.
d. Double stain for spores (Hauser’s method).
1. Prepare a film of any of the above organisms (providing a previous examination has shown that the spores are fully developed and the mother-cells have not disintegrated).
2. Fix, three times through the flame.
3. Stain with hot (steaming) carbol-fuchsin for 5 minutes.
4. Cautiously decolorize with 5 per cent. acetic acid until the pink color is nearly removed from the film.
5. Wash thoroughly in water.
6. Dry (blot).
7. Stain with Loeffler’s methylen blue, 3 minutes.
8. Mount and examine. The spores should appear crimson in blue bacilli.
Rererences. A. 171; H. 98; M. & R. 106; McF. 154; P. 46 & 2038; P. B. C. 15.
t
EXERCISE 26. FLAGELLA STAIN (Bunce).
GENERAL DIRECTIONS.
a. Make an agar streak of the organism to be stained.
b. After 18 to 24 hours, by means of the platinum needle re- move a portion ot the growth (being careful to avoid the culture medium) to a large drop of tap water on a perfectly clean cover- glass (16). Allow this to stand 5 minutes rather than spread, as there is less danger of breaking off the flagella.
c. Spread carefully 2 or 3 loopfuls of this drop on each of sev- eral clean cover-glasses and dry at room temperature.
d. Fix by passing the cover-glass while it is held in the hand, (not in the forceps, as over-heating will injure the preparation) through the top of the flame.
c. Flood the cover-glasses thus prepared with the following solu- tion (mordant): Liquor ferri sesquichloridi diluted with distilled water 1:20, 1 part; saturated aqueous solution of tannic acid, 3 parts. This mixture improves with age but should be filtered before using. Allow to act 1 minute.
f. Wash in water and dry between filter paper.
50 GENERAL BACTERIOLOGY
g. Stain with hot carbol-fuchsin for anout one minute.
h. Wash in water, dry and mount in balsam.
REFERENCES. A. 174; H.100;M. & R. 107; McF. 156; P. 205.
SprciaL Directions. Stain B. typhosus from cultures fur- nished, also try B. coli and B. subtilis.
EXERCISE 27. CAPSULE STAIN (Wetcx). GENERAL DIRECTIONS.
a. Spread film without the use of water.
b. Air dry.
c. Fix.
d. Apply glacial acetic acid, and drain it off immediately. Do not wash in water.
e. Apply Ziehl’s carbol-fuchsin which is to be renewed several times to remove acid.
f. Wash in 1 to 2% salt solution.
g. Examine in salt solution. (Balsam causes capsule to shrink).
REFERENCES. A. 170; H. 97; M. & R. 106; McF. 291; P. 203; P. B.C. 13.
SpEcIAL DirEcTIONS. Use pneumonic (‘‘rusty’’) sputum, blood of rabbit infected with the Bact. pneumoniae or a milk culture of a capsule bearing organism as Bact. pneumonicum (Fried.) Mig. or Bact. capsulatum (Stern.) Chester.
EXERCISE 28. STAIN FOR METACHROMATIC GRANULES (Ernst).
a. Stain a young culture of an organism such as Bact. diphtheriae with Loeffler’s methylen blue for about 3 minutes.
b. Wash in water.
c. Treat with a saturated solution of Bismarck brown for 30 see- onds.
d. Wash in water, mount in water and examine, or, dry, mount in balsam and then examine.
The granules should appear blue in a brown organism.
EXERCISE 29. MORPHOLOGY OF YEASTS AND MOULDS COMPARED WITH BACTERIA.
a. Mount some baker’s yeast (Saccharomyces cerevisiae) and examine in an unstained condition. Compare: Size; form; struc- ture and method of reproduction with the bacteria.
b. In same way examine a number of common moulds, e. g. Mucor, Penicillium and Aspergillus.
52 GENERAL BACTERIOLOGY
EXERCISE 30. GELATIN PLATE CULTURES.
EXPLANAToRY. Plate cultures are only possible with the lique- fiable solid media, gelatin and agar. In making them the bacteria are mixed with the medium while it is in a fluid state and spread out on a horizontal surface to cool. The dilution is such that the indi- viduals are separated from each other by several millimeters. In the solidified medium the organisms are fixed and their growths result in the formation of ‘‘colonies.’’ These vary in size and ap- pearance according to the peculiarities of the organism and the age of the culture, but are of the greatest service in the study and identi- fication of the various species. These cultures are prepared as fol- lows:
GENERAL Directions. Three gelatin tubes are marked Nos. 1, 2 and 3 and melted by placing them in a water bath at a temperature of 42° C. For this purpose a small cup of water placed on a tripod can be used (Fig.
& 13). They are inoculated by introducing the € material to be studied into tube No. 1. The quantity of this material varies. The amount clinging to the platinum needle will be suffi- cient if a pure culture be used, while in other cases several loops or even drops are necessary. The inoculated material is thoroughly mixed with the gelatin in No. 1. This is done by rolling the tube gently between the palms of Fra. 13. Method of met. the hands, instead of shaking, so as to prevent
ing gelatin. the introduction of air bubbles. With a ster- ile loop two loopfuls of fluid gelatin are now transferred from No. 1 to No. 2, and mixed. For method of handling tubes see Fig. 14. In like manner three or more loops from No. 2 are carried over to No. 3, which in turn is well mixed. The con- tents of each of the tubes are now poured into separate sterile Petri dishes. The process of pouring is performed as follows: The
Fie. 14. Another method of holding test-tubes.
54 GENERAL BACTERIOLOGY
Petri dish is placed on the desk; the gelatin tube is taken in the right hand, the cotton plug removed with the left hand; the mouth of the tube sterilized by flaming it once or twice, and, when the glass is cool, the gelatin is poured into the lower half of the dish while the cover is slightly raised (Fig. 15), but not inverted or laid on the table. The cover of the dish is then replaced, the test-tube filled with a solu- tion of corrosive sublimate, and the cotton plug returned. The gelatin is spread over the entire bottom of the dish by tipping it from side to side. It is then allowed to harden by placing the dish on the cooling apparatus, or leaving it on a horizontal surface at room temperature. A simple, inexpensive and effective cooling apparatus is a piece of soapstone, such as is sold at hardware stores (Fig. 16). In winter this can be cooled by hanging it out of doors, at other seasons by im- mersing it in cold water. The Fic. 16. Soapstone used for solidifying | three Petri dishes thus prepared gelatin in Petri dishes. should be properly labeled and placed under conditions where the gelatin will remain solid and yet growth will take place. The temperature of the laboratory should not be allowed to exceed 23° C. or gelatin cultures are in danger of melting while under examination. Within a few days colonies will make their appearance, in varying numbers, depending upon the dilution used.
Inasmuch as the first plate is generally too thickly seeded to be of much service, this gelatin tube is often replaced by a water blank, which is treated exactly as the gelatin tube No. 1, but is not, of course, “‘plated’’ but used simply to dilute the material.
Rererences. A. 130; H.65;M. & R.53; McF. 199; P. 224.
Fig. 15. Method of pouring plates.
tow
A t f Ren
SPECIAL DIRECTIONS.
a. Make three gelatin plate cultures, as directed above, and inoc- ulate with B. subtilis, introducing a minute portion of agar culture (13) into tube No. 1, two loops of No. 1 into No. 2, and three of No. 2 into No. 3. Label, and when the gelatin has solidified, place plates in cool chamber (14).
56 GENERAL BACTERIOLOGY
b. Also make a ‘‘blank’’ plate from an uninoculated gelatin tube, observing all precautions to prevent contamination. This will serve as a control or check on your other plates. If any colonies develop on this it will indicate carelessness.
EXERCISE 31. AGAR PLATE CULTURES.
GeNneEriL Directions. These are made in the same way as the gelatin plates except that the high melting point (96° C.) of agar makes it necessary to use boiling water to melt it. Inasmuch as the vitality of vegetative bacteria is destroyed at a temperature much above 42° C. it must be cooled down before it is inoculated, but as agar solidifies at 39-40° C. it must not, therefore, be cooled below that point. It is best to keep the melted agar at about 45° C. for 10 minutes before it is inoculated. For this purpose a water:bath should be so arranged that the temperature can be controlled by means of a thermo-regulator. A cheap and yet satisfactory arrangement is represented in Fig. 17. Inoeulate, make dilutions and H pour as in ease of gelatin, except that be- fore the agar is poured, it is well to slightly warm the Petri dishes by placing them in the incubator at 38° C. for a few minutes, otherwise the agar may solidify in lumps in the plate. In cooling, agar shrinks some- what, and in doing so water is expressed from the solid jelly. In the incubator this condenses on the under side of the cover of Fra. 11. Water bath for cool- the Petri dish to such an extent that drops
run down on to the culture surface, thus causing the developing superficial colonies to ‘‘run.’’ To obviate this the Petri dishes, when placed in the incubator, should be in- verted
Rererences. A.135;H.68;M.&R.57;N. 285; P. 225; P.B.C. 28.
SpeciaL Directions. a. Make three agar plates of B. coli; use one loop of bouillon culture (13) for tube No. 1 and proceed as in 30. b. Invert and place in incubator at 28° C.
EXERCISE 32. ROLL CULTURES (Esmarcn).
GENERAL Directions. These are essentially plate cultures in which the medium instead of being poured out into dishes is solidi-
58 GENERAL BACTERIOLOGY
fied in a thin, even layer on the inner surface of the test-tubes. This is best accomplished by means of a piece of ice placed in a dish on a piece of cloth, by which it can be kept in the desired position
: (Fig. 18). A hori- zontal groove is melt- ed in the ice by means of a test-tube filled with hot water. In this groove the test- tubes, inoculated as 41 in case of plate cul- tures, are rapidly | whirled until the me- } dium is thoroughly set. Both agar and gelatin can be used, although gelatin cannot be used successfully with those species which liquefy this medium. In the ease of agar the tubes should be placed in a horizontal position a few hours (over night) until the medium has become attached to the tube; afterwards they can be stored in the usual receptacles for tube cultures.
Rererences. <A. 137; HH. 69;M. & R. 56; McF. 206.
SpectaL Directicns. a. Melt.a tube of gelatin and without inoc- ulating it practice making a roll-culture as described above. Avoid tipping the tube enough to get medium on cotton plug. Remelt and roll again and again until the knack is acquired.
Fig. 18. Method of making "Roll. cultures. (Abbott).
b. Make two roll-cultures in gelatin of B. colt (18), using a water- blank instead of gelatin tube No. 1.
c. Make two agar cultures of B. subtilis in same way. d. Incubate b. in cool chamber, and c. at 28° C.
EXERCISE 33. STUDY OF PLATE CULTURES.
Macroscopic. <As the colonies appear, note: a. form, b. size, c. surface elevation, d. consistency, e. color. Both the surface and deep colonies should be described, as they are frequently very differ- ent. Drawings should always be made wherever they will be of value; study should be continued as long as changes are noticed. (See Chapter ITI.)
Microscopic. The colonies appearing on the plates are to be studied under a low power of the microscope. Use a $ in. (16 mm.)
i
60 GENERAL BACTERIOLOGY
objective. The Petri dishes can be inverted, and thus avoid the danger of exposing the culture to contamination from the air except with gelatin where liquefying organisins are present. Observe, a. structure of colony as a whole; b. character of margin. (See Chap- ter ITI.)
SpecraL Directions. Study, write descriptions and make draw- ings of all plate cultures. Use blank pages for description and sketch of cultures.
EXERCISE 34. USE OF DECOLORIZING AGENTS.
Make three cover-glass preparations from a 24 hour old eulture of B. subtilis, staining them with an aqueous solution of gentian violet. Mount in water and examine. While they are still under the microscope, place at one side of the cover-glass a few drops of one of the following solutions, and by means of a strip of filter paper at the opposite side draw the liquid under the cover glass until all the color is removed. In this way determine the relative value of aleohol (95%), acetic acid (5%), and nitric acid (30%) as decolor- izing agents.
EXERCISE 35. GRAM’S STAIN.
Expuanatory. This is a differential stain and one of the most useful. Some bacteria when stained by this method exhibit a dark violet color, others remain perfectly colorless, thus rendering pos- sible the differentiation of bacteria which are morphologically nearly or quite identical, and also greatly facilitating the demonstration of certain bacteria in animal tissue. Most of the pathogenic micrococei retain the violet stain, although there are important exceptions. The bacilli and spirilla may or may not remain colored.
GENERAL DIRECTIONS.
=}
. Spread film. . Air dry and fix. Stain with anilin-oil gentian violet 114 minutes. . Pour off stain and without washing. Apply Gram’s iodine solution (17, 6) 114 minutes.
Apply 96% alcohol 3 minutes, or until drippings do not stata white filter paper.
© af Ss
a
g. Wash in water,
62 GENERAL BACTERIOLOGY
h. Mount in water and examine. i. Dry and mount in balsam.
Rererences. A. 169; H. 89;M. & R. 102; McF. 150; P. 203.
SpeciaL Directions. Stain films of young cultures of B. coh and B. subtilis.
EXERCISE 36. TUBERCLE STAIN (Gaszetr).
Expuanatory. All of the differential methods of staining the tubercle bacterium depend upon the fact that this germ is very resistant towards the ordinary stains, and, in order to be stained at all must be treated with a dye containing a mordant and this either allowed to remain in contact with the micro-organism several hours or be appled hot. The latter method is the quicker and is usually employed, although it does not give as good results. When once stained this germ withstands the effect of decolorizing agents to such an extent that it is possible to remove the dye from all other objects on the cover-glass preparation (as in sputum) while it retains its own color. The application of a second dye, of a complementary color, readily distinguishes this germ from all others in the field. A few other bacteria have similar staining qualities. Red is the usual stain and blue the counter stain. Gabbett’s method is one of the simplest.
GENERAL DIRECTIONS.
. Spread film (sputum from tuberculous patient). . Air dry and fix.
. Stain with hot carbol-fuchsin 2 minutes.
. Wash in water.
. Treat with Gabbett’s solution 14 to 1 minute.
. Wash in water and examine.
. Dry and mount in balsam.
REFERENCES. <A. 167; H. 244;M. & R. 104; McF. 308; P. 304.
QQ meaqagoewea
Speciau Directions. Stain three samples of sputa which con- tain varying numbers of the tubercle bacteria.
64 GENERAL BACTERIOLOGY
CHAPTER IT
PHYSIOLOGY OF BACTERIA.
EXERCISE 37. PREPARATION OF SPECIAL MEDIA.
The following media will be necessary for the work outlined in this chapter:
a. DEXTROSE BouILton. To ordinary bouillon add 1% dextrose (c. P.), tube and sterilize in steamer, not in autoclave, 7 test-tubes and 2 fermentation tubes.
b. DExTROSE GELATIN. 1% dextrose (c. P.), tube and sterilize in steamer, 6 tubes.
c. Dextrose AGAR. 1% dextrose (c. P.), tube and sterilize in steamer, 5 tubes.
d. Lacroszs Acar. 1% lactose (c. P.), tube and sterilize in steamer, 2 tubes.
e. Litmus Souution. To 10 gms. of the dried material add 500 ec. of distilled water, digest in a warm place, decant clear liquid and add a few drops of nitric acid to produce a violet color. (Sutton.) Place in flasks or test-tubes and sterilize in steamer three times, 1 tube.
f. DExTROSE-FREE BrotH. This is prepared from beef by inocu- lating the meat infusion with an organism capable of fermenting sugar, such as B. coli, and allowing it to stand several hours at 38° C. (Between b. and c. Exercise 4.) The bouillon is then prepared in the usual manner.*
Or DunHAM’s SOLUTION. Sodium chloride 0.5 gm. Peptone (Witte) 1. gm. Water 100. gms.
g. NITRATE SOLUTION.
Boil until all is dissolved, filter, tube and sterilize, 4 tubes.
1§mith: Jour. Exp. Med., 1897, 2: 548,
66 GENERAL BACTERIOLOGY
Sodium chloride 0.5 gm. Peptone (Merck) 1. ‘“ Potassium nitrate 0.2 ‘‘ Water 1,000. gms. |
h. Litmus MILK.
1) Freshly separated milk, or if this is not available, new milk is placed in a separatory funnel in an ice chest over night to allow the separation of the cream and the milk then drawn off.
2) Litmus solution (e. above) is then added until medium is faintly blue.
3) Tube and sterilize in the steamer for 30-45 minutes on 3 or 4 consecutive days. During the summer months particularly very resistant bacterial forms abound in the milk, so that it is necessary to increase the number of applications or length of exposure. The efficiency of the sterilizing process should be tested by placing the milk in the incubator 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 ec. for 41. and 44.) 10 tubes of gelatin, 15 tubes of agar, 6 water-blanks and 5 potato tubes.
(If thought desirable the media required for Chapters IV. and V. [Exercise 58], may be prepared at this time; this would then com- plete all the media making required in Part I.)
i Filter, tube and sterilize, 3 tubes.
EXERCISE 38. EFFECT OF REACTION OF MEDIA ON GROWTH.
GENERAL DIRECTIONS.
a. Melt 6 tubes of gelatin and add, under aseptic precautions, to three of them, respectively, 0.1 ec., 0.3 ec., and 0.5 ec. of a normal solution of hydrochloric acid, and to the other three the same amounts of a normal sodium hydrate solution.
b. Thoroughly mix, solidify gelatin in ice water and then inocu- late (stab) each tube with the organism to be studied..
c. Make a control culture in a tube of neutral gelatin.
d. Incubate at 18° C. and note the effect of the chemicals on the rate, amount and character of the growth.
Rererences. L. & N. 35; MeF. 41.
SPECIAL Directions. Use B. subtilis and B. coli, Make sketches.
EXERCISE 39. 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.
68 GENERAL BACTERIOLOGY
b. Inoculate both with a pure culture of B. subtilis and incubate at 28° C. Explain changes which occur. c. Test extract of beef or syrup in the same way.
EXERCISE 40. 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 ineubator (28° C.), blood heat (38° C.).
c. By frequent observations as to luxuriance of growth, deter- mine the optimum temperature of growth for each.
REFERENCES. F.73;L.&N. 44; McF. 44.
SpeciaL Directions. Use a mesophilic bacterium as B. coli and a psychrophilic organism as Ps. violacea.
EXERCISE 41. 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, 5 test-tubes (16 mm. in diam.) containing exactly 10 ec. of standard bouillon. (Reaction + 1.5.)
c. After 15 minutes exposure at this temperature remove the cotton plug from one of the tubes, inoculate the broth with three loopfuls (standard size, 12) of the 48 hour old culture (a.), and carefully mix by slightly agitating the tube, without removing it from the hath.
d. After a further exposure of 10 minutes remove the tube from the bath and place it immediately in a vessel of ice cold water to cool. Then incubate at a temperature favorable to the development of the organism under observation.
e. Raise the temperature of the bath 5 degrees, i. c., to 50° 'C., inoculate another tube. Keep it at 50° for 10 minutes, remove. cool and incubate.
f. In the same manner expose the organism to the following temperatures : 55°, 60°, and 65° C. for a period of 10 minutes each.
g. In all cases incubate at least a week and take as the thermal death point the lowest temperature at which growth fails to appear.
70 GENERAL BACTERIOLOGY
(In more accurate work the temperature should be determined within 2° C.) REFERENCES. M. & R. 70; MecF. 246; P. 146; P. B. C. 32. Speciau Directions. Use B. coli or B. typhosus.
EXERCISE 42. COMPARATIVE EFFICIENCY OF DRY AND MOIST HEAT.
GENERAL DIRECTIONS.
a. Charge a water blank with culture of a spore-bearing bacillus, shaking it well to break up the clumps.
b. Sterilize eight cover-glasses by passing them several times through the flame, and place four in each of two sterile Petri dishes.
c. With a sterile loop place an equal quantity of the bacterial suspension (a@.) on each cover-glass, and dry by placing Petri dishes in the incubator with the covers slightly raised.
d. When dry place one Petri dish in the dry sterilizer (near the thermometer), and the other in the steamer.
e. Keep both sterilizers at a temperature of 100° C., and at the end of 5, 10, 20 and 40 minutes respectively, remove one cover-glass from each Petri, place it in a sterile Petri dish and pour a tube of liquefied gelatin or agar over it. Tip the dish from side to side to dislodge as many of the bacteria as possible from the cover-glass, solidify the medium and incubate.
SpreciAL Directions. Use an old (spore-bearing) culture of B. subtilis. Arrange data in the form of a table.
EXERCISE 43. EFFECT OF DESICCATION.
GENERAL DIRECTIONS.
a. Prepare five cover-glasses each of a spore-bearing and a non- spore-bearing culture, as directed in 42.
b. Place them in sterile Petri dishes, 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. 40.
SPecIAL Directions. Use a young culture of B. colt and an old (spore-bearing) culture of B. subtilis. Tabulate results.
72 GENERAL BACTERIOLOGY
EXERCISE 44. 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 ce. of a 5% solution of carbolic acid to one tube (No. 1) ; 0.6 ce. to another (No. 2) ; and 2 ce. to the third (No. 3).
c. Two hours later transfer three loopfuls from each tube to sterile bouillon and incubate all of the tubes at 38° C.
d. The carbolie acid does not prevent growth in No. 1 or its sub- culture. 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. 37; L. 107; McF. 45.
SPECIAL Directions. Use B. coli.
EXERCISE 45. 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 organism.
b. Sterilize a piece of mica or a cover-glass, by passing it several times through the flame and place this over several of the streaks. This is to shut out the air and should therefore be in perfect contact with the medium.
c. Make another plate in the same way, using an anaerobe,
Rererences. F. 60; L. & N. 41; L. 180; M. & R.19; MeF. 212; P. 151.
SpeciaL Directions. Use B. subtilis and an anaerobe. Sketch.
EXERCISE 46. EFFECT OF DIRECT SUNLIGHT.
GENERAL DIRECTIONS.
a. Make an agar plate of the organism to be studied (seeding rather thickly).
b. When agar has thoroughly set, invert the Petri and paste on under side a piece of black paper from which has been eut out a number of letters, c. g. stvdent’s initials.
c. Expose this dish, paper side up, to the direct sunlight for a number of hours (4-6).
74 GENERAL BACTERIOLOGY
d. Remove the paper and incubate. REFERENCES. F.71;M.& R. 20; L.77; McF. 41; P. 135.
Spectay Directions. Use B. prodigiosus (EHRENB.) FLUEGGE. Sketch.
EXERCISE 47. DETECTION OF GAS (SHaxe Currure).
GENERAL DIRECTIONS.
a. Melt a tube of dextrose agar or dextrose gelatin and inoculate with a gas-producing organism.
b. Thoroughly mix and solidify by placing in ice water.
c. Incubate over night.
Rererences. H.70;L.&N.89;M.&R. 78; McF. 49; P. 82.
SpEcIAL Directions. Use B.coli;incubate. Make sketch.
EXERCISE 48. QUANTITATIVE ANALYSIS OF GAS (Fermentation TUBE).
GENERAL DIRECTIONS. Pe
a. Inoculate the open arm of /*4_!
a fermentation tube with a gas-
producing organism. da! b. Incubate at 38° C.
c. By frequent observations
determine: 1. Whether growth takes place in the open or closed arm,
at j}—_| pt |
a a 244
4. e., whether it is aerobic or an- J NY aerobic. Fig. 19. Fermentation tube, showing 2. The rapidity and_ total method ob using Esspmeten
amount of gas formation. Use Frost’s gasometer. (Plate I.)
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 NaOH with the gas in the closed arm, then without removing the thumb return the gas to the closed arm, remove the thumb, when the r-edium will rise in the closed arm to take the place of the absorbed CO,. Measure. The remaining gas is considered as hydrogen; bring this into the open arm, remove the thumb and introduce a lighted match. Air mixed with the hydrogen present causes a slight explosion. Express the amount of CO, and H. in the form
- of a proportion O05 = Paes :
%;
76 GENERAL BACTERIOLOGY
REFERENCES. Smith: Wilder Quarter Century Book, 1893, p. 187; A. 212; McF. 49; M. & R. 79; P. 82.
SPECIAL Directions. Use B. colt; also try B. subtilis.
EXERCISE 49. DETECTION OF ACIDS AND ALKALIES (Worrtz).
GENERAL DIRECTIONS.
a. Melt a tube of lactose agar (or lactose gelatin) and add enough of a sterile blue litmus solution (37 e.) to give it a distinct color, cool to 42° C., inoculate it with an acid-producing organism and pour in the usual manner.
b. When the agar has solidified invert the dish and place it in the incubator.
REFERENCE. MceF. 51.
SpEcIAL Directions. Use sewage, putting a drop in a water blank and using a loop or two of this.
EXERCISE 50. QUANTITATIVE DETERMINATION OF ACIDS.
GENERAL DIRECTIONS.
a. Inoculate 5 test-tubes of dextrose bouillon (or milk) with an acid-producing organism.
b. Twenty-four hours later remove, with a sterile pipette, 5 ee. of the medium from one of the tubes and titrate with a twentieth normal potassium (or sodium) hydrate solution, using phenolphtha- lien as an indicator.
c. Make titrations as described above on each of the four suc- ceeding days, using the same amount of culture each day.
d. Plot the results, expressing the number of ce. of hydrate solu- tion as ordinates and the daily intervals as abscissae.
SpreciAL Directions. Use B. coli and incubate at 38° C.
EXERCISE 51. DETECTION OF NITRITES IN CULTURES.
GENERAL DIRECTIONS.
a. Make a culture of a reducing organism in a test-tube of the nitrate solution (37 g.).
b. Ineubate at 28° C. for 1 week, add 1 cc. of each of following solutions :
1) Sulphanilic acid (para-amido benzenesulphonic acid) 0.5 gm. Acetic acid (sp. gr. 1.04) 150 ee.
2) a-amido-naphthalene acetate. Boil 0.1 gram of solid a-amido- naphthalene in 20 ce. of water, filter the solution through a plug of
78 GENERAL BACTERIOLOGY
washed absorbent cotton, and mix the filtrate with 180 ec. of diluted acetic acid. All water and vessels used must be free from nitrites. (Leffmann 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. 226; McF. 53. SpecraL Directions. Use sewage.
EXERCISE 52. DETECTION OF AMMONIA.
GENERAL DIRECTIONS.
a. Make bouillon culture and incubate 24 to 48 hours.
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 analy- sis). A yellow to reddish brown color indicates the presence of ammonia. +
ReFereNcE. UL. & N. 78. SpecrAL Directions. Use sewage to inoculate medium.
EXERCISE 53. DETECTION OF SULPHURETTED HYDROGEN.
GENERAL DIRECTIONS.
a. Make a culture in a test-tube, or better, in 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 sa and can be best detected by frequent observations.
Rererence. L. & N. 76.
SpeciAL Directions. Use B. coli or sewage.
EXERCISE 54, DETECTION OF INDOL.
GENERAL DIRECTIONS.
a. Make two cultures in tubes of sugar-free broth (or Dunham’s solution).
b. Five days later add a few drops of concentrated sulphuric acid. The appearance of a pink color indicates that nitroso-indol has been formed (cholera-red reaction). If the pink or deep red color does not appear, add 1 ce. of sodium nitrite solution (sodium
80 GENERAL BACTERIOLOGY
or potassium nitrite 0.02 grams and distilled water 100cc.). The appearance of a red color indicates formation of indol.
REFERENCES. A. 223; H. 21; L. & N. 142; McF. 57; M. & R. 80. SpeciAL Directions. Use B. coli or sewage.
EXERCISE 55. DETERMINATION OF CHEMICAL ENZYMES IN CUL- TURES.
GENERAL DIRECTIONS.
a. Make two gelatin stab cultures of a rapidly liquefying organ- ism and incubate several days or until the gelatin has all been lique- fied and then add to each 7 ce. of a 5% solution of carbolic acid for each ec. of medium, shake thoroughly and filter.
b. Pour one into a tube of sterile gelatin and the other into a tube of milk and note changes.
REFERENCE. McF. 56. SpecraL Directions. Use B. subtilis or B. prodigiosus.
EXERCISE 56. VARIATION IN ENZYME PRODUCTION.
" Make stab cultures of Pseudomonas aeruginosa (SCHROETER) Mie. (B. pyocyaneus), or any slow liquefier, in ordinary neutral gelatin and also in dextrose gelatin. Compare rate of liquefaction in each.
EXERCISE 57. 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.
82 GENERAL BACTERIOLOGY
CHAPTER III
TAXONOMY
In order to become acquainted with a particular organism, to differentiate it from its congeners or to assign it a definite place in a system of classification, it must be studied under various conditions and its characters determined as indicated in the following table.
POINTS TO BE OBSERVED IN THE STUDY OF BACTERIA.
The following scheme gives the most important points to be noted in the description of an organism, together with some of the more common descriptive terms suggested by Chester and others.
MORPHOLOGICAL CHARACTERS.
a. Form and arrangement: Spherical, micrococcus, single and irregularly grouped ; diplococcus, streptococcus, tetracoccus, sarcina, rods, single, in chains and in filaments; spirals.
b. Size.
1. In terms of the micromillimeter; breadth, average and ex- treme length.
2. In terms of human blood cells.
c. Stain.
1. Aqueous solutions: stains easily or with difficulty ; uniformly or irregularly.
2. Special stains: Gram; tubercle; ete.
d. Motility.
1. Brownian movement.
2. Vital movement: sluggish or active; rotary or direct; most favorable temperature; age; media; ete.
3. Flagella: stained by Loeffler, Bunge or Van Ermengem’s method; distribution: monotrichous, lophotrichous or peritrichous.
e. Capsule: stained by Ziehl; Gram or Welch’s method; most favorable conditions ; broad or narrow; present in serum, milk or on agar streaks.
GENERAL BACTERIOLOGY 83
f. 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.
g. Vacuoles (plasmolysis).
h. Crystals.
4. Involution forms.
j. Pleomorphism.
1. Effect of various media.
2. Effect of reaction of media.
CULTURE CHARACTERS.
Puate-Cu.turss (Gelatin and Agar).
I. Surface Colonies.
1. Form: Punctiform, too small to be defined by the naked eye; circular; oval; fusiform, spindle-shaped, tapering at each end; cochleate, twisted like a snail shell (Fig. 20, A); conglomerate, an aggregate of similar colonies (Fig. 20, B) ; ameboid, very irregular like the changing forms of amebae (Fig. 20, C); rhizoid, of an irregular branched root-like character (Fig. 20, D); floccose, of a dense woolly structure; curled, filaments in parallel strands, like locks or ringlets (Fig. 20, FE); myceloid, a filamentous colony with the radiate character of a mould (Fig. 20, F) ; filamentous, an irreg- ular mass of loosely woven filaments (Fig. 20, G); rosulate, shaped like a rosette.
Fra. 20. Types of Colonies. A. Cochleate (B. coli, abnormal form). B. Conglomerate (B. Zopfii). C. Ameboid (B. Vulgatus). D. Rhizoid (B. mycoides). E. Curled (B. an- thracis). F. Myceloid (B. radiatus). G. Filamentous.
84 GENERAL BACTERIOLOGY
2. Size expressed in millimeters.
3. Surface elevation: Flat, thin spreading over the surface (Fig. 21, a); effused, spreading over the surface as a thin veilly layer, more delicate than the preceding; raised, thick growth, with
pny abrupt, terraced edges (Fig. 21,
GLA b) ; convex, surface segment of a
cezzzzzzzzzZZZ s eirele, but very flatly convex
(Fig. 21, c); pulvinate, surface
© ac {22 segment of a circle, but decided-
ly convex (Fig. 21, d) ; capitate,
SELL LD hemispherical (Fig. 21, e) ; wm-
a 2 bilicate, shaped like a navel (Fig.
a natty, Surteae Pgranct® Pogues: 21. £) ; wmbonate, bearing a knob e, Capitate; f, Umbilicate; g, Umbonate. in the center (Fig. 21, g).
4. Topography of surface: Smooth, surface even without any of the following distinctive characters: alveolate, marked by depres- sions separated by thin walls so as to resemble a honey comb; puiic- tate, dotted with punctures like pin-pricks; bullate, like a blistered surface, rising in convex prominenées, rather coarse; vesicular. more or less covered with minute vesicles due to gas formation, more minute than bullate; verrucose, wart-like, bearing wart-like promi- nences ; squamose, covered with scales; echinate, beset with pointed prominences; papillate, beset with nipple or mamma-like processes ; rugose, short, irregular folds due to shrinkage; contoured, an irreg- ular but smoothly undulating surface like the surface of a relief map; rimose, abounding in chinks, clefts, or cracks.
5. Microscopic structure.
A. Colony as whole: Power of refraction, weak or strong; amorphous, without definite structure; hyaline, colorless or clear; homogenous, structure uniform throughout; areolate, divided into rather irregular or angular spaces by more or less definite boun- daries (Fig. 22, 1); granular, finely or coarsely; grumose, clotted appearance, particles in clustered grains (Fig. 22, 2); moruloid, having the character of a morula divided into more or less regular seements (Fig. 22, 3) ; clouded, having a pale ground with ill-defined patches of deeper tint (Fig. 22, 4); gyrose, marked by wavy lines indefinitely placed (Fig. 22, 5); rivulose, marked by lines like the rivers of a map; rimose, showing chinks, cracks or clefts; marmo- rated, showing faint, irregular stripes, or traversed by vein-like markings as in marble (Fig. 22, 6); reticulated, in the form of a
GENERAL BACTERIOLOGY 85
network, like the vein of a leaf (Fig. 22, 7) ; filamentous, floccose, or curled, as defined under 1 above.
Fic. 22. Microscopie Structure of Colonies. A, Colony asa whole. B, Edge of Colony, 1, Areolate; 2, Grumose; 3, Moruloid; 4, Clouded; 5, Gyrose; 6, Marmorated; 7, Recticu- ty Repand; 9. Lobate; 10, Erose; 11, Auriculate; 12, Lacerate; 13. Fimbricate; 14,
111ate.
B. Edge of colonies: entire, without toothing or division; undulate, wavy; repand, like the border of an open umbrella (Fig. 22, 8); lobate, (Fig. 22, 9); ecrose, as if gnawed, irregularly toothed (Fig. 22, 10) ; awriculate, with ear-like lobes (Fig. 22, 11) ; lacerate, irregularly cleft, as if torn (Fig, 22, 12); fimbricate, fringed (Fig. 22, 13) ; ciliate, hair-like extensions, radially placed (Fig. 22, 14) ; filamentous, (Fig. 20, G); curled, (Fig. 20, FE).
6. Color (to be determined for both transmitted and reflected light) : transparent ; vitreous, transparent and colorless; oleaginous, transparent and yellow, olive to linseed oil colored; resinous, trans- parent and brown, varnish or resin colored: translucent ; paraffinous, translucent and white, porcelanous; opalescent, translucent, grayish- white by reflected light, smoky-brown by transmitted light ; nacreous, translucent, grayish-white with pearly lustre; sebaceous, translu- eent, yellowish or grayish white, tallowy ; butyrous, translucent and yellow ; ceraceous, translucent and wax colored; opaque; cretaceous, opaque and white; chalky, dull without lustre; glossy, shining; fluorescent ; iridescent.
7. Consistency: hard, friable; soft; viscid.
8. Changes in medium: Liqucfaction (gelatin), shape of liqui- fied area, character of the fluid, membrane and sediment see under Bouillon below ; color; odor; consistency.
II. Deep Colonies.
1. Form. 2. Size. 3. Character of surface. 4. Microscopic structure. 5. Consistency. 6. Changes in medium. Same as sur- face colonies.
Srap CuLTuRES (Gelatin or Agar).
L. Non-liquefying.
86 GENERAL BACTERIOLOGY
1. Line of puncture: filiform, uniform growth without any spe- cial characters (Fig. 23, 1) ; nodose, consisting of closely aggregated colonies; beaded, loosely placed or disjointed colonies (Fig. 23, 2) ; papillate, covered with papillae; echinulate, minutely prickly (Fig. 23, 3); villous, beset with undivided hair-like extensions (Fig. 23, 4); plumose, a delicate feathery growth; arborescent, beset with branched hair-like extensions (Fig. 23, 5).
2. Surface growth. Same as for plate cultures.
3 { + . EE 8
WY
Fig. 23. Types of Growth in Stab Cultures. A, Non-liquefying: 1, Filiform (B. coli); 2. Beaded (Str. pyogenes); 8, Echinate (Bact. acidi-lactici); 4, Villous (Bact. murisepti- cum); 5, Arborescent (B. mycoides). B, Liquefying: 6, Crateriform (B. vulgais, 24 hours); 7, Napiform (B. subtilis, 48 hours); 8, Infundibuliform (B. prodigiosus); 9, Sac- cate (Msp. Finkleri): 10, Stratiform (Ps. fluorescens).
II. Liquefying.
1. Shape of liquefied area: crateriform, saucer shaped (Fig. 23, 6); nvapiform, outline of a turnip (Fig. 23,7) ; infundibuliform, shape of a funnel, conical (Fig. 23, 8); saccate, shape of an elon- gated ‘sac (Fig. 23, 9); stratiform, liquefaction extending to the walls of the tube and then downward horizontally (Fig. 23, 10).
2. Condition of fluid: See Bouillon below.
StreAK CULTURES (Gelatin, Agar, Potato or Blood serum). 1. Form: filiform (Fig. 24, 1); nudose; beaded (Fig. 24, 3);
GENERAL BACTERIOLOGY 87
papillate; echinulate (Fig. 24, 2); effused (Fig. 24, 4); villous; plumose ; arborescent (Fig. 24, 5).
Fia. 24. Types of Streak Cultures: 1, Filiform (B. coli); 2, Echinulate (Bact. acidi- lactici); 3, Beaded (Str. pyogenes); 4, Effuse (B. vulgaris); 5, Arborescent (B. mycoides).
2. Size; in millimeters.
3. Surface elevation.
4. Topography of surface.
5. Color. Same as plate cultures. 6. Consistency. |
7. Changes in medium. J
BouILLON CULTURES.
1. Condition of fluid: clear; clouded, degree of, does or does not clear on standing.
2. Membrane: when formed; color; consistency; structure.
3. Sediment: amount; color; character; whether compact or flocculent ; on agitation appears granular, flaky or viscid.
4. Reaction.
MILK CULTURES.
I. Curd formed:
1. Time required to curdle.
2. Character of curd: hard or soft; massed or in fragments; changed or not on boiling.
3. Whey: amount; transparent or turbid.
4, Reaction: effect on litmus.
5. Digestion: time required; solution complete or incomplete ; reaction; character of solution, clear, or cloudy.
6. Gas bubbles.
7: Odor.
Il. Digestion without formation of curd.
III. No visible change even after boiling.
88 GENERAL BACTERIOLOGY
PHYSIOLCGICAL CHARACTERS. a. Effect of desiccation. b. Relation to temperature: minimum; optimum; maximum; ' thermal death point.
c. Relation to oxygen : under mica plate ; in hydrogen or nitrogen.
d. Relation to light, Buchner’s Experiment (46).
é. Relation to antiseptics and disinfectants.
f. Pigment production: relation of development to oxygen; rela- tion of development to character of medium; changes produced by alkali and acid; solubility ; spectrum analysis.
g. Gas production: rate, quantity and formula produced in dex- trose, lactose, and saccharose media.
A. Acid and alkali production: carbohydrates present; carbo- hydrates absent.
4. Relation of growth to acidity and alkalinity of medium ; growth in 1, 2,3 and 4% alkali; growth in 1, 2, 3, 4 and 5% acid.
j. Reduction of nitrates: to nitrites ; to ammonia.
k. Production of sulphuretted hydrogen.
l. Production of indol in sugar-free bouillon.
m. Enzyme production ; proteolytic ; diastatic.
n. Characteristic odor.
o. Pathogenesis:
1. Modes of inoculation by which its pathogenic properties are demonstrated.
2. Quantity of material required.
3. Duration of the disease and its symptoms.
4. Lesions produced and the distribution of the bacteria in the inoculated animals.
5. Which animals are susceptible and which are immune.
6. Variations in virulence and the probable causes to which they are due.
7. Detection of toxic or immunizing products of growth.
8. Agglutinating properties of serum of immune animals. ( Widal reaction. ):
9. Lysogeniec properties of serum of immune animals. (Pfeiffer’s phenomenon. )
REFERENCES. Chester, Reports Delaware Experiment Station, 1897, 1898 and 1899; A. 227; C.17; H. 105; P. B. C. (Cheesman’s Charts) ; Kendall, Rept. Am. Pub. H. Assn., 28: 481.
GENERAL BACTERIOLOGY 89
MIGULA’S SYSTEM OF CLASSIFICATION.
I. Cells globose in a free state, not elongated in any direction be- fore division into 1, 2, or 3
planes. CoccacEak Zopr emend. Mic. A. Cells without organs of mo- tion. a. Division in one plane, 1. Streptococcus BILLRoTH. b. Division in two planes, 2. Micrococcus (HAuLuIER }
Coun. c. Division in three planes, 3. Sarcina Goopsir. 4. Planococcus Miguua. 5. Planosarcina Micuua. II. Cells cylindrical, longer or shorter, and only divided in one plane, and elongated to twice the normal length be- fore the division. (1) Cells straight, rod-shaped without sheath, non- motile or motile by means of flagella. BaAcTERIACEAE MIGULA.
A. Cells without organs of motion, 6. Bactertum EHRENB.
B. Cells with organs of mo- tion (flagella). a. Flagella distributed over
the whole body, 7. Bacillus Coun. b. Flagella polar, - 8. Pseudomonas MIGuua. (2) Cells crooked, without sheath. SPIRILLACEAE MIGULA. 4, Cells rigid, not snake-like or flexuous. a. Cells without organs of motion (flagella), 9. Spirosoma MIGULA.
b. Cells with organs of motion (flagella). 1. Cell with 1, very rare- ly 2-3 polar flagella, 10. Microspira ScHROETER.
90 GENERAL BACTERIOLOGY
2. Cells with polar fla-
gella-tufts, 11. Spirillum WHRENB. B. Cells fiexuous, - 12. Spirochaeta HHRENB. (3) Cells inclosed in a sheath. CHLAMYDOBACTERIACEAE MIaua.
A. Cell contents without gran- ules of sulphur.
a. Cell threads unbranched. 1). Cell division always only in one plane, 13. Streptothrix Coun.
2). Cell division in three planes previous to the formation of co- nidia, i). Cells surrounded by very delicate, searcely visible sheath (marine). 14. Phragmidiothric Ene- LER. ii). Sheath clearly visible (fresh wa- ter), - 15. Crenothriz Conn.
b. Cell threads branched, 16. Cladothrix Conn.
B. Cell contents containing sul- phur granules. 17. Thiothrix WtINoGRap- SKY. (4) Cells destitute of a sheath, united into threads mo- tile by means of an un- dulating membrane. BuraciaTOACEAE TREVISAN.
Oniy one genus, - - 18. Beggiatoa TREVISAN.
GENERAL BACTERIOLOGY 91
BACTERIA ARRANGED IN CLASSES AND GROUPS. Saprophilic Class : Bacillus vulgatus Trevisan. Baeillus subtilis (Ehrenb.) Cohn.
Chromogenic Class: Bacillus prodigiosus (Ehrenb.) Fluegge.
Zymogenic Class: Bacterium acidi-lactici Hueppe.
Saprogeniec Class: Bacillus vulgaris (Hauser) Mig. Bacillus Zopfii (Kurth) Mig.
Phosphorescent Class : Bacterium phosphorescens (Cohn) Fischer.
Pathogenic Aérobes.
Erysipelas Group: Streptococcus erysipelatos Fehleisen.
Pus Coceus Group: Micrococcus pyogenes var. albus (Rosenbach) L. & N. Micrococcus pyogenes var. aureus (Rosenbach) L. & N
Malta Fever Group: Micrococcus melitensis Bruce. Diplocoeccus Group: Micrococcus gonorrheee (Baum) Fluegge. Micrococcus Weichselbaumii (Trevisan). Sarcina Group: Sarcina tetragena (Gaffky) Mig. Anthrax Group: Bacterium anthracis (Koch) Mig. Friedlander Group : Bacterium pneumonicum (Fried.) Mig. Bacterium aerogenes (Esch.) Mig. Bacterium capsulatum (Sternberg) Chester.
Swine Plague Group: Bacterium cholere (Zopf) Kitt. Bacterium bovisepticum (Kruse) Mig. Glanders Group: Bacterium mallei (Loeffler) Mig. Bacterium rhusiopathie (Kitt) Mig.
92
GENERAL BACTERIOLOGY
Diphtheria Group: Bacterium diphtheriz (Loeffler) Mig. Bacterium pseudodiphtheriticum (Loeffler) Mig.
Pneumonia Group: Bacterium pneumonie (Weichsel.) Mig.
Influenza Group:
Bacterium influenze (Pfeiffer) Lehm. and Neum. Tubercle Group:
Bacterium tuberculosis (Koch) Mig.
Bacterium tuberculosis var. avium (Kruse) Mig.
Colon Group: Bacillus coli (Escherich) Mig. Bacillus enteritidis Gaertner. Hog Cholera Group: Bacillus Salmonii (Trevisan) Chester. Bacillus icteroides Sanarelli. Typhoid Group: Bacillus typhosus Zopf. Bacillus dysenteriae Shiga. Bacillus pestis Lehmann and Neumann.
Pseudomonas Group : Pseudomonas eruginosa (Schroeter) Mig.
Cholera Group : Microspira comma (Koch) Schroeter. Microspira Metschnikovi (Gamaleia) Mig. Microspira Schuylkilliensis (Abbott) Chester.
Streptothrix Group: Streptothrix bovis (Harz) Chester. Streptothrix Madure Vincent.
Pathogenic Ansrobes.
Emphysema Group: Bacterium Welchii Mig. CGidema Group: Bacillus Feseri (Trevisan) Chester. Bacillus edematis Zopf. Bacillus botulinus v. Ermengem.
Tetanus Group: Bacillus tetani Nicolaier.
GENERAL BACTERIOLOGY 93
CHAPTER IV
SYSTEMATIC STUDY OF REPRESENTATIVE NON-PATHOGENIC BACTERIA
In making a systematic study of a bacterium it is necessary to determine as many as possible of the points indicated in the previous chapter (III.); and in the laboratory this becomes a regular rou- tine procedure—in the study of each germ. The organism is first inoculated into a number of the standard media. These cultures are frequently spoken of as a ‘‘set of cultures’’ and are usually com- posed of the following: Gelatin and agar plates, a gelatin stab, agar and potato streaks, a bouillon culture (or Dunham’s sol.), a milk culture and a dextrose gelatin or agar stab (or shake culture). These cultures are then incubated at the proper temperature for 24 hours. They are then examined, described and sketched. At the same time three cover-glass preparations are made, one each from the agar, bouillon and gelatin cultures and stained with the follow- ing dyes: agar with an aqueous solution, bouillon with Loeffler’s methylen blue, and the gelatin by Gram’s method. The bouillon culture is also examined in a hanging-drop for motility and the milk culture for capsules. From these microscopical preparations the morphological characters can usually be determined. The cultures are again placed in the incubator and 24 hours later (48 hours after inoculation) are again examined and any changes are noted and sketched. The cultures are now usually kept at the temperature of the room for about one week and then examined for the last time.
If the organism produces gas in dextrose media, fermentation tubes should be inoculated and the rate, amount and formula of the gas determined.
The descriptions and sketches are conveniently made on. the charts provided on the following pages.
94 GENERAL BACTERIOLOGY
EXERCISE 58. PREPARATION OF SPECIAL MEDIA.
Tube and sterilize the following media for work in Chapters IV. and V.:
80 tubes of ordinary or nutrient agar.
2 tubes of lactose agar.
10 tubes of dextrose agar or gelatin.
20 tubes of gelatin.
10 tubes of bouillon.
10 fermentation tubes of dextrose bouillon.
10 tubes of potato.
10 tubes of milk.
10 tubes of sugar-free bouillon, or Dunham’s solution.
10 water-blanks,
EXERCISE 59. SAPROPHILIC CLASS. 95
Bacillus vulgatus Trevisan.
Synonyms. Bacillus mesentericus vulgatus Fluegge; Potato bacillus.
Expuanatory. This is a widely distributed organism which was first described by Fluegge in 1886. Its spores are very resistant and ean almost invariably be found on potatoes. It can usually be ob- tained by boiling potatoes for a half an hour, halving them and incu- bating in a sterile moist chamber.
REFERENCES. Fluegge: Die Mikroorganismen, 1886; C. 271; L. & N. 323; Mig. 2: 556.
MORPHOLOGICAL CHARACTERS: SKETCHES.
1. FormM AND ARRANGEMENT: ay, BOUT O Ts eet «tees sieie wind es crime means EARN OKG an mameeeRKeTs4 axexee :
a. Aqueous gentian-violet
b: Loeffler’s meth ylem= Dl esi. 6scesis cs ioisiessrenis wie vis ee ents erties W's Ha wineveualaiaae 9's celdteiwlon attees asia GRVPETUN Ss SURATL: sy esrepele cis os uaa losers ar tcvtc a vovard ceseista ate anc eisig sone oosvavencs M6 de seco aucnetee tapeinane wae oa eerats VE Bi. Bpecial SIA Gis i cncews 14 cy temenein sire duaensiony 4 eames ey PReRdaeeReG AA LE LR Rei aces c¥
Ae, MOriEr Vis ic a tact ys oe Se aeremneees sa RES Uk ew Het a rote gat Na ajeeeunia Ma ddeoneneaenaae
a. Character of movement. bd. Flagella stain 6: (SPORES Siswidsesecatasiicerasiadiaumsins erleda nacga noe eaceiinins Te estAReee es BARES AGREE GOhastaee’ 6. SPECIAL CHARACTERS?!....+..065 ck AN NL nan te ae wR Soa eon ah LV Wed e clebend Ri ede ane
ine” Ca BOOS cocina Yh eiines vp geek PAK eer kieeNaAAN Chadeces dxmieniecaes Lp akemeR day
b. Involution forms c. Deposits or vacuoles.
d. Pleomorphism...... ..++
96 CULTURE CHARACTERS
Reaction of media (Fuller’s scale) + ........ OF —: 2 ames es a Gelatin plate: Grown 24 hours at............ °C. Sketches. (a) Surface Colonies. ; (b) Deep Colonies. 48 NGUES Abaisacinensdacnea, @rtenesinies Ce: COBY S. Ati is ccsiccsmsesce ventas nee °C. Agar plate: Grown 24hoursat.. ......... °C, Sketches. (a) Surface Colonies. (b) Deep Colonies.
SIS HOULES Ati, csiscimuaswse’ caw deere °C. Gi hay GEG ce: ise Woi¥ cio tis Meisiethalnveusiaaiers °c.
Special Media: (Such as litmus milk and blood serum.)
Baciutus VULGATUS 97
Gelatin Stab: Grown 24 hours at....seeeecereee Ce
°C.
°C
+++ DOUTS Ab... seeeeeshours at.......
z
AS hours Bhi. esoiwisss veacers”O,
Cay 6 ab coscnecen nates’.
Agar Streak: Grown 24 hours at...... cess scenes °C. Oo ol: ° oO 3 8 n nm Z 8 4 4 48 hours at.........0.. Pererer ria of 6 days at......++ sagaes vandaauea Ws
Potato: Grown 24 hours at ..cceccceeseeeecesneen er GO
°C.
°C
. hours at......5
secoseseshOurs At.....0+
48 hours at .....cesee eevee cess °C.
= C
CAAYS Bis wsesswvitencioweeennewes®
Bouillon: Grown 24 hours at........ ipsieisiasatestscoinine Ce
Cc °C.
POWs Bhacceiy® . hours at.......
iw
48 OUTS At ..sssevevenevereeero°Gn
CBYS Abi ai vic sieiicwswnneesiensea Cy
98 PHYSIOLOGICAL CHARACTERS
1. RELATION TO TEMPERATURE. <ccccnevrysigciae RE BP amig een geet Spend Lhaee wala We peaammady OPtiMUN « scans cs veces ex eee 9 Cie HMits): sc00i ecccwsesees webOneees S85 Gaesleoeend °C thermal death-point....... ....... ae .°C ; time of CxPpOSUre......e seer ees minutes; medium in which exposure iS MAdE.....sses cess eeee een enee i UncaGhesie ee aisihaiiereiesinue
2. RELATION TO FREE OXYGEN:...... ... doraisiois iobane ieareie se saialndalvermin's wider W aNich acelbaray oe doves
3. RELATION TO OTHER AGENTS, SUCH AS.......45 ee eee ineeaee seamed ¥E é desiccation, light, disinfectants, Ct@.:—.. sc... ceeeeeeneeneccuneenes ius sarees RAE
rate of development: 24 hours.. .......... per cent., 48 hours...........-- per cent.
TO NOULS sia sisiresieisiess aaiinwiene Per Conti, ianviciwccns ve oss WOUWES:. wisices seein per cent, reaction in Open AYM.........eeeeeee deHeS Eten eRe ae 1s Aga aD eames eolaeT
gas formula, H: COa: :...0..sceseereeeseees ee a ee avi Seaeniy eaeesoNen om
Os VACEOSE: sects a ninisisdiateieiogoiate nies ebicetoaisres C. SACCDATOSE..receccccvecncercaneeceecnetecess
6. ACID OR ALKALI PRODUCTION?! ....00 cecceeee teeeeeeee aan hve lnvee ctvacarsuevacnoveca Wier dealin 'sid or! ‘i
7. REDUCTION OF NITRATES:....-.+000005 to nitrites ...... piace iste aan iscereye ie eivrcssistwlecannteniy to ammonia...... gbiteeerdaveiersis {atest oagaory 8. INDOL PRODUCTION....-ssseceeeeres
48 hours.......+- 9. ENZYME PRODUCTION
WO e tence nee cere n eee eat eeee .
Proteolytic.....ecseeeeeeee aro javasbVb io aghvaievera a sleresneaNINSGahE.. avn wyevledeanteschva sidaibiaieielonntaaton siaiebartgunnen’s digestion of gelatin.....cssesseaseceeoes digestion of Casein......csee ceeseceeeee diastatic..... eiaia[aio'd tie Sai gris Haale ewes de camrelet ate ay ouaaecayaters aeey aercmrews v4 gee Seper oni
SAPROPHILIC CLASS—Continuvep. 99 Bacillus subtilis (Ehrenb.) Cohn. Synonyms. Vibrio subtilis Ehrenberg; Hay bacillus.
EXpLanatory. This is a well-known and widely distributed or- ganism. First described by Cohn in 1872. It is almost invariably found on hay, hence the common name. Its spores, like those of the “potato bacillus,’’ are very resistant to heat. A pure culture can usually be obtained by making an infusion of hay or straw and heating it to 80° C. for ten minutes.
Rererences. F. Cohn, Beitraege Zur Biologie, Bd. I, 1872, Heft 2, p. 175; C. 276; L. 170; L. & N. 317; Mig. 2: 515.
MORPHOLOGICAL CHARACTERS: SKETCHES.
1. FoRM AND ARRANGEMENT: uw. Bouillon.
uw. AQUEOUS GENtAN-Violeb...cccccescccnccsssccaveeeanecrentsencseesscssesceeenssennennsees
b: Loefiler’s meth ylen-DlUG hee aasneessaaesccomaeis Hee temties B58 i SSdinisiereeiaasy vada Sed etN cane
Be PPA IAI cinerea eesanansds obiswiaa dia smieewida eommeeiind penne inden inca ruaieanaes
d. Special stains A GPU 8 too x5 ep cee oy es ree cede Ge eee ee ee LS &. Character Of MOP ENC ticscas expnswses reeereceaewer is Léewe ees Coenen nie EF ial eae bv. Flagella stain
5. SPORES?...ccceeseesseeeeeees 6. SPECIAL CHARACTERS ?.:.ecseceeccenessansevnvneence ee re rr a. Capsules..... Kae nade Li Lbs ee ARG ARS SAGE DANES TET Le Renndleuee dances
v. Involution forms c. Deposits or VaCuoles...ssscecesescsereencneeneeeennees ote Gia ihravisnnai zie Sia leiaed we ctemreceintaea coisieta
d. Pleomorphism...... ...+++ sikigjajasoie's ais asiniaratane G-deavelwiesebene y aibreisve ong usieyaielbvapnccrajaieials sigtatacnuoteilaiota .
100 CULTURE CHARACTERS
Reaction of media (Fuller’s scale) + ........ OT. 8 igi siare os Gelatin plate: Grown 24 hours at...........+ ig OF Sketches. (a) Surface Colonies. (b) Deep Colonies. AB HOUTS Atawariwsneswinree wiawsoesiess °C. 6 days at.ccerrceeee Sia efe/ainleivinemts la davonnre °¢. Agar plate: Grown 24 hours at............ 5C, Sketches. (a) Surface Colonies. (b) Deep Colonies. 48 hours at...... Ses seer yoamcaa ner °C. 6 days at
: Special Media: (Such as litmus milk and blood serum.)
BaciuLus SuBTILIS
101
Gelatin Stab:
Grown 24 hours at.....
seneeen eee,
a
S| 3] Lay oF -j 3 n mn 5 5 ° ° 4 a
48 HOuLS At... see sseseeeceevee °C. 6 days at ....cesees eeveveaguweedlos Agar Streak: Grown 24 hours atecscccecssreness °C. o ro) ° ° 8 ro 4 4
ie
48 HOUrS Ati ..scceeeseceeeeeeees °C. 6 days at........eee0e in blorahtesia 3 Potato: Grown 24 hours at .........sseceeeeeeeens °C. ro) io) n mn 5 5 8 co) 4 4 £8 WOUYS Bb rcsewssvas eoeneanes ba 6 dAYS Bhisasexicecenrreat eters °C. Bouillon: Grown 24 hours at...ssssessseeeeneneee aC. ro) 3 ° ° n mn E| EI fo} So 4 A 48 HOUTS Bt ..ccsaceseceereeees 17 OF RdayGBb, cncanas tcaleranscaeien °C,
102 PHYSIOLOGICAL CHARACTERS
i, RenavioN TO TEMPERATURE i h.wiwiss torwewiea reeees 258 CORAING he MN CERES GERANKATT Fe OPEIMUNE ercsnidisiecosaned oe earerecenie 9©.3 HMits: seiacciesee SResnkewe LOuakeroteiwe teen Ge thermal death-point............. eee ee °C.: time of exposure............... minutes; medium in which exposure is made......... haiaibeaece ai Sia amidiere Necoeeue.ean a aoe Rees
2. RELATION TO FREE OXYGEN!....-. sesseeses Wagieiens “gidhelatawaiare ginleatenngate we waxies é Siotesiateleiieiaus
3. RELATION TO OTHER AGENTS, SUCH AS...+-.00e eee eee ee ee eee er
desiccation, light, disinfectants, Ct@.:—....eccsceeeeneenereeerenees Suspiaibias Haga-sashine een
reaction in open arm......... Ss a eiceeldig eR ce Nadlaea aes Mea Ea sate & Saremeoleeys
gas formula, H: CO2: i... cceeeceneeeee Aaiajacteverninrsaca wait abonanien apres apabcts qoonsiehcte ied doaynste
Wig TACKO SG ieesessessaisiscaia: de sistmisisierirelslalereiaiates oa So C. SaCChALOSe.....605 pranialsiaie vase a ideatate ees a/assiondiese
7. REDUCTION OF NITRATES!...:.e0ceeserseeeeerees eee e econ neeee ween n enone wane ne nene ven eneeeee
PO DMLICES - ovensaies 3 cone see ceeeeeeess60 aMMONIA...... eraiaisiaes arenas te oe Se ea :
4B NOUTS ie scencin aovine oer een ear cros 9. ENZYME) PRODUCTIONS :isiesca.000s asagriedan’s avieenewae se
proteolytic..... orb Fel sladleiewaleaiate ReinwesllewenGyenee! welds Glas Hee saedeeaies VeES Kets wh owes digestion of gelatin.........eseeeereeee digestion Of CaS€iN...cesseeesceeeeeneers GiAStAbiG ss ta cgisaiewast eae aia Bisiaacalics gta wrote Mullan Ruetonnistatnee ask Siereusans SGuaseabia,deveiavens eiate a a Gaeta bya
10. CHARACTERISTIC ODOR:..... Scalers fia wee aiaiaveetw era Caiedaiatand Wades ielaichece oneness ues Cree
eee eee ee ee ee cry eee eee ENO e ree n enna meee eee eee tee ee eee see eEe eee reer ee eee eee ee ee eee eee e cence sen eeee eee en eens ered ea sareresein le alma rmcwen mie eee eee Cee ee eee eee ee ee ee ee eee eee ce ee ee re re rire.
Deena e eee eee eee eees
EXERCISE 60. CHROMOGENIC CLASS. 103 Bacillus prodigiosus (Ehrenb.) Fluegge. Synonyms. Monas prodigiosa Ehrenb.; M. prodigiosus Cohn. Exrranatory. This organism was first described by Ehrenberg. It is the oldest known chromogenic bacterium. It is very commonly found in the air of Europe and has a very interesting history on account of its casual relation to bread epidemies—‘‘bloody bread,’’ ‘‘bleeding host,’’ ete. It occurs spontaneously in this country. It is slightly pathogenic. Introduced intraperitoneally into guinea pigs in large quantities it produces death. In- oculated into animals naturally immune to malignant oedema it renders them susceptible. Rabbits inoculated with anthrax are protected by a subsequent inoculation with this organism. It is grown with the streptococcus of erysip- elas to produce Coley’s Fluid for treatment of inoperable malignant tumors. Rererences. Ehrenberg, Verhandlunger der Berliner Akademie, 1839; C. 258; L.137; L. & N. 272; Mig. 2: 845.
MORPHOLOGICAL CHARACTERS: SKETCHES.
1, ForRM AND ARRANGEMENT:
a. Bouillon
Ww
BIQW! speiuaaa tesa, sent oa te Recmeweneet Heo ereML Reet Mie WA bbRT Rea Sy 1eeEweeaNads YRKaReS
b. Involution forms.....se...esene cr PAGRERRAN HIRORIAT Uoncouinn SoseeneRdasaeiwiRaneaee €. Deposits Or VACUOlES....ccceeseseeseeeeetseneseceevecensceseeseceseeseneeees sieietaeisia asta
4s PIOOMOLp MISTS. isicas ses cay eninsioins aaelea nivlaie a dada sonics daeiage aidaleisiey ene eceencceeeee
104 CULTURE CHARACTERS
Reaction of media (Fuller’s scale) + ........ OT) Sh 5 ak Guares ae Gelatin plate: Grown 24 hours at..........+. °C. Sketches. (a) Surface Colonies. (b) Deep Colonies. AS HOULS Abssccccsaes sees oveicdeenes °C. 6 days at..... sik Stelaaw stereneiabaly aiteeee eC: Agar plate: Grown 24 hours at....seccenes eC, Sketches. (a) Surface Colonies. (b) Deep Colonies. eer o4% 9¢:
SB ROUTS Biicrcs arcvecseessces exces °C. 6 GAY6 Abiccxiercivsnssenesaes
Special Media: (Such as litmus milk and blood serum.)
4
BaciLuus Propiciosus 105
Gelatin Stab: Grown 24 hours at.........seeeee °C. 3 3 F 4 Z Z 8 8 4 4 48 Hours at ...... cs eeeceeeeeeee °C. CA AY SINE siscectanitanaiedsounwansa ve: oC. Agar Streak: Grown 24 hours at.ecscesseeeeees °C. oO oO ° ° iy N 4 4 48 hours at......e cee eee wees °C, 6 Gays At.....csseceencneneeence 2s Potato: Grown 24 hours at.........ccseeeeeeeneees °C. io) fo) ’ Me S, E /\ : n Rn mm ee 3 s 3 8 4 4 48 HOULS Ab... eee cece eee neces °C. BAYS Atives cvaccwnesvoecoeseon °C. Bouillon: Grown 24 hours at... .cceccsccevereves °C. 3 3 rs a n n col ol =] 5 i) is) A A
48 HOULS Ate sscecsececeeenceens °C. 6 Gays atvisisescvcucessas sees ye °C,
106 PHYSIOLOGICAL CHARACTERS
1. RELATION TO TEMPERATURE! ..:-ccccuceseeenes aiask fa dneit dveiniale Sialeis aieia se iaea ee Mo SUR a HE Swab ewisiecs OP tM UM 444 osarseacnseisicicle s savin PO) ATU S 6 tisaiecarsincinisieinie ste wise TO.rees hie conaneAinnint °C;
thermal death-point...............4055 °C.; time of exposure...............minutes;
medium in which exposure is MAde........ceeeseeeceeeceeeeseeee catia’ vislois esearearen ta. 2%
5. GAS PRODUCTION IN SUGAR MEDIA?..++.eeee sevens CPE nen n eee e anon nae eee vere ene neserenenens
rate of development: 24 hours.. ........... per cent., 48 hours............. per cent. 72 NOULS....eee eee eects ce Per CODE: jcc ceca seeasenes SHOUTS. .cacdenaes oa +. percent. reaction in open arm...........8. Suds MedhcreatatartteseM ag phslersreseiaiens Cres Fs lasdteeSoyalave: aca yacovenseeaavter gas formula, H: COz::........ Rctaa Maen shevaceiave Fratnenseensivals a hoe sale Was anararasa ko Saal adavon dba &
db. lactose.......+ diniaiainateresipeataiats steve brats ase ce. saccharose......... ea ivtaisies iss 4 aroraialelevatalate’ais
PPOTEOLVLIG snes sarsisajeis oa screccisinenatebaae dmahinnss. a cpveidesauenk sania PANES isionioraite
digestion of gelatin..........ceceeceeees digestion of casein.....cecee ceeeeee nes
diastatic.......06- ENdiete @ SOARTRIAMS ede (an
10. GHARACTERISTIC ODOR?......cseeeseeeceeeseceeenees sates a teats sialvie h boerTNIRIA ae eMeeRleRENmSe 11. PATHOGENESIS (or other special characters) :....cccccceceecnaveveuveens sa Bastiat miaeeee *
dat O ORO ee Eee DRE REE Eee CHOOT OHH T OHO EEE DE HED TORO OHNE R OOOOH EEE EDEE EHH R DOES Hee eee ene eesensennEes
GENERAL BACTERIOLOGY 107
EXERCISE 61. VARIETY OF PIGMENTS.
Make agar or potato streak cultures of the following organisms, incubate at 28° C., study, describe and sketch.
3 20 6 Days SKETCHES
Bacillus indicus or
Sarcina aurantiaca or
Sarcina lutea or
Pseudomonas fluorescens (B. fluorescens) or
Pseudomonas aeruginosa (B. pyocyaneus) or
Pseudomonas violacea or
108 GENERAL BACTERIOLOGY
EXERCISE 62. SHPARATION OF BACTERIAL COLORING MATTER.
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 ce. of ether to each tube and shake vigorously until the red pigment 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 63. ZYMOGENIC CLASS. 109
Bacterium acidi-lactici Zopf.
Common Name. Lactic acid bacillus.
EXPLANATORY. This organism may be taken as a type of the bacteria causing sour milk, of which there are a very large number. It was first described by Hueppe in 1884. It is very widely dis- tributed.
REFERENCES. Hueppe, Mitteil. aus dem Kaiserl. Gesundheits- amte, 1884, Bd. II. p. 1837; C. 149; Cn. 189; L. 222; L. & N. 220. Mig. 2: 327.
MORPHOLOGICAL CHARACTERS: SKETCHES.
1. FoRM AND ARRANGEMENT: ie BOW ON oepcsiesecaderesiecieinaciessiefeaainaee wine sernieiniersdinie viele ce dieeteaed Weuwadla :
8. STAINING POWERS!...-++++eeeeeee stealiniesesGannetdceanes. sadn diehelse dae eRbitRRTe@Wens eave eee uw. Aqueous Gentian-Violet........scsceesseseeecceetseeeeersseesceeseteeeennene tee eeteenees bv. Loeffler’s methylen-blue
@; Grain’ sistaini cs sesrqancs ans damien wiser stance ewe died ool pdnne NY dia iss. dieiadcardso acae ove weaigoasaaeale
a. Character of movement......
Di Wael Sta ssieiesasse0'sisias sieceivietsg aisiaie cinisia er wistora stains Shaoauienianiend mowcedeamiers aes Oa eeeae.
ths CapsuleSscasceneni ssi Seitadennvmonese nieces Wincemnre: (dbeeeamisopiemmeenenenas HO ReomebE ens, b. Involution forms.....eese es eeee eeees eidaieiareie a Hida) in' ale aie tn So ele seiw aye BAIR WEE Seaithass iain e eraiwrareitiee
«. Deposits or vacuoles.....
d. Pleomorphism...... creseseresseeees
110 CULTURE CHARACTERS
Reaction of media (Fuller’s scale) 4 ........ OP go sscnaca oe
Gelatin plate: Grown 24 hours at............ °C, Sketches.
(a) Surface Colonies.
(b) Deep Colonies.
SE HOwre Rbissacisassccscs. wecrwnned °C.
GAAS (ats tscsnwceansarvermmaaesd °C.
Agar plate: Grown 24 hours at..........ee °C. Sketches.
(a) Surface Colonies.
(b) Deep Colonies.
AS DOULS At. eeeceeecencenseeseceees eC.
6 days at...ccesesee daeaweihatnees anvils
Special Media: (Such as litmus milk and blood serum.)
Bacterium Acipi-LActict 111
Gelatin Stab: Grown 24 hours at........... Bre tad OS
Cy
°.
. hours at. «ss hours at......-.°C
FE HOULE Ab acess pecscavne frre OF 6 days at....... nvvanenenerrenalar
Agar Streak: Grown 24 Hours ates eee OO, ‘s) “3 ° ° 4 4 tee AY 48 hours at.......++ asanoracstiha pane? Gs 6 days at...cssceseeees Gepahapsroieee °C. Potato: Grown 24 hours at......sesseeeeeee Seis °C. | oO | | AN : : \ : % 3 n m E 5 : Aol C.
48 hours at...... rayerecuscerse sont’ Ce 6 days at......... she gia Gioichclanatalcte® so
Bouillon: Grown 24 hours at......ceeesevenseneee a,
ora Ot Cc
. hours at seveeshOurs at.......%
ww,
48 hours at.c..seeveee Sasivoeasee Gs SUAS Alicnwtasvenatenns veorwne Oe
112 PHYSIOLOGICAL CHARACTERS
1. RELATION TO TEMPERATURE:...... Store dea irgpate
Coe ee beeen rene eer eee
optimum. ..... ...-..6. Mcieteiss °C.; limits.........65 Wiskeie since COs ssiciere Bi imcsio nts didceasnal a Oi thermal death-point........ srgthaece cunts °C.; time of exposure...............minutes; medium in which exposure is made......... wave iacreiale Ginesa. ova eewnsewt nie iid Rae ig Stas 2. BBGATION TO PRER ONYOEN toy ci ver ennemanyas sononneenes ritanneeriend ee
3. RELATION TO OTHER AGENTS, SUCH AS....... Dene emee nee eee e nae seenneresseneeee we eeeeeee
desiccation, light, disinfectants, CtC.:—.....cersecececeeenceecerserensteeeees searasaiaa $5)
4. PIGMENT PRODUCTION?....cccsecseesvesecveeeneeee weiafeteialw ats Gees sain Vamtinlsverrtna a canetelmeineainas 5. GAS PRODUCTION IN SUGAR MEDIA!..+ weesescssurees issd isan toes elasshsleignwie ores tahanerapofeioseisiesioeheenae wu. dextrose (1) Shake culture:.....cseeeceuecsecreenee areeasonia ind deineiaiaravaeaig 4 Gctiebedocarevh.ors oie jams
(2) Fermentation tube, growth in open arm..... a Aaeicietece- ne closed arm.........eeeeee
’ rate of development: 24 hours.........e.055 per cent., 48 hours............. per cent.
T2 WOULS) sae b4 ajeearers emieeiere PCY CONS... ces ee vcacceeres HOULS ee psisiasiercierny x per cent. POACTICN IN OVE ALM: toes ceiseeseesrceswe s08 is HREM eenCAeeD eaueeTS peAaeRES eo KoanEE 3
gas formula, H: CO2: :.....seeceeeseee cee wp eeRneS ineoke o40s tienes Hees Bier ED if
Div IRCLOB: scidemarerereiare Sa dinys nasoi masbopeleyainrs . Ge. SAC CHBNOSOaccrsieinieisis scdiereibielazes'e'« Marsiove-eWiasewiesreo
6. ACID OR ALKALI PRODUCTION? ..sece seeeecee seeeee stain Hroveesvacecets widen oun locale afar eiSyaceva aexe Sia
litmus MILK oc... cece cee eee cee cen ces eeneecenneees HiRes alelatsUSIgae nea De noINeTNCaTENe Ne tides
7. REDUCTION OF NITRATES!...ccsecccecccsecseuceteeseeresensees orelstavaSieheeceetaiatsiaevepiareid siete Beek tO DItTItES.....eereeees sete tence reeceeeer tO AIAMOIUMA,. cocccevccceccnnersostsersccees
8. INDOL PRODUCTION AS HOULSHs he.c09 os aes
9. ENZYME PRODUCTION? .. +s seseseeeeeeeenees alain bn elated Abieleied Oe MULa NLA ey ara Miele SRNR A TTENEMLES
PROCCOLY.GI1C esse sins asiesinmicirersisineinitigs Hes cenewesinas Seed suLEMNEe’ ap savaraverardiwrote wie aacalatchcaie eda’ WvGeiele digestion of gelatin. .........seeeseeeees digestion of casein......cseecsuecevenee WiAStai sy sasiessiisniccicne vere ated sig bad eieteamiarouans olania sicticennnte ys Ne ypreia ainaisins Ress ee ois Baas é
eee eee eee eee eee eee ee ee eee Co ee re ras PARE ES OEY SW EAS YEE See ern Pere Peer eee ee eee eee Cee r err reer errr reer rer err eee e rere ere) ce ee cis
114 CULTURE CHARACTERS
Reaction of media (Fuller’s scale) + ........ OF =" ovens as Gelatin plate: Grown 24 hours at.........+.+ °C. Sketches. (a) Surface Colonies. (b) Deep Colonies. AS TOUTS Abiicsiciaeeisies cians: wera veieees °C. 6 MAYS: Abi sicisrsaieieinnis sia descarareren aaivorcios °C. Agar plate: Grown 24 hours at. ......... ra Sketches. (a) Surface Colonies. (b) Deep Colonies. 48 OUTS At. cecccrecccssccencesvecees °C: CVO a YS Hei bis icie int ciguatagg alae wits oa sles vant oC,
Special Media: (Such as litmus milk and blood serum.)
BACILLUS VULGARIS
115
Gelatin Stab: Grown 24 bours at.s.scssceseneees2Ce
°C.
« hours at.......
a 8;
. hours at......-
48 hours at....... wiewtaiaateisiesieme®? Ge 6 days Ab ....-ceeseeeee aReie eens °
Agar Streak: Grown 24 hours at cscssseseecsnens °C. rs) oO ° °o @ ) @ mn n H cay 5 & 4 4 was
46 WOUPS OE canavagemoveneqegans =, 6 ays atis er seneaarriieweewane °C.
Potato: Grown 24 hours at
°C °0
-.. hours at....... hours at
48 hours at
sivabssatsiehe aia oustonsieiesnsuiiere a BR 6 dAYS Ateicessscreceeeeeeeneee
Bouillon: Grown 24 hours at
&Cz
.. hours at...... hours at
set ©,
4& HOULS At occ eseseeeeceeeeeeee °C, 6 days at
118 CULTURE CHARACTERS
Reaction of media (Fuller’s scale) 4 ........ OF SS: es naar
Gelatin plate: Grown 24 hours at..........5+ =C. Sketches.
(a) Surface Colonies. (b) Deep Colonies.
4S ours At.rcseeseeeerees eee essai °C, 6 days at...cceceee a aay ab ayauate aeakecataltc bs °C. Agar plate: Grown 24 hours at...........5 oC. Sketches. (a) Surface Colonies. ; (b) Deep Colonies. AB OUTS At... eeeeeeeeeeees selene vee °C. 6 GBYS Btessvsensunseweniencssesaeires °C.
Special Media: (Such as litmus milk and blood serum.)
BACILLUS ZOPFII 119
Gelatin Stab: Grown 24 hours at.......... sian °C.
oc. aC,
. hours at....... hours at.
e.
48 hours at.....ssseseeees ieaisevis oC; 6 days at ...csecseeeuee ofcksarsisnaye of.
Agar Streak: Grown 24 hours atssccccsseeeeeees a 8
iC, Cc
hours at . hours at.......°
&
XC . :
48 Hours at... .ceevececeeeeees 2, 6 GAYS At. .scceeccenecseenerece i
. Potato: Grown 24 hours at
8 c °C.
hours at . hours at......
ee
48 HOUYS At... sc cecseseeeeeseee OH | 6 CAS ates csisaiaiccascareansee °c.
Bouillon: Grown 24 hours at........+..- jialitinee eC),
Cc °C.
hours at ..-hours at.......
48 Hours At ..ccsereecerserseees °C. 6 days at..... Wot cmmioncmaptonw amie cc,
122 CULTURE CHARACTERS
Reaction of media (Fuller’s seale) + ........ OT ease bees : Gelatin plate: Grown 24 hours at...........+ *o,. Sketches. (a) Surface Colonies. (b) Deep Colonies. AB HOUTEIAG see. 108 aTawR °C, CAaYS Ate cco cnsmiaseseeereaaasmenn ce eG; Agar plate: Grown 24 hours at.. ......64- SG), Sketches. (a) Surface Colonies. (b) Deep Colonies.
IS NOUTS At. cccncusiasarssasracmoseed °c. 6 days at........ sora re ebreaeiesorsierommaeeittaie °C.
Special Media: (Such as litmus milk and blood serum.)
BAcTERIUM PHOSPHORESCENS
123 Gelatin Stab: Grown 24 hours at........ cononeerhs oF 3 3 3 3 Z Z 5 & 4 4 48 Hours Ateacey a xeewexcer ees °Cy CUBS Ab staves crncnswemancers oC. Agar Streak: Grown 24 DOUrs at .ssccsesseesseee "Ch, o oO Q g 5 8 4 4 48 hours at.....-.see..eee wees °C. 6 days at..cccceeceeeerenseerans °C.
Potato: Grown 24 hours at
we,
wai,
-. hours at.... hours at
AB HOUIS Ab. cceecerereeeeenee
GBS Atria vie diacwieaneieswccwan
Bouillon: Grown 24 hours at
°C. Cc
. hours at...... hours at
48 HOULS At. .cssececeescceecene
6 days at.
126 GENERAL BACTERIOLOGY
CHAPTER V
BACTERIOLOGICAL ANALYSIS
EXERCISE 66. COMPARATIVE ANALYSIS OF AIR (Kocx).
a. Plate three tubes of gelatin and expose by removing lid for 20 minutes in the following places: 1. Laboratory. 2. Cellar. 3. Out of doors.
b. Replace the lids and keep plates at 22° C. for several days.
c. Count the colonies. The counting is facilitated by the use of Plate II. on which the Petri dishes are to be placed. In counting a hand lens magnifying about 5 diameters should be used. Where possible all of the colonies on the plate should be counted, if this be impossible count a representative area and estimate the whole number.
d. Express the results in terms of the number of organisms which fall per square foot per minute. The area of the Petri dish can be read off directly from Plate II. in square centimeters, or it can be calculated by multiplying the square of the diameter by 0.785.
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 following method must be employed.
REFERENCE. H. 477.
EXERCISE 67. QUANTITATIVE DETERMINATION OF NUMBER OF BACTERIA IN AIR (Pernri-Sepewicx).
GENERAL DIRECTIONS.
a. A piece of glass tubing 6 mm. (14 in.) in diameter by 15 em. (6 in.) long is drawn out at one end-in a gas flame and sealed.
b. Fill this tube about one-third full with granulated sugar, insert a cotton plug next to the sugar, and one at the end of the tube (Fig. 25, A).
1380 GENERAL BACTERIOLOGY
QUANTITATIVE ANALYSIS.
a. After shaking the sample at least 25 times remove 1 ec. of the water by means of a sterile pipette and place it in the bottom of a sterile Petri dish. In the same way remove 14 cc. and 75 ce. Pipettes graduated to =,ths. may be used, or a 1 cc., or even a 5 cc. pipette may be used by counting the whole number of drops delivered and then taking the number of drops to make the required fraction. If the sample be supposed to be highly infected it should be diluted with sterile water before the cultures are made. Plates ought not to contain over 200 colonies.
. 6. Pour into the dishes fluid gelatin (not warmer than 42° C.) and tip them from side to ‘side until the medium and water are thoroughly mixed. Solidify and incubate at 22° C., or below.
c. In the same way make agar plates using ordinary agar or, better, 5% glycerine agar. Incubate at 22° C.
d. Count the colonies at the end of 48 hours as directed above (66 c.) and at intervals afterward until the maximum number of colonies is obtained. Express the results in the number of bacteria per cc. of water.
QUALITATIVE ANALYSIS.
a. Number of species. Examine carefully, under the low power of the microscope, the plates made above to determine the number of different species, describing each very briefly. Estimate also the total number of liquefying organisms per ce.
b. Tests for Fecal Bacteria (B. coli) as follows:
1. Fermentation tube test. Inoculate three fermentation tubes, containing 1% dextrose bouillon, with #5, 1, and 10 ee. of water and incubate at 38° C. Tubes which develop from 30 to 70% of gas should have lactose litmus agar plate cultures made from them and then the gas formula may be determined. For B. coli it will be about: CO, :H::1:20rC0, 1
iS
2. Indol test. Tubes of sugar-free bouillon or Dunham’s solu- tion inoculated and incubated at 38° C. for 4 to 5 days will show the presence of indol if B. coli be present.
3. Acid colonies. A lactose litmus agar plate should be made
132 GENERAL BACTERIOLOGY
(using about 1 ec. of water) also one from fermentation tube and kept at 388° C. Examine 24 hours later for acid colonies.
e. Pathogenic Bacteria. See Chapter X.
ReFerEeNces. A. 579; H. 457; McF. 234; M. & R. 133; P. 245; Prescott & Winslow, Elements of Water Bacteriology. For the determination of the various species present see Frankland’s Micro- organisms of Water; Fuller: Report Am. Public Health Assoc., 1899, 580; Chester.
SpeciaL Directions. Analyze a surface water (lake or river) and a deep well or a spring water.
EXERCISE 69. ESTIMATION OF NUMBER OF BACTERIA IN SOIL.
a. With a sterile knife collect a sample or sou in a sterile test-tube or Petri dish. Samples at various depths can be secured by means of an earth borer. (Fig. 28.)
b. Weigh out 1 gram and dilute 1000 times with sterile water.
c. Make three gelatin plate cultures using 1 ece., ¥% ee. and +, ee. 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. 609; H. 481; M. & R. 128; McF. pye.o8 Fraenk- 240; Cn. 3. el’s Soil Borer.
EXERCISE 70. QUANTITATIVE ANALYSIS OF MILK.
a. Obtain a sample of milk in a sterile vessel.
b. Dilute milk 1000 times with sterile water.
c. Make plates as under soil (69).
d. Count colonies and estimate number of bacteria per cc.
EXERCISE 71. EFFICIENCY OF PASTEURIZATION.
a. Place same milk as used in previous experiment in the bot- tles of a pasteurizing apparatus, such as Freeman’s, and pasteurize as per printed directions. Or, place the milk in ordinary milk bot-
134 GENERAL BACTERIOLOGY
tles or fruit jars, filling to a uniform level; these are then to be placed in a flat bottomed pail (Fig. 29) which is to be filled with water and heated to 71° C. (160° F.). Remove source of heat, cover and allow te stand 30 minutes. Remove bottles and cool as quickly as possible without danger to glass. Fic. 28. Home-Made Pasteurizers.
b. Determine bacterial content of pasteurized product by mak- ing plates. A dilution of 100 will probably be sufficient. Express results so as to indicate per cent. of organisms 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 observations.
Pasteurized milk should not have a permanently cooked taste.
REFERENCES. H. 485; Wis. Exp. il Station Bull. No. 44 and 18th. An. Rept. 185. Russell, Outlines of Dairy
cu
it
Fig. 30. The Freeman Pasteurizer. Bacteriology, (5th Edit.) 113.
EXERCISE 72, TESTING ANTISEPTIC ACTION OF CHEMICALS.
GENERAL DIRECTIONS.
a. Fill a number of test-tubes with a measured quantity of agar (5 ee).
b. Add to the agar varying but measured amounts of the sub- stance to be tested. If the antiseptic be not volatile, or affected by heat, sterilize.
c. Inoculate the tubes thus prepared, together with a control, with B. coli or M. pyogenes and make rolls.
d. Keep these cultures under observation in the 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 prevents growth.
SpectAL Directions. Test in this way carbolie acid (5%), aleohol (95 %).
Rererences. A. 619; H. 506; M. & R. 140; McF. 248.
136 GENERAL BACTERIOLOGY
EXERCISE 73. TESTING DISINFECTING ACTION OF CHEMICALS.
SUSPENSION METHOD.
a. Make a culture of the organism to be studied in tubes of bouillon containing 5 cc.
b. Incubate at 38° C. for 24 hours.
c. Add to this an equal amount (5 ec.) 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 or M. pyogenes. var. aureus.
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 ss directed in the experiment on desiccation (43).
c. When the cover-glasses are dry, they are to be immersed in the disinfectant for the stated periods of time; then removed, washed in sterile water 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 (4%) or formalin (10% ), using B. coli.
REFERENCES. A. 611; McF. 249; N. 518; P. 152: 8. 158.
PLATE IL
PLATE COUNTER (Modified from Jeffers).
For Counting CoLONIES OF BACTERIA.
The cross lines divide the figure into square centimeters. The numbers indicate the area of the various dises. The area of each sector (a. and b.) is one-tenth of the whole area. (See page 126.)
Facing page 187.
PART II
MEDICAL BACTERIOLOGY
138 MEDICAL BACTERIOLOGY
PART II—MEDICAL BACTERIOLOGY
CHAPTER VI
PATHOGENIC AEKROBES
EXERCISE 74. PREPARATION OF CULTURE MEDIA.
The following media will be necessary for the work outlined in the following chapters. This is exclusive of a few special media which are described under special heads and are to be made as a part of the exercises in which they are used.
100 tubes of agar.
12 tubes of dextrose agar.
100 tubes of gelatin.
12 tubes of dextrose gelatin.
30 tubes of bouillon.
10 fermentation tubes of dextrose bouillon.
35 tubes of potato.
35 tubes of litmus milk.
35 tubes of dextrose free broth or Dunham’s solution.
30 water blanks.
30 tubes of Loeffler’s blood serum. This is prepared as follows:
a. Collection of the blood. Sterilize Mason fruit jars, by suc- cessive washings in corrosive sublimate, distilled water, alcohol and ether (or a large pail may be used). These are to be carried to the slaughter house and the blood from a beef caught directly into them. The blood is then allowed to stand undisturbed for 15 to 30 minutes, or until the clot has firmly attached itself to the sides of the vessels, when they are to be covered and 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
140 MEDICAL BACTERIOLOGY
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 cylin- ders (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 14% 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 1% dextrose bouillon.
d. Sterilization. Fill sterile test-tubes (about 3 em. deep) with the serum mixture and place them im- ; mediately in a sloping position in an inspissator (Fig. 31), or steamer and heat to 95° C. for 1 hour on three con- secutive days. If a higher tempera- ture be employed bubbles are formed which rupture the surface of the me- dium in their escape. When steril- ized the tubes should be sealed with paraffin or otherwise.
REFERENCES. A. 110; H. 51; M. & R43; Mer, 187; NW. 463; 219, Pie: H+ Blood Serums Inspissator,
EXERCISE 75. ERYSIPELAS GROUP. 141 Streptococcus erysipelatos Frnteisen.
Synonyms. Streptococcus pyogenes ROSENBACH ; streptococeus.
EXpLaNATory. First described by Fehleisen. It is found in abscesses, pyemia, puerperal fever and erysipelas. It is frequently present in mixed or secondary infections, and occurs in the mouth and sputum and on the mucous membranes of the nose, urethra, vagina, etc.
Rererences. Fehleisen, Aetiol. des Erysipels, Berlin 1883; A. 279; C. 65; H. 165; K. & W. III, 303; L. & N. 135; Mig. 2, 6; M. & R. 184; McF. 262; P. 476.
MORPHOLOGICAL CHARACTERS: SKETCHES.
1, ForRM AND ARRANGEMENT:
a. Bouillon.
D \SIGH cicciew vs write ioveia al nsraittemaie ate tem ecnibmraneiniiiae Pa acinaiearaiere melon epremsts WK BU cbudaeiancienche bla vatordes
a. Aqueous gentian-violet b. Loeffler’s methylen-blue
G
6. Sprctat CHARACTERS! us ci secnass cerveennens Leresmewans Oxo YRNMENAS HERE mienmedd bape ann’
Gx Caps le Sis cae iesscasncenncdass rarer eee Ren clans des okt ner nw Aid ware ered b. Involution forms.......
c. Deposits or vacuoles.
d. Pleomorphism...... .scsccereeeeeveees
142 CULTURE CHARACTERS
Reaction of media (Fuller’s scale) + ........ OY — ...e0e ae
Gelatin plate: Grown 24 hours at............ °C, Sketches.
(a) Surface Colonies.
(b) Deep Colonies.
48 HOUTS Obese scssascieuses Geenge
eer (8 RAPS Gl etaracdiaiabcwaeredauess eC.
Agar plate: Grown 24 hours at............ af’, Sketches.
(a) Surface Colonies.
(b) Deep Colonies.
AB NOULS At. eee cccseeeeeeeecn eens
fae Os CCAS: Abi ites can tinenwaowuicaninsmeea ce °c.
Special Media:
(Such as litmus milk and blood serum.)
STREPTOCOCCUS ERYSIPELATOS 143
Gelatin Stab: Grown 24 hours at......... mained we Cy Ss) o 3 3 Fa a 3 a n mn E 5 ? iS) ro) 4 A AS NOUS Abie v:a:sscisiarcio sy aiesiaisisierats °C. CRAY PAE scsi nesses conneateounss oC. Agar Streak: Grown 24 hours at....seceeeeeeeee eC. o rs) 3 o a cm n n tol col 8 8 4 4 48 OUTS Atisicecccessswncssnss ee GANS Bb ccacawes csind Snemeaced © BG, Potato: Grown 24 hours at...........ceeeeeeeeeees “i. 3 3 3 : iV \ re] oe n n 3 5 ic) ts) 4 4 48 HOUTS At ...... eee eee ence ees a OF CGAY Gites wastrisiey vie etenigerese °C. Bouillon: Grown 24 hours at........-.0.seeeeeees °C, = ee / 3 2 Fe a 3 3 2 2 Bs 8 3 ° 4 4
48 hours at...... ipa gies tts eave Gy iC |) ene SC;
144 PHYSIOLOGICAL CHARACTERS
1. RELATION TO TEMPERATURE}...--.--.cccceccseseeseceenae aa pwn ni wig pasa ln are, sinipraie sibiats tale optimum............ aptaes sate ramsarote SCeP IM UtBsssineiassiseerripsie’s tOssees 4 SHidiietes anes oC 2 thermal death-point........ sreleveet ....°C.; time of exposure............... minutes; medium in which expOSure iS MACE..c.ceceeecescuceeneureeceseeceeteeserenss ePesisiemene
2. RELATION TO FREE OXYGEN:....... sre aidnlermanteeyiuleeciongwassre axouasaiataa te ocGianiisioss cad wsthararvaysseswinvn ofaetaioraudts
eee ee eee eee eee er rrr .
3. RELATION TO OTHER AGENTS, SUCH AS....+.-+ Panes eeneeees ween e noes we neneee deen eneeee eens
desiccation, light, disinfectants, Ct@.:—....cecseeeeeeeecnereceeeeeeeees eeniewasaes. iE
4. PIGMENT PRODUCTION:.....- Pence meen eee e eee seeeneneeeces weenie wn eevennee tune eeneee
5. GAS PRODUCTION IN SUGAR MEDIA!... ssseeeceeeeereereeees ones seees Seber renner eneerene .
uw. dextrose (1) Shake culture:..
(2) Fermentation tube, growth in open arm........ seeeees Closed arm.....s wea avers ‘ rate of development: 24 hours... .....+e.0+ per cent., 48 hours............. per cent.
22 NOULS vo-nsieneceagieneereensis PEL CONE... .csereecvereeceee OTS sce deicsescrecessscsers ans per cent, reaction in OPN ATM...cesccceeeceveaceeeeecncneees dcacqatanntacacn wariareyaiaes a hinlndrefenia tare sean) gas formula, H: CO2::.......65 6 ica sachgfiainsve arcana Scns tte dsiatesatolenctars;SiabeVererslavete cao
U; TACOS Cescns snes cooormenn aeememmaenn esis Gx SHCCHALOS Aas mii s ssinuogeaiees Caiawenye ais este 8
6. ACID OR ALKALI PRODUCTION: ....+. ceeeeeee sseeeees een eeneeene be eeetweenens se eeeee oeee
7. REDUCTION OF NITRATES!..-++ceeeseereeeee aidavicindtate ee einitawierne eeiewis fsulahkmiadwdtavracaaataee tO NitTItES.... cece ee eee eee {RHO RDE to ammonia...... areata picieretie Seer
8. INDOL PRODUCTION. .cssseceeeees ieaauhayaseieta utara actiees OPC One Tete noe dnasuieas nin tuid bis taiah Nesaasaiiosa
48 hours....seseeee pi aia creases ate eae sdunniaenvacess oan CRY Swawenernciansvemasie sce Saicaeoredeletele
PVOtEO]YtiC....ceeeeeeeeeeeee alaoruaisa STATA ose ueede saieisemewis aa Reaieeha digestion of gelatin..........ssees assets digestion of casein...... sheet e terete ees
diastatic.........- aessaie faye enagatetevetsin sisi iaapingd Lissgeeietis eae ERIE Hi deca R(STEGe 8 Menacate sath veur ‘
11. PaTHOGENESIS (or other special characters) :...........5 siaidiatseejehagabe jenotene’ eunsteieistetess elaytiaheansvs
So AT ER QUSREENS: Hie cee eee CRTC ee eT eee eee CEP ere Sere eee eee ee eee eee eee eer eer errr
EXERCISE 76. PUS COCCUS GROUP. 145
Micrococcus pyogenes var. albus (Rosrnsacu) L. & N.
Synonyms. Staphylococcus pyogenes albus RoSENBACH; Staphylococcus epidermidis albus WeLcu; white staphylococcus.
EXPLANATORY. First described by Rosenbach. One of the com- mon organisms found in pus. Occurs on the skin, in sputum, air, water, dust and soil.
REFERENCES. Rosenbach, Mikroorganismen bei dem Wundinfec- tionskrankheiten des Menschen. 1884; C. 75; K. & W. III, 105; L. & N. 180; Mig. 2, 87; McF. 255 ; P. 470.
MORPHOLOGICAL CHARACTERS: SKETCHES.
1. FORM AND ARRANGEMENT: :
i DGG ie ke catkins Akin Ia Semneaneeens HeRaMMaRN AA RAsatenadnaRaad :
c. Gelatin
Ds SUZBE sacciaaies agectsaeiie Hate
@. Aqueous Gentian-Violet.....sesceveceseversncssececacevsvesnattavaesoeeee siapazeiai cs Weiser be TGC er’ SM Sb V1 CH= DI Giarsisss sctictearssasiewtiees Salegainn aren bi sa aaR aaa dein eeakees See *
c. Gram’s stain
bd. Involution forms.......... Yheceearess Kees VH HT CHOMELARY LF RRAMANS TRANS MRRs Cae weenie ne
&, DepOSitsiOr: VACUOlEShirs weenaies Yaienies wdemmem eins Hageeeainiin’s vaisiescieieivieaia na eretere ace: nie auerdveieds
d. Pleomorphism...... .sereesceeneeees ia faaidla(elptacdssieatas inte: visas cope ncadn ‘is Seale wlnials. Whidaaiy aihlate se ielete
146 CULTURE CHARACTERS
Reaction of media (Fuller’s scale) 4 ........ OF = ae ees cts Gelatin plate: Grown 24 hours at............ °c, Sketches. a) Surface Colonies. (b) Deep Colonies. 48 hours at 6 days at....ccseses edad sanaentlassiers aes el Agar plate: Grown 24 hours at............ °C. Sketches. ja) Surface Colonies. (b) Deep Colonies. satay °C.
AS OUTS At...eserevee assie si eelaparsastetere °C. GAAYS Abs voiiscsneaasrraaenir ea
Special Media: (Such as litmus milk and blood serum.)
WMIcROCOCCUS PYOGENES var. ALBUS 147
Gelatin Stab: Grown 24 hours at..... vaeeesnene ety 3 ° rs 8 Zz n 3 3 4 4 48 HOULS At ..cseccceeseesencens SCY COSY Vat eccsiceraersecsewesaees of. o Agar Streak: Grown 24 hours atessrssecceereeee °C. 3 “ | o ° a/ Vd n n H ial 8 5 4 4 48 OUTS At. cseevececeeesenees °C. 6 CAS Ab... ssceccoonsceeresees aC. Potato: Grown 24 hours at....cceeesseeeeeeeen eens °C. io) oO n n cal e =] =] i) 8 |. 4 A Www 48 HOULS At... sec ee se cee e eee eee °C, 6 dayS at....ccscseoues aidiveinaisies MCS Bouillon: Grown 24 hours at......s..06. eiate eral °C. 3 a x ie] Nn n uy Me 5 =] ° ro) 4 4
48 HOUPS At ..csreceeceeeeeeeeee °C. 6 GAYS Wiis xsindiiccsaed saeeess °C.
148 PHYSIOLOGICAL CHARACTERS
1. RELATION TO TEMPERATURE:......... siete biaek Widiatavelale id oie Wid ae han LAIN Rated she Rae TN Ewes OPHMUDI 3 ceswiecaeacecdccnaaeae tok OA! 00) it eee iisteeeiesea HG: sisane abe tepens neat aashaieed °C; therma] death-point......... siaahebieked °C.; time of exposure....... seeceee Minutes; medium in which exposure iS MAdG.....cescssessccvevevncencrere aisdinGibisiavalsie sie wie he Deis
2. RELATION TO FREE OXYGEN! ..eseesececsseceeceeeesanes aban eecnenenes teen oteneeenreecceeees
3 RELATION TO OTHER AGENTS, SUCH AS......+45 sdatsis Ba cayeeaaen et eaarements £8 WxGeums Wee baa F
desiccation, light, disinfectants, Ct6.:—... 1c... ces ecccs cece eereeeeeseeeneesecceees
ee Breveneee Peace eee e re neeeeeees oer e eeneeeeeee esieisiersinte eee ne ensennes eis 6 Sieiwiateas ss tea eeeeeeecereres
4;. PIGMENT PRODUCTION Sip steins os ccarimanncain emeteaeae aay somulenen Cee teeta eee ne nees ve eeeeenes 5. GAS PRODUCTION IN SUGAR MEDIA:...... ean e cence senescence banca seenaeeones bee veeees eee
a, dextrose (1) Shake culture:
(2) Fermentation tube, growth in open arm.........+.+.+.- Closed armM....sseeeee sain
rate of development: 24 hours....-..s..eeee per cent., 48 hours...........05 per cent.
72 hours......... isis Rtas od DOL CON big. os seissis saiecies sss NOULS. ceaiiccie veemivn per cent. TEACHIOM IT ODEVIALIN cajacisjowsaseiers’ ds tara araiaparonien 5 cia dieleisinsiniernns He weemanaaalulsam Mpaginetsaiieele
gas formula, H: CO2::....... wae 48 vas sieerwdnys SEMA TS es a) Aan Mukmeree’ Seabury
D. laCtOS@....ccreccescenece cavestenveenes C. SACChALOSE..+.. ees ojadaats a sssea Wiasslalau tina mamiarelatereisid
6. ACID OR ALKALI PRODUCTION:..... Ree OOS eT eee
eee de ee nee nee pe eeeae ey Dee meee eee e eee n ence entre n eens ete eeeteee pees
7, REDUCTION OF NITRATES Dcccnsseses ve vsickuwies vecece ais Gide Hoss satelealoia as Seats Bp ladiosvigpeiaaeieasi COMM TIPS wes cemdwvanacsaawe tose reeeeeees FO BMMMONIA.ccecercveerecesececeecresraeece 8. INDOL PRODUCTION...+.ceeeeee siginle sie are steierdtanei ue sid bP MUR GEWAER UseNUlageans Men eaeties ae « ane 48 hours.......6- ee Cone ora ee 5 cena GAYS sesasng su cariiehe 1h ve Sa eaewislomiett 9. ENZYME PRODUCTION:.......+ sara eve is's biota iSinta wisp aiintvintane binds, Niall hats elanaincereereaatns Guise sate
proteolytic........s.e5- iors iors sak aibisiosersionssDdiesa oF bodalatsey’ earinh eae Mlotareied Aha Ea car neeaterse digestion of gelatin .........ccseeeeeees digestion of case@in.........0.- se eeee ages
GUASTAEIS siasionSoeiesievenielnien tesa iseaiutapley Rewer § na sieipitoneetieneten< sae sioainselas Aeviareeeare
10. CHARACTERISTIC ODOR:.......
11. PATHOGENESIS (or other special characters):..... ofa a sindas Cidiaeiedarmalnaiarn' es aratece Seeaieeewamnierien
SO beet ween sete teeta w nonce sane e ewer se aenenee wee eeee oe ee rr beeen ewan ensene Scene eens e een e esos eee ee ee ee ee ery Were nee mee w ern renee nese ee eens Pee ewe reenter eeee seneeee veer e nent e eens tebe eenene ere eet m ence ere eens Se ee ey
° pet eeneee OS
PUS COCCUS GROUP—Conrinvep, 149
Micrococcus pyogenes var. aureus (Rosenpacn) L. & N.
Synonyms. Staphylococcus pyogenes aureus RoSENBACH ; Golden pus coccus.
EXxpuanatory. First described in 1884 by Rosenbach. It is the most common organism in pus—80%.
REFERENCES. Rosenbach, Mikroorganismen bei dem Wundin- fectionskrankheiten des Menschen, 1884; A. 270; C. 89; Fl. 2, 96; H. 162; K. & W. III. 105; L. & N. 180; Mig. 2, 1385; M. & R. 182; McF. 256; P. 461.
MORPHOLOGICAL CHARACTERS: SKETCHES.
1. FoRM AND ARRANGEMENT:
Gi Bow] LOM aia, aiaiers ccccsisssi sisi siein eisreniia aida v pidiavatecaiecainidiars n' wyeisiatanaia wie 6 areida ieinteiaraidie’s ;
G@. AQueous entian-Violet.......cecseseeeeseeteceeesensecesseessseeeenseeereesensseeeeeees
b. Loeffler’s methylen-blue @s. Grams stalnic.sadicasiust Ssncnorieks eumnmaeees cadaueniess Rhelodedeansat Reese d. Special stains IMRT TM eaves teen Sig cre denice as csi oetnctnw va we Sasatinedlnne fends de enerataiaiela we gubeeidaewsleaieie dle Maw aLealeddaials ca a. Character of MOVEMENt.....sseerceeeeceeneeenees er er eee re bo. Flagella stain
5. SPORES:
G2 SPOT Ty GHA BA GT MRS taaasiiaie iu arate cicicinistarsis efaraisicsdiavachra’niainse wiaibiais o(e S.ciarsiivoraiamiais al guisleactgneutan guna > Capsules sicd.aaavaecis seawmeeneenaieeeneee seven eens. akaee demminsndaa eonaiaeesen damn aes a b. Involution forms....csereceteeesreees OPAC SC ROD CT OREO rice REE TOO cr Ocenre crate
c. Deposits or vacuoles
d. Pleomorphism...... .«.
150 CULTURE CHARACTERS
Reaction of media (Fuller’s scale) 4 ........ OF bvidines ce Gelatin plate: Grown 24 hours at...........5 °C. Sketches. (a) Surface Colonies. (b) Deep Colonies. 48 HOULS At...secesoeesses wadibiccereinsl eC. 6 dAYS At. rccsecvcceccecanreesseernee °C. Agar plate: Grown 24 hours at.........+6. ut Sketches. (a) Surface Colonies. (b) Deep Colonies. 48 HOULS) Bbc jercasewswounsigemasses °C. GAAS: Bhiiciswsresssiaeciweiaweasaeseiiee °C.
Special Media: (Such as litmus milk and blood serum.)
Micrococcus PyoGenss var. AUREUS
151
Gelatin Stab: Grown 24 hours at.......sseeeee0 °C. o ro) ° ° 3 vr a 3 g Z 8 8 4 4 48 HOULS At... cece eee eee e eee °C. 6 days at ....seeeee eisisisieeleisieieie OO: Agar Streak: Grown 24 hours at...... oKdewnines oc. 3 oO ° ° n mn : : 4 4 48 hours at ......ceceeeeeeeeens °C. 6 dayS At.....seseeee Saisaisiestens °C, “Potato: Grown 24 hours at..........--.seeeeeee eee °C. to) oO n n 3 5 E Z 48 NOUTS Bb <asievsitersssiesios sere °C; 6.dalyS abscsewnnseaces Ranisiniests °C; Bouillon: Grown 24 hours at......seeeeseerseeeee °C. ro) 3 | 3 é mn m 3 3 A 4
48 HOUTS At os csseseee eee eeneee °C, Cid ay Si Abii csnssccedeniselsammrness °C
152 PHYSIOLOGICAL CHARACTERS
OM ELTANL TI: <es5.5 oie, areiniols ed. siraieisieiettreso'e 2 SCs Timitssavdede veces Hiden REO have cassie a npisecstiineete We eCue thermal death-point...............0085 °C.; time Of CXPOSULC....ceeeeveeees minutes; medium in which exposure iS MAde.......ccsvessecvcnnevcccccescectevcesucnees i selena 2. RELATION TO FREE OXYGEN!...ccssecceeseeee scene oe WAKER ceuia ve uae Vai tEe sadieetennst2
S GAs PRODUCTION IN SUGAR SIRDIA iis taavevaiores siasreeeiele a wenciace Io adeaa ened iw. dextrose (1): Shake culture tccssciess cccvscsciees sasvnsviewaees «csieaeies sac Sisisidta we sisisce
(2) Fermentation tube, growth in cpen arm................ closed arm....ececseeeeee
reaction 1M OPEN ALM..ecsssseeceseceeeeee fede Toned Tanah au Bees sa sia coke be Bis-dts us sates a deb eeISSo NSS OT gas formula, H: COz2::...... leva onus Vad ivlores ami aveatne setae: 6 ¥ i ai Dade ators Sormearneinieeees
By TaCtOS@ wjeaig ganccnrsanesions ove Soicrersisceesat C. SACCHALOSE.......ee eee siuaivig'da ecusiipbotaatt pees
7. REDUCTION OF NITRATES:.......00+ ile lacs siaharabaud diode. wattadenseid o's lalntele Neeievialerosaraia thie anna ees Baaee TOT LITE Sadist va eentnndoteaan soe eeeeeead sis tO AMMONIA vecsergames se seirasisieies Vass
proteolytic......... aiats iether ieSeheestanarecy Meals, anardicueriaeodanl iidnties sowemtertae s Aegis diate ce rene ye ‘ digestion of gelatin...... sie enema aisraieas C digestion of casein......... aang ee Hass diastatic.........+5 ates vases Narseme thas exerwenais ee mae Srarara ee tincaeitie dies Ses:
10. CHARACTERISTIC ODORiveeeeeueeeereeeees
11. ParHOGENESIS (or other special characters) :.....ccccescseceseeenseeeeneeaeeeceeesanenaes
eee eee seenee em me reece eects eet eneee eee ay Dement eee ene eee vere eeceeaee bene eee or eccen Sees vee Coen neeen Dee ee eer eee c seen nee eee voesees Denne ew eee ee eer eters be eee eee eee eee eee ee eee ee eee ee ee ree eee een enn ee ve eneeee Seen meen ee emt ener ee ee ee ce ee ee rrr se eeeeee Pererr rere reer errr reer rere rr ee eee errr eee eee cer reer reer recy eaten Tableiadra leas B86 HEM aoe AOD
EXERCISE 77. MALTA FEVER GROUP. 153 Micrococcus melitensis Bruce.
ExpLaNnatory. This organism is the cause of Malta fever and is found especially in the spleen of the diseased.
REFERENCES. Bruce, Practitioner, Sept. 1887 and Ann. de Inst. Pasteur, 1893, 7, 289; Durham, Jour. Path. and Bact., 1898, 5, 377; H. 441; K. & W. IIT, 488; L. & N. 168; Mig. 2, 83; MeF. 581; M. & R. 452.
MORPHOLOGICAL CHARACTERS: SKETCHES.
1. FoRM AND ARRANGEMENT:
We, BO Wd On sss jdincs:sreserermsins Ho sare auncroressiarb ars nos eteiain veaiaieed ar svd stave Misieiaie @iaund saat ;
cv. Gelatin. ..
ep AGWCOUS BEN HaneViOl Ob sisncaseicd eeacuinjsorescad maeisceieinace msc sneanaerioadlewisaidiecs Solsmremeniiiie’s B:. LeOSHSr’ S Meth ylen-DIG siasecsieses sees sissicinniareise a vuaardnemoinads 44 aves reMeMiNS ENED alee c. Gram’s stain
d. Special stains
es, Qhraracterof MiOVEMeDibses so cc.aisseussessiers tueseisiorn disinissnisiajaie vip siaincssaisie'ncged arse oimidyeietarecaiesie d aie ehecaia
DB: Wlaeella Stainosiiia sedis co venicdandacaa agemiainn $404 edwin tanising shale noman shuts sale
§. SPECIAL CHARACTERS:.. tis CapSules.cscccceccecesceeeeren teen etree essence eee Peon ene Eee ee eee eee Eat Ebene Ente e tees be Tevelition POP niSr vids casein svar occnscas cenadener see veemrennes £1 lenxeenane endwnsaaaens & ¢. Deposits OF VactOles....ssceeesnce cere es nee eters eee ee tenet terete nese nets ere e nee eee ne
d. Pleomorphism...... .-+.+. SPRUE Re eee Oe meena bearer eT Nee das
154 CULTURE CHARACTERS
Reaction of media (Fuller’s scale) 4 ........ OF — sais sees Gelatin plate: Grown 24 hours at............ °C, Sketches. (a) Surface Colonies. (b) Deep Colonies. AS OUTS At... .ccecsees os seen eenees is Os 6 GAYS: Bbecsnvins veaseuineecn ce sciricnwns °C. Agar plate: Grown 24 hours at.. ......45- °€. Sketches. (a) Surface Colonies. (b) Deep Colonies. 48 hours at......4. seessoeees 6 days at
Special Media: (Such as litmus milk and blood serum.)
Micrococcus MELITENSIS 155
Gelatin Stab: Grown 24 hours at.....sesseeseeee Cn
S io)
2. 2,
@ e
g Z
3
4 4 48 hours at........ errr a oF 6 dBYS At svisisssisniven’s veisisisniane oC.
Agar Streak: Grown 24 hours at...eecereeree ee Gy Oo oO
°o °o
3 3
4 4 48 HOUTS Btisewses verecccde een °C. OVE Whawrseewserseusaey ses POs
Potato: Grown 24 hours at............ schon sissies’ Gs
°C
seceeees DOULS Abe 0010. -oees Ours at......
48 HOUES: ats ox seecseeiass oe ways °C. 6 days at.......065 bre weeparevetesatase®
Bouillon: Grown 24 hours at.........ee cece eens PC: 3
°
ai,
hours at . hours at.
48 HOUTS At ccessecesreerees wees’ Cs GAY SB tec Svcascrcnshacne siaskis. 8 on
156 PHYSIOLOGICAL CHARACTERS
1. RELATION TO TEMPERATURE:........ ix ki Buona fy Ry waepass at aamenag iene peekges vaavIRS ODPLIMUM esciioaseunins xa on Naseaa Cat MIMS sda nexwaios ev eaabOmiuiesiiquundnccne? Os
thermal death-point...................°C.; time of exposure.............. minutes;
medium in which exposure 18 MAe.....cscseccsnecccssen recs ceenenensectsnonesseeees
2, RELATION TO FREE OXYGEN! ..c.ccsscccccecceseesecevccacveuveeeassecceneanessaseetansnones
8. RELATION TO OTHER AGENTS, SUCH AS.csccccsscvecscenesnesneebeseesseeeesecesesececennes
desiccation, light, disinfectants, Ct6.:—........ccccece cece recs cnscseoncnscencaveceees
eee ee eee eee eee eee eee eee eee eee eee eee Creer eer ee eee eee ee eee eee cee rey
4,: (PIEMENT PROUVUOTION tiiicnncsinea Cusasasendeereaan: Sanwa Used oases ve8
5. GAS ‘PRODUCTION IN SUGAR MEDIA tis's, sc.sjesiasies a0 seiacizacinainne + eastuals aie eles e oletwials Taeewe- arabe es, AExtrOSe: (1) SHAKES CUM POS scans veameaswnass Meweadwcawe aawERemenes smarione ds seeiwees (2) Fermentation tube, growth in open arm................closed arm........... dees
rate of development: 24 hours.. ..........per cent., 48 hours.............per cent.
72 NOUTS...ceccreeee recess DOP CONG... cs ceeee ceecceeeesNOUPS..cceeeereceees per cent. TESCHONAN OPEN ATMs ai vigonnaiisaa sacciandwadl we seanetenieaT -MeeisIiaan ei waa wa ares
Cas formula; Ht COP trecasiavawacenes Sydieada ie eve Qepr os Sea oedeana weiieweuy deedae
Us VaCtOSOsscapes soi ations doemidass ies Us SACCHALOSCscmeas eetawcacess osaticeseaes a ees
6. ACID OR ALKALI PRODUCTION: ...c0 coeccsee ccensececnecsetneecee sen eneeeeeetereccnseeane
litmus Milk ss cepspsaccews as vanasrcetaies
7.. REDUCTION OF NITRATES? Hsscsiesnn Gs oy yaisiesiny gaia opine eateas i jeeenannagy ereon de aaa tence”
HONUGLITES iisiieasnoden ses aan sieiene de Laeeia- ce tO AMMONIA: vader semiainy sare eleiemae’s Wao cuca
8. INDOL PRODUCTION. .+.ceeceeeeeseeeees
48 TOURS Wak icuiciayinnaianie sii siepetnraannckaneH a leaetemat GAYS iasiey eniene ne calaestanees sees 92 NZ VME GRO D WC DIONE aie:s sia eidsastsin e100 kas ealoserocssesoieie 056 Giese tedsbiatebiy "sc sa a iain suave taawtainuaeg o ormtasr arate tulad
proteolytic.....
digestion of gelatin .......... cece ee eeee digestion of Casein......ceeecreceenenees
VBS EALETG i assis ste sossssageracecaie: oceavaie 1a: avoteeateeainraiatiry 4. ecmreswanrdrayrd-a connntee Saceas te ocera Savi glaveTe nies a Resleimernes
10: CHARACTERISTIC! ODOR ey sisiawissiariacdin cinsatninasat atecdios yiaraiplnagtanaretinerels Guldciowians <aneRrea ae see NS
11. PATHOGENESIS (or other special Characters):......cccccceesccceccueseuevecssuvcereceevese
OO ee a
ee ee ee ee ee ee eee ree ere rrr errr
OSS ea SO 0
DDO Os Hi ee reece ee em nee ns SHH benes er CHES OESEO REDE ORO®
eomeccoecaeces
EXERCISE 78. DIPLOCOCCUS GROUP. 157 Micrococcus gonorrhoeae (Baum) Fivecer. Synonyms. Gonococcus; Diplococcus gonorrhoeae Baum. EXxpuanatory. First described in 1879 by Neisser. It is con- stantly found in gonorrhoeal discharges and may produce disease on any mucous membrane; urethra, bladder, rectum, conjunctiva (causing ophthalmia neonatorum), and even cause arthritis (gonor- rheal rheumatism), endocarditis, salpingitis and general septicemia. REFERENCES. Neisser, Cent. f. d. Mediz. Wissensch., 1879, 497; Foulerton, Trans. Brit. Inst. of Prev. Med., 1897, 1, 40; A. 288; C. 72; H. 179; K. & W. III, 148; L. & N. 164; Mig. 2,188; M. & R. 189; McF. 275; P. 522. CULTURE CHARACTERS.
MORPHOLOGICAL CHARACTERS: SEETCHES.
1. FoRM AND ARRANGEMENT:
ti BRO UALLOM a sctisis-s syeys-sissecois:sieiscsieisieiessieS Wibie hateidias ste steyeinieie, ne:aciueteiaraiele wedteeeneete
B. STAINING POWERS!..csccsccceeenceusereecceeseneeese reid atuiee Si he a cvsachas enced orsage bieid oases geisidiarnioe
we Aqueous Centian-violet, ..rcecscrernverccerscesemiernesees HONE OUNEROREE ENE Neen peaees ds. Loefiler’s wrethylen-Dluticccccciesscsnencecee vi verseneiescvarean niaiaisieieioia miei Seawater
c. Grams stain
d. Special stains.
@. CapsulesS...creseceecarsces b. Involution forms..... c. Deposits or vacuoles
d. Pleomorphism...... ccossccvovercecsceceececcesseseeerenesseeseeneen ert e ener etc cecenr ees
158 CULTURE CHARACTERS
The Micrococcus gonorrhoeae does not grow on the ordinary arti- ficial media but may be cultivated on the following:
a. Blood agar. Blood drawn from the finger, under aseptic pre- cautions, into a capillary pipette is placed on the surface of agar either in tube or Petri dish. This blood is then inoculated with the material containing the organism (pus or pure culture) and smeared over the surface of the agar either with the loop, or better, by means of a sterile camel’s hair brush.
b. Wertheim’s method. Human blood-serum (from placenta or pleuritic or other effusion may be used) in a fluid and sterile condi- tion is placed in two or three test-tubes. These are heated to 40° C. and inoculated with the material containing the organism, making dilutions from one to another, if necessary. To each tube is then added an equal quantity of nutrient (ordinary or 2%) agar thor- oughly liquefied and cooled to 40° C. The two are then thoroughly mixed and quickly poured into Petri dishes and placed in the incu- bator at 38° C. Colonies appear in 24 hours.
c. Rabbit blood-serum may be used either in a fluid or solid condition.
DIPLOCOCCUS GROUP—Continvep. 159
Micrococcus Weichselbaumii (Trevisan).
Synonym. Diplococcus intracellularis meningitidis WEICHSEL- BAUM.
ExpuaNatory. First described in 1887 by Weichselbaum. It is found in the meningeal exudate of certain cases of epidemic cerebro- spinal meningitis and in nasal secretions in a number of eases.
REFERENCES. Weichselbaum, Fortschritte der Medicin, 1887; Councilman, Rept. Mass. State B. of H. 1898; A. 298; C. 64; H. 170; K. & W. III, 256; L. & N. 148; Mig. 2, 189; McF. 281; M. & R. 188, P. 516; 8. 310.
MORPHOLOGICAL CHARACTERS: SKETCHES.
1, FoRM AND ARRANGEMENT:
hs: BOUT OMe secssa ergs oh enhiarcre devas s'v.o deeye ohne: deoka velo's 47 aisayannvatctnca w auovaveloiayabakerniala s 6
ing ACROTS RTT ATE NIGER cos as cea snercscnunaich vas eas pees Gee meenntmaNeENueseedmeen bs Loeffler’s meth ylen-DlWe sci ie ccsiecs cesvinciesnccedes eennwnaniesg ess OeaeeRedlaces been ences
c. Gram’s stain
6. SPECIAL CHARACTERS: &. CAPSUICS ass cenceseressan¥es bv. Involution forms...... class wlWye Wisions Wiclels gialuie(stajeioraiserelo sisiaieveis siersiniessiers epueimereewiere ata Paes aie c. Deposits or vacuoles
d. Pleomorphism.
160 CULTURE CHARACTERS
Reaction of media (Fuller’s scale) + ........ OM Se ae enesd sos Gelatin plate: Grown 24 hours at............ °C, Sketches. (a) Surface Colonies. (b) Deep Colonies. AB HOULS Aterccssecccccses eveveneven °C. Gi GS: Bibs sccosraradiacinerdrsjarirennsaneas-coters °C; Agar plate: Grown 24 hours at.........066 °C. Sketches. (a) Surface Colonies. ; (b) Deep Colonies. eeeeeen °C,
AB. TOUTS Bhi edieeesia ee siearesaisndis ss aaa eC, GAPS Bt sisi seinsecnosnens nats
Special Media: (Such as litmus milk and blood serum.)
__. ~ ISELBAUMII
161
Gelatin Stab: Grown 24 hours at......seeeee eee OG,
seeeees DOUPS Ab. .0 °C
peed
PC,
. hours at,......
48 hours at........ § vgnins eine Os 6 GA78 Bb pasgecennivarersenestnly
Agar Streak: Grown 24 hours at...csseseeree ee OG,
ceeeeceeshours B6....06.°C
seceeeeesHours Bt. ......°C.
48 hours at........ee eee ee Pert OF 6 days at..... eampdsiosiee Got eee
Potato: Grown 24 hours at........cccee esses eee eee °C.
wea: DOUTS Bhaia PC,
C
seceeees hours at.......°C,
48 hours at.........5. sisbietee Ores OF
Od ays Ataris sieice dsinaesine's oeweeen” Oy
Bouillon: Grown 24 hours at.....ccecceesee een ee OG,
Pres Oe
soeeeees-DOUrS at.
C
°C,
-hours at.
48 hOUrS Ate wees ew eews ca oe ra er
$0096 Aas iuvmmnae GQ
162 PHYSIOLOGICAL CHARACTERS
1, RELATION TO TEMPERATURE!.........4
optimum ...... sxeraxemnfrinathe Netcoeanie SOS TANNA Gia fs ioie sien Resaseldvnie Viet BOs SiaNaersuenatanG aie trethle Ot thermal death-point...............0008 °C.; time Of EXPOSULE...seeseeeseees minutes; medium in which exposure is made...... palate ea Few ouarre ERs Ree agewaderiees
2. RELATION TO FREE OXYGENi.. sect eeeeeveneeveeees Cave nent eeee Peewee oer eeeeee eee neee anes
& REDACTION 00 OFHER AGENTS, SUCH AGiiiuscsdceviant cones idee niaddiad Feb erin sees cena ele
desiccation, light, disinfectants, ete :—..........65- MGA AAT RiaET EMER EN Ae ibe
4. PIGMENT PRODUCTION:...... sntaisiottiere Seicloinepusieesratennin a dotelstatenoetsiolsies BS ieisinisinneraoegeials) saaeleee agit eben
5s. GAS PRODUCTION) IN SUGAR MBDA sia: ssc eitiacaicicininineia'sinin's alaiasainsdetinls 4 Mia6isiesara insted a ainleie/ninia eras ee a, dextrose (1) Shake culture:......... sia eeReeS iba ioatdrsitne So ew Saelemueinatee ities ase
(2) Fermentation tube, growth in open arm......... coetarasays closed ALM... .seceeseeeee
rate of development: 24 hours.. ........... per cent.. 48 hours.....sseeeee per cent.
72 OUTS icwseads was teea ine PET CONG. cccsessccecrees ssDOUES: eee sa'nces vees percent. Yeaction im OPEN ALM. ..eceeesececeeve Tia ok Matte hare cave banioue Sawa Siedepa nc ousices nt
gas formula, H: CO2::........ ares saantene deh atoca ards apnrcesenre dha sanouse| ucten rate yf eV eaiava acininianwrestnte ‘
Ds lACtOSE. wesvcaevesiesien yecesed seceeneese Cs SACCHALOSCssssecrerceevereencs ate age wisvageenddiviers
6. ACID OR ALKALI PRODUCTION:....+1 s+ pee ee - Cece eee eeeeerterneeee sateen Oe eee neces neeenn
7. REDUCTION OF NITRATES:...+:eccssecceneeeees Aticdsas © wiucsio a aia aia araiouiall sae eared aw Sateen HO MITTILES. Lc cece cae ese e ee ee eee Sa spce@eeie tO AMMONIA rsarakan stat aged g S-Series. ae 8. INDOL PRODUCTION. csessssseceessecenveones BOUT OREO ere siaialarahetaaciinet iiraihayas lc Siacorshe
48 hours...... Fd dachc a giw nies feed conaa asad Os Svonecdues don Spec scvanaie vaaoreianuhess GAG Sims dadseinnsenes es Mateiateatns 9. ENZYME PRODUCTION: ..+ecseceeseeeene waseaee NORA RKEY ES EY “Hr SARs aie ws areas we tacekishtams
PTOCEOLYGIC. ..ccceereererenencanenees slaulacigaiiias aia Seaionouag ha e@haciate ares éaasa davonsees digestion of gelatin .....ccceseeeeeeseee digestion of casein...... Seal wiantenaia’ GiAStALICsiwsidstinantieeas aaaae se eRe srs romnbareseita Sees SoadGiediaion Globe sieges 8 ‘ $0, CwrsRecT ents 10l CONOR! a viseseneseeersacesneninris tienes eeu eh aber Wiis earelaaiawais
11. PATHOGENESIS (or other special Characters) :...cceesccecsseaereeveoese scenes J niehSgotadursiiye ais
seeeee eee reat nee etr eens rane ree eee eee ee eet e eee eeee rr rrr rrr eee ee eee ee eee ec een cee reese Woe emmewennene So ee rey on conse eee Cee eee eee eee eee eee ee eee ee ee ee eee cee ce rr er errs deen ees we aeeeuee oer eee eee eee ee Sere ee ee eee eee ee eC eee ee ee ee rrr Seer eee eee eee eee Cero eee ee eee rece eee eee eee eee eee re
vr aseetene Meee e ee eee Teter ener eee here COO OOO TOO OOE EROS OO ROO HORSES DOS HEE HC ens ene a een Eo eee HH oEte
EXERCISE 79. SARCINA GROUP. 163
Sarcina tetragena (Garr xy) Mic. Synonym. Micrococcus tetragenus GAFFKY.
EXpLaNnatTory. First deseribed in 1883 by Gaffky. It is found in phthisical eavities and in sputum, and it occasionally occurs in mixed infections as abscesses connected with carious teeth, about the neck, jaws, and middle ear; rarely elsewhere.
Rererences. Gaffky, Langenbeck’s Archiv, 1883, 28, 500; A. 826; C. 84; Fl. 2,155; H. 172; L. & N. 171; Mig. 2, 225; M. & R. 187; M. & W. 183; McF. 571; P. 472. ,
MORPHOLOGICAL CHARACTERS: SKETCHES.
1. ForRM AND ARRANGEMENT:
és; Bowlillotics.adacaiueseas cesacneseicesatans Sagia td abeb nana ae aces
&. AQUEOUS Gentian-ViOlet.....sccccsecsessseeessessassenecseetessesscnseeesseetsctecnsssas b. Loeffler’s. meth ylen-Dlue secs ccsees egos sceneveanes ve eaasiensinises aeemeseaeie’ soa. 4 Cron
c. Gram’s stain
. Special Stains.......ceccecrececeeeeeeeee een en eee sense eeecenereeeene GiaareT erie eiaisintay ee elon reas ihe) IC TST WIS Fes aaah Se ee sw ae cscvsavasabava gusss carafe ashi va’ as dssliota to (oIB%sale jo his nvafurend oheseier4 Gieigs sso araaiata.aa, acavateraaceta
a Ta eRe OF TO RR Es cc access dernicrscinidbancendanes (ai eeasasay esehetsaerenecisnund
dv. Flagella stain
6. GPECIAL CHARACTERS!...cecsecveveceeeerssscsesusseeeeensenas whence Wiebe Anenene EA Kaew CLE ‘ tis, CapSulésixisigaies remmaeaenua apieiaceeree’ aaaialaialgede> nn ewes ees anebehomelnns nideaRtens a b. Involution forms.........-. HOGREES ENEIGN ACER CaO eSETAee@ewserENs aNaMeOAaAS 3
c. Deposits Or VaCuoles...ccevceverseccesenseesteaee MeINAN te daleinenan samManiedelesayiaeaived
164 CULTURE CHARACTERS
Reaction of media (Fuller’s scale) + ........ OT — weer eene
Gelatin plate: Grown 24 hours at............ oC. Sketches.
(a) Surface Colonies.
(b) Deep Colonies.
AS OUTS Abecccrsoceeerees eoae sconataae Cs
6 days at..... Ce Or eco Orr °C.
Agar plate: Grown 24hours at. ........- er Sketches.
(a) Surface Colonies.
(b) Deep Colonies.
48 HOUTS Bt. re ereceveveresseeseneees °C.
6 days at...cscceuee Seared Sale BAAN °C.
Special Media: (Such as litmus milk and blood serum.)
SaRcina TETRAGENA 165 Gelatin Stab: Grown 24 hours at......seceeeseee °C. ro) ro) o ° rs a Z zg 8 5 4 4 48 HOULS At. .cssecseveeseceeeee °C. 6 GAYS Ab cccacvsaeasiesean owners oC. Agar Streak: Grown 24 hours At scccccssseseneee 8c. ro) oO ° o q a =] i=] 4 4 48 HOULS At .rcsceecccseseencees °C, 6 dAYS At.cssccccceseeeeeteerees eC; Potato: Grown 24 hours at.......scseeceeeee eens °C. rs) is) ° ° a n EI E ro) 8 M8 OUTS Abs iisiicrcciedeesiinasien PC: BOYS Btirewiiseiasrecisadaciereoes °C. Bouillon: Grown 24 hours at.......eceeeseeseeeee °C. rs) oO a rs n n w & 3 J io) ° a a 48 HOUPS Abs sssrsscenceeereaers °C. | Gidays Bbiscacisrinccisigeuiasnaianee °c.
166 PHYSIOLOGICAL CHARACTERS
1. RELATION TO TEMPERATURE! ...esseesseneeees Sener tere eee eeeere Abe eeeeneecr sere eeretesoen
2. RELATION TO PREE (OX VGN iiies.sicciya es aors nastasniociaverneac a domneicerocewied aad areaues Poem teens ee
3. RELATION TO OTHER AGENTS, SUCH AS..+..e05 beeen Steen noes seeeee AGaNdies Vee PEaMEAAS
desiccation, light; disinfectants, CbCs KH asas sess ceoarnsinstases aaulemslespaa ae ta-2s 23 84)8 as
5. GAS PRODUCTION IN SUGAR MEDIA t.c+ csceeeeeeerecs yea eee er ee re a. dextrose (1) Shake culture:.......ccccceeseseescvees i beter PRRs ERHetEA wean
(2) Fermentation tube, growth in open arm..... SO ror closed arm..... ahs esaticetayann
rate of development: 24 hours...........055 per cent., 48 hours..........++- per cent.
12 WOULSi.css:5 cee ee serie ore-re DEF COM by senmadeaind ceded BOWLS: sieaycae so sneeiee per cent, TEACTION 10 OPEN ALD. -cerveenceeseccevcrereceeeeweneeeav enn eeteceeeenes Biscstacataie avikis Ss Ncale
eas formitla, HPCO2! tina craesees asian aie sha: seabetacesdoniad agp diete Spiele isis Walgsoadinateasadndts
OU; T&CtOS Gin avaanmieceoes 14 Serene Ge SACCHALOSEs v% ances vi anys ceatesecetaree, wAneenibie
Hits Milk sswsus tierexnowmeneay RarswnnareeuNes mgt oubcraente 1G tees ciaremtatgs Se Be SEARS iste
LO DUGTILCS isis cen ivacedemacew eee saeneneesoneebO AMMONIA: wees oe oseSak tiara va ke GEA Beatie
8. INDOL PRODUCTION. ..eseseeeee
48 hours........ 9. ENZYME PRODUCTION
proteolytic..... SRA ara tare AS Pia ia dig oeia za alagibnditins,@ b.n Aateereeereian Gracia qe ns digestion of gelatin......csceseeeesucees digestion of CASCIN......c600 caceeeees ee ATA S CLG tare ays sea aessievessrek saptusra duinte's Wt aNaialsie lentes peishedesinndienetion ese dia vishsieaivaiateana satavln oy gears 10, CHARACTERISTIC ODOR?....+ tranha aeseaas bale ae atelier ee a Seay aia staat tts aGasrelchwred ais tar saben
11. PATHOGENESIS (or other special characters) :.........e6s SUNS a baaeia Rot iatinisitanre bie ste ae
EXERCISE 80. ANTHRAX GROUP. 167
Bacterium anthracis (Koc) Mic.
Synonyms. Bacillus anthracis Kocu ; anthrax bacillus.
EXpLaNatTory. First described by Robert Koch in 1876. Found in the blood and tissue in cases of anthrax or splenic fever.
REFERENCES. Koch, Cohn’s Beitraege zur Biologie der Pflanzen, 1876, 2, 277; Chester, Rept. Delaware Exp. Station, July, 1895; A. 492; C. 190; Fl. 2, 217; H. 184; K.& W. 11; L. & K. 287; L.& N. 307; Mig. 2, 280; M. & R. 300; M. & W. 156; McF. 469; P. 547; S. 328.
MORPHOLOGICAL CHARACTERS: SKETCHES.
1. FoRM AND ARRANGEMENT:
tee BOUWi lon hasudints tsi oasseitclae bas soak eadacenlo aise semua dekeameeteniien
«a. Aqueous gentian-violet
dD Toeffler’s te thy] en-us ia sein sisiticciasaiginiehe tiosainhuwinseite tials dg casa suvavaeradiaine ee aeecedureae sede r
GES CAD SIGS ses eoreesct fies erabeore 34 Deeieae AT ee nme aT eamibe dat Me aiaitee Reale ene yo NNER Ds TH OLULION: LOM Sa siiecwsirns Mak. Heed \eKeis Seca ertnnaigiase Wen aderains Dea wetuaanien aan tone
c. Deposits or vacuoles
si Pedr pliStiiccs cw senerseceesws os 14 peeeee es coereeeeres
168 CULTURE CHARACTERS
Reaction of media (Fuller’s scale) + ....... OF — savevsws Gelatin plate: Grown 24 hours at...........+ °C, Sketehes. (a) Surface Colonies. (b) Deep Colonies. M9 OUTS Biever ins aoevenenns as 6days at........ Ee Pe ee eee °c. Agar plate: Grown 24hoursat.. ......... °Cy Sketches, (a) Surface Colonies. i (b) Deep-Colonies. : ABOUTS Bt. cncccccccccscnsesseesece °C. 6 days at... aaedaeVnwasieetatesens °C,
Special Media: (Such as litmus milk and blood serum.)
Bactertum ANTHRACIS 169
Gelatin Stab: Grown 24 hours at.. RENEE
9. °.
hours at .. hours at........°C
48 hours at........ Guba °C, 6 days at ...ccevees aia pagaasapniatee oC.
Agar Streak: Grown 24 hours at...... teraateeatere *C.
Pi OF Cc
veceeesesDOULS Atseceee secvesessNOULS Ab.......°
48 WOUTS At csc ceeeereseseeeees "Cs 6 days At....eeseeeeee maneness e
Potato: Grown 24 hours at........csee cece ee eeeee °C.
°C, °C
. hours at.......
eveosesesNOUFS Ateose
48 hours at...... aieebke ened ag G days Otcsvsexernees eee be
Bouillon: Grown 24 hours at....scsseseeseeereene °C.
°C °C.
hours at . hours at.......
| ize
do ours At wcues seinen” Ce GGaYSiat. sce. .20s ssowt semis se °C;
170 PHYSIOLOGICAL CHARACTERS
1. RELATION TO TEMPERATURE:......... Sissies A Sauseetnas ae SN EALaG eo A RR OUNMMeS NER EE Te TAT
COMINUI siccaesceemnenss sawenee CG LIMES: ces carsiiaes aeeomebOncswacs wy douse. Os
thermal death-point........ -+-+..°O.; time of exposure..........,... minutes;
medium in which exposure iS MAdE.....escseceseeceeeceeee seen seen tteneteenneeeeeega DEERE FG BREE OE SCE aaaies vin canteen awit vce erdderkienswenenmes acer GQ ewiaannns
3. RELATION TO OTHER AGENTS, SUCH AS..csscssecceecerserccusessceesssessenerrectecsetenes
desiccation, light, disinfectants, C66 ts cs seesasacseadeneds v5 csamwieeGae is ves cvieaeons
eee ee eee eee eee ee eee eee eee ee ee ee cee)
4. PIGMENT PRODUETION Saws ncicwiaigjerosisGins Palzaielarctostr @ gure edamame s pnaaeeaitemlenn cadahenuisee
5. GAS PRODUCTION IN SUGAR MEDIA!.cocesseeseueeees BEE ODAC COON Ceti cor iy Eee ws dextrose: (1) Shake Cultures, cv ss wcsiwicays va vivarcnseaine as Melinda tie ve bev cewlsnde cee vader aa (2) Fermentation tube, growth in open arm..........00...-ClOS€G ATM... eee eee eee ees rate of development: 24 hours..............per cent., 48 hours.............per cent. 72 DOULS vsaewes so ses ve nece DOP COD ye vs cic san vieciesinee NOULS eee cataricsenss DeT- CONE: LEACLION LOD OTE ALM igiaisieigie § tipi voce acdin ie:016:0:4 pie nian, bra buailaid, nse Sie cigapandiaig’d Vo wweisintern Releiaaisis gas formula, H: COz:
Oy Teta Ste wp zariumemensseian math Ge SARCNUPUEGccaws ey an wdeniene +s eoranwmeaneees
6. AGID OR. ALKALE PRODUCTION wcjoieiere. ine itaicsdscierwS sicieis aie ei dlaeiaie wav oaajneiersinrerarsorae sivvenrewaceaaieala NGAUS MADD oars sicraseyaiarssatngereneianerereaeeicreies ais 3G dk PROSITE AST eM ATEN Fea aedacanede
i; REDUGBION OF NITRATES tiicswss sagan ss ix erdwctinaes seaeseamedines drlemeadmeas mreandeimsoeds, COMPS Siinetaracinii se araeesocniees vee DO WMMONIA Gs osencenewerds Saowudianeraneis
8; -INDOL, PRODUCTION :6 is ssacneasiayincien nadeia guieaieaieelng’e # aseisr een aierein Saieistaiowecle alee deul Geena 48 hours....... 4 Ligh WWE sa aerledeslomimraaets. GAYS) sxx wotienan eoxnaeeconnes
D, ENZYME. PRODUCTIONS aise’ svcnswincanesns eee nucamala ts Seen << saleenb ae adadmemeae tans daensee
DT OLCOM GIO o5,5 iss didescusigerpocata aia 'ei.eu Moleatacied ea nee iki bg pulse iabinaatee tenn omnimahaane
digestion of gelatin.........ccseeeeeeees digestion of Casein.....-cccesseeeeeeeee
LAS CA LLG sas caiuisiaiciciaseie aia terete Side aigustanjerne aos 4% ldmeatticatareiae ex esmbreaignns
(0;, . CHARACTBRISTIO ODOR T eseaweweenns reeckiiseeetanan moudreanwess sawed sony eas raeencadney
ii. PATHOGENESIS (or other special characters):......seeeeee
eeeRerTREPe reer eeeeereererreeere rrr Cote ece re reer eer reer yr Cee re err rere rer errr serra rere eee eee ee eer eres
eee eeeeer reece errr eee rece reece error er eer eee rere? eee rere reer rr cere errr errr rere rere ere ery
EXERCISE 81. FRIEDLANDER GROUP. 171 Bacterium pneumonicum (Frrep.) Mic.
Synonyms. Friedlander’s bacillus; bacillus pnevmonie Wricu- SELBAUM ; Pneumobacillus of Friedlander.
Expianatory. First described by Friedlandey in 1882. Found frequently in normal saliva, lungs, ‘‘rusty sputum’’ of pneumonia, and has been found in air and water.
REFERENCES. Friedlander, Virchow’s Archiv, 32, 319; C. 131; FI. 2, 342; H. 314; K. & W. III, 189; L. & N. 225; Mig. 2, 350; M. & R. 209; MeF. 300; P. 458.
MORPHOLOGICAL CHARACTERS: SKETCHES.
1. ForM AND ARRANGEMENT:
ez BOM NONisrasas whe chaiecelsactecince Maaibacaaman ae Gea eMeenA hws Hise eneeoemes
Gs, AQUGOUS FENCanAWIOlEticewss vaeaisucmwesis vigdsrssitterclis VE Te ies ncdarsiedsereg a vw tieteledeseauner wee
bv. Loeffler’s methylen-blue c. Gram’s stain
Gy SGA) BAG own ice ve oc apace was pereieddad nui sd cwagecoeas araeeanedne Feud sowie
a, CHBGaCter Of MOVER Et teas ca era ewewwne viwnpedow ewan ny cb ENDiwERS a Ma rtaraneuns ssa aeadone b> Mla ella bain saci sew achiever clove wees etuhak Waeeiwidioainies Wace Seapine sears reclaae Wee shauamanwans
5. SPORES:
6: SPRGIAL: CHARACTERS tas wisezasaeraiasennies maonnercanaaaeanoisapee¥ vaieweintiees cesduiens so awees tis CR BSUWIER) Jaana tei enaran ted Valienddt rea Rianes BLE Hebseind Lawnmbne anne Vee eiaiaawCe es b. Involution forms........5 Ldaibinre siesiniavarein we orbouersiniaaaiece peeled alias Bik aivininataald ae UeewleOn sine 4a
c. Deposits or vacuoles.
d. Pleomorphism.
172 CULTURE CHARACTERS
Reaction of media (Fuller’s scale) + ........ OR — icacadas Gelatin plate: Grown 24 hours at............ °C. Sketches. (a) Surface Colonies. (b) Deep Colonies. ASHOULS Ate sce ereesecceee ceceeeners °C. GAYS Ab... ccecccesecsecvesvecvenece °C. Agar plate: Grown 24 hours at.. ......... °C. Sketches. (a) Surface Colonies. (b) Deep Colonies. eipretdisteee °C:
AB HOULS At. ce ccecereceeceeenereseens °C. 6 UAVS: :Rbs sas irereises aeisinniveres a6
Special Media: (Such as litmus milk and blood serum.)
BaActTEeRIuM PN&UMONICUM 173
Gelatin Stab: Grown 24 hours at....ssseeeeneeee 9G,
-hours at........°C hours at. ech,
sane
48 hours at......... sie iacsiadiaats °C. 6 days Gs é-ccnd aes areiaceieis saslewiaen aC.
Agar Streak: Grown 24 hours at......sseeeerees a
°C. Cc
hours at » hours at.......°
48 HOULS Bb... cc seeeeseewceenes °C. 6 dayS At....ssceveees dsdiniarwinigiecnie °C.
Potato: Grown 24 hours at ......cseceeesceeneneees °C.
opie Gs
. hours at....
secceceesNOUrs Ateoeeee PS
48 HOUTS Ab... eee eee ences °C. 6 days at.rccscseee a 9)
Bouillon: Grown 24 hours at...csesecseeseeeeeees °C. is)
hours at . hours at.......°C
48 hours at...... caioanii carina Oy Cay Ss Abiicaicenseswesianniaioaw ed cr 6
174 PHYSIOLOGICAL CHARACTERS
L. REUATION TO TEMPERATURES c0 1 vassayences is ryvenweses ee eee siarsbealeinshna eon OPCIMUM si as vieiesenaaias woeaied 9 C8 MIS ccsans eesas vocees CO nwa arena sida ate eCs thermal death-point................08. °C.; time of CXPOSULE....eeseereenes minutes; medium in which exposure iS MAME... ee ece cece eee eeeeeee Misa oignica ansaleneno en Mo 5
2, RELATION TO FREE OXYGEN:.......66 waren we Rewnad Veale x sje/atebets hiiva ly puarsharmatarauneuiena Sa
6. GAS PRODUCTION IN SUGAR MEDIA:...... ; a a. dextrose (1) Shake culture:......seeesceeeeeeeeee ob Salis daayuyopesadiroud Mvsrauayertoniatsk Oaateisre raw,
(2) Fermentation tube, growth in open arm........... +++ Closed @YM........00e sae
rate of development: 24hours.. ...... ++. per cent., 48 hours......ereeees per cent.
TA WOULS): iisaars ve shined sit ciacejniely per cent.,......+ idle etotatiaites HOMES icetete oosicne per cent. reaction iN OPEN ATM...1e. se ceee sees neens Sine awieaneine 19 ORaRNREIIE AAR sharing ¥a-evenraieeas
gas formula, H: CO2::....... aa shares Se aidindteae Te tae seeMan deat oH delved
De TREIGEOs si iawnenadena resi bandas seseeee co. Saccharose....... sation Wiaenideannad vevnnmne
6. ACID OR ALKALI PRODUCTION sid siete etyanarelalsanrins Reta sats litmus milk..... Aisieeone aes Satheswaai ces Sdudadoarsoars rnrernjarsrecauaisraneenimienion « paniscaanderees Wales enka vate
T., REDUCTION OF NITRATHS?., ccsiuaes oi sacaischeaiaa ga Siedeiemsied een des Lidge woes api Bamalbied TO MITIFC Ss ssisees va ris ceecains sblecoin’s Wislesumance $OBMMONIG s« sineniccsosids aaneninnuaaes axeet 5
8. INDOL PRODUCTION...... sieMEMENEs LeMOO RRS ee peaaieie Sak Maekawa et 8 santa 455 tale
9, ENZYME PRODUCTION. ....+eeeseeees Sag Salbinentisiasege’s aa wibsaie itis: iste i ceiadiaieielaarew gelnnaieatannees proteolytic.......++ is Maivinnbarrace is ween iaat desraiaae Wa araias siioaweaie wamianineieaed saa Sgecpireies on digestion of gelatin...........6- Hoesen digestion of casein......sccescceeeenees
FRIEDLANDER GROUP—Continuzp. 175
Bacterium aerogenes (Esca.) Mic.
Synonyms. Bacterium lactis aerogenes Escuericu; Bacillus aerogenes KRUSE.
ExpLANATory. This organism was first described by Escherich, who isolated it from the milk-stools of infants. It is very similar to Bact. acidi-lactici, and often difficult to differentiate from B. coli. Found in milk, feces, air, water, ete.
RerereNnces. Escherich, Fortschritte der Medizin, 1885, No. 16- 17. C.128;L. & N. 221; Mig. 2, 396.
MORPHOLOGICAL CHARACTERS: SKETCHES.
1. FoRM AND ARRANGEMENT:
Gs Boutllonsspiniicadiciapsre diss ssneraiace avaiatiars 4diwrandyarerestenieaineres soamrsecamieedien sporumieet :
Ws AQUEOUS Pentian-Violebiic. vis cicnca dee veo ceacasasnnad se sdawienaa signe wells aaueawmUTeNess Bb. Loeffler’s methylen-Dlue.....sccecccesscrssessacesceccesseesssreceresesseneseneees sees c. Gram’s Stain. ..csecceeseceeeeenene svaGloeaanads Coie IRARG ENE Reema eNMSENRANeTIs ve ene @. Special stains. «ca ieissesr ins ceneveretnaa) as enaiaees FA rade a eT tare Eesenaeese nes Ay. MGWIGRES Gees sis cdean anne ciorninse eo inne + Connco eee ni td dadanaenceneintieeeeenennoae a. Character Of MOVEMENE...cccctecscceesvcccessceeerccreecenesseeereneessanstsenesaserees
b. Plagella stain.....-.ssceeccesevees is staiytci at seenicals ala ababictestanaiyhatenal eae stad ocoatevins was aun mRNA
G.. CRDS USS wiesaw txeidiand easaegreaied seeeabeinan ethine Gag Rs DOANE HAAS ERRRIUANelNd Gd Oe ‘ b. Involution forms...... sata ysenenes Wain ARMES oe Neen EY Math Meats enue tieteeie aa o €. Deposits Or VACUOlES...reeceee sec e cee e eee e en ene e nee Een Err ene EEE EEE enn EEE EE ESD .
x PISOMOLPPHISM, seis. dae-renivdieaer seins ie eeinataeiend saviesaimenade iads Hinemiea’ weMyaieS sees
176
CULTURE UHARAULEKS
Reaction of media (Fuller’s scale) + ........ or — ........
Gelatin plate: Grown 24 hours at............ oC. Sketches.
‘a) Surface Colonies.
(b) Deep Colonies.
48 hours at... 6c. ce, ee eee ee
ove *E. Gay S Btls ties che tieesitay cn iiigenden ea
Agar plate: Grown 24 hours at............ *. Sketches.
(a) Surface Colonies.
(b) Deep Colonies.
48 hours at
ioe ° Ce CUBYS Ab. .c i ceeeeceeceneeeeteeeceees °C.
Special Media:
(Such as litmus milk and blood serum.)
DACTERIUM AEROGENES 177
Gelatin Stab: Grown 24 hours ati..scccoeeeneee eC,
°C
seeeeessDOUPS Ab... cease seveenesNOurs Of....000.°C.
ho
AB HOULS Abo. cece eee e erences CL COBYS Ab sivacesucewvisadeaears oC.
Agar Streak: Grown 24 hours atsseessereeereees °C.
Cc °C.
deaecae se DOULS Ati cece? ieestewe DOORS Abe cewade
48 hours at.......+. ean Cx Chie | ee eee
Potato: Grown 24 hours at......ssecesenees Cr 5
°C. °C,
ha
sep evaee DOULS Abo vena hours at
C
48 hours at...-.... snuaeeveas nd? Ce 6 days Ativecscsssecnveveeressees2Oy
Bouillon: Grown 24 ours At...cceseceseeree eee eeGy G 3 ° ° a 3 n mn uy H a 3 ° ° 4 4
eae.
- dOUTS Ab.e eee ee iain Sia pera Of C.daYs Ati secsssccin gxee ceases °C.
178 PHYSIOLOGICAL. CHARACTERS
1. RELATION TO TEMPERATURE! .......ccceeeeeeenees ia es PEt min meea x oioed agua apa:siaia gibi ielerass ouativess OPUCIMIWIN o5:9 6:06 eeanersolainena patie 1G. MIS insane caecan sveeee Olt sane tah eres °C. thermal] death-point................605 °C.; time of exposure..... seoeeeiss Minutes; medium in which exposure: is MaAACwiw<ss ecsissessexceviieonas ivarwereewsrs cearewcsnes
2, RELATION TO FREE OXYGEN!...s.s+eeeee euidinie ne aie canoe PieGs MACNN A Ib aoe
3. RELATION TO OTHER AGENTS, SUCH AS..... eda iaie ace aia sienna: w/e |g inn lg ae (gal Vcacaiaotbin’w'Sceiwenseiasg
desiccation, light, disinfectants, ete.:—..... vate ence teen ereeneeeeeneeee vane nee eeees
4. PIGMENT: PRODUCTION aiaieiasinierncsie-sieisieiaseeiniere-aeeie eo -ayatorasesetacere paturereacainiate ets a werarecacavninie ale SAA i sites
(2) Fermentation tube, growth in open arm......... teen closed arm..... Risiesslatacasiiy rate of development: 24 hours.. ....eveeee per cent., 48 hours...........- -per cent. 12 HOULS oie sone cmaienicnes per Cent.,....eeeeseceeeeees POUWLS soe oe sieenaresies per cent. reaction in OPeN ALM ....eeseeeeeeeeeeee Seuibunanterssd aisieinehets Nae satay rset anenelbhsnsratclec gas formula, H: COa2::......... «. taesnee eeacincsnals ag Saeseene les isewtemtis caisiues: wheels
Oy GCHOS Ss 1c aeeneeemete sg sd 4 an semtanmenines c. saccharose......... ee Seareiansievatsie Ba tiaides seisieteeis
TO MIGTIGES: arsiaisaiose vinstieccnnciecsces were si aenrdicetesars TOAMMONL Asse viseweesiceey eeriees giasatahe ene ‘ 8. INDOL PRODUCTION......++5 cg deista stays Ginjalarsiela slap ueragua eay aceumeaae ois Wenlersrsisitee Ralenuph eens gis
ASNOULS jresace ieawaoes comes amines 5 te he jeanne ere Cay Siwvewta caveress 5 datesrinnawanes 9}. INZYME: PRODUCTION! swanicdsia si Mecteaniatatwnwuamaannees a Dae siatsteinjaja NisinietanetGie oe weeimlwinveuntace
proteolytic........ aaendeaia nies iach Mia laegiatnve Ge @haravesteadd OR aUOaNEaT be aE orbirecsaledenaiiesredacetn digestion of gelatin ............see0000 © digestion of casein...... tee ereeeeeeenees
diastatic..... etaree sataietayen sis a AoNeONGAS TH asignemaarers a4 sae Neste 14 eeaueREe see tassivaists ‘
10. CHARACTERISTIC ODOR?..+.eeseeee ceeeeees sick ae wihdenake HdEteaee ihe nsanmes caveinnees
11. ParHogeEnssis (or other special characters):.....+.+css.ses0e8 Widacersvaatenonmrendaraa tren nuns
ee Peart eee ee eens Se ck cs dee ceeee benee
FRIEDLANDER GROUP—ConmnveED. 179
Bacterium capsulatum (Sreryzerc) Curster. Synonyms. Pfeiffer’s capsule bacillus; Bacillus capsulatus STERNBERG.
EXPLANATORY. First described by Pfeiffer, who isolated it from the blood of guinea pigs which died spontaneously.
REFERENCES. Pfeiffer, Z. f. H. 1889, 6, 145; C. 129; L. & N. 228; Mig. 2, 349.
MORPHOLOGICAL CHARACTEERS: SKETCHES.
1. FORM AND ARRANGEMENT:
&. BONO tasesvsicsireeranesnaieneniadwiawsishey Os Lomederead ea eanemiwenn i
8: ‘SPAINING: POWERS) jccains saediginsitijesiaaisemeiantekinndaeiawa X sieeestoantwsiress sacndumeaii a nmese @. AGUCOUS BENIAR-ViIGlEtiasstemscsass beavis cea GONE MT Sawisaineeiess og dadaaraaranienans ane b. Loeffier’s methylen-blue
CL AG PANS SUA Gi ws 5 ine sis oenereso-sselp wind aja ineorainre bio sacl searmbavereiyio" Mucnaiauialatdbataun Wie sieteiels
Qs SSOCTAN SEATING oi cisytierra seca ites ‘iene sin ce rorataresace vie niareialniaiauaehioiiia Savana eweajonete laieleway aocalanatein are e
i, CAPSaleSusns se gieEsehs MMe MRS MatoeMeemMRER IRE Raninas MONT eRGa Medics cesta Radu 6 ibis Dro Lat LO Tah O TeRA Sz vsas sve iaie ecw’ setesay gesshesd: SRW aebsehstonsh gt bone) aidbonstavbiniainrg aes eracequraiecalarewinve uuneresaha chcatbianads
ec. Deposits or vacuoles...
Gs PU GOMORD HUSH 5 sisnsles soistciaisied ~sistaimstnidissaeievars n:pis easualbibiathen'a le tsiataathebuelapetbialé SlativaetA did abe
180 CULTURE CHARACTERS
Reaction of media (Fuller’s scale) 4 ........ OF = xahaveas
Gelatin plate: Grown 24 hours at............ 8G: Sketches.
(a) Surface Colonies.
(b) Deep Colonies.
48 HOULS At. creecesee scene eens as Helse °C.
OAAYVS: Bbsiciéccscwasieseeeds svemaeeanis 26;
Agar plate: Grown 24 hours at............ * Sketches,
(a) Surface Colonies.
(b) Deep Colonies.
AB HOULS At... eccseeeeeevceeees nono OF
6 days abictie icssowewvse ts seciecicainnss °C.
Special Media: (Such as litmus milk and blood serum.)
Bacterium CAPSULATUM
181
Gelatin Stab: Grown 2¢ hours at.......0.00000. °C 3
+s hours at........°
a.
seseeess hours at..
48 DOULS At. cscccsenseceesee eee Ly 6 days at ....ccseeee
Agar Streak: Grown 24 hours at ..cccceseeeee eS
veveecees DOULS Abr ......°C.
sesvesee NOUTS At......0°C,
i
48 HOULS Ab .veseesseveeeeee errs OF 6 days at........ a(aceiarais Sreiosugereinrs
°
Potato: Grown 24 hours at.....cccccssessusseneess? Cy
seeveeeeshOUFS Abo... .°C.
<
°C
. hours at.......
48 HOULS Ab... seccerevceeeeeee Ce 6 days at........-
Bouillon: Grown 24 hours at......cccceerereeeeee Cy
°C.
-.. hours at......
«.s-bours at.......°C.
SBthours atu vt gedmiws mare? Ce GAYS Abia candy iesesesiis seed Yan
182 PHYSIOLOGICAL CHARACTERS
1. RELATION TO TEMPERATURE! ...seceecceeeeees ee ee ee ee F ODEIMUM iapsvaracacon decade sere? O.8 [IMIS ccwndincieasend cine Ouse lve eal eegutcinae °C. thermal death-point.............. «.---°C.; time of exposure..... seceeeees Minutes;
medium in which exposure is made..... i's nieversrelscomnniase' eineined inibiateid udsane sata
2. RELATION TO FREE OXYGEN:..... ia ate ais Watgin abigearejereg oe Siejah stalelaipraiena tote as sian oaatauel ae ana waa 3. RELATION TO OTHER AGENTS, SUCH AS...... aamns Lexainnaeey Bie weumennes va tae (idcoiaao’
desiccation, light, disinfectants, €t¢ :—..cccecrsccsseeecencceeeceeseseneseaneeeneeees
4. PigMent PRODUCTION# «sas cecssewneness exorwerneny ai sisiealWeineaee ealeniciecereg sak coasewenuten
a, dextrose (1) Shake culture:....... cadsina de nkiboaybyaeas siaialnis DuIGLIEHAL Fe EOC aa hee as ais
(2) Fermentation tube, growth in open arm....... seeeeess Closed arm...... soiarerasigavy rate of development: 24 hours........-..605 per cent.. 48 hours,........0e0+ per cent. TA NOULS sai piscine sce vengon DOL COMPiis sage demanaaes vee DOWTS asics ses tats. araysla per cent. Teaction IM OPEN ATM....cccccecnseccavcceeecareesecesscetee asieewuRs alereaneinacy 16 a eee Gas formula; Hi CO2 tiawsesdes. « vvtwewaneawsg vacaieaaee nie sadkiees veeeecndeeeeeemets
Dy TACEOSG ieee cele scisisweiineunbonmnevieis s vele sare e. saccharose......... SiakeieGiaia eaaasia PRanagaiineaNs,
PO MAUTLIGES scvccesisse is niievnininaieres vaaimediniaieen £0 AMIMOD Lavine s cccesacwa teeaas saea saline 8. INDOL PRODUCTION...csesceseeeeseeeeves saewineaes cteteh cepa oe adiuceiidn Riadoumaiiee pannto’ 48 HOUTSse2: seeacamines vs aOAeNEA iia ta aves wie os CAYSwacew ow ats STARR ONS 4 teas 9. ENZYME PRODUCTION... seceeeneeveenes guided siete ndrieaela ON ee Cee proteolytic........66 ints digie asia NNN FL eemnabnbeaete utara iaeerein accesso tatiana eer feats digestion of gelatin .......scseeee we tnks digestion of casein...... adejeleditiaieinin’ays vee
GSS tAtiCs cisiaey oe Soninasis + oelunmae sen ew oy ewan SRC inn ER cern ee ee
10. CHARACTERISTIC ODORSicsasss iacaeaeesas midisiorane Aedes Me Ha KRiaN Sot ertrelNs Causey OF 4 SEWER
1. PATHOGENESIS (or other special characters) :.i sic. issis revi secdeeesens voeeee slihea a desist
beeen eeeeeee beeen eee eee eee eee eee eee eee eee eee ee Cee r eee cece eee Cer e er ee errr . PNR ener eee neem ee Reece eee eRe eee eH EHO ROO REARS DERE ES tmmmm eee RODEN EH Tee eee eer es peer e te eee eee beeen nen eee PPeEee Pee RE eeeeeere err erererrre reer eer Tres reece rere r eter cre rere eee reer reer eer rereecer ren) shee eee eee ee eee ee eee eee Pere Cee ee eee ee ee ec i sane Dre rece eee VDRO e eee eRe ee Ree eRe TED EE EHD H CHER OHDO SHEL OHH EHS HH OHHH E ECE S OHHH Ee Em E EET e eH e Ent? “
Denne e ete e eee een eee Pee eeeeEreererrerer eee ee reer rarer ret See e eee ee eee eee eee eee eee errr
EXERCISE 82. SWINE PLAGUE GROUP. 183 Bacterium cholerae (Zorr) Krrr.
Synonyms. Bacillus of chicken cholera; Bacillus of swine plague; Bacterium cholere-gallinarum CrooKsHanx; Bact. suicida Micuua; Bacillus septicemize hemorrhagice HvuEprs.
Expianatory. First deseribed by Koch in 1878. Found in blood, organs and excreta of chickens suffering with fowl cholera, and in swine suffering from swine plague.
Rererences. Koch, Wundinfectionskrankheiten, Septikaemie bei Kaninchen, 1878; Smith, Report on Swine Plague, Bureau of Animal Industry, U. 8. Dept. Agri., 1891; Smith & Moore, Bull. 6, B. A. 1, 1894; C. 185; H. 305; K. & W. II, 543; L. & N. 208; Mig. 2, 364; McF. 534.
MORPHOLOGICAL CHARACTERS: SKETCHES.”
1. FORM AND ARRANGEMENT:
bs BOWL OW ieaisictscsse arsiniviate v sine aasaiajala tie 0 ald aia deat eraipueraiy 44 aaaRORNKSEES EREMBAE
a. Character of movement
b. Flagella stain
6. SPECIAL CHARACTERS:.
Gs, CAPSiUless casein vice cymes ad sasen me aeasetieEn > wise b. Involution forms: ....eeserreeeeeeeees iy sy5/0aua) exe aninzy uovedncha savas ‘oi anciohStavala rewliceierey ee care e ce. Deposits or vacuoles..... ie Shas Ach ala’ winres asangunicinndinis ei ghelbyefajelere aus ap MIE AsEINe BY Sate lala acnaascnuws
Ge, PLOOMION PH UB rites isis sips sieiaisiniesaiaie 5% Hiern aiarslasara doe Sis aralatessiernnanieatinion sue diosaieees eeciend bine
184 CULTURE CHARACTERS
Reaction of media (Fuller’s scale) 4 ........ OF = aces sia Gelatin plate: Grown 24 hours at............ °C. Sketches. (a) Surface Colonies. (b) Deep Colonies. 48 hours at..... iedenntean i eDprespian man COR GC AGRA tinsciewenitar saccade veaain °C, Agar plate: Grown 24 hours at.. ......... °C. Sketches. (a) Surface Colonies. (b) Deep Colonies.
48 hours:a&t.+.+.
Special Media: (Such as litmus milk and blood serum )
BAcTERIUM CHOLERAE
185
Gelatin Stab: Grown 24 hours at....... wwieiacers
«ne
vee °C.
»ss. hours at. veeeeessDOUTS At. ...eee
ed
d8 hHOUFS Atisxcsweiveswer samesea? C.
6 days at
Agar Streak: Grown 24 hours at..scceseeeeee
na De
°C.
veseeuee+NOULS Abs. ecees sesceveeeNOULS Bteeeees °C.
es,
48 Hours at .ccecsccevcoccveeres £C.
6 days at....-.++ ajaleere eee eee
Potato: Grown 24 hours at..ccccsssessasceeeees
eras OF
. hours at.......°C.
seeceseeeDOULS Abeer eee PC
i
48 hours at..... eechatiis sain Ce
6 days at......... aia a Were vaceavoriea
°.
Bouillon: Grown 24 hours at....... icoiatadasaeannes
°c Lali eee hay
-hours at
seeeceeeshOUrs at......,
48 HOUTS Ab ssessceecosreevvcose Co
6 days at.
186 PHYSIOLOGICAL CHARACTERS
1. RELATION CO TEMPERATURE Sins sew swsrgenanionis 0 age smasesastes an ai deisindies venmeMeME ree Ts Ae0
thermal death-point........ diate °C.; time of exposure...............minutes:
Mediam in Whiclicexposure iS Made. . vsicssies seer visegnesewoeesinnavedien ce eed saw vin wee
2. RELATION TO FREE OXYGEN:..
8. RELATION TO OTHER AGENTS, SUCH AS.cssserserccnesscncsnscceecceeseesenerenceenctneneee
desiccation, light, disinfectants, ete :—.........
,
4; PIGMENT PRODUCTION Sess cscs os ccainaiseceiaeses wvcide’ cjuyinines wibnw ord gions ofp oc eidie wales sows noes
& GAS PRODUCTION IN SUGAR MEDIAN cs saiskas. cs wr esteusunnns Cotingavwnnnd doewene HG Haas TAS Pe CEXEFOSS AGL) SHAME: Cul GPS sass asa oie Biss aigicnavieia Mia Ho giclSinve inde 8 8 Sa Giesa ciate as Ga fereuaidee misloreieidasere
(2) Fermentation tube, growth in open arm.......-.eeeeeee closed arm..... sss asi eesaiias
rate of development: 24 hours..............-per cent.. 48 hours............-per cent.
12 NOUTS + sacecassesaegs sees DOP CONG yavicci ton sevsuacees NOULSicssessecnccees Percents TEACCIO WM OD ST ALT sia aieicosivinsgeds a: 6.5 vevacaugisee heb-a's bela Sista gwtaaigiats 4 6 SiPeidleletaiaw og actiasbioln rele A Ha
gas formula, H: CO2::...... dail GUTS PAVE META ES He MERE RTE UE SRERRRS 14S ReWRCERERY
0: JACEOSOcca.ae ne vt ee tanes ctekme Giantess Cy SRC CDATOS Cyaaiens ni eneaweae it ae sa enoher ean
6. ACID OR ALKALI PRODUCTION:......
8. INDOL PRODUCTION.........+ siadedaeigaynntaly Su, Sie SraanagigngnaTiie BO ome dairies errarnateisieie be Mea aiREtyTES AS NOUS is eanris es tn vay smeawaeins oeiaes saleReeies aaaman: AY Sie ven. \eeomeue of vheveaeeemias
9. ENZYME PRODUCTION: ....cceeees
digestion of gelatin ... ccc. cece eevee eeee digestion of casein ..... secon ae nen eeeee
CHAISE ALIGis wa-acecsrisierewinsoietie joetelhnaree, HANG late: Worle Ov H-ETGatuNeT GT MpOriAiamE NTE NpleutetleMerelaen: 10, CHABACTERIBTIO ODORE. x. iuiccon wuvsaspineceauwensas is camneeeaney doeeeNneNsia eeneersn 11. PATHOGENESIS (or other special Characters) :....ccseececeseeeeenenev ee eenee nese teen eee
peso nee neee creer er reese Ce errr Co rr aren eer rere nr, errr wernt ree wr reer reer cr
Verrrrr rr cere reer er eee eee CeCe errr rere eer eer eee eee eee ee eee eee eee eee ee eee
SWINE PLAGUE GROUP—Continvep, 187
Bacterium bovisepticurh (Kruse) Mic.
Synonyms. Bacillus of hemorrhagic septicemia; Bacillus bovi- septicus KRUSE.
Expuanatory. First described by Bollinger, 1878. It is the cause of hemorrhagic septicemia in cattle and in other animals.
REFERENCES. Bollinger, Ueber eine neve Wild und Rinder- seuche, Muenchen, 1878; C. 137; K. & W. II, 559; Mig. 2, 367.
MoRPHOLOGICAL CHARACTERS’ SKETCHES.
1. FORM AND ARRANGEMENT:
w. Bouillon.
fi AMUSO S MENT AM VIGIEh, dee seeds Hea debe wens isis Kr cneunees Ver dieeicnmn men’ iG Mihai b. Loeffler’s methylen-blue......sseeceeee eee eeeeeenees Gieaieilenalasen- emmvaasniminen enh seats
6. Gram’s Stain .iceeecesdieseeviess sve Lois wih OY ANTRRGIAS WR SOME we Rae ees oy Shean
we Clapenlés. icici csawerns so tenendanvemsierece Ohalsa nGiists: sabi AbelsteNteinonteslesecneommee’s Ys Hee b. Law olution £OMmMS. «secede seasncweeswasimmesies safonaumsoery AvmigaRTeds Pe hMEMRt Meets
c. Deposits Or VACUOLES. ...cerecereeere ene neeene sneer eee ene e nent ete eens eens estes neeeeeanes
d. Pleomorphism...... »
188 CULTURE CHARACTERS
Reaction of media (Fuller’s scale) + ....... OT — wee. eee Gelatin plate: Grown 24 hours at.........00+ =). Sketches, ° (a) Surface Colonies. (b) Deep Colonies. 45 NOUTS Abscccceccssnvens coceesseas °C. GSE Bhesacewiaes Pe ceutaniervennat “C, Agar plate: Grown 24 hours at.........065 es Sketches. (a) Surface Colonies. : (b) Deep Colonies. 48 HOUESAbisacsicawsewiwnnies sata. °C, - 6 days at........ Sasi disionietarcrad
veteteees °C.
Special Media: (Such as litmus milk and blood serum.)
Bacterium BoviseptTicum 189
Gelatin Stab: Grown 24 hours at.....sseeeseeees .
°C.
2°,
. hours at........ hours at.
ong
49 hours Ab ccs sccesesaceves asin Os BAYS Ab -.ccrvceuevesnns weareins oC.
Agar Streak: Grown 24 hours atesssssscesseeees °C.
Cc °C.
hours at - hours at.......
<
46 howre. Ab vise serencecar reas i 8) 6 days at.....0- jaastiesanvamaan OO,
Potato: Growti 24 hours Ot csisccscessnies i viases OF
°C.
- hours at.......°C. « hours at.......
Cc >
48 HOULS At... ee eceeerseeeevens °C. 6 days at...ccceeee a aravestona teases e
Bouillon: Grown 24 hours at...sccseseeesesseeees °C.
as be
hours at - hours at.......°C
48 hours at........ Sisinis seineaeS oC; 6 GAYS Abii iis cacodiee siseatrcatatans eG
190 PHYSIOLOGICAL CHARACTERS
1. RELATION TO TEMPERATURE!......0.5 aiaiaialg wiahbvacal nidieigs aataaisiaialonah aloe sa NenenbaneareRidasiner nace OPtIMUM si vssgemesincssewes seas °C.s limits. ces Teowsetainadas Oni eesaans onesie °C. thermal death-point.............0.0008 °C.; time of exposure...... wide Saevedls minutes; medium in which exposure iS MACE......cseeee recente nc en cen eee een eneseseerestneeene
2. RELATION TO FREE OXYGEN! ..cesssececcsenneteeecseeaeeeseeeneeeees Si gia ey Saarncavesayara ncaa. aobedbagalnvaty
8. RELATION TO OTHER AGENTS, SUCH AS.....ccccsccvesursnescesceceens ey aelesibainay pup adete
desiccation, light, disinfectants, et@.:—.........ceeeeeeeee Wellies ieiesinsnE sen, ten ae
5. GAS PRODUCTION IN SUGAR MEDIA!.c+-.sseeeveee see e ee ennees Seen eee ny eee
a. dextrose (1) Shake culture:....... pieaidesccid stain 9 ahiedertemrnts deaewcistaie's aa erga ae aeals
rate of development: 24 hours....-......55- per cent., 48 hours...... ssaseecavek per cent. 12 NOWES: « caisaisicutuiossas nd per Cent.,..ceseee eves bviaers NOUNS - sie siesitiermaies de per cent, reaction in OPEN AYM...eeeeeeeeeeee wielacnnarnrajpibia ders eS seabrapctansie,@ barerpinapaing aie's olnarasaraninie yc a gas formula, H: COz::...... seslierncexeen arnamieaes 4 4 rinsieielans eens eae sannweaeenees ¢ <
b, lactOSe...e.eeeeeeees TCR eros Tce Rit) ¢. saccharose.......- Sencar Sac eerie
1; REDUCTION OF NITRATES! secsiescdanes va Haienaas $4 ge eeedintieh oo oxen Ue Set eine alae ak Meas FOMMUTIPES ss curatagacaiesamiireewasee sessnnd CO @MMONI Aisi wwrcwadiaiis sdideauunued es ase
8. INDOL PRODUCTION. ..ereee seers digidgs BAG ee aig eala gible SOU Eula MT Cae SeMngaE BA's sais Sapa .
48 hours......eee daoursnatn guile Mama sosiouie deitesiaee atta AAY Sissons Cewey th eneameeed wawids
proteolytic....... aise leis iia sic alae als aaidlsieGidiomlei es Gaewe cograng ies ayedvacassdie wae Sibiate maniacs bere aie digestion of gelatin... csecceceeeeeeeee digestion of casein..... Warale, Rigisinioe gan diastatic......... niga acees gestae» 4 bisraietdintaianininierd wisleiupe wie sing n 8 yea siawiseta Saeatnanleemine 3a auelels 10. CHARACTERISTIC ODOR! seeseeceeeereeeees susisia erate fs avaldeasrchaNtersiacets a ceayaven She wysek Rca ni
11. PATHOGENESIS (or other special characters) :....se.+eeees sence nO aaU aie cudorsebate acai aaietesea
peace eons ee eee eee eee ee eee ee eee eee eee Cece eee eee ee ere re eer rrr Seen neneenener Pete e neat eee eee eee anes eee eee ee eee eee eee eee eee eee rece re ee ee ee ree ey eee eee eee ELE eee eee eee eee ee eee eee eee ery
sete eeoee ee eeee veterans DO Rema mee Ohm Ae ree ORE TL EE EEA PAOD S OO UE OEE UOC OEE HE ESET Hee Eee E ee ee SH EEe
EXERCISE 83. GLANDERS GROUP. 191
Bacterium mallei (Lozrrier) Mic.
Synonyms. Bacillus mallet LOEFFLER ; Bacillus of Glanders.
Expianatory. First described by Loeffler in 1886. Found in the nodules, ulcers, discharges, etc., of glanders or farcy.
REFERENCES. Loeffler, Arbeit. aus dem Kaiserl. Gesundheit- samte, 1886, 1, 141; A. 376; H. 256; K. & W. II, 707; L. & K. 300; L. & N. 384; Mig. 2,498; M. & R. 275; M. & W. 164; MeF. 359; P. 508.
MORPHOLOGICAL CHARACTERS: SKETCHES.
1. ForM AND ARRANGEMENT:
Gi: Bawill Ons enetsorentven can ncwsnsas onnvewnegsorwass sommeacerey vive caaen ;
c. Gelatin...
toy CO BANTN Stes LTA asa tayenevteita Wass win intoannn ls /00Ns slave eseinionalonav ceva etdyandcasilesecavnYoreBleie'sieevera Orbea a SeesaiaiuarMiihte'e', one G. Special Stains.......csccceseceseseeeeee eee en seen nese eens nese e eee eee nee nese Eee Eee EOS
i. MODIDITY tos0 5 3 ces cessed conacareusa wens smeea deus Weems A aaeagasabaan ots eourebeelesion rer
u. Character of movement D. Flagella stain......ccccceccaseecceratesses ssseeneesen eee neseeeeerenastestoenee aii SB ie
6. SPORTS Hwssave spr imwdtale ieee
Gs CAPS UleS vise sines vievens cae ns om sinieinine’ coinen Pe er ee ee Dedeeeies D. Involution fOrMS.....cccsecseercecercecseeeeenenaes ia otayatayecd a: ack ubedroreiavarenece-weaunectohaccuera bis ‘aa €. Deposits OF VACUOLES. cesses eee ece eres teeeeeneennees aL aNeiataavslomein'a tstareeevamhelenstone's melecsteg ie
d. Pleomorphism...... ...++ La Ne aavaemetie 8 Dalasi dara nalngaiiaaisleiinin Reenter ealerenieaites '
192 CULTURE CHARACTERS
Reaction of media (Fuller’s scale) 4 ....... OR 8 penieaets Gelatin plate: Grown 24 hours at..........+5 °C, Sketches. (a) Surface Colonies. (b) Deep Colonies. | AB HOULS At vrecseseceenes oe sinavve Cy 6 days at...cerccces Pr ear? azdea® Ce Agar plate: Grown 24 hours at........6.05 °C. Sketches. (a) Surface Colonies. (b) Deep Colonies. Gays at.rcssccccsceee deavesned e ee'ewe Sas °C.
MS HOUTS As iiecsnetssiew sas wanesaaes °C.
\
Special Media: (Such as litmus milk and blood serum.)
Bacterium Mau.et 193
Gelatin Stab: Grown 24 hours at.....seeereeeen °C, io) rs) o ° od Pe 3 3B n n H carl 3 3 8 iS 4 4
48 hours at........ wine Ree tet eC. 6 ABYS AE sxciicmasnis cv ansisiscineeo Oe
Agar Streak: Grown 24 hours at..cesseceeees fa en 3 oO
° oO
F : (\
mn mn
: :
4 4 48 HOULS Ab s25 ess coesienecieaies 2 OF 6 dayS At...cseveccceevvereveses Oe Potato: Grown 24 hours at...........0eeeeee aeanese Cs a io}
3 o
3 3
m mn
3 5
4 a 66 LOUrS Bhs ic owemeeex eres xa.04 °C. CUAYS Riis ccmeermenins cree” Ce
Bouillon: Grown 24 hours at....ssssseseeseeeen ee Ge
°C
2°:
as ie HOMES Bbeexnens sivas Ours. ty.
LJ
48 hours at... esesveeeeeseeee sel Oy Gays Abas sincedsciemvestsemnsn” Cy
194 PHYSIOLOGICAL CHARACTERS
1.
OPEUMUM has eeae vids casncmseanes Ck TIMI tSaasavaaeeess aaedes tOhaduantiomnteemanes eCus thermal death-point,..............06. °C.; time of CXPOSULE.....eee sree ees minutes; medium in which exposure iS MAde......ises sevens Ce nr deena winti tiie
2, RELATION TO FREE OXYGEN!.......005 Sasetie dees Wieraiiaitns Ceeeoeeeat Ot Miia hristeeD Aumiasie
3. RELATION TO OTHER AGENTS, SUCH AS....... in’oudaeeyalesesmcarala_o.a-ace ele laainialg o's ware oarmanerew VE aaa
desiccation, light, disinfectants, etc. :—...seceseeseeeeneeeees aictehetciard i aiceis ehateetesaea
5. GAS PRODUCTION IN SUGAR MEDIA!..+ ceseeeeeeeeeees steigiis auathiatnniadoel a ala aabnls ig venuighansiatiiaeais a, dextrose (1) Shake culture:..... dreiayaigpetets aca bopted/omiaiais anaes. an aioepresa ee ba delsmeamlalae wantet wriiee
(2) Fermentation tube, growth in open arm.............6.. closed arMeccscisesccesd a
rate of development: 24 hours... .......... per cent., 48 hours...........4. per cent.
TO HOMES: «sa taiesamcnie Dees PEL Cent, ain sseny weaceed ip MOUPS is Peek ideenaieas per cent, Teaction iM OPN ALM.....seseveecseeeees GM Riess A saurclanssapine Ne RpeRIEIG wi ek ib iutatene ae
gas formula, H: CO2::....... bias ean eee Seccorbcady ech reealle AN etc abiteurs Matar SIAN bie ldiasesseaia ace
Ds TRCtOS Crciscsiassisresaa sc w siecetay-aierarsieia spas oe, C, SACCHALOSE ksicccsiads ve vewiccersrane ieiadiviccents aaataiatige
7, REDUCTION OF NITRATES (64, 0s swewaswoues na sieeeaveds hae scteleeanies acoeularaceyaptiavten diel 4 ies i aie to nitrites.........46- aCaleine ie SABIAN NEI TS $0 AMMO DI Gasees ex se eatieiinns eoot8 See
8: INDOU PRODUCTION «tatiana sree dbemce ee Rea Ves ea Mile aiectinnie. | wiseaeesrane gine ys Suaiatemtee be ayelieanaied ESN OUTS sasdivedaivns chee do taser eUaad dusedemanied sade GAYS iosaian Aarebiederdtaeien 14 temo 9. ENZYME PRODUCTION? ...-.sseeeeeee (MMENTER CHARA TAREE Sal HRA HoNMReREN FLAT 2
proteoclytic.....ccecececrece aitiiminteiasetereideris ma memes Riga iae alae ae Sarmapiats Geode WES digestion of gelatin ip earactte iaiensee veeaiae digestion Of CasSein.......... ces eeee eens
diastatic....... wg siainiaralene "a atiowsas relat Oke apse (OaGS Uetouenedemed ae Wanetacaly iid teat sees
10. CHARACTERISTIC ODOR! vececesccerseeeercnteeeeret adimearaniasnets ‘Haiteueund aesiaseteia aredereies nina ae’
11. PATHOGENESIS (or other special Characters) :...scccsccccenceeeceeeveeesteeeeeeeeeen scenes
sede eee nnnes eee eee eee eee SS ee Tee ee eee eee. Cec ee er cer er ce er cerns seeeies errr ree ree ere eee ee eee eee ree eer ee eee rer STS Tere eee eee ee eer rrr rrr
err er ern Deena neocons Seng nn hn re re ray Cenk nee wmee cena enue eee eeeeetunees We eeneene Fen ee reece neem e eee e eee eeeneeenenteeenane we eeneneee eeeeeOEee eee eee eee eee ee eee eee eee eee cee eee eee eee Cece ere err reer rer rrr rrr
PTeeeeeeeE PRUE eOU CREME eee EEE CEE CECCOeErrrereer rrr r rrr r tite cece reer er eee eee errr errr aren .
GLANDERS GROUP—Conrinven. 195
ay)
Bacterium rhusiopathiae (Krer.) Mic.
Synonyms. Bacillus of swine erysipelas; Bacterium erysipe- latus-suis MIcuLA.
EXpLaNatTory. First described by Loeffler in 1882. Found in blood, internal organs, etc., of swine infected with the disease.
REFERENCES. Loeffler. Arb. aus dem Kaiserl. Gesundheitsamte, 1885, 1, 46; C. 352; K. & W. ITI, 711; L. & N. 300; Mig. 2, 431; MeF’. 552.
MORPHOLOGICAL CHARACTERS: SKETCHES.
1. FORM AND ARRANGEMENT:
a. Bouillon...
0, Agneous Pentian-violets co seein eeiaieeneneni nerds sees aS isenessaansine 14 peeee §: Loéffler’s. meth ylen-bl te is sicicinsecieccamienie's aie sevistameuickivie ve vienitisaelee ew He saiaareasleuds A Stuer
a, Capsules b. Involution forms..... aiesdelbaing Fes Adatenies' oe otamona Me Usshiantaonin vs ayeenanaer reKenawaan ee ‘ 6: Deposit or!vaciolesiiss s- ctacuss somiecs wat me vaneesnns eoeeeants Mutemea re eleneRT ETE
Gi PIEOMOLDP HAUS Wey asics ses voids, Nasa ais HERES TSE METRE Masai nace sadivin’s vb eesiaire scale oie Selene “
196 CULTURE CHARACTERS
Reaction of media (Fuller’s scale) 4 ........ OF — ...eeeee Gelatin plate: Grown 24 hours at... ...... =O, Sketches , (a) Surface Colonies. (b) Deep Colonies. 48 ROULS Abiicsiice cise vcw's cuvesenees °c. 6 dayS at...eee--ses diedinyacey oe iiateaisats °C, Agar plate: Grown 24 hours at.. ........ °c. Sketches, (a) Surface Colonies. (b) Deep Colonies.
AB OUTS Atsvevsvevvcrsceessrsnvscese °C. 6 days Atvevereseses saiaeacameeMeens °C.
4
Special Media: (Such as litmus milk and blood serum.)
DAUTERIUM KHUSIOPATHIAE 197
Gelatin Stab: Grown 24 hours at............066+ °C,
°C, oC
«...-hours at seoseessDOurs at...... 5.
48 HOULS At .sccseeseeeeveeee Perl Ory 6 days at .......... ebeicaiessate vsosece oC.
Agar Streak: Grown 24 hours at....... reeeereTy °C. oO rs) °o o @ 3 4A 4
48 HOULS At ..ccsccececeeees ewes °C. 6 days At...sercccceeenenseecees °C. : Potato: Grown 24 hours at......ccecee eee eee OC, fo} ry
- 8
+evnecaa MOVES Abeawxene®, «»..-hours at......
G
48 HOULS Ab... ce ceeeeeeeeeeeee a So 6 days Atroocescssavccuessavcees °C. Bouillon: Grown 24 hours at..... ewe peuotrenmnmiee Os oO 3 3 3 8 a n n uu H 3 5 ° ° 4 4
48 hours at....sescescveaees mente’ Oy Oday Satisiscwsavascsenacces vee Cy
498 PHYSIOLOGICAL CHARACTERS
ODMH acsnpaence tes <axeaae ne °C.; limits....... isigia keene AG coicvsiarne teersneidatas °O3 thermal death-point................665 °C.; time of exposure............... minutes: medium in which exposure is MaAde......s eee ee vce ee een even een eenees aioaiewelaciea ane vere ae
2 RELATION TO FREE OXYGEN?.......+ Sahat Sis Samuel RV a Oey dieignesnigg emg aiaios aie aap Ss ewlealEeteG avis
3. RELATION TO OTHER AGENTS, SUCH AS.....++ Penne eer eee e eres eeeeeeeEraseneeetererenirees
desiccation, light, disinfectants, etc.:—.......eeeee es ciaiiasoiatacisiciniate aisitie ti aloieteicsieeina oad
a ee Demon Oca near neem en esas eeaeeseeneeae Dect e eee nee n ar eee eee e eens Ameer eee ne ee ree sree ter reeeeeane ee eee rene cere eeene eee see eeeeees Scene eee e ener eneeee
a. dextrose (1) Shake culture:....... Saati ices steteermele ed deepened cia enindameeavieeaimieet Sater (2) Fermentation tube, growth in open arm........-.. snsdetns CLOSE A ATM ease 03 erence ‘ rate of development: 24 hours............05 per cent.. 48 hours........+++++ per cent,
V2 DOUES wis ssswieses se reeera DEN CON tiys cscaeee cateees «ps NOUTSiss aewaies von soe per cent. reaction in open arm........+++ Petes OTN MANGA ARNG Aare SoMtenaeay TaaMERredN gas formula, H: CO2::....... seieeaaraameste sianitass stpeadcareeheiad sion shea aissaiategetoaiasciteisra sianiaaeoia
Ves LACTOSE venice siete ainidaenecia eaianelae’ aieerinad c. saccharose......-- Sapa wenneatacaseetnthe 4 aretainsdiore
Dibra S aml ee aisrssawd eiaeeevewe wie esas ees Cauanermeatens awison ated yp eCpassavdiains ste - -acaiapejeieta ie .
7. REDUCTION OF NITRATES!..ccseeeesevceeserenes Enna a(einncitearalestan secposouaenaanes ey sGioesdsasapiediers wat.cias ag tONULItES sii pssesnwnies onweee coverceesceessbO AMMONIA». ceveenes aching ae 8. INDOL PRODUCTION......+++ seas es ARR Teas Esai e acererarn Bai die Geetha les ataiatar ease ciara ease slat heolt 48 hours.......- ssiareratenesee sg §-sjoletaloressCairielg hae gia oiediada dL UY Sriereisie: iais-a tale nansivarsiadies ae aie ot0 . 9. ENZYME PRODUCTION?...e..eeseeeee ApenwemenawReeartene iauis) BaRhaaeheeeRaeaeene gee ee
PTOUCOl TICS sxcivinieasa nines sa s0e cieaialen Guin v4 sree Ont Ki neaaninnts sea ae ararioGonia Oaaa ales F digestion of gelatin ....... Himecematades digestion Of CaSCiN.....cceee eee eeee eae IASC Cra temsnseias tate sade Desens ehuieeea EL AA oucanalMias dais siaigeaneebasbasr". "acetone sielt 10, CHARACTERISTIC ODOR ssseseceveereeeeseere ecbiaw's Wasa eae eye jeaoanennten aaa
11. PATHOGENESIS (or other special characters):..... sieia be arstotorsieneigs Ruane sclsas sairwinganiewe
EXERCISE 84. DIPHTHERIA GROUP. 199 Bacterium diphtheriae (Logrrier) Mic.
Synonyms. Bacillus diphtheriae Lorrrier; Klebs-Loeffler bacillus.
ExpLanatory. First deseribed in 1883 by Klebs. First culti- vated in 1884 by Loeffler. Found in the false membranesin cases of diphtheria, and in small numbers in spleén, liver, ete.; occasionally in healthy throats.
REFERENCES. Klebs, Verhandl. d. Kongress fuer innere Medi- zin, 1883, II; Loeffler, Mitth. aus dem Kaiserl. Gesundheitsamte, 1884, 2, 421; A. 386; C. 354; Fl. 2, 460; H. 194; K. & W. IT. 759; L. & K. 207; L. & N. 389; Mig. 2, 499; M. & R. 356; M. & W. 137; McF. 410; P. 229.
MORPHOLOGICAL CHARACTERS: SKETCHES.
1. ForRM AND ARRANGEMENT:
a. Bouillon
a. Aqueous gentian-violet U: Loeffler’s Methylen-DlUeicessa sane siwciineee neeiadavisvve's dna aavaawamome ne RemaeNiON co ees
Ga GRATIS SB GALI asic sradersseisin sis is v cinnctejotanssiins Wa sieraratainaialals o welatbialaeies Ri aie Giants pacman Meee RS
a. Character Of MOVCMECNt..cece reece eee crete ete e ete teen eee eee eee tree EEE bees ai Siareidieinie
‘Bis FUL ASSL ass GAM sis avessecsniyosess mince eiosevisecesednig bare: di4o vatoeaanvnjaacahsyyaroreyayea hugo lace alaighulndieimeale ais piel wealeipealeiees
ry CAPSS iaga ve noncusgs secedanmee tetrad eens ceases. eae Ke EOad ease ce ER omemnCaNN bs Lope b. Involution forms......+..++ SSialansinatied: Jel oe Bhvisianetare SterenSanlardinitie Sy Morena Npatelelnn astasbietilas eae en
c. Deposits or vacuoles.
a, Pleomorghism....<>
200 CULTURE CHARACTERS
e Reaction of media (Fuller’s scale) + ........ OT — weeweees Gelatin plate: Grown 24 hours at............ °C. Sketches. a) Surface Colonies. (b) Deep Colonies. AB HOUTS At. cc cccceresscee seoeeenees °C. RUBS Bicasasrcrcevanncnens cies vewes °C. Agar plate: Grown 24 hours at.. ......... °C; Sketches, (a) Surface Colonies. (b) Deep Colonies. AS OUTS At. sseeseceeeeeeneeveneeeees °C. 6 days at...cseeues SounewadaeeceseNs °C.
Special Media: (Such as litmus milk and blood serum.)
eee Wir HERIAE 201
Gelatin Stab: Grown 24 hours at....seseeeeee ee °C
) ° Lae é | 8 R & | 3 E | a A8 HOLS Bbc cccisness vewseancs OF 6 days at..... ese Be taraiwleatieatgais oCs Agar Streak: Grown 24 hours at.....seseeeeee ee °C. 3 on ° ° 3 € n n Eo 3 =] te) 4 A 48 HOULS At ..sseeeeeereeese waar Cs 6 days at...... ecashianBereneraiae eecees Potato: Grown 24 hours at....-.......-eeee eee eee °c. ro) Oo ° ° 4 2 n n bar] w & & 4 4 48 hours at........ vedaweweaayd °Cy CUA S Atrvasaiceasienaeamecians OCs Bouillon: Grown 24 hours at.......-eeeseeeeeeees PC. ia} 3 3 3 4 = 3 3 nN n iol iol 3 =] ) iS} 4 4
C
48 HOULS Abseseseccveveeeevoore Oe Gia yiS) AD: isiieeyts vis eiprmarmereaneyerie PCs
202 PHYSIOLOGICAL CHARACTERS
1, RELATION TO TEMPERATURE!......ssccessenee ee reread ion OPtiMUM o5 os i cw cornseawarees ee a °C.; limits..... oh Waeead Keaeee TOnsosiw es veeueeaas te PCs thermal death-point.................4. °C.; time of CXPOSULE......ee sree minutes; medium in which exposure is madeé.........5- adumnininhemeduenanne SiC eae eee a
2, RELATION TO FREE OXYGEN?:.......esee0s 86s srinsleteioes Hawise Sebaneaes Uiashee pioratnaisia nee Meee we aoe
3. RELATION TO OTHER AGENTS, 8UCH AS........ aaah Meme Ag saouinvalets xe cone ae x
desiccation, light, disinfectants, ete. :—....0...scccceeceeceeeveuseuceesuscuseeeunenss
5. GAS PRODUCTION IN SUGAR MEDIA l..6 cssseccccusccsuscceeseuvetenecenes ena ene e rere ses
a, dextrose (1) Shake culture:
(2) Fermentation tube, growth in open arm........ ern Closed. AM en ccr ies eee nee rate of development: 24 hours.......... ...-per Cent.. 48 hours....sesseeeee per cent.
TZ IVOUTS iearwovinns 3050 He asigiigss PCY CONt.,.ccreeeeernee sees DOULS. eee eee tes iaselce Sea per cent. reaction in OPEN ATM. cee seceeeeceeeees Sas ain eae eee ates sie gi anteianeeaie aa preigkerateinary arate gasiformila, Fos CO2i 2: sascncws, 26 28 teewun awk eee eeies we Sosawaws v4 Sessoms daa aie
vb, lactose... Sad Ghd ude hchec eas aygeo leoiatnateis 8 AVE digas) HAS AEC DATOS Ch acta via & salisiessdieaiots One pa inimeieINe nareinwls
6. ACID OR ALKALI PRODUCTION: «11.6 seeeeeee scence Peden eee e ene ee nanos Deemer eneeane sane
7, REDUCTION OF NITRATES foci snieianiia ce fi steenion ae aaseeisio meats 04 db ears terete Sia sibrareeteiats ve ePaysisield PO TUIGLICES: icc rise ay swine aie desidenratgen to ammonia..... saluieianars od aetna sen caee NEE i 8 INDO PRODUCTION: ys va sevewncar rs eo daedaaenersiniegl eoainemem av sae BOD OCT hee CMe a re AS WOUPS ie ch sa dk “RewANGS io eASvamE TSG fi aerial AY Sie caiie MISS AR RMTS Seow 9. ENZYME PRODUCTION. ...ecseeeeeees
proteolytic......... a digestion of gelatin diastatic..... AGH des RAORET 1G-Ny TEMMORTEN EE Se aD sreiodion’ a ais BAG aesiosers J PHSTAAOR EEGs #- Ranomnien
10. CHARACTERISTIC ODOR? .ncceec iss sacndangeabar’ venmes aa tyata lis hc tesrtiara crates tera toeaiets aise Sisiens
been beenee reer eee reer eee eee rere) ee eee eee eee ee err peereee date eee were e eee eene eee eee eee er ee eee ce eis ee er ee es OTe CeCe Cee eee eee ee eee er rrr RC re ine aor iene rca nare) tae eeeeeee yaa abo "4 a Se LENIN A beg akieacnle Be eae Soe FB OaINILE NEES eee ee renee eee Perret Cece eee eee ray es
DIPHTHERIA GROUP—Continven. 2038
Bacterium pseudodiphtheriticum (Lorrrrer) Mic.
Synonyms. Pseudodiphtheria bacillus of LOEFFLER; xerose ba- cillus of Nersser-KUsScHBERT.
EXPLANATORY. Isolated by Hoffman and others, from the healthy mouth and throat; by Neisser-Kuschbert and others from xerosis and other affections of the conjunctiva. This may be only a non-virulent variety of Bact. diphtheriae.
Rererences. A. 401; C. 355; H. 214; K. & W. II, 823; L. &N. 404; Mig. 2, 503; M. & R. 370.
MORPHOLOGICAL CHARACTERS: SKETCHES,
1. FORM AND ARRANGEMENT:
Gs BOUiO RM sescchs Saeowentils ay ad adie diesaiedionaais 5
iis: UBOUS SONTAG cadscscrsndancnenk dancguiuney es ie okuawRea te ere Nmeeewawscons b. Loeffler’s methylen-blue abe ater Maja eases tin vinlacatvle ews Napali atelahouinus, 6a aie MaMMRe Mala Me Meoeaieaan. Sean i Gratd siStaitiy casucsss te agiewtis wx sanigecams aeoeieeaemten He Mareara nes a Anlneididreremarne dd dw weua-nern
d. Special stains....
a. Capsules b. Involution forms..........) Fideane SY KARR TAINED TARE UND bs Ladeoenae enndes vc. Deposits or VaCUoles....ssseesecreeeee Beta tieieiats ieiesataiea eee
d. Pleomorphism...... ssssesoseeees Gn estanatntsrn ‘0% sscedStehn in wie ates che abbbrareswratans siaraesnalnntaric wave aeavain ere
204 CULTURE CHARACTERS
Reaction of media (Fuller’s scale) 4 ........ OF Seeks ee te Gelatin plate: Grown 24 hours at............ °C, Sketches. (a) Surface Colonies. (b) Deep Colonies. 4B OULS Atscevcccceee ices weet eee °C, Gay Scdtiesnccews Mdseannauws wed Wen Agar plate: Grown 24hoursat.......... °C. Sketches. (a) Surface Colonies. (b) Deep Colonies. GAYS Ab iicadie de ad temesaa geeennmuitien PCs
AS HOULS At. rceceeeeeesnecsnseevecees °C.
Special Media: (Such as litmus milk and blood serum.)
Bactrertum PSEUDODIPHTHERITICUM
205
Gelatin Stab: Grown 24 hours at.... se.Gs
erie sonwies 3 3 x x é 3 g g 5 5 4 4 48 HOUTS At.eescecceeeeeeee tine’ Oe 6 days atsxvinseonrsnsssneorace€s Agar Streak: Grown 24 hours at...-.sseeeeeee Orig OF 5 3 3 o if \ ra 4 d : 4 4 AS HOUTS Ati cenzec scien carameawey °C. 6 days at......+ siaidhnid aia arotere.cyous 2 OF Potato: Grown 24 bours at..... ceeeeeeeeeeeeees Pad OF is] fo) 3 : ule rs rs n n 3 3 io) ° 4 4 48 HOUWLrS at c2esecss cc seewseeees °C; COAST Wtiecigeraareisiseitenere dines 8CS Bouillon: Grown 24 hours at......+seeeseeseeeeee a Os io) 3 e ° ° a eo n n 3 3 ro) ES) _ ar 48 HOUYS Ab sseeseeeserenes wines 6 days at...... ia ance isin ated nna on
206 PHYSIOLOGICAL CHARACTERS
1. RELATION TO TEMPERATURE!....seeessees sis SSE dca’ RRMA 03 Ke CRE ERE Breer ae OPPIMUNY aicesccdeiewtveneoreses SC IMIES: owas Hane see eomsed LO nd oviaaintied vodamaae 528658 thermal death-point.........0..0es0eee °C.; time of exposure............... minutes; medium in which exposure iS Made... ese eeceee reese eer eeeee aieviossiaia wneaes earareceny ere
2. RELATION TO PREP OXYGEN: cides ssnvens ine venawe Aad (eee keouaU TREE ENG oN
hb OF NONE RO INS BEN SHS Le FETE Ae! Be Belew See ne ey eee ery
8. RELATION TO OTHER AGENTS, SUCH AS...eeseeees isa ac bans enue a Hiteisleletutere g DiaeluaicuminIAnS ae
desiccation, light, disinfectants, efc.:—....ccsseeeee stdhieaecneeatavicved eee Si wietarNareateisre witeets
eee eeae beens Scenes Sen ee senna set eeeeee eee rr Se en seen eees
5. GAS PRODUCTION IN SUGAR MEDIA!.....005 win 6 fanaa, sedv@iavoxerb fio elaesndavarolatene Win nisieteain ata sous aw. dextrose (1) Shake culture:..... site oy widaaee eiccton aise distoisisiaidis aYatalpibtatalieraie puis winhduatstots siteaad
(2) Fermentation tube, growth in open arm.....seeeuseeees closed arm.......... cate?
rate of development: 24 hours.. ....... ++. per cent., 48 hours..........54. per cent.
G2 OUT Srna esac saci pvcisiees PEF COME, ccwise sereasncedees hours hae extaies sees Der cent, Yeaction iN OPEN ALM. .ceecseeceerececeeee a fers aa Shejaseceiseeualetespists oe Rees asa Sieg Stacgons igstreichasninis
Pas Lormiilia, Hi COs 8 vies siejewariawacd sinyesaerasined na emeedarnwes, saidwre nies av eniensremacede .
Vs, TACEOS Crisco cs de $s o siidaennela eases se NOS SMOCCHALOSE ieisie's eo diesieseipars e's Seipiecensicieinens aia
7, REDUCTION OP NITRATES tescssca ds sa cecum peed sae cwiee se oa eee amis ae enemas eee Ree ootace
HOMMTEETES Sisaieieic taitevare vis 4 Melarcieied Mean tO AMMONIA... eecceccseeserecceeren reeves 8. INDOLYPRODUCTION( ce scsreea ie dio onsie wraeayiaterereinstd ela sasbciaiatesiieae. ine Pereete cee rer a Tere
48 hours..... sineiaretaie acatdin etnies disse Ga sis datiwle Maleate ae ee days..... Cidscrateatcte oun eeeeaise wi 9, BW2ZVME PRODUOTIONS ....c..ck2rvy haweoded Cenks Sieachowncy » —shaneen Voweenns Masaceas a sta Mee
PYOTCOLYV FIC. ssccns 05 vas eiwmumcerseseres dacaewee eid - ged San tatnene aebtaiste es Saeeearenen Gigestion- GLelatin s seers. s.ccx daca digestion of casein.......... aaleciateietstonte
diastatic..........06- Sion Sivarsilyneaiantds ea Sateyasniatt-a od narm@tesereuteid atarharenGamilets a doin apdepeaang cae ase
{0 CHARACTERISTIC (ODORS, iiss wiinciisy Giciwy ngrsieiseisiwapy be He aarnwed is as lee gale a Seiaaiangewenuigame
11. PATHOGENESIS (or other special characters):..........05 ohewuna Cen Beene ROR werent sehen
sence men ceen ees tenon ener eee ene oe eee) See ry . Senet eee eae e none vee eee e eee ene e eens Pernt eee mmmnee eee ee eenee ve Fete e eee eens ee ee rc ry peewee eens beeen eeee wee eee eens Bebe e en eeeseoeenene been entre eee ee eee ee ee eee ee eee ee ee eee er eee ee eee reer eee r rey ee ey eee eee eee ere e ee eee eee eee eee eee eee eee CeCe eee eee eee eee ree eee)
A DOAOS IE Kathy sew ws ale: eee eee eee eee cee eee eee eee eee eee eee eee eee rer
EXERCISE 85. PNEUMONIA GROUP. 2047 Bacterium pneumoniae (WricuseLbaum) Mic,
Synonyms. Fraenkel’s pneumococcus; diplococeus pneumonie WEICHSELBAUM ; Streptococcus lanceolatus GAMALELA.
EXpPLANaTory. First described by Sternberg in 1880. Found in saliva and in the nasal secretion of healthy persons—from 20 to 50 per cent. Usually present in ‘‘rusty sputum’’ of pneumonia patients.
REFERENCES. Weichselbaum, Wiener Med. Jahrbuecher, 1886; Welch, Johns Hop. Hosp. Bulletin, 1892, 3, 125; A. 321; C. 63; H. 310; K. & W. II, 823; L. & N. 143; Mig. 2, 347; M. & R. 208; McF. 289; P. 498.
MorPHOLOGICAL CHARACTERS: SKETCHES.
1. ForM AND ARRANGEMENT:
Gs. BOUTMONE. seassissesnrsioinie SidicivicisialeaaeS Biss tiicsa Se can ahead sersie ofsleraw eave lareutente i
a, AQUOS Pentian-vidleticss sereecnewic ds teeewrenest ese: pepe CORRE LE eeNeReneaG aK
b. Loeffler’s methylen-blue Gc MCU TLS SURAT toeia epnscho ehstavsrane i scatertic aassalace aisha lcvelavaislsiur arias gialaie Whatd We dk ai eeepdiordia eae goretiuectayan ea WE ds. Specialistains.siass a vesccotns cane nataebiiueuney savennaes ai asiseatnasiena na conn ewes t
Ae MODUL TT ite asia ses eoriesvincinte ale th DANO AR em nak See Reeaeed Ne Sa aReNdNeRIAS ei eeaaaR tee
uw. Character of movement.
b. Flagella stain........++« BA datalolai neha cetatonsis See: ap ies ares ahsalo: avend auanavesalavestevata eiblaraialeccuara seis Siete achat
G:- SPHOTAT, CHAR A OTERS tiie sisaisiassisce cidrsnnisioraioreibiasannsciaindaiate areintaedle Siansainienath eis cceaimiere Wie cmvbrcioutra or dimeldels
G. Capstlesiseisees ovaves ssssingnaadantea sates eeniecsan spew neat on temestionesicnenintwauaer aie Dy Tri VOlUbOT LORS Ss eeratecerstee We eer Kitice eine slateialaca ws oa iwia avaisiesiale avs areisiatecnigincele siecersigiosaie eaivin
c. Deposits or vacuoles
d. Pleomorphism...... ..cecee cree een eeeeeeee
208 CULTURE CHARACTERS
Reaction of media (Fuller’s scale) 4 ........ OF = Gees aan Gelatin plate: Grown 24 hours at............ °C. Sketches. (a) Surface Colonies. (b) Deep Colonies. PSHOULS Aticccvenesas os aeewceeen °C. Geb VS Aa Bi ssenratirseisnsireSisasnaaaresne as °C. Agar plate: Grown 24 hours at. ......... i OF Sketches. (a) Surface Colonies. (b) Deep Colonies. |! AS HOUTS Abi iiiecnteiecemncies sacwaninee eC.
BC AYS Abis saisisivesw dei wasswardameteceer °C.
Special Media: (Such as litmus milk and blood serum.)
Bacterium PNEUMONIAE
209
Gelatin Stab: Grown 24 hours at.........-.6.....°C.
eC.
md OF
veeeeeeNOUTS At.eceeeee . hours at
43 DOULS Abe. e eee cece cen eees at OF 6 days at
Agar Streak: Grown 24 hours at.....seseeeeeeee °C.
°c °C.
\
Sea
seceveeeshOUES Ab.....05
aa
seseeeessNOULS Abeeeeeee
48 HOULS At ..sseceeeeeseeseeeee PCy
6 days at.....- aaiareaelatatantesionreee °C.
Potato: Grown 24 hours at.......... sSeE ees °C.
C °C,
seeceeeesNOUFS Ateseeeee? (row BOUlS Bticnncin
<
48 hours at..... ohdyivavaea sae anda Os 6 days at.
i
Bouillon: Grown 24 hours at.........5+5 iesialage noun
°c 16)
hours at . hours at.......
48 HOUTS Btis< sciewreasscswsenss Cs 6 days at.
210 PHYSIOLOGICAL CHARACTERS
ODEMUM os i.e nensiecwens ves saicanee 2Gi9 LIMES: ceamwsaciicne acess TOs ndianinrsa denommnacar °C thermal death-point.............0eeeee °C.; time of exposure...... ....... minutes; medium in which exposure is made....... a Stara s/asaeeiarauaca Sin aietoreattgavetgss Test we wlapaane 2. RELATION TO FREE OXYGEN!....eeseeeees i ip ewentas (aisy Cewheimney. epened sakes 444 RARE 3 RELATION TO OTHER AGUNTS, SUCH AScsxisi co cedwans i 6 Whedews RR e ed eVeseorenda crane Kase
desiccation, light, disinfectants, etc.:—
4. PIGMEMT PRODUCTION fis cmesusxeseeunernees rea Kes SmeaW ed EDR ER ERAT Si Bate ReTaNGIC Aa Finis ath j
rate of development: 24 hours.. ...... ...-per cent., 48 hours.........0.65 per cent. 12 DOUTS wicies os c0 eo rewene DOE COlibaysisesaxine seaey on seus OWES sa seiie ee ox enes per cent. reaction in open arm......sseeeeee rarest FSSA AE AAAs SERIES Be RRA choy eaiind
Pas Gnmiala, Hes) COaix ss wsieinseiarers sacs stoaersveeisretuiera ds wkcgactices. aw tev dandiad va ween dcebins eee
Di AlACKOS Cian. vigor weseean euuwene ede tue Ge SACCHALOSC isis.s vs sinisicrdeeauns a viewwenens agrearee
AB OULS.. eee eee i shales ieee stiainiisieaiiieaa se GAY Sisiviec a edacddaw avnes ecapanaes ‘ 9. ENZYME PRODUCTION:......+065 a ois SARS G CeieehieTieWieGieied. ate satsemans wii@etecande is Bias eae
proteolytic...... sas igveceisigh avai sidielblanuinnsisiaiass sigeutsbvodaren Whoever Siehisheienind te Cateotsamnas digestion of gelatin..........6- aoreeetor digestion of Gasein........6.-cce eee meas
diastatic....... fe saeianag Fa Sate ee Soa semignee dises mae as a Soi Acidiahaitielulannsbeadiios ers
se eeenes ne ee any eee eee eee eee ee eee ee eee eee rere eee rer Peet eee ee ene e ne tesa eene ee ee ee cee cee reece Tee e eee eee errr errs
EXERCISE 86. INFLUENZA GROUP, 211
Bacterium influenzae (Preirrer) L. & N.
Synonym. Bacillus influenzae PFEIFFER.
Expuanatory. First described in 1892 by R. Pfeiffer. Found in the sputum and in nasal secretions of the diseased.
Rererences. Pfeiffer, Z. f. H. 1993, 13; 357; A. 371; C. 351; Fl. 2; 434; H. 316; K. & W. III, 359; L. & N. 202; Mig. 2, 506; M. & R. 430; MeF. 574; P. 320.
MORPHOLOGICAL CHARACTERS: SKETCHES.
1. FoRM AND ARRANGEMENT:
uw. Bouillon.,....eeeceee
8: STAINING POWERS ticnsis ccusscmnndemsecansaememienwearea dete widseeceenedsae “ w. Aqueous gentian-violet............ee a enewenoucee ence e neuen ee eeerecseeeneteeetsesneeees b. Loeffler’s methylen-blue......seeeeeeeeereeeies so cin ore a sinisvecebatbedreveteigiars ote dig eieteslineaiak. carn c
€
a, Character of movement.....-seeeseeeeeee Sortys sis HORT Cara RE a sieisiaieleiajaainie-g-e aise eiaidiaivinracia
bs: Pla zella Staines sesssssca aageeosteass teaciest wecce SEG sip siaieie wraie aie alo ots ea ai natn eaiee ss Saw
tis, Capsilesscicsies sicearecceris re
. Involution forms
o
c. Deposits OF VACUOlES.....sseeseeseteeeneneee saa iv ore ipteia aie lore: aes'ojete elotnarary Da WORSEN EE Sires
d. Pleomorphism...... veneee. cocveseecees Giaiasaiadaie ahucahauia Sete ex xtinopen Adon cette esate ee
212 CULTURE CHARACTERS
B. influenzae does not grow on the ordinary artificial culture media, but may be cultivated on agar slopes upon the surfaces 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 87. TUBERCLE GROUP, 218
Bacterium tuberculosis (Kocu) Mice.
Synonyms. Tubercle bacillus; Bacillus tuberculosis Kocu.
EXpLaNatory. First described by Koch in 1882. Found in diseased tissues of man and animals and in phthisical sputum.
REFERENCES. Koch, Berlin. Klin. Wochenschr., 1882, 15, 221; Smith, Jour. Exp. Med., 1898, 3, 451; A. 330; C. 356; FI. 2, 481; H. 225; K. & W. II, 78; L. & K. 251; L. & N. 410; Mig. 2, 492; M. & R. 236; M. & W. 148; McF. 305; P. 623.
MORPHOLOGICAL CHARACTERS: SKETCHES
1. FORM AND ARRANGEMENT:
a. Bouillon
B. STAINING POWERS!...cccseeecereeeeerecerereenenseeesene oe aeesieeate
a. Aqueous Gentian-Violet....csccsccecceveeeesceveves nelah COMI MeRaN ated se oak sGiisicnereraiiie ne i
b. Loeffler’s methylen-blue..........eseeseecees SiG RISSGICISS Hieies ids ticisiacniedatere cannes tachi’ beaeere
c. Gram’s stain d. Special Stains....ccccecsccceceeseeeeee Uisshia sioitvannubionstejaisia se slosPavdermmracammnte son a's cewisisrdespip eas dy MOPILATY Riccccssciamrcnpcmcnecamnesiniien” sieieramreiaa bins Wale shed arctadwis & she apaiaineieales Ua eS aeiawiainaie
ux Character of MOVeEMeN bisicicscisicsisscwine ces ctiiewials waaindeisieaiale Ssisteplwanwawaneds cue eeinta
a. Capsules......seeeee At reeseareasesseneeces eeseeeeee vos aiatsia eaem eine emai a ae aieeaeatiaeies Ore dL. Involution LOTM Si: + ccwenaws va oaeseien ve taasays ox Me Ke RineN es Meda RHS TUES owas RaE Oe ‘ c. Deposits or vacuoles........ a taENa7a ar tisrasayedaBA7a Ot Ses Siatpieipiois asbiauccsierstaie og danayiloa sincere entcleaadibare
gh, Wien ist. vc aa as sdae ch dagnmns 25 bake aden pad atdmainsd vevawankan te Leanne s
214 CULTURE CHARACTERS
Bact. tuberculosis does not grow upon the ordinary artificial media, but may be grown upon human and beef blood serum, and after it has been isolated for some time it may be grown upon bouil- lon, agar and potato to which 5% of glycerine has been added. Media which are suitable for the isolation of this organism and at the same time are easy to prepare are Smith’s dog blood serum (Jour. Exp. Med., 1898, 3, 456), and Dorset’s Egg Medium. The last is the simpler, is very satisfactory, and is prepared as follows: Perfectly fresh eggs are taken, shell broken at one end and the entire contents poured into a wide mouthed sterile flask. The yolks are broken with a sterile platinum wire and 25 cc. of water added to each four eggs, and then the flask is shaken until the contents are evenly mixed. The mixture is then strained through sterile cloth which removes the bubbles and makes a homogeneous medium. Run into sterile test-tubes, about 10 ce. in each, and incline in a serum inspissator or oven and heat up to 70° C. until coagulated. This usually requires four to five hours a day for two days. This is all of the sterilization usually needed. If heated higher the medium is hardened quicker but the tubercle germ does not seem to grow so rapidly. Before inoculating the tubes they should be sealed and placed in the incubator for several days. Cultures from tubercular lesions are made by tearing the tubercle out with sterile forceps, crushed as well as possible with the forceps, transferred to the egg- slopes with a sterile platinum loop; leave bits of tissue on medium, avoid breaking surface of medium. (Amer. Med., 1902, and Bull. 52, Part I., Bureau of Animal Industry, 1904.) The tubercle bac- terium is very sensitive to temperature variations and should there- fore be kept at a temperature varying at most only a degree or two from 38° C. It is also extremely sensitive towards desiccation, and, for this reason, the cotton plug should be well paraffined, or replaced by a cork through which a small cotton-plugged glass tube passes, and the atmosphere of the incubator kept saturated with moisture.
Bacterium TUBERCULOSIS 215
216 TUBERCLE GROUP—Uontinvep.
Bacterium tuberculosis var. avium (Kruse) Mic.
Synonyms. Bacillus of fowl tuberculosis; Bacillus tuberculosis avium KRUSE.
Expianatory. This organism was first separated from B.
tuberculosis by Maffucci, and is probably only a variety of the latter. It is pathogenic for fowl], but ordinarily not for other animals.
REFERENCES. Maffucci, Z. fur H., 1892, 11, 445; C. 356; K. & W. IT, 127; L. & N. 418; Mig. 2, 495.
MORPHOLOGICAL CHARACTERS: | SKETCHES,
1. FORM AND ARRANGEMENT:
is BOUT OM vsics peasart 02 6 dorset eons ae Hesa aleve aonteane ede miawinne NED Nataacas atin Aaiaree
G. Breen GORA GACT ERR rains veer seasdoney iaweieus of eivla Holgaiah footed SeaimaraveG Wax eeiied Gs ls MB STD OG ona e vx ewes sues ss uy nena eiademael ds eal RRRAGH A LOR ean aad Koumdedwanciaed be O, DavOlition TeeiSiewnseninceneay anunvey ri eewee IN Yi Ueekwedns pd dhe eaan1 a penednncunea dawns ‘ ¢. Deposits or vacuoles...sscsesenes aedaadinns Sid p iasatalachiaasa-aeactsataan’y wa viavastaaa4 Sabrieamebiad ear
Bacterium TUBERCULOSIS var. AVIUM 15
Culture requirements practically the same as for Bact. tuber- culosis.
218 CULTURE CHARACTERS
EXERCISE 88. COLON GROUP. 219
Bacillus coli (Escuenicu) Mie.
Synonyms. Bacterium coli commune Escu.; Colon bacillus.
EXPLANATORY. Escherich was the first to describe this organism which is widely known as a common inhabitant of the colon of man, and of some of the lower animals.
REFERENCES. Escherich, Darmbakt. des Siuglings, Stuttgart, 1886; A. 432; C. 205; H. 282; K. & W. II, 334; L. & N. 248; Mig. 2, 734; M. & R. 325; McF. 510.
MORPHOLOGICAL CHARACTERS: SKETCHES.
1. FoRM AND ARRANGEMENT:
Oe HOU M Othe c.csessawsciceasousaeee ds vedeveaenecncad ceaansiawaws danceeeaes
-. Gelatin. ..
tts, AOUBOUS BEMTANAVIOIOC wikis cersnamane sere, canasamacan so oc amaee ners eb AG Es PREREGAER TS & §. Loefler's Meth ylen-Dl Wein. weiss ciiasinecweieeo’s » saves cengis am ye te aeRmeweuees L4 Rises sass
c. Gram’s stain
d. Special stains
w. Character of MOVEMENE..crcceecsseeeteeeeeeeee een ceeseeeeese reece eenes b. Plagella stain.........ceesseeeeeeetene sieig;bh (pinta iuin ngs, be eislewatisletslcialate ag Poem REs AS Lage 5. SPORES2-ccecccssescccncccvensoneacceevenese eee rere ere rer tb Mpa sseRinis Gis win wteseninnd SAE Ree
6. SPECIAL CHARACTERS:......
@, CapsuleS..cc.ccccrree seoceerseccesseccessesecencens eoneenees b. Involution fOrMS.....csceeeeeee cones oes dra sien al stans azonabagaeyeisie, a. saa eww RintesatRta art Sarciiaiaqeiorereeed . ¢. Deposits or vacioles.:..e sc .s2ciinins saeneninses sHeaaT NS eadawieidics saeeiewnanee se eae aa
d. Pleomorphism...... cscceeescsesevceeonoes sereeisiatnieiaierniae siaaracstaiemale:deweatein Se ecreevoceccons
220 CULTURE CHARACTERS
Reaction of media (Fuller’s scale) + ........ OF > sii e's - Gelatin plate; Grown 24 hours at............ an Sketches. (a) Surface Colonies. (b) Deep Colonies. | AB HOULS Atereeeseseec sees ceeenenees °C. 6 days at..... isislasie avon feet te ne eees °C. Agar plate: Grown 24hoursat ........ ad Sketches. (a) Surface Colonies. (b) Deep Colonies. | ° a) MB hours At. 6 cccseeseascesernsensivns 8C; 6 days at....... Gateernindein as aimee eCs
Special Media: (Such as litmus milk and blood serum.)
BaciLuus Coit 221
Gelatin Stab: Grown 24 hours at.......06.0006.°C. 3
aa,
seeeees hours at... -hours at
ed
ABOUTS Abo csesseceveeerevee ee Oy 6 days at .......... er er
Agar Streak: Grown 24 hours at......eseeee eee °C.
veoveeeesDOurs at.......°C. secveeeesNOULS Abe00eee-°C,
48 OUrs abies oi scisicviceeecd gesin °C. 6 days at....... wiawistinwwseeamwel Os
Potato: Grown 24 hours at cccesonsaencvs ss is caases °C.
seeeshOUtS at.......°C.
seceeeeesDOULS Ateoeeeee °C.
C i
°
AS BOUES Blin cen menimnacas ined id 2 6 OAS Bhs cscs nineeces eres
Bouillon: Grown 24 hours at.............eeseeeee °C.
Pra
hours at -hours at.......°C
é
48 DOUYS at... ceeeeseeeeecevee °C. 6 days at.......... PMPs ee Cy
222 PHYSIOLOGICAL CHARACTERS
1. RELATION TO TEMPERATURE! .:...eeeeeeseeees sapsaise ates SOR alanis Skies Pata ae Nenedajeiae i ODTUAUIMN ...0 00s abesitineeevnneee soe OC Its) aaa senisinae icine bOMea aad Fe tawaaanie °C.3 thermal death-point.........eeeeeeeeee °C.; time of exposure.............. minutes; - medium in which exposure iS Made... ..cseceee sec ceee cece eeeeeeneeeeecenenears aides
2, RELATION TO FREE OXYGEN?..+-..: eaeaies a sha apeiclaalvintirs olprecslorertiosels tata wale ae WiaeeedtaraoirawINsicns
3. RELATION TO OTHER AGENTS, SUCH AS..s..ccesevveseeeeees Avie bicdobern teh cecanssrerens
desiccation, light, disinfectants, etC.:—.....ecccceeeeees ina liceatnavin alice siacaie erodes bale biy
Devens Bae eee e eee nets eeaseee Dae obec eee tiene et eee reese ecseereee eee eee ey Petes eee eee nny Stee e nee ween ee eeee oe e eee wna cence eee eee eneee so een ores wee e eer eneeees ee ee ere de neeee eee eee eee eee ee eee eee eee eee eee ee eee ey
4. PIGMENT PRODUCTION:......045 Dee a nee n neces eeees tees Nabe e ee meee nero nae eeeeen ee eeeeeeenne
5. GAS PRODUCTION IN SUGAR MEDIA!..ee...ee eee eee eee eee ee eee eee eee errr .
a. dextrose (1) Shake culture:
(2) Fermentation tube, growth in open arm..........+ +++. Closed arm.........65 eben rate of development: 24 hours...........+5+ per cent., 48 hours,..........6. per cent.
72 ours.....e.seee eS PEL CeNt.,.cccceeevvcceerecs hOUrSss.cjcesmsas cone per cent. TEACCION Tn OPEV Arias. vim, .Waiie ae shadow aay Vase ERTS CemeNaenHS i srenibedia te ais retical i gas formula, H: CO2::....... Sierdiard 4-4 Satara jormerererd wlashareraroinecd see ermneneiece acudaundiavacciosgreia Names ‘
1D; LACTOS Csicisiais ons vaccinate siniare aivirsinrais eeu etenes CS ACEH Arg seieeece use eteanegantey orsarate eiaieeee
6. ACID OR ALKALI PRODUCTION! ..-0+4 saceeeee osteeces ee cc cc ries
CO ee iy ere eee Cee Cee eee eee eee eee eee eee eee Tere ee eee eee eee cerry
7. REDUCTION OF NITRATES! iaescce snes cee sivnsinaennseinaeiianes ss ararefeces saa oli ieuhde dates’ daa Hi LO DITICESisienaacnscwwsss coe WeRRGe 18 GAS VSSHCO AM MONLAye. svsesactaraals oa smieiaaia sisi waa
proteolytic...... s -ViRde ems PON MATSON ECURE —GhRRTI as eGR OES DORE REM er digestion of gelatin.......ccseeeeeeecees digestion of Casein......sceecseeeeeeeeee
diastatic.........086 Mahia swisisneisioiew sivisinenysiveiate te ee Siipamebaiaiais: sieineaiensiaye sia Gie’s euanicninrala Neale a
10. CHARACTERISTIC ODOR: sig nis dabercisrcresitii
11. PATHOGENESIS (or other special characters) :.......0..eeeee dusishans wis gional eae nalesrrdenninai
ween wees eee eeeeeee eerie weeereee beeen rene enee on veneer eens . Denne reese on Steere ee eene ee eee ee ee Ps So ee ste ee reso ees eee ee Ce eee eee ee eee eee ee ee ee rn errr Daren ee nae eee ne eneeeee peeeeee eee eee Senn veneer eae AUTEN 6 dase wae Does Peete ee eee ee be eeeeee eer ee ee eee ee ee Pane eee ee nee rete etee
COLON GROUP—ContinveEp. 223
Bacillus enteritidis GarrtNER
ExpLanatory. Isolated by Gaertner from beef in meat poison- ing case, very closely related to preceding.
REFERENCES. Gaertner, Correspond. d. allg. Artze Vereins, Thuringen, 1888; C. 207; Fl. 2, 375; K. & W. II, 639; L. & N. 251; Mig. 2, 744; M. & R. 331; MeF. 517.
MORPHOLOGICAL CHARACTERS: SKETCHES,
1, FoRM AND ARRANGEMENT:
uw. Bouillon.
ie. AQUOOUS Cent laN-Fidlehesssnceies secer ener comer diouens so soe A Eee be Loefler’ smth yen bl US isisicsssevets.ete/cisicieictaccialbaraaie sia aigie electilainararey gen paymaetemlen fer Oe eRe
ec. Gram’s stain
5 SBORRS caswiecswns ravswasntaoos oiaiewemnanacsioh@ dats ds cauaemeamates peiNaaNesad Fa Dea! Beaeein vs 6. SPECIAL CHARACTERS?....+..05 dp itinsélapSapbiansista vie ate dausiavesstein’s «a@tossrareldica osasaye- oat ree iolintach’s agisidevarbvarare @, CapSulesS....cssecssseceenes ota ores cvdmeroiaiy ot i ewsineavtveran chaste estt axeiade ote Santa cualg neta vik rosaitrenageraes bv. Involution forms...... ais agoiahateea ace 6 aisiaidisrac ors Ziaveiia HBM GES SM ewWlale Buttle MleeniaGleg a eaatOTNeTeS
c. Deposits or vacuoles
d. Pleomorphism.
224 CULTURE CHARACTERS
Reaction of media (Fuller’s scale) + ...... OF — segue v9 Gelatin plate: Grown 24 hours at............ °C, Sketches. (a) Surface Colonies. (b) Deep Colonies. 48 HOULS Ateseessencceee aes maser Os GAGS Bb «cc's, 0:06 oiaie's Sisiejsisisigso's ere: aceretn °C. Agar plate: Grown 24 hours at..........5. °C. Sketches. (a) Surface Colonies. (b) Deep Colonies. oraigitiaees °C.
48 LOWES ties va28 caiwiswcarernaaywcmien 2G. GAYS Atirccccsevecceeceeeeee
Special Media: (Such as litmus milk and blood serum.)
BaciLLus ENTERITIDIS 225
Gelatin Stab: Grown 24 hours at....... RPA Lait: °C,
°
--hours at eis . hours at........°C
Ho) 3
AS DOMES AE cence nederie ness °C; 6 days at...... kale
Agar Streak: Grown 24 hours at... seeeeeeseeee °C:
aC °C
hours at soca SS DOUS Asc c sede
1
M8 NOULS Ab eee eee eee eee cece eee °C. 6 days at......+. OCC ONT ES
Potato: Grown 24 hours at........... ssiesniawariaaae® Gu.
°C wy
seeeeeee NOUFS Abeoweeee hours at.....
M8 OUTS Abs. cece eee ee wees 26. Gay Si atissens detec eats we.”
Bouillon: Grown 24 hours at............ epemvain °C,
°C.
eG
seeeeess hOUrS at.......
C
veawereeeHOUTS: Atenas
48 hours at... 1... eee eeee Brees Or 6 days at.......... awleoswapajronn’ Oy
226 PHYSIOLOGICAL CHARACTERS
OPTIMUM 60's tices cisicie wis oes cvecinn °C SIM IGS: seen. seid Quest bOnen ans xed Assieinsinensl Ces thermal death-point..............eeeee °C.; time of exposure..... shennan minutes; medium in which exposure iS MAdE.....cssecceveeeeveenenees Biginitls SG lereeeastanayatevess aes 2% RELATION TO FREE OSYGEN!. .ccccseseeees Sindl@ateiaia ein’ aiaieonlaieiialermiare Dee sueienisanoare a satbeelareente all
8. RELATION TO OTHER AGENTS, SUCH AS....sceeseeeeeeeee ee er eer
desiccation, light, disinfectants, etc.:—.......-.csceeee sladinairemaineremans wa kaneis Ore
5. GAS PRODUCTION IN SUGAR MEDIA!l.c+ cscccccceeseereeeeereesenseeseeuss Odean ec eeeceeseens
(2) Fermentation tube, growth in open arm.............64. closed arm........... rue rate of development: 24 hours..........0+- per cent., 48 hours............. per cent. W2NOUES, genercaeciieners scene per cent.,....0.66 Bihieautlneds HOULS: eejaiieaessa per cent.
reaction in open arm.....
gas formula, H: CO2::..... disiah \Gieiee wiearradalsnand Ke wacib ines ooh SGlamelnnts dabbles mannan D; lACtOSOsmaos sass +3 08 mete daca as ee Fe Gs SACCHALOS Cin cues iaieedweninay os caiticowenisiiins
6. ACID OR ALKALI PRODUCTION:....64 seeee Ce es
Litmiws Milk 25%. mmnanesoxexescanmeeade RSE Sea RUNMRa NENG MommANNGe sa, Riagumeites
POMPTIUFE Sis Lsrawicussorasnoies ye vious are daageancs TO BMIMON Lass 6c weriadane od serarmnnteneamts 8. INDOL PRODUCTION..:..eseeeeeeeeees caked veceeeees Geen iaiaiot Seiwilala sate daeadeas eta Seem Ge vente ISMOULS natigonces sah yRasainateslon HESdET gmat eeaialgs 9. ENZYME PRODUCTION:..
PVOteO PEG. 62 ie veces cpaceaenead estate ences anidigy shes Stage lavouecessiaeiekais aly edi abanapoesnneteienci® digestion of gelatin..........+++ Sree erie digestion of Casein........6. ceeeee enue
diastatic..... a astpbenne ae ete cata tcistera SNe Niet vO aiinini tnvoteie ibaa esuaterelte ante Seen eee ree nee
10: CHARACTERISTIC ODORS sauaag-adassinaaisndvany scan midais inipoeases cadeassaw’s tesnoaeeananas
11. PATHOGENESIS (or other special Characters) :...sccecsscsereeeeeeees Senne cremieemisaents
eee eee eee ee ee eee eee ee. hry seeeeee ee ee ee eee eee eee eee ee eee eee eee eee eee eee eee eee erry VELA VA ReOkage Es 88 64g eee ee ee eee eee eee e eee ee Ce ee ee ce
eee eee ee eee ee eee Cree ere ee eee eee e reer errr Cree eee ee ce ce rc
EXERCISE 89. HOG CHOLERA GROUP. 22%
Bacillus Salmonii (Trevisan) Cursrer.
Synonyms. Hog-cholera bacillus; B. suipestifer Kruse; Bact. eholera-suum Leum. & NEuUM.
ExpLanatory. First deseribed by Klein, 1884, first cultivated by Salmon and Smith in 1885. Occurs in blood, organs and intes- tinal contents of hogs suffering from hog cholera.
REFERENCES. Salmon and Smith, Rept. Bureau Anim. Ind., 1885-91; C. 210; H. 281; K. & W. III, 622; L. & N. 252; Mig. 2, 759; McF. 538.
MORPHOLOGICAL CHARACTERS: SKETCHES.
1. FoRM AND ARRANGEMENT:
te Bouillon jissssxcetdioak ue 2d enadenn as rim cnagieairanan ia vanicecanarmanaen
B. STAINING POWERS!.ccseecseeeeeneeeeenseeeeenens eMaCeae. wot vemetiees Seg Ns Sieiel csesansaearsiasstedesaiaie uh. AQUEOUS GENbIAN-VIOlCt. oc .cceeeeveeeeseeeeeeeeeteenenseneeereeeeenaeeees eioees Guormarareaaloud
b. Loeffier’s methylen-blue vs hous apbteoesesocaeteronedaverate jaaraardiineintessaian Poaw sthanovane
6, GRAINS Stat gus nee sania seanioeueaeeanweden srasevosd Seat Rial davesS saat nla eta ota tanstafeoes wrarenseatess i G. Special stains......scccccvesceesccsesserseneeneeeeseeeeeeeerneteeeennesseensseseessanness
€, MO DEIEV Hiwcdeans a tarinceaine i ranen oon’ BA espleaia OF EAI Gen eeI NENA St eeuReais Nee esamtere,
a. Character of MOVEMENE....erseseeeeeceees aetaleeieans So YonNeeea ee
D. Flagella stain.......cccceceeseeseseeeesens seesseenes fears beas Heme eRe Oni
eer eee errr eee PWrPPeeeeerCee Creer eee eer eee eee eee eee ee eee ee ee eee re eee ry
5. SPORESs: sieacaxcvelorweias ansersiniie aces cee Sia sisi $a\sicialonarsioiniaisiates ie aalataa tadegeieia Sisteaneawremea awe siete
6. SPECIAL CHARACTERS?..-++++cesuceeee a ae ote arslevetare ts ordin niaiaiemnensinie’s HearmeR sta eee ee ove Ut. CapSules...ccccccccceetenees tenes ence nee sen een eee ne beeen seen nencenseeeneneroesen eee eel F b. Involution fOriMS....ceere eves ceeee i Soya sovavearndea aroverasensvouocncovon ore ccaldvalarrlara's WA Ai SoatecwoneTatale ‘“
+, Deposits or vacuoles
7, Pleomorphism...... essere
228 CULTURE CHARACTERS
Reaction of media (Fuller’s seale) + ........ OF — saseanss Gelatin plate: Grown 24 hours at ........... °C. Sketches. (a) Surface Colonies. (b) Deep Colonies. 4S NOUS Btossniaaviinteee wane pens ba GWays ahi ccsics oven eisinbargyitela oe apiniee is Agar plate: Grown 24 hours at.. ......... °C. Sketches. (a) Surface Colonies. (b) Deep Colonies. Regus *Q.
ABTOULS At... eccceseceeenetcrneeeees °C. GB dAaYS Ab... ccccccsveravseces
Special Media: (Such as litmus milk and blood serum.)
BAcILLus SALMONI! 229
Gelatin Stab: Growu 24 hours at............0-. oe
°c °C.
e NOUNS Abicmewass hours at.....
4B Ours ab... cecceee cece neces ba 6 days at..... Sisavdaerniarsiece nies oC.
Agar Streak: Grown 24 hours at....... sisiainntageiee Ole
aah °C.
hours at .-hours at.
48 hours at..... PPeeerrerrr re rie oF 6 days at.......-+++ paeaeas Wass °C.
Potato: Grown 24 hours at...... a a's ahehinererciaizies Ye 8 °C.
oo PG,
-hours at...
1 aes awe OTES Bhi eewee ls
48 hours at......... aisyaleusesniieisie °C. 6 days at....ceccseeevee shinee
Bouillon: Grown 24 hours at....-..+---seeee sees °C.
°C,
og2C,
.» hours at...... hours at
‘
48 hours at........ enw pieenaa he GO GAYS Abin: weaasacises wera °C.
230 PHYSIOLOGICAL CHARACTERS
optimum....... Ph atirthousteid sists gacinn ? Cas LIM ItSedua 1 siete cuca TO cen vrnameevion woe? Cus thermal death-point........ SRE NS °C.; time of exposure........... +... Doinutes; medium in which exposure is madeé...........45 eer eres overeat
2. RELATION TO FREE OXYGEN!.........0. os avin Lao iers Ys Waheerielen wirtoutannfonatsanadtante satan
3. RELATION TO OTHER AGENTS, SUCH AS..ssevee seve eeereeee enw eeneeees See ree ea
desiccation, light, disinfectants, ete.:—........ lle asters furans We atayentve ness Neate Stata Hor
Oem e meee errno recene Onan e meen eae eer enenenee a eee ec ere] pene Co en ny Smee ee eee weeee Oem e ew eee sere e eran eee eeane Peden e weer een e ner e gene Oe ee ere ee iy Penne newer ee reees ae See eee ence erent e eet neenee
usa dextrose (1) Shake cultures... cciceceai ss sacdaesars Ration MiAGdSAd acct mee RNEneN ene (2) Fermentation tube, growth in open arm.......eeeeeeeee closed ArM....seseeeeeees rate of development: 24 hours.. ..........- per cent., 48 hours........se0es per cent.
TO WOUTS wiaranteaiews sain PCL CON sesccw scissor ae HOULS.5 sis cietsieisisncs ese per cent. reaction in open arm..... iis es ea nanaReae ais isos ainda ichaeaec ena a toieedasigiaes WA e iajatebie ty aterelarnais gas formula, H?'CO2! tivesceves. cas vesarmceceies txcnae Ie TAk SRRrOGNTR PE REHEeMiEs OES ‘
Ue lactoS Gra uesr tag aaeinaiats weeded Oy SACCHATOSEsiccay os vadicriene’s a ssid cis iotooutsGn eisiare
proteolytic....... erat ttoieue dat savaus shied Howamenenaes alms, wwaeavenwee nea Aaiessiince’s 9 avsaccaveiwletatstets digestion of gelatin..........ceeeeeeeene digestion of casein...... aise heehee was IASTAEL Cis srercitrsraid: ars ietenin: sieisinininaeamaastgmoeiates ae sais eamind raises sideline $4 ower 8 96 6
oer Peeeeeeeee eee ee reer rete rere errr ee erry
Se eee ee ee eS
HOG CHOLERA GROUP—Cowmnuzp. 931
Bacillus icteroides Sanaretxt.
EXpLaNatory. First described in 1897 by Sanarelli, and claimed by him to be the cause of yellow fever. Very closely related to preceding.
REFERENCES. Sanarelli, Ann. d. 1’Inst. Past., 1897; L. & N. 256; M. & R. 456; McF. 525; P. 609.
MORPHOLOGICAL CHARACTERS: SKETCHES.
1. FORM AND ARRANGEMENT:
is BOUT O Tiss asacseustsia viesinnawains voumumen siesund sansa NG DION. 44 Fema TE i
‘9 IS a ATNINGSP OW BRS iis dion diac aicisees SeamremeG mars MaMa samMNa ES AOMEUGRY aeaaebasaadiedies acerimaieccets we G, AQUEOUS Pentian-VIOletos occiecenirenia soso neinina ed's oan mae sine ose sielelsisiens enue sa baieNiaie ies bs TOS MMEer’ Ss MSthHYlON=b1lUSicoeiss simerisiadcesie vovoaaiewars sia, a.d view/etiatdlaraibidia/eu wsiaiviasgieiernidd gorerie> ie ed sis
c. Gram’s stain
a. Character of movement......
B, Plagella Staiticc cctcescacsoreeecessenaense a4 ernewenteerer eines never aiaaaanees@ea bes . 5. SPORES?...cccccerecnseeeecoeres siejalaseyaydla’ bale afoc! aLageibyelans austainmintarewia pele aahe ewaniercledy ateleawy siardioeewres 6, SpeOtat DHA RACTERS fiserscavcdandanedsnnains taceeeaers eameneNaa Mawes HGCA RRR DA vio ste a niare . Gb. CAPSULES... 0. ccc c ee eecee een n ete ee eee een eee e escent ne FEE Ree eH EOE Tete eee Een EEE Eee eet eS ‘ by Ta vGliti0d TOMS sveesans ievsees ci cer aeecane toeeseseen i eae rants 6 ioe tiwloieivin Mncslaloreis .
c. Deposits or vacuoles.
ad. Pleomorphism...... .+.+
232 CULTURE CHARACTERS
Reaction of media (Fuller’s scale) + ........ OWS estadiess Gelatin plate: Grown 24 hours at............ °G, Sketches. (a) Surface Colonies. (b) Deep Colonies. 48 hours at....sseeeeens chs was booms °C. GQ Oy! Bb is sine vise sivrareierseudinveter’ ebserevece en Agar plate: Grown 24 hours at. ......... °C, Sketches. (a) Surface Colonies. (b) Deep Colonies. sladamuavaye °C.
48 WOUTS Abs 6 2:55 sissies sede °Cs 6 WAYS: Aboswiewneer veisisisineiss ne
Special Media: (Such as litmus milk and blood serum.)
DACILLUS LCTEROIDES 233
Gelatin Stab: Grown 24 hours at......ceseeneee °C.
cha On °C
hours at. . hours at........
48 Hours atc... cesses eee eee eee °C, GCARYS Ab sauisseswinwe mieneeenes oC.
Agar Streak: Grown 24 hours at....seeereeeen n° Cy
ah,
ee
hours at . hours at.....
48 Hours aticsc.ceeccccessoeee °C. 6 days at...---s0s-0e panies seages aC:
Potato: Grown 24 hours at.......cceee eee e eee eee °C.
XY
°
..-hours at.......
seeseeeesDOULS Abeer PC,
48 OUTS Wb acca cass cai surcssiere cceteis *C. COAGS tie corsa ia sowie baie SHY 2
Bouillon: Grown 24 hours at......--eeesee seen eee LO
“15 wane Oe
. hours at....... hours at
48 HOUFS Ab... cece eee eee eee °C. 6 GAYS Aticn ssw neeniianadions oC;
234 PHYSIOLOGICAL CHARACTERS
1. RELATION TO TEMPERATURE?....- leseivrase sre ora ares atesntansleietetetels ararsicuninig: wygle etalelucaresareibietists wibisteislecsies optimum........... soidnibiaetesn es? Cot MIMTSaa civ meas cea ns bOneuentieiesecawecene Ose thermal death-point........ eas eee °C.; time of exposure...............-minutes;
medium in which exposure i8 Ma&de...ceesceeeeee seen ceenees 2. RELATION TO FREE OXYGEN! .ccsccreceneee nee reece eee eee eee eRe OEE e ED Eee eR Een ete GEES
3. RELATION TO OTHER AGENTS, SUCH AS.esseeseeeee
desiccation, light, disinfectants, CtC.:—...cccesesceecetert reeset eeeetenenetneeeeeges
4, PIGMENT PRODUCTION:...... si Raicederoakedinins ak a9 ayes saSeyeatuas tits oZoiatsiate euccohcralatelbiess afeiais s\erasiqreeyelens
5. (GAS PRODUCTION IN SUGAR MEDIAt.ce cscereeseeesen rene ene e eee e eee ene n nena ee ee bent e nes as Cextrose (1) SWake Cultureiic ic oc vacannad snag ve veopiea avenue i ciede ora coinewagunionaaomsegn
(2) Fermentation tube, growth in open arm..... exes CIOSED ATMaeisciios eoarens
rate of development: 24 hours..............per cent., 48 hours.............per cent. 12 ROUTS wee eeie since Wick DEF CODE, pias scans i saRT ee RMOUPS i ascaeweasearace DEF Cent
reaction in open arm.
seen neon
PasrFo rmmiiliay Ts CO Bes csnaveessarares iasesaG oc wlan Haieedreldieessgnisia a Mave) iniatoia « ere kaehatsiggaysateied a tare D, lactOse.......ecseeee die bedeiwae’s derine Cy GSACCHATOSE. orwicniero vies nea seniieeneeea nd nee sie
6. ACID OR ALKALI PRODUCTION:..
7. REDUCTION OF NITRATES: to nitrites ......... rere 8. INDOL PRODUCTION........ PARG APIS ag RRR R AS MERIeRH Hb aie er se wainielauli a Seles MaceNeS Saat AB OUTS:. 9 aingitssspremne atin hadi densa es Re PATROL AY Sisaresy. GS Co MAMeNeeARI OY URS
9. ENZYME PRODUCTION:
POCO] FG Cirdesiesesesharwarapasa ais are aisuns paar sramiemuainaemaunds acenniedac « HHeaaee aemleeaes ha: comnea digestion of gelatin.........eseseeeeeeee digestion of Casein......ceceveseceesvens diastatic....
10, “GHARACTERISTIC ODOR te ecsees yas cn oxi ga5 naiuAeSaG% Lent ORNs Os Seretenmaehat Tha teaaNT
11. PATHOGENESIS (or other special characters) :.........+
Seer eee eee er ee eee re ere ree eee eee ee ers UNO Rene enema eee e are e reer e ese eee een eeteenseeeee UURUCRUCECITEL EES CEeT EC eeEeeereer ere eee eee rrr errr ere rer cererrererr rr rece re rer errr errr errr errr rer es
POreeeee err eee eee re Seer ere errr rrerrer creer errr coerce reer eee eee ere ee eer ee eee e ee ee ree!
Tee eeeee eee eee eer ee er Meer e reer errr errr cere reece ee Creer err errr rrr rer rrr rere rere rere Seer reer errr yy
EXERCISE 90. TYPHOID GROUP. 235
Bacillus typhosus Zorr.
Synonyms. Typhoid bacillus; Eberth’s bacillus; Bacillus typhi abdominalis Aur.
EXPLANATORY. First described by Eberth in 1880, first culti- vated by Gaffky, 1884. It is found in the feces and urine of typhoid patients.
REFERENCES. Eberth, Virchow’s Archiv. 1880, 81, 58 and 1881, 83, 486 ; Gaffky, Mitth. aus dem Kaiserlichen Gesundheitsamte, 1884, 2, 372; A. 408; C. 213; H. 263; K. & W. II, 204, 166; L. & N. 232; Mig. 727; M. & R. 319; McF. 481; P. 402.
MORPHOLOGICAL CHARACTERS: SKETCHES.
1. ForRM AND ARRANGEMENT: We BOUUTOTG. 4 sartsiepemsins ee aistesaaitit Soares eircansianien sve maabaiieies ‘
eee eee eee eee eee See eee ee eee eee ee ee eee eee e ee eee eee eee eee ee ees
Gs AGUCOMS TENLIAN- VI OLE Bice wossieicisrs dies esievie scores tis ¥'ssieisiidjasaia sale acres eerdainn a4 waeearedineay FY b. Loeffler’s Meth ylen-blUes.«+ sciscccssiaccvswawassinass eveiecesaaseeveaansawaas Caan Gove s
(Gi RB ara SBE ALTA aia cintsiin 5sals LUN RDC NORaIR ebm aSTa Ion oie ais. HR RRNA RG ae, Bae aimed nnataielning eoeareinse- ae
G.. Character Of MOV EM Erte seis sissies siaieisiaiazee aie bras! wiaie speioie/eib bs wevslalelacdia’s cuoraiete Siseisiquien beieraneiane
b. Placelia stalin sd: sescous sonwiasaraeedeeny Ke veswanses Here uRnnuena cee hemes ea ei veenaeeRN
ee eer etree eee eee eee eee eee eee ee cere eee eee eer cere eee eee ee eee rer rer
dis CapsileSivevsce cress issaeeseeke Rises Tiae hal mines CeMEd pir eee eee nena eseen ees b. Involution formS....ssecerereeeeeeaee dintalesduate ola Retele nbd ouasaseamrediars dtaieraiaiiee a harmaucingies Cereeis c. Deposits or vactioles....scsececereseene PubemEkN LR Eae RAEI Regus we eaeEkayy RoNRIIA<
d. Pleomorphism...... ssessseresccavecnenenes 25% saaninan it sé eeeavanGu sade de es MME eROS aie
236 CULTURE CHARACTERS
Reaction of media (Fuller’s scale) 4 ........ OR = saaeiees< Gelatin plate: Grown 24 hours at............ °C. Sketches. (a) Surface Colonies. (b) Deep Colonies. AS DOULS Bbsseaciiag ss seed wews eSeaeE °C. GARY S: Abiswecevsissans: paahnaatnaeed °C. Agar plate: Grown 24 hours at. ......... ©C. Sketches. (a) Surface Colonies. (b) Deep Colonies.
48 OUTS Abs cscndiens ceesnedendas nee a OF Gay Sratueiishoaiass wcqaienaded < wee
Special Media: (Such as litmus milk and blood serum.)
Bacituus TypHosus 237
Gelatin Stab: Grown 24 hours at......ee00e0 020°C
°C °C.
hours at. .. hours at......
48 hours ato... .. cece eee eee eee 2C, 6 days at
Agar Streak; Grown 24 hours at.....csseeeee ees 25
ee 8 °C
hours at So APSMOUTS Abie nisi
°
C <
48 HOUPS Abas os sceecenwess core °C. CAAYS:Abiccasinsiee aie viasisenies
Potato: Grown 244 hours at..........0.5 uidintdgpeisee Os
xe Gi
seveeeeeNOUFS Abe. PC. «...-hours at.....
48 hOUTS at 2.6 secevqeavaess ees °C. GC GBYS Atace 2 scgewtnwn es Snes °C:
Bouillon: Grown 24 hours at........-....66eeeeee °C.
°C.
Prado
hours at - hours at.......
LU
48 hours at...... ened eabemcne wa has G days Stiicccwes x5 ipacienen ys °C.
238 PHYSIOLOGICAL CHARACTERS
1. RELATION TO TEMPERATURE:......... CALL eniR CERRRSREREAS EI RIRRRER RT LaNNMaeaES a8 OPtiMUM.....- eee eeeeeeeee gelete® Cal IMTS ea auedesssales aivepabOned sar assem sau °C; thermal death-point........ sdincignca Bek °C.; time of exposure...............minutes} medium in which exposure is made............ ipo aie is al pnsblstovebosaeda sieoveioiesoansesuai aoneencreiseeios
2. TBLATION TO PRES O20 GON taiessoes pee ane ered ed 1 Pere onneenens eunswae do daadaaweeereaneds
3. RELATION PO OTRER AGENTS, SUCH WS) cincensiis consensus 6) Ceenneeeers Cearemenearad 14044
desiccation, light, disinfectants, CtC.:—....cseceeseeeeeeeeees eratintes Mela earn ART esis
rate of development: 24 hours........... . per cent., 48 hours...........65 per cent.
(2 WOUTSsinind dscmaaiiieeares per cent.,........ side asians HOWYS: 565 sx Heese per cent.
Viti S SATE scsscretessvresih a Sieteweetn aaa Reet salematenewA Te See Lee eee eee ee ene ee een es ev
1; REDUCTION OF NITRATES Fc csaas oe 04 aeons ee a ia ey eo BENS Pee eee em ee renee wee e weno
8 ANDOU PRODUCTION ss ocgeuyads vee dggwinawings ogg women. Qube Nes A Eales! Jeon eet 48 hours......... + CaYSkecas ver soa SRONGTAA DEERE
9. ENZYME PRODUCTION:
proteolytic...... sia Ucwsolatgto Gare, wea sieigiaiasiicaarseiSea abaahaucembaiads oP sista sccibisiaiensiniae airettstiete digestion of gelatin.....s.csceeeeeeeeees digestion of casein........0.--eeee ahaha
OTaSta tite sapcemscaie soasiinvdie se siwronenee nes etiraneeniens Ge saaganeree stinsigisiescaraseiam sad tt Sie ee
1D, CHARACTERISTIC ODOR cwnncas casi venaeoriaavias aeraetaintet ¥ Peete nee eee eee neees 11. PATHOGENESIS (or other special Characters) :...sceccceeceveseseneueeeesenveceeecceenennee
eee eee e ene ennee errr ee eeerre rer TT rere eee err er ere cree e ee eee ee reer Cero ree er res) ene oe Pee eeeneee PPOeReeeeEer ere rer errr reer eee reer eee eee reer ere rer rrey eaeeee Sere ence eneeee ween Onn e eR D RHEE RHEE ESE EE PL PEEEOHOEHE SEED DOE O OED DOOR O ELAS EH aE EEEeE . Pent ee anes
reer ee ee ee TNE OOO eee Oe eRe HOO HEED OTE OEE OE HHO FOE Herre eseneneese
TYPHOID GROUP—Cons1nuzp. 239
Bacillus dysenteriae Surea.
Synonym. Bacillus of Japanese dysentery. SHIca.
EXPLANaToRY. First described by Shiga, who found it causally related to a dysentery epidemic; Flexner has more recently found it in the Philippines and elsewhere.
REFERENCES. Shiga, C. f. B., 1898, 23; 599 and 24: 817, 870 and 913; Eldridge, Public Health Repts., 1900, 15; p. 1, Flexner, Phil. Med. Jour. 1900, Sept. 1; A. 440; C. 228. K. & W. II, 317; M. & BR. 3850; McF. 519.
MORPHOLOGICAL CHARACTERS: SKETCHES.
1. FORM AND ARRANGEMENT:
| |
as BOUT Os coe coseges wameneeasars sae MER IRaayaieiain’s od <tidrnewianrtranaes barcidelecen-eerens :
a. Aqueous gentian-violet
bs Loeiiler’s meth ylen= blues ccsiiccceacsiasinsieusaisosiaicis we a porhemluncgeuy eh ee deetenabe dees bieas vad Gove
Ge Gara S SFA: sre ssais pis ape.ciscieg ede 8's ays Soisielelond oie ae BeleoRUsN eR DemEaiIS GWA GX savewWagiecisna vad eresoeen
Gi CAaPSUl CS wisi iaisiess sincincedwceietartaaine dD. Involution forms......e see. seen ee eee c. Deposits or vacuoles...
d. Pleomorphism...... s..++6 tseatietere ei aee
240 CULTURE CHARACTERS
Reaction of media (Fuller’s scale) + ........ OF SS eevee ees Gelatin plate: Grown 24 hours at....... sean es Sketches. (a) Surface Colonies. (b) Deep Colonies. 48 hours at.......0. i, Una tence anees °C. GAYS. AG sidcncnieedwcgedens cree Perec OF Agar plate: Grown 2lthoursat.......... eG: Sketches. (a) Surface Colonies. ; (b) Deep Colonies.
WS OUTS. Otis sss ieeacncrcvaaaie ae EOF 1G MAYS Ab suawiiivas niente sAgientegra °O.
Special Media: (Such as litmus milk and blood serum.)
Baciutus DYSENTERIAE 241
Gelatin Stab: Grown 2¢ hours at..........06066.°C.
°C.
veeee DOUTS At... PC, .es+e hours at..
48 HOULS Ab... eee eee e eee eee °C. GF i iS AE -cresiaveceatscntnnsceies iis a0,
Agar Streak: Grown 24 hours at.............065 eC.
°C, Cc
. hours at..... »s.-bours at.......°
ad ee,
AB OUTS Ab vcieeseeeee ever en ene Cy 6 days at.....ceeeeeee nee a¥eES eC.
Potato: Grown 24 hours at......s.eereee seataunigaiy inn da
°C eG,
seeeveseeNOULS Abscess hours at
C
48 hOurS At... cee eee eee e eee ee °C, 6 days at........05 eiasehatxovsasie disis e
Bouillon: Grown 24 hours at........sseeeeeseeeee *C.
°C aa Ge
.. hours at....... hours at
M8 HOULS Abie serecevevevene eee Oy 6 days at......... shoe Meissecee “(3
242 PHYSIOLOGICAL CHARACTERS
1. RELATION TO TEMPERATURE! ......c0cccsscseeeecteues vavecsseneenees aida dfeieldls Waraeyeiew nate Say OTM UMihessiasistictincenes cesarean Cue Mt) ace aces. eeanns LOleaah4 rete He REE °C thermal death-point..............00085 °C.; time of expostre.....eeseeeeees minutes; medium in which exposure is Made.......sseeeeeeeee a abiheshai cheb adeeaig a causneas nat eae
2. RELATION TO: RREE: OXVGENG 0% sens speci seas soindie duiseduauiedd 4 ve dcsleataa’ 48 tution penn ner eceee
8. RELATION TO OTHER AGENTS, SUCH AS...... ec aa Comoe 4 ietaieien 5 SeegIeaaatean ta REaTeTS
desiccation, light, disinfectants, Ct@.:—....-.ciccceeeeneeeeeeeeneenee epancheciatacetesea 8 seats
(2) Fermentation tube, growth in open arm.......... relish bed closed arm........... aad rate of development: 24 hours.. .......45- per cent., 48 hours..........645 per cent. TA NOUPSsvsav kas dasaenisien esDCL CONE... cess ee cece eee e ee DOOMS ye sivas sein seisiass per cent.
reaction in open arm......eeee om
gas formula, He COZ: sivag easels: swensaasi « sa atgeran Feet eee ee nent ener er enna es ae Oi TACTOSS: 2s ccaeneiminecs 6 tack tees c. saccharose....... saa Tasaiay Bieta se SEG Aiare's eieitant Saree
6. ACID OR ALKALI PRODUCTION:....55 vee ee cerry See nner
9.
proteolytic....... aioashutacavusaraiatera Steelers Sina tirarstnd aaa SHUN HasrInNEeaees hives aa eave (Noobs
digestion of gelatin... ..ccecseceeeeers digestion. of Case@lisacisecss<dsmadanadwe
diastatic.......+6 ChERGS selgteaes Pea Lieiehiaiee ae 230i eteldieiginiys av Gada ayes Stes aia aed wy eel Slaaia'e
10. CHARACTERISTIC ODOR! ...eeeseseesecceeeeeeaeeeneees i dd araeacateletete vie oieiareanibis s s earareeeereats 11. PATHOGENESIS (or other special characters) :......6+eseeeeee siraotatniacs Guoiaveneleialane ur Saaieiaews
eee e ene e nner eeeeeeene
Seno nnn on eee CeCe eT eee eer cee ce eee ecco eee eee eee eee eee Whew en eeeceeneeuverereseones eee e eee ene rec cee eee coreeartarnneeenees
HOODOO OHH Eee scereeeeD
eco vee tee erence eo enene + Fee meee neroeen ner ceuenereuserye nee
TYPHOID GROUP—Continvep. 843
Bacillus pestis (Lenm. & Nevm.) Cuesren.
Synonyms. Bacterium pestis Lnum. & Neum.; Bacillus pestis- bubonice Kruse; Bacillus of bubonic plague.
ExpLanatory. Described at about the same time independently by Kitasato and Yersin in 1894. Found in the buboes, and occa- sionally in the feces, urine and blood and, in the pneumonie form, in the sputum.
REFERENCES. Kitasato, Lancet, 1894, 2, 428; Yersin, Ann. Inst.
Past., 1894, 8, 662; A. 310; C. 215; H. 291; K. & W. II, 475; M. & RK. 435; L. & N. 213; Mig. 2, 749; McF. 559; P. 606.
MORPHOLOGICAL CHARACTERS: SKETCHES,
1. FoRM AND ARRANGEMENT:
Gi BOUIN ONG: cg sssaraieies seissaisrsjeroaiees ionwenee ee ;
a b C Gs Specwal Stain Gi ssporcancavsie’s dssayeie stare verre hw pis/avorentlovwauevais wv ovale leleteueyayetdie ia’ aiwenvaaveervecemntars ¥ wbiatesects Be! » WAM TT IVES baecs cis cactalee aicercteis oh crevstata pda ve esi, elenansha voids giaraunvebounle alate dlgreatacstarcutad aes We etree aed os aed oo
a. Character of movement.
Os Plaeellass Galt. cieaueves aeansronncmied aidaiiins dian aaredterisisteie's clasts Sie diocesan’ hea eelate soma yes
5. SPORES:..... teciapatnd byavsussc nsw Rm Seana ebdve Ov (a:010 eitearayn inst ne ye ate ataleta rane fate! Merde o sletalstone. te amverremtaeneetiess 5
c. Deposits or vacuoles..... withers
d. Pleomorphism...... ...+ sigtinsameter 586 ‘gs 5 5° oleh ajetobalgunverejatdeielalalataveia SSI aeas9,30/0)4 dokce ain locales
244 CULTURE CHARACTERS Reaction of media (Fuller’s scale) 4 ........ OLS sashes oe Gelatin plate: Grown 24 hours at ....... ... 9, Sketches. (a) Surface Colonies. (b) Deep Colonies. 48 HOUPSBtisiss wags 2h ext ores °C. 6 days at.....esseee salted dig ar cect? Oh | Agar plate: Grown 24hoursat ......... eG Sketches, (a) Surface Colonies. (b) Deep Colonies.
AS HOULS Ab... eee cece eee nett eee eeee J Oe GAYS! atic iis satsieneed os Sages Soe °C.
Special Media: (Such as litmus milk and blood serum.)
tg ae BACILLUS PESTIS 245 Gelatin Stab: Grown 24 hours at.......ceseeseee °C. 3 as) - 3 3 eI g g 8 8 4 4 48 OUTS Abii sicorwdetsaiannsian °C. OMA SAE . iiccasnanwpigiies server of. Agar Streak: Grown 24 hours at...... canals er Oo “o ° Py | 4 4 48 hours at... .cceeeeeeeee Pr OF 6 days at.....ceeeeee Po onne Ts oC.. Potato: Grown 24 hours at........ STERN TOE °C. io} re) ° oO n n bal ia 3 3 8 8 4 4 48 hours at......... Bregeioresciadneee °C. CA AY SYA bixissiaventimans abode °C. Bouillon: Grown 24 hours at.............65-s000. °C. oO 3 ° ° 3 3 n n 3 3 4 | 4 48 hours at..... aie etelasaetain aaesoee °C, GRAS AB tis cidicusiates ~adhaanzuavesecesanavie °C.
246 PHYSIOLOGICAL CHARACTERS
1, RELATION TO TEMPERATURE?.......0..0000 008 isdiand ~ Seagiaiainn ee cee uae Sue Seen he Gases OPTIMUM: ginny Gree aebuaedinaes SCS UIMHS an weneas war nay LO Heater sneanie et ons: eOe thermal death-point...... .........0-- °C.; time of ExXPOSUTE......e eee eee minutes; medium in which exposure iS MAdC......-.eeseeeceeeeveeaes i sahcotaysiasdinnaye austoyaiienye anata
2. RELATION TO FREE OXYGEN! .. cscs eeeeneeeee Uns lod-obemiarahan viele deeds tases seed abraenwtsiy Seaton
8. RELATION TO OTHER AGENTS, SUCH AS...ccsceseeeeseeeeeees
desiccation, light, disinfectants, etc.:—
5. GAS PRODUCTION IN SUGAR MEDIA!.+ sicessevceseenseenereenseeues Senne ene nen se eeenee oe
aw. dextrose (1) Shake culture:........cccceeeeveeees siuatt vaoknegay ce eb eeisnty Oe tsigl Baie See E
rate of development: 24 hours.. .........- per cent., 48 hours............. percent. T2 WOULS i245 oi Sa ase het PCT CON. eosccicncocme meses OUTS vricsisneaie eae ace per cent,
reaction in open arm...... achive aie ciaiabasiegais ese eaidvsvataleseteuocoia’ aye bslevaicnavalay niece viisigthenininnencd
gas formula, He COs ti esairnaem: 25.04 ahemowaecay anaes B:; LACTOSE sgars gs a8 cereeaans dwsowes 34 Oe vee G. SACCHALOSE.... 600s diatelePaele aS SG MARR Saisie igs
6. ACID OR ALKALI PRODUCTION! vesese seceeeee tener bie SER areas stale ttaie sini olaie-ocelamioannmbarsOeedse
7. REDUCTION OF NITRATES
to nitrites ..........ee Ants tanelacveiere
8. INDOL PRODUCTION........065 clinton ‘a Wai eiNcdvcabldiapavane ws ae et Aassaeeneia eis palate eigen elelsi-S denonaiaes AS HOUPS 4 sh meiies is oe cores egvatece sui oie dhe aires a gaat GAY Sissies ccomeecewas ca v4 cemeceees
proteolytic........ Nei Pine Nea NS eR MATS Geko Aa i sivcd ptanvle Rela deniovmseiaes reer are digestion of gelatin...........05 Jee aieeer digestion of CaS@in......ceeecseueeeeeens
diastatic........+ byciahalaih & eancavased Ais Ne aia nando ARGS GGONIRA Mbit se alewe gaan esas h cares
weed e neve reese bnew eeaee ‘ + Penne rere ee eee ee eee nee eee be rene nnee Deena e een e nn aneeoneeeeee een errr eer e rece Teer eer eee eee eee ee eee ee Tee eee eee Cece Terre errr eee eee eee eee eee eee ee ween e nen en ener eee eee RCE CeCe eee eee eee eee eee eee Cee e eee eee eee eee eee eee eee.
EXERCISE 91. PSEUDOMONAS GROUP. 24.4
Pseudomonas aeruginosa (Scurorrer) Mic.
Synonyms. Bacillus pyocyaneus GESSARD; Bacillus of blue- green pus.
EXpLanatory. First described in 1872 by Schroeter. Found in green pus, and 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., 1890, 627: Lartigau, Ibid., 1898, 595; A. 304; C. 321; H. 171; K. & W. III, 471; L. & N. 281; Mig. 884; M. & R. 186; M. & W. 160; McF. 269; P. 535; S. 454.
MORPHOLOGICAL CHARACTERS: SKETCHES.
1. FoRM AND ARRANGEMENT:
fee Bo uilloniesgacamese cuits Gam Sat nace Uananeu ae Saab oa Ens ;
Ce Gelatin. accnezaanerodadgecieanineas isomsenamnicanisls oomdreunmmman one cemeateae G.. OCBSE WME CBs Acie nsidiarararaiors sstarciosstenieWainaas esionts-crdicdiayting Mesiwameuatins Hae De» STZ WBS: er evese eed essawajaass eid bos Stain aga dra alnvel Baste usubertees ech By sceyaCr 8 StaaNS TOY abo Sic @higvetaianavatd oon. bie ala-oia isin ears axe éataieiaie Waly . 8. STAINING POWERS? isciseesees LiKhideareieiaeuiavedain rh MReRElayinse sesiineitnidie anh ga siealea male GQ» AQUEOUS Gentian=Violetis.ss dcvsearinowss townneoweased aa saiwasieie se 0 de elects oe eeeeeeesateeees b. Loeffler’s methylen-Dlue.... .cccecesecssersveneeeesscctveeeseeseeeeeseeeeeneeee wee aese
c. Gram’s stain
d. Special stains.
Mh LOBEL TT Wigs orton cu velysioners e's s.01a.seatare elo to.gi a. eselnttioioca bia adie cheneievalaneie oreimsereyactia sin iaieiaidca:don-@ serene wia.eia Siatese . uw. Character of movement........ Scone eodlie aaitieaah Gus eawan wa aummatatuGiats siuisesesiieldeties Gs Minwella stainisaascswisacvanseseauvy eevee eats ureweaa oak ina aes sgisuin ee iiisdoseosar 5. SPORES:.....+ aa ateiata BCR IoroTs srivevewwennee err ere soda Cota Sed pinieresss . Se Lapua: einiecohn seete Raa denna te AV ep eS Onbie a aebiainaee eed aaa Regie Lib ad oe GANAS YEO S WELEESE WRC a ie 6. SPECIAL CHARACTERS !..sssseees ee errs oe tka KRM Ee PEDO REE KUNE RRR Oe eiENs seeeee @. CaPSilessisscennarovaes paeeneaes ¥ ere ee te er ee teeee b. Involution forms...... ateieiens avarslelgwateiaaRaiatsiee einieiwaie ola male wateia sinwne Ss wear ale era seevceceee c. Deposits or vacuoles....- samieieie Ra wiareterase eects aints Wisyatvie Sareea eters wit bia Sab peraieia ciais sevceecoes
d. Pleomorphism...... sscccseseeececseeeeeee J ce'ee Deisleivie'ey. “es diets deelunlesinauesined ae eeeeesseee
248 CULTURE CHARACTERS
Reaction of media (Fuller’s scale) + ........ OF = catyrars sss Gelatin plate: Grown 24 hours at............ °C, Sketches. (a) Surface Colonies. (b) Deep Colonies. ASDOULS Atidiaias srchaada wealecoawn °C. 6 DAYS! Bibiiidaces hedatamieanmimrsoneve °C, | Agar plate: Grown 24 hours at.. ......... en Sketches. {a) Surface Colonies. (b) Deep Colonies.
48 OUTS Abi coisistiaisisnisreniated oa aotebioe °C. G CLAYS (iby 17 cig vieseainy saismsnims oe oC.
Special Media: (Such as litmus milk and blood serum.)
Me PseupoMonas AERUGINOSA 249 Gelatin Stab: Grown 24 hours at.......-.....-++ °C. = eS ¢ 4 wa n H cal =] =] 3 3 4 A 48 OULS Abe. vececns toes wersgge Cs 6 GAYS Absiniisas sesanicaecne nb tes el. Agar Streak: Grown 24 hours at......eeeee eee °C. fo) Oo a a n Lod col u =] A ° t=} 4 4 48 hours at........ ee OF 6 GAYS Ab... cece reece cena eeeee °C. Potato: Grown 24 hours at.......c.seeeeeeeees pao Ga 3 a3 3 3 n n u he J 2 3° iS) 4 4 48 NOULS Abs as sacwsasiee oe vases OF GHA RY Saab wissevesend ase cca spacansucicraihie BE aC. Bouillon: Grown 24 hours at.......---..scereeeee °C. 3 3 4 8 RN n wy H s 2 ° ° a 4 AS hours Aticcexs veeneeses ve nnn ay 6 WAYS Atiain cae actenecicnd 62
250 PHYSIOLOGICAL CHARACTERS
1. RELATION TO TEMPERATURE!.-....se0005 ibis as entenreg seas Sv ee tempeqae medi aes eaw eeweaans oe ODPM UM Ys 65 sais ciaiscriwieies seins OC FAIA TES sisraiere sinnedaiasteniaiense tO liga o-« ieainanies exer Cae
thermal death-point...... ......-.-065 °C.; time of exposure........eees + minutes;
medium in which exposure iS MAMe.....csceeeseeceeeeeeeecenceeeeneen saunas Re ACS
2. RELATION TO FREE OXYGEN!....06 esesseesee cannes beeen ener eee See be eee eeee weenie e aes
8. RELATION TO OTHER AGENTS, SUCH AS.ccsscseeeseueeeeseeereeeeee Decne teen tenant neta
desiccation, light, disinfectants, Ct i—.....ccc cece sent eee cece neeneeteaer seneetecees
(2) Fermentation tube, growth in open arm.............06- closed Arm ....csesseeeeee rate of development: 24 hours... .....se0.- per cent.. 48 hours..........065 per cent.
M2! OUTS, sw cdccisusretine sa haw aes PEL CONG. s:000 vere sbiead OUTS iss sea winscemeeee ied per cent, reaction in open arm..... wiaciteté Bi aaisoiniauere sae Voaialans aiaieiiierarsians Reema a sieiels ralsiatuiecas Sd welotra gas formula, Hi C O2t soc ccccsee ins « Wydsereetekadtate oareel er ee S Aas SMe nas oon Tae LE
Uy TAGKOSE: 9a, ania es yrwsruncan leaner ps 34 06 Us SACCHATOSEssssuveveaesaeases cae erate mith
6 AGID OR ALKALI PRODUCTION? ...... ceeccees teeceesaeeee BER GSG Sd le abana Sta dase wis inssibimiavesbainie.a
ee eee er ee re ere eee ee eer erry ee ee eas
litmus milk ........ csibesidiby Sameinee Kinase re Meumeeews wicietrenates aa Rus. Reems
8. INDOL PRODUCTION...-.+.seeeees LAURIN ca esc iae ie auauva ene ea GiiNs eras sues aie avaidedianetandsBie ash Waateinal 48 hours..... ade ete s cP ER opeWalEOd £8 spe OMe de awe a ele TOBY Saimiri wvesiesiaidieies sis Ceaientsinre
Qi TONZ VN PRO DUCTION cavriicraeie ciare wi shai titininjnenn(e's 4 oisressielnsioneeceib: <a: soesinisra iaaqurataie § Ateienpatesaiaiain 0 bia hola
10. CHARACTERISTIC ODOR:.....- ia altel cansppciarainin sioialdlauisigieieannoran ih CARMA RP AenS Ceruinn junk
11. ParHoGENEsIS (or other special characters):....0.0c..e eee whee Tie wow ReaaTnawNes cinders
Peet een eeenne ee ee ee cr ee eee ee eee ee eee crs on eee eee eee eee eee eee ee eer es Deve meee eee ntee eee enees
seen ene eee on eee eee rey Seen eee reser ener ew enen eens eat eeeee serene eens a oe Deen ee enee eenen oe oennes beer eeee Teme neem eee mee este eee e renee eeeeee a eeeeee VOCEUERUCSECEOCOSE ECE eee TT Eee ee CeCe ere eee eee e rere errr rr rer rrr rere ee reer reer ees
ee WORE RECE ST RE Cee reer ee eee c reer ee ee eee eee eee eee ec rer eer eer rere reerreny
EXERCISE 92. CHOLERA GROUP. 251
Microspira comma (Kocr) Scuroerer.
Synonyms. Spirillum cholerae-asiaticae Zorr; Vibrio cholerse Lrum. & Neum.; Comma bacillus; Cholera bacillus.
EXxpLaNaTory. First described by Koch in 1884. Found in the intestinal contents of cholera patients. It has also been isolated several times from water supplies.
REFERENCES. Koch, Berl. Klin. Wochenschr., 1884, no. 31 u. 32; A. 446; C. 335; FL 2, 527; H. 333; K. & W. III, 1; L. & N. 353; Mig. 2,960; M. & R. 407; M. & W. 152; McF. 442; P. 568.
MORPHOLOGICAL CHARACTERS: SKETCHES.
1. FORM AND ARRANGEMENT: \
Ge BOUM ON cacnscatescemenss cecum Pecan cakaaes as vadaredatat det dees :
2. STAINING BOWERS fais sies so cteeieiaineiais a4 weacasereitie bn deatettiem, 4 & onainaieee nies sie ielooteid-dinalais’s SlataeaNy
a. Aqueous gentian-violet
b. Loeffler’s methylen-blue... Gi BAT SUSHI rss vic sapaiciaseis ois we lsapederstasain g Wawa Monn bys breast hegre naaougenh eh VdinaRteGeees
d
6, SPRL CHAR ACTURS ck eesdendereres tases teeneereevaetsnaseasenenes err egT Terre anne Gey CAPS SS iesss acararaatsje Visissavels aiaiune 4 vive sceie'ateys ajelels\wisterwn iin'ss “algisidieiaiarece apbleiaju lel erasinnde-syearreemaa’ vo etre * b. Involution forms......+-.+ Seen Saaisies ae LaG, tcaansielomiaaesinntorewe te Ra,aneareies ata Neleealasaccvaia Siard aidia c. Deposits Or VaCuoleS...ccecseasesrceeeuncecuteneansceer iis Sie diass iascig'n dense bs piahein-gin'g-ovincnavece 3. 46
df. Plecmarpiietiionss se. seat nnn ss jawemmens 24 deen 7 £2 ERWURORMAR oh seaeaiees seamen eae .
252 CULTURE CHARACTERS
Reaction of media (Fuller’s scale) 4 ........ OF — ws... ne
Gelatin plate: Grown 24 hours at............ OF Sketches.
(a) Surface Colonies.
(b) Deep Colonies.
ASHOULS Abiaiieencwcswenas sreewinwsa °C;
6 days at........ alana estar eaiiaainmeinenuard °C,
Agar plate: Grown 24 hours at............ ea Sketches,
(a) Surface Colonies.
(b) Deep Colonies.
AS DOULS Absccescersentceceersereeens °C.
6 days aterrseseeesces Heieiiiel ne wsige'eaiga ee.
Special Media: (Such as litmus milk and blood serum.)
MicrospirA ComMMA 253
Gelatin Stab: Grown 24 hours at........6.666...°C.
isa coos HOUES Btiencncs.°O,, 4 i#avews MOUS Abacaescas® Cs Co
48 HOUFS Atiesrriaecsowssnssesen? Ce CCA YR AE ceases s:cvenieeserieine ors °
Agar Streak: Grown 24 hours at...ssseeeeeeeeee °C,
°C Src Or
ease
hours at seveesee hours at...
AB NOUS: Bb aicncacwssiadamngests °C. 6 days at.......- deer saree OF
Potato: Grown 24 hours at.....-csseeee eee e ees Lien
wma h.
.. hours at...
seseeeeesDOULS Abecseeee?Cy
AB HOUTSIAL wise se/ersigiarizeeeteree ns eC. 6 days at........+ Siuatocnietnioaants 8G:
Bouillon: Grown 24 hours at...........ceeee eee eC
°C °C.
hours at «hours at.......
| Ow
48 hours at.......- anemia Gs 6 days at..... duet satienanere’ eG:
254 PHYSIOLOGICAL CHARACTERS
1. RELATION TO TEMPERATURE?.......0.006+ apawkesaivnune eae ee OPLIMU I sracsains os ewenrsdverases Oss Wnts. sary dcuass: vaseinbOsannrenens ex teawen Oe thermal death-point........seseeeeeeers °C.; time of exposure.............. minutes;
medium in which exposure iS Made... scsccesceeeeeeeeeseeeeeneeresecentensesseseces
38. RELATION TO OTHER AGENTS, SUCH AS.csscsscneseceneeneees MdiRe@avunteiners.ceeee eRe desiccation, light, disinfectants, Ct¢.:—....sec.ceeeeeveeeeene i trasseisdleha lea greavaeia tiie
Ce ee rs eee ee eee eee eee ee beeen ences bene e ameter teen eeneeeee
4, PIGMENT PRODUCTION!.....s.005 Deen ean ence een enes Cede er eee e neers eae n enna reer ouee eee ees
a. dextrose (1) Shake culture:....... aiaviieaiate en dasacaneaeeia agen (2) Fermentation tube, growth in open arm..........5.s005 closed arm......sseee eas rate of development: 24 hours....-....00605 per cent., 48 hours........eeee per cent.
82 WOUPS dcisirstis os apeewsamates POY CON begs sewer cueeeas ces Hours.......eeee sess. Der cent.
TEaction in OPEN ALM. vessceceeverecrecevcrereveeeseees
Fas LOTMA, Te CO 2s ts aiccsaies wiats daisiasietalositiesng” siatieeseie' Sa aires Se ANCOR Cn aEe rr erer De TACEOSO si ssisetacisdianinieadd- 2G) estetiarsarneicieae c. Saccharose.......e suiaaan@raias ae amooaaamanes
6. ACID OR ALKALI PRODUCTION:...... +
ADEM US MATE aad eee bie tae sisaed- pes wieiecvisto elosmrocasarnjalnn eee bine Sea artlocnie wiaeo erawiddaenet ) < ataucceatuae ate 7. REDUCTION OF NITRATES!....c.ccsesessvevevees sess inks fasts Prion teaceneenet 3 ie onkidigiare Nini aacea cuit Seer TO NILTICES crecceeeeeeee Boer reeepeadees cs LOAMMONAvisss sanoawaness qesmadeces seas
8. INDOL PRODUCTION..+.++0« aeeey
proteolytic digestion of gelatin....ccecseseeeeeeeees digestion of casein........6. «5 Sees sa ae diastatic........45 Bocce Conny 1, TNO ACI BS emit e aR PAO RRRG As a. Ne MOORES Te areMeRS
10. CHARACTERISTIC ODOR!...sse eres teeeerene Beans hancement oi baehaneravesne ais svayachbeecera A ea Box 11, PATHOGENESIS (or other special Characters) :.....ccceccceccee senescence nce t eee eee teen noes
CHOLERA GROUP—Cont1nvep. 255
Microspira Metschnikovi (Gamatera) Mic.
Synonym. Vibrio Metschnikovt GAMALEIA.
Exputanatory. First described in 1888 by Gamaleia. Found in intestinal contents, in blood and in organs of chickens suffering from a disease resembling chicken cholera.
REFERENCES. Gamaleia, Ann. Inst. Past., 1888, 2, 482; A. 485; C. 334; H. 345; K. & W. III, 68; L. & N. 366; Mig. 2, 979; M. & R. 427; McF. 462; P. 593.
MORPHOLOGICAL CHARACTERS: SKETCHES.
1, FORM AND ARRANGEMENT:
Os Bowilloneas. « scsrcacsccicaisscnswes .
G@. AGUEOUS CENtAN=VIOl Eb sie veccincsseimeia sin sine wiseeinsiny aroame eda aetaar sieve iinialeaelarels 8% 00:8
b. Loeffler’s methylen-bDlue....secscececceeceseereecsensesees Slvird desis tatheaia ates caiavaseraianes’ vepieies
c. Gram’s stain
a. Character of MOVEMENT... secsecreceeceseeeeens og Sao aiatailetaiutdis Veieateraasaineles cae aialiieacesiite ite Os Fla@ellaStalnisss cemascsiws dnomeiioniesd ones abainaes seasews wanes Kacey comes MeneenOResS
aw. Capsules b. Involution forms...... casnoendeseeees RROD E RNIN Saag Vsiaicanssiees avai SisiaiOatat ia talnsy arataavaronee vc. Deposits or vacuoles..... Haieiaiee es atenieaai cine srisiaie OWA lamaiilaswieais i aU ldevessiuieia’eaieiodaigise’sicigare 5
d. Pleomorphism...... -.++++ eR Tn ee ACCC ‘
256 CULTURE CHARACTERS
Reaction of media (Fuller’s seale) 4 ........ OR = eaehiss Gelatin plate: Grown 24 hours at...........+ °C. Sketches. (a) Surface Colonies. (b) Deep Colonies. AS VOUTS At. vesicneds ceicads aie Heo ven °C. 6 days at..... sin beled eekwaiatas is eos °C. Agar plate: Grown 24 hours at.. .......6. eC. Sketches. (a) Surface Colonies. (b) Deep Colonies. 48 HOULS At. .ceseee cree eee de ene eG GC AYS Abs cccccceeseceeveetrseeteeees °C.
Special Media: (Such as litmus milk and blood serum.)
thigsiyeriid lt bCHNIKOVI 250
Gelatin Stab: Grown 24 hours ate... cseeeesee ee °C a ro) 8 e n n 3 | I 6 $ 4 4 | eae 5 18 DOWrS Bhewes ess venewes tava °C, 6 days at ....-...5 idutudaaiaiee aC. Agar Streak: Grown 24 hours at......cseeeneeee °C. io} oO | 3 3 : : 9 = x x +3 +4 \ @ e a a | ieee 48 HOULS. At wri: wdspictencnaiaace cen °C. 6 dayS At....eeeeeseee weaeimiltoce's och Potato: Grown 24 hours at........ seaaian gainsaniies °C. io} oO 3 3 ce 8 nm n E EI ro) i) 4 A 48 HOUIS At he ces peewecesasess 2Cs CLARY Sibi riceiecintanjeiaae vecipece °C. Bouillon: Grown 24 hours at......iscceereeeevece i is) 3 > 3 a S Z Z 3 2 iS) 3 4
K
46 WOUTE BE: coreeiieecarereanen | 6 days at........ eT e
258 -PHYSIOLOGICAL CHARACTERS
1, RELATION TO TEMPERATURE!.-....0esseeeenev ones soy) Aamabiaeasiat ae aa ayaa raaammtiaad EO rad Ore
thermal death-point...................°C.; time of exposure...............minutes;
optimum.. Seles ks <ebte paiuieas Cae LIMES bay te wcteeeee- aennaty ebOUA E
medium in which exposure iS MAME... ccseeeee eae ee sense eee eneeeeeneeereneeeeeeeees 2, RELATION TO FREE OXYGEN?...... adhih in ssossuee aGgta/avest aft o/saayendsalbterash Sa gia. eianalaso's slaksaia din bin ecefaineantiata 3. RELATION TO OTHER AGENTS, SUCH AS...-..05
desiccation, light, disinfectants, CtC.-—...cceescecerccnceeseeneeeesenne sees neneee ene
4. PIGMENT PRODUCTION:...... EE I A ER PETC UR PROT CTR ISTIC TCE
5: GAS) PRODUCTION IN SUGAR MEDIA thes. casaxss ostiameeaeainssacwanors sad rvtneinnes Wieenanas a. dextrose (1) Shake culture:....... Ki SER, Sse 5G TENG SrA ROTI Ke Rear as Ea Lacie ae
(2) Fermentation tube, growth in open arm.........6.2++. -ClOSEd ATM... cecceveeenes
rate of development: 24 hours.. -..-...+5- per cent., 48 hours.............per cent.
TAN OULSY + ideewiainecisnes’s oe oe PEL COLbignasaveaie sadcieserss MOULS) saieanis seeeeees DOF Cent,
reaction in OPEN ATM....s.seseeeceeeeereceee
gas formula, H: COz2: v. lactose.......... GaN bs demiciiasicgoeeies, Ce BACCHATOSS wy ectewiaiss sraataanveeeias Mewisidineein weds ve
6. ACID OR ALKALI PRODUCTION: ....00 ceesseee Seeceeceneoneeeereresaeessaseaeeeenarcunenees
THEM WS TAT cose: ye. orcb Ssbsssteeceiate Sia: Seg eieteraesNs aa RS Os Mia TO laa eae He avacaatesinoid ss
f.. REDUCTION OF NITRATEST.. 254 stcedeeddioes Santas 04 Veet aR Gah va Speen Bee TOMES. oe to nitrites ....... eee eee eee Fay wicfarehal wataraneans sve bOrAMAM ON Bixrisa see ainmsnuted Perciicintiogees
8. INDOL PRODUCTION....-...seeeee08 Satara ripipk VG FAD ATMA ¥ 8 Rheartias erie a Miah eran nie, acepaNE ie
48 hours......+
9. ENZYME PRODUCTION! ..ccseeceeeeeeeeeeeneeenees proteolytic....... sities bin New didiaronkainnarsiolecSG-ine Semaehad.s adobe ineduingesns diane Tahaan ryesine an-Dieanens
digestion of gelatin..........seeeee digestion of casein..... .... ceceeseeees
Giastatic....cccecveveeee ceeeereeeeeee eee e eee eee e eee enact nena e eee eee Greene
10. CHARACTERISTIC ODOR}. .eeeeeer serene
11. PATHOGENESIS (or Other Special Characters) :..cscccseecserceeneeneeeeeeneeneee sett eeeenee
Senay
ePPerrerrrererrrrr ree rrr err errerre reer rere errr rere ee ee cre ee errr rrr errr errr errr eee cere reer errr reer rr errr irre errr eee eee eee ere eee seer eerie)
dr eeeonne SoPCeerr Terr eererrerrre rrr rere reer career err rrr cere eee reece eer rrr ere creer)
rrr rrr errr errr err rrre Perr r errr r reer rer cer rrr rere rece eee ee eee eee reer rr)
CHOLERA GROUP —Continuzp. 259
Microspira Schuylkilliensis (Azsorr) Cuzsrer.
Synonym. Vibrio Schuylkilliensis ABBort.
EXxpLANAToRY. Isolated from the Schuylkill river water by Abbott in 1896. Very similar to preceding.
REFERENCES. Abbott, Jour. Exp. Med., 1896, 1, p. 419; A. 490; C. 334; M. & R. 428; McF. 465.
MORPHOLOGICAL CHARACTERS: | SKETCHEs.
1. FoRM AND ARRANGEMENT: |
uw. Bouillon
8: SPAINING: POWBRS Si.ccccua aia eanincmnmaswiaas oa da dbrccreniestisisietn a: Maveiaeandetedaiecinisib sw acdamuneeeniae MTA as sae
G: MAUGCOUS SON CANEVIO] CE a wsvieas vas i Kan dadwemar ener ceiainadenas soleoseuieieeseaien Oey O49 b. Loeffler’s, methylen-Dlwe: «sc cccaciscieerscseiedoiecs oe seewedavuieneles 64 seaeceeaayey gexed c. Gram’s stain
d. Special stains
a. Character of MOVeMENt.....cssereceveees Rneiaereyarane Badhaiiere orwidsadibac ouh Detanwmnkiaaaae:
bv. Flagella stain.
C85 Ca SUT OS esses as scecicicie acsiwcasass eiases dean la aise ie we Medan din. alton asoweta wiSie wesvencteanreiaheiaatdiniayd 4. dlalaatMiwiiraslats Os TAVO]UGION POLS. we ieieraiascatejeroiace niatecict vassnese Vinacwrebatasb eis dearadie nrajan we onaettmederea as eeeeeeeeas ce. Deposits or vacuoles,..... apie sMaee abn abaommcamith sansa Ree: ekaniioxiene 3 alawvetnsis
@, Pieomierph Wetticsase ev ssse0 cass dso 04 Wa cad eee Ths Bi eee Ne ceneea een dene oe
260 CULTURE CHARACTERS
Reaction of media (Fuller’s scale) + ........ OF — .....5. Gelatin plate: Grown 24 hours at............ 6 Sketches. (a) Surface Colonies. (b) Deep Colonies. AB OUTS Abe. cceces cece es veneneeees eC, 6 days at... cccccccserecteesseens cece °C. Agar plate: Grown 24 hours at.. ......... 2: Sketches. (a) Surface Colonies. ; (b) Deep Colonies. ere win
ASHOULS At. rcevecccserceceeeseveeses °C. OG DAYS Jat. issceiecwa vecitneams es
Special Media: (Such as litmus milk and blood serum.)
Micrcspirra SCHUYLEILLIENSIS 261
Gelatin Stab: Grown 24 hours at.ccsssseeeeeeees °C.
hours at. . hours at....
Se AS NOULSIAE comsnaireaccniengeed °C: Gila YS WE aaananinttaneeenawen aC. Agar Streak: Grown 2! hours at........-...065 °C, rs) o 3 o Pe a n wu 5 ie} a AB OUTS At occ ec ee cece eee e ees sO OG days al..ccnsenoesersceonene Potato: Grown 24 hours at....... cece eee eee oh or o | = | / 4 Pe 3 3 “* a uw co fa) =] ° ° ae 4 AS WOUIS Ob sc ccscaiecescceneas °C. 6 days at.....eeeeeeee smieuanetesins °C. Bouillon: Grown 24 hours at....s.seeeeeeeee sees ei, 3 3 | 3 3 t Bl ‘1 ae 8 e 3 2 na! 2 2 ° is} a a : yA -. PB limursiatec sc cs. cteeasagene °Cy | GiOayS Bis ccanuad cosaoaariad Cen
262 PHYSIOLOGICAL CHARACTERS
1. RELATION TO TEMPERATURE!......ccececeeccee seen ce teeeees a niss's die eso elds eteinne Hele side eid dice OPEMMUM Gis be saisicicrnedinieaes aatagars OC mM It sien: 9 apes: sorta TO lnciisey < semeanteeaas °C. thermal death-point..........s..eeeeee °C.; time of CXPOSULE.....eceeeneees minutes;
medium in which exposure iS MAde......sceeeeseeceeseeseeens ayidiesessitigrecahasiae ts
2. RELATION TO FREE OXYGEN:..... Dee eee n ee renee Cente eeerees Penne rene tree eran tenner ee
rate of development: 24 hours.. -.......65 per cent., 48 hours............. per cent. 12 MOUS siviaressisiars asia’ wataies +-per cent.,.....+ Ssinsesularec sets HOMES deielawuase slenicignee per cent. reaction in OPEN ATM......es cece seen eens i s/ae gees crajauneinia oes 6 isoidjciatens ‘aca dtectieie iayelihs aomalaana ayetars
gas formula, H: CO2::......... «5 j ARG SHO NSS MAAS MHRA Wels iG US aire apace BA See aie
Ve 1ACtOS Oeics cosa sasdeotee seein Ms seve Cs SACCHALOSE....eseeeeee ai stadilens inte wasan ead sieaie
HEMUS MUD. sss secieseawsees ames ewess Ce oeiewiie nor iucdis gaieees aia G ouaaRee > A uireaNtie eS
8. INDOL PRODUCTION ...-.eceeeeee AB HOUTSe2 heces cers 68 08% 9. ENZYME PRODUCTION?:..-..seeseees
POCEONY.b1 Gas tororssdegs' acolo Sans osaiersurararaacdiarniaina amawatsa rates aiearrcniewaataus dudipdarads ns euaietoicialssets digestion of gelatin.......cceceeeeneeens digestion of casein...... itgiaje cio ah tlaciocansicke
diastatic...... iis eiuintojerce w Oeeeiaraatelnatne Xie Sokeclacs sald Fs eau maer sais ANIOG His i negareteen
10, CHABAOTERIOTIO ODOR! cases suai ciunens sTsinyeea ahs aes cattihechcetaMe iets Resnlh Mkessate orale widiautrarereiarsipioere ant aa
11. PATHOGENESIS (or other special characters):..... ns silo cana acta aiacetecsvoeneate SYoUremekystefolas waaiechatg
been een ene eee eee eee eeCC Peer TC eT Terre eee eee Serer eee eee ee eee eee err rer rr tenes Pere ee eee et eeeees eee ees Oe ee cee rrr see e neem eer neneraceeeeeereeres ee ee ey Pee e eee renee et ean ne enene vances eee ee eee eee Cc ee ee re cry bene eee ee eee eee eee eee eee eee eee ee ee ere ee ey
EXERCISE 93. STREPTOTHRIX GROUP. 263
Streptothrix bovis (Harz) Cuester.
Synonyms. Actinomyces bovis Harz; Streptothrix actino- myces Rossi-Doria; Oospora bovis Sauv. et Rapats; ray fungus; actinomyces.
EXpLaNATory. First described by Bollinger. It occurs in actinomycosis or lumpy-jaw in cattle, hogs, horses and man. It probably leads a saprophytic life on plants, ete.
REFERENCES. A. 361; C. 361; H. 349; K. & W. II, 861; L.&N. 440; M. & R. 287; McF. 371.
MORPHOLOGICAL CHARACTERS: SKETCHES.
1. FORM AND ARRANGEMENT:
bis: TONAL TNO Ties inss debi sncao ei aegis Sis aps ara 3G6'o: 3 asa iaaa'Wieratalasatararelereceoavanzeiatelataades ore aemaraeaene }
. Aqueous gentian-violet
b Loeffier’s methylen-blue
ex Grams Stainton cracusens wicesueiwe Merncmtesnals ont tesms Caelaidiiedtiedaaies oa ohana viet ore
@: Character of movem enti: csisssaanpeweranmcacuaiie aeaecinmawance ie celaceraeadead satiarees
b. Flagella stain.......-..00.4-
65 SPR CIAT: CHARACTERS 2 ociccsriain cuarcaremaiiio ad eecssarmsaiis ainiaoitlepietiaina slebionietetoaea s wiaealeters aiare one is CADSIIES eciesiainre aciorare “daibinvers eiaicnesatuldly sinrsnareccla diay’ ofa.gh, wha andi ag Wawmaanteaaey tee eceamnind ores bv. Involution forms.......
c. Deposits or vacuoles
Gis, PYOOMAO EP DUS Wests scpec vasain's ci seaiajevolo's AMorasen sodaniaca’s oumabAasstenve VA GA Gvleceatongttnctoad valarorabanians tae agains
264 CULTURE CHARACTERS
Reaction of media (Fuller’s scale) + ........ OF = ssegeics Gelatin plate: Grown 24 hours at........ ... eC: Sketches, (a) Surface Colonies. (b) Deep Colonies. ¥ 48 OUTS Atiiiisewsss wae saeteeNeT °C. GAYS Hab: sd, satdawaii us av waleintingg sean 2G) Agar plate: Grown 24hoursat ........ 7c Sketches. (a) Surface Colonies. (hb) Deep Colonies. 48 HOULS ts <a ve cnewese gesese ve seem °C. GiAYS! Bbiceaas sd duetanletwnrtescancas °C,
Special Media: (Such as litmus milk and blood serum.)
StrREPTOTHRIXx Bovis 265
Gelatin Stab: Grown 24 hours at.............5-. oC 3 S| 3 3 if oe ‘| : Pe 4 3s i a Le n : 5 5 6 4 4 a : \ a XN = ieee 48 OWS Ot nekc is aanreasroionns i GuAWSTE, doskoee anaes Bierce an Agar Streak: Grown 24 hours at...-... seers °Cs = is) | % . ge i at aa = ‘ fe oad ¥ a s 3 n n { eI 5 8 S a | ep Sl : eS : ~~ ABV NOUTSAL ccs crew owed coe OC, 6 days Afi sc veeseeeis seems eee oC | | Potato: Grown 24 lours at..... cece eee eee °C 2 on > > | : | ps : : ( Bes Bey 3 3 mn n col e 23 =] 6 8 a es Su INGHEL SARL. sera Sao ne Redeeeane. fo a a | Giese Sah gious Pass See 2s I | Bouillon: Grown 24 hours at...sseceeeeesee eens ET Ges 3 5 | 3 > : i | x eS Y Rn n | 3 5 | ° °o eS 40 | 34 ed tand AB NOULS: Ab isie veiiigesiegvies veces °C. GC GYS Bibex 02 es Gre. a4 at neeeeeres °C.
266 PHYSIOLOGICAL CHARACTERS
OP EUMA TIN Assisi ys Lishatsieiardienicnicte OC URIS: sit pe ox chelate Mactnad iGO isiciicich zs a'eisiewateatsie we °C.- thermal death-point........ bisees cigemiehs °C.; time of CXPOSULE...ccceseseeee minutes; medium in which exposure is Made.....ccesesssececcnevneceesnees WRSRIMEREARD POSH
2. RELATION TO FREE OXYGEN?...sseseeceeesses Secs Bats Saiiciicts SOO Co eat eee
3. RELATION TO OTHER AGENTS, SUCH AS.....005 Seno e eee ennees Peete een e nner esernesenes
desiceation, light, disinfectants, CC.:—...ccecssccscenvensee creer eee seeeneceneeteaees
DOM eet ene e errr wernt ence e rene erate be ene ene nee nreeereeens ee eee es pene ene Deen eeeeee Poe eee meee eee e nee e neta rene eeenee Peete Coenen eee we ernnenneaes SO cc cin eee eee eee eee ees OeSERSTEL OSES TEESE eee eer eer ee ee eee eee eee Sy
eee eee eee ee eee eee eee ee eee errr thee e eee ee eer ences Sen eeeeeeeenae onee
5. GAS PRODUCTION IN SUGAR MEDIAL cess sseeeeceseeseeeceneeseues ‘sinielacibandinn at ajareisery Arete wu. dextrose (1) Shake culture:....... Meares er Teemees eer ee ee
(2) Fermentation tube, growth in open arm.........eseeees closed arm..........5- isis
rate of development: 24 hours.. .-.....,+.+ per cent., 48 hours............. per cent.
TS WOUTS srceceess ve nemisantes per cent.,......06 eiisilareiy Holy HOULS..--- es eeeeaeee per cent. reaction in OPEN ALM.....-seeceeseeeeeeee aid a(S specoseratineseaies vis sreniectedios ears eae eas caaars
gas formula, H: COa:: ....csceceeescecees sivcinng Sola greiezemle nyh- wi gasiain sn oneatnia ecalinsibnd als aie
bv. lactose.......-++ i ieleeae wements eeOERE c. Saccharose.....ees Braye mia's Wa srampecmiects te acai
6. ACID OR ALKALI PRODUCTION:...... tio iaiaseie cs NRE Rea ION SHE eraav StS aiiore Bnsereletecmassiens & siclaisia
Htmus Milky .scwsenaenaessaaieseiesmes sien slag Hapa sireurawe Ge auaishn Set 5 MvEedReiEe Gaede q
9. ENZYME PRODUCTION?.....eseeeseseeenes iota apoga ininnnea sei 2a seein aie senate Ay iatetera digcpnelstvaraniewladeisis s
proteolytic........ phe tsiareras tale ‘at talavas vee e 5. “ata gt Mn Bic cis ees oS caues wv aia aia’ digestion of gelatin .....scsseeseeeeeeee digestion of casein.......... cesses wees
TAS TATIG ecsdiare srsriea ata ais aia eosare.nceia’s wiobareyasaienstionteldle’n(es spa teissteers ovsts wid Rey Aeraraaninatand aa elebemiewreaiy wh lay
10. CHARACTERISTIC ODORi.-seeeee eee if Ga Sagem ewes 4 LO WAR RIES TERR ROMT ERS REET AS 11. PATHOGENESIS (or other special Characters) :...cccsccsecseccenecsceeeneeeesseneceneeseues
Pee e eee cere erent teen eenee err eee eee cr ee ee ee er ee eres
One n ewan eeeereeene ee ee ee ee ee ee ee eee ee err ee rn .
eRe eee eee eee eee eee Cree eee ee eee eee SPP CCC creer cee eee ee eee ere rns
STREPTOTHRIX GROUP—Convinvep. 267
Streptothrix Madurae Vincent.
ExpLaNnatory. First described by Vincent. Associated with a warty, ulcerative affection of the feet, but rarely of the hands.
REFERENCES. Vincent, Ann. Past. Inst., 1894; A. 365; C. 368; H. 356; K. & W. II, 839, III, 454; L. & N. 452; M. & R. 297; MeF. 378.
MORPHOLOGICAL CHARACTERS: SKETCHES.
1, FORM AND ARRANGEMENT:
Gz BO WUN OTs aiesesercia anszerciaseiiig siete aicvotsraianste shale 15 cies ue prastispeiaiecs Yoda eRe paletelon VR eigtelshs
c. Gelatin
Ws, AQUCOUS Lentian-Vidlet.scs coc descicsavens os soeeeieaas cee eeeislenswan eoere Dea edsieees wrt
b. Loeffler’s methylen-DlUe.....sceeceecsecceccuseecceseuceeseuseeetsectesees Paieleineeias te wiaiata
co Granils Stain sa cciecatas eecwamecsinen’s sxinaremoraws wr eames 2.251 Bisls e eisrasaaveis elo e pareraetaming are
is Dec al aA cas cased 5 ay OR aN HiS ao hens de end ba Chen nenensaeeeaa vateRsdasens
ws Capen lesan: cones esstosdisinjeieis
Be THVOMTELON EL OPIS oa d oiess1isa ass aestosaissate (od inl teoveiere satan 04 Seah wpardiareiapmnrd oe Wa cle eres Is.O% Fisdieyaiecameaveie’s c. Deposits or Vacuoles.....ccesesecccoes 1h. CORR R ERNE TS PVGRROAKA Sea Gaaabee AIRE
Wd. Pleomorphisw...... cecvcseseeeerecenneee nee e eee eb eee een e eee n nese eens ee eset en eees wee.
268 CULTURE CHARACTERS
Reaction of media (Fuller’s scale) 4 ........ OT SS spa shinee Gelatin plate: Grown 24 hours at ........... On Sketches. (a) Surface Colonies. (b) Deep Colonies. 48 hours at....eee eee de deed a anee CS C.days: Btevwece as Sin aaeentand eden ®Cs Agar plate: Grown 2ihours at. ......... a Sketches. (a) Surface Colonies. (b) Deep Colonies. AS HOUTS Btissiieus sca sanraaaias en en 2G. 6 days ab.cceeeee. eetews Rees eaetaety LOR
Special Media: (Such as litmus milk and blood serum.)
orsaimrocs ayx MADURAE 269
Gelatin Stab: Grown 24 hours at..sssseeseveer PC.
a Oo Pa OF
hours at -oos Hours at...cces
: 4
48 HOUTS Ab sc0ssacncces sence °C. 6 days at ...cceceeee sinienietpreieies of.
Agar Streak: Grown 24 hours at........ seanerels eC)
°C °C.
Sa ; ee
ae) 8 n u 3 ° 4
| \
. hours at.......
48 HOULS Ato... cere cece eee eee °c. 6 dAYS At... eee reer eee ee ee eee
Potato: Grown 24 hours at.........665 wists seaane’ Cs
°C iQ,
secceeeesDOULS Abeoereee hours at.
@
AS HOULS Ate Hiwwennarresroesre 2h Clays Btasivecnenens verntsiase 2G
Bouillon: Grown 24 hours at...........+ neeeaniad er
ee OULS Bhvwsaxee °C hours at
‘Sa : Re
48 HOULS Abiesesssecereveev eects OdkyS Atinsiisons wee sacewses OF
70 PHYSIOLOGICAL CHARACTERS
rate of development: 24 hours.. .......... per cent., 48 hours............. per cent. 12 hours.. suas waite tere werenets PCF Celitas cc Seyne vgeesaias x NOUS 2 os seeseciees va per cent. TEACHLON IN ODED ALIN xaiaesanie seed oegaeReGes Ds cs Kime Rey Pte SeeeG Yh eedelORAS Veena pas formula, Hes COZ eis secu. aici ceca encidawad es oeabeiees Gece Whale OSE aaseaceeten ais Di ACTOS ine rsiovaaine Va vautwe doleurindeloiot ss Gx SACCHALOSE vewrdiirs ac bieumenton seecemeniine wie
6. ACID OR ALKALI PRODUCTION:...
7. RREDUCTION OF NITRATES!..cesccsecceeseeneneesnereensarecees Sasoratcrachewr are suachivannnen deat saves to nitrites.........5055 ieee4 aearenenieins i 56 tO. AMMO avcsescas os peewee es Sage essai
8. INDOL PRODUCTION. ....cceeeeeeeeeeee peace Se Ree NS SOREN Gn Aeeeneie
ASM OUTS ek ico sestasor oie. sseed: a5 starr oeloanatais Bs-w a alewigprearcees f GAY Sista sinlimiyeds end olereleisveeleicuaihaiarere
proteodlytic........seccuree siaieiaietninigiasaiaecaie gyms Hgntie don Tie Wis Uideergeeadea nate Beatarntalt daletesioa asia saaate
digestion of gelatin......cseesseeeeeeens digestion of casein......
GIA STALIG sires prin By ed beateress 240% ROT renirn yer nn tad ¥ 10, CHARACTERISTIC. ODORS. ssiusieiee evecneansosawee teniwewavenis aia iar aeabatavanktaie(oreye wedi iaipeu ses
11. PATHOGENESIS (or other special characters) :..........0.005 shige Ws aieloaTeelemaaieeaea TaN
ig Se LES eNeie E49 OS TRE ONG MAAR TS FOS, DO n a rm nee ence neem ene e ee eee eet nese eeeeesaee eee eee eee eee ee eee eee eee ee eee cee eee eee Cee ere reer reer rere rer
PoeERUSUECOE EO CREREE UPC URESUCECOCEEUOeereeee reece re reer eee er ees ee ec res ceri er rere reer eeee reer rere ry
PWePeUrePeETEeEPe Eee reer reeeerrerecrrsereeer eure ce rrr cercr ere errerrver rer rerrerree rer rrr rr ry
POOESETERE PERE SES OREO ROO USE OOR ECE OeOCerreerer reer errr rec ere e rece r rec recrererereererrrr erry
MEDICAL BACTERIOLOGY 271
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, ete.
The first two methods are the most reliable. In the displace- ment method, hydrogen, carbon dioxide or illuminating gas may be used; hydrogen is best. This gas is readily prepared by the action of sulphuric acid (1:8) on zinc. Hither a Kipp gen- erator may be used or one of a simpler construction (Fig. 32). The gas should be washed, Ist, in lead nitrate to absorb the sulphuretted hydrogen, 2d, in silver sul- phate to absorb any arseniur- etted or phosphuretted hydro- gen, and 3d, in potassium hy- drate to remove sulphur and earbon dioxide.
The cultures should be made in dextrose media (which should preferably be freshly prepared and always boiled immediately be- fore being inoculated), either as test-tube or plate cultures. Novy’s anaerobic jars are perhaps the most satisfactory receptacles for the cultures. (For description of same, see N. 306.)
Fia. 82. Hydrogen Generator.
202 MEDICAL BACTERIOLOGY
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 receptacles; 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 ec. of air space, the tubes are put in place, then about 10 ec. of a normal sodium hydroxide solution are added to each gram of pyrogallic acid, and the apparatus immediately and hermetically sealed. A very convenient method has recently been outlined by Wright for test-tube cultures. It is as follows: After the culture medium has been inoculated in the usual manner, thrust the cotton plug into the test-tube so that the upper end of the cotton is about 2 em. below the mouth of the test-tube (it is usually desirable to eut off a part of the protruding portion before doing this). Fill the tube with py- rogallic acid. Add with a pipette enough of a 4% solution of sodium hydrate to dis- solve the acid. Close the tube immediately, making it air tight by inserting a rubber stopper in its mouth. Then invert, in the ease of solid media, and set aside for devel- opment. Fig. 33. Rickards has recently published a modification, which consists In for enitivating anaerobes. inverting the inoculated tubes, without the pyangallie acid galutions
. ‘x ‘ é rubber cork. plugs, into a glass in which is a layer of dry pyrogallol and then adding the hydroxide. Plate cultures are made by using Erlenmeyer flasks instead of Petri dishes.
Rererences. A. 206; L. & K. 98; M. & R. 68; M. & W. 117; McF. 216; P. 233; 8. 78; Wright, Jour. Boston Soc. of Med. Sci., 1900, 5, 114; Rickards, C. f. B., 1st Abt., Originale, 36; 557.
EXERCISE 94. EMPHYSEMA GROUP. 273
Bacterium Welchii Mic.
Synonyms. Bacillus aerogenes capsulatus Wrtcu; B. der Gasphlegmon Frarnxer; B, emphysematasus Kruse.
Expranarory. First described by ‘Welch in 1892. Occurs at autopsies in which gas bubbles are present in the larger vessels, accompanied by the for- mation 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.)
Rererences. Welch and Nuttall, Bull. Johns Hopkins Hospital, 1892, 3, 81; Welch and Flexner, Jour. Exp. Med., 1896, 1, 5; C. 183; H. 329; L. & N. 344; Mig. 392; M. & R. 402; Mel. 591; P. 545.
MORPHOLOGICAL CHARACTERS: SKETCHES.
1. FoRM AND ARRANGEMENT:
a. Bowillonis s xctesee sad bash arg secaion os eamnoaeneest cheeeeseeeen wees
8. STAINING POWERS!.....+-+006 , AQUEOUS Gentian-ViOl€t......cccecccceeceeeceeeeeeee sees renee eee eeeeeseeeeseeetereeenees b. Loeffler’s methylen-blue vs GOIN S SEBIN, oc eceas ce csmrsdeundataehecereres Ho rRioKECTEhe Sem aTE Pe eRe ETT
d. Special stains
42 “MOTIVES S.csces cae eeamen We esinnemnatey caaeene Ras dentaredonnt Wexs tetorcaamauaas Man ror oisteis isle w#. Character of mo Pena teicuesuuns 1 sermpeaeaas 104 Hed teue ka Vi Th doeneneenay taeeecsenanens
b. Flagella stain
Bs. (SPO RSS 2 weciseers viatesicesyaieisiors evs dusiraible, Ma vie aaceaptaaAn le de beavis aeatiefeannen me Be evesIam: Len abate SAE aa
6. SPECIAL CHARACTERS:
tes QOPSULC Sees: conacss acceereenes sesereeeenent sere ctees
b. Involution forms c. Deposits or vacuoles......+-.e0+ penees siahajaranaie tate ayelans eiooyaiin Se Roldladateinuine UntURAA Seis Uae
id. Pleomorphisim: iiss, dvecame sinowseniretonmeasinaesy oeurceowonnies te SdadEahes satisaed ts .
274 CULTURE CHARACTERS
Reaction of media (Fuller’s scale) 4 ........ OF, "ea aegase's Gelatin plate: Grown 24 hours at.........e0e °C. Sketches. (a) Surface Colonies. (b) Deep Colonies. 48 HOULS Ab. ecseceeevees serdesouwnaes Os 6 days at..... terteneeeeeeeeer neon ee sO, Agar plate: Grown 24 hours at.........665 °c. Sketches. (a) Surface Colonies. (b) Deep Colonies,
AB OUTS At. cersccccccerectsesconee °C. 6 days ateecseccssece dhiaemseneeakigs °C
Special Media: (Such as litmus milk and blood serum.)
DAUCLERIUM WELCHII
275
Gelatin Stab: Grown 24 hours Otecsscsseeeeeee ee Ge 3
saneeesMOULS AG. ..66..°C
Ok.
seeeees DOUPS At......-
48 hours ato... .sceeseneee ean. CCE Abixcccarcemmaneeaacrers ° Agar Streak: Grown 24 hours at....... sate Sovttes °C. oO o Py S a é n nm u q 8 é 4 4 48 NOUS Abscsa vosansreseswawsase Ce CUAYS Biss wacisiviciseninwa vis waren °C; Potato: Grown 24 hours at......esseceeeeees cacieen °C. 3 | Oo 3 3 F F a & a | n cal ~ =I 2 a ro) 4 A ae ee 48 hours at...... cuuisouaiouieeaw’ Os Fi 6 GAYS Bhinisisensiersreisrsieresitnaaren °C. Bouillon: Grown 24 hours at......seeecceseeeeees a oO 3 a 3 3 3 2 : 5 =] i) 6 = A
48 hours at...... nouceaudes kane’ Oe Gd ay Sati icinesenicwneesins saci °C.
276 PHYSIOLOGICAL CHARACTERS
1. RELATION TO TEMPERATURE!.... .e.eseee sibveyalgsaye.oj\eie| sretevare.deceretatalh aldara tasierelals i -Metodiaias oh emia OPUMUM tadicsrwareia eexcaweis awatead Get MIG hae vied asteiaysreisiese sina EO sisueisisierwigs six sraietate °C: thermal death-point..................- °C.; time of ExpOSUYE...secsceeeeeeee minutes; MEAN Ti Which Exposure. 1S: Mad Siw sveewewa ss ve-cariaieemwae Va Rw les. 00 8% gpvioedine vida
2. RELATION TO FREE OXYGEN! ... sess eseeueeeeneenees sivuelgueshiaig’g aoprd a. 0ldleselery wrvieldineioase-s 0 dmacealel
8 RELATION TO OTHER AGENTS, SUCH AS...c.scsesceeeceeneceneee diaiggeietdiess eistalguajshataseene apna tenses
desiccation, light, disinfectants, Ct.:—....sccseseceeeveeneeensenece eawaiey tv Kanone
rate of development: 24hours.. ...... ++.-per cent., 48 hours..........0 es per cent. TA HOUTS ssijsaivaessevaee sess PCT CONGigacnwaeseweviess vere HOULES. sae ee isin inte per cent. PEACCIOM LM OPEL ALIN cicsesiis Vaasa sind sinetains o's sigivcgiere os Hee paly oiginiele Ve sialsbiasbieiers balgubarerstaees
gas formula, H: CO2: t.csescccescercceencee dich 04 Hees TIGUE. NakOs Oi MOU ERERE a
b. lactose....... so setatnaeMettets savrienes CoSaAccharos@iss vcsusans ck canden see ete:
6. ACID OR ALKALI PRODUCTION?..++++ trees eee Teer e ences Se ees see enessoces
Doveeerenees Debt eee e een eee sen ee nen bee e eee enenene eee Cee ee eee eee er ere Ty
litmus milk...... cepistaraayS aia dare Ret shat SE sectagesiereie Seer Onc SGlaNdieaeiniedereaeiesiamens
8. INDOL PRODUCTION......++ ajecaveratatotniates' Mikiape eatiitana Sibuadas sraveem aierataberote slaceerevalaysie ereeinecesiins 6a Me ais annls
48 hours...... je aya rsibed iste) cuasasselavaustonsimagrmenmieaneleisie’s ditties fe days... Mex ne adiahsrosiek asttentesia 9, ENZYME PRODUCTION:.....+. siefeaser dsaiainie Had suaretaaes ease eer a is ioraiais elapnnasssbee Si usehale abel fieae wieses Hehe
weeereeee Saeco eee e semen eens eee eee e eee Cee ee eee eee ee eee ee cere er ee eee eee eee
proteolytic..... ai SS nig BUS Are iaPaya nines aig av elererarerBIRA GE HAEISS MELEE HE Ree Si drdrorateiaiaieietciciassinctia ois alaeinisie' eis
digestion of gelatin digestion Of CaSCIN.....scseeseesecceeees diastatic....... ig tj oe oie bc atu tig osdhs taba W Steen tra in ndSiSi6 a Gaaleha nieve oie Wleiale Maps eimatajemate stale ia dh setae reiaos ae Sartte seats he eae rere ee ee SEES CEES SES ES EOeee eee erate)
10. CHARACTERISTIC ODOR!....+seeeee teens ese singe a) Ha eslnemarelnse a vara ela alee weldunieN seme ates
11. PaTHoGeweEsts (or other special characters):......cesseeneee COOP OCC EOE OE oe IoC)
beeen een eeeenereee Dee eee e eee e ee eee ee cert enes wee teen ween eee, eee eee ereeee beeen eee rere reer) weno oe Serer ee eee eee eee eee ee weet ne emee Seen e eee e eee neeene Peeve ne ceeccencee eee Eee eee e eee eee ee eee ee eee eee eee ee ee eee
errr eer ee eee cee eee eee teeta n nee eee eee ers tree eoenee wetness bee eee ence weeny th ween ee ree eee Pee eee Ree CeCe R Ce eee CCC eC e eee eee eee eee eee eee ee rey
EXERCISE 95. OEDEMA GROUP. Q77
Bacillus Feseri (Trevisa.) Krrr.
Synonyms. Bacillus of symptomatic anthrax; Black-leg bacil- lus; Bacillus carbonis Mic. ; Bacillus anthracis-symptomatici Kruse.
EXPLANATORY. First described by Arloing, Cornevin and Thomas in 1880. It occurs in the subcutaneous tissue, muscles and serous exudate of animals suffering from symptomatic anthrax.
Rererences. Arloing, Cornevin and Thomas, Le Charbon symptomatique du boeuf, 2nd edit. Paris, 1887; A. 527; C. 296; FL. 2, 245; H. 328; K. & W. II, 601; L. & N. 339; Mig. 593; M. & R. 401; MeF. 583; P. 563.
MORPHOLOGICAL CHARACTERS: SKETCHES.
1. FORM AND ARRANGEMENT:
is ROUT O Me sestsssisivoztarevacdcw ais ois eocayas ele bosiateabave w statalnnautnetcbataigin\olaigreidtodg bie ateress Stes St
Ds SSL ZI DY. sccoc ie aeelete ay (ede. a6h leer asso ey sely coca ap Se Catansa abe concn ata ate sora TarakWla aie AgaeiW Nica toda a Reavis RE abe a ee 8: . STAINING BOWERS Si savieaciinmeainns ve slotaiedeaua ea eave esd <i aiateivieiauelaleinrs miele siginmlechaulenannat 4/002
we AGUCOUS TONMHANAVIOl EE sie os so rersiescwras soon siesawiwstwiane nedsislawiseu aes OY Fa Na ARE MORIA eee &
b. Loeffler’s methylen-blue
C. (GRAMS Stalin. ccinanaiwrG 5 aemmsmanics ws Hpseda eh ey on eae aeNeety srediscacuaals ou dacheecraens aa,
Us CAPSUUNESiiaceranjemiwas riestesiemnnesesenneabieadide: aneae seketepedwene ss Kitaileeeead Ii ete . by Yoordltlon (ORME actcessa 4 came 453 Ka reeaies cereeered tier rem ecen ne cemnoweveetianur ‘ bo Depesite or waee 6S: saccades sccunaess deainbsenay feckienennend venes deanna nemienedid ne
d. Pleomorphism
278 CULTURE CHARACTERS
Reaction of media (Fuller’s scale) 4 ........ OL SS sean os Gelatin plate: Grown 24 hours at ........... #6. Sketches. (a) Surface Colonies. : (b) Deep Colonies. M8 HOUTS Abs essa ve csiisede waeedad vows °C. 6 days at.rcrreseeee Rising Manse °C. Agar plate: Grown 24 hours at.. ......... °C; Sketches. (a) Surface Colonies. (b) Deep Colonies. a eielicaiava °C.
48 hours at........ cement ete veel, 6 days at..cccseeee Siainrajeiaswi oe
Special Media: (Such as litmus milk and blood serum.)
Bacituus FEseri 279
Gelatin Stab: Grown 24 hours at...ssssecseveees°Ce
see:
hours at..... tee nkicw POUES Be anaees Py
48 OWS Abswies essences vee es °C. 6 days At ..ccseeceees agorainiayi nie of.
Agar Streak: Grown 24 hours at....ccceeeeeeees °C.
aC.
--hours at......
seeeeeesHOULS tees. OC
43 WOUTS BU evwecrerreseuernevcs a : 6 GA98 Bb. cases erences a Be Potato: Grown 24 hours at...............06- Rageer °C. 3 ro) : 3 3 8 wm n ist ial 2 3 ro) 8 4 4 Cc
18: HOUES Bbins ar caemnoaays witanes °C. 6 GAYS Ab.oreccecccusccneventees i
Bouillon: Grown 24 hours at......seeecesceceeees 2C.
want? Cy
ora ©
seeeeees DOUFS Bb.... hours at.
48 HOULS At... resseceevnevseeee Cy BiGay Sabi pectin viesatan oerecand eH
280 PHYSICLOGICAL CHARACTERS
ty RELATION TO TEMPERATURES inciiwas os os noneedans dendneuuainde vy ugegen Reintie oes sneeese eRe 4 ODE MU Ml anetieres ps4 eeainaeae s One AMIGS: avis cones, ta vats COlnosnan sineases een "C4 thermal death-point...... ........... °C.; time of exposure....... eeeeess Minutes; medium in which exposure iS MAdE.........c eee eeeee veces fbi ia aravecaieieraisemadie Shar tceaneee
2, RELATION TO FREE OXYGEN?...... .- Ansatesatsleh Jamie at” <ovotohatethitesacecsiace sauduavaionabas-aitaraedeendesatdin w-aCaaredora
3. RELATION TO OTHER AGENTS, SUCH AS ......05 ree ee ree park eeRaERIES IES
désicestion, light, disinfectants, 6t@ t—.c asap cxvneeenve vrs sseiots tatasts AVR aaess So9
5. GAS PRODUCTION 18 SUGAR MEDIAtic« 44s 17 14 eevermemnawei nr os twdeganay eans ore tuo We Gextrose (1) Shake Culeurei sss ss adsiease ye aecnlatcatigle te vid Ldaiedeiny, es elerbaisow yada aaer
(2) Fermentation tube, growth in open arm................ closed arm......ccseeeees
rate of development: 24 hours.. -.......45 per cent.. 48 hours...........4. per cent.
72 NOUS: saciesace ss ea asna ve PCT CONbagiss seo dasasiuness OUP wissicinsieiariers per cent.
reaction iN OPEN ATM.........e0e cece eee afigivie’ aun och Sieeesiapeisuarass Watecerareteys te eR eERERE an ATES
gas formula; H? CO8s tics ccsaes. aan vs curse seqaie as anaemens a alesis Sa swiare me HA oA
1D; JACtOSCwan ass caved nadie teaeteenGaaye €.. SRCCHATOSOsacncanion Wis Mises Ws cidwimainsied sa
AS HOULSis.. 00s cor deans 9. ENZYME PRODUCTION
PHOLCOLY LIC sa-cia dione aiaiaGneiarersisrernichetioinroinn oh aarenisancanls Se dgialuisadnelag se deena athe’ WHERE dogs
digestion of gelatin..... ssstehguaVacoielans ater digestion of casein...... dicleae aweeeeirelaiein diastatic...... COT or i Tee e ene ne eaieralassinatbegeke Bteletne 10. CHARACTERISTIC ODOR¢scccccscccarsncesssceeesecenctsonscseasesnnenas we outa piciartass Oud Sam
11. PaTHoGENEsIs (or other special characters):.......... pity ieiasan are wisncess nis cu Canad ekvenaen
See eee eee eee ee eee eee eee eee eee eee Decne neon eee see aee eee veeeee Decne eee peewee nent ones Sennen eet emane ones ---- Cee es Serr wees eee eee eee eee eee eee eee eee ere eee Cee eee eee eee ee eee Cee eee eee eee ee Se vet eeeeeenees ee ee ee eee cee ee ery peeves Sennen ener ee nee eee e neers dere e ene eeeee ee ee ee ee eee errs eee eene
OEDEMA GROUP —Coxtixuen. 281
Bacillus oedematis Zorr.
Synonyms. Bacillus of malignant oedema Bacillus oedematis- maligni Zopr.
EXPLANATORY. First described by Pasteur in 1877. Widely distributed in soil and putrefying material. Few cases on record of infection in man.
Rererences. Zopf, Spaltpilze, 1885, 88; A. 522; C. 292; Fl. 2, 234; H. 326; K. & W. II, 619; L. & N. 341; Mig. 604; M. & R. 393; Mce.F. 587; P. 543; 8. 488.
MORPHOLOGICAL CHARACTERS: SKETCHES.
1. FORM AND ARRANGEMENT:
Ce “ES Ound OM ssivacase-a.ocacscerbitgater a aioraiers « Cacectsveunin earebiniere s Wis Mitac Gates waa end areeectatatade :
G8 GET 8 A Tals ayect car ceesnvace asian ee cegaaasa far bneio ausvbunabatevors vacacniasa petra wis iaiorapeusaderu bir ie Sct
2 Glee Sitti neessmndgnnaneeeece cen ii nendmanaunna inn wieemende cc
a. Aqueous gentian-violet bv. Loeffler’s methylen-blue
G: AT SURE. wa muwiny cernconseds ceveneandes 1 ew eeawes 19 UENRHe B44) Hi DLHWESwewee ss rows ad
es CAPSulesimeirs sccraciay apayedsainysyenre) aie sia ayoteayeisiaie aiviawiaisign ww aaawaulenn sway ReRleedeaieee ns Pree
b. Involution formsS......e.. veer eeeee aamaciante sie
e. Deposits or vacuoles.
Gz PISOMOLED HTS Misccce sav orssees sicine datas: oases ba navm aula Senin ereoe Maat aise a@awanteate gee ee: eee
282 CULTURE CHARACTERS
Reaction of media (Fuller’s scale) + ........ OF SS seervesue Gelatin plate: Grown 24 hours at.........055 oe). Sketches. (a) Surface Colonies. (b) Deep Colonies. hours Oeics a ssctvar saannwonnx aC. 6 days at..... coo be 2 Agar plate: Grown 24 hours at............ °C. Sketches. (a) Surface Colonies. (b) Deep Colonies. 48 HOULS Ate cserereeesrerees Diwivintete ois 8c. Gidays ‘at. niwwacssicceteaws stoners °C.
Special Media: (Such as litmus milk and blood serum.)
BACILLUS OEDEMATIS 283
Gelatin Stab: Grown 24 hours at...ssseseseeen ee Gy
°C
hours at...... 2) tote HOOT Ota ccncnc®O.
AB HOULS AL... ces eens eee e eee Cy 6 GAYS Ab cceccssceeetveeereees ole
Agar Streak: Grown 24 hours ab...e.eeeee eee PC. 3 3 3 Ps r a n mn I cal 5 6 4 4 _ G
48 HOULS At rss ceeceeceeeeee ens al OF 6 dayS at....-se eee wine atinsiessceies 2 ‘N Potato: Grown 24 hours at......... prsapaenereene Cs oO fo) 3
sevceeeesNOULS Abe... ee? seeeesshours at...
AB HOULS Ab... ee ee eee e teens °C. 6 days at.ccseesereeee Teewares i
Bouillon: Grown 24 hours at..... J smevaneaweninee le
i
«°C.
sees hours at.. seveeves DOULS Bb....-
$8 WOULS Bl ss saaas gan grsvenesa O days ateiciewexe.ieeieeseceis OF
284 PHYSIOLOGICAL CHARACTERS
OD BUDA Wane ayesopesardord aised deamereuaesiere o 9 SCS iMAC st wecaonses asenan LO i spdcasnasmend aeeate “Crs thermal death-point...... ws... eee °C.; time of exposure..... sesoeeees Minutes; medium in which exposure is made............ S gece ime as ealuterdeneiuiee SA eneeaRe wine 2. RELATION TO FREE OXYGEN! ...... ccsccseucecccessuees dies seam AKANE CRUARUE AD Ebi ii
3. RELATION TO OTHER AGENTS, SUCH AS ..-
desiccation, light, disinfectants, et@.:—.........ceeeeee eee SUGGES MareAtRetmRES ae
rate of development: 24 hours.. .........- per cent.. 48 hours............- per cent.
72 HOUPS. cars taateeseaen s per Cent.,......08 nae VoeaneR NOUPSimeaas ve actows per cent. reaction in OPEN AYM......... eee cece eens seer dauetecsions Stee gis Sch Bueenose lan isisinecis Usviae dati gas formula, Hs CO 2! tanec cece: stuaiaiaesiee denies ome Em yraetig idea ets cote diecena fo bab earaiaceie
Ui IACKOS a tcliscnaianriionis ae merce ercceeteians €. SACCHALOSE........ ce eee ee Sieidcnagiesbie wis sselupatate fs
TO MITES: caceascags ang newts 4 dans ated to ammonia........ ite Mea Ss GhiSd WE RRR 8: INDOEZERO DUCTION: Ai oi cxGiaite choi se RMA OTR RS, Ti husene aia yaa ober Pood Wiuagideauieen
AS AO UTS id oisenh \ seaniced icine, Naavaad Medan. Srenimiad, eave GAY Siieewh srertacabsennisarenente sna 9, ENZYME PRODUCTION Sica a4 decisis aad os etesas MARIGS © maaaes Heatheiiasas da eeRnaN IY
PLOLCOIYLIGy oy ag 5 saawhnaesonaua es abel gens feces Fee Ay 8 Lac hw TERE eam ERY aia diséstion.of CClativiscaeiwas vaveseecnwes digestion of casein.......... aptoleisansets 89 diastatic...... mia biduatvtonste SED Sioignie. tS ROHR IST Me HO ORAS A eniaisisiaie eae aiarsaiae «Gea Ts
10. CHARACTERISTIC ODOR! «cere eeeee eeeeee wibiaia ata and tig bos carats We segeevetienora Waretlarersave bails saa leceles 11. PaTHoGENEsIS (or other special characters):........00005 siuiaaiea tajaauaserae eicenet wisi twa esate
eee eee eee! ween eeeeee Deere eenees eee eee emen ne Dennen eee ee eee eens Web ee eee eee eee Eee RE eee ee ry Pde ene nem nee ene on ese re eee eertenetnereeteeeens Senn See . bene Dene wee eee ecee setae eeeeee ene er eee ee ey penne Tore eer reer ee eee eeE rere eee ere eer r ee anne Wee ee nee e ease eeees °
tenes beeen cree een eee ane ber eeemeereesvoerroins ee eee ee i rnin
OEDEMA GROUP—ConmTINvED. 285
Bacillus botulinus v. Ermencrem.
Expuanatory. Isolated by v. Ermengem from ham which had caused meat poisoning. Believed to be the cause of meat poisoning characterized by nervous symptoms of central origin, botulism.
REFERENCES. v. Ermengem, Z. f. H., 1898, 26, 1; C. 297; K. & W. II, 671; L. & N. 337; Mig. 616; M. & R. 398.
MORPHOLOGICAL CHARACTERS: SKETCHES.
1. FoRM AND ARRANGEMENT;
a. Bouillon......
w. AQUEOUS Gentian-Violet...rcceesevcccvcsceeersceees iiaresdticoicie oi Siaceld doa ieee iiacavessieie/ siege ve sisisy b. Loeffler’s methylen-Dlue...sseccscsssccerscceeccerecceveveses aaeesiay ite eeidlateeie’s O68 “eles &., Cram's Gti ssestexsenasne se reeensnr ee sommes Cine a enNeeN Laney fanen
d. Special stains...»
bv. Flagella stain........ 5. SPORES! .cceeeeess sree ere ee rr er ere As sdeieeshemietiieg i (piknadane Ase bemoiesnans se RNS Leb OR eR RARACAN EAA RRNA NRNRAD TANT We ROMER LAEVIS RRs e cheer’ 6, SPECIAL CHARACTERE fini sscnnntaes se ereiarsitiees eohiesaeid os bv beeen an tava ennee ofa a sstiatelace ° Us. CaPSUleS..carcaccccresscavenevececescnescseesseeese veessosuesnsseceestecessesseceenris on . TeVON LOIS cs sewn cn enead ese cncdenweneys Gancantad Kees ecenen seinen ie Ne ‘
c. Deposits or vactioles..c+s0e
d. PleoMOrphisM....ersevceeveveseversevcvrersecvsecaseecseesssseenevercecvercvecsssenecane
286 CULTURE CHARACTERS
Reaction of media (Fuller’s scale) + ........ OF aes sages Gelatin plate: Grown 24 hours at............ eC. Sketches. (a) Surface Colonies. (b) Deep Colonies. 48 hours at...... dials awnaer Seeionenes's °C, 6 days at.....e.ee a araeroccrathiarn aceneserasasais °C. Agar plate: Grown 24 hours at.. .......+- °C. Sketches. (a) Surface Colonies. (b) Deep Colonies.
AS HOUPSIAG: ts weiscicscrsemerasais asians eC, 6 days at..... sycharcraete erate date 54 Rare AGS °C:
Special Media: (Such as litmus milk and blood serum.)
BacItLtus BoTuLINus 287
Gelatin Stab: Grown 24 hours at.....sseeeeeeee °C, z lo} Cy ° o o n n iol u 3 3 & & 4 a
48 HOUS Abo... sees eeeeeeeseees °C. 6 days at ........un0s simibiesnatisirs oC.
Agar Streak: Grown)24 hours at............0-5s eC. fo) o j 3 °
8 3
7] nm
H H
6 8
4 4
Ke
48 hours at......eeee eee
Potato: Grown 24 hours at.. oO io} 3 3 3 3 wn nm 3 EI c=) io) 4 4 C.
AB NHOUTS Abies ss poccesaceisesane Ci 6 dB Satin ccivsmcovacmenncmaae ?
Bouillon: Grown 24 hours at....cesecseeeeeneenes i
os °C
hours at ase OURS! Obie oscars
48 hours At...sseseeeeeeee wee Oe | 6 days atc eae ee.
288 PI-YSICLOGICAL CHARACTERS
it, eA rion Th TEMPE A TURE ewcsewe ee samionnaies ieenemnatanes nes iisharasdibiels bheceiepeiies nGiewisie ni OPLIMUNicscccmmanace acaamtionss SCS IMIGS: ava wasps wae ae TOR ideale sidan Viewrataiale OF thermal death-point...... .......see. °C.; time of exposure....... seveees Minutes; medium in which exposure is made...... aia ate aie be srdaraiet en eas deelegind sixieeenrs oan 2. RELATION TO FREE OXYGEN?...... seseseeees Bele ie kor sleds osc spbienste asees sariiaiaige ot Reece waehwe ania a. HELATION TO OTHER AGENTS, SUCH AS cc cp yucenu paves coameeraismannden Be hemsewens Ka wang r desiccation, light, disinfectants, ete :—....-....eeeee tyes Ie Naa does deqeaOMAT is sou ‘
rate of development: 24 hours.. .......... per cent,. 48 hours............ per cent. TA NOUTS' nas ccewemaeies S45 PCY CON. cscs ale veensiawes WOWLSssxscesancna ss per cent. reaction in Open ATM. «1... cece eee eeeee sseeogiuitig tre semis sgesoinverarsietanstaices Li vie eodranuiesabe vee aeatnaetsen
gas formula; Ai COB nai siyes goss cxwteidien sé savieses. 444 ras ae yey saindis sy ta r
Us TACOS Stn ce crteateisiacs 35 0g 9 Sa Tear ias GC. BACCHALOSC sss ccssecrenccoeeses sceyavionisiaiss ss
6. ACID OR ALKALI PRODUCTION: ...6. seeevers ceeeevee Cn ne ceria
PTOteoOlytic....seseceeeerevee ire WR aONU UE RRS —eAenaulauee namie SARIN duiaeiaies Stared siatany digestion of gelatin......ccceseee ces e ees digestion of casein........... si aiantiata diastatic....... ORG eS ee HUNG TS Im Se@eNIR SE LS Se nL Re eEOURETS AReRRaeREH sigeiea ar tera 10, CHARACTERISTIC ODOR: oo .02 seice tes ys wens Sd sihibiec cs taeeaae Ye ratslasteatels (ache smnnaent main ead 11. PATHOGENESIS (or other special characters) :.....5 cisceceeeneneseenacs weeiion siiaitnatsiars says
EXERCISE 96. TETANUS GROUP. 989
Bacillus tetani Nico.arer.
Explanatory. Discovered by Nicolaier, 1884. First cultivated by Kitasato, 1889. Occurs in man and in animals suffering from the disease, and is widely distributed in nature, especially in soil.
REFERENCES. Nicolaier, Deutsche Med. Wochenschrift, 1884; Kitasato, Deutsche Med. Wochenschrift, 1889; A. 513; C. 302; FI. 2, 260; H. 320; K. & W. II, 566; L. & N. 332; Mig. 592; M. & R. 376; McF. 389; P. 385.
MORPHOLOGICAL CHARACTERS: SKETCHES.
1. FORM AND ARRANGEMENT:
Gx BOUMMO DN ciesistisisncsersiats «naan taediecgraeg +3 ee awl ealeapearatawies bs Houllelnnegtesy ww eaa
Bs STAINING BOW BRS: cisige'cz saesgrne aciateiaierisin's aris Siplnsetsiantbeiets -clstadrcpamalanen v4 SAGER Sect Mave oneed @ AQUCOWS Bentian-ViOletiiss sceasiossaee da coed Veeweeadals ey ETERS aRSEe Se aaNiaTedenes comma
b. Loeffier’s methylen-blue
c. Gram’s stain ....
a. Capsules........6+- b. Involution forms...... Ws did crore leis tachaisieisisiatesn te eae eiateies a eieince Svstefena. give. Shinto aroused ota ebebdvaisiateusi nea ace 6s. DGPOSIES OF VACUOIESs aie are sicsaveiarsrnicia s.o-ossieibsaiateraracd weasehatavaiaaiain'e wiaisiateleln stares « gradoaiaietestels Malelayeena OX
a. PleOMOrphismbsssiie: jek v dciaieceisislie Hs sideweaawasienwresie gs oo ag sdailceniues emEeiReA va HME RENEES
290 CULTURE CHARACTERS
Reaction of media (Fuller’s scale) 4 ........ C) er : Gelatin plate: Grown 24 hours at............ =f). Sketches. (a) Surface Colonies. (b) Deep Colonies.
48 hOULS At... sce eee cee cae CRONE SC. 6 days at..... aNeea NAMES
Agar plate: Grown 24 hours at.........505 °C; Sketches. (a) Surface Colonies. (b) Deep Colonies.
GS OUTS Aticcsinciervecscinaiavian sie ria OF 6 days at...s..eee a ilaibiniels saiiewareian °C.
Special Media: (Such as litmus milk and blood serum.)
Baciuuus TETANI
291
Gelatin Stab: Grown 24 hours at...-..660006605.°O.
°C,
- hours at.......
AB OULS Ab rsesseceeseeneeee eee Cy
6 days at .....ecseee
seveeee OUTS 86,.......°C. , Co
eer)
Agar Streak: Grown 24 hours at..... wiegar sera wate bd 64 5 5
° ° ‘ 3 /’ a, n n u con 5 8 4 A 48 hours at......... dctstedaispainave OF 6 days at.......eseeee auecereiesa digs °C.
Potato: Grown 24 hours at....... dareieleustatetalotain oats ee 3 Oo 3 > e a mn n E 5 } i) : ‘ :
48 hours at...... tere eeeetneees °C. CAAYS: Abas cit cei cieiginicigusiiseesene Os
Bouillon: Grown 24 hours at..........+ cdrsdisitionane °C. 3 3 3 3 a 3 n mR E : 3 ° 4 A
48 HOUTS Abs.ecrccceenececvesee Oy
6 GAYS Ati cma nenanmncs<e 65
292 PHYSIOLOGICAL CHARACTERS
1. RELATION TO TEMPERATURE)S..-seecreceesecreeertee sees un'4 iby aaanare id y9\9 a ibe ais Gin aid asasaieesase’otal OD EMU ss 4 vis wiaserestassins eared © CM TES iivcwiedaveeeisie tincranted CORaaiie saaeariaas weve Gee thermal death-point...............0eee °C.; time of exposure............... minutes; medium in which exposure is made...........0005 dienes nee stasis asFeatalaya ayers é beteegs
2. RELATION TO FREE OXYGEN! ...scesecseeeeeee eeneseeneees er ctdieoationaysie Ss Salseceet Beereineee Bey
3. RELATION TO OTHER AGENTS, SUCH AS...-..seseeenees id shcwichinrdcaoieine 6 draakesdietn be ieeetedvavecs sash
desiccation, light, disinfectants, etc.:—......... cece cece sees Vinkigubanutece geminata
a. dextrose (1) Shake culture:............00055 wake a5 SivcgimEeiey ceasvoeN disie ds Chiat eM aI alate
rate of development: 24 hours.. .......... per cent., 48 hours...........06 per cent. 72 OUTS sda susesetaainiernag a HEL CONE. saris civics aawanet MOUT Sis. sceasiraeionan s per cent. TEACHONAN, OME: ALM v4 acomeinya:pasea-avwaseveauorna ied s-9 doe Laiseovectse yo niesiaeaierpidcae Hunchaaeiros aft Gas formula, AY COs Missi cseass anaadncaeaninentuwas’ Shores otace> Usain eiavalala levnhevats ataanvatatstons eels
Ds TACO S's vies sierra gga vi Awanseslaarcaaiats ¢c. saccharose........ aia a eigiechricledss orate smaies ea
7. REDUCTION OF NITRATES
GO A TOPLbOS) iors sioeG eaeeisiieceea ad ioe
PLOtCOlYtiCscvesnessanes 4 He teesiesaeeeys if Simei 13 eh iSeeaatn wi SeANleR ean MARTA digestion of gelatin..........cceeeeeeees digestion of casein........... Naaeominalnid
Mi aASCA ECs. siicvnke wba daseta aod euntenan eater meer sag acc nesteaarrnG nese naires pip lover win lot
10. CHARACTERISTIC ODOR?:..-.0.cceeeeeeeereeeeeeee ce ray Weave eee necenee
Dawe e en eeee seen ween eee seme e ence renee eset en ones peer rene Se ene ny Dee e eee eeeenene . ere eee eee ee ey deco eee e eee ee er rr errs er ene enn see eee nooo ee tenes ere eee ea ee ee ee ee ee reel enone er eeneneeee etree eee eee eee eee eee eee eer eee Cer eee Tere ere eee eee re rer ees
PEUESERTRESUSESUUERECEE OS TOOOEEOCEe STORE Cer eCee rece Cereer reer ver teeter rere erere rer rere errr rrr
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294 MEDICAL BACTERIOLOGY
CHAPTER VIII
ANIMAL INOCULATION AND STAINING OF BACTERIA IN TISSUE
EXERCISE 97. ANIMAL INOCULATION.
Metuops or InocuuaTion. Animal inoculation is practiced to determine the pathogenic properties of an organism and also the character of the tissue changes produced. The animals commonly used are white mice and white rats, rabbits, guinea pigs and pigeons. Inoculations are usually made subcutaneously, intraperitoneally or intravenously, and in special cases into the pleural cavity, brain, eye, etc., ete. Mice require a holder, the inoculation being usually 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% earbolie 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 material injected.
b. For solid material a pocket is made which is stitched, or sealed with contractile collodion, after the material is introduced.
Intraperitoneal. Hither liquids or solid material may be intro- duced.
a. For liquids. The seat of inoculation is prepared as above, the syringe needle is then plunged directly into the peritoneal cavity.
b. For solid material. The animal is anesthetized; the hair is clipped or shaved from a portion of the median line, about half way between the pubis and the sternum; a slit is made in the skin with sterile instruments; the smallest possible opening is made along the linea alba into the peritoneal cavity and the material introduced; the wound closed and the body wall and the skin stitched separately. It is hardly necessary to add that the whole operation is carried out under the most strict aseptic precautions. Collodion sacs are intro- duced in this way.
296 MEDICAL BACTERIOLOGY
Collodion Sacs. The use of the collodion sac has recently become very common and deserves description as one of the necessary labor- atory procedures. The difficulty in making these sacs has been largely overcome by recent methods. One of these is the following: Small-sized test-tubes are selected. Thick collodion is then poured into the tube to a depth of two inches. The collodion is then poured out along one side of the tube into another tube and from this one to another and so on until the required number is obtained. The desired length of the sac can be secured in all of the tubes by tipping and rolling them, thus bringing the collodion into contact with the glass to the proper height. As the tubes are coated they are placed, mouth down, in a wire basket or test-tube rack as indicated in Fig. 34,1). In this way the extra collodion drains off and free access of air dries and hardens the collodion, leaving a thin coat covering the inner surface of the tube. The thickness of the coat depends on the consistency of the collodion. A ten per cent. collodion, in equal parts of alcohol and ether, makes a sufficiently thick coat for ordi- nary purposes. The drying may be stopped at any point by filling
Fig. 34. Preparation of Collodion Sac: 1, Tube inverted to allow the extra collodion
to drain off and the film to air-dry. 2, Sac ready for sterilization; (A) Surgeon’s knot; (B) Ends of cord; (C) Tongue of collodion. 3, Sac ready to be inoculated into animal.
the tube with water and after standing a few minutes the collodion shrinks and the sac may be easily removed. The sacs are then filled from one-fourth to three-fourths full with bouillon. They are then immersed in a test-tube of the medium. The sacs are held in posi- tion in the test-tube by means of the tongue formed by the collodion flowing out of the tube. This tongue is folded over the lip of the tube. (Fig. 34,2, C.) Before, however, the sac is put into the test- tube a piece of cotton or silk cord is placed around the sac near the
298 MEDICAL BACTERIOLOGY
top and held in position by means of a surgeon’s knot, loosely drawn. The cord should be quite stout so that the sac can later be tightly closed. The ends of the cord are brought outside of the tube as shown at B, Fig. 34,2. Sterilization may be accomplished either in the autoclave or by means of the intermittent method of sterilization.
The medium is inoculated by means of the platinum needles in exactly the same way in which tube cultures are ordinarily inocu- lated. The sac thus inoculated should be incubated for twenty- four hours and if the medium outside of the sac remains clear the sac may be used. Otherwise it would be discarded.
The tube is placed in a tumbler or test-tube rack. The sac is then pulled out of the tube until the cords can be drawn tight so as to close the sac and securely tied. With sterile scissors the end of the sac is cut off a few millimeters above the constriction. .If there is any moisture on the inside of the sac above the neck this must be removed with sterile filter paper and then a few drops of a thin solution of collodion is placed in the neck so as to hermetically seal the sac. The long and contaminated ends of the cord are now cut off, the sac dropped back into the test-tube, and the cotton stopper replaced. (Fig. 34, 3.) The sac is now ready to be placed in the body cavity of an animal. (Frost.)
The method of inoculation is especially useful in increasing the: virulence of attenuated forms, and in producing immunity in ani- mals to induce the agglutinating and lysogenic properties in the blood.
REFERENCES. Harris, C. f. B. L., 1902, 32: 74; Frost, Proc. Am. Pub. H. A., 1903, 28, p. 536.
Intravenous A rabbit is generally chosen for this purpose and the inoculation made into an ear vein. Of the three branches of the vena auricularis posterior, the ramus lateralis posterior is the smallest, but, due to the fact that it is the most firmly imbedded in connective tissue, it is much more easily entered than the others. The artery forceps (Fig. 35, @) are used to gorge the vessel and are, of course, removed before the mate- rial is injected. Avoid the introduction of air, which causes immediate death, and keep the animals un- der close observation for
Fia. 35. Dorsal view of right ear of rabbit. one hour. a, artery forceps; b, syringe.
300 MEDICAL BACTERIOLOGY
Inoculation into Lymphatic system. Fluid cultures, or suspen- sions 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 without injuring the lung.
Inoculation into the Anterior Chamber of the eye. Rarely prac- ticed. 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.
STERILIZATION OF INSTRUMENTS. These are best sterilized by boiling in a solution of soda or borax for 15 minutes. This is ac- complished in an especially designed apparatus or in an ordinary enamel stew pan. In case of emergencies the instruments may be dipped in benzine or alcohol and burned. This is less injurious to the instruments than heating in the direct flame.
Use blank, p. 308 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 identi- fied. Fig. 36 shows a simple eage made of galvanized iron with soldered seams. After use it is sterilized by boiling water in it. The wire door is covered with a cloth to prevent the tov rapid escape of steam.
Fic. 36. Wesbrook’s sterilizable, gal- The inoculated animals musi vanized-iron animal cage. be kept under constant observa- tion and the following conditions noted:
a. Temperature.
b. Loss of weight.
c. Peculiar position in cage.
d. Loss of appetite.
e. Condition of the coat or hair.
f. Condition of the secretions of the air passages, conjunctiva and kidneys; diarrhea or hemorrhage from the bowels.
g. The condition of the seat of inoculation.
302 MEDICAL BACTERIOLOGY
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.
A.
a. Inspect externally and note presence and character of any lesion.
b. Sterilize a suitable post-mortem board with corrosive sub- limate solution, 1 to 1000, place the animal belly upwards and tack the four legs fast to the board.
c. Wash the surface of the thorax and abdomen with corro- sive sublimate solution, make an incision through the skin at the pubis, introducing one blade of the scissors, and extend the incision as far as the chin.
d. 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.
a. 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 transversely 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.
b. 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 manner. Cut through the ribs with strong sterilized scissors along the sterilized tracks on the sides of the thorax, when the whole ante- rior wall of the thorax is easily lifted and entirely removed by severing the diaphragm connections.
c. When the thoracic and abdominal cavities are fully exposed, a careful examination of the organs and surroundings is made without disturbing them.
Plates (Petri-dishes) or roll cultures are prepared from the blood, liver, spleen, kidneys, and from any exudates present.
304 MEDICAL BACTERIOLOGY
The method is as follows:
(1) Heat a scalpel and scorch a small surface of the organ 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 ster- ilized 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 heart cavities, the surface being seared with a hot knife before opening.
As soon as the culture material is obtained, cover-glass speci- mens are prepared from each organ and from existing exudates.
Small pieces of each organ are also preserved for future exam- ination.
When the autopsy is finished the remainder of the animal should be burned? and the instruments should be sterilized (see p. 300). 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 micro- scopically, and by means of sub-cultures.
Use blank on p. 308 for preservation of data. Fig. 37 shows the method of making a post-mortem and the location of the most im- portant lymphatic glands.
ReFerences. The above is taken largely from Bowhill, 74; see also A. 230; N. 260; and other texts.
Common LABORATORY EXPERIMENTS.
The following inoculations are those most frequently made:
Streptococcus erysipelatos. Mice or rabbits, intravenous.
A. pyogenes var. aureus. Rabbit, intravenous.
Sarcina tetragena. Guinea pigs and white mice, subcutaneous.
Bacterium anthracis. Guinea pigs or rabbits, subcutaneous.
Bacterium cholerae. Rabbits and pigeons, subcutaneous.
Bacterium pneumoniae. Rabbits and mice, subcutaneous.
Bacterium pneumoniae. Rabbits and mice, subcutaneous with sputum.
Bacterium pneumonicum, Mice and young rats, intraperitoneal.
Bacterium tuberculosis. Guinea pigs, rabbits and field mice, subcutaneous or intraperitoneal.
*For small animals a muffle furnace does very well.
306 MEDICAL BACTERIOLOGY
Bacterium mallei. Male guinea pigs, intraperitoneal.
Bacterium diphtheriae. Guinea pigs, rabbits and fowl, subcuta- neous and intratracheal,
Bacillus pestis. Rats, mice, guinea pigs and rabbits, subcuta- neous.
Bacillus Salmon. Rabbits and mice, subcutaneous.
Bacillus tetant. Guinea pig, subcutaneous.
Bacillus tetant. White rat with garden earth.
Bacillus Welchit. Rabbit, intravenously, and then kill in 3 minutes. See p. 352.
Bacillus Welchii. Guinea pig, subcutaneous.
Microspira Metschnikovi. Pigeons, subcutaneous.
MEDICAL BACTERIOLOGY 307
-- SUBMAXILLARY
MEDIASTINAL Gras AND BRONCHIACS_ GLANDS GLANDS \ Se eee DEEP CERVICAL LUNGS ~.__ GLANDS LEFT LAID : ON RIGHT “g{4 @-fL-- 7------- AXILLARY AND SUBSCAPULAR GLANDS GALL s2=---=5 ===------ LIVER BLADDER ©-4--}------- ADRENAL GLANDS KIDNEY ===" 7-77" "FTN THM 4 wan. SPLEEN LUMBAR~____ "=~. RETRO PERITONEAL GLANDS 7 | GLANDS iiss Cop aeeaee SUPERFICIAL DEEP INGUINAL INGUINAL GLANDS GLANDS
BLADDER -~~
Fie. 37. Diagram showing method of making autopsy on guinea pig; ana also the most important glands (adapted from Delepine & Curtis).
308 MEDICAL BACTERIOLOGY
BLANK FOR ANIMAL EXPERIMENTS
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Autopsy findings:
MEDICAL BACTERIOLOGY 809
Bacteriological examination:
Histological Examination:
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310
MEDICAL BACTERIOLOGY
EXERCISE 98. PREPARATION OF TISSUE FOR EXAMINATION.
Portions of the diseased tissue, removed at autopsy, should be eut into cubes having edges about 5 mm. long and treated as follows: 1). Frxine. Use 15 or 20 times their volume of 95% alcohol
for 24 hours.
them near the top and the alcohol changed after 3 or 4 hours.
The specimens should be placed on cotton to keep
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.
| u. Absolute alcohol 6-24 hours.
| b. Xylene 6-24 hours.
c. Paraffin melting at 50° C. and kept in an oven or water-bath at a temperature a few degrees above the melting point of the paraffin 3-12 hours.
d. Embed. Pour melted par- afin into a paper box or other suitable receptacle and with warm forceps arrange block of tissue in proper position and cool rapidly by plunging into cold water.
|
3). SECTIONING.
B. Celloidin Method |
a. Mixture of ether and absolute alcohol (equal parts) 24 hours.
|
b. Thin celloidin (about 6%) 24 hours to several weeks.
| ce. Thick celloidin (about 12%) 24 hours to several weeks.
d. Remove block of tis- sue to a piece of wood fiber covered with thick cel- loidin, orient, dry a few minutes in air, then place in 80% aleohol for 6-24 hours.
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:
iC;
Freezing Method. uw. Place in 1% formalin 2 hours.
I
hb. Place tissue on
plate of freezing mi- crotome in water, or, better, first soak tis- sue in a syrupy so- lution of gum arabic and moisten plate with same _ before freezing.
|
Cut sections from 10-12 p thick.
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 sections are more secure if the cover-glasses are first smeared with a thin coat of
312 MEDICAL BACTERIOLOGY
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 be removed; this is done with xylene, and this in turn with absolute aleohol.
Rererences. A. 182; M. & W. 204-239; N. 531,
EXERCISE 99. STAINING SECTIONS. GENERAL HistoLogicaAL METHop. Hematoxylin and Eosin. a. Transfer sections from alcohol to distilled water. b. Stain in alum-hematoxylin 5 minutes. The stain may be prepared as follows (Boehmer) :
1. Hematoxylin crystals, - 1 gram. Absolute alcohol, 10 ee.
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. Acid aleohol 5 to 10 seconds.
d. Ammonia water (114%) until sections are a light blue.
e. Wash in water.
f. Counter-stain with eosin (#, to 144% in 60% alcohol) 3 min-
utes.
. Aleohol, 95%, two or three changes to dehydrate and remove excess of counter-stain. h. Clear in oil of origanum, or in Dunham’s mixture (white oil of thyme 4 parts, oil of cloves 1 part). 1. Balsam.
S
GENERAL BACTERIOLOGICAL METHODS.
A. Loeffler’s Universal Method. a. Take sections out of aleohol and place in Loeffler’s methylen blue for 5 to 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.
314 MEDICAL BACTERIOLOGY
e. Mount in balsam.
B. Weigert’s Method. a. Lithium carmine (carmine 3 gms., saturated aqueous solution of carbonate of lithium, 100 ec., a erystal of thymol, filt- ered), 5 minutes.
. Acid alcohol, 15 seconds.
Wash in water.
. Transfer to slide and blot.
. Ehrlich’s anilin water gentian violet 3 minutes.
. Blot.
. Place in potassium iodide and iodine solution (iodine 1 part, potassium iodide 2 parts, water 100 parts) 2 minutes.
h. Blot.
4. Decolorize in a mixture of anilin oil 2 parts and xylene 1 part,
2 to 5 minutes. j. Blot. k. Mount in balsam.
‘Qme ase
This stain can only be used with those organisms which take the Gram stain, namely: Str. erysipelatos, M. pyogenes var. albus, M. pyogenes var. aureus, Sar. tetragena, Bact anthracis, Bact. pneu- moniae, Bact. rhusiopathiae, Bact. tuberculosis, Bact. leprae, Bact. diphtheriae, Ps. aeruginosa, Bact. Welchii, B. Fesert, B. oedematis, B. tetani and Streptothriz bovis.
SprecraL BACTERIOLOGICAL METHODS.
Particular organisms may be stained as follows:
Pus micrococci. Loeffler’s or Weigert’s method.
Micrococcus gonorrhoeae. Loeffler’s method gives the best re- sults.
Sarcina tetragena. Loeffler’s or Weigert’s method.
Bacterium anthracis. Loeffler’s or Weigert’s method.
Bacterium pneumoniae (Pneumococeus). Weigert’s method.
Bacterium pneumonicum (Friedlander’s bacillus). The follow- ing method is recommended for staining the capsules in sections
(M. & W.):
a. Stain for 24 hours in the incubator in the following solution: Saturated alcoholic solution of gentian violet 50 ee. Distilled water - oe 100 ec. Glacial acetic acid 10 ec.
b. Wash out in 1% solution of acetic acid. c. Alcohol.
316 MEDICAL BACTERIOLOGY
d. Xylene. e. Canada balsam.
Bacterium cholerae (chicken cholera). Loeffler’s method.
Bacterium tuberculosis.
a. Weigert’s method (staining with anilin oil gentian violet 24
hours at room temperature, or 2 to 3 hours at 40° C.).
b. Ziehl-Neelsen’s Method. 1. Stain with carbol-fuchsin (12 to 24 hrs. room temper-
ature, 1 to 3 hrs. 40° C.).
2. Decolorize with nitric acid (10%) a few seconds, and then with alcohol (60 to 90%) until color is nearly all extracted.
3. Counter-stain with methylen blue.
4. Dehydrate with absolute aleohol (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 sections 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:
a. An aqueous solution of fuchsin 6 to 7 minutes.
b. Acid aleohol (nitric acid 1, aleohol 10) 14 minute.
c. Wash in water.
d. Counter-stain in a saturated aqueous solution of methylen blue.
e. Alcohol.
f. Xylene.
g. Balsam. The bacteria of leprosy stain readily by this method, tuber- cle bacteria do not.
Bacterium mallet.
Slow Method. a. Stain in Loeffler’s methylen blue 6 to 8 hours. b. Wash in distilled water. ce. Tannic acid solution (10%) 4 to 5 hours. d. Wash thoroughly in water. e. Dehydrate in absolute alcohol. f. Clear in xylene and mount. Quick method. a. Stain in carbol-methvlen blue 10 to 30 seconds.
318 MEDICAL BACTERIOLOGY
o>
. Wash in distilled water. Tannic acid solution (10%) 1% to 1 minute.
. Counter-stain with a weak solution of eosin until sections are red. e. Wash in water until pink. f. Dehydrate in absolute alcohol. g. Clear in xylene and mount.
Bacterium diphtheriae. Loeffler’s or better Weigert’s method.
Bacillus typhosus. a. Loeffler’s methylen blue or carbol-fuchsin 15 min. to 24 hrs. b. Wash slightly in distilled water. c. Place in 30% solution of tannic acid for 10 to 60 min. d. Dehydrate rapidly in alcohol. e. Clear in xylene. f. Examine. g iS)
as
. Mount in balsam. uch sections examined under a low power will be found to con- tain 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 Salmonti (hog cholera). Loeffler’s method. Bacterium Welchit (gas bacillus). Weigert’s and Loeffler’s methods. Bacillus Feseri (symptomatic anthrax). Use Pfeiffer’s stain: a. Dilute earbol-fuchsin 14 hour. b. Absolute alcohol slightly acidulated with acetic acid until section is a reddish violet tint. c. Xylene and examine. d. Mount in balsam. Bacillus oedematis (malignant oedema). Pfeiffer’s stain. Streptothriz bovis (actinomyces). . Ziehl’s carbol-fuchsin, 10 minutes. . Wash in distilled water. Pieric acid (cons. ale. solution). . Wash in distilled water. Wash in aleohol (50%). Dehydrate in absolute alcohol. . Clear in xylene. . Balsam. Tissue stained yellow, rays red. REFERENCES. M. & W. 239-286; N. 537.
FTQ re aawa
320 MEDICAL BACTERIOLOGY
CHAPTER IX
BACTERIOLOGICAL DIAGNOSIS
EXERCISE 100. 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, submaxillary and sublingual glands. In disease the normal character of the different parts may vary, or there may be various exudates and growths present.
CoLtLectTion. Material for bacteriological examination is best obtained by means of a sterile probang or by forceps. This material may be examined directly by means of cover-glass preparations or by means of cultures.
a. Method of Preparing Outfit. Wind asmall piece of absorbent cotton on the end of a wire (about 1 mm. in diameter and 14 cm. 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 1 hour. This with a tube of nutrient medium (usually Loeffler’s Blood serum) is placed in a box for transportation. Fig. 38.
b. 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 li
tubes securely stoppered and the outfit sent to the ee laboratory. theta Gulie
Tube 1 is a ster- ile swab; 2 is a
OrGANISMS CoMMONLY Founp. blood serum Bacterium diphtheriae.
The presence of this germ in the mouth usually results in the 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 be examined directly for the diphtheria bacillus by smearing on a cover-glass and staining by following methods:
322 MEDICAL BACTERIOLOGY
a. Loeffler’s methylen blue, or Roux stain.?
b. Gram’s stain.
c. Neisser’s stain: a. 1 gram methylen blue dissolved in 20 ec. of aleohol (96%), is added to 950 ce. of distilled water and 50 ec. of glacial acetic acid; b. 2 grams of bismarck brown dissolved in a liter of distilled water. Films are stained in a. 5 to 8 seconds, washed in water, stained in b. 3 to 5 seconds, dried and mounted. The Crouch? stain may be similarly employed.
Usually, however, mere microscopical examination is not suffi- cient, 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 to 38° C. for 12 to 24 hours and then examine the growth in cover-glass preparations. The diph- theria organism if present, should show:
a. Characteristic appearance with Loeffier’s methylen blue.
b. Positive Neisser stain. ,
c. Positive Gram stain.
Occasionally micro-organisms (pseudo-diphtheria bacilli among others) are met with that very closely resemble the Klebs-Loeffler bacillus and render a positive diagnosis doubtful. In such cases attention to following table will be helpful:
*Roux stain—Solution A: Dahlia 1, alcohol 10, and distitled water 90 parts; Solution B: Methyl green 1, alcohol 10, and distilled water 90 parts; mix 2 parts of A with 1 of B.
2 Crouch stain— Aqueous solution of dahlia (1%) 1 part, aqueous solution of methyl green (1%) 5 parts, and distilled water 4 parts.
324 MEDICAL BACTERIOLOGY
B. diphtheriae B. pseudo diphtheriticum 1) Form Slender, and of same di-| Thicker at center than ameter throughout ends, plumper, shorter and less variable than B. diphtherize 2) Size Average 1.2 to 2m Averaging 1 to 1.6 w& 3) Threads Not formed Not formed 4) Grouping Parallel grouping more or} Parallel but lie closer to- less characteristic but do gether not touch 5) Involution forms Common Rare 6) Motility Immotile Immotile 7) Stains a. Loeffler’s methylen Stains readily giving band- | Stains more regularly blue ed or polar stain Polar stain rare b. Gram. Positive Positive ce. Neisser Characteristic stain with | Not under 24 hours very young cultures, six hours 8) Spores Absent Absent 9) Alkaline potato Growth almost invisible Visible and cream colored 10) Sugar agar and gela- in 2 days tin stab cultures Full length of stab Only at upper part 11) Neutral litmus milk Acid reaction Alkaline reaction 12) Dextrose bouillon Acid reaction Alkaline reaction 18) Anaerobic cultures in H. Grows well No growth 14) Nitroso-indol reaction | After 7 days After 21 days. 15) Inoculation expert- ments (Guinea pig subcutaneous) Death 36-48 hours Non-pathogenic
Pus Micrococci. (Str. erysipelatos, M. pyogenes var. aureus and albus, Sar. tetragena. )
a. Stained cover-glass preparations are to be examined, and if micrococci are found make:
b. Smear cultures, or better, agar plate cultures and work up the- colonies as they appear.
Monilia candida (Organism of Trush).
The material is collected by removing a portion of the patches or membrane and examining it:
a. Under the microscope in a drop of glycerine.
b. Cover-glass preparations stained with carbol-fuchsin or Gram’s method.
c. By means of smear cultures on agar or blood serum, the result- ing growth being examined either in glycerine mounts, or stained cover-glass preparations.
REFERENCES. Em. 48; v. J. 95; Si. 122. See also various texts under special organism.
326 MEDICAL BACTERIOLOGY
EXERCISE 101.—EXAMINATION OF SPUTUM.
DerFinition. By this term is meant all of the material derived from the air passages by the act of coughing or hawking.
Metuop of CoLLEcTIoN. For diagnostic purposes it is best col- lected 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.
OrGANISMS Most ComMMONLY FOUND.
Bacterium tuberculosis. Place the sputum in a Petri dish over a black surface and select one of the little cheesy masses, if these be 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:
a. Gabbett, see Part 1, p. 62. b. Ziehl-Neelsen :
1. Carbol-fuchsin ten times through the flame (5 to 10 min.),
2. Nitric acid (30%) momentarily.?
3. Water.
4, Aleohol (60%) until red color disappears. It may be necessary to immerse preparation in acid a second time, but care must be exercised to prevent extraction of dye from tubercle bac- terium.
5. Loeffler’s methylen blue, 1 minute.
6. Mount and examine.
While the tubercle bacteria may be detected when present in considerable numbers with a 1-6-inch objective, when there are few present, a 75-inch oil immersion will be necessary, 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 conven- ience in a systematic search.
At least two preparations should be stained and thoroughly exam- ined before a negative result is pronounced.
The viscosity of sputa may be overcome and the bacteria con- centrated, where the number 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 de- posited with the sediment. This sediment can be obtained by allow-
*xRavenel recommends use of 5% nitric acid in 80% alcohol, claiming that there is no danger of decolorizing the tubercle bacilllus no matter how long the contact.
328 MEDICAL BACTERIOLOGY
ing the mixture to stand in a conical glass vessel or, more quickly, by the use of a centrifuge. 2) Hammond’s method:
a. Add 5% of crystallized carbolic acid (in the case of sputum add 5 times its bulk of a 5% solution of carbolic acid).
b. Place 15 ec. in the tubes of a centrifuge and whirl for 15 minutes.
c. Pour off supernatant fluid and treat precipitate with 3 cc. of a 5% KOH solution. Mix thoroughly and allow to stand 2 minutes.
d. Fill to 15 ec. mark with distilled water and whirl 20 minutes.
e. 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 addition of a small quantity of carbolic acid (5%).
Negative results are of positive diagnostic value only when re- peated examinations are made of different samples taken at different times.
REFERENCES. Em. 75.
Bacterium influenzae. This micro-organism is frequently pres- ent 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 with distilled water 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:
1) Microscopically by staining, with a weak carbol-fuchsin, smears from the purulent 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. REFERENCES. Em. 64.
Bacterium pneumoniae (pneumococcus). The sputum of patients suffering from pneumonia is usually of
330 MEDICAL BACTERIOLOGY
a rusty color, due to presence of blood (rusty sputum). The ‘‘pneu- mococeus’’ is readily seen in such material when stained by Gram’s method or with earbol-fuchsin and momentarily washed with alco- hol, as lancet-shaped organisms with outer ends pointed and sur- rounded by a clear area—the capsule. The capsule can be easily stained by Welch’s method. (See 27.)
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 is most susceptible and should be inoculated subcutaneously. Asa result of infection with this organ- ism the animal dies quickly with a typical septicemia, the micro- organisms 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 meth- ods of detection see 105.
Streptothrix bovis (actinomyces). This organism has been occa- sionally found in sputum and in such eases the peculiar morphology of the colonies is well brought out by Gram’s method. See 105.
REFERENCES. v. J. 114; Si. 245. See also various texts under particular organisms.
EXERCISE 102. EXAMINATION OF BLOOD.
CoLLECTION. 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 recepta- cles and hermetically sealed. For this purpose Sternberg’s bulb is excellent. The skin should first be sterilized by use of corrosive sub- limate or carbolie acid followed with alcohol.
It is usually well in any case to make cover-glass smears at the bed-side for microscopical 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 place a second cover-glass on this; this flattens the drop of blood out into a thin film. Immedi- ately and before coagulation 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 an- thrax, blood or portion of spleen should be removed with least. pos-
332 MEDICAL BACTERIOLOGY
sible danger from infection or distribution of bacilli and studied as follows:
a. Microscopical examinations of blood or of the spleen pulp of animals show (when stained with Loeffler’s methylen blue) large bacteria in chains (5 or 6 segments) presenting the bamboo appear- ance.
b. In hanging drop preparation large, homogeneous, immotile bacilli.
c. Agar plate cultures should also be made, and, from the separate colonies, subcultures; the gelatin stab being especially characteristic.
d. In important cases (as in man) guinea pigs, or white mice, should be inoculated, and, in case of death, organism isolated and identified.
Spirochaeta Obermeiert (relapsing fever). This organism is found in the blood only during a paroxysm. It is a long slender organism 6 or 7 times the diameter of a red blood corpuscle (45z). It has a brisk, vibratile movement in the direction of its long axis, and is very sensitive to reagents of all kinds. Even the addition of distilled water will cause it 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.
f. Examined.
Pus Micrococcit. These are occasionally found, and for method of detection see 105.
Bacteriwm mallet. Sometimes found in the blood of those suffer- ing with glanders. It may be detected in the blood-smears. For special methods see 105.
Bacterium pnewmoniae (pneumococeus). This germ is frequent- ly present in fatal cases 24 to 48 hours before death. The blood should be drawn with a sterile hypodermic syringe and about 1 ee. of blood mixed with a tube of melted agar at 43° C. and poured into a Petridish. Characteristic colonies appear in 24 to 48 hours,
334 MEDICAL BACTERIOLOGY
Bacterium tuberculosis. In case of miliary tuberculosis they may be very rarely found in sufficient numbers to be detected by staining methods, see sputum 101.
Bacterium influenzae. Canon claims to have stained and culti- vated this organ in blood, but this needs confirmation.
Bacillus col. This organism may be found in the blood. For methods of isolation and identification see feces 103.
Bacillus pestis. This germ occurs in the blood, in certain eases at least. Considerable skill in detecting it is required—due to its variable appearance. Broth tubes should be infected and animals inoculated. ; Bacillus Salmoni (hog cholera).
a. Make agar plate and streak cultures from spleen of dead ani- mal, 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 from 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 +, 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 ce. of water ; to remove hemoglo- bin, wash with water and stain with following solution for 1%4
minute : Borax ee 5.0 parts. Methylen blue - ee ee ee eee 0.5 parts. Water - 2 - = = = - = = + = 100 parts.
Wash, dry and mount in balsam (Manson).
REFERENCES. v. J. 45; Si. 100. See also texts under particular organisms.
Wipau Reaction. Dried blood method. This method is espe- cially valuable where patient is some distance from the laboratory. Collect the blood as follows: ‘‘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 needle,’? Remove two or three large drops of blood on a clean glass slide, aluminum 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:
336 MEDICAL BACTERIOLOGY
a. Make a hanging drop preparation from a 24 to 72-hour old agar, or bouillon, culture of Bacillus typhosus.
b. If the bacilli be 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.
c. Examine with a high dry power (1-6 in obj.) rather than with the oil immersion.
The dilution is made in the following way: Nine drops of sterile water are placed around the drop of dried blood. (The drops of water should be of about the same size as that of the original drop of blood.) The drops are all mixed together and allowed to soak up the blood for about two minutes. In this way an approximate dilu- tion of one to ten is obtained. One drop of this is added to the hanging-drop culture. This gives a dilution of one to twenty which is the one usually employed.
More exact dilutions of dried blood may be made by weighing out the blood and adding it to a measured amount of water.
Where possible the blood should be collected so that the clear serum may be separated and used for the test. This can be done in hospital work and wherever it is possible to get the blood to the laboratory a few hours after it is collected. For this purpose a glass
pipette is prepared by drawing out a glass tube, as indicated
in Fig. 39, which represents the pipette natural size. The
skin is cleaned and the blood drawn as indicated above and
when a large drop has collected on the skin one of the points
of the pipette is introduced when the blood is drawn up by
capillary attraction. The bulb ought to be about one-half
filled. The pipette is then placed in a horizontal position
until the blood has clotted, when it may be taken to the
laboratory. It should then be placed in the ice chest, still
in a horizontal position, for two or three hours. The end
which was used to draw up the blood is then scratched with
a file and broken off. By holding the tube in a vertical
Fre.39, Position the clear serum may now be dropped from the
viges opposite end: into a glass or porcelain capsule. The clear
serum is then taken up with a clean capillary pipette and a
drop placed in another capsule and then after rinsing out the same
pipette is used to add the requisite number of drops of bouillon or
salt solution to make the required dilution. The test is then made in exactly the same way as described for the dried blood.
338 MEDICAL BACTERIOLOGY
In a typical reaction the motility is almost immediately affected, and soon motion ceases altogether while the bacilli collect in clumps,
Fig. 40. Widal Reaction. I. B, typhosus before adding typhoid blood; II, A typical reaction.
4. e., become ‘‘agglutinated.’’ (Fig. 40.) The usual time limit is thirty minutes when the dilution is 1 to 50.
REFERENCES. v.J.45; 81.100. See also texts under particular organism.
EXERCISE 103. EXAMINATION OF FECES.
The material expelled from the rectum and comprising the sub- stances from the food and the secretions of the alimentary tract come under this head. The number of micro-organisms occurring here is enormous, and comprise a large number of species and among them several pathogenic forms particularly B. typhosus, Msp. comma, Bact. tuberculosis and Amoeba coll.
Bacillus typhosus. This organism occurs in the feces in the case of typhoid patients; but on account of the large number of other organisms its detection is very difficult. The following methods are the most serviceable :
A. Parterti’s MetHop. This method consists in adding Parietti’s solution (carbolic acid 5 grams, hydrochloric acid 4 grams, and dis- tilled water 100 ce.) to bouillon in the following manner: A num- ber of tubes of bouillon have a varying quantity of the above solu- tion added, e. g. 1 drop to one tube, 2 to another, 3 to another, and soon. These tubes are inoculated with a small quantity (one or two loops), of the feces and then placed in the 38° C. incubator. Twen-
340 MEDICAL BACTERIOLOGY
ty-four hours later the tube containing the largest amount of Pari- etti’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 their agglutinating power on typhoid blood. Instead of the lactose litmus agar one of the following media may be used:
B. Hiss’ Puate Mepium.’
10 grams 25 grams 5 grams 5 grams 10 grams
This contains: of agar.
of gelatin.
of beef extract (Liebig). of sodium chloride.
of dextrose.
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, dextrose added and the medium tubed and sterilized.
Make plate cultures in ordinary way and incubate at 38° C. for 18 hours, then examine the colonies microscopically. The colonies of B. typhosus have irregular outgrowths and fringing threads. The colonies of B. colt, on the other hand, are much larger and as a rule are darker in color and do not form threads.
The colonies may be further examined by the use of Hiss’ Tube
Medium.
5 grams 80 grams 5 grams 5 grams 10 grams 1000 grams
of agar-agar.
of gelatin.
of beef extract (Liebig). sodium chloride. dextrose.
water.
Made as plate medium except that it 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. C. Meprum or MAcConkKEy, as modified by Griinbaum.’
1 Jour. Exp. Med. 1897, 2: 677. 2 Brit. Med. Jour. 1902, Pt. 1, p. 1473.
342 MEDICAL BACTERIOLOGY
Twenty grams each of agar-agar and peptone are dissolved in one liter of boiling water, and the whole made alkaline by adding 4 cc. of a normal solution of sodium hydrate after having first brought the reaction to the neutral point of litmus.
Then add, while hot,
Sodium taurocholate - - - - - - - - - 5 grams. Lactose - - - - - - - - 10 grams. Neutral Red (5% watery solution) - - - - - 10 c¢
When the solution is complete the mass is filtered through cotton, tubed, and sterilized in the steam sterilizer once for twenty-five or thirty minutes.
D. Mep1um or DrIgALskI AND CoNRADI.'
To two liters of sugar-free broth add:
Peptone (Witte) 10 grams Nutrose - 10 grams ‘ Sodium chloride 10 grams these amsy be omitted
and dissolve by the aid of heat. The mixture is brought to the boil and sixty grams of agar-agar added, and the mixture kept boiling until the agar is dissolved. Then the reaction of the mass is made weakly alkaline to litmus by the addition of sodium hydrate (4% sol.) and filtered.
This being done, a mixture of litmus solution (6%) and lactose (¢. p.)
Litmus sol. - - - - - 260 ec. Lactose - - se ee - 30 grams.
is added while both solutions are hot, and ne whole boiled gently for five minutes. Then add a solution of water-free sodium carbonate (10%) in the proportion of 4 ce. (this may be omitted) followed by 20 ec. of a fresh solution of crystal violet (Griibler’s)—0.1 gram in 100 ee. water—tubed, and sterilized in the steam sterilizer for 20 minutes on three successive days.
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 (Asiatic cholera).
1. Microscopal 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
1 Zeit. f. Hyg., 1902, Heft ii, p. 283.
344 MEDICAL BACTERIOLOGY
of peptone water (Dunham’s solution) and incubated at 38° C. The surface growth 6-12 hours later is to be examined microscopi- cally 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.
B. dysenteriae. This organism has been isolated from the feces of dysentery patients by numerous investigators and from children suffering from summer diarrhoea by Duval & Bassett’? and others. The following method is recommended:
Agar plates are made from the bloody mucus in the feces or from scrapings of the ulcerated mucosa of the intestines. Agar plates are made and incubated at 38° C. for 12 hours and then the colonies which have appeared are marked with a pencil or pen and then the plate is incubated for several hours longer. The colonies which appear later are most likely to be colonies of B. dysenteriae. The suspected colonies are then put into dextrose agar and only those which fail to produce gas are tested farther. The crucial test is the Widal reaction which can be made with blood obtained from the patient or cadaver.
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.
Ameba coli.
a. A drop of the mucus portions of stool is placed on a glass slide, covered with a cover-glass and examined with a magnification of about 500 diameters (1-6 in objective). Examination should be conducted on a warm stage in order to get ameboid movements.
b. Preparations may be stained with methylen blue and carmine. The nucleus is stained with carmine.
c. Discharges may be hardened and stained by Mallory’s method as follows:
1. Fix tissues in aleohol.
2. Stain (paraffin) sections in a saturated aqueous solution of thionin for 5-20 minutes.
3. Wash in water.
4, Differentiate in a 2% aqueous solution of oxalic ocid 14-1
minute.
5. Wash in water.
1Duval and Bassett, Amer. Med., 1904, 4: 417.
346 MEDICAL BACTERIOLOGY
6. Dehydrate in aleohol (95%).
7. Clear in oil of bergamot.
8. Wash with xylene and mount in balsam.
Nuclei of Amebae brownish red, other nuclei blue.
REFERENCES. v. J. 199; Si. 206. See also texts under various organisms.
EXERCISE 104. 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, 101.
It is best to centrifuge the product and care must be taken to differentiate from the smegma bacterium. For this pur- pose stain cover-glass smears as follows (Bunge & Fran- teroth) :
. Absolute aleohol, 3 hours.
. Chromic acid, 15 minutes.
Stain in hot carbol-fuchsin.
. Decolorize in sulphuric acid (25%) 2-3 minutes.
Counter-stain with a saturated alcoholic solution of methylen
* blue.
The smegma bacillus is decolorized by this method.
The 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.
Pus Micrococct. 105.
Micrococcus gonorrhoeae. 105.
Bacillus typhosus. 108.
Spirochaeta Obermetert (relapsing fever). 102.
a
sao se
REFERENCES. v. J. 273; Si. 500, and texts under the various organisms.
EXERCISE 105. 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,
348 MEDICAL BACTERIOLOGY
a. If staphylococci alone are present search for the Pus Coceus Group. b. If streptococci suspect Str. erysipelatos. e. If diplococei or tetracocci.
1, Within the pus-cells test for M. gonorrhoeae or M. Weichsel-
baumii.
2. Free suspect Sar. tetragena.
d. If bacilli any of the following may be searched for:
1. B. coli. This organism is likely to be found especially in suppu- tative peritonitis and diseases of the urinary organs. 2. Bact. anthracis. 3. Bact. pneumoniae. 4. Bact. tuberculosis. 5. Bact. leprae. 6. Bact. mal- lei. 7. B. pestis. 8. Ps. aeruginosa. 9. Bact. Welchii. 10. B. oedematis. 11. B. tetani. :
e. Streptothrix bovis.
f. Ameba coli.
Pus Micrococci. These organisms are frequently present in pus and should be isolated and identified in pure cultures, as micro- scopical examinations alone will not suffice.
Streptococcus erysipelatos. 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. When once dried it should not be wet or moistened again as this would destroy the pus-cells, and hence the value of the material for diag- nosis.
a. Simple stain.
1. Loeffler’s methylen blue 3-5 minutes.
2. Wash in water.
3. Dry, mount in balsam and examine with +, in. oil immer- sion.
4. Look for a biscuit-shaped diplococeus within the pus cells.
b. Gram’s method.
. Anilin oil gentian violet 15 minutes.
. Wash in water.
. Treat with iodine solution 2 minutes.
. Decolorize with alcohol.
. Counter-stain with eosin, 4% minute.
. Wash, dry and mount in balsam.
Examine with oil immersion.
If the gonococci be present they will be stained brown.
If diagnosis be of great importance make cultures as follows:
aor wD Ht
oS
a. Make 6 or more streak cultures on blood agar, or better, make plates on Wertheim’s medium (p. 158). Grow at 38° C.
350 MEDICAL BACTERIOLOGY
b. 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 gonococeus is the only organism that:
1. Oceurs 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. Micrococcus Weichselbaumii (M. 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. The needle (4 cm. 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 gen- erally 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. At a depth of 3 or 4 em. in children and 7 or 8 in adults the needle enters the subarach- noid 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 on 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.
Microceus Weichselbaumii stains by Loeffler’s method and ap- pears 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 meth- ods of diagnosis see exercises indicated.
Bacillus coli. 1038,
Bacterium tuberculosis. 101.
Bacterium leprae. For method of staining, see 99.
Bacterium pneumoniae. Stain for capsule. Cultivate on blood- agar. 101.
Bacterium mallet.
a. Widal reaction. (If in man, typhoid and diphtheria must be excluded in ease of a positive reaction. )
352 MEDICAL BACTERIOLOGY
b. Examination of discharge.
1. Microscopical examination usually without result. 2. Cultures, glycerine agar and potato from pus.
c. Animal inoculations, Straus’ method.
Bacillus 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.
Pscudomonas aeruginosa (B. pyocyaneus). Easily recognized by its culture characters.
Bacterium Welchii (gas bacillus).
This germ is non-pathogenic for rabbits, but Welch and Flexner have shown that if a rabbit be inoculated intravenously with 0.5 to 1 ee. 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.
Bacillus oedematis (B. malignant oedema).
a. Make cover-glass preparations from fluid of affected parts.
b. Also make anaerobic cultures. If material contains spores it should be heated to 80° C. for 10 minutes before it is seeded.
Bacillus tetane.
a. Make cover-glass preparations from pus and search for drum- stick bacillus.
b. Make dextrose bouillon and agar-plate cultures and develop in hydrogen.
c. Inoculate animals with the discharge, and also with the bouil- lon culture, and watch for characteristic symptoms.
Streptothrix bovis (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.
Ameba coli. 108.
REFERENCES. v. J. 405; Si. 514 and 518. See also texts under the various organisms.
304 MEDICAL BACTERIOLOGY EXERCISE 106. DIAGNOSIS OF RABIES.
A. Microscopical Diagnosis.
a. The head of the animal is opened and the brain removed. In case the animal is some distance from the laboratory it is best to cut off the head, pack in ice and ship by express.
b. Thin pieces of the various parts of the brain, such as Ammon’s horns, cerebellum, cerebrum and medulla are fixed in equal parts of formalin and 95% aleohol for 12 to 18 hours. They are then treated as follows:
. 95% aleohol, 14 hour.
. 95% aleohol again for 1% hour.
Anilin oil until clear; one hour is usually sufficient. . Xylol 15 to 30 minutes.
. Melted paraffin, 214 hours.
. Cut to 3 or 4 microns.
aur wn re
7. Stain in hematoxylin and eosin.
Cell inclusions known as negri bodies, which are usnally found most abundant in Ammon’s horns, are the evidence of rabies.
The medulla is searched for infiltrations around the blood ves- sels. These changes are found in a few conditions other than rabies.
B. Smear Method.
a. Small pieces of the various parts of the brain are crushed out between two clean slides and treated as follows:
1. Fix the smears while still wet in methyl aleohol (neutral- ized with sodium carbonate) to which 4, % of picric acid has been added. Blot off excess of fixative.
2. Stain smears as follows: Saturated alcoholic solution of fuchsin, 3 ee. Saturated alcoholic solution of methylene blue, 2 ce. Distilled water, 30 ee.
Heat the slide over a flame until it steams, wash in tap water and blot. The stain does not keep long.
Williams, Amer. Jour. of Public Hygiene, Feb., 1908.
356 MEDICAL BACTERIOLOGY
The negri bodies are often found outside of the cell and can be recognized by their form, color, and internal structure.
C. Pasteur’s Method.
a. The medulla of the suspected animal is removed under aseptic precautions, as soon as possible after death.
b. Place a piece of the medulla about the size of a pea, in 4 or 5 ee. of sterile bouillon and thoroughly grind up the same.
c. Anesthetize 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 subecutan- eous tissue along the median line, while a crucial incision is made through the periosteum on one side of median line thus avoiding hemorrhage from the longitudinal sinus. The periosteum is then pushed back and a dise of the skull (14 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, 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 107. EXAMINATION OF MATERIAL FROM HUMAN 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. In all cases the sur- face 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.
358 MEDICAL BACTERIOLOGY
CHAPTER X
DETECTION OF PATHOGENIC BACTERIA IN WATER AND MILK SUPPLIES
EXERCISE 108. EXAMINATION OF WATER FOR PATHOGENIC BACTERIA.
It is rarely necessary to test water directly for either the typhoid or cholera organisms, as there is little chance of their being found except in the most grossly polluted waters. What is usually sought for is evidence of sewage pollution. If this is found the water is not regarded as potable. The more common methods of detecting fecal bacteria have already been given (Chapter V). The follow- ing methods are reliable and the detection of these germs in artifi- cially infected waters furnish most excellent practice for the student.
Bacillus typhosus. In the examination of water it is best to con- centrate the bacteria by filtering a large amount of the water through a Berkefeld filter and to use the slime on the filter to make the plates.
a. Parietti’s method, see 103.
b. Hiss’ method. Make plate cultures and incubate at 38° C. for 18 hours. Inoculate suspicious colonies into Hiss’ tube medium, fermentation tube and milk. Also make indol test and 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.
Microspira comma.
a. If there be a reason to believe that the spirilla are very numer- ous gelatin plate cultures can be made directly from the water, and the suspicious colonies worked up.
360 MEDICAL BACTERIOLOGY
b. Ordinarily the organisms are very sparse and large quantities must be used. 100-1000 cc. 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.
Here and in typhoid the agglutination of the germ with great dilution of a high potency serum is the crucial test.
Bacterium anthracis (Robert’s Method.)
a. Heat suspected water to 80° for ten minutes to kill water bacteria.
b. Make plates in agar and in gelatin and work up colonies.
c. Inoculate a guinea pig with several cubic centimeters of the water.
REFERENCES. Horrocks and Prescott & Winslow.
EXERCISE 109. EXAMINATION OF MILK FOR PATHOGENIC BACTERIA.
Bacterium diphtheriae.
Where Bacterium diphtheriae is suspected in milk, make a con- siderable number of streak cultures on Loeffler’s blood serum and incubate at 38° C. from 8 to 12 hours, stain and examine micro- scopically.
Bacterium tuberculosis. .
Hammond’s method of examining milk for B. tuberculosis. See Sputum, 101.
Animal Inoculation.
USE OF MAIL FOR TRANSMISSION OF BACTERIA.
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, 1893, forbidding the use of mails for the transmission of specimens of germs of cholera or other diseased tissues, is hereby modifiea to this extent: Specimens of diseased tissue may be admitted to the mails for transmission to United States, State or munic- ipal laboratories only when inclosed in mailing packages constructed in accordance with the specifications hereinafter enumerated. Upon the outside of every package shall be written or printed the words: ‘Specimen for Bac- teriological examination.’ No package containing diseased tissue shall be delivered to any representative 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 regulation, to receive such specimens.’’
APPENDIX A
A KEY TO THE IDENTIFICATION OF THE COMMON PATHOGENIC AND A FEW OF THE WELL KNOWN SAPROPHYTIC BACTERIA
This key has been compiled from the works of Migula and Chester, the latter of which contains a very complete key to practically all known bac- teria. To this, as well as to Migula, Sternberg and Kolle and Wassermann
(for the pathogenic bacteria), the student is referred for detailed descriptions of‘the various organisms.
Cells in their free condition globular (cocci). A. Cells without flagella. I. Division in only one direction of space forming chains (streptococci). 1. Grow on gelatin. a) Do not liquefy gelatin. 1) No surface growth in gelatin stabs. Str. erysipelatos Fehleisen. II. Division in two directions of space (micrococci). 1. Grow on gelatin. a) Colonies white. 1) Do not liquefy gelatin. M. Melitinsis Bruce. 2) Liquefy gelatin. M. pyogenes var. albus(Rosenbach)L. & N. b) Colonies yellow, and liquefy gelatin. M. pyogenes var. aureus (Rosenbach)L. & N. 2. Do not grow on gelatin. . M. gonorrhoeae (Baum) Fluegge. M. Weichselbaumii (Trevisan). III. Division in three directions of space (sarcinae). 1. Grow on gelatin. a) Colonies white. 1) Do not liquefy gelatin. Sar. tetragena (Gaffky) Mig. b) Colonies yellow. 1) Do not liquefy gelatin. Sar. lutea Fluegge. Sar. ventriculi Goodsir. 2) Liquefy gelatin. Sar. aurantiaca Fluegge.
Cells short or long, cylindrical, straight, without sheath, endospores present or absent, non-motile (bacteria).
361
362 APPENDIX A
A. Forms endospores. I. Grow at room temperature. 1. Gelatin liquefied. f Bact. anthracis (Koch) Mig. B. Without endospores. I. Aerobie and facultative anaerobic. 1. Grow well on gelatin and do not liquefy it. a) Gram’s stain negative. 1) Gas generated in dextrose media. i) Gas generated in lactose media. Bact. aerogenes (Esch.) Mig. Bact. capsulatum (Sternberg) Chester. ii) Little or no gas in lactose media. Bact. pneumonicum (Fried.) Mig. 2) No gas in dextrose media. Bact. cholerae (Zopf) Kitt. Bact. bovisepticum (Kruse) Mig. (see also B. pestis) b) Gram’s stain positive. 1) Gas generated in dextrose media. Bact. acidi-lactici Hueppe. Bact. phosphorescens (Cohn) Fischer. 2) No gas in dextrose media. Bact. rhinoscleromatis (Trevisan) Mig. 2. Gelatin liquefied slowly. Bact. mallei (Loeffler) Mig. Bact. rhusiopathiae (Kitt) Mig. 3. Do not grow well on gelatin at room temperature. a) Stain with basic aniline dyes but are readily decolorized by mineral acids when stained with carbol-fuchsin. 1) Grow well in bouillon at body temperature and stain by Gram’s method. Bact. diphtheriae (Loeffler) Mig. Bact. pseudodiphtheriticum (Loeffler) Mig. 2) Do not grow in bouillon or on ordinary media. Bact. leprae (Hansen) Mig. 3) Growth very limited on ordinary media. i) Gram’s stain positive. Bact. pneumoniae (Weichsel.) Mig. ii) Gram’s stain negative. Bact. influenzae (Pfeiffer) L. & N. b) Do not stain with aqueous solutions of basic aniline dyes and not easily decolorized by acids. Bact. tuberculosis (Koch) Mig. Bact. tuberculosis var avium (Kruse) Mig. II. Obligate anaerobic. Bact. Welchii Mig.
Cells short or long, cylindrical, straight, without sheath, endospores present or absent, motile, flagella distributed over whole body (bacilli).
APPENDIX A 363
A. Form endospores.
I. Aerobie or facultative anaerobic. 1. Potato cultures irregularly wrinkled. B. vulgatus Trevisan. 4, Potato cultures smooth. B. subtilis (Ehrenb.) Cohn. I. Obligate anaerobes. 1. Rods not swollen at sporulation. B. oedematis Zopf. 2. Rods spindle-shaped at sporulation. B. Feseri (Trevisan) Chester. B. botulinus v. Ermengen. 3. Rods clavate-capitae at sporulation. B. tetani Nicolaier.
B. Spore formation not observed.
I. Aerobic or facultative anaerobic. 1. Gelatin colonies roundish not distinctly ameboid. a) Gelatin not liquefied. 1) Gram’s stain negative. i) Generate gas in dextrose media. * Coagulate milk. § Produce indol. B. coli (Escherich) Mig. §§ Do not produce indol. B. enteritidis Gaertner. ** Do not coagulate milk. B. Salmonii (Trevisan) Chester. B. icteroides Sanerelli. ii) Gas not generated in dextrose media. B. typhosus Zopf. B. dysenteriae Shiga. B. pestis L. & N. b) Liquefy gelatin. 1) Generate gas in dextrose media. B. cloacae Jordan. 2) No gas generated in dextrose media, chromogenic, pig- ment reddish. B. prodigiosus (Ehrenb.) Fluegge.
2. Gelatin colonies ameboid or irregular. a) Do not liquefy gelatin. ‘B. Zopfii (Kurth) Mig. b) Liquefy gelatin. B. vulgaris (Hauser) Mig. Cells cylindrical, straight, without sheath, endospores known in only few species. Actively motile, flagella attached to the poles (pseudomonas).
A. Produce a greenish-bluish fluorescence in the culture media.. I. Gelatin liquefied.
364 APPENDIX A
1. Milk coagulated.
Ps. aeruginosa (Schroeter) Mig. 2. Milk not coagulated.
Ps. fluorescens (Fluegge) Mig.
II. Gelatin not liquefied.
1, Milk rendered alkaline.
Ps. syncyanea (Ehrenb.) Mig. 2. Milk reaction not changed.
Ps. putrida (Fluegge) Mig.
Cells cylindrical, more or less spirally curved, without endospores; actively motile, flagella attached to the poles (microspira). A. Liquefy gelatin. I. Produce indol in 24 hours. 1, Very pathogenic to pigeons.. Microspira Metschnikovi (Gamaleia) Mig. Microspira Schuylkilliensis( Abbott) Chester. 2. Not distinctly pathogenic to pigeons. Microspira comma (Koch) Schroeter. II. Little or no indol in 24 hours. Microspira Finklerii Schroeter
Cells in their ordinary form long branched filaments; cultures generally have a mouldy appearance. A. Gelatin liquefied. Streptothrix bovis (Harz) Chester. B. Gelatin not liquefied. I. No distinct pigment on gelatin or agar. Streptothrix farcinica Rossi-Doria. II. Growths on gelatin or agar become reddish. Streptothric madurae Vincent.
Threads without distinct sheaths. A. Without sulphur grains. Lepothria buccalis Miller. B. With sulphur granules, motile, not attached. Beggiato alba (Vaucher) Trevisan. Threads with sheaths. A. Without sulphur granules. I. Without pseudodichotomous branching. Crenothrix polyspora Cohn. II. With pseudodichotomous branching. 1. Growths on gelatin whitish but gelatin stained brown. Cladothria dichotoma Cohn. 2. Gelatin not stained brown, colonies floccose—filamentous. Cladothriv intrica Russell. B. With sulphur granules. Thiothrix tenuissima Winogradsky.
APPENDIX B
eee ent eeey seeneeerne eee nee Pence cote t meee eer ee eee ea seen rr eeeene ee ee cnn
Name of organism, source, habitat, etc.
REFERENCES.......665 SAVENGIS aa SE a wast isibnmsuinrojorseebovenanasloanaiarstsl tees eFo(Ocdin’ «aba aisuepSbolsaoiesoiviele-sinisiaie oSuiwvaietee Ol
MORPHOLOGICAL CHARACTERS: SKETCHES.
1. FORM AND ARRANGEMENT:
ths, BOW TOT .ssisiges saieriarereaisrea 2a ae RGN aidan Neen o4 PRIN :
B, AGaL veces sediaaaiaeuinly sees dog aed diets Hai a demande eke enewa tare aes
c. Gelatin. ...e..cseeeee isles inis u evelelo dainpatatslaeetbywtbveacs 2 itsistiymslanlyitrete we annette aa
2. S1zE: ie Sistas ecaSooe slay rasevase at vats SANbva alah eot a Gialave atau a Seve Shee or eat ataiotiys vis oe DEERE eesinrciete dieateiore
3. STAINING POWERS 4s .cessviscaresisisiens saieeangeaning fF yee gars siti aerate wseee einai BEG ae esigto a a@, AQUEOUS Gentian-Vidletycics cocseavees cece cecesmvind ss 64 dees e eas eoeen iieiatrasininincasdviea ia b. Loeffler’s methylen-blue.....ccscsceeeeeecaee saerdigteraias f sidisleierye ceainisiaiees ane Roniinile ds ieee
Gh, Grams! Stain Scccaai tv's sitteiceives ee ie cieisein bie aas take aymenees peetnnnion Jikan anthems ese cere
a. Character of MOVeMEeNL......secssseeeceseeeene seRGRilnd nrg nesiaua eae TeNes aseTs ag ee vb. Flagella stain......... FOr éemaeaeia ies. SRnERNEN EEA esi credavainystatare 5" eNssnicterninte es Mane 5. SPORES:...cccceccercececvence elaseiicui sdlolaiatdessiteless Vida esas» Waa ginrce dla td fora Wansiaviapetenatave eis ices We
a. Capsules........++-+ HRSG oF WRIAdleee’s sd aaetated dois” aasaniee siete Leewsnaee sien etoile iiss b. Involution forms...... Saisie awa wee elateais Fis HG .nisiaraean ala MYR TleR NOMS sie drare wenreecarine scarier c. Deposits or vacuoles....seesesseres
d. Pleomorphism...... «1.065
366 CULTURE CHARACTERS
Reaction of media (Fuller’s scale) + ........ OP Se aerediaceet
Gelatin plate: Grown 24 hours at..........05 1 Sketches.
(a) Surface Colonies.
(b) Deep Colonies.
48 hours at...... aietsingese waessewess eC;
CARYS labs sitasurewwineanniiinedan °C.
Agar plate: Grown 24 hours at.........06+ °C. Sketches,
(a) Surface Colonies.
(b) Deep Colonies.
48 HOULS At. ceeeeesseesee sieia nistoaateeid °C.
Grays Bes aisiccisicice valeinetaiense waeerwe ties *C,
Special Media: (Such as litmus milk and blood serum.)
APPENDIX B 367
Gelatin Stab: Grown 24 hours at........eecseeee 9G,
°C, °C
+. hours at doses NOULS Abas cs cses
48 HOUTS Abi ccavasanomassare oC: 6 days at.....
a
Agar Streak: Grown 24 hours at........seeeeeee ad OF
pear Ok seC
+.» hours at..
» oes hours at...
Y
C
48 hours at........05 secsvenenetG. 6 days at......++ # sats Bis lbvaints Siaeaiare a
Potato: Grown 24 hours at..............000e dena °C.
) val
«. HOUTS at.....ee
e-seecee hOUFS at.......°C
48 hours at ....seseeeees weemner Cy 6 MAYS Bhiissanence ee a
Bouillon: Grown 24 hours at.......sseseseseeeeee RCs
Cc whe
+s... hours at... hours at
48 HOPS Ab... escecvccevescenee Ce | GAYS ativeieadans ieee aniedioraes °Cy
368 PHYSIOLOGICAL CHARACTERS
ODEMUM crises nciicavieswanawscas SOs IMTS ov sinc tiersiscere ss sien d 1G: :pevsaneduvesrwnxe” Ge)
thermal death-point.........++++++++.-°C.; time of exposure............... minutes; medium in which expOSure iS MAde...cerrseccncceececsssscecsecererecscsseeee avacwalets e
2. RELATION TO FREE OXYGEN?....eeseeeee anaiee assists erateolererarares
8. RELATION TO OTHER AGENTS, SUCH AS.ssceeseeceeesceneeneees Sn .
desiccation, light, disinfectants, €60.:—....cecsseeeeeeneeeeee isiotehseaia’ a hdedbarela iigvesits, ord ie
a. dextrose (1) Shake culture:.......:cecceccuseereeeneeeneneenes A uniarealicauea nates aierenluiae ees’ (2) Fermentation tube, growth in open arm.............5+5 closed arm..... HinteanNaT rate of development: 24 hours... ........65 per cent.. 48 hours............. per cent.
92 NOUTSs53 sxanewsinsins og 4% DEL CON bigness a9 aaivasieseiaules HOUPSiesccciearer vst per cent.
reaction in OPEN ALM... .eccee eee eeeeeeenseeeeneeenen gas formula, H: CO2: :.......0e cece eee ee dcfdneteticun Soltek SaWloginaas Waimentemaaen tine Oi LACHOS O25 scurmrsiacinge ye da. 4 mi wantanaerevtiete c. saccharose...... ja byeiaedsar lavafe pede ida at A iisttraitiose
Wibma US MAE cjssencesieieciesameceieess se emeceee sae oe Sista marceaebmneareas eiaiaeiersl aeeceemienis
7. REDUCTION OF NITRATES!....:ccsccscceecssecseccnscsesnscceees isioie a caineiniaie pacbamnsnbne nace CO NILTIFES decreas sie sg we norawrenwie’s epiesetiewe ies GO AMMONIA... eee cere cece een e eee ea Wale
Proteolytic. ..c.cccwecseveeee le acaea iia i saan s'nanwiaroeeeacas a's se bun, seateeelwihemaaeane weurgetee’ digestion of gelatin.......cccceeeseneees digestion Of CaS@in......sesecsesececeees
GIAStatICs caxiensaiwiviens semseredaeeaiuerins e904 ayesha WAiesaiaw earedeimetuants PER RB ee ena eee Wee
10. CHARACTERISTIC ODOR:......+ dene Senn ee ee ey ere wens
11. PATHOGENESIS (or other special characters) :..........65 ainnbtareconains SO \necdavs Wlefeaiajedunaiaieaaibaee ‘
Ce ee re
peewee ere eee ce
APPENDIX B 369
C0000 renee Hee eeen recent eneeens ee eer ery Deen eee ere e area ners ee neeeeeeeeneeeeneees
Name of organism, source, habitat, etc.
eRe eee eee eee ee CeCe CeCe reer ere ere ree reer eee eee errr See ery .
One he Rem em an eee cate e eee nea ae eee e reer es eS OSES TESTES HE SEH Oe HOSE IESE ESET ERODE SESE H EEE EEO SEE H OOD
REFERENCES) «ssiciscsapiesissiniesiieie se veimesies soesratehe Beara Hiss HRT ava ea as. CUTEST OreTe Rte S eI ae ee RTS
MORPHOLOGICAL CHARACTERS: SKETCHES.
3. FORM AND ARRANGEMENT:
. Bouilloticnsiisinnyie sires acracaiians sickors Soainiticid Sretviatasaicis oe dorapeishetals Biase ies esse eiaisie _
|
3. STAINING POWERS:..-ee+-seeeeeee patsielacownnntes eaialdlgiotiinatee “va eaters bjeislaletd isa ar eanapientnsi we dete a. Aqueous gentian-Violet......scsceeeeeenesenreneeeeneeeeees slats v8 a8 RAN RecSueiew owl b. Loeffler’s methylen-blue.......ceseceserees swnmpeainhten ti oeaaes Wi sceleieis esinte Sislacobotnnsn severe.
ty Grom’ s StI nnn cc news seca cemess ie ee cttecenetaia ata 2G inblasa lay seoisidisiain ara.cyaia sities Reeaaaee wiaseevinte
@. Capsules.....cseseceereee ee ee ee Ce iio tases vicemiaoysiire aie ee wee ema ae b. Involution forms.....cceee seer eee srpivatusilared te xe silgilatnelals of Hessen ss vavaMetons ey c. Deposits or Vacuoles...scesesecersvenes ee fea ee ee venue
d. Pleomorphism...... - siaverainan sieisiates RMaI Leese Le dbiiteisioial nie ai eHldin come an awareauledas Walers
370 CUliewan, semaniaws ning
Reaction of media (Fuller’s scale) + ........ OR piusiewkis Gelatin plate: Grown 24 hours at............ °C. Sketches. (a) Surface Colonies. (b) Deep Colonies. 48 HOULS Ab...ccceceeecses coos eneees °c. 6 ay S sa bsiiisieisinnisrslaiacsseiviguerenraiey adorning °C. Agar plate: Grown 24 hours at............ eC; Sketches. (a) Surface Colonies. : (b) Deep Colonies. tee ae °C:
48 DOULERE nnconvaeaeas vorvoumntens °c. CGAYS! Abies nisansas demace
Special Media: (Such as litmus milk and blood serum.)
APPENDIX B 371
Gelatin Stab: Grown 24 hours at...sseseseere ees °C,
5) o) e, o% os eS n mn EI 3B ° ° 4 4
‘
4BOULS atic cdesesa essere reese oGr 6 days at...... ie icsavasetatsialecasevelecrin oC.
Agar Streak: Grown 24 hours at......eseeeeeees "Cs
°C.
Brad
hours at . hours at......
48 hours at... .....ceccceeseces 2; 6 GAYS At... cceceee een seeeeeenes cS
Potato: Grown 24 hours at
*y
4
wey °C
sraceegeeMOUES Abssons hours at.......
eC
AS NOULS! ab osc. cso. ce ees cae os °C. ' 6 dayS At.escecceceececeneoncees °
Bouillon: Grown 24 hours at...........esceeeeeee °C,
ora OF
°
hours at -hours at.......°C
48 hours at........ aisaieiwiae ees °C. 6 days at......... muda eameee °C,
372 PHYSIOLOGICAL CHARACTERS
L RELATION TO TEMPERATURE:...s+e0ee bere eeecenees Oe be eenceesnee eee ene ODUMUM sii cscccrcevece ce ceees #0 Ces limits: cccsescenee Sisijeieee GOceiecis ince cieiaians ate °C. thermal death-point................6605 °C.; time of exposure............ . minutes;
5. GAS PRODUCTION IN SUGAR MEDIA:... ..
uw. dextrose (1) Shake culture:.........ccceeeseeeseeuveneeeee (2) Fermentation tube, growth in open arm.............+45 closed Arm........cee sees rate of development: 24 hours.. .......... per cent., 48 hours...........45 per cent.
12 OUTS. wecanisesdaiiaine dunenve DEY CON joacs coed doremresaics sMOULSsonnecctecein enon per cent. reaction in OPEN ATM......ceeee ee eeee ie ahaa sate ca(o ite Seaaavalenaiae ' iuiwatclia ina bia SopatlNaaNe He Som etaLe TRS A
Pas formulas, Hi COs! iia series waawsezeleciineweicg oa eds eta es Sigieie teen 2 Saremincenee eeeteaale Us JACTOS Osc eicciisiies sn cas nese mts Gs SACCHALOSCs..jeieazs oe. eeis devas vem ie
6. ACID OR ALKALI PRODUCTION:..
8. INDOL PRODUCTION.......5 eaasa aaibrbsnieG oialsia uamtesetandeisinsie onneomeaaniae sebiuincecdwees VS wisideelSiuriishalangnatege 48 HOUPS ss omc eevsa enviar sGumawenwaweranen Glad sis ites CBY S's ssises dejnmeds easisien daineasnnnte 9.. INZYME) PRODUCTIONS seins cesvamemensnies tumesanwiale rei Ge": Taian aiheiaabictare ois Hisacrernant one PLOCEONYVbICs veces ceieesiecesiensees a cholatareyapare arocrels- "ele iaierayedy akeegiasexicn es Wt Senate ater digestion of gelatin............. Ger en digestion of case@in..........-csaeece ven diastatic...... See e oes icessab asic eines Bb SER St nceawigialetsan Moree sienna wee iexsiodh nen
10. CHARACTERISTIC ODOR?..-.+.0005 wiireh oe 11, PaTHOGENESIS (or other special characters):...........sseeeeee Ve neAMneaEs taal WeseMeT
APPENDIX B 373
Name of organism, source, habitat, etc.
Rae Vi pianaai EO iaedeh i bee bare date dnb abaNun ta ea pein intestine Or ncndara gs CELERON SOR ARES REFERENCES..... iesleeara sieisisaisranvdans sive Saisreeencierasaia staus aieyaatarets tar love Yearere piararaarelsiaaiaisls f kan hinnwmaa sank OE
ocean ewww eeneae Senn See e eee ee eens ee eeneee Tamme neta cee e eee e reer ea eet ne tense reteesaseene
MORPHOLOGICAL CHARACTERS: SKETCHES.
1. FoRM AND ARRANGEMENT: hs BOUT O Meck sasaisieis + dstsosernseeieie eavntavecrateetd i bara asathacalavevave seraveamaverameuvevaa slater a3
C. Gelatin. ...ccccccceeeeeereeees nsiaie-acdisselqrayeinvecaia’s cvonayoraiavarayars stared seatiaquasaiata ss seats
Ds | SUZ Be thinentcioes e Aawrarintaars mecinwwiein Giclees cemcinethiate sb faetaath NCS es class: meiengenta asain aarp nie ie’a.o 8 sia ens siete 8. STAINING POWERS: ....sesceeeeseeen sence eeees eeaiteenntanet cae anew apenas cnn & VateAaietdeatare u. Aqueous gentian-violet........6sseseeeeeee kesadien Wis nebnwaaiee ss Heennea Jor eronmen 6. Loeffler’s methylen-blue....... sieuisinanteatea asia Shajsinisin’cscterears sats lasiaiside curses pUecricn)
G.. Gra 'S StAlDisncceneassactenenenetts areeses Pid geiniuales tones bcos shies evan RAB Ke Sielaveioi
Ge MOTILITY hice te od emesis 0 6g dessin SS Gags lace MSNA Rasa uaa Sai tiocayerateleia yaaa Saideaa eee veeates
6. SPECIAL CHARACTERS:...++++++ asseie bisiole anaiaisionaiine gisseratcisisiet aia ele acoimnassustaimie erste, avnusleraenistad eats arr GU. CapsuleS...ccseeccereeseeees eo Wed ovahnce lo einiatasasaoasnisluin: pine Wovens ose cnusie’d SSIs wien ects ted on a acetone b. Involution forms.........-. Sige elerale atichuistays Warscnienutigd odedieherinaee. 6ayl sae acne cd c. Deposits or vacuoles..... asa sae SiG lawianeexlele Wiad ieeeMN ES Us ARE Sawh oe facie’ sa aeaare
d. Pleomorphism...... .. fee e er eanes ate ais cbiatare raters aMessth heskecisieie: tic tev aaeteannye samnneeah eee ne sevnees
Bt4 CULTURE CHARACTERS
Reaction of media (Fuller’s scale) 4+ ........ OT — ..ceeeee Gelatin plate: Grown 24 hours at...........5 Pe Sketches. fa) Surface Colonies. (b) Deep Colonies. 48 hours at...... asarainsrarsia. waieersteseiadae OCs GA BYS Abs giissinc se lspaicnesisie ss Heieareieiers °C. Agar plate: Grown 24 hours at..........55 °0. Sketches. (a) Surface Colonies. ; (b) Deep Colonies. sighaaiets °C.
AB HOUTS At. .cccccccccsecesereesees rhe OF 6 days at..ccccsoves aeiasinartats
Special Media: (Such as litmus milk and blood serum.)
APPENDIX B 375
Gelatin Stab: Grown 24 hours at.......66006065.°C.
C °C,
. hours at.......
‘ ;
+ Hours at....... 6%
48 NOUES, Abies csgiuisirssicesinsin °C. 6 days at..... sipaagecaaaiapsenteCs Agar Streak: Grown 24 hours at..........-- ge Cs 3 fo) 3 Py a a 3 a n n q u g : A A 48 LOUES AE woe 0c cs osniese ws sis °C. 6 days at.....-eseeeee Pnekeianee °C Potato: Grown 24 hours at.......cccecesee eee eeeee a OF
6 Ars OF
hours at....... hours at....
48 DOUTS Ab. os.ccsecieseecces oes °C. OAS Abies seca scene se cones a OF
Bouillon: Grown 24 hours at.......-sseeeeeeen eee i
°C
°C.
«oss hours at....... hours at
aw
48 HOUPS Abies i<cienc aes secre si 6 days at.......... eisai waisonmrag Ces
376 PHYSIOLOGICAL CHARACTERS
1, RELATION TO TEMPERATURE!......ceeeeseeseee scogeiGiaate sau aasenee recnranaiater ave Bta\aseso orelpcelorelaje’a's optimum............ Raisers ous otto 2 TMI TES isc sieiniace sapeiaind amined LOWS steiaisiess pencsraysiar Read 6 thermal death-point..........eeee eens °C.; time of exposure........... ... minutes; medium in which exposure is made.........0065 aeetnnngs 5 er amdnaey eR BAL GIGS Seay
2. RELATION TO FREE OXYGEN:....... Been ae ceaees Oe ence nee n neces eee e ene ee nen enees
3. RELATION TO OTHER AGENTS, SUCH AS. ecusssnnucnconccunvees Doce e eee eeenc eee eeetenteene
desiccation, light, disinfectants, etc.:—......... Sis Stesars hoot iis tot aie' a aloistedatane bie epives ovate
«#, dextrose (1) Shake cultwret..s.cicrceneensancessveras rab abate ssnadaedelshetesetncaia acavavenstersie Stee (2) Fermentation tube, growth in open arm............006. closed arm..... Ristiaeens age ‘ rate of development: 24 hours.. .......... per cent., 48 hours..........-6- per cent.
Pe MOUTS nn ds 04 nieces POY CONE... eeeeceeee singer HHOULS vomsie's vesicle sees. DEF Cent. Teaction in OPEN ATM... .ecssesenerrccuccnsenassaeeeessesseesessentes daireuiny TUR ew aN Baer gas formula, H: CO2: :.....cceeceeseneeneees eg dainwtrs sisi atides <iaSn Gates ala sis Sea aie ees OF
Os WaCtO Sivan? ssjanscisaare Gin tisnedisils cane +e. c Saccharose........ pana caeatensine prove Guabacusalncerars ©
6. ACID OR ALKALI PRODUCTION:...++4 ees salar eaGsoninns pisieieins eoraresinas sales aidearnini sis leswvepaatene distant
4. REDUCTION OF NITRATE to nitrites.......... wy giesebeiere.dlasene WOVEN ONT Biss oig op0in git. e- nerds oa wehalenanieais 8. INDOL PRODUCTION.......--+ A easels by aera TTY ic bg RS ARG ae CREE saga apAMaeMess Fo NaNSGRE AS NOUTS aeca5 vs qajesodeans none Emenee SERA W EIeen a CAVE taemmsinasy na cdeteniten CRN OEE
9. ENZYME PRODUCTION:,.....+ araiaiege alee « HRN i iaiauis oid valet tor das woo telavaaleie vole eaeare NE Sargigcntwre
proteolytic........05 eavate asa. carb daeanaea beesbahig: es FkR Sastre, Gates Sodas AEA eI eG RRO RaMaearerelEA digestion of gelatin....cscsseeseeevovers
diastatic..... eisieibed toa eteisrratiay oa ws greataurhgiea ys eet eee
10. CHARACTERISTIC ODOR! ...++-0+see sees Sedawnsies 11. PATHOGENESIS (or other special characters)
pS veee
Ae ne ne eR b OEE eee TERE EO OED ERE E EOE OEE HT OOD EHLERS DOE HOU OOH OO HE SENET ett e eee Seer irery
APPENDIX B By?
eee ee eee eee een e ence cence recent eer tennaee donee eer een eens nerene Sere C eee eee eee eee eee eee eee eee ee eee eee eee ee ee eee ee eee wa eees eee ery Deanne eeaee
RREFERENCES...ccsccccccecccsseseaness Se eo Rees ECT Aen merce toi Sat Venissannonmetines:
MORPHOLOGICAL CHARACTERS: SKETCHES.
1. Form AND ARRANGEMENT:
tis, TED OUTIL O Tis .ra ees touat os zie aprarasviavenieare Va er Suididiotssia wi AA Datta pisleiatenseteareeas :
@. AQUEOUS Gentinn-ViIOl€t..... ecscssceeeeceereeesesennsenectesceneesesennenes saves
b. Loeffler’s methylen-blue
ex Gram’ sistain: jjcsedaaeansecasieshwh hoses d. Special stains.......sseeseeee ose ataselosa nin ensuh anche soya faibs deb aroaacnepeuadayagolanana a/hvarnvee dete hnaiefe atciode A. MOTILITY:.......00 ce ccee se cees ones its Wis Dhbedioven dain Hete\aue acerbiayAiecaty sbi eeecadkecol olasd.s bia aiaya piajeibie’e sige es a. Character of movement............+ ai winliie velo ole ale stani@teinia’ ia « 410s ataievareinls 0 vb diated eeaiecine scenes
b. Plagella stain........ccse cee eee eee rene ee bia? (oid Seistoleysintawintins deoadintaleelaicteetaie seater wanes
6. SPECIAL CHARACTERS!..++eseseenes aid taser ya ars erg orev: ate Oe Sin ela teases orci a ate ayeiaarase aeuiass cig ease ‘
a, Capsules..... blind oitiseenclaleiaVuin sis emibetelotbin'6 slelba vba mintale trots calereletetoateters sepeiedanegtnaaraeees epteae r b. Involution fOrMs....sseeeeseeeeeeeeee oa Rann eS sihsceecdtas Sh Th ROO EN dahediodané mas c. Deposits or Vactioles....ssseceeeceeeeeeenececeseeneeee ce aSadesiaesasresd Sedgtetgiane Burau ie
@. Pleomorplistine.cxn acarsssncscaserrwecesd seareseees fea eenad pened sens hice sid acbiaysienagaed “
378 CULTURE CHARACTERS
Reaction of media (Fuller’s scale) 4 ........ (0) er
Gelatin plate: Grown 24 hours at............ °C. Sketches.
Sa) Surface Colonies.
(b) Deep Colonies.
48 hours at..... WEE LER Eee are red °C.
6 days at. .cceeeee aia ais capausteteiatais iano °C.
Agar plate: Grown 24 hours at............ a OF Sketches.
(a) Surface Colonies.
(b) Deep Colonies.
ABOUTS At. rceecceseesevccesenceeens °c.
6 GAYS Bitsy vic exidsintieis ie@eiaaeies say i OF
Special Media: (Such as litmus milk and blood serum.*
APPENDIX B 379
Gelatin Stab: Grown 24 hours at.............06- °C;
veeeee es DOUES Ab....6....°C,. sive ves ROUTS Atheacess PC,
$8 WOWES: Ate is i ocaiecinceceineay ad OF 6 days at ..........- silanes bre of.
Agar Streak: Grown 24 hours at......eseeeeeeee °C.
aay
-hours at.....
veeeceessMOULS Abe vee PC.
18 NOUNSEAE s iscrceied te nisosueens &C. 6 days at...... emeniwass« area?
Potato: Grown 24 hours at.......cceeceneeeeeenees °C.
seeceees DOULS AbeoeeeeePC,
hours at.......°C,
48 HOUrS ab ...... se eee eee eee eee °C. 6 dayS at...cccceseeee Gite dieters Cre
Bouillon: Grown 24 hours at.............6.. sue OE
ener, °C.
hours at ...bours at.
De ow
48 hours at........000 issareiaiasre? Coa 6 days at........ p sarsenneeies + sc)
380 PHYSIOLOGICAL CHARACTERS
1. RELATION TO TEMPERATURES.-sececsecsceneveneteneenes wedeasewass vi ciao henamitexitaye sian saa Cede OPtiIMUM....... eee eee eens muaiecattie SO elim its eaters: sates eeeey LOi4 Hash Ce tess °C.: thermal death-point..............eeeee °C.; time of exposure..... sete ranasines minutes; medium in which exposure is made..........+. a taille staid obiamtccke eenpa cel ieieratsotembadarws iy
2, RELATION TO FREE OXYGEN?..s+sereseeeeenee eisiaiehk $i x Gases sy ease ha paiciaises take onieiovaielorvis
8. RELATION TO OTHER AGENTS, SUCH AS..ccccsesececseecners Maina Seam ean Sepsis Steaeery
desiccation, light, disinfectants, CtC.:—......ssceeececnceneeeenenncnnens Sipsuas he oe
Co eee ee er Ane renee eens Jesse Peete eee eer ee ene tee
5. GAS PRODUCTION IN SUGAR MEDIA:...... Sh beeen e renee snes Se
a. dextrose (1) Shake culture:.........ccseeeeceneeeeneee SVs Gos RMaaegen Sta Bu elevate nistetedties Sas
gas formula, H: CO2: t..cccceeereveeeeeees dagaae away ce aiasehelsaInnrie Balesets chefs erssoisiate Us TACO SE rciiisassens vd 83 gd Sadenestagede > GC. SACCHALOSE...cecvecvconcvenecs SeeNeoes Soe
6. ACID OR ALKALI PRODUCTION:...06+ seeeeee este e eee eee enene Pee rer ene senor eeneeene eens
8. INDOL PRODUCTION.....++seeee ashlhze dtatbyadavaersiaisees § Syaaceeaio ein eiate a 44) st Necoretaie ise Aitauala Ssusevaiaheaten Saiisiete va
proteolytic....... gaaiecye ae de ua teawssiel dieind Wa Seelam awd HO Rawodh semanas OSes Maes eis digestion of gelatin..........c.ceeeee eee digestion of casein...... ainetehs caterer
diastatic...... dnwpeinmnae ang sods sai deirios nbs s aedterotsiniute le alee HasgnlanagetCe eames bisa ater atecne@ ees
eeer err errr rere errr reer reer errr reer ere eee Sy Deen amen e eee en enn eee eee eee e eee n eee
errr err errr errr reer eee ee Cer rrer rr Cece r rr rec ee eee r ee ee eee ee ee eee eee rin
APPENDIX FR 381
rr erry eon e ne ceenene Fnac e ner nee em ee ree ne rene eee renee erneesenrsees
MORPHOLOGICAL CHARACTERS: SKETCHES.
1. FoRM AND ARRANGEMENT:
Go Bouillon dccsiasacadcceeciienus se swocee pages cass dh dec dyantoavalonaale ay duviaooes pestounyge? ;
38. STAINING POWERS:....- Sys GA RRS SE eS sonatas he Seisttioatie sidiacgaaa arasasgeciciura nats ete sr ajor sara’ a. Aqueous gentian-violet..........eeeeeee draigiyecntlndh vaiarhed ei a1eie 9 Wve v atei@lalaleahe aiieleratae iemretoala b. Loeffler’s methylen-blue.......ccccsecevvessevenecncunes Cisicnatalgiacareisleiihe Revie inate eure late c. Gram’s stain.......... sheuaiardhatds slats paiareiateaieividisioidaciina waxy ielaseheiagd bie aNeReeARenEE aia Pelabarshahai d. Special stains........... ard niatsssiepeialecshons siainisiare ease hie ditet ee siersias npes seaweeuoneesase aiginiiniaians 4, MOTILITY:......... 0006 ee re ieisiarianes saealnaeenes ata PRR aia aE e's EK Sikisindasianmacsais F a. Characteref mOvemiel tieseisivivennend io wiawaweesncess i tea dexteace a batician Section de cele wae
b. Flagella stain....essseeeee sees cassava vse en esa en shavstetaelass/e vA crantreiSteyage ecdualdaisietuidiareietelata aise sis
6. SPECIAL CHARACTERS:....+.- saa pigeneameta soieara® wakes sda ee nade Maile alate devduers sera teeta @. Capsules....seceeeeeerees paaminiaeen axe Saleem SARS SBMA NGie sas faSiesstacaians eceiarecoisihia ase iainestse wits b. Involution forms.....- A AGhe hoc eaine cdariderusyarere neice a scdardeiva v's dbandidestnetevecels osialsleatie: Soiie Aa ea C. Deposits Or VACUOlIES...cesceeeceeesenseeeneceeseeeescnnrs aie A Bows Oted MMe YAoS
d. Pleomorphism...... sseseverscssesenneeceeens wate as oaiaibiaien sy tH Ia atarenaeets Ghar S Saez cctisrads aidan axe F
382 CULTURE CHARACTERS
Reaction of media (Fuller’s scale) 4 ......4+6 OF — seeeeeee
Gelatin plate: Grown 24 hours at..........4. EF Sketches. (a) Surface Colonies. (b) Deep Colonies. | ’ { { i 48 hours at...... aan dibca/siaie Ss Srreienneuieined °C. 6 days at.......e0e aiid aieiatviaraie waver SWereiw’® Os Agar plate: Grown 24 hours at..........es °C, Sretches. (a) Surface Colonies. (b) Deep Colonies.
AB DOUTS Ate rcceeseeseeceeeencn scenes 5G; 6 days at....e.e oa yasaisiaia vie’ Srarssayaresciaiare. ®Cu
Special Media: (Such as litmus milk and blood serum.)
AIDE RTD IK B 383
Gelatin Stab: Grown 24 hours at.
Sait easiness °C fo) iS)
2 B.
rey as
3 a
n n
~ col
3 =]
io) iS
a a
48 hours at........eeeeee Hapeiaces eC. 6 days at...... sah oveseiaracd es Dea of.
Agar Streak: Grown 24 hours at.............06- 2Cs 5 rs) 3 3
FE 5 F
4] 3
n nN
: :
6 5
A 4
48 OUTS Ab... cece cece eee ee eee 2G; 6 days at.
Potato: Grown 24 hours at
a 2
°
. hours at.......°C hours at «a Cy
AB NONYS Abies. saaiccsies es scimase °C. CARY S Ati. cserncuees socpeeniess os On
Bouillon: Grown 24 hours at
ty
2
‘ 2G:
Pre ©
hours at . hours at......
48 hours at ..ccseccceee ees ee eee °C. CUAGS Wiican ia vedasader raed eC,
384 PHYSIOLOGICAL UHARACTERS
1. RELATION TO TEMPERATURE!......--+e005 Raveena suntewersels Bagi OPHMOM sisiesccweas se siescmenan OC MITE, ois ueceeeretadie ales EO jscaiasice sccrornseinien ative ous thermal death-point............0eeeeee °C.; time of EXPOSULe......-.eee eee minutes; medium in which exposure is made.........005 saan eanahoneneuameeeaE Winrsuaiess Be
2. RELATION TO FREE OX VEEN I scadguis ceavevinns byaues serpeweny eyed esateenlods seer erases Hibs
8. RELATION TO OTHER AGENTS, SUCH AS...csccccsessesccsnsrteeceneensenetseceeneenetaneees
desiccation, light, disinfectants, etc.:—..... Misia: Midiciesderenpionesse warheeutiotsiant sd eae a oNee ‘
6 GAS PRODUCTION IN SUGAR MEDIA!.c+-ccssessees areas tere tatatits searenNistereeattis ah Carn cema eorsteslneae 2
rate of development: 24 hours...... seeeeees per Cent., 48 HOULS.. +. eee ee eee per cent. TA NOUES Ws iAieuietted awstats ve per cent.,......+.. arena HOUTSessictswemen’s see per cent. reaction in open arm..... saideiiaigageeesats ei gigeiarg ep sista rermeeeaisisinie ws sTe viaerets Saebaitere ARIS aNS
gas formula, H: CO2::...... sigminsternente’esie
Ob: 1CLOS6is.sc tarcwav ain tees sett
7. REDUCTION OF NITRATESi....++++ tO NILTIGES. ... cece eeeeee Fae 8. INDOL PRODUCTION.......+eeeeeeeeees shacareienawlacna Sinhalese AAs DeNaideeGlanamaaetee s eSteNe. Ges martes 48 WO UE Skis ceciece Se daesnieateisiess eae saiennion eed deemmemicaais vies GAY Sisistacs Se octets chore ebtiaiampaiate wverormals 9. ENZYME PRODUCTION?:..+.eeseeeeeeee a chatsuissassiee eisie' BuisieRGinsieiar, iereaPbehdpetuilh aaisidtolesinrsrstnacs bias Piers
Proteolytic. s..cereevere
digestion of gelatin............. PCAC digestion of casein..........- eieeeane iets ares CIA SEAT C aigiciene dies seniee AG 6 vein esa N eda secures Revo heareleed aw Pieusdararenalelpatensste ties caiece 10. CHARACTERISTIO ODOR:...+.- netaniadten RIAA Eien eaeade wee piwaleted aatausns aia gt acavaietsisis dhe
11. PATHOGENESIS (or other special Characters) i...cceeecceceueeteneceresnscueeeeeeeeestecens
eben eeereeeteeees eer Te rrrer ee Tee ee eee ee eee eee ery ee ce ee er is teen e ees eeeee PTPPTPTTETePTTereer rr eererrrerrr rr eerrrererrerrrrcer rrr err eee re er ree eee e eee eee eee cere cer)
eee eee errr ee eee eee eres
APPENDIX B 385
Ueno ee ene ee eee een eet ee eee cate Caen erteeeees Peereere ree eeeeee errr ere errr te Serre eee)
Name of organism, source, habitat, etc.
ee eee Cece ee Cee eee eee eee eC Cee eee er ere eee ee eee Pereeeere Tere reer reer eee er eee Pee Ce eee ee ee eee ere eee eee ee Serer!
REFERENCES.....-....005 60°sib' asa 8010 6 nio (0's whe oid ina’ Oieinjnie nle vinielaibye bees a)6'8 Se sie AiGels o-gia-sie: sd 0 weinerdale Feesineig
. Seen wea meee ence eseaae wee eee bebe meee m eet etteeeneconne ee neeene deco mareeeeeererreenees
MORPHOLOGICAL CHARACTERS: SKETCHES.
1. ForRM AND ARRANGEMENT:
@. Bouillon.....ecesecseeseeeneaes S65 catne Re re seentousess nee!
b. Agar......+ ssaia acd nse dapha pte elude aadiase aratatetece evn gia clini g tare istecorbje ¥dtanwiesstetarats
Gis OL AEINY. J sinreisisrsinisinss HD SINTOT aR atin ad atereineiesag Sear aWae? den ntienieuinenaed
Peek eee c meee ee ee te ta R ER HOHE SEER OEE EEE ESET ETE H OE ES POTEET SORE DOOR EEE ODE EEE EH EE Eee!
2: OU Wed lavas sicmussiaemasgewaasedsiegisieasaces detemeuesayeesmmesaut sx et O SIZES aviwauen 14 Swabavine’s cetera ava Ve CSIR % COMRANNS PURGE ST stin sine owen euwacadeaniade saan
S, BSeRWING POWERS tes cexncses wns ss eieensT Rl UDIRIEIR ieee Veswereeeencen anes
eee eereenseeveees
uw. AQUEOUS Gentian-Violet.....scccccvesecscecceveeeesveesseee
b. Loeffler’s methylen-blue........sseeeeseeves piiduetiwenss hand c. Gram’s stain........ aaaeawecaaees ad. Special stains........ widen ro aoe ih wiaatere arsed areas Sena ores 4. MOTILITY:.......00-eeeee bre evaheedrate ais Senge sieanion watees Sa CaRsMyoRaVRUarataDeRAPa Vag rINH NT MSGAOATs elnis wieseracnace «. Character of movérmei ieseies cs cecwwessis i enernniieis os cndeeeonensaies panaeneewececee
D. Plagella stain.......ccecseeeeeeereeee sabres Wie ab ieapa sibel apeiase Geaio o wuain’bs Rosi seaia alale ora tporeretevauleicioe
Gs “SPORMS toi isisirejiss cee sieswaivias vvdesnemmniaeened seins Wight siaecess ae suchas acaberes toe aa bereft 6 lovetatava shaeaareseety
6, SPHCLAT CHARACTERS tres vaccrtesiare's sis'eicreraieinn ed ov tis einer eps Ne opainaia eG aw MaNET MO eaaitetunace a a ee recon RRO ed ame ANNE ATLA CORN LPRER COORG in aaa ee AD Den Larva Tora LOVING siaiessis ie she cid iecein No sCSie ata assays Ines SS RTA WHA e as eiereanasa a. dvd.o s mrdwlewentares shresnersle c. Deposits Or VaCUOleS..rcceseceseeeeeesee eee ee sees e ee enebeeeeeneeneeeeneneteeneserescenes
d. Pleoomorphisma...... ccccrscscccesrecvereorsersevrestesseeressessonseesesserresseceesees
386 CULTURE CHARACTERS
48 hours at..... aediayavastalatess Seasedersvona arisen er 6 GAYS Ab.ccccceceevesvseeeeee
Reaction of media (Fuller’s scale) + ........ OF = esses eee Gelatin plate: Grown 24 hours at..........66 °C. Sketches. (a) Surface Colonies. (b) Deep Colonies. 48 HOULS Ate crecsscree 200 cevencees °C. 6 days at.....seeeee a area aR we Se °C. Agar plate: Grown 24 hours at............ °C, Sketches. (a) Surface Colonies. | (b) Deep Colonies. wuitecaed °C.
Special Media: (Such as litmus milk and blood serum.)
arnsuvesX B
387
Gelatin Stab: Grown 24 hours at.........ccsenee °C:
red 0 2¢
hours at . hours at..
F
48 hours at.......... pon gadgione Oo
CYA AGB2A bs warancecgesiangaes es ee eects ol,
Agar Streak: Grown 24 hours at......seee eee °C. oO oO ° 3 3 & 4 4 Lo ee AB WON ES AL... negated widciciedice oC, 6 days at......+- saat eeslaamaiees 1 OF Potato: Grown 24 hours at...... ccc cece sees ee eeeee =C, io) oO 3 3 3 @ n n w col 3 =I ) rc 4 4 wee Veacine cier queetinereer a Gays Atacssesstaias conn exe Gy Q oi men Bouillon: Grown 24 hours at...........- gt nicwseu he 3 3 3 3 é £ 2 8 8 4 4 48 hours at........ Sosiease nadia en GAYS Obseesins semecars owascwine °C.
388 PHYSIOLOGICAL CHARACTERS
1, RELATION TO TEMPERATURE(......ccceseeeeeens ee ochre nla aebintivninle aceraie Sraiavevareiaierale ejaie aibieee a, OP EMU vs cseceies sin aiscedisserdions ee iO LIMITS i weiiasceweaag eens EO scateckcdieta's Reeser eeermecsauait °C. thermal death-point..........c..eeeeee °C.; time of exposure....... iawn’ minutes; medium in which exposure iS MAC... .eceseeeseeveenesneeves aversrate coisiaisher ecteieeers alex
2, RELATION TO FREE OXYGEN:..... er ee re ee re is vie BaReiaRs aonhieoN bs
sailor a oe ernie ate Oe cactantpaetete vee Birt sein enterce pte wee he heawndac ss megane SONG Keaaee
8. RELATION TO OTHER AGENTS, SUCH AS.scsssseveceseceveeeeeveuveeesness jppeteeeranceh :
Aesiccation, light, disinfectants, ete.:—........ ecabaleoecaiee ivinie-a vakameate saseuadeanaiasced Seeleoese
Cee es ee esr eteneeneeeseeereees Dement meen nee seen terre een e eer ee ee eee ene seeeresesearen ene eessentne eee Co eeereneee ote e een ee eee eee meee een ee oe wane ne ereene Penn reece eee verte ere rer ee eeeeseeneneaee . See een ee eeeee te eeeee eee cement eene cee encanto rece nnee teense eens steeee Uber eee n eres eee .
4. PIGMENT PRODUCTION!.......cs008 eroracsiare awe sarees Sobats Sa wares se dinhiaratmenamiaies squeleeuee, Sa B: GAS PRODUCTION ON BUUAH MEDIA Lose neieueteas aeorsienkees beteenerees er eiani eoenwnne uw. dextrose (1) Shake culture:....... iehalaiGraceiad a KIMea Siowterela beni Cinalene Gee Haein shame neee
(2) Fermentation tube, growth in open arm.........eee0++ClOSEd ALM... ceeeveeees ‘
rate of development: 24 hours........ .++++-per cent., 48 hours.............per cent.
V2 NOUPS ss sacswaniedicsnses + POY CONG... se eeee eeeveeee ss MOUFS. +00 sevceceeses DEF Cent.
FOACHON. IN OPED ALM:ies cocanasncereswenadsenmense nen sor wwe SeeasnNss ecaleceNa Sete™
gas formula, H: COz::......5 aie e Actayy iatvia Hetero ereloeay ae
6. ACID OR ALKALI PRODUCTION 1iscre seeeecrecssesererecsursooumees bch esa diabie Syaiilacbleiwelntense ss
Pence ence Sey beeen econ teens ere e ee ee eee eee eee eee Se er rr
7. REDUCTION OF NITRATES to Nitrites.......:ee eee Reieia areraiaie,s Sinivicia viprerere:s MOCRIAMONI Bee's visjetviere.s eviais sinisis sia slnvistetsinnieners 8. INDOL PRODUCTION......seeeeeee slait Fis sieweuN ae e ea TeeIS TS Kewals Masee-eeiileareinal eid Tecan’ 48 hours..... sagieareutcetiiey Ha exe misters reece pACUABY Sivceseesir Cereal eres 9. ENZYME PRODUCTION:.....s+eeeee aeteee eu widinisiwie loseinis 5 Weerdaibtaa> sethiaxe Stein edebstawe didiaseahielsieNdalg ae hauls Proteoclytic......seseececeeees ia tied sodiahaiqunyanane tea side abec cenuined Bae npteidrelaoiaie;nie aioe ameMamad pores
digestion of gelatin.............08 lita digestion Of CAGEINs ta <ccrvensgss vevewens
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APPENDIX C
TABLES
COMPARISON OF THERMOMETERS. (From Gould’s Dictionary of Medicine)
a b 8 ‘ 4 ‘ is) 5 g a | 2 a/ 8) a lg fy S) (4 Fy iS) °S a iS) (4 212 100 80 122 50 40 32 0 0 210 98 9 791 120 48.9 39.1 30 -1.1 -0.9 208 97.8 78.2 118 47.8 38.2 28 2.2 -1.8 206 96.7 77.8 116 46.7 37.3 26 3.8 27 204 95.6 76.4 114 45.6 36 4 24 -4.4 3.6 202 | 94.4 5.6 112 44.4 35.6 22 -5 6 4.4 200 93.3 74.7 110 43 3 34.7 20 5 7 5,8 198 92 2 73.8 108 42.2 33.8 18 78 6.2 196 91.1 729 106 44.1 32.9 16 a) eral 194 90 72 104 40 32 14 -10 -8 192 88.9 M11 102 38.9 3.1 12 -11 1 8.9 190 87.8 70.2 100 87 8 30.2 10 ~12 2 9.8 188 86.7 69.3 98 36.7 29 3 8 -13.8 -10.7 186 85.6 68.4 96 35.6 28.4 6 144 -11.6 1 4 67.6 94 34.4 27.6 4 -15 6 -12.4 182 83.3 66.7 92 33.3 26.7 2 -16.7 -13.3 180 82.2 65.8 90 32.2 25.8 0 -17 8 -14.2 178 81.1 64.9 88 31.1 24.9 2 -18.9 -15.1 > 176 80 64 86 30 4 4 -20 -16 174 78.9 63.1 84 28.9 23.1 -6 21.1 -16.9 172 77.8 62.2 82 27.8 22.2 -8 -22 2 -17.8 170 6.7 61.3 80 26.7 21.3 -10 -23.3 -18.7 168 75.6 60.4 78 25 6 20.4 -12 24.4 -19.6 166 14:4 59.5 76 24.4 19.6 -14 -25 6 ~20.4 164 73.3 5B 7 7 23.3 18.7 -16 26 7 21.3 162 72.2 57 8 72 22 2 17.8 -18 a7 8 22.2 160 71.1 56.9 70 21.1 16.9 -20 ~23.9 23.1 158 70 56 68 20 15 -22 -30 -24 156 68.9 55.1 66 18 9 151 24 -31 1 24.9 154 67 8 54.1 61° 17.8 14.2 -26 -32.2 -25.8 152 66.7 53 3 62 16.7 13.3 -28 33.3 -26.7 150 65.6 52.4 60 15.6 12.4 ~30 ~34.4 -27.6 148 64.4 51.6 58 14.4 11.6 -32 -35 6 -28.4 146 63.3 50.7 56 13.3 10.7 -34 ~36 7 -29.3 144 62.2 49 8 54 12.2 9.8 -36 -37.8 -30.2 142 61.1 48 9 52 11.1 8.9 -38 -38.9 ~31.1 140 60 48 50 10 8 -40 ~40 -32 138 58.9 471 48 8.9 71 -42 41.1 ~32.9 136 7.8 46 2 46 7.8 6.2 44 ~42.2 -33.8 134 56.7 45.3 44 6.7 53 46 +43 3 4.7 132 55.6 44.4 42 56 4.4 48 44.4 ~35.6 130 54.4 43.6 40 4.4 3.6 50 ~45.6 -36.4 128 53.3 42.7 38 3.3 2.7 52 46.7 -37.8 126 52.2 41.8 3 6, O28 18 54 47.8 -38.2 124 51.1 40.9 34 11 0.9 ~56 -48.9 -39.1
To change Centigrade to Fahrenheit: (C x $) + 32 = F.
For example, to find the equivalent of 10° Centigrade, C = 10° (10°x 2) + 32 = 50°F.
To change Fahrenheit to Centigrade: (F. — 32°) X 8 =C.
For example, to reduce 50° F. to Centigrade, F. = 50° and (50° — 32°) xX § = 10°C. or — 40° F. to Centigrade, F. = — 40° (— 40° — 82°) = — 72°, whence — 72° x § = — 40°C.
389
390 APPENDIX C
COMPARATIVE LIST OF METRIC AND ENGLISH SYSTEMS.
Metre = 100 centimetres, 1000 millimetres, = 39.8704 inches.
Millimetre = 1000 microns, #, inch, approximately.
Inch = 25.399772 mm. (25.4 approximately).
Litre = 1000 millilitres or 1000 cc., 1 quart (approximately).
Cubic Centimetre = 74, of a litre.
Fluid ounce (8 fluid drachms) = 29.578 cc., (380 cc., approximately). Gram = 15.482 grains.
Kilogram = 2.204 avoirdupois pounds (24 pounds, approximately). Ounce, avoirdupois, =(48714 grains) =28.349 grams 30 grams, ap- Ounce, Troy or apothecaries, =(480 grains) =31.108 grams, t proximately.
A
INDEX
BBE, condenser, 34. Absorbent cotton, 14,
Acetic acid, decolorizing action, 60. Acid
acetic, 60.
alcohol, 312, 316.
carbolic, use in detecting enzymes, 80. in Pariette’s solution, 338.
production, 76. pyrogallic, 272 sulphanilic, 7 76.
Acids, determination of, 76. Acidity of Media, 8. Actinomyces bovis, 263. Aerobes, 72, 74.
pathogenic, 188.
Agar culture medium, 16,
dextrose, 64.
filtering of, 16. hanging-drop culture, 44. lactose, is
melting point of, 56. plate cultures, 56. preparation of, 16.
slopes, 16.
sterilization of, 16.
Air analysis, 126.
Alcohol,
pump, as a decolorizing agent, 60.
Alcoholic solutions of dyes, 28. Alkalies, detection of _in cultures, 76. Alkalinity of media, 8
Ameba coli,
344, 352.
Ammonia in cultures, 78.
An
An
An AD
AD
An An
aerobes, 72, 74. pathogenic. 92, 27
Wright’s Method ce Cultivating, 272.
alysis of Air, 126.
of gas,
of milk, 132.
of soil, 132.
of wa.er, 128.
ilin dyes, 28.
water, 30.
imal autopsy, 307. cage,
care of inoculated, 300. experiment, blank for, 308. inoculation, 294.
thrax bacillus, 1.7. group, 91, 167. tiseptic action, 72. tiseptics, testing of, 134.
Apparatus for cooling plates, 54.
for tubing media, 10
Arnold sterilizer. 10. Arrangement of bacteria, 91.
Asiatic cholera germ,
251.
Asparagin, 46. Aspergillus, 50. Aspirator, 126 Autoclave, 12. Autopsy, animal. 302.
cultures at, 304 human. 356. instruments, 300.
BAcits. 89.
acidi lactici, 105.
grogenes, 175. ‘ capsulatus. 273.
amylobacter, 46.
391
Bacill
us anthracis, 167. sym eae 277. botulinus, 285. bovisepticus, 187. capsulatum, 179. carbonis, 277. cuolere gallinarum, 183. suum, coli, biology of, 219, effect of chemicals on, 134. desiccation, 70 reaction of medium, 66 for removing sugars, 64. Gram’s stain for, 62. in water, 130. motility, 40, 50. plate cultures, 56, 58. production of acids, 76. gases, 7 hydrogen sulphide, 78. indol, 78. thermal death point, 68. diphtheriz, 199. dysenterie, 239, 344 edematis, 281, 314, 3i8, 352. matigni 381. enteritidis. 223. Feseri. 277, 314, 318. icteroides, 931, influenze, 211. mallei, 191. mesentericus Ve eetas 95. mycoides, 40, of blue-green cor 247. of bubonic plague, 243. of chicken cholera, 183. of glanders 191. of hemorrhagic septicemia, 187. of Japanese dysentery, 239. of malignant edema. 281. of swine plague, 182. of swine erysipelas, 195. of symptomatic anthrax, 277. pestis. 243. 306, 330, 334, 352. bubonier, 243. pneumonicer, 171. prodigiosus. 74. 80, 103, 108. proteus, 113. pyocyaneus. 247. Salmonii. 227. 306, 318, 334. septicemia hemorrhagic, 1 183. subtilis, 24. 25. 40. 46, 48 50, 54, 58, 62. 66. 70, 72, 76, 80, 99. suipestifer. 227. typhi abdominalis, 235. ee 50, 70, 235, 318, 336, 338,
tetani. 46. Sea, 306, 314, 352. tuberculosis. see Bact. tuberculosis. vulgaris, 113
vulgatus, 95.
Zopfii, 117.
Bacteria
arrangement in groups, 91.
eapsule stain for, 50, 314.
cell grouning of, 42.
classification, 89.
colonies of, 52.
omnanes with gras and moulds, 50. decolorizing.
determining “ae 38.
392
Bacteria drawing, 36. effect of chemicals on, 72. of desiccation on, 70. of direct sunlight on, 72. of moist and dry heat on, 70. flagella stain, 48. formation of ammonia by, 78. of enzymes by, 80. of gas by, 74. of indol by, 78. of sulphuretted hydrogen by, 78. involution forms, metachromatic granules, 50. movement of, 40 non-pathogenic, 938. pathogenic, 138, 271. reduction of nitrates by, 76. relation to oxygen, 72 staining, 30. in tissue, 312. spores of, 46. thermal death point of, 68. Bacteriacex, 89. Bacterium, 89. acidi-lactici, 109. erogenes, 175. anthracis, 46, 167, 304, 314, 330, 360. bovisepticum, 187. capsulatum, 50, 179. cholere, 183, 316. cholerse gallinarum, 183. suum, 227. coli commune, 219. phi he 46, 50, 199, 806, 314, 318,
36 diagnosis, 320. erysipelatos suis, 195. influenze, 211. lactis erogenes, 175. lepre, 314, 316, 350. mallei, 191, 306, 316, 332, 350. pestis, 243. phosphorescens, 121, pneumonia, 50, 207, 804, 314, 328, 332, 350. pneumonicum, 50. 171, 304, 314. sero Cary ae an, 322. rhusiopathie, 195, suicida, 183. tuberculosis, 2138, 304, 314, 316, 326, 834, 344, 346. 350, 360. Gabbett’s stain, 62. in sputum, 326. in tissue, 316. in urine, 346. yen he 814, 318, 352. Zopfii, 1 Baretta analysis, 126. diagnosis, 320. Beef extract, 6. Beggiatoa, 90. Beggiatoacer. 90. Bismarck brown, 28. Black-leg bacillus, 277. Blood, agar, 212. examination of, 330. serum, 138. Boebmer’s hematoxylin, 312. Bolton's potato tubes, 18. Bouillon cultures, 22, 87. dextrose, 64. lactose, 64, preparation of, 6. saccharose, 64 sugar-free, 64. Brownian movement, 40. Buccal secretion, 320. Buchner’s method of growing anaerobes,
bunge’s flagella stain, 48.
INDEX
CAGE for animals, 300. Calcium chloride, 6.
Canada balsam, 382.
Capsule stain, 50, 314.
Carbol-fuchsin, 28, 34.
Carbolic acid, 28, 72, 80.
Care of culture media, 18.
Cell grouping, 42.
Celloidin sections, 310.
Centrifuge, 328
Chain coccus, 141.
Chemicals, antiseptic action of, 134. disinfecting action of, 6. effect on bacteria, 72,
Chlamydobacteriacer, 90.
Cholera group, 92, 251. red reaction, 78, 344.
Chromogenic class, ‘91, 103.
Cladothrix, 90.
Cleaning glassware, 2: slides and cover-glasses, 26. solution, U8.
Coccacer, 89.
Coccus of Mata fever, 153.
Collodion sacs, 296.
Colon bacillus, sce 13. coli. group, 92, 219.
Colonies, 52, color of, 85. counting, 126. miscroscopic structure, 84. surface elevation, 84. ~ types of, 83.
Color of colonies. 85. production, variation in, 80.
Coloring matter, separation of, 108.
Concentration of media,
Condensed milk, 66.
Condenser, Abbé, 34.
Cooling apparatus, 54,
Cornet forceps, 32.
Corrosive sublimate, IX.
Cotton, 4. absorbent for filtering, 14.
Counting apparatus, 126.
Cover-glass preparations, 30.
Cover-glasses, cleaning of, 26.
Grenothrix, 90,
Culture characters of bacteria, 83. media, 6, 10, 138. of anaerobes. 271.
Cultures, agar, 22.
bouillon, 84. gelatin, stab. 22. plate, : hanging-drop, 44. incubation of, 24.
potato, 24 set of, 93. stab, 22. streak, 24. test-tube, 22.
JD)ECOLORIZING agents, 60. Desiccation, effect on bacteria, 70. Dextrose agar, 64. bouillon, 64. free bouillon, 64. , Belatin,. 62. Diagnosis. methods of, 320. of rabies, 354. Diaphragm. iris. 36. Diphtheria bacillus, see B. diphtheria. group, 92, 199. outfit, 82 0. Bip ereene sooner, 157. roup, 91, 157. eeneenitens meningitidis, 159. meumonie, 257. Disinfectants, 72, 136. Drawing bacteria, 36.
INDEX
Drigalski & Conradi’s medium, 342.
Dry air sterilizer, 4.
Dunham's clearing solution, 312. peptone solution, 64
Dyes, anilin, 28.
E&s. use in clearing media, 14. Ehrlich’s anilin oil gentian violet, 28. Embedding tissue, 310. Emphysema group, 92, 273. Endospores, study of, 46. Enzymes, 80 Eosin, 312, Ernst’s stain, 50. Erysipelas group, 91, 141. Esmarch’s roll cultures, 56. Examination of blood, 330. of buccal secretion, 320. of feces, 338 of sputum, 326. of transudates and exudates, 346. of urine, 346. Extract of beef, 6. Exudates, examination of, 346. Eyepieces, sce ocular.
Fecal bacteria, 130.
Feces, examination of, 338. Fermentation tube, 74. Filling tubes aa flasks, 10. a ae
Filtering atch, 14,
agar
Flagella stain, 48.
Folder filter, 8.
Forceps, Cornet, 32. cover-glass, 32 Stewart, 32.
Form types. study of, 40.
Frenkel’s soil borer, 132.
Fiiediander’s bacillus, 171. group, 91, 171.
Frost's gasometer, 74.
Frozen sections, 310.
Fuchsin, 28,
G ABBETT'S methylen blue, 30. tubercle stain, 62. Gas analysis, 74. formula, 74. Gasometer, 74. Gelatin, dextrose, 64. filtering of, 14. melting point of, 24. plate cultures, 52. preparation of, 14. reaction of, 14. sterilization of, 14. Gentian violet, 28) Glanders bacillus, 191. group, 91, 191. Glass slides. cleaning of, aos hollow ground, tumblers, 24. Glassware, cleaning of, 2. sterilization of, 4 Golden pus coccus, 149. Gonococcus. 157. Gonorrheal pus. 348. Gram’s iodine solution, 30. stain, 60. list of organisms taking this stain, 314.
Guinea pig, diagram, 307. inoculation of, 294. Groups, arangement of bacteria in, 91.
HAMMOND’ S method of concentrating puberils bacilli in milk and sputum,
O28. Hanging-drop eres 44, preparation, 38,
393
IIauser’s spore stain, 48.
Ilay bacillus, 24.
Ileat, effect of on bacteria, 68.
Hematoxylin stain, 312.
Hiss’ culture medium, 340.
Hog cholera bacillus, 227. group, 92, 227.
Hot air sterilizer, 4.
Hydrochloric acid, 2, 8.
Hydrogen generator, 271.
[MMERSION lens, 34. Impression preparations, 42.
Incubation of cultures, 24
Incubator, 24.
Indol test, 78, 130.
Influenza bacillus, 211, 328. group, 92, 211.
Inoculated animals, care of, 300.
Tnoculation of animals, 294. into ear, 298. into eye. 300. intraperitoneal, 294. intrapleural, 300. intravenous, 298. lymphathic, 300. subcutaneous, 294. subdural, 354.
Instruments, sterilization of, 300.
Involution forms. 46.
Todine solution, 30, 314.
Iris diaphragm, 34.
J SEFER’ S$ counting apparatus, modified, Plate II, p. 187.
KEY to identification of bacteria, 361. Kipp hydrogen generator, 271.
Klebs-Loefller’s bacillus, 199.
Koch’s method of air analysis, 126.
LABELS. 20.
Lactic acid bacillus, 109.
Lactose agar, 64 litmus agar. 76.
Lens paper, 36
Litmus milk. 66.
solution. 64.
Loeffler’s blocd serum, 138. methylen blue, 28. 7 stain for sections, 312.
Loop, platinum, 20.
M4c CONKEY'S medium, 340. Malaria, 826. Mallory's stain for pmebe. colt, 834. Malta fever group, 91, Media, care of, 12, 18. examination of, 12. labeling, 20. preparation, 6-18, 64, 94, 138. reaction of, 8 sterilization of, 10. Measuring bacteria. 36. Mesophilic bacteria, 68. Metachromatic granules, stain for, 50. Methylen blue, alkaline, 28 Gabbett's 30. Loeffler’s, 28. Mice, white, 294. Micrococcus, 89. gonorrhes, 157. 314, 346, 348. melitensis, 153. pyogenes, var. albus, 145, 314, also pus micrococci). var. aureus, 149, 314 (see also pus micrococci). Weichselbaumii, 159, 350. Micrometer. ocular, 36. stage, 36 Micron, 38. Microspira, 89. comma, 251, 342, 858,
(see
394 INDEX
Microspira
Metschnikovi, 40, 255.
Schulykilliensis, 259. Microscope, 34. Microscopic study of plate cultures, 58.
of bacteria, 40. Migula’s classification, 89. Milk anaylsis, 132, 360.
cultures,
litmus, 66.
pasteurization of, 132. Molecular movement, 40. Monilia candida, 324. Monas prodigiosa, 103. Morphology, 2, 82. Motility of bacteria, 40. Moulds, morphology of, 50. Mucor, 50.
NEEDLE, platinum, 20. Neisser’s diphtheria stain, 322. Nessler’s reagent, 78. Neutralization of media, 6. Nitrate solution, 64. Nitric acid, decolorizing, 60. Nitrites. detection of, 76. Nitroso-indol (cholera red), 78. Non-pathogenic bacteria, 93. Normal solutions, 8. Novy’s anaerobic jars, 271.
OSIJECTIVE, oil-immersion, 34. Ocular micrometer, 36.
Ocular, 34.
Oedema group, 92, 277.
Oil-immersion ojective, 34.
Oospora bovis, 263.
Oxalic acid, 344.
Oxygen, relation of growth to, 72.
PARAFFIN, sealing tubes with, 20. sections, 310. Parietti’s solution, 338. Pasteurization of milk, 132. Pathogenic aerobes, 91, 188.
anaerobes, 271.
bacteria, reaction of media for, 8.
best temperature for, 24. in water and milk supplies, 358.
Penicillium, 50. Peptone, 6. Petri dish cultures. 52. Petri- Sedgwick’'s air analysis, 126, Pfeiffer's cansule bacillus, 179.
stain, 318. Phenolphthalein, 8. Phosphorescent class, 91, 121. Photobacterium phosphorescens, 121. Thragmidiothrix, 90. Physiology of bacteria, 64.
Physiological characters of bacteria, 88.
Pigeons, inoculation of, 294 Pigments, separation of. 107. variety of, 108. Planococcus, 89. Planosarcina, 89. Plasmodium, malaria, 334. Plasmolysis, 83. Plate cultures, agar, 56. gelatin, 52. method of pouring, 54. study of, .58. Platinum needles, 20, Plugging flasks and tubes, 4. Pneumobacillus, 171. Pneumonia group, 92, 207. Post-mortem examination, 302. Potassium hydrate, 28. Potassium iodide, 30. Potato bacillus, 95. Potatoes, preparation of. 18. Preparation of agar, 14. of bouillon, 6. of cover-glasses, 30.
Preparation of gelatin, 14. of hanging-drop, of potatoes, 18. of staining solutions, 25S. of water blanks, 18 Proteus vulgaris, 113. Zenckeri, 117. Pseudodiphtheria bacillus, 203. Pseudomonas, 89. eruginosa, 80, 247, 314, 352. fluorescens, 40. group, 92, 247. violacea, 68. Psychrophilic bacteria, 68. Pus micrococci. 314, 324, 332, 346, 348. Pyrogallie acid, 272.
RALzRITS, 294.
Rabies, diagnosis of, 354.
Rats, white, 294.
Ravenel’s method of making agar, 16. on diagnosis of rabies, 354.
Ray fungus, 263.
Reaction of media, 8, 66.
Ribbert’s method of concentrating the tubercle bacteria in sputum, 326.
Rice cooker, 6.
Roll cultures, 56.
Rubber dam for sealing tubes, 20.
Russell’s water sampler, 128.
eo al cerevisie, 50.
Seed: physiological, 18. Saprogenic class, 91, 113. Saprophilic class, 91, 95.
Sarcina, 89. group, 91, 163. lutea, 0. tetragena, 168, 304, 314.
Sections, 310, 31 12,
Sedgwick's method of air analysis, 126.
Shake culture, 74.
Slides, cleaning of, 26.
Smegma bacillus, 346.
Spirillacee, 89.
Spirillum, 90. cholere Asiatice, 251. rubrum, R4
Spirochaeta, 89 Obermeieri, 532, 346.
Spirosoma, 9.
Spore stain, 48.
Spores, study ad, 46.
Sputum, centrifugalizing, 328. examination of for bacteria, 326. stain of for tubercle bacteria, 62, 326.
Soapstone for cooling plates, 54.
Sodium chloride, 6 hydrate, 8 nitrate solution, 64. nitrite, 78.
Soil analysis, 132.
Stage micrometer, 36.
Stain, anilin, 28. bottles, 30.
Bunge's for flagella, 48. Ernst’s. 50.
Gabbett’s, 62.
Gram's. 60.
Hauser's, 48.
Loeffler's, 312. solutions, 28. Weigert’s, 314.
Ziehl’s, 326.
Stab cultures, 22, 85.
Staphylococcus, epidermidis albus, 145, pyogenes albus, 145. pyogenes aureus, 149.
Steam pressure, 12 sterilizer, 10.
Sterilization, discontinuous, 10. of glassware, 4.
INDEX
Sterilization of instruments, 300. of media, 10. Sterilizer, hot air, 4. Sternberg’s bulbs, 330. Streak cultures, 24, 86. Streptococcus, 89. erysipelatos, 141, 304, 314, 348. lanceglatus, 307. pyogenes, 141. Streptothrix, 90. actinomyces, 263. bovis, 268, 314, 318, 330, 352. group, 92, 263. Madure, LOT. Sugar media, prot lzation of, 12. Sulphanilie acid, Sulphuric acid, 30. Sulphuretted hydrogen, 78. Sunlight, effect on bacteria, 72, Swine plague group, 91, 183. Swine erysipelas, 195. Syringe, sterilization of, 300. use of, 294,
TAXONOMY, 82. Temperature, effect on growth, 68. Testing antiseptic action, 134. disinfecting action, ‘a Test-tube, cleaning of, cultures, 22, agar, 24. blood serum, 24. gelatin, 22. potato, 24. study of, 26. staining of, 30. Tetanus group, 92, 289. Thermal death point, 68. Thermo-regulator, 12, 26. Thermostat, 24. Thionin, 344, Thiothrix, 90. Thrush, organism of, 324. Tin foil, for sealing tubes, 20. Tissue embedding, 310. fixing, 310. handling sections, 310.
preparation of for examination, 310.
sectioning, 310.
395
Tissue, staining of bacteria in, 312. Titration of media, 6. Transudates, examination of, 346. Tubercle bacillus, 213.
group, 92, 213.
stain, 62, 326. Tyndallization, 10. Trphoid bacillus, 235.
group, 92, 235,
URINE, examination of, 346.
VIBRIO cholere, 251. Metschnikovi, 255. ‘Schuylkilliensis, 259. subtilis, 99. Van Gehucten & Nélis’ method of diag- nosing rabies, 354.
W ATER analysis, 128, 358. reaction of media for, 8. bath, 56. blanks, 18. Weigert’s iodine solution, 314. stain, : Welch’s capsule stain, 50. Wetluein medium for the gonococcus, 158, Wesbrook’s animal cage, 300. hanging-drop culture, 44. White mice. 4. rats, 294. -staphylococcus, 145. Widal reaction. 334. , Weens method of cultivating anwrobes, 7
Wurtz’ lactose litmus agar, 76.
X EROSE bacillus, 203. Y BASTS, morphology of, 50.
7 EHL'S earbol-fuchsin, 28. Ziehl-Neelsen's stain, 326, Zymogenic class, 91, 109.