A laboratory guide in elementary bacteriology

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LIBRARY
NEW YORK STATE VETERINARY COLLEGE
ITHACA, N. Y.

This Volume is the Gift of

Dr, V. A. Moore

Cornell University

The original of this book is in
the Cornell University Library.

There are no known copyright restrictions in
the United States on the use of the text.

http://www.archive.org/details/cu31924000870109

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LABORATORY (ZUIDE

IN

ELEMENTARY BACTERIOLOGY

BY

WittiamM DODGE FROST

INSTRUCTOR IN BACTERIOLOGY, UNIVERSITY OF WISCONSIN

ILLUSTRATED

PREFACE.

The following pages constitute, substantially, the material which has been furnished the stu-
dents in Bacteriology at the University of Wisconsin, in mimeograph form, for several years. They
contain directions for the performance of certain fundamental exercises in Bacteriology.

Ina rapidly developing subject it is important that the various exercises be worded so as to lend
themselves readily to changes which become desirable from time to time. With this end in view the
exercises have been divided, where possible, into a general and a special part. The general directions
contain the essential part of the exercise which does not permit of any considerable variation, while
the special directions embrace such features as are most subject to modification, as for instance,
the particular organism to be used, the kind of medium, the incubation temperature, etc. Desirable
changes here are easily indicated when the exercise is assigned.

Some of the exercises can be performed in a few minutes, while others require several days
for their completion. No attempt has been made to group them according to their length, nor to
divide the text into lessons, but as far as possible they are arranged in the order in which they would
be logically used in the laboratory.

The right hand pages have been left for notes and drawings with the idea that notes in perma-
nent form are the only ones of value to the student in subsequent years.

The charts of the various organisms furnish a most satisfactory means for recording the observa-
tions made during the study of a germ and are especially convenient for reference.

Part I. is the work required of students taking the General Course in which special emphasis is
placed on the biology of bacteria. It is completed in the first semester. Part II. which is given
during the second semester includes the more specialized phases of the work, particularly as applied
to the student preparing for medicine.

References have been made to all of the leading English text-books and occasionally to original
sources. It is expected that the student will make constant use of these references.

My thanks are due Prof. H. L. Russell under whose general direction the work outlined here is
given, for valuable help in the selection and arrangement of the material and for generous council, I
am also indebted to Mr. E. G. Hastings, Assistant Bacteriologist to the Wisconsin Experiment Station,
for critical reading of manuscript and proof. .
WILLIAM DODGE FROST.
MapIson, WiIs., January, 1901.

(iii)

CONTENTS.

List of References...........-. 83 Sit cecusded 8 8-0 E SA MENOWE RE 400 Oi Swi eae e he spree A fds aeaiaon dash aupe Ree ise vi
List of Apparatus. ........0. cece cece eee ee eet e ne terete erent eters f nee nee tense tence enenes vii
Laboratory Rules........ cc eee cece ene ee een eee eee ene teen esaaea cert sree ttesanees . Vili

PART I.—GENERAL BACTERIOLOGY.

Cuarter I. MoreHoLtoGy AND ELEMENTARY TECHNIQUE.

EXERCISE. PAGE. | EXERCISE. _ PAGE.
I. Cleaning Glassware.............. 2 XX. Test-tube Cultures Illustrating
II. Plugging Flasks and Tubes....... 2 . Form Types s4 2 ssieseuws s+ oss 26
III. Sterilization of Glassware........ 2 XXI. Study of Test-tube Cultures 26
IV. Preparation of Bouillon ......... 4 XXII. Microscopical Study of - Forn
V. Filling Test-tubes and Flasks with TY POS ec asssecieeny cetera! wos eee ace 26
Culture Media ................ 6 XXIII. Drawing Bacteria...............- 28
VI. Sterilization of Culture Media.... 8 XXIV. Study of Cell Grouping .......... 30
VII. Preparation of Gelatin. ........ 10 XXV. Study of Involution Forms....... 30
VIII. Preparation of Agar............. Io XXVI. Gelatin Plate Cultures........... 32
IX. Preparation of Potatoes. ....... 12 XXVIL. Agar Plate Cultures ............. 34
X. Preparation of Water- blanks nigh as 12 XXVIII. Roll Cultures ......... .... 202008 34
XI. Care of Culture Media..>........ 14 |. XXIX. Study of Plate Cultures.......... 36
XII. Platinum Needles ............... 14 XXX. Use of Decolorizing Agents ...... 36
XIII ' Test-tube Cultures. ...5.,........ 14 XXXKI. Gram's Stain ..........0.02 sees 36
XIV. Incubation of Cultures. % . 16 XXXII. Tubercle Stain (Gabbett)......... 38
XV. Cleaning Slides and Cover- glasses. 18 XXXIII. Staining Endospores............. 38
XVI. Preparation of Staining Solutions. 18 XXXIV. Study of Endospores .... ....... 40
XVII. Simple Cover-glass Preparation... 20 XXXV. Flagella Stain...........---0..0. 40
XVIII. Use of Microscope .. .......... 22 XXXVI. Capsule Stain.... .............. 42
XIX. Hanging-drop Breparehion: ocr 24

CuarTer II. PxHystoLoGy oF BACTERIA.

EXERCISE. PAGE. | EXERCISE, PAGE.
XXXVIL. Preparation of Special Media..... 44 XLVII. Detection of Gas............... 50
XXXVIII. Effect of Reaction of Media on XLVIII. Quantitative Analysis of Gas . 50
GROWN onic cis oa Wnaewon nies 44 XLIX. Detection of Acids ............ 52
XXXIX. Effect of Concentration of Media L. Quantitative Determination :of Acids 52
on. Growth 2 66. ..608 senenes 46 LI. Detection of Nitrites in Cultures... 52
XL. Effect of Temperature Variations LII. Detection of Ammonia....... .. 52
on Rate of Growth............. 46 LILI. Detection of Sulphuretted Hy-
XLI. Determination of Thermal Death TORBEN cris. teas ss aipails va sistas 54
FOUR, coal veenmens bok eend omeee 46 LIV. Detection of Indol............... 54
XLII. Comparative Efficiency of Dry and LV. Determination of Chemical En-
Moist’Heéat 0.2 2 ss cesinee eg ee 48 zymes in Cultures ...........6. 54
XLIIL. Effect of Desiccation ............ 48 LVI. Variation in Enzyme Production... 54
XLIV. Effect of Chemicals on Bacteria... 48 LVII. Variation in Color Production.... 56
XLV. Relation to Oxygen............ +. 50 :
XLVI. Effect of Direct Sunlight ........ 50

CuapTerR III, Taxonomy.

PAGE,

Points to be observed in the study of Bacteria.. 57 | Classification of Bacteria (Migula)............. mao
CuapTeR IV. SysTEMATIc Stupy oF REPRESENTATIVE Non-PATHOGENIC BACTERIA.

EXERCISE. ; : PAGE. | EXERCISE. PAGE
LVIII. Preparation of Special Media..... 63 LXII. Bacterium phosphorescens ....... 68

~ LIX. Bacillus prodigiosus ore eee 64 LXIII. Bacillus acidi lactici:........... , 70
LX. Variety of Pigments ............. 66 LXIV. Bacillus vulgaris ...........0... 72

LXI. Separation of Bacterial Coloring
Mattef wa ieanas cetncamaaaa dick ce 67

(iv)

CHAPTER V. BACTERIOLOGICAL ANALYSIS.

EXERCISE. PAGE. | EXERCISE. PAGE.
LXV. Comparative Analysis of Air...... 80 LXIX. Water Analysis... .......-.0eeeeee 82
LXVI. Quantitative Determination of LXX. Quantitative Analysis of Milk. 84
Number of Bacteria in Air..... 80 LXXI. Efficiency of Pasteurization...... 84

LXVII. Relation of Bacteria in Air to Dust LXXII, Testing Antiseptic Action of Chem-
PartiGlesic: o.<cocawiae neon dunsits 82 ICAISE cwesvinnactavirs. dave ane danse one 84

LXVIII. Estimation of Number of Bacteria LXXIII. Testing Disinfecting Action of
in Soil.......... 5 ahaa RIPEN 82 Chemicals ............... see 86

PART II. MEDICAL BACTERIOLOGY.
CuHarter VI. PaTHOGENIC AEROBES. ,
EXERCISE. PAGE. | EXERCISE. . PAGE.
LXXIV. Preparation of Culture Media..... 88 LXXXVII. Bacterium tuberculosis ......... - 114
LXXV. Streptococcus pyogenes .......... go | LXXXVIII. Bacterium mallei....... ........ 116
LXXVI. Micrococcus pyogenes ........... g2 LXXXIX. Bacterium diphtheriae ........... 118
LXXVIL. Micrococcus melitensis .... 94 XC. Bacterium influenzae ............ 120
LXXVIIL. Micrococcus aureus.............. 96 XCI. Bacillus typhosus.... ........+.. 122
LXXIX. Micrococcus gonorrhoeae......... 98 XCII. Bacillus pestis ...............0- 124
LXXX. Micrococcus intracellularis ...... 100 XGIII. Bacillus suipestifer ............ +. 126
LXXXI. Sarcina tetragena................ 102 XCIV. Bacillus icteroides........ ..... 128
LXXXII. Bacterium anthracis............. 104 XCV. Pseudomonas aeruginosa......... 130
LXXXIII, Bacterium pneumoniae. ......... 106 XCVI. Microspira comma .............. 132
LXXXIV. Bacterium pneumonicum......... 108 XCVII. Microspira metschnikovi...,..... 134
LXXXV. Bacterium cuniculicida.......... 110 XCVIII. Microspira finkleri .......... 136
LXXXVI. Bacterium rhusiopathiae......... 112
CuHaPTeR VII. PATHOGENIC ANAEROBES.
EXERCISE. PAGE. | EXERCISE. PAGE.
XCIX. Bacterium welchii .............. 150 CI. Bacillus oedematis .... ......... 154
C. Bacillus chauvaei .......... .... 152 CII. Bacillus tetani.................. 156
CHAPTER VIII. ANIMAL INOCULATION AND STAINING OF BACTERIA IN TISSUE.

EXERCISE. . PAGE. ; EXERCISE. : PAGE.

CIII. Animal Inoculation.............. 162 CIV. Preparation of Tissue for Exami-
nation.. SRE DY 844 aa ERT 170
CV. Staining Sections. Sache he RONS te 172

CuarTeR IX. BACTERIOLOGICAL DIAGNOSIS.

EXERCISE. PAGE. | EXERCISE. PAGE.

CVI. Examination of Buccal Secretion.. 178 CXI. Examination of Transudates and
CVII. Examination of Sputum.......... 180 XAG a tO 2 a0. 5 ao: avecanatereis bee ve ar 194
CVIII. Examination of Blood............ 184 CXII. Diagnosis of Rabies............. 198

CIX. Examination of Faeces........... 188 CXIII. Examination of Material from Hu-
CX. Examination of Urine........... 192 man Autopsies ...............- 198

CuapTrer X. DETECTION OF PATHOGENIC BACTERIA IN WATER AND MILK SUPPLIES.

EXERCISE. PA EXERCISE. PAGE,

CXIV. Examination of Water for Patho- CXV. esainalion of Milk for Patho-
genic Bacteria ..............., 200 genic Bacteria................. 200

LIST OF TEXTS AND REFERENCE WORKS WITH ABBREVIA-
TIONS USED.

A.— Abbott: Principles of Bacteriology. Lea Bros. & Co. Philadelphia, 5th Edit., 1899.

B.— Bowhill: Manual of Bacteriological Technique. Oliver & Boyd, London, 1899.

F.— Fischer: Structure and Functions of Bacteria. Clarendon Press, New York, 19009.

Fr.— r Frankland: Micro-organisms of Water. Longmans, Green & Co., 1894.

G. - Gage: The Microscope. Comstock Pub. Co., Ithaca, N. Y., 7th Edit., 1899.

H.— Hewlett: Manual of Bacteriology. Blakiston, Son & Co., Philadelphia, 1898.

J. H.— Jordan's Translation of Hueppe: Principles of Bacteriology. Open Court Pub. Co., Chicago,
1899.

v. J v. Jaksch: Clinical Diagnosis. Charles Griffin & Co., London, 4th Edit., 1899.

K. & D.— Kanthack & Drysdale: Practical Bacteriology. MacMillan Co., New York, 1895.

L.— Lafar: Technical Mycology. Vol. 1. Lippencott Co., Philadelphia, 1898.

L.& K.— Levy & Klemperer: Clinical Bacteriology. Saunders & Co., Philadelphia, 1900. :
L. & N.— Lehmann & Neumann: Atlas and Essentials of Bacteriology. Wood & Co., New York, 1897.
M.-— Moore: Laboratory Directions for Beginners in Bacteriology. Ginn & Co , New York, 1900,
M. & R.— Muir & Ritchie: Manual of Bacteriology. MacMillan Co , New York, 2nd Edit., 1899.

M. & W.—Malloty & Wright: Pathological Technique. Saunders & Co., Philadelphia, 1897.

McF.— McFarland: Text-Book of Pathogenic Bacteria. Saunders & Co., Philadelphia, 2nd Edit., 1898.
N.— Novy: Laboratory Work in Bacteriology. Geo. Wahr, Ann Arbor, Mich., 2nd Edit., 1899.
Ne.— Newman: Bacteria. Putnam, New York, 1899.

P.— Park: Bacteriology in Medicine and Surgery. Lea Bros. & Co., Philadelphia, 1899.

P. B. C — Proceedings of the Bacteriological Committee from Jour. Amer. Pub. Health Assn. Vol. XXIL.
P. & M.— Peamain & Moor: Applied Bacteriology. Bailligre, Tindall & Cox, London, 1897.

s.— Sternberg: Manual of Bacteriology. Wood & Co., New York, 1893.
Si.— Simon: Clinical Diagnosis. Lea Bros. & Co., Philadelphia, 2d Edit., 1897.
w.— Woodhead: Bacteria and Their Products. Charles Scribner & Sons, New York, 1892.

Wm.— Williams: Manual of Bacteriology. -Blakiston, Son & Co., Philadelphia, 1898.
[vi]

LIST OF APPARATUS.

This list comprises the apparatus which is to be under the exclusive control of the student and
does not include the general laboratory outfit, such as sterilizers, incubators, microscopes, general

chemical supplies, etc.

FOR INDIVIDUAL USE,
A.

50 (4 oz.) cover-glasses, 18 mm. (3 in.) square
and 0.17 mm. thick (No. 2).
50 glass slides.
100 labels, 2 cm. square.
12 cm. platinum wire (No. 27).
1 pair cover-glass forceps (Cornet or Stew-
art).
1 pair fine pointed forceps.
2 slide boxes for 50 slides.
1 hanging-drop slide.
1 towel.
B.

- 1 flask, 1000 cc.
1 flask, 400 cc.
3 flasks, 250 cc.
1 flask, 100 cc.
200 test-tubes (15 x 120 mm.).
15 Petri dishes (10 cm).
2 fermentation tubes.
2 glass tumblers.
4 tin cans.
2 glass rods for platinum needles.
3 pipettes, 1 cc.
1 brass tube to hold pipettes (25 x 250 mm.).
8 stain bottles with pipettes, in block.
1 waste dish.
1 yard of muslin.
3 sheets of filter paper.
3 sheets of lens paper.

[vii]

FOR GROUP USE (About Four Students).

1 glass funnel, 12 cm.
1 glass funnel, 5 cm.
1 filtering flask with rubber stopper.
2 stirring rods.
1 pipette, 5 cc.
1 thermometer, 0-100° C.
10 cm. rubber tubing. 1 cm. dia.
1 Mohr stopcock.
1 anaerobic jar for plates.
lanaerobic jar for tubes.
1 potato knife.
1 Bunsen burner with tubing.
1 piece of wire gauze.
1 tripod with reducing rings.
1 rice cooker.
8 small wire baskets.
1 enamel pan.
1 roll of cotton wool.
¥% lb. absorbent cotton.
1 piece of Russia iron, 12 cm. square.
1 graduated cylinder, 300 cc.
1 graduated cylinder, 100 cc.
1 graduated cylinder, 25 cc.
1 evaporating dish, 10 cm.
1 disinfecting jar.
1 copper cup.
1 ring stand with clamp.
1 test-tube brush.

See Fig. 1.

LABORATORY RULES.

I. Food should not be eaten in the laboratory and lead pencils or labels should
not be moistened with the tongue.

II. All possible cleanliness should be observed in the care of apparatus, desk,
ete.

Ill. The platinum needles used in making cultures should be sterilized shortly
before and immediately after use and before they are laid down. When the
needles are covered with infectious material they should be held at the side of
the flame until dry before being sterilized; this will avoid the danger of scatter-
ing this material about the laboratory.

IV. If infectious matter should by accident come in contact with the hands or
be dropped on the table or floor, corrosive sublimate (1:1000) should be imme-
diately applied.

V. Solid material, culture media and corrosive sublimate should not be put
in the sink but in crocks provided for the purpose. Burnt matches, pieces of
paper, ete., should also be put in the crocks and not on the floor.

VI. All cultures of bacteria should be labeled with the name of the organism,
the name of the student and the date.

VII. Discarded cultures 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 infectious material
should be placed in a glass cylinder containing a disinfectant or potassium bi-
chromate and sulphuric acid.

VIII. When using the steam sterilizer see that there is enough water present
before lighting the gas and do not leave the laboratory until the gas has been
turned off. ‘is

IX. Before beginning an exercise read over the directions and look up some
of the references. Keep notes of everything done and the conclusions reached
on the right hand pages in this Guide. Make drawings wherever they will be
of value. Outline with pencil and fill in with India ink. The laboratory Guide
should be kept in the laboratory.

X. At the close of the day’s work the tables should be washed with corrosive
sublimate and the hands disinfected by washing in the sublimate solution (or a
germicidal soap) and then in soap and water.

[viii]

PART I.

GENERAL BACTERIOLOGY.

PART I.—GENERAL BACTERIOLOGY.

CHAPTER I.

MORPHOLOGY AND ELEMENTARY TECHNIQUE.

EXERCISE I. CLEANING GLASSWARE.

. GENERAL Direcrions. All glassware to contain culture media must be thoroughly
clean. New glassware should be washed in hot soap-suds' (a test-tube brush will be
needed for the test-tubes), rinsed in tap water and then placed for a few minutes in water
to which about 1% of hydrochloric acid has been added to remove free alkali frequently
present on new glass, and then thoroughly rinsed in tap water. It is then allowed to-
drain. Test-tubes and flasks are best dried by placing them on a drain board especially
prepared, or standing them mouth down in a box with a cloth bottom or on filter paper.

Glassware containing media (discarded cultures, etc.), is best cleaned by first stand-
ing in water for some hours, or by being steamed and pouring out the material while in
a liquid condition and then cleaning as above with the exception of the use of the hydro-
chlorie acid.

REFERENCES. A. 120; H. 39; K. & D. 81; M. & W. 74; N. 158; P. 223.

SPECIAL Directions. Clean as directed above, all flasks, test-tubes, fermentation
tubes and Petri dishes in your possession.

; EXERCISE II. PLUGGING FLASKS AND TUBES.

GENERAL DirEcTIONS. When the flasks, test-tubes and fermentation tubes are
thoroughly dry they are to be plugged with cotton. The cotton for this purpose should
be of the best non-absorbent quality, i. e., as free from foreign matter as possible. The
plugs should be sufficiently loose to permit the interchange of gases and at the same time
tight enough to support the weight of the vessel and its contents, to prevent their being
pulled out in handling the vessel. The cotton should be rolled into a cylinder of the
proper diameter and long enough to extend into the mouth about 24 em. (1in.) and pro-
ject sufficiently to protect the lips from dust. The plug should be pushed in straight and
not twisted; the surface next to the glass must be perfectly smooth, presenting no
creases for the entrance of dust.

REFERENCES. A.121; H. 39; M. & W. 74; M. & R. 56; McF. 107; P. 223.

SpeciaL Directions. Plug all test-tubes, flasks and fermentation tubes in your
possession.

EXERCISE Ill. 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

4 General Bacteriology.

be so arranged that a temperature of 150° C. can be quickly reached and readily main-
tained. In such a sterilizer all glassware to be used for the reception of culture media,
such as flasks, test-tubes, Petri dishes, etc., is submitted to a temperature of 140-150° C.
for 1 hour, or until the cotton plugs are slightly browned; this change being due to the
incipient charring of the cotton. The test-tubes are placed erect in square baskets made
of galvanized iron wire. When the air in the sterilizer has cooled to about 40° C. the
glassware can be taken out and stored ready for use. The Petri dishes are not to be opened
until used for culture purposes.

Rererences. A. 71 and 121; H. 32; L. & K. 74; M. & R. 36; N. 159; McF.
106; P. 228; S. 51.

SpectaL Directions. Ail glassware prepared in I. is to be sterilized for one
hour at 150° C. The small pipettes should be placed in brass tubes, provided for the
purpose, and also sterilized.

EXERCISE IV. PREPARATION OF BOUILLON.

GENERAL Directions. Any one of the three methods (A B or C) may be used.
They are arranged in order of preference, but method C is the most convenient, and
hence most used.

A.
a. From 500 grams (14 Ib.) of lean

beef, remove the fat and connective tis- .

sue and mince (Hamburg steak).

b. Add 1 liter of distilled water and
after thoroughly shaking set in ice chest
for 12 to 24 hours. :

c. Squeeze through a cloth and add
enough distilled water to make 1 liter
and place in vessel to cook. This may be
done either in a flask which is heated ina
water-bath or a sterilizer, or in a rice
cooker. In this case use 250% solution of
calcium chloride in outer vessel instead
of water as by this means the contents
of the inner vessel can be brought to a
rapid ebullition, something impossible by
the use of water alone.

d. Boil + hour and make up loss of
water.

e. Add to any of the above solutions:

B.

Secure meat as
under A a, add 1 liter
of distilled water,
weigh (see e below),
cook for } hour at
about 70° C., and pro-
ceed as directed under
e below.

Cc.

Weigh out three
grams of beef extract
(such as Liebig’s),
add 1 liter of water,
and then proceed as
directed under e be-
low.

1% (10 gms.) peptone (Witte) and +% (5 gms.) common salt (NaCl), then weigh
solution, with vessel, so that the water which is subsequently driven off in cooking can

be accurately replaced.

f. Heat until ingredients are in solution, then restore the water lost by evaporation.

g. Neutralize or render slightly alkaline.

great care.

work. For ordinary routine work B may be employed.

This is a very important step and calls for
Method A is more accurate and should be employed for special or research

6 General Bacteriology.

A.

1.) Titrate as follows: Pipette off 5 cc. of the fluid
into a4-inch evaporating dish, add 45 ce. of distilled
water, boil for three minutes, add 1 cc. of phenol-
phthalein (0.5% substance in 50% alcohol), and then
carefully run in, drop by drop, from a burette a twen-
tieth normal *solution of sodium hydroxide (yNa
OH) until the solution turns a faint pink color. Treat
two other samples in the same way. If the amount
of Na OH required is approximately the same in each
case the average can be taken as the amount necessary
to neutralize 5 cc. Calculate the amount necessary
to neutralize the whole (1000-15 ec.). Since this
amount would dilute the medium too much, a stronger
solution (normal) is used, hence,

2.) Neutralize by adding #yth of the volume eal-
culated above of a normal solution of sodium hydrox-
ide. Test the accuracy of the work at this point by
the addition of a few drops of phenolphthalein to a
ec. or so of the medium. If a faint pinkish tintis not
obtained, titration and neutralization must be re-
peated.

B.

Use a normal solution of so-
dium hydroxide ({NaOH). Add
to the hot solution a few ec. at a
time, at first, later afew drops,
stirring thoroughly with a glass
rod. After each addition, test
by placing a drop of the solu-
tion by means of the glass rod
on a strip of red litmus paper,
and then moisten the paper with
distilled water. The addition
should continue until the red
litmus paper is turned blue, but
no change occurs on blue litmus

paper..

If by mistake more alkali is added than is required, the reaction ean be corrected by

the use of a normal solution of hydrochloric acid.
h. Boil for 5 minutes and restore weight.
1. Test reaction and adjust if necessary.

j. Add 0.5 to 1.5% of a normal hydrochloric ania if neutralized by method A, oth-

erwise omit.

The amount of acid to be added varies with the purpose for which the

medium is to be used, e. g., in water analyses +1.5 (acid) is preferable, with the path-
ogenic bacteria a smaller amount of acid (+ 0.5) more nearly meets requirements.
k. Filter through moistened filter paper (Abbott p. 96), or absorbent cotton,

(VII. m).

If the filtrate is not perfectly clear, cool to 60° C.,

add the white of an

egg, thoroughly mix and boil for 5 minutes without stirring.
The filtrate (bouillon) should be of a light straw color, perfectly clear, and should

not give a precipitate on boiling.
REFERENCES.

SPECIAL DIRECTIONS.
Secure and put to soak meat for VII.

A. 90; M. & R. 48; McF. 124; N. 234; P. 212;

P. B.C. 18-24.

Prepare 1 liter of bouillon according to method C.

EXERCISE V. FILLING TEST-TUBES AND FLASKS WITH CULTURE MEDIA.

GENERAL DIRECTIONS.

In filling tubes be careful not to allow the media to touch

the neck of the vessels as this will cause the cotton to stick to the glass when the plugs
are removed. Place the culture fluid to be tubed in a funnel arranged with a delivery

* Normal solutions are prepared so that one liter at 16° C. shall contain the hydrogen equivalent

of the active reagent weighed in grams (Sutton).
hydrate is sufficiently accurate.

For present purposes a 4 % solution of sodium

8 General Bacteriology.

tube and stopcock (fig. 1), from which it can be run into sterile vessels. Test-tubes
should contain 6-10 ec. of medium (about 3 cm. deep). Flasks are to be filled about
three-fourths full.

SPECIAL Directions. Fill 15 test-tubes and preserve remainder
of bouillon in larger flasks.

EXERCISE VI. STERILIZATION OF CULTURE MEDIA.

EXPLANATORY. Toaccomplish this steam is used almost exclusively

either as streaming steam or under pressure. The unconfined steam is
applied in an apparatus known as a steam sterilizer. Of the various
patterns the Arnold is perhaps the most satisfactory. It is effective,
economical in the use of gas, and does not allow the escape of large
quantities of steam into the room, as a large part is condensed to be re- Fic. 1, Appara-
converted into steam. For student use the form shown in fig. 2 is very ea ald
convenient. The method of using these different forms is identical.
Always have plenty of water present before heating. The discontinuous method is most -
frequently employed. Exposure is made on three consecutive days for 20 minutes, be-
ginning to count time when the material reaches the temperature of the steam, which will
vary with different substances and the volume treated. Between successive steamings
culture media should be kept under conditions favorable to bacterial development (room
or incubator temperature).

For the employment of steam under pressure the
autoclave is essential. The lid should contain a thermom-
eter as well as a steam gauge, safety and outlet valve. <A
thermo-regulator is also desirable. The following table
gives the temperature corresponding to atmospheres of

-

_ pressure:
Atmospheres. Degrees C.
1 100
aoe Bb 1.5 112.2
Fic. 2. Simple sterilizer consisting 2 121.4
of a galvanized iron pail with a cover 9.
aand a false bottom 4. 5 128.8
3 135.1

This table is only true when all of the air in the apparatus is replaced by steam,
and hence the steam must be allowed to escape freely before the outlet valve is closed. A sin-
gle exposure of 20 minutes at a temperature of 120° C. (one additional atmosphere) is
. sufficient to kill all germ life. After the proper exposure, care must be taken not to allow
the steam to escape too rapidly, otherwise the culture media may be forced against the
. plugs owing to the unequal pressure.

GENERAL DIRECTIONS. Ordinary media may be sterilized by either method. Sugar
media cannot be sterilized in the autoclave as it must not be heated above 100° CO. The
solidifying property of gelatin is impaired if submitted to a temperature of 120° C.
longer than 15 minutes, and at a temperature above 120° C. momentarily.

REFERENCES. A. 55-73; M. & R. 87; McF. 109; N. 161; P. 213.

SpectaL Directions. Sterilize bouillon prepared in IV. for 20 miuutes in a
steam sterilizer on three consecutive days.

10 General Bacteriology.

N. B. Some time is required to raise the temperature of the media to that of the steam,
especially if the vessels are large.

All media should be carefully examined every day for a week or more, and if ‘‘specks’’
or the least cloudiness appears, the medium is not sterile and the process of sterilization
must be repeated.

All receptacles containing media should be labeled after sterilization. For this pur-
pose labels can be purchased, the size used: for glass slides, or gummed paper in sheets
can be cut into squares (2 cm.). The labels are to be attached to each vessel 1 em. from
the lip. The kind of medium and the date of preparation should be written across the
top, as ®9°/44°% leaving the rest of the label to be filled in when the medium is inoc-
ulated.

EXERCISE VII. PREPARATION OF GELATIN.

GENERAL DIRECTIONS.

ato d. Same as bouillon. (IV.)

e. Add 1% peptone; 0.5% salt and 10-15% * of best white gelatin, and weigh.

f. Heat until ingredients are dissolved.

g. Neutralize.

h. Boil 5 minutes and restore weight.

i. Test reaction.

j. If neutralized by method A add 5 ec. of a normal hydro-
ehlorie acid. In method B omit acid.

k. Cool and add egg and boil 5 minutes.

1. Filter. Arrange the apparatus shown in fig. 3. Use
absorbent cotton. The funnel and flask should first be heated
with warm water. Start the filter pump before pouring in the Qa
culture medium. This prevents the unfiltered gelatin from fy¢.3, apparatus for filtering

o °c o oe AUT,
q

passing between the cotton and the glass. media through absorbent cotton;
7 a, layer of cotton; 3, tubes for
ats Tube. (V.) making connection with air
n. Sterilize. pump; c, Bunsen valve to prevent
o. Label entrance of water into flasks.

REFERENCES. A. 95; H. 42; M. & R. 46; McF. 127; N. 153; P. B. C. 26.

SpeciaL Directions. Make 1 liter, using method A. Fill 30 test-tubes. Put
the remainder in flasks, sterilize in steam sterilizer or autoclave. Remember long exposure
to high heat injures the solidifying properties of gelatin.

EXERCISE VIII. PREPARATION OF AGAR.

GENERAL DIRECTIONS.

a. Add 15 grams of agar-agar threads (finely chopped) to 500 ec. of water and either
(1)boil until the agar-agar is dissolved (about } hour) and make up loss of water by evap-
- oration, or (2) dissolve in autoclave by heating up to 120° C., closing off gas and allow-
ing to cool.

* The amount to be varied according to the season of the year, 10 per cent in winter, 12-15 per
cent in summer, but it should be remembered that different quantities affect the appearance of the
culture.

12 General Bacteriology.

b. 1)-4) Same as a-d in the preparation of bouillon (IV.), except that only one-
half (500 ec.) of the amount of water is added to the beef or extract.

5) Add 1% peptone and 0.5% salt.

6) Heat until peptone is dissolved.

7) Neutralize.

8) Mix a. and b. (in case beef is used it will be necessary to cool a. to about 60°.
C. before mixing).

9) Boil 5 minutes and restore weight.

10) Test reaction. :

11) Addition of egg will be necessary only where extract is used.

12) Filter as in case of gelatin, (IV. m.)

13) Tube.

14) Sterilize in steam for 15 minutes on three suc-
cessive days or in autoclave for 20 minutes at 120° C.
After the last sterilization place most of the tubes in a
sloping position to harden (fig. 4), these are known as
agar slopes. Those solidified horizontally can be used for plate cultures.

15) Label.

REFERENCES. A. 100; H. 43; M. & R. 48; McF. 129; N. 235; P. B.C. 27; S.
43; Journal of Applied urossony: 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.

Fic. 4. Method of sloping agar.

EXERCISE IX. PREPARATION OF POTATOES. (BOLTON.)

GENERAL DIRECTIONS.

a. Select a number of rather large test-tubes (15020 mm.) place a
small wad of absorbent cotton in the bottom of each (fig. 5 a), plug and We)
sterilize as usual.

b. Wash a large potato, then with a cork borer slightly smaller than
the test-tubes punch out cylinders about 5-6 cm. long.

c. Divide these diagonally and trim to shape indicated in fig. 5 b.

d. Add a few drops of distilled water to each test-tube and place
pieces of potato in position.

e. Sterilize on three consecutive days for 30 to 45 minutes.

Unless the tubes are to be used immediately, they should be sealed.
(XI.) The dark color can be prevented by immersing the pieces between ¢ Fic.5. Bolton’s
and d@ in running water for from 12-18 hours. Potatostubes

REFERENCES. A. 104; M. & R. 54; McF. 134; N. 183; P. 216; P. B.C. 28; S. 47.

SpeciaL Directions. Prepare 15 test-tubes of potato, sterilize, label, and seal with
paraffin. (XI. 2.)

EXERCISE X. PREPARATION OF WATER-BLANKS.

GENERAL DtREcTIONS. Water-blanks are prepared by placing exactly 10 ce. of a
physiological salt solution (6 poe: per 1,000 ce. of water) in test-tubes and sterilizing in
autoclave 15 minutes at 120 ° C., or in steamer 15 minutes on three successive days.

SPECIAL DIRECTIONS. Prepare and sterilize 10 water-blanks.

14 General Bacteriology.

EXERCISE XI. 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 considerable 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 plugs, dipping same in melted paraffin (melting point about
50° C.) and then replacing them; (3) by cutting off the projecting cotton and
drawing over the mouth of the vessel a rubber cap (made for the purpose) which has
been sterilized in a solution of mercuric bichloride (1: 1,000, spoken of in the lab-
oratory as ‘‘sublimate solution’’); or (4) By use of a cap of tin-foil. In this case the
foil should be put on as soon as the tubes are filled, and sterilized with the medium.

All media should be carefully examined every day for a week or more, and if spots or
the least cloudiness appears, the medium is not sterile and the process of sterilization must
be repeated.

EXERCISE XII. PLATINUM NEEDLES.

GENERAL DIRECTIONS. These are made by > See
fusing a piece of No. 27 platinum wire (5 cm.
long) into a glass rod or tube (18cm. long). (Fig. ei , RENEE
6.) Hach student should have two such needles; ee
in one the wire should be straight (designated ‘‘needle’’) and the other bent to form
a ‘‘loop’’. This loop should be formed around a No. 10 wire. These instruments must

be sterilized shortly before and immediately after use by heating the wire to a glow in
the gas flame. Thehandle should also be passed through the flame two or three times.
Cool before using. If the habit of sterilizing is thoroughly acquired much trouble will
be avoided and possible danger prevented. These needles will be in constant use.

REFERENCES. A. 125; M. & R. 58; N. 172; P. B. C. 33, foot note.

EXERCISE XIII. TEST-TUBE CULTURES.

ExpLanaTory. The extreme minuteness and slight variation in the form of dif-
ferent bacteria render a thorough study of them by direct microscopic observation a dif-
ficult and well nigh impossible task. In their study, therefore, it is necessary to depart
from the usually accepted rules that govern the determination of the life history of other
forms of life and resort to special methods. The most successful of these are those
known as culture methods. According to these methods the bacteria are sown on vari-
ous food substances and upon these they develop forming masses easily visible to the
naked eye. The manner of their growth and the changes which they produce in these
media make it possible to detect differences which would otherwise escape attention.
The most common culture media, bouillon, gelatin, agar and potato have already been
prepared, and others will be described as needed.

Cultures may be made either in test-tubes (streak or stab cultures), or on glass
plates, as plate cultures. The plate culture is especially important and is used (a) to
obtain pure cultures; and (b) for ascertaining the character of the colonies as an aid to

16 General Bacteriology.

their diagnosis. The tube-cultures are serviceable in giving opportunity for a further
study of the characters as well as to furnish the most convenient method of maintain-
ing the cultures.

GENERAL DIRECTIONS. Bacteria when ob-
tained in ‘‘pure culture’’ are usually grown in
test-tube cultures. To make these a small portion
of a previous culture is transferred to fresh cul-
ture media by means of the platinum needles.

a. Stab Cultures are made in test-tubes con-
taining solid, transparent media, such as gelatin
and agar. The end of a sterile needle is infected
with the material tobe transferred. The needle is
then thrust into the medium to the bottom of the
test-tube and withdrawn. In this way the bacteria
are left along the entire length of the needle track.

' For method of holding tubes see fig. 7. They are
held in an inclined position to prevent the possi- Fic. 7. Method of holding test-tubes.
bility of infection.

b. Streak Cultures are cultures made by drawing the needle or loop over the surface
of the medium (test-tubes with media having sloped surfaces or plate cultures). Agar,
potato and blood serum are frequently used in this way, and occasionally gelatin.

c. Fluid Cultures (bouillon, milk, ete.), are inoculated by transferring the desired
material to them on either the needle or loop.

REFERENCES. A. 146; H. 51; M. & R. 60; McF. 146.

SprEcIAL DIRECTIONS.

a. Make a gelatin stab, an agar streak, a potato streak, and a bouillon culture of
Bacillus subtilis (EHRENB.) COHN (hay bacillus) and Bacillus coli (Escu.) Mic. (colon
bacillus) from agar cultures supplied.

b. Label each tube, writing the name of the organism, the date of inoculation and
your own name.

c. Place the gelatin in the cool chamber, and the other cultures in the incubator at
28° C. (XIV).

EXERCISE XIV. INCUBATION OF CULTURES.

ExeLaNnatory. Most bacteria grow at ordinary temperatures (22° C.), but their .
growth is usually hastened by a higher temperature (e. g. 28°-30° C.). The pathogenic,
or disease-producing bacteria grow best at the temperature of the human body (38° C.).
All bacteriological laboratories are, therefore, supplied with apparatus arranged for
maintaining constant temperatures, known as thermostats or incubators.

The non-pathogenic cultures are usually kept at 28° C., while the pathogenic ones
are kept at 38° C. All gelatin cultures, however, must be kept at a temperature several
degrees below the melting point of gelatin, i. e., not above 22° C. Ordinarily the temper-
ature of the locker, especially near the floor, will be found satisfactory. In avery warm
room, particularly in the summer, an artificially cooled chamber will be necessary.

Test-tube cultures are stored in the various incubators in tin cans or glass tumblers
with a layer of cotton in the bottom, while the Petri dishes are stacked in low piles.

REFERENCES. A. 136; H. 48; M. & R. 88; N. 178 & 248; P. 231; P. & M. 37.

18 General Bacteriology.

SPecIAL DIRECTIONS. :

a. Incubate all cultures of the non-pathogenic bacteria at 28° C., except the gelatin.
Keep these in the cool chamber. After growth has taken place, the cultures can be taken
from the incubator and kept at the room temperature.

b. Study and make diagrams of an incubator, a Reichert thermo-regulator, a Roux
thermo-regulator and Koch’s safety burner.

EXERCISE XV. CLEANING SLIDES AND COVER-GLASSES.

GENERAL DirREcTIONS. Slides can be sufficiently cleaned by washing in water or
alcohol and drying with a towel. ‘The cover-glasses for bacteriological work, however,
must not only be freed from visible dirt but must be rendered free from fat. One of the
best methods is the following: New cover-glasses are cleaned by washing in water and
drying from alcohol between driers (two blocks 20x10x2} mm. covered with several layers
of cotton cloth or chamois skin), and then heating them on a piece of sheet iron or in hot
air sterilizer for one hour at about 200° C. They are best kept in a clean Petri dish and
handled with forceps. (Novy). Old slides and covers having balsam on them should first
be dropped one by one into a cleaning solution (potassium bichromate 60, sulphuric acid
60, water 1000), and boiled for one-half hour and then treated as above.

SpeciAL DrrEcTIoNS. Clean } oz. of cover-glasses and place them in a clean Petri
dish. j
EXERCISE XVI. PREPARATION OF STAINING SOLUTIONS.

GENERAL Directions. The dyes most useful for staining bacteria are the basic
anilin dyes which come in powdered or crystalline form. (Gruebler’s dyes are standard.)
Those in most common use are Fuchsin, Methylen blue, Gentian violet and Bismark
brown. They keep well in powdered form, with perhaps the exception of Methylen
blue, but because of greater convenience and equally good keeping qualities, saturated
alcoholic solutions are kept in stock. These are made by adding the dry dye to 95%
alcohol to saturation and filtering. This form can not be used for staining bacteria. The
following solutions are required to begin work with:

1. Aqueous solution of Gentian violet.

Saturated alcoholic solution of Gentian violet, - - - 2.5 ee.
Distilled water, - - - - - - 47.5 ee.
2. Saturated aqueous solution of Bismark brown.
8. Ziehl’s carbol-fuchsin. ,
Saturated alcoholic solution of Fuchsin. - - ‘ - 5 ce.
Solution of carbolic acid (5%)- - = E ‘ 45 ec.
4. Loeffier’s Methylen blue.
Saturated alcoholic solution of Methylen blue, S(t - 15 cee.
Potassium hydrate (1:10,000), —- - @ Z 50 ec.
5. Ehrlich’s Anilin Oil-Gentian violet.
Saturated alcoholic solution of Gentian violet, - - - 6ee.
Absolute alcohol, - : - - - - 5 5 ee.
Anilin water, - - - - - ? 7 - 50cee.

Anilin water is prepared by adding 2~8 ec. of anilin oil drop by drop to 50 ce. of
water, thoroughly shaking and then filtering through moistened filter paper until per-
fectly clear.

20 General Bacteriology.

This stain should stand 24 hours and then be filtered. It does not keep well and must
not be used when more than 14 days old.
6. Gram’s Iodine solution.

Todine, - - - - - - - - 1 gm.
Potassium iodide, - - - - - - 2 gm.
Distilled water, - - - - - - - 300 ec

7. Gabbett’s Methylen blue solution.
Methylen blue (dry), - - - - - 2 gms.
Sulphuric acid, - - - - - - - 25 ec.
Distilled water, - - - - - - 75 ee.

8. Alcohol, 96%.

REFERENCES. A. 156; H.75; M. & W. 245; M.&R.
103; McF. 90; P. 200.

SpEecIaL DrrEcTIONS. Prepare the solutions of dyes
from the saturated alcoholic solutions (furnished) and
place them in 2 oz. bottles arranged with pipettes and neatly
labeled. ‘The bottles are conveniently kept in a block.
Fig. 8.

EXERCISE XVII. SIMPLE COVER-GLASS PREPARATION.

Fic. 8. Block for stain bottles.

GENERAL Directions. Bacteria may be studied under the microscope in a living
condition in a hanging drop preparation (XIX); buton account of their hyaline charac-
ter, which makes the examination difficult, the student should first learn to stain them
and later make the hanging drop preparation. With a few exceptions all bacteria can
be stained by the following process: <A small drop of distilled water is placed on a clean
cover-glass by means of the platinumloop. With a sterile needle a portion of the material
to be examined is secured and while the cover-glass is held in the fingers of the left hand
the bacteria on the needle are introduced into the water, thoroughly mixed and then
spread in a thin film over as much of the surface of the cover-glass as possible. When
the bacteria are taken from fluid media a drop of water will not be necessary. In this
case use a loop. The film is now allowed to dry. Ifthe drop is sufficiently small this
will be ashort process. It may be hastened by holding the cover-glass high over the
flame, but it should always be held in the hand to prevent over-heating, which spoils the
preparation.

When the film is thoroughly dry place the cover-glass in a pair of Cornet or Stewart

forceps and ‘‘fix’’ the bacteria in the flame. This is done by passing the preparation
through the upper portion of a gas flame, film side up. Three passages should be made,
each consuming about one second of time. The forceps are now placed on the table and
the film flooded with one of the anilin dyes. After the stain has acted for five or ten
minutes it is washed off into a waste dish with a stream of distilled water, and while
the cover-glass is still wet it is placed, bacteria side down, on a clean glass slide, being
careful to avoid air bubbles. The surplus water is then taken up by means of a small
piece of blotting or filter paper.

a preparation is now ready for microscopical examination. (For directions see
XVIII).

22 General Bacteriology.

The preparation can be made permanent either by allowing the water under the
cover-glass to dry before it is removed, or by floating it off with water and afterwards
drying. When dry a drop of Canada balsam, dissolved in xylene, is placed on the cover-
glass and this is then lowered on to the slide again.

Resume.

a. Spread film,
. Air dry,
Fix,
. Stain,
Mount,
Examine,
Mount in balsam, or,
Mount in balsam,
. Examine. :
‘‘The great mistake made by beginners is to take too much growth.’’ (M. & R.)
REFERENCES. A. 151; H. 71; L. & K. 104; M. & W. 89; M. & R. 95; MecF. 91;
N. 147; P. 198; P. B. C. 11; S. 25.

SPECIAL DirEcTIONS. Make cover-glass preparation from agar streak of B. subtilis

(XIII) staining with an aqueous solution of gentian violet for 5 minutes. ,

euehws ass

EXERCISE XVIII. USE OF MICROSCOPE.

GENERAL DirEcTIons. For bacteriological purposes a microscope with a magnifying
power of at least 500 diameters is needed. There should be a coarse adjustment (rack
and pinion) as well as a fine micrometer screw; and the following accessories: two eye
pieces, one 1 in. (25 mm.) and one 2 in.(50mm.); three objectives, one $ in. (16 mm.),
one+in. (4mm.), or¢ in. (3.5 mm.) and one oil immersion 7y in. or 7s 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. Liant. The proper angle at which the mirror should be placed is best determined
by removing the eye-piece and so arranging the mirror that the unobstructed light from
the window covers the whole field. The ideal light is that from a white cloud. Direct
sunlight should never be used.

b, ABBE CONDENSER. The purpose of the condenser is to furnish a large cone of light,
and as it is corrected for parallel rays the plane side of the mirror should always be used,
except when artificial light is employed. When highly stained objects are to be exam-
ined, the open diaphragm should be used, but when the structural rather than the color
picture is desired, it will be necessary to diminish the light by closing the diaphragm.
When the high powers are employed, raise the condenser as high as possible; for low
powers a lower position will give better definition.

c. Focusing. Turn the proper objective in place and rack down until the objective
nearly touches the cover-glass. This should be done while the eye is held at one side and
directs the movement. Then with the eye at the tube slowly move up with the micrometer
screw. Never rack down with the eye at the tube.

d, UsE or O1L-Iumersion. The oil-immersion objective is indispensable to the proper
study of bacteria. It is constructed upon the principle that a drop of fluid having the
same refractive index as the objective, prevents the dispersion of light, thus permitting
the use of lenses having a greater numerical aperture and longer working distance for

24 General Bacteriology.

the same degree of amplification than is possible with the dry system. In using an
immersion lens, place a small drop of oil on the preparation, then carefully lower the
objective until it touches the oil drop and nearly touches the cover-glass. Apply eye to
the ocular and focus upward very slowly with fine adjustment until the definition is clear.
At the close of the day’s work the oil must be removed from the objective and cover-
glass. This is best accomplished by wiping them with apiece of Japanese paper made for
the purpose. In case the oil should accidentally dry on the objective, it can be removed
by adding a little more oil and allowing it to stand for a few minutes; it can then be
wiped off with paper. If this method does not succeed, the objective should be taken to
the instructor. Great care must be observed since solvents of the oil are also sol-
vents for the lens mountings.

REFERENCES. See Gage; A. 190; H. 104; M. & R. 938; McF. 86; N. 123; P. 206.

SPECIAL DIRECTIONS.

a. Examine cover-glass preparations made in (XVID) first with 4 in. objective,
and then with the oil-immersion objective. If the specimen is satisfactory wipe off the
oil and mount in Canada balsam.

b. Practice making cover-glass preparations by staining specimens from each of your
cultures. Use Loeffler’s methylen blue for the gelatin and bouillon; aqueous solution of
gentian violet for agar, and carbol-fuchsin for potato. Examine, mount permanently and
hand to instructor for inspection.

EXERCISE XIX. HANGING-DROP PREPARATIONS.

GENERAL DIRECTIONS. These are made by adding a small portion of bacterial cul-
ture from solid media to a drop of water on a clean cover-glass, or in case of fluid media
by placing a loop of the culture medium on the cover-glass. A hollow ground glass
slide having the rim of the cavity previously coated with vaseline, is inverted and lowered
over the cover-glass enclosing the drop. With a careful, quick movement the prepara-
tion is now brought right side up.

Instead of the hollow ground glass-slide an ordinary glass-slide to which a small
section of a glass or rubber tube has been cemented can be used, and in some 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 (4 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.

REFERENCES. A. 195; H. 101; L. & K. 102; M. & W. 111; M. & R. 94; MeF. 88,
N. 142; P. 209. lead

SPECIAL DIRECTIONS.

a. Make hanging-drop preparation of B. subtilis from agar or bouillon. (XIII)

b. Make same preparation of B. coli. (XIII)

c, Make same preparation of organism supplied. (Micrococcus)

d. Make same preparation of water containing particles of india ink or carmine in
suspension. Study character of movement in all cases. Distinguish between vital and
molecular movement.

26 General Bacteriology.

In cases where vital movement is questionable, remove the cover-glass and place a
drop of formalin or chloroform in the bottom of the cell; replace the cover-glass, ex-
amine and note change in character of movement, if any.

EXERCISE XX. TEST-TUBE CULTURES ILLUSTRATING FORM TYPES.

a. Make test-tube cultures in bouillon, gelatin, agar and potato of the following
organisms:

Micrococcus (any species).

Sarcina lutea SCHROETER.

Pseudomonas fluorescens (FLUEGGE) Mic.

Bacillus mycoides FLUEGGE.

Microspira Metschnikovi Mic. (or any vibrio).

Spirillum rubrum v. ESMARCH.

b. Incubate all cultures, except gelatin, at 28° C.

EXERCISE XXI. STUDY OF TEST-TUBE CULTURES.

GENERAL Directions. As soon as growth becomes visible a systematic and careful
study of the cultures should be made. A detailed list of the points to be noted will be
found in Chapter III, and should be consulted in writing up the descriptions. The sum-
mary below will, however, be found useful.

_ For bouillon cultures note: 1) condition of fluid, 2) character of sediment, 3) pres-
ence or absence of membrane, and 4) characteristic odor.

For solid cultures (agar and potato slopes), note: 1) Form of growth, 2) size, 3)
surface elevation, 4) consistency, 5) color, 6) effect on media, and 7) characteristic odor.

For gelatin. stab cultures, note: 1) Effect on media, a. non-liquefying, i) line of
puncture, ii) surface, b. liquefying, i) shape of liquefied area, ii) condition of fluid, iii)
character of sediment, 2) characteristic odor.

The study should be continued from day to day as long as changes are noted. Make
drawings wherever they will be of service in elucidating the descriptions.

REFERENCES. P. B. C. (Charts by Cheesman.)

SPECIAL DIRECTIONS. Study and write careful descriptions and make necessary
drawings of all cultures made.

EXERCISE XXII. MICROSCOPICAL STUDY OF FORM TYPES.

a. Make cover-glass preparations from the agar streaks (XX) and stain with an
aqueous solution of gentian violet or with Loeffler’s methylen blue.

b. Examine with the oil-immersion objective, write the names of the organisms in’
their proper place in the table below.

Name. Sketch.
MOGI vs a sacusigane+scarinumasaten 4 Mee ooleibbeee adidas
Coceaceae (spherical)
STH ALM ss eos oe bhewh waeentwe hehe apes. Oo Soees. . den temieedle
DAG de diesen es cinsclsls Soppoae Rae alen GRR ats ook Syste dwedbles
Bacteriaceae (elongated)
SNA. es iveetuie arse vec eco

CUPVECH Se a54 saves concey aiweweeedan
Spirillaceae (spiral)

twisted

28 : General Bacteriology.

c. Make similar preparations from the gelatin and potato and note any variations in
form, size. ete. ?

d. All these preparations are to be mounted in balsam, sketched (XXIII) and
handed to instructor for inspection.

P EXERCISE XXIII. DRAWING BACTERIA.

GENERAL Directions. In drawing bacteria only a few organisms occuring in the
microscopic field should be sketched, but these should be made of considerable size so
that the exact outline may be indicated. Furthermore they should be drawn to scale and
individuals selected to give range in form and size. ;

To measure microscopic objects an ocular micrometer is used, and the first step will
be to determine its value. Place the ocular micrometer on the diaphragm in the ocular,
use a stage micrometer as an object and focus. The image of the scale on the stage mi-
crometer will appear imposed on that of the ocular micrometer. Make the lines of the
two micrometers parallel and then make any two lines of the stage micrometer coincide
with any two on the ocular micrometer, pulling out the draw-tube if necessary. Divide
the value of the included space or spaces on the stage micrometer by the number of
divisions on the ocular micrometer required to include them, and the quotient so obtained
will give the valuation of the ocular micrometer in fractions of the units of measure of
the stage micrometer (Gage). If result is notin terms of micron (/) it should be con-
verted to such, as this is the unit in micrometry.

In making drawings represent a micron by two millimeters on paper. This will give
a magnification of 2,000 (x 2,000).

REFERENCES. G. 100-108.

SPECIAL DIRECTIONS.

a. Determine the value of the ocular micrometer and fill out blanks in following
table:

No. of Microscope.............0000000. Mak6 sei cwstensesccton.
Ocular............ iM., OF ........00- mm
Value of single di-

Objective. Tube length. vision on scale
in 4,

4 in. (16 mm.)

+ in. (4 mm.)

Oil-immersion.

b. Make drawings of cover-glass preparations made in XXII in place provided in
table. .

30 General Bacteriology.

EXERCISE XXIV. STUDY OF CELL GROUPING.

Hanoine Drop PREPARATIONS.
a. Make hanging-drop preparations from bouillon cultures prepared above and also

from those supplied.
b. Examine with oil-immersion objective and assign organisms to their proper place,
as determined by cell grouping, in the following scheme:
Name. Sketch.

Isdlated) j- jg. i -HSkntereith JealdbatuldciaeaGecaineoeealn Spasanieiyseasinbotebtees ace

Bacilli, dew svezsaweeeeigds acdekngieesetiige ait d's £5, Ge cua Aaa G tatu
Filaments
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IMPRESSION PREPARATIONS. The exact relation of cell to cell as they develop in the
colony can frequently be determined with greater accuracy by studying a ‘‘contact prep-
aration’’ which is prepared as follows:

a. Melt a gelatin tube and slopeit, when solid make a streak culture of B. mycoides
FLUEGGE 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 arrangement of
adherent bacteria is not disturbed.

c. Thoroughly air dry the same, then fix and stain in the ordinary manner.

d. Examine the thinner layers noticing the arrangement of cells with reference to
each other and draw a sufficient number to illustrate this relationship.

AGcar Hanaine-Drop CULTURES.

a. Melt a tube of agar and cool to 48° 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 3mm. long of B. subtilis.

d. With the loop place a drop of liquid agar so as to cover up streak.
e. Seal cover-glass to hollow ground slide. Incubate and later examine and sketch.

EXERCISE XXV. STUDY OF INVOLUTION FORMS.

a. Grow Bacillus subtilis (EHRENB.) Mic. in bouillon and also in a solution con-
taining 0.1% asparagin, 10% sugar, and by means of stained cover-glass preparations
compare the individual organisms in each case in regard to theirform and size. The de-
generated or involution forms are more apparent by staining. Draw several cells illus-
trating a variety of involution forms.

b, Examine a culture of Bacterium diphtheriae (LOEFFLER) Mia. on Loeffler’s blood
serum. Read M. & R. 5.

32 General Bacteriology.

EXERCISE XXVI. GELATIN PLATE CULTURES.

EXPLANATORY. Plate cultures are only possible with the liquefiable solid media,
gelatin and agar. In making them the bacteria are mixed with the medium while it is
in a fluid state in such quantities that the individuals are separated from each other by
several millimeters when it is spread out on a horizontal surface to cool. As the medium
solidifies, the organisms become fixed and their growth results in the formation of ‘‘colo-
nies,’’? These vary in size and appearance according to the peculiarities of the organism
and the age of the culture, but are of the greatest service in the study and identification
of the various species. These cultures are prepared as follows:

GENERAL Directions. Three gelatin tubes are marked Nos. 1, 2 and 3 and melted
by placing them in a water bath at a temperature of 42° C. For this purpose a small
cup of water placed on a tripod can be used (Fig. 9). They are
inoculated by introducing the material to be studied into tube
q No. 1. The quantity of this material varies. The amount cling-

ing to the platinum needle will be sufficient if a pure culture is
used, while in other cases several loops or even drops are neces-
sary. The inoculated material is thoroughly mixed with the
gelatin in No.1. This is done by rolling the tube gently be-
tween the palms of the hands, instead of shaking, so as to pre-
vent the introduction of air bubbles. With a sterile loop three
loopfuls of fluid gelatin are now transferred from No. 1 to No.2,

L sore. and mixed. For method of handling tubes see Fig. 7. In like
Fic. 9. Method of melting gelas manner three or more loops from No. 2 are carried over to No.

tin. 3, which in turn is well mixed. The contents of the tubes Nos.
1-8 are now poured into separate sterile Petri dishes. :
The process of pouring is performed as follows: The
Petri dish is placed on the desk; the gelatin tube is
taken in the right hand, the cotton plug removed with
the left hand; the mouth of the tube sterilized by
flaming it once or twice, and when the glass is cool Fic. 10. Method of pouring plates.
the gelatin is poured into the lower half of the dish while the cover is slightly raised
(Fig. 10), but not inverted or laid on the table. The cover of the dish is then replaced,
the test-tube filled with a solution of corrosive sublimate, and the cotton plug returned.
The gelatin is spread over the entire bottom of the dish by tipping it from side to side.
It is then allowed to harden by placing the dish on the cooling apparatus or leaving it
on horizontal surface at room temperature. A simple, inexpensive and effective cooling
apparatus is a piece of soapstone, such as is sold at
hardware stores (Fig. 11). In winter this can be cooled
by hanging it out of doors, at other seasons by im-
mersing it in cold water, The three Petri dishes thus
prepared should be properly labeled and placed un-
der conditions where the gelatin will remain solid and
hai ais. Geapeiosc cued Rare ats a yet growth takes place. The temperature of the

tin in Petri dishes. 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 nunumbers, depending upon the dilution
used.

34 General Bacteriology.

Inasmuch as the first plate is invariably too thickly seeded to be of much service,
this gelatin tube is often replaced by a water blank, which is treated exactly as the gela-
tin tube No. 1, but is not of course ‘‘plated’’ but simply serves to dilute the material.

Rererences. A. 124; H.57; L. & K. 88; M. & W.108; M.& R.61; MeF. 140;
N.171; P. 224; 8S. 72.

SPECIAL DIRECTIONS.

a. Make three gelatin plate cultures, as directed above, and inoculate with B. sub-
tilis, introducing a minute portion of agar culture (XIII) into tube No. 1, two loops of
No. 1 into No. 2, and three of No. 2 into No. 3. Label, and when the gelatin has solidi-
fied, place plates in cool chamber (XV).

b. Also make a “‘blank’’ plate from an uninoculated gelatin tube, observing all pre-
cautions to prevent contamination. This will serve as a control or check on your other
plates. If any colonies develop on this it indicates carelessness.

EXERCISE XXVIII. AGAR PLATE CULTURES.

GENERAL DIRECTIONS. These are made in the same way as the gelatin plates ex-
cept 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 inoculating, but as agar solidifies at
39-40° C. it must not, therefore, be cooled below that point. It is best to keep the melted
agar at about 42° C. for 10 minutes before it is inoculated. For this purpose a water-

so 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. 11. Inoculate, make dilu-
tions and pour as in case of gelatin, except that before the agar
is poured, it is well to slightly warm the Petri dishes by placing
them in the incubator at 38° C. for a few minutes, other-
wise the agar may solidify in lumps in the plate. In cooling,
agar shrinks somewhat, and in doing so water is expressed from
the solid jelly. In the incubator this condenses on the under
side of the cover of the Petri dish to such an extent that drops
run down on to the culture surface thus causing the developing
Pred, Wher eth, ir codiice superficial colonies to ‘‘run.’’ To obviate this the Petri dishes,
agar. when placed in the incubator, should be inverted.
REFERENCES. H. 61; L. & K. 94; M.& R. 66; N. 285; P. 225; P. B.C. 28.
SpeciaL DirEcTIONS. a. Make three agar plates of B. coli; useone loop of bou-
illon culture (XIII) for tube No. land proceed as in XXVI. 0b. Place in incubator at
28° C. inverted.

EXERCISE XXVIII. ROLL=CULTURES (Esmarch).

GENERAL DrrEcTIons. These are essentially plate cultures in which the medium
instead of being poured out into dishes is solidified in a thin, even layer on the inner surface
: of the test-tubes. This is best accomplished by means of a

piece of ice placed in a dish on a piece of cloth by which it
can be kept in the desired position (Fig. 12). A horizontal
groove is melted in the ice by means of a test-tube filled with
; hot water. In this groove the test-tubes, inoculated as in case

ree ee is of plate cultures, are rapidly whirled until the medium is thor-
oughly set. Both agar and gelatin can be used, although gelatin cannot be used suc-

36 General Bacteriology.

cessfully with those species which liquefy this medium. In the case of agar the tubes
should be placed in a horizontal position a few hours (over night) until the medium has
become attached to the tube; afterwards they can be stored in the usual receptacles for
tube cultures.

REFERENCES. A. 131; M. & RB. 65; McF. 148.

SpeciaL DirEcTIoNS. a. Melta tube of gelatin and without inoculating it practice
making a roll-culture as described above. Avoid tipping the tube enough to get medium
on cotton plug. Remelt and roll again until the knack is acquired. ;

b. Make two roll-cultures in gelatin of B. coli (XIII), using a water-blank instead
of gelatin tube No. 1.

c. Make two agar cultures of B. subtilis in same way.

d. Incubate b. in cool chamber, and c. at 28° C.

EXERCISE XXIX. 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 different. Drawings should always be made wherever they will be of
value; study should be continued as long as changes are noticed. (See Chapter III,
I..A. a.-e.)

Microscopic. The colonies appearing on the plates are to be studied under a low
power of the microscope. Use a¥%in. (16 mm.) objective. The Petri dishes can be
inverted, and thus avoid the danger of exposing the culture to contamination from the
air except with gelatin where liquefying organisms are present. Observe, a. structure of
colony as a whole; b. character of margin. (See Chapter III. I. A. fdég.)

REFERENCES. P. B. C. (Cheesman’s Charts.)

SPECIAL Directions. Study, write descriptions and make drawings of all plate
cultures. Use blank pages for description and sketch of cultures.

EXERCISE XXX. USE OF DECOLORIZING AGENTS.

Make three cover-glass preparations from a.24 hour old culture of B. subtilis, stain-
ing them with an aqueous solution of gentian violet. Mount in water and examine.
While they are still under the microscope, place at one side of the cover-glass a few
drops of one of the following solutions, and by means of a strip of filter paper at the
opposite side draw the liquid under the cov er-glass until all the color is removed. In
this way determine the relative value of alcohol (95%), acetic acid (5%), and nitric acid
(80%) as decoloring agents.

EXERCISE XXXI. GRAM’S STAIN.

Expuanatory. This is a differential stain and one of the most useful. Some bac-
teria when stained by this method exhibit a dark violet color, others remain perfectly
colorless, thus rendering possible the differentiation of bacteria which are morphologically
nearly or quite identical, and also greatly facilitating the demonstration of certain bac-
teria in animal tissue. Most of the pathogenic micrococci retain the violet stain although

there are important exceptions. The bacilli and spirilla may or may not remain colored.
GENERAL DIRECTIONS.

a. Spread film.
b. Air dry and fix.

38 General Bacteriology.

Stain with anilin-oil gentian violet 5 minutes:
. Pour off stain and without washing:

Apply iodine solution 2 minutes (use several changes).

Decolorize with 96% aleohol until drippings do not stain white filter paper.
. Wash in water and counter-stain with Bismarck brown.
. Mount in water and examine.

Dry and mount in balsam.

REFERENCES. A. 162; H. 78; L. & K. 106; M. & W. 91; M. & R. 110; McF.
99; N. 287; P. 208.

SPECIAL DirEcTIoNnS. Stain films of young cultures of B. coli and B. subtilis. Also
a film of an organism supplied. ,

Sra wr as

EXERCISE XXXII. TUBERCLE STAIN (Gabbett).

EXPLANATORY. All of the differential methods of staining the tubercle bacterium
depend upon the fact that this germ is very resistant towards the ordinary stains and in
‘order to be stained at all must be treated with a dye containing a mordant and this
either allowed to remain in contact with the micro-organism several hours or be applied
hot. The latter method is the quicker and is usually employed, although it does not
give as good results. When once stained this germ withstands the effect of decoloriz-
ing agents to such an extent that it is possible to remove the dye from all other objects
on the cover-glass preparation (as in sputum) while it retains its own color. The appli-
cation of a second dye, of a complementary color, readily distinguishes this germ from
all others in the field. A few other bacteria have similar staining properties. (See
Part II.) Red isthe 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 } to 1 minute.

Wash in water and examine. ;

. Dry and mount in balsam.

REFERENCES. A. 162; M. & W. 92; McF. 214; P. 304.

SpeciaL Directions. Stain three samples of sputa which contain varying numbers
of the tubercle bacteria.

a wo Aaa ee

EXERCISE XXXII STAINING ENDOSPORES.

GENERAL DIRECTIONS.
A. Simple stain.

a. Prepare film as usual.

b. Fix by passing through flame 10 or 12 times instead of 3 times. (This prevents
the vegetative portion from taking the stain).

c. Stain 2-5 minutes in hot carbol-fuchsin.

d. Mount and examine.

\

40 General Bacteriology.

B. Differential stain (Hauser’s method).

a. Make cover-glass preparation of a spore-bearing culture, fix and stain with hot
carbol-fuchsin until spores are thoroughly colored. This must be determined by mount-
ing in water and examining under microscope.

b. Cautiously decolorize with acetic acid, 5%, until stain is removed from the vege-
tative portion only. This to be determined as above.

c. Wash in water and counter-stain with methylen blue.

d. Examine. Crimson spores will be seen in blue bacilli.

REFERENCES. Other methods, see A. 164-167.

SPEcIAL DirEcTIoNS. Stain by each method spores in cultures of B. subtilis or
other spore-bearing organisms.

EXERCISE XXXIV. STUDY OF ENDOSPORES.

a. Make cultures on peptoneless agar, or agar to which afew drops of calcium
hydrate has been added, of the following organisms and incubate at 28° or 88° C. depend-
ing upon the organisms:

Bacillus subtilis (EHRENB.) COHN.

Bacterium anthracis (Koca) Mie.

Bacillus amylobacter Van TyecHEM (or any clostridium form).

Bacillus tetani Nicouater. (or any ‘‘drumstick’’ bacillus).

Pseudomonas erythrosporus (Conn) Mia.

b. When the cultures are 48 hours old mount films without staining, examine and
note:

1) Form.
2) Size.
3) Color. :
4) Power to refract light.
5) Relation to mother-cell.
(1) Median or central.
(2) Intermediate.
(3) Terminal or polar.
(4) Clostridium form.
(5) Drumstick form.

c. Make drawings.

Reap: J. H. 26; L. 60; L.& N.76; M. &R. 6; N. 46; P. 46; P. B.C. 15;

8. 114.
EXERCISE XXXV. FLAGELLA STAIN (Bunge).

GENERAL DIRECTIONS.

a. Make an agar streak of the organism to be stained.

db. After 18 to 24 hours, by means of the platinum needle, remove a portion of the
growth (being careful to avoid the culture medium) to a large drop of tap water on a
perfectly clean cover-glass (XV.) and allow to stand 5 minutes rather than spread, as
there is less danger of breaking off the flagella.

ec. Spread carefully 2 or 3 loopfuls of this drop on each of several clean cover-glasses
and dry at room temperature.

d. Fix by passing the cover-glass through the top of the flame while it is held in the
hand, not in the forceps, as over heating will injure the preparation.

42 General Bacteriology.

e. Flood the cover-glasses thus prepared with the following solution (Mordant) :
Liquor ferri 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.

Jf. Wash in water and dry between filter paper.

g. Stain with hot carbol-fuchsin for about one minute.

h. Wash in water, dry and mount in balsam.

REFERENCES. M. & W. 108; McF. 104; P. 205. Other methods M. & R. 115;
McF. 101; A. 167.

SPECIAL Directions. Stain B. typhi from cultures furnished, also try B. colé and

B. subtilis.
EXERCISE XXXVI. CAPSULE STAIN (Welch).

GENERAL DIRECTIONS.

a. Spread film without the use of water.

b. Air dry.

c. Fix.

d. Apply glacial acetic acid, and drain it off immediately. Do not wash in water.

¢. Stain with anilin-oil gentian violet (Ehrlich) which is to be renewed several
times to remove acid.

f. Wash in 1 to 2% salt solution.

g. Examinein salt solution. (Balsam causes capsule to shrink.)

REFERENCES. A. 163; P. 203; P. B.C. 13.

SpecIAL DirEcTIoNS. Use pneumonic (“‘rusty’’) sputum, or blood of rabbit in-
fected with the pneumococcus.

CHAPTER II.
PHYSIOLOGY OF BACTERIA.

EXERCISE XXXVII. PREPARATION OF SPECIAL MEDIA.

The following media will be necessary for the work outlined in this chapter:

a. Gutucosr Bovumion. To ordinary bouillon add 1% glucose (c. P.), tube and
sterilize in steamer, not in autoclave, 2 test-tubes and 2 fermentation tubes.

b. GLucosE GELATIN. 1% glucose (c. P.), tube and sterilize in steamer, 6 tubes.

c. Guucose AGAR. 1% glucose (c. P.), i a 5 tubes.

d. Lacrosse AGAR. 1% lactose (c. P.), = i of 2 tubes.

e. Lirmus SotutTion. To 10 gms. of the dried material add 500 cc. of distilled water,
digest in a warm place, decant clear liquid and add a few drops of nitric acid to produce
a violet color. (Sutton). Place in flasks or test-tubes and sterilize in steamer three
times, 1 tube.

f. DuNHAM’s SOLUTION.

Sodium chloride 0.5 gm. a until all is dissolved, filter, tube and steril-

Peptone (Witte) 1. gms. tae; tL tribes,

Water 100.“
g. NITRATE SOLUTION.
Sodium chloride 0.5 gm.

Peptone (Merk) 1 gms.
Potassium nitrate 0.2 ‘‘ >? Filter, tube and sterilize, 3 tubes.
Water 1,000 7

h. Lirmus Minx.

1) Freshly separated milk (or if this is not available, new milk is placed in a sep-
aratory funnel in an ice chest over night to allow the separation of the cream and milk
then drawn off) is titrated with ¢y>Na0H and rendered slightly alkaline to phenolphtha-
lein by the addition of {NaOH. eK

2) Litmus solution is then added until medium is faintly blue.

3) Tube and sterilize in the steamer for 30-45 minutes on 38 or 4 consecutive days.
During the summer months particularly very resistant bacterial forms abound in the
milk so that it is necessary to increase the number of applications or length of exposure.
The efficiency of the sterilizing process should be tested by placing the flasks in the in-
cubator for several days to see if any change occurs, 2 tubes.

In addition to the above have 15 tubes of bouillon (9 to contain exactly 10 cc. XLI
& XLIV), 10 tubes of gelatin, 15 tubes of agar, 6 water-blanks and 5 potato tubes.

EXERCISE XXXVIN. EFFECT OF REACTION OF MEDIA ON GROWTH.

GENERAL DIRECTIONS.

a. Melt 6 tubes of gelatin and add, under aseptic precautions, to three of them, re-
spectively, 0.1 cc., 0.3 cc., and 0.5 ce. of a normal solution of hydrochloric acid, and to
the other three the same arnounts of a normal sodium hydrate.

46 General Bacteriology.

b. Thoroughly mix, solidify gelatin in ice water and then inoculate (stab) each
tube with the organism to be studied, making a control culture in a tube of neutral
gelatin.

c. Incubate at 18° C. and note the effect of the chemicals on the rate, amount and
character of the growth.

REFERENCES. L. & N. 87; McF..46.

SPECIAL DirEcTIONS. Use B. subtilis and B. coli.

EXERCISE XXXIX. EFFECT OF CONCENTRATION OF MEDIA ON GROWTH.

a. Pour about 2 ec. of ‘‘condensed milk’’ into each of two sterile test-tubes,. dilute
one with five times the volume of sterile water.

b. Inoculate both with a pure culture of B. subtilis and incubate at 28° C.
Explain changes which occur. ;
-¢. Test extract of beef or syrup in the same way.

EXERCISE XL. EFFECT OF TEMPERATURE VARIATIONS ON RATE OF GROWTH.

GENERAL DIRECTIONS.

a. Make four agar streak cultures of organism to be studied.

b. Incubate them at the following temperatures: Ice chest (7° C.), room (20° C.),
low incubator (28° C.), blood heat (38° C.).

c. By frequent observations as to luxuriance of growth, determine the optimum
temperature of growth for each.

REFERENCES. F. 73; L. & N. 98.

SpeciaL Directions. Use B. campestris and B. coli.

EXERCISE XLI. DETERMINATION OF THERMAL DEATH POINT.

GENERAL DIRECTIONS.

a. Make a bouillon culture of the organism to be tested.

b. 48 hours later heat a large water-bath to 45° C. Place in this, in close proximity
to a thermometer, a test-tube (16 mm. in diam.) containing exactly 10 cc. of standard
bouillon. (Reaction +1.5.) i

c. After 15 minutes exposure at this temperature remove the cotton plug from the
tube, inoculate the broth with three loopfuls (standard size, XII) of the culture prepared
above (a.), and carefully mix by slightly agitating the tube, without removing it from the
bath. ;

d. After a further exposure of 10 minutes remove the tube from the bath and place
it in a vessel of ice cold water to cool. Then incubate at a temperature favorable to the
development of the organism under observation.

e. In the same manner expose the organism to the following temperatures: 50°, 55°,
60°, and 65° C.

f. In all cases incubate at least a week and take as the thermal death point the low-
est temperature at which growth fails to appear. (In more accurate work the tempera-
ture should be determined within 2° C.).

REFERENCES. P. B. C. 32.

Specian Directions. Use B. coli or B. typhosus.

48 General Bacteriology.

EXERCISE XLII. COPMPARATIVE EFFICIENCY OF DRY AND MOIST HEAT.

GENERAL DIRECTIONS.

a. Charge a water blank with culture of a spore-bearing bacillus, shaking it well to
break up the clumps.

b. Sterilize eight cover-glasses by passing them several mee through the flame, and
place four in each of two sterile Petri dishes.

c. With a sterile loop place an equal quantity of the bacterial suspension (a.) on
each cover-glass, and dry by placing Petri dishes in the incubator with the covers slightly
raised.

d. When dry place one Petri dish in the dry sterilizer (near the thermometer), and
the other in the steamer.

e. Keep both sterilizers at a temperature of 100° C., and at the end of 5, 10, 20
and 40 minutes respectively, remove one cover-glass from each Petri, place it in a sterile
Petri dish and pour a tube of liquefied gelatin or agar over it. Tip the dish from side
to side to dislodge as many of the bacteria as possible from the cover-glass, solidify the
medium and incubate.

REFERENCES. L. 101; S. 146.

SpeciaL Directions. Use an old (spore-bearing) culture of B. subtilis. Arrange
data in the form of a table.

EXERCISE XLII. EFFECT OF DESICCATION.

GENERAL DIRECTIONS.

a. Prepare five cover-glasses each of a spore-bearing and a non-spore-bearing cul-
ture, as directed in XLII.

b. Place them in a sterile Petri dish, and dry in the incubator.

c. Next morning and every twenty-four hours later plate one of the cover-glasses.

d. In this way determine the length of time the organism in question can withstand
desiccation.

REFERENCES. F.77; L. & N. 938; McF. 46; S. 151.

Specisu DirEcTIoNS. Use a young culture of B. coli and an old (spore-bearing) cul-
ture of B. subtilis. Tabulate results.

EXERCISE XLIV. EFFECT OF CHEMICALS ON BACTERIA.

GENERAL DIRECTIONS.

a. Inoculate three tubes containing 10 ce. of sterile bouillon, with three loopfuls of
a 24-hour old broth culture of organism to be studied.

b. Add 0.1 ec. of a5% solution of carbolic acid to one tube (No. 1); 0.6 ee. to an-
other (No. 2); and 2 cc. to the third (No. 3).

c. Two hours later transfer three loopfuls from each tube to sterile bouillon and in-
cubate all of the tubes at 38° C.

d. The carbolic acid in No. 1 and its sub-culture does not prevent growth. In No.
2 no growth, but abundant in its sub-culture (acts as an antiseptic). In both No. 3 and
its sub-culture no growth (acts as a disinfectant) .

REFERENCES. F. 81; L. & N. 90; L. 107; McF. 46.

SPECIAL DirEcTIonS. Use B. coli.

50 General Bacteriology.

EXERCISE XLV. RELATION TO OXYGEN.

GENERAL DIRECTIONS.

a. Pour a tube of melted agar into a sterile Petri dish, and when the medium has
hardened make several parallel streaks with a platinum loop charged with an aerobic or-
ganism.

b. Sterilize a piece of mica or acover-glass, by passing it several times through the
flame and place this over several of the streaks. This is to shut out the air and should
therefore be in perfect contact with the medium.

c. Make another plate in the same way using an anaerobe.

Rererences. F. 60; L. & N. 95; L. 180; McF. Chap. VIII.

SpeciaL Directions. Use B. subtilis and an anaerobe.

EXERCISE XLVI. EFFECT OF DIRECT SUNLIGHT.

GENERAL DIRECTIONS.

a. Make an agar plate of the organism to be studied (seeding rather thickly).

b. When agar has thoroughly set, invert the Petriand paste on under side a piece of
black paper from which has been cut out a number of letters, e. g., student’s initials.

c. Expose this dish, paper side up, to the direct sunlight for a number of hours
(4-6).

d. Remove the paper and incubate.

REFERENCES. F. 71; L. &N. 101; L. 77; McF. 46; 8. 151.

SPECIAL DirEcTIONS. Use B. prodigiosus (Ehrenb.) Fluegge or B. typhosus.

EXERCISE XLVII. DETECTION OF GAS (Shake Culture).

GENERAL DIRECTIONS.

a. Melt a tube of glucose agar (or gelatin) and inoculate with a gas-producing
organism.

b. Thoroughly mix and solidify quickly by placing in ice water.

c. Incubate over night.

REFERENCES. L. & N. 153; M. & RB., 85.

SpecraL Directions. Use B. coli; incubate. Make sketch.

EXERCISE XLVI. QUANTITATIVE ANALYSIS OF GAS (Fermentation Tube).

GENERAL DIRECTIONS.

a. Inoculate the open arm of a fermentation tube with a gas-producing organism.

b. Incubate at 38° C.

c. By frequent observations determine: P

1. Whether growth takes place in the open or closed arm, i. e., whether it is aero-
bic or anaerobic.

2. The rapidity and total amount of gas formation. Use gasometer. (Plate I. B.)

3. Kinds of gas. When the culture has ceased producing gas, completely fill the
open arm with a 2% solution of sodium hydrate; place the thumb over the mouth of the
tube and thoroughly mix the Na OH with the gas in the closed arm, then without remov-
ing the thumb return the gas to the closed arm, remove the thumb, when the medium
will rise in the closed arm to take the place of the absorbed CO,. Measure. The re-

B. GASOMETER FOR
FERMENTATION
TUBE

ora e
ne \
coca IEn

gan
Ze=
Zen

>

TORRE
1RQarananaenl
RSIS

7
MA

ti-

meters. The numbers indicate the area o e various

A
sc

LT

=
=

ss
ot
oH
Hye

cross-lines divide the figure into square cen

A. PLATE COUNTER. See p

log

Het
rH
HH

Re
aa {J
a
ee) Ch,
1
ap O)
. oe
Ao
if.

|

. , el
LeU /o a
of,

See p. 50.

Ly
ae
Wh

L]
a
eet

52 General Bacteriology.

maining gas is considered as hydrogen; bring this into the open arm, remove the thumb
and introduce alighted match. Air mixed with the hydrogen present causes a slight ex-

plosion. Express the amount of CO, and H. in the form of a proportion. _H _ as,

CO.
Rererences. A. 203; McF. 54; M. & R. 86.
SprciaL Directions. Use B. coli, also try B. subtilis.

EXERCISE XLIX. DETECTION OF ACIDS (Wurtz).

GENERAL DIRECTIONS.

a. Melt a tube of lactose agar (gelatin can be used) and add enough of a sterile, blue
litmus solution to give it a distinct color, cool to 42° C., inoculate it with an acid-pro-
ducing organism and pour in the usual manner.

b. When the agar has solidified invert the dish and place it in the incubator.

REFERENCES. McF. 54.

SprcraL DirEcTIONS. Use B. coli and incubate at 38° C.

EXERCISE L. QUANTITATIVE DETERIIUNATION OF ACIDS.

GENERAL DIRECTIONS.

a. Inoculate 5 test-tubes of glucose bouillon (or milk) with an acid-producing
organism.

b. At periods 24 hours apart remove, with a sterile pipette, 5 ce. of the medium from
each and titrate with a twentieth normal potassium (or sodium) hydrate solution, using
phenolphthalien as an indicator.

c. Plot the results, expressing the number of cc. of hydrate solution as abscissae
and the daily intervals as ordinates.

SprciaL Directions. Use B. coli and incubate at 38° C.

EXERCISE LI. DETECTION OF NITRITES IN CULTURES.
GENERAL DIRECTIONS.

a. Make a culture of a reducing organism in a test-tube of the nitrate solution
(XXXVII. g.).

b. Incubate at 28° C. for 1 week, add 1 ce. of each of following solutions:
1) Sulphanilic acid (para-amido benzenesulphonic acid) 0.5 gm. Acetic acid (sp.
gr. 1.04) 150 ce.
2) @-amido-naphthalene acetate. Boil 0.1 gram of solid a-amido-naphthalene in
20 ce. of water, filter the solution through a plug of washed absorbent cotton, and mix

the filtrate with 180 cc. of diluted acetic acid. All water and vessels used must be free
from nitrites. (Leffman and Beam.)

The presence of a nitrite is indicated by a pink color.

c. A tube of the original medium should be incubated and tested as a control.
REFERENCES. A. 215; McF. 56.

SpeciaL Directions. Use Bacillus vulgaris. (Hauser.) Mig.

EXERCISE LI, DETECTION OF AMMONIA.

GENERAL DIRECTIONS.
a. Make bouillon culture and incubate.

54 General Bacteriology.

b. Place in neck of tube a piece of filter paper which has been dipped in Nessler’s
reagent (for formula see works on water analysis). A yellow to reddish brown color
indicates the presence of ammonia.

REFERENCES. -L. & N. 141.

SPECIAL DirEcTIONS. Use sewage to inoculate medium.

EXERCISE LIII. DETECTION OF SULPHURETTED HYDROGEN.

GENERAL DIRECTIONS.

a. Make a culture in a test-tube, or better, a flask of bouillon and incubate at
38° C.

b. Twenty-four hours later fasten in the flask, by means of the cotton plug, a strip
of filter paper moistened with lead acetate.

c. The presence of sulphuretted hydrogen is indicated by change of color from
brownish to blue. The color change is often slight and can be best detected by frequent
observations.

REFERENCES. L. & N. 188.

SpeciaL Directions. Use B. colt or sewage.

EXERCISE LIV. DETECTION OF INDOL.

GENERAL DIRECTIONS.

a. Make a culture in a tube of glucose-free broth* (or Dunham’s solution).

b. 24 hours to 1 week later add a few drops of concentrated sulphuric acid and 1 ec.
of sodinm nitrite solution. (Sodium nitrite, 0.02 gms. Distilled water, 100 gms.)

The presence of indol is indicated by the production of a deep red color.

Rererences. L. & N. 142; McF. 56; M. & R. 87.

SpeciaL Directions. Use B. colt.

EXERCISE LY. DETERMINATION OF CHEMICAL ENZYMES IN CULTURES.

GENERAL DIRECTIONS.

a. Make two gelatin stab cultures of a rapidly liquefying organism and incubate
several days or until the gelatin has all been liquefied.

b. Pour one into a tube of gelatin to which carbolic acid (zy ce. of a5% sol. per
ec. of medium) has previously been added. Mark the line which separates the liquid and
solid gelatin.

c. Add the other tube of liquefied gelatin to a tube of carbolized milk.

d. Make control cultures in the carbolic media with a pure culture of the organism
used above to show that the acid inhibits the growth and that the changes are not due
to the living organism.

REFERENCES. McF. 53.

’ SpectaL Directions. Use B. subtilis.

EXERCISE LVI. VARIATION IN ENZYME PRODUCTION.

Make stab cultures of Pseudomonas aeruginosa (SCHROETER) Mia. (B. pyocyaneus),
or any slow liquefier, in ordinary neutral gelatin and also glucose ais Compare
rate of liquefaction in each.

*This is prepared from beef by inoculating the meat infusion with an organism capable of fer-
menting sugar, such as B. coli, and allowing it to stand several hours at 38° C. The meat is then
strained and the bouillon prepared in the usual manner. This is recommended for testing for indol.

56 General Bacteriology.

EXERCISE LVIIl. VARIATION IN COLOR PRODUCTION.

Make an agar streak of B. prodigiosus. Incubate at 38° C. 24 hours later transfer to
fresh media. Continue the process of daily transplanting from cultures of previous day
until chromogenic property is lost, even at the room temperature.

‘CHAPTER IIL.

TAXONOMY.

POINTS TO BE OBSERVED IN THE STUDY OF BACTERIA.

The following scheme gives an idea of the points to be noted in the description of
an organism together with some of the more common descriptive terms.

CULTURE CHARACTERS.
1. GELATIN PLATE:

A. Surface colonies.

a. Form: Punctiform, too small to be defined by naked eye; circular; oval;
irregular; fusiform; cochlate, twisted like a snail shell; amoeboid, very irregular like
changing forms of amoebae; conglomerate, an aggregation of colonies.

b. Size, expressed in millimeters.

c. Surface Elevation: flat; spreading; thin; raised, growth thick with abrupt,
terraced edges; convex, surface segment of a circle but very flatly convex; pulvinate, sur-
face the segment ofa circle but decidedly convex; capitate, hemispherical; rough, irregular
elevations and depressions; contoured, like the undulating surface of a relief map; papil-
late, horn like projections; rugose, wrinkled; alveolate, depressions separated by. thin
walls; pitted; sulcate, ridged or furrowed. ,

d. Consistency: thin; membraneous, thin, dry, separating from medium; coria-
ceous, thick like leather or parchment; viscous, ropy; slimy; gelatinous; brittle.

e. Color: transparent; vitreous, transparent and colorless; oleaginous, trans-
parent and yellow, olive to linseed oil colored; resinous, transparent and brown, varnish
or resin colored; translucent; paraffinous, translucent and white, ‘porcelaneous; opales-
cent, translucent, grayish-white by reflected light, smoky-brown by transmitted
light; macreous, translucent, grayish-white with pearly lustre; sebaceous, trans-
lucent, yellowish or grayish-white, tallowy; butyrous, translucent or yellow;
ceraceous, translucent and wax colored; opaque; cretaceous, opaque and _ white;
chalky, dull without lustre; glossy, shining; fluorescent; iridescent.

f. Margin (To be determined by low power of microscope): entire; undulate;
repand; erose, finely eroded as if gnawed; lobed; articulate; laciniate, cut jaggedly into
deep narrow lobes; lacerate, cut variously into irregular segments; jfimbricate, edge
bordered by slender processes thicker than hairs; ciliate, tufted; floccose, wooly, filaments
in fleecy masses; curled, filaments in locks or ringlets: filamentous, consisting of loosely
placed, interwoven filaments, not so dense as floccose.

g. Internal structure (To be determined by microscope): homogeneous, uniform
throughout; concentrically zoned; marmorated, traversed by veins as in some kinds of
marble, marbled; finely punctate; areolate, marked out with small spaces, reticulate;
moruloid, having the character of a morula, resembling a mulberry; segmented; finely
granular; coarsely granular; grained, as in lumber; curled, composed of twisted bundles
of parallel filaments as in locks or ringlets; floccose; filamentous.

h. Change in Medium: consistency; color; odor.

58 General Bacteriology.

B. Deep colonies:

a. Form.

b. Size.

ce. Color.

d. Internal structure.
2, AGAR PLATES:

A. Surface colonies.

B. Deep Colonies.
38. GELATIN Stas CULTURES.

A. Non-liquefying.

a. Line of puncture: filiform, uniform growth without any special characters;
tuberculate; papillate, covered with papille; echinulate, minutely prickly; villous, beset
with long or short undivided hair-like extensions; arborescent, beset with branched hair-
like extensions; beaded, composed of small round more or less conjointed colonies;
banded longitudinally.

b. Surface: (Same as surface colonies gelatin plates 1 c.)

B. Liquefying.

a. Shape of liquefied area: crateriform, saucer shaped liquefaction of gelatin;
saccate, shape of an elongated sack, tubular; cylindrical; funnel formed; napiform, out-
line of a turnip; fusiform, outline of a parsnip; stratiform, liquefaction extending to
the walls of the tube and then downward horizontally.

b. Fluid: clear; turbid; flocculent.

c. Sediment: flocculent; stringy ; granular.

d. Membrane: character; color.

4. STREAK CULTURES:

. Form. )
Size.

Surface elevation.

. Consistency. . ; : : ;
Gctor: ( Same as for colonies on gelatin plates (1).

} Same points as in gelatin plate, (1).

Margin.
Internal structure.
. Change in medium. /
5. PoTaTto.
A. Growth apparent. (Same as plate cultures).
B. Growth not apparent.
6. BovmLLoN:
a. Character of fluid: clear; turbid; ete.
bv. Sediment.
c. Membrane.
7. MILK.
A. No visible change, even after boiling.
B. Curd formed:
a. Time required.
b. Character of curd: hard; soft.
c. Digestion.

FR mMeasecs

General Bacteriology. 59

d. Character of whey: clear; turbid; flocculent.
e. Reaction.
f. Gas.
g. Odor.
8. Buoop Serum: (Same as streak cultures).

MORPHOLOGICAL CHARACTERS.

a. Form.

b. Cell grouping.

c. Size.

1. In terms of the micromillimeter; breadth, average and extreme length.
2. In terms of human blood cell.
d. Stain.
1. Aqueous solutions; stains easily or with difficulty; uniformly or irreg-
ularly. ; :
2. Speéial stain; Gram; tubercle; ete.
e. Motility.
1. Brownian movement.
2. Vital movement; sluggish or active; rotary or direct; most favorable
temperature ; age; media; ete.
3. Flagella; stained by howtos, Bunge or Van Ermengem’s method; dis-
tribution, monotrichal, lophotrichal or peritrichal.

f. Capsule; stained by Ziehl, Gram or Welch’s method; most favoriile con-
ditions; broad or narrow; present inserum, milk or on agar streaks.

g- Spores; time required for formation; media; position in cell, center or end;
effect on shape of cell, clostridium, or drumstick; germination, time, temperature; stain,
Hauser or Moeller’s method; temperature limits.

h. Vacuoles (plasmoloysis).

1. Crystals.

j. Involution forms.

k. Pleomorphism.

1. Effect of various media.
2. Effect of reaction of media.

PHYSIOLOGICAL CHARACTERS.

. Effect of desiccation.
. Relation to temperature; minimum; optimum; maximum; thermal death

oe

point.
c. Relation tooxygen; under mica plate; in hydrogen.
d. Relation to light; (Buchner’s Experiment XLVI.).
e. Relation to antiseptics and disinfectants.

f. Pigment production; relation of development to oxygen; relation of de-
velopment to character of medium; changes produced by alkali and acid; solubility; spec-
trum analysis.

g. Gas production; rate, quantity and formula produced on glucose, lactose,

and saccharose media.

60 General Bacteriology.
®
h. Acid and alkali production; carbohydrates present; carbohydrates absent.
i. Relation of growth to acidity and alkalinity of medium; growth in 1.5, 3
and 4 % alkali; growth in 1.5, 8, 4 and 5 % acid.
. Reduction of nitrates; to nitrites; to ammonia.
. Production of sulphuretted hydrogen.
- Production of indol.
. Enzyme production; proteolytic; diastatic.
. Characteristic odor.
Pathogenesis:
1. Modes of inoculation by which its pathogenic properties are demonstrated.
2. Quantity of material required.
3. Duration of the disease and its symptoms.
4. Lesions produced and the distribution of the bacteria in the inoculated
animals.
5. Which animals are susceptible and which are immune.
6. Variations in virulence and the probable causes to which they are due.
7. Detection of toxic or immunizing products of growth.
8. Widal test.
9. Pfeiffer’s phenomenon.

eo 8 Bare.

REFERENCES: Chester, Report Delaware Experiment Station, 1897; A. 216; P. B.
C. (Cheesman’s Charts).

CLASSIFICATION OF BACTERIA. (MIGULA.)

I. Cells globose in a free state, not elongated
in any direction before divisions in 1,

2,or 3 planes. CoccacEkaAE ZOPH emend. Mia.
A. Cells without organs of motion.

a. Division in one plane, - - 1. Streptococcus BILLROTH.
b. Division in two planes, - - 2. Micrococcus (HALLIER) COHN.
c. Division in three planes, - - 3. Sarcina Goodsir.

B. Cells with organs of motion.

a. Division in two planes, - - 4. Planococcus Miguua.

b. Division in three planes, - - . Planosarcina Miqauua.
II. Cells cylindrical, longer or shorter, and only di-

vided in one plane, and elongated to twice the
normal length before the division.
(1) Cells straight, rod-shaped without sheath,
non-motile by means of flagella. BaCTERIACEAE MIGULA.
A. Cells without organs of motion, — - 6. Bacterium ENRENB.
B- Cells with organs of motion (flagella).
a. Flagella distributed over the whole

oO

body, - - - - 7. Bacillus Coun.
b. Flagella polar, - 2 - 8. Pseudomonas Miguua.
(2) Cells crooked, without sheath. SPIRILLACEAE MIGULA.

A. Cells rigid, not snake-like or flexuous.

General Bacteriology.

a. Cells without organs of motion (flag-

ella), - - =~ = = 9. Spirosoma Miquua.
b. Cells with organs of motion (flagella)

1. Cells with 1, very rarely 2-3

polar flagella, - - - 10. Microspira SCHROETER.
2. Cells with polar flagella-tufts, 11. Spirillum EHRENB.
B. Cells flexuous, - - - - 12. Spirochaeta EHRENB.
(3) Cells enclosed in a sheath. CHLAMYDOBACTERIACEAE MIGULA.

A. Cell contents without granules of sulphur.
a. Cell threads unbranched.
1). Cells division always only in one
* plane, - - - - 18. Streptothrix Coun.
2). Cell division in three planes pre-
vious to the formation of
condia.
i). Cells surrounded by very
delicate scarcely visible

sheath (marine), - —§- 14. Phragmidiothrix ENGLER.
ii). Sheath clearly visible
(fresh water), - - 15. Crenothrix Coun.
b. Cell threads branched, - - 16. Oladothric Conn.

B. Cell contents containing sulphur granules. 17. Thiothrix WINOGRADSKY.

(4). Cells destitute of a sheath, united into threads
motile by means of an undulating

membrane. BEGGIATOACEAE.
Only one genus. (The single species is

scarcely separable from Oscillaria) - 18. Beggiatoa TRAVISAN.

61

CHAPTER IV.

SYSTEMATIC STUDY OF REPRESENTATIVE NON-PATHOGENIC
BACTERIA.

EXERCISE LVIII. PREPARATION OF SPECIAL MEDIA.

Tube and sterilize the following media for work in Chapters IV and V:
80 tubes of plain agar.
2 tubes of lactose agar.
20 tubes of gelatin.
8 tubes of bouillon.
10 fermentation tubes of glucose bouillon.
8 tubes of potato.
8 tubes of milk.
8 tubes of Dunham’s solution.
10 water-blanks.

EXERCISE LIX. BACILLUS PRODIGIOSUS (Ehrenb.) Fluegge.

EXPLANATORY. This organism was first described by Ehrenberg (Erhandlunger der Berliner Akademie) in 1839 and named Mong
prodigiosa. It is the oldest known chromogenic bacterium. It is commonly found in the air of Europe and has a very interestinghi,
tory on account of its casual relation to bread epidemics—‘“‘bloody bread,” ‘bleeding host,” etc. It is questionable if it occurs Spontg
neously in this country. Itis slightly pathogenic. Introduced intraperitoneally into guinea pigs in large quantities it produces deat,
Inoculated into animals naturally immune to malignant oedema it renders them susceptible. Rabbits inoculated with anthrax are protecta
by a subsequent inoculation with this organism. It is grown with the streptococcus of erysipelas to produce Coley’s Fluid for —
of inoperable malignant tumors. a

REFERENCES. Lafar, 137-138. :

SKETCHES,

cultures.
Incubation
temp. (°C.)

MoRPHOLOGICAL CHARACTERS.

Age of

1, Form:
@, Bouillon: sce vececc vere dichnes cbid nase Gawcarsiew bo Ad cia hd Eee weinte Miaqes seid GEN VERA TH A ami Re RIE wale wie Semana Tis Ra Sabie [95 ie anise eit

ad, Other media

BAS ZO ie da teray ee we
3. Cell groupings...

atid ‘ATrANGEMEN tS! ai.c se iciencises vigeRaswa) aie wis tide ay apie ailtiondis ais nye Sueisinws wiaiRia ouassie: Wlars Subeahd SG cals ate avetaye e/Rdia SadceraCoee dub, Qddgonc ese gagvecd| B56 fF inuiare | ean Goinisles | ESM RMON

a, Aqueous gentian-violet.......... 0... cece ee cece eee ee eee eee eee 1 aiesanesalivd eaCSoaeione sls 9% late SERS GSS AERTS i ce ateactiret || tee sacellanese eee
6;..Loeffier’s: meth ylens blues 2 sccsacw a ssecncraine sews damint tie Aacd 20 a6 aoa canadian oe Denaeeewaoas ubie sedans daapa cede Ren Bid Baud spent ees

@: Grany’S Stain + wrceacaaw im peieg eaters $e jiica las Yel weoshateeatadiaa ye. gigas iene eMae us Se ewaw aarti maine: GR Gee ies dion ice’ cae mdall ela ator

4, Flagella stain
6. Spores:..

is Special Characters; SuGh AS .22..0dcnd id dee cseudeade 5 bea scedeesd ze gee, ae Sb sev drend nud ccrenngaren aya td lode attivhton sah agerdcanboad seajsopene cted dhe vanenstenbccrenewbil, des ucleedae and [reswinte edvaal ded aeaee
deposits, VaCuolesy sires ay se iain weakaincarse.ag Wis winulins de Wakiwteninn Geen ata ae SaeemlO es Fu ieee ae Renamer aIY Baines Sa SinatiA ad em wind tunel] alae atecete Posse ass She
pleomorphiciand:involution forms, capsules, tevicsciics scl clsjegie oldnriua te ag snes da done im aatens endaules yx av apace nenad waaeayed | Ay oi gaeall aeew ooze:

PHYSIOLOGICAL CHARACTERS.

i ‘Relationto temperature r:iictesnnarsraicanastiy se ks einen shad whadaw te aeremaaaniNamoaie maine aMaet, wLentie sk meine AUS Hage AR mtidatea Bans OA Mae Soaaeenie a ae aoe hme teas diate Ce
a: Relation to:free: Ox ye etl os ned seats cova ine sracate tatters sa aus BE ay Relays Sh SUR Wo NE BERG prsnsltA e Ae wieisicna 4-4 eayeidd. doa sesdsorepany gearelign be wyend bobs AG MAMebea Sa echo alma RM aaeaea Mamaia
3. Relation to other agents, such aS......... 0... cee cece eee eee eee eee een 8 Seid ienaaara ivggte seajesSele Denspupn end -Take ha ahaa. saliia tages Vee wae sncdajeritiptavar Wie Wed aahoteumbdann Sok -ave eS tara ats eat eee ete

desiccation, light, disinfectants, etc.:.......... 2... 5. eolecus aicenee Scclan ssa Renanarn east ne ase en aah yaalelove haan eecatpesiae
4. Pigment production:

5. Gas production in glucose media:

@: Shake Cultures ov xsi: as vheee- se se tees east agi ge os ye Shui Ad eee ee RIRe BERR G Rete ROE SEE i doar isda tengiauele en aie Syste Sia adhe toje teas aR yada zamemeEee ET
6, Fermentation tube, growth in: (1) opemarm:............ 0. cece eee ee eee waisisenegitney (2) closed arm: .....
(3) rate of development: 24 hours ............... percent., 48 hours ......... per cent., 72 hours ............. percent., .... .. hours

(4) reaction in open armi..... 2... ee eee cee eee cet eee ener sees eeee (5) gas formula: H:COg::
6. Acid or alkali production, litmus milk. .... 0... 6c. cece cece eee ee eee ene EEE ERLE RE en Ene EE EE Ooo cE ed Ee EE EEE Ebb bed bbb bebe cece cece cece cued 4
7. Reduction of nitrates; to nitrites eat ncansde tI rats a foe ee be
8. Indo] production; 24 hours ........ 62. .c cece cece ce eee ‘
fecal Sders 24, WOUTS soa pacus so be ccuswuwns coronene vane

g. Enzyme production : proteolytic .....

ar PathO@emesis: << 9-22rq i iesaus id me geeteeceaatees selseeg! Easinre mime it me seaRepietes 1a ie ne cnubdeudeh yee eae cocks

[64]

CULTURE CHARACTERS.

Reaction.

of Medium,
Incubation
Temp. (°C)

24..

.» Hours.

SKETCHES.

(1)

latin
late:

~ Surface
Colonies,

x Deep
Colonies.

~

(2)
ar
late :
Surface
Colonies.
Deep
Colonies.

(3)

atin.

sab.

(5)

Sy

[65]

EXERCISE LX. VARIETY OF PIGMENTS.
Make agar or potato streak cultures of the following organisms, incubate at 28°C., study, describe and sketch.

AGAR STREAK. 24 Hours. : 48 Hours. SKETCHES.

Bacillus
indicus
or

Sarcina
aurantiaca
or

Sarcina
lutea
or

Pseudomonas
fluorescens
(B. fluorescens)
or

Pseudomonas

aeruginosa

{B. pyocyaneus)
or

Pseudomonas
violacea
or
a

[66]

General Bacteriology. 67

EXERCISE LXI. SEPARATION OF BACTERIAL COLORING FATTER.

a. Make four agar streaks of Bacillus prodigiosus, which are to be kept in the dark
until the coloring matter is well formed.
b. Add about 10 cc. of ether to each tube and shake vigorously until the red pig-
ment has all been dissolved out.
c. Pour into a large test-tube and allow to stand over night in the dark, then
pipette off the colored portion.
d. Divide this into four parts and treat them as follows:
1. Evaporate on glass slide and examine crystals formed under microscope.
‘2, Add a few drops of hydrochloric acid, drop by drop.
3. Add a few drops of sodium hydroxide.
4. Stand in direct sunlight.

EXERCISE LXII.

BACTERIUM PHOSPHORESCENS Fischer.

GENERAL CONSIDERATIONS. Described by Fischer in 1887 (Zeitschrift fir Hygiene, Band II, p. 92). Found in Kiel harbor, dead
sea fish, oysters and occasionally on meat in shops. The production of light isshown in the dark, especially when the organism jy
grown on a medium made by boiling two salt herrings in a liter of water, adding 100 gms. of gelatin to the filtrate without neutraliza,
tion, tubing and then sterilizing (Lehmann). Phosphorescence can even be restored to attenuated cultures by growth on this Medium,

Inasmuch as oxygen is necessary to light production surface growths are best.
REFERENOES. Lafar 160-164.

MORPHOLOGICAL CHARACTERS.

Sultures.

Age of

Incubation
temp. (°C.)

SKETCHES,

1, Form:

a. Bouillon...

d. Other media

6 POs cnc navasns

. Cell groupings

and arrangements :2 39% ctstanaseme iene ve ce seteeinee memeuuies teed bai iasaQieineaebbchnetate aeacage)S ane Aoeged eC ues Ga chance elveraneein Macrae ees

a, Aqueous gentian-violet.....
5. Loeffler’s methylen-blue.... .

c. Gram’s stain

d. Special stains

OMA GAIA 92 cee cM ON ea NN Oh ae A A Sa set DOO Sass ese ae AT Ae NA Teer thE ed se teria as

a. Character of movement

6. Flagella stain

SOR a ceeded 5 oie vance ded a biSiuahges ay 1s Se Satara wie Sides acd aE athe @aentd etched ne DRA e RIAN ey aaa Rien ante mss Ege

desiccation, light, disinfectants, etestii. sis iwns cnisisdarsaeeneiae neley epinsiaasinweaverd’s Gs £6 tetas nedvse meee gout

4. Pigment production:...... 0... .c0. cee cette eee EEE eee nee ca ee cone EE EE Stuy ctbE bebe dEE bEbE bbeL bebe bebe babe babe bebbibee cbebuansat "
g Gapedueeaminemneeda?, ==CU~C~C~‘“‘C‘“‘C“‘(Sé~*C*‘“(‘ a SO
@, Shake culture .. 20.0... cee eee cee eee ee EEE EEE EERE EEE EEE DEE EEE be bbe bel
5. Fermentation tube, growth in: (x) open arm... ..eccceceeecesecceeceee cesveeee(@) 10800 AIM! ceecsoerecee
(3) rate of development: 24 hours ......,........ per cent., 48 hours .......... percent., 72 hours ........, seee POL Cent.) . consis cad
fa) weacilot It OPE APMG, .0.icincensi ater oxen nasrunwed nus goeenien aa v++00(5) gas formula, H :CO,::
6. Acid or alkali production, litmus milk. .... 1. ..1e see ee cece eee eee eee eet e cede ceae tenet end crite dabei bebe bbe ce
7. Reduction of iti Ps tea atta aay ane cdcrgcercnad ah eh dnaain ae daa tah en Noein mae natn. ae cree tees
8. Indo] production; 24 hours ........-.60. cee eee eee te eee 6 ABR OUES scsars davsins winded vase Mae weboeenpee ina a
fecal odor; 24 HOUTS .......66 06 ce eee eee econ een eens AB NOUTS (5:5 seers alsin? sdayownn al Seu hoy odcees
9. Enzyme production : proteolytic ........ 0... cece cece eee eee ener nsec ners lets veceucens
10, Characteristic Odor... .... 2.2.6 e cece cee eee ee ee ee ee eects neE tebe cite sien bee eens
11, Pathogenesis .........-6..scceeeee eens cere eee eee nee een tnee tbl bte ibe cbae eee.

[68]

CuLTURE CHARACTERS.

Reaction
es 24.....5 Hours. ABs aise Hours. Cra vrais Days. SKETCHES.
Temp. (°C)
(1)
Gelatin .
plate:
(a) Surface
Colonies.
(b) Deep
Colonies.
(2)
Agar
plate:
(a) Surface
Colonies.
(b) Deep
Colonies.
(3)
Gelatin
Stab.
a A. i /N
/\| 47
\| |
Agar . i AM |
Streak. \
N
a
(5) -
/
/ \ / a
{7 lL |/
/ \
Potato. |
NN,
(6)
Bouillon.
(7)
Special
Media.

[69]

EXERCISE LXIH. BACILLUS ACIDI LACTICI Hueppe.

‘GENERAL CONSIDERATIONS. First described in 1884 by Hueppe in Mitteil. aus dem Kaiserl. Gesundheitsamte, Bd. II, p. 1837, This
organism may be taken as a type of the bacteria causing sour milk.
REFERENCES. Lafar, 222-244.

; SZ | Sa SKETCHES;
os = =I
MORPHOLOGICAL CHARACTERS. BO | gs ;

1. Form:

@. Bouillon ............

2, Size ........
3. Cell groupings.......
and arrangements ..
in growths....
4. Staining powers:
a, Aqueous gentian-violet ..
8. Loeffler’s methylen-blue. .. ah GRA eee eaiaish Seta ASE Baas Re RIACRIASH ae Moe NAGA Aevetads TAR Sek deal do eamaye erates lemeinaierats.| denied
c. Gram’s stain...
da. Special stains .
5. Motility:...... 00... eee

a. Lineaeter a! MmoveMien tices de iv ingens sarees 01 peed een eraninnea i icamen renee ing. Ra th Remon areTEs sh oP wake saved mean vor eet xpos
Be PICO ASA ons eecavazas v4 490s Guaes wr baues SAK Ba eer) Cuan ERY 9-H CRRA A PRE 4 HE 05 Ua SRG SRR WH WR wed ceeds bewne vere
6. Spores..... diguibitabaes atanbighd Holand ane dha. secaiaraus asypauesialancland © Shatss voesialan aed rueeanen teal erara aa
7s DPOcial Characters, SUCHIAS! counter cs cen cantrauciWonn Sa ne van Juste Uvicw dake da pe pad duauyd nepmeMe ta sa deNeaa Te pulaeweey eee aeadenanlte-04 mprilloctes wane

deposits, vacuoles............. ag Fa de lawton args erblnctie wh oe eawtantne yee arcaanre akgh desks tamleeasaese Bead Gemiee tates de Avon nace ayers

Pleomorphic and involution fornis, capsules. Gidecisc <4. asmaaaneiwadaude ts dosadeamde i eloweues ee camer <aeeade ne ae denne sted 14 License,

PHYSIOLOGICAL CHARACTERS.

3. Relation to other agents, such as:.

desiccation, light, disinfectants, etc.

Gas production in glucose media:

a. Shake culture .........-...065 seeeeee ‘iidriaiiners Uae eeiinlai bg allowing a emacs Yee A Resaibid deat araiasetclat inne ah median soph: Seats Masitoa lb laaidid ded $a onssormetaialzns ndeonibeagusionatéee dab

b. Fermentation tube, growth in: (1) open arm:

(3) rate of development: 24 hours.......... ..per cent., 48 hours............ per cent., 72 hours

2

Acid or alkali production, litmus milk. .

Requetiok Of MN TARESS TO MIME ER cceicsn See sa ak eR IRAE anes ds be bd cenaumaraeceen ra wen , to ammonia.

8. Indol production; 24 hours....

fecal odor; 24 hours... 2.2... sce cece ee ee cece cence ey FB MOUTS eee eee ee eee bebe eee eee ce ga

Tr, Pathogenesis..... 0.60600 cee ce cece eee ee ee eee ee ete eens ete Eee tebe bt cabs cree ee ee ces

[70]

CULTURE CHARACTERS. aS

Reaction ; oy
pede OF ne Hours. SAB ag ie Hours. ere Days. SKETOHES.

Temp. (°C)

(1)
Gelatin
plate:
(a) Surface
Colonies.
(b) Deep
Colonies.

(2)
Agar
plate: :
(a) Surface
Colonies.
(b) Deep
Colonies.

(3)

Gelatin
Stab.

(4)

/
Agar H
streak.

(5)

Potato.

(6)

Bouillon.

(7)

Special
Media.

[71]

EXERCISE LXIV. BACILLUS VULGARIS (Hauser) Migula.
PROTEUS VULGARIS.

GENERAL CONSIDERATIONS. Described by Hauser in 1885 as Proteus vulgaris (Ueber Faulnis Bakterien). It is widely distributed
and is commonly found in putrefactive substances. It is one of several related species included under the old name of ‘‘Bacterium
termo.” While in small doses and under ordinary conditions it is harmless, at times and in large doses it may be pathogenic.

REFERENCES. Lafar 194-199. ‘

SE | 8a SKETCHES,
MoRPHOLOGICAL CHARACTERS. 85 | £8
1. Form:
@. Bouillon cicvssvcialecsnialse ewer aera enreed «xe neon eeeuedeenee ans eecie Sateen vids ee ayed Palags dy te Hee Maan tte eel oonte se Lae enentt
Os PRB Das i cyecccssisza.d Sicediege'e ha doyeecheneaitenrd thes be RSG Aids aR Ke Oa RE REE Ral a GOR ERE aes neal Reem ORT ees tadooneen bene GO [swe es
c, Gelatin ...., GIB aS Sec steak UA et th see Ras eke Sie Bena He Sip ean lesotsltte al eiaNe eld i esata Sncg MRSA Sie. Bite GARMIN ME UE MINUS Ra NIMREIE is ab cetera PTS RY RS [chinese
2s Othet medias sdieacdens tansy a vale Sera tatinie ak ween Ss ig ales Salers Boa oe Bectiaaee a eemeeta ae Acme meneame wade boa ec | ORR BR
BN SIZO i scoschivisemseptoniacieneddlaatts ve a biaiehls Gaevenaee hee Suite aicksins ec oeu sand tame Reims Vernisitna Wy IS ogmnngiee aati seiee tan alacluvielaaaterte ainsi pean Gal neue 3a
8. (Cell groupings). csi suze canwoeaa yp ya Saee Pedismtied cde hy jnsdacieaacaieceuise Te Te Geman aseNRRRANT ENE Es POhupieNRGS ExAe Taw HMR SEaNs [ade sag reser
and arrangements....... dinecusiaotdalhe cuca aageum~necats ddan dius same Bemebannondenauina widensine 2 cath iare ublnndae eta dana wach tated Mek ty 38 tates | Meee
WD GLOWING ais cisiisdineas araramrnanden ata te vind winded exe Coane alae MRR Ree ReaalH RAR Danid DERE ERR Twas ee hese eW eee eave [i or Cael nie EHR
4: Staining’ powers®:/siiacivieiswdas op eande sodeeaiassy. Hany wen epeecmeMane ne qusG eeaa Rew LUG bs oo Rae OUNe ta Raine Be aa nw lees dahuieta tes [246 Ree flere
@; Aqueous ‘gentian-violet! scx sees yemcied sy 26 daneostion ind nates aels scievaiata baseeeured ohUs seuceed eabwede Seah Gemeleema Rear ets a ee
Oe AOeM EL! s MSE MY] Cia) Sys caer its ica caregtassvorarus saesotecverrehasod dun u eS okeuplespaleasote lecdeaieen bon Nlacigheh dvacavain 7 debi daounia beasEd. nade uaabrnaal sees eave Ne oeltiee| RS é04elesues we
c, Gram’s stain........... 1 Seabed AER WN Yo RE RRRMRE RL ROR IA ed ReaHS ie ls niente SW alan a vaTnMS aie Pate psu (Stsaan aCRaatoeoce WE wey baal ee, casera] ntanone ES
@. Spécial stains siccrn setae sshautes dy sence eloads vanutaeneeres stor seranepanecneanae pws Ntaes amalnenia ae saa pmTaN@ terse fel a
Be NEY Sac ves waren pa wna eR RAe eas TG BL RRRHAGEAG HEROES Sah nicld ine nwdnad wkale 8b ne Kaa SNL Re HEN ROPE N PATO AN Les oh wie cre og aw ets
a, Character of movement ................. PC
b. Hlawella Stains, 9 tiocusiaitineaa vacant sig clacies 4 Sacptnidy aeiran lecsina dernlnginalesiclaaietgg ars Qhyian vase Gh Bei odladse sions Sosnaen od de lnmatriche leaseu detec
Bi SPOres scsi viesisiesehs Pa ea a Nicaea alah ie ars ls ARE Masi se oat eaten valventien descr ae sev dlapeciad Sareeoiie aus Muehbbiaa ene mos aseak leek le sel es ax oes
7+ Special characters) SUCH as? ai es ciseistienicistind £9 en: Majeigsaier advedebapatis a aa corht wsisicans en wapaiol gate alk culos payee Ve BikMEALAY Bo dckadioslunabemd fedie. “[sewetaes
deposits, vacuoles) «ccarcsnacsedimitmarneasiinaduans aempauconne nat gurggiemuly aaa@ea nie 4.6 Meslay beaded ad Goes Snes specu yacade sec

Gas production in glucose media:

aie Pate cscheniea bi oe se mene nme nnen ba ule Adie Ghee 1h ue daaly 4aneu eae enuldae ub eg ee aan haere

3’. Fermentation tube, growthin: (1) open arm:............ 00060 ce cece cece ence veuaas (2) Chose Ati cin satsseidadaw se peu wanse se seoneeiee stesso de eolreaaeases Sind daauateeeataees
(3) rate of development: 24 hours ............... per cent., 48 hours........... per cent., 72 hours..............percent., oo... .cecceecseMOULS. cee ce cece ,.per cent.
(4); PEACH ON: IOP EM AG OTS 5 58 sais: yo dies aissnies Ses vie cesior skin do atobeas daca wage dea od eacek seAE (5) gas formula, H : COg::
6. Acid or alkali production, litmus milk.... 21... 02... ccc eee ee eee teen eee eee cate ceeds ete ete bee setaatue bite tenensne ce
7. Reduction of nitrates; to nitrites ............ ccc cece e eee cee eee eee ene -» to ammonia.............., <
8. Indol production; 24 hours........ 0... cece cece cece e eee eee , 48 hours.... days
fecal odor; 24 Hours) wists saved eeirreas vee tcemae diet niet , 48 hours......... days
6. Enzymé production: proteolytic... si... econ essa aeerseuesseccenn see se. diastatic ............ me
40:, (Characteristicod omen) qachicssicieiaclacasine ss a ia lyy caaumninda ve oon eee Seema cc Aes oauect NTE sire RMN aie CSRs os ae
sy TABU Ech sane ne areaeimnotantoan Ws entices seh occu cian ovcaah ide ohiheggn deere dade area, adeeigd Gk 027, ce asthe

[72]

CULTURE CHARACTERS.

Reaction

of Medium,
Incubation
Temp. (°C)

SKETCHES,

(1)

selatin
plate:

(a) Surface

Colonies.

(b) Deep

Colonies.

(2)

Agar
plate:
(a) Surface

Colonies.

(b) Deep

Colonies.

(3)

Gelatin
Stab.

(4)

Agar
Streak.

(5)

Potato.

(6)

Bouillon.

(7)

Special
Media.

ee
7 \
\
NY

[73]

Name of organism ............. ..000 LSet ae ia aadiedaasl oa-oeS s ees io pi ea aN. erates

Source, habitat, etc. ........... i aacemaess ania aa di ahaligiesd ood nzga ancien uae ke esata cea seabird emeue et Sistas eae ON Gehan hie oye aPadeite iat yee

SKETCHEs,

Incubation
temp. (°C.)

MoRPHOLOGICAL CHARACTERS. BS

1. Form:

Ge Bout wiscss sisccoders aed aia waves tania vine aornee de bie Grecia edauerabaciand, ga whe Sraiginle MSG UTES C8 TG SANS RRB ATRIEE BF Fs Slee Meee aes EELS Ee «pee aienatecimele slat

Be Cell Pro IMRS acai cs cee voc ste neiaedascwais sous vedEaT saa ReKaeR EA TERE am N RROEEN Sead ee am de SokmeUE eee Smee ERE Be BE [A EERE feed rE
atid ST PaU@eRIENtS cc ci ores ve deems 14 to te oa swes Conn Ke ne epee Be amd pees cae ewes dameaRS ARE lekes RaNaReD Lee meaurmeR [Aid bane LORRY TRH
TPE RP OWUNS cr cacy ba on sennaader ced suis 16s oeRO RIG ARNE MARR DUET COERTTTE INO NAAE Dey HAMS EE RER Nts ne Hee Ht pews a wean ee | 359 waa duane Bice
4. Staining POWe rs? » seieisicsiecsedrerine sawecelemensaumemunenne gees errr CO OOOO ESCO CECE COCO es Crees Eee eee
a, Aqueous gentian-violet ........-.... cee cece eee eee : sinsaiaete Ste sac theater eS ahaa Ws el antec can daa aeasaasSe ha ia gistghaE GIdzeRas A aagra\g Taretelee | UR crea tesad| Nineare nae

&, Loeffler’s methylen-blue

G.. GraniS Stains sin scciaan salen dake TA RUEMR IA IE HERA SAREE ARIS almier $4 Se ne Heme eenMan i adeyuee aca teoded ehiens ageauede bays

a. Character Of movéitient secre gee seeasia’ ences acgseisidie o 5 is iene srecntine Nisnolapk aa Rig aig cae hada Misi aomne Ad ie MaMa a Swalita Haiste es eeabenten Ihe waters de [age

6. Flagella SEALIN Sane cnca sta ceee metaee cctautectabareeas cae uae cae Aa ach aerh createed eh Ga hab bean cnea ron ren actrees || Sago eta Bee,

ASPHSIES, VACHOIES :.5 1 eevs vawey dar ouaunad i6 VlSERBERs LF Ee ses eee Kes meee TameNHN Seen 414 HROF MEHMET WE TAR) WME han ee deme

pleomorphic and Tivalatioh [oris, cA pSales, Oh s coces exeemenwas ta ws Wades KREWE HORE EA ES Sends daenae epawes, waeeas + enee

PHYSIOLOGICAL CHARACTERS.

x. Relation to temperature: ......... 0.0. .cc eee eee eee ce Weupaiees, Saad Ha 1a Macagle gaal eipeie nba es sy Bm UY PRN. ND. HE WORD w niaewang 6 sd erkiie aves ndeod Halen es 4eladun phed amie nore min eR

5. Gas production in glucose media:

a ShakéiCulture i. : cai catcew se sets eacieg ca wines ns me tolerate ere aeaes aa ak Baad ated ce Vantein’ We als Sada caeeibiaue iepduisa.ges avers ceevarteranu NY Mg ie kesiatieaneeyerde. oe. ue molePeegnaes ese be mowoierty ennmcie nana
b. Fermentation tube, growthin: (1) opem arm: ..............ccceee cee cee eee nee eee (2) closed arm:................ a. Dah aaa vgptie
(3) rate of development: 24 hours............-.+. per cent., 48 hours............ per cent., 72 hours............ per cent., a - een we ,
(4). ‘Teaction in Open arms... sisi) seasicnin cede ss Hendnmanesa ose Sons chien ailaedactneaye ai (5) gas formula, H : CO, ::
6. Acid or alkali production, litmus milk. .... 4:0. 4.0. cess eee deee oe ne coed dette cee ee tded eae ceeeseea pore aes ones suse ebeddesd Hdde vues cee cece ee ee cece cece cece ceceene cou uniuvet
7. Reduction of nitrates; to nitrites MAMAS HRA hee ea ena ae sO Coen hb
é. Indol production; 24 hours nena cans Rata oma NG ad eo eee eae
fecal odory24, HOUTS: xqcicenns ts ne curr semmncanies oto 8 MOUNTS: crcarms oy ih getaadtasene wens Jane vacingwsecelds ea iva da ecmuaceaecercn days..
g. Enzyme production: proteolytic... 1.0.2... 0... seer eee cette eee eee eee eee ee cette es Giastatic..c. cecisescceis ce conneee

rx, Pathogenesis ........--..seee cence te eee cee eee nee eee teen ene cee teen eee ees

CULTURE CHARACTERS.

Reaction

pL Mvata 4... Hours. 48.0... Hours. 6...... Days. SKETCHES.
Temp. (°C)

(1)
Gelatin
plate:
(a) Surface
Colonies.
(b) Deep
Colonies.

(2)
Agar .
plate:
(a) Surface
Colonies.
(b) Deep
Colonies.

(3)

Gelatin
Stab.

(4)

kgs / \ li
Streak. : \

(5)

Potato.

(8)

Bouillon.

(7)

Special
Media.

[75]

Name of organism .... sontenead Hi amnsee Pata ae bebe n eee PAWS MG Saga See hi eR S ei PET ENE EE aS Rae ee eI RY 9 ee RE a
Source, habitat, etc. ........... Phi cebtduele @ oN ed Manche 2 ee as a ieheestalar is sae Seek eee ale Mie eines shia? eee heh Ruaawenre di Stasa thal fai
ie dicgerar te atone aya aire an Miele a nites Gs Geamlansl te tess Uestiec puree ten oat Ontianees Ie rae ede rsepuanae Drei s cease eee era EE
References: ace Gack esiedes Sui veaan ee wee ees) cana eees Aaah Teel ald Rann cmhel Sheng aati at maaae Set ee en

SKETCHES,

Incubation
temp. (°C.)

MORPHOLOGICAL CHARACTERS.

1. Form:

a. Bouillon

36

&, Cell ereupitie ss neces vj cayoe va va aves damn oi Seeen fo pa galny wang e eee tains ewe Hd es wha Ke RUE RRR EN cre Rene 4d Dee EHOe Lene teen he ge 27 E PTR RNEE | Abie Hare
and arrangements.......... 0... .c0eeecseeee sees eens ‘aaas pauaectevk bisnbaherahet uoretin ran tach tataarasy enctatSetonsatie Wabi aly cialaisee Htalaved al ARN maging Geese [3 sel npcsssais per sbseis 6s
GR STOWLHS 0 sc So durcsviaindateioas node badi, Patioadad daue este hace autsrna se momen RoR Ye ee SOMME Hemeaaee Weld kia dteidsoe Soe alban seine [pense oe eee
4, Staining powers: ....... sey 5 aOR ove EPA Some ibcst WV AIRS ESB A oS ENS QOEVS Sedat HaeRNGODIEIE DS G-RAUEE RAROIGR SESE GORI BAleaae we re | Senne feet aries

@. Aqueous gentian-violet..... 0.0... cece ce cece eee cee nee eee een neta cere ee ee nt ea rete este erent cee ccee eter ertereensctseee [eneereee soa aeee

bd. Loeffler’s methylen-blue...
GGA SS TAIR 55.6. sees, aysiga dss wining 3G SS SEM Qislsy H gear ay De eae Meek Daa eae ta ot eer
a2. Specialistains sof cacec vaaecewesies ana dacenwaweaap ues 6

2 MEG CAI AES piesictcnsoapes cessstoeto stance esses ake, ia eat ats doeev nape Nat tng cee a Pau ea agah su eaten A i canted apie Sudlaep aster dns Bt asst gy -dnehd ebbetRicdialara avo asticeneey|sicketsea ?

w

@; Characterof in 6vemietit cigs castes gdlvinccin versie sais wieswretis dinveosimut deals searone wishals ieggeatane ie iettcrncece aaa ava Ws eetlnas “Shien aata ws eRe ow: [Lacs Ne ene tae eee
6: Blagellastain s¢scn oaceaceee #6 gy swearesaaaeteaaan ay in on oh eee aida apepimig ae wi ss baGdl i glgserawRe oe ae data DaeShaiele ta sila lTsabarausllsgteee wes
65: SPOLES se ccsdiscasang Be waist apaiiaus 2nce gar when ea de Selon awE eeatabien aie Week suites ghaupt wade Baoan tee emnee aa MMi oven ais eeiagl| Menem el ated

N
wn
is]
®
Q
>
2
°
>
Bp
a
2
°
>
@
bet
iP
wu
Ss
°
im
~
a

GE POSITS; VACUO] ES! re toresa acess Hates (ais vis: wataien wiacia He Testateale va ai MMNCEIRRIDIS a Aatalclaedbida ie ny ovis bhatemaneaNabniniale aa gUingla wuglamnnyynde se ait lewis estarceell eiepens Salers

pleomorphic and involution forms, capsules, etC.. 1.0.0... cece cee eee eee eee rete tere rete eben ene tae eeen eres pacale Nae

PHYSIOLOGICAL CHARACTERS.

i, Relation. to temperature: ss sasvinscrc as de ction eee s sw Hawin sctiaisne He wis tein cinyans Mawhsniala Siewince dave Bahasa YeneamAiTe oe AP Amieeee ne Ree die VE MODS Wadee UG iaes Slneeue doe oda aeR Se

5. Gas production in glucose media:
@; Sha kere dl (ure ae aawanscnnrmnmias sana laeeven ve tains Boaters eh ae wrest a. ta telly aa ea ae reine nce aig ad ccs Dealt ddan ace oll BASae hte Aaa cheba aldose ibs
6. Fermentation tube, growth in: (1) open arm:........ 0 eee cee eee eee +++es.+-(2) closed arm: ..

(3) rate of development: 24 hours

8. Indo) production; 24 HOUWIS .2.~ 02.5 casas neracenneey © te o0e
fecal odor; 24 hours ............. sii ais enecen Votes se a

Bagyme production 2 BreteOlytie acy 14 ce ocwn scien vavuioany os 19 pew elewens ba EAE wae in ad wenws

Pathageneeis «a o.ceess heewes i a See oeS Ae an BNE os ce con aceene

CULTURE CHARACTERS.

Reaction

of Medium,
Incubation
Temp. (°C)

SKETCHES.

(1)
Gelatin
plate:
(a) Surface
Colonies.
(b) Deep
Colonies.

(2)
Agar
plate:
(a) Surface

Colonies. |

(b) Deep
Colonies.

(3)

Gelatiu
Stab.

(4)

Agar
Streak.

Potato.

(6)

Bouillon.

(7)

‘Special
Media.

[77]

Name Of Organism 0.0.0.0... c ee cece cen eek eee en en ne EEE TREE E TEER EERE EEE FEET EEE EES CRETE EEE Ree eee eee ayy,

Source, habitat, etc. ........... ccc cece eee piney Ses vete PRN ge ee ayer ets as pibsetees neem: doh a aceeaieds ee eee

References.... ........cc ccc eees cece eees asa Saree Smee aes baie talent a ROG ELIE ed ates PEAS ARR ROS cbecaailad eed. a

Incubation
temp. (°C.)

MorPHOLOGICAL CHARACTERS. BO

1. Form:
Wi TIOMUNON « svciivsdavatcwaiee yoda cated obs Gdeksd Wi WAASSRIE AE RARS 1A. Senee DEE ges Fs PCa ende sd veneers eae be caaeieh | 08 CoE eR eN aN te

d. Other media
@ SIZE: s; sdea nas emacenenk tet: seve aueosaw weddeuN SER deed Se oe Seah Meee a Palgeead aa pee GaGa ae GATE A inaetOM deen da pndeMNIE OEE
3. Cell groupings
andiarrangements) i ssciiccrandis swrea es ndieaieeeenveaven ones Ginsane dda atagdvonse-edane aegonie marae atom AG Tk BAST
TY BYOWLNE. gesy coe vide bean 2s cs chow us eens naam’ va Ras Wena ss Hew ReeN RTA EARREG ER PET EeMSe Hed era RRR ERE KOM) Bed Ts Ene DRIFT Bed EP
& Staining POWers! cove crap sces cous cowed vat awen news a a gnawed eg nett we nmin He coke wdawny Rou MER REE RE RE ARE Rh Oae ob sa eae dea eR ahenIe Rinne modi wade
@, Aqueous gentian-violet 0.0.00. ccc cece cee ce eee cette tee ete bebe seed nee e eee n een en rete este ee nese eres cee tne cseenees laren ces fall eerie ete $e
b, Loeffler’s meth Vlen=blue. ...jcc-sa ia cece: ace sak one ne.ng ob enue deeeis te enuineniobe 14 Se cage Ree AE aS MORE cane ecm hee Pod rr | Cre ier
Oy Grails Stain sss ve ss eeaerocancrmaini taste se aetna awe eis Mmm GrALeMnY we cid deme da dha h/aeide Saait Seimes Hamers SAN Saga | ea aeoligaeee ae

d. Special stains .......... 00: cece cece cee ete ete tee cent tere tee een een ne ee eee tees ene eee ene eere rete rere tater refer tences [tere cers

7. Special characters, such as:

deposits, vacuoles.

pleomorphic and involution forms, capsules, tC. ........ 06.0 ccc cece cece cee cene cess cute cet ceteteteettetsteettisteseses[esseeeea[eeee eens

PHYSIOLOGICAL CHARACTERS.

5. Gas production in glucose media:

a. Shake culture

8, Tndol prodwetions 24 HoUTSs excuse 14 1a case coun ove cnye eats
fecal Odor? 24 HOUTS =. vs cccinie ce es seedaege Hagin oy ee

9. Enzyme production: proteolytic............. 0... cece cece cee eee eee siavssaeere Sialeretine an oy oudea

CULTURE CHARACTERS.

Reaction

of Medium,
Incubation
Temp. (°C)

SKETOHES.

(1)

telatin

plate:
a) Surface

Colonies.

b) Deep

Colonies.

(2)
Agar
plate:
a) Surface

Colonies.

'b) Deep

Colonies.

(3)

Gelatin

Stab.

(4)

Agar
Streak.

(5)

Potato.

(6)

3ouillon.

(7)

‘pecial
Media.

[79]

CHAPTER V.
BACTERIOLOGICAL ANALYSIS.

EXERCISE LXV. COMPARATIVE ANALYSIS OF AIR (Koch’s [ethod).

a. Plate three tubes of gelatin and expose by removing lid for 20 minutes in
the following places: 1. Laboratory, 2. Cellar, 3. Out of doors.

b. Replace the lids and keep plates at 22° C. for several days.

e. Count the colonies; if the number of colonies is greater than 100, use the
counting plate figured in Plate I. A. and count a portion and estimate the whole number.

d. Calculate the area of the Petri dish by multiplying the square of the diameter

by 0.785.
e. Express the results in terms of the number of organisms which fall per

square foot per minute.

This method enables one to make a rough comparison of the number of organisms
occurring in the localities examined, but to determine the number per volume the fol-
lowing method must be employed.

REFERENCES. H. 390.

EXERCISE LXVI. QUANTITATIVE DETERMINATION OF NUMBER OF BACTERIA IN AIR
(Petri-Sedgwick Method).

GENERAL DIRECTIONS.
g a. A piece of glass tubing 6 mm. (2 in.)
Hf in diameter by 15 em. (6 in.) long is drawn out
at one end ina gas flame and sealed.
b. Fill this tube about one-third full :
with granulated sugar, insert a cotton plug next a;:-,
to the sugar and one at the end of the tube
(Fig.13).
c. Sterilize in the hot air sterilizer for A
1 and % hours at 130° C. (sugar melts at a
higher temperature) . . » ey
d. Fasten the tube, pointed end up, in ~
a clamp, remove the first cotton plug and con-
-nect with an aspirator. (Fig. 14).
Fic.14. Aspirator for é. Break off the pointed end of the tube
filtering air. and draw a measured quantity of air through Fic.13. Apparatus for fil-

the sugar. tering air through sugar.

A, ready for sterilization.

SPECIAL DIRECTIONS. B, point broken off and at-
a. Filter 50 liters of air. taghed ty aspirator,

b. Dissolve sugar in 10 ce. of sterile water and make plates, using 1 ec. of the
mixture. :
c. Incubate, count colonies as above and estimate the number of organisms per
liter of air. ;

REFERENCES. A. 551; H. 393; L. & K. 392; McF. 164; N. 449; §. 541.

82 General Bacteriology.

EXERCISE LXVII. RELATION OF BACTERIA IN THE AIR TO DUST PARTICLES.

a. Pour a tube of gelatin into a Petri dish and solidify.

b. Remove the lid and shake a dust-brush or cloth over it.

c. 18-24 hours later, examine under low power of microscope to determine the rela-
tion of the developing colonies to the dust particles.

EXERCISE LXVIII. ESTIMATION OF NUMBER OF BACTERIA IN SOIL.

a. With a sterile knife collect a sample of soil in a sterile test-tube
or Petri dish. Samples at various depths can be secured by means of an
earth.borer. (Fig. 15).

b. Weigh out 1 gram and dilute 1000 times with sterile water.

c. Make three gelatin plate cultures using 1 cc., $cc. and zy ce. of
this suspension. Incubate.

d. Count the colonies as they develop and estimate the number of
bacteria per gram of soil.

e. Many of the bacteria of the soil are anaerobic and can only be
grown in the absence of free oxygen. See Part II. Chap. VII. for methods of
cultivation.

REFERENCES. A. 556; H. 394; L. & K. 389; McF. 174; N. 444; S.
567.

Fig. 15. Fraenkel’s
Soil Borer.

EXERCISE LXIX. WATER ANALYSIS.

; QUANTITATIVE ANALYSIS.

a. Collect a sample of water in a sterile test-tube or bottle. Fig.
16 shows a form of apparatus used in taking samples of water at vari-
ous depths.

b. Make two gelatin plates using $cc. and 7y cc. of the water.

e. Count the colonies as they appear, and estimate the number
per ce.
‘d. Make agar plates and compare results with those obtained
above. :

e. Analyze asurface water (lake or river), a deep well and a

spring water.

QUALITATIVE ANALYSIS.

a. Detection of putrefactive organisms. Examine gelatin plates,
made above and (1) determine number of liquefying organisms per ce.
(2) search for the presence of proteus forms. (B. vulgaris.)

b. Detection of Faecal Bacteria. Pi Giatr Leip

1) Inoculate a fermentation-tube containing glucose bouillon (1%) with 1 ce.

of water.
2) Make litmus lactose agar plate using 1 ce. water.
3) Ineubate both at 38° C.
4) Compare growth obtained with that of B. coli.
REFERENCES. A. 526; H. 878; L. & K. 396; McF.169; M. & R. 79: N
P. 245; S. 553. For the determination of the various Species present ace Bratt
Micro-organisms of Water; Fuller: Report Am. Public Health Assoe., 1899, 580

. 422;
kland’s

84 General Bacteriology.

EXERCISE LXX. QUANTITATIVE ANALYSIS OF MILK.

. Obtain a sample of milk in a sterile vessel.

. Dilute milk 1000 times with sterile water.

. Make plates as under soil (LX VIII).

. Count colonies and estimate number of bacteria per cc.

a we

a

EXERCISE LXXI. EFFICIENCY OF PASTEURIZATION,

a. Place same milk as used in previous experiment in the bottles of a pasteurizing
apparatus, such as Freeman’s, and pasteurize as per printed directions, or place the milk
in ordinary milk bottles or fruit jars, filling
to a uniform level; these are then to be
placed in a flat bottomed pail which is to be
filled with water and heated to71° C.(160° F.),
remove source of heat, cover and allow to
stand 30 minutes. Remove bottles and cool
as quickly as possible without danger to glass.

b. Determine bacterial content of pas-
teurized product by making plates. A dilu-
tion of 100 will probably be sufficient. Ex-
press results so as to indicate per cent. of or-
ganisms destroyed by the process. Compare
the keeping qualities of the pasteurized pro-
duct with that of the raw milk by keeping

samples of both under similar conditions, .
e. g. in locker or ice chest, making frequent Fic. 17. A home-made pasteurizer (Russell.)
observations.

Pasteurized milk should not have a permanently cooked taste.

REFERENCES. Bull. Wis. Exp. Station No. 44. Russell, Outlines of Dairy Bacte-
riology, 95 (4th Edit.).

EXERCISE LXXH. TESTING ANTISEPTIC ACTION OF CHEMICALS.

GENERAL DIRECTIONS.

a. Fill a number of test-tubes with a measured quantity of agar (5 cc.).

b. Add to the agar varying but measured amounts of the substance to be tested. If
the antiseptic is not volatile, or affected by heat, sterilize.

ce. Inoculate the tubes thus prepared, together with a control, with B. colt and make
rolls. :

d. Keep these cultures under observation in the 28° C. incubator.

e. If no growth appears within 96 hours repeat the experiment, using smaller amounts
of the antiseptic. In this way determine the amount of chemical (in %) which just pre-
vents growth.

SpeciaL Directions. Test in this way carbolic acid (5 %), aleohol (95 %).
REFERENCES. A. 566; H. 411; N. 527; 8, 156.

86 General Bacteriology.

EXERCISE LXXHI. TESTING DISINFECTING ACTION OF CHEMICALS,
SUSPENSION METHOD.

a. Make a culture of the organism to be studied in tubes of bouillon containing 5 ce.
b. Incubate at 38° C. for 24 hours.
c. Add to this an equal amount (5 cc.) of the disinfectant to be tested, of double the
required strength. : ‘
_d, At the end of 5, 10, 20, 40, and 60 minutes make agar rolls, using two or three
loopfuls of the mixture for each roll.
e. In this way determine the time of exposure necessary to kill the organism used.
f. Test in this way the value of corrosive sublimate (1:1000) and Lysol (5 %), using
B. coli.

COVER-GLASS METHOD.

a. Make a bouillon culture of the organism to be studied and incubate at 38° C. for
24 hours.

b. By means of a burette, pipette, or loop, place the same sized drop on each of
several sterile cover-glasses and dry as directed in the experiment on desiccation (XLII.

c. When the cover-glasses are dry, they are to be immersed in the disinfectant for
the desired time, then removed and transferred to tubes of melted agar which are then
made into rolls.

d. Test by this method carbolic acid (5%), aleohol (95%) and formaldehyde (10%),
using B. coli.

REFERENCES. A. 558; N. 518; P. 152; 8. 158.

PART II.

MEDICAL BACTERIOLOGY. |

PART II.—MEDICAL BACTERIOLOGY.

CHAPTER VI.

PATHOGENIC AEROBES.

EXERCISE LXXIV. PREPARATION OF CULTURE MEDIA.

The following media will be necessary for the work outlined in the following chap-
ters. This is exclusive of a few special media which are described under special heads
and are to be performed as a part of the exercise in which they are used.

100 tubes of agar. -

12 tubes of glucose agar.
100 tubes of gelatin.
12 tubes of glucose gelatin.
80 tubes of bouillon.
30 fermentation tubes of glucose bouillon.
30 tubes of potato.
30 tubes of milk.
30 tubes of glucose free broth or Dunham’s solution.
380 water blanks.
30 tubes of blood serum:

a. Collection of the blood. Sterilize Mason fruit jars, by successive washings in
corrosive sublimate, distilled water, alcohol and ether (or sterile Erlenmeyer flasks may
be used). These are to be carried to the slaughter house and the blood from a beef caught
directly into them. They are then allowed to stand undisturbed for 15-30 minutes, or
until the clot has firmly attached itself to the sides of the vessel, when they may be
removed to the laboratory.

b. Separation of the serum from the blood clot. The clot is separated from the
sides of the vessel by means of a sterile knife or glass rod, and the vessel placed in the
ice chest. After standing 48 hours the clot will have shrunken away from the walls of .
the vessel leaving the clear serum on the top and at the sides. This can now be pipetted or
siphoned off. If the serum contains a large number of red blood corpuscles it can be
placed in rather tall cylinders (graduates) and allowed to stand 24 hours longer, when
the clear straw colored serum can be readily. separated. This may be preserved for a
long time by the addition of + % chloroform and kept in a tightly corked bottle in a
cool place.

c. Loeffler’s mixture. This consists of 3 parts of blood serum and 1 part of glu-
cose bouillon (1 %).

d. Sterilization. Fill sterile test-tubes (about 3 em. deep) with the serum and ster-
ilize either:

Medical Bacteriology. 89

(1) By heating to 60-65° C. for 1 hour on 5 successive days, and finally plac-
ing the tubes in a sloped position in inspissator (or sloping tray ina high temperature in-
cubator or steamer) and heated above the coagulating point of the serum (70° C.) In this
method the clear serum is used and not Loeffler’s mixture and a transparent medium ob-
tained. This method is not usually employed, but the following:

(2) Loeffler’s mixture is used and the tubes are immediately placed in a sloping
position in an inspissator, or steamer and heated up to 95° ©. for 1 hour on three con-
secutive days. If a higher temperature is employed bubbles are formed which rupture
the surface of the medium in their escape. When sterile the tubes should be sealed with
paraffin or otherwise.

REFERENCES. A. 106; H. 45; L. & K. 88; M. & R. 50; M. & W. 81; McF. 181;
N. 463; P. 219; S. 87 & 35.

EXERCISE LXXV. STREPTOCOCCUS PYOGENES Rosenbach.

First described by Rosenbach in 1884. It is found in abscesses, pyemia, puerperal fever, and erysipelas. It is frequently present jn
mixed or secondary infections and occurs in the mouth and sputum and on the mucous membranes of the nose, urethra, vagina, ate,

REFERENCES. Rosenbach: Mikroorganismen bei den Wundinfectionskrankheiten des Menschen, 1884. A. 268; H. 133; L. & K. 117;
M. & R. 168; M. & W. 124; McF. 190; P. 476; 8. 274.

ltures.

e of
u

MoRPHOLOGICAL CHARACTERS.

Incubation
temp. (°C.)

*e

1. Form:

@ Bouilotigs os acess as tiscahecug vase eepanreieane xe ais Medhats SSAA Sh Ge ace Shine 2a de MESS SAE RE Se thee FEMME Rome eauree eR ERA cols | pete eee

35 Cell Prot PINES ceca seve caves Maes Beem oe Ne SS aad brated ave addadiaued poke aan dead ead eenn b Dash tea on iesoe wecuieen ede Aaa Bec Re eR es

RE AUTRE GS ic 3:2 sca Se oh aevdedaskd aedeoihes oa Sdoi uAemh Vaan diiada Wade auulis vale Gawd emaRRe ag URI e ag SeadeNas en tale Lae anual nad asa

@s Aqueous Pentian-Viol etic: cs ys ulsins Ye Big septeidisen cis wiitiens oie aadrermacdiasne died aqraesd odusionjoe sade Akelounlsmndanus rb aa ceneeomen eens

ib; Loetier's methylenebl tess isa csaantsd ssacerdsiecyaisasad i-oaeevarafuat amavoa de ee Aaenceve amd taveanin oe he ausian var hades ad alee ea Scenes aba

€. Grain’ S\S tain egies eens siesina iad aa wees Gans Stee saw Se ae aan Nesige suis! tie saa Sagl ded ae F eadeeate ait an wpen cake

a, Character of movement.................00065 5. Sons dad ls scaoncgSed sata divcxGobah 0 Sea wus, Riel tceedeseh Gi emeHN Sex recaueaane oe NUTT Vina eRage BOE ala, ade

Py. PRA RIN SIAID scare astied bu 1g Haan sintieka anaes sane wmalore eee balybbcial wa ou neces maelans euaeeee wae de: puhew Wada Rens Bs Gs wubBee es Gate Ree

an

if SPOLES! sisisirsiy sewwicawnanin rasan fas ifs mltnat a sbaisroied aaah oie ies aveaieiarainsaiped'svohygaliig a7 ste AASy Saws Tie OEE TS RAGE Retaiege ebiclar ae dakaeabeoee a8 Souder

N
uw
ho]
®
°.
2.
p
tat
i)
oa
>
iat
»
a
o
o
a
we
a
c
is)
=
ry
2

deposits, vacuoles

pleomorphic and involution forms, capsules, ete. 0. ... ec ece cece cece cece sete sees ueneenee ees

PHYSIOLOGICAL CHARACTERS.

5. Gas production in glucose media:

as Shakeiulturess oe ni poncccisiianstiaynie dd amen endaundwnmaos so ae Meeaxomsios valeurs ox
&. Fermentation tube, growthin: (1) opemarm:.....................
(3) rate of development: 24 hours................ per cent., 48 hours............ per cent., 72 hours......... per cent. hours

(5) gas formula, H : COg::

$. Indo] production: 44 houre a .w0s pecas erreves vies ceews vie

fecal odor; 24 hours .......... 2c. cee cece eee e ee eens

Enzyme production: proteolytic

CULTURE CHARACTERS.

Reaction

gt Medians 24...... Hours. 48...... Hours. Pee Days. SKETCHES.

Temp. (°C)

(1)
Gelatin
plate :
(a) Surface
Colonies.

(b) Deep
Colonies.

(2)
Agar
plate:
(a) Surface
Colonies.
(b) Deep
Colonies.

(3)

Gelatin
Stab.

(4) ON N
\
Agar \ H
Streak.

Potato.

(8)

ouillon.

(7) i

_pecial
~ Media.

[91]

EXERCISE LXXVI. MICROCOCCUS PYOGENES (Rosenbach) Mig.

: STAPHYLOCOCOUS PYOGENES ALBUS; STAPHYLOCOCCUS EPIDERMIDIS ALBUS.

First described by Rosenbach, in 1884. One of the common organisms found in pus. Occurs on the skin, in sputum, air, water, dust

and soil. ; =
REFERENCES. Rosenbach: Mikroorganismen bei dem Wundinfectionskrankheiten des Menschen. 1884. McF. 184; P. 470; §, 9%
a ,
= at
we | S$ é SKETCHES,
, = a
MORPHOLOGICAL CHARACTERS. 85 g2
< -
1. Form:
> Bouillon sepscacione’satsdaitie voy sleicclgsaiglg gia buainigitig Seb ge nique ysis See eee asta hasan Salas ialcstcara odes inane: whe be eactesSace ana ha arial crate oa ae | das aelanmies
Di PRA sss sis iti ap St a aaldehal bbe Sdisishad dea sEiTn Noa dla aad bed dioetsdcAd 4 gh oectavadbed ad Gaatvee be Bs UA La GaNE PaNieioiae sea UR TE TERRE tela Ski [leamaaessa | Meateeeate
6s Greatness ras ay gels delaras ae’ slaetds yaireiges ds a amen vanoeuemm ds ov ellaomes avaealg oo ug aeelas Mobdaan soaheualad arene a | oavdarenl bewneeae
@s OMe Medias escent air. _adjunsciie: uecl Gece eA ebAAn Oe be dectasiaain ecmuduerke Go) kd waveiegd Secyacans wea etyedaas Nod ieee an aieas LE Ga bisa) even Seal Se areas
POUL G25 cd Ae trudebp eeaca fatal nbeed ha esas wrstecirih eats oe aparece ee Gian Matec «Sab eccat aaittidedai giacth Austin fasts wncaheay Se oa heme ee | eee ea
ariCellsgroupings aici sachet ances ao aten Aas haetgahn al son etatseee ai ted corte ae prea nag anni d oa ebaceie E daeyatatatgs De tetian a exe ol earn el Race cet drt
and ‘arrangementsiwe: av aandssamgnenessg wees eae niSemaeadN BT Gh ceded ue RURG Mew els obaats Meuee a ay Saeacan anes heat sete deel aechs Guaed Gabe onde
BRE REIN ete ca vices Sa se Saas aoecn ww RUS G4 ee BIE Hed RI RAGIna 6 HA Ad paein cresdiel whedbiehs pd by Ged GAL GR GSAX HE GARI od edie Seid AMA NAHE mane anes
4, Staining DOWEIS 3 cox nasasnanivhan cata saveteaeten aut iach sae MN Ann Rhea mieemeae ae 5 aistaibavslplina ni ieainioa aie ata ininaticla Si Ro-araueAe Nie Bela wisibteciaiein| ga a Aelia Becks
: a. Aqueous gentian-violet.!....... 0.0... cece cece eee cece ee id eibtanane werd Gleenalen Ge soUMENER SGOT ote RiIERIA Hate Me ae GiamMAd ear few BF Gavelleelad HERE
Be Teeter’ & wie lyg OR UG ses ia er ner emavneutamen v4 we eas WRU eee tam Ee COmER Raa mun ees han MOY eee A Geto | a Sa eon ada aoa
Cp GYAN 'S:Btalm ascecnams san eae th Hy atee Gabteneecematy wh de Wed aiaaurbiehigisiddauad da opr ammeuphestierandseiesititee wo ae rasaylahh dio qayiesis Aut acaba onda ek Wop cial eehave tires
dz SpecialiStainsycie crue ieuieesavaetskeaae wat, “aapiiwunetan: aieauandseatasaded wrea aia sat nie awed ober onae [leeleaerealls chaise se
Ha IMM OUR DE Yio as ee ssvacea eal ib ve devzcastna Fab tahetedet ckcnre ta cevovdeevn tarcteeconant ee ce eodenel avait eeeannh ud ty ss stars Worle cats sods eat a nataaata tated tne labenaynsh Gas Mlacealee”wnaee! IES Bare eae
@.. Character of movement enue scisareaigusaicne ns sanenige aparuelis ia-sip goa aumian ad Mesite S4-sa awobarnemne vary Saye nen GHaihty dese ides saad yu lle otemenae
B. PNAS CNA SEA sce ca vweraging Sy das ealmey wae ealnl aaen 48.4 BOMB Nuay ta HeKALIN TA Ed EA HORAK Wie KREG be Cae cmdins hyena oa de souaasnyl nianss oe laercance
6), SPOLeSnemenmeremen ae teineate ta ao Meret as Se mek hn sens eens Po octal se cick Ekta dasscrl aie ake Ryluost Alou arora wa Sa aaa ute near odlgeae ain aaa ced alas el Sts taneecs
7 ISPCClAL Characters; SUCH AS 5.52 srac.tieissesses inva om aon raise est nee tl atcee ans dada wala biain diate Adal te Reg ueeenst Mianoetn valde emeonmerntedetesny vent Bice alllicedhec ou laces
DS POSIES) VACUOLES raissiasciciais 3 sis sing gueccimc nt a tre sapsthansvenanstcearaccie! ie bal Sain gamegsha aiataueenanenteca na ge asoetateetieg Gecteetanle be Sy aewdindersciBananone alacnte aeOoave Wescegencerweel ecacen sess
pleomorphic and involution forms, capsules, etc.. 2... 0.6.6 ccc ccc eee eee cee ec ea eee neta eae sete cctetensnteceratsesscraefeccevessbereeces
PHYSIOLOGICAL CHARACTERS.
ut. (Relation €o temperature 'sevsjcis < c0 cuteacre ve Haves enemas aiaidiaise Mcseisi be Ye: vale uculglele waalaed uekseiatnie o9-Db HOM gal Wie widielsoaed Ws 4.4 baum aachoubawiee ad
2. Relationtortree Oxy Pen tenants sates oe pe geste ott eae ce a dieiaeaGuiont a deiceam ad's 5 bau aneth ers sh OUaW ae eulbela dvine waa Mn mec e
§.. RElBHOD:tG CtHEP AGENTS; SUCH AS ss cecics cciscins. ada dictermvteue Daarmnan hon wigh) BAaveruund Lee aelg Acne omesAMMAEee eetoash acme
desiccation, light, disinfectants, et0.isacaaus sas casie apse svesnuscnn Weeacinn node ws x2 dammed abbceimnawme ns. dvajewd cadinawer ang
4c. Pigment production tas ciaearis svwssaysvdmwianiens sa seie genie nsaeelay de se gtas oetlen ds mie Soaudiaedng ho ede euannneaueeae seleranatdne
5. Gas production in glucose media:
ai Shake: culture: ig xcdousdsds wecdaseaeee doo wtnas nereblnerautees jutactalh ea shed vane amin eee tac ag alin wen eae
6. Fermentation tube, growth in: (1) open arm:.... 0.0.0... cee eee cece eee cece eae (2) closed arm: ...
(3) rate of development: 24 hours ..............4 per cent., 48hours ......... percent., 72 hours ....... evade per cent., ............., hours .... 0.000 per cent.
(4) reaction in open arm:...... 2.0... eee eee eee eee tence ee eee eeee sees eeeeeeee(5) gas formula, H: CO, 33
6. Acid or alkali production, litmus milk. 0.0.2.0... cece cee cee cee cee cee ee eu cous eeeseteavunnenanees |
9, Reduction of nitrates; to nitrites..c ee cee adiny cea wenwes ex: G SE Ratt din Saas sonse aseapvbeeth atu aie A ty Westen moeneh do
8. Indo] production; 24 hours ............ 00 cece ee eee cee ee
feGal Odors 24 NOUTS! sic ia ss aaootmamidnedeendisenene
g. Enzyme production : proteolytic
to; Characteristic odoniiassascasnseareoie onsaningaivine ste aiaiyaa wn tganlngas 9 Ti jehe doesen Rance
11. Pathogenesis

CULTURE CHARACTERS.

Reaction
of Medium, 94
Incubation '

Temp. (°C)

Pasenscs Hours. 48...... Hours. 6,..... DAYs. SKETCHES.

(1)
Gelatin
plate:
(a) Surface
Colonies.
(b) Deep
Colonies,

(2)
Agar
plate:
(a) Surface
Colonies.
(b) Deep
Colonies.

(3)

Gelatin
Stab.

(5)

Potato.

(6)

Bouillon.

(7)

Special
Media.

[93]

EXERCISE LXXVII. MICROCOCCUS IMELTINESIS Bruce.

This organism is the cause of Malta fever and is found especially in the spleen of the diseased.

REFERENOES. Bruce: Ann. de!’ Inst. Pasteur, 1899, 8; 289. Durham: Jour. Path. and Bact., 1898, 5; 377. H. 361; M. & R, 449,

i le)
6B | Ba SKETCHRS,
os pa :
MORPHOLOGICAL CHARACTERS. 20 ceo
t. Form:
@s BOUIN ONS secs casnteis acnuaceewnnds af ts ceed Meaiaws veoh kiana
0; AG ar: cs ce drnaeaieive oepaeviie tealnanwaniad nde Reus wnedicig 35 ou Spalaemeeeeee s
6} Gelatin sowseieasnsae vee amen rveniaees xh Ua eae Heads Osea GR sa Ss Theda Heal amees He
at, ee DCA aaa wen ddd vars dates eiemaae ere pasd meni Wd Kens 1A SRAM AS
Big SABE ahi ic agers ssw eelntare Mover SAE Hiroe ORAS N A we cernlnn Ae aely Bed aed ae
3, Cell groupings: : ssicces ss se suis walsdaciod v5 oe Shou aisle nest dg ame genase ware setinnmain Hane MERRIER he He ot

and arrangements

Ue BROW ETS ve veidd Oi Goad eeadd cater eds V4 das Hes acre TORs wine sc gderoe Oe RARE BMA ReteereRA ess

dx Slate DOMES? ccs vows wanes nesanen wees dies cues Weedead Kise Hae Reer Tesi mawe LEE BETeSE RRR TURNER Rees CARR ND te gee 3 end tien
a, Aqueous gentian-violet ........ ... cece cee cece eee eee eee eens
6. Loeffler’s methylen-blue.............. cece cess cree ee te es

2 Grams Star tigr is sie e shade siv sie Sessa dpaeeeeas od ST HeisE Ae IOS DRA BERS

@z, Special Sains) ss .aciivs ciweiewice ne dee waiemessauueneeas. ng sawed vnwespinens ne ae Ssihog SE Puente 6G GEMM ae Ee OPEN Fee
%, Moa iiecaecaviengnus aces swung ide sanw iawa Soe Akane Res SHH 05 AF MER EE RE RRR RUSE NRO ve TEe SENSE RE Lh Haile pode GER AONE AAS OO CFKE ORNa Res Baa
@. Character of MOVEMENL.... 2.6... e eee cee cee tee cree eee rene cee nee tenet etn eeee ene eeteeetennereeer cere sete ceealaceceeeeleeee eens
6. Plagella stain. sisi .caicccah ae ows ea ee eeeenacas Ye, tabi Syste eaets ie tial ssasnarey ae sie a ooo: Nay evades wares cola ale Wace an's use’ dvdcel econ Se Rashida Ga

6, SPOreSiscassae nea cinend peewee wasie dies ania dees qiass eos voit sieialacein nop dip weve hioie se AURGM Gls Ue aD AHEAD ARATE BG HE Sat Wielein seein ee eeenaiie Gea Apieg dea [Gree nee

7. Special characters, such as: .......... 0.60. e00 ee Galop ie ieeshaicncacseh a matte eteyenecaitead Sa dee yi deli died ear: a did anegcbe le! cn SHEAR a? yb ac aa hcinn MSc doiealSeaaciaaes let liaayea oeuass

deposits, vacuoles............. -. oss tapsa ste eyunsate sep tope“s ‘sen coetergita patspe astndcvasines ais Sisk Ge ci oaTeI Re eaaTEIa win aes: MISS Reese ala eT cavthoaa eoaiataceel ancrap areata lee cpa

pleomorphic and involution forms, capsules, etc......... 0.6. ccee cece ence eee teen ee een enter tte tene rr te ceteceteneen lees tees lore eees

PHYSIOLOGICAL CHARACTERS.
1 Relation to Gem pe rats Oi <atjcisicniv ds se CRSD sen eer alan aerate,

2.

5. Gas production in glucose media:

a. Shake culture

(3) rate of development: 24 hours................ per cent., 48 hours............ per cent., 72 hours.............. per cent.,.............. hours.........

(4) reaction in open arm:........ 2.0. ce cece eee eee eee eee eee eee eeee ..(5) gasformula, H:COz ::

6. Acid oralkali production, litmus milky. occa cics smtaisanasrecs senaarveacaege

7. Reduction of nitrates; to mitrites.......0 0 1c cree eee eee ctr ete ete tenet et ee es p tO AMMONIA srs sicisne dined tg cane ganmabead weak SeR URE Ps te aeriO) meade elas see

8. Indol production; 24 hours..........-. sec eee eee eee eee feAB ROUES i coletd ssttieennlnan ra cmaieet vlan ble ebay seme oed,sicine oad utuataciebenge days

fecal odor; 24 hours

9g. Enzyme production: proteolytic..... 0... ..c cc eee ee eee e eee cee erent ee ttt cence renee

to. Characteristic odor...........

Ir.

CULTURE CHARACTERS.

Reaction

of Medium,
Incubation
Temp. (°C)

(1)

elatin

plate:
1) Surface

Colonies.

») Deep

Colonies.

SKETOHES.

(2)

\gar
plate:

a) Surface

Colonies.

b) Deep

Colonies.

(3)

yelatin
Stab.

(4)

Agar
Streak,

(5)

-otato.

(6)

‘ouillon.

(1)

oecial
Media.

[95]

EXERCISE LXXVIII. MICROCOCCUS AUREUS (Rosenbach) Mig.
STAPHYLOCOCCUS PYOGENES AUREUS; GOLDEN PUS COOCCUS.

First described in 1884 by Rosenbach. It is the most common organism in pus.—80%.
REFERENCES. Rosenbach: Mikroorganismen bei dem Wundinfektionskrankheiten des Menschen. A. 260; H. 180; L. & K. 115: u, &
R. 166; M. & W. 121; McF. 184; P. 461; 8. 265.

as

Incubation |.
temp. (°C.)

MORPHOLOGICAL CHARACTERS.

1, Form:
a, Bouillon

4, Call OvOU PINES 25 cc0e.a wi soanaunteves yas ea a sewer aewguy He eens See ee ce eees oe es ee ee
and arrangements. ........ 0.0. cece cess ence ee seenes
Tasrowthstssnusacacan stromans heeaeawnvameny

4 Staining POWETS: . 625. ccee cen cece nner cesnensnees

a. Aqueous gentian-violet

b. Loeffler’s methylen-blue

€. Gram ’S Stains... sscsesaesscai as aecdhenees
Gx, Special Stains i. seas ceva carers oats via
5 MOtiHltyS sc.aci cneccusantentse is ae ote eeeeten
a. Character of movement
b:. Plagellarstain: coc areqactsd vaceeaand igadied Uese ceed Boa ohtts Ue ie apeemetiot ss Macon ye Eee atau ye eee ae ataR
6; Sporesis, aavisvs vs a saeay daidpleye seh orien tianiaceemie es san Aa dmnwies Somrgenens Kobe Comms eegtater Seneayia bemted Mae aE Airco hall asad Redidckemidtes
9. Special charactérs; Such as? s.sc0.o:is is seeds cutee os Pate eB eee RN a ge ore es Beni ke yet Se ah Beane de gelding Siereleieand weet [laa = ae me
deposits, vacuolesivs svcrasau vesiniis-catmdnvainttinade babes siMbi euaaaamey Sele ates qed d oe ech aN Hud ceceN TOR AA Ter RATA oi Note oacell anise
plesmorphic and involution forms, capsules, CECH ictcais aie Git dave caters Sad aimee Se Rea Pabetctofancladnrals, Hd apniwenh aidalgiubalgsaiitoslgie atacand edema Wal ede Ligne
PHYSIOLOGICAL CHARACTERS.
z. Relation, to: temperatures... c.ccceses sa Seedigeneeed wens ee ue sana aae aa He pee daees Sans eeNe eH phaME EET ice sed deen biol greeter ve ts meepiaeeaete Uk “Omega an Paes yea
@, Relation to. freevoxyeen sen va: gasspeacwawanaees vere Menredi ence wena aciacacenindweeines Raed eweens darnanied AN ASU SAR lag WE TEE matnN Mas widaRanNeN aeTee wend Lica acacacn Bits nian Rane Heed SIE "
3. Relation to other agents, such aS:........ 0-106 eee e eee eee ee eee eee ne nen nee een eens ee ee ee ee a
desiccation, light, disinfectantsyéterstscasses qeeeaer os ot coca eer acaais 1958 AUER APRS TIE We Re LMI a ean ee ebenciteaaie ned : Peace eet ae oa tandice a aan eRe ae
Sg Eas ee alee cine GER A sts consiigtke teceneneeaseansas ed AON we
see | ~=~!C~C~S«S«7«7«S;«SC73<3<32CSC;737TS lee is
a, Shake Culture isisicccese cient 3.08 kdeganeatoreesn a eae easy GRAS AEE SE eae CB hiein ies Seen Paonbesidigra ayes awa qrakoaNe AG Vesinanal dele AUTEM S AD Ga SiSeTELA QEEIAS En Aslan WalviaoNta EE sansa
&. Fermentation tube, growthin: (1) Opem arm:...... 6... ce eee cee cee eee renee (2) closed) artic... osc wees evans exe a ane eer Rte athe vot sia
(3) rate of development: 24 hours ..........-.++. per cent., 48 hours........... per cent., 72 hours.............. per cent., ....... hatietahcea ies sateniass GATE a cea
(4) neactionin Open arith wees 22 eos bees eee coger em Keer ce oe gave ciaos anveee ieee y (5) gas formula, H:COe::
6s Acid or'alkaliproduction; litmus: MiG, 25. sesiass ae vewsineaietmataite: aa-ae ditine a telpiseoul ne ph ve-tiea ateutel ale arp elsig Berean denlanpen we Ok santeroassoek telewhoee auaade pheasedg canbe nny
7. Reduction of nitrates; to nitrites .... 2.6.66... ee cece eee cee eee ree eee eee tenn ee eee a DIO thes eee ees
8, Indol- production; 24 hours. ss .ysc¢ 23 ca een na amnasine se oe oe 448 BOUTS srnene iy an Oy tenets cheeses cede dgum tis banwev cus aeas eer arene GAYS occ ee ce te cere erent
fecal orlors 94 BOUTS cess tse ecaw 44 uae ne ee GhesdR OTe g ABOU : 6c'cx 2chsacs eaten sativachien tenisbelswlars alalindile veaoedesiadgenta tos Ls cnabae Lanes DAYS 4s rascn ves cone borer
9. Enzyme production: proteolytic..........ce sce eeee cette ee eee eee ttre rete teen eee GiaS tabi G its Gacy cateu ca ueeans Haas ead teataita detalacehls damien tons ainaineigiinn™
SAO wiegeatia Sakae neeyt Gaia mre Sehpee san an Pee T NA Sila HL ae eailtic eeiteMab dig seas sco diac sa Dalaman LE
16; Characteristic d Ott: pecasiscciase aime uiine ats cie ih si etaiwegalne ah oie insid sa ebweulila gay veetyien Geauo aac Shite 7 Ie oe ee ae
i Ne Raper tian Oe hee date nee eta aaa ee cttcaaa dacaasutae Oa tc ne herve eee. ie
Fa te eas en te ermine terecrnca ee ean Narre

CULTURE CHARACTERS.

Reaction

of Medium,
Incubation
Temp. (°C)

SKETCHES.

(1)
Gelatin
plate:
(a) Surface
Colonies.
(b) Deep
Colonies.

(2)
Agar
plate:
(a) Surface
Colonies.
(b) Deep
Colonies.

(8)

Gelatin
Stab.

(4)

Agar
Streak.

(5)

Potato.

(6)

Bouillon.

(7)

Special
Media.

[97]

EXERCISE LXXIX. MICROCOCCUS GONORRHOE4 (Neisser) Fluegge.
Gonococcus; DIpLococcus OF GONORRHOEA.
First described in 1879 by Neisser. It is constantly found in gonorrhoeal discharges and may produce disease on any mucous mem.

brane; urethra, bladder, rectum, conjunctiva (causing ophthalmia neonatorum), and even cause arthritis (gonorrhoea] rheumatism)

endocarditis, salpingitis and general septicaemia.
REFERENCES. Neisser: Ceut. f. d. Mediz. Wissensch., 1879, 497; Foulerton: Trans. Brit. Inst. of Prev. Med., 1897, 1:40; A. 277: 8, 145.

L. & K. 311; M. & R. 189; M. & W. 130; McF. 201; P. 522; 5, 283.

MoRPHOLOGICAL CHARACTERS.

1. Form:

Gx. BO UNO M925 sg sane is iL eas caso cosas Sag eA Seen eS ata ae a atin San eotin bi oak aye aR Genrer nigh eras ty cae OUR hes

3. Cell groupings................
and arrangements ....................
In GrowthSadsay wascowpes Sete acen

4. Staining powers: .............

a, Aqueous gentian-violet .... 0.0.0.0... 0... ee cee eee

b,, Loeffler’s*methylen- ble asec nencan nese anton atee cede g. 24 ag Eheiu Men apideimna h eitemerns Seaman HER

c. Gram’s sStain............

d@. Spécial stains: sis ssucsseieeeiiete dy ta Gasset paca cs anos

a. Character of movement.... .

6. Flagella statin cs cccscota sais ce cane sees ce sae wk Ys

OSS POLCSh 524 een be Ya esta we dleina i waderais ng Megala Danes find ose luhantined y acatnsaeiste cob Glee sternal atesony Glau mR anaes eisg eerie ESE

Fa “Specialacharacters,/SUCM-ASs sicwcssge st etgreiy aged be Cearoae n!eoaconsand poe Ea a Eee ws Gout UOMO OF OP Sena Matra Nae Re sre ab Gooagea NS Dalen eae ae bod

deposits, VaCUOleSsi isn is cas ricci admsihtecse he ae saa gap eens a ereene, hats te oe

pleomorphic and involution forms, capsules, etc .

I =

SKETCHES,

Incubation
temp. (°C.)

PHYSIOLOGICAL CHARACTERS.

1. Relation to temperature:.............

2. Relation to free oxygen: ...............

3. Relation to other agents, such as:................-22.0055

desiccation, light disintéctantsnete is secs 3. sb bel lst neem) We ach Manes Ava ta ante ctee aonieaxtereneneleiaa a

4. Piginentiproduetionyin siccchrscias onde wana dolecaga ican dole cu nc enue, dane cart

5. Gas production in glucose media:

Gz Shakescull tateiss. acanwov seas Saath see eRe PAs Bhs

6. Fermentation tube, growth in: (1)

(3) rate of development: 24 hours........

(4): “reaction in OpeM Anes g 2 ccs sem azaleas seas

6. Acid or alkali production, litmus milk ........

7. Reduction of nitrates; to nitrites ..................

8. Indol production; 24 hours ....................4.
fecal odor:24 hours 2.208 266 cere cos

9. Enzyme production: proteolytic...................

1o. Characteristic odor.......

11. Pathogenesis ....

open arm: ......

...48 hours

.. per cent., 72 hours.....

RAOSNOULS a be Se ey yer nuucedsan

..diastatic....

uci) KCLOSEG AMIN a nealae cn cialde ss yahiseac is ames Aaeehy \eeea ahaa nspea asah ee

te DET GEN bays cach

..(5) gas formula, H: COg::

see COATING NL Ae ony, partis We rset gate gage nce od ae ay

wee hours... cee eee percell

Pe ey men
uqean OAV Shs sei aya sade ae sega

Medical Bacteriology. 99

The Micrococcus gonorrhoeae does not grow on the ordinary artificial media but may
be cultivated on the following:

a. Blood agar. Blood drawn from the finger, under aseptic precautions, in a cap-
illary pipette is placed on the surface of agar either in tube or Petri dish. This blood
is then inoculated with the material containing the organism (pus or pure culture) and
smeared over the surface of the agar either with the loop or better by means of asterile
eamel’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 condition is placed in two or three test-tubes.
These are heated to 40° C. and inoculated with the material containing the organism,
making dilutions from one to another if necessary. To each tube is then added an
equal quantity of nutrient (ordinary or 2%) agar thoroughly liquefied and cooled to
40° C. The two are then thoroughly mixed and quickly poured into Petri dishes and
placed in the incubator at 88° C. Colonies appear in 24 hours.

¢. Rabbit blood-seram may be used either in a fluid or solid condition.

EXERCISE LXXX. MICROCOCCUS INTRACELLULARIS (Weichselbaum) Mig.

DIPLOCOCCUS OF CEREBRO-SPINAL MENINGITIS.

First described in 1887 by Weichselbaum. _ It is found in the meningeal exudate of certain cases of epidemic cerebro-spinal menip.
gitis and in nasal secretions in a number of cases. :

REFERENCES. Weichselbaum: Fortschritte der Medicine, 1887; Councilman: Rept. Mass. State B. of H. 1898; A. 285; H. 138; Mg
R. 172; M. & W. 135; P. 516; S. 310.

SKETCHES,

Cultures.
Incubation
temp. (°C.)

Age of

MORPHOLOGICAL CHARACTERS.

1. Form:

BBO Will OMa Se eeecesccse mec Sescosooa Sia eee tite tees ete ae STR PSA SBR CTRL ATs INA NA EO GAG a Sah EDGE PERE NP BES Ta AR SS RECHARGE Re gee Nemec aa SNe ee

GEG OVAL gcse w sim gsustcns woe epee ia soe aes RAT A RPO SSIES Ts ws ona C Toad AE Me ape REVO ee BREE Ddnsjace Stepan eae aan Ue ae 2

@. OU Media sos cs peice oss neg 4 vas sancioetn aee o CA SERS DSRS RMAEE NE DARE a) PERE Se oh leer Nea eaa eens ore |Metiaes enka wee
IB SUZ es. acks ss a Anse ea nao Ede eels na Need fr OS EONS oso ein ee a ands bid SRVSpS SG uN ea Rtas END ESS aeRE ere A SN th ag RRA a etn ates tsb Lesage tes ate aeniceceae
Bie COM: BRO UPN SS, cchsteectana israreoztoa me whe ccdave en's eye Maa ats abd age sae ata Rede pennlars oiaatac atest nlaie yt age atten andl apt are Siereue wea eanauaaie Aesmeine Naa tes resis | attire tetra | Meee
ANG PALFANG EINE ES sy 5 accyrres2 oes ase oesraw i ovepdis we OMB Gee Wao table telseloeeinactaln sain sralble a se masksitaeiedievede ya Ae Me eae naad Dota wales aia Satass elie Grud Data) eaeaterwtela
DAT GT OWUHS ooo tev occas, atk eeaiely pis wa edie Rigged SS Annet a ag Ree he Poe Saas ae yee el one edveen erage dle aera ore bitacieetie indians nd anaclehe a Raa sia isbe ics Pain ecm Ce Sa Ms
Af, ASEAN TUT DO WET S20 sca o nr, aoa teriense ys coy unc asd Soo Gish Donic igneous Gabsire 5 6 Ge DarSla epi e BG LEI He UR SITET Ta a EAE ENG, DB Tee tes TTR Beg te ie | Seer sea cette
ai, Aqueous gentian-vidl ets sc.<cceye weseeaesetieee Sede Radek ah ephee ny ot ao et aeeeh as amdat ERE on ge amen Os euNe le ee oe ReRE TE f. || erred] 88 Shee
6: oefler’ssinethylensbl ies a 2a.asarisiine ener atcccamaginarss nace nen aa we mea waleat sa he leusnarns aMayAN Rahal aadeson Atal ot delete th re eaten BE Cat

6: Granv’s Stain 74-33: ease co situa ase eee weet oS

i SPGCIALS PALS 5 54-feecdaa aly oases sues eck a ow eater ooh a MANN Se RSS GUTS HORE eS URE PO ne ad BS MTOR as Cae ness

5 2 MI OULU Vos zcassvettvn cneaBvecd H4s En TART Raia eA Letcue msn ese en ung aaa yD ive Se atecayosvasn a ors Fynieietn bs Pe eee Gisie eRaRGAUneN Omaees wm msraeaaie Me waRS [se mackie eanenneeS
a. Character ol mnOvem Ent acainwssccncuiseeen es ant aceite aeredeien aoe sie eatienesin ba eater ne SAA en ge tes Dae oL ad tas | Vamen neh fabeme es
by lapels tank aarss cuted ani op gerintisssins ab maid sche dia set eahastir Sead a acl elapa nate 3a area elalcraiang Bais ausay mew Rie sie ed diylels abacoanaNeen acne [asthe avaatccndl ite teslann

Ge [SPObeSi. coat tend te Reais Sa Gaket se aa deters eae ot te adideg eam nice tabenin ote nen ead deme ee eeu wenie eo aaa oir elise tees

7 SPECIAl TC HANAGCLE RS) SU CHM AS pees orest Gs se estysh vn Seavietie vale eed See Beane Ss tes aaePaa voc Pophsble ahs See balee ss aed avai lata need nraasd santos Genoa ead sum cea teed Re aig ahah | iene hea
deposits, vacuolesias cis cccecnaed-aeaene ns uate rset aaa Kaw ea Gade roo A NaN TE Mead dew Eh Tan oe Ta yale Meee ne gerautaa|\s catnlanie |lgmmataum

pleomorphic-and involution ‘forms. capsules; ete. sc cocasanacs coms wane oa seman: mee oe pean ape meals Ao ye Ania 45 wameae tied ey sage: arenes ers erste

PHYSIOLOGICAL CHARACTERS.

n /Relation totemperaturess: .cisay wesc wnat oid es aaieennind Redan amen tek wie aa ee Rae eae bine

2, Relation-to-freevOxy Cente sca ey ce eats waits ee erro A Ay gee es Se ES Be ce ae a tualranaae ge ih oie eels Ses Blgeana rato qheavy Ae esstdind. etic ahve bec aig lave ae inettae ceaeeloswa aceaei Aika Ho nO am
§,, Relation to othémage nts; SUCH-AaSs. sy aicas sac aimee ccsaaeie seed Sem uwiie Game sacndarad ne de Matt eiee enter ve
desiccation; light, disinfectants, €t@s osacac ncn aces Manwies bende: dot save cca denne aGiadn ae ne nactebyldeoneein ae encase

@:. Pigmient: producw One aceis ac(rcebrelnstaans nacido ae ma micas nh pitt atu saaieknssesicen bs ainaenra tere ne Ua Se MaRS RUA Oe eds OA OA ie ed oa oh Asay tht eons eee

5. Gas production in glucose media:
a. Shake: Culture: saradiesecs kes wnt a aicee ee aeua te AR OUA TAD ameniestam somes
6. Fermentation tube, growth in: (1) openarm:............0........0...... 4. ......(2) Closed arm: ............ en

(3) rate of development: 24 hours ............... per cent., 48 hours ......... per cent., 72 hours .. .. percent., .............. hours.......... per cel:

(4) ‘reaction incopen ATMs. ..s. sees cece note ese ns. Gueteag taareuad tone dasa aennpned(§) gas formala, Fes iCOo t4

6... Acid oralkali-productionNtmius: Mikey 40:5). crue sieseonasgiecrmaan erases Bugaa sa PeUae ie ie acgoeswlie deinbian Cauaeadeaeee

|

Reduction of nitrates; ‘to mitrites......61 cies sees ween eee e ee aie es ey eee weenie encod vey EO AMMONIA... sce os Gavassivccne cove sues uoee eauacceuceaucvuyeven sen steel

wo

Tadoll pnodudtions 21 HOWtS wo cca dscs seat cs catgna oe xa bey BO DOWNES occas ee se
fecal odor; 24 hours ..... Saeetas Gastidab emcee ca wen FON OUS Heap ama es it deee Woke oes etl anaes

Oo. Enzyiie: production } proteolytte sisi cc seec act aps heonsa we gemreneovada Pi ths esa abiatss wel AS ba HOt a ten te mevaels «hc a Ra a cohen = aun uepseeh ace fica apeee aoe aR

10.. Characteristic odor. ox. .cs003 s+ 0%

fx, Pabhameaesis ox ac saucy eS ercaseh as waaes eo vk aa ya Cow haemnadsaiwasenes

CULTURE CHARACTERS.

Reaction

of Medium,
Incubation
Temp. (°C)

SKETCHES.

(1)
a

Gelatin

plate:
(a) Surface
Colonies.
(b) Deep
Colonies.

(2)
Agar
plate:
(a) Surface
Colonies.
(b) Deep
Colonies.

(3)

Gelatin
Stab.

(4)

Agar
Streak,

(5)

Potato.

(6)

Bouillon.

(7)

Special
Media.

[101]

EXERCISE LXXXI.

MICROCOCCUS TETRAGENUS.

SARCINA TETRAGENA (Gaffky) Mig.

First described in 1883 by Gaffky. It is found in phthisical cavities and sputum and it occasionally occurs in mixed infections gs

abscesses connected with carious teeth, about the neck, jaws, and middle ear, rarely elsewhere.
REFERENCES. Gaffky: Langenbeck’s Archey, 1883, 28:500. A. 309; H. 189; M. & R. 171; M. & W.

133; McF.

448; P. 472;8. 314,

MORPHOLOGICAL CHARACTERS.

1. Form:

Bs Bou OM sense arya Cera ie ty eet ealerigtege TY Sa BO a eee

Oe ABT ene 2a eras PEAY ee ee Ae RED Laie ieie Se are a heen lp tog aNd aia tas |ticaa ale esl Acar os so adhe i ORE TEA core me NejoueTy

G2 (Gelatin in on eetecacew tana Men CaN SS RNAS APS

d. Other media......

PARISI Saran ene aeh nr heieee ire trae Ure te eae oe ae yore ea vane

9s Cell arouip tg Sica ayeyeieis ace nespeanesaia tie asia ave etn se eee mS Sade SE om ae 9k a

ANGATTANGEMEN tS: sane cu ecace ve ae emits aes qcitagy em ebiRa Maids ne meminen cea aeioeerete Ra GATaELEs HE ARR MEO oe ORME te eet a Oe

iM OFOWENS: ears doee spikes i dead Mee donee

os

a. Aqueous gentian-violet ........

b. Loeffler’s-methylenzbl tie’ os sicscscsece acta . «class we sdinoma. vie os) anna we aatha ane Winans Sig eS EG Se eating, @BeOAE gh aaagdE eae eats
Gs GRAMS: Stains ss ska case Se nt Gags habe oe ak Goss Face ae te Bs eee dd ene ea o we at Ea ems or eae eT

@Special:Stains$*s wicaads ese ugncus newadeunecadeae

J OLalMINg POWSIUSs: \ekeaG weigh 16 Aba dots eee eran Trib Sidadisstt eee dl arb senha Ad Saas ates Dae, ae eee Re Ae rgey pore. deel apts AUR EAE ATA ee

b:-Flagellastaim sacie vic quan kn Mewewenn wae meus eda 4 oe alanine 1h ae ES GMpe mena eatin oe on de apes

GO DOES Ara yiinna een Re ere a Re eh ean Osea oe Rak cea Sieh nates ern ean SANG

7: SpeGial Characters) sSUCM AS i i.ve.geqarrmne ne eansen, selnereiens wuadhe ne eM aRoRE sta Ana Manna MeOne aa daa ea SaROa an ae Aas Wan OS nies

deposits, vacuoles.............

pleomorphic and involution forms, capsules, etc

PHYSIOLOGICAL CHARACTERS.

”

2. Relation to free oxygen:.............

3. Relation to otheragents)jsueli Asie a: ese taaeeae. avin sade SRARe Se Wiel eae eas g alma Ae aie eahoas owALs smo Me we ea tis gueeney Sdeces be buen nea eacten: ilo eat

desiccation, light, disinfectants, etc.:.

ti Pigment productions... .ciscax sears tts ees ee

5. Gas production in glucose media:
a@: Shake culturé .22iccs0 sass cous

&. Fermentation tube, growth in: (1) open arm

(3) rate of development: 24 hours................

(4) reaction in open arm:....
6. Acid or alkali production, litmus milk. ......

Reduction of nitrates; to nitrites........

NI

a

Indol production; 24 hours................
fecal odor; 24 hours.
9. Enzyme production: proteolytic.........

to, Characteristic odor..

In, Pathogenesis. cscs. osc

Age of
Cultures.

Incubation
temp. (eC.)

SKETCHEs,

Relation to ‘temperatures: -2o.sicce oc sc seas oe ae ae yamiea cevisnees

Fe gaRE Gg RtSETe.N Sus Gueee sald Ae mre Aare (2) sChOSEG) SANE Actin ce Ree wy he Soin de Alias eae ese un tte anes Mem ananeiem

per cent., 48 hours....

seoeee-(5) gasformula, H:CO. ::

aces: PO MMOUNS Soe es ccasecgncs

Seare ks WB HOUTSx se sae 5

[102]

see per cent.,, 72 hours’... 1...

ty dee DETICCD Gia sean 3

wea se MOUTS..c02

wo... per cent,

2 36) LOY ATMO MIT Ais 5 sasvd deduce hak wooed ventas

CULTURE CHARACTERS.

Reaction
of Medium,
Incubation

24...... Hours.

SKETCHES.

(1)
Gelatin
plate:
(a) Surface
Colonies.
(b) Deep
Colonies.

Temp. (°C)

(2)
Agar
plate:
(a) Surface
Colonies.
(b) Deep
Colonies.

(3)

Gelatin
Stab.

(4)

Agar
Streak,

(5)

Potato.

(6)

3ouillon.

(7)

‘pecial
Media.

[103]

EXERCISE LXXXll.

First described by Robert Koch in 1876.

REFERENCES.
A. 448;

BACTERIUM ANTHRACIS (Koch) [lig.

BACILLUS OF ANTHRAX.

Found in the blood and tissue in cases of anthrax or splenic fever.

Koch: Cohn’s Beitraege zur Biologie der Pflanzen, 1876, 2; 2
H. 151; L. & K. 287; M. & R. 295; M. & W. 156; McF. 356; P. 547; 8. 328.

277.

Chester: Dept. Delaware Exp. Station, July, 1895

MORPHOLOGICAL CHARACTERS.

e of
ultures.

*8

Incubation
temp. (°C.)

SKETCHEs,

1. Form:

25 Bout Ont dsereayin crate eter os wate ha dec udeeatoice Seen ae oeretoelte mea NAG natarn ear edo heterieste ree nae aaa arabes esis Vin MaMa AR ESE

e. Gelatin ......

ds Other medias iaia. ce koa shesanauise oni dye baza AAT see obeine aenaeauee abe
Bi OLZ Oe he epi uaey ee a ae tia sens eecstyes h dan sea acta c oe re Gc on oso Sa eet ete Nard aon a asec tea caToctcb neh pebensnt ec eiors eda argc optncios Sem atdnas) «spleen aeN
Be UU RBs Sisk ccats ate agp ding Ad a rscee eee Wt a Be tare ames tees a Ry eels ta Seah is ie oe 9S ag Eadie os gee
an dsarrang enien tS eceyiat esas coin sc cae tier: Ces aie ad Ee olals Lake Se AE Sao aioe

MEMEO MY EINS cis Secesge ears 3h cecrye spas am dese asa nt aRe Soave cava ig a shades ata aida RIN OPS TEE tease Ears aaa Sel alia

4: Staining: powerss «scan scsagne yore xanrsoamecses

2, “Aqueous: gen tlanaviGlet idacaaas «ade ante neusen swe ising PRO SE Buea AE MEA a Gana on aU we ae Edina SRR diner vie ened areata aan wa LeSteersisies
o:- oetler’ sumethylen-b) ies. oo aactene sce anidie iysncalee Suid Soloimetee bd be aya ae aoe ak Dae Deal Le DE os Beate REO
(Gs HANNA Ss SEERA ND stoic loys age ot nape it clca VQ AD ies Wav PREAH cay orm whence einen eee kaha Svat ata oa RSET DI aacoaete lea ce a
ids SPeClal Staite aiawier can crece we mcm me A tu Notes eeate el etl te a tba hate Aap RAAT nhdae Mtn eveehrnstcte at aes ic rare ae
Se: MLO CAT ey i aca cisjate rts oigtce ois vs otras ted ave costliest eieoon coraye Enea ecm AR ee nag Cie a ae eh re crams nutes site wade a nN aa oaaie te alts

x Character Ob MOvement jo iecec cians cuciceer ot ah Scie Pusag eo ssote Rea Seale Pearson putes ahciare Magee Inet SAAC AAA ERR Ga eeeiad atta a osewaens

Os Fl agellaS tains ii4.go cea sacs 2 aides nce sree oaves aoe Aa

GIS DOMES «saci ait ass celiac We. ace wUeiialoe eiainnsas unaided eehacadiaarda act mtemeg eine BE Rebs mateo as tee A a as nda age oacaeeieeuatt anaes

9». Special ‘characters; Su Ghyas? wisn se sc ck aac asain tavadaliead sgrgaae hge-n's DaameanWotadhaci@ulouuts

deposits,

pleomorphic and involution ‘forms, capsules, Cte «neva ve ve anus eecien nish vewd ie duc song duodeace detpatetactn 4a ga ue auulaee atiehieuie oe

WACUGIESE SA gsune cits isd ivAavea iia set won et Wel ececse na tec eae aig ore es

PHYSIOLOGICAL CHARACTERS.

1. Relation to temperature:..... 2.0.0.0... cee cee cee eee es oan cvaea (btede rewvuba toe copianian Sala aicah ey Maca ate wes er Sielana ceSisise Blea any ECL eth RE Means AE nts Aik oiled See, anes ane dn 2 Raa fms dss Sabreie ae SANT OT ER

Be TeMas on te Free ey ROR a cniss oo naa eeenmnpacran ne oy aomin's ea eelweRe fas meng AMINE HELGA va ea Geen oo Ded ened bh diiebuhvauieweveuden ae

3, Relation:tootheragents; Such assiac.sseceees heen Sa ee & Ses va ho ka GS baad duces aa deoea ebay narnatla ents

desiccation; light, disinfectanits,: €t¢2).< o:.c.cs/.ccc0s vs ecceeeed baud oaewawnns eczema.

45. Pigment produchomiys cho lesdad wes ave aeer biceuaceieeetees wiles an ae

5. Gas production in glucose media:

a. Nae nCuleUner access cemisutsariedie Ua-oe sitah lacie ace. D.AaINe Bacay Aah ceeectire ate ae eid te Un pllge Slas anactoe nm ioe ade A pet gatien ce Ps

6. Fermentation tube, growth in:

(3)
(4)

rate of development: 24 hours ..........

CE) OPEM-ATM un caecessey nee eae Maes Kee eos
.... per cent., 48 hours..

LEACKION in OPeniarm pense cdg ewe ohaak acai use Qoaeh din abbas

6. Acid or alkali production, litmus milk............0..0 00.02 0.0000%

7. Reduction of nitrates; to nitrites ........

8; Indol production;.24 hours. +: ssa seas idasde ay aes cadios «

fecal odor; 24 hours ............

9. Bnzynieproduction: proteolytic: scccaasscemeae smouns ye Speen seed Seamed cdawanes

10, Characteristicodor:ccscsivaceewssdaasncasd sas

Pathogenesise iv eacaedwsecinnns cane mhiady sateen Aha eae ci Gok dos tcattiestenntawnaardandiees

sy ASI NOUTS a ac.gcichbs atinonage de sens sae osigaticwaaae ts cenewss

saved PEMICENt., Z2NHOUTS ie. ocd an once per cent.,

.......(5) gasformula, H:

a acoel dy LOMAMMONMA Ss 4 oss tdueaebavihd qualia seers

Sipaticietys peg ON OUTS ice Caden tated Nua rans talatiorn

ae ae

+. .shours.....

ioe 2) AClOSECa amin Sy dinee aeearam a Ant ee one AA aie alot, SE ae

ibeargne GAYS sianac gas oe es tanto

te Uiastaticn tas Fe an ie c. c atic met ial osan iscsi nama tannin dotiaunanaseniteza Na

CULTURE CHARACTERS.

Reaction

ieee ie 4...... Hours. Bi ciees» Hours. Ga deies Days. SKETCHES.

Temp. (°C)

_ dd)
yelatin
plate:
a) Surface
Colonies. |.
b) Deep
Colonies.

(2)
Agar
plate:
a) Surface
Colonies.
b) Deep
Colonies.

(3)

yelatin

Stab.

(4) N.- N

Agar
Streak. (

(5)

-otato.

(6)

fouillon,

(7)

pecial
Media.

[105]

EXERCISE LXXXII. BACTERIUM PNEUMONIAE (Weichselbaum) Mig.

PNEUMOCOCOUS; DIPLOCOCCUS OF PNEUMONIA; MICROCOCCUS LANCEOLATUS.

First described by Sternberg in 1880.
in “rusty sputum” of pneumonia.

REFERENCES. Weichselbaum: Am. Jour.

& K. 118; M.& R. 204; M. & W. 128; McF. 345;

Found in saliva and nasal secretion of healthy persons—about 20 per cent.

Med. Sci., July, 1886:
P. 498; S. 298.

MoRPHOLOGICAL CHARACTERS.

e of
Cultures:

Incubation
temp. (°C.)

A

1. Form:

a. Bouillon

Ge PING E OTA6 ais 2 sacral chy seaming Se ph NM. adie jr woaaesatc ora Braud lsaseea abe seomieie "ace Wc ataeio & ant eat ce ens chemwientne evan ae

By NOCD UPI Ss ay reid oe salen ener 4 Yk EHS AR VE NCEE A Oia Cte ee Ged we wa Aad GUILE AH wow wd 9 DLR ewe nn ad See
ANIGCATTAMBEIMENUS sup aee fami otic cde ates cigiaa) Wer aceeeattelaniis o 5 tua mee rte San SES AAS HR RNR ae MMe ATG Ge ais MES
DEAS PRO WILIS) cece 22 cs hese taunt caste an gh DAEs SUAS aE Ey pica ce ete aceel nstycate Rode fe ok peach een cach lay. ee atl bel ac eealee ae oom, ee
4s. (SCALING POWERS ius ses .acaiavierae:niarsbexie aiterediat ada Ba oy. alee isl avnaamanhouista vo He bdiabe Sea Sn eae Seren Sate erm ars Sa Anemt one acne tae Nila AOE
Be PGCE SOUT ATEN IGIEL in oxcsic ae vss Sees RRS tHCEAA WI SL eve ORE Cand oe Ao Bi buen Aa Rabas Obs SA Gd Gebapledayshanans cummin
Bp TO BUEN Te x ca it id ocaaaeeseensag GS bess, uusea.d oo busy 34 bu Sea oa ea eRe Weak de dh Wiener Stree
6, Gram S(Stai Mss 35 sevesciisna iitaieplaae seat ses Pave aupateher: Sa sheadenni eases

@. (Specialis tains sccvescmcecwhy ae ceeiasces darsnrmesanekln, oa

6. Motilityiixnccc ccs csnece

ia; Character Of MOVEM EN tis cc.sicy gate celirees ee Suds wane wes ue a anise Sameieoeticye us LO Mk UmobaLed wun aie weudedetnlectrcdgy uel oaelante aakomee
O:-Hlagellarstain,s:s:eenos: Base oumiaederaechiiiae ney sates miawactean Pe Pee eae ee och ancien sare a einen eh onda
6. Spores Syseteatlepeb/t toe wre. nbese 2 6 aecwits eS ete a sir on Pitea ark Wnfeisiee abe Sie tar ia ta Unease ameN IS Soe te guRE Oru OMe mA atd awe eatod Riera sana ibaa

Fe SPO) CHAACIGES, BUCH Het < anl'soaeeaeneaion ay ak ayes HON EER TREN Kp Sv anne SAGA4 Ab ddan ba xb wel vaeucanmnnde ke

COSTES VSCUG OS a caus ya Ke teen Wad Sena SAM alse mma @F ada lod bb Gk ue RU wey Shebunaare ee

pleomorphic and involution forms, capsules, etc... 2.0... 0.0.0... cee cee

Usually Present

Welch: Johns Hop. Hosp. Bulletin, 1892, 8; 125; A. 303. H 278; L

SKETCHEs,

PHYSIOLOGICAL CHARACTERS.

He Relation tortemperatures wise eee ue cepees salary cs Re atid clare had wood alae SedesaMbena meee

2. Relation to free oxygen: ....... 0... cee cee cece cee cece eee cene vee

3. Relation;:to -otheragenits,, Such ast ioe ocins aninin vc aaannide neler as Waeaw Weietacaue Le ae phiadanaadermeme a aon ie ge Rigen

desiccation, light, GISIMLCCLANESHELC the sahsste ote acess AS tet canine noe g sa” of atin ada tea Meyer ae

4 FipMient Productton Fe see poet acad Nustiecrnn ve 22 ae ates aa Medea Vek Sieet epee naan maak bacterae aecaatentandelee se, NT dive lun Be

5. Gas production in glucose media:

G@: SHAKSic itunes eet Seaweed oats cia dea cine. Se gitavlaweantecamane

6. Fermentation tube, growth in:
(3) rate of development: 24 hours..........

(4) reaction in open arm:..

(1) openarm: ...............

+++... per cent., 48 hours............ per cent., 72 hours

pace soe 2): Closed varies vse cece uscasau eeee area:

veoreesee (5) gas formula, H: COo::

6. Acid omalkali production Jitmus milky os scGace cess sidiose weg tcaulvoad WO oa rele wtntads cna mere eed ade aeons

7. Reductioniof nitrates; to; mitrites cc... sa.csceaeeeaian awedan owt ads wiana oa. oe-odlage beehce ie otters

8. Indol production; 24 hours .......... 0.0. cee cee cece ce ee

fecal odor; 24 hours

9. Enzyme production: proteolytic........ 0.6... eee eee eee eee

dona tiaie yp TAS PILI ater ican Gc la ot eetwavndes

10; (Characteristic Odor jaisenasnnnensssseeaeiel wt sz aieac enetueok dann smek Menke emamn soy ueuorned ae he ge:

TT g ~PathO@emesis b cicnivasn as andere sou saina se Samieatie hameodreuior ction elvemnennnenn es Gia &

Oe renee ins Sara Scent a

SERRE NEN per cent., ............hours......

vevees per cent,

.., to ammonia clad stats -e) DH FEN GNMaea ona so AE a mnameemaL eee ce cee re

2 bigaias HAMAS ce v2 ns cag cacavanaeeeien eae

CULTURE CHARACTERS.

Reaction

of Medium,
Incubation
Temp. (°C)

SKETCHES.

Wd)

yelatin

plate:
‘a) Surface

_ Colonies,

(b) Deep

Colonies.

(3)

Agar
plate:
(a) Surface

Colonies.

(b) Deep

Colonies.

(3)

Gelatin
Stab.

(4)

Agar
Streak.

(5)

Potato.

(6)

Bouillon,

(7)

Special
Media.

ee,

LY

[107]

(Pie Var M
a BEI RAEN G

pqd

OLLECE

EXERCISE LXXXIV. BACTERIUM PNEUSIONICUM (Friedlander) lig.

FRIEDLANDER’S BACILLUS.

First described by Friedlander in 1882. Found frequently in normal saliva, lungs, ‘rusty sputum” of pneumonia, and has been

found in air and water.

REFERENCES.

Friedlander: Virchow's Archiv, 32; 319; H. 278; L. & K. 119; M. & R. 211; McF.

352; P. 458; S. 296.

MorPHOLOGICAL CHARACTERS.

Incubation
temp. (°C.)

1. Form:

Ar BO UI Oars iecta wart cass ee cease see nec te Danek aabaracnbote eu Ae eo rie ates ie eso eames

CAGO ACM este vintnco emai aeksne

Biv Celle ROU PINGS sense: era eoatucave SaahetarateioweL tere oeieaig beta gmeaN ae Sate Se GA RAA ERM Gueiaaine hela ate nena ee up teen dave weMlee wer Alen deacons
ANGvaT RANG EMVETESS wise eeteiee vd cee ce tae etinant oa anaes aawisae be Raceusna asa ics tasuraia ea AaTAY seclelans Seabed a oad SS Me ammo se boa DvG elS
GIP TOW ENSibai pit ae eer ee oe cere, Sos Nactead leet fos aga cuetvaah aetna ih eben a A are Hun eee ati deren Aad So ate eR as clean cas el es on

ARES LAIN IME OW ETS setae mht hos Aare A Aeon eg aved. ond: aad veoh peers eee ey ealere bane Renate ce hal erated yee eae eed

a. Aqueous gentian-violet................

ds Loetler’s: nie thy len abl ees ae aw vy iavcse vasa a sen wie sa stot Me Same epee duos eG Eee by GG oe ee

We SPECIAN Stal NSais SoMa a eae aeNeeh Oech ee. GA Kerieatmdine

5. Motility. cautccazasaea:

i EB RACLE RT Sai CIE can oa Sicily. «x pe ongpuenaey xa caaa barea 44 9A SEAS Ga IS BY Sy el id Sw Pes as ol

9. Specialischaractersy Suchiass wc. ac cncvnnwes senaaweeunactense ns

deposits, vacuoles. ............

pleomorphic and involution tornisycapsullesetcis cach eacae ead shyace ba pan auretncittcns neacsovele.d ge Precnlasd dow hade me ocala be ee

SSS

SKETCHEs,

PHYSIOLOGICAL CHARACTERS.
oo Relation

COsLEM PE hATUNE Gijaaa sere wamlaminins ida When ogra marata net emus owe Nee oe Soko ne tele OSI RA pl lh

2. SIRELAION: HOUR CEONY OOM ire 8 ca. he xh Jctenecinine abla! aviler eecald Seaede ayaaste se tele

3. Relation to
desi:

4. Pigment prc

5. Gas produci:on in glucose media:

a. Shake culture ......

6. Feiineontation tube, growth in: (1)

(3) rate of development: 24 hours ...............

(gy MeaChioninOpenaVriitscesccsencacin sete, <a ce

ODENIA TING ties occa ieee ae Oe ciclon oe

MGA EMTS SU GHSASS sien aarti ro suay tie casts ene eens
tion, light; disinfectants, efCt j.csv0 oc cssme eens ayes gee ok eade Saati fete atv eRE Na ch aaadain steve agha WaveneHems~ be das aes Oar :

..(2) closed arm:

per cent., 48 hours .......... per cent., 72 hours Per ceMmts,, cscs

-+e....(5) gas formula, H :

6. Acid or alkali proctuction, litmus milk. hy A AAe s HON EON a TE Te HARP idoad gees Rn nagadaies unligated eh hoe uoee Gaeee cis

7@ SREAUEKONIOEINATES EO NILES 2 cc atteas sox ae, aan S 4 sea aea aN nee eens ay eoeeee oe

8. Indol production; 24 HOurSiss 1. cenaecareaeotersaten aacceve

fecal odor; 24 hours

9g. Enzyme production: proteolytic ....................

Characteristic odor. «occas s25.

Pathogenesis

SHB SNOUTS Aik eo dos an ace

a AB NOUTSIS: 53 se uk ceetevceaton

SACU AS EAL Chale eld eanatutl daar eR nie, oe a

ieee ove NOUS.

vee ve cece per cel,

CULTURE CHARACTERS.

Reaction

of Medium,
Incubation
Temp. (°C)

SKETCHES.

(1)

Gelatin

plate:
(a) Surface

Colonies.

(b) Deep

Colonies.

(2)
Agar
plate:
(a) Surface

Colonies.

(b) Deep

Colonies.

(3)

Gelatin
Stab.

(4)

Agar
Streak.

Potato.

(6)

Bouillon.

(7)

Special
_ Media.

[109]

EXERCISE LXXXV. BACTERIUI CUNICULICIDA Koch.

BACILLUS OF CHICKEN CHOLERA; BACILLUS OF SWINE PLAGUE; BAOILLUS SEPTIOAEMIAE HEMORRHAGIOAE.
First described by Koch in 1878. Found in blood, organs and excreta of chickens suffering with fowl cholera, and swine Suffering

from swine plague.

REFERENCES. Koch: Wundinfektionskrankheiten. Septikaemie bei Kaninchen, 1878; Smith: Report on Swine Plague, Burean of
Animal Industry, U. 8. Dept. Agri., 1891; Smith & Moore: Bull.

6, B. A. L, 1894; H. 268: McF. 409; S. 408.

MORPHOLOGICAL CHARACTERS.

SKETCHES,

e of
Itures.

u
Incubation
temp. (eC.)

te

1, Form:

a. Bouillon ...................

DIAL: ace 5c aise testis de eedEcdly nach HAYA. iy Bs NUN aS TERI Natale: SMA HIRI ae A SS WER eA RE ER ee ep eR eaaett eet aaNet 8

Gi Gelatin isvews cancae seen tees einas Gane paca mania tach arstehedelonsdiats cig acabunetatinn aeasdeG near. eipa ile cist head orhataneisiam ache oe a> Blk

[cA I a1 => cat 01s bf: eNaea tam everett Resear Per meen a ean rem oP rere it fre erere Tera tty ert eran RMR EAT crc circ Sarr ot ey an eA aot arc
De SIZE gaia vi no Wlalteecva ms tmchae Sak siore Bea siesaeinanierm bia ae Campa aia Beate Te slo waea (acer e a aia ce eeANG Gated asya fale a Sub I Aoeeels Reon aoe Die ele Gand Pee dre tinvets [a eagle
3. Cell groupings; 214s esnsuse sins soca contr oe saak gaasedade srummed exe tegen cig ties BOTS Mbeae mapa Ee Re bEN Jai ae Rae Ie HE Teas
and‘arrangéments: gos. ncy ede wey ace neta ee oh aaeen iG ye eee eee Se MERE BROS eG ap Fe SEE es Baigine Cee ee ee eRe BES

IMs GLOW ENS Nines ass dois Soda aiee SeaisaauSnle ykedk ale aasuuind oechetaldiearaate Ae Ab Sacahd samba deeatin aise Seated Mees ang emNN seh Rae aia ees aala ee aie

4. Staining powers:

@, Aqueous gentian-violet ssn cwcidyessu tate os va ani anyone asaiaarng 24 +a rgpite mba sien aesteiaes Sh eeias eisleba aac Tok Rea reeels gone
6, Loefiler’s meth ylen-bluéew 2:23 asic oe disinaene be ee Game teens a eae Ne BA EE aS Ra ei iG Kisienlg 2g lead on oat eee ON
ad. Special! stains «sa gs se-seeseye sess iernccen sx siea yas aitcnwis ai a aw aceaeiers tin sie -asapar a nrersites PERRO ee NG alg Siig HE Ha NTE NNEE bee nde I Alo oe

POS UNE TFE A er noemern ERT Tay air Ps ER? ersnten rnc sqeaca is iran tis rine Ratio Ne Ba UMN ae EN ret?

@: ‘Character of Movement cegecs sesee ye rs ead acta assem ac Ye ie Gamat ad cei ee aeun eases eatnaag He sau Tem amemee sa 58
Oss Plage) a Star igs s oi Socyiasessai ues tnaceeecs assis ia wees uctaeetep spein ss looal te ate eso ee wether Seco tp aac aceon eh Satis oee wave Map Seemaae eee Sk aFs
Ge AS POr eSiees Soa chara ahh sean esederes atest osc Apo ENR au ate Rais heh hearer A Nacht ate, hac den Va. Seas Moonee aa BGI nia tease Sala ro mee de

7: Special:characters, SUChias? .ivss suacun ee he va mone eivaahe Aavean we veers oils naubianlaiinga ye uy aeGieminaag Aaaited di lp elising Ae BREA as Se
Ge posits; WacCuolesics cae 5c si esouss cussccielsreie yes cease ley Ay ay a RNS ae gee Sa Ss cana rennet cemeipeee Ra aes, heed Desa oe

pleomorphic.and ‘involution forms, capsules, Gt@. 0... sc sccrd sp. oan veacned ad nd same eb ak swaiele aueted ow wtcnene 85 aig Seeloule ee Gaeta ess Fb

PHYSIOLOGICAL CHARACTERS.

Tf; CRelation-tortempera ture ts aos. wi cas charts eerie ey i IS vas Os naa sao tin: ahsiars ves tid dated. ed pa Mao eR Gen weed stares sewn é on

2; Reélation-to: free Oxy Pens cance cede sw dasewesats ox tease ame ssod wens sae Aa AaMaW Vawe apie uaa SEAS EOD RS ee Moo boiee

§; Relation to other agents; SUCH JaSte2. cis v6 say canta 24 aaemeredaiee awed Dade E MEATY Yas Gee abuekeeudu bb ea duban kee anions ”

desiccation; light disinfectants; €tCsi-xeecy ties vadidewe Bees Gales gop ad aasedde Ac oe AS edad beet ened ean

ae “Pigment produ ction tii eoiyesntens no aye cisnis oeeet tue. 30d b dechecteyet ene vs Ged ence hi Seeslue AN ete ee avs ch hese ea

5. Gas production in glucose media:
a, Shake culture
é. Fermentation tube, growth in: (tr)

(3)
(4)

rate of development: 24 hours................ per cent., 48 hours.....

6,. Acid or alkali production, litmus Mi)Ks 02.06. cc. esa eee chee ve ne Sees Dena eae nea oe o

7. Reduction of nitrates; to nitrites........ 0.6... eee eee eee eee eee

8. Indol production) 24 hoursie.s css seas ede ceewr yee ss ee ames

fecal odori2d: hourSsscisackeaeceahusues aeeawe oe aes

9: Enzyme production: proteolyti¢.. co.cc: crse seco reendan an yeoman weer s semana rete ds

Tos: Charactenisti@: 0d Otis ssecinancrnsceearere ne anes Reaver os eee ters

Il.

OPEN: ATM Sas ci eseweok ag 14 9S aE se Sie aes

++.....per cent., 72 hours.......

FEAChiOn 1M OPeMarMlivccscens soso ns Kaede mess He daceteR gine o ois diawio be

ps4 DIN OUTS epheue date cc ioc ds se aii pes tease teres ee bey

24 48 HOULS pore eye oe os

Patho@enesisweccy cise ay co nagesdsacimntnds Se slctacys sed mm tanaren Heeaee Guile Ninel deca ome

enaan eo\(2)) CLOS@CAIRIM) csek ae avers sino aoe

wig cage DEDCEN yaa hi geno eeemes hours...........per cent,

-++.+.:(5) gasformula, H:CO, ::

Seeeveialy COAMMIMO MANS 2 aca neg wataa's ae aasemeincwine sas seen eeeenee

......diastatic.... 2...

[110]

CULTURE CHARACTERS.

Reaction,

of Medium,
Incubation
Temp. (°C)

SKETCHES.

(1)
Gelatin
plate:
(a) Surface
Colonies.
(b) Deep
Colonies.

(2)
Agar
plate:
(a) Surface
Colonies.
(b) Deep
Colonies.

(3)

Gelatin
Stab.

(4)

Agar
Streak,

(5)

Potato.

(6)

Bouillon.

)

Special
Media.

[111]

EXERCISE LXXXVI. BACTERIUM RHUSIOPATHIAE (Kitt) lig.

BACILLUS OF SWINE ERYSIPELAS: ROUGET.

First described by Loeffler in 1882. Found in blood, internal organs, etc., of swine infected with the disease.

REFERENCES. Loeftler: Arb. aus dem Kaiserl. Gesundheitsamte, 1885, 1; 46; McF. 426; S. 420.

SKETCHEs,

Cultures.
Incubation
temp. (°C.)

Age of

MoRPHOLOGICAL CHARACTERS.

1. Form:
GEE RGA OT ve eee eras ae ne oak fe Specs cheese es Lala hevaten sR HE ae aa one 2s Gate HAS eam TENET EEE aoe RNR earn ye Te Py ype an

Bec Ae oct das Seas nse a celiac ats os AGE Be NE Sa ae en see 2 ns RTS SIR ecg reas Ment r a Se aR BIRR PANE DRI TESA 1G IE SY

ESV ARTI ice aoe as Se ee Ra BE tina of Sd RAEI SSPE SERTER a eo SeeE eset Rs bette Te fst Beas > hoot TRA BL USES ES SBCA RR erate NSE TIE RSIS SG

be CEG Te IVE CL IEA na, onesies cle ese ees ee ted ore cpe dete dns Tad Sea CGY Ca eth RNAI SRS es a aha ers enhta) Se ata Suere re sbtea tn Se lee SIRE SS Sa Psat
Ba SU sore ae setae ae erase ee hoe detects MA ence nent aueTaCgUe spl aeRNaaE Abad aller (esi eaans Seeissee are ie MOR OLE LED TENE TS ARR Nara Tee A auton esate) Sie SE | EES SY
SCO SOUP INES’ darsrea vo hc os, weadarsemnseus ees ay Ae hia Riad a oat ih ds) dele niet ethe nFSaheralvayarers eae lreresce pee wee AG SF CUREIR In Sy AE Nee Sse Nati Oat Sera
and arrangements icc ties dated eshahensgeeda ak wediciwmncehadae Sith Rea Sate sents SD La SAE eee eee Rae S cane etree nae ae ne pies
in growths............. 5 ese esl se steed stoke beg ESAs Gee Bae acd Bas A SA ERTE: TG RG CHAI REUNITE Gee eS TE Ase oA fapnralay ota 9G naesaie = eeie | BE Le RS Syamndeeles
4. Staining POWEIS2s sink esas aneneanerode ae sraaieiaiee en. shee pie be ame nemosnon RSM eeraiea sce Dicinieh wes Meee BERIT Marrs crest RANE Selah diate | ASAE SRS
a. Naeous: Pen tian-Viol Otis se. s sh yee vate ee Mites te aa alas ato trays Afalcbanta meting ats aeteieet noveeclcgemeia eS he LG Retest, Se Sats heigige Paeia istritl mie a
&. Loetler’s imetivleme blu Gscscc5c.c sive enc as 36 nds Sakeweags 14 AREER TE NG BONG CRD eon a nmeNRRY Fai TINS a REED WE MAE HNN Hee | PF SRA RRO TS
Gy GAIA SSE ATI dois seessmee scenere secs ave stcretecevshe peasadey dee BB vee? Gebenea vive de Yaad egsdfs ise atoaccek imme ae aaa Mare ween ea ag ise NREL Bi Sienna seis ae ga eee Besishd fitic eta az ae
de! SpeGiall SCAMS ans scene ws re Apeenlis aslo alg oom auc asaeae ctelosy nat mae Tae ADS. £i0.S Cisaclisa it ed Seem RSD ie Reem a wats Hy SP rs aoe aerate heheh eg

gM GHANM EH esate yscds a saat ntcbense BS Racneltoay Gens EARNS OU Se SUISSE rae oe seeetecae dal te aps ae eas tata ose AN kA ct ie Slee nents BE Se IMRAN HE AER AAG ia area eS

wn

a. Chawacteriof movement 4 .sda sete cs.cc dec Qanvas ness HAS RERE SY HE SESE CRM W LA OE Rae RaR Th ae Gane e TEES Tee GA ee eet tennis dae damnit Peiear|pineecntay!
& Mlageélla-stait’ <2 cccesa: savsxectiwsatdeed otanecer ett

Ot Spots. cecsenn ts gure ey eee

H.°S peclalicharac bens, UCM AS pac. bcay oe decunterg nea wesc eM Osee Ma MAB NN Oops a yitan neved ok sence a Seats aya aathaite:iubath Seaeanslag ee

deposits, vacuoleS...............ee cece eens

pleomorphic and involution forms, capsules, etc .......-. 6.05.00. cesses

PHYSIOLOGICAL CHARACTERS.

fz Relation: to:femiperature? ccc. as ug adackous oeceme der St (eee waaa se aie Beets Gear era eae vanes as gree adamemuldn Ay ante pes omawe we salam a Ronit:

a. Welatiow to free ORY PEI cc ve ov aece cess cance ya come anes ope ae bu wa ie veda HPIT RG ede ee PEE PERE ARE Dewey Ad EX ANA ER RENNER SEE Oa FT ROE TE AIA ARR ERAT Le RS eee RENEE COORD
3. Relation to other agents, such aS:........ 0.6.0. eeceee eee ees

desiccation, light, disinfectants, etc:..

4. Pigment production:

5. Gas production in glucose media:
i Slralces Gill Cues ei teaiaa Nek ee ah cease eke cuca eee I ieeeciar eS Bd Gales Mona gaa ie Saat ae
4. Fermentation tube, growthin: (1) open arm:..............0.0 -2e eee eee sees... (2) closed arm: at

(3) rate of development: 24 hours .....2...0...0. per cent., hours... 0.0505 percent, 72 hours... oe cece Per CONt., oo... sees eee MOUNTS peer cee . per cent,

(Qn action in Open nme: a teen eer func weil ese te Siok es eve cna nets (5) gas formula, H:COe::
6: Acid oralkaltproduction, Htmusanilke: acs. o3gasucnd Ub vaeielas ca de Lacege ee Miieasaoeviton vale ;
9, Reduction of mitrates; to nitrites’: cogs. x sa neense sansa sane gess pemes oveaianemsys oideesy CO AMMONIA. cc 4 bsy sedan se tae teeta
8: Indolproductiows 24 Hours witud secsien a vane sty ee wet hi sy 48 NOUNS nts Ve tadisc Ld So dad ne eaeeatecp Reeve Weve edemnione S NOS

feca] odor; 24 hours ...........- Soy Wada ens Saag dos ated OSNOUT Sista tyne day :

6: Enzyme production ® proteolytics.n:cna01 sranten ates oaiauhein mores ames opel: ... diastatic i

16,0 Characteristi@odor css 5 acxany eo fe oe eR bees

11, Pathogenesis, .... 2.24 2.6 cone eeee ee beeen seus

CULTURE CHARACTERS.

Reaction

of Medium,
Incubation
Temp. (°C)

eee

SKETCHES.

(1)

relatin

plate:
a) Surface

Colonies.

b) Deep

Colonies.

(2)

Agar
plate:
a) Surface

Colonies.

b) Deep

Colonies.

(3)

Felatin
Stab.

(4)

Agar
Streak.

(5)

otato.

(6)

3ouillon.

(7)

pecial
Media.

A
: \}
h \
er

[118]

EXERCISE LXXXVII.

BACTERIUM TUBERCULOSIS (Koch) lig.

BacILLUS OF TUBERCULOSIS.

First described by Koch in 1882. Found in diseased tissue of man and animals and phthisical sputum.

REFERENCES. Koch: Berlin. Klin. Wochenschr., 1882, 15

M. & R., 224; M. & W. 148; McF. 208; P. 263; 8. 375.

21; Smith: Jour. Exp. Med., 1898, 3: 451; A. 312; H. 189; L. & x 95).

MoRPHOLOGICAL CHARACTERS.

e of
lentes:

A
Incubation
temp. (°C.)

SKETCHEs,

1. Form:

(GMOS TONND A Voy 9 Pees he De CnUAR COL ir Beran arc tre We ate io tM REY Mee Pn eo POTN ee Ee Se OME TS eS ic eet are rer? ec

INGE circ aoe St cise oe ae eaves aes tae Se GY des a wig ge aaa UaER Hg SR SGM be SUED OE Mee EERO ONE RG ie RAE SO OCR RRS Salat eee

6) (Gelatittive aciyhitin i sanenke te Ae

Te Wad @\ 1 Vcd ce V0 bE: eer eset lee ira eee eae a one Mes TiCe ee aE EAA er aRT, 8 PENORI Rts ated ny Gehint ar Me Te HAG clamor RC ONE ee ree ie 7

BAS ZE ays Se sctleesier tee lewis ede WES RIAD RTI TRAE EE DENSE Ht 1G BE WOIe HSU RRA Tes PEAT SURE SETAE DAN ERD: OS Nie Soe es DEST

3. Cell: BrOuUpINGSsatenscaekecistns secnssiomeias +a ddan nate oe

‘and arrange CMments as at cheecrnam ae sean Aaah es Sacer eta tH ea eee Late eee ee MEL Na Bering heap ta cae tue eee Wide [Wy aye

TED HO WENS oe b.ck coh sys 5G ab Hb cGesnite GH garaged BO Hee dank Canmenr ge take

4. Staining powers: isis: ecasa teen ac cgi wean iad cis eee ee

a. Aqueous gentian-violet ................0...

bs Teo Mer’ ssnethy len-bl WG ae eae eect eee ery deena eee oe eh ee hee Nola alg pee a tag oh eee Se nt cao ease nr aradatuaeie
Gis GATTI S SEAM os ssid nese ene ae see pian RAPS Ce Entei EAA oh Mea CMDR ek Sol Steer RAE oe eV EA Rh Ge Sesten be aie t= tae oe Bokeh ea |

ad. Special/stains: .uweasececuseeieon Aiden Rote os ieee

& Motility ie ees dense wens sy +s see

wn

a@. Character of movements a csac0 ine tea: aeeenrgs peta hs da ba aw dee ay haa de ba Seawind Seated
Oz srlagellasStalny ssa ecen aes cto et canes Gas a ares foe ea oe eS at as aces Poo iach ate eran aaa datos ras eases re RT ANS cite
(6: SPORES: Oa cihsn es deca e teat unto oa ska ge Mile nnanal Woon RN iain aa ketonne mame mitienlatrtatmeccctin ola AY FE ete

9. (Special Characters, Suchias3 isco curses anshe we ae sean AeA y oevaaan ce Ue ke a3 OUR Sea dete was oma aoaedee aheas
ME PaSitS Vv ac uOles cn sak crete see ei manr ee enatene aicWie as eoenge oa octet ings, Bt. ys ala EY Syne oe aupybeSoanyeoubey dee he a nwchol ae

pleomorphic and involution forms, capsules, etc... 06-2... 2 cee eee cece eee eee

PHYSIOLOGICAL CHARACTERS.

1. Relation to temperature:.............

2, Relation to freeoxy gen? anc cumnanisais ve dqieiatecea sees paces gauwa oe

3. Relation to:other agents, Such asin is capa seceescen nena sa ae wr one aA8 SEES Ne we de ai wielgucd Hb ducal Bede cig

desiccation, light, disinfectants ete .c..5 60 ceseaie on an Sanders snags ambenee dewewe

4. = Bigaient production ders see 5 voters arcane sae cheba ncato ha Gite tieaigeiaa core asap dauslita gy meamher eae he

5. Gas production in glucose media:

Ga ShralkeevCull ture. aie ze) os as ices oa asa eae ON Done anesns taaye oor OMe Secs asiee

6. Fermentation tube, growthin: (1) openarm:.................

(3) rate of development: 24 hours.......

(4): TEACHION. In! OPEN Arnie c.u eaves i ie Ae ee reve va weiss sess cobs wa Sucuteen Sos
6; <Acid-or alkali production, litmusnillle ..cicc es cose ion has cee nome eeae Sauee <eieme eaa

7. Reduction of nitrates} ‘to nitrites ..2.0agc.s ase asavacsesaea aeani ns ooaeieesa jee yusun lent ci

8. Indo] production; 24 hours.................. 0.02008.
fecal odor; 24 hours ......... 0... 00.00.00 0005

g. Enzyme production: proteolytic....................

oeeseee per cent., 48 hours......

ease AS) HOURS tee Saane te Aine ctas

aad SHOUTS wich »

.......,diastatic....

sean cell 2)) Closed Ja riitegiera satiate wos ac Gide pace sede een. 2 baie ceueaceibanre

++ ..+eper cent., 72 hours............ PERICENS, seas orcs hours

.-(5) gas formula, H: COe::

1 gaee etn AYSiwets ons soemacaies isk

fe Marae er StI OG hate wae ns ee coos eeng Fpaw dade We eae enmes eine EIRLAL oe nes

¥e.. PSthOvenesl © aac cs ce waces ones 22 cs cite noaeuuie agen peay ghbds ences pa sane aan

spo WE MITT ands Ur ecasa sere Gots conyladle sess eesncul

Medical Bacteriology. 115

B. tuberculosis does not grow upon the ordinary artificial media, but may be grown
upon blood serum [see p. 89-(1) ] and bouillon, agar and potato to which 5% of glye-
erine has been added. The tubercle bacterium is very sensitive to temperature varia-
tions and should therefore be kept at a temperature varying at most only a degree or
two from 38° ©. Itis also extremely sensitive towards desiccation and for this reason the
cotton plug should be well paraffined or replaced by a cork through which a small cotton
plugged glass tube passes and the incubator kept saturated with moisture. For methods
of culture and isolation see Smith: Jour. Exp. Med., 1898, 3; 456.

EXERCISE LXXXVIIl. BACTERIUM MALLEI (Loeffler) Mig.

BACILLUS OF GLANDERS.

First described by Loeffler in 1886. Found in the nodules, ulcers, discharges, etc., of glanders or farcy.

REFERENCES. Loeffler: Arbeit. aus dem Kais.
McF. 248; P. 598;S. 396.

Gesundheitsamte, 1886, 1; 141; A. 339; H. 217; L. & K. 300; M. & R. 268; M. & w. 164.

MORPHOLOGICAL CHARACTERS.

ee

SKETCHES,

Cultures.
Incubation
temp. (°C.)

Age of

1, Form:

UB O AEN A cscs sects eed at ey lS AS ide Se LA and aa Cache as Sate bid he HSER WED RU AS PIR ER IRE it RAR, Save Garena a

Eb, FOL ALID ques as we cee ie semeae mee sissies (edie DIR Bean loa haa youd oe aa rere odes ae ene UE Sey eR tN Ree a Gps Sie ieee ere

d. Other media................

Bt “SIZO a wi ek cea whahovoctaiindws Weds npces ou aaa Aa ab Gaia Dawe geet Ie Mean GaGa pene tine SERRE cle ye cop nee as wenn ee SE aoe
9. Cell growping sien ches <sacane secs deeb waded nents 2424 serena ue ete homes Ga ae Mans Sole PERO AS BEAU EERE cee Reh de oe DENG whe oaNeR RET
ANG ALFANGEMIEN Sins ci warren dvds sie ao. tnieins oeechioiay trem Se senate ae Hee EA Aieae Ab art Oieineremeren eiditeeriain ob ay Heeadng EMER Wales heed ay oes
He LOWEN sees vk gars. eS Ved oe ay 8 Be Oe WEA vO wed aa de FA a penton s om ecees SATE LENE SEAR Seem ek es Er amie ETRe RE AE Eee HS

4. Btaining Powers 6 oc eas usc a sea cnen neha wads Dade d IGN 44 HESER OK AE OA Oe wR wena VETER N oe ae anes Wb pee ba ba p4 5K BREE Ee paso eR EE

a, Aqueous gentian-violet................. ce eee eee ee

6; Loeffler's: eth ylen-Dlue: ssc sence ceased hotel peoedad ee de ao Savomusisiple ie dale) geod o nine Gibbins se Dadae IG TE uate ER eer ay 282
Grain’ SiS tal ve \cx. dak disaghiacecs ae secy ore eases wae Wee aialeathe ne ain testes AMIE SE Han HOG REAacEMIAG A Gan TGR Hearne ure

@., Character’ ok movement ss; toca igs viehas ae ise onde dunyeantgansre av wSharae HE Seda bien Gok seid MRA See Eps ageseee bea Seewe sists Tha

bs Blagellavs tam case coriacs iain sone pin tina te awe
6; SPOTES sanve sath neagad ey eee Sem

9, Special charscters, S00h Bes. oo cna esau oad sekne ne Se Bh Gated Ag HORROONER AS RE Dan Be ee A EEE A ORIG UO RRA E IS AA GRACIA RE TAG
GEPOSIS VACUOLES sarees eau nach ane mays erty N sche, eo See aN alta asd es SateuNE Ciced au caeioncee Oe eek Rie GM Ta seine nlRCAce an 2 SENNA aaa paat ats

pleonmrphic and invelution forms, capsules, eb0.. icc canes teeninn eewess veNauee Be peak V8 Ne FAN CATA RE RR RE DOMMES RoR ae Es oA

PHYSIOLOGICAL CHARACTERS.

x; - Relation-to temperaturesias. cou.c2. vedere He deteuttues shake us anol wee NMRRE Guan d ve URGmEd embne aa lated ea we donate arena we

2: Relation to feevOxy Geis xc. ce. a2 He sacs sate arneanescmialaeia sla ns aecsapyernialane asiceaing Sethe th aq. nme a a Balle aa we eee Ig eel Hea eh Se Habe ve vlads .
3.. Relation-to otheragents, Such asi.22. exes tg vane ween Mahe e doe aie: dbginem Yaga ee Leino ie edie de Gaus Heals
desiccation, light disinfectants, CtGss.)i2ccsc st tj Gane dahmtak Swe webs ah esgaas andere Oe ecaimeea Amato s

a. Pipment productiottc sacecscstiarstai aa oh seas teaes pena cen anibe He09 Dead Aadia dkdawna cpa week Heme

5. Gas production in glucose media:

os SHANG W) CULE raceme t Nese Oo Fe Beams tices teas ITA ais ae ear OPEN ee Oh See ARS SH ae ae ttt a Nyt eee Aaa ”

6. Fermentation tube, growthin: (1) open arm:........ 0.0.0... cee ee eee cee eee

(3) rate of development: 24 hours ...............
(4) reaction in open arm:...............-.

6. Acid or alkali production, litmus milk. ......

7. Reduction of nitrates; PONT ERIES acute: aes amb nes eacrenoe see

8. Indo) production; 24 HOUTE senee. a6 en seiee sees cere Gey

fécaliodOr 24 MOUES se.cgia ni taste ol ig bs io Rated houses

9. Enzyme productions: proteolytic prc canatxesecosseeessanasieve com aneay tame teats

TO: eHATACbeRS TC OG OBR we i.2.505,5. 5% onda stn yiea booty 16. oe OFS DSIAN ee ecco

Tt: PathOgenSis: belcpiittiicoaronesmar ne emia geaiten

etn ABZ NOUTS i Ae Ba orn pee BeVah unten yc Vs) Sols
Ase SM OUTS Ha Aedpcicensvastiene ne aeeean ne

A(2) FOOSE ATE tyre ae Beene nie cerns aS oe eeu ae hana eaten eee
per cent., 48 hours .......... per cent., 72 hours ............. per cent., voce cece veveee HOUPS sc 5. c.ee++ per cel

.+-..6(5) gas formula, H:COeg::

= Shiau HUOAAUNIN ORLA as- se score nae mre Oa ehdeeae beens eo eee cece enae eely
FAY Gee ge ee dea Gane cece nee edie GAYS ac nase se cane wena ea ceeds gC
ih ation eee deena is arcu» UBS ciamecves vs ve nasa aen Sedmy

fados y CLAS tat Ci, ascscuede «

116]

CULTURE CHARACTERS.

Reaction

of Medium,
Incubation
Temp. (°C)

SKETCHES.

(1)

Gelatin

plate:
(a) Surface

Colonies.

(b) Deep

Colonies.

(2)

Agar
plate:

(a) Surface

Colonies.

(b) Deep

Colonies.

(3)

Gelatin
Stab.

(4)

Agar
Streak.

(5)
x

Potato.

(8)

Bouillon.

(1)

Special
Media.

[117]

EXERCISE LXXXIX. BACTERIUM DIPHTHERIAE (Loeffler) Mig.

BacILLUs OF DIPHTHERIA; KLEBS-LOEFFLER BACILLUS.

First described in 1883 by Klebs. First cultivated in 1884 by Loeffler. Found in the false membrane in cases of diphtheria and in
small numbers in spleen, liver, etc. ; occasionally in healthy throats.
REFERENCES. Klebs: Verhandl. d. Kongress fuer innere Medizin, 1883, II. Loeffler: Mitth. aus dem Kais. Gesundheitsamte, 1884,

2; 421.

A. 349; H. 162; L. & K. 207; M. & R. 353; M. & W. 137; McF. 284; P. 329; 8. 356.

MORPHOLOGICAL CHARACTERS.

Cultures.
Incubation
temp. (°C.)

Age of

SKETCHEs,

1. Form:

G, BOuUilOnyscacdent sa te hea Arod tans et BFS COWRA ea ONS 1 OTe Ayes leon’ Get dena we ae, oda eS RR As Eevtisieana rs Seer eras ta: lS e Sarees

G55 GOLA ELI Scis-chscscna to agerercrsapelbia erase gover eace ae ae abv aes hc aoe gmpeanin als Sie ae Tae so aaNet a tidaye ghd dL RIDE eee eNom ORE Bea aA NR anals. Sb gps [latest

i OMee medias: ys. cgee sy os seek Bebe ahead a ORE aed ae Ede fa Sees EES et eee a es os epee ares pa cee eat teamed
iS Ha Oar cie eet ait ale eceae VS gopel ae lesen, DOL a VIO Ne DIN AS 5 LU <ESTCUR VEL uate ce Legh GETS Sac me one Se eM are ASSIA ee eam see or Gress
3. Cell groupings os essa: se gee aia Some tee ve Hs ERROR RIE sce RG Re ARNE Rein RE NAAA ET RR a LER Roao eee pow euieMe Sanlu es D> GH

and arrangements ins weve eee enizaoria teh nig oot sinac amended Ha melameals ates

ITIP EOW ENS scacecetnde ce sehes ao hare AE AUER OE BAR RR Na a bed Sed te aaa SU nee ae adele zaaLeacin nent seme meee UE ea

4. Staining powers:

a. Aquéous gentian-violeti sess ecm came canes ue aia aeeN nS HORS ALAS TE SEDI AE Gd wan oald ee OVO aR NY Soe EES

b,.leoemer’s nrethylen=zblucin sx tas /cncssviaas fas alata aalomiann nah eoualsiae teas Paola he UMMA ce FEAT RE aan Betactn as ne eae eeraS

6 Grams Stainwenesdse- was eis as acne id ae Seok oeea ant een In ee Pdi ee eee eR RAG Adltn ails ates who saaieise nthe saan meres] ahs aenatinallla aman
GSS P ECVAIS PALMS 56.52. Sy Aensse esd, J sce cisosch ete ch 2 Sey aca byes depts ese ato ial tude Gh aceasta eeeseiet he ve nN SARIN ARP 4 cave wee AME ah
Bis MLO UGY ste riecc. ataxia ca rsurpais cays ASSIS Vena Te eG NE AVE WEIN Sena des tales main ACR iA ei etal 5 Culestare aut Wyiealiten sdk ae fechas eens meee NE a
@. Character-of movement cess sens emecantace ea atena cium 4 a8 dainael sh dann nda Bo SND We my eas GU GS Tura REE Ane Mane aaa
6. SS PORES, 5s. Fos.5, se seeasshs as ese 3 SS OS Na aiSd GS GS BES Ca a ENE STG Ur Bee RES EB a DEED. Stir erev at dina ye aie ape] ate tes

5: Special characters, Such Ase sac eewate sce cpacere sictane eater tai ates ae cues arora eee ca ah arm ap tess eee ere NSU HE RG IO Ea bE Eerdae se rain | Resa eaa ibe ee een

deposits, vacuoles.............

pleomorphicand! involationfornis; capsules; €tC vies aa hecee ake we eeng Ayes fa inate sa 3 RA re ee ee ng ed Ae eA We

PHYSIOLOGICAL CHARACTERS.

Tt, Relation to: temperatures: . 2. scuce sone as sestisaserenag geass

Ziv TREAT OMCOMTEE ONY REM: Ssh scege si ures a arabe ots Pe Ges hd Ws wi Ad cee eutia se te ancl pian eben ee eT Hek seanvchotaauseee ale beimeeese acletid ates pc Toreaeaee dete eae ed eee clube re ome openssl stove alemiorma eet

3. drelation: tooth emarents Such, asians cans sae Gani Zagato anyedta iy anahkas anise a athe scl uel antag ne eunlome wap ge Beate yeh dia cs unt gotnaute aerial eh ale 8 hace anil dag Mee

desiccation, light, disinfectants, etc.:.........

4e- PIP MEN tp Od UGLO IAs ook wind te edione Avot AneR My RE AatLAAOR SAE ENA @ MRL ces Ue else Soles i dan od Sued aps a shawrerme rang hhc ne Geucane

5. Gas production in glucose media:

@: Shake cultures. a2 Gacscey sense debieaes audi ae ge cule

&. Fermentation tube, growth in: (1)
(3)

(4)

open arm:.

rate of development: 24 hours................

Faction M-OpeNalMgwacieioces tate se caus shveeeas anes dzae wee guess abs

sposiien (2) Closed ATMA uae avs ge aly von Mowe ona set dundied Huds amma rioetae amen ane HMR

per cent., 48 hours............percent., 72 hours.............. DEF CONtiy cs ssess seas

......(5) gasformula, H: CO, ::

6. Acid of alkali productiom, [itimusiinilkey vo. naawee wae geek heen ate de Fee Mena a4 Vatyea iva adiwernaes

7. Reduction of nitrates; to nitrites........

8, Indel productions 24 hourtsij.2c secede ase aa ea ieee naan
feCal“ddor: Se NOUTSsuswed cas cae Hea dcwmmiey

g. Enzyme production: proteolytic.................0......

To 0 ChiapactenStic- Od Omscsnweve tase orate fey

peg P Sede scar dwar tes Bac ee se Methane wen deere ew
AB SNOUTS UCAS Ge hese enim eee aay

puis CAS LAN CHicarstraiaky da, caste hene conte

ri,. Pathogenesisi..9; adistet Gate Mchat eat inereancz a

[118]

ages cae ROULS ace

. se... per cent.

Foam Ona. 2 3: Noss atestnnst. ne eaten Ss Beenie emlau wemsmatia a pan aanamanmeina putes eee
theadee AAYS wasyrasnmedays ye ca ix eeancwneiel aM

CULTURE CHARACTERS.

Reaction

of Medium,
Incubation
Temp. (°C)

SKETCHES.

(1)

Gelatin

plate:
(a) Surface

Colonies.

(b) Deep

Colonies.

(2)

Agar
plate:
(a) Surface

Colonies.

(b) Deep

Colonies.

(3)

Gelatin
Stab.

(4)

Agar
Streak.

(5)

Potato.

(6)

Bouillon.

(7)

Special
Media.

[119]

EXERCISE XC. BACTERIUM INFLUENZAE (R. Pfeiffer) Lehm. & Neum.

BACILLUS OF INFLUENZA; LA GRIPPE.

First described in 1892 by R. Pfeiffer. Found in the sputum and nasal secretions of the diseased.

REFERENCES. Pfeiffer: Z. f. H. 1893, 18; 357; A. 334; H. 280; L. & K. 281; M. & R. 431; M. & W. 162;

McF.

446; P. 320; 8. 370,

MoRPHOLOGICAL CHARACTERS.

Age of
Cultures.

Incubation
temp. (°C.)

SKETCHEs,

1. Form:

N

10.

TI.

@3 Bouillon: cnweaads qekcordencneauoncseesesmet aes

By BOE otek pce ta dakaabaacnccais aawe cine hax awed 65.54 ca nue dau be bambre da 1s OURS WORE ERA: isla hE aR Qawe eR DERE et eee

6; Gelatiny se varuasenagieteca oeseiasee Gangs oy lees Ae ae Same esian oaategle

@. Other medias. cc samc neta wigs sa eastern sais Peed dace Vanes eastbound des
BADIZEs s veMened ged oY REREG TEBE RA Te eee a ae eRe Ghia sass seme
« Cell Groupings... .5 occ cnnsice wa cees ss

ANG ATFANGEMENtS wives shton ak are te else bh aMA DOS te sa hen bce eavaiomensias Gea Dahle eee Sh aahesli dats ch aiaG nema soins Tee

in GOW thSsisise gies se acice ids eal edawtne va ve atea able ga SAimay eres Fs eg aie ge aie Die hee etonent ae Wienninn APRN BS A ANI ae Vase a alte ee

@. Aqueous Gentian-Vidlet. occ iooccdcce ns so saiesa gene neways 06 9G GBS ROHS La igang ATT Sh een Se Renee RE Re ee IS) eng Rn
5. Loeffler’s methylen-blueioi. sic cs di cate cee ce cre neee ence ca ee nears se emibion ea aie galsed ea o Eb Sale be de 89Re a wens SERN oe aa ee eR
€) Gram’ s'stai nie: sc ac tn ies c aloes aan seeeenea a Sanveasen 4a HemaiAT en ehes be ohecinee nb aa Si aang UEaas Pasay yee nies

@: Spéciallistains: suse os gene sagecnea os a teins Weak Sy LAME Ne Melon Gada Fee a nsla na ane meme Bieri NA sealnee doe bie cam lsahendtn are gaa wie ie Gi

as, Character: of move m entre acces vachitaceeeeenvraean ch stindene Gavin Atm Sos eed eee ma Saes swe ee ealee

. Plagellastaine ¢acceneierasiasnade someacia dee owaira ee

WS DOMES 1g gciscssnecd neste caging aaa Cen aG tie tea lenis 3s Yr ain apa aie rad ee dice teaitctbe tes camenans ecient Wk ata eo das

Special characters,Such/ ase asics secure: nt cessed bad selina de Moiyeni ag Remade ed Seueaieavnseseieanaacnn ad
eposits, VacuoleSicsovis senkons save gave wees Hae ak, calendar oeae Raa A

pleomorphie and involution fornis; capsules, Cte: cs <5 spare cesses dese ong mriona nb eytiede + pmonseoedad Gumgen dene gma

iy MEEVEE Yi spac aca oee ru: eaceed avn in. Sens te ehegee bE Sven GG c Fi bcd, a a ROU AS oe leisln Sb Soa E EIN eR ace oeet is REN gee ERNE Rata acted ark eens eto hota es as Aes Ras

PHYSIOLOGICAL CHARACTERS.

Relation: -tontelmpe natures ssciss. aeinusesun tip. dyna vk facectcintustthrashentgs rusiesols Be gous: da lidlevateya) Daluliae aS castin’ uageen ede aera encase na eie Septevaue eAalowe es sewertey avant Jak dvadunt ddeuiye. stances ieuysieesnecmaratian ae

Relationitd freeiox yeens ace zak ve eeenes See ada Gia va nara gs We iyns dena Now lee oa eal daielneany ue ES ae aa emmiRauia 26 a8 Satine es Lig ae seen Gee Ae ? Sette onnans

Relationstoothermagents,.Suchvas®: jog) .g.z aces cee av euetea oar ae monk aateteraiad aaa arate Sat nnc ds aieys ra Leer aad Per pS Me uae INe Daten eager : Garant

‘desiceation; lights disinfectants;; €tC sxicuhe tay set aie Ae Uo as ess te osetia seg aANa a ainpene eee a ciubatyc wis azsaevagen nd Soehear avcimienNeeoiore hae a beeen

Pigiient production x. save snd syd aim on valida caine wlecewels pape tate ones aaeakiRa le Aoteadia ya nal hhey ea he neulie aba e nee Ae Ye ge gee tenets

Gas production in glucose media:

Gs Shialcerewl EU] spose ets cstes cnerecs eaters nh daa Ee Se a pie de hee, cia eeeaileindl elena

8. Fermentation tube, growthin: (1) opemarm:..... .........

(3)
(4)

rate of development: 24 hours ...............

per cent., 48 hours.....

FEAaction An OPeMAannss suiansd snes odenh wehraGavat eons UAahA Whe de nena

een Se) EVISE AMIN Gil wos w be Opag a be baoRe eb mnd ae Saad oe Oy cad enad aoe meneame

...... per cent., 72 hours.........

.......(5) gas formula, H:COe::

....per cent.

sia tees HOULSiss 36-6

vess seer cent.

Avid or alkali production, [ities MANU sacica ce ox a: eatrnn wasn we on once aus sndneGe Hs Oh 4a Hi Bedi causes daakave %

Reduction ofnitratess tomitites: cose se ee apoio eee te eat wad eaew Sede cat

Tndol productions 24 hours... voc. ceni veneers

fecal odor; 24 hours

Enzyme production? proteolyti¢cscwies.: vest veces ec sueenan pees Gene Ree fees dae laens

25 ABOUTS fcc vetat aca tee amas

....., to ammonia... ...

Characteristic od otisc seston es heck sas bees Gigs Woe ahne ar aredan bla yeas oa da beteue tte oe

PathogenesisSins ics cess: th eonoestuedae 12 Seek Verge ee ee Siemens arenes

iduning ee GAYS seacia tintianieasens eee ein eae

ee rueya 8 MOUNS stein tineag eet hae her danse la se ee AA loan gee eave trie ended rains Gomme LAYS wear Terai waives athena satel

OLAS tALiCs je: sisemieanetnise siombctanes nt wdc wh ke nea nan soaNOS tanto na Sa male one

’

Medical Bacteriology. 121

B. influenzae does not grow on the ordinary artificial culture media but may be cul-
tivated on agar slopes upon the surface of which blood has been smeared. The blood
from man, rabbits, guinea-pigs and frogs can be used, but that from pigeons is best.
The blood may be obtained from a needle prick and spread over the medium with a loop.
The skin should first be washed with alcohol and then ether and the first drops should
not be used. The sterility of these tubes should be tested by placing them in an incu-
bator for 24 hours previous to inoculation.

EXERCISE XCI.

BACILLUS TYPHOSUS Gaffky.

BacILLUS OF TYPHOID FEVER; EBERTH’S BACILLUS.

First described by Eberth in 1880, first cultivated by Gaffky, 1884. It is found in the faeces and urine of typhoid patients.

REFERENCES. Eberth: Virchow’s Archiv. 1880, 81;58 and 1881, 83; 486. Gaffky: Mitth. aus dem Kais. Gesundheitsamte, 1ggq 9.

B72; A.

369; H. 223; L. & K. 166; M. & R. 317; M. & W. 141; McF. 366; P. 402; 8. 337.

MoRPHOLOGICAL CHARACTERS.

Age of
Cultures.

Incubation
temp. (°C.)

SKETCHES,

1. Porm:
Ms BOUIN GMs nize bse ables Garner
6. Agar
CAG CLAW ecnctecamaratetheke uk Sanwa sone enon ant
da, Other media....
De ULC sashes Okey et sae nies
3. Cel] groupings........
and arrangements .
In GROWtHS \c.uies can ieee Bow Ane Sader ere atee & aa ehs
Ge Stain POW ETS} de soie yooh cieomno ia gies ae RAGS Ee Ce
a, Aqueous gentian-violet
b. Loeffler’s methylen-blue.....
@, Grain’s Stalin seg cnes nevi wees wees as
d. Special stains ..
5a. Motilitytirs cgi ccaees

a. Character of movement

6. Flagella stain

ie SOLES 2nd a ennnracn cate eno ce RIGS We See Wate ete Nea SoA Sa onian eae soo seni aide ge tones

7, Special characters, suchas: «021.0... -48:
He BOSIOS: VICUICIES 3.08 ous awneis sea venwomwy as yee nen es eenieeris% wees
pleomorphic and involution forms, capsules, etc. © ......-.. 0.00025.
PHYSIOLOGICAL CHARACTERS.
1. Relation to temperature: .........
2. Relation to free oxygen: .............-.
3. Relation to other agents, such as:
desiecdtion; light, disintectants; 206%. .0000je wi arti ceeds tensenep maser acids oe ewan 24 aedeloleagy batee be bee
4. Pigment production:............. _
Ree res -
5. Gas production in glucose media:
a. Shake culture......
b. Fermentation tubé,.growth in: (1) Opemsarm? oc. in coonce ny 25.0 ona Beek ncad ee cae (2)
(3) rate of development: 24 hours........ veeveee per cent., 48 hours............per cent., 72 hours............ per cent.,
(4) reaction in open arm: ...(5) gas formula, H: COo::
6. Acid or alkali production, litmus milk
7. Reduction of nitrates; to nitrites ...... cose. ese enseiein eee
8, Indol production; 24 NOWTS «20.25.4024 o2 sees eo a eis oh , 48 hours
fecal Odore2a HOUTS sc. wa ccnmorcnseeresd een ne ries (onal Gina Seer NN css ie
9. Enzyme production: proteolytic... neterline tates
to, Characteristic odor.......
11. Pathogenesis ....

GIOSEG a Pini acerca: Vode aenteoign Ha anise :

& Sep ibO- ANIM ONIA wens le ee A Be

per cent.

CULTURE CHARACTERS.

Reaction
of Medium,
Incubation
Temp. (°C)

a6 3S Hours.

SKETCHES.

(1)

Gelatin

plate:
(a) Surface

Colonies.

(b) Deep

Colonies.

(2)
Agar
plate:
(a) Surface

Colonies.

(b) Deep

Colonies.

(3)

Gelatin
Stab.

(4)

Agar
Streak.

Potato.

(8)

Bouillon.

(7)

Special
Media.

[123]

EXERCISE XCII. BACILLUS PESTIS Kitasato and Yersin.
BACILLUS OF BUBONIC PLAGUE. ¢

Described at about the same time independently by Kitasato and Yersin in 1894. Found in the buboes, and occasionally in the feaces

urine and blood and, in the pneumonic form, in the blood.

REFERENCES. Kitasato: Lancet, 1894, 2;428; Yersin: Ann. del’ Inst. Past., 1894, 8; 662; A. 292; H. 259; L. & K. 200; M, & R, 437.

McF. 433; P. 606.

MORPHOLOGICAL CHARACTERS.

Cultures.
Incubation
temp. (°C.)

Age of

1, Form:

Be OWT ca oe Sur sa vencnlene Wpeied ceded 24 Ma eG Geen ew te a we ety Kr oe Ga as red Kewanee ¥

DU INGAT si ahcnee es

SNOUT ae ay cs darrian a ic ee es to ae ain ace Seo GAR Men ee a Hs PONS GHEY AE GE GUT 4S GOOSEN TE HATE

d, Other medias. sc cciccccaccs:

Bie SUZ Ow son ereocpenin ne Annie a Ah cree late NAA a tap rolep Nk tale Aten Ree ae lcci ta Thane Lanier had eal ater
3 Cell BLOUPINES ashes cecke rs hs whe taming tren ae aes ear asaya GA ae utah AIR aEe a MAR tinraedun ik WA ered
ANCVATHANIGEMMENS 3) 37.5 e asain den, G aaah mayne ELIAS LULU fv DGG etapa ease lvotepepeciaa alae cates gs orate - seca os toes ee eR eee

AME TO WENS see ctcreeeciseigsiins veg Sims wrrcrican wie iehasie arnada ne ako etme eRleela sea SG Ad Heloa as wimenlg Ge ymecealove at etd esata suai ah BE Meme en

qo Staining POWE Sins sn sacks auton cu uy memes og ends

a. Aqueous gentian-violet.....................

B -OCMEer Suet nh Vle d= DIU Cie ce 3 uiadarctiee ato Se ree uss actvale igen ak Siecaeltes eredsicd aed pacpauaes Saree mae ewe mele

EMGTATI S| STAIR a aon «knots ane nace mene eenetenes oan ieee aneeer name

d. Special stains ..............

5: Motility: vss ex cneseia nes

@:, Chara Cher Ol MOVE Mie ME se arash carers Moa aaa soe har acAl Cog ca ae es dtc ih GPs tetha Hit sg ma ete Pah weer vocals creche hems ans testes etalon he

OI SPOLESS assis skaie ohrsrnaenns ono

7. Special characters, such as:...........-... cece eeee

GEPOSITS WACUOlES mache AM agiias om ARAL Se Toei Set ead nec eee eel pte day

pleomorphic and involution forms, capsules, etc...............

es |

SKETCHES,

PHYSIOLOGICAL CHARACTERS.

t.~ Relation fo températuresiccas os nce an Ghat saaatanoatan

2; Relation to:free oxy Pens c6 oscar can czinoans sees exec

5. Relation to otheragents, such ast svc ov ye nd bites sae ea ssn Gare Se ean Svea

desiccation, light, disinfectants, etc.:..........0.... 0.

4. Pigment productioniza. ccc. usceaeseis seas cada Hee awe

5. Gas production in glucose media:

a. Shake culture ......

é. Fermentation tube, growth in: (1) open arm:....

(3) rate of development: 24 hours ...............

(4) reaction in open arm:.................

6. Acid or alkali production, litmus milk. 2... .........

7. Reduction of nitrates; to mtrites........ 0.............

8. Indo] production; 24 hours

fecal odor; 24 hours ............... meee ta ae

g. Enzyme production : proteolytic ....................

tO; ‘Characteristic: odotws< a: ix cacneaimotenese aan

ir. Pathogenesis ........

per cent., 48 hours ....

Jip 4B NOUNS sors Sa hs ares oe ee ireeeae oo lhok onsen

eek WO WNOULB acerd pricey esd bps) Mid ee ans

+++. per cent., 72 hours ........

sees... (5) gas formula,

++...) to ammonia.............

occ hours... 00.06. per cemt,

CULTURE CHARACTERS.

Reaction
at Meginins OA. she: Hours ABs ceed Hours. | 6...... Days SKETCHES,
Temp. (°C)
(1)
Gelatin
plate:
(a) Surface
Colonies.
(b) Deep :
.Colonies.| ¢
(2)
Agar |
plate:
(a) Surface
Colonies.
(b) Deep
Colonies.
(3)
Gelatin
Stab.
“~ aN
ri \ 7 N
. , ALi 2
Agar | \ \
Streak. \ 1
y
~ (5) A
f
/ \ / ‘
\
4 \
Potato. ;
‘ =f \ y,
(6)
Bouillon.
(7)
Special
Media.

[125]

Macy

EXERCISE XCIII.

BaCILLus

BACILLUS SUIPESTIFER Kruse.

oF HoG CHOLERA.

First described by Klein, 1884, first cultivated by Salmon and Smith in 1885. Occurs in blood, organs and intestinal contents of hogs

suffering from hog cholera.

REFERENCES.

Salmon & Smith: Rept. Bureau Anim. Ind, 1885-91; H. 269; McF. 413; 8. 413.

MORPHOLOGICAL CHARACTERS.

e of
ultures.

SKETCHES,

Incubation
temp. (0C.)

t. Form:

Bs (BOMMIN 0 Nsasssae aes wee seers tociolstiets ota avore

OS NLD oo ssc yc scr saveo ks Dhar BS UE Foes eevee dig ONL TS De AOS a aio Sp aN Ns Sh iiSS GUNS Shae ISV ica atch BE TRIB ae ed GI GSTS TLE STE Tt te eo caesar ae
G5 Gelatin. a sists casa) crane haan cutee de sacaucgame @even Semeeie ween Semin Lem he Keene ve ee enon inige Gu umbend ys SA aRlenia Asani amas eeu

GQ OE OE We AIA oi 45.5 Asean 2 a eat ot cecal naire veh 0 ats YO a CONE eA? Sd AE AY SHRI BAUS eR OR AO Neda Nal airilon i Qan Se ceaine Ba sxeeiains aves

ANG AT TAN SEMEN US = rccahae dees canine J suo te ate ONG REQ HUUAES Sen ued (adr nd LE HEGRE: OS uae Estee aeG LORD abl SONGS SOUNDER EES

Es SUZ © rcicyz ng Sib pastries pea evecare bes ncn re eho Remeclore isl padentenege rau cheney seat

Geligrouping seahorse rake heey kale Anne ey DRCA AE durable Re eh aed atea A en EE Red eT. andeteeims

Ti SLOW UNG seated 4.5 oenpuiet atest ganna heen aatue nk uiitea Aids chaos uh as

. Staining powers:

@, Aqueous sentian=violett cc tee. we aaa acane ws Ws Be eed SOUS CE eee es
> Teoether’semethiylen; DIU GC xvkatic. oats cctanaaiie aia SecA esed Vie mwa ae moog HH URE RE ra DAS YER Home Me ME Be Eee enone

d; Special stains. cic vsaasenys sts ataatnos pauiorrme ms aayates

w

@ Chara GteriOi Mm Ovienie nitive sceg sie ele a 7a eee ae Se Ara sO oa el ee ae a turn Rane Roe Usa od rd SSeS ete ae ee

6. Flagella stain

D

7. Special characters, such as: ......

deposits, vacuoles............

pleomorphic and involution forms, capsules, ete. :. 02563 ce% vdecs eeereiw 25 08 vere ad Keene sis a eee Re dar be o

PHYSIOLOGICAL CHARACTERS.

desiccation, light, disinfectants, etc.:.........

Gas production in glucose media:

SME OUIIIE Veteran Sy-cca aon Beda dart tvaaslor aca apienene ene WA a nrctac re yeaah ia taeda Roo neue, Del aee wc heerte iy Wha te ee paar 9S Cn alae Und Decne mid | Mea Ne

Pigment produc Onin sate ioaGacnie dios women eink ae CATAS os eae:

GSMA KEIGU EU GE Ss a ead ah tua tie en ee EES. cae Soy ETE Ree

()

6. Fermentation tube, growth in:

(3) rate of development: 24 hours
(4) reaction in open arm:..........-.
6,

Reduction of nitrates; to nitrites........

NI

Indol production; 24 hours
fecal odor; 24 hours....

Enzyme production: proteolytic.............

Characteristic odor..............

Pathogenesis...........
bg ©

open aie.

Acid or alkali production, litmus milk. ..................

Relation: to: temperature ices conse acta ttsno maa aoe eaeinnedt wheat yuan wae AM ast nine) Beate

sg te G2) ClOS Od) arin 4 eee eaves
dbne bhe per cent., 72 hours.........,
..(5) gasformula, H:CO.::

eoery tO AMMONIA. 0. e505

pAB TOUTS A622 .28 pone.

.... 48 hours

AE PETICEN C5 a5 03 Anta ede

pi hOUTS...52c0.0.. percent

CULTURE CHARACTERS.

Reaction

of Medium,
Incubation
‘Temp. (°C)

SKETOHES.

(1)
Gelatin
plate:
(a) Surface
Colonies.
(b) Deep
Colonies.

(2)
Agar
plate:
(a) Surface
Colonies.
(b) Deep
Colonies.

(3)

Gelatin
Stab.

(4)

Agar
Streak.

(6)

Bouillon. .

(%)

Special
Media,

[127].

EXERCISE XCIV. BACILLUS ICTEROIDES Sanarelli.

First described in 1897 by Sanarelli, and claimed by him to be the cause of yellow fever.

REFERENCES.

H. 369; M. & R. 453; McF. 400; P. 609.

MORPHOLOGICAL CHARACTERS.

Cultures.
Incubation
temp. (°C.)

Age of

SKETCHEs.

1. Form:

BC BOUIOT ew aniadage pes macmansenolsaee yelctans iach a

OG NPAT aoc cee sink oF Sa Sete Din weened gram Daiolagae Dt oui e dina a ie MaAPA Oe AEE Pa Ne ee

6) Gelatin: 2 scevadiss cea sn Gav dae are as he pana nee ee

d.. Other media’ ciuwsseie sevens conncaei ehh 4 dated poset see ese Geis 23 ESS
Ba SIZ EH nse Nysins Bt yr OR AMN Be SAID Coseselvsrele cet clus le ns OS ubiess BMG RSMO RR: AE EAS tala Se ch ciadle siSaseiees ese MORE TG RANTS AS fe aeabites A STRUT INS ie MARR ima a A
9. Cell SP FOUPINES sos sie de as seg hoa teal eaian mia degein sy BASE Da GRAN EG ea Rae ae et ome eee REA Merelniad Ge dante aie tens ine th RATER Sea
and arrangements..... 0.2.0... eee nce tee ee eee ete renee rete teen ttt teen esas

UH GOO WE ss conde noes, Sadat ecsvautense or ta donee aie ie Dees Fs LEE Be eae aaa Aes ERM RENE Oy FFs BERT POTTER ES Keene ets

4: Staining POWESrStins 1g earn by ek chin aa een en se seas

a. Aqueous gentian-violet ..................5.

6 Tooeer's methylene blue ice cy os oes cccsad db ayo ng a4 po bak bAGW ORT HRS ENE ES £4 OP REHAY Bs Lids eRe RHE PN ROR I we Be mE

¢; ‘Grami’s' Stainless ecu saccuaranwrn ares

id. Special stains: sys os weiniv as ood iey sa newer sony sam aide ne sa dhewenbiiies eiteeehant as aan Sipe a auereadlane it aac Ginn Se BSNS Bee 08 Gate wie

wn

a. Character of movement........

6. Flagellastaineja. co. 2 secre ne acne ssneey

a

o SOLES Hs. sp hackaisaryoee Sasa ay

N

MEPORITS; VACWOIEG:s .1xeaye us ains a5 waar whancdiqude se ce nemsweare peeAntee eee aaws

pleomorphic and involution forms, capsules; €t@) veer ieee ove ners cane oaciea eo Cho) Fee aE hen aed, Sakae se eee

ve MOREY Fay. i6 5 dock coi d dadeeode vs a weg oan tek Rei US Gio aes A ee Se eE TS ta ee eH een sea ne ted RINE AE RESET REE HE De etek ee

Sead eR ARLEN S, GUO HE: an ene 7i40 thay dole sneebasphred Lede seme sae e dnd ebat hd af deded Lap euhtne whe as RANTS

PHYSIOLOGICAL CHARACTERS.

1. Relation to temperature:.....

B. “Relationstortree OxVRe na eri eons ornate sae Saami rea chen ish Me ot aalataade welatdedascrinee srday: sun ieaadngd «ttle atin es Pa ntaRMerag aa ]nay aavbIa Bedale al Gmomrdle dip Asie weebuaiae Se Mea madnve nine seeneles

3. Relation to other agents, such as:........-....-.5

dawcation, Ndt, disintectante: Bhetentac 52s eantn ned wher sine abasted aidanmue smi ed toate ol end Mee elas phe domme we Rates Sead ere teas ‘i Saat Rs HET ale TON

4. Pigment production icc: scagercesess cow

5. Gas production in glucose media:

Phe ESTA IGENC AI IIE LDL G ictadare eat re onc cia ec tne Shae 62) SRG DY HIG ROSS NE RAE TRIB REA te i aiaitls eV eo pst maa ms ae nee nee ot eters oe Gita cee

b. Fermentation tube, growthin: (1) open arm
(3) rate of development: 24 hours ..........

(4) reactionin open arm:.............-..

Hakka em ania, deManatiee its selina amen C2) CIOSCUUATINE oer area aaectac ot ewline ieeaaacemnies Weaken es Wineineae dye Danan ad anaemia eta

outs PET CONC; 48 NOUTS, cece ec s PEP CONE. 72 NOULSs 2. cadeacess se DEr Camby, aance ones

..(5) gas formula, H : COe::

6. Acid or alkali production, litmus milk.... 2.0.0... 066. c cece eee e te eee eee

7. Reduction of nitrates; to nitrites ....

8. Indol productions 24 HOUTS singe. ceeer sees sph bee eet ee

fecal odor; 24 hours ............

9. Enzyme production: proteolytic.........-.. 2.6.06. c eee eee eee ee

Toy. (CharacteristicOdOre. aang cessed Sater wie ans ae Oh Bs Ge Ae oe Sey Ya eae ela Gouna

Ts PathoOpeMesisss ras ose.ccnen ss mateacee pdson winced hain Ho Lasid tke ebasninlce demmalnsahen Ayes

s deer OFAMMDIN OD La siete. aati seasrccesitic.c aonaaeate xi rlonuae sam euscercns osama eet te ate meahons Bun Cento mag

FpitdORILOU TSA Re nenery as aes e a uagohet rine deel ink ht Aaah teen

sheng .., 48 hours

AGN AS FAL GE Sens ewan enciamine eae

[128]

vee eewshOUrS.... 006.05. per cent.

tele eds AVS! on cigaruansa lca ssanemaien telahye
saiera SAVES a Saath fone Soa as Se 2 ars Sh ayS ues esee de auasehet ie) er Sas a, martin eae LAV S A cgeciean is enwmiegeete rae Oa

CULTURE CHARACTERS.

Reaction

of Medium,
Incubation
Temp. (°C)

SKETCHES,

(1)

Gelatin

plate:
(a) Surface

Colonies.

(b) Deep

Colonies.

(2)
Agar
plate:
(a) Surface

Colonies.

(b) Deep

Colonies.

(3)

Gelatin

Stab.

(4)

' Agar
Streak.

(6)

Bouillon.

(7)

Special
Media.

[129]

EXERCISE XCV. PSEUDOMONAS AERUGINOSA (Schroeter) Mig.

BACILLUS PYOCYANEUS OR BACILLUS OF BLUE-GREEN PUS.

First described in 1872 by Schroeter. Found in green pus, and is widely distributed in nature.

REFERENCES. Schroeter: Cohn’s Beitraege zur Biologie, 1872, 1; 126. Barker: Jour. Am. Med. Asso., 1897, July 31. Jordan: Jour.
Exp. Med. 1899, 627. Lartigan, Ibid., 1898; 595; A. 287; H. 138; L. & K. 120; M. & R. 170; M. & W.160; McF. 197; P. 535; 8, 454,

SKETCHES,

Cultures.
Incubation
temp. (°C.)

Age of

MORPHOLOGICAL CHARACTERS.

1. Form:
LEV OMEN Te pcs foe eS euSeGe ay Sicsles os See e te op n S EBL AS Coy ea SUSERN ioe oe eee as a do In MONEE: Nw AE eg gONaioa cada RES SI RAIS, AS ed Mer | ite det cree

Oy AGAT wate sabe eee eaden 35 He a ae Pate Me ae aes Ga teehee Lae a Aearan Reais be te Waar en Mare tare wu makes os REM eREN lees Tale ee desea

es Gre] Bt Rieae sc tecesee: armeatieted obdk GRUsnG ge seeibitis, we Weep seereaasatit racers Sia aamcdeen vera metndent, Sanh .G Mee ve ore east cra mals oe wai vdageees alos ad ncoereed dees ety bbec bi ceesah RE NS aaa
@ OUMER INEGI ia aco hee el ale ats eee weit Nee a ae lanl a RE le ae eld ete Ces as del doe pe Mie ide cig (aetna esha nce [rae setae
BEGG OF SA San SSID Bette cis GeO ty en gk ia aa Secs Bk Bee AAs as Cos eee SUES GIES ine OR OI GRE GS USA a act Ae eeh aad ate Nacesh inate g gstiissne e/a schall anereaes ke ses ee
3: Celligrou ping icc axes cree weiner ae cack tae hag eke ae GeReEe PaO RE An emkle ae PRA an sem ee Anbiee wien ries ie GWE | Toe a Dowel ee de be
TOWNS cs Se ee eee cert, Se es oe acaiiin Ne Maton ase had wh) ge nie ee a ee ei a Cae trae oe ah 2 gies co er Avra eee Nant ha a walled ae Be
Ae Staiain Ge POW ELSE A oa sistke Seale wekepeEncy nee ee SSC agg LAGU Sena vecded oe Sheietoaul ada yav oe Oe tee inde ne eB Marcos Ae ve in pan cE eal aed oad enced | ceeectonsaqall wees eee
ae: Aqueous pemtlanviclet.. cc: 1414 sewn c2e ween nce ws nae aA SweRte a He wn ee BH CHE MRRUR Bs a oadew ROEDER PEIN Sb9E Kee enwaeeee [Eis dade
0: Woether sine thiylemeb] 0 Ore este ve tunes vec an eich pean od He oh ate nm ende an wn achat Sie ne we AAI S ae en ee ene nee eens |
GAIGRANUS S ba ls sn eedeto ween uk ee hee ene a eae ee, einai ae aethiops aie ape wang ai gumitee adie ce ote nmaathscnds ga teatgelg Auto a udebek Seb [ors ae ersrceall eyua oer

Me SPECLALSbAln Sis porecn tae eomeemine eR eibay Mee cecas crise eum aN nee ease iceashegs Stacia asa a ae Ale Sa nt ua Rg Oe Bou AthA Ma Bee. Sater

est IMT Ga UE AM ye 5 crit Aces tees nb acs a aot ed eat y0d eeepc Sy arb vend eicnealo oa Ne caaeee dah ADVE IA Stargate A axe Meee ORE

wn

@. Character oLimovements +. scnucacieaninet yess as auans poner the aenmaadiennets

bs FlagellaStainie. sedis ocwamavecs tetas eck wet wattn< ste sennieae oe wee Maes wa meee se a ae os Saeed TH BRAD ap doeewod Sowaean bt aba be

a

pS DOMES a5 i 8 Sees toee acca cae SA Sa ne etn eh a ys ca NT ular ad She nt ea oA Gao GG, Siceatan teh yaa Oa eevee eee

7,, Special characters, Such aS? .aisiaze cd aaa a gs Riad Re ves Mate We aioe tp oe Aaae Hawes eee Males oy Babee es

GE POSTIS . VACUOLES Hau tts camancresss cece) a cathein a edetanirante Sas meninle Jk innaeteheyae law Beek Gave Wemaie as aoe kms

pleomorphic and involution forms, capsules, et . 0.0.6... ee eee eee eee neces eee ee ees

PHYSIOLOGICAL CHARACTERS.

nr, “Relation to temperatures cn cie: cs ecerae caise-wads cine a Semana einen mieten as a heemin ae aie ay ais Rina en Adele we ed

2, Relatiom tor tree Oxy. Gels o3s ress soos eh auras as HEME ee a ane tne ela et ia Rh bee GB ee Tue dk
3. Relation'tovotheragents; SUCKS: giv daves ica pers eitele sd demhe Bod saad bee TH Teena tue en
desiccation, light, disintectants, etc.:.......... 0.0.0.0. cee ae

4. “PISIM ENE PTOAUELIONS:: seta nerg ov akan gated ean saa ace sh on vemes mn epTAhe ead vip eal Ze dan ad ge matnnee

5. Gas production in glucose media:

: SAK Cultures, cc tasiGs cna cnetic ne me maniie aeteeortema wae Ha Waiay saa aude arom lng RE AMY Wee eed oe od als

6. Fermentation tube, growthin: (1) opemarm: 0.2... 0... cece ee cece ce ee (2) closed arm:

(3) rate of development: 24 hours........ ....... percent., 48 hours..........., per cent., 72 hours............ per cent.,

(4) reaction in open arM:.... 1... ieee eect et eee teeters eee eee ee (5) gas formula, H : COs. #2

6. Acid or alkali production, litmus milk ....................-2...

cure earn hours.... .

" io nitrates; to nitri Lilche Rasa kas eee bees A
7. Reduction of nitrates; to nitrites Peete cee eee eee eeeeeee vey CO AMMONIA ...........

8. Indo] production; 24 hours...... At ae Se pemsea ues ae ea OG HOU ES skeen tees cE Bed

fecal od Greed NOUTS sss a: -cia serene rauiy wrale-eesacnay MO NOUNS) «cx udesie sae gece

5» Enzyme priduction: protec Pits cic coer eiony es neeee ra eenees¥eende Gey ade ce Sava AS CALLE space ws anes nichancetien et ti

10s.) Characteristic: Odor acscenaied tended caede as ve letlondacurd calemoboe nous BSE RUGS eacuimauode moe al ndeaccen

1%, Pt CSS sete sesh tote thes pu aoe PaO RP eon tees Seat at aaes =

CULTURE CHARACTERS.

Reaction

of Medium,
Incubation
Temp. (°C)

SKETCHES.

(1)

Gelatin

plate:
(a) Surface

Colonies.

(b) Deep

Colonies.

(2)
Agar
plate:
(a) Surface

Colonies.

(b) Deep

Colonies.

(3)

Gelatin
Stab.

Potato.

(8)

Bouillon.

(7)

Special
Media.

[131]

EXERCISE XCVI. MICROSPIRA COMMA (Koch) Schroeter.
COMMA BACILLUS; CHOLERA VIBRIO.
First described by Koch in 1884. Found in the intestinal contents of cholera patients and has also been isolated several times from
a water supply.

REFERENCES. Koch: Berl. Klin. Wochenschr. 1884, no. 31 u. 82: A. 401; H. 244; L. & K. 181; M. & R. 402; M. & W. 152; McF, 311:
P. 568; S500,

SKETCHES,

e of
Cultures.
Incubation
temp. (°C.)

MorpPHOLOGICAL CHARACTERS.

Age

1. Form:
iG BOWilOiy was cg cc soe yeas OS See ae he ee Pa Be Oa Ba a eed B eSina een eee dd gt CaS Glee Bee Ga eenin ease ametinnl couse. | eee tate
Oi INE 4 cacteteeieie, os aisha! seatey tao anbeana ee igh dlab aA Gaba RRIpRG NE Eetetie Andante tex SNERSAIN aa apendanaeht ohana ies: paslchalleA sas dentine alee la dai gos Be Rll area tnten PRONE TY
Gra Gy CUAILING, Se oi haart Renteare i ea tenc Bossy ue eants Seo eee Magis De kara eee se ae sate aman Se ATE Oi ahene He Shiva raae ee Sethu en oe aoe RS ene BEY ainte | ebalgtaa tell oases, teers
d, Other media........ sogcad ae ssyclaanacheg dete awensasie/elalabrornict ens nad akotarata hedslederaustewa otne. ev nah «i PeaveNslanvie Sbeuesayes vy ara ceamcorei@ cneoulntbaane aah hie rh deere lg eave wets | ones peer
Be SIPC wea ath Bg fG aA ge eae Bo Send aw Gaede be bee eteN whereie De aecaicermie se Re ey eth ee tees om Bi etic Sodiart eee anaes ae bach ae
ANG LAT PAMMEMENES sy5:3 ce wes ca ssralind Mee eA PEELE eR RAD AG BA Hees Mamet eae SNe ae Hoos amNnS 7AeR RAS Wa wean |anoen as Hallsen cen

THER OWENS Art ar clo elae es Rie laa, aie sloshed on iereneevnstels La iseuclaiae emits

s SLAM. POWETS! a4. as Ghee sraneet se wa xen ese oay

=

a. Aqueous gentian-violet.........

Ox Thoefilerscnmveth yle ac DIl es seve hic aon ae ett et acs ersutee aaatayannabarsyeieptadia laa tebeabie im a apn weaieaoea rebates oe asad ae esa aisle Ieee

@; Special’stains|: 2s cecissa de oeebess

oped) Caviar pins Agree eee ae is rari CONC Ue erent CHE ce ee nerds eri tures Tee Oe nC ani err ey at Mean ae eeeargre ee ee AE ACRE Oe ee ae ere

wa

a, Character of movement ....0.0.. 066.60. cee

bs PlaeellasSta nna sams cas tacen ar aura celeines emacs ake ras Peaasereheacnee

6; SPOreSies cerns

. Special characters, such as:...........

NI

deposits, vacuoles ....

pleomorphic and involution forms, capsules, etc..

PHYSIOLOGICAL CHARACTERS.

i: Relahionecto, temperature se... sesccss opens se neneeta) asieewiendase vaca ee

2. Relation to free oxyrens, vs j...e op ee kann
s. Relation to other agents, such as:............
desiccation; light, disinfectants, etev.c cocci vesagaeccie vase cans
4. Pigment production:..............
5. Gas production in glucose media:
a. Shake culture ......
6. Fermentation tube, growth in: (1) openarm:............0........0000... ses... (2) Closed arm:

(3) rate of development: 24 hours ............... per cent., 48 hours

RAE per cent., 72 hours ............. percent., .............. hours per cent,

Qh) TEACHOMUM OPE ADM... nis ss tenses cca were ee tengigle de aacieaokamonnea(§) ‘gasfornula, Hi + COg:: : i

6. Acid or alkali production, Jitmus milk. .... ..0........

ao RedwetroiGl rintratess toaitritesi in wasce cha rcesg ewns area ean eo oats emumet aw clases iste, OO BAMIIG NLA a7 ah 45 ie ok od meaivee A amaaye nooale

8. Indo] production; 24 hours ......... ; Bis aol eerie GH OM MIOMIT Sea 4 <3 pads cichs Se urahe Me AINE anes catwikr Sashes Sita days vad

RECA OH OTS SH TOUTS oc pada rtd wt oe obey AON we ete ae GO OUTS ucinman ee meta ued desan cnc BaeretosAt eee ts days oe |

QO: Enzyire: production 2 prove ov ytie: wees sc acesveteneate ea ee ae ies aise dees eybri a tuectierareantCl TAS CALI Cigna wend renee Ue aetna ee

10. Characteristic odor..... ee Teese antes cues satan ily

11. Pathogenesis

CULTURE CHARACTERS.

Reaction
of Medium,
Incubation
Temp. (°C)

SKETCHES.

(1)

Gelatin
plate:

(a) Surface

Colonies.

(b) Deep

Colonies.

(2)
Agar
plate:
(a) Surface

Colonies.

(b) Deep

Colonies.

(3)

Gelatin
Stab.

(4)

Agar
Streak.

(5)

Potato.

(6)

Bouillon.

(7)

Special
Media.

[133]

EXERCISE XCVII.

First described in 1888 by Gamaleia.
bling chicken cholera.
REFERENCES.

Gamaleia: Ann. dl’ Inst, Past. 1888, 2; 482.

MICROSPIRA [ETSCHNIKOVI Ilig.

VIBRIO METSCHNIKOVI.

A. 441; H. 256; M. & R. 426; McF. 332; P.

Found in intestinal contents, blood andorgans of chickens suffering from a disease regem.

593; 8. 511,

MORPHOLOGICAL CHARACTERS.

Age of
Cultures.

SKETCHES,

Incubation
temp. (eC.)

1, Form:

Gr IOUUNOR acdsee his Raate aiiyerselor Miseen ears terelercion: Maney beta nee

Dep E Se ese ie eae ae Ct Net f20 ACES (Rs VO SBE Cpab ge A Senco cSdencps car LeUI yt Aen na aan ae ep OLS TA van MeN ACE EG

Bi AGG AIAN aces ie enti dal a clea ipa sam ced sa 1a Salepluninns AEN He yiwd Honed eemea ee ey aie YA AY ANIERS oA RNR TAs ERAN DEE VA Ge

Es MOTMG reAIVE GEA sec sects st Oth as eOe ce teased ys Sok ee ctcoee oh saan eae REND cyanea ane ooeraiyee?

2 SUZ Oh bach 4 Sua ha heehee eo ih Ratuar th Oee art AS gets nade enrane aie ate eeemtiae ere once

3. Cell groupings...............

andvarrangem Cmts: pay jecatar eke yale satis wwe taclaan seid ciatchudaed hid (aiaute AE diesud Gaihardaga inva musik tives DING De Heh ae Oe

PA ES cass cesar oor arcoreivas od Oa dud Sh aC SaG wh oe hekananea needs Gialamas oa wameua ane aees Wau eaunoakmnrrad aus ya waeeue le canoetaslomue ass

4. Staining powers:

@: “Aqueous Pentianzviol@ts scacerecg. 4 Adee oq deaunee ie bee aed em tame erates eee
ic EAOIAT Se AV ler ieee: aye ay sek sacha acvans ah bake Sabie Spaced Ge GP tama mtGh eee Robe Aa Ee. Due eae Aa naan elas

(Gh Garten’ SIS CARMA Zeek cecisecatss saat bliss’ cgetchd SauceesessUs %/e lence gd RAINE sects cpcnaenytcidesoe ard Mb die a rupee izes lesene ete Sums

A SOPC ClAll’S CAINS says seiavasteaceesi aes naas dam taneatrea relies

53: M Ott y sepesmaniis vrldeacuie we apaniamncelos a muamee rey tse ae Guay rama gs dann ee eaten nt acre intial eh de els ad hs

Bs AAS AOE OS Ti VS IS Ug ales crea acai case oS ih Gach A BO Dank Cava Ad ty OO oer Baa eee ren

bs Plagellastainy erg ieacansy vhnoae ab ranks cdoeraaok

7 soPeClaliCHatacters Suc hv Ass wuseieavaceacvee uses Se untan deep acca ol Lee Gacy dendis Sa cae ea uaa SR Mee Rhee eke aR Bara MA OeeNT EE

GEDOSIES WacWOlesies creat sie. neta ete lott ot on a cmt n yaaa atari senior eaegees teeta tadaeds aioe mie eae

pleomorphic and involution forms, capsules, etc................

PHYSIOLOGICAL CHARACTERS.

1. Relation to HOHE MA URE S12 5.5 3 leaaydiach rose were. 72 Sracla ftv elton Lenser hein nes nvacais oe eeeeeded cata aitayeeaul ay WS ete Bate nme aoe leroy en i LT Oey i oa nate iaveaoee cyan gs jaye elo agle

2: Relation tontnee: Oxy oe tis inamaron ee dete eaves sa aeetonnre Sx tages Giga heveeats Stuse ates sseete ant Getcha wees Ad Divan aiee tip eccnae

3. Relation to other agents, such as:.................
desiccation, light, disinfectants, etc.:.........

4. Pigment: production tis sc crweiids 2 seoctoneis aoe anaiy on adeve ¢

5. Gas production in glucose media:

i SNAKE CUI TCL aio se ytd atow Peta ea Aw tis Mysto Guest vais vate Melaka eee wn by daul eae RE open ca ek OSS

6. Fermentation tube, growth in: (t) openarm:.........
(3)
(4)

6. Acid or alkali production, litmus milk. ......

7. Reduction of nitrates; to nitrites....

8. Indol production; 24 hours................

fecal odor; 24 hours...

g. Enzyme production: proteolytic.................

to. Characteristic odor... scckca2ns2

Din PAthOgemesis; ocreenscinne uncer eau vamos:

rate of development: 24 hours................ per cent., 48 hours............percent., 72 hours.........

Be Set (GO MOUTSH Ain sendadae Renton eter cee ene Con eae eye Se aE

tk geese Ov UMOUIS HW ssster ih tes Desde aycaeed weeine deaeeuae Sie tadetay

seeks 2) closed) “animes ctasnsca tins Aske seg iioty tid ) aia el selec ap momenta wees ee ea aan ee

vieeo..-.(5) gasformula, H:CO.::

ecceaigs TOMATO Marea netics cy con aya agate marcia wid a Seared dose ciauaatee aioli om ace aaa oA emne oTe

ener ACTASCALICS sm ease akon

a eseis DER COM si: atratinvrs sa ween

hours. .i¢ccce0 per cent.

: ayaa Mee ciel eae eee

CULTURE CHARACTERS.

Reaction
of Medium,
Incubation

-|Temp. (°C)

SKETCHES.

(1)

Gelatin

plate:
(a) Surface

Colonies.

(b) Deep

Colonies.

(2)

Agar
plate:
(a) Surface

Colonies.

(b) Deep

Colonies.

(3)

Gelatin
Stab.

(4)

Agar
Streak.

(5)

Potato.

(6)

Bouillon.

(7)

Special
Media,

oe

[185]

EXERCISE XCVIII. MICROSPIRA FINKLERI Schroeter.

SPRILLUM OF FINKLER AND PRIOR.

First described in 1884 by Finkler & Prior. Deutsche Med. Wochenschr., 1884, 632.
REFERENCES. A. 429;H. 257; M. & R. 428; McF. 326; P. 589; S 509.

SKETCHES,

Cultures.
Incubation
temp. (°C.)

Age of

MORPHOLOGICAL CHARACTERS.

1. Form:
Gs Bouillon awausisevameide do ae see swine eeaaaieR. Sak Baws SEARING ee apaieetn de aa” Sean Mueke Wey Roane aeamas Enodis pene eaemaaeal seat Mes sautececces

SING ainsi sds cy EA eRe teats Hise see gee yal mete SE ea eae onscieae Ata eae ota oe centcler te ates eo shctctae STO RAC PSTN STEM arcana Ne tee eo eeaehoc eas

FOIE AG ae eas ae ries Bae aes ve sco eS a GS eee ae REL. ALY ELAN ad ER bo Eivneion Aaeoaie nis Bagh vba htaears. woe [BEER eee Meus ae
Me UNCP WOU, wees aos cedn va se meuds un pacien te aa seas Wunee a ORME RG oN Ed EERE NERA » RAGA eRe Whe anane LARIEE wa Te pe Os Pee el eetA Hone wae
Bet Z Ci sceecta nicer cone oe cin Seeds maya yas eA AED we Pa PED Wie ORNL ES dec aga aS os gE REIEIG 5 aa Stan AUD CIEE S Sy DEUS AL Ue Gibal ene Te wl Esbestet
3. \Cell roupine sis Wnts ei ace tecacc an uty suse F sce ae bie uate weve uated otek oe A tiecion alec eaih bt, Aeiey ces amelie metaun a aetetia datcaierve hap val avdaa bebe at eet
ATG ATFANSEMENES 3 ye5 ios he taka ek vaca deey deve ee ones omusdod ate ptensteset vets ecard nl ot timeefeay tes -o fale Apalnldla vio urbe ma Ala boe arale ot iaie sel pene cese Seqleue Lae
in growths............. pr naan miniidsaih WRG AIA St rg Analy eee SNA MOU MRL ee aU ROR eats ang wis 38 mn aleynen aah nu Gen rd aie weeeneays gene dd 20 eesti le Rete eer ayn [[aaiem wate
ie SLAIN POW ELS Ss alee array vse cernrn FA sa atone ee ety ca aesases nee che a SG Ess Wo GER tae LamalEED Deuba thc Pualiash Leg bo bialh potbin 2 et ada tg eon eeneell Sia | I es
2 AQUEOUS: SENANAVIOLSE cada ciose Se oi etateyencesc causa oa co cstawc aia Sax css pots hee te ote avec eretorn ee omeedtiece minccsen lea arasteds sent cebe rsh cabocu on Shel uot meneesee
bs Woefler!s:meth ylen=b] ie cases cisscsaratcr aries oss ex densa htse/e cle orvdels Se age ctae vce uaeomc tare el aeons ar ang ate anna aD es ee iva apace So | esomee arate
GM GTAIN!S Stal; trois hctaucienlecos aalaectomisds awe cH reet as Be Sears ste Ruet ipsa Dacia antigenic ay Sloss cod OOSIEIRGG Du ceostaeeiouaTG oS SNORT DE AOE AH aR deat EE ROA teen ed
Els SSP ECVal Steam S oie sere ecco id asters eb cates ns, zac eSapahod Bs RSU RES Aen SeceUNU SPEIER Te RS a PE zeus crear ene spease vent lleeebsle nella enelstees
Cag) Koy a Ul Unenetena en a etesat See et ae AND RN dr Re eerie ne i eet orn oe Ree EET Ab A NAAT AAA SVN Aan eR Bre WRT AW rt] MIRREN EN Fen /AUA

a. Character of movement .................... Rib eseishe Parnes 1 ta Saletan Ue PA ga han Ouledz ae pw NP NOR A Min Ra Nua RS ws, UAT AT ta ie neetotU rated leat

b-Rlagellastaintcoiiila snegnen char games sigpmogs Marat aerate ganceanaes is, Nehyead 4 teiaitdim Gale ladieedethe eae sewed Meeieiote cee:

6, SPOTS! 4 asucesawaet eacdewe ay ak

7;.Specialscharacters :Suchyas? a. 2:c:mapiaisnaeina saetern ne ab Sh tie Gene Mi ae eT: gas eo ndenh ec oeinu aac ohekech

Geposits;, VACUOlES Hs a0 Aue auew dueuerviag das yaw sane add sige asc cone ea ek orn arena

pleomorphic and involution forms, capsules, etc ................. 00.

PHYSIOLOGICAL CHARACTERS.

1. Relation to temperature:..... ......

2:2 Relation to free Ox yeenthes 2g hoes Iwan okra Aue saeco te catia cher oats atsctt ia Rata alee ume eMac eer antec SANE Pe sg ne eas oe pe la ell Be Ca MEN adhe sul pasa

3. Relation to other agents, such as:............0.00.0.. 0.0.0...

desiccation, light, disinfectants, etc:........

de JRIBRIENE prod UGtIOT ein nae gts te Drees 25, clans ajehAl oul helena lc esa ae plore aacseenctOlaeoe nip ma ants

5. Gas production in glucose media:
Gs Shalkcevcul Cure ian wlan aia dare apatite Wes oe a sa uy mea WP A See Naya: Seo els Se aol ged aeiauce aie es eel : Sicon ah dasesa SACRE
&. Fermentation tube, growthin: (1) openarm:................ Hntni diame Serge yee na(2) CIOSEM) AMM x ciaia-sn comes vsure
(3) rate of development: 24 hours ......... .... per cent., 48 hours........... per cent., JANONTS. 152 ccaeee er wePEL COME. «cnc caseate saat hours............per cent.
Ca TOROTIOE I BPCN MEN on cu tea ah yieelng oy ov ears he wace waa an Gk patel mmc eho (5) gas formula, H:COe::
6. Acid or alkali production, litmus milk......

7. Reduction of nitrates; to nitrites .... SCRE AE ean Ae dat a STB AS Te ah ee LE Bane tus meets: GEO AIM ON Latins ied fos wants wa hoes Acute oa anes
8. Indol production; 24 hours................0... ae Smeets

ndol p 4 to AS ROUT Se csisseyirys sat cuca ede inewecng alent e staan ap iin can. 4 Hanis ceca tee AVS sppdatna drvalus. vaten nowt wvnces ies a
feca] odor; 24 hours ............ Asses BA Sees

4 s cto HOMNOUESH? sem sptcete ea giainnc das tan oue sciniyniee cau ndte, deych, Si a beinele ae dx GAYS che 44 nes osaneenekataues eee

9. Enzyme production: proteolytic............... Meeesecr asin Memb ea mS se couse mite. 22 CUASTAIC) inv awueg unis wasn aunartak wae ia dayaa tne wmean naventrimstan wah newest nate

10,. Characteristic odor: sc.0ci00 04 si gewaeeaaead ex

11. Pathogenesis..................

[136]

CULTURE CHARACTERS.

Reaction

er bes la Q4....45 Hours. AB cexpecines Hours. Bid.oaes Days. SKETCHES,

Temp. (°C)

(1)
Gelatin
plate:
(a) Surface
Colonies.
(b) Deep
Colonies.

(2)
Agar
plate:
(a) Surface
Colonies.
(b) Deep
Colonies.

(3)

Gelatin

Stab. kod ,

(4)

/
Agar H
Streak.

(5)
/ a ox

Potato.

(6)

Bouillon.

(7)

Special
Media.

[137]

Naine of organism

SOurCE, “habitat ete? cacy cena: wae eels cates es RR ee ea Rare cata PE nee eet Sphan Lee oR
RELETON GCG s e mrcemntnytiiag tice ae once a. i evs afew a aS Se Ran eee ge Jed humdi Re caesar ter se euioneue beeen
a | &
= sate
On 5a SKETCHES,
tr os A zs ~ me 3 3 a
MORPHOLOGICAL CHARACTERS. &5 | gs
A na
1. Form:
a. Bouillon........ BSD eb eee bad het esutee ek eth eee Seed ae A Se GAN ng eye Sacer cto Na le [avs nce esr aoe ae ate
Obs ANGE ABS Aoconsse a0 fa hanahestiat eshte anc pon theca Bache AVP Screed ped as ey PIA I alder one saleke Aha bakes ge extend cha needle et eects ae
Cie GLAND hd AAtynee Woh Rah WTAE LR Dea At hE GO ER eae ONE Syne NO eA bE Saget N a keke SEEN area eta e assem SG ADCS Ge AR re IEE | GE AE SS
a SOE IVT COLT Ase, reals aetna oe if MPSA Desehiea a ares tc estas aarp Pascoe errant batole leloyes AA Fas ade eenctts a/b: ap ua ees Sacynaaperaintagm ones sk aeeh aaa raas ctor | wee easy sad Ne S's |
Be | SV A Cr ssay sie Sigsu Wargo eee Oe Eat RON TRO EAR SG eR Li Seuss hse ead eect a GaN P RI a SUN CECA NOE Sete pePR. BV tee a rales een Mit seat dna Seni ts Se orale |
32 Celli PLOUPINGSa sy pacce ah any at waael Al wit dar ah UEd em seeamNe ree cleo sues Marin ant apa Rte PUTED Gane ee i ewe sepanaaatyi el Eee neato Oecd Mass ooo
ANNA SA PHAM PCTS MES a aacteacscaniriawetee KAM Gar caetsoesta con eberae as clecedzes neha ets Guna onthe aes Waseetc hata aon See been Seis hed es see ater aye sda et scaoa neha ana | eee seats | ea gusto
TNS BTOWENS Soaenntcii eaten la sts. ashen las mast ROhtas see seralin Senge Wk). Aaa PE Sea ernde encanta wei ed Syan ee Monriaean oA atr-capmd se nme Melle uve: @ oe | eeeenente
ee FAMINE OWE TS ites gas caeoe ie ae eaves eva teams se BEG Arata PREIS ae ees es Sah & cote Hy Wig Sapeenes I Ra CAA Setar ese llevan din ll cearten wal ayih il Sata Bs |
Gi AGU COUS © ENILIAN IGE bert ah SNS toe ae RS Eee SileinG tenia Mc Ratt Geiger eA Nita Matte seks ELL AMEE aA ANAM Sieg 4 salar th tll lated hecnnecte | neetiaiats
G6; soe Mer s:me tyler abies nera-s.caye terres ase Se seed serves seeyecsaecee aa a weaves tae eta pete iA Si IS gata ate anak chgtkivee coe | Mea derencte ase | feoestenctone
G5 HG WAT SESUA Diet tee. aa searhetnceee tat Races ee pees ate baa fad Winsiinsal Pi abewoel te, as vcedestoe ad aba nbn Pee ellaimitte aa as Utne eee aed imal de dis eed cetshs appre ist ace a ans waco lbs weg eee a |
id. Special StalnStseveasa sass pee evah ee ed. Kio Ree NEAR ES as uae oe eee a eahia sy wis alas eet tae soe ees eee ee |
BVO ERLE Y 22 icici metoene es ota Sa cia Ee Eada ak ak amie nel aas De RERUNS SE SDR TT OG Sah Some hadoe ALAIN Lis MA nis anata bmellts |
aaiGharactemofmovemient:cansiccime ant vaciath ob tarioty eae iid
&. Flagella stain ...........
Os SDOLES isg ceria te sarin oa a Mlertioae Ac etiam lntienie sit emai iantalm nor cianehaimsiehg ots odaas Teoma aan aoe Seman ex pee ener as
SES EGE ab a ene ee BER EE Ghee RA URE TSG Hie Sahat Siete oa calmed ioeomieon secon fides [Geert :
7. Special characters, such as:............
EPOSIES VACUOIES oa Nine 2 suice S60 A cele ebes pant gomenh Brag OE Ae taG as 6
pleomorphic and involution forms, capsules.ete . .. ...scsce eves seas cada ben sede even geek ouUydubyueeouteacas
PHYSIOLOGICAL CHARACTERS.
t. Relation to temperature:.............
2. Relation to free oxygen:
3. Relation to other agents, such as:..............-0000. 000s
desiccation, light, disintectants, etc.:..........0....00..
4.. Pigment: productiontiicoc2s7e20s tae Reehei ne cre cans
5. Gas production in glucose media:
CoS Wwallcere ail bn tetas tera is cent detent sas eens MG Meade Se trates yh eae eten Beer ne ee thee Ca ae ad A
&. Fermentation tube, growthin: (1) openarm:........ -seeeeeeee. (2) closed arm:
(3) rate of development: 24 hours........ Lo ae PETEeN EY AB ar Be err NG Sirs SL
3 p ent., 48 hours........ vas pemeent., 72 WOUTS!: ¥. 4.6400 Per Cents, osc-sccas oy HOWTS os ¥y vere wee per cea.

6.

NI

(4) reaction in open arm:

Acid or alkali production, litmus milk ....

Reduction of nitrates; to nitrites .......... i
Indol productions -2¢ NOUS «: sass ns eases veeearanties ox ve
feca] odor; 24 hours ....

Enzyme production: proteolytic.
Characteristic odor.

Pathogenesis .

-....-..(5) gas formula, H : COo::

.., to ammonia .,

Agden, a CHaS PatiCh Geddy ae ee os aan eicind ae

CULTURE CHARACTERS.

Reaction

of Medium,
Incubation
Temp. (°C)

SKETCHES.

(1)
Gelatin
plate:
(a) Surface
Colonies.
(b) Deep
Colonies.

(2)

Agar
plate:

(a) Surface |

Colonies.
(b) Deep
Colonies.

(3)

Gelatin
Stab.

(4)

Agar
Streak.

(5)

(6)

3ouillon.

(7)

“special
- Media.

[139]

Name of organism.............00.00 006: est bennernics

Sources habitat. ievGs ss x cle vaeeta teavs coe de eho BE gaan TR tu ae eam He ees Renee ee ne ar

ir yt
ny

MorPHOLOGICAL CHARACTERS.

7)

o

Q

62

o>

ie)
x

Incubation
temp. (°C.)

SKETCHES.

1. Form:

i, Boul VOM soi. bct cave ose oe ee eee gsr Sy USF ADR GP ERE NOE ren TREE tS, SSRIGPA GTA OSS

6. Agar .

6 Gelatin: cesar

a: Other wmediar. vi: cascutaiewnes a's

Bo SEl MSO UuplnpeSixs sso Gaeageoia tue are educa ud wi eanstseionans aude helen One

and arrangements................5-
in growths......

4. Staining powers: .

a, Aqueous gentian-violet......... See re eens
6. Loeffler’s methylen-blue.....................
Gs, GUAM’ S Sta nai. mcs re ees

da. Special stains

me MOLLY, cards eect

w

a, Character of movement ...............-

O63 Plag@ella: Stain inc-cxs yeas secs fate Sone)

a

» SPOTeS: 6.00 55 ed BUNGE Hie

Special characters, such as:..

N

deposits, vacuoless ic neces cee eae

pleomorphic and involution forms, capsules, etc.

PHYSIOLOGICAL CHARACTERS.
1, Relation to temperature t............. 06.0055

desiccation, light, disinfectants, etc.:..............

4. Pigment-prodiiction tics yeesics anes wee

5. Gas production in glucose media:
a. Shake culture .
6. Fermentation tube, growth in: (1) open arm:
(3) rate of development: 24 hours ....
(,) reaction in open arm:.....
6. Acid or alkali production, litmus milk.

Reduction of nitrates; to nitrites........

<i

3. Indo] production; 24 hours ........
fecal odor; 24 hours ...

9. Enzyme production : proteolytic

ro. Characteristic odor.

tt, Patho@en@sis: os: o cqseeaien cones

ea 48: HOUTSi5 252

., gS hours....

..(2) closed arm:

een rete adn (5) gas formula, H : CO. ::

--+. percent. 48 hours ......... per cent., 72 hours ............. percent., ........-.-..

. hours....

cece ee per cent,

ae

CULTURE CHARACTERS.

Reaction

of Medium,
Incubation
Temp. (°C)

SKETCHES.

(1)
Gelatin
plate:
(a) Surface
Colonies.
(b) Deep
Colonies.

(2)

Agar
plate:
(a) Surface

Colonies. |’

(b) Deep
Colonies.

(8)

Gelatin
Stab.

(4)

Agar
Streak.

(5)

Potato.

(6)

Bouillon.

(7)

Special
Media.

[141]

Name of organism .......... Side eet Ses

Source, habitat, etc. ..... Seaeoeee cae hataresca panmencauieer

HG wists CAE Were ae eet ar ar a Se ee at ae

MorPHOLOGICAL CHARACTERS.

1, Form:

Gs BOW lon sa cecisag oc, cast atch ound: fe we anc as he ceed eae

6. Agar..

Cx Gelatinies eahiat tc oinwke te eh Ge aot ales

Ge Other Myedtay -ocikietsss x caine sq ae. eas HeAm Rene ie De ndinhe wee nacre Lente Ade den ice wae

Ged Otel Wire clo) by 01) 04 = Parga tee ae aI Ce meee ee oR ee

and, arrangements: scenes ssc csmeceenalesaaicna sa msiess

IN PCrowths..02s seueoeseess

{i OLAIMING “POW ELS! Ma sseeesencrsveeed wes aaa ee ee

a, Aqueous gentian-violet .....

6. Loefler’s methylen-blue..............

(GRAM S'Staliis a eeiede yee adeay we sean REOG oo MUS SRG Ae OL ORE

x Special Sbrinstancaeaavedaws sano ceeh meear he sakes

ie MOLINE yrs Aen tate cara ce coletace toh bit Ge Neryh Sakina Omiuae A SAC Se ae MARS aan A ii nak Na Iver ola Ban 2 ee

a. Character of movement............

Ge FlagellasStaitenwwws ne cc ssc0.s0 ob ios castorate an cmrenny

Gs SPOTES cesar he ee

. Special characters, such as: .

NI

deposits, vacuoles....... epieiat, fave slseiggiceteerss

pleomorphic and involution forms, capsules, etc

PHYSIOLOGICAL CHARACTERS.

t, Relation to: temperature tics ces. sscqeacaseascam sens oe anv

desiccation, light, disinfectants, etc.:.....

4. Pignient productions. ccs csceen veces

5. Gas production in glucose media:

@. Shakecultun€:. os ceccsacssacve

6. Fermentation tube, growth in: (1) open arm:.

(3) rate of development: 24 hours................

(4) reaction in open arm:........
6. Acid or alkali production, litmus milk. ...

Reduction of nitrates; to nitrites........

NI

8. Indol production; 24 hours..........
fecal odor; 24 hours
9. Enzyme production: proteolytic.....

to, Characteristic odor..

ir, Pathogenesis. s.....044

pericent.,.48 hours ios oss.

deol B) RR BRINN oaks a ene scet sean so eee ealaanien

+f PEMCOM TS FE HOUNS <4 chao os ses. cc -. per cent.,

-«(§) gasformula, HCO =;

adie) LO FAN MIO a uicaccawii ens ccatees vise eteneanamner cert sada aoe cae eee

VOL MSTBUIES 5 oo e5 es scene

eee ee ae

Age of
Cultures.

Incubation
temp. (°C.)

CULTURE CHARACTERS.

Reaction

of Medium,
Incubation
Temp. (°C)

SKETCHES.

(1)
Gelatin
plate:
(a) Surface
Colonies.
(b) Deep
Colonies.

(2)
Agar
plate:
(a) Surface
Colonies.
(b) Deep
Colonies.

(3)

Gelatin
Stab.

(5)

Potato.

(6)

Bouillon.

(7)

Special
Media.

[143]

Dame Of OVTaMiSitivss, cess viwand ee eae ewes CPS LET Sey SOURED CATER veces eee nee ‘ so Mince eee
Source, habitat, ete. .......... tee ate pe eet cia tala AM aa oe Sats suiewe es ; eee ce Salnges eee hoa ee es Se ee are ee ais
Referencesiniwn vicwcatewin cine Leh need Da eiacemta nate seh aererect AYE Ie Taser e NO SER a NERS POG AD OR AINE Oe DAN 4
g | 89
sz | 34
MoRPHOLOGICAL CHARACTERS. &S | g8
1. Form:
@>BOUIOMES ss seate -saotslcbainds vet was its paken Gia aed cae lacke te cecion mete oe Nan ted nena ah dens Indl ai. cad Warnes aula hit rare idea ora amanda satel seipencaes | seared
Dr EAA Seats ie COM a Nh ae says Hee Sad eit eteccek det a ctety lasts ade hee A nase Sete ake ae en at nto sneer tae | Aes [see e ae
Gs Gellatk tay: areata vidas ena te sate ne Sse nes aes eel ia Hata es ats oR RAL ohaserate EES oe UE RU ee He A PEs oe Ma iste led artista ell oediiet ato

d. Other media .

Dy SN Cress specriesserisat a nackte

3. Cell groupings

and arrangements.............
in growths. .........0..

4. Staining powerst.iiccccccws se cewen ees
a.

b.

Aqueous gentian-violet

c.

d. Special stains .

5. Motility:.

a. Character of movement.....................

6. Flagella stain. ........

6,, SPOTS os ase aes

7 = SPCC lal CHATACLERS) SUC) ASH teri iuclven Ria inckhivan ee 3a Haoceertcntwesie ae sactinns os ope ia aman erenag ot vet cabcok duvtaeat Geeta

deposits, vacuoles

pleomorphic and involution forms, capsules, ete .

TsoettlerShime thy len=blue.acnas. deen eaves syste ci omceted comove spel oaersenal ame eden

GeraMS 3S Calle eye ae arcseeasgteenietctearomacen die

SKETCHES,

PHYSIOLOGICAL CHARACTERS.

1. Relation to temperature:..... a
2. Relation to free oxygen:............
3. Relation to other agents, such as:....
desiccation, light, disinfectants, etc:........

4. Pigment production:.
5. Gas production in glucose media:

a. Shake culture....

6. Fermentation tube, growthin: (1) open arm:..... .....(2) closed arm:

(
(4)

3)

6,

Reduction of nitrates; to nitrites ....

N

Indo] production; 24 hours.

oo

fecal odor; 24 hours .
Enzyme production: proteolytic...

10, Characteristic odor..

11. Pathogenesis

rate of development: 24 hours

reaction IM OPEN ALME...c ie ceneeee t

+. percent., 48 hours........... percent., 72 hours......... ...percent., ................,hours

.-(5) gas formula, H:COe::

Acid or alkali production, litmus milk

SAS MOMTSYsc2 cues ox tiee ones

sei MB OUTS srves thks At ein’ weresece nelaunclon aeteth minis

cece eeveee Per cent

CULTURE CHARACTERS.

Reaction

of Medium,
Incubation
Temp. (°C)

SKETCHES.

(1)
Gelatin
plate:
(a) Surface
Colonies.
(b) Deep
Colonies.

(2)
Agar
plate:
(a) Surface

Colonies.

(b) Deep

Colonies.

(3)

Gelatin
Stab.

(4)

Agar
: Streak.

(5)

Potato.

(6)

Bouillon,

(7)

Special.
Media.

[145]

NaING OL OTPANISM 245525) nos Adee aaeeceenn Bae ae ee Ag eee ean ae SG ER ane piers Get nln oie gn Greener ca Ee nla Aaa :
Souree; habitat et@scc. dete iad eS ale ae ac eda tise oR a hae a IR ae vee eget caren BP SSS ag le tae onal Cae enters eee oes
Ref6reniGess a scniile ead 8 aeee eee Sea ORGS a Se REAGAN SY Ene ew GiWAS eee Gina ade poets Be Faye REN RRR AY fe APO TE rt pore LER Ea TEs

Incubation
temp. (°C.)

SKETCHES,

1. Form:
Gx, BOUIN OM gate ra vet eatatntoc.y ta Paha

é; Gelatin ison v5 ys esas vi

GAOT ET MVEA ag 22 2A hee nie desis be Fadel Bede

BE SUZOe acres ate sysvahesmiess i rinter sake oie

3. Cell: rOUPiNgS; escese weayeseaerecddad oa na neeatncne: orem anne meme Bese we
and arrangements ........
TIO TOWENS yue-3 peg Batigenanuerena mat Aoeneenl

4. Staining powers: .

a. Aqueous gentian-violet

b,. Loefiler’s methylén- blues. i454 cee se ve es os Qaedine ee eedd es aw inamuaye ge eeen ge oe ses

c. Gram’s Stain............
id; Special StainSss.cs vs seremcas Ha Anant aot tea aie

65 MOC Nyy cee dake ys ne fonds vavwieds e+ es natinwapaccie ania ta vera

a. Character ohinovemen teased: ui teers kine ve ph eenalg Me anette fa the at sieutla lens Hela asiviere pW ala Rais ahs ometa aes aye eats a Aiayepa saan Apasts
Be We ella Stay 5 daca xi Vesey a Soave eam 9S a Ro a CE ay ve a ad a on ge Gate sa aes SE KS Se
GAS POLES: warnctaoacraeetisien as wien esta Soe Saertacnan Le a nome Abas telnet Loe lear ot mietais paateaita aera G seem e ee eae

4, Special chatacters, SUCH aS? «<2... 2.c crass esa mene es oe sais

GOpORe, VACROIES «jc cesnv ss wae Kull g a5 SaeeR Ye Hea wh Pade ts De Ae M4 ved genes eae peL ee eg La Ye weed Seu es oN 14

pleomorphic and involution forms, capsules, etc............

PHYSIOLOGICAL CHARACTERS.

1. Relation to temperature:.............

2. Relation to free oxygen: ...........
3. Relation to other agents, such a$?.... 0.0. 0s00se0%
desiccation, light, disinfectants, etc.:......

4. Pigment production:...............-.

5. Gas production in glucose media:
a; Shakerculturesatisc heshecces toed
(1)

24 HOUTS:s sie eces

6. Fermentation tube, growth in:
(3)
(4)

rate of development:

6. Acid or alkali production, litmus milk ............ 60... .00 2 eee eee eee

7. Reduction of nitrates; to nitrites ..............
8. Indol production; 24 hours.......... 0... ..00 sees

fecal odow:24: HOURS an.4 522. ceaeet eae wee ee ale 2
9. Enzyme production: proteolytic........ 2.65.02. - te eee ete eee

Characteristic odor.......

Pathogenesis ....

OPENGAMUE cme maeeGee Loans

.., 48 hours........

.... per cent., 48 hours............percent., 72 hours

reaction im Open ALN: os vewrovsion Hot ectenins a eemygriee deca chem eRe eE He

nap SAR OMITS Sieaa nainctie me ge as atte RRMA and ose oe

ne hale Rata (2), | ChOSE GI AMUN hn dnivenicmn sehen sc aie eea deutch

per cent.,
tise veneS) gas formula, H : COs: :

per cent.

a sys tOMATMIMMI OMIA a ee nhs os ae ye ewe wheres seen

PsA Si ine neds We sads saneweGees Male

ORS osc elocask vosexs dees eee

vow o'Glagtatic:«..

CULTURE CHARACTERS.

| Reaction
of Medium, 24

Incubation =

Temp. (°C)

saaieys Hoors. 48...... Hours. 6...... Days. SKETCHES.

(1)
Gelatin
plate:
(a) Surface
Colonies.
(b) Deep
Colonies.

(2)
Agar
plate:
(a) Surface
Colonies.
(b) Deep
Colonies.

(3)

Gelatin
Stab.

(4) PA NN
\
\

Agar
Streak. q

5) .
: * / a eN

Potato.

(6)

Bouillon.

(7)

Special
Media.

[147]

CHAPTER VII.

PATHOGENIC ANAEROBES.

Anaerobic bacteria may be furnished conditions, which permit of their development,
in a variety of ways and a very considerable number of pieces of apparatus have been
devised to secure this end. In a general way all of the methods may be grouped under
the following heads:

1. Displacement of air.

2. Absorption of oxygen.

3. Exhaustion of air.

4. Exclusion of air.

5. Miscellaneous methods, in the presence of reducing substances as litmus, or
a strongly aerobic germ, etc.

The first two methods are the most reliable. In the displacement method, hydro-'
gen, carbon dioxide or illuminating gas may be used; hydrogen is best. This gas is
readily prepared by the action of sulphuric acid (1:8) on zine. Either a Kipp generator
may be used or one of a simpler construction. The gas should be washed, 1st. in lead
nitrate to absorb the sulphuretted hydrogen, 2nd. in silver sulphate to absorb any
arseniuretted or phosphuretted hydrogen, and 8rd. in potassium hydrate to remove sul-
phur and carbon dioxide.

The cultures are made in media containing glucose (which should preferably be
freshly prepared and always boiled immediately before being inoculated), either as test-
tube or plate cultures. Novy’s anaerobic jars are perhaps the most satisfactory recep-
tacles for the cultures. (For careful description of same, see N. 306.)

In the second method (Buchner’s method) an alkaline solution of pyrogallic acid

_ is used to absorb the oxygen. The cultures may be placed in Novy jars or similar re-
ceptacles; for tube cultures a large wide mouthed bottle fitted with a rubber cork does very
well. The dry pyrogallic acid is placed in the bottom of the receptacles, about 1 gram
to every 100 ce. of air space, the tubes are put in place, then about 10 ce. of a
normal sodium hydroxide is added to each gram of pyrogallic acid, and the apparatus
immediately and hemetically sealed.

REFERENCES. A. 206; L. & K. 98; M. & R. 68; M. & W. 117; McF. 153; P.
233; S. 78.

EXERCISE XCIX. BACTERIUM WELCHII Mig.
: BACILLUS AEROGENES CAPSULATUS.

First described by Welch in 1892. Occurs at autopsies in which gas bubbles are present in the larger vessels, accompanied by the
formation of numerous small cavities in the liver containing gas. It has been found also in emphysematous phlegmons, in puerperal
sepsis, in peritonitis and in other conditions (M. & W.). Widely distributed in nature. (Welch.)

REFERENCES. Welch and Nuttall: Bull. Johns mii a ii 1892, 3; 81; Welch & Flexner: Jour. Exp. Med., 1896, 1;5; A. 140;
M. & W. 173; McF. 463; P. 545; S. 781.

SKETCHES.

Incubation
temp. (@C.)

MoRPHOLOGICAL CHARACTERS. BO

1. Form:
@. Bowillon Juccsscxvsese vac Repent biuret sh ea cays deren antida wate ea Seandnared ontatoteen he Semen eee aInnaaihs eek temiaisiaies | smc] deleWeeter

Be COU POU B IBS isis a eadinisiind sob asatsind Maio wissser a te wielens@u ds 4 dhadra Sein radioisaalbGe yanis gega He OEMS a woe Meeeb ta Bik oa kines
BH APPANBOMEDIE cc picn see gaeens dacs Meena rawdied eres uel shed rendoda ee ee tke aparewad anaes ei epde PERE ED MORE WON
ATO WES Sc cree sid aassassians Vasvsuisenelns acre ceereidraaghtnacta Hie by ameeaesion mendes MERE REG eins WARTS OE NG Aaah alayeaeaananl HE dewenee e erausreners

@ Staining Powers: 2s eccers dacmecin cs oe Seas cane do LISkSag be Hewes aot sasbs de MacwleMilad se eaun Heine Menard Tee mia ie Se dateieptle meio i

@, AQUEOUS FON EAT =VIOLEE ij cc nie a eb aigniaes ealeiaitiasaininie oo ie leis cde nae seldn inde heele As TR MANES 86 be Ree eet pu getgtne rly sem

&. Loeffler’s methylen-blue
) Gran’ s Stains sieisigitzetacioetyavisie Se wa ccliie ders anctapss usdig is easrsiais otsisied ve ible a aise wie RE iets SE Re wvemMae aaded ele Miasie wreeteuesiaten] sie! Geese ffeateleyens ee
@: Special Stains: ve gisns ponacoe vs te ores ceva vaeean bene FAIRE RDO RR ET Gren errr rere eee ee ete ib der aed secant lb Wantawece
Bie MGCL ty Sas sisaccsinc ue ub side wosded ganacnd Ue BE ht eine aes ow ie DERRAS Sakae amas Neke wote Heoies are ee oS A eR eRe Ed eeR ve eames [ae oe chee seeds
Gi Character of MNOVEMENE souls Nes2 Gi dh dane neGaawdyad Anaedee Suva syy waves wealnetora WE8G: bdfahe Ghesadubscicuduelaey dendpersaltienasert haces Sie Mek Gee a [SOLER So Mesa
b-. Plagella'stainsescac wacicir axa deaiesiaias peeians ie aida camer oa reine Ge ENA OG TL EAN Betex FORCE CRU IT, | Dee Sl Net er
Ox SPOreSiccein cansmes sa teeter wan prelialenas vig careaieeiid Mesiemad ae sel nels@g hase aE Daletesld oh Hpae wlNNes 9.9 vaeretha Ne Se SideareetREe avdalesie ats SaaeR eal alee Agia Sse RAMS

as Spectal CHATACTETS, SUCGHCAS! 4.41 Hadacaucdd te teditaaneseddenguuad es ii ei GM BRS ila, Ge ea eb a AG HER ALD DAES ALB DETR O eae bebe HAE Sasa

déposits, vacnolesiseciccsncsac: — wins eswtiia erin tates Sa ea Sean heed Seb neonate a4 Mate ae maecde bh uboh eadwen GA a ambainel ke aeames lesaceune

‘pleomorphic and dnvolution forms; capsulesy, eteisuiinss sic ciize xiarcisie dare aivisiesustais viaiate wicca cia semiereiaie BHC aiclige wictatha atin wiaickgiasied eaten ages [ls08 Pplenite

PHYSIOLOGICAL CHARACTERS.
1. Relation to temperature : Sees joka Sen tha hdd els slop a tae py “a ana “a i dear aphdd Tua PddUASAE (Sha Sve GiRNEin Cea Se avoia raean ose Maca AatS EINE <45 GAG BlecaLR 4H HAGE RARE REINS ahaa DIR REIS. ci-Re Ta Menmaeee SoeaiAO

5. Gas production in glucose media:
@. Shakesculture ci sisise se wets aeawsetitad sed wiped sadaietensitins ve nwueeSiarein dimen aitiby va ae Geena NEUTUle Rha wine se. aitlad He Ga sed edna gS ean’ visas by Wale banSard naan asain eH .
b, Fermentation tube, growth in: (1) open arm:
(3) rate of development: 24 hours.............-..
(4) reaction I OPE AP. coc sne nice coed erinwe tops besos on oe me Nena meReuMBE ae nee,
6. Acid or alkali production, litmus milk.

7, Réduction of nitrates: to nitrites .:ccs.2 2x2 sexy ca ve eamep Ree eee easy Ss de cnet eenaonaed vee .

&. Indel production; ap hourSicsansee ca oc vows senraee se oe tent

fecal Odor 24. hOUTS 3:23 sscsiecce ve aoven aieays eae aes

g. Enzyme production: proteolytic.............. Side auletaa 24 Migeae BY te ve Sut hesien nod oe aicdde i

CULTURE CHARACTERS.

Reaction
‘ of Medium,
: Incubation 24

Moe Hours, 48...... Hours. 6...... Days. SKETCHES.
Temp. (°C) :

(1)
Gelatin
plate:
(a) Surface
Colonies.
(b) Deep
Colonies.

(2)
Agar
plate:
(a) Surface
Colonies.
(b) Deep
Colonies.

(3)

Gelatin
Stab.

(4) AN AN

/
Agar /
_ Streak, (

(5)

Potato.

(6)

Bouillon.

(7)

Special
Media, , bh

[151]

EXERCISE C. BACILLUS CHAUVAEI Arloing, Cornevin and Thomas.
“BACILLUS OF SYMPTOMATIO ANTHRAX.
First described by Arloing, Cornevin and Thomas in 1887. It occurs in the subcutaneous tissue, muscles and serous exudate of ani.

mals suffering from symptomatic anthrax.

REFERENCES. Arloing, Cornevin and Thomas; Le Charbon symptomatique du baeuf, 2nd edit. Paris, 1887; A. 482; H. 304; McF
453; P. 563; S. 493. : |

ary
Sf | 64 SKETCHES,
MorpHoLocicaL CHARACTERS. 85 | 38
i. Form:
a. BO ti gcanse ni actos swiocan vees eadee en aled, Gwe Weed RevadGRiGR OUT) txaRwn SAgdslad Mer weed eur weNs TA RANSY CLAN IG WER SOON eee eet eres
Ds BAG sili sitiallin Maks CE ewidin a Leip an RRA RA TD Gm in ERNE SEAR HAA MERRY VO ROENS FG A EER ANS RRR SR ES ENE REM REE a ss ateaie ne HIER BIN [8 eS
6 GOVAN 6 oak ey cesin de ceine id Sau ois Sunaina doawRa ene U MAG DER blew Seas na APTA Riad HD. Dida wade warn came tackle RELATE Oe BREET s ORR NaS
ORME MELA siessicscessdisicine sins yradesejecidvateats is da Siodatd- So Seiaciaielod Haogaiaiierd me eaee. — RA RUTENAAMRR ARE we Qesee eae are ge Ae wee shatelete ff sieedts oe [ieein aE
Be SZC ia secsussicinre cigs avgpusiassinivia eoaticisao.m Ha nang s viva alenbiaers He WERE Alecia naTateain EaTelE Seopa aeainleSindananae, oysid epeedicane wl tactslnles a erualia’s EOAOTS ee
§. \Cell CroOupi ngs sais: views ideaciee og os ues Aawassaiiis ae nd va winsins se nelaiacn He Edw Maciel saree enteteint, wisinle miatsinn a: Ais cdvdjam aiaree tva(aiayy dharevasereie eeune Ginid A SUERTE A | agree
and arrangements): ieee cuss gademasars eh acamiy eo de edwdiinsiheeeahwee TeNeAseEtwRa sig swag eH aiereteieee aelauninathE Erlewine eae eeeallineitis HlSe [REESE
BEL TO WEDS aio aiss cscs case vac" ese agaussas s deniielnsslg-b-0°ti8-Seatecussassyelssadundhave Gon dee Svndoel d/n/aowiguchensid py sTBS Wigidigale Va RSE NE NY ce alovaleclgnays, te dg verde Nigh [sRcespiaen sce fe Risntlciere
As: Staining; POWETS? siaisisariesceccisue sinless aacstiesiaisia ntncterns aalae, snyaiden nia Sushaveed Ses ser sian susintelalardedidintG ald wrntald eepaiel pa Ddveresdiar bod oe: Biaielaie we SUGSA NG FB Reolkegee Leg [oie he otter] is seer at ie
4; Aqueous -wentian-violet jpicais seiacrcnes ws daypoomaaedulewrciew ba eels wagale stig Aalegias ss qo oasiiewchdanerG de daias wettaene sehamyhaaded| iat gegala denies
B, LCM Er's TELRYIEN-DIUE: cs nec ss ca wnenee dd cians cae 4 Oh deeds CARRE E RAEN 148s ORs CERES te ERD eS peeheaee Komen anea | Hee atin |e ah NSE
Gx Granvsistaln 24 )ced ia deca wairesieamaein cayledve ba atoelee nd tm codtiaverieeuap’ bed atasben, ad die eoage acdsesSlalatctemysto’ sn SOG REIMAN Te ae Marae age [NEG tical eaters
@. Special: stains ¢ sox sa sdaics aisles Ysictesiawsisiwis sseratsns w stevciaivaate via ila ecensinsicdate arasaial aed BN Awa ata UAROLE ne Nea leimay bd domsiole: abies inioneadstIADe |i La 4 eleledes[ niereitrels oe
§> Motility tssc.ues ie v5 cies ogee raneanies wis ik dae AGs Sey ata pe nh See eG wins cee Mee 55-45 cieeatehaR ion acuedion are Ne NaIelN BiRlanaialeai Bia Sietwakemcinaiehen [Os Wes team] aieiaiolns
a, Character of movement ..............0..00...e0 eee 4 Wad neenyeue wasn eS omo asi schdetwen nie MDS RENN ay emMommi nade oy ay geen (nine gsis
b: Wlagella stain: cocesnsccatenaaei aedeacae eae eeuh aide Ger a Addie head Sean Senne whe aame Re iy shag raid ints ayoats vk aecbae attoonenerenvel| coy gate I meade nt Bs
G: SSPOTEs is: coveesce see yeree guiemie ds &. sts) he ace Sargstwnsiainss geietene is’ o aChva aubtardatasayshartoslsvavetal iarbye a alae clemstarw: webualga wee Sey saat otrtoeg eranaih aie obit ceeetibatetlt [ve aly aayeilla dardone tod a
yf, Betas KH APARI ATS, BUR WANE ye casonaes 14: bs ween txuadadawes He aL Reua ae Wee He badd Nom wees Waed ois Maa aeewublig Gang aedyseids | yy ey ue. Mebamtely ee
Meposits, VACUOLES <i ics svg wuwa ds ww wewciwwinn si takideita: Ses SAS Mian ea Valea BE alge ite “aaa yalte Wy a0 dant A@A ATER AY ow AAEM GENT Lead wewe| NHR Me
pleomorphic and hivolution formis, capsules, Cte sss vw. nx.e vere seomaeanciiamatat-c seecasarnmine ne pa bee eesendaapiedine geaid wlesiiglagi"| oa ye asuenillenme ee oa
PHYSIOLOGICAL CHARACTERS.
g, ‘Relation todemperatirticcs: ccncian cers deeds isycenied a aeeebeenaten ie de auueg pahmmbe d4 ot Gets Hands HH OL OREEL LL eewee £2 da Keke ne td Soke mend ee corm
a, Relation:to free oxy eens vc yess socnnescanan edas eineds asracen beta candy sd aweleg Ween Meee eS eae Wt ERT ReRml SN cima SEMA G Kame ENN ladle eters aaeleieln ey ab ganeaiane awh toe Same aE
3.. Relation to,other agents, SUCh:aS2..:2..c02 ais sg teciasietdiniens cceiGsteiens \G gie Ssgieaid die baaiqandace nje-alsiad Uoledbiacg dedlduaverane. dnypia wioidiacelangednd die ‘siaptevetadrarara oid Weee'one agtaveenie'e asso g Taian a Ale OS SHRM EOMS CREE
desiccation, light; disinfectants, tC? icc cciuaa cckosias sa te ceretys She remeied UE TOME Hkerd Houacnerad we-cmUke A aRAR Weln GMS Bole aNaR wise GiGi aeelanata sus aieteds SD Urea ele eines
d:< Pigitien ty prod UGH OM tease. a sararcensatde de gage Se ea asthe atte SS scale ne Saab Soap am dealer alone ent aAwradiange ey oa Saas eeeemans at cannes PaRee S maken acjibelgetee Sea tio
5. Gas production in glucose media:
@; Shake culture sc soisicas ie xc wacdenaweoitocaw oes aces, Raa nay Nee Hes RNG Sea Wardens Sand eGeLE HOE RENE og GE RRSELT URAC Ben ela eS a4 BY RU TRUE AS Oe Saree ve vA ae aS
b. Fermentation tube, growthin: (1) opem arm:.....0.0........ 0 66 eee eee eee teens (2) COSC dainty hens sawevens sheen hg Se Giardaecuby donee adsh mnsbicieraieale Ne Ge ARNG BE rai vee
(3) rate of development: 24 hours ............... per cent, 48 hours........-.... Per Cent., 92 NOUS, noc ees i608 ay PEF CON be, as vsssecsine a Pi vis OUTS cane ce eee per cent,
(a) venctinn in Oper WEOtisidces iy i usadnwadeesse mers 44 taaanwewesnce Lane cawd wmews (5) gas formula, H:COe:: : ak
6. Ace orolkal production, tiie Wilh k eee urn sxewres vende t4 v9 03 de ge 44 powne aa CARES EF Hite U4 OY RONE USTs KAR + endo da ee epee ane poe eonaedbd oun on oo aaa EER :
7. Reduction of nitrates; to nitrites .... 2.0.0... 6. cece cece eee eee cee nee eee eens ¢ SOMO Aas. cidnes enseanmussons vivoiemiesuned pes Ama ene eee PO
§: Undol:productions24¢:hoursis oi s.ccnsaumimandante omaniarateaaeiding p QO OUTS carusaeinn smumuan sean amet edeaaut oe nig geselre eases oe pe lane ctaled deans GAYS: bs casancieeeaseanansens eae
fecal Odor 24 JhOUTS: geiciic svingiwiennsie sas sional gamers x PAB ROUIS somites va sy tan gasimurreaiady Maawdinnwieey se Head daedleian ed daunend cate AAYB ccs carens a4 dene nbipainceutseouen ee?
g. Enzyme production: proteolytic... 2.0.0.6... ec cce cence eee e ee ee cee cee eet eee eee GAS tatiChsicces aiedinseumierainuaans vows aacedalle Uy astaulagurlgatn os Ay Suna gaye eres KomeTTHE
10; CharacteristiG odo tas s:5 5a mos cucnceaeude saan asecenibeudctaiea ab, AGeatuadaln add saajelbcbioie ala neaibaa taviaunnan Sante aw eee # walicaiavhis aaieaibueis Aleing ot SY Wael Meaase Dy casian Po tMEAes vane HATED
Hi. “Patho genesisistccqiriei.sine ie isan toesn passed sian emieswiaei sine seve sailsgnaing vobiten ates sisldsaiueaauns Tea dikinrs BAMGNY AMWRreacaCieicie UU GM athe kha Gebe Fdsbedhin d-dansee sdide SimauceasigncugeReanees .

[152]

hi

CULTURE CHARACTERS,

Reaction
of Medium, 3
Incubation 4
Temp. (°C)

syaiaitnave - Hours. 48...... Hours. 6...... Days. SKETCHES

“(Q)

Gelatin

plate:
(a) Surface
Colonies.
(b) Deep.
Colonies.

(2)
Agar
plate:
(a) Surface
Colonies.
(b) Deep
Colonies.

(3)

Gelatin

= vie

(4)

/ \
Agar / \ /
Streak : i
f \
\ q

; (5)

Potato..

(6)

Bouillon.

[153]

EXERCISE CI, BACILLUS OEDEMATIS Liborius.

BACILLUS OF MALIGNANT OEDEMA.

First described by Pasteur in 1877. Widely distributed in soil and putrefying material. Few cases on record of infection of ma

REFERENCES. Z. f. H., 1886; 1:158; A. 476; H. 302; L. & K. 305; M. & R. 394; M. & W. 175; M. & W. 459; P. 543; 8.

MORPHOLOGICAL CHARACTERS.

Incubation
temp. (°C.)

SKETCHES,

1. Form:
'. BOUM OR sess cc sass cease has ane ate Weed sock acetone oe asinine Sipser ata dlasepdenaceghaiaearbipiaraao soa ade mia Whweays ¥ Deraia he BEM RRARRROEE
&. Agar...

9. Cell SOUP NGS sctosiaisine sceleiaisis telaerpiniaiay aialiane 43 SY eeeo aren cede See Sines dad ieUe YA AC mathe eaadatin we OY AREA ERA RaRS aN eee
‘aiid AT EAMG ern ONES: cis scetyes es cerbad: i We cedars igs cits Wb 50a sstecendassausy sale uaynsbys Snnvden Mtovasd Gnasaneug vege debver teavaedardesi@vayone de Aaubsdvduivandideana ba oeeSe

REL TORE ceca edeniene veces ptmndiiald aa Sie Sethian i ALY Wy hte Rae Rance ota Dk a ule ea wens Reale AEE HONE RD ROE WER

a, Aqueous gentian-violet

b. Loeffler’s methylen-blue

Be MEATS BEAM dio aabe 22 3) Lack bee Reh Ang TA eHOARRA TDD ah Ln salute qundmawsaueoneuee eumlad ay Mie AN Boe meRe La ea nata ahem 4

b. Flagella stain
: GOS cnsas cerns vane aden ess man pans mEORON eae Peedi ph Wr dundee edleananee adler eens CONMeeINd Aa ERMRMUAD ENeLEL Re

deposits, vacuoles. ........ 0.6. cececeecceseeeee seen eseeeeseeees id Rie Caeai ote otal aeinl Nanette salah A Wnsjaa gre sigosaens Te ates eee er Sea coe

pleomorphic and involution forms, capsules, etc ....

PHYSIOLOGICAL CHARACTERS.

Te Relation 0 tem Perati4re?: «2.5.35. vines wees oa vance bn Gaew naeana en TREK OES be mma veds
svi seaiiecn oae ci RAW dein ta Saco tarnlcsbin iach bes sicinnage aio gfnicjaetovar a wie wlaneiy 8 be AiRMAgismieg AAR wented Nay
2. Relation to free OXYGEN? cisecictnourionas ares ceawy nad goae as Te TE ROAR es Wiszatnioiesduiear ue

5. Gas production in glucose media:

a. Shake culture

Se -

6. Fermentation tube, growth in: (1) open arm: ......... ccc cee cee eee cee e cree cues (2) closed arm:

(3) rate of development: 24 hours................ per cent., 48 hours

shel atateaaasttes per cent., 72 hours............ per cent.,

Sia avoca Sava Seay oer da ocoraecusare cache sistas vstancta wis vis peace wvetaielackeadard ae Se ates (5) gas. formula, H: COs::

fecal odor; 24 hours ........
9. Enzyme production: proteolytic

1G: CHanacteTigthe OdO Tara: es conan 15 ex sentande neenerel vey beads Hi YS ie Swen 444 none 44 0E Neha dade

2x5. ‘Pathogenesis .... ...ccces+resecies se cena nese cant ne density Soa mie baiee deea ses

STENT Sp eee On a

5

MESES Ce RR TROS SHE Shegiend Goa auesace ela NEDA, SH amesaiehal G'S 1M aeaiave didtela aacenwte nance ”

na bY Gee per cen

» ++) to ammonia «2. cece cece ec ceee eee ey dee ena

CULTURE CHARACTERS.

Reaction
of Medium, 24
Incubation

wsattenats Hoors. 48...... Hours. 6...... Days. SKETCHES,
Temp. (°C) ,

. (1)

Gelatin
plate:

(a) Surface

x Colonies.

(b) Deep
Colonies.

(2)
Agar
plate:
(a) Surface
Colonies.
(b) Deep
Colonies.

(3)

Gelatin

Stab. | . Le Ue.

(4)

/
Agar A : H
Streak. (

Potato.

(8)

Bouillon.

(7)

Special
Media.

[155]

EXERCISE CII. BACILLUS TETANI Nicolaier.
Discovered by Nicolaier, 1884. First cultivated by Kitasato, 1889. Bocuse in man and animals suffering from the disease and wide
distributed in nature, especially in soil.

REFERENCES. Nicolaier: Deutszche Med. Wochenschrift, 1884; Kitasato: Deutsche Med. Wochenschrift, 1889; A. 469; H. 9
L. & K. 230; M. &R. 376; M. & W.171; McF. 274; P. 385; S. 482.

6 te)
o ees
33 3 B SKETCHES,
MorPHOLOGICAL CHARACTERS. &S gs
4 Lal
1. Form:
1 BOUIN OB ns 8 G2 Na caution We aoc staan STS DG AIS elGva ML aWe fvbecules Behe a he arpeard apersahtaarove etpannigal4 © ituige UuvnstewlebSn saleistousinsadiysldielars | eee Sears lene ah dost
Dis GE AT se cassette hepa Yee Sia baci hea a la Sah Se eissnar SSM Sah Gh Se BS atana acid aeN WH seavUanIGh rateCndedotied SAWN Meade dieu SoA aoLE okie nid eae oes laeu deen
Ge MOL UR a saeecpeveey serve trae Ga Ps wae oars sectarian ate i aayenn dl gn een ceeh eden i ue een eye eta ue nd etre lignans laa ct eee lk So godin aes
i OSE TV Nags cv pee ge «iat sizasopaaets aid REyaeda- ane yisdt ad de acoeve RATA Ge da dn RGR a Seeaa abil yeaa ee ee Dade Ea aun eae AES
BOUL ress $513 fie gees vsng ns Cec votevednta wo ode eaeaecat ete ate Dead Oe nC AR AES DSIRE A td Saha ay tees cyan Masta a a ahah deta aga
Bus Cell @roupinig sins: nce arunpadee vn aestsaeldsiie weve wed hemes axemeimery ge acpua Sede tenn dcmeen odabs men tua meme uaed ution agkanwe eamanane

Qs SPCC Ali StAI NS vancaywous rave dortvees ean ceveaan ace bie sexes AM eas atts ole elcid cree meneseg ie a aes aeieneneyala elses Saab men weeay cisiesala(e. be ae oe
My SURE scores vx btteoeatd acne ued yess aempianae wen iieloa ca ea siewadeaabdamimuau Wiey AGURAY 04 dcaike Babe bodaiew rnsa ey bh cecrecs
a. Character of movement ns cue exsevawen taleineay ai ye Sitsnaiete ainmdanae Sa ud apasinca bemseiorew vee $A ovtesp oimalavteinyeeRe Me
Wy PEA SBIR xen oy ty tty wise 54 cnscianed been Gases bieweaiwaree oh be wane Soopceugleit naahevnd wales tiwslny Exanewia ad SoawIOEdlee
Gis DOLE S sisaase: dis ehclgdtesasvonsompsenive to) Laat ius cer apa vin ates iecats sens eds taicenve is ie easton el Bon ge Salers erapdenrcaipeaceer nets EA

PHYSIOLOGICAL CHARACTERS.

Relation to temperature :

2. Relation to: freesoxy gems cn. casas sive etal iy da yalalgnaea resets euaed vaca meray ua siduadc we Sv andi glenda babaetinthe

3. Relation to other agents, such aS:............ 0... ccc ccee cece cece ceaecnaeuee suctnets ine (NGL Was (aterage teeansisilaiw Vidigtaleteue Meaq AMAR SENS AHEM wij dunvalaiesibcuags bd vigeheed SBA4N decks ye Ne Mea weds Ga daeealenas
deStccation, light, ina I EEIIDES, CLC 2s ot copes a hans Seiad aawisien ot de Weenies
4. Pigment production: ... 0.20... ..esese seen cece cece ee eee eee eee cee en ete eee cena een eee beet sean Geese see scae cube tcdataee ests eee bEes cobb tb be bebe ceecbee Gecueee ivi
5. Gas production in glucose media:
a. Shake culture ........... iis ase Se Tagine eeaiead sera aes onl hd laceaiesecaiciaeetaver Satins eaeiegnatnes iGtes aed meGele een? Sa Ae aia Nol eae ADE as Shae Sea lesaigy ate Ravsteth SRO
6. Fermentation tube, growth in: CE) OPEN ALINE eee eee eee eee eee eee cee eee se (2) Closed arm: oo... ec cee cece cane ce
(3) rate of development: 24 hours ............... percent., 48 hours ......... percent., 72 hours ............. BEF COL, ose cane sevnes ROBIE an wos ve per cel
(4) POACEON HI OPEN BLING casas senaccsesssecen ines can : --(5) gasformula, H:CO,::
6. Acid or alkali production, Jitmus milk. .................. 7
ve Reduction of nitrates, tormitrites ic. css sigan vovanad ia bs doewRte sa ainne ad oe navemnwanmans » to ammonia........ 6. eee
8. Indo] production; 24 hours ................00- ietdba “eee Sh OSDOUFS cas excite sessed aeee aaaks weed ce Fi cusihea: es fein hedale ais Mime extant te days
fecal Odor: 24 HOWE: 25 ssi ne se owe seater ned acme seals y ABOUTS wines verncies yds dkios cieiocaacaech yy WE Rete a-ak gaat mice ee GAYS visasiasea de valsaanead: ves vetees
9. Enzyme production: proteolytic: .... .6scisescans cus os oy deeeeereadeesseesvevcoenucueecves GitstatiCwnsanwesiiva dn nesiasacaereocame ibis STOR we nekoishs AIS Het Fiesta
to. Characteristic odor... 00.1... i eine eee eee ete eet ete ete cece bene eeee dana session setsueee sear teteuubeteitenererse. eee ee tat scenes
11. Pathogenesis .... 00.0.2. ceee eee ee eee eee cient eee eee ete ceed cee cu ee seueueee sues teteeperceseceneeees BA Ea crac sg ce

CULTURE CHARACTERS.

Ih : of ee :
Incubation 24...... Hours. : ee Hours. Giscivaa Days. SKETCHES.
Temp: (°C) :
Bee =
(1)
Gelatin
% plate:
(2) Surface
: Colonies.
(b) Deep
~ Colonies.
(2)
Agar »
plate:
(a) Surface
Colonies.
(b) Deep
Colonies.
(3)
Gelatin
Stab. 2
4
(4) “ N
JX
/ \y ls
Agar f] \ tl
Streak, \
ast we,
= (5) A
/ yf
U “ul
Potato.
j
a (6)
Bouillon.
(7)
Special 3
Media.

[157]

Namé of organism ............... 0.05. re

Source, habitat, etc. ...... a eae siemaie ace eee ee se eases bis o aapeaaeules be Hiden Sas tamawlekes siveleeiia Ss «oe Ra es 44 emase aeey
References.... ......e.0esee00s Pola rhe Rasetinee hy rashes ieeeus ets aes as as poetaaa ain hare etal tay hseauantiaa tetra ten patlee hte
‘35 a6 SKETCHES.
MoRPHOLOGICAL CHARACTERS. BS | 8

1, Form:

@. Bouillon

3. Cell groupings............ saci tane aarti ch Non tc ug cS wt paren a gees Sen ae aidiskesdin Skat Serra eRA AGE sae Baws Soteete wees | senate | aay ane
and ATTADPEMIEN TS, sassas Aranaveaeesaned Warde dah wea WA Oae SAR Doe ae AGG PAAR UE Lacnh delice o Sinanesais a es TheNae®’ tlh aumutaice sate. Ramanan [are eels

LTO WETS sccassfansis aisle ovelectevseiy Seat gutintives vv ave’ wlsaphers ecmtinteabtsibie Sia are e sstclencdMovcls taus tte Sveiniduacayasis Wabi yaaa vig ate albialaty Sena ava arene Sea: allele ahem etE ae.

4. Staining powers:

a, Aqueous gentian-violet
&. Loeffler’s methylen-blue........... esha S-atiecis doses ecu ras tent aes et ctney hey Sas OevNAsoed ele Ameena ve oases vada cusea ann OaaNe Nia NaRE haatacdee ese aeee

G2 \GiAMS' Stal Is: go cpeeenndcnccicavanig sae Vaghejars 31a Pateweogia He Hata mag d Gene lenstaee gid metic a Wreeteres 9a She aeserenn MiRIa retalaveng aig asia anim anelaReae Tahara ba asate [x fo pateleins

Bs Blagella Staitiveciciicca ai scmssticte grasa inoced poeleaies gag setipgaiad gxtablss nade etewd a @aeeinduie mn Foss MewEey waedenIN be af bismmiald Senge [lagers Hee

6; SSPOFES zeaiwy srssanes.ns ay 2eehs ences AO FlaeiaN To Re RaMOR aA Sinteeed iG OY PERRET eee Gren Beads Fe eReN 23 WY Wider aeeea as eaeRepleses

PHYSIOLOGICAL CHARACTERS.

1. Relation to temperature: ’

5. Gas production in glucose media:

a. Shake culture

7, Reduction of nitrates; to nitrites

8. Indol production; 24 hours............ 0.0. cece eee eee are
fecal odors24 NOUTSi.ay. iis dene Haak aca aakoreiiy amas ic

g. Enzyme production: proteolytic

CULTURE CHARACTERS.

Reaction
of Medium,
Incubation 24

anounced Hours. 48...... Hours, 6...... Days. SKETCHES,
Temp. (°C)

. (1) , da
~ Gelatin
plate:
(a) Surface
Colonies.
~ — (b) Deep
Colonies.

(2)
Agar
plate:
(a) Surface
Colonies.
(b) Deep
Colonies.

(3)

Gelatin

/ \
/
Streak. : : \

Bouillon.

[159]

Name of organism ............ 0c ec ee cence enee Sisipubatstanttln ecg etiieadecerele sts soahac os Saceoent hie Sen ete saci eRe P ALES ata

Source, habitat, etc. ......... Dea alb eiapinbaate knee Reames FaiheandeRie TAASKASERETR TES Rates ee Ee deme ws aes aden sl biped capennaatasl dle oe

SKETCHES,

Incubation
temp. (°C.)

MORPHOLOGICAL CHARACTERS. , BO

1. Form:

a, Bouillon

3. Cell PrOUPINES scans sasorny 24 ¥e tera Te Tess ae He eawies Ri Men SEAE AURORE eA Gme 0o5s TRANG SHEE HG REN aetnG EPEC eS pe oe SHSM Re,| TERE Feels Hmaloee

and ATraNGeMeENtS, .. zie occas wscic Scinjers die wierwisseie we: Siaieniessiaargiguaiate dine slbiee SEieiviecabihcneiniviel olga Saueler! ama Windelg, wate Came IERu eR Sales cuMtermiad [asap eee pies

a, Character of POST TTT NIG EC CRY SIN TPCT VCINREAA ARN TOR MISS SRO 1 MRD ETE aT APES SA mCOT ETT teste: bes a veatvord [ts aaa
Gi Plagella: Stain sv shisiny seawsscntinins is eames asainaaennetian ODEs ls RaGUAEMRS Seneca Mlatleg: aa alaeAld OR ae eee sien

6. SPORES orcad nhs Fi4k eRe wehaa Hh cEmy CURA PP KMRen es 90 Ae 4s BENE y ANON AEITE GDI Sete Kel aGed bi ba deue taneians

7, Special characters;/Such: ASe sce ieasede weve arenas eawideda pena overs nhwes sale tenes Us oa Hansa waeeNe xq ay

MeEPOSS, VRCUUIES s ucuey svexuec qos 4404 ewer pee ndonmanas ca tg even apemnweeneN tat BGR. Gekdece_ans

pleomorphic and involution forms, capsules, etc ..... 2.2.6.0. se sete eee cee eee eres teen cee ee ec ees

PHYSIOLOGICAL CHARACTERS.

5. Gas production in glucose media:

@s SHAKE Gul tu teha sip sisicists sp saaicionss pe visinis aise aedceorns bare Sioesatd oa ate golem Ragin ee eme ae Pe aus oh een ha

8. Indo} production; 24 hours

fecal odor 24¢ HOULS sas wees se ce eeeel aepeeeeag ve ay tuee

g. Enzyme production: proteolytic............ cece cee eee ce ee eee ee et renee ee tence eeee diastatic

qr> PAathOgetesis .iiisiecanasicrcrsawsancaane wick wes tcae mcahtelanials og aneieharentanh ceenaeN ee Maat

CULTURE CHARACTERS.

Reaction
of Medium,
Incubation
Temp. (°C)

SKETCHES.

(1)
~ Gelatin
~ plate:

(a) Surface
R Colonies.

(b) Deep
~ Colonies.

(2)

Agar
plate:
* (a) Surface
Colonies.
(b) Deep
Colonies.

(3)

Gelatin
Stab.

(4)

. ie
Streak.

A Bouillon.

4

a
j \
A \

|
SS oo

[161]

. CHAPTER VIII.
ANIMAL INOCULATION AND STAINING OF BACTERIA IN TISSUE.

EXERCISE Cill. ANISLAL INOCULATION.

MeEtTHops oF INOCULATION. Animal inoculation is practiced to determine the path-
ogenic properties of an organism and also the character of the tissue changes produced.
The animals commonly used are white mice and rats, rabbits, guinea pigs and pigeons.
Inoculations are usually made intraperitoneally, intravenously or subcutaneously, and
in special cases into the pleural cavity, brain, eye, etc., etc. Mice require a holder, the
inoculation being made at the root of the tail. Other animals can usually be held by an
assistant. :

Subcutaneous. The place selected is usually the abdominal wall. Pigeons are
inoculated in the pectoral muscles; the hair or feathers should be removed and the skin
washed with a disinfectant, e. g., 5% carbolic acid.

a. For liquids a sterilized hypodermic syringe is used. A fold of the skin is raised,
the needle of the syringe inserted and the requisite amount of culture injected.

b. For solid material a pocket is made which is stitched, or sealed with contractile
eollodion, after the material is introduced.

Intraperitoneal. Prepare seat of inoculation as above, then plunge needle directly
into the peritoneal cavity.

Intravenous. A rabbit is generally chosen for this purpose and the inoculation made
into the dorsal vein of the ear. Slight pressure at the base of the ear will render the
vein more prominent. Avoid the introduction of air, which causes immediate death, and
keep the animals under close observation for one hour.

Inoculation into Lymphatic system. Fluid cultures or suspensions of bacteria can
be injected into the lymphatics by way of the testicles, by plunging the point of the
needle into the substance of the testicle and injecting the desired amount of fluid.

Inoculation into the Pleural Cavity. Where necessary the needle is introduced into
the pleural cavity between the ribs. It is very difficult to perform this experiment with-
out injuring the lung.

Inoculation into the Anterior Chamber of the eye. Rarely practiced. The eye is
treated with a few drops of cocaine (2% solution) and then the needle is inserted
through the cornea just in front of its junction with the sclerotic, the needle passing
into the anterior chamber in a plane parallel to the plane of the iris. .

The following inoculations are those most frequently made:

Streptococcus pyogenes. Mice or rabbits, intravenous.

Sarcina tetragena. Guinea pigs and white mice, subcutaneous.

Bacterium anthracis. Guinea pigs or rabbits, subcutaneous.

—— pneumoniae. Rabbits and mice, subcutaneous.

—— pneumonicum. Mice and young rats, intraperitoneal.
tuberculosis. Guinea pigs, rabbits and field mice, any method of inoculation
will produce the disease.

164 Medical Bacteriology.

B. mallei. Male guinea pigs, infection of lymphatics.

—— diphtheriae. Guinea pigs, rabbits and fowl, subcutaneous and intratracheal.
Bacillus pestis. Rats, mice, guinea pigs and rabbits, subcutaneous. —
suipestifer. Rabbits and mice,- subcutaneous.

STERILIZATION OF INSTRUMENTS. These are best sterilized by boiling in a solution
of soda or borax for 15 minutes. This is accomplished in an especially designed ap-
paratus or in an ordinary enamel stew pan. In case of emergencies the instruments
may be dipped in benzene or alcohol and burned. This is less injuriousto the instru-
ment than heating in the direct flame. :

Use blank, p. 168, for preservation of data.

OBSERVATION OF INOCULATED ANIMALS. After inoculation the animals should be
placed in separate cages, or if placed together they must be described or marked so as
to be easily identified. They must also be kept under constant observation and the
following conditions noted:

Temperature.

Loss of Weight.

Peculiar position in cage.

Loss of appetite.

Condition of the coat or hair.

Condition of the secretion of the air passages, conjunctiva and kidneys; diarrhea
or hemorrhage from the bowels.

7. The condition of the seat of inoculation. ‘

The animals should be fed regularly, weighed at the same hour each day and the
temperature taken at the rectum.

Post Mortem EXAMINATION.

Perform the autopsy as soon as possible after death. When delay cannot be
avoided, place the animal in the ice-chest until such time as is convenient.

OaRowe

A.

1. Inspect externally and note presence and character of any lesion.

2. Sterilize a suitable post-mortem board with corrosive sublimate solution, 1 to
1000, place the animal belly upwards and tack the four legs fast to the board.

3. Wash the surface of the thorax and abdomen with corrosive sublimate solu-
tion, make an incision through the skin at the pubis, introducing one blade of the scis-
sors, and extend the incision as far as the chin.

4. Carefully dissect the skin away from the abdomen, thorax, axillary, inguinal,
and cervical regions, and fore and hind legs, and pin it to the board as far as possible
from the thorax and abdomen. It is from the skin that the chances of contamination
are greatest. .

B.

All incisions from now on are made with sterilized instruments.

1. Take an ordinary potato-knife, heat it quite hot, and place it on the abdomen in
the region of the linea alba until the fascia begins to burn; the knife is then held trans-
versely to this line over the center of the abdomen, making two sterilized tracks through

which the abdomen may be opened by crucial incisions: two burned lines are also made
along the sides of the thorax.

166 Medical Bacteriology.

2. Make a central longitudinal incision from the sternum to the genitalia with
sterile scissors, the abdominal wall being held up with sterilized forceps, or a hook to
prevent the viscera being injured. A transverse incision is made in a similar man-
ner. Cut through the ribs with strong sterilized scissors along the sterilized tracks on
the sides of the thorax, when the whole anterior wall of the thorax is easily lifted and
entirely removed by severing the diaphragm connections.

3. When the thoracic and abdominal cavities are fully exposed, a careful examina-
tion of the organs and surroundings is made without disturbing them.

Culture plates (Petri-dish) or roll cultures are prepared from the blood, liver, spleen, .
kidneys, and any exudates present.

The method is as follows:

(1) Heat a scalpel and scorch a small surface of the pen from which the cultures
are to be made.

(2) Heat the scalpel again and penetrate the capsule of the organ with the point,
and through the opening insert a stout sterilized platinum loop, push it into the tissues,
twist around, and obtain enough material from the center of the organ to make the
culture.

Cultures from blood are usually made from one of the dart cavities, the surface
being seared with a hot knife before opening. As soon as the culture material is ob-
tained, cover-glass specimens are prepared from each organ and existing exudates.

Small pieces of each organ are also preserved for future examination.

When the autopsy is finished the remainder of the animal should be burned and the
instruments should be sterilized. Wash the post-mortem board with sublimate solution.
The cover-glasses and other material likely to contain infectious matter must also be
sterilized when of no further use.

Cultures are to be incubated at 38° C., growth examined microscopically, and by
means of sub-cultures.

REFERENCES. The above is } taken largely from Bowhill, 74; see also A. 219; N.
260; and other texts.

168 Medical Bacteriology.
BLANK FOR ANIMAL EXPERIMENTS.

ATVI 555 cs pains ioe Socio NOK a eal ee ees ABE oo eececcetecees Weight...
Specimens received .o...0... occ cceccceccevecceececececscuesecteceeueevseetsesttgeteseenss o’elock.......... MM.
Organs

Experiment:

STV eA 5 5 pa cya eed aca casas de a le en Sin esc mane NIE easy M.
ARES cca a apn Dameaee Gs We esd aaeo sea iad bees dawie eda oa Sry gO ONTO, oa IML
Findings:

Bacteriological Examination:

Histological Examination:

neo adiaatedabapubaben new

170

Medical Bacteriology.

EXERCISE CIV. PREPARATION OF TISSUE FOR EXAMINATION.

Portions of the diseased tissue, removed at autopsy, should be cut into cubes hav-
ing edges about 5 mm. long and treated as follows:
1). Frxme. Use 15 or 20 times their volume of 95% alcohol for 24 hrs. The speci-
mens should be placed on cotton to keep them near the top and the alcohol changed
atter 3 or 4 hours, if they are not to be sectioned immediately carry to 80% alcohol.
Where larger sections are desired they should be left a longer time in the alcohol.
2). PREPARATION FOR SECTIONING.

A.
Paraffin Method.
|

a. Absolute Alcohol 6-24
hours. |
_b. Xylene 6-24 hours.

|
c. Paraffin melting at 50°C.
and kept in anoven or water-
bath at a temperature a few de-
grees above the melting point
of the paraffin.

|

d. Embed. Pour’ melted
paraffin into a paper box or other
suitable receptacle and with
warm forceps, arrange block
of tissue in proper position and
coolrapidly by plunging into cold
water.

|

B.

Celloidin Method.
|
a. Mixture of ether
and absolute alcohol (equal
parts) 24 hours.

|
b. Thin celloidin (about

6%) 24 hours to several
weeks.

|
c. Thick celloidin
(about 12%) 24 hours to
several weeks.

|

d. Remove block of |

tissue to a piece of wood
fiber covered with ‘‘thick’’
celloidin, orient, dry a few
minutes in air then place in
80% alcohol for 6-24 hours.

|

Cc.

Freezing Method.
a. Place in 1%

Formalin 2 hours.

|

b. Place tissue on
plate of freezing
microtome in water or
better first soak tissue-
in a syrupy solution
of gum-arabic and
moisten plate with
same before freezing.

8). SECTIONING. Cut sections from 10-12 » thick.

4). MANIPULATION OF SECTIONS.

a. Celloidin sections can be preserved in 80 % alcohol and are best stained by
placing the sections first in water and then in the stain. The various reagents are best
used in watch glasses and the sections transferred from one to the other by means of a
section lifter.

b. Paraffin sections should be fixed to the slide or cover-glass as follows: A water-
bath is heated up to a few degrees below the melting point of the paraffin, the sections
are placed on the water where they will straighten out and are then transferred to the
slide or more conveniently to the cover-glass by simply dipping the same into the water
and drawing up the section by means of the fine point of a pair of forceps or a needle,
draining off the water and drying the section in an incubator for a few hours. The sec-
tions are more secure if the cover-glasses are first smeared with a thin coat of egg
albumin. When the sections are once fixed to the cover the staining can be carried on
in the forceps as with ordinary cover-glass preparations. Before staining, however, the
paraffin must he removed; this is done with xylene and this in turn removed with absolute
alcohol.

REFERENCES. A. 173; M. & W. 204-239; N. 581.

172 Medical Bacteriology.

EXERCISE CV. STAINING SECTIONS.

GENERAL HISTOLOGICAL METHOD.

Hematoxylin and Eosin.

a. Transfer sections from alcohol to distilled water.
; db. Stain in alum-hematoxylin 2, 5 to 30 minutes. The stain may be prepared as
follows (Boehmer):

1. Hematoxylon erystals, - - - - - - 1 gram.
Absolute alcohol, - - - - - - - 10ce.

2. Alum, - - - - - - - - 20 grams.
Distilled water, - - - - - - - 200 ec.

Cover the solutions and allow them to stand over night. The next day mix them
and allow the mixture to stand for one week in a wide-mouthed bottle lightly plugged
with cotton. Then filter into a bottle provided with a good cork. The solution is now
ready for use but its staining powers improve with age.

c. Wash the sectionsin several changes of water until they have lost all traces of a
red tint.

d. Counter-stain with eosin (jy to + % in 60 % alcohol) 1 to 5 minutes.

e. Alcohol, 95 %, two or three changes to dehydrate and remove excess of counter:
stain.

f. Clear in oil of origanum or Dunham’s mixture, white oil of thyme 4 parts, oil
of cloves 1 part.

GENERAL BACTERIOLOGICAL METHODS.

A. Loeffler’s Universal Method.
a. Take sections out of alcohol into Loeffler’s methvlen blue for 5-30 minutes.
b. Decolorize in acetic acid (0. 1%) 10 to 20 seconds.
c. Dehydrate in absolute alcohol, two or three changes, a few seconds.
d. Clear in xylene.
e. Mount in balsam.

B. Weigert’s Method.
a. From aleohol to Ehrlich’s waitin water gentian violet 5-15 minutes.
b. Wash in 0. 6% salt solution.
c. Dry with filter paper.
d, Place in potassium iodide and iodine solution (iodine 1 part, potassium
iodide 2 parts, water 100 parts).
e. Dry with filter paper.
J. Decolorize in a mixture of anilin oil 2 parts and xylene 1 part, 2-5 minutes.
g. Clear in xylene.
h. Mount in balsam.
This stain can only be used with those organisms which take the Gram stain, namely:
S. pyogenes, M. pyogenes, M. aureus, Sar. tetragena, B. anthracis, B. pneumoniae, B.
rhusiopathiae, B. tuberculosis, B. leprae, B. diphtheriae, P. aeruginosa, B. Welchii, B.
chauvaet, B. oedematis, B. tetani and Strevtothrix actinomyces.

174 Medical Bacteriology.

SPECIAL BACTERIOLOGICAL MEtTHopS.

Particular organisms may be stained as follows:

Pyogenic micrococci. Loeffier’s or Weigert’s method.

Micrococcus gonorrhoeae. Loeffler’s method gives the best results.

Sarcina tetragena. Loeffler’s or Weigert’s method.

Bacterium anthracis. Loeffler’s or Weigert’s method.

Bacterium pneumoniae. Weigert’s method.

Bacterium pneumonicum. The following method is recommended for staining the cap-
sules in sections (M. & W.):

a. Stain for 24 hours in the incubator in the following solution:

Saturated alcoholic solution of gentian violet - - - - 50cc.
Distilled water - - - a - . - - 100cc.
Glacial acetic acid - - - - - - - 10ec.

b. Wash out in 1% solution of acetic acid.

c. Alcohol.

d. Xylene.

e. Canada balsam.

Bacterium cuniculicida. Loeffler’s Method.

Bacterium tuberculosis.

a. Weigert’s method (staining with anilin oil gentian vale 24 hours at room
temperature, or 2-8 hours at 40° C.).

bp. Ziehl-Neelsen’s Method.

1. Stain with carbol-fuchsin (12-24 hrs. room temperature, 1-3 hrs. 40° C.)

2. Decolorize with nitric acid (10%) a few seconds and then with alcohol (60-90%)
until color is nearly all extracted.

8. Counter-stain with methylen blue.

4. Dehydrate with absolute alcohol (a few seconds).

5. Clear with clove oil.

6. Xylene (and examine).

7. Mount in balsam.

Bacterium leprae. This organism is stained with the tubercle stain, unless the sec-
tions have been kept in alcohol for some time, in which case Weigert’s method can be
employed. To differentiate this organism from B. tuberculosis, stain as follows:

. An aqueous solution of fuchsin 6-7 minutes.
Acid alcohol (nitric acid 1, aleohol 10) + minute.
Wash in water.
Counter-stain in a saturated aqueous solution of methylen blue.
Alcohol.
Xylene.
. Balsam.
The bacteria of leprosy stain readily by this method, tubercle bacteria do not.
Bacterium mallei.
Slow Method.
a. Stain in Loeffler’s methylen blue 6-8 hours.
b. Wash in distilled water.
c. Tannic acid solution (10 %) 4-5 hours.

a Ms aces

176 ‘Medical Bacteriology.

d. Wash thoroughly in water.
e. Dehydrate in absolute alcohol.
jf. Clear in xylene and mount.

Quick Method.
. Stain in carbol-methylen blue 10-30 seconds.
. Wash in distilled water. .
Tannic acid solution (10 %) 4-1 minute.
. Counter-stain with a weak solution of eosin until sections are red.
Wash in water until pink. :
Dehydrate in absolute alcohol.
. Clear in xylene and mount.
Bacterium diphtheriae. Loeffler’s or better Weigert’s method.
Bacillus typhosus.
. Loeffler’s methylen blue or carbol-fuchsin 15 min.—24 hrs.
. Wash slightly in distilled water.
Place in 10% solution of tannic acid for 10-60 min.
. Dehydrate rapidly in alcohol.
Clear in xylene.
Examine.
. Mount in balsam.

Such sections examined under a low power will be found to contain heavily stained
masses, which under a high power prove to be clumps of bacilli. Not infrequently the
bacilli are difficult to detect in tissue from typhoid cadavers.

Bacillus suipestifer. Loeffler’s method.

Bacterium Welchii. Weigert’s and Loeffler’s methods.

Bacillus chauvaei. Use Pfeiffler’s stain:

a. Dilute carbol-fuchsin % hour.

b. Absolute alcohol slightly acidutated with acetic acid until section is a reddish
violet tint. :
c. Xylene and examine.

d. Mount in balsam.

Bacillus oedematis. Pfeiffer’s stain.
Streptothria actinomyces.
. Ziehl’s carbol-fuchsin, 10 minutes.
. Wash in distilled water.

Picrie acid (cons. ale. solution).
. Wash in distilled water.

Wash in alcohol (50%).

Dehydrate in absolute alcohol.
. Clear in xylene.
. Balsam.

Tissue stained yellow, rays red.

REFERENCES. M. & W. 2389-286; N. 537.

ea MPrasae

aeme aAaows

FeMeracsws

CHAPTER IX.

BACTERIOLOGICAL DIAGNOSIS.

EXERCISE CVI. EXAMINATION OF BUCCAL SECRETION.

DEFINITION. The secretion of the mouth, or saliva, is a mixed product derived in
part from the mucous glands within the mouth and also from the parotid, submax-
illary, and sublingual glands. In disease the normal character of the different parts may
vary or there may be various exudates and growths present.

COLLECTION. Material for bacteriological examination is best obtained by means of
a sterile probang or forceps. This material may be examined directly by means of
cover-glass preparations or by means of cultures.

1. Method of Preparing Outfit. Wind a small piece of absorbent cotton on the end
of a wire (about 1 mm. in diameter and 14 em. long). Thrust the other end of the
wire through the cotton plug of a test-tube or fasten in a cork and sterilize at 150° C. for
lhour. This with a tube of nutrient medium (usually Leoffler’s Blood serum) is placed
in a box for transportation.

2. Method of Using Outfit. The patient is placed in a good light and the probang
gently but firmly rubbed over the suspected area of the throat and then drawn gently
over the surface of the medium, both tubes securely stoppered and the outfit sent to the
laboratory. The organisms to be sought for are B. diphtheriae, the pyogenic cocci and
Monilia candida.

BacCTERIUM DIPHTHERIAE..

The presence of this germ in the mouth usually results in a formation of a pseudo-
membrane a portion of which is to be removed with a pair of forceps or by means of the
outfit described above. It should, 1) be examined directly for the ae bacillus by
smearing on a cover-glass and staining by following methods:

a. Loeffier’s methylen blue.

b. Gram’s stain.

ce. Neisser’s stain: a. 1 gram methylen blue dissolved in 20 ce. of alcohol (96%), is
added to 950 cc. of distilled water and 50 ec. of glacial acetic acid; b. 2 grams of bismark
brown dissolved in a liter of distilled water. Films are stained in a. 2 to 3 seconds, washed
in water, stained in 0. 3 to 5 seconds, dried and mounted.

2) Usually, however, mere microscopical examination is not sufficient, and culture
methods must be employed. In fact this method ought always to be used.

In this case make smears on Loeffler’s blood serum and incubate them at 36-38° C.
for 12-24 hours and then examine the growth in cover-glass preparations. The diphthe-
ria organism if present should show:

a. Characteristic appearance with Loeffler’s methylen blue.

b. Positive Neisser stain.

c. Positive Gram stain.

180

Medical Bacteriology.

3) Occasionally micro-organisms (pseudo-diphtheria bacilli among others) are met
with that very closely resemble the Klebs—Loeffler bacillus and render a positive diag-

nosis doubtful.

In such eases attention to following table will be helpful:

B. Diphtheriae

B. pseudo-diphtheriae

1) Form

2) Size

3) Threads
4) Grouping

5) Involution forms

6) Motility
7) Stains
a. Loeffler’s methylen blue

b. Gram
c. Neisser

8) Spores

9) Alkaline potato

10) Sugar agar and gelatin
stab cultures
11) Neutral litmus milk
12) Anaerobic cultures in H
13) Nitroso-indol reaction
14) Inoculation experiments
(Guinea pig subcutaneous)

Slender and of same diameter
throughout

Average 1.2-2

Not formed

Parallel grouping more or less
characteristic but do not touch
Common

Immotile

Stains readily giving banded
or polar stain

Positive

Characteristic stain with very
young cultures, six hours.
Absent

Growth almost invisible

Full length of stab
Acid reaction
Grows well

After 7 days

Death 36-48 hours.

Thicker at center than ends,
plumper and shorter and less
variable than B. diphtheriae
Averaging 1-1.6 @

Not formed
Parallel but lie closer together

Rare

Immotile

Stains more regularly
Polar stain rare
Positive

Not under 24 hours
Absent

Visible and cream colored in 2
days

Only at upper part
Alkaline reaction

No growth

After 21 days

Non-pathogenic

PyoGcEenic Micrococct.

1) Stained cover-glass preparations are examined and if micrococci are found make: |
2) Smear cultures, or better agar plate cultures and work up the colonies as they

appear.

Monta CANDIDA (Organism of Thrush).
The material is collected by removing a portion of the patches or membrane and ex-

amining it:

1) Under the microscope in a drop of glycerine.

2) Cover-glass preparations stained with carbol-fuchsin or Gram’s method.

3) By means of smear cultures on agar or blood serum, the resulting growth being
examined either in glycerine mounts or stained cover-glass preparations.

REFERENCES.

EXERCISE CVII.

v. J. 95; 8. 101.

See also various texts under special organism.

EXAMINATION OF SPUTUM.

Definition. By this term is meant all of the material derived from the air passages
by the act of coughing or hawking.
For diagnostic purposes it is best collected in a salt-
mouthed bottle (about 2 oz. capacity) which has been sterilized. The morning sputum
is best and before being collected the mouth should be rinsed out with water.

METHOD OF COLLECTION.

182 Medical Bacteriology.

BACTERIUM TUBERCULOSIS. Place the sputum in a Petri dish over a black surface
and select one of the little cheesy masses, if these are present, and smear it on a cover-
glass. Where these particles are not present a loop or two of the thick portion is used.
The cover-glass preparations are to be stained by one of the following methods:

1) Gabbett, see Part 1, p. 38.

2) Ziehl-Neelson:

a. Carbol-fuchsin ten times through the flame.

b. Nitric acid (30%) momentarily.

c. Water.

d. Aleohol (60%) until red color disappears. It may be necessary to immerse
preparation in acid a second time, but the greatest care must be exercised to prevent
extraction of dye from tubercle bacterium.

é. Loeffler’s methylen blue, 1 minute.

f. Mount and examine:

‘While the tubercle bacteria may be detected when present in considerable numbers
with a } in. objective when there are few present a 7s in. oil immersion will be neces-
sary, and this ought to be used to search all slides where the tubercle germ has not been
_ found with a lower power. A mechanical stage is a great convenience in a systematic
search.

At least two preparations should be stained and thoroughly examined before a meg
ative result is pronounced.

The viscosity of sputa may be overcome and the bacteria concentrated where the num-
ber is very small by 1) Ribbert’s method which consists in the addition of a 2% solution
of caustic potash and boiling. This dissolves the mucus and the bacteria are then depos-
ited with the sediment. This sediment can be obtained by allowing the mixture to

stand in a conical glass vessel or more quickly by the use of a centrifuge. 2) Ham-
mond’s method:

1. Add 5% of crystallized carbolie acid (in the case of sputum add 5 times its bulk
of a 5% solution of carbolic acid).

2. Place 15 ec. in the tubes of a centrifuge and whirl for 15 minutes.

3. Pour off supernatant fluid and treat precipitate with 8 cc. of a 5% KOH solution.
Mix thoroughly and allow to stand 2 minutes.

4. Fill to 15 ce. mark with distilled water and whirl 20 minutes.

5. Make cover-glass preparation of sediment (or purify same by repeated washings
and centrifugalizations with distilled water).

A centrifugal machine should be able to make at least 2,500 revolutions per minute.
This speed ought to be maintained for 15 minutes. Sputum may be preserved by ad-
dition of small quantity of carbolic acid (5%).

Negative results are of positive diagnostic value only when repeated examinations
are made of different samples taken at different times.

BACTERIUM INFLUENZAE. This micro-organism is frequently present in enormous
numbers (100 or more) and sometimes in almost pure cultures in the greenish purulent
masses in the sputum. It stains readily with the ordinary dyes, and when lightly stained
presents the bipolar stain. Carbol-fuchsin diluted 10 times is one of the best stains.
Gram’s stain is negative.

Sputum from suspected cases should be collected either by means of a probang or
in a bottle and examined:

184 Medical Bacteriology.

1) Microscopically by staining, with a weak carbol-fuchsin, smears from the puru-
lent masses. If a very small bacillus is in large clumps, which fails to retain stain by
Gram’s method, the evidence is strong that it is the influenza bacillus; the diagnosis
should be confirmed, however, by

2) Cultures on blood agar.

Animal inoculations are without effect.

BaCTERIUM PNEUMONIAE.

The sputum of patients suffering from pneumonia is usually of a rusty color due to
presence of blood. The ‘‘pneumococcus’’ is readily seen in such material when stained
by Gram’s method, or with carbol-fuchsin and momentarily washed with alcohol, as
lancet-shaped organisms with outer ends pointed and surrounded by a clear area—the
capsule. The capsule can be easily stained by Welch’s method. (See XXXVI.)

This organism is also frequently found in the sputum of healthy persons and small
numbers may be detected by means of animal inoculation. The rabbit or mouse are
most susceptible and should be inoculated intraperitoneally. As a result of infection
with this organism the animal quickly dies with a typical septicaemia, the micro-organ-
isms being found in great numbers in the blood current.

BACILLUS PESTIS. This micro-organism is frequently found in the sputum especially
in the pneumonic form of the disease—for methods of detection see CX.

STREPTOTHRIX ACTINOMYCOSES. This organism has been occasionally found in
sputum and in such eases the peculiar morphology of the colonies is well brought out
by Gram’s method. See CX.

REFERENCES. v. J. 114; S. 245. See also various texts under particular organisms.

EXERCISE CVII. EXAMINATION OF BLOOD.

For serum test (Widal reaction) the blood may be collected and dried (see below),
but in other cases where cultures are to be made the blood must be collected aseptically
in sterile receptacles and hermetically sealed. For this purpose Sternberg’s bulb is ex-
cellent. The skin should first be sterilized by use of corrosive sublimate or carbolic acid
followed with alcohol.

It is usually well in any case to make cover-glass smears at the bed-side for micro-
copical examination. These are best made as follows: Place a drop of blood about
the size of a pin-head on a perfectly clean cover-glass and then a second cover-glass on
this; this flattens the drop of blood out into a thin film. Immediately and before coag-
sulation can take place the two are drawn apart horizontally and the films allowed to dry.
(Cabot.)

BacTERIUM ANTHRACIS. In case of animals dead of suspected anthrax, blood or
portion of spleen should be removed with least possible danger from infection or distri-
bution of bacilli and studied as follows:

1. Microscopical examinations of blood or the spleen pulp of animals show (when
stained with Loeffler’s methylen blue) large bacteria in chains (5 or 6 segments) pre-
senting the bamboo appearance.

2. In hanging drop preparation large, homogeneous, immotile bacilli.

3. Agar plate cultures should also be made and from the separate colonies subcul-
tures; the gelatin stab being especially characteristic.

186 Medical Bacteriology.

4, In important cases (as in man) guinea pigs, or white mice, should be inoculated,
and in case of death organism isolated and identified.

SPIRILLUM OBERMEIERI. This organism is found in the blood only during a par-
oxysm. It is a long slender organism 6 or 7 times the diameter of a red blood corpuscle.
(45) They have a brisk vibratile movement in the direction of their long axis. They
are very sensitive to reagents of all kinds. Even the addition of distilled water will cause
them to disappear. Fresh blood is best, but dried smears may be used and stained
with fuchsin or by Gunther’s method:

a. Dried films are treated with acetic acid (5%) 10 seconds, this is removed by
blowing and holding film over flask of strong ammonia previously shaken.

b. Stained in Ehrlich’s gentian violet.

c. Washed with water.

d. Dried.

e. Mounted in balsam or xylene.

jf. Examined.

Pyvocenic Micrococcr. These are occasionally found and for method of detection
see CX. : -

BacTERIUM MALLEI. Sometimes found in the blood of those suffering with Gland-
ers. It may be detected in the blood-smears. For special methods see CX.

B. PNEUMONIAE. This germ is frequently present in fatal cases 24 to 48 hours be-
fore death. The blood should be drawn with a sterile hypodermic syringe and about
1 ce. of blood mixed with a tube of melted agar at 43°C. and poured into a Petri dish.
Characteristic colonies appear in 24 to 48 hours.

B. TuBERcULOSIS. In ease of miliary tuberculosis they may be very rarely found in
sufficient numbers to be detected by staining methods, see sputum CVII.

B. INFLUENZAE. Canon claims to have stained and cultivated this organism in blood,
but this needs confirmation.

B. cout. This organism may be found in the blood, for methods of isolation and
identification see Faeces CIX.

BacILLuS PESTIS. This germ occurs in the blood in certain cases at least but ap-
pears to require considerable skill in detecting it due to its variable appearance. Broth
tubes should be infected and animals inoculated.

BACILLUS SUIPESTIFER.

a. Make agar plate and streak cultures from spleen of dead animal, and work up
the colonies as they appear. ‘

b. Widal Reaction (for technique see below under B. typhosus).

PLASMODIUM MALARIAE.

a. Examination of fresh blood. A droplet of blood from finger or lobe of ear is
placed on a glass slide, covered with a cover-glass and then the cover-glass is ringed with
vaselin. Examination should be made with a 75 in. oil immersion.

b. Stained. Prepare films as directed above and stain with methylen blue and
eosin or treat films with a very weak acetic acid 2 or 3 drops to 30 cc. of water; to
remove haemoglobin wash with water and stain with following solution for } y minute:
Borax - - : 7 : : :

- - 5.0 parts.
Methylen blue - - - : - : - - 0.5 Bes
Water - - - eee - - = - '- 100.0 parts..

Wash, dry and mount in balsam (Manson).

188 Medical Bacteriology.

BACILLUS ICTEROIDES. Make agar streaks from blood or fragment of liver (where liver is
obtained it is best wrapped in cloth and kept in incubator at 88°C. for 12 hours before
cultures are made to encourage development of the micro-organisms, which are usually
only sparingly present in tissue). Keep the cultures at 38°C. for 12-16 hours and
then at 22°C. for same time; the characteristic appearance is a transparent, bluish
growth surrounded by an opaque zone. If this is not obtained other culver must be
prepared and a thorough study of the organisms isolated made.

REFERENCES. v.J. 45; 8. 79. See also texts under particular organisms.

Wipau Reaction. Directions for collecting samples of blood. ‘‘ Wash with boiled
water the part from which the blood is to be obtained (lobe of ear, end of finger, or toe
in infant). Prick deeply the skin with a clean needle.’’ Remove two or three large drops
of blood on a clean glass slide, alluminum foil, piece of isinglass or letter paper.

Allow the blood to dry. Then place in an envelope and send to laboratory and test
as follows:

a. Make a hanging drop preparation from a 24-72 hour old agar, or bouillon, cul-
ture of Bacillus typhosus.

b. If the bacilli are actively motile, remove the cover-glass, add to the culture a
small drop of a solution of typhoid blood (diluted from 10-50 times), return the cover-
glass to the slide and seal well with vaselin.

¢. Examine with a high dry power (% in. obj.) rather than with the oli immersion.

In a typical reaction the motility is almost immediately affected and soon motion
ceases altogether while the bacilli collect in clumps, i. e. become ‘‘agglutinated.’’

REFERENCES. v.J. 49; 8.79. See also texts under particular organism.

EXERCISE CIX. EXAMINATION OF FAECES,

The material expelled from the rectum and comprising the substances from the
food and the secretions of the alimentary tract come under this head. The number of
micro-organisms occuring here is enormous, and comprise a large number of species and
among them several pathogenic forms particularly B. typhosus. M. comma, B. tubercu-
losis and Amoeba coli.

BacILLUS TYPHOSUS. This organism occurs in the faeces in the case of typhoid pa-
tients, but on account of the large number of other organisms its detection is very diffi-
eult The following methods are the most serviceable:

Parietti’s Method. This method consists in adding Parietti’ s solution (carbolie
acid 5 grams: hydrochloric acid 4 grams, and distilled water 100 ec.) to bouillon in
the following manner: A number of tubes of bouillon have a varying quantity of the
above solution added, e. g, 1 drop to one tube, 2 to another, 3 to another, and so on.
These tubes are inoculated with a small quantity, (one or two loops), of the faeces
and then placed in the 38° C. incubator. Twenty-four hours later the tube containing
the largest amount of Parietti’s solution which shows growth probably contains B. coli
and B. typhosus if it is present. The organisms may be separated most quickly and
easily by the use of the lactose litmus agar plate. The blue colonies should be worked
up, and especially tested for its agglutinating power on typhoid blood. Instead of the
use of the lactose litmus agar plate, either Elsner’s or Hiss’ methods may be used.

190 Medical Bacteriology.

Esner’s Medium. Method of preparation:

Peel and cut up 500 gms. of old potatoes of medium size, add 1000 ce. of water and
boil 1 and 4 hours.

Mash potatoes thoroughly; strain through a cloth and add water to filtrate to make
a liter.

Add 15 % gelatin and boil 10 minutes. Cool to 60° C. and add white of one egg
and boil 15 minutes.

Filter through cotton, then paper. Titrate and make gelatin 2-3 % acid. Just before
tubing add 1 % potassium iodide (10 ce. of a solution in which 1 ce. contains 1 gram of
potassium iodide). Tube and sterilize three times.

Plates of this medium are made in the usual way and kept at 15-18°C. On this me-
dium the typhoid germ forms very finely granular, small, bright droplets resembling
condensed moisture, while the colon bacillus gives rise to larger, brown colonies, which
are more granular and spread more.

Hiss’ Plate Medium. This contains:

10 grams of agar.

25 grams of gelatin.
5 grams of beef extract (Leibig).
5 grams of sodium chloride.

10 grams of glucose.

1000 grams of water.

_ It is made by first dissolving the agar, salt and extract in the water, then the gelatin
is added and dissolved, the reaction changed by use of NaOH and phenolphthalein so
that it will contain not less than 2% normal acid, cleared with two eggs and filtered, glu-
cose added and the medium tubed and sterilized.

Make plate cultures in ordinary way and incubate at 38° C. for 18 hours, then ex-
amine the colonies microscopically. The colonies of B. typhosus have irregular out-
growths and fringing threads. The colonies of B. coli, on the other hand, are much large
and as a rule are darker in color and do not form ehyaada,

The colonies may be further examined by the use of Hiss’ Tube Medium.

5 grams of agar-agar.

80 grams of gelatin.
5 grams beef extract (Leibig).
5 grams sodium chloride.

10 grams glucose.

1000 grams water.

Made as plate medium except that is is to contain 1.5% normal acid.

Within 18 hours at 38° C. the typhoid bacilli produce a uniform clouding. The
colon bacilli do not produce uniform clouding and do produce gas.

All suspected cultures should be tested with typhoid blood (Widal reaction).

The typhoid organism may be isolated from the stools during the first two weeks of”
the disease.

MICROSPIRA COMMA.

1. Microscopical examination of ‘‘rice-water’’ discharges for spirilla lying parallel.

2. Culture methods. Gelatin or agar-plates should be made from the rice-like flakes;
other flakes should be inoculated into flasks of peptone water (Dunham’s solution) and
inoculated at 38° C. The surface growth 6-12 hours later is to be examined microscop-

192 Medical Bacteriology.

ically and by means of plates. Then test the peptone cultures for nitroso-indol (cholera
red reaction) by the addition of a few drops of sulphuric acid.

BACTERIUM TUBERCULOSIS. This organism has been found in the stools in cases
of intestinal ulcerations, and may come, in cases of phthisis, from ingested sputa.

AMOEBA COLI.

1. A drop of the mucous portions of stool is placed on a glass slide, covered with a
cover-glass and examined with a magnification of about 500 diameters (4 in. objective).
Examination should be conducted on a warm stage in order to get amoeboid movements.

2. Preparations may be stained with methylen blue and carmine. The nucleus
is stained with the carmine.

3. Discharge may be hardened and stained by Mallory’s method as follows:

a. Fix tissues in alcohol.

b. Stain (paraffin) sections in a saturated aqueous solution, of thionin for 5-20
minutes. :

c. Wash in water.

d. Differentiate in a 2% aqueous solution of oxalic acid 4-1 minute.

e. Wash in water.

f. Dehydrate in aleohol (95%).

g- Clear in oil of bergamot.

h. Wash with xylene and mount in balsam.

Nuclei of Amoebae brownish red, other nuclei blue.

REFERENCES. v. J. 199; Si. 228. See also texts under various organisms.

EXERCISE CX. EXAMINATION OF URINE.

For bacterial examination urine should be drawn with a sterile catheter into a sterile
bottle.

BACTERIUM TUBERCULOSIS.

For method of staining see under Sputum, CVII.

It is best to centrifuge the product and care must be taken to differentiate from the
Smegma bacterium. or this purpose stain cover-glass smears as follows (Bunge &
Franteroth.):

1) Absolute aleohol, 3 hours.

2) Chromic acid, 15 minutes.

3) Stain in hot carbol-fuchsin.

4) Decolorize in sulphuric acid (25%) 2-3 minutes.

5) Counter-stain with a saturated alcoholic solution of methylen blue.

The smegma bacillus is decolorized by this method.

Tubercle bacterium in urine is frequently present in clusters while the smegma
bacterium occurs singly. Injection of guinea pigs, smegma bacillus is non-pathogenic.

The following organisms have also been found in the urine. For methods of isolation
see references.

PYROGENIC MIcROcoccI.. CXI.

M. GONORRHOEAE. CXI.

B. TypHosus. CIX.

S. OBERMEIERI. CVIII.

REFERENCES. v. J. 273; Si. 504. and texts under the various organisms.

194 Medical Bacteriology.

EXERCISE CXI. EXAMINATION OF TRANSUDATES AND EXUDATES.

The material should be collected in sterile vessels under aseptic precautions. Make
several cover-glass preparations and stain one with Loeffler’s methylen blue and the
others with gentian violet or carbol-fuchsin. Mount and examine.

a. If staphylococci alone are present search for the pyogenic micrococct.

b. If streptococci suspect S. pyogenes.

c. If diplococci.or tetracocci.

1. Within the pus-cells test for M. gonorrhoeae or M. intracellularis.

2. Free. 8. tetragena:

d. If bacilli any of the following may be searched for:

1. B. coli. This organism is likely to be found especially in suppurative
peritonitis and diseases of the urinary organs. 2. B. anthracis. 8. B. pneumoniae.
4, B. tuberculosis. 5. B. leprae. 6: B. mallet. 7. B. pestis. 8. P. aeruginosa. 9.
B. welchti. 10. B. oedematis. 11. B. tetani.

e. Streptothrix actinomyces.

f. Amoeba coli.

PYOGENIC MICROCOCCI. These organisms are frequently present in pus and should be
isolated and identified in pure cultures as microscopical examinations alone will not
suffice.

STREPTOCOCCUS PYOGENES. This organism is not infrequently present and can be
readily identified by culture methods.

MicROcoccUS GONORRHOEAE. Pus should be collected in a sterile receptacle or
spread on cover-glasses and allowed to dry, but should not be allowed to dry and then wet
up again to spread, as this destroys the pus-cells, and hence the valueof the material for
diagnosis.

Stain:

1. a. Loeffler’s methylen blue 3-5 minutes.

b. Wash in water.
e. Dry, mount. in balsam and examine with #5 in. oil immersion.
d. Look for a biscuit-shaped diplococcus within the pus-cells.
2. By Gram’s method.

a. Anilin oil gentian violet 15 minutes.

bd. Wash in water.

c. Treat with iodine solution 2 minutes. .

d. Decolorize with alcohol.

e. Counter-stain with Bismark brown, $ minutes.

f. Wash, dry and mount in balsam.

g- Examine with oil immersion.

If the gonococci are present they will be stained brown.

If diagnosis is of great importance make cultures as follows: —

1) Make 6 or more streak cultures on blood agar or better make plates on Wertheim’s
medium (p. 99). Grow at 38° C.

2) Make a set of ordinary agar plates or streak cultures and keep at 38° C.

The gonococcus grows on the first two media but not on the plain agar. The
gonococcus is the only organism that:

196 Medical Bacteriology.

1) Occurs in groups (cell-colonies) in pus-cells.

2) Is decolorized by Gram’s method.

3) Does not grow on agar at room or blood heat. (Foulerton).

MICROCOCCUS INTRACELLULARIS.

Pus may be obtained by lumbar puncture which is performed as follows. The back —
of the patient and the operator’s hands should be made sterile. Theneedle (4cm. X 1
mm. for children) should be boiled 10 minutes. The patient should lie on the right side,
with the knees drawn up and the uppermost shoulder so depressed as to present the spinal
column to the operator. The puncture is generally made between the third and fourth
lumbar vertebrae. The thumb of the left hand is pressed between the spinous processes
and the point of the needle is entered about 1 em. to the right of the median line,
and on a level with the thumb nail and directed slightly upwards and inward toward the
median line. Ata depth of 3 or4em. in children and 7 or 8 in adults the needle enters the
subarachnoid space and the fluid flows usually by drops. This is allowed to drop into an
absolutely clean test-tube, which has previously been plugged and sterilized. From 5 to 15
ec. of the fluid is a sufficient quantity for examination. Cultures should be made at once on
blood agar and plain agar (M. & W. 371.). After standing some hours, the sediment
should be examined in cover-glass preparations, stained with Loeffler’s methylen blue
and by Gram’s method.

Micrococcus intracellularis stains by Loeffler’s method and appears as a diplococcus
in groups in the pus cells, is decolorized by Gram’s method, and grows on blood-agar
and feebly on ordinary agar at 38° C.

The following organisms are also found occasionally. For methods of diagnosis see
exercises indicated.
cour. COIX.

PNEUMONIAE. Stain for capsule. Cultivate on blood-agar. CVII.
TUBERCULOSIS. OVII.

LEPRAE-. For method of staining, see CV.

MALLET.

a, Widal reaction (If in man typhoid and diphtheria must be excluded in case of a
positive reaction).

b. Examination of discharge.

1. Microscopical examination usually without result.
2. Cultures, glycerine agar and potato from pus.

ec. Animal inoculation, Straus method.

B. PESTIS.

a. Make plate cultures from blood and buboes and work up colonies.

b. Make subcutaneous inoculation into guinea pigs from bubo, and if death ensues
search for B. pestis.

P. AERUGINOSA. Hasily recognized by its culture characters.

B. WELCHII.

This germ is non-pathogenic for rabbits but Welch and Flexner have shown that
if a rabbit is inoculated intravenously with 0.5 to 1 cc. of a bouillon culture and killed
after a lapse of 5 or 10 minutes and the animal kept at 18°-20° C. for 24 hours or at
30°-35° C. for 4 to 6 hours, the organism will multiply in the blood and produce large
quantities of gas in the vessels and organs. This effect is characteristic.

wo Oo ho

198 Medical Bacteriology.

B. OEDEMATIS.

a. Make cover-glass ee paratows from fluid of affected parts.

b. Also make anaerobic cultures. If material contains spores it should be heated
to 80° ©. for 10 minutes before it is seeded.

B. TETANI.

a. Make cover-glass preparation from pus and search for drumstick bacillus.

b. Make cultures in glucose bouillon and agar-plates and develop in hydrogen.

c. Inoculate animals with the discharge, and also with the bouillon culture, and
- watch for characteristic symptoms.

S. ACTINOMYCES.

a. Place one of the minute sulphur yellow nodules in a drop of glycerine on a glass
slide and then apply gentle pressure.

b. Even the low powers of a compound microscope will then show something of the
clustered arrangement which can be more carefully studied under a higher power.

ce. Intraperitoneal inoculation of guinea pig. One month later nodules on peritoneum.

AMOEBA COLI. CIX.

REFERENCES. v. J. 405; Si. 514 and 518. See also texts under the various or-
ganisms.

EXERCISE CXII. DIAGNOSIS OF RABIES.

a. The medulla of the suspected animal is removed under aseptic precautions, as
soon as possible after death. In case the animal is some distance from the laboratory it
is best to cut off the head, pack it in ice and ship by express.

b. Place a piece of the medulla about the size of a pea, in 4 or 5cc. of sterile bouillon
and thoroughly grind up the same.

c. Anaesthetize a rabbit with ether, clip the hair from between the eyes and ears
and disinfect with a carbolic acid solution.

d. Make a longitudinal incision through the skin and subcutaneous tissue along
the median line, while a crucial incision is made through the periosteum on one side of
median line thus avoiding haemorrhage from the longitudinal sinus. The periosteum is
then pushed back and a disc of the skull (3 inch in diameter) removed with a trephine
and the dura mater exposed.

e. With a sterile hypodermic syringe introduce 2 or 3 drops of the suspension of
medulla beneath the dura mater, stitch the skin, disinfect, dry and seal the wound with
collodion.

‘The rabbits apparently experience no inconvenience; the wound heals rapidly and
the rabid symptoms appear in from 15 to 30 days, although sometimes they may occur
earlier or much later.

EXERCISE CXIII. EXAMINATION OF MATERIAL FROM HUSAN AUTOPSIES.

At human autopsies smears from the organs should be made on cover-glasses and
afterwards stained and examined. Plate-cultures should also be made from the various
organs or instead parallel streaks over blood serum, agar-slopes or agar-plates. In all
cases the surface from which the material is to be obtained should first be burned to
avoid infection of cultures with extraneous germs. Portions of the various organs
should also be preserved and hardened in alcohol.

CHAPTER X.

DETECTION OF PATHOGENIC BACTERIA IN WATER AND MILK
SUPPLIES.

EXERCISE CXIV. EXAMINATION OF WATER FOR PATHOGENIC BACTERIA.

Bacittus Typuosus. In the examination of water it is best to concentrate the
bacteria by filtering a large amount of the water through a Berkefeld filter and use the
slime on the filter to make the plates.

a. Parietti’s method, see CIX.

b. Hiss’ method. Make plate cultures and incubate at 38° C. for 18 hours. Inocu-
late suspicious colonies into Hiss’ tube medium, fermentation tube, milk and make indol
test. Also try Widal reaction. :

c. Animal Inoculation. (Michigan method).

1) Inoculate suspected water into bouillon tubes or flasks, and incubate at 38° C.

2) Twenty-four to forty-eight hours later inoculate one ec. into the peritoneal cavity
of a white rat.

3) If animal recovers B. typhosus is not present. If animal dies hold autopsy and
isolate and study organism causing death.

MrcrosPira CoMMA.

a. If there is reason to believe that the spirilla are very numerous gelatin plate cul-
tures can be made directly from the water, and the suspicious colonies worked up.

b. Ordinarily the organisms are very sparse and large quantities must be used, 100-
1000 ec. are placed in flasks and 1% of peptone and 0.5% salt are added, the fluid made
alkaline and incubated at 38° C. for 6-24 hours. Then gelatin plate cultures are made from
the upper layers and the suspicious colonies worked up as above.

EXERCISE CXV. EXAMINATION OF [SILK FOR PATHOGENIC BACTERIA.
B. DIPHTHERIAE.

Where B. diphtheriae is suspected in milk, make a considerable number of streak
cultures on Loefflers’s blood serum and incubate at 38° C. for 8-12 hours and examine
growth microscopically very carefully for B. diphtheriae.

BACTERIUM TUBERCULOSIS (Koch) Mig.

Hammond’s method of examining milk for B. Tuberculosis. See Sputum, CVII.
Animal Inoculation.

Concerning the transmission of material containing Bacteria in Mails, see Postal Guide, 1898
Ruling No. 82, p. 901. Part of which is as follows: ‘That the order of the Postmaster General of
June 1, 1893, forbidding the use of mails for the transmission of specimens of germs of cholera or
other diseased tissues, is hereby modified to this extent: ‘Specimens of diseased tissue may be
admitted to the mails for transmission to United States, State or municipal laboratories only when
inclosed in mailing packages constructed in accordance with the specifications hereinafter enumer-
ated. Upon the outside of every package shall be written or printed the words: ‘Specimen for
Bacteriological examination.’ No package containing diseased tissue shall be delivered to any rep-
resentative until a permit shall have first been issued by the Postmaster General, certifying that
said institution has been found to be entitled, in accordance with the requirements of this regula-
tion, to receive such specimens.”

[200]

BBE condenser, 22.
Acids, detectlon of in cultures, 52;
quantitative determination of, 52.
Agar, glucose, 44; glycerine, 115;
hanging-drop culture in, 380; lac-
tose, 4.
Agar plate cultures, 34; character of
colonies on, 58. C
Agar slopes, 12.
Air, analysis, comparative, 80; quan-
titative, 80.
Amoeba colt, in faeces, 192; in exu-
dates, 198.
Ammonia, detection of in cultures, 52.
Anaerobic cultures, 149.
Animal inoculation, 162.
Anilin dyes, 18.
Anilin oil gentian violet, 18.
Antiseptic action, 48.
Antiseptics, method of testing, 84.
Aspirator, 80.
Autoclave, 8.
Autopsies, examination of material
from, 198.

ACILLUS acidi lactici, 10.
aerogenes capsulatus, 150,
amylobacter, 40.
of anthrax, 104.
campestris, 46.
of blue-green pus, 130.
of bubonic plague, 124.
chauvaet, 152; 172; 176.
of chicken cholera, 110.
coli, 16; 24; 84; 36; 46; 48; 52; 54;
in the blood, 186; in transu-
dates and exudates, 196.
ot diphtheria, 118.
of Friedlander, 108.
of glanders, 116.
of hog cholera, 126.
icterotdes, 128; 188.
of influenza, 120.
of malignant oedema, 154.
mycoides, 26.
oedematis, 154; 172; 176; 198.
pestis, 124; 164; 184; 186; 196.
prodigtosus, 50; 56; 64; 67.
pyocyaneus, 130.
rouget du porc, 112,
of septicaemia haemorrhagica, 110.
subtilis, 16; 22; 24; 380; 34; 36; 40; 46;
48; 50; 52; 54.
suipestifer, 126; 164; 186.
of swine erysipelas, 112.
of swine plague, 110.

Bacillus of symptomatic anthrax, 152.
tetant, 40; 156; 172; 198.
tuberculosis, 114.
of typhoid fever. 122.
typhosus, 42; 46; 50; 122; 188; 192;

200.
. vulgaris, 52; 72.

INDEX.

Bacteria, in air, 80; transmission
thiough the mail, 200.

Bacteriological analysis, 80; diagnosis,
178.

Bacterium anthracis, 40, 104; 162; 184.
cuniculicida, 110; 174.
diphtheriae, 30; 118; 164; 172; 1785

200.
influenzae, 120; 182; 186.
leprae, 172; 174; 196.
mallet, 116; 164; 174; 186; 196.
phosphorescens, 68.
pneumoniae, 106; 162; 172; 174; 184;
186; 196.
pneumonicum, 108; 162.
pseudo-diphtheriae, 180.
rhustopathiae, 112; 172.
tuberculosis, 114; 162; 172; 174: 182;
186; 192; 196; 200.
welchit, 150; 196.

Bismarck brown, 18.

Blank for animal experiments, 168.

Blood, examination of, 184.

Blood serum, character of growth on,
59; collection of, 184; Loeffler’s
mixture, 88.

Bouillon, character of growth in, 58;
glucose, 44; preparation of, 4.

Brownian movement, 24.

Buccal secretions, examination of, 178.

Bunge’s flagella stain, 40.

Weapoiane balsam, 22.
Capsule stain, 42.

Carbol-fuchsin, 18.

Cell grouping, study of, 30.

Chemicals, effect on bacteria, 48.

Cholera red, 192.

Cholera vibrio, 182.

Classification of bacteria, 60.

Cleaning glassware, 2.

Colon bacillus, 16. See B. colz.

Color production, variation in, 56.

Coloring matter, separation of, 67.

Comma bacillus, 182.

Concentration of media, effect on bac-
terial growth, 46.

Cover-glass preparation, 20.

Cover-glass, cleaning of, 18.

Culture characters, description of, 57.

Cultures, fluid, 16; incubation of, 16;
stab, 16; streak, 16, test-tube, 14.

Culture media, care of, 14; preparation
of 4; 10; 12; 44; 63; 88; steriliza-
tion, 8.

ECOLORIZING agents, use, 36.
Desiccation, effect, 48.
Diplococcus of cerebro-spinal men-
ingitis, 100.
of gonorrhoea, 98.
of pneumonia, 106.

[203]

Disinfectant, 48.

Disinfectants, testing, 86.
Drawing bacteria, 28.
Dunham’s solution, 44,

Dust, relation of bacteria to, 82.

BERTH’S bacillus, 122.
Ehrlich’s anilin oil gentian violet,
18,

Elsner’s medium, 190,
Embedding tissue, 170.
Endospores, staining, 38; study of, 40
Enzymes, 54.
Esmarch rolls, 34.

AECES, examination of, 188.
Fermentation tube, 50.

Filter for gelatin, 10.
Flagella stain, 40.
Fluid cultures, 16.
Form types, study of, 26.
Fraenkel’s soil borer, 82.
Frost’s gasometer, 50.
Fuchsin, carbol, 18; Ziehl’s, 18,

ABBETT’S methylen blue, 20; tu-

bercle stain, 38.

Gas analysis, 50; detection, 50.

Gasometer, 50.

Gelatin glucose, 44; preparation, 10;
sterilization, 10.

Gelatin plate cultures, character of
colonies on 57; preparation, 82.

Gelatin stab culture, character of
growth in, 58; inoculation, 16.

Gentian violet, 18. ;

Glassware, cleaning and_ steriliza-
tion, 2.

Glucose media, 44.

Golden pus coccus, 96.

Gonococcus, 98.

Gram’s, iodine solution, 20; stain, 36.

FEMATOXYLIN and eosin stain,
172.
Hanging-drop preparation, 24.
Hauser’s spore stain, 40.
Hay bacillus, 16.
Heat, effect on bacteria, 48.
Hiss’ media, preparation, 190; use, 190;
200.

MPRESSION preparation, 30.
Incubators, 16.
Indo, 54.
Involution forms, 30.
lodine solution, Gram’s 20; Weigert’s,
172.

LEBS-LOEFFLER bacillus, 118.
Koch’s method of air analysis, 80.

ABELS, 10.
Lactose agar, 44.
Litmus, lactose agar-plate, 52; milk,
44; solution, 44; 52.
Loeffler’s blood serum, 88; tissue
stain, 172.

AILING bacteria, 200.
Methylen blue, Gabbett’s, 20;

Loeffler’s, 20.
Micrococcus aureus, 96; 172.

gonorrhoeae, 98; 174; 192; 194.
tntracellularis, 100; 196.
lanceolatus, 106.
melttensts, 94.
pyogenes, 92; 172.
tetragenus, 102.
Micrometer, ocular and stage, 28.
Microscope, use, 22.
Microspira comma, 132; 190; 200.
Jfinkleri, 136.
metschnikovt, 26; 134 .
Milk, character of growth in, 58; ex-
amination for pathogenic bacteria,
200; litmus, 44; pasteurization, 84;
quantitative analysis, 84.
Monilia candida, 180. -
Morphological characters, 59.
Movement, study of, 24.

EISSER’S diphtheria stain, 178.
Nitrites, detection, 52.
Nitrate solution, 44.
Non-pathogenic bacteria, 63.

BSERVATION of inoculated ani-
mals, 164.
Oil-immersion objective, 22.
Oxygen, effect on bacteria, 50.

ARIETTI'S method, 188; 200.
Pasteurization of milk, 84.
Pathogenic aerobes, 88; anaerobes, 149;
bacteria in water and food sup-
plies, 200.
Petri dishes, 32.
Petri-Sedgwick’s air analysis, 80.

Index.

Phenolphthalein, 6.

Physiological characters, 59.

Pigment, production, 56; varieties, 66.

Pipettes, sterilization, 4.

Plasmodium malariae, 186.

Plate cultures, gelatin, 82; agar, 34;
study, 36,

Platinum needles, 14.

Plugging flasks and tubes, 2.

Pneumococcus, 106. :

Post-mortem, examination, 164.

Potato, character of grown on, 58; in-
oculation, 16; preparation, 12.

Proteus vulgaris, 72.

Pseudomonas aeruginosa, 54, 130; 196.
erythro sporus, 40.
fluorescens, 26.

Pyogenic micrococci, 180; 186; 192; 194.

ABIES, diagnosis, 198.
Reaction of media, 6; effect on
growth of bacteria, 44.
Roll cultures, 34.
Russell’s water sampler, 82.

ARCINA lutea, 26.

tetragena, 102; 162; 172; 174.

Sections, cutting, 170; staining, 172.

Shake culture, 50.

Slides, cleaning, 18.

Soap stone for cooling plate cultures,
32.

Soil, analysis, 82.

Spirillum of Finkler and Prior, 136.
obermetert, 186; 192.
rubrum, 26.

Sputum, 180.

Stab culture, 16,

Stain bottles, 20.

Staining solutions, 18,

Staphylococcus epidermidis albus, 92.
pyogenes albus, 92; — aureus, 96.

Steam sterilizers, 8.

Sterilization, culture media, 8; discon-
tinuous, 8; gelatin, 10; glassware, 2;
instruments, 164.

205

Sterilizers, Arnold, 8; hot air, 2; sim-
ple form, 8.

Streak cultures, character of growth
on, 58; inoculation, 16.

Streptococcus pyogenes, 90; 162; 194.

Streptothrix actinomyces, 172; 184; 198.

Study of bacteria, 57.

Sugar media, preparation, 44; sterili-
zation, 8; 44,

Sulphuretted hydrogen in cultures, 54.

Sunlight, effect on bacteria, 50.

AXONOMY, 56,
Temperature variations, effect on

bacteria, 46.

Test-tube cultures, inoculation, 14; 26;
study, 26.

Test-tubes, cleaning, 2; filling, 6.

Thermal death point determinations,
46.

Thermostats, 16.

Tissue, embedding, 170; hardening, 170;
staining, 172.

Transudates and exudates, examina-
tion of, 194.

Tubercle stain, 38.

Typhoid blood, Widal reaction, 188,

(a examination of, 192.

IBRIO metschnikovi, 134.
Vital movement, 24.

ATER analysis, 82; examination
for pathogenic bacteria, 200.

Water blanks, 12.

Weigert’s stain, 172.

Welch’s capsule stain, 42.

Wertheim’s medium for gonococcus,
99; 194.

Widal reaction, 188.

Wurtz’s lactose litmus agar plate, 52

IEHL’S carbol fuchsin, 18.
Ziehl-Neelsen stain, 182.