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Full text of "A labortory guide in bacteriology, for the use of students, teachers, and practitioners"

\=>' 



LIBRARY 

OF THE 

UNIVERSITY OF CALIFORNIA, 



ClaSS LIBRARY 



-. . 



r* 



A LABORATORY GUIDE IN 
BACTERIOLOGY 



A Laboratory Guide 
in Bacteriology 



FOR THE USE OF 



STUDENTS, TEACHERS, AND 
PRACTITIONERS 



BY 



PAUL G. HEINEMANN, Sc.B. 

Fellow in Bacteriology, the University of Chicago 




THE UNIVERSITY OF CHICAGO PRESS 
1905 



t+4- 



COPYRIGHT 1905 
THE UNIVERSITY OF CHICAGO 



PREFACE 

The considerations which led the writer to add this 
laboratory guide of bacteriology to the number of such 
guides already in existence were of various nature and 
may be briefly set forth here. 

Probably no branch of biological science has advanced 
so rapidly during the past few years as the science of 
bacteriology, and it is difficult even for an active labora- 
tory worker to keep abreast of this advance. A text- 
book or guide fixes the status of the science at the time 
of its writing, but almost before it leaves the press it 
becomes antiquated. Revisions, corrections, and addi- 
tions are necessary at short intervals in order to keep a 
publication of this kind approximately up to date. There 
is therefore, almost at any given moment room for a new 
publication to fill the want of a progressive instructor for 
a guide that gives the latest accepted rules and practices 
of the laboratory. The value of such a publication will 
be enhanced by a plan and arrangement of sufficient 
flexibility and latitude to allow the instructor and the 
student to enter such additions and corrections as serve 
to bridge over the time between editions. 

Medical students entering on a course in bacteriology 
often have had too little previous laboratory training in 
methods of precision. It is a matter of importance for 
the instructor to put himself in the attitude of mind of the 
student and to try to appreciate his difficulties in under- 
standing details. Many of the pieces of apparatus 
employed in a bacteriological laboratory are novel even 



144184 



vi PREFACE 

to the student trained in chemistry and biology, and it 
has been thought best to exhibit these, to the smallest 
detail, by means of illustrations a feature not sufficiently 
considered in other guides. 

The formulae for stains and the methods of staining 
have not been collected in one chapter, as is usually the 
practice, because this tends to confuse the student. 
They are described during the progress of the course, as 
occasion offers to put them to practical use. 

Culture-description charts have not been included in 
this volume. A beginner naturally makes incomplete 
descriptions and many alterations, and thus defaces the 
book and impairs its future utility. A sufficient number 
of loose charts perforated for binding should be furnished 
to the student at a nominal figure. 

A point of inestimable importance is how best to 
stimulate the student to consult textbooks and special 
monographs, and other references, as often and as freely 
as possible. This guide has been written with the aim 
of not only not interfering in any manner with the reading, 
through including such points and characteristics as might 
make a textbook superfluous in the judgment of the 
inexperienced, but also of making it necessary for the 
student to read the best textbooks with freedom and 
understanding. Cultural and morphological features are 
left entirely to the actual observation of the student, sup- 
plemented by instruction and the reading of textbooks. 

The course, as outlined, is identical with the medical 
course given at the University of Chicago, with a few 
additional chapters which may be used during courses 
for non-medical students. A chapter containing a fairly 
complete list of formulae for culture media employed in 



PREFACE vii 

advanced work has been added with the object of making 
them easily accessible to those engaged in advanced work. 
The laboratory guides of Novy, Eyre, Frost, Gorham, 
Kanthack and Drysdale, and Connell, and the American 
edition of the Manual oj Bacteriology of Muir and Ritchie, 
have been freely consulted. I take this occasion of 
expressing my deep gratitude to Professor Edwin O. 
Jordan and Dr. Norman Mac Leod Harris for their 
invaluable help and suggestions in the preparation of 
this guide. 

PAUL G. HEINEMANN. 
CHICAGO, ILL. 
June, 1905. 



INTRODUCTION 

The advent of bacteriology into the realm of the 
biological sciences not only brought with it a new 
conception of the nature of many complicated phe- 
nomena, such as fermentation and disease, but also 
placed in the hands of experimental workers a new 
tool. The method of sterilization, of asepsis, made 
it possible for the first time to attack problems 
hitherto incapable of solution, or even of approach. 
This development of bacteriological technique, of 
rigid and undeviating adherence to definite rules 
and principles, is not likely to be passed over lightly 
by the historian of nineteenth-century science. The 
art of practical medicine and theoretical medical 
research alike owe much of their recent brilliant 
success to a ready adoption of the new method. 

At the present time an active campaign is being 
set on foot by public health authorities against 
several widespread and serious diseases of the human 
race. In various parts of the world, malaria, tuber- 
culosis, and typhoid fever are being fought energeti- 
cally and with much success. In these systematic 
and organized movements the resources of bacteri- 
ology are being utilized as never before, and a full 
understanding of technical procedure and devices 
is deemed essential by all workers in this subject. 
The problems of water-supply and sewage disposal, 

ix 



x INTRODUCTION 

of urban infantile mortality, and of the control of 
contagious diseases are all bound up with the intelli- 
gent application of bacteriological methods. 

In the almost untilled field of industrial bacteri- 
ology there is need for a fuller appreciation of the 
value of bacteriological methods and principles. 
Many great industries are based wholly upon the 
proper selection and adaptation of micro-organisms, 
and a timely and discriminating utilization of their 
products. Loose and empirical methods have been 
in force in the past, but these must eventually give 
way to a more precise and truly bacteriological 
technique. 

Agricultural bacteriology is just now much in the 
public eye, and it would be gratuitous to prophesy 
the results that may reasonably be anticipated in 
this direction. Here again crude, rule-of-thumb, 
" practical" ways of doing things are being sup- 
planted by the scientific, the reasoning, and the 
precise. 

To the student, whether in medical, hygienic, or 
industrial bacteriology, proper technical methods 
of work must always have a peculiar value, since 
without their aid advance is impossible, and stumb- 
ling and disastrous missteps are certain. A com- 
prehensive outline of modern bacteriological methods, 
therefore, is a necessary adjunct to obtaining a true 
and full understanding of the underlying principles 
and tendencies of the science. The technique of 



INTRODUCTION xi 

bacteriology is one of its greatest contributions to 
both science and art, and the use of so valuable 
and simple a tool should be mastered not only by 
the biological teacher and investigator, but by prac- 
tical workers in medicine, hygiene, and many other 
fields. 

EDWIN O. JORDAN. 



TABLE OF CONTENTS 

PAGE 

CHAPTER I. Laboratory Rules . . . . . . i 

CHAPTER II. Cleaning, Preparing, and Sterilizing Glassware 8 

CHAPTER III. Preparation of Culture Media . . .11 

Exercise i. Preparation of nutrient agar-agar . .11 

Exercise 2. Preparation of glucose-agar . . -19 

Exercise 3. Preparation of peptone-gelatin . -23 

Exercise 4. Preparation of peptone-broth . -25 

Exercise 5. Preparation of potato . . . .27 

Exercise 6. Preparation of litmus milk . . .28 

CHAPTER IV. Preparation of Staining Solutions . . 30 

CHAPTER V. The Microscope . . . . -31 

CHAPTER VI. Collecting Bacteria from the Air . . -37 

CHAPTER VII. Exercises on Infection and Sterilization . 45 

Exercise i. Phenomena of infection . . -45 

Exercise 2. Phenomena of sterilization . . -45 

Exercise 3. Phenomena of sterilization (continued) . 45 

CHAPTER VIII. Study of Yeasts, Molds, and Torulae . 49 

Exercise i. Cultural studies . . . . -49 

Exercise 2. Study of the germination of spores . 51 

CHAPTER IX. Scheme for Routine Study of the Various 

Groups of Organisms . . , . . -53 

CHAPTER X. Method of Describing Cultures . . -57 

CHAPTER XI. Study of Certain Chromogenic Bacteria . 69 

Exercise i. Cultural studies . ' . . . .69 

Exercise 2. Study of pigments . . . . 71 

CHAPTER XII. The Pyogenic Group . . -73 

Exercise i. Subgroup A . . . . -73 
Exercise 2. Subgroup B . .... 76 



xiv TABLE OF CONTENTS 

PAGE 

CHAPTER XIII. The Intestinal Group . . . -79 

Exercise i. Colon group . . . . . -79 

Exercise 2. Hog-cholera, Bac. enteritidis or intermediate 

group . . 84 

Exercise 3. Typhoid-dysentery group . 85 

Exercise 4. Proteus group . . . . .89 

CHAPTER XIV. The Capsulated Group . . . .92 

CHAPTER XV. The Diphtheria Group . . . -94 

CHAPTER XVI. The Hemorrhagic Septicemia Group. . 97 

CHAPTER XVII. The Anthrax Group . . .98 

CHAPTER XVIII. The Spirillum Group . . . . 101 

CHAPTER XIX. The Group of Acid-Resisting Bacilli . 103 

CHAPTER XX. The Miscellaneous Bacteria . . . 105 

CHAPTER XXI. The Actinomyces Group . . . 106 

CHAPTER XXII. The Anaerobic Group of Bacilli . . 107 

CHAPTER XXIII. Isolation of Unknown Bacteria from a 

Mixture . . . . . . . . .112 

CHAPTER XXIV. Bacteriological Examination of Water, Air, 

and Milk . . . . . . . .114 

Exercise i. Bacteriological examination of water . 114 

Exercise 2. Bacteriological examination of air . .118 
Exercise 3. Bacteriological examination of milk . 119 

CHAPTER XXV. Influence of Disinfectants on the Growth 

of Micro-organisms 122 

Exercises i and 2. Hill's test-rods . .--,. . .122 
Exercise 3. Influence ot sunlight . *.^?^- I2 3 
Exercise 4. Influence of moist heat . . . .124 

APPENDIX I. Special Media 127 

APPENDIX II. Staining Solutions 132 

APPENDIX III. Frost's Culture Chart (modified) > .134 
INDEX 139 






CHAPTER I 
LABORATORY RULES 

1. Carefully familiarize yourself with the laboratory 
rules. Upon their careful observance depend good work 
and your own safety. 

2. Food must not be eaten in the laboratory; lead- 
pencils, labels, or fingers must not be moistened with 
the tongue. 

3. If any portion of a culture is spilled by accident 
upon the desk or floor, it should be immediately covered 
with a germicide (HgCl 2 i : 1000 or 5 per cent, carbolic 
acid). After this has acted for 10 or 15 minutes, wipe it 
up and throw the cloth or paper into a waste-jar. 

4. In case the hands should by accident come in 
contact with infectious material, they should be washed 
with one of the above-mentioned germicides, and then 
thoroughly scrubbed with soap and water. 

5. The platinum needles used in making cultures 
should be sterilized in a flame shortly before and imme- 
diately after use, and before they are laid down. When 
the needles are covered with moist infectious material, 
they should be held at the side of the flame until dry 
before being sterilized; this will avoid the danger of 
scattering this material about the desk. 

6. All possible care should be observed in the care 
of the apparatus, desk, etc. Solid materials must not 
be put into the sinks. Burned matches, paper, cotton, 
etc., are to be put in the crocks provided for that purpose, 
and not on the floor. 



2 LABORATORY GUIDE IN BACTERIOLOGY 

7. Discarded cultures should be killed in the auto- 
clav (5 minutes at I2o 0j ) before emptying into the crocks. 

8. When using the steam sterilizers and autoclav, 
see that the pan contains sufficient water before lighting 
the gas. 

9. In general, see that the air inlets of Bunsen burners 
are open before lighting, and relight if the flame strikes 
down. 

10. Always return stock-bottles to the proper places 
on the shelves. 

11. At the close of the day's work the desks should 
be washed off with corrosive sublimate, and the hands 
cleaned by thorough scrubbing with green soap and 
water. 

12. Before leaving the laboratory, see that the gas 
is shut off under all apparatus, that water faucets are 
closed, and that all glassware, etc., is replaced in the 
lockers. 

GENERAL DIRECTIONS 

1. After obtaining the key to the locker, examine the 
outfit, check the apparatus (Fig. i) on the furnished list, 
and return the same signed with your name. This signa- 
ture is a receipt in full for the material received and in 
good condition, and the student is personally responsible 
for the return of the same in good condition. 

2. Matches, peptone, gelatin, and filter-paper are 
furnished with the locker outfit in sufficient quantities 
to last through the course. Anything needed in excess 
must be furnished by the student. 

1 Degrees of temperature throughout this work are indicated 
according to the Centigrade system. 



LABORATORY RULES 3 

3. To facilitate study, students may work in pairs in 
the preparation of culture media and the cultivation of 
organisms; but each student is responsible for a first- 
hand and independent description of each organism 
studied, as well as for stained cover-slip preparations. 

4. Read carefully in the textbook and the laboratory 
guide the work for the day. This will greatly facilitate 
an intelligent and systematic plan of work. 

5. Keep careful, neat, systematic notes of each exer- 
cise, and have them ready for inspection at any time. 
In the case of exercises devoted to the preparation of 
culture media, a simple statement as to their completion 
is sufficient. 

6. Printed charts for description of cultures can be 
obtained on application to the laboratory assistant. 

7. Accurate sketches must be made when called for 
by the directions. 

8. All the notes pertaining to each exercise must be 
kept together. "University covers" of suitable size are 
recommended for this purpose. 

9. Store clean cover-slips in a Stender dish and cover 
with 96 per cent, alcohol. A soft linen cloth is necessary 
for wiping them. An ordinary clean handkerchief will 
do very well. Coarse material breaks them easily. 

LOCKER OUTFIT 

150 culture-tubes. 
10 potato culture-tubes. 
6 fermentation-tubes. 
15 Petri dishes (for plate-cultures). 

3 Erlenmeyer flasks, one 1000 c.c., two 500 c.c. each. 
2 glass funnels, one 4 inches, one 6 inches. 

4 bottles for staining-fluiols, 



LABORATORY GUIDE IN BACTERIOLOGY 




Retort-Stand 



Bunsen Burner 
FiO. i 



Culture-Tube Pipette 



LABORATORY RULES 




Petri Dish 





Tripod 




Watch-G lasses 




Erlenmeyer Flask 



Saltcellar 





Balsam Bottl 



Magnifier 
(Hand-Lens) 

FIG. i 




Slender Dish 



6 LABORATORY GUIDE IN BACTERIOLOGY 

i balsam bottle. 

i Stender dish (for cover-slips). 

3 watch-glasses, 
i saltcellar. 

1 glass rod. 

2 platinum needles (turn the end of one needle around 
a sharp pencil point so as to form a closed loop). 

8 tin cups or tumblers (cover the bottom of each with 
ordinary cotton). 

4 wire baskets (to hold culture-tubes). 
i Bunsen burner and rubber tubing. 

i saucepan and cover. 

3 graduates, one 500 c.c., one 100 c.c., one 10 c.c. 
i pinchcock. 

i pipette with rubber hose attached, 
i magnifier (hand-lens), 
i tripod. 

i retort-stand with three rings, 
i thermometer in case, 
i box of matches. 
40 grams of peptone. 
1 20 grams of gelatin. 
6 sheets of filter-paper, 
i key. 

This outfit may be changed and added to in the dis- 
cretion of the instructor. 

Obtain the following articles from the storeroom: 
50 glass slides. 

1 camel's-hair brush. 

50 round cover-glasses (18 mm.). 

2 towels (one should be boiled in dilute NaOH, rinsed 
in clean water, and kept clean for wiping slides and 

cover-glasses.) 



LABORATORY RULES 

1 test-tube brush. 
50 labels. 

2 slide-boxes. 

2 hollow-ground slides. 

i glass pencil. 

i pair of forceps. 

i, or better 2, pairs of cover-slip forceps. 



CHAPTER II 

CLEANING, PREPARING, AND STERILIZING 
GLASSWARE 

EXERCISE I. CLEANING GLASSWARE 

Culture-tubes, flasks, fermentation-tubes, and Petri 
dishes must be free from organic matter, acids, and 
alkalis. They are best cleaned as follows: 

a) Completely immerse them in a vessel containing 
soapsuds or soap-powder, boil for at least 10 minutes, 
then thoroughly brush them with the tube-brush; or, 
immerse them for an hour or more in the chromic-acid 
cleaning-mixture, consisting of 

Potassium dichromate 60 parts 

Water 300 parts 

Concentrated sulphuric acid 460 parts 

The sulphuric acid is to be added slowly with constant 
stirring. 

b) Rinse thoroughly in tap-water. 

c) Again use the tube-brush, and soap and water, if 
necessary. 

d) Rinse very thoroughly, and get rid of every trace 
of acid, soap, or alkali. 

e) Turn upside down in a basket, and heat for 20 
minutes in the hot-air sterilizer until perfectly dry. 

EXERCISE II. PLUGGING OF CULTURE-TUBES 

Directions for plugging. Take a small amount of 
ordinary, non-absorbent cotton of good quality, fold it 
once, and by means of a glass rod push it into the mouth 

8 



CLEANING AND PREPARING GLASSWARE 




of the tube to the depth of about J of an inch; or roll 
the cotton into cylinders of the same s^*^/^ 
diameter as the culure-tube and insert / ^ ~ 
it into the mouth (Fig. 2). The plug /f7r ^ 
should allow the interchange of air, 
and at the same time be tight enough 
to hold the weight of the tube, and 
with no crevices on the side. The 
glass rod should never be pushed 
through the cotton. 

EXERCISE III Flo . 3 

STERILIZATION OF GLASSWARE Plugged Culture-Tube 

Sterilization is the process of killing all micro-organ- 
isms. This may be accomplished by heat, by certain 
chemicals, or by nitration. Sterilization by heat may be 
accomplished 

1. By dry heat; this method is applied to sterilization 
of many kinds of apparatus. 

2. By moist heat; this method is applied largely to 

sterilization of culture media, 
and will be described in 
Chap. Ill, Exercise II. 

Hot-air sterilizers are boxes 
with double walls of sheet- 
iron. The bottom shelf 
should always be covered with 
a piece of sheet-asbestos, to 
prevent too rapid heating of 
the apparatus. By means of 
a Bunsen burner with three 
flames a heat of about 150 
may easily be maintained. The flames of the burner 




FIG. 3 
Koch's Hot-Air Sterilizer 



io LABORATORY GUIDE IN BACTERIOLOGY 

should enter the hole provided at the bottom of the 
sterilizer for that purpose. Care should be taken to 
avoid the possibility of the flame becoming luminous, 
as this would fill the box and cover the glassware with 
soot (Figs. 3 and 4). 

The plugged culture-tubes are then placed in the 




FIG. 4 
Lautenschlager Hot-Air Sterilizer 

sterilizer, and a temperature of 150 is maintained for 
about 30 minutes, or until the plugs are slightly charred. 
This can be recognized by a slight brownish color. The 
tubes are now not necessarily sterile, but the plugs have 
become set so as to fit the mouth of the tube, and may 
easily be removed and replaced. 



CHAPTER III 

PREPARATION OF CULTURE MEDIA 
Each student is required to make up the following 
amounts of culture media: 



Name of Medium 


Amount 


Number of Tubes 


Plain agar. ... 


IOOO 2 


7Q 


G lucose-agar . 


2CO 


20 


Gelatin. . 


JQQ or . 


2 ^ 


Potato 


a**** & 


*0 

IO 


Broth 


300 c.c. 


2C 


Dunham's solution. 


?OO C C. 


2cr 


Litmus milk. 


7OO C C 


2^ 






*3 






160 



EXERCISE I. PREPARATION OF NUTRIENT AGAR-AGAR 

1. Weigh the saucepan without the lid accurately 
and make note of the weight. 

2. Measure into the saucepan 1000 c.c. of tap- water, 
adding about 300 c.c. of water to allow for evaporation, 
and heat over gas. 

3. Cut and shred i<;g. of agar-agar (ij per cent.), 
add to the water, and boil slowly, with constant stirring, 
until perfectly dissolved, taking care not to overheat. 

4. Add 3 g. of extract of beef. When dissolved, 
remove the saucepan from the flame, and 

5. Slowly dust 10 ( i per cent.) g. of Witte's peptone 
on the surface, constantly stirring until perfectly dissolved. 
Avoid allowing the peptone to clump. 

NOTE. Agar-agar (called simply "agar") is a watery ex- 
tract of certain seaweeds found on the Pacific coast of Asia. 



12 LABORATORY GUIDE IN BACTERIOLOGY 

6. Adjust the reaction. 

Bacteria, especially pathogenic bacteria, grow prefer- 
ably in a medium which is neutral or slightly acid to 
phenolphthalein (alkaline to litmus). Extract of beef 
always contains a certain amount of acid, which has to 
be neutralized. Two methods may be employed to 
accomplish this purpose. 

First method. Gradually add a 4 per cent, solution 
of sodium hydrate (NaOH) until a sample on the end 
of the glass rod turns phenolphthalein paper a pale rose- 
pink color. If accidentally too much NaOH is added, 
it may be readjusted by means of a 5 per cent, solution 
of hydrochloric acid (HC1). This method is sufficiently 
accurate for ordinary purposes. The medium is then 
strongly alkaline to litmus. 

Second method. A more precise method is as follows: 
By means of a pipette measure 5 c.c. of the liquid into 
a porcelain evaporating-dish ; add 45 c.c. of distilled 
water and i c.c. of a solution of i part phenolphthalein 
in 200 c.c. alcohol (50 per cent.), then heat to the boiling- 
point and slowly add from a graduated burette enough 
-$ normal NaOH to neutralize. (The liquid must 
have a decided, stable, pale-pink color which does not 
vanish when heated.) Read the amount used from the 
burette, and calculate the amount of normal NaOH needed 
for 1000 c.c. Then add 0.5 per cent, normal HC1. 

Example. By reading the burette we find that it 
takes 1.2 c.c. -$ normal NaOH to neutralize 5 c.c. of 
the medium, diluted with 45 c.c. of distilled water. To 
neutralize i liter, it will take 200 times this amount, 
which amounts to 240 c.c. of the -^ normal NaOH. 
Divide 240 by 20, and the result (12 c.c.) is the amount 



PREPARATION OF CULTURE MEDIA 13 

of normal NaOH needed to neutralize i liter of medium. 
To this add 5 c.c. normal HCL 

The medium is then 0.5 per cent, acid to phenol- 
phthalein ( + 0.5 per cent. Fuller's standard), or fairly 
strongly alkaline to litmus. 

The same result may be obtained by deducting 5 c.c. 
from the amount of normal NaOH to be added per liter. 
If, therefore, in the above example 12 c.c. 5 c.c. = 7 c.c. 
normal NaOH is added, the resulting reaction of the me- 
dium is 0.5 per cent, acid without the addition of HCL 

A normal solution is the equivalent weight in grams 
(gram-molecule) of a chemical in 1000 c.c. of distilled 
water. In the case of monovalent elements combining 
to form the chemical, the molecular weight of the latter is 
taken; in the case of bivalent ones, the molecular 
weight is divided by 2 ; etc. 

7. Allow the liquid to cool to 60; test by placing 
the thermometer in it. 

8. While cooling, dissolve the whites of two eggs in 
75 c.c. of water, and stir well into the liquid while still at 
60. The egg albumen is added for the purpose of clari- 
fying the liquid. If added before the temperature has 
fallen to 60, it would partially coagulate, and thus not 
serve the purpose. On the other hand, if allowed to 
cool to about 40, the agar would solidify. Upon gradu- 
ally heating the medium the albumen coagulates, me- 
chanically incloses suspended particles, carries them to 
the top, and forms a superficial film. 

9. Heat again, without stirring, on a piece of asbestos 
over the flame. A film will gradually form and harden 
with a dry surface. 

10. Now adjust the weight by calculating the total 



i 4 LABORATORY GUIDE IN BACTERIOLOGY 



weight of the different ingredients, allowing 1000 g. for 
all the water used and 30 g. for the weight of the white of 
each egg and the weight of the pan. If the actual total 
weight is in excess of the amount calculated, boil again 



a 



FIG. 5 

Apparatus for Filtering 
Media 

a. Filter 

b. Large funnel 

c. Small funnel 

d. Rubber hose 

e. Pinchcock 
/. Pipette 

g. Culture-tube 




until the proper weight is reached. If, on the other hand, 
the total weight is less, make up the deficiency by adding 
water. 

Example 

TVater 

Agar 

Extract of beef 

Peptone 

Whites of two eggs 

Saucepan for example . . 



3000. O 


.100.00$ 


15-0 


...1.50 


3- 


... o . 30 


IO.O 


... I . 00 


60.0 




450.0 





Total. 



PREPARATION OF CULTURE MEDIA 



By placing weights to correspond to this amount on the 
left side of the scales, and the saucepan with contents 
on the other side, the weight can be readily adjusted. 

ii. Heat again to the boiling-point, and filter the 
liquid into culture-tubes to about one-third of their 
length (7 c.c.). 

Method of filtering media. Arrange two funnels, a 
pipette with hose, and a pinchcock on the retort-stand, 
as illustrated in Fig. 5. Place a strainer made of per- 
forated tin (Fig. 6) against the side of the saucepan, 





FIG. 7 
Strainer in Position 

near the top, as illustrated in Fig. 7, and pour the medium 
into the larger funnel holding the filter. 

For the purpose of filtering the medium either white 
filter-paper of the best quality or absorbent cotton may 
be used. A filter-paper folded in the following manner 
is very serviceable: 

i. Take a square piece of filter-paper twice as wide 



16 LABORATORY GUIDE IN BACTERIOLOGY 

as the depth of the funnel to be used (Fig. 8, a), and fold 
to half the size (Fig. 8, b) so as to make i cover 2, and 3 
cover 4 

2. Fold this to make i cover 2 and 3 cover 4 (result: 
Fig. 8, c). It consists of four layers and forms a square. 

3. Fold the upper part, consisting of two layers, 



a- 





FIG. 8 

Method of Folding Paper Filter 
(For reference letters see text) 



from i to 2 (Fig. 8, d). The shaded triangle, 2-3-4, 
now has six layers; the other 1-3-4, two layers. 

4. Fold the upper double layer so as to make 2 cover 
a point in the diagonal at 5, taking care to make a sharp 



PREPARATION OF CULTURE MEDIA 17 

point at 4 (result: Fig. 8, e). The shaded part is now 
eight layers deep. 

5. Turn the folder face down, and repeat the opera- 
tions exactly as in 3 and 4 (result: Fig. 8, /). 

6. Take up and open the large middle fold (result: 
Fig. 8, g). The two halves must now be symmetrical. 

7. Fold so as to make the lines 1-3 and 1-4 meet 
at the center line 1-2 (result: Fig. 8, h). 

8. Now pick up and fold backward so as to have i 
cover 2 in the back (result: Fig. 8, i). 

9. Cut through the line 1-2 and open up. The 
extreme ends will be found without a fold, and may be 
folded so as to make nine sharp edges. 

This filter is then evenly inserted into the funnel, 
spreading the folds at a distance 
from each other as nearly alike 
as possible. Great care should be 
taken to make the folds and the 
point sharp, as this insures rapid 
filtering of the liquid and prevents 
the filter from tearing. A wire 
rack may be used, but does not FIG. 9 

yield nearly so good results (Fig. 9). 

Another method, simple and yielding excellent results, 
is the use of absorbent cotton. Take a piece about 3 
inches square, and cover lightly the lower end of the 
funnel with it. Take a larger piece, also square, judging 
the size according to the size of the funhel; pull it gently, 
so as to make the layer of cotton thinner, without showing 
any open spaces; and spread this around the inner sur- 
face of the funnel. 

If a vacuum pump is available, the medium may be 




i8 LABORATORY GUIDE IN BACTERIOLOGY 

filtered rapidly by the use of suitable apparatus, as illus- 
trated in Fig. 10. At the connection with the vacuum 
pump a valve should be inserted or a flask with a rub- 




FIG. 10 
Filtering Media by Means of Vacuum Pump 

a. Liquid medium e. Reflex flask 

b. Absorbent cotton /. Rubber stopper with two holes 

c. Rubber stopper g. Connection with aspirator 

d. Filtered medium 

ber cork with two holes may take its place to prevent 
the water from entering the flask. 

Whichever method is employed, the filter must always 
first be soaked in hot water, or, better, kept in steam 
until used. 



PREPARATION OF CULTURE MEDIA 19 

The filtered liquid is collected in a smaller funnel 
(Fig. 5, c). A wire basket should be supported so as to 
be inclined to an angle of about 45, so as to allow the 
proper arrangement of the tubes. 

Remove the plug of a culture-tube and quickly pass 
the latter up the full length of the pipette (Fig. 5, /), which 
should be no less than 3 inches long. Open the pinch- 
cock, fill the tube one-third full, and then quickly with- 
draw the tube so as to 
leave no trace of the me- 
dium on the upper part of 
the tube, and replace the 
cotton stopper. If this 
precaution is neglected, 
the cotton stopper will stick 
to the glass, which will 
cause a great deal of an- 
noyance later on and ex- 
pose the medium to the 
danger of contamination. 
Fill thirty tubes in this 
manner. 

If an exact volume of 
medium is required in a 
test-tube, an apparatus 
such as is shown in Fig. FlG - " 

. Filling Definite Amount of Medium 

ii may be employed. into Culture-Tube 

EXERCISE II. PREPARATION OF GLUCOSE-AGAR 

Glucose-agar is especially used for the demonstration 
of gas-forming organisms. In such cases the glucose is 
decomposed, and gas appears in bubbles throughout the 
medium. 




20 LABORATORY GUIDE IN BACTERIOLOGY 

Preparation 

1. Note the weight of an Erlenmeyer flask (500 c c.). 

2. Weigh 3.75 (ij per cent.) g. of glucose into the 
flask. 

3. Filter 250 g. of plain agar into the flask, heat, and 
agitate until the glucose is completely dissolved. 

4. Fill twenty tubes in the same manner as above. 

NOTE. By tying a string across a wire basket near the top, 
the plain agar-tubes may be kept separate from the glucose-agar. 
A slip of paper indicating the medium and the date of its prepara- 
tion must always be inserted. These two media are very diffi- 
cult to distinguish by the eye, and failure to label them properly 
will lead to entirely unreliable results. 

The media should now be sterilized in the autoclav. 
The principle of this mode of sterilization is the applica- 
tion of steam under pressure. Certain bacteria, and 
some of these are very widely distributed in nature, have 
the faculty of forming spores. These spores are very 
highly resistant to heat and do not lose their vitality either 
by boiling or by application of heat under ordinary atmos- 
pheric pressure, although all vegetative forms are killed. 
By adding the pressure of one atmosphere to ordinary 
pressure, the boiling-point is raised to 121.40, which is 
sufficient to kill all spores during an exposure of 5 
minutes; media in flasks should be given 10 minutes. 

The autoclav consists of a strong cylinder, made of 
iron, with a bottom and removable lid. Inside is a 
basket, or rack with shelves, resting on three short sup- 
ports with a centrally located hole in the bottom. The 
lid fits closely (in some autoclavs with a large washer), 
and is provided with a steam-valve (Fig. 12, a), a safety- 
valve (Fig. 12, b), and a gauge (Fig. 12, c). The latter 
indicates the pressure and temperature. The top is 



PREPARATION OF CULTURE MEDIA 



21 



fastened by means of thumbscrews (Fig. 12, d-d). The 
heat is produced by two circular sets of Bunsen burners 
in a hollow cylinder at the bottom (Fig. 12, e). 

Other forms based on the same principles are also 
in use. 

Before using the autoclav the inside should be exam- 



Pic. 12 
Autoclav 

a. Steam-valve 

b. Safety-valve 

c. Gauge 

d-d. Thumbscrews 

e. Bunsen burner and 
opening 




ined. It must be clean, and there must be enough clean 
water in it to reach nearly to the bottom of the basket. 
Water containing large amounts of impurities is liable to 
foam when boiling, and thus wet the plugs and ruin the 



22 LABORATORY GUIDE IN BACTERIOLOGY 

medium. Place the material to be sterilized in the basket. 
Fit the lid of the autoclav by joining corresponding 
marks (usually a number or a letter) on the side of the 
lid and margin of the main body. Then tighten the 
thumbscrews by hand, always tightening those diamet- 
rically opposite to each other at the same time. See that 
the steam-valve is open, and then light the gas. Regu- 
late the safety-valve to blow off at the required pressure, 
if provided with the means of doing this. 

It is necessary to allow the steam to escape for about 
one minute, in order to drive all air out of the cylinder. 
Then close the steam-valve. The pressure will begin to 
increase, as indicated by the hand of the manometer. 
When the desired temperature is reached, shut off the 
gas from the outer ring of burners and regulate the gas 
pressure of the inner rings, to maintain the proper tem- 
perature. It is sufficient to expose the media to a tem- 
perature of 120 for 5 minutes. After this has been 
accomplished, shut off the gas and allow to cool to 
normal pressure, or 100 or below. The lid should not 
be released before this, as a sudden diminution of pressure 
causes the contents of the tubes suddenly to boil up and 
push the stoppers out. 

The steam-valve should be opened when the auto- 
clav has cooled to 100 or below. This will guard 
against the possibility of the top blowing off and injuring 
the operator, if the gauge does not register properly. 
On the other hand, the suction caused by the contrac- 
tion of the steam may hold the lid down, so that it cannot 
be removed until the pressure is equalized. 

When sterilizing media which contain carbohydrates 
(glucose-agar, for instance), it is important not to allow 



PREPARATION OF CULTURE MEDIA 23 

the temperature to rise beyond 120, nor to allow it to 
remain at that point any longer than 5 minutes. Car- 
bohydrates are easily broken up by heat. They are 
then valueless in the medium, and also generate an acid 
reaction as a result of chemical decomposition. 

In the preparation of agar more difficulties are met 
with than in the preparation of any other medium. The 
main difficulty is the fact that it takes a long time from 
30 to 45 minutes for the agar to dissolve, and this 
creates the danger of burning it by overheating. Great 
care mus't therefore be exercised in the preparation 
at all stages. 

EXERCISE III. PREPARATION OF PEPTONE GELATIN 

1. Weigh the saucepan and measure 35oc.c. of 
tap-water into it. This amount includes 50 c.c. allow- 
ance for evaporation. 

2. Dissolve 0.75 g. of extract of meat, and, when near 
boiling, add 3 g. of peptone. 

3. When boiling, add during the cold season 10 per 
cent. (30 g.), during the warm season 12 per cent. (36 g.), 
of the best gelatin (Gold label) by dissolving two or 
three leaves at a time, with constant stirring. 

4. When completely dissolved, adjust the reaction 
as directed in the preparation of agar. As gelatin con- 
tains considerable acid, it will take mere NaOH solu- 
tion in proportion than for agar. 

5. Then cool to 60, and stir into the mixture the 
white of one egg dissolved in 30 c.c. of water. Slowly 
heat over the flame on a piece of asbestos, without stir- 
ring, until the egg albumen is completely coagulated and 
forms a dry film on top. 



24 LABORATORY GUIDE IN BACTERIOLOGY 



6. Adjust the weight, and filter through paper or 
absorbent cotton previously moistened with hot water, 
and distribute into thirty culture-tubes. 

7. Sterilize in the autoclav for 5 minutes at 120. 
Care is necessary not to overheat this medium, because 
gelatin easily loses the property of solidifying when cool. 
In order to avoid this, the discontinuous or intermittent 
method of sterilization is often used. By this method 
the medium is exposed in a steam sterilizer to steam at 
the temperature of 100 for 20 minutes on three con- 
secutive days. The first day all vegetative forms are 





FIG. 13 FIG. 14 

Arnold Steam Sterilizer \ Arnold Steam Sterilizer 

The hood is taken off and the door \ a. Inner water compartment 

opened, showing inside arrangement b. Outer water compartment 

c. Perforated bottom 

d. Sterilizing chamber 

e. Sheet-copper walls 

killed. By keeping the medium at room or incubator 
temperature, spores, which may be present, will develop 
into the vegetative form and be killed by the second day's 
exposure. If after this any spores should happen to 



PREPARATION OF CULTURE MEDIA 25 

survive, they will develop during the next 24 hours, and 
the third exposure to steam will complete the sterilization. 
The apparatus used for this purpose is the "Arnold Steam 
Sterilizer." The usual form used is seen in Figs. 13 and 
14. Fig. 13 illustrates the appearance of the ordi- 
nary form with the hood off. Fig. 14 shows the inside 
arrangement, a and b being two compartments connected 
by small holes, and in which a certain amount of water 
has to be kept. The water contained in the inner com- 
partment (a) is brought to a boil by a Eunsen burner, 
the steam rising through a number of holes in the bottom 
(c) into the chamber (d). The steam condenses at the 
top of the chamber and returns between two sheet-copper 
walls (e, e) to the large compartment (b). A larger form 
of steam sterilizer, constructed on the same principle, is 
convenient for sterilizing large amounts of the media. 

EXERCISE IV. PREPARATION OF PEPTONE-BROTH (FRENCH : 
BOUILLON) AND DUNHAM'S PEPTONE-SOLUTION 

1 . Weigh the saucepan and measure 600 c.c. of tap- 
water into it and heat. 

2. Dissolve, when hot (but not boiling), 6 g. of pep- 
tone. 

3. When completely dissolved, replace the evapo- 
rated water and divide into two equal amounts (300 c.c. 
each). 

4. Filter one part twice through the same filter 
(paper) and distribute into twenty-five tubes. Sterilize 
in autoclav for 5 minutes at 120. This is Dunham's 
peptone-solution. 

5. Dissolve completely i g. of extract of beef in the 
other 300 c.c. 



26 LABORATORY GUIDE IN BACTERIOLOGY 



6. Adjust the reaction in the same manner as above. 

7. Fill into an Erlenmeyer flask and autoclav for 
10 minutes at 120. 

8. Keep the Erlenmeyer flask and contents for 24 
hours in a cool place, filter, and then distribute into 




FIG. 16 

Potato Cylinder 
showing diagonal for cutting 

twenty-five tubes, and autoclav these at 1 20 for 5 min- 
utes. This is "peptone-broth." 

NOTE. The reason for exposing broth to a heat of 120 
twice is this: The solution contains a considerable quantity of 
substances, which are precipitated by heat and appear as a sedi- 
ment after cooling. As it is important to have a perfectly clear 
broth in tubes, these substances are precipitated by the first 
heating, and, if tubed later, the second sterilization will not 
affect the appearance of the medium. 

Broth was originally prepared from chopped beef, 
and for many purposes this is preferable. The method 
of preparation is as follows: 



PREPARATION OF CULTURE MEDIA 27 

1. 500 g. (i pound) of lean minced beef, as free as 
possible from fat and tissues, is added to 1000 c.c. of 
tap-water and set aside on ice for 24 hours. 

2. Weigh a saucepan, and cook the beef and water 
for about one hour. 

3. Strain through cheese-cloth, and press all the 
liquid out in a meat-press (Fig. 15). 

4. Replace the water lost by evaporation. 

5. Dissolve 10 g. of Witte's peptone. 

6. Adjust the reaction. 

7. Filter into flasks, and sterilize in autoclav at 120 
for 10 minutes. It may then be distributed into culture- 
tubes. 

Fresh meat, as well as extract of meat, often contains 
a small amount of muscle-sugar (glycogen). If it is 
necessary to prepare a sugar-free broth, a pure culture 
of Bacillus coli is added to the mixture of chopped beef 
and water, and this is incubated at 37 for 24 hours. 
After this the preparation is the same as described. 

EXERCISE V. PREPARATION OF POTATO 

1. Select several large potatoes, and cleanse by brush- 
ing all dirt off carefully and washing in water. 

2. Punch out cylinders with a borer of suitable size, 
trim them, and cut them into two equal parts (Fig. 16). 
Cut with a sharp knife along the diagonal. 

3. Immerse the pieces in running water for 24 hours. 

4. Insert one half -cylinder into each potato-tube 
so that the wide end rests on the constriction, then pour 
a small amount of water into it. If the potato-tubes 
have no constriction, place a small wad of cotton in the 
bottom and moisten this with water (Figs. 17 and 18). 



28 LABORATORY GUIDE IN BACTERIOLOGY 



5. Sterilize in autoclav for 5 minutes at 120, or in 
Arnold for 3 consecutive days with 20-30 minutes' 
exposure. 

EXERCISE VI. PREPARATION OF LITMUS MILK 

i. Separate five-sixths of the cream from the milk. 





FIG. 17 FIG. 18 

Ordinary Style of Potato-Tube Potato-Tube 

a. Potato b. Cotton 

2. Add 7-10 per cent, of a solution of litmus (tincture 
of litmus). 

3. Distribute about yc.c. into each of twenty-five tubes. 

4. Sterilize in autoclav for 5 minutes at 120, or in 
Arnold for 3 consecutive days. 

NOTE. The prepared culture media should be carefully 
stored in a dark, cool place. If they are to be kept for a consid- 



PREPARATION OF CULTURE MEDIA 29 

erable length of time, they should be sealed either with paraffin 
or with a rubber cap. They should always be protected from 
dampness, as mold fungi are apt to alight on the cotton stoppers 
and send their filaments into the tube. Before inoculation, they 
should always be carefully examined, and those which show 
cloudiness or colonies, as well as those which have shrunk from 
evaporation, should be rejected. 



CHAPTER IV 

PREPARATION OF STAINING SOLUTIONS 
Saturated alcoholic solutions of stains are prepared 
by covering an arbitrary amount of stain with absolute 
alcohol. The solution is saturated as long as any of the 
stain remains undissolved at the bottom of the vessel. 
Other solutions necessary for the preparation of stains are: 

1. Solution of potassium hydrate in water i : 10,000. 

2. Solution of carbolic acid in water (5 per cent.). 

3. Anilin- water, prepared by shaking anilin-oil with 
water (2 per cent.) and filtering twice through the same 
paper. It should be perfectly clear. 

The staining-bottles usually employed have a capaci- 
ty of about 30 c.c. The following amounts will nearly 
fill them: 

1. Loeffler's methylene-blue : 

Saturated alcoholic solution of methylene-blue 9 c.c. 
Potassium hydrate in distilled water i : 10,000 21 c.c. 

2. Ziehl-Neelsen's carbol-f uchsin : 

Saturated alcoholic solution of f uchsin 3 c.c. 

5$ carbolic acid solution in water 27 c.c. 

3. Ehrlich's anilin-gentian- violet : 

Saturated alcoholic solution of gentian-violet 7.5 c.c. 

Anilin-water 22.5 c.c. 

This stain requires occasional filtering and is somewhat 
unstable. 

4. Gram's iodin solution: 

lodin o.i g. 

Potassium iodid 0.2 g. 

Dissolve in about 2 c.c. of water, and then add enough 
water to make the total measure 30 c.c. 

3 



CHAPTER V 
THE MICROSCOPE 

(Fig. 19) 

The compound microscope is a necessary adjunct to 
any kind of bacteriological work. For this work three 
objectives (Leitz No. 3, No. 6, or No. 7 and fa oil im- 
mersion, Zeiss No. A or No. AA, No. D or No. DD, and 
fa oil immersion) and two oculars (Nos. 2 and 4) are 
indispensable. For the intelligent manipulation of the 
microscope it is absolutely necessary to understand the 
underlying optical principles, which may be studied from 
special works on the subject. 

References 

S. H. Gage, The Microscope. 

Carpenter and Dallinger, The Microscope and its Revelations. 

For use in the laboratory it will be sufficient to call 
attention to some of the most important points to be 
observed. 

The usual pattern of microscope consists of two main 
parts: the stand, and the optical parts (Fig. 19) which 
are attached to the stand. 

The stand consists of a body-tube, draw-tube, coarse 
adjustment, fine adjustment (micrometer screw) in a 
pillar, nose-piece, stage with clips for holding the object, 
main pillar, and the horseshoe base. At the junction 
of the main pillar and the fine-adjustment pillar is the 
inclination joint. 

The draw- tube, regulating the focal length, which 
varies in different instruments, should be raised to 16 



a 




e 



FIG. 19 
Microscope (after E. Leitz) 



a. Ocular 

b. Place where virtual picture 

formed 

c. Body-tube 

d. Coarse adjustment 

e. Micrometer screw 
/. Inclination joint 
g. Horseshoe base 
h. Mirror 



*. Iris Diaphragm and Condenser 

k. Objective 

I. Nose-piece 

m-o. Picture as it appears to the eye 

p.-g. Object (cover-slip) 

r. Adjustment spring 

s. Stage 

/. Object (slide) 



THE MICROSCOPE 



33 



or 17 mm. If a nose-piece is attached, the width of 
this must be deducted from the tube-length. 

The optical parts are the oculars, the objectives, the 
substage condenser, and the mirror. The ocular is a 
combination of lenses, which slips into the top of the 
draw-tube and is nearest the eye. The objective is a 
combination of lenses which is screwed into the nose- 
piece and fits to the lower end of the draw-tube. The 
substage condenser fits under the stage. It concentrates 
the light on the object and is raised for high powers or 
lowered for low powers. At the lower end of this con- 
denser is the iris diaphragm, which is regulated by a 
small lever with a milled head, and serves the purpose 
of regulating the light supply. The mirror has two 
sides, a concave and a flat. 

In the manipulation of the compound microscope the 
following points should be strictly observed: 

1. Keep the instrument scrupulously clean. When 
not in use, lock it in the case or cover it with a bell-jar. 

2. When carrying the instrument, grasp it by the 
main pillar underneath the stage, not by the fine-adjust- 
ment pillar. The fine adjustment consists of a very 
delicate screw-thread, which is easily damaged. 

3. The lenses, condenser, and mirror, when needing, 
should be wiped with Japanese lens-paper, never with 
any coarse material. 

4. For cleaning use a damp cloth. For wiping the 
lenses use water or xylol. Never use alcohol, as this 
dissolves the cement holding the lenses in place and also 
injures the lacquer. 

5. Do not take the instrument apart. The working 
parts are of extremely delicate nature and easily injured. 



34 LABORATORY GUIDE IN BACTERIOLOGY 

Be careful not to drop the oculars or objectives. 

6. The inclination joint can be used only with dry 
lenses and dry objects, not with the oil-immersion or 
hanging-drop preparations. 

7. After placing the object on the stage, focus with 
a low power and the coarse adjustment. With high 
powers use the coarse adjustment first and the fine adjust- 
ment afterward. The free use of the fine adjustment 
saves the accommodation of the eye. As the eye is cap- 
able of accommodating itself to distances, it may with an 
effort distinguish a picture which is not in perfect focus. 
This effort is saved by using the fine adjustment. 

8. Always raise the draw-tube by means of the coarse 
adjustment before changing the objectives or examining 
a different preparation. 

9. Before focusing, obtain as good a light as possible 
by turning the mirror, and then regulate the supply by 
the diaphragm. Always use reflected light, never direct 
sunlight. 

10. When focusing an object, lower the draw-tube 
until the lens almost touches the cover-glass. This can 
easily be seen by looking at the instrument from one side 
and watching the reflection of the objective in the cover- 
glass. Then, with the eye at the ocular, slowly focus up. 
Do not focus down with the eye at the ocular, as the lens 
may then come into violent contact with the object, 
destroying the latter and injuring the lens. 

n. For living or transparent objects use as little 
light as possible. For stained or opaque objects more 
light is necessary. 

12. Do not use higher powers than is necessary. 

13. To use the oil-immersion lens, place a drop of 



THE MICROSCOPE 35 

clear cedar oil, free from dust and air-bubbles, on the 
cover-glass, which must be perfectly dry. In this case, 
by careful manipulation, the objective, after being 
brought in touch with the oil by means of the coarse 
adjustment, may be gradually lowered by the fine adjust- 
ment until the object is focused; or lower the objective 
until almost in touch with the cover-glass, and focus up. 
High powers always require the use of a homogeneous 
liquid between the cover-glass and the front lens of the 
objective, to avoid loss of light by refraction. As a bundle 
of rays disperses when entering a thinner medium from a 
denser one, there is not sufficient light entering the objec- 
tive to make objects discernible, when using high powers. 
By the insertion of a liquid (inspissated oil of cedar) of 
the same, or nearly the same, refractive index, a homo- 
geneous connection is established between the cover-glass 
and the objective, thus avoiding this loss and allowing a 
bundle of light of sufficient power to enter the objective. 1 

14. After using the oil-immersion lens, wipe the oil 
off with lens-paper. If the oil sticks to the lens, wipe it 
off with xylol, never with alcohol. At the same time 
wipe the oil off the cover-slip. 

15. The microscope should stand on a firm table, 
to avoid being shaken. The table should be low enough 
so as not to necessitate bending the body. 

1 6. Always keep both eyes open. This saves the 
eyesight. Beginners find this a difficult rule to apply, 
but with very little practice and persistence it is easily 
accomplished. Also use both eyes alternately. 

17. It is always well to move the object while bringing 

'For detailed description and diagrams see S. H. Gage The 
Microscope. 



36 LABORATORY GUIDE IN BACTERIOLOGY 

it into focus. It is then easy to feel if the lens touches 
the glass, and a moving object is seen much more easily 
than a stationary one. 

1 8. Use the plain mirror in combination with the 
condenser, and the concave mirror without the condenser 
or with artificial light. 

19. In preparing stained preparations, it often hap- 
pens that a small amount of stain remains on the upper 
side of the cover-slip. Care must be taken to focus for 
a plane below this. 



CHAPTER VI 
COLLECTING BACTERIA FROM THE AIR 

1. Sterilize all Petri dishes in the hot-air sterilizer for 
one hour at 160. 

2. Melt two tubes of plain agar and one of glucose- 
agar in the water-bath. The water-bath (Fig. 20) is a 
round copper vessel with a number of holes in the top. 




These holes are large enough to allow a culture-tube to 
slip in easily. A thermometer is passed through a rubber 
cork with a hole in its center, and inserted into one 
of the holes in the water-bath. Enough water is then 
poured into the apparatus to fill it about two-thirds, and 
the thermometer is lowered until the mercury bulb is 

37 



38 LABORATORY GUIDE IN BACTERIOLOGY 

completely immersed in the water. The culture-tubes 
are then slipped in, and the water is heated to 100. 

3. Singe the cotton stopper of the liquefied agar- 
tubes in the flame, remove the cotton stopper, pass the 
mouth and about one inch of the tube through the flame, 
and pour the contents into a sterile Petri dish, carefully 
lifting the cover (Fig. 21) and quickly replacing it. 

4. Repeat this operation with the second agar-tube. 

5. Place both Petri dishes containing the liquid agar 
on a level surface. 

6. When the agar is solidified, expose, by removing 
the cover, one dish to the air of the laboratory, and the 
other outside on the window-sill, for 10 minutes. 




FIG. 21 
Pouring Medium into Petri Dish 

7. Replace the cover and place in lockers. 

8. Cool the water-bath to 43 exactly, and mix the 
scrapings from under a finger nail with the liquid glucose- 
agar. Keep this also in the locker. 

9. Remove the plug of a tube of broth and place a 
hair in the liquid. Keep also in the locker. 

When inoculating liquefied agar media, it is necessary 
to do so at a temperature no higher than 43 nor lower 
than 40. Above 43 the organisms are liable to be 
injured by heat; below 40 the agar solidifies, and an 
even distribution is impossible. If gelatin is used, the 
latter precaution is not imperative, as gelatin solidifies at 



COLLECTING BACTERIA FROM THE AIR 39 

about 25. Observe and make notes on the appearance 
of these Petri dishes after 24 hours. By this time it will 
be observed that a number of spots of different sizes, 
shapes, colors, etc., have formed on the surface of the 
medium. Each of these spots probably consists of a 
large number of the same species of organism, and is 
called a colony. Besides bacteria, yeasts and mold spores 
are constantly present in the atmosphere and drop on the 
plate. These organisms, being of higher specific gravity 
than the air, are constantly falling, or are carried hither 
and thither by currents in the atmosphere. Small 
particles of dust also may alight on the medium, and then 
form a dark spot near the center of the colony. Dust 
particles are usually covered with bacteria, possibly of 
different species, and in this case the resulting colony may 
not be composed of one and the same species. These 
are not "pure cultures." 

Inoculate two or three colonies on agar-slants. 

NOTE. Agar-slants are prepared by allowing liquid agar to 
solidify in a slanting position. 

Method oj inoculation 

1. Singe the cotton plug of a tube containing the 
medium. It is imperative always to singe the cotton 
plugs of tubes before opening them, whether these tubes 
contain a culture or a sterile medium. The organisms 
from the air are constantly falling on the cotton, and 
bacteria may also be deposited on the cotton by handling 
it with the fingers. If these organisms were not killed by 
the process of singeing, they would drop on the medium 
after removal of the stopper, and thus ruin a pure culture. 

2. Hold the tube (or, if a transfer is made, both tubes 
side by side) between the thumb and the forefinger, so 



40 LABORATORY GUIDE IN BACTERIOLOGY 

that the end of the tube rests on one edge of the hand 
(Fig. 22), holding them at an angle of about 45. If held 
horizontally, the condensation water, always present at 
the lower end of the agar medium, will moisten the surface 
and destroy a characteristic growth along the needle-track. 
If held in a vertical position, contamination from the air 
cannot be avoided. 





FIG. 22 
Method of Inoculating Media 

3. Remove the cotton stoppers by taking hold of 
the singed portion only, and hold also by the singed part 
only between the other fingers. If the portion of cotton 
from the inside of the tube is touched by the fingers, or 
accidentally falls on the table or the floor, it becomes con- 
taminated and must be singed before replacing. 

4. Sterilize the straight platinum needle in the flame, 
holding it like a pencil. The platinum wire should be 
heated until red-hot, and the glass end passed slowly 
through the flame once or twice. 

5. After cooling the needle by plunging into the 
medium, take up a small portion of a colony or culture 
by a lateral movement on the end of the needle, and, 



COLLECTING BACTERIA FROM THE AIR 41 

when removing it from the tube, take care not to touch 
the walls of the tube. If any cotton accidentally sticks 
to the mouth of the tube, it should be burnt off with 
a hot platinum needle, and then the mouth of the tube 
passed through the flame before inserting the needle. 

6. Insert the needle with the culture into the sterile 
tube, and carefully draw along the surface of the slant, 
turning the needle during the operation and without 
puncturing the jelly. 

7. Sterilize the needle in the flame, as above, and 
replace the cotton plugs. It is not superfluous to repeat 
here that the platinum needle should always be flamed 
immediately after taking it up, and again before laying 
it down. 

A stab-culture is made in the same manner as a slant- 
culture, except that the medium is punctured centrally 
by a quick, steady movement of the needle. Care should 
be taken not to let the needle touch the bottom of the 
tube, the object being to make as narrow a puncture as 
possible, and by touching the glass the needle would bend 
and make a ragged opening in the medium on with- 
drawing. 

Inoculations of liquid media are made the same as 
agar-slants, except that the end of the needle is gently 
rubbed against the glass below the surface of the liquid. 
The medium is then shaken. After inoculation milk- 
cultures should never be shaken again, as this might 
destroy a characteristic shape of the coagulum or break 
up the cream-ring. 

Inoculations from liquid media are made with the 
looped needle. This may also be used, if a considerable 
amount of growth is required on a solid medium. The 



42 LABORATORY GUIDE IN BACTERIOLOGY 

contents of the loop are then spread over the whole sur- 
face. 

Describe the appearance of the colonies as to 

1. Naked-eye appearance: 
Color. 

Shape. 

Border. 

Size (approximately in millimeters). 

2. Appearance under low power of microscope. 

3. Give the approximate number of colonies on the 
plate. 

4. Examine as to motility in the hanging-drop. 
Preparation of a hanging-drop (Fig. 23) : 




FIG. 23 

Hanging-Drop 

(After Muir and Ritchie) 

1. Clean a cover-slip in alcohol. Pass several times 
through the flame so as to burn the last traces of grease 
off the surface. 

2. Place a loopful of pure water on the center of the 
cover-slip. 

3. Flame the straight platinum needle, and, after 



COLLECTING BACTERIA FROM THE AIR 43 

cooling, touch one of the colonies and mix lightly with 
the drop of water without spreading it. Take only a 
very minute amount of culture, so as to produce the faint- 
est visible cloudiness in the water. 

4. Smear vaselin around the depression in a hollow- 
ground slide, quickly invert the cover-slip over the 
depression, and gently press the margin on the vaselin. 

5. Examine in oil, using very little light. 
Brownian movement. Rub a small amount of carmine 

in a mortar with some water and make a hanging-drop 
preparation. When examining this through the oil- 
immersion lens, it will be observed that the small particles 
of carmine have a lively vibrating motion. This is called 
" Brownian" or "molecular movement," or "pedesis." 
The particles scarcely change their relative position. 
Actively motile organisms, on the contrary, widely change 
their relative positions. The movement of these may be 
slow, snake-like, or like a fish swimming; or they may 
dart rapidly across the field. 

Now observe and describe what changes have taken 
place in the tube of broth with the hair. Compare with 
a sterile tube, noting the turbidity, sediment, odor, etc. 
Also examine in hanging-drop. 

Examine the tube of glucose-agar containing the nail- 
scrapings. Describe the general appearance, and note 
whether gas-bubbles are present. 

Finally, from the stained preparations make sketches 
of the appearance of the culture medium, the shape of 
the colonies, and the morphology of the organisms. 

Make stained preparations of three different colonies, 
using the three stains; i. e., gentian- violet, methylene- 
blue, and carbol-fuchsin, 



44 LABORATORY GUIDE IN BACTERIOLOGY 

Method of making stained preparations 

1. Clean and flame a cover-slip, or, if preferred, 
a slide may be used for this purpose. Cover-slips, if 
handled by the fingers, should be held by the edges. Use 
as much as possible the forceps made for that purpose. 
After handling the forceps they should be sterilized in the 
flame. 

2. Place one loopful of water on the cover-slip. 

3. Take the minutest quantity of the colony or cul- 
ture on a platinum needle and mix gently with water 
until faintly cloudy. Burn the remainder of the culture 
off the needle. 

4. Spread over the cover-slip by two or three sweeps 
of the needle. The water should spread easily and not 
run together. If this takes place, the cover-slip has not 
been cleaned sufficiently. 

5. Dry by moving high over the flame. 

6. Pass three times back and forth through the flame 
rapidly. This process precipitates albuminous matter 
and causes the bacteria to adhere firmly to the glass. 

7. Cover with stain for 10-15 seconds. 

8. Wash in water and examine. If satisfactory: 

9. Float the cover-slip off the slide with water. Blot 
with filter-paper, dry in the air or high over the flame, 
and mount in Canada balsam. 

10. Label and preserve this preparation. 

Try to avoid the mistake, made by most beginners, 
of taking too much growth on the needle. For hanging- 
drop preparations less material should be used than for 
a stained preparation. 



CHAPTER VII 

EXERCISES ON INFECTION AND STERILIZATION 
EXERCISE I. PHENOMENA OF INFECTION 

1. Prepare three agar-plates as directed in Chap. VI. 

2. Touch the surface of the jelly in one plate with 
the tips of the fingers. 

3. Touch the surface of the jelly of another plate with 
the tips of the fingers, after washing the hands carefully, 

4. Catch a fly and allow it to walk on the surface of 
the jelly of the third plate for a few seconds. Release 
the fly and replace the cover. 

5. Place these three plates in a locker or thermostat 
for 24 hours. Observe and describe the results. Make 
hanging-drop and stained preparations of some of the 
colonies formed. 

EXERCISE II. PHENOMENA OF STERILIZATION 

1. Make an infusion of hay in a flask with cotton 
stopper. (See Appendix I.) 

2. Set the flask aside for 24 hours in a warm place, 
and observe the results. To what are these results due ? 

3. Expose a tube of unsterilized broth to steam in 
the "Arnold," another to steam in the autoclav, at 120 
for 5 minutes each. 

4. Set aside in a thermostat, and observe the results. 

EXERCISE III. PHENOMENA OF STERILIZATION (CON- 
TINUED) 

Action oj Berkejeld and cotton filters. Berkefeld filters 
are made of diatomaceous earth, and are porous so as 

45 



46 LABORATORY GUIDE IN BACTERIOLOGY 

to allow liquids to pass through, but no solids or organ- 
isms. 

i. Arrange a Berkefeld filter so as to connect with 
the suction pump, and filter a quantity of unsterilized 
broth (Fig. 24). 




FIG. 24 
Action of Berkefeld Filter 



a. Berkefeld filter 

b. Filtered liquid 

c. Side tube with cotton filter 



d. Intercepting flask 

e. Connection with aspirator 
/. Rubber hose 



2. Set aside, and observe the results. The filter (a), 
after being connected with the flask, is sterilized in the 
autoclav. The cotton plug at c prevents the air, which 
is sucked back, from carrying germs. The flask d will 



INFECTION AND STERILIZATION 



47 



prevent the water from running back and mixing with 
the filtered liquid. 

Now arrange a cotton filter as shown in Fig. 25. 
Vessel a, provided with a rubber stopper (b) with two 
openings, is arranged so as to have a glass tube (c) reach 
to the bottom. This tube is provided with cotton at 




a. Erlenmeyer flask 

b. Rubber stopper 

c. Glass tube 

d. Cotton filter 

e. Broth 



FIG. 25 
Action of Cotton Filter 

/. Bent glass tube with cotton filter 

at f. 

g. Erlenmeyer flask 
h. Tube connecting with aspirator 



the top (d) and some nutritive medium (broth, e) is 
placed inside. Through the opening a bent glass tube 
leads out, and this tube is also provided with a cotton 
filter at /. The whole apparatus is then sterilized in 



48 LABORATORY GUIDE IN BACTERIOLOGY 

the autoclav at 120 for 5 minutes, and connection is 
made through the flask (g) and the tube (h) with the 
aspirator. Now aspirate some air through the flask, 
disconnect at /, and set aside. Observe the results. Why 
does no growth take place ? Remove the cotton filter 
(d), and drop it into a flask containing sterile broth; 
place in the thermostat for 18-24 hours; note and 
examine the conditions then present. 



CHAPTER VIII 

STUDY OF YEASTS, MOLDS, AND TORUL/E 
EXERCISE I. CULTURAL STUDIES 

Read carefully the chapter on yeasts and molds in 
the textbook. Yeasts, torulae, and molds grow better in 
a medium of a slightly acid reaction than in a neutral or 
alkaline medium. Wort-agar or gelatin prepared as 
described (Appendix I) will answer very well for this 
purpose. 

1. Melt eight wort-agar tubes in the water-bath. 

2. Pour the contents of two tubes into sterile Petri 
dishes, and set aside to solidify. 

3. Allow the other six tubes to solidify so as to form 
a slanted surface. 

4. After the plates have cooled, expose them to the 
air in two different places for 10 or 15 minutes. 

5. Place in your locker. 

6. Examine after 24 or 48 hours. Describe what 
you see. 

7. Transfer several mold and yeast colonies to the 
slants prepared. 

8. Make transfer of a stock-culture of Saccharo- 
myces cerevisiae, or any other species of yeast. 

NOTE. Molds may easily be recognized by the filamentous, 
cotton-like form of the colonies. The hyphae extending into the 
air carry the spores (conidia). By gently touching these with a 
sterile platinum needle, the spores may be transferred to the 
agar-slant, and development will take place after 24 hours. 
Colonies of yeasts or torulae appear smooth, moist, opaque, 
elevated, and slightly yellowish-white, or sometimes reddish. 

49 



50 LABORATORY GUIDE IN BACTERIOLOGY 

These may be transferred in the same manner as colonies of 
bacteria. Molds require very careful handling for microscopical 
demonstration. They are best examined in water in the unstained 
condition. 

Method of preparing molds for microscopical exami- 
nation 

1. Transfer some of the growth to alcohol (50 per 
cent.). 

2. When thoroughly moistened, transfer some of 
the growth to a drop of glycerin on a slide. 

3. Spread carefully with a platinum needle. 

4. Cover with a slip and examine. 

5. If satisfactory, the preparation may be made per- 
manent by painting a ring of asphalt around the edge 
of the cover-slip. 

Molds may also be stained in the following manner: 

1. Place a small amount of mold on a slide. 

2. Cover with alcohol. 

3. Wash in water. 

4. Stain with gentian-violet or methylene-blue. 

5. Mount in glycerin. 
Study of yeasts 

1. Examine a small amount of yeast taken from an 
agar-slant in water under the high power of the micro- 
scope. Note the manner of reproduction by " budding." 

2. Prepare a culture in liquid wort of Sacch. cere- 
visiae. 

3. Pour the supernatant liquid of the 24-hour-old 
culture off, and distribute the sediment on a gypsum 
block. 

NOTE. Gypsum blocks may be prepared in the following 
manner: Gypsum (plaster of paris) is mixed with half its vol- 
ume of water and quickly placed in a cylinder of paper. When 



YEASTS, MOLDS AND TORUL/2 51 

perfectly dry, the paper is cut away and the block is placed in a 
suitable vessel (a Stender dish or a deep, narrow Petri dish will 
answer very well). The block and vessel are then sterilized in 
the hot-air sterilizer for one hour. 

4. Pour enough distilled water around the gypsum 
block to submerge about one-half of it. 

5. Set aside in a cool, dark place for 3 or 4 days. 

6. Examine a small portion of the film on the surface 
of the gypsum under the microscope in water. 

NOTE. Under very favorable conditions, and in the pres- 
ence of plenty of oxygen, yeasts will undergo spore-formation. 
The great porosity of the gypsum block, which admits free com- 
munication with the water, and the fact that the surface of the 
block is exposed to the air, offer most favorable conditions for 
spore-formation, which takes place in 3 or 4 days. 

EXERCISE II. THE STUDY OF THE GERMINATION OF 
SPORES 

The germination of spores may be studied from spores 
(conidia) of mold fungi. Although somewhat different 
from the germination of bacterial spores, mold spores 
illustrate the general process very well. 

1. Make a suspension of spores of Penicillium glau- 
cum (common green mold) in broth. 

2. Flame carefully a hollow-ground slide so as to 
exclude all other organisms, and paint vaselin around 
the hollow so as to form a complete ring. 

3. Take a loopful of the spore suspension and place it 
on a clean flamed cover-glass. 

4. Invert over the prepared hollow-ground slide, and 
gently press the edges on the vaselin so as to exclude all 
communication with the air. This prevents the hang- 
ing-drop from drying out, and also the invasion of for- 
eign organisms. 



52 LABORATORY GUIDE IN BACTERIOLOGY 

5. Observe under the microscope (No. 7 objective), 
and make sketches from day to day of the progress in 
the growth, until the complete organism is developed 
and spores are again formed. 



CHAPTER IX 

SCHEME FOR ROUTINE STUDY OF THE VARIOUS 
GROUPS OF ORGANISMS 

This scheme is to be followed strictly in the study of 
all organisms, except when special instructions are given, 
and the student should thoroughly familiarize himself 
with the different steps. The work of cultivation as 
described in steps 1,3, and 5 may be divided between two 
students working together, but all other procedures are 
to be followed by each individual. 

1. The inoculation of one slant-agar-tube from each 
individual stock-culture supplied. These inoculated agar- 
tubes are to be incubated at 37 for 24 hours, unless 
otherwise instructed. 

2. At the end of 24 hours make from each agar-tube: 

a) A physical description of the culture (see Chap. 
X). 

b) A hanging-drop examination. 

c) A Gram stained preparation. 

d) An ordinary stained preparation. 

Method of making preparations according to Gram 

a) Prepare a film of the organism to be examined, 
the same as in the ordinary stained preparation. 

b) Cover with gentian-violet for i minute. 

c) Wash in water, and remove the water by means 
of filter-paper, leaving the surface moist. 

d) Cover with Gram's iodin solution for 2 minutes. 

e) Pour Gram's iodin solution off and, without 
washing, place in a watch-glass, film side up, and cover 
with 96 per cent, alcohol. 

53 



54 LABORATORY GUIDE IN BACTERIOLOGY 

/) Allow to remain in alcohol, with occasional agita- 
tion, for at least 4 minutes, or until no more stain is 
taken up by the alcohol. 

g) Dry without washing, and mount. 

This stain is an important means of differentiating 
species of bacteria. 

In order to be able to judge of the effect of Gram's 
iodin solution on the stain, the preparation before 
mounting may be washed in water and counterstained 
with Bismarck brown. This method shows all foreign 
matter brown in contrast to the bacteria, and is especially 
adapted for staining of bacteria in tissues, sputum, etc. 

It is a positive Gram's stain if by application of this 
method either the organism loses none of the stain, or 
the stain is dark blue or dark slate-blue. It is a negative 
stain if either the coloration is completely gone, or only 
a light bluish tinge is left. 

All stained preparations and all Gram stains must be 
preserved for inspection. 

3. Transfer from agar-culture to the following media : 
Glucose-agar. 

Gelatin. 
Potato. 
Broth. 

Litmus milk. 
Dunham's solution. 

These transfers, excepting gelatin, are to be placed 
in the thermostat, unless otherwise ordered. 

4. An accurate description is to be made of each 
individual culture (see Chap. X), and outline sketches 
especially of the gelatin and the milk cultures. These 
descriptions should be made complete after 24 hours, 



ROUTINE STUDY OF VARIOUS GROUPS 



55 



and any changes should be noted after 48 hours and after 
6 days. (See culture charts.) 

5. Plates are to be made in agar from a 24-48-hour- 




FIG. 26 
Thermostat or Incubator 

old broth-culture of each bacterium, unless otherwise 
directed. These plates are to be described once after 
24-48 hours. (See Chap. X.) 

NOTE. The thermostat or incubator (Fig. 26) is a box 
made of copper and having double walls, between which water 



56 LABORATORY GUIDE IN BACTERIOLOGY 

circulates. The outer surface is usually covered with asbestos 
or linoleum, so as to hold the heat. The thermostat is provided 
with two doors, the inner one of glass so as to enable the observer 
to look inside without opening it, the other one covered like the 
walls. A thermometer reaches through the upper part, and sev- 
eral air-holes are provided in it, which permit free circulation 
of air. The heat is controlled by Bunsen burners with self- 
regulating devices. The gas supply is shut off automatically, if 
it should accidentally become extinguished. For class use large 
incubators are constructed on the same principles, with a number 
of separate compartments. 



CHAPTER X 

METHOD OF DESCRIBING CULTURES 
The following method of describing cultures should 
be carefully studied, and each suggestion should be 
considered in the description. It is of prime importance 
that all cultures in all media should be closely observed, 
and accurately described and sketched, as this is the 
only method which furnishes the proper means of study- 
ing and determining the different species of bacteria. 
By keeping this scheme in sight while making ?. descrip- 
tion, it will be possible after a little practice to make 
perfect descriptions without its aid. 

I. Morphological characters of bacterium. 
Size. 

Facility and mode of staining. 
Gram's stain. 
Special staining qualities. 
Motility. 

Present or absent. 
Sluggish or active movement. 
Flagella present or absent. 
Capsules present or absent. 
Spores present or absent. 
Involution forms. 

Make an accurate sketch of part of a field under 
the microscope. 
II. Plate cultures. 

i. Naked-eye appearance. 
a) Surface colonies. 
57 



5 8 LABORATORY GUIDE IN BACTERIOLOGY 

Approximate number. 

Shape: punctiform, lanceolate, oval, cir- 
cular, spindle-shaped, conglomerate, 
irregular, branched, filamentous, rosette- 
shaped. 

Color in transmitted and reflected light. 

Approximate diameter in millimeters. 

Elevation (and shape of elevation) or 
depression. 

Translucency. 

Moist appearance. 

Smoothness. 

Luster. 

Liquefaction (in gelatin plates only). 

Consistency: soft, viscid, hard, chalky. 
b) Deep colonies. 

Color. 

Shape. 

2. Microscopic appearance under low power (No. 
3 objective). Care must be taken to use very 
little light, the diaphragm should be almost 
closed. 
a) Surface-colonies. 

Shape. 

Color. 

Translucency. 

Thickness (in center and edges). 

Nucleation. 

Striation. 

Granulation (if present, whether coarse or 
fine). 

Homogeneity. 



METHOD OF DESCRIBING CULTURES 59 

Tuberculation (tubercle-like appearances al 
edge). 

Edge : entire or smooth, wavy, with pointed 
protuberances, serrate, dentate, lacer- 
ate (as if torn), fringed, hairlike shoots, 
curled. 
b) Deep colonies. 

Shape. 

Color. 

Translucency. 

Granulation. 

III. Agar-slant-culture. 

Limitation: confinement to needle-track or 
spreading. If spreading, in what shape ? 

Vigor: luxuriant or scant. 

Color: by transmitted and reflected light. 

Elevation or depression. Elevation more pro- 
nounced at edges or in center. 

Translucency. 

Moistness. 

Smoothness. 

Luster. 

Coloration of medium. 

Pigment production. 

Odor. 

Gas-formation: in culture or in medium. 

IV. Stab-culture in plain agar. 

1 . Surface-growth (describe like surface-colonies.) 

2. Stab-growth. 
Vigor. 
Extent. 
Color. 



60 LABORATORY GUIDE IN BACTERIOLOGY 

Granulation. 
Outgrowths. 
Coloration of medium. 
Cloudiness. 
Gas-formation. 
V. Stab-culture in glucose-agar. 

Describe like plain agar, and in addition always 
note presence or absence of gas-formation, and 
presence of cloudiness. 
VI. Stab-culture in gelatin. 

Describe like plain agar, and in addition always 
note presence or absence of liquefaction. If 
liquefaction is present, it may be saucer-shaped, 
turnip-shaped, conical, funnel-shaped, hori- 
zontal (extending the whole diameter of tube), 
sack-shaped. Cloudiness and presence of 
sediment in liquefied area, and color and shape 
of sediment, should be described. 
VII. Potato- culture. 

Describe like agar-slant, adding to it the eventual 

discoloration of the medium. 
VIII. Litmus milk culture. 

Reaction (acid or alkaline, as indicated by 

color). 
Coagulation : at ordinary temperature or upon 

heating. 

Whey: if present, clear or turbid. 
Liquefaction of coagulum (proteolysis, pep- 

tonization). 
Gas-formation. 

Decolorization of litmus (complete or incom- 
plete). 



METHOD OF DESCRIBING CULTURES 61 

Color of cream-ring. 
Odor. 

In milk the presence or absence of coagulation and 
proteolysis should always be noted. Com- 
pare your culture always with a sterile con- 
trol milk-tube. 
IX. Broth-culture. 

Cloudiness: degree and uniformity, scum: ring- 

or island-shaped. 

Precipitate, observed by shaking. Color, forma- 
tion, diffusibility, viscidity, amount. 
X. Solidified blood-serum. 

Describe like agar-slant, and note presence or 

absence of liquefaction. 
XI. Fermentation-tubes. 

Gas-formation in closed arm, percentage and 
relation of carbon dioxid to hydrogen expressed 

TT 

by the formula . 

LUp 

Growth in both arms or in one arm only, observed 

by cloudiness. 
Reaction: acid or alkaline. 

Directions jor filling out the first page of culture- 
charts (see Appendix III.) 
I. Name the group and organism. 
II. Read in the textbook or references given so as to 
indicate the source and habitat. 

III. Name the most important references, and read 
them. 

IV. Morphological characters. 

i. Describe the morphology as observed from 
the stained preparation opposite the medium 
from which obtained. 



62 LABORATORY GUIDE IN BACTERIOLOGY 

2. Size: approximate estimate in microns. 
Note whether large or small, thick or slender, 
round or square ends, etc. 

3. Arrangement of bacteria: in groups, chains, 
bunches, pairs (diplococcus) , sarcina form, 
threads, branching, etc. Also note different 
arrangements, if observed, in different media. 

4. Staining powers: It is sufficient to mark + 
for positive, - - for negative stains. Special 
stains must be described more fully. 

5. Motility: If absent, mark ; if present, +. 
In the latter case describe the character of 
the movement. 

6. Spores: Absence noted by ; presence, by 
+ . This information is to be gathered from 
the textbook, unless specially instructed. 
If so mention the method of spore-stain applied. 

7. Note any peculiar appearance in the micro- 
scopical picture, especially involution forms 
and the presence or absence of capsules. If 
capsules are present, note the method of 
demonstration. 

V. Physiological characters. 

1. Relation to temperature : What is the optimum 
temperature? (See textbook or references.) 

2. Relation to free oxygen: aerobe, anaerobe, 
facultative aerobe or anaerobe. 

3. Relation to disinfectants, light, desiccation, 
heat (thermal death-point). (Refer to text- 
book or references.) 

4. Pigment-production: If present, +; if absent, 
. In the former case, note the color, dif- 



METHOD OF DESCRIBING CULTURES 63 

fusibility, solubility, influence of acids and 
alkalis. 

5. Gas-production in glucose media: To be filled 
out only in case of actual observation. In 
fermentation-tubes note the growth in either 
arm or both arms, recognized by turbidity. 
Note the total percentage of the gas formed in 
24 and 48 hours. Reaction may be tested by 
the addition of litmus-solution. Gas formula 

expressed : . 

CO 2 

6. Acid or alkali production in litmus milk. 

7. The production of indol or nitrites, or both, 
is tested on the sixth day of observation in a 
culture in Dunham's peptone-solution or 
sugar-free broth. 

NOTE. Indol is a decomposition product of 
proteids and belongs to the aromatic series. 
Nitrites are the result of reduction from nitrates. 
The ability of organisms to produce these reactions 
are of great importance in their differentiation. A 
control test with a tube of sterile medium should 
always be made. 

8. Enzym-production : Froteolytic enzym-pro- 
duction noted by the liquefaction of gelatin or 
casein. Coagulative, by precipitation of casein, 
if acid-formation is absent, or present only in 
quantities less than 0.3 per cent. Diastatic, by 
the digestion of starch (potato). 

9. Characteristic odor. 

10. Pathogenesis : What pathogenic effect has the 
organism on man ? What effect on animals, 



64 LABORATORY GUIDE IN BACTERIOLOGY 

and which animals ? What diseases are 
caused by the organism in man or animal ? 

NOTE. It may be well here to call attention to the terms 
"proteolysis," "enzym -production," and " coagulation." 

" Proteolysis " is the breaking up of complex nitrogenous 
compounds (proteids), rendering them soluble. This process is 
also expressed by the terms " peptonization " and "liquefaction." 
The liquefaction of gelatin is one kind of proteolysis. Gelatin 
is composed of nitrogenous matter (albuminoid or gelatinoid), 
and it is for this reason mainly that gelatin stab-cultures are 
made. If the gelatin is liquefied, we conclude that the organism 
is capable of producing a " proteoly tic " enzym. In milk the 
process is more complex, and this medium, on account of its 
composition (fat, milk-sugar, casein, lactalbumin), offers excel- 
lent opportunities for the organism to develop different charac- 
teristics. Milk is one of the most important media. The casein- 
ogen, contained in milk in colloid solution, may be precipitated 
by an enzym or by an acid. This precipitate forms the coagu- 
lum. At least 0.4 per cent, of acid, which is largely lactic acid 
produced by splitting of milk-sugar (lactose), is required for 
precipitation, and this amount of acid will turn the blue litmus to 
a decided pink. A coagulum may also be produced by the presence 
of a " coagulative " or "rennet "-like enzym, which is the result 
of the metabolic activity of the organism. Such coagulation may 
take place in milk of an amphoteric or alkaline reaction, as well 
as in milk of a slightly acid reaction. The coagulum formed by 
any of the mentioned agents may gradually contract, and a straw- 
yellow, opalescent liquid is squeezed out, called "whey." If, 
now, the organism also produces a proteoly tic enzym, this will 
attack the coagulum and gradually dissolve it (proteolysis, pep- 
tonization). At first the coagulum shows a broken-up surface; 
lumps separate and settle to the bottom, and finally the coagulum 
may completely disappear. Theoretically, coagulation is always 
necessary before proteolysis sets in, but in the case of some organ- 
isms the proteolytic enzym is so powerful as to produce immediate 
dissolution of the casein. 

Another phenomenon frequently observed in litmus milk is 



METHOD OF DESCRIBING CULTURES 65 

the discolorization of the litmus, whether this be pink or blue. 
This is due to the fact that the organism takes up the oxygen 
necessary to maintain the coloration. It may be frequently 
observed that at the surface, where atmospheric oxygen has 
access, the color remains or is restored. The color may also be 
restored by shaking the milk vigorously, thus bringing it into 
intimate touch with the oxygen of the air. 

The production of a diastatic enzym (diastase, amylase) is 
demonstrated by gas-production on potatoes. This medium con- 
tains starch in large amounts. The starch is inverted into glu- 
cose by diastase (amylase), and this is then fermented with 
gas-production. 

Directions for filling out second page of culture-charts 
i . Note the reaction of the medium by the sign -f- for 
acid and for alkali reaction. 

2. The incubation temperature may be (a) 37 
(thermostat), (b) room temperature, (c) ice-chest. 

3. Plates are to be described only once after 24 or 
48 hours, according to growth. Make notes in the first 
column for gelatin; in the second, for agar-plate, writing 
across the full width of the page. Make sketches in the 
special column reserved for this purpose. 

4. The growth on the media 3, 4, 5, 6, 7, and 8 
(enter here: " glucose-agar " only unless otherwise 
instructed) to be made fully according to the outline in 
the spaces under 24 hours. In the spaces under 48 hours 
and 6 days note only the changes from the first day. 

5. Make sketches frequently and accurately, espe- 
cially from milk and gelatin media, and any other remark- 
able growth, in the column reserved for this purpose; 
also a sketch of each organism from a part of a field 
under the microscope. 

It is well to understand that these directions for fill- 



66 LABORATORY GUIDE IN BACTERIOLOGY 




3 



V 



l\ 



FIG. 27 
Streak-Cultures 




FIG. 28 
Stab-Cultures 



METHOD OF DESCRIBING CULTURES 67 



L 




FIG. 20 
Liquefaction of Gelatio 




123 

FIG. 30 

1. Gas-bubbles in glucose-agar 3. Coagulation and peptonization of milk 

2. Coagulation of milk 4. Complete peptonization of milk 



68 LABORATORY GUIDE IN BACTERIOLOGY 

ing out culture-charts are applicable in the described 
manner to studies of cultures furnished by the laboratory. 
For determining any species of unknown bacteria, original 
researches must be made to cover all points, necessarily 
without the possibility of gathering this information from 
textbooks or references. On the accuracy of observation 
and description depends the success of bacteriological 
work and species-determination. It is frequently neces- 
sary to employ special media, or inoculation of animals, 
or such reactions as the agglutination test, to determine 
properly what species one is dealing with. 

The student will do well to familiarize himself care- 
fully with all directions and explanations given in this 
and the previous chapters. Find a characteristic for 
each item mentioned; otherwise the descriptions will 
be incomplete; and follow the instructions for routine 
work with all possible accuracy. 



CHAPTER XI 

STUDY OF CERTAIN CHROMOGENIC BACTERIA 
EXERCISE I. CULTURAL STUDIES 

1. Read carefully the following references to chromo- 
genic organisms, and the production and chemistry of 
pigments: 

Sternberg, Manual of Bacteriology. 
Lehmann and Neumann. 

Members of this group are widely disseminated in the 
air, water, etc. A few representatives will be studied. 

2. Inoculate agar-slants from stock-cultures of Bacil- 
lus prodigiosus, B. pyocyaneus, B. violaceus, and Sarcina 
luiea. Inoculate three slants each of B. prodigiosus and 
B. pyocyaneus, and one each of Sar. lutea and B. violaceus. 

3. Label each tube with the name of the culture 
inoculated, the date of the stock-culture and the date of 
inoculation. Place the label on the side of the tube 
where the slanted surface of the medium is. It is then 
possible to study the growth and see the label at the same 
time. Glass pencils for marking are very convenient. 

4. Place one culture of each organism in the thermo- 
stat, one culture of B. prodigiosus and B. pyocyaneus in 
the locker, and leave the others exposed to sunlight. 

5. After 24 hours compare the growths of B. prodigio- 
sus and B. pyocyaneus, under the various conditions, in 
respect to 

a) Relative amount of growth. 

b) Relative amount of pigment produced. 

What conclusions can be drawn from this experiment ? 
69 



70 LABORATORY GUIDE IN BACTERIOLOGY 

6. Note the characteristics of the pigments : Are they 
diffused through the medium, or are they confined to 
the growth ? 

7. Make descriptions of agar-cultures ; also hanging- 
drop, stained, and Gram preparations. 

8. Transfer from 24-hour-old agar-cultures of all 
organisms to all media. (See routine study, Chap. IX.) 
Potatoes may be inoculated with the looped needle, as 
the surface is too rough to allow of a smooth inoculation 
with the straight needle. 

9. After all cultures have been in the thermostat for 
24 hours, make all descriptions as outlined in Chap. X. 
(Record after 48 hours and 6 days only observations of 
change.) 

CAUTION. The gelatin-cultures are sometimes placed by 
students in the thermostat along with others through oversight. 
This, of course, completely defeats the purpose of obtaining a 
stab-growth, as the gelatin will melt. In order to avoid this 
mistake, it is recommended to label one tin cup or tumbler 
" Gelatin" in large letters. This will serve as a constant reminder 
that gelatin has to be kept at room temperature. 

10. Make plate-cultures of the four organisms. 
Method oj making plates 

a) Melt two agar-tubes for each organism in the water- 
bath and cool to 43. 

b) Transfer 3-5 loopfuls (according to the intensity 
of the growth, to be judged by the degree of cloudiness) 
of the broth-culture to a sterile tube of Dunham's solu- 
tions. (Dilution i). 

c) Shake well, avoiding air-bubbles as much as 
possible. 

d) Transfer 4 or 5 loopfuls from this suspension to a 
melted agar-tube, and label this 2. (Dilution 2). 



CERTAIN CHROMOGENIC BACTERIA 71 

e) Shake this carefully by rolling the tube between 
the palms of the hand, or stir with the platinum needle, 
so as to avoid air-bubbles. 

/) Transfer 4 or 5 loopfuls of this agar-tube (2) to 
the second agar-tube (3), and mix as above. 

g) If thought necessary, more tubes may be treated 
in the same way, resulting in still higher dilutions. In the 
meantime the inoculated tubes should be replaced in 
the water-bath, so as to keep them liquid. 

h) Pour the contents of the tubes, one after the other, 
into sterile Petri dishes. 

i) Tip the Petri dishes very carefully, so as to dis- 
tribute the medium evenly over the bottom. 

k) Label them 2nd, 3d, dilution, etc., with the name 
of the organism and the date. 

/) Set aside on a level place to solidify. 

m) When solidified, place them bottom up in the 
thermostat, in order to avoid moistening of the surface 
by the condensation water dropping down from the 
cover. 

NOTE. If the surface were moistened, the colonies would 
run together and the characteristic appearance be destroyed. Gela- 
tin plates, on the contrary, are placed cover up. Condensation 
water does not form on these plates as gelatin may be liquefied 
by the organisms. The liquefied part would then fall from the 
medium on the cover and ruin the plate. 

The plates prepared in the above manner should be 
studied after 24 hours, or, if not sufficiently developed, 
after 48 hours, according to directions in Chap. X. 

EXERCISE II. STUDY OF PIGMENTS 

On the sixth day take agar-slant or potato-cultures 
of the four chromogenic bacteria, and proceed as follows : 



72 LABORATORY GUIDE IN "BACTERIOLOGY 

Pour 96 per cent, alcohol on the cultures of B. prodigi- 
osus, B. violaceus, and Sar. lutea. The pigment should 
dissolve. Filter the liquids into clean test-tubes, and, 
by adding a few drops of 5 per cent, hydrochloric acid, 
note the change in color. Then add an excess of a 2 
per cent, solution of sodium hydrate, and note whether 
or not the color returns and is changed again. Then 
pour chloroform on a culture of B. pyocyaneus. This 
will dissolve the bluish-green pigment (pyocyanin). 
Filter, and evaporate on the water-bath. When almost 
dry, place a small amount on a slide, and observe the 
small crystals of pyocyanin under the microscope. Note 
also the aromatic odor given off by the pigment as the 
solvent evaporates. 



CHAPTER XII 
THE PYOGENIC GROUP 

EXERCISE I. THE PYOGENIC GROUP (SUBGROUP A) 1 
MEMBERS 

Staphylococci, streptococci, Microc. tetragenus. 

Inoculate agar-slants from cultures (furnished) of 
Staphylococcus pyogenes aureus, Staph. pyogenes albus, 
and Streptococcus pyogenes. These organisms are patho- 
genic, and care must be taken to observe the rules of 
technique with the utmost accuracy. Any carelessness 
may be followed by the gravest consequences. In case 
of accident, such as the spilling of a culture or infecting 
the hands, thorough disinfection e. g., with a solution 
of mercuric chlorid (i : 1000) is absolutely necessary. 

After 24 hours' incubation of the three agar-slants, 
proceed with the other media as outlined in the routine 
study (Chap. IX). 

Special study. A rabbit will be inoculated intra- 
venously with a broth-culture of Staph. pyogenes aureus. 
The ear of the rabbit is carefully shaved, washed with 
mercuric chlorid solution, followed by alcohol. Then 
0.5 c.c. of a 24-hour-old culture in broth is drawn up 
into a hypodermic syringe, which has been previously 
sterilized by immersion in boiling water for 10 min- 
utes. The mode of holding a rabbit is as follows: 
The left arm of an assistant rests against the hind- 
quarters of the rabbit on the table, while the two hands 

1 This subdividing of the pyogenic group is an arbitrary 
measure designed simply to facilitate study, the commoner pyo- 
gens being studied first. 

73 



74 LABORATORY GUIDE IN BACTERIOLOGY 

gently hold the fore-legs. If the animal struggles, too 
much force should not be applied, as this might injure 
or kill it. The struggles may be overcome by wrapping 
the animal in a towel or any other similar piece of cloth. 
The needle is then inserted into the lumen of the lower 
vein (ramus lateralis posterior of the vena auricularis 
posterior), which has been pinched between the fingers, 
or by means of a forceps, so as to arrest the circulation. 
No air should be injected with the culture, as this will 
kill the animal. The hypodermic needle is then with- 
drawn and sterilized in boiling water for 15 minutes. 

After the death of the rabbit, study the lesions pro- 
duced by the organism, and make cultures on slant-agar, 
and smears from the heart's blood, spleen, and any foci 
of suppuration. 

AUTOPSY (SEE SKETCH, FIG. 31) 

1. Have the instruments sterilized in boiling water. 

2. Tie the animal by the extremities on a square 
board, with the abdomen upward. 

3. Note the presence of any external lesions, such as 
swellings, ulcerations, etc. 

4. Wash with a solution of mercuric chlorid (i : 1000) 
followed by alcohol. 

5. Lift the skin over the pubes with the forceps, and 
with the scissors make an incision along the median line 
well above the sternal notch; then diagonal incisions 
extending to the fore- and hind-legs. 

6. Cut the skin away carefully with a moderately 
sharp knife, avoiding opening the abdominal cavity, and 
pin to the board. 

7. Open the abdomen by a median incision from the 
pubes to the sternum. 



PYOGENIC GROUP 



75 



-STOMAC H 
h-SPLEEN 
-KIDNEY 



INGUINAL 
GLANDS 




FIG. 31 
Method of Autopsy of a Guinea-Pig (Diagrammatic) 



76 LABORATORY GUIDE IN BACTERIOLOGY 

8. Remove the anterior thoracic wall by cutting away 
the ribs from below upward on each side to the thoracic 
apex. 

The viscera are now thoroughly exposed. Cultures 
and smears should be made from different parts, 
especially from the heart's blood, peritoneal cavity, 
spleen, liver, and localized foci of suppuration. 

EXERCISE II. THE PYOGENIC GROUP (SUBGROUP B) 

MEMBERS 

Micrococcus lanceolatus. 
Micrococcus gonorrhoeae. 
Micrococcus intracellularis meningitidis. 
Micrococcus zymogenes. 

Prepared cover-slips of Micr. gonorrhoeae are furnished. 
Stain these with methylene-blue and Gram's method, 
study, and describe the microscopical appearance. This 
organism is extremely difficult to cultivate. It is a strict 
parasite, and requires special media, and a great amount 
of time and care, for artificial cultivation. For these 
reasons, only the morphology, as it appears in gonorrheal 
pus, and which is very characteristic, is studied. 

Inoculate agar-slants from stock-cultures of Micr. 
lanceolatus and Micr. zymogenes. 

REFERENCES (Micr. zymogenes) 

MacCallum and Hastings, Journal of Experimental Medi- 
cine, Vol. IV (1899), p. 521. 

Harris and Longcope, Centralblatt fur Bakteriologie und 
Parasitenkunde, Part I, Vol. XXX, No. 9 (printed in 
English). 

1. Routine study. Note particularly the microscopic 
appearance of both organisms and the action of Micr. 
zymogenes on milk and gelatin. 

2. Special study A. The staining of capsules from 



PYOGENIC GROUP 77 

a milk-culture of Micr. lanceolatus . Two methods may 
be applied for this stain : 

First method (Friedlander's method) : 

a) Prepare a stain by the following formula: 

Glacial acetic acid i part 

Saturated alcoholic solution of gentian- 
violet 5 parts 

Distilled water 10 parts 

b) Prepare a very thin film in the usual manner from 
a 24-hour-old milk-culture, taking care to spread very 
thinly. 

c) Cover with stain for 10-15 seconds. 

d) Wash in a solution of sodium chlorid (0.85 per 
cent.). 

e) Examine in a sodium chlorid solution. 

/) If satisfactory, float the cover-slip off with salt 
solution, dry, and mount in balsam. Under no conditions 
should plain water come in contact with this preparation 
at any stage; otherwise no capsules will appear. 

Second method (Welch's method): 

a) Prepare a very thin film in the usual manner from 
a 24-hour-old milk-culture. 

b) Cover with glacial acetic acid for 5 seconds. 

c) Wash acid off with carbol-fuchsin. 

d) Wash stain off with a 0.85 per cent, sodium 
chlorid solution. 

e) Examine in salt solution. 

/) If satisfactory, mount in balsam. 

Plain water should not come in contact with this 
preparation at any stage. The capsule should be ob- 
served as a lightly stained zone with well-defined outline 
around the deeply stained organism. 



78 LABORATORY GUIDE IN BACTERIOLOGY 

3. Special study B. Inoculation of mouse with Micr. 
lanceolatus. 

a) Fasten the mouse in the holder (Fig. 32). 

b) Shave a place on the back immediately above the 
tail. 

c) Wash with a solution of mercuric chlorid (i : 1000), 
followed by alcohol. 




FIG. 32 
Mouse-Holder 

d) Inject 0.2 c.c. of a milk-culture of Micr. lanceo- 
latus. 

e) When dead, perform an autopsy, and study the 
lesions in the usual manner. 

/) Make cultures in milk and on slant-agar from 
the heart's blood or the spleen. 

g) Make a capsule stain from the heart's blood, 
spleen, or other organs. 



CHAPTER XIII 
THE INTESTINAL GROUP 

This chapter is devoted to the study of the "intestinal 
group" of organisms. This collective group may 
conveniently be subdivided into four subgroups: 

Subgroup i : the colon group. This group includes 
different varieties of Bacillus coli, B. lactis aerogenes, and 
some species of B. acidi lactici. 

Subgroup 2 : the hog-cholera, B. enteritidis, or inter- 
mediate group. This group includes B. cholerae suis, B. 
paratyphosus (several varieties), B. enteritidis, and B. 
icteroides. The term " intermediate " is assigned to this 
group, because it resembles the colon group on the one 
hand, and the typhoid group on the other. 

Subgroup 3: the typhoid-dysentery group. This 
group includes B. typhosus, varieties of B. dysenteriae, 
and B. jaecalis alcali genes. 

Subgroup 4 : the proteus group. This group includes 
all varieties of proteus (Bact. termo) and B. cloacae. 

EXERCISE I. STUDY OF SUBGROUP I : THE COLON GROUP 

Inoculate agar-slants from stock-cultures of B. coli 
(two varieties) and B. lactis aerogenes. Also inoculate 
one tube of broth with B. coli. 

These two types of B. coli are differentiated by their 
ability to ferment carbohydrates: 

A. Those which ferment dextrose, lactose, and 
saccharose. 

B. Those which ferment dextrose and lactose, but 
not saccharose. 

79 



8o LABORATORY GUIDE IN BACTERIOLOGY 

REFERENCES 

Theobald Smith, The Wilder Quarter Century Book, 1893, 
p. 187. 

Jordan, Journal o/ Hygiene, Vol. I (1901), p. 295. 

Durham, Journal oj Experimental Medicine, Vol. V, p. 353. 

1. Routine study. Observe carefully the growth on 
potato of B. lactis aero genes. What is the cause of gas- 
formation in this case ? 

2. Special study A. In order accurately to test the 
action of micro-organisms on various carbohydrates, it 
is necessary to eliminate the small amount of sugar in 
ordinary broth introduced into it by meat-extract, which 
generally contains muscle-sugar (glycogen). This is 
accomplished by adding to freshly prepared broth a 
culture of B. coli, which completely decomposes many 
carbohydrates, including muscle-sugar. By this method 
a sugar-free broth is prepared, which may be used as a 
solvent for any sugar desired. 

Preparation of sugar-free broth for the fermentation- 
tube: 

1 . Dissolve 

In water 400 c.c. 

Extract of beef i g. 

Peptone 4 g. 

by heat. Broth made from chopped beef (500 g. to i 
liter) may also be used for this purpose. 

2. After cooling, inoculate with a broth-culture of 
B. coli prepared 24 hours previously. 

3. Set aside in the locker, or, better, in the thermostat, 
for 18-24 hours. 

4. Boil 5 minutes (to kill B. coli), and filter repeat- 
edly through the same paper until perfectly clear. 

5. Divide into three equal parts and dissolve 2 g. 



INTESTINAL GROUP 81 

(ij per cent.) dextrose, lactose, and saccharose, respec- 
tively, in each part, and filter again, if necessary. 

6. Fill fermentation-tubes, taking care to label each 
one properly, and sterilize in Arnold on 3 consecutive 
days for 20 minutes. 

All gas must be carefully tilted out of the closed arm 
of the tube while the fluid is warm. When sterilization 
is completed, inoculate one set of the fermentation-tubes 
with B. coli A, another set with B. coli B, and a third set 
with B. lactis aero genes. Inoculate with the straight or 
looped needle. 

After 24 hours, note the percentage of gas formed. 
This is done by means of Frost's fermentation-chart, a 
model of which is represented by Fig. 33. This chart is 
placed between the open and closed arms of the tube, 
resting on the neck and moved along until the extreme 
upper end of the closed arm is level with the top of the 
chart and parallel with the vertical lines. The percent- 
age may then be read off by the figures marked at each 
end of the chart. 

After taking note of the percentages, replace the fer- 
mentation-tubes in the thermostat, and repeat the meas- 
urement after another 24 hours' incubation. Many 
organisms are able to obtain the oxygen necessary for 
life by attacking compounds as, for instance, carbo- 
hydrates and decomposing them. In such cases a tur- 
bidity is developed in the medium. The closed arm offers 
strict anaerobic conditions, all the atmospheric oxygen 
having been driven out by the heat of sterilization. The 
bulb allows the organism to come in contact with atmos- 
pheric oxygen, and therefore offers aerobic conditions. 

Gas-production is by no means a constant phenom- 



82 LABORATORY GUIDE IN BACTERIOLOGY 

enon accompanying a fermentation. Carbohydrates are 
fermented by many organisms without gas-formation. 
The usual product in this case is an acid, mostly lactic 
acid. Such fermentations produce turbidity only, and 
no gas. It is therefore necessary, in describing them, 
to characterize the process as fermentation without 
gas-formation. '. 

Method oj analyzing the gas produced in the closed arm. 
The gas consists approximately of carbon dioxid and 
hydrogen, which may be proved by the following method: 
Fill the bulb with a 2 per cent, solution of sodium hydrate, 
and close the mouth with the thumb, taking care not to 
leave any air between the thumb and the liquid. Now 
slowly tilt the gas back and forth from the closed arm 
to the bulb five or six times, and finally allow it to collect 
again in the closed arm. The sodium hydrate combines 
with the carbon dioxid, and consequently on releasing the 
thumb the volume of gas will become smaller in propor- 
tion to the amount of carbon dioxid absorbed. Now 
measure the percentage again with the chart, and ascer- 
tain the proportion of gas left in the closed arm to the 
original amount. 

Example 

Total percentage of gas before addition of NaOH. ... 45 
Percentage left after absorption by NaOH 30 

Difference 15 

30 per cent, then represents the amount of hydrogen 
left, and 15 per cent, the amount of carbon dioxid ab- 
sorbed. The proportion is expressed by the formula 
H =3 o 2 

co a 15 r 



INTESTINAL GROUP 83 

The fact that this gas remaining in the closed arm is 
hydrogen may now be proved by tilting it into the open 
arm, which has previously been filled with water and 
closed by the thumb. By holding a burning match 
over it and quickly releasing the thumb, a slight explo- 
sion takes place from the sudden combination of the 
hydrogen with the oxygen of the atmosphere. 

Gas-formation by bacteria does not necessarily 
depend on the presence of carbohydrates. Nitrogen is 
often produced from nitrites, and hydrogen sulphid and 
ammonia from proteids, especially during the process of 
putrefaction. 

3. Special study B. Test for indol and nitrites. 
What is indol ? What biological activity of the organism 
does the presence of indol or nitrites, or both, indicate ? 

a) Test for nitrites: Add to a culture in Dunham's 
solution, or, better, in sugar-free broth, successively i 
drop of each of the following solutions: 

(1) Sulphanilic acid 0.5 g. 

Acetic acid (25$) 150 c.c. 

(2) Naphthylamine chlorid o. i g. 

Distilled water 20 c.c. 

Acetic acid (25$) 150 c.c. 

A yellowish-red or rose-color shows the presence of 
nitrites. 

b) Test for nitrites and indol combined. 

(1) Add to a culture in Dunham's solution, or sugar- 
free broth, i or 2 drops pure sulphuric acid. 

(2) Heat gently. Rose-color shows the presence of 
nitrites and indol. If no reaction takes place, add 

(3) A few drops of a solution of o.i g. potassium or 
sodium nitrite in 1000 c.c. water. Rose-color then 
indicates the presence of indol only. 



84 LABORATORY GUIDE IN BACTERIOLOGY 

Perform these tests with all the organisms of the 
intestinal group, and make control tests in sterile Dun- 
ham's solution. 

4. Special study C. Make capsule stain of B. 
lactis acrogenes from 24-hour-old milk-cultures. (For 
method see p. 77.) 

The study of B. coll is of special importance in con- 
nection with bacteriological analyses of water (see Chap. 
XXIV). The presence of this organism in large num- 
bers indicates sewage contamination, and consequently 
the danger of an admixture of pathogenic bacteria such 
as B. typhosus and B. dysenteriae. 

EXERCISE II. STUDY OF SUBGROUP II 
THE HOG-CHOLERA, B. ENTERITIDIS, OR INTERMEDIATE GROUP 

Use great care in handling members of this group. 

Inoculate agar-slants from stock-cultures of B. 
cholerae suis (bacillus of hog-cholera), B. enteritidis 
(Gartner's bacillus), and B. paratyphosus (two strains). 
REFERENCES 
B. cholerae suis: 

Moore, The Pathology of Infectious Diseases of Animals. 

McFarland, Textbook of Bacteriology. 
B. paratyphosus: 

Buxton, Journal of Medical Research, 1902, Vol. VIII, p. 201. 

Wells and Scott, Journal of Infectious Diseases, 1904, No. i. 

Gushing, Johns Hopkins Hospital Bulletin, 1900, p. 156. 

Durham, Journal of Experimental Medicine, Vol. V, p. 353. 

1. Routine study. Observe particularly the bluish- 
green coloration of the cream-ring in litmus milk, and 
make test for indol. 

2. Special study A. Inoculate plain sterile milk with 
B. cholerae suis. After 8-10 days a clearing of the milk 



INTESTINAL GROUP 85 

will be observed, due to a solvent action of the alkali 
produced by the organism upon the proteid content. 

3. Special study B. Inoculate fermentation-tubes the 
same as for B. coli. Measure and test gas. Compare 
the results with those obtained in the study of the colon 
group. What difference do you notice ? 

4. Special study C. Inoculation of a rabbit sub- 
cutaneously with B. cholerae suis. Subcutaneous inoc- 
ulations of rabbits are usually made in the following 
manner: An assistant, in a sitting position, places the 
rabbit back-down in his lap. The head projects beyond 
the knees of the assistant. The ears and hind-legs are 
grasped, and the animal is thus held in position. The 
hair is then cut off on a portion of the abdomen, and the 
place is treated with mercuric chlorid and alcohol in the 
usual manner. With sterilized fingers the skin is then 
pulled up, the syringe inserted, and the material injected. 

After the rabbit has died, study the lesions produced 
by the organism, and make smears from the site of the 
inoculation, the heart's blood, and other organs. Note 
the polar staining, i. e., stained portions at the two ends 
of the cell and an unstained area between. Make cul- 
tures on agar from the heart's blood and other internal 
organs. 

EXERCISE III. STUDY OF SUBGROUP III 
THE TYPHOID-DYSENTERY GROUP 

Use great care in handling members of this group. 

Inoculate agar-slants from stock-cultures of B. typho- 
sus, B. dysenteriae (Shiga), and B. jaecalis alcaligenes. 

i. Routine study. Study carefully the reaction on 
milk, and test for indol. Preserve glucose-agar-cultures 
for two weeks. 



86 LABORATORY GUIDE IN BACTERIOLOGY 

2. Special study A. Inoculate fermentation-tubes in 
the same manner as in the two preceding groups. Ob- 
serve the absence of gas-formation but note growth in 
both arms. Compare the results with those of the colon 
and intermediate groups. 

3. Special study B. The staining of flagella. Read 
carefully in the textbook the methods of staining flagella. 
To demonstrate the presence of flagella on B. typhosus, 
the following method will give good results (Loeffler's 
method) : 

a) Prepare the mordant. (What is a mordant ? See 
the textbook.) 

Tannic acid (25$ aqueous solution) 10 parts 

Ferrous sulphate (saturated aqueous solution) ... 5 parts 
Fuchsin (saturated alcoholic solution) i part 

b) Prepare a number (six) of cover-slips by carefully 
flaming them, and place them side by side on a piece of 
filter-paper. (This paper must be burned after using). 

c) Place 4 or 5 loopfuls of water on a clean slide. 

d) Make a light suspension in this water of B. 
typhosus from a 24-hour-pld agar-culture, taking par- 
ticular care to stir the suspension as little as possible. 

e) Place a loopful of water on each of the cover-slips. 
/) Carry over a loopful of the suspension on the slide 

to one of the cover-slips, from this to second, from the 
second to the third, etc. 

g) Spread carefully and allow to dry in the air. 

h) Cover with the mordant. 

i) Heat over a small flame for ij minutes while 
steam rises, or better heat for 5 minutes on a water-bath. 
Replace the evaporated stain, to prevent its drying on 
the cover-slip. 



INTESTINAL GROUP 87 

j) Wash thoroughly in water. 

k) Drain the water off with blotting-paper. 

/) Cover with anilin-gentian- violet or carbol-fuchsin. 

m) Heat as before over a small flame for ij minutes. 

n) Wash thoroughly in water. 

0) Examine in water. 

p) If satisfactory, mount in balsam. 

4. Special study C. A ggluiination. Dried-blood 
method of Johnston: A drop of blood of a typhoid- 
fever patient is obtained by pricking the lobe of the ear, 
previously carefully cleaned and washed with alcohol. 
The blood is taken up by a piece of sterile non-absorbent 
paper or on a sterile aluminum slide. This is sent to a 
laboratory, where the blood is dissolved in physiological 
salt solution in such a manner as to obtain an approxi- 
mate dilution of 1:25. This solution is then tested with 
a suspension of typhoid bacilli, a young culture of which 
is constantly kept on hand for this purpose. 

For laboratory tests the serum of an animal (either 
a rabbit or a guinea-pig) which has been injected with 
cultures of B. typhosus, previously heated for i hour at 
60. This process kills the organisms, but the toxins 
remain active. The animal is then bled in the following 
manner: One of the ears is shaved, and the skin is 
washed with alcohol. A small vein near the border is 
opened, and the blood is collected in a sterile glass vessel. 
If the animal does not bleed freely enough, it must be 
warmed, preferably by means of a hot-water bag. The 
blood is placed in the ice-chest, and the serum is col- 
lected after separation. 

The method of procedure with a serum obtained in 
the above-described manner is as follows: 



88 LABORATORY GUIDE IN BACTERIOLOGY 

a) Small quantities of the serum are diluted with 
sterile salt solution (0.85 per cent.) so as to represent 
dilutions of 1:5, 1:25, and 1:50. 

b) A suspension of a 24-hour-old agar-culture of B. 
typhosus in salt solution is prepared. This suspension 
should be faintly turbid and uniformly so. 

c) Three hanging-drop preparations are made by 
mixing a loopful of this suspension, with a loopful of the 
three serum dilutions, respectively. The final dilutions 
then are: 1:10, 1:50, and 1:100. 

d) Examine with the high power (dry lens), and 
observe the clumping of the bacilli, preceded by the loss 
of motility. 

e) Tabulate the results as to time and completeness 
of reaction. 

/) Make a control hanging-drop without serum to 
test the motility and the absence of clumps. 

Blood may also be obtained by puncturing the lobe 
of the ear and collecting it in a capillary glass tube with 
a small bulb. By holding the bulb down, fill three- 
fourths full with blood, and seal the ends in the flame. 
In 45 minutes the serum will have separated, and is 
to be tested in the above manner. 

The above-described method of agglutination test is 
known as the microscopic test. Another method, in 
which larger amounts of serum and suspension are 
required, is known as the macroscopic method. Small 
test-tubes are used, and definite amounts of bacterial sus- 
pensions are introduced by means of finely graduated 
sterile pipettes. The serum is then added in varying 
amounts so as to effect the desired dilutions (see table, p. 
117). The tubes are then incubated at 37, usually for 



INTESTINAL GROUP 89 

2 hours, or longer if desired. If complete agglutination 
takes place, the bacteria will have collected in clumps at 
the bottom, forming a sediment. The supernatant fluid 
is perfectly clear. By varying amounts of sediment and 
varying degrees of turbidity of the supernatant fluid, the 
degree of agglutination may be estimated. A control- 
tube of a bacterial suspension without addition of serum 
serves as a guide. Controls with normal serum should 
also be made. 

5. Special study D. 

a) Make cover-slip preparations of B. dysenteriae 
from glucose-agar-cultures 10-12 days old. Involution 
forms are then plentiful and can be studied. 

b) Also make cover-slip preparations of B. typhosus 
from glucose-agar-cultures 10-12 days old. Does it 
show the same picture ? 

EXERCISE IV. STUDY OF SUBGROUP IV 

THE PROTEUS GROUP 
REFERENCES 

Lehmann and Neumann. 

Jordan, State Board of Health of Massachusetts, 1890. 

Jordan, Journal of Hygiene, Vol. II. 

Inoculate agar-slants from stock-cultures of Proteus 
vulgaris, Prot. zenkeri, and Bacillus cloacae. 

1. Routine study. Observe particularly the action 
on milk and gelatin. 

2. Special study A . Make plates in gelatin and agar, 
and observe the colonies after 24, 48, and 72 hours. 
Note the appearance of the colonies of Prot. vulgaris 
and of Prot. zenkeri on both media. 

3. Special study B. Inoculate fermentation-tubes in 
the same manner as for the other intestinal groups. 



90 LABORATORY GUIDE IN BACTERIOLOGY 

Determine the percentage of gas formed and the gas 
formula. Compare the results with those of the other 
intestinal groups. How does the gas formula differ 
from those previously examined ? 

NOTE. In order to obtain a clear picture of the chief differ- 
ential characteristics of the four intestinal groups, it is recom- 
mended to tabulate the results in parallel columns, as outlined 
below. Express positive results by the sign + ; negative, by . 
Complete agglutination is expressed by + + ; slight, by -f . 



INTESTINAL GROUP 



9i 



Knnag aioHdAj, 

HXIM NOIXVNIXmOOV 


























FERMENTATION OF 
CARBOHYDRATES 


Growth in 
Closed Arm 
No Gas 


tpDT^S 






















3 sop B1 






















JJXJQ 
























Free Gas in 


ipatK; 




















asopB"^ 


















a,x 3a 




















m 

D 
H 


!P"llV F 11 ! 111 - 13 ^ 


















uoijieziuojdaj 


















uot)T7[n3ro3 


















II^IV 


















PPV 


















POTATO 


SBQ 


















JUBDg 


















juBunxn-! 



















Noixonaoaj loa.vi 


















KVHQ 




















Axnixop^ 




















AOO'iOHdnow 
























o 

Id 






ORGANISM 


a 

rt 

*o 

M 


.t 
1 

PQ 


B. lactis aerogenes 


B. cholerae suis 


B. enteritidis 


B. paratyphosus 


B. typhosus 


B. dysenteriae (Shiga) 


I 

.1 

13 

"rt 
.fl 

H 


Proteus vulgaris 


1 

S 

PH 


B. cloacae 


1 

09 


I. 

w * 


II 
THE 
INTERMEDIATE 
BAC. ENTERITIDIS 

OR 

HOG-CHOLERA 
GROUP 


III 
THE 
TYPHOID- 
DYSENTERY 
GROUP 


JS 

H 



CHAPTER XIV 

THE CAPSULATED GROUP 
MEMBERS 

Varieties of Bacillus mucosus capsulatus. 

B. lactis aero genes. 
REFERENCE 

Perkins, Journal o) Infectious Diseases. 1904, No. 2. 

Inoculate agar-slants from stock-cultures of B. mucosus 
capsulatus (Friedlander's pneumo-bacillus) and B. lactis 
aero genes. 1 

1. Routine study. Observe particularly the viscous 
condition of cultures on solid media, the consistency of 
liquid media, and the gas-formation on potato. 

2. Special study A. Staining of capsules from 24- 
hour-old milk-cultures (see p. 77). 

3. Special study B. Intraperitoneal inoculation of 
rabbit with B. mucosus capsulatus. 

Method of intra peritoneal inoculation. The rabbit is 
held in the same manner as described in Chap. XII, 
The hair is clipped close over the left lower abdominal 
quadrant. Then (after washing with mercuric chlorid 
i : 1000 and alcohol) pass the needle at first beneath the 
skin, then, holding it at about a right angle to the abdomi- 
nal surface, carefully press it through the abdominal wall, 
which is usually made tense by the resistance of the 
animal. Successful passage of the abdominal wall can 
be felt by the sudden loss of resistance to the needle's 
pressure. Then make the injection, and rapidly with- 

1 B. lactis aerogenes, if studied in the colon group, need not 
be studied again 

92 



CAPSULATED GROUP 93 

draw the syringe. If the contents of the needle have 
been properly emptied into the peritoneal cavity, no 
swelling takes place, as is noticed in subcutaneous inocu- 
lations. 

When the animal has died, perform an autopsy and 
study the lesions. Make cultures from the heart or 
internal organs in the usual manner, and make capsule 
stains from the heart's blood. 



CHAPTER XV 
THE DIPHTHERIA GROUP 
MEMBERS 

Bacillus diphtheriae. 
B. pseudodiphthericus. 
B. xerosis. 

Use great caution in handling members of this group. 
Inoculate agar-slants from stock-cultures of B. 
diphtheriae and B. pseudodiphthericus. 

1 . Routine study. Stain B. diphtheriae with Loeffler's 
methylene-blue instead of gentian-violet. The staining 
may be facilitated by the application of mild heat. 
Observe particularly the peculiar effects of the staining 
process, and make accurate sketches of what yon see 
under the microscope. 

2. Special study A. Neisser's method of staining to 
demonstrate the granules in the bacilli: 

a) Prepare two solutions. 

SOLUTION I 

Methylene-blue i g. 

Dissolve in alcohol 96$ 20 c.c. 

Add distilled water. . . 950 c.c. 

Glacial acetic acid 50 c.c. 

SOLUTION II 

Bismarck brown 2 g. 

Distilled water 1000 c.c. 

b) Prepare a film in the usual manner from a culture 
of B. diphtheriae on blood-serum (furnished). 

c) Stain in Solution I for 5 to 6 seconds. 

d) Wash in water. 

94 



DIPHTHERIA GROUP 95 

e) Counterstain with Solution II for 10-15 seconds. 
/) Wash in water, examine in water, dry, and mount 
in balsam. 

3. Special study B. Test for acid formation in a 
culture, one week old, in neutral glucose-broth by addi- 
tion of a few drops of litmus-solution. 

4. Special study C. Cultivation of B. diphtheriae 
on eggs. (Method of Wyatt Johnston). 

NOTE. This method is recommended as an emergency cul- 
ture test, the egg taking the place of Loeffler's blood-serum. 

a) Sterilize over the flame a small empty pot of meat- 
extract, or any other vessel of suitable size. 

b) Carefully break with sterile forceps the shell of 
a hard-boiled egg at the blunt end, taking care not to 
rupture the membrane lining the shell. 

c) Flame the exposed part, and carefully free the 
coagulated albumen from any membrane. 

d) Inoculate by gently rubbing some culture or throat 
swab on the exposed egg-albumen. 

e) Invert and set in the sterilized pot. 
/) Place in the thermostat, and 

g) Observe the appearance and make a stained 
preparation after 24 hours. 

5. Special study D. Study of B. xerosis. Obtain 
mucus from inner angle of the eyelids by stroking 
with a platinum loop. Make two film preparations, 
stain one by Gram's method and the other with methy- 
lene-blue. Try culture on slant-agar and if impure, 
plate out. Wherein does B. xerosis differ from the 
other members of the group ? 

6. Special study E. 



96 LABORATORY GUIDE IN BACTERIOLOGY 
EXPERIMENT I 

a) Procure a guinea-pig (or rabbit), and clip the hair 
over a small area on the surface of the abdomen. 

b) Cut a small opening in the skin, and separate the 
skin from the muscles below by pushing in sterile scis- 
sors. Expand these slightly and after closing again 
remove. This forms a small pocket. 

c) Carry i loopful of a 24-hour-old agar-culture into 
this pocket. 

EXPERIMENT II 

a) Heat a 24-hour-old broth-culture in the water- 
bath for 30 minutes at 60. 

b) Inject 0.25 c.c. of this heated culture subcutane- 
ously into another guinea-pig (or rabbit) 

c) Observe and compare in both animals the results 
by taking note of the ante-mortem phenomena and the 
lesions post-mortem. What difference is there in the 
activities of the two cultures inoculated, and to what are 
these differences due? 



CHAPTER XVI 

THE HEMORRHAGIC SEPTICEMIA GROUP 
MEMBERS 

Bacillus pestis (bacillus of bubonic plague). 
B. cuniculicida (bacillus of fowl-cholera, bacillus of rabbit 
septicemia, Bacillus der Rinderseuche, Bacillus der 
Schweineseuche, etc.). 
REFERENCES 

Textbook: Moore, The Pathology of Infectious Diseases of 
Animals. 

Inoculate agar-slants from stock-culture of B. cuni- 
culicida. For obvious reasons, B. pestis will not be 
studied culturally. 

1. Routine study. Stain with Loeffler's methylene- 
blue and anilin-gentian-violet. Observe "polar stain- 
ing." What is "polar staining"? 

2. Special study. Inoculation of rabbit subcutane- 
ously or by scarification. When dead, study in the 
usual manner, and observe particularly the hemorrhages 
produced in the serous membranes. Make cultures 
from heart's blood, where large numbers of bacilli will 
be found. Also make a stained preparation, and note 
the typical polar staining. . 



97 



CHAPTER XVII 

THE ANTHRAX GROUP 

MEMBERS 

Bacillus anthracis. 

B. subtilis, including several varieties. 

Great caution is necessary in handling B. anthracis. 
Inoculate agar-slants from stock-cultures of B. anthra- 
cis and B. subtilis. 

1. Routine study. Observe particularly the colonies 
formed on agar and gelatin plates, 

2 . Special study A . Make ' ' impression preparation ' ' 
(Klatschprdparat) from a surface colony on a gelatin- 
plate. 

Method 

a) Clean and flame a cover-slip carefully. 

b) Place on a colony of suitable size, and gently press 
down, taking care not to press so hard as to disturb the 
characteristic shape of the colony. 

c) Lift the cover-slip carefully with the forceps. 

d) Dry, fix, and stain with methylene-blue or by 
Gram's method. 

e) Examine under low and high power (dry lens), 
and sketch what you see. 

3. Special study B. Staining of spores. Read care- 
fully the different methods of staining spores. Why are 
special stains necessary ? 

Moeller's method: 

a) Prepare several (five or six) films in the usual 
manner from 24-hour-old agar-cultures of B. anthracis 

(or B. subtilis). 

98 



ANTHRAX GROUP 99 

b) Place in chloroform for 2 minutes. 

c) After drying in the air, cover with a 5 per cent, 
solution of chromic acid for 2 minutes. 

d) Wash thoroughly in water. 

e) Cover with carbol-fuchsin and heat for 5 minutes 
over the water-bath at 100, or over a small flame, sim- 
mering gently all the while. 

/) Decolorize with i per cent, sulphuric acid for 25- 
30 seconds. 

g) Wash thoroughly in water. 

h) Mount in water, and examine under the micro- 
scope to see if spores are cherry-red and the protoplasm 
colorless or faintly pink. 

i) Counterstain with methylene-blue for 10-15 
seconds without heat. 

;) Wash, examine in water, and then mount in balsam. 

NOTE. The body of the cell should appear blue; the spore, 
red. 

4. Special study C. Demonstration of filament- 
formation. 

a) Spread a loopful of a broth-culture of B. anthrads 
or B. subtilis on a clean cover-glass. 

b) Dry and fix in the flame. 

c) Cover with strong acetic acid (80 per cent.) for 
5-10 seconds. 

d) Wash in water. 

e) Stain with gentian-violet. 

/) Examine in water, dry, and mount in balsam. 
What is the object of applying acetic acid ? 

5) Special study D. Inoculate a guinea-pig subcu- 
taneously with 0.2 c.c. of a 24-hour-old broth-culture of 
B. anthrads, or insert a loopful of a 24-hour-old agar- 



ioo LABORATORY GUIDE IN BACTERIOLOGY 

culture in a "pocket" under the skin. When the animal 
is dead, perform an autopsy, and observe particularly the 
hemorrhagic and gelatinous edema under the skin; also 
the enlarged spleen and the hemorrhagic adrenals. 
Make a stained preparation from the heart's blood, and 
observe the lack of spores, and also the presence of 
capsules and degenerate forms, which do not stain. 
What prevents the formation of spores ? How would 
you distinguish B. anthracis from B. subtilis? 



CHAPTER XVIII 
THE SPIRILLUM GROUP 

MEMBERS 

Spirillum cholerae asiaticae. 

Sp. of Finkler and Prior. 

Sp. Metchnikom. 

Sp. tyrogenum. 

And a number of spirilla indigenous to water. 

Great caution must be exercised in manipulating the 
spirillum of asiatic cholera. 

Inoculate agar-slants from stock-cultures of Sp. 
cholerae asiaticae, Sp. oj Finkler and Prior, and Sp. 
Metchnikovi. 

1. Routine study. In addition to the usual media, 
inoculate an extra tube of Dunham's peptone-solution 
from each organism. Observe carefully from day to day 
the action of these three organisms on gelatin, and com- 
pare the results by tabulation. Observe the formation 
of coccoid involution forms on agar after 3 days. Also 
make plates in gelatin, observe the colonies from day 
to day, and compare. 

2. Special study A. Test for the nitroso-indol or 
cholera-red reaction. (See test for indol, p. 83.) Make 
two tests, using one of the cultures in Dunham's solution 
after 24 hours, the other after 6 days. Compare the 
results of these two tests. 

3. Special study B Stain for flagella by Loeffler's 
method (see p. 86). 

4. Special study C. Schottelius' enriching method. 



102 LABORATORY GUIDE IN BACTERIOLOGY 

a) Make a solution of i g. Witte's peptone in 100 c.c. 
of water. 

b) Distribute in three small Erlenmeyer flasks, and 
sterilize in autoclav. 

c) Inoculate one of these flasks with Sp. chol. asiat. 
and B. chol. suis or any other motile bacillus. 

d) Incubate at 37 for 18-24 hours. Make a stained 
preparation from the surface of the liquid. 

e) After that time, take one loopful from the surface, 
inoculate the second flask, and incubate as before. 

/) After 18-24 hours, make a stained preparation 
from the surface of the second flask, and examine for 
spirilla. 

g) Transfer a loopful from the surface of the second 
flask to the third one, and incubate as before. 

h) After 18-24 hours, again stain and examine for 
spirilla. By this time usually a film has formed which 
contains the spirilla in practically pure culture. How is 
this phenomenon explained, and what is its epidemiologi- 
cal value ? 

5. Special study D. Inoculate a pigeon intramuscu- 
larly with 0.5 c.c. of a broth-culture of Sp. Metchnikom. 
The breast of the pigeon is laid bare, washed with mercuric 
chlorid and alcohol, and the syringe is plunged into the 
muscle-fibers and discharged. After death, note the 
peculiar appearance, resembling that of boiled beef. 
Make stained preparations from blood and muscle-juice, 
and examine for spirilla. 



CHAPTER XIX 

THE GROUP OF ACID-RESISTING BACILLI 
MEMBERS 

Bacillus tuberculosis. 
B. leprae. 
B. smegmae. 

Moeller's grass bacilli, including a number of bacilli found 
on grass, dung, in butter, milk, etc. 

For obvious reasons, B. tuberculosis will not be studied 
culturally. For comparison, the culture characteristics of 
Moeller's grass bacillus are instructive. 

Inoculate an agar-slant from a stock-culture of 
Moeller's grass bacillus. 

1. Routine study. 

2. Special study A . Method of staining acid-resisting 
bacilli. 

a) Pick out purulent matter from the sputum oi a 
tuberculous patient and spread carefully on a cover- 
glass. 

b) Dry and fix as usual. 

c) Heat for one minute on a water-bath at 100, or 
over a small flame with carbol-fuchsin. 

d) Decolorize with acid alcohol (2 per cent. HC1 in 
80 per cent, alcohol) until the film in its thin parts has 
lost almost all its color. 

e) Counterstain with methylene-blue for 10 seconds 
(cold). 

/) Examine and mount in balsam. 

NOTE. Make a second preparation, substituting anilin- 
gentian-violet for carbol-fuchsin, and Bismarck brown for methy- 
lene-blue. 

103 



io 4 LABORATORY GUIDE IN BACTERIOLOGY 

3. Special study B. Observe the lesions in a 
guinea-pig or rabbit dead of tuberculosis, which has 
been inoculated about four weeks previously. Note the 
caseous matter at the site of the inoculation, the enlarged 
yellowish inguinal and axillary glands, the small gray 
or yellowish tubercles in the liver, lung, spleen, mesentery, 
etc. Stain for bacilli from such sites. Make cultures on 
glycerin-agar or dog-serum (see the textbook). 

4. Special study C. Stain Moeller's grass bacillus 
from agar-culture by the same method, omitting the 
counters tain. 

5. Special study D. 

a) Grind in a mortar, previously sterilized, a small 
amount of a culture of the grass bacillus, and mix with 
some sterile milk. 

b) Make a cover-slip preparation of this mixture, and 
stain for tubercle bacilli. At what conclusions as to the 
quality of the milk would you arrive, if you should find 
acid-resisting bacilli in a fresh sample of milk ? 



CHAPTER XX 
MISCELLANEOUS BACTERIA 
Inoculate agar-slants from stock-cultures of Bacillus 
mallei and Micrococcus melitensis. Exercise great 
caution in handling B. mallei. 

1. Routine study. Note especially the growth of 
B. mallei on potato. Also note the morphology of Micr. 
melitensis and the staining properties of B. mallei. Is B. 
mallei motile ? 

2. Special study. Intraperitoneal injection of B. 
mallei from a broth-culture into a male guinea-pig. Why 
do you select a male guinea-pig? What is Straus's 
method of diagnosis of glanders? 



105 



CHAPTER XXI 
THE ACTINOMYCES GROUP 
Inoculate agar-slants from Actinomyces boms (homi- 
nis) and Actin. asteroides. 

REFERENCES 

Stokes, American Journal of the Medical Sciences, Novem- 
ber, 1904. 
Wright, Jour, of Med. Research, May, 1905. 

1. Special study A. Transfer from agar-slants to 
broth and potato only, and make descriptions and 
stained preparations as usual. 

2. Special study B. Suspend a small amount of the 
potato-culture in a physiological salt solution, and 
examine under the low power. 

3. Special study C. 

a) Examine a sample of actinomycotic tissue (bovine) 
in the fresh state, for so-called " sulphur granules." 
Crush some in salt solution under a cover-slip and search 
for " clubs," using the low and high power dry lenses. 

b) Crush others, dry, fix, and stain by Gram, counter- 
stain with eosin or Bismarck brown. 

c) Write a full description of the biology of the organ- 
ism, illustrated by drawings. 

d) What other actinomycetes have been described 
as pathogenic? Give their names and the diseases 
which they produce. 



106 



CHAPTER XXII 
THE ANAEROBIC GROUP OF BACILLI 

MEMBERS 

Bacillus tetani. 

B. oedematis maligni. 

B. aero genes capsulatus. 

B. anthracis symptomatici. 

B. botulinus. 

And others. 

Study the different methods of anaerobic cultivation in 
the textbook. 

1. Special study A. Park's method. 

a) Boil three tubes of glucose-agar vigorously for 5 
minutes, to drive out the dissolved oxygen. (Why is 
the presence of glucose desirable ?) 

b) Cool to 43 and inoculate from stock-culture of 
B. tetani, B. oedematis maligni, and B. aerogenes capsu- 
latus. 

c) Solidify rapidly by immersion in cold water. 

d) Cover the medium with a thin layer of liquid paraf- 
fin. 

e) Place in thermostat. 

NOTE. The layer of parraffin effectively excludes the atmos- 
pheric oxygen, which would be inhibitory to the growth of the 
anaerobes. The oxygen necessary for their multiplication is 
derived from nutrient material of the medium. 

2. Special study B. Wright's modification of Buch- 
ner's method. 

a) Liquefy, as before, six glucose-agar tubes, the 
plugs of which have been replaced by absorbent cotton. 

107 



io8 LABORATORY GUIDE IN BACTERIOLOGY 

Cool three to 43, and inoculate while fluid. Let the 
other three become solid, and make stab-cultures. 

b) Sterilize the cotton stoppers in a flame, and with 
the forceps, sterilized in a flame, push the stoppers into 
the test-tubes for the distance of about i inch (2-3 cm.). 

c) Pour into the tubes (upon the cotton stoppers) 
2 c.c. of a saturated solution of pyrogallic acid in water, 
followed by 2 c.c. of a 2 per cent, solution of NaOH. 

d) Cork the tubes immediately with rubber stoppers, 
and keep upside down. 

e) Incubate at the required temperature. 

Upon what principle does this method depend ? 
Do the organisms grow both aerobically and anaerobi- 
cally ? 

When you have obtained a growth, stain the organ- 
isms with anilin-gentian-violet and by Gram's method. 
Describe and illustrate. 

3. Special study C. Cultivation by Buchner's 
method, using fruit-jars. 

a) Into a Mason fruit-jar of ordinary type deposit 
10 g. of pyrogallic acid. 

b) Smear vaselin around the mouth of the jar. 

c) Pour into the jar 100 c.c. of a i per cent, solution 
of NaOH. 

d) Then deposit in the jar culture-tubes previously 
inoculated. 

e) Tightly fasten the cover of the jar, and incubate 
at 37 for 48-72 hours. 

4. Special study D. Cultivation in hydrogen gas. 

a) Inoculate all media from stock-cultures obtained. 

b) Fit up apparatus as shown in Fig. 34. 

c) Place culture-tubes in a Novy jar (Fig. 34, a). 



ANAEROBIC GROUP OF BACILLI 



109 



d) Open the faucet (b) of the gas-generator (c), con- 
taining zinc and hydrochloric acid. The hydrogen gas 
generated passes through two jars, one of which con- 
tains concentrated sulphuric acid (d), the other a 10 per 
cent, solution of sodium hydrate (e). Gradually the 
Novy jar is rilled with hydrogen gas, which can be tested 
by holding a culture-tube over the opening (/), and then 
over a burning match or gas flame. As long as any 
detonation takes place the hydrogen is still mixed with 




-I 



FIG. 34 

Anaerobic Cultivation in Hydrogen Gas 
c. Novy jar e. Sodium hydrate solution 

b. Glass cock /. Opening of Novy jar 

c. Gas generator g. Stopper 

d. Sulphuric acid 

atmospheric oxygen. When finally the hydrogen in the 
Novy jar is pure, close it off by turning the stoppers (g) 
and (b), and place it in the incubator. The whole pro- 
cess occupies about 10 or 15 minutes. 

5. Special study C. Inoculation of a rabbit with 
B. aerogenes capsulatus. 

a) Shave the ear of the rabbit. 



no LABORATORY GUIDE IN BACTERIOLOGY 

b) Wash with mercuric chlorid solution and alcohol. 

c) Inoculate intravenously with 0.5 c.c. of a 24-hour- 
old milk-culture. 

d) After the culture has been fully distributed in the 
circulation, which takes at the most 3 minutes, kill the 
rabbit by a quick blow on the back of the neck. 

e) Put the rabbit in a warm place say, on top of the 
thermostat for 18 hours, or from 6 to 8 hours inside of 
the thermostat. 

/) After this time has elapsed, perform an autopsy. 
Note the crackling, on pressure, over the axillary or 
inguinal regions. The rabbit is swollen to a great 
extent. Skin the animal carefully, without opening the 
abdominal cavity; then quickly puncture the abdominal 
wall and bring a flame to the opening. Note that the 
escaping gas will burn with a blue flame. What is this 
gas ? Also note the disorganized condition of the liver, 
spleen, and kidney. 

g) Make capsule stains from the heart's blood or 
organs by Welch's method (modified). The modification 
of Welch's method is as follows : Proceed in the manner 
indicated on p. 77 and, after washing the acetic acid off 
with the stain (carbol-fuchsin or gentian-violet), dry with 
filter paper, heat the specimen for 5 to 10 seconds before 
washing off with the salt solution. Then proceed as 
before. 

6. Special study F. Staining of spores of B. tetani 
and B. oedematis maligni from 3 -day-old glucose-agar- 
cultures (see p. 98). 

7. Special Study G. Inoculation of a white mouse 
or guinea-pig with B. tetani or its toxin (o.oi c.c.) in the 
hind-leg or over the root of the tail (if a mouse). Note 



ANAEROBIC GROUP OF BACILLI in 

daily the condition of the animal, and when dead make 
cultures and cover-slip preparations from the site of the 
inoculation. 

8. Special study H. Inoculate a white rat with gar- 
den earth subcutaneously or in a pocket above root of 
tail. Note the condition of the animal daily. When 
dead, make cultures and cover-slip preparations from the 
site of the inoculation. Would you expect to find bacilli 
in the heart's blood or organs ? Give reason for your 
answer. 



CHAPTER XXIII 

ISOLATION OF UNKNOWN BACTERIA FROM A 
MIXTURE 

1. Make hanging-drop, stained, and Gram prepara- 
tions from the mixture. Note observations and results. 

2. Melt five or six agar-tubes, and cool to 43. 

3. Transfer 5 or 6 loopfulsof the mixture to a tube 
of liquid agar, from this to a second, and so on until all 
the melted tubes are inoculated. 

4. Pour into sterile Petri dishes, and mark them with 
successive numbers and the date. Place in the thermo- 
stat. 

5. After 24 hours examine the colonies under the 
low power, describe them in the usual manner, and 
transfer to agar-slants all those which show different 
appearances. 

6. Now proceed with the usual routine study. Make 
hanging-drop, stained, and Gram preparations, transfer 
to all the media, and describe the culture characteristics 
accurately. Make sketches in the usual manner. 

7. Special tests may become necessary after 24 or 
48 hours. Such tests may consist of 

Capsule stain. 

Spore stain. 

Stain for acid-resisting bacilli. 

Fermentation tests of all those which produce gas 
in glucose-agar. Also note the growth, or lack of growth, 
in the closed arm of the fermentation-tube. 

Test for acid in neutral broth. 

112 



ISOLATION OF UNKNOWN BACTERIA 113 

Test for agglutination. 

Test for indol. 

Anaerobic cultivation. 

Inoculation of animals. 

For final diagnosis consult your notes and textbook. 



CHAPTER XXIV 
BACTERIOLOGICAL EXAMINATION OF WATER, AIR 

AND MILK 
EXERCISE I. BACTERIOLOGICAL ANALYSIS OF WATER 

REFERENCES 

Horrocks, Bacteriological Examination of Water. 

Prescott and Winslow, Elements o} Water Bacteriology. 

Fuller and Johnston, Journal of Experimental Medicine, Vol. 
IV. 

Committee Report o} the American Public Health Association, 
Jour, o} Infectious Diseases, Suppl. I, May, 1905. 

A bacteriological examination of water is made for 
the purpose of determining 

1. Bacterial numbers. 

2. Bacterial species. 

3. Sewage contamination. 

Collection oj samples. Procure wide-mouthed, glass- 
stoppered bottles, having a capacity of at least 100 c.c. 
After careful cleaning and drying, wrap them in lead-foil, 
and sterilize in the hot-air oven for i hour at 160; then 
deposit them in a metal or wooden case. The samples 
from surface waters should be taken at least one foot 
below the surface, to avoid contamination with organisms 
from the air. If possible, samples should be plated on the 
spot or in the laboratory within an hour at the very latest. 
But when a greater interval of time must occur, the 
samples should be taken to the laboratory packed in ice, 
despite the probability of thus destroying a certain per- 
centage of the bacterial flora. 

Method oj examination. A number of pipettes of 
various sizes (i c.c., 2 c.c., 5 c.c., and 10 c.c.) are plugged 

114 



EXAMINATION OF WATER, AIR, AND MILK 115 

with cotton and sterilized in the hot-air oven. Then a 
number of Erlenmeyer flasks are filled with 101 c.c. of 
distilled water, and these are sterilized in the autoclav at 
1 20 for 5 minutes. About i c.c. of water is lost by evapo- 
ration during this process, so that the sterile flasks con- 
tain 100 c.c. each. 1 

Method oj procedure. 

1. With a sterile pipette remove i c.c. of water from 
one of the sterilized dilution flasks. 

2. Carry over to this flask i c.c. of the sample after 
a thorough shaking. The dilution is now i : 100. Mark 
with glass pencil. 

3. With a sterile 10 c.c. pipette remove 10 c.c. from 
another dilution flask, and add to the remainder 10 c.c. 
of the first dilution. We now have a dilution of i : 1000. 
(See dilution table, p. 117.) Make a number of dilutions 
in this manner, carrying the dilutions higher in proportion 
to the quality of the water to be examined. 

4. Melt a number of agar and gelatin tubes, corre- 
sponding to the number of dilutions made, and cool to 
43. Read in reference book carefully the chapters on 
the importance of using both agar and gelatin media. 

5. Mix carefully i c.c. of each dilution flask with a 
tube of liquified medium, respectively. 

6. Pour into Petri dishes. 

7. Incubate the agar-plates at 37, and keep the 
gelatin-plates at room temperature. The mixing of the 
diluted sample and agar may also be effected by pouring 
the sample into a Petri dish and mixing it with the liquid 
medium. This method is not as convenient, but slightly 

1 If work is carried on with dilutions of i : 100 only, the flask may 
conveniently be filled with 100 c.c. water instead of 101 c.c. It is then not 
necessary to remove i c.c. of water, as only 99 c.c. are left after sterilization. 



u6 LABORATORY GUIDE IN BACTERIOLOGY 

more accurate, as no loss takes place by adhesion to the 
tube. 

Estimation oj colonies. The colonies are then counted 
after 48 hours, by means of a colony-counter (Fig. 35). 
Plates should be counted which contain no more than 
200-300 colonies. If it is necessary to count plates with 
a large number of colonies, an estimate must be made 
by counting different sections of the plate-counter and 
averaging the result for the whole plate. 

Species determination. If the different species of 
bacteria are to be studied, the colonies must be examined 
by the naked eye and the low power. Then those which 
appear to be different are transferred to slant-agar tubes, 
and from these to the ordinary media. 

Sewage contamination. The presence of Bacillus 
coli and streptococci is sufficient indication of sewage 
contamination in water. 

Method oj examination jor B. coli and streptococci. 

1. i c.c. of the sample, or, if necessary, of the diluted 
sample, is added to a series of ten fermentation-tubes, 
containing sterile 2 per cent, glucose-broth. 

2. Place in thermostat. 

3. Examine after 12-18 hours. 

4. Examine a loopful of the sediment in a stained 
preparation. 

Example. If i c.c. of the sample is added to each 
fermentation-tube, and six show gas-formation, there 
would be six colon bacilli in each 10 c.c. if undiluted 
water is employed. By this method fairly accurate 
results may be obtained. 

Isolation is accomplished by plating in an agar 
medium containing 2 per cent, lactose and 10 per cent, 
litmus solution. 



EXAMINATION OF WATER, AIR, AND MILK 117 
DILUTION TABLES FOR AGGLUTINATION 



Number 


Amount of Serum 


Amount of Salt 
Solution 


Final Dilution 


I 


i part 






2. 


i part of No i 


9 parts 




2. 


i part of No 2 






4. . 


i part of No ^ 


9 parts 













Amount of Serum or 
Serum Dilution 


Suspension 


Fina 


1 Dilution 


2 part clear serum 


1 8 parts 




10 


i " " " 


19 " 




20 


5 parts dil., No 


IS " 




40 


4 No 


16 " 




5 


2.5 " " No 


17-5" 




80 


2 " " NO 


18 " 




IOO 


i " " No 


10 " 




2OO 


4 " " No. 2 


y 
16 " 




CQO 


2 " " No. 2 


18 " 




y** 

1000 


I " " No. 2 


19 " 




200O 


4 No. 3 


16 " 




5000 


2 " " No. 3 


18 " 




IOOOO 


i " " No. 3 


19 " 




2OOOO 


4 No. 4 


16 " 




50OOO 


2 No. 4 


18 " 




IOOOOO 


i " " No. 4 


19 " 




200OOO 



DILUTION TABLE FOR WATER OR MILK ANALYSIS 



Number 


Amount of Dilution 


Amount of 
Sterile Water 


Final Dilution 


I. . . 


Original 




I 


2. . 


ic.c. of No. i 


OQC.C. 


IOO 


3 


2OC.C. of No. 2 


yyv-.v,. 

Soc.c. 


500 


4 


IOC.C. Of No. 2 


9oc.c. 


IOOO 


5 


50. c. of No. 2 


95c.c. 


2000 


6 


2C.C. Of No. 2 


98c.c. 


5000 


7 


ic.c. of No. 2 


99c.c. 


IOOOO 


8 


5c.c. of No. 4 


95c.c. 


2OOOO 


9 


2c.c. of No. 4 


98c.c. 


50OOO 


10 


ic.c. of No. 4 


99c.c. 


IOOOOO 


ii 


5c.c. of No. 7. 


95c.c. 


2000OO 


12 


2c.c. of No. 7. 


98c.c. 


5OOOOO 


*3 


ic.c. of No. 7 


99c.c. 


I 000000 



n8 LABORATORY GUIDE IN BACTERIOLOGY 
EXERCISE II. BACTERIOLOGICAL EXAMINATION OF AIR 

An approximate determination of the number of 
bacteria in the air can be made by the following simple 
method: Place a certain amount (50 c.c.) of broth in 
an Erlenmeyer flask (Fig. 36, a). This flask is provided 
with a rubber stopper (b), provided with two holes, 
through which the two glass tubes (c with a wide opening 




FIG. 36 
Apparatus for Determining the Number of Bacteria in a Definite 



a. Erlenmeyer flask 

b. Rubber stopper 
c-d. Glass tubes 



Volume of Air 

/. Five-liter flask 
g-h. Glass tubes 
*. Pinchcock 



and d) lead. Cotton plugs are then inserted at c and d, 
and the apparatus is sterilized in the autoclav. A 
large bottle (/), containing 5 liters of water, is then 
provided with a rubber stopper, and also with two glass 
tubes (g and h)\ h is connected with a short piece of 
rubber hose and a pinchcock (i). When the Erlen- 



EXAMINATION OF WATER, AIR, AND MILK 119 

meyer flask and contents are sterilized, the tubed is con- 
nected, by means of the rubber hose , with g, and the 
plug at c is removed. By opening the pinchcock i, 5 
liters of air are aspirated through the broth in flask a. 
The flask is then disconnected, and i c.c. is plated in 
agar and i c.c. in gelatin. The former is incubated at 37, 
and the latter kept at room temperature. After 48 hours 
the colonies are counted, and the result is multiplied by 
50. This then represents the amount of bacteria in 5 
liters of air. 

EXERCISE III. BACTERIOLOGICAL STUDY OF MILK 

The method for determining the number of bacteria 
in milk is fundamentally the same as for water, except 
that dilutions must be carried higher, as milk generally 
contains much larger numbers of bacteria. 

Sterilization and pasteurization oj milk. Some of the 
important germs in milk are saprophytes (which under 
favorable circumstances produce disagreeable odors or 
tastes), and such pathogens as the bacillus of tuberculosis 
(which may be derived from the cow, or may be an acci- 
dental contamination), the typhoid bacillus, the bacillus 
of "summer complaint" in children (possibly identical 
with the bacillus of epidemic dysentery), the germs of 
cholera, diphtheria, and scarlet fever. All these, except 
B. tuberculosis, flourish in milk at its ordinary tempera- 
ture. 

None of the methods employed in sterilizing milk 
render it sterile in the bacteriological sense of the word, 
but by means commonly employed most of the non- 
sporing pathogenic bacteria are destroyed, along with a 
large number of saprophytes, thus rendering the milk 



i 2 o LABORATORY GUIDE IN BACTERIOLOGY 

comparatively safe and less subject to the ordinary 
fermentative changes. 

1. Sterilization at 100 for 30 minutes. Such milk, 
if chilled and kept at a low temperature, will remain 
unchanged for more than a week; but, by the heating, 
certain alterations have been produced in its taste and 
nutritive qualities which render it supposedly less fit for 
food than when pasteurization is employed. 

2. Pasteurizing milk. The changes occurring in 
milk, as above mentioned, begin at about 82. Sterili- 
zation at a low temperature is accomplished by raising 
the temperature to only 75 for a period of 20 minutes. 
This has been shown to be sufficient to kill the germs of 
typhoid, cholera, diphtheria, and pyogenic cocci. Spored 
organisms are not killed. As shown by Theobald 
Smith, tubercle bacilli, when suspended in distilled 
water, physiological salt solution, broth, and milk, are 
destroyed at 60 in 15-20 minutes; but, if milk contain- 
ing tubercle bacilli has its surface exposed to the air when 
heated to 60, the pellicle which forms on its surface 
may contain living tubercle bacilli after an exposure of 
60 minutes. 

Study of the effect of the above two methods of steri- 
lization as compared with each other and with unsteri- 
lized milk: 

1. From the fresh milk provided make three agar- 
plates, using i, 2, and 3 loopfuls, respectively. 

2. Fill about 10 c.c. into each of ten sterile culture- 
tubes, and keep one at room temperature and one in the 
thermostat. 

3. Treat four of these tubes in the following manner: 
Place water in a saucepan sufficient to cover completely 



EXAMINATION OF WATER, AIR, AND MILK 121 

the milk when the tubes are immersed in it. Raise the 
temperature to 75, and keep it there by regulating the 
flame. The tubes of milk are then immersed in the 
water, and kept there for 30 minutes, as it requires about 
10 minutes for the milk in the tubes to reach the tempera- 
ture of the water. The tubes are then taken out and 
cooled quickly by standing them in cold water. Place 
one of the tubes at incubator and the other at room 
temperature. Aerate the other two by shaking vigor- 
ously for ij minutes. Keep one of these at room tem- 
perature, the other in the thermostat. 

4. Place two more milk-tubes in the Arnold at 100 
for 30 minutes. Keep one at room temperature and one 
in the thermostat. 

5. The remaining two tubes autoclav at 120 for 
5 minutes, and place one in the thermostat and keep the 
other at room temperature. 

6. Note the conditions of these ten tubes after 2 or 
3 days. Compare the results, and tabulate them. Note 
especially coagulation, time elapsed before coagulation 
sets in, gas-formation, condition of whey, film, and odor. 
How are these differences explained ? 

Plates in lactose-litmus-gelatin should be made from 
each of these tubes, and the colonies studied and counted. 
Subcultures on agar-slants may also be made, and the 
usual media inoculated from these, if the individual 
species are to be studied. 



CHAPTER XXV 

INFLUENCE OF DISINFECTANTS ON THE GROWTH 
OF MICRO-ORGANISMS 

EXERCISE I 

1. Prepare fifty-seven Hill's test-rods. These are 
prepared in the following manner: Glass rods about 
two inches longer than ordinary culture-tubes are marked 
with hydrofluoric acid by a circle exactly one inch from 
the end. A wad of cotton is then wrapped around the 
middle of the rod, and this is inserted in a culture-tube. 
The rod is then pushed down until it nearly reaches the 
bottom. That part of the rod which is free at the upper 
end is used for labeling. The whole apparatus is then 
sterilized in the dry-air oven. 

2. Fill two wide-mouthed flasks, one with 100 c.c. 
of a 5 per cent, solution of carbolic acid, the other with 
100 c.c. of a i per cent, solution. 

3. Fill two similar flasks, one with 100 c.c. of a solu- 
tion of mercuric chlorid i : 1000, the other with a solution 
of i : 10,000. 

4. Fill two similar flasks, one with 100 c.c. of a 10 
per cent, solution of formalin (40 per cent, formaldehyde), 
the other with a i per cent, solution. 

5. Prepare 48-hour broth-cultures of Staphylococcus 
pyogenes aureus, Bacillus coli and B. typhosus from 
stock-cultures. 

6. Dip nineteen of these rods into each of these 
cultures respectively, to the depth of one inch ; set them 
aside in their tubes to dry over night in the thermostat, 
after marking each tube carefully. 



INFLUENCE OF DISINFECTANTS 123 

EXERCISE II 

We have now six flasks containing different solutions 
of disinfectants. 

1. Place in each one of these flasks nine of the pre- 
pared rods, three of which have been dipped in the 
Staph. pyogenes aureus culture, three in the B. coli 
culture, and three in the B. typhosus culture. 

2. Take three rods (one of each organism) out of 
each flask after the lapse of half a minute, wash by 
gently pouring sterile physiological salt solution over 
them into a dish containing mercuric chlorid solution 
i : 1000, and place each rod in a tube of sterile broth 

3. Repeat the proceedings of step 2 .with a second 
series of rods after 2 minutes. 

4. Repeat again after 5 minutes with the remaining 
series. 

5. Place all tubes (fifty-seven) in the thermostat. 
Three of these tubes have not been dipped into any one 
of the six flasks containing antiseptics, and are incubated 
with the others as controls. 

6. Observe the results carefully on each of the four 
successive days, and on the last day prove the relative 
growth by making agar-plates with i c.c. of each culture, 
and count the colonies after 24 hours. 

7. Tabulate the results, and state your conclusions. 

EXERCISE III. INFLUENCE OF SUNLIGHT 
EXPERIMENT I 

1. Inoculate a flask containing 100 c.c. of sterile 
water with B. coli. 

2. After thoroughly shaking, take i c.c. by means of 
a sterile pipette, and plate in agar. Place the plate in 
the thermostat. 



i2 4 LABORATORY GUIDE IN BACTERIOLOGY 

3. Expose the flask to sunlight for several hours. 

4. Make another plate with i c.c. of the suspension, 
and place in a thermostat. 

5. After 48 hours count both plates, and compare 
the results. 

EXPERIMENT II 

1. Melt a tube of agar and cool to 43. 

2. Inoculate with B. coli (or any other organism). 

3. Pour into a sterile Petri dish. 

4. After solidification, turn bottom side up, and paste 
a strip of black paper on the glass, covering part of the 
surface. 

5. Expose to direct sunlight for several hours, and 
note the result. 

EXERCISE IV. INFLUENCE OF MOIST HEAT 

Read the methods of determining the thermal death- 
point of bacteria in the textbook. 

1. Prepare six broth-cultures each of B. coli and B. 
subtilis. 

2. Place four cultures of each organism in the water- 
bath and heat. 

3. Remove one of each at 40, one of each at 60, 
one of each at 80, and keep one of each for 10 minutes at 
100. 

4. Place one tube of each organism in the autoclav, 
and heat to 120 for 5 minutes. 

5. Now place all twelve tubes in the thermostat, 
including one of each organism as a control. 

6. After 24 hours, make plates of each tube in agar, 
and place them in the thermostat. 

7. After 24 hours, count the colonies and compare 
the results. 



APPENDIX I 
SPECIAL MEDIA 

STANDARD METHOD OF PREPARING BROTH, NUTRIENT 

GELATIN AND NUTRIENT AGAR 
(Report of American Public Health Assocciation,Vo\. XXX, 1905.) 



BROTH 


GELATIN 


AGAR 


i. 




Boil 15 g. thread agar in 500 
c.c. water for half an hour 
and make up weight to 500 
g., or digest for 10 minutes 
in the autoclav at no? 
Let this cool to about 60? 


2. Infuse 500 g. lean meat 24 
hours with 1000 c.c. of dis- 
tilled water in refrigerator. 


Ditto. 


Infuse 500 g. lean meat 
24 hours with 500 c.c. of 
distilled water in refriger- 
ator. 


3. Make up any loss by evap- 
oration. 


Ditto. 


Ditto. 


4. Strain infusion through 
cotton flannel. 


Ditto. 


Ditto. 


5. Weigh filtered infusion. 


Ditto. 


Ditto. 


6. Add i % Witte's peptone. 


Ditto. 
And 10 % gold la- 
bel sheet gelatin . 


Add 2 % Witte's peptone. 


7. Warm on water-bath stir- 
ring till peptone is dis- 
solved, and not allowing 
the temperature to rise 
above 60. 


Warm on water- 
bath until pep- 
tone and gela- 
tin are dis- 
solved, not 
above 60. 


Warm on waterbath until 
peptone is dissolved, 
not above 60. 


8. 




To 500 g. of meat infusion 
add 500 c.c. of the 3% 
agar, keeping the tem- 
perature below 60. 



9. Heat over boiling water (or steam) for 30 minutes. 
10. Restore loss by evaporation. 

u. Titrate after boiling one minute to expel carbonic acid. 
12. Adjust reaction to + i.o % by adding normal hydrochloric acid or sodium 
hydrate as required. 

125 



126 LABORATORY GUIDE IN BACTERIOLOGY 

13. Boil 2 minutes over free flame, constantly stirring. 

14. Make up loss by evaporation. 

15. Filter through absorbent cotton and cotton flannel, passing the filtrate 

through the filter until clear. 

1 6. Titrate and record final reaction. 

17. Tube, using 10 c.c. in each tube in the case of gelatin and agar. 

1 8. Sterilize 5 minutes in the autoclav at 120, or for 30 minutes in streaming 

steam on 3 successive days. Put at once into ice- water till solidified. 

19. Store in the ice-chest in a moist atmosphere to prevent evaporation. 

Blood-serum 

i. Fresh ox blood collected in sterile jars (museum jars) 




FIG. 37 
Koch Inspissator 

is set in the ice-chest until the serum has separated. The 
serum is filtered if necessary. 

2. Take 3 parts of this freshly prepared serum and i part 
of, broth containing 1.5 per cent, glucose, and mix. 



SPECIAL MEDIA 127 

3. Tube in the usual manner, and place in the Koch 
inspissator (Fig. 37) two or three rows deep. 

4. Incline the inspissator to the proper angle, so as to 
produce a large sloping surface of the serum. 

5. See that the water-jacket contains sufficient water. 
Place a Bunsen burner below, slowly heat the water to the 
boiling-point, and boil for 5 minutes. Then turn out the gas. 

6. Repeat this process upon the two following days at 
the same hour. 

NOTE. Always place about 25 c.c. of water in the inspissator 
along with the tubes, so as to keep up a saturation of the air in 
the apparatus. By doing this, one avoids giving the serum a dry 
surface. See that the inspissator contains a rack made of wooden 
or metal strips, which keeps the tubes from touching the bottom 
and wall ; otherwise the serum will be overheated forming bubbles 
in the mass. 

Litmus -solution. Dissolve i part of Merck's pure extract 
of litmus in 100 parts of water, filter through paper, and steri- 
lize. 

Glycerin-broth. Add 6 per cent, of pure glycerin to 
ordinary broth. 

Glucose-gelatin. Add i per cent, glucose to ordinary 
gelatin. 

Litmus-lactose-agar (for plating). Add i per cent, of 
lactose to sugar-free agar, distribute 8 c.c. in culture-tubes, and 
add i c.c. of sterile litmus-solution to each tube before using. 

Litmus -lactose- gelatin (for plating}. Prepare gelatin in 
the usual manner, using 10-12 per cent, of gelatin, and dis- 
solve i per cent, of lactose. Distribute 8 c.c. into culture- 
tubes, and add litmus-solution to each tube before using. 

NOTE If i c.c. of the liquid to be analyzed is added, 14$ 
of gelatin should be used. 

Litmus -mannit-agar . Prepare like litmus-lactose-agar, 
substituting i per cent, mannit in place of lactose. 

Neutral-red agar. Add enough 0.5 per cent, neutral-red 



i 2 8 LABORATORY GUIDE IN BACTERIOLOGY 

solution to nutrient agar containing i per cent, glucose to 
produce a clear red color without adjusting reaction. 

Litmus-broth. Add litmus-solution to ordinary broth in 
the proportion of 10 per cent. 

Glycerin-agar . Add 6 per cent, pure glycerin to nutrient 
agar. 

Beerwort- gelatin Autoclav beerwort at 120 for 5 min- 
utes to precipitate proteids. After cooling, filter, dissolve 10 
per cent, gelatin, and clarify with white of egg. 

Beerwort-agar. Prepared like beerwort-gelatin, substi- 
tuting 1 1 per cent, agar in the place of gelatin. 

Blood-agar. A drop of blood, obtained with aseptic 
precautions, is smeared on the surface of slant-agar. 

Litmus-whey (Petruschk-y, modified by Durham). 
Casein is precipitated from milk with rennet-extract; the 
whey is neutralized with 4 per cent, citric-acid solution and 
heated on the water-bath for half an hour. It is then filtered, 
and litmus-solution is added until a decided blue color is 
obtained. 

Whey-gelatin. Add 10 per cent, gelatin to clarified whey. 

Alkaline blood-serum (Lorrain Smith). Add 1-1.5 P 61 " 
cent, of a 10 per cent, solution of sodium hydrate to blood- 
serum, tube, and sterilize in Arnold or in Koch's serum 
inspissator. 

Bread-paste medium. Bread is cut into slices, dried in 
the oven, and pulverized; it is then distributed in 100 c.c. 
flasks until the layer on the bottom is half an inch thick. 
Water is gradually run in to cover the surface of the bread 
Sterilize in Arnold. 

Mac Conkey's bile-salt-agar. 

Nutrient agar 100 c.c. 

Sodium taurocholate 0.5$ 

Peptone 2.0$ 

This is boiled, clarified, and filtered, and then 2 per cent, 
lactose is added, tubed, and sterilized in the medium. 



SPECIAL MEDIA 129 

Mac Conkey' s bile-salt broth. 

Sodium taurocholate 0.5$ 

Peptone 2.0% 

Glucose o . 5$ 

are dissolved in beef -broth by boiling; then filter and add 
litmus-solution. 

Parietti's solution 

Carbolic acid 5 c.c. 

Hydrochloric acid 4 c.c. 

Water 100 c.c. 

Eisner's medium. i pound of sliced potatoes is grated 
with a liter of water, and the juice expressed. After filtering 
for 24 hours, add i per cent, peptone, i per cent, potassium 
iodid, and 10 per cent, gelatin. 

Glycerinated potato. Prepare potatoes in the ordinary 
manner and soak in a 25 per cent, solution of glycerin. Use 
a glycerin -solution in the bottom of the tube. 

Hay-infusion. 10 g. of chopped hay are macerated with 
1000 c.c. of water in the water-bath for 3 hours. Filter and 
sterilize in autoclav for 10 minutes at 120. 

Wine-must. Wine-must is diluted with four times its 
weight of water. Dissolve 0.5 per cent, ammonium tartrate, 
macerate in the water-bath for i hour, filter, and sterilize in 
Arnold for 3 consecutive days. 

Winogradsky' s solution (for nitric organisms). 

Dissolve in water 1000 c.c. 

Potassium phosphate i . oo g. 

Magnesium sulphate o . 50 g. 

Calcium chlorid o . 01 g. 

Sodium chlorid 2 . oo g. 

Distribute 20 c.c. in flasks, and add to each flask a small 
amount of magnesium carbonate. Sterilize in Arnold for 3 
consecutive days; then add to each flask 2 c.c. of a sterile 
2 per cent, solution of ammonium sulphate. 



130 LABORATORY GUIDE IN BACTERIOLOGY 

Yeast-water. i liter of washed yeast or i pound of pressed 
yeast is boiled with 2 liters of water for i hour. The reaction 
is made neutral to phenolphthalein, and the solution is filtered 
until clear, and sterilized in Arnold for 3 consecutive days. 

Dextrose-yeast-water. 10-15 per cent, dextrose is dissolved 
in yeast-water without adjusting the reaction. 

Egg medium (Dorset). Eggs are broken into a flask, and 
the yolks are broken with a platinum wire. Then the flask 
is gently shaken, so as to mix the yolks with the whites without 
causing foam to form. 10 c.c. are distributed in culture- 
tubes, and then the medium is hardened in a sloping position 
in the Koch inspissator for 2 successive days for 4 or 5 hours 
at 70. 

Egg-yolk medium (Dorset). Add 5-10 c.c. of sterile water 
to the yolks of 3 or 4 eggs, and then pursue the same course 
as in the other egg medium. 

Egg-yolk medium (Capaldi). A few loopfuls of egg-yolk 
are added to a tube of liquefied agar cooled to 45-47. 

Enriching solution for B. typhosus (Hoffmann and Fischer). 
To peptone-meat-broth add i per cent, nutrose, 0.5 per 
cent, caffein, and i per cent, solution of crystal violet. 

Endo's medium. 

Nutrient agar (3$) 1000 c.c. 

Lactose 10 g. 

Alcoholic solution of fuchsin 5 c.c. 

Sodium sulphite (10$ solution) 25 c.c. 

Sodium hydrate (10$ solution) 10 c.c. 

Drigalski and Conradi's medium (Modified by Harris). 

Dextrose-free broth 2000 c.c. 

Nutrose 20 g. 

Agar 40 g- 

Boil, dissolve, neutralize to phenolphthalein, autoclav at 120 
for 5 minutes. Clarify with whites of four eggs and filter. Then 
add 



SPECIAL MEDIA 131 

Lactose 30 g. 

Litmus-solution 260 c.c. 

Crystal violet (o. \% aqueous solution) 20 c.c. 

Tube and sterilize once in Arnold. 

Uschinsky's non-proteid medium (FraenkePs modifica- 
tion) . 

Disodium hydrogen phosphate 20 g. 

Ammonium lactate 63 g. 

Asparagin 34 g. 

Sodium chlorid 50 g. 

Water 10,000 c.c. 

Phenol media. i part carbolic acid is added to 1000 
parts medium. 

Dog-blood serum. Serum of dog's blood is coagulated in 
slanted tubes in the Koch inspissator for 3 hours at 75. 

Nitrate broth. Add 5 parts of potassium nitrate to each 
1000 parts of ordinary broth. 

Nitrate solution. 5 c.c. of 2 per cent, aqueous potassium- 
nitrate solution are added to a solution of i g. of peptone in 
1000 c.c. of water. 



APPENDIX II 
STAINING SOLUTIONS 
Delafield's hematoxylin 

Hematoxylin crystals 4 g. 

Alcohol .25 c.c. 

Ammonia alum 50 g. 

Water 400 c.c. 

Glycerin 100 c.c. 

Methyl alcohol 100 c.c. 

Alum-hematoxylin 

1 . Hematoxylin 2 g. 

Absolute alcohol 100 c.c. 

2. Ammonia alum 2 g. 

Water 100 c.c. 

Mix i and 2 and add 

Glycerin 850 c.c. 

Glacial acetic acid 100 c.c. 

Allow to stand for one month before using. 
Bismarck brown 

Bismarck brown o . 5 g. 

Water 100 c.c. 

Sajranin 

Safranin 0.5 g. 

Water 100 c.c. 

Carbolic thionin-blue (Nicolle') 

Thionin-blue i g. 

Carbolic acid 2 . 5 g. 

Water 100 c.c. 

Alum carmin 

Alum 2 . 5 g. 

Carmin i o g. 

Water 100 c.c. 

132 



STAINING SOLUTIONS 133 

Lithium-carmin (Orth) 

Carmin 2 . 5 g. 

Saturated watery solution of lithium car- 
bonate 100 c.c. 

Add a few crystals of thymol. 
Kiihne's methylene-blue 

Methylene-blue i . 5 g. 

Absolute alcohol 10 c.c. 

Carbolic acid solution (5$) 100 c.c. 

Carbolic gentian-violet (Nicolle) 

Gentian-violet (sat. alcoh. sol.) 10 c.c. 

Carbolic acid i g. 

Water po c.c. 



APPENDIX III 

FROST'S CULTURE CHART (MODIFIED)' 

Group 

Name of organism 

Source, habitat, etc 



MORPHOLOGICAL CHARACTERS: 


i 


Incubation 
Temp. (C.) 


Sketches 


i . Form : 
a Broth 








b Agar 








c Gelatin 








d Other Media 








2. Size 








3. Cell groupings and arrangements in 
growths 








4 Staining powers 








d ^nilin-gentian-violet .. 








b Loeffler's methylene-blue 








c. Gram's stain 








d. Special stains 
5. Motility 








a Character of movement 








b Flagella stain 








6 Spores 








7. Special characters, such as 








deposits, vacuoles 
pleomorphic and involution forms, 
capsules etc 

















PHYSIOLOGICAL CHARACTERS 
i. Relation to temperature 



Relation to free oxygen. 

Relation to other agents, such as dessica- 

tion, light, disinfectants, etc 



1 This represents a model only. Open spaces and lines should be extended for 
practical use to allow sufficient room for descriptions. 

134 



FROST'S CULTURE CHART 



4. Pigment production 

5. Growth in carbohydrate media: 
a. Stab or shake culture 

6. Fermentation-tube: i. Growth in bulb. . . .2. In closed arm. . 

c. Percentage gas produced in: Dextrose. .. .Lactose . . .Saccharose 

After 24 hours. . 

After 48 hours 

TT 

Gas formula T^T~ 

CU 2 

d. Reaction in bulb 2. Closed arm 

6. Acid or alkali production, in litmus milk 

7. Reduction of nitrates; to nitrites to ammonia 

8. Indol production; 24 hours 48 hours 4 days 

fecal odor; 24 hours 48 hours 4 days 

9. Enzym production; coagulative proteolytic diastatic 

10. Characteristic odor 

11. Pathogenesis 



CULTURAL CHARACTERS OF: 



(I) 

Gelatin 
plate or 
roll tube 

a) Surface 
colonies 

b) Deep 
colonies 



(2) 

Agar plate, 

or roll 

tube 
a) Surface 

colonies 
6) Deep 

colonies 



Reaction of 

Medium 
Incubation 
Temp. (C) 



Description 



Sketches 



136 LABORATORY GUIDE IN BACTERIOLOGY 
CULTURAL CHARACTERS OF: 





Reaction, 
etc. 


24 Hrs. 


48 Hrs. 


6 Days 


Sketches 


(3) 

Agar streak 
and stab 












A 

/ \ 
/ \ 




A 

/ X 

/ \ 

/ \ 




(4) 
Potato 






















& 




^ x / 




(5) 










Broth 


i 











v| 




- 




(6) 


i 






Litmus milk 












L 




w 




(7) 










Gelatin stab 












^ 









FROST'S CULTURE CHART 
CULTURAL CHARACTERS OF: 





Reaction, 
etc. 


24 Hrs. 


48 Hrs. 


6 Days 


Sketches 


(8) 












Glucose-agar 
or special 












media 













INDEX 



Absorbent cotton filter 17 

Acid, chromic 8 

Acid-resisting bacilli 103 
Actinomyces asteroides, bovis, etc. 106 

Agar-agar 1 1 

Agar, beerwort 128 

Agar, bile-salt 128 

Agar, blood 128 

Agar, glucose 19 

Agar, glycerin 128 

Agar, litmus-lactose 127 

Agar, litmus-mannit 127 

Agar, neutral red 127 

Agar, preparation of n 

Agar-slants 39 

Agglutination 87 
Air, bacterial examination of 37, 114,118 

Air, bacteria from 37 

Alkaline blood-serum 128 

Alum carmin 132 

Alum hematoxylin 132 

Amylase 65 

Anaerobic cultivation 107 

Anaerobic group 107 

Analysis of gas 82 

Anilin-gentian-violet 30 

Anthrax bacillus 98 

Anthrax group 98 

Arnold steam sterilizer 24 

Aspirator 46 

Autoclav 20 

Autopsy 75 

Bacilli, acid-resisting 103 

Bacillus acidi lactici 79 

Bacillus aerogenes capsulatus 107 

Bacillus anthracis 98 
Bacillus anthracis symptomatici 107 

Bacillus botulinus 107 
Bacillus cholerae suis 79,84 
Bacillus cloacae 79, 89 

Bacillus coli 79 

Bacillus cuniculicida 97 

Bacillus der Rinderseuche 97 

Bacillus der Schweineseuche 97 

Bacillus diphtheriae 94 
Bacillus dysenteriae 79, 85 
Bacillus enteritidis 79, 84 
Bacillus faecalis alcaligenes 79, 85 

Bacillus Gaertner's 84 

Bacillus, grass 103 

Bacillus icteroides 79 
Bacillus lactis aerogenes 79, 92 

Bacillus leprae 103 

Bacillus mallei 105 



PAGE 

Bacillus mucosus capsulatus 92 

Bacillus oedematis maligni 107 

Bacillus of bubonic plague 97 

Bacillus of dysentery 79, 85 

Bacillus of rabbit septicemia 97 

Bacillus paratyphosus 79, 84 

Bacillus pestis 97 

Bacillus prodigiosus 69 

Bacillus pseudo-diphthericus 94 

Bacillus pyocyaneus 69 

Bacillus smegrnae 103 

Bacillus subtilis 98 

Bacillus tetani 107 

Bacillus tuberculosis 103 

Bacillus typhosus 79,85 

Bacillus violaceus 69 

Bacillus xerosis 94 

Bacteria, chromogenic 69 

Bacteria from air 37, 118 

Bacteria, pyogenic 73, 76 

Bacteria, unknown 112 
Bacterial examination of air 

37, 114, 118 

Bacterial examination, of milk 114, 119 
Bacterial examination, of water 114 
Bacterium termo 79 
Balsam bottle 4 
Balsam, Canada 44 
Basket, wire 4 
Beerwort-agar 128 
Beerwort-gelatin 128 
Berkefeld filter 46 
Bile-salt agar 128 
Bile-salt broth 129 
Bismarck brown 132 
Blood-agar 128 
Blood-serum 125 
Blood-serum alkaline 128 
Blood-serum, dog's 131 
Bouillon (see peptone broth) 25 
Bread-paste medium 128 
Broth, bile-salt 129 
Broth, glycerin 127 
Broth, litmus 128 
Broth, meat 26 
Broth, nitrate 131 
Broth r peptone 25 
Broth, preparation of 25 
Broth, sugar- free 27, 79 
Brownian movement 43 
Bubonic plague 97 
Buchner's anaerobic culture meth- 
od 107 
Butter, bacilli in 103 



139 



1 4 o 



INDEX 



Canada balsam 44 

Capaldi's egg-medium 130 

Capsulated group 92 

Capsule stain 76 

Capsule stain, Friedlander's 77 

Capsule stain, Welch's 77 
Capsule stain, Welch's modified no 

Carbol-fuchsin 30 

Carbolic gentian- violet 133 

Carbolic thionin-blue 132 

Carmin 43 

Carmin, alum 132 

Carmin, lithium 133 

Casein 64 

Caseinogen 64 

Cedar oil 35 
Cholera red reaction (see Indol) 101 

Chrompgenic group 69 

Clarifying media 13 
Cleaning glassware 

Cleaning mixtures 8 
Coagulation 64 
Coagulative enzym 64 
Colon bacillus 79 
Colon group 79 
Colonies, counting of 116 
Colony 39 
Condensation water 40 
Conradi's medium 130 
Cotton filter 47 
Cotton filter, absorbent 17 
Counting colonies 116 
Culture- charts description 57 
Culture media n 
Culture- tubes 3, 4, 14 
Culture-tubes, plugging of 
Culture-tubes, potato 3, 28 
Cultures, egg 95 
Cultures, plate 70 
Cultures, pure 39 
Death-point, thermal 124 
Decolorization of litmus 65 
Delafield's hematoxylin 132 
Description of colonies 57 
Description of cultures 57 
Dextrose (see under glucose) 
Dextrose yeast water 130 
Diagnosis of glanders (Strauss) 105 
Diastase 65 
Diastatic enzym 65 
Dilution tables 117 
Diphtheria bacillus 94 
Diphtheria group 94 
Diphtheria toxin 96 
Directions for filling out culture- 
charts 6 1 
Directions, general 2 
Discontinuous sterilization 24 
Disinfectants 122 
Dog's blood-serum 131 
Dorset's egg medium 130 
Dorset's egg-yolk medium 130 



PAGE 

Drigalski's medium 130 

Dung bacillus 103 

Dunham's peptone solution 25 
Dysentery bacillus 79, 85 

Egg cultures 95 

Egg media 130 

Ehrlich's anilin -gentian- violet 30 

Eisner's medium 129 

Endo's medium 130 
Enriching method of Schottelius 101 
Enriching solution (Hoffmann and 

Fischer) 130 

Enzym 64 

Enzym, coagulative 64 

Enzym, diastatic 65 

Enzym, production of 64 

Enzym, rennet-like 64 

Erlenmeyer flask 5 
Examination, bacterial of air 

37, 114,118 

Examination, bacterial of milk 1 14, 1 19 
Examination, bacterial of water 114 
Fermentation-tube 4 
Filament formation 99 
Filling culture-tubes 14 
Filter, absorbent cotton 17 
Filter, Berkefeld 46 
Filter, cotton 47 
Filter, paper 15 
Filter rack 17 
Filtering media 16 
Filtering media by vacuum 17 
Finkler and Prior's spirillum 101 
Flagella, staining of 86 
Flask, Erlenmeyer 5 
Fowl cholera, bacillus of 97 
Friedlander's capsule stain 77 
Friedlander's pneumobacillus 92 
Frost's fermentation-chart 81 
Frost's culture-chart (modified) 134 
Fruit-jar method of anaerobic cul- 
tivation 1 08 
Fuchsin 30 
Fuller's standard 13 
Gaertner's bacillus 84 
Garden earth, inoculation of 1 1 1 
Gas analysis 82 
Gas formula 82 
Gas generator 109 
Gasometer (Frost's) 81 
Gelatin, beerwort 128 
Gelatin, glucose 128 
Gelatin, liquefaction of 64 
Gelatin, litmus-lactose 127 
Gelatin, peptone 23 
Gelatin, whey 128 
General directions 2 
Gentian- violet 30 
Gentian-violet, carbolic 133 
Germination of spores 51 
Glanders 105 
Glassware, cleaning 



INDEX 



141 



PAGE 

Glassware, sterilization 9 

Glucose-agar 19 

Glucose-gelatin 127 

Glycerin-agar 128 

Glycerin-broth 127 

Glycerinated potato 129 

Glycogen 27 

Gonorrheal pus 76 

Gram's iodin solution 30 

Gram's stain 53 

Grass bacillus 103 

Green mold 51 
Gruber-Widal test for typhoid fever 87 

Gypsum blocks 5 

Hand -lens 5 

Hanging-drop 42 

Hay-infusion 129 

Hematoxylin alum 132 

Hematoxylin, Delafield's 132 

Hemorrhagic septicemia group 97 

Hill's test -rods 122 
Hoffmann and Fischer's enriching 



method 
Hog-cholera group 
Hot-air sterilizer 
Hydrogen gas-generator 
Immersion oil 
Impression preparation 
Incubator 
Indol 
Indol test 

Infection, phenomena of 
Influence of disinfectants 
Influence of moist heat 
Influence of sunlight 
Infusion, hay 



130 

79,84 

9 

109 



I! 

85 

45 

122 
124 
123 
129 

Inoculation (see various headings) 

79, 84 
79, 84 
24 
79 
102 
92 
73 



Intermediate group 
Intermittent sterilisation 
Intestinal group 
Intramuscular inoculation 
Intraperitoneal inoculation 
Intravenous inoculation 
Involution forms 
Iodin solution, Gram's 
Isolation of unknown bacteria 
Jar, Novy 
Klatschprdparat 
Kiihne's methylene-blue 
Labeling media 
Laboratory rules 
Lactose-litmus-agar 
Lactose-litmus-gelatin 
Liquefaction of casein 
Liquefaction of gelatin 
Lithium carmin 
Litmus-broth 
Litmus, decolorization of 
Litmus-lactose-agar 
Litmus-lactose-gelatin 
Litmus-mannit-agar 



30 

112 
1 08 

98 
133 

20 
I 

127 
127 

6 4 

6 4 
133 
128 

65 
127 
127 
127 



PAGE 

Litmus milk 2 8 

Litmus-solution 127 

Litmus- whey 128 

Loeffler's flagella stain 86 

Loeffler's methylene-blue 30 

Mac Conkey's bile-salt agar 128 

Mac Conkey's bile-salt broth 129 

Magnifier 5 

Mannit litmus agar 127 

Meat broth 26 

Meat-press 26 

Media, adjusting reaction of 14 

Media, adjusting weight of 13 

Media, clarifying 13 

Media, filling in tubes 14 

Media, filtering 14 

Media, filtering by vacuum 18 

Media, phenol 131 

Media, preparation of 1 1 

Media, preservation of 28 

Media, reaction of 12 

Media, special 125 

Media, standard methods 125 

Medium, bread-paste 128 

Medium, Drigalski-Conradi's 130 

Medium, Eisner's 129 

Medium, Endo's 130 

Medium, Uschinsky's 131 

Medium, wine-must 129 

Method of describing cultures 39 
Method of inoculation of media 57 

Methylene-blue, Kiihne 133 

Methylene-blue, Loeffler 30 

Micrococcus gonorrhoeae 76 
Micrococcus intracellularis menin- 

gitidis 76 

Micrococcus lanceolatus 76 

Micrococcus melitensis 105 

Micrococcus tetragenus 73 

Micrococcus zymogenes 76 

Microscope, description of 31 

Milk, acid-resisting bacilli in 104 
Milk, bacterial, examination of 114,119 

Milk, litmus 28 

Milk, pasteurization of 119 

Milk, sterilization of 119 

Moeller's grass bacillus 103 

Moeller's spore stain 98 

Moist heat, influence of 124 
Moist heat, sterilization by 20, 24 

Mold, green 5 1 

Mold spores 39 

Molds 49 

Molds, staining of 5 

Molecular movement 43 
Mordant 

Mouse, inoculation of 

Mouse- holder 78 

Muscle-sugar 27 

Needles, platinum 4 

Neisser's stain 94 

Neutral red agar 127 



I 4 2 



INDEX 



PAGE 

Nicole's carbolic gentian-violet 133 
Nicole's carbolic thionin-blue 132 

Nitrate broth 131 

Nitrate solution 131 

Nitrites 63 

Nitrites, test for 83 

Nitroso-indol reaction 101 

Normal solution 13 

Novy jar 108 

Oil, immersion 34 

Orth's lithium carmin 133 

Parietti's solution 129 
Park's anaerobic culture method 107 

Pasteurization of milk 119 

Pedesis 43 

Penicillium glaucum 5 1 

Peptone broth 25 

Peptone gelatin 23 

Peptone solution, Dunham's 25 

Peptonization 64 

Petri dishes 5 

Phenol media 131 

Phenolphthalein 12 

Phenomena of infection 45 

Phenomena of sterilization 45 

Pigments 7 1 

Plate cultures 70 

Platinum needles 4 

Plugging culture-tubes 8 

Pneumobacillus 92 

Pocket inoculation 96 
Polar staining 85, 97 

Potato, glycerinated 129 

Potato, preparation of 27 

Potato-tubes 28 

Preparation, Gram 53 

Preparation, impression 98 

Preparation of agar-agar 1 1 

Preparation of bouillon 25 

Preparation of broth 25 

Preparation of culture media 1 1 
Preparation of Dunham's peptone 

solution 25 

Preparation of gelatin 23 

Preparation of glucose- agar 19 
Preparation of stains (see various 

headings) 

Preparation of peptone solution 25 

Preparation of potato 27 

Preparation, stained 44 

Preservation of media 1 1 

Proteolysis 64 

Proteus group 89 

Pure culture 39 

Pus, gonorrheal 76 

Pyocyanin 72 

Pyogenic group A 73 

Pyogenic group B 76 

Rabbit septicemia 97 

Reaction, indol 83 

Reaction, nitrites 83 

Reaction, nitroso-indol 101 



Reaction of media 14 

Rennet-like enzym 64 

Rinderpest 97 

Routine study 53 

Saccharomyces cerevisiae 49 

Safranin 132 

Sarcina lutea 69 

Schottelius' enriching method 101 

Schweineseuche 97 

Soap-powder 8 

Solution, Dunham's peptone 25 

Solution, litmus 127 

Solution, nitrate 131 

Solution, normal 13 

Solution, Parietti's 129 
Solutions, staining 30, 132 

Special media 125 

Spirillum cholerae asiaticae 101 

Spirillum Metchnikovi 101 
Spirillum of Finkler and Prior 101 

Spirillum tyrogenum 101 

Spore-staining 08 

Spores, germination of 51 

Spores of bacteria 20 

Spores of yeast 51 

Spores of molds 39 

Stain, Friedlander's capsule 77 

Stain, Welch's capsule 77 
Stain, Welch's capsule modified no 

Stain, Gram's 53 

Stain, Loeffler's flagella 86 

Stain, Neisser's 94 

Stained preparation 44 

Staining acid-resisting bacilli 103 

Staining flagella 86 

Staining molds 51 
Staining, polar 85, 97 
Staining solutions 30, 132 

Staining spores 98 

Standard, Fuller's 13 
Standard method of preparing 

media 125 

Staphylococcus pyogenes albus 73 
Staphylococcus pyogenes aureus 73 

Starch 65 

Steam sterilizer 24 

Sterilization 9 

Sterilization, discontinuous 24 

Sterilization, intermittent 24 

Sterilization of glassware 9 

Sterilization of milk 1 19 

Sterilization, phenomena of 45 

Sterilizer, hot-air 9 

Sterilizer, Arnold steam 24 
Strauss method of diagnosis of 

glanders 105 

Streptococcus pyogenes 73 

Study, routine 53 

Subcutaneous inoculation 85 
Sugar-free broth 27, 79 

Sulphur granules 106 

Sunlight, influence of 123 



INDEX 



PAGE 


Tables, dilution 


17 


Tetanus toxin 


no 


Test for indol 


83 


Test for nitrites 


83 


Test-rods, Hill's 


122 


Thermal death-point 


124 


Thermostat 


55 


Thionin-blue 


132 


Torulae 


49 


Tube, culture 


4 


Tube, fermentation 


4 


Tube, potato culture 


28 


Tubes, plugging of 
Typhoid-dysentery group 79 
Uschinsky's medium 


8 
, 85 
I3 1 


Water, bacterial examination of 


114 



PAGE 

40 
77 
no 
64 
128 
128 



Water of condensation 
Welch's capsule stain 
Welch's capsule stain modified 
Whey 

Whey-gelatin 
Whey litmus 

Widal test (see Gruber-Widal test) 
Wine-must 129 

Winogradsky's solution 129 

Xylol 33, 35 

Yeast water 130 

Yeast water, dextrose 130 

Yeasts 39, 49, 51 

Yeasts, budding of 51 

Yeasts, spores of 51 

Ziehl-Neelsen's carbol-fuchsin 30 




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