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laboratory guide in bacteriology, fort

Cornell University

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

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http://www.archive.org/details/cu31924003244492

A LABORATORY GUIDE IN
BACTERIOLOGY

THE UNIVERSITY OF OHIOAGO PRESS
CHICAGO, ILLINOIS

THE BAKER & TAYLOR COMPANY
NEW YORK

THE CAMBRIDGE UNIVERSITY PRESS
LONDON AND EDINBURGH ie

THE MARUZEN-KABUSHIKI-KAISHA
TOKYO, OSAKA, KYOTO, FUKUOKA, SENDAI

THE MISSION BOOK COMPANY
SHANGHAI

A LABORATORY GUIDE

BACTERIOLOGY

FOR THE USE OF STUDENTS, TEACHERS
AND PRACTITIONERS

BY

PAUL G. HEINEMANN, Pz.D.

THIRD REVISED EDITION

THE UNIVERSITY OF CHICAGO PRESS
CHICAGO, ILLINOIS

CopyRIGHT 1905 By
THE UNIVERSITY OF CHICAGO

First Edition June 1905
_ Second Edition June rorr
Second Impression October 1911
_ Third Impression October ror3
Third Edition September 1915
3econd Impression September 1916
Third Impression July 1919

Composed and Printed By
The University of Chicago Press
Chicago, Illinois, U.S.A.

PREFACE TO FIRST EDITION

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 ad-
vanced so rapidly during the past few years as the
science of bacteriology, and it is difficult even for an
active laboratory worker to keep abreast of this ad-
vance. A textbook 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 additions 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 bacteri-
ology 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 atti-
tude of mind of the student and to try to appreciate
his difficulties in understanding details. Many of

Vv

vi PREFACE TO FIRST EDITION

the pieces of apparatus employed in a bacteriological
laboratory are novel even 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 num-
ber 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, 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 char-
acteristics 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 mor-
phological features are left entirely to the actual ob-
servation of the student, supplemented 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

PREFACE TO FIRST EDITION vii

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 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 of 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 MacLeod
Harris for their invaluable help and suggestions in the

preparation of this guide.
Pau. G. HEINEMANN
Carcaco, IL.

June, 1905

PREFACE TO SECOND EDITION

The influence of applied bacteriology is extending
rapidly, and while formerly the science of micro-
organisms was chiefly confined to the medical field, it
has become of vast industrial importance. The gigan-
tic interests of fermentation industries, of the dairy, of
agriculture, of municipal sanitation, and of water puri-
fication are largely controlled by bacteriologists. A
knowledge of the principles of bacteriology is becoming
more and more desirable to the student of general
science, and teachers of domestic science in some
institutions are required to take an elementary course.

The second edition of this laboratory guide has been
revised and enlarged with the view of meeting modern
requirements. The first edition was devoted chiefly to
medical bacteriology. This has been partly rewritten,
and in addition courses in general bacteriology, in the
bacteriology of water, in the bacteriology of milk, in
soil bacteriology, and a course on molds, yeasts, and
acetic-acid bacteria have been outlined. This change of
plan necessitated an entirely new arrangement of the
material. The preparation of culture media, of stains,
etc., has been incorporated in a separate part, and
reference to this part is made at the beginning of each
special part. The course in the bacteriology of water
is calculated to be combined with the physical, chemical,
and microscopic examination, the student being thus
prepared for sanitary water analysis. Similarly, the
course on the bacteriology of milk is to be given in

1x

x PREFACE TO SECOND EDITION

connection with the physical and chemical examina-
tion of milk. ;

The author hopes that, by the radical changes indi-
cated, the usefulness of the book may be extended, and
that the study of applied bacteriology may be en-
couraged. J am under special obligations to Professor
Edwin O. Jordan for advice and suggestions as to the
general plan of the book, to Dr. Mary Hefferan for
critical reading of the manuscript and the proof, and
to Dr. R. E. Buchanan for valuable advice in outlining
the course in soil bacteriology.

Paut G. HEINEMANN
June, 1911

PREFACE TO THIRD EDITION

In this third edition the general plan of the book
has not been changed. The progress of science
necessitated alterations and additions which have
been incorporated in the text. A fermentation chart
of cardboard has been added to the colony counter
in the back cover.

The author is under obligations to Professors
Edwin O. Jordan and Norman MacLeod Harris for
valuable suggestions.

Paut G. HEINEMANN

June, 1915

INTRODUCTION

The advent of bacteriology into the realm of the
biological sciences not only brought with it a new con-
ception of the nature of many complicated phenomena,
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 technic, 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, tuberculosis, and
typhoid fever are being fought energetically and with
much success. In these systematic and organized
movements the resources of bacteriology 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, of urban infantile mortal-
ity, and of the control of contagious diseases are all
bound up with the intelligent application of bacterio-
logical methods.

xl

xii INTRODUCTION

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 technic.

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, “prac-
tical” ways of doing things are being supplanted 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 stumbling and
disastrous missteps are certain. A comprehensive out-
line of modern bacteriological methods, therefore, is a
necessary adjunct to obtaining a true and full under-
standing of the underlying principles and tendencies
of the science. The technic of 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 in-
vestigator, but by practical workers in medicine,
hygiene, and many other fields.

Epwin O. JorDAN

TABLE OF CONTENTS

Part I. BaAcTERIOLOGICAL TECHNIC

Section
Section
Section
Section
Section
Section
Section
Section
Section

Section 10.

L
2
3
4
5.
6.
7
8
9
oO.

Laboratory Rules

- General Directions . ;
Preparation and Cleaning of Glascyate é
Methods of Sterilization
Preparation of Culture Media
Preparation of Staining Solutions .. .

. The Microscope

Scheme for Routine Study af acest
Method of Describing Cultures <
Directions for Filling out Culture Charts

Part II. GENERAL BACTERIOLOGY

Section
Section

Section
Section

Section
Section

Section
Section
Section

Part III.

Section
Section
Section
Section
Section
Section

I.
2.

ed

9.

Preparation of Culture Media

Collecting and Cultivating ee
isms fromthe Air . .

Study of Molds, Yeasts, aad Torulae
Bacteriological Examination of Water,
Air, and Milk a ¢
Exercises on Infection and Gieciieation ‘
Influence of Disinfectants, Light, and
Heat on the Growth of Micro-organisms
Study of Chromogenic Bacteria :
Study of Micrococci

Study of Intestinal Bacteria

IMPORTANT PATHOGENIC BACTERIA

Preparation of Culture Media
The Pyogenic Group

I
2. Ce
3. The Group of Colon-Typhoid Bacilli
4. .
5.
6

The Proteus Group
The Capsulated Group
The Diphtheria Group .

xiv

Section 7,
Section 8.
Section 9.
Section 10.

Section 12.
Section 13.
Section 14.

TABLE OF CONTENTS

PAGE
The Hemorrhagic Septicemia Group . 127
The Anthrax Group 127
The Spirillum Group . . : 129
The Group of Acid-proof Bacilli - 131

Section 11 Aand B. Miscellaneous Organisms . 132, 133
Pathogenic Trichomycetes . . . 134
The Group of Anaérobic Bacilli 134
Isolation of Unknown Bacteria from a
Mixture . 139

PartIV. THe BACTERIOLOGICAL EXAMINATION OF WATER
AND SEWAGE

Section 1.

Section
Section
Section
Section
Section
Section

WAKE YD

Part V. THE
Section 1.

Section 2.
Section 3.

Section 4.
Section 5.

Section 6.
Section 7.

Section 8.

Preparation of Culture Media and of
Dilution Flasks . :
Bacteriological Examination of Water
Examination of Sewage . . . 3

‘Determination of Anaérobes in Sewaee :

A Study of B. coli and Streptococci .
Isolation of B. typhosus from Water
Study of Reaction of Bacteria on Neutral-
red Broth . . . . . ..

144
145
148
149
149
150

I50

BACTERIOLOGICAL EXAMINATION OF MILK

Preparation of Culture Media and of
Dilution Flasks. 2. 1. 2. 2. 1 we
Quantitative Bacteriological Examina-
tion of Milk .

Examination of Market Milk for Tiibercle
Bacilli . . .

A Study of the Acid Reraen tation é Milk
Determination of B. coli and Streptococci
inMikk . .

Study of the Effects ‘of Bustainivation
and So-called Sterilization of Milk

A Qualitative and Quantitative Study of.

Anaérobes in Milk .
A Study of Some Organisms Garang: Ab-
normal Fermentationsin Milk . . .

154
154

155
155

157
158
158

159

TABLE OF *con’ TENTS XV

PAGE

Section 9. Examination of Milk for Molds and
Yeasts . . +» I6o

Section 10. Examination for Takootesin Mit - 160

Section 11. A Study of Groups of Bacteria in Milk . 162
Part VI. THE BACTERIOLOGICAL EXAMINATION OF SOIL
Section 1. Quantitative Determination of Bacteria

and Sporesin Soil . . 165
Section 2. A Study of the Beptanication é Proteins

by Soil Bacteria . . 168
Section 3. The Formation of Amido Gonicasnie: and

Ammonia . . 170
Section 4. The Formation of Nitrites from Avininnita

and Isolation of Nitrite Bacteria. . . 171
Section 5. The Formation of Nitrates from Nitrites

and Isolation of Nitrate Bacteria. . . 174

Section 6. The Assimilation of Free Atmospheric
Nitrogen and Isolation of the Bacteria . 175
Section 7. The Reduction of Nitrates to Nitrites and

Isolation of the Bacteria . . 177
Section 8. The Reduction of Nitrates to Free enue.
gen .. 177

Section 9. Growing Demin’ i in Sand and in Saad
Inoculated with Legume Bacteria . . 178

Part VII. Mo.ps, YEAsts, ToRULAE, AND ACETIC-ACID

BACTERIA

Section 1. Preparation of Culture Media . . . 181
Section 2. AStudyofMolds . . . .. . . 182
Section 3. AStudyof Yeasts . . - ee R85
Section. 4. Examination of Baker’s Yeast 4 % » Foo
Section 5. Examination of Yeast of iba: Bread 191
Section 6. AStudyofTorulae .. . . 192
Section 7. A Study of Acetic-Acid Bacteria. ye 5 tg?

APPENDIX

Dilution Tables . . . sie Gay oe sey Hae EON
Table of Weights and Hiessaces.. : - 198

Table of Centigrade and Fahrenheit atthemonicteed . 198
INDEX. «ee ee ee ee we ee ws 203

PART I
BACTERIOLOGICAL TECHNIC

SECTION 1
LABORATORY RULES

I. 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; pencils,
labels, or fingers must not be moistened with the
tongue.

3. If any portion of a culture is spilt by accident
upon the desk or floor, it should be covered immediately
with a germicide (HgCl, 1:1000, or carbolic acid in
5 per cent solution). After this germicide 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 come in contact with
infectious material, they should be washed with one
of the above mentioned germicides, and then scrubbed
with soap and water.

5. The platinum needles used in making cultures
should be sterilized in a flame before and after use, and
before they are laid down. When the needles are
covered with viscous material, as milk, for instance,
they should be held at the side of the flame until dry
before being sterilized. This will avoid the danger of
scattering infectious material about the desk.

6. All possible care should be observed in handling
apparatus, etc. Solid material should not be put into
sinks. Burned matches, paper, cotton, broken glass,

3

4 LABORATORY GUIDE IN BACTERIOLOGY

etc., should be put into crocks and not on the floor or
into the sink.

7. Discarded cultures should be killed in the auto-
clave (5 minutes at 120° C.) before being emptied into
the crocks.

8. See that the air inlets of Bunsen burners are open
before lighting, and relight if the flame strikes back.

9. Always return stockbottles to the proper places
on.the shelves.

to. At the close of the day’s work the desks should
be washed off with corrosive sublimate, and the hands
cleaned by thorough washing.

11. 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. Culture tubes containing media or cultures
should be replaced in their proper places, in order to
avoid settling of dust or other foreign material on the
stoppers. Dust and air draughts are frequently the
cause of contaminations, and in order to avoid these
the utmost cleanliness should be observed. A bacteri-
ological laboratory should present an orderly appear-
ance at all times. ,

SECTION 2
GENERAL DIRECTIONS
The following directions should be followed in all
the work outlined in this guide:
1. After obtaining the key to a locker, the student

should examine the outfit, check all apparatus, and see
that everything is in good condition.

BACTERIOLOGICAL TECHNIC 5

2. The student should familiarize himself with the
program before him for each day, as this will facilitate
intelligent and systematic work.

3. The student should procure description charts
and fill them out carefully according to directions.

4. Store cover slips in a stender dish and cover
them with alcohol. A soft linen cloth should be used
for cleaning.

The following outfit will be needed for each course.
Additions to this outfit will be designated as required
in the respective courses.

200 culture tubes.
20 potato tubes.
12 fermentation tubes.
20 petri dishes.
Erlenmeyer flasks, one 1,000 c.c., two 500 c.c. each.
glass funnels, one 4 inches, one 6 inches.
bottles for staining fluids.
balsam bottle.
stender dish.
staining dishes.
saltcellar.
glass rod.
platinum needles. Turn the end of one of the needles
around a sharp pencil point so as to form a closed loop.
tin cups or glass tumblers, the bottoms of which should be
covered with a layer of cotton.
wire baskets.
Bunsen burner.
saucepan and cover, or better, a double boiler.
graduates, one 500 c.c., one I00 c.c., and one I0 c.c.
pinchcock.
pipette and hose attached.
magnifier (hand lers).
tripod.

Re HW HH PNW

fo)

HHH HWHHA RE

LABORATORY GUIDE IN BACTERIOLOGY

6

Platinum Needles

Pinchcock

Fermentation Tube

Bottle for
Staining Fluid

Wire Basket

Pipette

Culture Tube

Bunsen Burner .

Retort Stand

Fie. z

BACTERIOLOGICAL TECHNIC 7

Petri Dish

(Hand Lens) Stender Dish

Fie. 1

8 LABORATORY GUIDE IN BACTERIOLOGY

1 retort stand with three rings.
1 thermometer in case.
1 box matches.
40 grams pepton.
120. grams gelatin.
50 grams agar.
6 sheets filter paper.
50 glass slides.
I camel’s-hair brush.
50 cover glasses.
2 towels.
1 tube brush.
50 labels.
1 box for slides.
3 hollow-ground slides.
1 glass pencil.
1 pair forceps.
2 pairs cover-slip forceps.

SECTION 3
PREPARATION AND CLEANING OF GLASSWARE

Culture tubes, flasks, fermentation tubes, and petr
dishes must be free from organic matter, acids, any
alkalis. They should be cleaned as follows:

1. Immerse them in a vessel containing soapsuds or
soap powder, boil 10 to 1§ minutes, then clean them
with a tube brush. Or immerse them for an hour in a
solution of potassium bichromate and sulphuric acid:

Potassium bichromate..............0. 60 parts
Water. ..os¢ecicseas utddatama ss ne tek 300 parts
Concentrated sulphuric acid........... 460 parts

The sulphuric acid should be added slowly with con-
stant stirring.
2. Rinse in tap water.

BACTERIOLOGICAL TECHNIC 9

3. Again use tube brush, and soap and water if
necessary.

4. Rinse again in water to remove every trace of
acid or soap.

5. Place the tubes in a wire basket, mouth down, and
heat in a hot-air sterilizer for 20 minutes or longer
until dry.

All other glassware should be treated in the same
manner, excepting fermentation tubes, which should
not be heated in the hot-air oven, as this would be
likely to cause breakage.

The tubes should be plugged with cotton. Non-
absorbent cotton is suitable for this purpose. The
cotton plug allows free communication with the air,
admitting oxygen, which is necessary for the growth
of many bacteria; at the same time the admitted air is
filtered germ-free and contamination of cultures is
avoided.

Various methods for plugging tubes are employed
in different laboratories. The simplest method is as
follows: Take a small amount of cotton and push it
gently into the tube with a glass
rod. The cotton should reach
into the tube for about 2 of an
inch and be sufficiently firm to
support the weight of the tube
(Fig. 2). The cotton may also be
rolled into a cylinder of thickness
equal to that of the tube and then
- pushed into the mouth.

The plugged culture tubes
should: be placed in a hot-air oven _ Plugged Culture Tube

Io LABORATORY GUIDE IN BACTERIOLOGY

at a temperature of 150° C. for about 30 minutes, or
until the plugs are ‘slightly browned. The tubes are
not necessarily sterile, but the plugs have become set
so as to fit the mouth of the tube, and may be removed
and replaced readily.

SECTION 4

METHODS OF STERILIZATION.

Sterilization is the process of removing all living
organisms. This may be accomplished by heat, by

Fic. 3
Berkefeld Filter
a. Berkefeld filter d. Intercepting flask
b, Filtered liquid e. Connection with aspirator

c. Side tube with cotton filter f. Rubber hose

BACTERIOLOGICAL TECHNIC II

certain chemicals, or by filtration. Chemicals are
used chiefly for sterilizing the skin, surgical instru
ments, and cultures which have been accidentally
spilt. Filters for sterilization are made of some porous
material, either infusorial earth or unglazed porcelain.
Substances which may be injured by heat are sterilized
in this manner. Positive or
negative pressure is necessary
for this kind of sterilization
(Fig. 3).

Sterilization by dry heat.
—Sterilization by dry heat is
applicable to the steriliza-
tion of most glassware. This
method of sterilization is car-
ried out by means of hot-air “wJ
sterilizers (Figs. 4 and 5). eh ae
These hot-air sterilizers are a cE
boxes with double walls of sheet iron. The bottom
shelf should always be covered with a piece of asbes-
tos, to prevent heating the apparatus too rapidly.
The temperature is maintained at 160° or more for one
hour. The flame enters a hole provided at.the bottom
of the box. Care should be taken to avoid the possi-
bility of the flame becoming luminous, otherwise the
glassware will be covered with soot.

Culture media and all substances liable to be in-
jured by heat of 160° C. or over must be sterilized by
the application of moist heat. Experience has taught
that hot steam has greater germicidal powers than air
of the same temperature. Hot steam, therefore, is the
most common means of sterilizing culture media.

12 LABORATORY GUIDE IN BACTERIOLOGY

Steam is applied in two ways. The first method is
that of exposing media to steam of 100° C. for 20 min-
utes. This is done in an apparatus generally known as
the Arnold steam sterilizer. The usual form is illus-
trated in Figs. 6 and 7. Fig. 6 shows the appearance

Fic. §
Lautenschlager Hot-Air Sterilizer

of the ordinary form with the hood off. Fig. 7 shows
the inside arrangement. The two compartments, @
and 6, are connected by small holes, and a certain
amount of water has to be kept here. The water is
brought to a boil and the steam rises through a number
of holes in the bottom (c) into the chamber (d). The
steam condenses at the top and returns between two

BACTERIOLOGICAL TECHNIC 13

sheet-copper walls (e, ¢) to the large compartment
(b). Larger forms on the same principle are in use
(Fig. 8). +

The media -to be sterilized are placed in the large
chamber (d, Fig. 7). The water is then heated until
steam is generated, and the action of the steam on the
media is continued for 20 minutes from the time steam

mneeee =|

—=— =f,
; Sf
4 5 1B ~~...
Fic. 6 Fic. 7
Arnold Steam Sterilizer Arnold Steam Sterilizer
The hood is taken off and a. Inner water compartment
the door opened, showing 6. Outer water compartment
inside arrangement ¢. Perforated bottom

d. Sterilizing chamber

e. Sheet-copper walls
begins torise. This process is repeated on two succeed-
ing days, so that the media have been exposed to the
steam for three days. On the first day all vegetative
forms are killed. The media are then kept at room or
incubator temperature, so that spores which may be
present and are not killed by the first exposure may
develop into vegetative forms and be killed by the
second exposure. If after this any spores should

14 LABORATORY GUIDE IN BACTERIOLOGY

survive, they will develop in the next 24 hours, and the
third exposure to steam will complete sterilization.
Sterilization is accomplished in a shorter time by
the use of steam under pressure. The autoclave is the.
usual apparatus used for this purpose. Certain bac-
teria, some of which are widely distributed in nature,

Fic. 8
Arnold Sterilizer

have the faculty of forming spores. These spores are
highly resistant to heat and do not lose their vitality
either by boiling or by application of steam under
ordinary atmospheric pressure. By adding the pres-
sure of one atmosphere to ordinary pressure, the boiling
point is raised to121.4° C. 120° C. is sufficient to kill
all spores during an exposure of 5 minutes, if the media
are in tubes. Larger amounts of media require a
proportionately longer exposure.

The autoclave consists of a strong cylinder made of

-

BACTERIOLOGICAL TECHNIC 15

iron. Some forms have a cover, others a door on one
side. A basket, or a set of shelves, is on the inside.
A gauge indicates the pressure and temperature. A
safety valve opens automatically when the desired

pressure is reached. Two
forms are illustrated in
Figs. g and Io.

Before using the auto-
clave the inside should be
examined. It must be
clean and contain a suffi-
cient quantity of clean
water. Water containing
impurities is liable to foam
up when boiling, wet the
plugs, and ruin the media.
If the lid is on top, it
should be fastened care-
fully by tightening the
thumbscrews. In order
to distribute the pressure
of the lid uniformly: the
diametrically opposite
screws should be tight-
ened simultaneously. The
valve should be open and
left open until the steam
has escaped for about one
minute. Then the valve

Fic. 9
Autoclave

a. Steam valve a, d. Thumbscrews
b. Safety valve e. Bunsen burnee
c. Gauge © and opening

is closed and when the desired pressure has been
reached, the gas should be turned down so as to
maintain pressure for the requisite length of time.

ed

16 LABORATORY GUIDE IN BACTERIOLOGY

At the end of this period the gas is shut off and the
pressure allowed to decrease gradually. The valve
should not be opened, nor the lid removed, until
atmospheric pressure has been restored, otherwise the
sudden release of pressure would cause the media to
boil suddenly and push
the plugs out of place.

; When large autoclaves
are used (Fig. 10) pro-
vision must be made for
proper circulation of air,
or “air cushions” form
and media will not be
sterilized. If an aperture
remains in the outlet the
discharge of air is facili-
tated. Attachment of a
vacuum pump is often
desirable to remove all air.
Blood serum or egg
media are the most diffi-
cult to sterilize. The tem-
perature of coagulation of
these media is relatively
low, and sudden heating
causes the mass to break
up, form bubbles, and be-
come useless for cultural purposes. The Koch inspis-
sator may be used, or, with certain precautions, the
autoclave. The Koch inspissator (Fig. 11) allows the
tubes to rest in an inclined position and to be heated
gradually to 75° C. This temperature is maintained

Fic. 10

Autoclave

BACTERIOLOGICAL TECHNIC 17

for one hour. This process has to be repeated for five
or six successive days, before sterilization is complete.
If the autoclave is to be used, the tubes are placed
in the autoclave in an inclined position.

Good results are obtained by this method:

Koch Inspissator

1. Close the lid and the steam outlet.

2. Admit steam. After reaching 3 pounds pressure,
keep this pressure for 5 minutes.

3. Increase the pressure slowly to 5 pounds and
keep there for 5 minutes.

18 LABORATORY GUIDE IN BACTERIOLOGY

4. Increase to 10 pounds and hold for 5 minutes.

5. Increase to 15 pounds and hold for 5 minutes.

6. Open the steam outlet and keep at 15 pounds
pressure for 20 minutes.

It is advisable to sterilize the tubes again on the
following day by slowly bringing the pressure to 15
pounds with the steam outlet slightly open and keep-
ing at this pressure for 20 minutes.

SECTION 5

PREPARATION OF CULTURE MEDIA

EXERCISE I. PREPARATION OF DUNHAM’S PEPTON
SOLUTION AND OF PEPTON BROTH (BOUILLON)

1. Weigh the saucepan, measure into it 1,000 c.c. of
tap water, and heat over a flame or a water bath.

2. Dissolve in this, when hot, but not boiling, 10
grams Witte’s pepton.

3. When dissolved, replace the evaporated amount
of water and divide into two equal parts of 500 c.c.
each.

4. One-half is then filtered until perfectly clear,
tubed, and sterilized in the autoclave for 5 minutes at
120°C. This is Dunham’s Pepton Solution.

5. Dissolve 1.5 grams extract of beef in the remain-
ing 500 c.c.

6. Adjust the reaction with phenolphthalein paper
or by titration against n. NaOH.

NotEe.—The reaction of culture media is a matter of vital
importance. Bacteria, especially pathogenic bacteria, grow

preferably in a medium which is neutral or slightly acid to
phenolphthalein. The neutral point of litmus is about 2 per

BACTERIOLOGICAL TECHNIC 19

cent more alkaline than the neutral point of phenolphthalein,
so that a medium which is neutral to phenolphthalein is about
2 per cent alkaline to litmus. It has been found that about 1
per cent acid to phenolphthalein is the most favorable reaction
for the growth of pathogenic bacteria. A medium of this reac-
tion is still alkaline to litmus.

7. After neutralization fill into an Erlenmeyer flask
and autoclave for 10 minutes at 120° C.
8. Keep the sterilized broth for 24 hours and then
filter until clear and distribute into culture tubes,
which have to be autoclaved again.

Note.—The reason for exposing broth to a heat of 120°C.
twice is this: The solution contains substances which are pre-
cipitated by heat and appear as a sediment after cooling. As it
is important to have a perfectly clear broth in tubes, these sub-
stances are precipitated by the first heating, and, if tubed later,
the second sterilization will not affect the appearance of the
medium.

For ordinary purposes it is sufficient to neutralize media by
means of phenolphthalein paper. This is prepared by soaking
filter paper in a 1 per cent solution of phenolphthalein in 50 per
cent alcohol and then allowing the paper to become dry. A 2 per
cent or 4 per cent solution of sodium hydrate is added to the’
medium to be neutralized until a faint, but decided, pink appears
on phenolphthalein paper.

A more precise method is as follows: Measure by means of a
pipette 5 c.c. of the medium into a white porcelain evaporating
dish, add 45 c.c. of distilled water and 1 c.c. of a 1 per cent solu-
tion of phenolphathalein in 50 per cent alcohol. Heat the mix-
ture to boiling and slowly add from a graduated burette 1-2oth
normal NaOH until a faint but decided and stable pink appears
in the liquid. The amount of NaOH is read from the burette
and the amount for neutralization of the whole volume calculated.
It is desirable to make another titration after the NaOH has
been added. The amount to be added to the medium has to be
varied according to the reaction desired. If it is to be neutral,

20 LABORATORY GUIDE IN BACTERIOLOGY

the above proceeding will accomplish the object. If it is desired
to have a medium which has a reaction of 1 per cent acid, a pro-
portionate amount should be deducted from the total amount of
NaOH calculated. ;
Example.—By reading the burette we find that it takes 3 c.c.
1-20th normal NaOH to neutralize 5 c.c. of the medium. This
means that 60 c.c. 1-20th n.NaOH will neutraiize 100 c.c. medium,
or 600 c.c. 1-20th n.NaOH will neutralize 1,000 c.c. medium.
To find the requisite amount of n.NaOH divide the above figure
by 20. Then.3 c.c. n.NaOH will neutralize too c.c. and 30 c.c.
n.NaOH will neutralize 1,000 c.c. If the reaction is to be
I per cent acid, deduct 10 c.c. from 30=20 c.c. If 20 c.c.
normal NaOH are added to each liter the reaction should be 1 per
cent acid. This should be ascertained by a second titration.
A normal solution is the equivalent weight in grams (Gram-
Molecule) dissolved in distilled water and made up to 1,000 c.c.
In the case of monovalent chemicals the molecular weight is
taken, if bivalent the molecular weight is divided by two, etc.
All media should be prepared with the utmost care

and should be perfectly clear.

EXERCISE 2, PREPARATION OF NUTRIENT AGAR-AGAR

Agar-agar (or called simply “agar”) is a watery
extract of certain seaweeds found on the Pacific coast
of Asia. A solution of agar containing about 1.5 per
cent forms a firm jelly, which melts near the boiling
point of water, and on cooling solidifies at about 39°.
Gelatin solidifies at much lower temperature, and can-
not be kept solid at body temperature. The use of
agar is, therefore, of great importance in the study of
pathogenic bacteria, a large number of which prefer
body temperature for growth.

1. Weigh a saucepan, or, if available, a double
boiler. Note the weight. ;

2. Measure 1,000 c.c. of tap water into the sauce-

.BACTERIOLOGICAL TECHNIC ar

pan. It is advisable to add about 200 c.c. of water to
this to allow for evaporation.

3. Cut and shred 15 grams of agar, add this to the
water, bring to the boiling point, and keep at this
temperature until the agar is completely dissolved.
Violent boiling should be avoided and the mixture
should be stirred, so as to prevent overheating.

.Note.—The agar may be soaked in cold water over night.
This removes some of the impurities and renders the agar more
readily soluble.

4. Add 3 grams extract of beef and 10 grams Witte’s
pepton.

5. Adjust the reaction.

6. Adjust the weight. Place 1,000 grams weight
and the weight of the saucepan on one side of the scales
and then add’enough water to make the saucepan with
the agar balance. If the weight is too high, it should
be boiled gently until the weight has been brought
down to the proper amount.

7. Make a paper filter as described below and
arrange a retort stand as illustrated in Fig. 12.

8. When at the boiling point filter the agar and dis-
tribute into culture tubes.

9. The tubed agar should be sterilized in the auto-
clave for 5 minutes.

For agar slants each tube should contain about 7 c.c.
or be filled one-third of the length of the tube. For
plating, the tube should be half filled and contain ro
c.c. Slants are prepared by allowing the agar after
sterilization to cool in a slanting position. If it is de-
sired to slant a large number a whole basket may be
put in a slanting position. When a few tubes only

22 LABORATORY GUIDE IN BACTERIOLOGY

are required they may rest with the plugged end on a
glass rod or rubber hose until the agar has solidified.
For the purpose of filtering media heavy filter -
paper (Schleicher and Schiill No. 598) of the best
quality only should be used. This is especially im-
portant when filtering agar or gelatin. After the filter

Fic. 12

Apparatus for Filtering
Media

a. Filter
b. Large funnel

‘ ¢. Small funnel
d. Rubber hose
e. Pinchcock
J. Pipette
g. Culture tube

has been folded and inserted into the funnel hot water
should be run through the filter, until it is soaked
and warm.

Method of folding filters (Fig. 13).—a and 6) Take
a square piece of filter paper twice as wide as the depth
of the funnel and fold to half the size so as to make
No. r cover No. 2. (Compare with Fig. 13.)

BACTERIOLOGICAL TECHNIC 23

c) Fold this to make 1 cover 2 and 3 cover 4 (result
Fig. 13c). It consists of four layers and forms a square.

d) Fold the upper part, consisting of two layers,
from 1 to 2 (Fig. 13d). The shaded triangle, 2-3-4,
now has six layers; the other, 1-3-4, two layers.

AL 2
@
4 é “3 ae 3
g a i 3 2 °. 2
Z
fp |
ra +4 76 4
/ 4 5
a

2
Zz
‘és he
Fic. 13

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

e) Fold the upper double layer so as to make 2
cover a point in the diagonal at 5, taking care to make
a sharp point at 4 (result Fig. 13¢). The shaded part
is now eight layers deep.

f) Turn the folded part face down and repeat

24 LABORATORY GUIDE IN BACTERIOLOGY

operations exactly on the other side as in d and e (re-
sult Fig. 13).

g) Take up and open the large middle fold (result

Fig. 13g). The two halves must now be symmetrical.
__h) Fold so as to make the lines 1-3 and 1-4 meet at
the center line 1-2 (result Fig. 13%).

4) Now pick up and fold backward so as to have
1 cover 2 in the back (Fig. 137).

j) 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 g sharp edges.

This filter is inserted evenly into the funnel, spread-
ing the folds at a distance from each other as nearly
alike as possible. Care should be taken to make the
folds and the point sharp, as this insures rapid filtration.
and prevents the filter from tearing.

If a vacuum pump is available, the medium may be
filtered rapidly by the use of suitable apparatus, as
illustrated in Fig. 14. At the connection with the
vacuum pump a valve should be inserted or a flask
arranged as in the illustration, to prevent the water
from entering the flask if the water pressure should be
reduced suddenly.

If some precautions are properly observed, chiefly
the making of a good filter with sharp edges and a
sharp point, and the soaking of this in hot water, there
is no difficulty in filtering agar or gelatin successfully
in a short time. There is some danger of the point
of the filter breaking when the hot medium is poured
on, This may be avoided by folding a second filter
of about two inches diameter and fitting this small

filter on the bottom and outside of the larger one.

BACTERIOLOGICAL TECHNIC 25

_ The basket which is to receive the tubes after filling
should be placed in an inclined position, as this facili-
tates the proper arrangement of the tubes. In filling
the tubes the pipette at the end of the rubber hose

Fic. 14
Filtering Media by Means of Vacuum Pump
a. Liquid medium e. Reflux flask
6. Absorbent cotton f. Rubber stopper with two holes
c.- Rubber stopper g. Connection with aspirator

d. Filtered medium

should be inserted to a depth of at least two inches
and when the proper amount has been discharged
should be removed carefully so as to avoid wetting
the mouth of the tube. A wet tube-mouth will cause

26 LABORATORY GUIDE IN BACTERIOLOGY

the cotton plug to stick to the glass, and later not only
occasion much annoyance to the person using the tube
but expose the medium in it to danger of contamina-
tion.

EXERCISE 3. PREPARATION OF DEXTROSE AGAR

Dextrose is added to agar for the demonstration of
gas-forming organisms. Dextrose is decomposed by
these bacteria with gas formation, the gas appearing
as bubbles in the medium.

Dextrose agar is prepared by adding a definite
amount of dextrose, usually 1 per cent, to filtered agar.

Note.—Dextrose agar cannot be -distinguished from plain
agar by appearance. It is therefore necessary either to label
the tubes or to separate dextrose agar tubes from plain agar
tubes in a basket by tying a piece of string or inserting a piece of
paper between.

EXERCISE 4. PREPARATION OF PEPTON GELATIN

x. Weigh the saucepan and measure 1,000 c.c. of
tap water into it. To this 200 to 300 c.c. of water
should be added to allow for evaporation.

2. Dissolve 3 grams extract of meat and 10 grams
pepton. 4

3. When boiling dissolve 10 per cent gelatin in
cold weather and 12 per cent in hot weather. The
gelatin must be of the best quality (gold label) and
should be dissolved slowly, taking a few leaves at a
time, and with constant stirring.

4. When completely dissolved, adjust the reaction
as directed in the preparation of agar. Gelatin con-
tains an appreciable amount of acid and it will require
more NaOH solution for neutralization than agar.

BACTERIOLOGICAL TECHNIC 27

5. Cool to about 60°C. Dissolve the whites of
two or three eggs, or about 10 grams of pure powdered
egg albumin in about 100 c.c. of tap water. Mix this,
solution with the gelatin and heat slowly to the boiling
point, placing a piece of asbestos under the pan unless a
double boiler is used. Boil gently until the egg white
or egg albumin has coagulated and a solid film has
formed which mechanically incloses the impurities.

6. Adjust the weight to 1,000 grams, allowing for
the weight of the pan. Filter and tube, as in the
preparation of agar.

7. Sterilize in the autoclave for 5-8 minutes at
120° C., or in the arnold for three successive days.

If sterilized in the autoclave, care should be taken
not to allow the temperature to go beyond 120°C.,
and gelatin should not be permitted to remain at this
temperature beyond the prescribed time. Gelatin is
readily decomposed by heat and then does not solidify
after cooling.

EXERCISE 5. PREPARATION OF LITMUS MILK

Milk is one of the most important culture media.
Only the cleanest milk obtainable should be used.
“Certified milk” is most suitable. In many cases com-
mercial milk powder may be used. If certified skim-
med milk or fat-free milk is available step 1 is omitted.

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

2. Add a sufficient quantity of tincture of litmus to
impart a decided blue color to the milk. If a solution
of Merck’s pure extract of litmus 1:100 is at hand
about 5 per cent of this will be sufficient.

3. Distribute in culture tubes and sterilize in the

28 LABORATORY GUIDE IN BACTERIOLOGY

autoclave for 5 minutes at 120°C. After sterilization
the blue is usually more or less lost, but returns upon
standing.

EXERCISE 6. PREPARATION OF POTATO

1. Select several large potatoes, and cleanse by
brushing the dirt off, cutting out the eyes and other
blemishes, and washing in water.

Fic. 15
Potato Cylinder Ordinary Style of Potato Tube Potato Tube
(showing diagonal for a. Potato b. Cotton
cutting)

2. Punch out cylinders with a borer of suitable size,
trim them, and cut each cylinder into two equal parts
on a diagonal line (Fig. 15). 2

BACTERIOLOGICAL TECHNIC 29

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

4. Trim the pieces of potato. so they slide down
the tube and insert one half-cylinder into each potato
tube. The wide end rests on the constriction of the
tube. Pour a small amount of water into it, and
fill about one-half of the part of the tube below the
constriction. Large culture tubes without constric-
tions may be used. A small amount of cotton should
be pushed to the bottom of these and the cotton soaked
with water. The potato then rests on the cotton
(Figs. 16, 17).

5. Sterilize in the autoclave for 8 to 10 minutes
at 120° C., or in the arnold for three consecutive days.

Note.—Potatoes usually harbor a spore-bearing bacillus, the
spores of which are highly resistant. Therefore a longer expo-
sure in the autoclave is necessary to insure sterilization.

SUBSTITUTE FOR POTATO

1. Dissolve 15 grams agar in 600 c.c. water and
filter.

2. Dissolve the following salts in 200 c.c. water:

Asparagin..... Rasinesaced Seldovia bea axsneriee SIe 5 grams
KAP O phew tact caasteeaxenee a esate weno 2 grams
Nag EERO jw eess Sane iatels oe tee cates eonedas 2 grams
MeSOjs ccstiee aS 4a8 tapes gach meats ates nl a 2 grams
Cah in sic cave weenie ry os Aa seg papeedncde tesa 2 grams
Ammonium lactate................0005 2 grams

3. Add the solution of salts to the hot agar.

4. Dissolve 10 grams pepton.

5. Adjust the reaction to the neutral point with
phenolphthalein.

6. Suspend 30 grams washed starch in water.

7. Add the starch suspension slowly with constant
stirring to the previous mixture.

30 LABORATORY GUIDE IN BACTERIOLOGY

The weight of the mixture should be 1,000 grams.
Tube hot, without filtration, through a pipette with a
wide aperture. Autoclave for five minutes at 120° C.,
and allow to cool in a slanting position.

EXERCISE 7. STANDARD METHOD OF PREPARING BROTH,
NUTRIENT GELATIN, AND NUTRIENT AGAR
STANDARD METHODS FOR THE EXAMINATION OF WATER AND

SEWAGE. AMERICAN PUBLIC HEALTH ASSOCIATION,
755 BOYLSTON STREET, BOSTON, MASS.

Broth Gelatin Agar

1. Boil ro or 15 g. thread agar
in 500 c.c. water for half an
hour and make up weight
to 500 g., or digest for 10
minal es in the autoclave.
Let this cool to about 60°.

Infuse s00 g. lean meat

2. Infuse soo g. lean meat 24 2
hours with 1,000 c.c. of dis- Ditto. energies es fae ot
tilled water in refrigerator. ate ger
3- Make up any loss by evap- . :
oration. Ditto. Ditto.
4. Strain infusion through A :
cotton flannel. Ditto. Ditto.
5. Weigh filtered infusion. Ditto. Ditto.
ioe Ditto. ‘a
6. Add 1 % Witte’s pepton. italic Add 2 % Witte’s pepton.
atin.

Warni on water
bath until pep-
ton and gela-
tin are dis-
solved, not
above 60°.

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

Warm on water bath until
pepton is dissolved, not
above 60°.

8. To 500 g. of meat infusion
add s00 c.c. of the 3%
agar, keeping the tem-
perature below 60°.

g. Titrate after boiling one minute to expel carbon dioxid.
to. Adjust reaction to 1% acid by adding normal hydrochloric acid or sodium
L diped as required.
Il. t over boiling water (or steam) bath for 40 minutes.
12. Restore loss by evaporation.

BACTERIOLOGICAL TECHNIC 31

EXERCISE 8. PREPARATION OF BROTH FROM
FRESH MEAT

The following method has ‘some advantages over
the previous method and is specially adapted to the
preparation of diphtheria toxin:

1. Clean one pound beef or veal of adhering fat,
etc., and grind in a meat Chopper.

2. Cover with one liter of water and digest over
night at room temperature.

3. Heat to 60° C. and digest at this temperature for
two hours.

4. Boil for 30 minutes.

5. Press the liquid from the meat in a meat press
(Fig. 18). Mix the meat with
some more water, press out
again, and bring the volume of
the two combined liquids to 1
liter.

6. Dissolve in this 20 grams
pepton and 5 grams -sodium
chlorid.

7. Adjust the reaction to
1.2 per cent acid with phenol-
phthalein as indicator.

8. Filter until perfectly clear
and sterilize in the autoclave.

Fic. 1g
Meat Press

EXERCISE 9. PREPARATION OF BLOOD SERUM

1. Fresh ox blood, horse blood, or dog’s blood,
collected in sterile containers, is set aside in an ice
chest until the serum has separated.

32 LABORATORY GUIDE IN BACTERIOLOGY

2. Three parts of serum are mixed with 1 part
broth containing 1.5 per cent dextrose.

3. The mixture is filtered and tubed.

4. Place in a Koch inspissator (Fig. 11).

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

6. Heat slowly to the boiling point and boil for 5
minutes. This process is to be repeated on three or
four successive days.

NotE.—25 c.c. water should be placed in the inspissator with
the tubes, so as to keep the air in the inspissator saturated.
Blood serum may be sterilized in a shorter time by the use of
the autoclave (see p. 16).

EXERCISE I0. PREPARATION OF BEERWORT MEDIA

Hopped beerwort may be obtained from a brewery.
Beerwort media are used chiefly for the cultivation of
yeasts and molds, these preferring media of the acidity
of beerwort. Beerwort should be autoclaved, cooled,
and filtered before tubing, otherwise a precipitate will
form in the tubes.

Liquid beerwort.—Place about 7 c.c. of beerwort in
culture tubes and sterilize at 120° C. for five minutes.

Beerwort gelatin——Dissolve 10 to 12 per cent gold
label gelatin in beerwort and sterilize at 120° C. in the
autoclave.

Beerwort agar.—Dissolve 1.5 per cent agar in beer-
wort and sterilize at 120° C. in the autoclave.

EXERCISE II. PREPARATION OF YEAST-WATER MEDIA

Yeast water.—One liter of washed yeast or one
pound of pressed yeast is boiled in two liters of water
for one hour. The reaction is made neutral to phenol-

BACTERIOLOGICAL TECHNIC 33

phthalein, the solution is then filtered and sterilized
in the arnold for three successive days.

Dextrose yeast water.—Dissolve 10 to 15 per cent
dextrose in yeast water without adjusting the reaction.

EXERCISE I2. PREPARATION OF MEDIA FOR THE DIF-
FERENTIATION OF B. coli AND B. typhosus
MacConkey’s bile-salt agar—

Nutrient agar... ... cece cee eee 100 C.C.
Sodium taurocholate......... Rpcood is 0.5 per cent
Pepton.......... da foasnbessasaerin sl a a.5 Suess 2 per cent

Boil and filter, add 2 per cent lactose, tube and steril-
ize in the arnold.

MacConkey’s bile-salt broth—

Sodium taurocholate.............. ©.5 per cent
PE PbO nei satiesctay docs eeaeanede ote essimteacanss 2 per cent
DP OXUPOSE I alas soe ween Aa eek A ened 0.5 per cent

Dissolve in beef broth by boiling, filter, and add
litmus solution. Sterilize in the arnold.

Aesculin bile-salt agar—

7.) a I5 grams
Commercial bile-salt..........4-. 2.5 grams
Pepton (Witte).......--.seeeeeee ro). grams
Distilled water. ......-..eee eee 1,000 C.C.

Boil until dissolved and neutralize with n.NaOH.
Cool to 60° C., add the whites of two eggs or a solu-
tion of egg albumin, bring to boiling point, and filter
when the albumin has coagulated. Neutralize again,
if necessary, and add to the hot filtrate 1 gram aesculin
and 1 gram iron citrate in scales. The final reaction
should be 0.6 per cent acid.

34 LABORATORY GUIDE IN BACTERIOLOGY

Drigalski and Conradi’s medium (modified by

Harris) —
Dextrose-free broth............... 2,000 C.C.
INUtPOSE 055: 3.6 ic 4 aches bo aes 20 grams
PAT ors ids ecards biaeee Asie a oes 40 grams

Boil, dissolve, neutralize to phenolphthalein, auto-
clave at 120° C. for 5 minutes. Clarify with whites of
four eggs (or powdered egg albumin), and filter. Then
add after dissolving separately in water:

EA CCOSE sioctec sce sesetideaclly seattin-d gee sieve Seaed 30 grams
Litmus solution (sée p. 35).....0+0005 260 C.c.
Crystal violet (0.1 per cent solution) 20 c.c.

Tube and sterilize in the arnold.
Parietti’s solution—

Car bolo acid), nics oc devaivonssistone ataeaevins 5 CC.
Hydrochloric acid. ..............000000- 4C.C
Wate Pe oc ecictcece a Si tieomtarglensuretees e pawmeese 100 €.Cc

Phenol media.—One part carbolic acid (phenol) is
added to 1,000 parts medium.

Hiss’s plating medium for colon-typhoid differ-
entiation—

Agar hus tain a acne Suan ae oe mat I5 grams
Gelatin 2.5 crises Caksik Gussie aeewn I§5 grams
Extract of meat........... Miele eden s 5 grams
INGO, ict artetadne 5 grates wevayesnltnotesn eis amc seasons 5 grams
DEXtTOSE 0s cb ce areaeaneneendeas waods Io grams
Distilled water..............00000e 1,000 C.c.

CAR ieee Soe daistetanicldanealeleunnceys 5 grams
eB AI ab see deen dade) dsninssantgetetaleasbie eo 80 grams
Extract of meat..............000 0 5 grams
NGC: aisle Picn daria aheetuieera wee e8 5 grams
DeXULOSE 46.4 sssesyraia sa oeiagannics bes Io grams

Distilled water..........ceceeeeees 1,000 C.C.

BACTERIOLOGICAL TECHNIC 35

Hesse’s medium—

AGaby cicadas sc aaa easeniee 5 grams
Peptomiers ie vig taniiek vor a0as ahaa 10 grams
Extract of meat............c0ceees 5 grams
NGG) siie, acastecs asitevscas tne vee Gases 8.5 grams
Distilled water..............0.000% I,000 €.Cc

POptoniseeea cc rtnadenacdenn iaae hice 20 grams
Gelatb ie ices wndiccenesivad ¢aa04 bid To grams
BALE sos chile sore Rachel Netstecn seas ete eee 20 grams
Dextrose or mannit............... Io grams
NaCl eis toreseadoa ns teen eae 5 grams
ME Che iamse sea cheats ee eee AS 5 grams
Distilled water..............000eee I,000 C.c.

Malachite green media.—Two to 3 c.c. of a 2 per
cent solution of ‘“Héchst 120” malachite green solu-
tion is added to broth (alkaline to litmus).

Ox-bile medium.—This culture medium is used for
the detection of B. coli in water. Dissolve 1 per cent
lactose and 1 per cent pepton in fresh ox bile, filter and .
fill in fermentation tubes and sterilize in the arnold for
three successive days.

EXERCISE 13. PREPARATION OF MEDIA FOR WATER
AND MILK EXAMINATION

Litmus solution.—Dissolve 1 part Merck’s pure
extract of litmus in too parts of water, filter, and
sterilize in the autoclave for 5 minutes at 120° C.

Litmus lactose agar and litmus dextrose agar.—
Dissolve 1 per cent lactose or dextrose in sugar-free
agar. The litmus should be added in the petri dish
before use from a sterile tube of litmus solution by
means of a sterile 1 c.c. pipette.

36 LABORATORY GUIDE IN BACTERIOLOGY

Litmus lactose gelatin and litmus dextrose gela-
tin.—Dissolve 1 per cent lactose or dextrose in sugar-
free gelatin, containing 13 per cent gelatin. The tubes
should contain 8 c.c. of the medium. After adding
1 c.c. litmus solution and 1 c.c. of the material to be
plated the medium will contain 10 per cent gelatin
and remain solid.

Mannit agar.—Dissolve 1 per cent mannit in sugar-
free agar. ;

EXERCISE 14. PREPARATION OF WHEY MEDIA

Litmus whey (Petruschky, 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 added until a
decided blue color is obtained. Sterilize in the auto-
clave.

Whey gelatin.—Add 10 per cent gelatin to clarified
whey.

Whey agar.—Add a few drops acetic acid to boiling
milk until the casein is precipitated. Neutralize, or
bring to a reaction of 1 per cent acid if desired, and
dissolve 1 per cent pepton, 2 per cent dextrose, and 1.5
per cent agar. Filter, tube, and sterilize in the
autoclave.

EXERCISE I§. PREPARATION OF GLYCERIN MEDIA

Glycerin broth.—Add 6 per cent pure glycerin to
ordinary broth.

Glycerin agar.—Add 6 per cent pure glycerin to
nutrient agar.

BACTERIOLOGICAL TECHNIC 37

Glycerin egg medium.—Add 6 per cent glycerin to
the egg mixture before heating.

Glycerinated potato.—Prepare potatoes in the usual
manner and soak for 24 hours in a 25 per cent glycerin
solution in distilled water.

Substitute for glycerinated potato.—Mix 6 per cent
glycerin with the substitute for potato described on p.
20.

EXERCISE 16, PREPARATION OF EGG MEDIA

Dorset’s egg medium.—Eggs are broken into a
flask and the yolks broken with a platinum needle or
glass rod. The flask is gently shaken until the yolks
and whites are thoroughly mixed. Foam formation
should be avoided. Distribute in culture tubes and
sterilize in a Koch inspissator or autoclave in the same
manner as blood serum.

Dorset’s egg-yolk medium.—Add 5 to 10 c.c. of
sterile distilled water to the yolks of three or four eggs,
and treat the same as in the above egg medium.

Capaldi’s egg medium.—A few loopfuls of egg yolk
are added to a tube of liquefied agar, previously cooled
to 45 to 47°C.

EXERCISE 17. MEDIA FOR THE STUDY OF SOIL
BACTERIA

Winogradsky’s solution—

IMSS Osi aos enciigseel ick os 20.58 GRIM 0.50 gram
CB Clb. os reyiidncsbingale Ret tee taicencada 0.OI gram
INA Chae ccnorottacacesnuih tes eae Ateneecanaa 2.00 grams

Distilled water...............55 1,000 cc.

38 LABORATORY GUIDE IN BACTERIOLOGY

Solution for nitrite formation—

PeSO jai cinceescoos dagruenians oes 0.4 gram
MESOgi cies tethers esau 0.5 gram
KG PO gj. cise tap evens a settle I gram
Na@h. noua re tiesadd soe boieneas 2 grams
(NH) SO} eccaeies4tacnsawces I gram
Distilled Waterss ee ..j5 5 sesieacueeseseis I,ooo CC.

Dissolve 20 grams agar in the above solution.
After solution add 1o grams precipitated CaCO.
Shake and tube. Sterilize in the autoclave.

Preparation of silica jelly—Mix 100 c.c. HCl,
specific gravity 1.10 Beaumé’s scale at 60° F., with
too c.c. sodium silicate, specific gravity 1.09 Beaumé’s
scale at 60° F. Place in collodion sacs and dialyze in
running water for 12 hours. Prepare the following
solution:

(NF) SO pecs dcaietataescee need 0.40 gram
MgSO geiens esece vcoeeteitteemgsih ae acess 0.05 gram
KAA PO jas snes stronger ae cess 0.10 gram
Na, COjne a. o¢ssaeesiaaaey cess vane 0.90 gram
CaCbvauwcneccminkawand ee siee 0.01 gram

Dissolve these salts in the smallest amount of dis-
tilled water possible. Heat both the salt solution and
the silica jelly to boiling, cool rapidly without stirring.
Mix and pour on petri dishes or tubes and sterilize in
the autoclave at 110°C. The tubes should be placed
in a slanting position.

Synthetic agar for plating—

Dextrose: siscnuia anand dis eveecee Io =. grams
MgSO ge iswina vaipaacs a irises eat 0.2 gram
KH PO 4s ogee aan se eee nals 0.5 gram
Peptons: <2 ciniuagaes si sxeass v.05 gram
Agar sic gece aavoreet ney Rae i4 20 grams
Distilled water................. I,000—SsGc.

Dissolve, filter, tube, and sterilize in the autoclave.

BACTERIOLOGICAL TECHNIC 39

Solution for testing the formation of nitrates—

MgSO ysis ic tios duaiseededea te 0.3 gram
FeSO js isis eeedtawlen tice bowed eee 0.4 gram
INA CT issn ccc ears ae itelnens 0.5 gram
K,HPOQ,........ Ne aenechneseidneste 0.5 gram
Fused NazCO;.............00008 I gram
NANOS. gcespaineutce naeimeata tani I gram
Distilled water.............0.005 1,000 C.c,

Dissolve 20 grams agar in the above solution.
Sterilize in the autoclave. The salt solution may also
be added to the silica jelly. For plating fill in culture
tubes.

Solution for testing the assimilation of atmospheric
nitrogen—

1. Solution

REPO pices teiaiakhieiniac eee’ I gram
MgSO js seeecsisers's ceteetonann aes 0.50 gram
Na@hy cg: iae dentetaekiene ies Sas 0.01 gram
FeSO gies scat vive seh aniteiees eas 0.01 gram
MGSO ji cahe ciiee oe eeu ne oad 0.01 gram
DOXthOS@ 5) 250.5.0, 2.0586 gavage 20 grams
Distilled water................. I,000sC.C.

Dissolve 20 grams agar in this solution if intended
for plating and tubing. Sterilize in the autoclave.

2. Mannit Solution

KAP O}s cares slinc-« eaatinmale cast 0.20 gram
CaCl: satnau.ovs secndaeaecoss 0.02 gram
MESO ywiccuies e445 ceed ese 0.20 gram
POC Gis siciaa cents eve wad Restrnt’ 24 0.01 gram
Mannites ssscicnc notkasaraeee en. 15 grams
NaOH solution until neutral to phenolphthalein

Distilled water................- Ijooo CC.

Dissolve 20 grams agar in this solution if intended
for plating and tubing. Sterilize in the autoclave.

40 LABORATORY GUIDE IN BACTERIOLOGY

Solution for testing the denitrification of nitrates
to free nitrogen (Giltay solution)—

Solution 1
DEXtOSEs 83 fa dakanaciudweucniades tenes Io grams
Distilled water................0 00008 250 C.C.
Solution 2
KNO senses seen sev eres Z.00 gram
MeSO ysis se ate nacan eee aioe es 2,00 grams
Cit CAC ye. iacs Wecna casednacaeets 0 5.00 grams
HG PO peisave:aschace sue odanlaneransconaae oes 2.00 grams
CA CIES secs oiadavda wiarauntacaMianconpunccas ie 3 0.2 gram
Fes Cloise svaiic canst ances 0.01 gram
Distilled water.............-..04. 250 Cc.

Make solution 1 neutral to phenolphthalein with
ro per cent NaOH or KOH solution. Mix solutions
r and 2 and make up to 1,000 c.c._ Dissolve 20 grams
agar in the solution, tube, and sterilize in the autoclave.

Synthetic agar for quantitative examination of soil—

DOXEP OS sesso a isteactetniesresp acces 10 grams
MgSO giceecsscr i rapeerwcngeaaienss 0.2 gram
KG PO gs oesednctied a eareagnaees 0.5 gram
Peptomie vxie's pag earaetinornnas.su's 0.05 gram
Agar ss cite oi Siva whi s css 20 #8©grams
Distilled water................ 1,000 Cc.

Pepton solution for ammonification—
Distilled water.................0.. 1,000 C.C,
PeptOniss: seeccsise nie eases ey hs ro grams
Dissolve and sterilize in the autoclave.

EXERCISE 18. PREPARATION OF MISCELLANEOUS
MEDIA

Blood agar.—Fresh blood obtained under aseptic
precautions is smeared over the surface of agar, or

BACTERIOLOGICAL TECHNIC 41

blood may be mixed with agar, previously Hquefied
and cooled to 50°, in various proportions. The agwr
should contain 2 to 3 per cent agar according to the
amount of blood to be added.

Bread-paste medium.-—Bread is cut into slices,
dried in an oven, pulverized, and distributed in 100 c.c.
flasks until the layer on the bottom of the flask is about
half an inch thick. Water is added gradually until
the surface of the bread is moist. Sterilize in the
arnold. ~-

Hay infusion.—1o grams of chopped hay are
macerated in 1,000 c.c. water in the water bath for
three hours. Filter and sterilize in the autoclave.

Wine must.—Wine must is diluted with four times
its volume of water. Dissolve o.5 per cent ammonium
tartrate, macerate in the water bath for 1 hour, filter
and sterilize in the arnold for three successive days.

Acid broth—Add 0.5 per cent acetic acid to
ordinary broth.

Calcium carbonate broth.—A few lumps of marble
added to ordinary broth.
NITRATE MEDIA

Nitrate broth.—Add 5 parts potassium nitrate to
each liter of ordinary broth.

Nitrate solution.—5 c.c. of a 2 per cent aqueous
potassium nitrate solution are added to a solution of
I gram pepton and 1 gram dextrose in 1,000 c.c. water.

42 LABORATORY GUIDE IN BACTERIOLOGY

EXERCISE 19. PREPARATION OF NON-PROTEIN MEDIA
(SYNTHETIC MEDIA)

Jordan’s non-protein medium—

Redistilled water...........-..0005 1,000 C.C.

Asparagin's scsi vaed sceeen aaesGaee 2 grams
MeSQ4 se darn taen se oer nice g martes I gram
K.HPO,..... er hid BMRA SHG lloras I gram

Dissolve and sterilize in the autoclave.
Uschinsky’s medium (Frinkel’s modification)—

Water ine sa-untssinnd ovine sw iuine eves 1,000 C.c.

ASPArapiny, scones oe Cees aea oe 4 grams
Ammonium lactate..............4- 6 grams
NapHPO,: <vcawieiansyeieesnd 2 grams
NaC bites ani he ae iateet vate cutee 5 grams

Dissolve and sterilize in the autoclave.

Raulin’s solution for the cultivation of molds—

Distilled water.............04. 1,500 c.c,
Cane sugar... . cc cece eee ees 7Oo grams
Tartaric acid... ...........005- 4 ‘grams
Ammonium nitrate............. 4 = grams
Potassium carbonate........... 0.6 gram
Ammonium phosphate.......... 0.6 gram
Magnesium carbonate.......... 0.4 gram
Ammonium sulphate........... 0.25 gram
Zinc sulphate...........0-0. eee 0.07 gram
Tron sulphate...........-..005. 0.07 gram
Potassium silicate.............. 0.07 gram

Notr.—Culture media should be stored in a dark, cool place.
If they are to be kept for a considerable length of time, the tubes
should be sealed with paraffin or with rubber caps. They should
be protected from dampness, as mold fungi are apt to alight on
the cotton stoppers and send their filaments into the tubes.
Before inoculation each tube should be examined, and those
which are cloudy or contain colonies of bacteria or molds should

BACTERIOLOGICAL TECHNIC 43

be rejected. Ifthe medium has shrunk from the effects of evapo-
ration it should be rejected, or the evaporated water may be re-
placed and the medium sterilized again.

SECTION 6
PREPARATION OF STAINING SOLUTIONS

Saturated alcoholic solutions of stains are prepared
by covering the anilin dye with absolute or 96 per
cent alcohol and allowing it to stand with frequent
agitation until no more stain is dissolved. Other
solutions for the preparation of stains are:

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

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

3. Anilin water, prepared by shaking 2 c.c. anilin
oil with roo c.c. water and filtering through filter
paper until clear.

The following four stains are most commonly in
use for morphological studies of bacteria:

1. Léffler’s methylene blue—

Saturated alcoholic solution of methylene blue......... 30 C.C.
Potassium hydrate in distilled water 1:10,000......... 70 C.C.

2. Ziehl-Neelsen’s carbol-fuchsin—

Saturated alcoholic solution of fuchsin................ Io C.c,

5 per cent solution carbolic acid in distilled water....... go c.c.
3. Ehrlich’s anilin gentian violet—

Saturated alcoholic solution of gentian violet.......... 25 c.c.

Anilin water (2 per Cent).......ccecescesccesceeeeecs 75 C.c.
4. Gram’s iodin solution—

TOUS oe soica ce aeenpeaere babe Ga Malelutiel od aha Sanam laeeadets I gram

Potassium iodids ssiepcrde ie oy Cais wires dt oie 2 grams

Dissolve in a few cubic centimeters of distilled

44 LABORATORY GUIDE IN BACTERIOLOGY

water. After solution add enough distilled water to
bring the volume to 300 c.c.
Other useful stains are the following:

Delafield’s hematoxylin—

Hematoxylin crystals.............. 4 grams
PICOION 0 csecinis ssasedeacdsannttaie. dares sian ove 25 Cc.
Ammonia alum............0-0000 50 grams
WA LED cscs. ntatetatiauu nas teeaueees 400 CAC.
GY COLIIN scsi se se tuentscsoeaynaacanrns Boe 100) C.C.
Methyl alcohol «05.54 saveeaaearee eo 100) CC.
Alum hematoxylin—
a. Hematoxylin...............0 00s 2 grams
Absolute alcohol................ 100 C.C.
2. Ammonia alum...............-. 2 grams
Water... csduanenigheecs seeks Too) C.C,
Mix 1 and 2 and add—
Glycerin. .......... dnusulshGuts at pass 850 cc,
Glacial acetic acid..............:.. 100 C.C.

Allow to stand for one month before using.

Bismarck brown—

Bismarck brown...............005 0.5 gram

WAtED: wasn c/w grsolaiuneuidane et oases I0o C.C.
Safranin—

Daltaning hes ayatimleamin ara sanu 0.5 gram

Watering bx eapaiengceg aveystetacs too cc.
Carbolic thionin blue (Nicollé)—

‘Thionin blues o.iesseavs evaceere ns I gram

Carbolic acid.............06 Dieses 2.5 grams

Water. acxunceneine any oper ia4 I0o C.C.

PAT UITL Snax avaremiana Bate a aerate se 2.5 grams
CarmMin ic seca vee eae aa KA I.0 gram
Woaltercsennewomiuesiees neees ey oe I0o) (C.C.

BACTERIOLOGICAL TECHNIC 45

Kiihne’s methylene blue—

Methylene blue................... I.5 grams
Absolute alcohol...............005 Io Cc.
Carbolic acid solution (5 per cent) .. 100 c.c.

Carbolic gentian violet (Nicollé)

Gentian violet (sat. alcoh. sol.)..... Io C.C.
Carbolic acid. .............0c ee eee I gram
WALER iss: cisternae penavige ee nee go C.c.

Pappenheim’s Stain—

Sat. aqueous solution of methyl green 3-4 parts
Sat. aqueous solution of pyronin..... I-1.5 parts

Mix previous to staining. Apply for 20 seconds.
The mixture is dependable for a few weeks only.

SECTION 7

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 75 oil
immersion, Zeiss No. A or No. AA, No. D or No. DD,
and 2 mm. oil immersion) and two oculars (Nos. 2 and
4) are indispensable. For the intelligent manipula-
tion of the microscope it is useful 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 Iis Revelations.

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

°

46 | LABORATORY GUIDE IN BACTERIOLOGY

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
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 concen-
trates the light on the object and is raised for high
powers or lowered for low powers. At the lower end
of this condenser is the iris diaphragm, which is regu-
lated 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 one.

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

1. Keep the instrument 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-

tl

Fic. 19

Microscope (after E. Leitz)
a. Ocular : Fe i. Iris diaphragm and condenser
b. Place where virtual picture isformed k. Objective
¢. Body tube. l. Nose-piece |
d, Coarse adjustment m-o. Picture as it appears to the eye
e. Micrometer screw ’-a. Object (cover slip)
J. Inclination joint yr. Adjustment spring

g. Horseshoe base s. Stage
k. Mirror t, Object (slide)

48 LABORATORY GUIDE IN BACTERIOLOGY

adjustment pillar. The fine adjustment consists of
a delicate screw-thread, which is easily damaged.

3. The lenses, condenser, and mirror, when necessary,
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 delicate nature and easily injured. 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
adjustment afterward. ‘The free use of the fine adjust-
ment saves the accommodation of the eye. As the
eye is capable 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. Raise the draw tube by means of the coarse
adjustment before changing the objectives or examin-
ing a different preparation.

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

to. When focusing an object, lower the draw tube
until the lens almost touches the cover glass. This

BACTERIOLOGICAL TECHNIC 49

can 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.

11. 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 are necessary.

13. To use the oil-immersion lens, place a drop of
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’
adjustment until the object is focused; or better,
lower the objective until almost in touch with the cover
glass, and focus up. High powers 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 enter-
ing a thinner medium from a denser one, there is not
sufficient light entering the objective to make objects
discernible, when using high powers without oil. By
the insertion of a liquid (inspissated oil of cedar) of
nearly the same refractive index as glass, a homogene-
ous connection is established between the cover glass
and the objective, thus avoiding loss of light and allow-
ing a bundle of light of sufficient power to enter the
objective.?

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

50 LABORATORY GUIDE IN BACTERIOLOGY

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.

16. 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 well to move the object while bringing
it into focus. It is then easy to feel when the lens
touches the glass, and a moving object is seen more
readily than a stationary one.

18. Use the plane mirror in combination with the
condenser, and the concave mirror without the con-
denser or with artificial light.

zg. In preparing stained preparations, it may hap-
pen 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.

SECTION 8
SCHEME FOR ROUTINE STUDY OF BACTERIA

This scheme is to be followed strictly in the study of
all organisms, except when special instructions are
given, and the student should familiarize himself with
the different steps.

1. The inoculation of one slant agar tube from each
vulture supplied. These inoculated agar tubes are to

BACTERIOLOGICAL TECHNIC 51

be incubated at 37° for 24 hours, unless otherwise in-
structed.

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

a) A physical description of the culture (see Section
9).

b) A hanging-drop examination.

c) A Gram stained preparation.

d) An ordinary stained preparation.

All stained preparations and all Gram stains should
be preserved.

3. Transfer from agar culture to the following
media:

Dextrose agar.

Gelatin.

Potato.

Broth. —

Litmus milk.

Dunham’s solution, if a test for indol is to be
made. z

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

4. An accurate description and sketches are to be
made of each culture (see Section 9). These descrip-
tions should be made complete after 24 hours, 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-
old broth culture of each culture, unless otherwise
directed. These plates are to be described once after
24-48 hours. (See Section 9.)

Nore.—The thermostat or incubator (Fig. 20) is a box
made of copper and having double walls, between which water

52 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 causing a drop in temperature by admitting

Fic. 20
Thermostat or Incubator

the cooler air. The outer door is covered with asbestos like
the walls, A thermometer reaches through the upper part,
provided with several air holes which permit free circulation
of air. The gas supply is shut off automatically, if it should
accidentally become extinguished. The necessary heat may also

BACTERIOLOGICAL TECHNIC 53

be obtained successfully by electric devices. For class use large
incubators are constructed on similar principles, with a number
of separate compartments for the accommodation of large num-
bers of students.

SECTION 9
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 impor-
tance that cultures should be closely observed, in all
media, and accurately described and sketched, as this
is the only method which furnishes. the proper means
of studying and determining the different species of
bacteria. By keeping this scheme in sight while mak-
ing a description, it will be possible after some practice
to make perfect descriptions without its aid. After
growth has taken place the culture should be compared
with a sterile tube of the same batch of culture medium.

I. Morphological characters of bacterium.
Size.
Facility and mode of staining.
Gram 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.

54 LABORATORY GUIDE IN BACTERIOLOGY

Make a sketch of part of a field under the
microscope.

II. Plate cultures.
1. Naked-eye appearance.
a) Surface colonies.

Tf colonies are too small to make observations with
the naked eye, this fact should be stated.

b)

Color by reflected light.
Approximate size.

Elevation or depression.
Translucency.

Moist appearance.

Smoothness.

Luster.

Liquefaction (in gelatin plates only).
Consistency: soft, viscid, hard, chalky.
Deep colonies.

Color.

Shape.

2. Microscopic appearance under low power
(No. 3 objective, Leitz). Use 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) or Homogeneity.

BACTERIOLOGICAL TECHNIC 55

Edge: entire or smooth, wavy, with
pointed protuberances, serrate, den-
tate, lacerate, fringed, hairlike
shoots, curled, tubercle-like appear-
ance (tuberculated).

b) Deep colonies.

Shape: punctiform, lanceolate, oval,
circular, spindle-shaped, conglom-
erate, irregular, branched, filamen-
tous, rosette-shaped.

Color.

Translucency.

Granulation.

III. Agar slant culture.
Limitation: confinement to needle track or
spreading. If spreading, in what shape?
Vigor: luxuriant, fair, or scant.
Color: by reflected light.
Elevation or depression, more pronounced at
edges or in center.
Translucency.
Moistness.
Smoothness.
Luster.
Coloration of medium.
Odor.
Gas formation: in culture or in medium.
IV. Stab culture in plain agar.
1. Surface growth (describe like agar slant).

56 LABORATORY GUIDE IN BACTERIOLOGY

2. Stab growth.
Vigor.
Extent.
Color.
Granulation.
Outgrowths.
Coloration of medium.
Cloudiness.
Gas formation.

V. Stab culture in dextrose agar.
Describe like plain agar stab and in addition
always note presence or absence of gas

formation. ‘

VI. Stab culture in gelatin.

Describe like agar stab, and in addition
always note presence or absence of lique-
faction. If liquefaction is present, it may
be saucer-shaped, turnip-shaped, conical,
funnel-shaped, horizontal (extending the
whole diameter of tube), sack-shaped.
Cloudiness and presence of sediment in
liquefied area, and color and shape of sedi-
ment, should be described.

VII. Potato culture.
Describe like agar slant, adding to it the
eventual discoloration of the medium, and
presence of gas bubbles.

VIII. Litmus milk culture.
Reaction (acid, alkaline, or neutral, as indi-
cated by color).

BACTERIOLOGICAL TECHNIC 57

Coagulation: present or absent.

Note.—If acid has formed but no coagulation is evident after
six days, heat gently and see whether coagulation then takes

place.

Whey: if present, clear or turbid.

Liquefaction of coagulum (proteolysis).

Gas formation.

Decoloration of litmus.

Color of cream ring.

Odor.

In milk the presence or absence of coagula-
tion and proteolysis should be noted. Com-
pare your culture with a sterile milk tube.

IX. Broth culture.

Cloudiness: degree and uniformity; scum
(ring- or island-shaped).

Precipitate, observed by shaking. Amount,
color, formation, diffusibility, viscidity,
amount of precipitate.

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 ex-
pressed in simple figures by the formula
2:3
Co,"

Growth in both arms or in one arm only,
observed by cloudiness.:

Reaction: acid or alkaline to litmus.

58 LABORATORY GUIDE IN BACTERIOLOGY

SECTION 10

DIRECTIONS FOR FILLING OUT CULTURE CHARTS

(See chart on pp. 59-60.)

First page—

I. Name the group and organism.

II. Indicate the source and habitat from books
and references.

II.

IV.

Name the most important references, and
read them.

Morphological characters.

I.

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

. Size: approximate estimate in microns.

Note whether large or small, thick or
slender, round or square ends, etc.
Arrangement of bacteria: in groups,
chains, bunches, pairs (diplococcus), sar-
cina form, filaments, branching, etc. Also
note different arrangements, if observed,
in different media.

Staining powers: Mark + for positive,
— for negative stains. Special stains
must be described more fully.

Motility: If absent, mark —; if present,
+. In the latter case describe the char-
acter of the movement.

Spores: Absence noted by — ; presence, by
+. If present, mention the method of
spore stain applied.

FROST’S CULTURE CHART (MODIFIED)

Group. .....cceeeneeeee
Name of organism’ .canc. conse saw sie oka vey oa eeeay ae aaee doves
SSOUECE ses arate inns desha coher oie fazer aeststniehs.wiahs Sicianalaye UERG SIAN NSE Ree Be Sue ate eee B4aKe iets
Habitats ove owcuvsinsics ae see se ors cea is Mah eke ailerete ce sevars tsi

Del tentGi ck acs ha enone Vanes eluneo eek NaN dees ee aisha aha vane

MorPHOLOGICAL CHARACTERS SKETCHES

Age of
Culture

Incubation
temp. (°C.)

a. Form: Rop: Coccus: |SPIRILLUM:
a, On agar.......e.eee aiaea| diaestersis mast

b. On other medias. 12 1)22]IIIIIIIIT
napa rere ie anna eee
3. Cell groupings. P asssuanebalenn

4. Staining powers....... paren ee oraitoratess oe
a. Anilin gentian violet....
b. Léffler’s methylene blue. se
624Gram: Statins isc. sacnies ected wot Sawieceonetactes:s ka
d. Special stains, such as: (1) igelle. ..(2) Spore...}...
(3) “Tubercle”. ...(4) Capsule. ..............- a
5. Motility: (a) Flagellar.......(b) Molecular.
c. Character of movement..........0..06-
6. Spores... +. sseeeeeeeree acdiues apdetecavectiebeczcel
7. Special characters, such a8i........+.. ef
capsules, vacuoles, granules, ...........- ae eae fo
pleomorphic and involution forms, etc..........+.- ih es RDA Ss

PHYSIOLOGICAL CHARACTERS

c. Percentage of gas produced
in: fermentation tube....... DEXTROSE Lacrose | SACCHAROSE

After 24 hours ........-
After 48 hours........

8. Indol production; 24 hours......... 48 hours.............dayS.......

fecal odor; 24 hours........... peneeseeos 48 hours......days....... :

9. Enzym production; (a) proteolytic...... (b) coagulative....(c) diastatic..
to. Characteristic odor........+-eeeseeee

ix, Agglutination..........sesees
12. Pathogenesis... ....¢++eeseeeeeee

CULTURE CHARACTERS OF... 0. ccc ee ct ee ence enter een eeeeneeeenees aroisesin

J Reaction

of Medium, | _ 24 48 6 Sketches
Incubation |Hours |Hours | Days
Temp. (°C.)

(x)

« Agar A
Streak 77
or Stab. 7 N

(2) \

Potato, or
Heinemann’s
Synthetic j

Potato S77
Medium. i Meet oo
(3)

Gelatin 7 N yy €
Stab. / \ / \
(4) 1 ! \

Litmus
Milk.

a oF 7

Broth
- (Bouillon)

(6)
Dextrose

Agar, or
Special

Miredluna: K ow
(7)

Agar plate,

o? TO)
tube.

* (a) Surface
Colonies.

(b) Deep
Colonies. Ky

(8)

Gelatin
plate, or
roll tube.

(a) Surface
Colonies.

(6) Deep

Colonies. ae, a,

‘BACTERIOLOGICAL TECHNIC 61

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?

2. Relation to free oxygen: aérobe, anaérobe,
facultative aérobe, or anaérobe.

3. Relation to disinfectants, light, desicca-
tion, heat (thermal death-point).

4. Pigment production: If present, +; if
absent, —. In the former case, note the
color, diffusibility, 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 tur-
bidity. 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: a ?

6. Acid or alkali production in litmus milk.

7. The production of indol or nitrites, or
both, is tested on the sixth day of observa-
tion in a culture in Dunham’s pepton
solution or sugar-free broth.

Note.—Indol is a decomposition product of proteins and
belongs to the aromatic series. Nitrites result from reduction of

\

62 LABORATORY GUIDE IN BACTERIOLOGY

nitrates, or from oxidation of ammonia. The ability of organisms
to produce these reactions is of great importance in their differ-
entiation. A control test with a tube of sterile medium should
be made.

8. Enzym production: Proteolytic enzym
production noted by ‘the liquefaction of
gelatin or casein. Coagulative, recognized
by precipitation of casein, if acid forma-
tion is absent, or present only in quantities
less than 0.4 per cent. Amylolytic, by
the digestion of starch (potato).

g. Characteristic odor.

10. Pathogenesis: What pathogenic effect has
the organism on man? What effect on
animals, and which animals? What dis-
eases are caused by the organism in man
or animal?

Notr.—The terms “proteolysis,” “‘enzym production,” and
“coagulation” are frequently confusing to the beginner. The
following brief,explanation will aid in an intelligent interpretation
of the reactions observed.

“Proteolysis” is the breaking up of complex nitrogenous
compounds (proteins), 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, the assumption is that the
organism is capable of producing a “proteolytic” or gelatinolytic
enzym. In milk the process is more complex, and this medium,
on account of its composition (fat, milk sugar, casein, lactalbu-
min), offers excellent opportunities for the organism to develop
different characteristics. Milk is one of the most important of
media. The casein, contained in milk in colloid solution, may be
precipitated by an enzym or by an acid. This precipitate forms
the coagulum. At least 0.4 per cent of-acid, which is largely

BACTERIOLOGICAL TECHNIC 63

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 red. 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 amphoteric or alkaline
reaction, as well as in milk of slightly acid reaction. The coagu-
lum formed by any of the mentioned agents may gradually
contract, and a straw-yellow, opalescent liquid will be squeezed
out, called “whey.” If the organism also produces a proteolytic
enzym, this will attack the coagulum and gradually dissolve it
(proteolysis, peptonization). At first the coagulum shows a
broken-up edge; lumps separate and settle to the bottom, and
finally the coagulum may disappear completely. Theoretically,
coagulation is always necessary before proteolysis sets in, but in
the case of some organisms the proteolytic enzym is so powerful
as to produce immediate solution of the casein. (See Figs. 2,
22, 23, 24.)

Another phenomenon frequently observed in litmus milk
is the decolorization of 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 contact with the oxygen of the air.

The production of an amylolytic enzym (diastase, amylase) is
demonstrated by gas production on potatoes. This medium con-
tains starch in large amounts. The starch is converted into
maltose by diastase (amylase). Maltose may then be split by
the action of an inverting enzym into dextrose, which is then
fermented with gas production.

Second page—

1. Note the reaction of the medium.

2. The incubation temperature may be (a) 37°C.
(thermostat), (6) room temperature (20° C.),! (c) ice
chest.

= Gelatin cultures are to be incubated at room temperature.

64 LABORATORY GUIDE IN BACTERIOLOGY

Fic. 21
Streak Cultures

ogtete
wee ORS od
Seed

UU

Fic. 22
Stab Cultures

BACTERIOLOGICAL TECHNIC 65

Fic, 23
Liquefaction of Gelatin

J

0
Q

Fic. 24

1. Gas bubbles in glucose agar 3- Coagulation and peptonization of milk
2. Coagulation of milk 4. Complete peptonization of milk

66 LABORATORY GUIDE IN BACTERIOLOGY

3. Plates are to be described only once, after 24 or
48 hours, according to growth. Make notes in column
7 for agar plate; in column 8, for gelatin plate. Make
sketches in the margins reserved for this purpose.

4. The growth on the media 1, 2, 3, 4, 5, and 6 is
to be described 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 descrip-
tion.

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

These directions for filling out culture charts are
applicable in the described manner to studies of cul-

tures furnished by the laboratory. For determining
any species of unknown bacteria, original researches
must be made to cover all points, without the possi-
bility of gathering this information from textbooks or
references. On the accuracy of observation and de-
scription depends the success of bacteriological work
and species determination. It is frequently neces-
sary to employ special media, or inoculation of animals,
or such biological reactions as the agglutination test,
to determine what species one is dealing with.

The student will do well to familiarize himself
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. .

PART II
GENERAL BACTERIOLOGY

SECTION 1
PREPARATION OF CULTURE MEDIA
The following culture media are to be prepared for
this work:
First SET

Wortid eats witedoeisie ois ccececersecare: 6 a daa saletste 20 tubes
Ten of these tubes should contain about 7 c.c. for slants, the
other ten about ro c.c. for plating.

Wort gelatin. ....... 2. eee ceca to tubes
Liquid wort................ cle Se ie aber Io tubes
Fermentation tubes...............0.- 6 tubes

Two of the fermentation tubes to contain z per cent dextrose,
two 1 percent lactose, and the remaining two 1 per cent saccha-
rose.

SECOND SET
Meat extract agar......... 0. cece eee 20 tubes
Ten of these for slants and ten for plating.
Pepton gelatin. ......... cece eee eee 15 tubes
BEGthiaetn dkranuaebey on dh ou wera 15 tubes
Dextrose agar. 6. +--+. eset eee seer eenes 15 tubes
Litmus milk... 0... eee eee 15 tubes
Synthetic potato........ cece eeeeeeeeee to tubes

The beerwort media are to be prepared during the
first week, the other media later.

69

7o LABORATORY GUIDE IN BACTERIOLOGY

SECTION 2

COLLECTING AND CULTIVATING MICRO-ORGANISMS
FROM THE AIR

EXERCISE I

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

2. Melt two tubes of plain agar, one of dextrose
agar, and two of beerwort agar, in a water bath. The
water bath (Fig. 25) is a round copper vessel with a
number of holes in the top. These holes are large
enough to allow a culture tube to slipin. A thermom-
eter is passed through a rubber cork with a hole in its
center, and inserted into one of the holes in the water
bath. Water is then poured into the apparatus until
the level is slightly higher than the media in the tubes
and the thermometer
lowered until the mer-
cury bulb is immersed
in the water. The cul-
ture tubes are then
slipped in, and the
water heated to 100°C.

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

Fic. 25
Water Bath flame, and pour the

GENERAL BACTERIOLOGY 71

contents into a sterile petri dish, carefully lifting the
cover (Fig. 26) and quickly replacing it.

4. Repeat this operation with the other tubes,
excepting glucose agar.

5. Place all petri dishes containing the liquid agar
on a level surface.

6. When the agar has solidified, expose, by removing
the cover, one dish of pldin agar and one of wort agar
to the air of the laboratory, and the other two outside
on the window-sill, for 5 minutes.

7. Replace the cover and place in incubator.

Fic. 26
Pouring Medium into Petri Dish

8. Cool the water bath to 43°C., and mix the
scrapings from under a finger nail with the liquid
glucose agar. Incubate at 37°C. |

g. Remove the plug of a tube of broth and place a
hair in the liquid. Incubate at 37° C.

Liquefied agar media should be inoculated at a
temperature no higher than 43° C. nor lower than 40° C.
Above 43°C. the organisms are liable to be injured
by heat; below 40° C. the agar solidifies, and an even
distribution is impossible. If gelatin is used, the
latter precaution is not imperative, as gelatin solidifies _
at about 25° C. .

Observe and, make notes on the appearance of these
petri dishes after 24 hours. By this time it will be

72 LABORATORY GUIDE IN BACTERIOLOGY

observed that a number of spots of different sizes,
shapes, colors, etc., have formed on the surface of the
medium.

The low power of the microscope will reveal a dark
part in most of these spots. This dark part is a dust
particle, which has carried micro-organisms to the
surface of the agar, and thus opportunity is given for
multiplication and formation of a “colony.” A colony
originating in this manner may or may not consist of
one species of organism. By studying the organisms
we will find that the colonies are composed of bacteria,
yeasts, torulae, or molds. All or any of these may be
carried by dust particles in the air.

EXERCISE 2
Inoculate two or three colonies on agar slants.

Method of inoculation—

1. Singe the cotton plug of a tube containing the
medium. Organisms in the air are constantly alighting
on the cotton, and bacteria may also be deposited on the
cotton by handling it with the fingers. If these organ-
isms are not killed by the process of singeing, they may
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 fore-
finger, so that the end of the tube rests on one edge of
the hand (Fig. 27), holding them at an angle of about
45°. If held horizontally, the condensation water,
usually present at the lower end of the agar slant, will
moisten the surface and destroy a characteristic growth

GENERAL BACTERIOLOGY 73

along the needle track. If held in a vertical position,
contamination from the air may take place.

3. Remove the cotton stoppers by taking hold of
the singed portion, and hold also by the singed part
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
contaminated and must be singed before replacing.

4. Sterilize the straight platinum needle in the
flame, holding it like a pencil. The platinum wire

Method of Inoculating Media

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
on the end of the needle by a lateral movement, and,
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.

74 LABORATORY GUIDE IN BACTERIOLOGY

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

7. Replace the cotton plugs and sterilize the needle
in the flame, as above.

A stab culture is made by puncturing the medium
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 a narrow
puncture, and by touching the glass the needle would

sugeet ee
c ===> ------ &
OT eee
Fic. 28
Hanging Drop
a. Hollow 6. Drop c. Cover slip d. Slide

bend and make a ragged opening in the medium on
withdrawing.

Inoculations of liquid media are made by rubbing
the end of the needle against the glass below the surface
of the liquid. The medium is then shaken. After
inoculation liquid cultures should not be shaken again,
as this might destroy a characteristic shape of the
coagulum, break up the cream ring, or defeat sedi-
mentation in broth.

GENERAL BACTERIOLOGY 75

Inoculations from liquid media are made with the
looped needle. This may also be used from a solid
medium, if a considerable amount of growth is required.
The contents of the loop are then spread over the whole
surface.

EXERCISE 3

Describe the appearance of the colonies as outlined
in Section 9.
EXERCISE 4

Examine as to motility in the hanging drop.
Preparation of a hanging drop (Fig. 28):

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

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

5. Examine in oil, using very little light.

EXERCISE 5

Molecular movement.—Rub a small amount of car-
min 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

76 LABORATORY GUIDE IN BACTERIOLOGY

particles of carmin have a lively vibrating motion.
This is called “molecular movement,” “Brownian
movement,” or “pedesis.” The particles scarcely
change their relative position. Actively motile or-
ganisms, on the contrary, change their relative posi-
tions. 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.

EXERCISE 6

Make stained preparations of three different
colonies, using the three stains: i.e., gentian violet,
methylene blue, and carbol fuchsin, also a Gram stain.

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 pur-
pose. After handling, the forceps should be sterilized
in the flame.

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

3. Take a small quantity of the colony or culiure
on a platinum needle and mix with water until faintly
cloudy. Burn the remainder of the culture off the
needle.

GENERAL BACTERIOLOGY 77

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

5. Dry by moving high over the flame.

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

Nore.—The same object may be accomplished by allowing
absolute alcohol to evaporate from the cover slip. This method
has some advantages, since the bacteria do not shrink from the
heat.

7. Cover with stain for 10-15 seconds.

8. Wash in water.

g. Blot with filter paper, dry in the air or high over
the flame, and mount in Canada balsam.

. 1o. Label and preserve this preparation.

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

EXERCISE 7

Method of making preparations according to Gram—

x. Prepare a film of the organism to be examined,
as for the ordinary stained preparation.

2. Cover with gentian violet for 1 minute.

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

4. Cover with Gram’s iodin solution for 2 minutes.

5. Pour Gram’s iodin solution off and, without
washing, place in a staining dish, film side up, and
cover with 96 per cent alcohol.

78 LABORATORY GUIDE IN BACTERIOLOGY

6. 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.

7. Dry without washing; and mount.

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

It-is a positive Gram 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.

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 bac-
teria in tissues, sputum, etc.

By mounting a Gram stain and a gentian violet
stain of the same organism on the same slide both
time and material are economized.

SECTION 3
STUDY OF MOLDS, YEASTS, AND TORULAE

EXERCISE I. CULTURAL STUDIES

Yeasts, torulae, and molds grow better in a medium
of acid reaction than in a neutral or alkaline medium.
Media prepared from hopped beerwort are generally
used for this purpose.

Make transfer of a stock culture of Saccharomyces
cerevisiae or any other species of yeast which may
have appeared on the plates prepared in the previous
section. Also transfer two species of molds from these
plates to slanted wort agar.

GENERAL BACTERIOLOGY 79

Note.—Molds may be recognized by the filamentous, cotton-
like form of the colonies. The hyphae extending into the air
carry spores (conidia). By gently touching these with a sterile
platinum needle, the spores may be transferred to an agar slant,
and development will take place. Colonies of yeasts or torulae
appear smooth, moist, opaque, elevated, and slightly yellowish-
white, or sometimes reddish. These may be transferred in the
same manner as colonies of bacteria. Molds require careful
handling for microscopical demonstration. They are usually
examined in water or glycerin in the unstained condition.

EXERCISE 2

Method of preparing molds for microscopical
examination— -

r. Transfer some of the growth to alcohol (so 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
permanent 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 and allow alcohol to evapo-
rate.

3. Wash in water.

4. Stain with gentian violet or methylene blue.

5. Mount in glycerin.

EXERCISE 3

Study of Yeasts—
1. Examine a small amount of yeast taken from an
agar slant in water under the high power of the micro-

80 LABORATORY GUIDE IN BACTERIOLOGY

scope. Note the manner of reproduction by “bud-
ding.”

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

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

Note.—Gypsum blocks may be prepared in the following
manner: Gypsum (plaster of paris) is mixed with half its volume
of water and quickly placed in a cylinder of paper. When dry,
the paper is cut away and the block is placed in a suitable vessel
(a stender dish or a deep, narrow petri dish, covered by an
inverted tumbler). The block and vessel are then sterilized in
the hot-air sterilizer for one hour at 110° to 115° C., or in the auto-
clave for 30 minutes.

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

5. Incubate at 25° C.

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

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

Nore.—Under favorable conditions, and in the presence of
oxygen, yeasts will develop spores. The porosity of the gypsum
block, which admits free communication with the water, and the
fact that the surface of the block is exposed to the air, offer
favorable conditions for spore formation, which takes place in
3 or 4 days.

EXERCISE 4. CULTURE STUDIES OF YEASTS AND MOLDS

Make transfers from all agar cultures of yeasts to
wort gelatin and liquid wort.. Mark these cultures
with labels or glass pencils on the side of the tube op-
posite to the slanted surface and just below the cotton

GENERAL BACTERIOLOGY 81

stopper. Incubate the gelatin cultures at room tem-
perature, all other cultures at 25 or 37° C. in the incu-
bator. Make descriptions after 24 hours, 48 hours,
and 6 days, as outlined in Section 9.

EXERCISE 5

Select two species of yeasts and inoculate three
fermentation tubes with each species, using the looped
needle, so as to have each species act on the three differ-
ent sugars. Measure the gas evolved after 24 hours
and after 48 hours by means of Frost’s fermentation
chart or gasometer (back cover). The chart is to be
placed between the open arm and the bulb and moved
until the extreme upper end of the closed arm is level
with the top of the chart and parallel with the vertical
lines on the chart. Express the results in percentages
as read from the gasometer.

Gas production is not a constant accompaniment
of fermentations. Carbohydrates are fermented by
many organisms without gas formation, the usual
product being an acid, often lactic acid. Such fer-
mentations produce turbidity, but no gas. Growth
therefore must be described and can be observed by
turbidity, forming either in the closed arm, the bulb,
or both. Observations are to be noted in the space
for this purpose on the first page of the description
charts.

Analysis of gas produced in the closed arm—

The gas consists chiefly of carbon dioxid and hydro-
gen, as may be proved by the following method: Fill
the bulb with a 2 per cent solution of NaOH, and close
the mouth with the thumb, taking care not to leave any

82 LABORATORY GUIDE IN BACTERIOLOGY

air between the thumb and the liquid. Now tilt the
gas back and forth slowly from the closed arm to the
bulb and back to the closed arm five or six times, and
finally allow the gas to collect again in the closed arm.
The NaOH combines with the carbon dioxid, and con-
sequently, on releasing the thumb, the volume of gas
will become smaller in proportion to the amount of
carbon dioxid absorbed. The percentage of gas is
measured again with the chart, and the relation deter-
mined of the gas left in the arm to the original amount.

Example—
Total percentage of gas before addition of NaOH 45
Percentage left after absorption by NaOH .... 30
AUT CRETCE ss 5s cdussaszvausvedaisindasoueyolassiasg cua -acoadnuouayeynes 15

30 per cent represents the amount of hydrogen and
I§5 per cent the amount of absorbed carbon dioxid.
The proportion is expressed by the formula

The fact that the gas remaining in the closed arm is
probably hydrogen may be proved by tilting it into the
bulb, previously filled with water and closed by the
thumb. Hold a burning match over the mouth and
release the thumb. A slight explosion takes place
from the combination of the hydrogen with the oxygen
of the atmosphere.

The gas produced by yeasts usually consists chiefly
of carbon dioxid; the gas produced by intestinal
bacteria consists chiefly of two-thirds hydrogen and
one-third carbon dioxid; and the gas produced by the
proteus group consists chiefly of one-third hydrogen

GENERAL BACTERIOLOGY 83

and two-thirds carbon dioxid. These proportions,
obtained by the above-described method, are but
approximations to the actual condition. Somewhat
different results have been reported by Keyes (Jour.
Med. Res., 1909, N.S. 16, p. 69). For his experi-
ments synthetic media and more precise methods for
the control of conditions were employed.

Gas formation by bacteria does not necessarily
depend on the presence of carbohydrates. Nitrogen
may be produced from nitrites and nitrates, or urea,
hydrogen sulphid, and ammonia from proteins during
the process of putrefaction.

EXERCISE 6. STUDY OF THE GERMINATION OF MOLD
SPORES

Transfer two species of molds from the air plates
to slant wort agar and incubate at 37° C.

After several days, when sufficient growth has taken
place, remove spores from the surface of the hyphae by
means of a straight needle and suspend these in a tube
of liquid beerwort or broth. Transfer a loopful of
this suspension to a cover slip and examine under the
microscope, magnifying about 600 times. If only a
few spores are discovered in a field invert this cover
slip over the hollow of a hollow-ground slide and keep
in place by painting a ring of vaselin around the hollow.
If there are too many spores on the cover slip dilute
the suspension with beerwort or broth. This hanging
drop is incubated and observed daily under the micro-
scope and sketches made of the appearance. In seven
to ten days the spores should have produced the whole
cycle of development of the mold and new spores
should have formed.

84 LABORATORY GUIDE IN BACTERIOLOGY

SECTION 4

BACTERIOLOGICAL EXAMINATION OF WATER, AIR
AND MILK
EXERCISE I. BACTERIOLOGICAL ANALYSIS OF WATER
References—
Prescott and Winslow, Elements of Water Bacteriology, New
York, 1914.
Savage, The Bacteriological Examination of Water Supplies,
London, 1906.
A bacteriological examination of water is made for
the purpose of determining—
1. Bacterial numbers.
2. Bacterial species.
3. Sewage pollution.

Collection of samples.—Procure wide-mouthed,
glass-stoppered bottles, having a capacity of at least
too c.c, After cleaning and drying, tie lead foil or
filter paper over the stopper, wrap the bottles indi-
vidually in paper and sterilize in the hot-air oven for 1
hour at 160° C.; then deposit them in a metal or wooden
case. Thesamples from surface waters should be taken
at least one foot below the surface, to avoid contamina-
tion with organisms from the air. If possible, samples
should be plated on the spot or in the laboratory within
an hour at the 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 partially altering the bacterial flora.

Method of examination.—A number of pipettes of
various sizes (1 C.c., 2 ¢.c., § c.c., and 10 c.c.) are plugged

GENERAL BACTERIOLOGY 85

with cotton and sterilized in the hot-air oven. Thena
number of Erlenmeyer flasks are filled -with 100 c.c. of
distilled water, and these are sterilized in the autoclave
at 120°C. for 5 minutes. During sterilization a small
variable amount of water is lost. This has to be
disregarded.

Method of procedure—

1. With a sterile pipette carry over to one of these
flasks 1 c.c. of the sample after shaking. The dilution
is now 1:100. Mark with a glass pencil.

2. With a sterile ro c.c. pipette remove ro c.c. from
another dilution flask, and add to the remainder 10 c.c.
of the first dilution. We now have a dilution of 1:1,000.
(See appendix.) Make a number of dilutions in this
manner, carrying the dilutions higher in proportion
to the quality of the water to be examined.

3. Melt a number of agar and gelatin tubes, corre-
sponding to the number of dilutions made, and cool to
43°C.

4. Transfer 1 c.c. of each dilution flask to a petri
dish.

5. Pour the contents of one agar tube on the petri
dish and mix this with the 1 c.c. of water by tipping
the dish back and forth.

6. Incubate the agar plates at 37° C. and keep the
gelatin plates at room temperature.

Estimation of colonies.—The colonies are then
counted after 48 hours, by means of a colony counter
(see back cover). Plates should be counted which
contain no more than 200-300 colonies. If it is neces-
sary to count plates with a large number of colonies,
an estimate must be made by counting different sec-

86 LABORATORY GUIDE IN BACTERIOLOGY

tions 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 ex-
amined 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.—If Bacillus coli and strep-
tococci are present in relatively large number, sewage
pollution is indicated.

Method of examination for B. coli and strepto-
cocci—

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

2. Place in thermostat.

3. Examine after 12-18 hours.

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

Example.—If 1 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 number of B. coli
per cubic centimeter may be estimated.

Formula for determining the number of B. coli
present in water:

NXD
—z = Number of B. coli in 1 c.c. water.

N=Number of tubes with gas; D=Dilution; I=Total
number of tubes inoculated.

GENERAL BACTERIOLOGY 87

The presence of streptococci is determined by mak-
ing Gram stains from each of the fermentation tubes
after two or three days. The number of streptococci
can be determined approximately by the same method
of calculation as for B. coli, by substituting the number
of tubes containing streptococci for N.

Isolation of B. coli and streptococci is accomplished
by plating from the fermentation tubes in lactose’
litmus agar.

EXERCISE 2. THE BACTERIOLOGICAL EXAMINATION
OF AIR

For precise methods see:

“Report of the Committee on Standard Methods for the
Examination of Air,” Am. Jour. Public Hygiene, 1910, 20, p.

6.
7 Rettger, Jour. Exp. Med., 1910, 22, p. 461.

An approximate determination of the number of
bacteria in the air can be made by the following simple
method: Place 50 c.c. of broth in an Erlenmeyer flask
(Fig. 29, a). This flask is provided with a rubber
stopper () with two holes, through which two glass
tubes (c with a wide opening and d) lead. Cotton
plugs are then inserted at ¢ and d, and the apparatus
is sterilized in the autoclave. A large bottle (f), con-
taining 5 liters of water, is then provided with a rubber
stopper, and also with two glass tubes (g and k); his
connected with a short piece of rubber hose and a
pinchcock (7). When the Erlenmeyer flask and con-
tents are sterilized, the tube d is connected, by means
of the rubber hose e, with g, and the plug at cis removed.
By opening the pinchcock 4, 5 liters of air are aspirated

88 LABORATORY GUIDE IN BACTERIOLOGY

through the broth in flask a. The flask is then dis-
connected, and 1 c.c. is plated in agar and 1 c.c. in
gelatin. The former is incubated at 37°C., 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.

;
0
Fic. 29 a
Apparatus for Determining the Number of Bacteria in a Definite Volume of Air
a. Erlenmeyer flask f. Five-liter flask
6, Rubber stopper g, kh. Glass tubes
c,d. Glass tubes #. Pinchcock

EXERCISE 3. 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 larger numbers of bacteria.

Sterilization and pasteurization of milk.—Some of
the germs in milk are saprophytes (which under favor-

‘GENERAL BACTERIOLOGY 89

able circumstances produce disagreeable odors or
tastes), and such pathogens as the bacillus of tuber-
culosis (which may be derived from the cow, or may be
an accidental contamination), the typhoid bacillus,
the bacillus of “summer complaint” in children (pos-
sibly 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 temperature.

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 milk
comparatively safe and less subject to ordinary fer-
mentative changes.

1. Sterilization at 100°C. for 30 minutes.—Such.
milk, if chilled and kept at a low temperature, will
remain unchanged for more than a week, but, by
heating, certain alterations have been produced in
taste and appearance.

2. Pasteurizing milk—The changes occurring in
milk, as above mentioned, begin at about 80°C. Pas-
teurization at a low temperature is accomplished by
raising the temperature to 60-65° C. for a period of 20
minutes. This has been shown to be sufficient to kill
the germs of tuberculosis, typhoid fever, cholera,
diphtheria, and pyogenic cocci. Spores 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° C. in 15-20 min-

90 LABORATORY GUIDE IN BACTERIOLOGY

utes; but, if milk containing tubercle bacilli has its
surface exposed to the air when heated to 60°C., 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
sterilization as compared with each other and with
unsterilized milk:

1. From the fresh milk provided make three agar
plates, using 1, 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
the milk when the tubes are immersed init. Raise the
temperature to 65° C., 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 ro minutes for the milk in the tubes to reach the
temperature 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. Aérate the other two by shaking
vigorously for 14 minutes. Keep one of these at room
temperature, the other in the thermostat.

4. Place two more milk tubes in the arnold at
too’ C. for 30 minutes. Keep one at room tempera-
ture and one in the thermostat.

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

GENERAL BACTERIOLOGY gl

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 coagu-
lation sets in, gas formation, condition of whey, film,
and odor. :

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 indi-
vidual species are to be studied.

SECTION 5

EXERCISES ON INFECTION AND STERILIZATION

EXERCISE I. PHENOMENA OF INFECTION

1..Prepare three agar plates.

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.

4. Catch a fly and allow it to walk on the surface of
the jelly of the third plate. 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 2. PHENOMENA OF STERILIZATION

1. Make an infusion of hay in a flask with cotton

stopper. (See p. 41.)
2. Set the flask aside for 24 hours in a warm place,
and observe the results.

g2 LABORATORY GUIDE IN BACTERIOLOGY

3. Expose a tube of unsterilized broth to steam in
the arnold, another to steam in the autoclave, at
120° C. for 5 minutes each

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

EXERCISE 3. PHENOMENA OF STERILIZATION (CON-
TINUED)

Action of Berkefeld and cotton filters.—Berkefeld
filters are made of diatomaceous earth, and are porous
so as to allow the passage of fluids, while retaining
suspended solids, bacteria, etc. Some bacteria, known
as “ultramicroscopic bacteria,” are so small that they
pass through Berkefeld filters.

1. Arrange a Berkefeld filter so as to connect with a
suction pump, and filter a quantity of unsterilized
broth (Fig. 3).

2. Set aside, and observe results.

The filter (a), after having been connected with
the flask, is sterilized in the autoclave. The cotton
plug c prevents the air, which is sucked back, from
carrying germs into the flask. The flask d is an
intercepting or reflux flask, which guards against the
broth becoming contaminated from water being sucked
back if the pressure suddenly diminishes.

EXERCISE 4

Arrange a cotton filter as shown in Fig. 30. Vessel
a, provided with a rubber stopper (6) with two holes, is
arranged so as to have a glass tube (c) reach to the
bottom. This tube is provided with cotton at the top
opening (d) and some nutritive medium (broth, e) is
placed inside. Through the other hole a bent glass
tube leads out, and this tube is also provided with a

GENERAL BACTERIOLOGY 93

cotton filter at f. The whole apparatus is then steril-
ized in the autoclave at 120° C. for 5 minutes, and
connection is made through the flask (g) and the tube
(2) with the aspirator. Now aspirate some air through
the flask, disconnect at f, and set aside. Observe the

Fic. 30
Action of Cotton Filter
a Erlenmeyer flask e. Broth
6. Rubber stopper f. Bent glass tube with cotton filter at f
¢. Glass tube g. Erlenmeyer flask
d. Cotton filter kh. Tube connecting with aspirator

results. Remove the cotton filter (d), and drop it
into a flask containing sterile broth; place in the
thermostat for-18-24 hours; examine and note the
conditions then present.

Results of the exercises in this chapter are to be
observed by noting the appearance of colonies or

04 LABORATORY GUIDE IN BACTERIOLOGY

turbidity, and by preparing stains with gentian violet
from small amounts of the material in the culture
media.

SECTION 6

INFLUENCE OF DISINFECTANTS, LIGHT, AND HEAT
ON THE GROWTH OF MICRO-ORGANISMS

For precise methods see:

Anderson and McClintic, Hyg. Lab. Bull. 82, 1912.
Amer. Jour. of Public Health, October, 1912.
Rideal and Walker, ibid., June, 1913.
North Dakota Agricultural College, Special Bulletin, July,
August, 1913.
EXERCISE I

1. Prepare fifty-seven Hill’s test rods. These are
prepared in the following manner: Glass rods about
two inches lohger than ordinary culture tubes are
marked with hydrofluoric acid or a glass cutter (dia-
mond or file) by a circle one inch from the end. A
wad of cotton is then wrapped around the middle of
the rod, and 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 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
too c.c. of a x per cent solution.

3. Fill two similar flasks, one with 100 c.c. of a solu-
tion of mercuric chlorid 1: 1,000, the other with a solu-
tion of 1: 10,000.

GENERAL BACTERIOLOGY 95

4. Fill two similar flasks, one with 100 c.c. of a 10
per cent solution of formalin (40 per cent formalde-
hyd), the other with a 1 per cent solution.

5. Prepare-48-hour broth cultures of Staphylococcus
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 ther-
mostat after marking each tube.

EXERCISE 2

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. 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
pouring sterile physiological salt solution over them
into a dish containing mercuric chlorid solution 1: 1,000,
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 incu-
bated with the others as controls.

6. Observe the results on each of the four successive

96 LABORATORY GUIDE IN BACTERIOLOGY

days, and on the last day prove the relative growth
by making agar plates with 1 c.c. of each culture,
and count the colonies after 24 hours.

7. Tabulate the results, and state conclusions.

EXERCISE 3. INFLUENCE OF SUNLIGHT

Experiment 1—

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

2. After thoroughly shaking, take 1 c.c. by means of
a sterile pipette, and plate in agar. Place the plate in
the thermostat.

3. Expose the flask to sunlight for several hours.

4. Make another plate with 1 c.c. of the suspension,
and place in a thermostat.

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

Experiment 2—

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

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 4. 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.

GENERAL BACTERIOLOGY 97

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

3. Remove one of each at 40° C., one of each at
60° C., one of each at 80° C., and keep one of each for
Io minutes at roo’.

4. Place one tube of each organism in the autoclave,
and. heat to 120° C. 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.

SECTION 7
STUDY OF CHROMOGENIC BACTERIA

EXERCISE I. CULTURAL STUDIES
References to chromogenic bacteria, and the pro-
duction and chemistry of pigments:

Fischer’s lectures, Fliigge, Die Mikroorganismen.
Lehmann and Neumann.

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

1. Inoculate agar slants from laboratory cultures of
Bacillus prodigiosus, B. pyocyaneus, B. violaceus, and
Sarcina lutea. Inoculate three slants each of B.
prodigiosus and B. violaceus, and one each of Sar.
lutea and B: pyocyaneus.

98 LABORATORY GUIDE IN BACTERIOLOGY

2. Label each tube with the name of the culture
inoculated, and the date of inoculation.

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

4. After 24 hours compare the growths of B.
prodigiosus and B. violaceus, under the various condi-
tions, in respect to—

a) Relative amount of growth.

b) Relative amount of pigment produced.

5. Note the characteristics of the pigments: Are
they diffused through the medium, or are they con-
fined to the growth ?

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

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

8. After all cultures have been incubated for 24
hours, make all descriptions as outlined on pp. 53 ff.

Caution.—Through oversight gelatin cultures are sometimes
placed by students in the thermostat. This 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.

g. Make plate cultures of the four organisms.

Method of making plates—
1. Melt two agar tubes for each organism in the
water bath and cool to 43° C.

GENERAL BACTERIOLOGY 99

2. 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-
tion, or sterile physiological salt solution.

3. Shake well, avoiding air bubbles as much as
possible. :

4. Transfer 4 or 5 loopfuls from this suspension to a
melted agar tube.

5. Shake this carefully by rolling the tube between
the palms of the hand.

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

7. More tubes may be inoculated in the same way,
resulting in still higher dilutions if this is desirable. In
the meantime the inoculated tubes should be replaced
in the water bath, so as to keep them liquid.

8. Pour the contents of the tubes, one after the
other, into sterile petri dishes.

9. Tip the petri dishes so as to distribute the medium
evenly over the bottom.

to. Label them with the name of the organism and
the date. ‘

11. Set aside on a level place to solidify.

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

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

100 }=LABORATORY GUIDE IN BACTERIOLOGY

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 Section 9.

EXERCISE 2. STUDY OF PIGMENTS

On the sixth day take agar slant or potato cultures
of the four chromogenic bacteria, and proceed as
follows: Pour 96 per cent alcohol on the cultures of
B. prodigiosus, 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.

SECTION 8
STUDY OF MICROCOCCI

* Make transfers from laboratory cultures of Staphylo-
coccus aureus and Streptococcus lacticus.
Follow the outline of routine work and make descrip-
tions of these two organisms.

GENERAL BACTERIOLOGY Ior

SECTION 9
_STUDY OF INTESTINAL BACTERIA

EXERCISE I

Make transfers from laboratory cultures of B. coli,
B. suipestifer, B. fecalis alkaligenes, and B. cloacae.

EXERCISE 2

In addition to the usual routine study prepare
fermentation tubes in the following manner: Meat
extract broth or meat infusion broth is inoculated with
a culture of B. coli to remove the muscle sugar and
incubated at 37° C. for 24 hours. The broth is then
boiled, the reaction made 1 per cent acid, and the broth
filtered until clear. Dissolve 1 per cent dextrose in
one third, 1 per cent lactose in another third, and x
per cent saccharose in the remaining third. Fill these
solutions into fermentation tubes and sterilize in the
arnold for three successive days. Any gas which accu-
mulates during sterilization in the closed arm must be
tipped out while the medium is hot. When cooled
down, inoculate the fermentation tubes with the four
organisms, incubate for 24 hours, and then measure the
-gas in the closed arm. Replace in the incubator and
measure the gas again after 48 hours. Finally deter-
mine the composition of the gas as described on p. 81.

- In addition to the exercises outlined for this course
demonstrations should be made of stains of tubercle
bacilli in sputum, stains of diphtheria bacilli, methods
of anaérobic cultivation of bacteria, and various
outfits used in municipal laboratories for diagnosis.

PART III
IMPORTANT PATHOGENIC BACTERIA

SECTION 1
PREPARATION OF CULTURE MEDIA

The following amounts of culture media will be
required in this work:

Name of Medium Amount Number of Tubes}

300 C.c. 25

I,000 g. 30

250 g. 20

300 g. 25

200 C.C. 25

— Io

300 C.c. 25

160

The following staining solutions should be made up
in sufficient quantities to fill ordinary staining bottles:

Léffler’s methylene blue.

Ziehl-Neelsen’s carbol fuchsin.

Ehrlich’s gentian violet.

Gram’s iodin solution.

The work in this course should begin with Part II,

Section 7, p. 97.

SECTION 2
THE PYOGENIC GROUP
EXERCISE I. THE PYOGENIC GROUP (SUBGROUP A)!

Members—

Staphylococci, streptococci, Microc. tetragenus.

Inoculate agar slants from laboratory cultures of
Staphylococcus aureus, Staph. albus, and Strepto-

tThis subdividing of the pyogenic group is an arbitrary
measure designed to facilitate study, the commoner pyogens

being studied first.
. 10g

106 LABORATORY GUIDE IN BACTERIOLOGY

coccus pyogenes. These organisms are pathogenic,
and care must be taken to observe the rules of technic.
Any carelessness may be followed by grave conse-
quences. In case of accident, such as the spilling of a
culture or infecting of the hands, disinfection—e.g.,
with a solution of mercuric chlorid (1:1,000)—is neces-
sary.

After 24 hours’ incubation of three agar slants,
proceed with the other media as outlined in the routine
study (pp. 50 ff.).

Special study.—Inoculate a rabbit intravenously
with a broth culture of Staph. aureus. The ear of the
rabbit is shaved, and washed with mercuric chlorid
solution, followed by alcohol. Then 1 or 2 c.c. of a
24-hour-old culture in broth is drawn up-into a hypo-
dermic syringe, which has been sterilized by immersion
in boiling water for 10 minutes. 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 hold the fore-legs. If the
animal struggles, force should not be applied, as this
might cause injury. The struggles may be overcome
by wrapping the animal in a towel or some other 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
syringe is then withdrawn and sterilized in boiling
water for 15 minutes.

IMPORTANT PATHOGENIC BACTERIA 107

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 ’
foci of suppuration.

DIRECTIONS FOR AUTOPSIES (SEE SKETCH, FIG. 31)
See Mallory and Wright, Pathological Technique.

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 (1:1,000)
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 along the fore- and hind-legs.

6. Cut the skin away with a moderately sharp
knife, avoiding opening the abdominal cavity.

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

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 exposed. Cultures and smears
should be made from the heart’s blood, peritoneal
cavity, spleen, liver, and localized foci of suppuration.

Gram stains are of special value inasmuch as staphy-
lococci are gram positive, while the tissues are more
or less decolorized.

108 LABORATORY GUIDE IN BACTERIOLOGY

KIDNEY--~ WW-STOMACH
ADRENAL. ty Mw --- fr P-SPLEEN
Stans I er ~E- [TA KIDNEY
\ IN
\ TESTIND s }
‘© ___+/-\LBLapper
i—_-_—_—— O, ;
INGUINAL O 29!
SAREE | VN NR
i Se ed” ae
Fic. 31

Autopsy of a Guinea-Pig(Diagrammatic)

IMPORTANT PATHOGENIC BACTERIA 10g

EXERCISE 2. THE PYOGENIC GROUP (SUBGROUP B)

Members—

Streptococcus pneumoniae.

Micrococcus zymogenes.

Micrococcus gonorrheae.

M. gonorrheae is difficult to cultivate on labora-
tory media. It is a parasite and requires special media
for cultivation. For these reasons it is sufficient to
study the characteristic morphology in smears made
from gonorrheal pus. Methylene blue or Pappenheim’s
stain (p. 45) and Gram stains should be made.

Inoculate agar slants from laboratory ‘cultures of
Str. pneumoniae and M. zymogenes.

References (M. zymogenes)—
MacCallum and Hastings, Jour. Exper. Med., 1899, 4, p. 521.
Harris and Longcope, Centralbl. f. Bakt., 1901, 30, Abt.
I, p. 353 (printed in English).

Birge, Johns Hopkins Hospital Bull., 1905, 16, p. 309.

1. Routine study.—Note the microscopic appear-
ance of both organisms and the action of M. zymogenes
on milk and gelatin.

2. Special study.—The staining of capsules from
a milk culture of Str. pneumoniae. Three methods
may be applied for this.stain:

First method (Friedlinder’s method)—

1. Prepare a stain by the following formula:

Glacial acetic acid..................04- I part

Saturated alcoholic solution of gentian
WiOleticagwnoduigdungee as werounisenee 5 parts

Distilled water......... 0. cee e cece eee 10 parts

2. Prepare a film in the usual manner from a 24-
hour-old culture in milk.

Iro LABORATORY GUIDE IN BACTERIOLOGY

3. Cover with the stain for 10 to 15 seconds.

4. Wash in water.

5. Dry and mount in balsam.

Second method (Welch’s method)—

1. Prepare a film from a 24-hour milk culture by
smearing a loopful thinly over a coverglass.

2. Cover with glacial acetic acid for 15 to 20 seconds.

3. Wash the acetic acid off with carbol fuchsin.

4. Wash off the stain with 0.8 per cent NaCl solu-
tion.

s. Dry and mount in balsam.

Third method (Rosenow’s method) (Jour. Am.
Med. Assoc., 1911, 56, p. 418)—

This method is applicable for staining capsules in
tissues as well as cultures. If the material is too thick
or viscid it must be diluted with a suitable amount of
distilled water. Cultures from agar, blood serum, etc.,
should be mixed on a cover slip with a loopful of serum.

a) Prepare a thin film, and dry in the air.

b) When dry, cover with a 5 to ro per cent solution
of tannic acid for 10 to 20 seconds.

c) Wash in water and dry with blotting paper.

d) Cover with carbol gentian violet or anilin gentian
violet for one-half to one minute. Carbol gentian
violet is prepared by mixing one part saturated alco-
holic solution of Griibler’s gentian violet with 4 parts
of an aqueous 5 per cent phenol solution.

e) Wash in water.

f) Stain with Gram’s iodin solution for one-half to
one minute.

-g) Decolorize in alcohol.

IMPORTANT PATHOGENIC BACTERIA IIt

h) Stain with a saturated alcoholic (60 per cent)
solution of Griiber’s eosin.

4) Wash in water and blot.

j) Clear and mount in balsam.

Whichever method is applied, the capsule should
appear as a lightly stained zone with a well-defined
outline around the deeply stained cell. Capsules are
rarely demonstrable unless the organisms are’ culti-
vated in media rich in proteins, or are mixed with
serum previous to staining.

FIG. 32
Mouse Holder

Special study.—Inoculation of a mouse with Str.
pneumoniae.

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

2. Shave a place on the back above the tail.

3. Wash with a solution of mercuric chlorid
(1: 1,000), followed by alcohol.

4. Inject 0.2 c.c. of a milk culture of Str. pneu-
moniae.

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

6. Make cultures in milk and on slant agar from
the heart’s blood or the spleen.

4. Make a capsule stain from the heart’s blood,
spleen, or other organs.

112 LABORATORY GUIDE IN BACTERIOLOGY

SECTION 3
THE GROUP OF COLON-TYPHOID ‘BACILLI

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

Subgroup 1: the colon group.—This group includes
different varieties of Bacillus coli and B. aérogenes.

Subgroup 2: the B. enteritidis group.—This group
includes B. suipestifer, B. paratyphosus, B. enteritidis,
and B. icteroides. The term “intermediate” is assigned
to this group, because it resembles in part the colon
group and the typhoid group.

Subgroup 3: the typhoid-dysentery group.—This
group includes B. typhosus, varieties of B. dysenteriae,
and B. fecalis alkaligenes.

EXERCISE I. STUDY OF SUBGROUP I
THE COLON GROUP

Inoculate agar slants from laboratory cultures of
B. coli, B. coli anaérogenes,? and B. aérogenes. Also
inoculate one tube of broth with B. coli for the prepara-
tion of sugar-free broth.

References—
Smith, The Wilder Quarter Century Book, Ithaca, 1893, p. 187.
Smith, Amer. Jour. Med. Sci., 1895, N.S. 110, p. 283.
Rogers, L. A., Clark, W. M., and Davis, B. J., “The Colon
Group of Bacteria,” Jour. Inf. Dis., 14, p. 411.

t There are many varieties of B. coli, distinguished from each
other chiefly by their ability to produce gas from various car-
bohydrates. Most varieties produce gas from dextrose and
lactose, some also from saccharose, and a few from dextrose only,
while there is one variety, known as B. coli anaérogenes, which
does not produce gas from any one of the three sugars.

IMPORTANT PATHOGENIC BACTERIA 113

1. Routine study.—Observe carefully the growth on
potato of B. aérogenes. This organism produces an
amylolytic enzym, which manifests itself by gas produc-
tion. Gas bubbles frequently appear in the growth
on potato.

2. Special study.—In order to test the action of
micro-organisms on various carboltydrates, it is neces-
sary 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 decomposes‘ many carbo-
hydrates, 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 fermenta-
tion tube:

1. Dissolve by heat:

Extract: Ff beefvieiccicncice eure 1.5 grams
Peptoniss ons cevadie' ey ead ween 5 grams
in 500 c.c. water. Broth made from chopped beef
(500 g. to 1 liter) may also be used for this purpose.
One per cent pepton should be dissolved in meat in-
fusion (see p. 31).

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

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

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

5. Adjust the reaction to 1 per cent acid.

6. Divide into three equal parts and dissolve 1 per
cent dextrose, lactose, and saccharose, respectively, in
each part, and filter again, if necessary.

114. LABORATORY GUIDE IN BACTERIOLOGY

7. Fill fermentation tubes, taking care to label
each one properly, and sterilize in the arnold on 3 con-
secutive days for 20 minutes or in the autoclave for
5 minutes.

All gas must be carefully tilted out of the closed
arm of the tube while the fluid is warm. When sterili-
zation is completed,’ inoculate one set of the fermenta-
tion tubes with B. coli, another set with B. coli anaéro-
genes, and a third set with B. aérogenes. Inoculate
with the straight or looped needle.

Directions for measuring gas formation in fermenta-
tion tubes and ‘for analyzing the gas are given on p. 81.
The reaction in the closed arm is not always the same
as in the bulb. This may be ascertained by adding a
small amount of litmus solution by means of a suitably
bent glass tube. ;

3. Special study.—Test for indol and nitrites.

a) Test for nitrites: Add to a culture in Dunham’s
solution, or, better, in sugar-free broth, successively 1
drop of each of the following solutions:

(1) Sulphanilic acid........ Swick ee (0.5) gram
Acetic acid (25 per cent).......... Igo C.c.

(2) a Naphthylamine chlorid......... o.1 gram
Distilled water...............005 20 C.C.
Acetic acid (25 per cent).......... Iso G.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, 1 or 2 drops pure sulphuric acid.

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

IMPORTANT PATHOGENIC BACTERIA II5.

(3) A few drops of a solution of o.1 g. potassium or
sodium nitrite in 1,000 c.c. water. Rose color then
indicates the presence of indol only.

The appearance of indol red depends on the pres-
ence of NO,. This is liberated by sulphuric acid
from nitrites, if these are produced by the organism.
If nitrites are not produced, a small amount of a nitrite
solution is added, which then furnishes the necessary
material for production of NO.

Perform these tests with all the organisms of the
intestinal group, and make control tests in sterile Dun-
ham’s solution or sugar-free broth.

4. Special study.—Make a capsule stain of B.
aérogenes from 24-hour-old milk cultures. (For
method see p. 109.)

The study of B. coli is of special importance in con-
nection with bacteriological ganalysis of water (see
Part IV). The presence of this organism in large
numbers indicates sewage contamination, and conse-
quently bacteria such as B. typhosus and B. dysen-
teriae may be present.

EXERCISE 2. STUDY OF SUBGROUP II
THE HOG-CHOLERA, B. ENTERITIDIS, OR INTERMEDIATE GROUP
Inoculate agar slants from laboratory cultures of B.
suipestifer, B. enteritidis (Gartner’s bacillus), and
B. paratyphosus.

References—
B. cholerae suis:
Moore, The Pathology of Infectious Diseases of Animals.
McFarland, Textbook of Bacteriology. ~
B. paratyphosus:
Buxton, Jour. Med. Res., 1902, 7, p. 201.

116 LABORATORY GUIDE IN. BACTERIOLOGY

Wells and Scott, Jour Infect. Dis., 1904, 1, Pp. 72.
Cushing, Johns Hopkins Hospital Bull., 1900, p. 156.
Durham, Jour. of Exper. Med., 1900-1901, 5, Pp. 353-

1. Routine study.—Observe the bluish-green colora-
tion of the cream ring in litmus milk, and make a
test for indol in Dunham’s solution or sugar-free broth.

2. Special study.—Inoculate plain sterile milk with
B. suipestifer. After 8-10 days it will be observed
that the milk is becoming transparent, due to a solvent
action of the alkali produced by the organism upon the
protein content.

3. Special study.—Inoculate fermentation tubes as
with B. coli. Measure and analyze the gas. Compare
the results with those obtained in the study of the colon
group.

4. Special study.—Inoculation of a rabbit sub-
cutaneously with B. suipestifer. Subcutaneous
inoculations of rabbits are made in the following
manner: An assistant, in a sitting position, places the
rabbit back down in his lap. The head projects be-
yond 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. The skin is then pulled up, the syringe in-
serted, and the material injected.

After the rabbit has died, study the lesions pro-
duced 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 cultures on agar from the heart’s blood and other
internal organs,

IMPORTANT PATHOGENIC BACTERIA 117

EXERCISE 3. STUDY OF SUBGROUP III
THE TYPHOID-DYSENTERY GROUP

Use great care in handling members of this group.

Inoculate agar slants from laboratory cultures of
B. typhosus, B. dysenteriae (Shiga), and B. fecalis
alkaligenes.

1. Routine study.—Study the reaction on milk,
and test for indol. Preserve glucose agar cultures for
two weeks for the observation of involution forms.

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

3. Special study.—The staining of flagella.—To
demonstrate the presence of flagella on B. typhosus,
the following method will give good results (Léffler’s
method):

a) The mordant:

Tannic acid (20 per cent aqueous solution). 10 parts
Ferrous sulphate (saturated aqueous solu-

HON) :..0c...adesewadetce seasaecs seuss 5 parts
Fuchsin (saturated alcoholic solution).... 1 part

Add one part 1 per cent NaOH solution for each
too parts of stain.

b) Prepare several cover slips by 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-old agar culture, taking care to
stir the suspension as little as possible. —

e) Place a loopful of water on each of the cover slips.

f) Carry over a loopful of the suspension on the

118 LABORATORY GUIDE IN BACTERIOLOGY

slide to one of the cover slips and from this to each of
the other cover slips.

g) Allow to dry in the air.

hk) Cover with the mordant.

4) Heat over a small flame for 13 minutes while
steam rises, or better, heat on a water bath for 5 minutes.
Replace the evaporated mordant to prevent its drying
on the cover slip.

j) Wash in water.

k) Drain the water off with blotting paper.

1) Cover with anilin gentian violet or carbol fuchsin.

m) Heat as before over a small flame for 13 minutes,
or better, on a water bath for 5 minutes.

n) Wash in water.

o) Dry and mount in balsam.

4. Special study.— Agglutination. — Dried-blood
method of Johnston: A drop of blood of a typhoid
fever patient is obtained by pricking the lobe of the ear,
previously 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 toa
laboratory, where the blood is dissolved in physiologi-
cal salt solution in such a manner as to obtain an
approximate 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. A loopful of the diluted blood is mixed with
a loopful of the suspension on a cover glass, this making
a dilution of 1:50. The cover glassis then inverted over
a hollow slide like a hanging drop and observation made
after two hours’ incubation at 37°C. For laboratory
tests the serum of an animal (either a rabbit or a guinea-
pig) which has been injected with cultures of B. typho-

IMPORTANT PATHOGENIC BACTERIA 119

sus, previously heated for 1 hour at 60° C., is used.
This process kills the organisms, but the toxins remain
active. The first injection is followed by another one
with dead cultures after four to five days, and after the
same intermission a culture of virulent bacilli is in-
jected. By this time the agglutinative power of the
blood is well developed. 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 ina sterile
glass vessel. If the animal is hung head down enough
blood can be collected in a short time. The blood is
placed in the ice chest, and the serum is collected after
separation.

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

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.

6) A suspension of a 24-hour-old agar culture of B.
typhosus in salt solution is prepared. This suspension
should be uniform and not heavy. It is desirable to
filter the suspension through absorbent cotton or sterile
filter paper to remove clumps of bacteria.

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 I: I00.

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.

I20 LABORATORY GUIDE IN BACTERIOLOGY

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

g) When the clumping is completed allow the drop
to dry in the air without stirring or spreading, fix in the
flame, or better with absolute alcohol, and stain with
gentian violet. A permanent preparation can be
obtained in this manner, showing the agglutinated
bacteria.

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

The above-described method of an agglutination
test is known as the microscopic test. Another
method, in which larger amounts of serum and suspen-
sion are required, is known as the macroscopic method.
Small test tubes are used, and. definite amounts of
bacterial suspensions are introduced, by means of
graduated pipettes. The serum is then added in vary-
ing amounts so as to effect the desired dilutions (see
table in appendix, p. 197). The tubes are then incu-
bated at 37°C., usually for 2 hours. After this they
are placed in an ice chest for sedimentation. If com-
plete agglutination has taken place, the bacteria will
have collected in clumps at the bottom, forming a
sediment. The supernatant fluidis 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.

IMPORTANT PATHOGENIC BACTERIA 121

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

SECTION 4
THE PROTEUS GROUP

Inoculate agar slants from laboratory cultures of
Bacillus proteus, Proteus zenkeri, and Bacillus cloacae.

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

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

3. Special study.—Inoculate fermentation tubes and
determine the percentage of gas formed and the gas
formula. Compare the results with those of the in-
testinal bacteria.

Nore.—In order to obtain a clear picture of the differential
characteristics of the three groups of colon-typhoid bacilli and the
proteus group, it is recommended to tabulate the results in
columns, as outlined below. Express positive results by the
sign +; negative, by —. Complete agglutination is expressed
by + +; slight, by +.

LABORATORY GUIDE IN BACTERIOLOGY

122

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IMPORTANT PATHOGENIC BACTERIA 123

SECTION 5
THE CAPSULATED GROUP

Members—
Bacillus capsulatus.
B. rhinoscleromatis.

Inoculate agar slants from laboratory cultures of
B. capsulatus (Friedlinder’s pneumo-bacillus) and B.
rhinoscleromatis.

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

2. Special study.—Staining of capsules from 24-
hour-old milk cultures (see p. 109).

3. Special study.—Intraperitoneal inoculation of a
rabbit with B. capsulatus.

Method of intraperitoneal inoculation.—The rabbit
is held in the same manner as described on p. 106.
The hair is clipped close over the left lower abdominal
quadrant. Then (after washing with mercuric chlorid
1: 1,000 and alcohol) pass the needle at first beneath the
skin, then, holding it at about a right angle to the
abdominal surface, push 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 withdraw
the syringe. If the contents of the needle have been
emptied into the peritoneal cavity, no swelling takes

I24 LABORATORY GUIDE IN BACTERIOLOGY

place, as is noticed in subcutaneous inoculations.
Care is necessary not to puncture the intestines.

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

SECTION 6

THE DIPHTHERIA GROUP

Members—
Bacillus diphtheriae.
B. hofmannii.
B. xerosis.

Use great caution in handling B. diphtheriae.

Inoculate agar slants from laboratory cultures of
B. diphtheriae and B. hofmannii.

1. Routine study.—Stain B. diphtheriae with Léf-
fler’s methylene blue instead of gentian violet. The
staining may be facilitated by the application of mild
heat. Observe the darkly stained granules and make
sketches of some of the bacilli.

2. Special study.—

Neisser’s Granule Stain (Gin’s Modification)—

I. 5 per cent alcoholic solution. Methy-

lene blue (Griibler)................. 20 C.C.
5 per cent glacial acetic acid........ 1,000 C.c,

2. to per cent alcoholic solution. Crys-
tal violet (Héchst)...............0. 10 C.c.
Distilled water..............e eee 300 C.c.

Two parts of 1 with one part of 2; call solution a.

bv AcidlactiC;s ci uczassinercuaenaweas s Ic.c.

Lugol’s solution... ............0008 99 C.c.

c. Chrysoidin (r gram in hot water).... 300.c.

IMPORTANT PATHOGENIC BACTERIA 125

‘Stain film for 2 seconds with solution @ and wash.
Apply solution 6 for 3 to 5 seconds and wash well.
Apply solution ¢ for 3 to 5 seconds, wash, dry, mount.

3. Special study.—Test for acid formation in a
culture, in neutral glucose broth, one week old, by addi-
tion of a few drops of litmus solution, or by titration
with Nyy NaOH solution.

4. Special study.—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 Léffler’s blood serum.

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

b) Wash a hard-boiled egg in mercuric chlorid
solution, followed by alcohol, and break the shell at the
blunt end with sterile forceps, without rupturing the
membrane lining the shell.

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

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

e) Invert and set in the sterilized pot.

f) Incubate at 37°C.

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

5. Special study—

Experiment 1—

a) Clip the hair over a small area on the surface of
the abdomen of a guinea-pig or rabbit.

b) Cut a small opening in the skin, and separate the
skin from the muscles below by pushing in sterile

126 LABORATORY GUIDE IN BACTERIOLOGY

scissors. Expand these slightly and after closing
remove. This forms a small pocket.

c) Carry 1 loopful of a 24-hour-old agar culture into
this pocket.

Experiment 2—

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

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

Observe and compare in both animals the results
by taking note of the ante-mortem phenomena and the
lesions post-mortem.

Experiment 3—

Prepare two guinea-pigs and inject subcutaneously
into one a lethal dose of diphtheria toxin and into the
other the same amount of toxin neutralized with a
sufficient amount of antitoxin. Compare the results
in the two guinea-pigs.

6. Demonstration of methods employed in munici-
pal laboratories for the diagnosis of diphtheria.

7. Study of B. xerosis. Obtain mucus from the
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 methylene blue. Make a
culture on slant agar and if impure, plate out. Trans-
fer cultures to all media from colonies on the plate and
study these in the usual manner.

IMPORTANT PATHOGENIC BACTERIA 127

SECTION 7
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.).

Reference—

Moore, The Pathology of Infectious Diseases of Animals.

_ Inoculate agar slants from a laboratory culture of
B. cuniculicida or another organism of this group. On
account of the enormous infectiousness of B. pestis it is
not desirable to study this organism in classwork.

1. Routine study.—Stain with Léffler’s methylene
blue and anilin gentian violet. Observe “polar staining.”

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

SECTION 8
THE ANTHRAX GROUP
Members—
Bacillus anthracis.
B. subtilis. 7

Great caution is necessary in handling B. anthracis.
Inoculate agar slants from laboratory cultures of
B. anthracis and B. subtilis.

128 LABORATORY GUIDE IN BACTERIOLOGY

1. Routine study.—Observe the colonies formed on
agar plates. Also prepare gelatin plates and study the
colonies.

2. Special study.—Make an “impression prepara
tion” (Klatschpraiparat) from a surface colony on a
gelatin plate.

Method—

a) Clean and flame a cover slip.

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 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).

3. Special study.—Staining of spores.

Méller’s method:

a) Prepare several (five or six) films from agar
or potato cultures of B. anthracis (or B. subtilis).

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 in water.

e) Cover with carbol fuchsin and heat for 5 minutes
on a water bath, or over a small flame.

f) Decolorize with 1 per cent sulphuric acid for 25-
30 seconds.

g) Wash in water.

h) Counterstain with methylene blue for 10-15 sec-
onds without heat.

4) Wash, dry, and mount in balsam.

Note.—The body of the cell should appear blue; the spore.
red.

IMPORTANT PATHOGENIC BACTERIA 129

4. Special study.—Demonstration of filament forma-
tion.

a) Spread a loopful of a broth culture of B. anthracis
or B. subtilis on a 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.

f) Wash in water, dry, and mount in balsam.

5. Special study.—Inoculate a guinea-pig subcu-
taneously with o.2 c.c. of a 24-hour-old broth culture of
B. anthracis, or insert a loopful of a 24-hour-qld agar
culture in a “pocket” under the skin. When the
animal is dead, perform an autopsy, and observe 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
well. Make a culture on agar from the heart’s blood
or from the spleen and study the culture in the usual
manner.

SECTION 9

THE SPIRILLUM GROUP

Members—

Spirillum of Finkler and Prior.

Sp. metchnikovit.

Sp. tyrogenum. 2

And a number of spirilla indigenous to water.

Inoculate agar slants from laboratory cultures of
Sp. of Finkler and Prior, and Sp. metchnikovii. The
spirillum of Asiatic cholera is studied in Section 11B.

~~

I30 LABORATORY GUIDE IN BACTERIOLOGY

1. Routine study.—In addition to the usual media,
inoculate an extra tube of Dunham’s pepton solution
from each organism. Observe from day to day the
action of these two organisms on gelatin, and compare
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.—Test for the nitroso-indol or
cholera-red reaction. (See test for indol, p. 114.)
Make two tests, using one of the cultures in Dunham’s
solution after 24 hours, the other after 6 days. Com-
pare the results of these two tests.

3. Special study.—Stain for flagella by Léffler’s
method (see p. 117).

4. Special study.—Schottelius’ enriching method,
designed to demonstrate the presence of spirilla in
water.

a) Prepare a solution of 2 grams Witte’s pepton and -
o.5 gram sodium chlorid in 100 c.c. of water.

6) Distribute in three small Erlenmeyer flasks, and
sterilize in the autoclave.

c) Inoculate one of these flasks with Sp. metch-
nikovii and B. suipestifer or any other motile bacillus.

d) Incubate at 37° C. for 18-24 hours.

e) After that time, take one loopful from the surface,
inoculate the second flask, and incubate as before.
Also make a stained preparation from the surface of
the solution.

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

IMPORTANT PATHOGENIC BACTERIA 131

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 a film has formed which contains
the spirilla practically in pure culture.

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

SECTION 10
THE GROUP OF ACID-PROOF BACILLI

Members—

Bacillus tuberculosis.

B. leprae. .

B. smegmae.

Méller’s grass bacillus, and a number of bacilli found on
grass, dung, in butter, milk, etc.

Cultural studies of B. tuberculosis consume a great
deal of time and are, therefore, impracticable in an
elementary course.

For comparison, the culture characteristics of
Méller’s grass bacillus are instructive.

Inoculate an agar slant from a laboratory culture of
Miller’s grass bacillus. |

1. Routine study.

132, LABORATORY GUIDE IN BACTERIOLOGY

2. Special study.—Method of staining acid-proof
bacilli.

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

b) Dry and fix as usual.

c) Heat with carbol fuchsin over a small flame for
one minute or on a water bath for two minutes.

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

e) Wash in water and counterstain with methylene
blue for ro seconds (cold).

f) Wash again and mount in balsam.

Note.—Make a second preparation, substituting anilin gen-
tian violet for carbol fuchsin, and Bismarck brown for methylene
blue.

3. Special study.—Stain Méller’s grass bacillus
from an agar culture by the same method, omitting the
counterstain.

4. Special study.—Make a smear from a culture
of Moller’s grass bacillus in milk and stain this smear
for acid-proof bacilli.

SECTION 111A

MISCELLANEOUS ORGANISMS: BACILLUS MALLEI,
BACILLUS MBLITENSES, BLASTOMYCES
DERMATITIDIS ;

B. mallei has been the cause of more accidents
among bacteriologists than any other organism, and
it is therefore not prudent to study this organism in
the laboratory unless there is thorough supervision.

IMPORTANT PATHOGENIC BACTERIA 133

The virulence of B. mallei seems not to diminish in
laboratory cultures, and careless handling may affect all
those who are engaged in work in the same place.
Those who desire to study this organism will find
directions in the following section.

Inoculate agar slants from laboratory cultures of
B. melitensis and Blastomyces dermatitidis.

Carry these cultures through the usual routine.

SECTION 11B

In this section the following organisms have been
included: Spirillum cholerae, Bacillus mallei, Bacillus
influenzae, Micrococcus meningitidis (meningococcus).

A separate section has been devoted to these organ-
isms so as to enable the instructor to omit them if he
deems it advisable. The Sp. cholerae and B. mallei
are dangerous organisms to be manipulated by ele-
mentary students, and unless there is sufficient super-
vision accidents of grave consequences are liable to
happen. The Sp. cholerae may be studied in the
usual routine manner and the test for the so-called
cholera-red reaction (indol reaction) and Schottelius
enriching method (see p. 130) added. 3B. mallei should
also be studied in the usual manner and a demonstra-
tion of its infectiousness made on guinea-pigs.

B. influenzae requires special media for study, blood
agar being the most suitable. The meningococcus
grows to some extent on ordinary media. Stains
with methylene blue and according to. Gram’s method
are instructive, showing the resemblance of this organ-
ism to the gonococcus.

134 LABORATORY GUIDE IN BACTERIOLOGY

SECTION 12

PATHOGENIC TRICHOMYCETES
Members—

Actinomyces bovis (hominis) and Actin. asteroides.

Leptothrix.

Cladothrix.

Nocardia.

1. Inoculate broth and potato from laboratory cul-
tures, and make descriptions and stained preparations
as usual. Other media néed not be inoculated.

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

3. Special study.—Examine a sample of actinomy-
cotic tissue (bovine) in the fresh state, for so-called
“sulphur granules.” Crush some of the material in
salt solution under cover slips and search for ‘‘clubs,”’
using the low and high power dry lenses, and stain by
Gram’s method; counterstain with eosin or Bismarck
brown.

SECTION 13
THE GROUP OF ANAEROBIC BACILLI

* Members—

Bacillus tetani.
B. edematis.
B. welchii.

B. chauvei,

B. botulinus.
And others.

Anaérobic cultivation involves the growth in an
atmosphere devoid of oxygen. This is accomplished
by removing the oxygen from air by chemicals, or

IMPORTANT PATHOGENIC BACTERIA 135

consuming the oxygen by burning paper, or by sub-
stituting hydrogen gas for air. The following methods
are most commonly in use. ‘

1. Patk’s method.

a) Boil three tubes of dextrose agar vigorously for 5
minutes, to drive out the dissolved oxygen.

b) Cool to 43°C. and inoculate from laboratory
cultures of B. tetani, B. edematis, and B. welchii.

c) Solidify rapidly by immersion in cold water.

d) Cover the medium with a thin layer of liquid
paraffin or sterilized mineral oil.

e) Incubate at 37° C.

Note.—The layer of paraffin or oil excludes atmospheric
oxygen which is inhibitory to the growth of anaérobes. The
oxygen necessary for their multiplication is derived from carbo-
hydrates in the medium.

2. Wright’s modification of Buchner’s method.

a) Liquefy, as before, six dextrose agar tubes,
plugged with absorbent cotton. Cool three to 43°C.
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 1 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 stop-
pers, and keep upside down.

' e) Incubate at the required temperature. Pyro-
gallic acid in alkaline solution absorbs oxygen, leaving
the cultures in an atmosphere of nitrogen.

at

136 LABORATORY GUIDE IN BACTERIOLOGY

3. Cultivation by Buchner’s method, using fruit
jars.

a) In a Mason fruit jar of ordinary type deposit
10 g. of pyrogallic acid. $

6) Smear vaselin around the mouth of the jar.

c) Pour into the jar 100 c.c. of a 1 per cent solution
of NaOH.

d) Deposit in the jar culture tubes previously in-
oculated.

e) Fasten the cover of the jar, and incubate at 37° C.
for 48-72 hours.

4. Cultivation in hydrogen gas.

a) Inoculate all media from laboratory cultures.

b) Fit up apparatus as shown in Fig. 33.

c) Place culture tubes in a Novy jar (Fig. 33, @).

d) Open the faucet (6) of the gas generator (c), con-
taining zinc and hydrochloric acid. The hydrogen gas
generated is purified by passing through two jars, one
of which contains concentrated sulphuric acid (d), the
other a ro per cent solution of sodium hydrate (e).
Gradually the Novy jar is filled with hydrogen gas,
which can be ascertained by holding a culture tube
over the opening (f) and then over a burning match
or gas flame. As long as a detonation takes place the
hydrogen is mixed with atmospheric oxygen.. When
the hydrogen in the Novy jar is pure, close it off by
turning the stoppers (g) and (8), and place the jar in
the incubator. The whole process occupies 10 or 15
minutes.

5. A simple and effective method of anaérobic culti-
vation is as follows: Culture tubes, after inoculation,
are placed in a desiccation jar or any other jar with

IMPORTANT PATHOGENIC BACTERIA 137

tight-fitting cover. Vaselin is smeared on the top of
the jar to insure a tight fit of the cover. A small piece
of absorbent paper wetted with a few drops of alcohol
is placed inside of the jar, the paper is lighted, and the
cover replaced. The burning paper absorbs the oxygen,
leaving the cultures in an atmosphere of nitrogen.
When growth has been obtained by any of the above
methods, transfers should be made to milk, and gentian

f e
é
(ae os
// a
WAY > 4.
| {pe
aS ©
AUS yr:
SRR 5
Fic. 33
Anaérobic Cultivation in Hydrogen Gas
a. Novy jar e. Sodium hydrate solution
b. Glass cock jf. Opening of-Novy jar
c. Gas generator g. Stopper

d. Sulphuric acid

violet and Gram stains prepared. Motility should be
determined by preparing a hanging drop. Anaérobic
bacteria in a hanging drop will concentrate toward
the center of the drop away from the oxygen of the air.

6. Special study.—Inoculation of a rabbit with’ B.
welchii.

a) Shave the ear of the rabbit.

b) Wash with mercuric chlorid solution and alcohol.

138 LABORATORY GUIDE IN BACTERIOLOGY

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

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

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

f) 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, without opening the abdomi-
nal 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. 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 modifica-
tion of Welch’s method is as follows: Proceed in the
manner indicated on p. 110, and, after washing the
acetic acid off with the stain (carbol fuchsin or gentian
violet), dry with filter paper and heat the specimen for
5 to 10 seconds before washing. Then proceed as be--
fore.

4. Special study.—Staining of spores of B. tetani
and B. edematis from 3-day-old glucose agar cultures
(see p. 128). ;

8. Special study.—Inoculation of a white mouse
or guinea-pig with B. tetani or its toxin (0.01 c.c.) in
the hind-leg or over the root of the tail (if a mouse).
Note daily the condition of the animal, and when dead

IMPORTANT PATHOGENIC BACTERIA 139

make cultures and cover-slip preparations from the
site of the inoculation.

9. Special study.—Inoculate a white rat with garden
earth subcutaneously or in a pocket above the root of
the tail. Note the condition of the animal daily.
When dead, make cultures and cover-slip preparations
from the site of the inoculation.

SECTION 14

ISOLATION OF UNKNOWN BACTERIA FROM A
MIXTURE

1. Make hanging-drop, stained, and Gram prepa-
rations from the mixture. Note observations and re-
sults.

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

3. Transfer 5 or 6 loopfuls of 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
‘thermostat at 37° C.

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 appear different.

6. Transfer to dextrose agar and litmus milk and
incubate at 37°C. for 24 hours. Retain dissimilar
cultures and proceed with the usual routine study.
Make hanging-drop, stained, and Gram preparations,
transfer to all media, describe the culture character-
istics, and make sketches.

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

140 LABORATORY GUIDE IN BACTERIOLOGY

Capsule stain.

Spore stain.

Stain for acid-proof bacilli.

Fermentation tests of all those which produce gas
in dextrose agar. js

Test for acid in neutral broth.

Test for agglutination.

Test for indol.

Anaé€robic cultivation.

Inoculation of animals.

For final diagnoses the systematic works of Migula,
Matzuschita, and Chester should be consulted.

PART IV

THE BACTERIOLOGICAL EXAMINATION OF
WATER AND SEWAGE

INTRODUCTORY

This work is designed to follow the physical, chemi-
cal, and microscopic examination of water. ‘The
physical examination usually applies to odor, color, and
turbidity. The chemical examination determines the
oxygen consumed, dissolved oxygen, free and albumi-
noid ammonia, nitrites, nitrates chlorin, and hardness,
The microscopic examination refers to algae, protozoa,
etc. Many factors influence the results obtained by
any of these examinations, and in order to gain a clear
insight into existing conditions judgment on the quality
of water should not be passed unless all these examina-
tions and a bacteriological examination have been
completed.

References—

Savage, The Bacteriological Examination of Water Supplies,
London, 1906.

Horrocks, The Bacteriological Examination of Water, London,
Igor.

eee and Winslow, Elements of Water Bacteriology, New
York, 1914.

Ohlmiiller and Spitta, Die Untersuchung and Beurteilung
des Wassers und Abwassers, Berlin, 1910.

Kinnicut, Winslow, and Pratt, Sewage Disposal, New York,
Igio.

Hazen, The Filtration of Public Water Works, New York,
1goo.

ee Methods for the Examination of Water and Sewage.
American Public Health Association, 755 Boylston
Street, Boston, Mass.

Bacteriological Standards .for Drinking Water. Reprint
No. 232 from the Public Health Reports, 1914, Wash-
ington, D.C., Government Printing Office, 1914.

143

144 LABORATORY GUIDE IN BACTERIOLOGY

Whipple, The Microscopy of Drinking Water, New York,
John Wiley & Sons, 1914. ,
Jackson, Biological Studies by the Pupils of W. T. Sedgwick,
1906, p. 292.
Fuller and Johnson, Jour. Exper. Med., 1899, 4, p. 610.
Don, Chisholm, Modern Methods of Water Purification, Lon-
don, 1911.
Apparatus needed in addition to the list given on
PB 5- :
Two hundred culture tubes.
Four wire baskets.
Twenty fermentation tubes.
Twelve Erlenmeyer flasks, about 150 c.c. each.
Six wide-mouth glass-stoppered bottles of about 125 c.c,
capacity.
Twenty petri dishes.
Twenty-five 1 c.c. pipettes.
Ten to c.c. pipettes.

SECTION 1

PREPARATION OF CULTURE MEDIA AND OF DILU-
TION FLASKS

Dextrose or lactose agar—5o tubes. Thirty-five
tubes should contain 10 c.c. of agar for plating; the
others about 7 c.c.

Nutrient gelatin—2o tubes. This gelatin should
contain 12 per cent gelatin. This percentage is
reduced to 10 per cent after the contents of a tube has
been mixed with the litmus solution and the water used
for plating if 10 c.c. gelatin are filled into each tube.

Litmus solution—zo tubes.

Lactose bile fermentation tubes—3o.

EXAMINATION OF WATER AND SEWAGE 145

Dilution flasks, filled with 100 c.c. filtered tap water
or better too c.c. 0.8 per cent NaCl solution, After
sterilization in the autoclave these flasks are assumed
to contain 99 c.c. each. .

Sugar-free meat infusion broth in bulk for fermenta-
tion tubes and in tubes—2o.

In addition to these media the media listed on p. 105
should be prepared.

The reaction of all media should be adjusted to 1
per cent acid to phenolphthalein.

SECTION 2
BACTERIOLOGICAL EXAMINATION OF WATER

EXERCISE I. COLLECTION OF SAMPLES

1. Tie a piece of filter paper or lead foil over 6 glass-
stoppered bottles of about 125 c.c. capacity. Sterilize
these in the hot-air oven for one hour at 160° C.

2. Before taking the sample from surface waters
remove the paper cap, dip the bottle about 12. inches
below the surface, remove the stopper under water,
replace the stopper as soon as the bottle is filled, wipe
dry with a clean cloth or absorbent cotton, and replace
the paper cap.

3. Pack the samples in ice in a suitable container.
They should be kept on ice until ready for examination
either in the laboratory or in the field.

4. Samples from pumps should be collected after
several pails of water have been wasted.

5. Samples from hydrants should be taken after
the water has been running freely for at least 15
minutes.

146 LABORATORY GUIDE IN BACTERIOLOGY

EXERCISE 2. EXAMINATION OF SURFACE WATERS

1. Secure three samples of surface waters from dif-
ferent sources.

2. Shake the samples and prepare dilutions.

Dilution 1. 1:10; remove 1o c.c. from a dilution flask con-
taining 100 c.c. sterile water, and replace these 10 c.c. by 10 c.c.

of the sample.

Dilution 2. 1:100; add 1 c.c. of the sample to a dilution
flask.

Dilution 3. 1:1,000; add rc.c. of the dilution 1: 10 to another
dilution flask.

3. Melt a number of dextrose or lactose agar tubes
in a water bath and cool to 43° C.

4. Place 1 c.c. of sterile litmus solution on each of
12 petri dishes.

5. Place 1 c.c. of the sample and 1 c.c. of each
dilution on the same petri dishes. |

6. Pour the contents of a tube of the liquefied agar
on each of the petri dishes and mix.

7. After the agar has solidified incubate at 37° C.

Note.—If working in pairs, it will be instructive to have one
student incubate the agar plates at 37° C., and the other student
at room temperature. The period of incubation at 37°C. is 48
hours, at room temperature 72 hours. It is also instructive to
plate the same samples and dilutions in gelatin, incubating these

at 20°C. for 48 hours, and comparing the number of colonics
with those appearing on agar.

8. After the plates have been removed from the
incubator count the colonies, using a colony counter.
Make differential counts of acid-forming colonies,
recognized by the reddening of the litmus, and non-
acid-forming colonies.

EXAMINATION OF WATER AND SEWAGE 147

EXERCISE 3. QUALITATIVE AND QUANTITATIVE DE-
TERMINATION OF B. COLI AND STREPTOCOCCI

1. Take one sample and inoculate 1o fermentation
tubes, containing dextrose or lactose broth, each with
1 c.c. of the sample. Similarly inoculate 10 fermenta-
tion tubes with 1 c.c. of each of the dilutions.

Note.—This experiment requires 40 fermentation tubes and
may be divided among several students. This same experiment

should be repeated with lactose bile in place of dextrose or lactose
broth and the results compared.

2. Incubate the fermentation tubes at 37° C.

3. Measure the amount of gas in the closed arm after
24 hours and replace in the thermostat.

4. Measure the gas again after 48 hours and deter-
mine the composition. See p. 81 for directions.

5. Make smears of each tube and apply Gram’s
stain to determine the presence of streptococci.

6. Calculate the number of B. coli and streptococci
present according to the formula given on p. 86. Sub-
stitute the number of tubes containing streptococci
for the letter N in the formula to determine the num-
ber of streptococci.

Nore.—Each fermentation tube containing gas in the closed
arm shows the presence of B. coli. On the assumption that at
least one of the organisms is present in the tube the approximate
number in 1 c.c. of water is calculated.

EXERCISE 4. EXAMINATION OF WELL WATERS

Secure three samples of well water, make dilutions
of 1:10 and 1:100 and examine in the same manner as
directed in Exercises 2 and 3. Compare the results
with those obtained in Exercise 3.

148 LABORATORY GUIDE IN BACTERIOLOGY

EXERCISE 5. EXAMINATION OF RAIN WATER, SNOW,
AND ICE

Secure samples of rain water or snow, and of ice.
Examine these samples, making dilutions up to 1: 1,000
in the manner directed.

SECTION 3
EXAMINATION OF SEWAGE

1. Secure two samples of sewage. Place one of
these in an ice chest. Examine the other sample in
the same manner as directed for water, with this
exception, that dilutions of 1:1,000, 1:10,000, 1:100,-
000, and 1£:1,000,000 are to be used for plating and
inoculation of fermentation tubes, omitting the lower
dilutions.

2. Keep the first sample of sewage in your locker.

3. After seven days examine both samples, i.e., the
one kept in the locker and the one kept in the ice chest,
in the same manner as the fresh sewage was examined.
Note the difference in the results of this examination
as compared to that of fresh sewage.

4. Replace the samples in the locker and ‘ice chest
and examine again after seven and after 14 days.

Norte.—TIi possible a chemical examination for free’ and albu-
minoid ammonia and for nitrites and nitrates should be made
simultaneously with the bacteriological examination. This will
help to give a clear understanding of the changes which have
taken place during the three weeks.

EXAMINATION OF WATER AND SEWAGE 149

SECTION 4
DETERMINATION OF ANAEROBES IN SEWAGE

1. Prepare dilutions of sewage, 1:100, 1:1,000,
and 1: 10,000.

2. Inoculate a series of ro litmus milk tubes for
each dilution with 1 c.c. of each dilution.

3. Heat these tubes in a water bath at 80°C. for
15 minutes. ,

4. Incubate at 37° C. for 48 hours, in an anaérobic
jar (see p. 137).

5. Determine the number of anaérobes present.

Notre.—Heating the tubes at 80° C. kills all vegetative forms
of bacteria, leaving only the spores alive. These develop the
thermostat and their presence is recognized by violent gas forma-
tion and breaking-up of the milk curd. The number of anaérobes
is determined by the same formula as the number of B. coli, sub-
stituting the number of milk tubes showing growth of anaérobes
for the letter N in the formula.

SECTION 5
STUDY OF B.-COLI AND STREPTOCOCCI

Isolate colonies of B. coli and Streptococcus by plat-
ing from the fermentation tubes in lactose litmus agar.
Carry the two organisms through all the media and
determine whether their characteristics are typical
according to the rules laid down in the Committee
Report of the American Public Health Association.
If they are not typical, they must be rejuvenated
according to Fuller and Johnson’s method. This

150 LABORATORY GUIDE IN BACTERIOLOGY

consists in transferring the cultures for three consecu-
tive days in broth and incubating at 20°C. From the
third transfer they are plated in gelatin and a colony
from this transferred to slant agar, from which sub-
cultures are made.

SECTION 6
ISOLATION OF B. TYPHOSUS FROM WATER

1. Prepare 10 tubes each of Endo’s medium, Dri-
galski and Conradi’s medium, aesc. bile-salt agar (p. 33).

2. Prepare plates from water containing B. typhosus
from both of these media.

3. Incubate at 37°C. for 24 hours and isolate the
colonies.

4. Carry the colonies through all media and apply
the agglutination test with typhoid immune serum.

SECTION 7

STUDY OF REACTION OF BACTERIA ON NEUTRAL-
RED BROTH

Prepare a number of fermentation tubes with dex-
trose or lactose broth and add enough of a1 per cent
neutral-red solution until a decided red color has been
imparted. Inoculate these tubes with a variety of
colonies from water or sewage plates and note the differ-
ence in color reaction after 24 and 48 hours’ incubation.

PART V

THE BACTERIOLOGICAL EXAMINATION OF
MILK

INTRODUCTORY

This work is designed to follow the physical and
chemical examination of milk. The physical examina-
tion usually consists in the determination of the specific
gravity,sediment, odor, and general appearance. The
chemical examination determines the fat percentage,
total nitrogen, casein, albumin, milk sugar, acidity,
total solids, solids not fat, and preservatives, chiefly
formalin. To determine the exact character of milk,
physical, chemical, and bacteriological examinations
should be made.

References—

Farrington and Woll, Testing of Milk and Its Products,
Madison, 1908.

Russell and Hastings, Outlines of Dairy Bacteriology, tenth
edition, Madison, Wis., H.L. Russell, 1914.

Ward, Pure Milk and the Public Health, Ithaca, 1909.

Hygienic Laboratory Bulletin No. 56, “Milk and Its Relation
to the Public Health, Washington, 1909.

“Report of the Committee on Standard Methods of the
Bacterial Analysis of Milk,” American Journal of Public
Health, 755 Boylston Street, Boston, Mass.

Lafar, Handbuch der technischen Mykologie, Jena, 1905 to
1908.

Additional apparatus needed.—The outfit given for
the bacteriological examination of water (p. 143) is
to be used in this work, with the addition of ten 5 c.c.
pipettes.

153

154 LABORATORY GUIDE IN BACTERIOLOGY

SECTION 1

PREPARATION OF CULTURE MEDIA AND OF DILU-
TION FLASKS
The same culture media will be used as in the bac-
teriological examination of water (see p. 144), with
this difference, that dextrose agar only is to be prepared,
instead of lactose agar. In addition about ten tubes
of whey agar are to be prepared.

SECTION 2

QUANTITATIVE BACTERIOLOGICAL EXAMINATION
OF MILK

1. Secure samples of raw market milk, of pasteurized
market milk, of certified milk, and of cream.

2. Prepare dilutions as follows:

Raw market milk, 1:1,000 and 1: 10,000.

Pasteurized market milk, 1: 100 and 1:1,000.

Certified milk, 1: 100. .

Cream, 1:1,000 and 1:10,000.

3. Plate these in dextrose litmus agar, making
duplicate plates from each dilution.

4. Incubate one set at 37° C., the other set at 20° C.
The incubation period at 37°C. is 48 hours, at 20° C.
72 hours.

5. Count the colonies and express the results in
numbers of bacteria per cubic centimeter of milk.

6. Make a differential count of acid-forming colonies
and non-acid-forming colonies.

EXAMINATION OF MILK 155

SECTION 3

EXAMINATION OF MARKET MILK FOR TUBERCLE
BACILLI

1. Secure a sample of milk and centrifugalize for
30 minutes at a speed of about 1,200 revolutions per
minute.

2. Mix the sediment and the cream in a sterile tube;
if too thick add a sufficient amount of sterile o.8 NaCl
solution. -

3. Make stains of the mixture for acid-proof bacilli.

4. Divide the: mixture into three parts and inject
subcutaneously into three guinea-pigs.

Three weeks is about the shortest time in which
tuberculosis will develop. The guinea-pigs should be
watched closely after this time, and if any of them die,
postmortem examinations of the lesions should be
made and smears from the affected organs should be
stained to demonstrate the presence of tubercle bacilli.
Three guinea-pigs are used for an experiment of this
nature, because it is impossible to avoid injecting other
bacteria present in milk, which may cause the death
of one or more of the guinea-pigs before tuberculosis
has developed.

SECTION 4
A STUDY OF THE ACID FERMENTATION OF MILK

1. Secure three samples of milk, one of raw milk,
one of pasteurized milk, and one of certified milk.
2. Divide each sample into three parts, and keep one

159 LABORATORY GUIDE IN BACTERIOLOGY

set in an ice chest, one set in the locker, and the third
set in a thermostat at 37° C.

3. Prepare plates in dextrose litmus agar from the
original three samples and incubate these at 37° C.

4. Remove every day, with a sterile 5 c.c. pipette,
5 cc. of the milk from all samples, determine the
acidity by titration with 1/20 N: NaOH and phenol-
phthalein as indicator. When the acidity becomes
constant titrations may be omitted.

5. Prepare plates from all samples every day for
one week, or until the numbers do not increase materi-
ally. It will be necessary to carry the dilutions up to
a million after a day or two of market milk, after three
or four days of pasteurized milk, and toward the end of
the week of certified milk.

6. Differential counts of acid-forming and non-acid-
forming bacteria should be made when this is possible.

7. After the week has passed, allow the samples
to stand for two weeks longer and make titrations and
plates every three days until the acidity and the num-
ber of colonies are constant.

The results of this study should be tabulated, as
they will illustrate the process of “natural souring”
of milk.

If milk is kept at 37° C. for three weeks or longer the
acidity often reaches more than 2 per cent and even
up to 3 percent. This is due to the activity of a group
of bacteria wholly different from the ordinary lactic
acid bacteria. These bacteria grow poorly on ordi-
nary media. Their presence may be demonstrated by
plating the sour milk in beerwort agar, or better, in
whey agar (see p. 36).

EXAMINATION OF MILK 157

SECTION 5

DETERMINATION OF B. COLI AND STREPTOCOCCI IN
MILK
1. Inoculate a series of ten dextrose broth fermen-
tation tubes with 1 c.c. of market milk diluted 1:10,
another series of ten fermentation tubes with a dilution
of 1:100, another series 1: 1,000.

2. Incubate these tubes at 37° C.

3. Measure the gas formed after 24 and 48 hours
and determine the composition of the gas after 48
hours. Directions for this work are given on p. 81.

4. Determine the number of B. coli present in 1 c.c.
of the milk according to the formula on p. 82.

5. Make smears from each fermentation tube and
stain with Gram stain.

6. Examine these smears for streptococci and
determine the number according to the same formula,
substituting the number of tubes containing strepto-
cocci for the letter N in the formula.

7. Plate out one of the fermentation tubes contain-
ing both B. coli and streptococci and carry some of the
colonies through all media.

Note.—The work of this exercise should be repeated with
pasteurized and certified milk. For pasteurized milk the undi-
luted sample and dilutions of 1:10 and 1:100 should be used, for
certified milk the undiluted sample and a dilution of 1:10.

Estimation of numbers of B. coli in milk may be made by

inoculation of fermentation tubes containing dextrose broth
with falling amounts of milk, i.e., 1 c.c.. 0.1 C.c.. 0.01 C.c.. etc.

158 LABORATORY GUIDE IN BACTERIOLOGY

SECTION 6

A COMPARATIVE STUDY OF THE EFFECTS OF PAS-
TEURIZATION AND SO-CALLED STERILIZA-
TION OF MILK*

1. Mix three quarts of raw milk and pour plates
to determine the number of bacteria and the number
of acid-forming bacteria.

2. Divide the mixed milk into three parts. Heat
one-third for 20 minutes at 65° C., boil one-third for
several minutes, and place all parts in an ice chest.

3. As soon as cooled, prepare plates of the milk
heated to 65° C. (pasteurized) and of the boiled milk.
Note the ‘“‘cooked’’ taste and odor of the boiled milk.

4. Check the keeping qualities of the three kinds of
milk by preparing plates on three successive days,
and after that every other day for six days.

It is of importance to make differential counts and
determine the ratio of acid-forming and non-acid-
forming bacteria in all plates.

SECTION 7

A QUALITATIVE AND QUANTITATIVE STUDY OF
ANAEROBES IN MILK

1. Secure a sample of raw milk, one of pasteurized
milk, and one of certified milk.

2. Prepare dilutions of the raw milk 1:10, 1: 100, and

t The term “sterilized milk” is commonly used for boiled milk.

In a bacteriological sense boiled milk is not always sterile, some
of the spores being able to survive boiling.

EXAMINATION OF MILK 159

1:1,000; of the pasteurized milk 1:10 and r1:100, and
of the certified milk 1:10.

3. Inoculate a series of 10 litmus milk tubes with
1 c.c. each of the raw milk, a series of 10 milk tubes
with 1 c.c. each of the dilution 1:10, and the same with
the other dilutions and the other milks.

4. Heat these tubes in a water bath to 80° C. for
15 minutes.

5. Incubate anaérobically for 48 hours at 37°C.

6. Anaérobes will give evidence of their presence
by the breaking-up of the curd formed, and by violent
evolution of gas. The number may be determined
according to the formula given on p. 86 by substitut-
ing the number of milk tubes with growth for the
letter N in the formula.

SECTION 8

A STUDY OF SOME ORGANISMS CAUSING ABNORMAL
FERMENTATIONS IN MILK

1. Place about 50 c.c. certified milk in each of 5
Erlenmeyer flasks.

2. Sterilize these flasks in the autoclave for 5
minutes.

3. Inoculate these flasks with laboratory cultures of
B. prodigiosus, B. cyanogenes, B. viscosus, Sarcina
lutea, and Torula amara.

4. Allow these to remain in the locker or in-an
incubator at 20°C. for three days; examine the con-
ditions as to color and consistency.

160 LABORATORY GUIDE IN BACTERIOLOGY

SECTION 9

EXAMINATION OF MILK FOR MOLDS AND
YEASTS
1. Prepare plates in beerwort agar from undiluted
milk and from dilutions of 1:10, and 1:100.
2. Incubate these at 37° C. and examine for molds
and yeasts.

SECTION 10
EXAMINATION FOR LEUKOCYTES IN MILK

Examination for leukocytes in milk is carried on in
many laboratories and the test was formerly considered
of much value. Recent work, however, has cast
doubt on its significance, since it has been found that
milk of unquestioned purity and soundness may contain
as high as 1,000,000 leukocytes per cubic centimeter,
and with modern methods it is not possible to distin-
guish between leukocytes and pus cells. Leukocytes
are always present in milk in variable numbers. Some
observers believe that the presence of leukocytes and
streptococci together in large numbers indicate pus
in the milk. The reliability of this test, however, has
not been sufficiently demonstrated.

EXERCISE I. STOKES’ METHOD

1. Centrifugalize 100 c.c. of milk.

2. Smear the sediment on a cover glass.

3. Examine under the oil-immersion lens and record
the number of leukocytes and streptococci.

EXAMINATION OF MILK 161

More than five cells in a field are sufficient to con-
demn the milk.

EXERCISE 2. STEWART’S METHOD, MODIFIED BY SLACK

1. Fill 2 c.c. of milk into a tube provided for the
purpose.

2. Centrifugalize at a speed of 2,500 to 3,000 revo-
lutions a minute.

3. The stopper is then removed and the sediment
smeared on a slide so as to cover 4 square centimeters.

4. Examine the slide under the oil-immersion lens
and count the cells and streptococci.

More than §0 cells are sufficient to condemn the
milk.
EXERCISE 3. DOANE-BUCKLEY METHOD, MODIFIED BY

RUSSELL AND HOFFMANN

. Heat sample of milk for 1 minute at 85° C.

. Centrifugalize 10 c.c. for 20 minutes.

. Remove cream and milk, leaving 0.5 c.c. milk.
. Mix and place in a blood counter.

. Allow to settle for 2 minutes.

. Count the cells in several hundred squares.
The average number of cells per square is multiplied
by 200,000 to arrive at number of cells per c.c. of milk.

An PW DN

EXERCISE 4. PRESCOTT AND BREED’S METHOD

1. Remove with capillary pipette with rubber bulb
one drop (0.01 c.c.) of the shaken sample of milk.

2. Spread over x1 square centimeter of a glass slide.

3. Dry by gentle heat.

4. Dissolve out the fat with xylol.

5. Fix smear in alcohol for a few minutes.

6. Overstain with methylene blue.

7. Decolorize with alcohol.

162 LABORATORY GUIDE IN BACTERIOLOGY

SECTION 11

A STUDY OF GROUPS OF BACTERIA IN MILK
(Ayers and Johnson)

1. Prepare plates from a sample of milk.

2. Pick off all colonies from a suitable plate and
inoculate each colony in a tube of litmus milk.

3. Incubate for 2 weeks at 37°C.

4. Note all reactions and classify in groups as
follows:

a) Acid-coagulating group.

b) Acid-forming without coagulation.

c) Inert group, ie., no change in the milk.

d) Alkali-forming group.

e) Peptonizing group.

This experiment should be made with raw milk and
the same milk pasteurized. The results should be
compared.

PART VI

THE BACTERIOLOGICAL EXAMINATION OF
SOIL

INTRODUCTORY.

For this work a knowledge of elementary bacteri-
ology and quantitative and qualitative chemistry is
indispensable.

- References—
Lafar, Handbuch der technischen Mykologie, Jena, 1905 to
1908, ;
Bulletin 94, lowa Experiment Station (Soil Samplers).

Apparatus needed in addition to the list given on
p. 5:

Two hundred culture tubes.

Twelve Erlenmeyer flasks, 150 c.c. each.

Six Erlenmeyer flasks, 500 c.c. each.

Twelve fermentation tubes.

One mortar and pestle (wedgewood or porcelain).

One soil sampler.

The media required may be made up as the work
‘ requiring them comes up. Besides the special media
which will be needed a set of media similar to those
given for medical bacteriology will be used. See p. 105.

SECTION 1
QUANTITATIVE DETERMINATION OF BACTERIA AND
SPORES IN SOIL
EXERCISE I. SECURING SAMPLES OF SOIL
Soil samplers are used and cylinders of soil removed
from the upper eight inches of soil. Cylinders of soil
should be taken in various parts of the field so as
165

166 LABORATORY GUIDE IN BACTERIOLOGY

to obtain an average representation of the soil. The
cylinders are wrapped in sterile paper provided for
that purpose and taken to the laboratory. Take
samples in this manner from various kinds of soil, viz.,
garden soil, sand, loam, clay, manured fields, etc.

EXERCISE 2. PREPARATION OF SAMPLES FOR BAC-
TERIOLOGICAL EXAMINATION

1. The samples from the same kind of soil are mixed
in a sterile mortar.

2. Place part of each of these composite samples
in a sterile glass tube.

3. Weigh accurately ro grams from each composite
sample and dry in a hot-air oven for one hour at 100° C.
Cool and place in a desiccator. After 24 hours weigh
again and calculate the percentage of dry matter
present.

EXERCISE 3. PREPARING SOIL SUSPENSIONS AND
DILUTIONS AND POURING PLATES

1. Prepare .sterile dilution flasks. Fill 100 c.c.
water in Erlenmeyer flasks of about 150 c.c. capacity.
Sterilize these in the autoclave for 10 minutes. As
there is a loss by evaporation during the process of
sterilization the remaining amount is approximately
99 C.c.

2. Weigh the tube containing the sample of soil.

3. Remove about 10 grams by shaking the tube or
by means of a sterile knife or spatula. Place in a
sterile mortar.

4. Weigh the tube again and determine the precise
amount of soil removed.

5. Grind the soil sample in the mortar with some

EXAMINATION OF SOIL 167

sterile water from one of the dilution flasks for one
minute.

6. Add some more water, so that the final volume
is 100c.c. The suspension should be perfectly uniform.

7. Shake the suspension vigorously for three
minutes.

8. Prepare dilutions from this suspension in the fol-
lowing manner: Add 1 c.c. suspension to a flask with
99 c.c. sterile water. Shake well. Remove 10 c.c.
water from another flask and add 10 c.c. of the dilu-
tion of 1:100 to this flask. This makes a dilution of
1:1,000. Then add 1 c.c. of the dilution of 1:100 to
another flask, making a dilution of r:10,000. 1 c.c.
from the dilution 1:1,000 added to another flask makes
a dilution of 1:100,000. Finally add 1 c.c. of the dilu-
tion r:10,o00 to another flask, making a dilution of
1! 1,000,000.

g. Pour duplicate agar plates from all dilutions and
incubate for four days at room temperature. Plates
should be prepared in both agar and gelatin. ,

ro. Examine the gelatin plates daily, and count the
colonies éarlier than four days if there are many
liquefiers present.

zz. After counting the colonies on both agar and
gelatin calculate the number present in one gram of
dry soil.

12. Examine the colonies and determine as near as
possible the group to which they belong according to
Chester’s Determinative Bacteriology. In most cases
stained preparations and hanging drops will give this
information. If not, cultures on slant agar must be
prepared and the organisms studied in the usual
manner by making subcultures.

168 LABORATORY GUIDE IN BACTERIOLOGY

EXERCISE 4. DETERMINATION OF THE NUMBER OF
SPORES IN SOIL

1. Add 1 c.c. of the soil suspension 1:1,000 to a
tube of melted gelatin.

2. Heat the tube in a water bath to 80°C. for 10
minutes. :

3. Pour the gelatin on a petri dish and incubate
at room temperature for three or four days.

4. Count the colonies and calculate the number
for one gram dry soil.

Each colony represents one spore originally present
in the soil.

SECTION 2

A STUDY OF THE PEPTONIZATION OF PROTEINS BY
SOIL BACTERIA

EXERCISE I. DIGESTION OF BLOOD SERUM

1, Place 200 c.c. blood serum in a 500 c.c. flask
and sterilize in the autoclave.

2. After cooling, add 5 c.c. of a suspension of garden
soil in water.

3. Incubate at 37° C. until all of the serum has been
liquefied.

EXERCISE 2. DETERMINATION OF THE CHANGES PRO-
DUCED IN BLOOD SERUM
1. Add a suitable quantity of distilled water to the
liquefied blood serum.
2. Filter to remove bacterial masses and impurities.
3. Neutralize with NaOH, using litmus as indi-
cator.

EXAMINATION OF SOIL 169

4. Boil the solution. If a precipitate forms, “it is
due to undigested albumins.

5. Filter and add ammonium sulphate to the filtrate
to saturation. Albumoses will be precipitated.

6. Allow the mixture to stand over night, then
filter.

7- Dissolve the precipitate in distilled water.

8. Add to the dissolved precipitate an excess of
NaOH and a few drops of copper sulphate solution.
This will produce the characteristic color of the biuret
reaction for the albumoses present.

g. Add to the filtrate left from No. 6 an excess of
NaOH and a few drops of copper sulphate solution.
This will give the biuret reaction for peptons.

EXERCISE 3. THE DIGESTION OF GELATIN

1. Prepare gelatin plates with a dilution of garden
soil suspension.

2. Isolate one of the liquefying colonies by trans-
ferring it to slant agar. Make subcultures and
determine the species.

3. Sterilize 200 grams of gelatin in a 500 c.c. flask
and inoculate this heavily with a culture of the lique-
fying organism obtained in the previous experiment.

4. Incubate at room temperature until the gelatin
is completely liquefied.

5. Determine the composition in the same manner
as in Exercise 1.

EXERCISE 4. THE DIGESTION OF CASEIN

1. Precipitate the casein in a quantity of milk by
means of dilute acetic acid.
2. Filter.

170 LABORATORY GUIDE IN BACTERIOLOGY

3. Wash the precipitate with dilute acetic acid until
the milk sugar has been removed.

4. Place in a 500 c.c. flask and add 300 c.c. distilled
water.

5. Neutralize with NaOH and sterilize in the auto-
clave.

6. Inoculate with soil suspension.

7. Incubate at 37°C. until all the casein has been
dissolved.

8. Determine the composition of the solution as in
Exercise 1.

9. Make plates in gelatin and isolate the organisms.

Note.—The precipitated casein has to be washed in order
to remove all traces of milk sugar, otherwise acid-forming bacteria
will multiply rapidly and crowd liquefiers out.

SECTION 3

THE FORMATION OF AMIDO COMPOUNDS AND
AMMONIA

EXERCISE I. THE FORMATION OF INDOL

1. Prepare 200 c.c. Dunham’s solution and place
in a 500 c.c. flask.

2. Sterilize in the autoclave.

3. Inoculate with a pinch of garden earth and incu-
bate at 37° C. for one week.

4. Remove a small amount with a sterile pipette and
add a few drops sulphuric acid. Follow this up with
a few drops of a o.oor per cent solution of potassium
or sodium nitrite.

The appearance of a red color indicates the presence
of indol.

EXAMINATION OF SOIL 171

EXERCISE 2. THE FORMATION OF AMMONIA

Experiment 1.—Formation of ammonia from pep-
ton—

1. Test the remainder of the solution in Exercise 1
for ammonia, ‘

2. Make a quantitative determination of ammonia
by distillation and nesslerization.

‘Experiment 2.—Formation of ammonia from urea—

1. Prepare a solution as follows:

ULES cis chess cAaieieniateo dey Choise I gram
Pepton sins s arsuceiewss tiene eres 0.2 gram
Distilled water.............0000 100) C.C.

This solution must not be sterilized, as heat breaks
up urea into ammonia and carbon dioxid.

2. Inoculate with some garden earth.

3. Incubate at room temperature for one week.

4. From time to time moisten a piece of filter paper
with Nessler’s solution and pour a few drops from the
flask on the paper. A yellow or brown color indicates
the formation of ammonia.

SECTION 4

THE FORMATION OF NITRITES FROM AMMONIA
AND ISOLATION OF NITRITE BACTERIA

EXERCISE I. THE FORMATION OF NITRITES

1. Prepare five flasks and place 50 c.c. of the solu-
tion on p. 38 in each. Number these flasks con-
secutively.

2. Inoculat? flask 1-with soil, and incubate at room
temperature.

172, LABORATORY GUIDE IN BACTERIOLOGY

3. As soon as growth has appeared transfer some of
the growth from the first flask to the second.

4. As soon as growth has appeared in the second
flask transfer some of the growth to the third flask.

5. Repeat the procedure until all five flasks have
been inoculated. This method of transferring repeat-
edly gradually weeds out all bacteria but those which
are able to grow in the solution, and these may thus
be obtained in pure culture.

6. Examine these bacteria by making stains, hang-
ing drop, spore stain, etc.

7. Test each flask for nitrites with the starch iodin-
test as follows. Prepare the following solutions:

a) Dissolve 2 grams starch in 100 c.c. of water by
boiling.

b) Dissolve one gram potassium iodid in 100 c.c. of
distilled water. :

The potassium iodid solution must be freshly pre-
pared. Add a few drops of diluted H.SO, to liberate
nitrous acid. Then add 1 c.c. of each of the solutions
to 5 c.c. of the fluid to be tested. If nitrous acid is
present, iodin will be liberated and will give a blue
reaction with starch.

EXERCISE 2. ISOLATION OF NITRITE BACTERIA

Experiment 1.—Agar method—

1. Prepare the agar medium described on p. 38. '

2. Fill 10 tubes, and sterilize in the autoclave.

3. Pour plates from this medium inoculated with
the contents of the flasks prepared in Exercise 1 in
various dilutions. :

4. Incubate at room temperature until growth has
appeared.

EXAMINATION OF SOIL 173

5. Transfer some of the colonies to slanted agar
prepared as described on p. 38.

6. Study the colonies by stains, hanging drop, etc.,
and identify the organism.

Experiment 2.—Silica jelly method—

1. Prepare some tubes with silica jelly as described
on p. 38.

2. Melt these tubes, inoculate several with a soil
suspension, others with the contents of the flasks
prepared in Exercise 1, others with colonies obtained
by the washed agar method.

3. Incubate the plates at room temperature and
study the colonies in the usual manner.

Collodion sacs for the dialysis of silica are made in
the following manner:

Depending on the size desired a culture tube or an
Erlenmeyer flask or any other suitable glass vessel can
be used. The finished sac is always much smaller
than the vessel used and this shrinkage must be con-
sidered in choosing the right size of vessel. Celloidin
is dissolved in a mixture consisting half of ether and
half of alcohol in such quantity that the solution con-
tains 6 per cent celloidin. Celloidin usually is covered
with water. This must be washed off with alcohol
before the celloidin is dissolved. A quantity of the
celloidin solution is then poured into the flask and the
flask rotated until the whole inside surface is covered
with the solution. The flask is then inverted and the
-celloidin allowed to drain off so that a thin coat re-
mains on the sides of the flask. The flask must then
be rotated and air blown into it until the celloidin is
almost dry. The process is then repeated so that the

174. LABORATORY GUIDE IN BACTERIOLOGY

inside surface of the flask is covered with two coats.
For large sacs three coats should be applied. When
the celloidin is nearly dry water should be run into
the flask until filled. This water should be changed
several times. Commencing at the top of the flask
the film is separated from the glass and by means of a
smooth instrument, for instance, a glass rod which
has been rounded off at the end, the film is slowly
removed from the glass. As soon as there is a fairly
large part of the film separated, the water may be
emptied out and water run in between the film and the
glass. In this manner the film is gradually removed
from the glass. If the celloidin is too dry before the
flask is filled with water, it will crack and will be
difficult to remove from the glass. The technic is not
difficult and with some practice and care sacs can be
made which will be large enough to hold two liters of
liquid.

SECTION 5

THE FORMATION OF NITRATES FROM NITRITES,
AND ISOLATION OF NITRATE BACTERIA

EXERCISE I. THE FORMATION OF NITRATES FROM
NITRITES

1. Prepare a solution as described on p. 39.

2. Prepare a series of five flasks and place 50 c.c. of
the solution in each.

3. Sterilize in the autoclave.

4. Inoculate the first flask with soil and incubate at
room temperature.

5. As soon as growth has appeared inoculate a
second flask, from this one a third, etc., as in Exercise 1,

EXAMINATION OF SOIL 175

Section 4. The bacteria will finally appear in pure
culture.

6. Determine the presence of nitrates. For this
determination proceed ‘as follows: _

a) Preparation of phenol sulphonic acid: 30 grams
phenol are dissolved in 370 grams concentrated
sulphuric acid in a round-bottom flask. Immerse
completely in a water bath and heat for six hours.

b) Dissolve 20 grams KOH in 20 c.c. distilled water.

c) Evaporate a small amount of the culture to dry-
ness on a water bath.

d) Add to the residue one c.c. phenol sulphonic
acid and rub with a glass rod.

e) Add enough KOH solution to make the solution
alkaline.

f) Dilute with distilled water.

If nitrates are present this will be indicated by the
appearance of a yellow color.

EXERCISE 2. ISOLATION OF NITRATE BACTERIA

These bacteria may be isolated with agar or silica
jelly in the same manner as in Section 4, Exercise 2.

SECTION 6

THE ASSIMILATION OF FREE ATMOSPHERIC NITRO-
GEN AND ISOLATION OF THE BACTERIA

EXERCISE I. BY LEGUME BACTERIA (TUBERCLE
BACTERIA)
Experiment 1—
1. Secure the roots of a clover plant, or any other
legume. Note the appearance and distribution of
the tubercles. :

176 LABORATORY GUIDE IN BACTERIOLOGY

2. Crush between two slides a small tubercle and a
large one.

3. Make mounted preparations and examine them
under the microscope.

Experiment 2—

1. Wash a number of tubercles in distilled water,
then soak in mercuric chlorid solution 1 :1,000 to-
sterilize the exterior of the tubercles.

2. Wash again in several changes of sterilized
distilled water.

3. Crush in a sterile petri dish.

4. Plate in agar, prepared as.described on p. 38.

5. Incubate at room temperature.

6.. After growth has appeared transfer to all ordinary
laboratory media.

7. Study characteristics in the usual manner.

EXERCISE 2. BY ORGANISMS OTHER THAN LEGUME
BACTERIA

Experiment 1—

1. Prepare 5 flasks and place 50 c.c. of the solution
on p. 39 in each flask.

2. Sterilize in the autoclave.

3. After growth has appeared inoculate a second
flask from the first, from the second to the third, ete.

4. Determine the gain in nitrogen by the Kjeldahl
method. This determination should be repeated at
regular intervals to determine the progress of nitrogen
assimilation.

Experiment 2.—These organisms may be obtained
in pure culture by plating in agar or silica jelly media.

EXAMINATION OF SOIL 177

SECTION 7

THE REDUCTION OF NITRATES TO NITRITES AND
ISOLATION OF THE BACTERIA

1. Prepare a flask of pepton broth and add 0.5 per
cent potassium nitrate.

2. Inoculate with a small quantity of soil.

3. Incubate for several days at room temperature.

4. Test by the starch iodin method as described in
Section 4, Exercise 1.

5. Obtain pure cultures by plating on nutrient
agar.

SECTION 8.
THE REDUCTION OF NITRATES TO FREE NITROGEN

1. Prepare several large fermentation tubes with
the medium described on p. 40.

2. Inoculate the closed arm of the fermentation tubes
with varying quantities of soil.

3- Incubate at 37°C.

4. When gas evolution has ceased determine the
percentage of gas.

5. Determine the composition of the gas.

6. These bacteria may be isolated by plating in
agar, or better in a medium prepared by adding agar
to the original solution used in the fermentation tubes.

7. Study the morphology of the bacteria in pure
culture.

178 LABORATORY GUIDE IN BACTERIOLOGY

SECTION 9

GROWING LEGUMES IN SAND AND IN SAND INOCU-
LATED WITH TUBERCLE BACTERIA

Secure some flower pots and fill them with sand.
Plant some species of legume and soak the sand in half
the number of pots with suspensions of legume bacteria.
The difference in growth between those planted in pure
sand and those planted in inoculated sand will be evi-
dent after a short time.

PART VII

MOLDS, YEASTS, TORULAE, AND ACETIC-ACID
BACTERIA

INTRODUCTORY

References—
Kloécker, Die Géhrungs Organismen (translated into English).
Hansen (translated by Miller), Practical Studies on Fermenta-
tion, London, 1896.
Jorgensen, Microérganisms and Fermentation, London, 1900.
Green, Soluble Ferments and Fermentation, Cambridge, 1901.
Lafar, Handbuch der technischen Mykologie, Jena, 1905 8.
Marshall, Microbiology, Philadelphia, P. Blakiston, Son &

Co., 1911.

Apparatus needed in addition to the list on p. 5:
Fermentation tubes............ce eee ceceees 24
Erlenmeyer flasks, 150 c.c. each.............. 24
Sténde’ Cishesis,; pes ccc cured peewee Io
Glass: tttmblers's cc ciccoie os weaesaus s haces ae Io

SECTION 1

PREPARATION OF CULTURE MEDIA

Prepare 25 tubes of each of the following media:

Meat extract agar, partly for slants and partly for
plating.

Dextrose agar.

Litmus milk.

Liquid beerwort.

Beerwort agar for plating.

Beerwort gelatin.

Also fill 12 Erlenmeyer flasks with 50 c.c. beerwort
each, and

Meat extract broth 5 tubes.

Beerwort may be sterilized in a cask in the follow-
ing manner: Fit a cork with two holes in the bunghole

181

182 LABORATORY GUIDE IN BACTERIOLOGY

and insert two glass tubes through the holes. The
tubes should reach to the bottom of the cask (see
Fig. 35). The open ends of both tubes should be
plugged with cotton and have a constriction below the
cotton, to prevent it from slipping down. One of the
tubes is connected with the air valve of an autoclave
and steam passed through the wort for 30 minutes. - The
process is repeated on the two following days. Sterile
wort may be obtained from the cask by blowing through
the tube e and collecting from the bent tube, which,
when not in use, should be protected from contamina-
tion by a sterile test tube with a cotton plug.

SECTION 2
A STUDY OF MOLDS

EXERCISE I. COLLECTING MOLDS FROM THE AIR

1. Expose two plates of wort agar and two of meat
extract agar to the air in different places,

2. Incubate at 37° C.

3. After molds have appeared transfer three different
colonies to slanted wort agar. Transfers are made by
touching the ends of the hyphae with a platinum needle
and streaking on the slanted surface of a tube of wort
agar. :

4. After full development transfer some of the spores
to liquid wort or broth. This is done by touching the
ends of the filaments with a platinum needle.

5. Examine a large loopful of this spore suspension
under the microscope, using a low magnification.

MOLDS, YEASTS, TORULAE, AND BACTERIA 183

6. If the spores are numerous dilute the suspension
until only a few appear in a loopful.

7. When the dilution is high enough to show but
a few spores under the microscope transfer a large
loopful to a cover slip.

er one

Fic. 34
: Sterilizing Wort in a Cask
w. Cask 6. Rubber stopper c. Cotton plugs d. Test tube
e. Glass tubes ff. Cotton stopper and connection with autoclave

8. Invert this cover slip over the ring of a Béttcher
Moist Chamber (Fig. 35).

g. Incubate at 37° C.

10. Observe the hanging drop every day and make
sketches. After 7 to 10 days the cycle of development
will be completed so that a new crop of spores has
appeared.

184 LABORATORY GUIDE IN BACTERIOLOGY

The Béttcher Moist Chamber consists of a slide, a
cover slip, and a glass ring (see Fig. 35). The glass
ring is held on the slide by a mixture of rosin and castor
oil. A few drops of sterile water are placed in the
bottom to maintain moisture in the hollow of the ring.
The inverted cover slip with the hanging drop is held
in position by smearing vaselin around the top of the
glass ring.

eeGuds
weweresrrrssis tte «
iw emrees souteeses Ae
= == Se lig anf a
Fic 35
Béttcher Moist Chamber
uv. Slide : b. Glass ring c. Cover slip
d, Hanging drop c. Sterile water
EXERCISE 2

Preparation of slides for microscopic study of molds,
also stained preparations (see p. 79).

EXERCISE 3. STUDY OF MOLDS IN CHEESE

1. Secure some Roquefort and some Camembert
cheese.

2. Pick out with a looped needle some of the dark
spots in the Roquefort cheese and some of the mold on
the Camembert cheese.

3. Make streaks of this material on wort agar plates.

4. Isolate the molds and study as in Exercises 1
and 2.

EXERCISE 4. STUDY OF THE AMYLOLYTIC ACTION OF
MOLDS

1. Prepare cultures of Aspergillus oryzae.

2. Add some starch to liquid wort in a flask and
sterilize.

MOLDS, YEASTS, TORULAE, AND BACTERIA 185

3. Inoculate: with the mold and test for sugar
formation by the Fehling test from day to day.

EXERCISE 5. CULTIVATION OF MOLDS IN RAULIN’S
SOLUTION

1. Preparation of Raulin’s solution (see p. 42).

2. Place about 50 c.c. of the solution in three Erlen-
meyer flasks and sterilize in the autoclave.

3. Inoculate the surface with molds from three
species of molds.

4. Incubate at 37° C.

5. After growth has appeared examine the molds
and make sketches.

EXERCISE 6. A STUDY OF MOLDS FROM LABORATORY
CULTURES
Study cultures of molds furnished in the same
manner as outlined in the first five exercises of this
section.

SECTION 3

A STUDY OF YEASTS
Nore.—All yeast cultures are to be incubated at 25° C. unless
otherwise directed.
EXERCISE I. STUDY OF YEASTS FROM THE AIR

1. Expose two plates of wort agar to the air in
different places, or if the plates exposed in the previous
section have colonies of yeasts these may be used.

2. Transfer three colonies, which appear to differ
from each other by microscopic examination, to tubes
of slant wort agar, and incubate.

186 LABORATORY GUIDE IN BACTERIOLOGY

3. After growth has taken place transfer from these
agar slants to the following media: Meat agar slant,
dextrose agar, litmus milk, wort gelatin, liquid wort.

4. After 24 hours’ incubation make stains with
gentian violet and by Gram’s method, examine the
unstained cells in water, and make sketches.

EXERCISE 2. A STUDY OF GAS EVOLUTION BY YEASTS

1. Prepare 2 per cent solutions of the following
carbohydrates in wort: Dextrose, saccharose, lactose,
mannit, levulose, maltose.

2. Fill three fermentation tubes from each of these
solutions and sterilize for three consecutive days in the
Arnold.

3. Inoculate each set with the three yeast cultures
studied in Exercise 1.

4. Measure the percentage of gas evolved after
24 and 48 hours.

5. Determine approximately the composition of the
gas formed (see p. 82), using a 4 per cent NaOH
solution. :

6. Tabulate the results, stating the amount and
composition of the gas formed from each carbohydrate
by the three species of yeasts.

EXERCISE 3. A STUDY OF FILM FORMATION

1. Prepare cultures in liquid wort from three species
of yeasts.

2. After growth has appeared inoculate three flasks
containing sterile wort with each one of the yeasts.
This should be done after pouring the supernatant
liquid off, inoculating with a loop from the sediment.

3. Incubate these flasks as follows: One set at 37° C.,

MOLDS, YEASTS, TORULAE, AND BACTERIA 187

the second set at room temperature, and the third set
at 25°C.

4. Note which temperature is most favorable to
film formation.

5. Examine under the microscope some cells from
the film and some from the sediment, also make per-
manent stained preparations. It will be observed
that there is a difference in morphology between the
cells from the film and those from the sediment.. Also
note the decolorization of the wort.

6. Determine by distillation approximately the
amount of alcohol produced.

EXERCISE 4. A STUDY OF SPORE FORMATION

. Prepare cultures in liquid wort.
. Prepare gypsum blocks as described on p. 80.
3. After growth has taken place in the tubes pour
off the supernatant fluid and smear the sediment on the
surface of the gypsum blocks.
4. Incubate the gypsum blocks at 25° C.
5. Examine a small amount from day to day until
spores are present, and make sketches.

no oe

EXERCISE 5. PREPARATION OF PURE CULTURES OF
YEASTS FROM ONE CELL

The preparation of pure cultures from one cell has
become of vast importance in breweries. It is rela-
tively difficult to prepare pure cultures of bacteria
from one cell, these being smaller than yeasts and,
therefore, more difficult to manipulate. Barber
(Jour. Infect. Dis., 1908, 5, p. 379) has succeeded in
devising a practical method for isolating single cells of
bacteria and preparing pure cultures from these.

188 LABORATORY GUIDE IN BACTERIOLOGY

Experiment 1.—The dilution method—

1. Prepare a culture of a species of yeast in liquid
wort. ‘

2. Examine a large loopful under the microscope
and dilute the culture with sterile wort until there is
an average of about one cell to the loopful.

3. Inoculate a series of ten tubes of liquid wort
with a loopful each of the diluted culture, or inoculate
with a drop from a sterile capillary pipette.

4. Incubate.

All those tubes in which growth has appeared con-
tain a culture originating from one cell.

Experiment 2.—Hansen’s gelatin method—

1. Prepare a culture of a mixture of two or three
species of yeast.

2. After growth has appeared inoculate a tube with
liquefied wort gelatin at a temperature of about 30 to
35°C.

3. Examine under the microscope and dilute with
liquefied gelatin until there are only one or two cells
to each loopful.

4. Prepare cover slips in the following manner:
Dip several cover slips into liquid paraffin. After the
paraffin has solidified draw lines through the paraffin
with a sharp steel needle, so as to form a number of
squares. Mark each square with numbers or letters
with the same needle. Dip the cover glass into hydro-
fluoric acid for a few seconds, wash in water, then
chloroform, ether, and alcohol until the glass is clean
and free from fat. The hydrofluoric acid will have
etched the glass so as to show the marks permanently.

5. Cover the slip with a thin coat of the diluted
wort gelatin containing the culture.

MOLDS, YEASTS, TORULAE, AND BACTERIA 189

6. After the gelatin has solidified, paint the top of
the glass ring of a Béttcher Moist Chamber with
vaselin and invert the cover slip, gelatin down. Do
not neglect to place a small amount of water inside the
moist chamber.

7. Examine under the microscope and make a
sketch corresponding to the figures on the cover slip
and make a mark for all single cells observed. It is
possible then to find the cells again by comparing the
cover slip with the sketch. '

8. Incubate at room temperature.

g. Observe from day to day the growth of colonies
from single cells.

to. When the colonies are large enough to be picked
up with a platinum needle, inoculate several tubes of
liquid wort with the colonies.

11. Incubate at 25°C. and after growth has
appeared transfer the sediment from each tube to a
flask containing liquid wort.

12. Incubate these flasks and study the cultures by
making microscopic examination and stains, study the
film and the sediment separately, inoculate fermenta-
tion tubes with the various carbohydrates, and inoculate
gypsum blocks. Also note the aroma, and determine
the amount of alcohol formed.

EXERCISE 6. A STUDY OF LABORATORY CULTURES
Prepare cultures from Saccharomyces cerevisiae,
Sacch. pastorianus, and Sacch. ellipsoideus on liquid
wort. After 24 hours’ incubation transfer to all media
and study these veasts in the same manner as described
as to gas formation, film and sediment formation, spore
formation, etc.

190 LABORATORY GUIDE IN BACTERIOLOGY

EXERCISE 7. EXAMINATION OF BREWER’S YEAST

i. Secure some brewer’s yeast.

2. Inoculate a tube of liquid wort and incubate at
25°C.

3. Prepare pure cultures from this mixture accord-
ing to Hansen’s method..

4. When pure cultures have been obtained, study
these in the same manner as previously directed.

SECTION 4

EXAMINATION OF BAKER’S YEAST

1. Prepare 5 flasks with 100 c.c. sterile water.

2. Secure a cake of pressed yeast.

3. Prepare a suspension of I gram in Ioo c.c.
sterile water.

4. Weigh out about one gram, evaporate on a water
bath, place in a calcium chlorid desiccator over night,
weigh again, and determine the percentage of moisture.

5. Remove 10 c.c. water from a flask containing
100 c.c. sterile water, and replace with ro c.c. from the
suspension.

6. Transfer 1 c.c. of the suspension to another dilu-
tion flask.

7. Transfer 1 c.c. from the second flask to a third
dilution flask. The dilutions prepared are 1:100,
I!I,000, I:10,000, I:100,000, not considering the
amount cf moisture in the gram of yeast suspended.

8. Pour meat extract agar and wort agar plates
from all dilutions.

9. Incubate the meat agar plates at 37° C. and the
wort agar plates at 25°C.

MOLDS, YEASTS, TORULAE, AND BACTERIA 1091

to. After.28 hours count the number of colonies
of bacteria and yeasts present on both plates. Cal-
culate the numbers in a gram of dry yeasts.

Bacteria do not multiply well on wort agar, the acid
reaction being unfavorable. The count on meat agar
will, therefore, be higher than on wort agar.

ir. Examine the yeast colonies under the micro-
scope, make stains, examine in water, and transfer the
different colonies to wort tubes.

12. Study the cultures prepared as before.

SECTION 5
EXAMINATION OF YEAST OF SALT-RISING BREAD

1. Make smears and stains from the yeast.

2. Inoculate litmus milk and incubate at 37° C, for
two or three days.

3. Make gentian violet and Gram stains, and note
the dark red color of the litmus.

4. Inoculate a flask containing 250 c.c. sterile milk
and incubate for seven days at 37° C.

5. Determine the amount of acid formed from day
to day with 1/20 N- NaOH and phenolphthalein as
indicator. Record the results.

6. Plate in meat extract agar and wort agar.

7. Study the colonies in both plates.

Meat extract agar is not suitable for the organism.
active in this yeast and probably no colonies will
appear. Beerwort agar is more suitable. The active
organism is a large bacillus, which forms an amount
of lactic acid often as high as 3 per cent. When used
for baking this acid combines with the soda added to

I92 LABORATORY GUIDE IN BACTERIOLOGY

salt-rising bread dough, and carbon dioxicis liberated,
which causes the bread to rise.

SECTION 6
A STUDY OF TORULAE

Experiment 1—

1. Transfer from laboratory cultures to wort agar.

2. Make stains, and transfer to all the other media.

3. Make descriptions of these organisms, and note
the differences from yeasts.

Experiment 2—

1. Prepare three piates of wort agar.

2. Expose these to tye air in different places.

3. Incubate at 25°C.

4. After 48 hours fish vor torula colonies, transfer
these to all the media, prepare stains, and describe the.
cultures. -Inoculate fermentation tubes with carbo-
hydrates, and inoculate gypsum blocks. Some torulae
produce gas from lactose; the true yeasts do not.
Torulae do not form spores.

SECTION 7

A STUDY OF ACETIC-ACID BACTERIA
EXERCISE .I

1. Secure some old vinegar with a film on top.

2. Prepare a suspension of the film in sterile water.

3. Plate out in wort agar, and incubate at 25° C.

4. Transfer the colonies to slant agar.

5. Inoculate flasks containing about 50 c.c. wort and
z per cent alcohol with the cultures.

MOLDS, YEASTS, TORULAE, AND BACTERIA 193

6. Incubate one flask at room temperature, one at
37°C., and one at 40°C.

7. Examine and note the degree of film formation
at the different temperatures and the differences in
morphology.

8. Add small amounts of alcohol from time to time.

9. Titrate daily with 1/20 N - NaOH and determine
the amount of acid produced.

EXERCISE 2

Tnoculate liquid wort to which 3 per cent saccharose
has been added with an alcohol-forming yeast. After
alcohol has been produced, inoculate with one of the
acetic-acid cultures and note the amount of acid
produced.

APPENDIX

APPENDIX

DILUTION TABLES FOR AGGLUTINATION

Number Amount of Serum Anipant ot Salt Final Dilution
Rictgerseacaeeus|| 2T3pare 9 parts Isto
Bisa I part of No. z 9 parts I:I00
3- 1 part of No. 2 9 parts I!1,000
4- 1 part of No. 3 9 parts 1:10,000
Amount of Serum or Serum Dilution Suspension Final Dilution

18 parts 1:10

I9 parts 1:20

15 parts Ti40

16 parts 1:50

17.5 parts 1:80

18 parts I:I00

19 parts 1:200

16 parts 1:500

18° parts 1:1,000

19 parts 132,000

16 135,000

18 parts 1:10,000

Tg parts 1: 20,000

16 parts 1:50,000

18 parts 1:100,000

19 parts 13200,000
DILUTION TABLE FOR WATER OR MILK ANALYSIS

Final Dilution

Amount of Dilution eet a
Original fe eee
toc.c. of No. r goc.c.
1c.c. of No. 1 goc.c.
2oc.c. of No. 3 8oc.c.
toc.c. of No. 3 goc.c.
sc.c. of No. 3 95c.c.
2c.c. of No. 3 98c.c.
rc.c. of No. 3 99C.c.
5c.c. of No. 4 Q5C.C.
2c.c. of No. 4 98c.c.
1c.c. of No. 4 ggc.c.
sc.c. of No. 7 Q5C.C.
2c.c. of No. 7 98c.c.
tc.c. of No. 7 ggc.c.

Est:

1:10
I:100
1:500
111,000
12,000
135,000
I:10,000
1:20,000
150,000
1:100,000
1200,000
1: 500,000
11,000,000

197

198 LABORATORY GUIDE IN BACTERIOLOGY

COMPARATIVE TABLES OF WEIGHTS AND MEASURES

zrinch = 2.54 centimeters
1 foot = 0.3048 meters
1 yard = 0.9144 meters

r mile = 1.61 kilometers

1 micromillimeter (micron, ~) = 0.coceor meters = 0.00003937 in.

1 millimeter = ©.00r meters= ©.03937
I centimeter = 0.or meters= 0.3037
i decimeter = o.I meters= 3.937

I meter = I meters= 39.37

1 kilometer =1,000 meters = 3,281

I grain 0.0648 grams

28.35 grams
31.10 grams
453.6 grams
373 grams

I ounce, avoirdupois
I ounce, troy
I pound, avoirdupois
I pound, troy

ouedad

I gram = 15.43 grains
1 kilogram = 2.205 pounds, avoirdupois
r kilogram = .679 pounds, troy

3785 cubic centimeters
946 _ cubic centimeters
473 cubic centimeters

29.57 cubic centimeters

1 gallon, U.S. liquid
1 quart, U.S. liquid
I pint, U.S. liquid

1 ounce, U.S. liquid

mio a

1,000 cubic centimeters = 1.057 U.S. liquid quarts

in.
in.
in.
in.
feet

=3.28 feet =r .0936 yds.
=1093.6 yds.

CENTIGRADE AND FAHRENHEIT THERMOMETERS

Cc. F, Cc. F
—18 —o0.4 — 6 21.2
-17 1.4 —- 5 23.0
—16 3.2 —4 24.8
15 5.0 3 26.6
—14 6.8 2 28.4
13 8.6 -—1I 30.2
—12 10.4 ° 32.0
II 12.2 I 33.8
—I0 14.0 2 35.6
—9 15.8 3 37-4
—8 17.6 4 39-2
ae 2 19.4 5 41.0

Cc wn aM

Io
II
I2
13
14
15
16
17

F.
42.
44.
46.
48.
50.
51.
53-
55.
57-
59-
60.
62.

AwmMoOnFfP aAWONFP DAO

OKRAWO KR AWAOKHRAWOKRAABWDOKRRAWONA’

APPENDIX

Cc. F.

46 114.8
47 116.6
48 118.4
49 120.2
50 122.0
51 123.8
52 125.6
53 127.4
54 129.2
55 131.0
56 132.8
57: 134.6
58 136.4
59 138.2
60 140.0
61 141.8
62 143.6
63 145.4
64 147.2
65 149.0
66 150.8
67 152.6
68 154.4
69 156.2
70 158.0
71 159.8
72 161.6
73: 163.4

OKnRAWOKA AWO KAR AWODRN

200 LABORATORY GUIDE IN BACTERIOLOGY

Endo’s Medium—

Pepton (Witte)...........-.000.- Io grams
Extract of meat.......... cece eee 3 grams
Na Clic:icasena nse cee ca maniacs 5 grams
Distilled water... .........0ceeee I,000 C.c

Dissolve by boiling. Titrate to o.2 per cent acid
to phenolphthalein. Place in autoclave at 15 pounds
pressure for 6 minutes. Cool and filter off precipitate.
Titrate again to 0.2 to 0.3 per cent acid.

Then add: .

Agar threads..... svlacaiichovaveracsdalucopelalo 30 grams

Boil until dissolved and make up to 1,000 c.c.

Saturated solution of basic fuchsin... .. x OF

Sodium sulphite solution (5 per cent
anhydrous) .........-.--eeeeeeeee 23.3 grams

Lactose (dissolved in 50 c.c. hot water) 10 grams

Tube and sterilize in autoclave for 10 minutes at
10 pounds pressure.

Russell’s Medium—

Make up 1,000 c.c. broth (meat extract) and make
reaction 0.9 per cent acid to phenolphthalein. Auto-
clave and filter. Add 15 grams thread agar and
dissolve by boiling.

Add 1 per cent litmus solution in proportion of
5 per cent.

Add sufficient n.Na,CO, to bring the solution to
the neutral point of the litmus (12-15 c.c.).

Add 1 per cent lactose and o.1 per cent dextrose.

Tube and sterilize for 10 minutes at 10 pounds
pressure.

The amount of Na,CO, must be accurate, as other-
wise the medium will turn greenish after sterilizing.

INDEX

INDEX

Abnormal fermentation in milk,
159.

Acetic-acid bacteria, 192.

Acid broth, 41.

Acid fermentation in milk, 155.

Acid-proof bacilli, 131, 132.

Actinomyces asteroides, 134.

Actinomyces bovis, 134.

Actinomyces group, 134.

Actinomyces hominis, 134.

Aesculin bile-salt agar, 33.

Agar-agar, 20.

Agar: beerwort, 32; bile-salt, 33;
blood, 40; dextrose, 26; gly-
cerin, 36; litmus dextrose, 35;
litmus lactose, 35; mannit, 36;
slants, inoculation of, 72; syn-
thetic for plating, 38; standard
method for preparing, 30; whey,
36.

Agglutination test, 118.

Air, bacterial examination of,
87; number of bacteria in, 87.

Albuminoid, 62.

Alcohol formation by yeasts, 187.

Alum carmin, 44; hematoxylin,
44.

Amido compounds formed by
soil bacteria, 170.

Ammonia, formed by soil bacteria,
I7I.

Ammonification, pepton solution
for, 40.

Amylase, 63.

Amylolytic: action of molds,
184; enzym, 62
Anaérobes: in milk, 158; in

sewage, 149.
Anaérobic: bacilli, 134; cultiva-
tion, 134; Buchner’s method,
136; in hydrogen gas, 136;

Park’s method, 135; -Wright’s
method, 135.
Analysis of gas in fermentation
tubes, 81.
Anilin gentian violet, 43.
Anthrax group, 127.
Arnold steam sterilizer, 12.
Asiatic cholera, spirillum of, 133.
Aspergillus oryzae, 184.
Assimilation of atmospheric nitro-
gen, 175.
Autoclave, 14.
Autopsy, 107.

Bacilli: acid-proof, 131; anaérobic,
134.

Bacillus: aérogenes, 112; anthra-
cis, 127; botulinus, 134; chau-
vei, 134; suipestifer, 101, 112,
115; cloacae, 101, 121; coli,

.95, 101, 1125; coli anaéro-
genes, 112; coli and strepto-
cocci in milk, 157; coli and
streptococci in water, 86, 147,
149; cuniculicida, 127; cyano-
genes, 159; der Rinderpest,
127; der Schweineseuche, 127;
diphtheriae, 124; dysenteriae,
112, 117; edematis, 134; enter-
itidis, 112, 115; fecalis alcali-
genes, 101, 112, 117; of Gart-
ner, 115; hofmannii, 124; icter-
oides, 112; influenzae, 133;
leprae, 131; mallei, 132, 133;
melitensis, 132; Moller’s grass,
131; capulatus, 123; of bubonic
plague, 127; of rabbit septi-
cemia, 127; paratyphosus,
112, 115; pestis, 127; prodi-
giosus, 97, 159; pyocyaneus,
97; rhinoscleromatis, 123; smeg-
mae, 131; subtilis, 127; tetani,
134; tuberculosis, 131; typho-
Sus, 95, 112, 117, 150; typho-
sus in water, 150; violaceus,

202

204 LABORATORY GUIDE IN BACTERIOLOGY

97; viscosus, 159; welchii, 134;
xerosis, 124, 126.

Bacteria: and spores in soil, 165;
chromogenic, 97; from the air,
87; intestinal, IOI; legume,
176; nitrate, 174} nitrite, 171,
172; number in air, 87.

Bacterial examination: of air,
87; of milk, 88, 151; of soil,
163; of water, 84, 141.

Bacteriological technic, 1.
Baker’s yeast, 190.

Beerwort: agar, 32; gelatin, 32;
no 32; liquid, 32; media,

Betketeld filter, 10, 92.

Bile-salt agar, 33; broth, 33.

Bismarck brown, 44.

Blastomyces dermatitidis, 132.

Blood agar, 4o.

Blood serum, 31.

Béttcher’s moist chamber, 183.

Bouillon, 18.

Bread paste medium, 41.

Brewer’s yeast, 190.

Broth: acid, 41; bile-salt, 33;
calcium carbonate, 41; glycerin,
36; meat, 31; nitrate, 41;
pepton, 18; sugar-free, ror,
113} standard method of, pre-
paring, 30.

Brownian movement, 76.

Buchner’s method of anaérobic
cultivation, 136.

Budding of yeasts, 80.

Calcium carbonate broth, 41.
Capaldi’s egg medium, 37.
Capaldi’s medium, 35.
Capsulated group, 123.

Capsule stain, 109; Friedlander’s,
109; Rosenow’s, 110; Welch’s
110; Welch’s modified, 138.

Carbol fuchsin, 43.

Carbolic: gentian violet, 45;
thionin blue, 44

Carmin: alum, 44; lithium, 45.
Casein, 62.
Chart, culture, 59.

Cheese, Camembert, 184; molds
in, 184; Roquefort, 184.

Cholera red reaction, 130.

Chromogenic group, 97.

Cladothrix, 134.

Cleaning: of glassware, 8; mix-
ture, 8.

Coagulation of milk, 62.

Coagulative enzym, 62.

Collecting: micro-organisms from
the air, 70; molds from the air,
78, 182; yeasts from the air, 78,
182; torulae from the air, 78,
192.

Collection of water samples, 84,
145.

Collodian sacs, 173.

Colonies, estimation of, 85.

Colony, 72; counter, 85.

Comparative table: of Centigrade
and Fahrenheit thermometers,
198; of weights and measures,
198.

Condensation water, 72, 99.

Conidia, 79.

Conradi’s medium, 34.

Cotton filter, 92.

Counter, colony, 85.

Cultivation, anaérobic, 134; of
molds, 182.

Culture chart, 59.

Culture media, 18; filtration of,
ory preparation of, 18; eaten
of, 18; titration of, 18

Culture of yeasts from one cell,
187.

Culture tubes, 9; plugging of, 9;
potato, 2

Cultures, egg, 125;
describing, 53.

method of

Death-point, thermal, 96.
Decolorization of litmus milk ,63.

INDEX

Delafield’s hematoxylin, 44

Denitrification, solution for test-
ing, 40.

Describing cultures, 53.

Determination: of anaérobes in
milk, 158; of anaérobes in
sewage, 149; of B. coli and
streptococci in milk, 157; of

coli and streptococci in
water, 86, 147, 149; of bacteria
and spores in soil, 165; of
species in water, 86; of spores
in soil, 165.

Dextrose: agar, 26; litmus agar,
35; litmus gelatin, 36; yeast
water, 33.

Diastase, 63.

Differentiation of B. typhosus and
B. coli, media for, 33.

Digestion: of blood serum by soil
bacteria, 168; of casein by soil
bacteria, 169; of gelatin by
soil bacteria, 169.

Dilution tables, 197.

Diphtheria: antitoxin, 126; ba-
cillus, 124; group, 124; toxin,
126.

Directions for filling out culture
charts, 58.

Discontinuous sterilization, 11.
Disinfectants, 94.
Doane-Buckley method, 161.
Dog’s blood serum, 33.

Dorset’s egg medium, 37.
Drigalski and Conradi’s medium,

34-
Dry heat sterilization, 11.
Dunham’s pepton solution, 18.
Dysentery bacillus, 112, 117..

Effect: of pasteurization of milk,
88; of sterilization of milk, 88.

Egg media, 37.

Ehrlich’s anilin gentian violet, 43.

Endo’s medium, 200

Enriching method of Schottelius,
130.

205

Enzym, 62; amylolytic, 62;
coagulative, 62; diastatic, 62;
gelatinolytic, 62; inverting,
63; production, 62; proteo-
lytic, 62; rennet, 63.

Erlenmeyer flask, 7.

Estimation of colonies, 85.

Examination: of air, 87; of ice,
148; of milk, 88, 151; of milk
for B. coli and streptococci, 157;
of milk for leukocytes, 160; of
milk for tubercle bacilli, 155;
of molds, 184; of rain water,
148; of sewage, 148; of soil,
163; of water, 86; of well
water, 147.

Exercises on infection and sterili-
zation, o1.

Fermentation: abnormal, in milk,
159; chart, back cover; tube,
6; tube, gas production in,
81.

Filament formation, 129.

Filling culture tubes, 22.

Film formation by yeasts, 186.

Filter: Berkefeld, ro, 92; cotton,
92; folding of paper, 22.

Filtration of culture media, 21.

Finkler and Prior, spirillum of,
129.

Flagella stain, 117.

Formation: of alcohol, 187; of
amido compounds, 170; of
ammonia, 171; of film by
yeasts, 186; of indol, 114, 170;
of nitrates, 174; of nitrites,
171; of spores by bacteria, 128;
of spores by molds, 83; of
spores by yeasts, 80, 187.

Formula for determining the
number of B. coli, streptococci,
and anaérobes in milk and
water, 86.

Fowl cholera, bacillus of, 127.

Friedlinder’s capsule stain, 109.

Friedlander’s pneumobacillus, 123.

Frost’s culture chart, 59.

206 LABORATORY GUIDE IN BACTERIOLOGY

Fuchsin, carbol, 43.

Fuller and Johnson’s rejuvena-
tion method, 149.

Gartner’s bacillus, 115.

Garden earth, inoculation with,
139.

Gas: analysis, 81; evolution by
yeasts, 186; formula, 82; gen-
erator, 137; production, 81.

Gelatin, 26; beerwort, 32; dex-
trose, 36; digestion of, 62;
liquefaction of, 62; litmus dex-
trose, 36; litmus lactose, 36;
pepton, 26; standard methods
of preparing, 30; whey, 36.

Gelatinoid, 62.

Gelatinolytic enzym, 62.

General bacteriology, 67.

General directions, 5.

Gentian violet: anilin, 43; car-
bolic, 45.

Germination of mold spores, 83.

Giltay solution, 40.

Glanders, 132.

Glassware: cleaning of, 8; sterili-
zation of, 11.

Glycerin: agar, 36; broth, 36; egg
medium, 37; media, 36,

Glycerinated potato, 37.

Glycogen, 113.

Gonococcus, 109.

Gonorrheal pus, 109.

Gram’s iodin solution, 43; stain,
77+

Granule stain, 124.

Granules, sulphur, 134.

Grass bacillus, 131.

Group: actin6myces, 134; an-
thrax, 127; capsulated, 123;
chromogenic, 97; colon, colon-
typhoid, 112; diphtheria, 124;
enteritidis, 112-15; hemorrhagic
septicemia, 127; hog cholera,
112-15; intermediate, 112-15;
of acid-proof bacilli, 131; of
anaérobic bacilli, 134; proteus,

1213; pyogenic, 105; typhoid-
dysentery, 112, 117.
Groups of bacteria in milk, 162.
Growing legumes in sand, 178.
Gruber-Widal test, 118.
Gypsum blocks, 80.

Hand lens, 7.

Hanging drop, 75.

Hansen’s method of pure culture
of yeasts, 187.

Hay infusion, 41.

Heat, influence of, on bacteria,
94.

Hematoxylin:
field’s,

Hemorrhagic septicemia group,
127.

Hesse’s medium, 35.

Hill’s test rods, 94.

Hiss’s plating medium, 34; tube
medium, 34.

Hog cholera group, 115.

Hopped beerwort, 32.

Horse’s blood serum, 33.

Hot-air sterilizer, 11. |

Hydrogen gas generator, 137.

alum, 44; Dela-

Ice, examination of, 148.
Impression preparation, 128.
Incubator, 51.
Indol, 114; formation of,
170; test for, 114, 170.
Infection, phenomena of, or.
Influence: of disinfectants, 94;
of moist heat, 94; of sunlight,
96.

Inoculation: of agar slants, 72;
of animals (see various heads).

Inspissator, Koch’s, 17.
Intermediate group, 112, 115.
Intermittent sterilization, 11.
Intestinal group of bacteria, 112.
Intramuscular inoculation, 131.
Intraperitoneal inoculation, 123.

114,

INDEX

Intravenous inoculation, 106.
Inverting enzym, 63.
Involution forms, 117, 121.

Todin: solution, Gram’s, 43;
starch test, 172.
Isolation: of B. typhosus in

water, 150; of legume bacteria,
176; of nitrate bacteria, 175;
of nitrite bacteria, 172; of
unknown bacteria, 139.

Jar, Novy, 137.
Jordan’s non-protein medium, 42.

Klatschpraparat, 128.
Koch’s inspissator, 17.
Kiihne’s methylene blue, 45.

Laboratory rules, 3. .
Lactalbumin, 62.

Legume, bacteria, 176.
Leptothrix, 134.

Leukocytes in milk, 160.

Liquefaction of blood serum, 57;
of casein, 62; of gelatin, 62.

Liquid beerwort, 32.
Lithium carmin, 45.

Litmus: decolorization of, 63;
dextrose agar, 35; dextrose
gelatin, 36; lactose agar, 35;
lactose gelatin, 36; mannit

agar, 36; milk, 27; solution, ae
whey, 36.
Léffler’s flagella stain, 117.
Léoffler’s methylene blue, 43.

MacConkey’s bile salt agar, 33.
MacConkey’s bile salt broth, 33.
Malachite green media, 35.
Maltose, 63.
Mannit agar, 36. :
Measures and weights, 198.
Meat: broth, 31; press, 31.
Media: adjusting reaction of, 18;
beerwort, 32; culture, 18; egg,
37; filling in tubes of, 22;

207

filtering, 21; for differentia-
tion of B. *typhosus and B.
coli, 33; for the study of soil
bacteria, 37; for water and
milk examination, 35; glycerin,
36; miscellaneous, 40; nitrate,
41; non-protein, 42; phenol,
34; preparation of, 18; reaction
of, 18; standard method of pre-
paring, 30; synthetic, 42;
titration of, 18; whey, 36.
Medium: bread paste, 41; Capal-
di’s, 35; Dorset’s egg, 37;
Drigalski and Conradi’s, 34;
Endo’s, 33; glycerin egg, 37;
Hesse’s, 35; Hiss’s plating, 34;
Hiss’s tube, 34; Jordan’s non-
protein, 42; ox bile, 35; Uschin-
sky’s, 42; wine must, 41.
Meningococcus, 133.

Method: of describing cultures,
53; of making Gram stain, 77;
of making plates, 98; of mak-
ing stained preparations, 76;
of microscopic examination of
molds, 79; of preparing agar,
standard, 30; of preparing
broth, standard, 30; of pre-
paring gelatin, standard, 30.

Methylene blue: Kiihne’s, 45;
Lofler’s, 43. ~
Microcdtcus gonorrheae, 109;

meningitidis, 133; tetragenous,
105; zymogenes, 109.

Micro-organisms from the air, 70.

Microscope, 45.

Milk: abnormal fermentation in,
159; acid fermentation in,
155; acid-proof bacilli in, 89;
anaérobes in, 158; bacterial
examination of, 88; B. coli and
streptococci in, 157; coagula-
‘tion of, 63; examination of,
for tubercle bacilli, 89; litmus,
27; leukocytes in, 160; molds
and yeasts in, 160; pasteur-
ized, 89, 158; reaction of, 62;
sterilized, 158; sugar, 62.

Miscellaneous: media, 40; organ-
isms, 132.

208 LABORATORY GUIDE IN BACTERIOLOGY

Méller’s grass bacillus, 131.
Moller’s spore stain, 129.
Moist chamber, Béttcher’s, 183.
Mold spores, germination of, 83.

Molds, 182; amylolytic action of,
184; in cheese, 184; in milk,
160; microscopic examination
of, 184; yeasts, torulae, and
acetic-acid bacteria, 179.

Molecular movement, 75.

Mouse: holder, 111; microscopic
examination of, 111.

Muscle sugar, ror.
Must, wine, 41.

Natural souring of milk, 156.
Needles, platinum, 6.
Negative Gram stain, 78.
Neutral-red agar, 33.
Nicollé’s carbolic gentian violet,
45; carbolic thionin blue, 44.
Nitrate: bacteria, 174; broth,
41; formation, 174; media, 41;
solution, 41.

Nitrates, test for, 175.

Nitrite: bacteria, 171, 172; for-
mation, I71I.

Nitrites, test for, 114.

Nitrogen, assimilation of, 175.

Nitroso-indol reaction, 130.

Nocardia, 134.

Non-protein media, 42.

Normal solution, 20.

Novy jar, 137.

Ox-bile medium, 35.

Ox-blood serum, 33.

Parietti’s solution, 34.

Park’s method of anaérobic culti-
vation, 135.

Pasteurizing milk, 89, 158.
Pedesis, 76.

Pepton: broth, 18; gelatin, 26;
solution, Dunham’s, 18, solu-
tion for ammonification, 40.

Peptonization, 62, 168.

Petri dishes, 7.

Phenol: media, 34; sulphonic acid,
175.

Phenolphthalein, 19.

Phenomena: of infection, 91; of
sterilization, 91.

Pigments, 100.

Plate cultures, 98.

Plating medium, Hiss’s, 34.

Plugging culture tubes, 9.

Pneumobacillus, 123.

Pocket inoculation, 125.

Polar staining, 116, 127.

Positive Gram stain, 78.
Potato, 28; glycerinated, 37;
substitute for, 29; tube, 28.
Preparation: Gram, 77; hang-

ing drop, 75; impression, 128;
of agar-agar, 20; of blood
agar, 40; of blood serum, 31;
of bouillon, 18; of broth, 18,

31; of culture media, 18; of
dextrose agar, 26; of Dunham’s
pepton solution, 18; of egg
media, 37; of gelatin, 26; of
glycerin media, 36; of litmus
milk, 27; of media for soil ex-
amination, 373 of media for
water and milk examination, 35;
of non-protein media, 42; Pres-
cott and Breed’s method, 161;
of silica jelly, 38; of soil samples,
166; of soil suspensions, 166; of
staining solutions, 43; of sugar-
free broth, 101; of whey media,
36; stained, 76.

Proteolysis, 62.

Proteolytic enzym, 62.

Proteus: group, 121;
121; zenkeri, 121.

Pure culture: of bacteria, 187;
of yeasts, 187.

Pus, gonorrheal, ro9.

Pyocyanin, 100.

Pyogenic group, 105.

vulgaris,

Rabbit septicemia, 127.
Rain water, 148.

INDEX

Raulin’s solution, 42, 185.

Reaction: indol, 114, 170; nitrites;
114, 170; nitroso-indol,
of bacteria on neutral red
broth, 150; of culture media,
18; of milk, 62.

Reduction of nitrates, 177.

Rejuvenation of cultures, 149.

Rennet enzym, 62.

Rinderpest, 127.

Roquefort cheese, 184.

Rosenow’s capsule stain, r10.

Russell’s medium, 200

Saccharomyces: cerevisiae, 78,
189; ellipsoideus, 189; pas-
torianus, 189.

Sacs, collodion, 173.

Safranin, 44.

Samples of water, collection of,
84.

Salt rising bread, 191.

Sarcina lutea, 97, 159.

Scheme for routine study, 50.

Schottelius enriching method, 130.

Schweineseuche, 127.

Sewage: anaérobes in, 149; con-
tamination of, 86; examination
of, 148. . ;

Sketches of streak and stab
cultures, 64, 65.

Silica jelly, 38.

Snow, examination of, 148.

Soil: bacteria and spores in, 165;
examination of, 163; samples,
165; samplers, 165.

Solution: Dunham’s pepton, 18;
for assimilation of atmospheric
nitrogen, 39; for denitrification
of nitrates, 40; for formation of
nitrates, 39; for formation of
nitrites, 38; Giltay, 40; Gram’s
jodin, 43; litmus, 35; nitrate,
41; normal, 20; Parietti’s, 34;
pepton. for ammonification, 403

Raulin’s, 42; Winogradsky’s, 37.

Solutions, staining, 43.

130;,

209

Species determination in water,
86.

Spirillum: cholerae, 133; group,
129; metchnikovii, 129; of
asiatic cholera, 133; of Finkler
and Prior, 129; tyrogenum, 129.

Spore: formation of yeasts, 80,
187; staining, 128.

Spores: germination of, 83; in
soil, 168; of bacteria, 128;
of molds, 83; of yeasts, 80.

Stain: flagella, 117; Friedlinder’s
capsule, 100; Gram, 77; Léffler’s
flagella, 109; Pappenheim, 45;
Rosenow’s capsule, 110; spore,
128; Welch’s capsule, 110;
Welch’s capsule, modified, 138.

Stained’ preparation, 76.

Staining: acid-proof bacilli, 132;
capsules, 109; solutions, 43.

Standard method: for preparing
agar, 30; for preparing broth,
30; for preparing gelatin, 30.

Staphylococcus: albus, 105; au-
TFeus, 95, 100, 105.

Starch iodin test, 172.

Steam sterilizers, 11.

Sterilization, 10; by. chemicals,
11; by dry heat, 11; by moist
heat, 11; discontinuous, 11;
intermittent, 11; of blood
serum, 16; of glassware, 11;
and pasteurization of milk, 88,
158; of milk, 89, 158; phe-
nomena of, gI.

Sterilized milk, 158.

Sterilizer: Amold steam, 12;

hot-air, 11.

Streptococci: in milk, 157; in
water, 86.

Streptococcus: lacticus, 100; py-
ogenes, 105. pneumeae, 109.
Study: of acetic-acid bacteria,
192; bacterial, of milk, 88, 162;
of molds, 83; of molds, yeasts,
and torulae, 78; of pigments,
100; of soil bacteria, 165; of

yeasts, 79.
Subcutaneous injection, 116.

210

Substitute for potato, 29.
Sugar-free broth, ror.
Sulphur granules, 134.
Sunlight, influence of, 98.
Surface water, 146.
Suspension of soil, 166.

Synthetic: agar for plating, 38;
media, 42.
Table: of Centigrade and Fah-

renheit thermometers, 198; of
weights and measures, 198.

Tables, dilution, 197.

Technic, bacteriological, 1.

Test: agglutination, 118; for
indol, 114, 170; for indol and
nitrites, 114, 170; for nitrates,
175; for nitrites, 114; Gruber-
Widal, 118; rods, Hill’s, 04;
starch’ iodin, 172; Widal, 118.

Thermal death-point, 96.

Thermostat, 51.

Thionin blue, 44.

Titration of media, 18.

Torula amara, 159.

Torulae, 78, 192.

Trichomycetes, 134.

Tube: culture, 9; termentation,
7; medium, Hiss’ §, 34; potato,
28.

Tubercle: bacilli in milk, 89;
bacceria, 175.

LABORATORY GUIDE IN BACTERIOLOGY

Typhoid-dysentery group,
117.

112,

Ultramicroscopic bacteria, 92.
Uschinsky’s medium, 42.

Water: and milk examination,
media for, 35; and sewage,
bacterial examination of, 84,
141; bacterial examination of,
84; bath, 70; of condensation,
72; samples, collection of, 84;
species determination in, 86.

Weights and measures, 198.

Welch’s capsule stain, 110; modi-
fied, 138.

Well water, 147.

Whey, 62; agar, 36; selitin, 36;
litmus, 36; media, 36;

Widal test, 118.

Wine must, 41.

Winogradsky’s solution, 37.

Yeast: baker’s, 190; brewer’s,
190; of salt-rising bread, ror;
water, 32; water, dextrose,
33:

Yeasts, 78, 185; alcohol forma-
tion, 187; budding of, 80;
film formation of, 186; from
the air, 78, 185; gas evolution
of, 186; spore formation of,
80, 187.

Ziehl-Neelsen’s carbol fuchsin, 43.

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