\=>'
LIBRARY
OF THE
UNIVERSITY OF CALIFORNIA,
ClaSS LIBRARY
•» -. .
r*
A LABORATORY GUIDE IN
BACTERIOLOGY
A Laboratory Guide
in Bacteriology
FOR THE USE OF
STUDENTS, TEACHERS, AND
PRACTITIONERS
BY
PAUL G. HEINEMANN, Sc.B.
Fellow in Bacteriology, the University of Chicago
THE UNIVERSITY OF CHICAGO PRESS
1905
t+4-
COPYRIGHT 1905
THE UNIVERSITY OF CHICAGO
PREFACE
The considerations which led the writer to add this
laboratory guide of bacteriology to the number of such
guides already in existence were of various nature and
may be briefly set forth here.
Probably no branch of biological science has advanced
so rapidly during the past few years as the science of
bacteriology, and it is difficult even for an active labora-
tory worker to keep abreast of this advance. A text-
book or guide fixes the status of the science at the time
of its writing, but almost before it leaves the press it
becomes antiquated. Revisions, corrections, and addi-
tions are necessary at short intervals in order to keep a
publication of this kind approximately up to date. There
is therefore, almost at any given moment room for a new
publication to fill the want of a progressive instructor for
a guide that gives the latest accepted rules and practices
of the laboratory. The value of such a publication will
be enhanced by a plan and arrangement of sufficient
flexibility and latitude to allow the instructor and the
student to enter such additions and corrections as serve
to bridge over the time between editions.
Medical students entering on a course in bacteriology
often have had too little previous laboratory training in
methods of precision. It is a matter of importance for
the instructor to put himself in the attitude of mind of the
student and to try to appreciate his difficulties in under-
standing details. Many of the pieces of apparatus
employed in a bacteriological laboratory are novel even
144184
vi PREFACE
to the student trained in chemistry and biology, and it
has been thought best to exhibit these, to the smallest
detail, by means of illustrations — a feature not sufficiently
considered in other guides.
The formulae for stains and the methods of staining
have not been collected in one chapter, as is usually the
practice, because this tends to confuse the student.
They are described during the progress of the course, as
occasion offers to put them to practical use.
Culture-description charts have not been included in
this volume. A beginner naturally makes incomplete
descriptions and many alterations, and thus defaces the
book and impairs its future utility. A sufficient number
of loose charts perforated for binding should be furnished
to the student at a nominal figure.
A point of inestimable importance is how best to
stimulate the student to consult textbooks and special
monographs, and other references, as often and as freely
as possible. This guide has been written with the aim
of not only not interfering in any manner with the reading,
through including such points and characteristics as might
make a textbook superfluous in the judgment of the
inexperienced, but also of making it necessary for the
student to read the best textbooks with freedom and
understanding. Cultural and morphological features are
left entirely to the actual observation of the student, sup-
plemented by instruction and the reading of textbooks.
The course, as outlined, is identical with the medical
course given at the University of Chicago, with a few
additional chapters which may be used during courses
for non-medical students. A chapter containing a fairly
complete list of formulae for culture media employed in
PREFACE vii
advanced work has been added with the object of making
them easily accessible to those engaged in advanced work.
The laboratory guides of Novy, Eyre, Frost, Gorham,
Kanthack and Drysdale, and Connell, and the American
edition of the Manual oj Bacteriology of Muir and Ritchie,
have been freely consulted. I take this occasion of
expressing my deep gratitude to Professor Edwin O.
Jordan and Dr. Norman Mac Leod Harris for their
invaluable help and suggestions in the preparation of
this guide.
PAUL G. HEINEMANN.
CHICAGO, ILL.
June, 1905.
INTRODUCTION
The advent of bacteriology into the realm of the
biological sciences not only brought with it a new
conception of the nature of many complicated phe-
nomena, such as fermentation and disease, but also
placed in the hands of experimental workers a new
tool. The method of sterilization, of asepsis, made
it possible for the first time to attack problems
hitherto incapable of solution, or even of approach.
This development of bacteriological technique, of
rigid and undeviating adherence to definite rules
and principles, is not likely to be passed over lightly
by the historian of nineteenth-century science. The
art of practical medicine and theoretical medical
research alike owe much of their recent brilliant
success to a ready adoption of the new method.
At the present time an active campaign is being
set on foot by public health authorities against
several widespread and serious diseases of the human
race. In various parts of the world, malaria, tuber-
culosis, and typhoid fever are being fought energeti-
cally and with much success. In these systematic
and organized movements the resources of bacteri-
ology are being utilized as never before, and a full
understanding of technical procedure and devices
is deemed essential by all workers in this subject.
The problems of water-supply and sewage disposal,
ix
x INTRODUCTION
of urban infantile mortality, and of the control of
contagious diseases are all bound up with the intelli-
gent application of bacteriological methods.
In the almost untilled field of industrial bacteri-
ology there is need for a fuller appreciation of the
value of bacteriological methods and principles.
Many great industries are based wholly upon the
proper selection and adaptation of micro-organisms,
and a timely and discriminating utilization of their
products. Loose and empirical methods have been
in force in the past, but these must eventually give
way to a more precise and truly bacteriological
technique.
Agricultural bacteriology is just now much in the
public eye, and it would be gratuitous to prophesy
the results that may reasonably be anticipated in
this direction. Here again crude, rule-of-thumb,
" practical" ways of doing things are being sup-
planted by the scientific, the reasoning, and the
precise.
To the student, whether in medical, hygienic, or
industrial bacteriology, proper technical methods
of work must always have a peculiar value, since
without their aid advance is impossible, and stumb-
ling and disastrous missteps are certain. A com-
prehensive outline of modern bacteriological methods,
therefore, is a necessary adjunct to obtaining a true
and full understanding of the underlying principles
and tendencies of the science. The technique of
INTRODUCTION xi
bacteriology is one of its greatest contributions to
both science and art, and the use of so valuable
and simple a tool should be mastered not only by
the biological teacher and investigator, but by prac-
tical workers in medicine, hygiene, and many other
fields.
EDWIN O. JORDAN.
TABLE OF CONTENTS
PAGE
CHAPTER I. Laboratory Rules . . . . . . i
CHAPTER II. Cleaning, Preparing, and Sterilizing Glassware 8
CHAPTER III. Preparation of Culture Media . . .11
Exercise i. Preparation of nutrient agar-agar . .11
Exercise 2. Preparation of glucose-agar . . -19
Exercise 3. Preparation of peptone-gelatin . -23
Exercise 4. Preparation of peptone-broth . -25
Exercise 5. Preparation of potato . . . .27
Exercise 6. Preparation of litmus milk . . .28
CHAPTER IV. Preparation of Staining Solutions . . 30
CHAPTER V. The Microscope . . . . -31
CHAPTER VI. Collecting Bacteria from the Air . . -37
CHAPTER VII. Exercises on Infection and Sterilization . 45
Exercise i. Phenomena of infection . . -45
Exercise 2. Phenomena of sterilization . . -45
Exercise 3. Phenomena of sterilization (continued) . 45
CHAPTER VIII. Study of Yeasts, Molds, and Torulae . 49
Exercise i. Cultural studies . . . . -49
Exercise 2. Study of the germination of spores . 51
CHAPTER IX. Scheme for Routine Study of the Various
Groups of Organisms . . , . . -53
CHAPTER X. Method of Describing Cultures . . -57
CHAPTER XI. Study of Certain Chromogenic Bacteria . 69
Exercise i. Cultural studies . ' . . . .69
Exercise 2. Study of pigments . . . . 71
CHAPTER XII. The Pyogenic Group . . -73
Exercise i. Subgroup A . . . . -73
Exercise 2. Subgroup B . .... 76
xiv TABLE OF CONTENTS
PAGE
CHAPTER XIII. The Intestinal Group . . . -79
Exercise i. Colon group . . . . . -79
Exercise 2. Hog-cholera, Bac. enteritidis or intermediate
group . . 84
Exercise 3. Typhoid-dysentery group . 85
Exercise 4. Proteus group . . . . .89
CHAPTER XIV. The Capsulated Group . . . .92
CHAPTER XV. The Diphtheria Group . . . -94
CHAPTER XVI. The Hemorrhagic Septicemia Group. . 97
CHAPTER XVII. The Anthrax Group . . .98
CHAPTER XVIII. The Spirillum Group . . . . 101
CHAPTER XIX. The Group of Acid-Resisting Bacilli . 103
CHAPTER XX. The Miscellaneous Bacteria . . . 105
CHAPTER XXI. The Actinomyces Group . . . 106
CHAPTER XXII. The Anaerobic Group of Bacilli . . 107
CHAPTER XXIII. Isolation of Unknown Bacteria from a
Mixture . . . . . . . . .112
CHAPTER XXIV. Bacteriological Examination of Water, Air,
and Milk . . . . . . . .114
Exercise i. Bacteriological examination of water . 114
Exercise 2. Bacteriological examination of air . .118
Exercise 3. Bacteriological examination of milk . 119
CHAPTER XXV. Influence of Disinfectants on the Growth
of Micro-organisms 122
Exercises i and 2. Hill's test-rods . .-»-,. . .122
Exercise 3. Influence ot sunlight . *.^?^- • • I23
Exercise 4. Influence of moist heat . . . .124
APPENDIX I. Special Media 127
APPENDIX II. Staining Solutions 132
APPENDIX III. Frost's Culture Chart (modified) > .134
INDEX 139
CHAPTER I
LABORATORY RULES
1. Carefully familiarize yourself with the laboratory
rules. Upon their careful observance depend good work
and your own safety.
2. Food must not be eaten in the laboratory; lead-
pencils, labels, or fingers must not be moistened with
the tongue.
3. If any portion of a culture is spilled by accident
upon the desk or floor, it should be immediately covered
with a germicide (HgCl2 i : 1000 or 5 per cent, carbolic
acid). After this has acted for 10 or 15 minutes, wipe it
up and throw the cloth or paper into a waste-jar.
4. In case the hands should by accident come in
contact with infectious material, they should be washed
with one of the above-mentioned germicides, and then
thoroughly scrubbed with soap and water.
5. The platinum needles used in making cultures
should be sterilized in a flame shortly before and imme-
diately after use, and before they are laid down. When
the needles are covered with moist infectious material,
they should be held at the side of the flame until dry
before being sterilized; this will avoid the danger of
scattering this material about the desk.
6. All possible care should be observed in the care
of the apparatus, desk, etc. Solid materials must not
be put into the sinks. Burned matches, paper, cotton,
etc., are to be put in the crocks provided for that purpose,
and not on the floor.
2 LABORATORY GUIDE IN BACTERIOLOGY
7. Discarded cultures should be killed in the auto-
clav (5 minutes at I2o0j) before emptying into the crocks.
8. When using the steam sterilizers and autoclav,
see that the pan contains sufficient water before lighting
the gas.
9. In general, see that the air inlets of Bunsen burners
are open before lighting, and relight if the flame strikes
down.
10. Always return stock-bottles to the proper places
on the shelves. •
11. At the close of the day's work the desks should
be washed off with corrosive sublimate, and the hands
cleaned by thorough scrubbing with green soap and
water.
12. Before leaving the laboratory, see that the gas
is shut off under all apparatus, that water faucets are
closed, and that all glassware, etc., is replaced in the
lockers.
GENERAL DIRECTIONS
1. After obtaining the key to the locker, examine the
outfit, check the apparatus (Fig. i) on the furnished list,
and return the same signed with your name. This signa-
ture is a receipt in full for the material received and in
good condition, and the student is personally responsible
for the return of the same in good condition.
2. Matches, peptone, gelatin, and filter-paper are
furnished with the locker outfit in sufficient quantities
to last through the course. Anything needed in excess
must be furnished by the student.
1 Degrees of temperature throughout this work are indicated
according to the Centigrade system.
LABORATORY RULES 3
3. To facilitate study, students may work in pairs in
the preparation of culture media and the cultivation of
organisms; but each student is responsible for a first-
hand and independent description of each organism
studied, as well as for stained cover-slip preparations.
4. Read carefully in the textbook and the laboratory
guide the work for the day. This will greatly facilitate
an intelligent and systematic plan of work.
5. Keep careful, neat, systematic notes of each exer-
cise, and have them ready for inspection at any time.
In the case of exercises devoted to the preparation of
culture media, a simple statement as to their completion
is sufficient.
6. Printed charts for description of cultures can be
obtained on application to the laboratory assistant.
7. Accurate sketches must be made when called for
by the directions.
8. All the notes pertaining to each exercise must be
kept together. "University covers" of suitable size are
recommended for this purpose.
9. Store clean cover-slips in a Stender dish and cover
with 96 per cent, alcohol. A soft linen cloth is necessary
for wiping them. An ordinary clean handkerchief will
do very well. Coarse material breaks them easily.
LOCKER OUTFIT
150 culture-tubes.
10 potato culture-tubes.
6 fermentation-tubes.
15 Petri dishes (for plate-cultures).
3 Erlenmeyer flasks, one 1000 c.c., two 500 c.c. each.
2 glass funnels, one 4 inches, one 6 inches.
4 bottles for staining-fluiols,
LABORATORY GUIDE IN BACTERIOLOGY
Retort-Stand
Bunsen Burner
FiO. i
Culture-Tube Pipette
LABORATORY RULES
Petri Dish
Tripod
Watch-G lasses
Erlenmeyer Flask
Saltcellar
Balsam Bottl
Magnifier
(Hand-Lens)
FIG. i
Slender Dish
6 LABORATORY GUIDE IN BACTERIOLOGY
i balsam bottle.
i Stender dish (for cover-slips).
3 watch-glasses,
i saltcellar.
1 glass rod.
2 platinum needles (turn the end of one needle around
a sharp pencil point so as to form a closed loop).
8 tin cups or tumblers (cover the bottom of each with
ordinary cotton).
4 wire baskets (to hold culture-tubes).
i Bunsen burner and rubber tubing.
i saucepan and cover.
3 graduates, one 500 c.c., one 100 c.c., one 10 c.c.
i pinchcock.
i pipette with rubber hose attached,
i magnifier (hand-lens),
i tripod.
i retort-stand with three rings,
i thermometer in case,
i box of matches.
40 grams of peptone.
1 20 grams of gelatin.
6 sheets of filter-paper,
i key.
This outfit may be changed and added to in the dis-
cretion of the instructor.
Obtain the following articles from the storeroom:
50 glass slides.
1 camel's-hair brush.
50 round cover-glasses (18 mm.).
2 towels (one should be boiled in dilute NaOH, rinsed
in clean water, and kept clean for wiping slides and
cover-glasses.)
LABORATORY RULES
1 test-tube brush.
50 labels.
2 slide-boxes.
2 hollow-ground slides.
i glass pencil.
i pair of forceps.
i, or better 2, pairs of cover-slip forceps.
CHAPTER II
CLEANING, PREPARING, AND STERILIZING
GLASSWARE
EXERCISE I. CLEANING GLASSWARE
Culture-tubes, flasks, fermentation-tubes, and Petri
dishes must be free from organic matter, acids, and
alkalis. They are best cleaned as follows:
a) Completely immerse them in a vessel containing
soapsuds or soap-powder, boil for at least 10 minutes,
then thoroughly brush them with the tube-brush; or,
immerse them for an hour or more in the chromic-acid
cleaning-mixture, consisting of—
Potassium dichromate 60 parts
Water 300 parts
Concentrated sulphuric acid 460 parts
The sulphuric acid is to be added slowly with constant
stirring.
b) Rinse thoroughly in tap-water.
c) Again use the tube-brush, and soap and water, if
necessary.
d) Rinse very thoroughly, and get rid of every trace
of acid, soap, or alkali.
e) Turn upside down in a basket, and heat for 20
minutes in the hot-air sterilizer until perfectly dry.
EXERCISE II. PLUGGING OF CULTURE-TUBES
Directions for plugging. — Take a small amount of
ordinary, non-absorbent cotton of good quality, fold it
once, and by means of a glass rod push it into the mouth
8
CLEANING AND PREPARING GLASSWARE
of the tube to the depth of about J of an inch; or roll
the cotton into cylinders of the same s^*^/^
diameter as the culure-tube and insert / ^ ~
it into the mouth (Fig. 2). The plug /f7r ^
should allow the interchange of air,
and at the same time be tight enough
to hold the weight of the tube, and
with no crevices on the side. The
glass rod should never be pushed
through the cotton.
EXERCISE III Flo. 3
STERILIZATION OF GLASSWARE Plugged Culture-Tube
Sterilization is the process of killing all micro-organ-
isms. This may be accomplished by heat, by certain
chemicals, or by nitration. Sterilization by heat may be
accomplished —
1. By dry heat; this method is applied to sterilization
of many kinds of apparatus.
2. By moist heat; this method is applied largely to
sterilization of culture media,
and will be described in
Chap. Ill, Exercise II.
Hot-air sterilizers are boxes
with double walls of sheet-
iron. The bottom shelf
should always be covered with
a piece of sheet-asbestos, to
prevent too rapid heating of
the apparatus. By means of
a Bunsen burner with three
flames a heat of about 150°
may easily be maintained. The flames of the burner
FIG. 3
Koch's Hot-Air Sterilizer
io LABORATORY GUIDE IN BACTERIOLOGY
should enter the hole provided at the bottom of the
sterilizer for that purpose. Care should be taken to
avoid the possibility of the flame becoming luminous,
as this would fill the box and cover the glassware with
soot (Figs. 3 and 4).
The plugged culture-tubes are then placed in the
FIG. 4
Lautenschlager Hot-Air Sterilizer
sterilizer, and a temperature of 150° is maintained for
about 30 minutes, or until the plugs are slightly charred.
This can be recognized by a slight brownish color. The
tubes are now not necessarily sterile, but the plugs have
become set so as to fit the mouth of the tube, and may
easily be removed and replaced.
CHAPTER III
PREPARATION OF CULTURE MEDIA
Each student is required to make up the following
amounts of culture media:
Name of Medium
Amount
Number of Tubes
Plain agar. ...
IOOO 2
•7Q
G lucose-agar .
2CO £
20
Gelatin. .
JQQ or .
2 ^
Potato
a**** &•
*0
IO
Broth
300 c.c.
2C
Dunham's solution.
?OO C C.
2cr
Litmus milk.
7OO C C
2^
*3
160
EXERCISE I. PREPARATION OF NUTRIENT AGAR-AGAR
1. Weigh the saucepan without the lid accurately
and make note of the weight.
2. Measure into the saucepan 1000 c.c. of tap- water,
adding about 300 c.c. of water to allow for evaporation,
and heat over gas.
3. Cut and shred i<;g. of agar-agar (ij per cent.),
add to the water, and boil slowly, with constant stirring,
until perfectly dissolved, taking care not to overheat.
4. Add 3 g. of extract of beef. When dissolved,
remove the saucepan from the flame, and
5. Slowly dust 10 ( i per cent.) g. of Witte's peptone
on the surface, constantly stirring until perfectly dissolved.
Avoid allowing the peptone to clump.
NOTE. — Agar-agar (called simply "agar") is a watery ex-
tract of certain seaweeds found on the Pacific coast of Asia.
12 LABORATORY GUIDE IN BACTERIOLOGY
6. Adjust the reaction.
Bacteria, especially pathogenic bacteria, grow prefer-
ably in a medium which is neutral or slightly acid to
phenolphthalein (alkaline to litmus). Extract of beef
always contains a certain amount of acid, which has to
be neutralized. Two methods may be employed to
accomplish this purpose.
First method. — Gradually add a 4 per cent, solution
of sodium hydrate (NaOH) until a sample on the end
of the glass rod turns phenolphthalein paper a pale rose-
pink color. If accidentally too much NaOH is added,
it may be readjusted by means of a 5 per cent, solution
of hydrochloric acid (HC1). This method is sufficiently
accurate for ordinary purposes. The medium is then
strongly alkaline to litmus.
Second method. — A more precise method is as follows:
By means of a pipette measure 5 c.c. of the liquid into
a porcelain evaporating-dish ; add 45 c.c. of distilled
water and i c.c. of a solution of i part phenolphthalein
in 200 c.c. alcohol (50 per cent.), then heat to the boiling-
point and slowly add from a graduated burette enough
-£$ normal NaOH to neutralize. (The liquid must
have a decided, stable, pale-pink color which does not
vanish when heated.) Read the amount used from the
burette, and calculate the amount of normal NaOH needed
for 1000 c.c. Then add 0.5 per cent, normal HC1.
Example. — By reading the burette we find that it
takes 1.2 c.c. -£$ normal NaOH to neutralize 5 c.c. of
the medium, diluted with 45 c.c. of distilled water. To
neutralize i liter, it will take 200 times this amount,
which amounts to 240 c.c. of the -^ normal NaOH.
Divide 240 by 20, and the result (12 c.c.) is the amount
PREPARATION OF CULTURE MEDIA 13
of normal NaOH needed to neutralize i liter of medium.
To this add 5 c.c. normal HCL
The medium is then 0.5 per cent, acid to phenol-
phthalein ( + 0.5 per cent. Fuller's standard), or fairly
strongly alkaline to litmus.
The same result may be obtained by deducting 5 c.c.
from the amount of normal NaOH to be added per liter.
If, therefore, in the above example 12 c.c. — 5 c.c. = 7 c.c.
normal NaOH is added, the resulting reaction of the me-
dium is 0.5 per cent, acid without the addition of HCL
A normal solution is the equivalent weight in grams
(gram-molecule) of a chemical in 1000 c.c. of distilled
water. In the case of monovalent elements combining
to form the chemical, the molecular weight of the latter is
taken; in the case of bivalent ones, the molecular
weight is divided by 2 ; etc.
7. Allow the liquid to cool to 60°; test by placing
the thermometer in it.
8. While cooling, dissolve the whites of two eggs in
75 c.c. of water, and stir well into the liquid while still at
60°. The egg albumen is added for the purpose of clari-
fying the liquid. If added before the temperature has
fallen to 60°, it would partially coagulate, and thus not
serve the purpose. On the other hand, if allowed to
cool to about 40°, the agar would solidify. Upon gradu-
ally heating the medium the albumen coagulates, me-
chanically incloses suspended particles, carries them to
the top, and forms a superficial film.
9. Heat again, without stirring, on a piece of asbestos
over the flame. A film will gradually form and harden
with a dry surface.
10. Now adjust the weight by calculating the total
i4 LABORATORY GUIDE IN BACTERIOLOGY
weight of the different ingredients, allowing 1000 g. for
all the water used and 30 g. for the weight of the white of
each egg and the weight of the pan. If the actual total
weight is in excess of the amount calculated, boil again
— a
FIG. 5
Apparatus for Filtering
Media
a. Filter
b. Large funnel
c. Small funnel
d. Rubber hose
e. Pinchcock
/. Pipette
g. Culture-tube
until the proper weight is reached. If, on the other hand,
the total weight is less, make up the deficiency by adding
water.
Example —
TVater
Agar
Extract of beef
Peptone
Whites of two eggs
Saucepan for example . .
3000. O
.100.00$
15-0
...1.50
3-°
... o . 30
IO.O
... I . 00
60.0
450.0
Total.
PREPARATION OF CULTURE MEDIA
By placing weights to correspond to this amount on the
left side of the scales, and the saucepan with contents
on the other side, the weight can be readily adjusted.
ii. Heat again to the boiling-point, and filter the
liquid into culture-tubes to about one-third of their
length (7 c.c.).
Method of filtering media. — Arrange two funnels, a
pipette with hose, and a pinchcock on the retort-stand,
as illustrated in Fig. 5. Place a strainer made of per-
forated tin (Fig. 6) against the side of the saucepan,
FIG. 7
Strainer in Position
near the top, as illustrated in Fig. 7, and pour the medium
into the larger funnel holding the filter.
For the purpose of filtering the medium either white
filter-paper of the best quality or absorbent cotton may
be used. A filter-paper folded in the following manner
is very serviceable:
i. Take a square piece of filter-paper twice as wide
16 LABORATORY GUIDE IN BACTERIOLOGY
as the depth of the funnel to be used (Fig. 8, a), and fold
to half the size (Fig. 8, b) so as to make i cover 2, and 3
cover 4
2. Fold this to make i cover 2 and 3 cover 4 (result:
Fig. 8, c). It consists of four layers and forms a square.
3. Fold the upper part, consisting of two layers,
a-
FIG. 8
Method of Folding Paper Filter
(For reference letters see text)
from i to 2 (Fig. 8, d). The shaded triangle, 2-3-4,
now has six layers; the other 1-3-4, two layers.
4. Fold the upper double layer so as to make 2 cover
a point in the diagonal at 5, taking care to make a sharp
PREPARATION OF CULTURE MEDIA 17
point at 4 (result: Fig. 8, e). The shaded part is now
eight layers deep.
5. Turn the folder face down, and repeat the opera-
tions exactly as in 3 and 4 (result: Fig. 8, /).
6. Take up and open the large middle fold (result:
Fig. 8, g). The two halves must now be symmetrical.
7. Fold so as to make the lines 1-3 and 1-4 meet
at the center line 1-2 (result: Fig. 8, h).
8. Now pick up and fold backward so as to have i
cover 2 in the back (result: Fig. 8, i).
9. Cut through the line 1-2 and open up. The
extreme ends will be found without a fold, and may be
folded so as to make nine sharp edges.
This filter is then evenly inserted into the funnel,
spreading the folds at a distance
from each other as nearly alike
as possible. Great care should be
taken to make the folds and the
point sharp, as this insures rapid
filtering of the liquid and prevents
the filter from tearing. A wire
rack may be used, but does not FIG. 9
yield nearly so good results (Fig. 9).
Another method, simple and yielding excellent results,
is the use of absorbent cotton. Take a piece about 3
inches square, and cover lightly the lower end of the
funnel with it. Take a larger piece, also square, judging
the size according to the size of the funhel; pull it gently,
so as to make the layer of cotton thinner, without showing
any open spaces; and spread this around the inner sur-
face of the funnel.
If a vacuum pump is available, the medium may be
i8 LABORATORY GUIDE IN BACTERIOLOGY
filtered rapidly by the use of suitable apparatus, as illus-
trated in Fig. 10. At the connection with the vacuum
pump a valve should be inserted — or a flask with a rub-
FIG. 10
Filtering Media by Means of Vacuum Pump
a. Liquid medium e. Reflex flask
b. Absorbent cotton /. Rubber stopper with two holes
c. Rubber stopper g. Connection with aspirator
d. Filtered medium
ber cork with two holes may take its place — to prevent
the water from entering the flask.
Whichever method is employed, the filter must always
first be soaked in hot water, or, better, kept in steam
until used.
PREPARATION OF CULTURE MEDIA 19
The filtered liquid is collected in a smaller funnel
(Fig. 5, c). A wire basket should be supported so as to
be inclined to an angle of about 45°, so as to allow the
proper arrangement of the tubes.
Remove the plug of a culture-tube and quickly pass
the latter up the full length of the pipette (Fig. 5, /), which
should be no less than 3 inches long. Open the pinch-
cock, fill the tube one-third full, and then quickly with-
draw the tube so as to
leave no trace of the me-
dium on the upper part of
the tube, and replace the
cotton stopper. If this
precaution is neglected,
the cotton stopper will stick
to the glass, which will
cause a great deal of an-
noyance later on and ex-
pose the medium to the
danger of contamination.
Fill thirty tubes in this
manner.
If an exact volume of
medium is required in a
test-tube, an apparatus
such as is shown in Fig. FlG- "
. Filling Definite Amount of Medium
ii may be employed. into Culture-Tube
EXERCISE II. PREPARATION OF GLUCOSE-AGAR
Glucose-agar is especially used for the demonstration
of gas-forming organisms. In such cases the glucose is
decomposed, and gas appears in bubbles throughout the
medium.
20 LABORATORY GUIDE IN BACTERIOLOGY
Preparation —
1. Note the weight of an Erlenmeyer flask (500 c c.).
2. Weigh 3.75 (ij per cent.) g. of glucose into the
flask.
3. Filter 250 g. of plain agar into the flask, heat, and
agitate until the glucose is completely dissolved.
4. Fill twenty tubes in the same manner as above.
NOTE. — By tying a string across a wire basket near the top,
the plain agar-tubes may be kept separate from the glucose-agar.
A slip of paper indicating the medium and the date of its prepara-
tion must always be inserted. These two media are very diffi-
cult to distinguish by the eye, and failure to label them properly
will lead to entirely unreliable results.
The media should now be sterilized in the autoclav.
The principle of this mode of sterilization is the applica-
tion of steam under pressure. Certain bacteria, and
some of these are very widely distributed in nature, have
the faculty of forming spores. These spores are very
highly resistant to heat and do not lose their vitality either
by boiling or by application of heat under ordinary atmos-
pheric pressure, although all vegetative forms are killed.
By adding the pressure of one atmosphere to ordinary
pressure, the boiling-point is raised to 121.40°, which is
sufficient to kill all spores during an exposure of 5
minutes; media in flasks should be given 10 minutes.
The autoclav consists of a strong cylinder, made of
iron, with a bottom and removable lid. Inside is a
basket, or rack with shelves, resting on three short sup-
ports with a centrally located hole in the bottom. The
lid fits closely (in some autoclavs with a large washer),
and is provided with a steam-valve (Fig. 12, a), a safety-
valve (Fig. 12, b), and a gauge (Fig. 12, c). The latter
indicates the pressure and temperature. The top is
PREPARATION OF CULTURE MEDIA
21
fastened by means of thumbscrews (Fig. 12, d-d). The
heat is produced by two circular sets of Bunsen burners
in a hollow cylinder at the bottom (Fig. 12, e).
Other forms based on the same principles are also
in use.
Before using the autoclav the inside should be exam-
Pic. 12
Autoclav
a. Steam-valve
b. Safety-valve
c. Gauge
d-d. Thumbscrews
e. Bunsen burner and
opening
ined. It must be clean, and there must be enough clean
water in it to reach nearly to the bottom of the basket.
Water containing large amounts of impurities is liable to
foam when boiling, and thus wet the plugs and ruin the
22 LABORATORY GUIDE IN BACTERIOLOGY
medium. Place the material to be sterilized in the basket.
Fit the lid of the autoclav by joining corresponding
marks (usually a number or a letter) on the side of the
lid and margin of the main body. Then tighten the
thumbscrews by hand, always tightening those diamet-
rically opposite to each other at the same time. See that
the steam-valve is open, and then light the gas. Regu-
late the safety-valve to blow off at the required pressure,
if provided with the means of doing this.
It is necessary to allow the steam to escape for about
one minute, in order to drive all air out of the cylinder.
Then close the steam-valve. The pressure will begin to
increase, as indicated by the hand of the manometer.
When the desired temperature is reached, shut off the
gas from the outer ring of burners and regulate the gas
pressure of the inner rings, to maintain the proper tem-
perature. It is sufficient to expose the media to a tem-
perature of 120° for 5 minutes. After this has been
accomplished, shut off the gas and allow to cool to
normal pressure, or 100° or below. The lid should not
be released before this, as a sudden diminution of pressure
causes the contents of the tubes suddenly to boil up and
push the stoppers out.
The steam-valve should be opened when the auto-
clav has cooled to 100° or below. This will guard
against the possibility of the top blowing off and injuring
the operator, if the gauge does not register properly.
On the other hand, the suction caused by the contrac-
tion of the steam may hold the lid down, so that it cannot
be removed until the pressure is equalized.
When sterilizing media which contain carbohydrates
(glucose-agar, for instance), it is important not to allow
PREPARATION OF CULTURE MEDIA 23
the temperature to rise beyond 120°, nor to allow it to
remain at that point any longer than 5 minutes. Car-
bohydrates are easily broken up by heat. They are
then valueless in the medium, and also generate an acid
reaction as a result of chemical decomposition.
In the preparation of agar more difficulties are met
with than in the preparation of any other medium. The
main difficulty is the fact that it takes a long time — from
30 to 45 minutes — for the agar to dissolve, and this
creates the danger of burning it by overheating. Great
care mus't therefore be exercised in the preparation
at all stages.
EXERCISE III. PREPARATION OF PEPTONE GELATIN
1. Weigh the saucepan and measure 35oc.c. of
tap-water into it. This amount includes 50 c.c. allow-
ance for evaporation.
2. Dissolve 0.75 g. of extract of meat, and, when near
boiling, add 3 g. of peptone.
3. When boiling, add during the cold season 10 per
cent. (30 g.), during the warm season 12 per cent. (36 g.),
of the best gelatin (Gold label) by dissolving two or
three leaves at a time, with constant stirring.
4. When completely dissolved, adjust the reaction
as directed in the preparation of agar. As gelatin con-
tains considerable acid, it will take mere NaOH solu-
tion in proportion than for agar.
5. Then cool to 60°, and stir into the mixture the
white of one egg dissolved in 30 c.c. of water. Slowly
heat over the flame on a piece of asbestos, without stir-
ring, until the egg albumen is completely coagulated and
forms a dry film on top.
24 LABORATORY GUIDE IN BACTERIOLOGY
6. Adjust the weight, and filter through paper or
absorbent cotton previously moistened with hot water,
and distribute into thirty culture-tubes.
7. Sterilize in the autoclav for 5 minutes at 120°.
Care is necessary not to overheat this medium, because
gelatin easily loses the property of solidifying when cool.
In order to avoid this, the discontinuous or intermittent
method of sterilization is often used. By this method
the medium is exposed in a steam sterilizer to steam at
the temperature of 100° for 20 minutes on three con-
secutive days. The first day all vegetative forms are
FIG. 13 FIG. 14
Arnold Steam Sterilizer \ Arnold Steam Sterilizer
The hood is taken off and the door \ a. Inner water compartment
opened, showing inside arrangement b. Outer water compartment
c. Perforated bottom
d. Sterilizing chamber
e. Sheet-copper walls
killed. By keeping the medium at room or incubator
temperature, spores, which may be present, will develop
into the vegetative form and be killed by the second day's
exposure. If after this any spores should happen to
PREPARATION OF CULTURE MEDIA 25
survive, they will develop during the next 24 hours, and
the third exposure to steam will complete the sterilization.
The apparatus used for this purpose is the "Arnold Steam
Sterilizer." The usual form used is seen in Figs. 13 and
14. Fig. 13 illustrates the appearance of the ordi-
nary form with the hood off. Fig. 14 shows the inside
arrangement, a and b being two compartments connected
by small holes, and in which a certain amount of water
has to be kept. The water contained in the inner com-
partment (a) is brought to a boil by a Eunsen burner,
the steam rising through a number of holes in the bottom
(c) into the chamber (d). The steam condenses at the
top of the chamber and returns between two sheet-copper
walls (e, e) to the large compartment (b). A larger form
of steam sterilizer, constructed on the same principle, is
convenient for sterilizing large amounts of the media.
EXERCISE IV. PREPARATION OF PEPTONE-BROTH (FRENCH :
BOUILLON) AND DUNHAM'S PEPTONE-SOLUTION
1 . Weigh the saucepan and measure 600 c.c. of tap-
water into it and heat.
2. Dissolve, when hot (but not boiling), 6 g. of pep-
tone.
3. When completely dissolved, replace the evapo-
rated water and divide into two equal amounts (300 c.c.
each).
4. Filter one part twice through the same filter
(paper) and distribute into twenty-five tubes. Sterilize
in autoclav for 5 minutes at 120°. This is Dunham's
peptone-solution.
5. Dissolve completely i g. of extract of beef in the
other 300 c.c.
26 LABORATORY GUIDE IN BACTERIOLOGY
6. Adjust the reaction in the same manner as above.
7. Fill into an Erlenmeyer flask and autoclav for
10 minutes at 120°.
8. Keep the Erlenmeyer flask and contents for 24
hours in a cool place, filter, and then distribute into
FIG. 16
Potato Cylinder
showing diagonal for cutting
twenty-five tubes, and autoclav these at 1 20° for 5 min-
utes. This is "peptone-broth."
NOTE. — The reason for exposing broth to a heat of 120°
twice is this: The solution contains a considerable quantity of
substances, which are precipitated by heat and appear as a sedi-
ment after cooling. As it is important to have a perfectly clear
broth in tubes, these substances are precipitated by the first
heating, and, if tubed later, the second sterilization will not
affect the appearance of the medium.
Broth was originally prepared from chopped beef,
and for many purposes this is preferable. The method
of preparation is as follows:
PREPARATION OF CULTURE MEDIA 27
1. 500 g. (i pound) of lean minced beef, as free as
possible from fat and tissues, is added to 1000 c.c. of
tap-water and set aside on ice for 24 hours.
2. Weigh a saucepan, and cook the beef and water
for about one hour.
3. Strain through cheese-cloth, and press all the
liquid out in a meat-press (Fig. 15).
4. Replace the water lost by evaporation.
5. Dissolve 10 g. of Witte's peptone.
6. Adjust the reaction.
7. Filter into flasks, and sterilize in autoclav at 120°
for 10 minutes. It may then be distributed into culture-
tubes.
Fresh meat, as well as extract of meat, often contains
a small amount of muscle-sugar (glycogen). If it is
necessary to prepare a sugar-free broth, a pure culture
of Bacillus coli is added to the mixture of chopped beef
and water, and this is incubated at 37° for 24 hours.
After this the preparation is the same as described.
EXERCISE V. PREPARATION OF POTATO
1. Select several large potatoes, and cleanse by brush-
ing all dirt off carefully and washing in water.
2. Punch out cylinders with a borer of suitable size,
trim them, and cut them into two equal parts (Fig. 16).
Cut with a sharp knife along the diagonal.
3. Immerse the pieces in running water for 24 hours.
4. Insert one half -cylinder into each potato-tube
so that the wide end rests on the constriction, then pour
a small amount of water into it. If the potato-tubes
have no constriction, place a small wad of cotton in the
bottom and moisten this with water (Figs. 17 and 18).
28 LABORATORY GUIDE IN BACTERIOLOGY
5. Sterilize in autoclav for 5 minutes at 120°, or in
Arnold for 3 consecutive days with 20-30 minutes'
exposure.
EXERCISE VI. PREPARATION OF LITMUS MILK
i. Separate five-sixths of the cream from the milk.
FIG. 17 FIG. 18
Ordinary Style of Potato-Tube Potato-Tube
a. Potato b. Cotton
2. Add 7-10 per cent, of a solution of litmus (tincture
of litmus).
3. Distribute about yc.c. into each of twenty-five tubes.
4. Sterilize in autoclav for 5 minutes at 120°, or in
Arnold for 3 consecutive days.
NOTE. — The prepared culture media should be carefully
stored in a dark, cool place. If they are to be kept for a consid-
PREPARATION OF CULTURE MEDIA 29
erable length of time, they should be sealed either with paraffin
or with a rubber cap. They should always be protected from
dampness, as mold fungi are apt to alight on the cotton stoppers
and send their filaments into the tube. Before inoculation, they
should always be carefully examined, and those which show
cloudiness or colonies, as well as those which have shrunk from
evaporation, should be rejected.
CHAPTER IV
PREPARATION OF STAINING SOLUTIONS
Saturated alcoholic solutions of stains are prepared
by covering an arbitrary amount of stain with absolute
alcohol. The solution is saturated as long as any of the
stain remains undissolved at the bottom of the vessel.
Other solutions necessary for the preparation of stains are:
1. Solution of potassium hydrate in water i : 10,000.
2. Solution of carbolic acid in water (5 per cent.).
3. Anilin- water, prepared by shaking anilin-oil with
water (2 per cent.) and filtering twice through the same
paper. It should be perfectly clear.
The staining-bottles usually employed have a capaci-
ty of about 30 c.c. The following amounts will nearly
fill them:
1. Loeffler's methylene-blue :
Saturated alcoholic solution of methylene-blue 9 c.c.
Potassium hydrate in distilled water i : 10,000 21 c.c.
2. Ziehl-Neelsen's carbol-f uchsin :
Saturated alcoholic solution of f uchsin 3 c.c.
5$ carbolic acid solution in water 27 c.c.
3. Ehrlich's anilin-gentian- violet :
Saturated alcoholic solution of gentian-violet 7.5 c.c.
Anilin-water 22.5 c.c.
This stain requires occasional filtering and is somewhat
unstable.
4. Gram's iodin solution:
lodin o.i g.
Potassium iodid 0.2 g.
Dissolve in about 2 c.c. of water, and then add enough
water to make the total measure 30 c.c.
3°
CHAPTER V
THE MICROSCOPE
(Fig. 19)
The compound microscope is a necessary adjunct to
any kind of bacteriological work. For this work three
objectives (Leitz No. 3, No. 6, or No. 7 and fa oil im-
mersion, Zeiss No. A or No. AA, No. D or No. DD, and
•fa oil immersion) and two oculars (Nos. 2 and 4) are
indispensable. For the intelligent manipulation of the
microscope it is absolutely necessary to understand the
underlying optical principles, which may be studied from
special works on the subject.
References —
S. H. Gage, The Microscope.
Carpenter and Dallinger, The Microscope and its Revelations.
For use in the laboratory it will be sufficient to call
attention to some of the most important points to be
observed.
The usual pattern of microscope consists of two main
parts: the stand, and the optical parts (Fig. 19) which
are attached to the stand.
The stand consists of a body-tube, draw-tube, coarse
adjustment, fine adjustment (micrometer screw) in a
pillar, nose-piece, stage with clips for holding the object,
main pillar, and the horseshoe base. At the junction
of the main pillar and the fine-adjustment pillar is the
inclination joint.
The draw- tube, regulating the focal length, which
varies in different instruments, should be raised to 16
a
e
FIG. 19
Microscope (after E. Leitz)
a. Ocular
b. Place where virtual picture
formed
c. Body-tube
d. Coarse adjustment
e. Micrometer screw
/. Inclination joint
g. Horseshoe base
h. Mirror
*. Iris Diaphragm and Condenser
k. Objective
I. Nose-piece
m-o. Picture as it appears to the eye
p.-g. Object (cover-slip)
r. Adjustment spring
s. Stage
/. Object (slide)
THE MICROSCOPE
33
or 17 mm. If a nose-piece is attached, the width of
this must be deducted from the tube-length.
The optical parts are the oculars, the objectives, the
substage condenser, and the mirror. The ocular is a
combination of lenses, which slips into the top of the
draw-tube and is nearest the eye. The objective is a
combination of lenses which is screwed into the nose-
piece and fits to the lower end of the draw-tube. The
substage condenser fits under the stage. It concentrates
the light on the object and is raised for high powers or
lowered for low powers. At the lower end of this con-
denser is the iris diaphragm, which is regulated by a
small lever with a milled head, and serves the purpose
of regulating the light supply. The mirror has two
sides, a concave and a flat.
In the manipulation of the compound microscope the
following points should be strictly observed:
1. Keep the instrument scrupulously clean. When
not in use, lock it in the case or cover it with a bell-jar.
2. When carrying the instrument, grasp it by the
main pillar underneath the stage, not by the fine-adjust-
ment pillar. The fine adjustment consists of a very
delicate screw-thread, which is easily damaged.
3. The lenses, condenser, and mirror, when needing,
should be wiped with Japanese lens-paper, never with
any coarse material.
4. For cleaning use a damp cloth. For wiping the
lenses use water or xylol. Never use alcohol, as this
dissolves the cement holding the lenses in place and also
injures the lacquer.
5. Do not take the instrument apart. The working
parts are of extremely delicate nature and easily injured.
34 LABORATORY GUIDE IN BACTERIOLOGY
Be careful not to drop the oculars or objectives.
6. The inclination joint can be used only with dry
lenses and dry objects, not with the oil-immersion or
hanging-drop preparations.
7. After placing the object on the stage, focus with
a low power and the coarse adjustment. With high
powers use the coarse adjustment first and the fine adjust-
ment afterward. The free use of the fine adjustment
saves the accommodation of the eye. As the eye is cap-
able of accommodating itself to distances, it may with an
effort distinguish a picture which is not in perfect focus.
This effort is saved by using the fine adjustment.
8. Always raise the draw-tube by means of the coarse
adjustment before changing the objectives or examining
a different preparation.
9. Before focusing, obtain as good a light as possible
by turning the mirror, and then regulate the supply by
the diaphragm. Always use reflected light, never direct
sunlight.
10. When focusing an object, lower the draw-tube
until the lens almost touches the cover-glass. This can
easily be seen by looking at the instrument from one side
and watching the reflection of the objective in the cover-
glass. Then, with the eye at the ocular, slowly focus up.
Do not focus down with the eye at the ocular, as the lens
may then come into violent contact with the object,
destroying the latter and injuring the lens.
n. For living or transparent objects use as little
light as possible. For stained or opaque objects more
light is necessary.
12. Do not use higher powers than is necessary.
13. To use the oil-immersion lens, place a drop of
THE MICROSCOPE 35
clear cedar oil, free from dust and air-bubbles, on the
cover-glass, which must be perfectly dry. In this case,
by careful manipulation, the objective, after being
brought in touch with the oil by means of the coarse
adjustment, may be gradually lowered by the fine adjust-
ment until the object is focused; or lower the objective
until almost in touch with the cover-glass, and focus up.
High powers always require the use of a homogeneous
liquid between the cover-glass and the front lens of the
objective, to avoid loss of light by refraction. As a bundle
of rays disperses when entering a thinner medium from a
denser one, there is not sufficient light entering the objec-
tive to make objects discernible, when using high powers.
By the insertion of a liquid (inspissated oil of cedar) of
the same, or nearly the same, refractive index, a homo-
geneous connection is established between the cover-glass
and the objective, thus avoiding this loss and allowing a
bundle of light of sufficient power to enter the objective.1
14. After using the oil-immersion lens, wipe the oil
off with lens-paper. If the oil sticks to the lens, wipe it
off with xylol, never with alcohol. At the same time
wipe the oil off the cover-slip.
15. The microscope should stand on a firm table,
to avoid being shaken. The table should be low enough
so as not to necessitate bending the body.
1 6. Always keep both eyes open. This saves the
eyesight. Beginners find this a difficult rule to apply,
but with very little practice and persistence it is easily
accomplished. Also use both eyes alternately.
17. It is always well to move the object while bringing
'For detailed description and diagrams see S. H. Gage The
Microscope.
36 LABORATORY GUIDE IN BACTERIOLOGY
it into focus. It is then easy to feel if the lens touches
the glass, and a moving object is seen much more easily
than a stationary one.
1 8. Use the plain mirror in combination with the
condenser, and the concave mirror without the condenser
or with artificial light.
19. In preparing stained preparations, it often hap-
pens that a small amount of stain remains on the upper
side of the cover-slip. Care must be taken to focus for
a plane below this.
CHAPTER VI
COLLECTING BACTERIA FROM THE AIR
1. Sterilize all Petri dishes in the hot-air sterilizer for
one hour at 160°.
2. Melt two tubes of plain agar and one of glucose-
agar in the water-bath. The water-bath (Fig. 20) is a
round copper vessel with a number of holes in the top.
These holes are large enough to allow a culture-tube to
slip in easily. A thermometer is passed through a rubber
cork with a hole in its center, and inserted into one
of the holes in the water-bath. Enough water is then
poured into the apparatus to fill it about two-thirds, and
the thermometer is lowered until the mercury bulb is
37
38 LABORATORY GUIDE IN BACTERIOLOGY
completely immersed in the water. The culture-tubes
are then slipped in, and the water is heated to 100°.
3. Singe the cotton stopper of the liquefied agar-
tubes in the flame, remove the cotton stopper, pass the
mouth and about one inch of the tube through the flame,
and pour the contents into a sterile Petri dish, carefully
lifting the cover (Fig. 21) and quickly replacing it.
4. Repeat this operation with the second agar-tube.
5. Place both Petri dishes containing the liquid agar
on a level surface.
6. When the agar is solidified, expose, by removing
the cover, one dish to the air of the laboratory, and the
other outside on the window-sill, for 10 minutes.
FIG. 21
Pouring Medium into Petri Dish
7. Replace the cover and place in lockers.
8. Cool the water-bath to 43° exactly, and mix the
scrapings from under a finger nail with the liquid glucose-
agar. Keep this also in the locker.
9. Remove the plug of a tube of broth and place a
hair in the liquid. Keep also in the locker.
When inoculating liquefied agar media, it is necessary
to do so at a temperature no higher than 43° nor lower
than 40°. Above 43° the organisms are liable to be
injured by heat; below 40° the agar solidifies, and an
even distribution is impossible. If gelatin is used, the
latter precaution is not imperative, as gelatin solidifies at
COLLECTING BACTERIA FROM THE AIR 39
about 25°. Observe and make notes on the appearance
of these Petri dishes after 24 hours. By this time it will
be observed that a number of spots of different sizes,
shapes, colors, etc., have formed on the surface of the
medium. Each of these spots probably consists of a
large number of the same species of organism, and is
called a colony. Besides bacteria, yeasts and mold spores
are constantly present in the atmosphere and drop on the
plate. These organisms, being of higher specific gravity
than the air, are constantly falling, or are carried hither
and thither by currents in the atmosphere. Small
particles of dust also may alight on the medium, and then
form a dark spot near the center of the colony. Dust
particles are usually covered with bacteria, possibly of
different species, and in this case the resulting colony may
not be composed of one and the same species. These
are not "pure cultures."
Inoculate two or three colonies on agar-slants.
NOTE. — Agar-slants are prepared by allowing liquid agar to
solidify in a slanting position.
Method oj inoculation —
1. Singe the cotton plug of a tube containing the
medium. It is imperative always to singe the cotton
plugs of tubes before opening them, whether these tubes
contain a culture or a sterile medium. The organisms
from the air are constantly falling on the cotton, and
bacteria may also be deposited on the cotton by handling
it with the fingers. If these organisms were not killed by
the process of singeing, they would drop on the medium
after removal of the stopper, and thus ruin a pure culture.
2. Hold the tube (or, if a transfer is made, both tubes
side by side) between the thumb and the forefinger, so
40 LABORATORY GUIDE IN BACTERIOLOGY
that the end of the tube rests on one edge of the hand
(Fig. 22), holding them at an angle of about 45°. If held
horizontally, the condensation water, always present at
the lower end of the agar medium, will moisten the surface
and destroy a characteristic growth along the needle-track.
If held in a vertical position, contamination from the air
cannot be avoided.
FIG. 22
Method of Inoculating Media
3. Remove the cotton stoppers by taking hold of
the singed portion only, and hold also by the singed part
only between the other fingers. If the portion of cotton
from the inside of the tube is touched by the fingers, or
accidentally falls on the table or the floor, it becomes con-
taminated and must be singed before replacing.
4. Sterilize the straight platinum needle in the flame,
holding it like a pencil. The platinum wire should be
heated until red-hot, and the glass end passed slowly
through the flame once or twice.
5. After cooling the needle by plunging into the
medium, take up a small portion of a colony or culture
by a lateral movement on the end of the needle, and,
COLLECTING BACTERIA FROM THE AIR 41
when removing it from the tube, take care not to touch
the walls of the tube. If any cotton accidentally sticks
to the mouth of the tube, it should be burnt off with
a hot platinum needle, and then the mouth of the tube
passed through the flame before inserting the needle.
6. Insert the needle with the culture into the sterile
tube, and carefully draw along the surface of the slant,
turning the needle during the operation and without
puncturing the jelly.
7. Sterilize the needle in the flame, as above, and
replace the cotton plugs. It is not superfluous to repeat
here that the platinum needle should always be flamed
immediately after taking it up, and again before laying
it down.
A stab-culture is made in the same manner as a slant-
culture, except that the medium is punctured centrally
by a quick, steady movement of the needle. Care should
be taken not to let the needle touch the bottom of the
tube, the object being to make as narrow a puncture as
possible, and by touching the glass the needle would bend
and make a ragged opening in the medium on with-
drawing.
Inoculations of liquid media are made the same as
agar-slants, except that the end of the needle is gently
rubbed against the glass below the surface of the liquid.
The medium is then shaken. After inoculation milk-
cultures should never be shaken again, as this might
destroy a characteristic shape of the coagulum or break
up the cream-ring.
Inoculations from liquid media are made with the
looped needle. This may also be used, if a considerable
amount of growth is required on a solid medium. The
42 LABORATORY GUIDE IN BACTERIOLOGY
contents of the loop are then spread over the whole sur-
face.
Describe the appearance of the colonies as to —
1. Naked-eye appearance:
Color.
Shape.
Border.
Size (approximately in millimeters).
2. Appearance under low power of microscope.
3. Give the approximate number of colonies on the
plate.
4. Examine as to motility in the hanging-drop.
Preparation of a hanging-drop (Fig. 23) :
FIG. 23
Hanging-Drop
(After Muir and Ritchie)
1. Clean a cover-slip in alcohol. Pass several times
through the flame so as to burn the last traces of grease
off the surface.
2. Place a loopful of pure water on the center of the
cover-slip.
3. Flame the straight platinum needle, and, after
COLLECTING BACTERIA FROM THE AIR 43
cooling, touch one of the colonies and mix lightly with
the drop of water without spreading it. Take only a
very minute amount of culture, so as to produce the faint-
est visible cloudiness in the water.
4. Smear vaselin around the depression in a hollow-
ground slide, quickly invert the cover-slip over the
depression, and gently press the margin on the vaselin.
5. Examine in oil, using very little light.
Brownian movement. — Rub a small amount of carmine
in a mortar with some water and make a hanging-drop
preparation. When examining this through the oil-
immersion lens, it will be observed that the small particles
of carmine have a lively vibrating motion. This is called
" Brownian" or "molecular movement," or "pedesis."
The particles scarcely change their relative position.
Actively motile organisms, on the contrary, widely change
their relative positions. The movement of these may be
slow, snake-like, or like a fish swimming; or they may
dart rapidly across the field.
Now observe and describe what changes have taken
place in the tube of broth with the hair. Compare with
a sterile tube, noting the turbidity, sediment, odor, etc.
Also examine in hanging-drop.
Examine the tube of glucose-agar containing the nail-
scrapings. Describe the general appearance, and note
whether gas-bubbles are present.
Finally, from the stained preparations make sketches
of the appearance of the culture medium, the shape of
the colonies, and the morphology of the organisms.
Make stained preparations of three different colonies,
using the three stains; i. e., gentian- violet, methylene-
blue, and carbol-fuchsin,
44 LABORATORY GUIDE IN BACTERIOLOGY
Method of making stained preparations —
1. Clean and flame a cover-slip, or, if preferred,
a slide may be used for this purpose. Cover-slips, if
handled by the fingers, should be held by the edges. Use
as much as possible the forceps made for that purpose.
After handling the forceps they should be sterilized in the
flame.
2. Place one loopful of water on the cover-slip.
3. Take the minutest quantity of the colony or cul-
ture on a platinum needle and mix gently with water
until faintly cloudy. Burn the remainder of the culture
off the needle.
4. Spread over the cover-slip by two or three sweeps
of the needle. The water should spread easily and not
run together. If this takes place, the cover-slip has not
been cleaned sufficiently.
5. Dry by moving high over the flame.
6. Pass three times back and forth through the flame
rapidly. This process precipitates albuminous matter
and causes the bacteria to adhere firmly to the glass.
7. Cover with stain for 10-15 seconds.
8. Wash in water and examine. If satisfactory:
9. Float the cover-slip off the slide with water. Blot
with filter-paper, dry in the air or high over the flame,
and mount in Canada balsam.
10. Label and preserve this preparation.
Try to avoid the mistake, made by most beginners,
of taking too much growth on the needle. For hanging-
drop preparations less material should be used than for
a stained preparation.
CHAPTER VII
EXERCISES ON INFECTION AND STERILIZATION
EXERCISE I. PHENOMENA OF INFECTION
1. Prepare three agar-plates as directed in Chap. VI.
2. Touch the surface of the jelly in one plate with
the tips of the fingers.
3. Touch the surface of the jelly of another plate with
the tips of the fingers, after washing the hands carefully,
4. Catch a fly and allow it to walk on the surface of
the jelly of the third plate for a few seconds. Release
the fly and replace the cover.
5. Place these three plates in a locker or thermostat
for 24 hours. Observe and describe the results. Make
hanging-drop and stained preparations of some of the
colonies formed.
EXERCISE II. PHENOMENA OF STERILIZATION
1. Make an infusion of hay in a flask with cotton
stopper. (See Appendix I.)
2. Set the flask aside for 24 hours in a warm place,
and observe the results. To what are these results due ?
3. Expose a tube of unsterilized broth to steam in
the "Arnold," another to steam in the autoclav, at 120°
for 5 minutes each.
4. Set aside in a thermostat, and observe the results.
EXERCISE III. PHENOMENA OF STERILIZATION (CON-
TINUED)
Action oj Berkejeld and cotton filters. — Berkefeld filters
are made of diatomaceous earth, and are porous so as
45
46 LABORATORY GUIDE IN BACTERIOLOGY
to allow liquids to pass through, but no solids or organ-
isms.
i. Arrange a Berkefeld filter so as to connect with
the suction pump, and filter a quantity of unsterilized
broth (Fig. 24).
FIG. 24
Action of Berkefeld Filter
a. Berkefeld filter
b. Filtered liquid
c. Side tube with cotton filter
d. Intercepting flask
e. Connection with aspirator
/. Rubber hose
2. Set aside, and observe the results. The filter (a),
after being connected with the flask, is sterilized in the
autoclav. The cotton plug at c prevents the air, which
is sucked back, from carrying germs. The flask d will
INFECTION AND STERILIZATION
47
prevent the water from running back and mixing with
the filtered liquid.
Now arrange a cotton filter as shown in Fig. 25.
Vessel a, provided with a rubber stopper (b) with two
openings, is arranged so as to have a glass tube (c) reach
to the bottom. This tube is provided with cotton at
a. Erlenmeyer flask
b. Rubber stopper
c. Glass tube
d. Cotton filter
e. Broth
FIG. 25
Action of Cotton Filter
/. Bent glass tube with cotton filter
at f.
g. Erlenmeyer flask
h. Tube connecting with aspirator
the top (d) and some nutritive medium (broth, e) is
placed inside. Through the opening a bent glass tube
leads out, and this tube is also provided with a cotton
filter at /. The whole apparatus is then sterilized in
48 LABORATORY GUIDE IN BACTERIOLOGY
the autoclav at 120° for 5 minutes, and connection is
made through the flask (g) and the tube (h) with the
aspirator. Now aspirate some air through the flask,
disconnect at /, and set aside. Observe the results. Why
does no growth take place ? Remove the cotton filter
(d), and drop it into a flask containing sterile broth;
place in the thermostat for 18-24 hours; note and
examine the conditions then present.
CHAPTER VIII
STUDY OF YEASTS, MOLDS, AND TORUL/E
EXERCISE I. CULTURAL STUDIES
Read carefully the chapter on yeasts and molds in
the textbook. Yeasts, torulae, and molds grow better in
a medium of a slightly acid reaction than in a neutral or
alkaline medium. Wort-agar or gelatin prepared as
described (Appendix I) will answer very well for this
purpose.
1. Melt eight wort-agar tubes in the water-bath.
2. Pour the contents of two tubes into sterile Petri
dishes, and set aside to solidify.
3. Allow the other six tubes to solidify so as to form
a slanted surface.
4. After the plates have cooled, expose them to the
air in two different places for 10 or 15 minutes.
5. Place in your locker.
6. Examine after 24 or 48 hours. Describe what
you see.
7. Transfer several mold and yeast colonies to the
slants prepared.
8. Make transfer of a stock-culture of Saccharo-
myces cerevisiae, or any other species of yeast.
NOTE. — Molds may easily be recognized by the filamentous,
cotton-like form of the colonies. The hyphae extending into the
air carry the spores (conidia). By gently touching these with a
sterile platinum needle, the spores may be transferred to the
agar-slant, and development will take place after 24 hours.
Colonies of yeasts or torulae appear smooth, moist, opaque,
elevated, and slightly yellowish-white, or sometimes reddish.
49
50 LABORATORY GUIDE IN BACTERIOLOGY
These may be transferred in the same manner as colonies of
bacteria. Molds require very careful handling for microscopical
demonstration. They are best examined in water in the unstained
condition.
Method of preparing molds for microscopical exami-
nation—
1. Transfer some of the growth to alcohol (50 per
cent.).
2. When thoroughly moistened, transfer some of
the growth to a drop of glycerin on a slide.
3. Spread carefully with a platinum needle.
4. Cover with a slip and examine.
5. If satisfactory, the preparation may be made per-
manent by painting a ring of asphalt around the edge
of the cover-slip.
Molds may also be stained in the following manner:
1. Place a small amount of mold on a slide.
2. Cover with alcohol.
3. Wash in water.
4. Stain with gentian-violet or methylene-blue.
5. Mount in glycerin.
Study of yeasts —
1. Examine a small amount of yeast taken from an
agar-slant in water under the high power of the micro-
scope. Note the manner of reproduction by " budding."
2. Prepare a culture in liquid wort of Sacch. cere-
visiae.
3. Pour the supernatant liquid of the 24-hour-old
culture off, and distribute the sediment on a gypsum
block.
NOTE. — Gypsum blocks may be prepared in the following
manner: Gypsum (plaster of paris) is mixed with half its vol-
ume of water and quickly placed in a cylinder of paper. When
YEASTS, MOLDS AND TORUL/2 51
perfectly dry, the paper is cut away and the block is placed in a
suitable vessel (a Stender dish or a deep, narrow Petri dish will
answer very well). The block and vessel are then sterilized in
the hot-air sterilizer for one hour.
4. Pour enough distilled water around the gypsum
block to submerge about one-half of it.
5. Set aside in a cool, dark place for 3 or 4 days.
6. Examine a small portion of the film on the surface
of the gypsum under the microscope in water.
NOTE. — Under very favorable conditions, and in the pres-
ence of plenty of oxygen, yeasts will undergo spore-formation.
The great porosity of the gypsum block, which admits free com-
munication with the water, and the fact that the surface of the
block is exposed to the air, offer most favorable conditions for
spore-formation, which takes place in 3 or 4 days.
EXERCISE II. THE STUDY OF THE GERMINATION OF
SPORES
The germination of spores may be studied from spores
(conidia) of mold fungi. Although somewhat different
from the germination of bacterial spores, mold spores
illustrate the general process very well.
1. Make a suspension of spores of Penicillium glau-
cum (common green mold) in broth.
2. Flame carefully a hollow-ground slide so as to
exclude all other organisms, and paint vaselin around
the hollow so as to form a complete ring.
3. Take a loopful of the spore suspension and place it
on a clean flamed cover-glass.
4. Invert over the prepared hollow-ground slide, and
gently press the edges on the vaselin so as to exclude all
communication with the air. This prevents the hang-
ing-drop from drying out, and also the invasion of for-
eign organisms.
52 LABORATORY GUIDE IN BACTERIOLOGY
5. Observe under the microscope (No. 7 objective),
and make sketches from day to day of the progress in
the growth, until the complete organism is developed
and spores are again formed.
CHAPTER IX
SCHEME FOR ROUTINE STUDY OF THE VARIOUS
GROUPS OF ORGANISMS
This scheme is to be followed strictly in the study of
all organisms, except when special instructions are given,
and the student should thoroughly familiarize himself
with the different steps. The work of cultivation as
described in steps 1,3, and 5 may be divided between two
students working together, but all other procedures are
to be followed by each individual.
1. The inoculation of one slant-agar-tube from each
individual stock-culture supplied. These inoculated agar-
tubes are to be incubated at 37° for 24 hours, unless
otherwise instructed.
2. At the end of 24 hours make from each agar-tube:
a) A physical description of the culture (see Chap.
X).
b) A hanging-drop examination.
c) A Gram stained preparation.
d) An ordinary stained preparation.
Method of making preparations according to Gram —
a) Prepare a film of the organism to be examined,
the same as in the ordinary stained preparation.
b) Cover with gentian-violet for i minute.
c) Wash in water, and remove the water by means
of filter-paper, leaving the surface moist.
d) Cover with Gram's iodin solution for 2 minutes.
e) Pour Gram's iodin solution off and, without
washing, place in a watch-glass, film side up, and cover
with 96 per cent, alcohol.
53
54 LABORATORY GUIDE IN BACTERIOLOGY
/) Allow to remain in alcohol, with occasional agita-
tion, for at least 4 minutes, or until no more stain is
taken up by the alcohol.
g) Dry without washing, and mount.
This stain is an important means of differentiating
species of bacteria.
In order to be able to judge of the effect of Gram's
iodin solution on the stain, the preparation before
mounting may be washed in water and counterstained
with Bismarck brown. This method shows all foreign
matter brown in contrast to the bacteria, and is especially
adapted for staining of bacteria in tissues, sputum, etc.
It is a positive Gram's stain if by application of this
method either the organism loses none of the stain, or
the stain is dark blue or dark slate-blue. It is a negative
stain if either the coloration is completely gone, or only
a light bluish tinge is left.
All stained preparations and all Gram stains must be
preserved for inspection.
3. Transfer from agar-culture to the following media :
Glucose-agar.
Gelatin.
Potato.
Broth.
Litmus milk.
Dunham's solution.
These transfers, excepting gelatin, are to be placed
in the thermostat, unless otherwise ordered.
4. An accurate description is to be made of each
individual culture (see Chap. X), and outline sketches
especially of the gelatin and the milk cultures. These
descriptions should be made complete after 24 hours,
ROUTINE STUDY OF VARIOUS GROUPS
55
and any changes should be noted after 48 hours and after
6 days. (See culture charts.)
5. Plates are to be made in agar from a 24-48-hour-
FIG. 26
Thermostat or Incubator
old broth-culture of each bacterium, unless otherwise
directed. These plates are to be described once after
24-48 hours. (See Chap. X.)
NOTE. — The thermostat or incubator (Fig. 26) is a box
made of copper and having double walls, between which water
56 LABORATORY GUIDE IN BACTERIOLOGY
circulates. The outer surface is usually covered with asbestos
or linoleum, so as to hold the heat. The thermostat is provided
with two doors, the inner one of glass so as to enable the observer
to look inside without opening it, the other one covered like the
walls. A thermometer reaches through the upper part, and sev-
eral air-holes are provided in it, which permit free circulation
of air. The heat is controlled by Bunsen burners with self-
regulating devices. The gas supply is shut off automatically, if
it should accidentally become extinguished. For class use large
incubators are constructed on the same principles, with a number
of separate compartments.
CHAPTER X
METHOD OF DESCRIBING CULTURES
The following method of describing cultures should
be carefully studied, and each suggestion should be
considered in the description. It is of prime importance
that all cultures in all media should be closely observed,
and accurately described and sketched, as this is the
only method which furnishes the proper means of study-
ing and determining the different species of bacteria.
By keeping this scheme in sight while making ?. descrip-
tion, it will be possible after a little practice to make
perfect descriptions without its aid.
I. Morphological characters of bacterium.
Size.
Facility and mode of staining.
Gram's stain.
Special staining qualities.
Motility.
Present or absent.
Sluggish or active movement.
Flagella present or absent.
Capsules present or absent.
Spores present or absent.
Involution forms.
Make an accurate sketch of part of a field under
the microscope.
II. Plate cultures.
i. Naked-eye appearance.
a) Surface colonies.
57
58 LABORATORY GUIDE IN BACTERIOLOGY
Approximate number.
Shape: punctiform, lanceolate, oval, cir-
cular, spindle-shaped, conglomerate,
irregular, branched, filamentous, rosette-
shaped.
Color in transmitted and reflected light.
Approximate diameter in millimeters.
Elevation (and shape of elevation) or
depression.
Translucency.
Moist appearance.
Smoothness.
Luster.
Liquefaction (in gelatin plates only).
Consistency: soft, viscid, hard, chalky.
b) Deep colonies.
Color.
Shape.
2. Microscopic appearance under low power (No.
3 objective). Care must be taken to use very
little light, the diaphragm should be almost
closed.
a) Surface-colonies.
Shape.
Color.
Translucency.
Thickness (in center and edges).
Nucleation.
Striation.
Granulation (if present, whether coarse or
fine).
Homogeneity.
METHOD OF DESCRIBING CULTURES 59
Tuberculation (tubercle-like appearances al
edge).
Edge : entire or smooth, wavy, with pointed
protuberances, serrate, dentate, lacer-
ate (as if torn), fringed, hairlike shoots,
curled.
b) Deep colonies.
Shape.
Color.
Translucency.
Granulation.
III. Agar-slant-culture.
Limitation: confinement to needle-track or
spreading. If spreading, in what shape ?
Vigor: luxuriant or scant.
Color: by transmitted and reflected light.
Elevation or depression. Elevation more pro-
nounced at edges or in center.
Translucency.
Moistness.
Smoothness.
Luster.
Coloration of medium.
Pigment production.
Odor.
Gas-formation: in culture or in medium.
IV. Stab-culture in plain agar.
1 . Surface-growth (describe like surface-colonies.)
2. Stab-growth.
Vigor.
Extent.
Color.
60 LABORATORY GUIDE IN BACTERIOLOGY
Granulation.
Outgrowths.
Coloration of medium.
Cloudiness.
Gas-formation.
V. Stab-culture in glucose-agar.
Describe like plain agar, and in addition always
note presence or absence of gas-formation, and
presence of cloudiness.
VI. Stab-culture in gelatin.
Describe like plain agar, and in addition always
note presence or absence of liquefaction. If
liquefaction is present, it may be saucer-shaped,
turnip-shaped, conical, funnel-shaped, hori-
zontal (extending the whole diameter of tube),
sack-shaped. Cloudiness and presence of
sediment in liquefied area, and color and shape
of sediment, should be described.
VII. Potato- culture.
Describe like agar-slant, adding to it the eventual
discoloration of the medium.
VIII. Litmus milk culture.
Reaction (acid or alkaline, as indicated by
color).
Coagulation : at ordinary temperature or upon
heating.
Whey: if present, clear or turbid.
Liquefaction of coagulum (proteolysis, pep-
tonization).
Gas-formation.
Decolorization of litmus (complete or incom-
plete).
METHOD OF DESCRIBING CULTURES 61
Color of cream-ring.
Odor.
In milk the presence or absence of coagulation and
proteolysis should always be noted. Com-
pare your culture always with a sterile con-
trol milk-tube.
IX. Broth-culture.
Cloudiness: degree and uniformity, scum: ring-
or island-shaped.
Precipitate, observed by shaking. Color, forma-
tion, diffusibility, viscidity, amount.
X. Solidified blood-serum.
Describe like agar-slant, and note presence or
absence of liquefaction.
XI. Fermentation-tubes.
Gas-formation in closed arm, percentage and
relation of carbon dioxid to hydrogen expressed
TT
by the formula .
LUp
Growth in both arms or in one arm only, observed
by cloudiness.
Reaction: acid or alkaline.
Directions jor filling out the first page of culture-
charts— (see Appendix III.)
I. Name the group and organism.
II. Read in the textbook or references given so as to
indicate the source and habitat.
III. Name the most important references, and read
them.
IV. Morphological characters.
i. Describe the morphology as observed from
the stained preparation opposite the medium
from which obtained.
62 LABORATORY GUIDE IN BACTERIOLOGY
2. Size: approximate estimate in microns.
Note whether large or small, thick or slender,
round or square ends, etc.
3. Arrangement of bacteria: in groups, chains,
bunches, pairs (diplococcus) , sarcina form,
threads, branching, etc. Also note different
arrangements, if observed, in different media.
4. Staining powers: It is sufficient to mark +
for positive, - - for negative stains. Special
stains must be described more fully.
5. Motility: If absent, mark — ; if present, +.
In the latter case describe the character of
the movement.
6. Spores: Absence noted by — ; presence, by
+ . This information is to be gathered from
the textbook, unless specially instructed.
If so mention the method of spore-stain applied.
7. Note any peculiar appearance in the micro-
scopical picture, especially involution forms
and the presence or absence of capsules. If
capsules are present, note the method of
demonstration.
V. Physiological characters.
1. Relation to temperature : What is the optimum
temperature? (See textbook or references.)
2. Relation to free oxygen: aerobe, anaerobe,
facultative aerobe or anaerobe.
3. Relation to disinfectants, light, desiccation,
heat (thermal death-point). (Refer to text-
book or references.)
4. Pigment-production: If present, +; if absent,
— . In the former case, note the color, dif-
METHOD OF DESCRIBING CULTURES 63
fusibility, solubility, influence of acids and
alkalis.
5. Gas-production in glucose media: To be filled
out only in case of actual observation. In
fermentation-tubes note the growth in either
arm or both arms, recognized by turbidity.
Note the total percentage of the gas formed in
24 and 48 hours. Reaction may be tested by
the addition of litmus-solution. Gas formula
expressed : .
CO 2
6. Acid or alkali production in litmus milk.
7. The production of indol or nitrites, or both,
is tested on the sixth day of observation in a
culture in Dunham's peptone-solution or
sugar-free broth.
NOTE. — Indol is a decomposition product of
proteids and belongs to the aromatic series.
Nitrites are the result of reduction from nitrates.
The ability of organisms to produce these reactions
are of great importance in their differentiation. A
control test with a tube of sterile medium should
always be made.
8. Enzym-production : Froteolytic enzym-pro-
duction noted by the liquefaction of gelatin or
casein. Coagulative, by precipitation of casein,
if acid-formation is absent, or present only in
quantities less than 0.3 per cent. Diastatic, by
the digestion of starch (potato).
9. Characteristic odor.
10. Pathogenesis : What pathogenic effect has the
organism on man ? What effect on animals,
64 LABORATORY GUIDE IN BACTERIOLOGY
and which animals ? What diseases are
caused by the organism in man or animal ?
NOTE. — It may be well here to call attention to the terms
"proteolysis," "enzym -production," and " coagulation."
" Proteolysis " is the breaking up of complex nitrogenous
compounds (proteids), rendering them soluble. This process is
also expressed by the terms " peptonization " and "liquefaction."
The liquefaction of gelatin is one kind of proteolysis. Gelatin
is composed of nitrogenous matter (albuminoid or gelatinoid),
and it is for this reason mainly that gelatin stab-cultures are
made. If the gelatin is liquefied, we conclude that the organism
is capable of producing a " proteoly tic " enzym. In milk the
process is more complex, and this medium, on account of its
composition (fat, milk-sugar, casein, lactalbumin), offers excel-
lent opportunities for the organism to develop different charac-
teristics. Milk is one of the most important media. The casein-
ogen, contained in milk in colloid solution, may be precipitated
by an enzym or by an acid. This precipitate forms the coagu-
lum. At least 0.4 per cent, of acid, which is largely lactic acid
produced by splitting of milk-sugar (lactose), is required for
precipitation, and this amount of acid will turn the blue litmus to
a decided pink. A coagulum may also be produced by the presence
of a " coagulative " or "rennet "-like enzym, which is the result
of the metabolic activity of the organism. Such coagulation may
take place in milk of an amphoteric or alkaline reaction, as well
as in milk of a slightly acid reaction. The coagulum formed by
any of the mentioned agents may gradually contract, and a straw-
yellow, opalescent liquid is squeezed out, called "whey." If,
now, the organism also produces a proteoly tic enzym, this will
attack the coagulum and gradually dissolve it (proteolysis, pep-
tonization). At first the coagulum shows a broken-up surface;
lumps separate and settle to the bottom, and finally the coagulum
may completely disappear. Theoretically, coagulation is always
necessary before proteolysis sets in, but in the case of some organ-
isms the proteolytic enzym is so powerful as to produce immediate
dissolution of the casein.
Another phenomenon frequently observed in litmus milk is
METHOD OF DESCRIBING CULTURES 65
the discolorization of the litmus, whether this be pink or blue.
This is due to the fact that the organism takes up the oxygen
necessary to maintain the coloration. It may be frequently
observed that at the surface, where atmospheric oxygen has
access, the color remains or is restored. The color may also be
restored by shaking the milk vigorously, thus bringing it into
intimate touch with the oxygen of the air.
The production of a diastatic enzym (diastase, amylase) is
demonstrated by gas-production on potatoes. This medium con-
tains starch in large amounts. The starch is inverted into glu-
cose by diastase (amylase), and this is then fermented with
gas-production.
Directions for filling out second page of culture-charts —
i . Note the reaction of the medium by the sign -f- for
acid and — for alkali reaction.
2. The incubation temperature may be (a) 37°
(thermostat), (b) room temperature, (c) ice-chest.
3. Plates are to be described only once after 24 or
48 hours, according to growth. Make notes in the first
column for gelatin; in the second, for agar-plate, writing
across the full width of the page. Make sketches in the
special column reserved for this purpose.
4. The growth on the media 3, 4, 5, 6, 7, and 8
(enter here: " glucose-agar " only unless otherwise
instructed) to be made fully according to the outline in
the spaces under 24 hours. In the spaces under 48 hours
and 6 days note only the changes from the first day.
5. Make sketches frequently and accurately, espe-
cially from milk and gelatin media, and any other remark-
able growth, in the column reserved for this purpose;
also a sketch of each organism from a part of a field
under the microscope.
It is well to understand that these directions for fill-
66 LABORATORY GUIDE IN BACTERIOLOGY
3
• V
l\
FIG. 27
Streak-Cultures
FIG. 28
Stab-Cultures
METHOD OF DESCRIBING CULTURES 67
L
FIG. 20
Liquefaction of Gelatio
123
FIG. 30
1. Gas-bubbles in glucose-agar 3. Coagulation and peptonization of milk
2. Coagulation of milk 4. Complete peptonization of milk
68 LABORATORY GUIDE IN BACTERIOLOGY
ing out culture-charts are applicable in the described
manner to studies of cultures furnished by the laboratory.
For determining any species of unknown bacteria, original
researches must be made to cover all points, necessarily
without the possibility of gathering this information from
textbooks or references. On the accuracy of observation
and description depends the success of bacteriological
work and species-determination. It is frequently neces-
sary to employ special media, or inoculation of animals,
or such reactions as the agglutination test, to determine
properly what species one is dealing with.
The student will do well to familiarize himself care-
fully with all directions and explanations given in this
and the previous chapters. Find a characteristic for
each item mentioned; otherwise the descriptions will
be incomplete; and follow the instructions for routine
work with all possible accuracy.
CHAPTER XI
STUDY OF CERTAIN CHROMOGENIC BACTERIA
EXERCISE I. CULTURAL STUDIES
1. Read carefully the following references to chromo-
genic organisms, and the production and chemistry of
pigments:
Sternberg, Manual of Bacteriology.
Lehmann and Neumann.
Members of this group are widely disseminated in the
air, water, etc. A few representatives will be studied.
2. Inoculate agar-slants from stock-cultures of Bacil-
lus prodigiosus, B. pyocyaneus, B. violaceus, and Sarcina
luiea. Inoculate three slants each of B. prodigiosus and
B. pyocyaneus, and one each of Sar. lutea and B. violaceus.
3. Label each tube with the name of the culture
inoculated, the date of the stock-culture and the date of
inoculation. Place the label on the side of the tube
where the slanted surface of the medium is. It is then
possible to study the growth and see the label at the same
time. Glass pencils for marking are very convenient.
4. Place one culture of each organism in the thermo-
stat, one culture of B. prodigiosus and B. pyocyaneus in
the locker, and leave the others exposed to sunlight.
5. After 24 hours compare the growths of B. prodigio-
sus and B. pyocyaneus, under the various conditions, in
respect to—
a) Relative amount of growth.
b) Relative amount of pigment produced.
What conclusions can be drawn from this experiment ?
69
70 LABORATORY GUIDE IN BACTERIOLOGY
6. Note the characteristics of the pigments : Are they
diffused through the medium, or are they confined to
the growth ?
7. Make descriptions of agar-cultures ; also hanging-
drop, stained, and Gram preparations.
8. Transfer from 24-hour-old agar-cultures of all
organisms to all media. (See routine study, Chap. IX.)
Potatoes may be inoculated with the looped needle, as
the surface is too rough to allow of a smooth inoculation
with the straight needle.
9. After all cultures have been in the thermostat for
24 hours, make all descriptions as outlined in Chap. X.
(Record after 48 hours and 6 days only observations of
change.)
CAUTION. — The gelatin-cultures are sometimes placed by
students in the thermostat along with others through oversight.
This, of course, completely defeats the purpose of obtaining a
stab-growth, as the gelatin will melt. In order to avoid this
mistake, it is recommended to label one tin cup or tumbler
" Gelatin" in large letters. This will serve as a constant reminder
that gelatin has to be kept at room temperature.
10. Make plate-cultures of the four organisms.
Method oj making plates —
a) Melt two agar-tubes for each organism in the water-
bath and cool to 43°.
b) Transfer 3-5 loopfuls (according to the intensity
of the growth, to be judged by the degree of cloudiness)
of the broth-culture to a sterile tube of Dunham's solu-
tions. (Dilution i).
c) Shake well, avoiding air-bubbles as much as
possible.
d) Transfer 4 or 5 loopfuls from this suspension to a
melted agar-tube, and label this 2. (Dilution 2).
CERTAIN CHROMOGENIC BACTERIA 71
e) Shake this carefully by rolling the tube between
the palms of the hand, or stir with the platinum needle,
so as to avoid air-bubbles.
/) Transfer 4 or 5 loopfuls of this agar-tube (2) to
the second agar-tube (3), and mix as above.
g) If thought necessary, more tubes may be treated
in the same way, resulting in still higher dilutions. In the
meantime the inoculated tubes should be replaced in
the water-bath, so as to keep them liquid.
h) Pour the contents of the tubes, one after the other,
into sterile Petri dishes.
i) Tip the Petri dishes very carefully, so as to dis-
tribute the medium evenly over the bottom.
k) Label them 2nd, 3d, dilution, etc., with the name
of the organism and the date.
/) Set aside on a level place to solidify.
m) When solidified, place them bottom up in the
thermostat, in order to avoid moistening of the surface
by the condensation water dropping down from the
cover.
NOTE. — If the surface were moistened, the colonies would
run together and the characteristic appearance be destroyed. Gela-
tin plates, on the contrary, are placed cover up. Condensation
water does not form on these plates as gelatin may be liquefied
by the organisms. The liquefied part would then fall from the
medium on the cover and ruin the plate.
The plates prepared in the above manner should be
studied after 24 hours, or, if not sufficiently developed,
after 48 hours, according to directions in Chap. X.
EXERCISE II. STUDY OF PIGMENTS
On the sixth day take agar-slant or potato-cultures
of the four chromogenic bacteria, and proceed as follows :
72 LABORATORY GUIDE IN "BACTERIOLOGY
Pour 96 per cent, alcohol on the cultures of B. prodigi-
osus, B. violaceus, and Sar. lutea. The pigment should
dissolve. Filter the liquids into clean test-tubes, and,
by adding a few drops of 5 per cent, hydrochloric acid,
note the change in color. Then add an excess of a 2
per cent, solution of sodium hydrate, and note whether
or not the color returns and is changed again. Then
pour chloroform on a culture of B. pyocyaneus. This
will dissolve the bluish-green pigment (pyocyanin).
Filter, and evaporate on the water-bath. When almost
dry, place a small amount on a slide, and observe the
small crystals of pyocyanin under the microscope. Note
also the aromatic odor given off by the pigment as the
solvent evaporates.
CHAPTER XII
THE PYOGENIC GROUP
EXERCISE I. THE PYOGENIC GROUP (SUBGROUP A)1
MEMBERS —
Staphylococci, streptococci, Microc. tetragenus.
Inoculate agar-slants from cultures (furnished) of
Staphylococcus pyogenes aureus, Staph. pyogenes albus,
and Streptococcus pyogenes. These organisms are patho-
genic, and care must be taken to observe the rules of
technique with the utmost accuracy. Any carelessness
may be followed by the gravest consequences. In case
of accident, such as the spilling of a culture or infecting
the hands, thorough disinfection — e. g., with a solution
of mercuric chlorid (i : 1000) — is absolutely necessary.
After 24 hours' incubation of the three agar-slants,
proceed with the other media as outlined in the routine
study (Chap. IX).
Special study. — A rabbit will be inoculated intra-
venously with a broth-culture of Staph. pyogenes aureus.
The ear of the rabbit is carefully shaved, washed with
mercuric chlorid solution, followed by alcohol. Then
0.5 c.c. of a 24-hour-old culture in broth is drawn up
into a hypodermic syringe, which has been previously
sterilized by immersion in boiling water for 10 min-
utes. The mode of holding a rabbit is as follows:
The left arm of an assistant rests against the hind-
quarters of the rabbit on the table, while the two hands
1 This subdividing of the pyogenic group is an arbitrary
measure designed simply to facilitate study, the commoner pyo-
gens being studied first.
73
74 LABORATORY GUIDE IN BACTERIOLOGY
gently hold the fore-legs. If the animal struggles, too
much force should not be applied, as this might injure
or kill it. The struggles may be overcome by wrapping
the animal in a towel or any other similar piece of cloth.
The needle is then inserted into the lumen of the lower
vein (ramus lateralis posterior of the vena auricularis
posterior), which has been pinched between the fingers,
or by means of a forceps, so as to arrest the circulation.
No air should be injected with the culture, as this will
kill the animal. The hypodermic needle is then with-
drawn and sterilized in boiling water for 15 minutes.
After the death of the rabbit, study the lesions pro-
duced by the organism, and make cultures on slant-agar,
and smears from the heart's blood, spleen, and any foci
of suppuration.
AUTOPSY (SEE SKETCH, FIG. 31)
1. Have the instruments sterilized in boiling water.
2. Tie the animal by the extremities on a square
board, with the abdomen upward.
3. Note the presence of any external lesions, such as
swellings, ulcerations, etc.
4. Wash with a solution of mercuric chlorid (i : 1000)
followed by alcohol.
5. Lift the skin over the pubes with the forceps, and
with the scissors make an incision along the median line
well above the sternal notch; then diagonal incisions
extending to the fore- and hind-legs.
6. Cut the skin away carefully with a moderately
sharp knife, avoiding opening the abdominal cavity, and
pin to the board.
7. Open the abdomen by a median incision from the
pubes to the sternum.
PYOGENIC GROUP
75
-STOMAC H
h-SPLEEN
-KIDNEY
INGUINAL
GLANDS —
FIG. 31
Method of Autopsy of a Guinea-Pig (Diagrammatic)
76 LABORATORY GUIDE IN BACTERIOLOGY
8. Remove the anterior thoracic wall by cutting away
the ribs from below upward on each side to the thoracic
apex.
The viscera are now thoroughly exposed. Cultures
and smears should be made from different parts,
especially from the heart's blood, peritoneal cavity,
spleen, liver, and localized foci of suppuration.
EXERCISE II. THE PYOGENIC GROUP (SUBGROUP B)
MEMBERS —
Micrococcus lanceolatus.
Micrococcus gonorrhoeae.
Micrococcus intracellularis meningitidis.
Micrococcus zymogenes.
Prepared cover-slips of Micr. gonorrhoeae are furnished.
Stain these with methylene-blue and Gram's method,
study, and describe the microscopical appearance. This
organism is extremely difficult to cultivate. It is a strict
parasite, and requires special media, and a great amount
of time and care, for artificial cultivation. For these
reasons, only the morphology, as it appears in gonorrheal
pus, and which is very characteristic, is studied.
Inoculate agar-slants from stock-cultures of Micr.
lanceolatus and Micr. zymogenes.
REFERENCES (Micr. zymogenes) —
MacCallum and Hastings, Journal of Experimental Medi-
cine, Vol. IV (1899), p. 521.
Harris and Longcope, Centralblatt fur Bakteriologie und
Parasitenkunde, Part I, Vol. XXX, No. 9 (printed in
English).
1. Routine study. — Note particularly the microscopic
appearance of both organisms and the action of Micr.
zymogenes on milk and gelatin.
2. Special study A. — The staining of capsules from
PYOGENIC GROUP 77
a milk-culture of Micr. lanceolatus . Two methods may
be applied for this stain :
First method (Friedlander's method) :
a) Prepare a stain by the following formula:
Glacial acetic acid i part
Saturated alcoholic solution of gentian-
violet 5 parts
Distilled water 10 parts
b) Prepare a very thin film in the usual manner from
a 24-hour-old milk-culture, taking care to spread very
thinly.
c) Cover with stain for 10-15 seconds.
d) Wash in a solution of sodium chlorid (0.85 per
cent.).
e) Examine in a sodium chlorid solution.
/) If satisfactory, float the cover-slip off with salt
solution, dry, and mount in balsam. Under no conditions
should plain water come in contact with this preparation
at any stage; otherwise no capsules will appear.
Second method (Welch's method):
a) Prepare a very thin film in the usual manner from
a 24-hour-old milk-culture.
b) Cover with glacial acetic acid for 5 seconds.
c) Wash acid off with carbol-fuchsin.
d) Wash stain off with a 0.85 per cent, sodium
chlorid solution.
e) Examine in salt solution.
/) If satisfactory, mount in balsam.
Plain water should not come in contact with this
preparation at any stage. The capsule should be ob-
served as a lightly stained zone with well-defined outline
around the deeply stained organism.
78 LABORATORY GUIDE IN BACTERIOLOGY
3. Special study B. — Inoculation of mouse with Micr.
lanceolatus.
a) Fasten the mouse in the holder (Fig. 32).
b) Shave a place on the back immediately above the
tail.
c) Wash with a solution of mercuric chlorid (i : 1000),
followed by alcohol.
FIG. 32
Mouse-Holder
d) Inject 0.2 c.c. of a milk-culture of Micr. lanceo-
latus.
e) When dead, perform an autopsy, and study the
lesions in the usual manner.
/) Make cultures in milk and on slant-agar from
the heart's blood or the spleen.
g) Make a capsule stain from the heart's blood,
spleen, or other organs.
CHAPTER XIII
THE INTESTINAL GROUP
This chapter is devoted to the study of the "intestinal
group" of organisms. This collective group may
conveniently be subdivided into four subgroups:
Subgroup i : the colon group. — This group includes
different varieties of Bacillus coli, B. lactis aerogenes, and
some species of B. acidi lactici.
Subgroup 2 : the hog-cholera, B. enteritidis, or inter-
mediate group. — This group includes B. cholerae suis, B.
paratyphosus (several varieties), B. enteritidis, and B.
icteroides. The term " intermediate " is assigned to this
group, because it resembles the colon group on the one
hand, and the typhoid group on the other.
Subgroup 3: the typhoid-dysentery group. — This
group includes B. typhosus, varieties of B. dysenteriae,
and B. jaecalis alcali genes.
Subgroup 4 : the proteus group. — This group includes
all varieties of proteus (Bact. termo) and B. cloacae.
EXERCISE I. STUDY OF SUBGROUP I : THE COLON GROUP
Inoculate agar-slants from stock-cultures of B. coli
(two varieties) and B. lactis aerogenes. Also inoculate
one tube of broth with B. coli.
These two types of B. coli are differentiated by their
ability to ferment carbohydrates:
A. Those which ferment dextrose, lactose, and
saccharose.
B. Those which ferment dextrose and lactose, but
not saccharose.
79
8o LABORATORY GUIDE IN BACTERIOLOGY
REFERENCES —
Theobald Smith, The Wilder Quarter Century Book, 1893,
p. 187.
Jordan, Journal o/ Hygiene, Vol. I (1901), p. 295.
Durham, Journal oj Experimental Medicine, Vol. V, p. 353.
1. Routine study. — Observe carefully the growth on
potato of B. lactis aero genes. What is the cause of gas-
formation in this case ?
2. Special study A. — In order accurately to test the
action of micro-organisms on various carbohydrates, it
is necessary to eliminate the small amount of sugar in
ordinary broth introduced into it by meat-extract, which
generally contains muscle-sugar (glycogen). This is
accomplished by adding to freshly prepared broth a
culture of B. coli, which completely decomposes many
carbohydrates, including muscle-sugar. By this method
a sugar-free broth is prepared, which may be used as a
solvent for any sugar desired.
Preparation of sugar-free broth for the fermentation-
tube:
1 . Dissolve —
In water 400 c.c.
Extract of beef i g.
Peptone 4 g.
by heat. Broth made from chopped beef (500 g. to i
liter) may also be used for this purpose.
2. After cooling, inoculate with a broth-culture of
B. coli prepared 24 hours previously.
3. Set aside in the locker, or, better, in the thermostat,
for 18-24 hours.
4. Boil 5 minutes (to kill B. coli), and filter repeat-
edly through the same paper until perfectly clear.
5. Divide into three equal parts and dissolve 2 g.
INTESTINAL GROUP 81
(ij per cent.) dextrose, lactose, and saccharose, respec-
tively, in each part, and filter again, if necessary.
6. Fill fermentation-tubes, taking care to label each
one properly, and sterilize in Arnold on 3 consecutive
days for 20 minutes.
All gas must be carefully tilted out of the closed arm
of the tube while the fluid is warm. When sterilization
is completed, inoculate one set of the fermentation-tubes
with B. coli A, another set with B. coli B, and a third set
with B. lactis aero genes. Inoculate with the straight or
looped needle.
After 24 hours, note the percentage of gas formed.
This is done by means of Frost's fermentation-chart, a
model of which is represented by Fig. 33. This chart is
placed between the open and closed arms of the tube,
resting on the neck and moved along until the extreme
upper end of the closed arm is level with the top of the
chart and parallel with the vertical lines. The percent-
age may then be read off by the figures marked at each
end of the chart.
After taking note of the percentages, replace the fer-
mentation-tubes in the thermostat, and repeat the meas-
urement after another 24 hours' incubation. Many
organisms are able to obtain the oxygen necessary for
life by attacking compounds — as, for instance, carbo-
hydrates— and decomposing them. In such cases a tur-
bidity is developed in the medium. The closed arm offers
strict anaerobic conditions, all the atmospheric oxygen
having been driven out by the heat of sterilization. The
bulb allows the organism to come in contact with atmos-
pheric oxygen, and therefore offers aerobic conditions.
Gas-production is by no means a constant phenom-
82 LABORATORY GUIDE IN BACTERIOLOGY
enon accompanying a fermentation. Carbohydrates are
fermented by many organisms without gas-formation.
The usual product in this case is an acid, mostly lactic
acid. Such fermentations produce turbidity only, and
no gas. It is therefore necessary, in describing them,
to characterize the process as fermentation without
gas-formation. '.
Method oj analyzing the gas produced in the closed arm.
—The gas consists approximately of carbon dioxid and
hydrogen, which may be proved by the following method:
Fill the bulb with a 2 per cent, solution of sodium hydrate,
and close the mouth with the thumb, taking care not to
leave any air between the thumb and the liquid. Now
slowly tilt the gas back and forth from the closed arm
to the bulb five or six times, and finally allow it to collect
again in the closed arm. The sodium hydrate combines
with the carbon dioxid, and consequently on releasing the
thumb the volume of gas will become smaller in propor-
tion to the amount of carbon dioxid absorbed. Now
measure the percentage again with the chart, and ascer-
tain the proportion of gas left in the closed arm to the
original amount.
Example —
Total percentage of gas before addition of NaOH. ... 45
Percentage left after absorption by NaOH 30
Difference 15
30 per cent, then represents the amount of hydrogen
left, and 15 per cent, the amount of carbon dioxid ab-
sorbed. The proportion is expressed by the formula
H =3o 2
coa 15 r
INTESTINAL GROUP 83
The fact that this gas remaining in the closed arm is
hydrogen may now be proved by tilting it into the open
arm, which has previously been filled with water and
closed by the thumb. By holding a burning match
over it and quickly releasing the thumb, a slight explo-
sion takes place from the sudden combination of the
hydrogen with the oxygen of the atmosphere.
Gas-formation by bacteria does not necessarily
depend on the presence of carbohydrates. Nitrogen is
often produced from nitrites, and hydrogen sulphid and
ammonia from proteids, especially during the process of
putrefaction.
3. Special study B. — Test for indol and nitrites.
What is indol ? What biological activity of the organism
does the presence of indol or nitrites, or both, indicate ?
a) Test for nitrites: Add to a culture in Dunham's
solution, or, better, in sugar-free broth, successively i
drop of each of the following solutions:
(1) Sulphanilic acid 0.5 g.
Acetic acid (25$) 150 c.c.
(2) Naphthylamine chlorid o. i g.
Distilled water 20 c.c.
Acetic acid (25$) 150 c.c.
A yellowish-red or rose-color shows the presence of
nitrites.
b) Test for nitrites and indol combined.
(1) Add to a culture in Dunham's solution, or sugar-
free broth, i or 2 drops pure sulphuric acid.
(2) Heat gently. Rose-color shows the presence of
nitrites and indol. If no reaction takes place, add —
(3) A few drops of a solution of o.i g. potassium or
sodium nitrite in 1000 c.c. water. Rose-color then
indicates the presence of indol only.
84 LABORATORY GUIDE IN BACTERIOLOGY
Perform these tests with all the organisms of the
intestinal group, and make control tests in sterile Dun-
ham's solution.
4. Special study C. — Make capsule stain of B.
lactis acrogenes from 24-hour-old milk-cultures. (For
method see p. 77.)
The study of B. coll is of special importance in con-
nection with bacteriological analyses of water (see Chap.
XXIV). The presence of this organism in large num-
bers indicates sewage contamination, and consequently
the danger of an admixture of pathogenic bacteria such
as B. typhosus and B. dysenteriae.
EXERCISE II. STUDY OF SUBGROUP II
THE HOG-CHOLERA, B. ENTERITIDIS, OR INTERMEDIATE GROUP
Use great care in handling members of this group.
Inoculate agar-slants from stock-cultures of B.
cholerae suis (bacillus of hog-cholera), B. enteritidis
(Gartner's bacillus), and B. paratyphosus (two strains).
REFERENCES —
B. cholerae suis:
Moore, The Pathology of Infectious Diseases of Animals.
McFarland, Textbook of Bacteriology.
B. paratyphosus:
Buxton, Journal of Medical Research, 1902, Vol. VIII, p. 201.
Wells and Scott, Journal of Infectious Diseases, 1904, No. i.
Gushing, Johns Hopkins Hospital Bulletin, 1900, p. 156.
Durham, Journal of Experimental Medicine, Vol. V, p. 353.
1. Routine study. — Observe particularly the bluish-
green coloration of the cream-ring in litmus milk, and
make test for indol.
2. Special study A. — Inoculate plain sterile milk with
B. cholerae suis. After 8-10 days a clearing of the milk
INTESTINAL GROUP 85
will be observed, due to a solvent action of the alkali
produced by the organism upon the proteid content.
3. Special study B. — Inoculate fermentation-tubes the
same as for B. coli. Measure and test gas. Compare
the results with those obtained in the study of the colon
group. What difference do you notice ?
4. Special study C. — Inoculation of a rabbit sub-
cutaneously with B. cholerae suis. Subcutaneous inoc-
ulations of rabbits are usually made in the following
manner: An assistant, in a sitting position, places the
rabbit back-down in his lap. The head projects beyond
the knees of the assistant. The ears and hind-legs are
grasped, and the animal is thus held in position. The
hair is then cut off on a portion of the abdomen, and the
place is treated with mercuric chlorid and alcohol in the
usual manner. With sterilized fingers the skin is then
pulled up, the syringe inserted, and the material injected.
After the rabbit has died, study the lesions produced
by the organism, and make smears from the site of the
inoculation, the heart's blood, and other organs. Note
the polar staining, i. e., stained portions at the two ends
of the cell and an unstained area between. Make cul-
tures on agar from the heart's blood and other internal
organs.
EXERCISE III. STUDY OF SUBGROUP III
THE TYPHOID-DYSENTERY GROUP
Use great care in handling members of this group.
Inoculate agar-slants from stock-cultures of B. typho-
sus, B. dysenteriae (Shiga), and B. jaecalis alcaligenes.
i. Routine study. — Study carefully the reaction on
milk, and test for indol. Preserve glucose-agar-cultures
for two weeks.
86 LABORATORY GUIDE IN BACTERIOLOGY
2. Special study A. — Inoculate fermentation-tubes in
the same manner as in the two preceding groups. Ob-
serve the absence of gas-formation but note growth in
both arms. Compare the results with those of the colon
and intermediate groups.
3. Special study B. — The staining of flagella. — Read
carefully in the textbook the methods of staining flagella.
To demonstrate the presence of flagella on B. typhosus,
the following method will give good results (Loeffler's
method) :
a) Prepare the mordant. (What is a mordant ? See
the textbook.)
Tannic acid (25$ aqueous solution) 10 parts
Ferrous sulphate (saturated aqueous solution) ... 5 parts
Fuchsin (saturated alcoholic solution) i part
b) Prepare a number (six) of cover-slips by carefully
flaming them, and place them side by side on a piece of
filter-paper. (This paper must be burned after using).
c) Place 4 or 5 loopfuls of water on a clean slide.
d) Make a light suspension in this water of B.
typhosus from a 24-hour-pld agar-culture, taking par-
ticular care to stir the suspension as little as possible.
e) Place a loopful of water on each of the cover-slips.
/) Carry over a loopful of the suspension on the slide
to one of the cover-slips, from this to second, from the
second to the third, etc.
g) Spread carefully and allow to dry in the air.
h) Cover with the mordant.
i) Heat over a small flame for ij minutes while
steam rises, or better heat for 5 minutes on a water-bath.
Replace the evaporated stain, to prevent its drying on
the cover-slip.
INTESTINAL GROUP 87
j) Wash thoroughly in water.
k) Drain the water off with blotting-paper.
/) Cover with anilin-gentian- violet or carbol-fuchsin.
m) Heat as before over a small flame for ij minutes.
n) Wash thoroughly in water.
0) Examine in water.
p) If satisfactory, mount in balsam.
4. Special study C. — A ggluiination.— Dried-blood
method of Johnston: A drop of blood of a typhoid-
fever patient is obtained by pricking the lobe of the ear,
previously carefully cleaned and washed with alcohol.
The blood is taken up by a piece of sterile non-absorbent
paper or on a sterile aluminum slide. This is sent to a
laboratory, where the blood is dissolved in physiological
salt solution in such a manner as to obtain an approxi-
mate dilution of 1:25. This solution is then tested with
a suspension of typhoid bacilli, a young culture of which
is constantly kept on hand for this purpose.
For laboratory tests the serum of an animal (either
a rabbit or a guinea-pig) which has been injected with
cultures of B. typhosus, previously heated for i hour at
60°. This process kills the organisms, but the toxins
remain active. The animal is then bled in the following
manner: One of the ears is shaved, and the skin is
washed with alcohol. A small vein near the border is
opened, and the blood is collected in a sterile glass vessel.
If the animal does not bleed freely enough, it must be
warmed, preferably by means of a hot-water bag. The
blood is placed in the ice-chest, and the serum is col-
lected after separation.
The method of procedure with a serum obtained in
the above-described manner is as follows:
88 LABORATORY GUIDE IN BACTERIOLOGY
a) Small quantities of the serum are diluted with
sterile salt solution (0.85 per cent.) so as to represent
dilutions of 1:5, 1:25, and 1:50.
b) A suspension of a 24-hour-old agar-culture of B.
typhosus in salt solution is prepared. This suspension
should be faintly turbid and uniformly so.
c) Three hanging-drop preparations are made by
mixing a loopful of this suspension, with a loopful of the
three serum dilutions, respectively. The final dilutions
then are: 1:10, 1:50, and 1:100.
d) Examine with the high power (dry lens), and
observe the clumping of the bacilli, preceded by the loss
of motility.
e) Tabulate the results as to time and completeness
of reaction.
/) Make a control hanging-drop without serum to
test the motility and the absence of clumps.
Blood may also be obtained by puncturing the lobe
of the ear and collecting it in a capillary glass tube with
a small bulb. By holding the bulb down, fill three-
fourths full with blood, and seal the ends in the flame.
In 45 minutes the serum will have separated, and is
to be tested in the above manner.
The above-described method of agglutination test is
known as the microscopic test. Another method, in
which larger amounts of serum and suspension are
required, is known as the macroscopic method. Small
test-tubes are used, and definite amounts of bacterial sus-
pensions are introduced by means of finely graduated
sterile pipettes. The serum is then added in varying
amounts so as to effect the desired dilutions (see table, p.
117). The tubes are then incubated at 37°, usually for
INTESTINAL GROUP 89
2 hours, or longer if desired. If complete agglutination
takes place, the bacteria will have collected in clumps at
the bottom, forming a sediment. The supernatant fluid
is perfectly clear. By varying amounts of sediment and
varying degrees of turbidity of the supernatant fluid, the
degree of agglutination may be estimated. A control-
tube of a bacterial suspension without addition of serum
serves as a guide. Controls with normal serum should
also be made.
5. Special study D. —
a) Make cover-slip preparations of B. dysenteriae
from glucose-agar-cultures 10-12 days old. Involution
forms are then plentiful and can be studied.
b) Also make cover-slip preparations of B. typhosus
from glucose-agar-cultures 10-12 days old. Does it
show the same picture ?
EXERCISE IV. STUDY OF SUBGROUP IV
THE PROTEUS GROUP
REFERENCES —
Lehmann and Neumann.
Jordan, State Board of Health of Massachusetts, 1890.
Jordan, Journal of Hygiene, Vol. II.
Inoculate agar-slants from stock-cultures of Proteus
vulgaris, Prot. zenkeri, and Bacillus cloacae.
1. Routine study. — Observe particularly the action
on milk and gelatin.
2. Special study A . — Make plates in gelatin and agar,
and observe the colonies after 24, 48, and 72 hours.
Note the appearance of the colonies of Prot. vulgaris
and of Prot. zenkeri on both media.
3. Special study B. — Inoculate fermentation-tubes in
the same manner as for the other intestinal groups.
90 LABORATORY GUIDE IN BACTERIOLOGY
Determine the percentage of gas formed and the gas
formula. Compare the results with those of the other
intestinal groups. How does the gas formula differ
from those previously examined ?
NOTE. — In order to obtain a clear picture of the chief differ-
ential characteristics of the four intestinal groups, it is recom-
mended to tabulate the results in parallel columns, as outlined
below. Express positive results by the sign + ; negative, by — .
Complete agglutination is expressed by + + ; slight, by -f .
INTESTINAL GROUP
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II
THE
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OR
HOG-CHOLERA
GROUP
III
THE
TYPHOID-
DYSENTERY
GROUP
JS
H
CHAPTER XIV
THE CAPSULATED GROUP
MEMBERS —
Varieties of Bacillus mucosus capsulatus.
B. lactis aero genes.
REFERENCE —
Perkins, Journal o) Infectious Diseases. 1904, No. 2.
Inoculate agar-slants from stock-cultures of B. mucosus
capsulatus (Friedlander's pneumo-bacillus) and B. lactis
aero genes.1
1. Routine study. — Observe particularly the viscous
condition of cultures on solid media, the consistency of
liquid media, and the gas-formation on potato.
2. Special study A. — Staining of capsules from 24-
hour-old milk-cultures (see p. 77).
3. Special study B. — Intraperitoneal inoculation of
rabbit with B. mucosus capsulatus.
Method of intra peritoneal inoculation. — The rabbit is
held in the same manner as described in Chap. XII,
The hair is clipped close over the left lower abdominal
quadrant. Then (after washing with mercuric chlorid
i : 1000 and alcohol) pass the needle at first beneath the
skin, then, holding it at about a right angle to the abdomi-
nal surface, carefully press it through the abdominal wall,
which is usually made tense by the resistance of the
animal. Successful passage of the abdominal wall can
be felt by the sudden loss of resistance to the needle's
pressure. Then make the injection, and rapidly with-
1 B. lactis aerogenes, if studied in the colon group, need not
be studied again
92
CAPSULATED GROUP 93
draw the syringe. If the contents of the needle have
been properly emptied into the peritoneal cavity, no
swelling takes place, as is noticed in subcutaneous inocu-
lations.
When the animal has died, perform an autopsy and
study the lesions. Make cultures from the heart or
internal organs in the usual manner, and make capsule
stains from the heart's blood.
CHAPTER XV
THE DIPHTHERIA GROUP
MEMBERS —
Bacillus diphtheriae.
B. pseudodiphthericus.
B. xerosis.
Use great caution in handling members of this group.
Inoculate agar-slants from stock-cultures of B.
diphtheriae and B. pseudodiphthericus.
1 . Routine study. — Stain B. diphtheriae with Loeffler's
methylene-blue instead of gentian-violet. The staining
may be facilitated by the application of mild heat.
Observe particularly the peculiar effects of the staining
process, and make accurate sketches of what yon see
under the microscope.
2. Special study A. — Neisser's method of staining to
demonstrate the granules in the bacilli:
a) Prepare two solutions.
SOLUTION I
Methylene-blue i g.
Dissolve in alcohol 96$ 20 c.c.
Add distilled water. . . 950 c.c.
Glacial acetic acid 50 c.c.
SOLUTION II
Bismarck brown 2 g.
Distilled water 1000 c.c.
b) Prepare a film in the usual manner from a culture
of B. diphtheriae on blood-serum (furnished).
c) Stain in Solution I for 5 to 6 seconds.
d) Wash in water.
94
DIPHTHERIA GROUP 95
e) Counterstain with Solution II for 10-15 seconds.
/) Wash in water, examine in water, dry, and mount
in balsam.
3. Special study B. — Test for acid formation in a
culture, one week old, in neutral glucose-broth by addi-
tion of a few drops of litmus-solution.
4. Special study C. — Cultivation of B. diphtheriae
on eggs. (Method of Wyatt Johnston).
NOTE. — This method is recommended as an emergency cul-
ture test, the egg taking the place of Loeffler's blood-serum.
a) Sterilize over the flame a small empty pot of meat-
extract, or any other vessel of suitable size.
b) Carefully break with sterile forceps the shell of
a hard-boiled egg at the blunt end, taking care not to
rupture the membrane lining the shell.
c) Flame the exposed part, and carefully free the
coagulated albumen from any membrane.
d) Inoculate by gently rubbing some culture or throat
swab on the exposed egg-albumen.
e) Invert and set in the sterilized pot.
/) Place in the thermostat, and
g) Observe the appearance and make a stained
preparation after 24 hours.
5. Special study D. — Study of B. xerosis. Obtain
mucus from inner angle of the eyelids by stroking
with a platinum loop. Make two film preparations,
stain one by Gram's method and the other with methy-
lene-blue. Try culture on slant-agar and if impure,
plate out. Wherein does B. xerosis differ from the
other members of the group ?
6. Special study E. —
96 LABORATORY GUIDE IN BACTERIOLOGY
EXPERIMENT I
a) Procure a guinea-pig (or rabbit), and clip the hair
over a small area on the surface of the abdomen.
b) Cut a small opening in the skin, and separate the
skin from the muscles below by pushing in sterile scis-
sors. Expand these slightly and after closing again
remove. This forms a small pocket.
c) Carry i loopful of a 24-hour-old agar-culture into
this pocket.
EXPERIMENT II
a) Heat a 24-hour-old broth-culture in the water-
bath for 30 minutes at 60°.
b) Inject 0.25 c.c. of this heated culture subcutane-
ously into another guinea-pig (or rabbit)
c) Observe and compare in both animals the results
by taking note of the ante-mortem phenomena and the
lesions post-mortem. What difference is there in the
activities of the two cultures inoculated, and to what are
these differences due?
CHAPTER XVI
THE HEMORRHAGIC SEPTICEMIA GROUP
MEMBERS —
Bacillus pestis (bacillus of bubonic plague).
B. cuniculicida (bacillus of fowl-cholera, bacillus of rabbit
septicemia, Bacillus der Rinderseuche, Bacillus der
Schweineseuche, etc.).
REFERENCES —
Textbook: Moore, The Pathology of Infectious Diseases of
Animals.
Inoculate agar-slants from stock-culture of B. cuni-
culicida. For obvious reasons, B. pestis will not be
studied culturally.
1. Routine study. — Stain with Loeffler's methylene-
blue and anilin-gentian-violet. Observe "polar stain-
ing." What is "polar staining"?
2. Special study. — Inoculation of rabbit subcutane-
ously or by scarification. When dead, study in the
usual manner, and observe particularly the hemorrhages
produced in the serous membranes. Make cultures
from heart's blood, where large numbers of bacilli will
be found. Also make a stained preparation, and note
the typical polar staining. .
97
CHAPTER XVII
THE ANTHRAX GROUP
MEMBERS —
Bacillus anthracis.
B. subtilis, including several varieties.
Great caution is necessary in handling B. anthracis.
Inoculate agar-slants from stock-cultures of B. anthra-
cis and B. subtilis.
1. Routine study. — Observe particularly the colonies
formed on agar and gelatin plates,
2 . Special study A . — Make ' ' impression preparation ' '
(Klatschprdparat) from a surface colony on a gelatin-
plate.
Method—
a) Clean and flame a cover-slip carefully.
b) Place on a colony of suitable size, and gently press
down, taking care not to press so hard as to disturb the
characteristic shape of the colony.
c) Lift the cover-slip carefully with the forceps.
d) Dry, fix, and stain with methylene-blue or by
Gram's method.
e) Examine under low and high power (dry lens),
and sketch what you see.
3. Special study B. — Staining of spores. Read care-
fully the different methods of staining spores. Why are
special stains necessary ?
Moeller's method:
a) Prepare several (five or six) films in the usual
manner from 24-hour-old agar-cultures of B. anthracis
(or B. subtilis).
98
ANTHRAX GROUP 99
b) Place in chloroform for 2 minutes.
c) After drying in the air, cover with a 5 per cent,
solution of chromic acid for 2 minutes.
d) Wash thoroughly in water.
e) Cover with carbol-fuchsin and heat for 5 minutes
over the water-bath at 100°, or over a small flame, sim-
mering gently all the while.
/) Decolorize with i per cent, sulphuric acid for 25-
30 seconds.
g) Wash thoroughly in water.
h) Mount in water, and examine under the micro-
scope to see if spores are cherry-red and the protoplasm
colorless or faintly pink.
i) Counterstain with methylene-blue for 10-15
seconds without heat.
;) Wash, examine in water, and then mount in balsam.
NOTE. — The body of the cell should appear blue; the spore,
red.
4. Special study C. — Demonstration of filament-
formation.
a) Spread a loopful of a broth-culture of B. anthrads
or B. subtilis on a clean cover-glass.
b) Dry and fix in the flame.
c) Cover with strong acetic acid (80 per cent.) for
5-10 seconds.
d) Wash in water.
e) Stain with gentian-violet.
/) Examine in water, dry, and mount in balsam.
What is the object of applying acetic acid ?
5) Special study D.— Inoculate a guinea-pig subcu-
taneously with 0.2 c.c. of a 24-hour-old broth-culture of
B. anthrads, or insert a loopful of a 24-hour-old agar-
ioo LABORATORY GUIDE IN BACTERIOLOGY
culture in a "pocket" under the skin. When the animal
is dead, perform an autopsy, and observe particularly the
hemorrhagic and gelatinous edema under the skin; also
the enlarged spleen and the hemorrhagic adrenals.
Make a stained preparation from the heart's blood, and
observe the lack of spores, and also the presence of
capsules and degenerate forms, which do not stain.
What prevents the formation of spores ? How would
you distinguish B. anthracis from B. subtilis?
CHAPTER XVIII
THE SPIRILLUM GROUP
MEMBERS —
Spirillum cholerae asiaticae.
Sp. of Finkler and Prior.
Sp. Metchnikom.
Sp. tyrogenum.
And a number of spirilla indigenous to water.
Great caution must be exercised in manipulating the
spirillum of asiatic cholera.
Inoculate agar-slants from stock-cultures of Sp.
cholerae asiaticae, Sp. oj Finkler and Prior, and Sp.
Metchnikovi.
1. Routine study. — In addition to the usual media,
inoculate an extra tube of Dunham's peptone-solution
from each organism. Observe carefully from day to day
the action of these three organisms on gelatin, and com-
pare the results by tabulation. Observe the formation
of coccoid involution forms on agar after 3 days. Also
make plates in gelatin, observe the colonies from day
to day, and compare.
2. Special study A.— Test for the nitroso-indol or
cholera-red reaction. (See test for indol, p. 83.) Make
two tests, using one of the cultures in Dunham's solution
after 24 hours, the other after 6 days. Compare the
results of these two tests.
3. Special study B— Stain for flagella by Loeffler's
method (see p. 86).
4. Special study C. — Schottelius' enriching method.
102 LABORATORY GUIDE IN BACTERIOLOGY
a) Make a solution of i g. Witte's peptone in 100 c.c.
of water.
b) Distribute in three small Erlenmeyer flasks, and
sterilize in autoclav.
c) Inoculate one of these flasks with Sp. chol. asiat.
and B. chol. suis or any other motile bacillus.
d) Incubate at 37° for 18-24 hours. Make a stained
preparation from the surface of the liquid.
e) After that time, take one loopful from the surface,
inoculate the second flask, and incubate as before.
/) After 18-24 hours, make a stained preparation
from the surface of the second flask, and examine for
spirilla.
g) Transfer a loopful from the surface of the second
flask to the third one, and incubate as before.
h) After 18-24 hours, again stain and examine for
spirilla. By this time usually a film has formed which
contains the spirilla in practically pure culture. How is
this phenomenon explained, and what is its epidemiologi-
cal value ?
5. Special study D. — Inoculate a pigeon intramuscu-
larly with 0.5 c.c. of a broth-culture of Sp. Metchnikom.
The breast of the pigeon is laid bare, washed with mercuric
chlorid and alcohol, and the syringe is plunged into the
muscle-fibers and discharged. After death, note the
peculiar appearance, resembling that of boiled beef.
Make stained preparations from blood and muscle-juice,
and examine for spirilla.
CHAPTER XIX
THE GROUP OF ACID-RESISTING BACILLI
MEMBERS —
Bacillus tuberculosis.
B. leprae.
B. smegmae.
Moeller's grass bacilli, including a number of bacilli found
on grass, dung, in butter, milk, etc.
For obvious reasons, B. tuberculosis will not be studied
culturally. For comparison, the culture characteristics of
Moeller's grass bacillus are instructive.
Inoculate an agar-slant from a stock-culture of
Moeller's grass bacillus.
1. Routine study.
2. Special study A . — Method of staining acid-resisting
bacilli.
a) Pick out purulent matter from the sputum oi a
tuberculous patient and spread carefully on a cover-
glass.
b) Dry and fix as usual.
c) Heat for one minute on a water-bath at 100°, or
over a small flame with carbol-fuchsin.
d) Decolorize with acid alcohol (2 per cent. HC1 in
80 per cent, alcohol) until the film in its thin parts has
lost almost all its color.
e) Counterstain with methylene-blue for 10 seconds
(cold).
/) Examine and mount in balsam.
NOTE. — Make a second preparation, substituting anilin-
gentian-violet for carbol-fuchsin, and Bismarck brown for methy-
lene-blue.
103
io4 LABORATORY GUIDE IN BACTERIOLOGY
3. Special study B. — Observe the lesions in a
guinea-pig or rabbit dead of tuberculosis, which has
been inoculated about four weeks previously. Note the
caseous matter at the site of the inoculation, the enlarged
yellowish inguinal and axillary glands, the small gray
or yellowish tubercles in the liver, lung, spleen, mesentery,
etc. Stain for bacilli from such sites. Make cultures on
glycerin-agar or dog-serum (see the textbook).
4. Special study C. — Stain Moeller's grass bacillus
from agar-culture by the same method, omitting the
counters tain.
5. Special study D. —
a) Grind in a mortar, previously sterilized, a small
amount of a culture of the grass bacillus, and mix with
some sterile milk.
b) Make a cover-slip preparation of this mixture, and
stain for tubercle bacilli. At what conclusions as to the
quality of the milk would you arrive, if you should find
acid-resisting bacilli in a fresh sample of milk ?
CHAPTER XX
MISCELLANEOUS BACTERIA
Inoculate agar-slants from stock-cultures of Bacillus
mallei and Micrococcus melitensis. Exercise great
caution in handling B. mallei.
1. Routine study. — Note especially the growth of
B. mallei on potato. Also note the morphology of Micr.
melitensis and the staining properties of B. mallei. Is B.
mallei motile ?
2. Special study. — Intraperitoneal injection of B.
mallei from a broth-culture into a male guinea-pig. Why
do you select a male guinea-pig? What is Straus's
method of diagnosis of glanders?
105
CHAPTER XXI
THE ACTINOMYCES GROUP
Inoculate agar-slants from Actinomyces boms (homi-
nis) and Actin. asteroides.
REFERENCES —
Stokes, American Journal of the Medical Sciences, Novem-
ber, 1904.
Wright, Jour, of Med. Research, May, 1905.
1. Special study A. — Transfer from agar-slants to
broth and potato only, and make descriptions and
stained preparations as usual.
2. Special study B. — Suspend a small amount of the
potato-culture in a physiological salt solution, and
examine under the low power.
3. Special study C. —
a) Examine a sample of actinomycotic tissue (bovine)
in the fresh state, for so-called " sulphur granules."
Crush some in salt solution under a cover-slip and search
for " clubs," using the low and high power dry lenses.
b) Crush others, dry, fix, and stain by Gram, counter-
stain with eosin or Bismarck brown.
c) Write a full description of the biology of the organ-
ism, illustrated by drawings.
d) What other actinomycetes have been described
as pathogenic? Give their names and the diseases
which they produce.
106
CHAPTER XXII
THE ANAEROBIC GROUP OF BACILLI
MEMBERS —
Bacillus tetani.
B. oedematis maligni.
B. aero genes capsulatus.
B. anthracis symptomatici.
B. botulinus.
And others.
Study the different methods of anaerobic cultivation in
the textbook.
1. Special study A. — Park's method.
a) Boil three tubes of glucose-agar vigorously for 5
minutes, to drive out the dissolved oxygen. (Why is
the presence of glucose desirable ?)
b) Cool to 43° and inoculate from stock-culture of
B. tetani, B. oedematis maligni, and B. aerogenes capsu-
latus.
c) Solidify rapidly by immersion in cold water.
d) Cover the medium with a thin layer of liquid paraf-
fin.
e) Place in thermostat.
NOTE. — The layer of parraffin effectively excludes the atmos-
pheric oxygen, which would be inhibitory to the growth of the
anaerobes. The oxygen necessary for their multiplication is
derived from nutrient material of the medium.
2. Special study B. — Wright's modification of Buch-
ner's method.
a) Liquefy, as before, six glucose-agar tubes, the
plugs of which have been replaced by absorbent cotton.
107
io8 LABORATORY GUIDE IN BACTERIOLOGY
Cool three to 43°, and inoculate while fluid. Let the
other three become solid, and make stab-cultures.
b) Sterilize the cotton stoppers in a flame, and with
the forceps, sterilized in a flame, push the stoppers into
the test-tubes for the distance of about i inch (2-3 cm.).
c) Pour into the tubes (upon the cotton stoppers)
2 c.c. of a saturated solution of pyrogallic acid in water,
followed by 2 c.c. of a 2 per cent, solution of NaOH.
d) Cork the tubes immediately with rubber stoppers,
and keep upside down.
e) Incubate at the required temperature.
Upon what principle does this method depend ?
Do the organisms grow both aerobically and anaerobi-
cally ?
When you have obtained a growth, stain the organ-
isms with anilin-gentian-violet and by Gram's method.
Describe and illustrate.
3. Special study C. — Cultivation by Buchner's
method, using fruit-jars.
a) Into a Mason fruit-jar of ordinary type deposit
10 g. of pyrogallic acid.
b) Smear vaselin around the mouth of the jar.
c) Pour into the jar 100 c.c. of a i per cent, solution
of NaOH.
d) Then deposit in the jar culture-tubes previously
inoculated.
e) Tightly fasten the cover of the jar, and incubate
at 37° for 48-72 hours.
4. Special study D. — Cultivation in hydrogen gas.
a) Inoculate all media from stock-cultures obtained.
b) Fit up apparatus as shown in Fig. 34.
c) Place culture-tubes in a Novy jar (Fig. 34, a).
ANAEROBIC GROUP OF BACILLI
109
d) Open the faucet (b) of the gas-generator (c), con-
taining zinc and hydrochloric acid. The hydrogen gas
generated passes through two jars, one of which con-
tains concentrated sulphuric acid (d), the other a 10 per
cent, solution of sodium hydrate (e). Gradually the
Novy jar is rilled with hydrogen gas, which can be tested
by holding a culture-tube over the opening (/), and then
over a burning match or gas flame. As long as any
detonation takes place the hydrogen is still mixed with
-I
FIG. 34
Anaerobic Cultivation in Hydrogen Gas
c. Novy jar e. Sodium hydrate solution
b. Glass cock /. Opening of Novy jar
c. Gas generator g. Stopper
d. Sulphuric acid
atmospheric oxygen. When finally the hydrogen in the
Novy jar is pure, close it off by turning the stoppers (g)
and (b), and place it in the incubator. The whole pro-
cess occupies about 10 or 15 minutes.
5. Special study C. — Inoculation of a rabbit with
B. aerogenes capsulatus.
a) Shave the ear of the rabbit.
no LABORATORY GUIDE IN BACTERIOLOGY
b) Wash with mercuric chlorid solution and alcohol.
c) Inoculate intravenously with 0.5 c.c. of a 24-hour-
old milk-culture.
d) After the culture has been fully distributed in the
circulation, which takes at the most 3 minutes, kill the
rabbit by a quick blow on the back of the neck.
e) Put the rabbit in a warm place — say, on top of the
thermostat — for 18 hours, or from 6 to 8 hours inside of
the thermostat.
/) After this time has elapsed, perform an autopsy.
Note the crackling, on pressure, over the axillary or
inguinal regions. The rabbit is swollen to a great
extent. Skin the animal carefully, without opening the
abdominal cavity; then quickly puncture the abdominal
wall and bring a flame to the opening. Note that the
escaping gas will burn with a blue flame. What is this
gas ? Also note the disorganized condition of the liver,
spleen, and kidney.
g) Make capsule stains from the heart's blood or
organs by Welch's method (modified). The modification
of Welch's method is as follows : Proceed in the manner
indicated on p. 77 and, after washing the acetic acid off
with the stain (carbol-fuchsin or gentian-violet), dry with
filter paper, heat the specimen for 5 to 10 seconds before
washing off with the salt solution. Then proceed as
before.
6. Special study F. — Staining of spores of B. tetani
and B. oedematis maligni from 3 -day-old glucose-agar-
cultures (see p. 98).
7. Special Study G. — Inoculation of a white mouse
or guinea-pig with B. tetani or its toxin (o.oi c.c.) in the
hind-leg or over the root of the tail (if a mouse). Note
ANAEROBIC GROUP OF BACILLI in
daily the condition of the animal, and when dead make
cultures and cover-slip preparations from the site of the
inoculation.
8. Special study H. — Inoculate a white rat with gar-
den earth subcutaneously or in a pocket above root of
tail. Note the condition of the animal daily. When
dead, make cultures and cover-slip preparations from the
site of the inoculation. Would you expect to find bacilli
in the heart's blood or organs ? Give reason for your
answer.
CHAPTER XXIII
ISOLATION OF UNKNOWN BACTERIA FROM A
MIXTURE
1. Make hanging-drop, stained, and Gram prepara-
tions from the mixture. Note observations and results.
2. Melt five or six agar-tubes, and cool to 43°.
3. Transfer 5 or 6 loopfulsof the mixture to a tube
of liquid agar, from this to a second, and so on until all
the melted tubes are inoculated.
4. Pour into sterile Petri dishes, and mark them with
successive numbers and the date. Place in the thermo-
stat.
5. After 24 hours examine the colonies under the
low power, describe them in the usual manner, and
transfer to agar-slants all those which show different
appearances.
6. Now proceed with the usual routine study. Make
hanging-drop, stained, and Gram preparations, transfer
to all the media, and describe the culture characteristics
accurately. Make sketches in the usual manner.
7. Special tests may become necessary after 24 or
48 hours. Such tests may consist of—
Capsule stain.
Spore stain.
Stain for acid-resisting bacilli.
Fermentation tests of all those which produce gas
in glucose-agar. Also note the growth, or lack of growth,
in the closed arm of the fermentation-tube.
Test for acid in neutral broth.
112
ISOLATION OF UNKNOWN BACTERIA 113
Test for agglutination.
Test for indol.
Anaerobic cultivation.
Inoculation of animals.
For final diagnosis consult your notes and textbook.
CHAPTER XXIV
BACTERIOLOGICAL EXAMINATION OF WATER, AIR
AND MILK
EXERCISE I. BACTERIOLOGICAL ANALYSIS OF WATER
REFERENCES —
Horrocks, Bacteriological Examination of Water.
Prescott and Winslow, Elements o} Water Bacteriology.
Fuller and Johnston, Journal of Experimental Medicine, Vol.
IV.
Committee Report o} the American Public Health Association,
Jour, o} Infectious Diseases, Suppl. I, May, 1905.
A bacteriological examination of water is made for
the purpose of determining —
1. Bacterial numbers.
2. Bacterial species.
3. Sewage contamination.
Collection oj samples. — Procure wide-mouthed, glass-
stoppered bottles, having a capacity of at least 100 c.c.
After careful cleaning and drying, wrap them in lead-foil,
and sterilize in the hot-air oven for i hour at 160°; then
deposit them in a metal or wooden case. The samples
from surface waters should be taken at least one foot
below the surface, to avoid contamination with organisms
from the air. If possible, samples should be plated on the
spot or in the laboratory within an hour at the very latest.
But when a greater interval of time must occur, the
samples should be taken to the laboratory packed in ice,
despite the probability of thus destroying a certain per-
centage of the bacterial flora.
Method oj examination. — A number of pipettes of
various sizes (i c.c., 2 c.c., 5 c.c., and 10 c.c.) are plugged
114
EXAMINATION OF WATER, AIR, AND MILK 115
with cotton and sterilized in the hot-air oven. Then a
number of Erlenmeyer flasks are filled with 101 c.c. of
distilled water, and these are sterilized in the autoclav at
1 20° for 5 minutes. About i c.c. of water is lost by evapo-
ration during this process, so that the sterile flasks con-
tain 100 c.c. each.1
Method oj procedure. —
1. With a sterile pipette remove i c.c. of water from
one of the sterilized dilution flasks.
2. Carry over to this flask i c.c. of the sample after
a thorough shaking. The dilution is now i : 100. Mark
with glass pencil.
3. With a sterile 10 c.c. pipette remove 10 c.c. from
another dilution flask, and add to the remainder 10 c.c.
of the first dilution. We now have a dilution of i : 1000.
(See dilution table, p. 117.) Make a number of dilutions
in this manner, carrying the dilutions higher in proportion
to the quality of the water to be examined.
4. Melt a number of agar and gelatin tubes, corre-
sponding to the number of dilutions made, and cool to
43.° Read in reference book carefully the chapters on
the importance of using both agar and gelatin media.
5. Mix carefully i c.c. of each dilution flask with a
tube of liquified medium, respectively.
6. Pour into Petri dishes.
7. Incubate the agar-plates at 37°, and keep the
gelatin-plates at room temperature. The mixing of the
diluted sample and agar may also be effected by pouring
the sample into a Petri dish and mixing it with the liquid
medium. This method is not as convenient, but slightly
1 If work is carried on with dilutions of i : 100 only, the flask may
conveniently be filled with 100 c.c. water instead of 101 c.c. It is then not
necessary to remove i c.c. of water, as only 99 c.c. are left after sterilization.
u6 LABORATORY GUIDE IN BACTERIOLOGY
more accurate, as no loss takes place by adhesion to the
tube.
Estimation oj colonies. — The colonies are then counted
after 48 hours, by means of a colony-counter (Fig. 35).
Plates should be counted which contain no more than
200-300 colonies. If it is necessary to count plates with
a large number of colonies, an estimate must be made
by counting different sections of the plate-counter and
averaging the result for the whole plate.
Species determination. — If the different species of
bacteria are to be studied, the colonies must be examined
by the naked eye and the low power. Then those which
appear to be different are transferred to slant-agar tubes,
and from these to the ordinary media.
Sewage contamination. — The presence of Bacillus
coli and streptococci is sufficient indication of sewage
contamination in water.
Method oj examination jor B. coli and streptococci. —
1. i c.c. of the sample, or, if necessary, of the diluted
sample, is added to a series of ten fermentation-tubes,
containing sterile 2 per cent, glucose-broth.
2. Place in thermostat.
3. Examine after 12-18 hours.
4. Examine a loopful of the sediment in a stained
preparation.
Example. — If i c.c. of the sample is added to each
fermentation-tube, and six show gas-formation, there
would be six colon bacilli in each 10 c.c. if undiluted
water is employed. By this method fairly accurate
results may be obtained.
Isolation is accomplished by plating in an agar
medium containing 2 per cent, lactose and 10 per cent,
litmus solution.
EXAMINATION OF WATER, AIR, AND MILK 117
DILUTION TABLES FOR AGGLUTINATION
Number
Amount of Serum
Amount of Salt
Solution
Final Dilution
I
i part
2.
i part of No i
9 parts
•2.
i part of No 2
4. .
i part of No ^
9 parts
Amount of Serum or
Serum Dilution
Suspension
Fina
1 Dilution
2 part clear serum
1 8 parts
10
i " " "
19 "
20
5 parts dil., No
IS "
40
4 No
16 "
5°
2.5 " " No
17-5"
80
2 " " NO
18 "
IOO
i " " No
10 "
2OO
4 " " No. 2
y
16 "
CQO
2 " " No. 2
18 "
y**
1000
I " " No. 2
19 "
200O
4 No. 3
16 "
5000
2 " " No. 3
18 "
IOOOO
i " " No. 3
19 "
2OOOO
4 No. 4
16 "
50OOO
2 No. 4
18 "
IOOOOO
i " " No. 4
19 "
200OOO
DILUTION TABLE FOR WATER OR MILK ANALYSIS
Number
Amount of Dilution
Amount of
Sterile Water
Final Dilution
I. . .
Original
I
2. .
ic.c. of No. i
OQC.C.
IOO
3
2OC.C. of No. 2
yyv-.v,.
Soc.c.
500
4
IOC.C. Of No. 2
9oc.c.
IOOO
5
50. c. of No. 2
95c.c.
2000
6
2C.C. Of No. 2
98c.c.
5000
7
ic.c. of No. 2
99c.c.
IOOOO
8
5c.c. of No. 4
95c.c.
2OOOO
9
2c.c. of No. 4
98c.c.
50OOO
10
ic.c. of No. 4
99c.c.
IOOOOO
ii
5c.c. of No. 7.
95c.c.
2000OO
12
2c.c. of No. 7.
98c.c.
5OOOOO
*3
ic.c. of No. 7
99c.c.
I 000000
n8 LABORATORY GUIDE IN BACTERIOLOGY
EXERCISE II. BACTERIOLOGICAL EXAMINATION OF AIR
An approximate determination of the number of
bacteria in the air can be made by the following simple
method: Place a certain amount (50 c.c.) of broth in
an Erlenmeyer flask (Fig. 36, a). This flask is provided
with a rubber stopper (b), provided with two holes,
through which the two glass tubes (c with a wide opening
FIG. 36
Apparatus for Determining the Number of Bacteria in a Definite
a. Erlenmeyer flask
b. Rubber stopper
c-d. Glass tubes
Volume of Air
/. Five-liter flask
g-h. Glass tubes
*. Pinchcock
and d) lead. Cotton plugs are then inserted at c and d,
and the apparatus is sterilized in the autoclav. A
large bottle (/), containing 5 liters of water, is then
provided with a rubber stopper, and also with two glass
tubes (g and h)\ h is connected with a short piece of
rubber hose and a pinchcock (i). When the Erlen-
EXAMINATION OF WATER, AIR, AND MILK 119
meyer flask and contents are sterilized, the tubed is con-
nected, by means of the rubber hose £, with g, and the
plug at c is removed. By opening the pinchcock i, 5
liters of air are aspirated through the broth in flask a.
The flask is then disconnected, and i c.c. is plated in
agar and i c.c. in gelatin. The former is incubated at 37°,
and the latter kept at room temperature. After 48 hours
the colonies are counted, and the result is multiplied by
50. This then represents the amount of bacteria in 5
liters of air.
EXERCISE III. BACTERIOLOGICAL STUDY OF MILK
The method for determining the number of bacteria
in milk is fundamentally the same as for water, except
that dilutions must be carried higher, as milk generally
contains much larger numbers of bacteria.
Sterilization and pasteurization oj milk. — Some of the
important germs in milk are saprophytes (which under
favorable circumstances produce disagreeable odors or
tastes), and such pathogens as the bacillus of tuberculosis
(which may be derived from the cow, or may be an acci-
dental contamination), the typhoid bacillus, the bacillus
of "summer complaint" in children (possibly identical
with the bacillus of epidemic dysentery), the germs of
cholera, diphtheria, and scarlet fever. All these, except
B. tuberculosis, flourish in milk at its ordinary tempera-
ture.
None of the methods employed in sterilizing milk
render it sterile in the bacteriological sense of the word,
but by means commonly employed most of the non-
sporing pathogenic bacteria are destroyed, along with a
large number of saprophytes, thus rendering the milk
i2o LABORATORY GUIDE IN BACTERIOLOGY
comparatively safe and less subject to the ordinary
fermentative changes.
1. Sterilization at 100° for 30 minutes. — Such milk,
if chilled and kept at a low temperature, will remain
unchanged for more than a week; but, by the heating,
certain alterations have been produced in its taste and
nutritive qualities which render it supposedly less fit for
food than when pasteurization is employed.
2. Pasteurizing milk. — The changes occurring in
milk, as above mentioned, begin at about 82°. Sterili-
zation at a low temperature is accomplished by raising
the temperature to only 75° for a period of 20 minutes.
This has been shown to be sufficient to kill the germs of
typhoid, cholera, diphtheria, and pyogenic cocci. Spored
organisms are not killed. As shown by Theobald
Smith, tubercle bacilli, when suspended in distilled
water, physiological salt solution, broth, and milk, are
destroyed at 60° in 15-20 minutes; but, if milk contain-
ing tubercle bacilli has its surface exposed to the air when
heated to 60°, the pellicle which forms on its surface
may contain living tubercle bacilli after an exposure of
60 minutes.
Study of the effect of the above two methods of steri-
lization as compared with each other and with unsteri-
lized milk:
1. From the fresh milk provided make three agar-
plates, using i, 2, and 3 loopfuls, respectively.
2. Fill about 10 c.c. into each of ten sterile culture-
tubes, and keep one at room temperature and one in the
thermostat.
3. Treat four of these tubes in the following manner:
Place water in a saucepan sufficient to cover completely
EXAMINATION OF WATER, AIR, AND MILK 121
the milk when the tubes are immersed in it. Raise the
temperature to 75°, and keep it there by regulating the
flame. The tubes of milk are then immersed in the
water, and kept there for 30 minutes, as it requires about
10 minutes for the milk in the tubes to reach the tempera-
ture of the water. The tubes are then taken out and
cooled quickly by standing them in cold water. Place
one of the tubes at incubator and the other at room
temperature. Aerate the other two by shaking vigor-
ously for ij minutes. Keep one of these at room tem-
perature, the other in the thermostat.
4. Place two more milk-tubes in the Arnold at 100°
for 30 minutes. Keep one at room temperature and one
in the thermostat.
5. The remaining two tubes autoclav at 120° for
5 minutes, and place one in the thermostat and keep the
other at room temperature.
6. Note the conditions of these ten tubes after 2 or
3 days. Compare the results, and tabulate them. Note
especially coagulation, time elapsed before coagulation
sets in, gas-formation, condition of whey, film, and odor.
How are these differences explained ?
Plates in lactose-litmus-gelatin should be made from
each of these tubes, and the colonies studied and counted.
Subcultures on agar-slants may also be made, and the
usual media inoculated from these, if the individual
species are to be studied.
CHAPTER XXV
INFLUENCE OF DISINFECTANTS ON THE GROWTH
OF MICRO-ORGANISMS
EXERCISE I
1. Prepare fifty-seven Hill's test-rods. These are
prepared in the following manner: Glass rods about
two inches longer than ordinary culture-tubes are marked
with hydrofluoric acid by a circle exactly one inch from
the end. A wad of cotton is then wrapped around the
middle of the rod, and this is inserted in a culture-tube.
The rod is then pushed down until it nearly reaches the
bottom. That part of the rod which is free at the upper
end is used for labeling. The whole apparatus is then
sterilized in the dry-air oven.
2. Fill two wide-mouthed flasks, one with 100 c.c.
of a 5 per cent, solution of carbolic acid, the other with
100 c.c. of a i per cent, solution.
3. Fill two similar flasks, one with 100 c.c. of a solu-
tion of mercuric chlorid i : 1000, the other with a solution
of i : 10,000.
4. Fill two similar flasks, one with 100 c.c. of a 10
per cent, solution of formalin (40 per cent, formaldehyde),
the other with a i per cent, solution.
5. Prepare 48-hour broth-cultures of Staphylococcus
pyogenes aureus, Bacillus coli and B. typhosus from
stock-cultures.
6. Dip nineteen of these rods into each of these
cultures respectively, to the depth of one inch ; set them
aside in their tubes to dry over night in the thermostat,
after marking each tube carefully.
INFLUENCE OF DISINFECTANTS 123
EXERCISE II
We have now six flasks containing different solutions
of disinfectants.
1. Place in each one of these flasks nine of the pre-
pared rods, three of which have been dipped in the
Staph. pyogenes aureus culture, three in the B. coli
culture, and three in the B. typhosus culture.
2. Take three rods (one of each organism) out of
each flask after the lapse of half a minute, wash by
gently pouring sterile physiological salt solution over
them into a dish containing mercuric chlorid solution
i : 1000, and place each rod in a tube of sterile broth
3. Repeat the proceedings of step 2 .with a second
series of rods after 2 minutes.
4. Repeat again after 5 minutes with the remaining
series.
5. Place all tubes (fifty-seven) in the thermostat.
Three of these tubes have not been dipped into any one
of the six flasks containing antiseptics, and are incubated
with the others as controls.
6. Observe the results carefully on each of the four
successive days, and on the last day prove the relative
growth by making agar-plates with i c.c. of each culture,
and count the colonies after 24 hours.
7. Tabulate the results, and state your conclusions.
EXERCISE III. INFLUENCE OF SUNLIGHT
EXPERIMENT I
1. Inoculate a flask containing 100 c.c. of sterile
water with B. coli.
2. After thoroughly shaking, take i c.c. by means of
a sterile pipette, and plate in agar. Place the plate in
the thermostat.
i24 LABORATORY GUIDE IN BACTERIOLOGY
3. Expose the flask to sunlight for several hours.
4. Make another plate with i c.c. of the suspension,
and place in a thermostat.
5. After 48 hours count both plates, and compare
the results.
EXPERIMENT II
1. Melt a tube of agar and cool to 43°.
2. Inoculate with B. coli (or any other organism).
3. Pour into a sterile Petri dish.
4. After solidification, turn bottom side up, and paste
a strip of black paper on the glass, covering part of the
surface.
5. Expose to direct sunlight for several hours, and
note the result.
EXERCISE IV. INFLUENCE OF MOIST HEAT
Read the methods of determining the thermal death-
point of bacteria in the textbook.
1. Prepare six broth-cultures each of B. coli and B.
subtilis.
2. Place four cultures of each organism in the water-
bath and heat.
3. Remove one of each at 40°, one of each at 60°,
one of each at 80°, and keep one of each for 10 minutes at
100°.
4. Place one tube of each organism in the autoclav,
and heat to 120° for 5 minutes.
5. Now place all twelve tubes in the thermostat,
including one of each organism as a control.
6. After 24 hours, make plates of each tube in agar,
and place them in the thermostat.
7. After 24 hours, count the colonies and compare
the results.
APPENDIX I
SPECIAL MEDIA
STANDARD METHOD OF PREPARING BROTH, NUTRIENT
GELATIN AND NUTRIENT AGAR
(Report of American Public Health Assocciation,Vo\. XXX, 1905.)
BROTH
GELATIN
AGAR
i.
Boil 15 g. thread agar in 500
c.c. water for half an hour
and make up weight to 500
g., or digest for 10 minutes
in the autoclav at no?
Let this cool to about 60?
2. Infuse 500 g. lean meat 24
hours with 1000 c.c. of dis-
tilled water in refrigerator.
Ditto.
Infuse 500 g. lean meat
24 hours with 500 c.c. of
distilled water in refriger-
ator.
3. Make up any loss by evap-
oration.
Ditto.
Ditto.
4. Strain infusion through
cotton flannel.
Ditto.
Ditto.
5. Weigh filtered infusion.
Ditto.
Ditto.
6. Add i % Witte's peptone.
Ditto.
And 10 % gold la-
bel sheet gelatin .
Add 2 % Witte's peptone.
7. Warm on water-bath stir-
ring till peptone is dis-
solved, and not allowing
the temperature to rise
above 60°.
Warm on water-
bath until pep-
tone and gela-
tin are dis-
solved, not
above 60°.
Warm on waterbath until
peptone is dissolved,
not above 60°.
8.
To 500 g. of meat infusion
add 500 c.c. of the 3%
agar, keeping the tem-
perature below 60°.
9. Heat over boiling water (or steam) for 30 minutes.
10. Restore loss by evaporation.
u. Titrate after boiling one minute to expel carbonic acid.
12. Adjust reaction to + i.o % by adding normal hydrochloric acid or sodium
hydrate as required.
125
126 LABORATORY GUIDE IN BACTERIOLOGY
13. Boil 2 minutes over free flame, constantly stirring.
14. Make up loss by evaporation.
15. Filter through absorbent cotton and cotton flannel, passing the filtrate
through the filter until clear.
1 6. Titrate and record final reaction.
17. Tube, using 10 c.c. in each tube in the case of gelatin and agar.
1 8. Sterilize 5 minutes in the autoclav at 120°, or for 30 minutes in streaming
steam on 3 successive days. Put at once into ice- water till solidified.
19. Store in the ice-chest in a moist atmosphere to prevent evaporation.
Blood-serum —
i. Fresh ox blood collected in sterile jars (museum jars)
FIG. 37
Koch Inspissator
is set in the ice-chest until the serum has separated. The
serum is filtered if necessary.
2. Take 3 parts of this freshly prepared serum and i part
of, broth containing 1.5 per cent, glucose, and mix.
SPECIAL MEDIA 127
3. Tube in the usual manner, and place in the Koch
inspissator (Fig. 37) two or three rows deep.
4. Incline the inspissator to the proper angle, so as to
produce a large sloping surface of the serum.
5. See that the water-jacket contains sufficient water.
Place a Bunsen burner below, slowly heat the water to the
boiling-point, and boil for 5 minutes. Then turn out the gas.
6. Repeat this process upon the two following days at
the same hour.
NOTE. — Always place about 25 c.c. of water in the inspissator
along with the tubes, so as to keep up a saturation of the air in
the apparatus. By doing this, one avoids giving the serum a dry
surface. See that the inspissator contains a rack made of wooden
or metal strips, which keeps the tubes from touching the bottom
and wall ; otherwise the serum will be overheated forming bubbles
in the mass.
Litmus -solution. — Dissolve i part of Merck's pure extract
of litmus in 100 parts of water, filter through paper, and steri-
lize.
Glycerin-broth. — Add 6 per cent, of pure glycerin to
ordinary broth.
Glucose-gelatin. — Add i per cent, glucose to ordinary
gelatin.
Litmus-lactose-agar (for plating). — Add i per cent, of
lactose to sugar-free agar, distribute 8 c.c. in culture-tubes, and
add i c.c. of sterile litmus-solution to each tube before using.
Litmus -lactose- gelatin (for plating}. — Prepare gelatin in
the usual manner, using 10-12 per cent, of gelatin, and dis-
solve i per cent, of lactose. Distribute 8 c.c. into culture-
tubes, and add litmus-solution to each tube before using.
NOTE — If i c.c. of the liquid to be analyzed is added, 14$
of gelatin should be used.
Litmus -mannit-agar . — Prepare like litmus-lactose-agar,
substituting i per cent, mannit in place of lactose.
Neutral-red agar. — Add enough 0.5 per cent, neutral-red
i28 LABORATORY GUIDE IN BACTERIOLOGY
solution to nutrient agar containing i per cent, glucose to
produce a clear red color without adjusting reaction.
Litmus-broth. — Add litmus-solution to ordinary broth in
the proportion of 10 per cent.
Glycerin-agar . — Add 6 per cent, pure glycerin to nutrient
agar.
Beerwort- gelatin — Autoclav beerwort at 120° for 5 min-
utes to precipitate proteids. After cooling, filter, dissolve 10
per cent, gelatin, and clarify with white of egg.
Beerwort-agar. — Prepared like beerwort-gelatin, substi-
tuting 1 1 per cent, agar in the place of gelatin.
Blood-agar. — A drop of blood, obtained with aseptic
precautions, is smeared on the surface of slant-agar.
Litmus-whey (Petruschk-y, modified by Durham). —
Casein is precipitated from milk with rennet-extract; the
whey is neutralized with 4 per cent, citric-acid solution and
heated on the water-bath for half an hour. It is then filtered,
and litmus-solution is added until a decided blue color is
obtained.
Whey-gelatin. — Add 10 per cent, gelatin to clarified whey.
Alkaline blood-serum (Lorrain Smith). — Add 1-1.5 P61"
cent, of a 10 per cent, solution of sodium hydrate to blood-
serum, tube, and sterilize in Arnold or in Koch's serum
inspissator.
Bread-paste medium. — Bread is cut into slices, dried in
the oven, and pulverized; it is then distributed in 100 c.c.
flasks until the layer on the bottom is half an inch thick.
Water is gradually run in to cover the surface of the bread
Sterilize in Arnold.
Mac Conkey's bile-salt-agar. —
Nutrient agar 100 c.c.
Sodium taurocholate 0.5$
Peptone 2.0$
This is boiled, clarified, and filtered, and then 2 per cent,
lactose is added, tubed, and sterilized in the medium.
SPECIAL MEDIA 129
Mac Conkey' s bile-salt broth. —
Sodium taurocholate 0.5$
Peptone 2.0%
Glucose o . 5$
are dissolved in beef -broth by boiling; then filter and add
litmus-solution.
Parietti's solution —
Carbolic acid 5 c.c.
Hydrochloric acid 4 c.c.
Water 100 c.c.
Eisner's medium. — i pound of sliced potatoes is grated
with a liter of water, and the juice expressed. After filtering
for 24 hours, add i per cent, peptone, i per cent, potassium
iodid, and 10 per cent, gelatin.
Glycerinated potato. — Prepare potatoes in the ordinary
manner and soak in a 25 per cent, solution of glycerin. Use
a glycerin -solution in the bottom of the tube.
Hay-infusion. — 10 g. of chopped hay are macerated with
1000 c.c. of water in the water-bath for 3 hours. Filter and
sterilize in autoclav for 10 minutes at 120°.
Wine-must. — Wine-must is diluted with four times its
weight of water. Dissolve 0.5 per cent, ammonium tartrate,
macerate in the water-bath for i hour, filter, and sterilize in
Arnold for 3 consecutive days.
Winogradsky' s solution (for nitric organisms). —
Dissolve in water 1000 c.c.
Potassium phosphate i . oo g.
Magnesium sulphate o . 50 g.
Calcium chlorid o . 01 g.
Sodium chlorid 2 . oo g.
Distribute 20 c.c. in flasks, and add to each flask a small
amount of magnesium carbonate. Sterilize in Arnold for 3
consecutive days; then add to each flask 2 c.c. of a sterile
2 per cent, solution of ammonium sulphate.
130 LABORATORY GUIDE IN BACTERIOLOGY
Yeast-water. — i liter of washed yeast or i pound of pressed
yeast is boiled with 2 liters of water for i hour. The reaction
is made neutral to phenolphthalein, and the solution is filtered
until clear, and sterilized in Arnold for 3 consecutive days.
Dextrose-yeast-water. — 10-15 per cent, dextrose is dissolved
in yeast-water without adjusting the reaction.
Egg medium (Dorset). — Eggs are broken into a flask, and
the yolks are broken with a platinum wire. Then the flask
is gently shaken, so as to mix the yolks with the whites without
causing foam to form. 10 c.c. are distributed in culture-
tubes, and then the medium is hardened in a sloping position
in the Koch inspissator for 2 successive days for 4 or 5 hours
at 70°.
Egg-yolk medium (Dorset). — Add 5-10 c.c. of sterile water
to the yolks of 3 or 4 eggs, and then pursue the same course
as in the other egg medium.
Egg-yolk medium (Capaldi). — A few loopfuls of egg-yolk
are added to a tube of liquefied agar cooled to 45-47°.
Enriching solution for B. typhosus (Hoffmann and Fischer).
—To peptone-meat-broth add i per cent, nutrose, 0.5 per
cent, caffein, and i per cent, solution of crystal violet.
Endo's medium. —
Nutrient agar (3$) 1000 c.c.
Lactose 10 g.
Alcoholic solution of fuchsin 5 c.c.
Sodium sulphite (10$ solution) 25 c.c.
Sodium hydrate (10$ solution) 10 c.c.
Drigalski and Conradi's medium (Modified by Harris). —
Dextrose-free broth 2000 c.c.
Nutrose 20 g.
Agar 40 g-
Boil, dissolve, neutralize to phenolphthalein, autoclav at 120°
for 5 minutes. Clarify with whites of four eggs and filter. Then
add—
SPECIAL MEDIA 131
Lactose 30 g.
Litmus-solution 260 c.c.
Crystal violet (o. \% aqueous solution) 20 c.c.
Tube and sterilize once in Arnold.
Uschinsky's non-proteid medium (FraenkePs modifica-
tion) . —
Disodium hydrogen phosphate 20 g.
Ammonium lactate 63 g.
Asparagin 34 g.
Sodium chlorid 50 g.
Water 10,000 c.c.
Phenol media. — i part carbolic acid is added to 1000
parts medium.
Dog-blood serum. — Serum of dog's blood is coagulated in
slanted tubes in the Koch inspissator for 3 hours at 75°.
Nitrate broth. — Add 5 parts of potassium nitrate to each
1000 parts of ordinary broth.
Nitrate solution. — 5 c.c. of 2 per cent, aqueous potassium-
nitrate solution are added to a solution of i g. of peptone in
1000 c.c. of water.
APPENDIX II
STAINING SOLUTIONS
Delafield's hematoxylin —
Hematoxylin crystals 4 g.
Alcohol .25 c.c.
Ammonia alum 50 g.
Water 400 c.c.
Glycerin 100 c.c.
Methyl alcohol 100 c.c.
Alum-hematoxylin —
1 . Hematoxylin 2 g.
Absolute alcohol 100 c.c.
2. Ammonia alum 2 g.
Water 100 c.c.
Mix i and 2 and add —
Glycerin 850 c.c.
Glacial acetic acid 100 c.c.
Allow to stand for one month before using.
Bismarck brown —
Bismarck brown o . 5 g.
Water 100 c.c.
Sajranin —
Safranin 0.5 g.
Water 100 c.c.
Carbolic thionin-blue (Nicolle') —
Thionin-blue i g.
Carbolic acid 2 . 5 g.
Water 100 c.c.
Alum carmin —
Alum 2 . 5 g.
Carmin i • o g.
Water 100 c.c.
132
STAINING SOLUTIONS 133
Lithium-carmin (Orth) —
Carmin 2 . 5 g.
Saturated watery solution of lithium car-
bonate 100 c.c.
Add a few crystals of thymol.
Kiihne's methylene-blue —
Methylene-blue i . 5 g.
Absolute alcohol 10 c.c.
Carbolic acid solution (5$) 100 c.c.
Carbolic gentian-violet (Nicolle)
Gentian-violet (sat. alcoh. sol.) 10 c.c.
Carbolic acid i g.
Water po c.c.
APPENDIX III
FROST'S CULTURE CHART (MODIFIED)'
Group
Name of organism
Source, habitat, etc
MORPHOLOGICAL CHARACTERS:
i
Incubation
Temp. (°C.)
Sketches
i . Form :
a Broth
b Agar
c Gelatin
d Other Media
2. Size
3. Cell groupings and arrangements in
growths
4 Staining powers
d ^nilin-gentian-violet ..
b Loeffler's methylene-blue
c. Gram's stain
d. Special stains
5. Motility
a Character of movement
b Flagella stain
6 Spores
7. Special characters, such as
deposits, vacuoles
pleomorphic and involution forms,
capsules etc
PHYSIOLOGICAL CHARACTERS
i. Relation to temperature
Relation to free oxygen.
Relation to other agents, such as dessica-
tion, light, disinfectants, etc
1 This represents a model only. Open spaces and lines should be extended for
practical use to allow sufficient room for descriptions.
134
FROST'S CULTURE CHART
4. Pigment production
5. Growth in carbohydrate media:
a. Stab or shake culture
6. Fermentation-tube: i. Growth in bulb. . . .2. In closed arm. .
c. Percentage gas produced in: Dextrose. .. .Lactose . . .Saccharose
After 24 hours. .
After 48 hours
TT
Gas formula T^T~
CU2
d. Reaction in bulb 2. Closed arm
6. Acid or alkali production, in litmus milk
7. Reduction of nitrates; to nitrites to ammonia
8. Indol production; 24 hours 48 hours 4 days
fecal odor; 24 hours 48 hours 4 days
9. Enzym production; coagulative proteolytic diastatic
10. Characteristic odor
11. Pathogenesis
CULTURAL CHARACTERS OF:
(I)
Gelatin
plate or
roll tube
a) Surface
colonies
b) Deep
colonies
(2)
Agar plate,
or roll
tube
a) Surface
colonies
6) Deep
colonies
Reaction of
Medium
Incubation
Temp. (°C)
Description
Sketches
136 LABORATORY GUIDE IN BACTERIOLOGY
CULTURAL CHARACTERS OF:
Reaction,
etc.
24 Hrs.
48 Hrs.
6 Days
Sketches
(3)
Agar streak
and stab
A
/ \
/ \
A
/ X
/ \
/ \
(4)
Potato
&
^x /
(5)
Broth
i
v|
-
(6)
i
Litmus milk
L
w
(7)
Gelatin stab
^
FROST'S CULTURE CHART
CULTURAL CHARACTERS OF:
Reaction,
etc.
24 Hrs.
48 Hrs.
6 Days
Sketches
(8)
Glucose-agar
or special
media
INDEX
Absorbent cotton filter 17
Acid, chromic 8
Acid-resisting bacilli 103
Actinomyces asteroides, bovis, etc. 106
Agar-agar 1 1
Agar, beerwort 128
Agar, bile-salt 128
Agar, blood 128
Agar, glucose 19
Agar, glycerin 128
Agar, litmus-lactose 127
Agar, litmus-mannit 127
Agar, neutral red 127
Agar, preparation of n
Agar-slants 39
Agglutination 87
Air, bacterial examination of 37, 114,118
Air, bacteria from 37
Alkaline blood-serum 128
Alum carmin 132
Alum hematoxylin 132
Amylase 65
Anaerobic cultivation 107
Anaerobic group 107
Analysis of gas 82
Anilin-gentian-violet 30
Anthrax bacillus 98
Anthrax group 98
Arnold steam sterilizer 24
Aspirator 46
Autoclav 20
Autopsy 75
Bacilli, acid-resisting 103
Bacillus acidi lactici 79
Bacillus aerogenes capsulatus 107
Bacillus anthracis 98
Bacillus anthracis symptomatici 107
Bacillus botulinus 107
Bacillus cholerae suis 79,84
Bacillus cloacae 79, 89
Bacillus coli 79
Bacillus cuniculicida 97
Bacillus der Rinderseuche 97
Bacillus der Schweineseuche 97
Bacillus diphtheriae 94
Bacillus dysenteriae 79, 85
Bacillus enteritidis 79, 84
Bacillus faecalis alcaligenes 79, 85
Bacillus Gaertner's 84
Bacillus, grass 103
Bacillus icteroides 79
Bacillus lactis aerogenes 79, 92
Bacillus leprae 103
Bacillus mallei 105
PAGE
Bacillus mucosus capsulatus 92
Bacillus oedematis maligni 107
Bacillus of bubonic plague 97
Bacillus of dysentery 79, 85
Bacillus of rabbit septicemia 97
Bacillus paratyphosus 79, 84
Bacillus pestis 97
Bacillus prodigiosus 69
Bacillus pseudo-diphthericus 94
Bacillus pyocyaneus 69
Bacillus smegrnae 103
Bacillus subtilis 98
Bacillus tetani 107
Bacillus tuberculosis 103
Bacillus typhosus 79,85
Bacillus violaceus 69
Bacillus xerosis 94
Bacteria, chromogenic 69
Bacteria from air 37, 118
Bacteria, pyogenic 73, 76
Bacteria, unknown 112
Bacterial examination of air
37, 114, 118
Bacterial examination, of milk 114, 119
Bacterial examination, of water 114
Bacterium termo 79
Balsam bottle 4
Balsam, Canada 44
Basket, wire 4
Beerwort-agar 128
Beerwort-gelatin 128
Berkefeld filter 46
Bile-salt agar 128
Bile-salt broth 129
Bismarck brown 132
Blood-agar 128
Blood-serum 125
Blood-serum alkaline 128
Blood-serum, dog's 131
Bouillon (see peptone broth) 25
Bread-paste medium 128
Broth, bile-salt 129
Broth, glycerin 127
Broth, litmus 128
Broth, meat 26
Broth, nitrate 131
Brothr peptone 25
Broth, preparation of 25
Broth, sugar- free 27, 79
Brownian movement 43
Bubonic plague 97
Buchner's anaerobic culture meth-
od 107
Butter, bacilli in 103
139
1 4o
INDEX
Canada balsam 44
Capaldi's egg-medium 130
Capsulated group 92
Capsule stain 76
Capsule stain, Friedlander's 77
Capsule stain, Welch's 77
Capsule stain, Welch's modified no
Carbol-fuchsin 30
Carbolic gentian- violet 133
Carbolic thionin-blue 132
Carmin 43
Carmin, alum 132
Carmin, lithium 133
Casein 64
Caseinogen 64
Cedar oil 35
Cholera red reaction (see Indol) 101
Chrompgenic group 69
Clarifying media 13
Cleaning glassware
Cleaning mixtures 8
Coagulation 64
Coagulative enzym 64
Colon bacillus 79
Colon group 79
Colonies, counting of 116
Colony 39
Condensation water 40
Conradi's medium 130
Cotton filter 47
Cotton filter, absorbent 17
Counting colonies 116
Culture- charts description 57
Culture media n
Culture- tubes 3, 4, 14
Culture-tubes, plugging of
Culture-tubes, potato 3, 28
Cultures, egg 95
Cultures, plate 70
Cultures, pure 39
Death-point, thermal 124
Decolorization of litmus 65
Delafield's hematoxylin 132
Description of colonies 57
Description of cultures 57
Dextrose (see under glucose)
Dextrose yeast water 130
Diagnosis of glanders (Strauss) 105
Diastase 65
Diastatic enzym 65
Dilution tables 117
Diphtheria bacillus 94
Diphtheria group 94
Diphtheria toxin 96
Directions for filling out culture-
charts 6 1
Directions, general 2
Discontinuous sterilization 24
Disinfectants 122
Dog's blood-serum 131
Dorset's egg medium 130
Dorset's egg-yolk medium 130
PAGE
Drigalski's medium 130
Dung bacillus 103
Dunham's peptone solution 25
Dysentery bacillus 79, 85
Egg cultures 95
Egg media 130
Ehrlich's anilin -gentian- violet 30
Eisner's medium 129
Endo's medium 130
Enriching method of Schottelius 101
Enriching solution (Hoffmann and
Fischer) 130
Enzym 64
Enzym, coagulative 64
Enzym, diastatic 65
Enzym, production of 64
Enzym, rennet-like 64
Erlenmeyer flask 5
Examination, bacterial of air
37, 114,118
Examination, bacterial of milk 1 14, 1 19
Examination, bacterial of water 114
Fermentation-tube 4
Filament formation 99
Filling culture-tubes 14
Filter, absorbent cotton 17
Filter, Berkefeld 46
Filter, cotton 47
Filter, paper 15
Filter rack 17
Filtering media 16
Filtering media by vacuum 17
Finkler and Prior's spirillum 101
Flagella, staining of 86
Flask, Erlenmeyer 5
Fowl cholera, bacillus of 97
Friedlander's capsule stain 77
Friedlander's pneumobacillus 92
Frost's fermentation-chart 81
Frost's culture-chart (modified) 134
Fruit-jar method of anaerobic cul-
tivation 1 08
Fuchsin 30
Fuller's standard 13
Gaertner's bacillus 84
Garden earth, inoculation of 1 1 1
Gas analysis 82
Gas formula 82
Gas generator 109
Gasometer (Frost's) 81
Gelatin, beerwort 128
Gelatin, glucose 128
Gelatin, liquefaction of 64
Gelatin, litmus-lactose 127
Gelatin, peptone 23
Gelatin, whey 128
General directions 2
Gentian- violet 30
Gentian-violet, carbolic 133
Germination of spores 51
Glanders 105
Glassware, cleaning
INDEX
141
PAGE
Glassware, sterilization 9
Glucose-agar 19
Glucose-gelatin 127
Glycerin-agar 128
Glycerin-broth 127
Glycerinated potato 129
Glycogen 27
Gonorrheal pus 76
Gram's iodin solution 30
Gram's stain 53
Grass bacillus 103
Green mold 51
Gruber-Widal test for typhoid fever 87
Gypsum blocks 5°
Hand -lens 5
Hanging-drop 42
Hay-infusion 129
Hematoxylin alum 132
Hematoxylin, Delafield's 132
Hemorrhagic septicemia group 97
Hill's test -rods 122
Hoffmann and Fischer's enriching
method
Hog-cholera group
Hot-air sterilizer
Hydrogen gas-generator
Immersion oil
Impression preparation
Incubator
Indol
Indol test
Infection, phenomena of
Influence of disinfectants
Influence of moist heat
Influence of sunlight
Infusion, hay
130
79,84
9
109
I!
85
45
122
124
123
129
Inoculation (see various headings)
79, 84
79, 84
24
79
102
92
73
Intermediate group
Intermittent sterilisation
Intestinal group
Intramuscular inoculation
Intraperitoneal inoculation
Intravenous inoculation
Involution forms
Iodin solution, Gram's
Isolation of unknown bacteria
Jar, Novy
Klatschprdparat
Kiihne's methylene-blue
Labeling media
Laboratory rules
Lactose-litmus-agar
Lactose-litmus-gelatin
Liquefaction of casein
Liquefaction of gelatin
Lithium carmin
Litmus-broth
Litmus, decolorization of
Litmus-lactose-agar
Litmus-lactose-gelatin
Litmus-mannit-agar
30
112
1 08
98
133
20
I
127
127
64
64
133
128
65
127
127
127
PAGE
Litmus milk 28
Litmus-solution 127
Litmus- whey 128
Loeffler's flagella stain 86
Loeffler's methylene-blue 30
Mac Conkey's bile-salt agar 128
Mac Conkey's bile-salt broth 129
Magnifier 5
Mannit litmus agar 127
Meat broth 26
Meat-press 26
Media, adjusting reaction of 14
Media, adjusting weight of 13
Media, clarifying 13
Media, filling in tubes 14
Media, filtering 14
Media, filtering by vacuum 18
Media, phenol 131
Media, preparation of 1 1
Media, preservation of 28
Media, reaction of 12
Media, special 125
Media, standard methods 125
Medium, bread-paste 128
Medium, Drigalski-Conradi's 130
Medium, Eisner's 129
Medium, Endo's 130
Medium, Uschinsky's 131
Medium, wine-must 129
Method of describing cultures 39
Method of inoculation of media 57
Methylene-blue, Kiihne 133
Methylene-blue, Loeffler 30
Micrococcus gonorrhoeae 76
Micrococcus intracellularis menin-
gitidis 76
Micrococcus lanceolatus 76
Micrococcus melitensis 105
Micrococcus tetragenus 73
Micrococcus zymogenes 76
Microscope, description of 31
Milk, acid-resisting bacilli in 104
Milk, bacterial, examination of 114,119
Milk, litmus 28
Milk, pasteurization of 119
Milk, sterilization of 119
Moeller's grass bacillus 103
Moeller's spore stain 98
Moist heat, influence of 124
Moist heat, sterilization by 20, 24
Mold, green 51
Mold spores 39
Molds 49
Molds, staining of 5°
Molecular movement 43
Mordant
Mouse, inoculation of
Mouse- holder 78
Muscle-sugar 27
Needles, platinum 4
Neisser's stain 94
Neutral red agar 127
I42
INDEX
PAGE
Nicole's carbolic gentian-violet 133
Nicole's carbolic thionin-blue 132
Nitrate broth 131
Nitrate solution 131
Nitrites 63
Nitrites, test for 83
Nitroso-indol reaction 101
Normal solution 13
Novy jar 108
Oil, immersion 34
Orth's lithium carmin 133
Parietti's solution 129
Park's anaerobic culture method 107
Pasteurization of milk 119
Pedesis 43
Penicillium glaucum 5 1
Peptone broth 25
Peptone gelatin 23
Peptone solution, Dunham's 25
Peptonization 64
Petri dishes 5
Phenol media 131
Phenolphthalein 12
Phenomena of infection 45
Phenomena of sterilization 45
Pigments 7 1
Plate cultures 70
Platinum needles 4
Plugging culture-tubes 8
Pneumobacillus 92
Pocket inoculation 96
Polar staining 85, 97
Potato, glycerinated 129
Potato, preparation of 27
Potato-tubes 28
Preparation, Gram 53
Preparation, impression 98
Preparation of agar-agar 1 1
Preparation of bouillon 25
Preparation of broth 25
Preparation of culture media 1 1
Preparation of Dunham's peptone
solution 25
Preparation of gelatin 23
Preparation of glucose- agar 19
Preparation of stains (see various
headings)
Preparation of peptone solution 25
Preparation of potato 27
Preparation, stained 44
Preservation of media 1 1
Proteolysis 64
Proteus group 89
Pure culture 39
Pus, gonorrheal 76
Pyocyanin 72
Pyogenic group A 73
Pyogenic group B 76
Rabbit septicemia 97
Reaction, indol 83
Reaction, nitrites 83
Reaction, nitroso-indol 101
Reaction of media 14
Rennet-like enzym 64
Rinderpest 97
Routine study 53
Saccharomyces cerevisiae 49
Safranin 132
Sarcina lutea 69
Schottelius' enriching method 101
Schweineseuche 97
Soap-powder 8
Solution, Dunham's peptone 25
Solution, litmus 127
Solution, nitrate 131
Solution, normal 13
Solution, Parietti's 129
Solutions, staining 30, 132
Special media 125
Spirillum cholerae asiaticae 101
Spirillum Metchnikovi 101
Spirillum of Finkler and Prior 101
Spirillum tyrogenum 101
Spore-staining 08
Spores, germination of 51
Spores of bacteria 20
Spores of yeast 51
Spores of molds 39
Stain, Friedlander's capsule 77
Stain, Welch's capsule 77
Stain, Welch's capsule modified no
Stain, Gram's 53
Stain, Loeffler's flagella 86
Stain, Neisser's 94
Stained preparation 44
Staining acid-resisting bacilli 103
Staining flagella 86
Staining molds 51
Staining, polar 85, 97
Staining solutions 30, 132
Staining spores 98
Standard, Fuller's 13
Standard method of preparing
media 125
Staphylococcus pyogenes albus 73
Staphylococcus pyogenes aureus 73
Starch 65
Steam sterilizer 24
Sterilization 9
Sterilization, discontinuous 24
Sterilization, intermittent 24
Sterilization of glassware 9
Sterilization of milk 1 19
Sterilization, phenomena of 45
Sterilizer, hot-air 9
Sterilizer, Arnold steam 24
Strauss method of diagnosis of
glanders 105
Streptococcus pyogenes 73
Study, routine 53
Subcutaneous inoculation 85
Sugar-free broth 27, 79
Sulphur granules 106
Sunlight, influence of 123
INDEX
PAGE
Tables, dilution
17
Tetanus toxin
no
Test for indol
83
Test for nitrites
83
Test-rods, Hill's
122
Thermal death-point
124
Thermostat
55
Thionin-blue
132
Torulae
49
Tube, culture
4
Tube, fermentation
4
Tube, potato culture
28
Tubes, plugging of
Typhoid-dysentery group 79
Uschinsky's medium
8
, 85
I31
Water, bacterial examination of
114
PAGE
40
77
no
64
128
128
Water of condensation
Welch's capsule stain
Welch's capsule stain modified
Whey
Whey-gelatin
Whey litmus
Widal test (see Gruber-Widal test)
Wine-must 129
Winogradsky's solution 129
Xylol 33, 35
Yeast water 130
Yeast water, dextrose 130
Yeasts 39, 49, 51
Yeasts, budding of 51
Yeasts, spores of 51
Ziehl-Neelsen's carbol-fuchsin 30
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