. . LIBRARY . .

Connecticut
Agricultural College.

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CLASS NO.....bi..b.>..D..i...9..kl..
COST i

DATE- i5>.iUL....L3 I9.a.3.

In^

A LABORATORY COURSE

IN

BACTERIOLOGY

For the Use of Medical^ Agricultural^ and
Industrial Students ,

BY

FREDERIC R GORHAM, A.M.

Associate Professor of Biology, Brown University; Bacteriologist,
Health Department, Providence, R. I.

WITH 97 ILLUSTRATIONS

PHILADELPHIA AND LONDON

W. B. SAUNDERS & COMPANY

J90J

Gey

Copyright, 1901,
By W. B. SAUNDERS & COMPANY.

371s.

ELEOTROTVPEO BV

WE8TCOTT & THOMSON. PHILADA.

PRESS OF
W. B. SAUNDERS & COMPANY.

PREFACE.

Bacteriology is essentially a laboratory study.
It is only by actual laboratory work that it can be
tauo-ht in such a manner as to serve any useful pur-
pose. It is also a subject of very general scientific
interest. Courses in bacteriology are no longer con-
fined to the medical schools, but are being intro-
duced into colleges and agricultural and industrial
schools. This volume has been prepared as a guide
to the practical details of laboratory work. It is
intended to present the subject in such a general
way as to lay a broad foundation for later specializa-
tion in any branch of bacteriology. By a judicious
selection the course can be made to conform to the
requirements of medical, agricultural, or industrial
students.

Brown University, August, 1901.

CONTENTS.

CHAPTER I.

PAGE

Microscopic Examination of Bacteria n

Manipulation of the Microscope, ii. — Measurement of Bac-
teria, II. — Examination of Living Bacteria, 13. — Ordinary
Examination, 13. — Hanging Drop, 15. — Examination of
Stained Bacteria, 16. — Ordinary Stains, 16. — Gram's Stain,
19. — Staining Bacteria in Tissues, 20.

CHAPTER II.

Morphology of Bacteria 22

Demonstration of Form, 22. — Demonstration of Motion, 24. —
Staining Flagella, 25. — Demonstration of Capsules, 31.

CHAPTER III.

Reproduction of Bacteria 34

Division, 34. — Spores, 35. — Staining Spores, 36. — Germina-
tion of Spores, 38.

CHAPTER IV.
Classification of Bacteria 39

CHAPTER V.

Sterilization 43

Steam, 46. — Autoclave, 46. — Hot-air, 47.

CHAPTER VI.

Preparation of Culture-media 50

Bouillon, 50. — Gelatin, 50. — Agar, 50. — Media from Meat-
extracts, 55. — Potato, 55. — Dextrose, Lactose, and Saccharose
Bouillon, 56. — Milk and Litmus Milk, 57. — Blood serum, 59.

8 CONTENTS.

CHAPTER VII.

PAGE

Cultures of Bacteria 60

Bouillon Cultures, 60. — Gelatin or Agar Cultures, 61. — Potato
and Blood-serum Cultures, 66. — Plate Cultures, 66. — Im-
pression or Adhesive Preparations of Colonies, 75. — Cultures
in the Fermentation-tube, 76. — Anaerobic Cultures, 78. —
Demonstration of Liquefying Ferment, 80. — Isolation of
Species, 81.

CHAPTER VIII.

Determination of Species 83

Morphology and Life-history of a Species, 83. — Determina-
tion of the Name of a Species, 95. — Classification of Bacteria
by Groups, 107. — Classification of Water Bacteria by Groups,
112.

CHAPTER IX.

Bacterial Analysis of Water, Milk, Air, and Soil . . .114
Water Analysis, 114. — Milk Analysis, 120. — Bacteria in the
Air, 121. — Bacteria in the Soil, 122.

CHAPTER X.

Pathogenic Bacteria 124

Pyogenic Organisms, 125. — Gonococctis, 129. — Anthrax, 130.
— Glanders, 133. — Diphtheria, 135. — Influenza, 141. — Ty-
phoid and Colon Bacilli, 144. — Pneumonia, 150. — Tubercu-
losis, 151.— Actinomycosis, 158. — Malaria, 159.

Appendix 163

Bacterial Measurements by Photography, 163. — Moulds and
Yeasts, 165.— Stains and Reagents used in the Study of
Bacteria, 170. — Table of Synonyms, 175.

Index >^3

A LABORATORY COURSE

IN

BACTERIOLOGY

A LABORATORY COURSE

IN

BACTERIOLOGY

CHAPTER I.
MICROSCOPIC EXAMINATION OF BACTERIA.

I. MANIPULATION OF THE MICROSCOPE.

1. Kxamine mounted slides of bacteria with the
low-power (f inch) and high-power (^ inch) objec-
tives and with different eyepieces.

2. Manipulate the condenser, diaphragm, and
mirror, in order to ascertain what combination
gives the best result.

3. (a) Affix the oil-immersion lens (^ inch) to
the microscope.

(d) Place a drop of immersion oil on the cover-
glass.

(c) Bring the lens to a focus in the drop of oil.

(d) Again manipulate condenser, diaphragm, and
mirror to determine what combination now is best.

II. MEASUREMENT OF BACTERIA.

The most accurate method of measurement is
by photography ;^ but as this requires special appa-

^ See Appendix, page 163.

11

12 MICROSCOPIC EXAMINATION OF BACTERIA.

ratus, the following fairly accurate methods are
recommended :

1. First Method.

(a) Place a micrometer eyepiece in the micro-
scope.

{b) Examine the bacteria to be measured, and
record their lengths in divisions of the eyepiece
micrometer.

(c) Remove the slide of bacteria.

(d) Place a stage micrometer on the stage of
the microscope, and determine the relation of the
divisions of the eyepiece micrometer to the divis-
ions of the stage micrometer.

ie) The length of the divisions of the stage mi-
crometer is a fixed quantity (usually y^^- mm.) ;
therefore we have the equations :

Length of bacteria = x divisions of eyepiece
micrometer ;

X divisions of eyepiece micrometer = y di-
visions of stage micrometer ;

I division of stage micrometer = y^ mm.
From these equations we can readily determine the
length of the bacteria in millimeters. The unit of
length for microscopic measurements is the thou-
sandth part of a millimeter ; it is called a micron or
micromillimeter, and is designated by the Greek
letter fi. Therefore in the above equations we can
substitute for y^ mm. lo //, and write the result in
so many fi.

2. Second Method.

{a) Adjust the *' camera lucida" to the micro-
scope.

EXAMINATION OF LIVING BACTERIA. I 3

ifi) Draw by means of the camera the exact size
of the bacteria to be measured.

(r) Remove the slide of bacteria, and replace it
with the stage micrometer.

(d) Over the figure of the bacteria now draw the
scale of the stage micrometer, and read off directly
the size of the bacteria in divisions of the stage
micrometer.

III. EXAMINATION OF LIVING BACTERIA.

I. Ordinary Examination.

(a) Place a drop of water on a clean slide.

(b) Pass the cotton plug of the tube containing
the culture to be examined through the flame ; ex-

FiG. I. — Method of holding tubes during inoculation

(McFarland).

tinguish the ignited cotton by blowing or pinching
out the flame.

{c) Hold the test-tube containing the culture be-

14 MICROSCOPIC EXAMINATION OF BACTERIA.

tween the thumb and finger of the left hand, allow-
ing the lower end of the tube to rest on the back
of the hand.

{d^ Hold a straight platinum needle between the
thumb and forefinger of the right hand, and ster-
ilize it by heating red-hot. Allow it to cool.

Fig. 2. — Platinum needles for transferring bacteria ; made
from No. 2-] platinum wire inserted in glass rods.

{e) Grasp the cotton plug between the third and
fourth finger of the right hand, and remove it ; in-
sert the platinum needle, and transfer an exceed-
ingly minute portion of the culture of bacteria to
the drop of water.

(/") Return the cotton plug to the tube, and ster-
ilize the needle.^

{g) Place a clean ^ cover-glass over the drop of
water, and examine with the \ inch objective.

^ »The needle should invariably be heated before and after
using. If this practice is not carefully followed, cultures
will be contaminated, and perhaps pathogenic organisms
spread about the room. It is well also to pass the handle
of the needle through the flame before beginning work
each day.

"^ Cover-glasses and slides may be cleaned in the follow-
ing solution :

Potassium bichromate, 6 gm. ;

Concentrated sulphuric acid, 6 c.c. ;

Water, loo c.c.

Wash in water and store in alcohol.

Or boil the cover-glasses in sulphuric acid, wash in

EXAMINATION OF LIVING BACTERIA.

15

(Ji) Manipulate condenser, diaphragm, and mir-
ror to determine the best adjustment for the exam-
ination of unstained bacteria.

«. Examination in the ** Hanging Drop.**

Fig. 3. — A "concave sHde " with "hanging drop"

(McFarland).

Fig. 4. — A slide with cell for hanging drop. The ring
may be made of glass or of zylonite, and is cemented on
the slide with Canada balsam.

{d) Paint a ring of vaselin around the hollow in

water, and keep in alcohol ; or wash in strong nitric acid
for some time, rinse in water, and store in alcohol. Very
often simply passing them several times through a Bunsen
flame will clean them sufficiently.

1 6 MICROSCOPIC EXAMINATION OF BACTERIA.

a *' concave slide;" or use a slide on which is
cemented a small glass ring, and vaselin the top
of the ring.

(b) On the centre of a clean cover-glass place a
small drop of water.

{c) With a sterile platinum needle add to the
drop of water a very small portion of the culture
to be examined.

{d) Invert the slide over the cover-glass, so that
the drop of water is covered by the concavity or is
inside the glass ring, but does not touch the sides
of either ; press down so that the chamber is sealed
tight by the vaselin.

{e) Invert carefully and examine.

The hanging-drop examination is for the purpose
of determining the motility of bacteria or for watch-
ing their reproduction. The preparation may be
kept for examination from day to day without loss
by evaporation.

IV. EXAMINATION OF STAINED BACTERIA.

I. Ordinary Stains.

{a) Prepare a clean cover-glass.^

(b) Place a drop of water on the glass.'

(c) With a sterile platinum needle transfer a
minute portion of a culture to the drop of water

• See footnote, page 14, for directions for cleaning covers.

* If the cultures are in bouillon or other fluid, it is often
unnecessary to use the drop of water in spreading them on
the cover-glass.

2

EXAMINATION OF STAINED BACTERIA.

17

and spread uniformly over the surface of the cover-
glass. ^

id) Allow the film to dry.

(e) When dry pass the cover-glass, smeared sur-

FiG. 5. — Cornet's cover-glass staining forceps.

Fig. 6. — Stewart's cover-glass forceps.

Fig. 7.— Kalte\'er's cover-glass staining forceps.

face upward, three times through a Bunsen or alco-
hol flame at about the rate of the pendulum of a
clock.

The heat coagulates the albuminous material

' The cover may be held, while staining, in one of the
special forceps devised for the purpose.
2

1 8 MICROSCOPIC EXAMINATION OF BACTERIA.

around the bacteria and fixes them firmly to the
glass.

(/") Place a drop of stain * on the cover-glass large
enouo:h to cover the film. Allow it to stain for
from two to ten minutes ; the length of time de-
pends on the stain, the strength of the staining
solution, and the kind of bacteria.^

Fig. 8. — Bottles for stains.

i^g) Wash in water, dry the unsmeared side on
filter-paper, mount, film side down, in a drop of
water on a clean slide, and examine with the \ inch
objective.

' Almost any of the anilin stains maj' be emploj^ed.
Gentian-violet, basic fuchsin, and methylene-blue are those
most commonly used. For methods of preparing these
staining solutions, see Appendix, page 170.

^ It ver\' rarely happens that bacteria are over-stained.
But if such is the case, either a new film must be prepared
or the stain drawn with weak acetic acid (i : 1000), or the
cover-glass swept through i per cent, sulphuric acid. If
the film is not sufiiciently stained, repeat the staining
process.

EXAMINATION OF STAINED BACTERIA. 1 9

{It) If the bacteria are evenly distributed^ and
properly stained, dry both sides thoroughly between
filter-paper and mount, film side down, in a drop
of Canada balsam. Label and preserve.
^--tC Gramas Stain.

The value of this method depends on the fact that
the mycoprotein of certain bacteria forms with an
anilin dye and an iodid a compound insoluble in
alcohol. There are many bacteria in which such
an insoluble compound is not formed, and this
method consequently has considerable diagnostic
value. - —

{a) To a drop of water on a clean cover-glass add
a very small amount of a culture of Bacillus subtilis.

{b) To the same drop add a very small amount of
a culture of Bacillus vulgaris, or any culture that
does not stain by this method. _^

{c) Dry, fix, and stain for -five minutes in anilin
gentian- violet.^

(d) Wash in water.

\e) Treat with Gram's solution^ for one minute.

1 In a successful preparation the bacteria are evenly and
not too thickly distributed over the surface. If too many
bacteria are present, either a smaller amount of the culture
must be used, or a little from the first cover-glass must be
added to another drop of water on a second cover-glass, and
soon until the proper dilution is reached.

2 See Appendix, page 171.

3 Gram's solution :

lodin, I part ;

Potassium iodid, 2 parts ;

Distilled water, 300 parts.

20 MICROSCOPIC EXAMINATION OF BACTERIA.

(f) Wash in 95 per cent, alcohol until no more
color comes away. c^

{£■) Dry and contrast-stain in safranin ^ (thirty
seconds), or Bismarck-brown ^ (two to three min-
utes), or eosin ^ (one minute).

{/i) Wash, dry, and mount.

3. Staining Bacteria in Tissues.

(i) First Methods

(a) Harden the tissue in absolute alcohol or Zen-
ker's fluid.^

(d) Dehydrate, embed, and section by the usual
methods.

{c) Stain as directed for cover-glass preparations,
but a little more deeply.

(d) Draw the color with dilute acetic acid
(i : 1000) until the bacteria alone are stained.

(e) Contrast-stain in eosin or any stain not re-
quiring acid for differentiation.

(/) Clear and mount.

(2) Second Method,

Stain the sections by Gram's method as follows :
(a) Stain in anilin gentian-violet for five minutes.
(d) Wash in water.

(c) Treat with Gram's solution for one minute.

(d) Wash in 95 per cent, alcohol until no more
color comes away.

^ Stock solution is a i per cent, solution of safranin in
equal parts of methylated spirit and water. For use, dilute
with 5 parts of water.

'^ Saturated solution in equal parts of alcohol and water.

•'' Aqueous solution, i : 1000.

* See Appendix, page 173.

EXAMINATION OF STAINED BACTERIA. 21

(e) Wash in water.

(/) Contrast-stain in an aqueous solution of
eosin (i : looo) for one-half to one minute.

{g) Wash in 60 per cent, alcohol for thirty'

seconds.

{Ji) Wash in absolute alcohol for thirty seconds.
(i) Clear in xylol and mount in balsam.

. (3) Third Method.

{a) Stain in Kuhne's methylene-blue ^ for one-
half to one hour, or in carbol-thionin-blue^ for five
minutes.

{p) Wash in water.

\c) Treat with 0.5 per cent, acetic acid till pale

green.

id) Wash in water, 60 per cent, alcohol, and
absolute alcohol, each for thirty seconds.

{e) Contrast-stain as in above methods, clear, and
mount.

(4) Foufth Methods

{a) Stain the dried preparations in a dilute aque-
ous solution of methylene-blue.

{h) Wash in water and dry.

{c) Stain in aqueous eosin solution (i : 1000) for
one to one and a half minutes.

(^) Dehydrate, clear, and mount.

t. ~

^ See Appendix, page 171.
- See Appendix, page 171.

CHAPTER II.

MORPHOLOGY OF BACTERIA.

Bacteria are minute, unicellular, vegetable or-
ganisms. They consist of a sharply defined mass of
protoplasm which reacts to anilin stains very much
like the nuclei of other cells, and outside of this
a more or less well-developed envelope. They are
classified according to their form into three main
groups, the spherical cocci, the rod-shaped bacilli,
and the curved or spiral spirilla.

a

b

•

^

g

^

®

©

</

0(9®00

Fig. 9. — Diagram illustrating the morphologj^ of the
cocci : a, coccus or micrococcus ; b, diplococcus ; c, d, strep-
tococci ; e, f, tetragenococci or merivSmopedia ; g, h, modes
of division of cocci ; /, sarcina ; y, coccus with flagella ; k^
staph 3'lococci (McFarland).

I. Demonstration of Form.

{a) Make hanging-drop and stained preparations
from cultures of cocci, bacilli, and spirilla.

{b) Examine with the ^ inch or with the oil-
immersion lens.

no

DEMONSTRATION OF FORM.

23

In the hanging-drop preparations are the indi-
vidual bacteria spherical, rod-shaped, or spiral?
If rod-shaped, are the ends pointed, rounded, or
square? Are the bacteria motile?^ Are they single

a

d

•"**,-.

Fig. 10. — Diagram illustrating the niorpholog}' of the
bacilli : a, b, c, various forms of bacilli ; d, e, bacilli with
flagella ; f, chain of bacilli, individuals distinct ; g, chain
of bacilli, individuals not separated (McFarland).

Fig. II. — Diagram illustrating the morpholog}- of the
spirilla : a, b, c, spirilla ; d, e, spirochaeta.

or united in pairs, fours, irregular masses, or chains?
Preserve the hanging-drop preparations for further
study.

(c) Repeat these observations on the stained prep-
arations.

^ See I II., page 24.

24

MORPHOL OGY OF BAC TERM .

Are the individuals stained uniformly or irregu-
larly, deeply or faintly ? In the rods are the ends
more deeply stained than the centers (polar stain-
ing) ?

(d) Measure the various kinds and make draw-
ings of them.

II. Demonstration of Motion.

(a) To a small drop of water on a cover-glass add
a very little carmine or Bismarck-brown.

Fig. 12. — Bacillus suipestifer, showing flagella.

(d) Mount in the same manner as a hanging-drop
preparation.

(c) Examine with the ^ inch objective.

Are the bits of pigment in motion ? Do they
change their position relative to one another, or do
they dance about in one place? This is the so-
called Brownian movement.

STAINING FLAG ELLA. 25

{d) Bxamine the hanging-drop preparations of
the last section in reference to this movement.

Do they all show the Brownian movement ? Are
some actively swimming about, changing their
position in relation to one another? All bacteria
exhibit the Brownian movement, but certain ones
are motile of themselves. They possess organs of
locomotion or fiagella, lash-like appendages, by the
movements of which they propel themselves along.
The flagella may be very numerous, extending
from all sides of the cell, or they may be collected
in a tuft at one end, or there may be a single one or
a pair (peritrichous, lophotrichous, monotrichous,
amphitrichous). The flagella may be demonstrated
by appropriate methods of staining.

III. Staining Flagella.

I. Pitfield's Method*

{a) Prepare a mordant as follows :

Tannic acid (10 per cent, solu-
tion, filtered), loc.c. ;

Corrosive sublimate (saturated

aqueous solution), 5 c.c. ;

Alum (saturated aqueous solu-
tion), 5 c.c. ;

Carbol-fuchsin ^ 5 c.c.

Allow to stand and draw off the clear fluid. The
mordant will keep one or two weeks.

^ See Appendix, page 172.

26 MORPHOLOGY OF BACTERIA.

{b) Prepare a stain as follows :

Alum (saturated aqueous solu-
tion), loc.c;

Gentian-violet (saturated alco-
holic solution), 2 c.c.

The stain will keep two or three days.

{c) On an absohUely clean cover-glass^ place a
drop of distilled water.

(d) With a sterile platinum needle add to the
drop of water, without stirring, a very small por-
tion of an actively motile culture.

{e) Stand on a water-bath at 60° C. for one-half
hour.

(y) Treat the film with the cold mordant for
twenty-four hours, or with hot mordant (steaming,
but not boiling) for three minutes.

{g) Wash thoroughly in running water and dry.

(Ji) Treat with the stain the same as directed
for the mordant.

(i) Wash, dry, mount, and examine with the oil-
immersion lens.

2. Modification of Pitfield's Method.

(a) Prepare solution A as follows :

Alum (saturated aqueous solu-
tion), 10 c.c. ;

Gentian-violet (saturated alco-
holic solution), I c.c.

' See footnote, page 14.

STAINING FLAG ELLA. 2/

{b) Prepare solution B as follows :

Tannic acid, i gm. ;

Distilled water, lo c.c.

{c) Filter when cold, mix the two solutions, and
use immediately.

{d) Prepare films as before.

{e) Treat with the above mixture, gently heating
until it almost boils, then set aside for a minute.

if) Wash, dry, and mount.

Fig. 13.— Microspira comma, showing the flagella ; x 1000

(Giinther).

3. Loffle/s Method.

{a) Prepare a mordant as follows :

Tannic acid (20 per cent, solu-
tion), filtered, 10 CO.;

Ferrous sulphate (cold, satu-
rated solution), filtered, 5 c.c;

Fuchsin (saturated alcoholic so-
lution), I c.c.

28

MORPHOLOGY OF BACTERIA.

Filter each time before using. This mordant will
keep two or three days.

(h) Prepare films as before.

{c) Dry and pass once through the flame.

(</) Cover the film with the mordant, and warm
over the flame for a few seconds only.

Fig. 14. — Bacillus typhosus, from an agar culture six
hours old, showing the flagella stained by Loffler's method ;
X 1000 (Frankel and PfeifFer).

(e) Wash in water, dip in absolute alcohol, and
again wash in water.

(/) Cover the film with anilin-water fuchsin,^
and warm over the flame from three to four min-
utes, taking care that the solution steams, but does
not boil.

^ See Appendix, page 171.

STAINING FLAG ELLA. 29

(^) Wash in water, dry, mount, and examine
with oil-immersion lens.

4. Modification of Loffler's Method*

{a) Prepare a mordant as follows :

Tannic acid (20 per cent, solu-
tion), loc.c. ;

Ferrous sulphate (saturated so-
lution), 10 c.c. ;

lyOgwood solution (i gram
boiled in 8 c.c. of water
and filtered), 3-4 c.c.

(fi) Prepare a stain as follows :

Anilin- water, ^ 100 c.c;

Sodium hydrate (t per cent.), i c.c. ;

Methylene-violet or methylene-

blue or fuchsin, 4-5 gm.

Filter.

(c) Prepare films as before.

{d) Heat with the mordant until steam rises, and
then move over flame for one minute.

{e) Wash in water, and if mordant does not dis-
appear, in absolute alcohol.

{/) Dry and heat with the stain until steam rises,
then leave in the warm stain one minute.

^g) Wash, dry, and mount.

^ See Appendix, page 171.

30

MORPHOLOGY OF BACTERIA.

5. Van Ermengfem's Method^

(a) Prepare solution A as follows :

Osmic acid (2 per cent, solu-
tion), I part ;
Tannic acid (10 to 25 per cent.

solution), 2 parts.

To each 100 c.c. of the tannic acid add 4 or 5
drops of glacial acetic acid.

Fig. 15. — Bacterium F^!lfcii4ngii in blood ; x 1000 (Frankel

and Pfeiffer).

* Various modifications of Van Ermengem's method have
been suggested. See Ccntralbl. f. Bakt., Erste Abteilung,
Bd. xxvii., Nos. 16-17, P- 597 : ^^^d La7icet, vol. ii., No. 14,
p. 874, 1898.

DEMONSTRATION OF CAPSULES. 3 I

{B) Prepare solution B as follows :

Gallic acid, 5 gm. ;

Tannic acid, 3 "

Fused acetate of sodium or

potassium, 10 "

Distilled water, 350 c.c.

(c) Prepare films as before.

(d^ Place in solution A for one-half hour ; or, if
the solution is warmed to 60° C, five minutes are
sufficient.

(e) Wash thoroughly in water, then in alcohol,
then again in water.

(y) Place for two minutes in solution of nitrate
of silver (0.25-0.5 per cent.).

(<^) Without washing, place in solution B for
one and one-half to two minutes, using fresh solu-
tion for each preparation.

(//) Wash thoroughly in water, and examine in
water. If the flagella are not sufficiently stained,
begin again at (y^).

Always change the silver nitrate solution as soon
as any precipitate appears.

IV. Demonstration of Capsules.

In certain forms the envelopes surrounding the
cells can be stained as follows :

I. Welch's Method;

{a) Prepare films without using water.

(B) Place in glacial acetic acid for a few seconds.

(c) Remove the acid with filter-paper.

^ Bulletin of the Johns Hopkins Hospital, p. 128, Decem-
ber, 1892.

32

MORPHOLOGY OF BACTERIA.

id) Wash in anilin-water gentian-violet repeat-
edly until all the acid is removed.

(<?) Wash in a ^ per cent, solution of sodium
chlorid and examine in the same solution.

2. Johne's Method*

(a) Prepare films as usual.

(b) Warm in a 2 per cent, solution of gentian-
violet until steam arises.

Fig. i6. — Bacterium pneumoniae of Friedlander, from the
expectoration of a pneumonia patient ; x looo (Frankel
and Pfeiffer).

(r) Wash in water.

id) Place in 2 per cent, acetic acid for six to ten
seconds.

(e) Wash in water.

(/) Dry and mount in balsam.

DEMONSTRATION OF CAPSULES. 33

3. Boni's Method.^

[a) Prepare a solution as follows :'
White of one ^'g<g ;

Glycerin, 50C.C. ;

Formalin, 2 drops.

Shake together and filter.

{h) Prepare films of the organism to be studied,
using the above solution in place of water.

(c) Spread very thin, and heat over flame until
the glycerin is evaporated.

id) Stain in Ziehl's carbol-fuchsin ^ for twenty
to thirty seconds.

{e) Wash in water and dry with filter-paper.

(/) Contrast-stain in Lofiier's methylene-blue'^
for four to six minutes.

{g) Wash, dry, and mount in balsam.

^ Centralbl. filr Bakteriologie, Erste Ab., Band xxviii.,
No. 20, p. 705.

See Appendix, page 172. =» See Appendix, page 171.

3

2

CHAPTER III.

REPRODUCTION OF BACTERIA.

The common method of reproduction among the
bacteria is by binary division. In some forms this
is the only mode of reproduction known. Under
favorable conditions an individual cell grows in
length, a transverse constriction appears in the
middle and gradually becomes deeper until two
new cells are formed. Separation may be com-
plete or there may be the formation of chains. In
the spherical forms division may take place in one,
two, or three planes, forming chains (streptococci),
or groups of two (diplococci), irregular groups
(staphylococci or micrococci), or cubical packets
(sarcina). In the rod-shaped forms division takes
place in but one plane, forming chains, pairs, or
single individuals (Figs. 9 and 10).

Some bacteria, besides having the power of re-
production by division, form endogenous spores.
These spores are developed from the plasma of the
cell, and have a dense wall that protects them from
injury by drying, enables them to withstand high
temperatures, and also causes them to resist the
action of stains.

I. Reproduction by Division.

{a) On a clean cover-glass place a large drop of
bouillon.'

* See page 50.

34

REPRODUCTION BY SPORES. 35

(fi) Add to the bouillon a very small portion of
a culture of Bacilhcs subtilis^ or any chain-forming
and spore-producing species.

(c) Mount as a hanging-drop preparation and
place in the incubator.

(d^ Examine at intervals of one-half hour.

Notice the elongation of the bacilli and the for-
mation of chains.

ie) Inoculate a bouillon-tube as directed on page
60.

(/") Place in the incubator and make stained
preparations at half-hour intervals.

Preserve both hanging-drop preparation and
bouillon-tube for further examination.

II. Reproduction by Spores.

1. After the formation of chains has taken place
in the above cultures, continue the examination at
intervals. The formation of spores within the rods
will be observed ; finally, the rods will disappear
and nothing but spores remain.

a b c d c f

Cg^ CZ o) O 00 C^=3 %=D

Fig. 17. — Diagram illustrating sporulation : a, bacillus
enclosing a small oval spore ; b, drumstick bacillus, with
the spore at the end ; c, Clostridium ; d, free spores ; e, and
f, bacilli escaping from spores.

2. Make stained preparations from the bouillon
culture when rods and spores within the rods and
spores alone are all present. Stain as follows ;

36

REPRODUCTION OF BACTERIA.

III. Staining Spores.

(i) First Method.

id) Prepare films in the usual manner.

ifi) Stain in warm Ziehl's carbol-fuchsin ^ for
twenty to thirty minutes, or in the same solution
steaming, but not boiling, for five minutes.

{c) Wash in water.

{d) Dip in acid alcohol (70 per cent, alcohol, 97

Fig. 18. — Bacterium anthracis, stained to show spores ;
X 1000 (Frankel and Pfeififer).

CO. ; hydrochloric acid, 3 c.c.) or in i per cent,
sulphuric acid a few seconds and wash in water.

(e) Examine in water.

Spores should be red and rods unstained or
faintly pink. If the spores are not sufficiently

' See Appendix, page 172.

STAINING SPORES. 37

stained, place again in the fuchsin. If the rods
are still stained, wash longer in the acid alcohol.

(/) Dry and pass three times through the flame.

{g) Stain in Loffler's methylene-blue ^ for three
to four minutes, or in gentian-violet^ one minute,
or in saturated aqueous solution of methylene-blue
for half a minute.

(Ji) Wash, dry, and mount.

Spores should be red, rods blue.

(2) Second Method.

(a) Prepare films in the usual manner.

{b) Place in chloroform for two minutes.

{c) Treat with 5 per cent, chromic acid for one
minute. The acid acts on the membranes of the
spores and permits the entrance of the stain.

(d) Wash thoroughly in water.

{e) Stain in hot Ziehl's carbol-fuchsin for three
minutes.

(y) Without washing decolorize in 5 per cent,
sulphuric acid.

^g) Wash in water and stain in Loflfler's methy-
lene-blue^ for two minutes or longer.

(h) Wash, dry, and mount.

(3) Third Method.

(ci) Suspend the spore-bearing bacteria in a nor-
mal salt solution ^ in a test-tube.

(^) Add an equal volume of Ziehl's carbol-fuchsin.

(r) Place the test-tube in boiling water for fifteen
minutes.

^ See Appendix, page 171. ^ See Appendix, page 171.

^ See Appendix, page 173,

38 REPRODUCTION OF BACTERIA.

{d) Spread a loopful on a cover-glass ; dry ; fix.
(e) Decolorize in a i\ per cent, (by volume) solu-
tion of hydrochloric acid in alcohol.

Fig. 19. — Bacillus chauvaei, showing spores ; x 1000
(Frankel and Pfeiffer).

if) Wash in water and contrast-stain in Loffler's
methvlene-blue.

{g) Wash, dry, and mount.

(4) To Observe the Germination of Spores,

{a) Make a hanging-drop preparation, using ster-
ile bouillon and some of the spores from the above
culture.

{b) Place on a warm stage and watch the growth
of rods from the sDores.

(c) Inoculate a sterile bouillon-tube with a large
number of spores from the above culture.

{d) Make stained cover-glass preparations from
this at intervals of one-half hour.

CHAPTER IV.

CLASSIFICATION OF BACTERIA.

The position occupied by the bacteria in the
vegetable world is shown by the table on page 40.

The Bacteria or Schizomycetes are classified into
families according to their form, and again into
genera according to certain other characters. The
following classification is that proposed by Migula
in his System der Bakterien^ 1900 :

Bacteria, Schizomycetes.

EuBACTERiA, cells contain no sulphur granules
or bacterio-purpurin.

1. Family Coccace^, spherical forms.
Genus :

1. Streptococcus, non-motile ; cells divide in one plane.

2. Micrococcus, non- motile ; cells divide in two planes.

3. Sarcma, non-motile ; cells divide in three

planes.

4. Planococcvs, motile ; cells divide in two planes.

5. Planosarcina, motile ; cells divide in three planes.

2. Family Bacteriace^, straight, rod-shaped
forms without envelope.

Genus :

1. Bacterium, non -motile.

2. Bacillus, motile ; flagella over whole surface.

3. Pseudoinonas, motile ; flagella polar.

39

40

CLASSIFICATION OF BACTERIA.

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CLASSIFICATION OF BACTERIA. 4 1

3. Family Spirillace^, curved, rod-shaped
forms without envelope.

Genus :

1. Spirosofna, non-motile ; cells rigid.

2. Microspira, motile ; one, rarelj^ two or three,

polar flagella.

3. Spirilluyn, motile ; polar tufts of flagella.

4. SpirochcEta, cells flexile.

4. Family Chlamydobacteriace^, cells with
envelopes.

Genus :

1. Chlainydothrix , unbranched threads ; cell-division

in one plane.

2. Crenothrix, unbranched threads ; cell-division

in three planes ; sheath visible.

3. Phragmidiothrix , unbranched threads ; cell-division

in three planes ; sheath scarcely
visible.

4. Sphcerotiltis, branched threads.

Thiobacteria, cells contain sulphur granules or
bacterio-purpurin ; red or violet color, never green.

1. Family Beggiatoace^, thread-forming, with-
out bacterio-purpurin.

Genus :

1. Thiotrix, attached threads ; non-motile,

2. Beggiatoa, unattached threads ; motile.

2. Family Rhodobacteriace.^, cells contain
bacterio-pupurin and sulphur granules ; red or
violet.

42 CLASSIFICATION OF BACTERIA.

I. Subfamily Thiocapsace^, cells divide in
three planes.

Genus :

1. Thiocystis.

2. Thiocapsa.

3. Thiosarcina.

II. Subfamily Lamprocystace^, cells divide
first in three, then in two planes.

Genus :
I. Lamprocystis.

III. Subfamily Thiopediace^, cells divide in
two planes.

Genus :
I. Thiopedia.

IV. Subfamily Amebobacteriace^, cells divide
in one plane.

Genus :

1. Amebabacter.

2. Thiothece.

3. Thiodictyoii.

4. Thiopolycocais.

V. Subfamily Chromatiace^.

Genus :

1. Chromathdu.

2. Rhabdochro7uatium.

3. ThiospirilUim.

CHAPTER V.

STERILIZATION.

Sterilization is the process of killing micro-
organisms. A body is said to be sterile when all
the bacteria in or upon it have been killed or re-
moved. Sterilization is effected by the application
of heat, by treating with certain chemicals, or, in
the case of fluids, by filtration.

When spores are not present, bacteria are killed
by exposure for twenty minutes to boiling water or
steam. To destroy spores, however, a much longer
exposure is necessary, in some cases several hours.
Steam under a pressure of thirty pounds will give
a temperature of 120° C. ; this will kill all organ-
isms and spores in fifteen minutes.

Dry heat is not so fatal to either bacteria or their
spores as is moist heat or steam ; therefore to steril-
ize by dry heat a higher temperature, sustained for
a longer time, is necessary ; 150° C. continued for
one hour will kill all ordinary bacteria and their
spores. Liquids and objects liable to be injured by
dry heat can be sterilized only by steam.

The method of discontmuoiis sterilization is em-
ployed in sterilizing objects which would be injured
by long exposure to a high temperature. The ob-
jects to be sterilized are first subjected to a temper-
ature sufficiently high to destroy all bacteria not

43

44 -S* TERIL IZA TION.

in the spore condition, say ioo° C. for twenty
minutes. They are then allowed to cool for
twenty-four hours, and again sterilized as before.
This is repeated several times. The intermissions
allow such spores as are present to develop into rods,
and these are killed by the subsequent heating.

Pasteurisation is the term applied to the partial
sterilization of milk, effected by subjecting it to a
temperature sufficiently high to kill all pathogenic
and most of the souring and spore-forming bacteria,
but not high enough to produce any physical
changes, such as are brought about when the milk
is sterilized. A temperature of 60° C. continued
for fifteen or twenty minutes is usually sufficient,
though temperatures as high as 85° C. are fre-
quently employed. Enhanced keeping qualities
and the destruction of pathogenic organisms are
the results of pasteurization.

The chemical substances most frequently em-
ployed for sterilization or disinfection ^ are solutions
of corrosive sublimate (i : 1000), carbolic acid (i :2o),
or formalin (i : 20). A substance that prevents
the development of bacteria but does not destroy
them is an antiseptic. One that destroys all germs
and spores is a germicide or disinfectant. There
are many gases, acids, salts, etc., that have anti-
septic or germicidal properties.

Bacteria may be removed from a liquid by pass-
ing it through a properly constructed filter of un-

^ Disinfection is the term applied to sterilization by means
of chemicals.

FIL TRA TION.

45

glazed porcelain. The most satisfactory filters in
use at present are the Chamberland and the Berke-
feld.

Fig. 20. — Filter : a,
porcelain bougie ; b, at-
tachment for suction -
pump ; c, reservoir ; d,
sterile receiver ; e. rubber
tube wired to bougie and
reservoir.

Fig. 21. — Filter : a,
porcelain bougie ; b, at-
tachment for suction-
pump ; r, reservoir ; d,
sterile receiver ; e. perfo-
rated rubber collar ; f,
glass chimney for draw-
ing the fluid around the
bougie (Page).

Bacteria in the air are unable to pass through a
cotton-wool filter, and consequently sterile flasks or

46

STERILIZA TION.

test-tubes stopped with a cotton-wool plug remain
sterile indefinitely.

I. Steam Sterilisation.

For objects liable to be injured by dry heat, such

as culture-media, fluids, in-
struments, etc. :

{a) Fill the water-tank
of the sterilizer^ and start
the flame.

(p) When the chamber is
filled with steam place the
objects to be sterilized
within and close the door.

(c) If the objects will not
be injured by prolonged
heating, allow them to re-
main for one hour.

id) If prolonged heating
is injurious, allow them to
remain twenty minutes,
and repeat the process
three times at intervals of
twentv-four hours.

Fig. 22. — Kitasato's fil-
ter : a, porcelain bougie ;
b, attachment for siiction-
pump ; c, reservoir ; d,
sterile receiver.

II. Steam Sterilisation under Pressure.

For rapid and effective sterilization. In this
method an "autoclave" is used.

{a) Adjust the safety-valve at the desired press-
ure, say thirty pounds.

See that an abundance of water is present, so that
the steam will not be superheated.

* An "Arnold " sterilizer is the form usually employed.

HOT' AIR STERILIZATION,

47

{B) Place the objects to be sterilized in the
chamber.

[c) Close the door and turn on the steam, first
allowing the air present in the chamber to escape.

{(l) Bring the temperature to, say 120° C. ; allow
it to remain there fifteen minutes.

Fig. 2^. — Arnold steam sterilizer, Boston Board of Health

pattern.

ie) Shut off the steam and allow the apparatus to
cool well below 100° C. before opening the door
or allowing the steam to blow off.

III. Hot-air Sterilisation.

For glassware and other objects not liable to be

48

STERILIZA TION.

injured by dry heat. The oven of an ordinary gas-
stove into which a thermometer can be inserted
makes an excellent hot-air sterilizer.

(a) Wash thoroughly, using, if necessary, the
cleaning mixture recommended on page 14, all

Figs. 24, 25. — Autoclaves for rapid steriUzation by steam

under pressure.

flasks, test-tubes, Petri dishes, etc., to be used in
the preparation of culture-media.

(b) Plug the flasks and test-tubes with cotton.^

*" Sliver" obtained from cotton -mills is excellent for
plugging test-tubes, etc.

HOT-AIR STERILIZATION.

49

In the case of the test-tubes, the plugs should be
tight enough so that the tubes can be lifted by
them.

ic) Place in the hot-air sterilizer, close the door,
and bring the temperature to 150° C. ; keep it there

Fig. 26. — Hot-air sterilizer.

for three-quarters of an hour, or until the cotton
plugs begin to turn brown.

(d') Allow the oven to cool before opening the
door. Place the glassware in a clean place free
from dust until used.
4

CHAPTER VI.
PREPARATION OF CULTURE^MEDIA.^

1. Bouillon.

11. Gelatin.

III. Agar.

Boil 30 grams of thread

agar in i liter of water for

half an hour. Make up

loss by evaporation to a

weight of 1000 grams.

I. Infuse finely chopped

Ditto.

I. Infuse finely chopped

lean beef for twenty hours

lean beef for twenty hours

with twice its weight of

with its own weight of

distilled water in the re-

distilled water in the re-

frigerator, say looo grams

frigerator, say 1000 grams

of meat, 2000 grams of

of meat, 1000 grams of

water.

water.

2. Make up weight of

Ditto.

2. Make up weight of

meat-infusion (and meat)

meat-infusion (and meat)

to original weight by add-

to original weight by add-

ing water — i.e., to 3000

ing water — /. ^.,.to 2000

grams.

grams.

3. Filter infusion through

Ditto.

3. Ditto.

cloth to remove meat.

4. Weigh infusion, say

Ditto.

4. Ditto, say 900 grams.

1800 grams.

5. Set infusion on water-

Ditto.

5. Ditto.

bath, keeping temperature

below 60° C.

6. Add peptone, i per

Ditto,

and sheet

6. Add peptone, 2 per

cent., 18 grams ; salt, 0.5

gelatin.

10 per

cent., 18 grams; salt, i

per cent., 9 grams.

cent., 180 grams.

per cent., 9 grams.

7. After ingredients are

Ditto,

probably

7, Ditto, probably + 4.5

dissolved, titrate ;"■' reac-

-f- 4.0 to

+ 50 per

to -f- 4.7 percent.

tion probably -|- 2.3 to

cent.

-f- 2,5 per cent.

8. Neutralize (Fuller's

Ditto.

8. Ditto.

method).^

To the 900 grams of
meat-infusion (containing
now peptone and salt)
add 900 grams of the 3
per cent, agar-jelly de-
scribed at the head of
this column.

For notes see page 51.

60

B O UIL L ON— GELA TIN— A GAR. 5 I

' Substantially as recommended by the Bacteriologic
Committee of the American Public Health Association.
Some minor changes, suggested by Dr. H. W. Hill, in the
Report of the Health Department of Boston for 1898, have
been incorporated without detracting from their value as
standard media.

' Acid media are denoted by the plus sign, and alkaline
media by the mi?ius sign ; the degree of acidity or alka-
linity is denoted by parts per hundred. Thus, a medium

marked +1.5 indicates that the medium is acid, and that

n
1.5 per cent, of - sodium hydroxid is required to make it

neutral to phenolphthalein.
' Following is Fuller's method of obtaining the degree of

reaction of culture-media :

n
(a) Prepare a ^- solution of sodium hydroxid.

91

(d) Prepare a — solution of hydrochloric acid.

{c) Transfer 5 c.c. of the medium to be tested to a porce-
lain evaporating-dish.

[d] Add 45 c.c. of distilled water.

{e) Boil for three minutes.

(/) Add I c.c. of a 0.5 per cent, solution of commercial
phenolphthalein in 50 per cent, alcohol.

(g) Titrate while still hot with the acid or alkali as re-
quired, and determine the reaction.

To determine exactly when the neutral point is reached,
notice that in bright daylight the first change that can be
seen on the addition of alkali is a ver\' faint darkening of
the fluid, which on the addition of more alkali becomes a
more evident color and develops into what may be de-
scribed as an Italian pink. A still further addition of
alkali suddenly develops a clear and bright pink color, and
this is the reaction always to be obtained.

{h) When the reaction has been obtained, calculate the
amount necessary to neutralize the bulk of the medium
or to produce the required reaction, and add the proper
amount of a normal solution of the acid or alkali.

52

PREPARATION OF CULTURE-MEDIA.

9. Heat over boiling water (or steam) bath thirty
minutes.

10. Restore weight lost by evaporation to origi-
nal weight of filtered meat-infusion, for bouillon
and gelatin, and to twice that weight for agar —
1800 grams in each case.

11. Titrate, reaction probably + 0.3 to + 0.5 per
cent.

12. Adjust reaction to final point desired, + 1.5
per cent.

Graniteware funnel.

Laver of absorbent
cotton.

Coil of wire to pre-
vent cotton from
entering tube of
funnel

Fig. 27. — Filter for culture-media.

13. Boil five minutes over free flame, with stir-
ring.

14. Add water if necessary to make up loss by
evaporation to 1800 grams.

15. Filter through absorbent cotton, passing
the filtrate through the filter repeatedly until
clear.

16. Titrate to determine whether or not the de-
sired reaction has been maintained.

B O UIL L ON— GEL A TIN— A GAR.

53

17. Tube and sterilize for fifteen minutes in the
steam sterilizer on three successive days.^

Some of the gelatin- and agar-tubes after the

Fig. 28.— Funnel for filling tubes with culture-media :

a, funnel containing the culture-media in liquid condition ;

b, pinch-cock by which the flow of fluid into the test-tube
is regulated ; c, rubber tubing (Warren).

^ Allow the chamber to fill with steam before placing the
media within. Do not leave the media in the sterilizer to
cool. Plunge gelatin-tubes into ice-water immediately after
each sterilization, in order to maintain a high melting-point.

54

PREPARATION OF CULTURE-MEDIA.

Fig. 29. — Providence Health
Department tube of heavy
glass, with etched surface for
vi^riting data. These tubes are
etched by dipping for thirty
seconds in "white acid."

Fig. 30, — Potato-tube
(Mallorj- and Wright).

last sterilization should be allowed to solidify with
slanting surfaces.

Potato. 55

IV. Media from Meat-extracts.

ia) Mix thoroughly the white of i ^<gg with
1000 CO. of water for bouillon or gelatin ; with 500
c.c. of water for agar.

(b) Proceed then as directed in the above table,
beginning with No. 4, substituting "white of ^^^
solution" for "infusion." At No. 6 add 0.5 per
cent, of Iviebig's extract of beef for bouillon or gel-
atin, i.o per cent, for agar.

V. Potato.

{a) Select large sound potatoes and wash thor-
oughly.

(b) Cut off the ends, and with a sterile cork-borer
cut out cylinders of the potato a little smaller than
the tubes in which they are to be placed. Handle
the potatoes under water as much as possible, to
prevent darkening of the surface.

{c) Cut the cylinders into two equal parts by a
diagonal cut.

id) Place in cold running water for twelve to
eighteen hours. This will usually render the
potato neutral.

(If necessary to change the reaction of the potato,
steam in a measured quantity of distilled water for
one-half hour. Titrate and add the required

amount of sodium hydroxid, and repeat the boil-
ing for thirty minutes.)

{e) Distribute in tubes in the bottom of which a
small amount of non-absorbent cotton or a short
piece of glass rod has been placed, and sterilize in

56

PREPARATION OF CULTURE-MEDIA.

the steam sterilizer for thirty minutes on three suc-
cessive days.

VI. Dextrose, I/actose, and Saccharose
Bouillon.

(a) After filtration of the meat-infusion, prepared
as above described, place in an Erlenmeyer flask,
and inoculate with a fluid culture of the Bacillus
coli or an allied gas-producer.

(b) Place in the incubator at 37.5° for twenty-
four hours.

Fig. 31. — The Smith fer-
mentation-tube.

Fig. 32. — A graduated fer-
mentation-tube.

This removes the meat-sugar.

(c) From this infusion prepare bouillon in the
ordinary way.

id) To the completed broth add i per cent, of
the required sugar.

(e) Distribute in test-tubes or in fermentation-
tubes,' and sterilize in the steam sterilizer on three
successive days.

^ A small test-tube inverted inside a large one will
answer for a fermentation-tube.

Milk and litmus milk.

57

VII. Milk and I^itmus Milk.

{a) Heat fresh milk for fifteen minutes in the
steam sterilizer.

{h) Place in the ice-box over night.

(c) Siphon off the milk, without cream or sedi-
ment.

HoUdw thitnhle sto/>per
Surfaces in contact GroujicL

Fig. 2)7i' — The Hill fermentation -tube.

{d) Titrate.

{e) If less than 2 per cent, acid to phenolphtha-
lein, place in tubes and sterilize for twenty minutes
on four successive days in the steam sterilizer. If
more than 2 per cent, acid, adjust to + 1.5 per cent.

by the addition of - sodium hydroxid.

58 PREPARATION OF CULTURE-MEDIA.

(J) A solution of litmus may be added just

glass 40 cm.

Fig. 34. — Siphon, with one-way valve for starting the
flow of serum. The end of the glass tube is turned over to
prevent the clot from entering.

Fig. 35. — Coagulator for blood-serum tubes. Providence
Health Department pattern, with wooden rack for holding
tubes away from sides. When in use, the four sides and
the space below are covered with asbestos boards.

previous to its distribution in tubes, sufficient to
give the milk a pale-blue color.

BLOOD-SERUM. 59

VIII. Blood-serum {Ldffler's).

{a) Receive freshly drawn beef blood in sterile
jars.

{b) Allow twenty minutes for coagulation to
begin, then with a sterile glass-rod break up any
adhesions between the coagulum and the jar.

(<f) Allow the jars to stand twenty-four hours in
the ice-box.

{d) Siphon off the clear serum. ^

{e) To 3 parts of serum add i part of i per cent,
dextrose bouillon.^

{/) Adjust to -h 0.8 per cent.

{g) Distribute in tubes and solidify with a slant-
ing surface by heating for three hours in a blood-
serum coagulator.

{h) Sterilize in the steam sterilizer for twenty
minutes on three successive days.

^ If the serum is not clear, filter through the coagulum
left after the filtration of bouillon, as suggested by Hill.

^ If 1.25 per cent, glycerin is added also, it seems to pre-
vent the tubes from drying.

CHAPTER VII.

CULTURES OF BACTERIA.

Owing to their small size and to their similarity
of form, the different species of bacteria cannot be
recognized by microscopic examination alone. Re-
course must be had to a study of their biologic
peculiarities. For this purpose it is necessary to
grow them on artificial culture-media. If a single
germ is planted on a suitable nutrient medium,
and is protected from contamination, it multiplies
rapidly and forms a colony. Such a colony is com-
posed of but one species, since all its members are
the descendants of a single germ. Cultures made
from such a colony are known as "pure cultures,"
since they contain but one species. From a study
of the behavior of these pure cultures, under differ-
ent conditions, the diagnostic characters of the
species are determined.

I. BOUILLON CULTURES.

1. Inoculate a tube of bouillon by touching the
culture to be studied with a sterile platinum needle,
and then dipping the needle in the bouillon.

2. Place at the room-temperature or in the incu-
bator, and examine from day to day, and note any
changes as follows :

60

''STAB'' CULTURES. 6 1

{d) Does the bouillon become uniformly clouded,
or only at the surface or bottom ?

{6) Does a pellicle form on the surface or a de-
posit at the bottom ?

Fig. 36. — Method of holding tubes during inoculation.

{c) Is the color, odor, or reaction of the bouillon
changed ?

II. GELATIN OR AGAR CULTURES.

I. <<Stab'^ Cultures/

(i) Sterilize a straight platinum needle and touch
it to the culture to be studied ; hold the gelatin- or
agar-tube upside down, remove the cotton plug,
and stab the needle carefully up through the cen-
ter of the medium nearly to the bottom of the

* " Stab " cultures are usualh^ made in gelatin-tubes;
"stroke" cultures, on agar-tubes.

62

CULTURES OF BACTERIA.

a

tube ; withdraw the needle carefully and replace
the cotton plug.

(2) Allow to grow at the room-temperature, if gela-
tin is used ; at either room-
or incubator-temperature,
if agar is used; examine
from day to day.

(3) Note in stab cult-
ures :

(a) Does the growth ap-
pear along the line of
puncture, at the surface,
or in both places ?

(d) Is the surface growth
abundant or scanty ; does
it spread over the whole
surface or is it confined to
the point of puncture ; is
its margin regular or in-
dented ; is it flat or raised,
dry or moist ; what is its
color, luster ?

(^) I s the medium
changed in color, odor,
or condition ?

(ci) In gelatin cultures,
if liquefaction takes place,
is it at the surface, deep,
or throughout the line of
puncture ? What is the
form of the liquefied area.

The following terms have been suggested by

Fig. 37.— fl!," Stab "cult
ure ; b, "stroke" cult
ure.

STAB'' CULTURES.

Fig. 38. — Various forms of gelatin "stab" cultures:- a,
Bacillus typhosus ; b, Bacterium anthracis ; c, Bacillus
mycoides ; d, Bacillus mesentericus ; e, Bacillus cedematis ;
f, Bacillus radiatus.

Chester^ for describing the characters of gelatin
stab cultures :

L Non-Iiquefyingf Cultures*

Line of puncture ma}' be —

Filiforyn, uniform growth without special charac-
ters.

Nodose, consisting of closeh" aggregated colonies.

Beaded, consisting of looseh^ placed, disjointed

colonies.

Papillate, beset with papillate extensions.

Echiiiate, beset with acicular extensions.

Villous, beset with short, undivided, hair-like ex-

tensions.

Plumose, a delicate feathery growth.

Arborescent, branched or tree-like, beset with branched
hair-like extensions.

^ Eleventh Annual Report of the Delaware College Agri-
cultural Experiment Station, Newark, Delaware, 1898-1899.

64

CULTURES OF BACTERIA.

IL Liquefying Cultures^

Line of liquefaction may be —

Crateriform, a saucer-shaped liquefaction.

Saccate, shape of an elongated sac, tubular,

cylindrical.
Infundibuliform, shape of a funnel, conical.
Napiform, shape of a turnip.

':=JFusiform:^ ' shape of a parsnip, narrow at either

end, broadest below the surface.
Stratiform, liquefaction extending to walls of the

tube and downward horizontally.

Fig. 39. — Microspira comma (Asiatic cholera) : gelatin
puncture cultures aged forty-eight and sixty hours
(Shakespeare).

3. ** Stroke" Cultures.

(i) Sterilize the platinum needle, touch it to the

Fig. 40. — Microspira Finkleri : gelatin puncture cultures
aged fortj-eight and sixt}- hours (Shakespeare).

Fig. 41 . — Microspira Metschnikovi : puncture culture in
gelatin forty-eight hours old (Frankel and Pfeiffer).
6 65

66 CULTURES OF BACTERIA.

culture to be studied, and draw it over the surface
of an agar- or gelatin-tube that has been solidified
with a slanting surface.

(2) Allow to grow as before, and examine from
day to day.

(3) Note :

{a) What are the size and shape of the streak ?
(h) What is the character of the margin ?
{c) Is the growth abundant or scanty, flat or
raised, opaque or transparent ? ^

(d) What is the color, luster ?

(e) Has the color, consistence, or odor of the
medium changed?

III. POTATO AND BLOOD=SERUM CULTURES.

Stroke cultures are employed in using potato or
blood-serum tubes. The methods of study are
essentially the same as described for gelatin or
agar cultures.

IV. PLATE CULTURES.

In making plate cultures the nutrient medium is
liquefied and a very small portion of the culture to
be studied mixed with it ; it is then poured into
sterile Petri dishes, solidified, and the colonies
allowed to develop. By this method the bacteria
are scattered through the medium, and the colonies
that develop are the descendants of a single germ.
This permits different species to be separated from
a mixture in pure culture, and the peculiarities of

^ For descriptive terms, see p. 70, et seq.

PLATE CULTURES.

67

''colonies" in distinction from "cultures" to be
determined.

1. Liquefy three gelatin- or agar-tubes^ by placing
in a water-bath. Gelatin-tubes must not be heated
above 40° C. Agar- tubes may be heated to 100°
C, but must be cooled to 40° C. before the inocu-
lations are made.

2. With a sterile platinum needle transfer a

Fig. 42. — Petri dish with colonies.

minute portion of a culture to the liquefied medium
in the first tube.

3. Shake the tube thoroughly without producing
bubbles in the medium, and transfer a loopful to the
second liquefied tube.

4. Shake the second tube, and transfer a loopful
to the third tube.

^ Bouillon-tubes or tubes of sterile water nia\^ be substi-
tuted for the first two gelatin- or agar-tubes when it is
desired to make but one plate.

68

CULTURES OF BACTERIA.

V. '■?

t'J./

<«iC:«i5SsSS?»«vv >i..ti^ .

r-0

Fig. 43. — Brown University electric water or paraffin

bath.^

^ A porcelain-lined dish fitted with a tin cover, provided
with four doors. In the figure the cover is raised from the
dish to show the thirty candle-power electric lamp which
projects through the cover into the water or paraffin below.
The lamp is provided with a regulating socket giving

PLATE CULTURES.

69

5. Shake the third tube, and pour the contents
of the three tubes into sterile Petri dishes, having
first passed the lip of each tube through the flame.

6. On the surface of the first Petri dish, before

/\^'/l'l\\\A^y

Fig. 44. — The various appearances of colonies of bacteria
under the microscope : a, colony of Bacterium parvum ;
d, colony of Bacillus pol3piformis ; c, colony of Bacillus
radiatus.

the medium has entirely solidified, press a sterile '
cover-glass or small piece of mica.

candle-powers from five to thirty. By adjusting the socket
to the different candle-powers the requisite temperature of
water or paraffin is secured and indicated by the thermom-
eter. When used as a water-bath, the dish is provided
with a wire rack to support the tubes to be melted. When
used for paraffin, little baskets of wire gauze, containing the
specimens to be embedded, are hung about the sides. It
is convenient to have the water-bath kept at a temperature
of 40° C. In this gelatin-tubes may be melted, or agar-
tubes, after being melted at a higher temperature, may be
cooled down and kept melted until used.

* Sterilize M' heating in the flame. Allow it to cool before
placing on the plate.

70 CULTURES OF BACTERIA.

7. Allow to develop as directed for tube cultures,
and examine from day to day, or oftener as necessary.

8. In plate cultures note :

{a) Is there a difference in the number of colonies
in the three dishes? The use of the three tubes
and plates is for the purpose of reducing the num-
ber of colonies, so that only a few will be present
in the third plate.

(fi) Is there more than one kind of colony present ?

Notice that colonies on the surface differ from
those below the surface, though of the same species.
If the culture from which the inoculations wxre
made is a pure culture, and if no germs have been
allowed to enter during the process of making the
plates, there should be but one kind of colony present.

(c) What are the size, shape, texture, and color
of the colonies, both surface and deep? What is
the character of their margins?

The following terms are those suggested by
Chester for the description of colonies :

I. Form.

Ptinctiform^ dimensions too small for defini-
tion.

Roujid^ of a more or less circular outline.

Irregular.

Elliptical.

Fiisifcn-m, spindle-shaped.

Cochleate^ spiral or twisted like a shell.

Ameboid^ very irregular, streaming.

Mycelioid, a filamentous colony with the
radiate character of a mould.

SURFACE ELEVATION.

71

Filamentoiis^ an irregular mass of loosely

woven filaments.
Floccose^ of a dense woolly structure.
Rhizoid^ of an irregular, branched root-like

character.
Conglomerate^ an aggregate of colonies of

similar size and form.
Toruloid, an aggregate of colonies like the

budding of the yeast plant.
Rosidate^ like a rosette.

II. Surface Elevation.

I. General character of a surface as a whole.

Flat^ thin, leafy, spreading over the sur-
face.

Effused, spread over the surface as a thin,
veily layer more delicate than the pre-
ceding.

Raised, growth thick with abrupt terraced

edges.
Co7ivex, surface the segment of a circle,

but very flatly convex.
Pulvinate, surface the segment of a circle,

but decidedly convex.
Capitate, surface vSemi-spherical.
2. Detailed characters of surface.

S^nooth, surface even, without any of the

following distinctive characters.
Alveolate, honeycombed.
Punctate, dotted with punctures.
Bnllate, blistered.
Vesicular, more or less covered with mi-

72 CULTURES OF BACTERIA.

nute vesicles due to gas formation ;
more minute than bullate.

Verrucous., bearing wart-like prominences.

Squa^nose., scaly.

Echinate^ beset with pointed prominences.

Papillate^ beset with nipple-like processes.

Rugose., short irregular folds due to shrink-
age.

Corrugated^ in long folds due to shrinkage.

Contoured., a smoothly undulating surface.

Rimose., abounding in clefts or cracks.

III. Microscopic Structure.

1. Refraction weak. Outline and surface of
relief not strongly defined.

2. Refraction strong. Outline and surface of
relief strongly marked.

{a) Dense, not filamentous colonies.

1. General.

Amoj'phous., without definite structure.

Hyaline^ clear and colorless.

Homogeneous^ structure uniform through-
out all parts of the colony.

Homochromous^ colony of uniform color
throughout.

2. Granulations or blotchings.

Finely gra7iular.
Coarsely graitular.
Grumous^ coarser than preceding.
Moruloid^ segmented.

Clouded^ having a pale ground with ill-
defined patches of a deeper tint.

EDGES OF COLONIES. 73

3. Colony marking or striping.

Reticulate., in the form of a network.
Areolate., divided into rather irregular or

angular spaces.
Gyrose^ marked by wavy lines.
Marmorated^ traversed by vein-like mark-
ings.

Rivulose, marked by lines like the rivers

on a map.
Rimose., showing cracks or clefts.
(b) Filamentous colonies.

Filamentotcs'^ as already defined.

Floccose, composed of filaments densely

packed.
Curled, filaments in parallel strands.

IV. lodges of Colonies.

Entire, without toothing or division.
Undulate, wavy.

Repajtd, like the border of an open um-
brella.
Erose, as if gnawed, irregularly toothed.
Lobate, divided into lobes.
Lobulate, minutely lobate.
Auriculate, with ear-like lobes.
Lacerate, irregularly cleft, as if torn.
Fimbriate, fringed.
Ciliate, hair-like extensions.
Tufted,

Filamentous,
Floccose,
Curled .^

> as already defined.

y

74 CULTURES OF BACTERIA.

V optical Characters.

Transparent.

Vitreous^ transparent and colorless.

Oleaginous^ transparent and yellow.

Resinous., transparent and brown.

Translucent.

Figs. 45, 46. — The various appearances of colonies of
bacteria under the microscope : a colony of Bacillus
muscoides (Liborius) ; b, colony of Bacterium anthracis
(Fliigge).

Porcelaneous, translucent and white.
Opalescent^ translucent, grayish white by
reflected light, smoky brown by trans-

mitted light.

IMPRESSION OR ADHESIVE PREPARATIONS. 75

Nacreous^ translucent, grayish white with
pearly luster.

Sebaceous^ translucent, yellowish or gray-
ish white.

Bittyrous^ translucent and yellow.

Ceraceous^ translucent and wax-colored.

Opaque.

Cretaceous^ opaque and white ; chalky.

Dull^ without luster.

Glistening.

Fluorescent.

Iridescent.

Phosphorescent.

id) Is there growth beneath the cover-glass or
mica plate? This determines, roughly, whether
they require oxygen for their growth or not (aerobic
or anaerobic).

ie) In the gelatin plates is the gelatin liquefied,
and what is the nature of the liquefaction ?

V. IMPRESSION OR ADHESIVE PREPARATIONS OF

COLONIES.

An entire colony of bacteria may be preserved as
a microscopic specimen.

(a) Slightly warm^ a clean cover-glass.

ifi) Lay it upon the surface of the gelatin or agar
containing the colonies. Apply sufficient pressure
to remove all air-bubbles, but not enough to disturb
the colony.

ic) Remove the cover, gently lifting it from one
side. The colonies will adhere to the gflass.

76

CULTURES OF BACTERIA.

(d) Dry, fix, stain, and mount as for ordinary
preparations.

Museum preparations of gelatin or agar tube or
plate cultures may be made by exposing the
cultures to formaldehyd vapor until the growth is
killed, and then sealing the tubes or plates tightly
with sealing-wax or paraffin.

Fig. 47. — Bacterium tuberculosis : adhesive preparation
from a fourteen-da}' blood-serum culture ; x 100 (Frankel
and PfeifFer).

VI. CULTURES IN THE FERMENTATION=TUBE.

This method is for the purpose of studying gas
formation, and for the study of the aerobic or anae-
robic properties of organisms.

{a) Prepare fermentation -tubes with dextrose, lac-
tose, or saccharose bouillon.

ifi) Inoculate the tubes by floating a little of the

CULTURES IN THE FERMENTATION-TUBE. //

culture to be studied ou the fluid in the bulb with
a sterile needle.

ic) In the case of gas formation, at the end of
every twenty-four hours, for several days, mark the
level of the fluid in the closed branch upon the
tube or measure it with a millimeter scale.

{d) Record the result in percentages of the length

Fig. 48. — Bacterium anthracis : colony three days old
upon a gelatin plate ; adhesive preparation ; x 1000 (Frankel
and PfeifFer).

of the closed branch. If i cm. of gas forms in a
10 cm. tube, 10 per cent, of gas is said to have
formed.

{e) To test the relative amount of carbon dioxid
and hydrogen present. Fill the bulb completely
with a 2 per cent, solution of sodium hydroxid.
Place the thumb over the mouth of the bulb, and

78 CULTURES OF BACTERIA.

run the mixture six or eight times through the
length of the tube, returning the remaining gas to
the closed branch before removing the thumb.
Measure the amount of gas remaining ; the differ-
ence between this and the former measurement
shows in millimeters the amount of carbon dioxid
absorbed by the alkali. The remaining gas, mostly
hydrogen, may be transferred to the bulb and
exploded by a flame. The proportion of hydrogen
to carbon dioxid is usually expressed in the form

of a fraction called the gas formula, — -.

The fermentation-tube affords a ready method of
determining the oxygen requirement of bacteria.
Growth, indicated by cloudiness, in the bulb only,
is to be found only among obligatory aerobes ;
in the closed branch only, among obligatory anae-
robes ; while growth in both, only among the
facultative anaerobes.

VII. ANAEROBIC CULTURES.

Growth under the mica plate or cover-glass^ and
in the fermentation-tube are methods for the deter-
mination of the aerobic properties of organisms.
For the growth of strictly anaerobic forms special
methods have been devised :

1. Place the cultures in a vessel from which the
air can be withdrawn and hydrogen substituted.

2. Buchner^s Method. — Use two test-tubes, one
inside the other. The outer one is partially filled

1 See page 75.

ANAEROBIC CULTURES.

79

with pyrogallic acid made alkaline with sodium
hydrate, and is sealed tightly with a rubber stopper.
The inner tube contains the culture. The oxygen
is absorbed by the mixture in the outer tube.

3. Wright^s Method. — Make an ordinary cult-
ure in a test-tube. Clip off any superfluous cotton
from the plug, and push the plug into the tube so

Fig. 49. — Novy's jars for anaerobic cultures.

that it lies i centimeter below the mouth. For
test-tubes 6 X ^ inches, run into the cotton plug,
from a pi pet, approximately Yz c.c. of a freshly
prepared solution of pyrogallic acid (i part of acid,
I part of water), and then approximately i c.c. of
a solution of sodium hydrate (i part of sodium
hydrate, 2 parts of water). Quickly insert a rubber
stopper into the tube.

4. Make a hanging-drop culture. On one side
of the cover-glass introduce a little pyrogallic acid,
and on the other side a little sodium hydrate, so
that it runs around and unites with the acid. Seal
with vaselin.

8o CULTURES OF BACTERIA.

5. Distribute the germs to be studied in bouillon
or in liquefied gelatin or agar, and draw some of
the solution into sterile pieces of glass tubing of
small caliber. When the tube is full seal the ends
in a flame.

6. Put large quantities of culture-medium in the
tubes and puncture deeply. The surface of the
medium is then covered with sterile oil.

7. Parkas Method. — Cover the culture-medium
with melted paraffin.^ Sterilize by the ordinary
methods, and when cool enough for inoculation,
but before the paraffin solidifies, inoculate through
the paraffin into the medium below.

Wright recommends^ the following precautions
in growing anaerobic bacteria :

1. The medium should contain i per cent, of glu-
cose, and should be boiled and cooled immediately
before inoculation.

2. The medium should be freshly prepared.

3. The reaction should not be more acid to
phenolphthalein than +1.5. With i per cent,
glucose bouillon, growth is better if the reaction is
nearer the neutral point of phenolphthalein than
+ 1.5.

VIII. DEMONSTRATION OF LIQUEFYING FERMENT.

(a) Inoculate several gelatin-tubes with Bacillus
prodigiosus.

(fi) Allow them to grow until all the gelatin is
liquefied.

* Melting point, 42° C.

"^Jour. Boston. Soc. Med. Sci., vol. v., No. 4, p. 114.

ISOLATION OF SPECIES. 8i

{c) Add yi c.c. of chloroform, or 5 per cent, car-
bolic acid, to each tube, shake thoroughly, and
filter.

(d) Add the filtrate, now containing no living
bacteria, to tubes of sterile gelatin. Note the
liquefaction that takes place, caused by the fer-
ment produced by the bacteria in the first set of
tubes.

IX. ISOLATION OF SPECIES.

Given a bouillon culture, containing three species
of bacteria, to isolate the species.^

{a) Liquefy three gelatin or agar tubes and
number i, 2, and 3 respectively.^

(h) Transfer a minute loopful of the bouillon cult-
ure to tube No. I.

(c) Shake thoroughly, and transfer 2 loopfuls
from tube No. i to tube No. 2.

(ci) Shake and transfer 3 loopfuls from tube No.
2 to tube No. 3.

{e) Flame the lips of the tubes and pour their
contents into sterile Petri dishes.

(y) Examine in twelve to twenty-four hours.

(^g) Select the dish in which the colonies are
well developed, and in which they have not run
together ; look for three kinds of colonies.

If more than three kinds are present, it shows

^ This method is applicable to the separation of species
from any fluid.

* Tubes of bouillon or sterile water may be substituted
for tubes Nos. i aud 2, in which case, of course, but one
plate can be made — i. e., from tube No. 3.
6

97'7S

82 CULTURES OF BACTERIA.

that others have been allowed to enter through
carelessness in manipulation.

(//) Record the appearance of the different colo-
nies, and inoculate tl^em as pure cultures in tubes
of culture-media. Study them as directed in the
following chapter.

CHAPTER VIII.
DETERMINATION OF SPECIES.

I. MORPHOLOGY AND LIFE=HISTORY OF A SPECIES.

The following points in the morphology and life-
history of any form of bacterium must be known
before it can be fully described or assigned a place
in any particular species.^

I. Source and Habitat.

II. Morphologic Characters.

1. Form.

2. Dimensions.

3. Manner of grouping and arrangement in the
growths.

4. Staining powers : [a) with aqueous stains; {d) by
Gram's method.

5. Presence or absence of capsule.

6. Presence or absence of flagella (motility).

7. Spore-formation.

8. Tendency to pleomorphism.

9. Involution and degeneration forms.

III. Biologic Characters.
A. Cultural characteristics, mode of growth in and
upon —

1. Bouillon.

2. Gelatin plates (single colonies, surface and deep).

3. Gelatin-tubes.

4. Agar plates (single colonies, surface and deep).

^ These are the points recommended by the Committee
of Bacteriologists of the American Public Health Associa-
tion.

84 DETERMINATION OF SPECIES.

5. Agar-tubes.

6. Potato.

7. Milk.

8. Blood-serum.

B. Biochefnic features.

1. Temperature relationship (activity of growth at
i8°-22° C. and at 36°-38° C. and thermal death-point).

2. Relation to free oxygen (aerobic and anaerobic
growth).

3. Relation of growth to acidity and alkalinity of
media.

4. Action upon gelatin (presence or absence of lique-
faction).

5. Action upon proteids (milk and serum).

6. Action upon carbohj^drates (fermentation and gas
formation).

7. Action upon nitrates.

8. Production of indol.

9. Production of acid or alkali.

10. Pigment formation.

11. Development of odor.
C Pathogenesis.

The following tests are of value in certain cases :

I. Morphologic.

1. Staining reactions with special stains.

2. Study of flagella by special stains.

3. Permanency of morphologic characters after long-
continued growth and successive transplantation upon
artificial media.

4. Photographic reproductions of isolated bacteria.

5. Cover-glass impressions.
II. Physiologic.

A. Cultural characteristics, mode of growth in or upon —

1. Litmus gelatin.

2. Loffler's blood-serum.

3. Synthesized media.

4. Photographic reproduction of characteristic cultures.

STUDY OF FORM AND GROUPING. 89

B. Biochemic.

1. Minimum, optimum, and maximum temperatures
of growth.

2. Growth in atmospheres of various inert gases (when
anaerobic power of growth has been determined).

3. Optimum reaction of media and reaction limits, acid
and alkaline (indicated by phenolphthalein).

4. Chemic properties and solubility of pigments pro-
duced, and spectroscopic observations upon the pigment.

C. Pathogenesis.

1. Inoculation of various species of animals, with
minute study of the pathologic changes produced.

2. Immunity-producing properties.

3. Agglutinating properties of specific sera.

4. Determination and isolation of toxic substances
(from non-pathogenic, as well as from pathogenic, bacteria).

A suitable blank should be prepared, on which
are recorded the observations made on each species.
The appended form (pages 86, 87) is substantially
the one recommended by the Bacteriologic Com-
mittee of the American Public Health Associa-
tion.

A further explanation in regard to some of these
points is required.

(a) Study of Form and Grouping. — Determine
and describe the morphology from the growth
obtained upon at least one solid medium and in at
least one liquid medium. Growth at 36°-38° C.
should in oreneral be not older than from twentv-
four to forty-eight hours, while growth at room-
temperature (i8°-22° C.) should be not older than
from forty-eight to seventy-two hours. Growth on
solid media may be studied from cover-glass prep-
arations ; in liquid media growth is best observed

90 DETERMINATION OF SPECIES.

in the hanging-drop, preferably in a fresh medium
inoculated with a very small amount of the culture
to be examined. It is desirable that the form and
grouping be determined in bouillon, gelatin, and
on agar, and that any variation found upon the
examination of the growth on other media be accu-
rately noted.

(b) Test for Motility. — For the study of motility
the hanging-drop preparations should be made from
young cultures grown at or near the optimum
temperature for only a few (six to eighteen) hours.

(c) Tests for Spores. — The tests for the presence
of spores are :

{a) Do colonies develop from cultures which have
been subjected to a temperature of 80° C. for ten
minutes ?

{b) Are there highly refracting bodies within the
bacteria in unstained preparations, and can they be
demonstrated by the spore-staining methods ?

Cultures to be tested should be grown for forty-
eight hours in bouillon, and when possible at
36°-38° C. Three loopfuls of this culture after
agitation are transferred to tubes containing 10 c.c.
of bouillon. This is exposed to a temperature of
80° C. for ten minutes, and then placed under con-
ditions favorable for the development of any of the
organisms which may have survived.

(d) Pleomorphism. — In regard to pleomorphism,
attention is called to the variations in size and shape
broug^ht about by the following^ conditions of
growth :

{a) At different temperatures.

ACIDITY AND ALKALINITY OF MEDIA. 9I

(h) Upon or in media of different composition.

{c) Upon or in media of different degrees of
acidity and alkalinity.

{d) In cultures of different ages.

(e) As well as to the variations in the size and
shape of different individual bacteria obtained from
one culture and appearing often in the same field
of view — i. e.^ subjected to exactly the same condi-
tions of growth.

(e) Determination of the Thermal Death-
point. — In determining the thermal death-point,
the facts required to be known are (i) the time of
exposure to heat, (2) the presence or absence of
moisture, (3) the presence or absence of spores, (4)
the age of the culture, (5) the amount of the culture
used for the tests, and (6) the character of the con-
taining vessel.

The temperature required to destroy the species
imder consideration is to be determined within 2
degrees C. ; thus, if samples are exposed to temper-
atures of 50°, 52°, 54°, 56°, 58°, and 60° C, and it
is found that development in a suitable medium
occurs after exposure to 56° C, but not after expos-
ure to 58° and 60° C, the thermal death-point is
to be given as 58° C. , although further study
might show it to be somewhat less than this.

(f) Relation to Free Oxygen. — For the
methods of determining the aerobic properties of
organisms, see under Anaerobic Cultures, on page
78.

(g) Relation to Acidity and Alkalinity of
Media. — In determining the relation of growth to

92 DETERMINATION OF SPECIES.

acidity and alkalinity of media, all that is necessary
is to add to tubes containing equal quantities of
any of the usual media a calculated amount of a
standardized solution of hydrochloric acid or of
sodium hydroxid to obtain the desired reaction. A
record should be made of cultures upon at least one
medium reacting +3 per cent., +1.5 per cent,
neutral, and —1.5 per cent, to phenolphthalein.

(h) Action upon Carbohydrates. — For the
methods of determining the action upon carbo-
hydrates and for measuring the gas production, see
under Cultures in the Fermentation-tube, page 76.

(i) Action upon Nitrates. — To determine the
power of certain bacteria to reduce nitrates to
nitrites and ammonia, incubate them for seven
days at 20° C. in the followino; :

Nitrate Broth.

Peptone, i gm. ;

Potassium nitrate, 0.2 gm. ;

Tap water, ^ 1000 c.c.

Submit the inoculated tubes and also several un-
inoculated control-tubes to the following tests for
nitrites :

Prepare two solutions :

I. Naphthylamin, o.igm. ; ^

Distilled water, 20 c.c.

Boil until the naphthylamin is dissolved ; cool,

' Better than distilled water because of the other salts
present, which favor the growth of the bacteria.

ACTION UPON NITRATES. 93

filter, and add the filtrate to 150 c.c. of dilute (i to
16) hydric acetate.

II. Sulphanilic acid, 0.5 c.c; v^

Dilute (i to 16) hydric acetate, 150 c.c.

Keep these solutions in separate glass bottles,
tightly stoppered, and mix in equal parts before
use.

To 3 c.c. of the solution to be tested, in a per-
fectly clean test-tube, add gradually 2 c.c. of the
test-solution. A red color develops of an intensity
in proportion to the amount of nitrites present.
The appearance of the color may be hastened by
heating.

If this test shows the presence of nitrites, test
one-half of the remaining solution for ammonia
with Nessler's solution.^

The presence of ammonia is shown by the im-
mediate development of a yellow color or precipi-
tate on the addition of a few drops of the test-solu-
tion.

When these tests are positive our inquiry has
been answered. When nes^ative the nitrates mav
have remained unchanged or may have been re-
duced to free nitrogfen. It is therefore necessarv to
determine whether the nitrates are still present or
not, as follows :

{a) Invert the tube containing the culture and
evaporate to dryness the small amount of culture
remaining on the inside of the tube.

* See Appendix, page 174.

94 DETERMINATION OF SPECIES.

(b) Add, in order, phenol-sulphouic acid, ^ water,
and sodium hydrate. A yellow color shows the
presence of nitrates.

( j) Acid or Alkali Production. — For the deter-
mination of acid or alkali production, cultures are
made on solid media to which have been added i
per cent, of dextrose, saccharose, or lactose, and a
sufficient quantity of litmus solution to produce a
blue tinge. A pink coloration of the colony or a
reddening of the surrounding medium indicates
acid production.

(k) Test for Indol Production. — For the deter-
mination of indol production, incubate the species
to be tested in the dextrose-free bouillon described
on page 56. Add to the tube of culture i c.c. of a
0.01-0.02 per cent, solution of sodium or potassium
nitrite. To each 10 c.c. of medium add one drop
of concentrated sulphuric acid. A red coloration
indicates indol. If no result is obtained at once, it
is well to allow the tube to stand for one hour.

Pathogenesis. — In Chapter VIII. are described
the various methods of determining the pathogenic
properties of bacteria.

1. When a given form grows only at or below
18° to 20° C. inoculation should be made into the
dorsal lymph-sac of a frog, using about i per cent,
of the body-weight of a bouillon culture seven
days old.

2. When a given species grows at 35° C. or up-
ward, inoculation should be made into the peri-

^ See Appendix, page 174.

DETERMINATION OF SPECIES. 95

toneal cavity of a white or ordinary house mouse,
using I per cent, of the body-weight of a forty-eight
hour bouillon culture. If the mouse is killed, it is
well to try the culture on guinea-pigs or rabbits.
A careful autopsy should be made in all cases, cult-
ures taken from the organs, and all pathogenic
changes noted.

n. DETERMINATION OF THE NAME OF A SPECIES.

When the points mentioned at the beginning of
this chapter have been determined, the name of
the species, if it has been described and named,
can be found by referring to the bacterial analysis
tables in Migula's System der Bakte7'ieii^ Stern-
berg's Manual of Bacteriology^ Fliigge's Die
Mikroorganisinen^ or Chester's "Studies in Sys-
tematic Bacteriology" in the ninth, tenth, and
eleventh Reports of the Delaware College Agri-
cultitral Experi77ient Station^ 189?? 1898, and 1899.

The following table, adapted from Migula, may
assist in the determination of the name of a species :

BACTERIA.
T. Family. Coccaceae.
I. Genus. Streptococcus.
I. Grow on gelatin.
I. Colonies white.

A. Do not liquefy gelatin.

(ci) No growth on the surface in gelatin
stab cultures.
S. pyogenes.
S. equi.
;•>. ^^•^titidis.

96 DETERMINATION OF SPECIES.

S. urinse.
S. acidi lactici.
{b) Growth on the surface and along the
puncture in gelatin stab cultures.
S. mastitidis.
S. mirabilis.
B. Liquefy gelatin.
S. septicus,
S. morbi brightii.
S. gracilis.
S. vermiform is.
S. albus.
2. Colonies colored.
S. cerasinus.
S. citreus.
II. Do not grow on gelatin.
S. giganteus.
2. Genus. Micrococcus.
I. Grow on gelatin.
I. White.

A. Do not liquefy gelatin.
M. candicans.
M. tardissimus.
M. tardus.
M. plumosus.
M. viticulosus.
M. stellatus.
M. tenuissimus.
M. albocereus (cereus albus).
M. urese.
M. coryzae.
M. salivarius.

DETERMINATION OF SPECIES. 97

M. lacticus.
M. acidi lactici.
M. phosphoreus.
M. catarrhalis.
M. bovis.
M. similis.
B. Liquefy gelatin.
M. pyogenes.
M. ovis.
M. foetidus.
M. conoideus.
M. liquefaciens.
M. radiatus.
M. faviformis.
M. ampins.
M. dissimilis.
2. Yellow.

A. Do not liquefy gelatin.
M. aurantiacus.

M. tardigradus.
M. Inteus.
M. cereus.
M. versicolor.
M. varians.

B. Liquefy gelatin.

M. aureus (S. pyogenes aureus).

M. beri-beri.

M. fuscus.

M. coronatus.

M. conjunctivitidis.

M. tardus.

M. flavus.

98 DETERMINATION OE SPECIES.

M. desidens.
M. conglomeratus.
M. citreus (S. pyogenes citreus).
M. citrinus.
M. corrugatus.
M. mollis.
3. Red.

A. Do not liquefy gelatin.
M. cinnabareus.

M. carneus.

B. Liquefy gelatin.
M. roseus.

M. rosaceus.
M. carnicolor.
M. fragilis.
4. Blue and violet.
M. violaceous,
M. cyaneus.
II. Do not grow on gelatin.
M. gonorrhoeae.
M. intracellularis.
M. subflavidus.
M. rugatus.
M. cuniculorum.
M. progrediens.
M. tenuis.
M. nitrosus.
M. foetidus.
3. Genus. Sarcina.
I. Grow on gelatin.
I. White.
Sarcina alba.

DETERMINATION OF SPECIES. 99

2. Yellow.

A. Do not liquefy gelatin.
Sarcina lutea.
Sarcina ventriculi.

B. lyiquefy gelatin.
Sarcina liquefaciens.
Sarcina aurautiaca.

II. Family. Bacteriaceae.
I. Genus. Bacterium.

1. Form spores.

I. Spores polar.
Bact. anthracis.
Bact. anthracoides.
Bact. subtile.

2. Spores central.
Bact. carotarum.
Bact. angulans.

3. Position of spores undetermined.

A. Grow on gelatin at the room-temperature.
(a) Colonies white.

1. Do not liquefy gelatin.
Bact. acidi lactici.
Bact. coprogenes.

2. Liquefy gelatin.

(i) Center of colonies plainly floc-
cose.

Bact. vermiculare.
(2) Colonies not floccose.

Bact. tricomii.

Bact. nephritidis.

Bact. sempervivum.

Bact. lacteum.

100 DETERMINATION OF SPECIES.

(b) Colonies colored.
Bact. bninneum.
B. Do not grow on gelatin at the room-
temperature.
Bact. termophilum.
II. Spore formation not observed.
I. Grow well on gelatin.
A. Form no pigment.

{a) Do not liquefy gelatin.

1. Stained by Gram's method.
Bact. pneumoniae.

Bact. proteus.
Bact. rhusiopathise.
Bact. murisepticum.
Bact. lacticum.
Bact. parvum.

2. Do not stain by Gram's method.
Bact. pneumonicum.

Bact. rhinoscleromatis.
Bact. faschingii.
Bact. endocarditidis.
Bact. cuniculicidi.
Bact. suicida.
Bact. palumbarium.
Bact. ribberti.
Bact. cuniculi.
Bact. pseudotuberculosis.
Bact. columbarum.
Bact. aerogenes.
Bact. aceti.
Bact. salivae.
3. Gram's stain undetermined.
Bact. capsulatum.

DETERMINATION OF SPECIES. 10 1

Bact. keratomalacise.

Bact. felis.

Bact. pyogenes.

Bact. welchii (aerogenes capsu-
latus).

Bact. bienstockii.

Bact. laerii.

Bact. ubiquitum.

Bact. candicans.

Bact. phosphorescens.

Bact. giardi.
{b) Liquefy gelatin.
Bact. bovis.
Bact. vignali.
Bact. varicosum.
Bact. buccale.

B. Colonies yellow.

{a) Do not liquefy gelatin.

Bact. erythrogenes.

Bact. citreum.

Bact. aurescens.
{b) Liquefy gelatin.

Bact. arborescens.

Bact. aquatile.

Bact. chlorinum.

Bact. aureum.

C. Colonies red.
Bact. mycoides.

Bact. pyocinnabareum.

D. Colonies blue or violet.
Bact. coeruleum.

Bact. amethystinum.

102 DETERMINATION OF SPECIES.

2. Do not grow well on gelatin at room-
temperature.
Bact. tuberculosis.
Bact. tuberculosis avium.
Bact. leprae.
Bact. syphilidis.
Bact. smegmatis.
Bact. mallei.
Bact. diphtheriae.
Bact. xerosis.

Bact. pseudodiphtheriticum.
Bact. vaginae.
Bact. influenzae.
Bact. pseudoinfluenzae.
2. Genus. Bacillus.
I. Form spores.

1. Spores polar.
B. ramosus.

2. Spores central or oblique.
B. subtilis.

B. megaterium.

3. Position of spores undetermined.
A. White or dirty white on gelatin.

{a) Do not liquefy gelatin.
B. spermophilinus.
B. polypiformis.
B. muscoides.
{b) Liquefy gelatin.

I. Stain by Gram's method.
B. mycoides.
B. gracilis.
B. granulosus.

DETERMINATION OF SPECIES. IO3

B. tetani (anaerobic).
2. Gram's stain undetermined.

B. cereus.

B. laevis.

B. vermicularis.

B. thalossophilus.

B. intricatus.

B. limophilus.

B. circulans.

B. pseudanthracis.

B. globigii.

B. mesentericus.

B. vulgatus.

B. sporogenes.

B. liodermos.

B. albolactus.

B. butyricus.

B. chauvsei (anaerobic).

B. radiatus (anaerobic).

B. cedematis.
B. Form a black pigment on gelatin.
B. aterrimus.
B. niger.
II. Spore formation not observed.

I. Grow on gelatin. Not phosphorescent.
A. Colonies white.

{a) Do not liquefy gelatin.

1. Stain by Gram's method.
B. zenkeri.

B. muripestifer.

2. Do not stain by Gram's method.
B. typhosus.

I04 DETERMINATION OF SPECIES.

B. coli.
B. pestis.
B. avium.
B. suipestifer.
B. zeae.
B. glacialis.
3. Gram's stain undetermined.
B. murium.
B. solanacearum.
B. phaseoli. ,

B. amylovorus.
B. sorghi.
B. zopfii.
{b) lyiquefy gelatin.

1. Stain by Gram's method.
B. dysenteriae.

2. Do not stain by Gram's method.
B. pseudotuberculosis.

B. ozsense.
B. vulgaris.
B. halophilus.

3. Gram's stain undetermined.
B. arthuri.

B. sulfureus.
B. liquidus.
B. diffusus.
B. nubilus.
B. reticularis.
B. diaphanus.
B. mirabilis.
B. gasoformans.
B. delicatulus.

DETERMINATION OF SPECIES. I05

B. cloacae.
B. hyalinus.
B. superficial is.

B. Colonies yellow.
B. arborescens.

C. Colonies red.
B. prodigiosus.
B. indicus.

B. plymouthensis.
B. rubescens.
^. Grow on gelatin. Phosphorescent.
B. phosphorescens.
B. fischeri.
B. phosphoricus.
3. Do not grow on gelatin.
B. equi.
3. Genus. Pseudomonas.
I. Grow on gelatin.

1. Colonies white. Form no pigment.
P. litoralis.

2. Form fluorescent pigment.

A. I}o not liquefy gelatin.
P. alba.

P. tenuis.

P. eisenbergii.

P. stewarti.

B. Liquefy gelatin.

P. aeruginosa (B. pyocyaneus).
P. fluorescens.
P. minutissima.

3. Colonies blue or violet.
P. ianthina.

I06 DETERMINATION OF SPECIES.

P. pseudianthina.
P. laurentia.
4. Phosphorescent species.
P. javanica.
11. Do not grow on gelatin.
P. europsea.
P. javaniensis.
III. Family Spirillacese.

1. Genus. Spirosoma.
Spirosoma nasale.
Spirosoma linguale.
Spirosoma aureum.
Spirosoma flavum.
Spirosoma flavescens.
Spirosoma attenuatum.
Spirosoma gregarium.

2. Genus. Microspira.

I. Not phosphorescent.

1. Do not liquefy gelatin.
Microspira canalis.
Microspira saprophiles.
Microspira tonsillaris.

2. lyiquefy gelatin.
Microspira comma.
Microspira metschnikovi.
Microspira finkleri.
Microspira sputigena.
Microspira marina.

II. Phosphorescent.
Microspira dunbari.
Microspira coronata.
Microspira annularis.

CLASSIFICATION BY GROUPS. 107

Microspira glutinosa.
Microspira delgadensis.
Microspira tuberosa.
Microspira degenerans.
Microspira luminosa.
Microspira caraibica.
Microspira papillaris.

III. CLASSIFICATION OF BACTERIA BY GROUPS.

Systematic bacteriology is at present in a state
of chaos. Many times the most that can be done
in attempting to classify a new species, is to refer
it to some group, the members of which have
certain characters in common, and are probably
the descendants of one ancestral type. Chester has
proposed ^ a synopsis of several groups of bacteria
which, slightly modified, is given below.

BACTERIUM.

I. Spore- formers.

r. No growth at room-temperature or below

22°-25° C.

Thermophilic Group.
Bact. termophilum type.
2. Grow at room-temperature.

A. Do not liquefy gelatin.

Bact. f^calis Group.
Bact. subtile type.

B. Liquefy gelatin.

* Eleveyith Anmial Report of the Delaware College Agri-
cultural Experiment Station for 1898-99.

io8 determination of species.

Anthrax Group.
Bact. anthracis type.
II. Spore formation not observed. Aerobic and
facultative anaerobic.

1. Do not liquefy gelatin.

A. Do not stain by Gram's method.

{a) Obligate aerobic.
Acetic Ferment Group.
Bact. aceti type.
(b) Aerobic and facultative anaerobic.

1. Gas generated in glucose bouillon.

a. Gas generated in lactose bouil-

lon.
Bact. aerogenes Group.
Bact. aerogenes type.

b. Little or no gas generated in

lactose bouillon.
Friedlander Group.
Bact. pneumonicum type.

2. No gas generated in glucose

bouillon.

a. Coagulate milk.
Fowl-cholera Group.

Bact. cuniculcida type.

b. Do not coagulate milk.
SwiNE-PLAGUE Group.

Bact. suicida type.

B. Stain by Gram's method.

Gas generated in glucose bouillon.
Lactic Ferment Group.
Bact. lacticum type.

2. Liquefy gelatin.

classification by groups. 1 09

Glanders Group.
Bact. mallei type.
3. Do not grow well on gelatin at room-tem-
perature.
A. Stain with basic aniline dyes, but are
easily decolorized by mineral acids when
stained with carbol-fuchsin.
{a) Grow well in bouillon at body-temper-
ature and stain by Gram's method.

Diphtheria Group.
Bact. diphtherise type.
(b) Do not grow in bouillon or on ordinary
media.

1. Rods slender.

a. Stain by Gram's method.

Leprosy Group.
Bact. leprae type.

b. Do not stain by Gram's method.
Influenza Group.

Bact. influenzae type.

2. Rods variable.

Root-tubercle Group.
B. Do not stain with aqueous solutions of

basic anilins and are not easily

decolorized by acids.
Tubercle Group.

Bact. tuberculosis type.

BACILLUS.
I. Spore formers.

I. Aerobic and facultative anaerobic. Rods not
swollen at sporulation.

no DETERMINATION OF SPECIES.

A. Liquefy gelatin slowly.

Urobacillus Group of Miquel.

B. Liquefy gelatin quickly.

{a) Potato cultures rugose.

Potato-bacillus Group.

B. mesentericus type.
{b) Potato cultures smooth.

B. suBTiLis Group.

B. subtilis type.
2. Obligate anaerobic.

A. Rods not swollen at sporulation.

Malignant Edema Group.

B. chauvsei type.

B. Rods clavate at sporulation.

Tetanus Group.
B. tetani type.
IL Spore formation not observed.
I. Aerobic and facultative anaerobic.
A. Gelatin colonies roundish, not distinctly
ameboid.
{a) Do not liquefy gelatin.

I. Do not stain by Gram's method.
a. Generate gas in glucose bouillon,
(i) Coagulate milk.

Colon Group.

B. coli type.
(2) Do not coagulate milk.

Hog-cholera Group.
B. suipestifer type.

CLASSIFICA TION BY GRO UPS. 1 1 1

b. Do not generate gas in glucose bouil-
lon.

Typhoid Group.
B. typhosus type.
2. Stain by Gram's method.

B. MURIPESTIFER GrOUP.
B. muripestifer type.
{b) Iviquefy gelatin and generate gas in glu-
cose bouillon.

B. CLOACA Group.

B. cloacae type.
B. Gelatin colonies ameboid or irregular.
{ci) Do not liquefy gelatin.

B. zoPFi Group.

B. zopfi type.
(fi) Iviquefy gelatin.

Proteus Group.
B. vulgaris type.

MICROSPIRA.

I. Not phosphorescent.

1. Do not liquefy gelatin, or only slightly.

MSP. SAPROPHILES GrOUP.

Msp. saprophiles type.

2. Liquefy gelatin.
A. Produce indol.

{a) Very pathogenic to pigeons.

Msp. metschnikovi Group.
Msp. metschnikovi type.

112 DETERMINATION OF SPECIES.

{h) Not distinctly pathogenic to pigeons.

Cholera Group.
Msp. comma type.
B. Do not produce indol, or very little, at
least after twenty-four hours.

Cholera-nostras Group.
Msp. finkleri type*

IV. CLASSIFICATION OF WATER BACTERIA BY

GROUPS.

Fuller and Johnson have applied the group
method of classification to the bacteria of water as
follows : ^

WATER BACTERIA.

I. Fluorescent. Group I.

II. Non-fluorescent.

1. Chromogenic.

A. Red. Group II.

B. Orange. Group III.

C. Yellow. Group IV.

D. Violet. Group V.

2. Non-chromogenic.

A. Gelatin liquefied.

{a) Characteristic colo-
nies on gelatin
plates.

^ Jour, of Exp. Med., vol. iv., Nos. 5-6, p. 609. Conn
has adopted a somewhat similar classification into groups
for the dairy bacteria. See Report of the Storrs (Connecti-
cut) Agrictiltural Experimeiit Statioji for 1899.

CLASSIFICATION BY GROUPS. II3

1. Proteus forms. Group VI.

2. Subtilis forms. Group VII.
{b) Non-characteristic

colonies on gelatin
plates.

1. Fermentation of

carbohydrates.

a. Gas production. Group VIII.

b. No gas production. Group IX.

2. Non-fermentation

of carbohydrates. Group X.
B. Gelatin not liquefied.

{a) Fermentation of car-
bohydrates.

1. Gas production. Group XL

2. No gas production. Group XII.
{b) Non-fermentation of

carbohydrates. Group XIII.

CHAPTER IX.

BACTERIAL ANALYSIS OF WATER. MILK, AIR,

AND SOIL.

WATER ANALYSIS.

The biologic examination of water is for the
purpose of determining the number and kinds of
organisms present. It serves to supplement chem-
ical analysis, and both are necessary to arrive at a
sound conclusion as to potability.

I. Quantitative Analysis of Water. — The num-
ber of bacteria present in water varies within wide
limits without affecting the value of the water ;
consequently no fixed standard can be of much
value in determining the quality. The proper
study of a water-supply should include the deter-
mination of its normal mean number of bacteria at
every season of the year. Any variation from the
mean can then readily be determined, and its cause
investigated. If simultaneous analyses of waters
from various sources are made, the preferable water
for drinking-purposes can easily be selected.
Beyond this the value of the water can be deter-
mined by quantitative analysis alone only within
wide limits. Water containing less than loo bac-
teria per cubic centimeter is presumably from a
deep source and uncontaminated by surface drain-
age. It can usually be recommended for drinking-

114

WATER ANALYSIS. II5

purposes. Water containing more than 500 per
cubic centimeter should be looked upon with sus-
picion.

3. Qualitative Analysis of Water. — The quali-
tative examination of water requires not only the iso-
lation of the several species present, but also their
cultivation and the determination of their patho-
genic or non-pathogenic properties. Such an
examination takes a long time, and under most
favorable circumstances it is very difficult to
recognize the presence of pathogenic forms.

The principal value of the qualitative analysis of
water is in the detection of contamination by sew-
age. Sewage is always liable to contain the evacu-
ations of patients sick with typhoid fever or other
transmissible diseases. The germs of typhoid fever
are not easily identified, but there are certain bacteria
common in human and other animal evacuations
and in sewage (B. coli, B. vulgaris, B. cloacae, B.
sporogenes, Bact. aerogenes) whose presence is
easily detected. Consequently the presence of such
forms, though harmless in themselves, always indi-
cates contamination.

3. lyaboratory Work in Water Analysis. — All
samples should be collected in sterile flasks, and
cultures should be made immediately to secure ac-
curate results. If transportation is necessary, the
samples should be packed in ice. Tap water should
be allowed to run a few minutes before the sample
is taken ; if spring or well water is to be examined,
the sample should be collected from about a foot
below the surface.

ii6

BACTERIAL ANALYSIS.

(a) Transfer i c.c. of the water to be examined,
by means of a sterile graduated pipet, to each of
three tubes of liquefied gelatin.^ Use a sterile pipet
for each transfer.

(d) Shake the tubes, flame the lips, and pour into
sterile Petri dishes. Place these in the dark at
20° C.

(c) Examine the plates from day to day, and
count the colonies that appear.

Fig. 50. — Simple microscope for counting colonies.

It is best to count all the colonies if possible ; but when
they are very numerous, some one of the various methods
devivSed for counting colonies must be emplo^^ed.

(i) Wolfhiigel's counting-plate consists of a glavSS plate
on which are ruled square centimeters. The Petri dish is

* If the water is suspected of containing large numbers
of bacteria, smaller quantities than i c.c. should be added
to each tube, or, better, dilute the water by the addition of
a known quantity of sterile water. INIany workers prefer
to mix the water and the gelatin in the Petri dish instead
of in the tube.

WATER A A' A LYSIS.

117

placed thereon and the number of colonies in several of the
square divisions counted, the average taken, and the

Fig. 51. — Wolfhiigel's apparatus for counting colonies of

bacteria upon plates.

number in the whole dish estimated. A lens is used in
counting the colonies.

Fig. 52. — Jeffer's plate for counting colonies in circular
dishes. The area of each division is i square centimeter.
8

Il8 BACTERIAL ANALYSIS.

(2) Colonies in circular dishes may be counted by means
of Jeffer's plate. In this each circle is marked with its
area in square centimeters, and each division equals i
square centimeter.

(3) The colonies appearing under the microscope in the
field of a low-power objective may be counted, in several
parts of the dish, the average taken, and the number in the
whole dish estimated by the equation :

Number of colonies in the field ^ {\ diam. of field) '
Whole number of colonies {\ diam. of dish) •^

{d) If a qualitative analysis is required, isolate
and cultivate the different kinds of colonies.'

4. Test for the Presence of Bacillus coli. —
(a) Prepare 10 fermentation-tubes of sterile bouil-
lon containing i per cent, glucose.

(b) To each tube add i c.c. of the water to be
tested.

{c) Place the tubes in the incubator at 37.5° C.
for three days.

id) Note the amount of gas which forms on each
of the three days.

If gas-forming bacteria are present, gas will col-
lect in the closed tube. The number of tubes
showing the presence of gas gives a rough idea of
the number of gas-producing bacilli present.

Bacillus coli, if present, will fill the closed tube
by the second day. Too little or too much gas
does not point to the presence of Bacillus coli.
Bacillus coli forms most of its gas during the first
twenty-four hours. The liquid in the bulb must

* See p. 112 for Fuller and Johnson's groups of water
bacteria.

WATER ANALYSIS. 119

be distinctly acid to indicate the presence of Bacil-
lus coli.

5. Isolation of Bacillus coli.— First Method

{a) Add 50 c.c. of the water to be tested to 50 c.c.
of sterile bouillon in a sterile flask.

(b) Place in the incubator at 37.5° C. for two
days.

The high temperature will destroy the common
water bacteria, but will encourage the growth of
the coli group.

(c) Test part of this culture for indol. Its pres-
ence indicates the presence of Bacillus coli.

(^) Make plates from this culture.

{e) If any non-liquefying colonies are whitish
with irregular leafy outlines and show lines more or
less radial, they are probably colonies of Bacillus
coli, and must be carefully studied in cultures.^

Second Method — {d) Add 70 c.c. of the water to
be tested to 30 c.c. of sterile bouillon.

(b) To the mixture add i c.c. of 5 per cent, car-
bolic acid.

[c] Incubate at 37.5° C. for twenty-four hours.
Carbolic acid restrains the growth of the ordinary
water bacteria, while the coli group and other
intestinal forms grow unhindered.

(<^) From the growth that results inoculate fer-
mentation-tubes containing i per cent, glucose
bouillon. Note the amount of gas that forms as
before.

{e) Make plates from the growth in the tubes,

* See page 60.

I20

BACTERIAL ANAL YSIS.

and study the colonies that resemble those of
Bacillus coli.

6. Milk Analysis.— The analysis of milk is
conducted in the same manner as is that of water,
but on account of the great number of bacteria in

Fig. 53. — Hesse's apparatus for collecting bacteria from the

air.

milk the samples must be diluted with sterile
water.

Bacillus typhosus and Bacillus coli are detected
in milk by the same methods as in water analysis.
Tuberculosis bacilli may be detected in milk by the
method described in the next chapter.^

^Seepage 157.

AIR ANALYSIS.

121

For the classification of bacteria in milk, see H.
W. Conn, Report of the Storrs (Connecticut) Agri-
cultural Experiment Station for 1899.

V'^fii

t;?: ?■■

!iir — -Ti?

Fig. 54. — Petri's sand-fil-
ter for air examination.

Fig. 55. — Sedgwick's ex-
panded tube for air exam-
ination.

7. Bacteria in the Air. — {a) Prepare 3 gelatin
plates and expose them to the air for four or five

122 BACTERIAL ANALYSIS.

minutes in different places. (In recitation-rooms
before and after class, out of doors, etc.)

(^) Cover and allow to grow.

(c) Examine from day to day, and make cultures
from the different colonies.

This is a rough method of determining the rela-
tive number of bacteria in the air. For more exact
results recourse must be had to special apparatus
for aspirating through bouillon or through sugar, as
described in the text-books.

8. Bacteria in the Soil. — Numerous species of

Fig. 56. — Frankel's instrument for obtaining earth from
various depths for bacteriologic examination.

bacteria occur in the soil ; some are of special
interest on account of their pathogenic properties.
Many are anaerobic, and this fact must be kept in
mind while studying them.

To determine the number of bacteria in a sample
of soil :

[a) Collect the soil without contamination from
bacteria from other sources.

(/;) Introduce a measured quantity into a tube of
liquefied gelatin. Crush with a platinum needle,
and mix thoroughly w^ith the medium.

{c) Make plates, count, isolate, and study in the
usual way.

SOIL ANALYSIS. 123

Another method, and one which does away with
the presence of particles of soil in the medium, but
which perhaps does not give such accurate results,
is to mix the sample thoroughly with sterile water
and then make plates from the water.

CHAPTER X.
PATHOGENIC BACTERIA.

The methods for the study of pathogenic bacteria
are exactly the same as those already described.
In certain cases only are special culture-media
necessary for their growth. Most pathogenic forms
grow better in the incubator at body-temperature,
37-5° ^' -^^^ ^^^ cases animal inoculations are
necessary for the determination of pathogenicity.

Fig. 57. — Micrococcus aureus, from an agar-agar culture

(Giinther).

Great care must be used in handling pathogenic
cultures in order to avoid accidents. Have at hand
a solution of corrosive sublimate (i : 1000) or carbolic
acid (i : 20), with which to flood any material that

124

PYOGENIC ORGANISMS. 1 25

may by accident be spilled on floor or table. Care-
fully sterilize everything that the pathogenic
material may have contaminated. Thoroughly dis-
infect the hands, instruments, and table with the
corrosive sublimate solution after completing the
work.

Fig. 58. — Streptococcus p3-ogenes (from a bouillon culture).

I. PYOGENIC ORGANISMS.

{d) Study, according to the schedule in Chapter

VIII., the morphology and biology of:

Micrococcus aureus (Staphylococcus p3'ogenes aureus).
Micrococcus citreus ( Staph jiococcus p^'ogenes citreus).
^Micrococcus pj'ogenes (Staphylococcus p3'ogenes albus).
Streptococcus p^^ogenes.
Sarcina tetragena (Micrococcus tetragenus).

(8) Select and weigh a well-grown rabbit. In-
oculate into the ear vein b}^ means of a hypodermic
syringe^ i c.c. of a twenty-four or forty-eight hour
bouillon culture of one of the above species, or of
pus taken directly from an abscess.

^ Keep the syringe for some time before the operation in
a 2 per cent, solution of carbolic acid. Wash it out five or
six times with sterile water or bouillon before the inocula-
tion ; or the sj^ringe and needle may be boiled for five
minutes before using.

126

PATHOGENIC BACTERIA.

h I r 1 t^

Fig. 59. — Koch's syringe.

(c) Record the daily weight of the animal until
death.

{d) Perform an autopsy on the dead rabbit, note

Fig. 60. — Method of making an intravenous injection into
a rabbit. Observe that the needle enters the posterior vein
from the hairy surface (McFarland).

the various pathologic changes, and make cultures
and cover-glass smears from all the organs and
from the blood. ^

*Sear the surface of the body-wall and of each viscus
with a red-hot spatula or old scalpel before cutting into

PYOGENIC ORGANISMS. 12/

(e) Preserve some of tissues in absolute alcohol
or in Zenker's fluid/

(y) Stain the smear preparations and examine
for the inoculated organism.

^g) Make pure cultures from whatever growth is

Fig. 6i. — Streptococcus pyogenes ; cover-glass preparation
from the pus of an abscess ; x looo (Frankel and Pfeiffer).

obtained in the cultures from the blood and organs,
and try to recover the original organism.

them ; sterilize the blades of the knives, scissors, and for-
ceps used in making the incisions by dipping them in
meth}^! alcohol and passing them quickly near enough to a
Bunsen flame to ignite the alcohol. Smears and cultures
from the blood are best made from the blood in the heart.
For the method of preparation of blood-smears, see page

59-

^ See Appendix, page 173.

128

PATHOGENIC BACTERIA.

Fig. 62. — Streptococcus p3'ogenes, seen in a section through
human skin ; x 500 (Frankel and Pfeiffer).

Fig. 63. — Sarcina tetragena in pus from a white mouse ;

> 615 (Heim).

GONOCOCCUS.

129

(fi) Section the preserved tissue and stain for
bacteria with carbol-thionin-blue, Kuhne's methy-
lene-bhie, or by Gram's method.

Fig. 64. — Mouse-holder, with mouse in position for inocu-
lation.

Other animals — guinea-pigs, rats, or mice — may
be used for inoculations. Inoculations may be sub-
cutaneous, peritoneal, or intravenous, according to
the organisms used or the nature of the experi-
ment.

II. GONOCOCCUS.

1. Examine cultures of Alicrococciis gonorrhoccB^
and stain the organism with Loffler's blue and by
Gram's method.

2. Examine gonorrheal pus as follows :
{a) Prepare films on cover-glasses.

{b) Pass three times through flame.
(c) Stain with Loffler's methylene- blue or with
any aqueous anilin stain one minute.
{d) Wash, dry, and mount.
{e) Examine with the oil-immersion lens.

130 PATHOGENIC BACTERIA.

Gonococci are of medium size, composed usually
of two hemispheres separated by a narrow un-
stained interval. Occasionally two pairs of cocci
form a *' tetrad." The cocci are usually within
the leukocytes.

(/) Stain another film by Gram's method. The
gonococci are decolorized.

III. ANTHRAX.

1. Study the morphology and biology of cultures
of Bacterhini anthracis.

2. Inoculate a guinea-pig as follows :

Fig. 65. — Gonococcus in urethral pus ; x 1000 (Frankel

and Pfeiffer).

{a) Remove the hair from a small area on the
abdomen.

ANTHRAX.

131

{b) With a snip of a pair of sterile scissors make
a little subcutaneous pocket.

(c) Introduce into this pocket spores and bacteria
from a pure culture by means of a platinum loop.

{a) At the autops>' prepare cultures, smears, and
sections from the oro-ans.

Fig. 66.— Bacterium anthracis ; colony three days old
upon a gelatin plate ; adhesive preparation ; x 1000 (Fran-
kel and Pfeiffer).

{e) Make cover-glass preparations from the blood
as follows :

1. Place a small drop of blood between two abso-
lutely clean cover-glasses, draw them apart and
allow the smears to dry.

2. Fix in equal parts of ether and alcohol for
thirty minutes, or in absolute alcohol five minutes,

132

PATHOGENIC BACTERIA.

or in vapor of formaldehyd two and a half minutes;
or heat in the thermostat at iio°-i20° C. for twelve
hours ; or heat on a brass plate for one hour at the
point where water boils.

Fig. 67. — Bacterium anthracis ; cover-glass preparation
from the spleen of a mouse (Mallory and Wright).

3. Stain in eosin {\ per cent, in 60 per cent,
alcohol) from one to five minutes.

4. Wash in water and dry.

5. Contrast-stain in aqueous methylene-blue from
one-half to one minute.

6. Wash, dry, and mount.

GLANDERS.

133

IV. GLANDERS.

1. Study the morphology and biology of the
glanders bacterium.

2. Inoculate a male guinea-pig intraperitoneally
with I c.c. of a bouillon culture.

3. Note in two or three days the great swelling

Fig. 68. — Bacterium anthracis, stained to show spores ;
X 1000 (Frankel and Pfeiffer).

and redness of the testicles, caused by a semipuru-
lent affection of the tunica vaginalis. This is a
diagnostic test for the glanders bacterium.

4. At the autopsy prepare smears, cultures, and
sections from all the organs and from the peritoneal
and scrotal nodules.

5. Stain sections as follows :
9

134 PATHOGENIC BACTERIA.

(a) Carbol-tliionin-blue for ten to fifteen minutes.

{b) Wash thoroughly in water.

(c) Dehydrate in anilin oil.

{d) Treat with equal parts of anilin oil and xylol.

{e) Pass through pure xylol to balsam.

t ^ ^ 's

Fig. 69. — Bacterium mallei, from, a culture upon glycerin
agar ; x 1000 (Frankel and Pfeiffer).

6. For the diagnosis of a suspected case of glan-
ders proceed as follows :

(a) Rub a large swab made of absorbent cotton
in the discharge from the nose or in the suspected
ulcer.

(d) Transfer the swab to 5 c.c. of sterile water
and shake thorousfhlv.

(c) Inoculate the resulting suspension intraperi-
toneally into a well-grown male guinea-pig.

DIPHTHERIA. 1 35

{d) In two to seven days scrotal inflammation
will develop if the glanders bacterium was present.
If the glanders bacterium was not present, after a few
hours or days of depression the guinea-pig recovers
completely. It may happen that some acute septic
organisms were present in the material injected.
In such cases the guinea-pig usually dies within
twenty-four hours, and the test is evidently of no
value and must be repeated.

{e) If the scrotal lesions appear, perform an
autopsy. Look particularly for nodular deposits in
the peritoneum and visceral layer of the tunica
vaginalis, infiltration of the scrotal tissue, and
edema extending into the groin and suprapubic
region.

(/") Transfer aseptically a portion of a nodule to
potato, and place at 37.5° C. In twenty-four to
forty-eight hours small, smooth, glistening, amber-
colored colonies of the glanders bacterium should
develop. The bacterium is a short, thick rod, with
rounded ends, usually slightly curved or bent,
sometimes elongated into threads. It stains faintly
in Loffler's methylene-blue.

(^) A positive diagnosis can usually be made
from the gross lesions, but the isolation of the
organism makes the diagnosis certain.

V. DIPHTHERIA.

I. Make swabbings from the throats of healthy
individuals and from several diphtheria patients in
hospital. Depress the tongue and rub a sterile

136

PATHOGENIC BACTERIA,

Fig. 70. Fig. 71.

Fig. 70. — Providence Health Department outfit for diph-
theria diagnosis. A pasteboard box containing a swab-
tube and a serum-tube, both with etched surface on which
to write the name and address of patients, etc.

Fig. 71. — Bacterium diphtheriae ; agar culture (photo-
graph by Dr. Henry Koplik).

swab made of non-absorbent cotton over the back
of the throat, tonsils, diphtheritic membrane, etc.
2. Rub the swab over the surface of a Loffler
blood-serum tube.

DIPHTHERIA.

137

3. Place the tube in an incubator at 37.5° C. for
sixteen hours or longer.

4. Examine the growth for the small grayish,
slightly elevated diphtheria colonies.

5. Prepare films from a suspected colony.

6. Pass three times through the flame and stain
in Lofiier's methylene-blue for one minute.

jf

4»!^

fet

Fig. 72. — Bacterium diphtheriae, from culture upon blood-
serum ; X looo (Frankel and Pfeiffer).

7. Wash, dry, mount, and examine with the ^2
inch oil-immersion lens. The characteristic diph-
theria bacteria can easily be detected.

8. Isolate the diphtheria organisms in pure cult-
ure by inoculating tubes from a single colony
which on examination proves to be diphtheria. If
a single colony cannot be found, touch the needle

138

PATHOGENIC BACTERIA,

once to the growth on the serum-tube and make a
series of strokes on 3 or 4 tubes. When the colonies
develop, those in the last tube will be sufficiently
distinct so that pure cultures may be made from
them. Another method is to inoculate a bouillon-
tube or a tube of sterile water or normal salt solu-
tion, and immediately from this make stroke cult-

FiG. ']i. — Bacterium diphtherise, colony twenty-four hours
old upon agar ; x 100 (Frankel and Pfeiffer).

ures on serum-tubes. The growth on these will
probably be in individual colonies. Either of these
methods obviates the necessity of making plates.

9. Study the morphology and biology of the
diphtheria bacterium obtained above.

10. The followinor stains are diag^nostic for the
diphtheria bacterium :

DIPHTHERIA.

139

Hunt'* s Stat?i.

{a) Prepare films as usual.

{b) Stain in aqueous metliylene-blue for one
minute.

{c) Wash in water and dry.

{d) Treat with a 10 per cent, solution of tannic
acid for one minute.

(e) Wash in water and dry.

{/) Stain in aqueous methyl-orange for one
minute.

{g) Wash, dry, and mount.

Nezsser's Stain.

{a) Prepare solution A as follows :

Methylene-blue,
Alcohol (95 per cent.),

Dissolve and add

Acetic acid,
Water,

{b) Prepare solution B as follows:

Bismarck-brown,
Boiling water,

{c) Treat films with solution A for one to three
seconds.

{d) Wash in water.

{e) Treat with solution B for three to five seconds.

(/) Wash, dry, and moimt.

Ti. Test the virulence of the diphtheria organism
isolated as follows :

I gm. ;

20 c.c.

50 c. c. ;

950 c. c.

•

2 gm.;

1000 c.c.

140

PATHOGENIC BACTERIA.

(a) Prepare a twenty-four to forty-eight hour
bouillon culture.

(d) Sterilize a hypodermic syringe and needle by
soaking in 2 per cent, carbolic acid and washing
out in sterile water or bouillon, or by boiling for
five minutes.

(c) Select and weigh a full-grown guinea-pig.

(d) While the pig is held on its back on the table

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Fig. 74.— vSlide, 7 x 2^ inches, for the routine examina-
tion of diphtheria cultures. Each square can be placed
under the lens of the microscope without disturbing the
equilibrium of the slide.

by an assistant, the hair is removed from a small
area on the ventral abdominal wall.

ie) With the thumb and forefinger of the left
hand pinch up a fold of the skin, and with the
right hand insert the hypodermic needle between
the skin and the muscular body-wall.

(/) Inoculate an amount of the culture equal to
I per cent, of the weight of the pig.

\g) Record the daily weight of the pig until
death.

INFLUENZA. I4I

(Ji) Perform an autopsy on the dead pig ; note
the pathologic changes, and prepare cuUures,
smears, and sections from the organs and from the
point of inoculation.

VI. INFLUENZA.

I. Prepare blood-agar tubes by smearing the sur-
face of a ordinary agar-tube with a drop of blood

Fig. 75. — Bacterium influenzae, from a gelatin culture ;
X 1000 (Itzerott and Niemann).

obtained aseptically from man, rabbit, guinea-pig,
pigeon, or frog.

2. Break up a distinctly purulent portion of
influenza sputum in i or 2 c.c. of bouillon, and
spread a loopful of the suspension over the surface
of the blood-agar tube.

142

PATHOGENIC BACTERIA.

3. Place in the incubator and examine at the
end of from eighteen to twenty-four hours.

4. The influenza colonies appear as minute color-
less, glassy, transparent points, resembling drops
of dew. They are barely visible to the unpractised
eye, and require a low magnifying power to be seen
clearly.

Fig. 76. — Bacterium influenzae: colonies on blood-agar ;
low magnifying power (Pfeiffer).

5. Study the morphology and biology of the
organisms in one of these colonies. They should
not grow on ordinary media, and should have the
morphology of the influenza bacteria.

6. Prepare smears from one of the purulent

INFL UENZA.

143

masses in the sputum. Stain in very dilute carbol-
fuchsin for five to ten minutes, or in Loffler's
methylene-blue heated . to the steaming-point.
The influenza bacteria are very small, short, with

Fig. 77.— Bacterium influenzae : cover-glass preparation
of sputum from a case of influenza, showing the bacteria
within the leucocytes ; highly magnified (Pfeiffer).

round ends, are often present in large numbers,
and are frequently within the pus cells. They may
occur in pairs, and then resemble cocci. The ends
are usually more deeply stained than the central
portions.

144

PATHOGENIC BACTERIA.
VII. TYPHOID AND COLON BACILLI.

Fig. 78. — Bacillus typhosus, from a twenty-four hour agar

culture ; x 650 (Heim).

Fig. 79.^Bacillus coli, from an agar culture ; x 1000

(Itzerott and Niemann).

I. Study the morphology and biology of cultures

TYPHOID AND COLON BACILLI.

H5

of Bacillus lyphosics and Bacillus coli. Some of the
dififerences between them are indicated below :

B. TYPHOSUS.

Rods usually slender.

Flagella more numerous, longer,
more wavy (10-20).

In artificial media growth gener-
ally slower and not so vigorous.

Growth on fresh acid potato a
nearly transparent film.

Very slight acid production in
ordinary media, followed sometimes
by a production of alkali.

Litmus milk — no change.

Milk not coagulated.

Fermentation of lacto.se very
slight if any.

Litmus lactose agar — no change
in color.

Glucose media — no gas forma-
tion.

No production of indol in ordi-
nary bouillon.

Does not grow in Maassen's as-
paragin-glycerin solution.

Agglutination-test positive.

B. COLI.

Rod inclined to be a little
thicker.

Flagella fewer and shorter
(8-10).

Growth faster and more vigor-
ous.

Growth on potato a brownish
pellicle.

Well-marked acid production.

Litmus milk — pink color.

Milk coagulated.

Fermentation of lactose pro-
nounced.

Litmus lactose agar — produc-
tion of red color.

Abundant gas formation.

Well-marked indol production
in ordinary bouillon.

Grows in Maassen's solution.

Agglutination-test usually nega-
tive.

2. Study sections of spleen, intestinal lesions,
mesenteric lymph-glands, etc., from a hnman
typhoid autopsy. Stain with carbol-thionin-blue
or carbol-fuchsin.

3. Widal Reaction in Typhoid Fever.— If the
blood-serum of a person suffering with typhoid
fever or of one who has recently recovered from it

146

PArHOGENIC BACTERIA.

be added to a bouillon culture of actively motile
typhoid bacilli, the bacilli lose their motility and

Fig. 80. — Bacillus typhosus : superficial colony two days
old on a gelatin plate ; x 20 (Heim).

Fig. 81. — Bacillus coli : superficial colony two days old,
on a gelatin plate ; x 21 (Heim).

soon aggregate in clumps. Dilutions of serum
I : 10 and i : 30 with a half-hour time-limit are

TYPHOID AND COLON BACILLI.

H7

those most commonly employed. In doubtful cases
a dilution of i : lOO with a one-hour time-limit is
recommended.

{a) Place 9 drops of a twenty-four-hour bouillon
culture of actively motile typhoid bacilli on separate

Fig. 82. — Bacillus typhosus, from an agar culture six
hours old, showing the flagella stained by Loffler's
method ; x 1000 (Frankel and Pfeiffer).

Spots on a clean cover-glass. Add i drop of serum
from the blood of the suspected typhoid case,^ mix

^ The blood may be collected in a capillary tube and after
coagulation the serum removed ; or dried blood may be
moistened with water and the resulting solution used as
serum. The latter method, however, does not permit accu-
rate dilution, Chester and Robin have recently devised a
pipet for delivering a measured drop of blood, so that dilu-

148

PATHOGENIC BACTERIA.

all together, and mount as a hanging-drop prepara-
tion.'

{b) Examine with the \ inch or with the oil-

FiG. 83. — The Chester and Robin pipet for delivering
uniform drops of blood for the Widal test. A glass and
rubber pipet, the bulb of which is enclosed between two
strips of metal held in place by a Hoffman clamp.

immersion lens. If the case is one of typhoid, after
some time, varying between a few seconds and a

tions may be made with a fair degree of accuracy from
dried blood. It consists of an ordinarj' medicine-dropper
of a given size, the bulb of which is enclosed on either side
by two narrow strips of metal (Fig. 83, r, ^.), and both placed
in a medium-sized Hoffman clamp. The inward movement
of the clamp by means of the screw a compresses the bulb,
while a slight turn in the opposite direction dilates it a
little and permits a small drop of blood to enter. In expel-
ling the blood the dropper is held vertically over a strip of
thick filter-paper, and the clamp is slowly compressed until
a single drop falls of its own weight. This drop is then
dried, and when the test is to be applied the blood-spot is
cut from the paper and drops of the diluting fluid are
added from an exactly similar pipet until the required
dilution is reached.

' It is convenient to use a slide on which two glass rings
have been cemented, so that one may be used as a control,
containing the culture of bacilli alone.

TYPHOID AND COLON BACILLI.

149

half-hour, the bacilli will be seen to become less
motile, and finally to cease all movement and

Fig. 84. — Slide with two cells for observing Widal reaction

with a control.

appear in clumps here and there throughout the
field.

Fig. 85.— Widal reaction : a, bouillon culture of Ba-
cillus typhosus ; b, the same after the addition of typhoid
serum.

This reaction can be reversed and made to serve
as a means of identifying the typhoid bacillus if
the serum of a person known to have typhoid fever
is at hand.

10

ISO

PATHOGENIC BACTERIA.

VIII. PNEUMONIA.

Bacterium pneumonia (Frankel's Pneumococcus)
AND Bacterium pneumonicum (Friedlander's
Pneumobacillus).

Fig. 86. — Bacterium pneumonige, from the heart's blood
of a rabbit ; x looo (Frankel and Pfeiffer).

1. Study the morphology and biology of cultures
of B. pneicmonice and B. pneiinionictim.

2. Make cover-glass preparations from pneu-
monia sputum. Stain in Loffler's methylene-blue
or carbol-fuchsin, diluted one-half.

3. Prepare sections of lung-tissue from cases of

TUBERCULOSIS.

151

lobar pneumonia, general infection, etc. Stain in
carbol-thionin-blue and by Gram's method,

4. Inoculate a guinea-pig or rabbit subcutaneously
or intravenously with virulent cultures or with

Fig. 87. — Bacterium pneumonicum, from the expectoration
of a pneumonia patient ; x 1000 (Frankel and Pfeiffer).

pneumonia sputum ; also with sputum from healthy
persons.

5. At the autopsy note the pathologic changes,
prepare smears from the blood and organs, make
cultures, and prepare sections.

IX. TUBERCULOSIS.

I. Examination of Cultures.

Study the morphology and biology of Bac-
terhnn tuberculosis.

152 PATHOGENIC BACTERIA.

(i) For cultures use blood-serum, agar, and bouil-
lon to which from 4 to 8 per cent, of glycerin has
been added.

(2) Stain films, prepared from cultures, with ordi-
nary stains^ and by the Ziehl-Neelson method as
follows :

(a) Prepare films as usual.

(b) Stain in Ziehl's carbol-fuchsin, steaming but
not boiling, five minutes ; cold, twenty minutes.

(r) Wash in 20 per cent, sulphuric acid for three
to five seconds.

(d) Wash in 60 per cent, alcohol until no red
color is left.

(e) Wash in water.

(y) Stain in aqueous solution of methylene-blue
for one minute.

i^g) Wash in water, dry, and mount.

The bacteria of leprosy, syphilis, and smegma
stain by this method. Bacterium leprae stains
much more quickly than B. tuberculosis, B. syphil-
idis is decolorized more quickly, especially in sul-
phuric acid, and B. smegmatis is decolorized by the
alcohol.

3. Examination of Tuberculous Sputum.
(i) Ziehi=NeeIson Method.

ia) Place the sputum in a shallow glass dish on
a black surface.

{U) Select several of the characteristic yellowish

^ The tubercle bacillus takes the ordinary stains very
slowly and faintly.

TUBERCUL OS IS. 1 5 3

particles, place between two cover-glasses, and
spread evenly.

{c) Draw the covers apart and allow the films to
dry.

{d) Pass three times through flame.

N.

^'

\

\ '5

I »s,

A --

/^

\

S

Fig. 88. — Tubercle bacteria in sputum (carbol-fuchsin and

methylene-blue).

{e) Stain in hot (steaming but not boiling)
Ziehl's carbol-fuchsin for five minutes or in the
cold solution for twenty minutes.

{/) Wash rapidly in water.

{g) Decolorize in 20 per cent, sulphuric, hydro-
chloric, or nitric acid, for three to five seconds.

154 PATHOGENIC BACTERIA.

{h) Wash in water.

{{) Contrast-stain in saturated aqueous solution
of methylene-blue or in Loffler's blue for one-half
minute.

(/) Wash in water, dry, and mount.

{k) Examine with the -^^ inch oil-immersion
lens. The bacteria should be red in a blue field.^

(2) Koch=Ehrlich Method.

{a) Stain cover-glass preparations in anilin fuch-
sin or anilin gentian-violet for twelve to twenty-
four hours.

{b) Decolorize for three to five seconds in 20 per
cent, nitric acid.

{c) Wash in water, in 60 per cent, alcohol for five
to ten seconds, and again in water.

{d) Contrast-stain in aqueous methylene-blue for
one minute.

{e) Wash, dry, and mount.

(3) Gabbers Method.

{a) Stain in steaming Ziehl's carbol-fuchsin for
one minute.

{b) Wash in water for two to three seconds.

{c) Stain in Gabbet's blue^ for thirty seconds or
longer.

{d) Wash in water, dry, and mount.

(4) Rosenberger's Method.

{a) Stain in carbol-fuchsin (cold) for five to ten
minutes.

(/;) Without washing stain for one to two minutes

^ For the detection of tubercle bacteria in sputum when
present in very small numbers see page 157.
2 See Appendix, page 172.

TUBERCUL OS IS. i 5 5

in sweet spirits of nitre to which has been added
enough alcoholic solution of malachite-green, Bis-
marck-brown, or methylene-blue to give a deep-
colored fluid.

{c) Wash in water, dry, and mount.

3. Inoculations.

{a) Inoculate a guinea-pig subcutaneously in the
abdominal wall with tuberculous material.

Fig. 89.— Tubercle bacteria in sputum (Frankel and

Pfeiffer).

{b) After four to six weeks, ^ when the inguinal
lymphatic glands have become enlarged, kill the
animal.

{c) With proper aseptic precautions make three
or four cultures on blood-serum from two or three

^ For class-work such an animal must be previously inoc-
ulated.

156 PATHOGENIC BACTERIA.

glands, spreading a large quantity of the material
on the surface of the tubes.

(^) Seal air-tight, place in the incubator, and
examine the growth that occurs.

4. Sections.

{a) Prepare sections of tubercular lesions from
the inoculated animals or from the human lung: in
acute phthisis.

{b) Stain by the Ziehl-Neelson, Koch-Ehrlich, or
Rosenberger methods.

5. Detection of Tubercle Bacteria in Urine.

{cl) Make smears from the deepest layer of sedi-
ment thrown down by the centrifuge, or in the
sedimenting glass.

{b) Stain in hot (steaming but not boiling)
Ziehl's carbol-fuchsin for one minute.

{c) Wash in water.

{d) Decolorize in 20 per cent, sulphuric acid until
pink.

(e) Wash in water, 95 per cent, alcohol for thirty
seconds, and again in water.

(/) Stain in Loffler's methylene-blue for twenty
seconds.

(^) Wash in water, dry, and mount.

The smegma bacillus, which frequently occurs in
urine, does not stain by this method, as it is
decolorized by alcohol. Rosenberger' s method also
gives excellent results with urine sediment.

TUBERCUL OS IS. 1 5 /

6. Detection of Tubercle Bacteria in Milk.
(i) First Method.'

{a) To 50 c.c. of suspected milk add 10 c.c. of
carbolic acid.

{b) Shake vigorously for two to five minutes.
Pour into a sedimenting glass, cover, and allow to
stand for twenty-four hours ; or use a centrifuge.

(c) With a pipet remove the deepest layer of sedi-
ment and prepare films.

(d) Dry and pass three times through flame.

(e) Pass through equal parts of ether and alcohol.
{/) Dry and pass three times through the flame,

and stain as directed for sputum.

(^) Mount and examine with the -1^2^ inch oil-
immersion lens.

(2) Second Method.

id) To 20 c.c. of the milk add i c.c. of a 50 per
cent, potash solution.

{U) Heat in boiling water until the mixture turns
yellowish brown.

[c) Add 20 c.c. of acetic acid, shake, heat again
for three minutes, and centrifuge.

(^) Wash the sediment with hot water, again
centrifuge, and make cover-glass preparations from
the second sediment.

{e) Stain as directed for sputum.

1 This method may also be applied to the detection of
tubercle bacteria in sputum when present in very small
numbers, using 10-15 c.c. of sputum, 10 c.c, of water, and 6
c.c. of carbolic acid.

158

PATHOGENIC BACTERIA.
X. ACTINOMYCOSIS.

1. Study the morphology and biology of cultures
of Actinomyces bovis.

2. Prepare sections from tissue containing colo-
nies of the parasite.

3. Stain by Gram's method or as follows :

^«^iftS

Fig. 90. — Actinomyces (von Jaksch).

{a) Stain deeply in saturated aqueous eosin solu-
tion for ten minutes.

[b) Wash in water.

(r) Stain in anilin-gentian-violet for two to five
minutes.

id) Wash in normal salt solution.^

{e) Treat with iodin solution (iodine, i part ;
potassium iodid, 2 parts ; distilled water, 100 parts),
for one minute.

{f) Wash in water and dry slightly with filter-
paper.

^ See Appendix, page 173.

MALAR/A. 159

{g) Clear in anilin oil, then in several changes
of xylol.

(h) Mount in balsam.

XI. MALARIA.

1. :Kxamination of Fresh Blood for the Ma-
larial Hemato^oon.

{a) Clean a slide and cover-glass.

{b) Place a small drop of the blood to be ex-
amined on the slide, cover, and seal with vaselin.

{c) Examine with the oil-immersion lens.

The ameboid movements of the Hemato^oon
malarise are visible at the room-temperature, but
become more active if the slide is warmed.

2. Stained Preparations.
(i) First Method.

{a) Place a small drop of blood between two
cover-glasses, slide the covers apart, and allow the
films to dry.

{b) Fix by placing in absolute alcohol for five
minutes or in the vapor of formaldehyd for two
and a half minutes, or by one of the methods given
under Anthrax (page 131).

{c) Stain in a i per cent, aqueous solution of eosin
for five minutes or, if formaldehvd is used for fixa-
tion, in an alcoholic (60-75 P^^ cent.) solution of
eosin for three to ten minutes.

{d) Contrast-stain in saturated aqueous solution
of methylene-blue for five minutes for the alcoholic
preparations, for one minute for the formaldehyd

i6o

PATHOGENIC BACTERIA.

9

^o

//

0 <D

o

Fig. 91. — Some of the principal forms assumed by the
Hematozoon of tertian fever in the course of its cycle of
development (after Thayer and Hewetson).

preparations, or in Elirlicli-Biondi fluid ^ for one-
half hour.

pjQ Q2.— Crescentic and flas^ellated forms of Hematozoon
malarise: i, flagellated form of tertian fever; 2, flagellated
form of quartan fever ; 3, crescents, and 4, flagellated form
of estivo-autumnal fever (after Thayer and Hewetson).

» See Appendix, page 173.

MALARIA. l6l

{e) Wash, dry, and mount. Ehrlich-Biondi
preparations are examined dry.

In these preparations the red corpuscles are
stained red, the malarial organisms and the nuclei
of the leukocytes blue.

(2) Whitney's Method.

{a) Spread the blood as usual ; dry thoroughly in
the air or by gentle heat.

{b) Treat for twenty seconds with the following
modification of Zenker's fluid :

Potassium bichromate, 2 gm. ;

Sodium sulphate, i gm. ;

Water, 100 c.c.

Saturate while warm with corrosive sublimate.
Add 5 per cent, of strong nitric acid at time
of using.

{c) Wash in water, and dr}^ with filter-paper.

{d) Cover with Ehrlich's triacid stain ^ for three
minutes.

{e) Wash, dry, and mount.

Unna's polychrome methylene-blue^ and the
Chenzinsky-Plein eosin and methylene-blue^ solu-
tion work well after this fixation, but not Loffler's
methylene-blue.

* See Appendix, page 172.
^ See Appendix, page 172.
^ See Appendix, page 173.

APPENDIX.

I. BACTERIAL MEASUREMENTS BY PHOTOG-
RAPHY.

Wilson and Randolph ^ recommend the following
procedure for accurately measuring bacteria :

(a) Prepare a drawing about four times the size
of the desired negative by ruling with ink two sets
of equidistant lines at right angles to each other,
making every tenth line somewhat heavier than
the others.

(d) Reduce this drawing by photography to such
a size that the rulings are exactly i millimeter
apart.

(c) Adjust the photographic apparatus so that an
amplification of looo diameters is secured. Obtain
this by measuring the image of a stage micrometer
on the ground-glass screen.

(d) With this adjustment make a photomicro-
graph of the bacteria to be measured.

(e) Superimpose the print of the photomicro-
graph of the bacteria on the print of the scale, or

^Journal of Applied Microscopy, vol. ii, No. ii, Nov.,
1899.

163

164

APPENDIX.

vice versa. The reading is directly in micromilli-
meters or fi.

Fig. q^.— Wilson and Randolph's method of measuring

bacteria by photography.

MOULDS AND YEASTS.

165

II. MOULDS AND YEASTS.

Moulds and yeasts frequently contaminate plate
and tube cultures, and inasmuch as these growths
are occasionally the cause of pathologic conditions,

Fig. 94. — Saccharomyces cerevisise.

it is advisable that the bacteriologist be acquainted
with certain typical forms.

Fig. 95. — Mucor raceraosus : a, spore-bearing head ; b,
spores ; c, branch ; d, resting spores (after Jelliffe).

11

1 66

APPENDIX.

1. Saccharomyces cerevisiae.

Examine cultures of this yeast, mount, and stain
exactly as directed for bacteria.

2. Mucor racemosus.

(i) Cultures of this mould may be made on the
surface of gelatin- or agar-tubes, but preferably in
plates. The developing colonies may be examined
directly with the low-power lens of the microscope.

Fig. 96. — Aspergillus repens : a, conidia-bearing head; b,
conidia ; c, peritheca ; d, sterigmata (after Jelliflfe).

(2) For examination with the high power, re-
move a small portion of the growth to a mixture of
glycerin and water on a slide ; spread out as thin
as possible, cover, and examine.

(3) The preparation may be stained by adding a
little eosin solution to the glycerin and water.

(4) Permanent preparations may be made as
follows :

(a) Transfer a small amount of the mould to a
slide.

MOULDS AND YEASTS.

167

(p) Drop a little alcohol upon it to remove the
air from the hyphae.

Fig. 97. — PenicilHum crustaceum : a, conidia with myce-
lium ; b, conidia cluster ; c, sterigma ; d, conidia (after
JellifFe).

(c) Treat again with water.

(^) Stain in methylene-blue.

(e) Mount in glycerin or glycerin-jelly.

1 68 - APPENDIX.

3. Aspergillus repens and Penicillium crus-
taceum.

These moulds may be examined exactly as
directed for Mucor.

4. Key for the Identification of the Yeasts
and Moulds that most frequently contaminate
Cultures, l

Mycelium growth absent:

I. YEASTS, SACCHAROMYCETACE^.

1. Growth white to dirty white,

S. cerevisice.
S. albicans.

2. Growth reddish,

S. glutinis.

3. Growth brownish to black,

^S. 7i2ger.
Mycelium growth present:

II. MOULDS.

I. Spores inside of sporangia.

Phycomycetes. Family, Mucorini.
{a) Mycelium of one kind,

1. Spore-bearing bodies on unbranched

hyphse,

Mucor.

2. Spore-bearing bodies on branched hyphae,

Cifxinelia.
{b) Mycelium of two kinds,

Rhisoptis.

^Adapted from "Some Laboratory Moulds," by S. E.
Jelliffe, in Journal of Pharfnacology, Nov., 1897.

MOULDS AND YEASTS. 1 69

2. Spores free, at the ends of modified hyphae,

Hyphomycetes.
(a) Hyphae pallid, loose, not collected into
fascicles,

MUCEDINE^.

1. Conidia undivided,

a. Hyphae short,

(i) Hyphae unbranched,

Oospora.

(2) Hyphae branched,

Monilia.

b. Hyphae elongated,

(i) Conidia aggregated,

* Fertile hyphae enlarged at apex,
t Conidia on simple sterigmata.

Aspergillus.
tt Conidia on compound sterigmata,

Sterig77iatocystis.
** Fertile hyphae not enlarged at apex,

Penicillmiii.
(2) Conidia separated or loosely aggre-
gated,

Botrytis.

2. Conidia once septate,

Cephalothecm ?;/ .
{J)) Hyphae brownish or black, not collected into
fascicles,

Dematie.e.
I. Conidia non-septate,

a. Hyphae short, only slightly different
from conidia,

Torn la.

I/O APPENDIX.

b. Hyphse distinct from conidia,

Honnodendron,
2. Conidia septate,

a, Conidia in chains,

Alternaria.

b. Conidia single.

Macro spoi^iu ni .

(c) Hyphae pallid or dark, collected into fasci-

cles,

Stilbe^.

(d) Hyphae pallid or reddish, collected in wart-

like masses,

TUBERCULARIE^.

Conidia elongated, septate,

Fiisarmm.

III. STAINS AND REAGENTS USED IN THE
STUDY OF BACTERIA.

I. Simple Anilin Stains.

Prepare saturated alcoholic solutions of gentian-
violet, methylene-blue, thionin-blue, basic fuchsin,
safFranin, or Bismarck-brown,^ by adding sufficient
stain to absolute alcohol to make a saturated solu-
tion, and leave some undissolved stain at the bot-
tom of the vessel. To these stock solutions alcohol
may be added from time to time, taking care that
some undissolved stain always remains. When re-
quired for use add 5 c.c. of the saturated alcoholic
solution to 95 c.c. of distilled water, and filter.

^Use the anilins prepared especially for microscopic
work by Griibler.

STAINS AND REAGENTS. 171

The watery solutions soon decompose, and must
be made only as required for use. Methylene-blue,
however, may be made up as a saturated aqueous
solution, as it is permanent. Always filter a stain
before use.

«. Kuhne^s Methylene-blue.

Methylene-blue, 1.5 gm.;

Absolute alcohol, 10 c.c. ;

Carbolic acid (i : 20), 100 c.c.

Stain films five minutes.

3. I^bflaer^s Methylene-blue.

Saturated alcoholic solution of

methylene-blue, 30 c.c;

Solution of potassium hydrate

in water (i : 10,000), 100 c.c.

4. Carbol-thionin-blue.

Thionin-blue, i gm. ;

Carbolic acid (i : 40), 100 c.c.

Dilute I volume of the stain with 3 of water,
when required for use. Stain from three to
five minutes.

5. Anilin-water Solution.

Anilin oil, 5 c.c. ;

Water, 100 c.c.

Shake together, allow to stand for five minutes,
and filter through a moistened filter.

6. Anilin-gentian-violet and Anilin-fuchsin.

Anilin-water, 10 parts;

Saturated alcoholic solution of

gentian-violet or fuchsin, i part.

172 APPENDIX.

7. Gramas lodin Solution.

lodin, I part;

Potassium iodid, 2 parts;

Distilled water, 300 "

8. ^iehPs Carbol-fuchsin.

Basic fiichsiii, i part;

Absolute alcohol, 10 parts;

Carbolic acid (i : 20), 100 "

9. Gabbet^s Blue.

Sulphuric acid (25 per cent.

solution), 100 c.c. ;

Methylene-blue, 2 gm.

Allow the diluted acid to stand twenty-four
hours or until it is cold before adding the
methylene-blue.

10. Unna^s Polychrome Methylene-blue.

Methylene-blue, i part ;

Potassium carbonate, i "

Water, 100 parts.

Must be ripened for months. The ripened
solution may be procured from Griibler.

II. ^Ehrlich^s Triacid Stain.

Saturated aqueous solution

of orange G, 120 parts.

Acid fuchsin, 80

Methyl-green, 100

Distilled water, 300

Absolute alcohol, 180

Glycerin, 50

STAINS AND REAGENTS. 173

Never shake the solution. Pipet from the top
what is needed for use.

i«. i^hrlicli-Biondi Stain.

Is best procured ready made from Griibler.

13. Chen^insky-Plein Stain.

Saturated aqueous sohition of

methylene-blue, 40 c.c. ;

Alcoholic (70 per cent.) solu-
tion of eosin (i : 200), 20 c.c. ;
Distilled water, 40 c.c.
The best results are obtained by staining for
twenty-four hours. Fairly good results may
be obtained by using warmed stains for
fifteen minutes.

14. Normal Salt Solution.

Distilled water, 100 c.c. ;

Sod i u m ch 1 or i d , O- 7 5 gni •

15. Acid Alcohol.

Alcohol (70 per cent.), 97 c.c. ;

c.c.

Hydrochloric acid,

16. Zenker's Fluid.

Potassium bichromate, 2.5 gm. ;

Sodium sulphate, i gm. ;

Corrosive sublimate, 5 gm. ;

Glacial acetic acid, 5 c.c. ;

Water, ^^100 c.c.

Do not add the acetic acid until ready for use.
Fix tissues from one to twentv-four hours.

174 APPENDIX.

Wash in running water from twelve to
twenty- four hours. Preserve in 80 per cent,
alcohol.

17. Cleaning Mixture, for Slides, Cover-
glasses, and Glassware.

Potassium bichromate, 6 gm. ;

Sulphuric acid, 6 c.c. ;

Water, 100 c.c.
Wash in water and alcohol.

18. Nessler's Solution.

A.

Potassium iodid.

35 gm. ;

Water,

200 c.c.

B.

Alercuric chlorid,

16 gm. ;

Water,

500 c.c.

Add B to A until faint show of excess is
indicated, then add 160 grams of solid
potassium hydrate. Dihite to i liter, and
add strong solution of mercuric chlorid,
little by little, until the red mercuric iodid
just begins to be permanent. Do not filter.
Solution should be pale straw-color. It is
improved by age.

19. Phenol-sulphonic Acid.

Sulphuric acid (pure and

concentrated), 148 c.c;

Distilled water, 12 c.c;

Pure carbolic acid, 24 gm.

TABLE OF SYNONYMS.

175

IV. TABLE OF SYNONYMS.

The scientific names of species used throughout
this work are those adopted by Migula. They are
preferable because they conform to the laws of
scientific nomenclature and priority. The follow-
ing table gives the synonyms of many of the
common species in alphabetic order :
Bacillus —

albolactus, Mig.; B. lactis albus, Loffler.

amylovorus (Burrill), De Toni ; M. amylovorus, Burrill.

aterrimus, L. et N.; B. mesentericus niger, Lunt.

avium, Mig.; B. diphtheriae avium, Kruse.

chauvaei, Arloing, Cornevin et Thomas; B. carbonis,

Mig.; B. anthracis symptomatici, Kruse.
coli, Mig.; Bact. coli commune, Escherich.
diaphanus, Mig.; Halibacterium pellucidum, Fischer,
dysenteriae (Kruse), Mig.; B. dysenterise liquefaciens,

Kruse.
equi, Mig.; B. equi intestinahs, Dyar et Keith,
fischeri, Mig.; Photobacterium fischeri, Beyerinck ; B.

phosphorescens indigenus, Kruse.
globigii, Mig.; B. mesentericus ruber, Globig.
indicus, Koch ; B. indicus ruber, Mig.
intricatus, Mig.; Cladothrix intricata, Russell,
limnophilus, Mig.; B. limosus, Russell,
mesentericus, Mig.; B. mesentericus fuscus, Flugge.
mirabilis, Mig.; Proteus mirabilis, Hauser.
murium, Mig.; B. typhi murium, Loffler.
niger, Mig.; B. lactis niger, Gorini.
ozaenae, Mig.; B. foetidus ozseucC, Hajek.
phosphorescens, Fischer ; Photobacterium indicum, Bey-
erinck ; B. phosphorescens indicus, Kruse.

1^6 APPENDIX.

Bacillus —

phosphoricus, Mig.; B. argenteo-phosphorescens III.,

Katz.
prodigiosuSjFliigge ; Monas prodigiosa, Ehrenberg ; Bact.

prodigiosum, Schroter ; M. prodigiosus, Cohn.
pseudotuberculosis, Mig.; B. pseudotuberculosis lique-

faciens, Kruse.
suipestifer, Kruse; B. of hog cholera, Salmon-Smith;

B. cholerae suum, Mig.
sulfureus, Mig.; Proteus sulfureus, Lindenborn.
typhosus, Mig.; B. typhi abdominalis, Mig.
vulgaris, Mig.; Proteus vulgaris, Hauser.
vulgatus, IMig.; B. mesentericus vulgatus, Fliigge.
zenkeri, Hauser ; Proteus zenkeri, Hauser.
zopfii, Mig.; Bact. zopfii, Kurth.

Bacterium —

aceti, Zopf; Ulvina aceti, Kiitzing ; Mycoderma aceti,

Thomsen.
acidi lactici, Mig.; B. acidi lactici I., Hiippe.
aerogenes, Mig.; Bact. lactis aerogenes, Escherich.
amethystinum, Mig.; B. membranaceus amethystinus,

Eisenberg.
anthracis, Mig.; B. anthracis, Koch,
anthracoides, Mig.; B. anthracoides, Hiippe et Wood,
aquatile, Mig.; B. aquatile, Frankland.
arborescens, Mig.; B. arborescens, Frankland.
aurescens, Mig.; B. aurescens, Frankland.
bienstockii, Schr5ter ; B. coprogenes parvus, Bienstock.
bovis, Mig.; Pneumobacillus liquefaciens bovis, Arloing ;

B. pneumonicus liquefaciens, Kruse.
brunneum, Mig.; B. brunneus, Adametz-Wichmann.
buccale, Mig.; Leptothrix buccalis, Robin,
candicans, Mig.; B. candicans, Frankland.

TABLE OF SYNONYMS. ly/

Bacterium —

capsulatum, Pfeiffer ; B, capsulatus, Pfeiffer, Koch.

carotarum, Mig.; B. carotarum, Koch.

chlorinum, Mig.; B. chlorinus, Frankland.

citreum, Mig.; B. citreus, Frankland.

cceruleum, Mig.; B. coeruleus, Smith.

columbarum, Mig.; B. diphtheriae columbarum, Loffler.

coprogenes, Mig.; B. coprogenes fcetidus, Fliigge.

cuniculicida, Koch ; B. cholerae gallinarum, Fliigge ;

Bact. septichaemiae, Schroter.
cuniculi, Mig.; B. cuniculi pneumonicus, Kruse.
endocarditidis, Mig.; B. endocarditidis capsulatus, Weich-

selbaum.
felis, Mig.; B. felis septicus, Kruse.
giardi, Mig.; B. phosphorescens giardi, Kruse.
keratomalacise, Mig.; B. septicus keratomalacise, Babes,
lacteum, Mig.; B. lactis III., Kruse.
lacticum, Mig.; B. lacticus, Kruse; B. lactis acidi, Lieb-

mann.
laerii, Mig.; B. viscosus I., van Laer; B. viscosus cere-

visiae, Kruse.
leprae, Mig.; B. leprae, Hansen,
mallei, Mig.; B. mallei, L5ffler.
murisepticum, Mig.; B. murisepticus, Fliigge; B. muri-

nus, Schroter.
mycoides, Mig.; B. mycoides roseum, Scholl-Holschew-

nikoff.
nephritidis, Mig.; B. nephritidis interstitialis, Letzerick.
palumbarium, Mig.; B. cholerae columbarum, Kruse.
phosphorescens, Fischer ; Photobacterium phosphores-
cens, Beyerinck.
proteus, Mig.; Proteus capsulatus septicus, Banti ; B.

capsulatus septicus, Kruse.

1/8 APPENDIX.

Bacterium —

pseudodiphthereticum, Mig.; Corynebacterium pseudo-
diphthereticum, L. et N.

pseudoinfluenzae, Mig.; B. pseudoinfluenzae, Kruse.

pseudotuberculosis, Mig.; B. pseudotuberculosis, Pfeiffer;
Streptobacillus pseudotuberculosis rodentium, Preisz.

pyocinnabarium, Mig.; B. pyocinnabarius, Kruse.

pyogenes, Passet ; B. pyogenes foetidus, Passet.

rhusiopathiae, Mig.; B. rhusiopathiae suis, Kitt; Bact. ery-
sipelatus suis, Mig.

salivae, Mig.; B. salivae minutissimus, Kruse.

sempervivum, Mig.; B. lactis XII., Kruse.

smegmatis, Mig.; B. smegmatis, Kruse.

subtile, Mig.; B. subtilis simulans I., Bienstock; B. fae-
calis I., Kruse.

suicida, Mig.; B. suisepticus, Kruse.

termophilum, Mig.; B. termophilus, Miquel.

tuberculosis avium, Mig.; B. tuberculosis avium, Maf-
fucci ; Mykobacterium tuberculosis avium, L. et N.

tuberculosis, Mig.; B. tuberculosis, Koch; Mykobacte-
rium tuberculosis, L. et N.

ubiquitum, Mig.; B. ubiquitus, Jordan. ,

varicosum, Mig.; B. varicosus conjunctivae, Hombert.

vermiculare, Mig.; B. vermicularis, Frankland.

vignali, Mig.; B. g, Vignal; B. buccalis minutus, Stern-
berg.

welchii, Mig.; B. aerogenes capsulatus, Welch.

xerosis, Mig.; B. xerosis, Neisser et Kuschbert.

Micrococcus —

albocereus, Mig.; Staphylococcus cereus albus, Passet.
amplus, Mig,; M. albicans amplus, Fliigge.
aurantiacus, Cohn ; Staphylococcus cereus aureus, Mig.

TABLE OF SYNONYMS.

1 79

Micrococcus —

aureus, Mig.; Staphylococcus pyogenes aureus, Rosen-
bach; M. pyogenes aureus, Mig ; M. pyogenes, L. et N.

cereus, Mig.; Staphylococcus cereus flavus, Passet.

citreus, Mig.; Staphylococcus pyogenes citreus, Passet.

citrinus, Mig.; Diplococcus citreus liquefaciens, Unna.

conglomeratus, Flugge ; M. citreus conglomeratus,
Fliigge; Diplococcus citreus conglomeratus, Eisen-
berg.

conjunctivitidis, Mig.; M. flavus conjunctivae, Gombert.

conoideus, Mig.; Staphylococcus salivarius pyogenes,
Biondi.

corrugatus, Mig.; Merismopedia mesenterica corrugata,
Dyar.

coryzae, Mig.; Diplococcus coryzae, Hajek.

cuniculorum, Mig.; M. pyaemiae cuniculorum, Schroter.

cyaneus, Cohn ; Bacteridium cyaneum, Schroter.

desidens, Mig.; M. flavus desidens, Fliigge.

faviformis, Mig.; M. lacteus faviformis, Fliigge.

fragilis, Mig.; Merismopedia fragilis, Dyar.

gonorrhoeae, Fliigge ; Gonococcus, Neisser.

intracellularis, Mig.; Diplococcus intracellularis menin-
gitidis, Weichselbaum ; Streptococcus intracellularis,
L. et N.

lacticus, Mig.; Sphaerococcus acidi lactici, Marpmann.

liquefaciens, Mig.; M. ureae liquefaciens, Flugge.

luteus, Cohn ; Bacteridium luteum, Schroter.

mollis, Mig.; Merismopedia mollis, Dyar.

phosphoreus, Cohn ; M. lucens, v. Tieghem ; M. pfliigeri,
Ludwig, ex parte; Photobacterium phosphorescens,
Beyerinck.

pyogenes, Mig.; Staphylococcus pyogenes albus, Rosen-
bach ; M. pyogenes albus, L. et N.

roseus, Fliigge ; Diplococcus roseus, Eisenberg.

1 80 APPENDIX.

Micrococcus —

rugatus, Mig.; M. endocarditidis rugatus, Weichsel-
baum.

salivarius, Mig.; Coccus salivarius septicus, Biondi.

stellatus, Frankland ; Coccus stellatus, Lustig.

subflavidus, Mig.; M. tetragenus subflavus, v. Besser.

tardigradus, Mig.; M. flavus tardigradus, Fliigge.

tardior, Mig.; Diplococcus flavus liquefaciens tardus,
Unna et Tommasoli.

tardissimus, Mig.; Diplococcus albicans tardissimus,
Fliigge.

tardus, Mig.; Diplococcus albicans tardus, Unna et Tom-
masoli.

tenuis, Mig.; M. pyogenes tenuis, Rosenbach.

tenuissimus, Mig.; M. cumulatus tenuis, v. Besser.

varians, Mig.; Merismopedia flava varians, Dyar.

MiCROSPIRA —

annularis, Mig.; Photobacterium annulare, Fischer,
canalis, Mig.; Vibrio saprophiles ^, Weibel.
caraibica, Mig.; Photobacterium caraibicum, Fischer,
comma, Schroter ; Spirillum choleras asiaticae, Fliigge ;

Vibrio choler?e asiaticae, Mig.; Vibrio comma, Mig.
coronata, Mig.; Photobacterium coronatum, Fischer,
degenerans, Mig.; Photobacterium degenerans, Fischer,
delgadensis, Mig.; Photobacterium delgadense, Fischer,
dunbari, Mig.; Vibrio dunbari, Mig.
finkleri, Schroter ; Spirillum finkleri, Mig.; Vibrio fink-

leri, Mig.; Vibrio proteus, Mig.
glutinosa, Mig.; Photobacterium glutinosum, Fischer,
luminosa, Mig.; Photobacterium luminosum, Fischer,
marina, Mig.; Spirillum marinum, Russell,
metschnikovi, Mig.; Vibrio metschnikovi, Gamaleia.
papillaris, Mig.; Photobacterium papillare, Fischer.

TABLE OF SYNONYMS. l8l

MiCROSPIRA —

sapiophiles, Mig.; Vibrio saprophiles, /9, Weibel.
tonsillaris, Mig.; Vibrio tonsillaris, Klein,
tuberosa, Mig.; Photobacterium tuberosum, Fischer.

PSEUDOMONAS —

aeruginosa, Mig.; Bact. aeruginosum, Schroter ; B. seru-
ginosus, Schroter ; B. pyocyaneus, Gessard.

alba, Mig.; B. fluorescens albus, Zimmermann ; B. fluor-
escens non-liquefaciens (?), Eisenberg.

eisenbergii, Mig.; B. fluorescens non-liquefaciens, Eisen-
berg

europaea, Mig.; Nitrosomonas curopaea, Winogradsky.

fluorescens, Mig.; B. fluorescens liquefaciens, Fltigge.

ianthina, Mig.; Bact. ianthinum, Zopf; B. janthinus,
Zimmermann.

javanica, Mig.; Photobacterium javanense, Eijkmann.

javaniensis, Mig.; Nitrosomonas javaniensis, Winograd-
sky.

laurentia, Mig.; B. violaceus laurentius, Jordan.

litoralis, Russell ; B. litoralis, Russell.

minutissima, Mig.; B. fluorescens liquefaciens minutissi-
mus, Unna et Tommasoli.

pseudianthina, Mig.; B. violaceus, Frankland.

tenuis, Mig.; B. fluorescens tenuis, Zimmermann.

Sarcina —

ventriculi, Goodsir ; Merismopedia goodsiri, Husem ;
Merismopedia ventriculi, Robin ; Sarcina fuscescens,
de Bary.

Spirosoma —

attenuatum, Mig.; Spirillum attenuatum, Warming,
flavescens, Mig.; Vibrio flavescens, Weibel.
12

1 82 APPENDIX.

Spirosoma —

flavum, Mig.; Vibrio flavus, Weibel.
gregarium, Mig.; Myconostoc gregariiira, Cohn.

Streptococcus —

cerasinus, Mig.; M. cerasinus siccus, List.

citreus, Mig.; M. citreus, List-Eisenberg.

cystitidis, Mig.; Diplococcus ureae pyogenes, Rovsing.

equi, Schiitz ; S. coryzae contagiosae equorum, Eisen-

berg.
giganteus, Mig.; S. giganteus urethrae, Lustgarten et

Mannaberg.
gracilis, Mig.; S. coli gracilis, Escherich.
mastitidis, Guillebeau ; S. mastitidis sporadicae, Guille-

beau ; S. agalactiae contagiosa^, Kitt.
pyogenes, Rosenbach ; S. erysipelatos, Rosenbach ; S.

conglomeratus, Kurth ; S. brevis, v. Lingelsheim ; S.

longus, V. Lingelsheim ; S. murisepticus, v. Lingels-
heim ; S. septo-pyaemicus, Biondi.
septicus, Mig.; S. septicus liquefians, Babes; S. septicus

liquefaciens, Babes.

INDEX.

Acid alcohol, 173

carbolic, 44, 124

phenol-sulphonic, 94, 174

production, 94

white, 55
Acidity, determination of, 52

relation to, of medium, 91
Actinomyces bovis, 158
Actinomycosis, 158
Adhesive preparations of colonies,

75
Aerobic organisms, 75, 76, 78
Agar culture-medium, 51

cultures, 61
Air, analysis of, 121

bacteria in, 121
Algae, brown, 40

green, 40

red, 40
Alkalinity, determination of, 52

relation to, of medium, 91
Alkali production, 94
Amebabacteriaceae, 42
Amebacter, 42

American Public Health Associa-
tion, 52, 83, 85
Ammonia, production of, 92

test for, 93
Amphitrichous, 25
Anaerobic cultures, 78

organisms, 75, 76, 78, 80
Analysis of air, 121

of milk, 120

of soil, 122

of water, qualitative, 115
quantitative, 114
Anilin fuchsin, 171

gentian-violet, 171

stains, 18

water, 171
Anthrax, 130
Antiseptics, 44

Arnold's steam sterilizer, 46, 47, 48
Arrangement of bacteria, 85

Asiatic cholera, 27, 64
Aspergillus repens, 166, 168
Autoclave, 46, 49
Autopsy, making, 126

Bacilli, form of, 22

groups of, 109

reproduction of, 34

species of, 102
Bacillus, 39, 102, 109

acidi lactici I., 176

aerogenes capsulatus, 178

aeruginosus, 180

anthracis, 176

symptomatici, 175

anthracoides, 176

aquatile, 176

arborescens, 176

argenteo-phosphorescens. III.,
176

aurescens, 176
brunneus, 176
buccalis minutus, 178
candicans, 176
capsulatus, 176

septicus, 177
carbonis, 175
carotarum, 177
chauvaei, 38
chlorinus, 177
cholerae columbarum, 177

gallinarum, 177

suum, 176
citreus, 177
cloacae, 115
cceruleus, 117
coli, colony of, 146

for removing sugars, 56

in water, 115

isolation of, 119, 120

study of, 144

test for, 118
coli communis, 175
coprogenes fcetidus, 177

183

1 84

INDEX.

Bacillus coprogenes parvus, 176
cuniculi pneumonicus, 177
diphtherias, 136, 137, 138

avium, 175

columbarum, 177
dysenterioe liquefaciens, 175
endocarditidis capsulatus, 177
equi intestinalis, 175
fsecalis I., 178
felis septicus, 177
fluorescens albus, 181
liquefaciens, 181

minutissimus, 181
non-liquefaciens, 181
tenuis, 181
foetidus ozasnae, 175
g, Vignal, 178
indicus ruber, 175
janthinus, 181
lacticus, 177
lactis III., 177

XII., 178

acidi, 177

albus, 175

niger, 175
leprae, 177
limosus, 175
litoralis, 181
mallei, 177

membranaceus amethystinus, 176
mesentericus, 63

fuscus, 175

niger, 175

ruber, 175

vulgatus, 176
murinus, 177
murisepticus, 177
muscoides, 74
mycoides, 63

roseum, 177
nephritidis interstitialis, 177
CEdematis, 63
of hog cholera, 176
phosphorescens giardi, 177

indicus, 175

indigenus, 175
pneumonicus liquefaciens, 176
polypiformis, 69
prodigiosus, 80
pseudoinfluenzas, 177
pseudotuberculosis, 178

liquefaciens, 176
pyocinnabarius, 178
pyocyaneus, 180
pyogenes foetidus, 178

Bacillus radiatus, 63, 69
rhusiopathiae suis, 178
salivae minutissimus, 178
septicus keratomalaciae, 177
smegmatis, 152, 156, 178
sporogenes, 115
subtilis, 19, 35

simulans I. 178
suipestifer, 24
suisepticus, 178
syphilidis, 152
termophilus, 178
tuberculosis, 178

avium, 178
typhi abdominalis, 176

murium, 175
typhosus, colony of, 146

gelatin culture, 63

in milk, 120

in water, 115

isolation of, 115

showing flagella, 28, 147

study of, 144

Widal reaction, 145
ubiquitus, 178
varicosus conjunctivce, 178
vermicularis, 178
violaceus, 181

laurentius, 181
viscosus I., 177

cerevisias, 177
vulgaris, 19, 115
xerosis, 178
Bacteriaceae, 39, 99
Bacteria, classification of, 39, 99

by groups, 107
cultures of, 60
form of, 22
groups of. 107
in air, 121
in milk, 120

classification of, by groups, 112,
121
in soil, 122
in tissues, 20
in water, 114

classification of, by groups, 112
measurement of, 11, 163
morphology of, 22
pathogenic, 124
reproduction of, 34
species of, 99
Bacteridium cyaneum, 179

luteuni, 179
Bacteriologic committee, 52, 83, 85

INDEX.

185

Bacterio-purpurin, 39, 41
Bacterium, 39, 99, 107
aerogenes, 115
aeruginosum, 180
anthracis, colony of, 74, t] , 131
gelatin culture, 63
showing spores, 36
study of, 130, 132, 133
coli commune, 175
diphtheriae, 135, 136, 137, 138
diagnosis of, 135, 138
isolation of, 137
slide for examination of, 140
test for virulence, 139
erysipelatos suis, 178
faschingii, 30
ianthinum, 181
influenzae, 141, 142, 143
lactis aerogenes, 176
leprae, 152
mallei, 133, 134
parvum, 69
pneumoniae, 32, 150
pneumonicum, 150, 151
prodigiosum, 176
septicasmiae, 177
smegmatis, 152, 156, 178
tuberculosis, 76, 151
zopfii, 176
Basic fuchsin, 18, 170
Bath, paraffin and water, electric,

68
Beef. Liebig's extract of, 55
Beggiatoa, 41
Beggiatoaceae, 41
Berkefeld filter, 45
Binary division, 34
Biologic characters of a species, 83,

84
examination of water, 114
Bismarck brown, 20, 170
Blood agar, 141

examination in anthrax, 131
in malaria, 159, 161
Whitney's method, 161
preparations, 131 159, 161
Blood-serum cultures, 66
Loffler's, 58
tubes, 58
Boni's method for capsules, 33
Boston Board of Health sterilizer,

47
Bouillon, 51

cultures, 60
dextrose, 56

Bouillon, lactose, 56

saccharose, 56
Brown algae, 40
Brownian movement, 24, 25
Brown University paraffin and water

bath, 68
Bryophyta, 40
Buchner's method for anaerobes, 78

Camera lucida, 12
Capsules, demonstration of, 31
Carbohydrates, 92
Carbol-fuchsin, Ziehl's, 172
Carbolic acid, 44, 124
Carbol-thionin blue, 171
Cell, 15

Chamberland filter, 45
Characeae, 40

Chenzinsky-Plein stain, 173
Chester, 95, 107

Chester and Robin pipet, 147, 148
Chlamydobacteriaceae, 41
Chlamydothrix, 41
Chlorophyceae, 40
Cholera, Asiatic, 27, 64, 180
Chromatiaceae, 42
Chromatium, 42
Cladothrix intricata, 175
Classification of bacteria, 39
by groups, 107

of dairy bacteria by groups, 112,
121

of water bacteria by groups, 112
Cleaning mixture, 14, 174
Clostridium, 35
Club mosses, 40
Coccaceae, 39, 95
Cocci, 22
Coccus salivarius septicus, 179

stellatus, 179
Colon bacillus, 144
Colonies, counting, 116, 117, 118

impression, or adhesive prepara-
tions of, 75
Colony, 60
Concave slide, 15
Condenser, 11
Conjugatas, 40
Conjugates, 40
Conn, 112
Contamination of water by sewage,

115
Cornet's forceps, 17
Corrosive sublimate solution, 44,

124

1 86

INDEX.

Corynebacteriumpseudodiphtheriti-

cum, 177
Cotton, 48

Cotton-wool filter, 45
Counting-plate, Jeffer's, 117, 118

Wolfhiigel's, 116, 117
Cover-glass forceps, 17
Crenothrix, 41
Cryptogamia, 40
Culture-media, 51
Cultures, agar, 61

anaerobic, 78

blood-serum, 66

bouillon, 60

gelatin, 61

in the fermentation-tube, 76

of bacteria, 60

plate, 66

potato, 66

pure, 60

stab, 61

siroke, 64
\

Dairy bacteria, classification of,

112, 121
Demonstration of arrangement, 85

of capsules, 31

of division, 34

of flagella, 25

of form, 22, 85

of liquefying ferment, 80

of motion, 24, 90

of pleomorphism, 90

of spores, 35, 90
Determination of acid production,

94
of acidity of media, 52

of alkalinity of media, 52

of alkali production, 94

of arrangement, 85

of form, 22, 85

of motility, 24, 90

of name, 95

of pleomorphism, 90

of species, 83

of thermal death-point, 91
Dextrose, 56
Diagnosis of diphtheria, 135

of glanders, 134

of influenza, 141

slide for diphtheria, 140
Diaphragm, 11
Diatomeoe, 40
Diatoms, 40
Dinoflagellates, 40

Diphtheria, 135

diagnosis, 135
slide, 140

diagnostic stains, 138

inoculations of, 140

isolation of bacterium, 137

outfit for diagnosis, 136

test for virulence, 139
Diplococcus, 22, 34

albicans tardissimus, 180
tardus, 180

citreus conglomeratus, 179
liquefaciens, 178

coryzae, 179

flavus liquefaciens tardus, 180

intracellularis meningitidis, 179

roseus, 179

ureas pyogenes, 181
Discontinuous sterilization, 43
Disinfectant, 44
Disinfection, 44
Division, reproduction by, 34
Drumstick bacillus, 33

Ehrlich-Biondi stain, 173

Ehrlich's triacid stain, 172

Electric paraffin and water bath, 63

Endogenous spores, 34

Eosin, 20

Estivo-autumnal fever, 160

Etched tubes, 55

Eubacteria, 39

Examination of gonorrheal pus, 129

of tuberculous sputum, 152
Extract of beef, Liebig's, 55
Eyepiece, 11

Facultative anaerobes, 78
Fermentation-tubes, 57

cultures in, 76
Ferment, liquefying, 80
Ferns, 40
Fever, estivo-autumnal, 160

malarial, 159

quartan, 160

tertian, 160
Filter, 45

cotton-wool, 45

for culture-media, 53

Kitasato's, 46

porcelain, 45
Filtration, 45

of blood-serum, 59

of culture-media, 53
Fission-algce, 40

INDEX.

187

Fission-fungi, 40
Fission-plants, 40
Fixing blood smears, 131, 159, i6i.
Flagella, 25
Flugge, 95

Forceps, cover-glass, 17
Form, 22, 85
Formalin, 44

Frankel's instrument for collecting
soil, 122

pneumococcus, 150 !

Friedlander's pneumobacillus, 150 '
Fuchsin, anilin, 171

basic, 18, 170
Fuller and Johnson's groups of

water bacteria, 112
Fuller's method of titration, 52
Fungi, 40

Gabbet's blue, 172

method for sputum, 154
Gas, 77

formula, 78

production, 77

tests for, Tj
Gelatin culture-medium, 51

cultures, 61
Gentian-violet, 18, 170

anilin, 171
Germicide, 44
Germination of spores, 38
Glanders, 133

diagnosis of, 134

inoculation of, 133

isolation of bacterium, 135
Gonococcus, 129, 130, 179
Gonorrheal pus, examination of,

129
Graduated fermentation-tube, 57
Gram's iodin solution, 19, 172

method, 19

stain, 19
Granules, sulphur, 39, 41
Green algae, 40
Grouping, study of, 85
Groups, classification of bacteria by,
107
of milk bacteria, 112, 121
of water bacteria, 112

Halibacterium pellucidum, 175
Hanging drop, 15
Hematozoon malarise, 159
Hesse's apparatus for collecting bac-
teria in air, 120

Hill fermentation-tube, 57

Horsetails, 40

Hot-air sterilization, 47

sterilizer, 50
Hunt's stain for diphtheria, 139
Hyphomycetes, 40 ,

Identification of species, 83
of yeasts and moulds, 168

Immersion lens, 11
oil, II

Immunity, production of, 85

Impression preparation of colonies,

75
Indol production, 94
Influenza, 141

diagnosis of, 141
Inoculation of anthrax, 130

of diphtheria, 140

of glanders, 133

of plates, 66

of pneumonia, 151

of pus, 125

of sputum, 151

of tuberculosis, 155

of tubes, 61
Inoculations, 129

intraperitoneal, 133

intravenous, 125, 126, 151

subcutaneous, 130, 140, 151, 155
Instruments, sterilization of, 127
Intraperitoneal inoculations, 133
Intravenous inoculations, 125, 126

151
Involution forms, 90

Iodin solution. Gram's, 19, 172
Isolation of Bacillus coli, 119, 120
typhosus, 115
of Bacterium diphtheriae, 137
influenzas, 141
mallei, 135
tuberculosis, 155
of species, 81

Jeffer's counting plate, 117, 118
Johne's method for capsules, 32

Kalteyer's cover-glass forceps,

17
Kitasato's filter, 46
Koch-Ehrhch method for sections.

156
for sputum, 154

Koch's syringe, 126
Kiihne's methylene-blue, 171

i88

INDEX.

Lactose, 56
Lamprocystaceae, 42
Lamprocystis, 42
Leprosy, 152
Leptothrix buccalis, 176
Lichenes, 40
Lichens, 40

Liebig's extract of beef, 55
Life-history of a species, 83
Liquefying ferment, 80
Litmus milk, 58
Liverworts, 40
Lofifler's blood-serum, 58

method for flagella, 27
modification of, 29

methylene-blue, 171
Lophotrichous, 25

Malaria, 159

Malarial hematozoon, 159

Measurement of bacteria, ll, 163

Meat-extracts, 55

Meat-sugar, 56

Media, culture-, 51

Merismopedia, 22

flava varians, 180

fragilis, 179

goodsiri, 181

mesenterica corrugata, 179

mollis, 179

ventriculi, 181
Methylene-blue, 18, 170
Kiihne's, 171
Loffler's, 171
Unna's polychrome, 172
Mica, 6q
Micrococci, form of, 22

reproduction of, 34

species of, 96
Micrococcus, 39. 96

albicans amplus, 178

amylovorus, 175

aureus, 124, 125

cerasinus siccus, 181

citreus, 125, 181

conglomeratus, 179

cumulatus tenuis, 180

endocarditidis rugatus, 179

flavus conjunctivcE, 179
desidens, 179
tardigradus, 179

gonorrhcece, 129

lacteus fiiviformis, 179

lucens, 179

pflUgeri, 179

Micrococcus prodigiosus, 176

pyasmiae cuniculorum, 179

pyogenes, 125, 178
albus, 179
aureus, 178
tenuis, 180

tetragenus, 125
subflavus, 179

urese liquefaciens, 179
Micrometer, eyepiece, 12

stage, 12
MicromilHmeter, 12
Micron, 12
Microscope, 11

simple, for counting colonies, 116
Microspira, 41, 106, 11 1

comma, 27, 64, 180

finkleri, 65

metschnikovi, 65
Migula, 39, 95
Milk, analysis, 120

classification of bacteria in, 112,
121

culture-medium, 58

litmus, 58

tubercle bacteria in, 157
Mirror, 11

Monas prodigiosa, 176
Monotrichous, 25
Mordant, 25, 27, 29
Morphologic characters of a species,

83, 84
Morphology of a species, 83

of bacteria, 22
Mosses, 40
Motility, test for, 90
Motion, 24, 90
Moulds, 165

cultures of, 166

examination of, 166

identification of 168

staining, 166
Mouse-holder, 129
Movement, Brownian, 24, 25
Mucor racemosus, 165, 166
Museum preparations, 76
Mycoderma aceti, 176
Myconostoc gregarium, 181
Mycoprotein, 19
Mykobacterium tuberculosis, 178

avium, 178
Myxomycetes, 40

Name, determination of, 95
Needle, platinum, 14

INDEX.

189

Neisser's stain for diphtheria, 139
Nessler's solution, 174
Neutrahzation, 52
Nitrates, 92

test for, 93
Nitrites, production of, 92

test for, 92
Nitrosomonas europoea, 181

javaniensis, 181
Normal salt solution, 173
Novy's jars, 79

Objectives, ii -
Obligatory aerobes, 78
anaerobes, 78

Paraffin bath, 68
Park's method for anaerobes, 80
Pasteurization, 44
Pathogenesis, 85, 94
Pathogenic bacteria, 124
Penicillium crustaceum, 167, 168
Peridineae, 40
Peritrichous, 25
Petri dish, 67
Petri's sand filter, I2i
Phaeophyceae, 40
Phanerogamia, 40
Phenolphthalein, 52
Phenol-sulphonic acid, 174
Photobacterium annulare, 180
caraibicum, 180
coronatum, 180
degenerans, 180
delgadense, 180
fischeri, 175
glutinosum, 180
indicum, 175
javanense, 181
luminosum, 180
papillare, 180
phosphorescens, 177, 179
tuberosum, 180
Photography, measuring bacteria

by, 163
Phragmidiothrix, 41
Pigment, production of, 85
Pipet, Chester and Robin, for ma-
larial blood, 147. 148
Pitfield's method for flagella, 25

modification of, 26
Planococcus, 39
Planosarcina, 39
Plants, 40
Plate, counting, Jeffer's, 117, 118

Plate, counting, Wolfhiigel's, 116,
117

cultures, 66
Platinum needles, 14

wire, 14
Pleomorphism, 90
Pneumobacillus, 150

liquefaciens bovis, 176
Pneumococcus, 150
Pneumonia, 150

inoculation of 151

Polar staining, 24

Polychrome methylene-blue,Unna's,

172
Porcelain filter, 45
Potato, 55

cultures, 66
Potato-tubes, 56
Production of acid, 94

of alkah, 94

of ammonia, 92

of gas, 77

of immunity, 85

of indol, 94

of liquefying ferment, 80

of nitrites, 92

of pigment, 85

of spores, 35, 90

of toxin, 85
Proteus capsulatus septiciis, 177

mirabilis, 175,

sulphureus, 176

vulgaris, 176

zenkeri, 176
Pseudomonas, 39, 105
Pteridophyta, 40
Pure cultures, 60
Pus, gonorrheal, staining of, 129

inoculation of 125
Pyogenic organisms, 125

Qualitative analysis of water,

IIS, 118
Quantitative analysis of water, 114
Quartan fever, 160

Reagents, 170

Red algae, 40

Reproduction by division, 34

by spores, 35

of bacteria, 34
Rhabdochromatium, 42
Rhodobacteriaceae, 41
i Rhodophyceae, 40

1 90

INDEX.

Rosenberger's method for sections,

156
for sputum, 154

Saccharomyces cerevisise, 165,

166
Saccharomycetaceae, 168
Saccharose, 56
Safranin, 20, 170
Salt solution, normal, 173
Sand filter, Petri's, 121
Sarcina, 34, 39, 98

fuscescens, 181

tetragena, 125, 128
Schizomycetes, 39, 40
Schizophyceae, 40
Schizophyta, 40
Sections, staining, 20, 129
of actinomycosis, 158
of glanders, 133
of tuberculosis, 156
Sedgwick's tube for air analysis,

121
Sewage, 115

bacteria, 115

contamination, detection of, 115
Slide for diphtheria, diagnosis, 140

for Widal reaction, 148, 149
Slime-fungi, 40
Sliver, 48

Smegma bacterium, 152, 156, 178
Smith fermentation-tube, 57
Soil analysis, 122

bacteria, 122
Species, determination of, 83

isolation of, 81

life-history of, 83

morphology of, 83

names of, 95
Sphaerococcus acidi lactici, 179
Sphcerotilus, 41
Spirilla, 22
Spirillaceae, 41, 106
Spirillum, 41

attenuatum, 181

cholerge Asiaticas, 180

finkleri, 180

marinum, 180
Spirochseta, 23, 41
Spirosoma, 41, 106
Spores, 34

endogenous, 34

germination of, 38

production of, 35, 90

reproduction by, 35

Spores, staining, 36

test for, 90
Sputum, examination of influenza,

43
of tuberculous, 152
inoculation of, 151
tubercle bacteria in, 153, 155, 157
Stab cultures, 61
Stain bottles, 18
Gram's, 19
Hunt's, 139
Neisser's. 139
Staining actinomycosis, 158
anthrax blood, 131
bacteria in tissues, 20, 129
blood for malarial organism, 159,
161
preparations, 131, 161
diphtheria bacteria, 137, 138
flagella, 25
gonorrheal pus, 129
influenza sputum, 143
malarial blood, 159
pneumonia bacteria, 150
sections, 20, 129

for actinomycosis, 158
for glanders, 133
for tubercle bacteria, 156
spores, 36

tubercle bacteria, 152
Stains, 18, 170
anilin, 170
ordinary, 16
Staphylococcus, 22, 34
cereus albus, 178
aureus, 178
flavus, 178
pyogenes albus, 125, 179
aureus, 125, 178
citreus, 125, 178
salivarius pyogenes, 179
Steam sterilization, 46
under pressure, 46
sterilizers, 46, 47, 48, 49
Sterilization, 43
by hot air, 47
by steam, 46

under pressure, 46
discontinuous, 43
intermittent, 43
of instruments, 127
of syringes, 125
Sterilizer, 46

Arnold, 46, 47, 48
hot-air, 50

INDEX.

191

Sterilizer, steam, 46, 47, 48, 49

Sternberg, 95

Stewart's cover-glass forceps, 17

Stonewort, 40

Streptobacillus pseudotuberculosis

rodentium, 178
Streptococci, 22, 34

species of, 95
Streptococcus, 39, 95

agalactiae contagiosas, 182

brevis, 182

coli gracilis, 182

conglomeratus, 182

coryzae contagiosas equorum, 182

erysipelatos, 182

giganteus urethrae, 182

intracellularis, 179

longus, 182

mastitidis sporadicae, 182

murisepticus, 182

pyogenes, 125, 127, 128

septicus liquefaciens, 182
liquefians, 182

septo-pyaemicus, 182
Stroke cultures, 64
Subcutaneous inoculations, 130, 14c,

151. 155
Synonyms, table of, 175
Syphilis, 152
Syringe, Koch's, 126
Syringes, sterilization of, 125

Tertian fever, 160
Test for ammonia, 93

for Bacillus coli, 118

for motility, 90

for nitrates, 93

for nitrites, 92

for spores, 90

for virulence, Bact. diphtheriae,

139
Tetragenococci, 22 *

Thallophyta, 40
Thermal death-point, determination

of, 91
Thiobacteria, 41
Thiocapsa, 42
Thiocapsaceae, 42
Thiocystitis, 42
Thiodictyon, 42
Thionin blue, 170
Thiopedia, 42
Thiopediaceae, 42
Thiopolycoccus, 42
Thiosarcina, 42

Thiospirillum, 42
Thiothece, 42
Thiothrix, 41

Tissues, staining bacteria in, 20,
129
in actinomycosis, 158
in glanders, 133
in tuberculosis, 156
Titration, 52
Toxin, production of, 85
Triacid stain, Ehrlich's, 172
Tubercle bacteria, cultures of,
152
in milk, 157

in sputum, 152, 153, 155. 157
in urine, 156

staining, Gabbet's method, 154
in sections. 156
Koch-Ehrlich method, 154
Rosenberger's method, 154
Ziehl-Neelsen method, 152
Tuberculosis, 151
inoculation, 155
Tuberculous sputum, examination

of, 152
Tubes, etched, 55
Typhoid bacilli, 115, 144
fever, 115, 144

Ulvina aceti, 176

Unna's polychrome methylene-blue,

172
Urine, tubercle bacteria in, 156

Van Ermengem's method for fla-
gella, 30
modification of, 30
Vaselin, 15

Vibrio cholerse Asiaticas, 180
comma, 180
dunbari, 180
finkleri, 180
flavescens, 181
flavus, 181
metschnikovi, 180
proteus, 180
saprophiles /3, 180

V. 180
tonsillaris, 180
Virulence, test for, Bact. diphtheriae,
139

Water analysis, qualitative, 115
quantitative, 114
bacteria, classification of, 112

192

INDEX.

Water bath, 68

collection of, for analysis, 115
contamination of, by sewage, 115
Welch's method for capsules, 31
White acid, 55

Whitney's method for blood, 161
Widal reaction, 145, 149

slide, 148, 149
Wilson and Randolph's method for

measuring bacteria, 163
Wire, platinum, 14
Wolfhiigel's counting plate, 116,
117

Wright's method for growing anae-
robes, 79

Yeasts, 165, 166
cultures of, 166
identification of, 168
staining, 166

Zenker's fluid, 20, 173
Ziehl-Neelsen method for sections,

156
for sputum, 152
Ziehl's carbol-fuchsin, 172
Zylonite, 15

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will be more than a mere translation of the German ; for, in addition to the matter contained
in the original, it will represent the very latest views of the leading American special-
ists in the various departments of Internal Medicine. The whole System will be under the
editorial supervision of Dr. Alfred Stengel, who will select the subjects for the American
edition, and will choose the editors of the different volumes.

Unlike most encyclopedias, the publication of this work will not be extended over a
number of years, but five or six volumes will be issued during the coming year, and the
remainder of the series at the same rate. Moreover, each volume will be revised to the
date of its publication by the American editor. This will obviate the objection that has
heretofore existed to systems published in a number of volumes, since the subscriber will
receive the completed work while the earlier volumes are still fresh.

The usual method of publishers, when issuing a work of this kind, has been to compel
physicians to take the entire System. This seems to us in many cases to be undesirable.
Therefore, in purchasing this encyclopedia, physicians will be given the opportunity of
subscribing for the entire System at one time; but any single volume or any number of
volumes may be obtained by those who do not desire the complete series. This latter
method, while not so profitable to the publisher, offers to the purchaser many advan-
tages which will be appreciated by those who do not care to subscribe for the entire work
at one time.

This American edition of Nothnagel's Encyclopedia will, without question, form the
greatest System of Medicine ever p«oduced, and the publishers feel confident that it
will meet with general favor in the medical profession.

i8

NOTHNAGEL^S ENCYCLOPEDIA

VOLUMES JUST ISSUED AND IN PRESS

VOLUME I

Editor, William Osier, M» D.,

F.R.CP,

Professor of Medichie in Johns Hopkins
University

CONTENTS

Typhoid Fever. By Dr. H. Curschmann,
of Leipsic, Typhus Fever. By Dr. H.
Curschmann, of Leipsic.

Handsome octavo volume of about 600 pages.
Just Issued

VOLUME n

Editor, Sir J. W, Moore, B. A., M. D„

F.R.CP,I., of Dublin

Professor of Practice of JSIedicine, Royal
College of Surgeons in Ireland

CONTENTS

Erysipelas and Erysipeloid. By Dr. H.
Lenhartz, of Hamburg. Cholera Asi-
atica and Cholera Nostras. By Dr.
K. vo.x LiEiiKRMEiSTER, of Tiibingeii.
Whooping Cough and Hay Fever. t!y
Dr. G. Sticker, of Giessen. Varicella.
By Dr. Th. von JI'rgensen, of Tiibingen.
Variola (including Vaccination). By
Dr. H. Immermann, of Basle.

Handsome octavo volume of over 700 pages.
Just Issued

VOLUME m
Editor, William P. Northrup, M. D.

Professor of Pediatrics, University and
Belleviie Aledical College

CONTENTS

Measles. By Dr. Th. von Jltrgensen, of
Tiibingen. Scarlet Fever. By the same
author. Rotheln. By the same author.

VOLUME vn

Editor, John H. Musser, M. D.

Professor of Clinical Medicine, University
of Pennsylvania

CONTENTS

Diseases of the Bronchi. By Dr. F. A.
HoFFM.\NN, of Leipsic. Diseases of the
Pleura. By L)r. Rosenbach, of Berlin.
Pneumonia. By Dk. E. Alfrecht, of
Magdeburg.

VOLUME vni

Editor, Charles G. Stockton, M. D.

Professor of Medicine, University of Buffalo

CONTENTS '

Diseases of the Stomach. By Dr. F.
RiECiEL, of Giessen.

VOLUME DC
Editor, Frederick A» Packard, M. D.

Physician to the Pennsylvania Hospital and
to the Children's Hospital, Philadelphia

CONTENTS

Diseases of the Liver. I'.y Drs. H.
Quincke and G. Hoppe-Seylek, of Kiel.

VOLUME VI
Editor, Alfred Stengel, M. D,

Professor of Clinical IMedicine, University
of Pennsyh'ania

CONTENTS

Anemia. By Dr. P. Ehrlich, of Frank-
fort-on-the-AIain, and Dr. A. Laz.\rus, of
Charlottenburg. Chlorosis. By Dr. K.
VON Noorden, of Frankfort-on-the-Main.
Diseases of the Spleen and Hemor-
rhagic Diathesis. By Dr. AL Litten,
of Berlin.

VOLUME X
Editor, Reginald H. Fitz, A» M., M,D«

Hersey Professor of the Theory and Prac-
tice of Physic, Harvard University

CONTENTS

Diseases of the Pancreas. By Dr. L.
OsER, of Vienna. Diseases of the Supra-
renals. By Dr. E. Neussek, of Vienna.

VOLUMES IV, V, and XI

Editors announced later

Vol. IV. — Influenza and Dengue. By Dr.
O. Leichtenstein, of Cologne. Malarial
Diseases. By Dr. J. Mannaberg, of

\'ienna.

Vol.V. — Tuberculosis and Acute General
Miliary Tuberculosis. By Dr. G. Cor-
net, of Berlin.

Vol. XL — ^Diseases of the Intestines and
Peritoneum, By Dr. H. Nothnagel,
of Vienna.

19

CLASSIFIED LIST

OF THE

MEDICAL PUBLICATIONS

OF

W. B. SAUNDERS & COMPANY

ANATOMY, EMBRYOLOGY, HIS-
TOLOGY.

Bohm, Davidoff, and Huber— A Text-
Book of Histology, 4

Clarkson— A Text-Book of Histology, . 5

Haynes — A Manual of Anatomy, ... 7

Heisler — A Text-Book of Embryology, . 7

Leroy — Essentials of Histology, .... 15

Nancrede — Essentials of Anatomy, . . . 15
Nancrede — Essentials of Anatomy and

Manual of Practical Dissection, .... 10

BACTERIOLOGY.

Ball — Essentials of Bacteriology, .... 15

Frothingham— Laboratory Guide, . . . 6

Gorham — Laboratory Course in Bacte-
riology, 22

Lehmann and Neumann — Atlas of
Bacteriology, 17

Levy and Klemperer's Clinical Bacte-
riology, 9

Mallory and Wright — Pathological
Technique, 9

McFarland — Pathogenic Bacteria, ... 9

CHARTS, DIET-LISTS, ETC.

Griffith — Infaiat's Weight Chart, .... 7

Hart — Diet in Sickness and in Health, . 7

Keen— Operation Blank, 8

Laine — Temperature Chart, 9

Meigs — Feeding in Early Infancy, ... 10

Starr — Diets for Infants and Children, . 12

Thomas — Diet-Lists, 13

CHEMISTRY AND PHYSICS.

Brockway — Essentials of Medical

Physics, 15

Wolff— Essentials of Medical Chemistry, 15

CHILDREN.
An American Text-Book of Diseases

of Children i

Griffith- Care of the Baby, 7

Griffith— Infant's Weight Chart, .... 7
Meigs — Feeding in Early Infancy, ... 10
Powell — Essentials of Diseases of Chil-
dren, 15

Starr — Diets for Infants and Children, . 12

DIAGNOSIS.

Cohen and Eshner — Essentials of Diag-
nosis 15

Corwin — Physical Diagnosis, 5

Vierordt — Medical Diagnosis, 14

DICTIONARIES.

The American Illustrated Medical

Dictionary 3

The American Pocket Medical Dic-

3

Dictionary, lo

tionary, . .
Morton— Nurses'

EYE, EAR, NOSE, AND THROAT.

An American Text-Book of Diseases

of the Eye, Ear, Nose, and Throat, . . i
De Schweinitz — Diseases of the Eye, . 6
Friedrich and Curtis — Rhinology, Lar-
yngology, and Otology, 6

Gleason — Essentials of the Ear, .... 15
Gleason — Essentials of Nose and Throat, 15
Gradle — Ear, Nose, and Throat, .... 22
Grunwald and Grayson — Atlas of Dis-
eases of the Larynx, i6

Haab and de Schweinitz — Atlas of Ex-
ternal Diseases of the Eye 16

Jackson — Manual of Diseases of the Eye, 8
Jackson — Essentials Diseases of Eye, . 15
Kyle — Diseases of the Nose and Throat, 9

GENITO-URINARY.

An American Text-Book of Genito-
urinary and Skin Diseases, 2

Hyde and Montgomery — Syphilis and
the Venereal Diseases, 8

Martin — Essentials of Minor Surgery,
Bandaging, and Venereal Diseases, . . 15

Mracek and Bangs— Atlas of Syphilis
and the Venereal Diseases, 16

Saundby — Renal and Urinary Diseases, 11

Senn — Genito-Urinary Tuberculosis, . . 12

Vecki — Sexual Impotence, 14

GYNECOLOGY.

American Text-Book of Gynecology, . a

Cragin — Essentials of Gynecology, ... 15

Garrigues— Diseases of Women, . ... 6

Long — Syllabus of Gi^necology, .... 9

Penrose — Diseases of Women, 10

Pryor — Pelvic Inflammations, 11

Schaeffer and Norris — Atlas of Gyne-
cology, 17

HYGIENE.

Abbott — Hygiene of Transmissible Dis-
eases, 3

Bergey — Principles of Hygiene, .... 22
Pyle — Personal Hygiene, 11

MATERIA MEDICA, PHARMA-
COLOGY, and THERAPEUTICS.
An American Text-Book of Applied

therapeutics, i

Butler — Text-Book of Materia Medica,

Therapeutics, and Pharmacology, . . 4
Morris — ^Ess.ofM. M. and Iherapeutics, 15
Saunders' Pocket Medical Formulary, . 11
Sayre — Isssentials of Pharmacy, .... 15
Sollmann — Text-Book of Pharmacology, 22
Stevens — Modern Therapeutics, .... 13
Stoney — Materia Medica for Nurses, . . 13
Thornton — Prescription-Writing, ... 13

20

MEDICAL PUBLICA TIONS

21

MEDICAL JURISPRUDENCE AND
TOXICOLOGY.

Chapman — Medical Jurisprudence and
Toxicology 5

Golebiewski and Bailey — Atlas of Dis-
eases Caused by Accidents, 17

Hofmann and Peterson— Atlas of Legal
Medicine, 16

NERVOUS AND MENTAL DIS-
EASES, ETC.

Brower — Manual of Insanity, 22

Chapin — Compendium of Insanity. ... 5

Church and Peterson — Nervous and 5

Mental Diseases 5

Jakob and Fisher — Atlas of Nervous

System, 17

Shaw — Essentials of Nervous Diseases

and Insanity, 15

NURSING.
Davis — Obstetric and Gynecologic Nurs-
ing,

Griffith — The Care ot the Baby, . .
Hart — Diet in Sickness and in Health,
Meigs — Feeding in Early Infancy, .
Morten — Nurses' Dictionary, . . .
Stoney— Materia Medica for Nurses,
btoney — Practical Points in Nursing,
Stoney — Surgical Technic for Nurses,
Watson— Handbook for Nurses. . . .

OBSTETRICS.

An American Text-Book of Obstetrics,
Ashton — Essentials of Obstetrics, .
Boisliniere — Obstetric Accidents
Borland — Modern Obstetrics, ,
Hirst — Text-Book of Obstetrics, .
Norris — Syllabus of Obstetrics, . .
Schaeffer and Edgar— Atlas of Obstet-
rical Diagnosis and Treatment, . . . .

6
7
7

ID
10
13
13
13
14

2

4
6

7
10

17

PATHOLOGY.

An American Text-Book of Pathology, 2
Durck and Hektoen — Atlas of Patho-
logic Histology', 16

Kalteyer — Essentials of Pathology', . . 15
Mallory and Wright — Pathological

Technique, 9

Senn — Pathology, and Surgical Treat-
ment of Tumors, 12

Stengel— Text-Book of Pathology, ... 12

Warren — Surgical Pathology, .... 14

PHYSIOLOGY.

American Text-Book of Physiology, . 2

Budgett — E.ssentials of Physiology, . . 15

Raymond — Text-Book of Physiology, . ir

Ste\A'art — Manual of Physiology, . . 13

PRACTICE OF MEDICINE.

An American Year-Book of Medicine

and Surgery, 3

Anders — Practice of Medicine 4

Eichhorst — Practice of Medicine, ... 6

Lockwood — Practice of Medicine, . . . 9

Morris — Ess. of Practice of Medicine, . 15

Salinger & Kalteyer — Mod. Medicine, 11

Stevens — Practice of Medicine, .... 13

SKIN AND VENEREAL.

An American Text-Book of Genito-
urinary and Skin Diseases, 2

Hyde and Montgomery — Syphilis and
the Venereal Diseases, 8

Martin — Essentials of Minor Surgery,
Bandaging, and Venereal Diseases, . . 15

Mracek and Stelwagon— Atlas of Dis-
eases of the Skin, i6

Stelwagon— Essentials of Diseases of
the Skin, 15

SURGERY.

An American Text-Book of Surgery, 2
An American Year-Book of Medicine

and Surgery, . . 3

Beck— Fractures, 4

Beck — Manual of Surgical Asepsis, ... 4

Da Costa — Manual of Surgery, 5

International Text-Book of Surgery, , 8

Keen— Operation Blank, 8

Keen — The Surgical Complications and

Sequels of Typhoid Fever, 8

Macdonald — Surgical Diagnosis and

Treatment, 9

Martin — Essentials of Minor Surgery,

Bandaging, and Venereal Diseases, . . 15

Martin — Essentials of Surgery, 15

Moore — Orthopedic Surgery, 10

Nancrede — Principles of Surgery, ... 10

Pye — Bandaging and Surgical Dressing, 11

Scudder — Treatment of Fractures, ... 12

Senn — Genito-Urinary Tuberculosis, . . 12

Senn— Practical Surgery, 12

Senn — Syllabus of Surgery, 12

Senn — Pathology and Surgical Treat-
ment of Tumors, 12

Warren — Surgical Pathology and Ther-
apeutics, 14

Zuckerkandl and Da Costa — Atlas of

Operative Surgery, 16

URINE AND URINARY DISEASES.
Ogden — Clinical Examination of the

Urine, 10

Saundby — Renal and Urinary Diseases, 11
Wolf — Handbook of Urine Examination, 22
Wolff — Examination of Urine, 15

MISCELLANEOUS.
Abbott — Hygiene of Transmissible Dis-
eases, 3

Bastin — Laboratory Exercises in Bot-
any 4

Golebiewski and Bailey — Atlas of Dis-
eases Caused by Accidents, . . . . 17
Gould and Pyle — Anomalies and Curi-
osities of Medicine, 7

Grafstrom — Massage, 7

Keating — Examination for Life Insur-
ance, 8

Pyle — A Manual of Personal Hygiene, . 11
Saunders' Medical Hand-Atlases, . 16, 17
Saunders' Pocket Medical Formulary, . 11
Saunders' Question-Compends, . . 14, 15
Stewart and Lawrence — Essentials of

Medical Electricity, 15

Thornton — Dose-Book and Manual of

Prescription-Writing 13

Van Valzah and Nisbet — Diseases of
the Stomach, . 13

THE LATEST BOOKS,

Bergey's Principles of Hygiene.

The Principles of Hygiene : A Practical Manual for Students,
Physicians, and Health Officers. By D. H. Bergey, A.M., M. D.,
First Assistant, Laboratory of Hygiene, University of Pennsyl-
vania. Handsome octavo volume of about 500 pages, illus-
trated.

Brower^s Manual of Insanity^

A Practical Manual of Insanity. By Daniel R. Brower, M.D.,
Professor of Nervous and Mental Diseases, Rush Medical Col-
lege, Chicago. i2mo volume of 425 pages, illustrated.

Gorham^s Bacteriology.

A Laboratory Course in Bacteriology. By F. P. Gorham,
M. A., Assistant Professor in Biology, Brown University. i2mo
volume of about 160 pages, fully illustrated.

Gradle on the Nose, Throat, and Ear.

Diseases of the Nose, Throat, and Ear. By Henry Cradle,
M. D., Professor of Ophthalmology and Otology, Northwestern
University Medical School, Chicago. Handsome octavo volume
of 800 pages, profusely illustrated.

SoIImann^s Pharmacology.

A Text-Book of Pharmacology. By Torald Sollmann, M. D.,
Lecturer on Pharmacology, Western Reserve University, Cleve-
land, Ohio. Royal octavo volume of about 700 pages.

Wolffs Examination of Urine.

A Handbook of Physiologic Chemistry and Urine Examination.
By Charles G. L. Wolf, M. D., Instructor in Physiologic Chem-
istry, Cornell University Medical College. i2mo volume of about

160 pages.

22

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