Cornell University Library

BOUGHT WITH THE INCOME
FROM THE

SAGE ENDOWMENT FUND
THE GIFT OF

Henry W. Sage

1891

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Cornell University Library
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BACTERIOLOGICAL DIAGNOSIS

FOR

PRACTITIONERS
HANDBOOK

OF

BACTERIOLOGICAL DIAGNOSIS

FOR

PRACTITIONERS

INCLUDING INSTRUCTIONS FOR THE CLINICAL
EXAMINATION OF THE BLOOD

BY

W. D’ESTE EMERY, M.D., B.Sc. Lonp.

LECTURER ON PATHOLOGY AND BACTERIOLOGY IN THE UNIVERSITY OF
BIRMINGHAM

PHILADELPHIA
P. BLAKISTON’S SON & Co.
ror2 WALNUT STREET

1902
PRINTED BY
H. K, LEWIS, 136 GOWER STREET,

LONDON, W.C.
PREFACE.

No practitioner who wishes to do his best for his
patients and to promote his own interests can afford to
neglect any means of clinical investigation which may
help him to arrive at a correct diagnosis, and offer hints
as to prognosis and treatment. Pre-eminent among the
more recent methods of investigation are those which
are applied by the bacteriologist; and it is no exaggera-
tion to say that in many of the infective diseases a
diagnosis which is made without a_bacterioscopic
examination is either mere guess-work or can only be
made so late that the patient has suffered unnecessarily
in health and the practitioner in prestige. In many
cases, however, the investigation requires a consider-
able amount of technical skill and access to a well-
equipped laboratory; the former may perhaps be pos-
sessed by the rising generation (for bacteriology is now
an integral part of the medical curriculum), but it
would be unfair to expect every medical man to add
the latter to his already expensive equipage. But in
many cases the diagnosis can be arrived at by very
simple means—a few slides, cover-glasses, and stains,
a good microscope (which ought to be considered as
essential as a stethoscope), and a very moderate amount
of technical skill will often enable the practitioner to
arrive at correct diagnosis in a very short time. This
little book is intended in the first instance to show
exactly when this may be done, and to provide clear,
succinct, and full descriptions of simple methods which
may be employed. ‘The descriptions of the operations
which the practitioner can carry out for himself are
mostly written in the imperative mood, and are in-
tended to be referred to constantly and carried out, step
by step, during the process. They represent the in-
vl PREFACE.

structions which would be given by a teacher when
watching a beginner making a simple bacteriological
examination for the first time.

It cannot be too strongly urged that the practitioner
should make the examination for himself whenever it is
possible for him to do so. The report which is sent
from a public laboratory may often be of very consider-
able value, but it must be remembered that the bac-
teriologist can only supply facts, and the inferences
which may be drawn from those facts will largely
depend upon a knowledge of the patient’s clinical
history and the method in which the material was
obtained. The bacteriologist is too often in the posi-
tion of a detective who has to unravel a mystery from
observations made by other people, and has no oppor-
tunity of making investigations for himself. A bacterio-
logical examination which is made by one person and
interpreted by another, or which is made on material
which has passed through more hands than one, loses
much of its value; and an investigation made on the
spot may be more valuable than one made by a bac-
teriologist of far greater experience at a distance.

The methods which are described in this little book
are not in all cases the ideal ones, and in some cases
they are somewhat different from those which are
generally used, but they are simple and efficient. Of
course, the simple examinations which are described
here would frequently be supplemented by more com-
plicated cultural ones by a trained bacteriologist.
The methods described here have been taught in
the post-graduate classes which were initiated some
two years ago in the University of Birmingham;
these have already been attended by about a hundred
practitioners, who have found these methods of great
assistance to them in their everyday practice. They
have been selected so as to provide examples of some
of the more important operations in constant use in
the bacteriological laboratory. The author takes this
opportunity of expressing his cordial thanks to Professor
Leith for his kind suggestions as to the general scope
of the book.
PREFACE, vil

No apology will be made for the numerous repetitions
which will be found in this book. They are essential
to its scope, which is to give clear accounts of the
processes with as little reference to other chapters as
possible. In the majority of cases each section is
complete in itself.

These instructions are followed by information as to
the interpretation of the results which may be obtained ;
and this information applies equally whether the medi-
cal man has made the examination for himself or has
obtained it ready made from a public laboratory. It too
commonly happens that practitioners feel themselves
aggrieved because they get a negative report (as to the
presence or absence of Widal’s reaction) on blood taken
during the first few days of an illness which turns out to
be typhoid fever, or are inclined to discredit bacterio-
logical examinations because diphtheria bacilli are found
in throats which exhibit no membrane and clear up in a
few days without serious symptoms.

In the second place, there are a good many cases in
which the investigation had better be made in a public
laboratory. In these the questions, of what to send,
and how to send it, are fully explained. This a most
important point. A bacteriologist is not a magician
who is able to weave a spell if he has a small portion of
his victim’s anatomy to work upon; and the materials
must be taken in the proper way if his results are not to
be useless or even misleading. This is well seen in the
examination of the blood for bacteria. In many cases the
blood is drawn in such a manner that it must necessarily
be contaminated from the skin during the operation
and is transmitted in vaccine tubes which were almost
certainly not sterile before being filled. Under such
circumstances the bacteriologist will probably report
the presence of streptococci or staphylococci, and the
practitioner who does not understand the fallacies of
the examination may be led to make a diagnosis which
will be disastrous to his own reputation and may be
injurious to the patient.

Lastly, it need scarcely be said that this is not
intended to be a substitute for any one of the numerous
vili PREFACE.

excellent works on the science of bacteriology which are
current at the present time. The practitioner is strongly
recommended to supplement the very meagre details
concerning the life-history and pathogenic action of the
bacteria which are dealt with here by a study of one
of these textbooks. Muir and Ritchie’s admirable
‘Manual of Bacteriology,” Crookshank’s ‘“‘ Bacteriology
and Infective Diseases,’ Hewlett’s ‘ Bacteriology,”
Klein’s ‘ Micro-organisms and Disease,” McFarland’s
“Textbook upon the Pathogenic Bacteria,” or Curtis’s
‘Essentials of Practical Bacteriology” are all suitable
for this purpose, and a perusal of any one of them will
be both pleasurable and profitable to every medical
man.

Several illustrations have been borrowed from sources
mentioned in the text and for the loan of these the
author wishes to express his best thanks to the respec-
tive authors and publishers. His best thanks are also
due to Messrs. Baird and Tatlock, Swift and Son,
Zeiss, Leitz, Hawksley, and Hearson, for kindly pro-
viding illustrations of apparatus made by them.
CONTENTS.

PART I.
APPARATUS AND PROCESSES.

The Bacteriological Microscope
Sterilisation of Apparatus
Preparation of Culture Media
Inoculation of Culture Media
Incubation of Cultures

Method of Examining Cultures
Gram’s Method of Staining
Examination of Films—Use of Microscope
Stains : : 7
Cleaning Slides and Cover-Glasses
Pipettes

PART II.

DIAGNOSIS OF CERTAIN DISEASES.

Diphtheria

Tetanus : -
The Pneumococcus, Pneumonia, &c.
Influenza

Anthrax .

Tubercle

Leprosy

Actinomycosis

Glanders .

Typhoid Fever

Gonorrhea

Cholera

Plague

Soft Sore

Ringworm

PAGE

12
22
25
29
32
34
37
39
40

43
55
60
64
65
72
79
80
82
83
99
105
107
109
III
X CONTENTS.

PART III.
COLLECTION AND EXAMINATION OF CERTAIN MORBID
MATERIALS.

PAGE
The Collection of Pathological Exudates_ . : - 118
The Pleura : . . : : 120
Fluids from Joints i . : . 124
Lumbar Puncture . ‘ : 126
The Collection of Pus. . ; + 135
Collection of Material at Post-mortem Examinations i 140
Examination of the Blood . . . . %I4r
Estimation of the Red Corpuscles : : 5 142
Estimation of the Number of Leucocytes_ . : » 52
Estimation of the Amount of Haemoglobin 3 y 155
Preparation of Films for Staining . : + I60
Fixation of Blood Films : 7 164
Staining Blood Films for the Investigation of ier Cells » 166
The Bacteriological Examination of the Blood . . 169
Examination for Bacteria in Films : : - I7I
Malaria . . . 173
Collection of Blood i Hyannaaton by Cultural Methods » 175
Examination of Blood by Cultural Method - 7 180
Section Cutting : é . - 182
Fixing Material for Cutting ‘i . ¢ 185
Section Cutting by the Freezing Method , < . 187
Staining and Mounting Sections . . : 190
The Paraffin Process . : t a » Ig2
Dehydration : : F ‘ E Ig2
Clarification . : : + 193
Infiltration with Paraffin : . 2 ; 193
Casting the Blocks 5 - : ‘i . 94
Cutting the Section A : 195
Staining and Mounting Paraffin Béetione : ‘ - 198
Appendix 7 3 i : i 205

Index : ‘ : ‘ : » 209
LIST OF ILLUSTRATIONS.

IG.
1. Bacteriological Microscope (Leitz)

2.

Bacteriological Microscope (Swift)

3 & 4. Portable Microscope (Swift)
5 & 6. Hot Air Sterilizer (Baird & Tatlock)

7
8.
Oe

TO.

Il.

12.

13.

14.

I5.

16,

17.

18.

IQ.

20x

21

22.

23%

24.

25.

26.

28.
29.
30.
BI.
32.
33+

Steam Sterilizer (Baird & Tatlock)
Platinum Needles (Crookshank)

Method of inoculating cultures

Hearson’s Incubator

Cornet’s Forceps (Baird & Tatlock)

Pipettes .

Petri’s Dish (Baird & Tatlock) .
Stab culture of anthrax bacillus (Crookshank)

Young colony of anthrax bacillus (x 16) (Crookshank)

Hanging-drop preparation (Crookshank)
Widal’s reaction (microscopic) .
Method of filling pipette for Widal’s reaction
Widal’s reaction (macroscopic)
Microsporon Audonini (Curtis)
Trichophyton endothrix (Curtis)
Trichophyton ectothrix (Curtis)
Thomas’ Hemocytometer (Zeiss)
Method of counting red corpuscles
Method of counting leucocytes

Gowers’ Hzmoglobinometer (Hamibsies)
Oliver’s Heemoglobinometer

Method of spreading blood-films
Malarial parasites

Pipette for collecting blood

Cathcart’s Microtome (Baird & Tatlock)

Moulds for embedding in paraffin (Baird & Tatlock) .

Clamp for Cathcart’s Microtome (Baird & Tatlock)

PAGE

4&5

7&8
Ir
22
23
26
32
41
70
70
71
89
92
97
98

114
II5
116
143
150
153
156
158
163
174
178
189
195
196
PLATE I

e

 

Fig 5

wv
tits IS)
te ott x a

Lig 2

 

Lig 5

WEE. delt.
DESCRIPTION OF PLATE I.

Fic. 1.—Diphtheria bacillus (long form) from a young
culture on blood-serum. Léffler’s blue; x 1000.

Fic. 2.—Diphtheria bacillus (short form) and Hoff-
mann’s bacillus. From young cultures on blood-
serum. Ld6ffler’s blue; x 1000.

Fic. 3.—Pneumococci in sputum from a case of pneu-
monia. Stained with dilute carbol fuchsin and
thoroughly washed; x 1000.

Fic. 4.—Anthrax bacilli. The lower portion of the
field shows bamboo-like chains and spores, and is
taken from cultures. Methylene blue; x 1000.
The portion showing spores is stained with carbol-
fuchsin, decolorised by brief immersion in methy-
lated spirit, and counterstained in methylene blue.

Fic. 5.—Pus showing streptococci. Stained by Gram’s
method, counterstained with eosin; x 1000.

Fic. 6.—Pus showing gonococci and_ staphylococci.
Stained by Gram’s method and counterstained
with dilute carbol-fuchsin; x 1000.

nS
PLATE II

as

—

he Pag

a

™
ae

ae

aN hy

—

N

Lig

Fig l

 

fig &

 

<o}
s

) Cc

ee ee
Soy ee ee es is
ee ee a
ge hg .

WEE. delt
DESCRIPTION OF PLATE II.

Fic. 1.—Tetanus bacillus, from a culture. Stained
with dilute carbol-fuchsin; x 1000.

Fic. 2.—Tubercle bacilli. Stained with carbol-fuchsin
and decolorised in dilute sulphuric acid; not
counterstained; x 1000.

Fic. 3.,—Influenza bacillus in sputum. Some are con-
tained within a leucocyte. Ldffler’s blue; x 1000.

Fic. 4.—Plague bacilli from a culture. A short chain
and some coccoid (involution) forms are shown.
Léoffler’s blue; x 1200.

Fic. 5.—Vibrio of Asiatic cholera. Dilute carbol-
fuchsin; x 1000.

Fic. 6.—Small colony of actinomyces, as it appears
in pus. No clubs are visible. Gram’s method;
x 600.
A HANDBOOK

BACTERIOLOGICAL DIAGNOSIS

 

PRACTITIONERS.
PART I.

APPARATUS AND PROCESSES.

 

THE BACTERIOLOGICAL MICROSCOPE.

The essentials which a microscope must possess in
order to render it available for bacteriological work
are :—

1. A fivm and rigid stand and stage.

2. A firm, accurate, and delicate fine adjustment.

A microscope which possesses these may be made
available for bacteriological work by the addition of the
necessary parts, but one that is deficient in these
respects is useless.

3. A convex and a flat mirror.

4. An Abbé’s condenser and iris diaphragm.

5. Three lenses, a low power (4 in. or better, 2 in,),
a high power (% in. or thereabouts), and a 4 in. oil
immersion.

If the practitioner already possesses a microscope

B
2 BACTERIOLOGICAL DIAGNOSIS.

made by a reliable firm, and in good condition, this
should be sent to a maker (not necessarily the maker of
the microscope in question) or to a bacteriologist for an
opinion as to whether or no it is sufficiently firm and

 

Fic. 1.—Bacteriological Microscope.

has a fine adjustment good enough to justify the
addition of the other parts. If this is the case it should
be fitted with an Abbé’s condenser and an iris dia-
phragm; the cost should not exceed 30s. or £2. But
BACTERIOLOGICAL MICROSCOPE, 3

it is useless to do so unless the stand is sufficiently
steady to carry an oil immersion lens: and it is. useless
to think of having an inefficient fine adjustment altered .
for bacteriological use. :

There are now many microscopes which are sold at a

 

Fic. 2.—Bacteriological Microscope.

comparatively small price and which will answer every

purpose. Of these Leitz’s IIb stand, with the lenses

above mentioned, a triple nose-piece (which is so great

an advantage that it might almost be called an essen-

tial) and two eye-pieces costs about £13 and may be
B2
4 BACTERIOLOGICAL DIAGNOSIS.

highly commended. The stand is very firm and strong,
the fine adjustment delicate, and the lenses altogether
admirable. The only objection to this microscope is
that it is rather heavy; this is an advantage for
laboratory work, but it is a disadvantage for a medical
man who may have to examine blood, &c., at the
patient’s bedside. Swift & Son manufacture a micro-

 

Fic, 3.—Portable Microscope.:

scope of much the same type at about the same price;
it has all the good points of the Leitz microscope and is
somewhat lighter. The same firm also supplies a
portable instrument, the cost of which (without lenses)
is £5. This is very ingeniously made, and will carry an
oil immersion lens quite well: it is perhaps the most
suitable stand for a practitioner who may have to do
BACTERIOLOGICAL MICROSCOPE. 5

work at a distance, for it packs into a very small
compass and is not heavy.

The cost of a jin. oil immersion lens is, roughly
speaking, £5. Beck supplies a good lens for £4, and
Messrs. Gowlland of Selsey, Sussex, are now making
one for £2 15s. 6d., of which I have formed a very high
opinion after having examined a considerable number.
The same firm also produces a microscope fitted with

 

Fic. 4.—Portable Microscope.

all requisites for bacteriological work for about £11,
and this I believe to be quite efficient.

The cost of the other lenses may be put down at 30s.
or less apiece. Leitz’s No. 3 (about 3 in.) costs 15s.,
and his No. 6 (about 4 in.) costs 30s.; these are both
extremely well adapted for bacteriological work.

With regard to eye-pieces, it is an advantage to
have two,a No. 2 and a No. 4. Higher powers may
be used, but it must be remembered that any increased
6 BACTERIOLOGICAL DIAGNOSIS.

gain in magnification brought about by the eye-piece is
attended by a loss of definition. The same applies to
the increased magnification obtained by pulling out- the
draw-tube. :

~The total cost of a microscope may be put down at
£15, and for this sum a thoroughly efficient instrument
can be obtained; while one that will answer every
purpose may be bought for decidedly less. The cost of
adapting a good stand will be about £6 tos. or £7 (30s.
or £2 for the substage arrangement and £5 for the lens)
or less if a cheaper oil immersion lens is obtained.

It need scarcely be said that there are many ad-
mirable microscopes other than those mentioned above,
which have simply been selected as favourable and
low-priced instruments of English and Continental
manufacture. Amongst others, the microscopes made
by Beck, Ross, and Watson are all good, and reasonable
in price.

STERILISATION OF APPARATUS, Etc.

Requisites :—1. A thermometer graduated to 200° C.
2. A hot air steriliser.
ov A-cubical biscuit tin the soldering of which has
been replaced by brazing. This must -be mounted
upon a tripod stand.
« ov A kitchen oven, preferably a gas oven.
- 3. A steam steriliser. .
ov A large kitchen steamer. This should be deep
‘enough to contain a litre flask holding a funnel.
‘4. A large Bunsen burner or spirit-lamp. . =
--Bacteria- and their spores are ubiquitous, and. jit: is
STERILISATION OF APPARATUS. 7

Necessary to sterilise all vessels and other apparatus,
and all culture media before use. The methods which
are adopted all depend upon the action of heat;
‘chemical antiseptics are rarely used in the bacterio-
logical laboratory for the sterilisation of apparatus, for
it would be impossible to remove them completely, and
the traces which might remain would prevent the
development of those germs which we wished to

 

Fic. 5.—Slot Air Steriliser (simple form).

cultivate. Two chief methods are in use, sterilisation
by dry heat and by steam; we exclude sterilisation by
steam under pressure as this requires special and
expensive apparatus.

. Dry Heat is used to sterilise all glass vessels (flasks,
Petri dishes, test-tubes, pipettes, &c.,) cotton-wool, and
metal instruments. The heat must be continued for at
least an hour and must not fall below 150° C. as indi-
8 BACTERIOLOGICAL DIAGNOSIS.

cated by the thermometer. Another method which is
less reliable than the use of the thermometer, but which
may be resorted to in an emergency, is to wrap the ap-
paratus loosely in cotton-wool and to proceed with the
heating (allowing the temperature to rise gradually) until
the outer part of the wool is slightly singed over the
whole of the exposed surface.

ci pa

 

Fic. 6.—Steriliser.

The special steriliser which is used in the bacterio-
logical laboratory consists of a copper or iron oven with
double walls and perforated metal shelves. There is a
hole in the top, which is fitted with a perforated cork,
through which the thermometer passes. The oven is
mounted on a stand and heated by means of a large
Bunsen or Fletcher’s burner.
STERILISATION OF APPARATUS. 9

An efficient steriliser may be made out of a cubical
biscuit box, but it will not stand much usage unless the
joints are brazed instead of being soldered: this can be
done by any tinsmith. It is much better to have the
the bottom of the box replaced by a sheet of copper,
and a steriliser made in this way will answer every
purpose and be fairly durable. A circular hole is cut
through the centre of the lid and fitted with a cork
bored so as to admit the thermometer. A false bottom
or a shelf an inch or so from the bottom will keep the
articles which are being sterilised from the heated
surface; the false bottom may be made from a sheet of
tin two inches longer in one of the sides than the
bottom of the box. The extremities of the longer sides
are to be turned down for a length of an inch, and
several holes cut in the plate.

Lastly, the kitchen oven may be pressed into service
if no other steriliser is at hand in an emergency. The
apparatus to be sterilised is to be placed on a layer of
cotton-wool on one of the shelves, and the temperature
is observed by means of the thermometer, which should
be thrust through the little window which permits of
the regulation of the temperature. Or the heat may be
continued until the cotton-wool is singed over the whole
of the exposed surface. This method is very convenient
for practitioners sending materials to a laboratory for
bacteriological examination.

A gas oven is even more convenient, as the tempera-
ture can be regulated to a nicety.

All glass apparatus must be thoroughly cleansed and
dried before sterilisation. The remaining steps differ
Somewhat in the different cases.

Flasks are plugged lightly with cotton-wool before
being placed in the steriliser. Bottles may be sterilised
Io BACTERIOLOGICAL DIAGNOSIS.

in the same way and are sometimes requisite for the
transmission of fluids to a public laboratory for an
examination which the practitioner is unable to carry
out at home. They may also be sterilised by boiling.
Test-tubes are treated in the same way as flasks. Petri
dishes are wrapped round with tissue-paper or filter
paper before being sterilised.

Before removing glass apparatus from the steriliser
remember to let the temperature fall gradually, or the
vessels may crack. In the case of'a proper steriliser or
of a biscuit tin heated by a burner the gas is turned out
and the whole apparatus allowed to cool before the door
is opened. In the case of a kitchen oven the best plan
is to let the fire go out, or to open the door very
gradually.

Cotton-wool is sterilised by being spread out in thin
layers on the shelves of the apparatus, and the heat
is continued until the outside is singed.

Metal instruments (knives, scissors, &c.) may be steril-
ised in the same way and at the same time. They
should be wrapped loosely in cotton-wool, and should
not be removed from their wrapping until the moment
at which they are to be used.

Steam is chiefly used for the sterilisation of culture
media before use, and for the destruction of cultures
when they are done with. The latter purpose, however,
is accomplished more speedily and safely by the ad-
dition of a few drops of commercial formalin to each
tube.

The proper steam steriliser consists of a metal
cylinder with a perforated diaphragm six or eight
inches from the bottom. It is enclosed in a thick layer
of felt or other non-conductor of heat, and is provided
with a lid. The space .between the bottom and the
STERILISATION OF APPARATUS. II

diaphragm is partly filled with water, which is boiled
by means of a Bunsen flame or Fletcher burner, the
apparatus to be sterilised being placed in the chamber
above so as to be exposed to the steam.

An ordinary steamer (such as is used for cooking
potatoes or fish) will answer every purpose. In pro-

 

Fic. 7.—Steam Sterilizer.

curing such a steamer for bacteriological use it is best
to choose one that will accommodate a litre flask
holding a funnel, as it is often a great convenience in
the filtration of fluids which become solid on cooling to
carry out the process in an atmosphere of steam.

Exact details of the way it is used will be given
subsequently.
I2 BACTERIOLOGICAL DIAGNOSIS.

PREPARATION OF CULTURE MEDIA.

Bacteria are grown in the same way as other plants.
A gardener who wishes to grow a plot of a particular
plant will first prepare a soil suitable for the growth
of that plant and free it as far as possible of all seeds,
roots, &c. He will then sow it with the seeds of the
plant in question and do what he can to expose them
to a suitable temperature. An exactly similar process
is adopted when we wish to cultivate the smallest of all
plants. The soil which we prepare is called the culture
medium, and differs in the case of different bacteria;
the process of freeing this soil from bacteria and their
spores is called sterilisation and we ensure a suitable
temperature by means of an incubator, the heat of
which is kept constant.

The culture media which are used for special pur-
poses are almost innumerable, but in the daily routine
of the laboratory and for diagnostic purposes, broth,
gelatin, agar-agar, and blood serum are all that are
really necessary in the vast majority of cases. The
blood-serum medium is difficult to prepare and can be
replaced by ascitic agar in some cases. These media
may all be bought from any firm of manufacturing
chemists or from any bacteriological laboratory; and
their purchase saves a great deal of work and is to be
recommended for those who only wish to use them
occasionally. They are sold in test-tubes which are
kept sterile by being plugged fairly firmly with cotton-
wool; this substance prevents the passage of bacteria
as long as it is kept dry. The tubes are best stored in
jars provided with tightly fitting lids, and it -is an
PREPARATION OF CULTURE MEDIA. 13

advantage to place a shallow layer of a solution of
perchloride of mercury (or other xon-volatile antiseptic)
in the bottom of each jar to prevent the medium from
drying up. It is scarcely necessary to add that not the
smallest trace of the lotion should be allowed to come
into contact with the cotton-wool plug of the tubes.
Or the medium may be kept from drying up by covering
the tubes with india-rubber caps sold for the pur-
pose.

Brotu is very easily made, and as it is the foundation
of many other media, the practitioner is strongly advised
to prepare it for himself.

Requisites :—1. Liebig’s extract of meat.

2. Peptone.

3. Common salt.

4. A dilute solution of sodium carbonate—about 1
per cent., but the exact strength does not matter.

5. A large flask, a stirring rod, and a large glass
funnel.

6. Test-tubes and cotton-wool plugs. The exact size
of the tubes is unimportant, but 6 in. x @ in. is
convenient. The plugs are best prepared from wool
which has been previously sterilised by dry heat, and
should be fairly firm. The tube with the plug i situ
must be sterilised by dry heat ready for use.

7. Litmus paper.

Method.—Take 1 litre of tap-water in the flask and
add 5 grammes of Liebig’s extract, 10 grammes of
peptone, and 5 grammes of common salt, and boil until
all are dissolved. Test the reaction by withdrawing a
drop of the fluid on the stirring-rod and applying it to a
piece of litmus paper. You will find that it is slightly
acid. Now add some of the solution of soda drop by
drop, testing after each addition, until the reaction of
14 . BACTERIOLOGICAL DIAGNOSIS.

the fluid is slightly alkaline.* Boil the fluid for half an
hour to coagulate any albumen which may be present.

Next filter the broth into a sterile flask, passing it
through a double thickness of white filter or blotting
paper, and plug the flask firmly with sterilised cotton-
wool,

If the broth is to be used for the manufacture of
gelatin or agar it is next sterilised in the flask, while
if it is to be used as it is as a culture medium, it is
decanted into tubes and then sterilised.

In decanting media into tubes be very careful not to
get the plug wet and not to let any of the medium
get on to the upper part of the tube; otherwise the plug
will stick to the tube, and there will be some danger of
bacteria from the air “growing through” the fluid
contained in the interstices of the plug and contami-
nating the culture. Ordinary non-absorbent (brown)
wool is better than the white absorbent wool, as it is
less easily wetted.

The broth (and other culture medium after being
melted) may be poured into the tubes in the following”
way. A sterilised funnel is united by a short length
of india-rubber tubing to a piece of glass tubing drawn
out to a point: the rubber tube is clipped by a spring
clip or a pair of pressure forceps. The funnel is now
mounted on a retort stand, filled with the medium, and
covered over with a piece of glass. The cotton-wool
plug is removed from a test-tube and the latter placed
so that the glass tube attached to the funnel reaches
nearly to the bottom. The clip is released and the

* If during the neutralizing process too mnch alkali is added, then
it is necessary to re-acidify with dilute hydrochloric acid and re-
neutralize. The sodium chloride formed makes no practical differ-
ence in the medium.
PREPARATION OF CULTURE MEDIA. 15

requisite quantity of broth (enough to fill the tube to
the depth of an inch and a half or two inches) is
allowed to run in; the clip is then re-applied and the
tube removed and plugged. This process is repeated
until enough tubes have been filled.

The tubes and the broth which remains over (after
having been poured back into the flask and the latter
plugged with cotton-wool) are now sterilised. The
vessels are placed in the steam steriliser and exposed to
steam for half an hour on three successive days; this
process is called intermittent sterilisation, and its
rationale is very simple. The first steaming destroys
all developed bacteria, and would sterilise the fluid
entirely if no spores were present. In the interval
between the first and second sterilisation most of the
spores which may be present will develop into mature
bacteria, and these will be killed by the second steam-
ing. The third sterilisation is to kill off any bacteria
which may not have developed from spores in the first
interval. A very similar process is adopted by the
gardener in freeing soil from weeds; the application of
chemical weed-destroyers or a thorough hoeing will
destroy developed plants but will not injure seeds
which may be contained in the soil, and these processes
are repeated, intervals being allowed to permit the
development of the plants until they reach the stage in
which they are vulnerable.

To recapitulate; mix the ingredients and heat until
they are dissolved, render slightly alkaline, boil half an
hour, filter. Then place in sterilised flask or into
test-tubes and sterilise in the steam steriliser for half an
hour on three successive days.

NuTRIENT GELATIN is broth which has been solidified
by the addition of from ten to fifteen per cent. of gelatin;
16 BACTERIOLOGICAL DIAGNOSIS.

the former amount is used in the winter, the latter in
the summer. For general purposes 124 per cent. may
be used in all cases.

The special advantages of gelatin as a culture
medium are two-fold. In the first place a great many
organisms grow in or on it in a characteristic way, so
that a bacteriologist may be able to identify the organ-
ism by inspection of the culture. This arises partly
from the fact that some bacteria produce a ferment
which digests gelatin just as pepsin does; these bac-
teria “liquefy” the gelatin, and the distinction between
the bacteria which have and those which have not this
property is very important for purposes of diagnosis.
Further, some bacteria liquefy rapidly and others
slowly, and this is another important point in the
identification of a germ.

In the second place, the gelatin medium may be
melted at a temperature (about 25° C.) at which
bacteria are not killed. This fact is made use of in the
isolation of bacteria from a fluid which contains several
species by the process known as “lating.” Suppose,
for instance, that we find by microscopic examination
that a specimen of pus contains two different species of
bacteria (perhaps a bacillus and a coccus) and we wish
to obtain the two organisms in pure culture so that we
can ascertain their nature and properties. We take a
tube of gelatin and melt it by placing it in warm water,
and then inoculate the medium with a minute quantity
of the pus.* We then shake it so as to distribute the
organisms throughout the melted fluid, and then pour
the latter into a flat dish (Petri’s plate) so that the
gelatin flows out into a thin film and then sets. If

* Another and preferable plan is to inoculate the medium first and
to melt it atterwards.
PREPARATION OF CULTURE MEDIA. 17

our dilution has been properly made we shall have
separated each organism from its neighbours, and each
separate germ will grow up into a ‘“‘ colony” which will
soon be visible to the naked eye. In all probability we
shall be able to see that these colonies are of two kinds;
one may liquefy and the other not, one may be coloured
and the other colourless, one may be round and the
other angular, &c. Samples of each sort of colony
are then transplanted to fresh culture tubes and again
incubated. An example of this process is given on p. 70.

A slight modification of this process enables us to
make an estimate of the number of living bacteria
which is present in a given fluid. To do this we have
to follow out the above process, adding a definite
measured quantity of the fluid to the culture tube of
liquefied gelatin. The number of colonies which de-
velop is counted, and this gives us the number of
bacteria in the sample of fluid. For example, if one-
tenth of a cubic centimetre diffused throughout a tube
of melted gelatin and poured out into a thin film
produced twenty colonies, it follows that one cubic
centimetre of the fluid contained two hundred bacteria.
This is a brief description of the essentials of the
method adopted in the quantitative examination of
water.

Requisites for the manufacture of gelatin :—

1. Broth.

2. Gelatin. (Coignet’s gold label gelatin is best, but
any good brand will do).

3. Dilute solution of sodium carbonate.

4. Litmus papers.

5. Flasks, stirring-rod, funnel, and plugged test-tubes
as for broth.

Method.—Measure the broth and add to it 12%

c
18 BACTERIOLOGICAL DIAGNOSIS.

grammes of gelatin for each 100 c.c.; allow to soak
for an hour or more, and then heat until the gelatin is
dissolved. Continue the heat and render the medium
faintly alkaline just as was done in the preparation of
broth. Now filter through a moistened filter paper.
To avoid the setting of the gelatin during the filtration
it is best to use a double jacketted funnel containing hot
water, but if this is not at hand the whole apparatus
(flask and funnel) may be placed in the steam steriliser
(the lid being kept off to avoid the drops of condensed
water which might otherwise fall into the funnel) or in
a waym (but not hot) oven and left at a temperature of
about 40° C. until the process is complete.

The gelatin which is made by the above process is
sufficiently clear for most purposes. A more sightly
medium may be-made by clarification of the above by
white of egg. To the medium (after neutralisation but
before filtration) add the white of one egg for each 250
or 300 c.c. of fluid and shake thoroughly. Now boil in
the steamer for half an hour and filter as before.

Test-tubes are filled with gelatin just in the same
way as with broth, and the process must be carried out
quickly to avoid solidification of the medium. Some
of the test-tubes are allowed to cool in the vertical
position, others lying in a sloping position so that the
upper surface of the gelatin forms an ellipse some three
inches long. The former tubes are inoculated by
driving a straight platinum needle charged with the
material containing the bacteria into the gelatin in the
axis of the tube; cultures made in this way are called
“stab-cultures.” The gelatin ‘“‘slopes” are inoculated
by drawing the charged needle along the surface of the
medium, care being taken not to plough it up; cultures
made in this way are called “ stroke-cultures.”
PREPARATION OF CULTURE MEDIA. 19

Acar is the name given to the dried strips of a
Japanese sea-weed. It forms a jelly which differs from
that containing gelatin in that it melts at a higher
temperature; nutrient agar as used in the laboratory
melts just below the boiling point of water and sets at
about 40°C. This is an advantage in the cultivation
of most pathogenic bacteria, for these grow (as a rule)
best at or near the temperature of the body, the
temperature to which they are exposed under natural
circumstances; and at this temperature gelatin would
melt. Agar is somewhat difficult to prepare unless the
practitioner has an autoclave, and may be bought with
advantage. But the following method is not very
difficult, and, as agar is perhaps the most generally
useful of all media, should be learnt.

Feqwisites :—1. Broth.

2. Agar-agar. This should be cut up into very small
pieces with a pair of scissors, or may be bought in
powder.

3. Solution of acetic acid. (Glacial acetic acid 2-4
c.c., water 500 C.C.).

4. A large beaker.

5. Other apparatus and materials as for gelatin.

Method.—Weigh out 2 grammes of agar for each 109
c.c. of broth to be used, and soak it in the dilute acetic
acid for a quarter of an hour. Now strain off the acid
and wash the agar in water until a small piece does not
redden blue litmus paper when pressed upon it. Place
the broth in a glass beaker and add the agar. Now
place the beaker upon a piece of wire gauze upon a
‘tripod stand, and apply a small Bunsen flame or spirit
lamp; this must be placed so that the flame impinges
on a point not far from the side of the beaker. As the
fluid is heated it will rise and a continual circulation

c2
20 . BACTERIOLOGICAL DIAGNOSIS.

will take place, so that the fragments will not stick to
the bottom and cause it to crack. When all is dis-
solved the hot liquid must be carefully neutralised. It
is then allowed to cool to about 50° C. and the white of
an egg added for each 500 c.c. of fluid and mixed
in thoroughly by being stirred with a glass rod. The
whole is then placed in the steamer for an hour, at the
end of which time the albumen should be completely
coagulated. The beaker and its contents are then
allowed to cool gradually, so that the coagulum (re-
taining all solid particles) may settle to the bottom
before coagulation is complete. Perhaps the best
method of accomplishing this is to place it in the oven
(taking care that the temperature does not exceed
too® C.) after the fire has been raked out at night.
In the morning the mass will be found to have solidified
and there will be a coagulum at the. bottom. The
beaker is then inverted and the mass ‘turned out”
just as a cook turns out a jelly, and the sediment is cut
off with a sharp knife. This avoids filtration, which is
‘very troublesome.

An alternative method is to filter the melted jelly
through moistened filter paper or through two thick-
nesses of butter-muslin. It is necessary to keep flask
and funnel in a steamer (the water of which is kept
boiling vigorously) during. the whole process, or the
jelly will solidify in the outflow tube of the funnel.

The agar is again melted and placed in test-tubes;
these are sterilised on three successive days and allowed
to set in a sloping position. For certain purposes
glucose, glycerine, &c., are added to the agar. The’
addition should be made to the melted medium just
before it is poured into the tubes.

SOLIDIFIED BLOOD-SERUM is very difficult to prepare,
PREPARATION OF CULTURE MEDIA. 2I

and is best purchased ready for use from a good
laboratory. For clinical purposes it is chiefly used in
the preparation of the diphtheria bacillus, for which,
however, it may be replaced by the following medium,
which is more easy to prepare.

ASCITIC AGAR :—Requisites :—

1. Clear ascitic, pleuritic, or hydrocele fluid.

2. A Io per cent. solution of caustic potash.

3. Glycerine.

4. Other materials and apparatus as for agar. (Broth
is not required, its place being taken by the pathological
exudate).

Method.—Measure the fluid and add to it 2 c.c. of the
potash solution for each 100 c.c. used. Mix thoroughly,
pour a little into a test-tube, and heat. If the fluid
does not coagulate proceed with the manufacture of the
medium in the manner described below. If it coagu-
lates add a little more potash, mix, and again test.
The addition of the potash must be repeated until no
coagulum forms on heating, all the albumen being
converted into uncoagulable alkali-albumen.

The remaining steps of the process-are the same.as
for ordinary agar, except that the neutralisation is
omitted and 6 c.c. of glycerine per 100 c.c. of the
medium is added whilst the agar is being dissolved. ~

Potato tubes are in occasional use, and are easy to
prepare. The process is as follows:—Take large and
sound potatoes and scrub them thoroughly with a nail-
brush under the tap. Peel them deeply enough to
remove the eyes completely. Then cut them into
cylinders a little less than 3 in. in diameter (if you are
using 2 in. test tubes) and as long as possible; this is
best done by means of a cork-borer, but. they may. be
shaped by means of a kmife if this is not at hand.
22 BACTERIOLOGICAL DIAGNOSIS.

Then cut each cylinder in half by a cut running
obliquely from end to end; the shape of each half
should be exactly like that of the medium in a sloped
gelatin tube. Place the halves in a large vessel of
tap-water and allow them to soak all night; it is a good
plan to use running water if possible.

After this has been done place each half (base
downwards) in a test-tube, having previously inserted
a small mass of absorbent cotton-wool and enough
water to saturate it. Plug the mouth of the tube with
cotton-wool and sterilise on three successive days.

INOCULATION OF CULTURE MEDIA.

The method in which this is done varies greatly
according to the end in view, and variations of the

a.

 

  

 

Fic. 8.—Platinum Needles.

process now to be described will he mentioned under
their appropriate headings. We will suppose that we
have to examine a specimen of pus, and wish to make
a stroke-culture of agar and a stab-culture in gelatin.
The following must be at hand :—

1. The pus.

2. A sloped agar tube and a stab gelatin tube.
INOCULATION OF CULTURE MEDIA. 23

3. A Bunsen’s burner or a spirit lamp with a tall
flame.

4. A pair of dissecting forceps.

5. Platinum needles. Each needle consists of a piece
of platinum wire about three inches long mounted in
the axis of a glass rod about six or eight inches in
length. The wire should be just thick enough not to
bend too easily. They are easily prepared. The rod
is selected and the length of platinum wire is held in an
ordinary pair of forceps. The end of the glass rod is
held in the flame until quite soft ; the end of the wire is
then heated to redness, and pushed into the rod to the
depth of about a quarter of an inch, taking care that it is

 

Fig. 9g.

kept in the axis. The whole is allowed to cool and is
ready for use.

For some purposes we use needles which terminate
in a small loop, so that they will retain a drop of fluid.
These are prepared in the same way as the straight
needles, the free end of the wire being subsequently
twisted round a French nail or other suitable object.

The method is as follows :—

1. Hold the culture tube you are going to inoculate
first between the index and middle fingers of the left
24 ‘ BACTERIOLOGICAL DIAGNOSIS.

hand, pointing the mouth of the tube slightly down-
wards (so that no dust shall drop into it) and to the
right. Tubes of solid: media should always be held in
this position during inoculation ; tubes of liquid media
are held in a similar way, but of course their mouths
must point upwards.

2. Put the projecting portion of the cotton-wool plug
of the test tube into the flame so as to: singe it; this is
to destroy any germs which may have been deposited
upon it from the atmosphere.

3. Sterilise the points of the forceps by passing them
slowly through the flame and then use them to remove
the plug. Place this between the ring and little fingers
of the left hand and put the forceps down.

4. Take the platinum needle in the right hand, heat
the whole of the wire to redness, and pass the lower
three or four inches of the glass rod slowly through the
flame. Remember that every portion of the needle
which goes inside the tube must be sterilised in the
flame. Allow the needle to cool; you should have
found out how long this will take by a previous
experiment.

5. Dip the tip of the needle into the pus; pass it into
the tube until it reaches nearly to the bottom of the
tube (now uppermost) and allow to rest upon the
sloping surface of the medium; now withdraw it gently,
allowing the tip of the wire to trail gently along the
whole length of the sloped surface. Do not touch the
medium with the glass shoulder of the needle.

6. Sterilise the needle as before. This step must
never be forgotten.

7. Take the cotton-wool plug in the forceps, put it in
the flame, and singe all parts of its surface. Then plug
the tube while the wool is still burning. Label it.
INCUBATION OF CULTURES. 25

To make a stab culture take the other gelatin tube
and proceed as before until you get to step 5. When
you have passed the needle into the tube drive it steadily
into the medium, taking care not to deviate from the
axis of the tube. Finish the process as before.

All this may seem involved. Asa matter of fact it is
very simple and need not take more than a minute to
perform. But every step must be carried out, and the
whole process must be learnt so thoroughly that it is
performed automatically whenever a culture is made. |

INCUBATION OF CULTURES.

The limits between which bacteria can live are very
wide; some grow best at one temperature, others at
another, the limits for the great majority of organisms
being about 16° C. and 40° C. In practice two tem-
peratures are all that is used for ordinary work. The
lower, or so-called ‘‘room temperature” is about 20° C.
(68° F.) and is of most use for those bacteria which
grow naturally outside the body, 7.¢., as saprophytes.
The higher, or body temperature, is about 37° C.
(98°6° F.), and is the best temperature for the majority
of germs which live within the body, ¢.e., the parasites.
It is obvious that gelatin cannot be incubated at this
high temperature as it melts at 25° C. or thereabouts;
but all other media are available.

The term ‘room temperature” must not mislead the
practitioner, for the temperature of most rooms is
rarely constant at or near 20° C. for periods sufficiently
long to permit of its use for incubating bacteria. In
the laboratory we use an incubator, the temperature of
which is regulated by means of an automatic regulator,
26 BACTERIOLOGICAL DIAGNOSIS.

and remains constant for long periods whatever be the
external temperature. It is hardly necessary for the
practitioner to purchase one of these. Careful search
in the house will usually reveal some cupboard or

 

Fic. 10.—Incubator.

corner in which the temperature will remain sufficiently
near 20° C. for a sufficiently long period; it is more
important that it should not rise above 22° C. than that
it should not fall below 18° C., as the former tempera:
INCUBATION OF CULTURES. 27

ture may melt the gelatin, while the latter will only
delay the growth of the colonies. It will probably be
necessary to find one such place in the hot weather
(¢.g., the cellar) and another one in the winter (¢g., a
cupboard not far from the hot-water pipes).

It is necessary that cultures which are being incuba-
ted should be kept in the dark, as light is inimical to the
development of nearly all bacteria.

Incubation at the body temperature presents more
difficulty. An incubator is almost essential when much
work has to be done, and of these Hearson’s is by far
the best. The smallest size costs about £6 10s. and
is a thoroughly reliable and durable affair. Cheaper
instruments are also procurable; the Edinburgh in-
cubator (Alex. Frazer, 22, Teviot Place, Edinburgh)
costs about £4 10s. complete. Foreign incubators can
be obtained at an even lower price, but are not very
durable.

Much can be done without the use of so expensive an
apparatus if the practitioner can find a room in which
the temperature keeps approximately constant through-
out the twenty-four hours. A tin biscuit box (or any
other metal box) is covered with cotton-wool on the top
and sides, the bottom being left bare, and mounted on a
tripod stand.. It is heated by means of an ordinary
night-light (two may be necessary if the weather is cold)
shielded from draughts by means of a wide lamp-
chimney or a tin cylinder made out of an ordinary
canister, The temperature is observed by means of a
thermometer projecting through a hole in the lid,
and the night-light raised or lowered until the
temperature reaches the desired figure. The whole
apparatus should be placed on a metal tray containing
a small quantity of water and put in the middle of the
28 BACTERIOLOGICAL DIAGNOSIS.

floor and away from any inflammable materials. This
will be found to answer admirably and can easily be
fitted up in an emergency.

It would be better to use a tin box specially made for
the purpose and having a door at the side and a per-
forated false bottom so that the culture tubes do not
rest directly on the metal exposed to the flame. This
latter had best be made of copper.

If the practitioner is fortunate enough to possess a
conservatory which is kept at a temperature approxi-
mating to that of the body, this will serve admirably.
The culture tubes must be kept in a box which will
exclude light.

In the absence of this a cupboard near the kitchen
fire or the hot water cistern may be found that will
answer the purpose; a thermometer should be placed
in it and examined from time to time throughout the
day, and if the temperature does not fall below. 30° nor
rise above 40° it will serve at a pinch, though a tem-
perature which is more constant near 37° is desirable.
It has to be remembered that we are not now speaking
of the incubation of cultures for purposes of research;
we are dealing with methods of cultivation which are
necessary for diagnostic purposes, and for these it is
usually sufficient if the temperature remains nearly
constant at the proper point for some eighteen hours.

‘The author once succeeded in making a diagnosis in
a case of supposed diphtheria by the following method.
The tube of medium was inoculated from the throat
and placed in a jam pot which was partly filled with
water at 37° C.; a thermometer was also inserted and
the whole placed near the fire. It was watched from
time to time and moved near the fire if the temperature
showed signs of falling, and vice vevsé. Next morning
METHOD OF EXAMINING CULTURES. 29

there was an excellent growth and the diagnosis was
made with certainty.

Lastly, the author has heard of a practitioner who
was in the habit of incubating cultures at the body
temperature by carrying them in an inner pocket during
the day and taking them to bed with him at night !

METHOD OF EXAMINING CULTURES.

Requisites.—1. Clean slides and cover-glasses. (These
must be the thinnest in ordinary use, 7.e., No. 1).

2. A platinum needle. (Straight or loop).

3. A Bunsen’s burner or a spirit lamp with a tall
flame.

4. The stain to be employed (see p. 37).

5. Canada balsam dissolved in xylol. This should
be bought ready for use. .

6. A pair of dissecting forceps.

7. Strips of white blotting- or filter-paper.

Process.—1. Sterilise the needle and place a small drop
of water (preferably distilled) in the centre of a clean
slide.

The disadvantage of using tap-water arises from the
fact that it leaves a deposit of salts when it has dried,
and these salts make the film “dirty.” The amount of
distilled water required is very small, and sufficient can
be obtained by holding a cold tumbler or saucer in the
steam from a kettle.

2. Take the culture-tube in the left hand between the
index and middle fingers with its mouth directed to the
right and (in the case of a culture on a solid medium)
slightly downward.
30 BACTERIOLOGICAL DIAGNOSIS.

3. Burn the surface of the plug inthe flame. Remove
the plug with the forceps (previously sterilised by being
passed slowly through the flame) and place it between
the ring and little fingers of the left hand. Lay the
forceps down.

In cases where you are examining the culture for
diagnostic purposes only, and do not care if it becomes
contaminated during the process, it is unnecessary to
take these precautions. The cotton-wool plug may
then be removed with the fingers and laid down on the
table. Asa matter of fact very few cultures do become
contaminated even if no precautions are taken.

4. Sterilise the needle in the flame and allow it to
cool.

5. Now introduce the needle into the tube and take
up a small portion of the growth, taking care not to
scrape up the surface of the medium as you do. so.
Most beginners fall into the mistake of taking up far
too much of the growth, and preparing a film which is
spread so thickly that the individual bacteria cannot
be distinguished.

6. Take the plug up in the forceps, burn its surface
in the flame, re-plug the tube and lay it down.

7. Stir the droplet of water which has been deposited
on the slide with the tip of the needle so that the bac-
teria which it carries are mixed with the water. Now
spread out the emulsion thus produced so as to form a
patch about half an inch in diameter. If it does not
spread out uniformly it is a sign that the slide is not
clean.

8. Sterilise your needle.

g. Allow the film to dry spontaneously. If you have
spread it out sufficiently this will take a very short time.

10. Fix the film by passing the slide slowly through
METHOD OF EXAMINING CULTURES. 31

the flame once or twice. This coagulates the albumen
present and the bacteria are now fixed down so firmly
that they will not be removed by subsequent washing.
The exact amount of heat which should be used cannot
be stated, as it varies according to the thickness of the
slide, &c., and can only be determined by practice. It
may be estimated roughly by pressing the finger upon
the upper surface of the slide close to the film, but not
touching it. The slide should be just uncomfortably
warm to the finger, but not hot enough to burn it,

11. Filter a few drops of the stain on to the surface
of the film and allow it to act for the requisite time.
Exact details will be given in each case.

12. Next wash the slide under the tap, blot it with
clean white filter paper, taking care to avoid rubbing,
and heat it gently over the flame until absolutely dry.

13. Place a drop of balsam on the film and apply a
clean dry cover-glass.

The preparation is now ready for examination.

This also is a process which sounds more complicated
than it really is. The steps are readily learnt, and the
whole process (excluding the time spent in staining,
which may be long or short), does not take more than
two or three minutes.

Most bacteriologists make their films on cover-glasses
and not on slides. The steps are just the same, except
that the cover-glasses, being much thinner, naturally
require much less heating to fix the film; they are
passed rapidly through the flame three times. It isa
great advantage to use Cornet’s forceps in working with
cover-glasses. These forceps are self-retaining, and
hold the cover-glass in a horizontal position so that
stains can be poured upon them whilst the forceps are
resting on the table. But the staining can be carried
32 BACTERIOLOGICAL DIAGNOSIS.

out equally well in a watch-glass, and the cover-glass
may be held in dissecting forceps.

It is far easier and more satisfactory in every way to
make the films on the slides. Beginners will find that
they will break large numbers of cover-glasses (which
must be thin), drop more on the floor, and will be in
constant doubt as to which is the film side. With
slides these difficulties do not occur, and the use of
forceps is quite unnecessary.

 

Fic. 11.—Cornet’s Forceps.

GRAM’S METHOD OF STAINING.

The method of staining described above is available
for all organisms, and therein consists its advantage.
But other things than bacteria are stained; pus-celis,
fragments of tissue, debris, &c., will all be coloured,
and may obscure, or even be mistaken for, bacteria.
Gram’s method possesses the enormous advantage that
by its use the bacteria are coloured, while other struc-
tures (with the exception of particles of keratin and
dividing nuclei) are not. Hence in a film stained in
such a way the bacteria are very distinct.

Gram’s method possesses another advantage. It is
a selective stain. Some bacteria retain the stain, whilst
others do not, and this fact is of great value in dia-
‘gnosis. The diphtheria bacillus, for instance, stains
GRAM’S METHOD OF STAINING. 33

when treated in the way we shall describe, and if an
organism which presents the character of that bacillus
does not stain by the process it must be of some other
species. We shall append a table of the most impor-
tant pathogenic bacteria which stain and which do not
stain in this way.

Requisites.—1. Aniline gentian violet, or carbol-gentian
violet (see p. 38).

2. Gram’s iodine solution (see p. 39).

3. Absolute alcohol or methylated spirit.

Process.—1. Spread, dry, and fix the film in the way
described above. Stain for. two or three minutes in the
aniline gentian violet or carbol-gentian violet.

2. Rinse in water to remove excess of stain and flood
with the iodine solution, and allow the letter to act for a
minute.

3. Wash off the iodine solution with alcohol, and
continue the application of the latter until no more
colour comes away. It is best to pour a little alcohol
on the slide and rock the latter from side to side for a
minute or so, then to pour off the alcohol and add a
fresh supply, and continue this until the alcohol comes
off colourless. ;

Dry and mount as before.

The following important bacteria stain by Gram’s
method :—

Staphylococci.

Streptococci.

Pneumococci. :

In fact all important pathogenic cocci except the
coccus of Malta fever and certain diplococci enumerated
below :—

The bacillus of anthrax.
diphtheria.

” Fy
34 BACTERIOLOGICAL DIAGNOSIS.

The bacillus of tetanus.
<5 5 tuberculosis.
9 19 leprosy.
The actinomycosis fungus.
The following important pathogenic bacteria do not
stain by Gram’s method :—
' ‘The gonococcus.
Diplococcus intracellularis meningitidis (Weich-
selbaum). ’ <
Still’s diplococcus oe posterior meningitis.
The pneumo-bacillus. &
The bacilli coli communis,
The bacillus of glanders.

” 3 typhoid fever.
i ie influenza.

” ” plague.

”. ” soft sore.

-The-vibrio of Asiatic cholera.

EXAMINATION OF FILMS. USE OF MICRO-
SCOPE.

Daylight is the best illuminant for ‘microscopic work,
and the light reflected from a white cloud opposite the
sun is best of all. Direct sunlight is, useless, but the
light obtained from a ground glass window on which
the sun is shining is very good.

For work at night the light from an incandescent gas-
burner at a distance of two or three feet is excellent,
but an ordinary paraffin lamp will answer quite well.

Having arranged for a suitable source of light turn
the flat mirror uppermost and move it about until
EXAMINATION OF FILMS. 35

a beam of light is thrown on to the condenser.
Remember :—

In examining stained specimens use a lJavge diaphragm.

In examining unstained objects use a small diaphragm.

_You are now about to examine a stained specimen.
Place the slide on the stage, putting the stained film in
the centre of the aperture, and turn on the low power.
Look down the eye-piece and move the mirror about
until the field is brilliantly illuminated. Focus the
microscope (using the coarse adjustment) until the
image is clearly defined. Now move the slide about
until there is a deeply stained area in the centre of the
field. This area will not necessarily be the best for
examination with a higher power, but it will serve to
catch the eye when focussing the lenses which focus at
a short distance from the object.

Now turn on the high power (the $in.). Remember
that the ‘ working distance” of all lenses is necessarily
less than their focal distance, and that a } in. lens
focusses at a distance from the object which is decidedly
less than one-sixth of an inch; so also with the other
powers. Lower the lens until it almost touches the
object and screw up the sub-stage condenser as high as
it will go. Look down the microscope and focus
slightly upwards, using the coarse adjustment, until you
catch a glimpse of colour; then focus very slowly until
the object is sharply defined.

After a little practice you will be able to focus down-
wards on to the film, keeping a sharp look out for the
first appearance of colour, but for beginners the fore-
going method is easier and safer.

Study the object with the high power, and move it.
about until you find an area where the bacteria are
neither too thickly nor too thinly spread and are well

D2
36 BACTERIOLOGICAL DIAGNOSIS.

stained. Make out as much of their appearance as you
are able to do with this power. Very much can be
done; tubercle bacilli, gonococci, and many other bac-
teria may be recognised with this power, and the
peculiar arrangement of diphtheria bacilli can be seen.

Apply the clips to keep the slide in place.

Now raise the tube of the microscope for a short
distance, using the coarse adjustment, and place a
small drop of cedar oil on the centre of the cover-glass.
Lower the tube (using the coarse adjustment) until the
nozzle of the lens touches the drop of oil; then put your
head on a level with the stage and continue to focus
downwards, going very carefully, until the lens almost
touches the cover-glass. Next look down the micro-
scope and focus upwards, using the fine adjustment,
until you begin to see colour; then go more slowly until
the film is well defined.

Beginners are strongly urged to adopt this method of
focussing an oil immersion lens until they have acquired
a considerable amount of practice. It takes a little
time, but this is well repaid by the absence of all
danger or injury to lens and cover-glass. After a time
you may lower the lens until it touches the oil and then
look down the microscope and continue to lower it with
the fine adjustment.

After use, wipe the front of the immersion lens with a
soft silk handkerchief kept specially for the purpase,
and put the microscope back into its case. If oil or
balsam should get dried on the lens wipe it with a
handkerchief just moistened with xylol or pure turpen-
tine and then wipe quickly with a dry handkerchief.
Never dip the point of a lens into xylol or alcohol.
Never remove the front combination of an oil immersion
lens for cleaning or any other purpose.
STAINS. 37

STAINS.

The following stains are all that is really necessary
for the vast majority of purposes :—methylene blue,
basic fuchsin, gentian violet, thionin, and water-soluble
eosin. Bismarck brown may also be obtained. Ten
grammes will last the practitioner for a long time, and
this amount costs from 7d. to 1s. They should be of
Gribler’s make, and can be obtained from R. Kanthack,
Berners St., W.; Messrs. Baird and Tatlock, Hatton
Garden and Renfrew St., Glasgow; A. Frazer, Teviot
Place, Edinburgh ; and from Messrs. Southall or Philip
Harris, Birmingham. Other firms will also supply
stains, but Griibler’s should always be specified.

They are conveniently kept in a saturated solution of
absolute alcohol. The following formule are the most
useful :—

1. A saturated watery solution of methylene blue.—This
does not keep very well, and a fresh amount should be
prepared after a month. It is mostly used for staining
blood-films. Léffler’s blue will serve every purpose in
bacteriological work.

2. Léffler’s methylene blue is prepared by adding 30 c.c.
of saturated solution of methylene blue (alcoholic) to
100 c.c. of a I in 10,000 solution of caustic potash.

The potash solution is prepared thus :—Take 1 c.c. of
a Io per cent. solution of caustic potash and make up to
100 c.c. with water; shake thoroughly and pour away
90 c.c; make up to 100 c.c. with water, and again shake.
A sufficiently close approximation is made by adding 1
minim of the ro per cent. solution to 2 oz. of water.

This stain keeps fairly well.
38 BACTERIOLOGICAL DIAGNOSIS.

3. Carbol-fuchsin is made by adding a saturated al-
coholic solution of fuchsin to carbolic acid lotion (1 in
20) until the fluid has lost its transparency. This
keeps well.

4. The above stain diluted with four or five times its
volume of water. Label ‘dilute cavbol-fuchsin.”

5. Aniline gentian violet, which is prepared as follows:—

First prepare aniline oil water by shaking water
(preferably distilled) with more aniline oil than it will
dissolve; a milky emulsion will result, and this must
be allowed to settle for a short time. {Then filter it
through a double thickness of filter paper which has
been previously moistened with water.

To g parts of the solution thus obtained add 1 part of
saturated solution of gentian violet.

This solution keeps badly, and it is of great import-
ance that it should be freshly prepared, as very
important inferences are drawn from results obtained
with it. The following keeps better, and answers every
purpose.

Carbolic gentian violet.—(A substitute for aniline gen-
tian violet).

Add 1 part of saturated aicpiane solution of gentian
violet to g parts of a 1 in 20 carbolic lotion.

6. Cavtol-thionin is made by adding 1 gramme of
‘thionin to 100 c.c. of a 1 in 4o solution of carbolic
acid.

This stain keeps fairly well, but it must always be
filtered immediately before use, as crystals which may
have a most delusive resemblance to long slender bacilli
are frequently deposited in it. A similar formation of
crystals also occurs if the stain be allowed to dry on the
slide.

7. Eosin is used in a 4 or 5 per cent. watery solution.
CLEANING SLIDES AND COVER-GLASSES. 39

‘This keeps well. Red ink (slightly diluted) will answer
most pufposes.

Stains should be filtered before use. Where much
.wotk is to be done it is convenient to keep them in
bottles which are closed with a perforated cork through
which a small glass funnel is placed. A filter paper is
kept permanently in this funnel, and the stain is filtered
directly on to the slide or cover-glass.

Gram’s iodine solution may be mentioned here, though
it is not a stain. It corisists of a solution of iodine 1
part, iodide of potassium 2 parts, water 300 parts.
It keeps indefinitely.

‘CLEANING SLIDES AND COVER-GLASSES.

Slides and .cover-glasses must be absolutely clean
when used in the bacteriological laboratory; it is
especially necessary that they should be free from the
slightest trace of grease, for this will prevent: fluid from
spreading out into a thin and uniform film.

Slides are best cleansed by dropping them one at a
time into strong nitric or sulphuric acid, and allowing
them to soak for an hour or more. They are then
washed in running water for another hour, soaked in
strong ammonia for an hour, and kept in alcohol. As
methylated spirit is not so good for this purpose the use
of alcohol is rather expensive; but it is not absolutely
necessary, and the slides may be stored in a solution of
ammonia (about 1 in ro) until required. When about
to be used they are to be wiped dry with an old linen
handkerchief kept specially for the purpose. This
handkerchief should be as old as possible, and should
have been washed until it has begun to fall to pieces.
40 BACTERIOLOGICAL DIAGNOSIS.

When no properly cleaned slides are at hand the
following method may be adopted, though it is not so
good. Dip the end of a clean handerchief in strong
‘spirit (absolute or rectified) and wipe the slide with it,
using a considerable amount of friction. Now dry it
with the special handkerchief mentioned above, heat it
thoroughly in a smokeless flame, and allow to cool
completely. Spread the film on the surface which was
exposed directly to the flame.

Cover-glasses are cleaned in the method advised for
slides, and should be stored in strong alcohol smelling
strongly of ammonia. They are wiped with the special
handkerchief immediately before use.

When cover-glasses are to be used for covering films
spread upon slides (as is generally the.case if the
method recommended in this book is adopted) it is
quite sufficient to wipe them carefully with a clean
handkerchief moistened with spirit and then to dry
them. .

After slides or cover-glasses have been cleaned the
utmost care must be taken that they do not come in
contact with the skin, or a thin film of grease will be
deposited upon them.

PIPETTES.

Glass pipettes for the collection of pathological fluid
for bacteriological examination are in daily use in the
laboratory, and are very necessary for practitioners who
wish to send fluids for examination. They are readily
made from a piece of quill glass tubing, and a few
should always be kept in stock against emergencies.
PIPETTES. 41

The pipette consists of a bulb about half an inch long,
each end of which is drawn out into a narrow tube
at least six inches long, tapering gradually to the
extremities (fig. 12, 4). To make such a pipette take a
piece of glass tubing about six inches long and a quarter
of an inch wide, and heat it in a luminous gas flame at a

‘point half an inch or so from the centre. Continue the

heat until the glass is thoroughly softened over at least
half an inch of its length, turning the tube round all the
time; then remove it from the flame and draw the two
ends apart with a steady uniform pull, so that the
heated portion draws out into a capillary tube several

 

 

b

SO

c

Fic. 12.—Pipettes.

inches in length. Repeat the process at a point about
half an inch from the tapering end of the larger por-
tion of the tube; heat the bulb and then seal off both
ends of the capillary portion before the bulb cools.

The pipettes are necessarily sterile, having been
drawn out of partially melted glass, and they will
remain sterile indefinitely. Of course the exterior of
the glass will become contaminated, and it should be
passed through the flame before use.

The ends of the tube being sealed up while the bulb
contains heated air, it follows that the bulb will contain
a partial vacuum on cooling. This fact is made use of
42 BACTERIOLOGICAL DIAGNOSIS.

in the collection of specimens. Suppose, for instance,
we wish to take some blood from a heart at a post-
mortem examination for investigation at a distance.
A point on the surface of the heart is first seared with
a hot iron to destroy any germs which might be present,
and the end of the pipette (still sealed) is thrust through
into one of the cavities. It is then broken off by. dex-
terous pressure against the heart-wall, and the: pipette
will fill slowly with the blood. Another. method is to
break off the tip of the pipette and to warm the bulb
before making the puncture. The fluid will rise as the
bulb cools; or both ends may be broken up and the fluid
drawn into the bulb by gentle suction.

Under any circumstances both ends of the pipette
must be sealed up in a flame (the flame of a wax match
will answer at a pinch) and the tube labelled.

Another variety of pipette which is much used for the’
transmission of blood, for the purpose of testing the
serum reaction for typhoid fever, is drawn out to a
point at one end only, the other being left wide and
separated from the bulb bya constriction (fig. 12 8).
The open end should be loosely plugged with cotton-
wool, and serves as a mouthpiece. The manufacture of
these pipettes presents a little difficulty, but a small
amount of practice will enable the practitioner to turn
out a perfectly serviceable one on occasion. These
pipettes are not so suitable as the others for the trans-
mission of fluids for cultural examination or injection
into animals.
DIPHTHERIA. 43

PART II.

DIAGNOSIS OF CERTAIN DISEASES.

DIPHTHERIA.

Diphtheria is a local disease with general symptoms.
The local symptoms are due to the local action of the
bacillus which causes the disease, while the general
symptoms are due to the toxin or poison which they
produce and which is carried in the blood-stream to the
brain, heart and other organs. Now the local symptoms
are comparatively unimportant, and it is to the general
symptoms caused by the toxin that diphtheria owes the
greater part of its high mortality. Diphtheria antitoxin
neutralises this toxin (much in the same way as an
alkali neutralises an acid) and prevents it from harming
the vital structures; but it does ot repair the harm
that the toxin has done. It is obvious, therefore, that
we must not make our diagnosis of diphtheria from the
general symptoms if the antitoxin treatment is to do
any good. The diagnosis is to be made from the local
symptoms, and this is what we can rarely do by ordin-
ary clinical methods at a stage sufficiently early to get
the full value of the antitoxin treatment.

The practitioner has a choice of two methods. He
may inject all patients who suffer from sore throats
which present the slightest resemblance to those seen in
diphtheria, or he may employ bacteriological methods
‘44 BACTERIOLOGICAL DIAGNOSIS.

of diagnosis. The former method may be applicable in
an epidemic of diphtheria, but suspicious throats are
common and antitoxin expensive. In most cases it is
necessary to have recourse to the second method.

Most sanitary authorities have now recognised that
it is their duty and privilege to provide for the bac-
terial investigation of supposed diphtheria free of charge
to doctor and patient, and supply outfits to be used for
taking the material and transmitting it to the labora-
tory. When the practitioner lives within easy reach of
the laboratory (so that the swabs may reach it quickly)
it is his bounden duty to avail himself of the oppor-
tunity thus afforded of getting a free opinion from a
specialist.

But the case of a practitioner living at a distance is
somewhat different. Suppose the patient is seen on the
first day of the illness, an unusual occurrence except in
an epidenric. The swab is taken, dispatched by post,
and reaches the laboratory on the second day. It is
inoculated, and the culture is incubated and examined
on the third day, the result reaching the practitioner
about noon on that day. Now the mortality of diph-
theria which is treated with antitoxin on the first day
is very small, certainly less than five per cent., while
the mortality in cases in which its use is not com-
menced until the third day is much higher, probably
from ten to fifteen per cent., or even higher. In other
words, from 5 to 10 patients out of every 100 lose their
lives if the doctor waits for the result of the bacterio-
logical examination. It is therefore highly advisable
that every practitioner should provide himself with a
bacteriological microscope, and should at least examine
a film prepared directly from the swab and stained in
the manner described below. He should also make
DIPHTHERIA. 45

cultures or send a swab to the laboratory for examina-
tion.

Swabs and outfits are provided by the laboratory
where the examination is made, or can be bought
from most manufacturing chemists and instrument
makers. A swab consists of a steel or copper
(aluminium would be better) wire, the extremity of
which is covered drumstick fashion with a tightly
fitting roll of cotton-wool. The other end is pushed
through a cork, and the whole is contained in a stout
glass tube. It is sterilised before use. These swabs
may be readily made at home. A test-tube is fitted
with a good cork through which is passed a stout steel
knitting needle. This should be long enough to pass
nearly to the bottom of the tube when the cork is in
place, and the end which is to be outside the tube
should be cut off short. The other must be roughened
by a few strokes of a file. A small piece of cotton-wool
(unmedicated) is then held between the thumb and
finger of the left hand, transfixed with the roughened
end of the wire, and twisted round it. The swab is
now placed loosely in the tube and sterilised by dry
heat (see ante, p. 7). It is allowed to cool in the
steriliser, and the cork is pushed home into the tube as
soon as it is cold enough to handle. These swabs will
keep indefinitely, and a stock of them should always be
kept at hand. After use the cotton-wool should be
burnt off in a Bunsen’s burner or spirit lamp, and
another piece applied and the whole re-sterilised.

If a practitioner should see a supposed case of diph-
theria when he is unprovided with a swab he can
readily extemporise one which will answer sufficiently
well out of some cotton-wool (non-medicated), a wooden
skewer or pen-holder, and a glass phial. The wool is
46 BACTERIOLOGICAL DIAGNOSIS.

wrapped round the tip of the skewer, and (after the
swab has been taken) the latter is placed in the phial in
such a position that the cotton-wool does not touch the
glass; the place between the skewer and the neck of
the bottle is plugged with cotton-wool. It is not abso-
lutely necessary to sterilise the swab, although it is a very
great advantage to do so if time permits.

The method of taking the swab is of great importance,
and must be carried out in full detail. It is necessary
that the patient should not have had an antiseptic
gargle or application for at Jeast two hours previously.
It is also advisable to allow him to drink some beef
tea or boiled water (not milk, for this may contain cer-

tain bacilli which closely resemble those of diphtheria) |.

immediately before the process. This will serve to
cleanse the parts.

Requisites.—1. A good light.

2. The swab in its tube.

3. A tongue depressor. The form which is hinged so
as to bend at a right angle is most convenient.

4. A vessel containing antiseptic lotion or boiling
water.

Method.—1. Place the patient so as to face the light.
If a small child he should be held on his nurse’s lap.

2. Loosen the cork in the tube so that the swab. may
be withdrawn with one hand and place it at a con-
venient spot on your right side.

3. Get the patient to open his mouth, insert the
tongue depressor (held in the left hand) and get a clear
view of the area chiefly affected. Do not proceed with
the process (if it can be avoided) until you have done
this.

4. Take the cork between the finger and thumb of
the right hand and pass the swab into the patient's
DIPHTHERIA. 47

mouth, taking great care not to touch his lips, tongue,
or palate. Press it firmly against the area which you
wish to examine and rotate it between the finger and
thumb so as to remove some of the secretion, and, if
possible, some of the membrane. Withdraw the swab,
again taking care not to touch any part of the mouth,
and replace it in the tube.

5. Withdraw the tongue depressor and place it in the
antiseptic lotion or boiling water.

‘ 6. Push the cork home into the tube.

Method of examining the swab.

This may be carried out by means of stained films
prepared directly from the swabs, or by means of cul-
tures. The former method is less useful that the latter,
but we shall consider it first, as it can be performed by
anyone who. possesses a microscope carrying a jy in.
oil immersion lens, and often gives valuable informa-
tion. Moreover, it does not, take long and but little
delay is caused.

Requisites.—1. ‘Clean slides and cover-glasses.

2. Stains—Lé6ffler’s blue or carbol-thionin, and aniline
gentian violet.

3. Gram’s iodine solution and alcohol—methylated
spirit will do.

4. Strips of white filter- or blotting-paper.

5. Balsam.

Method.—Prepare a film in the following way:—Rub
the swab on the middle of a clean slide so as to spread
some of the secretion into a thin layer on the surface.
Allow it to dry, and fix it by passing it slowly through
the flame until the upper surface is just too hot to
prevent your pressing your finger upon it in comfort.
Allow it to cool.

Now filter a few drops of Léffler’s blue or carbol-
48 BACTERIOLOGICAL DIAGNOSIS.

thionin on to the film and allow it to act for 2 minutes,
Wash under the tap.

Dry by pressing carefully with strips of blotting-paper
and then in the flame. Place a drop of balsam upon
the film and apply a cover-glass.

Prepare a second film and stain by Gram’s method
(Pp. 32).

The films are now examined microscopically (see
p- 34). We shall defer the description of the points
upon which a diagnosis is to be based until we deal
with the examination of cultures.

CuLturaL MeEtTuHops.

The diphtheria bacillus grows best at or near the
body temperature (about 37° C.) and flourishes on
almost all culture media. But agar is scarcely ever
used in growing it for diagnostic purposes: this
medium -serves well for the cultivation of a great
many organisms, some of which are almost always pre-
sent in the mouth, so that cultures made upon it are
usually very impure. We use a medium which permits
the development of the diphtheria bacillus and inhibits
that of most other organisms. The best are ascitic
a and solidified blood-serum.

.. The method in which the medium is dneentuied is as
elloars :—The tube of culture medium and the tube
containing the swab are held side by side between the
index and middle fingers of the left hand, the mouths
of the tubes pointing to the right and slightly down-
wards. The-plug of the culture tube is then singed,
removed by means of a pair of forceps, and placed be-
tween the ring and little fingers of the left hand. The
DIPHTHERIA. 4¢

2

cork and wire of the swab tube are now withdrawn and
the cotton-wool plug is inserted into the culture tube
and passed onwards until it reaches the sloped surface
of the medium. It is then rubbed gently on the latter
and twisted round and round so that every part of the
swab may come into contact with the medium. If there
is a piece of membrane special care should be taken to
see that it is rubbed on the surface, for it is here that
we are most likely to find the bacilli. The swab is now
withdrawn and replaced in the tube, and the cotton-
wool plug of the culture tube singed and replaced.

The tube thus inoculated must now be incubated for
about 18 hours at a temperature not exceeding 37° C.,
and is then ready for examination.

Hewlett has suggested a useful method which may
be carried out without any special apparatus, the white
of a hard-boiled egg being used as the culture medium.
Take a fresh egg and boil it for ten minutes or more
and allow it to cool. Now take a narrow-mouthed
wineglass (or a wide-mouthed bottle, which is better)
and rinse it out with perchloride of mercury lotion.
Sterilise a knife by passing it slowly through the flame,
and cut off the top of the egg, care being taken not to
cut into the yolk. Invert the egg into the wineglass
(which must be narrow enough to prevent the egg from
dropping down into it) taking care that none of the
lotion touches the cut surface. This is the culture
medium, and it is sterilised ready for inoculation. At
a pinch it may be incubated in a warm corner near the
fire, near the hot water cistern, or other warm place.
50 BACTERIOLOGICAL DIAGNOSIS.

EXAMINATION OF THE CULTURES.

I. Naked eye.—Each living diphtheria bacillus which
has been deposited upon the surface of the culture
medium and kept at a suitable temperature will develop
into a colony of bacilli; and these colonies are fairly
distinctive, being different from those which are formed
by most other organisms. The expert bacteriologist
can often give an accurate guess as to the presence or
absence of diphtheria bacilli by mere inspection of the
cultures. The colonies formed by diphtheria bacilli on
solidified blood-serum or on ascitic agar are small round
vaised spots; they are variable in size, but rarely exceed
that of the head of a medium sized pin. They are
white or grey in colour, and opaque. They do not tend
to run together so as to form a uniform film over the
surface of the medium, but remain discrete even when
closely packed. Some cocci form colonies which closely
resemble those of diphtheria, but they rarely become
elevated so high above the surface in the same space of
time.

‘II. Microscopical.—Prepare films by the method de-
scribed on page 29, following out all steps in the fullest
detail. Stain one of them (step 11) with Léffler’s blue
or carbol-thionin, allowing the stain to act for two
minutes, and the other by Gram’s method.

In removing some of the growth to make the film,
remember the facts just stated as to the characters
of the colonies of the bacillus, and select a colony
presenting those characters (especially that of elevation)
if one is present. If there is no apparent growth in
the tube take ‘‘sweeps” of the whole surface. This
is conveniently done by means of a platinum loop,
DIPHTHERIA. 51

shaped like a stirrup, the flat bar being drawn along the
surface of the medium from bottom to top just as a rake
is drawri along a flower bed.

Now examine your specimens in the way described
on page 34.

CHARACTERS OF THE DIPHTHERIA BACILLUS.

The following are the chief points which are con-
sidered in deciding whether a given stained slide does
or does not show the diphtheria bacillus.

1. The shape of the bacillus is very variable, and this
is a feature which often affords us great assistance; a
specimen in which all the bacilli present resemble each
other exactly in shape and size, is not from a case of
diphtheria. Diphtheria bacilli are narrow rods; they
are either straight or slightly curved in an arc of a large
circle or into an f shape (Plate I., figs. 1 and 2). Their
ends are usually rounded, but it is not uncommon to
find forms with one end or both sharply pointed.
Lastly, clubbed forms are to be met with in almost all
cultures, though they are most frequent in those which
have been incubated for several days; they may be
compared to a note of exclamation (1).

2. Their size.—Two well marked varieties occur. The
long form is about as long as a tubercle bacillus (to
compare it with an organism with which the practitioner
may readily become acquainted) or somewhat longer; it
is decidedly thicker. The short form is only about half
as long and thick in proportion.

We do not know anything as to the difference in
pathogenicity (if any) of the long and the short varieties
of the diphtheria bacillus. They appear to “breed

E2
52 BACTERIOLOGICAL DIAGNOSIS.

true” for long periods, and cases of diphtheria caused
by the one appear to have as high a mortality as those
caused by the other.

Hoffman’s bacillus is dealt with subsequently.

3. Their staining veactions——The diphtheria bacillus
stains readily with all the stains in common use for
bacteriological purposes. It usually (but not invariably)
stains irvegulavly, deeply stained portions alternating
with others which remain colourless. This gives rise to
a beaded appearance, and forms sometimes occur which
can hardly be distinguished from short chains of strep-
tococci. When a powerful stain is applied for a long
time this appearance may be lost.

The diphtheria bacillus stains deeply with thionin.
This often affords a certain amount of help in the
diagnosis, as many other bacilli do not stain nearly so
deeply in the same time.

It stains by Gram’s method. A beginner should
always test his results in this way. If suspicious bacilli
do not retain the violet stain they are not those of
diphtheria.

4. Theiy avvangement.—This is a most characteristic
feature, but it is one which is difficult to describe. The
old comparison to the strokes which form a Chinese
letter is a fairly good one; the bacilli lie in little groups,
some lying parallel to one another, and some at various
angles with these. The characteristic arrangement is
best seen in a specimen made from a pure culture of
the short form.

Before coming to a conclusion as to the presence or
absence of diphtheria bacilli from an examination of 4
stained film, make a very thorough search; if no bacilli
are seen make several more films and examine them.
When you see a group of bacilli examine it carefully

2
DIPHTHERIA. 53

noting each characteristic and comparing it with those
described above.

The beginner is strongly recommended to procure a
series of slides of diphtheria bacilli from a bacterio-
logical laboratory and to study them carefully.

Hoffman’s bacillus (Plate I., fig. 2) is a modified
form of the true diphtheria bacillus, and is frequently
met with in throat cultures. It is about as long as
the short form of diphtheria bacillus, but decidedly
plumper and is more uniform in shape and size.
These bacilli stain uniformly and deeply. They ex-
hibit the same arrangement, and are often grouped in
pairs. Clubbed forms do not occur.

The diphtheria bacilli which occur in films made
direct from the swab are similar to those seen in
cultures, but are often thicker; they may stain uni-
formly, and clubbed forms are rare. It is unusual to be
able to make an absolute diagnosis as to their nature,
but it may be done at times. Yet such an examination
is often useful. If suspicious bacilli are present you
should inject antitoxin forthwith; if no suspicious
bacilli are seen it is safe to wait for the result of the
cultural examination.

INTERPRETATION OF RESULTS.

The discovery of the diphtheria bacillus in the exudate may
mean :—

(a). That the patient is suffering from diphtheria.

(b). That he has suffered from diphtheria and is now
convalescent but is stil infectious. The bacilli may
persist for weeks or months and while they do so the
54 BACTERIOLOGICAL DIAGNOSIS.

patient must be isolated and treated with antiseptic
gargles.

(c). That he is in danger of acquiring diphtheria if
subjected to any influence which lowers his vitality, or
which would cause ordinary sore throat in any ordinary
person.

(d). It always means that the person may communi-
cate diphtheria to a susceptible subject.

The significance of Hoffman’s bacillus is not yet
settled. It is frequently found in the throat when the
patient is convalescing from an attack of diphtheria,
and sometimes in subjects who afterwards develop
diphtheria. It also appears to cause epidemics of sore
throat which do not present anything remarkable in
their clinical characters.‘ It is safest to regard any
patient who presents these bacilli as being infectious.
In other words, in the present state of our knowledge it
is best not to draw any distinction between Hoffman’s
bacillus and the true diphtheria bacillus, as far as infec-
tivity is concerned.

A negative result may mean :—

(a). That the patient is not suffering from diphtheria.

(b). That the swab did not touch the affected area.

We exclude errors in technique and observation.

A sterile culture may mean :—

(2). That an antiseptic was used too soon before
taking the swab.

(). That the diseased portion of the throat was not
touched. Other parts of the mouth contain numerous
bacteria, but many of them do not grow well on blood
serum or ascitic agar.

We again exclude errors arising in the laboratory.

Whenever the culture tube remains sterile the exam-
ination should be repeated.
TETANUS. 55

TETANUS.

The pathology of tetanus is very much like that of
diphtheria. In each disease the specific bacilli are
localised at or near the region at which they enter the
body, and form a toxin which is absorbed into the
blood and affects distant organs. In each case research
has shown that an antitoxin is formed which neutralizes
this toxin and prevents it from uniting with the cells of
the body, but which has not the power of turning it out
from such a combination. In other words tetanus
antitoxin, like that of diphtheria, is preventive but not
curative. But here, unfortunately, the resemblance
between the two diseases ceases. The local lesion in
diphtheria is obvious and its presence causes a good
deal of inconvenience to the patient; he sees a medical
man early, and the diagnosis of diphtheria is made
before much of the toxin has entered the blood. It
is different with tetanus. In this the local symptoms
are practically nil; there may be suppuration at the
region of inoculation but this is so common as not to
excite suspicions. The result is that the diagnosis is
not made until the appearance of the symptoms refer-
able to the nervous system indicates that the period at
which antitoxin might have been used with success has
gone by.

To illustrate this we will imagine that the local lesion
of diphtheria to be so slight as to be unnoticed by
doctor and patient. The result would be that the
disease would only be diagnosed when the severe
toxemic symptoms had manifested themselves, and
antitoxin would then be almost or quite useless. If it
56 BACTERIOLOGICAL DIAGNOSIS.

were not for the discomfort and pain caused by the
throat lesion of diphtheria the antitoxin treatment of
the disease would have probably been abandoned as
useless.

But tetanus may be diagnosed by means of a. bac-
teriological examination of the local lesion before toxic
symptoms have appeared, and in cases where this is
done we may safely look for results from tetanus anti-
toxin which are as good as those obtained from the
early use of diphtheria antitoxin ; for the experimental
evidence in favour of the one is every whit as great as
that in favour of the other.

Considerations of time would prohibit the bacterio-
logical examination of the multitude of small wounds
which are seen by the majority of medical men. But
a wound which clinical experience and bacteriological
research as to the occurrence of tetanus bacilli outside
the body, indicates as being one which is likely to
become infected with the bacillus in question, should be
submitted to a careful and prolonged search for the
bacillus, These are deep incised and lacerated wounds,
especially those of the hand and foot, and especially if
garden earth or horse-dung has been rubbed into the
tissues. Wounds made with splinters should be exa-
mined, especially if there is reason to think that the
splinters were dirty. The same reniark applies to deep
stabs with rusty nails, &c. Tetanus may follow a
wound which heals up by first intention, but this is
unlikely; suppuration or necrosis of the edges (though
not due to the tetanus bacillus itself) is present in the
majority of cases.
TETANUS. 57

EXAMINATION oF Pus FROM SUSPECTED CASES OF
TETANUS.

Requisites—1. Slides and cover-glasses.

2. A stiff platinum loop.

3. Bunsen’s burner or spirit lamp.

4. Léffler’s blue or carbol thionin.

5. Materials for Gram’s staining.

6. Balsam.

If cultures are to be taken add a pipette (see p. 41),
a deep tube of agar to which two per cent. of grape-
sugar has been added previous to sterilisation, a flask of
water, and a thermometer.

Method.—Scrape the deeper portions of the wound
with the platinum loop and spread out the secretion
thus obtained on the surface of a slide. Prepare several
of these slides, and fix the film by heat. Stain some by
the simple stain for two minutes and others by Gram’s
method.

The bacillus of tetanus is about as long as the
tubercle bacillus and is very slender. It stains by
Gram’s method. A very characteristic feature is its
method of spore-formation. The spores are spherical
bodies which are formed at the extvemities of the bacilli,
giving them the appearance of pins or drumsticks. The
spores do not stain by the ordinary stains, and appear as
colourless and highly refractile bodies (Plate II., fig. 2).

The cultures are made in agar to which 2 per cent. of
grape-sugar is added, and the needle or pipette used in
making the inoculation is plunged deep down into the
medium. The bacillus of tetanus is an anaérobe, 7.¢., it
grows only in the absence of oxygen. The stabs are
made deep in order to inoculate the material far away
58 BACTERIOLOGICAL DIAGNOSIS.

from the air, and the glucose is added to absorb any
oxygen which may be in the medium. To increase our
chances of obtaining this bacillus in pure culture the
material to be examined is to be heated to a tempera-
ture which will kill all developed bacteria, but which
will not be injurious to spores; the tetanus bacillus is
the only anaérobic organism with a spherical terminal
spore which is at all likely to occur in a wound.

Method.—The inoculations are to be made with a
pipette. If the pus which comes from the wound can
be drawn up into the capillary tube of a glass pipette
such as is described on page 41, the material should be
collected in this way. If this is not the case the wound
must be scraped with a sterilised platinum needle or
other suitable instrument and the material thus obtained
mixed with some boiled water (previously cooled) and
then sucked up into the pipette; the end of the latter is
then to be sealed in the flame, care being taken that the
material itself is not heated.

Having filled and sealed the pipette, heat some water
in a small flask or large test-tube until it reaches 80° C.,
as measured by the thermometer; insert the sealed end
of the pipette in the water and maintain the tempera-
ture for ten minutes. The thermometer is to be kept in
the water the whole of the time, and the flame is to be
taken away when the temperature rises above 80° C.
and reapplied when it falls below that point.

At the end of this time the pipette will contain no
living object other than spores. Break off its point and
insert it gently into the glucose agar, taking care to
keep exactly in the axis of the tube, until the tip of the
pipette reaches almost to the bottom of the test-tube.
Withdraw the pipette gradually, blowing out its con-
tents as you do so. The spores of the tetanus bacillus
TETANUS. 59

(if present) will now be inoculated deep down in the
medium, far away from the air. To reduce the supply
of oxygen still further it is a good plan to melt some
paraffin (a hard candle answers perfectly) and pour a
layer an inch thick over the surface of the medium.

The cultures thus made are to be incubated for a few
days at the body-temperature. After about forty-eight
hours the growth begins to appear in the deeper portions
of the tube as a series of delicate wavy outgrowths from
the central stab. These do not appear in the upper
portion of the medium, where the oxygen hinders their
growth. If the tube shows such a growth it should be
submitted to a microscopic examination. It is a good
plan to break the tube and to split up the cylinder of
medium with a knife; films are made from the growth
and stained as above. Spores are formed after about
thirty-six hours.

The other methods of cultural examination are far
more difficult.

INTERPRETATION OF RESULTS.

If bacilli having the above characters are found in
films, the diagnosis of tetanus must not be considered
as being absolutely proven, for there are other bacilli
which might be mistaken for those under discussion ;
but the probability that the patient will develop the
disease is so strong that steps should be taken acccord-
ingly. The wound should be scraped and thoroughly
treated with antiseptics, and antitoxin should be given.
If the deeper portion of the glucose agar stab shows
the tree-like growth which has been described and con-
tains slender drum-stick bacilli, the case is strength-
60 BACTERIOLOGICAL DIAGNOSIS.

ened, even although the upper part of the medium is
contaminated with other organisms.

The only way in which the bacilli can be recognised
with absolute certainty is by means of animal experi-
ments, and to this end the practitioner should transmit
to the laboratory some of the scrapings from the deeper
portion of the wound in a test-tube sterilised by boiling,
or, still better, by dry heat.

THE PNEUMOCOCCUS, PNEUMONIA, Etc.

The pnemococcus is a very important organism, and
one which plays a prominent part in the production of
disease. It may occur in the mouth in a healthy
person; hence its recognition in small quantities in
the sputum is not of diagnostic value.

The pneumococcus is a very common cause of disease
of the respiratory system. It causes :—

1. Acute lobay pneumonia. Opinions differ as to whether
it is the only cause of this disease, though it appears
most probable that this is the case.

2. Lobulay (byoncho-) pneumonia. This disease may
also be caused by streptococci, staphylococci, diphtheria
bacilli, influenza bacilli, plague bacilli, tubercle bacilli,
and others. The pneumococcus may also occur as a
secondary infection in lobular pneumonia due to any of
these.

3. Pleunsy, either the serous, fibrinous, or purulent
varieties. It is important to notice that the prognosis
of empyema is better when the. disease is due solely to
the pneumococcus than when other organisms (strep-
tococci, staphylococci, tubercle bacilli, &c.) are present,
PNEUMOCOCCUS. 61

and such cases usually recover without resection of ribs.
The bacteriological examination of the pus from a
pleural cavity may thus lead to results important as
to prognosis and treatment.

4. The pneumococcus may occur as a secondary in-
fection in almost any disease of the lung; for instance,
in the walls of a phthisical vomica.

The most important primary lesion due to the pneu-
mococcus outside the respiratory system are :—

1. Otitis media, of which it is a very common cause.

2. Brawny induration of the skin with or without sup-
puration. This is uncommon, but the author has seen
several cases.

Pneumococci may escape into the blood from any of
these lesions, and may appear in that fluid when there
is no obvious primary lesion from which it could have
gained access. The most common results are :—

1. Septicenia.

2. Ulcevative endocarditis. Many other bacteria may
cause this disease.

3. Meningitis. Pneumococcal meningitis may also be
due to direct spread from the middle ear.

4. Arthritis.

5. Peritonitis.

In actual practice we have most commonly to search
for the pneumococcus in sputum, pus, and blood. In
the latter case cultural methods are usually necessary,
and we shall defer its consideration for the present.

Sputum.—The examination of sputum may be made
in order to make a diagnosis as to the presence or
absence of pneumonia in a case in which the physical
signs are indeterminate, or to establish the nature of a
lobular pneumonia.

The patient must wash out his mouth with water,
62 BACTERIOLOGICAL DIAGNOSIS.

which should have been boiled and allowed to cool.
He must then spit into a clean wide mouthed bottle
also containing boiled water, and care must be taken
that the sputum used for the examination comes
directly from the lungs and is not merely mucus which
has collected in the mouth.

The mass of mucus forming a single “spit” is
agitated gently in the water to remove contaminations
from the bronchial tubes and mouth; the water is
poured off and more added, and the process repeated
several times. Then the mass of mucus is fished out,
placed in a watch glass, carefully opened with a pair
of scissors, and a piece about as big as a pea is removed
from the centre of the mass with a platinum loop. It
is placed on a clean slide, another slide pressed upon it,
and the two are slid apart. The films thus obtained
are allowed to dry, and fixed by heat in the usual
way.

One is stained in dilute carbol-fuchsin for about two
minutes and then washed very thoroughly in water.
The other is stained by Gram’s method.

The pneumococcus is a diplococcus, i.¢., the indi-
vidual cocci are arranged in pairs. Each coccus has
usually an oval or lancet shape, the sharp ends of the
two germs pointing away from one another (Plate I.,
fig. 3). Abnormal forms (round cocci, short bacilli, &c.)
are frequent. The pneumococcus has a capsule when it
occurs in the living body or in pathological exudates,
but not in most cultures. This capsule does not stain
readily, and appears in a properly stained specimen as
a clear halo round the two cocci.

Examine your Gram specimen first. The pneumo-
cocci should be clearly seen, and you should be able to
make out their shape and characteristic arrangement in
PNEUMOCOCCUS. 63

pairs. The rest of the slide will be unstained, so that
you will not be able to make out the capsule.

Now examine the carbol-fuchsin specimen. You will
see the cocci coloured a deep red, and you will also
notice that the general surface of the film is stained
pink, while there is a clear and colourless zone round
each pair of cocci. This is the capsule, which is
rendered distinct by ‘negative staining.” If the
carbol-fuchsin has been allowed to act too long the
capsule may be stained a faint pink.

INTERPRETATION OF RESULTS.

In cases of lobar pneumonia you will probably find
pneumococci in great quantity, and no other bacteria in
a specimen of sputum made in the manner described. If
you find many pneumococci in a case of lobular
pneumonia the disease may have been caused by
another germ, and the cocci in question may have
been nothing more than a secondary infection. The
carbol-fuchsin specimen should be searched for bacilli
resembling those of influenza, &c., and another should
be stained for the tubercle bacillus if the clinical aspect
of the case suggests the possibility of a tubercular origin
for the disease.

Pus is examined in the same way and presents similar
appearances. Most of the cocci are extra-cellular.

The method of making a film from pus is described in
a subsequent chapter. It is very similar to that
described above, but the upper slide should be allowed
to fall on the lower one by its own weight; the two
should not be pressed together.
64 BACTERIOLOGICAL DIAGNOSIS.

. INFLUENZA.

The diagnosis of influenza is often very difficult, and
a simple bacteriological examination should be made
in all cases. The cultivation of the specific bacillus
is by no means easy, but this does not matter, as an
examination of stained films will usually permit of a
diagnosis. :

The influenza bacillus occurs in the sputum and
occasionally in the blood. It may be searched for in
the latter situation, but the quest is a very difficult one.
In the sputum it occurs in vast quantities and is almost
free from other germs.

The method by which the sputum is collected is the
same as that employed in pneumonia; a mass of
greenish-yellow muco-pus is selected for examination,
squeezed between two slides, and the films dried and
fixed as in the case for the examination for the tubercle
bacillus. One is stained for five minutes with dilute
carbol-fuchsin or Léffler’s blue, the slide being slightly
warmed, and the other by Gram’s method.

EXAMINATION OF THE SPECIMENS.

The specimen stained with carbol-fuchsin or Léffler’s
blue should be taken first and examined under the oil
immersion lens. The bacilli (in a positive case) will
be seen in vast numbers as extremely minute rods; it
would take from twelve to sixteen of these rods to make
up the diameter of a red blood corpuscle. They fre-
quently occur within the pus cells, and when in this
ANTHRAX. 65

situation they may appear to have a capsule and pre-
sent a resemblance to pneumococci, from which, how-
. ever, they may be distinguished by their small size,
and by the fact that they do not stain by Gram’s
method (Plate II., fig. 3).

Now examine the Gram specimen. The bacilli are
not seen, though other bacteria (pneumococci, strepto-
cocci, staphylococci, &c.) may be visible.

INTERPRETATION OF RESULTS.

If minute bacilli having the above characters are
found in large numbers in sputum taken in the manner
described, the diagnosis of influenza may be made with
certainty.

If none are apparent and you are sure of your
technique (and also of the fact that the sputum came
from the lungs) the diagnosis of influenza is highly
improbable.

If the pulmonary symptoms in a case of influenza do
not clear up speedily the sputum should be examined
for the tubercle bacillus and the examination repeated
at intervals.

ANTHRAX.

Anthrax occurs in man in three forms. The most
common is cutaneous anthrax, or, as it is sometimes
called, malignant pustule. Pulmonary anthvax or wool-
sorter’s disease is much rarer, and intestinal anthrax
rarer still, The practitioner will find the greatest

F
66 BACTERIOLOGICAL DIAGNOSIS.

assistance from a bacteriological examination in the
cutaneous form of the disease; he may search for the
specific bacillus in the sputum in a supposed case of
wool-sorter’s disease, but he must be careful in his
interpretation of his result, as bacilli which might be
mistaken by an untrained observer relying on the
morphological appearances alone, sometimes occur in
the sputum. The search for bacilli in the faces in a
supposed case of intestinal anthrax must be relegated to
an expert.

The true nature of a case which is examined post-
mortem can easily be determined bacteriologically ; the
cut surface of the liver or spleen should be rubbed upon
a clean slide, and the films treated secundum artem.
They will probably show the bacilli in large numbers.
Sections may also be cut, or portions of the organs
fixed in the manner to be described subsequently and
forwarded to a bacteriological laboratory.

In the later stages of any infection with anthrax the
bacilli may be found in the blood. They may be
apparent on examination of stained films, or by cultural
‘methods similar to those used in the diagnosis of
malignant pustule.

The anthrax bacillus varies considerably in length,
but is always a large organism and may be considerably
longer than the diameter of a red blood corpuscle. It
is much thicker than the bacilli which have been dealt
with hitherto, and it is invariably straight. The ends
of these bacilli are cut sharply at right angles to the
sides of the organism, and may be even somewhat
concave; this is a most characteristic feature. The
anthrax bacillus stains by Gram’s method (Plate L.,
fig. 4).

In cultures the appearances are somewhat different.
ANTHRAX. 67

Here the bacilli are frequently arranged in long chains
which have an appearance which has been compared to
that of a bamboo; chains occur in the blood or in the
inflammatory exudate, but are usually much shorter
than those seen in cultures. But the most important
feature in cultivation of the anthrax bacillus is the
development of spoves which are oval highly refractile
bodies, which lie in or near the centre of the bacilli, one
in each. These spores are possessed of tough capsules,
which resist the action of the ordinary stains much in
the same way as the tubercle bacillus does. Thus it
happens that in films of a cultivation of the anthrax
bacillus which have been stained with such a dye as
methylene blue the spores are readily seen as colourless
and refractile oval areas in the centre of the bacilli, the
latter being stained blue. The spores themselves may
be stained by a modification of the process used for the
tubercle bacillus. The films are first stained by heated
carbol-fuchsin, which penetrates slowly through the
capsule; they are then decolorised by a very vapid
immersion in dilute sulphuric acid and examined micro-
scopically. If the red colour has been entirely removed
by the spirit they are ready to be counterstained by
methylene blue; if not they must be dipped in the spirit
once more and re-examined. When this process is
successful the spores are stained red and the bacilli
blue.

The presence of spores enables us to isolate the bacilli
from most of the organisms with which it is likely to be
contaminated by a very simple process. The spores
resist the action of heat just as they resist stains, and
for the same reason, and suitable temperature will kill
off all the non-sporing organisms and spare the spores.
The latter may then be inoculated at a suitable tem-

F2
68 BACTERIOLOGICAL DIAGNOSIS.

perature and will develop into bacilli. This process,
however, is not applicable to the examination of the
blood or morbid effusions, as the bacillus of anthrax
does not form spores in the living body. In this it differs
from the tetanus bacillus.

INVESTIGATION OF A SUPPOSED CASE OF MALIGNANT
PustTuLe.

Requisites—1. Several glass pipettes; if cultures are
not required one will be enough.

2. Clean slides and cover-glasses.

3. Bunsen’s burner or spirit lamp.

4. Léffler’s methylene blue; also the materials for
Gram’s staining.

5. Balsam.

6. Tubes of gelatin if cultivations have to be taken.

Method.—Break off the extreme tip of one of the glass
pipettes and insert into one of the vesicles around the
dark papule in the centre of the lesion; it may be
necessary to make a puncture with a sterilised needle
before this can be done. If the fluid does not rise
spontaneously into the pipette break off the other end
and suck gently, watching the column of fluid so that.
it does not get into your mouth.

Having obtained a drop or two of the fluid exudate
blow it out on to the surface of a clean slide and spread
it out into a film; prepare as many of these as you can..
Allow them to dry, and stain one with Ldffler’s blue,.
and some by Gram’s method.

Examine with the oil-immersion lens. Make a care-
ful search over the films, looking for large cigarette-
shaped bacilli, noting whether they are or are not
ANTHRAX. 69

arranged in chains. Examine the Gram specimens and
see whether the bacilli are to be seen in them also.

INTERPRETATION OF RESULTS.

If the case is really one of malignant pustule the
chances are very greatly in favour of your finding the
bacilli in large numbers, and the failure to do so tells
strongly against a positive diagnosis.

Cultuval methods—The fluid for examination is taken
in exactly the same way as that described above, but
the isolation of the organisms will be greatly facilitated
if antiseptic methods are employed to prevent con-
tamination with skin: bacteria. To this end the surface
of the lesion should be washed gently with carbolic or
perchloride lotion and then (very thoroughly) with
alcohol or methylated spirit-to remove the antiseptic.
The surface is then allowed to dry.

If the material is to be transmitted to a public
laboratory for examination (and this is the wisest course
to adopt) the fluid must be carefully sucked up into the
bulb and both ends of the pipette carefully sealed.

If the examination is to be made at home the best
way is to make two inoculations in gelatine. The first
should be a stab culture and may be made with the
pipette direct; or the fluid may be blown out into a
watch-glass or on to the surface of a slide (in either
case sterilised by being heated in the flame and then
allowed to cool) and the stab made by dipping the end
of a straight platinum needle into the fluid and then
driving it into the gelatin.

The other culture is made with the pipette; this is
driven into the gelatin in a tube and the contents blown
70 BACTERIOLOGICAL DIAGNOSIS.

out. The gelatin is then immersed in waym water
until it is melted and poured out into a Petri’s dish

 

Fic. 13.—Petri’s Dish.

(fig. 13) previously sterilised by dry heat and allowed
to cool.

Both cultures are incubated at a temperature of
about 20° C.

 

Fic. 14.—Stab culture of anthrax bacillus (Crookshank).

In about two days the gelatin stab tube will show a
very characteristic appearance if the anthrax bacilli are
ANTHRAX. 9

present in pure culture. The growth takes place in
lines which project nearly at right angles to the line of
inoculation, and grow more vigorously the nearer they
are to the surface. The result is the development of
a culture which has a strong resemblance to an inverted
fir-tree (fig. 14). In another day or two the gelatin
will begin to show a certain amount of liquefaction at
the surface.

The appearances in the plate-culture are perhaps not
quite so characteristic, but they are manifested in
impure cultures. The young colonies of anthrax bacilli

 

Fic. 15.—Young colony of anthrax bacillus (xX 15). (Crookshank).

have a whorled appearance which has been compared to
a barrister’s wig or to the head of Medusa (fig. 15). The
plate should be placed upon the stage of the microscope
and examined for these colonies with the low power.
If one is found a clean cover-glass should be pressed
upon it, lifted up with a needle so as to bring up the
colony with it, fixed by heat, and stained with carbol-
thionin or methylene blue. The colonies are most
characteristic after two days incubation; at a later
period the gelatin is liquefied and spores are formed.
72 BACTERIOLOGICAL DIAGNOSIS.

A culture which presents these cultural and morpho-
logical appearances may be considered to he one of
anthrax with almost absolute certainty, though other
tests (notably animal tests) would be applied in a
laboratory.

TUBERCLE.

The diagnosis of tuberculosis by bacteriological
methods (in the case of most morbid exudates) is within
the reach of every practitioner; cultural methods are
not used and the recognition of the bacillus is rapid,
easy, and certain.

The bacilli may be sought for in sputum, urine, pus,
feeces, or any other morbid material. We will first
describe the method of staining which should be
adopted, then the characters on which the recognition
of the bacillus depends, and lastly the methods by
which the films are prepared from the various materials.

STAINING THE TUBERCLE BacILuus.

Requisites.—1. Slides, cover-glasses, forceps, and bal-
sam.

2. A Bunsen burner or spirit lamp.

3. Dilute sulphuric acid—about 20 per cent.—con-
tained in a wide-mouthed bottle or ina jar. This must
be large enough to admit a slide but not large enough
to permit it to fall down to the bottom.

4. Carbol-fuchsin.
TUBERCLE. 73

5- Methylene blue. (Saturated watery solution or
Loffler’s blue).

6. A metal (iron or copper) plate. The exact dimen-
sions do not matter, but 8 x 4 x } in. is a convenient
size. It should be mounted upon a tripod. This slab
is not absolutely necessary, but it is a very great advan-
tage.

Method.—We will suppose that the film has been
prepared by one of the methods described subsequently
and fixed by heat.

1. Place it upon the metal plate and heat the latter
by the flame. Flood the slide with carbol-fuchsin and
let the heat continue until the stain steams, but do not
allow it to dry up; let this go on for from three to five
minutes. If the stain shows signs of drying up adda
little more; if it begins to boil slide it along the plate
away from the flame, or remove the latter for a short
time.

If you have no metal plate it is possible to hold the
slide with a pair of forceps, but in this case the film is
most conveniently made on a cover-glass.

Remember not to let the stain dry up.

2. Remove the slide from the plate with the forceps
and wash it under the tap or in a bowl of water.

3. Put it into the bottle containing the dilute acid.
After three or four minutes withdraw it and again wash.
If much pink colour comes back re-insert it in the acid
for a short time and again wash. ‘The process must be
repeated until the film only shows a slight pink tinge.

4. Now apply the methylene blue for a minute or so.

5. Wash, dry with blotting paper and then by gentle
heat. Apply a drop of balsam and cover.
74 BACTERIOLOGICAL DIAGNOSIS.

RECOGNITION OF THE TUBERCLE BACILLUS.

The tubercle bacillus is about half as long as a red
blood corpuscle is wide, or rather longer, and is very
slender. It is straight or slightly curved, and is variable
both in shape and in size (Plate II., fig. 2).

We recognise it by means of a staining reaction.
Tubercle bacilli contain a considerable amount of fat,
and this prevents them from staining readily with
ordinary stains. In the process described above we
used fuchsin, which is a very powerful stain, and added
a mordant (carbolic acid) which increases its penetra-
tive properties. Even with this, staining is very slow,
so that we heated the specimen.

The fat which prevents the bacilli from staining also
prevents the stain from being removed by such sub-
stances as acids and alcohol. In stage 3 of the above
process we aim at allowing the acid to act until it has
removed the fuchsin from everything except the tubercle
bacilli. The methylene blue is a counter-stain, and
colours all organisms, pus cells (especially their nuclei),
epithelial cells, and shreds of lung tissue; in fact every-
thing except the tubercle bacilli. The latter appear as
slender red rods which often show the irregular staining
which has been described as occurring in the diphtheria
bacillus.

Now “acid-fast” bacilli are very rare, though they
have been found in unexpected situations of late years.
Only three such bacilli need be taken into consideration
in dealing with human pathology.’ These are the
tubercle bacillus, the leprosy bacillus, and the smegma
bacillus. The bacillus of leprosy would rarely lead to
TUBERCLE. 75

mistakes in this country; it is recognised by the fact
that it is straighter and more uniform than the tubercle
bacillus, and by the fact that it resists decolorisa-
tion more powerfully than the tubercle bacillus. The
smegma bacillus may occur in the urine and lead to
mistakes unless the sample examined was drawn off per
catheter. It is distinguished by the fact that it is
readily decolorised by alcohol (absolute alcohol or
methylated spirit) while the tubercle bacillus is not.
In staining a film from the urine we decolorise in spirit
for a minute after the acid and before the methylene
blue, 7.2., between stages 3 and 4 in the above
description.

In searching for the tubercle bacillus the } in. lens
will serve, though an oil immersion lens is an ad-
vantage.

METHOD OF COLLECTING THE SPUTUM.

This is of some importance, and the method recom-
mended should be carried out in all cases.

Get the patient to wash out his mouth thoroughly
with warm water before going to bed. Let him spit
into a clean bottle, jar, or tin, and employ only the
sputum coughed up before food is taken in the morning.

METHOD OF PREPARATION OF THE FILM.

Sputum.—Pour the sputum into a watch-glass and
place the latter upon a dark surface. Examine it
closely, looking out for small yellow particles; these
consist of caseous material and will probably contain
76 BACTERIOLOGICAL DIAGNOSIS.

tubercle bacilli in large numbers. The advantage of
getting the patient to wash out his mouth and using
only fasting sputum is obvious, for particles of food
may present exactly the same appearance.

Having found such a mass pick it out by means of a
platinum loop or pair of forceps and transfer it to the
middle of a clean slide. Now place another slide on
the top of the first, squeeze them together and then
slide them apart. You should get two good uniform
films. Dry and fix.

If there are no caseous masses pick out a mass of the
sputum at random and proceed as before.

A better method is as follows:—Half fill an ordinary
medicine bottle with carbolic lotion (1 in 20) and adda
drachm or two of the sputum. Shake thoroughly for a
few minutes and place the bottle where you can give it
an occasional shake during the next few hours. Then
pour the milky emulsion which results into a conical
urine glass and allow it to stand for twelve hours or
more. Remove some of the deposit which will form
with a pipette and spread it into a thin film on a slide.
Dry and fix.

Urine is treated in a similar way; but here carbolic
acid (liquefied or in crystals) is added to the urine in
amount sufficient to convert it into a 1 in 20 solution.
This is allowed to deposit or (better) is centrifugalised.
Films are prepared from the deposit.

Remember that they should be passed through alcohol
after staining.

Pus is best carbolised in the same way as sputum; if
very thin it may be treated like urine. The tubercle
bacilli will rarely be found in pus unless it is examined
soon after the abscess is opened, but may be detected
by inoculation experiments for long periods.
TUBERCLE. ay

Cleay exudates are more difficult to examine, and, as
they usually contain bacilli in very small numbers only,
a negative result should not be given too much weight.
They may be allowed to sediment in a conical urine
glass, a few crystals of camphor being added to prevent
bacterial growth, carbolic acid being inadmissible as
it precipitates the proteids. The examination is best
made in a bacteriological laboratory, as decisive results
can only be obtained by animal experiments. Collect
the fluid in a bottle which has previously been boiled
in water for half an hour and allowed to cool. Cork
it with a cork which has also been boiled. Add no
antiseptic and forward it to the laboratory as soon as
possible.

Milk may be examined in the same way as urine,
films being made from the cream as well as from the -
deposit. These films are fixed, soaked in ether to
remove fat, and again fixed. They are then stained as
before, and it is advisable to pass them through alcohol.

When feces are to be examined the best plan is to
administer opium in amount sufficient to cause consti-
pation. The surface of the scybalous motions which
result are to be scraped off and stained in the usual
way.

INTERPRETATION OF RESULTS.

The finding of tubercle bacilli in the sputum is con-
clusive evidence of tuberculosis of the lungs, but no
information as to prognosis can be drawn from the
numbers which are present; they may occur in great
quantities in the sputum from patients who are doing
well, and the author has found enormous numbers in
78 BACTERIOLOGICAL DIAGNOSIS.

the sputum of a person who had presented no sym-
ptoms of the disease for eight years and was apparently
cured. But a person in whom the bacilli are present is
always in danger of a recrudescence of the disease, and
may be a source of infection. Absence of the bacilli does
not disprove the diagnosis of tuberculosis; bacilli do
not appear in the sputum until the lung-tissue in which
they occur breaks down, and are therefore absent in the
early stages of acute tuberculosis.

In some cases of ordinary chronic phthisis bacilli may
occur in the sputum in very scanty numbers and may be
missed unless a very careful search is made. Bacilli
should not be considered as being absent until well
stained films have been examined for at least half an
hour, and the examination repeated on several occa-
sions.

The finding of tubercle bacilli in the urine is prac-
tically absolute proof of tuberculosis of some part of
the urinary tract, probably the kidneys or bladder.
Absence of bacilli implies nothing unless the exam-
ination has been made very thoroughly and repeated
several times at intervals. Then it affords presumptive
evidence that the urinary passages are free from the
disease.

The same is true of the examination of pus. Tubercle
bacilli rarely occur in inflammatory exudates except in
very small numbers and can only be demonstrated by
animal experiments. If you examine pus from a
chronic abscess and find no organisms of any kind it is
almost certain that the process is a tubercular one; and
the negative evidence obtained by the failure to find
tubercle bacilli should not be allowed to carry much
weight. The same is true for the clear exudates.
LEPROSY. 79

LEPROSY.

The leprosy bacillus resembles that of tubercle, but it
is somewhat straighter and more uniform. It occurs in
leprous lesions in great profusion, and its discovery does
not present any difficulty. It has never been cultivated.

In a suspected case of leprosy, films should be made
from the nasal discharge, for the nasal cavities are very
frequently affected. Indeed, it seems highly probable
that the primary lesion through which the bacilli gain
access to the body is in the nose in most cases. A
small portion of one of the leprous nodules may also be
removed and films made by rubbing the cut surface
against a clean slide. If there is an ulcer films may be
made from the secretion from it.

Films should be stained by the method which we
have recommended for the tubercle bacillus. If bacilli
are present in large quantities the case is almost cer-
tainly one of leprosy, for tubercle bacilli are never found
in similar situations except in scanty numbers. If a
doubt should arise as to the identity of the bacilli,
advantage should be taken of the fact that the leprosy
bacillus retains the fuchsin even more firmly than the
tubercle bacillus when exposed to the action of an acid.
A film from the suspected material should be spread at
one end of a slide, and some sputum known to be rich
in tubercle bacilli at the other; the whole should be
stained by hot carbol-fuchsin and decolorised by being
immersed bodily in 25 per cent. sulphuric acid for half
an hour. If the tubercle bacilli are decolorised any
bacilli which have retained the red colour are almost
certainly those of leprosy. If the tubercle bacilli are
80 BACTERIOLOGICAL DIAGNOSIS.

not decolorised a fresh specimen should be prepared
and immersed in the acid for a longer period.

ACTINOMYCOSIS.

Actinomycosis is very closely allied to tuberculosis ;
the lesions appropriate to the two diseases are almost
identical in histological appearance, and the granuloma
which occurs in actinomycosis goes on to fibrosis or to
the formation of ‘‘cold abscesses’”’ just as a tubercle
may do. The formation of fibrous tissue is most
marked in cattle, and in them the disease is more
chronic ; suppuration is more common in man, and the
disease runs a more rapid course.

The pus from an actinomycotic abscess is often viscid
and contains a greater or smaller number of small
greenish, yellow, or brownish nodules. They are about
as large as the head of a very small pin, and are quite
opaque; under the low power of the microscope such
a granule has a coarsely granular appearance, and looks
something like a raspberry. If nodules presenting these
appearances are found in any specimen of pus, whatever
be its origin, a careful microscopic examination should
be made to determine its nature. This is not difficult.

Method.—Place some of the pus which contains these
granules on a clean slide and press another slide upon
it'so as to crush the granules, dry, fix, and stain by
Gram’s method.

Tumours removed or incised at an operation, or
organs removed at a post-mortem examination, should
have their cut surfaces rubbed upon the surface of a
slide and the film thus obtained treated in a similar
ACTINOMYCOSIS. ° 81

way ; or they may be scraped, and the scrapings spread
on a slide. Sections may also be cut, but are not
usually necessary for the diagnosis.

EXAMINATION OF THE SPECIMENS.

Actinomycosis is caused by the ray-fungus, an organ-
ism which derives its name from the star-shaped
colonies which it forms whilst growing in the tissues.
It consists of two chief parts; the central portion of the
colony is formed of a network of narrow filaments, which
have a radial arrangement at the periphery (Plate IT.,
fig. 6). In this part small bodies which have the appear-
ance of cocci may often beseen. The outer zone consists
of the clubs which (when present) are so characteristic.
These clubs are flask-shaped expansions of the sheath
of the radial filaments already mentioned, and are
arranged with their narrow extremities pointing in-
wards. They are not generally present in man, and
when present are often badly developed; they are much
more common and more perfect in the ox, where the
disease is more chronic, and pus formation rare.

The films should be carefully examined for the pre-
sence of these structures. Clubs are not likely to be
found in the pus, and their absence does not tell against
the diagnosis; the dense felted network of filaments
retaining Gram’s stain is what is to be looked for,
and its presence is quite sufficient for a diagnosis.
Fortunate specimens may show a complete colony, with
its irregular network in the centre and the radial ar-
rangement of the fibres on the periphery, or there may
be mere fragments of mycelium.
82 BACTERIOLOGICAL DIAGNOSIS.

GLANDERS.

Glanders is one of the infective granulomata and is
closely allied to tuberculosis: it differs, however, in
running a more rapid course and in the greater ten-
dency which the specific lesions exhibit to undergo
suppuration. It is caused by the bacillus mallei, an
organism which is nearly as long as the tubercle
bacillus and decidedly thicker. It stains readily with
all stains and is easily decolorised; it loses its stain
when treated by Gram’s method, and does not form
spores.

The bacteriological diagnosis of the disease is not
easy, and should be referred to a public laboratory. A
quantity of the discharge from a suspected case should
be taken with aseptic precautions and transmitted as
soon as possible in a test tube or bottle which has been
sterilised by dry heat or by boiling. Pus had better be
sent in a pipette.

Where abscesses are opened cultures taken direct
from the pus may possibly contain the bacillus in pure
culture. In this case it may be identified by the
characters of its growth upon potato. The colonies
have the colour and appearance of honey at first; they
grow very rapidly, coalesce, and the potato is soon
covered with a moist-looking film which afterwards
becomes brown, the surface of the medium in the
neighbourhood becoming greenish-brown. If cultures
from pus grown on potato exhibit these appearances
and contain a short and thick bacillus which does not
stain by Gram’s method, the case may be diagnosed
as being probably one of glanders, even although the
culture be not a pure one.
TYPHOID FEVER. 83

The injection of mallein would probably supply
information which would be obtained more easily and
quickly, and would be more conclusive.

TYPHOID FEVER.

Typhoid fever is caused by a bacillus which is
variable in length, though usually short (about half
as long as a tubercle bacillus) and thick, its length
being only about three times its breadth. It does not
form spores, and it does not stain by Gram’s method.
It is actively motile; when. a culture of the organism
in a fluid medium is examined under the microscope
the bacilli can be seen darting rapidly about in all
directions. It owes its motility to the possession of a
large number of long wavy flagella, which can only be
seen after special and difficult staining processes.

The bacillus coli communis, the most plentiful organ-
ism of the intestine in man and animals, bears a very
close resemblance to the typhoid bacillus, and can only
be distinguished therefrom by the application of several
cultural and chemical tests, the performance of which
takes a considerable amount of time. This renders it
very difficult to diagnose typhoid fever by methods
similar to those which are in use for the other diseases
mentioned, 7.¢., by the demonstration of the specific
organism. Suppose for instance, that we were to
attempt to determine the nature of a case of diarrhea
by a search for the typhoid bacillus in the stools. For
every typhoid bacillus which we should encounter we
should find a great many colon bacilli, and we should
only be able to distinguish the one from the other by

G2
84 BACTERIOLOGICAL DIAGNOSIS.

a prolonged and careful examination of pure cultures.
It is quite certain that the disease might be diagnosed
in this way; indeed, it has been done, but the task is
an extremely difficult one and the diagnosis would be
delayed for a considerable period.

In other regions in which the typhoid bacillus occurs
during an attack of typhoid fever the search is usually
facilitated by the absence of other organisms, especially
by the absence of the bacillus coli. The specific bacillus
occurs in the blood, spleen, spots, mesenteric glands,
liver, and frequently in the urine.

It may often be demonstrated in the blood, but a
large quantity must be taken, and the technique is diff-
cult. The results obtained in this way are of great
scientific interest, but the process is of no value as a
means of diagnosis. The same remark applies to the.
spots; it is in fact somewhat doubtful whether the
bacilli which appear to be present in this situation do
not actually come from the blood which is drawn in the.
process. :

The mesenteric glands and liver are of course not
available for the purposes of diagnosis.

The demonstration of the bacilli in the urine is some-
times quite easy, and might be of some diagnostic value.
But they do not always occur in this excretion; and the
bacillus coli may do so.

The only way in which typhoid fever can be dia-
gnosed with ease and certainty by a demonstration of the
specific bacillus is by an examination of material drawn
directly from the spleen by means of a hypodermic
needle. The organism occurs constantly in this situa-
tion, and its demonstration is not difficult. The neces- :
sary operation, however, is by no means devoid of risk,
and is now generally abandoned.
TYPHOID FEVER. 85

This brings us to the only method in which typhoid
fever is now diagnosed by the bacteriologist—the
Widal’s reaction. This reaction is a special example
of a general law which was discovered by Durham and
others, and which is to the effect that the blood serum of
a person who has been through an attack of a bacterial
disease will cause the specific organism of that disease
‘to collect into clumps. For instance, if we take a broth
culture of the vibrio of Asiatic cholera (which is turbid
and opalescent) and add to it a small quantity of blood-
serum from a patient who has recovered from an attack
of cholera we shall find that the culture becomes clear,
a sediment collecting at the bottom of the tube; and if
-we examine this sediment we shall find that it consists
of felted masses of the vibrios. This reaction is a
general one, and is given in most, if not all, bacterial
diseases. But Widal, Griinbaum, and others, working
independently about the same time, showed that whereas
in many diseases it is a reaction of wmmunity (t.e., does
not occur until late in or after the disease) in typhoid
fever it is a reaction of infection, and occurs so early in
‘the course of the disease that it is of great value in
diagnosis.

The test is applied by adding a small quantity of the
serum from the patient suspected to be suffering from
.the disease to a larger amount of a young culture of
typhoid bacilli, and watching whether the appearance
of the culture undergoes any change: it may be
-watched under the microscope or by the naked eye, the
technique differing in the two cases. The microscopic
method is rapid and requires a very small amount of
blood, and is now generally used. The macroscopic
method is perhaps somewhat easier for a beginner, but
it takes a longer time and requires a larger amount of
blood serum.
86 BACTERIOLOGICAL DIAGNOSIS.

The technique in either case is readily learnt and can
be carried out with very little difficulty if the materials
are at hand. But the test is one which is seldom advis-
able for the home worker to attempt, as it requires a
young culture of typhoid bacilli. We shall, however,
describe the process, as some may carry it out during
an epidemic, or if they are living where they have not
access to a public laboratory. The process has now
been greatly facilitated by the demonstration of the
fact (by Widal) that the reaction is given with dead
cultures of the bacilli. These cultures can be obtained
from any bacteriological laboratory, and will keep.
They are prepared by adding four or five cubic centi-
metres of normal saline solution to an eighteen-hours-
old culture of the typhoid bacillus on agar, scraping off
the growth, filtering to remove clumps, and heating the
emulsion to 60° C. for ten minutes to kill the bacilli.
A small quantity of carbolic acid should be added to
prevent putrefaction. With a supply of this culture on
hand the practitioner can apply the test at home with-
out much difficulty.

Where the blood is to be transmitted to a laboratory
it should be sent in a pipette specially made for the
purpose. This consists of a bulb terminating in a
capillary tube at one end and in a mouth-piece (which
is plugged with cotton-wool) at the other (fig. 12, ¢).
To procure the blood wash the patient’s skin and make
a deep puncture with a sterilised needle ; further anti-
septic precautions are unnecessary. When a large drop
of blood has collected upon the skin (and this may be
hastened by gently squeezing the region, or by allowing
the finger to hang down) the tip of the pipette is in-
serted and suction applied. The practitioner must aim
at filling the whole of the bulb with blood, and must
WIDAL’S METHOD. 87

avoid air-bubbles. This is because the test is easier to
carry out and is more conclusive if clear serum can be
obtained. The test may be applied to a dry drop of
blood, but in this case there is no possibility of making
an accurate dilution. The test is wonderful enough as
it is, but the practitioner should not expect too much
from it if he sends a few splashes of blood inside a
capillary tube of the pipette. This is so important that
we repeat: do not be satisfied unless the bulb is com-
pletely filled with blood. When this is accomplished
suck the fluid a little further up and seal the pointed
end of the pipette in a flame—the flame of a wax match
will answer.

If a pipette is not at hand it is advisable to manufac-
ture one out of any piece of glass tubing which may be
available. Failing this the best plan is to dry several
large drops of blood on a clean slide; but results ob-
tained from blood forwarded to a laboratory in this way
should be received with caution, for the dilution is
necessarily a matter of guess-work.

Do xot send blood in vaccine tubes. When this is
done the blood often adheres so firmly to the glass that
the whole tube has to be ground down in a mortar with
a drop or two of water.

METHOD OF PERFORMING W1DaL’s REACTION BY THE
Microscoric METHOD.

Requisites—1. A young culture (not more than eigh-
teen hours old) of typhoid bacilli on agar.

Where dead bacilli are to be used this is to be re-
placed by a culture obtained from the laboratory and
prepared in the manner described.
88 BACTERIOLOGICAL DIAGNOSIS.

2. A small funnel provided with a double thickness of
white filter paper. This is unncessary if dead cultures
are to be used.

3. Three clean watch-glasses.

4. A platinum loop. This should be made of fine
wire and have a loop (which must be completely closed)
about #4, in. in diameter.

5. A hollow-ground slide. This is an ordinary slide
having a well about half an inch in diameter sunk in its
centre. If it isnot at hand a cell may be built up on
an ordinary slide. Take a piece of thin card one inch
square and cut out a square half an inch in diameter
from its centre. Fix this perforated square down on
to the slide with vaseline or immersion oil.

6. Thin cover-glasses.

7. The microscope. The test can be carried out
quite well with a % in. lens.

Process.—1. Making the emulsion.—Pour a small quan-
tity of tap water into the culture tube, or better,
scrape off some of the growth and mix it with some
water in a watch-glass. In either case stir it round
with the platinum needle for a few minutes so that the
bacilli are evenly distributed throughout the water and
form an emulsion.

Next take the hollow-ground slide and paint a ring of
immersion oil round the well (fig. 16,5). If you are
using a built-up cell paint the top of the card with the
oil. In either case vaseline may be used.

Place a drop of the emulsion on a clean dry cover-
glass and invert the hollow ground slide over it; press
it down so that the oil round the well adheres to the
cover-glass ; now invert the slide and you will havea
hanging-drop specimen. The bacilli will be contained in
the droplet of water (fig. 16,4) which hangs from the
WIDAL’S METHOD. 89

lower surface of the cover-glass ; this will not dry up if
the seal made by the oil is perfect.

Place the specimen under the microscope and exa-
mine it with the low power, using the fine adjustment
and stopping down the diaphragm. Focus until the
surface of the cover-glass is distinctly seen, and then
move the slide about until the edge of the hanging-drop
runs across the centre of the field. Then turn on the
3 in. lens and open and shut the diaphragm until the
field is faintly illuminated; the exact amount of light
required can only be learnt by experience.

 

 

    

et eee eae cee

Fic. 16.—Hanging-drop preparation (Crookshank).

Now focus up and down very carefully, using the fine
adjustment, until you see a line running across the field
and dividing it into a lighter and a slightly darker
portion. This is the edge of the drop. Focus a little
deeper; you should see numerous small unstained
bacilli, and if these are not visible it probably indicates
that the illumination is not right. Open and shut the
diaphragm, keeping a sharp look out down the micro-
scope all the time. It may help matters to lower the
condenser for a short distance.
go BACTERIOLOGICAL DIAGNOSIS.

Having obtained a clear view of the bacilli examine
them for motility and absence of clumps, and see whether
they are present in proper proportion to the amount of
fluid.

If the culture is in good condition the bacilli should
be seen darting about in all directions, but if the move-
ment is but sluggish the reaction may still be obtained.
If the specimen is kept for a short time in a warm
place or in the incubator the movements will usually
become more rapid. It is hardly necessary to say that
when dead cultures are used there will be no move-
ments of translation, though the bacilli may show
oscillatory (Brownian) movements.

The specimen must be searched thoroughly for clumps
of bacilli, and if these are present the emulsion must be
filtered through a double thickness of white filter paper.
This examination for clumps is a most important part
of the process, and must be attended to whether dead
or living cultures are in use.

Next see that the emulsion is neither too thin nor
too thick. No definite rules can be given, but if there
are very few bacilli in the field a further supply of
growth must be added to the stock of emulsion, and a
further specimen examined. If the bacilli are thickly
crowded together the emulsion must be diluted with a
little water and re-examined.

When you are satisfied that the emulsion is right,
slide off the cover-glass and drop it into some antiseptic
lotion ; of course this is unnecessary if dead cultures
are used.

2. Making the dilution. You are now about to dilute
a drop of the serum from the patient with a known
multiple (in this case thirty times) of its bulk of the
emulsion which you have just prepared. Todo so you
WIDAL’S METHOD. gt

will take advantage of the fact that the platinum loop
if dipped into a fluid and pressed against a surface so
that every part of the loop touches that surface will
deposit a drop of fluid of definite size. You are about
to mix one loopful of the serum with twenty-nine loop-
fuls of the emulsion just prepared and examined.

Blow the blood from the pipette out on to a watch-
glass (to do this it will be necessary to break the tip of
the pipette) and tilt the latter so that the serum flows
away from the coagulum. Now take a loopful of the
serum and place it on another watch-glass, taking care
to put the loop flat on the surface of the glass; this is
done more easily if the wire is slightly bent, or if a flat
slide is used instead of the watch-glass.

Next heat the platinum loop in the flame; this is to
burn off any blood which might remain on it and con-
taminate the emulsion. Take up a loopful of the emul-
sion and place it on the watch-glass by the side of the
drop of serum, but not touching it. Repeat this until
you have placed twenty-nine drops of emulsion round
the serum. Mix the whole together by stirring them
thoroughly with the platinum loop, place a droplet of
the mixture on a clean cover-glass, and make a hanging-
drop specimen and examine as before.

If the blood comes from a case of typhoid fever (with
certain restrictions which will be discussed below) the
microscopic appearances will be quite different from
those seen in the drop of emulsion which was previously
examined. The bacilli will no longer swim about
rapidly in all directions; they will become paralysed,
and remain quite motionless. Further, they will col-
lect into clumps; each clump consisting of a larger or
smaller number of bacilli arranged in a felted net-
work resembling that seen in a heap of “ spellicans”’
‘Q2 BACTERIOLOGICAL DIAGNOSIS.

(fig. 17, b). This is the complete positive reaction; it
consists of two parts, clumping and paralysis, and is
given ozly (in the dilution used) by the blood of a
patient who is suffering or who has suffered from
‘typhoid fever. Ifthis is not the case the bacilli will
continue to move about just as before, and will not
collect into clumps.

In the process which has been described above, the
blood has been diluted to thirty times its volume, and
this is the best dilution to use for diagnostic purposes.
But the reaction is given earlier if a lesser dilution (one

Fic. 17.
a. Negative Widal’s reaction. b. Positive Widal’s reaction.

in ten) is used, though there is then a greater chance of
‘fallacy.

Sometimes the reaction takes place almost as soon as
‘the serum is added. At other times it is delayed, and
‘for these it is necessary to fix a time limit. With a
-dilution of one in thirty, one hour is a safe time limit to
.adopt, and if the reaction takes place after this the re-
sult should be looked upon with great suspicion, and the
‘test reapplied after a day or two.

Exactly similar processes are carried out if dead cul-
‘tures of typhoid bacilli are used, but here the emulsion
WIDAL’S METHOD. 93

should be sent out from the laboratory free from clumps,
and containing exactly the right number of bacilli, so as
to be ready for immediate use. But the practitioner is
urged not to trust to such an emulsion without making
a hanging-drop, and examining it just before making
the test.

If dead cultures are used it is advisable to use a
rather less degree of dilution than in the above process.
A dilution of one in twenty will answer perfectly. The:
time limit is the same.

INTERPRETATION OF RESULTS.

A positive result may mean :—1. That the patient is
suffering from typhoid fever.

2. That he has suffered from typhoid fever within a
certain period before the blood was taken. The hypothe-
tical substance which we believe to be the cause of the
reaction (agglutinin) continues to be formed or remains
in the blood for some time after complete convalescence
from typhoid fever; the reaction has been known to
persist for seven or eight years, and probably usually
does so for about two. This fact must be remembered
in interpreting the results obtained from Widal’s test.
If the patient has suffered from typhoid fever, or from
an obscure illness which might possibly have been
typhoid fever, a year or two previously, the positive
reaction should be regarded with suspicion.

In such cases the test should be carried out so that
the smallest dilution which will cause clumping can be
ascertained, and the test repeated in two or three days.-
If, for instance, we found that the blood clumps -only in.
a dilution of one in twenty on one day and in a dilution
94 BACTERIOLOGICAL DIAGNOSIS.

of one in a hundred three days later, this affords a cer-
tain proof that the reaction is due to a present attack of
typhoid fever, and is not due to one which took place
at a previous date. This investigation should be en-
trusted to an expert bacteriologist and plenty of blood
sent on each occasion.

A negative result may mean :—

1. That the patient is not suffering from typhoid
fever. :

2. That he is suffering from typhoid fever, but the
date is too early for the appearance of the reaction.
The reaction sometimes occurs on the fifth or sixth day,
usually after the tenth day, and in all but a very small.
number of cases before the end of the second week. If
the onset of the disease (as far as it can be fixed) is less
than this the examination should be repeated after two
or three days.

3. In a very small number of cases the reaction is
delayed still further, and if the patient dies may not
occur at all. These cases are usually severe ones and
do not present any difficulty in diagnosis. Sometimes
the reaction is delayed well into the convalescence in
mild attacks, but this is exceedingly rare.

Macroscopic MEtTHop.

The macroscopic method can be carried out witha
young living culture on agar, or with an emulsion of
dead bacilli. The technique is not so difficult as in the
previous process and no apparatus is required other:
than a piece of narrow glass tubing from which to make
pipettes.

Requisites—1. A young culture on agar and some
MACROSCOPIC METHOD. 95

normal saline solution; or a dead emulsion of typhoid
bacilli.

2. Special glass pipettes. These are to be made from
a piece of glass tubing which should not be more than
% in. in internal diameter and about ten inches long.
The central two inches of this tube are to be heated in
a flat gas flame until thoroughly softened, then with-
drawn, and then the two ends are to be drawn steadily
apart. The softened portion is to be drawn out into
a long narrow portion (rather thicker than a vaccine
tube) about six inches long and of nearly uniform dia-
meter. The full diameter of the tube should pass rapidly
into this narrow portion; it should not taper gradually
(fig. 12, ¢). The two pipettes thus obtained are to be
broken apart, leaving about three inches of the narrow
tubing attached to each. The wide end of the tube is
to be plugged fairly firmly with cotton-wool. This
serves a double purpose; it prevents any of the fluid
from getting into the mouth, and it offers a certain
amount of resistance to the escape of the fluid, and thus
renders the pipettes more easy to fill.

Process.—Prepare an emulsion just as before, making
a drachm or so, and filtering it. An emulsion of dead
bacilli obtained from a laboratory should be ready for
immediate use. Insert the tip of the pipette into the
blood serum (of which there should be several drops),
and aspirate it gently into the tube, avoiding air-
bubbles; if these gain access blow out the serum and
begin again. It is easier to see what you are doing
if you use a piece of india-rubber tubing (such as is
used with a hemocytometer) through which to apply
suction.

Having drawn up enough serum to form a column
about two inches in length in the narrow portion of the
96 BACTERIOLOGICAL DIAGNOSIS.

tube, lay the pipette on its side, and make a mark with
ink or with a grease pencil to show how high the serum
reaches. Now suck the column of fluid a little way into
the tube, and insert the tip of the pipette into the emulsion ;
suck the latter up the tube until it reaches to the mark.
This will give you the same amount of emulsion as of
blood serum; the two fluids will be separated by a short
column of air. Now withdraw the tip for a moment
and suck up another small quantity of air; dip it into
the emulsion and suck it up to the mark again. This
will give you twice the amount of emulsion as of
blood serum, the three portions of fluid being separated
by air (fig. 18). Repeat this process until you have
nine times the amount of emulsion as of serum; then
suck the fluid still further from the tip of the pipette,
and seal the latter ina flame. Probably by this time
the fluids will have mixed together; if not, tap the
tube gently (holding it upright) until the air-bubbles
which you have sucked up make their escape and the
fluid forms a continuous column.

Fill another tube to a similar height with emulsion
(for a control) and place the two side by side in an
upright position for twelve hours.

Now examine the fluid in the pipettes. If the emul-
sion has been made from a living culture the control
pipette (7.e., that to which no blood has been added)
will probably remain turbid; if an emulsion of dead
bacilli has been used it will have become clear, and the
bacilli will form a uniform even layer at the bottom of
the pipette.

Compare the control tube with the pipette to which
blood has been added. If the reaction is negative
the appearances will be exactly the same in each;
but if the reaction is positive the bacilli will fall to the
MACROSCOPIC METHOD. 97

bottom in flocculent granular masses, which are similar
whether living or dead bacilli have been used. In the
former case there is no difficulty in determining whether

 

Fic. 18.—Showing method of filling pipette.

a. Is the mark on the tube. 6. The serum. c,c,c. The columns
of emulsion separated by the air bubbles d, d,d. e. Is the cotton-
wool plug.

H
98 BACTERIOLOGICAL DIAGNOSIS.

the reaction is present or absent, for the fluid will be
clear if it is positive, turbid if it is negative (fig. 19). The
dead emulsion presents more difficulties; you have to
compare the granular deposit which occurs in a positive
reaction with the more uniform deposit which occurs in
the control tube. If there is any doubt the best plan is

 

 

 

Fic. 19.—Positive and negative Widal’s reaction (macroscopic
method). The fluid in the left hand pipette (negative) is still turbid.
The right hand pipette (positive) contains clear fluid and has a
sediment at the bottom. The lower portions of the pipette have
been removed.

to break off the tip of the pipette and blow out some of
the deposit; examine it under the microscope after
applying a cover-glass. In a positive case the bacilli
will be seen to be collected into clumps, in a negative
case they will remain discrete.

This process is a modification of that described by
GONORRHGA. 99

Wright (Brit. Med. Four., p. 355, 1898), to which the
reader is referred for fuller details.

It is advisable to use a dilution of one in ten if the
macroscopic method is used.

GONORRHGA.

Nothing is more certain than the fact that gonorrhcea
can only be diagnosed by bacteriological methods ; and
every practitioner is very strongly urged to practise
himself in these methods and to employ them in all
cases. Leaving out of account the confidence which
the certainty of a correct diagnosis inspires, there is
always the possibility that legal questions may arise,
and a practitioner who made a diagnosis of gonorrhcea
without employing the only means by which that dis-
ease can be diagnosed would make a poor show in
cross-examination. Lastly, a bacteriological examina-
tion will often tell us that the disease is merely lying
latent and is still infective when apparently cured.

The gonococcus chiefly affects mucous surfaces; the
urethra in the male, the urethra and cervix uteri in the
female, and the conjunctiva in both sexes. These are
the regions in which the primary lesion usually occurs,
and it may extend by continuity to more distant parts.

In the male it may involve the prostate, the vesicule
seminales, and the bladder. It is doubtful whether
gonorrheeal epididymitis is due to this organism or to
another.

In the female the inflammation of the urethra may
extend to the bladder. The inflammation of the cervix
may extend to the mucosa of the uterus and thence to

H2
100 BACTERIOLOGICAL DIAGNOSIS.

the Fallopian tubes (causing pyosalpinx), to the mouths
of the tubes (causing local adhesive peritonitis, which
probably results in sterility), or to the: peritoneum,
where it may cause general peritonitis.

The gonococcus may escape into the blood from any
of these lesions, and the results of this occurrence are
arthritis, ulcerative endocarditis and meningitis; the
two latter are rare.

The search for the gonococcus may have to be made
(1) in urethral pus from either sex, (2) in pus from the
cervix uteri, (3) in pus from the conjunctiva, (4) in pus
from the meninges, tubes, peritoneum, or other region,
whether removed by operative measures or at a post-
mortem examination, (5) in the blood, or (6) in the
urine. It is to be noticed that the gonococcus rarely,
if ever, attacks the vagina, and that in cases of vaginitis
the cervical secretion should be examined.

In the vast majority of cases cultural examinations
are quite unnecessary. This is fortunate, for the gono-
coccus does not grow readily on artificial media. The
organism has well marked morphological characters,
and the deductions drawn from these characters need
only be corroborated in cases of generalised infection or
of meningitis, in which the results are to be published
(as they should be), and must, therefore, be proved
beyond doubt. In such cases the services of a bacterio-
logist should be called in if possible: or the material
may be collected in pipettes with the most careful
precautions as to asepsis and forwarded at once to a
laboratory.
GONORRHEA. IoIl

MeEtTHoD oF MakinG THE FILMs,

The pus is to be spread out into thin films at the time
at which it is taken, and this is true whether the prac-
titioner intends to make the examination for himself, or
is about to send the material to a laboratory. Gonor-
rheeal pus should never be forwarded dried on a piece
of cotton-wool or enclosed in vaccine tubes.

The films are to be made thus:—Take, two clean
slides and place two or three platinum loopfuls of the
pus on the centre of one of them; sterilise the needle
and lay it down. Now take the other slide and apply
its centre to the pus and allow it to fall on to the, first
slide by its own weight; do not squeeze the slides
together. Then slide them apart, keeping each in its
own plane until they are entirely separated. This will
give you two excellent films. Allow them to dry and
fix them in the flame.

The films may also be made on cover-glasses, exactly
the same process being adopted, except that it will be
necessary to squeeze the two lightly together. The
fixation is accomplished by passing the cover-glasses
rapidly through the flame.

These are the methods by which films are spread in
all cases; the way in which the pus should be obtained
varies somewhat with the nature of the case.

In the male it is advisable to cleanse the meatus and
to reject the first drop of pus, taking the second with a
platinum loop and proceeding as before. Antiseptic
precautions are entirely unnecessary, as no attempt is
to be made to get cultures. If the patient is suffering
from phimosis, and there is a purulent discharge which
102 BACTERIOLOGICAL DIAGNOSIS.

may be due to gonorrhcea, chancre, soft sore, or to a
non-specific balanitis, a similar method is adopted, but
here many.films should be taken, as a prolonged search
may be required. If the patient suffers from a slight
discharge in the early morning the best plan is to give
him two clean slides. These are to be smeared across.
the meatus whilst wet with the discharge and allowed
to dry.

In the female it is necessary to obtain the pus directly
from the urethra; it may be expressed by the finger in
the vagina. The first drop should be rejected.

If the patient is suffering from cervicitis or endo-
metritis the pus should be taken direct from the cervix,
a speculum being used.

It is absolutely necessary that you should spread the
films at once, even if you are having the examination
made at a distance. It is next to useless to send pus
dried on linen, cotton-wool, a Volkmann’s spoon, or
even in a thick layer on a-slide. The diagnosis may be
made from material sent in this way, but the difficulties
are much greater, and in some cases the results are less.
certain.

Preparation of films from conjunctival pus presents.
no difficulties. The same is true of pus from the tubes
or other internal regions, whether it is exposed by
operative interference or at a post-mortem examination.

Instructions for the examination of the blood are
given subsequently. A considerable number of films
should be taken, as the cocci are present in but very
small numbers.

The urine may be examined in the female if a local
examination is not considered advisable, or in the male
to obtain evidences as to whether the disease is cured
or not. The morning urine should be examined. It
GONORRHEA. 103

should be mixed with a small quantity of carbolic lotion
or other antiseptic and allowed to settle for twenty-four
hours; it is much better to use a centrifuge if one is
available. In cases where we require evidences as to
cure after an attack of gonorrhcea the urine is examined
after gentle massage of the prostate.

STAINING oF FiLms.

One film is to be stained by a simple stain such as
methylene blue or carbol-thionin. The other is to
be stained by Gram’s method, and then in dilute carbol-
fuchsin for half a minute. Bismarck brown may also be
employed but is hardly as good.

Examination of films.—First take the specimen in which
the simple stain has been used and examine it with the
oil immersion lens. You will see that it shows number:
less cells with very irregularly lobed nuclei; these are
the pus cells or polymorphonuclear leucocytes. There
will also be some flat squamous epithelial cells.

The gonococci will be stained even deeper than the
cell nuclei, and will be mostly contained within the pus
cells. If you see a cell which contains numerous small
blue or violet granules bring it into the centre of: the
field and examine it more thoroughly, to see whether
the granules have the characters of the organism which
we are about to describe.

The gonococcus is a large diplococcus, each compon-
ent of the pair being shaped like a kidney, the hilum
being turned toward that of its fellow. Single forms
(which may be rounded) and tetrads are sometimes seen.
It does not stain by Gram’s method, and this is one of
its most important features. Another important point is
104 BACTERIOLOGICAL DIAGNOSIS.

its arrangement; during the height of an attack of
-gonorrheea it is almost entirely intracellular, being
contained within the polymorphonuclear leucocytes.
Further, several pairs occur in each cell, and the great
majority of cells are entirely devoid of cocci.

If the organism which you find possesses these char-
acteristics turn to the specimen which has been stained
by Gram’s method and counter-stained by carbol-
fuchsin (Plate I., fig. 6). In this all bacteria which
retain Gram’s stain will be coloured violet, while organ-
isms which do not retain it will be red. You must
therefore search for groups of diplococci contained
within the cells and possessing the above character-
istics. They will not be so prominent as in the other
specimen, for the cells, nuclei, &c., will be coloured red
also, and the contrast is not so great. But if the case
is one of gonorrhoea you will find them after a careful
search.

INTERPRETATION OF RESULTS.

You are justified in considering a case to be one of
gonorrheea if in films made from the pus :—

1. Large kidney-shaped diplococci are present.

2. These cocci occur within the pus cells, and at
least four pairs (Foulerton) in each affected cell.

3. The vast majority of cells are entirely free from
cocci.

4. The organisms in question do not stain by Gram’s
method. This is an absolutely essential point.

If cocci which answer to the above description,
except that they are not enclosed within cells, the case
may still be one of gonorrhoea. The gonococcus is
CHOLERA. I05

frequently extra-cellular during the early stages of an
attack of urethritis, and, though to a less extent, during
its involution, whilst cases sometimes occur in which
a considerable number of the cocci lie free during the
whole course of the disease.

CHOLERA.

The diagnosis of cholera can only be made on clinical
grounds alone during an epidemic, as other diseases
present almost identical symptoms and course. ‘The
importance of making a correct diagnosis arises less
from the interests of the patient than from those of the
general public; if the case is one of true Asiatic cholera
the sanitary authorities must be notified and the fullest
precautions taken to prevent the spread of the disease.
In all suspicious cases a quantity of the rice-water
stools (in a bottle sterilised by boiling or by dry heat
and securely packed) should be forwarded at once to a
public laboratory. Meanwhile the diagnosis may be
established with a fair amount of certainty by the
following simple tests :—

1. Take a platinum loopful of the dejecta and spread
it in a thin film on a clean slide; dry, fix, and stain
with carbol-fuchsin for three minutes; wash, dry, and
mount.

2. Prepare another film and stain by Gram’s method.

EXAMINATION OF THE FILMs.

The spirillum of Asiatic cholera is about half as long
as a tubercle bacillus, or rather longer, and much
106 BACTERIOLOGICAL DIAGNOSIS.

thicker. It is slightly curved, hence the name of the
“comma bacillus,” the comparison being to a German
comma; it looks very like a caraway seed (Plate IL,
fig. 5).

In the carbol-fuchsin specimen vast numbers of these
curved rods will be seen; probably few other organisms,
if any, will be present if the case is one of true cholera.
Two or more rods may often be found joined together
with their concavities turned in opposite directions,
giving the whole the appearance of a very elongated
spiral. In the stools (but not usually in cultures) the
individual rods have frequently a parallel arrangement,
presenting the appearance of ‘‘ shoals of fish swimming
up stream.”

If you see these appearances examine the Gram
specimen. Very few organisms will be visible, as the
cholera vibrio does not retain the stain when treated in
this way.

If vibrios having the above characters are present:
proceed as follows :—

3. Take two or three small flasks (preferably steri-
lised by heat) and add to each t00 c.c. of water, fF
gramme of peptone, and 4 gramme of common salt;.
boil thoroughly and allow to cool. This forms a culture
medium in which the cholera vibrio will grow very
rapidly and other organisms far more slowly.

Inoculate each flask with a loopful or two of dejecta ;
plug each with cotton-wool and incubate for 8 to 12
hours at 37° C. If cholera vibrios are present the
cultures will conform to the following tests :—

a. There will be a film on the surface. This will be
more marked after a few hours longer.

b. This scum will present the microscopic appear-
ances described above, except that the vibrios are
PLAGUE. 107

usually somewhat straighter than those which occur
in the stools and the “fish in stream” arrangement is
not marked. They will not stain by Gram’s method.

¢. The addition of a small quantity of pure strong
sulphuric acid will give a pink or crimson tint. This is
the “‘ cholera-red ” reaction and is caused by the action
‘of sulphuric acid on indol in the presence of a minute
quantity of a nitrite; many other organisms (e.g., the
bacillus coli) produce this colour aftey the addition of
a nitrite, very few without it. The cholera vibrio pro-
duces nitrites as well as indol.

INTERPRETATION OF RESULTS.

In a case in which the above phenomena are ob-
served, the inference that the patient is suffering from
true Asiatic cholera is so strong that the authorities
should be notified and the fullest precautions taken.

A case in which they are absent is almost certainly
not one of true cholera.

PLAGUE.

The bacteriological diagnosis of plague should be
made by an expert; not because it is difficult, but be-
cause so much hinges upon it, at least in this country.
A brief account of the method by which a practitioner
who was unable to obtain expert help might proceed
may not be out of place.

The plague bacillus is a short and rather thick rod
which occurs in vast numbers in the bubo, in the blood,
108 BACTERIOLOGICAL DIAGNOSIS.

and in the internal organs. It does not stain by Gram’s
method, and when stained by other processes it often
exhibits a characteristic olay staining, the ends of the
‘bacillus being coloured deeply, whilst the intervening
portion remains colourless (Plate II., fig. 4). It might
be mistaken for a diplococcus; it could not be mistaken
for the pneumococcus (to which it has some resem-
blance) as that organism stains by Gram. Degenerate
forms which resemble cocci, &c., often occur in cul-
tures, but are seldom met with in the body during
life. :
The diagnosis may be made by an examination of
fluid aspirated from the bubo or of the blood. In
bubonic cases the former method should always be
adopted, as the bacilli are present therein in vast
numbers and generally in pure culture; the amount of
fluid which has to be removed is very small, even if
cultures have to be taken.

When this is not the case two films should be made
in the way already described, fixed and stained, the
one by dilute carbol-fuchsin or Léffler’s blue and the
other by Gram’s method. If the bacilli are present
they will appear as short oval rods which may or may
not exhibit the polar staining; if the specimen has been
stained for the proper length of time (about 2 minutes)
most of them will do so, but in any case it will most
likely be present in a few. The Gram specimen will
not show these rods; there may be a few pus cocci
present as a secondary infection.

The blood is examined by any of the methods to be
described subsequently, and a very careful search made,
as the numbers of the bacilli may be comparatively
scanty.

If a careful examination of stained films made from
SOFT SORE. 109g

a bubo does not show the organisms having the above
characters, it is probably safe to say that the case is not
one of plague.

SOFT SORE.

A very strong case has been made out in favour of
the view that the bacillus described by Unna and by
Ducrey is actually the cause of soft sore, though the
proof does not rest upon the solid ground demanded by
Koch. The bacillus has never been cultivated outside
the body, and the chief proof of its pathogenicity rest
upon the fact of its invariable presence in true soft sores.
It is hardly correct to say that it is invariably absent
from other sores, unless, indeed, we extend somewhat our
conception of what a soft sore really is; for the author
has found bacilli which were indistinguishable from
these in sores which were exactly like those which
follow herpes preputialis, though more severe than is
generally the case. But it is quite possible that future
research will show that the bacillus varies greatly in
virulence, and that it is only under certain circum-
stances that it produces typical soft sores. At present
all we can say about the diagnostic value of the organ-
ism is that its presence in the secretion from an ulcer
affords strong evidence that the case is really one of
chancroid, and that its absence almost certainly nullifies
such a diagnosis. It is scarcely necessary to say that
syphilis and soft sore (or gonorrhcea and soft sore) may
be inoculated at the same time, and the lesions appro-
priate to both diseases may be present simultaneously.

The bacillus in question is a short straight rod, less
than a quarter the length of the tubercle bacillus, and
Ito BACTERIOLOGICAL DIAGNOSIS.

not much longer than the bacillus of influenza. It is
rather thick in proportion to its length, its length being
only about three times its breadth. It does not stain
by Gram’s method; it stains, indeed, with some diff-
culty, and powerful stains (such as dilute carbol-fuchsin
or Léffler’s blue) should be used. It is best demon-
strated in films made from the deeper parts of a typical
soft sore, for the superficial parts contain bacteria of all
sorts, and the recognition of Unna’s bacillus is not easy
unless it is obtained in large numbers.

The bacteriological examination for this bacillus has
most often to be made in cases of urethral sore or of a
sore concealed beneath a phimosis. The method of
obtaining the specimen is the same in both cases. A
fairly stiff platinum loop is inserted beneath the prepuce
or into the urethra, and moved gently about until the
most tender spot is found. This should be scraped as
forcibly as the patient will allow, and the loop with-
drawn, care being taken that the mass of secretion is
not wiped off in so doing. Several films should then be
made, the secretion being rubbed up on the slide with a
drop of water. They should be stained with either of
the stains mentioned above for five minutes or more; it
is an advantage to warm them gently. They are then
rinsed in water, dried, and mounted, and examined
thoroughly with an oil-immersion lens.

When buboes occur in the course of soft sore the pus
they contain should be examined for this organism as
soon as they are opened ; the interest in this is chiefly
scientific, for opinions are divided as to whether they
are caused by this germ or by pyogenic bacteria. In
three cases examined by the author, it was found (in
very scanty numbers) on one occasion, staphylococci on
another, and no bacteria of any sort in the third.
RINGWORM. IIt

RINGWORM.

Examination of the hair and scales from the skin are
of very great value to the dermatologist. It settles
conclusively the question whether a patient is or is not
suffering from ringworm, and often gives important in-
formation as to prognosis; and may enable us to say
whether the disease has probably been derived from a
human source or has been contracted from one of the
lower animals.

Where the mere diagnosis of ringworm is in question
an examination of the hair or scales after soaking in
liquor potassz is often sufficient. -The materials are
placed in a drop of the solution on a slide and covered
with a cover-glass; after a quarter of an hour or so the
specimen is carefully examined under the microscope,
using a 4 in. lens anda small diaphragm. The spores
appear as spherical or oval highly refractile bodies
which can hardly be mistaken for anything but fat
globules. This possible source of fallacy may be re-
moved by soaking the hair in ether before applying the
liquor potasse.

This is a rapid and simple method, but it does not
enable us to diagnose the nature of the fungus with
certainty, except in very typical cases, and when spores
are scanty they’are readily overlooked. It is a very
great advantage to employ some method of staining.
These are by no means difficult, though they are some-
what tedious. Two processes, both modifications of
Gram’s method, will be given.
II2 BACTERIOLOGICAL DIAGNOSIS.

Morris’s MetTuop oF STAINING Hairs, ScaLes, &c.

Requisites—1. Anilin gentian violet, carbol-gentian
violet, or carbol-fuchsin.

2. Gram’s iodine solution.

3. Anilin oil to which sufficient iodine has been added
to give it a deep mahogany colour.

4. Anilin oil.

5. Xylol. This is not necessary unless the specimens
are to be kept permanently.

6. Blotting paper.

7. Slides, cover-glasses, and balsam.

Process.—Take the hairs from the edge of the sus-
pected area, and cut off all the free portion except a
piece about a quarter of an inch long. The root and
the part of the shaft next to it are all that are to be
examined. Several of these hairs can be stained at the
same time.

Stain in a watch-glass full of stain, or on a slide for a
quarter of an hour or longer; it is an advantage to
warm the stain gently, and it will penetrate better if the
hairs have been previously washed in ether, though this
is not absolutely necessary.

Remove the hairs from the stain and place them ona
slide; if already on a slide pour off the stain. Dry
them with blotting paper and pour’ on the iodine
solution; allow it to act for five or ten minutes. It is
an advantage to blot the specimen after a minute or two
and apply a fresh lot of the solution. Blot thoroughly.

Now decolorise in the solution of iodine in anilin oil.
This should be poured off and replaced occasionally,
and the specimen examined under a low power of the

‘
RINGWORM. II3

microscope. When the decolorisation appears to be
complete pour off the solution and replace it by anilin
oil; allow this to act for a minute or two.

If the specimen is not to be kept permanently it may
now be mounted in balsam and examined at once. If
it is to be kept the anilin oil must be washed out by
several applications of xylol. Mount in balsam.

Adamson’s method gives even better results, but is
somewhat more tedious.

Requisites—1. Liquor potasse.

Dilute alcohol—about 15 per cent.
Anilin gentian violet or its substitutes.
Gram’s iodine solution.

Anilin oil.

. Xylol.

Blotting paper.

. Slides, cover-glasses, and balsam.

Prociss. —Prepare the fragments of hair as before,
rejecting the free portions. Place them on a slide, add
a drop or two of liquor potassz and apply a cover-glass.
Allow the liquor to act for a quarter of an hour or
twenty minutes.

Now place a large drop of dilute spirit at one edge of
the cover-glass and a piece of blotting paper at the oppo-
site edge; this will suck up the potash and the spirit will
run in and replace it. After a few minutes lift off the
cover-glass and wash the hairs gently in more spirit.
This will harden them. Dry. If epithelial scales are
being examined they may be fixed to the slide or cover-
glass by heat in the usual way.

Stain in anilin géntian violet for half an hour or less.

Pour off the stain, blot gently, and pour on Gram’s
iodine solution. Allow this to act for five minutes.
Blot again.

SW ANKE wD
II4 BACTERIOLOGICAL DIAGNOSIS.

Decolorise with anilin oil, pouring it off and applying
a fresh lot from time to time. The process may take an
hour or more, and the specimen should be left under the
microscope and examined occasionally.

When the decolorisation is complete (i.e., when the
color is seen to be present in the fungus only), blot
gently, and wash thoroughly with xylol. Mount in
balsam.

The specimens are to be examined under an 2} in.
objective. A higher power is unnecessary.

 

Fic. 20.—Microsporon Audouini.*

There are certainly three, and possibly more species
of ringworm which occur in England, and the fungus
of favus is closely allied and is demonstrated by the
same process.

The microsporon Audouini is the most common species
of ringworm fungus in this country, being responsible
for about eighty or ninety per cent. of all cases. It is
the small spored fungus, and it may be distinguished
by the fact that its spores are arranged in an irregular

* Figs. 20, 21, 22 are from Curtis’s ‘‘ Essentials of Bacteriology ”
(Longmans). :
RINGWORM. II5

mosaic and not in chains (fig. 20). It does not invade
the interior of the hair, but forms a sheath of spores and
mycelium outside the submerged portion of the hair;
this sheath projects for a short distance above the sur-
face of the scalp and may be seen with the naked eye.

This fungus usually attacks the scalp in children.
It is very rare in adults, and it rarely attacks other
regions than the head. It is thought by some to be
always caught from a human case of the disease, but
there are reasons for thinking that it may be derived by
infection from the horse or cat.

 

Fic. 21.—Trichophyton endothrix.

The most important clinical fact about the micro-
sporon Audouini is that ringworm caused by it is
extremely intractable and may run a prolonged course
in spite of the most skilful treatment.

The trichophyton endothrix attacks the interior of the
hairs, and forms long chains. Its spores are somewhat
larger than those of the preceding species, but the
difference is not great; the organism is most easily
recognised by the chain-like arrangement of its spores,

12
I16 BACTERIOLOGICAL DIAGNOSIS.

and by the fact that they lie within the hair, the cuticle
of which usually remains intact (fig. 21).

This fungus is a rare cause (in this country) of ring-
worm of the scalp, and the disease caused by it appears
to be somewhat easier to cure than that due to the
microsporon, though opinions on this point are divided.
It also attacks other regions of the body, causing tinea
circinata or’eczema marginatum.

It always appears to be derived from a human case,
and never by infection from animals.

 

Fig, 22.—Trichophyton ectothrix from a case of kerion.

The tvichophyton ectothrix (like the microsporon) forms
a sheath round the outside of the hair, to which it is
closely applied, like the bark to a tree. The spores are
about as large as in the preceding species, and are
arranged in chains ; this fact, together with the position
of the.fungus with regard to the hair, will enable a
diagnosis, to be made (fig. 22),

In some cases this organism does tend to invade the
hair, and in this case the name “endo-ectothrix’’ is
applied to it.
RINGWORM. 117

This fungus is a rare cause of ringworm of the scalp,
and the disease it causes is readily cured. It also
causes ringworm of the body and of the beard region,
which the endothrix does not attack. According to
Sabouraud (to whose work on these organisms we owe
most of our knowledge on the subject) kerion is always
caused by this organism, but this is not generally
accepted. It appears, however, to be a fact that sup-
purative lesions (folliculitis, kerion, &c.) are usually
caused by this fungus.

This species is often derived from one of the lower
animals, especially from the horse, cat, and dog.

Favus is caused by a closely allied organism, the
achovion Schénleiniti. This may be demonstrated by
either of the processes already described. It consists
of mycelial threads which are not continuous, but are
composed of short rods. The whole has an appearance
which has been compared to that of the metatarsal
bones, and this resemblance is increased by the fact
that sometimes several filaments radiate from one point,
forming the “ favic tarsus.”

The identification of the variety of organism which is
present does not usually present great difficulties. The
first point to be looked to is the arrangement of the
spores. If these are present in an irregular mosaic the
microsporon is present; if they form filaments some-
what resembling those of a streptococcus the organism
is a trichophyton. To settle which it is look to see
whether the cuticle is present, and whether the fungus
invades the interior of the hair. This can usually be
determined by focussing up and down until you see an
‘‘ optical section” of the hair in question. It is impor-
tant not to be deluded by the fact that the fungus which
lies on the outside of the-hair will appear to be inside it
if a surface view only is taken.
118 BACTERIOLOGICAL DIAGNOSIS.

PART III.

COLLECTION AND EXAMINATION OF
CERTAIN MORBID MATERIALS.

Methods of collection and examination of :—
Fluids from the pleural cavities.
Fluids from the synovial cavities.
Fluids from the meninges (lumbar puncture).
Pus.
Materials obtained at post-mortem examinations.
The blood, including :—
1. Enumeration of red corpuscles.
2. Enumeration of leucocytes.
3. Preparation of blood films.
4. Examination for the parasite of malaria.
5. Bacteriological examination.
Solid materials by sections.

THE COLLECTION OF PATHOLOGICAL
EXUDATES.

A bacteriological examination of the inflammatory
exudates which collect in the various cavities of the
body often yields important information as to the
nature of the morbid process, suggests treatment, and
influences our views as to the prognosis of the con-
dition. This is especially the case with the fluids
which collect in the pleura, the membranes of the brain
PATHOLOGICAL EXUDATES. IIg

and cord, and the joints. In some cases all the needful
information may be obtained by the examination of
stained films, cultures being unnecessary ; and in these
cases no anti- or aseptic precautions (other than those
which are dictated by the interests of the patient) are
necessary. But in the greater proportion of cases this
is not enough, and cultures must be obtained. To this
end it is absolutely essential that the most scrupulous pre-
cautions should be taken against contamination of the
fluid by the organisms which are constantly present in
the air and in the skin, or the results will be worthless.
The precautions taken must be as complete as those
which are used before an operation upon a joint.
Indeed, a fresh precaution has to be taken, for whereas
the presence in the skin of a small quantity of an anti-
septic would not be detrimental to a surgical operation,
it might, by getting into the fluid, nullify a bacterio-
logical examination. Hence the skin must be aseptic
and free from any antiseptic chemical.

The technique, as far as aseptic precautions are con-
cerned, is as follows:—The skin at the region to be
punctured is first thoroughly cleaned with soap, hot
water, and (if the patient can stand it) a nailbrush.
Then layer after layer of some reliable antiseptic lotion
is painted on, each layer being allowed to soak in before
the next is applied. The most suitable lotions for the
purpose are perchloride of mercury (1 in 1000) biniodide
of mercury, (1 in 500 of methylated spirit) or carbolic
acid (1 in 20).

After being allowed to act for at least ten minutes
the skin is to be thoroughly cleansed with methylated
spirit ; this should be rubbed in with a piece of cotton-
wool and should be poured copiously over the area.
The operation may now proceed.
120 BACTERIOLOGICAL DIAGNOSIS.

Where possible it is preferable to apply a dressing of
lint soaked in one of the above lotions (which need be
of only half the strength) for a few hours.

The puncture may be made by means of some sort of
exploring syringe, or by means of a hollow needle with-
out any means for aspiration. The former is used for
the removal of fluid from the pleura or synovial cavity,
the latter in performing lumbar puncture. But it is
necessary that the whole of the instrument used should
be rendered sterile by heat; chemical antiseptics are
quite inadmissible. In the case of a hollow needle no
difficulty occurs ; some hypodermic syringes, however,
will not stand boiling, and these are useless for the
purpose, as the apparatus which is to be used must be
boiled for at least five minutes.

We shall now deal with the most important cavities
of the body, describing the methods to be employed in
the investigation of the inflammatory exudates which
they may contain, and the inferences which may be
drawn from the results of the examination.

THE PLEURA.

There is but little to be said about the method to be
employed in the collection of fluid from the pleural
cavities. The most careful antiseptic precautions are
to be taken, and the region to be punctured should be
decided by consideration of the physical signs.

The examination of the fluid thus obtained may be
either microscopical, cultural, or by injections into
animals. If the latter are required (and inoculation
should be performed in all cases where a tubercular
THE PLEURA. 121

origin is suspected) a considerable quantity of the fluid—
an ounce or more—should be enclosed in a bottle which
has been sterilised by boiling, and forwarded at once.

Where the diagnosis is to be made by cultural
methods, and the cultures are not to be made on the
spot, the fluid is best stored or sent to a laboratory in
pipettes. These are to be filled from the syringe direct ;
the needle is to be removed and the end of the pipette
(sterilised by being passed through the flame) is passed
into the fluid and filled by gentle suction at the other
end. Each end is then sealed in a flame, care being
taken not to heat the fluid. Two or three such tubes
should be sent.

Cleary fluid from the chest rarely, if ever, shows any
micro-organisms on microscopical examination. Cul-
tures are usually sterile; where streptococci or pneu-
mococci are found the inflammation is likely to pass on
into suppuration. The great majority of these cases of
‘simple’ acute pleurisy are really due to the tubercle
bacillus, but their true nature can only be demonstrated
by inoculation experiments.

Where any question arises as to the nature of a case
of simple pleurisy the practitioner is recommended not
to attempt the examination himself, but to send samples
of the fluid in pipettes to a laboratory; if animal experi-
ments are necessary a larger quantity of fluid should be
sent in a sterilised bottle.

Puvulent pleurisies (empyemata) may be caused by
many organisms, the most common being the pnemo-
coccus, streptococci, staphylococci, and the tubercle
bacillus.

The pneumococcus is readily demonstrated by a
microscopical examination, the method to be employed
being the same as that previously described.
122 BACTERIOLOGICAL DIAGNOSIS.

The pus in these cases is thick and creamy, and of a
greenish colour; after it has stood for ‘some time a thin
layer of a greenish fluid appears upon the surface.

When an empyema is due to the pneumococcus alone,
no other organisms being present, the prognosis is dis-
tinctly better than in cases in which other organisms
are present, and the patient has been known to recover
after simple aspiration.

The streptococcus is also readily demonstrated by a
simple microscopical examination; it grows readily on
agar, forming small round colonies which do not tend
to coalesce and are more opaque in the centre than in
the periphery.

The pus is not generally very thick, and has a yellow
colour. It separates into two layers, the upper trans-
parent layer being much more abundant than is the case
with pneumococcic pus.

This is the commonest and most dangerous form of
empyema. ‘Thorough drainage is essential.

Staphylococcic empyemata, according to Netter, are
very rare; the single case in which he found the staphy-
lococcus alone was secondary to ulcerative endocarditis.
He also states that when this organism is found in the
pus tubercle bacilli are often present as well.

The prognosis of these cases, therefore, appears to be
bad.

The tubercle bacillus is responsible for a compara-
tively small number of cases; and the results of opera-
tive interference are not gratifying. The prognosis is
worse than in any other form of the disease.

The diagnosis may be made from a careful micro-
scopical examination, but to this end it must be careful,
as the bacilli are present in but scanty numbers.

If wo organisms are found after a thorough micro-
THE PLEURA. 123

scopical examination, the inference is that the case is
tubercular. Ifa cultural examination is also negative
the inference becomes almost a certainty.

Having these facts in view the practitioner is recom-
mended to proceed to examine cases of purulent pleurisy
in the following manner :—The pus is to be withdrawn
with a hypodermic needle or exploring syringe, and a
few drops deposited at once on the surface of a culture
tube of agar:* this is to be incubated at the body
temperature.

The microscopical examination is made in the manner
described for pus, a simple stain and also Gram’s stain
being used. The presence of streptococci, staphylo-
cocci, and pneumococci will be revealed; bacilli may
be present, and in this case it should not be forgotten
that the tubercle bacillus stains by Gram’s method. If
no organisms are found in these films, or if there are
organisms which resemble the tubercle bacillus in
general appearance, another specimen should be sub-
mitted to prolonged staining in hot carbol-fuchsin and
decolorisation in dilute sulphuric acid, and thoroughly
searched for the tubercle bacillus. If the result is
negative several other films should be searched.

The cultures are to be examined after twenty-four
hours’ incubation. The pneumococcus will produce
tiny colourless colonies on the surface of the agar; the
streptococcus forms similar small colourless colonies,
but these are distinctly more opaque in the centre;

* The practitioner is recommended to inoculate the culture
directly from the hypodermic needle. The cotton-wool plug is to
be withdrawn somewhat and the projecting end thoroughly singed
in the flame: the needle (still attached to the syringe) is then pushed
through the plug, and a drop or two of the fluid expelled directly on
to the surface of the medium. A similar device may be employed
where the fluid is collected in a pipette.
I24 BACTERIOLOGICAL DIAGNOSIS.

staphylococci form an even film like a streak of paint;
and the tubercle bacillus does not develop. Films
should be made from the cultures, stained and exam-
ined. The cultural examination is of great value, but
much can be made out by the examination of stained
films made directly from the pus.

FLUIDS FROM JOINTS.

The technique of the process of withdrawing these
fluids is exactly the same as in the case of pleurisy; the
needle will naturally be inserted at a point where there
is definite evidence of the presence of fluid, and where
it lies near the surface.

The bacteriological examination is conducted on
exactly similar lines. A few drops of the fluid should
be allowed to flow on to the surface of a sloped tube
of agar, and the culture obtained after twenty-four
hours’ incubation examined in the manner already de-
scribed. Films should also be made directly from the
fluid and some stained by Gram’s method and others
by a simple stain such as carbol-thionin.

A great number of organisms may be present; the
streptococci, staphylococci, the pneumococcus, gono-.
coccus, and tubercle bacillus are the most important.
The coccus which has been described by several ob-
servers as the cause of acute rheumatism cannot be
considered as of diagnostic importance at present; the
same remark applies to the bacillus which is probably
the cause of rheumatoid arthritis.

Streptococci are readily distinguished on microscopi-
cal examination and may be present even if the fluid
is perfectly clear. When they are present in a joint
FLUIDS FROM JOINTS. 125

which is not the seat of a perforating wound they indi-
cate a general infection with the streptococcus, ulcera-
tive endocarditis, &c., and the prognosis is most grave.
The author was enabled to diagnose a case of strepto-
coccic septicaemia a few hours after the onset of sym-
ptoms by finding numerous chains in a single drop of
clear fluid aspirated from the knee-joint. The clinical
aspect was at that time very similar to that of severe
rheumatism, and the case had been so diagnosed.

In such cases the use of anti-streptococcic serum
offers some hope to the patient, and should be tried.

Staphylococci are generally found in cases of arthritis
due to perforating wounds, or in the course of a general
infection. They may also occur along with the gono-
coccus in cases of gonorrheeal arthritis.

The pneumococcus occurs in general infection from a
primary focus in the lung, middle ear, &c., or the course
of ulcerative endocarditis.

The gonococcus occurs in some cases of gonorrhceal
arthritis; it may be present in pure culture, or it may
be mixed with other organisms, especially the pus cocci.
In other cases of gonorrheeal arthritis no bacteria are
found, either microscopically or on cultural examina-
tion, and in these the bacteria have probably died out
before the fluid was withdrawn.

The tubercle bacillus may be found in cases of tuber-
cular synovitis, but it is far more probable that the
most careful search will be unsuccessful. If bacilli
having the general appearance of this organism are
found in the Gram specimen, the carbol-fuchsin method
of staining should be applied to a fresh film.

Fluid from a joint may be sterile in cases of tuber-
cular synovitis, gonorrhceal arthritis, synovitis due to
an aseptic injury, rheumatism, gout, or rheumatoid
arthritis, &c.
126 BACTERIOLOGICAL DIAGNOSIS.

LUMBAR PUNCTURE.

Fluid may be removed from the spinal meninges for
a bacteriological or other examination by means of
Quincke’s lumbar puncture. The information furnished
by this means is often of very great value; in fact Osler
says that ‘during the past ten years no single measure
of greater value in diagnosis has been introduced.”
The process is simple, easy, and entirely devoid of
danger, and can be carried out without an anesthetic.

Requisites.-1. A suitable needle. In children the
spinal meninges will be reached at a depth of 3-4 centi-
metres (roughly 1-14 inches) while in adults the depth
may be twice as great. The needle should not be less
than 23 inches long for an infant and 4 inches for an
adult, and should be sharp and strong. An antitoxin
needle will answer every purpose. Aspiration is un-
necessary, and no syringe is required.

2. Materials for disinfection of the patient’s skin and
(if cultures are to be taken) the hands of the operator.
Hot water, soap, alcohol, ether, perchloride lotion (1 in
1000).

3. Apparatus for boiling the needle in a dilute solu-
tion of washing soda.*

4. Spray for local anesthesia. (If used).

5. A test-tube sterilised by dry heat and plugged
with dry cotton-wool.

6. If cultures are to be taken the tubes of medium
should be inoculated at the time of the operation if

* If possible the needle should be sterilised by dry heat previous
to the operation and kept in a tube plugged at both ends with
cotton-wool as in the method recommended for the collecticn of
blood for bacteriological examination.
LUMBAR PUNCTURE. 127

possible. The medium required will depend to a great
extent upon the nature of the organism which is ex-
pected. If there are no indications upon this point the
most suitable medium is solidified blood-serum, but in
default of this ordinary agar will answer well. If the
case is thought to be one of cerebro-spinal fever the
most suitable medium for the cultivation of the specific
organism (Weichselbaum’s diplococcus intra-cellularis)
is alkaline 5 per cent. glycerin-agar, and a couple of
tubes of this medium should be at hand, as well as
blood-serum or ordinary agar.

Process.—1. Preliminary.—As in removal of fluids for
bacteriological examination from other parts of the
body, it is better if the skin can be sterilised some hours
before the operation and a pad soaked in an antiseptic
fluid kept on the area until the last moment. This is
sometimes impracticable, and the process will be de-
scribed as if it were performed at a single visit.

Put the needle to boil in a weak solution of washing
soda, and proceed to the disinfection of the patient’s
back. When the needle has boiled for ten minutes
remove the vessel from the flame and allow it to cool
without removing the needle.

Place ihe patient on his left side and find the pro-
cesses of the second, third, and fourth lumbar vertebre.
A line drawn between the upper points of the iliac
crests usually cuts the spine at the upper edge of
the spinous process of the fourth lumbar vertebra.
Scrub the skin in this vicinity with soap and hot
water; wash the region with alcohol and then with
ether and allow it to dry; paint on several layers of
perchloride lotion, allowing each to soak in before the
next is applied ; cover the region with a piece of lint
soaked with lotion and proceed to disinfect your hands.
128 BACTERIOLOGICAL DIAGNOSIS.

Lastly, pour some alcohol on to the skin of the patient’s
back to wash off the excess of the antiseptic.

2. Opevation.—Position.—Get the patient (still lying
on his left side) to draw up his knees so as to flex his
back somewhat, and to turn partly over on to his face.
It is scarcely necessary to say that the operator must
not touch the patient, as his hands have now been
sterilised.

Identify the processes of the third and fourth lumbar
vertebre and mark the centre of the space between
them by means of the index finger or thumb of the left
hand. If local anesthesia is to be employed freeze the
skin round a point about one-third of an inch to the
right of the middle line, opposite the spot marked by
your left finger or thumb, Take the needle in the right
hand, holding it like a pen, and enter it at a point level
with the centre of the interspace, and 1 cm. (a little less
than one-third of an inch) to the right of the middle
line. Direct it forward, shghtly upwards, and slightly
inwavds, and press it in with a steady and uniform
pressure ; this must be applied accurately in the axis of
the needle, or the latter may bend and take a wrong
direction.

If the needle strikes against bone withdraw it almost
completely and push it on again after changing its
direction slightly. If bone is again encountered it may
be advisable to try again in the interspace between the
second and third processes.

3. Collection of fluid and inoculation of media._-The first
few drops of fluid which escape may be stained with
blood; in this case it should be rejected. Allow a few
drops of the fluid to flow divectly on to the surface of the
media without touching the glass. Collect also some of
the fluid (1-4 drachms) in the sterilised empty tube.
LUMBAR PUNCTURE. I29

If no fluid flows through the needle it is presumptive
evidence against the presence of acute meningitis. A
“dry tap” may, however, occur from plugging of the
needle with fibrin, or from its point coming in contact
with a nerve root (Osler), and in some cases of menin-
gitis the purulent exudation is too thick to flow through
the needle.’

4. Examination of the fluid.—a. Naked eye.— When
meningitis is present the fluid is always more or less
turbid, and some observers hold that the turbidity is
greater in proportion to the severity of the case. Osler
has pointed out that the fluid may be alternately turbid
and clear, being clear during the remissions, and turbid
during the exacerbations of the disease. Blood-stained
fluid may occur in meningitis or from hemorrhage into
the cerebral or spinal meninges apart from inflamma-
tion. The presence of clear fluid affords strong evi-
dence of the absence of meningitis, but in tubercular
meningitis the amount of turbidity may be very slight.

b. Micvoscopical.—Prepare films of the exudate in the
manner recommended on p. 131 if the fluid is thin and
watery ; if it is thick and purulent treat it like ordinary
pus. Stain by any of the methods recommended for
the examination of the blood (Jenner’s stain being most
convenient) and examine.

The presence of leucocytes (except in very small
numbers) indicates meningitis. If the bulk of the
leucocytes are lymphocytes (indicated by their small
size, large, circular, deeply staining nuclei, and ab-
sence of granules) the presumption is that the case
is one of tubercular meningitis. In meningitis due to
other bacteria the chief cell is the polynuclear leuco-
cyte; this may be recognised by its larger size,
its twisted (apparently multiple) nucleus, and, if

K
130 BACTERIOLOGICAL DIAGNOSIS.

the staining method has been appropriate, by the pre-
sence in its protoplasm of minute granules which stain
with eosin. The fluid may also contain red blood
corpuscles and shreds of fibrin.

This examination is hardly necessary except in cases
in which a tubercular origin is suspected.

c. Chemical.—Cerebro-spinal fluid removed from a
person who is not suffering from meningitis contains a
very minute amount of albumen, while when the
meninges are inflamed the quantity is greatly increased.
The method of testing these small amounts of albumen.
are hardly within the reach of practitioners; if a con-
siderable amount of fluid has been obtained a small
quantity should be tested by heat and acetic acid and
the amount of opacity noted.

d. Bactertological_The chief organisms which cause
acute meningitis are given in the following table, which
is modified from one given by Osler :—

Primary (i.e. not dependent on an obvious lesion elsewhere in the

body).
1. Cerebro-spinal fever—
‘ oe Weichselbaum’s diplococcus.

2. Pneumococcic—
a. Pneumococcic infection of meninges alone
not dependent on disease of distant parts of
the body. Pneumococ-
b, Pneumococcic infection of meninges occurring cus.
as part of a general septicemia without
obvious primary lesion.

Secondary.
A. To direct extension from local disease of the cranium, middle
ear, fossze, spinal column, &c.
Pneumococcus.
Staphylococci.
Streptococci, &c.
LUMBAR PUNCTURE. 137

B. To septicemic infection due to disease in a distant part of the
body.
a. Pneumococcic—
Secondary to pneumonia, endocarditis, &c.
Pneumococcus.
b. Pyogenic—
Secondary to abscesses, &c., and occurring as a part of a
general infection.
Staphylococct.
Streptococci, &c.
c. Gonorrhceal—
Secondary to gonorrhea.
Gonococcus.
d. Tubercular—
Secondary to tuberculosis of other regions.
Tubercle bacillus.
e. Miscellaneous—
Secondary to typhoid fever.
Typhoid bacillus.
Secondary to influenza.
Influenza bacillus.
Secondary to anthrax, &c.
Anthrax bacillus, &c.

Of these the organisms which are most likely to occur
are Weichselbaum’s diplococcus, the pneumococcus, the
tubercle bacillus, streptococci, and staphylococci. The
examination for these bacteria may be carried out by
means of stained films or cultures.

Preparation of films.—If the fluid is thick and purulent,
films should be prepared, dried, and fixed in the
ordinary way. If the fluid is thin and watery it should
be allowed to stand for some hours. A certain amount
of coagulation will take place and the sediment which
collects will contain the bulk of the micro-organisms.
This sediment should be removed by means of a pipette
or platinum loop and films prepared from it. The
subsequent examination will depend to some extent

K2
132 BACTERIOLOGICAL DIAGNOSIS.

upon the nature of the organism which is probably
present; for general purposes stain one or more films
with Léffler’s methylene blue (two minutes), wash, dry,
mount, and examine.

Streptococci and staphylococci will be readily re-
cognised by their morphological characters. Ifdiplococci
are present they may be pneumococci, Weichselbaum’s
diplococci, or gonococci. Stain a film by Gram’s
method and counterstain in dilute carbol-fuchsin in the
method described for the gonococcus. Pneumococci
will retain the violet stain, while Weichselbaum’s
organism and gonococci will be coloured red.

Weichselbaum’s diplococcus meningitidis intracellularis is
now generally considered to be the specific cause of
cerebro-spinal fever. It is a medium-sized diplococcus,
the components of the pairs being approximately hemi-
spherical in shape and having their flat surfaces turned
towards one another. This organism has a strong
resemblance to the gonococcus, and this likeness is
increased by the facts that it is decolorised by Gram’s
method and that it is mostly contained within the
polynuclear leucocytes of the inflammatory exudation.
The two differ in their cultural characters and in their
pathogenicity to animals. If any question should arise
as to which of the two is present in the meningeal
exudation in a case in which no cultures have been
taken, some help may be afforded by the fact that the
diplococcus meningitidis often occurs in the nasal secretion
in cerebro-spinal fever. It is scarcely necessary to say
that other evidence of gonorrhceal infection should be
sought for.

Still’s diplococcus of posterior basic meningitis can-
not be distinguished from the diplococcus meningitidis
by its morphological characters alone, and many bac-
LUMBAR PUNCTURE. 133

teriologists think the two organisms are in reality
identical.

The vavey causes of meningitis—The bacilli of typhoid
fever, anthrax, influenza, &c., may also be recognised in
the methylene blue specimen, and should be identified
(if possible) by a careful study of their morphological
appearances and reaction to Gram’s stain,

If no organisms are found in the methylene blue
specimens after a careful search, and if the characters
of the fluid are such as indicate that meningitis is
present, the presumption is that the case is one of
tubercular meningitis. Films should be stained in
the method already described (p. 72) and carefully
searched; the bacilli are present in very scanty num-
bers, and many films may have to be examined before
one is found.*

Cultural examination.—The tubes which have been
inoculated by allowing the fluid to drop directly on to
the surface of the medium are to be incubated for
twenty-four hours at the body temperature. Strepto-
cocci, staphylococci, pneumococci, and the rarer organ-
isms will probably have developed by this time, and
will have formed colonies such as have been previously
described. Weichselbaum’s diplococcus forms (on blood
serum) ‘‘round, whitish, shining, viscid looking colonies
with smooth, sharply defined outlines which attain a
diameter of one to one and a half millimetres in twenty-
four hours.” The colonies on agar are similar but
slightly larger, and the growth may become confluent.

* Lenharz adds a shred of clean cotton-wool to the fluid. This
sinks slowly to the bottom, and is withdrawn after some hours,
spread on a slide, dried and stained for tubercle bacilli. The author
has had no experience of this method, but Mr. Leedham-Green
informs him that it is of considerable value.
134 BACTERIOLOGICAL DIAGNOSIS.

If no colonies appear on blood-serum or agar at the
end of forty-eight hours the case is probably due to the
tubercle bacillus or the gonococcus. In some cases of
cerebro-spinal fever the diplococci in the exudate are
all dead, and cultures remain sterile.

INTERPRETATION OF RESULTS.

The discovery of Weichselbaum’s diplococcus indi-
cates that the case is one of cerebro-spinal fever. The
chief importance in making the diagnosis (apart from
the fact that it may throw light upon the occurrence of
several cases of meningitis within a short space of time
by proving the existence of an epidemic) arises from the
fact that the prognosis is decidedly better than in other
forms of meningitis.

Meningitis due to the pneumococcus may arise from
dissemination from pneumonia or other pneumonic
lesion, by spreading from the middle ear, &c., or may be
primary. The examination of the exudate throws no
light upon this point, and the cause of the infection
must be sought for on ordinary clinical lines.

Tubercular meningitis is proved by the presence of
tubercle bacilli in the fluid, and is indicated by sterile
cultures, absence of bacteria from the stained films, and
predominance of lymphocytes.

The other varieties of meningitis do not call for
special mention.

The chief value of lumbar puncture to the suvgeon is
that it enables him to diagnose a concomitant menin-
gitis (which would negative an operation) in cases of
lateral sinus thrombosis and cerebral abscess. The
fluid usually becomes bloody within twenty-four hours
of a fracture of the base of the skull or laceration of the
COLLECTION OF PUS. 135

brain. This may assist in the diagnosis of obscure
injuries, or of the cause of a case of unconsciousness in
which no history can be obtained.

Hemorrhage into the meninges is indicated by the
withdrawal of blood-stained fluid, but it must be
remembered that the first few drops may contain a
small quantity of blood which has entered the needle
during its passage through the tissues, while the rest is
clear. Blood-stained fluid may occur in meningitis,
and should be submitted to a full examination for
leucocytes and bacteria.

Transmission of fluid to public labovatory.—If the prac-
titioner is unable to examine the fluid the best method
to adopt is to collect it in a long and narrow test tube
(sterilised by dry heat) and to seal the neck of the tube.
Or he may forward it in a bottle sterilised by boiling
and plugged with a cork which has also been boiled,
but this method is not so good.

THE COLLECTION OF PUS.

When a simple microscopical examination has to be
made the collection of pus presents no difficulties, as
the few bacteria which may gain access from the skin
or the air will not lead to error. The case is otherwise
where cultural examinations have to be made, or where
the material has to be transmitted to a laboratory.
Here the material should be collected in a pipette.
This is to be at hand when the abscess is opened; both
ends are to be broken off and passed through the flame
two or three times. The pipette should then be held
by an assistant or put with the end which is to be
inserted into the pus projecting over the side of the
table so as not to come into contact with any object.
136 BACTERIOLOGICAL DIAGNOSIS.

When the abscess has been opened a considerable
quantity of pus should be allowed to flow out, and the
sterilised pipette is then to be passed through the
incision (care being taken to avoid contact with its
sides) and the pus carefully sucked up into the bulb.
The fluid thus obtained may be used to inoculate
cultures there and then, or both ends of the pipette
may be sealed in the flame and the pipette sent toa
laboratory.

Tue EXAMINATION OF Pus.

The organisms which may cause pus are extremely
numerous, the most important being streptococci,
staphylococci, the pneumococcus and the gonococcus,
the bacilli of typhoid fever, tuberculosis, and glanders,
the bacillus coli communis, the bacillus pyocyaneus
(the organism which produces blue pus), and the fungus
of actinomycosis. Inthe majority of cases the organism
which is present in a given sample of pus can be
determined by a microscopic examination of films
prepared in the usual way and stained by a simple
stain, such as carbol-thionin. A specimen should aiso
be stained by Gram’s method and the results compared.

When cultural examinations are required they had
better be carried out in a public laboratory. If the
practitioner should desire to carry them out for himself
he had better make stroke cultivations on agar in the
manner described on page 22, and incubate them for
twenty-four hours at the temperature of the body. The
appearances of the colonies will be similar to those
described as occurring in cultures made from the blood,
to which the reader is referred. It is to be noted,
however, that the gonococcus will not grow under
COLLECTION OF PUS. 137

such circumstances unless the surface of the medium
has previously been coated with blood.

Another method is to make gelatin plates. This isa
very simple matter if the materials are at hand.

Requisites.-—1. Two or three tubes of gelatin.

2. Two or three sterilised Petri’s dishes.

3. A platinum needle—a loop will be best.

Process.—Inoculate a gelatin tube in the manner de-
scribed on p. 22, and then melt it by immersion in
warm (not hot) water.

Distribute the pus throughout the melted gelatin by
rolling the tube between the hands, and by tilting it
from side to side. Do not shake, and do not let the
melted gelatin touch the cotton-wool plug.

Take a loopful of the gelatin and transfer it to a
second culture tube. Melt the gelatin in this and mix
as before. Proceed to inoculate a third tube from the
second one if you think it probable that the pus is very
rich in organisms.

Now take the first tube and singe the projecting part
of the wool plug and heat the mouth of the tube in
order to destroy any germs which may be upon it;
allow it to cool.

Place the Petri dish on the table in front of you and
raise the lid sufficiently to allow you to insert the end of
the test-tube; do this, and tilt the latter so that the
melted gelatin flows into the dish. Immediately replace
the lid, and tilt and roll the dish until the gelatin forms
an even film over its whole lower surface. Place it on
a flat table to set. Repeat the process with the other
tubes. Incubate at about 20° C. for two or three days.
Examine the dishes, placing them on the stage of the
microscope and using the low power. Each organism
will have grown into a small colony, which will resemble
138 BACTERIOLOGICAL DIAGNOSIS.

those which are described in the section on the blood.
There will be slight differences, but not enough to lead
to error if the examination of the colonies is supple-
mented by an inspection of stained films.

The pneumococcus, gonococcus, the fungus of actino-
myces, and the tubercle bacillus will not grow on these
plates; and the bacillus of glanders will grow feebly, if
at all.

In a day or two longer the plates will, in some cases,
be found to have undergone a decided change. If
liquefying organisms are present the colonies will soon
become depressed below the general surface of the
medium and will be surrounded by haloes which
consist of liquefied gelatin. This will happen with the
staphylococci and the bacillus pyocyaneus; not with
the streptococci, the typhoid bacillus, nor with the
bacillus coli. lee

The bacillus of blue pus can readily be distinguished
from the staphylococci by its morphological appearance
(it is a slender rod) and by the fact that the gelatin
round the colony is coloured blue or bluish-green, the
growth itself being nearly white.

wesc

INTERPRETATION OF RESULTs.

The information which is obtained by a study of the
bacteria in pus is of more scientific interest than of
practical importance. It is the situation of the collec-
tion of pus rather than the bacteria causing it which
influences treatment and prognosis. A list of the more
important results which are produced by the chief pyo-
genic bacteria may be of interest.

Staphylococci are the chief producers of localised
suppuration in the skin—such, for instance, as that
COLLECTION OF PUS. 139

which occurs in boils, carbuncles, impetigo, folliculitis,
&c. They may cause abscesses in any part of the
body, and may also give rise to general infections,
ulcerative endocarditis, &c.

Streptococci usually cause spreading inflammation of
the type of erysipelas or cellulitis. They are common
causes of osteo-myelitis and suppurative and septiczemic
or pyzemic processes connected with the puerperium.

In general infections which are due to streptococci
the use of anti-streptococcic serum may offer the only
hope for the patient.

The pneumococcus often produces suppuration in con-
nection with the respiratory system, especially empyema.
It also causes many cases of suppurative otitis media
and meningitis.

The bacillus of typhoid fever sometimes causes abscesses
in connection with the bones after (sometimes long
after) typhoid fever. It has been found in other sup-
purative conditions, ¢.g., empyema.

The tubercle bacillus gives rise to ‘‘cold abscesses,”
usually in connection with bone. The suppuration
which occurs in the walls of phthisical vomice are due
to other bacteria, chiefly streptococci and staphylococci.

The bacillus of glanders only causes suppuration in the
specific lesions of the disease when these run an acute
course.

The bacillus coli communis is the chief cause of sup-
puration occurring in connection with the abdominal
viscera, especially of peritonitis due to perforation of the
intestine and appendicitis. It frequently attacks the
urinary passages, causing cystitis, &c.

The bacillus pyocyaneus causes blue pus, usually in
connection with the skin or subcutaneous tissues.

The fungus of actinomycosis has been dealt with already.
140 BACTERIOLOGICAL DIAGNOSIS.

COLLECTION OF MATERIAL AT POST-
MORTEM EXAMINATIONS.

The saprophytic bacteria which occur in such vast
numbers in the skin and alimentary canal during life
undergo very rapid multiplication after death; hence,
in cases where bacteriological examinations have to be
made the sectio should be performed as soon as possible
after death.

The materials which should be examined in all cases
are the heart-blood, the spleen, and the liver, and the
following methods are to be employed.

The heart-blood should be collected in the method
which has been described previously (see p. 42), and
cultures may be made upon the spot, or the pipettes
sent to a laboratory. .

The spleen may usually be examined in the same way.
If it is so firm and hard that no fluid rises into the pipette,
it should be treated in the same way as the liver.

Cultivations should be made from the diver at the
time when the autopsy is performed. The organ
should be cut in half, anda small portion of the cut
surface deeply seared with a hot iron. This area is
then to be perforated with a stout platinum needle and
the culture media inoculated at once.

If the material has to be sent to a distance and no
culture tubes are at hand a different course must be
adopted. The simplest way is to cut out a cube of
liver substance from the centre of the organ, and to sear
every part of its surface with the flat of a red hot knife.
The block (which may be about as large as a lump of
sugar) must be dropped at once into a sterilised bottle
and sent to the laboratory where the examination is to
be made. Another plan is to sear the surface of the
EXAMINATION OF THE BLOOD. I41I

block and then to tie a piece of string round it and dip
it quickly into melted paraffin (a candle will do) and
allow the coating to set; the dipping is to be repeated
several times, and the specimen (string and all) may
then be packed without further precautions. In any
case it must reach the laboratory as soon as possible.

Where cultural examinations are not required small
portions of the organs should be placed in a suitable
hardening fluid as soon as possible.

Other solid organs are treated in the same way.
Fluids (pus, the contents of cysts, pericardial or other
fluid, &c.) should be collected in pipettes in the manner
adopted for the heart-blood.

EXAMINATION OF THE BLOOD.

A clinically complete examination of the blood in-
cludes :—

1. A determination of the number of red corpuscles
present per cubic millimetre. (Normal numbers being
5,000,000 in the adult male and about 4,500,000 in the
female).

2. A determination of the number of leucocytes pre-
sent per cubic millimetre (the normal is between 4,000
and 10,000).

3. A determination of the amount of haemoglobin
expressed as a percentage of the normal amount.

4. An examination of stained specimens to ascertain
the presence or absence of abnormal corpuscles, and the
relative proportions of the leucocytes present.

In addition to these it is sometimes necessary to
make :—

5. A determination of the presence or absence of
parasites.
142 BACTERIOLOGICAL DIAGNOSIS.

ESTIMATION OF THE RED CORPUSCLES.

The best apparatus for the estimation of the number
of corpuscles (whether red or white) is the Thoma-
Zeiss hemocytometer. It should be provided with two
pipettes, and costs about thirty-six shillings.

Examine the pipettes. Each has a small bulb con-
taining a little glass ball and a stem which is graduated
into several parts below the bulb, and has a single
transverse graduation above it.

The pipette intended for use in counting the leuco-
cytes may be distinguished by the fact that it has the
figure 11 over the single transverse graduation above
the bulb.

There are two sorts of pipettes used for counting the
red corpuscles. In the one form the stem below the
bulb is divided into ten parts, the upper one (nearest
the bulb) being marked 1, and the middle one o'5
(fig. 23, S). Inthe other one the same portion of the
stem is graduated into three portions numbered ;45,
zig, and 345; the figure mentioned first is placed
nearest the bulb. These pipettes are used in the same
way, and it is quite immaterial which is obtained; we
shall describe the use of the first form.

The rationale of the method is this:—Blood is sucked
up to one of the divisions on the lower part of the stem,
and then an inert diluting fluid is drawn up to the
single mark above the bulb, and the two mixed by
rotating the whole apparatus for a minute or two.
This gives us a dilution of blood of definite strength,
the exact amount of dilution depending upon the
amount of blood which was taken. Thus, if blood had
ESTIMATION OF RED CORPUSCLES. 143

been drawn up to the figure 1 we should have a dilution
of 1 in 100, while if blood had been drawn up to the
figure 5 the dilution would be -5 in 100, or 1 in 200, and

 

 

 

0100mm.
sho num,

 

 

 

 

8

 

Fic. 23.—Thoma’s Hemocytometer.

 

so on. In the case of the other form of pipette the
dilution is read off directly from the figures on the lower
stem.

The diluted blood thus obtained is spread out in a
film of a definite known thickness on the slide supplied
144 BACTERIOLOGICAL DIAGNOSIS.

on the instrument (fig. 23, 2). This is ruled in squares,
and the squares are of known size. The amount of
blood lying upon each square is thus known, and the
number of corpuscles which lie upon it being counted
under the microscope, all the data for the calculation
are obtained.

In blood examinations it is absolutely necessary that
all points in the technique should receive the most
careful attention, or the result will be worse than
useless. For this reason we shall describe each step in
the process at some length, and advise the practitioner
to make several estimations before placing any reliance
whatever on his results.

Requisites.—1. The hemocytometer.

2. A needle suitable for obtaining a small quantity of
blood. A Hagedorn’s needle is the very best that can
be used, and an ordinary triangular surgical needle will
answer very well.

3. Diluting fluid. There are a good many formule
for this, and some are rather complicated. Isotonic
saline solution (common salt ‘5 per cent.) will answer
perfectly ; it is advisable to add to it a small quantity
of some stain, methyl violet being the best, although
gentian violet will do very well. This colours the leuco-
cytes, so that they are readily distinguished from the
red corpuscles.*

4. A microscope having an 4 inch lens which will
focus through the thick cover-glass supplied with the
hemocytometer. Ifthe examination is not to be made
by the bedside a strong india-rubber band a little
shorter than the pipette should be carried.

* The following formula is better:—Distilled water, 160 c.c.;
glycerine, 30 c.c.; sodium sulphate, 8 grams; sodium chloride, 1
gram; methyl violet, a trace.
ESTIMATION OF RED CORPUSCLES. I45

PROCESS.

1. Pricking the patient—The blood may be procured
from the convex border of the lobe of the ear or from
the lateral surface of the last phalanx of the finger.
The advantage of the former situation is that the pain
is very slight, the skin being thin, and that the patient
cannot see what you are doing and is not likely to start
at the critical moment. It is to be recommended for
children and nervous women. The advantage of the
finger is that the skin is free from hairs, and these are
objectionable in the preparation of film preparations by
the cover-glass method; an additional advantage is that
the patient can put his hand into the position most
convenient to you and you have not to lean over him.

The area of the skin to be punctured is washed
with soap and water and then with pure water, and
wiped dry. No other sterilisation is necessary unless
you are also taking blood for bacteriological purposes.
The needle is sterilised by being passed slowly through
the flame of a spirit lamp or Bunsen’s burner; the area
of skin to be pricked is taken between the finger and
thumb of the left hand, and a rapid and fairly deep stab
made with the needle. The skin is then released and a
drop of blood allowed to exude; this is wiped away and
the next drop which oozes out is used for examination.

The skin must never be pinched when blood is being
withdrawn; the blood must always be allowed to flow
out naturally.

2. Filling the pipette—The degree of dilution is deter-
mined by the number of corpuscles per cubic millimetre
which you expect to find. If the patient is anaemic use

iL
146 BACTERIOLOGICAL DIAGNOSIS.

1 in 100; if he has approximately the normal number of
corpuscles, or if you have reason to think that they be
present in increased quantities, use a dilution of 1 in 150
or 1 in 200.

Having decided upon the degree of dilution insert the
tip of the pipette into the drop of blood lying on the
skin, take the bone mouth-piece attached to the india-
rubber tube in your mouth, and suck the blood up to
the appropriate mark. If air bubbles gain access blow
the blood out and begin again. If you over-shoot the
mark remove some of the blood by touching the tip of
the pipette against some absorbent cotton-wool. Now
remove the pipette from the blood and wipe off the
excess with your finger; prevent blood from flowing
out by placing the tip of your tongue in the aperture
of the bone mouth-piece. Place the tip of the pipette
in the diluting fluid; a small quantity should be poured
out into a watch-glass or other suitable vessel, so as to
avoid any possibility of allowing some blood to escape
into the stock bottle and invalidating a subsequent obser-
vation. Suck the diluting fluid slowly into the pipette
until it reaches the single mark above the bulb; it is
best to rotate the pipette between the finger and thumb
as you do so.

Now remove the pipette from the diluting fluid, pre-
venting escape of fluid from the bulb by placing the tip
of the tongue on the aperture of the mouth-piece as you
do so; then place the tip of the finger over the aperture
of the pipette (fig. 23, S) and proceed to mix the con-
tents by rotating the pipette and by turning it over and
over. It is hardly necessary to say that it is useless to
shake it.

If the examination is to be made at a distance remove
the india-rubber tube and stretch an india-rubber band
so as to close both apertures of the pipette.
ESTIMATION OF RED CORPUSCLES. 147

. 3. Preparation of the specimen.—The slide which is
supplied with the instrument consists of a thick and
perfectly flat slip of glass (fig. 23, 0) on which is
cemented a glass square having a round hole in its
centre (W). Inthe centre of the hole thus left there
is a circular disc of glass (B); this inner disc is made
of glass which is exactly #, of a millimetre thinner than
that of which the outer glass is constructed. When
the whole cell is covered with a perfectly flat cover-
glass (D) there will, therefore, be a space exactly 4, of
a millimetre between the lower surface of this cover-
glass and the upper surface of the central disc; this
space is to be filled with the diluted blood.

Slide and cover-glass are to be wiped clean with
a soft handkerchief moistened with water and then
thoroughly dried; there must not be the minutest
particle of dust on any part of the surface.

The slide and cover-glass being ready, mix the con-
tents of the pipette as you did before (this must always
be done immediately before making the specimen, no
matter how carefully it had been done a short time
previously) and blow out about half of the fluid in the
bulb; this is to wash the diluting fluid out of the lower
part of the stem. Nowclip the india-rubber tube firmly
between your finger and thumb so as to prevent the
access of air, and, therefore, the escape of fluid, and wipe
the tip of the pipette from all fluid; this may be done
with the forefinger. Place the tip of the pipette on the
centre of the central disc of the slide and relax your
pressure on the india-rubber tube so as to allow a small
drop of fluid to escape; this is perhaps the most difficult
part of the process, and the exact amount which must
be allowed to fall on to the slide can only be learnt by
experience.

L2
148 BACTERIOLOGICAL DIAGNOSIS.

Cover the slide in this way :—Place your finger at the
side on the glass square on the slide, and apply the
cover-glass, letting it rest against your finger; lower it
gently in place with a needle or other suitable object.
When it is in place press it gently with the needle at
each corner in succession, and look at it obliquely so
as to see the light reflected from the surface. If the
slide and cover-glass are in sufficiently close contact
you will see Newton’s rings (looking like the eye of a
peacock’s feather) round the point at which you are
applying pressure. If you do not see this the inference
is that there is some dust between the slide and cover-
glass; you must clean both and begin again.

If you have taken the right amount of fluid the drop
should extend exactly to the edge of the central glass
disc, but should not run over into the “moat” (7).
If this happens, or if there are any bubbles under the
cover-glass, you must begin again. Ifthe drop does not
quite extend to the edge of the central disc no great
harm is done.

4. Focussing the specimen.—This is somewhat difficult
for beginners, and merits a short description. Place
the slide under the microscope, taking care to get it
accurately centred, and examine it with the low power.
You will find that the central disc is ruled into squares
like a chess-board (c). Get these squares into the
centre of the field.

Do not forget you are dealing with an unstained
object; use a flat mirror and a small diaphragm. The
examination is often easier if artificial light is used.

Now turn on the high power (4 in. or 4 in.) and
screw it downwards until it almost touches the cover-
glass; look down the microscope and focus gently
upwards, using the fine adjustment and keeping a
careful lookout for the rulings.
ESTIMATION OF RED CORPUSCLES. 149

Some ¢ in. lenses focus too near the object to be of
any use. If this is the case you must either get an
objective specially for the purpose, or a cover-glass
which is hollowed out in the centre. These can be
bought from the same place as the hemocytometer.

If the rulings of the slide are indistinct they may be
darkened by rubbing them with a very soft lead pencil.

5. Counting the corpuscles —Move the slide about until
you have come to one corner (preferably the left upper
corner) of the ruled area. You will see that each
fifth space is marked off by a line running down its
centre; this is to guide the eye and facilitate counting.
If you exclude the spaces which are thus marked with a
double line the whole area will be marked out into
a series of large squares, each consisting of 4 x 4 = 16
smaller squares (fig. 24). It is convenient to count the
smaller squares in these groups of 16. A? least a hun-
dred of the smaller squares, 7.¢., six of the large groups
and four small squares, should be counted.

In counting one of the smaller squares it is convenient
to begin with the corpuscles which are lying in the
middle of the square, and then to count those which are
lying on the lines. In dealing with these you count
those which are lying on the upper and left hand lines
as being within the square, and those that are on the
lowey and vight hand lines as being without it; if you like
you may reverse this, but you must keep to the same
method throughout (see fig. 24).

A few white corpuscles will be met with in every
case, while if the blood was taken from a patient with
leucocytosis or leucocythemia there will be many.
‘They may be distinguished from the red corpuscles by
their greater refractivity, or, if a stain has been used in
the diluting fluid, by their being faintly tinged. It is
I50 BACTERIOLOGICAL DIAGNOSIS.

scarcely necessary to say that they should not be
counted.

6. The calculation.—The best way of calculating the
number of corpuscles present from the data thus
obtained is the following :—

First add up the number of corpuscles in all the
squares which you have counted and divide the sum by
the number of squares counted. This gives the average
in each square.

 

Fic. 24.—Showing method of counting red corpuscles: a, a, a, are
counted in square A; 5, 6, in B; ¢, in C.

Now the space enclosed between each square and the
cover-glass above it is ~5 of a millimetre deep, 4, of
a millimetre wide, and #, of a millimetre long; its
cubic capacity is therefore 2, x gy X a = aooo Of
a cubic millimetre. Therefore the gg55 part of a
cubic millimetre contains the number of corpuscles
which we have already found as the average.

But the square contained diluted blood ; if the amount
ESTIMATION OF RED CORPUSCLES. I51

of dilution was 1 in 100 the amount of blood contained
in the space over each square was ;3, part of -d,, of
a cubic millimetre.

Therefore, the number of corpuscles which has been
determined as being the average per square is contained
in zgoo Of zz Of a cubic millimetre of undiluted blood,
the dilution being taken as 1 in 100.

Hence the number of corpuscles in one cubic milli-
metre of undiluted blood is obtained by multiplying the
average per square by the number which expresses the
dilution (in this case 100) and then by 4000.

It may be expressed as a formula, thus:—

If ~ is the total number of corpuscles counted,

s is the number of squares counted,
and if the dilution is 1 in d.
Then the number of corpuscles per cubic millimetre

 

a:
is= x d X 4000.

Example.—Suppose that we have counted 100 squares
and have found that they contain 1200 corpuscles.
Then the average per square is 12.

Then 3355 Of a cubic millimetre of diluted blood
contains 12 corpuscles.

Or, <3o Of aebo Of undiluted blood contains 12 cor-
puscles, supposing the dilution was 1 in 100.

Therefore, one cubic millimetre of undiluted blood
contains 12 X 100 x 4000 = 4,800,000 corpuscles.

Or by the formula :—

Number of corpuscles per cubic millimetre :—

1200
100

The beginner is strongly advised to work out the
problem at full length until he has become absolutely
familiar with the reasons for all the steps.

 

X 4000 X 100 = 4,800,000.
152 BACTERIOLOGICAL DIAGNOSIS.

ESTIMATION OF THE NUMBER OF
LEUCOCYTES.

All the steps are similar to those just described at
full length, except that a different diluting fluid is used.

The diluting fluid is one which destroys (‘‘lakes’’)
the red corpuscles, but does not injure the leucocytes.
It consists of a +3 or *5 solution of acetic acid (glacial)
in water; it is better to add a small quantity of methyl
violet or gentian violet, so that the leucocytes are
stained and thereby rendered more prominent. This
solution is best prepared fresh or at any rate kept in
a well stoppered bottle.

The pipette is distinguished from that used for the
red corpuscles by its having the number 11 above
the bulb. This indicates that if blood be sucked up
to the mark 1 below the bulb, and diluting fluid up to
the transverse mark above the bulb, the dilution will be
1 in 10, and so on.

The blood should be sucked up to the mark 1 if a
great excess of leucocytes is not expected. If the case
is one of leucocytosis a greater dilution is better, whilst
if there is a great excess of leucocytes (such as occurs
in severe leucocythemia) it is best to use the red
corpuscles pipette with a dilution of 1 in 100, but
employing the acetic acid diluting fluid.

In counting the squares a different method has to be
employed, as a far greater number of squares must be
counted. Either you may count al/ the squares on the
slide and then make a fresh preparation and count all
the squares on it; or you may adopt the following
method, which is much quicker and, therefore, since it
ESTIMATION OF NUMBER OF LEUCOCYTES. 153

permits of more squares being counted in a reasonable
time, more accurate.

Having focussed the rulings on the slide move the
draw-tube of the microscope up and down until the
upper and lower limits of the field of the microscope
coincide exactly with two of the horizontal lines, and
count the number of spaces (each enclosed between two
horizontal lines) in the diameter of the field. Using a
4 in. objective it will be found possible to arrange
matters so that these are eight in number, and this will
be found convenient, though any other number will do.

 

Fic. 25.—Showing field of microscope adjusted so that its diameter
is equal to that of eight squares.

The essential thing is that the upper and lower borders
of the field shall coincide exactly with the rulings. We
will suppose that the number is eight. Then the dia-
meter of the field of the microscope is equal to eight
times the length of a side of a square, and its radius is
equal to four times the length of a side of a square.
The total area of the field of the microscope is therefore

22 ; 22
4X 4X 9? (7 x w, where w is taken as 7) or 50 and

a fraction. Practically, therefore, when we look down
the microscope after it has been adjusted in this
154 BACTERIOLOGICAL DIAGNOSIS.

way we are looking at 50 squares; and this fact enables
us to dispense entirely with the rulings and count over
the whole area of the disc with great rapidity. The
slide is placed in position and all the cells which are
seen in the field counted and the result noted down, or,
preferably, dictated to someone else. The slide is then
moved on until a perfectly fresh portion of the field
comes into view; it is advisable to go too far rather
than not far enough. For this purpose (as for a great
deal of blood work) a mechanical stage is a great
advantage. In this way 2000 squares may be counted
in avery short time; it is an advantage, however, to
count 1000 squares, #.e, 20 fields, and then to clean the
slide and to prepare a fresh preparation from the fluid
which remains in the pipette and count another 20
fields.

The calculation is just the same as for the red cor-
puscles, remembering that the dilution is very different.
The average per square will of course be less than
unity. The same formula is applicable.

Immediately after use the pipettes must be thoroughly
cleaned. The fluid which remains in the bulb must be
blown out; and for this purpose, as well as for the
subsequent washings, it is an advantage to reverse the
position of the india-rubber tube, so that the fluid may
be blown out through the upper part of the pipette, this
being the wider. The whole pipette must now be filled
with water (preferably distilled) and the water blown
out. This process is repeated, using absolute alcohol
and allowing it to run out of the pipette without blowing
it. Lastly, fill the whole pipette with ether, remove
the india-rubber tube, replace it with the tube of an
ordinary spray (such as is used for scent fountains,
throat sprays, &c.) and pump air through until the
ESTIMATION OF AMOUNT OF HZMOGLOBIN. 155

apparatus is absolutely dry. You can tell when this has
happened by the fact that the ball inside the bulb will
emit a clear ringing sound when the pipette is shaken.
It is useless to attempt to dry the tube by blowing
through it from the mouth.

If blood has coagulated within the apparatus it must
be digested out. Fill the whole with an artificial
digestion fluid (pepsin and very dilute hydrochloric
acid) and place it in a test-tube of the same fluid in a
warm place for twenty-four hours. Then try to clean it
as before and repeat the digestion if this is impossible.

ESTIMATION OF THE AMOUNT OF
HAMOGLOBIN.

There is no absolutely satisfactory apparatus for the
estimation of the amount of haemoglobin in the blood at
present on the market. Those which are chiefly used
in this country are Gowers’, and Oliver’s. Gowers’ is
the simplest and by far the cheapest form; the neces-
sary manipulations are very easily learnt, but it is not
easy to take exact readings. Indeed, the margin of
error is very considerable. Oliver’s hzemoglobinometer
is a little more difficult to use, but it is somewhat easier
to read; its price prevents it coming into universal
use. It is to be recommended where absolute results are
required; for clinical purposes when we wish to see
whether a patient is or is not improving under treatment
Gowers’ will answer quite well.

It consists of two tubes mounted in a small stand.
One of these tubes is filled with a jelly tinted to
represent the colour of normal blood of a certain
156 BACTERIOLOGICAL DIAGNOSIS.

degree of dilution. The other is graduated into a
hundred parts, the graduation being such that when
20 cubic millimetres of normal blood are diluted with
water up to the 100 mark the colour of the two tubes
should be exactly the same. A pipette measuring 20
c.m. and a dropping bottle (which is to be filled with
water) are also provided.

Method of use—Place a few drops of water (preferably,
but not necessarily, distilled) in the graduated tube.

    

(Cigna saeco

F

 

 

Fic. 26.—Gowers’ Hzmoglobinometer..

Draw the blood in the usual way. Apply the tip of the
measuring pipette to the drop and suck gently until the
blood reaches up to the mark. Now put the tip of the
pipette into the small quantity of water in the bottom
of the graduated tube and blow out the blood; suck
water up the pipette until it reaches above the mark
and blow it out; repeat this process until the blood is
thoroughly washed out from the tube.

Place the two tubes side by side on a sheet of white
paper in front of a well-lighted window which is not
ESTIMATION OF AMOUNT OF HMOGLOBIN. 157

exposed to direct sunlight; look at them by the light
which is reflected from this paper, and add water from
the pipette belonging to the dropping bottle, drop by
drop, until the colour in the two tubes is exactly the
same. Read off the height of the column of diluted
blood; this gives the percentage amount of hemoglobin.

Oliver’s hzmoglobinometer differs from that of
Gowers’ in that the degree of dilution is constant
and the colour of the diluted blood is read off by
comparison with a series of carefully graduated stan-
dards. It consists of (1) a capillary glass tube with
thick walls and ground ends, one of which is flat and
the other pointed; this tube is mounted in a metal
handle, the other end of which serves as a stirrer
(fig. 27, c); (2) a small cell with an opaque white
bottom, and provided with a cover-glass which has
a slight bluish tint (e); (3) a series of twelve coloured
glass discs mounted over an opaque white back-
ground (a); (4) certain small pink glass discs used
as riders; (5) a short glass pipette with an india-
rubber nipple at one end and a short length of india-
rubber tubing at the other (d); the latter fits over the
pointed end of the capillary tube mentioned first; and
(6) a small wax candle such as is used for Christmas
trees. A camera tube lined with a green material is
used to screen the eyes whilst the comparison is being
made.

Method of use:—Prick the patient in the usual way.
Apply the polished end of the capillary tube to the
drop of blood; this will completely fill the tube, being
drawn up by capillary attraction. When quite full
wipe both ends of the tube with the fingers and apply
the end of the glass pipette (previously filled with
water) to the pointed end of the capillary tube. Now
 

Hemoglobinometer,

.—-Oliver’s

Fic. 27
PREPARATION OF FILMS. 159

squeeze the nipple gently so as to force the blood and
(subsequently) the water drop by drop into the cell.
Interrupt the process occasionally and stir the contents
of the cell with the metal handle of the measuring tube.
Continue to add water until the cell is exactly full;
this is the first step which presents the slightest diffi-
culty. Apply the coverglass; this must not enclose any
air under it, nor cause any of the diluted blood to flow
into the moat round the cell.

The specimen is now ready for comparison with the
standards. It is to be taken into a dark room and
examined by the light of one of the candles. This is to
be placed in front of the observer at a short distance
from the specimen and standards, which must lie side
by side.

The viewing is best done by means of a camera
tube which folds into the box containing the whole
apparatus. It terminates in a diaphragm which is
perforated by two small holes, one of which is to be
placed over the centre of the specimen and the other
over the centre of the standard. The latter is to be
moved about until a disc is found which nearly or quite
corresponds in colour with the diluted blood in the cell.
If the correspondence is exact the process is at an end ;
the number against the disc in question represents the
percentage amount of hemoglobin. If there is no disc
which exactly matches the specimen the latter is placed
against the disc which is nearest to it but not so deep in
colour. For example, if we found that the specimen
was darker than the disc numbered 50, but paler than
that numbered 60, then it would be placed opposite to
50. Avslip of colourless glass is then applied over the
specimen, and riders over the standard disc, until an
exact match is obtained. If, in the case mentioned
160 BACTERIOLOGICAL DIAGNOSIS.

above we had to add a rider marked 5 to the standard
to bring about an exact match, the percentage amount
of hemoglobin in the blood would be 55.

It is an advantage to place cell and standards side by
side rather than one above the other, for the upper and
lower portions of the retina differ in sensitiveness to
colour, whilst the sides do not.

PREPARATION OF FILMS FOR STAINING.

The processes to be described are the same whether
we are making films for the purpose of investigating the
nature of the corpuscles and cells contained, or for the
detection of parasites. If, in the latter case, we are
searching for the malaria parasite very good films are
necessary, but if we are only looking for bacteria we
may use films that are quite useless for a study of the
blood itself.

We shall describe two methods, one with cover-
glasses and one with slides and cigarette papers.

I. METHOD WITH COVER-GLASSES.

Requisites—1. Perfectly clean cover-glasses. These
must have been cleaned with nitric acid, washed, and
then soaked in ammonia, then washed in absolute
alcohol and kept in a mixture of equal parts of absolute
alcohol and ether. Immediately before they are re-
quired for use they must be removed with a clean
pair of forceps and dried with an old and soft handker-
chief. They may then be passed through the flame.
PREPARATION OF FILMS. 161

2. Two pair of dissecting forceps.

3. A needle for taking blood.

4. A platinum loop having a diameter of about one-
tenth of an inch is sometimes useful, especially to a
beginner.

Method.—The patient having been pricked and the
first drop of blood wiped away, take up a cover-glass in
one pair of forceps, holding the glass by its corner, and
touch the drop of blood with it. In doing so take great
care not to touch the patient’s skin. There should now
be a small drop of blood in the very centre of the cover-
glass. This is the difficult step; you must not get too

*much nor too little blood, or the films will be useless.
Now take up the second cover-glass in the other pair of
forceps and place it on the top of the drop in sucha
position that the corners of the two cover-glasses do not
correspond. Release the upper cover-glass; it will ap-
proach the lower one, being drawn thereto by the
capillary attraction caused by the presence of a small
quantity of fluid between the two.

At this stage you will see whether you have taken the
right amount of blood or no. If you have, the drop will
spread out, still retaining its circular shape, until it
approaches the octagon formed by the intersecting
edges of the two cover-glasses; if you have taken too
little it will not reach so far; and if you have taken too
much it will extend further and the upper cover-glass
will float loosely on the lower.

It is necessary to lay great emphasis on the fact that
the cover-glasses must mot be squeezed together, but
must simply come together by capillary attraction.

When the drop has ceased to spread take hold of the
upper cover-glass with the forceps at a corner opposite
to that of the lower cover-glass which you are still

M
162 BACTERIOLOGICAL DIAGNOSIS.

holding with the other pair of forceps; draw the two
apart with a steady even pull; they should separate
easily, and, if all the stages of the process have been
properly carried out, leave you with two perfectly
spread films.

Here again you find whether you have taken the
right amount of blood. If you have taken too little
the cover-glasses will be very difficult to separate ;
may indeed be impossible to do so without breaking
them. Ifyou have taken too much they will separate
with great readiness and the blood will spread in
uneven smears instead of forming a uniform film.

A word is necessary concerning the method in which
the cover-glasses are to be pulled apart. They must
always be kept in the same plane; if this is not done,
and if the upper cover-glass is lifted from the lower one,
the film will resemble the marks left on a knife which
has been pressed upon butter and then lifted off; such
films are useless.

The blood may be taken by means of the platinum
loop, and this is a good plan, as all danger of smearing
the cover-glass upon the skin is avoided. If several
films are to be taken a number of platinum loops should
be provided, as the blood upon them soon coagulates.
The exact size of the loop can only be learnt by
experiment, and when one has been found to deliver
a drop of the right size it should be kept entirely for
this work and carefully protected from injury.

Il. MeTHOD WITH CIGARETTE Papers.

Requisites.—1. Perfectly clean slides.
2. Some fairly stiff cigarette papers cut in half
FIXATION OF BLOOD FILMS. 163

longitudinally. Paper which is decidedly ridged or
ribbed will not answer.

3. Needle.

Method.—The patient is pricked and the first drop of
blood wiped away as before. One of the half strips of
cigarette paper is now held in the right hand, the index
finger being placed above the strip and the edges held
between the thumb and index finger and the index and
middle fingers respectively; this converts it into a
gutter, the convex edge of which is downward. The
edge of this gutter which points away from you (and
which is formed by a machine-cut edge of the paper)

 

Fic. 28.—Method of spreading films with cigarette paper.

is now dipped into the drop of blood and a small
quantity picked up on its lower surface. This lower
surface is then placed on a clean slide parallel to one
of its shorter edges and about half an inch from it,
and pressed gently upon it so as to flatten out the
paper gutter; as this flattens out the edge of the drop
of blood on its under surface will follow it. The strip
of paper is now drawn towards the other end of the
slide with a steady uniform movement, and in doing
so the drop of blood is spread out into a long uniform
film. In this way a film } in. wide and 2 in. long can
be made on a single slide. ‘A fresh piece of paper is to
be used for each specimen.
M2
164 BACTERIOLOGICAL DIAGNOSIS.

The author is of opinion that it is best to adopt the
cover-glass method, as he has found that it presents
fewer difficulties for beginners; this is not the universal
experience, and it is a good plan to try both and adopt
that with which you get the best results.

FIXATION OF BLOOD FILMS.

If films are required simply for bacteriological pur-
poses (i.¢., to search them for bacteria) they may be
fixed by passing them three times through the flame,
just as if they were ordinary films. This, however, is
not to be recommended in the study of the cells of the
blood or of the parasite of malaria, though it answers
very well in skilful hands. Three methods should be
learnt, the method by heat, the alcohol and ether
method, and the formalin method. Of these the first is
necessary if Ehrlich’s triacid stain is to be used, but the
others are perhaps better for other stains. In addition
to these we must mention that if Jenner’s stain is used
a preliminary fixation is unnecessary, as the fluid fixes
the film and stains it at the same time. This is the
method of blood examination which is most suitable
for practitioners; and it is doubtful whether it is not
also the best for the most accurate and delicate
scientific research.

I. Metuop oF FIxaTion By HEat..

Slides or cover-glasses tobe fixed by this method
must be exposed to a temperature of 150° C. for
FIXATION OF BLOOD FILMS. 165

about five minutes—slides requiring a slightly longer
time than cover-glasses.

The ideal way is to use a dry air steriliser (see p. 8)
to place the films in it whilst cold, to heat up to 150°C..,
and then to turn out the gas. In the absence of this
apparatus a metal slab or plate such as has been
recommended for use in staining the tubercle bacillus
answers well. It is mounted upon a tripod stand
and the heat applied at one end. After a time the
temperature of various portions of the plate are tested
by the application of a few drops of water; the point at
which the drop assumes the “spheroidal state” (i.¢.,
takes the form of a sphere and does not wet the plate)
is about the right point to use. The slides or films are
placed at this point for the appropriate time.

II. Frxation py ALCOHOL AND ETHER.

This is very simple; the films are placed in a mixture
of equal parts of alcohol and ether for at least half an
hour.

This method of fixation is perhaps the best that can
be adopted for general purposes.

III. Fixation By ForRMALIN.

There are several methods by which the fixative
action of formalin can be used for blood work. Of
these the use of a mixture of one part of formalin with
nine parts of absolute alcohol answers perfectly. The
films are immersed in this for half a minute and then
washed thoroughly under the tap.

This method of fixation is both good and rapid.
166 BACTERIOLOGICAL DIAGNOSIS.

STAINING BLOOD FILMS FOR THE IN-
VESTIGATION OF THEIR CELLS.

There are a great many methods of staining blood
films, and all depend upon the division of stains into
two varieties, the acid and the basic. All the stains
which are used in this branch of histology are salts;
and in some of these salts the acid radicle does the
staining, in others the basic.

Acid stains are those in which the colouring property
resides in the acid of the salt. A familiar example is
picrate of potash, a yellow stain in which the picric
acid is the active ingredient. The acid stains in chief
use are eosin, acid fuchsin, and orange G. Substances
which stain with an acid stain after suitable exposure
to a mixture of an acid and a basic stain are called
oxyphile, or, from the frequent use made of eosin as an
acid stain, eosinophile.

Basic stains are those in which the colouring property
resides in the basic radicle of the salt; they include all
the stains which are in use for staining bacteria and
they all colour the nuclei of cells. The most important
are methylene blue, methyl green, and toluidin.

We shall describe three methods of staining, and
these are sufficient for all purposes of diagnosis. They
are:—1. Ehrlich’s method with his triacid stain; 2.
Jenner’s method; and, 3. Eosin and methylene used
separately.

1. Ehrlich’s stain consists of a mixture of acid
fuchsin, orange G, and methyl! green dissolved in water,
glycerine, and alcohol. It is difficult to prepare and
should be purchased from a reliable maker. Its use is
STAINING BLOOD FILMS. 167

very simple. The film is fixed by heat in the manner
already described and the stain is poured on to it and
allowed to act for five minutes. The film is then
washed, dried with blotting paper and then by gentle
heat, and mounted in balsam.

Nuclei are stained green, red blood corpuscles orange,
and eosinophile granulations bright red. The small
eosinophile granulations which are present in the poly-
morphonuclear cells (the neutrophile granulations of
Ehrlich) are stained violet. The basophile granulations
are unstained.

This stain is not suitable for the parasite of malaria,
nor for bacteria.

2. Jenner's stain consists of a solution of a compound
of eosin and methylene blue in methyl alcohol. It must
be bought ready prepared. Nothing could be more
simple than the way in which it is used; no preliminary
fixation is necessary, the film being allowed to dry and
flooded with the stain. After a period of from a minute
and a half to three minutes the stain is washed off
in distilled or vain water and the specimen dried and
mounted.

After the use of this stain nuclei are stained blue, red
corpuscles red, eosinophile granules red, and basophile
granules violet. A striking feature of this stain, and
one which distinguishes it from other staining methods
in which eosin and methylene blue are used is the
intense way in which the fine eosinophile granules
in the polymorphonuclear leucocytes take the eosin.
Observers who have not been familiar with Jenner’s
stain have mistaken these cells for the eosinophile
leucocytes; this mistake could not arise after one of
the latter cells had been seen for comparison, as its
granules are so much larger and more prominent.
168 BACTERIOLOGICAL DIAGNOSIS.

Jenner’s stain is suitable for a study of the parasite of
malaria, which it stains blue. It may be used for the
detection of bacteria.

3. Eosin and methylene blue used separately.

In this method the films are to be stained with the
eosin first and then with the methylene blue. Its
successful application requires a certain amount of
practice.

The eosin used must be in watery solution, and the
exact strength does not matter; four per cent. is a
convenient strength to use. Most specimens of red ink
(slightly diluted) will do quite well. The films are
to be stained in this solution for three or four minutes ;
no harm will result if they are left in much longer.
They are then washed and immersed in a saturated
watery solution of methylene blue. This is the diffi-
cult part of the process, for no general rule can be
given as to the length of time for which this stain must
be applied ; it may be ten seconds, or it may be two or
three minutes. The only safe way is to stain the film
for a quarter of a minute, wash it, and then examine it
under the low power of the microscope. If the film is
properly stained the nuclei of the leucocytes will be seen
as blue points which can be distinguish with great ease
with the 2 inch objective. If they are not visible the
methylene blue must be applied for about a quarter of
a minute more and the examination repeated. When
the nuclei are seen to be well stained the film is dried
and mounted.

This process gives results which resembles those
afforded by Jenner’s stain except that the fine eosino-
phile granulations in the polymorphonuclear cells are
always less obvious and often quite invisible. It is
also suitable for malarial parasites and bacteria.
EXAMINATION OF THE BLOOD. 169

The practitioner is recommended to practice this
method of staining, as it does not require any reagents
which are not to be found in every well stocked surgery.
The watery solution of methylene blue which is used as
a counterstain for the tubercle bacillus and some red ink
are all that are necessary.

THE BACTERIOLOGICAL EXAMINATION
OF THE BLOOD.

The bacteriological examination of the blood is not of
very great diagnostic importance, as it is only in a very
few diseases that pathogenic bacteria are present in the
circulation in such quantities as renders the search for
them in the minute amounts which are withdrawn for
examination at all promising.

The chief organisms which have been found in the
blood are :—

1 & 2. Streptococci and staphylococci.These are found
in cases of septicemia, pyeemia, ulcerative endocarditis,
&c.; they always indicate an extremely bad prognosis.
The chief importance which attaches to the discovery
of these organisms is that it absolutely settles the
diagnosis (always provided that there are no errors in
technique) and that it indicates whether the use of anti-
streptococcic serum is advisable or not; it is useless in
cases of septicemia, &c., which are not due to strepto-
cocci.

A word of warning is necessary in the interpretation
of results which indicate that staphylocci are present in
the biood. These organisms are constantly present in
the skin and may be found in film preparations or in
170 BACTERIOLOGICAL DIAGNOSIS.

cultures, unless the rigid antiseptic precautions are
taken. Streptococci are common contaminations of
cultures, but rarely occur in film specimens.

3. Anthrax bacilliitThese may be detected with ease
and certainty, but they are never found in the blood
until it is too late to save the patient.

4. Tubercle bactli.—These are only present in very
scanty numbers and are very difficult to detect. The
diagnosis of miliary tuberculosis is to be made by other
methods, chiefly by that of exclusion.

5. The pueumococcus is found in severe cases of
pneumonia (probably it might be found in most cases
if a sufficiently large quantity of blood were examined)
and in septicemia and ulcerative endocarditis when
due to this organism. When found in the blood by
ordinary methods it always indicates a bad prognosis
and suggests the use of anti-pneumococcic serum. The
identification of the organism is easy and certain if they
are present in quantities sufficient for them to be found
in films.

6. Typhoid bacilli are present in the blood in all cases
of typhoid fever, but their isolation is difficult and the
diagnosis of the disease is made by other methods.

7. The bacillus of glanders may be found in acute cases
of that disease, but its isolation and identification are
matters for an expert.

8. The influenza bacillus is present in some, or, accord-
ing to some authorities, all cases of influenza. It may
be searched for in films, but no importance should be
attached to a negative result.

g. The bacillus of plague—This organism is often
present in the blood in relatively large numbers, and
the disease can usually be diagnosed after a careful
search through a number of suitably stained films. But
EXAMINATION FOR BACTERIA. I71I

the investigation of a drop of fluid drawn from the bubo
(if one is present) permits of an easier and earlier diag-
nosis. The blood examination is of most value in the
pulmonary and septicemic forms of plague.

10. The spivillum of relapsing fever is easily found, for
it possesses well marked characters and is present in
great numbers. The diagnosis of relapsing fever can-
not be made until it has been demonstrated.

11. The gonococcus has been found in the blood ina
few cases of ulcerative endocarditis. Its detection by
cultural methods is very difficult, and the services of
a bacteriological expert should be called in if the
characteristic cocci are not found in blood films in a
case in which the diagnosis of gonorrhceal ulcerative
endocarditis is probable, as further information upon
this point is greatly needed. We may point out that
ulcerative endocarditis, septicemia, &c., supervening in
the course of an attack of gonorrhcea are not necessarily
due to the gonococcus. Any pathogenic bacteria may
enter through the lesion of the mucous membrane
which the gonococcus has caused.

12. The bacillus coli is present in some cases of septi-
cemia.

EXAMINATION FOR BACTERIA IN FILMS.

This is the easiest method in which bacteria may be
found in the blood, and it does not require such a rigid
antiseptic technique as is necessary if cultures are to be
taken. The films are prepared and fixed in one or
other of the methods which we have described, the only
point worthy of notice being that the skin must be very
172 BACTERIOLOGICAL DIAGNOSIS.

thoroughly cleaned ; it may be scrubbed with soap and
a nail brush, using plenty of hot water. The films
need not be very thin and even.

The method of staining will depend upon the organ-
ism which is likely to be found, and more especially
whether it stains by Gram’s method. This is so impor-
tant in this connection that a repetition of a previous
table will not be out of place.

Gram’s MeETHop.

Stained. Unstained.
Streptococci. Typhoid bacilli.
Staphylococci. Bacillus of glanders.
Bacillus of anthrax. Bacillus of influenza.
Bacillus of tubercle. Bacillus of plague.
Pneumococcus. Bacillus coli.

Spirillum of relapsing fever.
Gonococcus.

If the organism which is present appears in the first
list the staining process is simply that which we have
described previously, and the organism will be stained
dark blue or violet and the other structures will be
unstained.

If the bacteria which are present in the films do not
stain by Gram’s method the matter is more difficult, for
any stain which colours them will colour the nuclei of
the leucocytes also. Jenner’s stain may be used, or
the film may be stained by eosin and methylene blue
separately. The organisms will then be stained blue.
Carbol-thionin is even more suitable, as the colour
which it imparts to the nuclei of the leucocytes is not
deep and the red corpuscles are merely tinged. This is
MALARIA. 173

the stain which we recommend for general use, and in
cases in which the nature of the organism (if one be
present) is entirely unknown.

If bacteria are detected by any of these methods
their nature must be recognised by a consideration of
their morphological features and staining reactions.

MALARIA.

The blood in a suspected case of malaria may be
examined fresh or in stained films. Of these methods
the former is the better and should be used if possible;
an examination of stained specimens should also be
made and is convenient, as it can be performed away
from the patient and at leisure.

Fresh films are made by touching a drop of blood on
the patient’s finger with the centre of a perfectly clean
cover-glass so as to remove an extremely small quantity
of blood. This cover-glass is then allowed to fall on to
a clean slide so that the droplet of blood may be spread
out by capillary attraction and by the weight of the
cover-glass, just as is the case in the method of making
blood films already described. But the slide is not
separated from the cover-glass; they are examined just
as they are, a ring of vaseline being painted round the
edge of the cover-glass to prevent evaporation.

The specimen is examined with a } in. objective, and
a place found in which the corpuscles are spread in a
single layer; this part is then searched thoroughly with
a 4; in. oil immersion lens. The parasites are seen as
pale irregularly-shaped bodies, with indistinct margins
which occupy the interior of the red corpuscles, and
174 BACTERIOLOGICAL DIAGNOSIS.

show ameboid movements of greater or less rapidity.
When the parasites are older they occupy a larger
space in the corpuscles, and there are granules of dark
pigment around their periphery. These granules are
often the first indications of the presence of parasites in
the examination of an unstained specimen. At a still
later stage the granules will be found in the centre of
the corpuscle (the hemoglobin of which is now almost
entirely removed), and the parasite will show segmen-
tation into a larger or smaller number of spores by lines
which have a radial arrangement and give the whole

Fic. 29.-—Malarial parasites in the blood. The dark area shows the
parasite as it appears when stained with thionin.

 

 

an appearance resembling that of a marguerite daisy.
These are only found when a rigor is imminent.

Crescents are found in the aestivo-autumnal form
of malaria; they are crescentic bodies with rounded
“horns,” and contain a ring of pigment granules in the
centre. They cannot be mistaken for anything else,
and if a single one is found it affords conclusive proof
that the patient has been infected with malaria.

Films for staining are made in the ways already
described, and must be thin and perfect. They may be
fixed by any of the methods we have recommended, the
alcohol-ether and the alcohol-formalin methods being
perhaps the best. They may be stained by Jenner’s
COLLECTION OF BLOOD. 175

stain, or by eosin and methylene blue used separately ;
the parasites are stained pale blue and the corpuscles
bright red.

A simpler stain is that recommended by Rees (Prac-
titioney, March, 1901) involving the use of carbol-thionin
prepared by dissolving 1°5 grammes of thionin in 10 c.c.
of absolute alcohol and 100 c.c. of a five per cent.
solution of carbolic acid. This is to be kept for at least
a fortnight and diluted with four times its bulk of dis-
tilled water immediately before use. Staining is com-
plete in about ten minutes. Ordinary carbol-thionin
answers very well indeed. Thionin stains the red
corpuscles a faint green, nuclei blue, and the parasites
an intense purple.

In a suspected case of malaria the search should not
be abandoned in less than half an hour, or, in the case
of an inexperienced observer, much longer.

A fuller description of the parasite and the differences
between the forms which are present in the various
forms of the disease is beyond the scope of this work,
and the reader is referred to the admirable special
number of the Practitioney mentioned above.

COLLECTION OF BLOOD FOR EXAMINA-
TION BY CULTURAL METHODS.

This is a much more difficult matter and should not
be attempted unless and until the film method has
failed. The difficulty arises from the abundant bac-
terial flora of the skin; unless the most thorough
antiseptic precautions have been taken the results are
absolutely useless. ‘They are worse than useless, they are
176 BACTERIOLOGICAL DIAGNOSIS.

misleading. A case of ulcerative endocarditis, for
example, might be due to streptococci, but might be
attributed to staphylococci on the strength of an inade-
quate bacteriological examination. ' For this reason we
are chary of recommending this method of diagnosis
in any hands other than those of an expert, and must
urge the practitioner not to attempt it unless he is
prepared to carry out the most thorough disinfection of
the skin.

Requisites:—1. A glass pipette such as has been
already described. It should be sealed at one end
while the other end should be left open and plugged
with a loose fitting plug of cotton-wool. The tube must
be sterile ; if it has been recently made (and we advise
the practitioner to make these pipettes for himself) this
will be the case; if it has been kept in stock for some
time it should be sterilised in the dry air steriliser as an
additional measure of precaution. The bulb of the
pipette should hold at least 1 c.c., and is to be com-
pletely filled with blood.

2. Needle.

3. Soap, nail brush, and hot water.

4. Some reliable antiseptic lotion, such as carbolic
acid (1 in 20), perchloride of mercury (1 in 1000), or
biniodide of mercury (1 in 500 in methylated spirit).

5. Alcohol. Absolute alcohol is best, but methylated
spirit will do.

6. Ether or turpentine ; the latter is not so good.

7. Spirit lamp or Bunsen’s burner.

Process—Scrub the skin, in the region to be pricked,
with the nail brush, using plenty of soap and hot water
for ten minutes; then dry the skin and wash it with
ether to remove the fat. Next paint on layer after layer
of the antiseptic lotion: it is much better to apply a wet
COLLECTION OF BLOOD. 177

dressing of a more dilute lotion for a few hours. The
next step is a very important one, and consists in the
thorough washing of the skin with methylated spirit or
rectified spirit; this must be continued until every trace
of the antiseptic has been removed.

The skin is then to be punctured with a sterile
needle; the stab must be a deep one and the blood
must flow freely. The first few drops which escape
must be rejected.

The end of the pipette is then to be inserted in the
drop of blood and placed as near the puncture as pos-
sible and the blood sucked very gently into the bulb;
great care must be taken lest air should gain access at
the same time, for it might contain bacteria which
might lead to erroneous conclusions. When the bulb
is completely filled both ends of the pipette are to be
sealed up.

A much better plan is to use a hypodermic needle
and to plunge it directly into a vein. The antiseptic
precautions are the same as in the former method, and
the syringe and needle are to be boiled immediately
before use. The vein is made prominent by a bandage
applied firmly above the seat of puncture, just as if
venesection were to be performed, and the needle thrust
obliquely through the skin. If this method is adopted
there is much less chance of contamination, and the
difficulties of the operation are certainly no less.

Undoubtedly the simplest and best of all methods is
that described by James and Tuttle (Report of the Presby-
tevian Hospital, New York, 1898). ‘‘A piece of glass
tubing 44 inches in length and + inch in diameter is
drawn out to a tapered end and ground to fit the cap of
a rather fine hypodermic needle. The larger end of the
tube having been stopped with a cotton plug, the whole

N
178 BACTERIOLOGICAL DIAGNOSIS.

is then placed in a larger tube and both ends of this
are similarly plugged with cotton* (fig. 29).

 

Fro, 30.—-Pipette for collection of blood for bacteriological
examination.

* These may be obtained from F. Ash, Edmund Street, Bir-
mingham.
COLLECTION OF BLOOD. 179

‘‘The apparatus is then sterilised by dry heat. In
using it the inner tube with needle attached is removed ;
the skin over one of the most prominent veins of the
anterior surface of the forearm, near the bend of the
elbow is selected, a piece of rubber tubing or a few
turns of a bandage being passed round the arm above
with moderate pressure, in order to produce distension
of vessels. The needle is then plunged into the vessel
and generally blood begins to flow by the blood-pressure
itself, but any quantity desired may be obtained by
making gentle suction either by applying the mouth
directly to the end of the tube where it is stopped with
cotton, or through the medium of a small piece of rubber
tubing slipped over it.

“« By the above instrument vein punctures have been
made in about 150 cases of a variety of diseases. At
no time was any difficulty experienced in obtaining the
amount of blood desired, which was generally about
ic.c. In a few instances it was necessary to try two
punctures before securing a free flow through the
needle; in no case was there any local reaction what-
ever at the seat of puncture, nor did the patient
complain of pain and annoyance.”

The following points are worthy of attention :—Select
for the puncture a vein which is superficial (as shown by
the blue colour appearing through the skin) not merely
prominent, for a deep vein will often slip in front of the
needle. Insert the needle very obliquely, with the point
directed away from the body. Lastly, remove the ban-
dage as soon as possible after withdrawing the needle,
or there may be a considerable amount of hemorrhage
into the subcutaneous tissues. If this should happen
place a pad of lint over the puncture and bandage the
arm from the wrist upward. No harm will result.

N 2
180 BACTERIOLOGICAL DIAGNOSIS.

A great advantage of this method is that it does away
with the necessity for any elaborate process for the
sterilisation of the skin. The authors quoted above
found that the results which they obtained were as good
after simple cleansing of the skin with soap and water
as after the use of antiseptic dressing. Nor is this to
be wondered at when we think of the very small chance
of any bacteria being carried through the skin and
subcutaneous tissues into a vein by a very fine and
sharp needle.

The advisability of employing some such method in
which the blood is drawn directly from a vein in place
of the simple skin puncture is very apparent from the
researches of Kihnau (‘“ Zeitschft. f. Hyg. and Infct.,”
1890), who made parallel series of experiments by the
two methods. He found that in cases in which the
blood drawn directly from the vein remained sterile,
growth (mostly streptococci or staphylococci) occurred
in as many as ninety per cent. of cultures inoculated
from skin punctures though the most careful antiseptic
precautions were used.

EXAMINATION OF BLOOD BY CULTURAL
METHOD.

This is a matter which is best carried out in a proper
laboratory by an expert. If the practitioner attempts
to carry it out for himself his best plan is to make a
series of inoculations on the surface of agar.* The tubes

* The ideal method is to make the inoculation directly into a large
quantity of broth and to make sub-cultures on agar later. This
method has the advantage that the blood becomes largely diluted,

so that any bactericidal substances which it may contain are less
likely to injure the bacteria. It is, however, rather more difficult.
EXAMINATION OF BLOOD. 181

are inoculated directly from the syringe, tube, or pipette
containing the blood, the pointed end being pushed
through the cotton-wool plug (the surface of which
must be previously sterilised by having its projecting
portion burnt off in the flame) and the blood allowed
to flow drop by drop on to the surface of the
medium, the tube being turned so that each portion
of the medium is covered by a film of blood. If the
tube method is used and the blood has been allowed
to coagulate the tip of the glass tube carrying the
needle should be broken off with a sterile pair of
forceps or pliers. The culture tubes are then to be
inoculated for twenty-four hours at the body tempera-
ture and examined. If they are sterile they are to be
returned to the incubator and examined after a further
period.

If colonies appear they are to be carefully examined
with a lens, and their characters noticed. The organ-
isms which will be most likely to develop are strep-
tococci, staphylococci, anthrax bacilli, pneumococci,
typhoid bacilli, the bacillus of plague, or the bacillus
coli; the gonococcus may also develop, for it will
obtain the hemoglobin necessary for the development
from the blood itself.

Streptococct form small white colonies which show no
tendency to run together to form a film. The centre of
each colony is more opaque than its periphery.

Staphylococct form a more or less uniform film, the
colonies extending laterally and fusing together. The
growth is opaque, and is of a dead white, lemon, or
orange colour, according to the nature of the staphylo-
coccus present (albus, citreus, or aureus).

Anthvax bacills form small white colonies having the
“« barrister’s wig’ appearance already described.
182 BACTERIOLOGICAL DIAGNOSIS.

The colonies of the pmewmococcus are small flat white
points which do not tend to fuse together. They are
difficult to see when they are young, and in case of
doubt the tube should be returned to the incubator.

The colonies of the typhoid bacillus and the bacillus colt
are whitish and opalescent. They usually have an
angular or polygonal appearance when small, and tend
to run together when older if they are thickly set.
Their discrimination must be left to an expert.

The bacillus of plague forms white colonies which are
circular or have a crenated outline; they tend to run
together and form a uniform film over the surface of the
medium.

The gonococcus, if it develops, forms very minute
transparent colonies which have been compared to
droplets of dew. They do not become confluent.
This organism will not grow if transplanted on to
the surface of ordinary media unless a thick film of
blood be previously spread over it.

After cultures have been obtained they are to be
examined microscopically by the method described on
page 29, and the morphological appearances compared
with those of the pathogenic organisms which we have
enumerated. It is especially important to test whether
the organism which has been isolated, stains by Gram’s
method or not.

SECTION CUTTING.

The methods employed in section cutting are some-
what outside the scope of this work, inasmuch as
sections are rarely necessary for the purposes of bacterio-
SECTION CUTTING. 183

logical diagnosis. The presence of bacteria in the
tissues can usually be demonstrated by the simple
processes of smearing the cut surfaces of tissues on
clean slides or cover-glasses, and treating the films thus
obtained by the fixing and staining methods previously
described. If, for instance, we have to search for
tubercle bacilli in tuberculous glands it is usually
sufficient to smear the cut surfaces of the glands on
a slide, dry, fix by heat, and stain in the same way as
sputum is stained for the tubercle bacillus. If anthrax
bacilli were being looked for in the liver or other
organ, removed post-mortem, the same method of pro-
cedure would be adopted, except that Gram’s method
of staining would be used. So also for typhoid bacilli
in the spleen, where the film would be stained with a
simple stain such as thionin or Léffler’s methylene blue.

It seems advisable, however, to give a short general
account of the processes involved in section cutting, for
they are by no means difficult, and do not require very
expensive apparatus. Further, the same methods of
section cutting are used for investigating the nature of
tumours, &c., and this is done already by many practi-
tioners and should be done by still more.

Slices of organs or tissues which are to be cut have
first to be fixed. The process of fixation consists essen-
tially in the application of some agent which brings
about coagulation of the component proteids with as
little distortion of the morphological elements as pos-
sible; if this step were not carried out the subsequent
processes would be liable to cause alterations in the
shape, size, and appearance of the cells and fibres.
There are two chief methods or fixation, that involving
the use of chemical substances, and that involving the use
of heat. The processes which are used in fixing the
184 BACTERIOLOGICAL DIAGNOSIS.

tissues hayden them at the same time; this is necessary,
for fresh tissue would yield before the sharpest knife
and could not be cut into thin sections. These processes
are always carried out, no matter what method of
section cutting is to be adopted.

In cutting sections it is necessary that the material
should be sufficiently fivm and homogeneous in consistency.
The former is secured to some extent by the process of
hardening, but a properly hardened block is rarely firm
enough to permit of its being cut into sections without
further preparation. Further, it almost invariably hap-
pens that some parts of the material are firmer or
harder than others; and if such a substance were cut
the harder parts might be sufficiently firm whilst the
softer parts would simply crumble before the knife.
There are two methods of overcoming this difficulty—
freezing and embedding.

The freezing process is very simple, and it is one which
can easily be carried out at home. The sections which
it yields are usually quite sufficient for purposes of
histological research (the diagnosis of tumours, &c.),
but they are rarely sufficiently thin for a proper demons-
tration of the bacteria which they may contain. The
sections are cut more easily by the freezing than by the
paraffin process, but they are decidedly more difficult
to manipulate.

In the freezing process the block or tissue after fixing
and hardening is dipped, or better soaked for some
hours, in a thick solution of gum arabic. It is then
placed on the plate of a microtome and frozen until the
tissue assumes the consistency of fairly hard cheese and
can be cut into thin sections.

The embedding process should be called the infiltration
process; the tissue to be cut is infiltrated throughout
FIXING MATERIAL FOR CUTTING. 185

with some firm substance and not merely embedded
therein. Two embedding materials are in general
use—paraffin and celloidin. The latter will not be
described, as it is only necessary for special work and
for ordinary purposes cannot compare with paraffin for
beauty of results and facility of application.

In the paraffin process the tissue is infiltrated through-
out with hard paraffin (such as is used for the better
varieties of paraffin candles) so that every cell and every
fibre is permeated and supported on every side. To do
this requires a number of processes. It would be of no
use to immerse the block of tissue in the paraffin just as
it is, for the paraffin would not wet it, much less soak
into it. The water is first removed; and this is done
by soaking the material in absolute alcohol. But
alcohol does not dissolve or mix with paraffin; it is
therefore necessary to remove it by means of some fluid
which will mix with it on the one hand and paraffin on
the other. Of these there are many; xylol, chloroform,
benzine, cedar oil, and many more are in use for special
purposes. Chloroform answers most purposes and is to
be generally recommended. The block of tissue is now
ready to be soaked in melted paraffin; it is kept in a
bath of this substance until the chloroform has been
entirely driven off and replaced by the paraffin. The
whole is then allowed to cool, is shaped into suitable
blocks, and is then ready for cutting.

We shall now describe the processes in fuller detail.

FIXING MATERIAL FOR CUTTING.

These processes must be understood by all practi-
tioners, even although they do not intend to cut sections
186 BACTERIOLOGICAL DIAGNOSIS.

for themselves. It happens to every medical man to
find it necessary to send tumours, &c. to a laboratory to
obtain a pathological diagnosis; and in very many
cases the materials are treated in a way which abso-
lutely prevents good sections from being obtained.
Many fixing fluids are in use, and any of them may
be selected, but it is absolutely necessary that the
material to be investigated should be cut into small
pieces and put into a large bulk of the fluid at once.
This is especially necessary in the case of material
removed at a post-mortem examination, where the
tissues and organs have already undergone alteration.

As regards the size of the slices which are to be
placed in the hardening fluid, it is sufficient to say that
they should never exceed } inch in thickness, and if
perchloride of mercury is used should be even thinner.
The other dimensions of the block are of less importance.

The bulk of the fluid in which the block is placed
should be at least twenty times that of the block, and it

wis not advisable to place two blocks in the same vessel.

The fluids which we shall recommend for this purpose
are i—

1. Perchloride of mercury in normal saline solution.
This is prepared by dissolving common salt in water
in the proportion of seven grammes to a litre (about 34
grains to the ounce), and saturating this solution whilst
hot with perchloride of mercury. The solution must be
allowed to cool completely; as it does so crystals of the
mercury salt will separate out.

This fluid fixes completely in twenty-four hours, or
less, and gives most excellent results. Its powers of
penetration are not very great, so that slices of tissue
which are to be fixed in it should be thin.

The after-treatment of the blocks fixed in this fluid
SECTION CUTTING. 187

must be described briefly. They are allowed to remain
in the solution for twenty-four hours and no longer, and
are then washed for twenty-four hours in running water
to remove the perchloride of mercury. They are then
passed through the various strengths of spirit (as will be
described subsequently), a little tincture of iodine being
added to each to remove any mercury which may still
remain. The other steps are the same as those which
are used if other methods of fixation have been
adopted.

2. Formalin. This should be used in a 5 per cent.
solution in water. It yields very good results, and is
perhaps the fluid which can be most warmly recom-
mended to a practitioner who is going to send his
material to a public laboratory.* The fluid has very
great powers of penetration, and the slices may be much
thicker than we have recommended. The one objection
to the fluid is that it interferes somewhat with the way
in which the sections stain.

3. Alcohol is not a very good fixing fluid, as it tends to
cause a good deal of shrinkage. When it is used the
blocks should be cut small and placed at once in undi-
luted methylated spirit.

SECTION CUTTING BY THE FREEZING
METHOD.

Sections which are prepared by the freezing method
are rarely as thin as those prepared by one or other of

* Formalin should not be used for tissues which are to be searched
for the tubercle bacillus, as it prevents the decolorising action of the

acid.
188 BACTERIOLOGICAL DIAGNOSIS.

the infiltration processes, but are prepared very rapidly,
and are often sufficient for diagnostic purposes, where
rapidity is the first consideration.

The blocks of tissue must be hardened before being
cut, any of the above fluids being applicable; where
alcohol is used it must be washed out in water, as it will
not freeze. Where more rapid work is required the best
method is a modification of the old boiling process, as
revived by Mr. Strangeways Pigg. The slices of tissue
from which sections are to be cut are thrown at once
into boiling water and allowed to boil vigorously for two
or three minutes; the water must be actually boiling
when the tissues are added, and the bulk used should
be large as compared with the block. The tissues are
then rapidly cooled by being thrown into cold water,
and are then ready for cutting. The outer surface of
the block should be rejected.

This method of fixation leads to a little distortion of
the tissues and alters any blood which they may con-
tain, but it is very good for diagnosing tumours. It is
invaluable in the post-mortem room, and for diagnosis
of the nature of a tumour during operation. In skilful
hands a section may be cut, stained, mounted, and a
diagnosis made in ten minutes.

A microtome is necessary for the successful cutting of
sections, and the Williams and Swift pattern are those
in general use for the freezing process. We shall
recommend the practitioner who intends to take up
this branch of work to procure a Cathcart microtome,
which is exceedingly cheap (it costs about a guinea)
and answers admirably. The great advantage of this
machine is that it will serve for cutting sections in
paraffin as well as for frozen sections.

The blocks of tissue which are to be cut are dipped
SECTION CUTTING. 189

in a thick and syrupy solution of gum arabic; if time is
no object it is a great advantage to soak them in this
for several hours. A block is then placed on the
corrugated plate of the microtome and frozen by means
of the ether spray which impinges upon it. When the

qu on |
PUTO

 

ih

 

ii ——__—
iii

Fic. 31.—Cathcart’s microtome arranged for cutting frozen sections.
3. 8 8

mass is nearly frozen a section is taken off by means of
a razor which is ground flat on one side, or the special
knife which may be obtained with the apparatus: it is
better to moisten the upper surface of the knife with a
Igo BACTERIOLOGICAL DIAGNOSIS.

little of the gum. The section is carefully removed
with a camel’s hair brush and placed ina large vessel of
clean water so that the gum may be dissolved out of it,
and is then ready for staining. The block is then raised
by means of a very slight turn of the large milled head
under the apparatus and another section cut.

The mass must not be frozen too hard; if this has
been the case the necessary thawing will be hastened
by gently breathing on the block. If it begins to thaw
a few squeezes of the bellows will bring it to the proper
consistency.

STAINING AND MOUNTING FROZEN
SECTIONS.

These processes are best carried out in watch-glasses.
No attempt will be made to describe the methods by
which frozen sections may be stained for the purposes of
bacteriological research, for they are not so suitable as
paraffin sections for this purpose. We shall describe
the process which would be adopted if a rapid diagnosis
were required at an operation or in the post-mortem
room. The sections are to be stained in hematoxylin
(with or without eosin as a counterstain) and mounted-
in balsam.

The vequisites are:—Five watch-glasses containing
respectively hzmatoxylin, watery solution of eosin
{about one per cent.), alcohol (50 per cent.), absolute
alcohol, and clove oil; a saucer or other vessel contain-
ing water to which a few drops of ammonia have been
added; several strips of thin writing paper, each about
one inch wide and two inches long; some needles,
STAINING AND MOUNTING. Igt

which may be mounted in handles; slides, cover-
glasses, and balsam.

A section is to be removed from the bowl of water in
which it is floating by means of one of the strips of
paper; this must be inserted under it, and the section
“‘pinned” in place upon by one of the needles. A
special section-lifter may be used, but is not so good.
It is then transferred to the watch-glass containing the
hematoxylin solution, and the staining process is
allowed to go on for a minute or two, a fresh section
being manipulated whilst it is taking place. The first
section is then removed in the same way as before and
placed in the water containing the ammonia; it soon
turns blue, and when this is the case it is ready to be
transferred to the eosin, then into the dilute alcohol,
the absolute alcohol (where it should remain for a
minute or more) and finally into the oil of cloves. It is
then ready to be mounted in balsam. A convenient
way in which a section can be transferred to a slide is
as follows :—The section is carefully spread out whilst
in the oil of cloves, two needles being used for the pur-
pose, and a slip of paper insinuated beneath it. This
strip of paper is then drawn slowly out of the liquid,
and any folds or creases which may be in the section
straightened out with the needles, the excess of the oil
of cloves being allowed to drop off whilst this is taking
place. The strip of paper is then inverted (the section
remaining adherent to the under surface) and placed
upon a clean slide and pressed firmly upon it; the
pressure squeezes out the greater part of the oil so that
the section adheres to the slide and the paper can be
stripped cautiously from it. A drop of balsam is then
applied, the section covered with a cover-glass, and
examined under the microscope.
192 BACTERIOLOGICAL DIAGNOSIS.

Where time permits it is a great advantage to rinse
the section in distilled or clear rain water after re-
moving it from the hematoxylin.

The solution of hematoxylin is best bought ready
made, as its preparation is somewhat difficult. Dela-
field’s solution is about the best for general work.

A counterstain is not really necessary for diagnostic
purposes, and its omission hastens the process some-
what.

THE PARAFFIN PROCESS.

Tissues which are to be cut in paraffin may be hard-
ened in any of the fluids mentioned above. They are
then dehydrated, cleared in chloroform or other fluid
which mixes with alcohol and dissolves paraffin, and
finally soaked in a mixture of hard and soft paraffin
kept just at the melting point. This paraffin should
be obtained specially for the purpose; the Cambridge
paraffin is the best. It is made in two varieties, the
soft, which melts at 48° C., and the hard, which melts
at 55° C. The amounts of each which should be used
for embedding depends upon the external temperature ;
in very hot weather hard paraffin may be used alone,
while under average circumstances a mixture of equal
parts of each is best.

We shall now proceed to describe the various pro-
cesses seriatim.

DEHYDRATION.

This is very simple. The blocks of tissue are placed
in weak spirit for a few hours or for a day, then
INFILTRATION WITH PARAFFIN. 193

changed into stronger spirit and so on until absolute
alcohol is reached. The slower this process is carried
out the better will be the results; in practice the
strengths of the successive lots of spirit used may be
40 per cent., 75 per cent., and the strong methylated
spirit, and the block may remain in each for twelve
hours. Lastly, it goes into two successive lots of
absolute alcohol.

In all cases the amount of fluid must be greatly in
excess. It is useless merely to cover the block with the
spirit.

CLARIFICATION.

In the next step the alcohol is removed from the
tissue and replaced by some fluid which will dissolve
paraffin. Fats are dissolved out from the tissues at
this stage.

This step is also very simple. The blocks are passed
directly from absolute alcohol into chloroform and
allowed to remain there until they appear translucent.
It is not necessary to use a preliminary bath of a
mixture of alcohol and chloroform.

It is a good plan to place the bottle containing the
block in a warm place with the cork out for an hour or
so before proceeding further, as by so doing the last
traces of the alcohol will be removed.

INFILTRATION WITH PARAFFIN.

This is the stage which presents most difficulties to
the home-worker, for it is necessary to keep the block
oO
194 BACTERIOLOGICAL DIAGNOSIS.

of tissue soaked in paraffin which is just melted for at
least twelve, and more often twenty-four hours. To do
this properly involves the use of some sort of an incu-
bator. This might possibly be rigged up out of a tin
biscuit-box in the manner already described, though
considerably more heat would be necessary, as the
paraffin melts at about 50° C. But the writer has often
embedded the blocks by placing them in bottles con-
taining the paraffin at such a distance from the fire that
the paraffin is never completely melted, but always
shows a thin solid layer on the surface. To do this it
is only necessary to look at the bottle occasionally and
move it a little further from the fire if the paraffin is
completely melted and vice versé. ‘The process may be
stopped at night without any harm resulting, and if the
soaking only continues for a few hours at a time it is of
no consequence so long as the total period is made up.

CASTING THE BLOCKS.

Special metal moulds are used in the laboratory
(fig. 31). A pill box will do quite well. A small amount
of melted paraffin is poured into the box and the piece
of tissue is taken from the bottle containing the melted
paraffin with a pair of forceps (previously warmed so as
to prevent the paraffin from setting upon the points) and
placed in the paraffin in the pill box. It is necessary to
see that the surface from which’ sections are to be cut
should be placed downwards. The box is then filled
up with melted paraffin, and placed in a cool place or
surrounded with water. The moment a firm film has
formed over the surface the whole is plunged in cold
water to hasten the setting of the paraffin; the more
SECTION CUTTING. 195

rapidly this takes place the better will the block cut.
When the paraffin mass has hardened completely
throughout it is trimmed into shape, taking care
that the edges of the surface which is to be cut are
accurately parallel.

 

Fic. 32.—L-shaped moulds for embedding in paraffin.

CUTTING THE SECTIONS.

For cutting sections in paraffin no microtome can
be compared with the Cambridge Rocker, but very
excellent results can be obtained by the use of the
Cathcart microtome already mentioned. The paraffin
block containing the piece of tissue is mounted on
the freezing plate of the microtome (which must be
heated and the lower surface of the block pressed
upon it) and the sections cut in the manner -de-
scribed; a very sharp knife is essential and the
stroke must be quicker and sharper than is the case
when frozen sections are being cut. In another form of
the microtome a special inner tube is provided for
cutting sections by the paraffin process. The blocks are

02
196 BACTERIOLOGICAL DIAGNOSIS.

retained in place by a clamp and appear in the same
position as that occupied by the mass of frozen gum; as
the paraffin is not sufficiently hard to be gripped by this
clamp they must first be mounted on a piece of wood of
a suitable size and shape. This can be cut out of a
piece of firewood, and should have one surface left
rough; this surface must be dipped in melted paraffin,
and the under surface of the block partially melted in
the flame and pressed firmly upon it. The piece of
wood is then to be placed in the jaws of the clamp and
the screw tightened up.

 

Fic. 33.—Clamp for holding wooden block with the paraffin block.

In the Cambridge Rocker and in some other forms of
microtomes the sections adhere to one another at the
edges and form long ribbons as they are cut. In the
Cathcart microtome this is not the case, and each
section must be dealt with separately; it is to be
removed carefully from the knife blade with a camel’s
hair brush or a finely pointed pair of forceps and placed
upon the surface of a bowl of water just hot enough to
warm the paraffin without melting it. When this is
done the sections will spread out and lose all the creases,
and are then ready to be mounted on slides or cover-
glasses.
SECTION CUTTING. IQ7

It often happens that the sections roll up on the
knife. In this case they must be placed on the surface
of cold water and an attempt made to straighten them
out by careful brushing with a camel’s hair brush; when
fairly flat they are to be lifted up on a slide or piece of
paper (dipped into the water’ and insinuated below
them) and transferred to the hot water as before. But
the rolling of the sections may often be prevented by
sharpening the knife, by re-imbedding the tissues in
harder or in softer paraffin according to the weather, or
by varying the angle which the knife edge makes with
the glass runners of the microtome. These devices can
only be learnt by experience.

When the sections are flattened out on the surface of
the hot water they are ready to be mounted upon slides
or cover-glasses; slides are by far the best for beginners.
The slides (or cover-glasses) must be perfectly clean,
and are best kept in methylated spirit until they are
to be used, and the spirit not wiped off. Each slide is
then inserted separately into the water in an oblique
position and the section moved until it lies over the
centre; the slide is then raised out of the water and
carries the section out with it.

The excess of water is now to be removed by a piece
of blotting or filter paper, and the slide placed in the
warm incubator for a few hours. At the end of this
time the sections will adhere by atmospheric pressure
(like a boy’s leather sucker to a stone) and will not
come off in the subsequent processes. If an incubator
is not at hand the slides may be placed near the fire
(protected from dust) and kept at the body temperature
or a little higher for a few hours; the exact temperature
does not matter, and no harm will result if the paraffin
melts.
198 BACTERIOLOGICAL DIAGNOSIS.

In the older methods of fixing sections to the slides
various forms of cements had to be used, and were a
great disadvantage. They are quite unnecessary except
for sections of the central nervous system; if these are
being dealt with the slide must be coated with a very
thin layer of a solution of egg-albumen in water before
the section is laid upon it. The process is then exactly
the same as before.

STAINING AND MOUNTING PARAFFIN
SECTIONS.

We will suppose that the sections have been cut,
flattened out on hot water, and caused to adhere to
slides, and shall describe in general terms the steps
through which they must be taken before they are
ready for examination. In the first place, it is obvious
that the paraffin which permeates all parts of the section
and surrounds it on all sides must be removed; and this
is done by pouring xylol, benzine, or turpentine upon it.
At least two supplies of the fluid should be used, and it
should be allowed to act for at least two minutes, the
slide being rocked all the time. We have now removed
the paraffin, and the next step is to remove the xylol or
other solvent; this is done by means of absolute alcohol.
At least two lots should be used, and it should be
allowed to act for two minutes. The slide is then
washed in water, and is ready for staining. When
the section is wet with xylol it will be quite transparent;
this is because the refractive index of the xylol is almost
the same as that of glass and the rays of light which
come through the section are not bent. But when the
STAINING AND MOUNTING. IgQ

alcohol is added the section will suddenly become
opaque, and for the opposite reason.

If there is a milkiness on the section or slide when
the water is poured on it is a sign that the xylol has not
been completely removed; xylol will not mix with
water and forms an emulsion with it. If this should
happen you must give the section another dose of abso-
lute alcohol.

It is an advantage to wipe the surface of the slide (of
course avoiding the section) before going from one fluid
to another.

A cardinal rule in dealing with paraffin sections is
never to let the section get dvy from the moment the first
dose of xylol is added until the final mounting in
balsam.

The methods of staining which are in use are legion,
and it would be far beyond the scope of this book to
describe even a few of those which are used in histo-
logical work and to give indications for their use. It
will be sufficient to describe (1) a method suitable for
the diagnosis of tumours, &c., and for ordinary histo-
logical purposes, (2) a method of staining to demon-
strate bacteria which stain by Gram’s method, (3) a
method for bacteria which do not stain by Gram’s
method, and (4) the process for demonstrating tubercle
bacilli in the tissues.

(1). Staining sections for histological purposes :—

1. Xylol, two lots (to remove paraffin).

2. Absolute alcohol, two lots (to remove xylol).

3. Water (to remove the alcohol).

4. Stain with hematin (or hematoxylin) for ten
minutes or more according to the nature of the speci-
men and the condition of the stain. The exact length
of time can only be learnt by trial, but ten minutes will
be about right. Rinse in distilled water.
200 BACTERIOLOGICAL DIAGNOSIS.

5. Wash thoroughly in tap water, continuing the
washing until the sections have a decidedly blue tinge.
The hematoxylin compounds are very much like litmus,
being red in presence of acids and blue in presence of
alkalies; the sections are to be coloured blue, and the
necessary alkali is contained in the tap-water. It will
hasten the process to rinse them in a very dilute solu-
tion of ammonia.

6. Stain in watery eosin for a minute or so. This is
the counterstain. The hematin will stain all nuclei blue,
but will scarcely tinge anything else; the eosin is added
to stain other structures a pale pink and thus make
them more visible. It stains almost instantaneously.

7. Wash off the eosin under the tap.

The sections are now stained. But they are opaque
and not in a suitable condition to be examined under
the microscope, and are to be rendered transparent by
being mounted in balsam. Now this cannot be done in
the same way as was used in the mounting of films, for
the drying would cause the sections to shrivel and
obscure their structure. The water is to be removed,
it is true, but by the use of absolute alcohol; at least
two lots should be used, and the slide rocked. from time
to time. Then the alcohol (which will not mix with
balsam) is to be removed by the use of xylol, balsam
added, and the section covered with a cover-glass.
The remaining steps are therefore :—

8. Absolute alcohol, two lots (to dehydrate).

g. Xylol, two lots (to render the section permeable to
balsam).

10. Balsam and a cover-glass.

The last three steps are practically the same as the
first three, but in the reversed order, and similar pheno-
mena are seen. The section is opaque whilst wetted
STAINING AND MOUNTING. 201

with the alcohol and becomes transparent when the
xylol is added ; and this transparency is the proof that
the steps have been carried out properly. If the section
looks opaque when held against a perfectly dark back-
ground an additional dose of alcohol must be used, and
the xylol applied again.

Hematoxylin may be used in exactly the same way
as hematin in the above process; it stains more quickly,
but does not give quite such beautiful results.

(2). Gram’s method as applied to sections; suitable
for sections of diphtheritic membrane, organs containing
anthrax bacilli, streptococci, staphylococci, &c.

1. Xylol, two lots.

2. Absolute alcohol, two lots.

3. Water. These steps are always the same with
paraffin sections, no matter what stains are to be used
subsequently.

4. Aniline gentian violet—five minutes.

5. Gram’s iodine solution—three minutes or more.

6. Absolute alcohol or methylated spirit—snttl no more
colouy comes out, This step is best carried out as fol-
lows :—Hold the slide by one end, keeping the fingers
clean by using a duster or pair of dissecting forceps,
and pour a little spirit on the section; rock it gently
from side to side and notice the clouds of colour which
it takes up. After a little time pour off the spirit and
add a fresh lot; repeat the rocking, and pour off again.
Do this until the spirit comes away quite clean and
does not take up any colour from the section. This
may take a long or a short time, and no definite rules
can be laid down.

In some cases decolorisation can be carried out best
by the use of clove oil. This is applied when the spirit
is wet with absolute alcohol (for it will not mix with
202 BACTERIOLOGICAL DIAGNOSIS.

water) and must be entirely removed with the same
fluid before the section is mounted, or it will cause it to
fade. Clove oil is a very powerful decolorising agent,
and requires careful use, or the colour may be removed
from the bacteria.

7. Eosin—half a minute or more. This is a counter-
stain, and is used to demonstrate the structural elements,
which are not coloured by the gentian violet. It may
be omitted in some cases.

8. Absolute alcohol—two lots. To remove the water.

g. Xylol—two lots, or until the section becomes trans-
parent.

10. Balsam and a cover-glass.

This method of staining is very easy of application,
and the results are exceedingly beautiful; bacteria
which take the stain are coloured blue or violet, and
actively-dividing nuclei and keratin are stained in the
same way, while all other structures are stained
pink.

(3). Method for bacteria which do not stain by
Gram’s method ; suitable for sections of typhoid ulcers,
lymphatic glands containing plague bacilli, &c.

The problem before us in this case is not at all easy
of solution. Inthe first place, the stains which colour
the bacteria also colour the tissues, especially the cell-
nuclei; the bacteria are easy to stain, but it is difficult
to stain a section in which there is good differentiation.
In the second place, the stains which are used for
bacteria are all soluble in alcohol; but alcohol is used
to dehydrate the sections. The following method will
be found to serve fairly well in most cases, though it
requires a certain amount of practice for its successful
accomplishment.

I, 2, and 3. Xylol, alcohol, and water, as before.
STAINING AND MOUNTING. 203

4. Stain in carbol-thionin for ten minutes or quarter
of an hour.

5. Wash in running water for ten minutes or longer.
This removes the stain from the tissues before decolor-
ising the bacteria, and a fairly differentiated specimen
may be obtained if the processes of staining and wash-
ing are carried out for suitable lengths of time.

Unna’s polychrome methylene blue may be used in a
similar manner, and gives even better results. The
staining should be continued for about ten minutes and
decolorisation effected by very short immersion in dilute
acetic acid (about 4 per cent.) followed by a good wash-
ing in pure water.

6. Remove as much water from the section as you
can without actually drying it by the cautious use of
clean blotting paper. Then apply anilin oil until the
section becomes perfectly translucent. Anilin oil mixes
with water on the one hand and xylol on the other and
can be used for dehydration just as alcohol was; the
process is slower and several lots of the oil must be used.

7. Wash off all the anilin oil by successive applications
of xylol. The permanence of the preparation will depend
on the thoroughness with which this step is carried out.

8. Balsam and cover-glass.

(4). Staining sections to demonstrate the tubercle
bacillus. Applicable to the leprosy bacillus also.

1, 2, and 3. Xylol, alcohol, and water, as before.

4. Carbol-fuchsin heated until the steam rises for five
minutes or longer, care being taken that the section
does not dry up. Or the slide may be immersed in the
stain and kept in a warm place for twenty-four hours.

5. Dilute sulphuric acid until only a faint pink tinge
appears after washing. This will generally require an
immersion of ten minutes or more.
204 BACTERIOLOGICAL DIAGNOSIS.

6. Methylene blue for three or four minutes. Some
of the stain comes out in the alcohol, so that the section
must be stained more deeply than will be required
ultimately.

8. Rinse off the blue stain in water, and then remove
the greater part of the latter with blotting-paper; this
is to render the dehydration more rapid.

g. Absolute alcohol—two lots in rapid succession.

10. Xylol.

11. Balsam and cover-glass.
APPENDIX. 205

APPENDIX.

I.
The following tables may be useful if stains, &c.,
have to be prepared when the metric weights and mea-
sures are not at hand. The equivalents given are not

absolutely accurate, but are sufficiently near for most
purposes.

1 Litre. i 352 fluid ounces.
too C.C. . : 3% fluid ounces,
1 Gramme ‘ 15°43 grains.

1 Fluid ounce . 28'4 C.c.

1 Fluid drachm 3°5 C.C.

1 Grain. ; ‘064 gramme.

To convert grammes per litre into grains per ounce
multiply by the factor ,4. Thus 10 grammes per litre -
= 10 X 7 = 4'4 grains per ounce.

To convert cubic centimetres per litre into minims
per ounce multiply by the factor 42. Thus 25 c.c. per
litre = 25 x 32 = 12 minims per ounce. (A close
approximation is given by multiplying by 4.

To convert degrees Centigrade into degrees Fahren-
heit multiply by the factor 2 and add 32.

II.

NEISSER’S METHOD OF STAINING THE DIPHTHERIA
BAcILLus.

The following method has been placed in the Appen-
dix as there is some doubt as to its value. It isa
206 BACTERIOLOGICAL DIAGNOSIS.

method by which the so-called polar bodies are stained
with methylene blue, while the rest of the organism is
coloured a faint yellowish-brown with Bismarck brown.

Cultures should be made on blood-serum and should
not be less than nine nor more than twenty-four hours
old. Films are spread in the ordinary way and stained
for half a minute in

Methylene blue . 2 I gramme.
Alcohol (96%) . f. “ZO:
Glacial acetic acid . 50C.Cc.
Water : : + 950 C.c.

They are then washed and treated for half a minute
with
Bismarck brown. : 5 grammes.
Water ; ; . 1000 C.c,

The polar bodies are small spheres which are con-
tained in the bacilli, there being usually two in each
bacillus, one at each end. In a film specimen of the
true diphtheria bacillus stained in this way they appear
as very minute dark blue or black dots, which may
easily be mistaken for cocci; the bodies of the bacilli
are often almost invisible. According to some authori-
ties the presence of these granules in young cultures
of bacilli which present the morphological characters
of the diphtheria bacillus, is proof of their virulence,
whilst their absence proves the cultures to be of the
non-virulent ‘‘pseudo-diphtheria” bacillus. The method
can also be applied to films made directly from the
swabs, and recent researches seem to prove that the
results thus obtained are of considerable diagnostic
value.

If this method be adopted the films must be very
APPENDIX. 207

carefully searched, as it often happens that character-
istically stained bacilli may be seen in one part of the
field, while they are entirely absent elsewhere. This
is especially true of films made direct from the swabs.

The author is inclined to attach very considerable
importance to a positive result obtained with this method
of staining, but would not consider a negative result
as indicating the absence of the diphtheria bacillus.
INDEX.

Abscesses
cold
Acid-fast bacilli
Acid stains
Achorion Schénleinii
Actinomycosis
Adamson
Agar
Anaérobic cultures
Aniline gentian violet .
Anthrax -
bacillus of
cultures
in blood .
Arthritis
Ascitic agar .

Bacillus, acid-fast
anthrax
coli communis
diphtheria
glanders
influenza ‘
leprosy
plague
smegma
soft sore
tetanus .
tubercle

Basic stains

. 74, I2I, 122, 125, 133, 203

PAGE
136
139

74

166
II7

80

113

19

57

38

65

, 66
69, 181
181

124
124

74
66, 181

83, 171, 182
51, 205

82

64

79

107, 182

75

107

57

166
P
210 BACTERIOLOGICAL DIAGNOSIS.

Blood .
bacteriology of
films . :

fixation of
staining of

Blood-serum :

Bottles, sterilisation ot

Broth .

Capsules
Carbol-fuchsin
thionin
Cathcart microtome
Cerebro-spinal fever
fluid
Chancroid
Cholera
red .
Cleaning slides
hemocytometer
Cornet’s forceps
Corpuscles, red
white :
Cotton-wool, sterilisation of
Cover-glasses, cleaning
films on
Crescents, malarial
Cultivation of bacteria

Dehydration of tissues
Diphtheria :
bacillus of
Diplococcus of gonorrhea
pneumonia .
Still’s . 3
Weichselbaum’s
Dry heat, sterilisation by

Eczema marginatum
Ehrlich’s stain

PAGLE

Iq1

169, 171, 174
160

é 164
166, 171, 174
20

9

13

130, 134
129
10g
105
107

39

154

31

142
152

Io

39

31, 162
174

12

192
43

51, 205
99

62

132

130, 132
7

116
166
INDEX.

Embedding
Empyema
Endocarditis
Enumeration of bacteria
red corpuscles
white corpuscles
Eosin . -
Eosinophile substances
Examination of cultures
Exudates, collection of

Feces, tubercle bacilli in
Favus
Film preparations
Films, staining of
Fixation of films .
tissues
Forceps, Cornet’s
Formalin
Fracture of skull .
Freezing microtome
Frozen sections
Fuchsin

Gelatin

Gentian violet

Glanders

Glucose agar
Gonococcus

Gonorrhea . i
Gowers’ hemocytometer
Gram’s method

Hematoxylin
Hemocytometer
Hemoglobinometer
Hanging-drop preparation
Heart-blood, collection of
Hewlett

Hoffman’s bacillus

2I1
PAGE
184

: 121
51, 169
17

142
152

38

166

29

118

77

117

: 29, 160
31, 166, 172, 174
30, 164

183, 185

: 31
Io, 165, 187
134

184

190

38, 166

15

38

32, 170

» 20, 57
103, 171, 182
99

155

32, 172, 201

192
142
155
89
42
49
53
Pee
212 BACTERIOLOGICAL DIAGNOSIS.

Immersion lens, focussing
Incubation of cultures

Indol

Influenza ‘ ,
Inoculation of culture tubes
Instruments, sterilisation of
Intermittent sterilisation
Todine solution

James
Jenner’s stain
Joints, examination of

Kerion .
Kithnau

Large spored ringworm
Lenharz

Leprosy

Leucocytes
Liquefaction of gelatin
Liver, examination of
Loffler’s blue

Lumbar puncture
Lymphocytes

Malaria
Malignant pustule
Media, culture
Meningitis
Meningeal hemorrhage
Methylene blue
Microscope .

use of
Microsporon Audouini
Microtome
Milk, tubercle bacilli in
Morris . .

130, 134

37, 166

PAGE

36

25

; 107
64, 170
22

10

15

39

177
167
124

117
180

15

133

79

152, 167
16, 138
152

37

126

129

173
65

13

134

34
I14
188

77
112
INDEX. 213

PAGE

Nasal secretion in leprosy 79
cerebro-spinal fever 132

Needles, platinum 22
Neisser’s method of staining diphvhedia bacill 205
Oliver’s heemoglobinometer 157
Otitis media 61
Paraffin, embedding in 192
Perchloride, fixation by 186
Petri dishes 10, 70; 137
Phthisis : 77
Plague 107, 170, 182
Plate cultures 16, 70, 137
Pipettes . 40
Platinum needles 22
Pleurisy 7 ‘ ; : . 120
Pneumococcus . 60, 121, 130, 134, 160, 182
Pneumonia 60
Polar staining 108
Polar bodies 206
Polymorphonuclear leucocyte 129
Post-mortem examinations 140
Pus 135
Ray fungus . 81
Relapsing fever 171
Ringworm , ir
of beard region 117

Section cutting , 182
staining Igo, 198
Septiczemia . . 61, 169
Slides, cleaning 39
Small spored ringworm 114
Soft sore 10g
Spirillum of cholera 105
relapsing fever 171

Spores 57) 67
Sputum, influenza 64
214 BACTERIOLOGICAL DIAGNOSIS.

PAGE

Sputum, pneumococcus . 61
tubercle . 3 ‘ 75

Stab cultures i : . 18, 24
Staining blood films ‘ : ; 166
for bacteria : é oe 172

malarial 7 ei ‘i 174

Stains : . ; , : - 37, 166
Staphylococci in blood : : 2 ; 10g
cultures of “ ‘ j : 181

in empyema . ; : ; 122

in meningitis si : F : 130

in pus ‘ ‘ ‘ : 169

Steam sterilisation . . ‘ 3 : 10
Sterilisation : : ; . Io
by dry heat : : : 5 7

by steam ‘ ‘ : Io

intermittent , : . ‘ 15

of skin s 3 ; : 176
Strangeways Pigg . A 5 ‘ 188
Streptococci in blood : : : ‘ 109
cultures of : ; : 181

in joints : ; . : 125

in meningitis . : 130

in pleurisy . . : 121, 122

inpus . . : : 3 181

Stroke cultures. : : 18, 24
Swabs 3 5 : 45
Test-tubes, sterilisation of . F Io
Tetanus ; : 55
Thionin . ; ‘ 38, 175
Thoma : a ‘ : 5 , 142
Tinea circinata . : : : , 116
Trichophyton ectothrix ‘ : 116
endothrix . 5 8: 115

Tubercle 6 : 72
bacillus : z ; + 74

in blood : : : : 170

in empyema . ‘ ‘ 122

in faeces . : : : . 77
INDEX. 215

PAGE
Tubercle in joints 125
in meningitis 133

in milk . 77

in pus 76, 139

in urine 76

pleurisy 121

Tuttle : 177
Typhoid fever 83
Ulcerative endocarditis ‘ 2 61, 169
Widal’s reaction ‘ : , ‘ a 85
macroscopic method . F 94

microscopic method . 87
A Classified Catalogue of
Books on Medicine and the
Collateral Sciences, Phar-
macy, Dentistry, Chemistry,
Hygiene, Microscopy, Etc.

ut

P. Blakiston’s Son & Company, Pub-
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No. 8. 12-13-01.
SUBJECT INDEX.

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SUBJECT. PAGE
Alimentary Canal(seeSurgery) 19
Anatomy.

Anesthetics
Autopsies (see Patho! ogy) eat
Bacteriology (see Pathology).. 16
Bandaging (see Surgery)........
Blood, Examination ot
TAIN .scccrereccsssssrae cones
Chemistry. Physics
Children, Diseases of ..
Climatology.
Clinical Chart
Compends. eae
Consumption (see Lungs’
Cyclopedia of Medicine..
Dentistry ......ccsecsseceee ae
Diabetes (see Urin. Organs).. 21

 

 

 

   
 

 

SUBJECT. PAGE
Milk Analysis(see Chemistry) 4
Miscellaneous ....csescssessseeee 14

Nervous Diseases
Nose.....

  
 
 
 
  
    
 
 

noe: 20
ursing . 15
Obstetrics, 16
Ophthalmology.. 9
Organotherapy .... 14
Osteology (see Anatomy) 3
Pathology 16
Pharmacy... 16
Physical Diagnosis . 6
Physical Training 12
Physiology ...... 17
Pneumotherapy. 14
Poisons (see Toxicology) . 13
Practice of Medicine... 18
Prescription Books.. 18

Refraction (see Eye).
Rest...
Rh

 

 

 
  
 
  
 
 
 
 

Diagnosis.........00006 te:
Diagrams (see Anatomy) 3
Dictionaries, Cyclopedias. 8
Diet and Food. anise ka:
Dissectors.... mec
Ear 9
Electricity 9
_ Embryology. 3
Emergencies...... 19
Eye..... 9
Fevers. 9
Food 14
Gout .. 10
Gynecology . 21
Hay Fever, 20
Heart Io
Histology.. 10
Hydrotherapy. 14
Hygiene.... 11
Hypnotism 14
Insanity ... 4

Intestines (see Miscellaneous) 14
Latin, Medieal (see Miscella-
oT 4y 16

neous and Pharmacy).
Life Insurance..

   
   

Medical Jurisprudence..

 

 
   

Sanitary Science...
Spectacles (see Eye) ie
Spine (see Nervous Diseases) 14
Stomach (see Miscellaneous)... 14
Students’ Compend...........22, 23
Surgery and Surgical Dis-

. 19

   
     
  
 
 

Technological Books.

4
Temperature Charts..... 6
Therapeutics. . 12
Throat . 20
Toxicology 13

Tumors (see Surgery’ ee
U.S. Phe cee
Urinary Organs
Urine .....ceeceee

Veterinary Medicine.. os
Visiting Lists, Physicians’.
(Send for Spectal Circular.)
Water Analysis...
Women, Diseases

 

Self-Examination for Medical Students. 3500 Questions on
Medical Subjects, with References to Standard Works in which the
correct replies will be found. Together with Questions from State

Examining Boards. 3d Edition.

Paper Cover, 10 cts.
SUBJECT CATALOGUE OF MEDICAL BOOKS. 8

 

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MORRIS. Text-Book ot Anatomy. 2d Edition. Revised and
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“‘ The ever-growing popularity of the book with teachers and students

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BROOMELL. Anatomy and Histology of the Human Mouth

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CAMPBELL. Dissection Outlines. Based on Morris’ Anatomy.
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DEAVER. Surgical Anatomy. A Treatise on Anatomy in its
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MANN. Manual of Psychological Medicine and Allied
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SMITH. Electro-Chemical Analysis. 2d Ed. 28 Illus. fice
6 SUBJECT CATALOGUE.

 

SMITH AND KELLER. Experiments. Arranged for Student
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SUTTON. Volumetric Analysis. A Systematic Handbook for
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MEDICAL BOOKS. 9

 

EAR (see also Throat and Nose).

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10 SUBJECT CATALOGUE.

 

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STOHR. Histology and Microscopical Anatomy. Edited by
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MEDICAL BOOKS. 11

HYGIENE AND WATER ANALYSIS.

 

Special Catalogue of Books on Hygiene sent free upon application.

CANFIELD. Hygiene of the Sick-Room. A Book for Nurses
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CONN. Agricultural Bacteriology. Illus. /ust Ready. $2.50
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PARKES. Hygiene and Public Health. By Louis C. Parkes,
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PARKES. Popular Hygiene. The Elements of Health. A Book
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MEDICAL BOOKS. 18

 

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MEDICAL BOOKS. 15

 

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16 SUBJECT CATALOGUE,

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PATHOLOGY.

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BLACKBURN. Autopsies. A Manual of Autopsies Designed for
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MEDICAL BOOKS. 17

 

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2
18 SUBJECT CATALOGUE.

 

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MEDICAL BOOKS. 19

 

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MEDICAL BOOKS. 21

 

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22 SUBJECT CATALOGUE.

COMPENDS.

From The Southern Clinic.

“We know of no series of books issued by any house that so fully
meets our approval as these ?QuizCompends?. They are well ar-
ranged, full, and concise, and are really the best line of text-books that
could be found for either student or practitioner,’’

BLAKISTON’S ? QUIZ-COMPENDS?

The Best Series of Manuals for the Use of Students.
Price of each, Cloth, .80. Interleaved, for taking Notes, $1.00.

4&ap- These Compends are based on the most popular text-books
and the lectures of prominent professors, and are kept constantly re-
vised, so that they may thoroughly represent the present state of the
subjects upon which they treat.

4a@j~ The authors have had large experience as Quiz-Masters and
attaches of colleges, and are well acquainted with the wants of students.

&@~ They are arranged in the most approved ;form, thorough and
concise, containing over 600 fine illustrations, inserted wherever they
could be used to advantage.

&ay- Can be used by students of any college.

Aa They contain information nowhere else collected in such a
condensed, practical shape. Illustrated Circular free.

No.1. POTTER. HUMAN ANATOMY. Sixth Revised and
Enlarged Edition. Including Visceral Anatomy. Can be used
with either Morris’s or Gray’s Anatomy. 117 Tinstrarions and 16
Lithographic Plates of Nerves and Arteries, with Explanatory
Tables, etc. By Samugt O. L. Porrsr, m.p., Professor of the
Practice of Medicine, College of Physicians and Surgeons, San
Francisco ; Brigade Surgeon, U. S. Vol.

No. 2. HUGHES. PRACTICE OF MEDICINE. Part I. Sixth
Edition, Enlarged and Improved. By Danrex E, Hueugs, M.v.,
Physician-in-Chief, Philadelphia Hospital, late Demonstrator of
Clinical Medicine, Jefferson Medical College, Phila.

No. 3. HUGHES. PRACTICE OF MEDICINE. Part II.
Sixth Edition, Revised and Improved. Same author as No. 2.

No. 4. BRUBAKER. PHYSIOLOGY. Tenth Edition, with
Illustrations and a table of Physiological Constants. Enlarged
and Revised. By A. P. Brusaxker, M.D., Professor of Physiology
and General Pathology in the Pennsylvania College of Dental
Surgery; Adjunct Professor of Physiology, Jefferson Medical
College, Philadelphia, etc.

No.5. LANDIS. OBSTETRICS. Seventh Edition. By Henry G.
LanpIs, M.D. Revised and Edited by Wm. H. WELLs, m.p.,
Demonstrator of Clinical Obstetrics, Jefferson Medical College,
Philadelphia. Enlarged. 52 Illustrations.

No.6. POTTER. MATERIA MEDICA, THERAPEUTICS,
AND PRESCRIPTION WRITING. Sixth Revised Edition
‘U.S. P. 1890). By Samugr O. L. Potrsr, m.p., Professor of

ractice, College of Physicians and Surgeons, San Francisco;
Brigade Surgeon, U. S. Vol.

 
MEDICAL BOOKS. 23

 

? QUIZ-COMPENDS ?—Continued.

No.7. WELLS. GYNECOLOGY. Second Edition. By Wm. H.
WeLLs, m.p., Demonstrator of Clinical Obstetrics, Jefferson
Medical College, Philadelphia. 140 Illustrations.

No. 8. GOULD AND PYLE. DISEASES OF THE EYE
AND REFRACTION. Second Edition. Including Treatment
and Surgery, and a Section on Local Therapeutics. By GzorGE
M. Goutp, m.p., and W. L. Pytz,m.p, With Formulz, Glossary
Tables, and 109 Illustrations, several of which are Colored.

No. 9. HORWITZ. SURGERY, Minor Surgery, and Bandag-
ing. Fifth Edition, Enlarged and Improved. By OrviLLE
Horwitz, B.S., M.D., Glnical trolessoe of Genito-Urinary Surgery
and Venereal Diseases in Jefferson Medical College; Surgeon to
Philadelphia Hospital, etc. With 98 Formule and 71 Illustrations.

No. 10. LEFFMANN. MEDICAL CHEMISTRY. Fourth
Edition. Including Urinalysis, Animal Chemistry, Chemistry of
Milk, Blood, Tissues, the Secretions, etc. By Hm=nry LEFFMANN,
M.D., Professor of Chemistry in the Woman's Medical College of
Penna; Pathological Chemist, Jefferson Medical College Hospital.

No. 11. STEWART. PHARMACY. Fifth Edition. Based upon
Prof. Remington’s Text-Book of Pharmacy. By F. E, Stewart,
M.D., PH.G. ate Quiz-Master in Pharmacy and Chemistry, Phila-
delphia College of Pharmacy; Lecturer at Jefferson Medical
College. Carefully revised in accordance with the new U.S. P.

No. 1. BALLOU. VETERINARY ANATOMY AND PHY-
SIOLOGY. Illustrated. By Wm.R. Battovu, m.p., Professor
of Equine Anatomy at New York College of Veterinary Surgeons ;
Physician to Bellevue Dispensary, etc. 29 graphic Illustrations

No. 13. WARREN. DENTAL PATHOLOGY AND DEN-
TAL MEDICINE, Third Edition, Illustrated. Containing
a Section on Emergencies. By Geo. W. WarREN, D.D.S., Chiet
ot Clinical Staff, Pennsylvania College of Dental Surgery.

No. 14. HATFIELD. DISEASES OF CHILDREN. Second
Edition. Colored Plate. By Marcus P. Hatriexp, Profes-
sor of Diseases of Children, Chicago Medical College.

No. 15. THAYER. GENERAL PATHOLOGY. By A. E.
TuHayer, M.v., Cornell University Medical College. Illustrated.

No. 164. SCHAMBERG. DISEASES OF THE SKIN. Second
Edition. By Jay F. SCHAMBERG, M.D., Professor of Diseases of
the Skin, Philadelphia Polyclinic. Second Edition, Revised and
Enlarged. 105 handsome IIlustrations.

No. 17. CUSHING. HISTOLOGY. By H. H. Cusuine, m.p.,
Demonstrator of Histology, Jefferson Medical College, Philadel-
phia. Illustrated.

No. 18. THAYER. SPECIAL PATHOLOGY. Illustrated. By
same Author as No. 15.

Price, each, Cloth, .80. Interleaved, for taking Notes, $1.00.

Careful attention has been given to the construction of each sentence,
and while the books will be found to contain an immense amount of
knowledge in small space, they will likewise be found easy reading ;
there is no stilted repetition of words; the style is clear, lucid, and dis-
tinct. The arrangement of subjects is systematic and thorough ; there
is a reason for every word. They contain over 600 illustrations.
THE STANDARD TEXT-BOOK

Morris’ ANATOMY

SECOND EDITION

 

Rewritten. Revised. Improved
WITH MANY NEW ILLUSTRATIONS

Has been recommended as a text-book at more than
seventy of the most prominent medical schools in the United
States and Canada, and is considered by al] anatomists as a
standard authority. It contains many features of special
advantage to students. A complete Text-book. Edited by
HENRY Morris, F.R.C.S., Surgeon to, and Lecturer on
Anatomy at, Middlesex Hospital, assisted by J. BLAND
SUTTON, F.R.C.S., J. H. DAvres-CoLLey, F.R.C.S., WM. J.
WALSHAM, F.R.C.S., H. ST. JOHN Brooks, M.D., R. MAR-
cus GUNN, F.R.C.S., ARTHUR HENSMAN, F.R.C.S., FRED-
ERICK TREVES, F.R.C.S., WILLIAM ANDERSON, F.R.C.S.,
Pror. W. H. A. JAcoBsSON, and ARTHUR ROBINSON, M.R.C.S.

Octavo. With 790 Hlustrations, of which a large number
are printed in colors

CLOTH, $6.00; LEATHER, $7.00

“« The ever-growing popularity of the book with teach-
ers and students is an index of its value, and it may safely
be recommended to all interested.’—From Zhe Medical
Record, New York.

“©Of all the text-books of moderate size on human
anatomy in the English language, Morris is undoubtedly
the most up-to-date and accurate.’’—From Zhe Philadet-
phia Medical Journal.

‘THUMB INDEX IN EACH COPY