•IOLGGT

METHODS AND FOEMUL^E

METHODS AND FORMULAE

USED IN THE PREPARATION OP

ANIMAL AND VEGETABLE TISSUES

FOR MICROSCOPICAL EXAMINATION

INCLUDING THE STAINING OF

BACTERIA

BY

PETER WYATT SQUIRE

FELLOW OF THE LJNNEAN SOCIETY

LONDON
J. & A. CHURCHILL,

11, NEW BURLINGTON STEEET

1892
Copyright Registered.

PEEFACE.

THIS little book has grown out of an attempt to frame a
collection of trustworthy formulae most likely to be useful to
the practical microscopist. Interspersed with these have been
added such practical notes and comments as have suggested
themselves during a very extensive course of experimenting
on the various materials and processes here described.

The larger text-books do not always agree in the figures
given for the same formula. "Whenever possible, these have
been corrected by reference to the original or most authentic
source, but where the formula has been so vague and inde-
finite as to be little more than a suggestion, a detailed process
has been worked out experimentally.

Although intended primarily as a guide in staining pro-
cesses, the most approved methods of hardening, fixing,
embedding, clearing and mounting, are also described. Sec-
tion-cutting alone has been purposely omitted, as this art can
only be learned properly from personal tuition.

Although the best results in staining sections, &c., can only
be obtained after considerable practice, an attempt has been
made in every case, by the employment of solutions of definite

VI PREFACE.

strength for definite times^ to enable even a beginner to arrive
at an average result without loss of time in experimenting.

It is therefore hoped that the usefulness of the work as
a compact and inexpensive book of reference for the expe-
rienced worker, as well as a guide to those commencing
investigations, may justify its publication.

Very few "double-stains" have been given, as even when
good results could be obtained by using two dyes in the same
solution, better and more precise staining was effected when
the same dyes were used separately one after the other.

Amongst the stains but little known, which have given
very good results, may be mentioned Benzopurpurine as
a counter-stain to Hsematoxylin ; Congo Red for the central
nervous system ; and Rose Bengale for the detection of early
Amyloid. The last reaction has not been previously noticed.

For the pathological work, the co-operation of F. J.
Warwick, M.B. (Cantab), a practical microscopist of much
experience in this subject, has been most valuable.

Most of the methods given under Micro-organisms have
been translated from the original papers. Kiihne's methods
are taken from the translation by Dr. Vincent Harris.

P. W. S.

413, OXFORD STBEET.
April o, 1892.

CONTENTS.

PACK

HARDENING AND FIXING REAGENTS . . . . . 1 — 10

DECALCIFYING FLUIDS 11 — 13

EMBEDDING MEDIA 14 — 19

NUCLEAR STAINS 20 — 39

PLASMATIC STAINS , 39—42

SPECIFIC STAINS 43—48

SPECIFIC METHODS 48 — 51

STAINING CELLULOSE AND ITS MODIFICATIONS .... 52—56

STAINING SIEVE -AREAS 56

MICRO-ORGANISMS . . 57 — 77

Stains — Intermediate Reagents — General Methods —
Special Methods.

DEHYDRATION ... 78

CLEARING AGENTS 79 — 82

MOUNTING MEDIA 83 — 86

NORMAL OR INDIFFERENT FLUIDS . . . . . . .87

DISSOCIATING FLUIDS . . . 88—90

HARDENING AND FIXING REAGENTS.

FOR general histological and pathological work, the harden-
ing agents usually employed are Alcohol and the Bichro-
mate solutions. The others are only used for special applications.

Alcohol will not penetrate well pieces larger than a cubic
inch, while the Bichromates are applicable to material of any
size.

If rapid hardening is required, Alcohol, Erlicki's Fluid, or
Klein's Fluid is employed, but the two latter have a tendency
to make the tissues brittle if they remain in the solution too
long.

A relatively large quantity of hardening fluid, about 20
times the bulk of the object to be hardened, should always be
used, and the pieces should be as small as circumstances will
allow. It is best to suspend the object near to the top of a
tall vessel filled with the fluid, which is accomplished by
placing the pieces of tissue on absorbent wool enclosed in
a muslin bag, but this is not always considered to be neces-
sary. The pieces can simply be placed in a bottle with the
fluid, which should be circulated from time to time, and
changed when necessary.

As soon as the objects are sufficiently hard they should be
removed from the hardening solution and placed in 70 p. c.
Alcohol till required.

Of the special fixing agents Osmic Acid is the best, more

2 HARDENING AND FIXING REAGENTS.

particularly in combination with Chromic Acid and Acetic
Acid, as in Flemming's and Fol's solutions.

Fresh vegetable tissues (leaves, roots, and stems) are best
fixed in Alcohol. In the majority of instances 90 p. c.
Alcohol will answer the purpose ; but where the nuclei in
particular are to be examined, a stronger Alcohol (Absolute)
should be used.

Fresh- water alga), &c., can be fixed in Picric Acid or a
Chrom-Osmic solution (Flemming's or Fol's) ; but, if it is
desired to preserve the colour of the chlorophyll, the Acetate
of Copper solution is recommended.

Sections of fresh tissues are sometimes fixed and stained at
the same time by Acetic Acid mixed with Methyl Green or
Gentian Violet.

Most stems, barks, and roots can be cut after fixing in
90 p. c. Alcohol ; but when very hard they can be softened
by removal from the Alcohol into Thymol Water, or equal
parts of Thymol Water and Glycerine. In rare cases it is neces-
sary to use 2 to 5 p. c. of Caustic Potash in the Thymol Water.

(i.) ALCOHOL.

Suitable for all tissues except the nervous system and tissues
undergoing fatty infiltration or degeneration ; from these the
fat would be dissolved out to a considerable extent by the
Alcohol.

Glandular structures and tissues to be examined for Bac-
teria are placed at once into 90 per cent. Alcohol, or Absolute
Alcohol, but in other cases, where it is required to preserve
the normal structure of the tissues, they are at first placed in
50 per cent. Alcohol for a day or two, depending upon the
size of the pieces, then into 70 per cent. Alcohol for a similar
time, and subsequently into 90 per cent. Alcohol until the
hardening is complete.

HARDENING AND FIXING KEAGENTS. 3

For fixing nuclear figures the strongest Alcohol (Absolute)
should be used, and the pieces should be as small as possible.

Throughout this book, per cent, of Alcohol is given by
weight ; when Methylated Spirit is used the gravities may be
slightly different. The following formulae are sufficiently near
for all practical purposes.

Absolute Alcohol, about 98 per cent. Sp. gr. 0-797.
The commercial article. Alcohol Ethylicum B.P.

Alcohol, 9O per cent. Sp. gr. 0-823.

Absolute Alcohol, 14 vols. ; Distilled Water, 1 vol. : mix.

The strongest commercial Methylated Spirit (64 o. p.) is
practically the same strength as 90 p. c. Alcohol, and being
duty free is much cheaper and answers every purpose.

This, of course, only applies to Methylated Spirit free from
Mineral Naphtha. The ordinary , ' ' Eetailer ' s ' ' quality contains
Naphtha, and becomes turbid when mixed with water.

Alcohol, 84 per cent. Sp. gr. 0-838.
This is Eectified Spirit B.P. (56 o. p.).

Alcohol, 70 per cent. Sp. gr. 0-872.

Absolute Alcohol, 3 vols. ; Distilled Water, 1 vol.
Eectified Spirit, 6 vols. ; Distilled Water, 1 vol.
Methylated Spirit, 4 vols. ; Distilled Water, 1 vol.

Alcohol, 50 per cent. Sp. gr. 0-918.

Absolute Alcohol, 5 vois. ; Distilled Water, 4 vols.
Eectified Spirit, 5 vols. ; Distilled Water, 3 vols.
Methylated Spirit, 5 vols. ; Distilled Water, 3J vols.

(ii.) ALCOHOL AND ACETIC ACID.

Absolute Alcohol, 75 c.c. ; Glacial Acetic Acid, 25 c.c.
This is an excellent fixing agent for nuclei. Tissues are

4r HARDENING AND FIXING REAGENTS.

placed in it from 6 to 12 hours, and then transferred to 90 p. c.
Alcohol until hardened. When the hardening is completed,
transfer the tissue to 70 p. c. Alcohol till wanted.

(iii.) CHROMATE OF AMMONIUM.

5 p. c. aqueous solution.

Especially useful for delicate cell structures, as parenchyma
of secreting cells. Tissues are placed for 48 hours in this
reagent, thoroughly washed in Distilled Water and passed
through diluted to strong Alcohol.

(iv.) BICHROMATE OF AMMONIUM.
BICHROMATE OF POTASSIUM.

2 to 5 per cent, aqueous solution of either.

Simple Bichromate solutions are sometimes preferred to
Miiller's Fluid for hardening parts of the central nervous
system, but opinions vary as to their greater usefulness even
for this purpose. A two per cent, solution should be used to
start with and changed after 24 hours and again after 3
days, and then once a week for the next 6 weeks, increasing
the strength by one per cent, each week till it reaches five per
cent. In this way an entire brain may be hardened thoroughly
and satisfactorily in 8 weeks.

After hardening for a month in Miiller's Fluid, it is often
advantageous to finish the process by a fortnight's immersion
in five per cent. Bichromate solution.

(iv.«) MULLER'S FLUID.

Bichromate of Potassium, 2 grms. ; Sulphate of Sodium,
1 grm. ; Water, 1 00 cc.

A good hardening reagent for delicate structures where
the process is required to be gradual. It is well adapted for

HARDENING AND FIXING REAGENTS. 0

the eye. It penetrates evenly and thoroughly, so that large
pieces can be hardened in it, while at the same time it has
no tendency to over-harden and render the tissues brittle.

The disadvantages attaching to all the Bichromate solutions
are that they discolour the tissue more or less. A long time
is required for the hardening, and if tissues be kept in them
too long they sometimes produce netlike forms of coagula-
tion, or deposits of a dark granular precipitate in the cells
and intercellular substances ; they are apt to develop moulds.
They dissolve out calcareous deposits.

When the nervous system is stained by Weigert's method,
the tissue must be hardened in this or one of the other
Bichromate solutions.

It is unsuitable for hardening tissues to be examined for
the amyloid reactions or for bacteria.

Tissues ought to be placed in at least 20 times their bulk of
" Miiller." This must be changed after 24 hours, again after
3 days, and then once a week for the next 6 weeks. Even for
small pieces 6 weeks in all are generally required. The
tissues, after removal from " Miiller," are soaked in 50 p. c.
Alcohol from 1 to 2 days, then 70 p. c. Alcohol for a similar
time, and finally placed in 90 p. c. Alcohol, which should be
changed occasionally so long as it becomes coloured. In the
case of large masses, a preliminary washing in water will save
Spirit and do no harm.

(iv.J) MULLER'S FLUID AND SPIRIT.

Midler's Fluid, 3 ; 90 p. c. Alcohol, 1.

This is usually directed to be cooled before using it, but as there is only
a rise of 11° E. on mixing- them, the precaution is hardly necessary.

Used in the same cases as " Miiller," only its action is more
rapid. It takes about half the time of " Miiller" to harden
tissues. Whilst hardening in this, and during the subse-

6 HAKDENING AND FIXING REAGENTS.

quent washing in Alcohol, tissues should be kept in the
dark.

The other Bichromate solutions may be used with Spirit in
the same way.

(iv.<?) ERLICKI'S FLUID,

Bichromate of Potassium, 5 grms. ; Sulphate of Copper,
I grm. ; Water, 200 cc. This solution hardens quicker than
" Miiller," and is used in much the same way.

It is sometimes stated that this solution should always be prepared freth
when required, but this is not necessary as it undergoes no change.

This is the best fluid for hardening large organs quickly.
It will harden a spinal cord in 10 days, and is the most suit-
able fluid for human embryoes.

It tends to render the tissues brittle, and also gives the
sections a greenish tinge.

(y.) CHROMIC ACID.

10 p. c. aqueous solution is kept for stock ; for use it is
diluted with 20, 30, or 50 times its bulk of Water, accord-
ing to the consistence and size of the tissue. The larger or
firmer the tissue the weaker the solution.

The pieces to be hardened must not be larger than J cubic
inch ; the fluid must be changed every day for the first 3 days,
and then every third day. Tissues are hardened in from 1 to
14 days, according to their nature and size; they are then
well washed in Water and kept in 70 p. c. Alcohol in a dark
place to avoid deposits.

Chromic Acid is energetic, but it does not penetrate well,
and it makes the tissues brittle. Tissues thus hardened are
not well stained by Carmine ; Hsematoxylin or Safranine
should be used.

HARDENIKG AND FIXING REAGhXTS. 7

(v.a) KLEIN'S FLUID.

Chromic Acid solution (10 p. c.) 1 cc. ; mix with. Water
60 cc. ; then add 90 p. c. Alcohol, 30 cc.

The Alcohol is added to increase the penetration of the
Chromic Acid. This solution should be made fresh as re-
quired and kept from the light, as the Chromic Acid is
rapidly reduced by the Spirit.

Suitable for all tissues, but more particularly the nervous
system. The fluid must be changed every day for the first
three days, and then every third day. The hardening is com-
plete in about a week to a fortnight.

In all cases except the nervous system, the tissue after
hardening must be well washed in Water, and preserved in
70 p. c. Alcohol.

The nervous system should be transferred direct from
Klein's Fluid to the Alcohol.

(v.J) RABL'S FLUID.

Chromic Acid solution (10 per cent.), 7 cc. ; Water, 200 cc. ;
Formic Acid (sp. gr. 1*2), 5 drops.

Fresh tissues in small pieces are placed in it from 12 to 24
hours; they are then washed in Water and hardened in
Alcohol of gradually increasing strength.

This solution is especially useful for demonstrating cell
division (mitosis) and nuclei.

The following are used more particularly as fixing agents.

(vi.) CORROSIVE SUBLIMATE.

5 p. c. aqueous solution.

This is a capital fixing agent, and acts quickly.

8 HARDENING AND FIXING REAGENTS.

Tissues ought to be placed in the solution from 1 5 minutes
to 2 hours, and then in, 90 p. c. Alcohol to complete the
hardening.

After this the tissues must be washed in 70 p. c. Alcohol ;
if Water be used black specks are apt to form.

The action of the fluid can be accelerated by heating it to
38° C. (100-4° P.)

For glands and glandular structures the following is very
useful: — Corrosive Sublimate, 10 grins.; Alcohol (70 p. c.),
100 cc. ; Glacial Acetic Acid, 1 cc. Small pieces are
placed in this for an hour and the hardening completed in
Alcohol.

(vii.) NITRIC ACID (Altmann).

3 p. c. aqueous solution. Sp. gr. about 1-02. Used as a
fixing agent.

The pieces of tissue must be as small as possible, and placed
in the Acid until quite fixed ; this takes from 15 to 30 minutes.
Useful for fixing nuclei of cells, embryo es and the retina.

viii.) OSMIC ACID.

1 p. c. aqueous solution, which must be carefully preserved
from dust, as it is readily reduced (blackened) by small quan-
tities of organic matter. This solution is usually kept in the
dark.

Especially useful for delicate structures, when rapid fixing
is required.

For use it is diluted with 4 or 5 times its volume of
Distilled Water, or more generally in conjunction with
Chromic Acid as in Flemming's and Fol's solutions. Tissues
are placed in it from 8 to 10 hours, well washed in Distilled
Water, cut, and mounted in Farrant's solution.

Some authorities prefer to fix with the vapour ; this is done

HARDENING AND FIXING REAGENTS.

by suspending the tissue in a bottle containing some solution
or crystals of Osmic Acid.

Tissues containing Fat are blackened by Osmic Acid, tee
Specific Stains.

(viii.a) FLEMMING'S FIXING SOLUTION.

Osmic acid (1 p. c. solution), 80 cc. ; Chromic Acid (10 p. c.
solution), 15 cc. ; Glacial Acetic Acid, 10 cc. ; Distilled Water,
05 cc.

This and the following solution are used solely as fixing
agents. Tissues are placed in either for about twelve hours ;
they are then thoroughly washed in Water and hardened in
Alcohol of gradually increasing strength (see Alcohol].

Especially useful for fixing tissues undergoing cell division
(mitosis). This fluid need not be used in the dark.

Sections should be stained with Safranine, Hsematoxylin, or
Gentian Violet.

(viiU) POL'S SOLUTION.

Osmic Acid (1 p. c. solution), 4 cc. ; Chromic Acid (10 p. c.
solution), 5 cc. ; Glacial Acetic Acid, 10 cc. ; Distilled Water,
181 cc.

Used for the same purpose as the preceding.

Flemming's and Fol's solutions represent the maximum and
minimum strength of Chrom-Osmic mixtures for fixing pur-
poses ; various intermediate strengths are also employed.

(ix.) PICRIC ACID.

A saturated aqueous solution ( 1 in 75), or 1 p. c. solution in 70
p. c. Alcohol, are used for fixing and hardening. The action
is rapid, taking from 1 to 24 hours.

After hardening, the tissue is washed in 70 p. c. Alcohol

10 HARDENING ASD FIXING REAGENTS.

(not Water) and preserved in 90 p. c. Alcohol. Picric Acid
solutions dissolve out calcareous deposits.

(x.) FICRO SULPHURIC ACID (Kleinenberg).

Saturated aqueous solution of Picric Acid, 100 cc. ; pure
Sulphuric Acid, 2 cc. ; filter ; to each 100 cc. of nitrate add
300 cc. of Distilled Water.

As in Kleinenberg's formula four-fifths of the Picric Acid is thrown
out of solution, the same final result can be obtained by the following : —

Saturated Solution of Picric Acid, 20 cc. ; Distilled Water,
380 cc. ; Sulphuric Acid, 2 cc.

Especially useful for the rapid fixing of very soft tissues,
as sarcoma and myxoma ; also for preserving embryoes. It
is used for fixing and preserving the earth-worm.

Tissues are placed in the fluid from 3 to 12 hours ; they
are then washed in 70 p. c. Alcohol, and finally placed in
90 p. c. Alcohol.

Carmine is the best stain to use after this solution ; but if
Hsematoxylin be used, the sections must first be placed in
a solution of Carbonate of Lithium, and then washed in
Water.

(xi.) ACETATE OP COFFER.

Acetate of Copper, 1 grm. ; Distilled Water, 200 cc. ; Glacial
Acetic Acid, 1 cc. ; Glycerine, 200 cc. ; Corrosive Sublimate,
3 grms.

This solution is especially useful for fixing and mounting
green Algse.

DECALCIFYING FLUIDS.

In the case of bony structures or tissues so impregnated
with Lime Salts as to interfere with section cutting, the
material must be decalcified in one of the following solutions.
The general plan is to combine an Acid capable of dissolving
out the mineral matter with a hardening agent which will
prevent swelling of the tissues.

Hydrochloric Acid is generally used with Alcohol. Diluted
Nitric Acid may be used alone, but usually in conjunction
with Alcohol, Chromic Acid, or an equivalent quantity of
Potassium Bichromate.

The older the bone the stronger will be the acid required.

It is advisable to harden first in Alcohol or Bichromate
before decalcifying, except in the case of Ebner's solution
where the two processes go on together.

(i.) ARSENIC ACID.

4 p. c. aqueous solution, used at a temperature of 30° to.
40° 0.

It acts rapidly. Tissues after softening should be kept in
Alcohol.

(ii.) CHROMIC ACID.

0-1 to 0-5 p. c. aqueous solution.

Used for softening small pieces of bone, but when use
alone it causes much shrinkage in the tissues.
It is best used in conjunction with Nitric Acid.

12

DECALCIFYING FLUIDS.

(iii.) HYDROCHLORIC ACID.

1 to 10 p. c. in water of the strong acid (sp. gr. 1-16.)
Softens rapidly, but causes tissues to swell and become almost
structureless. This is remedied by the addition of Alcohol
as in the following : —

(iv.) EBNER'S SOLUTION.

90 p. c. Alcohol, 1000 cc. ; Water, 200 cc. ; Chloride of
Sodium, 5 grms. ; Hydrochloric Acid (sp. gr. 1'16), 5 cc.

In this solution the Spirit and Salt are added to prevent the
swelling of the tissue. This solution takes a long time to
soften, its action being very gradual, requiring about six
weeks. To each volume of the tissue 200 to 300 vols. of the
solution must be used ; when softening large bones it is neces-
sary to add a small quantity of Hydrochloric Acid from day
to day, to replace that which combines with the Lime Salts,
and examine the tissue with a needle from time to time, till
the process of softening is complete ; the tissue is then
washed in a large volume of Water to remove the acid, and
finally kept in 70 p. c. Alcohol.

(v.) HYDROCHLORIC ACID AND GLYCERINE.

Glycerine, 95 ; Hydrochloric Acid, 5.

This is especially useful for softening teeth ; it softens and
yet does not in any way interfere with the structure or bony
appearance ; it also acts as a preservative. Its action is slow,
taking several weeks.

(vi.) NITRIC ACID.

Aqueous or Alcoholic solution containing from 1 p. c. for
small bones, up to 5 p. c. for large heavy bones.

They are first placed in 90 p. c. Alcohol for two or three

DECALCIFYING FLUIDS. 13

days, then treated with the Nitric Acid solution, which is
changed daily for about a week ; as soon as decalcification
is complete, wash thoroughly in Water, and place in 70 p. c.
Alcohol.

(vii.) NITRIC ACID AND CHROMIC ACID.

Chromic Acid, 1 grm. ; Water, 200 cc. ; Nitric Acid 3 cc.

This is one of the best softening fluids for general use, the
Chromic Acid hardening the bone protoplasm, while the Nitric
Acid dissolves out the calcareous salts.

It takes about a fortnight, and the solution must be renewed
every third day. The tissue is then well washed in water and
transferred first to weak and then to strong Alcohol.

(viii.) PICRIC ACID.

A saturated aqueous solution. Useful for softening foetal
bone. The tissue should be suspended in twenty times its
bulk of fluid, which must be kept saturated with crystals
of Picric Acid. It takes about a month; the tissue when
softened must be first well washed in Water and then passed
from weak into strong Alcohol (see " Alcohol").

EMBEDDING MEDIA.

These are mostly used to support the tissues during the
•cutting with a microtome.

The simplest method is that adopted for firm vegetable
tissues. A cylinder of carrot is made to fit the microtome,
and then divided vertically. The piece of tissue is fitted in
between the two halves of carrot ; the carrot and tissue are
cut at the same time, the former, of course, transversely.
The direction of the latter will depend upon the position in
which it is placed in the carrot. Another method is to embed
in Paraffin, and fit the block of Paraffin to the microtome.

Neither of the above are suited for delicate or porous tissues.
They must be supported in the interstices by saturation with
the embedding material. For this purpose Grum, Celloidin,
and Paraffin are used. When it is desired to support in
position tissues which would otherwise fall to pieces when cut
into sections, Celloidin is generally used. Saturation with
Paraffin is mostly employed for serial cutting.

(i.) GUM (Mucilage).

Colourless Gum Acacia, 2 ; Cold Water, 3.
Dissolve the Grum in the Water, and to each ounce of the
solution add 1 0 grains of Carbolic Acid.

(ii.) GUM AND SYRUP.

Mucilage of Acacia, 50 cc. ; Simple Syrup B.P., 30 cc.

EMBEDDING MEDIA. 15

Method of Using.
Before using either of the above for embedding, the tissues
must be washed for several hours in frequent changes of
Water, to free them from Alcohol, and then placed for 24 hours
(preferably 3 or 4 days) in the Gum or Gum and Syrup, to
saturate them before freezing on the microtome.

(iii.) CELLOIDIN.

Celloidin dissolved in equal parts of Absolute Alcohol and
Ether, an 8 p. c. Solution being used for Thick Celloidin,
and diluted with an equal volume of the Ether- Alcohol for
Thin Celloidin.

Method of Using.

The tissues (previously hardened by one of the methods
already given), if not already in strong Alcohol, should be
placed there ; they are then immersed for 12 hours in equal
parts of Absolute Alcohol and Ether. After this they are
immersed in Thin Celloidin for 24 hours ; then for another 24
hours (preferably 2 or 3 days) in Thick Celloidin, so that it
may thoroughly permeate the tissues. The pieces are now
removed from the solution, and placed on small cubes of soft
wood, care being taken to see that the wood is quite dry.
Allow the Celloidin to harden for a few minutes, and then
place the wood with the tissue in 90 p. c. Alcohol for 12 to 24
hours. They are then ready for clamping in the microtome.

If material, saturated with Celloidin, is to be cut on the
freezing microtome, the piece when removed from the Thick
Celloidin, is placed with some of the Celloidin in a small paper
case. Allow a thin film to form on the surface by evaporation,
and then place the paper case with contents into 80 or 90 p. c.
Alcohol for 12 to 24 hours or longer to harden. When it
assumes the consistency of soft cheese take it out of the
Alcohol, remove the paper case, and place it in Water to wash

16 EMBEDDIKG MEDIA.

away superfluous Alcohol ; the piece is then dipped in Muci-
lage, and finally frozen on the microtome in the usual way.

(iv.) GLYCERINE GELATINE (Kleb's).

Best Gelatine (French), 10 parts; wash and allow this to
stand in Distilled Water till it swells up : pour off excess of
Water, melt at a gentle heat, and add 1 0 parts of Glycerine,
and 2 p. c. of Carbolic Acid to preserve it.

Method of Using.

The tissue, which has previously been well washed with
Water to remove Alcohol, is saturated with the melted
Glycerine Jelly, which is then allowed to set. This may now
be frozen between pieces of carrot on the microtome by means
of Gum ; or it may be clamped between carrot for cutting.

This method is useful for flat structures, such as the walls
of hollow organs, or membranes.

(v.) PARAFFIN.

Paraffin melting at between 45° 0. (113°F.) and 50° 0.
(122° F.) according to the temperature of the room, and the
nature of the material to be cut.

This medium is best adapted for embedding tissues that
have been stained in bulk.

Method of Using.

Some tissues are embedded in Paraffin, or other suitable sub-
stance, only as a means of holding them in the microtome ;
other tissues which are very porous or brittle require to be
saturated with the embedding material to hold the parts to-
gether during or after the cutting.

Embedding. — The tissue which has been dehydrated is dipped
for a second or two into melted Paraffin, and taken out again.
This gives the piece a coating of Paraffin. The tissue may

EMBEDDING MEDIA. 17

also be transferred direct from the Alcohol to the cell of
Paraffin. In either case the piece is put into the melted
Paraffin, which has been poured into a cell, and which has
cooled sufficiently to hold the piece in any position required.
Another method is to make a hole in a small block of Paraffin,
fit in the piece of tissue, and fill up with warmed Paraffin.

The cell to hold the Paraffin can be made in a variety of
ways. A small rectangular tray can be made by folding paper
or thin card ; a cylinder can be formed by rolling paper round
a cork (allowing the paper to project beyond the cork) and
fixing it with a pin run through the paper into the cork. Cells
can also be formed by means of two pieces of brass, each of
them being bent at right angles, and the cylinders can be cut
from brass-tubing.

When the sections have been cut, the Paraffin can be
removed by immersion in Naphtha, Benzol, Toluol, or Xylol,
all of which are good solvents of Paraffin.

For staining, the sections must then be passed through
Absolute Alcohol to remove the liquid hydrocarbon.

Saturation. — The piece of tissue, after staining in bulk, is
placed in strong Spirit and afterwards in Absolute Alcohol.
It is then transferred to Cedar Oil or Xylol, and when cleared
is placed in a suitable (not too large) quantity of Paraffin,
kept just above the melting point, until the tissue is saturated
with it. Both Cedar Oil and Xylol are convenient media,
as either mixes readily both with Absolute Alcohol and melted
Paraffin. If the pieces of tissue carry much oil into the
Paraffin, a second bath of the latter may be used. When the
saturation is complete (2 — 5 hours), the Paraffin should be
quickly cooled, and a piece containing the tissue can be cut
out with a warmed knife. It is then ready for mounting on
the microtome.

Another Method of Saturation. — The tissue after staining is

c

18 EMBEDDING MEDIA.

passed into 90 p. c. Alcohol, and then into Absolute Alcohol,
in which it must remain for not less than 3 hours. It is next
placed in ten times its bulk of Chloroform or Benzol until
thoroughly saturated; then small pieces of the Paraffin
are added to the Chloroform or Benzol until no more will
dissolve ; in this the tissue is allowed to remain for 2 or 3
hours. Gentle heat is then applied to drive off the solvent
and melt the Paraffin, without injuring the specimen. The
mass is allowed to set, and gentle heat is again applied to
the vessel containing the mass to loosen it. The superfluous
Paraffin is trimmed and the mass fused on to the socket of
the rocking microtome.

(vi.) RESIN AND WAX.

Eesin, 3 ; Yellow Wax, 1 ; melt together.

It is used for holding tissues (bone and teeth) whilst being
ground.

Pieces are cut as thin as possible with a saw, and one side
is ground on an oilstone; the piece is then placed in the
Resin and Wax (liquefied by heat) and cemented on the
ground side to a piece of glass. When hard the tissue is
rubbed down to the required thickness on an oilstone. The
Eesin and Wax can be removed from the tissue by Oil of
Turpentine, in which both are soluble.

PRESERVING SECTIONS.

Sections are best preserved (previous to staining and
mounting) in 50 p. c. Alcohol, or in a mixture of Glycerine
and Thymol Water (1 in 1500) in equal volumes. If kept
long in strong Alcohol they are apt to shrivel.

MANIPULATION OF SECTIONS.

Thin sections which have been kept for some time in
Alcohol are apt to get folded or crumpled. If these be

EMBEDDING MEDIA. 19

placed one at a time on the surface of some water, they will
open up immediately, with the assistance if necessary of a
pointed glass rod (glass needle.) Glass rods (about J-inch
diameter for a J-inch section) are also convenient to transfer
sections from one dish or bottle to another in the staining
process, the section being allowed to wrap itself round the rod.
When a section is being treated in strong Alcohol or any liquid
which tends to make it shrink or curl, it should be allowed
to remain wrapped round the rod, which prevents any change
of shape. In transferring a section from the rod to a slide,
it is only necessary to place on the slide a drop or two of the
liquid from which it is being removed. Lay the rod with the
adhering section on the wetted slide, and roll the section off
the rod flat on to the slide. Very delicate sections can be
manipulated in this way after a little practice.

Vegetable sections are usually thicker than Animal sections,
and can be manipulated with a lifter, or a pair of bent forceps
the ends of which are broad and flattened.

Washing sections in Alcohol, dehydrating, and clearing,
are conveniently performed in small wide-mouthed bottles.

STAINS.

These may be divided into Nuclear, Plasmatic, or
Specific, according to their power of staining the nuclei, the
whole tissue, or certain elements of the tissue only.

Nuclear Stains. — Hsematoxylin, Carmine and Cochineal,
Methylene Blue, Methyl Green, Safranine, Vesuvine (Bis-
marck Brown), Gentian Violet, Hoffmann's Blue (acidified),
and Fuchsine.

Plasmatic Stains. — Picric Acid, Benzopurpurine, Eosine,
Erythrosine, Orange, and Acid Fuchsine.

Specific Stains. — Osmic Acid, Chloride of Gold, Nitrate
of Silver, Nigrosine, Acid Fuchsine, Congo Eed, Dahlia
Violet, Methyl Violet, Iodine, Eose Bengale, Safranine, and
Victoria Blue.

Stains and methods for Micro-Organisms are treated
separately.

NUCLEAR STAINING.

There is no colouring matter which is a nuclear stain only.
All of them have more or less effect upon the ground sub-
stance also. The value, therefore, of any dye as a nuclear
stain depends upon the comparative affinity which it has for
nuclei and ground substance respectively.

Direct Nuclear Stains are those in which the nuclei are
dyed so rapidly, compared with the rest of the tissue, that no
particular treatment is necessary to differentiate them.

Indirect Nuclear Stains are those in which the whole
tissue is deeply stained, and the nuclei are differentiated by
subsequent removal of the colour from the ground substance.

NUCLEAR STAINS. 21

For histological and pathological work, Hsematoxylin is
beyond doubt the best stain for general use. The nuclei are
clearly denned, the manipulation is easy, and good results
may be obtained with less experience than by any other
method.

With Ehrlich's solution it is easy to stain the nuclei with-
out affecting the ground substance to any marked degree ;
whereas with Carmine and the Aniline dyes the two are
stained together, and it is less easy to remove effectually the
colour from the ground substance and leave the nuclei sharply
denned.

When the tissues have been hardened in the Chromates,
the advantage of Haematoxylin over Carmine in rapidity of
staining is very marked. Carmine, on the other hand, pos-
sesses advantages in staining the nervous system, to which
Hsematoxylin is inapplicable, except by the somewhat tedious
process of Weigert or one of its modifications.

H2EMATOXYLIN.

This is essentially a nuclear stain, as it selects the nuclei
first ; but if the solution be too strong, or the action be con-
tinued too long, it will also stain the ground substance. It
is very extensively employed, and always in conjunction with
Alum, except in the special uses to which it is put in Weigert' s
method for the nervous system, in Heidenhain's method for
staining in mass, and Benda's copper method.

Of the solutions, Ehrlich's and Delafield's are recommended
for sections, and Kleinenberg's for staining in mass.

Sections are counter-stained with Benzopurpurine, Eosine,
Ery thro sine, or Rubin and Orange.

Hsematoxylin has been objected to on the ground that the
solutions deteriorate by keeping, and that the stained sections

22 NUCLEAR STAINS.

are not permanent. These objections certainly do not apply
to Ehrlich's solution, a -quantity of which, made five years
ago, is now in perfect order, and sections are still good which
were stained and mounted four and five years since.

In order to change the violet colour of the sections into
blue it is usual to soak them for some hours in "tap water,"
on the supposition that the alkalinity of the Carbonate of
Lime, dissolved in most natural waters, was necessary to the
change. That this is not the case is evident from the fact that
sections stained in a well-matured Ehrlich's Hsematoxylin
solution (which of course is itself acid) will become blue in
pure Distilled Water, freed from even the slightest trace of
Ammonia. The change, however, is undoubtedly assisted by
a trace of Alkali, and when sufficient Lime Carbonate does
not exist in the water, uniformly good results can always be
obtained in a few minutes by using Distilled Water con-
taining 1O grains of Bicarbonate of Sodium to the
pint (20 ounces). Light is in no way essential to the
development of the blue colour, and direct sunlight should
be carefully avoided on account of its bleaching action.

After the blue colour is developed, transfer the sections to
70 p. c. Alcohol, as the colour fades much more quickly when
kept in water.

It is occasionally stated that previous to staining the
sections must be freed from every trace of acid, as otherwise
the colour will not be permanent. Considering that one of
the best stains is itself acid, and that Acidulated Water or
Alcohol may freely be used to remedy overstaining, it is much
more to the point that sections, after the staining process is
completed, should be carefully freed and preserved from any
trace of acidity, more particularly when using counter- stains.
Freshly prepared solutions of Haematoxylin stain badly and
diffusely. When exposed to the air in strong daylight they

NUCLEAR STAINS. 23

undergo a change technically called " ripening," after which
the staining is much more precise.

The ripening process appears to depend upon conversion of
part of the Hsematoxylin into Hsematein by absorption of
oxygen from the air in presence of traces of free Ammonia.
By dissolving Heematoxylin crystals in Proof Spirit with -J- of
their weight of Ammonium Carbonate, and exposing to the
air in a shallow dish, the " ripening " process will be more
complete in 24 hours than after 3 months' exposure in the
usual way.

As solutions, even when made from the same formula,
differ widely in their staining power according to the ripen-
ing which the Heematoxylin has undergone, no very definite
directions as to dilution and time of immersion can be given.
As a rough guide, it may be stated that a well-ripened
Ehrlich's solution, diluted 1 to 4 of Distilled Water, and
Delafield's diluted 1 to 10, will stain well in about 20
minutes.

Accidental overstaining may be remedied by immersing the
sections in 70 p. c. Alcohol, to which -jV °r ^ Per cent, of
strong Hydrochloric Acid has been added. When the over-
staining is very deep, J p. c. Acid may be used to save time,
but requires caution. The sections are subsequently washed
in a dilute aqueous solution of Bicarbonate of Sodium ( 1 grain
in 2 ounces).

(i.) BOHMER'S H-ffiMATOXYLIN.

(a) Haematoxylin, 1 grm. ; Absolute Alcohol, 10 cc.

(b) Alum (Ammonia), 10 grms. ; Distilled Water, 200 cc.
Mix the two solutions, and after a week filter.

This is probably the earliest formula, as Hsematoxylin
staining was introduced by Bb'hmer.

6*± NUCLEAR STAINS.

(ii.) EHBLICH'S IIJEMATOXYLIN".

Hsematoxylin, 2 grms/; Absolute Alcohol, 100 cc. Dis-
solve and add — Glycerine, 100 cc. ; Distilled Water, 100 cc. ;
Alum (Ammonia), 2 grms. ; Glacial Acetic Acid, 10 cc.

It should be exposed to daylight for at least a month
before use, removing the stopper at intervals.

(iia.) EHBLICH'S H.ZEMATOXYLIN (Ammoniated).

Haematoxylin, 2 grms. ; Ammonium Carbonate, 0 '4 grms.;
Proof Spirit, 40 cc. : dissolve the Ammonium Carbonate and
the Hsematoxylin in the Proof Spirit and expose to the
air in a shallow dish for 24 hours, make up the volume to
40 cc. with Proof Spirit (warming if necessary to dissolve any
separated crystals) and add Ammonia Alum, 2 grms., dis-
solved in Distilled Water, 80 cc. ; Glycerine, 100 cc. ; Rectified
Spirit, 80 cc. ; Glacial Acetic Acid, 10 cc. This solution re-
quires no " ripening" and is ready to be diluted for use (1 — 10).

(iii.) DELAFIELB'S 1LZEMATOXYLIN.

To 400 cc. of a saturated aqueous solution of Ammonia
Alum add 4 grms. of Haematoxylin dissolved in 25 cc. of
Absolute Alcohol-; leave the solution exposed to the light
and air in an unstoppered bottle for 3 or 4 days ; filter, and
add to the filtrate 10Q,cc. of Glycerine and 100 cc. of Methylic
Alcohol (Wood Spirit); allow the solution to stand in the
light- until it is a dark colour, re-filter and preserve in a
stoppered bottle. , ,

Ammonia Alum dissolves about 1 in 1 1 of Water.

HJEMATOXYLIN. ,C?<P

itebmatoxylin, 12 grms. ; Alum (Ammonia), 50 grms. ;
GlySRhe, 65 cc. ; Distilled Water, 130 cc. ; boil, and while
hot add 5 cc. of liquid Carbolic Acid. Allow the mixture to
stand in daylight for at least a month to ripen before using.

I

NUCLEAR STAINS. 25

It is five to ten times stronger in Hsematoxylin than
other solutions, and the greater part of the Alum (85 p.c.)
crystallizes out on cooling.

When this solution is employed sections must be immersed
for a shorter time, or it must be more diluted than the other
solutions.

(y.) KLEINENBERG'S H-ffiMATOXYLIN.

The peculiarity of this solution is that it contains a quantity
of Chloride of Calcium, which, when staining in bulk, sets up
diffusion currents between the alcohol in the material to be
stained and the alcoholic staining solution, so- enabling the
latter to penetrate more rapidly.

Great differences exist in the formulae given for this solution. It would
appear from the Quarterly Journal of Microscopic Science, 1879, p. 208, that
Kleinenberg himself latterly used the following process : —

"Prepare a saturated solution of Calcium Chloride in 70 p. c. Alcohol,
with the addition of a little Alum; after having filtered, mix one
volume of this with 6 to 8 volumes of 70 p. c. Alcohol. At the time of
using the liquid pour into it as many drops of a concentrated solution of
Hsematoxylin in Absolute Alcohol as are sufficient to give the required
colour to the preparation, of greater or less intensity according to desire."

In an older formula, given by Balfouf and Foster in their " Elements of
Embryology," and copied into various text-books, a saturated solution of
Alum in 70 p. c. Alcohol is used instead of 70 p. c. Alcohol alone, for
diluting the strong Chloride of Calcium solution ; but as 70 p. c. Alcohol
only dissolves 1 in 1000 of Alum, this serves no useful purpose. It is also
directed that the Calcium Chloride solution should be saturated with Alum ;
but this is quite impracticable. Double decomposition takes plfwe.to^such
an extent that when saturated with Alum the liquid actually solidifies
from separatioCof Sulphate of Lime.

As Kleinenberg' s above formula is still very indefinite in regard to the
quantity of Alum used, and as the extent of the decomposition will vary
with the fineness of the Alum powder, the following modification
is ^recommended as giving 'an exact and uniform result. * -

Hsematoxylin, 2J grms. ; Crystallized
grms. in 10 cc. of Distilled Water; Alum, 3 grms. in 16
cc. of Distilled Water ; Rectified Spirit, 240 cc.

26 NUCLEAR STAINS.

<

Dissolve the Calcium Chloride and the Alum in their re-
spective quantities of water, by the aid of heat ; mix the solu-
tions and immediately dilute with the Eectified Spirit ; after
an hour filter and add the Hsematoxylin.

The above quantities make a good working solution. Any
dilution which is required must be made with some more of
the filtered liquid to which Hrematoxylin has not been added.

Crystallized Chloride of Calcium is preferred to the ordinary anhydrous
variety, on account of its greater purity : the anhydrous Chloride is
always alkaline. "Whichever be used, a saturated solution in 70 p. c.
Alcohol will contain about 40 p. c. of CaCls.

As in the finished solution, the whole of the Aluminium exists as
Chloride, it has been proposed to avoid the double decomposition with
Alum, by simply adding to the Chloride of Calcium solution a definite
quantity of Aluminium Chloride. It is difficult, however, to obtain this
latter free from uncombined Hydrochloric Acid, so that the double decom-
position process is the best.

Kleinenberg adds the Heematoxylin just before use. Lee says it is
best to add it 24 to 48 hours previous to use, with the additional note that
the solution does not keep ; while Stirling says it should be kept for at
least a month before it is wanted.

The solution is principally used for staining in bulk, and as
a rule the larger the object the weaker in Haematoxylin
should the solution be made to stain slowly and uniformly.

(vi.) WEIGERT'S HJEMATOXYLIN for the central
nervous system. (See p. 48.)

Haematoxylin, 1 ; Absolute Alcohol, 10 ; Distilled Water,
90 ; aqueous solution of Lithium Carbonate (1 in 70), 1.

(vii.) RENAUT'S GLYCERINE HJEMATOXYLIN.

Saturate Glycerine with Potash Alum, and to it add drop
by drop a saturated solution of Haematoxylin in 90 p. c.
Alcohol to form a deeply coloured solution ; let it stand
exposed to daylight for at least a week, and filter.

Both this and the following solution are employed for

NUCLEAR STAINS.

27

mounting unstained sections. After some time the sections
absorb the colour and become stained.

(viii.) RENAUT'S EOSINATED GLYCERINE HJE-
MATOXYLIN.

Concentrated aqueous solution of Eosine, 30 cc. ; saturated
solution of Haematoxylin in Alcohol, 40 cc. ; saturated solution
of Potash Alum in Glycerine, 130 cc. ; mix and let it stand
in a beaker for 5 or 6 weeks, so that the Alcohol may evapo-
rate, but protected from dust, then filter.

If desired, the nitrate can be diluted with Glycerine.

(ix.) HEIDENHAIN'S H2EMATOXYLIN METHOD.

(a} Hsematoxylin, 1 grm. ; Distilled Water, 300 cc.

(b) Chromate of Potassium, 1 grin. ; Distilled Water,
200 cc. Small pieces which have been hardened in Alcohol
or Picric Acid are placed in solution (a) for 12 to 24 hours ;
and then transferred for a similar time to solution (I}. Wash
the pieces thoroughly in Water, then dehydrate in Alcohol
for embedding (saturation) in Paraffin. (See p. 17.)

This process is used for staining in bulk previous to serial
cutting.

(x.) BENDA'S COFFER HJEMATOXYLIN METHOD.

For use after hardening in Chromic Acid or Flemming's
Solution. Sections are placed for 24 hours in a 5 p. c. solu-
tion of Neutral Acetate of Copper, kept at about 40° C. (104° F.)
washed thoroughly in Distilled Water ; stained in a saturated
aqueous solution of Hsematoxylin, until the sections become
dark grey or blackish. Decolourise to a rather light yellow
with Hydrochloric Acid diluted 1 to 500 of Water. The sec-
tions must then be brought to a light blue colour by a second
immersion in the copper solution. Wash in Water, dehy-
drate, clear and mount in Balsam.

28 NTJCLEAK STAINS.

CARMINE.

The principal uses to which. Carmine is applied are —
(1) Staining in "bulk. (2) Staining sections of the nervous
system. For the former, Grenacher's Alcoholic Borax Carmine
is generally used, and for the latter Ammonia or Lithium
Carmine, Grenacher's Alum Carmine, or Czoker's Alum
Cochineal. (3) Staining Vegetable sections.

For sections other than the nervous system Lithium Car-
mine or Borax Carmine are useful stains.

Carmine under ordinary circumstances acts as a general
stain, affecting the ground tissue as well as the nuclei. By
subsequent treatment with Acidulated Alcohol or Acidulated
Glycerine the colour is discharged from the ground tissue
without seriously affecting the nuclei. Used in this way,
Carmine becomes a good nuclear stain. It is also used exten-
sively by some histologists in the form of Picro-carmine
Solution.

Sections stained with the Carmines (except Alum Carmine)
should not be washed in Water as the colour will be to a great
extent removed, nor in Acidulated Water as the Carmine is pre-
cipitated on the sections. Acidulated Alcohol containing ^ or
1 p. c. of Hydrochloric Acid (sp. gr. 1/16), in 70 p. c. Alcohol,
or Acidulated Glycerine containing 1 p.c. of Glacial Acetic
or of Formic Acid (sp. gr. 1*2), in equal parts of Glycerine and
Water, should be employed. The former if Balsam be used
for mounting, the latter if Farrant or Glycerine be employed.

STAINING IN BULK.

This method is used only in preparing material for serial
cutting.

Tissues are placed in Grenacher's Alcoholic Borax Carmine
from one to three days. They are transferred from the stain

NUCLEAR STAINS. 29

into ^ p. c. Acidulated Alcohol till permeated by the acid,
placed for a day in 90 p. c. Alcohol, and finally saturated with
Absolute Alcohol before embedding.

NERVOUS SYSTEM.

Sections of spinal cord which has been hardened in Alcohol,
are generally placed in Beale's Ammonia Carmine for two or
three days, washed in 90 p. c. Alcohol, cleared, and mounted
in Balsam. Much better staining however is obtained by
using a stronger Carmine Solution, such as Lithium Carmine
or an Ammonia Carmine five times the strength of Beale's
Solution, q.v. Sections are stained in this for 24 hours and
washed either in 90 p. c. Alcohol, or 70 p. c. Alcohol acidu-
lated with yV p. c. Hydrochloric Acid. They may also be
stained in either of the Alum Combinations as mentioned
below.

When the central nervous system has been hardened in
Chromates, a very prolonged immersion in Ammonia Carmine
Solution is necessary, but in order to obviate this delay the
following plan (due to Merkel) may be adopted :—

Sections are first placed in a solution of Chloride of Pal-
ladium (1 in 500) till they are of a straw colour (about 10 —
1 5 minutes) ; rinsed in Distilled Water ; stained in the Ammonia
Carmine Solution (where they quickly become an intense red),
washed in 90 p. c. Alcohol, dehydrated, cleared, and mounted
in Balsam. The medullary sheaths are stained yellow by the
Palladium Solution, while the Carmine stains the neuroglia,
axis cylinders, and ganglion cells a deep red.

Sections hardened either in Alcohol or the Chromates are
advantageously stained in Alum Carmine or Alum Cochineal.
Both of these solutions stain much more rapidly than Ammonia
Carmine. The colour approaches to a violet, and is quite
distinct from the red of Ammonia Carmine.

30 NUCLEAR STAINS.

Either solution will stain Alcohol-hardened tissue in 30
minutes. Tissues hardened in the Chromates require 3 to 4
hours. When washed in 90 p. c. Alcohol (without acid),
cleared and mounted in Balsam, good differential staining
results. If, however, sections be left in the stain for 24
hours as recommended by some writers, overstaining takes
place, and treatment with Acidulated Alcohol becomes neces-
sary. This method also yields very good results, perhaps
slightly better than the previous one, but occupies a much
longer time.

FOR GENERAL USE.

Sections are stained with Lithium or Borax Carmine for
15 minutes to 2 hours according to the nature of the tissue
and the depth of stain required, then treated with Acidulated
Alcohol to decolourise the ground tissue, and so bring out
the nuclei, washed in 90 p. c. Alcohol, cleared and mounted
in Balsam.

As an example, a section of skin stained in Lithium Car-
mine for 50 minutes will be well differentiated by treatment
with 1 p. c. Acidulated Alcohol for 10 — 15 minutes; when
stained in Alcoholic Borax Carmine for 2 hours, acid wash-
ing for 4 minutes is sufficient for most purposes.

If Alum Carmine be used for the same length of time,
treatment in Acidulated Alcohol is not required. The
stained sections are first washed in water, then in 90 p. c.
Alcohol, cleared and mounted in Balsam.

COUNTERSTAINING. See Plasmatic Stains.

(i.) AMMONIA CARMINE.

All Alkaline Solutions are good solvents for Carmine, arid

NUCLEAR STAINS. 31

a Solution in Ammonia is stated to have been introduced in
1858 by Gerlaeh, the formula being: —

Carmine, 1; Strong Liquid Ammonia, 1; Water, 100; the
mixture exposed to the air for 24 hours to allow excess of
Ammonia to evaporate, then filtered.

This solution easily undergoes change by the action of
moulds and putrefactive ferments ; but if time be given for
these changes to run their course (some authors say " some
months," others "two years") the solution, after nitration,
keeps well and stains more precisely than a fresh solution.
Beale's Solution containing Glycerine and Alcohol keeps
much better than a simple Aqueous Solution, and answers
every purpose. Frey's Solution is similar to Beale's. The
Ammonia Carmine stain washes out completely in Water,
also to some extent in 70 p. c. Alcohol, but not at all in
90 p. c. Alcohol.

(ii.) BEALE'S AMMONIA CARMINE.

Carmine, 10 grs. ; Strong Solution of Ammonia, 30 mins. ;
Distilled Water, 2 oz. ; Alcohol, J oz. ; Pure Glycerine, 2 oz.
Dissolve the Carmine in the Ammonia with the aid of heat ;
boil for a few seconds and let it cool; allow the excess of
Ammonia to evaporate, and add the Water, Alcohol, and
Glycerine ; filter.

This solution will keep for months. Sometimes a little Carmine is
deposited owing to escape of Ammonia, in which case 1 or 2 drops of
Liquor Ammonise may be added to the 4 oz. of Carmine Solution.

(iii.) STRONGER AMMONIA CARMINE.

Carmine, 1 grin. ; Strong Solution of Ammonia, 2 cc. ; Dis-
tilled Water, 16 cc. ; Alcohol, 8 cc. ; Glycerine, 16 cc. ; pro-
ceed as directed for Beale's Solution.

32 NUCLEAR STAINS.

(iv.) GRENACHEK, S ALUM CARMINE.

Carmine, 1 grm. ; Alum (Ammonia), 5 grms. ; Distilled
Water, 100 cc. Dissolve the Alum in the Water, add the
Carmine, and boil the solution for 20 minutes, allow it to cool,
filter, and add Distilled Water to make the volume 100 cc.

This solution is chiefly used for the nervous system, but for
ordinary staining it possesses the advantage that it is nuclear
without after-treatment with acid.

(v.) CZOKER'S ALUM COCHINEAL.

Powdered Cochineal, 1 grm. ; Alum, 1 grm. ; Distilled
Water, 100 cc.

Dissolve the Alum in the Water, add the Cochineal and
boil ; evaporate down to half of its original bulk, filter and
add -| cc. of Liquid Carbolic Acid to the solution.

This is an excellent nuclear stain, and is chiefly used for the
central nervous system after hardening in Chromium Salts.
It is rather better for this purpose than Alum Carmine.

(vi.) GRENACHER'S BORAX CARMINE.

Carmine, 0-5 grm. ; Borax, 2 grms. ; Distilled Water,
100 cc. Heat the mixture to boiling point ; then add a 5 p. c.
solution of Acetic Acid, drop by drop, until the purple
colour turns red ; allow the mixture to stand for 24 hours,
decant, filter, and add a few drops of Carbolic Acid to preserve
the solution.

About 17 cc. of the Acetic Acid will be required.

(vii.) GRENACHER'S ALCOHOLIC BORAX CAR-
MINE.

Carmine, 3 grms. ; Borax, 4 grms. ; Distilled Water
100 cc. Dissolve the Borax in the Water ; add the Carmine

NUCLEAR STAINS. 33

and heat gently ; then add 100 cc. of 70 p. c. Alcohol. Filter
the solution (if necessary) before use. This is the best solu-
lution for staining in bulk. The pieces of tissue should be
placed in the solution for 1 to 3 days, and then transferred to
Acidulated Alcohol (p. 28).

Both of the above Borax Carmines are used for staining in
bulk, but the Alcoholic solution is better adapted for the
purpose, and keeps better than the aqueous which is apt to
gelatinize after a time. It also makes a good stain for
sections.

Although Acetic Acid is added to the Aqueous Solution, it does not
remain uncombined, so that the staining has still to be supplemented by
the usual Acid treatment.

(viii.) OBTH'S LITHIUM CARMINE.

Carmine, 2-5 grms. ; Saturated Watery solution of Car-
bonate of Lithium, 100 cc. Digest the Carmine in the Lithium
solution and filter.

Saturated Solution of Lithium Carbonate contains 1 grin. Carbonate
of Lithium in 70 cc. of Distilled Water.

A very useful stain. Like other alkaline Carmine solu-
tions, it stains both nuclei and ground substance, but is
restricted to the nuclei by treating the sections for a short
time with Acidulated Alcohol p. 28).

Lithium Carmine stains alcohol-hardened spinal cord very
nicely and quickly. When the staining is deep, extraction
with Acidulated 70 p. c. Alcohol gives a sharper definition
than Acidulated 90 p. c. Alcohol.

PICRQ CARMINES.

Combinations of Carmine with Picric Acid have largely
been used as effective double-stains. Various formulae for
these solutions exist, in which the Carmine is held in solution

D

34 NUCLEAR STAINS.

by Ammonia, Soda, or Lithia ; but as the neutral picrates of
these alkalies have in themselves no staining power, and the
manner in which the Picric Acid is combined has never been
accurately determined, no scientific explanation of their
action can be given.

The following formula and method is that generally recom-
mended, and in a large number of comparative experiments
was found to give the best results.

(1.) AMMONIA FICIIO CARMINE.

Carmine, 1 grm. ; Strong Solution of Ammonia, 3 cc. ;
Distilled Water, 5 cc. Dissolve the Carmine in the Ammonia
and Water with a gentle heat, then add Saturated Aqueous
Solution of Picric Acid, 200 cc. ; heat to boiling and filter.
This solution gives good results when used as follows : —
Take a section which has been rinsed in Distilled Water and
lay it out flat on a glass slide, drain off the superfluous water,
then pour on to the section several drops of the Picro-Carmine
Solution, warm the slide over a spirit lamp to a heat that can
be borne by the hand when touched with the glass (if the
section be too strongly heated it will shrivel), keep it about
this temperature for 5 or 10 minutes, remove the excess of
stain by tilting the glass and wiping it with a cloth or filter
paper, leaving some of the stain in the section, then place
one or two drops of Formic Farrant (p. 85) upon the section
and apply the cover-glass. The staining of the section is
much improved after it has been mounted 2 or 3 days and
exposed to daylight.

A section of skin gives the most striking results by this
method. Nuclei and the transverse muscular fibres stain
red, the remainder yellow.

The following formula for Eanvier's Picro-Carmine, pub-
lished in " Lee's Microtomists' Vade Mecum," 1890, p. 82, was

NUCLEAR STAINS. 35

given to Lee by Yignal as used in the Laboratory of Histology,
of the College of France.

" Water, 1000; Picric Acid, 20; Carmine, 10; Ammonia, 50.

' ' Put them into a stoppered bottle, and leave them for 2 or 3
months in a warm place ; then place the solution in a large
crystallizing dish and allow it to putrefy. When the liquid
has become reduced to four-fifths of its original bulk, remove
the crystals that have formed at the bottom, dry them and
dissolve them in a little Warm Water. Filter the solution,
and examine it with the microscope to see if the Carmine is
really dissolved ; if not, add more Ammonia and Water, and
repeat the above process. When the Carmine is so combined
as to dissolve in the warm Water, then dissolve it, evaporate
the solution to dryness in a stove, and reduce the Picro-
Carminate of Ammonia to powder.

" For use dissolve 1 grm. of the powder in 100 cc. of Distilled
Water."

(ii.) SODA FICRO-CARMINE.

»ustic Soda, 1 grm. ; Distilled Water, 1000 cc. ; dissolve
add Carmine, 10 grms. ; boil, filter and make up the
volume to 1000 cc. with Distilled Water; mix this with an
equal volume of Distilled Water, and add 1 p. c. aqueous
solution of Picric Acid so long as the turbidity, thus pro-
duced, continues to disappear on agitation.

(iii.) PICRO LITHIUM CARMINE.

Lithium Carmine solution, 100 cc. ; saturated aqueous solu-
tion of Picric Acid, 200—300 cc.

ANILINE NUCLEAR STAINS.

Fuchsine (Magenta), Gentian Yiolet, Dahlia Violet, Methy-
lene Blue, Methyl Green, Hoffmann's Blue, Safranine, and
Vesuvine are indirect nuclear stains.

36 NUCLEAR STAINS.

The nuclei are differentiated from the ground substance
by Alcohol washing.

A washing in Distilled Water,* previous to the Alcohol, has
the effect of fixing the stain in the nuclei so that it is less
readily washed out in the Spirit. This is especially the case
with Methylene Blue and Methyl Green, which must be washed
thoroughly in water previous to being treated with Alcohol.
If this be not done the colour will be almost completely
removed by the Spirit. Treated in this way, however, the
nuclear staining is very fine.

Vesuvine may or may not be previously washed in Dis-
tilled Water, according to the depth of stain required. If
water be used the colour is stronger, but less easily removed
from the ground tissue. The Yiolets and Safranine are better
washed out at once in the Spirit without previous washing
with water.

The differentiation in 90 p. c. Alcohol will vary from 10
to 30 minutes, according to the nature of the section.

When sections are to be also treated with one of the Plas-

*

matic stains, the counterstaining will of course be doi^pre-
vious to the dehydration in Absolute Alcohol.

(i.) METHYLENE BLUE.

Methylene Blue, 0'25 grm. • Eectified Spirit, 20 cc. ; Distilled
Water, 80 cc.

To stain the nuclei, sections are placed in this solution for
5 minutes, rinsed thoroughly in Distilled Water, differentiated
in 90 p. c. Alcohol, dehydrated, cleared and mounted in
Balsam.

(ii.) METHYL GREEN.

Methyl Green, 0'5 grm. ; Eectified Spirit, 20 cc. ; Distilled
Water, 80 cc.

To stain the nuclei, sections are placed in this solution for

NUCLEAR STAINS. 37

5 minutes, rinsed thoroughly in Distilled Water" , then differen-
tiated in 90 p. c. Alcohol, dehydrated, cleared and mounted in
Balsam.

The above solution, to which 1 p. c. of Glacial Acetic Acid
has been added, is employed for fixing and staining nuclei
in fresh vegetable tissues.

Methyl Green has been used for obtaining the peculiar pink
reaction with amyloid tissue, but this is probably due to the
fact that most methyl-greens contain methyl violet, as an
impurity. Methyl green free from violet does not give the
reaction.

(ii.a] Ekrlich-Biondi Fluid.

This is usually given as follows : — Saturated Aqueous
Solution of Methyl Green, 5 cc. ; Saturated Aqueous Solution
of Orange, 10 cc. ; Saturated Aqueous Solution of Acid
Fuchsine, 2 cc. ; for use dilute with about 40 volumes of
Water.

As these concentrated solutions form a precipitate when mixed, it is
better to dilute each of them with 20 to 40 volumes of water before
mixing1.

The solution is conveniently made as follows: — Methyl Green, 0-5
grm. ; Distilled "Water, 100 cc. : Acid Fuchsine, 0'5 grm. ; Distilled
Water, 40 cc. : Orange, 2 grms. ; Distilled "Water, 200 cc. Mix the
solutions and filter before use.

In some cases (serial sections for instance) it is convenient to use all
the dyes in one solution, but in other cases it is better to stain the nuclei
with Methyl G-reen and counter-stain with Rubin and Orange.

Sections are usually placed in this stain for 12 hours, then washed,
dehydrated, cleared and mounted.

This triple stain has also been strongly recommended as a counterstain
to Hsematoxylin, but in that case the Methyl Green is not required —
Rubin and Orange (p. 42) answers equally well, if not better.

(ii.J) Iodine Green.

This is another name for Methyl Green. Formerly Methyl
Iodide was employed in the manufacture of this green, and

38 NUCLEAR STAINS.

hence the names Methyl Green and Iodine Green. Now
Methyl Bromide or Methyl Chloride is used in its place.

Iodine Green always contained large quantities of Violet,
which was most probably the cause of the amyloid reaction
assigned to this dye (see Methyl Green}.

(iii.) SAFRANINE.

Safranine, 0-5 grm. ; [Rectified Spirit, 20 cc. ; Distilled
Water, 80 cc.

A good nuclear stain, and especially useful for tissues
that are rapidly growing and dividing.

For nuclear staining, sections are placed in the solution for
5 to 10 minutes, differentiated in 90 p.c. Alcohol, dehy-
drated, cleared and mounted in Balsam.

(iv.) VESUVINE (Bismarck Brown).

Vesuvine, 0-5 grm. ; Eectified Spirit, 20 cc. ; Distilled "Water,
80 cc.

A good nuclear stain, which is not easily washed out, and
is permanent.

Sections are placed in this for 5 minutes, washed in 90 p.c.
Alcohol with or without previous treatment with water,
dehydrated, cleared and mounted in Balsam.

(v.) GENTIAN VIOLET.
DAHLIA VIOLET.

Gentian or Dahlia Violet, 0'5 grm. ; Eectified Spirit, 20 cc. ;
Distilled Water, 80 cc.

To stain the nuclei, sections are placed in either of these
Violet solutions for 5 minutes, then washed in 90 p.c. Alcohol,
cleared and mounted in Balsam.

Gentian and Dahlia as well as Methyl Violet are generally
included amongst the nuclear stains, but they are more gene-

PLASMATIC STAINS. 39

rally used as Specific Stains. Gentian and Methyl Violets for
micro-organisms ; Methyl Violet for amyloid degeneration, and
Dahlia Violet to demonstrate the " mastzellen."

Dahlia Violet has a strong red tint and Methyl Violet &
strong blue tint, while Gentian Violet is intermediate between
the two. Gentian and Dahlia are both very fine nuclear stains,
but Methyl Violet washes out too readily in spirit to be of
much use.

Gentian Violet may be made directly nuclear by adding 1
p. c. of Glacial Acetic Acid to the solution.

(vi.) PUCHSINE (Magenta).

Fuchsine, 1 grm. ; Distilled Water, 150 cc. ; Eectified Spirit,
50 cc. ; dissolve and add Glycerine, 200 cc.

This fluid was devised by Dr. Ferrier for staining blood
corpuscles. The sp. gr. is similar to that of Liquor Sanguinis.
The coloured corpuscles of non-mammalian vertebrates alter
but little in shape while they become stained (Rutherford?*
Histology. 1876).

(vii.) HOFFMANN'S BLUE.

Hoffmann's Blue, 1 grm. ; Eectified Spirit, 20 cc. ; Distilled
Water, 80 cc. ; Glacial Acetic Acid, 0-5 cc.

Immerse the sections for 10 minutes, rinse in Water, wash
in 90 p.c. Alcohol, dehydrate, clear, and mount in Balsam.

FLASMATIC STAINS.

These colour the whole tissue uniformly. They are used as
ground stains in connection with the nuclear and specific stains
for the sake of contrast.

After the treatment for nuclear or specific staining, tissues
if taken from Alcohol are placed for about a minute in Distilled

40 PLASMATIC STAIKS.

Water, then transferred to the Plasmatic stain and treated as
directed.

After Hsematoxylin, other Blues, and Green, use either
Benzopurpurine (brownish red), Eosine (yellowish red), Ery-
throsine (pink), Orange, Eubin S., or Eubin and Orange ;
after Carmine and other Beds, use Picric Acid (yellow).

(i.) BENZOPURPURINE.

Benzopurpurine, 0-25 grm. ; Eectified Spirit, 20 cc. ;
Distilled Water, 80 cc.

A good contrast stain to Hcematoxylin and other blue
nuclear stains. Sections are placed in this for 2 to 5 minutes,
then washed in 90 p.c. Alcohol, dehydrated, cleared and
mounted in Balsam.

(ii.) EOSINE.

Eosine (Water-soluble), 1 grm. ; Eectified Spirit, 40 cc. ; Dis-
tilled Water, 160 cc.

For counterstaining, the sections are placed in this solution
for 2 to 5 minutes, then washed in 90 p.c. Alcohol, dehy-
drated, cleared and mounted in Balsam.

There are two classes of Eosines, characterised by their more ready
solubility in water or in spirit. Each is useful according to the nature
of the mounting media to be employed.

It is frequently stated that, when mounting in Balsam, the
Eosine must be dissolved in the Alcohol used for dehydrat-
ing to prevent the stain washing out, but this is not at all
necessary if the " water-soluble " variety be used.

It is a specific stain for red blood corpuscles, which it colours
a copper Ted ; also for the giant cells of Leprosy and Tuber-
cle, staining them an orange-yellow.

PLASM ATIC STAINS. 41

(iii.) ERYTHROSINE.

Erythrosine, 1 grm. ; Rectified Spirit 40 cc. ; Distilled
Water, 160 cc.

For counterstaining, sections are placed in this solution for
2 to 5 minutes, then washed in 90 p.c. Alcohol, dehydrated,
cleared and mounted in Balsam.

(iv.) ORANGE.

Orange, 2 grms. ; Rectified Spirit, 20 cc. ; Distilled Water,
80 cc.

Sections are placed in this solution for 10 minutes, washed
in 90 p.c. Alcohol, dehydrated, cleared and mounted in
Balsam.

(v.) PICRIC ACID (ALCOHOLIC).

Picric Acid, 1 grm. ; 70 p.c. Alcohol, 100 cc.

Principally used for staining fibrous tissue (skin, bone,
&c.).

For counterstaining after Carmine omit the treatment with
Acidulated Alcohol (p. 28), and rinse the sections for a
couple of minutes in 70 p.c. Alcohol ; transfer to Picric Acid
about 5 minutes ; dehydrate in Absolute Alcohol 3 or 4
minutes ; clear in Cedar Oil ; mount in Balsam.

It has been stated that Picric Acid must be added to the
Alcohol used for dehydrating the sections, to prevent the
stain washing out, but this is not necessary.

(vi.) ACID RUBIN.

Acid Fuchsine, 1 grm. ; Rectified Spirit, 40 cc. ; Distilled
Water, 160 cc.

For counterstaining, sections are placed in this solution for
5 to 10 minutes, washed in 90 p.c. Alcohol, dehydrated,
cleared and mounted in Balsam.

42 TLASMATIC STAINS.

The dyes sold under the names Acid Rubin, Acid Fuchsine,
and Acid Magenta have practically the same composition and
give the same reactions, although different samples may vary
somewhat in the shade of colour.

(vii.) RUBIN AND ORANGE.

Acid Fuchsine, 1 grm. ; Orange, 6 grms. ; Rectified Spirit,
60 cc. ; DistiUed Water, 240 cc.

For counterstaining, sections are placed in this for 5 to 10
minutes, washed in 90 p.c. Alcohol, dehydrated, cleared and
mounted in Balsam.

When the elements of the ground tissue have the same
affinity for each of the two colours, the result is a rich orange-
red; where any selective affinity exists, either of the two
colours will be more pronounced.

SPECIFIC STAINS. 43-

SPECIFIC STAINS.

These may be defined as stains which, possess a peculiar
affinity for certain elements in the tissues. The more impor-
tant are Osmic Acid, Chloride of Gold, Nitrate of Silver, Vic-
toria Blue, Dahlia Violet, Methyl Violet, Iodine, Safranine,
Eose Bengale, Nigrosine, Acid Fuchsine, and Congo Eed.

OSMIC ACID.

Aqueous solution, 1 p. c. (see also p. 8).

To be kept in an opaque bottle, and away from the light.

Useful for staining fatty elements, medullated nerve fibre,
and elastic fibres. It is especially valuable for demonstrating
fatty degeneration of organs.

Very small pieces of tissue in the fresh state are stained
and fixed at the same time. It is also used in combination
with Chromic Acid, as in Flemming's and Fol's solutions.

Material for sections should be hardened in Miiller's Fluid,
and cut on the freezing microtome. Alcohol partially dissolves
the fat, and is, therefore, unsuited for the hardening, except
when this is of no consequence. Osmic Acid also stains any
fat which is left after treatment with Alcohol.

Sections are placed for 6 to 12 hours in J to iVp- c- solution,
carefully protected from the light, or for J to \ hour in 1 p. c.
solution ; washed in Distilled Water, and mounted in Farrant
or Glycerine.

CHLORIDE OF GOLD.

\ or 1 p. c. solution in Distilled Water.

Commercial Chloride of gold is not the pure Chloride AuCls, but the
crystallized double Chloride of Gold and Sodium, containing 50 p. o. of
Metallic Gold.

Commercial Chloride of Gold and Sodium is the above crystallized
double Chloride mixed with an equal weight of Chloride of Sodium, and
contains 25 p. c. of Metallic Gold.

44 SPECIFIC STAINS.

Stains connective tissue corpuscles, and cartilage cells, but
is principally used for tracing nerve endings. Can only be
used for fresh, tissues.

The methods used are very numerous and will be found
well abstracted in Lee's Vade-mecum.

For fixed connective tissue corpuscles the following is the
simplest.

(i.) A small piece of perfectly fresh tissue is placed in a
|- p. c. solution for half an hour, the solution being kept in
the dark ; the tissue becomes yellow ; it is then washed in
Distilled Water and exposed to daylight in Distilled Water,
acidulated slightly with Acetic Acid. In a couple of days the
tissue becomes purplish or violet brown, and must be mounted
in Glycerine.

(iii.) For tracing nerve-fibres the process known as Ran-
vier's Lemon-Juice Method is generally applicable.

The fresh, tissue is soaked in Lemon juice for 5 to 10
minutes (40 grains of Citric Acid in an ounce of water forms
a convenient substitute) ; it is then rapidly washed in Dis-
tilled Water and transferred to a 1 p. c. Gold Chloride solu-
tion for from 10 minutes to 1 hour, according to the nature
of the tissue. After washing in Water it is placed in 50 cc.
of Water containing 2 drops of Acetic Acid and exposed to the
light, or — if we do not wish to retain the superficial epithe-
lium— after treating with. Lemon Juice and Gold, the tissue
is placed for 24 hours in Formic Acid (Sp. gr. 1-2) diluted
with three times its volume of Water, and kept in the dark.

NITRATE OP SILVER.

J- p. c. solution in Distilled Water.

Used for staining the tesselated epithelium of serous mem-
branes, lymphatics and blood-vessels, the matrix of connective

SPECIFIC STAINS. 45

tissue and cartilage, cancerous growths, and the eye ; it also
hardens tissue.

The tissue must he fresh and the sections very thin. They
are first washed in Distilled Water to remove the Chlorides,
and are then placed in the Silver solution for half an hour
in the dark. After this they are washed in Distilled Water,
placed in ordinary Water, and exposed to diffuse light till
brown ; they must he mounted in Glycerine or Glycerine Jelly,
and kept in the dark.

' ' Fixing " with Hyposulphite of Sodium has been suggested,
to render the specimens permanent in light.

VICTORIA BLUE.

Victoria Blue, 0'25 grm. ; Eectified Spirit, 20 cc. ; Distilled
Water, 80 cc.

Stain the sections 3 to 5 minutes, rinse in Distilled Water
about 2 minutes, wash in 90 p. c. Alcohol 3 or 4 minutes,
dehydrate in Absolute Alcohol 3 or 4 minutes, clear in Cedar
Oil, and mount in Balsam.

This is especially useful for demonstrating elastic tissue,
staining the elastic fibres an intense blue ; it is, however,
somewhat readily extracted by Alcohol.

It stains best after hardening in Chromium Salts, but this
is not necessary. It also acts as a nuclear stain.

DAHLIA VIOLET.

Dahlia Yiolet, 2 grms. ; Eectified Spirit, 25 cc. ; Distilled
Water, 70 cc. ; Glacial Acetic Acid, 5 cc.

Specially useful for demonstrating the granules in Ehrlich's
Mastzellen. The tissue must be hardened in Alcohol, as is
the case with bacteria. Stain for some hours, then wash out
in Alcohol until nearly colourless, dehydrate, clear in Cedar
Oil, and mount in Balsam.

46 SPECIFIC STAINS.

The uniformly round granules of these plasma cells (granu-
lar cells) are stained by" Aniline dyes (especially the Violets)
in a similar manner to bacteria, and are chiefly distinguished
from the latter by the fact that the granules are always
grouped in the form of cell-like structures, and the Aniline
stain is removed from the granules, but not from bacteria, by
treatment with a weak solution of Carbonate of Potassium.

STAINS FOB, AMYLOID DEGENERATION.
METHYL VIOLET.

Methyl Violet, 0-5 grm. ; Water, 200 cc. ; Glacial Acetic
Acid, 5 c.c.

Stain sections for 20 minutes ; wash thoroughly in Distilled
Water, then in a mixture of equal parts Glycerine and Water
BO long as any colour is taken out (generally about 2 hours)
and finally mount in Farrant.

The amyloid material is stained pink and the normal tissue
a bluish- green colour.

When stained as usual without the addition of Acetic Acid,
the colour-contrast is not so striking. It may, however, be
improved by after-treatment with dilute acid, but it is better
to combine the acid with the stain.

Most Methyl and Iodine Greens give the same reaction
owing to the quantity of Methyl Violet which they contain.

IODINE.

Iodine, 1 grm. ; Iodide of Potassium, 2 grms. ; Distilled
Water, 300 cc.

Solution is most easily effected by using 10 cc. of the
Water to dissolve the solids, then diluting with the remainder
j>f the water.

SPECIFIC STAINS. 47

This is the same as Gram's solution for Micro-organisms.

An alternative formula is : —

Liquor lodi, B.P. 10 cc. ; Distilled Water, 140 cc.

Stain the sections for 5 minutes ; wash in Water and mount
in thick Farrant to which a fourth of its volume of Liquor
lodi has been added.

Under the microscope (a low power answers best) by trans-
mitted light, the section appears coloured various shades of
yellow, but by reflected light the amyloid substance appears
almost black while the normal tissue remains unaltered.

SAFBANINE.

Safranine, 1 grm. ; Eectified Spirit, 20 cc. ; Distilled Water,
80 cc.

Stain the sections for 2 minutes and wash in water. The
amyloid substance gives a bright orange-red colour contrast-
ing well with the normal pink.

If to be mounted, wash the sections in a mixture of equal
parts Glycerine and Water, frequently changed until no
more colour is removed (12 hours is generally sufficient),
then in pure Glycerine in the same way, and finally mount
in Farrant.

By passing rapidly through 90 p.c. Alcohol and Absolute
Alcohol into Cedar Oil, the section may be mounted in Balsam,
but the bright orange colour of the Amyloid is always im-
paired by contact with spirit.

ROSE BENGALE.

Eose Bengale, 1 grm. ; Eectified Spirit, 20 cc. ; Distilled
Water, 80 cc.

Stain the sections for five minutes, wash in 90 p.c. Alcohol,
dehydrate, clear and mount in Balsam.

It demonstrates very well the commencement of amyloid

48 SPECIFIC METHODS.

degeneration, staining it a bright red round the blood vessels.
"With old amyloid the results are good, but not so striking.

This was noticed for the first time by "Warwick in my laboratory,
when using Rose Bengale in some experiments on ground stains for
different tissues.

Used as above it is an excellent stain for elastic fibres.

STAINS AND METHODS FOB NERVE CENTRES.
NIGROSINE. Aniline Black. Aniline Blue Black.

Nigrosine, 2 grms. ; Water, 100 cc.

Particularly useful for staining unhardened sections of
brain ; but it will stain equally well sections of spinal cord
which have been hardened in Alcohol,

Sections of hardened material are placed in this for 10 to
15 minutes, washed in 90 p.c. Alcohol, cleared and mounted
in Balsam.

ACID FUCHSINE.

Acid Fuchsine, 2 grms. ; Water, 100 cc.

Sections of hardened material are placed in this solution
for 2 or 3 minutes, washed in 90 p. c. Alcohol, cleared
and mounted in Balsam.

CONGO RED.

Congo Red, 2 grms. ; Water, 100 cc.

Sections are placed in this solution for 2 or 3 minutes,
washed in 90 p.c. Alcohol, cleared and mounted in Balsam.

This dye, but little known, is one of the best stains which
I have tried for the central nervous system.

WEIGERT'S METHOD.

The brain and spinal cord must be hardened in one of the
Bichromate Solutions, and completed in Alcohol.
For embedding in Celloidin or Gum, see p. 15.

SPECIFIC METHODS. 49

The pieces embedded in Celloidin are taken .from the spirit
and placed for 1 or 2 days in a Saturated Aqueous Solution of
Acetate of Copper diluted with an equal bulk of water, kept
at about 40° C. (104° F.), and then transferred to 80 p.c.
Alcohol till required for cutting : or the sections can be cut
first, and afterwards immersed in the Copper Solution.

The sections are well washed in 90 p.c. Alcohol and
transferred to Weigert's Hsematoxylin (p. 26) for a few
hours to 2 days, according to the differentiation required, they
should be opaque, and a deep blue-black ; and then well
washed for 2 or 3 days in Distilled Water.

They are now decolourised for J to 2 hours in the following
solution : —

Borax, 2 grms. ; Ferricyanide of Potassium, 2-5 grms. ;
Water, 200 cc.

While in this solution the sections must be carefully
watched, and removed as soon as the grey and white sub-
stances are sharply defined.

They are again washed in Water for half an hour, dehy-
drated, cleared, and mounted in Balsam.

The white substance of Schwann is coloured a purple
black, the connective tissue, axis cylinders, ganglion cells,
and degenerated tracts in cases of sclerosis, an orange brown.

PAL'S METHOD.

A modification of Weigert's. Takes less time, and admits
of counter-staining.

The sections after being cut are placed in the following
solution : —

Haematoxylin, 0-75 grms. ; Distilled Water, 90 cc. ; Abso-
lute Alcohol, 10 cc. — to which is added, just before use, a
saturated solution of Lithium Carbonate in the proportion of

E

50 SPECIFIC METHODS.

3 drops to every 10 cc. of the Hsematoxylin solution. This is
practically Weigert's Hsematoxylin.

In this stain the sections are allowed to remain from 5 to
6 hours until they are opaque, and have a dark bluish-black
colour.

They are then removed to Distilled Water and thoroughly
washed till no more colour comes away. If the sections are
not sufficiently stained a little Carbonate of Lithium solution
is added to the "Water.

The sections are now placed in 0'25 aqueous solution of
Permanganate of Potassium from 15 to 20 seconds to diffe-
rentiate ; they are then placed in —

Pal's Solution. — Oxalic Acid, 1 grm. ; Potasrium Sulphite,
1 grm. ; Distilled Water, 200 cc. ; and allowed to remain till
the white and grey matters are distinctly defined. This takes
from 1 to 2 minutes ; should black spots appear, the sections
must be replaced in the Permanganate of Potassium solution,
and put back into Pal's solution ; they are then washed for
15 minutes in Water.

By this means the medullary sheaths are stained a bluish-
black colour, while the rest of the tissue remains unstained.

The nuclei may now be stained in Alum Carmine.

Finally dehydrate, clear, and mount.

FAL-EXNER METHOD.

This is the most rapid of all, and takes about 3 days both
to harden and mount.

The spinal cord or brain is cut into J-in. cubes and immersed
in ten times its bulk of \ p.c. solution of Osmic Acid for 2
days, the solution being changed each day ; the pieces are
then washed in Water, transferred to Absolute Alcohol, and
embedded in Celloidin or Paraffin.

As the sections are cut they are placed in Glycerine, then

ft

SPECIFIC METHODS. 51

washed in Water, treated in the Permanganate of Potassium
and Pal's solution, and counter-stained with Carmine ; dehy-
drated, cleared, and mounted in Balsam.

GOLGI'S SUBLIMATE METHOD.

Small pieces (about J in. cubes) of the tissue are hardened
(15 to 30 days) in about 200 cc. of Muller's Fluid (p. 4) which
should be frequently changed.

They are then transferred to aqueous solution of Corrosive
Sublimate (0-25 to 1 p.c.) which must be changed from time
to time as it becomes coloured by the Bichromate.

The sublimate treatment takes from 8 to 10 days or even
longer.

Sections of the material thus treated are well washed with
water.

Subsequent treatment with a weak solution of Sulphide of
Sodium darkens the stain and makes it sharper.

STAINING CELLULOSE AND ITS
MODIFICATIONS.

Unaltered Cellulose. — This is stained by Carminer
Hsematoxylin, and most of the aniline dyes.

It is coloured blue with Chlorzine Iodine (Schulze's Solution) ;.
also by treatment with Iodine followed by Sulphuric Acid
diluted 1 to 3 of water.

It is dissolved by Sulphuric Acid (undiluted).

Staining with Carmine. — Place a section in Grenacher's
Alcoholic Borax Carmine (p. 32) for 15 to 30 minutes, rinse
for a second in Distilled Water, transfer to 90 p. c. Alcohol ;
dehydrate, clear, and mount in Balsam.

The Carmine washes out quickly in Water, therefore the
section must not be left there any length of time, but it is
better to just rinse the sections in Water, than to transfer
them direct from the Carmine solution to the Alcohol.

Cellulose, nuclei, and protoplasm in general are stained.

For staining nuclei only, see " Nuclear Staining," p. 20.

Staining with Hsematoxylin. — Kinse the sections in
Distilled Water for a minute or two, place them for 5
minutes in Ehrlich's or Delafield's Solution (p. 24) diluted
1 to 9 of Distilled Water; rinse in Distilled Water and transfer
to Tap Water (if alkaline) or to a weak solution of Bicar-
bonate of Sodium (1 grain to 2 ounces), until the staining
acquires a blue tint ; dehydrate, clear, and mount in Balsam.

STAINING CELLULOSE. 53

Staining with Aniline Dyes. — Methyl Green, Methylene
Blue, Safranine and Fuehsine give good results. Place sec-
tions in J p. c. solutions of either of these for 3 or 4 minutes,
rinse in Water, dehydrate, clear, and mount.

Lignified Cellulose. — When the cellulose undergoes the
change known as lignification, its reactions are altered.
It is coloured (i.) yellow or yellowish-brown by Chlorzinc
Iodine ; (ii.) red with Phloroglucin followed by Hydrochloric
Acid ; (iii.) yellow with Aniline Chloride. The results with
Phloroglucin are more definite and reliable than with Chloride
of Aniline.

It is generally stated that Haematoxylin and Carmine do
not stain lignified tissue. With Carmine this is practically
true; but while it is probable that pure "Lignin" is un-
affected by Hsematoxylin, the tissue generally takes on a
modified stain, which is lighter in proportion to the extent of
the lignification. In young stems it is sometimes noticed that
Hsematoxylin has a special staining power for the " middle
lamella " both of wood and bast, which Carmine, on the other
hand, does not affect at all.

The Aniline Dyes mentioned under " Cellulose Staining"
will also stain equally well lignified tissues.

It is stained by Picric Acid, but the dye washes out easily
in Water or Alcohol. Good results however can be obtained
by carefu treatment.

DOUBLE STAINING MIXED TISSUES.

The most striking results are obtained by using Carmine
and Methyl Green. The staining with Carmine and Picric
Acid is also very effective, but as the Picric washes out very
rapidly, the sections should not remain in the Alcohol longer
than is required to dehydrate them. A thick section takes

54 STAINING CELLULOSE.

longer to dehydrate, but holds the stain well ; a thin section
dehydrates quickly, but also rapidly loses the stain.

The differentiation with Aniline Blue and Picric Acid is not
so sharp as the above, but more delicate gradations are
obtained, from blue through green to yellow.

The general distribution of the lignified tissue is best
observed with a low power, say an inch object glass, when
the different colours should show distinctly.

With Carmine and Methyl Green. — Sections of stems
and roots containing cellulose and lignified tissue are rinsed
in Distilled Water ; place them in Methyl Green Solution for
3 or 4 minutes ; rinse in Distilled Water ; wash in 90 p. c.
Alcohol for 5 or 10 minutes, which to a great extent removes
the Green from the cellulose portion, and thus obtains a purer
red with the Carmine ; place the sections in Grenacher's Alco-
holic Borax Carmine for 15 or 20 minutes; rinse quickly in
Distilled Water to remove adhering stain; pass through
90 p. c. Alcohol, dehydrate, clear, and mount in Balsam.

Acid Green is frequently employed in the place of Methyl
Green, but if the latter is free from Violet, or nearly so, it is
the better of the two.

With Carmine and Picric Acid. — Einse the sections in
Distilled Water ; place them in Grenacher's Alcoholic Borax
Carmine 15 to 20 minutes ; rinse for a second in Distilled
Water; transfer to Picric Acid (Alcoholic) for 5 or 10 minutes;
dehydrate in Absolute Alcohol for 3 to 5 minutes, clear in
Cedar Oil, and mount in Balsam.

With Aniline Blue and Picric Acid. — Binse the sections
in Water; place them in Hoffmann's Blue (p. 56) for 10
minutes; transfer to Picric Acid (Alcoholic) for 10 minutes;
dehydrate in Absolute Alcohol for 3 or 4 minutes, clear in
Cedar Oil, and mount in Balsam.

STAINING CELLULOSE. 55

This method shows very well the bordered pits in a vertical
section of pine stem.

CHLOR ZINC IODINE (Schnlze's Solution).

No good working formula has been published for this
reagent : the points being to obtain a clear concentrated
solution which does not crystallize on keeping, and which
dissolves sufficient Iodine.

The following will yield a permanent solution, which gives
good results, staining unaltered cellulose a violet-blue and
lignified tissue yellow. Starch is of course stained blue.

Evaporate 100 cc. of Liquor Zinci Chloridi (B.P.) to 70 cc. ;
dissolve in it 10 grms. of Iodide of Potassium; then add
0-2 grm. Iodine : shake at intervals till saturated.

PHLOBOGLUCIN.

PMoroglucin, 1 grm. ; Rectified Spirit, 20 cc. ; Distilled
Water, 80 cc.

Sections containing lignified tissue placed in this solution
for about 1 5 minutes, then treated with strong Hydrochloric
Acid, are stained a cherry-red colour. The depth and distri-
bution of the colour are in proportion to the extent of the
lignification.

CHLORIDE OF ANILINE.

Chloride of Aniline, 2 grms. ; Rectified Spirit, 65 cc. ; Dis-
tilled Water, 35 cc. ; Strong Hydrochloric Acid, 2 cc.

Sections containing lignified tissue placed in this solution
for 15 to 30 minutes are stained a yellow colour, but the
differentiation is not so good as with Phloroglucin.

56

STAINING SIEVE AREAS.

METHYL GREEN.

Methyl Green, 0-25 grm. ; Eectified Spirit, 20 cc. ; Distilled
Water, 80 cc.

PICRIC ACID (Alcoholic).

Picric Acid, 1 grm. ; 70 p. c. Alcohol, 100 cc.

STAINING SIEVE AEEAS.

These are best stained by Hoffmann's Blue, but Eosine is
frequently employed for this purpose. They can also be
demonstrated by Haematoxylin, using the process given above
for cellulose.

HOFFMANN'S BLUE.

Hoffmann's Blue, l|grm. ; Eectified Spirit, 20 cc. ; Dis-
tilled Water, 80 cc. • Glacial Acetic Acid, 0-5 cc.

Place the sections in the stain for 5 to 10 minutes, rinse in
Distilled Water in which the colour is partially removed,
mount in Glycerine; or dehydrate, clear, and mount in Balsam.

This is also a very good nuclear stain.

EOSINE.

Eosine, 2 grins ; Eectified Spir.it, 20 cc. ; Distilled Water,
80 cc. Place the sections in the stain for 5 to 10 minutes,
rinse in Water, wash in 90 p. c. Alcohol, dehydrate, clear,
and mount in Balsam.

MICBO-OEGANISMS.

STAINS.

In the investigation of micro-organisms the following solu-
tions of the Aniline Stains and of the Intermediate Eeagents
are employed.

Different samples of the same aniline dye will differ in their
behaviour to solvents. Some will make a clear solution,
others will leave more or less insoluble residue. The solu-
tions should therefore be filtered or decanted. It is safer to
filter aqueous solutions each time before use.

Aqueous Solutions of the dyes do not keep well, therefore
Alcohol has been added as a preservative.

(i.) VESUVINE (Bismarck Brownk.

(a) Glycerine Solution. — Saturated ^solution in equal
parts of Glycerine and Distilled Water.

(b) Aqueous Solution. — Yesuvine, 1 grm. ; Rectified
Spirit, 20 cc. ; Water, 80 cc.

(ii.) METHYLENE BLUE.

(a] Saturated Alcoholic Solution.

Methylene Blue, 15 grms.; Absolute Alcohol, lOOcc. ; shake
at intervals for one or two days until saturated, then filter.

Only a small percentage of the dye is soluble in Alcohol, yet the depth
of colour obtainable in the solution increases in proportion to the quan-
tity added up to 15 p. c., but not further.

(b) Aqueous Solution. — Methylene Blue, 1 grin. ; Recti-
fied Spirit, 20 cc. ; Distilled Water, 80 cc.

58 MICRO-ORGANISMS.

A freshly made 2 p. c. solution is 50 p. c. stronger in colour ; but ifc
crystallizes out in cold weather. Owing to the influence of some soluble
impurity, a 10 p. c. solution gives a weaker colour than a 2 p. c.

(c) Koch's Methylene Blue Solution. — Saturated Alco-
holic Solution of Methylene Blue, 1 cc. ; 10 p. c. solution of
Caustic Potash, 0-2 cc. ; Distilled Water, 200 cc.

This formula is taken from Koch's paper, "Die Aetiologie der Tuber -
culose in Berliner Klinische Wochenschrif t, " 1882, No. 15, p. 221. In
most English text-books, the quantity of alkali is increased ten times,
owing to the omission of a decimal point in the translation from which
these are copied.

(d) Lb'ffler's Solution. — Concentrated Alcoholic Solution
of Methylene Blue, 30 cc. ; Solution of Caustic Potash (1 in
10,000), 100 cc.

(e) Kuhne's Carbolic Methylene Blue.— Methylene
Blue, 1-5 grms. ; Absolute Alcohol, 10 cc. Eub in a mortar,
and add 100 cc. of a 5 p. c. Aqueous Solution of Carbolic Acid.

(/) Xiihne's Aniline Oil Solution. — Eub up in a mortar
as much Methylene Blue as will go upon the point of a knife,
with 10 cc. of Aniline, and pour the whole unfiltered into a
bottle ; after a time the undissolved pigment will settle at the
bottom.

(in.) GENTIAN VIOLET.

(a) Concentrated Alcoholic Solution. — Gentian Violet,
25 grms. ; Absolute Alcohol, 100 cc.

(5) Aqueous Solution. — Gentian Yiolet, 1 grm. ; Eectified
Spirit, 20 cc. ; Distilled Water, 80 cc.

(c) *Gentian-Violet Aniline- Water.— Aniline Water, 100
cc. ; Concentrated Alcoholic Solution of Gentian Yiolet, 1 1
cc. ; Absolute Alcohol, 10 cc.

(iv.) METHYL VIOLET.

(a) Concentrated Alcoholic Solution.— Methyl Yiolet,
25 grms.; Absolute Alcohol, 100 cc.

* " Erhlich-Weigert-Koch " formula.

MICRO-ORGANISMS. 59

(i) Aqueous Solution. — Methyl Violet, 1 grm. ; Eectified
Spirit, 20 cc. ; Distilled Water, 80 cc.

(c) Xuhne's Solution. — Methyl Yiolet, 1 grm. ; Distilled
Water, 90 cc. ; Alcohol, 100 cc.

(d) * Methyl- Violet Aniline -Water. — Aniline Water,
100 cc. ; Concentrated Alcoholic Solution of Methyl Violet,
11 cc. ; Absolute Alcohol, 10 cc.

VICTORIA BLUE.

Victoria Blue, 2 grms. ; Alcohol (50 p. c.), 100 cc.

(vi.) FUCHSINE (Magenta).

(a) Concentrated Alcoholic Solution. — Fuchsine, 25
grms. ; Absolute Alcohol, 100 cc.

(i) Aqueous Solution. — Fuchsine, 1 grm.; Eectified Spirit,
20 cc. ; Distilled Water, 80 cc.

(c} *Fuchsine Aniline-Water.— Aniline Water, 100 cc. ;
Concentrated Alcoholic Solution of Fuchsine, 1 1 cc. ; Absolute
Alcohol, 10 cc.

(d) Ziehl-Neelsen Solution, or Kuhne's Carbolic Fuch-
sine.— Fuchsine, 1 grm. ; Absolute Alcohol, 10 cc. Dis-
solve and add 100 cc. of a 5 p. c. Aqueous Solution of Carbolic
Acid.

(e] Magenta Solution (Gibbes'). — Magenta, 2 grms. ;
Aniline Oil, 3 grms. ; Eectified Spirit, 20 cc. ; Distilled Water,
20 cc.

(/) Gibbes' Double Stain. — Magenta, 2 grms. ; Methy-
lene Blue, 1 grm. Well triturate the mixture, and then add
slowly Aniline Oil, 3 cc., dissolved in Eectified Spirit, 15 cc. ;
subsequently add Distilled Water, 15 cc., and keep stain in
a stoppered bottle. . •

* " ErHich-Weigert-Koch " formula.

60 MICRO-ORGANISMS.

(vii.) SAFRANINE.

Kuline's Aniline Oil Solution. — Made in the same way
as that of Methylene Blue.

Babes' Solution. — Saturate Aniline Water with Safranine
at 60° C. (140° F.), and filter.

(viii.) METHYL GREEN.

Aniline Oil Solution. — Made in the same way as Methy-
lene Blue.

(ix.) BLACK BROWN.

Carbolic Black Brown. — Made in the same way as Car-
bolic Euchsine.

(x.) ORSEILLE (Wedl).

Glacial Acetic Acid, 5 cc. ; Absolute Alcohol, 20 cc. ; Dis-
tilled Water, 40 cc. : mix and add Orseille (Archil, from which
excess of Ammonia has been driven off), to form a dark red-
dish fluid.

Israel has more recently used a strong solution of Orcein in
Acetic Acid.

INTERMEDIATE EEAGENTS.

(i.) GUAM'S SOLUTION.

Iodine, 1 grm. ; Iodide of Potassium, 2 grins. ; Distilled
Water, 300 grms.

(ii.) ANILINE WATER.

Distilled "Water, 100 cc. ; Aniline, 5 cc. To be well shaken
and the emulsion filtered twice.

This is the formula generally given, but as the solubility of
Aniline is 1 in 27 of Distilled Water, a clear solution of prac-
tically the same strength can be obtained by mixing 3 cc. of
Aniline with 90 cc. of Distilled Water ; it can also be made
extemporaneously by pouring a few drops of Aniline into a
test-tube and adding Distilled Water gradually (with agita-
tion) until the turbidity disappears. Aniline Water soon
spoils, and therefore should be made fresh as required. With
the addition of 10 p. c. of Alcohol it keeps much better.

(iii.) WEAK ACIDULATED WATER.

Hydrochloric Acid, 1 cc. ; Distilled Water, 1000 cc.

(iv.) LITHIA WATER.

Saturated Aqueous solution of Lithium Carbonate (1 in 70),
1 cc. ; Water, 30 cc.

62 MICRO-ORGANISMS.

(v.) SULPHURIC ACID AND ALCOHOL.

Sulphuric Acid (sp. gr. 1-84), 10 cc. ; Alcohol (90 p. c.),
90 cc.

(vi.) SULPHURIC ACID.

Sulphuric Acid (sp. gr. 1-84), 10 cc. ; Distilled Water,
30 cc.

(vii.) NITRIC ACID.

Pure Nitric Acid (sp. gr. 1-42) 10 cc. ; Distilled Water,
30 cc.

(viii.) SULPHANILIC AND NITRIC ACID.

Sulphanilic Acid (saturated aqueous solution), 30 cc. ; Nitric
Acid (sp. gr. 1'42), 10 cc.

(ix.) DILUTED ACETINE (CHLORHYDRIN) BLUE.

Acetine Blue (Chlorhydrin Blue), 10 grms. ; Absolute
Alcohol, 10 cc. ; Distilled Water, 90 cc.

Chlorhydrin Blue mentioned by Kiihne is not obtainable
commercially. Acetine Blue appears to be the same article.

(x.) PLUORESCINE.

Alcoholic Solution. — Yellow Fluorescine, 1 grm., rubbed
in a mortar with 50 cc. of Absolute Alcohol; the whole poured
into a bottle and allowed to settle.

GEKEKAL METHODS.

WITH ANILINE VIOLETS.

It would appear that the first application of Aniline colours
to Bacteriological investigation was due to Weigert, who
stained in a simple aqueous solution of Methyl Yiolet.

To better fix the colouring matter, Erhlich used a saturated
solution of Aniline in Water as a mordant for the dye, the
dye being dissolved in this, or added in the form of a con-
centrated alcoholic solution till a slight opacity appears.

Weigert subsequently proposed definite volumes of the solu-
tions which have been generally adopted, with the further
addition (by Koch) of 10 per cent, of Absolute Alcohol to
make a more permanent solution. Koch states that the solu-
tion so made will remain good for 10 days, without requiring
to be filtered each time of using. Weigert later substituted
Gentian Yiolet for Methyl Violet, as the former stains more
quickly and does not wash out so readily in Alcohol.

Koch produces an isolated staining of bacteria in sections,
by staining them in aqueous solution of Methyl Violet,
Fuchsine, or Methylene Blue, and washing them in a saturated
aqueous solution of Carbonate of Potassium diluted with an
equal bulk of water ; this removes the colour from the nuclei,
leaving the bacteria stained : the sections are then dehy-
drated, cleared and mounted.

The general methods now adopted are : —

64 MICRO-ORGANISMS.

For Living Bacteria.

The fluid under examination is mixed on the slide with a
drop of an aqueous (free from Alcohol) solution of Gentian
Violet, Fuchsine, or Methylene Blue.

For Tissues and Cover-glass Preparations.

Stain for a few minutes in the Gentian-Violet Aniline-
Water (p. 58) ; wash in water, and all but decolourise the
ground substance in strong Alcohol ; counterstain, if re-
quired, and mount as usual.

Gram's Method.

Sections are removed from weak Alcohol, and placed in
Gentian- Violet Aniline- Water for 1 to 3 minutes. (Tubercle
sections 12 to 24 hours.)

The sections are quickly rinsed in Absolute Alcohol and
transferred into Gram's solution (p. 61), until they acquire
a brown colour; this takes about 1 to 3 minutes. The
sections are then washed in 90 p. c. Alcohol until they
are a pale yellow, dehydrated, cleared, and mounted in
Balsam.

The sections are frequently counterstained with Eosine or
Vesuvine.

Weigert's Modification of Gram's Method.

As the prolonged washing in Alcohol may remove colour
from the organisms, Weigert proposed the substitution of
Aniline for Alcohol, and conducts the process on a slide.
Place the section on a glass slide, and stain it with a few
drops of Gentian- Violet Aniline-Water ; remove the excess
of fluid, and apply for 2 minutes a few drops of Gram's solu-
tion. Eemove this by gently blotting it off ; then wash the
section by allowing Aniline to flow gently backwards and
forwards over it'; when no more colour comes away, pour off

MICRO-ORGANISMS. 65

the Aniline, and treat in a similar way with Xylol for about
a minute, and mount in Balsam.

Previous to this process for bacteria, the nuclei in the sec-
tions may be stained with Carmine (p. 28).

Kuline's Modification of Gram's Method.

Kiihne uses Fluorescine Alcohol after Gram, in the place of
Alcohol for washing, and adopts Weigert's Aniline for clear-
ing ; he also stains the nuclei with Carmine (nuclear staining,
p. 28), previous to commencing the following process.

Sections are stained for 5 minutes in the solution of Methyl
Violet, diluted one-sixth with a 1 p. c. aqueous solution of
Ammonium Carbonate, or in the Victoria Blue solution ; they
are then rinsed thoroughly in Water, and transferred to Gram's
solution from 2 to 3 minutes, again rinsed in water, and the
excess of stain extracted in Fluorescine Alcohol. They are
then passed through pure Alcohol, Aniline, Ethereal Oil, and
Xylol, and mounted in Balsam.

WITH METHYLENE BLUE.

Lbffler's Universal Staining Fluid.

Concentrated Alcoholic solution of Methylene Blue, 30 cc. ;
Solution of Caustic Potash (1 in 10,000 of water), 100 cc.
This mixture will not keep good for many days, and should
be filtered each day before use.

Loffler found that most bacteria stained better in this solu-
tion than in the weaker solution used by Koch for the Tubercle
bacillus.

Sections are placed in this solution for a few minutes
(Tubercle sections for some hours) ; remove excess of stain by
immersion in very weak Acetic Acid (J p. c.) for a few seconds,

66 MICRO-ORGANISMS.

dehydrate in Absolute Alcohol, clear in Cedar Oil and mount
in Balsam.

Kuhne's Methods.

The sections are transferred from Alcohol to Carbolic
Methylene Blue (p. 58), and allowed to remain in this for
half an hour (leprosy bacilli require longer — 2 hours).
The sections are next rinsed in Water, and then placed in
Weak Acidulated Water until they are of a pale blue colour
(this must be done carefully or too much colour will be
removed) ; they are then rinsed in Lithia Water (p. 61), and
transferred to a basin of pure Water. The sections are taken
up one by one upon the point of a glass needle and dipped
into Absolute Alcohol in which some Methylene Blue has been
dissolved. They are then transferred to Methylene Blue
Aniline Oil to dehydrate, rinsed in Aniline and then placed
for about a couple of minutes into some ethereal oil of low
density, such as Terebene, to clear, then into Xylol, and
mounted in Balsam.

The above method is one that will apply to the staining of
all micro-organisms, with the exception of the bacilli of
leprosy and mouse septicaemia ; these it stains insufficiently.

In order to show satisfactorily the structure of the tissue,
Kuhne recommends the following plan : —

After staining in Carbolic Methylene Blue, the sections are
decolourised in a diluted solution of Chlorhydrin Blue
(p. 62), which requires from 10 to 60 minutes. The sections
are then passed through Alcohol, Aniline, Ethereal Oil, and
Xylol, and, in order to double-stain, the sections are taken
from the Xylol and placed in Safranine Aniline Oil, diluted
to about 4 or 5 times its bulk with Aniline. After remain-
ing in this from 2 to 10 minutes they are passed again through
Ethereal Oil and Xylol before mounting.

MICRO-ORGANISMS.

WITH FUCHSINE.

Ehrlich's Method, see Tubercle Bacilli, p. 68.

Kuhne's Methods.

Method of Staining Bacteria not belonging to the Group of
Tubercle Bacilli in the Tissues with Fuchsine (Kuhne].

The bacilli of mouse septicaemia, anthrax, and other
bacilli, and several kinds of cocci may be stained by the
following method, which will not apply to the bacilli of
glanders and typhoid fever : —

Sections are first dehydrated in Alcohol and then stained
from 3 to 5 minutes in Carbolic Fuchsine, rinsed in water,
dipped for 1 minute into Alcohol, and extracted in Methyl
Green Aniline Oil, which requires from 1 5 minutes to 2 hours,
according to the thickness of the sections. The sections are
then passed through Ethereal Oil and Xylol.

Method of Staining with Black Brown and Fuchsine (Kuhne}.

Anthrax sections are stained for 5 minutes in Carbolic
Black Brown, rinsed in Lithia Water (p. 61), and then in 90
p. c. Alcohol ; the sections are placed for 5 minutes in Car-
bolic Fuchsine, and decolourised in Fluorescine Alcohol.

In this method Carbolic Black Brown acts as a mordant to
Fuchsine, so that the Anthrax bacilli do not lose the red
colour in the Fluorescine Alcohol.

SPECIAL METHODS.

TUBERCLE BACILLUS.

Koch's Original Method.

Sections or cover-glass preparations are placed in Koch's
Methylene Blue Solution (p. 58) and left in it for 20 to 24
hours, or J to 1 hour if warmed to 40°C. (104°F.) ; they are
then placed in aqueous solution of Vesuvine for 2 minutes ;
this removes the blue colour from the tissue, staining it
brown, while the bacilli remain blue ; the excess of colour is
washed out in Distilled Water ; if cover-glass preparations,
they are then dried and mounted in Balsam ; if sections, they
are dehydrated in Absolute Alcohol, cleared in Cedar Oil,
and mounted in Balsam.

Kocli-Ehrlich Method.

In G-entian- Violet Aniline- Water (p. 58), or Fuchsine
Aniline- Water (p. 59), place sections or cover-glass prepara-
tions for at least 12 hours (more rapid staining of the latter
is obtained by warming) ; then immerse in Nitric Acid (p.
62) for some seconds. Einse in 60 p. c. Alcohol for some
minutes, and then count erstain with Aqueous Solution of
Yesuvine (p. 38) after Violet, and with Aqueous Solution of
Methylene Blue (p. 36) after Fuchsine ; rinse in water, dehy-
drate, clear, and mount in Balsam.

Nitric Acid is apt to injure delicate sections. To avoid this

MICRO-ORGANISMS. Dtf

Watson Cheyne recommended that the sections, after stain-
ing in the Fuchsine Aniline Water, should be transferred to
Distilled Water, rinsed in Alcohol and placed in the following
contrast stain for one or two hours: — Saturated Alcoholic
Solution of Methylene Blue, 20 cc. ; Distilled Water, 100 cc. ;
Formic Acid (sp. gr. 1-2), 1 cc.

Ziehl Neelsen Method.

This method is applicable for staining the bacilli of tubercle
and leprosy, and at the same time affords a diagnostic sign,
for these are the only micro-organisms that can retain the
stain after treatment with acid.

The sections are removed from weak Spirit into Neelsen's
stain (Carbolic Fuchsine) (p. 59) for 10 or 15 minutes; then
decolourised in Sulphuric or Nitric Acid (p. 62), rinsed in
60 p. c. Alcohol and washed in a large volume of Water, to
remove the Acid.

Nitric Acid is apt to contain considerable traces of Nitrous
Acid, which has a bleaching action on f uchsine-stained bacilli.
Where this is suspected it is better to use Sulphanilic Nitric
Acid (p. 62), the Sulphanilic Acid destroying any free Nitrous
Acid that may be contained in the Nitric Acid.

For counterstaining, the sections may be treated as de-
scribed for nuclear staining with Methyl Green or Methylene
Blue (p. 36).

Dehydrate in Absolute Alcohol, clear in Cedar Oil, and
mount in Balsam.

Kuhne's Method.

Place sections for 10 minutes in Carbolic Fuchsine;
thoroughly rinse in water; decolourise in Fluorescine Alcohol;
then transfer to Ethereal Oil, Xylol and Balsam ; or, to coun-
terstain before mounting, sections may be transferred for 5 to
10 minutes into Methyl Green Aniline Oil, diluted with half its

70 MICRO-ORGANISMS.

bulk of pure Aniline Oil ; then pass into Ethereal Oil for
2 minutes, afterwards into Xylol, and mount in Balsam.

Gibbes' Method of Double Staining.
Cover-glass preparations are placed for 4 minutes in Gibbes'
double stain, which has been previously slightly heated;
sections should be placed in the stain at the ordinary tem-
perature for some hours. The cover-glass preparations or
sections are then washed in Methylated Spirit till no more
colour comes away, dehydrated, cleared in Cedar Oil and
mounted in Balsam.

LEPROSY BACILLUS.

The same staining methods may be employed as for the
tubercle bacillus.

As leprosy bacilli stain much more quickly in the Fuchsine,
the following plan has been suggested for distinguishing
them : —

Sections are stained for 1 to 3 minutes in the Aqueous Solu-
tion of Fuchsine (p. 59) ; then placed for 30 seconds in a
solution consisting of 90 p. c. Alcohol, 10 parts; Nitric Acid
(Sp. g. 1 '420), 1 part ; dehydrate, clear, and mount in Balsam.

Treated in this way the leprosy bacilli appear well stained,
while the tubercle bacilli are not stained at all.

ANTHRAX BACILLUS. (MILZBRAND BACILLUS.)

This may be stained with Yiolet by Gram's method
(p. 64) or one of the modifications ; with Fuchsine or Methy-
lene Blue by Kuhne's methods (pp. 66 and 67).

GLANDERS BACILLUS. (ROTZ BACILLUS.
BACILLUS MALLEI.)

Cover-glass preparations are stained with Methylene Blue
by Kuhne's method (p. 66) or by Loffler's method (p. 65).

MICRO-ORGANISMS. 71

Schutz's Method.

Sections (and cover-glass preparations) are stained for some
hours in Aqueous Solution of Methylene Blue, differentiated
in very weak Acetic Acid (J p. c.) dehydrated in Alcohol,
cleared in Cedar Oil, and mounted in Balsam.

SYPHILIS BACILLUS.

Lusgarten's Method.

Sections are placed in Gentian-Violet Aniline-Water (p.
58) for 12 to 24 hours at the ordinary temperature of the
room, then for 2 hours at 40° C.

The sections are transferred to Absolute Alcohol for a few
minutes, then placed for 10 seconds in 1'5 p. c. solution of
Permanganate of Potassium, and washed in Sulphurous Acid.
If the ground substance of the sections is not completely
decolourised, the second part of the process must be repeated.
After this the sections are dehydrated, cleared, and mounted
in Balsam.

These bacilli, after staining by the Koch-Erhlich or Ziehl-
Neelsen methods (unlike the tubercle bacilli), are easily de-
colourised by mineral acids.

Giacomi's Method.

Cover-glass preparations are stained for a few minutes in
a hot solution of Fuchsine, then placed in Water containing
a few drops of solution of Perchloride of Iron; then de-
colourised in strong solution of Perchloride of Iron. Some-
times a precipitate is formed with the Iron Solution, then the
decolourisation is completed in Alcohol. Counterstain with
Vesuvine.

PNEUMO-CQCCUS.

Gram's Method

For staining the cocci.

Sections which have been deeply stained in Gentian- Violet

72 MICRO-ORGANISMS.

Aniline-Water, are rinsed in Alcohol ; placed in Gram's Solu-
tion (p. 6 1 ) rinsed in Alcohol, cleared and mounted. The micro-
cocci become a dense blue on a faintly yellow ground substance.

Friedlaender's Methods

For staining the capsule as well as the cocci.

Cover-glass Preparations.

Cover-glass preparations of the sputum are treated for 3
minutes with a 1 p. c. solution of Acetic Acid. The excess of
Acid is then removed by filter-paper and the cover-glass is
allowed to dry ; the preparations are next placed in Gentian -
Violet Aniline-Water (p. 58) for half a minute; then washed
in Water, dried, and mounted in Balsam.

For Tissues the following Plan is used: —

Sections are placed for 24 hours in the following solution in
a warm place :— Concentrated Alcoholic solution of Gentian
Violet, 50 cc. ; Distilled Water, 100 cc. ; Glacial Acetic Acid,
10 cc. They are then placed from 1 to 2 minutes in 0-1 p. c.
Acetic Acid ; dehydrated, cleared, and mounted in Balsam.

BACILLUS OF DIPHTHERIA.

To stain these Lomer used his process, described p. 65.
They may also be stained by Gram's method (p. 64).
A piece of the false membrane is placed on a cover-glass
and treated as a cover-glass preparation.

BACILLUS OF ENTERIC FEVER.

Gaffky's Method.

The organ from which the sections are made must be
hardened in Alcohol.

The sections are placed for 20 to 24 hours in a deep blue
opaque solution, which must be freshly made by adding a

MICRO-ORGANISMS. 73

saturated Alcoholic solution of Methylene Blue to Distilled
Water ; they are then washed in Distilled Water (not acidu-
lated), thoroughly dehydrated in Absolute Alcohol, cleared in
Oil of Turpentine, and mounted in Balsam.

These may also be stained by Loffler's Method (p. 65).

It is usually stated that Gram's Method is not applicable
as it removes the stain from the bacilli, but Woodhead affirms
that if the sections containing these bacilli are allowed to
remain for 10 minutes in a 3- p. c. solution of corrosive sub-
limate, and then stained by Gram's method the bacilli are
deeply coloured.

ACTINOMYCOSIS.

(i.) Weigert's Method.

Sections are immersed for 1 hour m Orseille (p. 60) ; they
are quickly rinsed with Alcohol and counterstained with Gen-
tian Yiolet.

If it is desired to stain the mycelium also, the sections, after
being stained with Orseille, are submitted to Weigert's modi-
fication of Gram's method (p. 64).

(ii.) Plant's Method.

Sections are placed for 10 minutes in Gibbes' Magenta solu-
tion (p. 59) (or Carbolic Fuchsine) at 45° C. ; next they are
rinsed in Water and placed in saturated aqueous solution of
Picric Acid mixed with an equal volume of Absolute Alcohol
for 5 to 10 minutes ; they are then washed in Water, passed
through 50 p. c. Alcohol into Absolute Alcohol, cleared in
Cedar Oil, and mounted in Balsam.

(iii.) Modification of Plant's Method.

Sections are placed for 10 minutes in Carbolic Fuchsine
(p. 59) and decolourised for 24 hours in Fluorescine Alcohol.

74 MICRO-ORGANISMS.

Stain the nuclei with Ehrlich's Hsematoxylin (p. 24) and
counterstain faintly with Benzopurpurine (p. 40).

(iv.) Babes' Method.

Stain sections in Babes' Safranine solution (p. 60) for 2
minutes, rinse in Alcohol, and decolourise in Gram's solution
(p. 61).

Only the clubs remain stained.

MAKING COVER GLASS PREPARATIONS.

To thoroughly clean the cover-glass before use, wash it
in Solution of Potash, rinse it in Diluted Nitric Acid, and
afterwards in Alcohol.

In the case of blood, urine, and fluid discharges, a little is
taken upon the point of a platinum needle which has been
previously heated to redness, spread out upon the clean cover-
glass, allowed to dry, and the cover-glass passed three times
through the flame of a spirit-lamp to coagulate the Albumen.

In the case of sputum it should be rubbed to and fro over
the cover-glass with a glass needle (thin glass rod drawn out
to a point) until a uniformly thin layer of mucus is formed on
the cover-glass. It is then allowed to dry ; the drying can if
necessary be accelerated by a gentle heat, and in the case of
tough sputum a vertical stream of air from an air-ball syringe
is useful. When dry the preparation is passed three times
through the flame as above.

When examining tubercular sputum, the yellow caseous
masses should be selected.

MAKING COVER-GLASS IMPRESSIONS.

A perfectly clean cover-glass is placed upon the culture,
gently pressed, and then carefully raised. It is allowed to
dry, passed three times through the flame of a spirit-lamp,
stained by one of the methods, and mounted in Balsam.

MICLIO-ORGAKISMS. 75-

STAINING COVER-GLASS PREPARATIONS.

For micro-organisms (when special methods are not em-
ployed) the following plan may be adopted : — 2 or 3 drops
of aqueous solution of either Fuchsine, Methylene Blue,
Methyl Violet, or Gentian Violet, are placed on the cover-
glass preparation ; after a couple of minutes the superfluous
stain is washed off with Distilled Water; the cover-glass
can now be examined if it be placed, preparation downwards,
on a glass slip, and the excess of Water removed with blot-
ting-paper, or if a permanent preparation be desired the
excess of Water is removed with blotting or filter paper, the
cover-glass is then allowed to dry, and finally mounted in
Balsam.

In place of the above aqueous solutions, Vesuvine dissolved
in Glycerine (p. 57) may be used, the film is then washed and
mounted in undiluted Glycerine.

Cover-glass preparations may also be treated by any of the-
methods described for use with sections.

STAINING TUBERCULAR SPUTUM.

A cover-glass preparation of sputum (p. 74) is placed in Car-
bolic Fuchsine (p. 59) warmed in a watchglass until steam rises
for 3 minutes ; it is then rinsed in Water to remove the ad-
hering stain, decolourised in Nitric Acid (p. 62) (preferably
Sulphanilic Nitric Acid), washed in 90 p. c. Alcohol to re-
move the Acid, then placed in aqueous solution of Methylene
Blue (p. 36) for 1 minute, and well washed in Water to
remove excess of stain; dried and mounted in Benzol
Balsam.

If there be an objection to the use of Nitric Acid, then
Sulphuric Acid and Alcohol (p. 62) may be used as a sub-
stitute.

76 MICRO-ORGANISMS.

STAINING SPORES.

If the cover-glass preparation be heated for much longer
than in the ordinary method for bacilli, the spores will be
stained as readily as the bacilli.

After a cover-glass preparation is made, it is passed several
times (12) through the flame, or heated to a temperature of
210° F. for half an hour, or exposed to the action of strong
Sulphuric Acid for a few seconds ; then stained with aqueous
solution of Fuchsine, Methylene Blue, or Gentian Violet.

DOUBLE-STAINING SPORE-BEARING BACILLI.
Neisser's Method.

Cover-glass preparations treated in the ordinary way (not
specially heated as in the foregoing method) are immersed
for 20 minutes in the Fuchsine Aniline- Water (p. 59) heated
to 80° or 90° C. (176° or 194° F.) ; the cover-glass is then
rinsed in Water, Alcohol, or weak Acid, according to the
nature of the bacilli ; counterstained with aqueous solution of
Methylene Blue, rinsed in Water, dried, and mounted in
Balsam.

The spores are stained red, and the other part of the bacilli
blue.

STAINING PLAGELLA.

(i.) Koch's Method.

Cover-glass preparations are immersed in 1 p. c. aqueous
solution of Hsematoxylin ; they are then transferred to a 5
p. c. solution of Chromic Acid, or to Miiller's Fluid, dried
and mounted in Balsam.

The flagella are stained a brownish black.

MICRO-ORGANISMS. 77

(ii.) Crookshank's Method.

Cover-glass preparations are stained with a drop of concen-
trated alcoholic solution of Gentian Violet ; the cover-glass
is then rinsed in Water, allowed to dry, and mounted in
Balsam.

(iii.) Loffler's Method.

The stock solution is made by adding to 10 cc. of a 25 p. c.
Aqueous Solution of Tannin, 5 cc. of cold saturated Aqueous
Solution of Ferrous Sulphate, and 1 cc. of Aqueous or Alco-
holic Solution of Fuchsine or Methyl Yiolet.

As the treatment, however, varies with the different micro-
organisms, the reader is referred to an abstract of the paper
in Jour. R.M.S., 1890, p. 678.

DEHYDKATIOK

As none of the usual clearing agents, used previous to
mounting in Balsam, will mix with aqueous fluids, sections
must be dehydrated before clearing. The usual dehydrating
agent is Alcohol, and the only point which requires notice is
the strength to be employed.

Preparatory to clearing in Oil of Cloves or Bergamot, 90
p. c. Alcohol answers every purpose. But for Oil of Cedar
or the other members of the Xylol group, Alcohol of at least
96 p. c. must be used, and Absolute Alcohol is preferable.

Celloidin Sections. — Oil of Bergamot clears Celloidin
without dissolving it, and as this oil can be used after 90 p. c.
Alcohol, it possesses great advantages.

Cedar Oil also clears Celloidin, but as the film is quickly
acted upon by Absolute (98 p. c.) Alcohol, it is safer to use a
slightly hydrated Alcohol 96 p. c. (sp. gr. 0-807 at 60° F.),
made by adding 2 cc. of Water to 120 cc. of Absolute Alcohol.
This will clear into either Oil of Cedar or Xylol, and in 15
minutes (the maximum time required for dehydrating sections)
will not perceptibly attack the celloidin. These sections can
also be dehydrated in Absolute Alcohol, but it must be done
quickly (about 2 minutes) or the celloidin film will be per-
ceptibly affected.

The only other dehydrating agent that may be mentioned
is Aniline, used in special circumstances where strong Alcohol
must be avoided. This clears readily from 70 p. c. Alcohol, and
is replaced before mounting by Cedar Oil, Benzol, or X}4ol.

CLEAEING AGENTS.

BEFORE MOUNTING IN BALSAM.

Those most generally recommended are Creosote, Turpen-
tine, Xylol, and the Oils of Bergamot, Cedar, Cloves, and
Origanum. .

The object of using a clearing agent is to replace the
Alcohol in the dehydrated section by a liquid which has a
refractive index about the same as the Balsam into which it is
to be placed and which will mix readily with it.

1. The fluid should clear quickly from a somewhat hydrated
Alcohol.

2. It should not dissolve the aniline colours nor celloidin.
No one clearing agent is perfect in both respects, for it

happens that the more readily the agent mixes with 90
p. c. Alcohol, the more liable it is to dissolve the aniline
colours.

For clearing celloidin sections, Bergamot or Cedar Oil
are the best. See Dehydration.

OIL OF BERGAMOT.

This oil will clear directly from 90 p. c. Alcohol. Its
solvent action on Aniline Colours is very slight, except in the
case of Methyl Green and the Violet dyes, which may be
appreciably dissolved. Apart from this, its powerful odour
and high price are the only objections to its general use.

It clears Celloidin without dissolving it.

80 CLEARING AGENTS.

CLOVE OIL.

This will mix in any proportion with 90 p. c. Alcohol with-
out becoming turbid, and clears rapidly, but it dissolves the
aniline colours to a very considerable extent.

The same objection applies even more forcibly to "Wood-
tar Creosote, and Carbolic Acid.

XYLOL.

This, on the other hand, is practically without action on
aniline colours, but has so little affinity for water that it can
only be used after Alcohol of at least 96 p. c.

Taking therefore Clove Oil and Xylol as types, the follow-
ing have been classified as resembling the one or the other in
reference to their behaviour with 90 p. c. Alcohol, and their
action on aniline colours.

This strength of Alcohol has been chosen because it is that
of the Methylated Spirit generally sold in this country and
which for economy is largely used in washing and dehydrat-
ing the material for microscopic work. An agent, therefore,
which will clear from this strength of Alcohol is a deside-
ratum.

OIL OP TURPENTINE

has a comparatively low refractive index, resembles Xylol,
but causes shrinkage and renders the tissues brittle. It
becomes resinified on exposure to air.

TEREBENE.

It belongs to the Xylol group, but acts slightly upon ani-
line colours, and is open to the same objection as Oil of
Turpentine regarding shrinkage.

BENZOL and TOLUOL.

These only differ from Xylol in their volatility; Benzol
"being the most volatile, and Xylol the least so of the three ;

, CLEARING AGENTS. 81

the rapidity of evaporation being in the ratio of 4, 5,
and 9.

It is curious that, in two excellent books on this subject,
Xylol is described as being more volatile than Benzol in one
of them, and Toluol in the other.

Benzol has also been described as a solvent of the aniline
dyes.

All three are practically without solvent action upon aniline
colours.

OIL OF CEDAR WOOD.

Although an essential oil it resembles the type Xylol, but
evaporates slowly and not to dryness.

It has very little solvent action on the aniline colours. It
clears rapidly from Absolute Alcohol, but not well from
90 p. c. Alcohol. Sections can be left in it for several days
without becoming brittle. It is a convenient medium in which
to examine tissues before mounting them permanently.

It clears Celloidin without dissolving it.

OIL OF ORIGANUM.

This is Oil of White Thyme more or less adulterated ; it
dissolves the aniline colours to about the same extent as Oil
of Cloves. Another oil, probably Oil of Marjoram, is sold as
Oleum Origani Cretici, but this also dissolves the aniline
colours.

BEFORE MOUNTING IN GLYCERINE.

Liquid Carbolic Acid, Liquor Potassae, Alcoholic Solution
Potash, Liquor Ammonise, Solution of Chloral Hydrate, Eau
de Javelle, Eau de Labarraque, are all used for clearing

G

82 CLEARING AGENTS.

vegetable sections for examination. Wash, in Water, except
after Alcoholic Potash, when use 50 p. c. Alcohol, and mount
in Glycerine or Glycerine Jelly.

ALCOHOLIC SOLUTION OF POTASH.

Hydrate of Potassium, 1 grin. ; Distilled Water, 10 cc. ;
Eectified Spirit, 10 cc.

Dissolve the Potash in the Water and add the Spirit.

SOLUTION OF CHLORAL HYDRATE.

Chloral Hydrate, 30 grms. ; Distilled Water, 10 grms.

SOLUTION OF POTASSIUM HYFOCKLOEJTE
(EAU BE JAVELLE).

Chlorinated Lime, 20 grms. ; Carbonate of Potassium, 20
grms. ; Distilled Water, 200 cc. Triturate the Chlorinated
Lime with half the Water ; dissolve the Carbonate of Potas-
sium in the other half ; mix. After an hour, filter.

SOLUTION OF SODIUM HYFOCHLORITE (EAU
DE LABARRAQUE).

It is made in the same way as above, substituting Crystal-
lized Carbonate of Sodium, 40 grms., for the Carbonate of
Potassium, 20 grms.

Both these Hypochlorite Solutions are also bleaching
agents.

MOUNTING MEDIA.

When sections are mounted with Balsam, Dammar, Far-
rant, Glycerine and Q-um, or Glycerine Jelly, it is not a neces-
sity that the cover-glasses should be fixed with cement, as
these fluids solidify at the edges, and so fix the cover-glass.

Some cement (Caoutchouc by preference) must be employed
when Glycerine or aqueous solutions are to be used.

CANADA BALSAM.

A soft substance which is usually dried over a water-bath
until, when cooled, it is brittle. It is then dissolved in Benzol
or Xylol to form a somewhat thin solution ; about 200 grms.
of Balsam to 100 cc. of Benzol : the Xylol solution may be
rather thicker, as it evaporates more slowly. Neither of them
will affect the Aniline stains.

Mounts in Benzol Balsam harden in a day or two ; those
in Xylol Balsam remain soft for a week or two. I there-
fore much prefer to mount in Benzol Balsam.

Transfer the sections to a slide (using for thin sections a
glass rod, p. 19), remove excess of clearing agent by gently
pressing on the section with clean blotting-paper, add one or
wo drops of Balsam Solution, and apply the cover-glass.

DAMMAR SOLUTION.

Gum Dammar dissolved in Benzol or Xylol.
About 100 grms. of Dammar to 100 cc. of Benzol.

84 MOUNTING MEDIA.

DAMMAR AND MASTIC.

Gum Dammar, 100 grms. ; Oil of Turpentine, 200 cc. Dis-
solve and filter. Gum Mastic, 50 grms. ; Chloroform, 200 cc.
Mix the two solutions.

This is an old formula which is still used by some workers.

GLYCERINE.

Two or three volumes of Glycerine to one of Distilled Water
is a good medium in which to examine tissues transferred
from aqueous fluids. For mounting permanently, the objects
should be immersed in the above diluted Glycerine, which
may be increased in strength if desirable ; delicate objects
may be placed in a mixture of Glycerine, 1 ; Alcohol, 1 ;
Water, 1 ; which is then allowed to concentrate by evapo-
ration.

GLYCERINE JELLY.

Immerse 100 grms. of French Gelatine in Chloroform
.Water, and, when soft, drain away the excess of water.
Dissolve the softened Gelatine in 750 grammes of Glycerine
over a water-bath, and add 400 grammes of Chloroform
Water with which has been incorporated about 50 grammes
of fresh egg albumen, and mix thoroughly. Heat the mix-
ture to boiling for about five minutes to coagulate the albu-
men; make up the total weight to 1,550 grammes with
Chloroform Water. Filter in a warm chamber, but pro-
tected as much as possible from evaporation.

Tissues can be mounted in this direct from water, but it is
better to immerse them for a little while in equal parts of
Glycerine and Distilled Water. Place them on a slide ; re-
move superfluous fluid, and apply a few drops of the melted

MOUNTING MEDIA. 85

Jelly ; breathe on a cover-glass, place it on the fluid, apply
gentle pressure, and set aside to cool.

FARRANT'S MEDIUM.

Dissolve 1 grm. of Arsenious Acid in 200 cc. of Distilled
Water. In this fluid dissolve, at the ordinary temperature,
130 grms. of Gum Acacia, with frequent stirring; add 100 cc.
of Glycerine ; mix. Filter the solution through fine Swedish
paper upon which has been deposited a thin layer of Talc.

This reagent is recommended because tissues mounted in it
preserve their normal appearance, and it is more convenient
than Glycerine in that it dries at the edges, and fixes the cover-
glass.

Tissues may be mounted in this direct from Water, but it
is better to soak them for a short time in dilute Glycerine
before mounting.

Place the section on a slide, remove superfluous fluid, add
1 or 2 drops of the medium, and apply the cover-glass.

Formic Parrant.

Farrant's Medium to which 1 p.c. of Formic Acid (sp. g.
1*2) has been added.

GLYCERINE AND GUM.

Chloroform Water (1 in 200), 200 cc. Dissolve in this fluid
130 grms. of Gum Acacia, with frequent stirring, at the
ordinary temperature of the air; add Glycerine, 100 cc. ; mix.
Filter as directed for Farrant.

This may be used in the place of Farrant's Medium when
the presence of Arsenious Acid is objectionable.

IODINE MOUNTING FLUID.

Liquor lodi B.P., 100 cc. ; Water, 300 cc. ; Glycerine, 200

86 MOUNTING MEDIA.

cc. Mix, and dissolve in it 260 grms. Gum Acacia, with fre-
quent stirring, at the ordinary temperature.

This is Warrant's Medium, in which Iodine is substituted
for Arsenic. It is used for mounting tissues stained with
Iodine.

ACETATE OF POTASSIUM.

Acetate of Potassium, 250 grms. ; Water 100 cc. ; dissolve
by a gentle heat, and filter.

ACETATE OF COPPER.

Acetate of Copper, 1 grm. ; Glacial Acetic Acid, 1 cc. ; Cam-
phor Water, 250 cc. ; Glycerine, 250 cc. ; Corrosive Subli-
mate, 4 grms.

Tor preserving and mounting Green Algse. The Glycerine
is sometimes omitted with advantage, as in the case of Yolvox.

NOEMAL OE INDIFFEEENT FLUIDS.

These may be described as fluids which will not injuriously
affect tissues. They consist of —

(i.) BLOOD SEBUM.

Obtained by allowing blood to coagulate, and pouring off
the serum after a day.

(ii.) AQUEOUS HUMOUR.

Best obtained by puncturing the cornea of a freshly excised
ox's eye-ball.

(iii.) NORMAL SALINE SOLUTION. (0-75 p. c.)

Colourless Eock Salt or recrystallized Sodium Chloride,
7 1 grms. ; Distilled Water, 1000 cc.

DISSOCIATING FLUIDS.

These fluids are used to soften the cement substance of
connective tissue, epithelium, &c., in order that the various
elements of the tissue may be separated and examined.

(i.) IODISED SERUM.

Solution of Iodine (B.P.), 1 cc. ; Serous Fluid, 100 cc.

Should the brown colour fade, add a little more of the
Solution of Iodine.

Place a piece of the tissue to be dissociated, which may
be about -fa in. diameter, in about 5 cc. of the fluid in
a glass-stoppered bottle, taking care to maintain the brown
colour of the fluid by adding a little of the Iodine Solution.
After the tissue has been in this solution for a couple of days
it may be teased out with needles.

This fluid is useful for macerating white nerve fibres.

(ii.) DILUTE ALCOHOL,

Alcohol (90 p. c.), 1 part ; Distilled Water, 2 parts.
Takes about 24 hours to dissociate epithelium, connective
tissue, &c.

(iii.) BICHROMATE OP POTASSIUM,

Bichromate of Potassium, 1 grm. ; Water, 500 cc.
Used for epithelium and nerve cells of spinal cord. Takes
from 1 to 2 weeks.

DISSOCIATING FLUIDS. 89

(iv.) CHROMIC ACID.

Chromic Acid, 1 grm. ; Water, 1000— 5000 cc.
For connective tissue, unstriated muscle, and nerve tissue.
Takes from 24 hours for nerve tissue, to 7 days for uterine
fibroid.

(v.) NITRIC ACID.

20 p. c. aqueous solution.

Takes 24 hours to dissociate small pieces of muscular tissue ;
the muscle cells are then teased out, and may be mounted in
Glycerine.

(vi.) NITRIC ACID AND GLYCERINE.

Glycerine, 1 part ; Water, 3 parts ; Nitric Acid, 1 part.
In dissociating nervous structure takes from 3 to 4 days.

(vii.) OSMIC ACID.
0-1 p. c. aqueous solution.
Used for cerebral cortex. Takes 24 to 48 hours.

(viii.) METHYL MIXTURE (Schieffer decker's).

Methylic Alcohol, 5 cc. ; Glycerine, 50 cc. ; Distilled Water,
100 cc.

Used for retina and central nervous system and takes
several days.

(ix.) CHLORIDE OF SODIUM.

10 p. c. aqueous solution.

Used for dissociating white fibrous tissue.

Ix.) CAUSTIC POTASH.

30 to 50 p. c. aqueous solution.

It acts very rapidly.

Used for separating muscle cells, as in cases of myomata.

90 DISSOCIATING FLUIDS.

The tissues must be examined in this fluid, and cannot be
preserved. If water is added to the dissociated tissues, they
are soon dissolved.

(xi.) SCHULZE'S MACERATING MIXTURE.

Sections are placed in Nitric Acid sp. gr. 1-2, and about
2 or 3 per cent, of Chlorate of Potassium is added.

If the fluid be not heated, the sections should remain in it
for several hours ; but if the fluid be gently warmed until gas
is given off freely, the result will be attained in a few
seconds.

The tissue is washed in water, transferred to a slide, and
the disintegration completed with needles.

Used in Botanical Histology for dissolving the middle
lamella in vertical sections.

INDEX.

PAGE

Acetate of Copper ... 10, 86

Acetate of Potassium ... 86

Acetic Acid and Alcohol . . 3

Acetine Blue 62

Acid Fuchsine 41,48

Acid Magenta 42

AcidKubiu 41

Acidulated Alcohol .... 28

Acidulated Glycerine ... 28

Acidulated Water .... 61

Actinomycosis 73

Alcohol and Acetic Acid . . 3

Alcohol for Hardening ... 2

Altmann's Nitric Acid ... 8

Alum Carmine, Grenacher's . 32

Alum Cochineal, Czoker's . . 32

Ammonia Carmine . . . . 30

Ammonia Carmine, Stronger . 31

Ammonia Picro- Carmine . . 34

Amyloid 46

Aniline Black 48

Aniline Chloride 65

Aniline Nuclear Stains ... 35
Aniline Violets, General Me-
thods 63

Aniline Water 61

Anthrax Bacillus 70

Aqueous Humour .... 87

Arsenic Acid 11

Babes' Method 74

Babes' Solution 60

Beale's Ammonia Carmine . 31

Benda's Copper Heematoxylin 27

Benzol 80

Benzol Balsam 83

Benzopurpurine 40

Bergamot Oil 79

Bichromate of Ammonium . 4
Bichromate of Potassium . 4, 88
Bismarck Brown ... ,38

PAGE

Black Brown 60

Blood Serum ...... 87

Blue Black 48

Bohmer's Hsematoxylin . . 23

Borax Carmine 32

Canada Balsam 83

Carbolic Acid 80, 81

Carbolic Black Brown ... 6-^

Carbolic Fuchsine .... 59

Carbolic Methylene Blue . . 58

Carmine 28

Cedar OH 81

Celloidin 15

Celloidin Sections * . 78, 79

Cellulose Staining . . . 52

Chloral Hydrate . . . . 82

Chlorhydrin Blue . . . 62

Chloride of Aniline . . . 55

Chloride of Gold . . . 43

Chloride of Sodium . . 87, 89

Chlor-Zinc Iodine . . . 55

Chromate of Ammonium . . 4

Chromic Acid . . . . 6, 11, 89

Clearing Agents 79

Clove OH 80

Congo Red ....... 48

Corrosive Sublimate .... 7

Counter staining Carmine . . 40

Counterstaining Hsematoxylin 40

Cover Glass Preparations . . 74

Crookshank's Method ... 77

Czoker's Alum Cochineal . . 32

Dahlia Violet . .^ . . . 38, 45

Dammar and Mastic . . . . 84

Dammar Solution .... 83

Decalcifying Fluids .... 11

Dehydration 78

Delafield's Hsematoxylin . . 24

Diphtheria Bacnius .... 72

Dissociating Fluids .... 88

92

INDEX.

Eau de Javelle 82

Eau de Labarraque . . . . 82

Ebner's Solution 12

Ehrlich-Biondi Fluid ... 37

Ehrlich's Hsematoxylin ... 24
Ehrlich's Haematoxylin Am-

moniated 24

Ehrlich' s Method .... 63

Elastic Fibres 45, 48

Embedding Media .... 14

Enteric Fever Bacillus ... 72

Eosine . 40, 56

ErlicM's Fluid ...... 6

Erythrosine ....... . 41

Farrant's Medium .... 85

Fatty Elements.. ..... 43

Fixing Agents 7

Flagella Staining .... 76

Flemming's Solution ... 9

Fluorescine 62

Fol's Solution ..*... 9

Formic Farrant 85

Friedlander's Method ... 72

Fuchsine . . . . .'39, 59, 67

Fuchsine 'Aniline-Water . . 59

Gaffky's Method 72

Gentian Violet • .' . . . 38, 58

Gentian- Violet Aniline-Water 58

Giacomi's Method .... 71

Gibbes' Double 'Stain ... 59

Gibbes' Magenta Solution . . 59

Gibbes' Method 70

Glanders Bacillus .... 70

Glycerine ....... 84

Glycerine Gelatine . . . . 16

Glycerine" and Gum . . . . 85

Glycerine JeUy ; .... 84

Gold Chloride 43

Golgi'B Sublimate Method . . 51

Gram's Method . . . . 64, 71

Gram'^ Solution 61

Grenacher's Alcoholic Borax

Carmine 32

Grenadier's, Alum Carmine . 32

Grenacher's. Borax Carmine . 32

Gum . . . . 14

Gum and Syrup 14

Hgematoxylin 21

Haematoxylin, Ripening . . 23

Hamilton's Hsematoxylin . . 24

PAGK

Hardening Agents .... 1
Heidenhain's Hsematoxylin . 27
Hoffmann's Blue . . . . 39, 56
Hydrochloric Acid . . . . 12
Hydrochloric Acid and Gly-
cerine 12

Hypochlorite of Potassium . 82

Hypochlorite of Sodium . . 82

Intermediate Reagents ... 61

Iodine 46

Iodine Green 37

Iodine Mounting Fluid . . .85
Iodised Serum 88

Kleb's Glycerine Gelatine . . 16
Klein's Fluid ...... 7

Kleinenberg's Hsematoxylin . 25
Kleinenberg's Picro- Sulphuric

Acid .• . 10

Koch-Ehrlich Method ... 68
Koch's Method . . . .68,76
Koch's Methylene • Blue Solu-
tion 58

Kuhne's Methods. 65, 66, 67, 69
Kuhne' s Solutions . . 58, 59, 60

Leprosy Bacillus 70

Lignined" Cellulose . . . . 53

Lithia Water 61

Lithium Carmine 33

Living Bacteria, Staining of . 64

Loffler's Methods . . . . 65, 77

Loffler's Solution ..... 58

Lusgarten's Method . . . . 71

Maceratiug Mixture, Schulze's 90
Magenta .,.,»... 39, 59

Magenta, Acid 42.

Manip.ula.tion of Sections . . 18

Mastzellen 45

Methyl Qreqn , . . . .36, 56
Methyl- Qreen Aniline- Oil . . 60

Methyl Mixture 89

Methyl Violet 46, 58

Methyl- Violet Aniline -Water 59
Methylene Blue , . .30, 57, 65
Methylene-Blue Aniline-Oil . 58
Micro -Organisms ..... 57

Mounting Media 83

Muller's Fluid 4

Muller apd Spirit . , . . . . 5

IKDEX.

93

PAGE

Neisser's Method 76

Nerve Centres 29, 48

Nerve Endings 44

Nigrosine 48

Nitrate of Silver 44

Nitric Acid . . . 8, 12, 62, 89
Nitric and Chromic Acid . . 13
Nitric Acid and Glycerine . . 89
Normal or Indifferent Fluids . 87
Normal Saline Solution ... 87
Nuclear Staining ..... 20

Oil of Bergamot 79

Oil of Cedar . . r . . . . 81

Oil of Cloves .80

Oil of Origanum 81

Oil of Turpentine 80

Orange . 41

Orseille • 60/

Orth's Lithium Carmine . . 33
Osmic Acid 8, 43, 89

Pal-Exner Method .... 50

Pal's Method ....... 49

Pal's Solution 50

Palladium Chloride .... 29

Paraffin 16

Phloroglucin 55

Picric Acid . . . . 9, 13, 41, 56

Picro- Carmines 33

Picro- Lithium Carmine . . 35

Picro -Sulphuric Acid ... 10

Plasmatic Staining . . . . 39

Plant's Method ..... 73

Pneumo-Coccus 71

Potash, Alcoholic ..... 82

Potash, Aqueous . . . . 81, 89

Preserving Sections . . . . 18

Rabl's Fluid 7

Ranvier's Lemon juice Method 44
Renaut's Eosinated Hsema-

toxylin 27

Kenaut's Haematoxylin ... 26

PAGE

Resin and Wax 18

Rose Bengale 47

Rubin, Acid 41

Rubin and Orange .... 42

Safranine 38, 47, 60

Safranine Aniline-Oil ... 60
Saturation Processes . . 15, 17

Saline Solution 87

Schiefferdecker's Mixture , . 89
Schulze's Solutions . . 55, 90

Schutz' s Method 7t

Sections, Manipulation of . . 18

Silver Nitrate 44

Soda Picro -Carmine .... 35

Specific Stains 43

Spores, Staining of . . . . 76
^Sputum, Staining of . . . . 75

Staining in Bulk 28

Stains for Micro -Organisms . 57
Sulphanilic and Nitric Acid . 62
Sulphuric Acid Alcohol . . 62
Sulphuric Acid ..... 62
Syphilis Bacillus 71

Terebene 80

Toluol 80

Tubercle Bacillus 68

Turpentine Oil 80

Vegetable tissues, see Cellulose 52

Vesuvine • 38, 57

Victoria Blue 4o, 69

Wedl's Solution 60

"Weigert's Haematoxylin . . 26
Weigert's Method . . .48, 73

Xylol 80

Xylol Balsam 83

Ziehl-Neelsen Method
Ziehl-Neelsen Solution

59

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