GIFT OF
MICHAEL REESE

BIOLOGY
UBRARY

THE

MICROTOMIST'S VADE-MECUM

THE

MICROTOMIST'S VADE-MECUM

A HANDBOOK OF THE METHODS OF
MICROSCOPIC ANATOMY

BY

ARTHUR BOLLES LEE

THI

LONDON

J. & A. CHURCHILL

11, NEW BURLINGTON STREET
1893

BIOLOGY
LIBRARY

PREFACE

THE present edition of this work has been most thoroughly
revised, and has been brought up to date by the incorpora-
tion of all the new matter of any value that has appeared
since the publication of the second edition.

Notwithstanding the very great pains that have been
taken in selecting matter for omission,*" and in condensing
the text as far as practicable, the book turns out to be con-
siderably enlarged. The reason for this is that the last
three years have produced, together with the usual crop of
ignorant futilities, a large amount of really valuable new
matter, of which due account has been rendered.

Amongst this new matter I would direct the reader's
attention to the thoroughly scientific and successful work
on the Theory of Staining with Carmine and with Haema-
toxylin, done by Paul Mayer, summarised in Chapters X
and XI, and to the new formulae for stains resulting there-
from. The results are eminently satisfactory, both theo-
retically and practically, and constitute the most brilliant
and solid contribution to this branch of the subject that has
been made since the publication, fourteen years ago, of
Grenadier's epoch-making alum-carmine and borax-carmine,
a feat that has not since been equalled.

The Ehrlich-Biondi-Heidenhain staining mixture may also
be noticed as being in its way a most brilliantly successful
thing ; it has in the course of the last three years attained
an almost unexampled degree of popularity.

As in the last edition, great extension has been given to
the subject of Neurological Methods, which now cover over
sixty pages, exclusive of the Methylen Blue method and
other impregnation methods discussed in Part I. This

* The receipt for Beale's carmine has been rejected from the text.
Probably the older among my readers will note its disappearance with some
touch of emotion. Eheu, fugaces !

VI PREFACE.

great extension is in large measure due to the luxuriant
growth of the literature that has sprung up round the has-
matoxylin methods of Weigert and the bichromate-of-silver
method of Golgi, and which it has been deemed advisable
to treat with all possible fulness.

The Methylen Blue impregnation method has nearly an
entire chapter assigned to it. This important process has
by no means been brought to perfection, but much has been
done already, and the future is full of promise.

The progress that has been made during the last three
years is certainly gratifying, but I must once more complain
of the mischief tnat is done by the method-mongers. The
reckless publication of crude or needless histological methods
is a crying evil. The really useful matter that is published
is smothered in a sea of rubbish, and the investigator is
obliged to devote to the acquisition of such new technical
knowledge as he may require very much more time than
would suffice if he were only unimpeded. Some idea of the
magnitude of the evil may perhaps be gleaned from the fact
that this book has now been out of print for many months,
the mass of literature that it has been necessary to digest
for the purpose of a new edition being so great that the
appearance of the work has been delayed far beyond the
contemplated time.

It is useless to remonstrate with the persons of whom I
am complaining ; they cannot grasp the fact of their igno-
rance, and cannot be brought to see that nobody heeds
them. I would appeal earnestly, therefore, to those with
whom the power lies to put an end to this nuisance. It
surely is not too much to ask that the editors of the periodi-
cals devoted to these matters should refuse to publish pro-
cesses that are not demonstrably new, and that do not make
out a good claim to be useful.

NYON, SWITZERLAND;
August, 1893.

PREFACE

TO THE

FIRST EDITION

IN its primary intention this work appeals rather to the
instructed anatomist than to the beginner. Its aim is to put
into his hands a concise but complete account of all the
methods of preparation that have been recommended as useful
for the purposes of Microscopic Anatomy, and so furnish
him with a ready source of information on points of detail as
to which his memory or his knowledge may be at fault. This
object is attained by the mere collection of Formula set out
in Part I, and of special methods described in Part II. But
the book could obviously be made to subserve a further end
— that of a guide to the beginner. To this end I have added
a General Introduction and a series of introductory paragraphs
prefixed, where needful, to the different chapters. These
introductory portions, taken together, go far to make up a,
formal treatise on the art. And as a further aid to the
beginner I have added the collection of examples given in
Part II. These examples are of course not intended for
servile imitation, but rather as hints suggestive of the most
fitting processes.

The collection of Formulae here brought together is, I
believe, practically exhaustive ; no process having any claim
to scientific status having been rejected, nor any, I trust,
unwittingly omitted. It may be useful here to say a word as
to the reasons for this — perhaps apparently excessive — catho-
licity of treatment. Doubtless a large proportion of the
formulae given are quite superseded in modern practice ; but
that is not a sufficient reason for rejecting them. The inclu-
sion of all of them is justified by the consideration that some

vili PREFACE TO THE FIRST EDITION.

one or other of them may perhaps serve, in some way that
cannot now be foreseen, to suggest some new method of value.
Let me give an example. Who, ten years ago, would have
thought that the formula of Blanchard's " Liqueur saline
hy drargyrique " deserved reprinting in a treatise on histologic
technic ? Yet it is to the disinterment of that forgotten
formula by Lang that we owe the establishment of corrosive
sublimate as one of the most useful fixing agents in the
arsenal of the microtomist. Or who would have deemed
Thiersch's lilac borax-carmine (Formula No. 80 a), published
in 1865, to be of greater importance than any other stain
till then made known ? Yet that formula it was that directly
suggested Woodward's admirable aqueous borax-carmine,
and through this, if I am not mistaken, the aqueous and the
alcoholic borax-carmines of Grenacher, the latter of which is
now to be found on the table of every embryologist.

All my abstracts and translations have been made from
the original sources, except where it has been impossible for
me to obtain sight of these. Keferences to the sources are
given in all cases ; but I desire here to make special acknow-
ledgment of the great assistance rendered me by the Journal
of the Royal Microscopical Society — in many respects the
best edited periodical known to me.

GENEVA, SWITZERLAND ;
February, 1885.

CONTENTS

PART I

PARAGRAPHS

CHAPTER I.

INTRODUCTORY. — The General Method

CHAPTER II.

KILLING .....

Sudden Killing, 7, 8 ; Narcotisation, 9—20.

CHAPTER III.

FIXING .

CHAPTER IV.

FIXING AGENTS : MINERAL ACIDS AND THEIR SALTS

1—5

6—20

21—24

25—46

CHAPTER V.

FIXING AGENTS : CHLORIDES, ORGANIC ACIDS, AND
OTHERS . . . .

CHAPTER VI.

HARDENING AGENTS , ....

Introduction, 67, 68; Mineral Acids, 64 — 74; Salts,
75 — 82 ; Chlorides and others, 83—90.

CHAPTER VII.

STAINING

47—66

67—90

91—94

CHAPTER VIII.

ANILIN COLOURS GIVING INDIRECT NUCLEAR STAINS

(FLEMMING'S METHOD) AND DIRECT NUCLEAR STAINS . 95 — 112
General Directions, 96—100; Indirect Stains, 101 —
107 ; Direct Stains, 108—112.

CONTENTS.

CHAPTER IX.

METHYLEN BLUE AND OTHER ANILIN STAINS

Methylen Blue, 113—122 ; Plasma and Ground Stains,
123— 143.

PARAGRAPHS

113-143

CHAPTER X.

CABMINE AND COCHINEAL STAINS ....
Theory of Carmine Staining, 144 — 150 ; Aqueous Car-
mines, Acid, 151 — 158; Neutral and Alkaline, 159
— 167 ; Alcoholic Carmines and Cochineals, 168 —
173.

144—173

CHAPTER XL

H.EMATEIN (H^MATOXYLIN) AND OTHER ORGANIC STAINS *

Theory of Staining 'with Haematoxylin, 174 — 179 ;
Hsematein (Hsematoxylin), 180 — 199; other Organic
Stains, 200—209.

174-209

CHAPTER XII.

METALLIC STAINS (IMPREGNATION METHODS)

Silver, 212—219; Gold, 220—230; other Metallic
Stains, 231—234.

210—234

CHAPTER XIII.

COMBINATION STAINS ......

Combinations having Carmine for a Primary Stain, 237
— 249; Combinations having Hsematoxylin for a
Primary Stain, 250—257 ; other Combinations, 258
—270.

235—270

CHAPTER XIV.
IMBEDDING METHODS : INTRODUCTION

271—274

CHAPTER XV.

IMBEDDING METHODS: PARAFFIN AND OTHER FUSION

MASSES 275—296

Paraffin, 275—287; Soap, 288—291; Gelatin, 292—
296.

CHAPTER XVI.

COLLODION (CELLOIDIN) AND OTHER IMBEDDING METHODS .
Collodion or Celloidin, 297—306 ; other Evaporation
Masses and Freezing Masses, 307 — 322.

297—322

CONTENTS.

XI

CHAPTER XVII.

SEEIAL SECTION MOUNTING .....
Methods for Paraffin Sections, 324 — 333 ; Methods for
Watery Sections, 334—33^; Methods for Celloidin
Sections, 337—341.

PARAGRAPHS

323—341

CHAPTER XVIII.

CLEARING AGENTS

342—355

CHAPTER XIX.

INDIFFEEENT LIQUIDS : EXAMINATION AND PEESEEVATION
MEDIA .......

Aqueous Liquids, 356—378 ; Mercurial Liquids, 379 —
385 ; other Fluids, 386—402 ; Glycerin Media, 403
—413 ; Resinous Media, 417—429.

356—429

CHAPTER XX.

CEMENTS AND VAENISHES

430—456

CHAPTER XXI.

INJECTIONS : GELATIN MASSES .... 457 — 497

Carmine, 466—473 ; Blue, 474—481 ; other Colours,
482—497.

CHAPTER XXII.

INJECTIONS : OTHEE MASSES ..... 498 — 521
White of Egg, Gum, 498, 499 ; Glycerin, 500—506 ;
Aqueous, 507—512; Celloidin, 513, 514; other
Masses, 515 — 521.

CHAPTER XXIII.

MACEEATION AND DIGESTION .

Maceration, 522—548 ; Digestion, 549—554.

522—554

CHAPTER XXIV.

COEEOSION, DECALCIFICATION, AND BLEACHING

Corrosion, 555 — 558 ; Decalcification and Desilicifica-
tion, 559—574 ; Bleaching, 575—583.

555—583

xii CONTENTS.

PART II

SPECIAL METHODS AND EXAMPLES.

PABAGKAFHS

CHAPTER XXV.
EMBETOLOGICAL METHODS ..... 584 — 631

CHAPTER XXVI.
CYTOLOGICAL METHODS ..... 632 — 645

CHAPTER XXVII.
TEGUMENTAEY ORGANS ..... 646—660

CHAPTER XXVIII.

MUSCLE AND TENDON (NEEVE-ENDINGS) . . . 661 — 681

Striated Muscle, 661—670 ; Tendon, 671—675 ; Smooth
Muscle, 676—681.

CHAPTER XXIX.

RETINA, INNEE EAE, NEEVES .... 682 — 727

Retina, 682—685 ; Inner Ear, 686, 687 ; Nerves, 688

—727.

CHAPTER XXX.

CENTEAL NEEVOUS SYSTEM ..... 728—785
Hardening, 729—742 ; Imbedding and Cutting, 743 ;
Staining, 744—776; Mounting, 777, 778; other
Methods, 779—785.

CHAPTER XXXI.

SOME OTHEE HISTOLOGICAL METHODS . . . 786—811

Connective Tissues, 786 — 801 ; Blood, 802—806 ;
Glands, 807—811.

CHAPTER XXXII.

SOME ZOOLOGICAL METHODS ..... 811—861
Tunicata, 811 ; Moliusca, 813—821 ; Arthropoda, 822
—831 ; Vernies, 832—844 ; Echinodermata, 845—
848 ; Coelenterata, 849—855 ; Porifera, 856 ; Pro-
tozoa, 857—861.

APPENDIX 862—881

INDEX pp. 477-509

THE MICKOTOMIST'S VADE-MECUM.

CHAPTER I.
INTRODUCTORY.

1. THE methods of modern microscopic anatomy may be
roughly classed as General and Special. There is a General
or Normal method, known as the method of sections, which
consists in carefully fixing the structures to be examined,
staining them with a nuclear stain, dehydrating with alcohol,
and mounting series of sections of the structures in balsam.
It is by this method that the work is blocked-out and very
often finished. Special points are then studied, if necessary,
by Special Methods, such as examination of the living tissue
elements, in situ, or in " indifferent" media; fixation with
special fixing agents ; staining with special stains ; dissocia-
tion by teasing or maceration ; injection ; impregnation ; and
the like.

2. THE GENEKAL METHOD. — The first thing to be done with
any structure is to fix its histological elements. (This state-
ment applies equally to all classes of objects, whether it be
desired to cut them into sections or to treat them in any other
special way.) Two things are implied by the word " fixing;"
first, the rapid killing of the element, so that it may not have
time to change the form it had during life, but is fixed in
death in the attitude it normally had during life ; and second,
the hardening of it to such a degree as may enable it to resist
without further change of form the action of the reagents
with which it may subsequently be treated. Too much stress
can hardly be laid on this point, which is the most distinctive

1

2 INTRODUCTORY.

feature of modern histplogical practice ; without good fixation
it is impossible 10 <ret good stains, or good sections, or prepa-
rations good in any way.

The structure having been duly fixed by one of the pro-
cesses described in the chapter on FIXING AGENTS, is washed
in order to remove from the tissues as far as possible all traces
of the fixing reagent.

The kind of liquid with which washing out is done is not a matter of in-
difference. If corrosive sublimate (for instance) or osmic acid, or a solution
into which chromic acid or a chromate enters, have been used for fixing, the
washing may be done with water. But if picric acid in any form has been
used, the washing must be done with alcohol. The reason of this difference
is that the first-named reagents (and, indeed, all the compounds of the heavy
metals used for fixing) appear to enter into a state of chemical combination
with the elements of tissues, rendering them insoluble in water ; so that the
hardening induced by these agents is not removed by subsequent treatment
with water. Picric acid, on the other hand, produces only a very slight
hardening of the tissues, and does not appear to enter into any combination
whatever with their elements; as it is entirely removable by treating the
tissues with water or alcohol. If the removal be effected by means of water,
the tissue elements are left in a soft state in which they are obnoxious to all
the hurtful effects of water. Alcohol must therefore be taken to remove the
picric acid and to effect the necessary hardening at the same time. Instruc-
tions for washing out are given, when necessary, in the discussion of the
different fixing agents in the following parts of this work.

At the same time that the superfluous fixing agent is being
removed from the tissues, or as soon as that is done, the
water of the tissues must be removed. This is necessary for two
reasons ; firstly, in the interest of preservation, the presence
of water being the condition of all others that most favours
post-mortem decomposition ; and secondly, because all water
must be removed in order to allow the tissues to be impreg-
nated with the imbedding material necessary for section-
cutting, or with the balsam with which they are to be finally
preserved. (The cases in which aqueous imbedding and pre-
serving media are employed are exceptional, and will be
treated of in the proper places.) The dehydration is performed
as follows : the objects are brought into weak alcohol, and
are then passed through successive alcohols of gradually in-
creased strength, remaining in each the time necessary for
complete saturation, and the last bath consisting of absolute
or at least very strong alcohol.

In dealing with extremely delicate objects, it may be necessary to take

INTRODUCTORY. 3

special precautions in order to avoid injury to them through the violent
diffusion-currents that are set up in the passage from water to alcohol, or
from one bath of alcohol to another of considerably different density. Some
kind of diffusion-apparatus should be used in these cases. The objects may
be placed in a tube plugged at one end and closed at the other by a
diaphragm of chamois skin or other suitable membrane, the tube being
then immersed in a vessel containing the grade of alcohol that it is desired
to add to the liquid in the tube, and the whole allowed to remain until by
diffusion through the diaphragm the two liquids have become of equal
density. Or COBB'S differentiator (Proc. Linn. Soc., N.S.W., v, 1890,
p. 157 ; Journ. Roy. Mic. Soc., 1890, p. 821) may be employed. Or, more
conveniently in most cases, the apparatus described and figured by HASWELL
(Proc. Linn. Soc., N.S.W., vi, 1891, p. 433; Journ. Roy. Mic. Soc.,
1892, p. 696). This consists of two wash-bottles connected in the usual way
by tubing, and furnished, the one with an overflow-tube, and the other with a
feeding-tube leading from a reservoir connected with it by means of a
regulating tap or drop arrangement. The objects are placed in the first
bottle ; some of the same liquid as that containing the objects is placed in
the second bottle ; and alcohol of the grade that it is desired to add is led
into it from the reservoir. The mixture of liquids therefore takes place in
the bottle that does not contain the objects, and the mixture itself is
gradually led over to the objects through the siphon-tube connecting the
two bottles. Another apparatus for rapid dehydration, devised by CHEATLE,
will be found described in Journ. Pathol. and Bacterial., i, 1892, p. 253,
or Journ. Roy. Mic. Soc., 1892, p. 892.

It is sometimes stated that it is necessary that the last alcohol bath should
consist of absolute alcohol. This however is incorrect, a strength of 90
per cent., or at all events 95 per cent., being sufficient in almost all cases.
For the small amount of water that remains in the tissues after treatment
with these grades of alcohol is efficiently removed in the bath of clearing
agent, if a good clearing agent be employed. Oil of cedar will remove the
remaining water from tissues saturated with 95 per cent, alcohol ; oil of
bergamot will "clear" from 90 per cent, alcohol, and anilin oil will clear
from 70 per cent, alcohol.

It is not generally necessary that pure ethylic alcohol be employed for
dehydration ; methylated spirit will suffice in most cases.

I am not aware of any substance that can entirely take the place of
alcohol for dehydration and preservation. Acetone, and methylal, have been
lately (PARKER, Zool. Anz., 403, 1892, p. 376) substituted for alcohol in the
dehydration of methylen-blue preparations ; but the great boon of an efficient
substitute for alcohol in general work remains yet to be discovered.

Considered as a mere dehydrating agent, alcohol fulfils its functions fairly
well. But considered as a histological preservative agent, it is far less
satisfactory. If tissues be left in alcohol for only a few days before sub-
jecting them to the further stages of preparation, the injurious effects of a
sojourn in alcohol will perhaps not be very disagreeably evident. But it is
otherwise if, as must often be the case, they are put away for many weeks
or months before the final preparation can be carried out. The dehydrating

4 INTRODUCTORY.

action of the alcohol being continuously prolonged, the minute structure of
tissues is sometimes considerably altered by it ; they become overhard and
shrink, and become brittle, and their capacity for taking stains well becomes
seriously diminished. KULTSCHITZKY (Zeit. f. wiss. Mik., iv, 3, 1887,
p. 349) has proposed to remedy this by putting up objects, after fixation and
washing out with alcohol, in ether, xylol, or toluol. FLEMMING has lately
(Arch. f. tnik. Anat., xxxvii, 1891, p. 685) advised putting up objects after
fixation in a mixture of alcohol, glycerin, and water, in about equal parts,
pointing out that objects thus preserved may be at any moment either pre-
pared for sectioning by treatment with pure alcohol, or softened for dissec-
tion or teasing by a little soaking in water, and that they do not become so
hard and brittle as alcohol specimens, and retain their staining power much
better. After extensive experience of this plan, I can highly recommend it,
and would only further suggest that the action of the liquid seems to me to
be in many cases much improved by addition of a little acetic acid (say 0'5
to 0'75 per cent.).

The water having been thus sufficiently removed, the
alcohol is in its turn removed from the tissues, and its place
taken by some anhydrous substance, generally an essential
oil, which is miscible with the material used for imbedding.
This operation is known as Clearing. It is very important
that the passage from the last alcohol to the clearing agent be
made gradual. This is effected by placing the clearing medium
under the alcohol. A. sufficient quantity of alcohol is placed
in a tube (a watch-glass will do, but tubes are generally
better), and then with a pipette a sufficient quantity of clear-
ing medium is introduced at the bottom of the alcohol. Or you
may first put the clearing medium into the tube, and then
carefully pour the alcohol on to the top of it. The two fluids
mingle but slowly. The objects to be cleared being now
quietly put into the supernatant alcohol, float at the surface
of separation of the two fluids, the exchange of fluids takes
place gradually, and the objects slowly sink down into the
lower layer. When they have sunk to the bottom, the alcohol
may be drawn off with a pipette, and the objects will be found
to be completely penetrated by the clearing medium. (It may
be noted here that this method of making the passage from
one fluid to another applies to all cases in which objects have
to be transferred from a lighter to a denser fluid — for instance,
from alcohol, or from water to glycerine. It is a more exact
method than that of successive baths of mixture of alcohol
and clearing agent.)

The objects are now imbedded. They are removed from

INTRODUCTORY. 5

the clearing medium, and soaked until thoroughly saturated
in the imbedding medium. This is, for small objects, gene-
rally paraffin, liquefied by heat, and for large objects generally
a solution of collodion or " celloidin." The imbedding medium
containing the object is then made to solidify, as described
in the chapter on imbedding processes, and sections are made
with a microtome through the imbedding mass and the in-
cluded objects. The sections are then mounted on a slide by
one of the methods described in the chapter on Serial Section
Methods, the imbedding material is removed from them (in
the case of paraffin), they are stained in situ on the slide,
dehydrated with alcohol, cleared and mounted in balsam or
damar. Or they may be stained, washed, dehydrated, and
cleared in watch-glasses, and afterwards mounted as desired
— the imbedding medium being first removed if desirable.

It is not always desirable to remove the imbedding mass ; celloidin sec-
tions stain well without being freed from it, and are usually even dehydrated,
cleared, and mounted without removal of the mass, which becomes quite
transparent in balsam. This plan has the advantage, which is a very im-
portant one for large sections, of allowing the sections to remain during the
whole of the manipulations protected by a supporting mass that holds all
their parts together.

The plan of staining sections on the slide is of somewhat recent introduc-
tion ; before it had been worked out the practice was to stain structures in
ioto, before cutting sections. And in cases in which structures are suffi-
ciently small and permeable to allow of satisfactory staining in this way,
and if it be not essential to save time, this plan is sometimes as good as the
one described. In this case the object, after having been fixed and washed
out, is taken from the water, or while still on its way through the lower
alcohols (it should not be allowed to proceed to the higher grades of alcohol
before staining, if that can be avoided), and passed through a bath of stain
(generally alcoholic borax-carmine or other alcoholic stain) of sufficient
duration, then dehydrated with successive alcohols, passed through a clearing
medium into paraffin, cut, and treated as above described, the sections in this
case being mounted direct from the turpentine, naphtha, or other solvent
with which the paraffin is removed. If aqueous staining media be employed
(and it is sometimes very desirable for particular purposes to prepare speci-
mens with some aqueous stain) the structures should either be stained in ioto
immediately after fixing and washing out, or sections may be stained on the
slide, the objects being passed through successive baths of alcohol of gradu-
ally decreasing strength before being put into the aqueous stain (a precaution
which will not be necessary for chromic objects (see below, § 5) ).

It was stated in the first edition of .this work that " the
great majority of preparations are made by fixing either with

, INTRODUCTORY.

sublimate or a picric acid combination, washing oat with
alcohol, staining with alcoholic borax-carmine, imbedding in
chloroform-paraffin, cutting with a sliding microtome, and
mounting the sections in series in Canada balsam." That is
probably still the case, but the method can no longer claim to
be what it then appeared to be, the classical method of micro-
scopic anatomy. I suggest the following, as being quite as
easy to carry out, and as giving preparations far richer in
detail and more truthfully preserved : — Fix in Flemming's
chromo-aceto-osmic mixture; wash out with water; dehydrate;
clear with oil of cedar-wood ; imbed in paraffin ; mount sec-
tions on the slide with Mayer's albumen medium ; stain with
safranin, or double-stain with gentian- violet and eosin ; and
mount in balsam or damar. That, or something like that, is
now the practice of many of the most advanced workers ; and
I know of no method that seems to have equal claims to be
considered a classical method of general morphological kin-
vestigation.

As regards the method to be used for imbedding, I take it that the paraffin
method is the method par excellence for small objects (objects up to 5 or 7
millimetres diameter) ; whilst the collodion or " celloidin " method is the
method par excellence for large objects.

As regards the rival claims of the method of staining objects in toto before
section cutting, and that of staining the sections, I confess that I cannot see
any reason for preferring the practice of staining in toto, which I consider
only has a raison d'etre in the case of objects which are not to be cut into
sections.

3. The treatment of objects which can be studied without
being cut into sections is identical with that above described,
with the omission of those passages that relate to imbedding
processes. Its normal course may be described as fixation,
washing out, staining, treatment with successive alcohols of
gradually increasing strength, final dehydration with absolute
alcohol, clearing, and mounting in balsam. This method
which may be termed the dehydration method, is usually pre-
ferred, as a general method, to what may be termed the wet
methods, by which objects are prepared and preserved in
aqueous media. The chief reason for this lies in the great
superiority of the dehydration methods as regards the pre-
servation of tissues. The presence of water is the most
important factor in the conditions that bring about the

INTRODUCTORY. 7

decomposition of organic matter, and its complete removal is
the chief condition of permanent preservation.

It is of course not intended here to suggest that wet methods of prepara-
tion should be altogether discarded. They have great value, they are even
indispensable, for special ends ; and all that is intended to be suggested is,
that they should be regarded not as general but as Special methods.

4. In the preparation of entire objects or structures that are
intact and covered by an integument not easily permeable by
liquids, special care must be taken to avoid swelling from
endosmosis on the passage of the objects from any of the
liquids employed to a liquid of less density, or shrinkage from
exosmosis on the passage to a liquid of greater density. This
applies most specially to the passage from the last alcohol
into the clearing medium. A slit should be made in the
integument, if possible, so that the two fluids may mingle
without hindrance. And in all cases the passage is made
gradual by placing the clearing medium under the alcohol, as
above described. Fluids of high diffusibility should be em-
ployed as far as possible in all the processes. Fixing agents
of great penetrating power (such as picrosulphuric acid or
alcoholic sublimate solution) should be employed where the
objects present a not easily permeable integument. Washing
out is done with successive alcohols, water being used only in
the case of fixation by osmic acid, or the chromic mixtures or
other fixing solutions that render washing by water imperative.
Staining is done by preference with alcoholic staining media.
The stains most to be recommended are Grenadier's borax-
carmine, or one of Mayer's new carminic acid or haematein
stains (for all of which see STAINING AGENTS). Anilin
stains are rarely applicable to this class of preparations.
Aqueous stains are more seldom used, though there are
many cases in which they are admissible, and some in which
they are preferable.

Minute dissections are best done, if necessary, in a drop of
clearing agent. I recommend cedar-wood oil for this pur-
pose, as it gives to the tissues a consistency very favorable
for dissection, whilst its viscosity serves to lend support to
delicate structures. Clove oil has a tendency to make tissues
that have lain in it for some time very brittle. The brittle-
ness is, however, sometimes very helpful in minute dissections.
Another property of clove oil is that it does not easily spread

8 INTRODUCTORY.

itself over the surface of a slide, but lias a tendency to form
very convex drops. This property also makes it frequently a
very convenient medium for making minute dissections in.

5. Following Paul Mayer, I gave in the previous editions the following
reasons for employing, in these cases, alcoholic rather than aqueous staining
media. Since, in most cases, treatment with alcohol forms part of the fixing
process, alcoholic solutions are logically indicated for staining. For by means
of them it is possible to avoid the bad effects that follow on passing delicate
tissues from alcohol into water, violent diffusive currents being thereby set
up which sometimes cany away whole groups of cells ; swellings being
caused in the elements of the tissues ; and, if the immersion in the aqueous
medium be prolonged, as is generally necessary in order to obtain a thorough
stain, maceration of the tissues supervening. But alcoholic staining fluids
have still other advantages ; they are in general more penetrating ; with them
alone is it possible to stain through chitinous integuments ; and, if it be de-
sired to stain slowly, tissues may be left in them for days without hurt.

Applied to the case now under consideration, the preparation in toto of
objects protected by not easily permeable investments, this doctrine is evi-
dently a wise one. For such objects must necessarily be fixed by some highly
penetrating but not permanently hardening agent such as picric acid, and
must necessarily be washed out with alcohol ; and it is a good maxim for
tissues so fixed that an object that has once been in alcohol should not be
allowed to go back into water, if that can possibly be avoided.

But in the case of structures that have been well fixed in a strongly and
permanently coagulating medium such as chromic acid, this precaution is
much less necessary. Sections of tissues that have been fixed for twenty-
four hours in Flemrning's solution may be passed with relative impunity
from absolute alcohol into an aqueous stain, and from that back again direct
into absolute alcohol. It is this property of tissues fixed in chromic solution
that determines me to recommend the practice of staining sections, instead
of staining objects in toto. As regards the quality of the stain, aqueous
solutions are in general the best. No alcoholic carmine or hematoxylin, for
instance, will give a stain equal in precision and delicacy to those given by
alum-carmine or Bohmer's hematoxylin. In a recent publication Mayer
himself states that he has somewhat cooled in his preference for alcoholic
staining media.

For an excellent exposition of the principles underlying the practice above
recommended, the reader may consult with advantage the paper of Paul
Mayer, in Mitth. Zool. Stat. Neapel, ii (1881), p. 1, et seq. See also the
abstract in Journ. Roy. Mic. Soc. (N. S.), ii (1882), pp. 866—881, and that
in Amer. Natural, xvi (1882), pp. 697 — 706, in which two last some im-
provements are mentioned which have been worked out since the publication
of Mayer's paper.

SUDDEN KILLING.

CHAPTER II.
KILLING.

6. In the majority of cases, the first step in the preparation
of an organ or organism consists in exposing it as rapidly and
as completely as possible to the action of one of the Fixing
Agents that are discussed in the next chapter. The organ or
organism is thus taken in the normal living state ; the fixing
agent serves to bring about at the same time, and with suffi-
cient rapidity, both the death of the organism and that of its
histological elements.

But this method is by no means applicable to all cases.
There are many animals, especially such as are of a soft con-
sistence, and deprived of any rigid skeleton, but possessing a
considerable faculty of contractility — such as many Ccelen-
terata, Bryozoa, and Serpulida, for instance — which if thus
treated contract violently, draw in their tentacles or branchiae,
and die in a state of contraction that renders the preserved
object a mere caricature of the living animal. In these cases,
special methods of killing must be resorted to.

Sudden Killing.

7. Speaking generally, there are two ways of dealing with
these difficult cases. You may kill the animal so suddenly
that it has not time to contract ; or you may paralyse it by
narcotics before killing it.

The application of Heat is a good means of killing suddenly.
It has the great advantage of allowing of good staining sub-
sequently, and of hindering less than any other method the
application of chemical tests to the tissues. By it, the tissues
are fixed at the same time that somatic death is brought
about. And what is more, the fixation thus brought about
is extremely faithful, provided the operation be properly per-
formed.

10 KILLING.

The difficulty consists in hitting off the right temperature,,
which is of course different for different objects. I think that
a temperature of 80° to 90° C. will generally be amply suffi-
cient, and that very frequently it will not be necessary to go
beyond 60° C. An exposure to heat of a few seconds will
generally suffice.

Small objects (Protozoa, Hydroids, Bryozoa) may be brought into a drop
of water in a watch-glass or on a slide and heated over the flame of a spirit
lamp. For large objects, the water or other liquid employed as the vehicle
of the heat, may be heated beforehand and the animals thrown into it.

As soon as it is supposed that the protoplasm of the tissues is coagulated
throughout, the animals should be brought into alcohol (30 to 70 per cent,
alcohol) (if water be employed as the heating agent).

An excellent plan for preparing many marine animals is to kill them in
hot fresh water. Some of the larger Nemertians, are better preserved by
this method than by any other with which I am acquainted.

8. Animals that contract but slowly, such as Alcyonium and
Veretillum, and some Tunicates, such as Pyrosoma, are very
well killed by throwing them into some very quickly acting
fixing liquid, either used hot or cold. Glacial or very strong
acetic acid (van Beneden's method) is an excellent reagent
for this purpose ; it may be used, for example, with some
Medusae. After an immersion of a few seconds or a few
minutes, according to the size of the animals, they should be
brought into alcohol of at least 50 per cent, strength. See
" Acetic acid " and " Tunicata." Corrosive sublimate is another
excellent reagent for this purpose.

Narcotisation.

9. The secret of narcotisation consists in adding some
anaesthetic substance very gradually, in very small doses, to
the water containing the animals, and waiting patiently for
it to take effect slowly.

THE TOBACCO-SMOKE METHOD for Actinia) (HEETWIG, Die Actinien*
1879) used to be practised as follows : — A dish containing the animals in
water is covered with a bell-glass, under which passes a curved glass or
rubber tube, which dips into the water. Tobacco smoke is blown into the
water for some time, through the tube, and the animals are then left for
some hours in order that narcotisation may become fully established. The-
animals are irritated from time to time by touching a tentacle with a needle.
As soon as it is observed that an animal begins to react slowly, that is to-
gay as soon as it is found that the contraction of the tentacle does not begin

ETHER AND ALCOHOL. 11

until a considerable time after it has been irritated by the needle, the nar-
cotisation may be considered sufficient. A quantity of some fixing liquid
sufficient to kill the animals before they have time to contract is then added
to the water.

A space of several hours is necessary in order to thoroughly narcotise an

Actinia by this method.

i

10. Nicotin in solution may be used instead of tobacco smoke
(ANDRES, Atti R. Accad. del Lincei, v. 1880, p. 9 ; see Journ.
Roy. Mic. Soc., N.S., ii, 1882, p. 881). Andres employs a
solution of 1 gramme of nicotin in a litre of sea-water. The
animal to be anaesthetised is placed in a jar containing half a
litre of sea-water, and the solution of nicotin is gradually con-
ducted into the jar by means of a thread acting as a siphon.
The thread ought to be of such a thickness as to be capable of
carrying over the whole of the solution of nicotin in twenty-
four hours.

11. Chloroform may be employed either in the liquid state
or in the state of vapour. KOROTNEFF (Mitth. Zool. Stat.
Neapel, v, Hft. 2, 1884, p. 229; Zeitf. wiss. Mik., 2, 1885, p.
230) operates in the following manner with Siphonophora.
The animals being extended, a watch-glass containing chloro-
form is floated on the surface of the water in which they are
contained, and the whole is covered with a bell-glass. As
soon as the animals have become insensible they are killed by
means of hot sublimate or chromic-acid solution plentifully
poured on to them.

Liquid chloroform is employed by squirting it in small
quantities on to the surface of the water containing the
animals. A syringe or pipette having a very small orifice, so
as to thoroughly pulverise the chloroform, should be employed.
Small quantities only should be projected at a time, and the
dose should be repeated every five minutes, until the animals
are anaesthetised.

I have seen large Medusae very completely anaesthetised in
the state of extension in an hour or two by this method.
ANDRES finds that this plan does not succeed with Actiniae, as
with them maceration of the tissues supervenes before anaes-
thesia is established.

12. Ether and Alcohol may be administered in the same way.
ANDRES has obtained good results with Actiniae by the use of

12 KILLING.

a mixture (invented by SALVATORE LO BIANCO) containing 20
parts of glycerin, 40 parts of 70 per cent, alcohol, and 40 parts
of sea- water. This mixture should be carefully poured on to
the surface of the water containing the animals, and allowed
to diffuse quietly through it. Several hours are sometimes
necessary for this.

EISIG employs alcohol in the same way.

13. Methyl-alcohol.— COEI (Zelt. f. wiss. Mik., vi, 4, 1890,
p. 438) prefers methyl-alcohol to all other reagents. It has,
amongst other advantages, that of having but a slight action
on albumins. CORI recommends a mixture composed of 10 c.c.
methyl-alcohol (of 96 per cent, strength), 90 c.c. water (fresh
or sea-water), and 0'6 g. of sodium chloride (to be added only
when fresh water is taken, the addition of the salt having for
its object to prevent maceration).

14. Hydrate of Chloral, which was first recommended, I
belive, by Foettinger (Arch, de Biol., vi, 1885, p. 115), gives
very good results with some subjects. Foettinger operates
by dropping crystals of chloral into the water containing
the animals. For Alcyonella he takes 25 to 80 centigrammes
of chloral for each hundred grammes of water. It takes
about three quarters of an hour to render a colony suffi-
ciently insensible to allow of fixing. Foettinger has obtained
satisfactory results with marine and fresh-water Bryozoa,
with Annelida, Mollusca, Nemertians, Actiniae, and with
Asteracanthion. He did not succeed with Hydroids.

I am bound to state that I have never had the slightest success with Ne-
mertians.

VEBWORN (Zeit.f. wiss. Zool., xlvi, 9, 1887, p. 99; see also Journ. Roy
Mic. Soc., 1888, p. 148) operates differently for fresh-water Bryozoa. He
puts Cristatella for a few minutes into 10 per cent, solution of chloral, in
which the animals sooner or later become extended.

KUKENTHAL (Zeit.f. wiss. Mik., iv, 8, 1887, p. 878 ; Journ. Roy. Mic.
Soc., 1888, p. 509) has obtained good results with some Annelids, by means
of a solution of one part of chloral in 1000 parts of sea-water.

The chloral method gives rise to maceration with some
subjects, and has been found to distort nuclear figures.
(This, I suspect, will probably be found to be the case with
most of these methods.)

HYDROXYLAMIN. 13

15. Cocain (RICHARD; Zool. Anz., 196, 1885, p. 332) has
been found to give good results. Richard puts a colony of
Bryozoa into a watch-glass with 5 c.c. of water, and adds
gradually 1 per cent, solution of hydrochlorate of coca'in
in water. After five minutes, the animals are somewhat
numbed, and half a cubic centimetre of the solution is
added, and the tentacles are caused to contract by irritating
them with a needle. Ten minutes later the animals should
be found to be dead in a state of extension.

This method is stated to succeed with Bryozoa, Hydra,
and certain worms.

It has been pointed out (by GOBI, in the paper quoted above) that unfor-
tunately when fixing agents, such as sublimate solution, are added to the
animals, the cocain is thrown down on them as a white precipitate.

16. Hydroxylamin. — HOFEE (Zeit.f. wiss. Mik., vii, 3, 1890,
p. 318) has employed hydroxylamin as a paralysing agent
with success with the most varied animal forms. Either the
sulphate or the hydrochlorate of the base may be used. He
recommends that the hydrochlorate be taken. This, as found
in commerce, is usually contaminated with HC1. It should
be dissolved in water (spring or sea-water, according to the
habitat of the organisms — in no case distilled water) and
the solution exactly neutralised by addition of carbonate of
soda. A 1 per cent, solution should be made up, and further
diluted for use. The organisms are placed in the diluted
solution, which may be taken of a strength varying from
0*1 per cent, used for thirty minutes or less (as for Infusoria)
to 0*25 per cent., used for from fifteen minutes to one hour
(Hydra), 1 per cent., one half to two hours (Hirudo), or as
much as ten to twenty hours (Helix and Anodonta).

It should be remembered that hydroxylamin is an extremely
powerful reducing agent. Care must therefore be taken not
to treat the paralysed animals with easily reducible fixing
agents, such as osmic acid, chromic acid, sublimate, chlorides
of gold or platinum, &c., unless it have been possible first to
sufficiently wash out the hydroxylamin with water.

17. Chloride of Magnesium— TULLBEBG (Arch. Zool. Exper. et Gen.,
x, 1892, p. 11 ; Journ. Roy. Mic. Soc., 1892, p. 435) has obtained some
results with this salt. For Actiniae, a 33 per cent, solution of the salt is to
be very slowly added to the water containing the expanded animal, until the
vessel contains one per cent, of the salt (thus for one litre of sea-water

14 KILLING.

33 c.c. of the solution must be added) . The addition must be made gradually ;
but it must be effected within half an hour. Thirty minutes later the
animal will be found to be anaesthetised, and may be fixed.

For terrestrial and fresh-water Invertebrates, rather stronger solutions
should be used.

18. Poisoning by small doses of some fixing agent is sometimes a good
method. SALVATOEE LO BIANCO employs the following method for pre-
serving Ascidise in an extended state. A 1 per cent, solution of chromic acid
acidulated with acetic acid is poured on to the surface of the water contain-
ing the animals, and allowed to diffuse slowly through it. The operation
takes four or five days (v. GAEBINI, Manuale per la Technica Mod. del
Microscopic, p. 168).

Osmic acid, or Kleinenberg's solution, is sometimes employed in the
same way.

I have seen Medusae killed in a satisfactory manner by means of crystals
of corrosive sublimate added to the water containing them.

Morphia, Ourare, Strychnin, Prussic Acid, and other paralysing
drugs have also been employed.

19. Asphyxiation may be sometimes successfully practised.
Terrestrial Gastropods may be killed for dissection by putting
them into a jar quite full of water that has been deprived of
its air by boiling, and hermetically closed. After from
twelve to twenty-four hours the animals are generally found
dead and extended. The effect is obtained somewhat quicker
if a little tobacco be added to the water.

Good results are sometimes obtained with aquatic animals
by simply leaving them to exhaust the oxygen of the water in
which they are contained. I have sometimes succeeded with
Holothuriae and other Echinoderms in this way ; and WAED
(see Amer. Nat., xxv, 1891, p. 398) has succeeded with
Hydroids, Actiniae, and similar forms. If the animals be
found to be imperfectly expanded when narcosis has set in,
they may be got to expand by putting them back for a short
time into pure sea-water ; and as soon as they are expanded
should be quickly thrown into some rapidly-killing reagent.

Carbonic Acid Gas has been recommended (by FOL, Zool. Anz., 128,
1885, p. 698). The water containing the animals should be saturated with
the gas. The method is stated to succeed with most Coelenterata and
Echinodermata, but not with Molluscs or Fishes. It has not been found
successful at the Naples laboratory.

20. Marine Animals are sometimes successfully killed by
simply putting them into fresh water.

Warm Water will sometimes serve to immobilise and even
kill both marine and fresh-water organisms.

FIXING AGENTS. 15

CHAPTER III.
FIXING.

21. The Necessity of Fixing.— The meaning of the term
'" fixing " has been explained above ( § 2). It only remains
here to insist on the absolute necessity of the employment of
fixing agents, and to briefly illustrate this necessity by a
single example. If a portion of living retina be placed in
aqueous humour, serum, or other so-called "indifferent"
medium, or in any of the media used for permanent preserva-
tion, it will be found that the rods and cones will not preserve
the appearance they have during life for more than a very
short time ; after a few minutes a series of changes begins
to take place, by which the outer segments of both rods and
cones become split into discs, and finally disintegrate so as to
be altogether unrecognisable, even if not totally destroyed.
Further, in an equally short time the nerve-fibres become
varicose, and appear to be thickly studded with spindle-
shaped knots ; and other post-mortem changes rapidly occur.
If, however, a fresh piece of retina be treated with a strong
solution of osmic acid, the whole of the rods and cones will
be found perfectly preserved after twenty-four hours' time,
and the nerve-fibres will be found not to be varicose. After
this preliminary hardening, portions of the retina may be
treated with water (which would be ruinous to the structures
of a fresh retina), they may even remain in water for days
without harm; they may be stained, acidified, hardened,
imbedded, cut into sections, and mounted in either aqueous
or resinous media without suffering.

22. The Action of Fixing Agents consists in the coagulation
of certain of the constituents of tissues, of their albuminoids,
their gelatin, their mucin. Some fixing agents seem to have
further the property of combining chemically with the tissues,

16 FIXING.

so that they cannot be easily removed from them by washing.
This is a consideration of great importance in view of ulterior
operations, and most particularly in view of staining. Chromic
acid and its salts, osmic acid, the chlorides of palladium, of
gold, and of iron, are reagents that seem to combine chemi-
cally with the tissues, and render necessary a special after-
treatment and special modes of staining, whilst picric acid,
nitric acid, and corrosive sublimate do not appear to enter
into that kind of combination, and can be entirely removed
from the tissues by washing, and leave the tissues in a state
in which they are susceptible of any kind of staining.

Practically it amounts to this, that if you fix with a chromio
or osmic mixture, you cannot stain with carmine but can only
stain with haBmatoxylin, if you wish to stain your objects in
toto ; or you may make sections, and stain them with safranin
or some other coal-tar colour. Whilst if you fix with subli-
mate or a picric acid mixture, you may do as you like in the
matter of staining.

The after-treatment appropriate to each fixing agent is
indicated in the special paragraphs.

23. Choice of a Fixing Agent. — Indications concerning the
proper fixing agent to employ for the different tissues and
organs of the animal kingdom, will be found in Part II. The
following remarks are intended as hints for beginners only.

The chief fixing agents for general work are FLEMMING'S
mixture, HERMANN'S platinum-chloride mixture, osmic acid,
corrosive sublimate, and picro-sulphuric acid.

I recommend that Flemming's mixture should be used
wherever it is possible, as I believe it to be in general by
far the best fixing agent yet invented, with the exception
of Hermann's mixture, which is unfortunately too expensive
to be employed in large quantities.

But it will not always be found possible to use it. Its
low power of penetration, for instance, puts it out of court
in the case of very impermeable objects, such as are fre-
quently offered by the Arthropoda. For these, picro-sul-
phuric acid may be recommended.

For very small objects, such as may be mounted whole,
osmic acid is nearly as good as Flemming's mixture, and
is frequently much more convenient to use.

FIXATION. 17

For objects a little larger, and for much embryological
work on objects that it is convenient to have stained in
totOy corrosive sublimate may be recommended. I think it
is in general a much better preservative of the forms of
anatomical elements than either Altmann's nitric acid or
Kleinenberg's fluid.

24. The Practice of Fixation, — Hints and Cautions.— See that
the structures are perfectly living at the instant of fixation,
otherwise you will only fix pathological states or post-mortem
states.

Do all you can to facilitate the rapid penetration of the
fixing agent. To this end, let the structures be divided into
the smallest portions that can conveniently be employed,
and if entire organs or organisms are to be fixed whole, let
openings, as large as possible, be first made in them.

The penetration of reagents is greatly facilitated by heat.
You may warm the reagent and put it with the objects to be
fixed in the paraffin stove, or you may even employ a fixing
agent heated to boiling point (as boiling sublimate solution
for certain corals and Hydroids, or boiling absolute alcohol
for certain Arthropods with very resistant integuments).

Let the quantity of fixing agent employed be at least many
times the volume of the objects to be fixed. If this precaution
be not observed the composition of the fixing liquid may be
seriously altered by admixture of the liquids or of the soluble
substances of the tissues thrown into it. For a weak and
slowly acting fixing agent, such as picric acid, the quantity of
liquid employed should be in volume about one hundred times
that of the object to be fixed. Reagents that act very ener-
getically, such as Flemming's solution, may be employed in
smaller proportions.

Be careful to use the appropriate liquid for washing out the
fixing agent after fixation. It is frequently by no means a
matter of indifference whether water or alcohol be employed
for washing out. Sometimes water will undo the whole work
of fixation (as with picric acid). Sometimes alcohol causes
precipitates that may ruin the preparations. Instructions on
this head are given in the paragraphs devoted to the different
fixing agents.

Use liberal quantities of liquid for washing.

2

18 FIXING.

Change the liquid as often as it becomes turbid, if that
should happen.

The process of washing out is often greatly facilitated by
heat. Picric acid, for instance, is nearly twice as soluble in
alcohol warmed to 40° C. as in alcohol at the normal tempe-
rature (Fol).

In the case of marine organisms it may be stated as a general
rule that their tissues are more refractory to the action of
reagents than are the tissues of corresponding fresh-water or
terrestrial forms, and fixing solutions should in consequence
be stronger (about two to three times stronger, according to
Langerhans) .

Marine animals ought to be freed from the sea-water adhe-
rent to their surfaces before treating them either with alcohol
or any fixing reagent that precipitates the salts of sea-water.
If this be not done, the precipitated salts will form on the
surfaces of the organisms a crust that prevents the penetra-
tion of reagents to the interior, thus allowing maceration to
be set up, and hindering the penetration of staining fluids.
Fixing solutions for marine organisms should therefore be
such as serve to keep in a state of solution, and finally remove,
the salts in question. They should never be made with sea-
water as a menstruum, as some workers have inconsiderately
proposed. If alcohol be employed it should be acidified with
hydrochloric acid. Picro- sulphuric acid is a reagent that
fulfils the conditions here spoken of. (On this subject see
Paul Mayer, in Mitth. Zool. Slat. Neapel, ii (1881), p. 1
et seq. See also the abstract in Journ. Eoy. Mic. Soc. (N.S.),
ii (1882), pp. 866—881, and that in Amer. Natural., xvi (1882),
pp. 697—706.)

OSMIC ACID. 19

CHAPTER IV.

FIXING AGENTS. MINERAL ACIDS AND THEIR SALTS.

25. Osmic Acid. — The tetroxyde of osmium (OsOJ is the
substance commonly known as osmic acid, though it does not
possess acid properties. It is a substance that is exceedingly
difficult to keep in use for any length of time. It is extremely
volatile, and in the form of an aqueous solution becomes par-
tially reduced with great readiness in presence of the slightest
contaminating particle of organic matter. (It is generally
believed that the aqueous solutions are reduced by light alone,,
but this is not the case ; they may be exposed to the light
with impunity if dust be absolutely denied access to them.
It would even seem that the solutions are improved for some
purposes by exposure to sunlight, vide infra, the remarks on
gold chloride solutions. Some observations communicated to
me by Dr. Lindsay-Johnson go to prove that, if dust be
avoided, solutions keep better in the light, with occasional
"sunning," than in the dark.)

Great stress is laid by authors on the fact that the vapour of osmium ia
very irritating to mucous tissues. It is said that the slightest exposure to
it is sufficient to give rise to serious catarrh, irritation of the bronchial tubes,
laryngeal catarrh, conjunctivitis, &c. I have never myself suffered in this
way, but there is no doubt that many persons do, and such susceptible sub-
jects should be very careful in handling osmium in any form.

26. How to keep the Solutions. — After having carefully tried
several of the plans that have been recommended for keeping
the working solutions free from dust, I have come to the con-
clusion that the following is the most practical plan for
preventing them from " going bad " : — The solution of osmic
acid in chromic acid solution is not, like the solution in pure
water, easily reducible, but may be kept without any special
precautions. I therefore keep the bulk of my osmium in the
shape of a 2 per cent, solution of osmic acid in 1 per cent,
aqueous chromic acid solution. This solution serves for

20 FIXING AGENTS.

fixation by osmium vapours, and for making up solution of
Flemming (or solution of Hermann) which is the form in
which osmium is most generally employed. A small quantity
of osmic acid may also be made up in 1 per cent, solution in
distilled water, and kept carefully protected from dust for use
in special cases. Those who have to do a great deal of fixing
by means of the vapours may also keep a supply of the solid
oxide for this purpose.

COEI (Zeit. f. wiss. MiJc., vi, 4, 1890, p. 442) finds that
solutions in distilled water keep perfectly if there be added to
them enough permanganate of potassium to give a very
slight rosy tint to the liquid. From time to time, as the
solution becomes colourless, further small quantities of the
salt should be added, so as to keep up the rosy tint.

27, Fixation by the Vapours. — Osmic acid is frequently em-
ployed in the form of vapour, and its employment in this form
is indicated in most of the cases in which it is possible to
expose the tissues directly to the action of the vapour. The
tissues are pinned out on a cork which must fit well into a
wide-mouthed bottle in which is contained a little solid osmic
acid (or a small quantity of 1 per cent, solution will do).
Very small objects, such as isolated cells, are simply placed on
a slide, which is inverted over the mouth of the bottle. They
remain there until they begin to turnbrowii (isolated cells will
generally be found to be sufficiently fixed in thirty seconds,
whilst in order to fix the deeper layers of relatively thick
objects, such as retina, an exposure of several hours may be
desirable). It is well to wash the objects with water before
staining, but a very slight washing will suffice. For staining,
methyl-green may be recommended for objects destined for
study in an aqueous medium, and, for permanent preparations,
alum-carmine, picro-carmine, or hsematoxylin.

In researches on nuclei, it is possible and may be useful to
employ the vapours of a freshly-prepared mixture of osmic
and formic or acetic acid (Gilson, La Cellule, i, 1885, p. 96).

The reasons for preferring the process of fixation by vapour of osmium,
where practicable, are that osmium is more highly penetrating when em-
ployed in this shape than when employed in solution, and produces a more
equal fixation, and that the arduous washing out required by the solutions is
here done away with. In many cases delicate structures are better pre-
served, all possibility of deformation through osmosis being here eliminated.

OSMIC ACID. 21

28. Fixation by Solutions. — When employed in aqueous solu-
tions osmic acid is used in strengths varying from -fa per cent,
to 2 per cent. Solutions of •£• per cent, to 1 per cent, have-
been very largely used, but the tendency of modern practice
seems to be towards weaker solutions and longer immersion.
For Infusoria \ per cent, for a few seconds ; for Porifera -^
to rfo per cent, for some hours ; for Mollusca 1 to 2 per cent,
for twenty-four hours ; for epithelia -fa to \ per cent, for an
hour or two ; for meroblastic ova -^ per cent, for twenty-four
hours ; for medullated nerve-fibre ^ to 1 per cent, for from
twenty minutes to two hours ; for tactile corpuscles -J- to 1 per
cent, for twenty-four hours ; for retina J to 2 per cent, for
from ten minutes to twenty-four hours ; for nuclei TV to 2 per
cent, for two or three hours. Such figures as these will serve
to give a general idea of the practice, whilst more precise in-
structions will be given when dealing with the tissues in detail.
(The durations here quoted appear to me exaggerated, except
for very voluminous specimens.)

A little acetic or formic acid (0'5 to 1 per cent.) may fre-
quently with advantage be added to the solutions just before
using.

If solutions made with pure water be used, they must be
kept protected from the light during the immersion of tissues.
This precaution is not necessary if Flemming's or Hermann's
solution be used. If the immersion is to be a long one the
tissues must be placed with the solution in well-closed vessels,
as osmium is very volatile.

KOLOSSOW (Zeit.f. wiss. Mik., v, i, 1888, p. 51) has recommended a 0'5
per cent, solution of osmium in 2 or 3 per cent, solution of nitrate or acetate
of uranium, as having a greatly enhanced penetrating power.

He has more lately (op. cit., ix, 1, 1892, p. 39) recommended for the same
reason a mixture of 50 c.c. absolute alcohol, 50 c.c. distilled water, 2 c.c.
concentrated nitric acid, and 1 to 2 g. osmium. This mixture is said to keep
indefinitely in a cool place.

The principle of combining osmium solutions with alcohol is due to
EANVIEE ET VIGNAL (RANVIEB, Leg. d'Anat. Gen., " App. term, des muscles
de la vie org.," p. 76 ; Vignal, Arch, de Physiol, 1884, p. 181). They take
equal volumes of 1 per cent, osinic acid and 90 per cent, alcohol (freshly mixed).
They wash out in 80 per cent, alcohol, then wash with water and stain for
forty-eight hours in picro-carmine or hsematoxylin. Viallanes has applied
this method to the histology of insects.

29. After-treatment. — The osmium must be well washed out

/"

22 FIXING AGENTS.

before proceeding to any further steps in preparation ; water
should be used for washing. Notwithstanding the greatest
care in soaking, it frequently happens that some of the acid
remains in the tissues, and causes them to over-blacken in
time. To obviate this it is necessary to wash them out in
ammonia-carmine or picro-carmine, or to soak them for twenty-
four hours in a solution of bichromate of potash (Miiller's
solution or Erlicki's will do), or in 0*5 per cent, solution of
chromic acid, or in MerkeFs solution, or in a weak solution of
ferrocyanide of potassium or cyanide of potassium. The
treatment with bichromate solutions has the great advantage
of highly facilitating staining with carmine or haematoxylin.
Max Schultze recommended washing, and mounting perma-
nently hi acetate of potash; but I believe the virtues attributed
to this method are illusory. Fol has lately recommended
treatment with a weak solution of carbonate of ammonia.
But the best plan of all is to properly bleach the preparations
(see "Bleaching"). This is perhaps most coveniently done
(as recommended by FOL, BRASS, and OVERTON) by means of
peroxide of hydrogen, which regenerates the osmium to osmic
acid. OVERTON (Zeit. f. wiss. Mik., vii, 1, 1890) finds that
bleaching is completed in a few minutes in a mixture of 1 part
commercial peroxide of hydrogen with 10 to 25 parts 70 per
cent, alcohol. (The commercial peroxide, slightly acidulated
with HC1, will keep well in the dark ; but the mixture with
alcohol must be made fresh for use.)

The same stains recommended for objects fixed by vapour will be found
useful here, with the addition of ammonia-carmine, which is really very use-
ful for strongly fixed specimens. For sections, of course in both cases,
safranin and the other nuclear anilin stains may be employed with advantage.

Osmic acid stains all fatty structures black ; it must therefore be avoided
for tissues in which much fat is present ; or if not, the preparations must be
subsequently very thoroughly bleached.

30. Chromic Acid. — Chromic anhydride, Cr03, is found in
commerce in the form of red crystals that dissolve readily in
water, forming chromic acid, H2Cr04. These crystals are very
deliquescent, and it is therefore well to keep the acid in stock
in the shape of a 1 per cent, solution. Care must be taken
not to allow the crystals to be contaminated by organic matter,
in the presence of which the anhydride is readily reduced into
sesquioxide.

CHROMIC ACID. 28

Chromic acid is employed in solution either in water or in
alcohol.

The most useful strengths in which it is employed in aqueous
solution are from 0*1 to TO per cent, for a period of immersion
of a few hours (structure of cells and ova). For nerve-tissues
weaker solutions are taken, -^ih to £th per cent, for a few
hours. Stronger solutions, such as 5 per cent., should only
be allowed to act for a few seconds.

The object should be washed out with water before passing
into alcohol or staining fluids. Long washing in water is
necessary to prepare them for staining, except an anilin stain
be used. It is possible to wash out in alcohol, and this may
be useful in special cases, but in general I think the practice
is not to be recommended. It is well to wash for many hours
in running water.

Tissues that have been fixed in chromic acid may be stained
in aqueous solutions if desired, as water does not appear to
have an injurious effect on them; the acid appears to enter
into some chemical combination with the elements of the
tissues, forming with them a compound that is not affected
either physically or chemically by water. The best stain to
follow chromic acid is haematoxylin, or, for sections, some
anilin stain. But the previous washing out with water must
be very thorough if good results are to be insured ; it may
take days.

Chromic acid is not a very penetrating reagent, and for this
reason, as well as for others, is seldem used pure, but plays an
important part in the mixtures described below, of which the
chief is certainly the mixture of Flemming. A chief objection
to the use of chromic acid is that it precipitates certain of the
liquid albuminoids of tissues in the form of filaments or net-
works, which are often of great regularity, and simulate struc-
tural elements of the tissues. This objection applies to all
mixtures into which chromic acid enters.

Action of light on alcohol containing chromic objects. — When objects
that have been treated by chromic acid or a chromate are put into alcohol
for hardening or preservation, it is found that after a short time a fine pre-
cipitate is thrown down on the surface of the preparations, thus forming a
certain obstacle to the further penetration of the alcohol. Previous washing
by water does not prevent the formation of this precipitate, and changing
the alcohol does not prevent it from forming again and again. It has been
found by Hans Virchow (Arch.f. mik. Anat., Bd. xxiv, 1885, p. 117) that

24 FIXING AGENTS.

the formation of this precipitate may be entirely prevented by simply keep-
ing the preparations in the dark. The alcohol becomes yellow as usual (and
should be changed as often as this takes place), but no precipitate is formed.
If this precaution be taken, previous washing with water may be omitted, or
at all events greatly abridged.

The brownish-green colour of chromic objects may be removed by treat-
ing them with peroxide of hydrogen (Unna, in Arch. f. mik. Anat., Bd.
xxx, 1887, p. 47 ; cf . Journ. Roy. Mie. Soc., 1887, p. 1060 ; and see the in-
structions for bleaching osmic acid preparations at the end of the last §).

31. Chromo-acetic Acid (FLEMMING, Zellsbz. Kern. u. Zellth.,
p. 382).

Chromic acid . 0*2 to 0'25 per cent.
Acetic acid . . O'l per cent., in water.

Flemming finds this the best reagent for the study of the
achromatic elements of karyokinesis. (Flemming wrote this-
in 1882, and I doubt whether it would now hold good.) Stain
with hasmatoxylin (the preparations are not favourable for
staining with safranin or other coal-tar colours).

I can recommend as a good fixing and hardening mixture
for Annelids in general, and probably for other forms, the
following fluid due to EHLERS (I do not know whether it has
been published) : — To 100 c.c. of chromic acid of 0*5 to 1 per
cent, add from 1 to 5 drops of glacial acetic acid. The pro-
portion of acetic acid indicated is sufficient to counteract any
tendency to shrinkage due to the chromic acid.

32. Chromo-formic Acid (RABL, Morph. Jahrb., x, 1884, pp.
215, 216). — Four or five drops of concentrated formic acid are
added to 200 c.c. of 0*33 per cent, chromic acid solution. The
mixture must be freshly prepared at the instant of using. Fix
for twelve to twenty-four hours, wash out with water, harden
in alcohol, stain with hsematoxylin or safranin. For the study
of karyokinesis. This is acknowledged to be one of the very
best reagents for the purpose.

33. Chromic Acid and Spirit. — A mixture of 2 parts of ^th
per cent, chromic acid solution with 1 part of methylated
spirit was much used by Klein in his investigations into the
structure of cells and nuclei, and found to give better results
than the ordinary reagents (including even osmic acid).
Hsematoxylin was used for staining.

The addition of alcohol to augment the penetrating power
of chromic acid seems to be a step in the right direction, and

CHROMO-ACETO-OSMIC ACID. 25

it is matter for surprise that such mixtures are not more used.
The alcohol should be added to the acid in aqueous solution,
as if strong alcohol be added to crystals of chromic anhydride,,
a very violent reaction is set up. The mixture should be kept
in the dark.

34. Chromo-osmic Acid (MAX FLESCH, Arch.f. mik. Anat., xvi, 1879,
p. 300). — This mixture (osmium O'lO, chromic acid 0.25, water lOO'O), origi-
nally introduced for the preparation of the auditory organ of vertebrates, is-
of general application. It does not require to be kept in the dark. Objects
may remain in it for twenty-four or thirty-six hours without risk of the-
osmic acid over-blackening them. Memming found it to preserve nuclear
figures well, but the preparations are pale, and difficult to stain well. He-
finds that the action of the mixture is improved (for nuclear figures) by the
addition of acetic, formic, or other acid. This addition brings out the-
figures more sharply, and has the further advantage of allowing of a sharper
stain with hsematoxylin, picro-carmine, or gentian violet. He recommends,
the following formula, which may be considered to have superseded Max
Flesch's.

35. Chromic-aceto-osmic Acid (FLEMMING, FIKST or WEAK
formula, Zellsubstanz, Kern imd Zelltheilung, 1882, p. 381). —
Chromic acid . . 0'25 per cent.]
Osmic acid . . O'l per cent. >In water.
Glacial acetic acid . 0*1 per cent.J

The best results (as regards faithfulness of fixation) are ob-
tained with this mixture when it is allowed to act for only a
short time (about half an hour).

But it may, without inconvenience, be allowed to act for
many hours or even days. Wash out, very thoroughly, in
water. Stain with haematoxylin, if you wish to stain in toto
(staining in this way with other reagents is possible, but
very difficult, and not to be recommended). Stain sections
with safranin, or other anilin, or with haematoxylin or Kern-
schwarz.

To make up this mixture with the usual stock solutions,,
you take :

Chromic acid of 1 per cent. . . 25 volumes.
Osmic acid of 1 per cent. . .10 „
Acetic acid of 1 per cent. . .10 „
Water ...... 55 „

If you keep your osmium in 2 per cent, solution in chromic
acid of 1 per cent., as I have recommended, you will have to
take only 20 vols. of chromic acid, 5 of your, osmium solution,

26 FIXING AGENTS.

and 65 of water. See also the remarks on the deterioration
of these solutions by keeping, in the next §.

It has been already stated more than once that Fleinming's solution is,
with the exception of Hermann's solution, probably the very best fixing re-
agent in general yet discovered. It has, however, been criticised. Faussek
(Zeitschr. f. wiss. Zool., Bd. xlv, 1887, pp. 694, et seq.) found it totally in-
applicable to the histology of the intestine of insects. He states that it
caused the intima to disappear, and the cells to run together into a compact
mass. Arnold (Arch. f. mik. Anat., Bd. xxx, 1887, p. 205) states that it
does not preserve cell-bodies faithfully. And A. Kotlarewsky (Mitth. d.
naturf. Ges. Bern., 1887 ; cf. Zeit,f. wiss. Mik., iv, 3, 1887, p. 387) found
that it preserved the forms of nerve-cells (spinal ganglia) less faithfully
than any of the reagents tried. I have not, myself, been struck by any
decided defect in the preservation of cystoplasmic structures in iny prepara-
tions made by this reagent, but think it possible that the observations of
these authors may be well founded as regards the present formula, but take
it that that is merely a reason for preferring the stronger mixture set forth
below.

It is not necessary in all cases to observe the exact proportions of the in-
gredients in this mixture. FOL (Lehrb. d. vergl. mik. Anat., 1884, p. 100)
recommends the following ;

1 per cent, chromic acid 25 vols.

1 per cent, osmic acid 2 „

2 per cent, acetic acid . ... . . 5 „

Water . 68 „

That is to say, a mixture much weaker in osmium than Flemming's. In
the Traite des Methodes Techniques, &c., Lee et Henneguy, 1887, I recom-
mended this mixture, as giving better results in general, but am now inclined
to think that, at all events as regards fidelity of fixation, it is a step in the
wrong direction, and that, on the contrary, the stronger mixture of Flemming
(next §) is a step in the right direction. Fol's formula has the advantage
of allowing better staining with carmine, that is all.

A mixture still weaker than this in osmium, viz. with 1 vol. osmium solu-
tion instead of 2, has been recommended by GOBI (Zeit.f. wiss. Mik., vi, 1,
1890, p. 441), but I still adhere to the opinion above expressed.

36. Chromo-aceto-osmic Acid (FLEMMING, SECOND or STRONG
formula, Zeit.f. wiss. Mik., 1, 1884, p. 349).—

1 per cent, chromic acid . .15 parts.

2 per cent, osmic acid . 4 „
Glacial acetic acid . . 1 „

If 2 per cent, osmium solution should not be at hand, you may con-
veniently make the mixture by taking

10 per cent, chromic acid . . . .15 parts.

1 per cent, osmic acid 80 „

Glacial acetic acid 10 „

Water 95

CHKOMO-ACETO-OSMIC ACID. 27

If this mixture be kept in stock in large quantities, it may
go bad, probably on account of the large proportion of
organic acid contained in it. I therefore recommend that it
be made up from time to time from stock solutions, in which
the osmium is kept separate from the acetic acid. The pro-
portions being as follows :

Cr03 0-15

Os 0-08

Acid, acet I'OO

Aq 19-00

You may make up and keep separately —

(A) 1 per cent, chromic acid . .11 parts.
Distilled water . . . 4 „

Glacial acetic acid . . 1 „

and (B) a 2 per cent, solution of osmium in 1 per cent,
chromic acid solution, and when required, mix four parts of
A with one of B ; or, of course, if you prefer it, you may
keep the osmic and chromic acid ready mixed in the propor-
tions given, and add 5 per cent, of acetic acid at the moment
of using.

In any case, it is better not to make up very large quan-
tities of the mixture at once, as osmium being very volatile
it will be found that solutions that have been long in use
no longer contain the proper proportion of that ingredient,
and the hardening action being thus weakened the swelling
action of the acetic acid will be insufficiently controlled.

Merk (Denksch.d. Math. Naturw. CL d, K. Acad. d. Wiss. Wien, 1887 ;
cf. Zeit.f. wiss. Mik., v, 2, 1888, p. 237) proposes to make up separately (A)
2 per cent, chromic acid ..... 7'5 parts.

Water 3'5 „

Acetic acid 1 „

and (B), some 1 per cent, osmium solution, and to mix for use 12 parts of
A with 8 of B. But this plan leaves you in the old difficulty of keeping
your osmium in aqueous solution.

It does not appear necessary to observe the exact proportions of the ingre-
dients of these mixtures, a certain latitude is allowable. Thus CABNOY (La
Cellule, 1, 2, 1885, p. 211) has employed a mixture one third stronger in
osmium and twice as strong in chromic acid, viz.

Chromic acid of 2 per cent, (or even stronger) . 45 parts.
Osmic acid of 2 per cent. . . . . 16 „
Glacial acetic acid 3 „

In the Traite des Meth. Techniques, 1887, I treated this

28 FIXING AGENTS.

formula somewhat coldly, pointing out (what was the case)
that Flemming recommended it merely for a very special
purpose, the hunting for karyokinetic figures, and that he did
not recommend it for general purposes. Further experience
has shown that ifc is applicable to general purposes, and will
probably be found for most purposes considerably superior
to the weak formula. I should use it by the hogshead if it
were not somewhat expensive.'

Arnold, in the place quoted in the last paragraph, says that it is to be
avoided if you wish to demonstrate the structure of certain nuclei (of
wandering cells) ; and the other objections there quoted as applying to the
weak formula are intended to apply more or less to the present formula. It
will be well not to attach too much importance to them. Let delicate struc-
tures be fixed for twenty-four hours or more, washed in running water for
an hour, and in successive alcohols for twenty-four hours, sectioned, and
stained with safranin or gentian violet, and there will be little complaint of
defective preservation.

The strong mixture does not brown tissues more than the
weak mixture, but rather less.

Fat is blackened by these mixtures ; but the blackened fat can be entirely
dissolved out of the tissues by treating them for a few hours with turpentine
that has been exposed to sunlight for an hour or two (see Flemming in
Zeit.f. wiss. Mik., vi, 1, 1889, p. 39 ; and vi, 2, 1889. p. 178).

PODWYSSOZKI recommends (for glands especially) the following modifi-
cation :

1 per cent. Cro,3 dissolved in 0'5 per cent, solution of cor-
rosive sublimate 15 c.c.

2 per cent, osmium solution ...... 4 c.c.

Glacial acetic acid 6 to 8 drops.

This sublimate is said to augment the penetration of the osmium, but is
unfavorable to staining. The proportion of acetic acid is reduced in order
to avoid swelling of the tissue-elements (ZIEGLER'S Beitrage z. path. Anat.+
i, 1886; cf. Zeit.f. wiss. Mik., iii, 3, 1886, p. 405).

36a. Platino-aceto-osmic Acid (HERMANN'S solution). This
extremely important reagent is historically a modification of
Flemming' s solution, platinum-chloride being taken instead
of chromic acid. See next Chapter, § 51.

37. Nitric Acid (ALTMANN, Arch. Anat. u. Phys., 1881, p. 219).

Altmann employs dilute nitric acid, containing from 3 to 3£ per cent, pure
acid. Such a solution has a sp. gr. of about T02 ; an araeometer may con-
veniently be used to determine the concentration of the solution. Stronger
solutions have been used, but do not give such good final results.

His (ibid., 1877, p. 115) recommended a 10 per cent, solution. Flemming

OHBOMO-NITEIO ACID. 29

at one time employed solutions of. 40 to 50 per cent, for the ova of inverte-
brates. This of course has the advantage of a very rapid fixing action.

Nitric acid has the valuable property of hardening yolk without making
it brittle. But, for general purposes at all events, the pure nitric acid solu-
tions may be considered to be superseded by Perenyi's chromo-nitric acid
mixture (below, § 39).

38. Nitrate of Silver has been used for fixation, but is too
uncertain in its action to be recommendable.

39. Chromo-nitric Acid (PERENYI'S formula, Zool. Anzeig.,
v, 1882, p. 459).

4 parts 10 per cent, nitric acid.

3 parts alcohol.

3 parts 0*5 per cent, chromic acid.

These are mixed, and after a short time give a fine violet-
coloured solution.

The objects are immersed for four to five hours, and then
passed through 70 per cent, alcohol (twenty- four hours) strong
alcohol (some days), absolute alcohol (four to five days).
They are then fit for cutting. The advantage of the process
is, amongst others, that segmentation spheres and nuclei are
perfectly fixed, the ova do not become porous, and cut like
cartilage.

Chromo-nitric acid is not only an embryological reagent,
and a very important one, but also an admirable one for
general work. I have found it altogether excellent for pre-
serving marine organisms, especially large forms. Strong
alcohol need only be used if the objects are destined to be
sectioned.

Another advantage is that the fixing solution may be combined with a
stain. (In this case the albuminous envelopes of the ova must be carefully
removed, otherwise the stain will not penetrate.)

Some stains, such as fuchsin or anilin red, may be dissolved directly in
the fixing solution. Others, such as eosin, purpurin, anilin violet, must first
be " dissolved in three parts of alcohol, and then shaken into the liquid."

Picro-carmine and borax-carmine may be added to the liquid, but they
give rise to a precipitate, which must be removed by filtration before using.

Another formula given by Perenyi (Zool. Anzeig., 274, 1888, p. 139, and
276, p. 196) is as follows:

3 parts 20 per cent, nitric acid.

3 parts 1 per cent, chromic acid.

4 parts absolute alcohol.

For embryos of Lacerta. Fix for twenty minutes. Wash out for an hour
with 70 per cent, alcohol, and then with strong alcohol. Stain with Dela-

30 FIXING AGENTS.

field's hsBmatoxylin, and treat the stained material for three to five minutes
with 1 per cent, chromic acid.

40. Chromo-nitric Acid with Bichromate (KOLLMANN, Arch. /.
Anat. u. Phys., 1885, p. 296).

Bichromate of potash ..... 5 per 100.

Chromic acid 2 „

Concentrated nitric acid . . . . 2 „
For ova of Teleostea. Fix for twelve hours, wash with water for twelve
hours, then remove the chorion, and put the ova into 70 per cent, alcohol.

41. Picro-chromic Acid (Fol. Lehrb., p. 100).—

Picric acid, sol. sat. in water . . . .10 vols.
1 per cent, chromic acid solution . . . 25 „

Water 65 „

At the instant of using, you may add 0'005 of osmic acid, which makes
the action more energetic. Wash with water (hot, nearly boiling water is
best), and then with alcohol. Fol says, "This reagent hardens tissues.
admirably, without hindering staining in any way ; but it is not very pene-
trating and fixes slowly."

I have seen Fol's formula, with the addition of a trace of acetic acid
quoted as " liquid of Hansel " — I know not with what justification.

42. Chromic Acid and Platinic Chloride (Merkel's solution $
from Mitth. Zool. Stat. Neapel, 1881, p. 11). — Equal volumes,
of 1*400 solution of chromic acid and 1*400 solution of platinic
chloride (PtCl 4). Objects should remain in it for several
hours or even days, as it does not harden very rapidly. After
washing out with alcohol of 50 per cent, to 70 per cent.,
objects stain excellently, notwithstanding the admixture of'
chromic acid. This is a very delicate and admirable fixative.
If objects that have been fixed by osmium be put into it
for some hours, blackening is effectually prevented. See
below § 50.

Salt*.

43. Bichromate and Cupric Sulphate Mixture (KULTSCHITZKY,
Zeit.f. wiss. Mik., iv, 3, 1887, p. 348). — A saturated solution,
of bichromate of potash and sulphate of copper in 50 per cent,
alcohol, to which is added at the instant of using a little acetic
acid, five or six drops per 100 c.c.

To make the solution, add the finely powdered salts to the
alcohol in excess, and leave them together in total darkness,
for twenty-four hours.

Fix for twelve to twenty-four hours in the dark, otherwise-
the salts will be precipitated. Then treat with strong alcohoL
for twelve to twenty-four hours, and make sections.

PERMANGANATE OF POTASH. 31

The rationale of this mixture is, that it fixes tissues faith-
fully, without causing the production of the delusive reticular
precipitates of albuminoids which we have mentioned as being
produced by chromic acid — that is the part played by the
bichromate and sulphate ; and that it also fixes faithfully the
chromatin of nuclei — that is the part played by the organic
acid.

The Chromates are useful as hardening rather than fixing agents.
They have a very mild and even action on tissues, but are not at all pene-
trating and act very slowly. They may still be found useful for fixing
certain tissues, some of those of Mollusca, for example. For mixtures that
may be used for such a purpose, see the chapter on HAEDENINQ AGENTS.

44. Cupric Sulphate. — Cupric sulphate was recommended some few
years ago in one of the well-known handbooks. It was recommended (in a
place which I cannot now find) to be used for marine organisms in saturated
solution in sea-water, the organisms to be preserved in the solution itself
till wanted. It has been quite recently (Arch. d. Sci. phys. et nat., Juin,

1889, t. xxi, p. 556) recommended by BEDOT for the preparation of Siphono-
phora and other delicate pelagic animals. See " Siphonophora."

FBIEDLAENDER (Biol. CentralbL, x, 1890, p. 483 ; Journ. Roy. Mic. Soc.,

1890, p. 804) preserves this class of objects by inundating them with a
mixture of 125 parts cupric sulphate, 125 parts zinc sulphate, and 1000
parts water.

45. Alum. — Alum has been used for fixing purposes, and may therefore
be mentioned here. Although quite superseded for general work by other
reagents, it may possibly still be found useful for certain special purposes.
For instance, for the preservation of Medusae the following process has been
recommended (by Pagenstecher). Take two parts of common salt and one
of alum, and make a strong solution. Throw the animals into it alive, and
leave them there for twenty-four to forty-eight hours. Preserve in weak
alcohol. A saturated solution of alum in sea-water preserves very well the
forms of Salpidte, Medusae, Ctenophora, and other pelagic animals. It
constitutes a preservative medium in which the objects may remain till
wanted.

According to my experience, it is not to be recommended for any but the
very coarsest work. It should be noted, however, that Kanvier (Traite Tech.
nique, p. 279) found that it fixed cartilage-cells better than any other reagent.
He employed a solution of 0'5 per cent.

46. Permanganate of Potash (Du PLESSIS, Bull. Soc. Vaud. Sci. Nat.,
2, ser. xv, pp. 278—280, 1878).— A strong solution in water. I find this
reagent has very slight penetrating power, and, besides, macerates some
tissues. It is therefore not adapted for general use, but it preserves very
well the forms of cells, and has one great virtue — it kills, I fancy, more
rapidly than any other agent I have been able to find ; even 2 per cent, osmic
acid is not equal to it in this respect. I have found it sometimes very
valuable for the study of isolated and very contractile cells, such as some
spermatozoa.

32 FIXING AGENTS.

CHAPTER V.

FIXING AGENTS — CHLORIDES, ORGANIC ACIDS, AND OTHERS.

Chlorides.

47. Bichloride of Mercury (Corrosive Sublimate). — Corrosive
sublimate is stated in the books to be soluble in about sixteen
parts of cold and three of boiling water. It will probably
be found that the aqueous solution contains about 5 per cent,
of the sublimate at the temperature of the laboratory. It is
more soluble in alcohol than in water, and still more so in
ether. Its solubility in all these menstrua is augmented by
the addition of hydrochloric acid, ammonious chloride, or
camphor. With sodium chloride it forms a more easily
soluble double salt ; hence sea- water may dissolve as much
as 15 per cent., and hence the composition of the liquid of
Lang.

For fixing corrosive sublimate may be, and very frequently
is, used pure ; but in most cases a finer fixation will be ob-
tained if it be acidified with acetic acid, say about 1 per cent,
of the acid. I find that a saturated solution in 5 per cent,
acetic acid is a very good formula for marine animals. VAN
BENEDEN has recommended a saturated solution in 25 per
cent, acetic acid.

In either case, the most concentrated solution obtainable
should in general be taken. The cold saturated aqueous
solution will suffice in most cases; but for some very con-
tractile forms (coral polypes, Planaria), a concentrated solu-
tion in warm or even boiling water should be employed. For
Arthropoda the alcoholic solution is frequently indicated.
Delicate objects, however, may require treatment with weak
solutions. HARTING found solutions of 0*2 to 0'5 per cent,
suitable for blood-corpuscles, and PACINI'S fluids are much of
the same strength. For these see the chapter on Examination
Media.

Objects should in all cases be removed from the fixing bath

CORROSIVE SUBLIMATE. 33

as soon as fixed, that is, in other words, as soon as they are
seen to have become opaque throughout, which is practically as
soon as they are penetrated by the liquid. Small objects are
fixed in a few minutes. I have found that a " salivary33 gland
of the larva of Chironomus is thoroughly fixed in three seconds.

Wash out with water or with alcohol. I consider alcohol
almost always preferable. Alcohol of about 70 percent, may
be taken. The extraction of the sublimate is hastened by the
addition of a little camphor to the alcohol. Or a little tinc-
ture of iodine may be added to the liquid, either alcohol or
water, used for washing, and the liquid changed until it no
longer becomes discoloured by the objects. It is important
that the sublimate be thoroughly removed from the tissues,
otherwise they become brittle. They will also become brittle
if they are kept long in alcohol. ,

You may stain in any way you like. Carmine stains are
peculiarly brilliant after sublimate, owing to the formation of
mercuric carminate. It is not necessary that the objects be
thoroughly washed out before staining ; the staining processes
themselves may be made to constitute a part of the washing-
out process.

It must be remembered that the solutions must not be
touched with iron or steel, as these produce precipitates that
may hurt the preparations. To manipulate the objects, wood,
glass, or platinum may be used ; for dissecting them, hedge-
hog spines, or quill pens.

When properly employed, sublimate is undoubtedly a fix-
ing agent of the very highest order. It is applicable to most
classes of objects. It is perhaps less applicable, in the pure
form, to Arthropods, as it possesses no great power of pene-
trating chitin.

Before passing to the sublimate mixtures that have been
recommended, it must here be stated that for general purposes
there seems to be no use in adding anything except a little
acetic acid to the pure sublimate.

48. Corrosive Sublimate (LANG'S formula, ' Zool. Anzeiger,' 1878, i,
p. 14). For Planar ia. — Take —

Distilled water . . . 100 parts by weight.
Chloride of sodium . . 6 to 10 parts.
Acetic acid . . . . 6 to 8 ,,
Bichloride of mercury . . * 3 to 12 „
(Alum, in some cases . . •£).

3

34 FIXING AGENTS.

The Planaria are to be placed on their backs and the mixture is to be
poured over them. They die extended. After the lapse of half an hour
they are brought into alcohol, first of 70 per cent., then of 90 per cent., then
absolute, and in two days' time are sufficiently hardened.

Second formula (ibid., 1879, ii. p. 46). — Make a concentrated solution of
corrosive sublimate in picro-sulphuric acid, to which has been added 5 per
cent, of acetic acid.

A solution containing 5 g. sublimate, 0'5 g. sodium chloride, and 100 c.c.
water, has been quoted as " solution of GAULE."

49. Cyanide of Mercury (KEISEB, BibliotU. Zool, H. vii, 1 Halfte,
1891 ; Zeit.f. wiss. Mik., viii, 3, 1891, p. 363).— Saturated aqueous solution.
Keiser found that this solution, warmed to 45° to 50° C., and allowed to act
for from 15 to 60 minutes, and then washed out with 70 per cent, alcohol,
was the best of all fixing media for Acanthoccphali.

50. Chloride of Platinum (Platinic Chloride, PtClJ.— An
extremely valuable reagent, originally introduced for the study
of karyokinesis, but of general application. RABL, to whom
we owe the introduction of this agent, employs an aqueous
solution of 1*300. The objects remain in it for twenty-four
hours, and are then washed with water, hardened in alcohol,
and sectioned. Stain with Delafield's haematoxylin, or with
safranin.

The action of platinum chloride is similar to that of gold
chloride, with the advantage that there is no blackening of
the preparations. Rabl finds it give better results (for the
study of karyokinesis) than any other reagent except chro-
moformic acid (§ 32). It causes a slight shrinkage of the
chromatin elements, a condition that renders the granules of
Pfitzner and the longitudinal division of the elements very
distinctly visible (see Rabl's well-known paper in Morph.
Jahrb., Bd. x, 1884, p. 216).

Platinum chloride is an extremely deliquescent salt, and for
this reason had better be procured in solution. Ten per cent,
solutions are found in commerce.

For Merkel's solution (chromo-platinic mixture) see ante,
§42.

51. Platino-Aceto-Osmic Mixture (HEEMANN, Arch. f. mik.
Anat., xxxiv, 1889, p. 58). — The author obtained excellent
results by substitutng 1 per cent, platinic chloride for the
chromic acid in Fleinming's strong formula for chromo-aceto-
osmic acid (§ 36), the other .ingredients either remaining as

PERCHLOUIDE OF IRON. 35

before, or the osmium being diminished one half. Thus,
1 per cent, platinic chloride 15 parts, glacial acetic acid 1
part, and 2 per cent, osmic acid either 4 parts or only 2 parts.
Hermann found that protoplasmic structures are thus better
preserved than with the chromic mixture, which I think is
undoubtedly the case. It was noted above (§ 30) that a chief
objection to the use of chromic acid is that it precipitates
certain of the liquid albuminoids of tissues in the form of
filaments or networks, which are often of great regularity,
and simulate structural elements of the tissues. This pla-
tinum chloride does not do. On the whole, I take it that this
mixture is probably the finest fixative yet discovered. Unfor-
tunately it is also the most expensive.

The after-treatment and staining should be the same as for
objects treated with Flemming's solution.

The remarks in § 36, as to the deterioration of Flemming's
solution by evaporation of osmium apply with equal force to
Hermann's mixture.

52. Chlorides of Palladium, of Gold, and of Iron.

Palladium Chloride. — Palladium chloride has been recommended by
experienced workers. It is used in solutions of 1'300, 1*600, or 1'SOO
strength, for from one to two minutes. Cattaneo recommends it as being
the best of fixatives for Infusoria. Tissues are impregnated and coloured
brown by it. For small objects one or two minutes will suffice for fixation.

This salt is found in commerce in the solid state. To dissolve it, take 10
grammes of the salt, one litre of water, and four to six drops of hydrochloric
acid. Solution will be effected in twenty-four hours.

Gold Chloride. — When used for fixing (and not for the object of
staining by impregnation) gold chloride is generally used in solution of 4
per cent, strength, for a few minutes (30 at most). Weaker solutions (£th
per cent.) or stronger (1 to 2 per cent.) may also be used. Wash out with
water.

Gold chloride is one of the most faithful fixing agents we know of. But
it is not fitted for general work on account of the capricious fashion in which
it undergoes reduction in the tissues, rendering the impregnated elements
unsusceptible of staining.

Perchloride of Iron (For,, Zeit. f. wiss. Zool, Bd. xxxviii, 1883, p.
491 ; and Lehrb. d. vergl. mik. Anat., p. 102). — Fol recommends 1 vol. of
Tinct. Ferri Perchlor. P. B. diluted with 5 to 10 vols. of 70 per cent,
alcohol. This gives better results than the weaker (2 per cent.) mixture at
first recommended. Aqueous solutions do not give nearly so good results.

Fix for a short time only and wash with alcohol. The preparations are best
stained with pyrogallol (see the chapter on Impregnation Methods). Fol
recommends this process chiefly for Infusoria, and other ciliated objects, but

36 FIXING AGENTS.

also as a general zoological method. I hear from Naples that it has been
tried for the preservation of marine organisms, and found wanting. Its
chief value seem to lie in the pyrogallol staining method, which may be
found very useful for nerve-end organs, which are impregnated somewhat
selectively by it.

Perchloride of iron (the tincture diluted with 3 to 4 vols. of either alcohol
or water) has lately been recommended for fixing medullated nerve by Platner
(Zeit.f. wiss. Mik., vi, 2, 1889, p. 187).

Organic Acids.

53. Acetic Acid. — The place of honour amongst organic
acids considered as fixing agents appears rightfully to belong
to this old-fashioned reagent. In the first edition of this
work it was merely stated that acetic and formic acid " are
useful and well-known fixatives of nuclei. Flemming,, who
has made a special investigation of their action, finds (Zell-
substanz, &c.,p. 380) that the best strength is from 0'2 to 1 per
cent. Strengths of 5 per cent, and more bring out the nuclein
structures clearly at first, but after a time cause them to swell
and become pale, which is not the case with the weaker
strengths" (ibid., p. 103). It must now be stated that, thanks
to v. BENEDEN, the strong acid has become established as a
most precious fixative of the most varied zoological objects.
It is particularly applicable to very contractile objects, such
as many Vermes, Ccelenterata, and Nudibranchs ; it kills with
the utmost rapidity, and has a tendency to leave them fixed
in the state of extension. The modus operandi is in general
as follows : — Pour glacial acetic acid in liberal quantity over
the organisms, leave them until they are penetrated by it —
which should be in five or six minutes, as the strong acid is a
highly penetrating reagent — and wash out in frequent changes
of alcohol of gradually increasing strength. Some persons
begin with 30 per cent, alcohol, but this appears to me rather
weak, and I think 70 per cent, or at least 50 per cent, alcohol
should be preferred.

In the Traite des Neth. Techn., 1887, 1 stated that the reason
why glacial acetic acid was not more used was that it did not
faithfully preserve delicate histological and cytological detail.
I now believe that if the instructions above given be followed,
in particular as regards the employment of the glacial acid,
and the washing out with somewhat strong alcohol, the most

FORMIC ACID. 37

delicate detail will generally be found admirably preserved.
I see no reason why other energetic reagents should not be
combined with the glacial acetic acid if desired. Dr. LINDSAY
JOHNSON (in. litt.} has found that the best of all fixatives for
retina is a mixture of equal parts glacial acetic acid and 2 per
cent, osinic acid.

54. Acetic Alcohol (CARNOY, La Cellule, t. iii, 1, 1886, p. 6 ;
and ibid., 1887, 2, p. 276 ; v. BENEDEN et NEYT, Bull. Ac. roy. d.
sci. de Belg., t. xiv, 1887, p. 218; ZACHAEIAS, Anat. Anz., iii
Jahrg., 1, i888, pp. 24—27; v. GEHUCHTEN, ibid., 8, p. 237).
— CAENOY has given two formulae for this important reagent.
The first is —

Glacial acetic acid . . .1 part.
Absolute alcohol . . .3 parts.
The second is —

Glacial acetic acid . . .1 part.
Absolute alcohol . . .6 parts.
Chloroform . . . . 3 „
The addition of chloroform is said to render the action of
the mixture more rapid.

V. BENEDEN and NEYT take equal volumes of glacial acid
and absolute alcohol.
ZACHARIAS takes —

Glacial acetic acid . . . 1 part.
Absolute alcohol . . .4 parts.
Osmic acid .... A few drops.
Acetic alcohol is one of the most penetrating and quickly
acting fixatives known. It preserves nuclei admirably, and
admits of admirable staining in any way that may be pre-
ferred. It was imagined by all of the authors quoted for the
study of karyokinesis in the ova of Ascaris, — proverbially one
of the most difficult objects to fix, — but it is applicable to
tissues in general. You may wash them out with alcohol and
treat them afterwards in any way that may be preferred. It
will be well, however, to avoid treatment with water as much
as possible.

55. Formic Acid may be used dilute in the same way as acetic acid
(supra, § 53). It is probable that it might also take the place of acetic
acid in the concentrated form, but I am not aware of any experiments in
this direction.

38 FIXING AGENTS.

56. Picric Acid. — Picric acid should always be employed in
the form of a strong solution. (That is to say, strong solu-
tions must always be employed when it is desired to make
sections or other preparations of tissues with the elements in
situ, as weak solutions macerate ; but for dissociation prepara-
tions or the fixation of isolated cells, weak solutions may be
taken. Flemming finds that the fixation of nuclear figures is
equally good with strong or weak solutions.) The saturated
solution is the one most employed. (One part of picric acid
dissolves in about 75 parts of cold water ; in hot water it is
very much more soluble.) Objects should remain in it for from
a few seconds to twenty-four hours, according to their size.
For Infusoria one to at most two minutes will suffice, whilst
objects of a thickness of several millimetres require from
three to six hours' immersion.

Picric acid should always be washed out with alcohol, as
water is hurtful to tissues that have been prepared in it. For
the same reason, during all remaining stages of treatment,
water should be avoided; staining should be performed by
means of alcoholic solutions, the only exceptions to this rule
being in favour of picro-carmine, which, probably on account
of the picric acid contained in it, does not appear to exert so
injurious an influence as other aqueous stains, and of methyl
green, and some few other aqueous stains that are themselves
weak hardening agents. It is one of the advantages of
picric acid that, by sufficiently prolonged soaking, it can with
certainty be entirely removed from any tissue by means of
alcohol.

Tissues fixed in picric acid can, after removal of the acid
by soaking, be perfectly stained in any stain. Mayer's para-
carmine, Grenacher's alcoholic borax-carmine, or Mayer's
haemacalcium may be recommended.

The most important property of picric acid is its great
penetration. This renders it peculiarly suitable for the pre-
paration of chitinous structures. For such objects alcohol of
70 per cent, to 90 per cent, should be taken for washing out,
and staining should be done by means of Mayer's cochineal
or hsemacalcium.

In very many if not most cases it is advantageous to employ picric acid
in the manner suggested by Kleinenberg (see below), that is, in combination

PJCRO-SULPHCRIC ACID. 39

with sulphuric acid ; or with nitric acid, or hydrochloric acid, as suggested
by P. Mayer (see below).

57. Picro-sulphuric Acid (KLEINENBERG, Quart. Journ. Mic.
Sri., April, 1879, p. 208; MAYER, Journ. Roy. Mic. Soc. (N.S.),
ii (1882), p. 867). — By picro-sulphuric acid, without any-
qualifying term, I understand a fluid made (following Mayer,
1. c.) as follows : — Distilled water, 100 vols. ; sulphuric acid,
2 vols. ; picric acid, as much as will dissolve (this will be about
0*25 vol. ; as the picric acid is much less soluble in sulphuric
acid solution than in water). This may also, in any case in
which confusion is likely to arise, be called " concentrated "
or " undiluted picro-sulphuric acid."

By " liquid of Kleinenberg " I understand a mixture sug-
gested by Kleinenberg (1. c.), and best made by diluting the
concentrated picro-sulphuric acid prepared as above with three
times its volume of water.

(Kleinenberg also directed the addition of as much creosote as would mix.
This was done with the idea of eliminating the swellings produced in some
objects by the liquid, but it has been found not to have the effect attributed
to it, and has been abandoned. FOL (Lehrb., p. 100) states that the same
end may be attained by adding one third vol. of 1 per cent, chromic acid.)

Of these two formulae the one commonly employed is that
given by Kleinenberg, — the dilute mixture ; undiluted picro-
sulphuric acid being reserved for objects requiring special
treatment, chiefly Anthropods. I may as well say at once
that in my opinion this practice is erroneous, for I hold that
Kleinenberg's solution is much weaker than is desirable in the
majority of cases, and should be reserved for special cases,
such perhaps as that for which it was originally proposed, the
embryology of the earthworm ; and the concentrated solution
should be the one taken for general work. This particularly
applies to marine organisms.

The treatment is the same in either case. " The object to
be preserved should remain in the liquid for three, four, or
more hours; then it should be transferred, in order to harden
it and remove the acid, into 70 per cent, alcohol, where it is
to remain five or six hours. From this it is to be removed
into 90 per cent, alcohol, which is to be changed until the
yellow tint has either disappeared or greatly diminished."

Warm alcohol extracts the acid much more quickly than

40 FIXING AGENTS.

cold, with which weeks may be required to fully remove the
acid from chitinous structures. I call attention here to what
was said as to washing out under the head of picric acid, viz.
that washing out must never be done with water. This is a
most important point, and one that is not sufficiently attended
to. You may stain as directed above for picric acid. You
may, of course, stain sections with alcoholic solutions of
safranin or the like.

The advantages of picro-sulphuric acid as a fixing agent are, that it kills
tissues very rapidly, that it has great penetrating power, that it can be
totally soaked out of the structures with alcohol (it is much more easily
removed from the tissues than pure picric acid), leaving them in a good con-
dition for staining, and, in the case of marine organisms, that it effectually
removes the different salts of sea-water that are present in them.

It has many disadvantages. For vertebrata it should be used with caution,
on account of the swelling caused by sulphuric acid in connective tissue.
For structures that contain much lime it is not to be recommended, for it
dissolves the lime and throws it down as crystals of gypsum in the tissues.
(For such structures the picro-nitric or picro-hydrochloric acid is to be pre-
ferred.) In numberless cases it produces swellings and maceration. For
the preservation of delicate, watery organisms, such as Medusae, it is an
abomination. For cytological researches it should be avoided, as its action
on both cytoplasm and nuclei is frequently most injurious. On the whole,
I find that for such objects as Arthropoda it is valuable on account of its
great penetrating power, the possibility of removing the acid entirely by
washing, and the facility thereby given for staining in toto. But for
general work, I consider that it is one of the most overrated reagents that
ever came into favour through the prestige of authority.

58. Picro-nitric Acid (MAYER, Mitth. Zool Stat. Neapel, 1881, p. 5 ;
Journ. Eoy. Mik. Soc. (N.S.), ii, 1882, p. 868).— Prepared in the same way
as picro-sulphuric acid except that instead of 2 vols. sulphuric acid you take
5 vols. pure nitric acid (of 25 per cent. N205) Mayer now dissolves the
picric acid in the nitric acid water, so that the formula runs :

Water 100 vols.

Nitric acid (of 25 per cent. N205) • • • 5 »

Picric acid, as much as will dissolve.
The fluid is used undiluted.

The properties of this fluid are very similar to those of picro-sulphuric
acid, with the advantage of avoiding the formation of gypsum crystals, and
the disadvantage that it is much more difficult to soak out of the tissues.
" Mayer recommends it strongly, and states that with eggs containing a large
amount of yolk material, like those of Palinurus, it gives better results than
nitric, picric, or picro-sulphuric acid."

59. Picro-hydrochloric Acid (MAYER, ibid.). — Prepared in the same
way as picro-sulphuric acid, except that instead of 2 vols. of sulphuric acid

ALCOHOL. 41

you take " 8 vols. of pure hydrochloric acid of 25 per cent. HCl." Mayer
now dissolves the picric acid in the hydrochloric acid water, so that the for-
mula runs :

Water 100 vols.

Hydrochloric acid (of 25 per cent. HCl) . . 8 „
Picric acid, as much as will dissolve.
The fluid is used undiluted.
The properties of this fluid are similar to those of picro-nitrie acid.

59 a. Picro-chromic Acid. See ante, § 41.

60. Picro-chromo-sulphuric Acid (KEISEE, Biblioth. ZooL, H. vii, 1
Halfte, 1891 ; Zeit.f. wiss. Mik., viii, 3, 1891, p. 363).— Keiser found the
following mixture excellent for Acanthocephali :

Picric acid ....... 1 gramme.

Cone, sulphuric acid ... . . . 10 grammes.

Chromic acid ...... 1 gramme.

Water 1000 grammes.

To be warmed to 55° C., allowed to act for 15 to 20 minutes, washed out
for 5 to 10 minutes with hot water, and afterwards for some days in 60 per
cent, alcohol.

61. Picro-osmic Acid. — FLEMMING (Zells. Eern-u.-Zellth., p. 381) has
experimented with mixtures made by substituting picric for chromic acid in
the chromo-osmic mixtures (ante, §§ 34 and 35). The results are identical
so far as regards the fixation (of nuclei) ; but staining is rendered more
difficult.

62. Picric Alcohol (GAGE, Proc. 'Arner. Soc. Micr., 1890, p. 120 ;
Journ. Roy. Hie. Soc., 1891, p. 418).— Alcohol (95 percent.), 250 parts;
•water, 250 parts ; picric acid, 1 part. Fix for about 24 hours, wash out for
a day in alcohol of 67 to 70 per cent., and then for a day or longer in
alcohol of 75 to 82 per cent.

Other Fixing Agents.

63. Alcohol. — For fixing, only two grades of alcohol are
found generally useful — very weak alcohol on the one hand,
and absolute alcohol on the other hand. Absolute alcohol
ranks as a fixing agent because it kills and hardens with such
rapidity that structures have not time to get deformed in the
process by the energetic dehydration that unavoidably takes
place. Dilute alcohol ranks as a fixing agent in virtue of
being of such a strength as to possess a sufficiently energetic
coagulating action and yet contain enough water to have but
a feeble and innocuous dehydrating action. The intermediate
grades do not realise these conditions, and therefore should
not be employed alone for fixing. But they may be very
useful in combination with other fixing agents (such as cor-

42 FIXING AGENTS.

rosive sublimate, chromic acid or nitric acid) by greatly
enhancing their penetrating power; 70 per cent, is a good
grade for this purpose.

One-third Alcohol.— The one grade of weak alcohol that is
found generally useful for fixing is one third alcohol, or
RANVIER'S ALCOHOL, known in France as " Alcohol au tiers/'
which is the name given to it by Ranvier himself; in Ger-
many as " Drittelalcohol " or " Ranviersche alcohol dilutus ;"
in Italy, as " alcool al terzo." It consists of two parts of water
and one part of alcohol o/90 per cent, (and not of absolute
alcohol, as was stated by an oversight in the first edition —
an error which I have seen copied in more than one place).
See the Traite Technique of Ranvier, p. .241, et passim.

Care should be taken that the alcohol is of the strength
specified, as the effects of this reagent depend to a remark-
able degree on its strength.

Objects may be left for twenty- four hours in this alcohol ;
not more, unless there be no reason for avoiding maceration,
which will generally occur after that time. You may con-
veniently stain with picro-carmine, alum-carmine, or methyl
green.

This classical reagent is a very mild fixative. Its harden-
ing action is so slight that it is seldom indicated for the fixing
of objects that are intended to be sectioned. Its chief use is
for extemporaneous and dissociation preparations.

Absolute Alcohol. — This is also a very valuable reagent It
preserves very well the structure of nuclei, which is by no
means the case with one-third alcohol. It has over the latter
also the advantage of superior penetrating power, being
indeed one of the most penetrating of known fixing agents.
Mayer finds that boiling absolute alcohol is often the only
means of killing certain Arthropoda rapidly enough to avoid
maceration brought about by the slowness of penetration of
common cold alcohol (especially in the case of Tracheata) .

It is important to employ for fixing a very large proportion
of alcohol. Alum-carmine is a good stain for small specimens
so fixed. For preservation, the object should be put into a
weaker alcohol, 90 per cent, or less.

Absolute alcohol is found in commerce. It is a product that it is almost
impossible to preserve in use, on account of the rapidity with which it

CHLORIDE AND ACETATE OF COPPEE. 43

hydrates on exposure to air. Fol recommends that a little quicklime be
kept in it. This absorbs part at least of the moisture drawn by the alcohol
from the air, and has the further advantage of neutralising the acid that is
frequently present in commercial alcohol.

Another plan that I have seen recommended is to suspend strips of gelatin
in it. It is stated that by this means ordinary alcohol may be rendered
absolute.

Ranvier adopts the following plan for preparing an alcohol absolute enough
for all practical purposes. Strong (95 per cent.) alcohol is treated with
calcined cupric sulphate, with which it is shaken up and allowed to remain
for a day or two. It is then decanted and treated with fresh cupric sulphate,
and the operation is repeated until the fresh cupric sulphate no longer
becomes conspicuously blue on contact with the alcohol ; or until, on a drop
of the alcohol being mixed with a drop of turpentine, no particles of water
can be seen in it under the microscope. The cupric sulphate is prepared by
calcining common blue vitriol in a porcelain capsule over a spirit lamp or
gas burner until it becomes white, and then reducing it to powder (see
Proc. Acad. Nat. Sci. Philad , 1884, p. 27 ; Science Record, ii, 1884, p. 65 ;
Journ. Roy. Micr. Soc. (N.S.), iv, 1884, pp. 322 and 984).

Acidulated Alcohol (PAUL MAYER, Mitth. Zool. Stat.Neapel, ii, 1881,
p. 7). — To 97 vols. of 90 per cent, alcohol, in which is dissolved a small
quantity of picric acid, add 3 vols. pure hydrochloric acid. Leave the
specimens in the mixture only just long enough to ensure that they are
thoroughly penetrated by it. Wash out with 90 per cent, alcohol, the dis-
appearance of the yellow stain of the picric acid being a sign that all the
acid is removed.

The use of this mixture is for the preparation of coarse objects it is intended
to preserve in alcohol. The object of the acid is to prevent both that glueing
together of organs by the perivisceral liquid, which is often brought about
by the coagulating action of pure alcohol, and the precipitation on the surface
of organs of the salts contained in sea- water, which is a hindrance not only
to the penetration of the alcohol, but also to subsequent staining.

Whitman (Journ. Roy. Mic. Soc. (N.S.), ii, 1882, p. 870) states that
"acid alcohol as above prepared loses its original qualities after standing
some time, as ether compounds are gradually formed at the expense of the
acid." He also states that 70 per cent, alcohol may be taken instead of 90
per cent, for washing out.

Acetic Alcohol. See ante, § 54, and the chapter on Cytological
Methods in Part II.

64. Chloride and Acetate of Copper (Ripart et Petit's formula,
CARNOY, La Biologie Cellulaire, p. 94).

Camphor water (not saturated) . . .75 grammes.

Distilled water 75

Crystallised acetic acid .... 1 gramme.

Acetate of copper 0'30 „

Chloride of copper . . . 0 30 „

This is a very moderate and delicate fixative. I consider that it has not
sufficient hardening power for objects that are intended to be dehydrated

44 FIXING AGENTS.

and mounted in balsam, but is frequently excellent and sometimes indis-
pensable for objects that are to be studied in as fresh a state as possible in
aqueous media. Objects fixed in it stain instantaneously and perfectly with
methyl green. Osmic acid may be added to the liquid to increase the fixing
action. For cytological researches this is a most invaluable medium.

65. Acetate of Uranium (ScHENK, Mitth. a. d. Embryol. Inst. Wien,
1882, p. 95 ; cf. GILSON, La Cellule, 1, 1885, p. 141).— This reagent is very
similar in its properties to picric acid. It has a mild fixing action, and a high
degree of penetration, which may make it useful for Arthropoda. It may be
combined with methyl green, which it does not precipitate.

66. Iodine. — Iodine possesses considerable hardening properties, and a
very high degree of penetration ; and, in point of fact, iodised serum, which
is generally employed as an "indifferent liquid," that is, one which is
supposed to exert no action whatever on tissues, is, in reality, a feeble
hardening agent, and forms a most admirable fixing agent for delicate
tissues. It is so classed by Kanvier (see Iodised Serum, Chap. XIX).
KENT (Manual of the Infusoria, 1881, p. 114; Journ. Roy. Mic. Soc. (N.S.),
iii, 1883, p. 730), has found it to act in a manner almost identical with osmic
acid, and in some instances even more efficiently (for fixing Infusoria). His
instructions are as follows : — " Prepare a saturated solution of potassic iodide
in distilled water, saturate this solution with iodine, filter, and dilute to a
brown-sherry colour. A very small portion only of the fluid is to be added
to that containing the Infusoria."

Or you may use the solution of LUGOL, of which the formula is as
follows :

Water . . . 100 parts.

Iodide of potassium 6 „

Iodine ........ 4 „

Iodine certainly kills cells very rapidly, without deforming them. Per-
sonally I have found it very useful for the examination of spermatozoa.
Unfortunately I am not acquainted with any nuclear stain that will work
well with it.

Very small objects may be instantaneously fixed by means of vapour of
iodine. Crystals of iodine may be heated in a test-tube till the vapours
are given off; then on inclining the tube the heavy vapours may be made to
flow over the objects arranged on a slide. The slide should then be warmed
to about 40° C. for 2 or 3 minutes in order to evaporate the iodine from the
objects, which may then be mounted or otherwise treated as desired (OvEE-
TON, Zeit.f. wiss. Mik., vii, 1, 1890, p. 14).

THE PRACTICE OF HARDENING. 45

CHAPTER VI.
HARDENING REAGENTS.

67. The Obligation of Hardening. — Methods of imbedding
liave now been brought to such a degree of perfection that
the thorough hardening of soft tissues that was formerly neces-
sary in order to cut thin sections from them is now, in the
majority of cases, no longer necessary ; by careful infiltration
with paraffin or some other good infiltration mass, most soft
objects can be satisfactorily cut with no greater an amount of
previous hardening than is furnished by the usual passing of
the tissues after fixing through successive alcohols in order to
prepare them for the paraffin bath. But there are some
exceptions. Such are, for instance, the cases in which it is
desired to cut very large sections, such as sections of the
entire human brain. Such an organ as this cannot be duly
infiltrated with alcohol in a few hours, and it is doubtful
whether it can be duly infiltrated with paraffin or any other
imbedding mass in any reasonable time- And certain organs
that are either extremely delicate or inaccessible, such as
retina or cochlea, will require to be specially hardened in
order to give the best results. The processes employed for
hardening such specimens as these will be described when
treating of the organs in question. In this chapter, which
may be considered as parenthetical, I confine myself to such
general statements concerning the employment of the usual
hardening agents as appear likely to be generally useful.

68. The Practice of Hardening — Hints and Cautions. — Employ
in general a relatively large volume of hardening liquid, and
change it very frequently. The exact proportions may be
made out by experiment for each reagent and each class of
objects. If the volume of liquid be insufficient its composition
will soon become seriously altered by the diffusion into it of
the soluble substances of the tissues ; and the result may be

46 HARDENING AGENTS.

a macerating instead of a hardening liquid. Further, as soon
as, in consequence of this diffusion, the liquid has acquired a
composition similar in respect x)f the proportions of colloids
and crystalloids contained in it to that of the liquids of the
tissues, osmotic equilibrium will become established, and dif-
fusion will cease. That is to say, the hardening liquid will
cease to penetrate. This means, of course, maceration of in-
ternal parts. On the other hand, it appears that a certain
slight proportion of colloids in the hardening liquid is favor-
able to the desired reaction, as it gives a better consistency
to the tissues by preventing them from becoming brittle.
Hence the utility of employing a certain proportion of harden-
ing agent.

Hardening had better be done in tall cylindrical vessels, the
objects being suspended by a thread at the top of the liquid.
This has the advantage of allowing diffusion to take place as
freely as possible, whilst any precipitates that may form fall
harmlessly to the bottom.

Always begin hardening with a weak reagent, increasing
the strength gradually, as fast as the tissues acquire a con-
sistence that enables them to support a more energetic action
of the reagent.

Let the objects be removed from the hardening fluid as soon
as they have acquired the desired consistency.

As to the choice of a hardening reagent, if you wish, above
all, for a rapid and energetic action, take chromic acid. If
you wish for a more moderate and more equable action, take
a chromic salt, or one of the compounds of which the chromic
salts are the principal ingredients.

Mineral Acids.

69. Chromic Acid. — Chromic acid is generally employed in
strengths of -J-th per cent, to J per cent., the immersion lasting
a few days or a few weeks, according to the size and nature
of the object. Mucous membrane, for instance, will harden
satisfactorily in a few days; brain will require some six
weeks.

Large quantities of the solution must be taken (at least 200
grammes for a piece of tissue of 1 centimetre cube, Ranvier).

In order to obtain the best results you should not employ

PICRO-CHROMIC ACID. 47

portions of tissue of more than an inch cube. For a human
spinal cord you should take two litres of solution, and change
it for fresh after a few days. Six weeks or two months are
necessary to complete the hardening.

The solution should be taken weak at first, and the strength
increased after a time. The objects should be removed from
the solution as soon as they have acquired the desired con-
sistency, as if left too long they will become brittle. (These
precautions are peculiarly necessary in the case of chromic
acid.) They may be preserved till wanted in alcohol (95 per
cent.). It is well to wash them out in water for twenty-four
or forty-eight hours before putting them into the alcohol. I
think it is frequently useful to add a little glycerin to the
hardening solution, there is less brittleness and, I think, less
shrinkage.

The reader's attention is called to the statements made in
§ 30 concerning the action of light on the alcohol containing
chromic objects.

Further directions for the employment of chromic acid will
be given in the special paragraphs. Chromic acid is a most
powerful and rapid hardening agent (by it, you may obtain
in a few days a degree of hardening that you would hardly
obtain in as many weeks with bichromate, for instance.) It
has the defect of a great tendency to cause brittleness.

70. Chromic Acid and Spirit (UBBAN PEITCHAED, Quart. Journ. Mic.
Sci., 1873, p. 427). — Chromic acid, 1 part ; water, 20 parts ; rectified spirit,
180 parts. Dissolve the chromic acid in the water first, and then add the
spirit (violent action will ensue if the dry chromic acid be added directly to
the spirit). The colour of the solution soon becomes brown. If, after a
few days, it turns semi-gelatinous, it should be changed for fresh. From a
week to ten days is required to harden such tissues as retina, cochlea, &c.,
for which this fluid is particularly well adapted.

71. Chromo-osmic Acid (MAX FLESCH.) Chromo-aceto-osmic
Acid (FLEMMING.) — Either of these mixtures may be used for
prolonged hardening, and are admirable. The weak form of
Flemming's solution is the one that should generally betaken
for hardening purposes. (See §§ 34 and 35.)

For delicate objects perhaps even better results may be
obtained by means of Chromic Acid and Platinic Chloride
(MEEKEL'S Solution). See § 42, ante.

72. Picro-chromic Acid. — This fixative may be found useful for

48 HARDENING AGENTS.

hardening objects that are only penetrable with difficulty. Some Tunicata,
for instance. See ante, § 41.

73. Osmic Acid. — Osmic acid is much more useful as a fixing agent than
as a hardening agent. Long immersion in osmic acid is sure to cause black-
ening, and may cause brittleness in the tissues. The strengths employed
for hardening vary from ^th per cent, to 1 per cent., and the tissues are left
in the solutions for twelve to twenty-four hours, seldom more. The chief
use of osmium in hardening is to enhance the energy of action of certain
mixtures into which it enters as a component, and serves to materially shorten
the time necessary for hardening. See the further information as to the
employment of this reagent given above, §§ 25, 26, 27, 28, 29.

74. Nitric Acid. — Nitric acid is taken of a strength of from 3 per cent,
to 10 per cent, or more, and may be allowed to act for two or three weeks.
It gives, thus employed (10 per cent, to 12 percent.), very tough preparations
of brain. It is also conveniently used by employing a very short immersion
and completing the hardening with alcohol, in which case it is properly con-
sidering as a fixing agent. See the information given under this head, § 37,
ante.

Salts.

75. Bichromate of Potash. — Perhaps the most important of
all known hardening agents, sensu stricto. It hardens slowly,
much more so than chromic acid, but it gives an incompar-
ably better consistency to the tissues, and it has not the same
tendency to make them brittle if the reaction be prolonged.
They may remain almost indefinitely exposed to its action
without much hurt.

The strength of the solutions employed is from 2 to 5 per
cent. As with chromic acid it is extremely important to
begin with weak solutions and proceed gradually to stronger
ones. About three weeks will be necessary for hardening a
sheep's eye in solutions gradually raised from 2 to 4 per cent.
Spinal cord requires from three to six weeks; a brain, at least
as many months.

After hardening, the objects should be well soaked out in
water before being put into alcohol. They had better be kept
in the dark when in alcohol (see above, § 30) . If you wish to
have a good stain with carmine, especially ammonia-carmine,
which is admirable for portions of nervous system so hardened,
you should not put the objects into alcohol at all, even for a
second, until they have been stained.

You may stain either with carmine or haematoxylin.

Bichromate objects have an ugly yellow colour which cannot be removed
bv soaking in water. It is said that it can be removed by washing for a

ERLICKI'S SOLUTION. 49

few minutes in a 1 per cent, solution of chloral hydrate. Gierke, however,
says that this treatment is prejudicial to the preservation of the tissues.

76. MtUler's Solution.—

Bichrotnate of potash . . 2 — 2J parts.

Sulphate of soda ... 1 part.

Water . . . .100 parts.

The duration of the reaction is about the same as with the
simple solution of chromio salts.

This fluid was very highly in vogue for many years, but
seems lately to be much less used. I fancy that the supe-
riority of this mixture over the simple bichromate solution
is not illusory, and is due to the formation in it of a trace of
free chromic acid. Fol says that for mammalian embryos, for
which it has been recommended, it is worthless.

77. Erlicki's Solution ( Warschauer med. Zeit., xxii, Nos. 15

and 18). —

Bichromate of potash . . . 2*5 parts.
Sulphate of copper . . . TO part.
Water lOO'O parts.

Here the addition of the cupric sulphate is intelligible.
This salt is itself a hardening agent of some energy, and may
well serve to reinforce the somewhat slow action of the bi-
chromate. As a matter of fact, " Erlicki" hardens very much
more rapidly than either simple bichromate or Miiller's solu-
tion. A spinal cord may be hardened in it in four days at
the temperature of an incubator, and in ten days at the
normal temperature (FoL, Lehrb. d. vergl. mik. Anat., p. 106).
I believe it to be one of the best hardening agents known for
voluminous objects. Human embryos of several months may
be conveniently hardened in it.

Nerve-centres that have heen hardened in Erlicki's fluid frequently contain
dark spots with irregular prolongations, simulating ganglion-cells. These
were at one time taken to be pathological formations, but they are now known
to consist of precipitates formed by the action of the hardening fluid. They
may be removed by washing with hot water, or with water slightly acidified
with hydrochloric acid, or by treating the specimens with 0'5 per cent,
chromic acid before putting them into alcohol (TscHisCH, Virchow's Arch.,
Bd. xcvii, p. 173 ; EDINGEE, Zeit.f. wiss. Mik., ii, 2, p. 245 ; LOEWENTHAL,
Rev. med. de la Suisse romande, 6me anne*e, i, p. 20).

4

50 HARDENING AGENTS.

78. Bichromate and Platinic Mixture (LINDSAY JOHNSON'S
Mixture; communicated by Dr. Lindsay Johnson). —

Bichromate of potash (2'5 per cent.) . 65 parts.
Osmic acid (2 per cent.) . . 15 „

Platinic chloride (2 per cent.) . 15 „

Acetic or formic acid . . . 5 „

This mixture was imagined for the preliminary hardening
of retina, being allowed to act for two hours only, and then
being followed by final hardening in pure bichromate solution.
But I have no doubt that it will prove applicable to other
structures, and give excellent results as a slow and gentle
hardening agent. For this purpose I would suggest that the
osmium might be diminished or omitted, the proportion of
Pt. C14 increased, and that of acetic acid diminished. The
function of the osmic acid in the present formula is to
enhance the hardening energy of the mixture. Dr. Lindsay
Johnson writes me that "it greatly reduces the length of time
necessary for hardening, three days being the time from
removal of the organ to its being in celloidin under dilute
spirit."

" If the osmium has a tendency to blacken, this may be
entirely prevented and a beautiful delicate chestnut-brown
deepening towards Bartholozzi-red tint obtained by adding
ten parts of 5 per cent, solution of nitrate of uranium,
which forms a layer of uranium on the top of the reduced
platinum and osmium (one or both)."

79. Bichromates and Alcohol. — Mixtures of either bichro-
mate with alcohol may be employed, and have a more rapid
action than the aqueous solution. Thus HAMILTON takes for
hardening brain a mixture of 1 part methylated spirits with
3 parts of solution of Miiller (see the chapter on the Central
Nervous System in Part II. ; see also KULTSCHIZKY'S Mixture,
ante, § 43). Preparations should be kept in the dark during
the process of hardening in these mixtures.

80. Bichromate of Ammonia. — A review of the literature of the sub-
ject shows that this salt is in considerable favour, for what precise motive is
not apparent. Its action is very similar to that of the potassium salt. Fol
says that it penetrates somewhat more rapidly, and hardens somewhat more
slowly. It should be employed in somewhat stronger solutions, up to 5 per
cent.

81. Neutral Chromate of Ammonia is preferred by some anatomists.

PLATINIC CHLORIDE. 51

It is used in the same strength as the bichromate. Klein has recommended
it for intestine, which it hardens, in 5 per cent, solution, in twenty-four
hours.

82. Sulphate of Copper. — This salt is seldom used alone, perhaps
because it does not give a sufficiently favorable consistency to the tissues
hardened by it. I take from the Lehrbuch of Fol (p. 106) the following
formula, which was first published by REMAZ, then modified by GOETTE, and
is said to be useful for hardening the ova of Amphibia :

2 per cent, solution of sulphate of copper . . 50 c.c.
Alcohol of 25 per cent. . . . . .50 c.c.

Eectified wood vinegar 35 drops.

Chlorides and others.

83. Platinic Chloride Mixture (MEKKEL'S Solution). — The
formula of this admirable reagent has been given above, § 42.
It is an admirable hardening medium for delicate objects.
Merkel states that he allowed from three to four days for the
action of the fluid for the retina ; for Annelids Eisig employs
an immersion of three to five hours, and transfers to 70 per
cent, alcohol ; for small leeches Whitman finds " one hour
sufficient, and transfers to 50 per cent, alcohol/'

Whitman recommends, for the hardening of pelagic fish
ova, a stronger mixture (due, I believe, to Eisig) , viz. :

0'25 per cent, solution of platinum chloride . 1 vol.

1 per cent, solution of chromic acid . 1 „

The ova to remain in it one or two days (WHITMAN, Methods
in Micro. Anat., p. 153).

84. Palladium Chloride (F. E. SCHULTZE, Arch. mik. Anat., iii, 1867,
p. 477). — This reagent was recommended by Schultze partly as giving to
tissues a better consistency than chromic acid or Miiller's solution, and partly
on account of a special faculty for penetrating organs rich in connective tissue
that he attributes to it. It is an impregnation reagent, staining certain ele-
ments of tissues in various tones of brown. For the somewhat lengthy
details of the manner of employing it, the reader is referred to the paper
quoted.

85. Chloride of Zinc is only employed for brain, see post, Part II

(GlACOMINl).

86. Picric Acid taken alone is a weak hardening agent, little used. It
should be employed in saturated solution. But it is a useful ingredient in
mixtures, serving to enhance the penetrating power. See Picro-chromic
Acid, ante, § 72; also Gage's Picric Alcohol, § 62.

87. Acetate of Lead. — Both the neutral acetate (sugar of lead) and the
basic acetate have been used for hardening nerve tissues. ANNA KOTLA-

52 HARDENING AGENTS.

BEWSKT found that nerve-cells hardened in 10 per cent, solution of sugar of
lead were admirably preserved. See her "Inaug.-Diss." in Mitth. d. naturf.
Ges. Bern., 1887, and Zeit.f. wiss. Mik., iv, 3, 1887, p. 387.

88. Alcohol. — When used alone, alcohol is inferior as a
hardening agent to most of the reagents discussed above ;
but when judiciously employed to complete the action of a
good fixing agent, it renders most valuable services. 90 to
95 per cent, is the -most generally useful strength. Weaker
alcohol, down to 70 per cent., is often indicated. Absolute
alcohol is seldom advisable. You ought to begin with weak,
and proceed gradually to stronger, alcohol. Large quanti-
ties of alcohol should be taken. The alcohol should be
frequently changed, or the tissue should be suspended near
the top of the alcohol, in order to have the tissue constantly
surrounded with pure spirit (the water and colloid matters
extracted from the tissue falling to the bottom of the vessel).
Many weeks may be necessary for hardening large specimens.
Small pieces of permeable tissue, such as mucous membrane,
may be sufficiently hardened in twenty-four hours.

89. Iodine may be used in combination with alcohol, and render service
through its great penetrating power. See the method of BETZ, post, Part II.

90. Pyridin. — Pyridin has been lately recommended as a hardening
agent (by A. DE SOUZA). It is said to harden, dehydrate, and clear tissues
at the same time. They may be stained after hardening by anilin dyes dis-
solved in the pyridin, or passed through water and stained by the usual pro-
cesses. It is said to harden quickly, and to give particularly good results
with brain. See Comptes Rendus hebd. de la Soc. de Biologic, 8 ser., t. iv,
No. 35, p. 622; Zeit.f. wise. Mile., v, i, 1888, p. 65 ; Journ. Eoy. Mic. Soc.,
1888, p. 1054.

THE KINDS OF STAINS. 53

CHAPTER VII.

STAINING.

91. The Kinds of Stains. — Stains are either General or
Special (otherwise called Specific, or Selective, or Elective).
A general stain is one that takes effect on all the elements of
a preparation. A special, specific, selective, or elective stain
is one that takes effect only on some of them, certain elements
being made prominent by being coloured, the rest either
remaining colourless or being coloured with a different
intensity or in a different tone. To obtain this differentiation
is the chief object for which colouring reagents are employed
in microscopic anatomy.

Two chief kinds of this selection may be distinguished, —
histological selection, and cytological selection. In the
former an entire tissue or group of tissue-elements is promi-
nently stained, the elements of other sorts present in the
preparation remaining colourless or being at all events
differently stained, as in a successful impregnation of nerve-
endings by means of gold chloride. This is the kind of
stain that is generally meant by a specific stain. In the
latter, the stain seizes on one of the constituent elements of
cells in general, namely, either the nucleus or the extra-
nuclear parts.

Stains that thus exhibit a selective affinity for the sub-
stance of nuclei, or nuclear stains, form at present by far the
most important class of stains — in zootomy at any rate.
What the zootomist wants, and the histologist too, in the
great majority of cases, is either to differentiate the intimate
structures of cells by means of a colour reaction, in order to
study them for their own sakes, or to have the nuclei of
tissues marked out by staining in the midst of the unstained
material in such a way that they may form landmarks to

54 STAINING.

catch the eye, which is then able to follow out with ease the
contours and relations of the elements to which the nuclei
belong; the extra-nuclear parts of these elements being
expressly left unstained in order that as little light as possible
may be absorbed in passing through the preparation. Pos-
sibly this may be an irrational procedure, but it has hitherto
been found in practice to be the most efficient for general
work.

To these two chief kinds of selective stains may be added
a third group, the plasmatic stains, consisting of those few
stains that take effect on cytoplasm, or formed tissue, or
ground substance only, leaving nuclei unstained. In this
book, therefore, stains are looked upon as being (1) General
or Ground stains ; (2) Selective stains ; the latter group
being subdivided into (a) Nuclear, (6) Plasmatic, (c) Histo-
logically Selective, or Specific. This classification, however,
is not followed in the arrangement of the special paragraphs,
it being more practical to follow an order based on the
chemical nature of the staining agents, and on convenience
of exposition.

Some writers have divided stains into nuclear, general, and selective.
This arrangement appears to me faulty because every nuclear stain is eo ipso
selective, and because it ignores the subdivisions of selective stains.

92. The Methods of Staining. — Colouring matters possessing
so great an affinity for certain elements of tissues that they
may be left to produce the desired electivity of stain, without
any special manipulation on the part of the operator, are un-
fortunately rare. In practice, selective staining is arrived at
in two ways. In the one, which may be called the direct
method, you make use of a colouring reagent that stains the
element desired to be selected more quickly than the elements
you wish to have unstained ; and you stop the process and fix
the colour at the moment when the former are just sufficiently
stained and the latter not affected to an injurious extent, or
not affected at all, by the colour. This is what happens —
for instance, when you stain the nuclei of a preparation by
treatment with very dilute haematoxylin ; you get, at a certain
moment, a fairly pure nuclear stain ; but if you were to pro-
long the treatment, the extra-nuclear elements would take up

THE STATE OF THE TISSUES TO BE STAINED. 55

the colour, and the selectivity of the stain would be lost. It
may be noted of this method that it is in general the method
of fast stains (" echte Farbung"), and that it renders great
services in the colouring of specimens in toto, — a procedure
which is not possible with the chief stains of the other class
(the anilins). It is the old method of carmine and hsema-
toxylin staining.

The second, or indirect, method, is the method of overstain-
ing followed by partial discoloration. You begin by staining
all the elements of your preparation indiscriminately, and
you then wash out the colour from all the elements, except
those which you desire to have stained, these retaining the
colour more obstinately than the others in virtue of a certain
not yet satisfactorily explained affinity. This is what happens
— for instance, when you stain a section of one deep red in
all its elements with safranin, and then treating it for a few
seconds with alcohol, extract the colour from all but the
chromatin and nucleoli of the nuclei. It is in this method
that the coal-tar colours find their chief employment. It is
in general applicable only to sections, and not to staining
objects in toto (the case of borax-carmine is probably only a
seeming exception to this statement). It is a method, how-
ever, of very wide applicability, and gives the most brilliant
results that have hitherto been attained.

93. The State of the Tissues to be Stained. — It is generally
found that precise stains can only be obtained with carefully
fixed (i. e. hardened) tissues. Dead, but not artificially hard-
ened tissues stain indeed, but not generally in a precise
manner. Living tissue elements in general do not stain at
all, but resist the action of colouring reagents till they are
killed by them.

Staining " intra vitam." — Some few substances, however,
possess the property of staining living cells without greatly
impairing their vitality. Such are — in very dilute solutions
— cyanm (or quinolein), methylen blue, Bismarck brown,
anilin black, and, under certain conditions, dahlia, and gentian
violet, with perhaps methyl violet and some others whose
action is not yet sufficiently established by experiment. Congo,
even in strong solution, is not toxic to some organisms, and
stains some structures (see SCHOLTZ, Centralb. f. d.med. Wiss.,

56 t STAINING.

1886, p. 449; also Journ. Roy. Mic. 8oc., 1886, p. 1092).
Living Rotifera are in part successfully stained by it during
life. (The paper of Martinotti, Zeit.f. wiss. Mik., v. 3, 1888,
p. 305, may be consulted on this subject.)

As to the employment of these reagents, it may be noted
that they must be taken in a state of extreme dilution, and in
neutral or feebly alkaline solution — acids being of course
toxic to cells. Thus employed, they will be found to tinge
with colour the cytoplasm of certain cells during life (never,
so far as I know, nuclear chromatin during life ; — if this stain,
it is a sign that death has set in). The stain is sometimes
diffused throughout the general substance of the cytoplasm,
sometimes limited to certain granules in it (which have been
taken, perhaps without sufficient reason, to be identical with the
granules of Altmann (Altmann's Studien uberdie Zelle, 1886).
Methylen blue has the valuable point that it is perfectly
soluble in saline solutions, and may therefore be employed
with marine organisms by simply adding it to sea-water. The
others are not thus soluble to a practical extent, but I find
that gentian and dahlia become so if a trace of chloral hydrate
— 0*25 per cent, is amply enough — be added to the saline
solution. Any of these reagents may be rubbed up with
serum, or other "indifferent " liquid.

Methylen blue may be fixed in the tissues, and permanent
preparations made, by one or other of the methods described
in Chap. IX. Bismarck brown stains may be fixed with 0*2
per cent, chromic acid, and the preparations may be stained
with safranin, care being taken not to expose them too long
to the action of alcohol.

I may say that personally I have found gentian, dahlia, and
methylen blue, added to indifferent liquids, extremely useful
in the examination of tissue-cells. Quinolei'n and Bismarck
brown are well-known aids to the study of Infusoria. Me-
thylen blue has a specific affinity for sensory nerves, and is an
extremely important reagent (see post, Chap. IX, and Part II).

94. Choice of a Stain. — The following may be recommended
with confidence for general work : — For sections, Fie mining's
method, with safranin or gentian for a single stain, or Hen-
neguy's permanganate modification of the same (Chap. VIII),
and the same with hsematoxylin (§ 106), or Flemming's

CHOICE OF A STAIN. 57

orange method (§ 258), or gentian andeosin (Chap. XIII) for
a double stain.

For staining in toto Grenacher's alcoholic borax-carmine
(§ 169), or Mayer's carmalum (§ 151), unless the object
be so impermeable as to require a more highly alcoho-
lised stain, in which case take Mayer's para-carmine
(§ 168), or for chromic acid objects Mayer's haemacalcium
(§ 182).

For fresh dissociated tissues or small entire objects, methyl
green, if it is not important to have permanent preparations ;
if it is, take alum-carmine.

Picric acid may be used for double staining after carmine
or hsematoxylin.

Many others of the numerous stains discussed in the follow-
ing chapters render most valuable services, and will be
found recommended in the special paragraphs as occasion
dictates.

I would add one word of advice to the beginner : never use
a double stain where a single one will do. To do so is too
often to go farther and fare worse.

And a word of caution to beginners and others :

You are not likely to succeed in staining, especially in the
beautiful processes of staining with coal-tar colours, unless you
see to it that you are working with chemicals of the proper
quality. You cannot ensure this by going to a generally
trustworthy house for chemical products — at all events, not
in the case of coal-tar colours. It is not sufficient that these
should be what they are commercially described to be ; they
may be pure, and yet not give good stains. They must (in
the case of anilins, at all events) be the identical products
used in their work by the authors who have described and
recommended them (see[the note on the numerous safranins in
the market, § 101). I therefore feel constrained to advise
everybody to get his reagents — at all events his anilins —
from the well-known chemists Grubler and Miinder. Griibler
has all the tried reagents in stock, and supplies only such as
have been found by experiment with tissues to furnish the
desired stain. He also makes up fixing and staining solu-
tions, injection and imbedding masses, &c., according to the
classical formulae, and sends them out neatly packed and ready
for use. From experience I can most highly recommend these

58 STAINING.

preparations, which are in nine cases out of ten better than
those the observer is likely to make for himself. They may
be ordered from the price list, or by quoting the numbers of
the formulae in this work. The address is : Herrn Dr. G.
GRUBLER, Chemiker, Baiersche Strasse 12, Leipzig. Griibler
can correspond in English.

His preparations can be obtained in London from Dr. R.
KANTBACK, 21, Golden Square, Eegent Street, W., who is also
the authorised agent for the microscopes and apparatus of
Zeiss, also for the microtomes of Becker, and the Bacterio-
logical Apparatus of F. & M. Lautenschlaeger, &c.

Mimder's address is : Herrn Dr. G. MUNDER, Mikroskopisch-
chemisches Institut, Gottingen.

Messrs. SQUIRE & SONS, Chemists, 413, Oxford Street,
London, W., also make a speciality of microscopical reagents,
and I am told furnish excellent products.

ANILIN COLOURS GIVING INDIRECT NUCLEAR STAINS. 59

CHAPTER VIII.

ANILIN* COLOURS GIVING INDIRECT NUCLEAR STAINS
(FLEMMING'S METHOD), AND DIRECT NUCLEAR STAINS.

A. Indirect Nuclear Stains.

95. VEEY few anilins give a precise nuclear stain by the
direct method (§ 92). Two of them — methyl green and Bis-
marck brown — are pre-eminently nuclear stains. Many of
the others — for instance, safranin, gentian, and especially
dahlia — may be made to give a nuclear stain with fresh tis-
sues by combining them with acetic acid ; but in ninety-nine
cases out of a hundred are not so suitable for this kind of
work as the two colours first named, which practically form a
class apart.

Again, very few anilins give a pure plasmatic stain Cone
leaving nuclei unaffected). The majority give a diffuse stain
which in some few cases becomes, by the application of the
decoloration or indirect method (92), the most precise and
splendid stain as yet obtainable by any means.

The indirect staining method, or Flemming's method, and
the direct nuclear stains will form the subject of the present
chapter, and the remaining anilins will be treated of in the
next chapter.

The following list shows the colours treated of in the two
chapters.

In Chap. VIII. — A. ( Colours giving indirect nuclear stains,
FLEMMING'S method.) — Safranin, gentian violet, dahlia, Victoria
blue, anilin green, Magdala red (naphthalin red, rose de
naphthaline), mauvein, rouge fluorescent, solid green, fuchsin
(anilin red, rubin, rosein, magenta, solferino, corallin), orange,
Bismarck brown, methyl violet, tropasolin 000, No. 2, (orange
II, chysaure'in, |3 naphthalorange), rocellin (echtroth, or-

* The word " anilin " is here used in the popular sense, to include all
coal-tar colours.

60 ANILIN COLOURS GIVING INDIRECT NUCLEAR STAINS.

seillin No. 3, rubidin, La Rawvarienne), benzoazurin. B.
(Colours giving direct nuclear stains.) — Methyl green (me-
thanilin green, vert lumiere, lichtgriin, griinpulver, vert en
cristaux), Bismarck brown (Manchester brown, phenylen
brown, vesuvin, La Phenicienne), methyl violet (methylanilin,
anilin violet, Paris violet, inchiostro di Leonardi), methyl
violet 6 B, fuchsin.

In Chap. IX. — A. Methylen blue. B. (Plasma and
ground stains.)— Bleu lumiere (Parma blue, toluidin blue,
lichtblau), bleu de Lyon (bleu de nuit, Griinstichblau), in-
dulin (nigrosin, bengalin, anilin blue-black, blackley blue,
artificial indigo), quinolein blue (cyanin, chinolinblau), ben-
zoazurin, anilin blue, violet B., anilin black (blue-black, ni-
granilin,noir de Colin), Saiirefuchsin (fuchsin S., acid fuchsin),
Congo red (Congoroth), benzopurpurin, delta purpurin, Bieb-
richer scharlach (Biebrich scarlet), eosin (primerose, ery-
throsin, pyrosin B., rose B. a 1'eau), Bengal rose, picric acid,
metanil yellow (metanilgelb), saiiregelb (echtgelb), tropaeolin
0., croce'in, gold orange, iodine green (Hofmann's griin),
thiophen green, anilin green, malachite green.

General Directions for the Indirect or " Flemming " staining

Method*

96. Staining. — Sections only can be stained by this method.

The solutions employed are made with alcohol, water, or
anilin, according to the solubility of the colour. There seems
to be no special object in making them with alcohol if water
will suffice, the great object being to get as strong a solution
as possible. The sections must be very thoroughly stained in
the solution. As a general rule they cannot be left too long
in the staining fluid. With the powerful solutions obtained
with anilin a few minutes or half an hour will frequently
suffice, but to be on the safe side it is frequently well to
leave the sections twelve to twenty-four hours in the fluid.
Up to a certain point the more the tissues are stained the
better do they resist the washing-out process, which is an

* Historically the principle of this method is due to HEBMANN and
.BOETTCHEE ; but it is universally known by the name of Flemming, to
whom is due the credit of having greatly improved the method in its prac-
tical details.

WASHING OUT. 61

advantage. For researches 011 nuclei the solutions made with
anilin had better be employed only with preparations well
fixed in chromo-aceto-osmic acid, as the basic anilin oil may
easily attack chromatin if not specially well fixed.

97. Washing out. — Washing out is generally done with
alcohol, sometimes pure, sometimes acidulated (with HC1).
The stained sections, if loose (celloidin sections), are brought
into a watch- glassful of alcohol; if mounted in series on a
slide they are brought into a tube of alcohol (washing out
can be done by simply pouring alcohol on to the slide, but it
is better to use a tube or other bath). It is in either case
well to just rinse the sections in water before bringing them
into alcohol.

The sections in the watch-glass are seen to give up their
colour to the alcohol in clouds, which are at first very rapidly
formed, afterwards more slowly. The sections on the slide
are seen, if the slide be gently lifted above the surface of the
alcohol, to be giving off their colour in the shape of rivers
running down the glass. In a short time the formation of
the clouds or of the rivers is seen to be on the point of ceasing ;
the sections have become pale and somewhat transparent, and
(in the case of chrom-osmium objects) have changed colour,
owing to the coming into view of the general ground colour
of the tissues, from which the stain has now been removed.
(Thus chrom-osmium-safranin sections turn from an opaque
red to a delicate purple.) At this point the washing out is
complete, and must be stopped instantly.

It is generally directed that absolute alcohol be taken for
washing out. This may be well in some cases, but in general
strong (95 per cent.) spirit is found to answer perfectly well.
The hydrochloric acid alcohol process had better only be
employed with tissues well fixed with " Flemming," as with
tissues imperfectly fixed it may cause swellings. Further,
the acid extracts the colour much more quickly from resting
nuclei than from kinetic nuclei, which is an advantage or a
disadvantage according to the end in view.

As a rough and ready guide to the beginner, it may be
stated that washing out should be done with pure alcohol
whenever it is desired to have resting nuclei stained as well
as dividing nuclei ; the other processes serving chiefly to
differentiate mitoses.

62 ANIL1N COLOURS GIVING INDIRECT NUCLEAR STAINS.

The length of time necessary for washing out to the precise
degree required varies considerably with the nature of the
tissues and the details of the process employed ; all that can
be said is that it generally lies between thirty seconds and
two minutes.

In more than one of the methods presently to be described treatment
with chromic acid or with iodine forms part of the washing out process.
The rationale of this is somewhat obscure ; the most probable point of view
appeal's to be that the chromic acid acts as a mordant on the chromatin and
helps it to retain the stain. It is known on the one hand that chromic acid
precipitates safranin from its solutions, so that by admitting a special
affinity of chromic acid on the other hand for chromatin, and especially for
chromatin in the kinetic state, the explanation is hypothetically complete.

The iodine in Bizzozero's (Gram's) process also appears to act as a fixative
of the colour.

It has been ascertained by HENNEGUY that permanganate of
potash acts as a mordant for coal-tar colours. For the
manner of employing it see § 106.

98. Substitution. — It was stated above that washing out is
generally done with alcohol. There exists another mode of
washing out that is both of practical importance and of great
theoretical interest, one anilin stain may be made to wash
out another. Thus methyleii blue and gentian violet are dis-
charged from tissues by aqueous solution of vesuvin or of
eosin ; fuchsin is discharged from tissues by aqueous solution
of methylen blue. The second stain " substitutes " itself for
the first in the general ' ' ground " of the tissues, leaving, if
the operation have been successfully carried out, the nuclei
stained with the first stain, the second forming a " contrast"
stain. It appears from the interesting paper of KESEGOTTI
in Zeit. f. wiss Mik., v, 3, 1888, p. 320, that it may be stated
as a very general rule that colours that do not give a nuclear
stain by the indirect method will wash out those that do.
Thus he found that —

Safranin, Dahlia, Methyl Violet, Gentian Violet, Eubin,
Victoria Blue, Magenta, Basic Fuchsin, are washed out by
the following :

Congo, Methyl Green, Iodine Green, Nigrosin, Methylen
Blue, Orange, Ponceau, Acid Fuchsin, Aurantia, Cyanin,
Eosin, Methylic Eosin, Magdala Ked, Bordeaux, Vesuvin.

CLEARING. 63

Eesegotti obtained the best results by washing out methyl
violet or dahlia with eosin or acid fuchsin (Saiirefuchsin).

FLEMMING has obtained important results by first double-
staining sections with safranin followed by gentian violet,
and then treating them with concentrated aqueous solution
of orange, which, in virtue of its acid qualities, washes out
most of the gentian (see the details of the process in the
chapter on Combination Stains, § 258).

It has been made out by KUHNE (Centralbl. f. Bakteriol. u. Parasiterik.,
xi, 1892, p. 756 ; Journ. Roy. Mic. Soc., 1892, p. 708) that saturated or
strong solutions of malachite green in anilin oil will wash out fuchsin,
methylen blue and crystal violet from sections (the process described by
Kiihne is one for the staining of bacteria in sections, and I cannot say how
far it is applicable to histological ends).

The student will note that the colours in the second of Eesegotti's lists
may be turned to account for washing out and producing a contrast stain at
the same time ; he should also take note that this washing out is a true
chemical decoloration, and if pushed too far will invade the nuclei as well
as the rest of the tissues.

99. Clearing. — The washing out of the colour may be
stopped by putting the sections into water ; but the general
practice is to clear and mount them at once.

You may clear with clove oil, which will extract some more
colour from the tissues. Or you may clear with an agent that
does not attack the stain (cedar oil, begamot oil, xylol, toluol,
naphtha, &c. ; see the chapter on Clearing Agents). If you
have used pure alcohol for washing out, you had perhaps
better clear with clove oil, as pure alcohol does not always,
if the staining have been very prolonged, extract the colour
perfectly from extra-nuclear parts. But if you have not
stained very long, and if you have used acidulated alcohol
for washing out, clove oil is not necessary, and it may be
better not to use it, as it somewhat impairs the brilliancy
of the stain. A special property of clove oil is that it helps
to differentiate karyokinetic figures, as it decolours resting
nuclei more rapidly than those in division.

Some colours are much more sensible to the action of
clove oil than others ; and much depends on the quality of
this much adulterated essence. New clove oil extracts the
colour more quickly than old.

Series of sections on slides are conveniently cleared by
pouring the clearing agent over them.

64 ANILIN COLOURS GIVING INDIRECT NUCLEAR STAINS.

When the clearing is accomplished to your satisfaction,
mount in damar or balsam, or stop the extraction of the
colour if clove oil have been used by putting the sections
into some medium that does not affect the stain (xylol, cedar
oil, &c.).

100. General Results. — The results depend in great measure
on the previous treatment of the tissues. If you have given
them a prolonged fixation in Flemming's strong chromo-aceto-
osmic mixture, and have washed out after staining with acid
alcohol and cleared with clove oil, you will get, with some
special exceptions, nothing stained but nucleoli and the
chroinatin of dividing nuclei, that of resting nuclei remaining
unstained. If you have given a lighter fixation, with
Flemming's weak mixture or some other fixing agent not
specially inimical to staining, and have washed out after
staining with pure alcohol, you will get the chromatin of
resting nuclei stained as well. With some of these stains —
Victoria, for instance — it is easy to get cytoplasm stained,
in a lighter tone than the nuclei, by merely washing out
lightly.

It may be noted here that most of the dyes discussed in
this chapter give a stain of a somewhat dead or dull quality,
so much so that in some cases chromosomes and nucleoli come
out quite opaque. Safranin and anilin green, however, do
not do this, but leave the structures beautifully transparent.
This is an advantage with thick sections, and sometimes for
other reasons ; but this transparency of the elements is un-
fortunately favorable to the production of diffraction lines
which may be a hindrance to good definition in delicate
work.

101. Safranin — one of the most important of these stains,
on account of its great power, brilliancy, and superior per-
manence in balsam, and also on account of the divers degrees
of electivity that it displays for the nuclei and other consti-
tuent elements of different tissues.

The great secret of staining with safranin is to get a good
safranin. It is needful here to insist most urgently on what
was said above, § 94, sub. finem. Before thinking of working
with this important reagent, you should go to Griibler or to
Miinder and order the safranin you want, specifying whether

SAFRANIN. 65

you want it for staining nuclei or for .staining elastic fibres,
or for what other purpose you may require it.

There are presumably at least a score of sorts of saf ranin in the market,
differing to a considerable extent in colour, weight, solubility and histological
action. Some are easily soluble in water and not so in alcohol, some the re-
verse, and some freely soluble in both. Fourteen brands, supplied by
Griibler and by Miinder, have been studied by RESEGOTTI (Zeit. f. wiss. Mik.,
v, 3, 1888, p. 320). They all gave positive results with the chromic acid
method, to be detailed below ; although Griibler had explained that the
brands XX, XXBN, TB, had not given positive results (with the usual
methods). Resegotti obtained his best results with the brands " Saf ranin
wasserloslich," " Saf ranin spiritusloslich," " XX," " XXBN," " TB," fur-
nished by Griibler, and with the brands "Rein," ?0£' " FII," and "Cone.,"
supplied by Miinder.

Staining.

The majority of safranins are not sufficiently soluble in
water, so that solutions in other menstrua must be employed.

A solution much used some time ago is that of PFITZNER
(Morph. Jahrb., vi, p. 478, and vii, p. 291), composed of saf-
ranin 1 part, absolute alcohol 100 parts, and water 200 parts,
the last to be added only after a few days.

The solution of FLEMMING (Arch. f. mik. Anat., xix, 1881, p.
317) is a concentrated solution in absolute alcohol, diluted
with about one half of water.

The solutions of BABES (Arch. f. mik. Anat., 1883, p. 356)
are (A) a mixture of equal parts of concentrated alcoholic
solution and concentrated aqueous solution (this is very much
to be recommended), and (B) a concentrated or supersaturated
aqueous solution made with the aid of heat.

Some people still employ simple aqueous solutions.

Lastly, there is the anilin solution of BABES (Zeit. f. wiss.
Mik., iv, 4, 1887, p. 470). It consists of water 100 parts,
anilin oil 2 parts, and an excess of safranin. The mixture
should be warmed to from 60° to 80° C., and filtered through
a wet filter. This solution will keep for a month or two.

ZWAARDEMAKER (Zeit. f. wiss. Mik., iv, 2, 1887, p. 212)
makes a mixture of about equal parts of alcoholic safranin
solution and anilin water (saturated solution of anilin oil in
water; — to make it, shake up " anilin oil," which is nothing
but pure anilin, with water, and filter).

Any of these stains may be used with any of the following

5

66 ANILIN COLOURS GIVING INDIRECT NUCLEAR STAINS.

washing-out processes. Of course you will have to stain
longer in the weaker solutions. As to the anilin solutions,
see ante, § 96.

Washing out and Clearing.

For general directions for washing out and clearing, see
above, §§ 97 and 99.

FLEM MING'S first method (1. c. in last §). — Wash out with
pure alcohol, followed by clove oil. This method stains
resting chromatin as well as " mitoses."

FLEMMING'S second method (Zeit.f. wiss. Mik., i, 3, 1884, p.
350). — Wash out until hardly any more colour comes away, in
alcohol acidulated with about 0*5 per cent, of hydrochloric
acid, followed by pure alcohol and clove oil. (You may use
the HC1 in watery solution if you prefer it.) The strength
given appears unnecessarily high, and I therefore generally
use with good results an alcohol of about 0*2 per cent, of HC1.*
(Objects are supposed to have been well fixed — twelve hours
at least — iuihe strong chromo-aceto-osmic mixture, and stained
for some hours.) PODWYSSOZKI (Beitr. z. path. Anat. v. Ziegler
u. Neuwerk, i, 1886; Zeit. f. wiss. Mik., iii, 3, 1886, p. 405)
prefers to stain for half an hour only, and wash out with O'l
per cent of HC1 in alcohol. In each of these ways you get
"mitoses" and nucleoli alone stained (if the fixation have
been performed as above directed).

FLEMMING'S orange-method (very important) is given in the
chapter on "Combination Stains" (§ 258).

PODWYSSOZKI (1. c.) gives another method, which consists in
washing out (for from a few seconds to two minutes) in a
strongly alcoholic solution of picric acid, followed by pure
alcohol. Same results (except that the stain will be brownish
instead of pure red).

BABES employed for washing out after staining in the
aqueous or alcoholic solutions above mentioned, pure alcohol
followed by oil of turpentine. For sections stained in the
anilin solution he recommends treatment with iodine, accord-
ing to the method of GRAM (see what is said as to the process

* In Flemming's latest work (on the achromatic structures of the cell) he
has been using a lower strength than this even, viz. O'l per cent, at most, see
Arch.f. mik. Anat., xxxvii, 1891, p. 249.

GENTIAN VIOLET. 67

of Gram in the paragraph on gentian violet, ^o,9£, § 102). This
process has also been recommended by PRENANT (Int. Monats-
schr. f. Anat., &c., iv, 1887, p. 368), who notes that the treat-
ment with the iodine solution should be somewhat longer, and
the treatment with alcohol somewhat shorter than with gentian
violet sections.

MABTINOTTI and KESEGOTTI (Zeit.f. wiss. Mik., iv, 3, 1887, p. 328) recom"
mend washing out with a freshly prepared mixture of one part of O'l per
cent, aqueous solution of chromic acid with nine parts of absolute alco-
hol, followed by pure alcohol and bergamot oil. In my experience this
method does not give better results (I think less good) than that of wasWng
out in the simple aqueous solution of chromic acid of Bizzozero followed by
alcohol (see the paragraph on gentian violet, next §). The latter is certainly
a most useful method. It should be mentioned that Martinotti and Rese-
gotti's results refer to lightly stained alcohol-fixed objects, and not to
chromo-aceto-osmic objects, which may make a great difference.

GARBINI (Zeit. f. wiss. Mik., v, 2, 1888, p. 170) has recommended that
sections be dehydrated after staining in methylic alcohol (wood spirit) in
which safranin is only very slightly soluble, and decoloured in a mixture of
two parts of clove oil with one part of cedar oil. I have not been able to
obtain good results by this method.

The reader will remember that safranin may be washed out by substitu-
tion (see ante, § 98). In preparations made with chromo-aceto-osmic acid,
safranin stains, besides nuclei, elastic fibres, the cell-bodies of certain horny
epithelia, and the contents of certain gland-cells.

It has been shown by OHLMACHEE (Journ. Amer. Med. Ass., vol. xx, No.
5, Feb. 4, 1893, p. Ill) that if tissues be treated with solutions containing
iodine or picric acid after staining with safranin, there may be produced in
the tissue-elements a precipitate of a dark red substance of a crystalline
nature but of lanceolate, semi-lunar, falciform or navicellar forms. This
precipitate is formed both in normal and pathological tissue, and occurs
either in the nuclei or in the cytoplasm. It is formed readily in carcino-
matous tissues ; and Ohlmacher makes out a strong case in favour of the
conclusion to which he has come that many of the bodies that have been
described as " coccidia," " sporozoa," or other " parasites " of carcinoma
are nothing but particles of this precipitate. This refers, amongst much
other work, to that of PODWYSSOZKI and SAWTSCHENZO. Forewarned is
forearmed ; but if the formation of Ohlmacher's precipitate should prove to-
be a very general phenomenon, it will be necessary to conclude with him that
to follow safranin staining with treatment by solutions containing iodine or
picric acid is not only unscientific but positively dangerous. Of course this
is not intended to discredit the use of safranin when washed 'out with hydro-
chloric acid, alcohol, or the like.

102. — Gentian Violet. — One of the most important of thesa
stains. It may be used in aqueous solution, or in alcoholic
solution diluted with about one half of water (FLEMMING,.

68 ANIL1N COLOURS GIVING INDIRECT NUCLEAR STAINS.

Zells. Kern. u. Zellth., 1882, p. 384), and the stain may be
washed out with pure alcohol or (FLEMMINQ, Zeit. f. wiss. Mik.,
1, 1884, p. 350) with acidulated alcohol, as directed below for
safranin. But by far the best way of using it is, in general,
that due to BIZZOZERO (Zeit. f. wiss. Mik., in, 1, 1886, p. 24).
The tissues may be hardened either in alcohol or in a chromic
mixture, but must in the latter case have been well washed
out with water. The staining solution is borrowed from that
of EHRLICH for bacteria, and consists of —

Gentian violet ..... 1 part.

Alcohol . . . . . .15 parts.

Anilin oil . . . . . 3 „

Water : 80 „

The sections are stained in it for five or ten minutes or
longer (for objects from Flernming's solution it will frequently
be advisable to stain for as many hours). After staining,
rinse the sections with alcohol, and bring them into a 0*1 per
cent, aqueous solution of chromic acid. After from thirty to
forty seconds bring them into alcohol, which begins the wash-
ing out of the colour. After thirty or forty seconds in the
alcohol put them back for thirty seconds into the chromic acid
(this is done in order to fix the colour more completely in the
nuclei) . Then bring them back into alcohol for thirty to forty
seconds, in order to wash out more colour and dehydrate them
at the same time. Then treat with clove oil, which will extract
more colour, and after a short time must be changed for fresh,
in which the sections remain until they are seen to give up
no more colour, when they are removed and mounted in
damar.

You might give a longer treatment with alcohol, and a shorter treatment
with clove oil, but you would get a slightly diffe rent result. Alcohol washes
out colour freely from kinetic nuclei as well as from resting nuclei, whereas
clove oil acts much more energetically on the latter than on the former, and
thus serves to differentiate dividing nuclei.

In some cases, especially those of tissues whose nuclei have
a tendency to give up the colour too freely, better results are
obtained by combining the foregoing method with that of
GRAM for the staining of bacteria (Fortschr. d. Medicin, ii,. 1884,
No. 6; British Med. Journ., Sept. 6th, 1884, p. 486; Journ.
Eoy. Hie. Soc. [N.S.], iv, 1884, p. 817).

DAHLIA. 69

In Gram's method the sections are treated, after staining,
with a solution composed of —

Iodine ..... 1 gramme.
Iodide of potassium . ... 2 grammes.
Water 300 „

In Bizzozero's adaptation of this process the series of opera-
tions is as follows : — Stain in the gentian, wash for five
seconds in alcohol ; two minutes in the iodine solution ; twenty
seconds in alcohol ; thirty seconds in the chromic acid solu-
tion ; fifteen seconds in alcohol ; thirty seconds in the chromic
acid again ; thirty seconds in alcohol ; and treatment with
changes of clove oil until final decoloration.

NISSEN (Arch. f. mifc. Anat., 1886, p. 338) employs this pro-
cess with omission of the treatment with chromic acid.

In resting nuclei the nucleoli alone are stained, or the
chromatin if stained is pale ; in dividing nuclei the chromatin
is stained with great intensity, being nearly black in the
equatorial stage.

This exceedingly powerful stain is quite as precise as that
of safranin, to which it is perhaps even preferable for much
work with very thin sections (thick sections with closely packed
nuclei may easily come out too dark). It lends itself admir-
ably to double staining with eosin, with which it affords one
of the most useful and beautiful double-stains known (see
§ 258).

The stain keeps fairly well in damar, though not so well as
that of safranin. Flemming found that after a year it had
fadad a little, though not so much as hasmatoxylin stains (v.
Zells. Kern. u. Zellth., p. 384).

Gentian violet in acid solution stains the nuclei of fresh tissues, and dis-
solved in indifferent media is sometimes very useful for staining intra vitam
(see above, § 93).

- Gentian may be washed out by substitution, whence the important
method of Flemming, § 258, which see.

, 103. Dahlia (FLEMMING, Arch. f. mik. Anat., xix, 1881,
p. 317). — Stain in an aqueous solution, either neutral or
acidified with acetic acid, and wash out with pure alcohol.
The stain is paler in the nuclei than with gentian or safranin.
The cytoplasmic granulations of certain cells are sharply
stained.

70 ANILIN COLOURS GIVING TNDIEECT NUCLEAR STAINS.

Dahlia is also a useful nuclear stain for fresh tissues (v. EHRLICH, Arch.
f. mik. Anat., xiii, 1876, p. 263). For these the aqueous solution must be
acidulated with (7*5 percent.) acetic acid; or you may stain in a neutral
solution, and wash out with acidulated water. Dehydrate with alcohol and
mount in turpentine colophonium. It is also useful for staining intra vitam.
See above, § 93.

For the staining of Ehrlich'g " plasma cells," see post, Part II.

104. Victoria Blue ( Victoriablau) . (LUSTGARTEN, Med. Jarhb.
It. Ges. d. Aerzte zu Wien, 1886, pp. 285 — 91). — Stain (speci-
mens strongly fixed in "Flemming" some hours, lightly-
fixed specimens a few minutes) in saturated aqueous solution.
Wash out in pure alcohol (about one minute, more or less).
You may clear with clove oil, but you had perhaps better take
cedar or bergamot oil, as clove oil washes out the colour very
freely.

A most brilliant and useful nuclear stain, and one that I
think should be particularly recommended to the beginner, as
it is particularly easy to work with. Chromatm and nucleoli
blue. Cytoplasm, if well washed out, colourless ; if less washed
out, green or greenish blue. The " spongioplasm " is very
finely brought out by this method.

Victoria has a special affinity for elastic fibres. For this
object Lustgarten recommends an alcoholic solution of the
dye diluted with two to four parts of water. Fixation in
chrom- osmium, or at least in a chromic mixture, is, I believe,
a necessary condition to this reaction. And you must stain
for a long time.

Victoria has also a special affinity for mucus- cells, from
which it is not washed out by alcohol.

105. Anilin Green. — Use precisely as directed for Victoria blue, supra.
An extremely delicate and absolutely precise nuclear stain, nucleoli being
peculiarly brilliantly stained by it. I am unfortunately unable to trace the
history of the colour used by me, which may be identical with the Solidgriin
of Flemming (Arch. f. mik. Anat., xix, 1881, pp. 317 and 742). It is well to
be even more careful in the use of clove oil than in the case of Victoria blue.
This colour seems not so generally useful as Victoria, as it does not give so
bold a stain.

Like Victoria, anilin green has also a special affinity for mucus-cells.
See the paragraphs on " mucus-cells " in Part II.

106. Henneguy's Permanganate Method (Jonrn. de I' Anat. et
de la PhysioL, xxvii, 1891, p. 397).— This method is based on
the fact, discovered by HENNEQUY, that permanganate of
potassium is a mordant for many anilin dyes, and will

HENNEGUY'S PERMANGANATE METHOD. 71

enable a good stain to be procured in cases in which the usual
methods fail.

Sections (from tissues fixed either in the strong solution of
Flemming for from two to six hours, or in other reagents
such as sublimate, liquid of Perenyi, liquid of Kleinenberg,
alcohol, &c.) are treated for five minutes with 1 per cent,
solution of permanganate of potassium. They are then
washed with water and stained (for about half the time that
would have been taken if they had not been mordanted with the
permanganate) in safranin, rubin, gentian violet, vesuvin, or
the like. The stain that succeeds the best is a safranin solu-
tion prepared with anilin water and absolute alcohol (see
above, § 101). After staining they are washed out with
alcohol, followed by clove oil in the usual way. This is the
delicate part of the process. The progress of the decolora-
tion must be watched under the microscope, in order that it
may be stopped at the proper moment. It goes on in general
slowly, and the slower it proceeds the more selective will be
the resultant stain. The decoloration sometimes continues
even after the sections have been mounted in balsam, espe-
cially if all traces of clove oil have not been removed before
mounting. It may thus happen that preparations which are
insufficiently washed OUD at the moment of mounting show a
perfectly differentiated stain twenty-four or forty-eight hours
afterwards.

If safranin have been taken as the stain the preparations
show protoplasm of an orange- grey tint, which shows up the
most delicate structures, in particular the achromatic figures
of cytodieresis. Chromosomes and nuclear membranes are of
a brilliant red, attractive spheres and centrosomes less
strongly stained, but still sharply brought out against the
rest of the cytoplasm.

If it be desired to have the details of protoplasmic structures
still more markedly brought out, the safranin stain may be
preceded by haematoxylin staining (0'5 per cent, solution of
haematoxylin in 90 per cent, alcohol ten minutes, wash
with water, treat for ten minutes with 2 per cent, solution of
bichromate of potash, and wash with distilled water before
treating with the permanganate). But with preparations
that have been fixed in Flemming this treatment will hardly
be necessary.

72 AXILIN COLOURS GIVING INDIRECT NUCLEAR STAINS.

The mordanting action of permanganate of potassium on
anilin stains is so energetic that if it have been overmuch
prolonged before staining with safranin, or, still more, with
rubin, it becomes almost impossible to wash out the sections
properly; it may be necessary to leave them for nearly a
month in clove oil.

Heneguy's preparations are certainly most successful, and
I most highly recommend the method for all preparations in
which it is desired to have the achromatic cell-structures
stained at the same time as the chromatic elements. And I
think it may be recommended to all workers who feel the
want of a means of slowing the sometimes inconveniently
rapid decoloration of sections treated by the F lemming
method.

107. Other Nuclear Stains by the Indirect Method.— The fore-
going paragraphs nearly exhaust the list of colours giving good nuclear
stains by the indirect process. FLEMMING (Arch. f. mik. Anat., xix, 1881,
pp. 317 and 742) mentions the following :

MAGDALA RED (NAPHTHALIN RED, ROSE DE NAPHTHALINE). — Nearly if
not quite as good a stain as any of the foregoing, and superior to all except
safranin in respect of permanency. This and the following should, as far
as is yet made out, be used in alcoholic solution diluted with about one half
of water, and be washed out with pure alcohol, followed by clove oil.

Mauvein and Rouge Fluorescent are good stains, but colour some
nuclei more deeply than others in the same preparation.

Solid Green (perhaps the same as the anilin green discussed above) is
very elective for nucleoli.

Fuchsin (meaning the basic fuchsins, a series of Rosanilin salts having
very similar reactions and found in commerce under the names of FUCHSIN,
ANILIN RED, RUBIN, ROSEIN, MAGENTA, SOLFERINO, COEALLIN). — A good
but somewhat weak stain, by the alcohol method. Good results are obtained
by substitution in the following manner (GBASEE, Deutsche Zeit. f. Chi-
rurgie, xxvii, 1888, pp. 538—584 ; Zeit. f. wiss. Mile., v, 3, 1888, p. 378).
You either employ the colour as directed for methyl violet (post, § 111), or
you stain for twelve to twenty-four, hours in a dilute aqueous solution, wash
out for a short time in alcohol, stain for a few minutes in aqueous solution
of methylene blue, and dehydrate with alcohol. A double stain. Chromatin
and nucleoli red, all the rest blue.

ZIEHL'S CARBOLIC FUCHSIN has been recommended as supe-
rior to safranin by SCHENK (Ueber Conservirung von Kerntheil-
ungsfiynren, Bonn, 1890). I do not know where the original
formula was published, and take this from Zeit.f. wiss. Mik.
(vii, 1, 1890, p. 39).

CARBOLIC FUCHSIN. 73

The stain is made either by taking —

Fuchsin . . . 1 gramme,

Acid, carbol. crist. . . 5 grammes,
Alcohol . . . . 10
Aq. dest. . . 100

or by saturating a 5 per cent, aqueous solution of carbolic
acid with concentrated alcoholic solution of fuchsin (the
saturation of the carbolic solution with fuchsin is made mani-
fest by the formation of a metallic-looking pellicle on the
surface of the liquid). The stain is washed out with alcohol
followed by clove oil.

ORANGE, precise but weak.

BISMARCK BROWN is not very satisfactory with chromic objects. With
alcohol objects it gives a good chromatin stain, but cannot be thoroughly
removed from cytoplasm by any means yet discovered.

KAISEE (Biblioth. Zool., H. vii, 1 Halfte, 1891 ; Zeit /.
wiss. Mik., viii, 3, 1891, p. 363) has obtained good results
with Bismarck brown in the following way : — Stain for forty-
eight hours, and at a temperature of 60° C., in saturated
solution of Bismarck brown in 60 per cent, alcohol (the solu-
tion to be made in boiling alcohol), and wash out (until all is
decoloured except the karyokinetic figures) in 60 per cent,
alcohol, containing 2 per cent, hydrochloric acid or 3 per
cent, acetic acid.

To these may be added —

METHYL VIOLET, perhaps best used according to the method of Eesegotti
given in § 101 ; and (according to GRIESBACH, Arch. f. mik. Anat., xxii,
p. 132.

TROP.EOLIN 000, No. 2 (ORANGE n ; CHRYSAUREIN, /3 NAPHTOLORANGE),
a fine dark orange stain, and

ROCELLIN (ECHTROTH, ORSEILLIN No. 3, KUBIDIN, LA RAUVARIENE), a

cherry -red stain.

BENZOAZURIN has been lately recommended by MARTIN (see Zeit. f. wiss.
Mik., vi, 3, 1889, p. 193). Stain for an hour or so in dilute aqueous solu-
tion and wash out with HC1 alcohol.

B. Direct Nuclear Stains.

108. As regards the direct nuclear stains, the reader is reminded
that, as was stated in § 95, many if not most of the anilins give a nuclear
stain of greater or less purity if they are used in solutions acidified with
acetic acid. Under the present heading, only those are mentioned which
give in all respects, alike as regards precision and permanence, simplicity of
manipulation and other qualities, a really valuable stain. The very exist-

74 DIRECT NUCLEAR STAINS.

ence of methyl green and Bismarck brown is a sufficient reason for being
silent, in this connection, with regard to the rest.

109. Methyl Green. — This is the most common in commerce,
of the " anilin " greens. It appears to go by the synonyms
of Methylanilin green, Vert Lumiere, Lichtgriin, Griinpulver.
When first studied by Calberla, in 1874 (Morphol. Jahrb., iii,
1887, p. (325), it went by the name of Vert en cristaux. It is
commonly met with in commerce under the name of more
costly greens, especially under that of Iodine green. It is im-
portant not to confuse it with the latter, nor with Aldehyde
green (Vert d'Eusebe), nor with the phenylated rosanilins,
Paris green, and Vert d'alcali or Veridine.

The chief use of methyl green is as a nuclear stain for fresh
or recently fixed tissues. For this purpose it should be used in
the form of a strong aqueous solution containing a little acetic
acid (about 1 per cent, in general). The solutions must always
be acid. (One of the latest books on the subject I have
seen gives a formula for a methyl green solution for
staining nuclei in which no acid is mentioned. Such a solu-
tion would not give the pure nuclem reaction which it is the
special function of this reagent to produce.) You may wash
out with water (best acidulated) and mount in some acid
aqueous medium containing a little of the methyl green in
solution. The mounting medium, if aqueous, must be acidu-
lated.

Employed in this way, methyl green is a pure nuclear stain,
in the sense of being a precise colour-reagent for nuclein.
For in the nucleus it stains nothing but the chromosomes, or
nuclein elements : it does not stain either nucleoli of any sort,
nor caryoplasm, nor achromatic filaments. Outside the nucleus
it stains some kinds of cytoplasm and some kinds of formed
material, especially glandular secretions (sericin, for instance).
But the nuclein elements are invariably stained of a bright
green (with the exception of the nuclein of the heads of some
spermatozoa), whilst extra-nuclear structures are in general
stained in tones of blue or violet.

The following paragraph, translated from the paper by Calberla above
quoted, appeared inadvertently without comment in the first edition, and has
since been repeated without comment in several places.

" He then found that ' the nuclei of subcutaneous connective tissue and

METHYL GREEN. 75

those of vessels and nerve-sheaths stained rose-red, cells of the coriuni
reddish white, and the cells of epidermis greenish blue to pure blue.' "

It should have been added that Calberla here says what he presumably
did not mean to say. Methyl green certainly never stains nuclei red, and
Calberla's observation should be taken to . refer to the results of a double
stain with methyl green and eosin, which is mentioned in the somewhat ob-
scurely expressed passage from which the quotation is taken.

Calberla's misstatement is repeated, and made one of the grounds of an
important theoretical deduction by Griesbach, in Zeit. f. wiss. Mik., iii, 3,
1886, p. 365.

Besides being a perfectly pure chromatin stain, methyl
green has other advantages. Staining is instantaneous ;
overstaining never occurs. The solution is very penetrating,
kills cells instantly without swelling or other change of form,
and preserves their forms for at least some hours, so that it
may be considered as a delicate fixative. Osmic acid (0*1 to
1 per cent.) may be added to it, or it may be combined with
solution of KIPART and PETIT (this, by the way, is an excellent
medium for washing out in and mounting in) .

Alcoholic solutions may also be used for staining. They
also should be acidulated with acetic acid.

The stain does not keep easily. It is difficult to mount it
satisfactorily in balsam, because the colour does not resist
alcohol (unless this be sufficiently charged with the colour) ;
and of preparations mounted with excess of colour in the
usual aqueous media the most fortunate only survive for a
few months. Dr. HENNEGUY however writes to me that it
keeps well in BRUN'S glucose medium (see § 402).

It was first pointed out, I believe, by Heschl (Wiener med. Wochenschr.,
2, 1879), that methyl green is a reagent for amyloid degeneration. His
observations were confirmed by Curschmann (Virchow's Arch., t. 79, 1880,
p. 556), who showed that it colours amyloid substance of an intense violet,
but this (as pointed out by SQUIBE, Methods and Formulas, &c., Churchill,
1892, p. 37) may be due to its containing methyl violet as an impurity.

Undoubtedly methyl green is one of the most valuable
stains yet known. It is the classical nuclein stain for fresh
tissues.

110. Bismarck Brown (Manchester Brown, Phenylen Brown,
Vesuvin, La Phenicienne) . — A fairly pure nuclear stain that
will work either with fresh tissues or with such as have been
hardened in chromic acid.

76 DIRECT NDCLEAE STAINS.

The colour is not very easily soluble in water. You may:
boil it in water, and filter after a day or two (WEIGERT, in
Arch. f. mile. Anat , xv, 1878, p. 258). You may add a little
acetic or osmic acid to the solution. MAYSEL (ibid., xviii,.
1880, pp. 237, 250) dissolves the colour in acetic acid (this
solution does not give a permanent stain). Alcoholic solutions
may also be used. Paul Mayer recommends a saturated solu-
tion in 70 per cent, alcohol ; or CALBERLA'S glycerin-and-
alcohol mixture, or dilute glycerine (say of 40 per cent, to 50
per cent.) may very advantageously be employed.

The watery solutions must be frequently filtered. The
addition to them of carbolic acid has lately been recommended,
vide Journ. Roy. Hie. Soc., 1886, p. 908. Bismarck brown
stains rapidly, but never overstains. The stain is permanent
both in balsam and in glycerin.

The chief use of this colour is for staining objects in toto ;
but it may also be employed for staining sections by the de-
coloration method (see above, § 107).

As has been noted above (§ 93), Bismarck brown has the
property of staining certain cellular elements during life (for
this purpose it is necessary to see that the colour employed
be pure and neutral) .

111. Methyl Violet (Methylanilin = anilin-violet = Paris violet = in-
chiostro di Leonardi). — The following process has been recommended by
OETH (Amer. Mon. Micr. Journ., i, 1880, p. 143 ; Journ. Roy. Mic. Soc.,
N.S., i, 1881, p. 137) : Sections are to be soaked in water, and then brought
into the following solution :

Anilin violet 1 part.

Acetic acid 300 parts.

Mount, without washing out, but simply draining, in acetate of potash
(acetate 2 parts, water 1 part).
. The stain will probably fade within a year or two.

This process does not appear to be of more than very limited applicability.
The following, however, due to GEASER (Deutsche Zeit.f. Chirurgie, xxvii,
1888, pp. 538—584 ; Zeit. f. wiss. Mik., v, 3, 1888, p. 378) may, be very
generally useful.

Sections are stained for from twelve to twenty-four hours in a (presum.
ably aqueous) solution so dilute that at the end of that time the sections
will have taken up all the colour from the liquid. They are then washed
out for a short time in acidulated alcohol, and then in pure alcohol (followed
presumably by clearing and mounting in balsam). Schiefferdecker, whose
account is here quoted, says that the results, as regards nuclear figures, are.

METHYL VIOLET. 77

even finer than with safranin. The method is applicable to objects fixed in
" Flemming."

A useful stain for fresh tissues is also obtained by using: dilute acetic acid
in the manner recommended above (by EEHLICH) for Dahlia, § 103.

Methyl violet 6 B has been recommended by KEOMAYEK for demonstrating
the fibrillar structure of epithelium, see Part II, "Epithelium."

112. Amyloid matter appears red in preparations stained with methyl
violet. This appears to be an optical effect, see the curious experiments of CAP-
PEEELLI, in Archivio per le scienze mediche, iii, No. 21, p. 1.

78 METHYLEN BLUE, AND OTHER ANILINS.

CHAPTER IX.
METHYLEN BLUE, AND OTHER ANILINS.

A. Methylen Blue.

113. The Uses of Methylen Blue. — This colour seems to be
in a fair way to become one of the most important reagents
in the histological laboratory. Its importance as a stain for
micro-organisms in tissues is well known to all pathologists.

As a histological reagent, it is used for sections of hardened
central nervous tissue, in which it gives a specific stain of
medullated nerves (post, Part II). It is a valuable specific
reagent for plasma-cells (for which see also Part II). It
possesses the property of washing out the stain of certain
other anilins, with which it gives valuable double-stains (post,
Chapter XIII). It stains a large number of tissues intra
vitam, with little or no interference with their vital functions.
And last, not least, it can be made to furnish stains of nerve-
tissue, intercellular cement-substances, lymph-spaces, and the
like, that are essentially identical with those furnished by a
successful impregnation with gold or silver. The results are
quite equal in most cases to those of gold or silver impregna-
tion, and are obtained with far greater ease and certainty. I
call especial attention to these processes, which seem to be
effecting a revolution in histological technique.

114. Staining in toto during Life. — Small and permeable
aquatic organisms may be stained during life by adding to
the water in which they are confined enough methylen blue
to give a scarcely perceptible or at least very light tint to the
water. If transparent organisms be taken, they may be
examined alive without further manipulation at any desired
moment. Microscopically examined, they will be found after
a time to be partially stained— that is, it will be found that
certain tissues have taken up the colour, others remaining

METHYLEN BLUE. 79

colourless. If now you put back the animals into the dilute
solution and wait, you will find on examination after a suffi-
cient lapse of time that further groups of tissues have become
stained. Thus it was found by EHELICH (Abh. Jc. Akad. Wiss.
Berlin, February 25, 1885) to whom the principle of the
methods under consideration is due, that on injection of the
colour into living animals axis cylinders of sensory nerves
stain, whilst motor nerves remain colourless. The motor
nerves, however, will also stain, though later than the sensory
nerves. It might be supposed that by continuing the staining
for a sufficient time, a point would be arrived at at which all
the tissues would be found to be stained. This, however, is
generally not the case. It is always found that the stained
tissues only keep the colour that they have taken up for a
short time after they have attained the maximum degree of
coloration of which they are susceptible ; as soon as that
point is attained they begin to discharge the colour even more
quickly than they took it up. And it is very often found that
the elements which have stained first will have lost much or
all of their colour by the time that those which stain later
have attained their maximum coloration. It may even happen,
as I have observed, that the whole of the stainable tissues of
an animal may run through the total gamut of coloration and
decoloration until the animal has become as colourless as
when first put into the tinted water, and that without any
apparent change in its vital activities.

It follows that a total stain of all the tissues of an organism
can hardly be obtained under these conditions, but that a
specific stain of one group or another of elements may be
obtained in one of two ways. If the tissue to be studied be
one that stains earlier than the others, it may be observed
during life at the period at which it alone has attained the
desired intensity of coloration, and the remaining tissues
are not yet coloured at all, or not coloured enough to be an
obstacle to observation. If it be one that stains later than
the others, it may be studied during life at the period at
which the earlier stained elements have already passed their
point of maximum coloration and have become sufficiently
decoloured not to be an obstacle to the observation of the
later stained ones; the latter being either at the point of
maximum coloration or at a point of desired intensity either

80 METHYLEN BLUE, AND OTHER ANILINS.

earlier or later than the maximum. Or the observer may fix
the stain in either of these stages and preserve it for leisurely
study by means of one of the processes given below under
the heading " Preservation of the Preparations"

The proper strength of the very dilute solutions to be
employed in the manner here considered must be made out by
experiment, for each object. I think the tint is practically a
sufficient guide, but it may be stated that when in doubt a
strength of 1*100000 may be taken, and increased or di-
minished as occasion may seem to require. ZOJA (Rendic. R.
1st. Lombardo, xxv, 1892 ; Zeit. f. iviss. Mik., ix, 2, 1892,
p. 208) finds that for Hydra the right strength is from
1-20000 to MOOOO.

The stain is somewhat capricious. It is not possible to
predict without trial which tissues will stain first in any
organism. It is to be remarked that the stain penetrates
Very badly, which is perhaps the chief cause of its seeming
capriciousness, and, I take it, the chief determining condition
of the order in which tissues stain. Gland-cells generally
stain early ; then, in no definable order, other epithelium-
cells, fat-cells, plasma-cells, ' Mastzellen/ blood and lymph
corpuscles, elastic fibres, smooth muscle, striated muscle.
There are other elements that stain in the living state, but
not when the staining is performed by simple immersion of
intact animals in a dilute staining solution in the manner we
are considering. Chief amongst these are nerve-fibres and
ganglion-cells, which remain unstained in the intact organism,
most likely, so far as I can see, for the simple reason that the
stain is not able to penetrate them.

115. Staining Nerve-tissue during Life. — It is commonly
believed that methylen-blue stains of the class we are con-
sidering are the product of a vital reaction of the tissues, and
cannot be obtained with dead tissue. It would, however,
appear to be more correct to say that methylen blue has the
property of staining living tissue, not because of its being
living, but in spite of its being living. DOGIEL (Arch. f. tnik.
Anat., xxxv, 1890, pp. 305 et seq.) found that muscle-nerves
of limbs of the frog could be stained as much as from three
to eight days after the limbs had been removed from the
animal. He concludes, indeed, that the reaction shows that

METHYLEN BLUE. 81

the nerves were still living at that time. But it seems more
natural to conclude with APATHY (Zeit. f. wiss. Mile., ix, 1,
1892, pp. 15 et seqq.) that nerve-tissue can be stained after
life has ceased.

APATHY has directly experimented on this point, and sums
up the necessary conditions as follows : — The tissue need not
be living, but must be fresh ; nothing must have been ex-
tracted from it chemically, and its natural state must not have
been essentially changed by physical means. For example,
the tissue must not have been treated with even dilute
glycerin, nor with alcohol, though a treatment for a short
time with physiological salt solution is not very hurtful;
it must not have been coagulated by heat.

Another common belief concerning the methylen-blue
nerve-reaction is that the presence of oxygen is necessary to
the reaction. It is therefore the usual practice to dissect out
the organ to be investigated after having exposed it to the
action of methylen blue by injection or immersion, and leave
it for some time exposed to the air. APATHY has also inves-
tigated this point, and finds (loc. cit., p. 25) that the practice is
in some cases correct, but the belief erroneous. It has been
explained above that shortly after a tissue has attained the
maximum degree of coloration of which it is susceptible it
begins to give up its colour again to the surrounding liquid.
The larger the volume of liquid with which the tissue is sur-
rounded, the faster will this washing-out process go on ; and
in order that it may not go on with excessive rapidity, wash-
ing out the stain from the nerve-fibres as well as from the
earlier stained elements (which alone it is desired to wash
out, so as to leave a differentiated specific stain of nervous
elements), it is in many cases desirable to have the process
go on in presence of as little liquid as possible. Another
consideration that justifies the practice is that by exposure
to air the preparations take up a trace of ammonia (derived
from the ammonium carbonate of the air) ; and Apathy has
experimentally established that this is an important factor
in the sharpness of the stain. Oxygen has nothing to do
with it.

116. Staining Nerve-tissue by Injection or Immersion. — The
practice of the earlier workers at this subject was (following

6

82 METHYLEN BLUE, AND OTHER ANIL1NS.

EHRLICH) to inject naethylen blue into the vascular system or
body-cavity of a living animal, wait a sufficient time for it to
take effect on the organ to be stained, then remove the organ
for further preparation and study. And there appears to
have been a belief with some workers that it was an essential
or at least a desirable condition to the production of the
stain that it should have been brought about by injection of
the colouring matter into the entire animal. It is now known
that this is immaterial, and that the reaction can equally well
be obtained by removing the organ and subjecting it to a
bath of the colouring matter in the usual way. So that treat-
ment with the colour by means of injection or by means of
immersion in a solution may be taken to be a matter of con-
venience only.

117. The Solutions employed.— The solutions used for injec-
tion are generally made in salt solution (physiological, or a
little weaker) ; those used for staining by immersion are made
either in salt solution or other "indifferent" liquid, or in pure
water. Very various strengths of solution have been em-
ployed. The earlier workers generally took concentrated
solutions. Thus AENSTEIN (Anat. Anz., 1887, p. 125) injected
1 c.c. of saturated solution into the vena cutanea magna of
frogs, and removed the organ to be investigated after the
lapse of an hour. BIEBERMANN (Sitzb. d. k. Akad. Wiss. Wien,
Math. Nat. Cl, 1888, p. 8) injected 0'5 to 1 c.c. of a nearly
saturated solution in O6 per cent, salt solution into the thorax
of crayfishes, and left the animals for from two to four hours
before killing them. MAYER (Zeit.f. wiss. Mik., vi, 4, 1889,
p. 423) took a strength of 1 : 300 or 400 of 0'5 per cent, salt
solution. This can be introduced into the system either by
means of a syringe or other injecting apparatus, or by auto-
injection through the heart. Even rabbits support this ope-
ration if artificial respiration be maintained. The solutions
of RETZIUS are of the same strength. But the tendency of
more recent practice is decidedly towards the employment of
weaker solutions. APATHY (Zeit. f. iviss. Mik., ix, 1, 1892,
pp. 25, 26, et seqq.} finds that it is not only superfluous, but
positively disadvantageous, to take solutions stronger than
1 : 1000.

For staining by immersion similar solutions to those used

METHYLEN BLUE. 83

for injecting may be employed, but they should, if anything,
be still weaker. DOGIEL (Arch. f. mik. Anat., xxxv, 1890,
p. 305; Zeit. f. wiss. Mik., vii, 4, 1891, p. 509) places objects
in a few drops of aqueous or vitreous humour, to which are
added two or three drops of a -^ to -^ per cent, solution of
methylen blue in physiological salt solution, and exposes
them therein to the air. In thin pieces of tissue the stain
begins to take effect in five or ten minutes, and attains its
maximum in from fifteen to twenty minutes. For thicker
specimens — retina, for instance — several hours may be neces-
sary, the preparation being kept just moist by occasional
treatment with a drop or two of indifferent liquid or methylen-
blue solution, added by turns.

118. APATHY'S Methods. — As a good example of this kind
of work, I subjoin a short account of the procedure recom-
mended by Apathy (Zeit. f. wiss. Mik., ix., 1, 1892, p. 15)
for Hirudinea. A portion of the ventral cord is exposed,
and if it be considered desirable, dissected out, but the sinus
and pigmented connective tissue around it had better not be
removed till the staining and fixation are completed. If,
however, it be desired to stain as many ganglion-cells as
possible as well as fibres, the lateral nerves, as well as the
connectives, should be cut through near a ganglion. The
preparation is then treated with the stain. This is, for the
demonstration chiefly of fibres in Hirudo and Pontobdella,
either a 1 : 1000 solution in 0'5 to 0*75 per cent, salt solution,
allowed to act for ten minutes ; or a 1 : 10,000 solution allowed
to act for an hour to an hour and a half; or a 1 : 100,000
solution allowed to act for three hours (Lumbricus requires
twice these times ; Astacus and Unio require three times ;
medullated nerves of Vertebrates four times). For the
demonstration of ganglion-cells the stain is allowed to act
three or four times as long (the connectives and lateral nerves
having been cut as directed above).

The staining having been accomplished, the preparations
from the 1 : 1000 solution are washed in salt solution for an
hour ; those from the 1 : 10,000 solution for a quarter of an
hour; those from the 1 : 100,000 solution need not be washed
at all. They are then treated with one of the ammoniacal
fixing and differentiating liquids described below in the

84 METHYLEN BLUE, AND OTHER ANILINS.

paragraph on "Preservation of the Preparations" (§ 119).
This is done by pouring the liquid over them, and leaving
them in it without moving them about in it for at least an
hour, and by preference in the dark. The further treatment
is as described in § 119.

The object of the ammonia in these liquids is to differentiate
the stain — to produce an artificial " secondary differentiation."
It acts by washing out the absorbed colour from certain
elements, others resisting its action longer, much as HC1
alcohol washes out a borax-carmine stain. In this case the
elements that are washed out are the protoplasmic parts of
nerve-fibres, and their " interfibrillar " and " perifibrillar "
substance, the " primitive fibrils " still retaining the colour
strongly. It is of theoretical interest to remark that accord-
ing to Apathy the coloration thus obtained is a true stain of
the " primitive fibrils," not an impregnation. The " primitive
fibrils" are sharply stained of a violet-blue, showing no
granular precipitate, and the " interfibrillar " and "perifi-
brillar " substance, as well as nuclei, are either colourless or
very lightly stained. The usual methods, on the other hand,
give an "inverse "reaction, the "primitive fibrils" remain-
ing colourless, whilst the interfibrillar substance and proto-
plasm of the nerve-fibres are impregnated with a finely
granular greenish-black or violet precipitate, and the nuclei
are usually stained.

119. Preservation of the Preparations. — There are consider-
able difficulties in the way of obtaining permanent preparations
of methylen-blue stains, as the stain is so very unstable that,
as above explained, it begins to discharge after a short time,
even in the living and not yet totally impregnated tissue.
The colour may, however, be fixed, and more or less permanent
preparations be made, by one or other of the following
methods :

DOGIEL (Arch. /. mik. Anat., xxxiii, 4, 1889, pp. 440 et seq.),
following ARNSTEIN (Anat. Anzeig., 1887, p. 551), brings the
preparations, in order to fix the colour, into saturated aqueous
solution of picrate of ammonia, in which they are allowed to
remain for half an hour or more, and are then removed,
washed in fresh picrate of ammonia solution, and studied in
dilute glycerin, or mounted permanently in glycerin saturated

METHYLEN BLUE. 85

with picrate of ammonia. More recently (Zeit. f. wiss. Mik.,
viii, 1, 1891, p. 15) lie has recommended an increased duration
of the picrate of ammonia bath up to eighteen or twenty-four
hours, and mounting, without washing out, in chemically pure
glycerin, free from acid. There is a defect in this process,
namely, that picrate of ammonia has a very injurious action
of a macerating nature on some tissues. This may, however,
be avoided by adding to the fixing-bath 1 to 2 per cent, of a
1 per cent, osmic acid solution. (If it be desired to harden
the tissues so that sections may be cut, the proportion of
osmium solution should be increased fourfold.)

S. MAYER (Zeit. f. wiss. Mile., vi, 4, 1889, p. 422) preferred
a mixture of equal parts of glycerin and saturated picrate of
ammonia solution, which served to fix the colour and mount
the preparations in. This was also in principle the method
followed by RETZIUS (Intern. Monatsschr. Anat. u. Phys., Bd.
vii, H. 8, 1890). DOGIEL, after careful study, quite refuses to
admit that this is in any way an improvement.

Other workers have employed saturated solution of iodine
in iodide of potassium (so ARNSTEIN), or picro-carmine (so
FEIST, Arch. f. Anat. u, Entwickel., 1890, p. 116; cf. Zeit. f.
wiss. Mik., vii, 2, 1890, p. 231), the latter having the advan-
tage of preserving the true blue of the stain if it be not
allowed to act too long, and the preparation be mounted in
pure glycerin.

Picric acid has been used by LAVDOWSKY, but this, too, after
careful study, is rejected by DOGIEL.

APATHY (Zeit.f. wiss. Mik., ix, 1, 1892, p. 30) has found, as
stated above, that free ammonia is a capital factor in the
differentiation of the stain. He brings preparations (after
washing in salt solution if the staining have been performed
with a strong methylen-blue solution, or without washing if
it have been done with a very dilute solution) either into a
concentrated aqueous solution of picrate of ammonia free
from picric acid, and containing five drops of concentrated
ammonia for every 100 c.c.; or, which is generally preferable,
into a 1 to 2 per cent, freshly prepared solution of neutral
carbonate of ammonia saturated with picrate. They remain
in either of these solutions, preferably in the dark, for at
least an hour. They are then brought into a small quantity
of saturated solution of picrate of ammonia in 50 per cent.

86 METHYLEN -BLUE, AND OTHER ANILINS.

glycerin, where they remain until thoroughly saturated. They
are then removed into a saturated solution of the picrate in a
mixture of 2 parts 50 per cent, glycerin, 1 part cold saturated
sugar solution, and 1 part similarly prepared gum-arabic solu-
tion. When thoroughly penetrated with this they are removed
and mounted in the following gum- syrup medium (1. c., p. 37) :
Picked gum-arabic . . .50 grms.

Cane-sugar (not candied) . 50 „

Distilled water . . . . 50 „

Dissolve over a water-bath and add 0'05 grm. thymol. This
mounting medium sets quickly and as hard as balsam, so that
no cementing of the mounts is necessary. Farrant's medium
(with omission of the arsenious acid) will also do. In neither
case should either ammonium picrate or methylen blue be
added to the medium.

None of the preceding methods can be said to be anything
like perfectly satisfactory. The stain is generally not pre-
served in its true blue colour, but turns to a grey, varying in
tone from reddish brown to bluish or greenish black. The
preparations seldom keep even in that state for more than a
very few months, and it is not satisfactory to be obliged to
mount preparations only in aqueous media. A strong solution
of platinum chloride is said to give a fixation that will resist
the treatment necessary for imbedding either in celloidin or
paraffin (see FEIST, Arch. f. Anat. und Entw., 1890, p. 116;
Zeit. /. wiss. Mik., vii, 2, 1890, p. 231) ; but the precipitate
it gives is a flocculent one, and the preparations are not very
satisfactory.

The method of PARKER (Zool Anzeig., No. 403, 1892, p. 375)
is therefore a most welcome step forwards. The stain is fixed
(1) (in the form of a finely grained purplish precipitate) by
cold concentrated aqueous solution of corrosive sublimate.
The preparations are dehydrated (2) in a solution composed
of 1 grm. of sublimate and 5 c.c. of methylal (pure methylal
washes out the stain to a certain extent). The methylal
solution is now removed by means of a mixture (3) of two
parts xylol, one part pure methylal, and one part of the
dehydrating mixture (2). After a short time the preparations
are placed in (4) a considerable quantity of xylol. Here they
should remain till all the methylal is replaced by xylol and
the corrosive sublimate completely washed out. This will

METHYLEN BLUE. 87

take some four or five days, as sublimate is very little soluble
in xylol. Mount in balsam, or if sections be desired, imbed
in paraffin in the usual way. Sections should be fixed to the
slide with Schallibaum's collodion, and not with Mayer's
albumen, which discharges the colour. The preparations will
keep for several weeks, but the finer details are likely to fade
after a month.

The times required for a ganglion of the ventral chain of a
crayfish are for (1) ten minutes, (2) fifteen minutes, (3) ten
minutes, (4) four or five days.

FEIST, in the paper quoted above, recommends JOLIET'S
gum-glycerin imbedding method; but in most cases the
method of Parker will doubtless be preferable.

Preparations preserved by the old methods (I do not know
whether it is the case with preparations preserved by Parker's
method) are extremely sensitive to the influence of light.
Diffused daylight is less injurious to them than the light con-
centrated on them by the illuminating apparatus of the
microscope during observation. Apathy finds that lamplight
is particularly injurious, especially the intense lamplight used
with high powers ; which he attributes partly to the yellow
rays, partly to the heat.

120. Methylen-Blue Impregnation of Epithelia, Lymph- spaces,

&c. (DOGIEL, Arch.f. mik. Anat., xxxiii, 4, 1889, p. 440, etseq.). — Suitable
pieces of tissue (thin membranes by preference) are brought fresh into a 4
per cent, solution of methylen blue in physiological salt solution. After a few-
minutes therein they are brought into saturated solution of picrate of
ammonia, soaked therein for half an hour or more, then washed in fresh
picrate of ammonia solution, and examined in dilute glycerin.

If it be wished only to demonstrate the outlines of endothelium cells, the
bath in the stain should be a short one, not longer than ten minutes in
general ; whilst if it be desired to obtain an impregnation of ground-sub-
stance of tissue so as to have a negative image of juice-canals or other spaces,
the staining should be prolonged to fifteen or thirty minutes, and it is
advisable to remove the endothelial covering of the objects operated on
before putting them into the stain.

If it be desired to preserve the preparations permanently, they had better
be mounted in glycerin saturated with picrate of ammonia. (For an im-
provement in the method of preservation given in a later paper see supra,

§ 119.)

The effect is practically identical (except as regards the colour) with that
of a negative impregnation with silver nitrate.

121. MAYER'S (S.) Impregnation Methods (Zeit. f. wiss.

88 METHYLEN BLUE, AND OTHER ANILINS.

iJc.j vi, 4, 1889, p. 422). — Mayer's experiments were made
contemporaneously with those of Dogiel, cover much the same
ground, and give the same results. Mayer stained tissues for
about ten minutes in a 1 : 300 or 400 solution of methylen
blue in 0'5 per cent, salt solution, rinsed in salt solution, and
put up in the glycerin-picrate of ammonia mixture given
above, § 119. He finds it may be stated that by this method
all the essential results of a nitrate of silver impregnation may
be produced by means of methylen blue. The images are either
positive or negative. In stratified epithelia and in endothelia
(for instance, in the testicular canals of the rat, in the
vascular system, in smooth muscle) cement-substance is
stained. In the cornea, ground-substance is stained, giving a
negative image of the corneal corpuscles ; and in like manner
a negative image is sometimes obtained of the intra-sarco-
lemmar nerve-endings in striated muscle of frogs and rats.
But methylen blue sometimes, as does sometimes silver
nitrate, gives positive images of the cornea. If the stain be
brought about by injection of the colour into the vascular
system, the positive impregnation is the more frequent ;
whilst if it have been brought about by the immersion of
the cornea, a negative image is more frequently obtained. In
medullated nerves, striking images of the cruciform figures in
the constrictions of Kanvier are obtained, just as with silver
nitrate.

To sum up, almost any reaction that can be obtained with
chloride of gold, or with nitrate of silver, can be obtained
with methylen blue, and with much greater ease and
certainty.

122. Other Uses of Methylen Blue. — Methylen blue is also
used, chiefly in conjunction with other colouring matters, for
staining fixed and hardened tissues, especially preparations of
central nervous system. These uses will be described in their
proper places.

B. Other Anilins (Plasma and Ground Stains).

123. Bleu Iiumiere is stated to be a plasma stain not affecting nuclei.
I have not been able to make out whether it is identical with Parma blue,
which is one of the numerous toluidin blues. If it is, Frey recommends a
•solution in water of 1 : 1000, in which tissues stain in a few minutes, and

INDULIN. 89

may be mounted either in glycerin or balsam. " Lichtblau " is possibly a
synonym of this colour. The principal use of such a colour is for making
double stains.

123 a. Carmine Blue (Bleu Carmin Aqueux). — This colour is much
recommended by GILSON, into whose laboratory it was introduced by
JANSSENS. See La Cellule, vii, 1891, p. 347.

124. Bleu de Lyon (Bleu de Nuit, Griinstichblau).— I quote this
colour here, although 1 am not sure to what extent it is a pure plasmatic
stain. It is said to be very useful for double-staining with carmine.

125. Indulin (Nigrosin, Bengalin, Anilin Blue-black, Blackley
Blue, Artificial Indigo). (Introduced by Calberla, see Morpli. Jahrb.,
iii, 1877, p. 627.) — Indulin dissolves into a dark blue solution in warm
water or in dilute alcohol. For staining, the concentrated aqueous solution
should be diluted with six volumes of water. Sections will stain in the
dilute solution in five to twenty minutes ; they may be washed in water or
in alcohol, and examined either in glycerin or oil of cloves.

The peculiarity of this stain is that it never stains nuclei ; the remaining
cell- contents and intercellular substance are stained blue. In its general
effects it resembles quinolein blue, and is exactly the opposite of methyl
green. The stroma of tendinous tissue, for instance, stains of a fine blue,
the connective tissue that surrounds the bundle hardly at all, and the
tendon-corpuscles of Ranvier remaining perfectly colourless, stand out as
white stellate figures on a blue ground.

126 Quinolein Blue (Cyanin, Chinolinblau ; v. Kanyier, Traite,
p. 102).— Quinolein should be dissolved in alcohol of 36° strength (i. e. 90
per cent.), and the solution diluted with an equal volume of water. (If the
alcohol were taken dilute in the first instance the blue would not dissolve.)
The solutions employed for staining should be very weak, as quinolein
stains very powerfully.

After staining, wash and mount in glycerin. When first mounted, nuclei
will be seen to be stained a fine violet, nerves of a grey-blue, smooth muscle
blue, protoplasm blue, fat deep blue. But after twenty-four hours in the
glycerin the aspect of the preparation is changed ; the nuclei have become
colourless ; the protoplasm remains blue, and is seen to contain granulations
stained intensely blue; nerves remain grey-blue, but frequently contain
granulations stained blue. Quinolein, in a word, has the property of
staining fatty matters an intense blue.

If the stained preparations be treated with solution of potash of 40 per
cent, strength, the differential reaction is produced immediately ; the nuclei
are unstained, protoplasm, nerve, and muscle-tissue are pale blue, and fatty
matters deep blue.

Quinolein is useful for staining Infusoria, which in dilute solution it
stains during life. On this point see the methods of Certes (post, Part II).

127. Benzoazurin may be made to give either a diffuse or a

90 METHVLEN BLUU, AND OTHER ANILINS.

nuclear stain, according to MAKTIN (see Journ. Roy. Hie. Soc.,
1890, p. 114).

128. Anilin Blue. — Used alone, without special precautions,
this is a diffuse stain hardly to be recommended at the
present time. See, however, the method of HEIDENHAJN,
Arch. f. mik. Anat., vi, 1870, p. 404. It is useful for double-
staining.

129. Violet B. (S. MAYER, Sitzb. d. k. k.Akad. d. Wiss., Wien, iii Abth.,
February, 1882).— Used in solutions of 1 grm. of the colour to 300 grms. of
0*5 per cent, salt solution, and with fresh tissues that have not been treated
with any reagent whatever, this colour gives a stain so selective of the ele-
ments of the vascular system that favorable objects, such as serous mem-
branes, appear as if injected. The preparations do not keep well ; acetate of
potash is the least unsatisfactory medium for mounting them in.

130. Anilin Black (Blue-black, Nigranilin, Noir de Colin).— Has

a special affinity for ganglion-cells, and is much used in the study of the cen-
tral nervous system. See post, Part II.

According to CEETES, anilin black dissolved in sea or fresh water is useful
for the study of Infusoria, to which it is in no way toxic.

131. Saurefuchsin (Fuchsin S., Acid Fuchsin). — A f uchsin in which
the colouring principle is an acid, instead of being a base as in ordinary f uch-
sin. It is only made by the " Badische Anilin und Soda Fabrik." It may be
obtained from Grubler or the other providers of histological reagents. It is
a powerful diffuse stain, having a special affinity for axis-cylinders, and is
chiefly used for staining nerve-centres. See the chapter on Nerve-Centres,
in Part II.

132. Congo Red (Congoroth) (see Griesbach, in Zeit. f. wiss. Mik., iii,
3, 1886, p. 379). — Also an "acid" colour, in the sense in which that appel-
lative is given to Saurefuchsin. The aqueous solution, however, has a
neutral or alkaline reaction. It becomes blue in presence of the least trace
of free acid (hence Congo is a valuable reagent for demonstrating the presence
of free acid in tissues ; see the papers quoted, 1. c.). A diffuse stain, much of
the same nature as that of Saurefuchsin, and like it seems to be at present
chiefly useful in staining axis-cylinders. See the chapter on Nerve-Tissue,
in Part II. It may also be used for staining some objects during life. See
ante, § 93.

133. Benzopurpurin.— According to Griesbach (1. c.), another
" acid " colour very similar in its results to Congo red. It
is probably one of the best diffuse stains yet made known,
and as a contrast stain to hasmatoxylin, will very likely take
the place of eosin in mauy cases. It is a very strong stain,
therefore a very weak solution should be taken, and only
allowed to act for a minute or so. ZSCHOKKE (ibid., v, 4,

EOSIN. 91

1888, p. 466) says that Benzopurpurin B. is 'the very best
contrast stain to ha3matoxylin known to him. It seems to
him preferable to eosin on account of its not being affected
by alcohol or the usual clearing agents. Weak aqueous
solutions should be used for staining, which is effected in a
few minutes, and alcohol for washing out. Deltapurpurin, a
more purple red, has similar properties, and may be used in
the same way. The solution (aqueous) should be moderately
concentrated, and allowed to act for a minute or two.

134. Biebricher Scharlach (BIEBEICH SCAELET), a diffuse bright red
stain, may be useful as a contrast stain. See Griesbach, Arch. f. miJc. Anai.>
xxii, p. 132.

135. Eosin, the potassium salt of a bromide of phthalei'n, is
found in commerce under the synonyms of Primerose Soluble,
Erythrosin, Pyrosin B., Rose B. a TEau. The preparations
indicated by these names are not quite identical in their pro-
perties, but vary according to the different modes of manu-
facture. Most of them are soluble both in alcohol and in
water, but some only in alcohol ("Primerose a I' Alcohol").

Eosin is of very great importance as a secondary or contrast
stain. It has already been mentioned that it washes out
many of the anilins that give nuclear stains by the indirect
method (see § 98) . Combined with a blue anilin, or with
hasmatoxylin, it gives instructive and durable double stains,
which are among the most beautiful that can be produced.
These will be treated of in the chapter on double stains.
Here it may merely be mentioned that the majority of the
authors who recommend these combinations proceed by stain-
ing first with the eosin and then with the nuclear stain, and
that the majority also employ alcoholic solutions of eosin. I
am by no means convinced that this practice is the best. As
regards the anilins, at all events, good results are certainly
obtained by staining first with the nuclear stain (gentian
violet, for instance) and washing out (sections) for a few
seconds or a minute or two in a tolerably strong aqueous solu-
tion of eosin, and then dehydrating with alcohol and mounting
in balsam. If the eosin is too much extracted by the alcohol
or clearing agent, these should be used charged with a little of
the colour. Eosin stains may also be kept in glycerin, if this
be perfectly neutral or, better, slightly alkaline (which may

92 METHYLEN BLUE, AND OTHER ANILINS.

be brought about by the addition of 1 per cent, of sodium
chloride), and also charged with a little eosin.

136. Bengal Hose (GEIESBACH, Zool Anz., No. 135, 1883, p. 172).—
Bengal rose, or "Kose bengale," or "Bengal rosa," is an eosin dye. It is the
bluest of the eosin dyes as yet known, approaching in hue to fuchsin, but
possessing far greater brilliancy and purity of hue. In aqueous solution it
is useful for staining chromic acid objects, especially spinal cord, in which
the grey matter stains of a deep bluish red, and stands out boldly from the
less deeply coloured white matter. It is also useful for double and treble
stains, as will be explained below.

137. Picric Acid. — Not used alone as a stain, but one of the
most useful of all colouring agents as a secondary or ground-
stain. Nothing is easier than to stain with an alcoholic solu-
tion of picric acid tissues of which the nuclei have previously
been stained by borax-carmine, alum-carmine, haematoxylin, or
an anilin stain. It should be borne in mind that picric acid
has considerable power of washing out other anilin stains ;
and that in combination with hydrochloric acid it very greatly
enhances the power with which this acid washes out carmine
stains. It does not otherwise affect any of the usual stains,
and may be most highly recommended as a useful though fre-
quently inelegant stain.

138. Metanil Yellow (Metanilgelb).— This colour has lately been
studied with great minuteness by GEIESBACH (Zeit. f. wiss. Mile., iv, 4,
1887, p. 448 ; see also Journ. Roy. Mic. Soc., 1889, p. 464). It does not
appear to be worthy of having so much time spent on it from the practical
point of view, the interest of Griesbach's work lying rather in the region of
chemical theory. The practical outcome is that metanil yellow is a diffuse
stain with a certain affinity for various elements belonging to the group of
the connective tissues, and that with some other colours it gives sharply
differentiated double stains of certain preparations. These will be mentioned
in the proper places.

139. Sauregelb (Echtgelb), Tropseolin O., Crocem, Gold Orange,
are all of them more or less diffuse yellow or orange stains, having certain
affinities for certain tissues, and may occasionally be found very useful for
double-staining, being good stains in their way (see GBIESBACH, Arch. f.
mik. Anat., xxii, p. 132).

140. Iodine Green (" Hofmann's Griin "), see GRIESBACH, Zool.
Anz., No. 117, vol. v, 1882, p. 406. — The high praise accorded
to this stain by Griesbach has not been justified by the ex-
perience of other workers. The colour is now no longer

MALACHITE GREEN. 93

manufactured for industrial purposes, but may be obtained
of excellent quality from C. A. F. Kuhlbaum's Chemische
Fabrik, Berlin, S.O. (Zool. Anz., No. 130, 1883, p. 56).

141. Thiophen Green (Thiophengriin), see KEAUSE, Intern. Monats-
sclir.f. Anat., &c., iv, 1887, Heft 2.

142. Anilin Green has a special affinity for mucous gland,
cells. We shall return to this subject in the chapter on
glands.

143. Malachite Green is a colour concerning which I have
not been able to obtain sufficient information. It appears to
be useful as a plasma-stain.

94 CAKMiNE AND COCHINEAL STAINS.

CHAPTER X.

CARMINE AND COCHINEAL STAINS.

144. The Theory of Carmine Staining. — I take the following
from the important paper of MAYBE, " Ueber das Farben mit
Carmin, Cochenille, und Hamatein-Thonerde," in Mitth. a. d.
Zool. Station zu Neapel, Bd. x, Heft 3, 1892, p. 480. The
rationale of staining with carmine has hitherto been obscured
by the erroneous notion that carmine is nothing but carminic
acid with at most certain impurities. This is not the case.
According to the analysis of LIEBEEMANN (Ber. d. Chem. Ges.,
Jahrg. 18, 1886, pp. 1969 — 1975) carmine is a very peculiar
alumina-lime-protein compound of carminic acid, a true chemi-
cal compound from which at all events aluminium and
calcium can no more be absent than sodium from salt.
Analysis gives about 17 per cent, of water, 20 per cent, nitro-
genous matters, 56 per cent, carminic acid, at least 3 per
cent, alumina, and 3 per cent, lime, together with a small
proportion of magnesia, potash, soda, phosphoric acid, and a
trace of wax. Mayer has come to the conclusion that in the
processes of histological staining (not of industrial dyeing)
the active factors of the compound are, besides the carminic
acid, only the alumina, and, in some cases, the lime. The
other bases are inactive ; the nitrogenous matters, so far as
they have any influence at all, are an obstacle, as it is they
that give rise to the well-known putrefaction of the solutions.

Having arrived at these conclusions, it seemed logical to
admit that carminic acid, instead of carmine, should be taken
as the basis of staining solutions. This had already been pro-
posed by DIMMOCK, whose paper (Amer. Natural., xviii, 1884,
pp. 324-7) I quoted at length in the first edition of this work.
But Dimmock's proposals were not very successful, for the
reason that he had omitted from his solutions the essential
element, the alumina. He stained, for instance, with pure

CARMINIC ACID. 95

alcoholic solution of carminic acid, or of carminate of am-
monia. Such solutions stain, but stain weakly and diffusely.

145. Carminic Acid occurs as a purple-brown mass, easily
soluble both in water and in alcohol. It is (according to
NIETZKI, Chemie der organischen Farlstojfe, Berlin, 1889,
pp. 231 — 234) a weak (LIEBERMANN says a strong) dibasic acid,
which forms soluble salts with the alkaline metals, insoluble
violet-coloured ones with the earthy and heavy metals. Very
little is known concerning the chemical nature of these salts.

The alumina salt (carminate of alumina) may be obtained
by precipitating a solution of carminic acid or of carminate
of ammonia by means of acetate of alumina. It has the
remarkable property of being soluble not only in acids and
acid salts, such as alum, but also in alkalies and alkaline salts,
such as borax, provided that only water or weak alcohol be
employed as the menstruum. It is also precipitated from the
above-named solutions by chloride of aluminium, but only in
part ; whilst if alum be taken no precipitate is produced, the
carminate of alumina remaining in solution. Hence the
composition of the staining fluid given below under the name
of Carmalum.

When chloride of aluminium is taken, a precipitate of
carminate of alumina is formed, as stated above. But this
precipitate will redissolve if more chloride of aluminium be
cautiously added. This gives the staining fluid described in
§ 152, which may be convenient in cases in which it is not
desirable to work with a fluid containing alum.

Both of these solutions stain in a violet tone, something like
alum-carmine. A redder tone may be obtained by adding
calcium chloride to the Carmalum solution. But this is not
advisable, for calcium chloride added to Carmalum precipi-
tates the solution with formation of gypsum. Of course, this
does not occur with the aluminium chloride solution; but for
other reasons the addition does not give satisfactory results
with the chloride of aluminium solution mentioned above.
But it does gives good results when combined with an alcoholic
chloride of aluminium solution, and thus -solves at once the
problem of obtaining a red stain and an alcoholic staining
fluid. This is described below under the name of Paracarmine.

If the foregoing explanations of the rationale of carmine

96 CAEMINE AND COCHINEAL STAINS.

staining be compared with the remarks on the theory of
staining with haematoxylin given below (§ 174) an interesting
parallelism will be observed. In both processes, it is not the
colouring matter alone which is active, but the colouring
matter combined with alumina. The stain is always got with
carminic acid -f alumina, or with hasmatem + alumina;
other substances, such as lime, occasionally playing a part.

146. The foregoing considerations deal with the theory of
staining with Carmine; we have now to consider the Theory
of Staining with Cochineal. According to MAYEE, whose
earliest researches are confirmed by his latest (Mitth. ZooL
Stat. zu Neapel, x, 3, 1892, p. 496), the active principle of
extract or tincture of cochineal (as used in histology) is not
free carminic acid, but carminic acid chemically combined
with a base which is not lime, but some alkali. The pure
aqueous extract contains only traces of lime, the alcoholic
none at all. The watery extract made with alum, or cochineal-
alum carmine (§ 154), owes its staining power to the formation
of a carminate of alumina, the general properties of which
have been discussed above when treating of the theory of
carmine staining (§ 145). The tincture made with pure
alcohol, on the other hand, contains only the above-mentioned
carminate of some alkali. This carminate alone stains weakly
and diffusely (like carminic acid alone). But if in the tissues
treated with it it meet with lime salts, alumina or magnesia
salts, or even metallic salts capable of combining with it and
forming insoluble coloured precipitates in the tissues, then a
strong and selective stain may result. As a matter of fact,
the simple cochineal tincture of Mayer given in § 172 does
give splendid results with certain objects (i. e. such as contain
the salts in question). But it is unfortunately equally certain
that such objects are rather rare than otherwise, and that with
the majority of objects the stain is a very poor one.

But if the necessary salts be added to the tincture itself,,
then a solution ought to result containing the necessary
elements for affording a strong and selective stain with all
classes of objects. This proves to be the case ; whence
Mayer's new formula, § 173.

147. General Remarks. — What are the carmine stains useful
for ? Is it for staining fresh tissues ? With the exception

PUEE CAEMINIC ACID. 97

of aceto-carmine, no. Is it for staining sections ? Again,
no ; for, in nine cases out of ten, sections are better stained
by some of the anilin stains than they can be in any carmine
stain. Is it for staining entire objects ? — for staining in the
mass ? Yes ; for in many, if not in most cases, that can be
done more satisfactorily by means of carmine than by meansi
of any other known agent. So that until a coal-tar colour
shall have been discovered that can beat alum-carmine and
borax-carmine on their own ground, these must still hold
their sway. As soon as that shall have been done — and it may
be done any day — carmine stains will become as extinct as the
dodo.

In view of the far greater simplicity and precision of the
methods proposed by Mayer, it is probable that they, or
at all events methods conforming to the principles above
laid down, will gradually take the place of the old methods.
Not, indeed, that some of the old stains will not hold their
ground. Alum-carmine will remain a superb stain, and
borax-carmine remains superior to paracarmine in the power
and brilliancy of its stain; but the superior precision of
Mayer's methods will doubtless be acknowledged. And it
is perhaps not too much to hope that fewer formulae of the
old roundabout, happy-go-lucky sort, with commercial car-
mine or hsematoxylin as the chief ingredient, will henceforth
be published.

148. Pure Carminic Acid may be obtained from E. MERCK
of Darmstadt, or from Dr. Gr. GEUBLER (12, Bayersche Strasse,
Leipzig). The price at present is from 2s. to 4s. per 10 grms.

149. Hints. — Overstains may in all cases be washed out with weak HC1
(e. g. O'l per cent.). HENNEGUT (Journ. de I'Anat. et de la Physiol., xxvii,
1891, p. 400) states that overstains may be completely removed by means of
permanganate of potash. Sections thus treated may then be after-stained
with anilin colours (see § 106). All carmine stains, with the exception of
aceto-carmine, are permanent in balsam. Aqueous mounts should be acid
(except for alum-carmine), and the best plan is to let the mounting medium
contain 1 per cent, of formic or acetic acid. Formic acid is to be preferred.

Kemember that none of the acid stains, except alum-carmine used as
directed, § 155, nor any of Grenadier's fluids can be used with calcareous
structures that it is wished to preserve.

Grenadier's alcoholic borax-carmine may be recommended to the beginner
as being the easiest of these stains to work with.

150. Classification of the Formulae. — In the treatment of the for-

7

98 CARMINE AND COCHINEAL STAINS.

mulae given in the following pages I have been guided by the considerations
set out in § 147. The best stains for staining in the mass are fully treated,
the old-fashioned solutions proposed for staining sections being thrown into
the background. The formulae set out below are arranged according to the
nature of the menstruum. This gives us two great groups, aqueous carmine
solutions and alcoholic carmine solutions. Taking first the group of aqueous
solutions, I have arranged the formulae comprised in it according to the
reaction of the solutions.

The first subdivision comprises the acid solutions, i. e. those with free acid or
acid salts. The second subdivision comprises the so-called neutral carmines
and the alkaline solutions. These last are scantly treated, the very fact of a
staining solution having an alkaline reaction and at the same time an aqueous
menstruum implying that it must have a deleterious effect on tissues. Such
stains should be reserved for tissues that have in the process of harden-
ing been overcharged with chrome salts to such an extent as to no longer
take up other stains, and for maceration preparations, for which they may
sometimes be found useful. The alcoholic group is too small to require sub-
division.

A. AQUEOUS CARMINE STAINS.
a. Acid.

151. Mayer's Carmalum (Mitth. Zool. Stat. zu Neapel, 10, 3,
1892, p. 489). — Carminic acid, 1 grm.; alum,10grms.; distilled
water, 200 c.c. Dissolve with heat. Decant or filter. Add some
antiseptic, either a few crystals of thymol, or 1 per cent, salicylic
acid, or 5 per cent, salicylate of soda. The solution will then
keep. A clearish red fluid with a violet tinge. It stains well in
bulk even osmium objects. If washed out with distilled water
only, the plasma will remain somewhat stained. If this be
not desired, wash out carefully with alum solution, or, in
difficult cases, with weak acid. The general effect is that of
an alum-carmine stain. A notable difference between the
two is that carmalum stains well in bulk, which alum-carmine
is not very suitable for when used in the ordinary way ; but
see § 155.

A weaker solution may be made by taking from three to
five times as much alum and five times as much water, and
dissolving in the cold, which may be convenient. This is a
very close equivalent of alum-carmine, giving, however, a
somewhat redder stain. It should be noted that with either
solution the objects to be stained should not have an alkaline
reaction.

152. MAYER'S Aqueous Carminate- of- Alumina- Solution (Mitth.

ALOM-OAEMINE. 99

Zool Stat. zu Neapel, 10, 3, 1892, p. 490).— Carminic acid,
1 grrn.; chloride of aluminium, 3 grms. ; water, 200 c.c. Add
an antiseptic, as for carmalum.

Use as carmalum. The stain is of a blue- violet colour,
very powerful, and elective. But it is not so pure a stain as
that of carmalum, plasma being more strongly coloured. It
is recommended only as a substitute for carmalum in cases
in which the latter is counter-indicated on account of the
presence of alum or the like.

153. Alum- Carmine ((TEENACHER'S formula, Arch. mik. Anat.,
xvi, 1879, p. 465). — An aqueous solution (of 1 to 5 per cent,
strength, or any other strength that may be preferred) of com-
mon or ammonia alum is boiled for ten or twenty minutes
with J to 1 per cent, of powdered carmine. (It is perhaps
the safer plan to take the alum solution highly concentrated
in the first instance, and after boiling the carmine in it dilute
to the desired strength.) When cool filter.

This stain must be avoided in the case of calcareous struc-
tures that it is wished to preserve (but see § 155).

TIZZONI (Bull. Sc. Med. Bologna, 1884, p. 259) and PISENTI (Gazz. degli
Ospetali, No. 24 ; Zeit. f. wiss. Mik., ii, 1885, p. 378) add a small percent-
age of sulphate of sodium, with the object of enhancing the energy of the
stain. It should not be forgotten that sodium sulphate is a substance that
exercises a very peculiar action on nuclei (see PFITZNEB, Morph. Jahrb., xi,
1, 1885).

GEIEB (Mem. Soc. Hal. Sci., t. vi, No. 9, 1887 ; Zeit. f. wiss. Mik., vii,
1, 1890, p. 47) gives a modification of Grenadier's formula which does not
appear to me rational.

It has already been sufficiently repeated that alum-carmine
is one of the best stains to be found outside the coal-tar
colours. It is particularly to be recommended to the beginner,
as it is easy to work with ; it is hardly possible to overstain
with it (except muscle). Its chief defect is that it is not
very penetrating, and therefore quite unsuitable for staining
objects of considerable size in the mass. This defect may,
however, be overcome by employing the method of Zoltan
von Eoboz (infra, § 155), or by employing the acid formula
of Henneguy (§ 156), if it be not convenient to use Mayer's
carmalum.

The stain is permanent in balsam, and in aqueous media
if not acid.

100 CAEMINE AND COCHINEAL STAINS.

154. Cochineal Alum-Carmine (PAKTSCH, Arch. f. mik. Anat.,
xiv, 1877, p. 180). — Powdered cochineal is boiled for some
time in a 5 per cent, solution of alum, the decoction filtered,
and a little salicylic acid added to preserve it from mould.

Another method of preparation has been given by CZOKOR
(Arch.f. mik. Anat., xviii, 1880, p. 413). Mayer has care-
fully examined both, and finds that Partsch's is the more
rational, the proportion of alum in it being exactly right,
whilst in Czokor's it is insufficient. Partsch's fluid has also
the advantage of keeping better.

The formula known as Klein's cochineal fluid (which appears to have been
first published in the Ann. and Mag. Nat. Hist., viii, 1881, p. 232) is iden-
tical with that of Czokor.

Both these solutions are to all intents and purposes " alum-
carmines. J) They give a stain that is practically identical
with that of alum-carmine made from carmine, with perhaps
even more delicate differentiations (but that depends so much
on the quality of the carmine, the quality of the cochineal,
and the nature of the objects to be stained, that no absolute
rule can be stated). On the whole it seems to be a mere
matter of convenience whether the one or the other should be
preferred. The cochineal fluids should be used in exactly the
same way as the carmine fluid.

It has been lately recommended by HERRICK (Journ. Comp.
Neur., Cincinnati, vol. i, 1891, p. 134) as a "vast improve-
ment" to make the solution with sulphate of aluminium
instead of alum (quoted from Mayer's paper, Mitth. Zool. Stat.
Neapel, 10,3, 1892, p. 496).

155. Alum-Carmine with Osmic Acid (ZOLTAN VON ROBOZ, in
lilt.). — To 50 or 60 grins, of water is added alum-carmine
until the mixture is of an almost red rose colour ; about ten
drops of a y^Vo" solution of osmic acid are then added. (The
mixture should have an appreciable smell of osmic acid.) The
objects to be stained remain in the mixture for from twenty-
four to forty-eight hours in the dark. It is hardly necessary
to wash them, as the stain is perfectly precise without that.
It is important to perform the staining in a well-closed vessel,
in order to prevent the evaporation of the osmium.

I have used this stain with the most diverse objects, and can
most highly recommend it. The result is a sharp nuclear

AOETO-CARMINEV 101

double stain (resting chromatin and nucTebli purple, kinetic
chroma tin red, the rest brown) . I find it valuable tor staining
soft tissues in the mass. As the staining bath contains but
very little alum, it may be used with calcareous structures,
and is, indeed, for such objects as Pluteus about the best stain
that I know of. It is difficult to understand why this admir-
able method has not been more used.

156. Acetic Acid Alum- Carmine (HENNEGUY, Traite des Meth.
techn.j LEE et HENNEGUY, p. 88). — Excess of carmine is boiled
in saturated solution of potash alum. After cooling add 10
per cent, of glacial acetic acid, and leave to settle for some
days. The deposit of carmine and alum that forms during
that time is removed by filtration.

For staining, enough of the solution is added to distilled
water to give it a deep rose tint. Stain for twenty-four to
forty-eight hours, and wash for an hour or two in distilled
water. (It is important that the water should be distilled in
order to avoid the formation of crystals.) Dehydrate with
alcohol and mount in balsam. You can mount in glycerin,
but the preparations do not keep so well as in balsam.

The advantage of this carmine is that it has great power
of penetration, and stains deep-seated layers of tissue just as
well as the superficial ones. The colour of the stain is a some-
what inelegant violet, but this can be changed to a warmer
tone by treating the objects with dilute HC1, as for borax-
carmine objects.

157. Alum-Carmine and Picric Acid. — Alum-carmine objects
may be double-stained with picric acid. LEGAL (Morph.
Jahrb., viii, p. 353) combines the two stains by mixing ten vols.
of alum-carmine with one of saturated picric acid solution.

GILSON'S picro-alum-carmine (the stain mentioned by BOL-
sius in La Cellule, vii, 1, 1891, p. 6, kindly communicated by
Prof. GILSON) is made by boiling 10 per cent, of cochineal for
about twenty hours in saturated solution of alum (in a Lie-
big's refrigerator or some flow-back arrangement for prevent-
ing diminution of volume by evaporation), and adding 10 per
cent, of saturated solution of picric acid.

158. Aceto-Carmine (Acetic Acid Carmine) (SCHNEIDER'S for-
mula, Zool. Anzeig., No. 56, 1880, p. 254). — To boiling acetic

102 CARMINE AND COCHINEAL STAINS.

a-cid of ^:p'er cent: '-strength add carmine until no more will
dissolve, and filter. (Forty-five per cent, acetic acid is the
strength that dissolves the largest proportion of carmine.)

To use the solution you may either dilute it to 1 per cent,
strength, and use the dilute solution for slow staining, which
is the method to be preferred for making glycerin prepara-
tions; or a drop of the concentrated solution may be added to
a fresh preparation under the cover-glass.

This is a very important reagent, which in certain cases
renders services that no other reagent can render. If you use
the concentrated solution it fixes and stains at the same time,
and hence is most valuable for the study of fresh objects. It
is very penetrating, a quality that enables it to be used where
ordinary reagents would totally fail. The stain is a pure
nuclear one. Unfortunately the preparations cannot be pre-
served, and for this and other reasons the stain is of very
restricted application.

ZACHAEIAS (see Zeit.f. wiss. Mik., v, 3, 1888, p. 371) adds
to this solution wood vinegar (acetum pyrolignosum) in the
proportion of 1 drop to 10 c.c.

j3. So-called "Neutral" and Alkaline.

159. As to Picro-carmine. — The term " picro-carmine " is commonly
used to denote a whole tribe of solutions in which carmine, ammonia, and
picric acid exist uncombined in haphazard proportions. KANVIEK, to whom
we owe the invention of picro-carmine, claims that when prepared by his
process it results as a definite chemical substance, a double salt of picric and
carminic acid and ammonia, or picro-carminate of ammonia. I am not
prepared to say whether this is or is not the case, but I think it may be
safely said that Ranvier's method of preparation is the only one yet pub-
lished that will give with certainty a product having the desired qualities.

Prepared in this way picro-carmine gives very delicate differentiations, and
has the great merit of being less hurtful to most tissues than other aqueous
alkaline carmines. It is a single or a double stain according to the manner
of using it. If the preparations be washed, after staining, with water, it is
a single stain, the colour of the carmine alone appearing ; if they be washed
quickly in alcohol it is a double stain, the yellow coloration of the picric
acid not being dissolved by the alcohol as it is by water. Of course the
washing with alcohol must not be overdone, or the yellow coloration may
be entirely removed. It should be understood that the chief value of picro-
carmine does not lie in its capacity of affording a double stain. The double
stain, if that is all that is wanted, can be just as well or better obtained by
staining first with borax-carmine, or the like, and after-staining with picric
acid. The essential point about picro-carmine is that it is a fairly neutral

RANVIKR'S PICRO-CARMINE. 103

fluid. Ranvier was, in fact, led to add picric acid to ammoniacal solution of
carmine by the desire of neutralising the ammonia, that is all.

The good qualities (especially that of precision and delicacy of stain)
above attributed to picro-carmine apply in their entirety only to Ranvier's
picro-carmine or " picro-carminate of ammonia." The other pseudo-picro-
carmines are in general so inferior as not to take rank as good stains at all.
The reader is in consequence warned against an over-confident faith in them,
and is especially warned against the so-called picro-carmine sold by the
opticians. The true Ranvier's " picro-carminate of ammonia " can only be
prepared with certainty by one process as yet known. This I proceed to
give, and amongst the other formulae set out one or two that may by chance
give rise to the formation of a picro-carminate, and should at all events
afford a useful staining solution (which the majority of these formulae only
do by chance).

For slow staining, dilute solutions may advantageously have 1 or 2 per
cent, of chloral hydrate added to them.

Overstains may be washed out with hydrochloric acid, say 0'5 per cent.,
in water, alcohol, or glycerin.

Preparations should be mounted in balsam, or if in glycerin this should
be acidulated with 1 per cent, of acetic or, better, formic acid.

160. RANVIEE'S Picro-carmine or Picro-carminate of Ammonia.

— The method of preparation employed in the Laboratory of Histology of
the College de France, kindly communicated to myself and Henneguy for
our Traite des Meth. techn. (q. v., p. 451) by M. VIGNAL, one of the
assistants there, is as follows :

Take-
Water . 1000 parts.

Picric acid 20 „

Carmine . . . . . . . 10 „

Ammonia ....... 50 „

Put them into a stoppered bottle and leave them for two or three months
in a warm place. Then put them into a large crystallising dish and let
them putrefy. When the liquid has become reduced by evaporation to four
fifths of its original volume, 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 whether the carmine is
really dissolved. If not, add water and ammonia, and let the solution
putrefy again ; evaporate and examine as before. When you have got your
carmine combined, evaporate the solution to dryness in a stove, and reduce
the picro-carminate to powder.

For staining, dissolve 1 grm. of the powder in 100 grms. of water,
and add a crystal of thymol to prevent the development of mould.

RANVIEE'S Original Formula (Traite, p. 100) was as follows : — To a
saturated solution of picric acid add carmine (dissolved in ammonia) to satu-
ration. Evaporate down to one fifth the original volume in a drying oven ;
and separate by filtration the precipitate, poor in carmine, that forms in the
liquid when cool. Evaporate the mother-liquor to dryness, and you will
obtain the picro-carminate in the form of a crystalline powder of the colour

104 CARMINE AND COCHINEAL STAINS.

of red ochre. It ought to dissolve completely in distilled water ; a 1 per
cent, solution is best for use.

161. Picro-carmine ( Weig erf s formula, Virchow's Archiv, Bd. Ixxxiv, pp.
275, 315; ZooL Jahrb., 1881, p. 40).— Two grins, of carmine are soaked
for twenty-four hours (in a vessel protected from evaporation) in 4 grms.
of ammonia; 200 grms. of concentrated solution of picric acid are then
added, and the whole put away for twenty-four hours more. Small quan-
tities of acetic acid are then added " until the first slight precipitate appeal's
even after stirring." The whole is again put away for twenty -four horn's
more, when it will be found that there has formed a precipitate that can
only partially be removed by filtration ; ammonia is then added drop by drop
at intervals of twenty-four hours, until the solution becomes clear. If the
solution stains too yellow, acetic acid is added ; if it overstains red, a little
ammonia is again added. All badly staining samples of picro-carmine may
be improved in the same way by addition of acetic acid or ammonia.

162. Other FormulsB for Picro-carmine. — GAGE, Am. M. Mic.
Journ., i, 1880, p. 22 ; Journ. Roy. Mic. Soc., vol. iii, p. 501 (very elaborate,
and has not afforded me a soluble carmine). FOL, Lehrb. d. vergl. mik.
Anat., p. 195. RUTHERFORD, Pract. Hist., p. 173. PAUL MAYER, Afitth.
ZooL Stat. Neapel, ii, p. 20. BABEE, Mon. Micro. Journ., xii, p. 48.
PEBGENS, Carney's Siologie cellulaire, p. 92. HOYER, Biol. Centralb., ii,
1882, p. 17. BIZZOZEBO, Zeit. /. wiss. Mik., 1885, p. 539. KLEMENSIE wics,
Sitzb. Akad. Wiss. Wien, Ixxviii, 1878, iii, Juni; Zeit.f. wiss. Mik., i, 1884,
p. 501. CUCCATI, Zeit.f. wiss. Mik., vi, 1, 1889, p. 42.

For Soda-Picro-carmine see LOWENTHAL, Anat. Anzeig., ii, 1887, No. 1,
p. 22 ; Anon.. Journ. Roy. Mic. Soc., 1888, p. 518 ; SQUIRE'S Methods and
Formulae, &c., 1892, p. 35.

163. Ammonia-Carmine. — In my opinion there is no valid excuse for
using ammonia-carmine at all at the present day. The most that can be
said of it is that it gives a good general stain of preparations of central ner-
vous system that have been hardened in chromic acid or a chromic salt. But
for central nervous system the stains described in Part II under the head of
" Special Stains " are what is generally wanted.

If, however, such a stain be used, care should be taken to get rid of the
free ammonia as completely as possible. This may be done by boiling until
the excess of ammonia has evaporated. (So long as free ammonia is present
large bubbles are formed in the fluid, and the latter shows a dark purple
colour. When the free ammonia has evaporated small bubbles appear, and
the solution takes a brighter red tint.)

One per cent, each of carmine and ammonia in distilled water is a good pro-
portion.

But a safer mode of preparation is that of E/ANVIEE, given below.

Staining is best done slowly, in dilute solutions, as many drops of the
stain being added to distilled water as will give a liquid of a pale rose tint.
Sections should remain in the liquid for twenty-four hours. In this way
chromic objects stain well. For sections of encephalon the hardening ought
to have been done in a chromic liquid, and all treatment with alcohol ought

RANVIER'S NEUTRAL CARMINE. 105

£o be carefully avoided until staining is accomplished ; alcohol should not
even be used for wetting the section knife.

Objects stained in strong solutions must be washed out in water, but
not too long, as the stain will wash out too much in time. The colour may
be "set," when required, by treatment with alcohol or a little acetic acid.
Objects stained in the dilute sections recommended will not require washing
out. Overstains may be reduced and made sharp by treatment with HC1
alcohol, as for borax-carmine. The stain is permanent in balsam, or iir
aqueous media containing a little (about 1 per cent.) of acetic or (better)
formic acid.

164. KANVIEE'S "Neutral" Ammonia-Carmine (kindly communi-
cated by Dr. MALASSEZ, see Traite des Methodes techniques, &c., of Lee and
Henneguy, p. 82). — Make a simple solution of carmine in water with a
slight excess of ammonia, and expose it to the air in a deep crystallising dish
until it is entirely dried up. It should be allowed to putrefy if possible.
Dissolve the dry deposit in pure water, and filter.

165. Other Ammonia-Carmines. — BEALE, How to Work, &c.,4th ed.,
pp. 109, 304. BETZ, Arch. mik. Anat., ix, 1873, p. 112 ; GIEEKE, Zeit. f.
wiss. Mik., i, 1884, p. 76 (these two are methods for the preparation of
putrefied carmine, and are highly recommendable, but excessively lengthy).
HUXLEY and MAETIN, Pract. Elemen. Biol., p. 268. FEET, Le Microscope,
p. 167. HOYEE, Biol. Centralb., ii, 1882, p. 17. BOHN (not " BOHM "),
Arch. Anat. u. Phys. (Anat. Abth.), 1882, p. 4. HEIDENHAIN, Arch. f. mik,
Anat., vi, 1870, p. 402.

166. Lithium-Carmine (ORTH, Berlin, klin. Wochenschr., 28, 1883,
p. 421). — Two parts and a half of carmine are dissolved in ninety-seven
parts and a half of saturated solution of carbonate of lithium.

The solution stains with equal readiness alcohol objects and chromic
objects. The stain is diffuse, but becomes restricted to nuclei on treatment
with hydrochloric acid (1 per cent, in 70 per cent, alcohol). The colour is
permanent both in balsam and glycerin. The only advantage of this solu-
tion is the readiness with which it stains tissues that refuse to stain in any
other medium.

HAUG (Zeit.f. wiss. Mik., vii, 1, 1890, p. 47, and viii, 1, 1891, p. 52) has
three elaborate modifications that appear to me wild and uncalled for.

j. Other Aqueous Carmines (Acid and Alkaline).

167. Schweigger-Seidel's Acid Carmine (EANVIEE, Traite, p. 99).
Hamann's Acid Carmine (Intern. Mon. f. Anat. u. Hist., i, 5, 1884 j
Zeit. f. wiss. Mik., ii, 1885, p. 87). Neutral Borax- Carmine (NiziT
FOBOW, Zeit.f. wiss. Mik., v, 3, 1888, p. 337). Neutral Borax-Carmine
(G-EENACHEE, Arch. f. mik. Anat., xvi, 1879, p. 466). HAUG'S modifications
of this, Zeit. f. wiss. Mik., vii, 2, 1890, p. 151, and viii, 1, 1891, p. 52.
"Woodward's Borax-Carmine (see Monthly Micr. Journ.,\\\, 1872, p. 38 ;
Am. Quart. Micr. Journ., i, 1879, p. 220 ; Journ. Roy. Mic. Soc., ii, p. 613)k

106 CARMINE AND COCHINEAL STAINS.

H. Gibbes* Borax-Carmine (see Journ. Roy. Mic. Soc., iii, 1883, p. 390).
Delage's Osmium-Carmine (Arch, de Zool. exp. et gen., iv, ser. 2,
1886 ; Zeit. f. wiss. Mik., iii, 2, 1886, p. 240). Reliefs Carminroth
(see Zeit. f. wiss. Mik., i, p. 91). Perl's Soluble Carmine (see FEET,
Das Mikroskop., 7 Auf., and Zeit.f. wiss. Mik., i, p. 91). Carminic Acid
(see DIMMOCZ, Amer. Natural., xviii, 1884, pp. 324-7 ; and Journ. Roy.
Mic. Soc., 1884, pp. 471-4). Boric Acid Carmine (ARCANGELI, see
Proc.-verb. Soc. Toscana Sc. Nat., 1885, p. 283; and Zeit.f. wiss. Mik.,
1885, p. 377). Boric Acid Alum-Carmine, ARCANGELI, ibid. Sali-
cylic Acid Alum-Carmine, ARCANGELI, ibid. Salicylic Acid Car-
mine, ARCANGELI, ibid. Picric Acid Carmine, ARCANGELI, ibid.
Picric Acid Carmine, MINOT (see WHITMAN'S Methods in Mic. Anat.,
p. 42). Uranium-Carmine, GIERKE, Zeit.f. wiss. Mik., i, 1884, p. 92 ;
SCHMAUS, ibid., viii, 2, 1891 ; cf . Milnchener med. Wochenschr., 1891, No. 8,
p. 147. Carbonate of Soda Carmine, CUCCATI, Zeit. f. wiss. Mik., iv, 1,
1887, p. 50.

B. ALCOHOLIC CAEMINE STAINS.

168. HAYEK'S Paracarmine (Alcoholic Carminate-of-Alumina-
and-Lime Solution) (Mitth. Zool. Stat. zuNeapel,x, 3, 1892, p.
491). — Carminic acid, 1 grm.; chloride of aluminium, 0' 5 grm,;
chloride of calcium, 4 grms.; 70 per cent, alcohol, 100 c.c. Dis-
solve cold or warm, allow to settle, and filter. A light red
liquid, specially adapted for staining in bulk, and the nearest
approach to a substitute for Grenacher's alcoholic borax-
carmine that has yet been discovered.

Objects to be stained should not have an alkaline reaction.
Wash out sections, or objects intended to be sectioned, with
pure 70 per cent, alcohol. Objects intended to be mounted
whole may be washed out with a weak solution of aluminium
chloride in alcohol, or if this be not sufficient, with 5 per
cent, common acetic acid (or 2f5 per cent, glacial acetic acid)
in alcohol.

For staining bulky objects with large cavities, such as
Salpa, the solution should be diluted (with alcohol); and as
this may cause precipitates to form during the staining,
especially if the objects are not very clean, it is advisable to
slightly acidify the dilute solutions.

Paracarmine gives a nuclear stain of a red colour, though
not so fiery red as that of borax-carmine. Its points of
supe-riority over borax-carmine are that it is not alkaline,
therefore less hurtful to tissues; that it is more highh

ALCOHOLIC BORAX-CARMINE. 107

alcoholic, therefore more penetrating ; that it has less tendency
to form granular precipitates in the interior of objects, and
that it keeps perfectly without precipitating.

169. Alcoholic Borax- Carmine (G-KENACHER, Arch. f. mik.
Anat., xvi, 1879, p. 466, et seq.). — Take a concentrated solution
of carmine in borax solution (2 to 3 per cent, carmine to 4 per"
cent, borax) ; dilute it with about an equal volume of 70 per
cent, alcohol, allow it to stand some time, and filter. Or the
mixture of carmine and borax solution is allowed to stand for
two or three days and occasionally stirred ; the greater part of
the carmine will dissolve. To the solution is added an equal
bulk of 70 per cent, alcohol ; the mixture is allowed to stand
for a week, and then is filtered. If on keeping more carmine
is deposited, it must be refiltered.

Preparations should remain in the stain until they are
thoroughly penetrated (for days if necessary), and then be
brought (without first washing out) into alcohol acidulated
with 4 to 6 drops of hydrochloric acid to each 100 c.c. of
alcohol. They are left in this until they are thoroughly pene-
trated, and may then be washed or hardened in neutral alcohol.
Four drops of HC1 is generally enough. Three drops I find
not quite sufficient. The stained objects should remain in the
acidulated alcohol till they acquire a bright transparent look
(three to six hours) .

For delicate objects, and for very impermeable objects, it
may be well to increase the proportion of alcohol in the stain ;
it may conveniently be raised to about 70 per cent. The
washing out, or decoloration, will be enormously facilitated
if picric acid be added to the acidulated alcohol, but in this
case the proportion of HC1 should be reduced. It should not
exceed that of 1 drop of HC1 to 100 c.c. of alcohol, and the
decoloration should be carefully watched, as the stain may
easily be entirely washed out in this mixture. For this reason
the process is not to be recommended in general ; I merely
mention it because it is well that the student should be
acquainted with the reaction.

This stain is probably by far the most popular of any for
staining in the mass. It is easy to use, and gives a most
splendid coloration. But it is not so penetrating as is
commonly supposed, and has the defect of sometimes forming

108 CABMINE AND COCHINEAL STAINS.

precipitates in the cavities of bulky objects which cannot be
removed by washing out. And it must be remembered that
the fluid is alkaline, and therefore not suitable for delicate
cytological work. I believe that some otherwise excellent
cytological work has been vitiated by over-confidence in this
reagent, and will have to be done over again.

170. Alcoholic Hydrochloric Acid Carmine. — In view of the above-
mentioned defects of borax-carmine — defective penetration, and a tendency
to form insoluble granular precipitates in the interior of objects — it is
desirable to possess a powerful staining medium more highly alcoholised and
of acid reaction. Hydrochloric acid carmine possesses these qualities, and
may be useful in the case of objects for which Mayer's paracarmine may
not suffice. It may, for instance, be frequently useful in work on Arthropoda,
especially the marine forms.

GBENACHEE'S receipt (Arch. f. tnik. Anat., xvi, 1879, p. 468) will be
found extremely troublesome by those who are not expert at neutralising.
The following method, due to PAUL MAYEE (quoted from GASSINGS
Manuals per la Technica moderna del Microscopic, first ed., p. 46), is easy
and gives excellent results. Take 100 grms. of alcohol (either absolute or of
any weaker grade), 1 or 2 drops of HC1, and an excess of carmine, and boil
until you get a clear solution, taking care that there remain an excess of
carmine. This ought to give a nuclear stain, without the aid of HC1 for
washing out. If overstaining or diffusion should occur, wash out with
alcohol, very slightly acidulated with HC1.

BEASS (Zeit. f. wiss. Mik., ii, 1885, p. 303) takes 100 c.c. of 70 per cent,
alcohol, 15 drops of HC1, and an excess of carmine. An old formula of
PAUL MAYEE'S (M. T. Zool. Stat. Neapel, iv, 1883, p. 521 ; Journ. Roy.
Mic. Soc. [N. S.], iv, 1884, p. 317), which gives a more powerful stain than
the preceding, is as follows :

Four grms. carmine are dissolved in 100 c.c. of 80 per cent, alcohol with the
addition of 30 drops of concentrated pure hydrochloric acid, and heated for
about half an hour in the water-bath ; the solution is filtered whilst still
hot, and the superfluous acid is carefully removed by the addition of caustic
ammonia, added until the carmine begins to be deposited. This solution
stains very rapidly (embryos of lobsters are stained in about a minute) and
intensely, though diffusely ; the preparations must be washed out with HC1
alcohol if a nuclear stain is required.

The heating of alcohol of so high a grade as 80 per cent, being trouble-
some, not to say dangerous, the process may be modified by dissolving the
carmine in 15 c.c. of water acidulated with the HC1, adding 95 c.c. of 85 per
cent, alcohol, and then neutralising with ammonia.

If it be desired to dilute any of these solutions it should be done with
alcohol, not water, and alcohol should be taken for washing out.

ALCOHOLIC COCHINEAL. 109

OTHER ALCOHOLIC CARMINE STAINS.

171. Alcoholic Boric Acid Carmine (FEANCOTTE, Bull. Soc. Beige
Mic,, 1886, p. 48). — Carmine, 0*4 grm. ; boric acid, 5 grms. ; water, 25 c.c. ;
90 per cent, alcohol, 75 c.c. Boil and filter.

DutilleuPs Pier o-borax-Car mine (Bull. Sci. Dep. Nord, xvi, 1885,
p. 371). — This is quoted here as having the distinction of being the very
worst stain I have ever tried.

Baumgarten makes a " borax-picro-carmine " by adding crystals of
picric acid to Grenadier's solution until it assumes a blood-red colour (Journ.
Roy. Mic. Soc., 1888, p. 676 ; 1889, p. 149).

172. Alcoholic Cochineal (MAYER'S Old Formula, Mitth. Zool.
Stat. Neap., ii, 1881, p. 14). — Cochineal in coarse powder is
macerated for several days in alcohol of 70 per cent. For
each gramme of the cochineal there is required 8 to 10 c.c. of
the alcohol. Stir frequently. Filter, and the resulting clear,
deep red solution is fit for staining.

The objects to be stained must previously be imbibed with
alcohol of 70 per cent., and alcohol of the same strength must
be used for washing out or for diluting the staining solution,
as water, or alcohol of a different strength, gives rise to tur-
bidity and precipitation of colouring matter (the fluid holding
in solution matters that are only soluble in alcohol of exactly
that degree of concentration). The washing out must be re-
peated with fresh alcohol until the latter takes up no more
colour. Warm alcohol acts more rapidly than cold. Over-
staining seldom happens ; it may be corrected by means of
70 per cent, alcohol, containing -^ per cent, hydrochloric or
1 per cent, acetic acid.

Small objects and thin sections may be stained in a few
minutes, larger animals require hours or days. In the latter
case large quantities of the solution must be employed. Very
thin sections and delicate objects are best stained in a very
dilute solution.

A nuclear stain, slightly affecting protoplasm. The colour
varies with the reaction of the tissues, and the presence or
absence of certain salts in them. Crustacea with thick chi-
tinous integuments are generally stained red, most other
organisms blue. The stain is also often of different colours
in different tissue elements of the same preparation. Glands

110 CARMINE AND COCHINEAL STAINS.

or their secretion often stain grey-green. In embryos of
Lumbmcus Kleinenberg found the vessels to stain red, their
contents of an intense blue.

Acids lighten the stain and make it yellowish red. Caustic
alkalies turn it to a deep purple.

The best stains are obtained in the case of objects that have
been prepared with chromic or picric acid combinations, or
with absolute alcohol. Osmic acid preparations stain very
weakly unless they have been previously bleached (§ 575).
The acid must be carefully washed out before staining, or a
diffuse stain will result. • The stain is permanent in oil of
cloves and balsam.

The object for which this stain was imagined is to obtain
an alcoholic staining fluid whose high penetrating power
allows it to be employed in the case of organisms, such as
Arthropoda, whose chitinous investments are but very slightly
permeable by aqueous solutions of carmine.

I have treated this stain at considerable length because I
am convinced that it ought to be better known. It is very
useful in many cases (Annelids, for instance), and indispen-
sable for Arthropoda. On the other hand, it gives a very
poor stain with unsuitable objects, and the majority of the
objects with which the histologist has to do are in this case.
For this reason MAYEK has been led to devise the fluid de-
scribed in the following paragraph, which, containing in
itself the salts necessary for producing a rich and energetic
selectivity of stain, gives good results with all classes of ob-
jects. He almost appears to intend it entirely to take the
place of the old fluid. But I think the old fluid cannot yet
be discarded. It has over the new fluid the (for some cases
considerable) advantage of being more highly alcoholic ; and
it does not contain free acid, so that it can be used with
calcareous structures which it is wished to preserve — which
the new fluid cannot.

173. MAYER'S Alcoholic Cochineal, New Formula (Mitth. ZooL
Btat.Neapel, 10,3, 1 892, p. 498) .—Cochineal, 5 grins. ; chloride
of calcium, 5 grms. ; chloride of aluminium, 0'5 grm. ; nitric
acid of 1'20 sp. gr., 8 drops ; 50 per cent, alcohol, 100 c.c.
Powder the cochineal finely and rub up in a mortar with the
salts, add the alcohol and acid, heat to boiling-point, leave to

NEW ALCOHOLIC COCHINEAL. Ill

cool, leave for some days standing with frequent agitation,
filter.

Use as the old tincture, the objects being prepared and
washed out with 50 per cent, alcohol. The stain is like that
of paracarmine, but not quite so strong and not so sharp.
Mayer only recommends it as a succedaneum of paracarmine.
The object for which it was devised has been explained in
the last paragraph, where also some limitations to its useful-
ness have been indicated.

For the chemical theory involved in the composition of
these tinctures see also the last paragraph, and supra, § 146.

112 H^MATEIN AND OTHER ORGANIC STAINS.

CHAPTER XI.

H^MATEI'N (HJIMATOXYLIN) AND OTHER ORGANIC STAINS.
A. Hsematoxylin and Hsematein.

174. Theory of Haematoxylin Staining. — It appears to be now
thoroughly well established (see NIETZKI, Chemie der organ-
ischen Farbsto/e, Berlin, Springer, 1889, pp. 215—217) that
the active colouring principle of hsematoxylin dyes is h&ma-
tein ; and further, that the haematein of the usual histological
staining solutions is a product of the oxidation of their con-
tained haematoxylin by means of the air to which they are
exposed (see MAYER, " Ueber das Farben mit Hamatoxylin"
in Mitth. a. d. Zool. Station zu Neapel, Bd. x, Heft 1, 1891,
pp. 170 — 186 ; UNNA, " Ueber die Reifung unserer Farbstoffe,"
in Zeit.f. wiss. Mik., viii, 4, 1892, p. 483). This change is
known as " ripening/' and until it has taken place the solu-
tions are not fit to use for staining.

Hitherto it has been the practice to rely (quite uncon-
sciously so far as the chemical theory is concerned) on the
spontaneous absorption by the solutions of oxygen from the
air to effect this " ripening," but it has now been discovered
(by both MAYER and UNNA independently) that nothing is
easier than to bring about the reaction artificially ; all that is
necessary being, for instance, to add to a solution of haerna-
toxylin containing alum a little neutralised solution of per-
oxide of hydrogen. The solution becomes almost instan-
taneously dark blue, " ripe," and fit for staining, thus
definitively confirming the truth of the hypothesis.

Mayer goes further. A solution of pure uncombined
haematein would not afford a. selective stain such as we
require in histology ; it would be at most a dye. The usual
solutions (I am not here speaking of Weigert's or similar
processes) all contain alum, and Mayer holds that the active
agent in them is a compound of haematein with alumina

THEORY OF H^IMATOXYLIN STAINING. 113

(" Hgemate'in-Thonerde " — Haematein-alumina) . He holds that
this salt is precipitated in the tissues ( chiefly in the nuclei) by
certain organic and inorganic salts there present (for instance,
phosphates) ; perhaps also by other organic bodies belonging
to the tissues. Such salts are known to exist in living
tissues. By the process of " fixing" they are fixed there, —
either in the same chemical state, as when such an agent as
alcohol is used for fixing ; cr as fresh compounds formed by
the action of the fixing agent itself, as when such a fixing
medium as sublimate is used.

The chief factor in the composition of these stains is,
therefore — after the colouring substance itself, — alumina, to
which, however, may be added some other bodies, such as
lime, which may play a secondary role (as, for instance, chlo-
ride of calcium does in Kleinenberg's solution) .

It follows that the first difficulty with which the worker
with haBmatoxylin stains has to contend is that of getting his
hasmatoxylin duly oxidised into haematein, in order to the
formation of the desired hsematein- alumina compound, or
lake. If this be done by the hitherto customary process of
leaving the solutions to " ripen " by the action of the air, a
second difficulty arises ; the oxidising process continuing, the
solutions become " over-ripe," the hgematein, through further
oxidation, passes over into colourless compounds, and the
solutions begin to precipitate. They are therefore, in reality,
a mixture in constantly varying proportions of " unripe,"
"ripe," and "over-ripe" constituents (the first and last being
useless for staining purposes), and, in consequence, their
staining power is very inconstant.

These difficulties are only very imperfectly got over by
any of the very various methods that have been proposed
until lately. Now, however, thanks to the important dis-
coveries of MAYER and UNNA, a great step in advance has
been taken.

The great point in Mayer's work is that not haematoxylin,
but hffimatein, should be taken in the first instance for making
the staining solutions. This at once relieves us from the
tedious and uncertain process of " ripening" in the old way.
We have a ripe solution to begin with. A discovery of
Unna's, to be mentioned below, affords a means of preventing
•ripemog " brought about by excessive oxidation.

(ff . ^ 8 '

Vv f*A. ••^n\V\k\ 4

114 H^MATEIN AND OTHER ORGANIC STAINS.

In face of the very great advantages offered by the mode
of preparation worked out by MAYER I have no hesitation in
saying that the old methods ought to be entirely discarded,
and Mayer's formula only, or at least only such formulae as
conform to the principles laid down by him, used instead. If
this be done the methods of Unna will come to be of but
secondary importance ; but they may still be occasionally
useful, and for that reason, as well as for their theoretical
interest, I proceed to set them out in the following paragraphs.
For MAYER'S haematein methods see §§ 179 et seq.

175. UNNA'S Ripening Method (Zeit. f. iviss. Mik., viii, 4,
1892, p. 483). — It has been explained above that the sponta-
neous "ripening" of haematoxylin solutions is due to the
absorption by them of oxygen from the air, and the conse-
quent oxidation of their haematoxylin into haematein. UNNA
has found (as MAYER had found before him) that a freshly
prepared light red alum solution of haematoxylin can be in-
stantly ripened — that is, converted into a dark blue solution
of haematein of even much greater staining power than spon-
taneously ripened solutions — by the simple addition of a little
neutralised peroxide of hydrogen. Such an instantaneously
ripe solution may be conveniently prepared as follows :—
Make a solution of crystallised hasmatoxylin in alcohol, and
another solution of about five times as much alum in water,
and mix the two solutions in a test-tube. Then pour a little
commercial peroxide of hydrogen solution into a watch-glass,
and add a crystal of soda. Remove the soda as soon as a
blue litmus paper remains blue in the solution, pour the solu-
tion into the test-tube, and heat for a moment over [a flame.
The staining bath is now ripe, and must be used for staining
at once, as precipitation of the haematem begins to set in the
moment the state of ripeness is attained.

176. UNNA'S Half-ripe Constant Stock Solution (ibid.). — It is
inconvenient to have to make a fresh solution of haomatoxylin
every time it is desired to prepare a ripened solution for
staining. Unna, therefore, sought for a means of preventing
the continuance of spontaneous oxidation of haomatoxylin
solutions, which, as explained above, leads in course of time
to over-ripeness. The spontaneous oxidation being arrested at
any desired stage, a constant half-ripe solution is obtained,

UNNA'S H JIM ATE IN STAIN. 115

which may be at any moment converted into a totally ripe
one by the process given in the last §.

This result maybe attained by simply adding a reducing
agent to the solution. Various reducing agents are available
for this purpose (see the original paper) ; the most convenient
method is the simple addition of a little sulphur. The fol-
lowing formula is recommended :

Haematoxylin ..... 1

Alum 10

Alcohol 100

Water 200

Sublimed sulphur .... 2

If the sulphur be added to the haematoxylin solution only
when the latter has become somewhat strongly blue, i. e.
after two or three days' time, the stage of oxidation attained
by the solution will be fixed by the sulphur. The solution
in this state may be used for staining. It will not give so
energetic a stain as the solutions totally ripened with peroxide
of hydrogen, and the stain will be somewhat more diffuse, but
washing out with acids will not be necessary. If an energetic
and more purely nuclear stain be desired, the solution should
be treated with neutralised peroxide of hydrogen, as described
in the last § ; sections should be stained in it at once, and
quickly brought into glacial acetic acid (where they should
only remain for a moment), or into HC1 alcohol, for washing
out.

177. General Remarks. — We have the coal-tar colours for
staining sections, and we have carmine and cochineal for
staining in the mass. What, then, do we want haematoxylin
for? The answer is that we sometimes want it for staining
in the mass on account of the faculty it has of readily staining
tissues that have been treated with chromic and osmic mix-
tures. This it does in general better than any carmine or
cochineal. We want it also for some special purposes, such
as staining the Nebenkern and achromatic figure of nuclei,
and for nerve researches and other special histological
objects.

None of the old solutions of haamatoxylin are perfectly
stable ; only one or two are fairly so.

116 HJBMATBIN AND OTHER ORGANIC STAINS.

It has been explained above that when freshly prepared
they stain badly and diffusely ; they ought either to be
"ripened" by Unna's method( § 175) or to be allowed to
" ripen" spontaneously before use. This takes, according to
the nature of the solution, a few hours, or days, or months.
On the other hand, kept solutions easily go bad by precipi-
tating, or becoming acid, or becoming mouldy.

Most of the solutions, when in good staining order, have a
great tendency to over-stain. Over-stains may be corrected
by washing out with weak acids (e. g. (H to O2 or even 0*5
per cent, of hydrochloric acid), but this is not favorable to
the permanence of the stain. If acids be used, it is well to
re-neutralise afterwards with ammonia or bicarbonate of
soda (O'l per cent.).

A better plan is, perhaps, to wash out with alum solution ;
but this frequently requires great patience.

The stain is fairly permanent in balsam, but is sure to fade
a little, and may fade a great deal. If acids have been used
after staining great care should be taken to wash them out
thoroughly before mounting. In aqueous media the stain
cannot be relied on to keep (this refers to the old solutions :
Mayer finds that his haematein preparations have kept well
both in aqueous media and, with certain precautions, in
balsam).

178. HaBmatoxyliii stains in different tones of blue or of red,
according to the composition of the staining solution.

According to WATNEY (see Phil Trans., 1882, p. 1075 ; KBATJSE, Intern.
Zeit. f. Anat. u. Hist., 5, p. 154 ; and M. FLESCH, Zeit.f. wiss. Mik., 1885,
p. 358, from whom I quote) the colour is an intense blue if the solution has
been made with freshly prepared alum, whilst a red tone is obtained if the
solution has been made with old alum. The reason of this is that alum
that has been long kept almost always contains free acid. The red solutions
exhibit a great affinity for connective tissue, and for the granules of "plasma-
cells ; " whilst the blue solutions show a special affinity for mucin and
chromatin. MAYER (Mitth. Zool Stat. Neapel, 10, i, 1891, p. 177) has a
different explanation of this reaction. He finds that solutions containing
only hsematein and aluminium chloride stain mucin with special energy ;
whilst solutions which also contain from 10 to 20 per cent, of magnesium
chloride, nitrate of ammonia, chloride of lithium or of calcium, of sodium,
or of ammonium, do not do so, but are pure nuclear stains.

It has been discovered by LANGHANS (see MAX FLESCH, loc. cit.) that it is
possible to obtain these two elective reactions with one and the same solution.
All that is necessary is to stain with the solution of Delafield, mount the

CONCERNING HJ1MATEIN. 117

preparations in balsam, and expose them for some time to the light. The
reaction is not obtained with glycerin mounts.

In order to get a blue stain in preparations that have come
out red through the acidity of the staining bath, it is usual to
treat them with weak ammonia, in the belief that the blue
colour is restored by neutralisation of the acid that is the
cause of the redness. According to MAYEE, the ammonia
acts not by neutralising the acid, but by precipitating the
alumina, which carries down the hgematein with it (if 110
alumina were present the colour would be purple, not blue).
The same result can sometimes be obtained by merely washing
out with common tap-water, which is usually sufficiently
alkaline, and can be obtained with certainty by treatment, with
bicarbonate of soda or acetate of soda or potash. And this
is the preferable course, as ammonia is certainly a dangerous
thing to treat delicate tissues with. Of course this is a dif-
ferent question from that of neutralising with an alkali tissues
that have been treated with an acid to correct over-staining.
Here the neutralisation may be indicated in the interest of
the preservation of the stain, or of the tissues themselves.

179. Concerning Hsematein. — The following is taken from the
two papers of Mayer quoted above, §§ 144, 174. Hsematein
occurs as a dark green metallic mass, red by transmitted light,
which may be rubbed up into a violet powder or small red
crystals. It is entirely, though with difficulty, soluble in
distilled water and in alcohol, giving a yellowish-brown
solution, which remains clear on addition of acetic acid.
Alkalies dissolve it with a blue-violet tint.

It is now found in commerce ; but Mayer has hitherto only
been able to procure it in a pure state from G-EIGY AND Co.,
in Bale. But there is also found in commerce an ammonia-
compound of haematein — Hdematein-Ammoniak, also known
in commerce as Haematemum crystallisation; this may be
obtained in a sufficiently pure state from E. MERCK, of
Darmstadt.

This is somewhat more easily soluble in both water and
alcohol than hsematein is, and does quite as well for staining
purposes. The histologist can easily prepare it for himself as
follows :

Dissolve 1 grm. of haematoxylm with the aid of heat in 20

118 KEMATEIN AND OTHER ORGANIC STAINS.

c.c. of distilled water, filter if necessary, add 1 c.c. of
caustic ammonia (of O875 sp. gr.), and bring the purple liquid
into a capsule of sucli dimensions that its bottom be not covered
to a depth of more than half a centimetre. Let the liquid
evaporate at the ordinary temperature and protected from
dust. The dry product will be haematein-ammonia, about
equal in weight to the haematoxylin taken in the first in-
stance. The evaporation should not be hastened by heat, as
this may give rise to the formation of substances that are
insoluble in alcohol. The preparation should not be touched,
until it is dry, with any other instruments than such as are
made of glass, porcelain, or platinum.

B. Alumina Hsemate'in Stains.

180. HAYEK'S Haemalum (Haematein-alum Solution, Hsema-
laun; MAYER, Mitth. a. d. Zool. Stat. zu Neapel, 10, 1, 1891,
p. 172; see also Zeit. f. wiss. Mik., viii, 3, 1891, p. 338, a
very exact report, but somewhat short considering the im-
portance of Mayer's paper). — One grm. of the colouring matter
(either haematein or the ammonia salt) dissolved with heat
in 50 c.c. of 90 per cent, alcohol, and added to a solution of
50 gr. of alum in a litre of distilled water. Allow the mix-
ture to cool and settle, filter if necessary, and add a crystal
of thymol to preserve from mould.

A dark liquid of about the tint of Grenacher's borax-car-
mine. It 'answers to Bohmer's haematoxylin,* stains at least
as well, either at first, for it is ripe from the beginning, or later.
It may be used either in the concentrated form or diluted.
Concentrated, it stains almost instantaneously (sections have
been stained by merely pouring the liquid over them).
Diluted twentyfold with distilled water it will still stain
through the tentacles of a Tubularia in an hour. (Spring
water or tap water containing lime must not be used for
diluting ; perhaps weak solution of alum in distilled water is
the best means of all.) After staining, sections may be
washed out either with distilled or common water. The
solution is admirable for staining in bulk. Large objects will,

* As with Bohmer's formula, it is not necesssaiy to conform exactly to
the proportions given, and a rough and ready hsBmaluin solution may be at
any time extemporised by adding a few drops of alcoholic solution of
hsematein to an alum solution of any desired strength.

MAYER'S H^IMACALCIUM. 119

however, require twenty-four hours' staining, and should be
washed out for the same time (this should be done with 1 per
cent, alum- solution if a sharp nuclear stain be desired).

Mayer's preparations have kept well in glycerin (care
being taken not to have it acid), also in balsam. It is to be
noted that if oil of bergamot be used for clearing, it must be
thoroughly removed by means of oil of turpentine befoFe
mounting ; and that oil of cloves is dangerous.

Haemalum may be mixed with alum-carmine, Saurefuchsin,
or the like, to make a double-staining mixture ; but it seems
preferable to use the solutions in succession.

181. MAYER'S Acid Haemalum (ibid., p. 174, note). — This
is haemalum with 2 per cent, glacial acetic acid (or 4 per
cent, common acetic acid). To be used as the last, wash-
ing out with ordinary water in order to obtain a blue-
violet tint of stain. It has the advantage of being a perhaps
even more precise nuclear stain than the last formula ; and
further, the solution does not precipitate through decompo-
sition caused by the ammonia of the air and the alkali of
the glass of bottles, which the simple hsemalum solution does
to a certain extent (though this does not affect its staining
power).

182. MAYER'S Hsemacalcium (ibid., p. 182). — Hasmatein (or
its ammonia-salt), 1 grm.; chloride of aluminium, 1 grm. ;
chloride of calcium, 50 grms. ; glacial acetic acid, 10 c.c. (or
common acetic acid, 20 c.c.) ; 70 per cent, alcohol, 600 c.c.
Eub up finely together the first two ingredients, add the
acid and alcohol, dissolve either cold or with heat ; lastly add
the chloride of calcium.

A reddish-violet liquid, imagined as a substitute for KLEIN-
ENBERG'S solution, in view of the many disadvantages of the
latter (see below, § 184). To be used in the main as Kleinen-
berg's. If the objects stain in too red a tone they should be
treated with a solution (of about 2 per cent.) of chloride of
aluminium in alcohol, or with a 0'5 to 1 per cent, solution of
acetate of soda or potash in absolute alcohol, or with bicar-
bonate of soda (see above, § 178).

The solution is not perfectly stable, but in course of time
(Mitth. a. d. Zool. Stat. Neapel, 10, 3, 1892, p. 499) turns
blue and precipitates. To avoid this the mixture should be

120 HJEMATEIN AND OTHER OUGANIC STAINS.

made up in two separate bottles, each containing half of
the alcohol and of the acid, and one containing besides all
the calcium chloride, the other all the haematein and all the
aluminium chloride ; equal quantities being taken from each
when required for staining.

With certain objects this solution does not penetrate well,
the stain being confined to their superficial parts. This may
be remedied by acidifying the solution, or, which is better,
by leaving the objects for some time before staining in acid
alcohol. Anyway, objects ought NOT to have an alkaline re-
action. If these precautions be taken, it will not be necessary
to use acid for washing out. For some objects also (e. g.
Hydroida) the penetrating effect is enhanced by diluting the
solution with one third vol. of glycerin, or by increasing the
proportion of aluminium chloride up to about eight times
that of the haematein.

This solution is not recommended as giving as good results
as haemaluin, — as a stain it is distinctly inferior ; and Mayer
is of opinion that no alcoholic haematein solution can be made
to give as precise a stain as the aqueous solutions, for he
holds that the watery nature of the menstruum is of itself of
a beneficial effect so far as regards the quality of the stain.
He recommends it merely as a substitute for Kleinenberg's
(in cases in which an alcoholic haematem stain seems indi-
cated), as being convenient, easy to prepare, and constant in
its effects, none of which qualities belong to Kleinenberg's
formula (see below, § 184).

183. MAYER'S Nitrate of Ammonia Haematein Stain (Mitth.
a. d. Zool. Stat. zu Neapel, 10, 1, 1891, p. 183, note) ; HENKING
(Zeit. f. wiss. Mik., viii, 3, 1891, p. 340) has proposed for this
the name of Haemammon). — Haemalum, 10 c.c. ; 70 percent,
alcohol, 10 c.c. ; nitrate of ammonia, 5 grms. Dissolve ; let
the solution stand overnight for the excess of alum to crystal-
lise out, and filter.

Very good for small objects (Mayer has not tried large
ones), but does not give so good a stain as hasmalum ; so
that, of course, its only raison d'etre is that, like haemacalcium,
it is an alcoholic solution.

184. Kleinenberg's Hsematoxylin (Quart. Journ. Mic. Sci., Ixxiv,
1879, p. 208). — Prepare a saturated solution of calcium chloride in 70 per

KLEINENBERG'S H^MATOXYLIN. 121

cent, alcohol, with the addition of a little alum ; after having filtered, mix a
volume of this with from 6 to 8 volumes of 70 per cent, alcohol. At the
time of using the liquid pour into it as many drops of a concentrated solu-
tion of haamatoxylin in absolute alcohol as are sufficient to give the required
colour to the preparation, of greater or less intensity according to desire.

MAYER (Mitth. a. d. Zool. Stat. zu Neapel, 10, 1, 1891, p. 174) has an
elaborate criticism on this formula, from which I extract the following : —
The object of the chloride of calcium is explained by Kleinenberg to be fEo "
setting up of diffusion currents between the alcohol in the tisssues and the
external staining medium, so as to facilitate the penetration of the latter.
Mayer himself does not admit this; he holds that it acts chemically, and that
its effect is to make the stain more precise. I gather (though this is not
quite clear) that he holds that it acts in this way both as a calcium salt, and
also, after double decomposition, with the alum, as aluminium chloride, the
presence of this salt being the most important factor in the production of
the stain. Now Kleinenberg's formula does not efficiently provide for this
double decomposition and formation of aluminium chloride. For cold 70
per cent, alcohol dissolves hardly any alum,* so that if the alcohol preserved
its strength when the calcium chloride is dissolved in it, it would dissolve
hardly any alum, and consequently hardly any aluminium chloride could be
formed. But as calcium chloride contains about 50 per cent, of water of
crystallisation the alcohol becomes gradually weaker, and in consequence
dissolves more chloride of calcium, until at last you get a dense liquid con-
taining 70 per cent, of the salt. If now alum be added to this, you get a
precipitate of gypsum which envelops the crystals of alum, and protects
them so that they no longer dissolve. And at the end the liquid contains
only a mere trace of aluminium chloride. If you take hot alcohol you will
dissolve more alum and obtain more aluminium chloride, but you will also de-
compose much of your alum by the heat, and thus get a strongly acid liquid,
not fit for staining (the sulphuric acid set free from the alum combines
with the calcium chloride, forming gypsum and setting free hydrochloric
acid).

A further point is the vagueness of Kleinenberg's formula as regards the
amount of hsematoxylin to be added to make the staining bath. If too much
be taken, as often happens, precipitates will be formed on the surface of the
objects, hindering the penetration of the stain, and obliging you to wash out
with acidified alcohol to remove them.

It was suggested long ago by MAYER (Mitth. a. d. Zool. Stat. zu Neapel,
2, 1880, p. 13) that it might be more convenient to avoid the douple decom-
position by taking aluminium chloride in the first instance instead of alum.
And, accordingly, two formulae founded on this principle have been given
by DIPPEL (in his Handbuch der Mikroskopie). But these formulae both
omit the calcium chloride, and give in consequence a blue- violet liquid which
furnishes a diffuse stain (as is the case with all solutions containing a rela-
tively large proportion of aluminium salt [chloride or nitrate] with relatively
little haematei'n). This makes it necessary to over-stain strongly and wash out

* Boiling 70 per cent, alcohol does not dissolve 0'5 per cent, of alum,
almost all of which falls out on cooling.

122 H&MATBIN AND OTHER ORGANIC STAINS.

with acid. A good staining solution should not be blue-violet ; if it be, it
should be corrected by cautious addition of acid. But it is better to avoid
having an excess of aluminium salt.

Mayer then gives some hints concerning the ingredients of staining solu-
tions such as we are here considering. Aluminium chloride should not be
damp. It should have a strongly acid reaction. The alcohol and the vessels
used should not have an alkaline reaction. The test for this is that a weak
solution of haematein in the alcohol should not turn purple ; or that a solu-
tion of 1 part each of hsematein and aluminium chloride in 100 parts of
alcohol being added to the alcohol, in the ratio of 1 : 100, should not precipi-
tate in the course of twenty-four hours. The calcium chloride should have
a neutral or feebly alkaline reaction, and should not be damp.

In view of the above criticism, the correctness of which, in the main at
least, will be acknowledged by all who have worked with Kleinenberg's
solution, I think it is quite time that it should be discarded, and Mayer's
haemacalcium used instead in all cases in which a powerful, alcoholic ha3ma-
tein stain is indicated (e. g. for staining in bulk large impermeable objects
that have been fixed in osmic or chromic mixtures).

If, however, it be used, the following points should be observed : — The solu-
tion should be allowed to ripen before using (Kleinenberg's practice was the
contrary of this, and, I conceive, erroneous). Small objects are best stained
slowly with a very dilute solution. If it be required to dilute a solution
already prepared for staining, this should not be done with alcohol, which
may easily cause precipitates to form on the tissues, but with the above-
described solution of alum in calcium chloride solution. Over-stains should
be washed out with acidulated alcohol. Either oxalic or (\ per cent.) hydro-
chloric acid may be used, and the specimens allowed to remain in them until
they begin to acquire a reddish hue. The acid is then removed by pure
alcohol, which restores the pure blue of the stain. It must be very
thoroughly removed in order to ensure the permanence of the stain.

For large or impermeable objects immersion for days in a very strong
solution may be necessary for staining. Osmium and chromic acid objects
stain sufficiently.

185. Delafield's Hsematoxylin (Zeit. f. wiss. Hik., ii, 1885, p. 288).—
The history of this formula is as follows : — It had long been in use in the
Institute of Pathology at Heidelberg, when it was communicated by Pfitzner
to Flemimng, who published it and particularly recommended it in his
Zellstz., &c., p. 388, 1882. Flemming then attributed the formula to
Grenacher, and in consequence the stain went for years by the name of
" Grenadier's ha3inatoxylin." Later on Flemming discovered that this
attribution was erroneous, and attributed the formula to Prudden ; and in
consequence it was thenceforth known for some time as " Prudden's hsema-
toxylin." In 1885 matters were set right by Prudden's explaining that the
stain was the invention of Delafield, and publishing the correct formula here
quoted (in the formula as published by Flemming the proportions are some-
what different).

To 400 c.c. of saturated solution of ammonia- alum* add
* Ammonia-alum dissolves in about 11 parts of water.

BOHMEE'S HJ:MATOXYLIN. 123

4 grms. of hgematox. crist. dissolved in 25 c.c. of strong alcohol.
Leave it exposed to the light and air in an unstoppered
bottle for three or four days. Filter, and add 100 c.c. of
glycerin and 100 c.c. of methylic alcohol (CH40). Allow the
solution to stand until the colour is sufficiently dark, then filter
and keep in a tightly stoppered bottle.

This solution keeps well, — it may be said to keep for years.
It is well to allow it to ripen for at least two months before
using it.

This famous solution is certainly one of the best hasmatoxy-
lin stains yet published. It is extremely powerful, and when
properly used very precise. For staining, enough of the
solution should be added to pure water to make a very dilute
stain ; and even then care should be taken not to leave objects
too long in the fluid.

BUTSCHLI (Unters. ub. mikroscopische Schdume u. das Proto-
plasma, &c., 1892; Zeit. f. wiss. Mik., ix, 2, 1892, p. 197)
recommends, under the name of "acid haBmatoxylin," solu-
tion of Delafield very strongly diluted, and with enough acetic
acid added to it to give it a decidedly red tint. This gives a
sharper and more differentiated nuclear stain than the usual
solution.

186. Bohmer's Haematoxylin (Arch. f. mik. Anat., iv, 1868,
p. 345; Aerzt. Intelligenzbl., Baiern,1865, p. 382). — Make (A)
a solution of haematox. crist. 1 part, alcohol (absolute) 12
parts, and (B) alum 1 part, water 240. For staining, add two
or three drops of A to a watch-glassful of B.

The staining solution ought not to be made up at the moment
of using, but should be made up beforehand and allowed to
ripen for some days (Mayer says at least two months). The
alcoholic solution of haematoxylin may be kept in stock; it
becomes brown, but does not lose its properties.

Washing out may be done with a 0*5 per cent, solution of
alum in water, or with acids if desired.

This formula gives perhaps a finer stain than Delafield's,
but, on account of the uncertainty of the ripening process,
is not so certain in its results.

187. Ranvier's Haematoxylin (Comptes rend. Ac. Sc., 2 sem., t. xcv,
p. 1375). — If solutions prepared according to the formula of Bohmer be kept
for some weeks they will be found to furnish an abundant precipitate.

124 H/EMATEIN AND OTHER ORGANIC STAINS.

Ranvier re-dissolves this precipitate in 1 per cent, solution of alum, and
employs the solution for staining sections of epidermis that have been
hardened in bichromate of potash. The sections should remain twenty-four
hours in the liquid. Nuclei are then found stained light violet, the granules
of ele'idin dark violet. CONTEJEAN (Bull. Soc. Philomath. Paris, 8, 3»
1891, p. 117) has given a receipt for preparing this product by precipitating
solution of Bohiner with baryta water. (Cf. Mayer, in Mitth. a. d. Zool.
Stat. zu Neapel, 10, 3, 1892, p. 500.) The stain is a very fine one ; but
critically examined, these methods appear to be only roundabout methods
of getting a solution of hrematein in alum.

188. Ehrlich's Acid Hsematoxylin (Zeit. f. iviss. Mik., 1886,
p. 150). — The ordinary (alum) haematoxylin staining solutions
easily decompose, giving rise to a blue precipitate which is
formed by the splitting up of the alum into free sulphuric acid
and a basic, lake-forming compound of alumina. By adding
to a solution an appropriate acid this decomposition may be
prevented. The end may be attained by acetic acid. Take —

Water 100 c.c.

Absolute alcohol . . . 100 „

Glycerin 100 „

Glacial acetic acid . . . 10 „
Haematoxylin .... 2 grammes.
Alum in excess.

Let the mixture ripen in the light (with occasional admission
of air) until it acquires a dark red colour. It will then keep,
with a perfectly constant staining power, for years, if kept in
a well-stoppered bottle. Sections are stained in a few minutes.
The stain is also very appropriate for staining in bulk, as
over-staining does not occur.

In order to get a blue stain with this acid solution, the
stained objects should be treated as directed supra, § 178,
but common tap-water may suffice.

For double-staining, either acid colouring matters (" Farb-
sauren"), such as eosin, or " basic colouring matters" ("Farb-
basen"), may be added to the solution.

This is one of the most important of the alum hsema-
toxylins.

188a. Ammoniated Hsematoxylin (SQUIRE, Methods and For-
mulae, &c., p. 24). — "Haematoxylin, 2 grms. ; ammonium
carbonate, 0'4 grm. ; proof spirit, 40 c.c. Dissolve the am-
monium carbonate and the haematoxylin in the proof spirit

IODINE H^EMATOXYLIN. 125

and expose to the air in a shallow dish for twenty-four hours ;
make up the volume to 40 c.c. with proof spirit (warming if
necessary to dissolve any separated crystals), and add ammonia
alum, 2 grms., dissolved in distilled water, 80 c.c. ; glycerin,
100 c.c. ; rectified spirit, 80 c.c.; glacial acetic acid, 10 c.c.
This solution requires no ' ripening/ and is ready to be
diluted for use (1—10)."

This ha3matoxylin is the one most recommended by Squire
after extensive experimentation. I regret that up to the
present I have not had time to make a trial of it, but have no
doubt that it is excellent. Critically examined it is seen to
correspond to Mayer's acid haemalum, with the addition of
alcohol and glycerin. I apprehend that the formula was
arrived at without knowledge of Mayer's work on the subject,
in which case the agreement between the two experimenters
is very satisfactory. I am doubtful as to the advisability of
the addition of alcohol and glycerin to alum-haBmatein solu-
tions. I suppose they help to make them keep, but do they
not impair the quality of the stain ?

189. Benaut's Glycerin Hsematoxylm ("Glycerine Hema-
toxylique ; " Arch, de PhysioL, 1881, p. 640).— Make a satu-
rated solution of alum in strong glycerin. Add drop by drop
about a quarter of a volume of concentrated solution of haema-
toxylin in alcohol. (If you add an excess of hsematoxylin the
liquid will become turbid, and you must then add more solu-
tion of alum in glycerin until the turbidity disappears.)
Filter, and leave the solution exposed to the light and air for
some weeks, until it can be perceived by the smell that it no
longer contains any alcohol. Filter.

Sections are stained in this solution in a few minutes. But
it can be used in another way, which is an original and really
valuable method. If sections or other objects be mounted
in a drop of the solution, they will, after a few weeks, be found
stained and the glycerin decoloured. Such objects keep the
stain for years.

190. Iodine Haematoxylin (CuccATi, Zeit. f. iviss. Hik., v, 1,
1888, p. 55). — Dissolve 25 grammes of chemically pure potassic
iodide in 25 c.c. of distilled water. Pour this solution gradu-
ally and with constant agitation into 75 c.c. of absolute alcohol

126 H^MATEi'N AND OTHER ORGANIC STAINS.

contained in a stoppered bottle. Close the bottle thoroughly.
Rub up in a mortar 75 eg. of crystallised haematoxylin with
6 grammes of chemically pure roche alum,* and add 3 c.c. of
the iodine solution. Keep the mixture agitated, and add gra-
dually the rest of the iodine solution ; then replace the whole
in a well-stoppered bottle. Agitate for some time, in order to
get the alum to dissolve, and let stand for ten to fifteen hours.
Then shake well and filter, taking the usual precautions
against evaporation of the alcohol, and preserve in a well-
stoppered bottle.

Objects should be left in the liquid for ten hours, then
well washed in water and mounted in glycerin, or washed in
alcohol and mounted in balsam.

The solution is stated to be perfectly stable and to give a
pure chromatin stain, and to be well adapted for staining in
the mass, as it never over-stains.

191. Iodine Haematoxylin (SANFELICE, Journ. de Hicrogr.,
xiii, 1889, p. 335; Journ. Eoy. Mic. 8oc., 1889, p. 837).—
Dissolve 0*70 grm. hasmatoxylin in 20 grms. absolute alcohol,
and 0*20 grm. alum in 60 c.c. distilled water. Add the first
solution, drop by drop, to the second. Expose the mixture to
the light for three or four days, add ten to fifteen drops of
tincture of iodine, agitate, and allow to stand for some days.
Stain for twelve to twenty-four hours, and wash out for the
same time in 90 per cent, alcohol, acidulated with acetic
acid. The fluid keeps well.

Mayer (Mitth. Zool. 8tat. Neapel, 10, 1, 1891, pp. 178,
182) remarks on this that it is essentially aBohmer's solution
with somewhat more alcohol, and stains well when ripe, the
function of the iodine being to prevent the formation of pre-
cipitates of colouring matter on the surface of the objects,
and not to directly increase the penetrating power of the
stain.

192. Other Aqueous Alum-Haematoxylins. — AENOLD, see Quart.
Journ. Mic. Sci., 1878, p. 86. MITCHELL, see Journ. Roy. Mic. Soc., 1884,

* Roche alum, or Roman alum (allume di rocca, alun de roche, alumen
rubrum verum, and other synonyms), is an alum originally imported from
Civita Vecchia, and much esteemed by dyers from being nearly free from
iron-alum. That now sold for it in England is ordinary alum coloured with
Venetian red, Armenian bole, or rose-pink (alumen rubrum spurium). See
COOLEY'S Cyclopaedia of Pract. Receipts, s. v. " Alum, Roman."

HKIDENHAIN'S H^MATOXYLIN. 127

p. 811 — a complicated method of treating logwood. HICKSON, see Quart.
Journ. Mic. Sci., 1885, p. 244 — a still more complicated method. COOK, see
Journ. of Anat. and Phys., 1879, p. 140 — a sulphate of copper solution.
HATJG, see Zeit. f. wiss. Mik., viii, 1, 1891, p. 51 — an acetate of alumina
solution.

c. Other Hdemate'in Stains.

193. Heidenhain's Haematoxylin (Arch. /. mik. Anat., 1884,
p. 468, and 1886, p. 383).— Stain for twelve to twenty-four
hours in a \ per cent, solution of haematoxylin in pure water
(distilled water only should be used). Soak the objects for
the same length of time in a 0'5 per cent, solution of neutral
chromate of potash. Wash out the excess of chromate with
water, and treat further as desired.

The above is a slightly modified form of the original pro-
cess, in which staining was done in a stronger hsernatoxylin
solution (O5 to 1 per cent.), and bichromate was used for
washing out instead of neutral chromate. The more recent
process gives a sharper chromatin stain.

The stain succeeds best with alcohol or picric acid objects,
but it will succeed with chromic objects if they have been very
well washed.

Objects that have been fixed in corrosive sublimate ought
to be very carefully washed out with water (many hours in
running water), as neutral hasmatoxylin forms a black preci-
pitate with the excess of sublimate that remains after wash-
ing out with alcohol (see TOENIEE, in Arch. f. mik. Anat., 1886,
p. 181).

The stain is black to grey (haematoxylin forming with
chromic salts a black compound) . It is a sharp stain, remark-
ably rich in detail.

The process is one well adapted to staining in the mass.
Perhaps its greatest advantage lies in the fact that you can
decolour the objects to any extent by prolonging the washing
in the chromate.

The method may be varied by washing out after staining
with alum solution (1 per cent.) instead of a chromate. In
this case the stain will be blue.

194. Apathy's Modification of Heidenhain's Process (Zeit. f.
wiss. Hik., v, 1, 1888, p. 47). — This is an alcoholic method.

128 ILEMATEJN AND OTHER OEGAN1C STAINS.

Stain in a 1 per cent, solution of heematoxylin in 70 or 80 per
cent, alcohol. Wash out (for " thin" sections, i. e. sections of
10 to 15 n, half the time of staining — for "thicker" sections
of 25 to 40 JJL twice the time of staining) in 1 per cent, solution
of bichromate of potash in 70 to 80 per cent, alcohol.

The bichromate solution is conveniently prepared by mixing
one part of a 5 per cent, aqueous solution with about four
parts of 80 to 90 per cent, alcohol. The mixture should be
made immediately before using, and should be kept from the
light (light precipitates it) during the process of decolora-
tion, and should also be changed for fresh several times
during the process. After the differentiation of the colour
has been accomplished, the objects should be thoroughly
washed (still in the dark) in several changes of 70 per cent,
alcohol.

Preparations made in this manner are more transparent and
better preserved than those made by Heidenhain's process.

For staining celloidin series of sections, Apathy also (Zeit.
f. wiss. Mik.,vi,2, 1889, p. 170) recommends the following pro-
cedure : — Stain in the haematoxylin solution as above for ten
minutes ; then remove the excess of haematoxylin fluid from
the sections by means of blotting-paper, and bring the series
for five to ten minutes into 70 per cent, alcohol containing
only a few drops of a strong (5 per cent.) solution of bichro-
mate. This must be done in the dark. If the heematoxylin
be not removed with blotting-paper as described, the celloidin
will take the stain. The sections should appear steel-blue to
steel-grey.

195. MALLOEY'S Phospho-molybdic Acid Hsematoxylin (Anat.
Anzeig., 1891, p. 375 ; see also Zeit.f. wiss. Mik., viii, 3, 1891, p. 341).—
One part of 10 per cent, phospho-inolybdic acid solution, 1 part haematoxylin,
100 parts water, and 6 to 10 parts chloral hydrate. Let the solution ripen
for a week in sunlight, and filter. This stain is recommended for prepara-
tions of central nervous system, but may perhaps be useful in other cases.
Sections should be stained for from 10 minutes to 1 hour, and washed out in
two or three changes of 40 to 50 per cent, alcohol. Dehydrate and mount
as usual. The celloidin remains colourless. The stain is blue, and in its
general effect something like a nigrosin stain. Besides ganglion-cells and
glia-cells, axis-cylinders are stained. It is necessary that the solution be
saturated with haematoxylin in order to obtain the best results ; if a good
stain be not obtained at once, more hiematoxylin must be added.

196. Iron Haematoxylin (BUTSCHLI, Unters. fiber mikrosko-

IRON H^MATOXYLIN. 129

pische Schdume u. das Protoplasma, &c., 1892; Zeit. f. wiss.
Mik., ix, 2, 1892, p. 197). — Sections treated with a weak
brown aqueous solution of ferric acetate, washed with water,
and stained in O5 per cent, aqueous solution of hsematoxylin.
This treatment gives a blue-black or brown-black stain of
extraordinary intensity. The process was used by Biitschli
for staining sections, 1 /j. in thickness, of Protozoa.

197. Iron Haematoxylin (M. HEIDENHAIN, "Uber Kern und
Protoplasma," in Festschr. Herrn Geheimr. A. v. Kolliker, fyc.j
gewidm., 1892, p. 118; Zeit. f. wiss. ffik., ix, 2, 1892, p. 204).
— Sections are treated from half an hour to at most two or
three hours with a 1*5 to 4 per cent, solution of aminonio-sul-
phate of iron: (NH4)2Fe2(SOJ4. They are then washed with
water, and stained for half an hour to twelve hours in an
aqueous solution (of about 0'5 per cent.) of hsematoxylin.
They are then rinsed with water, and again treated with the
iron solution, which slowly washes out the stain. As soon as
a satisfactory differentiation has been obtained, the prepara-
tions are washed for a quarter of an hour in running water,
and mounted. The results differ according to the duration
of the treatment with the iron and the stain. If the baths
have been of short duration, viz. not more than half an hour
in the iron and as much in the stain, blue preparations will be
obtained. These show a very intense and highly differen-
tiated stain of all nuclear structures, achromatic as well as
chromatic, cytoplasmic structures being pale. If the baths
in the iron and in the stain have been prolonged, and the
subsequent differentiation in the second iron bath also duly
prolonged, black preparations will result. These show
chromosomes stained, central and polar bodies stained in-
tensely black, cytoplasm sometimes colourless, sometimes
grey, in which case achromatic spindle-fibres and cell-plates
are stained, connective-tissue fibres black, red blood-corpuscles
black, micro-organisms sharply stained.

198. Weigert's Haematoxylin. — This method, which is the
inverse of Heidenhain's (§ 193), is only applicable to nerve-
tissues, and therefore will be described in the chapter on
« Nerve Methods" in Part II.

199. Minot's Haematoxylin Methods (Zeit.f. wiss. Mik., iii, 2, 1886,

9

130 HJIMATEIN AND OTHER ORGANIC STAINS.

p. 177). — Minot's account of these is as follows : — " They may be employed
with sections of tissues hardened in various ways, and need not be confined
to Muller's fluid or chromic acid specimens (as in Weigert's process). The
sections are soaked first in a salt solution for ten to fifteen minutes. The
following aqueous salt solutions seem to be the most valuable : — Alum, 2 per
cent. ; chromic acid, 1 per cent. ; bichromate of potassium, 5 per cent. ; ace-
tate of copper, nearly saturated. After soaking in one of these, the section
is passed quickly through distilled water, and placed at once in Weigert's
hsematoxylin (1 part of the crystals in 10 parts alcohol plus 90 parts water),
and may be left a short time for direct coloration, then washed and
mounted, or a longer time until they become black, and are to be washed out
by Weigert's iron solution (water 100, borax 2, ferricyanide of potassium 2^).
"The sections ought to be moved about constantly in the iron solution, other-
wise the colour will be extracted irregularly. The copper hsematoxylin goes
out very rapidly, so that with that stain it is better to dilute the iron solu-
tion with twice its bulk of water before placing the sections in it. After
the iron solution the sections must be washed very thoroughly in water, to
avoid further fading out, from which one is not entirely secure until the sec-
tions are actually mounted in balsam.

"These methods are all merely modifications of Weigert's, Heidenhain's,
and Bohmer's methods."

D. Other Organic Stains.

200. Alizarin, so far as I am aware, is only used for Nerve-Centres.
See Part II.

201. Purpurin is useful for cartilage and muscle. It is solu-
ble in a boiling aqueous solution of alum, from which it normally
precipitates on cooling, but may be prevented from doing so by
the addition of a certain proportion of alcohol. The employ-
ment of an alum solution as a vehicle for the colouring matter
has the advantage, at least so far as cartilage is concerned,
of fixing the cellular elements at the same time that they are
stained. (Ranvier found that alum in a solution of 5 — 1000
was the best of all fixing agents for cartilage-cells — Traite,
p. 279.)

KANVIER'S formula (Traite technique, p. 280). — 200 grms.
of water and 1 of alum are boiled in a porcelain capsule ;
purpurin rubbed up in water is added, and the boiling con-
tinued. The purpurin being dissolved to saturation (this is
ensured by taking care to have an undissolved excess in the
capsule), the solution is filtered hot into a flask containing
60 c.c. of alcohol (36° Cartier = 90 per cent.).

There is thus obtained a solution of an orange-rose colour,
presenting a marked degree of fluorescence.

PURPURIN. 131

(As regards the quantity of alcohol to be taken, Duval
writes that it should always be one fourth in volume of the
total mixture — Precis de Technique histologique, p. 221.)

The solution does not keep well for more than a few weeks.

Sections of fresh cartilage are to be placed in a small
quantity (only a few cubic centimetres) of the solution, and
after remaining there twenty-four to forty-eight hours are
washed in water and mounted in glycerin. The stain is
nuclear, the matrix remaining almost colourless. Duval (loc.
cit.) states that this stain has a special selective action on
sections of central nervous system (especially spinal cord)
obtained from tissues hardened in bichromate of ammonia
(2 — 1000), and mounted, after staining for forty-eight hours,
in Canada balsam. The nerve cells and processes, axis-cylin-
ders, and fibres of connective tissue are unstained; but the
nuclei of connective tissue and of the capillaries are stained
red.

GTRENACHEK'S formula (Arch. f. mik. Anat., xvi, 1879, p. 470).
—In 50 cubic centimetres of glycerin (pure or diluted with
very little water) dissolve from 1 to 3 per cent, powdered
alum; add a knife-pointful of purpurin, and boil. (Alcohol
must not be added.) Let the orange-coloured fluorescent
solution stand for two or three days, and then filter.

A nuclear stain ; ten to thirty minutes generally suffice to
produce good staining. The solution is stable, which Grenacher
finds that Ranvier's solution is not, the latter precipitating
after a few days.

202. Indigo. — Indigo is employed in histology in the form of solutions
of so-called indigo-carmine, or sulphindigotate of soda or potash. The
simple aqueous solution gives a diffuse stain, and is therefore not capable of
being usefully employed alone. It is, however, of great use when employed
to bring about a double stain in conjunction with carmine. Though it has
no selective preference for nuclei or protoplasm, it possesses to a high
degree the property of imparting different hues and intensity of stain to
different tissues ; and the nuclei being brought out by carmine, prepara-
tions are obtained of a diagrammatic clearness that is not afforded by car-
mine alone.

Indigo-carmine is found in commerce. The reader who may desire to
prepare it himself will find the necessary directions in Arch. /. mik. Anat.y
x, 1874, p. 32, and in Journ. Roy. Micr. Soc., ii, 1879, p. 614

203. Thiersch's Oxalic Acid Indigo-carmine (see Arch. f. mik.
Anat., i, 1865, p. 150).

204. Tincture of Saffron (H. BLANC, Zool. Anzeig., 129, 1883, p. 23).

132 HJKMATEI'N AND OTHER OEGANIC STAINS.

— Dissolve 5 grms. of saffron in 15 c.c. of absolute alcohol; allow the
solution to settle for a few days, filter, and dilute with one half of water.
After staining wash out to the desired degree with 80 per cent, alcohol, then
dehydrate with absolute alcohol, and mount in balsam.

205. LEVEN (The Microscope, ix, 1889, p. 88 ; Journ. Roy. Mic. Soc.,
1889, p. 467) has been using saffron for the study of the regeneration of
muscle. He stains in a solution containing saffron 1 part, absolute alcohol
100 parts, water 200, and washes out in acidulated alcohol (0'5 per cent.
HC1). Karyokinetic figures dark red, muscle nuclei pale with dark red
nucleoli. Leucocytes stain strongly.

206. Orchella (Orseille) (WEDL, Arch. f. path. Anat., Ixxiv,
p. 143; Journ. Roy. Mic. Soc., ii, 1879: for an account of
this substance vide COOLEY'S Cyclopsedia,subvoce "Archil").
— French orchella extract, from which the excess of ammonia
has been removed by gentle warming in a sand-bath, is poured
into a mixture of absolute alcohol 20 c.c., acetic acid (concen-
trated, of 1*070 sp. gr.) 5 c.c., and water 40 c.c., until a
saturated dark red stain is obtained, which must then be
filtered once or twice. Sections are washed with water,
drained, and treated with the stain. Mount in levulose. A
protoplasmic stain, nuclei remaining colourless. Connective-
tissue cells stain deeply, the intercellular substance less
deeply. Epithelia, if horny or calcareous, are not stained.
The basic substance of bone and teeth take the stain, and so
do ganglion-cells and their processes.

This colour ought to be useful for double-staining. FOL
(Lehrb., p. 192) advises staining for an hour in Wedl's solu-
tion, then rinsing with alcohol, and staining in a complemen-
tary stain.

207. Orcein (ISRAEL, Virchow's Archiv, cv, 1886, p. 169; Journ. Roy.
Mic. Soc., 1887, p. 514). — Orcein (not " orcin," as erroneously printed in
the last edition of this work) is a vegetable dye obtained from tinctorial
lichens, which unites in itself the staining properties of the basic and acid
stains, and also the combination of two contrast colours. Israel stains sec-
tions in a saturated acetic acid solution, washes in distilled water, and passes
rapidly through absolute alcohol to thick cedar oil, in which the preparations
remain definitively mounted. Nuclei blue, protoplasm red.

For the specific staining of elastic tissue by means of this reagent, a
subject which has been carefully worked out by TANZER and UNNA, see the
paragraphs on the Connective Tissues in Part II.

208. Kernschwarz (PLAINER, Zeit.f. wiss. Mik., iv, 3, 1887,
p. 350; Journ. Roy. Mic. Soc., 1888, p. 675). — Kernschwarz

KRRNSCHWAEZ. 133

is a black liquid of unknown composition, prepared in Russia.
It may be obtained from Griibler (address § 94). Sections
(sections only, this colour behaving like saf ranin, for instance)
may be stained in a tolerably strong dilution of the concen-
trated liquid, and washed out (it may be for some hours) in
an alkaline aqueous liquid. Dilute ammonia will do, but it is
better to take a not quite saturated solution of carbonate of
lithium (you may take a saturated solution, and dilute it with
three, four, or more volumes of water). The result is a
nuclear stain in the cytological sense; nuclear figures of
division are stained deeply, resting chromatin less deeply or
not at all, cytoplasm unstained or faintly grey. A peculiarity
of this stain, on which much stress was laid by Platner in his
first announcement of the colour, is that it stains also the
Neberikern (which safranin and the other anilins do not do
when employed in the usual way) .

After some experimentation I feel bound to say that I do not think Kern-
schwarz has the importance seemingly claimed for it by Platner. It cer-
tainly stains the Nebenkern, but it does so less rather than more effectively
than haematoxylin, not to mention Henneguy's permanganate-safranin
method (§ 106), or Flemining's orange method, § 258. Platner seems to
have come to this conclusion himself, his latest work on the Nebenkem
making no further mention of Kernschwarz, and having been done apparently
entirely with hsematoxylin. And, as a nuclear stain, Kernschwarz seems to
me inferior to hsematoxylin, to say nothing of the chromatin-staining anilins.
Only in the event of its being found available for staining in the mass (which
may be possible, though I have not succeeded in it) will Kernschwarz, as it
seems to me, be found to be an important reagent.

209. Other Stains. — Litmus, red cabbage, bilberry juice ("myrtillus"),
black currant juice (" ribesine "), and walnut juice (" nucina ") have been
recommended by Lawson Tait, Lavdowsky, Fol, and Leon respectively.
They do not appear to call for further notice here.

134 METALLIC STAINS (IMPREGNATION METHODS).

CHAPTER XII.

METALLIC STAINS (IMPREGNATION METHODS).

210. The Characters of Impregnation-Stains. — By impregna-
tion is understood a mode of coloration in which a colouring
matter is deposited in certain elements of tissues in the form
of a more or less finely granular or sometimes even flocculent
precipitate — the impregnated elements becoming in conse-
quence opaque. By staining, on the other hand, is understood
a mode of coloration in which the colouring matter is retained
by the tissues in a state of solution, showing no visible solid
particles under the microscope, the stained elements remain-
ing in consequence transparent. But it should be understood
that it is not correct to draw a hard and fast line between
the two kinds of coloration. Some of the metallic salts
treated of in this chapter give, besides an impregnation, in
some cases a true stain. And some of the dyes that have
been treated of in preceding chapters give, besides a stain,
a true impregnation. Methyl en blue, for instance, will give
in one and the same preparation an impregation and a stain ;
and critical examination of most fairly successful gold-chlo-
ride preparations will show that the coloration is in places of
the nature of a finely divided solid deposit, in others a per-
fectly transparent stain.

Thus is justified the alternative title that this chapter has
always borne — Metallic Stains, or Impregnation Methods.
Impregnations are distinguished as negative and positive.
In a negative impregnation, intercellular substances alone are
coloured of a deep black or brown or violet, according to the
method employed ; the cells themselves remaining colourless
or very lightly tinted. In a positive impregnation the cells
are stained and the intercellular spaces are unstained. (This
explanation is the more needful as a directly contrary state-
ment is made in a recent Lehrbuch.)

ACTION OF LIGHT ON SOLUTIONS OF METALLIC SALTS. 135

Negative impregnation is primary because it is brought
about by the direct reduction of a metal in the intercellular
spaces. Positive impregnation is secondary (in the case of
silver nitrate) because it is brought about by the solution in
the liquids of the tissues of the metallic deposit formed by a
primary or negative impregnation, and the consequent stain-
ing of the cells by the new solution of metallic salt thus
formed. These secondary impregnations take place when the
reduction of the metal in the primary impregnation is not
sufficiently energetic (see on these points His, Schweizer
Zeit.f. Heilk., ii, Heft 1, p. 1 ; GIERKE, Zeit. f. wiss. Mik., i,
p. 393; RANVIER, Traite, p. 107).

There still exists considerable obscurity as to the nature of
the black or brown deposit formed in the intercellular spaces
in cases of primary impregnation with a silver salt ; v. Reck-
linghausen held that the silver salt combined with a hypo-
thetical intercellular cement-substance ( Kittsubstanz) , forming
a compound that blackens under the influence of light. Other
authors refuse to believe in the intercellular cement, and hold
either that the coloured lines represent stained cell-mem-
branes, or that the metallic salt combines with the albuminous
and saline liquids that surround the cells, and is precipitated
in simple intercellular spaces. SCHWALBE (Arch. f. mik. Anat.,
vi, 1870, p. 5) thinks that two cases should be distinguished;
the black lines that are obtained by the action of very weak
solutions for a very short time being due to a true precipitate
formed by reduction of metal in the intercellular liquids, the
brown lines that are obtained by exposing tissues for a longer
period to the action of more concentrated solutions being due
to the formation of a compound of metal and cement-sub-
stance that becomes brown on exposure to light. (For the
history of these questions see GIERKE'S Fdrberei zu mikro-
skopischen Zivecken.)

211. Action of Light on Solutions of Metallic Salts. — Stock
solutions of metallic salts are generally kept in the dark, or
at least in coloured bottles, under the belief that exposure
to light spoils them by precipitating the metal in a state of
reduction. It has been pointed out above (§ 25) that in the
case of osmic acid not light, but dust is the reducing agent ;
and that solutions may be exposed to light with impunity if

136 METALLIC STAINS (IMPREGNATION METHODS).

dust be absolutely denied access to them. I Lave now good
evidence to the effect that the same is the case with other
metallic solutions ; and the interesting point is raised,
whether such solutions are not positively improved for im-
pregnation purposes by exposure to light ! My able friend
Dr. LINDSAY JOHNSON has been examining this question both
from a histological and from a photographic point of view,
and writes me as follows :

" One may (I find by experiment) state as a rule without
exception that all the solutions of the chlorides and nitrates
of the metals will keep indefinitely in clean white stoppered
bottles in the sunlight ; and as far as osmium, uranium, gold
and silver, and platinum are concerned, actually improve or
ripen by a good sunning. All photographers tell me their
papers salt more evenly by old well-sunned silver nitrate than
by a fresh solution kept in the dark ; and I go so far as to
say that this is one of the reasons why gold stains are so un-
satisfactory/' I have not been able myself to perform any
experiments for the purpose of confirming the hypothesis of
ripening by sunning, but I must say that it appears to me
very plausible and deserving of further inquiry.

Silver.

212. Silver Nitrate : Generalities. — This is the most commonly
used salt of silver. The general principles of its employment
are so well stated by RANVIEE (Traite, p. 105) that I cannot
do better than abstract his account.

Silver nitrate may be employed either in solution or in the
solid state. The latter method is the less frequently em-
ployed, but is easy and gives good results. It is useful for
the study of the cornea and of fibrous tissue, but is not suit-
able for epithelia. For the cornea, for instance, proceed as fol -
lows : — The eye having been removed, a piece of silver nitrate
is quickly rubbed over the anterior surface of the cornea,
which is then detached and placed in distilled water ; it is
then brushed with a camel's-hair brush in order to remove the
epithelium. The cornea is then exposed to the action of light.
On subsequent examination it will be found that the silver
nitrate, which was dissolved by the liquid that bathes the
surface of the cornea, has traversed the epithelium and soaked

SILVER NITRATE. 137

into the fibrous tissue, on the surface of which it is reduced
by the action of light. The cells of the tissues will be found
unstained.

Silver nitrate is generally employed in solution in the fol-
lowing manner : — A 1 per cent, solution is taken, to which
two, three, or four volumes of water are added according to
circumstances. The mode of employment varies in its details
according to circumstances, a point which is very important
to observe. In the case of a membrane such as the epiploon,
the membrane must be stretched like a drum-head over a
porcelain dish,* and washed with distilled water, in order to
remove the albuminates and white blood-corpuscles that are
found on its surface ; it is then washed with the solution of
silver nitrate. In order to obtain a powerful stain it is neces-
sary that this part of the operation be performed in direct
sunlight, or at least in a very brilliant light. As soon as the
tissue has become white, and has begun to turn of a blackish
grey, the membrane is removed, washed in distilled water,
and mounted on a slide in some suitable examination
medium.

If the membrane were left in the water the cells would
become detached, and would not be found in the finished
preparation.

If the membrane had not been stretched as directed the
silver would be precipitated not only in the intercellular
spaces, but in all the small folds of the surface, and the forms
of the cells would be disguised.

If the membrane had not been washed with distilled water
before impregnation there would have been formed a deposit
of silver on every spot on which a portion of an albuminate
was present, and these deposits might easily be mistaken for
a normal structure of the tissue. It is thus that very often
impurities in the specimen have been described as stomata of
the tissue.

If the solution be taken too weak — for instance, 1*500 or
I'lOOO, or if the light be not brilliant — a general instead of an
interstitial stain will result ; nuclei will be most stained, then
protoplasm, and the intercellular substance will contain but
very little silver.

* The Hoggans' histological rings, for which see below, § 216, will be
found much more convenient.

138 METALLIC STAINS (iMPEEGNATION METHODS).

In general, in a good " impregnation," the contents of cells,
and especially nuclei, are quite invisible.

Eanvier notes that when tissues are to be impregnated by
immersion they should be constantly agitated in the silver-
bath in order to avoid the formation on their surfaces of de-
posits of chlorides and albuminates of silver, which would give
rise to deceptive appearances.

Impregnation with silver may be followed by treatment
with picro- carmine (or other carmine stain), which will bring
out the nuclei, provided the impregnation has not been
overdone.

It should be noticed that impregnations only succeed with
Jresh tissues, and cannot be made to succeed with tissues
preserved in any way.

213. Silver Nitrate : the Solutions to be employed (RANVIER).
— The solutions generally employed by Ranvier vary in
strength from 1'300 to 1'500. Thus 1'300 is used for the
epiploon, pulmonary endothelium, cartilage, tendon ; whilst a
strength of 1'500 is employed for the study of the phrenic
centre, and for that of the epithelium of the intestine. For
the impregnation of the endothelium of blood-vessels (by in-
jection) solutions of 1*500 to 1*800 are taken.

M. DUVAL (Precis, p. 229) recommends solutions of 1, 2, or
at most 3 per cent.

V. RECKLINGHAUSEN used, for the cornea, a strength of
from 1—400 to 1—500 (Die Lymphgefasse, &c., Berlin, 1862,
p. 5).

ROBINSKI (Arch, de Physiol., 1869, p. 451) used solutions
varying between O'l and 0*2 per cent., which he allowed to
act for thirty seconds.

REICH (Sitzb. d. wien. Akad., 1873, Abth. iii, April;
Zeit. f. wiss. Mik., i, p. 397) takes solutions of from 1 — 600
to 1 — 400 for the study of the endothelium of Vessels by
injection.

ROUGET (Arch, de Physiol., 1873, p. 603) employed solutions
as weak as 1 — 750, or even 1 — 1000, exposing the tissues to
their action several times over, and washing them with water
after each bath.

The HERTWIGS take, for marine animals, a 1 per cent, solu-
tion (Jen. Zeit.f. Naturk., xvi, pp. 313 and 324).

SILVER NITRATE. 139

The HOGGANS (Journ. of Anal, and PhysioL, xv, 1881, p. 477)
take, for lymphatics, a 1 per cent, solution.

TOURNEUX and HERMANN (ROBIN'S Journal de VAnat., 1876,
p. 200), in their fine studies on the epithelia of Invertebrates,
employed a solution of 3' 1000 strength, and in some cases
weaker solutions. The tissues were allowed to remain in the
silver-bath for one hour, and were washed out with alcohol of
36° strength.

HOYER (Arch. f. mik. Anat., 1876, p. 649) takes a solution
of nitrate of silver of known strength, and adds ammonia to
it until the precipitate that is formed just redissolves, then
dilutes the solution until it contains from 0*75 to 0*50 per
cent, of the salt.

This ammonio-nitrate solution is intended principally for the
impregnation of the endothelium of vessels by injection, but
can also be used for the impregnation of membranes by pour-
ing on. It has the advantage of impregnating absolutely
nothing but endothelium or epithelium ; connective tissue is
not affected by it. It is also said to give a sharper localisation
of the stain than the ordinary solutions.

DEKHUYSEN (Anat. Anz., iv, 1889, No. 25, p. 789; Zeit. f.
wiss. Mik., vii, 3, 1890, p. 351) has applied to tissues of
terrestrial animals the method of HARMER for marine animals
(see below, § 217). He washes a portion of mesentery of a
frog in a 1*3 per cent, solution of nitrate of potash, and
brings it for from three to six minutes into a 0*25 per cent,
solution of silver nitrate containing 3 per cent, of nitric acid.
After that time it is brought into pure 3 per cent, nitric acid,
thence after a few minutes into 96 per cent, alcohol, and then
into clove oil, in which it is reduced in diffused light in a few
minutes. The method is stated to have the advantage of
giving an excellent fixation of tissues, and of allowing a good
nuclear after-stain with haematoxylin, safranin, or methyl
green.

ALFEROW (Arch, de PhysioL, 1874 ; Laboratoire d'histologie
du College de France, 1874, p. 258; DUVAL, Precis, p. 230)
recommends the soluble silver salts of organic acids, viz. the
picrate, lactate, acetate, and citrate, as giving better results
than the nitrate. He employs them in solutions of 1*800, and
adds to the solution employed for staining a small quantity of
the acid of the salt taken (10 to 15 drops of a concentrated

140 METALLIC STAINS (IMPREGNATION METHODS).

solution of the acid to 800 c.c. of the solution of the salt).
The object of the free acid is to decompose the precipitates
formed by the action of the silver salt on the chlorides, car-
bonates, and other substances existing in the tissues, leaving
only the albuminate, which is a more resistent compound.

214. Silver Nitrate : Reduction. — Reduction may be effected
in other media than distilled water.

V. RECKLINGHAUSEN washed his preparations in salt solution
before exposing them to the light in distilled water (Arch. f.
path. Anat., xix, p. 451). Physiological salt solution (0'75per
cent.) is commonly used for these washings.

MULLER (Arch. f. path. Anat., xxxi, p. 110), after impregna-
tion by immersion for two or three minutes in a 1 per cent,
solution of nitrate of silver in the dark, adds to the solution
a small quantity of 1 per cent, solution of iodide of silver
(dissolved by the aid of a little iodide of potassium). After
being agitated in this mixture the preparations are washed
with distilled water, and exposed to the light for two days in
a 1 per cent, solution of nitrate of silver (see also GIERKE, in
Zeit.f. wiss. Nik., i, 1884, p. 396).

ROUGET (Arch, de PhysioL, 1873, p. 603) reduces in glycerin.

SATTLER (Arch. f. Mik. Anat., xxi, p. 672) exposes to the
light for a few minutes in water acidulated with acetic or
formic acid. THANHOFFER (Das Mihroskop., 1880) recom-
mends this method. He employs a 2 per cent, solution of
acetic acid.

KRAUSE brings his preparations, after washing, into a light
red solution of permanganate of potash. Reduction takes
place very quickly, even in the dark. The method does not
always succeed (see GIERKE, in Zeit. f. wiss. Mik., i, 1884,
p. 400).

OPPITZ brings his preparations for two or three minutes
into a 0*25 or 0'50 per cent, solution of chloride of tin.
Reduction takes place very rapidly (GIERKE, 1. c.).

JAKIMOVITCH (Journ. de I' Anat., xxiii, 1888, p. 142 ; Journ.
Roy. Mic. Soc., 1889, p. 297) brings nerve preparations, as
soon as they have become of a dark brown colour, into a
mixture of formic acid 1 part, amyl alcohol 1 part, and
water 100 parts. The objects exposed to the light in this
mixture for two or three days at first become brighter, a part

S1LVEU IMPREGNATION OF MARINE ANIMALS. 141

of the reduced silver being dissolved ; hence the mixture must
be renewed from time to time. When all the silver has dis-
solved, a darker colour is permanently assumed. The nerve-
cells are left in this mixture for five to seven days.

215. After-Blackening. — LEGROS (Journ. de I'Anat., 1868,
p. 275) washes his preparations after reduction in hyposulphite
of soda, which prevents after-blackening. According to
DUVAL (Precis, p. 230) they should be washed for a few
seconds only in 2 per cent, solution, and then in distilled
water.

216. The Hoggans' Histological Rings are vulcanite rings
made in pairs, in which one ring just fits into the other, so as
to clip and stretch pieces of membrane between them. They
will be found described and figured in Journ. Roy. Mic. Soc.,
ii, 1879, p. 357, and in ROBIN'S Journ. de VAnat., 1879, p. 54.
They may be obtained, in sets of various sizes (that of seven
eighths of an inch being the most convenient for 3x1 slides),
of Burge and Warren, 42, Kirby Street, Hatton Garden,
London, E.G., price ten shillings the dozen pairs.

This useful little apparatus has lately been reinvented by
Eternod (Zeit. f. wiss. Mik., iv, 1, 1887, p. 39), and is made
according to his designs by Demaurex, Bandagiste, Fusterie,
Geneva (Switzerland).

The Hoggans' histological rings were described by me in
the first edition of this work, p. 375, and in the Traite d.
Meth. techn., LEE et HENNEGUY, p. 138 ; so that I am in no
way responsible for the waste of time involved in this reinven-
tion on the part of the inventor, and the editors of the Zeit.
f. wiss. Mik. and other journals.

217. Silver Impregnation of Marine Animals. — On account of
the considerable quantity of chlorides that bathe the tissues
of marine animals, these cannot be treated directly with ni-
trate of silver.

HERTWIG (Jen. Zeit., xiv, 1880, p. 324) recommends fixing
them with a weak solution of osmic acid, then washing with
distilled water until the wash-water gives no more than an in-
significant precipitate with silver nitrate, and then treating
for six minutes with 1 per cent, solution of silver nitrate.

HARMER (Mitth. Zool. Stat. Neapel, v, 1884, pp. 44 to 56)

142 METALLIC STAINS (IMPREGNATION METHODS).

has discovered that many marine animals will live for some
time (half an hour) in a 5 per cent, solution of nitrate of
potash in distilled water. By washing them in this way they
may be freed from the greater part of their chlorides, and
may then be treated with silver nitrate in the usual way. This
method gave good results with Loxosoma and Pedicellina, with
Medusae, Hydroids, Sagitta and Appendicularia.

VOSMAER has been able by this means to demonstrate the
epithelium of Chondrosia and Thenea, which Sollas was unable
to see ; and MEYER has obtained good results with Annelids
and ova of Teleostea. Few animals resist the action of nitrate
of potash so well as Loxosoma and Pedicellina, but die in the
solution in a few minutes. Their tissues, however, suffer but
little change, and give good impregnations. Harmer thinks
that for these animals other solutions having the same density
as sea- water might be substituted for the nitrate of potash,
and recommends a 4'5 per cent, solution of sulphate of soda.

See also DEKHUYSEN'S method, ante, § 213.

218. Impregnation of Nerve Tissue. — For this subject, which
includes the important bichromate-and-silver method of GOLGI,
see Part II.

219. Double-staining Silver-stained Tissues. — The nuclei of
tissues impregnated with silver may be stained with the usual
reagents, provided that solutions containing free ammonia be
avoided, as this would dissolve out the silver. These stains
will only succeed, however, with successful negative impreg-
nations, as nuclei that have been impregnated will not take
the second stain.

Impregnation with silver may be followed by impregnation
with gold. In this case the gold generally substitutes itself
for the silver in the tissues, and though the results are sharp
and precise, the effect of a double stain is not produced.

Gold.

220. The Characters of Gold Impregnations. — Gold chloride
differs from nitrate of silver in that it generally gives positive
(§ 210) impregnations only. It only gives negative images,
so far as I know, when caused to act on tissues that have first
received a negative impregnation with silver, the gold sub-
stituting itself for the silver. In order to obtain these images

CHAKACTERS OF GOLD IMPREGNATIONS. 143

you first impregnate very lightly with silver ; reduce ; treat
for a few minutes with a 0*5 per cent, solution of gold chloride,
and reduce in acidulated distilled water.

This process, however, is in but little use, and except for
the staining of cytoplasm for cytological researches and for
certain special studies on the cornea, and on connective tissue^
the almost exclusive function of gold chloride is the impreg-
nation of nervous tissue. For this tissue, gold chloride
exhibits a remarkable selectivity, in virtue of which it justly
ranks as a most valuable reagent for the study of nerve end-
organs and the distribution of nerves.

For all the objects above named gold chloride is capable of
furnishing preparations that for beauty and clearness cannot
be surpassed, if even they can be equalled by any other means.
A successful gold preparation shows at a glance, with dia-
grammatic clearness, a wealth of minute detail which perhaps
can only be painfully, glimpsed by other means. But not
every gold preparation is successful. I think there is no use
in blinking the fact that very few are successful (one of the
most experienced authorities in the matter told me lately that,
as to nerve end-organs at all events, one preparation in ten
thousand is successful). I took up in the first edition of this
work the doubtless unpopular position that " with all possible
precautions gold chloride is uncertain in its action, and that
the results obtained by means of it need to be controlled by
the employment of other methods/3 and illustrated that posi-
tion at considerable length.

That this position was the correct one is now generally
admitted. It is acknowledged to be abundantly evident that
the very best gold preparations give images that are only
worthy of credence as to what they show, and furnish abso-
lutely no evidence whatever as to the non-existence of any-
thing that they do not show ; for you can never be sure that
the imbibition of the salt has not capriciously failed, or its
reduction capriciously stopped at any point. That the images
frequently do stop capriciously short in the representation of
reality there is abundant evidence. One such case has been
treated by me ex professo in Recueil Zool. Suisse, i, 1884, p.
685 (Les organes chordotonaux des Dipteres, et la methods du
cldorure d'or).

.The authors of some of the methods about to be described

144 METALLIC STAINS (IMPREGNATION METHODS).

claim for them that they give permanent preparations. 1
warn the reader against indulging in the hope that, with all
possible precautions, his preparations will retain all their
beauty for more than a few weeks. A successful gold pre-
paration is certainly a thing of beauty, but it is exactly the
opposite of a joy for ever. The able histologist whose ex-
perience 1 have taken the liberty of quoting above tells me
that " as to permanence, they are —

" ' Like the snowfall on the river.' "

Still, the greater the care taken in preparation, and
particularly the greater the care taken to ensure thorough
reduction of the gold, the longer will be the life of the pre-
parations.

Careful attention to the devices to this end detailed in
the following paragraphs will do much; and possibly LINDSAY
JOHNSON'S suggestion (supra, § 211) of the utility of " sun-
ning " the solutions before use may prove an unexpected help.

221. As to the Commercial Salts of Gold. — It is necessary to
remind the histologist that " all is not gold that glitters."
Many things are not what they seem, and gold chloride is
one of them, as will appear from the following quotation from
SQUIKE'S Methods and Formula*, &c. (p. 43), an excellent
authority on the chemistry of histological reagents :

" Commercial chloride of gold is not the pure chloride,
AuCl3, but the crystallised double chloride of gold and
sodium, containing 50 per cent, of metallic gold.

" Commercial chloride of gold and sodium is the above
crystallised double chloride mixed with an equal weight of
chloride of sodium, and contains 25 per cent, of metallic
gold."

222. The Two Types of Method. — Gold methods may be
divided into .two groups. The one, chiefly concerned with the
study of peripheral nerves or nerve end- organs, is charac-
terised by employing either perfectly fresh tissues or tissues
that have been subjected to a special treatment by organic
acids ; the other, concerned with the study of nerve-centres,
is characterised by the employment of tissues hardened in the
usual way.

The hitherto classical rule, that for researches on nerve-

LO WIT'S METHOD. 145

endings the tissues should be taken perfectly fresh, seems not
to be valid for all cases. For DEASCH (Sitzb. k. k. Acad.
Wiss. Wien, 1881, p. 171, and 1884, p. 516; and Abhand.
math.-phys. Cl. d. K. Sack. Ges. d. Wiss., xiv, No. 5, 1887;
Zeit.f. wiss. Mik., iv, 4, 1887, p. 492) finds that better results
are obtained with tissue that have been allowed to lie after
death for twelve, twenty-four, or even forty-eight hours in a
cool place. He even suspects that the function of the organic
acids in the methods inspired by Lowit's method, is to bring
the tissues into somewhat the state in which they are naturally
found at a certain moment of post-mortem process — a state,
namely, in which the nerves have a special susceptibility for
impregnation with gold.

223. Cohnheim's Method (Virchow's Arch., Bd. xxxviii, pp.
346—349; Strieker's Handb., p. 1100;.— This, the archetype
of the gold methods, was as follows : — Fresh pieces of cornea
(or other tissue to be operated on) are put into solution of
chloride of gold of 0'5 per cent, strength until they are
thoroughly yellow, and then exposed to the light in water
acidulated with acetic acid until the gold is thoroughly reduced,
which happens in the course of a few days at latest. They
are then mounted in acidulated glycerin.

The method in this, its primitive form, often gave splendid
results, but was very uncertain, giving sometimes a nuclear
or protoplasmic stain, sometimes an extra-cellular impregna-
tion similar to that of nitrate of silver. And the preparations,
thus obtained are anything but permanent.

224. Lowit's Method. — The principle of this process is that
in order to facilitate the penetration of the gold and its sub-
sequent reduction in the tissues, the tissues are made to swell
up by treatment with formic acid before being brought into-
the gold-bath, and formic acid is employed to assist the reduc-
tion after impregnation.

The following directions as to this method, which may serve
as a type of the modern methods of research on nerve-endings,,
are taken from FISCHEK'S paper on the corpuscles of Meissner
(Arch./. mik.Anat., xii, 1875, p. 366).

Lowit's method was first published by him in the Wien*
Sitzgsber., Bd. Ixxi, Abth. iii, 1875, p. 1.

Small pieces of fresh skin are put into dilute formic acid

10

M6 METALLIC STAINS (IMPREGNATION METHODS).

(one volume of water to one of the acid of 1'12 sp. gr.), and
remain there until the epidermis peels off. They then are
put for fifteen minutes into gold-chloride solution (1J per
cent, to 1 per cent.), then for twenty-four hours into dilute
formic acid (1 part of the acid to 1 — 3 of water), and then for
twenty-four hours into undiluted formic acid. (Both of these
stages are gone through in the dark.) Thin sections are then
made and mounted in dammar or glycerin. Successful prepa-
rations show the nerves alone stained, but it is not possible
always to control the results.

225. Ranvier's Formic Acid Method^ (Quart. Journ. Mic. Sci.
[N.S.], Ixxx, 1880, p. 456).— The method of Lowit has been
modified by many workers by omitting the final treatment
with undiluted formic acid, and also in some other details.
Ranvier proceeds as follows : — Reflecting that the action of
the one third formic acid in which Lowit placed his tissues
must be hurtful to the final ramifications of the nerves, he
combines the formic acid with a fixing agent designed to
antagonise its altering action, and takes for this purpose the
chloride of gold itself. The tissues are placed in a mixture
of chloride of gold and formic acid (4 parts of 1 per cent, gold
chloride to 1 part of formic acid) which has been boiled and
allowed to cool (Ranvier's Traite, p. 826). They remain in
this until thoroughly impregnated (muscle twenty minutes,
epidermis two to four hours) ; the reduction of the gold is
effected either by the action of daylight in acidulated water,
or in the dark in dilute formic acid (1 part of the acid to
4 parts of water).

The object of boiling the mixture of gold chloride and
formic acid is this, that " by boiling in the presence of the
acid the gold acquires a great tendency to reduction, and
for this reason its selective action on nervous tissues is
enhanced."

226. Ranvier's Lemon-juice Method (Traite, p. 813). — Instead
of combining the formic acid with gold chloride in order to
mitigate its action, recourse maybe had to a less injurious
acid than formic acid. Ranvier finds that of all acids lemon-
juice is the least hurtful to nerve-endings. He therefore soaks
pieces of tissue in fresh lemon- juice, filtered through flannel,
until they become transparent (five or ten minutes in the case

KANVIER'S LEMON-JUICE METHOD. 147

of muscle). They are then rapidly washed in water, brought
for about twenty minutes into 1 per cent, gold chloride solu-
tion, washed again in water, and brought into a bottle con-
taining 50 c.c. of distilled water and two drops of acetic acid.
They are exposed to the light, and the reduction is complete
in twenty-four or forty-eight hours. The preparations thus
obtained are good for immediate study, but are not permanent
on account of their over-blackening with time, the reduction
of the gold being incomplete. In order to obtain perfectly
reduced, and therefore permanent, preparations, the reduction
should be done in the dark in a few cubic centimetres of dilute
formic acid (1 part acid to 4 of water). The reduction is
complete in twenty-four hours.

227. Viallanes' Osmic Acid Method (Hist, et dev. des Insectes,
1883, p. 42). — The tissues are treated with osmic acid (1 per
cent, solution) until they begin to turn brown, then with
one fourth formic acid for ten minutes ; they are then put
into solution of chloride of gold of 1'5000 (or even much
weaker) for twenty-four hours in the dark, then reduced
in the light in one fourth formic acid. According to my
experience this is a very excellent method, both the fixation
by osmic acid and the great dilution of the gold solution
being features likely to be of advantage in many cases.

228, Other Methods. — The numerous other methods that have
been proposed differ from the foregoing partly in respect of
the solutions used for impregnation, but chiefly in respect of
details imagined for the purpose of facilitating the reduction
of the gold, and rendering it as complete as possible.

Thus BASTIAN modified Cohnheim's original method by
employing a solution of gold chloride of a strength of 1 to
2000, acidulated with HC1 (1 drop to 75 c.c.), and perform-
ing the reduction in a mixture of equal parts of formic acid
and water, kept warm, heat being an agent that furthers
reduction.

HENOCQUE (Arch, de I'Anat. et de la Physiol., 1870, p. Ill)
impregnates in a 0*5 per cent, solution of gold chloride,
washes in water for twelve to twenty -four hours, and reduces,
with the aid of heat, in a nearly saturated solution of tartaric
acid. The tartaric acid solution must be contained in a well-
stoppered bottle. The best temperature for reduction is 40°

148 METALLIC STAINS (IMPREGNATION METHODS).

to 50° C. Reduction is effected very rapidly, sometimes in a
quarter of an hour.

This process has been described as the method of CHRSCHT-
SCHONOWIC (Arch.f. mik. Anat., vii, 1872, p. 383).

HOYER (Arch. f. mik. Anat., ix, 1873, p. 222) proceeds as
follows : — (For corneal nerves.) The double chloride of gold
and potassium has the following advantages over the simple
gold chloride. It is more easy to be obtained of unvarying
composition, it is more perfectly neutral, and its solutions are
more perfectly stable. It is used in solutions of the same
strength as chloride of gold, viz. 0'5 per cent. Corneas
must be very thoroughly imbibed with the solution. Small
corneas (rabbit, guinea-pig) require half to one hour, human
corneas two to five hours (in an acidulated solution). It is
better to err on the side of too prolonged immersion rather
than the contrary. In order to demonstrate the intra-epithe-
lial ramifications of nerves, the gold is partially reduced by
exposure for sixteen to twenty-four hours in (one or two
ounces of) distilled water, and there is added to the water one
or two drops of a pyrogallic acid developing solution, such
as is used in photography (vide GERLACH, Die Photographie
als Hulfsmittel der mikroskopischen Forscliung, Leipzig, 1863).
Or instead of treating them with the developing solution, the
corneas may be removed to a warm concentrated solution of
tartaric acid, and remain there at the temperature of an
incubating stove until the gold is fully reduced.

I have myself used the double chloride of gold and sodium
with good results.

CIACCIO (Journ. de Microgr., vii, 1883, p. 38; Journ. Roy.
Hie. Soc. [N.S.], iii, 1883, p. 290) prefers the double chloride
of gold and cadmium.

GERLACH, whose preparations of nerve-centres are said not
to have been equalled since, proceeded as follows (Strieker's
Handb., 1872, p. 678) : — Spinal cord is hardened for fifteen to
twenty days in a 1 to 2 per cent, solution of bichromate of
ammonia. Thin sections are made and thrown into a solution
of 1 part of double chloride of gold and potassium to 10,000
parts water, which is very slightly acidulated with HC1. They
remain there from ten to twelve hours, and having become
slightly violet, are washed in hydrochloric acid of 1 to 2 : 3000
strength, then brought for ten minutes into a mixture of 1

OTHER GOLD METHODS. 149

part HC1 to 1000 parts of 60 per cent, alcohol, then for a few
minutes into absolute alcohol, and thence into clove oil, for
mounting in balsam.

FLECHSIG (Die Leitungsbahnen im Gehirn, 1876 ; Arch. f.
Anat. u. Phys., 1884, p. 453) reduces in a 10 per cent, solution
of caustic soda.

NESTEROFFSKY treats impregated preparations with a drop
of sulphydrate of ammonium, and finishes the reduction in
glycerin (quoted from Grierke's Fdrberei z. mik. Zwecken) .

BOHM reduces in PRITCHARD'S solution.

PRITCHARD'S SOLUTION consists of amyl alcohol, 1 per cent. ;
formic acid, 1 per cent. ; and water, 98 per cent.

MANFRED: treats fresh tissues as follows (Arch, per le Sci.
med., v, No. 15) : — Gold chloride, 1 per cent., half an hour;
oxalic acid, 0*5 per cent.; they are then warmed in a water-
bath to 36°, allowed to cool, and examined. Mount in gly-
cerin. Sunny weather is necessary.

He treats tissues previously hardened in 2 per cent, solu-
tion of bichromate of potash, as follows (ibid.) . They are put
for half an hour into solution of arsenic acid, or into 1 per
cent, acetic acid. They are then put into 1 per cent, gold
chloride for half an hour, washed in water, and reduced in
sunlight in 1 per cent, arsenic acid solution, which is changed
for fresh as fast as it becomes brown. Mount in glycerin.
Sunny weather is necessary.

BOCCARDI (Lavori Istit. Fisiol. Napoli, 1886, i, p. 27; Journ.
Roy. Hie. Soc., 1888, p. 155) recommends oxalic acid of 0*1
per cent, or of 0*25 to 0'3 per cent., or a mixture of 5 c.c. pure
formic acid, 1 c.c. of 1 per cent, oxalic acid, and 25 c.c. of
water. Objects should remain in this fluid in the dark not
longer than two to four hours.

KOLOSSOW (Zeit. f. wiss. MiJc., v, 1, 1888, p. 52) impregnates
for two or three hours in a 1 per cent, solution of gold chloride
acidulated with 1 per cent, of HC1, and reduces for two or
three days in the dark in a O'Ol per cent, to 0'02 per cent,
solution of chromic acid.

UNDERWOOD (Journ. Brit. Dental Ass., xi, 1890, p. 696 ;
Journ. Roy. Mic. Soc., 1890, p. 815) gives the following : —
Wash sections in bicarbonate of soda (strength not given) ;
treat them for half an hour to an hour with 1 per cent, solu-
tion of chloride of gold, neutralised if acid by bicarbonate of

150 METALLIC STAINS (IMPREGNATION METHODS).

soda ; wash in water ; reduce until the sections turn crimson
(about an hour) in 1 per cent, formic acid kept " fairly hot "
on a water-bath, in the dark ; wash for half an hour in cold
water; dry the sections (sic) and mount them in glycerin
jelly (" specimens mounted in balsam always go wrong").

Dr. LINDSAY JOHNSON writes to me that besides the " sun-
ning" of the impregnating solution recommended above
(§ 211), the following precautions should be taken : — " The
tissue must be well washed in distilled water, and the gold
carefully acidulated with a neutral acetate or forrniate, or
acetic or formic acid, at least twenty-four hours before using ;
and then afterwards the tissue must be washed until no re-
action occurs to test-paper."

For the details of the application of the methods of which
the principles have been set forth above, and for those of the
important processes of impregnation of central nerve organs,
the reader is referred to those chapters of Part II. which treat
of nerve-tissues and organs.

229. Ulterior Treatment of Impregnated Preparations, — Pre-
parations may be mounted either in balsam or in acidulated
glycerin (1 per cent, formic acid).

Theoretically they ought to be permanent if the reduction
of the metal has been completed effected.

In practice, all are doomed to destruction in course of time
by after-blackening, and few will be found to survive more
than a few months. Ranvier states that this can be avoided
by putting the preparations for a few days into alcohol, which
possesses the property of stopping the reduction of the gold.
But this must be taken to mean that by this means the period
of usefulness of the preparations may be prolonged for some
time, not indefinitely.

Blackened preparations may be bleached with cyanide or
ferrocyanide of potassium. REDDING employs a weak solution
of ferrocyanide ; CYBULSKY a 0'5 per cent, solution of cyanide.
But the results are far from being perfectly satisfactory.

Preparations may be double-stained with the usual stains
(safranin and methyl green being very much to be recom-
mended), but nuclei will only take the second stain in the
case of negative impregnation.

230. Impregnation of Marine Animals. — For some reason that

OTHEH METALLIC STAINS. 151

I am unable to explain, the tissues of marine animals do not
readily impregnate with gold in the fresh state. It is said by
FOL that impregnation succeeds better with spirit specimens.

Other Metallic Stains.

231. Perchloride of Iron. — This reagent, introduced by POLAILLON
(Journ. de VAnat., iii, 1866, p. 43), sometimes gives most useful results,
especially in the study of peripheral nerve-ganglia, in which it stains the
nervous tissue alone, the connective tissue remaining colourless. The
method consists in impregnating in perchloride of iron, and reducing in
tannic, gallic, or pyrogallic acid.

The HOGGANS, who have done very good work with this reagent, proceed
as follows (Journ. Quekett Club, 1876 ; Journ. Roy. Mic. Soc., ii, 1879,
p. 358) : — The tissue (having been first fixed with silver nitrate, which is
somewhat reduced by a short exposure to diffused light) is dehydrated in
alcohol, and treated for a few minutes with 2 per cent, solution of perchloride
of iron in spirit. It is then treated with a 2 per cent, solution of pyrogallic
acid in spirit, and in a few minutes more, according to the depth of tint
required, may be washed in water and mounted in glycerin.

FOL (see ante, § 52) fixes in perchloride solution, and .treats the prepara-
tions for twenty-four hours with alcohol containing a trace of gallic acid.

POLAILLON (1. c.) reduces in tannic acid.

This method is not applicable to chromic objects.

I should add that in my own experience I have found it very useful in
certain special cases.

232. Pyrogallate of Iron (ROOSEVELT, Med. Rec., ii, 1887, p. 84 ;
Journ. Roy. Mic. Soc., 1888, p. 157). — A stain composed of 20 drops of
saturated solution of iron sulphate, 30 grms. water, and 15 to 20 drops pyro-
gallic acid.

233. Osmic Acid. — Everybody knows that osmic acid stains tissues.
Most people, I should think, would be heartily glad if it did not. Meanwhile,
to make the best of this willy-nilly stain, you may sometimes find it useful
to treat the tissues with weak pyrogallic acid, which will very quickly turn
them of a fine greenish black, sometimes giving useful differentiations.

This method was first published by me in 1887 (La Cellule, t. iv, fasc. 1,
p. 110), and I have since republished it twice, with qualified recommendation.
I have found it occasionally very useful, but have hesitated to recommend it
for general work because I have usually found the stain too energetic and
too little discriminating. It frequently happens that everything in a pre-
paration comes out blacker than ink. The process has lately been re-invented
by KOLOSSOW (Zeit.f. wiss. Mik., ix, 1892, p. 38) in a somewhat complicated
form. He finds that the reaction obtained as I have described is not energetic
enough, and recommends treating first with the osmium mixture given in
§ 28, and then with an elaborate " developing " mixture composed of water,
alcohol, glycerin, tannin, and pyrogallic acid. For the reasons above stated,
I fail to see the utility of this modification of the method (Kolossow himself

152 METALLIC STAINS (IMPREGNATION METHODS).

says that a simple 5 per cent, aqueous solution of tannin gives excellent
results, sometimes superior to those obtained by the " developer "). See
my note in Zeit.f. wiss. Mik., ix, 1892, p. 185 ; also op. cit., p. 316.

HEEMANN (Arch.f. Mik. Anat., xxxvii, 1891, p. 569 ; Zeit.f. wiss. Mik.,
viii, 3, 1891, p. 367) has been using with success crude pyroligneous acid for
reducing the osmium in preparations fixed in his platino-aceto-osmic mixture
(§ 36A). The acid should be allowed to act for twelve to eighteen hours.

ALLEN (Quart. Journ. Mic. Sci., xxxiv, 1892, p. 75) has been treating in
the same way objects fixed in FLEMMING'S solution, and attributes the method
to VON MAHBENTHAL.

The method of BBOSICKE (Centralb.f. d. med. Wiss., 1879, p. 873 ; Zeit.
f. wiss. Mik., i, 1884, p. 409) consists in treating osmium objects for twenty-
four hours with a solution of 1 part of oxalic acid in 15 parts of water-
This gives a Burgundy-red stain. It is necessary that the objects be washed
and brought into the oxalic acid as soon as possible after the treatment with
osmium, as if the osmium has once begun to blacken them, the oxalic acid
is powerless afterwards to redden them.

234. Palladium Chloride (F. E. SCHTJLTZE, see ante, 52 and 84).
Prussian Blue (see LEBEE, Arch. f. Ophthalm., xiv, p. 300 ; EANVIEE,
Traite, p. 108). Cupric Sulphate (see LEBEE, ibid.). Lead Chromate
(see LEBEE, ibid.). Sulphides (see LANDOIS, Centralb. f. d. med. Wiss.,
1885, No. 55 ; and GIEEKE, in Zeit. f. wiss. Mik., i, 1884, p. 497).
Molybdate of Ammonium (Merkel ; Krause) (see GIEEKE, Zeit.f. wiss-
Mik., i, 1884, p. 96). Impregnation with Fats, Altmann's Method
(see post, " Corrosion ").

PICRIC ACTD COMBINATIONS. 153

CHAPTER XIII.

COMBINATION STAINS.

235. The Classes of Multiple Stains. — I distinguish two
classes of multiple stains. In the one a pure nuclear stain,
taking effect on all the nuclei of all the tissues of a prepara-
tion, is combined with a stain taking effect on all the extra-
nuclear parts of all the tissues. Borax-carmine followed by
indigo-carmine is a typical example of such a combination.
In the second class, a stain taking effect on the totality of the
elements of any one tissue exclusively is combined with a stain
or stains of another colour taking effect on the totality of the
elements of the other tissues.

The first class, aiming at enhancing the usefulness of a pure
nuclear stain by improving the definition of extra-nuclear
parts, has a legitimate scientific end in view, and is capable
of rendering service in research. It will, therefore, here be
treated much more fully than the second class, which is com-
posed of much less generally useful, and too frequently merely
ornamental stains.

236. Picric Acid Combinations.— I follow FLEMMING (Zeit. f.
wiss. MiJc., i, 1884, p. 360) in pointing out that picric acid is
perhaps the most generally useful of all secondary stains. It
gives useful plasma stains with most of the nuclear stains,
and particularly with carmine and haematoxylin. It may be
used with the most delicate of these stains, even the delicate
coloration of alum-carmine being in no wise injured by it.
The modus operandi is as simple as possible ; it consists merely
in adding picric acid to the alcohols employed for dehydrating
the objects after staining with a nuclear stain.

Care must be taken in adding picric acid to alcohol acidu-
lated with HC1 (see ante, § 169) ; in fact, this practice had
better in general be avoided, and the picric acid only added
to the pure alcohol used after washing out.

154 COMBINATION STAINS.

Combinations having Carmine for a Primary Stain.

237. Picro-carmine. — Picro-carmine is a double stain, if
care be taken not to wash out the picrin beyond the point
desired.

238. Borax-carmine and Pier o- Car mine. — A. very beautiful
and precise ^double stain may be obtained by means of this
combination. I add to a watch-glassful of Grenacher's
alcoholic borax-carmine a few drops of picro-carmine.

The mixture will precipitate in the course of a few hours,
but the stain will be obtained nevertheless.
BAUMGARTEN'S borax-picro-carmine (§ 171).

239. Carmine and Picric Acid. — This combination has been
already sufficiently explained above (see § 236).

LEGAL' s and GILSON'S alum- carmine and picric acid mixture
(§ 157).

Either of these combinations may be further combined with
haematoxylin, giving very rich differentiations. The stain
referred to by BOLSIUS in La Cellule, viii, 1, 1891, p. 6, should
be understood to be obtained by means of a mixture of equal
parts of haematoxylin solution and alum -carmine, followed by
treatment with picric acid, and not by means of hasmatoxylin
and picric acid alone, as reported in Journ. Roy. Mic. Soc.,
1891, p. 828, which would not give the colorations there
described.

240. Orth's Picro-lithium Carmine. — See the place quoted, § 166. I
consider the stain to be superfluous.

241. Seller's Carmine followed by Indigo-Carmine (Am. Quart.
Mic. Journ., i, 1879, p. 220 ; Journ. Roy. Mic. Soc., ii, 1879, p. 613).—
Stain in borax-carmine, wash out with HC1 alcohol, wash out the acid, and
after-stain in an extremely dilute alcoholic solution of indigo-carmine.

I find this method gives good results when applied to sections, but very
bad results if it be attempted to stain in the mass with the indigo. The
indigo over-stains the superficial layers before it has penetrated to the deeper
layers. The method may be considered to be superseded by the carmine and
anilin blue method, infra, § 243, which is more convenient.

242. Merkel's Carmine and Indigo-Carmine in One Stain
KEL, Unters. a. d. anat. Anst. Rostock, 1874 ; Month. Mic. Journ., 1877,
pp. 242 and 317).

(A) Take 2 grms. of carmine, 8 grms. of borax, and 128 c.c. of water (or half
a drachm of carmine, two drachms of borax, and four ounces of water). Rub

CARMINE AND ANILIN BLUE. 155

up in a mortar, allow the fluid to stand some time, decant, filter, and keep
in a stoppered bottle.

(B) Take 8 grms. of indigo-carmine, 8 grms. of borax, and 128 c.c. of water
(or two drachms indigo-carmine, two drachms borax, and four ounces water).
Mix, decant, filter, and preserve as before.

Before using, mix A and B in equal proportions.

The objects to be stained must be thin ; all traces of chromic acid or
chromates must have been carefully washed out from them, and they must
be soaked in alcohol before staining. Stain for fifteen or twenty minutes
(MAX FLESCH finds it better to stain for several hours, see Zeit. f. wiss.
MiJc., 1885, p. 350). Wash out with saturated aqueous solution of oxalic
acid for a rather shorter time ; wash the acid out with water, and mount as
desired.

The oxalic acid is necessary for fixing the indigo-carmine, which, being
very soluble in water, would otherwise be washed out. Unfortunately it
precipitates carmine, so that successful preparations are not easily obtained,
the carmine being generally either precipitated or turned into a straw-colour.

Authors (MEEKEL, loc. cit. ; NOEBIS and SHAZESPEABE, Amer. Journ.
Med. Sc., January, 1877 ; MEEKEL, Mon. Mic. Journ., 1877, p. 242 ; MAESH,
Section Cutting, p. 85 ; BAYEEL, Arch. f. mik. Anat., xxiii, 1885, pp. 36,
37 ; MACALLUM, Trans. Canad. Instit., ii, 1892, p. 222 ; Journ. Roy. Mic.
•Soc., 5, 1892, p. 698) are unanimous in stating that successful preparations
show a most richly differentiated and yet very precise colouring. According
to Bayerl, the stain is quite specifically elective for red blood-corpuscles,
which are'stained of an apple-green. The ground substance of cartilage and
bone stains blue, their cells red.

The stain is not perfectly permanent. Bayerl recommends that benzin be
used for clearing, in lieu of clove oil, which oxidises the stain and injures it.

This method has recently been recommended for nerve-centres. For
Bayerl's application of it to ossifying cartilage see Part II.
.. I have put this method in small type because, though admirable for certain
special purposes, it is not at all to be recommended for general work.

243. Carmine and Anilin Blue (or Bleu Lumiere, or Bleu de
Lyon). — DUVAL (Precis de technique microscopique, 1878, p. 225)
proceeds as follows : — Stain with carmine " in the ordinary
way;" dehydrate, and stain for a few minutes (ten minutes
for a section of nerve-centres) in an alcoholic solution of
anilin blue (ten drops of saturated solution of anilin blue
soluble in alcohol to ten grammes of absolute alcohol, for
sections of nerve-centres). Clear with turpentine, without
further treatment with alcohol, and mount in balsam.

The sections should appear of a fine dark violet when taken
from the anilin ; they are extremely transparent under the
microscope — nerve-cells and axis-cylinders reddish violet,
blood-vessels bluish violet, and so sharply marked out that

156 COMBINATION STAINS.

the preparations have the aspect of injections. The connective
elements are stained of a nearly pure blue, so that it is easy
to distinguish them from the nervous elements.

Applicable to all kinds of tissues, but especially to sections
of nerve-centres.

Eecent authors recommend, instead of anilin blue, bleu de
Lyon, dissolved in 70 per cent, alcohol acidulated with acetic
acid (MAURICE and SCHULGIN), or bleu lumiere, which has
hardly any effect on nuclei.

The solutions of both these colours should be extremely
dilute. They may be used for staining in the mass.

244. Carmine and Malachite Green. — MAAS (Zeit. f. wiss. Zool., x,
4, 1890, p. 527 ; Zeit.f. wiss. MiJc., viii, 2, 1891, p. 205) recommends borax-
carmine followed by weak alcoholic solution of malachite green, with a final
washing out with stronger alcohol.

245. Carmine and Methyl Q-reen (MAX FLESCH, Zool. Anz., 12$,
1882, p. 554). — Sections of cartilage, skin, and glands, made from tissues
hardened in Muller's solution and alcohol, were stained with picro.carmine,
and subsequently (not " previously," as erroneously stated in Journ. Roy.
Mic. Soc. [N.S.], ii, 1882, p. 883) with an aqueous solution of commercial
methyl green made of such a strength that the sections are just distinguish,
able in a watch-glassful of the solution when placed on a light ground.

The method is easy, gives good differentiations, but the stain does not
appear likely to be permanent. Mount in balsam.

246. Picro-carmine and Iodine Green (STIRLING, Journ. Anat. and
Physiol, xv, 1881, p. 349, et seq.).

Iodine green has a specific action on adenoid tissue and mucous glands,
which it stains of a bright green. But as methyl green has a similar action,
the method appears to be in general superfluous. See " Mucus Glands," in
Part II.

247. Picro-carmine, Rosein, and Anilin Blue; or Picro-car-
mine, Anilin Violet, and Anilin Blue ; or Picro-carmine, Anilin
Violet, and Iodine Green ; or Picro-carmine, Bosein, and Iodine
Green (HENEAGE GIBBES, Journ. Roy. Mic. Soc., in, 1880, p. 392). — H.
Gibbes says of these methods that their great utility consists in their
power of differentiating glandular structures according to their secretions.
In a section of a dog's tongue " the ordinary mucous glands will be found
to have taken on a purple colour, while the serous glands which supply the
secretion to the taste-organs stain a totally different colour." (See also
"Mucus Glands," in Part II.) Most of the differentiations aimed at in the last
six sections will probably be better obtained by means of the EHELICH-
BIONDI stain given below, § 259.

248. Carmine and Picro-nigrosin (PIANESE), (see Journ. Roy. Mic.
Soc., 1892, p. 292).

BENAUT'S ELEMATOXYLIC EOSIN. 157

249. Carmine and the Metallic Stains. — These combinations
have been sufficiently spoken of in the passages devoted to
gold and silver impregnation-methods. It will suffice here to
call renewed attention to ZOLTAN v. ROBOZ, alum-carmine and
osmic acid (§ 155).

Combinations having Hsematoxylin for a Primary Stain.

250. Hsematoxylin and Picric Acid. — This excellent combina-
tion has been treated of- above (§ 236). See also § 239.

251. Haematoxylin and Eosin. — This is a well-known com-
bination, and one of the most instructive that have yet been
imagined, though many workers prefer the combination of
haematoxylin with Benzopurpurin (§ 133). Objects may be
stained with haematoxylin (either in the mass or as sections),
and the sections stained for a few minutes in eosin. I think
it is better to take the eosin weak, though it has been recom-
mended (STORE, see Zeit.f. wiss. Mik., i, 1884, p. 583) to take
it saturated. Either aqueous or alcoholic solutions of eosin
may be used.

This method is most particularly recommendable for embryo-
logical sections, as vitellus takes the eosin stain energetically,
and so stands out boldly from the other germinal layers in
which the blue of the ha3matoxylin dominates.

LIST (Zeit.f. wiss. Mik.,u, 1885, p. 148) stains for twenty-four
hours in a solution of three or four drops of Renaut's hsema-
toxylic glycerin (§ 189) in 250 c.c. of water, and then for a
few minutes in a mixture of one part of O5 per cent, aqueous
solution of eosin with three parts of absolute alcohol.

BUSCH (Verh. Berl. Phys. Ges., 1877; GIEKKE, Zeit.f. wiss.
Mik.j i, 1884, p. 505) treats sections for some days with 0*5
per cent, solution of chromic acid, or 1 per cent, solution of
bichromate of potash, washes, and stains first in an aqueous
solution of eosin and then in hasmatoxylin. This process has
been recommended for the study of the margin of ossification.

It should be noted that sections should be very well washed
before being passed from eosin into haematoxylin or the
reverse, as eosin very easily precipitates haematoxylin.

252. Renaut's Haematoxylic Eosin (Foi/s Lehrbuch,~p. 196).—
Renaut has given from time to time several formulas for this

158 COMBINATION STAINS.

stain. This one, communicated to Fol by Renaut, is the
latest, and I suppress the others.
Take-
Concentrated aqueous solution of potassic eosin

(eosine a la potasse) . . . . .30 c.c.

Saturated solution of haematoxylin in alcohol
(ought to have been kept some time and to
have precipitated) . . . . . .40 c.c.

Saturated solution of potash alum in glycerin

(of a density of about 1'26) ... 130 c.c.
Mix, and let the mixture stand five or six weeks in a vessel
covered with a sheet of paper pierced with holes until the
alcohol is evaporated, then filter.

For staining, the solution may be used as it is or diluted.
Staining goes on very slowly, and at first the colour is not
held by the tissues, but disappears on washing. After some
days or weeks, however, it becomes localised and fixed in the
tissues. You may then mount in balsam, taking care to
employ alcohol charged with a sufficient quantity of eosin.
But it is frequently preferable to proceed by mounting the
objects in the staining fluid diluted with one to two volumes
of glycerin. After a few weeks this mounting medium will
have become perfectly colourless through the absorption of
the colour by the tissues.

The stain has a specific action on the cells of salivary and
gastric glands. Mucus-cells become pale blue ; salivary
ferment-cells (crescent-cells of Gianuzzi) intense rose.

There is no doubt as to this being a very fine and useful
stain. See also Comptes Rendus, 1879, p. 1039 (Ire ser.),
and Arch, de PhysioL, 1881, p. 640.

253. Haematoxylin and Benzopurpurin (ZSCHOKKE). — For this
and the combination with Delta Purpurin, see ante, § 133.

254. Haematoxylin and Rubin and Orange. — Mr. A. PRINGLE
informs me that this is an admirable combination. You
should stain in EHELICH'S hsematoxylin, wash in distilled or
even acidified water, blue the stain with tap water, or water
with a trace of alkali added to it, then stain in the rubin and
orange.

255. Heematoxylin and Iodine Green (STIRLING, Journ. of Anat.
and PhysioL, xv, 1881, p. 353). Heematoxylin and Nitrate of Kosa-

H^MATOXYLIN AND SAFRAN1N. 159

nilin (Liar, Zeit. f. iviss. Mik., ii, 1885, p. 149). See "Mucus-cells," in
Part II.

256. Hsematoxylin and Safranin. — This celebrated combina-
tion, which was used to such good effect by RABL in his
classical researches on nuclei (Morph. Jalirb., x, 1884, p. 215),
does not strictly belong to this subdivision, the safranin being
the primary stain, though used after the hasmatoxylin. You
stain very lightly with haematoxylin, so lightly that the stain
would not be of any use by itself (Rabl uses very dilute Dela-
field's solution, for twenty-four hours) ; wash out first with
water, and then with alcohol acidulated with HC1, then stain
for some hours in (Pfitzner's) safranin, and wash out with pure
alcohol. Eabl certainly was not far wrong when he wrote,
" This method is unequalled by any other." For richness of
detail in both nucleus and cytoplasm and tissue this method
has indeed hardly been equalled.

Foi (Festschr. R. Virchow gewidmet, &c., i, 1891, p. 481 ;
Zeit. f. wiss. Mik., ix, 2, 1892, p. 227) prefers using the
haematoxylin and safranin combined in one solution. You
take —

Aq. dest. ..... ca. 100

Bohmer's haematoxylin ... .25

Safranin, usual 1 per cent, water and
alcohol solution . .... 20

Stain sections in a few drops of the mixture for from one
to three minutes, wash in water, dehydrate, and mount; or
before dehydrating treat with a weak alcoholic solution of picric
acid, or, for some cases, of orange. This mixture was
specially used for staining marrow in an investigation into
the development of blood.

257. Hsematoxylin and Metallic Stains. — The conditions
under which haematoxylin can usefully be employed for
staining impregnated tissues have been discussed under the
heads of Gold and Silver. It only remains here to remind
the reader that haematoxylin works very well after osmic
acid.

Other Combinations.

258. The Anilin Double Stains. — These very important com-
binations are so numerous that only a small proportion of
them can be mentioned here.

' '^x

160 COMBINATION STAINS.

As regards sections stained by the indirect or Flemming's
method, considerable latitude is allowable in the manipula-
tions. Some persons stain first in the secondary stain (eosin,
for instance), then wash and stain in the primary stain (by
"primary" stain I constantly mean the nuclear stain), and
wash out until the colour of the secondary stain reappears.

Another method, which will, I think, frequently be found
preferable as allowing a stronger primary stain, is »to stain
first with the primary, say gentian violet, and then with the
secondary, say eosin. Care must be taken not to wash out
the first stain as omnpletely as if you were going to mount it
at once, else the operations required by the second stain may
result in entirely removing the colour of the first.

Foremost amongst these processes must be mentioned
FLEMMING'S Safranin, Gentian, and Orange Stain (Arch. f. mile.
Anat.f xxxvii, 1891, p. 249; ibid., p. 685; Zeii.f. wiss. Mik.,
viii, 2, 1891, p. 223, and viii, 3, p. 343).— This is a substitu-
tion method. You stain (for as much as two or three days if
you want a very powerful stain) in strong alcoholic safranin
solution diluted with anilin water (§ 101) ; rinse in distilled
water ; wash out in absolute alcohol, containing at most O'l
per cent, of hydrochloric acid, until hardly any more colour
comes away ; stain for one to three hours in gentian (either
a simple very strong aqueous solution, or, if you prefer, you
may stain by the method of Gram, § 102) ; wash for a short
time in distilled water ; treat with concentrated, or at least
fairly strong, aqueous solution of orange, which in virtue of
its acid properties washes out most of the gentian. After at
most a few minutes, whilst pale violet clouds are still being
given off from the sections on agitation, bring them into abso-
lute alcohol until hardly any more colour comes away, clear
in clove or bergamot oil, and mount in damar or balsam
before the last pale clouds of colour have ceased to come
away (the orange should be the orange G, introduced into
commerce by Meister, Lucius, and Bruning, of Hochst, and
also prepared by the Actiengesellschaft fur Anilinfabrication,
Berlin ; it may be obtained from GRUBLER, § 94).

This is not a triple stain in the sense of giving three
different colours in the result ; the orange does not act as a
separate stain, but as an agent for the differentiation of the
gentian stain. Chromatin and nucleoli are purple-red ; achro-

THE EHELICH-BIONDI MIXTURE. 161

matic spindle-fibrils grey to grey-brown, or, in very favorable
cases, violet, and are very clearly brought out ; attractive
spheres, centrosomes, polar corpuscles, and "Zwischen-
korper" reddish in light stains, brown- violet to black-brown
in strong ones.

Lists of some colours that give good results as primary and
as secondary stains are given in § 98. Besides the combina1
tions there recommended I would particularly recommend
that of Gentian Violet and Eosin. Stain and wash out (not
too far) by Bizzozero's method (§ 102), and then stain for a
few seconds, or as much as two to five minutes, in fairly strong
aqueous solution of eosin, dehydrate rapidly with alcohol,
clear, and mount ; or Benzopurpurin may be taken instead of
eosin (see § 133).

Attention should be paid to the decolourising action of the
secondary stain on the primary (see § 98). This reaction may
be utilised, as proposed by BAUMGAETEN (infra, § 268). RE-
SEGOTTI (Zeit. f. wiss. Mile., v, 3, 1888, p. 323) recommendrs
staining for five minutes (this is for tissues fixed by alcohol)
in strong solution of Methyl Violet or Dahlia, then washing out
for one or two minutes in very weak solution of (alcoholic)
Eosin or of Saurefuchsin in alcohol, then dehydrating and
clearing.

In the following paragraphs are given some other methods
of proved utility.

259. The EHELICH-BIONDI Mixture (or EHELICH-BIONDI-HEI-
DENHAIN Mixture) (Pfluger's Arch., xlii, 1888, p. 1; Zeit. f.
wiss. Mik., v, 4, 1888, p. 520). This well-known stain is
somewhat troublesome to prepare. It may be obtained ready
made from Dr. GEUBLEE (12, Baiersche Strasse, Leipzig).

The receipt is as follows : — To 100 c.c. saturated aqueous
solution of Orange add with continual agitation 20 c.c. satu-
rated aqueous solution of Saurefuchsin (Acid Fuchsin) and
50 c.c. of a like solution of Methyl Green.

Dilute the mixture with 60 to 100 volumes of water. The
dilute solution ought to redden if acetic acid be added to it ;
and if a drop be placed on blotting-paper it should form a
spot bluish green in the centre, orange at the periphery. If
the orange zone is surrounded by a broader red zone, the
mixture contains too much fuchsin.

11

162 COMBINATION STAINS.

Stain sections for six to twenty-four hours. Wash out with
alcohol, clear with xylol, and mount in xylol balsam.

According to M. HEIDENHAIN'S most recently published in-
structions (" Ueber Kern u. Protoplasma," in Festschr. Herrn
Geh. A. v. Kolliker gewidm., &c., 1892, p. 115 ; see Zeit.f. wiss.
Mik., ix, 2, 1892, p. 202) the Orange to be used should be
" Orange G ; " the Acid Fuchsin or Saurefuchsin should be
" Rubin S " (" Rubin " is a synonym of Fuchsin) ; and the
Methyl Green should be "Methylgriin 00." And it is
absolutely necessary that these ingredients be those prepared
under those names by the Actienfabrik filr Anilin fabrication
in Berlin.

The strong solutions directed to be taken readily precipi-
tate on being mixed. To avoid this it is recommended by
SQUIRE (Methods and Formulae, &c., p. 37) to dilute them
before mixing.

If (HEIDENHAIN, Arch. f. mik. Anat., xxxv, 1890, p. 173;
Zeit. f. wiss. MiJc., vii, 3, 1890, p. 357) the alcohol used for
washing out be slightly- alkaline, the stain of the fuchsin will
become relatively pale in the result, and the stain of the
methyl green relatively strong ; whilst a slightly acid reaction
of the alcohol will produce the opposite effect. The energy
of coloration of the fuchsin is often found to become weakened
in kept solutions ; 'it may be restored by adding very dilute
acetic acid until a marked intensification of the red tint of
the mixture is perceived. See § 639.

I have unfortunately not found time to make, as I should
have wished, a thorough trial of this now favourite stain.
But I have collected a good deal of trustworthy evidence con-
cerning it, all my informants concurring in the conclusion
that it is really an excellent formula. My able friend Ero-
fessor GILSON has tried it with a variety of objects, and has
been good enough to give me the following details. The
nuclear staining is very sharp and good, the chromatic
elements being coloured of a somewhat slaty blue. Cyto-
plasm is of a more or less violet or more or less orange red,
and caryoplasm is of -the same colour as the cytoplasm, but of
a lighter tone. Cell-membranes, nuclear membranes, achro-
matic fibrillar structures, the Nebenkern when present — in a
word, all the denser protoplasmic structures, are stained of
the same colour as the hyaloplasm, but darker. The stain

METHYL GREEN AND EOSIN. 163

possesses the usual qualities of mixtures of Methyl Green and
Acid Fuchsin, the Orange apparently coming into play only
in special cases. The stain works well, and may be put into
the hands of beginners. It is on the whole an excellent
formula, and will give valuable results in many cases, but
possesses no special magic, and is not likely to supersede
those amongst other staining methods that extended experi-
ence has found to be valuable. It may not be amiss here to
repeat the warning that there neither is nor can be such a
thing as a universal reagent in histology — " nouvel objet,
nouvelle methode !"

Grilson adds that for cytological purposes the proportions
of the ingredients should be varied according to circumstances.
For cells whose nuclein is less chroniophilous than the aver-
age, the proportion of methyl green should be increased, and
in the opposite case the proportion of acid fuchsin should be
increased.

For the special purpose of obtaining a minutely precise stain of attraction
spheres and other cytoplasmic elements, HEIDENHAIN recommends that the
mixture be acidified. For the somewhat minute details of the process and
of the results, see § 639 in " Cytological Methods " in Part II.

The stain will work with chrome-osmium mixtures, but I
believe does not give so fine a coloration with them as with
sublimate.

Dr. LINDSAY JOHNSON writes me, ' ' For nerve tissues, brain
and cord, retina and cochlea, &c., nothing in my opinion can
beat the Ehiiich-Biondi stain to which about one third of
twenty per cent, (saturated) solution of nigrosin is added"
(see the paragraphs on " Retina " in Part II). Dr. Johnson also
points out that it has the advantage of giving a stain whose
colours are photographically correct ; he says " it is the stain
par excellence for photography, except where there are fibrous
or cartilaginous tissues, which it stains red."

I cannot say to what extent the stain is permanent.
Heidenhain says that the preparations made according to hris
acid method mentioned above are absolutely permanent.

260. Methyl Green and Eosin (CALBERLA, Morph. Jahrb., iii, 1877,.
Heft 3, p. 625). — Mix 1 part of eosin with 60 parts of methyl green, and .
dissolve the mixture in warm 30 per cent, alcohol.

Sections stain in this solution in five or ten minutes; they should be
quickly washed in successive alcohols, and mounted in balsam or glycerin.

164 COMBINATION STAINS.

261. Methyl Green and Eosin (LIST, Zeit. f. iviss. Mik., ii, 1885,
p. 147). — Stain for a few minutes in a mixture of three parts of absolute
alcohol with one part of aqueous solution of eosin (0'5 per cent.), wash, and
stain for five minutes in 0*5 per cent, aqueous solution of methyl green.
Wash, dehydrate, clear, and mount in balsam. The preparations do not keep
well in glycerin.

The method may be varied (1. c., p. 150) by diluting the methyl green
solution with fifty volumes of water, and staining for twenty-four hours.

It may also be varied by diluting the original methyl green solution with
three volumes of absolute alcohol.

The preparations should not be left in the alcohol used for dehydrating
after the colour of the eosin has begun to reappear.

261a. Methyl Green and Eosin (RHUMBLEE, Zool. Anzeig., No. 411,
1893, p. 47). — Make a mixture of equal parts of 1 per cent, aqueous methyl
green solution, of a solution of 0'8 grm. of eosin in 50 per cent, alcohol
(quantity not stated), and of absolute alcohol. Stain (sections or small
pieces of material fixed with picro-sulphuric acid or alcohol) for half an hour ;
wash out first with water, then with successive alcohols, for not more than
a quarter of an hour, clear and mount. The novelty contained in this paper
consists in the somewhat astonishing statement that the stain serves to
differentiate living substance from dead or inorganic substance. " All sub-
stance that was living at the time of fixation being stained of a sharp red,
all dead substance or inorganic substance of a sharp green."

262. Methyl Green and Bismarck Brown (Lisx, Zeit.f. wiss. Mik.,
ii, 1885, p. 145). — Stain for a few minutes in Weigert's Bismarck brown
(§ 110), wash, and stain in 0'5 per cent, aqueous solution of methyl green.
Olear with bergamot oil or xylol, and mount in balsam.

Or, dilute the Bismarck brown for staining with three volumes of absolute
alcohol, wash out with strong alcohol, and stain for a few minutes in the
methyl green solution diluted with three volumes of absolute alcohol.

Or, stain for twenty-four hours in the Bismarck brown solution diluted
with fifty volumes of water, and then for twenty-four hours in the methyl
green solution diluted with fifty volumes of water.

263. Methyl Green and Nitrate of Rosanilin (LIST, 1. c.).— Stain
for a few minutes in 0'5 per cent, aqueous solution of methyl green, wash,
and stain (1. c., iii, 1886, p. 393) for ten to fifteen minutes in O'OOOl per
cent, aqueous solution of nitrate of rosanilin, wash out (rapidly) with abso-
Inte alcohol, and clear.

264. Anilin Green and Bismarck Brown (Lisx, I c.).— To be used
in the same way as methyl green and Bismarck brown supra (§ 262), and
giving much the same results.

265. Anilin Green and Eosin (SCHIEFFEBDECKEB, Arcli.f. Mik. Anat.,
xv, 1878, p. 30).— Stain in a solution made by adding a few drops of aqueous
solution of eosin to a watch -glassful of alcohol for from half an hour to
several hours, wash with water, and stain for a few minutes in 1 per cent.

OTHER COMBINATIONS. 165

aqueous solution of anilin green. Wash with water, wash out with alcohol,
and clear with clove oil.

This combination possesses a rich selectivity, especially for connective and
glandular tissues.

LIST (1. c., § 262) recommends this combination, which he employs in the
following manner : — Stain for a quarter of an hour in the eosin solution
there quoted, rinse with alcohol, and stain for a quarter of an hour in 0'5
per cent, aqueous solution of anilin green diluted with three volumes-oir
absolute alcohol. Wash out with absolute alcohol and (as soon as the colour
of the eosin begins to reappear) clear with bergamot oil or xylol.

266. Dahlia and Eosin (SCHIEFFERDECKEB, 1. c.).— Proceed as for
anilin green and eosin (§ 265), using a 1 per cent, aqueous solution of
dahlia.

267. Methyl Violet and Eosin (SCHIEFFERDECKER, 1. c.).— Proceed as
before, using a 1 per cent, aqueous solution of methyl violet.

268. Baumgarten's Fuchsin and Methylen Blue (Zeit. f. wiss. MiJc.,
i, 1884, p. 415). — Stain sections (of chromic objects) for twenty -four hours
in a stain made by adding 8 to 10 drops of concentrated alcoholic solution
of fuchsin to a watch-glassful of water. Rinse with alcohol, and stain for
four or five minutes in concentrated aqueous solution of methylen blue, wash
out with alcohol for five to ten minutes, and clear with clove oil. Nuclei
red, tissues blue, the fuchsin having been driven out of the tissues by the
methylen blue, a result which is not attained by washing with alcohol alone,
either pure or acidified.

269. Safranin and Indigo-Carmine (KossiNSKi, Zeit. f. wiss. Mik.t
vi, 1, 1880, p. 61). — Stain sections ten to twenty minutes in saturated
aqueous solution of indigo-carmine, wash with water and with alcohol, and
stain with safranin (0"5 per cent, in dilute alcohol), dehydrate and mount.

Safranin and Nigrosin is a combination also recommended by the same
author. Stain for three to five minutes in O'l per cent, aqueous solution of
nigrosin, and proceed as before.

Other Combinations.

270. Baumgarten's Triple Stain (Bull. Soc. Belg. Mic., 7, 1887, and
14, 1888, p. 146 ; Journ. de Microgr., 1888, p. 415 ; Journ. Roy. Mic. Soc.,
1887, p. 676, and 1889, p. 149). Garbini's Safranin and Anilin Blue
(Zool Anz., 1886, p. 26 ; and Zeit. f. wiss. Mik., v, 2, 1888, p. 170). (Both
these are much too complicated to be generally recommendable.) Iodine
Green and Eosin (STIRLING, Journ. Anat. and Physiol., xv, 1881, p. 354).
Rose Bengale and Iodine Green (GRIESBACH, Zool. Anzeig., 135, 1883,
p. 172). Rosem and Anilin Blue, Rosein and Anilin Green, Anilin
Violet and Anilin Blue, Anilin Violet and Anilin Green (see H.
GIBBES, Journ. Roy. Mic. Soc., 1880, p. 391). Metallic Stains and
Anilin Colours. — Most of the 'coal-tar colours above quoted may be
employed after a metallic impregnation. The combination of safranin with
gold chloride, first recommended by Pfitzner, is a classical stain. Eosin may
usefully be made to follow nitrate of silver impregnation. And other com-
binations may be found useful on occasion.

166 IMBEDDING METHODS.

CHAPTER XIV.
IMBEDDING METHODS — INTRODUCTION.

271. A Word on Microtomes. — It is no part of the purpose of
this work to discuss instruments, yet a word on this subject
may be helpful to the student. The freezing microtome so
generally employed in England is less than any other form
adapted to the wants of the zoologist. Very thin sections can
be obtained by it more readily than with any other microtome,
but they are of little use when obtained. The relations of the
parts of the organs are deranged b}7 the freezing and by the
thawing, and the aqueous nature of the process prevents it
from being readily applicable to the mounting of series of sec-
tions. The microtome of the zoologist, therefore (I am not
writing merely for pathologists or for dilettanti), must be an
imbedding microtome. The two most important points to be
attended to in the choice of such a microtome are the object-
holder and the knife motion. The object-holder should never,
as in some forms, be a well in which the imbedded object is
raised by a screw; the principle of construction should always
be that the object-holder be raised in its entirety by the screw,
not the object alone. The knife motion should always be
mechanical, the knife being guided by a mechanism giving
the required precision of stroke. And the object-holder should
be fitted with mechanical motions allowing of the orientation
of the object in all three directions of space. This is a most
important point ; such an object-holder is absolutely necessary
in many delicate morphological researches.

Amongst microtomes fulfilling these conditions various
forms will be found almost equally convenient. Zeiss makes
a good one ; Schanze, of Leipzig, makes a good one ; Reichert,
of Vienna, makes a good one. All these are relatively cheap,
and, being at the same time perfectly efficient for easy work,
may be recommended. Amongst more precise instruments

A WORD ON MICEOTOMES. 167

the first place in order of date belongs to the THOMA sliding
microtome. This is made in several sizes by E. Jung,
Mechaniker in Heidelberg. For zoological and general histo-
logical work I recommend the medium size (No. 2a or 4), with
the newest Naples object-holder and newest form of knife and
knife-holder.

This instrument is described in Journ.Roy. Mic. Soc. (N.S7),~
vol. iii, p. 298 ; the new Naples object-holder (which I consider
essential for the zoologist) is described and figured p. 915.

The BECKER microtome is in many respects an improvement
on the Thoma model. It is essentially on the same principle,
but possesses a mechanical arrangement for moving the knife-
carrier; that is, the knife-carrier is not only guided by a
mechanical arrangement, as in the Thoma model, but is put
in motion by mechanism. This, I think, is certainly an
advantage. Another improvement is that the slides are made
of glass instead of metal; this allows one to dispense with
the use of oil to the slides, which in the Thoma model gives
rise to inequality in the thickness of sections. A minor point
is that the instrument is somewhat cheaper than the Thoma
form. It is made by Aug. Becker, Gottingen. Descriptions
of two forms (Spengel and Schieffer decker) will be found in
Journ. Roy. Mic. Soc., 1886, pp. 884 and 1084. The Naples
object-holder can be fitted to the Becker microtome.

The instruments above described are ' ' all-round " micro-
tomes ; by which is meant that they may be used either with
a square-set knife or an obliquely-set knife, and will cut
either celloidin sections or frozen preparations (if a freezing
apparatus be added to them) just as well as paraffin sections.
They will not, according to my experience, cut series of
paraffin sections with quite the same infallible regularity,
certainly not with the same rapidity as the instruments next
to be mentioned. But they give results of almost the highest
attainable quality, and in view of their adaptability to celloidin
or other semi-soft preparations, I think that one of them, the
Becker by preference, should be the instrument chosen by the
worker who desires not to be entirely confined to the paraffin
method, and who cannot conveniently possess more than one
microtome.

The beautiful Cambridge rocking microtome (furnished by
the Cambridge Scientific Instrument Company, St. Tibb's

168 IMBEDDING METHODS.

Row, Cambridge, price £5, or by Messrs. Swift and Son, or
by Jung) is only adapted for cutting paraffin sections (Mr.
Swift has shown me an arrangement for inclining the knife
so as to give it the position required for cutting celloidin ; but
I feel pretty sure that this will prove a failure in practice).
This instrument is extremely simple and extremely rapid, and,
what is more important, cuts more level series of sections than
any other microtome I am personally acquainted with. It
should be fitted with the improved moveable object-holder of
Henneguy and Vignal (Compt. Rend. Soc. BioL, 1885, p. 647),
or some equivalent arrangement allowing the precise orienta-
tion of the object. (This, as well as the entire instrument, is
manufactured in France by Dumaige, 9, Rue de la Bucherie,
Paris, or Messrs. Swift on request will furnish such an
arrangement, or it may be obtained, with or without the
entire instrument, from Jung, of Heidelberg.)

It has been objected to this instrument by Schiefferdecker
(see Zeit. f. wiss. Mik., ix, 2, 1892, p. 171, a description and
criticism of the instrument as made by Jung) that it does not
cut plane sections, but sections having the form of segments
of a cylinder. This is true ; but it does not therefore follow,
as Schieffer decker concludes, that the instrument is inappli-
cable to many morphological purposes, and especially to
embryological research. In practice, the slight deviation of
the sections from a plane figure is found to be quite inappre-
ciable, and therefore unimportant. The chief defect of the
instrument from the point of view of the morphologist is that
it will not cut through a sufficiently lengthy series of sections
without having the object-holder readjusted. Now readjust-
ment of the object-holder is a ticklish piece of work, even
with the precise and well-situated Naples object-holder
mentioned above in connection with the Thoma microtome ;
much more is this the case with the relatively imperfect and
ill-situated object-holders supplied with the Cambridge micro-
tome. An ideal microtome ought to be able to section at
least a centimetre of tissue without readjustment ; the Cam-
bridge will only manage about a millimetre.

For object-holders see the price list of JUNG ; also a paper
in Zeit.f. wiss. Mik., vii, 2, 1890, p. 165.

Lastly, I would call attention to the MINOT microtome made
by E. Zimmermann, Mechaniker, 37, Halle'sche Strasse,

IMBEDDING METHODS. 169

Leipzig- Gohlis. A description and figures of this instrument
will be found in Zeit. f. wiss. Mik., ix, 2, 1892, p. 176. It is
worked on the sewing-machine principle : the knife is fixed
as in the Cambridge instrument, and the object is made to
impinge on it by means of a rotary motion given to a wheel
by the hand, and converted by a crank and slide lever into _a
vertical one given to a slide carrying the object. This micro-
tome cuts with very great rapidity, and those who have worked
with it speak very highly of it. I have not tried it myself,
but make no doubt the recommendation is justified, though,
looking to the construction of the slide, I doubt whether it
will afford sections quite so true as those given by a carefully
handled Becker. The object-holder does not appear to be so
scientifically constructed as the Naples one. Like the Cam-
bridge instrument, this microtome is only adapted for paraffin
work, and for this reason and the others stated above I do
not feel satisfied that it should be preferred to the Becker or
Thoma by those who have to be content with a single
instrument.

272. Imbedding Methods. — The beautiful processes known
as Imbedding Methods are employed for a twofold end.
Firstly, they enable us to surround an object, too small or
too delicate to be firmly held by the fingers or by any instru-
ment, with some plastic substance that will support it on all
sides with firmness but without injurious pressure, so that by
cutting sections through the composite body thus formed, the
included object may be cut into sufficiently thin slices with-
out distortion. Secondly, they enable us to fill out with the
imbedding mass the natural cavities of the object, so that
their lining membranes or other structures contained in them
may be duly cut in situ ; and, further, they enable us to
surround with the supporting mass not only each individual
organ or part of any organ that may be present in the
interior of the object, but each separate cell or other anato-
mical element, thus giving to the tissues a consistency they
could not otherwise possess, and ensuring that in the thin
slices cut from the mass all the details of structure will precisely
retain their natural relations of position. Such a process of
imbedding is at the same time practically a process of harden-
ing in so far as it gives to tissues a degree of firmness that

170 IMBEDDING METHODS.

could otherwise only be obtained by the employment of
chemical processes such as prolonged treatment with chromic
acid and the like.

These ends are usually attained in one of two ways. Either
the object to be imbedded is saturated by soaking with some
material that is liquid while warm and solid when cold,
which is the principle of the processes here called Fusion
Imbedding Methods; or the object is saturated with some
substance which whilst in solution is sufficiently fluid to
penetrate the object to be imbedded, whilst at the same time,
after the evaporation or removal by other means of its solvent,
it acquires and imparts to the imbedded object sufficient firm-
ness for the purpose of cutting. The collodion process suffi-
ciently exemplifies this principle. If a piece of soft tissue be
dehydrated, and soaked first in ether and then in collodion,
and if the ether contained in the collodion be allowed slowly
to evaporate, the tissue and surrounding mass of collodion will
acquire a consistency such as to admit of thin sections being
cut from them. The methods founded on this principle are
here called Evaporation Imbedding Methods.

The now antiquated egg-emulsion process, in which a mass
that is liquid whilst cold is coagulated by heat, or other
agency, belongs to a third class, which may be described as
Coagulation Methods.

In any of these cases the material used for imbedding is
technically termed an " imbedding mass" — Einbettungsmasse
— masse df inclusion. Imbedding methods are spoken of by
French writers as methodes d'inclusion, or methodes d'enrob-
age.

273. Imbedding Manipulations. — Before proceeding to de-
scribe in detail the more important imbedding methods, it is
necessary to give an account of the manipulations of the pro-
cess of imbedding in general.

Imbedding in a melted mass such as paraffin is performed
in one of the following ways. A little tray or box or thimble
is made out of paper, some melted mass is poured into it ; at
the moment when the mass has cooled so far as to have a
consistency that will not allow the object to sink to the
bottom, the object is placed on its surface, and more melted
mass poured on until the object is enclosed. Or the paper

IMBEDDING MANIPULATIONS.

171

tray being placed on cork, the object may be fixed in position
in it whilst empty by means of pins and the tray filled with
melted mass at one pour. The pins are removed when the
mass is cold.

In either case when the mass is cold, the paper is removed
from it before cutting.

To make paper trays, proceed as follows. Take a piece oF
stout paper or thin cardboard, of the shape of the annexed
figure (Fig. 1); thin (foreign) post-cards do very well indeed.
Fold it along the lines a a' and b b', then along c c' and d d',
taking care to fold always the same way. Then make the
folds A A', B B', C C', D D1 ', still folding the same way. To
do this you apply A c against A a, and pinch out the line
A A', and so on for the remaining angles. This done, you
have an imperfect tray with dogs' ears at the angles. To
finish it, turn the dogs' ears round against the ends of the box,
turn down outside the projecting flaps that remain, and pinch
them down. A well-made post-card tray will last through
several imbeddings, and will generally work better after
having been used than when new.

et/

Af

\

7

Nx

s

\

s

\

/

C

"A~ ~B

C 3>

7"

X

X
X

/

X

/

N

.1

s

N

C

FIG. 2.

a V

FIG. 1.

To make paper thimbles, take a good cork, twist a strip of

172

IMBEDDING METHODS.

paper several times round it so as to make a projecting collar,
and stick a pin through the bottom of the paper into the
cork. For work with fluid masses, such as celloidin, the
cork may be leaded at the bottom to prevent it from floating
when the whole is thrown into spirit or other liquid for hard-
ening (Fig. 2).

LEUCKHAKT'S Imbedding Boxes are made of two pieces of
type-metal (Fig. 3). Each of these
pieces has the form of a carpenter's
" square " with the end of the shorter
arm triangularly enlarged outwards.
The box is constructed by placing the
two pieces together on a plate of glass
which has been wetted with glycerin
and gently warmed. The area of the
box will evidently vary according to
the position given to the pieces, but
the height can be varied only by using
different sets of pieces. In such a box
the paraffin may be kept in a liquid
state by warming now and then over a
spirit lamp, and small objects be placed
in any desired position under the microscope (Journ. Roy.
NIC. Soc. [N.S.], ii, p. 880).

SELENKA has described and figured a simple but perhaps
more efficacious apparatus having the same object. It con-
sists of a glass tube, through which a stream of warm water
may be passed and changed for cold as desired, the object
being placed in a depression in the middle of the tube (see
Zool. Anz.t 1885, p. 419). A modification of this method is
described by ANDEEWS in Amer. Natural., 1887, p. 101 ; cf.
Zeit.f. wiss. Mik., iv, 3, 1887, p. 375.

For small paraffin objects the following procedure is very
useful. The object is removed from the paraffin solution, the
superfluous fluid is removed by means of blotting-paper, and
the object placed on a cylinder of paraffin. A piece of stout
iron wire is now heated in the flame of a spirit lamp, aud with
it a hole is melted in the end of the cylinder; the specimen is
pushed into the melted paraffin, and placed in any desired
position. The advantages of the method lie in the quickness
and certainty with which it can be performed.

FIG. 3.

CHOICE OF A METHOD. 173

I strongly recommend the reader not to neglect this simple
method, which is capable of sometimes rendering services
which no other method can. Those who have to do work
with objects so small that their position can only be made out
with the aid of a powerful lens ought to know how to arrange
an object with a heated needle under a dissecting microscope^
or on the object-carrier of the microtome.

[In the first edition this procedure was attributed to
KINGSLEY. It appears to have been first published by BORN,
see " Die Plattenmodellirmethode/' in Arch. f. mik. Anat.,
1883, p. 591.]

There remains the watch-glass method. Melt paraffin in a
watch-glass, and throw the object into it; or place the object
in the watch-glass, add solid paraffin, and heat. After the
mass has hardened, cut out a block containing the object
(this is of course applicable to other masses, such as celloidin) .
If paraffin be used you may, instead of cutting out a block,
turn out the whole mass of paraffin by simply warming rapidly
the bottom of the glass, To facilitate the removal of the
mass some persons lubricate the watch-glass before pouring
in the mass. To do this a drop of glycerin should be
smeared over it and wiped off with a cloth until hardly a
trace of it remains.

As regards the merits of the watch-glass process, I wish to
say that, as regards small objects at all events, I consider it
the very best process of any.

274. Choice of a Method. — Amongst the very various methods
of imbedding that have been proposed two are pre-eminently
important — the paraffin method for small objects, and the
celloidin or collodion method for large objects.

The subject of the respective merits of paraffin and celloidin
still affords matter for discussion to some persons. The case,
however, seems to be a very simple one. Celloidin does not
afford by a long way the thinnest sections that are obtainable
with small objects. For such objects it is, therefore, not equal
to the demands made by modern minute anatomy, and paraffin
must be taken. On the other hand, paraffin (as at present
employed) will only cut very thin sections with small objects;
with objects of 7 millimetres diameter you cannot get with
paraffin thinner sections than you can with celloidin; and if

174 IMBEDDING METHODS.

you try to cut in paraffin objects of somewhat greater size,
10 mm. and upwards, it will probably happen that you will not
get perfect sections at all, blocks of paraffin of this size having
a tendency to split under the impact of the knife ; so that for
large objects celloidin gives better results.

I have not been able to satisfy myself that the preservation
of the tissues is better in celloidin sections than in paraffin
sections; so that — convenience apart — the case remains as
above stated, — paraffin for small sections, celloidin for large
ones.

To this must be added aqueous masses, such as gum or
gelatin, for very special cases.

It will be the purpose of the next chapter to describe the
paraffin method, and to mention some other masses that can
be employed in a similar manner, the celloidin method and
the other methods that remain to be described being treated
of in Chap. XYI.

PENETRATION OR CLEANING. 175

CHAPTER XY.

IMBEDDING METHODS — PARAFFIN AND OTHER FUSION MASSES.

275. Penetration or Clearing. — The first stage of the paraffin
method consists in the penetration or infiltration of the
object by some substance which is a solvent of paraffin. The
process may be called a clearing process, since the chief
substances used for infiltration are also "clearing" agents.

The process of penetration or clearing should be carefully
performed with well-dehydrated objects in the manner de-
scribed in a former chapter.

Penetration liquids being merely liquids that are, on the
one hand, miscible with alcohol, and on the other hand good
solvents of paraffin, are as numerous as could be wished.
Amongst them may be mentioned essence of turpentine, clove
oil, bergamot oil, creasote, benzol, xylol, toluol, naphtha, oil
of cedar wood, chloroform, and anilin oil.

Turpentine penetrates well and mixes readily with paraffin.
I do not, however, recommend it, because in my experience
it is of all others the clearing agent that is the most hurtful
to delicate structures.

Clove oil penetrates well, and preserves delicate structures
well ; but it mixes very slowly with paraffin, and quickly
renders tissues brittle.

Benzin has been recommended by BEASS (Zeit. f. wiss.
Mik., ii, 1885, p. 301).

Toluol (or toluen) has been recommended by HOLL (Zool.
Anz., 1885, p. 223).

Naphtha has been recommended by WEBSTER (Journ. Anat.
and Physiol., xxv, 1891, p. 278). For large specimens it has
the advantage of being very cheap. Dr. Webster writes me
that a quality known as "Persian naphtha" is best for fine
work, but the common pure naphtha is sufficient for ordinary
work.

Xylol is said by HEIDENHAIN (see Zeit. f. wiss. Mik., ix, 2,

176 IMBEDDING METHODS.

1892, p. 200) to be a cause of shrinkage in cells; he employs
oil of bergaraot.

Chloroform mixes well with paraffin, and after evaporation
in a paraffin bath (in the manner described in the next para-
graph) leaves behind a pure and very homogeneous paraffin,
having but little tendency to crystallise. But it is deficient
in penetrating power, so that it requires an excessive length
of time for clearing objects of any size ; and it must be very
thoroughly got rid of by evaporation in the paraffin bath, or
by successive baths of paraffin, as if the least trace of it
remains in the paraffin used for cutting it will make it soft.
Chloroform ought, therefore, to be reserved for small and easily
penetrable objects.

Cedar-wood oil is, according to my continued experience,
for the reasons stated by me in Zool. Anz., 1885, p. 563, in
general the best clearing agent for paraffin imbedding. It
penetrates rapidly, preserves delicate structure better than
any clearing agent known to me, does not make tissues brittle,
even though they may be kept for weeks or months in it, and
has the great advantage that if it be not entirely removed from
the tissues in the paraffin bath it will not seriously impair the
cutting consistency of the mass ; indeed, I fancy it sometimes
improves it by rendering it less brittle.

In some difficult cases anilin oil is indicated (see § 353).

276. The Paraffin Bath. — The objects having been duly
" penetrated " or " cleared," the next step is to substitute
melted paraffin for the penetrating or clearing medium.

Some authors lay great stress on the necessity of making
the passage from the clearing agent to the paraffin as gradual
as possible, by means of successive baths of mixtures of clearing
agent and paraffin kept melted at a low temperature, say 35° C.
With oil of cedar or toluol, at all events, this is not necessary.
All that is necessary is to bring the objects into melted paraf-
fin kept just at its melting-point, and keep them there till they
are thoroughly saturated ; the paraffin being changed once or
twice for fresh only if the objects are sufficiently voluminous
to have brought over with them a notable quantity of clearing
agent.

The practice of giving successive baths first of soft and then
of hard paraffin appears to me entirely illusory.

THE PARAFFIN BATH. 177

It is important to keep the paraffin dry — that is, protected
from vapour of water during the bath.

It is still more important to keep it as nearly as possible at
melting-point. If it be heated for some time to a point much
over its normal inelting-point, the melting-point will rise, and
you will end by having a harder paraffin than you set out
with. And as regards the preservation of tissues, of course
the less they are heated the better.

The duration of the bath must, of course, vary according to
the size and nature of the object. An embryo of the size of a
pea ought to be thoroughly saturated after an hour's bath, or
often less, if cedar oil has been used for clearing. In any
case, the preparations should be cooled (see below, § 279) as
soon as saturated. If left for many hours in a warm bath, as
is sometimes done, delicate structures may be seriously
injured. Indeed, the great point to be attended to in paraffin
work of the finer order is to minimise the action of heat. It
is therefore important both to employ a paraffin of the lowest
melting-point that will give good sections (see below, §§ 280,
285), and to abbreviate the warm bath as much as possible.

If chloroform or other volatile agent be taken, choice may
be made of two methods : either, as in Griesbrecht's method,
the chloroform containing the objects is heated to the melting-
point of the paraffin, and the paraffin gradually added, and
the mass kept at the melting-point of the pure paraffin until
all the chloroform is driven off ; or, as in Butschli' s method,
the objects are simply passed direct from chloroform into a
solution of paraffin in chloroform, in which they remain until
thoroughly impregnated (half to one hour), and which is then
evaporated at the melting-point of the paraffin. Butschli
recommends a paraffin solution melting at 35°. (Such a solu-
tion is made of about equal parts of chloroform and paraffin
of 50° melting-point.) Or, in the case of larger objects,
instead of evaporating the chloroform (which is often a very
long process, as the chloroform must be completely driven off,
or the mass will remain too soft for cutting), Butschli simply
transfers them from the bath of paraffin solution to a bath of
pure paraffin.

Giesbrecht's method (Zool. Anz., 1881, p. 484), more fully
stated, is as follows :

Objects to be imbedded are saturated with absolute alcohol,

12

178 IMBEDDING METHODS.

and then brought into chloroform (to which a little sulphuric
ether has been added if necessary, in order to prevent the
objects from floating). As soon as the objects are saturated
with the chloroform, the chloroform and objects are gradually
warmed up to the melting-point of the paraffin employed, and
during the warming small pieces of paraffin are by degrees
added to the chloroform. So soon as it is seen that no more
bubbles are given oil from the objects, the addition of paraf-
fin may cease, for that is a sign that the paraffin has entirely
displaced the chloroform in the objects. This displacement
having been a gradual one, the risk of shrinkage of the tissues
is reduced to a minimum.

277. Stoves and Water-baths. — It is important that the paraffin
should not be exposed to a moist atmosphere whilst it is in the liquid state.
If a water- bath be used for keeping it at the required temperature, provision
should be made for protecting the paraffin from the steam of the heated
water.

A very convenient apparatus for this purpose is that of Paul Mayer, which
•will be found described at p. 146 of Journ. Roy. Mic. Soc., 1883. It may
be procured from the Zoological Station at Naples (address — " Direzione della
Stazione Zoologica, Napoli "), or from M. Paul Rousseau, 17, Rue Soufflot,
Paris. See also Amer. Natural., 1886, p. 910 ; and Journ. Roy. Mic. Soc.,
1887, p. 167.

Other similar forms of paraffin-heating apparatus are described in several
places in the same journal, as also in Zeit.f. wiss. Mik.

But whenever the worker has gas at his disposition, it will be found
infinitely preferable to employ a regulating stove or thermostat. I recom-
mend the form described in FOL'S Lehrbuch, p. 121. Other descriptions of
similar apparatus will be found also in the above-named journals.

278. Imbedding IN VACUO. — There are objects which, on account of their
•consistency or their size, cannot be penetrated by paraffin in the ordinary
way, even after hours or days in the bath. For such objects the method of
imbedding in a vacuum renders the greatest service. It not only ensures
complete penetration in a very short time — a few minutes — but it has the
further advantage of preventing any falling in of the tissues such as may
easily happen with objects possessing internal cavities if it be attempted to
imbed them in the ordinary way.

The principle of this method is that the objects are put through the
paraffin-bath in vacuo. In practice this may be realised by means of any
arrangement that will allow of maintaining paraffin at the necessary tempe-
rature for keeping it fluid under a vacuum.

The apparatus of HOFFMANN will be found described and figured at p. 230
of Zool. Anz., 1884. In this arrangement the vacuum is produced by
means of a pneumatic water aspiration pump, the vessel containing the
paraffin being placed in a desiccator heated by a water-bath and furnished
•with a tube that brings it into communication with the suction apparatus.

IMBEDDING AND COOLING. 179

This arrangement is very efficacious and very simple if the laboratory pos-
sesses a supply of water under sufficient pressure.

In order to obtain the requisite vacuum without the aid of water under
pressure, a simple little apparatus has been designed by FEANCOTTE (Bull.
Soc. Belg. Micr., 1884, p. 45). In this the vacuum is produced by the con-
densation of steam.

POL (Lehrb., p. 121) employs the vacuum apparatus of Hoffmann, buJL
simplifies the arrangement for containing the paraffin. The paraffin is con-
tained in a stout test-tube furnished with a rubber stopper traversed by a
tube that puts it into communication with the pump. The lower end of the
test-tube dips into a water-bath. You pump out the air once or twice, wait
a few minutes to make sure that no more bubbles rise, then let the air in,
turn out the object with the paraffin (which by this time will have become
abnormally hard), and re-imbed in fresh paraffin.

See also a paper by PEINQLE, in Journ. Path, and BacterioL, 1892,
p. 117 ; or Journ. Eoy. Mic. Soc., 1892, p. 893.

279. Imbedding and Cooling. — As soon as the objects are
thoroughly saturated with paraffin they should be imbedded
by one of the methods given above (§ 273). If the watch-
glass method be followed the paraffin-bath will naturally
have been given in the watch-glass used for imbedding, and
no special imbedding manipulation will be necessary. In any
case the important point now to be attended to is that the
paraffin be cooled as rapidly as possible. The object of this is
to prevent crystallisation of the paraffin, which may happen
if it be allowed to cool slowly, and to get as homogeneous a
mass as possible.

Yery small objects may be taken out of the paraffin with a
needle or small spatula, and put to cool on a block of glass,
then imbedded in position for cutting on a cone of paraffin
by means of a heated needle in the manner described above
(§ 273). In the use of the needle it should be noted that it
is important to melt as little paraffin as possible at one time, in
order that that which is melted may cool again as rapidly as
possible.

If the watch-glass method be adopted, float the watch-glass
with the paraffin and objects on to cold water. Do not let it
sink till all the paraffin has solidified. When cool, cut out
blocks containing the objects ; do this with a slightly warmed
scalpel.

If paper trays be taken, cool them on water, holding them
above the surface with only the bottom immersed until all the
paraffin has solidified, as if yon let them go to the bottom at

180 IMBEDDING METHODS.

once you will probably get cavities filled with water formed
in your paraffin. Or you may put them to cool on a block of
cold metal or stone.

SELENKA recommends cooling the mass by passing a stream
of cold water through the imbedding tube described above
(§ 273).

The objects having been mounted on the carrier of the
microtome in position for cutting, pare the blocks square to
the knife, and sufficiently close down to the objects, and go
round them with a lens. If any bubbles or cavities or opaque
spots be present, prick with a heated needle till all is smooth
and homogeneous. Minutes spent in this way are well in-
vested.

It is well to cut within a few hours of imbedding if the
structures be at all delicate, as paraffin may continue to
crystallise slowly to a certain extent even after rapid cooling.
But this danger is very greatly diminished if the mass have
been properly cooled.

280. Cutting and Section-stretching. — Paraffin sections are
cut dry — that is, with a knife not moistened with alcohol or
other liquid. By this means better sections are obtained, but
a difficulty generally arises owing to the tendency of sections
so cut to curl up on the blade of the knife. It is sometimes
difficult by any means to unroll a thin section that has curled.
To prevent sections from rolling, the following points should
be attended to :

First and foremost, the paraffin must not be too hard, but
must be taken of a melting-point suitable to the temperature
of the laboratory (for the winter season the temperature of
the laboratory being between 15° and 17° C., a paraffin
melting at about 45° C. should be taken; for hot summer
weather, laboratory at 22° C., a paraffin of 48° C. melting-
point).

The exact degree of hardness necessary must be determined
by experiment. If, after cutting has begun, the paraffin be
found to be too hard, it may be softened by the following
simple expedient (FoL, Lehrbuch d.vergl.mikr. Anat., p. 123) : —
A lamp provided with a parabolic reflector is set up near the
microtome in such a position that the heat-rays of the flame
are thrown by the reflector on to the imbedded object. The

CUTTING AND SECTION-STRETCHING. 181

right temperature is obtained by adjusting the distance of
the lamp.

If, on the contrary, the paraffin be found too soft, it may
be hardened by exposing it to the cooling influence of a lump
of ice placed in the focus of a similar reflector.

It is often sufficient to moderate the temperature of the
room by opening or closing the window, stirring the fire,
setting up a screen, or the like.

Secondly, the knife should be set square, for the oblique
position produces rolling, and the more the knife is oblique
the more do the sections roll.

Thirdly, it is better to cut ribbons than disconnected sec-
tions; ribbons of sections will often cut perfectly flat even
when the same mass will only give rolled sections if cut dis-
connectedly.

Special masses having less tendency to roll than pure
paraffin have been proposed. Thus a mass composed of four
parts of hard paraffin and one of vaselin has been recom-
mended. I recommend, however, that all such mixtures be
avoided.

Mechanical means may be employed. The simplest of these
and perhaps the best is as follows :

During the cutting the edge of the section that begins to
curl is caught and held down on the blade of the knife by
means of a small camel-hair brush with a flat point, or by a
small spatula made by running a piece of paper on to the
back of a scalpel. Or the section is held down by means of
an instrument called a "section-stretcher." This consists
essentially of a little metallic roller suspended over the object
to be cut in such a way as to rest on its free surface with a
pressure that can be delicately regulated so as to be sufficient
to keep the section flat without in any way hindering the
knife from gliding beneath it.

See the descriptions of various forms of section-stretchers, Zool. Anzeig.,
vol. vi, 1883, p. 100 (SCHULTZE) ; Mitth. Zool. Stat. Neapel, iv, 1883, p. 429
(MAYEE, ANDRES, and GIESBEECHT) ; Arch.f. mile. Anat., xxiii, 1884, p. 537
(DECKEB) ; Bull. Soc. Beige Mic., x, 1883, p. 55 (FBANCOTTE) ; The Micro-
scope, February, 1884 (GAGE and SMITH) ; WHITMAN'S Meth.inMic.Anat.,
1885, p. 91 ; Zeit.f. wiss. Mik., iv, 2, 1887, p. 218 (STEASSEE) ; as well as
Journ. Roy. Mic. Soc., iii, pp. 450, 916, and other places.

Another plan is to allow the sections to roll, but to control

182 IMBEDDING METHODS.

the rolling. To this end, the block of paraffin is pared to the
shape of a wedg*e five or six times as long as broad, the object
being contained in the head or broad part, and the edge
turned towards the knife. The sections are allowed to roll
and come off as coils, the section of the object lying in the
outermost coil, which will be found to be a very open one —
indeed, very nearly flat. Lay the coil on a slide with this end
downwards, warm gently, and the part containing the object
will unroll completely and lie quite flat.

The most efficacious plan for unrolling sections is perhaps
the combined treatment with fluid and heat (&ASKELL, Quart.
Journ. Mic. Sci., xxxi, 1890, p. 382; M. DUVAL, Journ. de
VAnat. et de la PhysioL, 1891, p. 26; HENNEGUY, ibid., 1891,
p. 398; GULLAND, Journ. of Anat. and PhysioL, 1891, p. 56;
and others). The rolled sections are either floated- on to the
surface of warm water or warm alcohol contained in a watch-
glass or suitable dish, which causes them to flatten out, and
are then transferred to a slide to be mounted in any desired
manner. Or the slide has a layer of water spread over it,
the sections are laid on the water, and the slide is heated (to
about 45° to 50° C.) until the sections flatten out, which
happens in a few instants. The method can be made avail-
able for fixing series of sections to the slide ; the further
details necessary for the successful accomplishment of this
are given in Chap. XVII, § 324.

Minute examination of paraffin sections sometimes reveals
certain distortions and dislocations or even ruptures of delicate
elements. I have often noticed that in certain regions of my
sections all the karyokinetic figures are drawn up to one side
(always the same side) of the nucleus, leaving the rest of the
nucleus empty and vacuolar in appearance. The achromatic
fibrils of the division spindle are frequently ruptured, and I
have not rarely found isolated chromosomes lying far from
the nucleus in the body of cells, or even outside the cells
themselves. These phenomena have generally been ascribed
to " shrinkage " caused by the action of the fixing agents or
the processes of dehydration or imbedding. HEIDENHAIN
(Ueber Kern u. Protoplasma, in Festschr. Herrn Geheimr. A. v.
Koiliker gewidm., 1892 ; see Zeit. f. wiss, Mik., ix, 2, 1892,
p. 200) thinks that they are often caused by excessive tilt of
the under surface of the microtome knife. If this be found

CHAIN OK " RIBBON " SECTION-CUTTING. 183

to be the case, the knife should be readjusted by means of a
piece of cardboard placed in the jaws of the clamp. I would
suggest that another cause of these defects is to be found in
imperfection of the edge of the knife. If the knife be blunt,
or have a rounded or curled edge caused by untrue honing or
stropping, it will of course act in respect to minute structures
as a plough rather than a cutting instrument, and thus produce
the appearances described.

281. Chain or " Ribbon " Section-cutting. —If a series of
paraffin sections be cut in succession and not removed from
the knife one by one as cut, but allowed to lie undisturbed
on the blade, it not unfrequently happens that they adhere
to one another by the edges so as to form a chain which may
be taken up and transferred to a slide without breaking up,
thus greatly lightening the labour of mounting a series. The
following appear to me to be the factors necessary for the
production of a chain.

Firstly, the paraffin must be of a melting-point having a
certain relation to the temperature of the laboratory. Small
sections can always be made to chain when cut from a good
paraffin of 45° C. melting-point in a room in which the ther-
mometer stands at 16° to 17° C. (The temperatures quoted
apply to the case of rooms heated by an open fire, and pro-
bably would not apply to the case of rooms heated by closed
stoves, such as are usual in Germany.) At 15° C. the paraffin
will be found a trifle hard. At 22° C. the proper melting-
point of the paraffin will probably be found at about 48° C.,
but my observations at these temperatures are less extended.
Secondly, the knife should be set square. Thirdly, the block
of paraffin should be pared down very close to the object, and
should be cut so as to present a straight edge parallel to the
knife edge; and the opposite edge should also be parallel to
this. Fourthly, the sections ought to be cut rapidly, with
the swiftest strokes that can be produced. For it is the
sharp impact of the knife that slightly heats, and therefore
slightly softens the near edge of the paraffin, and thus causes
the sections to cohere. It is evident that this condition can
only be conveniently realised by means of a sliding micro-
tome ; but it is by no means necessary to have recourse to
special mechanical contrivances, as in Caldwell's automatic

184 IMBEDDING METHODS.

microtome or Strasser's ribbon microtome. The Thoma
microtome well flooded with oil is sufficient. But the au-
tomatic microtomes are certainly most advantageous for
this purpose, and amongst them the Cambridge Rocking
Microtome, and the Minot, may be quoted as giving admi-
rable results.

Various plans, such as coating the edges of the paraffin
with softer paraffin, or with Canada balsam, or the employ-
ment of specially prepared paraffin, have been recommended,
with the idea that they help the sections to stick. None of
these devices is necessary. For the prepared paraffin of Spee,
Brass, and Foettinger, see below, § 286.

282. Collodionisation of Sections. — Some objects are by nature
so brittle that, notwithstanding all precautions taken in
imbedding and previous preparation, they break or crumble
before the knife, or furnish sections so friable that it is im-
possible to mount them in the ordinary way without some
impairment of their integrity. Ova are frequently in this
case. The remedy for this state of things consists in cover-
ing the exposed surface of the object just before cutting each
section with a thin layer of collodion, which serves to hold
together the loose parts of even the most fragile sections in
a wonderfully efficacious way ; and the same treatment applied
to tissues which are not specially fragile will enable the ope-
rator to cut sections considerably thinner than can be obtained
in the usual way. BUTSCHLI has obtained in this manner
sections of less than I fi in thickness.

The primitive form of the process was to place a drop of
collodion on the free surface of each section just before
cutting it. But this practice has two defects ; the quantity
of collodion employed sensibly softens the paraffin, and the
thick layer of collodion when dry causes the sections to roll.

MARK (Amer. Natural., 1885, p. 628 ; cf. Journ. Roy. Mic.
Soc., 1885, p. 738) gives the following directions :

" Have ready a little very fluid collodion in a small bottle,
through the cork of which passes a small camel-hair brush,
which just dips into the collodion with its tip. The collodion
should be of such a consistency that when applied in a thin
layer to a surface of paraffin it dries in two or three seconds
without leaving a shiny surface. Collodion of this consistency

COLLODIONISATION OF SECTIONS. 185

does not produce a membrane on the paraffin in drying, and
therefore has no tendency to cause sections to roll. It has
further the advantage that it penetrates to a certain depth
below the surface of the preparation, and fixes the deeper
layers of it in their places. The collodion must be diluted
with ether as soon as it begins to show signs of leaving a shiny
surface on the paraffin.

" Take the brush out of the collodion, wipe it against the neck
of the bottle, so as to have it merely moist with collodion, and
quickly pass it over the free surface of the preparation. Care
must be taken not to let the collodion touch the vertical sur-
faces of the paraffin, especially not the one which is turned
towards the operator, as that will probably cause the section
to become stuck to the edge or under surface of the knife.
As soon as the collodion is dry, which ought to be in two or
three seconds, cut the section, withdraw the knife, and pass
the collodion brush over the newly exposed surface of the
paraffin. Whilst this last layer of collodion is drying, take
up the section from the knife and place it with the collodion-
ised surface downwards on a slide prepared with fixative of
Schaellibaum. Then cut the second section, and repeat the
manipulations just described in the same order. A skilful
operator can cut ribbons of sections, collodionising each
section."

HENKING (Zeit.f. iviss. Mik., iii, 4, 1886, p. 478) objects to
the above process that the ether of the collodion softens the
paraffin, and proposes a solution of paraffin in absolute
alcohol. The solution is made by scraping paraffin into
absolute alcohol.

For extremely brittle objects, such as ova of Phalangida,
the same author recommends a thin (light yellow) solution of
shellac in absolute alcohol.

HEIDER (Embryonalentw. v. Hydrophilus, 1889, p. 12; cf.
Zeit. f. wiss. Mik., viii, 4, 1892, p. 509) employs a solution
made by mixing a solution of gum mastic in ether, of a
syrupy consistency, with an equal volume of collodion, and
diluting the mixture with ether until quite thin and liquid.

283. Clearing and Mounting. — The sections having been
obtained are generally mounted on a slide in serial order by
one of the methods described in the chapter on " Serial Section

186 IMBEDDING METHODS.

Methods/' All that now remains is to get rid of the paraffin
and mount or stain as the case may be. The following
solvents of paraffin have been recommended for freeing sec-
tions from the paraffin with which they are infiltrated : — Tur-
pentine, warm turpentine, a mixture of 4 parts of essence of
turpentine with 1 of kreasote, kreasote, a mixture of turpen-
tine and oil of cloves, benzin, toluol, xylol, thin solution of
Canada balsam in xylol (only applicable to very thin sections),
hot absolute alcohol, naphtha, or any other paraffin oil of low
boiling-point. Any of these may be used, but naphtha and
xylol are probably in most respects the best. Toluol also
works very well.

If the slide be warmed to the melting-point of the paraffin,
a few seconds will suffice to remove the paraffin if the slide
be plunged into a tube of naphtha or toluol. The sections
may be mounted direct from the naphtha, or the slide may
be brought into a tube of alcohol to remove the naphtha for
staining.

284. Recapitulation of the Paraffin Method, as recommended
to be practised. — Put into a small test-tube enough oil of
cedar to cover your object. On to the oil pour carefully the
same quantity of absolute alcohol. Take your (already de-
hydrated) object and put it carefully into the alcohol. Leave
it until it has sunk to the bottom of the cedar oil. Then put
it into paraffin kept at melting-point in a watch-glass. Let
the paraffin be of the very lowest melting-point that will give
sufficiently thin sections, and to this end work in a cool place,
so as not to be obliged to go above 45° C, if possible (see
also next section). After a time change the paraffin (i.e. put the
object into a fresh watch-glass with clean paraffin) once, or
twice if the object be at all large. As soon as the object is.
thoroughly soaked with paraffin float the watch-glass on cold
water. When cool, cut out a block of paraffin containing the
object, and fix it with a heated needle on a cone of paraffin
already mounted on the object-carrier of the microtome.
Pare it square, and as close down to the object as possible on
all sides except the one turned towards the knife ; this had
better have a wall of a millimetre or two, or more, according
to the size of the object, left standing. Set the knife square.
Set the block square to the knife-edge. Cut sections in

PREPARED PARAFFIN. 187

chains or ribbons, collodionising them if necessary. Fix
them in serial order on a slide according to one of the methods
given in Chap. XVII. Warm, and remove the paraffin with
naphtha. Stain or mount.

Paraffin Masses.

285. Pure Paraffin. — It is now pretty generally admitted
that pure paraffin forms an imbedding mass greatly superior
for ordinary work to any of the many fatty mixtures that used
to be recommended. I have only to repeat here that a
paraffin melting a£45° C. is that which in my experience gives
the best results so long as the temperature of the laboratory is
between 15° and 17° C.; whilst for a temperature of 22° C. a
paraffin melting at 48° is required. If the temperature of
your laboratory have risen much above 22° C. you had better
give it up, for good section-cutting with paraffin under such
conditions is next to impossible.

Paraffin of the melting-points named is easily found in
commerce. Intermediate sorts may be made by mixing hard
and soft paraffin. Two parts of paraffin melting at 50°
with one of paraffin melting at 36° C. give a mass melting
at 48° C.

Many workers of undoubted competence prefer masses
somewhat harder than those recommended, viz. of melting-
points varying between 50° and 55° C. for the normal tem-
perature of the laboratory. Some authors still recommend
masses melting at 60° C. or higher. I can only repeat that I
am convinced that, besides being most hurtful to tissues} such
masses have no raison d'etre whatever in temperate climates.

Paraffin had better be obtained from Griibler, or one of the
known dealers in microscopic reagents. Gaule recommends
that the bluish transparent sorts be taken. I should say,
transparent by all means, but, if possible, rosy rather than
bluish. New paraffin is bluish; if kept long, which is well,
it generally becomes rosy.

286. Prepared Paraffin (Pure) .— GRAF SPEE (Zeit.f. wiss.
ii, 1885, p. 8) recommends the following preparation of com-
mercial paraffin as giving a mass particularly favorable for
ribbon-section cutting. Paraffin of about 50° C. melting-
point is taken and heated in a porcelain capsule by means of

188 IMBEDDING METHODS.

a spirit lamp. After a time disagreeable white vapours are
given off, and the mass shrinks a little. This result is arrived
at in from one to six hours, according to the quality of the
paraffin. The mass then becomes brownish yellow, and after
cooling shows an unctuous or soapy surface on being cut.
The melting-point will be found to have risen several degrees.
This mass may be obtained ready prepared from Griibler.

BRASS (loc. cit., p. 300) recommends the use of paraffin that
has been kept for some years, as such has less tendency to
crystallise than new paraffin. In this I concur.

FOETTINGER recommends (Arch, de Biol, vi, 1885, p. 124) a somewhat
complicated treatment with caustic potash, in which I have no faith (it was
tried by one of the writers of the Traite des Meth. techn., during the pre-
paration of that work).

287. Paraffin Mixtures and other Similar Masses. — Of these
the only ones that I think can be recommended for a moment
are the following : — SCHULGIN'S paraffin with a little cerisin
(this is evidently what Schulgin means by " ceresin"). Or,
instead of cerisin, white wax (see Zool. Anz., 1883, p. 21) ;
or the mixture of BRASS (Zeit. f. wiss. Mik., ii, 1885, p. 301),
who recommends four to six parts of white wax to 100 of
paraffin. Sections may be cleared with benzin.

Cacao Butter, pure, is still used by some histologists. It melts some-
what under 35° C. The objects may be prepared by penetration with clove
oil, which may also be used for removing the mass from the sections.

Bayberry Tallow (Myrtle Wax, Vegetable Wax, Japan Wax).—
See Journ. Roy. Mic. Soc., 1885, p. 73§ ; ibid., 1888, p. 151 ; MILLER and
BLACKBURN, New York Med. Rec., 1885, p. 429; Amer. Mon. Mic. Journ.,
1887, p. 164 ; Journ. Roy. Mic. Soc., 1887, p. 1048 ; FRANCOTTE, Bull Soc.
Beige de Mic., 1887, p. 140 ; Zeit.f. wiss. MiL, iv, 2, 1887, p. 230; Journ.
Roy. Mic. Soc., 1887, p. 681. Bayberry tallow has the, for some objects,
valuable quality of working with alcohol as a solvent instead of such solvents
as chloroform or benzol, which undoubtedly alter certain tissues more than
alcohol. But for general work it has not succeeded in taking the place of
paraffin.

Soap Masses.

288. Utility of Soap Masses. — Soap masses certainly have
many good points. The solvent is alcohol ; the mass is highly
transparent, very penetrating, and a good mass cuts far better
than even paraffin. The mass may be cut either dry or with

TRANSPARENT SOAP. 189

alcohol. As to the' preservation of tissues, the mass is alka-
line, which is against it ; yet some workers still prefer soap
to paraffin,, and it has lately been recommended by so expe-
rienced a worker as Chun, for Siphonophora (certainly as
delicate a class of objects as any that exist), on the ground of
its producing less shrinkage than paraffin.

289. Transparent Soap (POLZAM, Morph. Jalirl., iii, 1877,
3tes Heft, p. 558). — The following account is taken from
Salensky's paper on the gemmation of Salpa (loc. cit.).

Take good white soap (" gewohnliche Kernseife"), cut it
up into thin slices, and put them to dry in the sun for some
days until they become white. The slices are then to be
rubbed up to a fine powder, which is mixed with spirit to
the consistency of porridge. Now mix the porridge with
alcohol and glycerin in such proportions that the whole shall
contain, for every ten parts by weight of the soap, 22 parts of
glycerin and 35 parts of alcohol (90 per cent.). Let the whole
simmer until there is obtained a perfectly transparent, syrupy,
somewhat yellow fluid.

The objects, previously dehydrated in alcohol, are imbedded
in this mass in the usual manner.

The mass may be removed from the sections either by means
of water or of very dilute alcohol.

290. Transparent Soap (KADYI, Zool. Anz., 37, 1879, vol. ii,
p. 477). — Twenty-five grms. of shavings of stearate of soda
soap (any stearate of soda soap will do, but the most to be
recommended is the sort known in commerce as "weisse
Wachskernseife") are heated in a retort with 100 c.c. of 96
per cent, alcohol over a water-bath until the whole is dissolved.
Filter if necessary. If a drop of the solution be now poured
into a watch-glass it will be seen that it almost immediately
solidifies into a white mass. This is not what is wanted, and
is a sign that the solution does not contain water enough.
Small quantities of water are therefore added by degrees to
the solution, and the effect tested from time to time by pouring
a drop of the mixture into a watch-glass. The mass will be
seen to become more and more pellucid until a point is reached
at which it is almost perfectly transparent, with merely the
slightest blue opalescence. The preparation of the mass is
then complete.

190 IMBEDDING METHODS.

It is not possible to state a priori the exact proportion of
water that should be added, as this naturally depends on the
amount of water already present in the sample of soap taken.
In very many cases it will be found that for about 120 grms.
of soap solution 5 to 10 grms, of water will be required.

It is necessary to be very cautious in adding the water, as
if too much be taken the mass solidifies more slowly or not at
all. The greatest amount of elasticity and consistency is
possessed by the mass at the moment in which it contains
exactly the minimum amount of water necessary to make it
transparent.

The reasons for this process are explained as follows : — Stearate of soda
soap is soluble in divers proportions in warm alcohol. On cooling, the solu-
tion either solidifies into a homogeneous and pellucid mass, or into a white
granular mass ; or, in certain cases, does not solidify at all. The result in
each case depends on the proportion of water present in the solution. For
instance, if 5 to 6 parts of a tolerably dry soap be dissolved in 100 parts of
96 per cent, alcohol, a solution is generally obtained that solidifies into a
transparent mass. But such a mass is too soft, and its melting-point too
low ; it melts by the heat of the finger. If now, in order to get a harder
mass, you add more soap, you will get a solution that solidifies on cooling
into a white granular mass ; and it is only after adding to it a certain
(small) quantity of water that you will obtain a solution that solidifies on
cooling into a transparent mass. If you add more water than is just
absolutely necessary to this end the mass will have too high a melting-point,
and will solidify more slowly ; and if still more water be added the solution
will not solidify for hours, or, indeed, not at all. The more soap you have
in your alcoholic solution the more water must you add in order to get a
transparent mass, and the more may you add without depriving the solution
of the faculty of solidifying. Besides the mass prepared in the proportions
given above, useful masses may be made for certain purposes with 10, 20,
30, 40 per cent., or more or less of soap in alcohol. Weisker has employed
a mass composed of about equal parts by weight of soap and alcohol.
Such a mass is transparent, but yellow and oily, and takes a long time to
solidify. When cool it is very tough. It requires a considerable tempera-
ture to liquefy it, and has less penetrating power than the more alcoholic
masses. It is, however, very suitable for hard, and especially for chitinous
structures.

The mass recommended above boils at about 60° to 70° C.
Objects should be imbedded in it in a watch-glass or in paper
cases in the usual way. Whilst cutting, wet the knife and
the mass with strong alcohol (one advantage of this method
is that the knife remains perfectly clean). The sections are
brought into 96 per cent, alcohol, which frees them from

GELATIN MASSES. 191

the mass instantaneously if warmed, and after a time if left
cold.

291. Other Soap Masses (FLEMMING, Arch. f. mik. Anat., 1873, p.
123 ; PFITZEE, Ber. deutscli. bot. Ges., 1887, p. Ixv ; Journ. Roy. Mic. Soc.,
1888, p. 316 ; POLI, Journ. de Micr., xiii, 1889, p. 337 ; Journ. Roy. Mic.
Soc., 1889, p. 835).— 90 per cent. ; alcohol, 1 part ; glycerin, 1 part ; glycerin
soap, 1 to 2 parts. Heat the glycerin and spirit on a water-bath to 60° of
70° C., and drop the soap into it in small pieces.

Gelatin Masses.

292. Gelatin Imbedding is a method that has the advantage
of being applicable to tissues that have not been in the least
degree dehydrated, and may render great service in the study
of very watery objects.

The modus ^operandi is, on the whole, the same as for other
fusion masses, with the difference that the objects are prepared
by penetration with water instead of alcohol or a clearing
agent. After the cooling of the mass it may sometimes be
cut at once, but it is generally necessary to harden it. This
may be done by treatment for a few minutes with absolute
alcohol (KAISER), or for a few days with 90 per cent, alcohol
(KLEBS) or chromic acid (KLEBS), or it may be frozen

(SOLLAS).

The mass is removed from the sections by means of warm
water.

293. Klebs' Gelatin (Glycerin Jelly) (Arch. f. mik. Anat., v,
1869, p. 165). — A concentrated solution of isinglass is mixed
with half its volume of glycerin.

294. Kaiser's Gelatin (Bot. Centralb., i, 1880, p. 25; Journ.
Roy. Mic. Soc., iii, 1880, p. 504). — One part by weight of the
finest French gelatin is left for about two hours in 6 parts by
weight of water; 7 parts of glycerin are added, and for
every 100 grms. of the mixture 1 grm. of concentrated carbolic
acid. The whole is warmed for ten to fifteen minutes, stir-
ring all the while, until the whole of the flakes produced by
the carbolic acid have disappeared. Filter whilst warm
through the finest spun glass which has been previously washed
in water and laid whilst wet in the filter.

295. Gerlach's Gelatin (GERLACH, Unters. a. d. Anat. Inst.
Erlangen, 1884; Journ. Roy. Mic. Soc., 1885, p. 541).— Take

192 IMBKDDING METHODS.

gelatin, 40 grms. ; saturated solution of arsenious acid, 200
c.c. ; glycerin, 120 c.c. Clarify with white of egg. The
mass may be kept for years in a well-stoppered bottle. The
objects to be prepared for imbedding by a bath of one third
glycerin.

296. BRUNOTTI'S Cold Gelatin Mass (Journ. de Botan., vi,
1892, p. 194; Journ. Roy. Mic. Soc., 1892, p. 706).— Twenty
grms. gelatin dissolved with heat in 200 c.c. distilled water,
and 30 to 40 c.c. of glacial acetic acid with 1 grm. corrosive
sublimate added after filtering. At the temperature of
15° C. the mass has the consistence of a thick syrup. Objects
are prepared by soaking in some of the mass diluted with two
to three vols. of water, then imbedded in the undiluted mass.
The mass is then hardened in spirit or bichromate of
potash, picric acid, or the like. No heat at all is required in
this process.

COLLODION, CELLOIDIN, AND PHOTOXYLIN. 193

CHAPTER XVI.
COLLODION (CELLOIDIN) AND OTHER IMBEDDING METHODS.

297. Collodion, Celloidin, and Photoxylin.— The collodion
method is due to DUVAL (Journ. de I'Anat., 1879, p. 185).
Celloidin, recommended later on by MERKEL and SCHIEFFER-
DECKER (Arch. f. Anat. u. Phys., 1882, p. 200), is merely a
patent collodion.

It is stated to be a preparation of pure pyroxylin, and is
patented for Germany and England under the name of
" Schering's Celloidin." It is manufactured by the Chemische
Fabrik auf Actien (vorm. E. Schering), Berlin, N. Fenstrasse
11, 12. It may be obtained through the post by writing to
SCHERING'S " Griine Apotheke," Wittick and Benkendorf,
Berlin, N. Chaussee-Strasse, No. 19, or from GRUBLER, or the
other dealers in histological reagents.

It is stated to be prepared with the purest pyroxylin, and
to be always of a uniform composition. It is sent out in the
form of tablets of a tough gelatinous consistency and slightly
milky-white transparency. These tablets contain 20 per cent,
of pure pyroxylin. Celloidin is entirely soluble, in all pro-
portions, in ether and alcohol. It is free from acids. It is
not detonant. If ignited it burns like paper ; heated in a
test-tube it carbonises without exploding.

In order to make a 2 per cent, collodion, take one tablet of
celloidin (which contains 40 grins, of the dry pyroxylin), and
such a quantity of alcohol and ether that the whole shall
weigh 2000 grms. For a 3 per cent, collodion you take such
a quantity of alcohol and ether that the whole shall weigh
1333 grms. ; and for a 4 per cent, collodion such a quantity
that the whole shall weigh 1000 grins. The relative portions
of alcohol and ether may be taken according to discretion.
To prepare a medicinal collodion according to the Prussian
Pharmacopoeia you take for each tablet 720 grains of ether
and no alcohol, as the celloidin already contains the prescribed

13

194 COLLODION AND OTHER IMBEDDING METHODS.

proportion of alcohol. The tablets cost three marks (= three
shillings) each. A single tablet would, I think, suffice for
imbedding many hundreds of embryos.

There is a strife of opinion amongst authorities as to the
relative merits of celloidin and common collodion. DUVAL, in
a restatement of the method (Journ. de Micr., 1888, p. 197),
teaches that celloidin has no real advantage over common
collodion, whilst the latter has the advantage of being more
transparent. SCHIEPFEEDECKER, also restating the method,
declares that celloidin has " many points of superiority "
(Zeit. f. wiss. Mik., v, 4, 1888, p. 504). On carefully reading
Schiefferdecker's paper, however, it appears that the only
reason alleged in support of this superiority is that it is more
" convenient " to make a thick mass by making a strong
solution of celloidin than by allowing common collodion to
concentrate by evaporation.

Personally I incline to Duval's point of view. The supe-
riority of celloidin, if it exists, is mainly of the nature of a
mere matter of convenience. Otherwise there is but little to
choose between the two, and therefore in this work the terms
collodion and celloidin are used indifferently.

Photoxylin (KRYSINSKY, VIRCHOW'S Archiv, cviii, 1887,
p. 217 ; BUSSE, Zeit. f. wiss. Mik., ix, 1, 1892, p. 47) is a dry
substance, of the aspect of cotton wool, and chemically nearly
related to celloidin. It can be obtained either from SOBERING
or G-RUBLER. It gives a clear solution in a mixture of equal
parts of ether and absolute alcohol, and should be used in
exactly the same way as celloidin. It has the very great
advantage of affording a mass which after hardening in 85
per cent, alcohol remains perfectly transparent.

298. Preparation of Objects. — The objects must first be
thoroughly dehydrated with absolute alcohol. They are then
soaked till thoroughly penetrated in ether, or, which is better,
in a mixture of ether and absolute alcohol. DUVAL (1. c.)
takes for this purpose a mixture of ten parts of ether to one
of alcohol ; SCHIEFFERDECKER (and the majority of workers) a
mixture of equal parts of ether and alcohol ; TUBBY (in Nature,
November 17th, 1892, p. 51) advises a mixture of four parts
of ether and one of alcohol. But the point is one of no
great importance.

THE COLLODION BATH. 195

This stage may be omitted if the objects are of a sufficiently
permeable nature, and they may be brought direct from
alcohol into the collodion bath.

299. The Collodion Bath. — The next step is to get the objects
penetrated with thick collodion. The secret of success here_
is to penetrate them first with thin solutions, then with the
definitive thick one. (A thin solution may be taken to mean
one containing from 4 to 6 per cent, of dry celloidin ; a thick
solution one containing 10 to 12 per cent.)

If collodion be taken the thin solution may be made by
diluting it with ether. If photoxylin or celloidin be taken
the solutions are made in a mixture of ether and absolute
alcohol in equal parts. Apathy recommends that celloidin be
allowed to dry in the air until it becomes yellow, transparent,
and of a horny consistency, and that it be then dissolved in
the alcohol and ether (sulphuric, free from acid). The solu-
tions thus prepared are free from the excess of water that is
present in the undried celloidin, and give after hardening a
mass that is more transparent and of a better consistency for
cutting (Zeit. f. wiss. Mik., vi, 2, 1889, p. 164).

BUSSE (op. cit., ix, 1, 1892, p. 47) gives the following pro-
portions for the successive baths : — No. 1, 10 parts by weight
of photoxylin or perfectly dried celloidin to 150 parts of the
ether and alcohol mixture ; No. 2, 10 parts of photoxylin or
celloidin to 105 of the mixture ; No. 3, 10 parts to 80 of the
mixture (already used solution may be employed for the first
bath).

The objects ought to remain in the first bath until very
thoroughly penetrated; — days, even for small objects, — weeks
or months for large ones (human embryos of from six to
twelve weeks, for instance). If the object contain cavities,
these should be opened to ensure their being filled by the mass.

When the object is duly penetrated by the thin solution, or
solutions if more than one have been employed, it should be
brought into the thickest one. SCHIEFFERDECKER (1. c.) recom-
mends that this be done by allowing the thin solution to con-
centrate slowly (the stopper of the containing vessel being
raised, for instance, by means of a piece of paper placed
under it), and making up the loss from evaporation with thick
solution.

196 COLLODION AND OTHER IMBEDDING METHODS.

300. Imbedding. — At this stage, if it has not been done
"before, the objects must be imbedded — that is, arranged in
position in the receptacle in which they are to be hardened.
For the usual manipulations see § 273. I recommend the
paper thimbles or cylindrical trays, Fig. 2, as being very
convenient for collodion imbedding. The bottoms, however,
should be made of soft wood in preference to cork ; cork is
elastic, and bends in the object-holder of the microtome, de-
forming the mass and object. The box should be prepared
for the reception of the object by pouring into it a drop of
collodion, which is allowed to dry. The object of this is to
prevent bubbles coming up through the wood or cork and
lodging in the mass.

Objects may also be imbedded on a piece of pith or leather,
which should also be prepared with a layer of dry collodion.

Watch-glasses, square porcelain water-colour moulds, and
the like, also make convenient imbedding receptacles. Care
should be taken to have them perfectly dry. Any of these
receptacles or supports may be set with the mass under a
glass shade, allowing of just enough communication with the
air to set up a slow evaporation. Or porcelain moulds or
small dishes may be covered with a lightly fitting cover.

If it be desired to mark the position of the object in the mass in order to
facilitate the subsequent orientation of it on the object- holder of the micro-
tome, recourse may be had to the method described by ETCLESHEIMEE in
Amer. Nat., xxvi, 1892, p. 354 (see also Journ. Boy. Mic. Soc., 1892,
p. 563). The object is imbedded in one of the metal boxes described in
§ 273. The box has its ends and sides perforated at regular intervals by
small opposite holes. Silk threads are passed through these holes from side
to side, stretched, and kept tight by sticking them to the sides of the box by
means of a drop of celloidin, leaving a length of a couple of inches hanging
loose. The loose ends are soaked in thin celloidin solution with which lamp-
black has been mixed. The object is arranged in position on the framework
formed by the taut threads in the box, the mass is poured in, and the whole
is hardened. After hardening, the celloidin holding the ends of the threads
is dissolved by means of a drop of ether, and the lampblacked ends are pulled
through the box. This leaves adhering to the bottom of the mass a series
of black lines which form useful orientation points.

301. Hardening. — This is logically the next step, but as a
matter of fact is frequently begun before. For the different
processes of the collodion method so run into one another tbat
it is difficult to assign natural lines of demarcation between
them.

HAEDENING. 197

The objects being imbedded, and in the stage at which we
left them at the end of § 299, the treatment should be as
follows : — As soon as the added thick collodion (of which only
just enough to cover the object should have been taken) has
so far sunk down that the object begins to lie dry, fresh thick
solution is added, and the whole is left as before. Provision
should be made for slow evaporation, either in one of the ways
above indicated, or, which is perhaps better, by setting the
objects under an hermetically fitting bell- jar, which is lifted for
a few seconds only once or twice a day. I have sometimes
found it advantageous to set the objects under a bell-jar
together with a dish containing alcohol, so that the evapora-
tion is gone through in an atmosphere of alcohol. This is
especially indicated for very large objects.

When the mass has attained a consistency such that the
ball of a finger no longer leaves an impress on it, it should be
scooped out of the dish or mould, or have the paper removed
if it has been imbedded in paper, and be submitted to the
next stage of the hardening process. (If the mass is found
to be not quite hard enough to come away safely it should be
put for a day or two into weak alcohol, 30 to 70 per cent.)

Several methods are available for the definitive hardening
process. One of these is the chloroform method, due to VIAL-
LANES (Rech. sur I* Hist, et le Dev. des Insectes, 1883, p. 129).
I recommend this method for small objects, because I find it
more certain and more rapid than the alcohol method, and
preferable on account of a superior consistency it gives to the
mass. (ScHiEFFEKDECKER does not find this, v. Zeit.f. wiss. Mik.,
v, 4, 1888, p. 506.) For large objects the method is inferior
to the alcohol method, because the rapid hardening of the
external layers is an obstacle to the diffusion necessary to the
hardening of the inner layers.

The method consists in bringing the objects into chloroform.
" Under the influence of this reagent the collodion coagulates
into a mass having the consistence of wax, but having also
an elasticity that renders it unbreakable, and having besides
the precious quality of being admirably transparent, and
possessing exactly the index of refraction of glass/'

In some cases a few hours' immersion is sufficient to give
the requisite consistence. In no case have my specimens re-
quired more than three days. But the length of time required

198 COLLODION AND OTHER IMBEDDING METHODS.

varies in a very inexplicable way, so that no rule can be given.
The collodion frequently becomes opaque on being put into
the chloroform, but regains its transparency after a time.

Small objects may be hardened by chloroform without pre-
liminary hardening by evaporation. All that is necessary is
to expose the mass to the air for a few seconds until a mem-
brane has formed on it, and then bring it into chloroform. If
the mass is in a test-tube this may be filled up with chloroform,
and left for two or three days. By this time the collodion
mass will be considerably hardened, and also somewhat shrunk,
so that it can be shaken out of the tube. It is then brought
into fresh chloroform in a larger vessel, where it remains for
about six days, after which time it is generally ready for
cutting.

Good chloroform is a necessity, as the reaction cannot be
obtained with samples of chloroform that are not free from
water.

The following rapid method, communicated to me by Prof.
GILSON, has the advantage of being the most expeditious of
any. The object is dehydrated, soaked in ether, and brought
into a test-tube with collodion or thin celloidin solution.
The tube is dipped into a bath of melted paraffin, and the col-
lodion allowed to boil (which it does at a very low tempera-
ture) until it has become of a syrupy consistence. The mass
is then turned out, mounted on a block of hardened celloidin,
and the whole hardened in chloroform or in a mixture of chlo-
roform and cedar oil for about an hour. It is then cleared in
cedar oil (if hardened in pure chloroform; special clearing
will not be necessary if it has been hardened in the mixture).
It may now be fixed in the microtome and cut, using cedar oil
to wet the knife, and cover the exposed surface of the object
after each cut.

The more commonly employed hardening method is the
alcohol method. The objects are thrown into alcohol and left
there until they have attained the right consistency (one day
to several weeks). The bottle or other vessel containing the
alcohol ought not to be tightly closed, but should be left at least
partly open.

The strength of the alcohol is a point on which the practice
of different writers diifers greatly. Some take very weak
alcohol ; so KOLLETT, one third alcohol (see ROLLETT'S Unters.

PRESERVATION. 199

lib. d. Ban d. quergestr. Muskelf., 1885; or Zeit. f. wiss Mik.,
iii, 1, 1886, p. 93). Others take equal volumes of absolute
alcohol and water. SCHIEFFERDECKEE (Zeit. f. iviss. Mik., v,
4, 1888) recommends alcohol of 50 to 60 per cent. ; THOMA,
alcohol of 82 per cent. (0*842 sp. gr. ; see Journ. Roy. Mic. 80 c.,
1883, p. 305); DUVAL, alcohol of 36° (= 90 per cent., Journ.
de Microgr., 1888, p. 197). The question may now be con^
sidered to be finally settled by experiments, specially directed
to the clearing up of this point, made by BUSSE (Zeit. /. wiss.
Mik., ix, 1, 1892, p. 49). Busse finds that alcohol of about
85 per cent, is the best, both as regards the cutting consistency
and the transparency of the mass. Care must be taken to keep
the mass moist whilst cutting, as it dries by evaporation very
quickly.

Lastly, the mass may be frozen. After preliminary hard-
ening by alcohol it is soaked for a few hours in water in
order to get rid of the greater part of the alcohol (the alco-
hol should not be removed entirely, or the mass may freeze
too hard) . It is then dipped for a few moments into gum
mucilage in order to make it adhere to the freezing plate,
and is frozen. The sections are brought into warm water.
If the mass have frozen too hard, cut with a knife warmed
with warm water.

A paper has been lately written by FLORMAN (Zeit. f. wiss. Mik., vi, 2,
1889, p. 184) to recommend that the definitive hardening should be done
without the aid of alcohol or chloroform, by simply cutting out the blocks,
turning them over, and carefully continuing the evaporation process in the
way described above. No doubt the author is right in claiming for this pro-
cess a superior degree of hardening of the mass ; but I doubt whether it is
possible to carry the hardening much beyond the point attained by the
chloroform or alcohol method without incurring a degree of shrinkage that
must be destructive of the scientific value of the preparations.

302. Preservation. — The hardened blocks of collodion may
be preserved till wanted in weak alcohol (70 per cent.). They
may also be preserved dry by dipping them into melted
paraffin (APATHY, Zeit. f. wiss. Mik., v, 1, 1888, p. 45).

Reference numbers may be written with a soft lead pencil
on the bottom of the paper trays, or with a yellow oil pencil
on the bottom of the watch-glasses in which the objects are
imbedded. On removal of the paper from the collodion after
hardening the numbers will be found impressed on the
collodion.

200 COLLODION AND OTHER IMBEDDING METHODS.

303. Cutting. — If the object has not been stained before
imbedding, it will form so transparent a mass with the
collodion that the arrangement of the object and sections in
the right position may be rendered very difficult. It is,
therefore, well to stain the collodion lightly, just enough to
make its outlines visible in the sections. This may be done
by adding picric acid or other suitable colouring matter
dissolved in alcohol to the collodion used for imbedding, or
to the bergamot oil used for clearing.

To fix a collodion block to the microtome, proceed as fol-
lows. Take a piece of soft wood, or, for very small objects,
pith, of a size and shape adapted to fit the holder of the
microtome. Cover it with a layer of collodion, which you
allow to dry. Take the block of collodion, cut a slice off the
bottom, so as to get a clean surface ; wet this surface first
with absolute alcohol, then with ether (or allow it to dry), place
one drop of very thick collodion on the prepared wood or pith,
and press down tightly on to it the wetted or dried surface
of the block of collodion. Then throw the whole into weak
(70 per cent.) alcohol for a few hours (or even less), or into
chloroform for a few minutes, in order that the joint may
harden.

Dr. LINDSAY JOHNSON informs me that he finds it very
convenient to take for this purpose the cement used by metal
turners for fastening metal objects on to boxwood chucks.
The exact composition of this cement varies somewhat, but
an average one is — beeswax, 1 part ; rosin, 2 parts. To use
it, you must get the block of celloidin perfectly dry at the
bottom, then warm the object-holder slightly, if possible,
over a flame; drop on to it a few drops of melted cement,
and press on to it the block of celloidin, which will be firmly
fixed as soon as the cement is cool — that is, in a few
seconds.

For objects of any considerable size it is important not to
use cork for mounting on the microtome, especially if the
object-holder be a vice ; for cork bends under the pressure of
the holder, and the elastic collodion bends with it, deforming
the object. I have seen large embryos so deformed in this
way that the sections obtained were true calottes, segments of
a sphere. If the object-holder be of the cylinder type, as in
the later forms of the Thoma microtome, the above-described

CUTTING. 201

accidents will be less likely to happen, and a good cork may
be used ; but even then, I think, wood is safer.

Sections are cut with a knife kept abundantly wetted with
alcohol (of 50 to 85 or even 95 per cent.). Some kind of
drip arrangement will be found very useful here. Apathy
recommends that the knife be smeared with yellow vaselin ;
it cuts better, is protected from the alcohol, and the mobility
of the alcohol on the blade is lessened.

The knife is set in as oblique a position as possible.

Very brittle sections may be collodionised as explained
above (§ 282).

The sections are either brought into alcohol (of 50 to 85 or
95 per cent.) as fast as they are made ; or if it be desired to
mount them in series they are treated according to one of the
methods described below, in the chapter on " Serial Section
Methods."

The above process is the one that has been until lately the
usual one ; but some workers prefer not to cut in the wet
way with alcohol, as above described, but to clear before
cutting. BUMPUS (Amer. Natur., xxvi, 1892, p. 80 ; see Journ.
Roy. Mic. Soc., 1892, p. 438) advises clearing the mass, after
hardening in chloroform, with white oil of thyme or other
suitable clearing agent (see below, § 305). After clearing,
the under surface of the block of mass is washed with ether,
and cemented with thick celloidin solution to a block of wood
for cutting in the manner described above, the whole being
thrown into chloroform for a few minutes to harden the joint.
The knife is wetted with the clearing oil, and the same oil is
employed for covering the exposed surface of the object after
each cut. Similar recommendations are made by EYCLESHEIMEE
(op. cit., pp. 354, 563), carbolic acid, or glycerin, or the
mixture given § 305 being suggested for clearing. Professor
GILSON writes me that he has for some time past adopted the
practice of clearing before cutting with cedar oil, as described
above, § 301. I have not had the opportunity of trying this
modification of the usual process. It certainly wears the
look of being, at least for objects that have been stained
before imbedding, distinctly an improvement.

304. Staining. — The sections may now be stained as desired,
either loose, or mounted in series on slides or on paper as

202 COLLODION AND OTHER IMBEDDING METHODS.

described below. It is not in general necessary, nor indeed
desirable, to remove the mass before staining, as it usually
either remains colourless or gives up the stain on treatment
with alcohol. But some of the anilin dyes and some other
colours stain it strongly, and are not removed with suffi-
cient completeness by the processes of dehydration and
clearing. If it be desired to employ these, the mass may be
removed by treating the sections, either loose in a watch-glass,
or mounted on a slide with Mayer's albumen (which will,
however, generally hold the stain just as obstinately as the
collodion), with absolute alcohol or ether.

305. Clearing and Mounting. — You may mount in glycerin
without removing the mass, which remains as clear as glass
in that medium.

You may mount in balsam, also without removing the mass,
which does no harm, and serves the useful purpose of holding
the parts of the sections together during the manipulations.
Dehydrate in alcohol of 95 or 96 per cent, (not absolute, as
this attacks the collodion). NIKIFOEOW (Zeit. f. wiss. Mik.}
viii, 2, 1891, p. 189) recommends a mixture of equal parts of
alcohol and chloroform. Clear with a substance that does not
dissolve collodion. The clearing agents most recommended
are origanum oil (01. Origan. Oretici should be taken, not 01.
Orig. Gallici ; but see, as to this reagent, the remarks in
Chap. XVIII, § 348), bergamot oil (said to make sections
shrink somewhat), oil of sandal-wood, lavender oil, oil of cedar-
wood (safe and gives excellent results, but acts rather slowly),
chloroform, xylol or benzin (may make sections shrink if not
well dehydrated), or Dunham's mixture of three or four parts
of white oil of thyme with one part of oil of cloves. (As to
oil of thyme, see also "Origanum Oil" in Chap. XVIII, § 348.)

Some specimens of clove oil dissolve collodion very slowly, and may be
used, but I would not be understood to recommend it. The action of origanum
oil varies much, according to the samples ; some sorts do not clear the collo-
dion, others dissolve it, others pucker it. MINOT (Zeit.f. wiss. Mik., iii, 2,
1886, p. 175) says that Dunham's mixture "clarifies the sections very readily
and softens the celloidin just enough to prevent the puckering, which is so
annoying with thyme alone."

Carbolic acid has been recommended. WEIGEET (Zeit. f. wiss. Mik., iii,
4, 1886, p. 480) finds that a mixture of 3 parts of xylol with 1 part of
carbolic acid (anhydrous) clears well. But it must not be used with the

DOUBLE IMBEDDING IN COLLODION AND PAKAFFIN. 203

basic anilin stains, as it discolours them. For these, anilin oil may be used
with the xylol, in the place of carbolic acid.

Anilin oil clears well (it will clear from 70 per cent, alcohol), but unless
thoroughly removed the preparation becomes yellowish brown. It may be
removed by soaking in chloroform for twenty-four hours (see VAN GIBSON,
Amer. Mon. Mic. Journ., 1887, p. 49, or Journ. Roy. Mic. Soc., 1887,
p. 519, for a review of these clearing agents ; see also § 353).

Beech wood kreasote has been recommended (by M. Flesch).

EYCLESHEIMEE (Amer. Nat., xxvi, 1892, p. 354 ; Journ. Roy. Mic. Soc.,
1892, p. 565) advises a mixture of equal parts of bergamot oil, cedar oil, and
carbolic acid.

306. Double Imbedding in Collodion and Paraffin. — The best-
hardened collodion masses are of a more elastic consistency
than is desirable for fine section cutting. This defect may be
to a certain extent remedied as follows : — Dip the block of
hardened collodion into chloroform, and imbed it simply in
paraffin.

KULTSCHIZKY'S Celloidin-Paraffin Method. — A more thorough-
going procedure is the following infiltration method (Zeit. f,
wiss. Hik., iv, 1, 1887, p. 48) :— After the collodion bath, the
object is soaked in oil of origanum (Oleum Origanivulg.}. It
is then brought into a mixture of origanum oil and paraffin,
heated to not more than 40° C., and lastly into a bath of pure
paraffin.

The mass may be preserved in the dry state, and may be
cut dry.

RYDER (Queen's Micr. Bull., 1887, p. 43; Journ. Roy. Mic.
Soc., 1888, p. 512) has modified the process by substituting
chloroform for the origanum oil. He states that sections
chain more easily than even in ordinary paraffin imbedding.
They may be cleared for mounting either with chloroform or
with Weigert's xylol and carbolic acid mixture (supra, § 305 ;
Ryder says "equal parts," but Weigert's formula is as above
given).

IDE (La Cellule, vii, 1891, p. 347 ; see also Zeit. f. wiss.
Mik., ix, 2, 1892, p. 213) has employed with success the
following method : — The object is imbedded in collodion in a
tube by GILSON'S process (supra, § 301) ; the collodion is
boiled for forty minutes, then brought for fifteen minutes
(this is for small objects) into chloroform heated to 30° C.
containing one fourth part of paraffin dissolved in it, then for
ten minutes into pure melted paraffin.

204 COLLODION AND OTHER IMBEDDING METHODS.

Other Evaporation Masses.

307. Joliet's Gum and Glycerin Method (Arch. Zool. exp. ei
gen., x, 1882, p. xliii; Journ. Roy. Mic. Soc. [N.S.], ii, 1882,
p. 890) . — Pure gum arable dissolved in water to the consistency
of a thick syrup. (Solutions of gum sold under the name of
strong white liquid glue [" colle forte blanche liquide afroid"
may also be used ; they have the advantage of having a uni-
form consistency.*) Pour a little of the solution into a watch-
glass, so as not quite to fill it, add from 6 to 10 drops of pure
glycerin, stir until thoroughly mixed.

Between the limits of 6 to 10 drops of glycerin the propor-
tions most suitable to the nature of the object and to the
season of the year must be found by experimental trials. In
the winter or in rainy weather less glycerin should be taken
than in the summer or dry weather.

It is often well to soak the object in glycerin before putting
it into the mass. In this case less glycerin should be added
to the gum, in proportion to the amount of glycerin contained
in the object.

The object is imbedded in the mass in the watch-glass, and
the whole left to dry for from one to four days. When it has
assumed a cartilaginous consistency, a block containing the
object is cut out, turned over, and allowed to dry again until
wanted for use. A stove, or the sun, may be employed for
drying, but it is best to dry slowly at the normal temperature.
The block may be preserved in good condition almost indefi-
nitely, the gum, when mixed with a sufficient quantity of
glycerin, never becoming hard or brittle. It is generally
better to wait till the blocks have assumed such a consistency
that they cannot be easily bent. It is after having waited
almost a week that the author always obtained the best sec-
tions. The gum is dissolved out from the sections by means
of a drop of water on the slide. The sections are then covered,
and a drop of glycerin being added, the preparation is com-
plete as soon as the water has evaporated.

This mass has the advantage of being transparent. It
can be cut dry. Joliet employed it for Pyrosoma. A similar
mass was employed by Hertwig for Ctenophora (Jen. Zeitsch.,

* It is highly probable that these commercial preparations contain gelatin,
and perhaps some other gum besides gum arabic.

VON KOCH'S COPAL METHOD. 205

xiv, 1880, pp. 313, 314; Journ. Roy. Mic. Soc. [N.S.], ii,
p. 278).

It would probably be advantageous to add some preserva-
tive substance.

308. ROBERTSON'S Grape-sugar Method (Journ. ofAnat. and Physiol.,
xxiv, 1890, p. 230 ; Zeit.f. wiss. MiJc., vii, 1, 1890, p. 33).

Grape-sugar ...... 5 parts.

Dextrin 10 „

Boric acid .... 1 part.

Mix and add three drachms of water for each quarter of an ounce of the
mixture, and dissolve by heating to boiling-point. After cooling, add 6
drops of carbolic acid for each ounce of the solution. The directions for
using this mass are practically identical with those given by Joliet for his
gum and glycerin mass, last section.

The mass above described is advised for fresh tissues. For hardened
tissues a mass is given containing two parts of soft soap (sapo mollis) instead
of the 1 part of boric acid ; the rest as before.

309. Strieker's Gum Method (Hdb. d. Gewebel., p. xxiv). — A concen-
trated solution of gum arable. The object may be prepared in alcohol and
imbedded in the gum in a paper case. The whole is thrown into alcohol, and
after two or three days may be cut.

I have seen masses of admirable consistency prepared by this simple
method.

310. Hyatt's Shellac Method (Am. M. Micr. Journ., i, 1880, p. 8 ;
Journ. Roy. Mic. Soc.} iii, 1880, p. 320). — Prepare the object by soaking
in alcohol, and then put it for a day or two into a clear alcoholic solution of
shellac. Take a cylinder of soft wood, split it, and make a groove in one or
both of the half-cylinders sufficiently large to admit the object without
pressure. Imbed in the groove with plenty of thick shellac solution, and
tie together the two halves of the cylinder with thread. In a day or two
the shellac will be quite hard ; the cylinder is then fixed in a microtome, is
soaked with warm water, and sections made. Should the shellac prove so
opaque as to interfere with a proper examination of the sections, a drop of
borax solution will immediately remove this difficulty.

This process is intended for the purpose of making sections through hard
chitinous organs consisting of several pieces, such as stings and ovipositors,
retaining all the parts in their natural positions.

311. Von Koch's Copal Method (Zool. Anz., 2, vol. i, 1878,
p. 36). — Small pieces of the object are stained in bulk and
dehydrated with alcohol. A thin solution of copal in chloro-
form is prepared by triturating small fragments of copal in a
mortar with fine sand, pouring on chloroform to the powder
thus obtained, and filtering. The objects are brought into
a capsule filled with the copal solution. The solution is now

206 COLLODION AND OTHER IMBEDDING METHODS.

slowly evaporated by gently heating the capsule on a tile by
means of a common night-light placed beneath it. As soon
as the solution is so far concentrated as to draw out into
threads that are brittle after cooling, the objects are removed
from the capsule and placed to dry for a few days on the tile,
in order that they may more quickly become hard. When
they have attained such a degree of hardness that they cannot
be indented by a finger-nail, sections are cut from them by
means of a fine saw. The sections are rubbed down even
and smooth on one side with a hone, and cemented, with this
side downwards, to a slide, by means either of Canada balsam
or copal solution. The slide is put aside for a few days more
on the warmed tile. As soon as the cement is perfectly hard
the sections are rubbed down on a grindstone, and then on a
hone, to the requisite thinness and polish, washed with water,
and mounted in balsam.

The process may be varied by imbedding the objects un-
stained, removing the copal from the sections by soaking in
chloroform, decalcifying them if necessary, and then staining.

It is sometimes a good plan, after removing the copal, to
cement a section to a slide by means of hard Canada balsam,
then decalcify cautiously the exposed half of the specimen,
wash, and stain it. In this way von Koch was able to demon-
strate the most delicate lamellae of connective tissue in Isis
elongata.

This method was imagined in order to enable the hard and
soft parts of corals to be studied in their natural relations.
It is evidently applicable to the study of any structures in
which hard and soft parts are intimately combined. It is
certainly a method of the very greatest value.

312. Ehrenbaum's Colophonium and Wax Method (Zeit.f. wiss.
Mik., 1884, p. 414). — Ehrenbaum recommends that the objects
be penetrated by a mass consisting of ten parts of colophonium
to one of wax. The addition of wax makes the mass less
brittle. Sections are obtained by grinding in the usual way.
The mass is removed from them by means of turpentine fol-
lowed by chloroform.

313. Weil's Canada Balsam Method (Zeit. f. wiss. Mik., v, 2, 1888,
p. 200). — Balsam heated till brittle when cold, then dissolved in chloroform.
Heat the objects in the mass on a water-bath. For further details see
Journ. Roy. Mic. Soc., 1888, p. 1042.

FREEZING MASSES. 207

White of Egg Masses.

314. The White of Egg Method consists in imbedding in a white of
egg emulsion, and hardening by alcohol or by the combined action of alcohol
and heat. The method had certainly a raison d'etre at one time, as
giving results which could not then be obtained by other means. But the
method was extremely cumbrous, had many defects, and must now be con-
sidered to be entirely superseded. See Zeit. f. wiss. Mik., 1884, p. 223.
(EFNGE) ; Morph. Jahrb., Bd. ii, 3tes Heft, 1876, p. 445 (CALBEELA) ;
Zool Anz., 6, vol. i, 1878, p. 130 (SELENKA) ; Journ. Roy. Mic. Soc., 1883,
p. 304 (THOMA) ; and BECKER'S ZurAnat. d. ges. u. kranJcen

Congelation Masses.

315. The Freezing Method. — Fresh tissues may be, and are,
frequently frozen without being included in any mass, and in
certain cases very satisfactory sections can be obtained in this
manner. But the formation of ice crystals frequently causes
tearing of delicate elements, and it is better to infiltrate the
tissues with a mass that does not crystallise in the freezing
mixture, but becomes hard and tough. Gum arabic affords
such a mass. Some workers use common gum water, which
is either poured into the well of the microtome or round the
object on the object plate, according to the form of microtome
used.

316. Syrup and Gum Congelation Mass (HAMILTON, Journ. of
Anat. and Phys., xii, 1878, p. 254). — Hamilton cuts sections
(of hardened brain) in a Rutherford's freezing microtome.
The hardening reagent having been soaked out by water, the
tissues are prepared for freezing in the following manner,
which it is important to observe, otherwise it will be found
that the crystals of ice so break up the delicate nervous tissue
as to render it totally useless for minute examination. The
tissues are to be well soaked in syrup. The sugar somewhat
retards the freezing, and besides, seems to alter the manner
of crystallisation, so that instead of the ice being spicular in
form it becomes granular, and does no injury to the parts.

The syrup requires to be of a particular strength, viz. double
refined sugar, 2 ounces ; water, 1 fluid ounce.

Wash the superfluous syrup from the surface, and put into
the ordinary mucilage for an hour or so before cutting. Imbed
in the freezing microtome with mucilage in the usual way.
Float the sections into water.

208 COLLODION AND OTHER IMBEDDING METHODS.

317. Gum and Syrup Congelation Mass (CoiE, Methods of
Microscopical Research, 1884, p. xxxix ; Journ. Roy. Mic. Soc.
[N.S.], iv, 1884, p. 318).— Gum mucilage (B. P.), 5 parts;
syrup, 3 parts. (For brain and spinal cord, retinae, and all
tissues liable to come in pieces put 4 parts of syrup to 5 of
gum.) Add 5 grains of pure carbolic acid to each ounce of
the medium.

(Grum mucilage [B. P.] is. made by dissolving 4 ounces of
picked gum acacia in 6 ounces of water.)

The syrup is made by dissolving 1 pound of loaf sugar in
1 pint of water and boiling.

This medium is employed for soaking tissues previous to
freezing. They may remain in it for " any length of time,
all the year round " if desired.

The freezing is conducted as follows : — the gum and syrup
is removed from the outside of the object by means of a cloth ;
the spray is set going and a little gum mucilage painted on
the freezing plate ; the object is placed on this and surrounded
with gum mucilage ; it is thus saturated with gum and syrup,
but surrounded when being frozen with mucilage only. This
combination prevents the sections from curling up on the one
hand, or splintering from being too hard frozen on the other.
The mass ought to cut like cheese. Should freezing have
been carried too far, wait for a few seconds.

318. Dextrin Congelation Mass (WEBB, The Microscope, ix,
1890, p. 344; Journ. Roy. Mic. 8oc., 1890, p. 113).— Thick
solution of dextrin in solution of carbolic acid in water
(1 in 40). Use heat for making the solution if desired. This
medium is much cheaper than the gum and syrup mass, and,
according to Webb, possesses superior cutting qualities.

319. Gelatin Congelation Mass (SOLLAS, Quart. Journ. Mic.
Soc., xxiv, 1884, pp. 163, 164; Journ. Roy. Mic. Soc. [N.S.],
iv, 1884, p. 316). — "Instead of gum one uses gelatin jelly.
This is prepared and clarified in the usual manner. It should

set into a stiff mass when cold The tissue to be cut is

transferred from water to the melted jelly, and should remain
in it till well permeated."

The sections are transferred to a slide a? soon as cut. On
touching the glass they adhere to it. When enough sections
have been thus arranged they are covered with a drop of

OIL OF ANISEED CONGELATION MASS. 209

glycerin ; a cover is put on, and the mount closed with any
suitable cement. In process of time the glycerin will per-
meate the gelatin and convert it into glycerin jelly; this
may be hastened by placing the slide in an oven kept at about
20° to 30° C.

320. Gum- Gelatin Congelation Mass (JACOBS, Amer. Natural. ,
1885, p. 734; Journ. Roy. Mic. Soc., 1885, p. 900).— Gum
arabic, 5 parts; gum tragacanth, 1 part; gelatin, 1 part.
Dissolve in enough warm water (containing one sixth of
glycerin) to give a mass of the consistency of thin jelly when
cold.

321. White of Egg Congelation Mass (ROLLETT, Derikschr. math,
naturw. Kl k. Acad. Wiss. Wien, 1885 ; Zeit.f. wiss. Mile., 1886, p. 92). —
Small portions of tissue brought in the white of a freshly laid egg on to the
freezing stage, frozen, and cut. The knife must be well cooled.

322. Oil of Aniseed Congelation Mass (KUHNE, Centralb. f.
BaJcteriol., xii, 1892, p. 28 ; Journ. Boy. Mic. Soc., 1892, p. 706). — Soak in
oil of aniseed for twelve to twenty-four hours, freeze and cut, and remove
the oil from the sections by means of alcohol.

14

210 SERIAL SECTION MOUNTING.

CHAPTER XVII.
SERIAL SECTION MOUNTING.

323. Choice of a Method.— All the following methods are
excellent if properly carried out. I recommend for general
work the following : — For paraffin sections that have been
already stained, Schallibaum's collodion. For paraffin sections
that are to be stained on the slide, Mayer's albumen, unless
the stain to be employed be one that will stain the albumen
or if the sections be badly folded, in which case take one of
the water or alcohol methods given in the next section. For
collodion objects, one of the forms of Summers' ether-vapour
process. For very large collodion sections, Weigert's process.

Methods for Paraffin Sections.

324. The Water or Alcohol Method.— The principle of this
method is due to GAULE (Arch.f. Anat. u. Phys. \_Phys. Abth.~\,
1881, p. 156), who practised it as follows : — A slide is moistened
with alcohol, the sections are arranged on it by means of a
camel-hair brush, also moistened with alcohol; the slide is
slightly warmed so as to cause the sections to stick to the
slide ; a cover is put on, and a solution of Canada balsam in
xylol (equal parts of each) run underneath it. If the sections
are not thicker than y1^ mm. they will be clear at once, and
nothing remains but to refill the cell day by day as the xylol
evaporates, in order to have a perfect mount. If, however,
the sections are thicker than -fa mm. they will contain
more paraffin than the xylol balsam can dissolve. In that
case the excess of paraffin must be removed by means of a
drop of pure xylol (the sections being first melted on to the
slide as before), and the mount is completed by means of
xylol balsam.

Both the moistening with alcohol and the heating are
necessary for the attachment of the sections to the slide ; the

THE WATER OR ALCOHOL METHOD. 211

effect is not obtainable by means of one of these manoeuvres
alone.

This simple process is very useful for the preliminary exa-
mination of trial sections whilst cutting. It is often suffi-
cient to put on a cover, warm, withdraw the melted paraffin
by means of a cigarette paper, and run in a drop of clearing^
agent.

In the primitive form given above, G-aule's method is use-
ful enough for the purpose of mounting a small series of sec-
tions that do not require to be stained or otherwise further
manipulated on the slide. Later workers have by improve-
ments in the details of the process brought it to a state of
great perfection, so that it may now be considered a fairly
safe process for extensive series of sections, and will allow of
staining on the slide.

SUCHANNEK (Zeit. f. wiss. Mik., vii, 4, 1891, p. 464) pointed
out that the slides must be absolutely free from grease in order
that the alcohol (50 per cent.) or distilled water, which may
be used instead, may spread out in a thin and uniform layer.
Secondly, that the slides should not be warmed to more than
40° C., it being important both that the alcohol should evapo-
rate slowly and that the paraffin should only be softened , not
melted, until the evaporation is complete. HOYER had already
for this reason advised slow evaporation at the temperature of
the laboratory.

GULLAND (Journ. of Anat. and PhysioL, xxvi, 1891, p. 56;
Journ. Roy. Mic. Soc., 1892, p. 161) floats sections on to the
surface of warm water (not warm enough to melt the paraffin),
or alcohol if preferred, in a dish, and thence floats them into
position on the slide. The slide is drained, and the water
evaporated from it at a low temperature as described above
(Gulland says c< a little under 50° C.," which of course is only
a low temperature relatively to a paraffin of what I consider
an undesirably high melting-point). When the water of the
sections has evaporated completely they become more trans-
parent, and look dry. The fixation is then complete, the
paraffin may be melted and removed by means of any desired
solvent, and the sections may be mounted, or be stained in
any medium, or otherwise manipulated as desired. Thin sec-
tions will generally be fixed in about an hour ; thick ones will
require six hours or more. Here it may be pointed out that

212 SERIAL SECTION MOUNTING.

the degree of adhesion seems to depend very much on the
nature of the sections.

SCHIEFFERDECKER (Zeit. f. wiss. Nik., ix, 2, 1892, p. 202)
finds that the larger and thinner sections are, the better do
they stick, and vice versa. And Mr. ANDREW PRINGLE writes
me that he finds that tissues thoroughly fixed in chrome solu-
tions, so that their albuminoid substances have become quite
insoluble, do not adhere sufficiently without the aid of some
substance to fix them to the slide. He prefers arranging
sections on the slide with cold water, and then warming until
the sections flatten out.

HEIDENHAIN (" Feschr. Herrn. v. Kolliker gewidm.," &c.,
1892; see Zeit.f. wiss. Mik., ix, 2, 1892, p. 201) also proceeds
in this way, and advises that the heating be not carried to
above 35° C., at which temperature several hours at least are
necessary to ensure fixation. He also finds that water is pre-
ferable to alcohol, which is too mobile on the slide and eva-
porates too quickly. DURHAM'S method (Quart. Journ. Mic.
Sci., xxxiii, 1891, p. 116; Journ. Roy. Mic. Soc., 1892, p. 293)
is the same as the last described, with the exception that he
uses 70 per cent, alcohol instead of water.

325. Schallibaum's Collodion Method (Arch. f. mile. Anat.,
1883, p. 565). — One part of collodion is shaken up with three
to four volumes (according to the consistency of the collodion)
of clove oil or lavender oil. This should give a clear solution.
A little is spread thinly on a slide with a small brush. After
arranging the sections on the prepared surface, warm over a
water-bath, gently, until the clove oil has evaporated (five to
ten minutes). The sections are then found to be fixed, and
can be treated for days with turpentine, chloroform, alcohol,
and watery fluids, without becoming detached. The advan-
tage of this method is that it allows of staining on the slide.
If after staining any cloudiness should appear between the
sections, dehydrate the slide and treat it several times with
absolute alcohol and turpentine, warming it gently the while ;
or brush the space between the sections repeatedly with a
brush moistened with clove oil. This cloudiness only arises
from the collodion solution having been taken too concentrated,
or having been laid too thick on the slide.

I find it is not necessary to evaporate over a water-bath.

SCHALLIBAUM'S COLLODION METHOD. 213

It is sufficient to hold the slide over a spirit lamp until the
paraffin has melted and the clove oil has collected in drops
between the sections. Schallibaum has stated elsewhere that
long evaporation of the slide is necessary if the sections are
to be secured firmly enough to allow of staining on the slide.
That is not so. What is necessary is that the paraffin and
clove oil be thoroughly removed from contact with the sec-
tions ; and that can be done in a second (as was shown me by
Professor v. Korotneff). Warm the slide over a flame, and
whilst the paraffin is still melted hold it close before your lips
and blow down on it vigorously. The paraffin and clove oil
are scattered right and left over the slide, leaving the sections
high and dry.

Personally I do not consider Schallibaum' s method so safe
as Mayer's albumen (and some other methods) for objects
that are to be stained on the slide. I recommend it for
already stained objects, because it is found to work very
pleasantly. Its great defect is that it does not readily lend
itself to any device for the flattening out of folded sections.
I recommend xylol or naphtha for clearing, in preference to
turpentine.

Good collodion is essential in this process.

STRASSER (Zeit. f. wiss. Mik.y iv, 1, 1887, p. 45) recommends
a mixture of 2 parts collodion, 2 parts ether, and 3 parts
castor oil; or (ibid., vi, 2, 1889, p. 153) 2 parts of collodion
with one of castor oil, the sections being painted over with a
thicker solution, viz. collodium concentratum duplex 2 to 3
parts, castor oil 2 parts, and the slide being plunged at once,
without warming, into a bath of turpentine, in which it
remains till the paraffin is dissolved (two to ten hours, some-
what less if the whole be put in a stove). The turpentine
suffices to harden the collodion (benzin, benzol, and chloro-
form have the same effect) .

GALLEMAERTS (Bull. Soc. Beige de Micro., xv, 1889, p. 56 ;
Zeit. f. wiss. Mik., vi, 4, 1889, p. 493), following DRASH, em-
ploys a saturated solution of gun-cotton in acetone, diluted to
the requisite thinness with absolute alcohol.

GAGE prefers preparing slides with a layer of pure collodion,
which is allowed to dry, and is rendered adhesive at the instant
of using by brushing with clove oil.

SUMMERS (Amer. Mon. Hie. Journ., 1887, p. 73; Zeit. f. wiss.

214 SERIAL SECTION MOUNTING.

.j iv, 4, 1887, p. 482) also employs a dry layer of collodion,
which he renders adhesive after the sections are arranged on
it, by wetting with a mixture of equal parts of alcohol and
ether. As soon as the mixture has evaporated, the sections
are found to be fixed.

326. Strasser's Collodion-Paper Method (Zeit.f. wiss. Mile., iii, 3,
1886, p. 346). — This is an extremely complicated modification of Weigert's
collodion method for celloidin sections (post, § 340). It is certainly too
elaborate to have any chance of supplanting, for ordinary work, the classical
methods of preparing series on slides. And it is only adapted for use with
Strasser's very complicated " Schnitt-Aufklebe-Mikrotom," or automatic
section-fixing microtome. The descriptions of the process and the microtome
already published by Strasser would fill a small volume, and cannot be
properly abstracted in the space at my disposal here. See the original
papers in Zeit.f. wiss. Mik., iii, 3, 1886, p. 346; vi, 2. 1889, p. 154 ; vii, 3,
1890, p. 290 ; ib., p. 304; and ix, 1, 1892, p. 8 ; see also a very short abstract
of the last paper in Journ. Roy. Mic. Soc., 1892, p. 703, in which is a figure
of the "Schnitt-Aufklebe-Mikrotom."

327. The Shellac Method (GIESBRECHT, Zool Am., 1881, p. 484).—
Prepare a stock of slides covered with a thin and even film of shellac. This
is done as follows : — Make a not too strong solution of brown shellac in abso-
lute alcohol, filter it thoroughly ; warm the slides, and spread over them a
layer of shellac by means of a glass rod dipped in the solution and drawn
once over each slide. Let the slides dry.

Just before beginning to cut your sections take a prepared slide and brush
it over very thinly with kreasote applied by means of a brush ; this forms a
sticky surface on which the sections are now arranged one by one as cut,
care being taken to bring them on to the slide with as little surrounding
paraffin as possible.

When all the sections are arranged the slide is heated on a water-bath for
about a quarter of an hour at the melting-point of the paraffin ; this causes
the paraffin to run down into a thin layer, and allows the sections to fall
through it and come into close contact with the shellac film, whilst at the
same time it evaporates the kreasote.

The slide is allowed to cool, and the sections are now found to be firmly
fixed in the shellac. The paraffin is dissolved away by dropping turpentine
on to the sections, which are then mounted in Canada balsam. There is no
danger of the sections being floated away by the turpentine, because turpen-
tine does not dissolve shellac.

In the note in the Zool. Am. above quoted, the shellac solution is stated
to be prepared with common brown shellac (choosing, of course, by prefer-
ence the paler sorts), on account of the insolubility of white shellac in
alcohol. In the Mitth. d. Zool. Stat. of Naples, of the same year, " bleached
white shellac" is recommended to be dissolved as before, in absolute alcohol.
In the Journ. Roy. Mic. Soc. (N.S.), vol. ii, 1882, p. 888, it is stated (on

hose authority is not clear) that the solution is made by mixing 1 part of

MAYER'S ALBUMEN. 215

bleached shellac with 10 parts absolute alcohol, and filtering. In the same
place it is added that " Dr. Mark uses the bleached shellac in the form in
which it is prepared for artists as a ' fixative ' for charcoal pictures. It is
perfectly transparent, and a film of it cannot be detected unless the surface
is scratched. He attaches a small label to the corner of the slide, which
serves for the number of the slide and the order of the sections, and at the
same time marks the shellac side otherwise not distinguishable." (The
latter object is better attained by gumming a paper square, or spinning a ring
with ink, in the centre of the unprepared surface of the slide. The disc or
ring then serves at the same time for centring the group of sections.)

The account given in the Mitth. d. Zool. Stat. further varies in one other
detail from that given in the Zool. Anz. It directs that the shellac slides
be brushed before cutting with oil of cloves, instead of kreasote, the slide
being slightly warmed before brushing.

The white shellac of commerce is sometimes not easily soluble in alcohol.
KINGSLEY (see WHITMAN'S Methods in Microscopical Anat., p. 117) recom-
mends that brown shellac be taken, and bleached by exposure to the sun.

CALDWELL (Quart. Journ. Mic. Soc. [N.S.], Ixxxvii, 1882, p. 336)
simplifies the method by merely brushing over the slide (thinly) at the
moment of using with a strong solution of shellac in anhydrous kreasote.
(To make the solution, warm the kreasote.)

In both the foregoing methods it often happens that the shellac becomes
granular or cloudy on the slide. P. MAYER attributes this to the kreasote
or clove oil, and proposes to remedy it by employing carbolic acid instead
(Amer. Natural, 1882, p. 733 ; Zeit. f. wiss. Mik., iv, 1, 1887, p. 77 ;
Journ. Roy. Mic. Soc., 1885, p. 910). Powdered white shellac is heated
with crystallised carbolic acid till it dissolves, and the solution filtered
warm.

But more recently (Intern. Monatschr. f. Anat., &c., 1887, H. 2 ; Zeit. f.
wiss. Mik., iv, 1, 1887, p. 77) the same author, on the ground that hot car-
bolic acid attacks some tissues, recommends another method. Slides are pre-
pared with alcoholic shellac according to Griesbrecht's plan. The sections
are arranged on the dry film and gently pressed down on to it, then exposed
for half a minute to vapour of ether.

Chloroform softens shellac ; therefore chloroform balsam is not a safe
mounting medium for sections fixed by these methods.

These methods do not allow of staining on the slide.

I feel bound to say that I am at a loss to understand by what virtue
it is that the shellac method continues to survive, as it certainly seems to do,
in the face of far more convenient and efficient processes.

328. Mayer's Albumen (Mitth. Zool. Stat. Neapel, iv, 1883;
Journ. Roy. Mic. Soc. [N.S.], iv, 1884, p. 317; Intemat.
Monatschr. f. Anat., 1887, Heft 2 ; Journ. Eoy. Mic. Soc., 1888,
p. 160). — White of egg, 50 c.c.; glycerin, 50 c.c.; salicylate
of soda, 1 grm. Shake them well together, and filter into a
clean bottle.

FOL (Lehrb., p. 134) takes whipped white of egg, filters it

216 SERIAL SECTION MOUNTING.

through a Bunsen filter, and adds the glycerin and a little
camphor or carbolic acid.

I find it convenient to beat up the egg with a little water
before adding the glycerin and filtering; the salicylate
should be dissolved in the water in the first instance.

According to my experience carbolic acid is perfectly
efficient as a preservative, but is not to be recommended
because it precipitates a great deal of the albumen.

A thin layer of the mixture is spread on a cold slide with a
fine brush, and the sections laid on it and warmed for some
minutes on a water-bath. As the parafiin in the sections
melts it carries the albumen away from them, and this is one
of the advantages of the method. The sections may be treated
with turpentine, alcohol, and aqueous or other stains without
any danger of their moving.

It is not necessary to warm the slide ; the parafiin can be
removed in the cold by putting the slide into toluol, xylol, or
the like. But the slide should be very thoroughly treated with
alcohol after removal of the paraffin, in order to get rid of the
glycerin, which will cause cloudiness if not perfectly removed.

The function of the glycerin is merely to keep the layer of
albumen moist.

This method allows of the staining of sections on the slide
with anilin stains, which is seldom practicable with Schalli-
baum's method, as the collodion stains with most anilin stains,
and does not yield up the colour to alcohol, which the albumen
generally does.

This method can be combined with the water process for
flattening out sections (§ 280), as described by HENNEGUY
(Journ. de VAnat. et de la PhysioL, 1891, p. 398). A drop of
water is spread by means of a glass rod on a slide prepared
with white-of-egg mixture, the sections are arranged on it,
the whole is warmed (not to melting-point of the paraffin)
until the sections flatten out, the water is then evaporated off
at a temperature of about 40° C., and as soon as it has entirely
disappeared the paraffin is melted, and the slide further
treated as above described.

According to my experience the albumen method is abso-
lutely safe, and is the one that should in general be preferred
for staining on the slide, more especially for staining with
anilins by Flemming's method.

FRENZEL'S GUM METHOD. 217

It has been stated (VossELER, Zeit.f. wiss. Mik., vii, 4, 1891,
p. 457) that Mayer's albumen generally "goes bad," and
loses its adhesive power after about six months' keeping.
The only remedy for this is to make up fresh mixture every
few months, as no change of antiseptics has had any effect in
improving its keeping qualities.

329. Flogel's Gum Method (Zool. Anz., 1883, p. 565).— Make
a solution of one part gum arabic in twenty parts water, filter,
and add a little alcohol to prevent the formation of mould.
Slides are prepared by pouring the solution over them, and
draining. (It is important that the slides be so perfectly
clean as to be evenly wetted all over by the gum solution.)
Sections may now be cut and laid on the gum surface before
it has become dry, and floated into the proper position ; this
is the best plan for sections of y^- mm. thickness, and for
large sections. For thinner and small sections it is best to
take slides that have completely dried, arrange the sections
dry on the gum film, and then breathe on it until the gum has
become sticky.

A very neat method for cases in which it is not required to
treat the slide with watery fluids.

WADDINGTON (Journ. Quek. M. Club, vi, 1881, p. 199 ; Journ. Roy. Mic.
Soc. [N.S.], i, 1881, p. 704) gives the following process for preparing
" arabin," a purified gum arabic which lias the advantage of not presenting
a granular appearance under the microscope as ordinary gum arabic does.

Dissolve clear and white gum arabic in distilled water to the consistency
of thin mucilage. Filter. Pour the filtrate into rectified alcohol, and si mice
well ; the arabin separates as a white pasty mass. Place it on filter-paper,
and wash with pure alcohol until the washings are free from water. Dry.

The white powder thus obtained should be dissolved in distilled water and
filtered twice. It may then be placed on slides, which are drained, dried,
and put away till wanted. In this condition it may be preserved indefinitely.

330. Frenzel's Gum Method (Arch. f. mik. Anat., Bd. xxv,
1885, p. 51).— Gum arabic is dissolved in water to the consist-
ency of a thin mucilage, and to this is added aqueous solution
of chrome-alum. An excess of the latter does no harm.
Finally add a little glycerin and a trace of alcohol (1. c., p. 142) .
The slide is prepared with this in the usual way, the sections
(either cut dry or in the wet way) are gently pressed on to it
with a brush and slightly melted on, and heated for at most a,
quarter of an hour at a temperature of 30° to 45° C., which

218 SERIAL SECTION MOUNTING.

suffices to render the gum insoluble. This layer has the ad-
vantage of not staining with the majority of staining fluids;
fuchsin and safranin are the only ones that stain it to a
harmful degree. In the other anilins, and in carmine or
haematoxylin, it does not stain. Watery stains (it is stated)
may be used with it.

331. Born and Wieger's Quince-Mucilage (Zeit. f. wiss. Mik.,
1885, p. 346). — To two volumes of the ordinary pharmaceutical
quince-mucilage add one volume of glycerin and a trace of
carbolic acid. Spread in a thin layer on a carefully cleaned
slide, and arrange the sections on the moist surface. Heat
for twenty minutes at a temperature of 30° to 40° C. After
removal of the paraffin by turpentine the slide is brought
for half an hour into absolute alcohol. You may then mount,
or pass through successive alcohols, and stain. Alkaline
staining fluids must be avoided, as they soften the mucilage
and cause the sections to become detached.

\. GEAVIS'S Agar-agar (Bull. Soc. Beige de Micr., xv, 1889, p. 72 ;
Zeit. f. wiss. Mile., vi, 4, 1889, p. 494).— Solution of agar-agar in 1000
parts of water, to be used as in last section. The formula seems to me
superfluous.

333. GEAY'S Qelatin Process (The Microscope, ix, 1889, p. 325 ; Journ.
Boy. Mic. Soc., 1890, p. 117).— Solution of gelatin in 100 parts of water.
Use as gum arabic solution, taking care not to melt the paraffin ; let the slide
dry spontaneously overnight, and remove the paraffin with a suitable solvent ;
remove the solvent with alcohol, and then treat for five minutes with 2 per
cent, solution of potassium bichromate to render the gelatin insoluble. Stain
as desired, or mount.

Methods for Watery Sections.

334. Fol's Gelatin (Foi, Lehrb., p. 132).— Four grammes of
gelatin are dissolved in 20 c.c. of glacial acetic acid by heating
on a water-bath and agitation. To 5 c.c. of the solution add
70 c.c. of 70 per cent, alcohol and 1 to 2 c.c. of 5 per cent,
aqueous solution of chrome-alum. Pour the mixture on to
the slide and allow it to dry. In a few hours the gelatin
passes into the insoluble state. It retains, however, the
property of swelling and becoming somewhat sticky in pre-
sence of water. The slide may then be immersed in water
containing the sections, these can be slid into their places,
and the whole lifted out ; the sections will be found to be fixed
in their places.

APATHY'S OIL OF BERGAMOT METHOD. 219

This method is especially useful for sections made under
water, large celloidin sections amongst others.

335. POLI (Malpighia, ii, 1888, 2, 3 ; Zeit. f. wiss. Mik., v, 3,
1888, p. 361) arranges sections on a layer of melted Kaiser's
gelatin (supra, § 294), adds glycerin, and covers. See. also
supra, § 333.

336. Frenzel and Threlfall's Gutta-percha (or Caoutchouc)
Method (Zool. Anz., 1883, pp. 51, 301, and 423). — This extremely elegant
method is not perfectly safe; the gutta-percha film being liable to tear ; and
is now, I believe, very generally abandoned.

Methods for Celloidin Sections.

337. Summers' Ether Method (Amer. Hon. Mic. Journ., 1887,
p. 73; Zeit. f. wiss. Mik., iv, 4, 1887, p. 482; Journ. Roy.
Mic. Soc., 1887, p. 523). — Besides the method given above
(§ 325), which is applicable to celloidin sections, but is
needlessly complicated, Summers recommends the following
simpler method : — Place the sections in 95 per cent, alcohol
for a minute or two, arrange on the slide, and then pour over
the sections sulphuric ether vapour, from a bottle partly full
of liquid ether. The celloidin will immediately soften and
become perfectly transparent. Place the slide in 80 per cent,
alcohol, or even directly in 95 per cent, if desired. The
sections will be found to be firmly fixed, and may be stained
if desired.

SCHIEFFERDECKEB (Zeit. f. iviss. Mik., v, 4, 1888, p. 507) re-
commends that the slide be one that has been previously pre-
pared with a layer of collodion, if it is desired to stain on the
slide ; but if not, a clean slide is perfectly sufficient. The
slide may of course be treated with ether vapour in a prepara-
tion glass or similar arrangement.

338. Apathy's Oil of Bergamot Method (Mitth. Zool. Stat.
Neapel, 1887, p. 742; Zeit. f. wiss. Mik., v, 1, 1888, p. 46,
and v, 3, 1888, p. 360; Journ. Roy. Mic. Soc., 1888, p. 670).
— Cut with a knife smeared with vaselin (§ 303) and wetted
with 95 per cent, alcohol. Float the sections, as cut, on
bergarnot oil (must be green, must mix perfectly with 90 per
cent, alcohol, and must not smell of turpentine). The sections
spread themselves out on the surface of the oil ; before they
sink, each one is pushed by means of a needle into its place

220 SERIAL SECTION MOUNTING.

on a slip of tracing-paper dipped into the oil. (A good size
for the paper is about as broad as the slide, and three times
as long as the cover.) When the requisite number of sections
has been arranged on the paper, you drain the paper, dry the
under side of it with blotting-paper, turn it over, and gently
press it down with blotting-paper on to a carefully dried slide.
Kemove the paper by rolling it up from one end. The sections
remain adhering to the slide, and may have the remaining
bergamot oil removed from them by means of a cigarette
paper. If they are already stained, nothing remains but to
add balsam and a cover.

In the case of unstained or very small objects, it is well to add a little
alcoholic solution of safranin to the bergamot oil. The celloidin of the sec-
tions becomes coloured in it in a few seconds, and makes them readily visible.
The colour disappears after mounting in a few days.

If the sections are to be stained, the slide after removal of
the bergamot oil is exposed for a few minutes to the vapour
of a mixture of ether and alcohol, then brought into 90 per
cent, alcohol, and after a quarter of an hour therein may be
stained in any fluid that contains 70 per cent, alcohol or more.

If it be desired to stain in a watery fluid, care must have
been taken when arranging the sections to let the celloidin of
each section overlap that of its neighbours at the edges, so
that the ether vapour may fuse them all into one continuous
plate. This will become detached from the slide in watery
fluids, and may then be treated as a single section.

339. Apathy's Series-on-the-Knife Method (Zeit.f.
vi, 2, 1888, p. 168). — The following is in some respects more
convenient than the oil of bergamot method. The knife is
well smeared with yellow vaselin rubbed evenly on with the
finger, and is wetted with alcohol of 70 to 90 per cent. As
fast as the sections are cut they are drawn with a needle or
small brush to a dry part of the blade, and there arranged in
rows, the celloidin of each section overlapping or at least
touching that of its neighbours. The rows are of the length
of the cover-glass, and are arranged one under the other so
as to form a square of the size of the cover-glass. When a
series (or several series, if you like) has been thus completed,
the sections are dried by laying blotting-paper on them
(there is no risk of their becoming attached to it, as they are

WEIGERT'S COLLODION METHOD. 221

held down by the vaselin). The series is then painted over
with some of the thickest celloidin solution used for imbed-
ding, is allowed to evaporate for five minutes in the air, and
is then either wetted with 70 per cent, alcohol, and allowed
to remain whilst cutting is proceeded with, or (if no more
sections are to be cut, or if the knife is now full) the knife is
removed and brought for half an hour into 70 per cent, alcohol. -
This hardens the celloidin around the sections into a con-
tinuous lamella, which can be easily detached by means of a
scalpel, and stained, or further treated as desired. It is well
to bring it at once on to a slide, moisten the edges of the
celloidin plate with ether and alcohol mixture, so that it may
not become detached, and bring the whole into the staining
solution.

340. Weigert's Collodion Method (Zeit. f. wiss. Mik., 1885,
p. 490). — Sections are cut wet with alcohol. Care should be
taken not to have so much alcohol on the knife as to cause
the sections to float. Prepare a slip of porous but tough
paper (Weigert recommends " closet paper "), of about twice
the width of the sections. Soak it in alcohol, take it by both
ends, stretch it slightly, and lower it on to the section that
is on the knife. The section will adhere to the paper, and is
taken up by moving the slip horizontally or slightly upwards,
away from the edge of the knife. Take up the first section
towards the end of the paper that you hold in your left hand,
and let the remaining sections follow in order from left to
right. After each section has been taken up, the slip is
placed, whilst the next section is being cut, with the sections
upwards on a moist surface prepared by arranging several
layers of blotting-paper, covered with one layer of closet
paper, in a plate, and saturating the whole with alcohol.
When all the sections have been arranged on the slip, you
pass to the next stage of the process, the collodionisation of
the series.

This is done in two steps. The first of these consists in
transporting the series on to a plate of glass prepared with
collodion. The plate is prepared beforehand by pouring on
to it collodion and causing it to spread out into a thin layer,
as photographers do, and allowing it to dry. (A number of
the plates may be prepared and kept indefinitely in stock;

222 SERIAL SECTION MOUNTING.

microscope slides will do for series of small sections.) Take
one of these plates ; lay the slip of paper with the sections
on the plate, the sections downwards ; press it down gently
and evenly, and the sections will adhere to the collodion, then
carefully remove the paper. (Do not place more than one or
at most two lines of sections on the same plate, for those first
placed run the risk of becoming dry whilst you are placing
the others.) This finishes the first stage of the collodionising
process.

Now remove with blotting-paper any excess of alcohol that
may remain on or around the sections, pour collodion over
them, and get it to spread in an even layer. As soon as this
layer is dry at the surface you may write any necessary indi-
cations on it with a small brush charged with methylen blue
(the colour will remain fast throughout all subsequent manipu-
lations).

The plate may now either be put away till wanted in 80 per
cent, alcohol, or may be brought into a staining fluid. Wei-
gert recommends his hsematoxylin process, but other watery
stains may be used. The watery fluid causes the double sheet
of collodion to become detached from the glass, holding the
sections fast between its folds. It is then easy to stain, wash,
dehydrate, and mount in the usual way, merely taking care
not to use alcohol of more than 90 to 96 per cent, for dehy-
dration. Weigert recommends for clearing the above-
described mixture of xylol and carbolic acid (§ 305). Both
the dehydration and the clearing take rather longer with the
collodionised series than with free sections.

The series should be cut into the desired lengths for
mounting whilst in the alcohol. It is perhaps safer to lay
them out for cutting on a strip of closet paper saturated with
alcohol.

It is hardly necessary to comment on the great value of this
beautiful method.

It is suggested by STRASSER that gummed paper might be an improvement
on the glass plates used in this process — especially for very large sections.
See ante, § 326.

Other Methods.

341. Giacomini's Collodion-Gelatin Method.— See the chapter on
" Nerve-centres " in Part II.

INTRODUCTORY REMARKS. 223

CHAPTER XVIII.
CLEARING AGENTS.

342. Introductory Remarks. — Clearing agents are liquids
one of whose functions it is to make microscopic preparations
transparent by penetrating amongst the highly refracting
elements of which the tissues are composed, the clearing liquids
themselves having an index of refraction not greatly inferior
to that of the tissues to be cleared. Hence all clearing agents
are liquids of high index of refraction. The same substances
have also a second function, which consists in getting rid of
the alcohol in which preparations are generally preserved, and
facilitating the penetration of the balsam or other resinous
medium in which preparations are, in most cases, finally
mounted. Hence all of the group of bodies here called
" clearing agents " must be capable of expelling alcohol from
tissues, and must be at the same time solvents of Canada
balsam and the other resinous mounting media.

It is important to note again, notwithstanding some repeti-
tion, the manner of employing these agents. The old plan
was to take the object out of the alcohol and float it on the
surface of the clearing medium in a watch-glass. This plan
was faulty, because the alcohol escapes from the surface of
the object into the air quicker (in most instances) than the
clearing agent can get into it ; hence the object must shrink.
To avoid or lessen this cause of shrinkage, clearing is now
generally done by the method suggested by Giesbrecht, which
consists in putting the clearing medium under the alcohol
containing the object. This is done in the following manner.
Take a test-tube, and pub into it enough alcohol to contain
the objects (a watch-glass will often do well, but a test-tube
is safer). With a pipette carefully put under the alcohol a
sufficient quantity of clearing medium (or carefully pour the
alcohol on to the clearing medium) . Then put the objects into

224 CLEARING AGKJSTS.

the alcohol. They will sink down to the level of separation
of the two liquids at once; and after some time they will be
found to have sunk to the bottom of the clearing medium.
They may then be removed by means of a pipette, or the
supernatant alcohol drawn off and the preparations allowed to
remain until wanted.

The chief clearing agents are essential oils. A classifica-
tion of these is given below (No. 343, Stieda and Schieffer-
decker).

The penetration of all clearing media may be hastened by
using them warm. Directions for clearing are given when
necessary under the heads of the different organs and tissues.
It will suffice here to advise the beginner to keep on his table
the following : — Oil of cedar, for general use ; clove oil, for
making minute dissections in cases in which it is desirable to
take advantage of the property of that essence of forming very
convex drops on the slide, and of imparting a remarkable
brittleness to soft tissues; carbolic acid, for rapidly clearing
imperfectly dehydrated objects.

It frequently happens that the essential oil with which ob-
jects are being treated in a watch-glass or on a slide becomes
cloudy after a short time, and fails to clear the tissues. This
is owing to a combination between the essential oil and mois-
ture, derived,,! think, rather from the air than from the ob-
jects themselves. The cloudiness can usually be removed by
warming (as pointed out by HATCHETT JACKSON, Zool. Anzeig.,
1889, p. 630), but this remedy is not always successful, for in
certain states of the atmosphere the cloudiness will persist,
notwithstanding continued warming. It is for this reason
that I advise that clearing be done, whenever possible, in
shallow well-corked tubes, under which conditions the pheno-
menon rarely occurs.

343. Classification of Clearing Agents (STIEDA).— Stieda's
experiments with essential oils led him to establish the follow-
ing classification :

A. The turpentine group, capable of clearing in a short time
perfectly dehydrated sections, but clearing watery sections
only after many hours or not at all.

01. Terebinthinae.
01. Absinthii.

01. Balsam. Copaivae.
01. Cortic. Aurantiorum.

CLASSIFICATION OF CLEARING AGENTS. 225

01. Cubebarum, 01. Lavandulae.

01. Fceniculi.

01. Millefolii florum.

Ol. Sassafras.

01. Juniperi.

01. Menthae crispae.

01. Cumini.

Ol. Cajeputi.

01. Cascarillas cortic.

01. Sabinae.

01. Citri.

Ol. Origani vulgaris.

This, then, for Stieda, is the Index Expurgatorius of clearing
media.

B. The oil-of-cloves group, clearing very rapidly sections
that have been dehydrated, and clearing watery sections
"somewhat more slowly" and with a certain amount of
shrinkage.

01. Gaultheriae.
01. Cassias.
01. Cinnamomi.
Ol. Anisi stellati.

01. Cardamomi.
01. Coriandri.
01. Carui.
01. Eoris marini.

01. Bergamotti.

But Stieda found kreasote preferable to any of these.

He relates that kreasote was suggested to him by a paper
of KUTSCHIN, tlber den Ban des Riickenmarks des Neunanges,
Kasan, 1863. Kutschin rinsed his sections in water, brought
them on to slides, drew off the water by means of blotting-
paper, and added a drop of kreasote at the side. When
clear, he covered and closed the mounts with a border of
dammar.

Stieda modified this process by mounting in dammar instead
of kreasote.

He then tried experiments to ascertain whether oil of cloves
could be applied in the same manner — that is, to the clearing
of non-dehydrated sections. He found that it could, though
its employment requires longer time. Sections brought from
water into kreasote clear in a few minutes, whilst in oil of
cloves they require from half an hour to an hour or more;
and this slowness of the process exposes them to the risk of
shrinkage.

To the group of good clearing agents should be added —
cedar-wood oil, sandal-wood oil, carbolic acid.

NEELSEN and SCHIEFFERDECKER (Arch.f. Anat. u. Phys., 1882,
p. 206) examined a large series of ethereal oils (prepared by
Schimmel and Co., Leipzig), with the object of finding a

15

226 CLEARING AGENTS.

not too expensive substance that should combine the properties
of clearing quickly alcohol preparations, not dissolving out
anilin colours, clearing celloidin without dissolving it, not
evaporating too quickly, and not having a too disagreeable
smell.

The following is a list of twenty-four products examined
by them. It seems worth while to give it, although the
authors only found three amongst the number that fulfil the
conditions ; as to know that they have been found wanting in
some of these respects may perhaps save somebody a wild-
goose chase.

Oils of Anise, Amber, Birch tar, Cajeput, Calmus, Cassia,
Cedar-wood, Citrons, Dill, Field thyme, Fir needles, Mint,
Cumin, Niobe, Origanum, Palmarosa, Peppermint, Penny-
royal, Kosernary, Sassafras, Spikenard, Thuja, Sandal-wood,
Caraway.

Of these, the following three fulfil the conditions and can
be recommended : — Cedar-wood, Origanum, Sandal-wood.

It would be important to possess a list of the exact indices of refraction of
the substances used for clearing. I have, unfortunately, not been able to
obtain sufficient information of a trustworthy nature for the compilation of
such a list. Cedar oil has nearly the index of crown glass (this is true of the
oil in the thick state to which it is brought by exposure to the air, not of the
new, thin oil, which is less highly refractive), it therefore clears to the same
extent as Canada balsam. Clove oil has a much higher index, and therefore
clears more than balsam. Turpentine, bergamot oil, and kreasote have much
lower indices, and therefore clear less.

344. Cedar Oil (NEELSEN and SCHIEFFERDECKER, 1. c., last sec-
tion) . — For finest cedar- wood oil, price per kilo varies from
fifteen to twenty shillings, say about sevenpence halfpenny per
ounce for small quantities, or about the price of clove oil. Very
thin, colour light yellow, odour slight (of cedar-wood), evapo-
rates slowly, is not changed by light, is miscible with chloro-
form balsam, and with castor oil. Clears readily tissues in
95 per cent, alcohol, without shrinkage, does not extract
anilin colours. Celloidin sections are cleared in five to six
hours.

Cheap, but requires an inconvenient length of time for the
clearing of celloidin sections.

The observer should be careful as to the quality of the
cedar oil he obtains. I have examined the clearing properties
of a sample obtained from the celebrated firm of Rousseau,

CLOVE OIL. 227

Paris. This sample was absolutely colourless. It totally
failed to clear absolute alcohol objects after many days.

The authors think that a laboratory supplied with cedar oil
and origanum oil is fully equipped for all possible cases (the
origanum oil being used merely to take the place of cedar-
wood oil for the special case of celloidin sections). See below,
§348.

Cedar oil is very penetrating, and for this and other reasons
is one of the best media, if not the very best, for preparing
objects for paraffin imbedding.

345. Clove Oil. — Samples of clove oil of very different
shades of colour are met with in commerce. It is frequently
recommended that only the paler sorts should be employed
in histology. A word of explanation is here necessary.
Doubtless it is, in general, best to use a pale oil, provided it
be pure, but it is not always easy to obtain a light-coloured
oil that is pure. Clove oil passes very readily from yellow
to brown with age, so that in choosing a colourless sample
you run great risk of obtaining an adulterated sample, for
clove oil is one of the most adulterated substances in commerce.

Two important properties of clove oil should be noticed here.
It does not easily spread itself over the surface of a slide, but
has a tendency to form very convex drops. This property
makes it a very convenient medium for making minute dis-
sections in. The second property I wish to call attention to
is that of making tissues that have lain in it for some time
very brittle. This brittleness is also sometimes very helpful
in minute dissections.

These qualities may be counteracted if desired by mixing
the clove oil with bergamot oil.

Clove oil has, I fancy, the highest index of refraction of all
the usual clearing agents ; it clears objects more than balsam.
It dissolves celloidin (or collodion), and therefore should not
be used for clearing sections cut in that medium,, without
special precautions. Notwithstanding the opinion of Schieffer-
decker, I consider this to be one of the best of clearing
agents, and very valuable on account of the properties to
which attention has been called above. New clove oil washes
out anilin colours more quickly than old. It is well to possess
trustworthy samples of both new and old oil.

228 CLEARING AGENTS. •

346. Cannel Oil. — Greatly resembles clove oil, but is in
general thinner. An excellent medium, which I particularly
recommend.

347. Oil of Bergamot. — SCHIEFFERDECKER (Arch. Anat. u.
Phys., 1882 [Anat. Abth.], p. 206) finds that this oil has
many good qualities ; it clears 95 per cent, alcohol prepara-
tions and celloidin preparations quickly, does not attack anilin
colours, but the strong odour is disagreeable ; it is as dear as
oil of cloves, twice as dear as oil of origanum, and three times
as dear as oil of cedar. He considers its action preferable to
that of oil of cloves, but, all things considered, gives the palm
to cedar and origanum. I think that this is a very valuable
medium, and though I do not agree with Schieiferdecker in
thinking its action superior to oil of cloves, I think it should
always be kept at hand.

Bergamot oil is, I believe, the least refractive of these
essences, having a lower index than even oil of turpentine.

SUCHANNEK (Zeit. f. wiss. Mik., vii, 2, 1890, p. 158) says
that bleached, colourless bergamot oil will not take up much
water, whereas a green oil will take up as much as 10 per
cent.

348. Oil of Origanum (NEELSEN and SCHIEFFERDECKER, Arch.
Anat.u.Phys., 1882, p. 204).— Price per kilo 15 marks ( = 155.).
Thin, light brown colour, odour not too strong, agreeable,
does not evaporate too quickly, is not changed by light, is
rniscible with chloroform balsam and with castor oil. Ninety-
five per cent, alcohol preparations are cleared quickly, and so
are celloidin sections, without solution of the celloidin. Anilin
colours are somewhat extracted.

For work with celloidin sections care should be taken to
obtain 01. Origani Cretici (" Spanisches Hopfendl"), not 01.
Orig. Gallici (v. GIBSON; see Zeit. f. wiss. Mik., iv, 4, 1887,
p. 482). Specimens of origanum oil vary greatly in their
action on celloidin sections, and care should be taken to
obtain a good sample.

SQUIRE, in his Methods and Formula?, &c., p. 81, says that
origanum oil (meaning, doubtless, the commercial product) is
nothing but oil of white thyme more or less adulterated, and
that the product sold as 01. Origani Cretici is probably oil of
marjoram.

KREASOTE. 229

349. Sandal-wood Oil (NEELSEN and SCHIEFFERDECKER, ibid.).
— "Finest East Indian sandal-wood oil/' price per kilo 50
marks (=£2 10s.). Somewhat thicker than the last two,
light yellow, odour faint, agreeable, evaporation hardly per-
ceptible, unchangeable by light, miscible with chloroform
balsam and with castor oil. Ninety-five per cent, alcohol^
preparations cleared quickly, celloidin more slowly, anilin
colours unaffected.

Very useful ; its worst fault is its high price.

350. Turpentine. — Generally used for treating sections that
have been cut in paraffin, as it has the property of dissolving
out the paraffin and clearing the sections at the same time ;
but many other reagents (naphtha, for instance) are prefer-
able for this purpose (see ante, § 283). If used for alcohol
objects it causes considerable shrinkage, and alters the struc-
ture of cells more than any other clearing agent known to
me, unless used in the thickened state, a method which is
much liked for some purposes in Germany. Thickened tur-
pentine ("Verhartzes Terpentinol" of German writers) is
prepared by exposing rectified turpentine in thin layers for
some days to the air. All that is necessary is to pour some
turpentine into a plate, cover it lightly so as to protect it from
dust without excluding the air, and leave it until it has attained
a syrupy consistency. Turpentine has, I believe, the lowest
index of refraction of all the usual clearing agents except
bergamot oil ; it clears objects less than balsam.

351. Carbolic Acid. — Best used in concentrated solution in
alcohol. Clears instantaneously, even very watery prepara-
tions. This is a very good medium, but it is better avoided
for preparations of soft parts which it is intended to mount
in balsam, as they generally shrink by exosmosis when placed
in the latter medium. It is, however, a good medium for
celloidin sections (see above, § 305).

352. GAGE'S Mixture (Proc. Amer. Soc. Micr., 1890, p. 120;
Journ. Roy. Micr. Soc., 1891, p. 418). — Carbolic acid crystals
melted, 40 c.c. ; oil of turpentine, 60 c.c.

352 a. Kreasote. — Much the same properties as carbolic acid.
Beech-wood kreasote is the sort that should be preferred for

230 CLEARING AGKNTS.

many purposes, — for clearing celloidin sections (for which it
is a very good medium) amongst others.

353. Anilin Oil. — This is a very important reagent on
account of its ability to clear excessively watery objects.
Common anilin oil will readily clear sections from 70 per cent,
alcohol, and with certain precautions objects may be cleared
from watery media without the intervention of alcohol at all.
This property renders anilin valuable in certain cases as a
penetrating medium for preparing for paraffin imbedding.
For ordinary work the usual commercial anilin will suffice ;
and it is immaterial whether it be colourless or have become
brown through oxidation. For difficult work, it is well to
use a perfectly anhydrous oil. This may easily be prepared
by distilling the common oil (SOCHANNEK, Zeit.f. wiss. Mik.,
vii, 2, 1890, p. 156). It should be distilled over into a per-
fectly dry bottle containing a few pieces of caustic potash
(this, being quite insoluble in anilin, may remain in the anilin
without being in any way a hindrance to its employment).
The first 10 to 12 c.c. of liquid that come over will contain
some water, and should be thrown away. The remainder of the
distillate should be preserved over the potash, the bottle being
closed with a rubber stopper and kept protected from light
as far as possible. It is used for preparing for paraffin in the
usual way, alcohol objects clearing quickly in it, whilst quite
watery objects will make the oil cloudy and require it to be
changed once or twice. SUCHANNEK recommends that the
objects be not brought direct from the anilin into paraffin,
but first soaked for some hours in toluol or xylol — the anilin
in this process taking the place of the usual alcohol rather
than that of the usual clearing agent.

As above pointed out (§ 305), anilin is used for clearing
celloidin sections, and is sometimes found very valuable for
this purpose.

354. Xylol, Benzol, Toluol, Naphtha, Chloroform.— Too volatile
to be recommendable as general clearing agents, but may be
used for celloidin sections or for paraffin sections. For these
I greatly recommend naphtha.

355. Absolute Alcohol (SEILEE, Journ. Roy. Mic. Soc., 1882, p. 126).—
Absolute alcohol is recommended by Seiler for preparing objects for mount-

ABSOLUTE ALCOHOL. 231

ing in balsam, the balsam being in this case dissolved in warm absolute
alcohol (see No. 419). The method is said by Seiler to give very good results ;
but it is obvious that it is only applicable to cases in which it is not desired
to make a preliminary examination of the cleared objects (for the sake of
selecting the best or the like) before mounting them.

I have several times tried this process, with results in nowise distinguish,
able from those obtained by ordinary methods, and cannot recommend it for
ordinary purposes.

232 INDIFFERENT LIQUIDS.

CHAPTER XIX.

INDIFFEEENT LIQUIDS — EXAMINATION AND PRESERVATION

MEDIA.

356. Introductory, — I comprehend under this heading all the
media in which an object may be examined. The old distinc-
tion of "indifferent" liquids, and those which have some
action on tissues, appears to be misleading more than helpful ;
inasmuch as it is now well understood that no medium is
without action on tissues . except the plasma with which they
are surrounded during the life of the organism; and this
plasma itself is only ' ' indifferent " whilst all is in situ ; as
soon as a portion of tissue is dissected out and transferred to
a slide in a portion of plasma the conditions become evidently
artificial.

It does not appear necessary to create a separate group for
mounting media, as all preservative media may be used for
mounting.

For directions as to making permanent mounts in fluid
media, see the early sections of Chap. XX.

357. Water. — To preserve it from mould, a lump of thymol or camphor
should be kept in the supply. Water may be employed without inconveni-
ence, and sometimes (on account of its low index of refraction, with great
advantage) for the examination of all structures that have been fixed with
osmic or chromic acid, or some salt of the heavy metals ; but it is by no
means applicable to the examination of fresh tissues — that is, tissues that have
not been so fixed. It is important that the beginner should bear in mind
that water is very far from being an " indifferent " liquid ; many tissue-
elements are greatly changed by it (nerve-end structures, for instance), and
some are totally destroyed by its action if prolonged (for instance, red blood-
corpuscles).

358. Theory of Indifferent Liquids. — In order to render water
inoffensive to such tissues as these it must, firstly, have dis-
solved in it some substance that will give it a density equal to
that of the liquids of the tissue, so as to prevent the occurrence

IODISED SERUM. 233

of osmosis, to which process the destructive action of pure
water is mainly due. Salt solution is a medium suggested by
this necessity. But salt solution by no means fulfils all the
conditions implied in the notion of an " indifferent " liquid.
In so far as it possesses a density approaching to that of the
liquids of the tissues, one cause of osmosis is eliminated; but
there remains another, due to the difference of composition oF
the liquids within the tissues and that without. Cell contents
are a mixture of colloids and crystalloids; salt solution contains
only a crystalloid, whose high diffusibility causes it to diffuse
over into the colloids of the tissues. In order to reduce the
consequent osmotic processes to a minimum, it is necessary
that the examination medium contain, in addition to a due
proportion of salt or other crystalloid, also a due proportion
of colloids. By adding, for instance, white of egg to salt
solution this end may be attained, and, as a matter of fact,
the liquids recommended as indifferent are found invariably
to contain both crystalloids and colloids. Thus (as stated by
Frey) vitreous humour contains 987 parts of water to about
4*6 of colloid matters and 7*8 of crystalloids (common salt)?
In 1000 parts of the juice of fruits are contained about 3'8
parts of colloid matter (albumen), 5*8 of salt, and 3'4 of urea.
In blood-serum, 8'5 of colloids and 1 of crystalloid substance
are found.

359. Salt Solution ("normal salt solution/' " physiolo-
gical salt solution "). — 0*75 per cent, sodium chloride in water.
Carnoy recommends the addition of a trace of osmic acid.

360. Iodised Serum. — Iodised serum was first recommended
by Max Schultze (Virchow's Archiv, xxx, 1864, p. 263). I
take the following instructions concerning it from Ranvier
'(Traite, p. 76).

The only serum that gives really good results is the amniotic
liquid of mammals. A gravid uterus of a sheep or cow
having been obtained (in large slaughter-houses such can be
obtained without difficulty), an incision is made through the
wall of the uterus and the foetal membranes. A jet of serum
issues from the incision, and is caught in a flask prepared for
the purpose. Flakes of iodine are then added, and the flask
is frequently agitated for some days. Two points should be
noted. A perfectly fresh amnios must be taken, for the

234 INDIFFERENT LIQUIDS.

merest incipience of putrefaction will spoil the preparation.
The flask should have a wide bottom, so that the serum may
form only a shallow layer in it ; otherwise the upper layers
will not be sufficiently exposed to the action of the iodine.

Another method is as follows : — Serum is mixed with a
large proportion of tincture of iodine; the precipitate of
iodine that forms is removed by filtration, and there remains
a strong solution of iodine in serum. This should be kept in
stock, and a little of it added every two or three days to the
serum that is intended for use.

Ranvier explains that at the outset serum dissolves very
little iodine ; but if an excess of iodine be kept constantly
present in the solution, it will be found that after two or
three weeks iodides are formed, and allow fresh quantities of
iodine to dissolve ; so that after one or two months a very
strongly iodised serum is obtained. It should be dark brown.
Such a solution is the most fitting for the purpose of iodising
fresh serum in the manner directed above, and for making
the different strengths of iodised serum that are required for
different purposes. In general, for maceration purposes, a
serum of a pale brown colour should be employed.

361. Artificial Iodised Serum (FKEY, Le Microscope} p. 131).
Distilled water . . . . .135 grms.

White of egg 15 „

Sodium chloride . . . . .0*20 grm.

Mix, filter, and add1 —
Tincture of iodine .... 3 grms.

There is formed a precipitate, which is removed by filtering
through flannel ; and a little iodine is added to the filtrate.

362. Kronecker's Artificial Serum (from VOGT et YUNG, Traite
d'Anat. comp. prat., p. 473 : I have been unable to discover
the original source).

Common salt ...... 6 grms.

Caustic soda ...... O06 grm.

Distilled water .... 1000 grms.

363. MIGULA'S Glycerized Blood-serum (ses the paper in Zeit.
/. wiss. Mik., vii, 2, 1890, p. 172; also Journ. Roy. Mic. Soc.,

1890, p. 804).

CALCIUM CHLORIDE. 235

364. Aqueous Humour, Fruit Juice, Simple White of Egg. —
Require no preparation beyond filtering. They may be iodised
if desired.

365. Syrup. — An excellent medium for examining many
structures in the fresh state. To preserve it from mould,
chloral hydrate may conveniently be dissolved in it (1 to 5-
per cent.). I have used as much as 7 per cent., and found no
disadvantage.

Carbolised Syrup. — Carbolic acid may be employed instead
of chloral ; 1 per cent, is sufficient.

Either of these syrups may be used as a mounting medium,
but they are not to be recommended for that purpose, as there
is always risk of the sugar crystallising out.

A good strength for syrup is equal parts of loaf sugar and
water. Dissolve by boiling.

366. Saliva. — Saliva has been recommended with the idea of its being
innocuous to delicate structures ; it is of course a macerating agent (see
MACEBATING AGENTS, Artificial Saliva).

367. Carbolic Acid. — 1 per cent, in water. Is a mounting medium.

368. Kreasote. — 5 per cent, in water.

369. Thwaites' Kreasote Fluid (see BEALE, How to Work, &c., p. 55).

370. Beale's Naphtha and Kreasote (ibid., p. 56).

371. Quekett's Wood-naphtha Fluid (ibid.).

372. Alum Sea-water. —A saturated solution of alum in sea- water is
useful for the examination and preservation of the tissues of many marine
organisms (Medusae, Siphonophora, Ctenophora, Pelagic Tunicata). The
animals may be killed in the fluid, which is a fair fixing agent.

373. Acetate of Alumina (GANNAL'S SOLUTION, BEALE, ibid.).

Acetate of alumina ..... 1 part.
Water 10 parts.

374. Acetate of Potash (MAX SCHULTZE, Arch. mik. Anat., vii, 1872,
p. 180). — A nearly saturated solution in water. It is used by letting a drop
run in under the cover-glass to the object, which is in water. After twenty-
four hours the mount may be closed. The index of refraction is lower than
that of glycerin.

This medium has been frequently recommended as having the property of
preventing the blackening of objects that have been 'treated with osmium ;
but it seems extremely doubtful whether this is really the case.

375. Calcium Chloride (Micro. Diet., art. " Calcium, chloride "). —
Either about 1 part of the salt to 2 of water, or a saturated solution may be
used. A lump of camphor should be added to the solution to preserve it.

236 PRESERVATIVE LIQUIDS.

As this salt is very hygroscopic, its solution presents the advantage of not
drying up, so that it is not necessary to close the mounts until it is desired
to put them away.

376. Chloral Hydrate. — 5 per cent, in water (LAVDOWSKY,
Arch.f. mik. Anat., 1876, p. 359).

Or, 2*5 per cent, in water (BRADY, British Copepods).
Or, 1 per cent, in water (MuNSON, Journ. Roy. Mic. Soc.,
1881, p. 847).

377. Hydrochlorate of Chinolin appears to be a powerful
antiseptic (see ROSENTHAL, in Biol. Centralbl., ix, 1890, p. 767;
or Zeit.f. wiss. Mik., viii, 3, 1891, p. 342).

378. Alcohol. — Not very recommendable for mounting, as if taken weak
it is not a very efficient preservative, and if taken strong it attacks the
cement of mounts.

CABPENTEE (The Microscope') recommends a strength of 1 part to 5 of
water.

The chief use of alcohol for preservation purposes is of
course for preserving specimens in till wanted for further
preparation and study. See, on this point, the remarks in
Chap. I, § 2.

Mercurial Liquids.

379. Corrosive Sublimate Solution (HABTING'S FLUID, Micro. Diet.,
art. " Preservation," p. 640).— One part of sublimate to from 200 to 500 of
water. (For blood-corpuscles of frog 1 — 400, of birds 1 — 300, of mammals
1 — 200.) " Harting recommends this as the best preservative for the cor-
puscles of the blood, nerve, muscular fibre, &c."

380. Pacini's Fluids (Journ. de Mic., iv, 1880; Joum. Roy. Mic. Soc.
[N.S.], ii, 1882, p. 702). — Pacini remarks that "bichloride of mercury coagu-
lates and precipitates the albuminous matter that exists in the interstitial
fluids of the tissues," and therefore in order to prevent this coagulation it is
well to associate with it salt for certain preparations, or acetic acid for others.
On this principle are prepared the following classical fluids of Goadby and
Pacini :

FLUID No. 1 is identical with that of Harting given above, viz. 1*200
sublimate in water. Pacini uses it for removing, when desired, the salt or
acid from preparations that have been placed in one of the other solutions.
FLUID No. 2 :

Bichloride of mercury .... 1 part.
Common salt ...... 2 parts.

Water 200 „

Of general employment, but especially useful for blood-corpuscles of cold-
blooded animals, as it has a less density than the following fluid. It pre-

PACINI'S SOLUTIONS. 237

serves spermatic fluid, epithelia, nerves, and muscle-fibres. It is also used
for fixing Infusoria, a small quantity being added to the water containing
them.

FLUID No. 3 :

Bichloride of mercury .... 1 part.

Common salt 4 parts.

Water 200 „

For blood-corpuscles of warm-blooded animals.
FLUID No. 4 :

Bichloride of mercury . . . . 1 part.

Acetic acid ... 2 parts.

Water . . . ... . . 300 „

" Serves best for the nuclei of animal tissues, but it swells up the fibres
and distorts the forms of the cells."

FLUID No. 5 (FEEY, Le Microscope, 1867, p. 233).— In the place here
quoted, Frey speaks of the liquids of Pacini as differing from those of Goadby
through their containing glycerin in lieu of alum. He gives the following
directions. Take —

Sublimate 1 part.

Sodium chloride 2 parts.

Glycerin (25° Baume) . . . 13 „

Water 113 „

Allow the mixture to remain undisturbed for at least two months. At
the end of that time take for use 1 part, mix with 3 parts of water, and
filter. This mixture is said to be a good preservative of all delicate tissues.
FLUID No. 6 (ibid.) :

Sublimate ...... 1 part.

Acetic acid 2 parts.

Glycerin (25° Baume) . . . . 43 „

Water 115 „

This mixture is to be employed in the same way as the last. It is said to
destroy red blood-corpuscles, but to preserve white blood-corpuscles.

381. Modifications of the foregoing Sublimate Solutions. — The
following formulae are quoted by Frey from Cornil as being in use at the
Pathological Institute of Berlin :

1. Sublimate 1 part.

Sodium chloride 2 parts.

Water 100 „

For the more vascular tissues of warm-blooded animals.

2. Sublimate 1 part.

Sodium chloride 2 parts.

Water 200 „

For similar tissues of cold-blooded animals.

3. Sublimate 1 part.

Sodium chloride . . . . . 1 „
Water 300 parts.

For pus-corpuscles and analogous elements.

238 PRESERVATIVE LIQUIDS.

4. Sublimate 1 part.

Water 300 parts.

For blood-corpuscles.

5. Sublimate ...... 1 part.

Acetic acid 1 „

Water . . . . . ... 300 parts.

For epithelia, connective tissue, and pus-corpuscles, when it is desired to
demonstrate the nuclei.

6. Sublimate 1 part.

Acetic acid 3 parts.

Water • . 300 „

For ligaments, muscles, and nerves.

7. Sublimate 1 part.

Acetic acid ...... 5 parts.

Water 300 „

For glandular tissues.

8. Sublimate 1 part.

Phosphoric acid 1 „

Water 30 parts (sic).

For cartilaginous tissues.

382. Goadby's Fluids (Micro. Diet., art. "Preservation").
IST FLUID:

Bay salt (coarse sea salt) . . 4 ounces.

Alum 2 „

Corrosive sublimate ... 2 grains.
Boiling water .... 1 quart.
This is found to be "too strong" for most purposes, and
therefore the following is recommended for general purposes.
2ND FLUID :

Bay salt ..... 4 ounces.

Alum 2 „

Corrosive sublimate ... 4 grains.
Water ...... 2 quarts.

" Schultze recommends it for preserving Medusae, Echino-
dermata, Annelid larvae, Entomostraca, Polythalamia, and
Polycystina, and advises the use of glycerin afterwards to
produce transparence."

3RD FLUID. — When carbonate of lime exists in the prepara-
tions, the alum must be omitted. The following formula is
recommended :

Bay salt ..... 8 ounces.

Corrosive sublimate ... 2 grains.
Water ...... 1 quart.

EIPAET AND PETIT's FLUID. 239

4TH FLUID. — " Marine animals require a stronger fluid of
this kind, made by adding about 2 ounces more salt to
the last."

383. Owen's Fluid (quoted from VOGT et YUNG, Traite
d'Anat. comp. pratique, p. 19).

Corrosive sublimate . . . 0*014 grm.
Alum ...... 79 grms.

Salt 137

Water 1680 „

Said to be very useful for the preservation of soft-bodied
animals.

384. Gilson's Fluid (CARNOY'S Biologie cellulaire, p. 94).
Alcohol of 60 per cent. . . .60 c.c.

Water 30 „

Glycerin . . . . . 30 „
Acetic acid (15 parts of the glacial

to 85 of water) . . . 2 „

Bichloride . . . . O'lS grm.

A really excellent medium for the study of fine cellular
detail with well-fixed objects.

385. Gage's Albumen Fluid (Zeit. f. wiss. Mik., 1886, p.
-223).

White of egg .... 15 c.c.

Water 200 „

Corrosive sublimate . . . 0'5 grm.

Salt 4 grms.

Mix, agitate, filter, and preserve in a cool place. Recom-
mended for the study of red blood-corpuscles and ciliated cells.

Other Fluids.

386. Chloride and Acetate of Copper (RIPART et PETIT'S fluid,
Brebissonia, 1880, p. 92 ; CAKNOY'S Biol. cell., p. 95).

Camphor water (not saturated) . 75 grms.
Distilled water .... 75 „
Crystallised acetic acid ... 1 grm.
Acetate of copper .... 0*30 „
Chloride of copper . . . 0*30 „

This is certainly a most valuable medium for work with
delicate fresh tissues. It may be used in combination with

240 PRESERVATIVE LIQUIDS.

methyl green, which it does not precipitate. The most
delicate elements are perfectly preserved in it ; the addition
of a drop of osmic acid or corrosive sublimate does not cause
the least turbidity, and enhances its fixing action.

387. Tannin (CARNOY, 1. c.).

Water 100 gnns.

Powdered tannin . . . . 0*50 grm.

388. Picro-carmine. — Picro-carmine has been recommended by Ranvier
as a medium for teasing fresh tissues in, in the belief that it possesses suffi-
cient fixing action to preserve the form of cells. Carnoy finds that cells live
in it for a considerable time, and become gorged with water and deteriorated
to a considerable degree. Unfortunately, too, picro-carmine cannot be com-
bined with a good fixing agent, as it is precipitated by alcohol and by acids,
and especially by osmic acid.

389. Methyl Green. — See under STAINING AGENTS. The
aqueous solution is sometimes very useful as an examination
medium for fresh tissues. It should be taken fairly concen-
trated, in which state it has sufficient fixing power, which is
enhanced by the addition of a trace of osmic acid.

390. Wickersheimer's Fluid (Zool. Anz., 1879, p. 670; cf. Journ.
Roy. Mic. Soc., 1882, p. 427 ; id., 1880, p. 355 ; and Entomol. Nachr., 1880,
p. 129). — This once famous fluid appears to be quite unsuccessful for histo-
logical purposes.

391. Meyer's Salicylic Vinegar Preservative Solutions (Arch,
mik. Anat., xiii, 1876, p. 868).— "Salicylic vinegar" is a
solution of 1 part of salicylic acid in 100 parts of pyroligneous
acid. The pyroligneous acid should be of 1*04 specific gravity,
and should be of a pale yellow colour. This product is found
in commerce, and may be obtained from Herrn J. M. Andreas,
Droguerie-Handlung, Frankfurt-a.-M.

IST FLUID :

One vol. salicylic vinegar to 10 vols. of the following dilute
glycerin, viz. glycerin 1 vol., water 2 vols.

For various larvae, Hydrae, Nematodes, &c.

2ND FLUID :

One vol. salicylic vinegar to 10 vols. of the following dilute
glycerin, viz. glycerin 1 vol., water 4 vols.

For Infusoria.

392. Noll's Salicylic Vinegar and Gum Medium (Zool. Anz.,
1883, p. 472). — A mixture of equal vols. of Meyer's second
fluid (ante, last formula) and Farrant's medium (post, 395).

GUM AND GLYCERIN MEDIUM. 241

This mixture never becomes turbid, and does not dry up.
The covers may be luted with asphalt or any other cement.
The fluid answers admirably for delicate Crustacea and their
larvae ; the preparations do not shrink, and are not too much
cleared. It also answers well for hardened and stained
preparations of Hydroids, small Medusae, and other Coelen-
terates.

393. Dean's Medium (see Micro. Diet, art. "Preservation"). — Appears
to be now superfluous.

394. Hoyer's Gum with Chloral Hydrate or Acetate of Potash
(Biol. Centralb., ii, 1882, pp. 23-4; Journ. Roy. Mic. Soc.
[N.S.], iii, 1883, pp. 144-5).— A high 60 c.c. glass with a
wide neck is filled two thirds full with gum arabic (in pieces),
and then either a solution of chloral (of several per cent.)
containing 5 — 10 per cent, of glycerin is added, or acetate of
potash or ammonia. The gum with frequent shaking dissolves
in a few days, and forms a syrupy fluid, which is slowly
filtered for twenty-four hours. The clear filtered fluid will
keep a long time, but if spores of fungi begin to develop a
little chloral can be added and the fluid refiltered. The
solution with chloral is for carmine or haematoxylin objects —
that with acetate for anilin objects.

395. Gum and Glycerin Medium (F ARRANT' s medium; BEALE,
How to Work, &c., p. 58).

Picked gum arabic ... .4 ounces.

Water 4 „

Glycerin 2 ,,

To be kept in a stoppered bottle with a lump of camphor.
This medium is quoted by Frey as consisting of equal
parts of gum, glycerin, and saturated aqueous solution of
arsenious acid.

The Micrographic Dictionary gives the following directions :

Gum arabic 1 ounce, glycerin 1 ounce, water 1 ounce,

arsenious acid 1£ grains; dissolve the arsenious acid in tlio

water, then the gum (without heat), add the glycerin, and

incorporate with great care to avoid forming bubbles.

Another method for making this medium is given by A. F.
STANLEY KENT in Journ. Roy. Mic. Soc., 1890, p. 820.

396. Gum and Glycerin Medium (LANGERHANS' formula,

16

242 PRESERVATIVE LIQUIDS.

modification of FARRANT'S medium, Zool. Anzeig., ii, 1879,
p. 575).

Gummi arab. ...... 5'0

Aquae ....... 5*0

To which after twelve hours are added —

Glycerini 5'0

Sol. aquosa acid, carbol. (5-100) . . lO'O
Marine animals may be preserved in this by simply run-
ning in a drop under the cover, and next day or later adding
what is necessary to make up for evaporation, and closing
the mount. Shrinkage is very slight, and most colours keep
well.

397. FARIS'S Glycero-Gum (The Microscope, x, 1890, p. 59;
Journ. Eoy. Mic. Soc., 1890, p. 414). — Gum arabic, 2 ounces;
glycerin, 1/5 ounces; water, 1'5 ounces; thymol, 1 grm.
Mix, dissolve with heat, and filter.

398. Gum and Glycerin Jelly (SHIMER, The Microscope, ix,
1889, p. 138; Journ. Eoy. Mic. Soc., 1890, p. 411).— Equal
parts of glycerin jelly (Foi/s second formula, post, § 412),.
Farrant's solution, and glycerin.

399. COLE'S Gum and Syrup Medium (see above, § 317).

400. APATHY'S Gum and Syrup Medium (see above, Chap*
IX, § 119). — This medium is recommended by Apathy in a
general way, and not merely for the special purpose for which
it is quoted in § 119. It sets very hard, and, combined with a
paper cell (see Chap. XX), may be used for ringing glycerin
mounts.

401. Fabre-Domergue's Glucose Medium (La Nature, No. 823,
9 Mars, 1889, supp.).

Glucose syrup diluted to twenty-
five degrees of the areometer
(sp. gr. 1-1968) . . . 1000 parts.

Methyl alcohol .... 200 „

Glycerin 100 „

Camphor, to saturation.

The glucose is to be dissolved in warm water, and the other
ingredients added. The mixture, which is always acid, must
be neutralised by the addition of a little potash or soda.
This medium is said to preserve without change almost all

GLYCERIN. 243

animal pigments. If it really performs this, its great value is
evident.

402. BRUN'S Glucose Medium (from FABRE-DOMERGUE'S Pre-
miers Principes du Microscope et de la Technique microscopique,
Paris, 1889, p. 123).

Distilled water .... 140 parts.
Camphorated spirit . . . 10 „

Glucose . 40 „

Glycerin 10 ,,

Mix the water, glucose, and glycerin, then add the spirit,
and filter to remove the excess of camphor which is pre-
cipitated on mixing. I am indebted to Dr. HENNEGUY for
calling my attention to this liquid, which is an important one.
It is preferable to glycerin because it preserves the colour of
preparations stained with anilin dyes, methyl green included.

402 a. Levulose as a Mounting Medium. — Levulose is recommended
as a mounting medium by BEHBENS, KOSSEL, u. SCHIEFFEBDECKEB (Das
MikrosJcop. u. d. Meth. d. miJc. Unters., Braunschweig, 1889). It is un-
crystallisable, and preserves well carmine and coal-tar stains (haeinatoxylin
stains fade somewhat in it). The index of refraction is somewhat higher
than that of glycerin. Objects may be brought into it out of water.

Glycerin Media.

403. Glycerin. — Glycerin diluted with water is frequently
employed as an examination and mounting medium. Dilution
with water is sometimes advisable from an optical point of
view, on account of the increased visibility that it gives to
many structures by lowering the index of refraction of the
glycerin. But from the point of view of efficacious preserva-
tion it is always advisable to use undiluted glycerin, the
strongest that can be procured.

Long soaking of tissues in glycerin of gradually increased
strength is a necessary preliminary to mounting in all cases
in which it is desired to obtain the best possible preparations,
and to ensure that they shall keep well. If this soaking is
done on the slide (the cover being removed and the object
treated with fresh glycerin every one or two days) , it is well
to take the precaution recommended by Beale, of luting the
edges of the cover so as to make the preparation air-tight, as
glycerin is so highly hygroscopic that a drop of it exposed to
the air rapidly diminishes in strength to a very considerable

244 PRESERVATIVE LIQUIDS.

degree. In order to facilitate the removal of the cover in
this process, the slide may be gently warmed by passing it
two or three times through the flame of a spirit lamp. No
preparation can be considered to be made secundum artem
until every part of the object has been thoroughly impregnated
with strong pure glycerin.

The shrinking that frequently occurs when delicate struc-
tures are brought into glycerin may generally be cured by this
treatment; cells which at first appear hopelessly collapsed
gradually swell out to their normal forms and dimensions.

For closing glycerin mounts, the edges of the cover should
first (after having been cleansed as far as possible from
superfluous glycerin) be painted with a layer of glycerin jelly ;
as soon as this is set a coat of any of the usual cements may
be applied. This has of course been for the last twenty years
one of the commonplaces of hisfcological technic, but that
has not prevented somebody from recently describing the pro-
cess at great length as new.

Glycerin dissolves carbonate of lime, and is therefore to be
rejected in the preparation of calcareous structures that it is
wished to preserve.

The already high index of refraction of glycerin (Price's glycerin, n =
1*46) may be raised to about that of crown glass by dissolving suitable sub-
stances in the glycerin. Thus the refractive index of a solution of chloride
of cadmium (CdCl2)* in glycerin may be T504 ; that of a saturated solution
of sulphocarbonate of zinc in glycerin may be 1'501 ; that of a saturated
solution of Schering'sf chloral hydrate (in crusts) in glycerin is I'olO; that
of iodate of zinc in glycerin may be brought up to 1'56.J The clearing
action of glycerin may thus be greatly increased, and the full aperture of
homogeneous objectives brought to bear on objects mounted in one of the
above-named solutions.

The sulphocarbolate of zinc solution § may be prepared by taking equal
parts by weight of Price's glycerin and sulphocarbolate of zinc crystals,
mingling the two, and applying sufficient heat to boil the glycerin. The
solution can be made in about an hour, but no fear need be had about boil-
ing too long, as the longer this is done the less liability will there be for the
solution to deposit crystals on the bottom of the bottle when cooled, which
it will do if the temperature is only kept up long enough to dissolve the

* Journ. Roy. Mic. Soc., ii, 1879, p. 346.

f Ibid. (N.S.), i, 1881, p. 943.

J Ibid., p. 366.

§ Ibid., iii, 1880, p. 1051.

DEANE'S GLYCERIN JELLY. 245

crystals. Filter while hot. The index may be brought up to T525 if de-
sired, by evaporating the solution somewhat, or by adding more carbolate.

404. Barff's Boroglyceride (see Journ. Boy. Mic. Soc., 1882, p. 124).
— This preparation may be obtained (price Is. per bottle) from The Kreo-
chyle Company, Viaduct House, Farringdon Street, E.C., or all wholesale
chemists.

405. Glycerin and Alcohol Mixtures. — These most useful
fluids afford one of the best means of bringing delicate objects
gradually from weak into strong glycerin. The object is
mounted in a drop of the liquid, and left for a few hours or
days, the mount not being closed. By the evaporation of the
alcohol the liquid gradually increases in density, and after some
time the mount may be closed, or the object brought into pure
glycerin or glycerin jelly.

1. CALBERLA'S LIQUID :

Glycerin . . . . . .1 part.

Alcohol . . . . . . 1 „

Water 1 „

A most valuable examination fluid. As already pointed out
(§ 2), this liquid is in many cases to be preferred to alcohol
for keeping fixed objects in until required for dissection or
other further preparation.

2. I strongly recommend the following for very delicate
objects :

Glycerin 1 part.

Alcohol . . . . . . 1 „

Water 2 parts.

3. H^NTSCH'S LIQUID :

Glycerin 1 part.

Alcohol ...... 3 parts.

Water 2 „

4. JAGER'S LIQUID (quoted from VOGT and YUNG'S Traite
d'Anat. comp. prat., p. 16) :

Glycerin 1 part.

Alcohol . . . . . . 1 „

Sea water . . . . . .10 parts.

406. Deane's Glycerin Jelly (from FREY'S Le Microscope, p.
231). — 120 grammes glycerin, 60 grammes water, 30 grammes
gelatin. Dissolve the gelatin in the water, and add the gly-

246 PEESERVATIVE LIQUIDS.

cerin. This, and the following glycerin jellies, must of course
be used warm.

407. Lawrence's Glycerin Jelly (DAVIES, Preparation and
Mounting of Microscopic Objects, p. 84). — "He takes a quan-
tity of Nelson's gelatin, soaks it for two or three hours in cold
water, pours off the superfluous water, and heats the soaked
gelatin until melted. To each fluid ounce of the gelatin,
whilst it is fluid but cool, he adds a fluid drachm of the white
of an egg. He then boils this until the albumen coagulates
and the gelatin is quite clear, when it is to be filtered through
fine flannel, and to each ounce of the clarified solution add
6 drachms of a mixture composed of 1 part of glycerin to 2
parts of camphor water."

408. Scale's Glycerin Jelly (How to Work, &c., p. 57). —
Gelatin or isinglass, soaked, melted, and clarified if desired,
as in the last formula. To the clear solution add an equal
bulk of strong glycerin.

409. Brandt's Glycerin Jelly (Zeit.f. Mik., ii, 1880, p. 69;
Journ. Roy. Mic. Soc., iii, 1880, p. 502).— Melted gelatin 1
part, glycerin 1J parts.

The gelatin to be soaked in water and melted in the usual
way. After incorporating the glycerin, the mixture is to be
filtered. This is a point of vital importance, as the gelatin of
commerce is always mixed with particles of dust and minute
threads. Swedish filtering-paper does not allow the fluid to
pass through sufficiently, and flannel produces more threads
than before. The following simple apparatus is found effec-
tive. A wide-necked bottle is broken in two, and the upper
part taken. The neck is stopped with a cork having two holes
bored in it. In the first hole a glass tube, about 20 cm. long,
is inserted so as to project a little into the inside of the bottle,
and on the outside it is bent sharply to one side and drawn
out into a point of about 1J to 2 mm. diameter. In the
second hole a funnel-shaped filter is inserted so that the coni-
cal part is inside the bottle and the tube projects a few centi-
metres beyond the cork and the neck of the bottle. The
apparatus is then placed so that the wide opening of the
bottle and of the funnel is uppermost, and some spun glass
is pressed into the lower conical part of the filter. In using

FOI/S GLYCERIN JELLIES. 247

the apparatus the funnel is filled with glycerin gelatin, and
the bottle with hot water, which runs off slowly through the
tube in the first hole and is constantly replenished.

Some drops of carbolic acid should be added to the fluid
product of the filtering. For mounting, use warm by melt-
ing a small portion on the slide, the object having been pre-
viously soaked for some time in a small bottle of the medium
warmed with a suitable apparatus.

410. Kaiser's Glycerin Jelly (Bot. Cent., i, 1880, p. 25;
Journ. Roy. Mic. Soc., iii, 1880, p. 504). — One part by weight
finest French gelatin is left for two hours in 6 parts by weight
distilled water, 7 parts of glycerin are added, and for every
100 grammes of the mixture 1 gramme of concentrated car-
bolic acid. Warm for ten to fifteen minutes, stirring all the
while, until the whole of the flakes produced by the carbolic
acid have disappeared. Filter whilst warm through the finest
spun glass laid wet in the filter. Use for mounting as above.

I prepared some of this jelly many years ago, and find it is
still perfectly clear.

411. Seaman's Glycerin Jelly (Amer. Mon. Mic. Journ., ii,
1881, p. 45; Journ. Roy. Mic. Soc. [N.S.], i, 1881, p. 534).-
Dissolve isinglass in water, so that it makes a stiff jelly when
at the ordinary temperature of the room, add one tenth as
much glycerin, and a little solution of borax, carbolic acid,
or camphor water. Filter whilst warm through muslin, and
add a little alcohol.

412. Fol's Glycerin Jellies (Lehrb., p. 138).

1. Melt together one volume of Beale's jelly (§ 408) and
one half to one volume of water, and add 2 to 5 per cent, of
salicylic acid solution, or carbolic acid or camphor.

2. Gelatin 30 parts.

Water 70 „

Glycerin 100 „

Alcoholic solution of camphor . 5 ,,

Prepare as before, adding the camphor last.

3. Gelatin 20 parts.

Water 150 „

Glycerin 100 „

Alcoholic solution of camphor . . 15 „

248 PRESERVATION MEDIA.

413. SQUIRE'S Glycerin Jelly (SQUIRE'S Methods and
Formulae, &c., p. 84). — Soak 100 grms. of French gelatin in
chloroform water, drain when soft, and dissolve with heat in
750 grms. of glycerin. Add 400 grms. of chloroform water
with which has been incorporated about 50 grms. of fresh egg-
albumen ; mix thoroughly, and heat to boiling-point for about
five minutes. Make up the total weight to 1550 grms. with
chloroform water. Filter in a warm chamber.

414. Stephenson's Biniodide of Mercury and Iodide of Potas-
sium (Joui-n. Boy. Mic. Soc., 1882, p. 167).— Interesting as giving a solu-
tion which when saturated has an index of T680, the highest index of any
known aqueous fluid. I have experimented both with strong and weak
solutions, and doubt whether much practical advantage can be derived from
them. Tissues are well preserved, but the preparations are ruined by a
precipitate which forms in the fluid.

415. Monobromide of Naphthalin. — See Journ. Boy. Mic. Soc., 1880,
p. 1043 (ABBE and TAN HEUECK), and Zool. Anz., 1882, p. 555 (MAX
FLESCH).

416. THOMPSON'S High Refractive Medium, — See Journ. Roy.
Hie. Soc., 1892, p. 902.

Resinous Media.

417. Resins and Balsams. — Kesins and balsams consist of a
vitreous or amorphous substance held in solution by an essen-
tial oil. By distillation or drying in the air they lose the
essential oil and pass into the solid state. It is these solidified
resins that should, in my opinion (and that, I believe, of the
best microscopists), be employed for microscopical purposes ;
for the raw resins always contain a certain proportion of
water, which makes it difficult to obtain a clear solution with
the usual menstrua, is injurious to the optical properties of
the medium and to its preservative qualities, and, further,
especially hurtful to the preservation of stains. I therefore
recommend that all solutions* be made by heating gently the
balsam or resin in a stove until it becomes brittle when cold,
and then dissolving in an appropriate menstruum.

FOL (Lehrb., pp. 138-9) is of a different opinion.

Solutions made with volatile menstrua, such as xylol and
chloroform, set rapidly, but become rapidly brittle. Solutions
made with non- volatile -media, such as turpentine, set much
less rapidly, and pass much less rapidly into the brittle state.

CANADA BALSAM. 249

As to the old dispute about the respective merits of damar
and balsam, the case appears, after all, to lie in a nutshell.
Damar gives the better definition of delicate detail ; balsam
has greater clearing action, and affords perhaps more solid
mounts.

It may be remarked here that for some of the purposes for
which these media are employed, Oil of Cedar may be founcl
preferable. The mounts need not be closed (except for im-
mersion work), as the oil soon sets hard enough to keep the
cover in place.

418. Canada Balsam. — Prepare with the solid balsam as
above described. The usual menstrua are xylol, benzol,
chloroform, and turpentine. Dissolve the solid balsam in one
of these to the required consistence. The turpentine solution
is to be preferred only in cases where it is desired to have a
medium that sets very slowly, or in view of the better pre-
servation of certain stains. For most other purposes the xylol
solution is the best. If time be an object, a benzol solution
should be preferred, as it sets much quicker than the xylol
solution.

HEYS states that if the chloroform solution be poured into
long, thin, half-ounce phials, corked up, and set aside for at
least a month, the medium will be clearer and set much
quicker than if the balsam is mixed with the chloroform at
the time it is required for use (Trans. Mic. Soc., Jan., 1865,
p. 19; BEALE, p. 51).

SAHLI (Zeit. f. wiss. Mik., 1885, p. 5) recommends oil of
cedar as a menstruum.

419. Seller's Alcohol Balsam (Ptoc. Amer. Soc. Mic., 1881, pp. 60-2;
Journ. Roy. Mik. Soc. [N.S.], ii, 1882, pp. 126-7).—" Take a clear sample
of Canada balsam and evaporate it in a water- or sand-bath to dryness ; i. e.
until it becomes brittle and resinous when cold. Dissolve this while warm
in warm absolute alcohol and filter through absorbent cotton."

The advantage of this medium is stated to be that objects may be mounted
in it direct from absolute alcohol, without previous treatment with an essen-
tial oil or or other clearing agent; Seiler considers that by this means
" shrivelling is avoided, as well as the solution of fat in the cells."

The process is not very easy to carry out, and I cannot recommend it for
general work.

420. Damar (Gum Damar, or Dammar, or d'Ammar). — The
menstrua are the same as for balsam, and the solution should

A^

bVE*

250 PRESERVATION MEDIA.

be prepared in the same way. The most beautiful of all
these mounting media is the solution of damar in xylol. Heat
is not necessary to make the solution.

Minute directions (which I think unnecessary) for pre-
paring a working solution are given by MAETINOTTI in Zeit. f.
iviss. Mik., iv, 2, 1887, p. 156, and in Malpighia, ii, 1888,
p. 270 ; cf. also Journ. Roy. Mic. Soc., 1889, p. 163.

FLEMMING, PFITZNER, and a writer signing C. J. M., all
employ a mixture of benzol and turpentine (see Arch. mik.
Anat.j xix, 1881, p. 322 ; Sci. Gossip, 1882, p. 257 ; Journ.
Roy. Mic. Soc. [N.S.], iii, 1883, p. 145 ; Morphol. Jahrb., vi,
1880, p. 469; Journ. Roy. Mic. Soc. [N.S.], ii, 1882, p. 583).

MAX FLESCH notes hereon (Zool. Jahresber. f. 1880, p. 51)
that at Wiirzburg the ordinary dammar varnish of arists is
employed.

JAMES (Engl. Mech., 1887, p. 184; Journ. Roy. Mic. Soc.,
1887, p. 1061) also gives some, I think, superfluous formulae
for damar solutions; and still another new method is given,
op. cit., 1890, p. 680.

A formula for a damar and mastic medium is given by
SQUIRE, in his Methods and Formulas, &c., p. 84.

421. Balsam-Damar. — It has been recommended to mix equal volumes
of benzol balsam and turpentine damar, but this medium seems to me
superfluous.

422. Colophonium. — A solution of colophonium in turpen-
tine has been recommended by Kleinenberg. I find it works
very pleasantly. The palest kind of colophonium should of
course be taken.

This medium sets very slowly, so that ample time is afforded
for arranging objects in it. Kleinenberg warns against the
employment of absolute alcohol as a solvent; the prepara-
tions are beautiful at first, but soon become spoiled by the
precipitation of crystals or of an amorphous substance.

The turpentine solution keeps perfectly limpid, gives very
good definition, and is altogether so excellent a medium that
I am surprised that it is not more used. It should be recom-
mended to beginners.

423. Venice Turpentine for Mounting (VossaLER, Zeit. f. wiss.
Mik., vi, 3, 1889, p. 292, et seq.). — Vosseler strongly recommends this
medium as having considerable advantages over Canada balsam or damar.

COPAL VARNISH. 251

Commercial Venice turpentine is mixed in a tall cylinder glass with an equal
volume of 96 per cent, alcohol, allowed to stand in a warm place for three
or four weeks, and decanted. It is stated that preparations may be mounted
in this medium without previous clearing with essential oils or the like.
The index of refraction being lower than that of the above-named balsams,
delicate details are more distinctly brought out. Stains keep well in the
medium, and Vosseler states that he possesses preparations made fifteen
years ago that are perfectly well preserved.

This medium is also recommended by SUCHANNEK (ibid., vii, 4, 1891,
p. 463). He advises that it be prepared with equal parts of Venice turpen-
tine and neutral absolute alcohol (obtained by treating commercial absolute
alcohol with calcined cupric sulphate and quicklime). The mixture should
be agitated frequently and kept in a tile stove for a day or two until clear
and sufficiently inspissated.

424. Copal Varnish. — I have seen tissues very instructively
mounted in this medium, which is probably worthy of further
study. " Berry's Hard Finish/7 which is an easily obtainable
copal varnish, has been highly praised for mounting purposes
(see Journ. Roy. Hie. Soc., 1887, p. 1064).

425. Castor Oil. — This has been lately recommended as a mounting
medium for certain delicate tissues (sections of eyes of Cephalopods) by
GEENACHER (Abhandl. naturf. Ges. Halle-a.-S., Bd. xvi; Zeit.f. wiss. Mik.,
1885, p. 244). This was with the idea that its low refractive index (n =
T49, whilst Canada balsam n= 1'54) would give a useful augmentation of
visibility for the more refractive elements of the tissues.

With the objects with which I have experimented I have not found this
to be the case.

426. Photographic Negative Varnish (for mounting large sections
without cover-glasses). — See WEIGEBT, Zeit.f. wiss. Mik., iv, 2, 1887, p. 209.

427. Styrax and Liquidambar. — See Journ. Roy. Mic. Soc., 1883, p.
741 ; ib., 1884, pp. 318, 475, 655, and 827 ; and the places there quoted.
Also Bull. Soc. Beige de Mic., 1884, p. 178 ; and FOL, Lehrb., p. 141.

428. Tolu Balsam (see Zeit.f. wiss. Mile., iv, 4, 1887, p. 471).

429. Frankincense (see Journ. Roy. Mic. Soc., 1892, p. 901).

252 CEMENTS AND VARNISHES.

CHAPTER XX.
CEMENTS AND VARNISHES.

430. Thanks to the efforts of the dilettanti to outshine one
another with neatly gaudy " rings," microscopical literature
contains a goodly show of receipts for cements and varnishes.
I have collected such as appear likely to be useful, rejecting
all that relates merely to ornament.

Two, or at most three, of the media given below will
certainly be found sufficient for all useful purposes. For
many years I have used only one cement (Bell's). I recom-
mend this as a cement and varnish ; gold size may be found
useful for turning cells ; and Miller's caoutchouc cement may
be kept for occasions on which the utmost solidity is required.

Marine glue is necessary for making glass cells.

Carpenter lays great stress on the principle that the cements
or varnishes used for fluid mounts should always be such as
contain no mixture of solid particles ; he has always found
that those that do, although they might stand well for a few
weeks or months, yet always became porous after a greater
lapse of time, allowing the evaporation of the liquid and the
admission of air. All fluid mounts should be ringed with
glycerin jelly before applying a cement ; by this means all
danger of running -in is done away with.

The above passage stands as it stood, italicised as here, in the 1st and 2nd
editions. It was translated and amplified, in a special paragraph, in the
Traite des Meth. techniques. I may therefore be excused from hunting
up the name of the anatomist who recently published as new this old, old
method, or the pages of the journals which reproduced his paper without
protest.

The reader who requires more information concerning
microscopical cements and varnishes than can be given in
this chapter may consult with advantage the papers of
AUBERT, The Microscope, xi, 1891, p. 150, and Journ. Roy.

BELL'S CEMENT. 253

Mic. Soc., 1891, p. 692; BECK, The Microscope, xi, 1891,
pp. 338, 368, and Journ. Roy. Mic. Soc., 1892, p. 293 ; and
the last edition of BEHREN.S' Tabellen zum Gebrauch bei mikro-
skopischen Arbeiten (Bruhn, Braunschweig, 1892).

431. Gelatin Cement (MARSH'S Section-cutting, 2nd ed., p.
104). — Take half an ounce of Nelson's opaque gelatin, soak_
well in water, melt in the usual way, stir in 3 drops of kreasote,
and put away in a small bottle. It is used warm.

When the ring of gelatin has become quite set and dry,
which will not take long, it may be painted over with a
solution of bichromate of potash made by dissolving 10 grains
of the salt in an ounce of water. This should be done in the
daytime, as the action of daylight is necessary to enable the
bichromate to render the gelatin insoluble in water. The
cover may then be finished with Bell's cement.

This process is particularly adapted for glycerin mounts.

432. The Paper Cell Method. — According to my experience,
the best way to make a fluid mount safe is the following : — By
means of two punches I cut out rings of paper of about a
millimetre in breadth, and of about a millimetre smaller in
diameter than the cover-glass. Moisten the paper ring with
mounting fluid, and centre it on the slide. Fill the cell thus
formed with mounting fluid; arrange the object in it; put
the cover on ; fill the annular space between the paper and
the margin of the cover with glycerin jelly (a turn-table may
be useful for this operation) ; and as soon as the gelatin has
set turn a ring of Bell's or other cement on it.

For greater safety, the gelatin may of course be treated
with bichromate according to Marsh's plan.

433. Comparative Tenacity of Cements (see BEHRENS, Zeit. f.
wiss. Mik., ii, 1885, p. 54, and AUBERT, Amer. Mon. Mic.
Journ., 1885, p. 227 ; Journ. Roy. Mic. Soc., 1886, p. 173).—
Behrens gives the palm to amber varnish ; Aubert places
Miller's caoutchouc cement at the head of the list, Lovett's
cement coming halfway down, and zinc white cement at the
bottom, with less than one quarter the tenacity of the caout-
chouc cement.

434. Bell's Cement. — Composition unknown. May be ob-

254 CEMENTS AND VARNISHES.

tained from the opticians or from J. Bell and Co., chemists,
338, Oxford Street, London.

This varnish flows easily from the brush, and sets quickly.
For glycerin or other fluid mounts, the cover should be ringed
as above described with glycerin jelly before applying the
varnish. This precaution is especially necessary with glycerin.
This is the best varnish for fluid mounts known to me. It is
soluble in ether or chloroform. It is not attacked by oil of
cedar.

435. Miller's Caoutchouc Cement. — Composition unknown.
May be obtained from the opticians. A very tenacious and,
which is frequently an important point, a quickly drying
cement.

436. Asphalt Varnish (Bitume de Jude'e). — Unquestionably
one of the best of these media, either as a cement or a varnish
provided it be procured of good quality. It can be procured
from the opticians or from the oil-shops. KITTON (Month. Mic.
Joum., 1874, p. 34) recommends asphalt dissolved in benzol
with the addition of a small quantity of gold size.

437. Brunswick Black.— Best obtained from the opticians.
A receipt for preparing it is given in BEALE, How to Work, &c.,
p. 49.

' ' If a little solution of india rubber in mineral naphtha be
added to it, there is no danger of the cement cracking when
dry." Carpenter states that without this addition it is brittle
when dry. Brunswick black is soluble in oil of turpentine.
A most useful cement, works easily and dries quickly. It can
be recommended for turning cells.

438. Brunswick Black and Gold Size (EULENSTEIN, BEALE, How
to Work, &c., p. 49). — Equal parts of Brunswick black and
gold size with a very little Canada balsam.

439. Gold Size. — Receipts for preparing it may be found in
the Micrographic Diet, or in COOLEY'S Cyclopaedia ; but it is
certainly best to obtain it from the opticians or oil-shops. It
is soluble in oil of turpentine. A good cement, when of good
quality, and very useful for turning cells.

440. Marine Glue. — Found in commerce. Carpenter says
the best is that known as Gr K 4.

It is soluble in ether, naphtha, or solution of potash. Its

TUEPENTINE. 255

use is for attaching glass cells to slides, and for all cases in
which it is desired to cement glass to glass.

Receipts for preparing it may be found in BEALE, p. 49, or
in COOLEY'S Cyclopaedia.

441. Harting's Gutta-percha Cement (see BEALE'S How to
Work, &c., p. 49). — Marine glue serves the same purpose, viz*
that of attaching cells to slides.

442. India-rubber and Lime French Cement. — See BEALE, p.

58.

443. Knotting (Journ. Boy. Hie. floe., 1882, p. 745).—
" Patent knotting " from oil and colour stores, exposed to
the air until it has become of the proper consistency; — for
mending cells and for preventing running-in of the finishing
varnish (Northern Microscopist, ii, 1882).

444. Turpentine, Venice Turpentine (CsoKOE, Arch. mik.
Anat.j xxi, 1882, p. 353; PAEKEE, Amer. Hon. Mic. Journ., ii,
1881, pp. 229-30; Journ. Eoy. Mic. Soc. [N.S.], ii, 1882, p.
724). — Venice turpentine (Terebinthina veneta) is the liquid
resinous exudation of Abies larix. It is seldom met with in
a pure state. The following are the directions for preparing
and using it given by Parker :

Dissolve true Venice turpentine in enough alcohol, so that
after solution it will pass readily through a filter, and, after
filtering, place in an evaporating dish, and by means of a
sand-bath evaporate down to about three quarters of the
quantity originally used. (The best way to tell when the
evaporation has gone far enough is to drop some of the
melted turpentine, after it is evaporated down to about three
quarters its original volume, into cold water; if on being
taken out of the water it is hard and breaks with a vitreous
fracture on being struck with the point of a knife, cease eva-
poration and allow to cool.)

Or (CSOKOE), common resinous turpentine of commerce is
put in small pieces to melt over a water-bath, then poured
into a suitable vessel and allowed to cool. It should form a
brittle, dark brown mass, not yielding to the pressure of a
finger. It is sometimes useful, in order to attain the right
degree of hardness in the cold mass, to add a little resinous

256 CEMENTS AND VARNISHES.

oil of turpentine to the melted mass, and then to evaporate
for several hours over the water-bath.

This cement is used for closing glycerin mounts; it is
applied in the following manner: — Square covers are used,
and superfluous glycerin is cleaned away from the edges in
the usual way.

The cement is then put on with a piece of wire bent at
right angles (No. 10 — 12 wire is taken, and copper is the
best, as it gives to the turpentine a greenish tinge) ; the short
arm of the wire should be just the length of the side of the
cover-glass. The wire is heated in a spirit lamp, plunged
into the cement, some of which adheres to it, and then
brought down flat upon the slide at the margin of the cover.
The turpentine distributes itself evenly along the side of the
cover, and hardens immediately, so that the slide may be
cleaned as soon as the four sides are finished. It is claimed
for this cement that it is perfectly secure, very handy, and
never runs in. Parker saw this cement, or a similar one
known as Venedischer Damarlack, exclusively used for gly-
cerin mounts in the Pathological Laboratory at Vienna.

This is an extremely valuable method. It is very rapid and
very safe. The cement sets hard in a few seconds.

445. Colophonium and Wax (KKONIG, Arch. f. mik. Anat.}
1886, p. 657 ; Journ. Roy. Mic. Soc., 1887, p. 344).— Seven to
nine parts of colophonium are added piecemeal to two parts
of melted wax, the whole filtered and left to cool. For use,
the mass is melted by placing the containing vessel in hot
water. The cement is not attacked by water, glycerin, or
caustic potash.

446. Amber Varnish. — As above mentioned, BEHRENS finds
this cement to possess an extreme tenacity. He does not
give the composition of his varnish, which was procured from
B. Pfannenschmidt at Dantzic. The following is from COOLEY'S
Cyclopaedia, art. (t Varnish : "

" Take of amber (clear and pale) 6 Ibs., fuse it ; add of hot
clarified linseed oil 2 gallons, boil it until it " strings" well,
then let it cool a little and add of oil of turpentine 4 gallons
or q. s."

Other receipts, 1. c.

STIEDA'S WHITE ZINC CEMENT. 257

447. Amber and Copal Varnish (HEYDENREICH, Zeit. f. iviss.
Mik., 1885, p. 338). — An extremely complicated mode of
preparation. The varnish may be obtained from Ludwig
Marx, at 110, Moskowskaja Sastawa, St. Petersburg; or 79,
Gaden, Vienna ; or 1, Romerthal, Mayence.

448. Shellac Varnish (BEALE, p. 28). — Shellac should be_
broken into small pieces, placed in a bottle with spirit of
wine, and frequently shaken until a thick solution is obtained.
The Micro. Dictionary says that the addition of 20 drops of
castor oil to the ounce is an improvement.

Untrustworthy, but useful for protecting balsam mounts
from the action of oil of cedar.

For a method of preparing chemically pure shellac (a some-
what important matter), see WITT, Zeit. f. wiss. Mik., 1886,
p. 199.

For SEAMAN'S shellac cement for attaching metal to glass,
see Journ. Roy. Mic. Soc., 1888, p. 520.

449. Sealing-wax Varnish (Micro. Diet., " Cements "). — Add
enough spirit of wine to cover coarsely powdered sealing-wax,
and digest at a gentle heat. This should only be used as a
varnish, never as a cement, as it is apt to become brittle and
to lose its hold upon glass after a time.

450. Tolu Balsam Cement (CAKNOY'S Biol. Cell., p. 129).
Tolu balsam . . . . .2 parts.
Canada balsam . . . . .1 part.
Saturated solution of shellac in chlo-
roform ...... 2 parts.

Add enough chloroform to bring the mixture to a syrupy
consistence. Carnoy finds this cement superior to all others.

451. Stieda's White Zinc Cement (Arch. f. mik. Anat., 1866,
p. 435). — Rub up oxide of zinc with turpentine, and add,
stirring continually, for every drachm of the zinc oxide 1
ounce of a solution of damar in turpentine (of the consist-
ency of thick syrup) . This gives a white cement like Ziegler's.
For a red cement take, instead of zinc, cinnabar, and take 2
drms. of the metal for each ounce of the damar solution.
If the cement has become too thick with age, dilute with
turpentine, ether, or chloroform.

17

258 CEMENTS AND VARNISHES.

452. Ziegler's White Cement. — Composition unknown. Is
very much used on the Continent.

453. Kitten's White-lead Cement (Month. Mic. Journ., 1876,
p. 221). — Equal parts of white-lead, red-lead, and litharge
(all in powder), ground together with a little turpentine until
thoroughly incorporated, then mixed with gold size. The
mixture should be thin enough to work with a brush. No
more of the cement should be made than is required for
present use, as it soon sets and becomes unworkable; but a
stock of the materials may be kept ready ground in a bottle.

454. Lovett's Cement (Journ. Roy. Mic. Soc., 1883, p. 786). —
Two parts white-lead, 2 parts red oxide of lead (minium), 3
parts litharge. To be ground very fine, mixed dry, and kept
so in a bottle. When required for use mix a little of the
powder with gold size to the consistency of paint, taking care
that no grit gets into it.

455. Apathy's Cement for Glycerin Mounts (Zeit. f. wiss.
Mik., vi, 2, 1889, p. 171). -Equal parts of hard (60° C. melt-
ing-point) paraffin and Canada balsam. Heat together in a
porcelain capsule until the mass takes on a golden tint, and
no longer emits vapours of turpentine. On cooling this forms
a hard mass, which is used by warming and applying with a
glass rod or brass spatula. One application is enough. The
cement does not run in, and never cracks.

456. AspinalTs Enamel. — STANLEY KENT (Journ. Roy. Mic.
Soc., 1890, p. 821) finds this of great use, both for ringing
slides and making cells.

KOBIN'S GLYCERIN-GELATIN VEHICLE. 259

CHAPTER XXI.

INJECTIONS — GELATIN MASSES.

457. Introduction. — Injection masses are composed of a
coloured substance, technically termed the colouring mass, and
of a substance with which that is combined, technically termed
the vehicle.

The following formulae are grouped according to the nature
of the vehicle. A note on the employment of nitrite of amyl
for provoking the dilatation of vessels will be found at § 500.

458. Robin's Gelatin Vehicle (Traite, p. 30). — Take some
gelatin, of the sort known as '< colla de Paris." (This
gelatin is found in commerce in the form of thin sheets,
marked with lozenge-shaped impressions of the cords which
supported them whilst drying.) Soak it in cold water, then
heat in water over a water-bath. One part of gelatin should
be taken for every 7, 8, 9, or even 10 parts of water; it is a
common error to employ solutions containing too much gelatin.
The solution is now to be combined with one of the colouring
masses given below.

This vehicle, like all gelatin masses, is liable to be attacked
by mould if kept long; camphor and carbolic acid do not
suffice to preserve it.

Chloral hydrate added to the mass will preserve it (HOYER).
A sufficient dose, at least 2 per cent., should be employed (see
below, §§ 472, 473).

459. Robin's Glycerin-Gelatin Vehicle (Traite, p. 32).— Dis-
solve in a water-bath 50 grms, of French gelatin (" colle de
Paris") in 300 grms. of water in which has been dissolved
some arsenious acid-; add of glycerin 150 grms., and of car-
bolic acid a few drops. Unlike the pure gelatin vehicles, this
mass does keep indefinitely.

The colouring masses recommended for combination with
the vehicles above described are made as follows :

260 INJECTIONS.

460. Carmine Colouring Mass (Traite, p. 33). — Rub up in a
mortar 3 grms. of carmine with a little water and enough
ammonia to dissolve the carmine. Add 50 grms. of glycerin,
and filter.

Prepare 50 grins, of acid glycerin (containing 5 grms. of
acetic acid for every 50 grms. of glycerin), and add it by
degrees to the carmine-glycerin, until a slightly acid reaction
is obtained (as tested by very sensitive blue test-paper, moist-
ened and held over the mixture).

One part of this mixture is to be added to 3 or 4 parts of
the gelatin injection vehicle (ante, Formula 458), or of the
glycerin-gelatin (No. 459), or glycerin-alcohol vehicle de-
scribed below (No. 502).

461. Ferrocyanide of Copper Colouring Mass (ibid., p. 34).
Take—

(1) Ferrocyanide of potassium (concentrated

solution) 20 c.c.

Glycerin . . . . . . 50 „

(2) Sulphate of copper (concentrated solu-

tion) . . . . . . 35 „

Glycerin . . . . . 50 „

Mix (1) and (2) slowly, with agitation; at the moment of
injecting combine with 3 volumes of vehicle.

462. Blue Colouring Mass (Prussian Blue) (Robin's modifica-
tion of Beale' s formula, ibid., p. 35).

Take—

(A) Sulphocyanide of potassium (sol. sat.) 90 c.c.
Glycerin . . . . . . 50 „

(B) Liquid perchloride of iron at 30° . 3 „
Glycerin . . . . . . 50 „

Mix slowly and combine the mixture with 3 parts of vehicle.
It is well to add a few drops of HC1.

463. Cadmium Colouring Mass (ibid., p. 36).

Take-
Sulphate of cadmium (sol. sat.) . . 40 c.c.
Glycerin . . . . . . 50 „

and

Sulphide of sodium (sol. sat.) . . 30 „
Glycerin . . . . . . 50 „

Mix with agitation and combine with 3 vols. of vehicle.

RANVIER'S CARMINE-GELATIN MASS. 261

464. Scheele's Green Colouring Mass (ibid., p. 37).
Take-

Arseniate of potash (saturated solution) 80 c.c.

Glycerin . . . . . . 50 „

and

Sulphate of copper (saturated solution) 40 „

Glycerin . . . . . 50 ,,

Mix and combine with 3 vols. of vehicle.

465. Anilin Colouring Masses (ibid., p. 37). — Most of the
anilin colours have, for injections, the great fault of being
soluble in alcohol ; fuchsin is soluble in water, in alcohol, and
in glycerin ; it therefore cannot be employed with a gelatin
or glycerin vehicle. Anilin blue, violet, yellow, may be com-
bined with these vehicles after dissolving in a small quantity
of alcohol; and (alcohol being avoided for hardening purposes)
the injected organs may be preserved in glycerin.

Carmine-Gelatin Masses.

466. Ranvier's Carmine-Gelatin Mass (Traite technique, p.
116). — Take 5 grms. Paris gelatin, soak it in water for half
an hour, or until quite swollen and soft ; wash it ; drain it ;
put it into a test-tube and melt it, in the water it has absorbed,
over a water-bath. When melted add slowly, and with con-
tinual agitation, a solution of carmine in ammonia, prepared
as follows : — 2J grms. of carmine are rubbed up with a little
water, and just enough ammonia, added drop by drop, to dis-
solve the carmine into a transparent solution.

When the carmine has been added to the gelatin you will
have about 15 c.c. of ammoniacal solution of carmine in
gelatin, if the operations have been properly performed. This
solution is to be kept warm on the water-bath, whilst you
proceed to neutralise it by adding cautiously, drop by drop,
with continual agitation, a solution of 1 part of glacial acetic
acid in 2 parts of water. (When the mass is near neutrality,
dilute the acetic acid still further.) The instant of saturation
is determined by the smell of the solution, which gradually
changes from ammoniacal to sour. As soon as the sour smell
is perceived, the addition of acetic acid must cease, and the
liquid be examined under the microscope. If it contains a

262 INJECTIONS.

granular precipitate of carmine, too much acid has been added,
and the mass must be thrown away.

Ranvier states that by practice the operator learns to attain
to perfect neutralisation almost infallibly in this way, and
that this is the only way to attain to it. Trust must not be
put in certain formulae that profess to indicate the propor-
tions of ammonia and acetic acid necessary for neutralisation,
on account of the variation in strength of the solutions of am-
monia kept in laboratories. The method proposed by Frey of
determining beforehand the quantity of a known acetic so-
lution that is necessary for neutralisation of a given quantity
of the ammonia employed, is not infallible because it often hap-
pens that commercial gelatin is acid ; in which case the pro-
posed method would cause the operator to overpass the point
of saturation.

The mass having been perfectly neutralised is strained
through new flannel.

467. How to Neutralise a Carmine Mass (VILLE, Gaz. hebd.
d. Sci. med. de Montpellier, Fev., 1882; maybe had separately
from Delahaye et Lecrosnier, Paris). — Ville is of Ranvier' s
opinion that the method of titration recommended by Frey is
defective, but for a different reason. When carmine is treated
with ammonia a certain proportion of the ammonia combines
with the carmine to form a transparent purple compound, and
the rest of the ammonia remains in excess. It is this excess
that it is required to neutralise precisely. In Frey's method a
quantity of acid sufficient for the neutralisation of the whole
of the ammonia employed is taken; hence, naturally, the point
of neutralisation is overstepped, and a granular mass is the
result.

As to the acidity accidentally found in commercial gelatin,
that source of error is easily eliminated. Instead of soaking
the gelatin in water, it should be placed in a large funnel with
a narrow neck, or, better, in a stopcock funnel, and the whole
should be placed under a tap, and a stream of water arranged
in such a manner that the gelatin be constantly completely
immersed. Washing for an hour or so in this way will
remove all traces of acids mechanically retained in the
gelatin.

As to the neutralisation of the colouring mass, Ville is of

HOW TO NEUTRALISE A CARMINE MASS. 263

opinion that the criterion of neutrality given by Kanvier — the
sour smell that takes the place of the ammoniacal odour — can-
not be safely relied 011 in practice. He considers it greatly
preferable' to employ dichroic litmus paper (litmus paper
sensitized so as to be capable of being used equally for the
demonstration of acids and bases).

To prepare such a paper, the tincture obtained by decoction:
of cake litmus is slightly acidified by an excess of sulphuric
acid. By this means the excess of alkali, or of alkaline
carbonates, that is always present in litmus decoction, and
which diminishes its sensibility as a reagent, is neutralised.
The decoction is then heated and agitated with an excess of
precipitated carbonate of baryta, and filtered.

The solution of litmus thus obtained is exposed to the air
in wide vessels until its intense blue colour has given place to
a reddish tint. Strips of white unsized paper are then
dipped in it, and dried in the shade on stretched threads, in
a place free from vapour of ammonia.

A shorter method consists in adding very dilute sulphuric
acid, drop by drop, to the ordinary laboratory tincture of lit-
mus, until the colour changes to red. Then, by adding suc-
cessively traces of alkali and very dilute sulphuric acid, the
reddish dichroic tint may be obtained, and the paper pre-
pared with the solution as before.

The paper is used in the same way as ordinary litmus paper.
A strip is moistened with distilled water and held as close as
possible to the injection mass kept melted on a water-bath.
It becomes blue at first, very rapidly and decidedly; but as
fast as fresh quantities of acid are added this reaction
becomes less evident, and at a certain moment the change of
colour becomes very slow in making its appearance. It is then
that the addition of acid should cease, and the operation is
ended.

Very delicate sensitized paper may also be prepared with
other reagents than litmus — for instance, with Nessler's re-
agent* or with alcoholic solution of haematoxylin, or with a

* Nessler's reagent may be prepared as follows : — Mercuric chloride, in
powder, 35 grins. ; iodide of potassium, 90 grms. ; water, 1750 c.c. Heat
gently till dissolved in a large basin ; then add of stick caustic potash 320
grms., and 50 c.c. of saturated solution of mercuric chloride (WANKLYN).
From COOLEY'S Cyclopaedia, s. v. " Nessler's Test."

264 INJECTIONS.

solution made by adding a trace of dilute sulphuric acid to
" liqueur orange No. 3" (a liquid found in commerce, and
used for detecting acids) ; the solution takes on a gooseberry-
red colour.

The preparation of the injection mass is facilitated by em-
ploying acetic acid and ammonia of known strength. For the
acetic acid it is sufficient to keep the glacial acid in a well-
stoppered bottle. But this will not suffice for the ammonia,
which is notably lowered in strength through the mere pour-
ing from one bottle into another. Ville has imagined an ap-
paratus which allows of withdrawing a known quantity
without permitting any access of air to the stock solution.
Description and figures, 1. c.

With the exception of the processes above described, Ville
prepares the injection mass exactly as Ranvier.

468. Gerlach's Carmine-Gelatin Mass (see Arch. f. mik. Anat.,
1865, p. 148; and Ranvier's Traite, p. 113).

469. Thiersch's Carmine-Gelatin Mass (see ibid.).

470. Carter's Carmine-Gelatin Mass (see BEALE, p. 113).

471. Davies' Carmine-Gelatin Mass (see his Prep, and Mount-
ing of Mic. Objects, p. ]38).

472. Hoyer's Carmine-Gelatin Mass (Biol. Centralb., 1882,
p. 21). — Take a concentrated gelatin solution and add to it a
corresponding quantity of neutral carmine staining solution
(see § 163). Digest in a water-bath until the dark violet-red
colour begins to pass into a bright red tint. Then add 5 — 10
per cent, by volumes of glycerin, and at least 2 per cent,
by weight of chloral, in a concentrated solution. After
passing through flannel it can be kept in an open vessel
under a bell-glass.

473. Fol's Carmine-Gelatin Mass (Zeit.f. wiss. ZooL, xxxviii,
1883, p. 492).

The following method of preparation has the advantage of
producing masses that can be kept in the dry state for an
indefinite length of time. (Fol finds that the addition of
chloral hydrate to wet masses is not an efficient preservative.)

One kilog. of Simeon's photographic gelatin* is soaked for

* This gelatin may be obtained either from the ordinary furnishers of

FOI/S CARMINE-GELATIN MASS. 265

a couple of hours, until thoroughly soft, in a small quantity
of water. The water is then poured off and the gelatin melted
over a water-bath, and one litre of concentrated solution of
carmine in ammonia is poured in with continual stirring.
(The carmine solution is prepared by diluting strong solution
of ammonia with three or four parts of water and adding
carmine to saturation ; the undissolved excess of carmine is~
removed by filtration just before the solution is added to the
gelatin.)

To the mixture of gelatin and carmine, which should have
a strong smell of ammonia, sufficient acetic acid is added to
turn the dark purple colour of the mixture into the well-known
blood-red hue. Exact neutralisation is not necessary. The
mass is set aside until it has become firm, and is then cut up
into pieces, which are tied up in a piece of tulle or fine netting.
By means of energetic compression with the hand under
water (it must be acidulated water, 0*1 per cent, acetic acid,
otherwise the carmine will wash out : cf. Journ. Roy. Mic. Soc.,
iv, part 3, 1884, p. 474) the mass is driven out through the
meshes of the stuff in the shape of fine strings, which are
washed for several hours in a sieve placed in running water in
order to free them from any excess of acid or ammonia. The
strings are then again melted, and the molten mass is poured
on to large sheets of parchment paper soaked with paraffin,
and the sheets are hung up to dry in an airy place. When
dry the gelatin can easily be separated from the sheets, and
may be cut into long strips with scissors and put away, pro-
tected from dust and damp, until wanted for use. In order
to get the mass ready for use, all that is necessary is to soak
the strips for a few minutes in water and melt them over a
water-bath.

The process may be simplified, without giving very greatly
inferior results, as follows (Lehrb., p. 13). Gelatin in sheets
is macerated for two days in the above-described carmine
solution, then rinsed and put for a few hours into water acidu-
lated with acetic acid. It is then washed on a sieve for several

articles used in photography, or direct from Simeon's Gelatin-fabrik,
Winterthur, Switzerland. Two sorts, a hard and a soft, are sold ; the softer
is to be preferred on account of its lower point of fusion. Probably the
photographic gelatins of Hinrichs, of Frankfurt, and of Coignet, of Paris,
would answer equally well ; as also the best English preparations.

266 INJECTIONS.

hours in running water, dried on parchment paper, and pre-
served as above.

This injection mass is very well spoken of.

Blue Gelatin Masses.

474. Robin's Prussian Blue Gelatin Mass (see above, No. 462).

475. Ranvier's Prussian Blue Gelatin Mass (Traite, p. 119).
— Twenty-five parts of a concentrated aqueous solution of
soluble Prussian blue (prepared as directed below) mixed
with 1 part of solid gelatin.

The mixture of the Prussian blue with the vehicle is effected
in the following manner :

Weigh the gelatin, soak it in water for half an hour or an
hour, wash it, and melt it in a test-tube, in the water it has
absorbed, by heating over a water-bath. Put the solution
of Prussian blue into another test-tube, and heat it on the
same water-bath as the gelatin, so as to have the two at the
same temperature. Pour the gelatin gradually into the
Prussian blue solution, stirring continually with a glass rod.
Continue stirring until the disappearance of the curdy pre-
cipitate that forms at first. (Some gelatins produce a per-
sistent precipitate; these must be rejected; but it must be
borne in mind that the precipitate that invariably forms in
even the best gelatins disappears if the heating be continued.
It is essential to remember this when preparing Prussian blue
and gelatin mass.) As soon as the glass rod has ceased to
show blue granulations on its surface on being withdrawn
from the liquid, it may be concluded that the Prussian blue
is completely dissolved. Filter through new flannel, and
keep the filtrate at 40° over a water-bath until injected.

The soluble Prussian blue for the above mass is prepared
as follows :

476. Soluble Prussian Blue for Injection Masses (RANVIER,
ibid.). — Make a concentrated solution of sulphate of peroxide
of iron in distilled water, and pour it gradually into a con-
centrated solution of yellow prussiate of potash. There is
produced a precipitate of insoluble Prussian blue. (An
excess of prussiate of potash ought to remain in the liquid ;
in order to ascertain whether this is the case take a small

BRUOKE'S SOLUBLE BERLIN BLUE. 267

quantity of the liquid and observe whether a drop of sulphate
of iron still precipitates it.) Filter the liquid through a felt
strainer, underneath which is arranged a paper filter in a
glass funnel. The liquid at first runs clear and yellowish
into the lower funnel ; distilled water is then poured little
by little on to the strainer ; gradually the liquid issuing from
the strainer acquires a blue tinge, which, however, is not
visible in that which issues from the lower filter. Distilled
water is continually added to the strainer for some days until
the liquid begins to run off blue from the second filter. The
Prussian blue has now become soluble. The strainer is turned
inside out and agitated in distilled water ; the "Prussian blue
will dissolve if the quantity of water be sufficient.

The solution may now be injected just as it is, or it may be
kept in bottles till wanted, or the solution may be evaporated
in a stove, and the solid residuum put away in bottles.

For injections, if a. simple aqueous solution be taken, it
should be saturated. Such a mass never transudes through
the walls of vessels. Or it may be combined with one fourth
of glycerin, or with the gelatin vehicle above described.

477. Soluble Prussian Blue (GUIGNET, Journ. de Microgr.,
1889, p. 94; Journ. Roy. Mic. Soc., 1889, p. 468}.— Guignet
gives two methods :

1. To a boiling solution of 110 grammes of ferridcyanide
of potassium are added gradually 70 grammes of crystallised
sulphate of iron. After boiling two hours it is filtered, and
the precipitate washed with fresh water until the washings
are strongly blue. It is then dried at 100° C.

2. A saturated solution of oxalic acid is mixed to a pasty
consistence with an excess of pure Prussian blue. The liquid
is filtered and allowed to stand for two months until all the
blue is precipitated. It is then filtered and washed with
weak spirit in order to remove any oxalic acid, then dried.

A similar result may be at once obtained by precipitating
the oxalic solution with 95 per cent, alcohol, or with a con-
centrated solution of sodium sulphate, and then washing the
precipitate with weak spirit.

478. Briicke's Soluble Berlin Blue (Arch. f. mik.Anat., 1865,
p. 87). — Briicke first prepared it by taking a 10 per cent,
solution of ferrocyanide of potassium, and precipitating by

268 INJECTIONS.

means of a dilute solution of sesquichloride of iron (taken in
such a quantity as to contain just half as much chlorine as is
necessary for the decomposition), and washing the precipitate
on the filter until solubility is attained.

Later on he employed a greater excess of ferrocyanide, and
took just so much dilute solution of chloride of iron that the
weight of the dry chloride employed came to y1^ or -J- of
that of the ferrocyanide. The precipitate was washed on a
filter (using the filtrate to wash with) until nothing but a
clear yellow liquid filtered off, then washed with water until
the water began to run off blue, then dried, pressed between
blotting-paper in a press, the resulting mass broken in pieces
and dried by exposure to the air.

A cheaper method is the following :

Make a solution of ferrocyanide of potassium containing
217 grammes of the salt to 1 litre of water.

Make a solution of 1 part commercial chloride of iron in
10 parts water.

Take equal volumes of each, and add to each of them twice
its volume of a cold saturated solution of sulphate of soda.
Pour the chloride solution into the ferrocyanide solution,
stirring continually. Wash the precipitate on a filter until
soluble, and treat as above described.

The concentrated solution of the colouring matter is to be
gelatinised with just so much gelatin that the mass forms a
jelly when cold.

479. Thiersch's Prussian Blue Gelatin Mass (Arch. f. mik.
Anat., i, 1865, p. 148).
Take—

(1) A solution of 1 part gelatin in 2 parts water.

(2) A saturated aqueous solution of sulphate of iron.

(3) A saturated aqueous solution of red prussiate of
potash.

(4) A saturated aqueous solution of oxalic acid.

Now (A) mix 12 c.c. of the iron solution with one ounce of
the gelatin solution at the temperature of 25° R.

Then (B) mix, at the same temperature, 24 c.c. of the prus-
siate solution with two ounces of the gelatin solution.

(c) To the latter mixture add first 24 c.c. of the oxalic
acid solution, stir well, and then add the gelatin and iron

FOL'S BEKLIN BLUE GELATIN MASS. 269

mixture (A). Stir continually, keeping the temperature at
from 20° to 25° R. until the whole of the Prussian blue is
precipitated. Finally, heat over a water-bath to about 70°
R. and filter through flannel.

480. Fol's Berlin Blue Gelatin Mass (Zeit. f. wiss. Zool.,
xxxviii, 1883, p. 494). — A modification of Thiersch's formula,
No. 479. 120 c.c. of a cold saturated solution of sulphate of
iron are mixed with 300 c.c. of the warm gelatin solution.
In a separate vessel 600 c.c. of the gelatin solution are mixed
with 240 c.c. of a saturated solution of oxalic acid, and
240 c.c. of a cold saturated solution of red prussiate of potash
are added to the mixture. The first mixture is now gradually
poured into the second, with vigorous shaking, the whole is
warmed for a quarter of an hour over a boiling water-bath,
the mass is allowed to set, is pressed out into strings through
tulle or netting, as described for the carmine mass, supra,
§ 473, and the strings are washed and spread out to dry on
the prepared paper. (It is necessary to dry the strings
without remelting in this case, because the mass does not
readily melt without the addition of oxalic acid.) In order
to prepare the mass for injection, the strings are put to swell
up in cold water, and then warmed with the addition of
enough oxalic acid to allow of complete solution.

481. Hover's Soluble Berlin Blue Gelatin Mass (Arch. f. mik.
Anat.y 1876, p. 649). — The filtered and not too much washed
precipitate of soluble Berlin blue is brought in a little water
on to a Graham's dialyser, and the external water changed
until the solution begins to pass through the parchment.
Dilute the solution and filter through filter-paper, an opera-
tion which becomes easy after dialysis. The solution may be
injected pure (for lymphatics, for instance) or may be com-
bined with gelatin. To do this, warm the solution almost to
boiling-point, and add gradually a warm, thin solution of
gelatin until coagulation begins to set in. Strain through
wetted flannel.

Gelatin Masses of other Colours.

482. Robin's Cadmium Gelatin Mass (see § 463).

483. Thiersch's Lead Chromate Gelatin Mass (Arch. /. mik.
Anat.y 1865, p. 149).

270 INJECTIONS.

Make —

(A) A solution of 1 part gelatin in 2 parts water.

(B) A solution of 1 part neutral chromate of potash in 11
parts water.

(c) A solution of 1 part nitrate of lead in 11 parts water.

Mix 4 parts of the gelatin solution with 2 parts of the lead
solution, and in another vessel mix 4 parts gelatin solution
with 1 part of the chromate solution. Heat both the mixtures
to 25° R. ; mix them together with continual stirring until all
the chromate of lead is precipitated ; heat over a water-bath
to 70° R., and filter through flannel.

484. Hover's Lead Chromate Gelatin Mass (ibid., 1867, p.
136).
Take-
One volume of a solution of gelatin containing 1 part of

gelatin to 4 of water.
One volume of cold saturated solution of bichromate of

potash.
And one volume cold saturated solution of sugar of lead

(neutral plumbic acetate).

Filter the gelatin solution through flannel, and mix in the
bichromate solution. Then warm almost to boiling-point, and
add gradually the (warmed) sugar of lead solution. Allow
the mass to cool down to body temperature, and inject at
once. Another mode of preparation is as follows : — Mix the
sugar of lead solution with part of the gelatin solution, mix
the bichromate solution with the remaining gelatin solution,
heat the latter mixture, and pour into it the former mixture
(gradually), stirring continually.

If the solutions are mixed at a low temperature a lumpy
granular precipitate is formed. Further, when solution of
sugar of lead is added to a hot solution of bichromate of
potash a rich orange-red precipitate is obtained; whilst if the
solutions be mixed cold the precipitate is bright yellow.

If the solutions of the two salts be kept ready prepared,
the injection mass may be mixed in less than a quarter of an
hour. Its advantages are that, on account of the extremely
fine state of division of the precipitate, the mass is almost
transparent, and runs so freely that even lymphatics may be
perfectly injected with it, whilst its intensity of colour makes

TREY'S WHITE GELATIN MASS. 271

the vessels much more distinct than the very pale mass of
Thiersch (No. 483). It is also easier to manage than
Thiersch's mass, as it does not solidify so quickly. It shows
well in the vessels by reflected, as well as by transmitted
light.

485. Fol's Lead Chromate Gelatin Mass (Lehrb., p. 15).

486. Hoyer's Silver Nitrate Yellow Gelatin Mass (Biol. Cen-
tralbl, ii, 1882, pp. 19, 22 ; Journ. Eoy. Mic. Soc. [N.S.], iii,
1883, p. 142). — " A concentrated solution of gelatin is mixed
with an equal volume of a 4 per cent, solution of nitrate of
silver and warmed. To this is added a very small quantity
of an aqueous solution of pyrogallic acid, which reduces the
silver in a few seconds ; chloral and glycerin are added as
before" (see ante, HOYER'S formula for carmine-gelatin,
No. 472).

This mass is yellow in the capillaries and brown in the
larger vessels. It does not change either in alcohol, chromic
or acetic acid, or bichromate of potash, &c.

487. Hoyer's Green Gelatin Masses (ibid.}. — Made by mixing
a blue mass and a yellow mass.

488. Thiersch's Green Gelatin Mass (Arch.f. mik. Anat., 1865,
p. 149) . — Made by mixing the blue mass, § 479, and the yellow
mass, § 483.

489. Robin's Scheele's Green Gelatin Mass (see § 464).

490. Hartig's White Gelatin Mass (FEEY, Le Microscope,
p. 190). — Dissolve 125 grammes of acetate of lead in so much
water that the whole shall weigh 500 grammes.

Dissolve 95 grammes of carbonate of soda in so much
water that the whole shall weigh 500 grammes.

Take equal volumes of the two solutions, and add two
volumes of gelatin solution.

491. Frey's White Gelatin Mass (ibid.). — Put into a tall glass
cylinder 125 to 185 grammes of cold saturated solution of
chlorate of baryta. Add drop by drop, very carefully, sul-
phuric acid. Allow the precipitate that forms to settle for
twelve hours, then decant almost all the clear supernatant
liquid. The remaining mucilaginous mass containing the-

272 INJECTIONS.

precipitate is to be mixed with an equal part of concentrated
gelatin solution.

Frey states that this is a very finely grained mass. Injected
organs may be preserved in chromic acid.

492. Teichmann's White Gelatin Mass (ibid.,p. 191). — "Take
3 parts of nitrate of silver dissolved in the gelatin solution,
and add 1 part of common salt."

The mass is very fine-grained, and is not decomposed by
chromic acid; the disadvantage of it is that it blackens under
the influnce of light and of sulphurous solutions.

493. Fol's Brown Gelatin Mass (Zeit. f. wiss. Zool., xxxviii,
1883, p. 494). — 500 grms. of gelatin are soaked, and allowed
to swell up, in 2 litres of water in which 140 grms. of com-
mon salt have previously been dissolved ; the mass is melted
over a water-bath, and a solution of 300 grms. of nitrate of
silver in a litre of water is gradually added, with vigorous
shaking. (If it be desired to have an extremely fine-grained
mass, both the solutions should be diluted with 3 or 4 volumes
of water.) The mass is pressed out into strings as before
(§ 473), and the strings are stirred up in clear daylight with
the following mixture : — 1 \ litres of cold saturated solution
of potassic oxalate to 500 c.c. of cold saturated solution of
sulphate of iron. As soon as the whole mass is thoroughly
black the operation is at an end. The strings are then washed
for several hours, remelted, and poured on to the prepared
paper.

494. Miller's Purple Silver Nitrate Gelatin Mass. — See Amer.
Hon. Mic. Journ.j 1888, p. 50; Journ. Roy. Mic. Soc., 1888, p.
518; Zeit.f. wiss. Mik., v, 3, 1888, p. 361.

495. Robin's Mahogany Gelatin Mass. — See § 461.

496. Ranvier's Gelatin Mass for Impregnation (Traite, p. 123).
— Concentrated solution of gelatin, 2, 3, or 4 parts ; 1 per
cent, nitrate of silver solution, 1 part.

497. Fol's Metagelatin Vehicle (Lchrb., p. 17). — The opera-
tion of injecting with the ordinary gelatin masses is greatly
complicated by the necessity of injecting them warm. FOL
proposes to employ metagelatin instead of gelatin.

If a slight proportion of ammonia be added to a solution of

FOI/S METAGELA.TIN VEHICLE. 273

gelatin, and the solution be heated for several hours, the
solution passes into the state of metagelatin, that is, a state
in which it no longer coagulates on cooling. Colouring masses
may be added to this vehicle, which may also be thinned by
the addition of weak alcohol. After injection, the prepara-
tions are thrown into strong alcohol or chromic acid, which
sets the mass.

18

274 INJECTIONS.

CHAPTER XXII.

INJECTIONS — OTHER MASSES.

498. Joseph's White-of-Egg Injection Mass (Carmine) (Ber.
naturw. sect. Schles. Ges., 1879, pp. 36 — 40; Journ. Roy. Mic.
Soc. [N.S.], ii, 1882, p. 274).— « Filtered white of egg, diluted

with 1 to 5 per cent, of carmine solution This mass

remains liquid when cold; it coagulates when immersed
in dilute nitric acid, chromic or osmic acid, remains trans-
parent, and is sufficiently indifferent to reagents."

For Invertebrates.

499. Bjeloussow's Gum Arabic Mass (Arch.f. Anat. u.Phys.,
1885, p. 379). — Make a syrupy solution of gum arabic and a
saturated solution of borax in water. Mix the solutions in
such proportions as to have in the mixture 1 part of borax to
2 of gum arabic. Rub up the transparent, almost insoluble
mass with distilled water, added little by little, then force it
through a fine-grained cloth. Repeat these operations until
there is obtained a mass that is free from suspended gela-
tinous clots. (If the operation has been successful, the mass
should coagulate in the presence of alcohol, undergoing at the
same time a dilatation to twice its original volume.)

The vehicle thus prepared may be combined with any
colouring mass except cadmium and cobalt.

After injection the preparation is thrown into alcohol, and
the mass sets immediately, swelling up as above described,
and consequently showing vessels largely distended.

Cold-blooded animals may be injected whilst alive with this
mass. It does not flow out of cut vessels. Injections keep
well in alcohol. Glycerin may be used for making them
transparent.

If it be desired to remove the mass from any part of a pre-
paration, this is easily done with dilute acetic acid, which
•dissolves it.

BEALE'S PRUSSIAN BLUE GLYCERIN MASS. 275

Glycerin Masses (cold).

500. Nitrite of Amyl as a Vaso -dilatator. — Glycerin masses
are certainly very convenient, and give very good results from
the scientific — not from the aesthetic — point of view. They
have a great defect for the injection of fresh specimens — that
is, those in which rigor mortis has not set in ; they stimulate
the contraction of arteries. In these cases it may be advisable
to use nitrite of amyl as a vaso-dilatator. The animal may be
anassthetised with a mixture of ether and nitrite of amyl, and
finally killed with pure nitrite. Or, after killing in any way,
a little nitrite of amyl in salt solution may be injected before
the injection mass is thrown in. In any case it is advisable
to add a little nitrite to the mass just before using. The
relaxing power is very great (see OVIATT and SARGENT, in St.
Louis Med. Journ., 1886, p. 207; and Journ. Roy. Mic. Soc.,
1887, p. 341).

501. Scale's Carmine Glycerin Mass (How to Work, &c.,
p. 95). — Five grains of carmine are dissolved in a little water
with the aid of about five drops of ammonia, and added to
half an ounce of glycerin. Then add half an ounce of glycerin
with eight or ten drops of acetic or hydrochloric acid, gradu-
ally, with agitation. Test with blue litmus paper, and if
necessary add more acid till the reaction is decidedly acid.
Then add half an ounce of glycerin, two drachms of alcohol,
and six drachms of water.

502. Robin's Carmine Glycerin Mass (Traite, p. 33) .—Consists
of the following vehicle :

Glycerin 2 parts.

Alcohol ...... 1 part.

Water 1 „

Combined with one third or one fourth its volume of the
carmine colouring mass, ante, formula No. 460.

503. Beale's Prussian Blue Glycerin Mass (How to Work, &c.,
p. 93).

Common glycerin ... 1 ounce.
Spirits of wine ....!„
Ferrocyanide of potassium . 12 grains.

Tincture of perchloride of iron . 1 drachm.
Water .... 4 ounces.

276 INJECTIONS.

Dissolve the ferrocyanide in one ounce of the water and
glycerin, and add the tincture of iron to another ounce.
" These solutions should be mixed together very gradually
and well shaken in a bottle, the iron being added to the solu-
tion of the ferrocyanide of potassium. Next, the spirit and
the water are to be added very gradually, the mixture being
constantly shaken/'

" The ivater " spoken of in the last sentence appears to
mean the remaining three ounces of water that were not
mixed with the glycerin at first.

Injected specimens should be preserved in acidulated gly-
cerin, otherwise the colour may fade.

504. Beale's Acid Prussian Blue Glycerin Mass (ibid-., p. 296).
Price's glycerine ... 2 fluid ounces.
Tinct. of sesquichloride of iron . 10 drops.
Ferrocyanide of potassium . . 3 grains.

Strong hydrochloric acid . . 3 drops.
Water ..... 1 ounce.

Proceed as directed above, dissolving the ferrocyanide in
one half of the glycerin, the iron in the other, and adding
the latter drop by drop to the former. Finally add the water
and HC1. Two drachms of alcohol may be added to the
whole if desired.

I consider this a most admirable formula. I possess some
of this mass prepared many years ago, in which not the
smallest flocculus has made its appearance. The Prussian
blue appears to be in a state of true solution. The mass
runs well, and has not so much tendency to exude from cut
capillaries as might be Supposed.

505. Ranvier's Prussian Blue Glycerin Mass (Traite, p. 120). —
Consists of the Prussian blue fluid, § 476, mixed with one
fourth of glycerin.

506. Other Colours. — Any of the colouring masses, §§ 460
to 465, or other suitable colouring masses, combined with the
vehicle, § 502.

Aqueous Masses.

507. Ranvier's Prussian Blue Aqueous Mass (Traite} p. 120).

TAGUCHI'S INDIAN INK. 277

— The soluble Prussian blue, § 476, injected without any
vehicle. It does not extravasate.

508. Miiller's Berlin Blue (Arch. f. mile. Anat., 1865, p. 150).
— Precipitate a concentrated solution of Berlin blue by means
of 90 per cent, alcohol.

The precipitate is very finely divided ; the fluid is perfectly
neutral, and much easier to prepare than the formula of
Beale.

509. Mayer's Berlin Blue (Mitth. zool. Stat. Neapel, 1888,
p. 307). — A solution of 10 c.c. of tincture of perchloride of
iron in 500 c.c. of water is added to a solution of 20 gr. of
yellow prussiate of potash in 500 c.c. of water, allowed to
stand for twelve hours, decanted, the deposit washed with
distilled water on a filter until the washings come through
dark blue (one to two days), and the blue dissolved in about
a litre of water.

510. Emery's Aqueous Carmine (ibid., 1881, p. 21). — To a
10 per cent, ammoniacal solution of carmine is added acetic
acid, with continual stirring, until the colour of the solution
changes to blood-red through incipient precipitation of the
carmine. The supernatant clear solution is poured off, and
injected cold without further preparation. The injected
organs are thrown at once into strong alcohol to fix the
carmine. For injection of Fishes.

511. Letellier's Vanadate of Ammonia and Tannin (Journ.
Roy. Mic. Soc., 1889, p. 151). — Vanadate of ammonia is
soluble in warm, and tannin in hot water. The two solutions
are kept apart until required for use, when they are mixed
according to the tint required. A black mass, very fine.
The walls of vessels are stained black by it.

512. Taguchi's Indian Ink (Arch. f. mik. Anat., 1888, p. 565 ;
Zeit. f. wiss. Mik., 1888, p. 503). — Chinese or (better) Japanese
ink well rubbed up on a hone until a fluid is obtained that
does not run when dropped on thin blotting-paper, nor form a
grey ring round the drop. Inject until the preparation
appears quite black, and throw it into some hardening liquid
(not pure water).

278 INJECTIONS.

I believe this will be found useful for many purposes,
especially for work amongst Invertebrates, as well as for
lymphatics, juice-canals, and the like.

Celloidin Masses.

513. Schiefferdecker's Celloidin Masses (Arch. Anat. u. Phys.>
1882 \_Anat.AUh.'])p. 201). (For Corrosion preparations.)

1. Asphalt-celloidin is the best of these injections. To pre-
pare it —

Pulverise asphalt in a uiortar, and put it for twenty-four
hours into a well-closed vessel with some ether, shaking occa-
sionally. After the twenty-four hours pour off the ether into
another vessel, and dissolve in it small pieces of celloidin until
the solution is of the consistency of one of the thicker fatty
oils. (The undissolved asphalt may be employed for colouring
a fresh quantity of ether, in which substance it is not very
soluble.)

2. Vesuvianin Celloidin Brown Injection. — Make a concen-
trated solution of Vesuvianin in absolute alcohol, and dissolve
celloidin in it. (This colour is not fast.)

3. Opaque Blue Celloidin Injection. — Dissolve celloidin in
equal parts of absolute alcohol and ether, and add pulverised
Berlin blue.

4. Opaque Red Celloidin Injection. — Proceed as above (3),
taking pulverised cinnabar instead of Berlin blue. The last
two pigments should be rubbed up in a mortar with a little
absolute alcohol, and the paste added to the celloidin mass.
Be careful not to take more pigment than is absolutely neces-
sary, or the injection will become brittle. To filter (if this
be thought necessary), strain the mass through flannel wetted
with ether. Syringes must be free from grease, which would
render the mass brittle. The nozzles to be filled with ether.
Inject quickly, as the mass soon sets on contact with watery
tissues. Clean syringes and nozzles with ether.

Corrosion of the Preparations. — The injected organs are
thrown into unrectified hydrochloric acid, where they remain
(the acid being changed from time to time if necessary) until

HOYER'S SHELLAC MASS. 279

all the tissues are destroyed. Wash under a slow stream of
water from a tap furnished with an india-rubber tube. Leave
for some weeks in water, rinse, and put up in glycerin, or a
mixture of glycerin, alcohol, and water in equal volumes.

514. Hochstetter's Modification of Schiefferdecker's Mass
(Anat. Anz., 1886, p. 51 ; Journ. Roy. Mic. Soc., 1888, p. 159).
— Kaolin is rubbed up with ether, to which cobalt blue, chrome
yellow, or cinnabar is added. To this, celloidin solution of
the consistence of honey is added.

Other Masses.

515. Budge's Asphaltum Mass (Arch. f. mik. Anat.,xiv, 1877,
p, 70). — A large quantity of asphaltum has benzol poured on
it, and is allowed to stand for several days, and then preserved
for use. Before injecting add one third to one half benzol
and filter. Chloroform and turpentine may also be used as
solvents. Used for injecting the juice-canals of cartilage by
the method described 1. c., or by puncture.

516. Hoyer's Shellac Mass (Arch. f. mik. Anat., 1876, p. 645).
. — Place a quantity of good shellac in a wide-necked flask, and
add just enough alcohol (of about 80 per cent, strength) to
cover the shellac. Leave it for twenty-four hours, and then
warm it in a water-bath to complete the solution. When
cool, dilute, if necessary, with alcohol to the consistency of a
thin syrup, and strain thrpugh moderately thick muslin. The
solution thus obtained may be coloured by the addition of
anilin colours in (filtered) concentrated alcoholic solution, or
of granular pigments suspended in alcohol. Of these, cinnabar
gives the finest coloration, and may be employed for corro-
sion preparations (anilin colours may also be used for this
purpose, but then they are not permanent) . Berlin blue and
yellow sulphide of arsenic are useful. A mixture of the two
gives green. Freshly precipitated sulphide of cadmium gives
a fine permanent yellow. The pigments should be rubbed up
to fine powder with water, and alcohol added ; let the mix-
ture settle, pour off the dilute alcohol, and add strong alcohol.
.Shake in a flask, by which means the coarser particles are
brought to the bottom of the liquid, and at this moment pour
off the supernatant fluid which contains the finer particles

280 INJECTIONS.

only. Add this to the shellac solution and strain through
muslin. For very minute injections dilute the mixture with
alcohol, filter through filter-paper on a covered funnel, and
evaporate down to the desired consistency. Common moist
water-colours, such as are sold in tin tubes, may be employed ;
they are to be well washed through several changes of water
to get rid of the medium with which the pigments are mixed,
and then suspended in alcohol as above directed. (These are
to be recommended for injections into the blood of living
animals.)

The shellac solution is not attacked by hydrochloric acid ;
hence its applicability to corrosion preparations. To correct
the brittleness of the corroded mass it is well to add to the
injection-fluid some 5 per cent, of a filtered alcoholic solution
of Venetian turpentine. This may also be of use for prepara-
tions that are not to be corroded. For corrosion, concen-
trated (fuming) hydrochloric acid may be used, and small
objects left in it for one day, large ones many days or even
weeks.

For hardening injections, of which it is desired to cut
sections, chromic acid may be used, or a mixture of chromic
and hydrochloric acid (1 part of each to 250 — 500 parts water) .
Sections are best mounted in glycerin.

This method, with some slight modifications of detail, has
lately been recommended by BELLARMINOW (Anat. Anz., 1888,
p. 650; see also Zeit. f. wiss. Mik., v, 4, 1888, p. 523, and
Journ. Roy. Mic. Soc., 1889, p. 150).

517. Hoyer's Oil-colour Masses (Internal. Monatschr.f. Anat.t
1887, p. 341; see also Zeit. f. wiss. Mik., 1888, p. 80, and
Journ. Roy. Mic. Soc., 1888, p. 848). — 5 grms. artist's Berlin
blue oil-colour are rubbed up with 5 grms. thickened linseed
oil, and mixed with about 30 grms. of lavender oil, fennel oil,
thyme oil, or rosemary oil, allowed to stand for twenty-four
hours in a well-stoppered vessel, and decanted. Shake before
using.

For injection of the vessels of the spleen and other difficult
objects.

Good results were also obtained with chrome-yellow oil-
colour.

NATURAL INJECTIONS. 281

518. Pansch's Starch Mass (see Arch. f. Anat. u. Entw., 1877,
p. 480; 1880, pp. 232, 371 ; 1881, p. 76; 1882, p. 60; 1883,
p. 265 ; and a modification of the same by GAGE, Amer. Mon.
Mic. Journ., 1888, p. 195; and Journ. Roy. Mic. floe., 1888,
p. 1056).

519. Teichmann's Linseed-oil Masses (see fl. B. Math. KT.
Krakau Akad., vii, pp. 108, 158; Journ. Roy. Mic. floe., 1882,
pp. 125 and 716).

520. Olive Oil for Corrosion Preparations (see below, § 558).

521. Natural Injections (ROBIN, Traite, p. 6). — To preserve
these throw the organs into a liquid composed of 10 parts of
tincture of perchloride of iron and 100 parts of water.

282 MACE RATION AND DIGESTION.

CHAPTER XXIII.

MACERATION AND DIGESTION.

Maceration.

522. Methods of Dissociation. — It is sometimes necessary, in
order to obtain a complete knowledge of the forms of the
elements of a tissue, that the elements be artificially separated
from their place in the tissue and separately studied after
they have been isolated both from neighbouring elements and
from any interstitial cement-substances that may be present
in the tissue. Simple teasing with needles is often insufficient
to effect the desired isolation, as the cement-substances are
often tougher than the elements themselves, so that the latter
are torn and destroyed in the process. In this case recourse
must be had to maceration processes, by which is here meant
treatment with media which have the property of dissolving
or at least softening the cement-substances or the elements
of the tissue that it is not wished to study, whilst preserving
the forms of those it is desired to isolate. When this soften-
ing has been effected the isolation is completed by teasing,
or by agitation with liquid in a test-tube, or by the method
of tapping, which last gives in many cases (many epithelia,
for instance) admirable results which could not be attained
in any other way. The macerated tissue is placed on a
slide and covered with a thin glass cover supported at the
corners on four little feet made of pellets of soft wax. By
tapping the cover with a needle it is now gradually pressed
down, whilst at the same time the cells of the tissue are segre-
gated by the repeated shocks. When the segregation has
proceeded far enough, mounting medium may be added, and
the mount closed.

The student will do well not to neglect this simple method,
which is one that it is most important to be acquainted with.

A good material for making wax, feet is obtained (VossELER,

ALCOHOL. 283

Zeit. f. wiss. Mik.j vii, 4, 1891, p. 461) by melting white wax
and stirring into it one half to two thirds of Venice turpen-
tine. Care must be taken if the operation be performed over
a naked flame, as the turpentine vapours are inflammable.

523. Iodised Serum. — The preparation of this reagent has_
been given in Chap. XIX. The manner of employing it for
maceration is as follows : — A piece of tissue smaller than a
pea must be taken, and placed in 4 or 5 c.c. of weakly iodised
serum in a well-closed vessel. After one day's soaking the
maceration is generally sufficient, and the preparation may be
completed by teasing or pressing out, as indicated above ; if
not, the soaking must be continued, fresh iodine being added
as often as the serum becomes pale by the absorption of the
iodine by the tissues. By taking this precaution, the macera-
tion may be prolonged for several weeks.

It is obvious that these methods are intended to be applied
to the preparation of fresh tissues, the iodine playing the part
of a fixing agent with regard to protoplasm, which it slightly
hardens.

524. Artificial Iodised Serum (FKEY, Le Microscope, p. 131 ;
RANVIEK, Traite, p. 77).

The formula has been given in Chap. XIX. Ranvier states
that he has been unable to obtain good results, for purposes
of maceration, by this method.

525. Alcohol. — Kanvier employs one-third alcohol (1 part of
36° alcohol to 2 parts of water) . Epithelia will macerate well
in this in twenty-four hours. Ranvier finds that this mixture
macerates more rapidly than iodised serum.

Other strengths of alcohol may be used, either stronger
(equal parts of alcohol and water) or weaker (J alcohol, for
isolation of the nerve-fibres of the retina, for instance —
Thin).

All observers are agreed that one-third alcohol is a mace-
rating medium of the highest order; LIST (Zeit. f. wiss. Mik.,
1885, p. 511) states that for glandular structures it should be
used with precaution, on account of swellings that it produces
in the cells, and that Muller's solution, or osmic acid, should
be preferred foy€«ch objects.

* f. • 'V~V

284 MACERATION AND DIGESTION.'

526. Salt Solution. — 10 per cent, solution of sodium chloride
is a well-known and valuable macerating medium.

527. MOLESCHOTT and Piso BORME'S Sodium Chloride and
Alcohol (MOLESCHOTT'S Untersuchungen zur Naturlehre, xi, pp.
99—107; RANVIER, Traite, p. 242).— 10 per cent, solution of
sodium chloride, 5 volumes ; absolute alcohol, 1 volume.

For vibratile epithelium, Ranvier finds the mixture inferior
to one-third alcohol.

528. Chloral Hydrate. — In not too strong solution, from 2 to
5 per cent., for instance, chloral hydrate is a mild macerating
agent that admirably preserves delicate elements. LA.VDOWSKY
(Arch. f. mik. Anat., 1876, p. 359) recommends it greatly
for salivary glands. HICKSON (Quart. Journ. Mic. Sci., 1885,
p. 244) recommends it for the study of the retina of
Arthropods.

529. Caustic Potash, Caustic Soda. — These solutions must be
employed strong, 35 to 50 per cent. (Moleschott) : so employed
they do not greatly alter the forms of cells, whilst weak solu-
tions destroy all the elements. (Weak solutions may, how-
ever, be employed for dissociating the cells of epidermis,
hairs, and nails.) The strong solutions may be employed by
simply treating the tissues with them on the slide. To make
permanent preparations, the alkali should be neutralised by
adding acetic acid, which forms with caustic potash acetate
of potash, a well-known mounting medium (see BEHRENS,
KOSSEL, and SCHIEPFERDECKER, Das Mikroskop, i, 1889, p. 156).
It has been found by S. H. and S. P. GAGE (Proc. Amer. Soc.
of Microscopists, 1889, p. 35; Zeit.f. wiss. Mik., vii, 3, 1890,
p. 349) that instead of acetic acid, 60 per cent, acetate of
potash solution, employed in considerable quantity and if
desired with addition of 1 per cent, of acetic acid, may be
used, the preparations either being mounted therein, or in
glycerin or glycerin jelly. They may be stained if the ace-
tate be first washed out by treatment for twenty-four hours
with alum solution.

530. Sulphocyanides of Ammonium and Potassium (STIRLING,
Journ. Anat. and Phys., xvii, 1883, p. 208). — Ten per cent,
solution of either of these salts is an admirable dissociating
medium for epithelium. Macerate small pieces for twenty-

LANDOIS'S SOLUTION. 285

four to forty-eight hours, stain with fuchsin, eosin, or picro-
carmine.

If a crystalline lens be macerated as above its fibres become
beaded or moniliform.

531. Saliva, Artificial (for embryology of nerve and muscle)
(CALBERLA'S formulae, Arch. f. mik. Anat., xvi, 1879, p. 471,
el seqf). — After having made trial of various different mace-
rating agents, with the object of obtaining isolation of the
developing muscle and nerve of embryos of Amphibia and
Ophidia, Calberla found that the best results were obtained
by means of Czerny's mixture of saliva and solutio Mulleri.
This led him to imagine an artificial saliva, which on trial
gave results as good as those obtained by natural saliva, or
even better.

Second formula (the first formula is suppressed, as being
more complicated, and not giving better results) :

Potassium chloride .... 0*4
Sodium chloride ..... 0'3
Phosphate of soda .... 0*2
Calcium chloride ..... 0*2

M

This is dissolved in 100 parts of water, saturated with
carbonic acid, and the solution combined with water and
solutio Mulleri, one volume of the solution being combined
with half a volume of Miiller's solution and a volume of
water.

In either case the Miiller's solution may be replaced by a
2J per cent, solution of chromate of ammonia. The best
results were obtained when the solutions were saturated with
the CO2 just before using.

The tissues are isolated by teasing and shaking, and speci-
mens mounted in concentrated acetate of potash.

532. LANDOIS'S Solution (Arch.f. mik. Anat., 1885, p. 445).
Saturated sol. of neutral chromate of ammonia 5 parts.
Saturated sol. of phosphate of potash . 5 „

Saturated sol. of sulphate of soda . . 5 „

Distilled water 100 „

To be used in the same way as chromic acid : — Small pieces
of tissue are macerated for one to three, or even four or five

286 MACERATION AND DIGESTION.

days, in the liquid, then brought for twenty -four hours into
ammonia carmine diluted with one volume of the macerating
liquid.

GIERKE particularly recommends this liquid for all sorts of
macerations, but especially for the central nervous system, for
which he finds it superior to all other agents. It is also re-
commended for the same purpose by NANSEN (v. Zeit. f. wiss.
Mik., v, 2, 1888, p. 242).

533. Permanganate of Potash, — Has an action similar to that
of osmic acid, but more energetic. Is recommended, either
alone or combined with alum, as the best dissociating agent
for the fibres of the cornea (ROLLETT, Strieker's Handbuch,
p. 1108).

534. Chromic Acid. — Generally employed of a strength of
about 0'02 per cent. Specially useful for nerve tissues and
smooth muscle. Twenty-four hours' maceration will suffice
for nerve-tissue. About 10 c.c. of the solution should be
taken for a cube of 5 mm. of the tissue (Ranvier).

535. Bichromate of Potash. — 0*2 per cent.

536. Muller's Solution.— Same strength.

537. Muller's Solution and Saliva (see above, § 531).

538. BROCK'S Medium (for nervous system of Mollusca,
Intern. Monatsch. /. Anat., i, 1884, p. 349). — Equal parts of
10 per cent, solution of bichromate of potash and visceral
fluid of the animal.

539. MOBIUS'S Media (quoted from Zeit. f. wiss. Mik., iii, 3,
1886, p. 402).

1. One part of sea water with 4 to 6 parts of 0*5 per cent,
solution of bichromate of potash.

2. 0*25 per cent, chromic acid, O'l per cent, osmic acid,
0*1 per cent, acetic acid, dissolved in sea water. For Lamelli-
'branc'hiata. Macerate for several days.

540. GAGE/S Picric Alcohol (Proc. Amer. Soc. of Microscopists,
1890, p. 120; Zeitf. wiss. Mik., ix, 1, 1892, pp. 87, 88).— 95
per cent, alcohol, 25Q parts; \\rater, 750; picric acid, 1.'
Recommended for most tissues, but especially for epithelia
and smooth and striated muscle. This is also much recom-

NITRIC ACID. 287

mended by HOPKINS in the same place. A few hours' macera-
tion is generally sufficient.

541. Osmic Acid. — Ol per cent., for from a few minutes to
a fortnight (cortex of cerebrum — Kindfleisch) . Maybe fol-
lowed by maceration in glycerin.

542. Osmic and Acetic Acid (the HERTWIGS' Liquid, Das Ner-
vensystem u. die Sinnesorgane der Medusen, Leipzig, 1878, and~
Jen. Zeitschr., xiii, 1879, p. 457 ; Journ. Roy. Hie. Soc.j iii,
1880, p. 441, and [N.S.] iii, 1883, p. 732).

0'05 per cent, osmic acid . . .1 part.

0'2 „ acetic acid . . . 1 „
Medusae are to be treated with this mixture for two or
three minutes, according to size, and then washed in repeated
changes of 0*1 per cent, acetic acid until all traces of free
osmic acid are removed; they then remain for a day in 0*1
per cent, acetic acid, are washed in water, stained in Beale's
carmine (in order to prevent the osmium from over- blacken-
ing, and to assist the maceration), and are preserved in
glycerin.

For Actinise, the osmic acid is taken weaker, O04 per cent. ;
both the solutions are made with sea water; and the washing
out is done with 0*2 per cent, acetic acid. If the maceration
is complete, stain with picro-carmine ; if not, with Beale's
carmine.

543. BELA HALLER'S Mixture (Morphol. Jahrb., xi, p. 321). —
One part glacial acetic acid, 1 part glycerin, 2 parts water.
Specially recommended for the central nervous system of
Mollusca (Rhipidoglossa), A sufficient degree of macera-
tion is obtained in thirty to forty minutes, the cells showing
less shrinkage than with other liquids.

544. Nitric Acid. — Most useful for the maceration of muscle.
The strength used is 20 per cent. After twenty-four hours'
maceration in this, isolated muscle-fibres may generally be
obtained by shaking the tissue with water in a test-tube.
Preparations may afterwards be washed with water and put
up in strong solution of alum, in which they may be preserved
'for a long time ( HOPKINS, Proc. Amer. Soc. of Microscopists,

1890, p. 165 ; Zeit. f. wlss. Mik., ix/1, 1892, p. 86).

Maceration is greatly aided by heat, and at a temperature

288 MACERATION AND DIGESTION.

of 40° to 50° C. may be sufficiently complete in an hour
(GAGE).

545. Nitric Acid and Chlorate of Potash (KUHNE'S method,
Ueber die peripherischen Endorgane, &c., 1862 ; BANVIER,
Traite, p. 79). — Chlorate of potash is mixed, in a watch-glass,
with four times its volume of nitric acid. A piece of muscle
is buried in the mixture for half an hour, and then agitated
with water in a test-tube, by which means it entirely breaks
up into isolated fibres.

546. Sulphuric Acid (RANVIER, Traite, p. 78). — Sulphuric
acid has been employed by Max Schultze for isolating the
fibres of the crystalline.

Macerate for twenty-four hours in 30 grins, of water, to
which are added 4 to 5 drops of concentrated sulphuric acid.
Agitate.

ODENIUS found very dilute sulphuric acid to be the best
reagent for the study of nerve-endings in tactile hairs. He
macerated hair-follicles for from eight to fourteen days in a
solution of from 3 to 4 grains of "English sulphuric acid" to
the ounce of water.

Hot concentrated sulphuric acid serves to dissociate horny
epidermic structures (horn, hair, nails).

547. Oxalic Acid. — Maceration for many days in concen-
trated solution of oxalic acid has been found useful in the
study of nerve- endings.

548. SCHIEFFERDECKER'S Methyl Mixture (for the retina)
(Arch.f. mi/c. Anat., xxviii, 1886, p. 305). — Ten parts of gly-
cerin, 1 part of methyl alcohol, and 20 parts of distilled water.
Macerate for several days (perfectly fresh tissue).

Digestion.

549. BEALE'S Digestion Fluid (Archives of Medicine, i, 1858,
pp. 296 — 316). — The mucus expressed from the stomach
glands of the pig is rapidly dried on glass plates, powdered,
and kept in stoppered bottles. It retains its properties for
years. Eight tenths of a grain will dissolve 100 grains of
coagulated white of egg.

To prepare the digestion fluid, the powder is dissolved in

GEDOELST'S METHODS. 259

distilled water, and the solution filtered. It filters readily.
Or the powder may be dissolved in glycerin. The tissues to
be digested may be kept for some hours in the liquid at a
temperature of 100° F. (37° C.).

550. BRUCKE'S Digestion Fluid (from CARNOY'S Biologie cellu-
laire, p. 94).

Grlycerinated extract of pig's stomach. . 1 vol.
0'2 per cent, solution of HC1 . . . 3 vols.
Thymol, a few crystals.

551. BICKFALVI'S Digestion Fluid (Centralbl. f. d. med. Wiss.,
1883, p. 833). — One grm. of dried stomachal mucosa is mixed
with 20 c.c. of 0*5 per cent, hydrochloric acid, and put into an
incubator for three or four hours, then filtered. Macerate
the tissue in the solution for not more than half an hour to an
hour.

552. KUSKOW'S Digestion Fluid (Arch. f. mik. Anat., xxx.
p. 32; cLZeit.f. wiss. Mik., iv, 3, 1887, p. 384). — One part of
pepsin dissolved in 200 parts of 3 per cent, solution of oxalic
acid. The solution should be freshly prepared, and the
objects (sections of hardened Ligamentum Nuchae) remain in
it at the ordinary temperature for ten to forty minutes.

553. SCHIEFFERDECKER'S Pancreatin Digestion Fluid (Zeit. f.
wiss. *Mik., iii, 4, 1886, p. 483). — Solution of pancreatin in
water. Schiefferdecker employs the " Pankreatinum siccum"
prepared by Dr. Witte, Eostock. A saturated solution is
made in distilled water, cold, and filtered. Pieces of tissue
(epidermis) are macerated in it for three to four hours at
about body temperature. Nuclei are preserved, and the
forms of prickle-cells well shown.

554. KUHNE'S Methods (see Unters. a. d. Phys. Inst. Univ.
Heidelberg, i, 2, 1877, p. 219). — Trypsin is prepared by ex-
tracting ox-pancreas with alcohol and ether, and evaporating
to dryness. One part of the product is heated for three to
four hours at a temperature of 40° C., with 5 to 10 parts of
O'l per cent, solution of salicylic acid, and the solution forced
through linen and filtered cold.

554 a. GEDOELST'S Methods (see La Cellule, iii, 1887, p. 117,
and v, 1889, p. 126; also Zeit. f. wiss. Mik., vii, 1, 1890,
p. 57).

19

290 CORROSION, DECALCIFICATION, AND BLEACHING.

CHAPTER XXIV.

CORROSION, DECALCIFICATTON, AND BLEACHING.

Corrosion.

555. Caustic Potash, Caustic Soda, Nitric Acid. — Boiling, or
long soaking in a strong solution of either of these, is an effi-
cient means of removing soft parts from skeletal structures
(appendages of Arthropods, spicula of sponges, &c.).

556. Eau de Javelle (Hypochlorite of Potash) (NOLL'S METHOD,
Zool. Anzeig., 122, 1882, p. 528).— Noll remarks that the
usual method of preparing the skeleton of siliceous sponges
and similar structures by corroding away the soft parts by
means of caustic potash has many disadvantages, of which a
principal one is that the spicula are not preserved in their
normal positions. He therefore proceeds as follows : — A piece
of sponge is brought on to a slide and treated with a few drops
of eau de Javelle, in which it remains until all soft parts are
dissolved. (With thin pieces this happens in twenty to thirty
minutes.) The preparation is then cautiously treated with
acetic acid, which removes all precipitates that may have
formed, and treated with successive alcohols and oil of cloves,,
and finally mounted in balsam.

The same process is stated to be applicable to calcareous
structures. I feel convinced, however, that if the structures
are delicate, they will suffer, or be entirely destroyed/

557. Eau de Labarraque (Hypochlorite of Soda) may be used
in the same way as eau de Javelle. Looss (Zool. Anz., 1885,
p. 333) finds that either of these solutions will completely
dissolve chitin in a short time with the aid of heat. For this
purpose the commercial solution should be taken concentrated
and boiling. A formula for making it is given in § 581.

If solutions diluted with 4 to 6 volumes of water be taken,
and chitinous structures be macerated in them for twenty-

DECALCIFICATION OF BONE. 291

four hours or more, according to size, the chitin is not dis-
solved, but becomes transparent, soft, and permeable to stain-
ing fluids, aqueous as well as alcoholic. The most delicate
structures, such as nerve-endings, are stated not to be injured
by the treatment. The method is applicable to Nematodes
and their ova, an object well known for the resistance they
oppose to ordinary reagents.

This is undoubtedly a valuable method.

558. ALTMANN'S Corrosion Method (Arch.f. mik, Anat., 1879, p. 471).
— Whilst almost all animal tissues are very quickly destroyed by eau de
Javelle, yet fats, and particularly fats hardened by osmic acid, withstand its
action for a long time. If, then, you introduce some fat or other into a
tissue, harden it with osmic acid and corrode the tissue with eau de Javelle,
you will obtain a mould in osmium-blackened and hardened fat, of the spaces
you had filled with the fat introduced.

The method may be of much use in certain special researches, such as those
on the choroid, iris, and pigmented organs. I recommend the reader to
carefully study the article, which does not well bear abstracting. A good
abstract will be found in Journ. Roy. Mic. Soc., 1879, p. 610, with plate.

Decalcification and Desilicification.

559. Decalcification of Bone. — I take the following historical
sketch from Busch's article " On the Technique of the Histo-
logy of Bone " (Arch. f. mik. Anat., xiv, 1877, p. 481 ; see
also the paper of HAUG, in Zeit. f. wiss. Mik., viii, i, 1891, p. 1).

The most widely used agent for decalcification is hydro-
chloric acid. Its action is rapid, even when very dilute, but
it has the disadvantage of causing serious swelling of the
tissues. To remedy this chromic acid may be combined with
it, or alcohol may be added to it. Or a 3 per cent, solution
of the acid may be taken and have dissolved in it 10 to 15
per cent, of common salt. Or (Waldeyer) to a 10100 per cent,
solution of chloride of palladium may be added -^ of its
volume of HC1.

Chromic acid is also much used, but has a very weak decal-
cifying action and a strong shrinking action on tissues. Fo
this latter reason it can never be used in solutions of more
than 1 per cent, strength, and for delicate structures much
lower strengths must be taken.

Phosphoric acid has been recommended for young bones.

Acetic, lactic, and pyroligneous acids have considerable

292 CORROSION, DECALCIFICATION, AND BLEACHING.

decalcifying power, but cause great swelling. Picric acid
has a very slow action, and is only suitable for very small
structures.

560. Nitric Acid (Buscn, 1. c.). — To all other agents Busch
prefers nitric acid, which causes no swelling and acts most
efficaciously, whilst at the same time it does not injuriously
attack tissue- elements.

One volume of chemically pure nitric acid of sp. gr. 1'25 is
diluted with 10 vols. water. It may be used of this strength
for very large and tough bones ; for young bones it may be
diluted down to 1 per cent.

Fresh bones are first laid for three days in 95 per cent,
alcohol; they are then placed in the nitric acid, which is
changed daily, for eight or ten days. They must be removed
as soon as the decalcification is complete, or else they will
become stained yellow. When removed they are washed for
one or two hours in running water and placed in 95 per cent,
alcohol. This is changed after a few days for fresh alcohol.

Young and foetal bones may be placed in the first instance
in a mixture containing 1 per cent, bichromate of potash and
y1^ per cent, chromic acid, and decalcified with nitric acid of
1 to 2 per cent., to which may be added a small quantity of
chromic acid (-^ per cent.) or chromate of potash (1 per cent.).
By putting them afterwards into alcohol the well-known
green stain is obtained.

Staining agents. — Sections of bone treated in the last-
described manner are stained five or ten minutes in a weak
aqueous solution of eosin. The ground-substance and small
cells of cartilage remain colourless, the nuclei of the large
cells are stained red, and so is periosteum, bone-tissue, and
the cellular contents of the medullary spaces. Hsematoxylin
may be used in conjunction with eosin (before or after it) to
obtain double-stains, which, however, are seldom successful.

Sections are dehydrated in absolute alcohol, and mounted
(without clearing by oil of cloves or the like) in a benzol-
solution of Canada balsam.

561. Nitric Acid and Alcohol. — 3 per cent, of nitric acid in
70 per cent, alcohol. Soak specimens for several days or
weeks. I do not know who first recommended this admirable
medium.

PHLOKOGLUOIN. 293

THOMA (Zeit.f. wiss. Milt., viii, 2, 1891, p. 191) gives the
following method. — Take 5 vols. of 95 per cent, alcohol and 1
vol. pure concentrated nitric acid. Leave bones in this mix-
ture, changing the liquid every two or three days, until
thoroughly decalcified, which should happen even with large
bones in two or three weeks. Wash out until every trace of
acid is removed (i. e. for some days after no acid reaction is
obtained with litmus paper) in 95 per cent, alcohol containing
an excess of precipitated chalk. This may take eight to four-
teen days, after which the tissues will stain well and may be
treated as desired.

562. Phloroglucin (ANDEER, Centralbl. f. d. med. Wiss., xii,
xxxiii, pp. 193, 579 ; Intern. Monatschr., i, p. 350 ; Zeit.f. wiss.
Mik., 1885, pp. 375, 539; Journ. Roy. Hie. Soc., 1887, p. 504;
HAUG, Zeit.f. wiss. Mik., viii, 1, 1891, p. 8; FEEREBI, ibid., ix>
2, 1892, p. 236; Bull. R. Accad. Med. di Roma, 1892, p. 67).—
This is the most recent of the decalcification methods. It has
the advantages of being the most rapid of any, and of pre-
serving the tissues very well (with the exception of blood).

Phloroglucin by itself is not a solvent of lime salts ; its
function in the mixtures given below is so to protect the
organic elements of tissues against the action of the mineral
acids that these can be used in a much more concentrated
form than would otherwise be advisable.

HAUG advises the following procedure : — Bring 1 grm. of
phloroglucin into 10 c.c. of pure, not fuming nitric acid
(1*4 sp. gr.), and warm very slowly and carefully with gentle
agitation. There is formed a clear solution of (presumably)
a nitrate of phloroglucin. Dilute the solution with 100 c.c.
of distilled water, and add 10 c.c. of nitric acid. This gives
a solution containing 20 per cent, of acid, which is the proper
proportion. More water may be added to the solution, to
make it up to 300 c.c., if nitric acid be also added in the
proportion given. But the dilution must not be carried
beyond this point, in order that the preservative action of
the phloroglucin be not overmuch weakened. The process of
decalcification in this solution is extremely rapid, and there-
fore should be carefully watched. Foetal and young bones
become quite soft in half an hour; small pieces of old and
hard bones (femur, temporal bone) in a few hours. Teeth

294 COEEOSION, DECALCIFJCATION, AND BLEACHING.

take longer, and may require, if time be an object, a solution
made with 35 to 45 per cent, of nitric acid. Wash out for
two days in running water. The tissues stain well.

The solution may be made with hydrochloric acid instead
of nitric acid, 30 per cent, ot acid being taken, and 0*5 per
cent, of sodium chloride added.

For slow decalcification a 2 to 5 per cent, nitric acid solu-
tion may be used, or a mixture containing of phloroglucin
I part, nitric acid 5, alcohol 70, and distilled water, 30 parts.

For the labyrinth, FERRERI advises a mixture containing
1 grm. of phloroglucin, 10 grms. of hydrochloric acid, 100 of
water, and 200 of 70 per cent, alcohol, to be changed once a
week during thirty to forty days.

563. Hydrochloric Acid (see above, § 559). — RANVIER says that it
may be taken of 50 per cent, strength, and then has a very rapid action. To
counteract the swelling action of the acid, sodium chloride may be added.
Two formulae of this sort have been given by VON EBNEE (see HATJG'S paper
quoted in the last section). The first is, 100 c.c. cold saturated solution of
sodium chloride in water, 100 c.c. water, and 4 c.c. hydrochloric acid. Pre-
parations to be placed in this, and 1 to 2 c.c. of hydrochloric acid added daily
until they are soft. The second is, 2'5 parts of hydrochloric acid, 500 of
alcohol, 100 of water, and 2'5 of sodium chloride. HAUG prefers the pro-
portions of 1-0 to 5'0 of acid, 70 of alcohol, 30 of water, and 0'5 of salt.

564. Hydrochloric Acid and Chromic Acid (BAYEEL, Arch. f. mik.
Anat., 1885, p. 35). — Equal parts of 3 per cent, chromic acid and 1 percent,
hydrochloric acid. For ossifying cartilage. HAUG recommends equal parts
of 1 per cent, hydrochloric acid and 1 per cent, chromic acid (1. c.).

565. Hydrochloric Acid and Nitric Acid (HOPEWELL SMITH, Journ.
Hoy. Mic. Soc., 1892, p. 433).— Place teeth in 12 parts of 10 per cent, HC1,
and after 15 hours add 1'5 parts of HN03, and after 48 hours add 1'5 parts
more of HNO3. After 75 to 80 hours remove and wash for half an hour in
a solution of 5 grms. of lithium carbonate to an ounce of water.

566. Hydrochloric Acid and Glycerin. — Glycerin, 95 ; hydrochloric
acid, 5. Recommended for softening teeth in SQUIRE'S Methods and For*
mulse, p. 12.

567. Picric Acid should be taken saturated.

Pier v -sulphuric acid should of course be avoided on account of the forma-
tion of gypsum.

Picro-nitric or Picro-hydrochloric Acid. — The reader will perhaps reflect
that the last two fluids appear likely to be very useful for decalcifications.
Mayer points out that the action is very rapid, and that the copiously evolved
C02 often produces, mechanically, lesions in tissues ; so that in many cases
in which calcareous structures are concerned chromic acid is to be preferred,

HYDROFLUORIC ACID. 295

the more so as it more effectually hinders any collapsing of the structures
that might result from the withdrawal of their supporting calcareous
elements.

Picric acid fluids are good media, as though their action is slow they pre-
serve tissues well, and leave them in a good state for subsequent staining.

568. Phosphoric Acid.— 10 to 15 per cent. (HAUG, 1. c., in § 559).
Somewhat slow, staining not good.

669. Lactic Acid. — 10 per cent, or more. Fairly rapid, preserves well,
and may be recommended (HAUG, 1. c.). •

570. Chromic Acid is employed in strengths of from O'l per cent, to 2
per cent., the maceration lasting two or three weeks (in the case of bone).
It is better to take the acid weak at first, and increase the strength gradually.
In any way the action is extremely slow, and it is therefore better to take
one of the mixtures of chromic acid with a more energetic agent.

571. Chromic and Nitric Acid. — Dissolve 15 grms. pure chromic acid
in 7 oz. of distilled water, to which 30 minims of nitric acid are afterwards to
be added. Macerate for three or four weeks, changing the fluid frequently
(Marsh).

FOL takes 70 volumes of 1 per cent, chromic acid, 3 of nitric acid, and 200
of water (Lehrb., p. 112).

See also § 564. It remains to be added that even with the addition of
nitric or hydrochloric acid the action is excessively slow, frequently requiring
months to be complete.

572. Arsenic Acid. — 4 per cent, aqueous solution, used at a temperature
of 30° to 40° C. (SQUIBE'S Methods and Formulae, &c., p. 11).

573. Glycerin. Alum-Carmine. — It should be remembered that these
commonly used reagents dissolve carbonate of lime ; they must therefore be
avoided in the preparation of structures containing delicate calcareous ele-
ments that it is wished to preserve (calcareous sponges, larvaB of Echino-
dermata, &c.).

Desilicification.

574. Hydrofluoric Acid (MAYER'S method, Zool. Anz., 1881, No. 97,
p. 593). — The objects from which it is desired to remove siliceous parts are
brought in alcohol into a glass vessel coated internally with paraffin (other-
wise the glass would be corroded by the acid). Hydrofluoric acid is then
added drop by drop (the operator taking great care to avoid the fumes, which
attack mucous membi'anes with great energy). A Wagnerella borealis may
thus be completely desilicified in a few minutes. Small pieces of siliceous
sponges will require a few hours, or at most a day. The tissues do not
suffer ; and if they have been previously stained with acetic acid carmine the
stain does not suffer ; at least, this was so in the case of Wagnerella.

This dangerous method is best avoided. As regards sponges, I would
point out that if well imbedded, good sections may be made from them with-

296 CORROSION, DECALCIFICATION, AND BLEACHING.

out previous removal of the spicula. The spicula appear to "be cut ; probably
they break very sharply when touched by the knife. Knives are of course-
not improved by cutting such sections.

Bleaching.

575. MAYER'S Chlorine Method (Mitth. Zool. Stat. Neapel, ii,
1881, p. 8). — This is a process imagined for the purpose of
getting rid of the blackening that often occurs as a conse-
quence of treatment by osmic acid.

The specimens are put into alcohol (either of 70 or 90 per
cent.). Crystals of chlorate of potash are added until the
bottom of the vessel is covered with them. A few drops of
concentrated hydrochloric acid are then added by means of a
pipette, -and mixed in by shaking the vessel as soon as the
green colour of the evolving chlorine has begun to show itself.
Warm if necessary ; but most objects, even large ones, may be
bleached in half a day without the employment of heat. The
tissues do not suffer.

Instead of hydrochloric acid, nitric acid may be used ; in
which case the bleaching agent is the freed oxygen, instead
of chlorine.

The first method may be used for the purpose of removing
pigment from the eyes of insects.

576. MARSH'S Chlorine Method (Section Cutting, p. 89). —
Marsh generates chlorine in a small bottle by treating crystals
of chlorate of potash with strong HC1, and leads the gas (by
means of a piece of glass tubing bent twice at right angles)
to the bottom of a bottle containing the sections in water.
(See a fig. of the apparatus in Journ. Roy. Mic. 8oc., iii, 1880,.
p. 854.)

577. Chlorine Solution (SABGENT'S method). — Hydrochloric acid, 1O
drops ; chlorate of potash, \ dr. ; water, 1 oz. Soak for a day or two. Wash
well.

This method is intended for " bleaching insects ; " it will be seen that it is-
only applicable to the preparation of hard pails, as soft tissues would be de-
stroyed by the solution.

578. Kreasote (POUCHET'S method, Journ. de I'Anat., 1876, p. 8, etseq.).
— I gather from the paper here quoted that most of the granular animal
pigments are soluble in kreasote. Other solvents are mentioned in this
paper (" On the Change of Coloration through Nervous Influence"), but
this appears to be the only one capable of general histological application.

GRENACHER'S MIXTURE FOR EYES OF ARTHROPODS. 297

579. Nitric Acid has -a similar action. PARKER (Bull. Mus.
Comp. ZooL, Cambridge, U.S.A., 1889, p. 173; see Zeit. f. wiss.
Mik., viii, 1, 1891, p. 82) says that for eyes of scorpions the
usual 5 to 10 per cent, solutions are not strong enough. He
treats sections, fixed to the slide with Schallibaum's medium,
for about a minute with a solution of up to 50 per cent, of nitric
.acid in alcohol, or, still better, with a 35 per cent, solution of
a mixture of equal parts of nitric and hydrochloric acid in
alcohol. To make the solution, the acid should be poured
slowly into the alcohol (not vice versa), and the mixture kept
cool.

580. Peroxide of Hydrogen (Oxygenated Water) (POUCHET'S
method, M. DUVAL, Precis, &c., p. 234). — Macerate in- glycerin
to which has been added a little oxygenated water (5 to 6
drops to a watch-glass of glycerin). (Oxygenated water may
be procured from perfumers or hair- dressers, by whom it is
sold as a hair dye under the name of " Aureoline," " Golden
hair-wash," or the like.)

The brownish-green colour communicated to tissues by
chromic solutions may be changed to yellow by means of oxy-
genated water (see § 30).

Osmium preparations may be bleached in the same way.

581. Eau de Labarraque. Eau de Javelle (see §§ 556, 557). — These
are bleaching agents. For the manner of preparing a similar solution see
Journ. de Microgr., 1887, p. 154, or Journ. Roy. Mic. Soc., 1887, p. 518.
It is, shortly, as follows : — 8 parts of caustic soda are dissolved in 100 parts
of distilled water, and chlorine is passed through to saturation. During the
passage of the chlorine the solution must be surrounded with a mixture of
salt and ice, otherwise the temperature rises, and chloride and chlorate of
soda are produced. The resulting solution contains 7'45 per cent, of hypo-
chlorite of soda. It is green ; and the more effectual the cold, the greener is
the colour. The energy of the decolourising action is proportional to the
greenness of the solution. Of course the method cannot be used for bleach-
ing soft parts which it is desired to preserve.

582. Chloroform helps to clear strongly pigmented chitin,
and combined with nitric acid will decolourise it entirely
(see below, in the chapter on Arthropods, Part II).

583. GRENACHER'S Mixture for Eyes of Arthropods and other
Animals (Ahli. nat. Ges. Halle-a.-S., xvi; Zeit. f. wiss. Mile.,
1885, p. 244).

298 COREOSION, DECALCIFICATION, AND BLEACHING.

Glycerin 1 part.

80 per cent, alcohol .... 2 parts.
Mix and add 2 to 3 per cent, of hydrochloric acid.
Pigments dissolve in this fluid, and so doing form a stain
which suffices in twelve to twenty-four hours for staining the
nuclei of the preparation. You may, if you like, first stain
the objects with borax-carmine, and then put them into the
liquid, the pigment being washed out more rapidly than the
carmine. But the progress of the decoloration must be care-
fully watched.

PART II.

SPECIAL METHODS AND EXAMPLES.

SUPERFICIAL EXAMINATION. 301

CHAPTER XXV.
EMBRYOLOGICAL METHODS.

584. Artificial Fecundation. — This practice, which affords
the readiest means of obtaining the early stages of develop-
ment of many animals, may be very easily carried out in the
case of the Amphibia anura, Teleostea, Cyclostomata, Echino-
dermata, and many Vermes and Coelenterata.

In the case of the Amphibia, both the female and the male
should be laid open, and the ova should be extracted from
the uterus and placed in a watch-glass or dissecting- dish, and
treated with water in which the testes, or, better, the vasa
deferentia, of the male have been teased.

Females of Teleostea are easily spawned by manipulating
the belly with a gentle pressure ; and the milt may be ob-
tained from the males in the same way. (It may occasionally
be necessary, as in the case of the Stickleback, to kill the
male, and dissect^out the testes and tease them.) The sper-
matozoa of fish, especially those of the Salmonidae, lose their
vitality very rapidly in water ; it is therefore advisable to add
the milt immediately to the spawned ova, then add a little
water, and after a few minutes put the whole into a suitable
hatching apparatus with running water.

Artificial fecundation of Invertebrates is easily performed
in a similar way. It is sometimes possible to perform the
operation under the microscope, and so observe the penetra-
tion of the spermatozoon and some of the subsequent phe-
nomena, as has been done by Fol, the Hertwigs, Selenka, and
•others, for the Echinodermata and other forms.

585. Superficial Examination. — The development of some
animals, particularly some Invertebrates, may be to a certain
extent followed by observation of the living ova under the
microscope. This may usefully be done in the case of various
Teleosteans, such as_the Stickleback, the Perch, Macropodus,

302 EMBRYOLOGIOAL METHODS.

and several pelagic forms, and with Chironomus, Asellus
aquaticus, Ascidians, Planorbis, many Ccelenterata, &c. I
advise the student to carefully draw the different stages so
observed, for such drawings are most important aids to the
study of the same stages by the section method.

Some ova of Insecta and Arachnida which are completely
opaque under normal conditions become transparent if they
are placed in a drop of oil; if care be taken to let their
surface be simply impregnated with the oil, the normal course
of development is not interfered with (BALBIANI).

586. Preparation of Sections. — Osmic acid, employed either
alone or in combination with other reagents, is an excellent
fixing agent for small embryos, but not at all a good one for
large ones. It causes cellular elements to shrink somewhat,
and therefore brings out very clearly the slits that separate
germinal layers, and any channels or other cavities that may
be in course of formation.

In virtue of its property of blackening fatty matters,
myelin amongst them, it is of service in the study of the
development of the nervous system.

Chromic acid is indispensable for the study of the external
forms of embryos ; it brings out elevations and depressions
clearly, and preserves admirably the mutual relations of the
parts ; but it does not always preserve the forms of cells
faithfully, and is a hindrance to staining in the mass.

Picric liquids have an action which is the opposite of that
of osmic acid; they cause cellular elements to swell some-
what, and thus have a tendency to obliterate spaces that
may exist in the tissues. But notwithstanding this defect,
the picric compounds, and especially Kleinenberg's picro-
sulphuric acid, are amongst the best of embryological fixing
agents.

For imbedding, the celloidin-chloroform method of Viallanes
gives excellent results, and so does paraffin. This latter is
preferable in so far as it lends itself better to the rapid pro-
duction of series of sections, and allows of the use of the
Cambridge Rocking Microtome, or the Minot, which is per-
haps the microtome par excellence of the embryologist.

As to staining, my eminent fellow- worker, Dr. Henneguy,
writing the chapter on embryological methods for the French

RECONSTRUCTION OF EMBRYOS FROM SECTIONS. 303:

edition of this work, advised staining in the mass with borax-
carmine or alum-carmine (Henneguy's acetic acid formula,
§ 156) ; or, as an alternative, the staining of sections by
Flemming's method. The improvements that have in recent
times been worked out in this method give still greater weight
to the latter recommendation.

587. Reconstruction of Embryos from Sections. — The study of
a series of sections of any highly differentiated organism of
unknown structure is so complicated that it is often necessary
to have recourse to elaborate methods of geometrical or of
plastic reconstruction in order to obtain an idea or a model
of the whole. These methods have now been brought to so
high a degree of complexity that a volume rather than a
paragraph would be necessary to describe them. See BOBN,
"Die Plattenmodellirmethode," in Arch. f. mik. Anat., 1883,
p. 591, and Zeit. f. wiss. Mik., v, 4, 1888, p. 433 ; STEASSER, in
Zeit.f. wiss. Mik., iii, 2, 1886, p. 179, and iv, 2 and 3, pp. 168
and 330 ; KASTSCHENKO, in Zeit. f. wiss. Mik., iv, 2 and 3,
1887, pp. 235-6 and 353, and v, 2, 1888, p. 173 (abstracts of
all these papers may be found in Journ. Roy. Mic. Soc. of
the years quoted).

A simple, but in many cases quite efficient plan, has been described by
FOL (Lehrb., p. 35) as follows : — Before cutting your sections, you make
an outline drawing of your object, under the magnification that you intend
to employ for the reconstructed drawing, and in a plane perpendicular to
that of the intended sections. For instance, if you intend to make trans-
verse sections of an embryo, begin by making a profile drawing of it, that
is, a drawing of the outline of an ideal sagittal section of it. Then make
your series of sections, and make drawings of them all under the same
magnification as the sagittal drawing. Then trace over your sagittal draw-
ing a series of equidistant parallel lines in positions corresponding to the
sections that have been made. If your sections are one hundredth of a
millimetre thick, and your drawing be magnified one hundred times, the
lines should be one millimetre apart (if you intend to reconstruct the whole
of your sections, but the operation may frequently be abridged by only
reconstructing say every fifth or every tenth section).

You have now to fill in your outline drawing with details borrowed from
the drawings of the sections. You may help yourself greatly in the follow-
ing way : — A plate of glass, of a size suitable to the intended drawings, is
covered with a layer of gelatin, and dried. On this is ruled a series of
parallel lines, very close together, and ruled with differently coloured inks,
the colours recurring in regular order. The plate is then cut into two un-
equal pails by a diamond, on a line perpendicular to the coloured lines.

304 EMBRYOLOGICAL METHODS.

Lay one of the parts of the plate on the outline drawing so that the cut
edge covers the line that corresponds to the first section you are going to
fill in ; then lay the other part of the plate on the drawing of the section in
such a position that the limits of the drawing correspond to the same coloured
lines that cover the limits of the outline drawing on the other part of the
plate already placed.

Trace on the plate that covers the drawing of the section the outline of
the internal organs. Lay it against its fellow-plate on the outline drawing,
making the coloured lines correspond, and you will easily be able to mark
off accurately on the outline drawing a series of dots that correspond in
position to the outlines of the internal organs. This operation having been
repeated for each of the sections that you desire to bring into your recon-
struction, nothing remains but to join your dots by lines, and you will have
filled up your outline drawing with a representation of the internal organs
in the same plane.

If any reader think this process complicated, he needs but to spend five
minutes in trying it with a piece of tracing paper, and will find it to be in
reality extremely simple.

Another simple plan is to gum the drawings of the sections on cardboard
of a thickness proportional to the thickness of the section and the magni-
fication, cut out all the cavities o£ the drawing with a knife or fret-saw,
and gum all the fretwork thus obtained together. This gives, of course, a
model of the object.

In simple cases it may be sufficient to adopt the plan
described by SCHAFFER (Zeit.f.wiss. Mik., vii, 3, 1890, p. 342).
Careful outlines of the sections to be reconstructed are drawn
on tracing-paper with the aid of the camera lucida, super-
posed, and held up against the light for examination by
transparence. EYCLESHEIMEE'S method of producing orienta-
tion lines in celloidin imbedding mass has been described in
Chap. XYI.

Mammalia.

588. Rabbit. — The rabbit may conveniently be taken as a
type for this kind of work.

Dissection. — For the study of the early stages the ova must
be sought for in the tubas a certain number of hours after
copulation. The dehiscence of the follicles takes place about
ten hours after the first coitus. The tubss and cornua having
been dissected out should be allowed to cool, and remain until
the muscular contractions have ceased. Then, with the aid
of fine scissors or a good scalpel, all the folds of the genital
duct are carefully freed from their peritoneal investment.

The tubas are then (if the ova are still within them, which

EABBIT. 305

is the case up to the end of the third day after coition) laid
out on a long slip of glass, and slit up longitudinally by
means of a pair of fine, sharp scissors. By means of needles
and forceps the tubal mucosa is spread out so as to smooth
out its folds as much as possible, and is carefully looked over
with a strong lens or with a low power of the microscope.
When the ova are found a drop of some "indifferent" liquid
is dropped on each, and it is carefully taken up with the
point of a scalpel, a cataract needle, or a small pipette. They
may be examined in the peritoneal fluid of the mother if the
animal has been killed, or in its aqueous humour, or in
amniotic liquid, or in blood-serum, or in Kronecker's or other
artificial serum.

If you have not been able to find the ova with the lens or
the microscope, scrape off the epithelium of the tubal mucosa
with a small scalpel, mix it with a little indifferent liquid,
and look for the ova under the microscope by transmitted
light.

Another method, employed by Kolliker, consists in in-
jecting solution of Miiller or weak osmic acid into the oviduct
by means of a small syringe, and collecting the liquid that
runs out in a series of watch-glasses, in which the ova can
very easily be found by the microscope.

The same doe may be made to serve for two observations, at some hours'
or days' interval. A longitudinal incision of 8 to 10 centimetres length is
made on the median or a lateral line of the abdomen ; an assistant keeps
the intestines in their place ; a ligature is placed at the base of one of the
uterine cornua, beneath the neck, and a second ligature around the mesome-
triuin and mesovarium. The ovary, the tuba, and the cornu of that side are
then detached with scissors. The abdomen is then closed by means of a few
sutures passing through the muscle-layers and the skin. The animals sup-
port the operation perfectly well, and the development of the ova of the
opposite side is not in the least interfered with. When it is desired to study
these the animal may be killed, or may be subjected to a second laparotomy
if it be desired to preserve it for ulterior observations.

During the fourth, fifth, and sixth days after copulation the
ova of the rabbit are free in the uterine cornua ; they are
easily visible to the naked eye, and may be extracted by the
same manipulations as those of the tubes. After the sixth
day they are at rest in the uterus, but have not yet contracted
adhesions with the mucosa, so that they can still be extracted
whole. At this stage the parts of the cornua where the ova

20

306 EMBRYOLOGICAL METHODS.

are lodged are easily distinguishable by their peculiar aspect,
the ova forming eminences of the size of a pea. The cornua
should be cut up transversely into as many segments as there
are eminences, care being taken to have the ova in the centre
of the segments. You then fix each segment by means of
two pins on the bottom of a dissecting dish, with the meso-
metrial surface downwards and the ovular eminence upwards.
The dissecting dish is then filled up with serum or liquid of
Miiller, or O'l per cent, solution of osmic acid, or Kleinenberg's
picro-sulphuric acid, or nitric acid, or acetate of uranium
solution. With a small scalpel a longitudinal incision is made
on the surface of the ovular eminence, not passing deeper
than the muscular layer; the underlying uterine mucosa is
then gently dilacerated with two pairs of small forceps, and
the ovum set free in the liquid.

From the moment the ova have become adherent to the
uterine mucosa they can no longer be extracted whole. The
embryo being always situated on the mesometrial surface, the
ovular eminence is opened by a crucial incision, and the strip
of mucosa to which the embryo remains adherent is fixed
with pins on the bottom of the dish. ED. v. BENEDEN (see
Arch, de Biol., v, fasc. iii, 1885, p. 378) has been able by
operating in this way in serum of Kronecker, and keeping the
whole at blood temperature, to observe the circulation of the
embryo for hours together. (If this be desired to be done,
the crucial incision should not be too extended, so as to leave
the terminal sinus intact.)

Preparation. — In order to make permanent preparations
of the different stages of fecundation and segmentation,
y. BENEDEN (Arch, de Biol., i, 1, 1880, p. 149) recommends
the following process : — The living ovum is brought into a
drop of 1 per cent, osmic acid on a slide, and thence into
solution of Miiller (or bichromate of ammonia or solution of
Kleinenberg). After an hour the liquid is changed, and the
whole is put into a moist chamber, where it remains for two
or three days. It is then treated with glycerin of gradually
increasing strength, and at last mounted in pure glycerin
acidified with formic acid. Ova may be stained with Beale's
carmine or picro-carmine, after removal from the osmic acid
and careful washing.

In order to bring out the outlines of blastoderm-cells the

RABBIT. 307

living ovum may be brought into one third per cent, solution
of nitrate of silver. After remaining there for half a minute
to two minutes, according to the age of the vesicle, it is
brought into pure water and exposed to the light. The pre-
parations thus obtained are instructive, but blacken rapidly,
and cannot be permanently preserved.

After the end of the third day the blastodermic vesicle can"
be opened with fine needles, and the blastoderm washed,
stained, and mounted in glycerin or balsam, v. Beneden
has also obtained good preparations by means of chloride of
gold.

For embryonic areas and more advanced embryos Kolliker
recommends putting the ovum into 0*5 per cent, solution of
osmic acid until it has taken on a somewhat dark tint,
which happens in about an hour, and then treating it with
successive alcohols for several hours. If the ovum be ad-
herent to the uterine mucosa the portion of the membrane to
which it is fixed should be left, stretched out with pins, in
O'l per cent, solution of osmic acid for from four to six hours.
The blastodermic vesicle can then easily be removed, and
immersed for a few hours more in 0'5 per cent, solution of
osmic acid, and finally be brought into alcohol. For sections
Kolliker fixes with osmic acid. v. Beneden treats the ova
for twenty -four hours with I per cent, solution of chromic
acid, then washes well, and brings them through successive
alcohols. Chromic acid has the advantage of hardening
thoroughly the vesicle, and maintaining at the same time
the epiblast-cells perfectly adherent to the zoha pellucida.
v. Beneden also recommends the liquid of Kleinenberg.
Henneguy writes that he frequently employs it for embryonic
areas and embryos of various ages, always with excellent
results. Fol's modification of the liquid of Flemming, and
Hanvier and Vignal's osmic acid and alcohol mixture (§ 28),
also give excellent results. For staining, Henneguy recom-
mends borax-carmine or Delafield's hgematoxylin for small
embryos; for large ones, Henneguy's acetic acid alum-car-
mine is the only reagent that will give a good stain in the
mass.

For sections, pure paraffin. Cut in series and mount in
balsam.

PIERSOL (Zeit.f. wiss. Zool, xlvii, 2, 1888, p. 155) has been

308 EMBEYOLOGICAL METHODS.

lately using for fixation either Kleinenberg's solution or, for
young stages, Altmann's 3 per cent, nitric acid. Staining
and cutting as above.

Aves.

589. Superficial Examination.— Excellent instructions on this
head are given in FOSTER and BALFOUR'S Elements of Embryo-
logy, to which, as it is certain to be in the student's hands, he
may be referred. What follows here is given merely as being
of more recent publication.

If it be desired to observe a living embryo by transmitted
light, the egg should be opened under salt solution, as de-
scribed below. A little of the white is then removed through
the window, the egg is lifted out of the liquid, and a ring of
gummed paper is placed on the yolk so as to surround the
embryonic area. As soon as the paper adheres to the vitel-
line membrane, which will be in a few minutes, a circular
incision is made in the blastoderm outside the paper ring.
The egg is put back into the salt solution, and the paper
ring removed, carrying with it the vitelline membrane and
the blastoderm, which may then be brought into a watch-
glass or on to a slide and examined under the microscope

(DUVAL).

590. Gerlach's Window Method (Nature, 1886, p. 497; Journ.
Roy. Mic. Soc., 1886, p. 359). — Remove with scissors the shell
from the small end of the egg ; take out a little white by
means of a pipette ; the blastoderm will become placed under-
neath the window thus made, and the white that has been
taken out may be replaced on it. Paint the margins of the
window with gum mucilage, and build up on the gum a little
circular wall of cotton wool ; place on it a small watch-glass
(or circular cover-glass), and ring it with gum. When the
gum is dry, the cover is further fixed in its place by means
of collodion and amber varnish, and the egg is put back in
its normal position in the incubator. The progress of the
development may be followed up to the fifth day through the
window.

A description of further developments of this method, with figures of
special apparatus, will be found in Anat. Anz., ii, 1887-) pp. 583, .609 ; see
also Zeit.f. wiss. Mik., iv, 3, 1887, p. 369.

AVES. 309

591. Preparation. — During the first twenty-four hours of
incubation it is extremely difficult to separate the blastoderm
from the yolk, and they should be fixed and hardened together.
In later stages, when the embryo is conspicuous, the blasto-
derm can easily be separated from the yolk, which is very
advantageous. To open the egg, lay it on its side and break
the shell at the broad end by means of a sharp rap ; then
carefully remove the shell bit by bit by breaking it away
with forceps, working away from the broad end until the
blastoderm is exposed. The egg should be opened in salt
solution, then lifted up a little, so as to have the blastoderm
above the surface of the liquid ; the blastoderm is then treated
with some fixing solution dropped on it from a pipette (1 per
cent, solution of osmic acid, or Ranvier and Vignal's osmic
acid and alcohol mixture, iodised serum, solution of Kleinen-
berg, 10 per cent, nitric acid, &c.). By keeping the upper
end of the pipette closed, and the lower end in contact with
the liquid on the blastoderm, the blastoderm may be kept well
immersed for a few minutes, and should then be found to be
sufficiently fixed to be excised. (Of course, if you prefer it,
you can open the egg in a bath of any fixing liquid [10 per
cent, nitric acid being convenient for this purpose] of such a
depth as to cover the yolk ; and having exposed the blasto-
derm, leave it till fixed [fifteen to twenty minutes] ; but I
think the procedure above described will generally be found
more convenient.)

The egg is put back into the salt solution, and a circular
incision made round the embryonic area. The blastoderm
may then be floated out and got into a watch-glass, in which
it may be examined, or may be brought into a hardening
liquid.

Before putting it into the hardening fluid, the portion of
vitelline membrane that covers the blastoderm should be
removed with forceps and shaking.

In order to counteract the turning up of the edges of the
blastoderm that generally happens during the process of
hardening, it is well to get the blastoderm spread out on the
convex surface of a watch-glass, and leave it so during the
hardening.

For hardening, FOSTER and BALFOUR recommend solution of
Kleinenberg for five hours, followed by alcohol. Or chromic

310 EMBRYOLOGICAL METHODS.

acid, a solution of O'l per cent, for twenty-four hours, followed
by a solution of O3 per cent, for twenty-four hours more, then
by 70 per cent, alcohol for a day, 90 per cent, alcohol for two
days, and lastly absolute alcohol. They also recommend a 0'5
per cent, solution of osinic acid, in which the embryo remains
for two hours and a half in the dark, and after washing is
brought into absolute alcohol.

HENNEGUY prefers the osmic acid and alcohol mixture of
Ranvier and Vignal, or Flemming's mixture followed by
successive alcohols.

Staining and imbedding may be performed by the usual
methods.

Up to about the fiftieth hour embryos may be mounted
entire, in glycerin or balsam.

592. M. Duval's Orientation Method (Ann. d. Sc. nat. Zool.,
1885). — In the early stages of the development of the ova of
Aves, before the appearance of the primitive streak, it is
difficult to obtain a correct orientation of the hardened cica-
tricula, so as to be able to make sections in any desired
direction. Duval, starting from the fact that during incuba-
tion the embryo is almost always found to be lying on the
yolk in such a position that the big end of the egg is to the
left, and the little end to the right of it, marks the position
of the blastoderm in the following way.

With a strip of paper 5 millimetres wide and 50 millimetres
long you construct a sort of triangular bottomless box. You
lay this on the yolk, enclosing the cicatricula in such a posi-
tion that the base of the triangle corresponds to what will be
the anterior region of the embryo, and its apex to the posterior
region ; that is to say, if the big end of the egg is to your
left, the apex of the triangle will point towards you. You
now, by means of a pipette, fill the paper triangle with 0*3
solution of osmic acid. As soon as the preparation begins to
darken you put the whole egg into weak chromic acid, remove
the white, and put the rest into clean chromic acid solution
for several days. After hardening you will find on the sur-
face of the yolk a black triangular area, which encloses the
cicatricula and marks its position ; you rut out this area with
scissors and a scalpel, and complete the hardening with
chromic acid and alcohol.

AMPHIBIA. 311

Another way of hardening is to place the egg, after the action of the
osniic acid, in a solution of chromic acid which is then raised to boiling-
point on a water-bath ; after cooling, the blackened region is cut out, and
the hardening completed in the usual way with chromic acid and alcohol.

593. KOLLEE'S Method (Arch.f. mik. Anat., xx, 1881, p. 182).— Chromic
acid, O'l per cent., twenty-four hours ; idem, 0'2 per cent., twenty -four
hours ; and so forth, with daily increments of O'l per cent, up to 0'5 per
cent. When hard, remove the blastoderm together with a segment of the
yolk. Water, twenty-four hours. Stain and imbed.

594. VIALLETON'S Method (Anat. Anzeig., vii, 1892, pp. 624— 627 5
Journ. Roy. Mic. Soc., 1892, p. 889). — Egg opened in salt solution, blasto-
derm excised and removed to a glass plate, then treated with 1 per cent,
nitrate of silver solution, washed with water, and put into 70 per cent, alco-
hol for six to twelve hours in the dark. Borax-carmine, alcohol, dammar.

Reptilia.

595. General Directions. — The methods described above for
the embryology of birds are applicable to the embryology of
reptiles. During the early stages the blastoderm should be
hardened in situ on the yolk ; later the embryo can be iso-
lated, and treated separately with Kleinenberg's solution and
alcohol (STRAHL, Arch.f. Anat. u. Phys., 1881, p. 123).

596. KUPFFEE'S Method (ibid., 1882, p. 4). — The ova are opened and
the albumen removed under osniic acid of -^ per cent. The yolk is put for
twenty-four hours into an ample quantity of ^ per cent, chromic acid solu-
tion ; the blastoderm is removed, washed out in water, and put for three
hours into Calberla's liquid (aa glycerin, water, and alcohol), and finally
hardened in 90 per cent, alcohol.

597. SAEASIN'S Method (SEMPEE'S Arbeiten, 1883, p. 159).— Fix with
chromic acid or hot water, and harden with alcohol. Stain with Bismarck
brown, alum-carmine, or hsematoxylin, or picro-carmine. Imbed in collodion,
and collodionise the sections as cut.

Amphibia.

598. Preliminary. — In order to prepare the ova of Amphibia
for section cutting, it is essential to begin by removing their
thick coats of albumen. This may be done by putting them
for two or three days into 1 per cent, solution of chromic acid,
and shaking well ; but ova thus treated are very brittle, and
do not afford good sections. A better method is that described
by WHITMAN (Amer. Natural., xxii, 1888, p. 857), and by
BLOCHMANN (Zool. Anz., 1889, p. 269). Whitman puts the
fixed eggs into a 10 per cent, solution of sodium hypochlorite

312 EMBRYOLOGICAL METHODS.

diluted with 5 to 6 volumes of water, and leaves them there
till they can be shaken free, which happens (for Necturus) in
a few minutes. Blochmann takes eau de Javelle (potassium
hypochlorite), and dilutes it with 3 to 4 volumes of water,
and agitates the eggs, previously fixed with solution of
Flemming, for fifteen to thirty minutes in it. The ova are
afterwards preserved in alcohol in the usual way. Some other
means of attaining the same end are given in the following
paragraphs.

599. Axolotl. — The ova are easier to prepare than those of
the Anura, because the yolk is separated from the albuminous
layer by a wide space filled with a liquid that is not coagulated
by reagents. Put the eggs for a few hours into picro-sulphuric
acid, then pierce the inner chorion with fine scissors or needles,
and gently press out the ovum. Harden in alcohol.

600. Triton (ScoTT and OSBORN, Quart. Journ. Hie. Sci.,
1879, p. 449). — The albumen is here present in the form of
several concentric coats, which are very delicate. Incise each
of them separately with fine scissors, turn out the ovum, and
fix it. Solution of Kleinenberg is the reagent that gives the
best results.

601. Triton (HERTwiG,/e?i. Zeit.f. Naturw., 1881-2, p. 291).—
Put the eggs into a mixture of equal parts of 2 per cent,
acetic acid and O5 per cent chromic acid. After ten hours
incise the membranes, opening one end of the inner chorion,
and turn out the embryos and bring them into successive
alcohols.

602. Salamandra (RABL, Morphol Jahrb., xii, 2, 1886, p.
252). — Fix in chloride of platinum of 0*25 to 0'3 per cent.,
kept warm for from three to twenty-four hours, according to
the size of the embryos, wash well with water, treat with
successive alcohols, make sections, and stain on the slide.

603. Rana (0. HEETWIG, Jen. Zeit. f. Naturw., xvi, 1883,
p. 249). — The ova are thrown into nearly boiling water (90°
to 96° C.) for five to ten minutes. The albuminous envelope
of the ovum is then cut open, and the ovum extracted under
water. The ova are then brought into 0*5 per cent, osmic
acid, or into alcohol of 70, 80, and 90 per cent. Chromic acid

TELEOSTEA IN GENERAL. 313

makes ova brittle ; they ought not to remain in it for more
than twelve hours. Chromic acid destroys or attacks the
pigment of the ova, whilst alcohol preserves it, which is fre-
quently important for the study of the germinal layers.

MORGAN (Amer. Nat., xxv, 1891, p. 759 ; Journ. Roy. Mic.
Soc., 1892, p. 284) has the following. During the periods in
which it is difficult or impossible to remove the inner jelly-
membrane the eggs can be freed as follows : — Each egg is cut
•out with scissors from the general jelly-mass, and put for
from one to twelve hours into saturated solution of picric acid
in 35 per cent, alcohol containing " the same amount of sul-
phuric acid as in Kleiuenberg's solution/' Wash for several
hours in several changes of alcohol, beginning with 35 per
cent., and increasing the strength gradually up to 70 per cent.
About the second day in the 70 per cent, alcohol the inner
membrane begins to swell, and on the third or fourth day
may be pierced by a needle, and the egg removed and placed
in 80 per cent, alcohol (see also WHITMAN, Meth. of Research,
p. 156; and SCHULTZE, Zeit.f. wiss. ZooL, v, 1887, p. 177).

Pisces.

604. Teleostea in General. — The ova of many of the bony
fishes can be studied by transmitted light in the living state ;
but those of the Salmonidse must be hardened and removed
from their envelopes for the study of the external forms of
the embryo.

To this end the ova may be put for a few minutes into
water containing 1 to 2 per cent, of acetic acid, and thence
into 1 per cent, chromic acid. After three days the capsule
of the ovum may be opened at the side opposite to the embryo,
and be removed with fine forceps. The ovum is pnt for
twenty-four hours into distilled water, and then into successive
alcohols. Embryos thus prepared show no deformation, and
their histological elements are fairly well preserved. But the
vitellus rapidly becomes excessively hard and brittle, so as
greatly to interfere with section cutting.

The following processes give good results as regards section
cutting.

Put the ova for a few minutes into 1 per cent, osmic acid ; as
soon as they have taken on a light brown colour bring them

314 EMBBYOLOG1CAL METHODS.

into Miiller's solution. Open them therein with fine scissors —
the vitellus, which immediately coagulates on contact with air,
dissolves, on the contrary, in Miiller's solution — and the germ
and cortical layer can be extracted from the capsule of the
ovum. They should be left in clean Miiller's solution for a
few days, then washed with water for twenty-four hours, and
brought through successive alcohols.

Another method is as follows : — The ova are fixed in solution
of Kleinenberg containing 10 per cent, of acetic acid. After
ten minutes they are opened in water containing 10 per cent,
of acetic acid, which dissolves the vitellus. The embryos are
put for a few hours into pure solution of Kleinenberg, and
are then brought through alcohol of gradually increasing
strength.

605. KOLLMANN'S Method (see § 40).

606. PEKENYI'S Method (see § 39).

607. KOWALEWSKY'S Method (Zeit. f. wiss. ZooL, xliii, 1886, p. 434>
—Fix for an hour and a quarter in a mixture of 8 volumes of picro-sulphuric
acid with one of 1 per cent, chromic acid. Wash out for twelve hours with
20 per cent, alcohol, and pass the ova very gradually through alcohol of 20,
28, 35, 43, 50, 60, and 70 per cent, strength, the last to be changed frequently
until all the picric acid is extracted. Before staining, the capsules of the
ova should be opened. Stain with borax-carmine or haematoxylin, and im-
bed in paraffin.

608. BOYEE'S Methods. — See Bull. Mus. Comp. Zool., Harvard, xxiii,
1892, p. 93 ; Journ. Eoy. Mic. Soc., 1892, p. 699.

609. KABL-RUCZHABD'S Method (Arch.f. Anat. u. Entw., 1882, p. 67).
— Fix in 10 per cent, nitric acid for fifteen minutes. Remove the mem-
branes to avoid deformation of the embryos, and put the ova back into the
acid for an hour. Wash out in 1 to 2 per cent, solution of alum for an hour,
and harden in alcohol.

Modification of this method by GORONOWITSCH (see Morph. Jahrb., x,
1884, p. 381).

610. Pelagic Fish Ova (WHITMAN'S method ; Amer, Natural., xvii,
1883, pp. 1204-5 ; Journ. Roy. Mic. Soc. [N.S.], iii, 1883, p. 912, and
Methods of Research, &c., p. 152). — Fix by treatment first for five to ten
minutes with a mixture of equal parts of sea water and ^ per cent, osmie
acid solution, and then for one or two days with a modified Merkel's solution
(due to Eisig), consisting of equal parts of 0'25 per cent, platinum chloride
and 1 per cent, chromic acid. Prick the membrane before transferring to
alcohol. Whitman found that the usual Merkel's fluid caused maceration of
the embryonic portion of the egg. Picro-sulphuric acid causes the embryonic
cells to swell, and in many cases to become completely disorganised. Tho

CEPHALOPODA. 315

osmic acid treatment is necessary in the case of segmenting ova because the
Merkel's fluid does not kill rapidly enough, so that eggs placed in it may
even pass through one or two stages of cleavage before dying. This fluid
arrests the process of blackening by the osmium, or will even bleach the ob-
jects if blackening has set in. See also AGASSIZ and WHITMAN, in Proc.
Amer. Acad. Arts and Sciences, xx, 1884. For later stages the authors re-
commend the method of Perenyi. And see the experiments detailed by
COLLINGE, Ann. and Mag. Nat. Hist., x, 1892, p. 228 ; Journ. Boy. Mic~.
Soc., 1892, p. 883.

Tunicata.

611. Distaplia. — DAVIDOJT (Mitth. Zool. Stat. Neapel, ix, 1,
1889, p. 118) lias some important observations on the fixation
of the ova of D. magnilarva. The best reagent is a mixture
of 3 parts of saturated solution of corrosive sublimate and 1
of glacial acetic acid. The ova to remain in it for from half
an hour to an hour, and be then washed for a few minutes in
water and brought through successive alcohols. Another
reagent, almost as good, consists of 3 parts of saturated solu-
tion of picric acid and one of glacial acetic acid, the objects
to remain in it for three to four hours, and then be brought
into 70 per cent, alcohol.

612. Amaroecium (MAURICE and SCHULGIN, Ann. Sci. Nat.
Zool., xvii, 1884). — Stain in borax-carmine, wash out, and
stain for fifteen to twenty hours in very weak solution of bleu
de Lyon in 70 per cent, alcohol with a few drops of acetic
acid. In sections the epiblast and hypoblast appear chiefly
blue, the mesoblast-cells, on the contrary, appearing almost
entirely red.

Mollusca.

613. Cephalopoda (Ussow, Arch, de BioL, ii, 1881, p. 582).
— Segmenting ova are placed, without removal of the mem-
branes, in 2 per cent, solution of chromic acid for two minutes,
and then in distilled water, to which a little acetic acid (one drop
to a watch- glassful) has been added, for two minutes. If an
incision be now made into the egg-membrane the yolk flows
away and the blastoderm remains ; if any yolk still cling to
it, it may be removed by pouring away the water and adding
more.

WATASE (Journ. of Morphol., iv, 1891, p. 249; Journ. Roy.
Mic. Soc., 1892, p. 152) kills the ova in the macerating mix-

316 EMBRYOLOGTCAL METHODS.

ture of the Hertwigs (§ 542), and as soon as the blastoderm
turns white and opaque removes it under dilute glycerin.
Treatment with liquid of Perenyi is recommended for surface
views of cleavage.

614. Gastropoda (HENNEGUY). — Ova of Helix may be fixed
for from four to six hours in Mayer's picro-nitric solution
(§ 58). The carbonate of lime that encrusts the external
membrane is thus dissolved, and the albuminous coat of the
egg is coagulated. The egg is opened with needles, the
albumen comes away in bits, and the embryo can be removed.
Treat with successive alcohols, and imbed in paraffin.

615. Limax (early stages) (MARK, Bull. Mus. Comp. Zool., Harvard
Coll., vi, 1881). — The ova are treated with acetic acid of 1 to 2 per cent, for
four or more hours. The two external membranes are incised with fine
scissors, and the egg squeezed out in its albumen membrane. This is dis-
sected off on a slide, the egg is separated from the albumen, stained, and
mounted in glycerin.

For later stages, or for making sections, osmic acid is used instead of
acetic acid, and the egg is hardened within its albuminous coats.

HENCHMAN (Bull. Mus. Oomp. Zool., Harvard, xx, 1890, p.
171; Journ. Roy. Mic. Soc., 1891, p. 274; Zeit.f. wiss. Mik.y
viii, 2, 1891, p. 216) finds that the best way of obtaining em-
bryos is to keep some twenty-five or thirty adults (of L.
maximus) in a large tin pail with a cover perforated with
small holes. They should be fed on cabbage, and the vessel
kept very clean. Eggs are generally found in the morning
in bunches of thirty to forty. As they are more abundant in
the early stages of confinement it is better to obtain a few
slugs often than many at once. In a moderately warm room
hatching occurs between the twenty-second and twenty-
seventh day. The eggs must be carefully protected from
desiccation.

Kill with 0*33 per cent, chromic acid, or with liquid of
Perenyi. It is best to remove only the outer envelope before
putting into the chromic acid, the inner membrane being
removed after two or three minutes therein. Where Perenyi
is used the membranes must be removed first, as the albumen
will else coagulate in such a way as to prevent the removal of
the embryos.

See also SCHMIDT, Studien zur Entwickelungsgesch. d. Pul-
monaten, Dorpat, 1891.

ARTHROPODA. 317

Arthropoda.

616. Fixation of Ova. — In most cases the ova of Arthropods
are fixed by heat in a more satisfactory way than by any other
means. This may be followed either by alcohol or some
watery hardening agent. If it be desired to avoid heating,
picro-sulphuric acid or liquid of Perenyi may be tried.

Removal of Membranes. — This is frequently very difficult,
and it may often be advisable not to attempt to remove them,
but to soften them with eau de Javelle or eau de Labarraque
(see the methods of Looss and LIST).

MORGAN (Amer. Natural., xxii, 1888, p. 357; Zeit. /. wiss.
Mik.j vi, 1, 1889, p. 69) recommends (for the ova of Peri-
planeta) eau de Labarraque diluted with five to eight volumes
of water, and slightly warmed. Thus used, it will soften the
chitin membranes sufficiently in thirty to sixty minutes if
employed before fixing. Fixed ova take longer. The fluid
must, of course, not be allowed to penetrate into the interior
of the ovum.

617. HENKING'S Methods. — For the whole subject of the
technique of the embryology of Insecta see an elaborate
paper by this specialist in Zeit. f. wiss. Mik., viii, 2, 1891,
p. 156. Henking agrees with other workers at this subject,
that in the majority of cases heat is the only available fixing
agent that will give fair results. He kills ova by plunging
them into hot water, or by pouring hot water on to them in
a watch-glass, and then removing into 70 per cent, alcohol.
But, as might be expected, he finds that the preservation of
structures by this method is far from being perfectly satisfac-
tory, cell-contours being not at all sharply brought out by it,
and achromatic cell-structures being but imperfectly pre-
served. He finds that in some cases ova may be fixed with
liquid of Flemming, which, as may be supposed, gives incom-
parably better results in these respects. Suitable ova may
be put into liquid of Flemming (Henking does not say which
formula) for half an hour, then for two hours into the same
diluted with three volumes of water, then treated with alcohol
as usual. Boveri's picro-acetic acid was found not to pene-
trate the membranes.

Henking thinks that eau de Javelle for softening mem-

318 EMBBYOLOGICAL METHODS.

branes is best avoided. Membranes should either be dissected
away or left in situ, and cut with the rest of the egg, accord-
ing to the nature of the case. The great obstacle to section
cutting is the brittleness of the yolk. This difficulty may be
overcome as follows : — After fixing and treating with alcohol,
prick the chorion and stain with borax-carmine. Put the
stained ova for twelve hours into a mixture containing 20 c.c.
of 70 per cent, alcohol, one drop of concentrated hydrochloric
acid, and a knife-pointful of pepsin (it is not necessary that
all the pepsin should be dissolved). The ova may then be
treated with alcohol, oil of bergamot, and paraffin, and (with
some exceptions, amongst which is Bombyx mori) will be found
to cut without crumbling.

The contents of fresh ova may conveniently be studied by
means of the following fluid :

Distilled water . . . .80 c.c.

Glycerin . . . . . . 16 „

Formic acid . . . . 3 „

1 per cent, osmic acid . . . 1 „
Dahlia 0'04 grm.

The eggs are simply teased in a drop of the liquid, and a
cover-glass put on. If it be desired to preserve the prepara-
tion, nothing more is necessary than to lute the cover-glass.

618. Lepidoptera (BOBRETZKY, Zeit. f. tciss. Zool., 1879, p.
198). — Ova (of Pieris cratsegi and Porthesia chrysorrhoea) are
slightly warmed in water and put for sixteen to twenty hours
into 0'5 per cent, chromic acid. The membranes can then be
removed, and the ova brought for a few hours into absolute
alcohol, stained with carmine, and cut.

619. Blattida (PATTEN, Quart. Journ. Mic. Sd., 1884, p. 549) .
— The ova or larvae are placed in cold water, which is gra-
dually raised to 80° C. You leave off heating as soon as the
ova have become hard and white. Pass very gradually
through successive alcohols, beginning with 20 per cent. ;
stain with Kleinenberg's haematoxylin or Mayer's cochineal
(only alcoholic stains will traverse the chorion). The ova
may remain in the haematoxylin for five or six days, and be
washed out in alcohol containing one drop of HC1 per 20
grms., in which they should remain for several days, and then

DIPTERA. 319

be soaked in pure alcohol until they have regained their
violet colour. Penetrate with benzol and imbed in paraffin.

WHEELER (Journ. of MorpTi., iii, 1889, p. 292 ; Journ. Roy.
Mic. Soc., 1890, p. 250) dissects out ovarian ova in salt solu-
tion and fixes in liquid of Perenyi (fifteen minutes), then
treats with alcohol, and stains with borax-carmine. Laid
eggs may be killed by Patten's method. After heating, the
two lips of the crista of the capsule may be separated with
fine forceps and pieces of the walls torn away, and the eggs
pushed out of the compartments formed by their choria and
hardened as desired. Good results are also obtained by
heating to 80° C. for ten minutes in liquid of Kleinenberg,
and preserving in 70 per cent, alcohol. This causes the en-
velopes to dilate and stand off from the surface of the egg,
so that they can easily be dissected away.

CHOLODKOWSKY (Mem.Acad.Imp. /S^.PetersZmr^xxxviii, 1891 ;
Zeit. f. wiss. Mik., ix, 1, 1892, p. 80) recommends cutting off
the ends of the cocoons and fixing for twelve hours in liquid
of Perenyi, or for a few minutes in a solution of 1 part iodine,
1 part iodide of potassium, and 300 parts water, heated to
boiling-point.

HEYMONS (Zeit. f. wiss. ZooL, liii, 1892, p. 434; Zeit. f.
wiss. Mik., ix, 3, 1893, p. 343) finds that Cholodkowsky's
methods are good for the study of general relations of parts,
but not satisfactory for the preservation of delicate detail.
For young embryos it is better to incise the cocoon at the end
by which it inheres in the body of the mother, bring it for
two minutes into water heated to 90° C., and open in Flem-
ming, in which the embryo should be dissected out.

620. Diptera (HENKING, Zeit. f. wiss. Zool., xlvi, 1888, p. 289 ;
Zeii. f. wiss. Mik., 1889, p. 59). — Ova still contained within
the fly may be fixed by pluoging the animal for some time
into boiling water, then dissecting out and bringing them into
70 per cent, alcohol. Laid eggs may have boiling water
poured over them, or be put into solution of Flemming in a
test-tube which is plunged into boiling water until the eggs
begin to darken (about a minute). Cold solution of Flemming
easily causes a certain vacuolisation of the contents of the
•ova. Open the ova at the larger end, stain with borax-car-
mine for fifteen to thirty hours, and cut in paraffin.

320 EMBRYOLOGICAL METHODS.

See also (for Chironomus) RITTER, Zeit. f. wiss. ZooL, L>
1890, p. 408; Zeit. /. wiss. Mik.,viii, 1, 1891, p. 87 (strings
of ova fixed with hot 30 per cent, alcohol containing a little
sublimate, and stained in the mass by immersion for several
days in picro-carrnine).

621. Aphides (WiLL, Semper' s Arbeiten, 1883, p. 223).— Sections to be
made through the entire animals containing the ova and embryos. The
animals are killed in water of 70° C., and brought into alcohol. The cuticle
may then be pricked with a needle, and the animals stained in the mass with
borax-carmine or hsematoxylin. You may imbed in collodion and collodio-
nise the sections as cut.

622. Araneina (BALFOUB, Quart. Journ. Mic. Sci., 1880, p. 167). —
Balfour hardened the embryos in bichromate of potash, after placing them
for a short time in nearly boiling water. After removal of the membranes
they were stained as a whole with hsematoxylin.

KISHINOUYE (Journ. Coll. Sci. Imp. Univ. Japan, iv, 1891,
p. 55; Zeit. /. wiss. Mik., ix, 2, 1892, p. 215) fixes in water
warmed to 70° or 80° C., puts into 70 per cent, alcohol, and
after twenty-four hours therein pierces the membranes and
passes through stronger alcohol. Stain with alcoholic cochi-
neal or picro-carmine, and imbed in paraffin.

See also LOCY, Bull. Mus. Comp. ZooL, Harvard, xii, 3, 1886 ; Zeit. f.
wiss. Mik., iii, 2, 1886, p. 242. Fix by hot water. The liquid of Perenyi
may also be used ; it has the advantage of not making the yolk so granular.

623. Phalangida (BALBIANI). — The ova of Phalangium opilio are en-
closed in a chorion covered with yellow corpuscles which renders them quite
opaque. They may be cleared by treating them with water containing a
little solution of caustic potash and raised to boiling-point. The ova are
then laid on blotting-paper, and the chorion is removed by rubbing them
gently with a small brush. The vitelline membrane remains intact and
transparent, and the embryo may be studied through it.

624. Phalangida (HENKING, Zeit. f. wiss. Mik., iii, 4, 1886, pp. 470
et seq.). — Fix with boiling water or " Flemming." Preserve the ova in 90
per cent, alcohol. To open the chorion, bring them back into 70 per cent,
alcohol, which causes them to swell up so that the chorion can easily be
pierced with needles, and the ovum turned out.

625. Astacus (REICHENBACH, from Zeit. f. wiss. Mik., 1886, p. 400).^-
Fix in water gradually warmed to 60° or 70° C. (if the chorion should burst,
that is no evil), harden for twenty-four hours in 1 to 2 per cent, bichromate
of potash orO'5 per cent, chromic acid, wash out for the same time in running
water, and bring into alcohol. Remove the chorion, remove the embryo
from the yolk by means of a sharp knife, and stain with picro-carmine and
mount in balsam.

LUMBRIOUS. 321

626. Amphipoda (Orcliesiia) (ULIANIX, Zeit.f. wiss. Zool., xxxv, 1881,
p. 441). — Ova in the earliest stages of development were treated for two hours
with picro-sulphuric acid (Kleinenberg's formula). This causes the chorion
to swell and burst. Ova in later stages, in which the embryo is surrounded
by a cuticular membrane, which encloses an albuminous liquid, must have
this membrane torn with needles and the albuminous liquid allowed to ooze
out before placing in the picro-sulphuric acid.

627. Maturation of Ova, and other early stages. — These should be
studied by the methods given in the chapter on " Cytological Methods."

Vermes.

628. Taenia (v. BENEPEN, Arch, de Biol., ii, 1881, p. 187). —
Ova in which a chitinous membrane has formed around the
embryo are impervious to reagents. They may be put on a
slide with a drop of some liquid and covered. Then, by with-
drawing the liquid by means of blotting-paper, the cover may
be made to gradually press on them so as to burst the mem-
branes, and the embryo may then be treated with the usual
reagents. •

629. Planaria (!IJIMA, Zeit. f. wiss. Zool., xl, 1884, p. 359). —
The capsule containing the ova (of fresh-water Planaria) is
opened with needles on a slide, in a drop of 2 per cent, nitric
acid. The ova are extracted and covered (the cover being
supported by paper, or by wax feet). After half an hour
they are treated with successive alcohols under the cover,
and finally mounted in glycerin. For sections, the whole of
the contents of a capsule is hardened in the mass in 1 per
cent, chromic acid and cut together.

630. Lumbricus (KLEINENBEEG, Quart. Journ. Hie. Sci., 1879,
p. 207). — Fix with Kleinenberg's picro-sulphuric acid, or,
which is not quite so good, with vapours of osmium, pass
through successive alcohols, stain with Kleinenberg's hserna-
toxylin, and cut in paraffin.

WILSON (Journ. of Morph., iii, 1889, p. 445 ; Journ. Eoy.
Mic. Soc., 1890, p. 402) finds that liquid of Perenyi is by far
the best fixing reagent, being in most respects superior even
to Flemming's. Fix for fifteen to sixty minutes, wash out in
70 per cent, alcohol, stain with borax-carmine, wash out in
acid alcohol, treat for a few minutes with ammoniacal alcohol
to neutralise the acid, and after-stain for twelve hours with

21

322 EMBRYOLOGIOAL METHODS.

very dilute Kleinenberg's haernatoxylin. Imbed in paraffin,
giving a short paraffin bath (not more than ten to fifteen
minutes).

For study of entire specimens in the early stages, Perenyi's
liquid, followed by alcohol, water, very dilute iodine solution,
and glycerin, gives the best results.

631. Ascaris. — See the chapter on " Cytological Methods."

Echinodermata, Coelenterata, and Porifera.

See the paragraphs treating of these groups in Chap.
XXXII.

For the maturation and fecundation of the ova of the Echi-
nodermata, see also the chapter on " Cytological Methods."

SUBJECTS FOR STUDY. 323

CHAPTER XXVI.

CYTOLOGICAL METHODS.

632. The Methods of Study.— There are three ways of ob-
taining knowledge of cell-structure — study of living cells,
study of fresh unhardened cells, and study of hardened
material in sections. Of these the last is the most fruitful ;
and I advise the beginner to keep as close as possible to the
method of Fleniming and Rabl, utilising unhardened material
chiefly for the purpose of controlling the results obtained by
the study of sections, and reserving the study of living cells
chiefly for establishing the sedation of already observed phe-
nomena.

633. Subjects for Study. — One of the best objects for this
purpose is the tail of young larvas of Amphibia, both Anura
and Urodela.

In the living animal the epithelial cells and nuclei (in the
state of repose) are so transparent as to be invisible in the
natural state. They may, however, be brought out by cura-
rising the larva ; or, still better, by placing the curarised
larva for half an hour in 1 per cent, chloride of sodium solu-
tion. Normal larvae may be used for the study of the active
state of the nucleus, but much time is saved by using curare.

Curare. — Dissolve 1 part of curare in 100 parts water, and
add 100 parts of glycerin. Of this mixture add from 5 to 10
drops (according to the size of the larva), or even more for
largo larvae, to a watch-glassful of water. From half to one
hour of immersion is necessary for curarisation. The larvae
need not be left in the solution until they become quite
motionless ; as soon as their movements have become slow
they may be taken out and placed on a slide with blotting-
paper. If they be replaced in water they return to the normal
state in eight or ten hours, and may be re- curarised several
times.

324 CYTOLOGICAL METHODS.

Etherisation. — Three per cent, alcohol, or 3 per cent, ether,
may be used in a similar way. These reagents cause no
obstruction to the processes of cell-division, and are useful,
but their action as ansesthetics is inconstant.

Indifferent Media. — One per cent, salt solution, iodised serum,
syrup, cold water ( + 1° C.), and warm water (35°— 40° C.).
The tail may be excised from the living animal and studied
for a long time in these media (PEREMESCHKO, Arch. f. mik.
Anat., xvi, 1879, p. 437).

Perhaps (FLEMMING, ibid., pp. 304 et seq.) the very best
subject for these studies is Salamandra. The adult offers
for study the thin transparent bladder ; in the larva the gills
and caudal "fin" may be studied in the living state. The
gills are difficult to fix in position for observation, and are
obscured by pigment. In the fin there is always a spot, near
to the hind limbs, that is free from pigment ; and on lightly
coloured larvae other such spots may be found on the ventral
half of the fin and on the lateral line. On a flat-finned larva
it is possible to study these spots with high-power glasses.

The larva may be fixed in a suitable cell, or wrapped in
moist blotting-paper, or may be curarised ; or the tail may be
excised. (It is preferable to cut through the larva close in
front of the hind limbs.)

A favorable object tor preparation is found in the gill-plates,
delicate laminae that are to be found attached to the gill-car-
tilages on the mouth side.

The lungs, parietal peritoneum, and mesentery of the larvae
are also very favorable objects for preparations (see FLEM-
MING, Arch. f. mik. Anat., xxxv, 1890, p. 275 ; and xxxvii,
1891, pp. 249 and 685). To prepare the lungs the larva,
which should be of not more than 4 cm. length, should be
killed by immersion in chromo-aceto-osniic acid, the body-
cavity cut into, and the viscera gently drawn out and exposed
to the action of the liquid, care being taken not to let the
lungs get into folds. After fixation they should be carefully
got on to a slide, and a small strip removed from their mar-
gins on either side by means of a scalpel, after which the two
walls may be separated from each other, and utilised as thin,
flat preparations.

Another excellent object is the intestine of the adult, of
which suctions may be made by the paraffin method, as recom-

STUDY OF FRESH AND LIGHTLY FIXED CELLS. 325

mended in the important paper of M. HEIDENHAIN, " Uber Kern
und Protoplasma," in Festschr. z. 50 jdhr. Jubil. d. H. Prof.
Geheimr. v. Kolliker (also in separate reprint), Engelmann,
Leipzig, 1892, p. 111. This organ offers for study, besides
the large epithelium-cells of the intestinal crypts, numberless
examples of leucocytes, an extremely favorable object.

Larvae may be bred from adults kept in confinement, and
supplied with a vessel of water, in which they will place the
larvae of their own accord. In May gravid females may be
killed and the larvae extracted. The larvae must be kept in
frequently changed water and fed every day or two. Aquatic
worms may be used for feeding them, e. g. Tubifex rivulorum.

It is extremely important that they should be fed regularly
and abundantly, for, if not, cell-divisions in the tissues become
rare, and may even cease altogether.

Other classical subjects of study will be found mentioned
in the following paragraphs.

634. Stains for Living Cells. — It is sometimes of the very
greatest importance to be able to stain a cell in the living
state, even though it be but feebly and imperfectly. Methylen
blue, dahlia, or gentian violet may be used in solution in pure
water, or in an indifferent liquid ; the addition of a trace of
chloral hydrate will enable you to obtain a clear solution of
the last two in saline media. It is sometimes advisable to
rub them up with serum, as recommended by v. LA VALETTE
ST. GEOEGE. These methods are most important for the study
of the Nebenkern.

The student will remember that no known reagent will
stain any part of the nucleus whilst alive. The " Nebenkern"
stains sometimes, but feebly. Most frequently the colour is
only taken up by certain granules of the cytoplasm, which
may or may not be identical with the " granules" or "bio-
blasts" of ALTMANN.

These matters have already been discussed in the paragraph
headed " Staining ' intra vitam,' " § 93.

635. Study of Fresh and Lightly fixed Cells.— -It has been
rightly pointed out by Flemming that so-called " indifferent"
liquids must not be believed to be without action on nuclei.
Iodised serum, salt-solution, serum, aqueous humour, lymph,
better deserve the name of weak hardening agents. Between

326 CYTOLOGICAL METHODS.

these, and such energetic hardening agents as Flemming's
.mixture, come such light fixing agents as picric acid or very
dilute acetic acid. These it is whose employment is indicated
for the study of fresh isolated cells.

A typical example of this kind of work is as follows : — Tease
but a piece of living tissue in a drop of acidulated solution of
methyl green (0'75 per cent, of acetic acid). This is a deli-
cate fixing agent, killing cells instantly without change of
form. Complete the fixation by exposing the preparation for
a quarter of an hour to vapour of osmium, and add a drop of
solution of Bipart and Petit and a cover.

Or you may fix the preparation, after teasing, with vapour
of osmium for half a minute to two minutes, then add a drop
of methyl green, and after five minutes wash out with 1 per
cent, acetic acid, and add solution of Kipart and Petit and
cover.

Or you may kill and fix the cells by teasing in solution of
Bipart and Petit (to which you may add a trace of osmium
if you like), and afterwards stain with methyl green.

HENKING'S mixture, which has been given above (§ 617),
may also be found useful.

FLEMMING (Arch. f. mile. Anat.} xx, 1881, p. 3) found the
following method useful for the study of the division of the
ova of Echinodermata : — The ova are stained on the slide by
adding the stain at the edge of the cover. Safranin or other
nitro- or anilin colours may be used. As soon as the entire
ovum is of a dark colour the stain is drawn off with blotting-
paper, and acetic acid of 1 per cent, added. Schneider's
acetic carmine (Zool. Anzeig., 1880) (see § 158) is very con-
venient, and gives good results. (For the details of the
manipulation by which these reagents are added and drawn
off on the slide it is well to consult the article quoted, p. 6.)

Another good method is as follows : — Segmenting ova are
treated with a mixture of 40 to 50 parts of concentrated
nitric acid with 60 to 50 parts water. Wash with water until
all the yellow stain of the nitric acid has disappeared ; stain
with Schneider's acetic carmine, and mount in glycerin. (The
preparations cannot be said to be permanent, as after a time
the stain darkens in such a way as to render the nuclear
figures unrecognisable.)

Other fixing agents, such as picric acid or weak sublimate

SOME M1CEOCHEMIOAL REACTIONS. 327

solution, may of course be used, and in some cases doubtless
should be preferred. Other stains, too, such as Bismarck
brown or Delafield's haematoxylin, may be used as occasion
dictates ; and of course other examination media than solution
of Ripart may be employed. But, for general purposes, the
methyl-greeii-osmium-and-Ripart's-medium method gives such
good results, and is so very convenient, that it may well be
called the classical method for the study of fresh cells. I
think great credit is due to CARNOY for his frequent insistence
on the excellence and handiness of this method.

Other fixing agents and stains that are applicable to this
kind of work will be found discussed in the course of the
following paragraphs.

636. Some Microchemical Reactions. — Methyl green is a test
for chromatin, in so far as it colours nothing but the chro-
matin in the nucleus. It is, however, not a perfect test, for
the intensity of the coloration it produces varies greatly in
different nuclei, and may in certain nuclei be extremely weak,
or (apparently) even altogether wanting. In these cases
other tests must be applied in order to establish with certainty
the presence or absence of that element. The following sug-
gestions are taken from CARNOY, who is, I believe, the only
writer — on the zoological side, at all events — who has insisted
on the necessity of applying microchemical methods in a
systematic manner to the study of cells.

Chromatin is distinguished from the lecithins and from
albuminoids by not being soluble, as these are, in water and
in weak mineral acids, such as 0*1 per cent, hydrochloric acid.
It is easily soluble in concentrated mineral acids, in alkalies,
even when very dilute, and in some alkaline salts, such as car-
bonate of potash and biphosphate of soda. In the presence of
10 per cent, solution of sodium chloride it swells up into a
gelatinous mass, or even, as frequently happens, dissolves en-
tirely (Biol. Cell., pp. 208-9). It is only partially digestible
(when in situ in the nucleus) in the usual laboratory digestion
fluids.

Tlip solvents of chroraatin that are the most useful in prac-
tice are 1 per cent, caustic potash, fuming hydrochloric acid,
or cyanide of potassium, or carbonate of potash. These last
generally give better results than dilute alkalies. They may be

328 OYTOLOGICAL METHODS.

employed in solutions of 40 to 50 per cent, strength. If it be
desired to remove all the chromatin from a nucleus the reac-
tion must be prolonged, sometimes to as much as two or three
days, especially if the operation be conducted on a slide and
under a cover-glass, which is the safer plan.

It must be remembered that these operations must be per-
formed on fresh cells, for hardening agents bring about very
considerable modifications in the nature of chromatin, render-
ing it almost insoluble in ammonia, potash, or sodic phosphate,
&c. Hydrochloric acid, however, still swells and dissolves it,
though with difficulty.

Partial digestion may render service in the study of the
chromatic elements of nuclei. Chromatin resists the action
of digestive fluids much longer than the albumens do ; so that
a moderate digestion serves to free the chromosomes from
any caryoplasmic granulations that may obscure them, whilst
at the same time it clears up the cytoplasm.

In the last edition the term " nuclein " was used throughout this section
in all the places where the term " chromatin " has been used in the above
paragraphs. It is now known that there exists a whole series of nucleins,
differing chiefly in respect of their richness in phosphorus and proteids. At
one end of the chain is nucleic acid, with 9 to 11 per cent, of phosphorus, and
without any proteid (this compound occurs in nature in the heads of sper-
matozoa) ; in the middle are what are generally termed the nucleins, con-
sisting of proteid with varying amounts of nucleic acid ; and at the other
extreme are nucleins which are nearly all proteid, containing only 0*5 to 1
per cent, of phosphorus, and are in fact the same substances which have
received the name of " nucleo-albumin ;" they may also be termed the
artificial plastins.

These substances have both been isolated from the most diverse tissues of
the animal body, and have been prepared artificially. A corresponding series
of nucleins exists within the nucleus itself. There are those that contain
most nucleic acid ; these are readily soluble in alkalies, and precipitable with
difficulty by acid : chromatin is one of these. There are others more in-
soluble in alkalies and poorer in nucleic acid : these are the plastins, the
pyrenin of nucleoli being one of them. And there are others even poorer in
nucleic acid : these are the nucleo-albumins (which exist also in the cyto-
plasm) ; the paralinin, or nuclear sap, appears to be in part composed of
these, in part of phosphorus-free compounds.

There appears to be some doubt whether chromatin is or is not nucleic
acid itself. The principal reactions in which it resembles nucleic acid are
given by HALLIBURTON (Goulstonian Lectures on the Chemical Physiology
of the Animal Cell, 1893, p. 574 of the Report in the British Medical
Journal, No. 1681, March 18th, 1893, from which place also I have con-
densed the above remarks on the chemistry of the nucleins) as follows : —

CYTOLOGIOAL FIXING AGENTS. 329

" 1. It does not give Millon's nor the xantho-proteic reactions. 2. It is easily
soluble in alkalies, soluble with difficulty in acids. 3. It is soluble in an
acetic acid solution of potassium ferrocyanide. 4. After treatment with
concentrated copper sulphate solution for twenty-four hours it loses its
affinity for stains. It is not, however, dissolved by the copper sulphate as
Schwartz stated. 5. It has a great affinity for anilin dyes, especially for
basic dyes like methyl green. If a mixture of methyl green and acid
f uchsin is employed, nucleic acid is stained green. The nucleins next richest
in phosphorus are stained a blue- violet tint, whereas the phosphorus-poorest
are coloured red. Now in the dividing nucleus, when the amount of chro-
rnatin is at its maximum, the nucleus stains green ; whereas in the resting
nucleus, where there is more pyrenin, a blue colour is observed."

These considerations appear to justify the employment of the term
" chromatin " for the element of the nucleus that stains with methyl green,
the term " nuclein " having obtained a wider extension.

See also KOSSEL, in BEHEENS, KOSSEL, und SCHIEFFERDECKER'S Das
Mikroskop, &c., ii, p. 47 ; the same, in Verli. d. physiol. Ges., Berlin, Oct.
21st, 1892 ; and MALFATTI, Ber. d. naturw. med. Verelnes in Innsbruck,
1891-2.

For the microchemical detection of phosphorus in tissue-elements see the
paper of LILIENFELD and MONTI, in Zeit. f. physiol. Chemie, xvii, 1892,
p. 410 (Report in Zeit.f. wiss. Mik., ix, 3, 1893, p. 332) ; also a short notice
in HALLIBUETON'S Goulstonian Lectures, 1893 (see Brit. Med. Journ.,
March llth, 1893, p. 505).

637. Cytological Fixing Agents. — The following is in great
part taken from the numerous papers of FLEMMING in the
Arch. f. mik. Anat. from the year 1879 onwards, and from his
Zellsubstanz, Kern- und Zelltheilung*

Osmic acid (y1^- to 2 per cent.) preserves the form of the
entire cell, but swells the nuclei and rounds off nucleoli. It
renders the nuclear (( reticulum" undiscernible. Picric acid,
either concentrated or dilute, and chromic acid, 0*1 to 0*5 per
cent., are to be preferred to alcohol and other agents for the
study of the cells of Vertebrates. Shrinking and distortion of
the nuclear figures (and, with picric acid, swellings of them)
are to be expected, but other agents have the same defect to
a much greater degree ; alcohol especially causes entanglement
of the filaments. Acetic acid does the same, and causes
swelling besides. Stronger chromic acid solutions cause
shrinking. Neither of these reagents is harmless as regards
the nuclei of red blood-corpuscles. The salts of picric acid
(potash-, soda-, and baryta-salts) are most harmful. Weak
(i. c. not more than 1 per cent.) acetic, hydrochloric, or nitric
acid, combined with clearing in glycerin and staining, may be

330 CYTOLOGICAL METHODS.

useful for bringing out reticula and nucleoli. Chloride of
gold preserves the forms well, but generally leaves the nuclear
structures unstained. Nitrate of silver is hopelessly uncon-
trollable in its action. Alcohol has much the effect of chromic
acid, but often causes a much greater shrinking of the nuclei.
Bichromate of potash and chromate of ammonia bring out very
sharply the appearance of a reticulum, but these appearances
cannot be accepted as true (1. c., p. 334, et seq.).

" Those who seek to xtudij cdl-dii'ixion by means of bichromate
of potash or other chromic salt* arc hopelessly in the wrong
road." And this because of the injurious action of the
bichromate, not on the body of the cell, which it preserves
well, but on the chromatin structures. Chromic salts are
excellent reagents for general histological work, but not for
nuclear structures. They dissolve nucleoli, destroy nuclear
" net works," and swell up and distort karyokinetic figures to
such a degree that the appearances obtained from them are
merely unnatural caricatures of the true structure.

Altmann's nitric acid method is excellent for the purpose
of hunting for cell- divisions in tissues; but the minute struc-
ture of the figures is not so well preserved as it is by means
of chromic or picric acid. The same must be said of Kleinen-
berg's picro-sulphuric acid method. (I am not alone in hold-
ing that this is a most untrustworthy cytological reagent; see,
for instance, HOLL [Sitzb. h. Acad. Wiss. Wien, xcix, 1890,
p. 311 ; Zeit.f. wiss. Mik., ix, 1, 1892, p. 89], who found that
it frequently reduced chromosomes to the state of mere lumps,
" Krumeln.")

There are two fixing mixtures which may be said to be
classical for cytological studies, FLEMMING'S chromo-aceto-osmic
acid mixture, §§ 35, 36, and HERMANN'S platino-aceto-osmic acid
mixture, § 51. As to the former of these, Flemming has the
following explanations : — Attempts to omit the chromic acid
from the formula did not give good results. The omission of
acetic acid (as in Max Flesch's formula, § 34) causes the
figures to be far less sharply brought out. The presence of
acetic or formic acid in all osmium solutions is favorable to
the precision of subsequent staining with ha3matoxylin, picro-
carmine, or gentian- violet. But mixtures of osmic and acetic
acid without chromic acid (Eimer) do not give such good
results as the chromo-aceto-osmic acid mixture. Mixtures

CYTOLOOICAL FIXTXO AGENTS. 331

of picric acid with osmic acid or with osmic and acetic acid
(proportions of the latter as in the chromo-acetic-osmic mix-
ture [§ 35], but of picric acid about 50 per cent.) fix quite as
well as the chromic mixtures, but precise staining is even
more difficult than with pure osmic acid preparations. Flem-
ming concludes that the beneficial effects of the osmium in
all these mixtures are to be ascribed to the instantaneous
rapidity with which it kills, the function of the other acids of
the mixture being to render the structures distinctly visible.

Mixtures containing osmic acid should therefore be em-
ployed whenever it is desired to fix the chromatic figures as
faithfully as possible; whilst pure chromic acid should be taken
whenever very sharp staining is the more important point.

For the study of the achromatic figures he recommends the
chrorno-acetic acid mixture (§ 31), followed by staining in
haematoxylin (anilins do not give so good results for this
purpose) .

For the study of polar corpuscles he recommends the
osmium mixtures, or pure chromic acid followed by staining
with gentian-violet.

The above account stands nearly as it stood in the first
edition. The state of things at present is as follows : — It is
admitted by all competent observers that the chromo-aceto-
osmic mixture is, with at most one or two possible exceptions,
by far the best fixing agent for nuclei. But some observers
have stated that it does not always preserve the cell-body
well. This is a question that has been already discussed in
§§35 and 36. I will only add here that after considerable
experience I see no reason to distrust Flemrning's mixture as
a preservative of any kind of protoplasm, provided it be used
in the proper way. It must be taken of the proper strength,
it must be used with very small objects, so that it may act on
all parts of them with its full strength, and not be filtered and
diluted through thick walls of tissue before coming into con-
tact with the object of study; and it must be allowed to act
for the proper time.

This brings us to another point. There are two chromo-
aceto-osmic mixtures — the old weaker one, and the new stronger
one. Flemming recommended the strong one primarily as
affording a means of differentiating kinetic chromatin from
resting chromatin. He did not recommend it as a reagent for

332 CYTOLOGICAL METHODS.

general work. Whether of these two solutions should be
used for general work ? According to my experience, the
strong solution does preserve both nuclear structures and
caryoplasmic structures quite as faithfully at least as the old
formula, and some structures most decidedly much better.
Of course the one and the other should be taken according
to the nature of the object you are dealing with ; but I think
it may safely be stated as a general rule that if you take the
strong mixture, and fix thoroughly in it, you are not likely to
go far wrong. And what is meant by a thorough fixation ?
Half an hour may be taken to be generally enough ; but for
very delicate things, such as the Nebenkern and the achro-
matic figure, at least eighteen hours ought to be given.

It only remains to point out that this doctrine is at variance
with that expressed in the first edition and in the Traite, and
with the earlier recommendations of Flemming ; but I feel
some confidence that it will not be called in question by the
majority of workers at this subject. Of course it goes without
saying that further precise evidence on the matter is very
much to be desired.

As to the platino-aceto-osmic mixture of HERMANN, it has
already been explained in § 51 that the point of superiority
over Flemming's mixture that is claimed for it lies in a more
faithful preservation of cytoplasm and achromatic structures.
That the alleged superiority really exists appears to be the
general opinion of those who have worked with this reagent.
Flemming (Arch. f. mik. Anat., xxxvii, 1891, p. G85; Zeit. /.
/r/.s.v. Mik., viii, 3, p. 343) agrees that it gives a peculiarly
sharp demonstration of spindle fibres, centrosomes, and polar
corpuscles ; but thinks that the chromo-aceto-osmic mixtures
give a somewhat more faithful preservation of the chromatic
elements.

Two or three of the fixing agents proposed by other writers
may also rank as first-class reagents for this kind of work.
There is RABI/S chromo-formic acid (§ 32). Fix in this for
twelve to twenty-four hours, wash out well with water, and
pass into alcohol. And there is the same observer's platinum
chloride solution (§ 50). In Rabl's paper in Anat. Anz., iv,
1889, p. 21, he recommends that Saltinmntlra larvae be fixed
(for twenty-four hours) in a solution of from one tenth to one
eighth per cent, strength. In his earlier work he used solu-

CYTOLOGICAL FIXING AGENTS. 333

tions of 1 — 300 strength. Platinum chloride has the pecu-
liarity of causing a slight shrinkage of the chromatin, which
helps to bring into evidence the granules of Pfitzner and the
longitudinal division of the chromosomes.

There remain to be mentioned several fixing agents with which very
important work has been done. These, however, are, I think, not quite first-
class reagents for the purpose, the brilliant results that have been obtained
with them having been obtained rather in spite of their defects than on
account of their good qualities. For instance, acetic alcohol is a reagent
with which some of the most important work in recent cytology has been
done — namely, much of that on the maturation and fecundation of the ovum
of Ascaris. It is evident that for such an extraordinarily impenetrable
object as the ovum of Ascaris, the employment of some such highly pene-
trating fluid as acetic acid is imperatively indicated, notwithstanding certain
defects that it may have.

CARNOY (La Cellule, iii, 1, 1886, p. 6) used at first a mixture of three
parts of absolute alcohol with 1 of glacial acetic acid ; later (ibid., iii, 2, 1887)
the chloroform mixture (§ 54). From five to fifteen minutes is enough for
even the most resistent ova.-

VAN BENEDEN and NEYT (Nouvelles Rech. sur la Fee. et la Division
mitosique, 1887) employed a mixture of equal parts of absolute alcohol and
glacial acetic acid, or even pure acetic acid.

Acetic alcohol may be washed out with either pure alcohol, or with dilute
glycerin (Calberla's formula would be a good one in many cases). For
further details see ante, § 54.

M. HEIDENHAIN (Ueb. Kern u. Protoplasma, 1892, p. 113) has been
using corrosive sublimate, on account of its convenience, and, above all, on
account of the great facility it affords for the employment of any kind of
stain. The beautiful figures of attraction spheres and other cytoplasrnic
structures given in this paper show that the most brilliant results may be
obtained by this means. But I would remark that the figures of nuclear
structures appear to me less convincing. The author figures and describes
under the name of " Lanthanin," an acidophilous caryoplasmic substance ex-
hibiting a minutely reticular arrangement. The figures remind me of
appearances which I frequently obtained in certain nuclei when working
with sublimate some years ago, and which I regarded as artefacts, and in
consequence was led to the abandonment of sublimate as a cytological fixa-
tive. I do not find in Heidenhain's paper that he has instituted control
experiments to show that the reticular arrangement of his " lanthanin " is
preformed in the nucleus, and would point out the need of such experiments
before either the existence of the lanthanin reticulurn or the fidelity of the
reagent can be deemed established.

ALTMANN (Arch.f. Anat. u.Entwickel., 1892, p. 223 ; Zeit. f. wiss. Mik,
ix, 3, 1893, p. 331) has a new fixative for resting nuclei, viz. a 2*5 per cent,
solution of ammonium molybdate to which is added about O25 per cent, of
chromic acid. I have no personal knowledge or other information concern-
ing this method.

334 CYTOLOG1CAL METHODS.

Lemon juice (fresh, filtered) has been warmly recommended as a fixative
for nuclei by VAN GEHUCHTEN (Anat. Anzeig., iv, 1889, p. 52). Fix for
five minutes, wash well with water and stain with methyl green, and exa-
mine in liquid of Ripart and Petit.

Heat has been recommended, but I believe it to be altogether objection-
able. HENKING (Zeit. f. wiss. Zool., xcix, 3, 1890, p. 503 ; Zeit. f. wiss.
Mik., vii, 2, 1890, p. 211) has found that it totally destroys achromatic
structures.

638. Chromatin Stains, — For fresh or lightly fixed tissues
methyl green is the most generally useful nuclear stain. For
the properties of this reagent see ante, § 109. It may be
used either alone, or in the form of Ehrlich-Biondi mixture
(see below).

Bismarck brown is another useful stain for such objects.
It may be used in aqueous solution with acetic acid or with
hydrate of chloral, or dissolved in dilute glycerin. Alum-
carmine may occasionally be useful. Ehrlich's ha3inatoxylin
or Mayer's haemalum will render services for osmium objects.
Methyl violet, employed according to the method of GRASEK
(§ 111), may also be found a very useful stain.

For sections of hardened tissues we have the choice be-
tween the finer liaeinatein stains (haemalum, or Ehrlich's or
Bohmer's hgematoxylin) and those obtained by means of
safranin, gentian violet, Victoriablau, and some other anilins,
used according to the indirect or Flemming's method. This
has been so fully explained in Chap. VIII that it is only
necessary to refer the reader back to the paragraphs in
question. Flemming's orange method has been given in § 258,
and the Ehrlich-Biondi mixture in § 259, and will be further
considered in the next section.

BABES'S supersaturated safranin stain (Arch. f. mik. Anat.,
xxii, 1883, p. 361) may also occasionally be useful. It is us
follows : — A supersaturated solution of safranin in water is
warmed to 60° C. and filtered warm. On cooling, it becomes
turbid through the formation of small crystals. Sections tire
placed in a watch-glass with some of this turbid solution, and
the whole is warmed for a few seconds (till the liquid becomes
clear) over a spirit-lamp. Allow the whole to remain for one
minute, and wash out with water, and treat with alcohol and
turpentine in the usual way. Tissues which do not take on
the stain at once must be warmed over and over again. Clove
oil must be avoided for clearing.

STAINS FOR PLASMA AND ACHROMATIC STRUCTURES. 335

Kenewed attention is called to OHLMACHER'S observations
on the employment of iodine or picric acid with safranin
(§ 101).

M. HEIDENHAIN'S iron haematoxylin stain has been described,
§ 197. It should be noted that the description of the results
given by Heidenhain applies to preparations fixed in subli-
mate, and will presumably not apply to chromic sections.

639. Stains for Plasma and Achromatic Structures. — Foremost
amongst these is the EHELICH-BIONDI mixture, described in
§ 259. To the account there given there remain to be added
some minutiae regarding the application of the stain to
cytological objects, particularly as regards the study of
attraction-spheres and other achromatic elements. M.
HEIDENHAIN (Uber Kern und Protoplasma, Engelmann, 1892,
p. 116; see also Zeit.f. wiss. Mile., ix, 2, 1892, p. 202, a very
full report) gives the following instructions. Preparations
made with the usual mixture are liable to fade; by acidifying
the mixture a stronger and more sharply selective stain is
obtained, which does not fade. But too much acid must not
be added, as this would cause a staining of the interfilar
substances. Heidenhain' s directions are as follows :

Take some of the "commercial stock solution" (by which
appears to be meant the mixture as given in § 259), and dilute
it with distilled water in the proportion of about 6 parts of
the mixture to 400 of water. Fill two beakers with distilled
water, and add to each a few drops of the diluted solution. The
coloured liquid thus obtained will show, at the same time, a
red tint owing to the rubin, a yellow tone owing to the
orange, and a greyish tone derived from the methyl green.
Now add to one of the beakers, with continual agitation, drop
by drop, a 1 : 500 solution of acetic acid until the colour of the
liquid turns to a strong crimson, the former yellow tone dis-
appearing, and the grey of the methyl green becoming less
marked. The two beakers serve as standards for the degree
of acidity that should be given to the staining bath. The
dilute solution that you made by diluting the original stock
solution* is now acidified with the dilute acetic acid, added
drop by drop with continual agitation, and from time to time

* " Das aus der Stanimlosung dureh Verdiinnung gewonnene Quantum."
The passage is somewhat misty.

336 CYTOLOGICAL METHODS.

a few drops are taken from it and added to a beaker of
distilled water until there is obtained the crimson tint of the
first test-beaker. Addition of acid should then cease, and
the staining bath is ready. If the preparations should not
turn out quite satisfactory a small further quantity of acid
may be added.

Before staining, sections should be treated for a couple
of hours with O'l per cent, acetic acid, then for ten to
fifteen minutes with officinal tincture of iodine, and be rinsed
with alcohol before bringing into the stain, in which they
should remain for twelve to eighteen hours. The treatment
with acid is necessary in order to ensure having the sec-
tions acid on mounting in balsam. The primary object of the
iodine is to remove any sublimate from the preparations
(Heidenhain's descriptions refer to sublimate objects, but
Ehrlich-Biondi mixture will work with chrome objects) ; but
it also enhances the power of staining of the chromatin with
methyl green, and produces a more selective staining of
protoplasmic elements.

Instead of acidifying with acetic acid, the mixture may be
modified by increasing the proportion of acid fuchsin, a sug-
gestion which is, I gather, also due to HEIDENHAIN (I do not
know whether it has been published). Dr. Griibler has sent
me a mixture of the three colours in the dry state in such pro-
portions that you have only to dissolve 0'4 grin, of the mixture
in 100 c.c. of water, and add 7 c.c. of a 0'5 per cent, solution
of Sauref uchsin . I greatly regret that I am unable to make
trial of this preparation before going to press.

BENDA ( Verh. ph ysiol. Ges. zu Berlin, Dec. 18th, 1891, Nos. 4
u. 5 ; see also Zeit.f. WISH. Mik., viii, 4, 1892, p. 516) proceeded
as follows (testes of Mammalia) : — Sections stained for twenty-
four hours in anilin- water safranin solution, then for about
half a minute in a solution of 0!5 grm. " Lichtgrim F. S." or
Saureviolett (Griibler) in 200 c.c. of alcohol, dehydrated, and
mounted in balsam. Chromatin red, archiplasma bright green
(or violet), centrosomata and " Spitzenknopf " (of spermato-
zoa) sometimes green, sometimes red. The chroinatoid
" Nebenkorper " of HERMANN stains red.

The achromatic structures of the ova of Axc<tri* are best
demonstrated, according to VAN BENEDEN and NEYT (None.
liech. sur la Fecund. < t La Uiv. mitorique), by fixing with acetic

CELL-GRANULES. 337

alcohol (ante, § 637), and bringing the ova into one- third
glycerin in which is dissolved a little malachite green. The
" spheres attractives" stain green. See also BOVERL'S Zellen-
Studien (in Jen. Zeitschr. f. Naturw., xxi, -1887, p. 423, or
sold separately).

The osmium and pyroligneous acid method of HERMANN has been de-
scribed in § 233. It is said to give a so abundant reduction of the osmium
that no other stain is necessary. In the same place HEBMANN also recom-
mends a modification of the haBmatoxylin impregnation method of PAL
(§ 692) : — Testes of Proteus put for twelve to eighteen hours into hsema-
toxylin, 1 grm. ; absolute alcohol, 70 c.c. ; water, 30 c.c., in the dark, and
washed out for the same time in 70 per cent, alcohol, also in the dark. Sec-
tions having been made are treated with dilute pale rose-coloured solution
of permanganate of potash till they turn ochre-coloured, rinsed in water,
and washed out for removal of the brown peroxide of manganese in Pal's
oxalic acid mixture diluted with 5 to 10 volumes of water. They are finally
stained for three to five minutes in safranin.

The iron-haematoxylin stain of BUTSCHLI and that of M.
HEIDENHAIN have been given in §§ 196, 197.

For BENDA'S copper-hasmatoxylin, see Arch. f. mik. Anat., xxx, p. 49 ;
also Journ. Boy. Mic. Soc.. 1888, p. 158, or Zeit. /. wiss. Mik., v, 1888,
p. 499.

FLEMMING'S orange method has been given § 258.

HENNEGUY'S permanganate method has been given § 106.

RABI/S method (platinum chloride, hsematoxylin, safranin).
—See Morph. Jahrb., x, 1884, p. 215.

PLATNER'S. — See Arch. f. mik. Anat., xxxiii, 1889, p. 125.
His Kernschwarz has been discussed in § 208.

640. Nucleus of BALBIANI (" Noyau Vitellin," " Cellule Embryo-
gene") (Zool. Anz., 1883, p. 659).— This may be observed in the fresh state,
without the addition of any reagent, in the ova of some animals, amongst
others a great number of Arachnida and Myriapoda. It may be brought out
more distinctly by treating the ova with a mixture of equal parts of acetic
acid and 1 per cent, osmic acid, to which is added a little sodium chloride.
This mixture does not render ova so granular as pure dilute acetic acid.

641. Cell-granules. — These for the most part undoubtedly
metabolic products are best studied in gland-cells and blood-
and lymph-corpuscles, and in certain elements belonging to
the group of connective tissues, and the reader is therefore
referred to Chap. XXXI for the appropriate methods.

For ALTMANN'S "Bioblasts," see, besides that author's

22

338 CYTOLOGICAL METHODS.

Studien iiber die Zelle, 1886, his Die Elementarorganismen,
Leipzig, 1890, and a paper in Arch. f. Anat. u. 'EntwickeL,
1892, p. 223; also Zeit.f. wiss. Mik., vii, 2, 1890, p. 199 ; ix,
3, 1893, p. 331; and L. and R. ZOJA, in Mem. R. 1st. Lorn-
bardo di Sci. e Lettere, xvi, 3, vii, p. 237. Omitting minutiae
and variations, it may be said that these granules may be
demonstrated by fixing for twenty-four hours in a mixture of
equal parts of 5 per cent, bichromate of potash and 2 per cent,
osmic acid, imbedding in paraffin, staining sections for a
minute on the slide held over a flame with a solution of 20
grms. of acid fuchsin in 100 c.c. of anilin water (§ 101), and
washing out with saturated alcoholic solution of picric acid
diluted with 2 volumes of water, heat being used as before to
aid the differentiation, and finally clearing with xylol and
mounting in balsam.

642. Mounting. — For fresh objects you have so large a
choice of mounting media that you may take whatever liquid
gives you the best optical results. Sections of hardened tissue
should always (if possible) be mounted in damar (or colopho-
nium), not balsam, as the slightly lower index of damar or
colophonium solutions gives more powerful images of very
delicate details. If you have to deal with objects so delicate
that you have cause to fear mechanical injury to them on
putting them into damar, they may be mounted in thickened
turpentine (or cedar oil, but turpentine is preferable on ac-
count of its lower index). Rabl has lately been using methyl
alcohol as an examination medium for dehydrated objects;
but the preparations do not keep in this. Castor oil may be
tried (GRENACHER). I have not had good results with it.

643. Other General Cytological Methods.
STRASBURGER'S Methods (see Arch.f. mik. Anat., xxi, 1881, p. 477).
USKOFF'S Nitric Acid Method (see Arch. f. mik. Anat., xxi, 1882,

p. 292).

PFITZNER'S Sulphate of Soda Method (see Morpliol. Jahrb., xi, 1885,
p. 54).

TIZZONI (Bull delle Sci. Med. di Bologna, 1884, p. 259).

BAUMGARTEN (Zeit.f. wiss. Mik., i, 1884, p. 415).

BIZZOZERO (ibid., iii, 1886, p. 24).

BABES (ibid., iv, 4, 1887, p. 470).

ZWAARDEMAKER (ibid., iv, 2, 1887, p. 212).

NISSEN (Arch.f. mik. Anat., 1886, p. 338) stains material fixed in Flem-
ming and sectioned, by the method of GRAM (see above, § 102).

SPERMATOLOGICAL METHODS. 339

SCHOTTLANDEE (Arch, f. mik. Anat., xxxi, 1888, p. 426 ; Zeit. f. wiss.
MiJc., v, 4, 1888, p. 515 — cell-division in the corneal endothelium).

644. Cytology of the Ovum (Ascaris).

VAN BENEDEN (Arch, de Biol., iv, 1883, p. 279).

CAENOY (La Cellule, ii, 1, 1886, pp. 17, 18 ; iii, 1, 1886, p. 6 ; iii, 2,
1887).

VAN BENEDEN et NEYT (Nouv. Rech. sur la Fee., &c., separate, 1887, or
Bull. Acad. roy. d. Sc. de Bruxelles, 1887, iii ser., xiv ; also Journ. Roy.
Mic. Soc., 1888, p. 508).

BOVEEI (Zellen-Studien, in Jen. Zeit. f. Naturw., xxi, 1887, p. 423, or
separate ; also Journ. Roy. Mic. Soc., 1888, p. 664).

ZACHABIAS (Anat. Anz., iii, 1888, p. 24 ; also Journ. Roy. Mic. Soc., 1888,
p. 663).

VAN GEHTJCHTEN (ibid., p. 237).

KULTSCHITZKY (Arch. f. mik. Anat., xxxi, 1888, p. 567) recommends for
fixing a mixture of 3 parts of acetic ether with 1 part of absolute alcohol.
Clears for mounting in concentrated acetic acid, and mounts in a mixture of
acetic acid and balsam. An excellent resume of the technical part of these
papers is to be found in Zeit.f. wiss. Mik., v, 3, 1888, p. 367.

Other Ova.

HEETWIG, 0. (Morph. Jahrb., x, 1884, p. 338, Rana).

HEETWIG, 0. and E. (Jen. Zeit. f. Naturw., xix, 1885, p. 124).— Fecunda-
tion of Strongylocentrotus (Echinodermata). Careful study of cell-anaes-
thetics. Abstracts in Zeit. f. wiss. Mik., iii, 4, 1886, p. 505, and Journ.
Roy. Mic. Soc., 1887, p. 835.

BOVEEI (Zellen-Studien, iii [Echinodermata], in Jen. Zeit. f. Naturw.,
xxiv, 1890, p. 314, or separate).

645. Spermatological Methods. — One of the great difficulties here
met with consists in getting a sufficiently rapid fixation of spermatids and
spermatozoa, some of which are probably the most rapidly contracting ele-
ments that exist. For fresh material, teased on the slide, strong solution of
permanganate of potash is very useful, being the most rapidly fixing agent
that I know of. Tincture of iodine is also useful. For staining, gentian
violet or dahlia should be used as far as possible, as they have a special
affinity for spermatic cells. For mounting aqueous preparations, solution
of Bipart and Petit, or some such mercurial fluid as those of Pacini, are in-
dicated.

As to the Nebenkern, which is here so important an element, see ante,
§639.

Besides the papers quoted for cytological methods, and besides the nume-
rous publications of v. LA VALETTE ST. GEOBGE, in Arch. f. mik. Anat., see
the following :

CAENOY (La Cellule, i, 1885, p. 209, Arthropoda).

GILSON (ibid., i, 1885, pp. 40, 56, 58, 87, 121, 141, and ii, 1886).— Here is
a useful fixing medium for fresh teased preparations — iodised osmic acid

340 CYTOLOGICAL METHODS.

made by adding a little of a concentrated solution of iodine in iodide of
potassium to 2 per cent, osmic acid ; and some other formulae useful for
their special objects (most various forms of Arthropoda).

SWAEN ET MASQUELIN (Arch, de BioL, iv, p. 752). — Selacians, Sala-
mandra, Bos.

FLEMMING (Arch. f. mik. Anat., xxix, 1887, p. 387 ; abstracts in Zeit. f.
wiss. Mik., v, 2, 1888, p. 236, and Journ. Roy. Hie. Soc., 1888, p. 146).

PBENANT (Intern. Monatschr.f. Anat., iv, 1887, p. 358 ; abstracts in Zeit.
f. wiss. Mik., v, 1, 1888, p. 84 ; and Journ. Roy. Mic. Soc., 1888, p. 844
[Mammalia] ).

KENSON (Arch, de BioL, iii, 1882, p. 302).

BENDA (Arch. f. Anat. u. Phys. ; Phys. Abth., 1886, p. 186).— For his
modification of Weigert's hsematoxylin stain, see below.

JENSON (Arch, de BioL, iv, 1883, p. 11, et passim [Selacians and Inverte-
brates] ). — Concentrated solution of oxalic acid for hardening.

PICTET (Mitth. a. d. zool. Stat. zu Neapel, Bd. x, Hft. 1, 1891, p. 89).—
Instructive series of figures showing the very different forms assumed by
spermatozoa of Strongylocentrotus under treatment by twenty -three different
reagents. The most faithfully-preserving examination fluid was found to
be a 5 to 10 per cent, solution of chloride of manganese containing a small
quantity of dahlia dissolved in sea water.

For some reactions of divers stains on the nucleus and Nebenkem of
spermatozoa, see LECLERCQ, Bull. Acad. Roy. Belg., Ix, 1890, p. 138;
Journ. Roy. Mic. Soc., 1890, p. 816.

BALBIANI (Leg. sur la Gener. des Vertebres, 1879, p. 244). — Double-stain
sections with picro-carmine and methyl green. The latter stages of differ-
entiating cells stain blue, less advanced ones lilac ; and the cells which form
the walls of the tubes stain only red.

KYDEE, in WHITMAN'S Meth. of Research, p. 52. — Sections of hermaphro-
dite gland of Lamellibranchiata stained for two or three hours in a mixture
of equal parts of concentrated alcoholic solution of safranin and methyl
green, diluted with 8 volumes of water, washed out for five to ten minutes
in alcohol, and mounted in balsam. Nuclei of ova, red ; heads of sperma-
tozoa, bluish green.

If sections of a gland of Helix be stained with methyl green and eosin,
the ova are stained rose colour, and the spermatozoa bluish green (HENNEGUY,
in Traite des Meth. Techn., LEE et HENNEGUY, p. 343).

AUEBBACH (Sitzb. k. Acad. Wiss. Berlin, xxxv, 1891, p. 713 ; see Journ.
Roy. Mic. Soc., 1891, p. 714 ; Zeit.f. wiss. Mik., ix, 1, 1892, p. 81) has made
some observations which seem to point to a qualitative chemical difference in
the composition of the nuclei of male and female sexual products. Sections
of male and female sexual products, prepared in precisely the same way,
were fixed together on slides and stained with pairs of the following dyes
(used either mixed or in succession) :

SPERMATOLOGICAL METHODS. 341

Blues. Reds.

Methyl green. Carmine.

Emerald green. Eosin.

Victoria blue. " Echtroth."

Methylen blue. Fuchsin.

(Sometimes) Haematoxylin. Orange,

Orange with fuchsin.

Rosanilin sulphate.

It was found that the chromatin (and also the nucleoli) of the female ele-
ments constantly took the red stain, whilst that of the male elements con-
stantly took the blue stain. Auerbach constantly arrives at the generalisa-
tion that female chromatin is erythrophilous, male chromatin cyanophilous.

His procedure for staining was to make aqueous solutions of the two
colours of approximately equal intensity of tint, and either to stain in them
successively or to mix equal volumes of them for a staining bath.

The subject appears to call for further research.

342 TEGOMENTARY ORGANS.

CHAPTER XXVII.

TEGUMENTARY ORGANS.

646. Epithelium. — One of the chief methods of obtaining
preparations giving" instructive surface views of epithelia is
the nitrate of silver method. For this see ante, § 212, et seq.,
in the chapter on " Impregnation Methods." The reader
may also consult with advantage the admirable instructions
given by RANVIER in his Traite technique, p. 246, et seq.,
and the memoir of TOUENEUX and HERMANN in the Journ.
de I'Anat., 1876, p. 200.

The perchloride of iron and pyrogallic acid method of the
HOGGANS, § 281, and the osmic acid and pyrogallic acid pro-
cess, § 233, may also be found useful here. But in many
cases impregnation with methylen blue will doubtless be
found preferable (see § 113, et seq., and especially §§ 120 and
121).

KKO MAYER'S process for demonstrating his intra- and inter-
cellular fibrils of epithelia (Arch.f. mik. Anat., xxxix, 1892,
p. 141 ; Zeit.f. wiss. Mik., ix, 1, 1892, p. 84, and ix, 3, 1893,
p. 355) is as follows : — Sections are stained for five minutes
in a mixture of equal volumes of anilin water (§ 101) and
concentrated aqueous solution of methyl violet 6 B. They are
well washed in water, and treated with solution of iodine in
iodide of potassium until they become blue-black (one to
thirty seconds). They are again washed with water, dried
with blotting-paper, and treated with a mixture of 1 vol. of
anilin to 2 vols. of xylol until sufficiently differentiated, when
they are brought into pure xylol. Very thin sections will
require more xylol in proportion to the anilin, viz. 1 : 3 or
1:4; thicker ones may require more" anilin, viz. 3:5 or 3:3.
Gentian or Krystallviolett will do instead of methyl violet,
but not quite so well. For a discussion of the points of dif-
ference between the fibrils of Kromayer and the fibres of
HERXHEIMER, see EHRMANN, and JADASSOHN, Arch. f. Der-

EPITHELIUM. 343

matol. u. Syphilis, 1892, 1, p. 303; Zeit. f. wiss Mils., ix,
1893, p. 356.

Sections are easily made by the usual methods. The best
hardening agent for skin appears to be Mailer's solution.
This was the conclusion of F. E. SCHULTZE in 1867 (Arch. f.
mik. Anat., p. 145), and it is that of TIZZONI, the author of
important researches on this organ (Bull, delle Sc. med. di
Bologna, 1884, p, 259), and of BEEN (Arch. f. mik. Anat.,
xxxix, 1892, p. 581). Simple bichromate of potash solution
will do about as well.

For glandular epithelium it is frequently better to employ
a chromic acid liquid, or osmic acid (see, for example, KANVIER,
loc. cit., p. 258, et seq.), or absolute alcohol (BLAUE, Arch. f.
Anat. u. Phys., 1884, p. 231) ; "Kleinenberg" is not so good.

Prickle-cells and Intercellular Canals. — Besides maceration,
which is one of the most important of the methods for the
study of these objects, impregnation may be useful. MITRO-
PHANOW (Zeit. f. wiss. ZooL, 1884, p. 302, and Arch. f. Anat.
u. Phys., 1884, p. 191) recommends the following process : —
Wash with distilled water the tail of an axolotl larva ; put it
for an hour into 0*25 per cent, solution of gold chloride with
one drop of hydrochloric acid to a watch- glassful of the solu-
tion ; wash, and reduce in a mixture of one part of formic
acid with six parts of water.

Macerating Media. — For soft epithelia, mild macerating
agents, such as iodised serum, one-third alcohol, saliva, or
Schultze's mixture of saliva and solution of Miiller, or a
mixture of saliva with three to four volumes of physiological
salt solution (BIZZOZERO, Intern. Monatschr. f. Anat., 1885, p.
278) ; — for hard epithelia, energetic dissociating agents, such
as 40 per cent, solution of caustic potash.

MINOT (Amer. Natural., xx, 1886, p. 575 ; cf. Journ. Roy.
Mic. Soc., 1886, p. 872) recommends maceration for several
days in 0'6 per cent, solution of sodium chloride containing
O'l per cent, of thymol, which allows the isolation of the
epidermis of embryos, and is useful for the study of the
development of hairs.

Another method, given by MITROPHANOW (see Zeit. f. wiss.
Mik., v, 4, 1888, p. 513), is as follows : — An embryo of axo-
lotl is fixed for a quarter of an hour in 3 per cent, nitric acid,
and then brought into one-third alcohol. After an hour the

344 TEGUMENTARY ORGANS.

epidermis begins to come away in places ; and if the embryo
be put for twenty-four hours into stronger spirit, it will come
away almost entirely.

For the purpose of separating the epidermis from the
^orium, LOEWY (Arch. f. mik. Anat., xxxvii, 1891, p. 159;
Zeit.f. wiss. Mik., viii, 2, 1891, p. 222) recommends macerat-
ing for twenty-four to forty-eight hours, at a temperature of
about 40° C., in 6 per cent, pyroligueous acid. Acetic acid
of ^ per cent. (PHILIPPSON) is also good.

The elements of hairs and nails may be isolated by pro-
longed maceration in 40 per cent, potash solution, or by
heating with concentrated sulphuric acid.

Horny tissues stain well in safranin or gentian violet
.(REINKE, Arch.f. mik. Anat., xxx, 1887, p. 183 ; Zeit.f. wiss.
Mik., iv, 3, 1887, p. 383).

647. Tactile Hairs. — RANVIER (Traite, p. 914) recommends
for the study of the nerve-endings the boiled formic acid and
gold-chloride method, § 225. A tactile hair having been
isolated with its bulb, and its capsule incised, is put for about
an hour into the formic acid and gold-chloride mixture, the
gold is reduced in slightly acidulated water, hardening is
completed in alcohol, and longitudinal and transverse sections
are made. For DRASCH'S method see Zeit. f. wiss. Mik., iv, 4,
1887, p. 492.

648. Skin Nerves. — WOLTERS (Arch. f. Dermatol. u. Syphilis,
1892 ; Zeit. f. wiss. Mik., ix, 3, 1893, p. 360) employs the
haematoxylin method, § 726, post.

649. Intra-epidermic Nerve-fibres. — May be studied by the
gold method. EANVIER (Traite, p. 900) recommends the
boiled formic acid and gold-chloride method, § 225.

He also (p. 910) recommends this method for the study of
the tactile menisci of the pig's or mole's snout.

Pieces of skin are impregnated as directed § 225, and
after reduction are brought into alcohol, which completes the
hardening, and stays the further reduction of the gold.
Sections are then made.

For the study of the tactile menisci of the snout, Ranvier
also recommends the lemon juice and gold-chloride method,
§226.

CORPUSCLES OF HERBST AND GRANDRY. 345

The methylen blue impregnation method, § 113, et seqq.,
should also be employed in the study of these nerve-endings.

650. Innervation of the Muzzle of the Ox (CYBULSKY, Zeit. f.
wiss. ZooL, 1883, p. 635; cf. Journ. Roy. Mic. Soc., 1885, p.
555). — Cybulsky employs the method of HENOCQUE given
above, § 228.

651. Tactile Corpuscles (FISCHER, Arch. f. mik. Anat., 1875,
.p. 366). — Fischer employed the gold method of Lowit — see

§ 224. Kanvier (Traite, p. 918) also recommends this method,
as well as his two gold methods, §§ 225, 226. Pieces of skin
are first impregnated whole, then hardened by alcohol and
sectioned. He finds (as do other authors) that osmic acid
and picro-carmine are invaluable aids to the study of these
structures, and to that of the corpuscles of Pacini. Purpurin
and hsematoxylin may also be used for after-staining. See
RANVIEE, Traite, p. 919; and see also LANGERHANS, Arch,
f. mik. Anat., 1873, p. 730; and KULTSCHIZKY, ibid., 1884,
p. 358.

652. Corpuscles of Herbst and Corpuscles of Grandry ( CARRIERE,
Arch.f. mik. Anat., 1882, p, 146) .—Take fresh beaks of ducks,
remove the skin and papilla3 from the margins, and put pieces
into formic acid (50 per cent.) for twenty minutes or until
transparency is attained ; remove the corneous layer of epi-
thelium; rinse in water; gold chloride 1 per cent, (twenty
minutes) ; rinse in water ; Pritchard's solution (amyl-alcohol
1 per cent., formic acid 1 per cent., water 98 per cent.) from
midday till next morning (in the dark) ; rinse in water ; treat
with alcohol, imbed in paraffin, and make sections.

It is important to take only small quantities of gold
chloride, not more than about 10 c.c. of the solution to
" quite a number" of pieces of skin and papillae. On the
other hand, large quantities of Pritchard's solution should be
employed.

DOGIEL (Arch. f. Anat. u. Entwickel, 1891, p. 182; Zeit. f.
wiss. Mik., viii, 4, 1892, p. 520) prefers the methylen blue
method (Chap. IX). Four per cent, solution of methylen
blue, warmed to 40° C., is injected into blood-vessels of the
heads of ducks or geese ; pieces of skin are removed from the
beaks, sectioned in pith, and the sections brought on to slides

346 TEGUMENTARY ORGANS.

and moistened with aqueous or vitreous humour from the
animal, and left for a few minutes exposed to the air (it is
well to add to the aqueous or vitreous humour a few drops of
'TIT Per cent, methyl blue solution). After about ten to
thirty minutes the nerve-endings are seen to be stained, and
the sections are then brought into picrate of ammonia, and
treated as described in § 119.

653. Corpuscles of Krause (Cornea and Conjunctiva bulbi)
(DoGiEL, Arch.f. mik. Anat., xxxvii, 1891, p. 602; Zeit. f. wiss.
Mik., viii, 4, 1892, p. 519). — A fresh bulb should be enucleated
in toto, and incised along a line running parallel to the equator
and 5 to 8 mm. behind the corneal margin.- The anterior
segment thus obtained is freed from the ciliary body, lens,
&c., the conjunctiva being left in situ on the cornea. It is
then cut into pieces which are stained for an hour to an hour
and a half in inethylen blue -f aqueous humour, as described
in § 117, and further treated for examination and preserva-
tion as described in § 119.

See also LONGWORTH'S methods, Arch. f. mik. Anat., 1875,
p. 655.

654. Papillae Foliatae of the Babbit (HERMANN).— See Zeit. f.
wiss. Mik., v, 4, 1888, p. 524.

655. Olfactive Organs of Vertebrates (DooiEL, Arch. f. mik.
*Anat., 1887, p. 74).

656. Organs of a "Sixth Sense" .in Amphibia (MITROPHANOW).
— See Zeit.f. wiss. Mik., v, 4, 1888, p. 513. This paper con-
tains some details as to staining with " Wasserblau," for
which see also Biol. Centralb., vii, 1887, p. 175.

657. Nerve-endings in Tongue of Frog (FAJERSTAJN [FEUER-
STEIN], Arch, de Zool. exper. et gen., vii, 1889, p. 705; Zeit.
f. wiss. Mik., vii, 3, 1890, p. 357). — Amongst other methods
for the study of the terminal discs, the methylen blue method
is recommended. Solution of 1 part methylen blue to 800 of
0*6 per cent, salt solution should be injected through the
abdominal vein (not through the vena cutanea magna), the
animal being curarised or etherised. After injecting, care
must be taken that air has access to the mouth. Injection
through the dorsal lymph-sac will also succeed, and simple

COENEA. 347

pouring of the stain into the mouth sometimes gives good
results. See also Chap. IX.

658. Cornea. — There are three chief methods for the study of
the corneal tissue — the methylen blue method, the silver
method, and the gold method.

For the methylen blue method see Chap. IX, particularly
§§ 120 and 121.

Negative images of the corneal cells are easily obtained by
the dry silver method (KLEIN) . The conjunctival epithelium
should be removed by brushing from a living cornea, and well
rubbing the corneal surface with a piece of lunar caustic.
After half an hour the cornea may be detached and examined
in distilled water.

In order to obtain positive images of the fixed cells the
simplest plan (RANVIER) is to macerate a cornea that has been
prepared as above for two or three days in distilled water.
There takes place a secondary impregnation, by which the
cells are brought out with admirable precision.

The same result may be obtained by cauterising the cornea
of a living animal as above, but allowing it to remain on the
living animal for two or three days before dissecting it out,
or by treating a negatively impregnated cornea with weak
salt solution or weak solution of hydrochloric acid (His) .

But the best positive images are those furnished by gold
chloride. RANVIER prefers his lemon-juice method to all
others for this purpose (see § 226). It is important that the
cornea should not remain too long in the gold solution, or the
nerves alone will be well impregnated.

Ranvier also recommends this method as being the best for
the study of the nerves.

ROLLETT (Strieker's Handbuch, p. 1115) recommends a
double impregnation with silver followed by gold for obtaining
gold-stained negative images. A cornea having been treated
for a short time only with O5 per cent, silver nitrate solution,
and the silver reduced, is treated with 0'5 per cent; gold-
chloride solution. The brown stain of the silver disappears
immediately the preparation is placed in the gold solution ;
after a few minutes the preparation is exposed to the light in
acidulated water. Reduction of the gold rapidly takes place,
and in the place of the former brown stain of the silver the

348 TEGUMENTAKY OEGANS.

ground-substance shows the well-known blue of reduced gold.
The cells are, however, visible, being recognisable by their
granular appearance and pale yellow tint.

KENAUT (Gomptes Rend., 1880, ler sem., p. 137) gives the
following process for corneal corpuscles : — Cornea of frog:
formic acid, 20 per cent., ten minutes ; gold chloride, 1 per
cent., twenty-four hours; formic acid, 33' 3 per cent., twenty-
four hours.

659. Cornea, other Methods (ROLLETT, Strieker's Handb., p.
1102). — Rollett strongly recommends the following plan : — A
fresh cornea is placed (in humor aqueus) in a moist chamber,
and exposed to the action of iodine vapour. As soon as it has
become brown the epithelium may easily be peeled off. If the
reaction is not complete the cornea may be put back into the
iodine chamber. When sufficient iodine has been absorbed
the preparation may be examined, and it will be found that
the network of corneal cells is brought out with an evidence
hardly inferior to that of gold preparations. The method
never fails, which is not the case with the gold method. It
is admirable as a fixing method.

For dissociation of the fibres Rollett recommends macera-
tion in a solution of permanganate of potash or a mixture of
this solution with alum. As soon as the tissue has become
brown it is shaken in a test-tube with water, and breaks up
into fibres and bundles of fibres.

TARTUFERI (Anat. Anz., v, 1890, p. 524; Zeit.f. wiss. Mik.,
vii, 3, 1890, p. 365) has the following method for demonstrat-
ing corneal cells and the ramifications of their processes : — A
cornea is placed for three or more days in a solution of 15
grammes of hyposulphite of soda to 100 c.c. of distilled water,
kept at a temperature of about 26°, then removed for two
days into water containing very finely powdered chloride of
silver (if left longer the corneal cells will not be stained, but
innumerable elastic fibres will be demonstrated). The pre-
parations are said to be permanent.

For cell-division in the membrane of Descemet, see SCHOTT-
LANDER'S methods, ante, § 643.

660. Crystalline (Hardening of) (LowE, Arch.f. mik. Anat.,
1878, p. 557). — A fresh bulb is placed in a vessel containing

CRYSTALLINE (HARDENING OF). 349

several litres of 1 per cent, bichromate of potash solution,
which is frequently changed for stronger solutions until the
strength of a cold saturated solution is attained. The bulb
must remain in this for at least a year and a half, in order
that the crystalline may attain the right degree of hardness.
For Maceration, use Max Schultze's sulphuric acid solution,
supra, § 546.

350 MUSCLE AND TENDON.

CHAPTER XXVIII.

MUSCLE AND TENDON (NERVE-ENDINGS).
Striated Muscle.

661. Sections (ROLLETT, Denkschr. math, naturw. Kl. k. Acad.
Wiss. Wien, 1885; Zeit. f. iviss. Mik., 1886, p. 92).— Besides
the usual section methods, the following methods of Rollett
should be noted: — (1) The method mentioned above, § 321,
of freezing living tissue in white of egg. (2) The same
method applied to recently fixed muscle. (3) For hardened
muscle, Rollett prefers celloidin. He stains for several hours
in Renaut's haematoxylic glycerin diluted with water to a
very light violet tint. Dehydrate with alcohol, clear with
origanum oil, and mount in dammar.

662. Dissociation.-— See Chap. XXIII.

LANGERHANS' methods for Amphio.vus (Arch.f. mik. Anat., 1875, p. 291).
— For isolation of the muscle-plates macerate the fresh animal in 20 per
cent, nitric acid.

For isolation of the nervous system macerate an animal for three days in
20 per cent, nitric acid, then place it for twenty-four hours in water, and
shake forcibly. The whole of the nervous system may thus be separated,
almost down to the finest peripheral terminations of nerves.

663. Muscle-cells. — For the study of these and allied sub-
jects see, inter aliat BEHRENS, KOSSEL, und SCHIEFFERDECKER,
Das Mikroskop, &c., vol. ii, pp. 154 — 161 ; also, for the ap-
plication of the gold method to the study of muscle-cells,
SCHAFER, Proc. Roy. Soc., xlix, 1891, p. 280; or Journ. Roy.
Mic. Soc., 1891, p. 683.

664. Sarcolemma. — Besides the places quoted in last section,
see SOLGER, Zeit. f. wiss. Mik., vi, 2, 1889, p. 189 (small
pieces of fresh muscle teased and examined in cold saturated
solution of carbonate of ammonia).

NERVE -END INGS. 351

665. Nerve -endings. — For the study of nerve-endings in
muscle, both motor and sensory, the four chief methods are
the methylen-blue method, the gold method, the silver method,
and the bichromate of silver method of Golgi.

666. Nerve-endings — the Methylen-blue Method. — The prin-
ciples of the impregnation of nerve-tissue with methylen blue
have been explained in Chap. IX.

BIEDERMANN'S procedure for the muscles of Astacus has been
indicated in § 117 (see also Zeit. f. wiss. Mik., vi, 1, 1889, p.
65). After impregnating as there directed the carapace
should be opened, and the muscles exposed to the air in a
roomy moist chamber for from two to six hours, in order that
the stain may differentiate. The abdominal and caudal
muscles are those which give the best results.

For Hydrophilus piceus, Biedermann proceeded by injecting
O5 c.c. of methylen-blue solution between the ultimate and
penultimate abdominal rings, in the ventral furrow, and keep-
ing the animals alive in water for three to four hours. After
this time the thorax should be opened by two lateral incisions,
and the muscles of the first pair of legs (which are the most
suitable) removed and exposed to the air for three or four
hours in a moist chamber, and finally examined in salt
solution.

GERLACH (Sitzb. k. math.-phys. Cl. k. bayer. Akad. Wiss.
Munchen, 1889, ii, p. 125; Zeit. f. wiss. Mik., vii, 2, 1890, p.
220) injected frogs, either through the abdominal vein or
through the aorta, with 4 to 5 c.c. of a 1 : 400 solution in 1
per cent, salt solution, and examined pieces of muscle (pre-
ferably the head and eye muscles) in serum of the animal,
afterwards fixing the preparations with picrate of ammonia
and mounting in glycerin jelly.

The procedure of DOGIEL has been given in § 117.

667. Nerve-endings — the Gold Method. — FISCHER (Arch. f.
mik. Anat., 1876, p. 365) used the gold method proposed by
LOWIT ( Wien. Sitzgsber., Bd. Ixxi, Abth. 3, 1875, p. 1), and
employed by himself in his researches on the tactile cor-
puscles ( Arch. f. mik. Anat., xii, p. 366) . See ante, § 224.

BIEDERMANN, in the paper quoted in the last section, recom-
mends for Astacus a similar procedure, the preliminary treat-

352 MUSCLE AND TENDON.

ment with formic acid being omitted, and the muscles being put
for a couple of days into glycerin after reduction in the acid.

The procedure of TRINCHESE (Mem. R. Accad. 1st. Bologna,
5, ii, p. 279; Zeit.f. wiss. Mik., ix, 2, 1892, p. 238) is also
practically identical.

RANVIER (Traite, p. 813) finds that for the study of the
motor terminations of Batrachia the best method is his lemon-
juice and gold-chloride process (§ 226). The delicate elements
of the arborescence of Kiihne are better preserved by this
method than by the simple method of Lowit.

For the study of the motor plates of reptiles, fishes, birds,
and mammals, he finds (ibid., p. 826) that his formic acid and
gold- chloride method, § 225, gives preparations infinitely
superior to those obtainable by the method of Lowit ; but the
lemon-juice method is still better, especially for lizards and
mammals. The branches of the terminal arborescence are
more regular than in preparations obtained by the formic
acid process.

668. Nerve-endings — the Silver Method. — RANVIER finds that
the silver nitrate method of Cohnheim is also useful. He
employs it as follows (ibid., p. 810) : — Portions of muscle
(gastrocnemius of frog) having been very carefully teased out
in fresh serum are treated for ten to twenty seconds with
nitrate of silver solution of 2 to 3 per 1000, and exposed to
bright light (direct sunlight is best) in distilled water. As
soon as they have become black or brown they are brought
into 1 per cent, acetic acid, where they remain until they have
swelled up to their normal dimensions (the swelling induced
by the acid serving to make up for the shrinkage caused by
the nitrate of silver) . They are then examined in a mixture
of equal parts of glycerin and water.

This process gives negative images, the muscular substance
is stained brown, except in the parts where it is protected by
the nervous arborescence, which itself remains unstained.
The gold process gives positive images, the nervous structures
being stained dark violet.

669. Nerve-endings — the Bichromate of Silver Method. — The
osmium bichromate and silver method of Golgi has been suc-
cessfully applied by RAMON Y CAJAL to the study of the ter-
minations of nerves and of tracheae in the muscles of insects,

NERVE-ENDINGS. 353

and is doubtless susceptible of still wider applications. The
process used by him is given below, § 714.

670. Nerve-endings — other Methods.

BEEMER (Arch. f. mik. Anat., 1882, p. 195). — Impregnate according to
the method of Fischer, ante, § 224, and soak for two or three weeks in
glycerin containing 20 per cent, of formic acid. Mount in glycerin contain-
ing 1 per cent, of formic acid.

CIACCIO (Joum. de Micrographie, 1883, p. 38). — Lemon'juice followed
by 1 per cent, solution of double chloride of gold and cadmium, and reduc-
tion partly in the light and partly in concentrated formic acid in the dark.

WOLFF (Arch. f. mik. Anat., 1881, p. 355). — Study of the living muscle
in salt solution.

GAEL SACHS (ibid.}. — Dilute acetic acid followed by very weak picric acid
for twenty-four hours. After mounting in glycerin, this gives very trans-
parent preparations.

KEAUSE (Intern. Monatschr. f. Anat. u. Hist. ; Zeit. f. wiss. Mik., 1885,
p. 547). — A muscle is put for three or four hours into concentrated solution
of oxalic acid, then boiled for two minutes in water, treated for twenty-four
hours with O'l per cent, osmic acid, and mounted in glycerin.

NEGEO (Zeit.f. wiss. Mik., v, 2, 1888, p. 240).— Make the following solu-
tion :

Concentrated solution of ammonia alum . . 180 parts.
Griibler's saturated alcoholic solution of hsema-

toxylin ........ 2 ,,

Mix, and let the mixture stand for a week exposed to the air, and add 25
c.c. each of methyl alcohol and glycerin. A drop of this is brought on to a
fresh preparation of muscle of Tropidonotus natrix, Lacerta viridis, or
Rana, and in a few minutes the stain is washed out and the preparation
mounted in balsam.

BOCCAEDI (see Zeit. f. wiss. Mik., iv, 4, 1887, p. 492). — Preparations
treated according to Ranvier's lemon-juice method or his formic acid method
are washed in distilled water, and put for a couple of hours into oxalic acid
of from O'l to 0'25 or 0'30 per cent., or, better, into a mixture of —
Pure formic acid ....... 5 c.c.

1 per cent, oxalic acid 1 „

Distilled water 25 „

Then washed and mounted in glycerin.

KUHNE (Zeit.f. Biol., xxiii, v, 1887, p. 1 ; Zeit.f. wiss. Mik., iv, 4, 1887,
p. 495). — This paper contains a critical review of the different gold methods,
of which the following are the principal conclusions :

(1) The method of Lowit is particularly applicable to thin entire (un-
teased) muscles. ,

(2) Preliminary treatment with weak (0'5 per cent.) formic acid, and re-
duction in dilute glycerin containing one-fourth to one-fifth volume of
formic acid, in the dark, is a good process for muscles of warm-blooded
animals.

23

354 MUSCLE AND TENDON.

(3) The same process, with omission of the preliminary acidification, is to
be recommended for cold-blooded animals.

(4) The method of GOLGI (Mem. delle R. Accad. di Sci. di Torino, ii,
32) is applicable to all classes of objects. It is as follows : — Acidification
with 0'5 per cent, arsenic acid, impregnation in 0*5 per cent, solution of
double chloride of gold and potassium, and reduction in 1 per cent, arsenic
acid in sunlight.

(5) A modification of the last (apparently due to MATS) consisting in im-
pregnation in a mixture containing 0*5 per cent, arsenic acid, O25 per cent,
chloride of gold and potassium, and O'l per cent, osmium, and reduction as
before, is stated to be particularly applicable to Reptilia. (This process has
been further modified by MARSHALL [Quart. Journ. Mic. Sci., 1890, p. 73 ;
Journ. Roy. Mic. Soc., 1890, p. 404], who impregnates [after previous im-
mersion for a few seconds in 1 per cent, acetic acid] in a mixture of 20 parts

1 per cent, acetic acid, 4 parts 1 per cent, gold chloride, and 1 part 1 per
cent, osmic acid. He had found that the arsenic acid of Mays' formula dis-
integrated the muscle-fibre.)

Kiihne also remarks that teasing ought to be done in the gold solution.
Specimens should be removed from the gold every few minutes, and from
the reducing medium every hour, so that the right duration both of impreg-
nation and reduction may be hit off. Preliminary acidification is unfavor-
able to the preservation of the arborescence, and the treatment with acids
after impregnation is best abbreviated as much as possible. Mounting in
formic acid glycerin is not favorable for the preservation of detail. This
is best studied in arsenic acid. Entire muscles are best mounted in balsam.

See also an older method of MAYS in Zeit.f. Biol, 1884, p. 449 ; Zeit. /.
wiss. Mik., 1885, p. 242.

Tendon.

671. Corpuscles of Golgi (RANVIER, Traite, p. 929).— Take
the tendon of the anterior and superior insertion of the gemini
muscles of the rabbit. Free it as far as possible from adherent
muscle-fibres. Treat it according to the formic acid and gold
method (§ 225), and after reduction of the gold scrape the
tendon with a fine scalpel, in order to remove the muscle-fibres
that mask the " musculo-tendinous organs."

672. Corpuscles of Golgi (in the tendons of the motores
bulbi oculi) (v. MARCHI'S methods, Archivio per le Scienze
Mediche, vol. v, No. 15). — The enucleated eyes, together with
their muscles, were put for not less than three days into

2 per cent, bichromate of potash. The muscles and tendons
were then carefully dissected out, stained with gold chloride
and osmic acid (Golgi' s method, supra, § 670), and by the
methods of MANFREDI, given in § 228.

Mount all these preparations in glycerin (balsam clears too

SMOOTH MUSCLE — ISOLATION OP FIBEES. 355

greatly). The methods only succeed completely during fine
sunny weather.

673. Corpuscles of Golgi (CATTANEO, Arch. ital. de Biol., x,
1888, p. 337). — The method here recommended is the arsenic
acid method of Golgi that has been described above, § 670,
sub voce " KUHNE " (4) .

See also RUFPINI (Atti E. Ace. Lincei Roma, 1892, p. 442;
Zeit.f. wiss. Mik., ix, 2, 1892, p. 237), who recommends the
method of Fischer.

674. Corpuscles of Golgi (CiACCio, Mem. R. Ace. Sci. Bologna
(4), t. x, 1890, p. 301 ; Zeit.f. wiss. Mik., vii, 4, 1891, p. 507).

—For Amphibia the usual gold methods are not satisfactory,
because the ground-substance of the tendon takes the stain
at the same time as the nerve- endings. Pieces of tendon
should be put into 0*1 per cent, hydrochloric acid or 0*2 per
cent, acetic acid until quite transparent. They should then
be put for five minutes into a mixture of 0*1 per cent, gold
chloride and O'l per cent, potassium chloride. After that
they are put back into the acetic acid, and remain there for
a day in the dark, and for two or three hours more in the
sunlight. When they have become somewhat violet they are
put for a day into 0*1 per cent, osmic acid, and finally mounted
in Price's glycerin acidulated with 0*5 per cent, of acetic or
formic acid.

675. The Methylen-blue Method. — I find no mention of the
application of this method to the study of the relations of
nerve and tendon, for which it would seem a priori to be pecu-
liarly suitable.

Smooth Muscle.

676. Smooth Muscle — Isolation of Fibres (SCHWALBE, Arch.f.
mik. Anat., 1868, p. 394). — Maceration in weak chromic acid
solution (0-02 per cent, proved a generally useful strength).
This is a better reagent than osmic acid, 1 per cent, acetic
acid (Moleschott), weak sulphuric acid, pyroligneous acid
(Meissner), 20 per cent, nitric acid (Reichert), 32 to 35 per
cent, potash solution (Moleschott), as it preserves better than
any of these the finer structure of the cells.

GAGE'S methods. — See Journ. Roy. Mic. Soc., 1887, p. 327;
and §§ 529, 540, and 544, ante.

356 MUSCLE AND TENDON.

MOBIUS, liquid for maceration of the muscle of Oardium (see
above, § 539).

BALLOWITZ, muscle of Cephalopoda, see Arch. f. mik. Anat.,
xxxix, 1892, p. 291 ; Zeit.f. wiss. Mik., ix, 3, 1893, p. 344.

677. Bladder of Frog, Innervation of (WOLFF, Arch. f. mik.
Anat., 1881, p. 362). — A frog is killed and a solution of gold
chloride of 1 : 20,000 injected into the bladder through the
anus. (If the injection flows out on removal of the syringe,
tie the frog's thighs together.) Now open the frog, dissect
away the attachments of the bladder, ligature the intestine
above the bladder, and cut away the abdomen of the frog so
as to have in one piece bladder, rectum, and hind legs. (All
this time the bladder must be kept moist with weak gold
solution.) The bladder and the rest are now put into gold
solution of 1 : 2000 for four hours ; the bladder is then ex-
cised, slit open, and pinned (with hedgehog spines) on to a
cork (outside downwards). Place it under running water
until all the epithelium is washed away. Use a pencil if
necessary. Put for twenty-four hours into gold solution of
1 : 6000. Wash in pure water, and put away in the dark "for
some time" in acidulated water, and finally reduce in fresh
water in common daylight. The muscles should be pale blue-
red; medullated nerves dark blue-red; sympathetic nerves
and ganglia carmine-red. KANVIER (Traite, p. 854) recom-
mends one or the other of his two gold processes. The
bladder of frogs should be carefully distended by injection of
the lemon juice or gold chloride and formic acid through the
cloaca.

The methylen-blue method would appear to be very much
indicated for this subject, but has not been tried so far as I
am aware.

678. Musculus dilatator Pupillae (DOGIEL, Arch.f. mik. Anat.,
1886, p. 403). — An enucleated eye is divided into halves, and
the anterior one with the iris brought for some days into a
mixture of two parts one-third alcohol and one part 0'5 per
cent, acetic acid. The iris can then be isolated, and split
from the edge into an anterior and posterior plate, and these
stained according to the usual methods.

679. Iris (KOGANEI, Arch.f. mik. Anat., 1885, p. 1).— The
pigmented epithelium can be removed by brushing with a

TEST FOB SMOOTH MUSCLE. 357

small brush after prolonged maceration in solution of Miiller.
The pigment may also be bleached by chlorine water, which,
however, should only be allowed to act for a few hours, until
the pigment has become of a light brown ; complete de-
coloration may be obtained by prolonging the reaction for
twenty-four hours, but then the tissues suffer. (See Journ.
Roy. Mic. Soc., 1886, p, 874.)

See also CANFIELD, in Arch.f. mik. Anat., 1886, p. 121 ; and
DOSTOIEWSKY, ibid., p. 91 (sections stained with haema-
toxylin and eosin, or [LiST, Zeit.f. wiss. Mik.f iii, 4, 1886, p.
514] with Renaut's hsematoxylic glycerin).

680. Stomach of Triton (see STILLING and PFITZNER, in Arch,
mik. Anat., 1886, p. 396).

681. Test for Smooth Muscle (RETTERER, Comptes Rend. Soc.
Biol., iv, 1887, p. 645; Journ. Roy. Mic. Soc., 1888, p. 843).—
If a specimen of tissue be fixed in a mixture of ten vokimes
of 90 per cent, alcohol and one volume of formic acid, well
washed, and stained for twenty-four to thirty-six hours with
alum- carmine, the cytoplasm of smooth muscle will be found
to be stained red, whilst connective-tissue cells remain un-
stained, and are swollen.

358 RETINA, INNER EAR, NERVES.

CHAPTER XXIX.

RETINA, INNER EAR, NERVES.

Retina.

682. Fixation and Hardening. — For section cutting, the
retina of small eyes is best prepared by fixing the entire un-
opened bulb with osmium vapour. According to RANVIER
(Traite, p. 954) you may fix the eye of a triton (without
having previously opened the bulb) by exposing it for ten
minutes to vapour of osmium. The sclerotic being very thin
in this animal, such a duration of exposure is generally suffi-
cient. Then divide it by an equatorial incision, and put the
posterior pole for a few hours into one-third alcohol.

Somewhat larger eyes, such as those of the sheep and calf,
may be fixed in solutions without being opened. But it is
generally the better practice to make an equatorial incision,
and free the posterior hemisphere before putting it into the
liquid.

The older practice was to use strong solutions of pure osmic
acid ; but most of the best recent work has been done with
chromic mixtures.

KUHNT (Jen. Zeit.f.Naturw., Bd. xxiv, H. 1, 1889, p. 177;
Zeit.f. wiss. Mik., vii, 1, 1890, p. 65) finds that for isolation
preparations the best way is to fix for twenty to twenty-eight
hours in f per cent, osmic acid, then put the preparation for
fourteen days into water in a well-closed vessel, changing the
water once at the middle of the time, and at the end of it bring-
ing the preparation for three to four weeks into a mixture
of 80 parts water, 12 parts alcohol, and 8 parts glycerin.

For sections he prefers fixing for two days in solution of
Flemming, hardening for three days in alcohol, and imbedding
in celloidin.

BARRETT (Quart. Journ. Mic. Sci., 1886, p. 607) recommends
fixing the entire unopened bulb in a liquid containing 0*2 per

FIXATION AND HARDENING. 359

cent, of osmium and ^ per cent, of chromic acid in water, for
twenty-four to thirty-six hours, with subsequent hardening
for fourteen days in alcohol containing 2 per cent, of car-
bolic acid. He finds that the preservation of specimens so
treated is far superior to that of specimens hardened in pure
alcohol.

SCHIEFFEKDECKER (Arch. f. mik. Anat., 1886, p. 305) used
either Miiller's solution, or chromic acid of ^ per cent., or a
mixture of 1 part of wood- vinegar (Acetum Pyrolignosum)
with 3 parts of distilled water, which he states gives particu-
larly instructive preparations without any serious alteration
of the elements.

CUCCATI (Mem. R. Accad. jSci. 1st. di Bologna ; Zeit.f. wiss.
Mik., v, 1, 1888, p. 86) fixes large eyes (the lens and vitreous
body having been removed) for two days in a solution of —
1 per cent, osmic acid . . .14 grms.,
1 per cent, chromic acid . . . 25 „
Acetic acid ..... 1 drop,
which should be renewed after the lapse of twenty -four
hours.

See also DENNISSENKO (Arch. f. mik. Anat., 1881, p. 395).
Dr. LINDSAY JOHNSON writes me that he gets the best results
by fixing for three minutes with a mixture of equal parts of
acetic acid and 2 per cent, osmic acid, injected into the
vitreous or the cavity of the eye. After that the eye is
washed in alcohol for a quarter of an hour, and put for two
hours into the mixture, § 78 ; it is washed in running water,
and suspended in a large volume of 2 '5 per cent, bichromate
of potash for two days, then passed gradually through suc-
cessive alcohols, beginning with 20 per cent., and ending with
absolute.

CHIEVITZ (Arch.f. Anat. u. PTiys., Anat. Abth., 1889, supp.,
p. 139; Zeit.f. wiss. Mik., vi, 4, 1889, p. 511) uses nitric acid
of 2*5 per cent, strength. Entire heads of frogs may be fixed
in it for a couple of hours, washed out for a night in water, and
put up in alcohol. Bulbi of birds should be opened in the acid,
and the retina carefully exposed by removing the cornea, iris,
lens, and vitreous, or by separating the sclera and choroidea
from it. The duration of these operations affords a sufficient
fixation.

ANQELUCCI (Moleschott's Untersuchungen, &c., xiv, 1890,

360 RETINA, INNER EAR, NERVES.

p. 231 ; Zeit.f. wiss.Mik., ix, 1, 1892, p. 85) recommends that
eyes be put for from half an hour to three hours into 3 per
cent, nitric acid, then for ten days into solution of Miiller,
and lastly into alcohol.

683. Staining. — Dr. LINDSAY JOHNSON, writing to me, recom-
mends, above all things, the Ehrlich-Biondi mixture, either
alone or with addition of nigrosin (see § 259). Used in the
latter way, the nuclear cells are stained pale brown, and
nucleoli a darker brown; whilst the Miiller fibre layers,
molecular layers, and rods are stained a beautiful green,
varying from yellowish to bluish green according to thickness
of specimen and strength and length of action.

KAMON Y CAJAL employs the rapid chromate of silver method
of Grolgi, as given below (§ 715).

KUHNT (see last section) employs Pal's modification of
Weigert's haematoxylin process.

DOGIEL employs the methylen-blue method, as given in
§§ H7, 119.

SCHAFFER (Sitzb. k. Accid. Wiss. Wien, xcix, 1890, 3, p. 110;
Zeit.f. iviss. Mik., viii, 2, 1891, p. 227) recommends mordant-
ing sections in 1 per cent, chromic acid for some hours,
washing for a short time only with water, staining for twenty
hours in Kultschitzky's acetic acid hsematoxylin (post, § 697),
and differentiating for twelve hours in Weigert's ferricyanide
solution.

KRAUSE (1. c., § 685) obtains instructive preparations by treating fresh
retina with perchloride of iron or of vanadium in 1 per cent, solution, and
then with a 2 per cent, solution of tannic or pyrogallic acid. These reagents
only stain the granule layers and the nuclei of the ganglion-cells. The ele-
ments of the other layers may then be stained with Sauref uchsin, or some
other anilin.

LENNOX (Arch. f. Ophthalm., xxxii, 1 ; Zeit. f. wiss. Mik., iii, 3, 1886,
p. 408; and Journ* Boy. Mic. Soc., 1887, p. 339) has been applying
Weigert's hrematoxylin method to the retina, with some remarkable results.

CUCCATI (1. c., last section) stained with concentrated aqueous solution of
Saurefuchsin, and mounted in balsam.

See also BERNHEIMEB, Sb. k., Akad. Wiss. Wien, 1884; or Journ. Roy.
Mic. Soc., 1886, p. 167 ; and KAMON Y CAJAL, Rev. trim, de Hist. Norm, y
Path., i, 1888, p. 1 ; Anat. Anz., 1889, p. Ill ; Zeit.f. wiss. Mik., v, 3, 1888,
p. 373, and vi, 2, 1889, p. 204.

684. Sections. — Some workers recommend celloidin ; but
I see no reason whatever for not employing paraffin. Sections

INNER EAR. 361

may be mounted in dammar or (FLEMMING) in glycerin.
RAMON Y CAJAL (1. c. last section) mounts in dammar or colo-
phonium varnish without a cover-glass.

685. Dissociation Methods. — For maceration preparations
you may use weak solutions (0'2 to 0'5 per cent.) of osrnic
acid for fixation, and then macerate in 0'02 per cent, chromic
acid (M. SCHULTZE), or in iodised serum (M. SCHULTZE), or in
dilute alcohol (LANDOLT), or in Miiller's solution, or (RANVIER,
Traite, p. 957) in pure water, for two or three days. THIN
(Journ. of Anat., 1879, p. 139) obtained very good results by
fixing for thirty-six to forty-eight hours in one-third alcohol,
or in 25 per cent, alcohol, and then staining and teasing.

SCHIEFFEEDECKEE (1. c. § 682) macerates fresh retina for
several days in the methyl mixture, § 548.

KRAUSE (Intern. Monatsch. f. Anat. u. Hist., 1884, p. 225 ;
Zeit.f. wiss. Mik., 1885, pp. 140, 396) recommends treatment
for several days with 10 per cent, chloral hydrate solution.
Barrett finds that this process preserves the rods and cones
admirably.

Inner Ear.

686. SCHWALBE (Beitr. z. Phys., 1887; Zeit. f. wiss. Mik.,
iv, 1, 1887, p. 90; Journ. Roy. Mic. 8oc., 1887, p. 840).— Fix
(cochlea of guinea-pig) for eight to ten hours in " Flemming,"
wash in water, decalcify (twenty-four hours is enough) in 1
per cent, hydrochloric acid, wash the acid out, dehydrate,
and imbed in paraffin.

PRENANT (Intern. Monatsschr. f. Anat. u. Physiol., ix, 1,
p. 6; Zeit.f. wiss. Mik., ix, 3, 1893, p. 379).— For sections,
open the cochlea in solution of Flemming or of Hermann, and
fix therein for four to five hours. Avoid decalcification as far
as possible, as it is inimical to good preservation of elements ;
but if necessary, take 1 per cent, palladium chloride. Make
paraffin sections and stain with safranin, or with methyl violet
B, or with anilin green and orange, or with Renaut's eosin-
hgematoxylin.

Isolation preparations of the stria vascularis may be made
by putting a cochlea for a day into 1 per cent, solution of
osmic acid, then for four to five days into O'l per cent, solution ;
the stria may then be got away whole.

362 NERVES.

687. Other Methods.— WALDEYER, Strieker's Handb., p. 958 (decalci-
fication either in O'OOl per cent, palladium chloride containing 10 per cent,
of HC1, or in chromic acid of O25 to 1 per cent.).

UBBAN PBITCHARD (Journ. Roy. Mic. Soc., 1876, p. 211). — Decalcifica-
tion in 1 per cent, nitric acid.

LAVDOWSKY (Arcli.f. mik. Anat., 1876, p. 497).— Fresh tissues (from the
cochlea) are treated with 1 per cent, solution of silver nitrate, then washed
for ten minutes in water containing a few drops of 0'5 or 1 per cent, osinic
acid solution, and mounted in glycerin.

MAX FLESCH (Arch.f. mik. Anat., 1878, p. 300).

TAPANI (Arch. Ital. de Biol., vi, p. 207).

POLITZER, 4(Die anatomische u. histologische Zergliederung
d. menschlichen Gehororganes," Stuttgart (Enke), 1889 (see
Zeit.f. wiss. Mik.,vn, 3, 1890, p. 364).— The reviewer (HAUG)
here calls attention to the admirable qualities of phloroglucin
as a decalcification agent for this object (see § 562).

EICHLER (Abh. d. math.-phys. Cl. d. k. Sachsischen Ges. d.
Wiss., Bd. xviii, 1892, p. 311 ; Zeit.f. wiss. Mik., ix, 3, 1893,
p. 380). — Detailed account of manipulations for injection of
blood-vessels of the labyrinth.

Dr. LINDSAY JOHNSON greatly recommends the Ehrlich-
Biondi stain (see §§ 259, 683).

Nerves.

688. WETGERT'S Method for Medullated Nerves (Fortschr. d.
Med., 1884, pp. 113, 190; 1885, p. 136; Zeit.f. wiss. Mik.,
1884, pp. 290, 564; 1885, pp. 399, 484).— The ordinary
methods of staining with haematoxylin depend on the produc-
tion of an aluminium lake of haematoxylin. Weigert's method
depends on the formation of another lake, a chromium or
copper lake. In consequence of the formation of these lakes
haematoxylin acquires the property of staining the myelin of
nerves in a quite specific way.

In Weigert's process the formation of these lakes takes
place in the tissue itself. The details of the process have
been considerably modified, both by other workers and by
Weigert himself. I give here the 1885 method, a knowledge
of which is necessary to the understanding of the subsequent
modifications. It is as follows : — The tissues are to be hard-
ened in bichromate of potash (the solutions of Muller or
Erlicki will do as well, so far as I know). The hardening
need only be carried to the point at which the tissues have

WEIGERT'S METHOD. 363

acquired a brown, not a green, coloration (but green tissues
may be used, provided they have once passed through the
brown stage) . The preparation is then (but this is not neces-
sary) imbedded by infiltration with celloidin, and the celloidin
block fastened on -cork and hardened in the usual way. The
hardened block is put for one or two days into saturated
solution of neutral acetate of copper diluted with one volume of
water, the whole being kept at the temperature of an incubat-
ing stove. By this treatment the tissues become green, and
the celloidin bluish green. The mordantage of the tissues is
now terminated, and the preparation may be kept, till wanted
for sectioning, in 80 per cent, alcohol.

Sections are made with a knife wetted with alcohol, and are
brought into a stain composed of —

Haematoxylin . . . . 0'75 to 1 part.

Alcohol . . . . . .10 parts.

Water 90 „

Saturated solution of lithium car-
bonate ...... 1 part.

(A trace of any other alkali may be added in the place of lithium carbonate.
The object of adding a little of some base is to " ripen " the hsematoxylin
solution.)

The sections remain in the stain for a length of time that
varies according to the nature of the tissues : — Spinal cord,
two hours ; medullary layers of brain, two hours ; cortical
layers, twenty-four hours.

They are then rinsed with water, and brought into a de-
colourising solution composed of —

Borax ...... 2*0 parts.

Ferricyanide of potassium . . 2*5 „

Water 200-0 „

They remain in the solution until they are decoloured to
the right degree — that is, until complete differentiation of the
nerves (half an hour to several hours) — and are then rinsed
with water, dehydrated with alcohol, and mounted in balsam.
They may be previously stained, if desired, with alum-carmine
for the demonstration of nuclei.

For very difficult objects, such as pathological nerves, the decolouring
solution should be diluted with water, and the immersion in it prolonged.
GELPKE (Zeit. f. wiss. Mik., 1885, p. 489) states that for transverse sections
of atrophied nerves the solution should be diluted with fifty volumes of

364 NERVES.

water, and the immersion be prolonged to twelve hours at the least ; for
longitudinal sections it should be diluted with ten volumes of water.

The process is applicable to tissues that have been hardened in alcohol
or in anj other way, provided that they be put into a solution of a chromic
salt until they become brown, before mordanting them in the copper solu-
tion.

As above stated, it is not necessary that the mordantage be done in bulk
with tissues imbedded in celloidin. MAX FLESCH (Zeit.f. wiss. Mik., iii, 1,
1886, p. 50) finds that this practice is unfavorable to subsequent staining
with other reagents than hsematoxylin, and prefers (following LICHTHEIM)
to make the sections first, bring them on closet-paper into the mordant, and
after mordanting bring them on a spatula into 70 per cent, alcohol, and
thence into the stain.

In the process given above, a copper lake is formed in the tissues. In the
earlier form of the process the mordantage with the copper salt was omitted,
and the stain depended on the formation in the tissues of a chromic lake.
The results were not quite so good, and the process may be taken to be
superseded by the copper process. A modified form of the former, due to
MAX FLESCH, has some advantages, and is given in the next section.

If very many large sections have to be prepared, and if the staining solu-
tion be thrown away after using, the process may be found somewhat
expensive. The following method for regenerating the staining solution is
given by FANNY BEELINERBLAU (Zeit. f. wiss. Mik., 1886, p. 50) : — About
2'5 to 5 per cent, of baryta water is added to the used solution ; it is well
shaken and allowed to stand for twenty-four hours ; carbonic acid (obtained
from the action of crude hydrochloric acid on marble) is led through it, it is
allowed to stand for twenty-four hours more, and then filtered.

PANETH (ibid., 1887, p. 213) makes the stain with extract of logwood
instead of pure hsematoxylin. One part of commercial extract of logwood is
dissolved in 90 parts of water and 10 of alcohol. To the filtered solution is
added 8 drops of concentrated solution of lithium carbonate for each 100 c.c.
Sections require from eighteen to twenty-four hours in the stain at the
normal temperature.

BREGLIA (ibid., vii, 2, 1890, p. 236 ; see also Journ. Roy. Mic. Soc., 1890,
p. 817) stains with liquid extract of logwood or Pernambuco wood, prepared
by extracting 7 to 10 grms. of the wood for 5 or 6 days with 100 c.c. of alco-
hol of 90 to 95 per cent.

The results obtained by Weigert's method are most splendid.
The blue-black nerves stand out with admirable boldness on
a golden ground. The method is applicable to the study of
peripheral nerves as well as to nerve-centres, and is likely to
be of great utility in Vertebrate embryology.

Nerve-tissue is not the only tissue stained by the process,
which can be usefully applied to lymphatic glands and to
skin (see SCHIEPPEEDECKER. in Anat. Anz., ii, 1887, p. 680).

PAL'S METHOD. 365

Modifications of Weigert's Method.

689. MAX FLESCH (Zeit.f. wiss. Mik., 1884, p. 564).— Sec-
tions, made either by the celloidin process or otherwise, are
put for a few minutes or longer into O5 per cent, chromic
acid solution; they are then rinsed in water, and brought
into the stain, where they take on a sufficient coloration"
much more rapidly than the tissues will if stained in the
mass. Decoloration is done in the usual way. No stove is
required in this process, and either brown .or green material
may be used. Flesch finds (1. c., 1886, p. 51) that this method
is to be preferred to Weigert's copper method in all cases in
which it is important to produce differential staining in nerve-
cells, and especially in the study of peripheral ganglia, and
also for producing differential staining of the medulla of
central and peripheral nerves, whilst the copper process is
most certainly superior for the demonstration of fine nerve-
fibres.

690. BENDA (Verhandl. Physiol Ges. Berlin, 1885-6, Nos. 12, 13, 14 ;
Zeit. f. wiss. Mik., iii, 3, 1886, p. 410 ; Arch. f. Anat. u. Phys., 1886, p.
562). — The tissues are to be fixed and hardened for at least three days in
saturated solution of picric acid, washed out for several days in water,
hardened in alcohol, imbedded in paraffin, and sections made. (The treat-
ment with water after picric acid seems to me to be most inimical to the
good preservation of the tissues, and I cannot understand why Benda should
not have used alcohol.) The sections are mordanted by treating them for a
few minutes or hours with a salt of iron, for which purpose concentrated
solution of ammonio-sulphate of iron is recommended. After being well
washed in several changes of water, they are stained for about ten minutes
in 1 per cent, aqueous solution of haematoxylin, then decoloured by treatment
for about five minutes in O'Oo per cent, aqueous solution of chromic acid,
rinsed in water, dehydrated, cleared, and mounted in balsam.

This method is stated to be peculiarly adapted for the demonstration of
very fine fibres and their relations to ganglion-cells.

691. HAMILTON (Journ. of Anat. and Physiol., xxi, 1887, p. 444; Journ.
Roy. Mic. Soc., 1888, p. 1051) mordants his preparations with a copper sul-
phate solution, and makes sections by the freezing method, after imbedding
in collodion.

BEEVOE (Brain, 1885, p. 227 ; Journ. Roy. Mic. Soc., 1886, p. 898) gives
a very slight modification of Weigert's original process.

692. PAL (Wien. med. Jahrb., 1886; Zeit.f. wiss. Mik., iv,
1, 1887, p. 92; Med. Jahrb., 1887^p. 589; Zeit. f. wiss. Mik.,
1888, p. 88) describes the following process : — After staining

366 NERVES.

in the haematoxylin solution the sections are washed in water
(if they are not stained of a deep blue, a trace of lithium car-
bonate must be added to the water) . They are then brought
for twenty to thirty seconds into 0*25 per cent, solution of
permanganate of potash, rinsed in water, and brought into a
decolouring solution composed of —

Acid. Oxalic, pur. . . TO

Potassium Sulphite (Kalium Sul-

furosum [S03K2] ) . . 1-0

Aq. dest. . . . 20OO

In a few seconds the grey substance of the sections is de-
colourised, the white matter remaining blue. The sections
should now be well washed out, and may be double-stained
with Magdala red or eosin, or (better) with picro-carmine or
acetic-acid-carmine.

For further details as to the somewhat elaborate minutiae
of the process see the papers quoted, or BEHRENS, KOSSEL, and
SCHIEFFERDECKER'S Das Mikroskop, i, p. 199.

BEEVOR (Journ. Roy. Mic. Soc., 1892, p. 898) says of this
process that it is more difficult to carry out than Weigert's,
and that it does not show the different forms of fibres so well,
but is useful on account of the double stain.

693. The process of PAL has been modified by SCHAFER (I
quote from BEEVOR, 1. c., not knowing where the method was
first published) as follows : — " The material is hardened from
four to six weeks, and after cutting the sections are put into
MARCHI'S fluid (1 part of a 1 per cent, solution of osmic acid,
and 2 parts of a 3 per cent, solution of potassium bichromate) .
Then wash quickly in water and stain with the haematoxylin
solution (haematoxylin 1 grm., acetic acid 2 c.c., water 100 c,e.).
Develop afterwards as by Pal's method. This is a very
good method for quickening the hardening process, and for
sections which have been too long in alcohol after potassium
bichromate."

694. FLECHSIG (Arch. f. Anat. u. Phys., Physiol. Abth., 1889, p. 537 ;
Zeit. f. wiss. Mik., vii, 1, 1890, p. 71 ; Journ. Roy. Mic. Soc., 1890, p. 538)
stains sections for three to eight days at a temperature of about 35° C. in a
solution of redwood (Rothholz, Brazil-wood). This is prepared (after BBANCA)
by dissolving 1 grm. of extract pur. of Japanese ledwood in 10 grms. of
absolute alcohol, and adding 900 grms. of water, 5 grms. of saturated solu-
tion of Glauber's salt, and 5 grms. of saturated solution of tartaric acid.

KULTSCHITZKY'S METHOD. 367

The sections are then washed in water and differentiated by the method of
Pal.

BBEGLIA (1. c. in last section) mordants sections for ten to fifteen minutes
in a mixture of 15 c.c. of 90 per cent, alcohol with 3 to 7 c.c. of saturated
aqueous solution of neutral acetate of copper (ARMANNI). They are then
brought for five or ten minutes into a mixture of 3 parts of water with 1
part of saturated aqueous solution of lithium carbonate. They are then
stained for eighteen to twenty-four hours in the extract of logwood solution
given in the last section, to which if desired one-fifth to one-third of the
lithium carbonate solution may be added. They are then differentiated in
Weigert's decolourising solution. Instead of the acetate of copper, you may
take a 4 to 7 per cent, solution of sulphate of copper, and mordant therein
for twenty minutes ; or carbonate of copper, subacetate of lead, perchloride
of iron, or other salts may be used.

695. VASALE (Rivista sper. di Freniatria, xv, 1889, p. 902;
Zeit.f. wiss. Milt., vii, 4, 1891, p. 517) finds that Weigert's
process can be considerably abbreviated as follows. He
stains first for three to five minutes in the hsematoxylin, then
treats the sections for the same length of time with saturated
aqueous solution of neutral acetate of copper, in which they
become black. They are then washed with water and dif-
ferentiated by moving them about in Weigert's decolourising
liquid diluted with one-half of water.

696. Another rapid modification is that of LISSAUEE, de-
scribed by Sachs (Centralb.f. Nervenheilk., xv, 1892, p. 330;
Zeit.f. wiss. Mik., ix, 3, 1893, p. 391). Sections of material
hardened in liquid of Miiller are heated (the mordantage
with copper being omitted) in 1 per cent, solution of chromic
acid until the solution begins to form bubbles. They are
then rinsed with water, and again heated, until bubbles begin
to form, in Weigert's hsematoxylin solution. They are then
differentiated by Pal's process. The method is said to succeed
even with material that has not been very well hardened, and
to give good results with brains that have been only a short
time in liquid of Miiller.

697. KULTSCHITZKY (Anat. Anz.} 1889, p. 223, and 1890, p.
519 ; Zeit.f. wiss. Mik., vi, 2, 1889, p. 196, and vii, 3, 1890, p.
367) has given two forms of his well-known process, of which
the following is the later : — Specimens are hardened for one
to two months in solution of Erlicki, imbedded in celloidin or
photoxylin, and cut. Sections are stained for from one to
three hours, or as much as twenty-four, in a stain made by

368 NERVES.

adding 1 grm. of haematoxylin dissolved in a little alcohol to
100 c.c. of 2 per cent, acetic acid. They are washed out in
saturated solution of carbonate of lithium or sodium. By
adding to the carbonate of lithium solution 10 per cent, of a
1 per cent, solution of red prussiate of potash, and decolouris-
ing therein for two or three hours or more, a finer differen-
tiation is obtained. After this the sections are well washed
in water and mounted in balsam.

\. Eossi (Zeit. f. wiss. Mik., vi, 2, 1889, p. 182) also recommends a
process stated to be simpler than Weigert's. The tissues are hardened in a
liquid composed of water, 100 c.c. ; chromic acid, 0*75 grm. to 1 grm. ;
acetate of copper, 5 grrns. Human cord requires six to eight days ; the cord
of the dog, three to four ; brain of dog, fifteen to eighteen. They are then
dehydrated and imbedded in celloidin. Sections are stained in a liquid made
by adding 7 to 8 drops of a 5 per cent, solution of hsematoxylin in absolute
alcohol to about 30 c.c. of alcohol. After two hours they are brought into a
mixture of 8 drops of hydrochloric acid with 100 c.c. of absolute alcohol,
and washed therein until the white substance is seen to be differentiated
from the grey. They are washed out for twenty minutes or more in water,
dehydrated, and mounted. The sections may be double-stained with borax-
carmine.

699. WOLTEKS (Zeit. f. wiss. Mik., vii, 4, 1891, p. 466) pro-
ceeds as Kultschitzky, except that he stains in a solution con-
taining 2 grms. of hasmatoxylin (dissolved in a little alcohol)
to 100 c.c. of 2 per cent, acetic acid, and kept warm by plac-
ing it on the top of a stove kept at 45° C. for twenty-four
hours, after which time the sections are dipped in solution of
Miiller, and differentiated by the method of Pal. This is
an intense myetin stain ; medullated fibres blue-black ; ground
light ; ganglion-cells yellow.

700. A second method of WOLTERS, which gave good results
with the cerebellum and some peripheral nerves, is as follows :
— Sections from material hardened in Miiller and cut in
celloidin are mordanted for twenty-four hours in solution of
acetate of aluminium, or, which is better, in a mixture of —

10 per cent, vanadium chloratum . . 2 parts,
8 per cent, aluminium aceticum liquidum . 8 „
washed for five or ten minutes in water, stained in the above-
mentioned haematoxylin, and differentiated in Weigert's de-
colourising mixture. A myelin stain in general, with superb
differentiation of the protoplasmic processes of the cells of
Purkinje.

WEIGERT'S NEW METHOD. 369

For details of the numerous mordants that were unsuccess-
fully tried, see the paper quoted.

Wolters's axis-cylinder stain is given in § 726.

701. MEKCIEE (Zeit.f. wiss. MiJc., vii, 4, 1891, p. 480) has a modification
which consists in adding glycerin to the stain (which possibly may serve to
usefully increase its penetrating power), and employing after the usual dif-
ferentiating fluid a second differentiating fluid composed of 2 parts of 10 per
cent, potash solution, 10 pails of water, and 1 part of sulphuric ether. I
cannot find that this process has been recommended by any other worker.

702. HAUG'S modification of Weigert's process is given in § 755.

703. WEIGEKT'S New Method (Deutsche med. Wochenschr.,
42, 1891, p. 1184; Zeit. f. wiss. MA?., viii, 3, 1891, p. 392).—
The material is to be hardened in bichromate and imbedded
in celloidin in the usual way. The hardened blocks of cel-
loidin are brought into a mixture of equal parts of a cold
saturated solution of neutral acetate of copper and 10 per cent,
aqueous solution of potassio-tartrate of sodium (C4H406KNa
-f 4H20, salt of Seignette). They are left in the mixture for
twenty-four hours in an incubator. (Large specimens [Pons]
will require forty-eight hours, the mixture being changed for
fresh at the end of twenty-four hours.) They are then brought
for twenty-four hours into aqueous solution of neutral acetate
of copper, either saturated or diluted with 1 volume of water,
being kept as before in the incubator. They are then rinsed
with water and brought into 80 per cent, alcohol, in which
they may either remain till wanted or be cut after half an
hour.

The sections are stained for from four to twenty-four hours
at the temperature of the room in a freshly prepared mixture
of 9 vols. of (A) a mixture of 7 c.c. of saturated aqueous solu-
tion of carbonate of lithium with 93 c.c. of water, and 1 vol. of
(B) a solution of 1 grm. of hsematoxylin in 10 c.c. of alcohol (A
and B may be kept in stock, but A must not be too old) . The
sections must be loose ones, not such as have been seriated in
celloidin, and must not be thicker than 0*025 mm. The stain
is poured off and the sections are washed in several changes
of water poured on to them. They are then treated with 90
per cent, alcohol followed by carbolic-acid-and-xylol mixture
(for a short time only), or by a mixture of 2 parts of anilin oil
with 1 of xylol, then pure xylol and xylol balsam (not chloro-
form balsam, which injures the stain).

24

370 NERVES.

Medullated fibres dark blue on a light, sometimes rosy
ground. If it be wished to have the ground particularly
colourless, take instead of the second wash-water a mixture
of -J- to J volume of common (not glacial) acetic acid with 100
volumes of water. Thick sections or series in celloidin require
a special differentiation. They may be differentiated either
with the above-mentioned acetic acid mixture, or in the usual
borax-ferricyanide mixture diluted with water. In the latter
case the ground will be yellow.

If the impregnation with the copper be imperfect (as, for
instance, may happen if the treatment with the copper salt
be performed at the normal temperature instead of in an in-
cubator) some instructive differentiations of ganglion-cells
may be obtained, the processes of the cells of Purkinje in
the cerebellum, for instance, being very sharply brought out ;
but such preparations have a tendency to after-blackening,
which does not happen with those that have been thoroughly
impregnated with the copper.

The advantages of the improved method are that differen-
tiation after staining is not necessary ; that the annoying pre-
cipitates formed on the surface of the preparations by the
copper in the old method do not appear ; that the divers
manipulations are simpler and easier; the preparations are
equal in beauty to those of Pal, and can be obtained with
greater certainty.

704. GOLGI'S Nerve Methods— Bichromate and Silver Nitrate
Process, or "Slow" Method. — I take the following resume of
this method from the interesting paper of Grolgi's pupil, REZ-
ZONICO, "Sulla struttura delle fibre nervose del midollo
spinale," in the Archivio per le scienze mediche, iv, No. 4
(1879), p. 85.

1. Take pieces of perfectly fresh spinal cord, and soak
them in a 2 per cent, solution of bichromate of potash for a
period of time varying according to temperature. (In summer
eight to fifteen days may suffice, in winter about a month is
necessary.)

2. Wash them, and put them into a 0*75 per cent, solution
of nitrate of silver. The period of immersion therein depends
on the temperature : in summer the reaction will be complete
throughout the tissues in two or three days ; in winter, eight,
ten, or more days.

GOLGl's BICHROMATE OF SILVER METHOD. 371

3. Dehydrate small pieces with, alcohol (make sections if
necessary), clear in oil of turpentine, tease in the turpentine,
and mount in dammar.

4. The preparations are then left to themselves in order
that the secondary impregnation may take place. In direct
sunlight eight to ten days will complete the process; in
diffused daylight (or in the dark ?), twenty, thirty, or forty
days.

A somewhat greater precision of the reaction is obtained by treating the
fresh tissues with osmic acid (by means of insterstitial injection) before
putting them into the bichromate. In this case a much shorter immersion
in the bichromate will suffice (four, six, or eight days).

By this means may be demonstrated in the medullated fibres of the spinal
cord a chain of conical funnels, set one within another, and embracing the
axis-cylinder with their narrow aperture, and the external surface of the
following funnel with their greater aperture ; and it is seen that they con-
sist of a fine spiral fibre wound into the form of a funnel. (The appear-
ance of rings and strainers is due to imperfect action of the silver.)

705. For the Study of Peripheral Nerves Golgi modifies the
process as follows (1. c., 1879, p. 238) :

1. Pieces of nerve are immersed in the bichromate solution,
for from four, six, or eight hours to one day, or at most two
days.

2. From time to time pieces are removed into the nitrate
of silver; they remain there for from twelve to twenty-four
hours.

3. They are washed with several changes of alcohol.

4. Tease in the alcohol, dehydrate, clear with turpentine,
mount in dammar.

5. Reduce in direct sunlight ; in summer a few days suffice,
in cold weather some weeks are necessary.

Does not give quite such fine results as the osmium bichro-
mate and silver method, or "rapid" method, next to be
described ; but the preparations keep indefinitely.

706. The Osmium, Bichromate, and Silver Method, or Rapid
Method (ibid., p. 237). — A perfectly fresh piece of nerve is
thrown into the following liquid :

2 per cent, solution of bichromate of

potash 10 parts.

1 per cent, solution of osmic acid . 2 „
After about an hour's immersion the piece of nerve may

372 NERVES.

be cut into lengths of 4 to 1 cm., which are put back into the
liquid.

Four hours after the first immersion of the nerve in the
mixture begin to put the pieces into nitrate of silver solution,
transferring a certain number of pieces every three hours,
so as to be sure that some of them shall have had a bi-
chromate bath of a proper duration. (This duration may,
roughly speaking, be said to lie between six and twenty-four
hours.)

The strength of the nitrate of silver solution is O50 per
cent. The duration of the silver-bath must not be less than
eight hours ; it may be indefinitely protracted.

Dehydrate, clear with turpentine, mount in dammar.

The method is more expeditious and easier of application
than the bichromate and silver nitrate method, and the results
are somewhat more precise, but the preparations do not keep in
dammar (without special precautions, to be discussed below).

These two methods serve for the demonstration in peripheral rnedullated
nerve-fibres of the funnel-shaped coils of sustaining filaments discovered by
Rezzonico in the medullated fibres of the spinal cord.

In all methods for the demonstration of the funnels it is important to
observe the utmost delicacy of manipulation, and in particular, the fibres
must not be stretched; their stretching is a weak point in the methods of
Kanvier (Traite, p. 718).

Modifications of Golgi's Chromate of Silver Method.

707. Critique of GOLGI'S Method. — The above-described
methods have been found extremely valuable in the most
various departments of nervous anatomy. They have given
brilliant results in the study of peripheral nerves and their
origins or terminations, and in the study of the relations of
fibres and cells in the central nervous system. It has been
found, at the same time, that they have the defect of con-
siderable uncertainty in the production of the desired reac-
tion, and a still greater uncertainty in the preservation of the
stain. These defects have given rise to a most elaborate
discussion, which unhappily has not as yet led to very satis-
factory results.

Golgi's method is apparently (but this is by no means
certain) a double decomposition method, based on the com-
bination of the bichromate of potassium in the tissues with

MODIFICATIONS OF GOLGl's METHOD. 373

the silver nitrate, and formation of a brown precipitate of bi-
chromate of silver (K2Cr207 + 2AgN03 = Ag2Cr207 + 2KN03;
the precipitate is brown by reflected light, but appears black
by transmitted light). The problem is to preserve this pre-
cipitate in the tissues free from chemical or molecular change.
And the problem is not an easy one ; without special precau-
tions the preparations will not resist the processes necessary
for imbedding, will not always resist those necessary for
merely mounting in balsam, and even then may easily " go
bad" after they have been mounted for a short time.

708. SEHRWALD, in an elaborate paper (Zeit. f. wiss. Mik.,
vi, 4, 1889, p. 443), develops the theory that the deterioration
of the preparations is due to solution of the precipitate in the
reagents employed in the ulterior processes of preparation —
in water, in chlorides (which may be present in alcohol), in
the xylol, in the paraffin, in the very balsam itself. He made
elaborate attempts (fruitless, but instructive) to turn the diffi-
culty by transforming the soluble chromic salt of silver into
some insoluble compound. He tried to replace the acid of
the salt ; he tried to replace the metal of the salt ; he tried to
make a sulphide of it ; he tried to reduce it to the metallic
state with photographic developing solutions (and had some
success with hydroquinone, but does not recommend the pro-
cess on account of its giving rise to deceptive precipitates
that simulate organised elements). None of these experi-
ments gave the desired result ; and the upshot of the paper is
that it is better not to attempt to modify the state of the pre-
cipitate, but rather so to modify the preparation liquids as to
reduce their solvent action on the precipitate. This may be
done by supersaturating them with bichromate of silver before
using. The powdered salt should be added to each of them
in excess, including the clearing media, the medium used for
fixing the sections to the slide, the paraffin itself, the solvent
used for removing it, and the balsam, and made to dissolve
by the aid of heat. By this means thin sections may be pre-
pared and mounted in balsam without injury to the stain.
Sehrwald does not say how long they will keep.

One of the annoyances of Golgi's process is that it fre-
quently gives rise to the formation at the periphery of the
preparations of voluminous precipitates that are destructive

374 NEEVES.

of the clearness of the images. SEHRWALD (1. c., p. 456) finds
that this evil can be avoided by putting the tissues into
gelatin solution before bringing them into the silver-bath. A
10 per cent, solution of gelatin in water may be made. The
tissues are imbedded in this in a paper imbedding box with
the aid of a little heat (the gelatin melting at a sufficiently
low temperature), and are brought therein into the silver-
bath. After the silvering the gelatin is removed by warm
water saturated with chromate of silver.

709. SAMASSA (op. cit., vii, 1, 1890, p. 26) points out that
bichromate of silver is not soluble either in absolute alcohol,
toluol, xylol, paraffin, or Canada balsam, nor otherwise chemi-
cally affected by them ; and that therefore Sehrwald's ex-
planation is untenable. He thinks that the true explanation
of the deterioration of the preparations is that the precipitate
forming the impregnation is little by little floated away from
the tissues by the mechanical force of the diffusion currents
set up on the passage of the preparations through the dif-
ferent reagents, and particularly those long-continued ones
caused by the slow drying of balsam under a cover-glass.
He recommends, therefore, simply that the preparations be
preserved without a cover.

710. GREPPIN (Arch. f. Anat. u. Entw., Anat. Abth., 1889,
Supp., p. 55; Zeit. f. wiss. Mik.,vii, 1, 1890, p. 66) finds that
by means of hydrobromic acid, suggested by NEUMANN, pre-
parations made by the slow method may be rendered suffi-
ciently resistent to bear mounting under a cover. After
silvering, sections are made with a freezing microtome and
treated for thirty to forty seconds with 10 per cent, solution
of hydrobromic acid, and may then be well washed in several
changes of water and mounted in the usual way. If they be
cleared in clove oil and exposed therein to sunlight for ten or
fifteen minutes, they will take on a violet tone, and details
will be more strongly brought out. It is sometimes well to
treat them, after the 10 per cent, acid, with 40 per cent, acid
for twenty to thirty seconds. They may also be treated by
Pal's modification of Weigert's haematoxylin process.

711. OBREGIA ( Virchow's Archiv, cxxii, 1890, p. 387; Zeit.
f. wise. Mik., viii, 1, 1891, p. 97; Journ. Roy. Mic. Soc., 1891,

MODIFICATIONS OF GOLGl's METHOD. 375

pp. 536, 830 : the Journal here appears to have been track-
ing itself in its own snow; the two reports are literatim iden-
tical, the second one being copied from the Amer. Mon. Micr.
Journ., 1891, p. 210, which took it verbatim from the Journal's
first one) gives the following. Sections of silvered material
are made, either without imbedding or after imbedding either
in paraffin or celloidin, care being taken in either case not to
use alcohol of a lower grade than 94 or 95 per cent. They
are brought from absolute alcohol into a mixture of eight to
ten drops of 1 per cent, solution of gold chloride with 10 c.c.
of absolute alcohol, which should be prepared half an hour
beforehand and exposed to diffused light until the sections
are placed in it, when it should be put into the dark. After
fifteen to thirty minutes therein, according to their thickness,
the sections are quickly washed in 50 per cent, alcohol, then
in water, then treated for five or ten minutes with 10 per
cent, solution of hyposulphite of soda. They are lastly
washed well with water, and may then be mounted at once
in balsam under a cover, or if desired may be previously
stained with carmine or hgematoxylin, or Pal's modification of
Weigert's process, or the like.

Obregia thinks that the reason why Sehrwald did not suc-
ceed in substituting gold for the silver in his preparations
(vide supra] is that he took the gold salt in aqueous solution.

This method is also applicable to material treated by Golgi's
sublimate process, § 760.

712. FICK (Zeit.f. wiss. Mik., viii, 2, 1891, p. 168) does not
admit Samassa's theory of the deterioration of sections through
diffusion currents (supra). He points out that bichromate of
silver is soluble in water, especially with the aid of heat ; and
after an elaborate series of experiments, concludes that the
water of the reagents or damp confined by the cover-glass is
the cause of the ruin of the preparations. Watery fluids
should be avoided, and sections should be mounted without a
cover, or on a cover raised free of contact with the slide by
means of wax feet, or the like. Or sections mounted without
a cover may be later on provided with one if the balsam be
first rendered perfectly anhydrous by careful heating.

This last method is also recommended by HUBER (Anat.
Anz., vii, 1892, p. 587; Joum. Roy. Mic. Soc., 1892, p. 707).

376 NERVES.

713. There remain to be mentioned some variations in the
details of the process, chiefly connected with the strengths of
the solutions to be used and the duration of the impregnation-
baths.

MONDINO (Arch, per le Sci. Med., viii, p. 45 ; Zeit. f. wiss.
Mik., 1885, p. 547) recommends that the preparations be left
in the bichromate mixture longer than directed by Golgi —
viz. from one to eight days ; and also that they be left for
over twenty-four hours in the silver-bath.

For some minutiae concerning the application of the bichromate and silver
method to cerebro-spinal nerves, see PETEONE, in Internat. Monatschr. f.
Anat., v, 1, 1888, or Zeit.f. wiss. Mik., v, 2, 1888, p. 238.

714. RAMON Y CAJAL, who has done a great deal of im-
portant work by Golgi's method, has always used the rapid
process. In Zeit. f. wiss. Mik., vii, 3, 1890, p. 332, he gives
it as follows : — Fresh muscle of insects put into a mixture of
20 parts 3 per cent, bichromate solution and 5 parts 1 per
cent, osmium solution for twelve to twenty-four hours, then
into 0'75 per cent, nitrate of silver for one day, then alcohol
of 40 degrees (presumably Baume, i. e. about 90 per cent.),
then clove oil followed by resinified turpentine and (if I under-
stand rightly) balsam. For the times and strengths used by
him in his researches on the cerebral cortex of mammals, see
his paper in La Cellule, vii, 1891, p. 125, or Zeit. f. wiss. Mik.,
ix, 2, 1892, p. 239 ; also Journ. Roy. Hie. Soc., 1892, p. 154.
He generally hardened for from five to eight days at a
temperature of about 25° C., and found it useful to adopt
Sehrwald's gelatin process (supra, last section) for avoidance
of peripheral precipitates. He prefers not to adopt Greppm's
treatment with hydrobromic acid, nor Obregia's treatment
with gold chloride, finding that although they serve to
render the preparations permanent, they obscure the finer
relations of fibres.

For embryos of the fowl he employs the same process ;
see his paper in Anat. Anz., v, 1890, p. 85, or Zeit. f. wiss.
Mik., vii, 2, 1890, p. 235.

715. In a paper on the structure and relations of the sympa-
thetic ganglia (which I have not seen, and quote from Zeit. f.
wiss. Mik., 1. c.) RAMON Y CAJAL describes a process of "in-
tensive" or "double" impregnation. After hardening for

MODIFICATIONS OF GOLGl's METHOD. 377

three days (embryos of fowl) in the osmium-bichromate mix-
ture, the preparations are put for thirty-six hours into nitrate
of silver solution (0*5 to O75 per cent.). They are then
brought back into the same osmium-bichromate mixture, or
into a weaker one containing only 2 parts of osmic acid solu-
tion to 20 of the bichromate. After treatment with this they
are washed quickly with distilled water, and put for a seconcf
time into the silver solution for thirty-six to forty-eight hours.
It is important to hit off the proper duration of the first
impregnation in the bichromate. If it has been too long
(four days) or too short (one day), the second impregnation
will not succeed. In this case a third impregnation must be
resorted to, the objects being again treated with the weak
osmium-bichromate mixture, and afterwards again with the
silver solution.

For retina the process is given as follows : — Put the retina
(batrachians and reptiles) with the sclerotic for twenty-four
hours into the weak osmium-bichromate mixture. Then put
it into 1 per cent, silver solution for twenty-four hours, then
back into the osmium mixture for twenty-four hours, then for
twenty-four hours again into the silver-bath.

716. OYARZUN (Arch.f. mik. Anat., xxxv, 1890, p. 380) used
the first-given process of Ramon y Cajal in his researches on
the fore-brain of Amphibia. He gives the times as twenty-
four hours in the osmium mixture, and the same in the silver-
bath. This is for frogs. For Triton and Salamandra twenty
hours is enough. Thirty to forty hours is too much.

717. VAN G-EHUCHTEN (La Cellule, vii, 1891, p. 81 ; viii, 2,
p. 225; see Journ. Roy. Mic. Soc., 1892, p. 153, and Zeit. f.
wiss. Mik., ix, 2, 1892, p. 237), who has done some very im-
portant work on nerve-centres with Grolgi's process, also
follows the first-given modification of Ramon y Cajal, with
this exception, that he employs formic acid to assist the action
of the silver-bath (as suggested by Ramon y Cajal in an early
paper). He treats the tissues (spinal cord and cerebellum in
small pieces) with the osmium mixture for two to two and a
half days at the ordinary temperature (or for less time at a
temperature of 35° to 40° C.) . They are then brought into
the silver-bath, to which is added a little formic acid (1 to 2
c.c. to a litre, or 1 drop to 100 c.c. will do; care must be taken

NERVES.

not to add too much). Twenty-four hours in the bath is
enough, but preparations may stay in it longer (in one case
as much as two months) without hurt, provided they be kept
in the dark. Wash, treat with alcohol for a quarter of an
hour, then for the same time with absolute alcohol, and the
same time with celloidin solution, after which they are
hardened for about the same time in 70 per cent, alcohol, cut
and mounted as usual.

718. SALA (Zeit. f. wiss. ZooL, Hi, 1, 1891, p. 18; Zeit. f.
wiss. Mik., viii, 3, p. 389), in a paper written in G-olgi's labora-
tory, gives the rapid process as follows (for the pes hippocampi
majoris of small mammals) : — Four to five days in 2 per cent,
bichromate of potash ; twenty-four to thirty hours in a bichro-
mate and osmium mixture containing 8 parts of the bichro-
mate solution to 2 of the osmium solution ; and a silver-bath
of 0'75 per cent, strength. He finds Greppin's hydrobromic
acid variation not merely useless, but hurtful. And he
thinks that Sehrwald's process for imbedding the material in
paraffin with the object of getting very thin sections is a
mistake. The chief quality of G-olgi's process is that it
admits of the following of nerve-cell processes for a very great
distance. Evidently this cannot be done with very thin sec-
tions. It is better simply to wash the preparations taken from
the silver-bath with water, fix them to a cork with gum, put
the whole into alcohol for a few hours to harden the gum,
and cut with a microtome without imbedding.

Other Nerve Methods.

719. Structure of Medullated Nerve. — In order to demonstrate
the axis-cylinder and the sheath of Schwann, the myelin may
be removed. This may be done by boiling in caustic soda,
and then neutralising ; by boiling in a mixture of absolute
alcohol and ether, and adding caustic soda; by boiling in
glacial acetic acid; by boiling in fuming nitric acid, and
adding caustic potash ; or by treating with eau de Javelle.

720. Neuroceratin Structures (GALLI, Zeit. f. wiss. Mik., iii, 1, 1886,
p. 467). — An ischiatic nerve is excised, and fixed and hardened for eighteen
to twenty minutes in solution of Miiller. Small portions of the nerve are
then further treated for one or two days with solution of Miiller diluted with
2 parts of water, then for a quarter of an hour with glycerin containing 1 or

MAECHl'S METHOD. 379

2 drops of glacial acetic acid for each cubic centimetre, and finally (without
previous washing with water) are stained for fifteen to twenty minutes in
aqueous solution of China blue (the best China blue for this purpose is that
supplied by the Badische Anilin- und Soda-Fabrik, at Stuttgart). They
are washed out in alcohol, cleared in essence of turpentine, and mounted in
dammar.

For the results see the paper and the plate, 1. c.

PLATNER (Zeit. f. wiss. Mik., vi, 2, 1889, p. 186) obtains a specific stain
of the neuroceratin framework of medullated nerve in the following way :—
Small nerves are fixed and hardened for several days in a mixture of 1 part
of Liq. Ferri Perchlor. (Ph. G. Ed. ii) and 3 to 4 parts of water or alcohol.
They are then to be washed out in water or alcohol till no traces of iron
remain in them (the reaction of the washings with rhodanide of potassium
being a good test of this), and are stained for several days or weeks in a con-
centrated solution of "Echtgriin" (dinitrosoresorcin, not " dinitroresorcin,"
as erroneously in Platner's paper) in 75 per cent, alcohol ; after which they
are dehydrated, imbedded, and sectioned.

See also the papers of GEDOELST in La Cellule, iii, 1887, p. 117, and v,
1889, p. 126 (good details of digestion methods) ; also the report in Zeit. f.
wiss. Mik., vii, 1, 1890, p. 57.

Further, in general, RANVIER, Traite. p. 718, et seq. ; REZ-
ZONICO, Arch, per le Sci. Med., 1879, p. 237 ; TIZZONI, ibid.,
1878, p. 4 (a process of boiling in chloroform for an hour or
two, then staining and mounting in glycerin) ; BOVERI, Zeit.
f. iviss. Mik., iv, 1, 1887, p. 91 ; JAKIMOVITCH, Journ. de I'Anat.,
xxiii, 1888, p. 142, or Zeit. f. wiss. Nik., v, 4, 1888, p. 526
(instructions for impregnating the axis-cylinder with silver,
followed by reduction in formic acid and amyl alcohol) ;

SCHIEFFERDECKER, in BEHRENS, KOSSEL, U. SCHIEFFERDECKER,

Das Mikroskop, Bd. ii, p. 227.

720 a. MARCHI'S Method for Degenerate Nerves. — I have not
been able to find out where this was first published, and take
the following from the report by Schiefferdecker of a paper
by EIJKMAN, in Zeit.f. wiss. Mik., ix, 3, 1893, p. 350. Nerves
are first hardened for a week in solution of Muller, and then
put for a few days into a mixture of 2 parts solution of
Muller and 1 part 1 per cent, osmic acid solution. The treat-
ment with the chrome salt deprives the medullary sheath of
normal fibres of the faculty of impregnating with osmium,
whilst the degeneration products in diseased sheaths retain
that faculty. In consequence the sheaths in normal nerves
acquire a yellow coloration, those of degenerated tracts a
black one.

380 NERVES.

Axis-cylinder Stains.

721. Amongst the chief methods for the study of the re-
lations of axis-cylinders or non-medullated nerve-fibres are
the gold methods given in Chaps. XII in Part I, and XXVII
and XXVIII in Part II. The method of Freud for nerve-
centres, which will be given in the next chapter, is also a
good one for the study of the topography of peripheral nerves.
But perhaps even more useful are the methods of GOLGI given
above, and the very important methylen-blue impregnation
methods that have been described in Part I, Chap. IX. The
following are also serviceable methods.

722. KTJPFFEE'S Method (Sitzb. math. phys. Kl. k. Bayr. Acad. Wiss.,
1884, p. 446 ; Zeit.f. wiss. Mile., 1885, p. 106).— A nerve is stretched on a
cork and treated for twenty-four hours with 0'5 per cent, osmic acid. It is
then washed in water for two hours and stained for twenty-four to twenty-
eight hours in saturated aqueous solution of Saurefuchsin ; after which it is
washed out for from six to twelve hours (not more in any case) in absolute
alcohol, cleared in clove oil, imbedded in paraffin, and cut. Sections are said
to show the axis-cylinder as a bundle of fibrils (stained red) floating in an
albuminous liquid.

723. NISSL'S Method (Miinchener med. Wochenschr., 1886, p. 528 ; Zeit.
f. wiss. Mik.,'\\\, 3, 1886, p. 398). — Bichromate objects to be treated as follows :
— Alcohol of 95 per cent. ; aqueous solution of Congo red, of 5 : 400 strength,
seventy-two hours ; alcohol of 95 per cent., five to ten minutes ; alcoholic
solution of nitric acid of 3 per cent, strength, six hours ; alcohol, fire
minutes ; clove oil ; balsam.

724. ALT (Munch, med. Wochenschr., 1892, No. 4 ; Zeit. f. wiss. Mik.,
ix, 1, 1892, p. 81) stains for a couple of hours in solution of Congo in abso-
lute alcohol, and washes out with pure alcohol. The results are said to be
specially adapted to the study of peripheral axis-cylinders. SCHIEFFERDECKER,
reporting on the method, does not recommend it. SQUIRE (using a 2 per
cent, aqueous solution) says it is one of the best stains he has tried.

725. Similar to this is one of the methods of REHM (Miinchener med.
Wochenschr., 1892, No. 13 ; Zeit. f. wiss. Mik., ix, 3, 1893, p. 390). Sec-
tions are stained for a few minutes in concentrated aqueous solution of
Congo, washed in alcohol, and treated for ten minutes, until they become
blue, with alcohol acidulated with hydrochloric or nitric acid, then cleared
with origanum oil and mounted. A sharp axis-cylinder stain with consider-
able richness of other detail.

But REHM prefers the following simple process : — Sections of alcohol-
hardened material are placed for one to two days in an aqueous solution of
haematoxylin of 0'5 per cent, strength, then washed out in aqueous solution
of carbonate of lithia (strength not given) until no more colour comes away

IODIDE OP PALLADIUM METHOD. 381

from them, dehydrated and mounted. Axis-cylinders, cells, and processes,
grey-black. The sections may be after-stained for a few minutes with O'l
per cent, aqueous solution of Bismarck brown.

726. The method of WOLTEES for axis- cylinder and cell
staining (his myelin stains have been given above as modifi-
cations of Weigert's) is as follows (Zeit. f. iviss. Nik., vii, 4^
1891, p. 471) :

The material (either central or peripheral nervous tissue) is
"hardened in liquid of KULTSCHITZKY, § 43, followed by alcohol,
as there described. It is imbedded either in celloidin or
paraffin, and cut. The sections are mordanted for twenty-
four hours in the chloride of vanadium and acetate of alu-
minium mixture used for Welters' myelin stain, ante, § 700,
washed for ten minutes in water, and stained for twenty-four
hours in the haematoxylin mixture, § 699. They are washed
out until they are of a light blue-red colour (it is not possible
to specify exactly the time required) in 80 per cent, alcohol
acidulated with 0'5 per cent, of hydrochloric acid. Remove
the acid thoroughly by washing with pure alcohol, dehydrate,
clear with origanum oil, and mount.

A sharp axis-cylinder stain, myelin being coloured only if
the differentiation in the acid alcohol is insufficient. It is
applicable to peripheral nerves, and also to the central
nervous system. In this, besides axis-cylinders, nerve-cells
and their processes are stained; pyramid cells, Purkinje's
cells and their processes, and glia cells are sharply brought
out. Fine images of epithelium-cells are also obtained; in a
word, besides axis-cylinders, every species of cells present in
the preparations are stained. This, and a superior certainty,
are the advantages which Wolters claims for his method over
that of GOLGI, to which it is in other respects comparable.

727. PALADINO'S Iodide of Palladium Method (Eendic. E.
Accad. Scienze Fis. e Mat., Napoli, iv, 1890, p. 14, and 1891
(1892), p. 227; Zeit.f. wiss. Mik., vii, 2, 1890, p. 237, and ix,
2, 1892, p. 238; Journ. Roy. Hie. Soc., 1890, p. 817, and 1892,
p. 439). — Pieces of material hardened in bichromate, chromic
acid, or corrosive sublimate, and not more than 5 to 8 mm. in
thickness, are put for two days into a large quantity (at least
150 to 200 c.c. for each piece) of O'l per cent, solution of

382 NERVES.

chloride of palladium (which may be made as directed in
§ 52). They are next put for twenty -four hours into a 1 per
cent, solution of iodide of potassium (the later paper quoted
says the iodide solution should be of 4 — 100 strength, and
that a relatively small volume of it should be taken, other-
wise the iodide of palladium, which is rapidly formed in the
tissues, may be again extracted by the liquid : small pieces of
tissue should not remain in it for more than one or two hours) .
Dehydrate ; imbed, if necessary, in paraffin by the chloroform
method; mount in balsam.

A brown stain, being both a myelin stain and an axis-
cylinder and cell-process stain, and applicable both to
central and to peripheral nervous structures. It is very well
spoken of.

HAEDENING BY THE FREEZING METHOD. 383

CHAPTER XXX.

CENTRAL NERVOUS SYSTEM.

728. Introductory. — For small objects, such as the spinal cord and
encephalon of very small Mammalia and of inferior Vertebrata, the ordinary
methods of microscopic anatomy are very often sufficient. The cord and
encephalon of Batrachia, for instance, may be treated as follows : — Corrosive
sublimate, half an hour or so ; alcohol of from 50 to 70 per cent., about two
hours ; then borax-carmine, or Mayer's paracarmine ; paraffin ; and sections
mounted in balsam. The same organs of somewhat larger animals, such
as the cat or rabbit, can also be prepared without recourse to any very
special methods. They may be fixed and hardened in liquid of Erlicki, or
other bichromate solution, well washed out with water, stained and imbedded,
or first imbedded in collodion and stained after sectioning with alum-carmine,
ammonia-carmine, or Heidenhain's haematoxylin, or, if it be desired to study
the topography of nerve-fibres, with Weigert's hsematoxylin. Even the
cord of man may be treated by these methods ; or rather, could be so treated
if it did not almost always come into the hands of the anatomist in a state
of post-mortem softening that necessitates special precautions in the
hardening process.

But the voluminous encephala of man and the larger Vertebrates cannot
be thus simply treated. They require specially modified methods for
hardening, for the manipulation of sections, and for staining.

These methods have lately been described with great completeness in the
works of BEVAN LEWIS (The Human Brain : Histological and Coarse
Methods of Research, London, Churchill) and OBEBSTEINER (Anleitung
beim Studium des Baues d. nervosen Centralorgane im gesunden u. Jcranken
Zustande, Leipzig, Toeplitz). These very welcome additions to the litera-
ture of the subject relieve me from the obligation of treating the subject
with all the minuteness that might be desired by specialists ; the more
so as they show that so to treat the subject requires a volume, not a
chapter.

Hardening.

729. Hardening by the Freezing Method. — This is in many cases a
very good method, and in particular may be of service for the histological
study of the cortex.

If it be desired to freeze an organ that has been already hardened by
reagents, the freezing may be done by means of a freezing mixture of ice
and salt ; but in this case the preparation should first be penetrated by a

384 CENTRAL NERVOUS SYSTEM.

mucilaginous or gelatinous freezing mass (§ 316, et seq.), in order to avoid
the formation of ice crystals in the tissues. But in the case of fresh tissue,
the ether freezing method is to be preferred. This method allows of rapidly
producing any desired degrees of hardness, and maintaining them or allow-
ing them to diminish as occasion may require, and is perhaps the only
method by which satisfactory sections of unhardened nerve-tissue can be
obtained.

The sections should be floated on to water, treated for a minute on the
slide with 0*25 per cent, osinic acid solution, and stained or otherwise treated
as desired.

For a detailed description of these manipulations see BEVAN LEWIS'S The
Human Brain.

730. Generalities on Hardening by Reagents. — If large pieces
of nerve-tissue are to be hardened, it is necessary to take
special precautions in order to prevent them from becoming
deformed by their own weight during the process. Spinal
cord or small specimens of any region of the encephalon may
be cut into slices of a few millimetres thickness, laid out on
cotton wool, and brought on the wool into a vessel in which
they may have the hardening liquid poured over them. The
wool performs two functions : it forms an elastic cushion on
which the preparations may lie without being distorted by
their own weight ; and it allows the reagent to penetrate by
the lower surfaces of the preparations as well as by their ex-
posed surfaces. A further precaution, which is useful, is to
hang up the preparations, lying on or in the cotton wool, in a
glass cylinder or other tall vessel ; by hanging them near the
top of the liquid the processes of diffusion and the penetra-
tion of the reagent are greatly facilitated.

If the preparations are placed on the bottom of the vessel,
they should never be placed one on another.

If it be desired to harden voluminous organs without divid-
ing them into portions, they should at least be incised as deeply
as possible in the less important regions. It is perhaps better
in general not to remove the membranes at first (except the
dura mater), as they serve to give support to the tissues. The
pia mater and arachnoid may be removed partially or entirely
later on, when the hardening has already made some pro-
gress.

The spinal cord, the medulla oblongata, and the pons Varolii
may be hardened in toto. The dura mater should be removed
at once, and the preparation hung up in a cylinder-glass,

HARDENING BY REAGENTS. 385

with a weight attached to its lower end. The weight has the
double function of preventing any part of the preparation
from floating above the level of the hardening liquid (a thing
that easily happens where somewhat dense liquids, such as
Miiller's solution, are used) , and of preventing the torsions of
the tissues that may otherwise be brought about by the elastic
fibres of the pia mater and arachnoid.

The cerebrum should be very delicately laid out on a layer
of cotton wool, or, if possible, hung up in it. Plugs of the
wool should be put into the fissure of Sylvius, and between
the operculum and the median lobe, and as far as possible
between the convolutions. Unless there are special reasons
to the contrary, the brain should be divided into two sym-
metrical halves by a sagittal cut passing through the median
plane of the corpus callosum. Betz recommends that after a
few hours in the hardening liquid the pia mater should be
removed wherever it is accessible, and the choroid plexuses
also. I have found this by no means easy, and think it is
an operation that can only be recommended for experienced
hands.

The cerebellum should be treated after the same manner.

The temperature at which the preparations are kept in the
hardening solution is an important point. The hardening
action of most solutions is greatly enhanced by heat. Thus
WEIGERT (Centralb.f. d. med. Wiss., 1882, p. 819; Zeit.f. wiss.
Mile., 1884, p. 388) finds that at a temperature of from 30° to
40° C. preparations may be sufficiently hardened in solution
of Miiller in eight or ten days, and in solution of Erlicki in
four days ; whilst at the normal temperature two or three
times as long would be required.

But it is not certain that this rapid hardening always gives
the best definite results. SAHLI, who has made a detailed
study of the hardening action of chrome salts, is of opinion
that it does not, and thinks it ought for this reason to be aban-
doned (see Zeit.f. iviss. Mik.t 1885, p. 3).

On the other hand, the slowness of the action of chromic
salts at the normal temperature is such that decomposition
may easily be set up in the tissues before the hardening and
preserving fluid has had time to do its work. For this reason
voluminous preparations that are to be hardened in the slow

25

386 CENTEAL NERVOUS SYSTEM.

way should be put away in a very cool place — best of all in
an ice-safe.

731. The Reagents to he employed. — The hardening agents
most used are the chromic salts. Chromic acid was much
used at one time, but most workers now agree that its action,
though much more rapid than that of the salts, is much more
uneven, and frequently causes a disastrous friability of the
tissues. Osmic acid is excellent — according to BEVAN LEWIS,
it is the best of hardening agents ; but its employment is un-
fortunately very restricted, as it can hardly be used for objects
of more than a cubic centimetre in size.

It has already been noted that the liquid of Erlicki has a
more rapid action than the other solutions of chromic salts ;
for this reason it is one of the most commonly employed
solutions. SAHLI, however (1. c.), after having studied the
action of the usual solutions, concludes that the best harden-
ing agent for fresh tissues is pure bichromate of potash, in
3 or 4 per cent, solution, the hardening being done in a cold
place. And he does not approve of the addition of sodium
sulphate (Miiller), and rejects the liquid of Erlicki on account
of the precipitates it so frequently gives rise to (see § 77).

OBERSTEINEK is of the same opinion, recommending pure
iDichromate for general hardening purposes; whilst for the
study of the most delicate structural relations he recommends
fixing in Fol's modification of Flemming's liquid (§ 35) for
twenty-four hours, followed by washing with water and harden-
ing in 80 per cent, alcohol.

In view of the slowness of penetration of chromic salts it is
often advisable to treat preparations for twenty-four hours or
more with alcohol of 80 to 90 per cent, before putting them
into the hardening liquid, in order to avoid maceration of the
deeper layers of tissue.

For the question as to how far certain so-called pathological alterations
of ganglion-cells should be attributed to putrefactive changes or to the
influence of reagents see Neurologisches Centralb. for the years 1884
and 1885.

As to the so-called " pigment spots " produced by the liquid of Erlicki
see supra, § 77.

732. Strengths of the Reagents. — All hardening reagents
(except osmic acid) should at first be taken as weak as is con-

STRENGTHS OF THE REAGENTS. 387

sistent with the preservation of the tissue,, and be changed by
degrees for stronger.

Osmic acid may be taken of 1 per cent, strength, and will
harden small pieces of tissue sufficiently in five to ten days

(EXNER).

Bichromate of potash should be taken at first of not more
than 2 per cent, strength ; this is then gradually raised to 3
or 4 per cent, for the, cord and cerebrum, and as much as 5
per cent, for the cerebellum. Obersteiner begins with 1 per
cent., and proceeds gradually during six to eight weeks to 2
or 3 per cent. (This is at the normal temperature; at a tem-
perature of 35° to 45° C. the hardening can be got through
in one or two weeks.)

Bichromate of ammonia should be taken of half the
strength recommended for bichromate of potash, or even
weaker at first ; it may be raised to as much as 5 per cent, for
cerebellum towards the end of the hardening.

Chromic acid is not much used alone. See § 69. It forms part of some
of the mixtures mentioned below. A very little chromic acid (say one to
two drops of 1 per cent, solution for each ounce) added to bichromate
solution will do no harm, and will quicken the hardening.

Nitric acid has been and still is employed in strengths of 10 to 12 per
cent., and gives particularly tough preparations. Perhaps a weaker solution
might give good results, but I cannot find that any such have been tried.

Neutral acetate of lead in 10 per cent, solution affords an excellent
preservation of ganglion-cells, according to ANNA KOTLAREWSKI (see Zeit.
f. wiss. MiJc., iv, 3, 1887, p. 387).

TEZEBINSKI (Virchoiv's Arch., 1887, p. 1 ; Zeit.f. wiss. Mik., iv, 4, 1887,
p. 497) finds that as regards the faithful preservation of ganglion-cells (of
the spinal cord of the rabbit and dog) the best results are obtained by
hardening for eight days in 10 per cent, solution of corrosive sublimate,
followed by hardening in alcohol containing 0'5 per cent, of iodine.

DIOMIDOFF (ibid., p. 499) also obtained very excellent results by harden-
ing small pieces of brain (as suggested by GAULE, OGATA, and BECHTEREFF)
for from five to nine days (not more in any case) in 7 per cent, sublimate
solution, and then putting the tissues for twenty-four hours into 50 per
cent, alcohol, and for the same time into 70 per cent, and 96 per cent,
alcohol successively. (This process produces artificial "pigment spots,"
similar to those produced by solution of Erlicki ; they may be dissolved
out by prolonged treatment with warm water, or in five minutes by strong
solution of LUGOL.) The tissues are of a good consistence for cutting.
Chloride of zinc has been recommended for some purposes (see below,
§ 780).

The next following paragraphs give in detail some methods of hardening
recommended by some of the most competent workers.

388 CENTRAL NERVOUS SYSTEM.

733. Spinal Cord (KRAUSE, Arch.f. mik. Anat., 1875, p. 226).
— Solution of Mull er, twenty-four hours. Then chromic acid,
1 per cent. On the fourth day the solution must be changed
for fresh. A few days later (when the cord appears hard)
the chromic acid is removed by means of water, and the cord
put into spirit, followed by absolute alcohol, which must be
changed at least twice.

CIAGLINSKI (Zeit.f. wiss. Mik., viii, 1, 1891, p. 19) recom-
mends hardening for three or four months in solution of
Miiller, renewed every day during the first week, and after-
wards as often as it becomes turbid. Liquid of Erlicki
will harden in as many weeks, but has the defect that if
preparations are left longer in it they are injured by the
formation of the above-mentioned precipitates (last section
and § 77).

734. Encephalon (M DUVAI/S methods, EOBIN'B Journal de
I'Anatomie, 1876, p. 497).— First Method.— Place the fresh
tissues in solution of bichromate of potash 25, water 1000 ;
change the liquid after the first twenty-four hours, and again
after three or four days. After two or three weeks place the
preparations in chromic acid of 3 per 1000, change the liquid
every day for the first week, and after that every eight days
until the middle of the second month, after which time it is
no longer needful to change the liquid. The preparations
must remain at least two months in the chromic acid ; the
longer they remain in it the better. A few fragments of cam-
phor should be added to the liquid in order to prevent the
growth of mould.

Second Method. — Place the fresh tissues in a mixture of equal parts of
glycerin and acetic acid ; after twenty-four hours remove them to Miiller's
solution, and after forty-eight hours more to chromic acid. (The strength
of the solution is not indicated.) Change the chromic acid once or twice,
and the preparations will be fit for cutting in about eight or ten days.
(Small encephala [rat, bat] need not be extracted from the cranium provided
this be largely opened before immersing them in the glycerin, nor need
they be extracted before cutting, as the cranium will be found to be com-
pletely decalcified.)

This method is highly expeditious, and furnishes hardened tissues of a
singularly homogeneous consistence, quite without fragility, but it should
only be employed for the purpose of obtaining general views of structural
relations, as the anatomical elements are somewhat changed by it : cells and
axis-cylinders swell.

BEAIN. 389

735. Cerebrum (RUTHERFORD ; from BEVAN LEWIS). — Place
small portions of cerebrum in methylated spirit for twenty-
four hours in a cool place. After that put them into the fol-
lowing solution :

Chromic acid .... 1 part.

Bichromate of potash ... 2 parts.

Water 1200 „

Change the liquid at the end of eighteen hours, and then
once a week. Should the tissue not be sufficiently tough for
cutting at the end of six weeks place it in a -i per cent, solu-
tion of chromic acid for a fortnight, and then in rectified
spirit.

736. Cerebrum (BEVAN LEWIS, The Human Brain, p. 102). —
Methylated spirit, twenty-four hours in a cool place. Miiller's
solution, three days in a cool place. Then change the liquid ;
and after three days more change it again, or, preferably,
substitute a 2 per cent, solution of potassium bichromate. At
the end of the second week a solution of double the strength
may be added; and if, at the termination of the third week,
the mass is still pliable and of the consistence of ordinary
rubber, it is as yet unfit for section cutting, and the reagent
should be replaced by a solution of chromic acid.

737. Brain (HAMILTON, Journ. of Anat. and PhysioL, 1878,
p. 254). — Take a fresh brain and make a series of incisions
into different parts, still keeping- everything in situ ; or slice
it into any number of segments about one inch thick, but of
the whole length or breadth of the organ, as may be desired.
Do not remove the membranes ; they form a protection for the
superficial layers, and do not interfere with the hardening
process. The large segments are placed flat in a large vessel
padded with cotton ; do not put them one above the other.
Cover them with the following fluid :

Miiller's fluid ... 3 parts.

Methylated spirit . . 1 part.

(Heat is evolved on mixing these liquids, and the mixture
must be allowed to cool before pouring it over the brain
tissue.) Put the preparations away in an ice-safe. Turn the
segments over next day. Change the solution in a fortnight
or three weeks ; or if on examining a section of one of the
pieces it is found that the hardening reagent has penetrated

390 CENTRAL NERVOUS SYSTEM.

to the interior, they may be at once removed to the following
mixture :

Bichromate of ammonia . . 1 grm.,

Water . . . .400 c.c.,

in which they remain for one week. Then change the solu-
tion to one of 1 per cent, for one week ; and let this be followed
by a solution of 2 per cent, for another week, or longer if
required. The pieces will now be sufficiently hard for cutting ;
they may be kept permanently in solution of chloral hydrate,
twelve grains to the ounce.

Probably the chloral hydrate serves to attenuate the yellow
coloration produced by the chromic liquids.

This is a process particularly adapted to the preparation of
large segments of brain. The consistence is very tough and
firm.

738. Entire Encephalon (DEECKE, Journ. Roy. Mic. Soc., 1883,
p. 449). — "To harden the entire brain so that the inside and
the outside shall be hardened equally and properly, Dr. Deecke
finally adopted bichromate of ammonia in | to 1 per cent,
solution, according to the consistence of the brain. When
nominally soft he adds say ^ to ^ per cent, of chromic
acid to the solution, and always £ to J of the whole volume
of alcohol. It is then placed in a refrigerator and the fluid
changed frequently. After a month add a little more alcohol
from week to week until the alcohol is 90 per cent. This is
changed as often as it is discoloured. The treatment requires
from twelve to eighteen months."

739. BETZ'S Methods (Arch. f. mik. Anat., 1873, p. 101).— The spinal
cord, medulla oblongata, and pons Varolii are treated as follows : — The
dura mater is removed, and they are hung up in a cylinder containing 75 to
80 per cent, alcohol, to which is added enough iodine to produce a light
brown coloration. After from one to three days the preparation will be
found to be somewhat surf ace -hardened ; it is taken down, and the pia
mater and arachnoid are removed. If the pia mater does not come away
completely enough the preparation is put back for some days into the
alcoholic iodine. The membranes having been removed, the preparation
is put back into the original fluid, which is found to have become colourless
owing to absorption of the iodine by the tissues. Fresh quantities of a
strong solution of iodine in alcohol are from time to time added to the
liquid in order to keep it at its original strength of iodine (as shown by the
colour). If the membranes have been carefully removed it will be found
that after about six days the preparation ceases to take up further quantities
oi. iodine. The preliminary hardening may now be considered complete.

BETZ'S METHODS. 391

The preparation is now brought into a 3 per cent, solution of bichromate
of potash. (A small weight is attached to it to prevent any portion of it
from floating above the surface of the liquid. After a day or two it will
have lost much of its alcohol, and will sink to the bottom of the vessel,
which is equally undesirable ; this must be watched for, and the preparation
hung up or otherwise supported.) The vessel is put away in a cool place.
As soon as a brown turbidity is seen in the liquid, together with a brown
deposit on the preparation, the hardening may be considered to be complete/
The preparation must be at once washed with water, and put away until
wanted in a £ to 1 per cent, solution of bichromate.

Cerebellum. — Must be quite fresh, and before placing in the iodine the
membranes and vessels must as far as possible be very carefully removed.
(If the pia mater does not come away freely, the organ must be macerated
for a few hours in iodine solution in which other preparations have been
kept, and which is diluted before using for this purpose.) The membranes
having been removed, the cerebellum is placed (supported on cotton wool,
with which the different organs are so propped up as to preserve their
natural position) in solution of iodine for two or three days, and fresh
iodine solution frequently added.

The pia mater is now removed from the rest of the preparation, which is
put back for seven to fourteen days into the iodine solution. If at the
expiration of this time it be found that the cerebellum can be supported on
the finger by the vermiculus alone without bending, the preliminary harden-
ing is complete, and it is brought into a 5 per cent solution of bichromate,
where it remains until fit for cutting.

Cerebrum. — The cerebrum is divided into two halves along the median
line of the corpus callosum, and put into the iodine solution. After a few
hours the pia mater is removed from the fissure of Sylvius and from the
corpus callosum, and if possible the choroid plexus is removed likewise.

The preparation is now put away in the iodine solution in a cool place
(in summer in a cool cellar), and fresh iodine added as soon as the liquid is
seen to lose colour (which must be watched for). After twenty-four to
forty-eight hours the remaining pia mater is carefully removed by means
of scissors and forceps from the fissures and convolutions, and one half-
volume of fresh iodine solution is added to the liquid. (To facilitate the
penetration of the liquid, wads of cotton wool are stuffed into the fissure
of Sylvius, between the operculum and the median [central] lobe, in the
direction of the descending cornu, and between the convolutions). After
twenty-four to seventy -two hours the brain is brought into fresh solution
of iodine in 70 per cent, alcohol, where it remains until the hemispheres are
hard enough to be supported on two fingers without bending. (This will
not be before ten to fourteen days.) It is then put into 4 per cent, solution
of bichromate and left to acquire its definitive hardness. If an excessive
brown deposit make its appearance, and the brain be found notwithstanding
to be not hard enough for cutting, it must be rinsed with water and the
bichromate solution changed. When ripe for cutting the brain ought to
show an almost equal intensity of yellow-brown stain over the whole surface
of a cut made through the total thickness of a hemisphere.

392 CENTRAL NERVOUS SYSTEM.

Brains that are not fresh require for hardening longer time and stronger
alcohol.

Instead of the iodine solution, it is possible to use for the preliminary
hardening a mixture of equal volumes of chloroform and ether ; but this
mixture is not to be recommended, on account of its solvent action on proto-
plasm and on the processes of ganglion-cells.

The methods of Betz are particularly adapted to the hardening of volu-
minous specimens, and of tissues that are in a state of post-mortem softening.

740. Osmic Acid (ExNEB, Sitzb. It. Akad. Wiss. Wien, 1881, Ixxxiii,
3 Abth. ; BEVAN LEWIS, The Human Brain, p. 105). — A small portion of
brain, not exceeding a cubic centimetre in size, is placed in ten times its
volume of 1 per cent, osmic acid. The solution should be replaced by fresh
after two days, a proceeding which may advantageously be repeated at the
end of the fourth day. In from five to ten days the piece is usually
hardened throughout, and may be washed with water, treated with alcohol,
and imbedded. The sections may be treated by a drop of caustic ammonia,
which clears up the general mass of the brain substance, leaving medullated
fibres black. B. Lewis says that this method exhibits a wealth of structure
which no other method displays. The sections may be mounted in soluble
glass. The chief value of this method is for tracing the course of medul-
lated fibres.

741. BELLONCI (Arch. Hal. de BioL, vi, p. 405) employed this method in
his researches on the optic nerve of mammalia. He used an osmic acid
solution of 0'5 to 1 per cent., hardened for only fourteen to twenty hours,
made sections, and treated them for three or four hours with 80 per cent,
alcohol, and then with ammonia.

742. Nervous Centres of Reptiles and Batrachia (MASON, Central
Nervous System of Certain Reptiles, &c. ; WHITMAN'S Methods, p. 196). —
Iodised alcohol, six to twelve hours ; 3 per cent, bichromate, with a piece of
camphor in the bottle, and to be changed once a fortnight until the harden-
ing is sufficient (six to ten weeks).

BUECKHAEDT (Das Centralnervensystem von Protopterus, Berlin, 1892 ;
Zeit.f. wiss. Mik., ix, 3, 1893, p. 347) recommends a liquid composed of
300 parts of 1 per cent, chromic acid, 10 parts of 2 per cent, osmic acid, and
10 parts of concentrated nitric acid, in which brains of Protopterus are
hardened in twenty-four to forty-eight hours.

Imbedding and Cutting.

743. The Methods of Imbedding. — The paraffin infiltration
method can only be used for the smaller objects of this class.
Human spinal cord (which is quite at the upper limit as re-
gards size) can be properly penetrated with paraffin by taking
the precaution of first cutting it up into slices of not more
than a few millimetres — preferably not more than one — in
thickness. The largest objects of this class, such as entire

THE METHODS OF IMBEDDING. 393

hemispheres of man, cannot be properly penetrated by any
known imbedding mass ; and the anatomist must be content
with simple superficial imbedding — a proceeding which is
here of the greatest service. For intermediate objects — those
whose size varies between that of a small nut and a walnut —
it appears to me that no hesitation as to the proper course is
possible; such objects should be treated by the collodion
method, which is at once the safest, the most convenient, and
the most advantageous, as regards the ulterior treatment of
sections.

BEVAN LEWIS recommends the ether freezing method for fresh brain.
For hardened brain he recommends some of the old-fashioned wax-and-oil
and other fatty mixtures, a doctrine at which I am surprised.

HAMILTON recommends freezing in the gum and syrup mass given above,
§ 316. He also recommends a method of penetrating with collodion, which
is hardened in the usual way, the hardened mass being cut with an ice-and-
salt freezing microtome (see Journ. of Anat. and Physiol., 1887, p. 444 ; or
Journ. Roy. Mic. Soc., 1888, p. 1051).

For sections of entire human brain, DEECKE (1. c., § 738) proceeds as fol-
lows : — The brain to be cut is placed upon the piston of the microtome (a
Eanvier model) and held in situ by several pieces of soft cork. It is then
imbedded in a cast of paraffin, olive oil, and tallow, which, after it has
become hard, is held in position by a number of small curved rods attached
to, and projecting upwards from, the piston to the height of about an inch.
Before cutting, and as it proceeds, the cast is carefully removed from around
the specimen to the depth of about half an inch (which is easily done by the
use of a good-sized carpenter's chisel), so that the knife never comes in
contact with the cast.

Cutting is done under alcohol, the entire microtome being immersed in a
copper basin. The sections are floated, with the aid of a fine camel-hair
brush, on to sheets of glazed writing-paper. They are removed thereon
successively into staining, washing, and clearing fluids. After clearing,
they are brought on the paper on to a slide, and the paper is gently pulled
away from them ; they are then mounted in chloroform- or benzol-balsam.

It should be noted that the membranes should not be removed from the
brain ; they present no obstacle to cutting if this is done with a slight
sawing movement, or with a series of short cuts, instead of one sweep of
the knife. By this plan the sections are much more perfect and uniform in
thickness, and the loss in a series of from four to five hundred to the inch
through the entire cerebrum of man may not amount to more than 2 or
3 per cent.

OSBOKN (Proc. Acad. Nat. Sci. Philadelphia, 1883, p. 178, and 1884,
p. 262 ; WHITMAN'S Methods, p. 195) found advantage in employing Ruge's
egg-mass (for the brain of Urodela). He recommends that the mass be
injected into the ventricles.

The paraffin and collodion methods have already been sufficiently described
in Part I.

394 CENTRAL NERVOUS SYSTEM.

CIAQLINSKI (Zeii. /. wis8. Mik., viii, 1, 1891, p. 22) gives the following
process for imbedding spinal cord : — Dehydrated segments of hardened cord
are left for twenty-four hours in anilin oil, and then put for two or three
hours into xylol. They are then brought into a mixture of equal parts of
xylol and melted paraffin, and kept therein for twenty hours at a tempera-
ture of 30° C. They are then put through a twenty-four hours' bath of
pure paraffin to which a few drops of cedar oil have been added (said to
make the paraffin more elastic), the paraffin taken being of 45° C. melting-
point for winter work, and 52° C. for hot summer weather. Lastly, they
have a short bath of pure paraffin without cedar oil.

FEIST (ibid., viii, 4, 1892, p. 492) gives a useful hint for
marking the right and left sides of spinal cord. He imbeds
with each segment of the cord a small cylinder (of about 1
square millimetre in section) of hardened liver, stuck verti-
cally in the imbedding mass (either celloidin or paraffin)
against the side of the cord that it is desired to mark.

Staining.

744. The Kinds of Stains. — By a "general stain" is meant
one by which it is intended to demonstrate as far as possible
all the histological elements of a preparation.

It does not seem possible to divide the stains used in the
study of the central nervous system accurately into the two
categories of general and special. But an incomplete division
is better than none at all, so I have grouped the methods as
general and special, meaning by the latter term those that
are principally intended for the demonstration of the relations
of nerve-fibres and ganglion-cell processes, and that make no
claim at all to the demonstration of other elements.

The nuclear stains that are so important in other branches
of histology are here much less useful, partly because the
nuclei of nerve-cells happen to be remarkably poor in chro-
matin, and partly because the chief point of interest is not
the relations of nuclei, but the relations of fibres to one another
and to cells.

A. General Stains.

745. Ammonia-carmine is old-fashioned, but may be used for general
views. Scale's formula is a good one, especially where prolonged staining
is required. But in view of the greatest precision of stain, the process of
GIEEKE, or that of BETZ, should be preferred (see § 165).

Picro-carmine is also an excellent reagent. It has much the same action

GENERAL STAINS. 395

as ammonia-carmine, but gives a better demonstration of non-nervous
elements.

Chromic objects stain very slowly in both these media. Sections may,
however, be stained with them in a few minutes if they be put into a watch-
glass with the stain, and the whole be kept on a wire-net over a water-bath
heated to boiling-point (OBEBSTEINEB). See also § 163.

746. HENLE (Handb. d. Nervenlehre, 1871) gives the following, after
MEBKEL. In order to do away with the slowness of staining of tissues
hardened in chromates, sections should be placed in solution of chloride of
palladium (1 in 300 to 1 in 600) till they are of a straw colour (one or two
minutes), rinsed in water, and stained in strong ammonia-carmine. Myelin,
yellow ; axis-cylinders, nerve-cells, and neuroglia, deep red.

747. SCHMAUS (Munch, med. Wochenschr., 1891, No. 8 ; see Zeit. f.
wiss. Mik., viii, 2, 1891, p. 230, and Journ. Boy. Mic. Soc., 1892, p. 439)
obtains a ground-stain and axis-cylinder stain by means of a uranium
soda-carmine solution.

748. UPSON (Neurolog. Centralb., 1888, p. 319 ; Zeit. f. wiss. Mik., v,
4, 1888, p. 525) employs a stain made by adding to 5 c.c. of strong Gren-
acher's alum-carmine 1 to 3 drops of phospho-rnolybdenic acid. In this,
sections stain in from two to ten minutes.

He also employs a stain made by saturating alum-carmine with zinc
sulphate. Sections stain in this in from half an hour to twelve hours.
This is said to give very good differentiations of the elements of peripheral
nerves.

A third method of the same author consists in staining with carminic acid,
and afterwards treating with a mordant. See the papers quoted.

FBEEBORX (Amer. Mon. Mic. Journ., 1888, p. 231 ; Journ. Roy. Mic.
Soc., 1889, p. 305) also recommends carminic acid, followed by treatment
with 10 per cent, solution of chloride of iron.

749. Borax-carmine may be used. It is chiefly useful when employed
for double-staining with indigo-carmine or an anilin blue to follow. I have
obtained some superb stains with Seller's borax-carmine and indigo-carmine
process (§ 241). MEBKEL'S mixture of borax-carmine and indigo-carmine
(§ 242) has been strongly recommended by MAX ELESCH (Zeit. f. wiss. Mik.,
1884, p. 566, and 1885, p. 349), who says that it gives extremely rich and
instructive images.

DUVAL (Journ. de I'Anat., 1876) speaks very highly of the double-stain
quoted ante, § 243.

750. Alum-carmine (Grenadier's or Csokor's) may be used as a nuclear
stain (OBERSTEINER). The stain principally takes effect on non-nervous
nuclei.

751. KEHM (Munch, med. Wochenschr., 1892, No. 13 ; Zeit. f. wiss. Mik.,
ix, 3, 1893, p. 389) gives the following: — Sections (of alcohol-hardened
material) stained for five minutes in 1 per cent, ammonia-carmine, and
washed out in 70 per cent, alcohol acidified with 1 per cent of nitric acid ;
the acid removed by pure alcohol ; the sections stained for half a minute

396 CENTRAL NERVOUS SYSTEM.

in O'l per cent, solution of rnethylen blue, differentiated in alcohol, cleared
in origanum oil, and mounted in colophonium. Nuclei and axis-cylinders,
red. This stain is said to prove that the nuclei of the ganglion-cells of the
rabbit contain only one true nucleolus, instead of two or more, as has been
hitherto believed.

752. For a plasma-stain REHM (1. c.) gives a method modified from
NISSL. Sections of alcohol-hardened material are stained for half a minute
to a minute in a hot O'l per cent, solution of methylen blue, washed in
96 per cent, alcohol till no more colour comes away, cleared with origanum
oil, and mounted in balsam or benzin-colophonium. Nerve-cells, dark blue ;
connective-tissue cells lighter, and greenish.

753. To obtain a sharper distinction between nerve-cells and connective-
tissue cells, REHM stains as before in the hot methylen-blue solution for
not more than half a minute, and washes out as before with 96 per cent,
alcohol. The sections are then stained for fifteen to thirty minutes in a O'l
per cent, solution of fuchsin in 96 per cent, alcohol, washed out for a minute,
until no more red colour comes away, in alcohol, cleared in clove oil, and
mounted. Nerve-cells blue-red, their nuclei being unstained ; nuclei of
connective tissue and of vessels, brilliant red.

This distinction is not obtained in embryonic tissues.

Nuclei of connective tissue and vessels may also be brought out by
staining for a few minutes in 1 per cent, aqueous solution of eosin, fol-
lowed by a few minutes in warm O'l per cent, aqueous solution of dahlia,
dehydration, and mounting (the nuclei blue, all else red). Or 1 per cent,
aqueous solution of nigrosin may be taken instead of the eosin, and O'l per
cent, alcoholic solution of fuchsin (half an hour) instead of the dahlia
(nuclei red, all else grey-blue).

754. Haematoxylin may be used as a general stain. Bevan
Lewis recommends the formula of Kleinenberg, or a formula
that he attributes to Minot, which is essentially the same as
Bohmer's. BERNHEIMER'S formula, which is quoted in regard
to the retina, § 683, is practically the same thing.

MALLORY'S Phospho-molybdic Acid Haematoxylin Stain has
been given in § 195.

SCHIEFFERDECKER and YoBis find that celloidin sections of
material hardened in Miiller stain well in it.

755. HAUG (Zeit.f. wiss. Mik., vii, 2, 1890, p. 153) gives the following
modification of WEIGEBT'S process, as being more convenient to carry out,
and serving to demonstrate all the elements of spinal cord with equal clear-
ness. Small segments of fresh cord are put into saturated aqueous solution
of neutral acetate of copper. After two days (or, for large specimens, longer)
they are put for a day or a day and a half into a 5 per cent, or even saturated
solution of bichromate of potash. They are next rinsed with water and put
for thirty-six to forty-eight hours into 70 per cent, alcohol in the dark.
They are then treated for the same time with absolute alcohol, also in the

GENERAL STAINS. 397

dark ; then imbedded, either in paraffin or celloidin, and cut. The sections,
after removal of the paraffin (if that have been used), are treated with
alcohol, and then with water. They are stained for a quarter or half an
hour in a well-ripened mixture of 1 part of haematoxylin, 30 parts of
alcohol, 1 part of ammonia alum, and 300 parts of water (or they may be
treated for a quarter of an hour to an hour with saturated solution of car-
bonate of lithia, and then for an hour, in total darkness, with simple satu-
rated aqueous solution of hsematoxylin). After staining to a deep blaclc
they are rinsed in water and differentiated until they turn red (a quarter of
an hour at most), either in 70 per cent, alcohol acidified with 0*5 to 1 per
cent, of hydrochloric acid or in a solution containing 0*5 part of oxalic acid,
O'l to 0'25 of hyposulphite of soda, and 100 of water. They are then
washed with pure water until they turn blue or bluish grey ; after which
they may either be mounted, or first counterstained by momentary immer-
sion in a strong solution of neutral carmine. If desired, sections after
staining may be differentiated in Weigert's borax-ferricyanide liquid, instead
of the liquids here given.

756. Alizarin has been recommended by BENCZUE (see THANHOFFEB'S
Das Mikroskop; or Zeit. f. wiss. Mik., 1884, p. 97). It is directed that
sections be stained for twenty-four hours in a concentrated solution of
alizarin in alcohol.

Anilin blue alone is useful for the demonstration of ganglion-cell pro-
cesses (see ZuFPLN'GEE, in Arch. f. mik. Anal., 1874, p. 255).

757. Anilin blue-black was first recommended by SANKEY
(Quart. Journ.Mic. Sci., 1876, p. 69). He stained in a 0'5
per cent, solution, and, in order to obtain a differential stain,
washed out for twenty to thirty minutes in solution of chloral
hydrate. BEVAN LEWIS (Human Brain, p. 125) considers this
to be one of the most valuable stains for nervous centres. He
stains sections for an hour in 0*25 per cent, aqueous solution,
and clears and mounts (in the case of brain or cord sections) ;
for the cortex of the cerebellum he washes out for twenty to
thirty minutes in 2 per cent, chloral solution. SANKEY and
STIRLING have also used anilin blue-black in a much weaker
solution, which Bevan Lewis does not recommend. YEJAS,
however (Arch. f. Psychiatric, xvi, p. 200), obtained good
results by staining from eighteen to twenty-four hours in a
solution of 1 in 3000.

GriERKE (Zeit. f. wiss. Mik.j 1884, p. 379) was not able to
obtain good results with anilin black procured in Germany,
and finds that the treatment with chloral is injurious to the
preservation of the tissues. MARTINOTTI (ibid., p. 478) comes
to the same conclusion.

LUYS (Gaz. med. de Paris, 1876, p. 346) greatly recommends

398 CENTRAL NERVOUS SYSTEM.

the anilin colour known as Noir Colin. He stains for three to
four minutes in a 0*1 per cent, solution.

JELGEBSMA (Zeit.f. wiss. Mik., 1886, p. 39) finds that anilin
blue-black gives excellent results, provided that the English
preparation of the colour be alone employed. He makes solu-
tions of 1 : 100, 1 : 800, and 1 : 2000, of which the first stains
sections in a quarter of an hour, the second in five hours, the
third in twelve hours. The stain takes effect on ganglion-
cells and their processes, and on axis-cylinders, but does not
demonstrate neuroglia or connective tissue.

SCHMAUS (Munch, med. Wochenschr., No. 8, 1891, p. 147 ;
Zeit.f. wiss. Mik., viii, 2, 1891, p. 230) recommends English
blue-black in 0'25 per cent, solution in 50 per cent, alcohol,
with the addition of a little picric acid ; sections to be stained
for an hour. The addition of picric acid has the advantage of
leaving celloidin almost colourless, whilst it stains strongly in
pure aqueous solutions of blue-black.

MARTINOTTI (1. c., 1884, p. 478) finds that picro-nigrosin
gives very good results, especially for pathological objects.
He stains for two or three hours or days in a saturated
solution of nigrosin (§ 125) in saturated solution of picric
acid in alcohol, and washes out in a mixture of 1 part of
formic acid with 2 parts of alcohol until the grey matter
appears clearly differentiated from the white to the naked
eye.

758. KAISER (Zeit.f. wiss. Mik., vi, 4, 1889, p. 471) advises, for celloidin
sections of spinal cord, naphthylamin brown (obtainable from Griibler).
Sections are stained for a few hours in a solution containing 1 part of
naphthylamin brown, 200 parts of water, and 100 parts of alcohol, washed
with alcohol, cleared with origanum oil, and mounted. Chromophilous
ganglion-cells, dark brown ; chromophobous cells, light on a dark ground.
Black-ground illumination is recommended. The stain has the advantage
of not over-staining, and not staining celloidin to an injurious extent.

759. The EHRLICH-BIONDI mixture of methyl green, orange,
and Saurefuchsin, which has been given in the chapter on
" Combination Stains," § 259, is a good stain for nerve-
centres, especially (Dr. LINDSAY- JOHNSON informs me) if there
be added to it about one-third of 20 per cent, (saturated)
solution of nigrosin.

760. GOLGI'S Sublimate Method. — Last, but not least, as a
general stain we have the sublimate method of GOLGI (Archivio

GENE HAL STAINS. 399

per le Scienze Nediche, 1878, p. 3). This method, which may
be said to be in principle identical with the bichromate of
potash and silver nitrate method of the author, consists, like
the latter, of two processes : 1, hardening in bichromate ; 2,
treatment with bichloride of mercury.

For hardening, use either a solution progressively raised
in concentration from 1 per cent, to 2 4 per cent., or Mutters
solution. Take small pieces of tissue (not more than 1 to 2
c.c.), large quantities of liquid, and change the latter fre-
quently, so as to have it always clear. Fifteen to twenty
days' immersion will suffice, but twenty to thirty should be
preferred.

The tissues are then passed direct from the bichromate
into the bichloride of mercury. The solutions of the latter
employed by Golgi varied from 0*25 per cent, to 0*50 per
cent. : he cannot say which strength is to be preferred. The
immersion in the bichloride must be much longer than the
immersion in the nitrate of silver bath of that process : for
the latter, twenty-four to forty-eight hours suffice ; but in the
bichloride an immersion of eight to ten days is necessary in
order to obtain a complete reaction through the whole thick-
ness of the tissues. During the bath the bichromate will
diffuse out from the tissues into the bichloride, which must
be changed every day ; at the end of the reaction the pre-
parations will be found decolourised, and offering the aspect
of fresh tissue. They may be left in the bichloride for any
time.

In Bendiconti B. 1st. Lombardo di Sci. Milano, 2, xxiv, 1891, pp. 594,
656 (see Zeit. f. wiss. Mik., viii, 3, 1891, p. 388) Golgi says that for the
study of the diffuse nervous reticulum of the central nervous system the
best results are obtained by keeping the preparations in 1 per cent, sub-
limate for a very long time, two years being not too much in some cases.

Before mounting, the sections that have been cut must be
repeatedly washed with water (if it be wished to mount them
permanently), otherwise they will be spoilt by the formation
of a black precipitate. In the last place quoted Golgi says
that after washing they may be toned by putting them for a
few minutes into a photographic fixing-and-toning bath, after
which it is well to wash them again, and stain them with
some acid carmine solution. Mount in balsam or glycerin ;
the latter seems the better preservative medium.

400 CENTRAL NERVOUS SYSTEM.

The result of this process is not a true stain, but an " appa-
rently black reaction/' the tissues appearing black by trans-
mitted light, white by reflected light. Golgi thinks that
there is formed in the tissue elements a precipitate of some
substance that renders them opaque. The elements acted on
are (1) the ganglion- cells, with all their processes and rami-
fications of the processes. These are made more evident than
by any other process except the bichromate and silver-nitrate
process. An advantage of the mercury process is that it
demonstrates nuclei, which is not the case with the silver
process. (2) Connective-tissue corpuscles in their charac-
teristic radiate form. But the reaction in this case is far
less precise and complete than that obtained by the silver
process. (3) The blood-vessels, and particularly their mus-
cular fibre cells.

The method gives good results only with the cortex of the
cerebral convolutions, hardly any results at all with the
spinal cord, and very scanty results with the cerebellum.
And, on the whole, the method shows nothing more than
can be demonstrated by the silver-nitrate method, but it is
superior to it as regards two points : the reaction can always
be obtained with perfect certainty in a certain time, and
the preparations can be perfectly preserved by the usual
methods.

TAL (Gazz. degli Ospitali, 1886, No. 68) finds that if sections made by
this process be treated with solution of sodium sulphide, a much darker
stain is obtained. Sections may then advantageously be double-stained with
Magdala red.

Golgi's method may be combined with Weigert's nerve stain (see PAL,
Wien. med. Jahrb., 1886 ; Zeit.f. wiss. Mik., v, 1, 1887, p. 93).

761. Cox (Arch. f. mik. Anat., xxxvii, 1891, p. 16 ; Journ. Roy. Mie.
Soc., 1891, p. 420) finds the sublimate and bichromate may be used together,
and give a uniform impregnation. He used a fluid consisting of 20 parts
5 per cent, bichromate, 20 parts 5 per cent, sublimate, 16 parts 5 per cent,
simple chromate of potash, and 30 to 40 parts of water. The mixture
should be as little acid as possible. The duration of the impregnation is
from two to three months. There is considerable difficulty in preserving
sections, which must be made with a freezing microtome, alcohol being
avoided, treated with solution of sodium carbonate, and mounted without a
cover.

762. MAGINI (Boll Accad. Med. di Roma, 1886 ; Zeit. f. wiss. Mik.,
1888, p. 87) recommends a development of Golgi's process in which zinc
chloride is used in place of sublimate. Portions of tissue of 2 to 3 cm. cube

SPECIAL STAINS. 401

are hardened for at least two or three months in Miiller's solution. They
are well washed with distilled water, and brought into a O'o to 1 per cent,
solution of chloride of zinc. This is changed for fresh every day for seven
to ten days (until it does not become yellower than bichromate solution).
Sections are then made, washed quickly with alcohol, imperfectly cleared
(see § 779) with kreasote, and mounted in dammar. This process is said to
demonstrate better than Golgi's the finer structure of ganglion-cells and
their processes.

763. FLECHSIG (Arch. f. Anat. u. Phys., Physiol. Abth., 1889, p. 537 ;
Zeit. f. wiss. Mik., vii, 1, 1890, p. 71) has a process for combining Golgi's
impregnation with v. BRANCA'S redwood stain (ante, § 694) and a gold
chloride impregnation, which seems to me too complicated to be trustworthy.

764. MONTI (Atti d. R. Accad. dei Lincei Roma, Rendic., v, 1889, 1 sem.,
p. 705 ; Zeit. f. wiss. MiJc., vii, 1, 1890, p. 72) describes a brown impregna-
tion brought about by the combined action of bichromate of potash and sul-
phate of copper. The method does not appear to have been yet brought to
a practical form.

|3. Special Stains.

765. The most important of the methods for the study of
nerve-tracts, plexuses, and the finer relations of nerve-fibrils
and ganglion-cell processes are those that have already been
given in the last Chapter, together with some of those given
in this Chapter, and classed as general stains.

As myelin stains we have the processes of Weigert, §§ 688
and 703, and the modifications of the same by Max Flesch,
§ 689 ; Benda, 690 ; Pal, § 692 (very important) ; Schafer,
§ 693 ; Kultschizky, § 697; Wolters, §§ 699, 700, and others;
and as processes less exclusively directed to the demonstra-
tion of the myelin element, and claiming more particularly to
serve for the demonstration of axis-cylinders and cell processes,
the chromate of silver impregnation methods of Golgi, §§ 704
to 706 (of the highest importance, and of the widest applica-
bility), with the modifications of the same by Sehrwald, § 708;
Greppin, § 710 ; Obregia, § 711; Ramon y Cajal, § 714, and
others, amongst which I would particularly call attention to
the details of the process as carried out by Ramon y Cajal ;
and amongst the others later described that of Wolters,
§ 726, and the iodide of palladium method of Paladino,
§ 727.

There remain to be mentioned some other methods, based
for the most part on somewhat different principles.

26

402 CENTRAL NERVOUS SYSTEM.

766. The methylen blue impregnation method has been
described in Chap. IX, and its great importance has been
insisted on.

767. The Gold Method is one of the most important methods
for the study of the finer nerve-fibrils of the spinal cord.
The instructions given by G-ERLACH (Strieker's Handb., p.
678) have been already quoted, ante, § 228, p. 148.

BOLL (Arch. f. Psych, u. Nervenk., iv, p. 42 ; GIERKE, Zeit.
f. wiss. Mik., 1884, p. 403) makes the following observations
on Gerlach's process : — The hardening in bichromate ought
not to be prolonged further than is absolutely necessary, for
after eight days the elective susceptibility of the tissues for
gold impregnation begins to diminish, and after fifteen days
has almost disappeared. The knife should not be wetted
with alcohol; the tissues should never be allowed to come
into contact with alcohol. Only a small quantity of gold
solution, relatively to the volume of the sections, should be
taken. .The impregnation should last for twelve hours.

Gierke (1. c.) says, " Gerlach's gold preparations have never
had their equal for the demonstration of very fine nerve-
fibrils. Weigert's Saurefuchsin preparations are the only
ones that can be compared with them in this respect."

SCHIEPFERDECKER (Arch, f. mik. Anat., 1874, p. 472) also
recommends gold chloride for demonstrating fine networks in
transverse sections. Strength 1 : 5000 or 10,000. Time about
one to three hours. After washing in water the sections are
put for twenty-four hours into acetic acid of J to 1 per cent.,
then mounted in balsam.

According to the same author, chloride of palladium may
advantageously be used for the demonstration of the longitu-
dinal fibres, and (of course, therefore) for staining longitudinal
sections. A solution of 1 : 10,000 strength is taken; the sec-
tions remain in it till light brown (about three to five hours).
For some further methods of the same author see l.c.,1878,p.38.

MERKEL'S chloride of palladium and carmine method has
been given above, § 746.

768. FREUD'S gold method (Arch. /. Anat. u. Phys., 1884,
p. 453) may be used for controlling the results obtained by
Weigert's process, and for obtaining an impregnation of axis-

SPECIAL STAINS. 403

cylinders. This method is based on that of FLECHSIG (see
§ 228), which I suppress, as it gives less certain results. Freud
proceeds as follows : — Sections of material hardened in solu-
tion of Erlicki (which may be followed by alcohol without
hindrance to the impregnation) are washed with water, and
put for three to five hours into 1 per cent, solution of gold
chloride. After again washing with water they are treated
for three minutes with a solution of 1 part of caustic soda in
5 or 6 of water. They are then drained (not washed) and
brought into 10 or 12 per cent, solution of iodide of potassium.
After from five to fifteen minutes therein they are washed
with water, dehydrated, and mounted in balsam. (In the case
of objects that stain easily it is useful to dilute the gold solu-
tion with a volume or two of alcohol ; the stain is more highly
selective.)

BECKWITH (Journ. Roy. Hie. Soc., 1885, p. 894) modifies this process by
intercalating a treatment for thirty minutes with 10 per cent, solution of
carbonate of potash between the soda and the iodide.

769. ZIEHEN'S gold and sublimate method (Neurol. Centralb.,
x, 1891, No. 3, p. 65; Zeit. f. wiss. MiL, viii, 3, 1891, p. 385)
gives the following derivative of G-olgi's sublimate method as
an improvement on it : — Small pieces of fresh material are
thrown into a large quantity of a mixture of 1 per cent, sub-
limate solution and 1 per cent, chloride of gold solution in
equal parts. They remain therein for at least three weeks,
preferably for several months (up to five), by which time they
will have become of a metallic red-brown colour. They are
gummed on cork and sectioned without imbedding. The
sections are treated either with solution of Lugol (§ 66)
diluted with four volumes of water, or with dilute tincture of
iodine, until duly differentiated, which will require more or
less time according to the thickness of the sections. They
are then washed and mounted in balsam. The result is a
bluish-grey impregnation ; both medullated and non-medul-
lated nerve-fibres are stained, also nerve- and glia-cells and
their processes.

770. UPSON'S Methods (MERCIER, in Zeit.f. wiss. Hik., vii,4,
1891, p. 474 : I pass over the older methods given in Neurol.
Centralb., 1888, p. 319; and Zeit. f. wiss. Mik., 1888,
p. 525).

404 CENTRAL NERVOUS SYSTEM.

The Gold and Iron Method.

Material must be very carefully hardened for from four to
six months in bichromate of potash (not solution of Miiller,
which does not allow of the same precision of stain) . A 1 per
cent, solution should be taken at first, and should at first be
frequently changed. Only after some weeks should the
strength be gradually increased up to 2 or 2*5 per cent. All
the hardening should be done in the dark. Over-hardened
material is not available. The surface of section of a properly
hardened piece of material should show microscopically no
marked difference of colour between the white and the grey
matter. If the white substance appear very dark, almost
black, and the grey substance whitish grey, good results will
probably not be obtained ; a medullary stain will probably
result, whereas the method ought to give an axis-cylinder
and cell-process stain.

After hardening; the specimens are washed with water, and
put for two or three days into 50 per cent, alcohol, changed
as often as necessary, and then into 95 per cent, alcohol, in
which they should remain until they show a decidedly green
coloration (two to four weeks), the alcohol being changed as
often as precipitates occur. Sections are made either under
water or by the celloidin process (in the former case they
should not be allowed to remain in contact with the water
longer than is absolutely necessary, and should be put back
into alcohol for two or three days before passing to the next
stage) ,

The next stage consists in a bath of from one to two hours'
duration in 1 per cent, solution of chloride of gold acidified
with 2 per cent, of hydrochloric acid. After this the sections
are rinsed with water, and treated for half a minute with a
freshly prepared solution of ferricyanide of potassium in 10
per cent, potash solution (a lump of ferricyanide not half so
big as a pea to 5 c.c. of the potash solution). They are then
washed for half a minute more in pure 10 per cent, potash
solution, and after that for some time in distilled water.

The following reducing mixture (which must be freshly
made up at the very instant of using it for each section) is
now to be prepared :

EPSON'S METHODS. 405

Acidum Sulfurosuin . . .5 c.c.

Tinctura lodi (3 per cent, strength) « 10—15 drops.
Mix, and add — ,

Liquor Ferri Chloridi .... 1 drop.

You must mix quickly in a graduated glass. The section
should now be brought on a piece of filter-paper into a watch-
glass, and the reducing mixture should be poured over it
evenly at one pour, just as developing solution is poured on
to a photographic negative. The section should remain in
this bath until it is of a fine rosy red tint, not a second
longer, or it will become reddish black and useless. As soon
as the rose-red colour appears the section is removed into dis-
tilled water. The water is changed once, the section is
placed on a slide aad brought into absolute alcohol, and after
five to fifteen minutes therein into clove oil and mounted in
balsam. The sections must be kept in the dark.

(The treatment with ferricyanide of potassium sometimes
gives rise to the formation later on of precipitates of Berlin
blue in the tissues ; and as this treatment is not essential it
may be omitted, the sections being simply treated with pure
potash solution as directed.)

771. Upson's Methods — the Gold and Vanadium Method. —
Sections (made as before) are placed in the following solution :
1 per cent, chloride of gold solu-
tion ...... 5 c.c.

Saturated solution of vanadate of

ammonium ..... 10 drops.

Hydrochloric acid . . . . 3 „

After two hours therein they are washed with distilled
water, and put for half to one minute into a mixture of —
10 per cent, solution of caustic potash . 5 . , .
10 per cent, solution of permanganate

of potash . . . . . . 10 drops.

Vanadate of ammonium . . .a trace.

They are rinsed in distilled water, and treated until they
become red, as in method a, with the following freshly pre-
pared reducing fluid :

a. 3 per cent, tincture of iodine, to which
has been added chloride of tin until of

406 CENTRAL 1S7ERYOUS SYSTEM.

a white or yellowish tint (may be kept

in stock) 15 drops.

b. Distilled water 3 c.c.

c. Saturated solution of phosphate of iron
in distilled water (the usual pharma-
ceutical solution) .... 3 — 5 drops.

d. Sulphurous acid . . . .3 c.c.

Mix a, 6, and c, and add d, which will cause a voluminous
precipitate, at which instant the mixture is to be poured over
the section, or the section brought into it, as in method a,
the remaining treatment being exactly as there described.

The principle of the two methods is the decoloration of
the impregnated elements (especially glia-cells) up to a cer-
tain point by means of the potash, or potash and ferricyanide,
or the vanadate and permanganate. It is important to
take care that this decoloration be not overdone, as if car-
ried too far the axis-cylinders and ganglion-cells will be
decolourised.

772. WEIGEET'S Sdurefuchsin method mentioned above appears to be
definitively superseded by his bsBinatoxylin method. See, however, Centralb.
f. d. tned. Wiss., 1882, pp. 753, 772; Fortschr. d. Med., 1884, Nos. 4 and 6 ;
Zeit.f. wiss. Mik., 1884, pp. 123, 290.

773. SAHLI (Zeit.f. wiss. Mik., 1885, p. 1) gives the following method :
— Sections of tissue hardened in bichromate to the degree required for
Weigert's hsematoxylin process are washed for not more than five or ten
minutes in water, and stained for several hours, until they are of a dark blue
colour, in concentrated aqueous solution of methylen blue. They are then
rinsed with water, and stained for five minutes in saturated aqueous solu-
tion of Saurefuchsin. If now they be rinsed with alcohol and brought into
a liberal quantity of water, the stain becomes differentiated, axis-cylinders
being shown coloured red and the myelin sheaths blue. If, instead of rins-
ing with pure alcohol, alcohol containing from 0*1 to 1 per cent, of caustic
potash be taken, the stain differentiated in water, and the sections cleared
with cedar oil and mounted in balsam dissolved in cedar oil, still finer
images are obtained. Axis-cylinders are red as before, but the myelin
sheaths are blue in some places, red in others. Sahli thinks that this
reaction points to some difference of kind in the nerve-tubes that exhibit it.

The same author (1. c., p. 50) also gives a method for obtaining a specific
stain of nerve-tubes by means of methylen blue alone. Sections of material
hardened as before are stained for a few minutes or hours in the following
liquid :

Water . 4f) parts.

Saturated aqueous solution of methylen blue .24 „
5 per cent, solution of borax . . . 16 „

(Mix, let stand a day, and filter.)

NIKIFOROW'S METHOD. 407

The sections are then washed either in water or alcohol until the grey,
matter can be clearly distinguished from the white, are cleared with cedar
oil, and mounted in balsam. Nerve-tubes are stained blue, ganglion-cells
greenish, nuclei of neuroglia blue. Micrococci are stained, if any be present
in the tissues. The preparations are not perfectly permanent.

774. Safranin followed by methylen blue gives a very special stain of
spinal cord. The method is due to ADAMKIEWICS (Sitzb. k. Acad. Wiss.
Wien, Math. Naturw. Kl., 1884, p. 245; Zeit. f. wiss. Mik., 1884, p. 587).
Sections are washed first with water, then in water acidified with a little
nitric acid, and stained in concentrated solution of safranin. They are then
treated with alcohol and clove oil till no more colour comes away, and are
brought back again into water, washed in water acidified with acetic acid,
stained in methylen blue, and cleared as before. The process is said to de-
monstrate the existence of " chromoleptic zones " which surround the grey
matter. Myelin ("erythrophilous substance" of Adamkiewics) is red, nuclei
of nerves, of neuroglia, and of vessels violet. The erythrophilous substance
of pathological nerve-tubes does not take the stain, so that the method is
valuable for the study of degenerative changes.

775. NIKIFOROW (Zeit. f. wiss. Mik., v, 3, 1888, p. 338) modifies the
foregoing method as follows : — The tissue is to be hardened in a chrome
salt. It is not to be washed in water afterwards. Sections are brought
direct from alcohol into the safranin stain, either concentrated aqueous, or
Babes's solution, or a solution in 5 per cent, carbolic acid. Stain for twenty-
four hours. Bring the sections into alcohol until the colour is sufficiently
extracted for the grey substance to be distinguished from the white. Then
put them into a weak (O'l to 0'2 per cent.) solution of chloride of gold or
platinum or any metallic salt. They are to be left in this until the grey
substance just begins to get a violet tone (not longer). They are then well
washed in water, and put into alcohol until the rosy violet of the grey sub-
stance becomes distinctly marked off from the red of the white substance.
Clear with clove oil, wash out the clove oil thoroughly with xylol (this is
absolutely necessary for the permanence of the preparations), and mount in
balsam. Nikiforow says that this method gives much sharper and more
certain results than that of Adamkiewics.

776. Purpurin, according to DUVAL, has a special action on nerve-tissue
(especially spinal cord) that has been hardened in bichromate of ammonia.
Nerve-cells and their processes, axis-cylinders and connective-tissue fibrils,
remain unstained, whilst the nuclei of connective tissue and of capillaries
stain red (see § 201).

Mounting .

777. General Mounting Methods. — Under this head it is to
be noted that it is often advisable to fix sections to the slide
before applying balsam, or other medium, and a cover. For
celloidin sections you may use one of the usual methods de-
scribed in Chap. XVII, or for large sections the gelatin
method of Fol, or Minot's shellac method. This is as follows :

408 CENTRAL NERVOUS SYSTEM.

— You arrange the sections on the slide, and cover them with
a layer of 10 to 12 per cent, solution of shellac, then warm to
30° or 40° C., until the shellac appears dry, clear with clove
oil, and mount in balsam (Zeit. f. wiss. Mik., 1886, p. 175).
This process is applicable either to paraffin or collodion sec-
tions.

778. Serial Section Mounting. — Besides the ordinary methods for
mounting small sections, there are certain processes specially adapted to the
mounting of very voluminous sections. One of these is GIACOMINI'S collodion-
gelatin method, which is certainly a valuable one. But it is hardly more
than a macroscopic method principally adapted for the study of the coarser
topography of the brain, and as such may be passed over here, though I
would willingly include it did our shortening space allow. Descriptions of
the process are to be found in Gazzetta delle Cliniche, Nov., 1885 ; Zeit. f.
wiss. Mik., 1885, p. 531 ; and Traite des Meth. techn., LEE et HENNEQUY,
p. 392.

A further reason for passing over this process is that the same end may
be attained, in many cases more effectually, by the beautiful methods of
WEIGEET (§ 340), STKASSEB (§ 326), and APATHY (§§ 338, 339), which see.

A useful method for small sections is that of OSBORN, who arranges his
sections, wet with alcohol, in order on the slide, covers them with a cigarette
paper, and treats them with the clearing agent through the paper.

For DEECKE'S method see above, § 743.

Other Methods for Nervous Centres.

779. The Half-clearing Method.— Since the days of Lockhart Clarke,
to whom the method is due, most observers have been struck by the wealth
of minute structure revealed by half-cleared sections. Detail that is invisi-
ble in perfectly cleared sections stands out in sculptural relief during the
period in which the tissues are only about half cleared. It is of course most
important to be able to preserve preparations permanently in this state.

MEBZEL (Arch. f. mik. Anat., 1877, p. 622) gives the following instruc-
tions : — Sections are dehydrated with alcohol of about 94 per cent, (in which
they must remain for at least ten minutes), and then cleared with xylol, in
which they are examined.

Certain elements of the tissues retain more obstinately than others the
small quantity of water that they bring with them out of the 94 per cent,
alcohol. Now, as xylol is absolutely immiscible with water, it can exercise
no clearing action on these, and they stand out boldly in the picture by virtue
of the difference between the index of refraction of their contained water
and that of the xylol. The axis-cylinders are at first all that is visible ; after
a time the ganglion-cells appear with their processes. Nuclei, vessels, and
whatever else may be in the preparation are totally invisible. Merkel thinks
this method superior to all others for the study of the distribution of nerve-
fibres. The preparations are not permanent, though they may be kept for
some weeks by mounting them in Canada balsam. When a preparation
(either in xylol or balsam) has become so transparent as to be of no further

GIACOMINI'S "DRY" PROCESS. 409

use (which will always eventually happen), it may be reprepared by putting
it back into the alcohol, and thence again into the xylol.

BEVAN LEWIS (The Human Brain, p. 122) proceeds as follows : — A sec-
tion is placed, saturated with spirit, on a slide. When the spirit has nearly
all evaporated, a drop of oil of anise is allowed Ito flow over the section (not
to float it up), and the clearing is watched on the stage of the microscope ;
then, just when the desired image is arrived at, a drop of balsam is allowed
to run over the section, and a cover put on.

Instead of oil of anise, Lewis has frequently employed glycerin followed
by mounting in glycerin jelly, with the same results.

BYBOJI BEAMWELL (Edinb. Med. Journ., Oct., 1886) also recommends
the process. He uses clove oil.

780. GIACOMINI'S "Dry" Process for Preserving Brains (Arch, per
le Scienze Mediche, 1878, p. 11). — Although this is in intention a macro-
scopic method, it appears worth while, both on account of its thorough success
and on account of its suggestiveness, to give a description of it here.

The object is to make " dry " preparations of the encephalon ; by which
is meant preparations that can be permanently preserved in the air. The
methods hitherto employed were not successful because they consisted in
making preparations that were "dry" in the literal sense of the word — that
is, deprived of their natural water; and since brain-substance contains 88 per
cent, of water, such preparations could not of course be obtained without so
great an amount of shrinkage as to most seriously diminish the scientific
value of the result. The principle of Griaconrini's method is, on the contrary,
to retain the natural water of the tissues, or an equivalent for it, by means
of impregnation with a hygroscopic substance, — glycerin.

The process consists of two divisions : — 1, hardening ; 2, impregnation
with glycerin.

1. For hardening may be used zinc chloride, bichromate of potash,
chromic acid, nitric acid, or alcohol.

Chloride of zinc gives the best results. Perfectly fresh brain is put into
a saturated aqueous solution of the salt (if there be reason to fear that the
tissues are somewhat softened through having been left too long after the
death of the subject, it is well first to inject 600 grms. of the solution
through the internal carotid arteries). After forty-eight hours' immersion
(during which time the floating brain must be turned over three or four
times, so that all parts of it may duly come into contact with the liquid) the
surface of the brain will have attained a consistency that will allow of the
removal of the arachnoid and pia mater. The meninges having been removed,
the encephalon is put back into the solution for two or three days more,
during which time it will be seen that, increasing in specific gravity, it tends
towards the bottom of the vessel containing it. When this is seen to happen
it must be removed into commercial alcohol, as if allowed to remain longer
in the chloride of zinc solution it would take up too much water.

In the alcohol it may remain for an indefinite time, or it may be removed
if desired after ten or twelve days. (During the alcohol-bath it must be
frequently turned over in order that no malformation may arise from con-
tinuance of pressure on the same part.)

410 CENTRAL NERVOUS SYSTEM.

It is then removed into glycerin (either pure or with 1 per cent, of carbolic
acid). It floats at first, but gradually sinks as the alcohol evaporates. As
soon as it has sunk just below the surface it may be removed and exposed
to the air.

It is set aside to " evaporate " in a convenient place for a few days. As
soon as the surface has become dry, it is varnished with india rubber or
(better) with marine glue varnish diluted with a little alcohol. This com-
pletes the process.

If it be desired to make dissected preparations, the necessary dissection
should be made on removing the encephalon from the alcohol before putting
into glycerin.

Bichromate of potash may be used for hardening in solutions gradually
increasing in concentration from 2 to 4 per cent. The liquid must be fre-
quently changed ; the immersion must be of not less than a month's duration.
Six to eight days will suffice for the alcohol-bath, or this may be altogether
omitted.

Nitric acid is used in solutions of from 10 to 12 per cent, for twelve to
fifteen days. (Encephala float in this liquid, and must therefore be fre-
quently turned over. It is this reagent that gives the toughest prepara-
tions.)

Concerning the value of the process, Golgi (from whose abstract I take
the foregoing account) states that after a series of experiments he is able to
affirm that for preservation of the volume, the colour, the finer relations of
the parts, and the physiognomy proper to the organ, the process is far
superior to any hitherto known. I am able to add that I saw specimens of
Giacomini's preparations at the Milan International Exhibition of 1881,
and think it would be hard to over-praise their beauty of aspect.

It should be added that histological detail is preserved to a remarkable
extent by this process, and that excellent sections may be cut at any time
from the hardened brains. And as the preparations take up as little room
as possible, there seems no reason why the process should not be generally
adopted in medical schools, lunatic asylums, and similar institutions.

The method may also be applied, with most perfect success, to the pre-
servation of small animals entire, such as Batrachia, K-eptilia. It is well to
inject them with the zinc solution.

Another " dry " method has been given by MAX FLESCH (Mt. Naturf.
Ges. Bern , 1887, p. xiii). He hardens in alcohol, and then brings the
brains through Calberla's mixture (§ 405) into glycerin containing (1 part
to 3000 of) sublimate. He does not appear to varnish his preparations.
See Journ. Roy. Mic. Soc., 1888, p. 507.

781. Dissociation Methods. — Maceration of nervous tissue is a method
that rendered service years ago for the isolation of ganglion-cells with their
processes, &c. These details can, however, now be studied in situ by means
of the excellent methods of special staining or impregnation that have been
given in the foregoing pages, so that the maceration methods can hardly
claim now to be useful for purposes of research, and may at most claim to
serve as means of demonstrating to beginners the general characters of
ganglion-cell processes. See, however, the following :

NEUROGLIA. 411

STILLING'S method (Arch. f. mik. Anat., 1880, p. 471 ; Bau. d. nervosen
Centralorgane, 1882).

CABBIEBE'S (Arch.f. mik. Anat., 1877, p. 126).

SCHIEFFEEDECKEE'S (ibid., 1878, p. 38; also BEHBENS, KOSSEL, und
SCHIEFFEEDECKEE, Das Mikroskop, Bd. ii, p. 227).

FBEEBOBN'S (Amer. Mon. Mic. Journ., 1888, p. 231 ; Journ. Roy. Mic.
Soc., 1889, p. 298).

782. BEVAN LEWIS'S Compression Method for Fresh Tissue (Mon.
Mic. Journ., 1876, p. 106 ; Human Brain, p. 146) is as follows : — Vertical
sections, as thin as possible, are made from a piece of a convolution of
brain from which the membranes have been removed. The sections are
made by free hand by means of a section-knife flooded with spirit. The
sections are got on to a slide and treated with Muller's solution for a few
seconds. A cover is then put on and steadily pressed down so as to flatten
out the sections into an almost transparent film. The slide is then rinsed in
water and placed for thirty to forty seconds in a bath of methylated spirit.
It is then removed, one edge of the cover-glass steadied with the finger, the
blade of a penknife inserted under the opposite edge, and the cover gently
lifted. The section, which remains adherent either to the slide or to the
cover, is washed with water, stained in any way that may be desired*
dehydrated, and mounted in balsam.

For staining the cells of the cortex, Lewis prefers a 1 per cent, solution of
anilin black.

He prefers to dehydrate by drying under a bell-glass in the presence of
concentrated sulphuric acid. Clear with chloroform in preference to clove
oil. Lewis finds that this process results in far less rupture and tearing of
nerve-cells and processes than would be imagined ; he considers that " the
result is equivalent to the most delicate teasing of tissue, the processes being
gradually unravelled from their dense networks, and the structural elements
universally displayed to the best advantage."

v. THANHOFFEE (Zeit.f. wiss Mik., iv, 4, 1887, p. 467) describes a similar
process.

783. Neuroglia (GIEKKE, Arch. f. mik. Anat., 1885, p. 444).
— Maceration in the usual media, chromic solutions, iodised
serum, &c., but especially in the liquid of Landois (§ 532).
For sections, harden for six to ten weeks in bichromate of
ammonia, first of 1*5 per cent., and gradually raised to 3 per
cent. Avoid paraffin for imbedding, and imbed in celloidin
or cut without imbedding. Stain with ammonia- carmine, or
picro-carminate of soda, or alum-carmine, or Heidenhain's
haematoxylin. Other details in this valuble paper.

784. Myelon of Reptilia and Amphibia.— Besides the usual methods
which will suggest themselves as being applicable to this class of objects,
see MASON'S methods, supra, § 742 ; SCHMIDT, Zeit. f. wiss. Mik., 1885,

412 CENTRAL NERVOUS SYSTEM.

p. 389 ; v. LENHOSSEZ, Arch. f. tnik. Anat., 1886, p. 379 (minute directions
for dissecting out the spinal ganglia of Rand) ; KOEYBUTT-DASZKIEWICS,
ibid., 1889, p. 51 ; or Zeit.f. wiss. Mik., vi, 2, 1889, p. 203; OSBOEN, Journ.
Roy. Mic. Soc., 1885, p. 536 ; or WHITMAN'S Methods, p. 195.

785. APATHY'S double stain for differentiating nervous tissue and con-
nective tissue (Zeit. f. wiss. Mik., vi, 2, 1889, p. 170) consists of staining
with Heidenhain's hsematoxylin, and after-staining with very weak alum
hsematoxylin.

MARCHI'S Method for Degenerate Medullated Nerve-fibres.— See
§ 720A.

CONNECTIVE TISSUE. 413

CHAPTER XXXI.

SOME OTHER HISTOLOG1CAL METHODS.
Connective Tissues.

786. Connective Tissue.— S. MAYER (Sitzb. k. Akad. Wiss.,
Ixxxv, 1882,, p. 69) recommends, for staining fresh tissue, a
solution of 1 gramme of "Violet B" (Bindschedler and
Busch, Bale) in 300 c.c. of 0'5 per cent, salt solution. In this
liquid connective-tissue cells stain rapidly and energetically.
Elastic fibres and smooth muscle also stain, but of different
tints.

FBEEBOBN (Amer. Mon. Mic. Jonrn., 1888, p. 231 ; Jowrn. Roy. Mic.
Soc., 1889, p. 305) recommends (for sections) picro-nigrosin, made by mix-
ing 5 c.c. of 1 per cent, aqueous solution of nigrosin with 45 c.c. of aqueous
solution of picric acid. Stain for three to five minutes, wash with water, and
mount in balsam. Connective-tissue fibres bright blue, nuclei blackish, all
the rest greenish yellow. Sections may be after-stained for five or six
minutes in a mixture of 1 c.c. of saturated alcoholic solution of eosin and 49
c.c. of alcohol. The results are as before, except that the yellow colour is
replaced by red.

For RANVIER'S method of artificial oedemata for the study
of areolar tissue, see his Traite, p. 329.

For FLEMMING'S observations on the development of connective-tissue
fibrils, see Festschr. R. Virchow gewidmet, &c., 1, 1891, p. 215 ; Zeit. f.
wiss. Mik, ix, 2, 1892, p. 225.

For PIANESE'S very complicated double stain with carmine and picro-
nigrosin, see Journ. Roy. Mic. Soc., 1892, p. 292.

Note also the EHBLICH-BIONDI stain, § 259.

787. Fat. — DEKHUTSEN (see FLEMMING, in Zeit. f. uriss. Mik., 1889,
pp. 39, 178) has discovered that fat that has been stained black by treat-
ment with chromo-aceto-osmic acid (not with pure osmium) is dissolved in
the course of a few hours in turpentine. It is dissolved also in xylol, ether,
and kreasote. Flemming finds that very good demonstration preparations
may be made by treating fatty tissue with chromo-aceto-osmic acid, staining
with safranin or gentian, and then treating for a few hours with turpentine
until all the fat is dissolved. The optical hindrance caused by the high

414 SOME OTHER HISTOLOGICAL METHODS.

refraction of the fat being thus eliminated, nuclei and cytoplasm may be
studied to far greater advantage than in the usual preparations.

788. Granule-cells (" Plasmazellen " and " Mastzellen ").— In
1874 there were described by WALDEYER (Arch. f. mik. Anat., Bd. xi) cer-
tain special cells existing between the bundles of connective tissue, besides
the flat cells, and lymphatic and fat cells. They are large round cells contain-
ing large granules ; Waldeyer called them plasma cells (" Plasmazellen ").
Later on, EHELICH (ibid., xiii, 1877) distinguished in the same tissue and in
other places certain cells containing large granules, which have a superficial
resemblance to Waldeyer's plasma cells, but which differ from them in
staining reaction (Verhandl. Berl. Physiol. Ges., January 17, 1879 ; Reichert
u. Du Bois Raymond's Arch., 1879, p. 166). Ehrlich named these food
cells (" Mastzellen"), intending to express thereby the opinion that these cells
are derived from fixed connective-tissue cells by a transformation brought
about by exalted nutrition.

If (KOKYBUTT-DASZKIEWICZ, Arch, f. Mik. Anat., xv, 1878, p.
7) frogs be kept for two months (in snmmer) without food,
then placed in a reservoir of running water and well fed for
four weeks, " plasma-cells " will be found in abundance,
especially in the neighbourhood of the nerves.

It is a pretty general character of these elements that they
take the stain of anilin colours and retain it on treatment with
alcohol with greater energy than other tissue cells. And
further (at least so far as regards the true Mastzellen of
Ehrlich) that in successful preparations they show the nucleus
unstained, the general mass of cytoplasm unstained or but
slightly coloured, and, in the cytoplasm, the characteristic
granules very intensely stained.

The staining reactions of the .granules are very similar to
those of bacteria. In order to distinguish them from these
it may be observed that the stain of anilins is removed from
the granules, but not from bacteria, by treatment with a
weak solution of carbonate of potassium, (SQUIRE, Methods and
Formulae, p. 46). EHELICH distinguishes no less than five kinds
of granulations, recognisable by certain differences in their
staining reactions. It will be remembered that according to a
generally accepted principle of classification, due to Ehrlich
(Arch.f. Anat. u. Phys., Phys. Abth., 1879, pp. 167 and 572),
anilin dyes are divided into two chief classes, viz. the " basic "
dyes, or those in which the colouring matter plays the part of
a base combined with a colourless acid ; and the " acid "
dyes, in which the colouring matter plays the part of an acid

EHRLICH'S " MASTZELLEN. 415

in the compound ; to which may be added a small group of
" neutral " dyes. The basic dyes are in general nuclear
stains, — that is, they exhibit a marked affinity for chroinatin,
as methyl green, for instance ; and are also in general
specific bacteria stains. The acid dyes have opposite affinities
and are in general plasma stains, as eosin, for instance. The
cell-granules in question have been classified by Ehrlich,
according to their respective degrees of affinity for one or the
other of these two groups of dyes, into (1) a granulations, or
eosinophilous granulations, being such as in a mixture of dyes
select the most acid stain there present ; (2) the )3 granula-
tions, or amphophilous granules, being such as in a mixture
of basic and acid dyes take up both ; (3) the 7 or basophilous
granules, being such as take up basic dyes only (such are the
granules of Mastzellen; (4) the § granulations, also baso-
philous ; and (5) the e granulations, or neutrophilous granules,
being such as take up neutral anilin dyes, such as methyl blue
and " acid " fuchsin (acid f uchsin is a weakly acid dye). I do
not propose to enter minutely into the subject of these re-
actions, as it is one that cannot profitably be treated apart
from the histology of the elements in question, and will con-
fine myself to mentioning a few well-known methods.

789. EHRLICH'S "Mastzellen" (Arch. f. mik.Anat., 1876, p.
263). — First Method. — Stain with neutral dahlia, and wash
out with acidified water, and mount as directed supra, § 103.
If there be any Mastzellen in the preparation they will be
brought out by the superior energy with which they take the
stain.

Second Method. — It may be desirable to obtain a specific
stain of the plasma-cells alone, which may be done as
follows :

The tissues must first be well hardened in strong alcohol
(chromic acid and its salts must be avoided). They are then
placed for at least twelve hours in a staining fluid composed
of—

Absolute alcohol .... 50 c.c.

Aqua . ... . . . 100 c.c.

Acid. acet. glacial . . . . 12^ c.c.

— to which has been added enough dahlia to give an almost
saturated solution. After staining, the preparations are

416 SOME OTHER HISTOLOGICAL METHODS.

transferred to alcohol, which washes out the stain from all
but the plasina-cells, and may then be mounted in the resin-
turpentine solution.

Mucus-cells and fat-cells are also sometimes stained by
these solutions.

Other Media. — In a similar way other soluble anilins may
be employed (in the form of a fluid containing 7J per cent,
of acetic acid), — primula, iodine violet, methyl violet, pur-
purin, safranin, fuchsin ; of these, methyl violet gives the
best results.

790. Plasma-cells (NORDMANN, Beitr. z. Kenntniss d. Mast-
zellen, Inauguraldiss., Helmstedt, 1884). — Nordmann finds it
useful to employ a solution of vesuvin containing 4 to 5 per
cent, of hydrochloric acid. Sections should remain for a few
minutes in the solution, and then be dehydrated with absolute
alcohol. The paper quoted contains a detailed discussion of
the microchemical reactions of granule-cells.

791. ic Mastzellen." — SCHIEFFERDECKER (SCHIEFPERDECKER AND
KOSSEL'S Gewebelehre, p. 329) recommends the following,
after ORTH. A piece of mesentery of a rat is. brought into a
solution of gentian violet in anilin water (§ 101), which is
carefully heated over a flame until vapour begins to be given
off, and then allowed to remain for a couple of hours (or the
heating may be omitted, and the preparation allowed to stand
for twenty-four hours). It is then rinsed in water, washed
out until almost colourless in hydrochloric acid alcohol, rinsed
in water, counterstained (if desired) in carmine, and mounted
in balsam. Nuclei red, granules blue.

792. Plasma Cells and Mastzellen. — UXNA, in his paper, ZeiL
f. wiss. Mik., viii, 4, 1892, p. 475, gives the following :

A. For Plasma Cells.

Methylen blue ..... TO
Caustic potash ..... 0*05

Distilled water 10OO

Add a few drops of this to ten, fifty, or one hundred vols.
of anilin water (§ 101) in a watch-glass, and stain (alcohol
•material, or at most sublimate and alcohol material, not
chromic material) for half an hour, several hours, or over-

STAINS FOR PLASMA CELLS. 417

night. Dehydrate rapidly in absolute alcohol, differentiate
in creosol (details not given), rinse in xylol, and mount in
balsam.

B. General Stain, also bringing out Plasma Cells.
Methylen blue . . . . . 1-0
Carbonate of potash . . . . 1-Q
Distilled water . . . . . 10OO

Alcohol 20-0

Heat on a water-bath until reduced to lOO'O. Use for
staining undiluted, or diluted with one vol. of anilin water.
Differentiate (details not given) with glycol, styron, or
creosol. Mastzellen are not differentiated.

c. Stain giving Red Mastzellen with Blue Plasma Cells.
Methylen blue . . . . . I/O

Kali Carbon, (natron carbon, ammon.

carbon) ...... 1*0

Aq. dest. (Aq. carbolisata, chloroforma) 10OO
Dilute about 100-fold, stain slowly, treat with 70 to 80 per
cent, alcohol, differentiate in styron, and bring through
berganiot oil or xylol into balsam. In this process the
granules of the Mastzellen stain red in consequence of the
formation of methylen red in the staining bath.

In a paper "Ueber Plasmazellen, insbesondere bei Lupus"
(for which see Monatschr. f. praJct. Dermatol., xii, 1891, p. 296,
or Zeit. f. wiss. Mik., ix, 1, 1892, p. 92), UNNA gives some
directions concerning the process of differentiation with
creosol. Creosol only differentiates the stain, it does not
dehydrate the sections. Sections should, therefore, first be
dried with blotting-paper, and should then be treated with
absolute alcohol for a few seconds, or with anilin oil before
applying the creosol. The time required for differentiation
in the creosol is from a few minutes to a few hours. When
the proper stage of differentiation is attained, it should be
fixed with xylol and the sections mounted. The method
shows Mastzellen of a cherry- red tone.

In a paper, 1. c.; xiii, 1891, p. 364 (see Zeit. f. wiss. Mik., xi, 1, 1892,
p. 89), VAN DEE SPEK and UNNA describe some further experiments. Main-
taining the method of staining given above under A, they find that besides
the differentiating agents given under B, a lengthy series of other reagents

27

418 SOME OTHER HTSTOLOGICAL METHODS.

are available. In all of them Mastzellen are distinguishable from plasma
cells by the reddish coloration of their granules.

793. Plasma Cells and Mastzellen. — BEKGONZINI (Anat. Anz.,
vi, 1891, pp. 595—600; Zeit. f. wiss. Mik., ix, I, 1892, p. 95)
gives the following : — Mix 1 volume of 0'2 per cent, solu-
tion of Saurefuchsiii with 2 volumes of a like solution of
methyl green, and 2 volumes of a like solution of gold-
orange, and filter through cotton wool. Stain alcohol or sub-
limate sections (after washing in water) for three to four
minutes, wash for one or two minutes in water, bring into
absolute alcohol for two minutes, clear in bergamot oil or
pure creosote, wash in turpentine, and mount in balsam.

One sort of gold-orange precipitates methyl green, and there-
fore cannot be used in this mixture. Orange G may be used
instead (thus giving something very like Ehrlich-Biondi mix-
ture), but in this case the acidophilous granules will stain grey-
ish instead of red or orange. Basophilous granules ought to be
green, weakly acidophilous granules red, strongly acidophilous
ones orange or brown, nuclei always green. Other details 1. c.

794. Plasma Cells. — JADASSOHN (Arch, f. Dermatol. u. Syphilis,
Erganzungsheft 1, 1892, p. 58 ; Zeit. f. wiss. Mik., ix, 2, 1892, p. 226) re-
commends staining for nottoo long in a 1 : 2000 strongly alkaline or borax
solution of Thionin, and washing out with acidulated water. Unfortunately
this colour is, I believe, no longer obtainable in commerce.

795. Elastic Tissue. — Two of the most salient characters of
elastic fibres are that they have a great affinity for osmium,
staining with much more rapidity than most other tissue-
elements, and that they are not changed by caustic soda or
potash. A further character is that they have a great affinity
for certain anilin dyes, especially Victoria blue.

For a review of the older methods of BALZER, UNNA, LUST-
GARTEN, and HERXHEIMER, see the paper by MARTINOTTI in Zeit.
f. wiss. Mik., iv, 1, 1887, p. 31.

LUSTGARTEN'S process, which is simple, and gives very good
results, has been given in § 104. The colour used by him was
called " Victoriablau 4 B," and this is probably an important
detail.

But perhaps the best method is that of MARTINOTTI (1. c.).
Fix in a chromic liquid, wash, stain for forty-eight hours in
strong (5 per cent. Pfitzner's) solution of safranin, wash,
dehydrate, clear, and mount in balsam. Elastic fibres are

UNNA'S ORCEIN METHOD.

stained of an intense black, the rest of the preparation show-
ing the usual characters of a safranin stain.

The staining will be performed quicker if it be done at the temperature
of an incubating stove (GEIESBACH, ibid., iv, 1887, p. 442). And FEEBIA
(ibid., v, 3, 1888, p. 342) says that clearer preparations will be obtained if
the sections be left for a long time, say twenty-four hours, in the alcohol, or
be treated for a short time with very dilute alcoholic solution of caustic
potash. This decolourises more completely the ground of the preparations.

Another safranin method, which seems to have the fault of requiring a
too minute attention to details, is that of MIBELLT, see Mon. zool. italiano,
1, p. 17, or Zeit.f. wiss. Mik., vii, 2, 1890, p. 225 (the report mJourn. Roy.
Mic. Soc., 1890, p. 803, is vitiated by a misprint).

UNNA'S Orcein Method (Monatschr. f. prakt. DermatoL, xii,
1891, p. 394; Zeit.f. iviss. Mik., ix, 1, p. 94) is as follows : —
Dissolve O'l grm. of Griibler's orcein in 20 grms. of 95 per
cent, alcohol and 5 grms. of water, and O'l grm. of concen-
trated hydrochloric acid in a like mixture of alcohol and water.
Take six to ten watch-glasses, and pour into each 10 drops of
the stain. Acidify them with the acid mixture, beginning with
5 or 10 drops for the first glass and increasing in the proportion
of 1 drop for each glass in succession. Put one or two sections
into each watch-glass and stain for twelve hours. Examine in
a drop of glycerin and choose the successful sections ; they
should show the elastic fibres of a saturated shiny brown on a
lighter ground. Counter-stain nuclei if desired.

See also ZENTHOEFER, in Unna's DermatoL Studien, 1892,
or Zeit.f. wiss. Mik., ix, 4, 1893, p. 509.

KOPPEN (Zeit. f. wiss. Mik., vi, 4, 1889, p. 473; and vi, 1,
1890, p. 22) gives the following : — Stain sections of alcohol
material for fifteen to twenty-four hours in a freshly prepared
mixture of five parts concentrated solution of crystal- violet
("Kry stall violett"), and 105 parts of 5 per cent, aqueous
solution of carbolic acid. Treat for two minutes with the
usual solution of iodine in iodide of potassium, then for five
minutes with 10 percent, salt solution, and for fifteen seconds
with 1 per cent, hydrochloric acid, Wash out in absolute
alcohol, clear with terebene followed by xylol, and mount in
balsam. Elastic fibres dark violet, stratum corneum of
cutis and sheath of Henle of hairs being also stained.

BURCI (see Journ. Roy. Mic. Soc., 1891, p. 831; and 1892,
p. 292) stains sections for a minute or two in saturated alco-

420 SOME OTHEIl HISTOLOGICAL METHODS.

holic solution of Aurantia, dehydrates in absolute alcohol,
clears in clove oil, and mounts in balsam.

WOLTERS (see Zeit. f. wiss. Mik., ix, 3, 1893, p. 360) mor-
dants sections for twenty-four hours in a mixture of two parts
10 per cent, chloride of vanadium, and eight parts 8 per cent,
acetate of aluminium, stains for twenty-four hours in an in-
cubator in Kultschizky's haematoxylin, and differentiates in
Weigert's ferricyanide mixture, or, after a short treatment
with solution of chloride of iron, in water.

For a somewhat complicated method of MARTIN OTTI'S, with nitrate of silver,
which is, I think, of more theoretical than practical interest, see Zeit. f. wiss.
Mik., v, 4, 1888, p. 521.

For the methods of UNNA and TAENZEE with nitrate of rosanilin salts,
see Monatsch. f. prdkt. Dermat., vi, 1887 ; or BEHRENS, KOSSEL, u. SCHIEF-
FEEDECKER, Das Mikroskop, 1, p. 204.

796. Bone, Non-decalcified (RANVIER, Traite, p. 297).—
Ranvier points out certain precautions that it is necessary to
take in the preparation of sections of dry bone. In general,
the bones furnished by " naturalists," or procured in ana-
tomical theatres, contain spots of fatty substance that prevent
good preparations from being made. Such spots are formed
when bones are allowed to dry before being put into water
for maceration ; when a bone is left to dry the fat of the
medullary canals infiltrates its substance as fast as its water
evaporates.

Bones should be plunged into water as soon as the sur-
. rounding soft parts have been removed, and should be divided
into lengths with a saw whilst wet. The medulla should
then be driven out from the central canal by means of a jet
of water ; spongy bones should be submitted to hydrotomy.
This may be done as follows : — An epiphysis having been
removed, together with a small portion of the diaphysis, a
piece of caoutchouc tubing is fixed by ligature on to the
cut end of the diaphysis, and the free end of the piece of
tubing adapted to a tap through which water flows under
pressure.

As soon as the bones, whether compact or spongy, have
been freed from their medullary substance they are put to
macerate. The maceration should be continued for several
months, the liquid being changed from time to time. As
soon as all the soft parts are perfectly destroyed, the bones

BONE, NON-DECALCIFIED. 421

may be left to dry. When dry, they should be of an ivory
whiteness, and their surfaces exposed by cutting of a uniform
dulness.

Thin sections may then be cut with a saw and prepared by
rubbing down with pumice-stone. Compact pumice-stone
should be taken and cut in the direction of its fibres. The
surface should be moistened with water and the section of
bone rubbed down on it with the fingers. When both sides
of the sections have been rubbed smooth in this way, another
pumice-stone may be taken, the section placed between the
two, and the rubbing continued. As soon as the section is
thin enough to be almost transparent it is polished by rubbing
with water (with the fingers) on a Turkey hone or lithographic
stone. Spongy bone should be soaked in gum and dried
before rubbing down (but see VON KOCH'S copal process, ante,
§ 311, and EHRENBAUM'S colophonium process, § 312).

WEIL (Zeit. f. wiss. Mik., v. 2, 1888, p. 200) gives a process
similar to the foregoing, rough sections or portions of bone
and teeth being penetrated with chloroform balsam, which
is hardened in an oven, or over a water-bath, and then
ground.

ROSE (Anat. Anz., vii, 1892, pp. 512 — 519 ; Zeit.f. wiss. Mik.,
ix, 4, 1893, p. 506) points out some precautions that it is
well to take. He penetrates first with a mixture of cedar oil
and xylol, then with pure xylol, and imbeds in solution of
dammar in chloroform or xylol. The method can be combined
with Golgi's impregnation.

NEALEY (Amer. Hon. Mic. Joum., 1884, p. 142; Journ. Roy.
Mic. Soc., 1885, p. 348) says that perfectly fresh portions of
bone or teeth may be ground with emery on a dentist's lathe,
and good sections, with the soft parts in situ, obtained in half
an hour.

WHITE (Journ. Roy. Mic. Soc., 1891, p. 307) recommends
the following : — Sections of osseous or dental tissue should
be cut or ground down moderately thin, and soaked in ether
for twenty-four hours or more. They should then be put for
two or three days into a thin solution of collodion stained
with fuchsin (made by dissolving the dye in methylated spirit,
adding the requisite quantity of ether, then the pyroxylin) .
The sections are then put into spirit to harden the collodion.
After this they are ground down to the requisite thinness

422 SOME OTHER HISTOLOGICAL METHODS.

between two plates of old ground glass, with water and
pumice powder, and mounted, surface dry, in stiff balsam or
sty rax, care being taken to use as little heat as possible.
Lacunas, canaliculi, and dentinal tubuli are found infiltrated
by the coloured collodion.

MATSCHTNSKY (Arch.f. mik. Anat., xxxix, 1892, p. 151 ; Zeit.
f. wiss. Mik., ix, 3, 1893, p. 353) prepares sections by soaking
in gum and rubbing down as recommended by B-anvier.
When sufficiently thin, the gum is removed by means of water,
the sections dried, put for half an hour into benzin or ether,
and stained in a saturated aqueous solution of some anilin
dye, from one to seven days according to the age of the bone.
They are then washed in water, further ground down, put for
twenty-four hours into alcohol, and mounted by the aid of
heat in thickened balsam. The ^result is a stain of the ground
substance, of varying degrees of intensity.

HOPEWELL-SMITH (Journ. Brit. Dent. Ass., xi, 1890, p. 310 ;
Journ. Roy. Mic. Soc., 1890, p. 529) says that for preparing
sections of teeth showing odontoblasts in situ the best plan is
to take embryonic tissues. A lower jaw of an embryonic
kitten or pup may be taken, and hardened in solution of
Miiller followed by alcohol, then cut with a freezing micro-
tome. The knife cuts quite easily the thin cap of semi- calcified
dentine and bone.

797, Bone and Teeth, Decalcification Methods. — Besides the
processes given in Chap. XXIV, attention may be called to
the following :

HOPEWELL-SMITH (Trans. Odont. Soc., xxiv, 1891, p. 20;
Journ. Roy. Mic. Soc., 1892, p. 433). — Harden a newly-ex-
tracted tooth in solution of Miiller (three to four weeks)
followed by alcohol. Wash in water, and seal up apical fora-
men with collodion. Decalcify as directed in § 565. Wash
with distilled water. Divide with a razor into several pieces,
wash them in water, and put them for twelve to fifteen hours
into gum mucilage (B.P.). Cut with a freezing microtome, and
stain with orange-rubin, gold-chloride, borax-carmine, or by
Weigert's method. Dehydrate, clear with cedar oil, and
mount in balsam.

798. LKPKOWSKY ( Anat. Anz., vii, 1892, p. 274; Zeit. f. wiss.
Mik., ix, 3, 1893, p. 355) recommends the following as giving

BONE, DECALCIFIED. 423

a superior preservation of the structures. Sections of fresh
teeth, not thicker than 0*75 mm., are made with a saw, and
put into a mixture of six parts 1 per cent, gold chloride and
three parts pure formic acid, for twenty-four hours. Wash
with distilled water, and reduce for twenty-four hours in gly-
cerin gum solution. Pass through water and alcohol and
imbed in celloidiu or paraffin, and cut. The dentinal canaliculi
are shown stained.

Bone can be treated in the same way.

For UNDEKWOOD'S gold process for teeth see § 228.

799. VIVANTE (Intern. Monatssch. f. Anat. u. Phys., ix, 1892,
p. 394 ; Zeit. f. wiss. Mik., ix, 3, 1893, p. 351) has made out that
thin specimens of bone can be successfully treated by Golgi's
bichromate of silver process. He places portions of frontal bone
of four to six months calves, which are not more than 3 to
4 mm. thick, for eight days in solution of Miiller, then in the
osmium bichromate mixture, and then in the silver solution.
After impregnation the specimens should be decalcified, which
may be done by putting them for twenty days into von
Ebner's mixture (§ 563), after which they should be well
washed with water and brought into solution of carbonate of
soda, and finally imbedded in paraffin.

Another method recommended by Vivante is as follows : — Fix for five or
six days in solution of Flemming, wash and decalcify with von Ebner's
mixture, wash, treat with carbonate of soda, imbed in paraffin, cut, and stain
the sections for an hour in 0'2 per cent, solution of quinolei'n blue (§ 126).
Wash out in equal parts of alcohol and water followed by pure water, dry
the sections in a stove at 40° C. (both alcohol and glycerin must be avoided),
clear with bergamot oil, and mount in damar. Nuclei violet, protoplasm and
ground substance different shades of blue.

800. Bone, Decalcified (FLEMMING, Zeit. f. wiss. Mik., 1886,
p. 47). — Sections of delcacified bone are made with the free
hand. They are soaked in water, and brought in a drop of
water on to a glass plate, where they are spread out flat. The
excess of water is removed with blotting-paper, and the
sections are covered with another glass plate, to prevent them
from rolling. The whole is brought into a plate and covered
with alcohol. After the lapse of half an hour the sections
have become fixed in the flat position, and may be brought
into absolute alcohol without risk of their rolling. To mount
them, wash them with fresh alcohol (which may be followed

424 SOME OTHER HISTOLOG10AL METHODS.

by ether) ; lay them again flat on glass, and cover them with
a double layer of blotting-paper and a somewhat heavy glass
plate, and let them dry for a day in the air or in a stove.
When they are dry, put a drop of melted balsam on a slide,
and let it spread out flat and cool. Prepare a thin glass
cover in the same way, put the section on the prepared
slide, cover it with the prepared cover, put on a clip, and
warm.

By this process sections can be very expeditiously pre-
pared, which show the lacunar system injected with air in
quite as instructive a manner as non-decalcified sections.

KOLLIKER (Zeit. f. wiss. Zool.j xliv, 1886, p. 662) recom-
mends the following process for the demonstration of the
fibres of Sharpey in decalcified bone. Sections are treated
with concentrated acetic acid until they become transparent,
and are then put for one quarter to one minute into a con-
centrated solution of indigo-carmine, then washed iu water
and mounted in glycerin or balsam. In successful prepara-
tions the fibres of Sharpey appear stained of a pale or dark
red, the remaining bone-substance blue.

801. Cartilage (and Decalcified Bone). — For an excellent
discussion (especially as regards staining) of the methods
that have been recently recommended for these objects, see
the exhaustive paper of SCHAPPEE in Zeit. f. wiss. Mik., v. 1,
1888, which gives in sufficient detail all the methods in ques-
tion. The following appear to be the best : —

RANVIER'S purpurin method. This has been given in detail
in § 201.

SCHAFFER'S safranin method. This method is due in its
principle to BOUMA (Centralb.f. d. med. Wiss., 1883, p. 866).
I give it in the form to which it has been brought by the
careful study of Schaffer (Zeit. f. wiss. Mik., v. 1, 1888, p. 17) .
Sections of bone decalcified with nitric acid (chromic acid
may be used, but the stain will be less brilliantly contrasted)
are stained for half an hour to one hour in 0*05 per cent,
aqueous solution of safranin, washed with water, put for two
or three hours in 0*1 per cent, solution of corrosive sublimate,
and examined in glycerin. In order to make permanent
preparations, the sections on removal from the sublimate
are rinsed with alcohol, pressed on to a slide with filter-

CARTILAGE STAINS. '125

paper, cleared for a long time in bergamot oil or clove oil, and
mounted in xylol balsam.

This is a double stain; cartilage, orange; bone, uncoloured
(or green in chromic objects) ; marrow, red.

BAYERI/S method for ossifying cartilage (Arch. f. mik. Anat*,
1885, p. 35) is as follows : — Portions of ossified cartilage are
decalcified as directed § 564, cut in paraffin, and stainetf
in Merkel's borax-carmine and indigo-carmine mixture, as
directed § 242, and mounted in balsam.

MAX FLESCH (Zeit.f. wiss. Mik., 1885, p. 351) particularly
recommends this process for the study of the development of
dental tissue.

MOERNER (Skandinavisches Arch. f. PhysioL, i, 1889, p. 216;
Zeit.f. wiss. Mik., vi, 4, 1889, p. 508) gives several stains for
tracheal cartilage, chiefly as microchemical tests. 1. Stains
for the trabeculse ("Balkennetz"). — a. Sections (of alcohol
material, I gather) put for half an hour into concentrated
aqueous solution of Tropseolin 000, No. 2 (Schuchardt's),
and washed out in water until only the trabecula3 remain
coloured, b. Stain for a few minutes in concentrated aqueous
solution of Sulphindigotate of Potash (not soda), and wash out
as above.

2. Stains for " Chondrinballen" (groups of cartilage- cells).

a. Stain for half a minute to two minutes in 0*15 per cent,
aqueous solution of methyl violet, rinse in water, and wash
out in 10 per cent, acetic acid until the trabeculse are de-
coloured. " Chondrinballen" blue on a colourless ground.

b. Stain as above, using a 0*15 per cent, solution of anilin red,,
and wash out as before.

3.. Combined methods, a. Stain as in 1 a, then as in 2 a,
dehydrate quickly, and clear in clove oil. b. Stain as in 1 6,
then as in 3 a, but using anilin red instead of methyl violet.

See a critique of these methods by WOLTERS in Arch. f. mik..
Anat., xxxvii, 1891, p. 492; Zeit.f. wiss. Mik., viii, 3, 1891,
p. 383 ; also, on the whole subject of cartilage, see SCHIEFFER-
DECKER'S Gewebelehre, p. 331.

Blood.

802. It might be supposed that for the study of blood it
would suffice to prick a finger, place a drop of blood on a
slide, cover, and examine it. But this is by no means the-

426 SOME OTHER HISTOLOGECAL METHODS.

case. " Blut ist ein ganz lesonderer Saft" * and will not
yield up its secrets to such simple wooing, The technique of
blood is most elaborate ; see, for instance, the voluminous
work of HAYEM, Du sang et de ses alterations anatomiques, pp.
1035, with 126 figures, Paris, Masson, 1889 (a report of over
twenty pages on this important work is contained in Zeit. f.
wiss. Mik., vi, 3, 1889, p. 330, et seq.) ; LOWIT, Sitzb. k. Acad.
Wis8.Wien,3, Ixxxviii, 1883; xcii, 1885; xcv, 1887; Zieg-
ler's Beitr. z. path. Anat., x, 1891, p. 214; Zeit. f. wiss. Mik.,
vi, 1889, pp. 74, 76; viii, 3, 1891, p. 371; Arch. f. mik. Anat.,
xxxviii, 1891, p. 524; Zeit. f. wiss. Mik., ix, 2, 1892, p. 233;
Studien zur Physiol. u. Path. d. Blutes u. d. Lymphe, Jena,
Fischer, 1892; EHELICH, Zeit. f. klin. Medicin, i, 1880, 3,
p. 558; Zeit. f. wiss. Mik., i, 1884, p. 382, and other papers ;
MUELLER, Sitzb. k. Acad. Wiss. Wien, xcviii, 3, p. 219; Zeit.
/. wiss. Mik., ix, 3, 1893, p. 365 ; GEIESBACH, Zeit. f. iviss. Mik.,
vii, 3, 1890, p. 326; and many other investigators.

It is out of the question for me to attempt to abstract these
memoirs in the space at my disposal, so I must confine myself
to giviDg a few methods that may be useful to the general
student, referring the specialist to the original papers.

803. Fixing and Preserving Methods.— The time-honoured
process of drying drops of blood over a flame gives rise to
great deformation of the elements, and should be abandoned
as far as possible. It is better to mix the blood at once
with some fixing and preserving medium, and study it as a
fluid mount.

Most recent authors ( BIONDI, Mosso, MAX FLESCH) are
agreed that by far the most faithful fixing agent for blood-
corpuscles is osmic acid. A drop or two of blood (Biondi re-
commends two drops exactly) is mixed with 5 c.c. of osmic
acid solution, and allowed to remain in it for from one to
twenty-four hours. The exact degree of concentration of the
osmium solution is a somewhat important point, and must be
made out by experiment for each form. As a rule it should
be strong, 1 to 2 per cent. According to Biondi, 2 per cent,
is best. Fixed specimens may be preserved for use in acetate
of potash solution (MAX FLESCH, Zeit. f. wiss. Mik.,v, 1, 1888,
p. 83).

* Goethe's Faust, i, 4, line 1387.

STAINS FOR BLOOD. 427

G-RIESBACH also (op. cit., p. 328) prefers osmic acid, not
only as being a first-rate fixing agent, but because it can be
combined with certain stains without decomposing them. He
mentions methyl green, methyl violet, crystal violet, saf-
ranin, eosin, Saurefuchsin, rhodamin, and iodine in potassic
iodide.

Rossi (Zeit. f. wiss. Mik., vi, 4, 1889, p. 475) advises a mix^
ture of equal parts of 1 per cent, osmic acid, water, and
strong solution of methyl green, permanent mounts being
made by means of glycerin cautiously added.

The mercurial liquids of PACINI (§ 380), especially Nos. 2
and 3, are good. HAYEM (op. cit. ; see also Zeit.f. wiss. Mik.,
vi, 3, 1889, p. 335) has a similar formula, viz. sub-
limate 0'5, salt 1, sulphate of soda 5, and water 200. This
should be mixed with blood in the proportion of about 1:100.
Eosin may be added to it. LOWIT'S formula (Sitzb. k.Acad.
Wiss. Wien, xcv, 3, p. 129; Zeit. f. wiss. Mik.,vi, 1, 1889,
p. 75) consists of 5 c.c. cold saturated sublimate solution,
5 grms. sulphate of soda, 2 grms. salt, and 300 c.c. water.
Mosso finds, however, that both of these are too weak in
sublimate.

Of course other well-tried fixing fluids, such as Flemming's
solution, or Hermann's, may also be used for blood.

804. Stains for Blood. — Blood prepared as above can be
satisfactorily stained with many of the usual reagents.

Eosin stains rose-red all parts of blood-corpuscles that
contain haemoglobin (see WISSOWSKY, Arch.f. mik. Anat.,
1876, p. 479) ; parts that do not contain haemoglobin, such as
the nucleus, remaining unstained. This suggests double-
staining with eosin and haematoxylin.

WISSOWSKY (1. c.) stains in a solution of equal parts of
eosin and alum in 200 parts of alcohol, and then with haema-
toxylin.

MOOEE (The Microscope, 1882, p. 73; Journ. Roy. Mic. Soc.,
1882, p. 714) stains for three minutes in a similar solution
without the alum, washes, and stains for two minutes in a
1 per cent, aqueous solution of methyl green. Eed corpuscles,
red ; nuclei and white corpuscles, bluish green.

MERKEL'S carmine and indigo-carmine stain has been dis-
cussed above (§ 242).

428 SOME OTHER HISTOLOGICAL METHODS.

Fresh (unfixed) blood is perhaps best treated as follows
(BIZZOZERO and TORRE, Archivio per le Scienze mediche, vol. iv,
No. 18, 1880, p. 390) :— Dilute a drop of blood with 0'75 per
cent, salt solution in which has been dissolved a little methyl
violet. This liquid in no wise affects the form of the elements,
stains intensely the nucleus of the red corpuscles, and, in the
white, stains the nucleus intensely, and the protoplasm less
intensely. May be used for the study of bone-marrow and
spleen.

For the staining of the blood-plates of BIZZOZERO, this
observer (Arch. f. path. Anat. u. Phys. ; Zeit. f. iviss. Mik.,
1884, p. 389) employs a 0'02 per cent, solution of me-
thyl violet in salt solution, or a 1 : 3000 solution of gentian
violet.

TOISON (Journ. 8ci. med. de Lille, fev., 1885; Zeit. f. wiss.
Mik.f 1885, p. 398) recommends that blood be mixed with the
following- fluid :

Distilled water .... 160 c.c.

Glycerin (neutral, 30° Baume) . 30 c.c.

Pure sulphate of sodium . . 8 grammes.

Pure chloride of sodium . . 1 gramme.

Methyl violet 5 B . . . 0'25 „
(The methyl violet is to be dissolved in the glycerin with
one half of the water added to it ; the two salts are to be
dissolved in the other half of the water, and the two solu-
tions are to be mixed and filtered.) White blood-corpuscles
stain in this medium in five or ten minutes ; the maxi-
mum of coloration is attained in from twenty to thirty
minutes. White blood corpuscles, violet ; red blood- corpuscles>
greenish.

FERRIER'S liquid is said to have a sp. gr. similar to that of
liquor sanguinis. Fuchsin, 1 grm. ; water, 150 c.c.; rectified
spirit, 50 c.c.; dissolve, and add glycerin, 200 c.c. (from
Squire's Methods and Formulae, p. 39).

LECLEBQ'S fuchsin followed by malachite green, or Congo followed by
gentian and eosin, see Bull. Soc. Beige de Mic., xvi, 1890, p. 61 ; or Journ.
Roy. Mic. Soc., 1890, p. 675.

DEKHUYSEN'S methylen blue and acid fuchsin mixture, see Verhandl.
Anat. Gesellsch., 1892, p. 90 ; or Journ. Boy. Mic. Soc., 1893, p. 116.

GRIKSBACH, besides the stains mentioned in the last§ as being
capable of being combined with an osmic acid fixing solution,

DEMONSTRATION OF BLOOD-PLATES OF BIZZOZERO. 429

recommends a double stain by means of a mixture of solutions
of methyl green and rhodamin added to osmic acid solution
(1 per cent., the dyes in concentrated solution, but the pro-
portions of the mixture not given).

It goes without saying that the EHRLICH-BIONDI mixture
(§§ 259 and 639) will be found a most valuable reagent in
many haematological researches. LOWIT (Ziegler's Bietr. z.
path. Anat., &c., x, 1891, p. 214; Zeit. f. wiss. Mik., viii, 3,
1891, p. 371) obtained instructive results by staining sub-
limate preparations for one to two minutes in a concentrated
solution, and examining in water or glycerin.

For details as to the reactions of the granules of leucocytes and of Lowit's
" pyrenogenous " corpuscles, see the original paper ; also EHSLICH'S
" Methodologische Beitr. z. Physiol., &c., der Leucocyten," in Zeit. f. klin.
Med., i, 1880, 3, p. 558 ; cf. Zeit. f. wiss. Mik., i, 1884, p. 382, and later
papers of Lowit in Anat. Anz., vi, 1891, p. 344, and Arch. f. mik. Anat.,
xxxviii, 1891, p. 524 (Zeit. f. wiss. Mik., ix, 2, 1892, p. 233).

FOA (Festschr. R. Virchow gewidm., &c., 1891, i, p. 481 ;
Zeit. f. wiss. Mik., ix, 2, 1892, p. 227) proceeds as follows : —
Preparations (either coagulated blood or small pieces of haema-
topoietic organs) are fixed in a solution of 2 grms. sublimate
in 100 grms. of liquid of Miiller (the sublimate fixes the
figured structures of protoplasm and nuclei, the Miiller fixes
the haemoglobin). They are imbedded in paraffin, and sections
made and stained for one to three minutes in the following
mixture :

Aq. dest., about ..... 100 grms.

Bohmer's haematoxylin .... 25 „
1 per cent, alcoholic aqueous safranin solution 20 „
Wash out in water, followed by weak alcoholic solution of
picric acid, dehydrate, and mount in balsam. Several other
methods are also mentioned in the same paper.

The elaborate paper of MUELLER mentioned in § 802 is too rich in detail
to bear abstracting here. A novelty in it is the impregnation of cover-glass
preparations with gold chloride by Ranvier's formic acid process.

805. Demonstration of Blood-plates of Bizzozero (KEMP, Studies
fr. the Biol. Lab. Johns Hopkins Univ., May, 1886, iii, No. 6;
Nature, 1886, p. 132). — The mere demonstration of the blood-
plates of Bizzozero is easy enough. A somewhat large drop
of blood is placed on a slide, and quickly washed with a small
stream of normal salt solution. The blood-plates are not

430 SOME OTHER HISTOLOGICAL METHODS.

washed away, because they have the property of adhering to
glass ; and on bringing the slide under the microscope they
will be seen in large numbers. If it be desired to make
permanent preparations of them, they should first be fixed.
This is done by putting a drop of osmic acid solution on the
finger before pricking it.

For BIZZOZEEO'S recent methods for the numeration of these elements
and for the study of their regeneration, see his paper in Festschr. R. Vir-
chow gewidm.y &c., 1, 1891, p. 459 ; or the report of the methods in Zeit. /.
VJ188. Mik., ix, 2, 1892, p. 229.

For the application of some digestion methods to the study of blood-plates,
see LILIENFELD, Arch. f. Anat. u. Physiol., Physiol. Abth., 1892, p. 115 ;
or Zeit.f. wiss. Mik., ix, 3, 1893, p. 363.

806. BIONDI'S Section Method for Blood (Arch. f. mik. 'Anat., xxxi,
1888, p. 103). — None of the foregoing methods are perfectly satisfactory as
regards the preservation of the elements of blood without deformation, and
at the same time in a perfectly permanent manner. Biondi's ingenious pro-
cess does this.

Blood is fixed with osmic acid solution as described above, § 803. Four
or five drops of the mixture of blood and osmium solution are then mixed
with agar-agar jelly melted at 35° to 37° C. The whole is allowed to cool,
and the mass is put to harden in alcohol of 85 per cent. After a few days
the mass will have attained a consistence that allows of its being imbedded
in pith and cut with a microtome. The sections are treated according to
the usual methods. The best stains are obtained with methyl green,
methylen blue, fuchsin, and safranin. Methyl green and eosin is also a
good combination. After staining, the sections are cleared and mounted in.
balsam in the usual way.

Thinner sections can be obtained if the agar-agar mass be imbedded in
paraffin in the usual way, instead of in pith.

Instructions, too long to be abstracted here, are given for the preparation
of a suitable agar-agar mass. It may be obtained ready prepared from
Herrn Konig, 29, Dorotheenstrasse, Berlin. Celloidin, and others of the
usual imbedding masses, were tried, but without success. SCHIEFFEE-
DECKER, however, says that celloidin may be employed (see his Gewebelehre,
p. 389).

For further details the English reader may consult the Journ. Roy. Mic.
Soc., 1888, pp. 313, 659.

This is undoubtedly a valuable method, and is capable of extension to the
study of other animal fluids besides blood.

Glands.

807. Mucin.— It has already been stated that the blue
solutions of hsematoxylin have a special affinity for mucin.
For the demonstration of mucus gland-cells the following
process is, therefore, recommended by FLEMMING (Zeit. f.

MUCIN. 431

wiss. Mik.j 1885, p. 518) : — Stain sections first with haeina-
toxylin of Heidenhain, and afterwards with haematoxylin
of Delafield or Bohmer. The mucus cells are shown stained
violet.

HOYER, who has made a special study of the staining reac-
tions of mucin in tissues (Arch. f. miJc. Anat., xxxvi, 1890,
p. 310; see also Zeit. f. wiss. Mik., viii, I, 1891, p. 67), has
the following conclusions :

The mucin of mucus cells and goblet cells, both of Verte-
brates and Invertebrates, stains with basic tar colours, but
not with acid tar colours (see above, § 788) . More or less
specific stains of mucin are obtained, for instance, with
hydrochlorate or nitrate of rosanilin, commercial fuchsin,
Griibler's "neutral" fuchsin (" n. Unna"), magenta, Magdala
red, iodine green, methyl green, dahlia, methyl violet, gen-
tian, iodine violet, crystal violet, Victoria blue. A similar
reaction is obtained with alum-haematoxylin solutions, whilst
carmine behaves like the acid coal-tar dyes, and affords no
stain .

HOYEE obtained his best results by means of thionin (violet
of Lauth), which gives a double stain, the tissues blue, the
mucin elements ruddy violet. This dye is unfortunately no
longer to be found in commerce ; but the dye called amethyst,
prepared by Geigy and Co., of Bale, is a good succedaneum,
and so are toluidin blue (obtainable from Griibler) or the
phenylen blue of Oehler in Offenbach, and the p-phenylen blue
of the Hochst manufactory. Like thionin, all these give
metachromatic stains.

Results less brilliant than those given by the above-men-
tioned stains, but nevertheless excellent, are obtained by
means of methylen blue or Bismarck brown. Methylen green
and safranin also give good reactions, but are somewhat in-
constant in their effects. Methylen blue is particularly
useful from its power of bringing out the merest traces of
mucin.

All of these colours may be used in the same way. Speci-
mens should be fixed for two to eight hours in 5 per cent,
sublimate solution, imbedded in paraffin, cut, and the sections
stained for five to fifteen minutes in a very dilute aqueous
solution of the dye (two drops of saturated solution to 5 c.c.
of water).

432 SOME OTHER HTSTOLOGICAL METHODS.

It is theoretically interesting to observe that hyaline carti-
lage, the jelly of Wharton, and the Mastzellen of Ehrlich give
the same reactions with basic dyes as mucin does, even their
metachromatic reactions being identical.

These conclusions had already been in part formulated by
SUSSDOKP (Deutsche Zeit.f. Thiermed., xiv, pp. 345, 359; see
Zeit.f. wise. Mik., vi, 2, 1889, p. 205).

See also the important series of papers by BIZZOZERO,
" Sulle ghiandole tubulari del tubo gastro-enterico" &c., in the
Atti R. Accad. di Sci. di Torino, 1889 to 1892; reports in
Zeit.f. wiss. Mik., vii, 1, 1890, p. 61 ; and ix, 2, 1892, p. 219.
As regards the safranin reaction, it is well to note that it is
not obtained with all brands of the dye ; that of Bindschedler
and Busch, in Bale, gives it, whilst safranin 0 of Griibler does
not. For the distinctive reactions of old and young mucin
see the original, or the last- quoted report of the Zeit.f. wiss.
Mik.

808. " Reticulum -"' of Mucus Cells. — This was first demonstrated
bv SCHIEFFERDECKER (Arch. /. mile. Anat., 1884, Heft 3) by means of
anilin green. This reaction has given rise to a long polemic between
SCHIEFFERDECKER and LIST, for which see Zeit. f. wiss. Mile., 1885, pp.
51, 222, 223. According to List, the reticula in question stain in an equally
specific way in methyl green, in Bismarck brown, in nitrate of rosanilin (of
O'OOOl per cent., ten to fifteen minutes ; see op. cit., iii, 3, 1886, p. 393), and
in List's double-stains (§§ 262, 263). Schiefferdecker, on the contrary,
maintains that the reticula demonstrated by the methods of List are not
identical with those demonstrated by anilin green.

809. Goblet Cells. — So far as these contain inuciii they give
the reactions above described (see FLEMMING, Zeit.f. wiss. Mik.,
1885, p. 519; and PAULSEN, ibid., p. 520). But the reactions
appear to be different for different animals. Thus PANETH
(Arch. f. mik. Anat., xxxi, 1888, p. 113, et seq.) found that in
the small intestine of the mouse the contents of the goblet
cells did not stain with Bohmer's haematoxylin. And the
goblet cells of the small intestine of man did not stain with
safranin.

BANVIEE, in a paper too long to be abstracted here (Comptes rend., 1887,
3, p. 145 ; see also Zeit.f. wiss. Mik., v, 2, 1888, p. 233), describes a specific
reaction of perruthenic acid (Ru04) on goblet cells. By treating the
pharyngeal mucosa of the frog first for ten to twelve hours with vapour of
osmium, and then for three minutes with vapours of perruthenic acid, the
goblet cells are brought out witli remarkable distinctness. The contained

OTHER METHODS FOB GLANDULAR STRUCTURES. 433

mucigen is stained black, but the vacuoles are unstained. Since perruthenic
acid is very rapidly reduced by organic matter, Ranvier regards this reaction
as a proof that the vacuoles do not contain any organic substance, but pro-
bably only water and inorganic salts.

For detailed instructions for the study of goblet cells, see
LIST, in Arch. f. mik. Anat., xxvii, 1886, p. 481.
See also ante, §§ 246, 247, 252, 255.

810. Liver. — See hereon the important papers of KANVIEK,
" Les membranes muqueuses et le syst. glandulaire," in the
Journ. de Microgr., ix, x, 1885-6 ; IGACUSCHI, in Arch. f. path.
Anat., xcvii, p. 142, or Zeit. f. wiss. Mik., 1885, p. 243 (gold
process for study of fibrous networks) ; KUPFFER, Sitzb. Ges.
f. Morph., &c., Miinchen, Juli, 1889, or Zeit. f. wiss. Mik., vif
4, 1889, p. 506 (haematoxylin stain for demonstration of ulti-
mate bile-ducts, and application of Golgi's silver bichromate
method to the same object and to the study of fibrous net-
works) ; OPPEL, Anat. Am., v, 1890, p. 143; vi, 1891, p. 165;
and Zeit.f. iviss. Mik., vii, 2, 1890, p. 222; viii, 2, 1891, p. 224
(also concerning the application of Golgi's process to the
above objects).

811. Other Methods for Glandular Structures. — Amongst
numerous important papers that cannot be quoted here, see
KANVIEE, "Le mccanisme de la secretion," in Journ. de Microgr.,
x, 1886-7, and the valuable papers of HEIDENHAIN inPfluger's
Archiv.

The peculiar applicability of the Ehrlich-Biondi stain, § 259,
to this kind of work hardly needs pointing out.

Indigo, used as in § 241 or 242, may also be found very
useful.

For salivary glands see § 252.

28

434 SOME ZOOLOGICAL METHODS.

CHAPTER XXXII.

SOME ZOOLOGICAL METHODS.

Tunicata.

811. Fixation of Tunicata. — A method of SALVATOEE Lo BIANCO
for killing simple Ascidians in an extended state has been
given above, § 18. In the paper quoted below* this plan is
recommended for Ciona, Ascidia, and Rhopalea. But many
other forms, such as Clavellina, Perophora, Phallusia, Molgula,
Cynthia, &c., should first be narcotised by treatment for from
three to twelve hours with chloral hydratef (1 : 1000 in sea
water), then killed in chromo-acetic acid (chromic acid of 1
per cent. 100 parts, acetic acid 10 parts).

The compound Ascidians with contractile zooids are difficult
to manage if one does not go the right way to work. The
best process known to me is the following (due to VAN BEN-
EDEN, kindly communicated to me by Dr. C. Maurice). Place
the corms in clean sea water, and leave them alone for a few
hours, in order that the zooids may become fully extended.
Seize the corms with your fingers, and plunge them suddenly
into glacial acetic acid. Leave them there for two, four, or
six minutes, according to the size of the corms (which of

* A valuable paper giving an account of a number of the processes
employed in the Naples Zoological Station for the preservation of marine
animals has been published by SALVATOBE Lo BIANCO in Mitth. zool. Stat.
Neapel, ix, 1890, p. 435. Keferences to the work of S. Lo BIANCO in the
remainder of this chapter are to that paper. An abstract of it is contained
in Amer. Natural, xxiv, 1890, p. 856, and Journ. Roy. Mic. Soc., 1891,
p. 133, and a very full account in Zeit.f. wiss. Mile., viii, 1, 1891, p. 54.

f A new narcotising process recently incroduced by my friend Dr.
BBOCHEE, of Geneva, has been warmly recommended to me as being more
certain in its action than those hitherto employed. It consists in treatment
with monobromated camphor, which, in the case of small organisms at all
events, may be exhibited by simply placing a few crystals in the water in
which they are contained.

FIXATION OF MOLLUSCA. 435

course you will have taken care to select of as small a size as
possible) . Take them out of the acid with your fingers (or in
some manner that may dispense with the employment of steel
instruments, which would blacken the tissues) and bring them
into 50 per cent, alcohol. Wash them thoroughly in that, and
then bring them in the usual way through successively stronger-
alcohols.

I most strongly recommend this process, which gives
admirably preserved preparations quite free from any opacity
either in the tissues or the tunic. The acid will not hurt the
fingers if they be washed immediately.

S. Lo BIANCO recommends for this group the chloral
hydrate process, followed by fixation with sublimate or
chromo-acetic acid.

Small pelagic Tunicates are very easily fixed with osmic
acid or acid sublimate solution, with the exception of An-
chinia. The not very numerous preparations I have made of
this exceedingly delicate form have all been unsatisfactory.
And some other similar forms may be found difficult. I have
had a striking failure with Salpa virgula, which I fixed with
" Flemming," and got a very poor preparation. The very
similar S. pinnata is fixed perfectly in this medium.

Molluscoida.

812. Bryozoa. — For some methods of killing and fixing see
§§ 7, 14, and 15. S. Lo BIANCO employs for Pedicellina
and Loxosoma the chloral hydrate method, fixing with sub-
limate. For Flustra, Cellepora, Bugula, Zoobothrium, he
employs the alcohol method of EISIG, § 12.

BROCHEE'S monobromated camphor, last section, may be
useful for this group.

Mollusca.

813. Fixation of Mollusca. — Two groups at least amongst
the Mollusca offer considerable difficulties in the way of
fixation — Lamellibranchiata and Gastropoda.

If it be attempted to take living and normal Lamellibran-
chiata from the water they are contained in, in order to throw
them into a fixing solution, they invariably withdraw their
siphon and foot, shut their valves, and die in a state of con-
traction. And if it be attempted to open the shell by force

436 SOME ZOOLOGICAL METHODS.

after death, the mantle is generally injured, and it is im-
possible to get the foot and siphon into the extended state.
DE CASTELLARNAU (La Estacion Zoolog. de Napoles, Madrid,
1885) advises that they be killed by the method of EISIG and
ANDEES described for ActiniaB in § 12. Before dying, the
animals protrude largely their feet, siphons, branchiae, and
tentacles, and die with their shells open. They may be fixed
as soon as insensibility has supervened, by bringing them into
picro-sulphuric acid, or some other rapidly killing fixing
agent.

In order to demonstrate the absence of the supposed aquiferous pores in
the root of Lamellibranchiata, FLEISCHMANN (Zeit.f. wiss. ZooL, xlii, 1885,
p. 376) proceeds as follows : — A mussel is quickly seized at a moment when
the foot is fully extended, and the two valves of the shell are forcibly
pressed together, so as to prevent any flowing back towards the interior of
the liquid contained in the foot. The foot may then be fixed by holding it
for a few minutes in hot sublimate solution.

The same methods recommended for Lamellibranchiata
sometimes give good results with Gastropoda. The asphyxia-
tion method has been described in § 19.

S. Lo BIANCO advises that Lamellibranchiata, Prosobran-
chiata, and, amongst the Heteropoda, Atlantidae, be narcotised
with 70 per cent, alcohol, § 12. Opisthobranchiata ought not
to give much trouble, and I recommend sudden killing with
liquid of Perenyi, or the acetic acid method, § 811.*

For Pteropoda in general, liquid of Perenyi. Creseis is a
difficult form. S. Lo BIANCO advises the alcohol method,
§ 12.

Note the hydro xylamin method of HOFEE, § 16; also
BEOCHEB'S monobromated camphor, § 811.

814. Terrestrial Gastropods. — The asphyxiation method has
been described in § 19.

The quantity of mucus that exists in the integument of Gastropoda is
often a serious obstacle in the way of preparation. MARCHI (Arch. f. mik.
Anat., 1867, p. 204) finds that if a living Limax be thrown into moderately
concentrated salt solution it will throw off enormous quantities of mucus,
and die in a few hours. The epidermis will be found well preserved. If

* Aplysia may first be narcotised by subcutaneous injection of about 1 c.c.
of a 5 to 10 per cent, solution of hydrochlorate of cocain (ROBERT, Bull.
Scient. de la France, &c., 1890, p. 449 ; Zeit. f. wiss. Mik., ix, 2, 1892,
p. 216).

EYES OF CEPHALOPODA AND HETEROPODA. 437

the animal be thrown into osmic acid or Miiller's solution, if I understand
the writer justly, no secretion of mucus will occur.

815. Eyes of Gastropoda (FLEMMING, Arcli.f. mik. Anat., 1870,
p. 441).— The first difficulty here is to obtain the excision of
an exserted eye. It is impossible to sever the exserted
peduncle in a living animal without ite retracting at least
partially before the cut is completed. Never mind that ; make
a rapid cut at the base, and throw the organ into very dilute
chromic acid, or 4 per cent, bichromate ; after a short time
it will evaginate, and remain as completely erect as if alive.
Harden in 1 per cent, osmium, in alcohol, or in bichromate.

CARRIERE (Zool. Anz., 1886, p. 221) gives the following in-
structions : — Remove the eye, together with a portion of the
tentacle, and fix it by exposing it for some minutes to vapour
of osmium. Make sections according to the usual methods,
and fix them on a slide with Schallibaum's collodion. Stain
them with picro-carmine ; or first depigment them by very
careful treatment with very dilute eau de Javelle, and then
stain with picro-carmine. Mount in dammar. Successful
preparations show the tissues perfectly preserved ; but Car-
riere has only been able to make the depigmentation process
succeed with Helix pomatia ; with Prosobranchiata he failed.

816^ Eyes of Cephalopoda and Heteropoda (GRENACHER, Abh.
naturf. Ges. Halle-a.-S. Bd. xvi; Zeit. f. wiss. Mik., 1885,
p. 244). — Fix in picro-sulphuric acid, or in a saturated solu-
tion of corrosive sublimate in picro-sulphuric acid (this mix-
ture is especially useful for Octopus, Eledone, and Sepia, but
does not succeed with the pelagic forms, such as Loligo,
Ommatostrephes, and Rossia). Depigment the specimens with
hydrochloric acid (in preference to the nitric acid used by
Grenacher in former researches). The mixture § 583 may also
be used. The operation of depigmentation may be combined
with that of staining; if you stain with borax-carmine and
wash out in the last-mentioned mixture the pigment will be
found to be removed quicker than the stain is washed out.
Bub this process is delicate, and requires a practised hand.
The operation of depigmentation may be carried out on sec-
tions, but it is better to use portions of retina of 2 to 5 mm.
in thickness. Grenacher mounted his preparations in castor
oil, see § 425.

438 SOME ZOOLOGICAL METHODS.

Similar methods are recommended by the same author for
the eyes of Heteropoda (see Abh. naturf. Ges. Halle-a.-S., 1886 ;
Zeit.f. wiss. Mik., 1886, p. 243).

817. Eyes of Chitonidae (MOSELEY, Quart. Journ. Mic. Sci.,
1885, p. 40). — Moseley worked by decalcifying the shell and
making sections. He places fragments of shell (of which the
tissues have previously been hardened in strong alcohol) in
100 to 200 c.c. of distilled water, and adds drop by drop con-
centrated nitric acid until gas is freely given off, which gene-
rally happens when from 3 to 4 per cent, of acid have been
added. If the decalcification is not complete at the end of
twelve hours the objects should be removed to fresh distilled
water, and the operation repeated. This process is said to
give better results than the various processes of slow decalci-
fication.

818. Eyes of Pecten and other Forms, see PATTEN, in Mitth.
tool. Stat. Neapel, vi, 4, 1886, p. 733.

819. Shell. — Sections of non-decalcified shell are easily
obtained by the usual methods of grinding, or, which is often
a better plan, by the methods of v. Koch or Ehrenbaum,
§§311,312. For sections of decalcified shell, MOSELEY, who
has had great experience of this kind of work, particularly
recommends the method of decalcification given above, § 817.

820. Injection of Acephala (FLEMMING, Arch. f. mik. Anat.,
1878, p. 252). — To kill the animals freeze them in a salt-and-
ice mixture, and throw them for half an hour into lukewarm
water. They will be found dead, and in a fit state for in-
jection. Chloroform and ether are useless (but see § 16).
The injection-pipe may be tied in the heart ; but when this
has been accomplished there remains the problem of occlud-
ing cut vessels that it is impossible to tie. To this end, after
the pipe has been tied, the entire animal is filled and covered
up with plaster of Paris. As soon as the plaster has hardened
the injection may be proceeded with.

821. Maceration Methods for Mollusca. — For the study of
ciliated epithelium the following methods are recommended
by ENGELMANN (Pfliiger's Arch., xxiii, 1880, p. 505) :

Cyclas cornea (intestine), maceration in osmic acid of 0'2

GENERAL METHODS FOR ARTHROPODA. 439

per cent, (after having warmed the animal for a short time to
45° to 50° C.). Also, concentrated boracic acid, solution.

The Intra-cellular Processes of the Cilia. — The entire intra-
cellular fibre apparatus may be isolated by teasing fresh epi-
thelium from the intestine of a Lamellibranch (e. g. Anodonta)
in either bichromate of potash of 4 per cent., or salt solution
of 10 per cent. To get good views of the apparatus in situ iiT
the body of the cell, macerate for not more than an hour in
concentrated solution of boracic or salicylic acid. Very
dilute osmic acid (e. g. 0*1 per cent.) gives also good results.
The " lateral cells " of the gills are best treated with strong
boracic acid solution (5 parts cold saturated aqueous solution
to 1 part water).

BELA HALLER'S Mixture, see § 543.

BROCK'S Medium, § 538.

MOBIUS'S Media, § 539 ; the second of these is much re-
commended by DROOST (Morphol. Jahrb., xii, 2, 1886, p. 163)
for Cardium and My a.

See also the media recommended by PATTEN (Mitth. Zool.
Stat. Neapel, vi, 4, 1886, p. 736). Sulphuric acid, 40 drops
to 50 grammes of water, is here recommended as a most
valuable macerating and preservative agent. Entire molluscs>
without the shell, may be kept in it for months.

Arthropoda.

822. General Methods for Arthropoda. — It may safely be
stated that, as general methods for the study of chitinous
structures, the methods worked out by Paul Mayer (see §§4
and 5, and also 57 and 172) are superior to all others. It is
absolutely necessary that all processes of fixation, washing,
and staining should be done with fluids possessing great
penetrating power. Hence picric acid combinations should
in general be used for fixing, and alcoholic fluids for washing
and staining. Concentrated picro-sulphuric acid is the most
generally useful fixative, 70 per cent, alcohol is the most
useful strength for washing out, and tincture of cochineal in
alcohol of 70 per cent. (§ 172) is a very generally useful
staining fluid. Mayer's haemacalcium (§ 182) may some-
times be preferable, and alcoholic carmine and borax-carmine
will occasionally give more satisfactory results.

440 SOME ZOOLOGICAL METHODS.

Alcoholic picro-sulphuric acid may be indicated for fixing in
some cases.

Some forms are very satisfactorily fixed with sublimate.
Such are the Copepoda and the larva? of Decapoda. It is
sometimes indicated to use the sublimate in alcoholic solution.
Some Copepoda, however (Copilia, Sapphirina), are better
preserved by means of weak osmic acid, and so are the Ostra-
coda. In many cases the osmic acid will produce a sufficient
differentiation of the tissues, so that further staining may be
dispensed with ; Copilia and Phyllosoma are examples of
forms that may be prepared in this simple manner. The
pyrogallic process (§ 233) may often prove helpful in the
study of such forms.

823. Methods for Clearing and Softening Chitin. — The employ-
ment of eau de Javelle or eau de Labarraque, as suggested
by Looss, for making chitin transparent and permeable to
reagents has been described above, § 557.

LIST (Zeit. f. wiss. Mik., 1886, p. 212) has obtained good
results with Coccidae by treating them (after hardening) for
eighteen to twenty-four hours with eau de Javelle, diluted
with four volumes of water. After washing out with water,
the objects may be dehydrated with alcohol and imbedded in
paraffin, the chitin being sufficiently softened to allow of their
being penetrated and good sections being obtained. You may
stain before imbedding, with alum-carmine or picro-carmine
(five to six days).

The same methods are applicable to the preparation of the
ovaof Insecta — for instance, Periplaneta (see MORGAN, Am.Mon.
Hie. Journ., ix, 1888, p. 234).

824. Other Depigmentation Methods. — Besides the depig-
mentation processes discussed in Chap. XXIV, the following
methods are available.

SAZEPIN'S Method for Antennae of Chilognatha (Mem. Acad.
Imp. St. Petersb., xxxii, 9, 1884, pp. 11, 12).— Sazepin
treated antennae that have been dehydrated with alcohol by
steeping them in chloroform. The reaction is slow, the chitin
becomes gradually less opaque, but the pigment does not
entirely disappear. In order to remove the last trace of
it, it will be sufficient if a drop of fuming nitric acid be
now added to the chloroform. The mixture must be occa-

EYES OF ARTHROPODS. 441

sionally agitated, in order to prevent the acid from floating
on the chloroform. The reaction is complete in twenty-four
hours.

Employed in this manner, nitric acid has no injurious
action on tissues.

825. Eyes of Arthropods. — LANKESTER and BOURNE (Quart.
Journ. Mic. Sci., 1883, p. 180) prepared the eyes of Limulus
as follows : — Alcohol, turpentine,, paraffin; sections made and
carefully depigmented under the microscope with nitric acid
of 5 to 10 per cent., then mounted in balsam, some after
staining with borax-carmine, others unstained. Non-depig-
mented sections also mounted in the same manner.

HICKSON (ibid., 1885, p. 243) prepared the eye of a fly as
follows : — Remove the posterior wall of the head, and expose
the rest, with the eyes in situ, for twenty minutes to vapour
of osmium. Wash for a few minutes in 60 per cent, alcohol.
Harden in absolute alcohol. Make sections. To depigment
them, mount them on a slide with Mayer's albumen, remove
the paraffin with turpentine, treat them with absolute alcohol,
and invert the slide over a capsule containing 90 per cent,
alcohol to which a few drops of strong nitric acid have been
added. Nitrous vapours are freely given off, and the pigment
dissolves. The reaction may be stopped at any moment by
washing with pure alcohol.

For dissociation preparations, put the eye or the optic
nerve for twenty-four hours into 5 per cent, solution of chloral
hydrate, tease, and mount in glycerin. If the elements of the
teased tissues be fixed to the slide by means of Mayer's
albumen, they may be washed with alcohol and stained in situ,
or they may be depigmented before staining.

The methods of PARKER for eyes of scorpions have been
given, § 579.

For the eye of Homarus he gives the following staining
method (Bull. Mus. Comp. Zool., Cambridge, U.S.A., xx, 1890,
p. 1; Zeit. f. wiss. Mik., viii, 1, p. 82). Paraffin sections
fixed to slide with Schallibaum's collodion, and passed
through alcohol into water, are treated for half a minute with
0*1 per cent, caustic potash solution, well washed with water,
and stained for about three hours in Weigert's haematoxylin
solution at a temperature of 50° C. ; washed in water, de-

442 SOME ZOOLOGICAL METHODS.

hydrated, and mounted in balsam. No differentiation of the
stain is necessary.

See also the somewhat similar methods of VIALLANES (Ann.
d. Sci. Nat., xiii, 1892, p. 354; Journ. Roy. Mic. Soc., 1893,
p. 260).

826. Nerve and Muscle of Arctiscoida (DOYERE, Arch. f. mik.
Anat.f 1865, p. 105). — A score or so of Milnesium tardigradum
are collected (it is well to have a large number, as the process
by no means succeeds with all individuals) and put into a
test-tube with water that has been deprived of its air by
boiling. A drop of oil is run on to the surface of the water,
so as thoroughly to exclude the air. After twenty-four to
forty-eight hours the animals will be found, not dead, but
fixed and extended in a cataleptic state; the circulation of the
perivisceral fluid has ceased, the pigment of the cuticle has
disappeared or collected into patches that are no hindrance to
observation, the entire animal has gained in transparency,,
and the nervous and muscular systems stand boldly out. The
animals are examined in boiled water, unless it be wished to
study the phenomena of resuscitation, in which case spring
water should be used.

827. Sarcolemma of Insecta (THANHOFFEE, ibid., 1882, p. 27).
— In order to demonstrate the two plates of the sarcolemma,
digest muscle (of an insect) either in the stomach of a living
animal (by wrapping it in gauze and introducing it through
a fistula) or in artificial gastric juice (in the former case
several hours, in the latter half to one hour, at the tempera-
ture of the room in summer). Examine in gastric juice.

828. Phalangida (ROSSLER, Zeit. f. wiss. ZooL, xxxvi, 1882,
p. 672). — The animals are killed in boiling water; the water
is allowed to boil up several times, so that the albumen of the
tissues may be coagulated; they are then brought into alcohol,
first of 70, then 90 per cent., then absolute, until all water is
removed from them. They are then imbedded in soap. The
soap is remelted and allowed to cool once or twice, in order
to get the objects thoroughly penetrated. Sections are then
made, and stained on the slide with some colouring matter
dissolved in absolute alcohol.

Paraffin was tried for imbedding, but gave no good results,

TEEMATODES. 443

on account of the brittleness of the tissues, caused by the
preliminary treatment with turpentine or oil of cloves. I
fancy that this difficulty would be easily overcome by clearing
with cedar oil instead of clove oil, as I have constantly recom-
mended for the special purpose of avoiding brittleness in
tissues.

829. Macrotoma plumbea (SOHMER, Inaug. Diss., 1884, p. 4;
Zeit.f. wiss. Mik., 1885, p. 234). — Fix with boiling water ac-
cording to the method of Rossler, last section, and harden for
several hours in picro-sulphuric acid diluted with 5 volumes
of water. Penetrate with chloroform and imbed in paraffin.

830. Aphidae (see ante, § 621).

831. Other Methods for Arthropoda. — For Embryological
methods see Chap. XXV, §§ 616—627.

For Spermatological methods see Chap. XXVI, § 645.

Vermes.

832. Cestodes. — This group must of course be chiefly studied
by the usual section methods. It is only necessary here to
remind the reader that, as pointed out by VOQT and YUNG
(Trait. d'Anat. comp. prat., p. 204), the observation of the
living animal may be of service, especially in the study of the
excretory system. And, as shown by PINTNER, taeniao may be
preserved alive for several days in common water to which a
little white of egg has been added.

LONNBERG (Centralb. f. Bakteriol. u. Parasitenk., xi, 1892,
p. 89; Journ. Roy. Mic. Soc., 1892, p. 281) has kept Tri&no-
phorus nodulosus, a parasite of the pike, alive for a month in
a slightly acid pepsin-peptone solution contain ing from 3 to 4
per cent, of nutritive matter, and less than 1 per cent, of
NaCl.

The methylen blue -intra vitam staining method should be
found useful with this group.

833. Trematodes (FISCHER, Zeit.f. wiss. ZooL, 1884, p. 1). —
Opisthotrema cochleare may be mounted entire in balsam, after
treatment with absolute alcohol, picro-carmine, or haema-
toxylin or ammonia-carmine, and clearing with clove oil.
For sectioning, Fischer recommends imbedding in a mass
made by dissolving 15 parts of soap in 17*5 parts of 96 per

444 SOME ZOOLOGICAL METHODS.

cent, alcohol. This mass melts at about 60° C., penetrates
very rapidly, and solidifies very quickly. The sections should
be studied in glycerin.

WRIGHT and MACALLUM (Journ. o/Morph., i, 1887, p. 1) find
that Sphyranura is for most purposes best fixed in liquid of
Flemming, and stained with alum-cochineal.

Cercarise. — SCHWAEZE (Zeit. f. wiss. ZooL, xliii, 1886, p. 45)
found that the only fixing agent that would preserve the his-
tological detail of these forms was cold saturated sublimate
solution warmed to 35°— 40° C.

834. Turbellaria. — Methyleo blue will in some cases be
found useful for the study of living specimens.

Fixiug is difficult, arid generally unsatisfactory.

For Ehabdoccela BRAUN (Zeit. f. wiss. Mik.y iii, 1886, p. 398)
proceeds as follows : — For preparing entire animals, the spe-
cimens are got on to a slide, lightly flattened out with a cover,
and killed by running under the cover a mixture of three
parts of liquid of Lang with 1 per cent, osmic acid solution.
Other fixing media than that described were not satisfactory.
(BoHMia, however, commenting on this, says that for some of
the tissues, such as muscle and body parenchyma, nitric acid
and picro-sulphuric acid are very useful.) Sections may be
made by the usual paraffin method.

DELAGE (Arcli. de ZooL exp. et gen., iv, 2, 1886 ; Zeit. f. wiss. Mik., iii,
2, 1886, p. 239) strongly recommends fixation (of Rhabdoccela Acrela) by the
osmium-carmine mixture, § 167. Concentrated solution of sulphate of iron
is also an excellent fixing medium. The animals (Convoluta) die in it fully
extended. Liquid of Lang was not successful.

For staining, he recommends either the osmium-carmine stain or impreg-
nation with gold (^ formic acid, two minutes ; 1 per cent, gold chloride, ten
minutes ; 2 per cent, formic acid, two or three days in the dark. It is well
to allow an excessive reduction to take place, and then lighten the stain by
means of 1 per cent, solution of cyanide of potassium).

BOHMIG, commenting on the above, says that he has obtained very in-
structive images with Plagiostomidae fixed with sublimate and stained with
osmium-carmine.

VON GRAFF (Die Organisation d. Turbellaria Accela, Leipzig,
1891; see Zeit. f. wiss. Mik., ix, 1, 1892, p. 76) has the fol-
lowing remarks : — Chromo-aceto-osmic acid, followed by hae-
matoxylin, is good for the skin ; but even this method will not
afford a satisfactory preservation of the Rhabdites, which in
Acoela and Alloiocoela seem to be destroyed by swelling, whilst

NEMERTINA. 445.

in terrestrial and fresh-water Planaria, Polyclada, and most
Khabdoccela they are better preserved. The same method is
also good for the parenchyma of Amphichoerus cinereus, Convo-
luta paradoxa, and C. sordida. Sublimate is not good for
these forms, but it is good for Convoluta Eoscoffensis. For
some forms it is important to avoid picro-carmine, which
destroys the central parenchyma. The nervous system may
be investigated by the methods of DELAGE.

For Dendroccela sublimate solutions, sometimes hot, appear
indicated for fixing. CHICHKOPF (Arch, de Biol., xii, 1892,
p. 438 ; Journ. Roy. Hie. Soc., 1893, p. 262) recommends the
following for fresh-water Dendroccela : — 2 per cent, sublimate
solution, 6 parts; 15 per cent, acetic acid, 4 parts; pure nitric
acid, 2 parts ; 14 per cent, chloride of sodium, 8 parts ; and
2 per cent, alum, 1 part. The animals are said to die in it with-
out contraction. Note also the mixtures of LANG, § 48. The
staining method of that author with picro-carmine and borax-
carmine (Fauna u. Flora d. Golfes von Neapel [Polycladidea'],
1884, p. 30) seems to be now somewhat antiquated. Mayer's
tincture of cochineal, § 172, may be found useful for the study
of glands, for which purpose the Ehrlich-Biondi stain should
also be employed.

835. Nemertina. — After considerable experience of this
difficult group I have to say that I know of no method of
fixation that will certainly give good results. My best results
have always been obtained with cold saturated sublimate
solution, acidified with acetic acid. I have tried most of the
energetically hardening fixing agents, such as the osmic and
chromic mixtures, and do not recommend them for this
group, for they seem (the chromic mixtures and perchloride
of iron in particular) to act as irritants, and provoke such
violent muscular contractions that the whole of the tissues
are crushed out of shape by them. And, besides, they do
not kill as quickly as sublimate.

I have found it a good plan to decapitate the animals (in
the larger forms), cut them up quickly into lengths (not too
long), and throw these sharply into the sublimate, the mus-
cular contractions being less energetic in segments that are
no longer in connection with the cerebral ganglia.

Perhaps a better method than this will be found in the

446 SOME ZOOLOGICAL METHODS.

simple process, suggested to me by Prof. DU PLESSIS, of fixing
with hot (almost boiling) water. On the few occasions on
which I have tried it the animals have died in extension,
without vomiting their proboscis ; and I think it is certainly-
worth trial, especially for the larger forms.

I have tried FOETTINGEE'S chloral hydrate method (§ 14).
My specimens died fairly extended, but vomited their pro-
boscides. According to S. Lo BIANCO narcotisation with a
solution of 0*1 to 0*2 per cent, in sea water is found successful
at Naples. For the smaller forms Brocher's monobromated
camphor, § 811, may be found useful.

DE CASTELLAENAU (Estacion Zool. de NapoleSj p. 137) says
that Nemerteans can be successfully narcotised by Eisig's
alcohol method, described § 13, and I think the process may
be a good one for some of the larger forms.

DENDY (see Journ. Roy. Hie. Soc., 1893, p. 116) has suc-
ceeded with Geonemertes by exposing it for half a minute to
the vapour of chloroform.

Intra vitam staining with methylen blue will be found
useful in some cases. For the application of the methylen-
blue method to the study of the nervous system see BURGEE,
in Mitth. Zool 8 tat. Neapel, x, 1891, p. 206.

For staining fixed specimens in toto I hold that it is well-
nigh necessary to employ alcoholic stains, for even the most
delicate species are not satisfactorily penetrated by watery
stains in any reasonable lapse of time. Borax-carmine or
Mayer's alcoholic-carmine may be recommended; not so
cochineal or haematoxylin stains, on account of the energy
with which they are held by the mucin which in general
exists in such great abundance in the skin of these animals.

Sections by the paraffin method, after penetration with oil
of cedar (chloroform will fail to penetrate sometimes after the
lapse of weeks).

836. Nematodes. — The extremely impermeable cuticle of
these animals is a great obstacle to preparation. According
to Looss (Zool. Anz., 1885, p. 318) this difficulty may be
overcome by treating the animals (or their ova, which are in
the same case) with eau de Javelle or eau de Labarraque, in
the manner described in § 557.

For fixing, most recent authors recommend sublimate

GEPHYREA. 447

solutions ; chromic solutions seem to have a tendency to make
the worms brittle.

Staining is frequently difficult, and sometimes alcoholic
carmine, § 170, is the only thing that will give fair results.

BEAUN (see Journ. Roy. Mic. Soc., 1885, p. 897) recommends
that small unstained Nematodes be mounted in a mixture oj
20 parts gelatin, 100 parts glycerin, 120 parts water, and 2
parts carbolic acid, which is melted at the moment of using.
Canada balsam, curiously enough, is sometimes found to make
Nematodes opaque.

837. Acanthocephali. — It is very difficult to kill Echino-
rhynci so as to have the animals duly extended and the tissues
well preserved. Neither corrosive sublimate nor strong osmic
acid will, as a rule, attain this end, even after preliminary
intoxication with tobacco smoke or chloroform, the animal
thus treated dying contracted.

HAMANN, however (Jen. Zeit. f. Naturw., xxv, 1890, p. 113;
Zeit. f. wiss. Mi~k., viii, 2, 1891, p. 209), has succeeded with
sublimate, and also with alcohol containing a little platinum
chloride.

SAEFFTIGEN (Morphol.Jahrb., x, 1884, p. 120 ; Journ. Roy. Mic.
Soc. [N.S.], v, 1885, p. 147) obtained the best results by
killing gradually with O'l per cent, osmic acid; the animals
placed in this contract during the first hours, but stretch out
again and die fully extended.

Another method of killing is treatment with 0*1 per cent,
chromic acid; Echinorhynci live for days in it, but event-
ually die fully extended.

KEISER'S methods, see §§ 41 and 61.

838. Gephyrea. — VOGT and YUNG (Anat. comp. prat., p. 373)
direct that Sipunculus nudus be kept for some days in per-
fectly clean basins of sea water, in order that the intestine of
the animals may be got free from sand, which would be an
obstacle to section cutting, and then anaesthetised with chloro-
form, under which treatment they die extended, and may be
fixed as desired.

WARD (Bull. Mus. Comp. Zool., Cambridge, Harvard Coll.,
xxi, 3, p. 144) found the best plan was to put the animals
into a shallow dish with sea water and pour 5 per cent,
alcohol in a thin film on to the surface of the water. After

448 SOME ZOOLOGICAL METHODS.

four to eight hours, if the animals make no contractions on
being stimulated, they may be removed to 50 per cent,
alcohol.

S. Lo BIANCO says killing with O5 per cent, chromic acid
or with O'l per cent, chloral hydrate in sea water may be
tried, but either method is uncertain. Phascolosoma and
Phoronis should be treated by the alcohol method.

APEL (Zeit. f. wiss. ZooL, xlii, 1885, p. 461) says that
Priapulus and Halicryptus can only be satisfactorily killed by
heat. The animals may either be put into a vessel with sea
water and be heated on a water-bath to 40° C. ; or they may
be thrown as rapidly as possibly into boiling water, which
paralyses them so that they can be quickly cut open and
thrown into one third per cent, chromic acid, or picro-
sulphuric acid.

839. Rotatoria. — By far the most important method for the
study of this group consists in the observation of the living
animals. Great difficulty exists in the way of getting them
to keep sufficiently quiet. VOGT and YUNG (Anat. comp. prat.,
p. 420) say that a drop of solution of any of the soluble salts
of strychnin run under the cover sometimes renders service.
WEBER (Arch, de Biol., viii, 4, 1888, p. 713) finds that
strychnin, prussic acid, and curare act too strongly; of all
the reagents he tried, 2 per cent, solution of hydrochlorate of
cocain gave the best results. Warm water gave him good
results for large species, such as those of Hydatina and
Brachionus.

HARDY (Journ. Roy. Hie. Soc., 1889, p. 475) recommends
thick syrup added drop by drop to the water. HUDSON (ibid.,
p. 476) mentions weak solution of salicylic acid.

HOFER'S hydroxylamin method has been given, § 16, and
TULLBERG'S chloride of magnesium method, § 17. BROCHER'S
monobromated camphor, § 811, should be tried; as should
also the processes of EISMOND and of JANSEN, § 858. Methylen
blue, § 114, may be found useful.

Permanent preservation of Rotifers has hitherto been
considered by those who have tried it to be well-nigh impos-
sible. ROUSSELET (Journ. Quek. Mic. Club, v, 2, 1893, p. 205;
Journ. Roy. Mic. Soc., 1893, p. 262) has, however, lately had
a considerable measure of success. The animals should be

CLEANSING INTESTINE OF LUMBRICQS. 449

narcotised by means of cocain of 1 to 2 per cent, strength
added to the water in a trough containing them. They
should be watched under the microscope until the cilia have
just ceased to vibrate, and should then be fixed by running
strong solution of Flemming down the sides of the trough by
means of a pipette. They are left for fifteen minutes, washed
in five or six changes of distilled water, and permanently
mounted in distilled water containing a trace of solution of
Flemming (about 8 drops to the ounce). For minutiaa
concerning the different species see the original.

Annelida.

840. Cleansing Intestine of Lumbricus (KUKENTHAL, Journ.
Roy. Mic. Soc., 1888, p. 1044). — Put the animals into a tall
glass vessel which has been filled up with bits of moistened
blotting-paper. They gradually evacuate the earthy particles
from the gut, and fill it instead with paper.

VOGT and YUNG (Traite d'Anat. Comp. Prat., v) recommend
coffee-grounds instead of paper ; paper becomes rather hard
when imbedded, whereas coffee-grounds cut fairly well.

841. Lumbricus may be anaesthetised by putting the animals
into water with a few drops of chloroform. PERRIEE has
pointed out that it is better not to let the chloroform act
directly in solution on the animals, but to put them into
water in a shallow dish, set up a watch-glass with chloroform
in the corner of it, and cover the whole. In half an hour the
worms will be more or less narcotised, and if allowed to
remain will die in a state of extension.

CERFONTAINE (Arch, de Biol., x, 1890, p. 327; Zeit.f. luiss.
Mik., viii, 2, 1891, p. 210) much recommends curare, ad-
ministered by interstitial injection of a dose of about 2 c.c. of
a 1 : 500 solution. The animal should afterwards be put into
water, and after a quarter of an hour will be found dead.

In order to kill Criodrilus lacuum, COLLIN (Zeit. f. wiss.
Zool., xlvi, 1888, p. 474) puts the animals into a closed vessel
with a little water, and hangs up in it a strip of blotting-
paper soaked in chloroform. KUKENTHAL (Die mik. Technik,
1885 ; Zeit. f. wiss. Mik., 1886, p. 61) puts Annelids into a glass
cylinder filled with water to the height of 10 centimetres, and
then pours 70 per cent, alcohol to a depth of 1 to 2 centi-

29

450 SOME ZOOLOGICAL METHODS.

metres on to the water. The animals will be found suffi-
ciently narcotised for fixation in from four to eight hours.
For OpheliaddB he also employs 0*1 per cent, of chloral hydrate
in sea water.

Many marine Chaetopoda may be successfully narcotised
(S, Lo BIANCO) in sea water containing 5 per cent, of alcohol,
or by means of the mixture, § 12.

The Polychseta sedentaria offer the difficulty of a complex
and very contractile branchial apparatus. They may some-
times be satisfactorily fixed by bringing them rapidly into
corrosive sublimate. Cold, not hot solutions should be taken,
as heat frequently shrivels up the branchiae. The species of
Polychseta errantia that offer a contractile branchial appara-
tus, as Eunice and Onuphis, may be treated in the same way.

S. Lo BIANCO advises killing Chastopteridae, Sternaspidae,
Spirographis, Protula, by putting them for half an hour into
1 per cent, chromic acid. I have satisfied myself that good
show specimens can be obtained in this way ; but I doubt the
histological preservation of the parts being so good as with
sublimate specimens. Some of the sedentaria may be got
protruded from their tubes by leaving them for some hours in
Ol per cent, chloral hydrate in sea water (S. Lo BIANCO).

See also the methods §§ 14 to 19, and § 811.

I can recommend as a good fixing and hardening mixture
for Annelids in general the following fluid, due to EHLERS
(I do not know whether it has been published elsewhere) : —
To 100 c.c. of chromic acid of 0'5 to 1 per cent, add from 1 to
5 drops of glacial acetic acid. The proportion of acetic acid
indicated is sufficient to counteract any tendency to shrinkage
due to the chromic acid.

842. Blood-vessels of Annelids (KUKENTHAL, Zeit. f. wiss.
Mik., 1886, p. 61). — The animals should be laid open and put
for two or three hours into aqua regia (4 parts of nitric acid
to 2 of hydrochloric acid) . The ramifications of the vessels
will then be found to be stained black, the rest of the
preparation yellow.

843. Nerves of Annelids.— The methylen-blue method and
the bichromate of silver method of Golgi (the rapid method).
For the latter see v. LENHOSSEK (Arch. f. mik. Anat., xxxix,
p. 102 ; Zeit.f. wiss. Mik., ix, 3, 1893, p. 342).

HOLOTHURIOIDEA. 451

844. Hirudinea. — For the methods of killing see those
given for Lumbricus in § 841, also §§ 14 to 19, and § 811.

WHITMAN (Meth. inmic. Anat., p. 27) recommends that they
be killed with sublimate. This reagent kills leeches with
such rapidity that they die in general without having time to
change the attitude in which they were found at the moment
when the liquid came into contact with them.

Injection. — WHITMAN (Amer. Natural., 1886, p. 318) states
that very perfect natural injections may often be obtained
from leeches that have been hardened in weak chromic acid
or other chromic liquid. He considers that these injections
are the best for the purpose of the study of the circulatory
system by means of sections.

Of course Hirudinea (or any other Annelids) on which it is
desired to make artificial injections must be killed by some
procedure that leaves the tissues in a state that will allow the
injection to run freely.

JAQUET (Mitth. Zool. Stat. Neapel, 1885, p. 298) advises
that leeches be put into water with a very small quantity of
chloroform ; they soon fall to the bottom of the vessel and
remain motionless, They should be allowed to remain a day
or two in the water before injecting them.

Echinodermata.

845. Holothurioidea. — These animals are difficult to fix on
account of their contracting with such violence under the in-
fluence of irritating reagents as to expel their viscera through
the oral or cloacal aperture. It has been recommended that
they be seized by the middle of the body and firmly squeezed
in the hand, and so plunged in a fixing liquid (acetic acid, for
instance), or that they be anaesthetised (in the case of Synapta
and Cucumama) by adding ether to the water in which they
are contained. So far as my experience goes, I am bound to
say that I know no better way of killing them than that of
simply putting them into fresh water, in which they generally
die without contraction, and with their tentacles extended.
Of course the histological preservation of the parts is
detestable.

S. Lo BIANCO puts Holothurids into pure sea water until
they have expanded their tentacles, then seizes them with

452 SOME ZOOLOGICAL METHODS.

forceps behind the tentacles, so as to mechanically render
impossible their withdrawal, and immerses the anterior part
of the body in acetic acid, whilst at the same time an assistant
injects 90 per cent, alcohol through the anus.

VOGT and YUNG (Anat. Comp. Prat., p. 641) say that Cucu-
maria Planci (C. doliolum, Marenzeller) is free from the vice
of expelling its intestines under irritation ; but they recom-
mend that it be killed with fresh water, or by slow intoxication
with alcohol, chromic acid, or sublimate added to the sea
water in which it is contained.

Synapta may be allowed to die in a mixture of equal parts
of sea water and ether or chloroform (S. Lo BIANCO).

846. Asteroidea. — There are great difficulties in the way of
fixation here, too. It is quite possible to obtain a fixation of
the ambulacral feet, branchiae, and tentacles in the extended
state, by throwing the animals into boiling water, and then
bringing them into a fixing liquid. But this method has the
fault that the fixing liquid so employed only penetrates
extremely slowly into the interior of the animal, and therefore
does not give a good fixation of internal organs.

HAMANN (Beitr. z. Hist. d. Echinodermen, ii, 1885, p. 2) finds
it preferable to inject the living animal with a fixing liquid.
The cannula should be introduced under the integument at
the extremity of a ray, and the liquid injected into the body-
cavity. The ambulacral feet and the branchiae are soon dis-
tended by the fluid, and as soon as it seems to have penetrated
sufficiently the animal is thrown into a quantity of the same
reagent.

The study of the eyes presents points of special difficulty.
In order to study them in sections, with the pigment preserved
in situ, the eye should be removed by dissection, should be
hardened in a mixture of equal parts of 1 per cent, osmic acid
and 1 per cent, acetic acid, and imbedded in a glycerin gum
mass, or some other mass that does not necessitate treatment
with alcohol (which dissolves out the pigment, leaving the
pigmented cells perfectly hyaline). For maceration use one-
third alcohol, the aceto-osmic mixture failing to preserve the
rods of the pigmented cells.

847. Ophiuridea. — Should be killed in fresh water if it be

LARVJ3 OF EOHINODERMATA. 45S

desired to avoid rupture of the rays (DE CASTELLARNAU, La
Est. Zool. de Napoles, p. 135).

848. Larvae of Echinodermata. — I am greatly obliged to my
able friend Dr. BAREOIS for kindly writing down for me (for
the Traitv des Meth. techn., from which they are translated)
the following instructions, which are the outcome of a pro-
longed and minute study of the metamorphoses of the Echino-
dermata.

Pluteus. — In order to a fruitful study of the metamorphoses
of the Echinoidea and Ophiuridea it is necessary to obtain
preparations that offer the advantages presented by the study
of the living larvse; and especially such as give distinct
images of the different organs, and show the calcareous
skeleton preserved intact (a point of considerable importance,
since this skeleton frequently affords landmarks of the greatest
value). These preparations should further possess the fol-
lowing points : — They should give clear views of the region
of formation of the young Echinoderm (which is generally
opaque in the living larva). And they should possess suffi-
cient stiffness to allow of the larva being turned about in any
desired way, and placed in any position under the microscope.

It is not very easy to obtain preparations fulfilling these
conditions, on account of the difficulty of obtaining a selective
stain whilst preserving the integrity of the calcareous skeleton.
The following method is recommended : — Pluteus larvae are
fixed in a cold saturated solution of corrosive sublimate, in
which they remain not more than two or three minutes. They
are then washed with water, and brought into dilute Mayer's
cochineal (§ 172). This should be so dilute as to possess a
barely perceptible tinge of colour. The objects should remain
in the stain for from twelve to twenty-four hours, being care-
fully watched the while, and removed from the stain at the
right moment and mounted in balsam, or, which is frequently
better, in oil of cloves or cedar-wood. This method is per-
fectly satisfactory for the study of the chief phases of meta-
morphosis.

Auricularia and Bipinnaria. — The method described above
is equally applicable to these forms, and seems to be altogether
the best method for the study of the metamorphosis of Bipin-
naria. The earlier stages of the metamorphosis of Auricularia

454 SOME ZOOLOGICAL METHODS.

are better studied by fixing with osmic acid, staining with
Beale's carmine, and mounting in glycerin.

Larvse of Comatula. — The best method for the study of the
embryonal development of Comatula consists in fixing with
liquid of Lang, and staining with dilute borax-carmine. It
is important (for preparations that are not destined to be
sectioned) to use only dilute borax-carmine, as the strong
solution produces an over-stain that cannot easily be reduced.

Narcotisation by chloral hydrate before fixing is useful,
especially for the study of Pentacrinus larvae and of the young
Synaptse formed from Auricularia. Without this precaution
you generally get preparations of larvae either shut up
(Pentacrinus) , or entirely deformed by contraction (young
Synaptse) .

See also MACBRIDE on the development of Amphiura squa-
mata, Quart. Journ. Mic. Sci., xxxiv, 1892, p. 131 ; Journ. Roy.
Mic. Soc., 1893, p. 117 (osmic acid followed by liquid of
Miiller and alcohol ; decalcification with nitric acid in alcohol ;
staining with Mayer's paracarmine or haemalum).

849. Anthozoa. — Narcotisation. — For suitable narcotisation
methods see §§ 9 to 19, and § 811.

Fixation. — In LeAttinie. Fauna u. Flora d. Golfes v. Neapel
the following hints are given : — Hot corrosive sublimate often
gives good results. In the case of the larger forms the solu-
tion should be injected into the gastric cavity, and a further
quantity of the liquid be poured over the animals.

Freezing sometimes gives good results. A vessel containing
Actinias is put into a recipient containing an ice-and-salt
freezing mixture and surrounded by cotton wool. After
freezing, the block of ice containing the animals is thawed in
alcohol or some other fixing liquid.

The Zoantharia with calcareous skeletons are difficult to
deal with on account of the great contractility of the polyps.
Sublimate solution, which ought very often to be taken boil-
ing, sometimes gives good results. DE CASTELLARNAU (LaEst.
Zool. de Napoles, p. 132) says that this process succeeds well
with Dendrophyllia, Antipathes, Astroides, Cladocora, and
Caryophyllia.

The Alcyonaria have also extremely contractile polyps.
In the last edition I suggested for their fixation either hot

ANTHOZOA. 455

sublimate solution or glacial acetic acid (§ 53). S. Lo BIANCO
has since recommended essentially similar processes. GARBINI
(Manuals, p. 151) says that the polyps may be fixed in the
state of extension by drenching them with ether, and then
bringing them into strong alcohol.

WILSON (Mitth. Zool. Stat. Neapel, 1884, p. 3) kills Alcyo-
naria with a mixture of 1 part of strong acetic acid and 2
parts of concentrated solution of corrosive sublimate, the
animals being removed as soon as dead and hardened for two
or three hours in concentrated sublimate solution.

BEAUN (Zool. Anz., 1886, p. 458) recommends that for both
Zoantharia and Alcyonaria a little osmic acid be added to the
sublimate employed for fixation. For Alcyonium palmatum,
Sympodium coralloides, Gorgonia verrucosa, Caryophyllia cya-
thus, and Palythoa axinellse he proceeds as follows : — The
animals are left for a day or two in a glass vessel, so that the
polyps may become thoroughly extended. They are then sud-
denly drenched with a mixture of 20 to 25 c.c. of concentrated
solution of sublimate in sea water with four to five drops of 1
per cent, osmic acid. This is allowed to act for five minutes.

(This method also gives good results with Hydra and some
Bryozoa and Rotifers.)

SCHULTZB (Biol. Centralb., 1887, p. 760) says that for
Pennatulidae with large polyps the gradual addition of fresh
water is a good plan.

Sections. — For preparing sections, besides the usual methods
for sectioning decalcified specimens, we have the valuable
methods of von Koch and Ehrenbaum, §§ 311 and 312, which,
being applicable to undecalcified specimens and furnishing
preparations showing at one and the same time soft parts and
hard parts in situ, render most inestimable services.

Maceration. — For the HEETWIGS* well-known method (Jen.
Zeit., 1879, p. 457) see § 542. The tissues should be left to
macerate in the acetic acid for at least a day, and may then
be teased in glycerin.

LIST (Zeit.f. wiss. Mik., iv. 2, 1887, p. 211) recommends
dilute liquid of Flemming. Tentacles of Anthea cereus and
Sagartia parasitica treated for ten minutes with a mixture of
100 c.c. of sea-water with 30 c.c. of Flemming's liquid (the
strong solution, § 36), then washed out for two or three hours
in 0'2 per cent, acetic acid, and teased in dilute glycerin, give

456 SOME ZOOLOGICAL METHODS.

fine dissociations of the connective, sensory, and urticant cells
of the ectoderm, and after removal of the epidermis allow of
the demonstration of ganglion-cells and the supporting
lamella. Picro-carmine may be used for staining.

850. Hydroidea, Polypoid Forms. — For suitable narcotisation
methods see those quoted in last section.

Fixation. — In general the polyps may be very well killed in
saturated sublimate solution, in which they should be plunged
for an instant merely, and be brought into alcohol. The
solution should be employed cold in general for Grymnoblastea,
hot for most Calyptoblastea.

Ether attentively administered gives good results with Cam-
panularidae. Hydra is very easily killed by treatment with a
drop of osmic acid on a slide. BRECKENFELD (Amer. Hon. Mic.
Journ., 1884, p. 49) obtains good results by heating the ani-
mals in a drop of water on a slide for from three to five seconds
over a petroleum lamp. The methods for sections are the
usual ones.

The methylen-blue method of intra vitam staining is appli-
cable to this group, see, inter alias, ZOJA, 1. c., § 114.

851. Medusae: Fixation. — There is some difficulty in properly
fixing the forms with contractile tentacles, which easily roll
up on contact with reagents. The best results I have had with
these forms have been obtained by means of VAN BENEDEN'S
acetic acid method, § 53, followed by alcohol. A similar
method, with the difference that a mixture of chromic acid
and alcohol is used for washing out instead of pure alcohol, is
recommended by DE CASTELLARNAU (La Est. Zool. de Napoles, p»
133) for Oceania, Lizzia, Bougainvillia, Podocoryne, Syncoryne,
&c. The secret of success with the long-tentacled forms lies
in a trick of manipulation. Put sufficient acetic acid into a
deepish dish, hold it in your left hand (or, better, in both
hands if you have an assistant), and keep it moving in a
circle so 'as to communicate a vortex motion to the liquid.
Take up a medusa in a spoon with as little sea-water as
possible, and throw it into the moving liquid, and keep the
liquid steadily swirling round so as to cause the tentacles to
trail out at full length behind the animal antil it is thoroughly
fixed, then pass carefully into alcohol. Do not, unless you
are very expert, try to fix more than one medusa at a time ;

MEDUSA. 457

it is also better to keep the specimens separate, even in the
alcohol, as, if several are together, it generally happens that
their tentacles become entangled. The method is due to
S. Lo BIANCO. Oceania conica and Tiara may usefully,
according to S. Lo BIANCO, be narcotised with 3 per cent,
alcohol in sea water before fixation. Liquid of Kleinenberg,
which I have seen much used for the fixation of these and
similar forms, is, in my opinion, histologically a very objec-
tionable reagent for the purpose.

Trachymedusae and Acalephae may be fixed in the usual
way in chromic or osmic mixtures. Osmic acid may conve-
niently in some cases be added to the sea water containing
the animals, which should be removed to fresh water as soon
as they begin to turn brown.

852. Medusae : Sections.— I am not acquainted with any per-
fectly satisfactory method of sectioning these extremely
watery organisms. Paraffin and collodion will afford good
sections of some organs, but are certainly not satisfactory as
all-round methods for this group. Some modification of the
method employed by the HERTWIGS (Nervensystem der Medusen,
1878, p. 5) might be successful. They imbedded in liver
with the aid of glycerin gum, and hardened the objects and
the mass in alcohol. I should think better results would be
obtained by one of the freezing methods given in §§ 315 to
320.

853. Medusae : Maceration. — The methods of the HERTWIGS,
§ 542, have deservedly become classical for the study of the
tissues of this group.

Amongst other advantages of this process it should be
noted that the reduction of osmic acid by albuminates is
greatly hastened by the presence of acetic acid, which in the
case of animals so transparent and poor in cells as Medusae is
an advantage for the study of the nervous system ; for gan-
glion-cells and nerve-fibrils reduce osmium quicker than
common epithelium-cells. They become greenish brown, and
are easily distinguished from surrounding tissues. Doubtless,
in many cases the pyrogallic acid reaction, § 233, would give
better results.

The isolation of the elements of the macerated tissues is

458 SOME ZOOLOGICAL METHODS.

best done by gently tapping the cover-glass (which may be
supported on wax feet). This gives far better results than
teasing with needles. A camel-hair pencil also sometimes
renders good service.

854. Siphonophora. — This group contains some of the most
difficult forms to preserve that are to be found in tho whole
range of the animal kingdom. You have not only to deal
with the very great contractility of the zooids, but with the
tendency to general disarticulation of the swimming- bells and
prehensile polyps.

The cupric sulphate method of BEDOT, § 44, is practised
as follows : — Bedot directs that a large quantity of 15 to 20
per cent, solution of the salt be suddenly added to the sea
water containing the animals. As soon as they are fixed
(which happens in a few minutes) a few drops of nitric acid
are to be added and mixed in (this is in order to prevent the
formation of precipitates), and the whole is left for four to
five hours. The specimens are then to be hardened before
bringing them into alcohol. Bedot recommends that this be
done with solution of Flemming. The strong solution is the
one that should be taken, and it should be added to the solution
of sulphate containing the Siphonophore, about two volumes
of it being taken for one of the sulphate solution. The whole
should be left for at least twenty-four hours. After harden-
ing in the mixture a few drops of 25 per cent, alcohol should
be added to the fluid with a pipette, being dropped in as far
as possible from the colony, which should be disturbed as
little as possible ; and further alcohol, of gradually increasing
strength, should be added so gradually that the strength of
70 per cent, be not attained under fifteen days at least.
Ninety per cent, alcohol should be used for definite pre-
servation.

I have tested this method. I do not find that the his-
tological preservation is superior to that obtained by means
of the usual processes ; but the method is certainly a valuable
one in so far as it enables one to preserve specimens with all
their swimming -bells and polyps in situ, a result which is not
obtained by means of the usual methods.

The cupric and zinc sulphate mixture of FRIEDLANDER has
been given, § 44.

SPONGING. 459

S. Lo BIANCO employs for the majority of Siphonophora a
mixture of 10 c.c. of saturated solution of corrosive sublimate
with 100 c.c. of 10 per cent, solution of copper sulphate. This
is used as in Bedot's process. Diphyes, Rhizophysa, and Phy-
salia, however, are killed with sublimate solutions ; Velella
with chromo-picric acid, or a mixture of 100 c.c. of sublimate
solution with 50 c.c. of 1 per cent, chromic acid ; Porpita by
poisoning with liquid of Kleinenberg.

KOROTNEFF'S method of paralysing with chloroform has
been given in § 11. I would only add that I have seen
Physophora very successfully killed by the careful adminis-
tration of ether.

855. Ctenophora : Fixation. — The small forms are very easily
prepared by means of osmic acid. The large forms are for
the most part extremely difficult to deal with on account of
the extraordinary delicacy of the tissues. S. Lo BIANCO em-
ploys for Callianira a mixture of I part 0'5 per cent, chromic
acid, 1 part concentrated pyroligneous acid, and 2 parts con-
centrated sublimate solution. For Beroe Forskalii he recom-
mends the cupric sulphate mixture, last section (I would point
out that the cupric sulphate method is very unsuitable for
many forms, as it makes them opaque, and so heavy that they
easily break). The majority of forms he treats with a mix-
ture of 100 parts of 1 per cent, chromic acid and 2 parts 1 per
cent, osmic acid, Cestus Veneris with chromo-acetic acid,
rolling it up like a watch-spring in order that it may not
become deformed by its own weight.

SAMASSA has succeeded in making sections of Ctenophora
by means of the double-imbedding method, § 306 (see Arch.
f. mik. Anat., xl, 1892, p. 157; Zeit. f. wiss. Mik., 1893,
p. 340).

Porifera.

856. Spongiae : Fixation. — The smaller forms (Calcispongiae)
can be fairly well fixed by the usual reagents, osmic acid being
one of the best. For the larger forms no satisfactory fixing
agent has yet been discovered, so far as I can ascertain.
The tissues of this group are very watery, very delicate, very
friable after hardening, and macerate with the greatest
facility. For all but very small specimens, absolute alcohol

460 SOME ZOOLOGICAL METHODS.

is apparently the best fixing agent. If any watery fluid be
preferred, care should at all events be taken to get the sponges
into strong alcohol as soon as possible after fixation, on account
of the rapidity with which maceration sets in in watery fluids.
FIEDLER (Zeit.f. wiss. Zool., xlvii, 1888, p. 87) has been using
(for Spongilla), besides absolute alcohol, an alcoholic sublimate
solution, and the liquids of Kleinenberg and Flemming, with
good effect.

Staining. — On account of the great tendency to maceration
above referred to, I hold (notwithstanding many recommen-
dations of watery stains that are to be found in the literature
of the subject) that alcoholic stains be alone employed for
staining sponges, and I particularly recommend Mayer's tinc-
ture of cochineal as giving the best results personally known
to me.

Sectioning. — Calcareous sponges may be decalcified in alco-
hol slightly acidified with hydrochloric acid, and then im-
bedded in the usual way. Siliceous sponges may be desi-
licified by Mayer's hydrofluoric acid method mentioned ante,
§ 574. But in view of the really dangerous nature of this
operation, I feel bound to recommend that it be avoided.
Fair sections may be obtained from sponge tissues well im-
bedded in paraffin without previous removal of the spicula.
The spicula appear to be cut ; probably they break very
sharply when touched by the knife. Of course you will not
use your best knives for cutting such sections.

Preparation of Hard Parts. — Siliceous spicules are easily
cleaned for mounting by treating them on a slide with hot
concentrated nitric or hydrochloric acid, or solution of potash
or soda. The acids mentioned are very efficient, but it must
be pointed out that they will attack the silex of some delicate
Bpicules. Thus DEZSO found that the small stellate spicules
of the cortex of Tethya lyncurium are completely dissolved
by boiling hydrochloric acid. Potash solution is therefore
frequently to be preferred, notwithstanding that, in my expe-
rience, it does not give such clean preparations.

According to NOLL, eau de Javelle is preferable to any of
these reagents (see § 556).

Impregnation with Silver (see § 217).

Larvae of Spongiae. — SCHULTZE (Zeit. f. wiss. Zool., xxxi,
p. 295) places the ova and Iarva3 of Sycandra raphanus in

METHODS FOB QUIETING INFUSORIA. 461

hanging-drop moist chambers, oxygenated by means of a few
fronds of green algae. He also (ibid., xxxiv, 1880, p. 416)
found that the best sections of the more advanced sessile larvae
of Plakina were obtained by selecting larvae that had settled
down on thin fronds of algae, and treating them, together with
the fronds, with osmic acid, staining with alum-carmine, and.
bringing into paraffin in the usual way.

Protozoa.

857. Introductory. — Since the Protozoa may be considered
as free cells, and their peculiar organs known as "nucleus"
and " nucleolus," " macronucleus " and " micronucleus," &c.,
present in the main the same reactions as cell-nuclei, it is
evident that the reagents and methods of cytology are in great
part applicable to this group. One of the most generally
useful of these reagents will be found in the acid solution of
methyl green; it is the reagent that allows of the readiest and
best demonstration of the presence and form of the nucleus
and nucleolus (BALBIANI et HENNEGUY, Compt. rend. Soc. de
BioL, 1881, p. 131). The EHELICH-BIONDI mixture, § 259, will
of course in many cases be indicated.

Amongst useful reagents not mentioned in the following
descriptions of the methods employed by different authors, I
call attention to the weak solutions of alum, potash, and borax,
which serve to demonstrate the striations of the cuticle and
the insertions of the cilia of Infusoria.

858. Methods for quieting Infusoria. — The narcotisation
methods, §§ 14 to 18, are available for this purpose, as is also
the monobromated camphor process of BROCHEE, § 811, which
I believe has been found very successful with Yorticellidae.

According to SCHURMAYER (Jen. Zeit., xxiv, 1890, pp.402 —
470; Zeit. f. wiss. Mik,, vii, 4, 1891, p. 493) nitrate of strychnin
in weak solution, O'Ol per cent, or less, gives good results with
some forms, amongst which are Stentor and Carchesium.
Antipyrin in concentrated solution (O'l per cent.) or cocain of
O'Ol per cent, seems only to have given good results as regards
the extension of the stalk in stalked forms.

EISMOND (Zool. Anz.y No. 352, Dec., 1890, p. 723) has
proposed a mechanical means of slowing the movements of
small organisms (small worms and Crustacea as well as

462 SOME ZOOLOGICAL METHODS.

Ciliata). He directs that a drop of thick aqueous solution of
cherry-tree gum be added to the water containing the
organisms (gum arabic and the like, it is stated, will not do).
The objects remain fixed in their places, with cilia actively
moving, and all vital processes retaining their full activity.
I am greatly obliged to Dr. GRUBLER for having been at much
pains in making inquiry for me, concerning the cherry-tree gum
that should be used. It appears that this gum is a somewhat
insoluble one, and it is difficult to get hold of a sample that
will give a good solution. Further, the solutions will not
keep, and must be made up fresh every day. In the face of
these difficulties it would seem that the method is at present
a far from perfect one. It should, however, be stated that
CEETES (Bull. Soc. Zool. France, xvi, 1891, p. 93; Journ. Eoy^
Mic. Soc., 1891, p. 828) has found that the method gives
excellent results. He has also found that an intra vitam
stain may be obtained by adding methyl blue or " violet
dahlia No. 170," to the gum solution.

A similar process of inhibiting movements whilst preserving
life has been worked out by JENSEN (after STAHL ; see BioL
Centralb., xii, 1892, 18, 19, p. 556; Zeit. f. wiss. Mik., ix, 4,
1893, p. 483 ; Journ. Roy. Mic. Soc., 1892, p. 891). A solution
of 3 grms. of gelatin in 100 c.c. of ordinary water is made by
the aid of heat. This makes a jelly at the normal tempera-
ture. It is slightly warmed, and a drop of it is mixed in a
watch-glass with a drop of water containing the organisms.
This plan is said to afford great facilities for the vivisection
of Infusoria.

858. Staining intra vitam. — The possibility of staining In-
fusoria intra vitam was discovered independently and almost
simultaneously by BRANDT (Verh. d.physiol. Ges. Berlin, 1878),
by CERTES (Soc. Zool., 25 janv., 1881), and by HENNEGUY (Soc.
philom., 12fev., 1881).

CERTES found that living Infusoria stain, while continuing"
in life fora certain time, in weak solutions of cyanin, Bismarck
brown, dahlia, violet 5 B, chrysoidin, nigrosin, methylen blue,
malachite green, iodine green, and other tar colours, and haema-
toxylin. The solutions should be made with the liquid that
constitutes the natural habitat of the organisms. They should

PRESERVING 1NFUSOKIA. 463

be very weak, that is, of strengths varying between 1 : 10,000
and 1 : 100,000. For cyanin, 1 : 500,000 is strong enough.

The " nucleus " may be stained in the living organism by
dahlia and malachite green. Bismarck brown only colours
the (t nucleus " of certain species (Nychtoterus, Opalina — HEN-
NEGUY). The "nucleus" frequently behaves differently in
allied species.

A double stain of the nucleus (green) and protoplasm
(violet) may be obtained by the simultaneous employment of
dahlia and malachite green.

Examination in a coloured medium in which the organisms
do not stain, but show up on a coloured background in a
manner that produces somewhat the effect of dark-ground
illumination, is sometimes helpful. CEETES (Bull. Soc. Zool.
de France, xiii, 1888, p. 230) recommends solution of anilin
black for this purpose ; Infusoria will live in it for weeks.
FABEE-DOMEEGUE (Ann. de Microgr., ii, 1889, p. 545 ; Journ.
Roy. Mic. Soc., 1889, p. 832) recommends concentrated solu-
tion of diphenylamin blue.

859. Fixing and Preserving. — PPITZNEE (Morph. Jahrb., xi,
1885, p. 454) used concentrated solution of picric acid run in
under the cover.

BLANC (Zool. Anz., 1882, p. 22) advises liquid of Kleinen-
berg diluted with about a volume of water, and acidified
with acetic acid.

KOESCHELT (Zool. Anz., 1882, p. 217) recommends 1 per
cent, osmic acid, or for Amoebae, 2 per cent, chromic acid.

LANSBEEG (ibid., p. 336) advises the same reagents, but
recommends bringing the organisms into the fixing liquid
with a pipette, instead of running in the fixing liquid under
the cover.

CATTANEO'S Methods (Bollettino Scientifico, iii and iv; Journ.
Roy. Mic. Soc., 1885, p. 538) are as follows :— Fix for a few
minutes with -J- per cent, aqueous solution of chloride of palla-
dium. This is the best fixing agent, as it hardens in a few
minutes without blackening the structures. Double chloride
of gold and cadmium also fixes well, and brings out nuclei
even better than the palladium. Solution of iodide of mercury
and potash (J per cent.) is useful for bringing out proto-
plasmic networks, as it stains the granules of

464 SOME ZOOLOGICAL METHODS.

black. Corrosive sublimate in 5 per cent, solution gives good
results. Osmic acid causes darkness and opacity in the pre-
parations. Nitrate of silver (in £ to 1 per cent.) solution
may be used, the objects being washed out with solution of
acid sulphate of soda. Chromic, picric, and picro-sulphuric
acids and bichromate of potash are only second-rate fixing
agents for this purpose.

BRASS (Zeit.f. wiss. Mik.y i, 1884, p. 39) employs for fixing
unicellular organisms the following liquid :

Chromic acid . . . . .1 part.

Platinum chloride . . . 1 „

Acetic acid . . . . . . 1 ,,

Water .... 400 to 1000 parts.

For protozoa that are opaque through accumulation of
nutritive material, he proceeds as follows : — The organisms are
treated for three or four minutes with liquid of Kleinenberg,
and then for some time with boiling water. They are then
brought into water containing a small proportion of ammonia,
in which they reassume their natural forms and dimensions.
The ammonia is then neutralised by addition of a little acetic
acid, and the preparation is stained with borax-carmine or
ammonia-carmine. After washing, the objects are mounted
in dilute glycerin. This treatment is said to afford extremely
transparent preparations,

Brass also obtained good results with sublimate solution.
CERTES (Comptes rend., 1879, 1 sem., p. 433) makes perma-
nent preparations as follows : — Fix with osmic acid of 2 per
cent. (In the case of very contractile Infusoria, place a drop
of the solution on the cover-glass, and place it on the drop of
water that contains them. But generally speaking it is best
to employ only the vapour of the solution, exposing the
organisms to its action for not more than from ten to thirty
minutes.)

The objects having been covered, the excess of liquid is
removed by means of blotting-paper, and the following stain
is allowed to flow in :

Glycerin ...... 1 part.

Water . . . . . . 1 „

Picro-carmine . . . . . 1 „

(Eosin may also be used. Soluble anilin-blue does not give
such good results.) The stain should be placed at the edge

DEMONSTRATION OF CILIA. 465

of the cover, and the slide put away in a moist chamber, in
order that the water may evaporate very slowly and be
changed very gradually for the glycerin mixture; if this
precaution is not taken, shrinkage may occur. When the
exchange has taken place, strong glycerin may be added, and
gradually substituted for the dilute glycerin.

Certes states that the organisms thus prepared are fixed
perfectly in their natural form, and allow of the study of the
minutest detail of cilia, flagella, and the like, with the highest
powers ; the green coloration of Euglenae and Paramecia is
preserved. The nulear structures are sharply brought out
by the picro-carmine.

SAVILLE KENT and BEETHOLD (Manual of the Infusoria;
Journ. Roy. Mic. Soc., 1883, p. 451) prefer a brownish-yellow
solution of potassium iodide to osmic acid for fixing.

The employment of vapour of iodine has been described,
§ 66.

Du PLESSIS (VoGT et YUNG, Trait. Anat. Comp. Prat., p. 92) recommends
fixation with 0'2 per cent, solution of corrosive sublimate. Let the prepara-
tion dry up, and if the organisms have preserved their shape, stain and
mount in balsam.

FOL (Lehrb., p. 102) fixes delicate marine Infusoria (Tintinnodea) with
the perchloride of iron solution, § 52, added to the water containing them, and
stains with gallic acid as directed § 231, and states that this is the only
method that has given him good results, especially as regards the preservation
of cilia.

KUNSTLEE (Journ. de Microgr., 1886, pp. 17 and 58). — For Monadina.
Fixation by means of a drop of very concentrated osmic acid solution (a
grm. of osmium dissolved in only a few c.c. of distilled water).

LONGHI (Bull. Mus. Zool. Univ. Genova, 4, 1892 ; Zeit*
/. wiss. Nik., ix, 4, 1893, p. 483) has obtained good results by
means of sulphate of eserin. A 1 : 10 per cent, solution is
taken, and to each 10 c.c. of it one drop of 1 per cent, subli-
mate solution is added.

See also the methods of FABRE-DOMERGUE, Ann. de Microgr. y
ii, 1889, p. 545, and of SCHEWIAKOFP, Biblioth. Zool., v, 1889,
p. 5; Journ. Eoy. Hie. Soc., 1889, pp. 832, 833; ZOJA, Boll.
Sci. Pavia, 1892; Zeit.f. wiss. Mik., ix, 4, 1893, p. 485.

860. Demonstration of Cilia (WADDINGTON, Journ. Eoy. Mic.
Soc., 1883, p. 185). — Solution of tannin, or a trace of alcoholic
solution of sulphurous acid.

30

466 SOME ZOOLOGICAL METHODS.

861. Stains forFlagella. — The celebrated method of LOFFLEE
has run through several forms (Centralb. f. Bacterial., vi,
1889, p. 209 ; vii, 1890, p. 625; Zeit. f. wiss. Mik., vi, 3, 1889,
p. 359; vii, 3, 1890, p. 368; Journ. Roy. Mic. Soc., 1889,
p. 711; 1890, p. 678), of which that given here is the latest.
To 10 c.c, of a 20 per cent, solution of tannin are added 5 c.c.
of cold saturated solution of ferrous sulphate and 1 c.c. of
(either aqueous or alcoholic) solution of f uchsin, methyl violet,
or " Wollschwarz." The mixture will require for some forms
the addition of a few drops of 1 per cent, solution of caustic
soda; e. g. for typhoid bacilli, 1 c.c. ; for Bacillus subtilis, 28
to 30 drops; for bacilli of malignant oedema, 36 to 37 drops.
Some other forms will require besides the addition of a trace
of sulphuric acid to the soda solution, so for cholera bacteria,
half a drop to 1 drop ; for Spirillum rubrum, 9 drops.

Cover-glass preparations are made and fixed in a flame in
the usual way, special care being taken not to over-heat.
Whilst still warm the preparation is treated with mordant
(i. e. the above-described mixture), and is heated for half a
minute until the liquid begins to vaporise, after which it is
washed in distilled water and then in alcohol. It is then
treated in a similar manner with the stain, which consists of
a saturated solution of fuchsin in anilin water, the solution
being preferably neutralised to the point of precipitation by
cautious addition of 0*1 per cent, soda solution.

TEENKMANN (Centralb., vi, 1889, p, 433; Zeit. f. wiss. Mik.,
vii, 1, 1890, p. 79) mordants for several hours at the normal
temperature in a 1 per cent, solution of tannin in 0*5 per cent,
hydrochloric acid, and stains for several hours in carbolic
fuchsin ; and gives also two other similar methods.

BEOWN (The Observer, iii, 1892, p. 298; Journ. Roy. Mic.
Soc., 1893, p. 268) mordants for several hours in a mixture of
30 gr. tannin, 12 drops anilin oil, and 1 fl. oz, of alcohol,
which may, if required, be alkalised by addition of a trace of
caustic soda (so for Spirillum undula and Bacillus ulna), or
may be acidified for others with a little hydrochloric acid.
The cover is stained by the process of heating over a flame
for a few minutes with any anilin-water solution of fuchsin,
methyl violet, dahlia, methyl green, &c., neutralised with
caustic soda as in Loeffler's process, or with a solution of
rosanilin in anilin water.

APPENDIX.

862, The Usual Alcohols,

Strength.

Absolute

95
per cent.

90
per cent.

Made approximately as under.

See § 63. The so-called " ab-
solute alcohol " of commerce
is generally of about 98 per
cent, strength.

This is the average strength of
the common strong commer-
cial alcohol, which ranges in
general from 94 per cent, to
96 per cent, according to
temperature. The strength
of this, or of the following,
should be determined by
means of an areometer (Gay
Lussac's being very con-
venient), so as to form a start-

' ing-point for the following
mixtures.

May be made approxi-
mately by taking 100
vols. 95 per cent, alco-
hol, and 5' 5 vols. water.

Observations.

This grade is not neces-
sary for ordinary work.

This is the usual grade
for dehydrating before
clearing. It is the
highest grade that
should be used for de-
hydrating celloidin sec-
tions.

This is the usual strength
of the strongest com-
mercial Methylated
Spirit, which (if free
from mineral naphtha)
maybe taken instead of
pure alcohol for com-
mon work. If naphtha
be present the alcohol
becomes turbid on the
addition of water. Oil
of bergamot will clear
from this grade.

468

APPENDIX.

Strength.

85
per cent.

70
per cent.

Made approximately as under.

Made by taking 100 vols.
90 per cent, alcohol,
and 6'& vols. water.

May be made of 100 vols.
90 per cent, alcohol
or methylated spirit,
and 31 vols. water ; or
100 vols. 85 per cent,
alcohol, and 23 vols.
water.

50
per cent.

" One-
third
alcohol"

Made by taking 100 vols.

90 per cent, alcohol,

and 84' 7 vols. water;

or 100 vols. 70 per cent.

alcohol, and 41'7 vols.

water.
Made by taking 1 vol. of

90 per cent, alcohol,

and 2 vols. water.

Observations.

Eectified Spirit B.P. is a
little weaker than this,
viz. 84*5 per cent.

Only exceptionally power-
ful clearers, such as
anilin oil, will clear
from this grade ; see §
353. This is the proper
grade in general for
preserving organisms
and tissues in (but see
p. 3 sub. fin.) ; higher
gradesshould notgene-
rallyjbe used unless it
is desired to harden.
This is the proper
grade for washing out
borax-carmine stains,
corrosive sublimate
after fixing, &c.

This is the strength of
Proof Spirit.

See § G^.

863, Histological Reagents and Apparatus. — The great
importance of working with trustworthy chemicals has been
insisted on in § 94, where also the addresses of some chemists
who make a speciality of histological reagents have been
given. As regards Dr. GRUBLEE'S products, so often quoted
in the foregoing pages, I would add that they should either
be ordered from him direct (address, Herrn Dr. G. Grubler,

APPENDIX. 469

Chemiker, 12, Baiersche Strassej Leipzig, Germany), or, if
ordered through any agent, should be ordered to be sent in
the original packages, signed and dated by him. This is in
order to ensure the due freshness of the products ; many of
them will not keep well for very long.

Glass and other apparatus can be obtained as well as chemicals from the
above-quoted houses. I would call attention to the varied usefulness of the
"Siebdosen " or sieve-dishes of ZIMMEKMANN and SUCHANNEK (vide Zeit. f.
wiss. Mik., vii, 2, 1890, p. 159). They consist of a covered glass capsule
into which is fitted a " sieve " made of a watch-glass pierced with holes and
supported on legs. . It is evident that the arrangement is very handy, not
only for staining, washing out, treatment with vapours, &c., but for any
operation in which it is desirable to have specimens supported in the upper
layers of a quantity of reagent. They are sent out in a very neat form by
Dr. Griibler.

Want of space compels me to suppress the lists of suggested reagents
given in the last edition under the headings "The Laboratory table " and
" The Zoologist's Travelling Case." Either collection may still be obtained
from Dr. Griibler, the latter in appropriate bottles, fitted into a case
measuring 1 foot 4 inches X 5^ inches X 4^ inches, at the price of about
£2 5s., or a case somewhat larger, yet not too heavy to be carried in the
hand, at about £3.

864. Cleaning Slides and Covers. — The readiest way known to me of
freeing slides from balsam, damar, and cement is to wet with water and
scrape with an old knife, using afterwards, if necessary, one of the solvents
mentioned below.

HANAMAN, Journ. Roy. Mic. Soc., i, 1878, p. 295 ; American Naturalist,
xii, p. 573. — To a cold saturated solution of bichromate of potash add ^ of
its bulk of strong sulphuric acid (care must be taken on account of the heat
and vapours evolved).

HENEAGE GIBBES, ibid., iii, 1880, p. 392.— Place the cover-glasses in
strong sulphuric acid for an hour or two, wash well until the drainings give
no acid reaction ; wash first with methylated spirit, and then with absolute
alcohol, and wipe carefully with an old silk handkerchief.

SEILEE, ibid., p. 508. — New slides and covers are placed for a few hours
in the following solution :

Bichromate of potash ...... 2 ounces.

Sulphuric acid 3 fluid ounces.

Water 25

Wash with water. The slides may be simply drained dry ; the covers
may be wiped dry with a linen rag.

Slides and covers that have been used for mounting either with balsam or
a watery medium are treated as follows : — The covers are pushed into a
mixture of equal parts of alcohol and hydrochloric acid, and after a few days
are put into the bichromate solution and treated like new ones. The slides

470 APPENDIX.

are scraped free of the mounting medium with a knife and put directly into
the bichromate solution.

FOL (Lehrb., p. 132) recommends either a solution containing 3 parts of
bichromate, 3 of sulphuric acid, and 40 of water ; or simply dilute nitric
acid.

GABBINI (Manuale, p. 31) puts slides for a day into 10 per cent, sulphuric
acid, then washes, first with water and then with alcohol.

BEHEENS (Zeit. f. wiss. Mik., 1885, p. 55) treats slides first with con-
centrated nitric acid, then with water, alcohol, and ether.

JAMES (Journ. Roy. Mic. Soc., 1886, p. 548) treats used slides with a
mixture of equal parts of benzin, spirit of turpentine, and alcohol.

KNAUEB (Centralbl f. Bakt., x, 1891, p. 8 ; Zeit. f. wiss. Mik., ix, 2,
1892, p. 187 ; Journ. Roy. Mic. Soc., 1891, p. 833) recommends boiling for
twenty or thirty minutes in 10 per cent, lysol solution, then rinsing with
cold tap water till clear.

NIAS (Journ., pag. cit.) finds it is sufficient to boil with washing soda, and
rinse.

865. G-um Mucilage for Labels, &c. — The Journ. of the Chemical
Soc. says that the adhesive qualities of gum may be very much exalted by
the addition of aluminium sulphate (the so-called " patent " alum) to the
mucilage. " 2 grms. of crystallised aluminium sulphate, dissolved in 20
grms. of water, is added to 250 grms. strong gum arabic solution (2 grms.
in 5 grms. water). Ordinary solutions of gum arabic, however concen-
trated, fail in their adhesive power in many cases, such as the joining
together of wood, glass, or porcelain ; prepared, however, according to the
above receipt, the solution meets all requirements " (from Public Opinion,
Feb. 19th, 1886).

FOL (Lehrb., p. 148) advises that slides be prepared for labelling by
spreading over one end a layer of aluminium-chloride gelatin dissolved in
acetic acid, and allowing it to dry before putting on the label.

Why do not the glass makers furnish slides with roughened (ground) end-
surfaces for the reception of labels ?

For four other receipts for gums and pastes for labels, see ELIEL, in
Engl. Mechan., 1887, p. 535 ; Amer. Mon. Mic. Journ., 1887, p. 93 ; Zeit.
f. wiss. Mik., v, 1, 1888, p. 69.

VOSSELEB (Zeit. /. wiss. Mik., vii, 4, 1891, p. 459) recommends, for
attaching protective cardboard ridges to slides, a syrup-thick solution of
bleached shellac in alcohol.

866. Green Light. — The suggestion of the employment of green light
in microscopy is, I believe, due to ENQLEMANN (Pfliiger's Arch., 1880, p.
550). He strongly recommends the use of green light for delicate observa-
tions, as giving sharper definition, allowing finer detail to be seen, and tiring
the eyes less than white light. Green glass of sufficiently good quality is
found in commerce. The glass is best put between the mirror and the object,
e. g. on the diaphragm. Blue glass (cobalt or amrnoni*o-sulphate of copper)
is also useful, but less so than green. Red light is most hurtful. " The
explanation of these points, so important in practice, may be found in the
results obtained by Lamansky in his researches on the * Limits of Sensi-

APPENDIX. 471

bility of the Eye to the Different Colours of the Spectrum* (Arch. f.
Ophthalm., xvii, p. 123, 1871)." I would add that for lamp-light work,
especially fine work with high powers, either green or blue glass is, accord-
ing to my experience, a sine qua non if the best attainable results be
desired. I always use blue cover-glasses, putting from one to four of them
on the diaphragm of the condenser. For some unexplained reason I find I
get better results by means of several superposed thin glasses than by one
thick one. Spectral blue glass can be obtained from Mr. Pillischer, the
optician, in Bond Street.

867. Object-holder to Cambridge Microtome. — Since § 271
was written the Cambridge Sci. Inst. Co. have brought out
an adjustable object-holder to their rocking microtome, fulfil-
ling the required conditions as regards facility of orientation*

868. Obregia's Serial Section Method. — This method, which
was originally described in the Neurologisches Centralb. for
1890, and is given in the third edition of WOODHEAD'S Prac-
tical Pathology, had escaped my attention. It has been lately
recommended for class purposes, as being very safe and con-
venient, by GULLAND (Journ. of Path., February, 1893).
Slides, or glass plates of any size, are coated with a solution
made of —

Syrupy solution of powdered candy-
sugar made with boiling distilled
water . " . . . .30 c.c.

Absolute alcohol . . . . . 20 „

Transparent syrupy solution of pure

dextrin made with distilled water . 10 „
They are dried slowly for two or three days until the
surface is just sticky to the moist finger. Sections are
arranged and heated for a few minutes to a temperature
slightly above the melting-point of the paraffin. The paraffin
is removed by some solvent, such as xylol or naphtha, and
this is in turn removed by alcohol. The alcohol is poured
off, and the sections are covered with solution of celloidin or
with a solution of 3 per cent, of photoxylin in a mixture of
equal parts of ether and absolute alcohol. The plates are
left to evaporate in a horizontal position, and when the sec-
tions are required the sheet of collodion is cut into ribbons,
which are floated off in water, and further treated as desired,
e. g. as in Weigert's process, § 340. (It is well to divide the
sheet of collodion into ribbons by running the point of a knife

472 APPENDIX.

down it as soon as evaporation has produced a very slight
solidification, and the evaporation must not be artificially
hastened.)

869. The Water and Albumen Section-fixing Method. — A careful
and detailed description of the method shortly described after HENNEQUT
(§ 328) is given by OHLMACHEE (Journ. Amer. Med. Ass., April, 1893), who
has independently worked out the same process.

870. Monobromated Camphor for Narcotisation. — Since
writing the note calling attention to this new method in
§ 811 I have learnt that the method is not applicable to
marine organisms, as the camphor is not sufficiently soluble
in salt water. But for the most diverse fresh-water forms
it has proved highly convenient.

871. Gilson's Mercuro-nitric Fixing Mixture. — I regret to
find that by an unaccountable oversight I have omitted to
mention this liquid, the formula of which has been several
times published. I am indebted to Prof. GILSON for kindly
sending me the latest formula, which is as follows :

Nitric acid of 46° strength (this
would be sp. gr. 1*456, or 80 per

cent., nearly) .... 78 c.c.

Glacial acetic acid . . . . 22 „

Corrosive sublimate ... 95 grms.

60 per cent, alcohol . . . 500 c.c.

Distilled water .... 4400 „

When required for marine -animals add a few crystals of
iodine, which will prevent the formation of precipitates of sea
salts. If in any case the preparations should show a granular
precipitate, due probably to an abundance of phosphates in
the tissues, the precipitate may be removed by washing with
water containing a little tincture of iodine (not iodide of
potassium, which would precipitate the sublimate).

This mixture affords in general a faithful and delicate fixa-
tion, and gives to tissues an excellent consistency. Objects
may remain in it for a considerable time without hurt.
Tissues are left in a state very favorable for staining. The
liquid has a high degree of penetration. A treatment for a
few days with it will serve to remove the albumen from the
ova of Batrachians.

APPENDIX. 473

872. GILSON'S Pier o-alum- Carmine (§ 157). — Professor Gilson writes
me that the formula given in § 157 is erroneous as containing too much
picric acid, and has been good enough to send me an amended receipt, with
the remark, however, that all these formulae seem to have lost their raison
d'etre by the publication of Mayer's carminic acid stains. As that is really
quite the opinion I had come to myself on the matter, I suppress the new
formula, merely making use of this opportunity to reiterate the opinion
that for the present Mayer's stains are victorious all along the line.

873. Chloral Hydrate Jelly (kindly communicated by Prof.
GILSON). — 1 vol. of gelatin, melted secundum artem, and 1 vol.
of Price's glycerin. Mix, and add 1 vol. of chloral hydrate
(i. e. add crystals of chloral until the volume of the mixture
has increased by one half) ; warm till dissolved. This gives
a very highly refractive aqueous mounting medium, which is
found useful for opaque tissues that it is desired not to
dehydrate.

A similar medium is published by G-EOFFROY, Journ. de
Botan., 1893, p. 55 (see Zeit.f. wiss. Mik., ix, 4, 1893, p. 476).
He dissolves, by the aid of as little heat as possible, 3 to 4
grins, of gelatin in 100 c.c. of 10 per cent, aqueous solution
of chloral hydrate.

874. Sulphurous Acid as a Bleaching Agent. — Prof. GILSON
writes me that alcoholic solution of sulphurous anhydride (S02)
is very convenient for the rapid decoloration of bichromate
objects. A few drops suffice.

875. ZACHAEIAS'S Carmine Stain. — Notwithstanding the remarks on
carmine stains in general just made (§ 872), it seems worth while to call
attention to a process due to ZACHARIAS (Verh. d. Vers. D. Naturf. u.
Aerzte, Bremen, 1891, p. 121 ; recommended by SCHUTZ, Monatschr. f.
prakt. Dermat., xiv, 1892, p. 397 ; Zeit. f. wiss. Mik., ix, 4, 1893, p. 476),
as it involves a somewhat new principle. Tissues, after fixation in any of
the usual media, " Flemmrag " included, are stained for half an hour to an
hour in aceto-cannine (§ 158), are well washed in water, and brought for
five hours into 1 per cent, solution of sulphate of protoxide of iron. After
thorough washing they are dehydrated and mounted in balsam. Karyo-
kinetic figures dark black, the ground of a delicate neutral tone.

876. Rapid Modification of Weigert's Stain. — The following is
given by KAISER in Zeit. f. wiss. Mik., ix, 4, 1.-93, p. 468 : — Sections
treated from three to five minutes with 1 per cent, solution of bichromate of
potash, washed in Weigert's haematoxylin fluid (§ 688), and warmed over a
flame in a fresh quantity of the stain until bubbles are formed, rinsed with
water, and differentiated by the method of Pal, § 692 (sulphite of sodium
being, however, taken instead of the potassium salt). As will be seen on

474 APPENDIX.

reference to § 696, this modification of Weigert's process is essentially
identical with that of LISSAUEE.

Another process given by KAISER, loc. cit., is as follows : — Sections to be
mordanted for a few minutes in a mixture of 1 part liquor ferri sesqui-
chlorati, 1 part distilled water, and 3 parts rectified spirit, then stained
in Weigert's stain changed several times until a heavy precipitate is
formed, then washed in water and differentiated as before. They are then
to be rinsed in ammoniated water and stained in a Ol per cent, solution of
fuchsin in rectified spirit, or in a solution of 1 part of naphthylamin brown
in 100 of spirit and 200 of water. The fuchsin will stain in about half a
minute, the naphthylamin brown in from three to five minutes. Mount as
usual. Medullated nerves blue, non-medullated red or brown. The re-
action will be enhanced if the sections be warmed in the stain.

877. Preservation of Golgi's Bichromate of Silver Preparations.
— KALLIUS (Anat. Hefte, ii, 1892, p. 269; Zeit. f. wiss. Mik.,
ix, 4, 1893, p. 477) has worked out the following process.
Take 20 c.c. commercial hydroquinone developing solution
and 230 c.c. distilled water (the hydroquinone solution may be
made up with 5 grins, hydroquinone, 40 grms. sodium sul-
phite, 75 grms. carbonate of potassium, and 250 grins, dis-
tilled water). At the instant of using, further dilute the
solution with one third to one half its volume of absolute
alcohol, and put the sections into it for several minutes ; they
become dark grey to black. In order to ascertain whether
reduction is complete, throw a section into a solution of
hyposulphite of soda (about 10 parts to 50 of water) :
chromate of silver will quickly dissolve, whilst metallic silver
will not be attacked. As soon as reduction is complete the
sections are put for ten to fifteen minutes into 70 per cent,
alcohol, then brought for five minutes into the above-given
solution of hyposulphite of soda, and thence into a large
quantity of distilled water, where they should remain for
twenty-four hours or more. Lastly, dehydrate in the usual
way, and mount under a cover. All the details which in the
undeveloped preparations were brown are now black on a
light ground. After-staining with carmine, &c., may be em-
ployed. Other developers were tried, and gave satisfactory
reductions, but they caused a red or brownish discoloration
of the preparations.

To the references to the paper of HUBFR given in § 712,
add that to Zeit. f. wiss. Mik., ix, 4, 1893, p. 479. The
reader will of course understand that the balsam is to be

APPENDIX. 475

heated on the slide, with the section in it, until it imme-
diately sets hard on cooling.

878. Methylen Blue for Impregnation Purposes. — APATHY has a
note in Zeit. f. wiss. Mik., ix, 4, 1893, p. 466, to the effect that the best
methylen blue for impregnation — in fact, the only one that will give exactly
the results described by him (see § 118) — is that obtained from E. MERCK,
of Darmstadt, and quoted in his price list as " medicinisches Methylenblau,"
and described on the label as " Anilin-blau, Methylen, chemisch rein und
chlorzinkfrei."

879. Mounting Methyl Green Stains. — Attention was called
in § 109 to the difficulty of mounting methyl green stains in
balsam on account of the imperfect resistance of the colour
to alcohol. SQUIRE says, in his Methods and Formulse, p. 36,
that a thorough washing with water previous to the treat-
ment with alcohol has the effect of fixing the colour in the
nuclei, so that it is less easily removed by the spirit.

880. GOODALL'S Hapid Method for Preparing Spinal Cord (Brit.
Med. Journ., May, 1893, p. 947 ; Journ. Boy. Mic. Soc., 1893, p. 405).—
Cut sections of fresh tissue with a freezing microtome ; float them on to
water, and as soon as possible drain and float them on to pyridin. After a
quarter of an hour wash in water ; stain with 0'25 per cent, aqueous solu-
tion of anilin blue-black, followed by picro-carmine ; dehydrate and clear
in pyridin ; mount in balsam thinned with pyridin. See also ante, § 90.

881. Demonstration of Living Trichinae (BABNES, Amer. Mon. Mic.
Journ., xiv, 1893, p. 104 ; Journ. Roy. Mic. Soc., 1893, p. 406).— A piece
of trichinised muscle of the size of a pea should be placed in a bottle in a
mixture of 3 gr. of pepsin, 2 dr. of water, and 2 minims of hydrochloric
acid. The whole should be kept at body temperature for about three hours
with occasional shaking. The flesh and cysts being dissolved, the fluid is
poured into a conical giass, and allowed to settle ; the trichinae are drawn
off from the bottom with a pipette, got on to a slide with water, and
examined on a hot stage.

INDEX.

The numbers refer to the Paragraphs, not to the Pages.

A.

ABBE, mounting medium, 415.
Absolute alcohol, uses and preparation,

2, 63, 355, 862.
Acanthocephali, 49, 60, 837.
Acephala, 813, 820, 821 ; and see Mol-

lusca.

Acetate of alumina, for mounting, 373.
Acetate of copper, 64, 386.
Acetate of lead, 87, 732.
Acetate of potash, 374, 394, 529.
Acetate of uranium, 65.
Acetic acid for fixing, 53, 54, 811, 849

851.

Aceto-carmine, 158, 875.
Acetone, for dehydration, 2.
Achromatic figure, 639.
Acid anilin dyes, 788.
Acid, free, test for in tissues, 132.
Acid fuchsin, 95, 98, 131 ; for nerve

tissue, 722, 772, 773.
Acidophilous cell-elements> 788, 793.
Acids, see Acetic, Chromic, Formic,

Hydrochloric, &c.
Acidulated alcohol, 63.
Actiniaria, 9, 10—14,17, 19, 542,849.
ADAMZIEWICS, stain for nerve-centres,

774.

Adipose tissue, 36, 787.
Agar-agar, section fixative, 332.
AOASSIZ and WHITMAN, pelagic ova,

610.
Albumen, imbedding masses, 314, 321 ;

injection mass, 498; section- fixing

process, 328 j removal of, from ova,

598 et seq., 871.

Alcohol, its action on tissues, 2 ; fixing,
63 ; absolute, 63; " one third,"
63; acetic, 54; acidulated, 63;
picric, 62 ; for hardening, 88, 89 ;
macerating, 525; preserving tis-
sues, 2, 378 ; clearing, 355 ; table
of the usual grades, 862.

Alcohol-balsam, 419.

Alcoholic carmines, 168 — 171.

Alcoholic cochineal, 172, 173.

Alcoholic hffimatem stains, 182 — 184,
190, 194.

Alcyonaria, 7, 849.

Alcyonella, 14.

Alcyonium, 849.

ALFEBOW, silver impregnation, 213.

Alizarin, 200, 756.

ALLEN, osmium impregnation, 233.

ALT, Congo red, nerve stain, 724.

ALTMANN, fixing methods, 37, 637;
bioblasts, 634, 637, 641; impreg-
nation and corrosion methods, 558.

Alum, for fixing, 45 ; preserving, 372 ;
solubility in alcohol, 184; am-
monia-alum, solubility in water,
185 ; roche-alum, 190.

Alum-carmine, 153—157; ditto, with
acetic acid, 156 ; with osmic acid,
155 ; with picric acid, 157.

Alum-cochineal, 146, 154.

Alumina, acetate, 373.

Amaroecium, 612.

Amber varnish, 446, 447.

Ammonia, bichromate, 80, 732, 737,
738 ; neutral chroraate, 81.

Ammonia-alum, 185.

Ammonia-carmine, 163 — 165, 745.

478

INDEX.

The numbers refer to the Paragraphs, not to the Pages.

Ainmoniated hsematoxylin, 188a.

Ammonio-nitrate of silver, 213.

Ammonium, molybdate, for impregna-
tion, 234; for fixing nuclei, 637;
sulphocyanide, 530; vanadate,511.

Amphibia, for cytological study, 633 ;
embryology, 82, 598 et seq.

Amphioxus, 662.

Amphipoda, embryology, 626.

Amphiura, 848.

Amyl nitrite, 500.

Amyloid matter, 109, 112.

Anchinia, 811.

ANDEEB, phloroglucin, 562.

ANDBES, Actiniae, 10, 11, 12, 849;
section-stretcher, 280.

ANDBEWS, imbedding apparatus, 273.

ANGELUCCI, retina, 682.
.Anilin, for clearing, 305.

Anilin dyes, generalities, 92, 95 ; classi-
fication into acid and basic, 788 ;
classified list, 95 ; Hermann-Bott-
cher or Flemming staining process,
95—100, 258.

Anilin black, 93, 95, 130, 757.

Anilin blue, 95, 128, 756.

Anilin blue-black, 125, 757.

Anilin green, 95, 100, 105, 142, 808.

Anilin oil for clearing, 305, 353.

Anilin red, 107.

Anilin violet, 111.

Anilin water, 101, 275, 305.

Aniseed imbedding mass, 322.

Annelida, 14, 840—844; embryology,
630.

Anodonta, 16.

Anthea, 849.

Anthozoa, 849.

Antipathes, 849.

Antipyrin for narcotisation, 858.

APATHY, celloidin imbedding, 299;
preserving the blocks, 302 ; serial
sections, 338, 339; cement, 455;
haematoxylin stain, 194; double-
stain for nervous tissue, 785;
raethylen blue impregnation, 115,
117 — 119,878; mounting medium,
400.

APEL, Sipunculus, and Halicryptus,
838.

Aphides, preparation and embryology,
621.

Aplysia, 813, note.

Appendicularia, 217.

Aqua Javelli, 556, 581, 616.

Aqueous stains recommended, 5.

Aquiferous pores of Molluscs, 813.

Arabin, 329.

Araneina, embryology, 622.

ABCANGELI, carmines, 167.

Archil, 206.

Archiplasm, archoplasm, 639.

Arctiscoida, 826.

Areolar tissue, 786.

ABMANNI, logwood nerve stain, 694.

ABNOLD, critique of Fleinming's mix-
ture, 35, 36 ; hsematoxylin stain,
192.

ABNSTEIN, inethylen blue method, 117,
119.

Arsenic acid for decalcification, 572.

Arthropoda, fixing, 4, 5, 57, 822;
general methods, 822 ; clearing
and softening chitiu, 823 ; depig-
mentation, 823—825; eyes, 825;
nerve and muscle, 826; sarco-
lemma, 827; embryology, 616 —
627 ; spermatology, 645.

Artificial fecundation, 584.

Artificial indigo, 125.

Artificial oedemata, 786.

Artificial pigment spots, 77, 731, 732.

Artificial saliva, 531.

Artificial serums, 524, 361, 362.

Ascaris, ova, 54, 639, 644.

Ascidians, 18, 811.

Asphalt varnish, 436.

Asphyxiation as a killing method, 18.

Astacus, embryology, 118, 625.

Asteracanthion, 14.

Asteroidea, 846.

Astroides, 849.

Attraction spheres, 639.

AUBEET, cements, 430, 433.

AUEEBACH, stains for male and female
chromatin, 645.

Aurantia, 98.

Aureoline, 580.

Auricularia, 848.

Aves, embryology, 589 — 594.

INDEX.

479

The numbers refer to the Paragraphs, not to the Pages.

Axis cylinder, 719 et seq. ; stains, 721

et seq.
Axolotl, ova, 599.

B.

BABER, picro-carmine, 162.

BABES, cytological methods, 638, 643 ;
saf ranin stain, 101 ; supersatur-
ated do., 638.

BALBIANI, "noyau vitellin," 640; liv-
ing ova, 585 ; ova of Phalangida,
623 ; Protozoa, 857 ; spermato-
logical methods, 645.

BALFOUR, embryology of Aves, 589,
590 ; of Araneina, 622.

BALLOWITZ, muscle of Cephalopoda,
676.

Balsam, 417 — 419, 421, 428.

BALZEE, elastic tissue, 795.

BARFF, Boroglyceride, 404.

BARNES, observation of Trichinae, 881.

BARRETT, retina, 682, 685.

BARROIS, embryology of Echinoder-
mata, 848.

Basic anilin dyes, 788.

Basophilous cell-elements, 788, 793.

BASTIAN, gold method, 228.

Batrachia, central nervous system, 742,
784.

BATJMGARTEN, cytological methods,
643 ; f uchsin and methylen blue
stain, 268 ; picro-borax-carmiue,
171 ; triple stain, 270.

Bayberry tallow, 287.

BAYERL, stain for cartilage, 242, 801 ;
decalci6cation, 564.

BEALE, carmine stain, 165; digestion
fluid, 549; glycerin jelly, 408;
injections, 501, 503, 504; perser-
vative fluids, 370.

BECHTEREFF, hardening brain, 732.

BECK, cements, 430.

BECKER, microtome, 271.

BECKWITH, gold-staining nerve cen-
tres, 768.

BEDOT, fixing process, 44, 854.

BEEVOR, Weigert's haematoxylin, 691.

BEHN, hardening skin, 646.

BEHRENS, cements, 430, 433; amber
varnish, 446 ; cleaning slides, 864.

BELA HALLER, macerating mixture,
543.

BELLONCI, preparation of brain, 741.

BELL'S cement, 430, 434.

BENCZUR, staining nerve-centres, 756.

BENDA, haematoxylin, 639 ; double-
stain for sperm-cells, 639; sper-
matological method, 645; Wei-
gert's haematoxylin, 690.

BENEDEN, van, acetic acid fixing
method, 53, 811 ; acetic alcohol,
54 ; Ascidians, 811 ; corrosive
sublimate liquid, 47; cytological
methods, 644; embryology of
Mammalia, 588, of Taenia, 628.

BENEDEN, van, and NEYT, cytological
methods, 54, 637, 639, 644.

Bengal rose, 95, 136.

Bengalin, 125.

Benzol, for clearing, 354.

Benzo-azurin, 107, 127.

Benzo-purpurin, 95, 133.

Bergamot oil, 347.

BERGONZINI, staining mixture for
plasma cells, 793.

Berlin blue, 478.

BERLINERBLATJ, F., regeneration of
Weigert's haematoxylin, 688.

BERNHEIMER, heematoxylm for retina,
683.

Beroe, 855.

BERRY'S Hard Finish, 424.

BEVAN LEWIS, see LEWIS, BEVAN.

BIANCO, S. Lo, Molluscoida (Bryozoa),
812; Mollusca, 813; Nemertina,
835 ; Gephyrea, 838 ; Chaetopoda,
841 ; Holothurioidea, 845 ; Antho-
zoa, 849 ; Medusse, 851 ; Siphono-
phora, 854; Ctenophora, 855;
Alcyonaria, 849; Tunicata, 18,
811; narcotising mixture, 12.

Bichloride of mercury, see Corrosive
sublimate.

Bichromate of ammonia, 80, 732, 737,
738.

Bichromate of potash, for fixing and
hardening, 40, 43, 75—79, 731—
737, 739; for maceration, 535;

480

INDEX.

The numbers refer to the Paragraphs, not to the Pages.

bleaching bichromate objects, 75,
874.

Bichromate of silver impregnation,
704 et seq.

BICKFALYI, digestion fluid, 550.

Biebricher Scharlach, 95, 134.

BIEDEEMANN, methylen blue method,
117, 666; nerve-endings in mus-
cle, 666, 667.

Bilberry juiee stain, 209.

Biniodide of mercury medium, 414.

Bioblasts, Altmann's, 634, 637, 641.

BIONDI, staining mixture, 259, 639;
methods for blood, 803, 806.

Sipinnaria, 848.

Bismark brown, 93, 95, 107, 110, 638.

Bitume de Judee, 436.

BIZZOZEEO, cytological method, 643;
gentian violet, 102; picro-carmine,
162; maceration, 646; mucus cells,
807 ; blood, 804, 805.

BJELOUSSOW, injection, 499.

BLACKBURN, vegetable wax, 287.

Black currant stain, 209.

Blackley blue, 125.

Bladder of frog, 677.

BLANC, saffron stain, 204; infusoria,
859.

Blattida, embryology, 619.

BLAUE, glandular epithelium, 646.

Bleaching, 575 et seq. ; bichromate ob-
jects, 75, 874 ; Erlicki objects, 77 ;
osmic objects, 29.

Bleu carmin aqueux, 123a.

Bleu de Lyon, 95, 124.

Bleu de nuit, 124.

Bleu lumiere, 95, 123.

BLOCHMANN, ova of Amphibia, 598.

Blood, 802—806.

Blood-vessels of Annelids, 842, 844.

Blue-black, 130.

BOBBETSKY, embryology of Lepido-
ptera, 618.

BOCCARDI, gold method, 228, 670;
motor plates, 670.

BOHM, gold method, 228.

BOHMEE, haematoxylin, 186*

BOHMIG, rhabdocoela, 834.

BOHN, carmine, 165.

BOLL, gold staining nerve- centres, 767.

BOLSIUS, picro-alum carmine, 157,
872 ; do. with hsematoxylin, 239.

Bone, decalcification, 559 et seq., 797,
798; preparation, 796—801.

Borax - carmine, GEE N ACH EE' s, 167,
169 ; GIBBS', 167 ; NIZIFOEOW'S,
167; WOODWARD'S, 167; BATTM-
GAETEN'S, 171 ; HAUG'S, 167 ;
with indigo-carmine, 241, 242.

Bordeaux, 98.

Boric-acid carmine, 167.

BOEN, paraffin imbeding, 273 ; section
fixing, 331 ; reconstruction of sec-
tions, 587.

Boroglyceride, 404.

BOTTCHEE-HEBMANN staining process,
95—100; p. 60, note.

Bougainvillea, 851.

BOFMA, stain for cartilage, 801.

BOVEEI, ovum of Ascaris, 639, 644 ;
medullated nerve, 720.

BOTEE, embryology of Teleostea, 608.

Srachionus, 839.

BEADY, chloral hydrate medium, 376.

Brain, see Central nervous system and
Neurological methods.

BBAHWELL, brain, 779.

BEANCA, redwood nerve stain, 694.

BEANDT, glycerin jelly, 409 ; Protozoa,
858.

BEASS, alcoholic carmine, 170 ; bleach-
ing osmic objects, 29 ; paraffin
imbedding, 275, 286, 287 ; Proto-
zoa, 859.

BEATTN, Alcyonaria, &c., 849 ; Rhabdo-
ccela, 834 ; Nematodes, 836.

Brazil-wood nerve stain, 694.

BBECKENFELD, Hydra, 850.

BBEGLIA, Weigert's haematoxylin, 688,
694.

BEEMEE, motor plates, 670.

BEIDE, MAC-, see MACBEIDE.

BEOCHEE, narcotisation method, 811,
870.

BEOCK, macerating medium, 538.

BHOSICKE, staining method, 233.

BROWN, stains for flagella, 861.

BEUCKE, digestion fluid, 550 ; injec-
tions, 478.

BRTTN, mounting medium, 109, 402.

INDEX.

481

The numbers refer to the Paragraphs, not to the Pages.

BRTJNOTTI, gelatin imbedding mass,

296.

Brunswick black, 437.
Bryozoa, 7, 14, 15, 812.
BUDGE, injection, 515.
Bugula, 812.

BUMPUS, dry celloidin imbedding, 303.
BUECI, elastic tissue, 795.
BURCKHABDT, nervous system of Pro-

topterus, 742.
BUBGEK, nervous system of Nemertina,

835,
Buscii, eosin and hamiatoxylin, 251 ;

decalcification, 559, 560.
BUSSE, photoxylin imbedding, 297;

celloidin do., 299, 301.
BUTSCHLI, paraffin imbedding, 276;

haematoxylin stains, 185, 196.

C.

Cacao-butter imbedding mass, 287.

CAJAL, RAMON Y, silver chromate im-
pregnation, 714, 715, 717; ter-
minations of nerves and trachea,
669, 714; retina, 683, 684, 715;
sympathetic ganglia, 715.

CALBEELA, methyl green, 109 ; methyl
green and eosin, 260; indulin,
125 ; glycerin mixture, 405 ; mace-
rating mixture, 531; imbedding,
314.

Calcispongiae, 856.

Calcium chloride mounting medium,
375.

CALDWELL, serial sections, 327.

Callianira, 855.

Cambridge rocking microtome, 271,
867.

Camphor, monobromated, for narcoti-
sation, 811, 870.

Canada balsam, for mounting, 417 —
419, 421 ; imbedding method, 313.

CANFIELD, iris, 679.

Cannel oil, 346.

Caoutchouc, for section fixing, 336;
cement, 435.

CAPPAEELLI, dichroism of methyl
violet, 112.

Carbolic acid, 305, 351, 367.

Carbolised alcohol, 682.

Carbolised syrup, 365.

Carbonic acid narcotisation method,
19.

Carchesium, 858.

Carcinoma corpuscles, 101.

Carmalum, 145, 151.

Carmine, analysis of, 144 ; generalities
on, 144 — 150 ; theory of staining
with, 144—146 ; practice of stain-
ing, 149 ; classification of the for-
mula3, 150; for what purposes use-
ful, 147.

Carmine blue, 123 a.

Carmine double stains, 236—249.

Carmine stains, acetic, 156, 158, 167,
875; alcoholic, 168—173; alum-
carmine, 153 — 157 ; ammonia-car-
mine, 163 — 165 ; borax-carmine
(aqueous), 167 (alcoholic), 169,
171; boric acid, 167, 171; Car-
malum, 145, 151 ; Carmiuroth,
167 ; chloride of aluminium, 145,
152; cochineal, 154, 172, 173;
hydrochloric acid, 170; lithium,
166; neutral, 164, 165, 167;
osmium-carmine, 167 ; Paracar-
mine, 168; picro-carmine, 159 —
162 ; picro-alum-carmine, 157,
872 ; putrefied carmine, 165 ; sali-
cylic acid, 167 ; soda carmine,
167 ; uranium carmine, 162, 167 ;
and see the names of the respec-
tive authors, as also the separate
entries, "Alum-carmine," " Borax-
carmine," &c.

Carminic acid, 144—146, 148.

Carminroth, 167.

CABNOY, acetic alcohol, 54; chromo-
aceto-osmic acid, 36 ; cement, 450 ;
cytological methods, 635 — 637,
644; salt solution, 359; tannin
solution, 387 ; spermatology of Ar-
thropods, 645.

CABPENTEE, preservative media, 378 ;
cements and varnishes, 430.

CABBIERE, corpuscles of Herbst and
Grandry (gold method), 652 ; eyes
of Gastropods, 815; maceration
of nervous tissue, 781.

31

482

INDEX.

The numbers refer to the Paragraphs, not to the Pages.

CABTEB, injection mass, 470.

Cartilage, 801.

Caryophyllia, 849.

CASTELLABNAF, de, Actiniae, 849;
Eucharis, 748 ; Lamellibran-
chiata, 813, Medusse, 851; Ne-
mertina, 835; Ophiuridea, 847;
Zoantharia, 849.

Castor oil for mounting, 425.

CATTANEO, corpuscles of Golgi, 673;
Infusoria, 52, 859.

Caustic potash and soda, for macera-
tion, 529 ; for corrosion, 555.

Cedar oil, for minute dissections, 4 ;
for paraffin imbedding, 275 ;
clearing, 99, 344; for mounting,
417, 418.

Cell-granules, 641, 788 et seq.

Cell - researches, see Cytological
methods.

Cellepora, 812.

Celloidin, 297 et seq. ; the dry method,
303 ; clearing sections, 305 ; in-
jection masses, 513, 514.

Cells, paper, for mounting, 432.

Cements, 430 et seq.; generalities,
430; comparative tenacity of, 433;
APATHY'S, 455; BELL'S, 434;
colophon ium, 445 ; French, 442 ;
HABTING'S gutta-percha, 441 ;
KITTON'S, 436, 453; Knotting,
443; LOYETT'S, 454; MAESH'S
gelatin, 431 ; MILLEB'S, 435 ;
STIEDA'S, 451; Tolu balsam,
450 ; turpentine, 444 ; ZIEGLEE'S,
452.

Central nervous system, introduction,
728; hardening, 90, 729—742,
780, 880 ; imbedding and cutting,
743; general stains, 745—764;
special stains, 765—776; mount-
ing, 777, 778; the half-clearing
method, 779; dry processes for
„ preserving brains, 780; dissocia-
tion methods, 781, 782 ; neuroglia,
783 ; myelon of reptiles, &c., 784;
and see Nerves, Retina, &c. ; and
for list of Authors reported see
Neurological methods.

Cephalopoda, eyes, 816; ova, 613.

Cercarise, 833.

CEBFONTAINE, curarising Lumbricusf
841.

CEETES, Protozoa, 130, 858, 859.

Cestodes, 832.

Cestus, 855.

Chsetopoda, 841.

Chffitopteridae, 841.

Chain section cutting, 281.

CHEATLE, dehydration apparatus, 2.

Cherry gum, 858.

CHICHKOFF, Turbellaria, 834.

CHIEVITZ, retina, 682.

China blue, 720.

Chinolin, blue, 126; hydrochlorate,
377.

Chironomus, salivary gland, 47 ; em-
bryology, 620.

Chitin, clearing and softening, 823
et seq.

Chitonidaj, 817.

Chloral hydrate for narcotising, 14;
preservative solutions, 365, 376,
394, 873 ; for preserving injection
masses, 458 ; for maceration, 528,
685 ; for bleaching bichromate
objects, 75; Gilson's mounting
medium, 873; Geoffroy's ditto,
ibid.

Chlorate of potash for maceration, 545.

Chloride-of-aluminium carmine, 145,
152.

Chloride of calcium mounting medium,
375.

Chloride of copper fluid, 64, 386.

Chloride of gold, see Gold chloride.

Chloride of magnesium for narcotisa-
tion, 17.

Chloride of palladium, 52, 84, 559, 746,
767.

Chloride of platinum, 23, 42, 50, 78,
83.

Chloride of vanadium, 683, 726.

Chloride of zinc, for hardening brain,
780.

Chlorine bleaching methods, 575 — 577.

Chloroform, for paraffin imbedding
275, 276 ; for celloidin ditto, 301 ;
for bleaching, 582 ; for narcotisa-
tion, 11.

INDEX.

483

The numbers refer to the Paragraphs, not to the Pages.

CHOLODKOWSKY, embryology of Blat-
tida, 619.

Chondrosia, 217.

Chromate of ammonia, 43, 81.

Chromate of lead stain, 234.

Chromate of silver impregnation, 704
et stq.

Chromates as fixing agents, 43.

Chromatin, reactions of, 53, 97, 109,
636, 638 ; male and female, 645.

Chromic acid, generalities, 30 ; fixing,
30; hardening, 69, 732; killing,
18; maceration, 534; decalcifica-
tion, 559, 560, 570, 571 ; chromic
acid with acetic acid, 31 ; with
alcohol, 33, 70; with nitric acid,
39, 40, 571 ; with platinum chlo-
ride, 42 ; with osmic acid, 34, 71 ;
with acetic and osmic acid, 35, 36,
71.

Chromic objects, action of light on,
30 ; bleaching, 30.

Chromo-acetic acid, 31 ; chromo-aceto-
osmic, 23, 35, 36, 637; chromo-
formic, 32 ; chromo- nitric, 39, 40 ;
chromo-osmic, 34, 71; chromo-
picric, 41, 72 ; chromo-platinic, 42,
83.

CHBSCHTSCHONOWIC, gold method, 228.

Chrysaurein, 95, 107.

CHUN, imbedding Siphonophora, 282.

CIACCIO, gold process, 228; motor
plates, 670 ; corpuscles of Golgi,
674.

CIAGLINSKI, spinal cord, 733, 743.

Cilia of Infusoria, 860, 861.

Ciliated epithelium, 821.

060*4,814.

Cladocora, 849.

Clavellina, 811.

Cleaning slides and covers, 864.

Clearing, generalities, 2, 99.

Clearing agents, 99, 342 et seq. ;
STIEDA'S experiments on, 343 ;
NEELSEN and SCHIEFFEBDECKEB'S,
343—349 ; celloidin sections, 305;
paraffin ditto, 283.

Clove oil, 4, 99, 345.

COBB'S differentiator, 2.

Cocain for narcotising, 15.

Coccidse, 823.

Cochineal, theory of staining with,

144—146; use of, 147; HEB-

BICK'S 154; CZOKOB'S, 154;

KLEIN'S, 154; MAYEB'S, 172,

173; PABTSCH'S 154.
Cochlea, 559, 562, 686, 687.
Ccelenterata, 849—856.
COHNHEIM, gold staining, 223 ; silver

staining, 668.
COLE, freezing method, 317 ; gum

medium, 399.
COLLIN, Criodrilus, 841.
COLLINGE, pelagic fish ova, 610.'
Collodion imbedding, 297 et seq ; sec-

tion-fixingTprocess, 325, 326, 340.
Collodionising paraffin sections, 282.
Colophonium, cement, 445 ; imbedding

mass, 312 ; mounting medium,

422.

Comatula, 848.
Combination stains, 235 et seq ; and

see Stains, combination.
Congelation imbedding methods, 315,

322.

Congo red, 93, 95, 98, 132, 723—725.
Conjunctiva, 653.
Connective tissue, 138, 786 et seq.
CONTEJEAN, Ranvier's hsematoxylin,

187.

Convoluta, 834.
COOK, hamatoxylin, 192.
Copal, imbedding process, 311 ; varnish

for mounting, 424.
Copepoda, 822.
Copilia, 822.
Copper, chloride and acetate fixing

mixture, 64.
Copper sulphate fluids, fixing, 43, 44,

64 ; preserving and hardening, 64,

82 ; impregnation process, 234.
Coral, 311, 849.
Corallin, 95, 107.
COBI, narcotisation, 13, 15; Flem-

ming's mixture, 35; preserving

osmic acid solutions, 26.
Cornea, 121, 228, 653, 658, 659.
Corpuscles, tactile, 651 et seq / corpus-
cles of Golgi, 671 — 674 ; of Herbst

and Gramlry,652; of Krause,653.

484

INDEX.

The numbers refer to the Paragraphs, not to the Pages.

Corrosion, 555 et seq. ; ALTMANN'S
methods, 558.

Corrosive sublimate, fixing liquids, 36,
47, 48 ; LANG'S liquids, 48 ; pre-
servative liquids, 379 — 385.

Covers and slides, cleaning, 864.

Cox, Golgi's mercuric impregnation,
761.

Creosote, see Kreasote.

Creseis, 813.

Criodrilus, 841.

Cristatella, 14.

Crocein, 95, 139.

Crystal violet, 98, 795.

Crystalline, 660.

Ctenophora, 45, 307, 855.

CUCCATI, picro -carmine, 162; haenia-
toxylin, 190; retina, 682, 683;
soda-carmine, 167.

Cucumaria, 845.

Cupric fixing mixtures, 43, 44, 64,
854 ; hardening ditto, 82 ; pre-
servative ditto, 64, 386 ; impreg-
nation process, 234, 764.

Curare for narcotisation, 17, 633, 841.

CUESCHMANN, amyloid matter, 109.

Cyanide of mercury, for fixing, 49.

Cyanin, 93, 95, 98, 126.

CIBULSKY, gold method, 229 ; muzzle
of ox, 650.

Cytological methods, 632 et seq.;
methods of study, 632; subjects
for ditto, 633; observation of
living cells, 633, 634; staining
ditto, 634; fresh cells, 635; micro-
chemical1 reactions, 636; fixing,
64, 637; cytological stains, 638,
639 ; mounting, 642 ; Nebenkeru,
Archoplasmakugel, sphere attrac-
tive, 197, 208; nucleus of Balbiani,
640 ; cytology of the ovum, 644 ;
spermatology, 645; methods of
ALTMANN, 637, 641 ; BABES, 638,
643 ; BAUMOABTEN, 643 ; BEND A,
639 ; BENEDEN, VAN, 644 ; BENE-
DEN, VAN, and NEYT, 637, 639,
644; BIZZOZERO, 643; BOVEEI,
644; CAENOT, 635—637, 644,
645; FLEMMING, 633, 635, 637,
639, 645 ; GEHUCHTEN, VAN, 637,

644; GILSON, 645; GBENACHEB,
642 ; HEIDENHAIN, M., 633, 637,
639; HENNEGUY, 106; HERMANN,
637; the HERTWIGS, 644; KULT-

SCHITZKY, 644; NlSSEN, 643;

PEEEMESCHKO, 633 ; PFITZNEE,
643; PLATNEE, 639, 643; RABL,
637, 639, 642; SCHOTTLANDEE,
643 ; STEASBUBGEB, 643; TIZZONI,
643 ; USKOFF, 643 ; v. LA VALETTE
ST.GEOEGE,634,644; ZACHARIAS,
644 ; ZWAAEDEMAKER, 643.

CZEENY, macerating mixture, 531.

CZOKOE, cochineal, 154 ; turpentine
cement, 444.

D.

Dahlia, 93, 95, 98, 103, 789.

Dammar, 420, 421.

DAVIDOFF, ova of Distaplia, 611.

DAVIES, injection, 471.

DASZKIEWICS, KORTBUTT, myelon of
Reptilia, &c., 784; plasma-cells,
788.

DEANE, mounting medium, 393 ; gly-
cerin jelly, 406.

Decalcification, 559 et seq.

Decapoda, 822.

DECKER, section stretcher, 280.

DEECKE, hardening brain, 738; im-
bedding and cutting, 743.

Definition, affected by transparency of
stain«, 100; ?reen light for, 866.

Degenerate nerves, 720 a.

Dehydration, 2, 3.

DEKHUYSEN, fat, 787; blood, 804;
silver staining, 213.

DELAFIELD, hsematoxylin, 185.

DELAGE, osmium-carmine, 167; sul-
phate of iron for fixing, 834 ;
Khabdoccela, 834.

Deltapurpurin, 95, 133.

DENDY, Geonemertes, 835.

Dendrocoela, 834.

Dendrophyllia, 849.

DENNISSENKO, retina, 682.

Depigmentation, 575 et seq. ; 823 et
seq.

INDEX.

485

The numbers refer to the Paragraphs, not to the Pages.

Desilicification, 574.

DE SOUZA, pyridin, 90.

Dextrin freezing mass, 318.

DEZSO, Tethya, 856.

Differentiators, 2.

Diffusion apparatus, 2.

Diffusion currents, to avoid, 2.

Digestion, 549 et seq.

DIMMOCK, carniinic acid, 144, 167.

Dinitrosoresorcin, 720.

DIOMIDOFF, hardening brain, 732.

Diptyes, 854.

DIPPEL, haeinatoxylin, 184.

Diptera, embryology, 620.

Dissections, minute, 4.

Dissociation, 522 et seq; of nervous
tissue, 781.

Distaplia, 611.

DOGIEL, olfactive organs, 655; iris,
678 ; methylen-blue impregnation
method, 115, 117, 119, 120, 652,
653; corpuscles of Herbst and
Grandry, 652 ; retina, 683.

DOMEBGUE, FABBE, mounting media,
401, 402.

DOSTOIEWSKY, iris, 679.

Double imbedding in celloidin and pa-
raffin, 306.

Double stains, see Stains, Combination.

DOYEBE, nerve and muscle of Arctis-
coida, 826.

DBASCH, gold staining, 222; tactile
hairs, 647.

DRASH, serial section method, 325.

DROOST, Mollusca, 821.

DUNHAM, clearing celloidin sections,
305.

DU PLESSIS, fixing method, 46 ; Nemer-
tians, 835 ; Protozoa, 859.

DUBHAM, alcohol section-fixing pro-
cess, 324.

DUTILLEUL, picro-borax-carinine, 171.

DUVAL, silver staining, 213, 215 ; car-
mine and anilin blue, 243 ; col-
lodion imbedding, 297 et seq. ;
embryology of Aves, 592 ; harden-
ing eucephalou, 734 ; purpurin,
201, 776 ; paraffin sections, flatten-
ing, 280; staining nerve-tissue,
749.

E.

Ear, inner, 34, 686, 687.

Eau de Javelle, 556, 581, 598, 616.

Eau de Labarraque, 557, 581, 598,
616.

EBNEB, VON, decalcification, 563.

Echinodermata, 19, 635, 845, 848.

EcMnorhyncus, 837.

Echtgelb, 95, 139.

Echtgriin, 720.

Echtroth, 95, 107.

EDINGER, nerve-centres, 77.

Egg-emulsion imbedding masses, 314,
321.

EHLERS, fixing fluid for Annelids, etc.,
31,841.

EHBENBATTM, imbedding method, 312.

EHBLICH, dahlia, 103; haematoxvlin,
188; methylen blue for nerves,
114; plasma-cells, 788, 789,804;
gentian violet, 101; blood, 802,
804 ; classification of auiliu dyes,
788.

EHBLICH-BIONDI-HEIDENHAIN stain,
259, 639, 759.

EHBMANN, structure of epithelium, 646.

EICHLEB, labyrinth, 687.

EIJKMAN, degenerate nerves, 720a.

EISIG, fixing mixture, 83; narcotising,
13, 835.

EISMOND, quieting Infusoria, 858.

Elastic tissue, 104, 795.

Eledone, eyes, 816.

ELIEL, gum for labels, 865.

Embryology, general methods, 584 —
&87',ot'Agelena, 622; Amaroecium,
612; Amphibia, 82, 590—603;
Amphipoda, 626; Aphides, 621;
Araueina, 622; Arthropoda, 616
et seq. ; Ascaris, 54, 644; Astacus,
625; Aves, 589—594; Axolotl,
599; Blattida, 619; Cephalopoda,
613 ; Distaplia, 611 ; Diptera,
620; Echinodermata, 635, 848;
Gastropoda, 614, 615; Lacerta,
39, 596; Lepidoptera, 618; Limax,
615; Lumbricus, 630; Mammalia,
588; Mollusca, 613 et seq. ; Or-
chestia, 626; Phalangida, 623,

486

INDEX.

The numbers refer to the Paragraphs, not to the Pages.

624; Pisces, 604 et seq. ; Planaria,
629; Porifera, 856; rabbit, 588;
Ranat 603; Reptilia, 595—597;
Salamandra, 602; Tania, 628;
Teleostea, 40, 584, 604—610;
Triton, 600, 601; Tunicata, 611,
612 ; Vermes, 628 et seq.

Embryos, reconstruction from sections,
587.

EMEET, injection, 510.

Encephalon, 728 et seq. ; and see cen-
tral Nervous System, Neurological
Methods, etc.

Endosmosis, to avoid, 4.

ENGELMANN, ciliated epithelium, 821 ;
green light in microscopy, 866.

Entire objects, preparation of, 4.

Eosin, 95, 98, 135 ; eosin and anilin
green, 265; — and dahlia, 258,
266 ; — and gentian, 258 ; — and
hsematoxylin, 189, 251, 252 ; -
and iodine green, 270; — and
methyl green, 260, 261a; — and
methyl violet, 267; — and silver
nitrate, 270 ; eosin stains for
blood, 804.

Epidermis of Mollusca, 814, 821.

Epithelium, 120, 121, 646 et seq.;
ciliated, 821 ; glandular, 646.

EBLICKI, hardening fluid, 77, 731.

Erythrosin, 135.

Eserin, for preserving Infusoria, 859.

Essence, see Oil, and Clearing agents.

ETEENOD, histologicai rings, 216.

Ether, for Preserving, 2.

Etherisation, 12.

EULENSTEIN, cement, 438.

Eunice, 841,

Examination media, 356 et seq., 767.

EXNBE, hardening brain tissue, 732,
740.

Exosmosis, to avoid, 4.

EYCLESHEIMEE, orientation of celloidin
objects, 300 ; dry method of cut-
ting same, 303 ; clearing, 305.

Eyes, of Arthropoda, 579, 583, 825 ; of
Cephalopoda and Heteropoda, 816;
of Chitonidse, 817; of Gastropoda,
815; of Pecten, 818; of Verte-
brates, 682 et seq.

F.

FABBE-DOMEBGUE, glucose preserva-
tive medium, 401 ; infusoria, 858,
859.

FAJEESTAJN, nerve-endings in tongue
of frog, 657.

FAEIS, glycero-gum, 397.

FABEANT'S medium, 395, 396, 398.

Fat, 36, 787.

FAUSSEK, critique of Flemrning's mix-
ture, 35.

Fecundation, artificial, 584.

FEIST, methylen blue method, 119;
orientating spinal cord, 743.

FEEEIA, elastic tissue, 795.

FEEEIEE, examination liquid for blood,
804.

FEEEEBI, phloroglucin decalcification,
562.

FICK, Golgi's silver chromate method,
712.

FIEDLEE, Spongilla, 856.

FISCHEE, soap imbedding mass, 833;
Trematodes, 833; tactile corpus-
cles, 651; motor plates, 667.

Fixing, generalities, 2, 6 et seq., 21 et
seq.

Fixing agents, 25 et seq.; action o^', 22;
choice of, 23; methods of using,
24; washing out, 22, 24; acetic
acid, 53 ; alcohol, 63 ; ALTMANN'S
methods, 37 ; alum, 45 ; bichloride
of mercury, 47, 48 ; bichromate,
43; CAENOY'S, 36, 54; chromates,
43; chromic mixtures, 31 — 36, 39
— 42; GOBI'S, 35; cyanide of mer-
cury, 49 ; cytological fixing agents,
637; Du PLESSIS'S method, 46;
FLEMMING'S,*<?eFLEMMING; MAX
FLESCH'S, 34, 35 ; FOL'S, 41, 52 ;
GILSON'S, 871 ; gold chloride, 52 ;
heat, 7, 24 ; HEEMANN'S, 51 ;
iodine, 66; KLEINENBERG'S method,
57; KULTSCHITZKY'S, 43 ; LANG'S,
48; MAYEE'S, 58, 59, 63; MEE-
KKL'S fluid, 42 ; nitric acid, 37 ;
nitric and chromic acid, 39, 40;
nitrate of silver, 38 ; osmic acid,
25 — 29; PEEENYI'S liquid, 39;

INDEX.

487

The numbers refer to the Paragraphs, not to the Pages.

picric acid fluids, 41, 56—62; Infusoria, 52, 231, 859 ; narcotisa-

platino-acet-osuaic mixture, 51 ; tion method, 19 ;

RIPART and PETIT'S solution, 64.
FLAGELLA, stains for, 861.
FLECHSIG, gold method, 228, 768;

redwood nerve stain, 694 ; other

other nerve stains, 763.
FLEISCHMANN, Limax, 813.
FLEMMING, chromo-acetic acid, 31;

chrorno-aceto-osrnic acid, 23; weak

solution, 35; strong ditto, 36;

cytological methods, 633, 635, 637,

644, 645; damar solution, 420;

double stains, 98, 236, 258 ; eyes

of Gastropods, 815 ; fixing liquids,

31, 35— 36a, 51, 53, 56, 61 ; im-
bedding mass, 291 ; preservation

in glycerin mixtures, 2 ; injection

of Molluscs, 820; staining method,

96—100, 101—103, 105, 107, 258;

spermatological methods, 645 ;

fatty tissue, 36, 787; bone, 800;

mucous cells, 807; goblet cells,

809 ; connective tissue fibrils, 786.
FLESCH, MAX, chromo-osmic mixture,

34, 71 ; dichroisni of haematoxylin

stains, 178 ; double stain, 245 ;

inner ear, 687 ; modification of

Weigert's haeinatoxylin stain, 688,

689 ; staining nervous centres,

749 ; dry preservative process,

780; dental tissue, 801; blood,

803.

FLOGEL, serial sections, 329.
FLORMAN, ctlloidin imbedding, 301.
Flustra, 812.
FoA, hffiinatoxylin and safranin stain,

256; blood, 804.

FOETTINGER, chloral hydrate narcotisa-
tion method, 14; prepared paraffin,

286.
FOL, albumen fixative, 328 ; bleaching

osmic objects, 29; chromo-aceto-

osmic acid, 35; cleaning slides,

864; decalcification, 571 ; gelatin

fixative, 334; glycerin jelly, 412 ;

gold-impregnation of marine ani-
mals, 230 ; gumming labels, 865 ;

imbedding in vacuo, 278; injection

masses, 473, 480, 485, 493, 497;

n oven,

277; perchloride of iron fixing

and staining process, 52, 231 ;

picro-carmine, 162 ; picro-chromic

acid, 41, 57; reconstructing sec_r_

tions, 587 ; ribesin, 209.
Formic acid for fixing, 55.
FOSTER and BALFOTTR, embryology of

Aves, 589, 591.

Fowl, embryology of, 589—594.
FRANCOTTE, alcoholic carmine, 171;

section-stretcher, 280 ; vacuum

imbedding, 278; vegetable wax

ditto, 287.

Frankincense, mounting medium, 429.
FREEBORN, staining nervous centres,

748; dissociation of ditto, 781 j

connective tissue, 786.
Freezing microtome, masses for, 315—

322.

French cement, 442.
FRENZEL, serial section methods, 330,

336.

FREUD, gold process for nervous cen-
tres, 768.
FREY, ammonia-carmine, 165; injection

mass, 491 ; iodised serum, 361 ;

Parma blue, 123 ; indifferent

liquids, 358.

FRIEDLAENDER, fixing mixture, 44.
Frog, bladder, 677; tongue, 657.
Fuchsin, 95, 107; ZIEHL'S carbolic,

107.
Fuchsin, S., 95, 98, 131; and see

Saurefuchsin.

G.

GAGE, picro-carmine, 162 ; section-
stretcher, 280 ; preservative fluid,
385 ; injection, 518 ; smooth mus-
cle, 676 ; section fixing, 325 ;
clearing mixture, 352 ; picric al-
cohol, 62, 540; preservation of
potash or soda preparations, 529.

GALLEMAERTS, section-fixing process,
325.

GALLI, stain for ueuroceratin funnels,
720.

488

INDEX.

The numbers refer to the Paragraphs, not to the Pages.

GANNAL, mounting medium, 373.

GABBINI, safranin staining, 101;
double stain, 270 ; Alcyonaria,
849, cleaning slides, 864.

GASKELL, paraffin sections, water
method, 280, 324.

Gastropoda, embryology, 614, 615 ;
eyes, 815 ; preparation, 18, 813 et
seq.; killing, 19, 814.

GAULE, section-fixing process, 324;
hardening brain, 732; fixing liquid,
48; choice of paraffin, 285.

GEDOELST, digestion, 554a ; medullated
nerve, 720.

GEHUCHTEN, TAN, cytological methods,
637, 644 ; nerve centres, 717.

GEIGY & Co., chemical products, 179.

Gelatin, imbedding masses, 292—296,
319, 320; injection masses, 457 et
seq.,- cement, 431; section fixa-
tives, 333, 334.

GELPKE, Weigert's hsematoxylin, 688.

General method described, 2.

Gentian violet, 93, 95, 98, 102, 258.

GEOFFKOY, mounting medium, 873.

Geonemertes, 835.

Gephyrea, 838.

GEELACH, gold method, 228, 767;
gelatin imbedding, 295 ; injection,
468 ; embryology of Aves, 590 ;
nerve-endings in muscle, 666.

GIACOMINI, gelatin process for serial
sections, 778 ; dry process for pre-
serving brains, 779.

GIBBES, HENEAGE, borax-carmine, 167 ;
combination stains, 270 ; cleaning
slides, 864.

GIEEKE, ammonia-carmine, 165, 745 ;
maceration of nerve-tissue, 532;
staining nervous centres, 745, 757,
767 ; action of chloral on ditto, 75 ;
neuroglia, 783 ; uranium carmine,
167 ; impregnation, 234.

GIBSBKECHT, serial section method,
327; section-stretcher, 280; paraf-
fin imbedding, 276.

GIBSON, VAN, clearing celloidin sec-
tions, 305.

GILSON, acetate of uranium fixing
liquid, 65; bleaching bichromate

objects, 874 ; carmine blue, 123a ;
celloidin imbedding, 301, 303;
chloral hydrate jelly, 873; cyto-
logical methods, 645 ; double im-
bedding, 306; Ehrlich-Biondi
stain, 259; iodised osmic acid,
645; mercuro-nitric fixing mix-
ture, 871 ; preservative fluid, 384 ;
picro-alum carmine, 157, 872.

Glandular structures, 807 et seq.

Glucose preservative media, 401, 402.

Glue, marine, 440.

Glycerin, 403 — 413 ; glycerin mounts,
to close, 430—432; glycerin and
gum, 395 — 397; glycerin jelly,
406 — 413 ; preservative mixture,
2, 405.

Glycero-gum, 397.

GOADBY, preservative fluids, 382.

Goblet cells, 809.

GOETTE, hardening liquid, 82.

Gold chloride, for fixing, 52 ; for im-
pregnation, generalities, 220 — 222,
229; composition of the commer-
cial salt, 221 ; methods of BASTIAN,
228 ; BECKWITH, 768 ; BOCCABDI,
228,670; BOHM, 228; CAEEIEBE,
652 ; CHESCHTSHONOWIC, 228 ;
CIACCIO, 228, 670, 674; COHN-
HEIM, 223; CYBTJLSKY, 229;
DEASCH, 222; FLECHSIG, 228;
FBEUD, 768 ; GEELACH, 228, 767 ;
GOLGI, 670; HENOCQUE, 228;

HOYEE, 228; IGACUSCHI, 810;

KOLOSSOW, 228; LOWIT, 224;
MANFBEDI, 228 , MAYS, 670 ;
NESTEBOFFSKY, 228 ; RANVIEB,
225,226; REDDING, 229; SCHIEF-
FEBDECKEB, 767; UpsoN, 770,
771; VIALLANES, 227; ZIEHEN,
769.

Gold orange, 95, 139, 793.

Gold size, 439.

GOLGI, chromate of silver, Slow method,
704, 705; chromate of silver,
Rapid method, 706; critique of
the same, 707 et seq. ; variations,
— CAJAL, RAMON Y, 714, 715, 717 ;
FICK, 712; TAN GEHTJCHTEN,
717 ; GEEPPIN, 710 ; HUBEB,

INDEX.

489

The numbers refer to the Paragraphs, not to the Pages.

712, 877 ; KALLIUS, 877 ;
KUPFFEB, 810; LENHOSSEE, 843 ;
MONDINO, 713 ; NEUMANN, 710 ;
OBEEGIA, 711 ; OPPEL, 810 ;
OYAEZUN, 716; PETBONE, 713;
SALA,718; SAMASSA, 709; SEHB-
WALD, 708, 709, 711 ; corpuscles
of Golgi, 671 — 674 ; mercuric im-
pregnation of nervous centres,
760.

GOODALL, spinal cord, 880.

Grorgonia, 849.

GOEONOWITSCH, embryology of Tele-
ostea, 609.

GBAFF, VON, Turbellaria, 834.

GUAM, stain for bacteria, 101, 102.

Granule cells, 788 et seq.

Granules, protoplasmic, 93, 641, 788
et seq.

Grape sugar imbedding mass, 308.

GEASEE, staining process, 111.

GEAVIS, section fixative, 332.

GBAT, serial sections, 333.

Green light in microscopy, 866.

GEENACHEE, alum-carmine, 153; alco-
holic carmine, 170 ; bleaching
mixture, 583 ; borax - carmine,
aqueous, 167 ; alcoholic, 169 j cas-
tor oil for mounting, 425 ; haerna-
toxylin, 185 ; purpurin, 201 ; eyes
of Mollusca, &c., 816.

GEEPPIN, Golgi's silver chromate
method, 710.

GEIEB, alum-carmine, 153.

GEIESBACH, benzo-purpurin, 133; Ben-
gal rose, 136; Biebricher Schar-
lach, 134; blood stains, 802 —
804; Congo red, 132; crocein,
139; double stain, 270; elastic
tissue, 795; iodine green, 140;
metanil yellow, 138; methyl green,
109; gold orange, 139; rocelliu,
107; Sauregelb, 139; tropseolin,
107, 139.

GBUBLEB, histological reagents, 94,
863; cherry gum, 858; Ehrlich-
Biondi-Heidenluvin mixture, 639.

Grunpulver, 109.

GUIGNET, soluble Prussian blue, 477.

GULL AND, flattening and mounting

paraffin sections, 280, 324 ; syrup
and dextrin process, 868.

Gum arable pure, 329 ; preservative
media, 392—400 ; imbedding
masses, 307—309, 315—317, 320 ;
injection mass, 499 ; mucilage for
labels, 865 ; fixative for sections,
329, 330.

Gum damar, 420, 421.

Gutta-percha, cement, 441 ; section-
fixing process, 336.

H.

Hsemacalcium, 182.

Haemalum, 180, 181.

Hsemammon, 183.

Haematein, generalities, 174 — 179 ;
properties, 179 ; produced by
"ripening," 174—176; artifically
prepared, 179; where obtainable,
179 ; formulae for haematein stains,
180, et seq. (alumina -haemateiii
stains, 180 — 192, other hacmatein
stains, 193—199) ; MATEE'S haema-
lum, 180, 181; his hsemacalciuin,
182 ; his haemammon, or nitrate of
ammonia haematein, 183 ; and
see " Haematoxylin."

Haematein-ammouiak, 179.

Haematein crystallisatum, 179.

Haematoxylic eosin, 189.

Haernatoxylin, generalities, 174 — 179;
theory of staining with, 145, 174
—176, 178, 179 ; ripening of solu-
tions, 174 — 176; dichro'ism of the
stains, 178 ; affinities for plasma-
granules and for mucin, 178 ; the
practice of staining, 177 ; how to
" blue " stains, 178 ; formulae for
stains, — alum solutions, 180 — 192;
stains depending on the formation
of other lakes, 193 — 199, and 688
—703, 726,755; APATHY'S, 194;
ABNOLD'S, 192 ; BENDA'S, 690 -r
BEBLINEE - BLAU'S, 688 ; BOH-
MEE'S, 186 ; BBANCA'S, 694 ;
BKEGLIA'S, 688, 694 ; BUT-
SCHLI'S, 185, 190; COOK'S, 192 j

CUCCATI'8, lyO ; DELAFIELD'S,.

490

INDEX.

The numbers refer to the Paragraphs, not to the Pages.

185; DIPPEL'S, 184; EHBLICH'S,
188; FLECHSIG'S, 694; GBENA-
CHEB'S, 185 ; HAIJG'S, 755 ; HEI-
DENHAIN'S, R., 193; HEIDEN-
HAIN'S, M., 197; HICKSON'S,
192; KAISEE'S, 876; KLEINEN-
BEEG'S, 184 ; KULTSCHITZKY'S,
697; KUPFFEB'S, 810; LISSAU-
EE'S, 696; MALLOEY'S, 195;
MAYEB'S, 180—183; MEECIEE'S,
701; MiNOT's,199; MITCHELL'S,
192; PAL'S, 692; PETJDDEN'S,
185; RANVIEB'S, 187 ; RENAITT'S,
189; Rossi's, 698; SANFELICE'S,
191; SCHAFEB'S, 693; SQUIBE'S,
188a; WEIGEET'S, 688 — 703;
WOLTEE'S, 699, 700, 726 ; and see
the separate entries such as " Iron
Haeinatoxylin," " Haemacalcium,"
&c. ; and see " Hsematein."

HAENSEL, liquid of, 41.

Hairs, tactile and others, 546, 646,
647.

Halicryptus, 838.

HALLEE, BELA, macerating fluid, 543.

HALLIBUBTON, chemistry of the cell,
636.

HAMANN, carmine, 167 ; Echinoderms,
846 ; acanthocephali, 837.

HAMILTON, freezing process, 316;
WEIGEET'S luematoxylin stain,
691 ; hardening brain, 737 ; freez-
ing ditto, 743.

HANAMAN, cleaning slides, 864.

HANTSCH, glycerin medium, 405.

Hardening agents, 67 et seq. ; acetate
of lead, 87; alcohol, 88; bichro-
mate of ammonia, 80 ; of potash,
75, 76 ; ditto with cupric sulphate,
77 ; chloride of palladium, 84 ; of
platinum, 83; chromate of am-
monia, 81 ; chromic acid fluids, 69
— 72 ; nitric acidj 74 ; osmic acid,
73,732,740; EELICKI'S solution,
77; MBBKEL'S solution, 83 ; MUL.
LEE'S solution, 76; generalities,
67 to 69 ; hardening nerve-centres,
729 et seq.; PBBKNYI'S solution,
39; picric acid, 86; pyridin, 90,
880.

HAEDY, Rotifers, 839.

HABMEB, silver staining marine ani-
mals, 217.

HAETIG, white injection mass, 490.

HASTING, preservative fluid, 379 ;
cement, 441.

HASWELL'S Dehydration apparatus, 2.

HATJG, lithium-carmine, 166; borax-
carmine, 167; haematoxylin, 192,
755 ; decalcification, 559, 562, 568,
569 ; inner ear, 687 ; modification,
of Weigert's nerve stain, 755.

HAYEM, blood, 802, 803.

Heat, killing by, 7 ; fixing by, 24, 637.

HEGMONS, embryology of Blattida,
619.

HEIDEE, collodionising sections, 282.

HEIDENHAIN, R., carmine, 165 ; haema-
toxylin, 193; anilin blue, 128;
triple stain, 259 ; glands of small
intestine, 811.

HEIDENHAIN, M., cytological methods,
633, 637, 638, 639; lanthanin,
637 ; hsernatoxylin (iron), 197 ;
discussion as to the Ehrlich-Biondi
stain, 259 ; paraffin imbedding,
275; cause of "shrinkage" in
paraffin sections, 280; water sec-
tion fixing process, 324.

Helix, eyes, 815 ; ova, 614 ; sexual
glands, 645 ; narcotisation, 16, 19.

HENCHMAN, embryology of Limax,
615.

HENKING, collodionsing sections, 282 ;
embryology of Diptera, 620 ; of
Phalangida, 624; of Insecta in
general, 617; examination liquid
for ova, &c., 617.

HENLE, staining nervous tissue, 746.

HENNEGUY, alum-carmine, 156; em-
bryology of Aves, 591 ; of Axolotl,
599; of Gastropoda, 614; of
Mammalia, 588; of Teleostea,
604 ; sexual gland of Helix, 645 ;
Protozoa, 857, 858; washing-out
carmine stains, 149 ; permanga-
nate staining method, 10G, 149;
mounting methyl green stains,
109 ; mounting paraffin sections,
280, 324, 328.

INDEX.

491

The numbers refer to the Paragraphs, not to the Pages.

HENOCQUE, gold process, 228.

HERMANN, silver staining, 213 ; papillae
foliatae of rabbit, 654; platino-
aceto-osinic mixture, 23, 51, 637 ;
osmium impregnation, 233 ; cyto-
logical stains, 639.

HEEMANN-BOTTCHEB staining process,
p. 60, note.

HEEEICK, cochineal alum-carmine, 154.

HEBTWIG, silver staining, 213, 217;
macerating methods, 542 ; Actiniae,
9, 849; Ctenophora, 307; Medusae,
852, 853; embryology of Rana,
603 ; of Triton, 601 ; of Strongy-
locentrotus, 644; tobacco-smoke
method, 9.

HEEXHEIMEB, elastic tissue, 795 ; epi .
thelium, 646.

HESCHL, amyloid matter, 109.

Heteropoda, eyes, 813, 816.

HEUECK, VAN, mounting medium, 415.

HEYDENEEICH, amber varnish, 447.

HEYS, balsam, 418.

HICKSON, haematoxylin, 192 ; macera-
tion, 528 ; eye of Musca, 825.

Hirudinea, 16, 118, 844.

His, fixing, 37 ; cornea, 658.

HOCHSTETTEB, injection, 514.

HOFEE, narcotisation method, 16.

HOFFMANN, vacuum imbedding, 278.

" Hofmann's Griin," 140.

HOGGAN, silver staining, 213; histo-
logical rings, 216 ; perchloride of
iron stain, 231.

HOLL, toluol for imbedding, 275 ; note
concerning liquid of Kleinenberg,
637.

Holothnrioidea, 18, 845.

Homarus, eyes, 825.

HOPEWELL SMITH, decalcification, 565,
796, 797.

HOPKINS, maceration, 540, 544.

Horn, 046.

HOYEE, ammonia-carmine, 165 ; neutral
ditto, 165; picro-carmine, 162;
silver staining, 213; gold ditto,
228 ; gum arable mounting
medium, 394; gelatin injection
masses— carmine, 472; Berlin blue,
481; lead chromate, 484; silver

nitrate, 486; green, 487; shellac

mass, 516; oil-colour masses, 517 ;

chloral for preserving masses, 458 ;

water section-fixing process, 324 ;

reactions of niuciu, 807.
HuBEE,Golgi's silver chromate method,

712, 877.

HUDSON, Rotifers, 839.
HUXLEY and MAETIN, ammonia-car-
mine, 165.

HYATT, imbedding method, 310.
Hydatina, 839.
Hydra, 15, 16, 114, 849, 850.
Hydrate of chloral, for narcotising, 14 ;

and see Chloral.

Hydrochlorate of Chinolin, 377.
Hydrochlorate of cocam, 15.
Hydrochloric acid for decalcification,

559, 563—566.
Hydrofluoric acid for desilicification,

574.
Hydrogen peroxide, for bleaching, 29,

30, 580.

Hydroidea, 7, 15, 16, 19, 850.
Hydroxylamin, 16.
Hypochlorite of potassium, see Eau de

Javelle.
Hypochlorite of sodium, see Eau de

Labarraque.

I.

IDE, double imbedding, 306.

IGACUSCHI, gold process for liver, 810.

IIJIMA, embryology of Planaria, 629.

Imbedding, defined, 2 ; generalities on,
2, 272 et seq ; imbedding in vacua,
278 ; double imbedding in celloidin
and paraffin, 306; and see also
Celloidin, Paraffin, &c.

Imbedding masses, albumen, 314, 321 ;
aniseed, 322; liayberry tallow,
287; celloidin, 297 et seq.; col-
lodion, ibid.; colophonium, 312;
copal, 311; dextrin, 318; egg
emulsion, 314; freezing masses,
315—322; gelatin, 292—296;
grape sugar, 308; gum, 307, 309;
gum and syrup, 316; paraffin,

492

INDEX.

The numbers refer to the Paragraphs, not to the Pages.

285—287 ; photoxylin, 297 ; shel-
lac, 310; soap, 288, 291, 833;
vegetable wax, 287 ; wax, 287.

Impregnation, distinguished from
staining, 118, 210 ; negative and
positive ditto, 210 ; primary and
secondary ditto, 210.

Impregnation methods, 113—121, 210
et seq. ; and see Gold chloride,
Nitrate of silver, Methylen blue,
&c.

Inchiostro di Leonardi, 111.

India-rubber cement, 442.

Indian ink injection, 512.

Indifferent liquids, 356 et seq.

Indigo, artificial, 125.

Indigo- carmine, 202; THIEESCH'S
staining method, 203; SEILEB'S,
241 ; MEEKEL'S, 242.

Indirect staining method, 96 et seq.

Indulin, 95, 125.

Infusoria, 857 et seq. and see Protozoa.

Injection of leeches, 844.

Injection of Mollusca, 820.

Injections, 457 et seq. ; injection masses,
albuminous, JOSEPH'S, 498 : aque-
ous, BJELOUSSOW'S gum, 499;
EMEBY'S carmine, 510; LETEL-
LIEB'S vanadate of ammonia, 511 ;
MAYBB'S blue, 509; MULLEB'S
blue, 508 ; RANVIEB'S blue, 507 ;
TAGFCHI'S Indian ink, 512 : col-
lodion, HOCHSTETTEB'S, 514 ;
SCHIEFFEEDECKEB'S, 513 : fatty,
HOTEE'S oil colour, 517 ; TEICH-
MANN'S linseed oil, 519 -.gelatinous,
BBUCKE'S Berlin blue, 478 ; CAB-
TEE'S carmine, 470; DAVIES'S car-
mine, 471; FOL'S carmine, 473;
FOL'S black (or brown), 493;
FOL'S blue, 480; FBEY'S white,
491; GEBLACH'S carmine, 468;
GUIGNET'S Prussian blue, 477;
HAETIQ'S white, 490; HOYEB'S
carmine, 472; HOYEB'S Berlin
blue, 481; HOYEB'S chrome yellow,
484 ; HOYBB'S silver yellow, 486 ;
HOYEB'S green, 487; MILLEB'S
purple, 494; RANVIEB'S carmine,
466; RANVIEB'S Prussian blue,

475, 476; RANVIEB'S silver nitrate,
496 ; ROBIN'S vehicles, 458, 459 ;
ROBIN'S anilin masses, 465;
ROBIN'S cadmium, 463 ; ROBIN'S
carmine, 460 ; ROBIN'S mahogany,
461 ; ROBIN'S Prussian blue, 462 ;
ROBIN'S Scheele's green, 464;
TEICHMANN'S white, 492 ;
THIEBSCH'S carmine, 469 ;
THIEBSCH'S Prussian blue, 479;
THIEESCH'S lead chromate, 483 ;
THIEBSCH'S transparent green,
488; VILLE'S carmine, 467:
glycerin, BEALE'S carmine, 501 ;
B BALE'S Prussian blue, 503, 504 ;
RANYIEB'S Prussian blue, 505;
ROBIN'S carmine, 502; ROBIN'S
cadmium, 463; ROBIN'S mahogany,
461 ; ROBIN'S Prussian blue, 462 ;
ROBIN'S Scheele's green, 464 : re-
sinous, BUDGE'S asphaltum, 515 ;
HOYEB'S shellac, 516 ; HOYEB'S
oil colour, 517 : starch, PANSCH'S
518.

Injections, natural, 521.

Inner ear, 34, 559, 562, 686, 687.

Insecta, see Arthropoda.

Insecta, embryology, 617.

Intra-epidermic nerve-fibres, 649.

Intra-vitam staining, 93, 114, 115,
858.

Iodide of palladium nerve stain, 727.

Iodine, for fixing, 66 ; for hardening
89 ; LUGOL'S solution, 66 ; iodine
as a mordant, 97.

Iodine green, 95, 98, 140.

Iodine haematoxylin, 190, 191.

Iodised osmic acid, 645.

Iodised serum, 360, 361 ; ditto for
macerations, 523, 524.

Iris, 678, 679.

Iron, perchloride, for fixing, 52; for
staining, 52, 231 ; pyrogallate of,
232; sulphate of, 44, 854; iron-
haematoxylin stains, 196, 197.

ISEAEL, orcein, 207.

J.

JACKSON, cloudiness in clearing media,
342.

INDEX.

493

The numbers refer to the Paragraphs, not to the Pages.

JACOBS, freezing1 mass, 320.

JACQUET, injection of leeches, 844.

JADASSOHN, structure of epithelium,
646 ; thionin for plasma-cells,
794.

JAGER, glycerin medium, 405.

JAKIMOVITCH, silver staining, 214;
rnedullated nerves, 720.

JAMES, cleaning slides, 864 ; damar
solutions, 420.

JANSSENS, carmine blue, 123 a.

Japan wax, 287.

JAVELLE, eau de, see Eau de Javelle.

JELGERSMA, staining nervous centres,
757.

Jellies, glycerin, 406—413.

JENSEN, immobilisation of Infusoria,
858.

JENSON, spermatological methods, 645.

JOLIET, imbedding method, 307.

JOSEPH, injection mass, 498.

JOHNSON, LINDSAY, keeping osmic
solutions, 25; sunning solutions of
metallic salts, 25, 211 ; platinic
hardening mixture, 78 ; gold im-
pregnation, 228 ; Ehrlich-Biondi
stain, 259, 687, 759 ; retina, 682,
683; mounting methyl green
stains, 879; inner ear, 687;
cementing celloidin blocks, 303.

Juice-canals, 120, 121.

JUNG, microtomes, 271.

K.

KADYL, imbedding mass, 290.

KAISER, glycerin jelly, 292, 294, 410;
naphthylamin brown for spinal
cord, 758; Bismarck-brown stain,
107 ; other nerve stains, 876.

KALLITTS. Golgi's silver chromate
method, 877.

KANTHACK, histological reagents and
apparatus, 94.

KASTSCHENKO, reconstruction from sec-
tions, 587.

KEISER, fixation of Acanthocephali,
49, 60.

KEMP, blood-plates, 805.

KENT, S., Infusoria, 66, 859.

KENT, A. F. S., making Farrant's
medium, 395 ; cement, 456.

Kernschwarz, 208.

Killing tissues and organisms, 6 — 20 ;
killing by heat, 7 ; by rapid fixing
agents, 8 ; by narcotisation, 9 ; by
nicotin, 10; by chloroform, 11,
12 ; by ether and alcohol, 12 ; by
chloral, 14 ; by cocain, 15 ; by car-
bouic acid, 19 ; by asphyxiation,
19; by chromic acid, osmic acid,
" Kleinenberg," or sublimate, 18 ;
by fresh water, 20; by warm water,
20; by hydroxylamin, 16; by
chloride of magnesium, 17 ; by
curare and other drugs, 18.

KINGSLEY, imbedding method, 273.

KISHINOUYE, embryology of Araneina,
622.

KITTON, asphalt, 436 ; cement, 453.

KLEBS, glycerin jelly, 292, 293.

KLEIN, fixing mixture, 33 ; hardening
intestine, 81 ; cornea, 658 ; cochi-
neal, 154.

KLEINENBERG, colophonium for mount-
ing, 422 ; embryology of Lum-
bricus, 630 ; picro-sulphuric acid,
57 ; haematoxylin, 184.

KLEMENSIEWICS, picro-carmine, 162.

KNAUER, cleaning slides, 864.

Knotting, for cement, 443.

KOCH, VON, copal imbedding method,
311.

KOGANEI, iris, 679.

ROLLER, embryology of Aves, 593.

KOLLIKER, ovum of rabbit, 588 ; bone,
800.

KOLLMANN, fixing mixture, 40.

KOLOSSOW, gold method, 228 ; osmium
mixture, 28, 233.

KOPPEN, elastic tissue, 795.

KOROTNEFF, killing Siphonophora, 11.

KORSCHELT, Infusoria, 859.

KORYBUTT-DASZKIEWICS, rayelon of
reptiles, 784; plasma-cells, 788.

KOSSEL, chemistry of the cell, 636.

KOSSINSKI, double stains, 269.

KOTLAREWSKY, central nervous system,
35, 87, 732.

494

INDEX.

The numbers refer to the Paragraphs, not to the Pages.

KOWALKWSKY, embryology of Teleos-
tea, 607.

KBAUSE, corpuscles of, 653 ; molybdate
of ammonium, 234 ; motor plates,
670 ; silver staining, 214 ; retina,
683, 685 ; spinal cord, 733 ; thio-
phen green, 141.

Kreasote, for bleaching, 578; for
clearing, 352a ; preservative fluids
368—370.

KEOMATEB, methyl violet for epithe-
lium, 111, 646.

KBYSINSKY, photoxylin imbedding,
297.

KBONECKEB, artificial serum, 362.

KBONIG, cement, 445.

KUHNE, digestion fluids, 554 ; macera-
ting mixture, 545 ; motor plates,
670.

KUHNE, H., aniseed freezing mass,
322; staining method, 98.

KUHNT, retina, 682, 683.

KUKENTHAL, Annelids, 14, 840—842.

KTJLTSCHITZZY, fixing mixture, 43 j
double imbedding, 306 ; preserva-
tive fluids, 2 ; ovum of Ascaris,
644 ; tactile corpuscles, 651 ; stain
for nervous centres, with hserna-
toxylin, 697.

KUNSTLEB, Protozoa, 859.

KuPFFEB,embryological methods, 596;
axis cylinder, 722 ; liver, 810.

KTJSKOW, digestion fluid, 552.

KTJTSCHIN, clearing method, 343.

L.

LABABBAQTTE, eau de, see Eau de

Labarraque.
Labelling slides, 865.
Labyrinth of ear, 559, 562, 686, 687.
Lacerta, ova of, 39.
Lactic acid for decalcification, 559,

569.

LAMANSKY, sensibility of the eye, 866.
Lamellibranchiata, aquiferous pores,

813 ; epithelium, 821 ; eyes, 818,

820; sexual gland, 645; shell,

311—313, 817, 819.

LANDOIS, impregnation methods, 234;

macerating mixture, 532.
LANDOLT, retina, 685.
LANDSBEBG, Infusoria, 859.
LANG, fixing liquids, 48 ; methods for

Platyhelrnia, 48, 834.
j LANGEBHANS, Amphioxus, 662; gutn

and glycerin medium, 396 ; tactile

corpuscles, 651.
LANGHANS, dichroism of haernatoxyliu

stains, 178.

LANKESTEB, eyes of Limulus, 825.
LAVDOWSKY, bilberry juice stain, 209 ;

myrtillus, 209 ; chloral hydrate

medium, 376; macerating fluid,

528 ; inner ear, 687 ; methylen

blue impregnation, 119.
LAWBENCE, glycerin jelly, 407.
Lead, acetate, 87, 732 ; chromate,

234.

LEBEB, impregnation methods, 234.
LECLEBCQ,spermatological stains, 645;

blood stains, 804.
Leech, 844.

LEGAL, alum-carmine, 157.
LEGBOS, silver staining, 215.
Lemon juice for fixing nuclei, 637.
LENHOSSEK, VON, myelon of reptiles,

784 ; nerves of annelids, 843.
LENNOX, retina, 683.
Lens, crystalline, 660.
LEON, nucina, 209.
LEONABDI, inchiostro di, 111.
Lepidoptera, embryology, 618.
LEPKOWSKY, gold method for teeth

and bone, 798.
LETELLIEE, injection, 511.
LETJCKHABT, imbedding boxes, 273.
LEVEN, saffron stain, 205.
Levulose for mounting, 402.
LEWIS, BEVAN, see under Neurological

methods.
Lichtblau, 123.
Lichtgriin, 109.
LICHTHEIM, staining with Weigert's

hsematoxylin, 688.
LIEBEEMANN, analysis of carmine, 144;

carminic acid, 145.
Light, action of, on alcohol containing

chromic objects, 30 ; on metallic

INDEX.

495

The numbers refer to the Paragraphs, not to the Pages.

green,

in micro-

salts, 25, 211 ;
scopy, 866.

LILIENFELD, blood- plates, 805.
LILIENFELD and MONTI, test for phos-
phorus in tissues, 636.
Limax, 814 ; embryology, 615.
Limulus, 825.
LINDSAY JOHNSON, see JOHNSON,

LIXDSAY.

Linseed oil injection, 519.
Liquid of Miiller, of Erlicki, of Kult-
schizky, of Merkel, &c., see the
names of the respective authors.
Liquidambar, 427.
LISSAUEE, hsematoxylin nerve stain,

696, 876.

LIST, hsematoxylin and eosin, 251 ;
ditto and nitrate of rosanilin,
255 ; other combination stains,
261 — 265 ; macerations, 525 ; An-
thozoa, 819 ; Coccidae, 823 ; mucus
cells, 808 ; goblet cells, 809.
Lithium-carmine, 166.
Litmus stain, 209.
Liver, 810.
Lizzia, 851.

Lo BIANCO, S., see BIANCO, S., Lo.
LOCY, embryology of Agelena, 622.
LOEFFLER. stains for flagella, 861.
LONNBERG, Tri&nophorus, 832.
LOEWE, crystalline, 660.
LOEWENTHAL, nerve-centres, 77; picro-

carmine, 162.

LOEWY, macerating skin, 646.
Loliffo, eyes, 816.
LONGHI, eseriu for Infusoria, 859.
LONG WORTH, corpuscles of Krause,653.
Looss, softening chitin, 557 ; Nema-

todes, 836.

LOVETT, cement, 454.
LOWIT, gold method, 224, 667 ; blood,

802—804.

Loxosoma, 217, 812.
LUGOL, solution of, 66.
Lumbricus, 118, 840—843; embryo-
logy, 630.

LUSTGAETEN, elastic tissue, 104, 795.
LFYS, noir Colin, 757.
Lymphatics, 120, 121 ; and see Injec-
tions.

M.

MAAS, carmine and malachite green,
244.

MACALLUM, Sphyranura, 833 ; double
stain, 242.

MAC BBIDE, development ofAmphiura,
848.

Maceration, 522— 548, 662; nervous
tissue, 781 ; epithelium, 646 ; mol-
lusca, 821.

Macrotoma, 829.

Magdala red, 95, 98, 107,

Magenta, 95, 98, 107.

MAGINI, stain for nervous centres,
762.

Magnesium chloride, for narcotisation,
17.

MAHBENTHAL, VON, osmium impreg-
nation, 233.

Malachite green, 95, 98, 143, 639.

MALASSEZ, ammonia-carmine, 164.

MALFATTI, chemistry of the cell, 636.

MALLOEY, phospho-molybdic acid hse-
matoxylin, 195.

Mammalia, embryology, 588.

Manchester brown, 110.

MANFEEDI, gold method, 228.

MAECHI, VON, Limax, 814 ; corpuscles
of Golgi, 672 ; degenerate nerves,
720a.

Marine animals, killing, 20; fixing,
24, 57 ; silver impregnation, 217 ;
gold ditto, 230.

Marine glue, 440.

MARK, collodionising sections, 282 ;
embryology of Limax, 615 ; sec-
tion-fixing, 327.

MAESH, bleaching, 576; gelatin ce-
rneut, 431; decalcification mix-
ture, 571.

MABSHALL, May's gold process, 670.

MAETIN, benzoazurin, 107, 127.

MAETINOTTI, safranin straining, 101;
damar solutions, 420 ; anilin black,
757; picro-nigrosin, 757; elastic
tissue, 795 ; iiitra vitam staining,
93.

MASON, nervous system of Reptiles,
&c., 742.

496

INDEX.

The numbers refer to the Paragraphs, not to the Pages.

Mastzellen, 788—794.

Maturation of ovum, 644; and see
Cytological methods.

MATSCHINSKY, bone, 796.

MAUEICE and SCHULGIN, double stain,
243 ; embryology of Amarcecium,
612.

Mauvein, 95, 107.

MAX FLESCH, see FLESCH.

MAYEE, PAUL, acidulated alcohol, 63 ;
alcoholic carmine, 170; prep, of
Arthropods, 5, 58, 63, 822 ; albu-
men fixative for sections, 328 ;
bleaching, 575; Bismark brown,
110 ; carmine staining, theory of,
144—147, 174; Carmalum, 151;
Chloride-of-aluminium carmine,
152; Paracarmine, 168; cochineal}
old formula, 146, 172 ; new formula,
146, 173; desilicification, 574;
general principles of preparation,
5, 24; haematoxylin (hsematein)
staining, theory of, 145, 174 —
179; Hsemalum, 180, 181 ; Hsema-
calcium, 182 ; Haemammon or Ni-
trate-of-ammonia Hsematein, 183 ;
preparation of the Ammonia-salt
of Hsematein, 179; dichroism of
haematein stains, 178; blueing
them, 178 ; critique of Kleinen-
berg's hsematoxylin, 184; injec-
tion, 508 ; picro- carmine, 162 ;
picro-sulphnric acid, 55 ; picro-
hydrochloric acid, 59 ; picro-nitric
acid, 58; section-stretcher, 280;
section fixing methods, 327, 328 ;
shellac fixative, 327; water-bath
for paraffin, 277.

MAYEE, S., " violet B." for connective
tissue, 129, 786; methylen blue
method, 117, 119, 121.

MAYS, nerve-endings (gold process),
670.

MAYSEL, Bismarck brown, 110.

Medullated nerve, 688—727 ; and see
Neurological methods.

Medusas, 11, 18, 45, 542, 851—853.

MEECIEE, hsematoxylin nerve stain,
701; Upson's nerve-impregnations,
770, 771.

MERCK, carminic acid, pure, 148 ;
chemical products, 179.

Mercury, bichloride, 47, 48 et seq. ;
and see Corrosive sublimate ; bin-
iodide, mounting medium, 414 ;
cyanide, for fixing, 49.

MEEK, making up chromo-aceto-osmic
acid, 36.

MEBKEL, chromo-platiriic liquid, 42,
71, 83 ; impregnation method,
234; carmine and indigo stain,
242, 749; celloidin imbedding,
297; half-clearing brain sections,
779 ; staining nervous tissue, 746.

Metagelatin, 497.

Metallic salts, action of light on solu-
tions, 25, 211.

Metallic stains, 210 et seq.

Metanil yellow, 95, 138.

Methanilin green, 109 ; methanilin
violet, 111.

Methyl alcohol, for narcotising, 13;
for safranin staining, 101.

Methyl green, 95, 98, 109, 879 ; com-
bination stains with, 259 — 263.

Methyl mixture for maceration, 548.

Methyl violet, 93, 95, 98, 107, 111,
258.

Methylal, for dehydration, 2, 119.

Methylated spirit, 2, 862.

Methylen blue, for hardened central
nervous system, 751 — 753, 773,
774; for plasma cells, 792; for
impregnation, 95, 113 etseq., 878 ;
generalities, 113 ; staining in toto
during life, 114; staining nerve-
tissue during life, 115 — 121; influ-
ence of oxygen and of ammonia, 1 15 ;
staining by injection or by immer-
sion, 116 ; the solutions employed,
117, 118 ; preservation of the stain,
119; impregnation of epithelia,
lymph - spaces, &c., 120, 121 ;
stains for hardened nervous tissue
(REHM), 751—753 ; (SAHLI), 773 ;
(ADAMKIEWICS), 774 ; methods of
APATHY, 115, 117, 118, 119, 878 ;

ABNSTEIir, 117, 119 ; BlEDEE-
MANN, 117, 666; BiJEGEE, 835;

DOGIEL, 115, 117, 119, 120, C52,

INDEX.

497

The numbers refer to the Paragraphs, not to the Pages.

653 ; EHELICH, 114; FAJEBSTAJN,
C57; FEIST, 119; GEELACH, 666;
LAVDOWBKY, 119; MAYER, S.,
117, 119, 121; PABKEB, 119;
KETZITJS, 117, 119 ; ZOJA, 114.
MEYEB, salicylic solutions, 391.
MIBELLI, elastic tissue, 795.
Microchemistry of the cells, 636.
Microtomes, 271, 867.
MIGTJLA, glycerised serum, 363.
MILLEB, vegetable wax, 287; injec-
tion, 494.
Milnesium, 826.

MINOT, hsematoxylin stains, 199; clear-
ing celloidin sections, 305 ; mace-
rating skin, 646 ; carmine, 167 ;
serial sections, 777.
MINOT, microtome, 271.
MITCHELL, haematoxylin, 192.
MITBOPHANOW, gold process, 646 ;
maceration, ibid. ; sense organs of
Amphibia, 656 ; prickle-cells, 646.
MOBIUS, macerating media, 539.
MOEENEE, tracheal cartilage, 801.
MOLESCHOTT, potash or soda solution,
529; MOLESCHOTT and Piso
BOBME'S macerating fluid, 527.
Molffttla, 811.

Mollusca, 813 — 821 ; aquiferous pores,
813; embryology, 613 et seq.;
eyes, 815—819; fixation, 813;
injection, 820; maceration, 538,
543, 821 ; removing mucus from,
814; shell, 311—313, 817, 819.
Molluscoida, 7, 14, 15, 812; and see

Bryozoa.

Molybdate of ammonium, 234, 637.
Monadina, 859.
MONDINO, medullated nerves (Golgi's

method), 713.

Monobromide of naplithalin, 415.
MONTI, sulphate of copper impregna-
tion method, 764; test for phos-
phorus in tissues, 636.
MOOBE, stain for blood, 804.
Mordants for anilin dyes, 97, 101, 106.
MOBGAN, ova of Arthropods, 616, 823 ;
removing albumen frum ova, 603.
Morphia for narcotising, 18.
MOSELEY, shell, 819 ; Chitonidce, 817.

Mosso, blood, 803.

Motor plates, 665 et seq.

Mounds of Doyere, 826.

Mounting media, see Examination

media.

Mucilage for labels, 865.
Mucin, 178, 807 et seq.
Mucus, removing, from Mollusca, 814.
Mucus glands, 104, 105, 252, 807 et

seq.

MUELLEB, blood, 302, 804.
MULLEB, hardening liquid, 76, 536;

silver staining, 214; injection

mass, 508.
MUNDEB, G., his histological reagents,

94.

MITNSON, chloral hydrate fluid, 376.
Musca, eyes, 825.
Muscle, dissociation of, 544 et seq.t 662 ;

nerve- endings in, 665 et seq. ;

smooth, 676 et seq. ; sections, 661.
Myelin, 719.

Myelon, see Central nervous system.
Myrtillus, 209.
Myrtle wax, 287.

N.

Nails, 646.

NANSEN, maceration of nerve-tissue,
532.

Naphtha, for clearing, 275, 283, 354,
preservative fluids, 370, 371.

Naphthalin, monobromide, 415.

Naphthalin red, 95, 107.

Naphtholorange, 107.

Naphthylamin brown, 758.

Naples Zoological Station methods, 5,
sub. Jin., 811.

Narcotisation, 9—19, 811, note, 870.

NEALEY, bone, 796.

Nebenkern, 208, 634, 639, 645.

NEGBO, motor plates, 670.

Nematodes, 557, 836.

Nemertina, 7, 835.

Nerves, degenerate, 720a.

Nerves, medullated, 115 et seq., 688 —
727; and see Neurological Me-
thods.

32

498

INDEX.

The numbers refer to the Paragraphs, not to tho Pages.

Nerve-endings in muscle, 665 et seq. ;
in skin and others, 648 et seq.,
657 ; methylen blue method, 115
et seq. ; and see Neurological
Methods.

Nervous system, central, see Central
Nervous System and Neurological
Methods.

NESSLEE, test for ammonia, 467, note.

NESTEBOFFSKY, gold process, 228.

NEUMANN, Golgi's silver chromate
method, 710.

Neuroceratin, 704, 706, 720.

Neuroglia, 783.

Neurological methods, ADAMKIEWICS,
774; APATHY, 115, 117, 118, 119,
785, 878 j ANGELUCCI, 682 ; ALT,
724; ABMANNI, 694; ABNSTEIN»
117, 119; BABBETT, 682; BECH-
TEBEFF, 732 ; BECKWITH, 768 ;
BEEVOB, 691, 692; BELLONCI, 741;
BEBNHEIMEB,683; BENCZUB, 756 ;
BENDA, 690; BEBLINEBBLAU, 688;
BETZ, 165, 739, 745; BIEDEB-
MANN, 117, 666, 667 ; BOCCABDI,
670; BOLL, 767; BOVEBI, 720;
BBAMWELL, 779; BBANCA, 694;
BBEGLIA, 688, 694 ; BBEMEB,

670 ; BUBCKHABDT, 742 ; CA JAL,

RAMON Y, 669, 683, 684, 714, 715,
717 ; CABBIEBE, 652, 781 ; CAT-
TANEO, 673 ; CHIEVITZ, 682 ;

ClACCIO, 228, 670, 674 ; ClAGLIN-

SKI, 733, 743 ; Cox, 761 ; CUCCATI,
683; CYBULSKY, 650; DASZ-
KIEWICS, 784 ; DEECKE, 738, 743 ;
DENNISSENKO, 682; DIOMIDOFF,
732 ; DOGIEL, 115, 117, 119, 120,
652, 653, 655, 683 ; DUVAL, 734,
749,776; EHBLiCH,114; EIJKMAN,
720a; EXNEB, 732, 740; FAJEB-
STAJN, 657 ; FEIST, 119, 743 ;
FICK, 712; FISCHEB, 651, 667;
FLECHSIG, 228, 694, 763, 768;
FLESCH, MAX, 34, 71, 687— G89,
749, 780 ; FBEEBOEN, 748, 781 ;
FEEUD, 768 ; GALLI, 720 ; GAULE,
732; GEDOELST, 720; VAN GE-
HUCHTEN, 717; GELPKE, 688;
GEELACH, 228, 666, 767; GIA-

COMINI, 778, 780; GIKBKE, 75,
532, 745, 757, 767, 783 ; GOLGI,
(chromate of silver impregnation,
Slow Method) 704, 705, (Rapid
Method) 706, (critiques and varia-
tions) 707—718, 810, (corpuscles
of) 671 — 674, (gold impregna-
tion) 670, (sublimate impregna-
tion) 760, 761; GOODALL, 880;
GBEPPIN, 710; HAMILTON, 316,
691,737, 743; HAUG, 687, 755;
HENLE, 746; HEEMANN, 654;
HUBEB, 712, 877; IGACUSCHI,
810; JAKIMOVITCH, 720; JEL-
GEESMA, 757 ; JOHNSON, LINDSAY,
682, 683, 759 ; KAISEE, 757,
876 ; KALLIUS, 877 ; KOBY-
BUTT - DASZKIEWICS, see DASZ-

KIEWICS ; KOTLABEWSKY, 35,

87, 732 ; KBAUSE, 670, 683, 685,
733 ; KiiHNE, 670 ; KUHNT, 682,
683 ; KULTSCHIZKY, 651, (hamia-
toxylin stain) 697 ; KUPFFEE,
722; LAVDOWSKY, 119, 687;
LENHOSSEK, 784, 843; LENNOX,
683 ; LEWIS, B., 728, (hardening)
731, 736, 740, 743, (staining) 757,
(half clearing method) 779, (com-
pression method) 782; LICH-
THEIM, 688; LISSAUEE, 696; LOE-
WENTHAL, 77 ; LONGWOETH, 653 ;
LOWIT, 224, 667, 670; LTTYS,
757; MAGINI, 762; MALLOEY,
195; MANFEEDI, 228, 672;
MABCHI, 672, (degenerate nerve)
720 a; MAESHALL, 670; MABTI-
NOTTI, 757 ; MASON, 742 ; MAYEE,
S., 117, 119, 121; MAYS, 670;
MEBCIEE, 701, 770, 771; MEB-
KEL, 746, 749, 779; MITEO-
PHANOW, 646, 056; MONDINO,
713 ; MONTI, 764 ; NANSEN, 532 ;
NEGEO, 670; NESTEEOFFSKY, 228 ;
NEUMANN, 710; NIKIFOEOW,
775; NISSL, 723; OBEESTEINEB,
728, (hardening) 731, (staining)
745, 750; OBEEGIA, 711 ; OGATA,
732; OSBOEN, 743, 778, 784;
OVABZUN, 716; PAL, 692, 760;
PALADINO, 727; PANETH, 688;

INDEX.

499

The numbers refer to the Paragraphs, not to the Pages.

PETEONE, 713 ; PLATNEB, 52, 720 ;
POLITZEB, 687; PBENANT, 686;
PEITCHABD, 687; RAMON Y CAJAL,
see CAJAL; RANVIEE, (gold me-
thods) 225, 226, 647, 649, 667,
668, 671, 682, 685, 720; REHM,
725, 751—753; RETZIUS, 117,
119; REZZONICO, 704, 720; Rossi,
G98; RUTHERFOED, 735; RTJF-
FINI, 673; SACHS, 696, 670;
SAHLI, 730, 731, 773; SALA, 718 ;
SAMASSA, 709; SANKEY, 757;
SCHAFEE, 693; SCHAFFEE, 683;
SCHIEFFEEDECZEE, 584, 682, 685,
720, 767, 781; SCHMAUS, 747,
757; SCHMIDT, 784; SCHULTZE,
MAX, 685; SCHWALBE, 686;
SEHEWALD, 708, 709, 711; DE
SOUZA, 90, 880; SQTJIBE, 724;
STILLING, 781; STIBLING, 757;
TAFANI, 687; TAL, 760; VON
THANHOFFEE, 782; THIN, 685;
TIZZONI, 720; TEINCHESE, 667;
TEZEBINSKI, 732 ; TSCHISCH, 77 ;
UPSON, 748, (gold and iron me-
thod) 770, (gold and vanadium
method) 771; VASALE, 695;
VEJAS, 757; WALDEYEE, 687;
WEIGEBT, (hsematoxylin impreg-
nation) 688—703, (the 1885 me-
thod) 688, (the 1891 method) 703,
(variations) 689 — 702, 755,
(mounting sections) 305, 340, 426,
703, (hardening) 730, (Siiure-
fuchsin method) 772, (retina)
683; WOLFF, 670, 677; WOL-
TEES, 699, 700, 726; ZIEHEN,
769 ; ZOJA, 114 ; ZUPPINGEB, 756 ;
and see Impregnation Methods,
Ketiua, Inner Ear, Nerves, Cen-
tral Nervous System, &c.

N. MI utilisation of carmine masses, 467. !

Neutrophilous cell-elements, 788.

NEYT, see BENEDEN, VAN.

NIAS, cleaning slides, 864.

NIETZKI, carminic acid, 145; IKUMIUI-
te'in, 174.

N'u-otin for narcotisation, 10.

Nigranilin, 95, 130.

Nigrosin, 95, 98, 125.

NIKIFOBOW, borax- carmine, 167 ; stain
for nervous centres, 775 ; clearing
celloidin sections, 305.

NISSEN, gentian violet staining pro-
cess, 102; cytological methods,
643.

NISSL, axis cylinder, 723.

Nitrate of ammonia hajmatei'n stain,
183.

Nitrate of silver, for fixing, 38; for
impregnation, generalities, 212 ;
methods of ALFEBOW, 213 ; COHN-
HEIM, 668; DEZHUYSEN, 213;
DUVAL, 213, 215 ; HABMEB, 217 ;
the HEBTWIGS, 213, 217; the
HOGGANS, 212, 213, 216; HOYEB,
213; JAKIMOVITCH, 214; KEAUSE,

214; LEGBOS,215; MiJLLEB, 214;

OPPITZ, 214; RANVIEB, 212,
213 ; VON RECZLINGHAUSEN, 213 ;
REICH, 213; ROBINSKI, 213;

ROUGET, 213, 214; SATTLEB,214;

VON THANHOFFEB, 214; TOUB-
NEUX, 213 j VOSMAEB, 217; and
see GOLGI.

Nitrate of uranium, fixing mixture,
28 ; use with osmium, 78.

Nitric acid for fixing, 37—40, 637;
for hardening, 74, 780 ; for ma-
ceration, 544, 545 ; for decalcifi-
cation, 560, 561; for bleaching,
579.

Nitric and chromic acid mixtures, 39,
40.

Nitrite of ainyl as a vaso-dilatator for
injections, 500.

Noir Colin, 95, 130, 757.

NOLL, corrosion, 556 ; sponge spicules,
856 ; salicylic acid medium, 392.

NOBDMANN, plasma-cells, 790.

Normal salt solution, 359.

NOBBIS and SHAKESPEABE, carmine
and indigo stain, 242.

Nucina, 209.

Nuclein, reactions of, 636, and see
Chromatin.

Nucleus, see Cytological methods.

Nychtoterus, 858.

500

INDEX.

The numbers refer to the Paragraphs, not to the Pages.

0.

OBEBSTEINEB, central nervous system,
728; hardening, 731; staining,
745, 750.

OBBEGIA, Golgi's silver chromate me-
thod, 711 ; serial section method,
868.

Oceania, 851.
Octopus, eyes, 816.

ODENIUS, tactile hairs, 546.

OGATA, hardening brain, 732.

OHLMACHEE, reactions of safranin with
iodine and picric acid (pseudo-
carcinoma corpuscles), 101 ; sec-
tion fixing process, 869.

Oil, of aniseed, imbedding mass, 322 ;
of bergamot, 347; cannel, 346;
cedar wood, 344 ; cloves, 4, 343,
345 ; origanum, 305 ; sandal-
wood, 349 ; turpentine, 343 ; and
see Clearing agents.

Olfactive organs of Vertebrates, 655.

Ommatostrephes, eyes, 816.

One-third alcohol, 63, 525, 862.

Onuphis, 841.

Opalina, 858.

Opheliadae, 841.

Ophiuridea, 847.

OPPEL, liver, 810.

Opisthobranchiata, 813.

Opisthotrema, 833.

OPPITZ, silver staining, 214.

Optic nerve, 741.

Orange, 95, 98, 107, 258, 259.

Orcein, 207, 795.

Orchella, 206.

Orchestia, embryology, 626.

Origanum oil, 305, 348.

Orseille, 206.

Orseillin, 95, 107.

OETH, methyl violet, 111; lithium-
carmine, 166; picro-lithium car-
mine, 240.

OSBOBN, embryology of Triton, 600;
brain of Urodela, 743, 784 ; serial
sections, 778.

Osmic acid, for fixing, 2], 23, 25 — 29 ;
for hardening, 73, 732, 740, 741 ;
for macerating, 541, 542 ; for im-

pregnation, 233; blackening of
preparations with, 29, 36, 575;
how to keep solutions, 25, 26, 28,
35,36; GOBI'S solution, 26; Ko-
LOSSOW'S solution, 28 ; osmic acid
and alcohol, 28; and chromic
acid mixture, 34, 73 ; and chromic
and acetic acid mixture, 35, 36;
and iodine mixture, 645 ; acetic
acid mixture, 28, 542 ; osmic and
oxalic stain, 233 ; osmic and pyro-
gallic ditto, 233, 639.

Osmium-blackened fat, 36, 787.

Osmium-carmine, 167.

Osmosis, to avoid, 4, 358.

Ostracoda, 822.

Ova, pelagic, 83, 610.

OVEBTON, bleaching osmic objects, 29 ;
fixing with iodine vapours, 66.

OVIATT, injection method, 500.

Ovum, maturation of, 644; and see
Cytological methods.

Ovum of Ascaris, 54, 644 ; other ova,
see Embryology.

OWEN, preservative liquid, 383.

Oxalic acid for maceration, 547.

Oxygenated water, 29, 30, 580.

OrABzuN, Golgi's silver chromate im-
pregnation, 716.

P.

PACINI, preservative liquids, 380.

PAQENSTECHEE, fixing fluid, 45.

PAL, haematoxylin stain, 692 ; staining
nervous centres, 760.

PALADINO, iodide of palladium nerve
stain, 727.

Palinurus, ova, 58.

Palladium chloride, 52, 84, 234, 559,
746, 767.

Palladium iodide nerve stain, 727.

Palythoa, 849.

Pancreatin digestion fluids, 553.

PANETH, making Weigert's haema-
toxylin, 688 ; goblet-cells, 809.

PANSCH, injection-mass, 518.

Paper, imbedding trays, to make, 273 ;
paper cells, 432.

Papilla) foliutiu, 654.

INDEX.

501

The numbers refer to the Paragraphs, not to the Pages.

Puracarmine, 145, 168.

Paraffin, imbedding processes, 272 et
seq. ; review of, 284; paraffin
masses, 285 — 287; solvents of,
273, 283 ; ovens, 277.

Paris green, 109.

Paris violet, 111.

PAEZEE, turpentine cement, 444.

PAEKEE, G. H., dehydration methods,
2; methylen blue method, 119;
eyes of scorpions, bleaching, 579 ;
eyes of Homarus, 825.

Parma blue, 95, 123.

PAETSCH, cochineal stain, 154.

PATTEN, embryology of Blattida, 619 ;
eyes of Molluscs and Arthropods,
818 ; maceration of Mollusca, 821.

PAUXSEN, goblet cells, 809.

Pecten, eyes of, 818.

Pedicellina, 217, 812.

Pelagic ova, 83, 610.

Pennatulidaj, 849.

Pentacrinus, 848.

Pepsin digestion fluids, 449—552.

Perchloride of iron, for fixing,' 52 ; for
impregnation, 52, 231.

PEEEMESCHKO, cytological methods,
633.

PEEENYI, fixing fluids, 39.

PEB&ENS, picro- carmine, 162.

Periplaneta, ova of, 616, 823.

PEEL, soluble carmine, 167.

Permanganate of potash, 46, 97, 106,
214, 533, 658; for fixing, 46 ; for
washing out carmine stains, 149 ;
for mordanting, 106, 149 ; for re-
ducing silver impregnations, 214.

Perophora, 811.

Peroxide of hydrogen, 29, 30, 580.

PEREIEE, narcotising Lumbricus, 841.

Perruthenic acid, 809.

PETIT, see RIPAET and PETIT.

PETBONE, cerebro-spinal nerves, 713.

PFITZEE, imbedding mass, 291.

PFITZNEB, safranin solution, 101 ; gold
chloride and safranin, 270 ; damar
solution, 420 ; Opalina, 859 ; cyto-
logical methods, 153, 643.

IMialan-ida, 828; ova of, 282, 023,
624.

Phallusia, 811.
Phascolosoma, 838.
Phenicienne, la, 110.
Phenylen brown, 110.
PHILLIPSON, macerating skin, 646.
Phloroglucin, 562, 687.
Phoronis, 838.
Phospho-molybdic-acid hsematoxylin,

195.
Phosphoric acid for decalcification, 559,

568.

Phosphorus in tissues, test for, 636.
Photoxylin imbedding method, 297,

299.

Phyllosoma, 822.
Physalia, 854.

Physiological salt solution, 359.
Physophora, 854.
PIANESE, double-stain for connective

tissue, 786.
Picric acid, fixing fluids, 2, 56—62;

hardening ditto, 86; staining with,

137, 236—240 ; decalcification

with, 559, 567.
Picric alcohol, 62, 540.
Picro-alum-carmine, 157, 872.
Picro- carminate of ammonia, 159, 160.
Picro-carminate of soda, 162.
Picro-carmine, 159—162, 388.
Picro-chromic acid, 41, 72.
Picro-chromo-sulphuric acid, 57, 60.
Picro-hydrochloric acid, 59, 567.
Picro-lithiuin carmine, 240.
Picro-nigrosin, 757, 786.
Picro-nitric acid, 58, 567.
Picro-osmic acid, 61.
Picro-sulphuric acid, 57, 567.
PlCTET, examination liquid, 645 ;

spermatological methods, ibid.
PIEESOL, embryology of Mammalia,

588.
Pigment spots, artificial, 77, 731,

732.

PINTNEB, Taeniae, 832.
PISENTI, alum-carmine, 153.
Plagiostoniida?, 834.
Plakina, larvae, 856.
Planaria, 48, 629.
Plasma-cells, 178, 788—794.
Plastic reconstruction of sections, 587.

502

INDEX.

The numbers refer to the Paragraphs, not to the Pages.

Plastin, 636.

Platinic mixture, MERKEL'S, 83 ; LIND-
SAY JOHNSON'S, 78.

Platino-aceto-osinic mixture, 23, 51,
637.

Platino-osrao-bichroumte mixture, 78.

Platinum chloride, 23, 42, 50, 78, 83,
85.

PLATNEB, Kernschwarz, 208; Neben-
kern, 639; medullated nerve, 52,
720.

'Platyhelmia, 48, 832—835.

PLESSIS, DU, see DU PLESSIS.

Plutus, 155, 848.

Podocoryne, 851.

PODWYSSOZKI, fixing mixture, 36;
safrauin, 101 ; carcinoma cor-
puscles, 101.

Poisoning methods, 18.

POLAILLON, impregnation method, 231.

POLI, serial sections, 335 ; imbedding
mass, 291.

POLITZER, inner ear, 687.

Polychseta, 841.

Polycladidea, 834.

POLZAM, imbedding mass, 289.

Ponceau, 98.

Porifera, 856.

Porpita, 854.

Potash, acetate, mounting medium,
374.

Potash solution for maceration, 529.

Potassium bichromate, 75 et seq.

Potassium hypochlorite, 556, 581, 598,
616.

Potassium permanganate, see Perman-
ganate of potash.

POUCHET, bleaching methods, 578,
580.

PRENANT, safranin staining, 101 ; sper-
matological methods, 645, cochlea,
686.

Preservative media, 2, 356 et seq.

Priapulus, 838.

Prickle-cells, 646.

Primrose, 135.

PKINGLE, the water section-fixing pro-
cess, 324; vacuum imbedding,
278 ; double stain, 254.

PRITCHARD, hardening fluid, 70; re-

ducing solution, 228 ; inner ear,
687.

Prosobranchiata, 813.

Protopterus, 742.

Protula, 841.

Protozoa, introductory, 857; killing,
7, 859; quieting, 857; staining
intra vitain, 858; cilia, 860; fla-
gella, 861; other methods, 857—
859.

PHTJDDEN, haematoxylin, 185.

Prussian blue, soluble, 476, 477; im-
pregnation with, 234 ; injection-
masses, see Injections.

Prussic acid, 18.

Pteropoda, 813.

Purpurin, 201, 776.

Putrefied carmine, 165.

Pyrenin, 636.

Pyridin for hardening and clearing,
90, 880.

Pyrogallate of iron stain, 232.

Pyroligneous acid, for hardening, 682.

Pyrosiu, 135.

Pyrosoma, 7, 307.

Q.

QUEKETT, preservative fluid, 371.
Quieting Infusoria, 857.
Quince-mucilage fixative, 331.
Quiuolcin, 93, 95, 126.

B.

Rabbit, embyrology, 588.

UABL, fixing methods, 32, 50; embyros
of Salamandra, 602 ; staining
method, 256 ; cytological methods,
637, 639.

RABL-RUCZHABD, embryology of
Teleostea, 609.

RAMON Y CAJAL, see CAJAL.

Rana, embryology, 603.

RANVIEE, alcohol, absolute, prepara-
tion of, 63; one-third ditto, 63;
itri'olar tissue, 786; bladder of
frog, 677; bone, 796; carmine,

INDEX.

503

The numbers refer to the Paragraphs, not to the Pages.

neutral, 164; cartilage, 45, 801;
cornea, 658 ; corpuscles of Golgi,
G71 ; decalcification, 563 ; epithe-
lium, 646; gland-cells, 809—811;
gold chloride impregnation, 225,
226 (and see Motor plates, Nerve-
fibres, &c.); hrematoxylin, 187;
injection masses, carmine, 466 ;
Prussian bine, 475, 476, 507;
silver nitrate, 496 ; glycerin, 505 ;
aqueous, 507 ; iodised serum, 360 ;
maceration, 360, 523 — 534; me-
dullated nerve, 720 ; motor plates,
667—668 ; mucus cells, 809 ;
nerve-fibres, intra-epidermic, 649 ;
neutral carmine, 164; nitrate of
silver impregnation, 212, 213, 668;
perruthenic acid, 809; picro-car-
mine, 159, 160 ; purpurin, 201 ;
quinoleiin, 126 ; retina, 682, 685 ,
tactile hairs, 647.

RANVIEB and VIGNAL, osmium mix-
ture, 29.

Rauvarienne, la, 107.

Reagents, how to procure, 94; selected
collections of, 863.

RECKLINGHATJSEN, VON, impregnation
methods, 210, 213, 214.

Reconstruction of objects from sections,
587.

REDDING, gold process, 229.

Redwood nerve stain, 694.

REHM, nerve stains, 725, 751, 752, 753.

REICH, silver nitrate, 213.

REICHENBACH, embryology of Antaeus,
625.

REINKE, staining horny tissues, 646.

REMAK'S copper hardening fluid, 82.

RENAUT, haeraatoxylin, 189 ; haema-
toxylic eosin, 252; cornea, 658.

RENSON, spermatological methods, 645.

Reptilia, embryology, 595 et seq. ;
myelon, 742, 784.

RESEQOTTI, coal tar stains, 98, 101, 107,
258 ; staining by Substitution, 98 ;
on the kinds of safranin, and on
safranin staining, 101.

Resin, mounting media, 417, 422, 423;
imbedding masses, 311 — 313;
cement, 444, 445.

Retina, 21, 682 et seq. / fixing, 53, 78,

682; staining, 117, 683, 715;

sections, 684; dissociation, 685.
RETTEBEB, smooth muscle, 681.
RETZIUS, methylen blue method, 117.
REZZONICO, medullated nerve, 704r

720.

Rhabdoccela, 834.
Rhizophysa, 854.
Rhopalea, 811.
RHUMBLEB, methyl green and eosin

vital stain, 261a.
Ribbon section-cutting, 281.
Ribesin, 209.
RICHABD, Bryozoa, 15.
RINDFLEISCH, maceration, 541.
Ringing wet mounts, 431, 432.
RIPABT and PETIT, preservative fluid,

64, 386.
Ripening haematoxylin solutions, 174—

176.
RITTEB, embryology of Chironomus,

620.

ROBEBT, fixing Aplysia, 813, note.
ROBEBTSON, imbedding mass, 308.
ROBIN, injection-masses, 458 — 465,

521 ; and see Injection-masses.
ROBINSKI, silver nitrate, 213.
ROBOZ, Zoi/rAN VON, alum-carmine,

155.

Rocellin, 95, 107.
Roche alum, 190, note.
Rocking microtome, 271, 867.
ROLLETT, carminroth, 167; freezing

process, 320 ; celloidin imbedding,

301 ; cornea, 533, 658, 659 ; sec-

tions of muscle, 661.
ROOSEVELT, pyrogallate of iron, 232.
Rose B. a Teau, 135.
Rose de naphthaline, 95, 107.
ROSE, preparation of bone, 796.
Rosein, 95, 107.
ROSENTHAL, hydrochloratc of chinolin,

377.
Rossi, stain for nervous centres, 698 ;

blood, 803.

ROSSLEB, Phalangida, 828.
Rotatoria, 93, 839.
Rouge fluorescent, 95, 107.
ROUGET, silver nitrate, 213, 214.

504

INDEX.

The numbers refer to the Paragraphs, not to the Pages.

ROUSSELET, permanent preservation of

Rotifers, 839.
Rubidin, 95, 107.
Rubin, 95, 98, 107, 259.
RUFFINI, corpuscles of Golgi, 673.
RUGE, imbedding mass, 314.
RTJNGE, imbedding mass, 314.
RUTHEEFOED, picro-carmine, 162 ;

hardening fluid, 735.
RYDEE, double imbedding, 306 ; sexual

gland of Lamellibranchiata, 645.

S.

SACHS, motor plates, 670 ; haematoxy-
lin nerve stain, 696.

SAEFFTIGEN, Echinorhyncus, 837.

Saffron for staining, 204.

Safranin, 95, 98, 100, 101, 775 ; HEN-
NEGUY'S method, 106; BABES',
638; FLEMMING'S new method,
258 ; for elastic tissue, 795 ; for
cartilage, 801.

Sargartia, 849.

Sagitta, 217.

SAHLI, balsam, 418; nerve-centres,
730, 731 ; stain for, 773.

SALA, Golgi's silver chromate impreg-
nation, 718.

Salamandra, embryology, 602; cyto-
logy of, 633, 637.

Salicylic vinegar, 391, 392.

Saliva, artificial, 531.

Salivary glands, 252, 528.

Salpa, 45, 811.

Salt solution, 359; for maceration,
526, 527.

SAMASSA, Golgi's silver chromate me-
thod, 709 ; sections of Ctenophora,
855.

Sandal-wood oil, 349.

SANFELICE, hajmatoxylin, 191.

SANKEY, staining nervous centres, 757.

Sapphirina, 822.

SAEABIN, embryology of Reptilia, 597.

Sarcolemma, 664, 827.

SAEGENT, injection method, 500;
bleaching, 577.

SATTLEE, silver staining, 214.

Siiurefuchsin, 95, 98, 131 ; for nerves,
722, 772, 773.

Sauregelb, 95, 139.

SAVILLE KENT, see KENT.

SAWTSCHENKO, carcinoma corpuscles,
101.

SAZEPIN, antennae, 824.

SCHAFEB, muscle-cells, 663 ; Weigert's
nerve stain, 693.

SCHAFFEB, cartilage, 801 ; retina, 683 ;
reconstructing sections, 587.

SCHALLIBAUM, serial sections, 325.

SCHENK, fixing fluid, 65; carbolic
fuchsin, 107.

SCHEBING'S celloidin, 297.

SCHEWIAKOFF, Protozoa, 859.

SCHIEFFEBDECKEB, cartilage, 801 ;
celloidin imbedding, 297—301 ;
double stains, 265 — 267 ; injection
masses, 513; levulose for mount-
ing, 402; methyl mixture, 548,
685 ; pancreatin digestion fluid,
553; serial sections, 324, 337; re-
tina, 548, 682, 685 ; skin and lym-
phatics, 688; gold staining ner-
vous centres, 767 ; palladium ditto,
ibid. ; maceration of ditto, 781 ;
medullated nerve, 720, 724 ; Mast-
zellen, 791 ; mucus cells, 808 ;
muscle cells, 663 ; the water sec-
tion-fixing process, 324.

SCHMATJS, uranium carmine, 167;
staining nervous tissue, 747, 757.

SCHMIDT, embryology of Limax, 615 ;
myelon of reptiles, 784.

SCHNEIDEE, aceto-carmine, 158.

SCHOLTZ, intra vitam staining, 93.

SCHOTTLANDEB, cytological methods,
643.

SCHULGIN, double stain, 243 ; embry-
ology of Amaraecium, 612; pa-
raffin muss, 287.

SCHULTZE, F. E., palladium chloride,
841, 234; embryology of Spongiae,
856; Pennatulidse, 849; section-
stretcher, 280; hardening skin,
646.

SCHULTZE, MAX, acetate of potash
mounting medium, 374 ; sulphuric

INDEX.

505

The numbers refer to the Paragraphs, not to the Pages.

acid macerating mixture, 546;
retina, 685 ; iodised serum, 360.

SCHUEMAYEB, Infusoria, 858.

SCHUTZ, Zacharias' carmine stain, 875.

SCHWALBE, smooth muscle, 676 ; coch-
lea, 686 ; impregnation, 210.

SCHWANN, sheath of, 719.

SCHWAEZE, Cercaria, 833.

SCHWEIGGEB-SEIDEL, carmine, 167.

Scorpions, eyes of, 579.

SCOTT and OSBOBN, embryology of
Triton, 600.

Sealing-wax varnish, 449.

SEAMAN, cement, 448; glycerin jelly,
411.

Sections, chain or ribbon, 281 ; col-
lodionisation, 282; serial mount-
ing, 323 et seq. ; section-stretch-
ing and section-stretchers, 280 ;
unrolling, 280 ; shrinkage in, 280 ;
reconstruction, 587.

SEHEWALD, Golgi's silver chromate
method, 708, 709, 711.

SEILEE, cleaning slides, 864; mount-
ing method, 355, 419; double stain,
241.

SELENKA, imbedding method, 273, 279,
314.

Sepia, eyes, 816.

Serial section mounting, 323 et seq.,
777, 778.

Serum, artificial, 361, 362, 524 ; iodised,
360, 361, 523, 524.

Shell, 311—313, 817, 819.

Shellac, cement, 448 ; imbedding mass,
310; fixative for sections, 327,
777 ; varnish, 448.

SHIMEE, mounting medium, 398.

Shrinkage in elements of sections, 280.

Siebdosen, 863.

Sieve-dishes, 863.

Silver chromate impregnation, 704 et
seq., and see GoLGi.

Silver nitrate, see Nitrate of silver.

Siphonophora, 11, 854.
Xipu.iculus, 838.

Skin, hardening-, 646 ; nerves of, 648.

Slides, cleaning, 864 ; labelling, 865.

SMITH, HOPEWELL, decalcifi cation and
sections of teeth, 565, 796, 797.

Soap, imbedding masses, 288 et seq. ;
833.

Soda, solution for maceration, 529.

Soda, hypochlorite, see Eau de Labar-
raque.

Soda-carmine, 167.

Solferino, 95, 107.

SOLGEE, sarcolemma, 664.

Solid green, 95, 105, 107.

SOLLAS, gelatin imbedding (freezing
method), 319.

Solution of EELICKI, of MULLEB, of
MEEZEL, &c., see the names of
the respective authors ; normal
salt, 359; macerating salt, 526,
527.

Solutions of metallic salts, how to keep,
25, 211.

SOMMEB, Macrotoma, 829.

SOUZA, DE, pyridin, 90.

SPEE, prepared paraffin, 286.

SPEK, VAN DEE, and UNNA, plasma-
cells, 792.

Spermatological methods, 645.

Spermatozoa, preparation, 645.

Sphyranura, 833.

Spicules of sponges, 856.

Spinal cord, 728, 743, 767, 880 et seq.

Spirographis, 841.

Spongise, 856.

Spongilla, 856.

SQUIEE, histological reagents, 94 ; oil
of origanum, or white thyme,
348; methyl green, 109; com-
mercial salts of gold, 221 ; glycerin
jelly, 413 ; damar medium, 420 ;
granule cells, 788 ; mounting me-
thyl-green stains, 879; decalcifi-
cation, 566, 572 ; Congo red for
nerves, 724 ; picro-carmine, 162 ;
haematoxylin, 188a.

STAHL, immobilisation of Infusoria,
858.

Staining, generalities, 91 — 94 ; choice
of stain, 94 ; on slide, 2 ; in toto,
4, 5; intra vitam, 93, 114, 115,
634,858; reagents, how to obtain,
94 ; staining by substitution, 98 j
directions for the Indirect Method,
96 — 100 ; staining nerve-tissue,

33

506

INDEX.

The numbers refer to the Paragraphs, not to the Pages.

see Nerves, Central Nervous Sys-
tem, Neurological Methods, &c.

Stains, General, Selective (Nuclear,
Plasmatic, and Histologically
Specific), defined, 91 ; aqueous
and alcoholic stains, 5; effect of
transparency and opacity of stains,
100 ; Generalities, 91 et seq., and
see Anilin black, Anilin blue, Car-
mine, Cochineal, Eosin, Gold
Hsematoxylin, Indulin, Iron, Or-
chella, Purpurin, Saffron, Saf ranin
Silver, &c.

Stains, combination, in general, 235 et
seq.; anilin Hue and carmine, 243 ;
borax-carmine and indigo, 241,
242; ditto and picro- carmine, 238;
carmine and anilin blue or bleu de
Lyon, 243 ; ditto and malachite
green, 244 ; ditto and methyl
green, 245; carmine and picric
acid, 236—240 ; carmine and
metallic stains, 155, 249 ; dahlia
and Saurefuchsin, 258; Ehrlich-
Biondi-Heidenhain mixture, 259,
639 ; eosin and anilin green, 265 ;
ditto and dahlia, 258, 266 ; ditto
and hffimatoxylin, 251, 252 ; ditto
and iodine green, 270; ditto and
methyl green, 260— 261a; ditto
and methyl violet, 267; ditto and
gentian violet, 258; fuchsin and
methylen blue, 268 ; gentian violet
and eosin, 258 ; ditto and safranin
and orange, ibid. ; gold chloride
and other stains, 229 ; hcematoxy-
lin and delta- or benzo-purpurin,
131, 251, 253; ditto and eosiii,
251, 252 ; ditto and iodine green,
255 ; ditto and nitrate of rosanilin,
255; ditto and picric acid, 236;
ditto and rubin and orange, 254 ;
ditto and safranin, 256; iodine
green and Bengal rose, 270 ; ditto
and hsematoxylin, 255; lithium-
carmine and picric acid, 240 ;
methyl-green combinations, 245,
259—263 ; methyl-violet and eo-
sin, 258, 267; picro -carmine, 159
et seq., 237; ditto and iodine green,

246, 247 ; ditto and methyl green,
246 ; ditto and borax-carmine,
238 ; ditto and other anilins, 247 ;
safranin and anilin blue, 270;
ditto and gentian and orange, 258 ;
ditto and hsematoxylin, 256 ; ditto
and gold chloride, 270 ; ditto and
indigo, 249 ; ditto and methy-
len blue, 774 ; ditto and nigrosin,
269; ditto and Saurefuchsin and
methylen blue, 773.

Starch injection mass, 518.

Stentor, 858.

STEPHENSON, mounting medium, 414.

Stei'naspidse, 841.

STIEDA, cements, 451 ; clearing media,
343.

STILLING, macerating brain, 781.

STILLING and PFITZNEB, stomach of
Triton, 680.

STIRLING, double stains, 246, 255;
sulphocyanides of ammonium and
potassium, 530 ; anilin blue-black,
757.

STOHE, double stain, 251.

Stomach of Triton, 643, 680.

Storax, 427.

Stoves and water-baths, 277.

STEAHL, embryology of Reptilia, 595.

STEASBTJRGEB, cytological methods,
643.

STEASSEE, plastic reconstruction, 587;
section-stretcher, 280; serial sec-
tions, 325, 326, 340.

STEICKEE, imbedding mass, 309.

Strychnin for killing, 18, 858.

Styrax, 427.

Sublimate, corrosive, see Corrosive sub-
limate.

Substitution of stains, 98, 102, 107.

SFCHANNEK, the water section- fixing
process, 324; bergamot oil, 347;
anilin oil for clearing, 353 ; Venice
turpentine, 423 ; Sieve-dishes, 863.

Sulphate of copper fluids, 44, 82 ; im-
pregnation, 234.

Sulphate of iron for fixing, 834.

Sulphindigotate of soda or potash, 202.

Sulphocyanides, 530.

Sulphuric acid for maceration, 546.

INDEX.

507

The numbers refer to the Paragraphs, not to the Pages.

Sulphurous acid, for Infusoria, 860;

for bleaching, 874.
SUMMERS, serial sections, 325, 337.
" Sunning " solutions of metallic salts,

25, 211.

STJSSDORF, mucus cells, 807.
SWAEN and MASQUELIN, spermato-

logical methods, 645.
Sycandra, 856.
Symp odium, 849.
Synapta, 845.
Syncoryne, 851.
Syrup, imbedding masses, 308, 316,

317 ; examination media, 365.

T.

Tactile corpuscles, 651 et seq.

Tactile hairs, 647.

Tcenia, 832 ; embryology of, 628.

TAENZEE, elastic tissue, 795.

TAFANI, inner ear, 687.

TAGUCHI, injection, 512.

TAIT, LAWSON, litmus stain, 209.

TAL, Golgi's mercury stain, 760.

Tauniu solution, 387; for Infusoria,
860.

TAETUFEEI, impregnation, corneal
cells, 659.

Tegumentary organs of Vertebrates,
646 et seq.

TBICHMANN, white injection, 492 j oil
mass, 519.

Teleostea, embryology of, 604 et seq.

Tendon, 671 et seq,

Tethya, 856.

THANHOFFEB, VON, silver staining,
214; sarcolemma, 827; brain-
tissue, 782.

Thenea, 217.

Thermostats, 277.

THIEESCH, carmine iujectiou, 469 ;
Prussian blue ditto, 479 ; lead
chromate ditto, 483 ; green ditto,
488 ; oxalic acid indigo-carmine,
203.

Thimbles, paper, to make, 273.

THIN, retina, 685.

Thionin, for plasma-cells, 794; for
inucin, 807.

Thiophen green, 141.

THOMA, microtome, 271 ; celloidin im-
bedding, 301 ; decalcification, 561 ;
egg imbedding mass, 314.

THOMPSON'S high refractive medium,
416.

THSELFALL, section-fixing method,
336.

THWAITES, preservative fluid, 369.

Thyme, oil of, 305.

Tiara, 851.

Tintinnodea, 859.

TIZZONI, alum-carmine, 153; cytolo-
gical methods, 643; medullated
nerves, 720 ; hardening skin, 646.

Tobacco-smoke killing method, 9.

TOISON, blood, 804.

Tolu balsam, mounting medium, 428 ;
cement, 450.

Toluidin blue, 123.

Toluol, 275, 283, 354 ; for preserving, 2.

Tongue of frog, 657.

TOENIER, hsematoxylin staining, 193.

TOTJBNEUX and HEEM ANN, silver stain-
ing, 213 ; epithelium, 646.

Trays, paper, to make, 273.

Trematodes, 833.

TEENKMANN, stains for flagella, 861.

Tricenophorus, 832.

Trichina?, 881.

TEINCHESE, nerve-endings in muscle,
667.

Triton, eye, 682 ; stomach, 643, 680 ;
ova, 600, 601.

Tropseolin, 95, 107, 139.

Trypsin digestion fluid, 554.

TEZEBINSKI, spinal cord, 732.

TSCHISCH, nerve-centres, 77.

TUBBY, celloidin imbedding, 298.

TULLBEBG, narcotisation with mag-
nesium chloride, 17.

Tunica ta, 811 ; embryology of, 611,
612.

Turbellaria, 834.

Turpentine, for clearing, 350; for
mounting:, 423 ; cement, 444.

U.

ULIANIN, embryology of Amphipoda,
626.

508

INDEX.

The numbers refer to the Paragraphs, not to the Pages.

UNDERWOOD, gold process, 228.

Unio, 118.

UNNA, bleaching chromic objects, 30 ;
ripening of hsematoxylin solutions,
174, 175, 176 ; instanteously ripe
hsematoxylin stain, 175 ; half-ripe
stock solution, 176; plasma-cells
and Mastzellen, 792; elastic tissue,
795.

UPSON, gold and iron method, 770;
gold and vanadium method, 771 ;
other stains, 748.

Uranium, acetate, 28, 65.

Uranium, nitrate, 78.

Uranium - carmine, GIERKE and
SCHMAUS, 167.

Urodela, embryology of, 598—602.

USKOFF, nitric acid in cytology, 643.

Ussow, embryology of Cephalopoda,
613.

V.

Vacuum imbedding, 278.

VALETTE, ST. GEORGE v. LA, sperrna-

tological methods, 634.
VAN BENEDEN, see BENEDEN, VAN.
VAN GIBSON, clearing with anilin oil,

305.

VAN HETJRCK, mounting medium, 415.
Vanadateof ammonium, 772 ; injection

mass, 511.

Vanadium, perchloride, 683, 726.
Varnish, 430 et seq. ; amber, 446, 447 ;
asphalt, 436; Brunswick black,
437; copal, 424, 447; negative,
426; sealing-wax, 449; shellac,
448.

VASALE, hsematoxylin nerve stain, 695.
Vegetable wax, 287.
VEJAS, staining nervous centres, 757.
Velella, 854.
Venice turpentine, for mounting, 423 ;

cement, 444.
Veretillum, 8.
Veridin, 109.
Vermes, 832—844; embryology, 628

et seq.
Vert d'alcali, 109.

Vert d'Eusebe, 109.
Vert en cristaux, 109.
Vert lumiere, 109.
VERWORN, narcotisation, 14.
Vesuvin, 95, 98, 110.
VIALLANES, fixation of Insecta, 28;
gold method, 227 ; celloidin im-
bedding, 301 ; brain of Arthro-
pods, 825; osmic acid mixture,
28.

VIALLETON, embryology of Aves, 594.
'Victoria blue, 95, 98, 104, 795.
VIGNAL and RANVIER, osmium mix-
ture, 28; picro-carmine, 160.
VILLE, carmine injection, 467.
Violet B., 95, 129, 786.
VIRCHOW, action of light on chromic

objects, 30.
VIVANTE, bone, 799.
VOGT and YUNG, Cucumaria, 845;
Rotifers, 839; Sipunculus, 838 j
cleansing intestine of Lumbricus,
840.
VON EBNER, decalcification mixture,

563.
VON LENHOSSEK, nerves of Annelids,

843 ; myelon of reptiles, 784.
VON MAHRENTHAL, osmium impreg-
nation, 233.

VOSMAER, epithelium of sponges, 217.

VOSSELER, Mayer's albumen fixative,

328; Venice turpentine, 423; wax

feet, 522; cardboard ridges for

slides, 865.

W.

WADDINGTON, arabin, 329 ; Infusoria,
860.

Wagnerella, 574.

WALDEYER, inner ear, 687; plasma-
cells, 788 ; decalcification, 559.

Walnut-juice stain, 209.

WARD, narcotisation methods, 19;
Sipunculus, 838.

Washing out, 2, 22, 24.

Washing osmic acid objects, 29.

WATASE, embryology of Cephalopoda,
613.

Water, fresh or warm, for killing, 20.

INDEX.

509

The numbers refer to the Paragraphs, not to the Pages.

Water-baths, 277.

WATNEY, dichroism of hsematoxylin
stains, 178.

Wax imbedding masses, 287.

Wax feet, 522.

WEBB, dextrin freezing mass, 318.

WEBSTER, naphtha for paraffin im-
bedding, 275.

WEBEE, Rotifers, 839.

WEDL, orchella stain, 206.

WEIGERT, Bismarck brown, 110;
picro-carmine, 161 ; haematoxylin
nerve methods, 688—703, 755
(the 1885 method, 688; the 1891
method, 703; variations, 689—
702, 755) ; ditto for retina, 683 ;
clearing celloidin sections, 305,
703 ; mounting serial sections, 305,
340, 426, 703 ; varnish for mount-
ing without cover, 426 ; hardening
nerve-centres, 730 ; Sauref uchsin
for ditto, 772.

WEIL, section method, 313 ; bone, 796.

WHEELER, embryology of Blattida, 619.

WHITE, sections of bone or dental
tissue, 796.

White lead cement, 453.

White of egg, section-fixing process,
328 ; imbedding methods, 314,
321 ; injection-mass, 498.

White zinc cement, 451.

WHITMAN, fixing mixture, 83 ; ova of
Amphibia, 598 ; pelagic ova, 610 ;
Hirudinea, 844.

WICKERSHEIMER, preservative fluid,
390.

WILL, Aphides, 621.

WILSON, Alcyonaria, 849 ; embryology
of Lumbricus, 630.

WISSOWSKY, blood, 804.

WITT, shellac, 448.

WOLFF, motor plates, 670 ; bladder of
frog, 677.

WOLTERS, haematoxylin nerve stains,
699, 700, 726; cartilage, 801;
skin nerves, 648; elastic tissue,
795.

WOODWARD, borax-carmine, 167.

WRIGHT, Sphyranura, 833.

Xylol, for clearing, paraffin sections,
354 ; ditto celloidin sections, 354 ;
for half-clearing brain sections,
779 ; for paraffin imbedding, 275 ;
for preserving tissues, 2.

Z.

ZACHARIAS, acetic alcohol, 54; acetic
carmine, 158, 875; ovum of As-
caris, 644.

ZENTHOEFER, elastic tissue, 795.

ZIEGLER, white cement, 452.

ZIEHEN, gold and 'mercury impregna-
tion method, 769.

ZIEHL'S carbolic fuchsin, 107.

ZIMMERMANN, microtomes, 271.

ZIMMERMANN Sieve-dishes, 863.

Zinc chloride, for hardening brain, 780 ;
impregnation method, 762.

Zinc white cement, 451.

Zoantharia, 849.

ZOJA, methylen blue, 114 ; bioblasts of
Altmann, 641 ; Hydra, 850.

ZOLTAN VON ROBOZ, alum-carmine,
155.

Zoobothrium, 812.

ZSCHOZKE, deltapurpurin, benzopur-
purin, 133, 253.

/iUPPLNGER, ganglion cells, 756.

ZWAARDEMAKER, safranin stain, 101 ;
cytological method, 643.

PRINTED BY ADLARD AND SON,
BARTHOLOMEW CLOSE, B.C., AND 2O, HANOVER SQUARE, W.

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