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THE

MICROTOMISTS VADE-MECUM

First Epirion F Marcu, 1885.

Smconp Do. ; i Aprit, 1890.
THIRD Do. , : SEPTEMBER, 1893.
Fourtu Do. SEPTEMBER, 1896.
Firru Do. Jung, 1900.
SrxTH Do. ; Jung, 1905.

SeventH Do. Aveusr, 1913.

THE

MICROTOMIST’S VADE-MECUM

A HANDBOOK OF THE METHODS OF
MICROSCOPIC ANATOMY

BY

ARTHUR BOLLES LEK

SEVENTH EDITION

PHILADELPHIA
P. BLAKISTON’S SON & CO.
1012 WALNUT STREET
1918

PRINTED IN ENGLAND,

PREFACE.

AN important: improvement in histological technique, made
known since the publication of the last Edition, has been
effected by Giison’s new mounting media. For these media—
camsal balsam and euparal—afford a ready and safe means of
mounting direct from alcohol, without the intervention of
essential oils or other clearing agents, which are often contra-
indicated; and of conferring on unstained or insufficiently
stained elements just the required degree of visibility, thus
enabling us to see details which are invisible in the usual
mounts.

Some important improvements have also been made in the
silver fibril stains of Bretscoowsky and Ramon y CasaL, which
have now become less capricious methods for the study of
neurofibrils, and valuable aids to the study of other objects.
T have given these methods at length, abstracting the whole of
Raudén’s methods from the latest original source. The sec-
tions relating to neurofibrils are thus almost entirely re-written,
and so are those relating to blood and blood-parasites.

If these are the only novelties of much importance that
have offered themselves, yet I have found a large amount of
less important matter that it has seemed desirable to include
(the Index shows more than 700 new entries). I have been
able to find room for this, without increasing the size of the
book, partly by striking out some superfluous matter (mostly
of merely theoretical interest), and partly by rigorous con-
densation of the text and not a little typographical compres-
sion. To my satisfaction I find that this condensation and
compression is not to be regretted, for the text has in many

vi PREFACE.

places thereby become easier to understand, and information
desired at any time will be more readily found than before.

I have been careful not to carry the typographical com-
pression so far as to make the text trying to the eyes.

Bauey, suR CLARENS, SWITZERLAND;
August, 1913,

CONTENTS.

PART I.
PAGE
CHAPTER I.
INTRODUCTORY 1
CHAPTER II.
KILLING ; : i » 1
CHAPTER III.
FIxIneg AND HARDENING 19
CHAPTER IV.
FIxine AND HaRDENING AGENTS; MINERAL ACIDS aND THEIR
SaLts : ‘ 28
CHAPTER V.
Fixine AND HARDENING AGENTS; CHLORIDES, ORGANIC ACIDS
AND OTHERS F ‘ : : : . 45
CHAPTER VI.
DE-ALCOHOLISATION AND CLEARING AGENTS ‘ . 68
CHAPTER VIL.
ImpeppIne MrtHops—INTRODUCTION 75

CHAPTER VIII.

IMBEDDING METHODS: PARAFFIN AND OTHER FUSION Masses. 81
Paraffin, 81; Gelatin, 100.

vill CONTENTS.

PAGE
CHAPTER IX.

COLLODION (CELLOIDIN) AND OTHER IMBEDDING Mrtuops Lue
Collodion or Celloidin, 102; other Cold Masses, 115; Grind-
ing Masses, 115; Freezing, 117.

CHAPTER X.
SERIAL SEecTIon Mounting 119
Methods for Paraffin Sections, 119; Methods for Watery
Sections, 125; Methods for Celloidin Sections, 125.

CHAPTER XI.
STAINING 129

CHAPTER XII.

CARMINE AND COCHINEAL STAINS. 141
Theory of Carmine Staining, 141; Aqueous Carmines, Acid,
142; Neutral and Alkaline, 146; Alcoholic Carmines and
Cochineals, 118.

CHAPTER XIII.

Hamatnin (H@MATOXYLIN) STAINS 152
Theory of Staining with Hematoxylin, 152; Iron-hematein
Lakes, 155 ; Aluminium-hematein Lakes, 159; other Hzema-
tein Compounds, 165.

CHAPTER XIV.

NvucLEAR STAINS WITH COAL-TAR DyzEs 169
Progressive Stains, 170; Regressive Stains, 173.

CHAPTER XV.

PLASMA STAINS WITH COAL-TAR DyEs dye

CHAPTER XVI.

MetHyLen BLUE . 199

CHAPTER XVII.

Merauiie Srains (IMPREGNATION Meruops) 219
Silver, 21-4; Gold, 218; other Metallic Stains, 220.

CONTENTS. 1x

PAGE

CHAPTER, XVIII.

OTHER STAINS AND COMBINATIONS : , 228
Other Organic Stains, 228; Carmine Caureseene: 229 ;
Hematein Combinations, 231.

CHAPTER XIX.

EXAMINATION AND PRESERVATION MEDIA ‘ 234.
Aqueous Liquids, 235; Mercurial Liquids, 239 ; ther Fluids,
239; Glycerin Media, 241; Jellies, 242; High Refractive
Liquids, 243; Resinous Media, 244.

CHAPTER XX.

CEMENTS AND VARNISHES . ; ‘ 249

PART II.
SPECIAL METHODS AND EXAMPLES.

CHAPTER XXI.
INJECTIONS: GELATIN Masses (WARM) . , 257
Carmine, 259; Blue, 261; other Colours, 262.
CHAPTER XXII.

INJECTIONS: OTHER MASSES (COLD) . 264

CHAPTER XXIII.

MacERATION, DIGESTION, AND CORROSION F . 270
Maceration, 270; Digestion, 276; Corrosion, 277.

CHAPTER XXIV.
DECALCIFICATION, DESILICIFICATION, AND BLEACHING . 279
Decalcification, 279; Desilicification, 283; Bleaching, 284.
CHAPTER XXV.

EmBRYOLOGICAL MerHops . 287
Mammalia, 291; Aves, 295; Reptilia, 299 ; sketegitiey 300;
Pisces, 303; Tunicata, 306; Bryozoa, 306; Mollusca, 307;
Arthropoda, 309; Vermes, 314.

CHAPTER XXVI.

CytToLtogicaL METHODS : ; : ‘ j . 317

x CONTENTS.
CHAPTER XXVII.
TEGUMENTARY ORGANS

CHAPTER XXVIII.

MuscLe anp Tenpon (NERVE-ENDINGS) .

Striated Muscle, 337; Blectric Organs, 339; Tendon, 340;

Smooth Muscle, 341.

CHAPTER XXIX.

Connective Tissue, 344; Elastic, 346; Plasma Cells, 349;

Fat, 351; Bone and Cartilage, 353.

CHAPTER XXX.
BLoop AND GLANDS .
Blood, 359; Glands, 368.

CHAPTER XXXI.

NeERvovus System: GENERAL METHODS

CHAPTER XXXII.

Nervotus System: CyrrotocicaL MrtHops
Cells, 386 ; Cells and Fibres, 389; Medullary Sheath, 404,

CHAPTER XXXIII.

Mye.in Starns (WEIGERT AND OTHERS) .

CHAPTER XXXIV.

Axis-CYLINDER AND DENDRITE STAINS (GOLGI AND OTHERS) .

CHAPTER XXXvV.

NEUROGLIA AND SENSE ORGANS
Neuroglia, 438; Retina, 442; Inner Ear, 445,

CHAPTER XXXVI.

MerrnHops ror INVERTEBRATES
Tunicata, 448; Molluscoida, 449; Mollusca, 449 ; ere

454; Vermes, 458; Echinodermata, 467 ; Cudlentersts, 470;

Porifera, 474; Protozoa, 475.

APPENDIX .

PAGE

331

337

359

386

407

419

448

484

THE MICROTOMIST’S VADE-MECUM.,

CHAPTER I.
INTRODUCTORY.

1, The General Method.—The methods of modern microscopic
anatomy may be roughly classed as General and Special.
There is a General or Normal method which consists in care-
fully fietng 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; dissociation by teasing
or maceration ; injection ; impregnation ; and the like.

There is a further distinction which may be made, and
which may help to simplify matters. The processes of the
preparation of tissues may be divided into two stages, Pre-
liminary Preparation and Ulterior Preparation. Now the
processes of preliminary preparation are essentially identical
in all the methods, essential divergences being only found in
the details of ulterior preparation. By preliminary prepara-
tion is meant that group of processes whose object it is to
get the tissues into a fit state for passing unharmed through
all the ulterior processes to which it may be desired to submit
them. It comprehends the operations of (1) killing; (2)
fixing; (3) the washing and other manipulations necessary
for removing the fixing agent from the tissues, and substi-

1

2 CHAPTER 1.

tuting for it the preservative liquid or other reagents which
it is desired to employ. Ulterior preparation comprehends
the processes sketched out in Q¥ 3 ef seq.

2, Preliminary Preparation. —The first thing to be done with
any structure is to fix its histological elements. (‘his 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 re-
agents with which it may subsequently be treated. Without
good fixation it is impossible to get good stains or good
sections, or preparations good im any way.

The structure having been duly fixed by one of the pro-
cesses described in the chapter on Fixing Agents, is, except
in special cases, 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 indifference. 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 certain other
agents, such as picric acid, have been used, the washing should 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) enter into a state of chemical combination with the elements of
tissues, rendering them insoluble in water; so that the hardeniny
induced by these agents is not removed by subsequent treatment with
water. Picric acid, on the other hand, produces ouly a very slight
hardening of the tissues, so that the tissue elements are left in a state
in which they are obnoxious to all the hurtful effects of water. Alechol
should therefore le taken to remove the picric acid and to effect the
necessary hardening at the same time.

These operations having been duly performed, two roads
become open. The object may be further prepared by what
may be termed the wet method, in which all subsequent
operations are performed by means of aqueous media. Or it
may be further prepared by the dehydration method, which
consists in treatment with successive alcohols of gradually

INTRODUCTORY. 3

increasing strength, final dehydration with absolute alcohol,
imbibition with an essential oil or other so called clearing
ayent which serves to remove the alcohol, and lastly either
mounting at once in balsam or other resinous medium or
imbedding in’ paraffin for the purpose of making sections.
The dehydration method is the course which is generally
preferred, chiefly because of its great superiority as regards
the preservation of tissues. For the presence of water is
the most important factor in the conditions that bring about
the decomposition of organic matter, and its complete removal
is the chief condition of permanent preservation.

3. Dehydration.—The further course of preparation by the
dehydration method is as follows :—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: first, in the
interest of. preservation, as above explained ; and secondly,
because all water must be removed in order to allow the
tissues to be impregnated with the imbedding material
necessary for section-cutting, or with the balsam with which
they are to be finally preserved. This dehydration is per-
formed as follows:—The objects are brought into weak
alcohol, and are then passed through successive alcohols of
gradually increased 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 delicate objects, it may be necessary to take 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 may conveniently be used in these
cases. The objects may be placed with some of their liquid in a tube
corked at one end and closed at the other by a diaphragm of muslin or
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, Cons’s differentiator (Proc. Linn. Soc., N.S.W., v, 1890,
p. 157; Journ. Roy. Mie. Soc., 1890, p. 821) may be employed. Or, the
apparatus of Hasweui (Proc. Linn. Soc., N.S.W., vi, 1891, p. 433;
Journ, Roy. Mic. Soc., 1892, p. 696). Or that of CHEaTLE, described in
Journ. Pathol. and Bacteriol., i, 1892, p. 253, or Journ. Roy. Mic. Soc.,

A CHAPTER 1.

1892, p. 892. See also ScHuLrzE (Zeit. wiss. Mik., ii, 1885, p. 537) ; and
Susuxy, ¢bid., 1909, p. 211; Koster (cbid., xvii, 1900, p. 29-4).

The “ Siebdosen,” or sieve-dishes of STEINACH, ZIMMERMANN, and
SucHANNEK (vide Zeit. wiss. Mik., iv, 1887, p. 433, and vii, 1890, p. 158),
ave useful for many purposes. ‘They are sent out in a very neat form
by Griibler and Co. See also TiscHATKIN, ébid., xxiii, p.45. FAIRCHILD §
perforated porcelain cylinders for washing (bid., xii, 1896, p. 301) seem
to be a very neat idea. See also the similar device of SCHAFFER (ibid.,
xvi, 1900, p. 422; Journ. Roy. Mic. Soc., 1900, p. 894). For EwaLps
section-washing apparatus, see Zeit. Biol., xxxiv, 1897, p. 26+.

That of ScHouBeEn (cbéd., xx, 1903, p. 168) is simple and efficient ; as
also that of KrirGBaum (ibid., xxvii, 1910, p. 504).

A capillary siphon for the aspiration of liquids in the fixing, staining.
and washing of suspended blood-corpuscles, sperm-cells, protozoa, and
the like, is described by Ewaxp, cbid., p. 253.

It is sometimes stated that it is necessary that the last
alcohol-bath should consist of absolute alcohol. ‘This, how-
ever, is incorrect, a strength of 95 per cent. being sufficient
in most 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.

I am not aware of any substance that can entirely take
the place of alcohol for dehydration and preservation. Ace-
tone and methylal have been substituted for alcohol in the
dehydration of methylen-blue preparations (Parker, Zool.
Anz., 403, 1892, p. 376), and aunilin oil can be made to
dehydrate watery sections if they be first mopped up with
blotting-paper ; but a really efficient substitute for alcohol
in general work remains yet to be discovered.

4. Preservation—Considered as a mere dehydrating agent,
alcohol fulfils its functions fairly well. But considered as a
lnstological preservative agent, it is far less satisfactory. If
tissues be left in alcohol for only a few days before further
preparation, injurious effects will perhaps not be very dis-
agreeably evident. But it is otherwise if they are put away
in it for many weeks or months before the final preparation
is carried out. The dehydrating action of the alcohol being
continuously prolonged, the minute structure of tissues is

INTRODUCTORY. 5
sometimes considerably altered by it ; they become over-hard
and shrink, and become brittle, and their capacity for taking
stains well becomes seriously diminished. Kutrscurrzxy
(Zeit. wiss. Mik., iv, 1887, p. 349) has proposed to remedy
this by putting up objects after fixation and washing out
with alcohol in ether, xylol, or toluol. Fiemine (Arch.
mik, Anat., xxxvii, 1891, p. 685) advises 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 prepared for sectioning by
treatment with pure alcohol or softened for dissection 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 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.).

For material that is intended only for section-cutting, T
find that by far the best plan is to clear (next §) and imbed
at once in paraffin. ‘This affords, as far as I can see, an
absolutely perfect preservation. Cedar-wood oil is, I find,
nearly, if not quite, as good as paraffin, so far as the preser-
vation of the tissues is concerned, but of course it is not .so

handy for storage.

5. Removal of Alcohol; Clearing—The water having been
sufficiently removed, as described in § 3, 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 or mounting.
This operation is generally known as Clearing. It is very
important that the passage from the last alcohol to the clear-
ing agent be made gradual. This is effected by placing the
clearing medium wider 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 sufl-
cient quantity of clearing 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

6 CHAPTER T.

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 after some further lapse of time the objects will be
found to be completely penetrated by the clearing medium.

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 glycerin.

This is a convenient stage for carrying out minute
dissections, if any such have to be done, a drop of clearing
agent being a most helpful medium for carrying out such
dissections (see § 9).

At this point the course of treatment follows one of two
different roads, according as the object is to be mounted
direct in balsam (§ 8), or is first to be sectioned (§ 6).

6. Imbedding, and Treatment of Sections.—The ebjects are
now imbedded. They are removed from the clearing medium,
and soaked until thoroughly saturated in the imbedding
medium. This is, for small objects, generally paraffin,
liquefied by heat, and for large objects either paraffin or a
solution of collodion or “celloidin” (in this latter case the
clearing may be omitted and the tissues be imbedded direct
from the alcohol). The imbedding medium containing the
object is then made to solidify, and sections are made with a
microtome through the imbedding mass and the included
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 ¢n situ on the shde,
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,

Or, the material may be stained in bulk, before cutting the
sections. In this case the object, after having heen ficed
and washed out, is taken from the water, or while still on its
way through the lower alcohols (it should not be allowed to

INTRODUCTORY. 7

proceed to the higher grades of alcohol before staining, if
that can be avoided), and passed through a bath of stain,
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 chloroform, xylol, or other solvent with which the
paraffin is removed. If aqueous staining media be applied
(and this is sometimes desirable), the structures should either
be stained in toto immediately after fixing and washing out, or
sections may be stained on the slide, the objects, if delicate,
being passed through successive baths of alcohol of gradually
decreasing strength before being put into the aqueous stain.

In my opinion it 1s generally advisable not to stain in
bulk material that is intended to be sectioned; by staining
it as sections the staining can be much better controlled, and
many excellent stains can in this way be employed that are
not available for staining in bulk; and of course sections can
be stained much more rapidly than material in bulk.

Balsam mounts of which the stain has faded, or which it
may be desired to submit to some other staining process, or
mount in some other medium, may often with great advan-
tage be re-strained and re-mounted. All that is necessary is
to put the slide into a tube of xylol or benzol till the cover
falls off (about two days), wash well for some hours in clean
xylol, and pass through alcohol into the new stain. Since
this was pointed out to me by Dr. Henneguy I have
unmounted and re-stained a large number of old prepara-
tions, and have sueceeded in every case with series of
sections mounted on Mayer’s albumen, or by the water
method. For shellac-mounted series, see EH. Mryzr, Biol.
Centralb., x, 1890, p. 509, or last edition.

The most convenient vessels, I find, in which to perform the various
operations of staining, differentiating, dehydrating, clearing, etc., on the
slide, are flat-bottomed corked glass tubes. I have mine made 10 centi-

metres high and 27 millimetres internal diameter. Each of these will
then take two slides, English size, placed back to hack.

7. Resumé of the General Method—To sum up, you may
either fix, wash out, stain, wash, dehydrate, clear, imbed, cut
sections, clear and mount them in balsam; or fix, wash,
dehydrate, clear, imbed, cut, stain, wash, dehydrate, clear,
and mount—according to choice.

8 CHAPTER I.

8. Preparation of Entire Objects, or of Material that is not to
be sectioned.—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.

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 described ($ 5). Fluids of high diffusibility should be
employed as far as possible in all the processes. Fixing
agents of great penetrating power (such as picric acid or
alcoholic sublimate solution) should be employed where the
objects present a not easily permeable integument. Wash-
ing 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 recom-
mended are Grenacher’s borax-carmine, or one of Mayer’s
alcoholic carminic acid or hamatein stains. Aqueous stains
are more rarely indicated, though there are many cases in
which they are adiniasible, aoa some in which they are
preferable.

9. Minute Dissections—These are best done, if necessary, in a
drop of clearing agent. I recommend cedar-wood oil for
this purpose, as it gives to the tissues a consistency very
favourable for Aiswwotion, 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,

INTRODUCTORY. 9

The brittleness is, however, sometimes very helpful in
minute dissections. Another property of clove oil is that it
does not easily spread itself over the surface of a slide, but
has a tendency to form very convex drops, and this also
makes it frequently a very convenient medium for making
minute dissections in,

If it be desired to dissect in a watery fluid, such as
elycerin, it may be well to prepare the slide by spreading on
it a thin layer of Maynr’s albumen, and on this place a
small drop of gtycerin, or other dissecting medium. As soon
as the dissection has been accomplished, a cover may be let
fall, horizontally, on to the preparation to keep the parts
in place, and a weight placed on it. Then the mount may
be filled up with glycerin, or other mounting medium, run in
under the cover, and closed, if desired, or instead of the
albumen a solution of gelatin may be taken, and hardened
in formol with the objects on it. For a balsam mount, after
clove or cedar oil, ScHAutiBAum’s collodion may be taken,
and the organs fixed in situ on this by adding xylol.

10. Instruments.—I or all that concerns the mechanism and
manipulation of the Microscope, see vol. i of Carrenrrr’s The
Microscope, eighth edition, 1891; paying particular attention
to all that is said concerning the English and the Continental
Models, pp. 254 to 261, the Substage, pp. 184 to 189, Con-
densers, pp. 289 to 316, and Tube Length, pp. 158 to 159.

For information concerning the principles of construction
and manipulation of the Mierotome, see also CarpEnrer’s
The Microscope. Microtomes are instruments for the
accurate production of thin slices of tissues. They are used
both for cutting tissues that have acquired a certain favour-
able consistency through having been tubedded in paraffin,
and also for cutting tissues that have been imbedded
in softer masses, such as collodion, and tissues that have not
been imbedded at all. Not all microtomes are equally well
adapted for all these three classes of work. The microtome
of the zoologist should at all events be one that is well
adapted for cutting imbedded material.

Now there are two methods of imbedding in general use
—the paraffin method and the celloidin method. In the
paraffin method the object is cut dry, frequently with the

10 CHAPTER 1.

knife set syware to the line of section. In the celloidin
method, as in the cutting of unimbedded tissues, it is
generally cut wet, and always with the knife set slanting.
Some microtomes that are well adapted for the paraffin
method are ill adapted for the celloidin method or the
cutting of unimbedded material, and vice versd. It may be
well to possess the two sorts of instrument; but if only one
can be afforded it should be such as will give good work in
either way.

Microtomes fall further into two classes according as
the knife and the surface of section of the ohject arc (A)
in a horizontal plane, or (s) in a vertical plane. The
former offer greater facility for the orientation of the plane
of section, which is an important point for the zoologist and
embryologist. Amongst these may be mentioned (a) The
“Sliding” Microtomes, in which the knife is carried on a
sledge and moved against the object (those of THoma,
Scuanze, Rercurrt, and others). The ‘soma, of medium
size, as made by R. Jung, Hebelstrasse, Heidelberg (No. 56
of his catalogue for 1911, which may be obtained from Mr.
C. Baker, 244, High Holborn, London), is very suitable for
the zoologist. It works equally well with either paraffin or
celloidin, and can be adapted as a freezing microtome. But
this (as is the case with the others mentioned) will not
always furnish work of the highest accuracy; for the knife
being only clamped at one end is liable to spring, and to give
sections of unequal thickness. This defect is remedied in
(b), a type of sliding mierotomes in which the knife is
clamped at both ends and is a fixture, the object being carried
on a sledge and moved against it (CAMBRIDGE NcIeNTIFIC
Instrument Company’s Jarge microtome, the Minor precision
microtome, Luirz’s, pe Groov’s, Juna’s “'Tetrander.” This
last seems to he near perfection ; see the description by
Mayer in Zett, wiss, Mik., xxvii, 1910, p. 52; but is more
cumbrous than is desirable for ordinary work.

Class a also includes some instruments in which the knife
is carried on a horizontal arm and swung against the object
by a rotary movement (June, Roy, Fromme, Reiceerr,
Tarn, and others). I know nothing of these personally, but
doubt their constant accuracy.

Class Bo contains some very fine instruments, admirably

INTRODUCTORY. Ae

adapted for the production of continuous ribbons of sections
by the paraffin method, but not so well adapted for celloidin
or other work in the wet way, or for soft objects. Amongst
these are the Rocking Microtome, made by Tue Screnriric
Instrument Co., Cambridge, or by Swirt & Son, or by Juna,
or by van per Srap, Amsterdam; the Mryor, made by
Bausca & Lome, or by Becker (Gottingen), or by Zimmer-
mann (21, HEmilienstrasse, Leipzig); the Remnonp-Givray,
made by J. W. Gizray, Delft.

For descriptions of the multitudinous models on the
market see the reports in the Zezt. wiss. Mik. and Journ. Roy.
Micr. Soc., and the price lists of the instrument makers.

11, Staining Reagents and Chemicals.—You are not likely
to succeed in staining, especially in 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 coal-tar colours, 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, s. v. Safra-
nin). I therefore advise the reader to get his reagents—at
all events his dyes—from the well-known chemists Guisier
& Hotizorn, who have specialised in the matter. They may
be ordered froin the price list, or by quoting the numbers of
the formule in this work. The address is: G. Gripier &
Hottzorn, Chemiker, Leipzig, Germany. Their preparations
can be obtained in London from Mr. Cuariys Baxer, 244,
High Holborn, W.C., or H. F. Ancus & Co., 838, Wigmore
Street, Cavendish Square. (Both of these firms also supply
the microtomes and accessory apparatus of the best makers,
bacteriological apparatus, etc.) Griibler & Hollborn’s pre-
parations can be obtained in the States from Himmr &
Amend, 205-211, Third Avenue, New York; Paun Weiss,
Optician, 1620, Arapahoe Street, Denver, Colorado ; and
Epwarp P. Dorery & Co., 3613, Woodland Avenue, Phila-
delphia.

CHAPTER JI.
KILLING.

12. In the majority of cases, the first step in the prepara-
tion 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 sufficient 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, which if thus treated
contract violently, and die in a state of contraction that
renders them unfit for study. In these cases special methods
of killmg must be resorted to. Speakine generally, there
ave 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.

Sudden Killing.

13. Heat.—The application of Heat affords a means of
killing suddenly. By it the tissues are more or less fixed at
the same time that somatic death is brought about.

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

Small ehjects (Protozoa, Hydroids, Bryozoa) may be brought inte a

KILLING. 13

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 coagu-
lated 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.

14. Slowly Contracting Animals.—Animals that contract
but slowly, such as Aleyoniwm and Veretillum, and some
Tunicates, such as Pyrosoma, are very well killed by throw-
ing them into some very quickly acting fixing liquid, either
used hot or cold. Glacial or very strong acetic acid (VAN
Brneprn’s method) is an excellent reagent for this purpose ;
it may be used, for example, with some Meduse. 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. Lemon juice employed in
this way has given me very good results with small Annelids
and Hirudinea. Corrosive sublimate is another excellent
reagent for this purpose.

Narcotisation.

15. Narcotisation is performed by adding some anesthetic
substance very gradually, in very small doses, to the water
containing the animals, aud waiting patiently for it to take
effect slowly.

The Tobacco-smoke Method for Actiniw, of Lo Branco (Jenu
Zeit. Naturw., Bd. xiii, 1879, p. 467; Mitth. Zool. Stat. Neapel, Bd. ix,
1890, p. 499), is:—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
overnight. As soon as it is observed that the contraction of u tentacle
does not begin until a considerable time after it has been irritated by
a needle, a quantity of some fixing liquid sufficient to kill the animals
before they have time to contract is added to the water.

16. Nicotin in solution may be used (Anpius, Atty R. Accad.
det Lincei, v, 1880, p. 9). Andres employs a solution of

14 CHAPTER If.

1 gramme of nicotin in a litre of sea water. ‘Che animal is
placed in a jar containing half a litre of sea water, and the
solution of nicotin is eradually conducted into it by means
of a thread, acting as a syphon, of such a thickness as to be
capable of carrying over the whole of the solution of nicotin
in twenty-four hours. See also Mitth. Zool. Stat. Neapel,
Bd. ui, 1880, p. 123.

17. Chloroform may be employed either in the liquid state
or in the state of vapour. ‘The animals being extended,
watch-glass containing chloroform may be floated on the
surface of the water in which they are contained, and the
whole covered with a bell-glass. As soon as they have
become insensible they are killed by means of hot sublimate
or chromic acid solution plentifully poured on to them.
(Kororverr, Mitth. Zool. Stat. Neapel, v, 1884, p. 233).

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 anzsthetised.

I have seen large Medusz very completely anwsthetised
in extension in an hour or two by this method. ANbrEs
finds that it does not succeed with Actiniew, as with them
maceration of the tissues supervenes before anesthesia is
established.

Preyer (Mitt. Zool. Stat. Neapel, Bd. vii, 1886, p. 27)
recommends chloroform water for star-fishes.

Wappineron employs a mixture of equal parts of 1 per
cent. sol. of cocaine (or eucain) and saturated sol. of chloro-
form in water (sea or fresh), according to the habitat.

18. Ether and Alcohol may be administered in the same way.
Anpres has obtained good results with Actiniw by the use
of a mixture (invented by Sanvarors to Bianco) containing
20 parts of glycerine, 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,

KILTING. 15

Eisia (Hauna u. Flora Golf. Neapel, 16, 1887, p. 259)
benumbs Capitellides by putting them into a mixture of one
part of 70 per cent. alcohol with 9 parts of sea water.

Ousvercren (Zeit. wiss. Mik., xix, 1903, p. 800) makes a
saturated (7 to 8 per cent.) solution of ether in sea or soft
water, and uses it either concentrated or diluted to about 1
per cent., and finds it to succeed with all classes of aquatic
animals.

Cort (Zeit. wiss. Mik., vi, 1890, p. 438) recommends a
mixture composed of 10 ¢.c. methyl-alcohol (of 96 per cent.
strength), 90 ¢c.c. water (fresh or sea water), and 0°6 erm. of
sodium chloride (to be added only when fresh water is
taken, the addition of the salt having for its object to
prevent maceration). It may be well to add to this mixture
a very few drops of chloroform (for Crestutella ; Zeit. wiss.

Zool., lv, 1893, p. 626).

19. Chloreton (Aceton Chloroform) is recommended for inver-
tebrates and larve of Rana by Ranpoten (Zool. Anz., xxiii,
1900, p. 436). Krucker (Zeit. wiss. Zool., xev, 1910, p.
383) takes solutions of 4 to 1 per cent. for Oligocheta.
SuLima (Zeit. Biol. Techn., Strasburg, i, 1909, p. 379) takes
a mixture of 99 parts of sea water and 1 of 10 per cent. sol.
of chloreton in absolute alcohol, for Scylliwm and Anguilla.

20. Hydrate of Chloral—Fourrincer (Arch. de Biol., vi,
1885, p. 115) 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 sufficiently insensible. He has obtained
satisfactory results with marine and fresh-water Bryozoa,
with Annelida, Mollusca, Nemertians, Actinie, and with
Asteracanthion. He did not succeed with Hydroids.

Lo Bianco (Mitth. Zool. Stat. Neapel, Bd. ix, 18:0, p.
442) employs for various marine animals freshly prepared
solutions of chloral in sea water, of from one tenth to one
fifth per cent. strength.

T have never had the slightest success with Nemertians.
VERWORN (Zeit. wiss. Zool., xlvi, 1887, p. 99) puts Cristutella for a

16 CHAPTER IL.

few minutes inty 10 per cent. solution of chloral, in which the animus
sooner or later become extended.

KUkentuan (Jena Zeit. Nuturw., Ba. xx, 1887, p. 511) has obtained
good results with some Aunelids by means of a solution of one part of
chloral in 1000 parts of sea water.

The chloral method gives rises to maceration with some
subjects, as I can testify, and has been said to distort nuclear
figures.

21. Cocaine (Ricnarps, Zoul. Anz., 196, 1885, p. 332).—
Richards puts a colony of Bryozoa into a watch-glass with 95
c.c. of water, and adds gradually 1 per cunt. solution of
hydrochlorate of cocaine in water. After five minutes the
animals are somewhat numbed, and half a cubic centimetre
of the solution is added; and 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 is the best method for Rotifers (RouSsELET).
It has also been recommended for .Ap/ysia.

It has been pointed out (by Corr, in the paper quoted § 1) that,
unfortunately, when fixing agents, such as sublimate solution, are
added to, the animals, the cocaine is thrown down on them as a white
precipitate. This precipitate, however, may be redissolved afterwards
in alcohol (Ersi@).

Coeaine solutions cannot be depended on to keep for more than a few
days.

22. Eucain. Harris (Journ. Roy. Alice. Soc., 1900, p. 404)
recommends a 1 per cent. solution of eucain hydrochloride, as
giving far better results, with Vorticellide, Rotatoria, and
Vermes. Rousseney (ibid.) reports favourably as to its action
on Flosculariw. It is stated to be perfectly stable in aqueous
media. It dissolves in sea water to about 0°5 per cent.

23. Hydroxylamin.—Horer (Zevt. wiss. Mik., vii, 1890, p. 318).
Either the sulphate or, preferably, the hydrochlorate may be used.
This should he dissolved in water (spring or sea water, according to the
habitat) and exactly neutralised by addition of carbonate of soda. The
organisms are placed ina solution diluted to about O1 per cent., for
thirty minutes or less (as for Infusoria), to 025 per cent., 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 (Heliw and Anodonta),

Hydroxylamin is a powerful reducing agent, and should therefore be
well washed out before treating with easily reducible fixing agents.

KILLING. 17

24. Chloride or Sulphate of Magnesium.—Tutnpera (Arch.
Zool. He pér. et Gen., x, 1892, p. 11). For Actinie, a 33 per
cent. solution of the chloride should be very slowly added to
the water containing the expanded animal, until the vessel
contains 1 per cent. of the salt (thus for one litre of sea
water 33 c.c. of the solution must be added). The addition
must be completed within half an hour, and thirty minutes
later the animal may be fixed.

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

RepenzaucH (Amer. Natural, xxix, 1895, p. 399) takes the
sulphate, either added in crystals to the sea water containing
the animals until a saturated solution is obtained, or in the
shape of a saturated solution into which they are thrown
(Annelids.)

See also Mayer, Biol. Bull. Wood’s Hole, xvii, 1909,
p. 3841 (puts direct into sol. of 2 strength).

25. Poisoning by small doses of some fixing agent is sometimes
good. Lo Branco kills Ascddia and Rhopalea in an extended state
(Mitth. Zool. Stat. Neapel, ix, 1890, p. 471) by pouring a little 1 per cent.
chromic acid on to the surface of the water containing them, and
allowing it to slowly diffuse into it. About twelve to twenty-four hours
is necessary. He kills Ciona ina similar way with a mixture of one
part of 1 per cent. chromic acid and nine parts of 49 per cent. acetic

acid.
Osmic acid, or Kleinenherg’s solution, is sometimes employed in the

same way.
I bave seen Meduse killed in a satisfactory manner by means of

crystals of corrosive sublimate added to the water containing them.
Morphia, Curare, Strychnin, Prussie Acid, and other paralysing
drugs, have also been employed.

26. 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 they 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

2

18 CHAPTER II.

Holothurie and other Echinoderms in this way. Warp (see
Amer. Nat., xxv, 1891, p. 398) has succeeded with Hydroids,
Actinie, and similar forms, and Urxxit (Mitth. Zool. Stat.
Neapel, xii, 1896, p. 463) with Echinids.

Marine animals are sometimes successfully killed by simply
putting them into spring water.

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

Carbonic Acid Gas has been recommended (by Fot, 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. I have had most excellent results
with small Annelids and Hirudinea. It is not necessary to
employ a generator for obtaining the gas. It suffices to take
an ordinary “soda-water”’ syphon, and squirt its contents
into the water containing the animals.

Narcotisation is very rapidly obtained with very small
animals, but much more slowly with larger ones. For
instance, Stylaria proboscidea, I find, is paralysed in a few
seconds; a small Nephelis of 15 or 20 millimetres in length,
will require about five minutes; and a large Nephclis, of
from 10 to 15 centimetres, will require as many hours.

Urxxtin (Aatth. Zool. Stat. Neapel, xii, 1896, p. 463) has
paralysed Echinids very rapidly with carbonic acid, likewise
a small Teleostean fish; whilst Scylliwm and Crustaceans
were affected much more slowly, and mussels not at all.

27. Peroxide of Hydrogen.——Voik (Zool. Anz., xix, 1896,
p. 294) kills Rotatoria by means of one or two drops of a
3 per cent. solution added to 1 c.c. of the water containing

them.

CHAPTER III.
FIXING AND HARDENING.

28. The Functions of Fixing Agents—The meaning of the
term ‘ fixing” has been explained above (§ 2). Here
is an example showing the necessity of fixation. 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 preservation, 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
unrecugnisable, 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.

This example shows that one of the objects aimed at in
fixing is to impart to tissues the degree of hardening neces-
sary to enable them to offer such mechanical resistance to
post-mortem change and to the processes of after-treatment
as not to suffer change of form. Another important
function of fixing is to render insoluble elements of cells and

20 GHAPTER lI.

tissues that would otherwise be more or less dissolved out
by the liquids employed in the after-treatment. A. third
and highly important function of fixing-agents consists 1m
producing optical differentiation in structures. By coagu-
lating the elements of tissues and cells, fixing agents alter
their indices of refraction, raising them in varying degrees.
They do not act in an equal degree on all the constituent
elements of cells and tissues, but raise the index of some more
than that of others, thus producing optical differentiation
where there was little or none before. Compare the aspect
of the epithelium of the tail of a living tadpole, observed in
water, with its aspect after the action of a little diluted
solution of Flemming. In the living state the protoplasm
of its cells has a refractive index little superior to that of
water, and consequently so low an index of visibility that
hardly any structure can be made out in the object. But as
soon as the protoplasm has been sufficiently coagulated by
the reagent the refractive indices of some of its elements
will have been raised to above that of balsam, the chromatin
of the nuclei will be brought out, and other structures
be revealed where none was visible before.

29. The Action of Fixing Agents consists in coagulating and
rendering insoluble certain of the constituents of tissues.
This is effected sometimes without any chemical action being
involved, as when alcohol is employed, which acts by simple
withdrawal of the water of the tissues. But in the majority
of cases the fixing agents enter into chemical combination
with certain of the elements of the tissues. The compcunds
thus formed are sometimes unstable and soluble, so that they
are removable by washing, as is the case with several of
those formed by picric acid. It is found in practice, how-
ever, that those formed by chromic acid and its salts, and
the salts of the heavy metals, as mercury, iron, platinum,
gold, and silver, are mostly insoluble.

The insolubility of these bodies is an advantage in that
it ensures that the tissues shall not be robbed of their
essential constituents, nor deprived of their desired con-
sistency and optical differentiation, by the reagents
subsequently employed. It is also sometimes an advantage
in that certain of the compounds in question have the

FIXING AND HARDMNING. 21

property of combining with certain colouring matters, and
thus affording important stains which could not otherwise be
obtained ; or in other words, of acting as mordants.

But it is sometimes a disadvantage, inasmuch as these
same compounds which render possible the production of
some stains are hindrances to the production of others.
Tissues that have been fixed with osmic or chromic acid or
its salts are in general not easily to be stained with carmine
or similar colouring matters, unless the metals have been
previously removed by special chemical treatment; though
they may generally be stained with hemalum, or, after
sectioning, with iron hematoxylin or tar colours.

According to Fiscaner (fivirung, Firbuny, und Baw des
Protoplasmax, Jena, G. Fischer, 1899), the coagulation which
constitutes fixation is, in the case of the liquid and semi-
liquid constituents of tissues, always a phenomenon of
precipitation, ‘lhe more solid constituents (such as fibrils
that are visible during life, nucleoli, and the like) he admits
may be acted on by fixing reagents without the formation of
any visible precipitates. But all the liquid ones, in so far
as they are fixed at all, are visibly precipitated in special
precipitation forms, which vary according to the precipitant.
Kach fixing agent gives its own characteristic fiaution image,
which may be more or less lifelike, but can never be
absolutely so. Fischer gives copious descriptions of the pre-
cipitation forms of the chief organic compounds found in
tissues, and of the precipitation powers of the chief fixing
agents, which the reader will do well to study.

It seems to be a consequence of Fischer’s theory of fixa-
tion by precipitation that the most energetic fixing agents
should always be found amongst the most energetic precipi-
tants. But on the showing of his experiments this is not
so. Jor instance, it is allowed on all hands that osmic acid
is a most energetic fixative. But Fischer finds (op. cit.
pp. 12—14, 27) that it is a very incomplete and weak
precipitant. Or, to take a contrary instance, he finds that
picric acid is an energetic precipitant of the majority of cell
constituents ; but surely every cytologist must admit that it
is not a highly energetic fixative.

It would seem to follow, from these instances and from
other similar ones, that Fischer’s tables of precipitating

22 CHAPTER III.

power cannot be taken as a measure of the fixing power of
the reagents. And further, the study of the fixation image®
of tissues afforded by osmic acid, formaldehyde, and other
reagents, seems to show that the coagulation brought about
by them is in part accompanied by the formation of visible
precipitates, but in part not so, and that they may do their
work to a larger extent than he seems to admit through a
homogeneous coagulation. But from his very suggestive obser-
vations it certainly appears that the formation of visible
precipitates is a very wide-spread, if not universal concomitant
of fixation ; and that the wider the precipitating power of a
fixative (i.e. the greater the number of organic liquids that
it can precipitate), the greater will be the number of artefacts
to which it can give rise.

30. The Characters of the Usual Fixing Agents—A good
fixing agent should first of all preserve all the elements it is
desired to fix. But that is not enough; it should also give
good optical differentiation, and should have sufficient power
of penctration to ensure that small pieces of tissue be equally
fixed by it throughout. No single substance or chemical
compound fulfils all that is required of a good fixing agent ;
hence it is that all the best fixing agents are nuatures.
Osmic acid, for instance, fulfils some of these conditions, but
not all of them. It kills rapidly and preserves admirably
the elements of cytoplasm, but nuclei not so well. But the
optical differentiation that it gives, though sometimes good,
is often very inferior. For osmic acid, by coagulating in
nearly equal degrees alike the spongioplasm (the plastin
reticulum) and the hyaloplasm (the enchylema) of the cell-
body, and the chromatin of nuclei, raises alike the refractive
indices of all of them; so that if the fixing action have been
in the least degree overdone, the cells acquire a homogeneous
aspect in which the finer details are obscured by the general
refractivity of the whole. If now, instead of using it pure,
it be used in combination with acetic acid, a better differentia-
tion is obtained ; for acetic acid, whilst enhancing, or at all
events not interfering with the fixation of chromatin, serves
to facilitate penetration and to counteract the excessive action
of the osmic acid on the protoplasm, so that the cells come
out less homogeneous and with more detail observable in

FIXING AND HARDENING. 23

them. A still better effect is obtained if to the osmic acid
there be added not only acetic acid, but also chromic acid.
For osmic acid has the property of blackening tissues, thus
rendering them opaque. Chromic acid counteracts in a
considerable degree this blackening action. It also helps
probably to bring out the chomatin of nuclei, which is
insufficiently fixed by the other two ingredients, and probably
also helps in other ways to bring about optical differentiation ;
so that in the result a much clearer picture is obtained.

I take it that it has been established by experience that,
as a general rule, in order to get the most complete fixation,
fixatives should have an acid reaction. Consequently, if
they have not naturally an acid reaction, they should be
acidified, e.g. osmic acid should be acidified with acetic acid
or the like. As a matter of fact, it will be found that
acetic acid is very largely employed in mixtures. It is
generally held that it acts in them as an agent for facilita-
ting penetration and producing differentiation, as explained
above, and also for ensuring the fixation of nuclein (if the
other ingredients are not adequate thereto); but this is
probably not all. Fiscuer (in the work quoted above, pp. 10,
27, and other places) holds that its function in these mixtures
is chiefly that of a neutraliser or acidifier (Ausiiurer) for
ensuring that the other ingredients shall have an acid, or at
least a neutral medium to do their work in. For the pre-
cipitating power, that is, in his view, the fixing power of a
reagent, varies according to the reaction, acid or alkaline, of
the things to be fixed; and a feebly acid reaction is the one
most favourable for ensuring precipitation. Many things
that are quite unprecipitable by certain reagents whilst in
alkaline or neutral solution are immediately precipitated by
them if the solution is rendered acid. ‘ Many kinds of cell
contents,” he says (op. cit., p. 10) “indeed the majority,
have an alkaline reaction, and are thereby quite inaccessible
to the precipitating action of certain agents, such as osmic
acid, or bichromate ; and the action of certain other fixatives,
such as platinum chloride and chromic acid, is more or less
hindered by the presence of free alkalies. For neither the
chromic acid (of solution of Flemming) nor the platinum
chloride (of solution of Hermann) would be adequate to act
as acidifiers to the osmic acid of the mixtures. They cannot

24: CHAPTER IIT.

do so, first, because they themselves become combined
with the tissues much more quickly than the slowly working
osmic acid, and secondly, because they themselves have only
an extremely weak acid reaction.” Hence the function of
the organic acid is to bring into play the precipitating power
of the other ingredients. ;

In default of special reasons to the contrary, fixing
mixtures may conveniently be made to contain from 1 to »
per cent. of acetic acid. But for some purposes they should
be neutral, or even alkaline. See, for instance, Neurofibrils.

I think the beginner should avoid such things as liquid of
Fiemmine and similar mixtures. He may take, instead,
Bourn’s piero-formol. :

Corrosive sublimate (acidified) is a good all-round fixative,
with excellent penetration, but is not quite so easy to
manage.

Picric acid gives a fair though weak fixation, with very
good penetration, is easy to manage, and does not make
tissues brittle, which sublimate easily may do. Pure formol
is not bad, and very easy to manage.

Speaking generally, osmic acid, chromic acid, hichromates, chloride
of platinum, and the majority of the compounds of the heavy metals,
are hindrances to staining; whilst heat, alcohol, trichloracetic acid,
formol, corrosive sublimate, nitric acid, picrie acid, and acetic acid, are
neutral, or even favourable, in this respect.

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

Fixation is generally performed by immersion of the
objects in the fixing liquid. In this case, everything should
be done 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 object 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

FIXING AND HARDENING. 2D

for certain Arthropods with very resistent integuments).
But this should only be done as a last resource.

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,

But fixation may also be performed by injection of the fixing liquid
into the objects, thus ensuring a more rapid and thorough penetration
of voluminous objects. See for this practice the methods of fixation by
injection of Goner, Dr QuEeRvaAIN, Many, and others, given under
Nerrous System.

Bravus and DRUENER (Jena. Zett. Naturw., Bd. xxix, 1895, p. 435)
fix fishes by injection through the bulbus aortw. The vessels are first
washed out with normal salt solution, and the fixing liquid is then
thrown in.

Koimer (Anat. Anz., xlii, 1912, p. 47) fixes thus even large mammals
(Chimpanzee, Goat). He first washes out with RINGER’s solution.

It is well not to leave specimens in fixing liquids longer’
than is sufficient to obtain the desired reaction. Sublimate,
for instance, soon makes tissues brittle. But long immersion
may be necessary to produce the desired optical differentia-
tion with some reagents.

Careful washing owt (by which is meant the removal from
the tissues of the excess of uncombined fixative) is necessary
in order to get tissues to stain properly. But it is not always
equally imperative. Alcohol and formaldehyde do not require
washing out before staining ; acetic and picric acid only for
some stains; sublimate will allow of staining even if not
washed out, but allows of a sharper stain if well washed out ;
all osmic, chromic, and platinic liquids require very thorough
washing out.

It is important 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

26 CHAPTER III.

alcohol causes precipitates that may ruin the preparations.
Objects fixed in alcohol, formol, acetic acid, picric acid, oY
nitric acid require to be washed out with alcohol, or at least
with some hardening liquid, whilst those that have been
fixed with osmic or chromic acid, or with one of the other
compounds of the heavy metals, require in general to be
washed out with water. Sublimate, however, is best washed
out with alcohol.

Use liberal quantities of liquid for washing.

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

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

32. Fixation of Marine Animals. —The tissues of marine
organisms are as a general rule more refractory to the action
of reagents than those of corresponding fresh-water or terres-
trial forms, and fixing solutions should in consequence be
stronger (about two to three times).

Marine animals ought to be freed from the sea water adhe-
rent to their surface 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. 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.
If alcohol be employed, it should be acidified with hydro-
chlorie or some other appropriate acid. — Picro-nitrie acid is
a fixing reagent that fulfils the conditions here spoken of.
(On this subject see Mayer, in Mitth. Zool. Stat. Neapel, ii
(1881), p. 1, et seq.)

33, Hardening.—The process of hardening is distinguished
from that of fixing as being directed to the attainment of a
degree of consistency sufficient to allow of soft tissues being
cut into sections without imbedding. It is an after-process,
and only ranks as a special method.

Methods of imbedding have now been brought to such a
degree of perfection that the thorough hardening of soft
tissues that was formerly necessary in order to cut thin

FIXING AND HARDENING. oe

sections from them is, in the majority of cases, no longer
necessary. 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.

The reagents employed for hardening are for the most
part of the same nature as those employed for fixing. But
it does not follow that all fixing agents can be employed for
hardening. Corrosive sublimate, for instance, would be most
inappropriate as a hardening agent.

34, The Practice of Hardening.—Himploy in general a rela-
tively large volume of hardening liquid, and change it very
frequently. If the volume of liquid be insufficient, its com-
position will soon became seriously altered by the diffusion
into it of the soluble substances of the tissues; and the
resulé may be 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 of the
proportions of colloids and crystalloids contained in it to
that of the liquids of the tissues, osmotic equilibrium will
become established, and diffusion will cease; that is to say,
the hardening liquid will cease to penetrate. his means,
of course, maceration of internal parts. On the other hand,
it appears that a certain slight proportion of colloids in the
hardening liquid is favourable 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 hardening agent.

Hardening had better be done in tall cylindrical vessels,
the objects being suspended by a thread, or muslin bag, or
otherwise, 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 ; or, they may be laid on a layer of cotton-wool, or
filter-paper, or spun glass.

In general, begin hardening with a weak reugent, 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.

CHAPTER LV.

FIXING AND HARDENING AGENTS—MINERAL ACIDS AND
THEIR SALTS.

35. Osmic Acid.—The tetroxide of osmium (OsOy,) 18 the sub-
stance commonly known as osmic acid, though it does not
possess acid properties. It is extremely volatile, and in the
form of an aqueous solution become partially 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 7f
dust be absolutely dented access to them.

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 fixation by osmium vapours, and for
making up solution of Flemming, 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 in a drop-bottle with grooved
stopper, from which quantities can be obtained when
required without removing the stopper.

Cort (Zeit. wiss. Mik., vi, 1890, p. 442) finds that solutions
in distilled water keep perfectly if there be added to them
enough permanganate of potassium to give avery 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.

Buscw finds that the addition of sodium iodate hinders
reduction (Newrol, Centralb., xvii, 1898, p. 476).

Pinrner finds that a slight addition of corrosive sublimate

FIXING AND HARDENING AGENTS. 29

has the same effect, e.g. ten drops of 5 per cent. solution of
sublimate added to 100 ¢.c. of 1 per cent. solution of osmic
acid.

For the so-called “regeneration ” of reduced solutions,
see previous editions.

Osmic acid is found in commerce in the solid form in sealed tubes.
The assigned weights should be checked, as they may vary greatly
(this does not apply to the tubes sent out by Gribler and Hollborn, or
Kahlbaum).

Fixation by the Vapouwrs——This 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 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
turn brown (isolated cells will generally be found to be
sufficiently fixed in thirty seconds: whilst im 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,
picrocarmine, or hematoxylin.

In researches on nuclei, it 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).

An apparatus for conveniently employing the vapours is described
by ANDREWS (Zeit. wiss. Mik., xiv, 1898, p. 448).

The reasons for preferring fixation by the vapour are that osmic acid
is more highly penetrating in vapour than in solution; that the
arduous washing out required by the solutions is done away with; and
that all possibility of deformation through osmosis is eliminated.

Vixation by Solutions —Qsmic acid is now very seldom
used pure in the shape of solutions. When, however, it is
so employed it is used in strengths varying from ,', per
cent, to 1 percent. I should say myself that, as a rule, not

30 CHAPTER IV.

more than 0-1 per cent., and never more than 0°5 per cent.,
should be used.

On account of its feeble penetrating power the objects to
be fixed should be as small as possible.

A little acetic or formic acid (0°5 to 1 per cent.) muy
generally with advantage be added to the solutions just
before using.

The solutions should be kept protected from the light
during the immersion of tissues. (‘I'his 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. The objects may
be deemed to be fixed as soon as they have become brown
throughout.

After-Treatment.—The excess of osmic acid must be well
washed out before proceeding to any further steps in
preparation ; water should be used for washing. | Notwith-
standing the greatest care in soaking, it frequently happens
that some of the acid remains in the tissues, and causes
them to blacken in time, and in any case hinders staining.
To obviate this blackening it has been advised to wash them
out in ammonia-carmine or picro-carmine (not very effectual),
or to soak them for twenty-four hours in a solution of bichro-
mate of potash (Miiller’s solution or Erlicki’s will do), or in
0-5 per cent. solution of chromic acid, or in Merkel’s
solution. ‘The treatinent with bichromate solutions has the
great advantage of highly facilitating staining with carmine
or hematoxylin. Max Schultze recommended washing, and
mounting permanently in acetate of potash. Fol, treatment
with a weak solution of carbonate of ammonia. But the best
plan of all is to properly bleach the preparations (see
“ Bleaching”). This may be done by means of perovride of
hydrogen. Overton (Zeit. wiss. Mik., vii, 1890, p. 10) finds
that it is completed in a few minutes in a mixture of 1 part
commercial peroxide with 10 to 25 parts 70 per cent.
alcohol. (The commercial peroxide, slightly acidulated with
HCl, will keep well in the dark; but the mixture with
alcohol must be made fresh for use.) According to Brrsror
(Amer, Natural, xxvii, 1893, p. 176) the peroxide acts best
in the san. Binwr (Journ, de V Anat. et de la Physiol., xxx,
IS94, p. 449) has successfully used permanganate of potash.

FIXING AND HARDENING AGENTS. 31

Mann (Methods, etc., p. 83) takes a solution of 0:25 per
cent., and treats the browned tissues with 1 part of saturated
solution of sulphurous acid to 9 of normal salt solution. . . .
Moncxesere and Beran (Arch. Mik. Anat., liv, 1899, p. 135)
have succeeded in satisfactorily restoring the staining
susceptibility of osminm material by means of sulphurous
acid (obtained by adding hydrochloric acid to bisulphite of
sodium, 2 to 4 drops of the acid added to 10 c.c. of a 2 per
cent. solution of the salt).

Fou (Lehrb., p. 174) recommends a weak aqueous solution of ferri-
cyanide of potassium.

I find the sulphate of iron solution used in Benda’s hematoxylin
stain has a marked bleaching effect, and so also, though in a less
degree, the iron alum of Heidenhain’s process.

ALTMANN (Die Elementarorganismen, pp. 33 and 35) puts sections
overnight into gold chloride of 2 per cent., and reduces in formic
acid in the sun, and removes the gold by iodised alcohol.

But perhaps the best plan is the chlorine method of Mayer,
or his magnesium peroxide, for both of which see Bleaching.

The same stains recommended for objects fixed by the
vapours will be found useful here. Tor sections, of course,
in both cases safranin and other anilin stains may be em-
ployed with advantage, as may hematoxylin.

In general osmic acid, especially when used in the form
of vapour, fixes protoplasm very faithfully, nuclei badly. It
is pre-eminently a fixative of the hyaloplasm or enchylema of
cells. The penetrating power of the solution is very low, so
that if any but very small pieces of tissue be taken the outer
layers become over-fixed before the reagent has penetrated
to the deeper layers. Over-fixed cells have a certain
homogeneous, glassy, or colloid look, and are unfit for study,
and attention should be confined to cells four or five layers
deeper down, which will generally be found to present the
required intensity of fixation. In these the fixation is
admirable, with no shrinkage and next to no swelling of
anything.

86. Osmic Mixtures—RANVIER ET ViGNAL (RANVIER, Leg.
@ Anat. Gén., p. 76; VIGNaL, Arch. de Physiol., 1884, p. 181) take equal
volumes of 1 per cent. osmic acid and 90 per cent. alcohol (freshly

mived). They wash out in 80 per cent. alcohol, then wash with water
and stain for forty-eight hours in picro-carmine or hematoxylin.

32 CHAPTER Iv.

Nicotas (Intern. Monatsschr., 1891, p. 3) adds 3 per cent. of osme
acid to nitric acid of 3 per cent. I have employed a similar mixture
and not had good results, though I find the mixture keeps perfectly.

Buscu (Neurol. Centrulb., xvii, 1898, No. 10, p. 476; Zeit. wiss. Mik.
xv, p. 373) finds that the penetration of osmic acid is enhanced by
combining it with iodate of sodium, which by hindering its too rapid
decomposition in the tissues ensures « more energetic action in the
deeper layers. He adds 8 per cent. of sodium iodate to a 1 per cent.
solution of osmic acid.

Unna (Monatschr. prakt. Derm., xxvi, 1898, p. 602) adds 1 per cent.
of alum toa 1 per cent. solution. For some mixtures of KoLossow,
see Sth ed., or Zeit. wiss. Mikr., v, 1888, p. 51, and ix, 1892, p. 39.

37. Chromic Acid —Chromic anhydride, CrO,, is found in
commerce in the form of red crystals that dissolve readily in
water, forming chromic acid, H,CrO,. 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 is generally employed in aqueous solution.
Some observers (Kinin; Ursan Prircuarp; Perinyt) have re-
commended alcoholic solutions; but this is evidently irrational.
For in the presence of alcohol chromic acid has a great
tendency to become reduced to chromous oxide or sesquioxide,
neither of which appears to have any fixing power.

The most useful strengths in which it is employed in aqueous
solution are from 0:1 to 1-0 per cent. for a period of immersion
of a few hours (structure of cells and ova). For nerve tissues
weaker solutions are taken, 4, to ¢ per cent. for a few
hours. Stronger solutions, such as 5 per cent., should only
be allowed to act for a few seconds.

Washing out.—The general practice is to wash out very
thoroughly with water (by preference running water, for
many hours), before bringing into alcohol or any staining
liquid. For if the objects are put direct into alcohol it is
found that after a short time a fine precipitate is thrown
down on the surface of the preparations, thus forming an
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

FIXING AND HARDENING AGENTS, 33

from forming again and again. It has, however, been found
by Hans Vircuow (Arch. mik. Anat., xxiv, 1885, p. 117) that
it may be entirely prevented by simply keeping the prepara-
tions 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.

Mayer (Grundziige, Ist ed., p. 28) proceeds as follows :—
The fixed material is merely rinsed in water and brought
direct into 70 per cent. alcohol. It is washed therein, pre-
ferably in the dark, until after several changes the alcohol
remains colourless. It is then either passed through higher
alcohols and imbedded in paraffin, the chromous oxide (or
whatever chrome compound it may be that is present in the
tissues) being removed from the sections after these are
made ; or this necessary removal is performed at once. If
this be preferred, the material is brought into sulphuric acid
diluted with twenty volumes of water, or into nitric acid
diluted with ten volumes of water. After at most a few
hours therein, it will have become of a light greyish green,
and on removal of the acid may be readily stained. If it be
preferred to treat the sections, it is sufficient to put them into
the usual hydrochloric acid alcohol (four to six drops of HCl
to 100 c.c. of 70 per cent. alcohol), in which after a short
time they become almost white, and will stain excellently
with any of the usual stains. So also Epincgr (Zezt. wiss,
Mtk., i, 1884, p. 126; nitric acid 1:20 for five minutes).
Unna (Arch. mik, Anat., xxx, 1887, p. 47) holds that the
chrome is present in the tissues in the form of chromic
chromate, and removes it by treatment with peroxide of
hydrogen. Ovzrron (Zeit. wiss. Mtk., vii, 1890, p. 9) employs
a weak solution of sulphurous acid, which converts it into a
sulphate. See also the directions for bleaching osmic acid
preparations, § 35.

Tissues that have been fixed in chromic acid may be
stained in aqueous solutions, as water does not have an
injurious effect on them.

The best stain for chromic material that has not been
treated by Mayer’s special process, or by a similar one, is
hematoxylin, or, for sections, the basic tar colours.

3

34, CHAPTER IV.

Chromic acid is not a very penetrating reagent, and for
this reason, as well as for others, is now seldom used pure
for fiwing.

For prolonged hardening it is generally employed 17
strengths of + per cent. to 4 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
portions of tissue of more than an inch cube. Fora 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.

I think it is frequently useful to add a little glycerin ;
there is less brittleness.

The solution should be taken weak at first, and the strength
increased after atime. 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. 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. After a time
they generally become green in the alcohol. They may be
bleached if desired.

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.

88. Chromic Acid and Alcohol (URBAN PRITCHARD, Quart.
Journ. Mic. Sci., 1873, p. 427)—Chromic acid, 1 part; water, 20 parts ;
rectified spirit, 180 parts. For hardening such tissues as retina,
cochlea, ete.

A mixture of 2 parts of + per cent. chromic acid solution with 1 part
of methylated spirit was once much used by KLEIN (Quart. Journ. Mic.
Sci., 1878, p. 315).

Both these mixtures are irrational (see § 37). A still more irrational

FIXING AND HARDENING AGENTS. 35

mixture with oxalic acid has been propounded by Grar (Cont. Path.
Inst. New York State Hosp., 1898, No. 15; see Grundziige, 4th ed.,
p. 34).

39. Chromo-acetic Acid (FtumMine, Zellsbz., Kern. uw. Zellth.,
p. 382),

Chromic acid . . 0'2 to 0:25 per cent.
Acetic acid. . Ol per cent. in water.

Flemming found this the best reagent for the study of
the achromatic elements of karyokinesis. You can stain with
hematoxylin, or the basic anilin dyes.

The following has been recommended for Annelids by
Eaxers:—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 acetic
acid is said to be sufficient to counteract any shrinkage due
to the chromic acid.

Similar to this is the “chromo-acetic acid, No. 1,” of Lo
Bianco (Mitth. Zool. Stat. Neapel, ix, 1890, p. 443), viz. 1
part 50 per cent. acetic acid and 20 parts 1 per cent. chromic
acid, which is found very useful for fixing marine animals,

40. Chromo-formic Acid (Rast, 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. Used by Rabl for the study of karyo-
kinesis.

41. Chromo-osmic Acid (Max Fiescu, Arch. mik. Anat., xvi, 1879,
p. 300).—Osmic acid. 0:10 ; chromic acid, 0°25; water, 100:0. Lo Branco
(Mitth. Zool. Stat. Neapel., ix, 1890, p. 443) employs for marine animals
a mixture of 1 part 1 per cent. osmic acid and 50 parts 1 per cent.
chromic acid.

42. Chromo-aceto-osmic Acid (FLemmine, Zellsubstanz, Kern
und Zelltheilung, 1882, p. 381). Firsr or Weak formula:

Chromic acid. ; . 0°25 per cent.
Osmic acid . : . O1 3 in water.
Glacial acetic acid . . ol

a”
Meves (Encycl. mikr. Techn., 1, p. 475) sometimes adds
1 per cent. of sodium chloride.

36 CHAPTER IV.

Fou (Lehrb. d. vergl. mik. Anat., 1884, p. 100) recommends the
following variant :

1 per cent. chromic acid ; : . 25 vols.
; per cent. osmic acid : ie 2 Ry

2 per cent. acetic acid : : » 58s
Water . ' : » 68 55

—that is to say, a mixture ies weaker’ in osmium than Flemming’s.

A mixture still weaker than this in osmium, viz. with 1 vol. osm
acid solution, instead of 2, has been recommended by Cort (Zett. Wiss.
Mik, vi, 1890, p. 441).

mic

Szconp or Srrone formula (Zeit. wiss. Mik., |, 1884,

p. 349) :
1 per cent. chromic acid. . 15 parts.
2 per cent. osmic acid : . 4s,
Glacial acetic acid. é . I part.

If this mixture be kept in stock in large quantities, it
may go bad, on account of the large proportion of organic
acid contained in it. I therefore recommend that the osmic
and chromic acid be kept ready mixed in the proportions
given, and 5 per cent. of acetic acid added at the moment of
using.

Weaker Formura.—More recently, Fiemmine has been
making up the mixture with only 2 parts of the osmic acid
instead of 4, and has spoken of this modification as “weaker
osmium mixture” (Mzves, in Encycl. Mikr. Techn., p. 476).

Meves (loc. cit.) takes for delicate objects 15 parts of
chromic acid of only 0°5 per cent., 2 or 4 of osmic acid of
2 per cent., and 1 of acetic acid, and thus gets less
shrinkage.

PopwyssozkI recommends (for glands especially) the following modi-
fication :
1 per cent. CrO, dissolved in 0°5 per cent. solution of

corrosive sublimate . . 15 ee.
2 per cent. osmic acid solution . i : . 40.
Glacial acetic acid : : . 6 to 8 drops.

The sublimate is said to augment ‘the penetr ation of the osmium, but
is unfavourable to staining (ZIEGLER's Beitrdge z. path. Anat., i, 1886 ;
Zeit. wiss. Mik., iii, 1886, p. 405).

The first or weak liquid is the better for very small
objects, the second or strong one for larger ones, as it has
better penetration. These liquids may be allowed to act for
many honrs or days, or according to some workers even

FIXING AND HARDENING AGEN'S. 37

weeks or months; but this exaggerated fixation is clearly
only justifiable in very special cases, if at all. Wash out
very thoroughly in water (running, 24 hours, or treat as
directed for chromic acid § 37). Stain with alum hema-
toxylin if you wish to stain in toto (staining in this way
with other reagents is possible, but difficult). Stain sections
with safranin or other basic coal-tar colour, or with iron
hematoxylin.

For fixing with the strong mixture you need only take a
bulk of liquid of some 4 times the volume of the objects (but
with the weak mixture the proportion should be increased).
Both of them are first-rate fixatives of cellular structures,
both as regards their preservation and as regards their
optical differentiation. But they must be properly used, and
not applied to objects for which they are not fitted. For
instance, their power of penetration is extremely bad ; they
will not fix properly, even in a loose-celled tissue, through
more than a layer of about five cells thick. They are
therefore suitable only for very small objects or for very
small pieces of tissue, such as suffice for cytological or
histological work. The strong liquid especially has not the
character of a general reagent. As a matter of fact it was
recommended by Fremarne in the first instance merely for a
very special purpose, the hunting for karyokinetic figures,
and not for general purposes. It is still very much used, but
in my opinion unadvisedly. In most cases, Bouin’s picro-
formol will do all that it is intended to do, without its
disadvantages.

It may be used for prolonged hardening, e.g. of small
pieces of nervous tissue, and is very good for that purpose.

Fat is blackened (or browned) by it (see § 35).
Chromatin is mordanted by it for basic anilin dyes, enabling
them to give peculiarly sharp and powerful stains.

43. Osmic Acid and Bichromate.—ALTMANN (Die Elementarorgan-
ismen, Leipzig, 1890), takes for his “ bioblasts” a mixture of equal parts
of 5 per cent. solution of bichromate of potash and 2 per cent. solution
of osmic acid. The bichromate ought not to contain any free chromic
acid.

Lo Branco (Mitth. Zool. Stut. Neapel, ix, 1890, p. 443) employs for
marine animals a mixture of 100 c.c. of 5 per cent. solution of bichro-
mate and 2 c.c. of 1 per cent. osmic acid.

fa)
38 CHAPTER IV.

Horn, (Arch. Anat. Phys., Anat. Abth., 1896, p. 31),
recommends a mixture of 80 ¢.c. of 3 per cent. bichromate,
20 c.c. of 1 per cent. osmic acid, and 2 ¢.c. of glacial acetic
acid.

44. Osmic, Bichromate, and Platinic Mixture (Luvpsay JoHN-
soy’s Mixture).—Latest formula, 1895, communicated by Dr.
Lindsay Johnson :

Bichromate of potash (2°5 per cent.) . 70 parts.

Osmic acid (2 percent.) . ' . 10 = 4,
Platinum chloride (1 per cent.) » ID x
Acetic or formic acid : : eB gs

Henneauy, who has worked a great deal with this reagent,
and recommends it highly, says (Lecons sur la Cellule, p. 61)
that it is well only to add the acetic or formic acid just before
using, as it frequently reduces the osmium and platinum
very rapidly and energetically. He finds that it contracts
the more spongy sorts of protoplasm less than mixture of
Fremuinc. I think highly of it—for certain objects.

45, Platino-aceto-osmic Acid (Hurmann’s solution (Arch. mek.
Anat., xxxiv, 1889, p. 58). One per cent. platinum chloride
15 parts, glacial acetic acid one 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.

The after-treatment and staining should be the same
as for objects treated with Flemming’s solution. RenGeL
(Zeit. wiss. Zrol., lxiti, 1898, p. 454) washes out for half an
hour to an hour with saturated aqueous sol. of picric acid,
which he thinks facilitates the staining, especially of nuclei.

The action of this fixative is, roughly, similar to that of
Flemming’s. Like Flemming’s, it mordants chromatin for
staining with “ basic ” colours, with which it affords equally
fine nuclear stains. But, owing to the platinum in it, it
diminishes more than Flemming’s the colorability of tissues
with “acid” colours, so that it is ewtremely dificult to
obtain good plasma slains after its action. It causes a
notable shrinkage in chromatin. It gives a full fixation of
cytoplasm, to which it gives a much more fine-grained
aspect than liquid of Flemming: does.

FIXING AND HARDENING AGENTS. 39

A mixture of BorRrex’s (from CAULLERY and Mesnix, Arch. Protis-
tenk, vi, 1905, p. 281), consists of 2 parts of osmic acid, 2 of platinum
chloride, 3 of chromic acid, 20 of acetic acid, and 350 of water.

46. Rawitz (Zeit. wiss. Mikr., xxv, 1909, p. 386) takes 4 parts of
Kahlbaum’s Phospho-Tungstie acid, 5 of alcohol, and 1 of acetic acid,
added just before use, fixes for 24 hours, and washes out the sections
before staining with water containing a little calcium acetate.

47. Nitric Acid (Atrmann, Arch. Anat. Phys, 1881, p. 219)—
Altmann employs for fixing embryos dilute nitric acid, containing from
3 to 33 per cent. pure acid. Such a solution has a sp. gr. of about 102.
Stronger solutions have been used, but do not give such good final
results. After extensive trial I find ALTMaNn’s solution to be a second-
rate reagent, giving a weak and thin fixation.

His (béd.,1877, p. 115) reeommendeda10 per cent. solution. Flemming
at one time employed solutions of 40 to 50 per cent. for the ova of
Invertebrates.

TELLYESNICZKY (Arch. mik, Anat., lii, 2, 1898, p. 222) thinks that “for
general cell-fixing” the proper strength is 2 per cent. to 23 per cent., as
stronger grades act too energetically on the superficial layers.

Maver has had good results with 5 per cent. solution.

Nitric acid has the valuable property of hardening yolk without making
it brittle.

Pure water should in no case be used for washing out; the pre-
parations should be brought direct into alcohol. Some persons take
absolute, but I should say 70 per cent. is more generally indicated.
Rabl has employed a 1 or 2 per cent. solution of alum.

For prolonged hardening, strengths of from 3 to 10 per cent. are
sometimes employed. A strength of 12 per cent., allowed to act for two
or three weeks, is said to afford very tough preparations of the en-
cephalon.

Benpa (Verh. Anat. Ges., 1888; Ergeb.d. Anat., i, 1891, p. 7) fixes for
twenty-four to forty-eight hours in 10 per cent. nitric acid, and then
hardens in bichromate of potash.

Fol’s Mixture (verbally communicated to me).—Three vols. of nitric
acid, with 97 vols. of 70 per cent. alcohol.

48. Chromo-nitrie 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.

Fix for 4 to 5 hours and pass into alcohol of 70 per cent.

This mixture has been criticised (see previous editions) as irrational,
the alcohol reducing the chromic acid and itself becoming etherised by
the nitric acid. Some workers reject it, especially for ova, for which it
is specially intended. But others speak highly of it. I myself have
used it extensively for preparing objects for dissection and museum

40 CHAPTER LV.

specimens, and found it admirable for these purposes. But preparations
made to test its value from a cytolovical point of view have given me
only second-rate results. It is now little used.

49. Chromic Acid and Platinum Chloride (Murxe.’s Macula
lutea des Menschen, Leipzig, 1870, p. 19).—Equal volumes of
1-400 solution of chromic acid and 1°400 solution of platinum
chloride. Objects should remain in it for several hours or
even days. After washing out with alcohol of 50 per cent.
to 70 per cent., objects stain excellently. If objects that
have been fixed by osmic acid be put into it for some hours,
blackening is said to be effectually prevented.

This is an excellent hardening medium for delicate objects.
Merkel allowed from three to four days for the action of the
fluid for the retina; for Annelids Hisig 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.

A similar mixture, with the addition of 0°25 to 0-1 per
cent. of acetic acid, is recommended by Brass for Protozoa ;
and Lavpowsxky has used for nuclei a mixture of 10 parts of
1 per cent. chromic acid, 5 of 1 per cent. platinum chloride,
and 100 of 5 per cent. acetic acid.

Whitman recommends for the hardening of pelagic fish
ova, a stronger mixture (due, I believe, to Hisig), vizi—

0°25 per cent. solution of platinum chloride . 1 vol.

1 per cent. solution of chromic acid F wi. Ae es
The ova to remain in it one or two days (Wurman, Methods
in Micro. Anat., p. 153).

Salts.

50. Chromates.—The chromates are amongst the oldest and
best tried of hardening agents. The bichromate of potash
especially was at one time universally employed for hardening
all sorts of tissues.

Fremaine (Arch. mihk, Anat., xviii, 1880, p. 352) poited
out that though it preserves cytoplasm well it causes
chromatin to swell, and therefore should not be employed
for the study of nuclei. But, duly corrected with acetic acid,
it affords a correct and fine fixation of nuclei; wlulst pre-

FIXING AND HARDENING AGENTS. 4

serving hyaloplasm and its inclusions, secretions, etc., much
better than chromic acid.

For an elaborate study of the action of chrome salts on
nucleus and cytoplasm, see Burcxuarot, La Cellule, xii, 1897,
p. 335. He finds that the bichromates of sodium, ammonium,
magnesium, strontium, and zinc have the same destructive
action on nuclei that the bichromate of potassium has; but
that the bichromates of barium, calcium, and copper have
not. He concludes that acetic acid ought always to be added,
not only to ensure the correct fixation of nuclei, but also to
enhance penetration and the good preservation of cytoplasm.

The following is recommended by him as a good combina-
tion for the fixation both of cytoplasm and nucleus:

Bichromate of barium, 4 per cent. solution . 60 vols.
Bichromate of potassium, 5 per cent. solution 30 ,,
Glacial acetic acid . : : e FD ba ge

(Instead of the barium you may take 4 per cent. solution
of bichromate of calcium, or 6 per cent. solution of bichromate
of copper.)

For the demonstration of the achromatic figure of cell
division he recommends—

Chromic acid, 1 per cent, solution ; . 60 vols.
Bichromate of potassium, 5 per cent. solution 30 ,,
Glacial acetic acid. ; : : a OD 35

51. 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 incom-
parably better consistency to the tissues. 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, or be treated as directed
for chromic acid, § 87. They had better be kept in the dark
when in alcohol (see § 37), (Boum and Orrgx [Taschenbuch,

42 CHAPTER IV.

3 Auf., 1896, p. 22] fix in the dark.) If you wish to have
a good stain with carmine you should not put the vbjects ito
alcohol at all, even for a second, until they have been stained.

You may stain either with carmine or hematoxylin, a
well as with tar colours.

Bichromate objects have an ugly yellow colour which cannot be re-
moved by mere soaking in water. It is said that it can be removed by
washing for a few minutes in al per cent. solution of chloral hydrate.

Prof, Gitson writes me that alcoholic solution of sulphurous anhy-
dride (SO,) is very convenient for the rapid decoloration of bichromate
objects. A few drops suffice. See also § 37, and “ Bleaching.”

To facilitate staining with hematoxylin, WoLFF (Zeit. wiss. Mik., xv,
3, 1899, p. 311) first stains in Boehmer’s hematoxylin for twenty-four
hours, and then for a few minutes in the same hematoxylin to which
has been added 1 drop per watch-glassful of 5 per cent. solution of
oxalic acid.

The simple aqueous solution of bichromate is hardly to be
recommended as a fizing agent, because not only does it not
preserve nuclei properly, but also because it penetrates very
slowly. The first of these defects may be overcome entirely,
the second to some extent by addition of acetic acid; whence
the liquid of Teriyusniczky, next §.

52. Acetic Bichromate (TeLLyesniczky, Arch. mik. Anat., lu,
1889, p. 242)

Bichromate . . : ; ‘ 3 grins.
Glacial acetic acid . ; : 5 cc.
Water . . 100

: ‘ : 53
Smaller objects to remain in the fluid for one or two days,

larger ones longer. Wash well in plenty of water, and pass
through alcohols of increasing strength, beginning with 15
per cent.

Mixtures of bichromate with osmic acid have been given above, §§ 43
and 44.

53. MUiiEr’s Solution.—

Bichromate of potash 5 ‘ . 2-23 parts.
Sulphate of soda : ‘ . 1 part.
Water . : : . 100 parts.

The duration of the reaction is about the same as with
the simple solution of chromic salt.

FIXING AND LLARDKENING AGENTS. 43

Recent authors find the action of this liquid to be identical
with that of plain bichromate, and doubt whether the
sulphate in it has any effect whatever as regards its harden-
ing properties. Fol says that for mammalian embryos, for
which it has been recommended, it is worthless.

54. Exzicxt’s Solution (Warschauer med. Zeit., xxii, Nos. 15
and 18; Drogrés Médical, 1897, No. 31).—

Bichromate of potash . : 2°5 parts.
Sulphate of copper : : 1:0 part.
Water , , . 100°0 parts.

Here the addition of the cupric sulphate is intelligible, for
this salt is itself a hardening agent of some energy. As a
matter of fact, “ Erlicki’’? hardens very much more rapidly
than either simple bichromate or Miiller’s solution. 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
(Fox, Lehrb. d. vergl. mik. Anat., p. 106). Human embryos
of several months may be conveniently hardened in it.

Nerve-centres that have been hardened in Erlicki’s fluid frequently
contain dark spots with irregular prolongations, simulating ganglion-
cells. These are now, known to consist of precipitates formed by the
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°52 per cent. chromic acid before putting them into alcohol
([scuiscH, Virchow’s Arch., Bd. xevii, p.173; EDINGER, Zeit. wiss. Mik.,
ii, p. 245; LozwrentHaL, Rev. méd. de la Suisse romande, 6me année,
i, p. 20).

55. KuLtTscHITzky’s Solution (Zeit wiss. Mtk., iv, 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 im the dark. Then treat with
strong alcohol for twelve to twenty-four hours.

56. Dekhuyzen’s Liquids (C. R. Acad. Sci., cxxxvii, 1903, pp. 415
and 445).—(a) 250 ¢.c. of 2°5 per cent. sol. of bichromate in sea-water,
25 c.c. of 63 per cent. nitric acid, and 54 c.c. of 2 per cent. osmic acid.
For’ general use with marine animals.

(B) $1731 ¢.c. of the bichromate sol. and 269 of 2 per cent. sol. of
osmic acid. For objects containing calcareous elements that it is desired
to preserve.

These liquids are stated to be isotonic with sea-water.

44, CHAPTER IV.

57. Bichromate and Sublimate (Kuirscuirzky, Arch. f. mik. Anat.,
xlix, 1897, p. 8).—T wo parts bichromate, } part corrosive sublimate,
50 parts 2 per cent. acetic acid, and 50 parts 96 per cent. alcohol. The
mixture should be filtered after twenty-four hours. Tissues of verte-
brates may remain in it for four to six days. LavpowsKy (Zeit. wiss.
Mik., xvii, 1900, p. 301), takes 500 c.c. of 1 per cent. acetic acid, 20 to 25
g. bichromate, and 5 to 10 ¢.c. saturated solution of sublimate in water.

58. Bichromate of Ammonia.—This salt is in considerable favour
for hardening. Its action is very similar to that of the potassium salt.
Fol says that it penetrates somewhat more rapidly, and hardens some-
what more slowly. It should be employed in somewhat stronger solu-
tions, up to 5 per cent.

59. Neutral Chromate of Ammonia is preferred by some. Itis 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.

60. Bichromate of Calcium. Sonnensropt (Arch. mikr. Anat.,
lxxii, 1908, p. 416), fixes ovaries of Gallus in 20 parts of 2 per cent.
sol. of calcium bichromate with 10 of 2 per cent. sol. of sublimate and
1 of acetic acid.

61. Bichromates and Alcohol.—Mixtures of bichromate of potash
ov ammonia with aleohol may be employed, and have a more rapid action
than the aqueous solution. Thus Hamiuton takes for hardening brain
a mixture of 1 part methylated spirits with three parts of solution of
Miiller; see also KuLtscuitzKy’s Mixture, ante, § 55). Preparations
should be kept in the dark during the process of hardening in these
mixtures.

62. Sulphurous Acid.—WappinaTon (Journ. Roy. Mic. Soc., 1883,
p. 185) uses a saturated solution of sulphurous acid in alcohol for fixing
infusoria. OvERTON (Zezt. wiss. Mzk., vii, 1890, p. 9) uses the vapours
of an aqueous solution for fixing alge.

CHAPTER V.

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

Chlorides.

68. Bichloride of Mercury (Corrosive Sublimate) —Corrosive
sublimate is soluble in about sixteen parts of cold and three
of boiling distilled water. It is more soluble in aleohol
(1: 3) or in ether (1: 4) than in water. 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 over 15 per cent.

The simple aqueous solutions should always be made with
distilled—not spring—water. The HgCl, in them is partly
split up by hydrolysis into Cl, H, and (HgCl),, or HgClOH
(see Chem. Centralb., 1904, i, p. 571; the statements of
Mayw [Methods, pp. 22, 77] are incorrect). These solutions
should give an acid reaction with litmus paper, whilst those
made with strong sodium chloride solution are neutral.

For fixing, corrosive sublimate may be used pure ; but in
most cases a finer fixation will be obtained if it be acidified
with acetic acid, say about 1 per cent. of the glacial acid. I
find that a saturated solution in 5 per cent. glacial acetic
acid is a very good formula for marine animals; for others
I should take the acid weaker. KatsEr’s solution consists of
10 g. sublimate, 3 g. glacial acetic acid, and 800 g. distilled
water (from Zeit. wiss. Mtk., xi, p. 378). Van Benupen has
used a saturated solution in 25 per cent. acetic acid, and
Lo Branco (Mitth. Zool. Stat. Neapel, ix, 1890, p. 443) a
mixture of 2 parts saturated solution with 1 part of 49 per
cent. acetic acid.

It is sometimes advisable to take the most concentrated

46 CHAPTER V.

solution obtainable. For some very contractile forms (coral
polypes, Planaria), a concentrated solution in warm or even
boiling water should be employed. For Arthropoda alcoholic
solutions are frequently indicated. Delicate objects, however,
may require treatment with weak solutions.

Objects should in all cases be removed from the fixing
bath as soon as fixed, that is, as soon as they are seen to have
become opaque throughout, which may be in a few minutes
or even seconds.

Wash out with water or alcohol. Alcohol is almost always
preferable. Alcohol of about 70 per cent. may be taken,
and (Mayer, Intern. Monatsschr. Anat. Phys., iv, 1887, p. 43)
a little tincture of iodine may be added to the liquid, either
alcohol or water, used for washing, enough to make it of a
good port-wine colour, and the mixture be changed until it
no longer becomes discoloured by the objects. APATHY
(Mikrotechnik, p. 148) takes a 0°5 per cent. solution of iodine
in strong alcohol, leaves the objects in it (suspended) until
they have become of about the colour of the solution, and
then washes for twenty-four hours in pure alcohol.

In obstinate cases solution of iodine in iodide of potassium (e.g.
Luaou’s) may be taken. Mayer (Zett. wiss. Mik., xiv, 1897, p. 28) makes
it by dissolving 5 grammes of iodide of potassium in 5 c.c. of distilled
water and mixing this witha solution of 0°5 gramme of iodine in 45 c.c. of
90 per cent. alcohol, but seldom uses the mixture concentrated, merely
adding as much of it as is required to the alcohol or water containing
the objects. The important point is, that the iodine and iodide be em-
ployed together. Theiodine may be washed out in obstinate cases with
magnesia water. Similarly ApitHy (Mitth. Zool. Stat. Neapel, xii,
1897, pp. 729, 730).

It has been objected to this process that iodine in potassic iodide pre-
cipitates corrosive sublimate instead of dissolving it. That is true, but
the precipitate is soluble in excess of the precipitant.

The iodide of potassium process should be employed with care, for the
iodide may partly redissolve the precipitated compounds formed by the
sublimate with the albuminoids. ete., of the tissues, and it may be well
not to begin adding the iodine till the objects have been brought into
fairly strong alcohol, 70 or 80 per cent.

It is important that the sublimate be thoroughly removed
from the tissues, otherwise they become briti/le, and will not

stain so well. They will also become brittle if they are kept
Jong in alcohol.

FIXING AND HARDENING AGENTS. 47

It may happen that if the extraction of the excess of sub-
limate from the tissues in bulk has been insufficient, crystals
may form in the sections after they have been mounted in
balsam. This may easily be prevented by treating the
sections themselves with tincture of iodine for a quarter of
an hour before mounting. Some workers hold that this does
away with the necessity of treating the tissues in bulk with
iodine, which is frequently a very long process. Thus, Mann
(Zeit, wiss. Mik., xi, 1894, p. 479) prefers treating the sections
rather than the tissues in bulk, on the ground that the
iodine makes them soft, so that they shrink on coming into
paraffin. ScHaprer (Anat. Anz., xiii, 1897, p. 463), however,
has shown that neglect to extract the sublimate from the
tissues in bulk may give birth to serious artefacts, which
appear to arise during the imbedding process. So also
Lovrz (Arch, Anat. Micr., vii, 1905, p. 71). Humennatn
(Zeit. wiss. Mik., xxv, 1909, p. 398) removes the iodine from
sections by means of sodium thiosulphate.

You may stain in any way you like. Carmine stains are
peculiarly brilliant after sublimate.

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, hedgehog spines, or quill pens, or
cactus spines.

When properly employed, sublimate is for general work
undoubtedly a most useful fixing agent. 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
penetrating chitin. For cytological work it is, according to
my experience, not to be trusted, and only to be recom-
mended where more precise fixing agents are counter-indi-
cated by reason of their lack of penetration, or the like.
Amongst other defects it has that of frequently causing very
serious shrinkage of cells.

64. Sublimate with Salt—A solution containing 5 g. sublimate,
05 g. sodium chloride, and 100 c.c. water has been quoted as “solution
of GAULE.”

A one-half per cent. aqueous solution of sodium chloride saturated
whilst hot with sublimate was much recommended by HEIDENHAIN
(Festschrift f, Koelliker, 1892, p. 109),

48 CHAPTER V.

The addition of sodium chloride allows a stronger solution to be
obtained than can be made with pure water, and also, it is stated,
enhances the penetration of the sublimate. But the fixation-precipitates
(§ 29) formed by the double salt are (according to SpuLER, Ency-l. mike.
Technik., p. 1274) for the most part soluble in water, thus giving rise to
imperfect preservation.

Concentrated (i. e. over 20 per cent.) solution in sea-water is recom-
mended for some marine animals.

STOELZNER (Zeit. wiss. Mikr., xxiii, 1906, p. 25) recommends saturated
solution of sublimate in sugar solution of 43 per cent., as isotonic (for
warm-blooded animals).

Liquid of Lang (Zool. Anzeiger, 1878, i, p. 14) —For Planaria.—

Distilled water . : : . 100.
Chloride of sodium. 6 to 10.
Acetic acid é ‘ 5 6 to 8.
Bichloride of mereury : 3 to 12.
(Alum, in some cases 24)

65, Alcoholic Solutions—Ardruy (Mikrotechnik, p. 111)
recommends a solution of 3 to 4 grammes of sublimate
and 05 gramme sodium chloride in 100 ¢.c. of 50 per
cent. alcohol, for general purposes.

OHLMACHER (Journ. Exper. Medicine, ii, 6, 1897, p. 671) takes—

Absolute alcohol : : j 80 parts.
Chloroform , : 7 5 » TS 55
Glacial acetic acid : : ae oer

Sublimate to saturation (about 20 per cent.).

“ Ordinary pieces” of tissue are sufficiently fixed in fifteen to thirty
minutes. Entire human cerebral hemispheres, subdivided by Meynert’s
section, take eighteen to twenty-four hours.

For liquids containing a much higher proportion of acetic acid, see
Acetic Alcohol.

66. Aceton Solution —Hexp (Arch. Anat. Phys., Anat. Abth.,
1897, p. 227), fixes nerve-tissue in a 1 per cent. solution of
sublimate in 40 per cent. aceton, and washes out through
increasingly concentrated grades of aceton.

67. Phenol Solution.—PaprennHerm (Arch. Path. Anat., clvil, 1899,

p. 23) shakes up carbolic acid with aqueous sublimate solution and
filters.

68. Ciaccio (Arch. Ital. Anat. Embr., vi, 1907, p. 486) has an irrational
mixture of sublimate, iodine, and formol.

69. Mercuro-nitric Mixtures—Frenzet (Arch. mik. Anat.,
xxvi, 1885, p, 282) recommends a half-saturated solution of

FIXING AND HARDENING AGENT'S. 49

sublimate in 80 per cent. alcohol, to which is added nitric
acid in the proportion of 1 drop to lee. or 2c¢.c. Objects
of the size of a pea to be fixed in it for five or ten minutes,
then hardened in the same sublimate alcohol without the
acid, and finally in 90 per cent. alcohol. It is said that the
nitric acid renders after-treatment with iodine unnecessary.

Gutson’s Mixture (Gitson, in litt. 1895).

Nitric acid of 46° strength (this

would be sp. gr. 1-456, or 80 per

cent., nearly) . . . . ldce.
Glacial acetic acid : . a ee
Corrosive sublimate ; : . 20 grm,
60 per cent. alcohol : ; » 100 cc.

Distilled water . ‘ ; . 880 ,,

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, this may be removed by washing with
water containing a little tincture of iodine. _

I find that it affords in general a faithful and delicate
fixation, and gives to tissues an excellent consistency.
Objects may remain in it for a considerable time without
hurt. It 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. This liquid may be recom-
mended to beginners, as it is very easy to work with, For
some objects, as I found, the proportion of sublimate may
be increased with advantage.

Kosranucki and Srepiecki (Arch. mik. Anat., xlviti, 1896,
p. 181) take a mixture of saturated sublimate solution and
3 per cent. nitric acid in equal parts, or a mixture of equal
parts of sublimate solution, 3 per cent. nitric acid, and
absolute alcohol, fix for twenty-four hours, and wash out in
iodine-alcohol.

Perrunkewitscu (Zool. Jahrb. Abth. Morph., xiv, 1901, p.
576) takes water 300, absolute alcohol 200, glacial acetic
acid 90, nitric acid 10, and sublimate to saturation. Both
this and Gilson’s have been much used lately.

70. Picro-sublimate Mixtures —Rasi’s (Zeit wiss. Mihk., x1,
1894, p. 165), Sublimate, saturated solution in water, 1
A

50 GHAPTER V.

vol.; a similar solution of picric acid, 1 vol. ; distilled
water 2 vols. Embryos may be left in it for twelve hours

washed for two hours in water, and brought into weak
alcohol,

Mann’s (op. eét., xi, 1895, p. 480) —1 per cent. of pieric acid with or
without 1 per cent. of tannin in a saturated solution of sublimate m
normal salt solution.

The same author's Aleoholic Picro-sublimate (Anut. Anz, vill, 1895.
pp. 441—443) consists of absolute alcohol 100 ¢.c., picric acid 4 yrins.,
sublimate 15 grms., tannin 6 to 8 grms. The tannin is added in order
to prevent excessive hardening.

Pacaut (Arch. Anat. Mier., viii, 1906, p. 438), takes 200 parts of
saturated solution of sublimate and picric acid, 6 of 3 per cent. platinum
chloride, and 5 of 163 per cent. solution of chromic acid.

O. vom Rarn (Anat. Anz., xi, 1895, p. 268) takes cold
saturated solution of picric acid, 1 part; hot saturated
solution of sublimate, 1 part; glacial acetic acid, 4 to 1 per
cent. Also the same with the addition of 10 per cent. of 2
per cent. osimic acid solution,

Fisu (Lrans. Amer. Mier, Soc., xvii, 1896, p. 143) takes 1 litre of
water, 1 g. picric acid, 5 g. sublimate, and 10 g. acetie acid.

Lenuossgx (Intern. Monatschr. Anat. Phys., xxiv, 1907, p. 293) takes
saturated sublimate 75 ¢.c., acetic acid 5, 50 per cent. alcohol 25, and
picric acid to saturation.

71, Osmio-sublimate Mixtures.—Mann’s (Zeit. wis. Mik., xi,
1894, p. 481) consists of afreshly prepared mixture of equal
parts of 1 per cent. osmic acid solution and_ saturated
solution of sublimate in normal salt solution (for nerve-
centres).

Drinen’s (Jena, Zeit. Naturw., xxviii, 1894, p. 294) con-
sists of 1 part of 1 per cent. osmic acid solution added to 20

parts of a solution of 5 per cent. each of sublimate and
glacial acetic acid in water.

O. vom Rarn’s, see last §.

72. Chromo-sublimate,— Lo Bianco (dlitth. Zool. Stat.
Neapel, ix, 3, 1890, p, 443). Concentrated sublimate solu-
tion, 100 parts, 1 per cent. chromic acid, 50 parts.

Mann (Verh. Anat. Ges., 12, 1898, p. 39) takes for nerve-celis equal
parts of 5 per cent. sublimate and 5 per cent. chromic acid.

73. Sublimate and Bichromate —Zm@nker’s Mixture (Miinchener

FIXING AND HARDENING AGEN'I's. 51

med, Wochenschr., 24, 1894, p. 534; quoted from Mercier,
Zeit. wiss. Mik., xi, 4, 1894, p. 471). Five per cent. of
sublimate and 5 per cent. of glacial acetic acid dissolved in
solution of Mttizr. Fix for several hours, wash out with
water, treat the tissues in bulk, or the sections with alcohol
containing tincture of iodine.

See also Retrerer, Journ. Anat. Phys., xxxiii, 1897, p.
463, and xxxvii, 1901, p. 480.

If the objects be allowed to remain too long in the fluid
there may be formed precipitates, which it is very difficult to
remove. Spuier (Hnceycl. mik. Technik., [st edition, p. 1280)
says that they may be avoided by removing the objects as
soon as penetrated, and completing the hardening in liquid
of Miusr.

Dauucren’s modification, consisting of equal parts of
Miiller’s solution and saturated sublimate solution with 5 per
cent. of glacial acetic acid, gives fewer precipitates (Spur,
lc.)

a. (Zeit. wiss. Mik., xx, 1904, p. 413) omits the acetic
acid, and adds, immediately before use, 5 per cent. of formol.

Maximow (tb., xxvi, 1909, p. L179) adds 10 per cent. of
formol and sometimes 10 per cent. of osmic acid of 2 per cent.
(fix in the dark).

FoA (Quart. Journ. Mic. Sci., 1895, p. 287) takes equal parts of
saturated solution of sublimate in normal salt solution, and of liquid of
Miller, or 5 per cent. solution of bichromate.

Brwsipy (Proc. Canadian Inst., v, 1897, p. 77; Zect. wiss. Mik., xvii,
1900, p. 233) takes equal parts of saturated solution of sublimate in 96
per cent. alcohol and 2 per cent. solution of bichromate in water.
Wash out in 50 per cent. alcohol.

Hover (Arch. Mikr. Anat., liv, 1899, p. 97) takes 1 part 5 per cent.
sublimate and 2 of 3 per cent. bichromate.

Koun (¢b., lxx., 1907, p. 273) takes 5 parts 5 per cent. sublimate, 15
parts 34 per cent. bichromate, and 1 part acetic acid.

ARNOLD (Arch. Zellforsch., iii, 1909, p. 433) takes 2'5 parts bichromate,
1 of cupric sulphate, 10 of acetic acid, and 100 of saturated solution of
sublimate.

74, Sublamin (Hthylendiamin Sulphate of Mercury) is recom-
mended in 5 per cent. solution by KLINGMULLER and VBEIEL, Zett. wiss,
Mikr., xxi, 1904, p. 58.

75, Platinum Chloride—The substance used and intended by
the authors who have recommended this reagent is not the

52 CUAPTER V.

true platinic chloride, or tetrachloride, PtCl,, but the com-
pound H,PtCl,, that is, platinochloric, or hydro-chloro-
platinic acid, by custom called platinum chloride. It occurs
as brown-red crystals, easily soluble in water and very
deliquescent. For this reason it had better be stocked in
the form of a 10 per cent. solution, kept in the dark (weak
solutions—0O'5 per cent.—may be kept in the light).

It appears that some authors have stated that they were
using platinous chloride, PtCl,, but that is not possible, as
this salt is not soluble in water.

Rast (Morph. Jahrb., x, 1884, p. 216) employed an
aqueous solution of 1:300. ‘The objects remained in it for
24 hours, and were then washed out with water. Well-
washed preparations give good chromatin stains with the
“basic”? tar colours; but I find, as do others, that plasma-
staining with the “acid” colovrs is rendered extremely
difficult. It causes a certain shrinkage of chromatin.

It is now almost always employed in the form of mixtures.
For these see §§ 44, 45, 49, 76, as well as the mixtures given
under Picric acip and Formo..

76. Rast (Zeit. wiss. Mikr., xi, 1894, p. 165) takes for
embryos of vertebrates, and also for other objects, 1 vol. of
1 per cent. platinum chloride, 1 of saturated sublimate, and
2 of water.

Lennossek (Arch. Mikr. Anat., li, 1898, p. 220) takes 20
parts of 1 per cent. platinum chloride, 20 of 5 per cent.
sublimate, and 1] of acetic acid.

77. Palladium Chloride (ScHULZE, Arch. mik. Anat., iii, 1867, p.
477).—Used by Schulze as a hardening agent in a 1: 800 solution,
acidified with hydrochloric acid.

CaTTANEO has used it in solutions of 1:300, 1-600, or 1:800 strength,
for from one to two minutes, for Infusoria.

FRENKEL (Anat. Anz., viii, 1893, p. 538) recommends for connective
tissue a mixture of 15 parts 1 per cent. palladium chloride, 5 parts 2 per
cent. osmic acid, and a few drops of acetic acid.

78. Iridium Chloride (Eisrn, Zeit. wiss. Mik., xiv, 1897, p. 195).—
Solution of one half or one fifth per cent., acidified with 1 per cent. of
glacial acetic acid.

With the ovotestis of the snail, I have obtained about the worst fixa-
tion I have ever seen, but with the testis of Tritun much better results.

79, Osmium Chloride (HIsEn, Jow n. of Morph., xvii, 1900).—Solution

FIXING AND HARDENING AGENTS. 53

of 3 to 3 per cent. From specimens I have seen I should say it is
useless.

80. Perchloride of Iron (Fou, Zezt. wiss. Zool., xxxviii, 1883, p. 491,
and Lehrb. d. vergl. mik. Anat., p. 102).—Fol recommends 1 vol. of
Tinct. Ferri Perchlor. B.P. diluted with 5 to 10 vols. of 70 per cent.
alcohol.

The tincture diluted with 3 to 4 vols. of either alcohol or water has
been recommended for fixing medullated nerve by PLATNER (Zezt. wiss.
Mik., vi, 1889, p. 187).

81. Iron Alum.—StrRone (Journ. comp. Neur., xiii, 1903, p. 296) fixes
(and decalcifies) heads of young Acanthias in 9 parts of 5 per cent.
solution of iron alum with 1 of formol, for about two weeks.

82. Chloride of Zine is sometimes used for hardening brain (see
Part II). Gitson (La Cellule, vi, 1890, p. 122) has used it as a fixative
for the silk glands of Lepidoptera, as follows :

Glacial acetic acid ‘ ; 5 ce.
Nitric acid of 46° (or 80 er bent, newly) : Deaths
Alcohol of 80 per cent. , ‘ . 100 ,,
Distilled water . : A : F . 300 = ,,
Dry chloride of zine. ‘ : : . 20 grms.

83. Iodine.—KeEnt (Manual of the Infusoria, 1881, p. 114) uses it for
fixing Infusoria. 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 LUGOL’s solution :

Water . : : : . 100 parts.
Iodide of potassinm A z : 6
Todine . ‘ : ‘ 4,

Or for small marine animals, a solution of iodine in sea-water.

Personally I have found it very useful for the examination of sperma-
tozoa.

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 aslide. The slide
should then be warmed to about 40° C. for one to three minutes in
order to evaporate the iodine from the objects, which may then be
mounted or otherwise treated as desired (OVERTON, Zezt. wiss. Mik., vii,
1890, p. 14).

Organic Acids, and other Agents.

84. Acetic Acid—Flemming, who has made a special
investigation of its action on nuclei, finds (Zellsubstanz, etc.,
p- 380) that the best strength is from 0-2 to i per cent.

54 CHAPTER V.

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). The strong acid is, however, a
valuable fixative of certain objects, which it kills with the
utmost rapidity, and leaves fied in a state of extension.
The modus operandi of Van Benepen is 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 80 per cent. alcohol, but this appears to me rather
weak, and I think 70 per cent. or at least 50 per cent. should
be preferred.

Other energetic reagents may be combined with the
glacial acetic acid if desired. Dr. Linpsay Jonnson (in, litt.)
has found that one of the best fixatives for retina is a
mixture of equal parts glacial acetic acid and 2 per cent.
osmic acid. §. Lo Branco adds to the “concentrated ”*
acid one tenth of a 1 per cent. solution of chromic acid.
He finds that even this small proportion of chromic acid
serves to counteract in a marked degree the softening action
of the acetic acid.

Acetic acid, used alone, is only a fixative for a limited
time. If its action be prolonged, it becomes a swelling
agent. Its function in mixtures is, besides that of filling,
the valuable one of counteracting the shrinking action of
the ingredients with which it is combined, and by its swelling
action enhancing the penetration of the mixture; whilst by
clarifying tissues it adds in the optical differentiation of their
elements.

The proportions in which it should enter into mixtures in
general seem to me to be from 0°5 per cent. to 5 per cent. of
the glacial acid; higher strengths, such as 25 per cent. to
100 per cent., being only indicated in cases in which the
highest possible penetration is the chief consideration.

Throughout this work, wherever acetic acid is mentioned,

* The acid referred to as “concentrated” by Lo Branco in his

Metodi (Mitth, Zocl. Stat. Neapel, xi, 3, p. 435) isan acid of approximately
49 per cent. (sp. gr. 1060).

FIXING AND HARDENING AGENTS. 55

it is the glacial acid that is meant unless the contrary is
stated.

All liquids containing a large proportion of this acid (e. g.
$$ 85, 86) should only be allowed to act for a very short
time.

85. Acetic Alcohol (Carnoy, La Cellule, ii, 1886, p. 6; and
ibid., 1887, p. 276; v. Bunnpen et Nuyr, Bull. Ac. Sev. Belg.,
xiv, 1887, p. 218; Zacwarias, Anat. Anz., ii, 1888, pp.
24—27; v. Genucuren, tbid., 8, p. 227).—Carnoy has given
two formule: for this important reagent. The first is—

Glacial acetic acid : . 1 part.

Absolute alcohol ; ‘ . 38 parts,
The second is—

Glacial acetic acid : . 1 part.

Absolute alcohol : ; . 6 parts.

Chloroform . wo8

* S a
The addition of chloroform is said to render the action of

the mixture more rapid.

V. Benepen and Neyr take equal volumes of glacial acid
and absolute alcohol.

Zacuartas takes—

Glacial acetic acid ; ; . I 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 both nuclei and cyto-
plasm, and admits of staining in any way that may be
preferred. It was employed by all of the authors quoted
for the ova of Ascaris—proverbially one of the most difficult
objects to fix,—but I have found that it is applicable to
many other objects. Wash out with alcohol, and avoid
aqueous liquids as far as possible in the after-treatment.

86. Acetic Alcohol with Sublimate—Carnoy and Leproun (La
Cellule, xiii, 1, 1887, p. 68, due to Gitson).
Absolute alcohol : 1
Glacial acetie acid : : . ol
Chloroform . 1

Sublimate to saturation.

vol.

a?

I?)

56 OHAPTER V.

(The mixture does not keep long, forming ethyl acetate,
which precipitates.)

Isolated ova of Ascaris, even though furnished with a
shell, are fixed in twenty-five to thirty seconds. Entire
oviducts take about ten minutes. The liquid is therefore
one of the most penetrating and rapidly acting of any, if not
the most.

Wash out with alcohol until all traces of odour of the
acetic acid have disappeared (I myself wash out with alcohol
containing tincture of iodine). I consider this a very fine
reagent.

For Ohlmacher’s mixture see § 65.

Mrneazzini’s Mixture (Ricerche Lab. Anat. Roma, iii, 1893, p. 47).—
Two vols. saturated aqueous solution of sublimate, one of absolute
alcohol, and one of glacial acetic acid.

87. Trichlor-acetiec Acid (HoLtmarEN, Anat. Hefte, xviii, 1901, H.
2).—5 per cent. solution in water. Fix (nerve-cells) for 8 to 24 hours,
wash out with alcohol. See also HEIDENHAIN, Zeit. wiss. Mikr., xxii,
1905, p. 821, and xxv, 1909, p. 405, who makes a mixture of 6 per cent.
sublimate solution with 2 per cent. of trichlor-acetic and 1 per cent. of
acetic acid, which he calls “ Subtriessig.”

88. Trichlor-lactiec Acid (HoLMGREN, Anat. Anz., xx, 1902, p. 435).—
As the last. Gives rise to serious swelling.

89. Salicylie Acid (HEIDENHAIN, Arch. mik. Anat., liv, 1899, p.
186).—Saturated solution in one-third alcohol. A trial has given me
simply atrocious results.

90. Chloride and Acetate of Copper (Ripart et Petit’s Liquid,
Carnoy, La Biologie Cellulaire, p. 94).—

Camphor water (not saturated) . 75 grms.
Distilled water. : ’ ee
Crystallised acetic acid . : . | grm.
Acetate of copper ; , . 030 ,,
Chloride of copper é ‘ . 0°30 ,,

This is a very moderate and delicate fixative, extremely
useful for objects that are to be studied in as fresh a state as
possible in aqueous media. Objects fixed in it stain instan-
taneously and perfectly with methyl green. Osmic acid
may be added to the liquid to increase the fixing action,
For cytological researches a valuable medium,

FIXING AND HARDENING AGENTS. 57

91. Nitrate of Copper (GiLson, from GELDERD, La Cellule, xxv,
1909, p. 12).—Nitrate of copper 200, formol 500, sea-water 200. Seven
parts of this solution to be diluted with 100 of sea-water. For Crustacea.

92, Acetate of Uranium (ScHENK, Mitth. Embryol. Inst. Wien,
1882, p. 95; ef. Grison, La Cellule, i, 1885, p. 141) has a mild fixing
action, and a high degree of penetration, and may be combined with
methyl green.

FRIEDENTHAL (Svtzb. Ges. Nat. Freunde Berlin, 1907, p. 209) recom-
mends equal parts of saturated solution of the acetate and trichlor-
acetic acid of 50 per cent.

93, Picric Acid.—Picric acid in aqueous solution should
be employed in the form of a strong solution whenever it
is desired to make sections or other preparations of tissues
with the elements iu siéx, as weak solutions macerate; but
for dissociation preparations 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 86
parts of water at 15° C.; 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 several hours.

Picric acid should always be washed out with alcohol, that
of 70 per cent. being mostly indicated. Staining should be
performed by means of alcoholic solutions, or if with aqueous,
then with such as are themselves weak hardening agents,
such as hemalum, carmalum, methyl green.

Washing out is facilitated by heat, the extraction being
about twice as rapid at 40° C, as at the normal temperature
(Fon).

It has been found by Jeuinex (Zeit. wiss. Mik., xi, 1894,
p. 242) that the extraction is greatly quickened by the
addition of a base to the wash-alcohol. He recommends
carbonate of lithia. A few drops of a saturated solution of
the salt in water are added to the alcohol; a precipitate is
formed. ‘The objects are put into the turbid alcohol, which
becomes clear and yellow in proportion as the picrin is
extracted. Further quantities of carbonate are added from
time to time until the colour has been entirely extracted.

58 CHAPTER. V.

Tissues fixed in picric acid can be perfectly stained in
any stain. It is seldom necessary to remove the picric acid
by washing out before staining, Paracarmine, Borax-
carmine, or Haemacalcium may be recommended for entire
objects.

The most important property of picric acid is its great
penetration, This renders it peculiarly suitable for the
preparation of chitinous structures.

94. Picric Alcohol (GAGE, Proc. Amer. Soc. Mier., 1890, p. 120).—
Alcohol (95 per cent.), 250 parts ; water, 250 parts; picric acid, 1 part.

95. Picro-acetic Acid.— Boveri (Zellenstudien, 1, 1887, p. 11) dilutes
a concentrated aqueous solution of picric acid with two volumes of water
and adds I' per cent. of acetic acid. According to my experience, the
results are miserable.

ZIMMER’s mixture (from DrrcENnER, Zool. Jahrb. Abth. Morph., xxvii,
1909, p. 634)—Saturated aqueous solution of picric acid, 10 parts ; abso-
lute alcohol, 9; acetic acid, 1.

96. Picro-sulphurie Acid (KLEINENBERG, Quart. Journ. Mie. Scz.,
April, 1879, p. 208; Mayrr, Mitt. Zool. Stat. Neapel. ii, 1880, p. 2).—
Maver takes distilled water, 100 vols.; sulphuric acid, 2 vols.; picric
acid, as much as will dissolve.

Liquid of KLEINENBERG is made by diluting the concentrated picro-
sulphuric acid prepared as above with three times its volume of water.

Thold that the concentrated solution is generally preferable. This
particularly applies to marine organisms.

Wash out with successive alcohols, beginning with 70 per cent., never
with water.

Warm alcohol extracts the acid much more quickly than cold, without
which weeks may be required to fully remove the acid from chitinous
structures.

This liquid may still be useful for Arthropoda, on account of its great
power of penetrating chitin; and for some embryological purposes.
For a fuller account see early editions.

97. Picro nitric acid (MAyrER, Mitth. Zool. Stat. Neapel, 1881

p. 5).—
Water . ‘ ‘ ’ : . 100 vols.
Nitric acid (of 25 per cent. N,O,) F , 5)
Picrie acid, as much as will dissolve.

Properties of this fluid 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 states that with eggs containing a large amount of yolk material,
like those of Palinnrus, it gives better results than nitric, picric. or

FIXING AND HARDENING AGENTS. 59

picro-sulphuric acid. I myself consider it distinctly superior to picro-
sulphuric for most things.

98. Picro-hydrochloric Acid (Mayrzmr, zbid.).—
Water . : é é . : : . 100 vols.
Hydrochloric acid (of 25 per cent. HCl) . 8
Picric acid, as much as will dissolve.

”

99. Picro-chromic Acid (Fot, Lehrb., p. 100).—

Picric acid, sol. sat. in water ‘ . 10 vols.
1 per cent. chromic acid solution . 3 2B,
Water. ; : . 65

‘ : 3
I have seen Fol’s formula, with the addition of a trace of

acetic acid, quoted as “liquid of Haensel.”

Lo Bianco takes equal parts of picro-sulphuric acid and
chromic acid of 1 per cent.

Rawitz (Lettfaden, 1895, p. 24) takes 1 part of picro-nitric
acid, and four parts 1 per cent. chromic acid. Wash out in
70 per cent. alcohol.

100. Picro-osmic Acid.—FLemMine (Zells. Kern u. Zellth., p. 381)
has experimented with mixtures made by substituting picric for chromic
acid in the chromo-osmic mixtures (§ 42), and finds the results identical,
so far as regards the fixation of nuclei. The fixation of cytoplasm is in
my preparations decidedly inferior.

O. vom RatHi(Anat. Anz., xi, 1895, p. 289) adds to 200 c.c. of saturated
aqueous solution of picric acid, 12 ¢.c. of 2 per cent. solution of osmic
acid, and 2 c.c. of glacial acetic acid.

Rawitz (Leitfaden, p. 24) takes picro-nitric acid, 6 vols.; 2 per cent.
osmic acid, 1 vol. Fix for 3 to3 hours. Transfer direct to 70 per cent.
alcohol.

101. Picro-platinic and Picro-platin-osmic Mixtures.—O. vom
Ratu (toe. ctt., last §, pp. 282, 285) makes a picro-platinic mixture with
200 c.c. saturated aqueous solution of picric acid, 1 g. of platinic chloride
(dissolved in 10 c.c. of water), and 2 c.c. of glacial acetic acid.

The picro-platin-osmic mixture, which is, in my opinion, much
superior, is made by adding to the foregoing 25 c.c. of 2 per cent. osmic
acid.

Other Picric Mixturgs. See §§ 70 and 110 to 112.

Other Mixing and Hardening Agents.

102. Alcohol.—For fiving only two grades of alcohol should
be employed—very weak, or absolute. Absolute alcohol

60 CHAPTER Y.

ranks as a fixing agent because it kills and hardens with
such rapidity that structures have hardly time to get
deformed in the process; very weak, because it possesses a
sufficiently energetic coagulating action and yet contains
enough water to have but a feeble 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 by enhancing their penetrating power; 70 per cent.
is a good grade for this purpose.

Tuble for diluting alevhol (after G.ay-Lussac).—To use this table, find
in the upper horizontal row of figures the percentage of the alcohol that
it is desired to dilute, and in the vertical row to the left the percentage
of the alcohol it is desired to arrive at. Then follow out the vertical
and horizontal rows headed respectively by these figures, and the figure
printed at the point of intersection of the two rows will show how many
volumes of water must be taken to reduce one hundred volumes of the
original alcohol to the required grade.

ORIGINAL GRADE.
Weaker grade
required. '
90 85 80 75 70 65 60 55—i«#‘
p. 100. | p. 100. | p. 100. | p. 100. | p. 100. | p. 100. | p. 100. | p. 100. | p. 100.
|
p. 100, |
656 | |
80 1379) 6-83 |
75 21:89 1448 7-20
70 31°05 | 23:14, 15°35] 7 64
65 ane 33:03 2466] 1637] 8:15
60 5865 4448 35:44 26-47| 17°58! 8°76
55 67-87 | 57-90, 4807 | 3832) 2863 1902) 9-47
|
50 8471, 73-90 68-04) 52-43 41°73) 8125 20-47] 10°35
45 10534 93:30 81°38] 69°54) 5778 4609 34-46] 2290) 11-41
40 —|13080 17°84 104°01| 90°76) 77:58 | 64-48 51-43| 38-46) 25-55
35 163-28 148-01 132 88 117-82 |102°84, 8793. 73-08! 58°31) 43-59
30 206 22 18897 171-5 |183°61 18604 118.94 101 71| 8454 |_ 67-45

Alcohol is an easily oridisable substance, Chromic acid,

FIXING AND HARDENING AGEN'IS, GL

for instance, easily oxidises it, first into aldehyde, and then
into acetic acid. It follows that alcohol should not be com-
bined in mixtures with oxidising agents of notable energy.
Further, alcohol is a reducing agent, and therefore should
not be combined with easily reducible substances. These
remarks particularly apply to chromic acid, see §§ 37, 38, 48.

For fixing, alcohol is a very third-class reagent, only to
be used alone where better ones cannot be conveniently
employed, though it enters as a useful ingredient into many
mixtures, in which it serves to enhance the power of pene-
tration, For hardening it is an important one. 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, aleohol. Large
quantities of alcohol should be taken. ‘I'he alcoho] should
be frequently changed, or the tissue should be suspended
near the top of it (§ 31). 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.

103. Absolute Alcohol.—This is sometimes valuable on
account of its great penetrating power. Mayer finds that
boiling absolute alcohol is often the only means of killing
certain Arthropoda rapidly enough to avoid maceration.

It is important to employ for fixing a very large pro-
portion 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.

As to the supposed superiority of absolute alcohol over
ordinary strong alcohol, see last §; and amongst authors
upholding its superiority, see besides Ranvier, Mayzr
(Mitth. Zool. Stat. Neapel, ii, 1880, p. 7); Brien (Zool.
Jahrb., Abth. Morph., x, 1897, p. 569); and van Reus
(cbid., iti, 1888, p. 10).

Absolute alcohol is a product that it is almost impossible to preserve
in use, on account of the rapidity with which it hydrates on exposure to
air. Fol recommends that a little quicklime be kept init. This absorbs
part at least of the moisture drawn by it from the air.

62 CHAPTER V.

Another plan that I have seen recommended is to suspend strips of
gelatin in it. But it is probably rendered very acid thereby.

Ranvier prepares a sufficiently “absolute ’ alcohol as follows :—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 18
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 flame until
it becomes white, and then reducing it to powder (see Proc. Acad. Nat.
Sci. Philad., 1884, p.27; Journ. Roy. Mic, Soc., 1884, pp. 322 and 984).

Test for the presence of water (Yvon, C. R. Acad. Sci., 1897, p. 1181).—
Add coarsely powdered calcium carbide; the merest trace of water will

cause an evolution of acetylene gas, and on agitation the alcohol will
become turbid.

104. One-third Alcohol—The grade of weak alcohol that is
generally held to be most useful for fixing is one-third
alcohol, or Ranvier’s Atconon. It consists of two parts of
water and one part of alcohol of 90 per cent. (and not of
absolute alcohol). See the Traité Vechnique of Ranvier,
p. 241, eb passim.

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 reagent is a very mild fixative. Its hardening action
is so slight that it is not at all indicated for the fixing of
objects that are intended to be sectioned. Its chief use is
for extemporaneous and dissociation preparations.

105. Acid Alcohol (MayER, Mitth. Zool. Stat. Neapel, ii. 1881, p. 7) —
To 97 vols. of 90 per cent. alcohol add 3 vols. pure hydrochloric (or
nitric) acid. Wash out with 90 per cent. alcohol.

The use of this mixture is principally for the preparation of museum
specimens.

Lo Branco (Mitth. Zool. Stat. Neapel, ix, 1890, p. 443) takes 50 per
cent. alcohol with 5 per cent. of hydrochloric acid.

106. Pyridin.—Pyridin has been recommended as a hardening agent
(by A. DE Souza). It hardens, dehydrates, and clears at the same time.
It is said to harden quickly, and to give particularly good results with
prain. See Comptes Rendus hebd. de la Soc. de Biologie, 8 sér., t. iv,
1887, p. 622.

FIXING AND HARDENING AGENTS. 63

This substance is strongly alkaline, and, either pure or diluted with
water, dissolves many albumens and fats. It causes considerable shrink-
age of nuclei (not so much of cytoplasm). It is now in much use in
certain neuro-fibril stains, see BrzLscHowsky and Ramon. It is

soluble in water and in alcohol. Pure, it will harden and dehydrate
small brains in a week.

107, Acuronn is said to harden very rapidly. Scuouz
(Zeit. wiss. Mikr., xxii, 1905, p. 415) fixes small objects in
warm acetone for half an hour to an hour and brings them
direct, or through alechol and ether, into celloidin.

Similarly Fuss (Arch. path. Anat., clxxxv, 1906, p. 5),
using it cold, and Linrwarew (ibid., ccvi, 1911, p. 36) for
erythrocytes, in which it preserves the hemoglobin.

108. Formaldehyde, Formic Aldehyde, Methyl Aldehyde
(Formol, Formalin, Formalose).—Formaldehyde is the chemical
name of the gaseous compound HCOH, obtained by the
oxidation of methyl-aleohol. ‘ Formol,” “Formalin,” and
“ Formalose ”’ are commercial names for the saturated (40 per
cent.) solution of this in distilled water. ‘This quickly loses
in strength through contact with air, and laboratory solutions
rarely contain more than 38 per cent. of formaldehyde.

Much confusion has been caused by indiscriminate use of
the terms “formaldehyde” and “formol.”’ The proper way is
evidently either to state the strengths of solutions in terms
of formaldehyde, and say so; or to say “ formol—or formalin
—with so many volumes of water.” The majority of writers
seem to state in terms of furmol.

Solutions of formaldehyde sometimes decompose partially
or entirely, with formation of a white deposit of paraformal-
dehyde. Fisu says that to avoid this the solution should be
kept in darkened bottles in the cool, or, according to some,
it suffices to add glycerin to them.

The solutions almost always have an acid reaction, due to
the presence of formic acid; but that is, as a rule, rather an
advantage. But some observers hold that neutral or feebly
alkaline solutions fix better than acid ones. Solutions may
be neutralised by the usual methods: it will generally suffice
to make them up with spring water.

It was said above that formaldehyde possesses certain
hardening and preserving qualities. It hardens gelatine,

6-4. CHAPTER V.

for instance, and certain albuminoids; but others, on the
contrary, are not hardened by it, but sometimes even
rendered more soluble than they are naturally. For some
theoretical considerations concerning its action on tissues,
see F, Brum, in Anat. Anz., xi, 1896, p. 718; Benepgecen, in
Arch. Anat. wu. Phys., Abth., 1897, p. 219; Gurora, in Intern.
Monatschr. Anat., xiii, 1896, p. 108; Zeit. wiss. Mck., xiii, p.
311; Ssdsrine in Anat. Anz., xvii, 1900, p. 274; and Brum,
in Encycl. Mik. Technik., p. 393. It seems to be generally
admitted that this action consists in the formation of methylene
compounds with the substances of the tissues.

I find that, used pure, it is far from a first-class fixative.
For it over-fixes and shrinks some things, and swells and
vacuolates others. But notwithstanding this it is frequently
very convenient on account of its compatibility with the most
various stains. It has a high degree of penetration, and is
a valuable ingredient in many miatures.

It is a powerful reducing agent, and therefore zncompatible
with such reagents as chromic acid or osmic acid and the
like, which it very rapidly decomposes.

For fixing I find that a strength of about 4 per cent.
(1 vol. formol to 9 of water, or to 8 of water if the formol
has been long kept) is generally about right; and this is
the strength used by most writers. Mayrr takes 1 of
formol to 9 of sea-water, for marine animals. Few workers
use much stronger solutions. Only one (Hoyer, Anat. Anz.,
ix, 1894, p. 236, Erganzungsheft) seems to have used
concentrated solutions. JI think this exaggerated, for I
have found enormous over-fixation with solutions of 1 to
2 vols. of water. Wash ont with alcohol (of 50 per cent.
or more), not water.

For hardening, the same strengths may be taken.
Hardening is more rapid than with alcohol. For prolonged
hardening, considerable volumes of liquid should be taken,
and the liquid should be renewed from time to time; for
the formaldehyde fixes itself on the tissues with which it
comes in contact, deserting the solution, which thus becomes
progressively weaker. The specimens should be suspended
in the liquid or otherwise isolated from contact with the
containing vessel. ‘The hardening obtained is gentle and
tough, giving an elastic and not a brittle consistency. It

FIXING AND HARDENING AGENTS. 65

varies greatly. with different tissues. Mucin is not pre-
cipitated and remains transparent. Tat is not dissolved.
Micro-organisms retain their specific staining reactions,
Formaldehyde is said to harden celloidin as well as gelatin,
and to be useful for celloidin-imbedding (Bium, Anat. Anz.,
xi, 1896, p. 724).

Several of the following mixtures are irrational, becoming
reduced more or less quickly, but may give good results all
the same.

109. Alcoholic Formol (Lavpowsky, Anat. Hefte, iv, 1894,
p- 361.)—Water 40 parts, 95 per cent. alcohol 20, formol 6,
acetic acid 1; or water 30, alcohol 15, formol 5, acetic
acid 1.

GuLtanp (Zeit. wiss. Mikr., xvii, 1900, p. 222) takes (for
blood) 1 part formol and 9 parts of alcohol.

Bugs (Trans. Roy. Soc. Edinburgh, xli, 1905, p, 792) takes
1 of formol, 90 of alcohol of 70 per cent., and 3 of acetic
acid.

TeLivesniczKy (Eneyel. mikr, Techn., i, p. 472) takes 5 of
formo], 100 of alcohol of 70 per cent., and 5 of acetic acid,

110. Picro-Formol—P. Bouin (Phénomines cytologiques
anormaue dans L’ Histoyencse, ete., Nancy, 1897, p. 19)
recommends—

Picric acid, saturated aqueous sol. . 78 parts,
Formol . 25,
5

Acetic acid . : F F 33
Wash out with alcohol, first of 50 per cent., then 70 per
cent. till the picric acid is mostly removed. I consider this
to be for most purposes the most valuable fixative yet made
known. I have satisfied myself that the proportions are
exactly what they should be and cannot be changed without
hurt. It is rather a strong fixative, and should not be
allowed to act for more than 18 hours. If a weaker
mixture be desired, dilute the whole with water. The
penetration is great, the fixation equable, delicate detail
well preserved, staining qualities admirable, especially with
iron-hematoxylin and Saiirefuchsin. See also GARNIER,
Bibl. Anat., v, 1898, p. 279.

The formule of Grar (State Ho:p. Bull, New York, 1897; Journ. Roy.
5

66 CUAPTIEL V.

Mic, Sov. 1898, p. 492) ure in my view too weak, and suffer by the
omission of the acetic acid.

Morravux (Bibl. Anit., 1910, p. 265) takes 15 parts formol, 85 of
trichlor-acetic acid of 3 per cent., and picric acid to saturation.

111. Picro-platinie Formol (M. and P. Bouin, Bibl. Avat.,
1898, f. 2, p. 2).—

Platinum chloride, 1 per cent. sol. . 20 parts.
Picric acid, saturated sol. . : i, 20> 3
Formol . ; : ; . 10. ,z,
Formic or acetic agia ‘ . 5

I find this excellent, but the mixture fae not keep more
than a day or two.

Bouin also (Arch. Biol., xvii, 1900, p. 211) simply
substitutes formol for the osmic acid in Herstann’s mixture,
§ 45,

112. Sublimate Formol (M. and P. Bourn, loc. e/t.).—A similar
mixture, in which sublimate of 1 per cent. is substituted for the plati-
num chloride.

Another formula of the same authors (Arch. Biol., xvii, 1900, p. 211)
is 1 part of formol to 3 of saturated aqueous sublimate. Rinse with
water and bring into alcohol of 70 per cent.

SpuLeErR (neyel. mik. Vechnik., 1st ed., p. 1280) adds to sublimate of 3
per cent. or more 1 per cent. of glacial acetic acid and 10 per cent. of
formol.

Mann (Verh. Anat. Ges., 1898, p. 39) takes for nerve-cells 23 g. sub-
limate, 1 g. picric acid, 5 ¢.c. formol, and 100 c.c. water, or (Methods,
etc., p. 97) for all tissues 24 9. sublimate, 20 ¢.c. formol, and SU c.c. water.

Branca (Journ. Anat. et Phys., xxxv, 1899, p. 767) adds 10 parts of
formol and 1 of acetic acid to 60 parts of saturated solution of picric
acid in saturated aqueous sublimate.

Nowak (Anat. Anz., xx, 1901, p. 244) takes 30 parts of saturated sub-
limate, 30 of 1 per cent. chromic acid, 27 of water, 3 of acetic acid, and
10 of formalin.

113. Formol-Muller—This is the name given by Onre
(Berl. klin. Wocehenschr., 1896, No. 13) to a mixture of 1
part of formol with 10 of liquid of Miiller ($ 53). It should
be freshly made up. Fix for three hours in the stove, or
twelve at normal temperature, wash out with running water.
Much used, especially for nervous tissues.

Morier (Zeit. wiss. Zool., [xvi, 1899, p. 85) takes 1 vol.
of formol and 4 of 38 per cent. bichromate (for the intestine
of mammals).

FIXING AND HARDENING AGENTS. 67

Huip (Abk. Stichs, Ges, Wiss., xxxi, 1909, p. 196) takes
3 per cent. sol. of bichromate with 4 per cent. of formol
and 5 per cent. of acetic acid (for inner ear). See also
Moret and Bassat, Journ. Anat. Phys., xlv, 1909, p. 632,

114. Chromic Acid Formol—Lo Branco fixes marine animals
for half to one hour in 10 parts of 1 per cent. chromic
acid with 1 of formol and 9 of sea-water, and passes
into graded alcohols.

Marcuovx (from Psrez, Arch. Zool. Hupér., v, 1910,
p. 11) takes 11 parts 1 per cent. chromic acid, 1 of acetic
acid, 4 of water, and 16 of formol (added just before
using).

115. Copper Formol.—Neuts (Bull. Acad. Se. Belg., 1899 [1900],
p. 726) fixes spinal ganglia for twenty-four hours in 1 litre of 7 per cent.
formol with 5 ¢.c. of acetic acid, 20 g. of cupric sulphate, and sublimate
to saturation.

SrapPers (La Cellule, xxv, 1909, p. 356) used (for Sympoda) a mixture
of Grnson’s: 100 parts of formol of 5 per cent. with 2 of nitrate of
copper.

Srrone (Journ. Comp. Neur., xiii, 1903, p. 296) fixes the head of
Acanthias by injecting a mixture of equal parts of formol and 5 per
cent. solution of bichromate of copper.

116. Nitric Acid Formol.—WILHELMI (Fauna u. Flora Golf. Neupel,
xxxii, 1909, p. 15) fixes Triclads in APATHY’s mixture of equal parts of
6 per cent. nitric acid and 6 per cent. formol, and brings them direct
into strong alcohol.

117. Acetone Formol.—Bine and ELLERMANN (Arch. Anat. Phys.
Phys. Abth., 1901, p. 260) fix medullated nerves in 9 parts of acetone
with 1 of formol.

CHAPTER VI.
DU-ALCOHOLISATION AND CLUARING AGENTS.

118. Introduction.—De-alcoholisation agents are liquids
employed for the purpose of getting rid of the alcohol
which has been employed for dehydrating tissues (§ 3), and
facilitating the penetration of the paraffin used for imbed-
ding, or the balsam or other resinous medium in which
preparations are, in most cases, finally mounted. Hence
all of them 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. The
majority of them are essential oils.

Clearing agents are liquids whose function it is to make
microscopic preparations transparent by penetrating amongst
the highly refracting elements of which the tissues are com-
posed, the clearing liquids themselves having an index of
refraction superior, or equal, or, at all events, not greatly
inferior to that of the tissues to be cleared. Hence all
clearing agents are liquids of high index of refraction.

The majority of de-alcoholisation agents being also liquids
of high refraction, it follows that they serve at the same time
for de-alcoholisation and for clearing; and in consequence
it has come about that de-alcoholisation agents are generally
spoken of as clearing agents. But that practice is not
strictly correct, for not all clearing agents are solvents
of the resins, and not all de-alcoholising agents can serve
as clearers. I shall, however, still in many cases continue
to use the term “ clearing” to signify “ de-alcoholising,” for
the sake of brevity.

Neevsen and Scuigerrerpecker (Arch. Anat. Phys., 1882,
p. 206) examined a large series of ethereal oils (prepared
by Schimmel] and Co., Leipzig), with the object of finding a
not too expensive substance that should combine the proper-

DE-ALCOHOLISATION AND CLEARING AGENTS, 69

ties of clearing quickly alcohol preparations, not dissolving
out anilin colours, clearing celloidin without dissolving it,
and not evaporating too quickly.

Of these, the following three fulfil the conditions :—Cedar-
wood, Origanum, Sandal-ivood.

To these should be added the others recommended in the
following paragraphs.

See also the paper of Jorpawn (Zeit. wiss. Mik., xv, 1898,
p. 50), as to the behaviour of some essential oils towards
celloidin.

119, The Practice of De-alcoholisation or Clearing.—The
old plan was to take the object out of the alcohol and float
it on the surface of the de-alcoholising or 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 de-alcoholising or clearing agent
can get into it; hence the object must shrink. To avoid
this cause of shrinkage, the operation is now generally done
by the method suggested by Mayer and Giesbrecht, which
consists in putting the clearing medium wnder the alcohol
containing the object, as described § 5. The objects should
not be considered to be perfectly penetrated by the clearing
medium until the wavy refraction-lines caused by the mix-
ture of the two liquids at their surface have ceased to form,
and they should not be mounted or imbedded until they
have first been soaked for some time in a fresh quantity of
clearing medium, to remove. any alcohol that has got into the
first bath.

The penetration ofall clearing media may be hastened
by using them warm.

It frequently happens that the essential oil with which
objects 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 essen-
tial oil and moisture, derived, I think, rather from the air
than from the objects themselves. The cloudiness can
usually be removed by warming (as pointed out by
Harcusrr Jackson, Zool. Anzeiy., 1889, p. 630), but in certain
moist states of the atmosphere it may persist, notwithstand-
ing continued warming. It is for this reason that I advise

70 CHAPTER VI.

that clearing be done, whenever possible, in shallow corked
tubes, under which conditions the phenomenon rarely occurs.
In any case, be careful not to breathe on the liquid.

120. Choice of a De-alcoholisation or Clearing Agent—I
advise the beginner to keep on his table the following :—
Oil of cedar, for general use and for preparing objects for
imbedding in paraffin; clove oil for making minute dissec-
tions in (§ 122), and for much work with safranin, etc.; oil
of bergamot, which will clear from 90 per cent. alcohol, and
which does not extract coal-tar colours; carbolic acid, for
rapidly clearing very imperfectly dehydrated objects.

For special clearers for celloidin sections see Chapter IX.

121. Cedar Oil (NeeLsen and Scurerrerpecker, loc. cit.,
§ 118).—Clears readily tissues in 95 per cent. alcohol aith-
out shrinkage; does not extract anilin colours. Celloidin
sections are cleared in five to six hours.

The observer should be careful as to the quality of the
cedar oil he obtains. JI have examined the clearing proper-
ties of a sample, obtained from a celebrated firm, which
totally failed to clear absolute alcohol objects after many
days.

Cedar oil is very penetrating, and for this and other
reasons is, in my experience, the very best of all media for
preparing objects for paraffin imbedding. I find it to be less
hurtful to cells than any other medium known tome. Tissues
may remain in it for any length of time without hurt. If it
should become milky through keeping, filter.

122. 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. 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 oi] is one of the most adulterated
substances in commerce.

Clove oil does not easily spread itself over the surface of

DE-ALCOHOLISATION AND CLEARING AGENTS. i.

a slide, but has a tendency to form very convex drops. This
property makes it a very convenient medium for making
minute dissections in. It also has the property of making
tissues that have lain in it for some time very brittle. This
brittleness is also sometimes very helpful in minute dissec-
tions,

These qualities may be counteracted if desired by mixing
the clove oil with bergamot oil.

This is one of the most useful of clearers. According to
Benrens (Tahellen, 3 ed., 1898, p. 33), it will clear from
alcohol of 74 per cent.

Tt has a high index of refraction, and clears objects more
than balsam mounting media, It dissolves celloidin (or col-
lodion), and therefore should not be used for clearing
sections cut in that medium, without special precautions.
New clove oil washes out basic tar colours more quickly than
old.

123. Cinnamon (or Cassia) Oil greatly resembles clove oil, but is in
general thinner, and is more highly refractive. An excellent medium,
which I particularly recommend.

124. Oil of Bergamot (Scuievrerpecker, Arch, Anat. Phys.,
1882 [Anat. Abth.], p. 206).—Clears 95 per cent. alcohol
preparations and celloidin preparations quickly, and does
not extract anilin colours.

Bergamot oil is, I believe, the least refractive of these
essences, having a lower index than even oil of turpentine.

Sucwannek (Zeit. wiss. AMik., vii, 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.

Van ver Srricut (Arch. de Biol., xii, 1892, p. 741) says
that bergamot oil will, with time, dissolve out the fatty
eranules of certain ova.

125. Oil of Origanum (Nerisen and ScurerrerpecKer, Arch.
Anat. Phys., 1882, p. 204)—Ninety-five per cent. alcohol
preparations are cleared quickly, and so are celloidin sec-
tions, without solution of the celloidin. Anilin colours are
somewhat extracted.

For work with celloidin sections care should be taken to

72 CHAPTER VI.

obtain Ol. Origani Cretici (“Spanisches Hopfendl”’), not Ol.
Orig. Gallici (v. Gieson; see Zeit. wiss. Mik., iv, 1887, p.
482). Specimens of origanum oil vary greatly in their action
on celloidin sections.

Squire, in his Methods and Formule, etc., p. 81, says that
origanum oil (meaning the commercial product) is nothing
but oil of white thyme more or less adulterated (see next §),
and that the product sold as Ol. Origani Cretici is probably
oil of marjoram.

126. Oil of Thyme—F isu (Proc. Amer. Mic. Soc., 1893 ;
Zeit. wiss. Mik., xi, p. 503), following Bumpeus, says that for
most of the purposes for which origanum oil has been recom-
mended, oil of thyme will do just as well if not better. The
ved oil is just as efficient as the white for clearing.

Schimmel and Co., in their Report of October, 1895, p. 69,
state that in France white oil of thyme is adulterated with
oil of turpentine to the extent of as much as 50 per cent.

127. Oil of Gaultheria.—Used by Unna (Monatschr. prakt. Derm.,
Ergiinaungsh, 1885, p. 53) for thinning balsam. The artificial oil,
methyl salicylate, is recommended by GUEGUEN (Comp. Rend. Soc.
Biol., v, 1898, p. 285) both as a de-alcoholisation and clearing agent and
as a solvent of paraffin. The refractive index is 153. It is, unfor-
tunately, very sensitive to water.

128. Sandal-wood Oil (NEELSEN and SCHIEFFERDECKER, loc. cit.).
—Very useful, but its high price is prohibitive.

129. Oil of Cajeput—Now much used. I have used it
myself and found it to clear well, but to be rather thin.
Carnoy and Lesrun (La Cellule, xiii, 1897, p. 71) have found
it useful for clearing celloidin sections. It dissolres celloidin
very slowly and clears without shrinkage.

130. Oil of Turpentine —Generally used for dissolving out the
paraffin from sections; but many other reagents, such as xylol, benzol,
are preferable for this purpose. If used for alcohol objects, it canses
considerable shrinkage, and alters the structure of cells more than any
other clearing agent known to me. Turpentine has, I believe, the
lowest index of refraction of all the usual clearing agents except
bergamot oil; it clears objects less than balsam,

131, Terpinol (liquid, from Schimmel and Co.) is recom-

DE-ALCOHOLISATION AND CLEARING ACENTS, 73

mended by Mayer, Zeit, wiss. Mikr., xxvi, 1910, p. 523.
Clears from alcohol of 90 per cent., or even 80 per cent.

132. 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.

Gaae’s Mixture (Proc. Amer. Soc. Mier., 1890, p. 120)—Carbolic
acid crystals melted, 40 c.c.; oil of turpentine, 60 c.c.

133. Creosote —Much the same properties as carbolic acid.
Beech-wood creosote is the sort that should be preferred for
many purposes,—amongst others, for clearing celloidin sec-
tions, for which it is a very good medium.

134, Anilin Oil1—Common anilin oil will readily clear
sections from 70 per cent. alcohol, and with certain pre-
cautions (for which see the paper of SucwanneK quoted
below) objects may be cleared from watery media without
the intervention of alcohol at all. This renders it valuable
in certain cases as a 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. For directions
for preparing this see Sucnannex, Zeit. wiss. Mik., vii, 1890,
p. 156, or the third edition of this work.

Anilin is chiefly used for clearing celloidin sections. It
ought however to be soaked out before mounting by some-
thing else (chloroform or xylol for instance for some hours),
as if not removed it will brown both the tissues and the
mounting medium.

135. Xylol, Benzol, Toluol, Chloroform.—'oo volatile to be
recommendable as clearing agents in which it is desired to
evamine specimens, but very useful for preparing paraffin
sections for balsam. Of the three first-mentioned liquids,
benzol is the most volatile, then toluol, and xylol is the least
volatile, in the proportion of 4: 5: 9 (Squiru, Methods and

74 CHAPTER VI.

Formule, p. 20). Chloroform is injurious to some delicate
stains, but is in other respects an excellent de-alcoholisation
agent, as it will take up a good deal of water, if any be left
in the preparations. I consider it too volatile to be safe to
use before balsam. Xylol is the best of these in that

respect.
Both xylol and toluol are liable to become acid if kept in

only partially filled vessels.

CHAPTER VII.
IMBEDDING METHODS—INTRODUCTION.

136. Imbedding Methods—The processes known as Im-
bedding 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 instrument, 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 in-
cluded object may be cut into sufficiently thin slices without
distortion. Secondly, they enable us to fill out with the im-
bedding 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 not only to
surround with the supporting mass each individual organ or
part of any organ that may be present in the interior of the
object, but also to fill with it each separate cell or other
anatomical element, thus giving to the tissues a consistency
they could not otherwise possess, and ensuring that im the
thin slices cut from the mass all the minutest details of
structure will precisely retain their natural relations of
position.

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
Inbedding Methods; or the object is saturated with some
substance which whilst in solution is sufficiently fluid to
penetrate the object to be imbedded, whilst, after the evapo-
ration or removal by other means of its solvent, it acquires
and imparts to the imbedded object sufficient firmness for
the purpose of cutting. The methods founded on this
principle are here called Evaporation Imbedding Methods,

76 CHAPTRR VII.

In any of these processes the material used for imbedding
is technically termed an “ imbedding mass.”

There are two chief methods of imbedding—the paraffin
method and the celloidin or collodion method,

The paraffin method is the one in most use; for it is the
more rapid, requiring only hours where the celloidin process
requires days or weeks ; and it is the one which the most
readily affords very thin sections. But this only apples to
fairly small objects: with objects of much over half an inch
in diameter you cannot easily get with paraffin much thinner
sections than you can with celloidin ; and if you try to cut
in paraffin objects of still greater size, say an inch and
upwards, it will frequently 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. This
defect is, however, much reduced by the employment of a
softer paraffin than is usual. In this way Srrasser (Zeit.
wise, Mik., ix, 1892, p. 7) has obtained series of frontal
sections 80 « thick through the entire human brain, in
paraffin blocks measuring 10 x 15 cm. And Mayer, with
the ''etrander microtome, has obtained series of only 7°5 mu
with a surface of 44 x 3 cm.

For very large objects celloidin is safer, because it does
not split, and presents advantages for the manipulation of
the sections obtained. For all classes of objects it has
the advantages of affording a transparent mass (which
facilitates orieutation of the object), and of producing less
shrinkage than paraffin (paraffin unavoidably shrinks on
cooling to at least 12 per cent.). It is for these two reasons
that celloidin is so frequently preferred by embryologists—
even for small objects.

Aqueous masses, such as gum or gelatin, may render great
service in cases in which it is desired to arotd dehydrating
tissues, and to apply cheinieal tests to them.

137. Imbedding Manipulations—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, and the object placed in
the midst of it. Or, the paper tray being placed on cork,
the object may be fixed in position in it whilst empty by:

IMBEDDING METHODS. 77

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
apnexed figure (Fig. 1); thin (foreign) post-cards do very
well indeed. Fold it along the lines « a’ and 0’, then along
cc and dd’, taking care to fold always the same way.

a b
| |
| |
WN onlin tomer | Seemerene hy
eaeat eae
NY | | Y
Ba | | “
¢ seme ee j“¥_-___je'
A B|
|
| | a5
| |
| |
| |
lo p! '
[ney fiee eam [Re ete | eee
d a re d
tam Rl “Se
pa | | SS
ee ae, San Ul os oe a2 (Peyewnnesit tere
|
L |
aw b
Fig. 1.

Then make the folds 4 A’, B B’,C C’, D L’, still folding the
same way. ‘To do this you apply 4 ¢ against 4 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.

Another method of folding the paper (Mayur) is described
in the Grundziige, Luz and Mayer, 4th ed., p. 77.

78 CHAPTER VIL.

GIESBRECHT now makes trays of photographie films, which
being transparent facilitate orientation under the dissecting
microscope.

To make paper thimbles, take a good cork, twist a strip of
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 loaded at the bottom by means of a nail
or piece of lead, to prevent it fron floating when the whole
is thrown into spirit or other liquor for hardening (Fig. 2).

Leuckuani’s Imbedding Boxes are made of two pieces of
type-metal (Fig. 3). Hach 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 glycerine
and gently warmed. The area of the
box will vary according to the position
given to the pieces, but the height can
be varied only by using different sets
of pieces. ‘Two sets will be sufficient
for most work; one set of one centi-
metre in height, and one of two centi-
metres, each being eight centimetres
in length, and three in breadth. To
make the box paraffin-tight, so that it
will hold the melted paraffin long enough in the liquid
state to permit of the objects being carefully orientated in it,
Mayer ((Mitth. Zool. Slat. Neapel, iv, 1883, p. 429) first
smears the glass plate with glycerin, then arranges the metal
“squares,” and then fills the box with collodion, which
is poured out again immediately. As the ether evaporates,
a thin layer of collodion remains behind, which suttices to
keep the paraffin from running out. Even without the
collodion, the mere cooling of the paraftin by the metal will
generally suffice to keep it in long enough for orientation, if
it is not in a superheated state when it is poured in.

In such a collodionised box the paraftin may be kept in a
liquid state by warming now and then over a spirit lamp, and

IMBEDDING METHODS. 79

smail objects be placed in any desired position under the
microscope (Journ, Roy, Mie, Soc. [N.S.], u, p. 830).

A lighter form of “squares,” made of brass, and devised
by Anpres, GiesBeecHt, and Mayer, is described lve. cit.
(see Journ. Roy. Mic. Suc., 1883, p. ¥13). A more compli-
cated sort is described by Witson in Zett. wiss. Mik., xxvii,
1910, p. 228, for use with imbedded threads to serve as
orientation guides (see “ Orientation”).

Franki (Zeit. wiss, Mik., xin, 1897, p. 438) builds up
boxes with rectangular blocks of glass, which may be found
convenient, but are more expensive than the metal squares.

SELENKA has described and figured another sort of apparatus having
the same object. It consists 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., 1885, p. 419). A simple modification of this apparatus, which
anyone may make for himself, is described by ANDREWS in Amer.
Natural., 1887, p. 101; and a more complicated imbedding and orienting
box, seldom necessary, is described by JoRDAN, in Zett. wiss. Mck., xvi,
1899, p. 32.

To imbed in a watch-glass, the object, previously saturated
with paraffin, is put into a (preferably very concave) watch-
glass containing molten paraffin. After this has been solidi-
fied by cooling (see next chapter), a block containing the
object is cut out of it, and mounted on the object-holder of
the microtome (this is, of course, applicable to other masses,
such as celloidin).

For imbedding very small objects in this way certain precautions may
be necessary in order not to lose them. SamrTeR (Zezt. wiss. Mik., xi,
1894, p. 469) saturates small unstained objects with paraffin that has
previously been strongly coloured with alkanna extract, and then imbeds
them in pure paraffin. RHUMBLER (ibdd., xii, 1895, p. 312, and xiii, 1896,
p. 303) stains previously the objects themselves with eosin dissolved in
strong alcohol, and removes the stain from the sections with weak
alcohol. See also ibid., xili, p. 200, a paper by ScHYDLOWSKI; and in
Zeit. wiss. Zool., lviii, 1897, p. 144, a process of BORGERT.

Borerrt (Zeit. wiss. Zool., lviii, 1897, p. 144) allows paraffin to solidify
in a watch-glass, bores a hole in it, and places the objects in the hole
with a little benzol, and puts the whole for a short time into a stove.

A watch-glass provided at the bottom with a groove or trough, in
which small objects may be made to collect, is described by Lerrvrs,
Journ. App. Mic., v, 1902, p. 280 (see Journ. Roy. Mic. Soc., 1903, p. 233).

LAUTERBORN (Zeit. wiss. Zool., lix, 1895, p. 170) brings the objects

80 CHAPTER VIL.

through chloroform into paraffin in a small glass tube, and after cvoling
breaks the tube and so obtains a cylinder of paraffin with the objects
ready for cutting.

Hoyer (Arch. mik. Anat., liv, 1899, p. 98) performs all the operations
in a glass cylinder (5 cm. long and 7 mui. wide), open at both ends, Lut
having a piece of moist parchment paper tied over one of the openings.
It is then not necessary to break the cylinder; by removing the parch-
ment paper the paraffin can be pushed out of it in the shape of a
cylinder containing the objects imbedded at one end of it.

MAYER (Zett. wiss. Mikr., xxiv, 1907, p. 180) takes the gelatin capsules
used by chemists; after cooling in water the gelatin swells and is easily
removed.

Meves (Arch. mikr. Anat., xxx, Ath. ii, 1912, p. $5) employs wedge-
shaped capsules made by G. Pohl, Schénbauw, Bez, Dantzig.

CHAPTER VIII.
IMBEDDING METHODS—PARAFFIN AND OTHER FUSION MASSKS.

138. Saturation with a Solvent.—The first stage of the
paraffin method consists in the saturation of the object with
some substance which is a solvent of paraffin. The process
is sometimes called “ clearing,” since many of the substances
used for infiltration are also “clearing ”’ agents.

The process of saturation should be carefully performed
with well-dehydrated objects in the manner described in
§ 119.

Saturation liquids being liquids that are, on the one hand,
miscible with alcohol, and on the other hand good solvents
of paraffin, are not quite as numerous as could be wished.

According to GRazre (Chem. Centralb., 1906, p. 874), at
20° C. petroleum ether (1 c.c.) dissolves 200 mg. of paraffin ;
chloroform 246; benzol 285; carbon tetrachloride 317.
And according to ApAruy, at 20° U. benzol dissolves 8 parts
per cent., chloroform 10, toluol 10, xylol 12, oil of turpen-
tine 8, cedar oil 4 tu 6, bergamot oil 0°5 to 3, creosote and
clove oil hardly any. Acetone, according to Mayr, dissolves
hardly any.

Turpentine I do not recommend, because in my experience
it is of all others the clearing agent that is the most hurtful
to delicate structures.

Clove oil mixes very imperfectly with paraffin, and quickly
renders tissues britile.

Oil of bergamot mixes still more imperfectly with
paraffin.

Benzol has been recommended by Brass (Zeit. wiss. Mik.,
ii, 1885, p. 301), and is now much used.

Toluol (or toluen) has been recommended by Hott (Zool,
Anz., 1885, p, 223),

6

82 CHAPTER VIII.

Xylol is said by M. Heiwennaiw (Kern wnd Protoplasma,
p- 114) to be a cause of shrinkage in cells. So it is, if you
use it to de-alcoholise the specimens. But used after otl of
cedar, ov the like, it is very good, as it is one of the best of
solvents of paraffin.

Chloroform 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. The process of removal
requires a very long time, in some cases days. It ought
therefore to be reserved for small and easily penetrable
objects.

Naphtha has been recommended by WessTER (Jowin. Anat. and
Physiol., xxv, 1891, p. 278).

FIEtp and Martin (Zeit. wiss. Mik., xi, 1894, p. 10) recommend a
light petroleum known as “ petroleum-wther.” It is highly volatile,
and thus a cause of shrinkage.

Sulphide of carbon has been recommended by HEIDENHAIN (Ze/t.
wiss. Mik., xviii, 1901, p. 166) as being a very powerful solvent of
paraffin. Most workers have found it to be much too disagreeable and
dangerous a reagent for ordinary work. and not necessary even for
delicate work.

Carbon tetrachloride has been recommended by PLECNIK (op. e7t., xix.
1903, p. 328) and PRanTER (zb7d., p. 329) on the ground of not dissolving
out osmium-blackened fats.

Maver finds it no better than henzol.

As a general thesis, the best of all these are cedar oil,
benzol, and chloroform.

Cedar-wood oil is, according to my continued experience,
for the reasons stated by me in Zool, Anz., 1885, p. 563, for
general work the very best clearing agent for paraffin im-
bedding. 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,

IMREDDING METHODS. 83

139. The Paraffin Bath—The objects having been duly
saturated with a solvent, the next step is to substitute
melted paraffin for the saturating medium.

Some authors lay great stress on the necessity of making
the passage from the saturating agent to the paraffin as
gradual as possible, by means of successive baths of mixtures
of solvent and paraffin kept melted at a low temperature,
say 35° C. With oil of cedar, at all events, this is not
necessary. I simply put the objects into melted paraffin
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 volu-
minous to have brought over with them a notable quantity
of clearing agent. If the objects have been for a very long
time—months or years—in the cedar oil, so that this has
become thick, I remove it partially or entirely by soaking
in xylol (80 minutes to several hours) before putting into
the paraffin. But with fresh oil of cedar I find no advantage
in doing so.

GrusBrecHY’s method (Zool. Anz., 1881, p. 484), is as
follows :—Objects to be imbedded are saturated with
chloroform, and 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 tu the chloroform. So soon as it is seen that
no more bubbles are given off from the objects, the addition
of paraffin 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.

Mayrr (Grundziige, Lun and Mayer, 1910, p. 84) first
saturates the objects with benzol, and then adds to the
benzol some small pieces of paraffin, and lets them dissolve
in the cold. After several hours (up to eighteen) the whole
is brought in an open vessel on to the cold water-bath, the
bath is then warmed gradually so as to attain a temperature
of 60°C. in about two hours, and as fast as the benzol
evaporates melted paraffin is added to it. Lastly, the
paraffin is changed once before the definitive imbedding,
He rarely leaves objects overnight in the water-bath.

Apdtuy (Mikrotechnik, pp. 149, 150) first clears with oil

84 CHAPTER VIII.

of cedar, then brings the objects (by the process described
§ 119) into a solution of paraffin in chloroform saturated at
the temperature of the laboratory. The objects remain in
the chloroform-paraffin solution for from one to three hours,
without warming, until all the cedar oil is soaked out of
them. The whole is then warmed on the water-bath or
oven to a few degrees above the melting-point of the paraffin
intended to be used for imbedding, and the object is brought
into a mixture of equal parts of paraffin and chloroform,
being suspended therein near the top on a bridge made of
hardened filter paper (or in a special apparatus to the same
end, not yet described). It remains in this mixture, at the
temperature of the oven, for one to three hours, and lastly
is brought (still on the paper bridge or in the apparatus)
into pure paraffin, where it remains for half an hour to two
hours.

Denne (in litt., 1907) points out that the objects ought at
first to be at the bottom of the mixture. For this mixture
is not a true solution, and the lower section of the contents
of the tube is comparatively free from paraffin while the
upper part is nearly pure paraffin. He moves the holder
up in the tube at intervals, and the infiltration proceeds
gradually with the minimum risk of shrinkage. Lastly, he
removes the objects, on the holder, to the top of a tube
of pure paraffin.

The practice of giving successive baths first of soft and
then of hard paraffin, which has been frequently advised,
appears to me entirely illusory.

It is important to keep the paraftin 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 melting-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. Overheating, as
well as prolonged heating, tends, amongst other things, to
make tissues brittle.

The duration of the bath must, of course, vary according
to the size and nature of the object. An embryo of 2 to 3
millimetres in thickness ought to be thoroughly saturated

IMBEDDING METIODS. 85

after an hour’s bath, or often less. Many workers habitually
give much longer baths, I think often longer than necessary.
But some objects, such as ova of Crustacea, may require
three or four days. (Heiecks, Jena. Zeit., xxxviii, 1904,
p. 506; Mayvzr, Grundziige, Luz and Mayr, 1910, p. 85;
Brinkmann, Mitth. Zuol. Stat. Neapel, xvi, 1903, p. 367,
three to five days for uterus of Selachians: Mijuiur, Arch.
mikr, Anat., xix, 1906, p. 3, for lungs of mammals; Poso,
Esperienze microtechniche, Napoli, 1910, p. 29, five to twelve
days for uterus and placenta of Lomo.) I take as a guide,
generally, the length of time the object has taken to clear
in the cedar oil, assuming that the warm melted paraffin
ought to penetrate at least as quickly as the cold oil;
and then allowing somewhat longer, say as much again,
in order to be on the right side.

140. Water-baths and Ovens.—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 tempera-
ture 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,
or “ Naples water-bath,” which will be found described at p. 146 of
Journ. Roy. Mic. Soc., 1883, or CARPENTER’S T'he Microscope, p. 452.
An extremely simple stove, which anyone can make for himself, is
described in Centralbl. Bakt., xlv, 1907, p. 191 (see Journ. Roy. Mic. Soc.,
1908, p. 109). For others, see the price-lists of the instrument makers,
especially June, and GxrUBLER and HoLuporn; and the descriptions in
the technical journals.

141. Imbedding 1n 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 under a vacuum (strictly, under diminished
atmospheric pressure) 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. It is realised by means
of any arrangement that will allow of keeping paraffin melted under a
vacuum.

That of HorrmMann is described and figured at p. 280 of Zool. Anz.,
1884. In this arrangement the vacuum is produced by means of a
pneumatic water aspiration pump, the vessel containing the payrafiin
being placed in a desiccator heated by a water bath and furnished with
a tube that brings it into communication with the suction apparatus.

86 CHAPTER VIII.

Francorre (Bull. Soc. Belg. Méic., 1884, p. 45) produces the reyuisite
vacuum by the condensation of steam.
' Fou (Lehrb., p. 121) employs the vacuum apparatus of Hoffmann, but
simplifies the arrangement for containing the paraffin. The paraffin is
contained 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, then turn out the object with the paraffin
(which by this time will have become abnormally hard), and re-imbed in
fresh pavrafiin.

See also PrinGLE, in Journ. Path. wid Bacteriol., 1892, p. 117; or
Journ. Roy. Mic. Soc., 1892, p. 893; Kouster, in Zeit. wiss. Mik. xviii,
1901, p. 170; Bure, Zett. wiss. Mik., xxvi, 1909, p. 200; FUHRMANN,
ibid, xxi, 1904, p. 462; Koumer and Wotrr, ibid., xix, 1902, p. 148;
GEMMILL, Journ. Roy. Mic. Soc., 1911, p. 26.

142. Imbedding and Orientation —.lx soon ux the objects are
thoroughly saturated with paraffin they should be émbedidled
by one of the methods given above (§ 137), and the paraffin
cooled as described next §,

But it may be desirable to have the object fixed in the
cooled paraffin in a precisely arranged position, and, above
all, in a precisely marked position. Very small objects may
be oriented as follows:—The object is removed from the
melted paraftin, and placed on a cylinder of solid paraffin.
A needle or piece of stout iron wire is now heated in the
flame of a lamp, and 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 advan-
tages of the method lie in the quickness and certainty with
which it can be performed. In using the needle it is
important to melt as little paraffin as possible at oue time, in
order that that which is melted may cool again as rapidly
as possible.

Kerr (Quart. Jowrn, Alicr. Se., xlv, 1901, p. +) employs
an electrically heated needle.

The method of Parren (Zezt. wiss. Mik., xi, 1894, p. 13) is useful
when one desires to orient large numbers of small objects. You get
some writing paper of the sort that is made with two sets of raised
parallel lines running at right angles to each other (“linen cloth paper”).
Small strips are cut from this, and at suitable intervals along them small
drops of a mixture of collodion and clove oil, of about the consistency of
thick honey, are arranged close together along one of the ribs that run
lengthwise. The objects to be im)edded are cleared in clove oil or oil of

INMBEDDING METHODS. 87

bergamot. They are taken one by one on the point of a knife, and after
the excess of oil has been drawn off, are transferred each to a drop of
the collodion mixture, in which they will stay in any required position,
When half a dozen or more objects have been oriented in reference to
the cross lines (which are to be parallel to the section planes) the whole
thing is placed in turpentine. This washes out the clove oil and fixes
the objects very firmly to the paper. The paper with the attached
objects is now passed through the bath of paraffin and imbedded in the
usual way, After cooling on water the block is trimmed and the paper
peeled off, leaving the objects in the paraffin close to the under-surface
of the block. This surface is now seen to be marked by the orienting
lines of the ribbed paper, and also by any record numbers which may
before imbedding have been written with a soft pencil on the paper.

Knowen (Journ, Morph., xvi, 1900, p. 507) takes smooth paper and
engraves parallel lines on it with a needle, and takes xylol instead of
turpentine.

A somewhat more complicated form of this process has been
described by Woopwortu, Bull. Mus. Comp. Zool., xxxviii, vol. xxv,
1893, p. 45.

A similar process has also been described by FreLD and Marrin in
Zeit. wiss. Mik., xi, 1894, p. 11, small strips of gelatin beg used instead
of paper.

MAYER also (Grundziige, Ler and Mayer, 1910, p. 89) takes strips of
photographic gelatin, and lets the collodion set in benzol.

Horrmann (Zeit. wiss. Mik., xv, 1899, p. 312, and xvii, 1901, p. 443)
takes, instead of the ribbed paper, glass slips ruled with a diamond, and
completely imbeds the objects in large drops of clove oil collodion
(equal parts), allowed to stand for twenty-four hours in an open vessel.
The drops are caused to set in xylol. See also SamTur, <bid., xiii, 1897,
p. 441; Jorpan, zbid., xvi, 1899, p. 33; and Prter, Verh. Anat. Ges.,
xili Vers., 1899, p. 134.

Entz (Arch. Protistenk., xv, 1909, p. 98) orients in clove oil collodion
on a cover-gluss coated with paraffin, and puts the whole into chloroform
in which the mixture sets into a sheet which can be detached.

DENNE (Journ. Appl. Mic., iii, 1902, p. 888) imbeds on disks of paper
held at the bottom of glass tubes containing the paraffin by bent wires,
by means of which a cylinder of paraffin containing the object may be
lifted out as soon as cool.

Wison (Zeit. wies. Mik., xvii, 1900, p. 169) makes orientation lines by
imbedding alongside the objects strands of osmium-blackened nerve-
fibres. See also a further development by Wilson, zbzd., xxvii, 1910,
pp. 228 and 231.

143. Cooling the Mass.—Whatever method of imbedding
and orientation in the molten paraffin has been employed,
the important point now to be attended to is that the paraffin
be cooled rapidly. The object of this is to prevent crystalli-

88 CHAPTER VIII,

sation of the paraffin (which may happen if it be allowed to
cool slowly) and to get as homogeneous a mass as possible.

If the definitive imbedding has been done in a watch-
glass, hold it on the top of cold water until all the paraffin
has solidified, and then let it sink to the bottom. When
thoroughly cool, cut out blocks containing the objects. If
the watch-glass has been smeared with a drop of a mixture
of equal parts of glycerine and water before putting the
paraffin into it, the solidified paraffin will generally detach
itself in a single cake and float up in a few minutes, or
hours at any rate. Do not attempt to remove it entire
by warming the bottom of the watch-glass. Similarly with
the paper trays or metal imbedding boxes. Or you may
put them to cool on a cold slab of metal or stone.

SELENKA cools the mass by passing a stream of cool water through
the imbedding tube described above (§ 137). Mayrr cools the mass in
the paraffin-tight moulds (§ 187) by passing cold water through a special
movable water-bath, which allows of the arrangement of the objects by
transmitted light under a dissecting microscope, see Mitth. Zool. Stat.
Neapel, iv, 1883, p. 429 ; Intern. Monatsschr. Anat. Hist., iv, 1887, p. 39.
A complicated apparatus for the same purpose is described by MrissnER
(Zeit. wiss. Mik., xviii, 1902, p. 286). Similarly, Hawn, zbéd., xxv, 1908,
p. 184, and Kappmrrs, ¢bid., xxiv, 1907, p. 254.

The paraffin blocks with the objects are now mounted on
the carrier of the microtome in position for cutting, and
pared to the proper shape (next §). If any bubbles or
cavities or opaque spots be present, prick with a heated
needle till all is smooth and homogeneous. The same should
be done if any cavities present themselves in the course of
cutting. In bad cases, re-imbed.

144. Shape and Orientation of the Block of Mass to be cut.—
These differ accordingly as the cutting is done with a slanting
knife or a square-set knife (see next $). In the first case,
the block is best trimmed to a three-sided prism, and
orientated as in Fig. 4, so that the knife enters it at the
angle a and leaves it at the angle c. When the section is
cut it will adhere to the knife only by the angle c, and can
thus most readily be removed by means of a brush or needle.
The object itself should come to lie in the block close to the
line b c, so that the knife at first cuts only paraffin, and

IMBEDDING METHODS. 89

that if the section begins to roll it may be caught and held
down by a brush or section-stretcher before the object itself
is reached. For the square-set knife the block is best
trimmed to a four-sided prism, and orientated as in the first
case, so that the. knife first touches one angle, if only
isolated sections are to be cut. But if ribbons (§ 148) are
to be cut, the block must be orientated with one of its sides
parallel to the knife-edge, and the opposite side must be
strictly parallel to this one.

An object which is not approximately isodiametrical but
gives a section which is wider in one direction than another
should be orientated end on, that is, so as to present its
narrowest diameter to the knife-edee: for it is in this
position that it will offer the least re-
sistance to the blade, and tend the least
to make the edge bend away or dig into
it. This is specially important with
longitudinal sections of worms, Amphi-
oxus, embryos of vertebrates, and the
like. Most especially with a square-set
knife should the narrowest diameter of
the object be presented to the knife ;
and only when the object is particularly
hard, or otherwise difficult to cut, should
it be turned so as not to let the whole
of that diameter be attacked at once by the knife, but only
"a corner of it. And as far as possible arrange that the
hardest part of an object be the last to be touched by the
knife.

For Noacx’s simple apparatus for accurately orientating
small blocks, see Zett. wiss. Mik., xv, 1899, p. 488, or Journ.
Roy. Mic. Soc., 182, 1899, p. 550.

For Etrrnop’s machine for trimming blocks to true cubes, see Zezt.
wiss. Mih., xv, p. 421, and for that of ScHAFFER, zbid., xvi, 1900, p. 417.

145. Knife Position —The position to be given to the knife
may be considered under two heads, viz. its slant and its tilt.
By the slant of the knife is meant the angle that its edge
makes with the line of section: that is with the line along
which it is drawn through the object (or along which the
object moves across it in the case of microtomes with fixed

90 CUAPTER VIIT.

knives). The position is transverse when the edge makes an
angle of 90° with the line of section, or the knife in that
case is said to be set square. It is oblique or slanting when
it makes a smaller angle with that line. he difference
between the effect of the two positions is that the oblique
position affords a more acute-angled wedge than the trausverse
one.

It does so for the following reasons :—Neglecting for the
moment the distinction between the cutting-facets and the
surfaces of the blade (which are distinct usually because they
are not ground to the same angle),* it is clear that the knife
itself is a wedge, the angle of which depends on the relation
between the height of its base and the distance from the
base to the edge. With the same base the angle becomes
more acute the greater the distance from edge to base.
Now by slanting the knife we can effect what is equivalent
to an increase in the distance from edge to base; for we can
thus increase the distance between the point of the edge
which first touches the object, and the point of the back
(strictly, of the back edge of the under cutting-facet) which
last leaves it. When the knife is set transversely, the line
along which any point of it traverses the object is the
shortest possible from edge to base of the wedge, and the
effective angle of wedge is the least acute obtainable with
that knife. But if it is set as obliquely as possible, the line
along which any point of it traverses the object traverses the
knife from heel to toe, that is, along the greatest possible
distance from edge to base, and therefore affords practically
a much more acute-angled wedge than in the first case; and
so on, of course, for intermediate positions. (Nee the sterco-
metrical constructions of these relations by ScHIEFFERDECKER,
op. ctt., p. 115; and also with more instructive figures,
Apdruy, “Ueber die Bedeutung des Messerhalters in der
Mikrotomie,” in Sitzber. med.-naturw. Section d. Sieben-

* The edge of a microtome knife is composed of two plane surfaces—
the upper and lower cutting-facets, which meet one another at an acute
angle, the cutting-edge, and posteriorly join on to the upper and lower
surfaces of the blade (see some good figures of differently shaped knives
in BEHRENS, KosseL und SCHIEFFERDECKER, Das Mikroskop., p. 115,
et seq.; and in APATHY'S paper quoted below). It will be seen that the
two facets together form a wedge welded on to the blade by the base.

IMBEDDING MBETIODS. 9]

birgischen Museumvereins, Bd. xix, Heft 7, p. 1 (Kolozsvar,
1897, A. K, Ajtai).

For honing knives see SsoBoLew, Zeit. wiss. Mik., xxvi, 1909, p. 65;
Lenpvat, ibed., p. 203; Funex, ¢bid., xxvii, 1910, p. 75.

Very large objects are best cut with the slanting knife,
and so are all objects of very heterogeneous consistency,
such as tissues that contain much chitin or much muscular
tissue ; and better with a slowly working sliding microtome
than with a quick-working Rocker or the like. Soft masses
such as gelatin or celloidin cut wet, can only be cut with the
slanting knife. The slanting position causes less compressivu
of sections than the transverse one. It has the defect of
producing rolling in paraffin sections more easily than the
transverse position. The latter is the proper position for
cutting ribbons of sections from paraffin.

By the t/t of the knife is meant the angle that a plane
passing through its back and edge makes with the plane of
section: or, practically, the greater or less degree of eleva-
tion of the back above the edge (it is not to be confounded
with the inclination of the long axis of the knife to the
horizon ; any accidental inclination that this may have is a
matter of no moment).

The question of the proper tilt to be given to the knife
under different circumstances has been investigated by
Apdruy, loc. cit. supra. He concludes—(1) The knife should
always be tilted somewhat more than enough to bring
the back of the under cutting-facet clear of the object.
(2) It should in general ke less tilted for hard and brittle
objects than for soft ones; therefore, ceteris paribus, less for
paraffin than for celloidin. (38) The extent of useful tilt
varies between 0° and 16° or occasionally 20°. (4) Exces-
sive tilt causes rifts (longitudinal) in the paraffin, also
furrows that in bad cases spht up the section into narrow
ribbons. It also makes sections roll. Also it may cause
the knife not to bite, thus causing sections to be missed. Or
it may give an undulatory surface to the sections, owing to
vibrations set up in the knife, which may be heard as a deep
humming tone. Further, I would add, excessive tilt may
cause the knife to act as a scraper, carrying’ away portions
of tissue bodily from their places. Hxcessive tilt may often

92 CHAPTER VIN.

be recognised by the knife giving out a short metallic sound
just as it leaves the object. For knives with plane under-
surfaces it is seldom advisable to give less than 10° tilt.
Knives with concave under-surfaces, on the contrary, may
require to be placed almost horizontal. Jung’s knife-holders
give mostly a tilt of about 9°, which is ouly enough for
cutting ribbons with hard paraffin.

A knife with too little tilt will often cut a second section,
or fragments of one, without the object being raised, showing
that during the first cut the object was pressed down by the
knife, and recovered itself afterwards. his fault is denoted
by the ringing tone given out by the knife on passing back
over the object before the latter is raised. Such a knife
gives out a dull rattling sound whilst cutting. ‘Too little
tilt causes folding or puckering of sections, and does not
allow of the cutting of the thinnest possible sections, as the
edge does not bite enough. It is thus frequently a cause
of sections being missed, or coming off thicker at one end
than the other.

A slanting knife should have more tilt given to it than a
square-set one.

Ribbon section-cutting ($ 148) requires a relatively hard
paraffin and less tilt. With celloidin it is very important
to avoid insufficient tilt, as the elastic celloidin yields before
an insufficiently tilted knife and is not cut.

The tilt of the knife is given to a certain extent by the
knife-holder sold with the microtome. With plane-concave
knives it can be regulated to a certain extent by simply
turning the blade over. It is more accurately regulated by
means of mechanical contrivances, of which the most simple
are the horseshoe-shaped wedges of Neumayer (see Jung’s
price-list). A pair of these, each ground to the same angle,
is taken, and one of them placed (thin end towards the
operator) under, and the other (thick end towards the
operator) over, the clamping-arm of the knife-holder.
Three pairs, having different degrees of pitch, are supplied,
and are sufficient for most work. Other contrivances to the
same end consist of knife-holders that permit of rotating
the knife on its long axis, and, though more costly, will be
found a great convenience where much section-cutting has
to be done. For these see Jung’s price-list, and various

IMBEDDING METHODS. 93

recent papers in Zeit. wiss. Mrk., also that of ApArny, in the
paper quoted above (very complicated), and especially the
description of the two latest of Jung, viz. his model J and
model n, by Mayer and ScHogsst, in Zeit. wiss. Mtk., xvi,
1899, p. 29 (see figure of model / in Jowrn. Roy. Mic. Soc.,
132, 1899, p. 546). Also Carpentrr’s The Microscope,
p. 463.

146. 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,
see § 151.

If, after cutting has begun, the paraffin be found to be too hard, it
may be softened by placing a lamp near the imbedded object. But
then, the paraffin being warmed most on the side nearest the lamp,
becomes softer on that side, and the sections have a tendency to hecome
compressed and puckered-in on that side.

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.

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.

For other devices for warming or cooling the paraffin see HELD, Arch.
Anat. Phys., Anat. Abth., 1897, p. 315; VAN WALSEM, Zett. wiss. Mik.,
xi, 1894, p. 218; LenpDENnFeLD, ibéd., xviii, 1901, p.18; Krauss, ibid.,
xxv, 1908, p. 299; Foot and STROBELL, Biol. Bull. Wood's Hole, ix, 1905,
p. 281.

Secondly, the knife should be set square, for the oblique
position encourages 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 flat, when the same
mass will only give rolled sections if cut disconnectedly.

Rolling may often be lessened or suppressed by cutting
the sections thinner,

94. CHAPTER VIII.

Mechanical means may be employed. The simplest of
these 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, which is much better, 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, 18838, p. 100 (ScHULTZE); Mitth. Zool. Stat. Neapel, iv,
1883, p. 429 (MayEr, ANDRES, and GIESBRECHT); Arch. m7k. Anat..
xxiii, 1884, p. 537 (DECKER); Bull. Soc. Belg. Mic., x, 1883, p.55 (FRAN-
corre); The Microscope, February, 1884 (Gace and SmitH); Wutt-
MAN'S Meth. in Mic. Anat., 1885, p. 91; Zezt. wiss. Mik., iv, 1887, p. 218
(STRASSER) ; zbid., x, 1893, p.157 (Born). The best are those of Mayer
and Born.

I find that Mavyer’s, beautifully made by June, works
admirably and is most valuable.

Another plan is to allow the sections to roll, but to control
the rolling. To this end, the block of paraffin is pared to the
shape of a wedge five or six times as long as broad, the
object being contained in the broad part, and the edge
turned towards the knife (see Fig. 4). 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 he a very
open one—indeed, very nearly flat. Lay the coil on a slide
with this end downwards, warm gently, and the part con-
taining the object will unroll completely and lie quite flat.

Anite (Glandole duodenali, Napoli, 1908, p. 51) and
Vasrarini-Cresi (Mon. Zool. Ital., 1906, p. 164) lay a strip
of wet filter-paper on the block.

A defect opposite to that of the rolling of sections is the
compression and the crumpling or puckering of sections,
indicating that the paraffin has been compressed by the knife
instead of being merely cut true by it. Such sections,

IMBEDDING METHODS. 95

besides showing creases or folds, have a smaller area than
that of the block from which they are cut. This is a bad
fault, for the compression may obliterate important cavities
or efface important limits between cell-layers, etc. It may
be caused by a badly cutting knife, and is very easily caused
by the paraffin being too soft. To prevent it, correct the
knife or cool the paraffin, or re-imbed in harder paraffin.

Very large sections tend to form folds on the knife, and are difficult to
remove from it. Mayer (Grundziige, Len and Mayer, p. 94) gets
them to wrap themselves round a glass or gelatin tube laid on the block
just in front of the knife-edge and rolled forwards as it progresses.
When cut, the section is rolled off on to the surface of water.

147. Cutting Brittle Objects (Collodionisation).—Some objects
are by nature so brittle that they break or crumble before
the knife, or furnish sections so friable that it is impossible
to mount them in the ordinary way. Ova are frequently in
this case. A remedy for this state of things consists in
covering the exposed surface of the object just before cut-
ting each section with a thin layer of collodion, which serves
to hold together the loose parts; and will enable the
operator to cut sections considerably thinner than can be
obtained in the usual way.

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.

Marx (Amer. Natwral., 1885, p. 628; cf. Jowrn. 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. It must be diluted with
ether as soon as it begins to show signs of doing so,

“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

96 CHAPTER. VIIT.

surfaces 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.”

HENKING (Zeit. wiss. Mik., iii, 1886, p. 478) takes instead of collodion
a solution of paraffin in absolute alcohol.

For extremely brittle objects, such as ova of Phalangida, he recom-
mends a thin (light yellow) solution of shellac in absolute alcohol.

HEIDER (Embryonalentw. v. Hydrophilus, 1889, p. 12; cf. Zeit. wiss.
Mik., viii, 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.

Rabu (zbid., xi, 2, 1894, p. 170) employs superheated parafin (of about
100° C.). This has the advantage of filling up any cavities there may be
in the objects, and also of preventing the sections from rolling. A com-
plicated development of this process is described by LENDENFELD in
Zeit. wiss. Mtk., xviii, 1901, p. 18.

APATHY (Mikrotechnik, p. 183) employs a 1 per cent. solution of
celloidin, allows the sections to roll, and unrolls them by the water-
process (§ 149).

JORDAN (Zeit. wiss. Mik.) adds 5 drops of oil of cedar to 15 c.c. of the
solution of celloidin, and finds that rolling is prevented.

148. 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 or ribbon which may be
taken up and transferred to a slide without breaking up,
thus greatly lightening the labour of mounting a series.
For the production of a ribbon, the paraffin must be of a
melting-point having the right relation to the temperature of
the laboratory, see § 151. Secondly, the knife should be set
square. Thirdly, the block of paraffin should be trimmed so
as to present a straight edge parallel to the knife edge; and

IMBEDDING MW'TLHODS. 97

the opposite edge should also be parallel to this. It is by
no means necessary to have recourse to special mechanical
contrivances, as in the so-called ribbon microtomes; the
Thoma microtome is sufficient. But the automatic microtomes,
and amongst them the Cambridge Rocking Microtome and
the Minot, are certainly most advantageous for this purpose.
If the paraffin is very hard, it is necessary for sections of
10 pw, and advisable for thinner ones, to coat the block with
softer paraffin. To do this, take paraffin of about 40° C.
melting-point, melt it, heat it to about 80° on the water-
bath, dip the block into it for an instant, and rapidly turn
it over so that the fluid paraffin may run down away from
the top part as much as possible. Allow it to cool, and pare
away again the soft paraffin from the two sides that are not
to be arranged parallel to the knife. Or, as I frequently
prefer, simply plaster a wall of soft paraffin (superheated)
on to the fore and aft faces of the block with a small
spatula. Large blocks may have two coatings given them.
It sometimes happens that the ribbon becomes electrified during the

cutting, and twists and curls about in the air in a most fantastic and
undesirable manner. It may be got flat by warming slightly.

149. Section Flattening—The sections having been ob-
tained may be cleared and mounted at once if they are
quite perfect, that is, neither rolled nor creased nor com-
pressed. But should they in the least degree show any
of these defects, they must first be unrolled or smoothed, o1
expanded to their proper dimensions.

The most efficacious plan is combined treatment with tuid
and heat. ‘The sections are either floated on to the surface
of warm water or warm alcohol contained in a suitable dish,
which causes them to flatten out perfectly, and are then
transferred to a slide, by floating them into position, or
otherwise. Or the slide has a layer of water spread over it,
the sections are laid on the water, and the slide is heated
(to somewhat beluw the melting-point of the paraffin) until
the sections flatten out, which happens in a few seconds.

A special water-bath for flattening sections is described by Nowak
in Zeit. wiss. Mik., xii, 1896, p. 447.

150. Clearing and Mounting.—The sections having been
duly smoothed by one of these processes, and duly fixed to
@

98 CHAPTER VIL.

the slide (Chapter X), unless it is desired to keep them
loose, all that now remains is to get rid of the paraffin and
mount or stain as the case may be. Many solvents have
been recommended for this purpose :—Turpentine, warm
turpentine, a mixture of four parts of essence of turpentine
with one of creasote, creasote, a mixture of turpentine 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. Of these xylol and toluol are generally in most
respects the best. Benzol and chloroform are too volatile
for safe manipulation.

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 xylol or toluol. For thin sections,
10 to 15 pr, it is not necessary to warm at all. The sections
may be mounted direct from the xylol, or the slide may be
brought into a tube of alcohol to remove the solvent for
staining.

Paraffin sections can be stained without removal of the paraflin, so

that after-treatment with alcohol can be suppressed, but this is only
very exceptionally advantageous.

151. Pure Paraffin.—It is now almost universally admitted
that pure paraffin is superior for ordinary work to any of
the many mixtures with wax and the like that used to te
recommended. Paraffin varies enormously in hardness ac-
cording to the temperature of its surroundings. It should
therefore be taken of a melting-point suitable to the
temperature of the laboratory. A paraffin melting at 50° C,
or a little harder, is that which in my experience gives the
best results so long as the temperature of the laboratory is
between 15° and 17°C. For higher temperatures a harder
paraffin is required, and for lower temperatures a softer one,

Many workers of undoubted competence prefer masses
somewhat harder than this; so, for instance, Heidenhain
(58°), Apathy (55°), Rabl (56°), Mayer (58° to 60° in
summer; in winter about 56°, but never less than 50°).
Mayer points out that at Naples the temperature during
five months of the summer and autumn is over 22° GC, in
the laboratory, sometimes over 30°. Temperatures such as

y

IMBEDDING METHODS. 99

these are seldom realised in the British Isles, and, whilst I
quite admit that such hard paraffin may have its raison
@étre for Naples, I hold that for that very reason it is
in general unnecessarily hard for cooler climates.

My recommendation of a relatively soft paraffin refers to
work with the Thoma sliding microtome. Microtomes with
fived knives, such as the Cambridge, the Minot, or the
Reinhold-Giltay, will give good results with much harder
paraffin, and, in fact, require such.

Stout knives of hard steel will take a harder paraffin than
thin ones of soft steel ; but the latter may be preferable for
soft masses.

For thin sections a harder paraffin is required than for
thick ones,

Hard objects require a harder paraffin than soft ones.

Brass (Zeit. wiss, Afik., uu, 1885, p. 3800) recommends
paraffin that has been kept for some years, as it has
less tendency to crystallise than new paraffin.

Paraffin of various melting-points is easily found in com-
merce. Intermediate sorts may be made by mixing hard
and soft paraffin. I find that two parts of paraftin melting
at 50° with one of paraffin melting at 36° C. give a mass
melting at 48° C., and a mixture of one part of that melting
at 53° with one part of that melting at 45° gives a mass
melting at 50° C.

According to E, Burcuarpr (Jena Zeit. Naturw., xxxiv,
1900, p. 719) mixtures of paraffins of different melting-points
give better results than an unmixed paraftin of the same
melting-point as the mixture. He recommends 10 parts of
40° paraffin + 1 of 45° + 1 of 52° 4+ 1 of 58° + 6 of 60°.

For methods for ascertaining melting-points see Kissling,
Chem. Centralb. ii, 1901, p. 407,

152. Overheated Paraffin.—Sprz (Zeit. wiss. Mik.., ii, 1885, p. 8) takes
paraffin of about 50° C. melting-point and heats it in a porcelain capsule
by means of a lamp until it has become brownish-yellow, and after
cooling shows an unctuous or soapy surface on being cut. This mass
may be obtained ready prepared from Griibler. The object of this pre-
paration is to make the mass stickier, in view of cutting ribbons.

Van Watsem (Verh. Akad. Wetensch. Amsterdam, 1899, p. 182) still
recommends the addition of 5 per cent. of yellow wax to paraffin of 52°
to 57° melting-point (for large sections of central nervous system).

JouNnstron (Journ. Appl. Micr., vi, 1903, p. 2662) adds 1 per cent. of

100 CHAPTER VIL.

india-rmbber in very small pieces, dissolved by heating to 100° C. for
twenty-four hours, or several days to 60° C. Clear with xylol. For
very brittle objects.

153. Soap Masses.—These have never been much used, and are now
entirely discarded. But see early editions, or Porzam (Morph. Juhrb.,
iii, 1877, p. 558); Kapyz (Zool. Anz., 1879, vol. ii, p. 477); DOLLKEN
(Zeit. wiss. Mtk., xiv, 1897, p. 32).

Gelatin Masses.

154. Gelatin Imbedding is a method that has the ad-
vantage of being applicable to tissues that have not been
in the least degree dehydrated.

The modus operandi is, on the whole, the same as for other
fusion masses, with the difference that the objects are pre-
pared by saturation with water instead of alcohol or a
clearing agent. After the cooling of the mass it may some-
times 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 (Kaisur), or for afew days with 90 per cent.
alcohol (Ktrss) or chromic acid (Kies) or formaldehyde
(Nicozas), or it may be frozen (Soutas).

The mass can be removed from the sections by means of
warm water.

155. Glycerin Gelatins, KLess’ (Arch. mk. Anat., v, 1869, p. 165).
—A concentrated solution of isinglass mixed with half its volume of
glycerin.

Kaiser's (Bot. Centralb., i, 1880, p. 25)—One part by weight of
gelatin is left for about two hours in 6 parts by weight of water ; 7 parts
of glycerin are added, and for every 100 grm. of the mixture 1 grm. of
concentrated carbolic acid. The whole is warmed for ten to fifteen
minutes, stirring all the while, until the whole of the flakes produced by
the carbolic acid have disappeared.

GERLACH’S (Unters. u. d. Anat. Inst. Erlangen, 1884; Journ. Roy. Mic.
Soc., 1885, p.541).—Take gelatin, 40 grm.; saturated solution of arsenious
acid, 200 ¢.c.; glycerin, 120 ¢.c. Clarify with white of egg. The objects
to be prepared for imbedding by a bath of one third glycerin.

ApitHy (Mitth. Z. Stat. Neupel, xii, 1807, p. 718, and Zect. wiss.
Mikr., xxix, 1918, p. 472) soaks small objects first in glycerin and water
(equal parts) and then for at least 24 hours at 40° C. in a solution of
1 part of gelatin in 3 of glycerin and 6 of water. They are then
arranged in some of this in an imbedding box, and the whole is warmed
(over calcium chloride) in a stove at 45° to 60° C. until the mass has
evaporated down to one half, losing 5 of its 6 volumes of water (as I

IMBEDDING METHODS. 101

understand—the description is not clear). Blocks are then cut out
and hardened in absolute alcohol (suspended therein) for several days
(1 day per millimetre of thickness), cleared in terpinol (1 day per
millimetre), and cut with a knife wetted with the same. Said to give
sections of 3», without the least shrinkage.

Bruworrr’s Gold Gelatin Mass (Journ. de Botan., vi, 1892,
p. 194).—Twenty germs. 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. Objects
are prepared by soaking in some of the mass diluted with
two to three volumes 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.

Nicotas’s Method (Bibiogr. Anat., Paris, 3 année, 1896,
p. 274).—Preparations «re first soaked for one or two days
in a 3 per cent. to 4 per cent. aqueous solution of gelatin
kept at 25° C., then for the same time in a 10 per cent.
solution, and then for two or three days more in a 20 per
cent. to 25 per cent. solution containing 8 per cent. to 10
per cent. of glycerin and kept at 35°C. They are then
imbedded in some of the same mass in paper trays, and as
soon as the gelatin has set are thrown into a mixture
of formol 1 part, water 7. After a few days therein
the gelatin has become hard and insoluble, and may be cut
or preserved for months in weak formol solution, or dilute
alcohol or glycerin, or even in pure water. Sections must
be very gradually passed through. successive alcohols for
dehydration, as they curl up very easily. They, however,
flatten out at once on being brought from absolute alcohol
into cresylol, and may then be mounted in balsam. To
mount in glycerin is of course easy.

Burzynsxi (Polw. Arch. Biol. Med. Wss., i, 1901, p. 39)
finds that alkaline formol hardens gelatin better than acid.

Gasket (Journ. Path. Bact., July, 1912, p. 58) soaks in
pure gelatin, melted s.a., for two to five hours at 87° C.,
and hardens the mass in vapour of formol, for three or more
days. To cut, he freezes. He mounts in glycerin jelly, to
avoid dehydration and shrinkage.

CHAPTER IX.
COLLODIUN (CNLLOIDIN) AND OTHER IMBEDDING METHODS.

156. Introduction.—Collodion (or celloidin) masses do not
require the employment of heat. ‘They do not require that
the objects should be cleared before imbedding, and that is
an advantage in the case of very large objects. They are
more or less transparent, which facilitates orientation. And
they are specially indicated for very large objects, for the
soaking in collodion, being quite inoffensive to the most
delicate elements, may be prolonged if necessary for weeks.
Lastly, the mass being quite transparent after mounting, it
is not necessary to remove it from the sections before staining
and mounting them ; it may remain, and fulfil the function of
an admirable support to the tissues, holding in their places
brittle or detached elements that without that help would
fall to pieces and be lost.

There are disadvantages. One is that the process is a
very long one; as usually practised, it requires some three
days for the imbedding of an object that can be imbedded
in paraffin in an hour. Another is that it is impossible to
obtain with eclloidin sections quite so thin as those furnished
by paraffin.

In the older celloidin method the mass 1s cut wet, before
clearing. I strongly recommend the more recently intro-
duced practice of clearing before cutting, and cutting dry as
described in §§ 168-170.

157. Collodion, Celloidin, and Photoxylin—The collodion
method is due to Duvan (Journ. de V Anat., 1879, p. 185).

Celloulin, recommended later on by Merxer and Scuterrer-
pecker (Arch. Anat. Phys., 1882, p. 200), is merely a patent
collodion. It may be obtamed from Griitr, or the other
dealers in histological reagents. It is sent out in the form
of tablets. These tablets may, if desired, be dissolved at

COLLODION AND OTHER IMBEDDING mrruops. 105

once in ether, or a mixture of ether and alcohol, to make a
collodion of any desired strength. But it is better, as
recommended by ApAruy, to cut them up into thin shavings,
which should be allowed to dry in the air until they become
yellow, transparent, and of a horny consistency, and that these
be then dissolved in alcohol and ether (sulphuric, free from
acid). The solutions 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. wiss. Atk., vi, 1889, p. 164).

Imbedding masses of excellent quality can be prepared
with ordinary collodion, but celloidin furnishes more readily
solutions of known concentration. Otherwise there is but
little to choose between the two, and therefore in this work
the terms collodion and celloidin are used indifferently.

According to Unna (Monatschr. ». Dermatol., xxx, 1900, pp. 422 and
476; Zeit. wiss. Mck., xviii, 1901, p. 32) a more inelastic, and therefore
better, mass is obtained by adding to celloidin 2 per cent. of oil of
turpentine, stearate of soda, or (best of all) castor-oil. Celloidin with
this addition has been put on the market under the name of “ Celloi-
dinum inelasticum,” by the Chemische Fabrik vorm. E. Schering, in
Berlin.

Photoxwylin (Krysinsxy, VircHow’s Archiv, evili, 1887, p. 217;
Bussg, Zeit. wiss. Mik., ix, 1892, p. 47) is a dry substance, of the aspect
of cotton-wool, and chemically nearly related to celloidin. It can he
obtained from GrRUBLER. 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 advantage of affording a mass
which after hardening in 85 per cent. alcohol remains perfectly trans-
parent. Some writers say that it gives a better consistency, but others
deny this (APATHY, ¢.9.).

TSCHERNISCHEFF (Zedt. wiss. Mik., xvii, 1900, p. 449) recommends
Collozylin (10 grms. dissolved in 10 grms. of eugenol or clove oil, with
the addition of 50 ¢.c. of ether and 1 of absolute alcohol).

The Older Celloidin Afethod.

158. Preparation of Objects.—The objects must first be
very thoroughly dehydrated with absolute alcohol. ‘They ave
then soaked till thoroughly penetrated in ether, or, which is
better, in a mixture of ether and absolute alcohol. Duvar
(loc. cit.) takes for this purpose a mixture of ten parts of
ether to one of alcohol; Scuterrerpecker (and the majority

104 CHAPTER LX.

of workers) a mixture of equal parts of ether and alcohol ;
Tuspy (in Nature, November 17th, 1892, p. 51) advises a
mixture of four parts of ether and one of alcohol. Fisu
advises acetone, see next §. Mann (Methods, etc., p. 172)
takes equal parts of ether and methyl alcohol. So also
Paviow, Zeit. wiss, Mikr., xxi, 1904, p. 15.

This stage may be omitted if the objects are of a suffi-
ciently permeable nature, and they may be brought direct
from alcohol into the collodion bath.

159. The Collodion Bath—The secret of success here is to
infiltrate the objects 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 celloidin [dried as
described in § 157]; a thick solution, one containing 10 to
12 per cent.)

If collodion be taken, the thin solutions 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.

The dried celloidin shavings dissolve very slowly in the
mixture. Exscunia (Zeit. wiss. Mikr., x, 1893, p. 445), has
found that solution is obtained much quicker if the shavings
be first allowed to swell up for twenty-four hours in the
necessary quantity of absolute alcohol, and the ether be
added afterwards.

Busse (op. cit., ix, 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).

I generally use only two solutions. one weak one, and one
strong one corresponding approximately to Busse’s No. 2.
His No. 3 is so thick that excessive time is required to obtain
penetration by it.

Maywn (Methods, p. 172) uses solutions of 24 and 10 per
cent.

AvAtny (Brurens, Tabellen., 198, p. 82) takes 2 per cent.
and 4 per cent. for the first baths, 8 per cent. for the last,

COLLODION AND OTHER IMBEDDING METHODS. 105

Mysrs (Arch. Anat. Phys., Anat. Abth., 1902, p. 370)
takes 14 per cent., 6 per cent., and 16 per cent.

See also NeumayveEr, Zeit. wiss. Mik., xxv, 1908, p. 38; Dr Veccut,
ibid., xxiii, 1906, p. 312; and FUHRMANN, Zeit. wiss. Zool., lxxviii, 1905,
p. 524.

FisH (Journ. Appl. Microscop., ii, 1899, p. 323) first infiltrates with
acetone (which he says may be used as a fixing and dehydrating agent
at the same time), then with a 4 per cent. solution of pyroxylin (gun-
cotton) in acetone; and, lastly, in an 8 per cent. acetone solution of the
same. See for other solutions §§ 170 and 171.

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).

When tlie 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. This may be done (as first
described in this work, Ist edit., 1885, p. 194) by allowing
the thin solution to concentrate 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.

Aparuy (Mikrotechnik, p. 121) holds that it is preferable
to transfer to fresh thick solution, as he finds that a better
consistency after hardening is thus obtained.

160. Imbedding.—The objects must now, if it has not been
done before, be imbedded—that is, arranged in position in
the thick collodion in the receptacle in which they are to
be hardened. For the usual manipulations see § 187. If
paper thimbles be taken for imbedding, the bottoms should
be made of soft wood in preference to cork, see § 165.
They should be prepared for the reception of the object by
pouring into them 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 im the mass.
Watch-glasses, deep porcelain water-colour moulds, and the
like, also make convenient imbedding receptacles. Care
should be taken to have them perfectly dry.

It not infrequently happens that during these manipula-

106 CHAPTER IX.

tions bubbles make their appearance in the mass. Before
proceeding with the hardening these should be got rid of
by exposing the whole for an hour or two to the vapour
of ether in a desiccator or other well-closed vessel. Care
should be taken that the ether (which may be poured on
the bottom of the vessel) does not wet the mass (Busse,
Zeit. wiss. Mik., viii, 1892, p. 467).

161. Orientation.—Celloidin being more or less transparent,
it is seldom necessary to recur to special aids to orientation.

ApAtuy (Zeit. wiss. Mtk., v, 1888, p. 47) arranges objects
on a small rectangular plate of gelatin, placed on the bottom
of the imbedding-recipient. The gelatin is turned out with
the mass after hardening, and cut with it. The edges of
the gelatin form good orientation lines. .

Hatie and Born (Zeit. wiss. Mik., xii, 1896, p. 364) use
plates of hardened white ot egg, in which a shallow furrow
for the reception of the objects has been cut by means of a
special instrument. See also § 142.

For the complicated method of Eycizsnymer (Amer. Nat.,
xxvi, 1892, p. 354) see previous editions.

See also the article “ Rekonstruction ” in the Encyel. mik.
Technik.

162. Hardening, Preliminary.—The objects being imbedded,
the treatment should be as follows:—The receptacles or
supports are 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. 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 he dry, fresh thick solution is added,
and the whole is left as before. (If the first layer of
collodion has become too dry, it should be moistened with a
drop of ether before adding the fresh collodion.) | Provision
should be again made for slow evaporation, either in one of
the ways above indicated, or—which is perhaps better—by
setting the objects under a hermetically fitting bell-jar,
which is lifted for a few seconds only once or twice a day.
I have frequently found it advantageous to set the objects

COLLODION AND OTHER IMBEDDING METHODS. 107

under a bell-jar, together with a dish containing alcohol, so
that the evaporation is gone through in an atmosphere of
alcohol. This is especially indicated for very large objects.
The whole process of adding fresh collodion and placing
the objects under the required conditions of evaporation is
repeated every few hours for, if need be, two or three days.

When the mass has attained a consistency such that the
ball of a finger (not the nail) 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.)

163. Hardening, Definitive—Several methods are available
for the definitive hardening process. One of these is the
chloroform method, due to Viatnanus (Rech. sur ? Hist. et le
Deév des Insectes, 1888, p. 129).

It consists in bringing the objects into chloroform. In
some cases a few hours’ immersion is sufficient to give the
requisite consistence. In no case have my specimens
required more than three days. 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 if need be. 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 a few more days until it is ready
for cutting. But sufficient hardening is sometimes obtained
in a few hours.

Good chloroform is a necessity.

The above processes are excellent, but I regard them as
primitive forms of the chloroform method. I now almost
always harden in vapour of chloroform, All that is neces-

108 CHAPTER IX.

sary is to put the liquid mass (after having removed bubbles
as directed in § 160), with its recipient into a desiccator on
the bottom of which a few drops of chloroform have been
poured. The action is very rapid, and the final consistency
of the mass at least equal to that obtained by alcohol
hardening.

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 prac-
tice of different writers differs greatly. Bussg (Zeit. f. wiss.
Mikr., ix, 1, 1892, p. 49) has found, as I also have done,
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 masses hardened in this grade
of alcohol moist while cutting, as they dry by evaporation
very quickly.)

Some workers use lower grades, 70 to 80 per cent., or
even lower. Avatruy (Microtechnik, p. 185) mentions “ gly-
cerin-alcohol,” but without giving details. Bium (Anat. Anz.,
xi, 1896, p. 724) mentions “ weak spirit with formol added
to it,” saying that formol hardens celloidin.

Lastly, the mass may be frozen. After preliminary hardening by
alcohol, it is soaked for a few hours in water, in order to get rid of the
greater part of the alcohol (the alcohol 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. If the mass have frozen too hard, cut with a knife warmed
with warm water.

FiLormMan (Zeit. wiss. Mzk., vi, 1889, p. 184) recommends that the
definitive hardening should be done without the aid of alcohol or chloro-
form, by simply cutting out the blocks, turning them over, and care-
fully continuing the evaporation process in the way described above. I
described this process myself in the first edition of this work. I doubt
whether it is possible in this way to carry the hardening much beyond
the point attained by the chloroform or alcohol method without incur-
ring a very undesirable degree of shrinkage.

164. Preservation.—The hardened blocks of collodion may
be preserved till wanted in weak alcohol (70 per cent.), or

COLLODLON AND OTHER IMBEDDING METHODS. 109

dry, by dipping them into melted paraffin (ApAtHy, Zevt.
wiss. Mikr., v, 1888, p. 45), or, after rinsing with water, in
glycerine-jelly, which may be removed with warm’ water
before cutting (ApAray, Mitth. Zool. Stat. Neapel, xii, 1897,
p. 372).

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.

165. Cutting.—If the object has not been stained before imbedding, it
may form so transparent a mass with the collodion that the arrange-
ment 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 oil used for
clearing.

To fix a collodion block to the microtome 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 or the infiltrated and hardened but not
imbedded object, and 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 or
object. Then throw the whole into weak (70 per cent.)
alcohol for a few hours, or even less, or, better, into chloro-
form, or vapour of chloroform, for a few minutes, in order
that the joint may harden.

Linpsay Jonson prefers a mixture of 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 col-
lodion, 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 best not to use

110 CHAPTER La.

cork for mounting on the microtome, 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. If
the object-holder be of the cylinder type, a good cork may
be used ; but even then, I think, wood is safer. GacE has
recommended bits of glass cylinders. JrnineK (Zeit. wiss.
Mik., 11, 1894, p. 237) recommends a sort of vulcanite
known as “ Stabilit,’” which is manufactured for electrical
insulation purposes. It is supplied in suitable blocks by
Jung, and by Grisizr. Wood is liable to swell in alcohol
so that it no longer fits into the object-holder. Baxncock
(Journ. R. Mier. Soc., 1901, p. 339) uses a block of hard
paraffin, with the surface corrugated.

Sections (from such masses as have not been cleared before
cutting) are cut with a knife kept abundantly wetted with
alcohol (of 50 to 85 or even 95 per cent.). APATHY recom-
mends that the knife be smeared with yellow vaseline ; 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

147.

; 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 Chapter X.

Masses that have been cleared before cutting with cedar
oil or the like may be cut dry, § 170.

166. Staining.—'Tle sections may now be stained as desired,
either loose, or mounted in series on slides or on paper as
described in Chapter X,. 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 if it be desired, the mass may
be removed by treating the sections with absolute alcohol or
ether.

167. Clearing and Mounting—You may mount in glycerin
without removing the mass, which remains as clear as glass
in that medium.

COLLODION AND OTHER IMBEDDING METHODS. 111

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). Nikirorow (Zeit.
wiss. Mck., viii, 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 recom-
mended are origanum oil (Ol. Origan. Cretict, it is said, should
be taken, not Ol. Orig. Gallici; but see as to this reagent
the remarks in § 125), 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 benzol (may make sections
shrink if not well dehydrated), or Dunham’s mixture of 3
or 4 parts of white oil of thyme with 1 part of oil of
cloves. (As to oil of thyme, see also $$ 125, 126.)

Fisa (Proc. Amer. Mik. Soc., 1893) advises a mixture of
one part of red oil of thyme with three parts of castor oil,
the latter being added in order to counteract the volatility
of the thyme oil. But later (June, 1895), writing to me,
Dr. Fish says he has substituted the white oil of thyme for
the red, and finds it an advantage in orientating. See also

§ 126.

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 collodion, others dissolve it, others pucker it. Minor
(Zeit. wiss. Mik., iii, 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. WEIGERT (Zeit. wiss, Mik., iii,
1866, 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
basic anilin stains, as it discolours them. For these anilin oil may be
used with 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,
see § 134. See van GiEson, Amer. Mon. Mic. Journ., 1887, p. 49, or
Journ. Roy. Mic. Soc., 1887, p. 519, for a review of these clearing agents.

Beech-wood creasote has been recommended (by M. Flesch).

EycLEsHYMER (Amer. Nat., xxvi, 1892, p. 354) advises a mixture of
equal parts of bergamot oil, cedar oil, and carbolic acid.

112 CHATTER LX.

‘© For oil of cajeput see § 129; and for this and other
clearers see also Jorpan, Zeit. wiss. Mik., xv, 1898, p. 51,
who recommends, amongst other things, oil of Linaloa, which
remains colourless.

The Newer Cellotdin Method.

168. The New Method, by Clearing before Cutting.—This pro-
cess is due, I believe, in the first instance to E. Mgyzr (Biol.
Oentralb., x, 1890, p. 508), who advised soaking blocks before
cutting for twenty-four hours in glycerin. Bumpus (Amer.
Anat., xxvi, 1892, p. 80) advises clearing the mass, after
hardening in chloroform, with white oil of thyme or other
suitable clearing agent (see § 167). 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 EycLusHymer (op. cit., pp. 354,
563), carbolic acid, or glycerin, or the mixture given § 167,
being suggested for clearing; and Ginson has for a long
time past adopted the practice of clearing before cutting
with cedar oil, as described in the next §.

Fisu (loc. cit., § 167) also advocates the practice of clear-
ing in the mass, recommending the clearing mixture there
given. Similarly Gace, Trans. Amer. Mik. Suc., xvii, 1896,
p. 361.

All the authors above quoted cut in the wet way, that is
to say, with a knife wetted with the clearing liquid.

169. Gitson’s Rapid Process (communicated, April, 1892).
—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
collodion allowed to boil (which it does at a very low
temperature) until it has become of a syrupy consistence.
(It should be boiled down to about one third of its volume.)
The mass is then turned out, mounted on a block of hardened
celloidin, and the whole hardened in chloroform or in a mixture
of chloroform 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,

COLLODION AND OTHER IMBEDDING METHODS. 113

using cedar oil to wet the knife, and cover the exposed
surface of the object after each cut,

This process is very much more rapid than the old process:
small objects can be duly infiltrated in an hour, where days
would be required by the old process. As collodion boils at
a very low temperature very little heat is required, and there
is no risk of the tissues suffering on that head.

170. The Dry Cutting Method.—I recommend the following
as a further improvement. JInfiltrate with collodion or
celloidin either by Gitson’s process, or by soaking in the
cold in the usual way, § 159. Imbed as usual. Harden in
vapour of chloroform for from one hour (generally sufficient
for small objects) to overnight. This is done by putting the
object (definitively imbedded in the final thick solution, but
without any preliminary hardening in the air) into a Steinach’s
sieve-dish or into a desiccator, on the bottom of which a tea-
spoonful of chloroform has been poured. (The objects may
remain for months in the chloroform vapour if desired.) As
soon as the mass has attained sufficient superficial hardness,
it is, of course, well to turn it out of its recipient; and turn
it over from time to time, in order that it may be equally
exposed on all sides to the action of the vapour. When fairly
hard throw it into Gitson’s mixture. This should be at first
a mixture of one part of chloroform with one or two parts of
cedar oil. From time to time more cedar oil should be
added, so as to bring the mixture up gradually to nearly pure
cedar oil. As soon as the object is cleared throughout, the
mass may be exposed to the air, and the rest of the chloro-
form will evaporate gradually. The block may now either
be mounted on the holder of the microtome, § 165, and cut
at once, or may be preserved indefinitely without change in
a stoppered bottle. Out dry, the cut surface will not dry
injuriously under several hours. The cutting quality of the
mass is often improved by allowing it to evaporate in the air
for some hours.

The hardening may be done at once in the chloroform and
cedar oil mixture, instead of the chloroform vapour, but I
find the latter preferable. And clearing may be done in
pure cedar oil instead of the mixture, but then it will be
very slow, whereas in the mixture it is extremely rapid.

114 CHAPTER IX.

Srepanow (Zeit. wiss. Mik., xvii, 1900, p. 185) soaks and imbeds in a
solution of celloidin in a mixture of eyual parts of ether and clove oil,

hardens in alcohol or vapour of chloroform, or in benzol, and cuts either
wet or dry.

See also TSCHERNISCHEFF, ¢bid., p. 449.
JorvAN, zbid., p. 193, imbeds in a mixture of 5 parts of 8 per cent.
celloidine solution with 1 of oil of cedar, hardens first in vapour of

chloroform and then in a mixture of 5 parts of chloroform with 1 of oil
of cedar, and cuts wet or dry.

171. Double Imbedding in Collodion and Paraffin.—This is
sometimes, though rarely, employed for objects of which it is desired to
have very thin sections, and which are too brittle to give good sections
by the plain paraffin process.

Kuttscuitzxy’s Method (Zeit. wiss. Mik., iv, 1887, p. 48), —After
the collodion bath, the object is soaked in oil of origanum (Olcum
Origuni vulg.). 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 Mier. Bull., 1887, p. 43; Journ. Roy. Micr. Soc., 1888,
p. 512) modified the process by substituting chloroform for the
origanum oil.

Ine (La Cellule, vii, 1891, p. 347, and viii, 1, 1892, p. 114) imbeds in
collodion ina tube by GiLson’s process (§ 169); 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 paraftin.

Fretp and Martin (Bull. Soc. Zool. de France, 1894, p. 48) make a
solution of dried celloidin in a mixture of equal parts of absolute alcohol
and toluene, of about the consistency of clove oil. This solution is
saturated with paraffin, added in shavings at a temperature not exceed-
ing 20° to 23° C. The tissues are prepared by soaking in some of the
mixture of alcohol and toluene, and are then penetrated with the
celloidin-paraffin solution. The mass is hardened in a saturated solution
of paraffin in chloroform or in toluene, and is finally imbedded in pure
paraffin in the usual way.

SrEPANOw imbeds in paraffin after clearing with benzol, last §.

JORDAN, after imbedding as in last §, passes through a bath of
paraffin dissolved in chloroform into pure paraffin.

WILHELM! (Fauna Flora Golf. Neapel, xxxii, 1909, p. 17), following
ApAvuHy, imbeds in celloidin, hardens in chloroform, then adds benzol
to the chloroform, and passes through pure benzol (half an hour to an
hour) into paraffin, and cuts dry.

Similarly, BRECKNER, Zezt. wiss. Mck., xxv, 1908, p. 29.

STERLING (Jena Zezt., 1909, p. 253) soaks for two or three days in
equal parts of clove oil and collodion, puts for a couple of hours (until
clear) into xylol, and imbeds in paraffin.

See also DAHLGREN, Journ. Appl. Microsc., 1898, p. 97; Sapussow,

COLLODION AND OTHER IMBEDDING MBTHODS. 115

Mitth. Zool. Stat. Neapel, xii, 1896, p. 353; Muyegr, ibid., xiv, 1901, p.
295; MirropHanow, Arch. Zool. Expér. [3], 3, 1896, p. 617; Feperrctr,
Anut. Auz., xxxi, 1907, p. 602; Borpagr, Bull. Sci. France Belg., xxxix,
1905, p. 385; Ganpourt, Zezt. wiss. Mih., xxv, 1909, p. 421; Mayer,
tbid., xxiv, 1907, p. 182.

Other Cold Masses.

172. Joliet’s Gum and Glycerin Method (Arch. Zool. Hupér. et
Gén., x, 1882, p. xliii)—Pure gum arabic dissolved in water
to the consistency of a thick syrup. Pour a little of the
solution into a watch-glass, and add from 6 to 10 drops of
pure glycerin. In the winter or in rainy weather less
glycerin should be taken than in the summer or dry weather.

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 contain-
ing 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.

173. STRICKER’s Gum Method (Hdb. d. Gewebel., p. xxiv) —A con-
centrated solution of gum arabic. The object is imbedded in the gum
in a paper case. The whole is thrown into alcohol, and after two or
three days may be cut. The alcohol should be of about 80 per cent.
(MAYER).

I have seen masses of sufficiently good consistency prepared by this
simple method.

174. RoBeRTSoN’s Grape-sugar Method, see Journ. of Anat. and
Physiol., xxiv, 1890, p. 230.

175. Hyart’s Shellac Method, see Am. M. Mic. Jcurn., i, 1880, p. 8;
Journ. Roy. Mic. Soc., iii, 1880, p. 320. For sections through hard
chitinous organs consisting of several pieces, such as stings and ovi-
positors, retaining all the parts in their natural positions.

176. Brunorti’s Cold Gelatin Mass has heen given, § 155.

Masses for Grinding Sections.*
177. G. von Kocn’s Copal Method (Zool. Anz., i, 1878, p.
36).—Small pieces of the object are stained in bulk and

* For the manipulations of section-grinding, see CARPENTER’S The
Microscope.

116 CHAPTER EX.

dehydrated with alcohol. A thin solution of copal in chloro-
form is prepared by triturating small fragments of copal ina
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
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 away 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
unstained, 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.

This method was invented in order to enable the hard and
soft parts of corals to be studied in their natural relations,
and is valuable for this and similar purposes.

178. EnrrenBaum’s Colophonium and Wax Method (Zeit. wis.
Mik., 1884, p. 414).—Ehrenbaum recommends a mass con-
sisting 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 followed by chloroform,

179. Jounstonr-Lavis and Vosmaurr’s Balsam Method (Journ.
Roy. Mie, Svc., 1887, p. 200).—Aleohol material is carefully

COLLODION AND OTHER [MBEDDING METHODS, 117

and gradually saturated, first with benzol, and then with thin
and thick solution of benzol-balsam. It is then dried for a
day in the air and for several days more in a hot-air bath.
When hard it is ground in the usual way.

180. Writ's Canada Balsam Method, see Zett. wis:. Mik., v, 1888,
p. 200.

181. GIESBRECHT’S Shellac Method.—For hard parts only, spines
of Eshinws, shell, ete., see Morph. Juhrb., vi, 1880, p. 95, or the abstract
in Lex und Maver, Grundziige.

Congelation Masses.

182. The Methods of Freezing.—For the requisite manipula-
tions, and means of producing the requisite degree of cold,
see Carpenter’s The Microscope (ether spray); Jouneg, Zeit.
wiss. Mik., xiv, 1897, p. 370 (liquid carbonic acid); Wotrr,
tbid., xxv, 1908, p. 175 (ethyl chloride); Krauss, ibid.,
p. 289 (solid carbonic acid); Juna, Verh. Ges. Naturf. Aertze,
Ixix, 1898, p. 129 (ethyl chloride); Brissy, C. R. Soc. Bivl.,
Ixii, 1907, p. 1115 (liquid air).

Fresh tissues may be, and are, frequently frozen without
being included tn any mass. But the formation of ice
crystals frequently causes tearing of delicate elements, and
it is better to infiltrate the tisswes with a mass that does not
crystallise in the freezing mixture, but becomes simply hard
and tough, such as one of those given below.

When sections have heen obtained, it is difficult to
manipulate them. Oxr (Zeit. wiss. Mik., xxiii, 1906, p. 327)
puts them into a 1 per cent. solution of gelatin, brings them
therein on to a slide, hardens for an hour in vapour of
formaldehyde, and soaks for a few minutes in formol of 10
per cent. Anrrscaxow (tbid., xxvii, 1910, p. 73) puts them
into aleohol of 50 per cent., gets them on to a slide prepared
with Mayer’s albumin, presses down with paper, puts into
alcohol of 98 per cent., and thence through lower grades
into water.

183. Gum and Syrup Masses.—Hamitton (Journ. of Anat.
and DVhys., xii, 1878, p. 254) soaked tissues in syrup made
with double refined sugar, 2 ounces; water, 1 fluid ounce ;

118 CHAPTER IX.

then washed the superfluous syrup from the surface, and
put into ordinary gum mucilage for an hour or so, and then
imbedded in the freezing microtome with mucilage in the
usual way.

Coie (Methods of Microscopical Rescarch, 1884, p. XXxix)
takes gum mucilage (B. P.), 5 parts; syrup, 3 parts. (For
brain and spinal cord, retina, and all tissues liable to come
in pieces put 4 parts of syrup to five of gum.) Add 5 grains
of pure carbolie acid to each ounce of the medium.

(Gum 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.)

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. Should freezing have been carried
too far, wait for a few seconds.

Wess (The Microscope, ix, 1890, p. 844; Journ. Roy. Afic.
Soc., 1890, p. 118) takes thick solution of dextrin in solution
of carbolic acid in water (1 in 40).

184. Gelatin (SoLLAS, Quart. Journ, Mic. Soc., xxiv, 1884, pp. 163,
164). Gum Gelatin (Jacoss, Amer. Natural., 1885, p. 734). White
of Egg (Routuerr, Denshkschr. math. naturw. Kl. hk. Acad. Wiss. Wien,
1885 ; Zett. wiss. Mik., 1886, p.2)—Small portions of tissue brought in
the white of a freshly laid egg on to the freezing stage, frozen and cut.
Oil of Aniseed (Kuunn, Centralb. f. Bukteriol., xii, 1892, p. 28; Journ.
Roy. Micr. Soc., 1802, p. 706; V. A. Moorn, Amer. Mon. Mie. Journ.,
1894, p. 873; Journ. Roy. Mic. Sec., 1895, p. 247). Anethol (anise
camphor), STEPANOW, Zeit. wiss. Mik., xvii, 1900, p. 181.

For details of these see previous editions.

For DéuiKeEn’s method of solidifying formol by means of resorcin,
see Zeit. wise, Mik., xiv, 1, 1897, p. 33.

CHAPTER X.
SERIAL SECTION MOUNTING.

185. Choice of a Method.—I recommend the following :—
For general work with paraffin sections, the combined water
and albumen method, § 188. For very delicate work, the
water method. For collodion sections, the albumen method ;

for large collodion sections, Granam Kern’s seems the most
convenient,

Methods for Paraffin Sections.

186. The Water or Dessication Method.—Gauiu (Arch. nat.
Phys., Phys. Abth., 1881, p. 156); Sucuannex (Zeit. wise. Mik-.,
vil, 1891, p. 464); Guttanp (Journ. Anat. and Phys., xxvi,
1891, p. 56) ; Scuierrerpecker (Zeit. wiss, Alth., ix, 1892, p.
202) ; Herpennain (Kern und Protoplasma, p. 114) ; Nussaum
(Anat. Anz., xii, 2, 1896, p. 52); Maver in the Grundziige,
Lee und Mayzr, 1898, p. 118; De Groor (Zeit. wiss. Mik,
xv, 1898, p. 62), and others—The principle of this method
is that the sections are made to adhere to the slide without
the intervention of any cementing substance, being brought
into intimate contact with the glass by being slowly drawn
down by the evaporation of a layer of water on which they
are floated. It is now practised, with unessential variations,
as follows:

(a) For sections that are large and not wwmerous. The
sections are flattened out on water by one or other of the
processes described in $149. The slide is then drained and
put away to dry until every trace of water has completely
evaporated away from under the sections. This drying may
be performed at the temperature of the laboratory, in which
case many hours will be necessary (to be safe it will generally
be necessary to leave the sections overnight). Or it may be

120 OHAPTER X.

performed in a stove or on a water-bath at a temperature a
few degrees below the melting-point of the paraffin (best not
above 40° C.), in which case fixation will be much more rapid,
large thin sections being often sufficiently fixed in an hour,
though thick ones will require half a dozen hours or more.
The paraffin must not be allowed to melt before the sections are
perfectly dry, the sections are sure to become detached if it
does. Perfectly dry sections have a certain brilliant trans-
parent look that is easily recognisable. As soon as dry the
paraffin may be removed and they may be further treated
as desired. ‘To remove the paraffin all that is requisite is
to put the slide into a tube of xylol or other good solvent,
which in a few seconds, or minutes at most, removes the
paraffin perfectly. Most workers first melt the paraffin, but
I find this is not necessary.

(b) For series of numerous small sections. Clean a slide
perfectly, so that water will spread on it without any tendency
to run into drops (see below). Breathe on it, and with a
brush draw on it a streak of water as wide as the sections
and a little longer than the first row of sections that it is
intended to mount. With a dry brush arrange the first row
of sections (which may be either loose ones or a length of a
ribbon) on this streak. Breathe on the slide again, draw on
it another streak of water under the first one and arrange
the next row of sections on it, and so on until the slide is
full. Then breathe on the slide again, and with the brush
add a drop of water at each end of each row of sections, so
as to enable them to expand freely; then warm the slide so
as to flatten out the sections, taking care not to melt the
paraffin. Some persons do this by holding it over a small
flame for a few seconds. I prefer to lay it on a slab of
thick glass, warmed, watching the flattening of the sections
through a lens if necessary. As soon as they are perfectly
flat, draw off the excess of water from one corner of the
mount with a dry brush, and put aside to dry as before (a).

In order to succeed in this method it is absolutely essential
that the sections be perfectly expanded and come into close
contact with the slide at all points. And to ensure this it
is necessary that the slide should be perfectly free from
grease, so that the water may wet it equally everywhere.
The test for this is, firstly, to breathe on the slide; the

SERIAL SECTION MOUNTING. 121

moisture from the breath should condense on it evenly all
over, and disappear evenly. Secondly, streaks of water
drawn on it with a brush should not run. ‘To obtain a slide
that will fulfil these conditions, clean it well in the usual
way, place a drop of water on it and rub it in thoroughly
with a damp cloth and try the tests. If this does not
suffice, take a turn of a corner of the cloth round a finger
and rub it with a piece of chalk, then damp the cloth and
rub the slide with it, finishing up with a clean part of the
cloth and clean water (Du Groot, loc. cit. supra). If after
performing this operation twice the slide still refuses to take
the water thoroughly, it should be rejected as incorrigible ;
for there are apparently some sorts of glass that can never
be got to wet properly. Mayer finds carbonate of magnesia
or soda useful.

Gupernatscu (Zeit. wiss. Mikr., xxiv, 1908, p. 358) washes
the slide well with potash soap, and arranges the sections
on it whilst still wet. Hux.iy (id¢d., 1906, p. 330) passes it
two or three times over the flame of a Bunsen burner.

Tap water seems preferable to distilled water; it seems to spread
better and give a stronger adhesion. Nuspaum adds a trace of gum
arabic (one or two drops of mucilage to a glass of water); APATHY
(Microtechnik, p. 126) adds 1 per cent. of Mayer's albumen (§ 187); and
Hennecuy (Legons sur la Cellule, 1896, p. 62) takes a 1:5000 solution
of gelatin, with a trace of bichromate of potash, added just before
using, and dries the slides exposed to light. Similarly, BuRcHARDT
(Jena Zett., xxxiv, 1900, p. 719).

Some workers have used alcohol (50 per cent. or 70 per cent.) instead
of water; but this I believe to be now generally abandoned.

This is the most elegant method of any, as there is
nothing on the slide except the sections that can stain, or
appear as dirt in the mount. ‘Tissues do not suffer from
the drying, provided the material has been properly
imbedded. Sections stick so fast by this method that they
will stand watery or other fluids for weeks, so long as they
are not alkaline. When successfully performed it is quite
safe, provided that the sections are of a suitable nature.
They must be such as to afford a sufficient continuous
surface, everywhere in contact with the slide. Sections of
parenchymatous organs stick well; sections of thin-walled
tubular organs stick badly. Sections of chitinous organs

122 CHAPTER X.

are very unsafe. he larger and thinner sections are, the
better do they stick, and vice vers. Sections from chromic
or osmic material adhere less well than sections from alcohol
or sublimate material.

By taking a staining solution instead of pure water for expanding,
the sections can be got to stain at the same time, and so be brought into
balsam without passing through alcohol; see Maygr, Mitth. Zool. Stat.
Neapel, xii, 1896, p. 820; Scumory, Path.-hist. Untersuchungsmethoden,
1897, p. 38; Smiru, Journ. Anat. Phys., xxxiv, 1899, p. 151.

187. Mayer’s Albumen (Aitth. Zool. Stat. Neapel, iv, 1883 ;
Internat. Monatschr. f. Anat., iv, 1887, p. 42).—White of egg,
50 c.c.; glycerin, 50 c.c.; salicylate of soda, 1 grm. Shake

1em well together, and filter into a clean bottle. he filter-
ing may take days or a week, but the preparation does not
spoil meanwhile.

Francorre shakes up the albumen with a few drops of
acetic acid before adding the other ingredients, and finds
the filtering greatly quickened. So do I. Be careful with
the acid. Si

A very thin layer of the mixture is spread on a slide with
a fine brush and well rubbed in with the finger (I prefer a
small rubber ‘“‘ squeegee”). ‘Ihe sections are laid on it and
pressed down lightly with a brush (if they will bear it).
The slide may then be warmed for some minutes on a water-
bath, and the paraffin removed with a solvent.

It is not necessary to warm the slide at all; the paraffin
can be removed in the cold if desired by putting the slide into
toluol, xylol, or the like. But the shde must, in any case,
be treated with alcohol after removal of the paraffin, in order
to get rid of the glycerin, which will cause cloudiness if not
perfectly removed.

This method allows of the staining of sections on the slide
with perfect safety, both with alcoholic and aqueous stains,
provided they be not alkaline.

According to my experience, the albumen method is abso-
lutely safe, provided that alkaline fluids be avoided in the
after-treatment. It has the defect that certain plasma
stains (not chromatin stains) colour the albumen very
strongly, and cannot be removed from it, and that sections
are not expanded by it.

SERIAL SECTLON MOUNTING. 123

It sometimes happens that the mixture after it has stood
for some time becomes turbid, and at last coagulates, pass-
ing into a caseous state; or it may undergo a hyaline
coagulation, drying up like amber. But up to the very last
it does not ¢n general lose its adhesive properties. I have,
however, found it to do so, after keeping for five or six
years, so that, to be on the safe side, it may be well to make
it up fresh every six months.

Heivennain (Zeit. wiss. Mikr., xxii, 1905, p. 331) makes it
up with 1 erm. of blood albumen dissolved in 25 ¢.c. of water,
and an equal volume of 50 per cent. alcohol.

188. The Albumen and Water Method (Hennecuy, Jowrn. de
PAnat. et de la Physiol., 1891, p. 398).—A drop of water is
spread on a slide painted with Mayer’s white-of-egg mixture,
the sections are arranged on it, the whole is warmed (not to
the 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 sufficiently disappeared,
which at that temperature will be in about ten to fifteen
minutes, the slide is further treated as described last §.

This is a most valuable method. It is quicker than the
water method, and, for difficult material, safer.

See also OHLMACHER, Journ. Amer. Med. Assoc., April, 1893.

The so-called “ Japanese ' method, attributed to IkEDA by REINKE
(Zeit. wirs. Mik., xii, 1895, p. 21), 2s merely that of HENNEGUY.

Maww (Anat. Anz., viii, 1898, p. 442) shakes up white of egg with
water, coats slides with it and dries them. He flattens sections on water
at 40° C., lifts them out on a prepared slide, and dries for five minutes
at 35° C.

189. Garlic-water—HoLLANDE (Arch. d’ Anat. Mier., xiii, 1911, p.
171) gives the following as more adhesive than albumen :—50 g. of crushed
and chopped garlic are rubbed up with 80 ¢.c. of chloroform-water
(Codex, A.C.) and filtered after twenty-four hours. Use as albumen.

190. ScHALLIBAUM's Collodion (Arch. mikr. Anat., xxii, 1883, p. 565).
—One part of collodion shaken up with 3-4 parts of clove or lavender
oil. Use as albumen. Sections can be treated with alcohol (not absolute)
and divers staining fluids. I do not find it safe for this. Rasx, how-
ever (Zeit. wiss. Mrk., xi, 1894, p. 170), finds that it is if you take 2 parts
of collodion to 3 of clove oil, and make up fresh every four or five days.

124 CHAPTER X.

191. Oxrucia’s Method for Paraffin or Celloidin S2ctions
(Newrologisches Centralb., ix, 1890, p. 295; GuLLanp, 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 ’ 2 : : . 380 cc.
95 per cent. alcohol : ; - 20 ;,
Transparent syrupy solution of pure

dextrin made by boiling with dis-

tilled water 5 : ; sy LOS. 5

They are dried slowly for two or three days until the
surface is just sticky to the moist finger. Puruffin 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, and this in turn by absolute
alcohol. The alcohol is poured off, and the sections are
covered with solution of celloidin. The plates are left to
evaporate for ten minutes in a horizontal position, then
brought into water, in which the sheet of celloidin with the
sections soon becomes detached, and may be further treated
as desired, e.g. as in Weigert’s process, § 198. The evapo-
ration must not be artificially hastened.

DIMMER (Zedt. wi:s. Mik., xvi, 1899, p. 44) coats the slides with a
solution of about 16 parts of gelatin in 300 of warm water, and dries
them (two days), and proceeds in other respects as above.

A good method for large sections, equally applicable to
paraffin sections, to celloidin sections, and to sections of
material that has not been imbedded at all.

For BLocuMman’s modification of Weigert’s process, by means of
which large sections can be preserved wnmounted, see Zeit. wiss. Mik.,
xiv, 1897, p. 189.

192. STRASSER’S Collodion Paper Method (ibid., iii, 1886, p.346).—
This is an extremely complicated modification of Weigert’s method for
celloidin sections, and is only adapted for use with SrRAssER’s automatic
ribbon-microtome. See Zezé. wiss. Mvh., iii, 1886, p. 346 ; vi, 1889, p. 154;
vii, 1890, pp. 290 and 304; ix, 1892, p. 8; xii, 1895, p. 154; and xiv, 1897,
p. 39; also Schomnemann, ¢bid., xix, 1903, p. 333; STRassER, cbdd..
p. 3387; and Ruppricut, ¢bdd., xxviii, 1912, p. 281.

SERIAL SECTION MOUNTING. 12:

we

Methods for Watery Sections.

193. Fou’s Gelatin (Fou, Lehrd., 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 mix-
ture on to the slide and allow it to dry. In afew hours the
gelatin passes into the insoluble state. It retains, however,
the property of swelling and becoming somewhat sticky in
presence 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.

This method is specially intended for sections made under
water, large celloidin sections amongst others.

Similarly, Roppricat, loc. czt., last §, with the needless com-
plication of a seriation on Strasser’s collodionised paper.

Swrasser (loc. ctt., last $) also employs a dry gelatin film
which he makes sticky by means of carbol-xylol.

Methods for Celloidin Sections.

194. The Albumen Method.—I find that celloidin sections
may be mounted on Mayer’s albumen, and have the celloidin
removed, if desired, by putting them into ether-alcohol.
Care must be taken to press them down very thoroughly
on to the albumen ; and it is well not to have them too wet.

Similarily Jorpawn (Zeit. wiss. Mik., xv, 1898, p. 54), and
Arourinsky, bid. xvii, 1900, p. 87. See also Jorpan (ibid.,
192-194) ; Dantscuaxkorr, tlid., xxv, 1908, p. 35; Maximow,
ibid., xxvi, 1909, p. 184; AnirscuKow, tbid., xxvii, 1910,
p. 68; Wapsur, ibid., xxix, 1912, p. 186 ; Roupascaxin, Anat.
Anz., xxxi, 1907, p. 30. Weber paints over the series on
the albumen with a layer of thin collodion, and puts into
alcohol of 50 per cent., then into a mixture of equal parts of
chloroform and absolute alcohol. After staining, pure
absolute alcohol must be avoided.

195. Summers’ Ether Method (Amer. Mon. Mic. Journ.,1887,
p. 73).—Place the sections in 95 per cent. alcohol for a

126 CHAPTER X.

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 colloidin will immediately soften and
become perfectly transparent. Place the slide in 80 per
cent. alcohol, or even directly in 95 per cent. if desired. I
have not myself found this method safe.

Instead of pouring the ether vapour over the slide, it may,
of course, be treated with ether vapour in a preparation glass
or similar arrangement, which I think preferable.

Gaar (Proc. Amer. Soc. Mic., 1892, p. 82) advises that the
slide be one that has been previously coated with a 0°5 per
cent. solution of white of egg and dried; the collodion
adheres much more strongly to an albuminised surface.

AUBURTIN (Anat. Anz., xiii, 1897, p. 90) arranges on a clean slide,
dehydrates the sections with blotting-paper and treatment with absolute
alcohol, then drops on to them a mixture of alcohol and ether which
dissolves out the celloidin from the sections, then allows the thin col-
lodion thus formed to evaporate into a thin sheet on the slide. Then
70 per cent. alcohol and other desired reagents.

Similarly, Marer (Miinch. med. Wochenschr., lvii, 1910, No. 12; Zett.
wiss. Mik., xxvii, 1910, p. 385), but adding a treatment for ten to fifteen
minutes with sulphide of carbon.

See also MyrErs, Areh. Anat. Phys., Anat. Abth., 1902, p. 371 (com-
plicated).

196. ApAtHy’s Oil of Bergamot Method (Mitth. Zool. Stat.
Neapel, 1887, p. 742; Zeit. wiss. Mik., v, 1888, pp. 46 and
360, and vi, 1889, p. 167).—Cut with a knife smeared with
yellow vaseline and wetted with 95 per cent. alcohol. Float
the sections, as cut, on bergamot oil (must be green, must mix
perfectly with 90 per cent. alcohol, and must not smell of
turpentine), or on carbolxyol (Mikrotechnik, p. 176). The
sections flatten themselves out on the surface of the oil; and
are then transferred to a slide which (ApAtuy, Mikrotechnik,
pp. 127 and 176) has been previously collodionised and
dried.

If the sections are to be stained, the slide after removal
of the bergamot oil, by a cigarette paper, 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.

SERIAL SECTION MOUNTING. 127

If it be desired to stain in a watery fluid, care must have
been taken when arranging the sections to let the colloidin
of each section overlap that of its neighbours at the edges,
so that the ether vapour may fuse them all into one con-
tinuous plate. This will become detached from the slide in
watery fluids, and may then be treated as a single section.
Terpinol may be taken instead of bergamot oil.

197, ApAruy’s Series-on-the-Knife Method (Zeit. wiss. Mik.,
vi, 1888, p. 168)—The knife is well smeared with yellow
vaseline, rubbed evenly on, 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 everlapping or at least touching that of its neighbours.
When a series (or several series, if you like) has been thus
completed, the sections are dried by laying blotting-paper
on them, and the series is painted over with some of the
thinnest celloidin solution used for imbedding, is allowed to
evaporate for five minutes in the air, and the knife is then
removed and brought for half an hour into 70 per cent.
alcohol. ‘his hardens the celloidin around the sections into
a continuous lamella, which can be easily detached by means
of a scalpel, and stained, or further treated as desired.

198. Wetcerv’s Collodion Method (Zeit. wiss. Mikr., 1885,
p. 490).—Shides, or larger plates of glass, are prepared by
coating them with collodion in a thin layer, as photographers
do, and allowing them to dry (they may be kept thus in
stock). Sections (cut wet with alcohol) are got on to one of
these (by a roundabout process, not essential), and arranged
in order, and gently pressed down with paper.

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
indications on it with a small brush charged with methylen
blue (the colour will remain fast throughout all subsequent
manipulations).

The plate may now be either put away till wanted in 80
per cent. alcohol, or may be brought into a staining fluid,

128 OHAPTER X.

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 dehydration.
Weigert recommends for clearing the mixture of xylol and
carbolic acid (§ 167).

The series should be cut into the desired lengths for
mounting whilst in the alcohol.

A good method for large and thick sections.

For Brocnman’s modification see § 191.

SrrassER takes gummed paper instead of the glass plates used in this

process. See the papers quoted § 192.
See also WINTERSTEINER (Zeit. wiss. Mik., x, 1893, p. 316) and Kuzo

(Arch. mik. Anat., xx, 1907, p. 178).

199. Obregia’s Method—Slides are prepared as directed
(§ 191), the sections are arranged on them and covered with
celloidin or photoxylin and evaporated as described, § 191.

For Diumer’s modification see also § 191.

200. Collodion Film Method—Granam Kerr (in litt., 1908)
seriates on Kodak films. A film has the emulsion removed
by hot water. The sections are arranged on a dry film, and
the applications of a drop of absolute alcohol and ether (or
an atmosphere of alcohol and ether) suffices ta weld them
into a mass with the film. The sheet may then be stained
and mounted, or rolled up and stored in cedar oil.

Other Methods for Celloidin Sections—See $$ 193 (For) and
182 (Ont).

CHAPTER XI.
STAINING.

201. The Molecular Processes involved in Staining. — The
question whether the phenomena of staining and of industrial dyeing
are chiefly of a chemical order, as held by some, or chiefly of a physical
order, as held by others, is outside the province of this book. See
(besides works on chemistry, amongst which may be mentioned Brnuz-
pikt and Kwecut, The Chemistry of the Coal-tar Colours, London,
1889) FiscHEer’s Fixirung, Firbung und Bau des Protoplasmus, Jena,
G. Fischer, 1899; PAPpPENHEIM’s Gruudriss der Farbchemie, Berlin, A.
Hirschwald, 1901; and the articles in Encyel. mk. Technik.

202. Histological Staining: Specific, Nuclear, and Plasmatic.
—Stains are either general or special (otherwise called Spe-
cific, 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. ‘I'o obtain this differentiation is the chief
object for which colouring reagents are employed in micro-
scopic 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 prominently
stained, the elements of other sorts present in the prepara-
tion 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 stam that
is generally meant by a specific stain. In the latter the
stain seizes on one of the constituent elements of cells in
general, for instance, either on the chromatin of the nucleus,

9

130 CHAPTER XI.

or on one or other of the elements that go to make up the
cytoplasm.

Stains that thus exhibit a selective affinity for the sub-
stance of nuclei—nuclear or chromatin stains—form a class
of stains of peculiar importance for the embryologist or
zootomist. For they enable him 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 catch the eye, which is then able to follow out with ease
the contours and relations of the elements to which the
nuclei belong.

To these must be added another group of stains of the
ereatest importance to the cytologist and histologist, the
plasmatic stains or plasma stains. These take effect es-
pecially on elements of cells and tissues other than the
chromatin—for instance, on the reticulum of cytoplasm,
or on its granules, or on polar corpuscles, etc., or on the
formed material of tissues.

203. Dyes: Basic, Acid, and Neutral.—The colouring matters,
or dyes, employed either in industrial dyeing or in histo-
logical staining are almost always salts, They are known
as “ basic,” “ acid,” or “neutral” dyes. By a“ basic” dye
is meant one which is either a base or a compound in which
a so-called “ colour base” (or molecular group to which the
compound owes its colouring properties) is combined with a
nou-colouring acid. For instance, fuchsin or magenta is a
basic dye. It is the hydrochloride of rosanilin, and its
colouring properties are due to the rosanilin which exists as
a base in the compound, and not to the hydrochloric acid of
the compound. By an “acid” dye is meant a compound in
which a so-called ‘ colour-acid”’ is combined with a non-
colouring base. The dye known as acid fuchsin or acid
magenta (Siurefuchsin) is an “acid” colour. It is the
soda-salt of di- or tri-sulphoconjugated rosanilin, that is of
rosanilin di- or tri-sulphonic acid, and its colouring pro-
perties are due to the rosanilin which exists as an acid in
the compound, and not to the soda. Or, to take a simpler
case, picrate of ammonia is an “acid” colour, and _ its
colouring properties are evidently due to the picric acid in
it, and not to the ammonia.

STAINING. 131

It is important to keep clearly in mind that in speaking
of dyes the terms “ acid” or “ basic ” refer to the characters
of the colour acids or colour bases, and not to those of the
salts. An “acid” dye may have a neutral or alkaline
reaction (e. g. picrate of ammonia), and vice versd.

Basic dyes are generally easily soluble in alcohol, less
easily in water; whilst the contrary is the case for acid
dyes. The free colour bases or colour-acids are generally
less soluble in water than their salts, for which reason they
are so little used that they are hardly to be found in com-
merce. It follows that such histological formule as depend
on setting free a colour-acid from its salt (e.g. as by precipi-
tating it from eosin by means of alum, as advised by Ranvier
and Wissotzky) are irrational. Colour-bases or colour-acids
may themselves be colourless.

‘The stain given by acid dyes is fast to acids, and may be
intensified by them; whilst basic dyes are washed out by
acids, but intensified by alkalies.

“ Neutral” dyes are compounds of a colour-base with a
colour-acid. They are seldom or never prepared industrially,
the only example that I can find mentioned in Benepixr and
Knecav’s Chemistry of the Coal-tar Colours being artificial
indigo. They are prepared for histological purposes by
mixing the aqueous solutions of a basic ‘and an acid dye.
For instance, by mixing the acid picrate of ammonia with
the basic hydrochloride of rosanilin, you can bring about the
formation of sal ammoniac and picrate of rosanilin, which is
a “ neutral” colouring matter. They are generally insoluble
in pure water, and hence precipitate when the mixture is
made, but may be got to redissolve by adding an excess
of the acid colour, or of the basic, and are always soluble
in alcohol. They can also, as is often done, be formed
in the tissues themselves by staining first with an acid dye,
and then bringing the preparation, without first washing out,
into a basic dye.

ExRuicH and Lazarus (“ Die Anemie,” Wien, 1898, p. 26) state that
the basic dyes methyl-green, methylen-blue, amethyst violet (also
pyronin and rhodamin), and the acid dyes Siéurefuchsin, Orange G,
and Narcein, are peculiarly favourable for making neutral mixtures.

See further as to the * neutral” colours, Rosin, “Ueber eine neue
Gruppe der Anilinfarbstoffen,” in Berliner klin. Wochenschr., xii, 1898,

152 CHAPTER XI.

p. 251; Zeit. f. wiss. Mék., xvi, 2, 1899, p. 223; Journ. Roy. Mie. Soc.,
1899, p. 547; Hneyel. mik. Technik, 1903, p. 1028; PAPPENHEIM, op. czt,
supra; HEIDENHAIN, Anat. Anz., xx, 1901, p. 36,

204. The Chromatophily of Tissue-Elements.—The elements
of tissues are distinguished as “basophilous,” “ acido-
philous,” and “neutrophilous,” according as they seem to
show a natural affinity for basic, acid, or neutral colouring
matters respectively | According to a generalisation due to
Kuniicw (Zeit. hlin. Med., 1, 1880, p. 555; Reicuerr anp
Du-Bois Reyvmonn’s Arch. Anat. Phys., Phys. Abth., 1879, p.
571), the basic ‘colours are in general chromatin stains—that
is, they have a special affinity for the element of nuclei
known as chromatin, so that they are mostly sharp nuclear
stains, and chromatin is basophilous. The acid colours, on
the other hand, are, according to him, in general plasma
stains—that is, they have a special affinity for cytoplasm and
intercellular substances, which are therefore acidophilous.
The neutral colours exhibit special affinities for certain cell-
contents, and the elements affected by these are said to be
newrophilous,

I think that that is a generalisation which requires some
explanation and qualification. In practical histology we
have to take account not only of the affinities for dyes of
cellular clements in a physically and chemically unaltered
state, but of the alterations in these affinities brought about
by the action of fixatives and mordants. Now most fixing
agents either diminish or increase the chromatophily of
tissues; so also do all mordants, some of which may
even invert the natural chromatophily of tissues (see S$ 205,
207). Then, too, we have to take account also of the
resistance of the stain to the liquids employed for washing,
for dehydration, for clearing; in short, we have to take into
account the way in which the dye. behaves when employed
as a regressive stain (f £09). This is of pecuhar importance
in the case of the coal-tar colours, seeing that they are
largely used for the regressive staining of sections destined
to be dehydrated by alcohol and mounted in balsam. Now
Ehrlich’s experiments take no account of these conditions,
(He worked with “cover-glass preparations”’ of isolated cells,
such as blood and lymph cells, and-was thus able to avoid

STAINING. 138

the prolonged washing necessary for most sections, and to
suppress altogether the dehydration by alcohol, his cover-
glass preparations being simply dried after staining in a
stove.) In consequence, his chemical categories of basic
colours and acid colours fail to correspond always in practice
to the technical categories of chromatin stains and plasma
stains.

For instance, orange is an acid colour; but used as a
regressive stain I find it will give a very sharp stain of
chromatin: it cannot, therefore, be classed as a mere plasma’
stain, though it is also a very good plasma stain.. Siinre-
fuchsin is a very acid colour. It behaves in general as a
decided plasma stain. But used as a regressive stain it
sometimes, under conditions which I am not able to specify,
gives a very vigorous stain of chromatin. Safranin is a
basic colour, but by the use of appropriate mordants it can
be made to behave as a plasma stain. Methylen blue is a
basic colour. But, as is well known, when employed according
to the method worked out by Ehrlich for the so-called intra-
vitam staining of nerves, it affords a stain that is essentially
plasmatic, such staiming of nuclei as may occur in this
process being an accidental epiphenomenon. Nigrosin is,
according to Ehrlich, an acid colour,.and should therefore be
esseiitially a plasma stain. Yet I find that, used as a
regressive stain in the same way as safranin, it gives a
vigorous chromatin stain, cytoplasm being only faintly
coloured. Bordeaux is an acid colour, but it stains chro-
matin as well as cytoplasm. Further, both carminic acid
and hematein are acid dyes, but combined with the mordant,
alum (as in alum-carmine or alum-hematoxylin), they give
nuclear stains. Indeed, it is not too much to assert that
there is hardly any colour, either basic or acid, that may not
be made to afford either a chromatin stain or a plasma stain,
according to the way in which it is employed. ‘There is,
in practice, no absolute chromatophily of tissue-elements.

205. Substantive and Adjective Staining; Mordants—In the
industry of dyeing, colouring matters are divided into two
classes, according to their behaviour with respect to the
material to be dyed. Certain dyes are absorbed directly
from their solutions by the material immersed therein, and

134 CHAPTER XT.

combine with it directly. In this case the material is said to
be substantively dyed, and the colouring matter is called a
substantive colouring matter.

Other dyes do not combine directly with the material to
be acted on, but this material must first be charged with
some substance known as a mordant (generally a metallic
salt or hydrate) before it will combine with the colouring
matter. These are known as adjectire colouring matters.*

Mordants are bodies which have the property of com-
bining on the one hand with the elements of tissues and on
the other with the colouring principle of the dyes used,
forming with the latter insoluble coloured compounds (known
as Lakes), which are retained in the tissues. It follows that
basic dyes require mordants of an acid character and acid
dyes mordants of a basic character. The mordant may be
introduced into the tissues either before the dye or at the
same time with it.

It may seem at first sight that the majority of histological
stains are obtained by substantive staining of the tissues.
But on reflection it will be seen that many of the histological
stains that are obtained without intentional mordanting of
the tissues should yet in strictness be attributed to the class
of adjective stains. For whenever there is reason to suppose
that the stain obtained results from a combination of the
colouring matter with some metallic salt or hydrate that is
not a constituent of the living tissue, but has been brought
into it by the fixing or hardening reagents, it must be ad-
mitted that these reagents play the part of mordants though
only intentionally employed for another purpose. This would
appear to be the case with the stains, or some of them,
obtained after fixation with corrosive sublimate, picric acid,
salts of iron, of platinum, of palladium, of uranium, and, for
certain tissue elements and certain colours, chromium. And
further, the mordanting substance may not only be present
unintentionally in the fixing or hardening agents, it may be
present unintentionally, or with imperfect realisation of its
import, in the staining solutions themselves. Such is un-
doubtedly the part played by alum in most of the stains in

* For an excellent popular exposition of this subject see BENEDIKT

and Knrcut’s Chemistry of the Coal-tar Colours (George Bell and
Sons).

STAINING. 135

which it figures as an ingredient. Iodine also plays in some
staining processes a part which seems only explicable on
the supposition that it acts as a mordant. And in some
processes an acid dye is made to act as a mordant for a
subsequently employed basic dye.

In some staining processes, however, mordants are inten-
tionally resorted to in order to fix the stain. Mordanting
has long been intentionally employed in some hamatein
staining processes, such as the iron-alum process of Brnpa
and M. Herpennain. More lately it has been resorted to for
staining with tar colours.

By combining with the elements of tissues, mordants confer on them
an artificial chromatophily. This may take place to so great an extent
that the original affinities of the tissues for dyes are not only masked
but may be seemingly altered into their opposites, thus producing an
“inversion” of their chromatophily. Thus by means of appropriate
acid mordants certain basic anilins, which by the usual methods of
regressive staining are pure chromatin stains, may be made to afford a
pure plasma stain—one not affecting chromatin at all, thus giving an
“inversion ” of the usual stain.

It would seem that the nuclear stains obtained by carmine and hema-
toxylin should in strictness be classed as inversion stains. The colouring
principle of carmine, carminic acid, is an acid hody, and consequently 2
plasma stain. But in the form of carmine it is combined with the basic
oxide alumina, which by mordanting the acid body nuclein confers on it
a basic character and renders it acidophilous, thus producing an inver-
sion of the natural staining affinity. So also with the acid dye hematein.
So that we see that whilst basic dyes substantively employed are nuclear
stains and acid dyes substantively employed are plasma stains, yet
basic colours with acid mordants may give a plasma stain and acid
colours with basic mordants may ‘give a nuclear stain.

206. Metachromasy.—Adjective stains are of the colour of the lake
formed by the mordant and the colouring principle of the dye. Sub-
stantive stains are for the most part of the colour of the solution of the
dye employed. But this is not always the case. There is a very small
group of dyes, mostly basic tar-colours, which have the property of
staining different elements of tissues in different colours, one of these
being the same as that of the solution of the dye, and the other, or
metachromatic colour, being the same as that of the free colour-hase
of the dye. For instance, the red dye, safranin, colours nuclei red, but
mucin and the ground-substance of cartilage orange. The blue dye,
methyl violet, stains normally blue; but amyloid matter, Mastzellen,
mucin, and cartilage, are stained red by it. Similarly with thionin,
dahlia, gentian violet, toluidin blue, etc.

In these cases the effect appears to be due to chemical reaction, But

136 CHAPTER XI.

in other cases a similar effect has been shown to be due to the presence
of impurities in the dyes; so with iodine green and methyl green, which
mostly contain an admixture of methyl violet.

It has been held by some that metachromasy is an optical pheno-
menon, but that is clearly only true of certain cases.

207. The Preparation of Tissues for Staining.—It is generally
found that precise stains can only be obtained with carefully
fixed (i.e. hardened) tissues. Dead, but not artificially
hardened 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 (see, however, next section).

It appears probable, as was first pointed out, I believe, by
Mayer (Mitth. Zool. Stat. Neapel, 11, 1880, p. 17), that most
of the histological stains obtained with fixed tissues are
brought about in one of two ways. Hither they result from
the combination of the colouring agent with certain organic
or inorganic salts,—phosphates, for instance, that existed in
the tissue elements during life and were thrown down in situ
by the fixing or hardening agent employed, as seems to
happen when such a fixing agent as alcohol is employed.
Or they result from the combination of the colouring agent
with certain compounds that did not pre-exist in the tissues,
but were formed by the combination of the constituents of
the tissues with the chemical elements brought to them by
the fixing agent, as seems to happen when such a fixing
agent as chromic acid is employed—the compounds in
question being probably chiefly metal albuminates. It
follows that correct fixation and careful washing out are
essential to the production of good stains.

Objects which have been passed through alcohol generally
stain better than those which have only been in watery
fluids.

But long preservation of tissues in alcohol is generally
unfavourable to staining.

208. Staining “intra vitam.’—-Some few substances (which
are almost always basic dyes) possess the property of staining
—or rather, tongeing—living cells without greatly impairing
their vitality. Such are—in very dilute solutions—eyanin
(or quinolein), methylen blue, Bismarck brown, aunilin black,

STAINING. 137

Congo red, neutral red, Nile blue, Janus green, and, under
certain conditions, dahlia and eosin, gentian violet, with
perhaps methyl violet, and some others.

These reagents are best employed in a state of consider-
able dilution, and in neutral or feebly alkaline solution—
acids being of course toxic to cells. ‘hus employed, they
will be found to tinge with colour the cytoplasm of certain
cells during life ; never, so far as I can see, nuclear chromatin
during life; if this stain, it is a sign that death has set in.
The stain is sometimes diffused throughout the general sub-
stance of the cytoplasm, sometimes limited to certain granules
in it.

It has been asserted by some observers that the nucleus
may be stained during the life of the cell by means of
Bismarck brown, Congo red, methylen blue, neutral red,
Nile blue, and safranin. But it is by no means clear from
the statements of these writers that the coloration observed
by them is localised in the chromatin of the nucleus. It
would rather appear to be a diffuse coloration brought about
by mechanical and momentary retention of the dye in the
nucleus—which is a very different thing from a true nuclear
stain. And in some of the cases reported it is by no means
certain that the coloured nuclei were really in the living
state. See hereon the article by Fiscar. (“ Firbungen,
intravitale ”) in Mneycl. mik. Tecknik.

I have myself made a considerable number of observa-
sions on the subject of intra-vitam staining, and have come
to the same conclusion as Gaxeorti (Zeit. wiss. Alik., xi,
1894, p. 172), and many recent writers, namely, that most
of the so-called “intra-vitam ” stains are either not true
stains or that the stained substances are not really living.
The coloration appears mostly, if the cell that shows it has
remained in a state .of unimpaired vitality, to be due to
simple absorption or imbibition of the colouring matter
by the cell, not to a molecular combination of the colouring
matter with any of the constituents of the cells.

And when a more or less fast stain has been obtained, it
is generally found that this is limited to cell-contents that
do not appear to form an integral part of the living texture
of the. cell—to food-granules, or katabolic products, or the
like,

138 CHAPTER XT.

Fiscuen (Anat. Hefte, xvi, 1901, p. 417, and op. cit. supra)
concludes in favour of the vitality of certain of the stained
granules.

Apart, however, from this question, it must be conceded
that these so-called “ vital stains” are frequently very useful.
According to my experience, methylen blue is the most
generally useful of them. It has (with Bismarck brown,
Congo red, and neutral red) the valuable point that it
is sufficiently 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 ample 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. XVI. Bismarck brown stains may be fixed with 0:2
per cent. chromic acid or with sublimate solution (Mayer),
or 1 per cent. osmic acid (Loisen, Journ. de V Anat. et de la
Phys., 1898, No. 2, p. 212—a work that contains a good deal
of information on the subject of infra-vitam stains), and the
preparations may be stained with safranin, care being taken
not to expose them too long to the action of alcohol. For
the study of cell-granules, neutral red is perhaps the best.

Fiscuet (‘ Unters. ueb. vitale Faerbungen,” Leipzig.
1908) finds that alizarin is specific for nerves. Add excess
of alizarin to boiling water, boil and filter, and add 1 vol. of
the filtrate to the water containing the organisms (Cladocera).
The stain takes several hours.

For sulphorhodamin, which is selective for many organs
(kidney, liver, uterus, skin, lymph-glands, etc.), see
ANDREEW, in Virchow’s Arch., cciv, 1911, p. 447.

See also Gottman, Proc. Roy. Sue., Ixxxv, 1912, p. 146
(trypan blue, isamin blue, diamin blue, etc.) ; and GoLpMann,
‘“Die aeussere u. innere Sekretion, etc.,’ Tésrncey, 1909,
and “ Neue Untersuch., etc.,’’ ibid., 1912.

209. The Practice of Staining.—-Selective staining is arrived
at in two ways. In the one, which is called the progressive
wethod, you make use of a colouring reagent that stains the

STAINING. 1389

elements 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 alum hematoxylin :
you get, at a certain moment, a fairly pure nuclear stain ;
but if you were to prolong the treatment, the extra-nuclear
elements would take up the colour, and the selectivity of the
stain would be lost. This is in general the method em-
ployed for the colouring of specimens in bulk—a procedure
which is not possible with most of the regressive strains. It
is the old method of carmine and hematoxylin staining.

The second, or regressive method, is the method of over-
staining followed by partial decoloration. 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 re-
taining the colour more obstinately than the others, in
virtue of their chemical or physical constitution. This
is what happens, for instance, when you stain a section
of one deep red in all its elements with safranin, and then,
treating for a few seconds with alcohol, extract the colour
from all but the chromatin and nucleoli of the nuclei. This
method is in general applicable only to sections, and not to
staining objects in bulk (the case of borax carmine, with a
few others, is an exception). It is a method, however, of
very wide applicability, and gives, perhaps, the most brilliant
results that have hitherto been attained. It frequently enables
us to obtain a powerful stain of certain elements that would
not be sufficiently brought out by the progressive method.

Tissues are stained either in bulk or in sections. For
accurate work, such as is necessary in cytology and fre-
quently in histology, it is greatly preferable, sometimes
even necessary, to stain the sections, as by this means
only is accurate control of the staining process under the
microscope possible.

Staining solutions are mostly made with either ater or
alcohol as a menstruum, Water is generally preferable so
far as the quality of the stain is concerned; but alcohol is

140 CHAPTER XI.

frequently indicated, both on account of its greater power of
penetration and as being less injurious to tissues. It is a
good rule not to let staining baths contain more than fifty
per cent. of alcohol.

Better results (as regards the quality of the stain, not as
regards the preservation of the tissues) are generally obtained
by prolonged staining in very dilute solutions, rather than
by a short bath in a strong one.

210. Choice of a Stain —The following may be recommended
to the beginner for general work:—J’or sections, Mayer’s
hemalum; or, for chromosmium objects more especially,
Benpa’s or Huiprnwain’s iron hematoxylin.

For staining in tofo Grenacher’s alcoholic borax-carmine,
or Mayer’s carmalim, or hemalum, unless the object be so
impermeable as to require a very highly aleoholised stain, in
which case take Mayer’s paracarmine, or for chromic acid
objects Mayer’s hamacaleium.

For fresh tissues or small entire objects, methyl green, if it
is not important to have permanent preparations; if it is, take
carmalum or alum-carmine (but both of these may give
precipitates with marine animals).

Most of the carmine and hematoxylin solutions, properly
used, give stains that are indefinitely permanent—at least
in balsam. But most of the stains obtained with coal-tar
dyes fade much in a few months or years. The most
permanent are safranin, gentian violet, Bismark brown, and
picric acid, which fade very little. Victoria blue, I find, is
also fairly permanent.

CHAPTER XII.
CARMINE AND COCHINEAL STAINS.

211. Carmine —Carmine is by no means merely carminic
acid with at most certain impurities. According to the
analysis of Lizpermann (Ber. d. Chem. Ges., Jahrg. 18, 1886,
pp. 1969—1975) it is a very peculiar aluwnina-lime-protein
compound «f carminic acid, a true chemical compound from
which at all events alwminiwm and calcium can no more be
absent than sodium from salt. It results from the researches
of Mayer (Mitth. Zool. Stat. Neapel., x, 1892, p. 480) that in
the processes of histological staining (not of industrial dyeing)
the active factors of the compound are, besides the carminic
acid, always 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.

This being so, it follows that carminic acid may, if desired,
he taken as the basis of stuining solutions instead of carmine.
Staining solutions thus prepared do not give essentially better
stains than those made with carmine; but have the advan-
tage of being of more constaut composition. For carmine is
a product which varies greatly from sample to sample.

Carminic acid of sufficient purity is furnished by Grisier
and Ho.isorn (or C. A. F. Kautzaum, in Berlin). It is .
soluble in water and weak alcohol (that of 70 per cent. only
dissolves Jess than 3 per cent.) It cannot be used alone for
staining, as it only gives in this way a weak and diffuse stain.

212. Cochineal— According to Mayzr (Mtth. Zool. Stat.
Neapel, x, 1892, p. 496), the active principle of extract or
tincture of cochineal (as used in histology) is not free car-
minic acid, but carminic acid chemically combined with a
base which is not lime, but some alkali. ‘The watery extract

142 CHAPTER XL.

made with alum, or cochineal-alum carmine (§ 216), owes its
staining-power to the formation of carminate of alumina
(last §). he 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. And if the necessary salts
be added to the tincture itself, there results a solution
containing the necessary elements for affording a strong and
selective stain with all classes of objects. Hence Mayer’s
new formula, § 236,

213. General Remarks.—Carmine stains are chiefly used for
staining entire objects, or tissues in bulk. In most cases
this can be done more satisfactorily by means of carmine
than by means of any other known agent. For most
hematein solutions have a disastrous tendency to overstain ;
and the tar-colours are generally inapplicable to staining in bulk.

Grenacher’s alcoholic borax-carmine may be.recommended
to the beginner as being the easiest of these stains to work
with: or para-carmine, for objects which require a highly
alcoholic solution. Carmalum, or one of the alum-carmines,
is also an easy and safe reagent.

Overstains may in all cases be washed out with weak HCl
(e.g. Ol per cent.). Alum-solution will often suffice, or,
according to Hennecuy (Journ. de V Anat. et de la Physiol.,
xxvil, 1891, p. 400), permanganate of potash. All carmine
stains, with the exception of aceto-carmine, are permanent
in balsam. The alum-carmines are fairly permanent in
glycerin. None of the acid stains, nor any of Grenacher’s
fluids, should be used with calcareous structures that it is
wished to preserve, unless they be taken in a state of extreme
dilution.

A. Aqueous CarMINE Stains.
a, Acid.

214. Alum-carmine (Grunacuer, Arch, mik, Anat., xvi, 1879,
p. 465). —An aqueous solution (of 1 to 5 per nent strength,

CARMINE AND COCHINEAL STALNS. 1438

or any other strength that may be preferred) of common or
ammonia alum is boiled for ten or twenty minutes with }$ 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
structures that it is wished to preserve.

Tizzoni (Bull. Sc. Med. Bologna, 1884, p. 259), Pisenti (Gazz. degli
Ospetalt, No. 24; Zett. wiss. Mik., ii, 1885, p. 378), and GRiEB (Mem.
Soc. Ital. Sci., t. vi, No. 9, 1887; Zezt. wiss. Mik., vii, 1, 1890, p. 47)
have given modifications of Grenacher’s formula which do not appear
to me rational.

Mayer (zbid., xiv, 1897, p. 29) makes a stronger stain by taking 2
grms. carmine, 5 grms. alum, and 100c.c. water, and boiling for an hour.

Alum-carmine is an eacellené stain. It is particularly
to be recommended to the beginner, as it is easy to work
with; it is hardly possible to overstain with it. Its chief
defect is that it is not very penetrating, and therefore
unsuitable for staining objects of considerable size in bulk.

215. Acetic Acid Alum-Carmine (Hennucuy, in Trazté des
Méth. techn., Len et Hunnecuy, 1887, p. 88).—Excess of car-
mine 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, then filter.

For staining, enough of the solution is added to distilled
water to give it a deep rose tint. In order to ensure rapid
diffusion it is well to bring the tissues into the stain direct
from alcohol. Stain for twenty-four to forty-eight hours,
and wash for an hour or two in distilled water. Mount in
balsam. You can mount in glycerin, but the preparations
do not keep so well.

The advantage of this carmine is that it has much greater
power of penetration than the non-acidified alum-carmine.

216. Cochineal Alum-carmine (Partscu, Arch. 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 Czoxor

14-4 CHAPTER XII.

(ibid., xviii, 1880, p. 413).—Mayer 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
also keeps better.

Rasr (Zeit. wiss. Mtk., xi, 2, 1894, p. 168) takes 25 grms.
each of cochineal and alum, 800 c.c. of water, and. boils down
to 600 c.c. He prefers this because it is not so purely nuclear
a stain as the others.

These solutions give a stain that is practically identical
with that of alum-carmine made from carmine, with perhaps
even more delicate differentiations.

Rawitz (Zeit. wiss. Mih., xxv, 1909, p. 392) takes cochineal + grms.,

nitrate of aluminium (or ammonio-sulphate of cobalt) 4 grms., water
100 c.c., and glycerin 100 ¢.c. Only for sections.

217. Mayni’s Carmalum (Mitth. Zool. Stat. Neapel, x, 1892,
p. 489).—Carminic acid, 1 grm.; alum, 10 grms.; distilled
water, 200 c.c. Dissolve with heat (if necessary). Decaut
or filter. Add some antiseptic, either J c.c. formol, or 0-1
per cent. salicylic acid, or 0°5 per cent. salicylate of soda.
The solution will then keep. 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, followed in either case with water.
The general effect is that of an alum-carmine stain.

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.

With either solution the objects to be stained should not
have an alkaline reactiun.

Rawitz (Anat. Anz., xv, 1899, p. 488) takes 2 grms. carminic acid, 20
grms. ammonia-alum, 150 c.c. water, and 150 ¢c.c. glycerin. A strongly
staining solution, which is said to kecp well. Only for sections.

All solutions prepared with alum tend to precipitate. Carmalum
made up with 500 c.c. of water instead of 200, and with glycerin or 10
per cent. of formol or pyroligneous acid added, keeps well.

218. MayER’s Aqueous Aluminium-Chlo:ide-Solution (Mitth.
Zool. Stat. Neapel, x, 1902, p. 490)—Carminic acid, 1 grm.; chloride
of aluminium, 3 grms.; water, 200 ¢c.c. Add an antiseptic, as for car-
malum. a

CARMINE AND COCHINEAL STAINS. 145

Use as carmalum. The stain is of a blue-violet colour, very powerful,
and elective, but not so purely nuclear as carmalum. It is recommended
only as a substitute for carmalum in cases in which the latter is counter-
indicated on account of the alum in it or the like.

219. Alum-Carmine and Picrie Acid.—Alum-carmine objects
may be double-stained with picric acid. Lecar (Morph.
Jahrb., viii, p. 353) combines the two stains by mixing ten
vols. of alum carmine with one of saturated picric acid
solution. I find this very recommendable.

219a. Aceto-Carmine (Acetic Acid Carmine) ScHNEIDER (Zool.
Anzezg., 1880, p. 254).—To boiling acetic acid of 45 per cent. strength
add carmine until no more will dissolve, and filter. (Forty-five per cent.
acetic acid is, according to Schneider, 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; or a drop of the concen-
trated solution mav be added to a fresh preparation under the cover-
glass. If you use the concentrated solution it fixes and stains at the
same time, and hence may render service for the study of fresh objects.
It is very penetrating. The stain is a pure nuclear one. Unfortunately
the preparations cannot be preserved, and for this and other reasons
the stain is of very restricted applicability.

A similar stain has been prepared with formic acid by PIANESE (see
Zeit. wiss. Mik., x, 4, 1894, p. 502).

For BuRcHARDT’s pyroligneous-acid carmines see Arch. mik. Anat.,
lili, 1898, p. 232; and Jena. Zect. Naturw., xxxiv, 1900, p. 720.

220. Iron Carmine.—I recommend trial of the following,
which I have already published in the Traité des Méth.
Techniques, Len et Henneauy, 1902. Sections (I have not
tried material in bulk) are mordanted (a few hours will
suffice) in sulphate of iron (Benda’s Liquor ferri, as for iron
hematoxylin), washed, and stained for an hour or so in 0°5
per cent. solution of carminic acid in alcohol of 50 per cent.
Wash in alcohol of 50 per cent.; no differentiation is
necessary. When successful, an almost pure chromatin
stain, quite as sharp as iron hematoxylin, but somewhat
weak.

221. Iron Carmine.—PFEIFFER VON WELLHEIM (Zeit. wiss. Mik.,
xv, 1898, p. 123) mordants for six to twelve hours in a very weak solu-
tion of chloride of iron in 50 per cent. aleohol, washes in 50 per cent.
alcohol, and stains as above. Overstains may he corrected with 0°1 to
0°5 per cent. HCl alcohol. I find this good, but not so good as the last.

10

146 CHAPTER XII.

222. Iron Carmine (ZACHARIAS, Zool. Anz, 1894, p. 62).—Stain for
several hours in an aceto-carmine (made by boiling 1 grm. of carmine
with 150 to 200 c.c. of acetic acid of 30 per cent., for twenty minutes, and
filtering). Rinse the objects with dilute acetic acid, and bring them
(taking care not to touch them with metallic instruments) into a 1 per
cent. solution of ammoniated citrate of iron. Leave them, for as much
as two or three hours if need be, till thoroughly penetrated and blackened
(with sections this happens in a few minutes). Wash for several hours
in distilled water. A chromatin and plasma stain.

223. Iron Carmalum (pe Groot, Zeit. wiss. Mik., xx, 1908,
p. 21).—Dissolve 0°1 grm. of ferric alum in 20 c.c. distilled
water and add 1 grm. carminic acid. Dissolve, add 180 c.c.
of water, warm, add 5 grms. potash alum, dissolve, cool,
filter, and add 2 drops of hydrochloric acid. To be used as

carmalum, and said to give a stronger strain.

224. Iron Cochineal (SPULER, Encyclopwdiv d. mik. Technik, 1908,
p. 153. and 1910, p. 240)—Stain for 48 hours in a stove, in extract of
cochineal (made in a highly complicated way), wash with water, put
into solution of ferric alum of # per cent. strength for 24 hours or
more. If the stain is not sufficiently intense, the whole process may he
repeated.

PETER (Zezt. wiss. Mik., xxi, 1904, p. 314) stains material in bulk for
48 hours (sections 18 to 24) in an incubator, in a similar extract, acidi-
fied with HCl, treats with iron-alum of 24 per cent. for one hour to one
day (sections half to two minutes), then alcohol, xylol, paraffin, or
balsam. Chromatin black, protoplasm grey, yolk granules red.

HANSEN (cbid., xxii, 1905, p. 85) stains sections or entire objects in a
solution of 5 to 10 grms. cochineal, 8 grmns. ferric alum, 250 ¢.c. water,
and 25 ¢.c. sulphuric acid of 10 per cent., boiled for fifteen to twenty
minutes.

B. So-called “ Neutral” and Alkaline.

225. Ammonia-Carmine.—Best made by the method of RANVIER.
Make a simple solution of carmine in water with a slight excess of
ammonia, and expose it to the air in a deep erystallising dish until it is
entirely dried up. It should be allowed to putrefy if possible. Dissolve
the dry deposit in pure water, and filter.

Van WIJHE (Vers. Akad., Amsterdam, viii, Deel, p. 507) takes an old
strong solution of carmine in ammonia (or boils carmine with ammonia
and peroxide of hydrogen), then precipitates it by adding alcohol to
excess, washes the precipitate with alcohol, and dries it.

226. Soda-Carmine appears to be still used by some for central
nervous system (see Cuccati, Ztt. wiss. Mtk., iv, 1887, p. 50). It can
be obtained from GRUBLER & HOLLBORN (Natron-Carm7n),

CARMINE AND COCGHINEAL STAINS. 147

226a. OnTH’s Lithium-Carmine (see early editions) macerates

strongly, and is superfluous. For that of Bust, see Zezt. wiss. Mik.,
xxili, 1906, p. 322.

227. Magnesia-Carmine (Maymr, Zeit. wiss. Mik., xiv, 1897, p. 28).
—Take 1 grm. carmine, 0:1 grm. magnesia usta, and 50 c.c. distilled
water, boil for five minutes, filter, and add three drops of formol. This
is the stock solution. A weuk solution may be made by boiling 0-1 grm.
carmine for half an hour in 50 ¢.c. of magnesia water (made..by leaving
Ol grm. of magnesia usta in contact with 100 ¢.c. of spring water for a
week with frequent agitation, and decanting when required for use).
Said to be less injurious to tissues than the other alkaline carmines.

228. 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 wncombined in hap-
hazard proportions. These solutions do not contain a double
‘salt of picric and carminic acid and ammonia, or picro-car-
minate of ammonia. They are always alkaline, and frequently
injurious to tissues. The ratson d’étre of picro-carmine does
not lie in its capacity of affording a double stain, but in that
the picric acid in it is supposed to neutralise the ammonia,
which it only does imperfectly. See Mayer in Zeit. wiss.
Mik., xiv, 1897, p. 18.

229. RANviER’s Picro-carmine, Original Formula (Traité, p. 100).
—To a saturated solution of picric acid add carmine (dissolved in
ammonia) to saturation. Evaporate down to one fifth the original
voliime in a drying oven, and separate by filtration the precipitate that
forms in the liquid when cool. Evaporate the mother liquid to dryness,
and you will obtain the picro-carmine in the form of a crystalline
powder of the colour of red ochre. It ought to dissolve completely in
distilled water; a 1 per cent. solution is best for use.

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 acid, or better, formic
acid.

RANVIER’s Newer Formula does not give a more constant product
(see previous editions).

230. Van W1sHE dissolves 0°5 per cent. of the dry ammonia-carmine,
§ 225, in a 1 per cent. solution of neutral picrate of ammonia, boils until
the vapour ceases to blue reddened litmus paper, and adds 1 per cnt,
of chloral hydrate. Gives an almost neutral preparation,

148 CHAPTER XII.

231. Maver’s Picro-magnesia Carmine (Zeit. wiss. Mik., xiv, 1897,
p. 25) is relatively constant and innocuous to tissues. It consists of
1 vol. of the stock solution of magnesia-carmine (§ 227), and 10 vols. of
a 0°6 per cent. solution of picrate of magnesia, or of equal parts of the
weak solution and the picrate solution. The picrate may be obtained
from GRUBLER & Honipory, or the solution may be made by heating
0:25 grms. of carbonate of magnesia in 200 c.c. of 0°5 per cent. solution
of picric acid, allowing to settle, and filtering.

De Groor’s picro-magnesia carmine (cbid., xxix, 1912, p. 184) contains
ammonia, which is bad, and seems to me superfluous.

232. Other Formule for Picro-carmine and Other Aqueous
Carmines (Acid and Alkaline).—I have tried most of them, and
found no real advantage in any of them (see previous editions).

B, ALCOHOLIC CARMINE STAINS.

233. Alcoholic Borax-carmine (GRrenNacHER, Arch. mik. Anat.,
xvi, 1879, p. 466, et seq.)—-Make a concentrated solution of
carmine in borax solution (2 to 3 per cent. carmine to 4 per
cent. borax) by boiling for half an hour or more (or allowing
it to stand, with occasional stirring, for two or three days) ;
dilute it with about an equal volume of 70 per cent. alcohol,
allow it to stand some time and filter.

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 of 70 per
cent. acidulated with 4 to 6 drops of hydrochloric acid to each
100 c.c. of alcohol. They are left in this until they have
taken on a bright transparent look (which may require days),
and may then be washed or hardened in neutral alcohol.
Four drops of HCL is generally enough. Three drops I find
not quite sufficient.

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 50 percent. It should
not exceed 60 per cent. in any case (Mayer).

This stain used to be the most popular of any for staining
in bulk. It is easy to use, and gives a most splendid colora-
tion. But it is not so penetrating as is commonly supposed,
and has the defect of sometimes forming precipitates in the
cavities of bulky objects which cannot be removed by washing
out. And the fluid is alkaline, and therefore may not be
suitable for certain delicate work.

CARMINE AND COCHINEAL STAINS. 149

234. Mayen’s Paracarmine (Mitth. Zool. Stut. Neapel, x, 3,
1892, p.491).—Carminic acid, 1 grm.; chloride of aluminium,
05 grm. ; chloride of calcium, 4 grms.; 70 per cent. alcohol,
100 c.c. Dissolve cold or warm, allow to settle, and filter.

Objects to be stained should not have an alkaline reaction,
nor contain any considerable amount of carbonate of lime
(spicules or skeletal parts of corals, etc.) which would give
rise to precipitates. Wash out sections or objects intended
to be sectioned, with pure 70 per cent. alcohol. Objects in-
tended 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 2°5 per
cent. glacial acetic acid) in alcohol. This may also be done
with section material, if it is desired to obtain a more purely
nuclear stain.

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.

Instead of calcium chloride, which is very hygroscopic,
strontium chloride may be taken.

Paracarmine is less hurtful to delicate tissues than borax
carmine ; it is more highly alcoholic, therefore more pene-
trating; and has less tendency to form precipitates in the
interior of objects. But, in my hands, it does not give quite
so fine a stain.

934a. Alcoholic Hydrochloric-Acid Carmine.—GRENACHER’S re-
ceipt (Arch. f. Mik. Anat., xvi, 1879, p. 468) is troublesome. That of
Maver (Mitth Zool. Stat. Neapel, iv, 1883, p. 521; Intern. Monatsschr.
f. Anat., etc., 1897, p. 43) is better: Carmine 4 grms.; water, 15 c.c.;
hydrochloric acid, 30 drops. Boil till the carmine is dissolved, add
95 c.c. of 85 per cent. alcohol, and neutralise by adding ammonia until
the carmine begins to precipitate.

If it be desired to dilute the solution, it should be done with alcohol,
not water, and alcohol of 80 to 90 per cent. should be taken for washing
out.

A very powerful stain, which I have found useful. If it be desired to
have a purely nuclear stain, the alcohol must be very slightly acidulated
with HCl.

For a complicated receipt of LonwENTHAL see Zeit. wiss. Mik., xix,
1902, p. 56.

150 CHAPYER XII.

235, Alcoholic Cochineal, Mayex’s Old Formula (Mil/h. Zool.
Stat. Neapel. i, 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.

The objects tobe stained must previously be saturated
with alcohol of 70 per cent., and alcohol of the same strength
must be used for washing out or for diluting the staining
solution. The washing out must be repeated with fresh
alcohol until the latter takes up no more colour. Warm
alcohol acts more rapidly than cold. Overstaining 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,

A nuclear stain, slightly affecting protoplasm. The colour
varies with the reaction of the tissues, and the presence or
absence of certatn salts in them. Crustacea with thick
chitinous 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
or their secretion often stain grey-green.

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

All acids must be carefully washed out from the objects
before staining, or a diffuse stain will result. The stain is
permanent in oil of cloves and balsam.

Very penetrating and especially useful for Arthropoda.

It has over the new fluid (next $) the advantage of being
more highly alcoholic ; and it does not contain free acid, so
that it can be used with caleareviis structures which it is
wished to preserve—which the vew fluid cannot. For
specimens of Plu/eas, for instance, I find it excellent. But it
only gives good results with such objects as contain the
necessary salts, § 212.

236. MayER’s Aleoholie Cochineal, New Formula (Aitth. Zool.
Stat. Neapel, x. 1892. p. 498). —Cechineal, 5 erms.; chloride of caleium,
5 grms.; chloride of aluminium, 0-5 grm.; nitric acid of 120 sp. er.,
8 drops; 50 per cent. alcohol, 100 cc. Powder the cechineal and rub

GARMINE AND COCHTNEAL STAINS. 151

up with the salts, add the alcohol and acid, heat to boiling-point, leave
to 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. Mayer only recommends it as a succedanewm
of paracarmine.

Since this fluid contains in itself all the necessary salts (§ 212), it
gives good results with all classes of objects.

CHAPTER XIII.
HHMATEIN (HEMATOXYLIN) STAINS.

237. Introduction Hematoxylin is a dye extracted from
logwood. It is a substance that oxidises very readily, thus
becoming converted into hematein, or, as often happens, into
other more highly oxidised products. It appears to be now
thoroughly well established (see Ninrzx1, Chemie der organ-
ischen Farbstoffe, Berlin, Springer, 1889, pp. 215—217, and
Maver, Mitth. Zool. Stat. Neapel, x, 1891, p. 170) that the
colouring agent in solutions of logwood or hematoxylin is not
the hematoxylin itself, but hamatein formed in them (or, in
some cases, one of the higher oxidation products).

Hematein is an acid body, a “ colour acid ” ($$ 208, 205).
Substantively employed, it is a very weak plasma stain. But
combined with appropriate mordants it becomes basophilous,
and can be made to give a powerful nuclear stain, or at the
same time a nuclear and a selective plasma stain. The
mordants employed in histology are aluminium, chrome, iron,
copper, and (rarely) vanadium and molybdenum. Aluminium
and iron are the mordants most employed, the former fur-
nishing lakes used for progressive staining of material in
bulk, the latter forming in most cases in the tissues a lake
that requires differentiation, and is only applicable to the
staining of sections.

The presence of a sufficient amount of hematein in stain-
ing solutions was formerly brought about by allowing solu-
tions of hematoxylin to oxidate spontaneously by exposure
to air. The change thus brought about in the solutions is
known as “ ripening,” and until it has taken place the solu-
tions are not fit to use for staining.

It was discovered by Mayrr and Unya independently (see
Mayer in Mitth. Zool. Stat. Neapel, x, 1891, pp. 170—186 ;
Unna in Zeit, wiss. Mtk., viii, 1892, p. 483) that nothing is

HAMATEIN (H®MATOXYLLIN) STAINS. 1538

easier than to bring about this change artificially ; all that is
necessary being, for instance, to add to a solution of hama-
toxylin containing alum a little neutralised solution of peroxide
of hydrogen or other powerful oxidising agent.* The
solution becomes almost instantaneously dark blue, “ ripe ”
and fit for staining. Other methods of “ ripening,” or of
preparing hematein separately, are given further on, and
constitute a great progress. For under the old practice of
leaving staining solutions to “ripen” by the action of the
air, it is necessary to wait for a long time before the reaction
is obtained. During all this time, it may be weeks or
months, there is no means, except repeated trial, of ascertain-
ing whether the solution at any moment contains sufficient
hematein to afford a good stain. And here a second difficulty
arises : the oxidising process continuing, the solutions become
“ over-ripe ”; the hematein, 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
“ overripe ” constituents (the first and last being useless for
staining purposes), and, in consequence, their staining power
is very inconstant.

Logically, therefore, as concluded by Mayer, not hema-
toxylin, but hematein, should be taken in the first instance
for making the staining solution. This at once relieves us
from the tedious and uncertain process of “ripening”’ in the
old way. We have thus a ripe solution to begin with.

But this is not always indicated ; for such solutions may
easily over-oxidise, either in the bottle or on contact with
the tissues. So that it is sometimes preferable to start
from hematoxylin. In this case, it should not be done
by dissolving the hematoxylin straight away in the other
ingredients of the staining solution. The solutions should
be made up from a strong stuck sulution made by dissolving
hematoxylin crystals in absolute alcohol: one in ten is a
good proportion. This solution should be kept for a long
time—months, at least, a year if possible; it gradually
becomes of a vinous red, and should not be used till it has

* Re-invented lately (Zect. wiss. Mik., xxix, 1912, p. 69) by Piazza,
who adds to Boehmer’s solution about 20 per cent., to Delafield’s about
7 per cent., to Ehrlich’s about 12 per cent. of peroxide of hydrogen.

154 CHAPTER NII.

become quite dark. It has then become to a great extent
oxidised into hematein, and the staining solutions made up
from it will be at once fairly ripe.

Hematein (or hematoxylin) affords a stronger stain than
carmine, and gives better results with tissues fixed in osmic
or chromic mixtures. The alum solutions are indicated for
staining in bulk, iron hematoxylin for sections.

238. Hematoxylin is found in commerce in the form of
crystals, either colourless or browned by oxidation, easily
soluble in either water, glycerin, or alcohol.

Hematein is found in commerce as a brown powder, en-
tirely, 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.

Mayer (Zeit. wise. Mik., xx, 1903, p. 409) prepares it as follows:
1 grm. of hematoxylin is dissolved by boiling in not more than 1) c.c.
of distilled water, and to the solution is added a hot solution of 0-2 grm.
of iodate of sodium in about 2 ¢.c. of water. Mix well and cool by
placing the recipient in cold water. After a couple of hours bring the
mixture on to a filter, wash thereon with cold water to remove the
excess of iodide of sodium that has formed, then dry the residue.

There is also found in commerce an ammonia-compound
of hematein—hematein-Ammoniak, also known as Hema-
teinum crystallisatum ; this may be obtained in a sufficiently
pure state from Grisrur & Hortzorn.

This is somewhat more easily soluble in both water and
aleohol than hematein is, and does quite as well for staining
purposes. It can be made as follows:

239. Hemateate of Ammonia (MayER, Mitth. Zool. Stat. Neapel,
x, 1891, p. 172).—Dissolve 1 grm. of hematoxylin with the aid of heat
in 20 ¢.c. of distilled water, filter if necessary, add 1 c.c. of caustic
ammonia (of 0°875 sp. gr.), and bring the liquid into a capsule of such
dimensions that its bottom be covered to a depth of not more than half
a centimetre. Let the liquid evaporate at the ordinary temperature
and be protected from dust. The dry product will consist of haemateate
of ammonia, about equal in weight to the hematoxylin taken in the
first instance. 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

HAMATEIN (HAMATOXYLIN) STAINS. 155

platinum. The product is not of perfectly constant quality. It ought
to dissolve easily in water or alcohol, and the solution should not
become turbid on addition of acetic acid ; if it does, it is over-oxidised.

240. Iron Hematoxylin, Generalities—This method is due
to Benpa (Verh. Phys. Ges., 1885—1886, Nos. 12, 18, 14;
elreh, Anat. Phys., 1886, p. 562 ; third ed. of this work, p. 365).

The method was independently worked out about the same
time by M. Hemrnuain. The method is almost universally
practised in the form given by Heidenhain, not on account
of any essential difference between the two, for there is
none, but chiefly because Heidenhain has given more precise
instructions concerning the process.

After carefully comparing Heidenhain’s process with
Benda’s later process (next §), I find that the two give an
absolutely identical stain ; that is to say, that if you mor-
dant in Benda’s Mquur ferri, next §, and differentiate in the
same, you will get exactly the same effect as by mordanting
in ferric alum and differentiating in the same. But you may
vary the results somewhat by varying the differentiation.
Benda has pointed out (Verb. Anat. Ges., xv, 1901, p. 156)
that you may differentiate either by an agent which simply
dissolves the lake—such as acetic or hydrochloric acid; or
by an oxidising agent, such as chromic acid, or the Mquor
ferrt or the ferric alum. The former, he thinks, are the
best for the demonstration of nuclear structures, the latter
for cytoplasmic structures. For these he greatly recom-
mends Weicert’s borax-ferricyanide mixture, as being the
easiest and safest to employ.

For myself, I find that differentiation in the iron salt
(§ 241 or § 242) is sufficient for almost all purposes. Acetic
acid of 30 per cent. acts much too quickly to be sate, and
causes swelling of the tissues.

Van Girson’s picro-siurefuchsin has been recommended
as a differentiation fluid by Benda (Deutsch. med. Wochen-
schr., 1898, No. 30). I find it gives very delicate differ-
entiations, but acts very slowly, requiring nearly as many
hours as the iron alum solution does minutes. The addition
of the siurefuchsin to the picric acid is, I find, not necessary,
and may prove an injurious complication.

In these processes hematoxylin is generally used for the

156 CHAPTER XIII.

stain, not hematein, the iron salt oxidising it into hama-
tein, or into a higher oxidation product. I have obtained
some good stains with hematein, but also some very bad
ones; presumably the solutions easily over-oxidise on con-
tact with the iron salt.

The hematoxylin is generally dissolved in water. I
frequently prefer alcohol, of 50 per cent., as less injurious
to tissues.

The method is a regressive one. It has been proposed to
stain progressively, which I have tried, and had extremely
bad results.

The differentiation requires to be carefully timed. For
this reason the method is only applicable to sections, which
should be thin, best not over 10 p.

lron hematoxylin is one of the most important of stains.
It enables us to stain elements which cannot be selectively
stained in any other way. The stain is very powerful, and
of a certain optical quality that is peculiarly suited to the
employment of high powers; it will allow of the use of
deeper eye-pieces than other stains. It will take effect on
any material, and is quite permanent. Further details as
to the characters of the stain are given in § 242.

241. Benpa’s later Iron Hematoxylin (Verb. d. Anat. Ges.,
vii, 1, 1893, p. 161).—Sections are mordanted for twenty-
four hours in liquor ferri suphurici oxidati, P.G.,* diluted
with one or two volumes of water. They are then well
washed, first with distilled water, then with tap water, and
are brought into a 1 per cent. solution of haematoxylin in
water, in which they remain till they have become thoroughly
black. They are then washed and differentiated. The
differentiation may be done either in 80 per cent. acetic
acid, in which case the progress of the decoloration must
be watched ; or in a weaker acid, which will not require
watching ; or in the sulphate solution strongly diluted with
water.

I find that if the iron solution be taken for the differen-

* This preparation consists of sulphate of iron, 80 parts ; water, 40 ;
sulphuric acid, 15; and nitric acid, 18, and contains 10 per cent. of Fe.
Doubtless the ferr? persulphatis liquor B. P. will do instead ; the point
is, to have a per-salt, and not a proto-salt.

HAMATEIN (HEMATOXYLIN) STAINS. 157

tiation, it should be taken extremely diluted (of a very pale
straw-colour, about 1: 380 of water), and the progress of
the differentiation watched; as if it be only diluted about
tenfold, for instance, the decoloration is extremely rapid.
See also last §.

I also find that Benda’s mordant is unnecessarily, some-
times harmfully, strong, and that the Liquor ferri may be
diluted tenfold with advantage. The duration of the bath
in the mordant is also for most purposes excessive as
directed by Benda. I find that three to six hours in the
solution diluted tenfold is generally sufficient, with favour-
able material.

242. Hurpengain’s Jron Hematoxylin (M. Heripennarn,
“Uber Kern und Protoplasma,” in Festschr. fiir Kélliker,
1892, p. 118).—Sections are treated from half an hour to at
most two or three hours with a 1:5 to 4 per cent. solution of
ferric aluin (ammonio-ferric sulphate). By this is always
meant in histology the double salt of ammonium and sesqui-
oxide of iron (NH,).Fe, (SO,),,1n clear violet crystals; the
double salt of the protoxide, or salt of Mour in green
crystals, will not serve. If the crystals have become yellow
and opaque, they have gone bad, and should be rejected.
They ought to be kept in a stoppered bottle, and the solution
should be made in the cold (Arch. mik. Anat., xliii, 1894,
pp. 431, 435). The sections are then washed with water
and stained for half an hour in un aqueous solution (of
about 0°5 per cent.) of hematoxylin. They are then rinsed
with water, and again treated with the iron solution, which
slowly washes out the stain. The progress of the differen-
tiation ought to be controlled under the microscope. The
sections should to this end be removed from time to time
from the alum solution, and put into tap-water whilst they
are being examined. This is favourable to the stain. As
soon as a satisfactory differentiation has been obtained, the
preparations are washed for at least a quarter of an hour in
running water, but not more than an hour, and mounted.
The results differ according to the duration of the treat-
ment 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

158 CHAPTER XIII.

obtained. These show a very intense and highly: differen-
tiated stain of nuclear structures, cytoplasmic structures
being pale. If the baths in the iron and in the stain have
been prolonged (twelve to eighteen hours), and the sub-
sequent differentiation in the second iron bath also duly
prolonged, black preparations will result. These show
chromosomes stained, central corpuscles stained intensely
black, cytoplasm sometimes colourless, sometimes grey, im
which case achromatic spindle-fibres and cell-plates are
stained, connective-tissue fibres black, red blood-corpuscles
black, micro-organisms sharply stained, striated muscle very
finely shown.

Later (Zeit. wiss, Mik., xiii, 1896, p. 186) Heidenhain gives
further instructions for the employment of this stain in the
study of central corpuscles. All alcohol should be removed
from the tissues,* by means of distilled water before bringing
them into the mordant. This should be a 24 per cent.
solution of ferric alum, not weaker. Leave the sections
therein (fixed to slides by the water method, § 186) for six
to twelve hours, or at least not less than three. Keep the
slides upright in the mordant, not lying flat. Wash out well
with water before staining. Stain in a“ ripened” hema-
toxylin solution, 7. e. one that has stood for four weeks [of
course, if you make it up with the ripened brown alcoholic
solution recommended § 237 sub. fin., this will be superfluous].
Stain from twenty-four to thirty-six hours. Use the same
staining solution over and over again until it becomes spoilt ;
for the solution after having been used gives a more ener-
getic stain, owing to its containing a trace of iron brought
over by the sections. Differentiate in a 24 per cent. solu-
tion of ferric alum. Rinse for ten minutes in running water,
clear with xylol, not with any essential oil, and mount in
xylol-balsam. See also under ‘‘ Centrosomes.”’

BriEeLaAszewics (Bull. Acad. Cracovie, 1909; 2 serié, p. 152) differen-
tiates with very weak solution of calcium chloride; GUARNIERI (Mon.
Zool. Ital., xvii, 1906, p. 44) with saturated solution of picric acid.

GuRWITSCH (Zett. wiss. Mik., xviii, 1902, p. 291) floods sections on
the slide with mordant, warms on a water-bath till bubbles are given off

* Why? I find my iron-alum solution, as well as the liquor ferri
sulph. owtd., last §, mix clear with alcohol without the least precipitate
forming,

H@MA'TEIN (H®MATOXYLIN) SPAaiNs. 159

or the mordant becomes turbid, then stains with the hematoxylin in
the same way. The whole process takes about ten minutes.

HELD (Arch. Anat. Phys., Anat. Abth., 1897, p. 277) adds to the
staining bath a very little of the iron-alum solution until a scarcely
perceptible precipitate is produced. A dangerous practice. I find it is
not even safe to add a little of an over-used bath (supra).

Francorre (Arch, Zool. Exp‘r., vi, 1898, p. 200) mordants with tartrate
of iron, MALLorY (Journ. Exper. Med., v, 1900, p. 15) with chloride.

243. Iron Hematoxylin (BUrscH1i, Unters. iiber mikroskopische
Schéume u. das Protoplasma, etc., 1892, p. 80).—Sections treated with a
weak brown aqueous solution of ferric acetate, washed with water, and
stained in 0°5 per cent. aqueous solution of hematoxylin. A stain of
extraordinary intensity, used by Biitschli for sections, 1 in thickness,
of Protozoa.

244, Weigert’s Iron Hematoxylin Mixture (Zezt. wiss. Mik., xxi,
1904, p. 1).—Mix one part of a 1 per cent. solution of hematoxylin in
aleohol of 96 per cent. with one of a solution containing 4 c.c. of liq.
ferri sesquichlor., 1 ¢.c. of officinal hydrochloric acid (sp. gr. 1124) and
95 of water. The mixture may he kept for some days (until it begins to
smell of ether), but is best used fresh. Stain sections for a few minutes;
no differentiation is necessary.

For an earlier process of WEIGERT’s (Allg. Zeit. Psychiatr., 1894, p.
245) see last edition.

More and Bassa (Journ. Anat. Phys., xlv, 1909, p. 632) stain in
bulk in Weigert’s mixture with the addition of 1 ¢.c. of 4 per cent. solu-
tion of acetate of copper.

245, JANSSENS’ Iron Hematoxylin (““Hématoxyline noire”; La
Cellule, xiv, 1897, p. 207).—A similar mixture to that of DELAFIELD,
ferric alum being taken instead of ammonia alum, the rest as in Dela-
field’s. A progressive stain, nuclear: for yeast cells.

246. HANSEN’s Iron Hematoxylin (Zezt. wiss. Mik., xxii, 1905, p. 55).
—A solution of 10 grms. ferric alum in 150 ¢.c. water is added to a
solution of 1°6 grm. hematoxylin in 75 ¢.c. water, the mixture heated
to boiling-point and cooled without access of air. Filter before use.
To get a pure nuclear stain, add dilute sulphuric acid.

247. Aluminium Hematein (Alum Hematoxylin) Generalities.
—The mordant and dye are generally combined in a single
staining bath, giving a progressive stain. The stain is in
different tones of blue or red according to the composition of
the staining solution. Neutral or alkaline solutions give a
blue stain; acid solutions give a red one. Jn order to get a
blwe stain in preparations that have come out red through

160 CHAPTER XIII.

the acidity of the staining bath, it is a common practice 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 Mayer, the ammonia
acts, not by neutralising the acid, but by precipitating the
alumina, which carries down the hematein with it (if no
alumina were present the colour would be purple, not blue).*
The same result can generally 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 different 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.

Squire (Methods, p. 22) finds that sections can be blued in
a few seconds by treatment with a 1: 1000 solution of bicar-
bonate of soda in distilled water. Mayer holds that acetate
of potash is the most inoffensive reagent to take; a strength
of 0°5 to 1 per cent. may be taken.

Several of these solutions have a great tendency to over-
stain. Over-stains may be corrected by washing out with
weak acids (e.g. O'l to 0:2 or even 0°5 per cent. of hydro-
chloric acid, or with oxalic or tartaric acid), but this is not
favourable to the permanence of the stain. Carnoy (La
Cellule, xii, 2, 1897, p. 215) recommends iodised water. If
acids be used, it is well to neutralise afterwards with ammonia
or bicarbonate of soda (01 per cent.).

Bicarbonate of soda may be used for neutralisation with
70 per cent. alcohol as the vehicle (von WiusrinaHausEn,
Mitth. Zool. Stat. Neapel, x, 1891, p. 41).

Over-staining may be avoided by staining very slowly in
dilute solutions. The purest chromatin stains are obtained
by staining for a short time (sublimate sections half an hour,
say) in solutions of medium strength, such as hemalum
diluted ten to twenty-fold with water. The stain obtained

* FIscHER, in his Fixirung, Firbung u. Bau des Protoplasmus, pp.

156, 157, does not admit this explanation. He proposes another one of
a highly speculative nature.

HAEMATEIN (HAMATOXYLIN) STAINS. 161

either with very strong solutions, or with the slow stain of
the dilute solutions, is at the same time a plasma-stain, which
of course may or may not be desired. Maver says that very
dilute solutions will give a pure nuclear stain if they have
been diluted with alwm-solution, or have been acidified.
Chromosmium material will not yield a pure chromatin
stain unless it is very fresh; it is consequently next to im-
possible to obtain the reaction with paraffin sections of such
material ; they constantly give a plasma-stain in addition to
the chromatin stain, which is not the case with sublimate
material.

The stain is fairly permanent in balsam, but is very liable
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 solu-
tions: Mayer finds that his hematein preparations have kept
well for at least some months in glycerin, if not acid, and,
with certain precautions, in balsam). Turpentine-balsam
should not be used.

Formule §$ 248 to 259 give aqueous solutions; and
$$ 260 to 263 alcoholic ones.

248. Mayrn’s Hemalum, Newer Formula (Zert. wiss. Mik., xx,
1903, p. 409) —Hamatorylin, 1 grm.; water, 1 litre. Dis-
solve, and add 0:2 germ. of iodate of sodium (NaIQ,) and
50 grms. of alum, dissolve and filter.

This is an amended formula. The original one (Mitth. Zool. Stat.
Neapel, x, 1891, p.172) was: One grm. of hematein (or the ammonia salt,
§§ 238, 239) dissolved with heat in 50 ¢.c. of 90 per cent. alcohol, and
added to a solution of 50 gr. of alum in a litre of distilled water.

This solution does not keep very well, but may be made
more stable by adding 50 grms. of chloral hydrate and 1 grm.
of citric (or acetic) acid.

It stains equally well, either at first, or later. Con-
centrated, it stains sometimes almost instantaneously, or in
any case very rapidly. (Spring water or tap-water contain-
ing 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

11

162 CHAPTER XIII.

distilled or common water. It is admirable for staining in
bulk. Large objects will, 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). All alum must be care-
fully washed out of the tissues before mounting in balsam ;
and it is well to blue the stain with tap-water or otherwise,
§ 257. The stain is generally a nuclear one; in any case
such may be obtained by washing out with alum-solution.
Mayer’s preparations have kept well in glycerin (care being
taken not to have it acid), also in balsam. If oil of bergamot
be used for clearing, it must be thoroughly removed by
means of oil of turpentine before mounting, and oil of cloves
is dangerous. It is best (Mayer, in litt.) to use only xylol,
benzol, or chloroform, and to mount in xylol-balsam or
chloroform-balsam or benzol-balsam.

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

249. Maver’s Acid Hemalum (Mit/h. Zool. Stat. Neapel, x,
1891, p. 174).—This is hemalum with 2 per cent. glacial
acetic acid (or 4 per cent. common acetic acid). To be used
as the last, washing out with ordinary water in order to
obtain a blue-violet tint of stain. The solution keeps better.

250. Unna’s Half-ripe Constant Stock Solution (Zezt. wiss. Mik.,
vili, 1892, p. 483).

Hematoxylin . ‘ 4 1
Alum. ; . 10
Alcohol . : . i j 3 ; ‘ . 100
Water . . ‘ . ‘ : ; . 200
Sublimed sulphur , , 2

If the sulphur be added to the hematoxylin solution only when the
latter has become somewhat strongly blue, 7. e. after two or three days’
time, the stage of oxidation attained by the solution will be fixed for
some time by the sulphur, and according to Unna the solution will
remain * constant” in staining power. MAYER (Mitth. Zool. Stat. Neapel,
xil, 1896, p. 309) finds that the sulphur process does not preserve the
solutions for long, whilst glycerin does ; see below, “ GLYCHAEMALUM.”

251. MayeEr’s Glychemalum (Mitth. Zool. Stat. Neapel, xii, 1896,
p. 310) Hematein (or hremateate of ammonia) 0-4 grm. (to be rubbed
up in a few drops of glycerin) ; alum, 5 grmns.; glycerin, 30; distilled
water, 70. The stain is not purely nuclear, but may be made so by

HAMATEIN (ERMATOXYLIN) STAINS. 163

washing out with alum solution or a weak acid. The solution keeps
admirably.

Rawirz (Leitfaden, 2nd ed., p. 63) takes 1 grm. hematein, 6 grms.
ammonia alum, 200 grms. each of water and glycerin.

Or (Zeit. wiss. Mik., xxv, 1909, p. 391) 1 grm. hematein, 10 grms.
of nitrate of aluminium, 250 grms. each of water and glycerin.

252. Hansen’s Solution (Zool. Anz., 1895, p. 158).—See fourth edition.
Hansen oxidises a mixture of alum and hematoxylin by means of per-
manganate of potash. I find it does not keep. See also MAYER in
Mitth. Zool. Stat. Neapel, xii, 1896, p. 309, or the Grundaziige, LEE and
Mayer, 1901, p. 171.

253. Harris’s Solution (Micr. Bull., xv, 1898, p. 47; Journ. App.
Mice., iii, p. 777) —Alum-hematoxylin solution ripened by addition of
mercuric oxide. Mayer (Grundziige, 1901, p. 171) finds the formula
“gives too much hematein.”

254. Bohmer’s Hematoxylin (Arch. mik. Anat., iv, 1868, p. 345;
Aerzt. Inteiligenzbl., Baiern., 1865, p. 382)—Make (A) a solution of
hematox. cryst. 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 alcoholic solution of hematoxylin ought to be old and dark
(§ 237).

255. Delafield’s Hematoxylin (Zeit. wiss. Mik., ii, 1885, p.
288; frequently attributed erroneously to GrunacHeR or
Pruppen).—T’o 400 c.c. of saturated solution of ammonia-
alum (that is about 1 to 11 of water) add 4 germs. of
hematox. cryst. dissolved im 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 (CH,O). Allow
the solution to stand (uncorked) until the colour is sufficiently
dark, then filter.

This solution keeps for years. It is well to allow it to
ripen for at least two months before using it.

For staining, enough of the solution should be added to
pure water to make a very dilute stain. It is an extremely
powerful stain.

It is still much used. I find that when well ripened—for
years rather than months—it is quite a first-class stain.

Burson (Unters. tib. mikroscopische Sehiiume wu. das Protoplasma,

164 CHAPTER XT.

etc., 1892) recommends, under the name of “ acid hematoxylin,” solution
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 differ-
entiated nuclear stain than the usual solution.

Marrinorri (Zett. wiss. Mk., xxvii, 1910, p. 31) makes it up with 0-2
per cent. of hematezn, and less alum (2 per cent.).

256. Ehrlich’s Acid Hematoxylin (Zeit. wiss. Mik., 1886, p.
150).—Water 100 c.c., absolute alcohol 100, glycerin 100,
glacial acetic acid 10, hematoxylin 2 grms., 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 constant staining power, for yeurs, if
kept in a well-stoppered bottle. It is very appropriate for
staining in bulk, as over-staining does not occur. I find it
excellent.

Many (ibid., xi, 1895, p. 487) makes up this stain with an
equal quantity of hematein instead of haematoxylin.

Mayer (Grundztiye, Lex and Mayrr, first edition, p. 154) —
finds that this is too much and makes the mixture overstain ;
0 4 grm. of hematein is quite enough.

257. BURCHARDT'’S Pyroligneous Acid Hematoxylin (Areh. mzk.
Anat., lili, 1898, p. 232) would seem to be superfiuous at least.

258. Unna’s Oxidised Hematoxylin (from Marrtnortt, Zeit.
qiss. Mik., xxvii, 1910, p. 31)—Hematoxylin 0:5, alum 2,
water 60, alcohol 10, glycerin 20, peroxide of hydrogen
solution 10, carbonate of soda 0:05.

Marrinortt, loc. cit., makes it up with hematein (02 grms).

259. ArArny’s Hematein Mixture I A (Mu/th. Zool. Stat.
Neapel, xii, 1897, p. 712). —Make (a) a solution of 9 per cent.
alum, 3 per cent. glacial acetic acid, and 0:1 per cent.
salicylic acid in water, and (8) a 1 per cent. solution of
hematoxylin in 70 per cent. alcohol, preserved for six to eight
weeks in a bottle not quite full. Mix one part of a with one
of B and one of glycerin. Stains either sections or material
in bulk. Apathy uses it for staining neuro-fibrils.

260. KLEINENBERG'S Hematoxylin (Quart. Journ. Micr. Sez. Ixxiv,
1879, p 208),—Highly irrational and very inconstant in its composition

ILEMATIN (HAMATOXYLIN) STAINS. 165

and its effects ; see early editions; also the criticism of Mayer (Mitth.
Zool. Stat. Neapel, x, 1891, p. 174), and that of Squire in his Methods
and Formule, p. 25, and the alternative formule of SqureE (loc. cét.)
and of von WIsTINGHAUSEN (Mitth. Zool. Stat. Neapel, x, 1891, p. 41).

261. Mayer’s Hemacaleium (Jitth. Zool. Stat. Neupel, x,
1891, p. 182).—Heematein (or hemateate of ammonia, $$ 238,
239), 1 grm.; chloride of aluminium, 1 erm.; 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. Rub up
finely together the first two ingredients, add the acid and
alcohol, dissolve either cold or with heat; lastly add the
chloride of calcium.

If the objects stain in too red a tone they may be treated
with a solution (of about 2 per cent.) of chloride of
aluminium in 70 per cent. alcohol, or with a 0°5 to 1 per
cent. solution of acetate of soda or potash in absolute
alcohol; but washing with neutral alcohol will generally
suttice.

With certain objects this solution does not penetrate well.
This may be remedied by acidifying the solution, or, which
is better, by leaving the objects for some time before stain-
ing in acid alcohol. Anyway objects ought nor to have an
alkaline reaction. If these precautions be taken, it will not
be necessary to use acid for washing out.

The solution is not recommended as giving as good results
as hemalum, and Mayer recommends it merely as a substi-
tute for Kleinenberg’s, in cases iv which an alcoholic heematein
stain seems indicated, as being easy to prepare, and constant
in its effects.

262. Mayer’s Hemastrontium (Grundzige, Lee and Mayer,
1910, p. 166).—1 grim. hematein, 1 grm. aluminium chloride,
50 erm. strontium chloride, 600 ¢.c. alcohol of 70 per cent.,
and (if desired) 0°25 g. citric acid. Prepare and use as
hemacalcium.

263. De Groort'’s Aleoholic Hemalum (Zeit. wiss. Mtk., xxix, 1912,
p. 182).—Mix 20 cc. of glycerin with 240 of alcohol of 70 per cent.
Take 4 c.c. of the mixture, 2 ¢.c. of hydrogen peroxide, and 0°5 grin. of
hematoxylin, and dissolve with heat. Add 60 ¢.c. of the mixture, 4 ¢rms.
of calcium chloride, and 2 gris. of sodium bromide. Dissolve, add 3 grms.

166 CHAPTER XIII.

of alum, heat and add 100 c.c. of the mixture. When the alum is dissolved
add 02 erm. of ferri-cyanide of potassium; dissolve and add 3 grins.
more of alum and the rest of the mixture. Said to stain almost as well
as hemalum. Wash out with alcohol of 70 per cent.

264. Other Alumina-Hematein Solutions—A large number of
suppressed receipts will be found given in the earlier editions.

265. R. HEIDENHAIN’s Chrome Hematoxylin (Arch. mk. Anat.,
xxiv, 1884, p. 468, and xxvii, 1886, p. 383).—Stain for twelve to twenty-
four hours in a 3 per cent. solution of hematoxylin in distilled water.
Soak for the same time in a 0°5 per cent. solution of neutral chromate
of potash. Wash out the excess of chromate with water.

Objects that have been fixed in corrosive sublimate ought to be very
carefully washed out with iodine, or the like, as neutral hematoxylin
forms a black precipitate with any excess of sublimate that may remain
in the tissues (see TORNIER, in Arch, mik. Anat., 1886, p. 181).

The process is adapted to staining in bulk. You can decolour the
objects to any extent by prolonging the soaking in the chromate.
Bichromate will do instead of the neutral chromate.

266 ApitTHy’s Modification of Heidenhain’s Process (Zett. wiss.
Mik., v, 1888, p. 47).—This is an alcoholic method. Stain in a 1 per
cent. solution of hematoxylin in 70 or 80 per cent. alcohol. Differentiate
sections of 10 to 15 p, half the time of staining, sections of 25 to 40 p
twice the time of staining, in 1 per cent. solution of bichromate of
potash in 70 to 80 per cent. alcohol, and wash out in alcohol of 70 per
cent. All these processes should be done zi the dark.

For celloidin series of sections, Apathy (/béd, 1889, p. 170) stains in
the hematoxylin solution as above for ten minutes; then removes the
excess of hematoxylin fluid from the sections by means of blotting-
paper, and brings 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
bichromate.

267. ScHULTZE’s Chrome Hematoxylin (Zezt. wiss. Mik., xxi, 1904,
p. 5).—The tissues to be fixed for twelve or more hours in a bichromate
or chromic acid solution, preferably an osmium-bichromate mixture or
liquid of Flemming. Then to be washed out for twenty-four hours in
50 per cent. alcohol in the dark and stained for twenty-four hours or
more in 0°5 per cent. hematoxylin in alcohol of 70 per cent., then
washed out in alcohol of 80 per cent.

268. HANSEN'S Chrome Hematoxylin (/bid., xxii, 1905, p. 64).—
Ten germs. of chrome alum boiled in 250 ¢.c. of water till green, and
1 erm. hematoxylin (dissolved in 15 ¢.c. of water) added ; to the mixture
when cold add 5 ¢.c. of sulphuric acid of 10 per cent. and (drop by drop)
a solution of 0°55 grm. of bichromate of potash in 20 c¢.c. of water.
Filter before use. Wash out with water free from air.

HAMATEIN (HAMATOXYLIN) STAINS. 167

269. Vanadium Hematoxylin (H#IDENHAIN, Encyclop. mck.
Technik., 1903, p. 518).—Add 60 ¢.c. of a 6 per cent. solution of hema-
toxylin to a 0°25 per cent. solution of vanadate of ammonium (quantity
not stated; should be 30 c.c., see COHN in Anat. Hefte, xv, 1895, p. 302).
The mixture to be used after three or four days; it will not keep over
eight days. To be used with sections of sublimate material. A strong
plasma stain for special purposes, especially mucus glands.

270. Benpa’s Copper Hematoxylin (Arch. mik. Anat., xxx, 1887,
p. 49).—See fourth edition. According to my experience, not to be
compared with iron hematoxylin, and superfluous.

271. Mattory’s Phospho-molybdic Acid Hematoxylin (Anat.
Anz., 1891, p.375).—One part 10 per cent. phospho-molybdic
acid solution, 1 part hematoxylin, 100 parts water, and 6 to
10 parts chloral hydrate. Let the solution ripen for a week
in sunlight, and filter. Chiefly for central nervous system.
Sections should be stained for from ten minutes to one hour,
and washed out in two or three changes of 40 to 50 per cent.
alcohol. It is necessary that the solution should be saturated
with hematoxylin in order to obtain the best results; if a
good stain be not obtained at once, more hematoxylin must
be added. Water must never be used for diluting it.

See also RipperRt (Centralb. allg. Path., vii, 1896, p. 427; Zeit. wiss.
Mik., xv, 1898, p. 93), PATBLLANI (Mon. Zool. Ital., xiii, 1902, p. 6), and
GoLovIN (Zeit. wiss. Mik., xix, 1902, p. 184).

SaRGENT (Anat. Anz., xv, 1898, p. 214) quotes this stain, preceded by
mordanting for twenty-four hours in 5 per cent. sulphate of copper, as
Kerwyon’s.

Koonts (Arch. mik. Anat., lix, 1901, p. 211) takes hamatoxylin, 1 part;
molybdic anhydride, 1:5; water, 100; H,O,, 0°5, or a crystal of HgO.

Poxice (Arch. Zool. Napoli, iv, 1909, p. 300) takes 0°35 grm. hama-
toxylin, 10 drops phospho-molybdic acid of 10 per cent., 10 grms. chloral
hydrate, and 100 grms. alcohol of 70 per cent.

272. Mattory’s Phospho- tungstic Hematoxylin (Journ, Hap.
Med., v, 1900, p. 19; Zeit. wiss. Mik., xviii, 1901, p. 178) :

"Heematoxylin , . ‘ ; 0-1
Water : ‘ . 80°0
10 per cent. selatio of (Mprox’ 8) phospho-
tungstic acid . : . 20:0
Denatdle: of hydrogen (U. 5: Ph. Nea : 0:2

(Dissolve the hematoxylin, add the acid, then the peroxide.)
Stain sections two to twenty-four hours, wash out with water.

168 CHAPTER X11.

A polychromic stain, nuclei blue, intercellular substances pink.
I consider this a fine stain.

273. Donaaeio’s Tin Hematoxylin (Ann. Nevrol. Napoli, xxii,
1904, p. 192) —A 1 per cent. solution of hematoxylin is poured slowly
into an equal volume of 20 per cent. solution of pink-salt (ammonio-
chloride of tin). Keep in the dark.

274, Osmium Hematoxylin—ScuvuLtzeE (Zeit. wiss. Mik., xxvii,
1910, p. 465) treats tissues for twenty-four hours or more with osmic
acid of 1 per cent., washes well with water, and puts for a couple of
days into ripened 0°5 per cent. solution of hematoxylin in alcohol of 35
to 50 per cent. Wash out for a day or more with alcohol of 7 per cent,
Intense plasma stain.

CHAPTER XIV.
NUCLEAR STAINS WITH COAL-TAR DYES.

275. Introduction.—Very few coal-tar dyes give a precise
nuclear or chromatin stain by the progressive method (§ 209).
Two of them—methyl green and Bismarck brown—avre pre-
eminently progressive chromatin stains. Many of the others
—for instance, safranin, gentian, and especially dahlia—may
be made to give a progressive nuclear stain with fresh tissues
by combining them with acetic acid; but in general are not
so suitable for this kind of work as the two colours first-
named.

Again, very few coal-tar dyes give a pure plasmatic stain
(one leaving nuclei unaffected). The majority give a diffuse
stain, which in some few cases becomes by the application
of the regressive method (§ 209) a most precise and splendid
chromatin stain.

But plasma staining is generally done by the progressive
method,

The basic anilin dyes were at one time greatly in vogue
for the staining of chromatin in researches on the structure
of nuclei. ‘I‘hey have been little used for that purpose since
the working out of the iron hematoxylin process, which
gives a more energetic stain. But they may still be useful
as a means of controlling the iron hematoxylin process, which
frequently stains all sorts of things besides chromatin, which
does not occur with the best tar colour stains.

The acid and neutral anilin dyes afford some of our best
plasma stains.

I recommend—for staining nuclei of fresh tissues, methyl
green; for staining nuclei of fixed tissues by the regressive
method, safranin for a red stain; and gentian violet or
Thionin for a blue one; as a plasma stain for sectious,
Saurefuchsin ; for entire objects picric acid.

170 CHAPTER XIV.
A. Progressive Stains.

276. Methyl Green.--This is the most common in commerce
of the “anilin” greens. It appears to go by the synonyms
of Methylanilin green, Griinpulver, Vert Lumiere, Lichtgriun ;
these two last are in reality the name of another colour.
When first studied by Calberla, in 1874 (Morphol. Jahrb., ui,
1887, p. 625), 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
important-not to confuse it with the latter, nor with aldehyde
eveen (Vert @ Huscbe), nor with the phenylated rosanilins,
Paris green, and Vert d’ Alcali, or Veéridine.

Methyl green is the chloromethylate of zinc and penta-
methyl-rosanilin-violet. It is obtained by the action of
methyl chloride on methyl violet. The commercial dye
always contains unconverted methyl violet as a consequence
of defective purification. It is sometimes adulterated with
anilin blue (soluble blue). It is also sometimes adulterated
with a green bye-product of the manufacture—the chloride
of nona-methyl-para-leukanilin (see Benepixr and Knecut’s
Chemistry of the Coal-tar Colours). For tests for purity see
Mayer, Mitth. Zool. Stat. Neapel, xii, 1896, p. 312, and
Fiscuer, Fiairung, Farbung, wu. Bau des Protoplasmas, p. 89.

Methyl green is extremely sensitive to alkalies. It is
therefore important to use it only in acidified solutions and
to use only acid, or at least perfectly neutral fluids for
washing and mounting.

This is an extremely important histological reagent. Its
chief use is as a chromatin stain for fresh, unfiacd 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. If
the tissues have been previously fixed with acetic acid you
will not get a chromatin stain. The same applies to fixation
with acetic acid sublimate: whilst pure sublimate will allow
of a chromatin stain (BurckHarpr, La Cellule, xii, 1897,
p. 864). 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 mediun, if
aqueous, must be acidulated.,

NUCLEAR STAINS WITH COAL-TAR DYES. 171

Employed in this way, with fresh unfixed tissues, methyl
green is a pure chromatin stain, in the sense of being a pre-
cise colour reagent for chromatin. For in the nucleus it
stains nothing but chromosomes or chromatin elements; it
does not stain plasmatic nucleoli (unless indeed these contain
chromatin), nor caryoplasm, nor achromatic filaments. Outside
the nucleus it stains some kinds of cytoplasm and some kinds
of formed material, especially glandular secretions (silk for
instance, and mucin). The chromatin elements are invariably
stained of a bright green (with the exception of the nuclem
of the head of some spermatozoa), whilst extra-nuclear struc-
tures are in general stained in tones of blue or violet. But
this metachromatic reaction is probably due to the methyl-
violet impurity, and is not obtamed with a chemically pure
methyl green.

Staining is instantaneous ; overstaining never occurs. The
solution is very penetrating, kills cells instantly without swel-
ling or other change of form, and preserves their forms for at
least some hours, so that it may be considered as a delicate
fixative. It may be combined without precipitating with
divers fixing or preserving agents. Osmic acid (of 0:1 to 1
per cent.) may be added to it, or it may be combined with
solution of Rirarr and Prrir (this 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 sufficiently (unless this be charged with the colour).
The resistance of the colour to alcohol is, however (at all
events if it be used in the Euricn-Bronp1 combination), con-
siderably increased by treating the sections for a few minutes
with tincture of iodine before staining (M. Hripenuaiy).

Of preparations mounted with excess of colour in the usual
aqueous media, I find the most fortunate only survive for a
few months. Dr. Henrecuy, however, writes to me that it
keeps well in Brun’s glucose medium.

It was first pointed out, I believe, by HEscun (Wiener med. Wochenschr,
2, 1879), that methyl green is a reagent for amyloid degeneration. His
observations were confirmed by Curscumann (Virchow’s Arch., vol.
Ixxix, 1880, p. 556), who showed that it colours amyloid substance of an

172 CHAPTER XLV.

intense violet ; but this,as pointed out by Squire (Methods aud Formule,
etc., Churchill, 182, p. 37), is undoubtedly due to its containing methyl
violet as an impurity.

277. 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, or otherwise.

The colour is not very easily soluble in water. You may
boil it in water, and filter after a day or two (Weicerrt, in
alrch. mik. Anat., xv, 1878, p. 258). You may add a little
acetic or osmic acid to the solution. Maysen (ibid., xviii,
1830, pp. 237, 250) dissolves the colour in acetic acid (this
solution does not give a permanent stain). Alcoholic solu-
tions may also be used, e.g. saturated aqueous solution
diluted with one third volume of 90 per cent. alcoholic ; or
CaLBerta’s glycerin-and-alcohol: mixture, or dilute glycerin
(say of 40 per cent. to 50 per cent.) may very advan-
tageously be employed.

The watery solutions must be frequently filtered (but then
much of the colour is retained on the filter). The addition
to them of carbolic acid has been recommended (vide Journ.
Roy. Mic. Soc., 1886, p. 908). Bismarck brown stains rapidly,
but never overstains. The stain is permanent both in balsam
and in glycerin.

This colour may be used as a chromatin stain for fresh
tissues in the same way as methyl green. Huerta (Arch. Biol.,
xiii, 1893, p. 423) employs for ova of Ascaris a mixture of
0-25 parts vesuvin, 0°25 malachite green, 10 of glycerin and
100 of water, and washes out with weak glycerin.

The chief use of this colour is for progressive staining ;
but it may be employed for staining by the regressive method
(see § 289), and also for infra-vitem staining ($ 208) (for
this purpose it is necessary to see that the colour employed
be pure and neutral).

278. Methyl Violet (Methylanilin Violet, Anilin Violet, Paris
Violet)—GRASER (Deutsche Zeit. Chirurgée, xxvii, 1888. pp. 538—S84 ;
Zeit. wiss. Mék.,v, 3, 1888, p. 378) stains sections from twelve to twenty-
four hours in a 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. The method is applicable to objects fixed in Flemming’s
mixture.

NUCLEAR STAINS WITH COAT-TAR DYKES. 173

279. Other Progressive Stains.—Most of the basic tar colours used
for regressive staining will also give by the progressive method a nuclear
stain of greater or less purity if used in solutions acidified with acetic
acid. Amongst these may be mentioned tidzonin, which need not even
be acidified ; also, for fresh tissues especially, gentian violet, duhlia, and
tolaidin blue.

B. Regressive Stains.

280. The Practice of Regressive Staining : The Staining Bath.—
Sections only, or material that is thin enough to behave like
sections, such as some membranes, can be stained by tlis
method.

The solutions employed are made with alcohol, water, or
anilin, or sometimes other menstrua, 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. Indeed, the solutions
made with strong alcohol are found not to give quite such
good results as those made with water or weak alcohol.
Alcohol of 50 per cent. strength, however, may be said to
constitute a very generally desirable medium. 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 usually 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 advantage. Some workers, indeed,
prefer weak solutions ; so Herprnnain, Encyel. mik.
Technik, i, pp. 483, 434; but the nature of the fixing agent
should be taken into account.

Material fixed in chromic or chromo-osmic mixtures gives
a sharper and more selective stain than material fixed in
sublimate or the like. In fact, to ensure the best results, only
material fixed in chromic mixtures (ov Hermann’s fluid) should
Le employed.

During the staining the tissues become overstained, that
is, charged with colour in an excessive and diffuse manner.
The stain must now he differentiated by removal of the excess
of colour.

174: GHAPTER XIV.

281. Differentiation—This is generally done with alcohol,
sometimes neutral, sometimes acidulated (with HCl). 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 (differentiation 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, or even to wash them
well in it, 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 trans-
parent, and (in the case of chrom-osmium objects) have
changed colowr, owing to the coming into view of the general
ground colour of the tissues. (Thus chrom-osmium-safranin
sections turn from an opaque red to a delicate purple.) At
this point the differentiation is complete, or nearly so.

It is generally directed that absolute alcohol be taken for
differentiation. This may be well in some cases, but in
general 95 per cent. is found to answer perfectly well.
Herpennain (Encyel., i, p. 434) takes methyl alcohol.

The hydrochloric-acid-alcohol extracts the colour much
more quickly from resting nuclet than from kinetic nuclet.
Therefore, washing out should be done with neutral alcohol
whenever it is desired to have resting nuclei stained as well
as dividing nuclei; the acid process serving chiefly to
differentiate karyokinetic figures.

The proportion of HCl with which the alcohol should be
acidified for the acid process should be about 1 : 1000, or
less ; seldom more.

The length of time necessary for differentiating 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. he acid process is vastly more rapid
than the neutral process, and therefore of course more risky.

NUCLEAR STAINS WITH COAL-TAR DYES. 175

There exists also a method of differentiation known as substétution—
one stain being made to wash out another. Thus methylen blue and
gentian violet are discharged 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 has been
successfully carried out, the nuclei stained with the first stain, the
second forming a “contrast” stain. In the paper of REsscorri in Zezt.
wiss. Mik., v, 1888, p. 320, it is stated as a very general rule that colours
that do not give a nuclear stain by the regressive method will wash out
those that do. But Resecorrr used the second colour in alcoholic
solution ; so that it remains uncertain how far the differentiation should
be attributed to the second colour itself, and how far to the alcohol
used as a vehicle. The same remark applies to Benpa’s Safranin-and-
Lichtgriin process.

282. Clearing —After due differentiation, the extraction 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 or anilin, which will extract
some more colowr from the tissues. Or you may clear with
an agent that does not attack the stain (cedar oil, bergamot
oil, xylol, toluol, etc. ; see the chapter on Clearing Agents).
If you have used neutral alcohol for washing out, you had
perhaps better clear with clove oil, as neutral 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 nuclet more rapidly than those in division.

Some colours are much more sensitive 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; and aniline than clove oil.

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

After clearing you may either mount at once in damar or
balsam ; or, stop the extraction of the colour, if clove oil
have been used, by putting the sections into some medium

176 CHAPTER XLV.

that does not effect the stain (xylol, cedar oil, etc.). Chloro-
form should be avoided, either as a clearer or as the men-
struum for the mounting medium.

283. 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 differentiated after staining with
acid alcohol and cleared with clove oil, you will get, with
some special exceptions, nothing stained but nucleoli and the
chromatin 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 differentiated after
staining with neutral alcohol, you will get the chromatin of
resting nuclei stained as well. Hither process may also stain
mucin, the ground-substance of connective tissues (especially
cartilage), the bodies of Nissl in nerve-cells, and the yolk of
ova.

284. HENNEGUY'S Permanganate Method (Journ. del’ Anat. et dela
Physiol., xxvii, 1891, p. 397).—Sections are treated for five minutes with
1 per cent. solution of permanganate of potassium. They are then
washed with water and stained (for ahout 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, and are differentiated
with alcohol, followed by clove oil in the usual way.

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

285. OHLMACHER’S Formaldehyde Process (Medical News, Feb-
ruary 16th, 1895) —Ohlmacher states that formaldehyde is a powerful
mordant for tar colours. Tissues may either be mordanted separately
by treatment for a short time (one minute is enough for cover-glass —
preparations) with a 2 per cent. to 4 per cent. formalin solution; or the
formalin may be combined with the stain. One gramme of fuchsin or
methylen blue dissolved in 10 c.c. of absolute alcohol may be added to
100 ¢.c. of 4 per cent. formalin solution. Sections are said to stain in
half a minute and to resist alcohol much more than is the case with
those treated by the usual solutions,

286. Safranin.—One of the most important of these stains,
on account of its power, briliancy, and permanence in balsam,

NUCLEAR STAINS WITH COAL-TAR DYKS. 177

and the divers degrees of electivity that it displays for the
nuclei and other constituent elements of different tissues.

The great secret of staining with safranin is to get «u good
safranin. In ordering it, from Griibler & Hollborn or else-
where, it is well to specify whether 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 safranin 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 reverse, and some freely solubie in both. The brand I
have been using for a long time, which gives good results, is
the “ Safranin O” of Gritbler & Co.

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

Privaner (Morph, Jahrb., vi, p. 478, and vii, p. 291) advised
a solution of safranin 1 part, absolute alcohol 100 parts, and
water 200 parts, the last to be added only after a few days.

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

Bases (ibid., 1883, p. 356) used (a) a mixture of equal
parts of concentrated alcoholic solution and concentrated
aqueous solution (this is very much to be recommended), or
(B) a concentrated or supersaturated aqueous solution made
with the aid of heat,

Some people still employ simple aqueous solutions.

The anilin solution of Bases (Zett. wiss. Mik., iv, 1887, p.
470) 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.

ZWAARLEMAKER (tbid., iv, 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 with water, and filter). This, I find, will
keep for many months, perhaps indefinitely.

I myself use equal parts of saturated solution in anilin
water, and saturated solution in absolute alcohol.

Differentiation. —For general directions see §§ 281 and 282.

12

178 CHAPTER XLV.

Fieauine’s acid ditferentiation (Zeit. wiss. Mik., 1, 1884,
p. 350).—Differentiate, 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 HCl in watery solution if you prefer it.)
Or you may use a lower strength, viz. O'l per cent. at most
(see Arch. mik. Anat., xxxvii, 1891, p. 249); and this I find
is generally preferable.

Objects are supposed to have been well fixed—twelve
hours at least—in the strong chromo-aceto-osmic mixture,
and stained for some hours. In this way you get kinetic
chromatin and nucleoli alone stained.

Popwyssozx1 (Beitr. z Path. Anat., 1, 1886, p. 289)
differentiates (for from a tew 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).

Bazes recommends treatment with iodine, according to the
method of Gram (see next section). ‘This process has also
been recommended by Prenanr (Zut. Monatsschr. Anat., ete.,
ivy, 1887, p. 368).

Tt has been shown by OHLMACHER (Journ. Amer. Med. Assoc., vol. xx,
No. 5, February 4th, 1893, p. 111) that if tissues be treated with 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, semilunar, falciform, or navicellar forms.
The precipitate is formed both in normal and pathological tissue,
readily in carcinomatous tissues ; and Ohlmacher concludes that many
of the bodies that have been described as “ coccidia,” “ sporozoa,” or
other “ parasites” of carcinoma are nothing but particles of this pre-
cipitate.

See also the differentiation process of MartTinoTTi and RESEGOTTI
(Zeit. wiss. Mik., iv, 1887, p. 328) for alcohol-fixed material; and of
GARBINI (Zeit. wiss. Mik., v, 2, 1888, p. 170).

In preparations made with chromo-aceto-osmic acid,
satranin stains, besides nuclei, elastic fibres, the cell bodies of
certaim horny epithelia, and the contents of certain gland-
cells (mucin, under certain imperfectly ascertained condi-
tions).

The stain is perfectly permanent.

287. Gentian Violet may be used in aqueous solution, or as
directed for safranin.

NUCLEAR STAINS WITIL COAL-TAR DYES. 179

In some cases it may be useful to employ the method
devised by Gram for the differentiation of bacteria in tissues
(Lortschr. d. Medicin., i, 1884, No. 6; British Med. Journ,
Sept. 6th, 1884, p. 486; Journ. Roy. Mic. Soc. [N.S.], 1v,
1884, p. 817). In this the sections are treated, after stain-
ing, with a solution composed of—

Iodine. : : ; : 1 gramme,
Todide of potassium . : F 2 grammes,
Water. . 300

22

for two or three minutes, until they become black. ‘They
are then differentiated with neutral alcohol, until they turn
erey, and are then finally differentiated with clove oil.

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

Gentian violet is an exceedingly powerful stain, quite as
precise as safranin.

The stain keeps well. It is more or less dichroic, possibly
owing to the fact that the dye is not a pure substance, but
a mixture of “ Krystallviolett ” and methyl violet.

Hermann (Arch. mik. Anat., xxxiv, 1889, p. 58) first stains for twenty-
four hours or more in safranin, differentiates incompletely with alcohol,
then stains for three to five minutes in the anilin-water gentian solution,
treats with the iodine solution for one to three hours, and finally
differentiates with absolute alcohol.

288. Thionin.—The hydrochloride of thionin, or violet of
Lauth, is a colour chemically nearly allied to methylen blue.
Its action is so selective from the first that it may almost be
considered to be a progressive stain. If you stain for only
a short time (a few minutes) im a concentrated aqueous
solution, hardly anything but the chromatin will be found
to be stained. If the staining be prolonged, plasmatic
elements will begin to take up the colour. After a short
stain no special differentiation is required; all that is
necessary is to rinse with water, dehydrate, and mount.
After a strong stain you differentiate with alcohol in the
usual way, with this advantage, that the stain is so highly
resistant to alcohol that there is no risk whatever of over-
shooting the mark; the stain will not be more extracted in

180 CHAPTER XIV.

an hour than that of gentian or dahlia is in a minute, so
that the process may be controlled under the microscope
if desired. For this reason I think this stain may be useful
to beginners, but I myself prefer gentian. It is a very
powerful stain.

Thionin is a specific stain for mucin, g.v. Some observers have found
the stain to fade. Wo.Lrr (Ze?t. wiss. Mik., xv, 1899, p. 312) says that
to avoid this, preparations should be mounted in a little solid colo-
phonium or balsam melted over a flame. FELIzar and BRANCA (Journ.
Anut Phys., xxxiv, 1898, p. 590) mount without a cover. HENNEGUY °
(in lit.) clears with acetone.

Kine (Anat. Record, iv, 1910, p. 236) stains with a saturated solution
in carbolic acid of 1 per cent., and finds the stain permanent.

NiIcoLie’s “thionine phéniquée” consists of 1 part of saturated
solution in alcohol of 50 per cent., und 5 parts of 2 per cent. aqueous
solution of carbolic acid.

289, Other Regressive Stains.—'l'he following may be
useful :—

Dahlia, according to Fiemmine (Arch. mik. Anat., xix,
1881, p. 317), best used in aqueous solution, either neutral
or acidified with acetic acid, and differentiated with neutral
alcohol. A pure blue stain, which keeps well. See also
Scuupere, in Zeit. wiss. Zool., xxiv, 1903, p. 7, and Ixxxvii,
1907, p. 557.

Victoria Blue (Victoriablau) (Lustaarren, Med. Jahrb. k.
Ges. d. Aerzte zu Wien, 1886, pp. 285—291).—This dye
( Victoriablau 4 A’) has a special affinity for elustic 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, and for cartilage.

This stain keeps very well.

With Toluidin Blue I have had some superb stains of chromatin,
unfortunately accompanied by a diffuse staining of cytoplasm.

Mann (Zett. wiss. Mik., xi, 1894, p. 489) states that he has had good
results by staining with it after eosin.

See further as to the micro-chemical properties of this dye, Harris,

NUGLEAR STAINS WITH COAL-TAR DYES. 181

The Philadelphia Medical Journal, May 14th, 1898. It much resembles
methylen blue.

Metzner (Nagel’s Handb. Phys., ii, 1907, p. 915) mordants sections,
before staining, for three quarters of an hour in iron alum.

Magdala Red (Naphthalin Red, Rose de Naphthaline).

Fuchsin (meaning the basic fuchsins, a series of Rosanilin salts
having very similar reactions, and found in commerce under the names
of Fucusin, ANILIN RED, Rusin, Rosrrn, Magenta, SoLrerino,
CORALLIN).—GRASER (Deulsche Zeit. Chirurgie, xxvii, 1888, pp. 5388—
584; Zeit. wiss. Mik., v, 1883, p. 378) stains for twelve to twenty-four
hours in a dilute aqueous solution, washes ont for a short time in alcohol,
stains for a few minutes in aqueous solution of methylen blue, and
dehydrates with alcohol. A double stain. Chromatin and nucleoli red,
all the rest blue.

Zinu’s Carbolie Fuchsin (Zeit. wiss. Mtk., vii, 1890, p. 39)
consists of fuchsin 1 grm., acid. carbol. crist. 5 grms.,
alcohol 10 grms., aq. dest. 100 grms. ‘The stain is
differentiated with alcohol followed by clove oil.

Kres:fuchsin (Rotxic, Arch. mik. Anat., Ivi, 1900, p. 354).—Its
aqueous solution is red, and stains mucus, cartilage, keratin, and nuclei
red, whilst its alcoholic solution is blue and stains elastin blue. See
also under “ Connective tissues.”

Bismarck Brown has this advantage, that being sufficiently resistant
to alcohol it may be utilised for staining entire objects.

Kaiser (Biblioth. Zool., H. 7, 1 Halft, 1891; Zeit. wiss. Mik., viii, 1891,
p. 363) stains for forty-eight hours, and at a temperature of 60° C. in
saturated solution of Bismarck brown in 60 per cent. alcohol (the solution
to be made in boiling alcohol), and washes 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.

Methyl Violet. See ante, § 278

Benzoazurin (MARTIN, Zeit. wiss. Mik., vi, 3,1889, p. 193). Stain for
an hour or so in dilute aqueous solution, and wash out with HC] alcohol.

Methylen Blue.

Nigrosin (Errera, Proc.-Verb. Soc. Belge de Mik., 1881, p. 134) gives
a good stain which resists alcohol’well.

Methyl Green is sometimes useful in certain mixtures (see next
chapter).

CHAPTER XV.

PLASMA STAINS* WITH COAL-TAR DYES.

290. Introduction—By a plasma stain is meant one that
stains the extra-nuclear parts of eclls and the formed material
of tissues, or one of these.

The plasma stains described in this chapter are for the
most part those obtained by means of “acid” dyes ($ 2083);
but some of them are obtained by means of ‘‘neutral’’ dyes
(§ 203), and a few by “basic”’ dyes.

The mode of staining is generally progressive, almost
always so when acid colours, used substantively ($ 205), are
employed. But the regressive method, with differentiation,
is sometimes made use of, especially when a mordant has
been used with the dye.

In some processes, e.g., Flemming’s orange method, a
basic and an acid dye (or vice versa) being employed in suc-
cession, there is formed in the tissues a neutral colour (¢ 203)
which effects the desired stain. These may be considered as
adjective stains, the first colour serving as a mordant for
the second. Not any two dyes taken at haphazard will
behave in this way: they must be such as to form by com-
bination a suifable neutral lake (cf. § 203). The basic dye
may be made the primary stain, as in Flemming’s process :
or the contrary. ,

In such stains as Reinke’s orange method, or the Ehrlich-
Biondi mixture, and many others, ene or more neutral colours
are formed in the mixture and stain progressively.

Iam not acquainted with any plasma stain that is

* This chapter includes only such stains as are used in ordinary work
on tissues in bulk or sections, stains for special purposes being treated
under “ Nervous tissue,” ‘‘ Blood,” ete. It includes some double or
triple stains that affect nuclei as well as plasma, but in different. hues.

PLASMA STAINS WITH COAT-TAR DYES. 183

-thoroughly satisfactory for delicate work. I recommend for
sections Saurefuchsin, either alone or in the form of Hhrlich-
Biondi mixture, or Hhrlich’s triacid: for material in bulk,
picric acid (but only for rough work).

291. Saurefuchsin (Acid Fuchsin, Fuchsin §, Acid Rubin, Rubin
S, Saurerubin, Acid Magenta, Magenta $).—The chemical deserip-
tion of this acid colour has been given (§ 203): it must not
be confounded with basic fuchsin, as seems to have been
done by some writers.

This dye is highly soluble in water, less so in alcohol. I
use a 05 per cent. solution in water and allow it to act on
sections for a few minutes in the case of easily stainable
material, or twenty-four hours or more for chrom-osmium
material, The stain is fast to neutral alcohol. It is very
sensitive to alkalies, so that overstains can easily be removed
by washing for a few minutes in tap-water. Acids strengthen
the stain, so that it is frequently useful to treat sections after
staining for a few seconds with acidulated water. A good
stain should show the reticulum of cytoplasm, together with
nuclear spindles and asters, stained red, and connective tissue
strongly brought out. It may be advisable to acidify the

staining bath very slightly. Successful stains are admirably
sharp.

292. Pyronin.—A basic dye, red, only used (as far as I can
find) in mixtures. Parrrnnuim (Arch. Path. Anat., clxvi, 1901,
p- 427) takes two parts 1 per cent. solution of methyl green
and one part 1 per cent. solution of pyronin, stains sections for
five minutes, rinses, and differentiates in a solution of resorcin
or hydroquinon in absolute alcohol. According to Corrr and
Ferrara, Aon. zool. Ital., xvi, 1905, p. 319, this mixture
generally stains chromatin green and cytoplasm red, but,in
Flemming or Hermann material the reverse. It seems to me
a coarse plasma stain, but likely to be sometimes useful.

Unna’s CARBOL-PYRONIN-METHYL GREEN modification (Lneycl.
Mik. Techn., 1910, ii, p. 412: I am indebted for the formula
to Dr. Gaupiitz) is as follows: Stain for five to ten minutes
at 30° to 40°C. in methyl green 0°15 parts, pyronin 0°25,
alcohol 2°5, glycerin 20, and carbolic acid of 0°5 per cent. to
make up 100 volumes. Cool rapidly, rinse, dehydrate, and

184 CHAPTER XV.

pass through bergamot oil, or xylol or benzol (not clove-oil)
into balsam. Brings out bacteria (red) in organic liquids.
The mixture may be had from Griibler & Hollborn.

293. Orange G—This is the benzenazo-beta-naphthol-
disulphonate of soda (to be obtained from Griibler & Hollborn,
and not to be confounded with about a dozen other colours
that are on the market under the name of “ Orange,” with
or without asuffix). As indicated by its chemical description,
this is an “acid” colour.

It is easily soluble in water, less so in alcohol. Use as
directed for Siiurefuchsin. Almost, if not quite, as precise a
stain as Sdurefuchsin. It does not overstain, but may wash
out other dyes.

294, Saurefuchsin and Orange G.—I have had good results
by mixing the aqueous solutions of these two dyes, but unfor-
tunately have not noted the proportions. Squire (Methods
and Formule, p. 42) takes 1 grm. Siiurefuchsin, 6 grms.
Orange G. in 60 c.c. of alcohol, and 240 ¢.c. of water. See
also under “ connective tissues.”

295. Kuriicu-Bionpr Mixture (or Euruiicu-Bronpi-Hrt-
DENHAIN Mixture) (Pfliiger’s Arch., xiii, 1888, p. 40).

To 100 c.c. saturated aqueous solution of orange add with
continual agitation 20 c.c. saturated aqueous solution of
Saiurefuchsin (Acid Fuchsin) and 50 c.c. of a like solution of
methyl green.

(According to Krause [ Arch. mii. Anat., xlii, 1893, p. 59], 100 parts of
water will dissolve about 20 of Siiurefuchsin [Rubin 8], 8 of orange G.
and 8 of methyl green.) The solutions must be absolutely saturated,
which only happens after several days.

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.

According to M. Humrnnain (“Ueber Kern u. Proto-
plasma,” in Festschr. f. Kolliker, 1892, p. 115) the orange to
be used should be “ Orange @”’; the Acid Fuchsin or Siiure-

PLASMA STAINS WITH COAL-TAR DYES. 185

fuchsin should be “ Rubin 8 ” (“ Rubin” is a synonym of
Fuchsin) and the methyl green should be “ Methylgriin OO.”
And it is absolutely necessary that these ingredients be those
prepared under those names by the Actienfabrik fiir Anilin-
fabrikation in Berlin. They can be obtained from Gribler
& Hollborn, either separately, or as a mixture of the three
dyes in powder (which I do not recommend).

The strong solutions directed to be taken readily precipi-
tate on being mixed. ‘To avoid this it is recommended by
Squire (Methods and Formulx, etc., p. 37) to dilute them
before mixing.

Other proportions for the mixture have been recommended by KRAUSE
(Loe. cit. supra), viz. 4 ¢.c. of the Siurefuchsin solution, 7 of the orange G
and 8 of the methyl green; the mixture to be diluted 50 to 100-fold with

water. THomi (Arch. mk. Anat., lii, 1898, p. 820) gives the proportions
2:5:8, and dilutes 100-fold.

Stain sections (N.B. sections only) for six to twenty-four
hours. Dehydrate with alcohol, clear with xylol, and mount
in xylol balsam.

In the intention of the observers who have elaborated this
stain it is a progressive stain, and not a regressive one. It
does not require any differentiation, and the sections should
be got through the alcohol into xylol as quickly as possible
in order to avoid any extraction of the methyl green, which
easily comes away in the alcohol. Drtnzr (Jena. Zert.,
xxix, 1894, p. 276) stains for ten minutes in the concentrated
solution, treats for one minute with alcohol containing 0-1
per cent. of hydrochloric acid, and then with neutral
alcohol.

The best results are obtained with sulbiimate material ;
chrom-osinium material, and the like, give a much inferior
stain. Preparations made with the usual mixture, as given
above, are lable to fade; by acidifying the mixture a
stronger and more sharply selective stam is obtained, which
does not fade. But too much acid must not be added,
as this would cause a staining of the interfilar substances.
According to the Eneyel. mik, Technik, you may add 15
to 24 drops of 0-2 per cent. acetic acid to 100 c.c. of the
diluted solution.

Another process of acidification is given by M. Herpennain (Ueber

186 CHAPTER XV.

Kern und Protoplasma, p. 116); for this see fourth edition. See also
Israen (Praktikum Path. Hist.,2 Aufl., Berlin, 1893, p. 69); TRAMBUSTI
(Ricerche Lab. Anat. Roma, v, 1896, p. 82; Zeit. wiss. Mik., xiii, 1896, p.
357): and THOME (op. cit. supra). Eisen (Proc. Calif. Acad. [3], i,
1897, p. 8) acidifies with oxalic acid.

After acidification the solution must not be filtered, and if
it has been kept for some time a little more acid must be
added.

Before staining (M. Herpennaiy, loc. cit.), sections should
be treated for a couple of hours with 0-1 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. The treatment with acid is necessary in order to
ensure having the sections acid on mounting in balsam. The
primary object of the iodine is to remove any sublimate
from the preparations, but it also is said to enhance the power
of staining of the chromatin with methyl green, and to
produce a more selective staining of protoplasmic elements.

The stain is a very fine one when successful. But it is
very capricious. The correct result should be a precise
chromatin stain combined with a precise stain of the plastin
element of cytoplasm by the Saurefuchsin. Now the least
defect or excess of acidity causes the plasma stain of the
Siiurefuchsin to become a diffuse one, instead of being
sharply limited to the plastin element. It is difficult to
dehydrate the sections without losing the methyl green.
For this reason the stain will only work with very thin
sections: to be quite sure of good results, the sections should
be of not more than 3 , in thickness, and if they are over
5 the desired results are almost hopeless. The stain
keeps very badly. I admit that the method has its raison
@ tre for the very special objects for which it was imagined
—for the researches on cell-granulations for which Exriica
employed the three colours, or for the researches on the
plastin element of cytoplasm for which Martin Hempennain
employed the mixture; for the study of gland cells; and
for similar objects. But to recommend it, as has been done,
as a general stain for ordinary work, is nothing but mis-
chievous exaggeration. Tor it is far from having the
qualities that should be possessed Ly a normal section stain.

PLASMA STAINS WITH COAL-TAR DYES. 187

Workers have at length found this out, and it is now but
little used except for the special purposes above indicated.

296, Huruica’s “ Triacid” Mixture.—This name would seem
to indicate that the mixture contains three “acid” colours,
which is not the case, methyl green being a strongly “basic”
colour. Ehrlich explains in a letter to Mayer (see also
Eariich and Lazarus, Die Anemie, 1898, p. 26) that it is
so called ‘‘ because in it all the three basic groups of the
methyl green are combined with the acid dye-stuffs.’? A
very pretty conundrum !

The latest receipt (op. cit., p. 28) is as follows:

Prepare separately saturated solutions of orange G,
Siurefuchsin, and methyl green, and let them clarify by
settling. Then mix, in the order given, using the same
measure-glass, 13 to 14 c.c. of the orange, 6 to 7 of the
Siiurefuchsin, 15 of distilled water, 15 of alcohol, 124 of
the methyl green, 10 of alcohol, and 10 of glycerin. After
adding the methyl green, shake well, but do not filter.

The mixture keeps well. I find its qualities and defects
to be much those of the Ehrlich-Biondi mixture. The stain
seems more powerful but Jess delicate, and the methyl green
in it appears to have more resistance to alcohol, so that it is
better adapted for ordinary work.

Maver (Grundziige, Lpp & MAyYER, p. 197) has simplified the formula
thus: Take 1 g. methyl green, 2 g. orange, 3 g. Saurefuschin, and dis-
solve in a mixture of 45 c.c. water, 10 c.c. glycerin, and 20 c.c. aleohol of
90 per cent.

Moret and Douenis (C. R. Soe. Biol., liv, 1902, p. 1255) mix 1 vol. of
the solution with one of 8 per cent. formalin and add 0:1 per cent. of
acetic acid, and state that thus the methyl green is hetter fixed in the
nuclei.

297. PIANESE’S Saurefuchsin-malachite Green (from MULLER,
Arch. Zellforsch., viii, 1912. p. 4) consists of 05 grm. malachite green,
0-1 grm. Saurefuchsin, and 0:01 grm. Martius yellow in 150 ¢.c. water
and 50 ¢.c. aleoho). Stain for 24 hours, differentiate with alcohol, con-
taining 1 to 2 drops of HCl per 200 ¢¢.

£98. Picric Acid.—FPicric acid gives useful plasma stains
after carmine and hematoxylin. The modus operanda con-
lists merely in adding picric acid to the alcohols employed
for dehydrating the objects.

188 CHAPTER XV.

Picrie acid has considerable power of washing out other
anilin stains; and im combination with hydrochloric acid t
very greatly enhances the power with which this acid washes
out carmine stains. It should, therefore, not be added to
the acidulated alcohol taken for differentiating borax-carmine
stains, or the like, but only to the neutral alcohol used after-
wards. It has the great quality that it can be used for
staining entire objects, and is much indicated for such
objects as small Arthropods or Nematodes, mounted whole.

It can in some cases be employed by dissolving it in the
solution of another dye (see Picro-carmine, Lecay’s alum-
carmine, § 219, etc.) ; or (for sections) by dissolving it in the
xylol or chloroform used for clearing.

Though picric acid is a useful ground stain, it is at most a
rough one, being very diffuse. It stains, however, horn,
chitin, muscle and erythrocytes, with special energy.

According to Frénnica (Zeit. wiss, Mik., xxvii, 1910,
p. 319) picraminic acid (from Griibler & Hollborn) has some
advantages over picric acid.

299. Vaw Ginson’s Picro-Saurefuchsin (from Zett. wiss. Mik.,
xiii, 1896, p. 344).—To a saturated aqueous solution of
picric acid is added a few drops of saturated aqueous
solution of Sdurefuchsin, until the mixture has become
garnet-red. Or (Trans. Amer. Mier. Soc., xix, 1898, p.
105) to 100 parts of the picric acid solution add 5 parts of
1 per cent. solution of Siiurefuchsin. After staining (sections
only), rinse with water, dehydrate, and clear in oil of
origanuin.

OutmacHer (Journ. Eaper. Med., ii, 1897, p. 675) adds 0°5
per cent. of Siurefuchsin to a saturated solution of picric
acid which has been diluted with an equal quantity of water.
He uses this after previous staining with gentian violet.

Ramon y Casati recommends 0'l grm. of Siéurefuchsin to
160 of saturated solution of picric acid (ScHarrer, Zeit. wiss.
Zool., \xvi, 1899, p. 236).

Hansun (Anat, Anz., xv, 1898, p. 152) adds 5 c.c. of 2 per
cent. solution of Siurefuchsin to 100 c.c. saturated solution
of pieric acid, and for staining adds to 3 c.c. of the mixture
one third of a drop of 2 per cent. acetic acid, stains for a
few minutes or hours, rinses in 8 ¢.c. of water with 2 drops

PLASMA STAINS WITH COAL-TAR DYES. 189

of the acidified stain added, dehydrates, clears with xylol,
and mounts in xylol-balsam. Connective-tissue red, elastin
and all other elements yellow.

Wuicerr (Zeit. wiss. Mtk., 1904, p. 3) adds 10 parts of 1
per cent. Siurefuchsin to 100 of saturated picric acid.

See also Métuur, op. cit., xv, 1898, p. 172.

This stain is generally used as a contrast stain to follow
hematoxylin, ApAruy (Behrens’ Tubellen, 3rd ed., p. 129)
takes for this purpose 1 grm. of Séurefuchsin in 500 c.c. of
saturated solution of picrate of ammonia.

WitHetm (Fauna Flora Golf. Neapel, xxii, 1909, p. 18)
takes 0°2 erm. Saéurefuchsin, 0°8 grm. picrate of ammonia,
10 grm. absolute alcohol, and 89 grm. water.

HK. and T. Savini (Zeit. wiss. Mik., xxvi, 1909, p. 31) use
a formula due to Benpa. Ninety-five volumes of saturated
solution of picrate of ammonia are mixed with 5 volumes of
1 per cent. solution of Saurefuchsin. For use, two to four
drops of saturated solution of picric acid are added to 10 e.c.
of the mixture. This neither overstains nor attacks the
primary stain.

300. FLEMMING’s Orange Method (Arch. aik. Anat., xxxvii, 1891,
pp. 249 and 685).—Stain sections of Flemning or Hermann material in
strong alcoholic safranin solution diluted with anilin water (§ 286) ;
differentiate in absolute alcobol, containing at most 0:1 per cent. of
hydrochloric acid, until hardly any more colour comes away; stain for
one to three hours in gentian violet (§ 287); wash for a short time in
distilled water; treat with concentrated, or at least fairly strong,
aqueous solution of orange G. After at most a few minutes, whilst
pale violet clouds are still being given off from the sections on
agitation, bring them into absolute 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 must be orange G.

WINIWARTER and Sarnmonr (Zedt. wiss. Mik., xxv, 1908, p. 157,
and Arch. Biol., xxiv, 1909, p. 15) stain for 24 hours in the gentian,
wash out after the orange for 2 to 3 hours in 100 c.c. absolute alcohol
with three to four drops of HCl, and differentiate finally with oil of
cloves.

This is not a triple stain in the sense of giving three different
colours in the result ; it is a nuclear and plasmatic stain in mixed tones;
the orange, apparently, combines with the gentian to form a “ neutral”
dye, soluble in excess of the orange (§ 203) which thus differentiates the
stain.

190 GHAPIBR XV.

See also FLEMMING in Arch. Anal. Phys. Anat. Abth., 1897, p. 175.
Never popular, this clumsy and uncertain provess is now little used.

801. Retnku’s Orange Method (Arch. mik. Anat., xliv, 2, Lsv4,p. 262).
—To a concentrated ayueous solution of gentian violet are added “a few
drops” of a like solution of orange G. The solution precipitates in part,
owing to the formation of an imperfectly soluble * neutral” colour, but
becomes almost clear again if an excess of water be added. The
solution is not to be filtered, but the sections are to be stained in tue
mixture made almost clear by addition of water. It is said that the
“neutral” solution may be preserved for future use by adding to it one
third of alcohol. After staining (sections previously stained with
safranin), you differentiate rapidly with alcohol and clear with clove oil.

I have tried this process and obtained exactly the same results as with
Flemming’s process, and so have other workers.

ARNOLD'S Orange Method (Arch. Zellforsch., iii, 1909, p. 434).—
Sections (of chrome material) are treated for five minutes with solution of
equal parts of iodine and iodide of potassium in alcohol of 40 per cent.,
then washed and stained for 4 hours in saturated solution of safranin in
alcohol of 75 per cent. : then washed and put for 5 to 15 minutes into
solution of seven parts of methylen blue, 0'5 of carbonate of soda and 100
of water, washed, dehydrated, and treated until pale blue with solution of
orange G. in oil of loves. Cytoplasmic reticulum blue on orange ground,
nucleoli and centrosomes red. Instead of the safranin, basic fuchsin
may be taken.

302. Bonney’s Triple Stain (Virchow’s Arch., cxciii, 1908,
p. 547, and elsewhere).—Stain sections (of acetic alcohol or
sublimate material, not chrome or formol material) for two
minutes in a solution of 0:25 parts methyl violet and 1 part
pyronin in 100 of water. Wipe slide dry, and flood twice
with the following: 2 per cent. aqueous solution of orange G,
boiled and filtered, is added drop by drop to 100 c.c. of
acetone, with agitation, until there is formed a flocculent
precipitate, which redissolves on further addition of the
orange. Wash rapidly in pure acetone, and pass through
xylol into balsam. Chromatin violet, cytoplasm red, con-
nective tissue yellow, keratin violet. Not adapted for blood
films.

303. Bordeaux R.—.\n “acid” dye, giving a general stain
taking effect both on chromatin and cytoplasm, and, I con-
sider, a very good plasma stain. I use for chrom-osmium
material a 1 per cent. solution, and stain for twelve to
twenty-four hours. ‘I'he stain is sufficiently fast.

PLASMA STAINS WITH COAL-TAR DYES. 191

304. Bordeaux R, Thionin, and Methyl Greea (GriBunG, Zedct.
wiss. Mik., xiii, 4, 1896, p. 460).

305. Congo Rad (Congoroth) (see Griuspacu, in Zeit. wiss.
Mik., iii, 1866, p. 379).—An “acid” colour. Its solution
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 loc.
cit.). A stain much of the same nature as Siurefuchsin.
It is useful for staining some objects during life (see ante, §
208). Carwoy (La Cellule, xii, 1897, p. 216) has had very
good results with it after hematoxylin of Dutariutp. He
used 0°5 per cent. solution in water. Note that this colour
is not to be confounded with other Congos, as Congo yellow,
or brilliant Congo. It is one of the azo dyes.

306. Congo-Corinth.—Also an acid dye. HEIDENHAIN (Zezé. wiss.
Mik., xx, 1903, p. 179) recommends Congo-Corinth G (or the allied
colour Benzopurpurin 6 B) (Elberfelder Farbwerke). Sections must be
made alkaline before staining, by treating them with very weak sal
ammoniac or caustic soda, in alcohol. After staining, pass through

absolute alcohol into xylol. Used after alum hemotoxylin, the stain of
which it does not cause to fade.

307. Benzopurpurin.—According to GRiIEsBACH (loc. cit., § 305),
another ‘“‘acid” colour very similar in its results to Congo red. See
also ZSCHOKKE (cbid., v, 1888, p. 456), who recommends Benzopurpurin
B, and says that weak aqueous solutions should be used for staining,
which is effected in a few minutes, and alcohol for washing out.
Deltapurpurin may be used in the same way.

See last § as to the necessity of alkalising the sections, which Heiden-
hain states is necessary with all dyes of this group.

308. Blauschwarz B and Brillantschwarz 3 B (HuIDENHAIN, op.

cit., § 306, p. 183).—Acid azo dyes. To be used in 1 per cent. solution
with sections of sublimate material, staining for five to ten minutes.
Then stain is a basic dye, such as toluidin blue or safranin.

309. Neutral Red (Neutralroth) (Huruicu, Allg. med. Zeit.,
1894, pp. 2,20; Zeit. wiss. Mik., xi, 1894, p. 250; GaLzort,
ibid., p. 193).—A “basic” dye. The term“ neutral” refers
to the hue of its solution. Its neutral red tint is turned
bright red by acids, yellow by alkalies. The stain in tissues
is in general metachromatic, nuclei being red, cell-bodies
yellow (cf. Rosin, in Deutsche med. Wochenschr., xxiv, 1898,
p. 615; Zeit. wiss, Mik., xvi, 2, 1899, p. 238). Up to the

192 CHAPTER XV.

present this colour has chiefly been employed for intra-vitam
staining. ‘ladpoles kept for a day or two in a solution of 1:
10,000 or 100,000 absorb so considerable a quantity of the
colour that all their tissues appear of a dark red. ‘The stain
is limited to cytoplasmic granules (Harzicu), and to the
contents of mucus cells (Ganzorri).

According to Eurticu and Lazarus (Spec. Pathol. und
Therapie, herausgeg. von NovrunaGet, villi, 1, 1898, p. 1;
Zeit. f. wiss. Mik., xv, 3, 1899, p. 338) it may be used for
intra-vitam staining of tissues in the same way as inethylen
blue, by injection or immersion with contact of ar. It is
especially a granule stain, Similar results are recorded by
Arnoip (Anat. Anz., xvi, 1899, p. 568, and xxi, 1902, p. 418).
See also Hariica and Lazarus, Anemie, 1, 1898, p.85; Lose.
(Journ. de V Anat. et de la Physiol., 1898, pp. 197, 210, 217)
(intra-vitam staining of sponges) ; and ProwazexK (Zett. wiss.
Zool., \xii, 1897, p. 187) (intra-vitam staining of Protozoa).
I myself have had very good results with it as an intra-
vitam stain.

According to Gotovine (Zeit. wiss. Mih., xix, 1902, p. 176),
the stain may be fixed in the tissues by means of sublimate,
chromic acid, bichromates, picric acid, or platinum chloride,
followed by molybdate of ammonium.

It has also been found useful for staining, in hardened
material, the corpuscles of Nissi (g. v.) im nerve-cells. 5.
Mayer (Lotos, Prague, 1896, No. 2) states that it also stains
degenerating wyelin. The solutions that have been employed
for staining fixed material are strong aqueous ones, | per
cent, to concentrated.

310. The Eosins, found in commerce under the names of
Eosin, Saffrosin, Primerose Soluble, Phloxin, Bengal Rose, Ery-
throsin, Pyrosin B, Rose B, a Iau, etc., are all “acid”
phthalein colours. ‘They are not quite identical in their
properties. Most of them are soluble both in alcohol and
in water, but some only in alcohol (“Primerose «@ PAlcvol ”).

They are all diffuse stains, formerly much used as con-
trast stains, less so now. Hansen (Anat. Hefte, xxvii, 1905,
p. 620) adds 1 drop of acetic acid of 2 per cent. to 9 c.c. of
1 per cent. eosin, which makes the stain more selective.

For Bengal Rose see Grinspacu, Zool. Anz., 1883, p. 172.

PLASMA STAINS WITH COAL-TAR DYES. 193

Kosin is a specific stain for red blood-corpuscles, and also
for certain granules of leucocytes (see under “ Blood aa

The yolk of some ova takes the stain strongly, so that it
is useful in some embryological researches.

811. Euectice’s Indulin-Aurantia-Eosin, or Acidophilous
Mixture, or Mixture C, or Mixture for Eosinophilous Cells (from
the formula kindly sent me by Dr. Grisrer).—Indulin,
aurantia, and eosin, of each two parts ; glycerin, thirty
parts. This gives a very thick, syrupy solution. ‘'l'o use it,
cover-glass preparations may be floated on to it ; or sections
on slides may have a few-drops poured on to them, the slide
being laid flat till the stain has taken effect (twenty-four hours
for Flemming material). I find that with Flemming material
it gives a powerful and good stain, which is much more
resistant to alcohol than that of the Enruicu-Bionpi mixture,
and is, therefore, much more adapted to ordinary work.
The stain keeps well.

Israet (Prakttk. Path. Hist., Berlin, 1893, p. 68) gives a more
complicated receipt.

312. Methyl Green and Eosin (CAaLBERLA, Morph. Jahrb., iii, 1877,
Heft 3, p. 625; List, Zect. wiss. Mck., ii, 1885, p. 147; Banpranz, Ann.
Microgr., Paris, vii, 1895, p. 245; RHUMBLER, Zett. wiss. Zool., 1xi, 1895,
p. 38).—See carly editions.

313. Methylen Blue and Eosin (CHENzINSKY, quoted from Zezt.
wiss. Mck., xi, 2, 1894, p. 269).

Methylen blue, sol. sat. in water . ; ; . 40
Eosin, 0°5 per cent. in 70 per cent. alcohol . . 20
Distilled water, or glycerin . F . 40

This solution will only keep for about eight days.

PIANeEsE (cbid., xi, 1894, p. 345) adds a considerable proportion of
carbonate of lithia.

See also the mixture of BREMER (Arch. mik. Anat., xlv, 1895, p. 446).

I have tried CHENZINSKY’S mixture as a tissue stain, without good
results; but see Rosin, Berliner klin. Wochenschr., 1898, p. 251; Zezt.
wiss. Mik., xvi, 1899, p. 223, and xvii, 1900, p. 333.

See also Laurent (Centralb. allg. Path., xi, 1900, p. 86 ; Zeit. wiss. Mik.,
xvii, 1900, p. 201).

814. MaLuory’s Eosin and Methylen Blue (Journ. med. Research,
January, 1904).—Sections of ZENKER material (other sublimate material
not so good) are stained for half to three quarters of an hour at 56° C.
in 5 per cent. aqueous solution of eosin, rinsed and flooded with solution

13

194 CHAPTER XV.

of one part of methylen blue, and one of potassium carbonate in 100 of
water, diluted with about seven parts of water. After forty minutes
they are flooded (not washed) with water, and differentiated for about
5 minutes in alcohol of 95 per cent. Absolute alcohol, xylol, balsam.

315. Other Eosin and Methylen-blue Stains—For some very
important ones see under “ Blood.”

316. Light Green (Lichtgrin §. F.).—An “acid” colour,
soluble in water or alcohol, and a good plasma stain.

Benpa (Verh. physiol. Ges. Berlin, Dec. 18th, 1891, Nos.
4 u. 5) stains sections for twenty-four hours in anilin-water
safranin solution, then for about half a minute in a solution
of 0°5 grm. Lichtgriin or Saureviolett (Griibler) in 200 c.c.
of alcohol, dehydrates and mounts in balsam. ‘his process
gives a very elegant stain, but requires very thin sections,
and there is always risk of the safranin being washed out.
The Lichtgriin stain unfortunately does not keep at all well.

See also Prenanr, Arch. mik. Anat., vii, 1905, p. 430, and
Gutnyssg, C.R. Suc. Biol., 1x1, 1907, p. 1212.

317. Janus Green (MIcHAELIS, Arch. mik. Anat., lv, 1900, p. 565).—
Used in solution of 1: 30,000 for staining certain granules (pancreas,
salivary glands, etc.) in the fresh state.

318. Malachite Green (syn. Solid Green, Victoria Green, New
Green, Benzoyl Green, Fast Green).—A basic colour, which has been
used as a plasma stain for the ova of Ascarts by VAN BENEDEN and
Neyrt. These authors used it for glycerin preparations; it can hardly
be got into balsam.

Fuemmine (Arch. mik. Anat. xix, 1881, p. 324) attributes to it a
special affinity for nucleoli.

319. Iodine Green (“ Hormann’s Grun”), see GRIESBACH (Zool.
Anz., No. 117, vol. v, 1882, p. 406).—Stain essentially that of methyl
green, but plasma often violet through the presence of a violet impurity
(Mayer, Mitth. Zool. Stat. Neapel, xii, 1896, p. 311; see also earlier
editions). It is now only used by botanists.

320. Thiophen Green (Thiophengrtin), see Krauss, Intern. Mon-
atsschr, Anat., eéc., iv, 1887, Heft 2.

821. Coerulein S., a green “acid” dye, is recommended for the
staining of muscle-fibrils by M. v. LenHosstxK (Anat. Anz., xvi, 1899, p.
339).—See also HEIDENHAIN, ¢bid., xx, 1901, p. 37, and Rawrrz, ibid.,
xxi, 1902, p. 554.

I'LASMA STAINS WITH GOAL-TAR DYES. 195

822. Quinolein Blue (Cyanin, Chinolinblau; v. RANvigER, Trazté,
p. 102).—Quinolein is said by Ranvier to have the property of staining
fatty matters an intense blue.

It is useful for staining Infusoria, which in dilute solution it stains
during life. See the methods of CERTES.

From the reactions mentioned by Ranvier it would seem that his “bleu
de quinoléine” is not the preparation that usually goes under that name.
See Enruicn, in Arch. mik. Anat., xiii, 1877, p. 266.

323. Indulin and WNigrosin.—Indulin, WNigrosin, Indigen,
Coupier’s Blue, Fast Blue R, Fast Blue B, Blackley Blue, Guernsey
Blue, Indigo substitute are the names of brands of a group of dyes,
mostly “acid,” related to the base violanilin. According to BEHRENS
the name Indulin is generally given to a bluish brand, and that of
Nigrosin to a blacker one.

Nigrosin, used with sublimate material, I find stains both nuclei and
cytoplasm, the chromatin strongly. It will not give the stain at all
with chrom-osmium material.

According to CALBERLA (Morph. Jahrb., iii, 1877, p. 627) the concen-
trated aqueous solution of Indulin should be diluted with six volumes
of water. Sections will stain in the dilute solution in five to twenty
minutes. He also says that it never stains nuclei; the remaining cell-
contents and intercellular substance are stained blue. This seems to
me to be, roughly, correct.

324. Safranin and Nigrosin (or Indigo-Carmine) (KossInsx1,
Zeit. wiss. Mik., vi, 1880, p. 61).—See early editions.

325. Picro-Nigrosin, Pritzur (Deutsch. Botan. Gesellsch., 1883,
p: 44) dissolves nigrosin in a saturated solution of picric acid
in water, and uses it for fixing and staining at the same
time, on the slide. See also under “ Connective Tissues.”

$26. Anilin Blue.—Under this title are comprised various
“basic” derivatives of the base rosanilin. They occur under
the names Spirit Soluble Blue (Bleu Alcool), Gentian Flue 6 B,
Spirit Blue 0, Opal Blue, Bleu de Nuit, Blue Lumiere, Parma
Bleu, Blue de Lyon. Some authors give the name Bleu de
Nuit and Griindstichblau as synonyms of Bleu de Lyon. The
Encyel. mik. Technik. says it is ‘ Anilinblau B— 6 B,” with
many synonyms, or designations of brands, Parma blue being
“Anilinblau R or 2 R.” I find it a fairly good stain, giving
very good differentiations of nerve-tissue and of cartilage
(as has already been pointed out by Baumearrgw and by
Jacopy). Maurice and Scuurein stain in bulk with it
after borax-carmine, using a very dilute alcoholic solution,

196 CHAPTER XV.

Baumecarren and Jacosy stain sections in a 02 per cent.
alcoholic solution.

Tonxorr (Arch. mik. Anat., lvi, 1900, p. 894) adds a little
tincture of iodine to the solution of the dye, or mordants
the sections with iodine.

Srropansky (Intern. Monatsschr. Anat., xxi, 1904, p. 20)
uses it in water with picric acid.

327. Carmine Blue (Bleu Carmin Aqueux, from Meister, Lucius,
and Brunig, at Hochst-a-M.).—Janssens (La Cellule, ix, 1893, p. 9)
states that this colour possesses a special affinity for the parts of cyto-
plasm that are undergoing cuticular differentiation. He uses it in
alcoholic solution acidified.

328. Methyl Blue—Under this title are comprised some
other derivatives of the base rosanilin. They are “acid”
colours. Here belong Methyl Blue, Cotton Blue, Water Blue
(Wasserblau), Methyl Water-Blue, China Blue (Chinablau),
Soluble Blue.

Amongst these Water Blue (Wasserblau) possesses some
useful properties. According to MirrorHanow (quoted from
Zeit. wess, Mik., v, 18&8, p. 518),-used in concentrated
aqueous solution it gives a very good double stain with
safranin. It is very resistant to alcohol. Using the Wasser-
blau first, and then the safranin, I have had some interesting
results. The Wasserblau must be used first. With chrom-
osmium material, twelve to twenty-four hours in the blue,
and four or five in the safranin, may not be too much. My
stains have not kept well.

Mann (Methods, etc., p. 216) uses a mixture of 85 parts
1 per cent. solution of eosin, 45 of methyl blue, and 100 of
water. He has also (Zeit. wiss. Mik., xi, 1894, p. 490) used

a similar mixture for nerve-cells.

329. Anilin Blue-black.—A preparation cited under this name has
been recommended by Bevan Lewis and others for nervous tissue.
The dye used by them cannot now he identified. Dr. GRUBLER writes
me that the anilin blue-black of his list is the oxyazo colour blue-
black B or AzoscHwaRz; but that dye had not been discovered when
Bevan Lewis wrote. See also HEIDENHAIN in Zezt. wiss. Mik., xx,
1903, p. 185, and xxv, 1909, p. 407.

330. Violet B (or Methyl Violet B) (S. Maver, Sitzb. k. k.
Akad, wiss. Wien, ii Abth, February, 1882)—Used in
solutions of 1 grim, of the colour to 800 grms, of 0°5 per cent,

PLASMA STAINS WILE COAL-'TAR DYES. 197

salt solution, and with fresh tissues that have not been
treated with any reagent whatever, this colour gives a stain
so selective of the elements of the vascular system that
favourable objects, such as serous membranes, appear as if
injected. The preparations do not keep well; acetate of
potash is the least unsatisfactory medium for mounting them
in, or a mixture of equal parts of glycerine and saturated
solution of picrate of ammonia (Anat. Anz, 1892, p. 221).
See also under “ Plasmafibrils.”’

The allied dye, Crystal Violet, has been employed for stain-
ing sections, e.g. by Knomaver and others. Bunpa (Neurol.
Centralb., xix, 1900, p. 792) stains in a mixture of 1 vol.
saturated sol. of the dye in 70 per cent. alcohol, 1 vol. 1 per
cent. sol. of hydrochloric acid in 70 per cent. alcohol, and
2 vols. of anilin water, the liquid being warmed until vapour
is given off, then cooled and the sections dried with blotting-
paper, treated one minute with 30 per cent. acetic acid,
dehydrated with alcohol and cleared with xylol.

331. Kresyl Violet.—An oxyazin dye, giving metachromatic stains.
HERXHEIMER (Arch. mik. Anat., lili, 1899, p. 519, and liv, p. 289) stains
sections of skin with Kresyl-echtviolett. Nuclei blue, plasma reddish.
Similarly Ficx (Centralb. allg. Path., xiii, 1902, p. 987; Zeit. wiss. Mik..
xx, 1903, p. 223), staining for three or four minutes in a concentrated
aqueous solution, and differentiating in alcohol until the connective
tissue has become colourless. Keratohyalin violet-red to salmon-
coloured.

832. Sdureviolett, see § 316.

333. Benzoazurin may be made to give either a diffuse or a nuclear
stain, according to MaRTIN (see Zezt. wiss. Mik., vi, 1889, p. 193).

834, Rawitz’ “Inversion” Plasma Stains.—It has been discovered
by Rawirz that by means of appropriate mordants certain basic anilins,
which by the usual methods of regressive staining are pure chromatin
stains, may be made to afford a pure plasma stain, thus giving an “in-
version” of the usual stain. The stain, in my opinion, is a vile one.
For details see fourth edition, or Rawirz (Sitzb. Gesnaturf. Freunde,
Berlin, 1894, p. 174; Zeit. wiss. Mzk., xi, 1895, p. 503; and his Ledtfaden
f. hist. Untersuchungen, Jena, 1895, p. 76).

335. Artificial Alizarin (Rawitz, Anat. Anz., xi, 10, 1895, p. 295).—
A double stain by means of artificial Alizarin, or Alizarin-cyanin,
requiring the use of special mordants supplied by the colour manu-
facturers, and very complicated. See fifth edition.

198 CHAPTER Xv.

Rawitz (Zeit. wiss. Mit., 1909, pp. 393 and 395) also recommends
a solution of 1 grm. of Saure-Alizarinblau BB (or Sauregrun G)
(both from Héchst), 10 grms. ammonia alum, 100 c.c. glycerin, and
100 c.c. water.

Sztrz (¢bid., xxix, 1912, p. 289) fixes in a mixture of 15 c.c. 1 per
cent. platinum chloride, 15 ¢.c. formol, and 30 c.c. saturated solution of
sublimate, makes paraffin sections, and stains them with Heidenhain’s
iron-hematoxylin. They are then treated for five to six hours with
5 per cent. solution of aluminium acetate, rinsed, and stained for five
to six hours with Benda’s sulphalizarinate of soda (given under ‘ Mito-
chondria”), and got into balsam. A red plasma stain, affecting plasma-
fibrils. For intra vitam stains with alizarin see § 208 (FIscHEL), and
Nixsson, Zool. Anz., xxxv, 1909, p. 196.

336. For Bewpa’s Alizarin Stains, sce under ‘‘Centrosomes,”’

“ Mitochondria,’ and “ Neuroglia.”

CHAPTER XVI.
METHYLEN BLUE.

337. Methylen Blue is a “ basic ” dye, being the chloride or
the zine chloride double salt of tetramethylthionin. It
appears that some persons have confounded it with the
“acid” dye methyl blue, to which it has not, histologically,
any resemblance.

Commercial methylen blue sometimes contains as an im-
purity a small quantity of a reddish dye, which used to be
taken to be methylen red. This impurity is present from
the beginnning in many brands of methylen blue, is
frequently developed in solutions of the dye that have been
long kept (so-called “ripened” solutions), and is still more
frequently found in kept alkaline solutions. According to
Nocut (Centralb. Bakteriol. xxv, 1899, pp. 764-769 ; Zeit.
wiss. Mik., xvi, 1899, p. 225) it is not methylen red, nor
methylen violet either, but a new colour, for which Nocur
proposes the name “ Roth aus Methylenblau.”

According to Micuzuts (Centralb. Bakteriol., xxix, 1901,
p. 768, and xxx, 1901, p. 626; Zeit. wiss. Mik., xviii, 1902,
p. 305, and xix, 1902, p. 68) confirmed later by Nocut, Revver,
and Gizmsa, this dye is Methylenazur, an oxidation-product
of methylen blue, already described by Burnruszn in 1885.
It is an energetic dye, of markedly metachromatic action,
and to it are due the metachromatic effects of methylen blue
solutions (methylen blue itself is not metachromatic).

The presence of this dye as an impurity in methylen blue
is not always an undesirable factor; on the contrary, it
sometimes affords differentiations of elements of tissues or of
cells that cannot be produced by any other means. Methylen
blue that contains it is known as polychrome methylen blue,
and is employed for staining certain cell-granules. Unna
(Zeit. wiss. Mik., viii, 1892, p. 483) makes this as follows;

200 CHAPTER XVI.

A solution of one part of methylen blue and one of carbonate
of potash in twenty of alcohol and a hundred of water is
evaporated down to a hundred parts. (It may be used at
once, or after diluting with an equal volume of anilin water,
for sections, which after staining may be differentiated with
elycol, creosol, or Unna’s glycerin-ether mixture—all of
which, as well as the polychrome methylen blue, can be
obtained from Griibler & Hollborn.) Micnants (op. cit.)
makes it as follows: 2 gr. of medicinal methylen blue are
dissolved in 200 c.c. of water, and 10 c.c. of 74; normal solu-
tion of caustic soda added. Boil for a quarter of an hour; after
cooling add 10 ¢e.c. of 7; normal sulphuric acid, and filter.

Methylenazur is isolated from methylen blue by the prolonged action
of an alkali or of silver oxide. It seems also that it is formed in certain
mixtures of methylen blue with eosin (RomANoWSKY, LAVERAN, GIEMSA
and others), by means of the eosin, which in these mixtures acts chemi-
cally, and can be replaced by resorcin, hydroquinon,and the like. It is best
procured from Griibler & Hollborn, who supply it pure as ~ Azur I,”
and mixed with an equal quantity of methylen blue as “ Azur II.” See
further as to this dye under “Stains for Blood.”

There are several sorts of methylen blue sold, the most
important being—“ methylen blue, according to Euruicy ” ;
“methylen blue, according to Kocu”; “methylen blue BX,
according to 8. Mayer”; “ Methylenblau, medic. pur.”

The colour to be employed for intra-vitam nerve staining
should be as pure as possible. ApAtuy (Zeit. wiss. AMik., ix,
1893, p. 466) writes that the best—in fact, the only one that
will give eaactly the results described by him—is that of KH.
Mercx, of Darmstadt, described as “ medicinischis Methylen-
blau.” Dogien (Hneycl. mik. Technik., 1st edition, p. 811)
has had his best results with ‘‘ Methylenblau n. Ehrlich,” or
“BX,” obtained from Gritbler & Hollborn.

338. The Uses of Methylen Blue.—As a histological reagent
it is used for sections of hardened central nervous tissue, in
which it gives a specific stain of medullated nerves. It
gives more or less specific stains of the basophilous granula-
tions of ‘* Mastzellen” and plasma-cells, and the granules of
Nisst in nerve-cells, also mucin. It is much used—in the
form of mixtures affording methylen azur—in the study of

MEVHYLEN BLUE. 201

blood, blood parasites, and similar objects. For all of these
see the respective sections in Part I]. Further, it stains a
large number of tissues infra vitam, with little or no inter-
ference 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, and are obtained with greater ease and
certainty ; with this difference, however, that gold stains a
larger number of the nervous elements that are present ina
preparation, sometimes the totality of them; whilst methylen
blue stains only a selection of them, so bringing them more
prominently before the eye, and allowing them to be traced
for greater distances. These two uses form the subject of
this chapter.

339. 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 it a very light tint. After a time they will be found
to be partially stained—that is, it will be found that certain
tissues have taken up the colour, others remaining colourless.
If now you put back the animals into the tinted water and
wait, you will find after a further lapse of time that further
groups of tissues have become stained. Thus it was found
by Euruica (Biol. Centralb., vi, 1886, p. 214; Alh. k. Akad.
Wass. Berlin, February 25th, 1885) 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 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, and then
begin to discharge the colour even more quickly than they
took it up. According to HEuriicu this decoloration is
explained as follows: methylen blue, on contact with reduc-
ing agents in alkaline solution, can be reduced to a colourless

202 CHAPTER XVI.

body, its “ leucobase.” Now living or recently dead tissue
elements are, or may be, both alkaline and very greedy of
oxygen, and thus act on the dye as reducing agents. The
leucobase thus formed is easily reoxidised into methylen blue
by oxidising substances, or acids, or even by the mere
contact of air—which latter property is taken advantage of
in practice.

It follows that a total stain of all the tissues of a living
intact organism can hardly be obtained under these con-
ditions, 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 studied during life at the period at which it alone
has attained the desired intensity of coloration. If it be
one that stains later than the others, it may be studied at
the period at which the earlier stained elements have already
passed their point of maximum coloration and have become
sufficiently decoloured, the later stained ones being at a
point of desired intensity. 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 § 349.

.The proper strength of the very dilute solutions to be
employed for the staining of living organisms must be made
out by experiment for each object. JI think the tint is
practically a sufficient guide, but it may be stated that when
in doubt a strength of J : 100,000 may be taken, and in-
creased or diminished as occasion may seem to require.
Zosa (Rendic R. Ist. Lombardo, xxv, 1892; Zeit. wiss. Mik.,
ix, 1892, p. 208) finds that for Hydra the right strength is
from 1 : 20,000 to 1 : 10,000.

The stain is capricious. It is not possible to predict
without trial which tissues will stain first in any organism.
The stain penetrates very badly, which is no doubt one cause
of its capriciousness. 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

METHYLEN BLUE. 203

remain unstained in the intact oryanism. To get these
stained, it is necessary to isolate them sufficiently, as explained
in the following sections.

340. Staining Nervous Tissue during Life——It was made out
by Euruicu (op. cit., last §) that by injecting a solution of
methylen blue into the vessels or tissues of living animals
and shortly afterwards cutting out and examining small
pieces of their tissues, these will be found to be intensely
stained in some of their elements (chiefly nervous). If the
tissues are mounted under a cover-glass, the stain will fade
in a short time; but if the cover-glass be removed, so that
oxygen can have access to the tissues, the stain will be
restored, as explained last §. The chief elements stained
in this way are peripheral nerves, and amongst these more
especially axis-cylinders of sensory nerves.

Ehrlich held that the stain so obtained is a product of a
vital reaction of the tissues, and that it cannot be obtained
with dead material. Doaiet, however (Arch. mtk. Anut.,
xxxv, 1890, pp. 805 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 the nerves
were still living at that time. But it seems more natural to
conclude with ApArHy (Zeit. wiss. Mik., ix, 1892, pp. 15
et scq.) that nerve-tissue can be stained after life has ceased.
ApArny 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 extracted
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. Muicuattow (ibid., xxvii,
1910, p. 7) prefers tissues that have Jain from one and a half
to two hours after the death of the subject in Ringev’s salt
solution.

As above explained, the primary stain obtained by injecting
methylen blue, or immersing tissues in it, only lasts a
very short time. In order to get it to last long enough for

204: CHAPTER XVI.

study, it must be re-blued by oxidation (see last §). It is
therefore the usual practice to dissect out the tissues to be
examined, and leave them for some time exposed to the air.
This is done in order that they may take up from the air
the necessary ovygen. Another consideration that justifies
the practice is that by exposure to air the preparations take
up a trace of ammonia, and ApAtuy hag experimentally
established that this is an important factor in the sharpness
of the stain. Khrlich also (op. cit.) holds that an allaline
reaction of the tissues is a necessary condition to the stain.
Apathy further holds that the stain is a regressive one, easily
washed ont by the surrounding liquid; and in order to
prevent this washing-out being excessively rapid, it is
desirable to have it go on in presence of as little liquid as
possible.

341, The Modes of Staining.—The practice of the earlier
workers at this subject was (following Earutce) to imyect
methylen blue into the vascular system or body-cavity of a
living animal, wait a sufficient time, then remove the organ
for further preparation and study. And there appears to
have been a belief with some workers that it was essential
that the stain should have been brought about by injection
of the colouring matter into the entire animal. It is now
known that the reaction can often be equally well obtained
by removing an organ and subjecting it to a bath of the
colouring matter in the usual way. But in some cases it
seems that injection is preferable, if not necessary.

342. The Solutions employed.—The solutions used for aujec-
tion are generally made in salt solution (physiological, or a
little weaker) ; those for staining by immersion, either in
salt solution or other “indifferent”? liquid, or in pure water.
The earlier workers generally took concentrated solutions.
Thus Arnstein (Anat. Anz., 1887, p. 125) injected 1 c.c. of
saturated (7. e. about 4 per cent.) solution into the vena cutanea
magna of frogs, and removed the organ to be investigated
after the lapse of an hour. Bisprrmann (Sitzb. Akad. Wass.
Wien, Math. Nat. Cl, 1888, p. 8) injected 0°5 to 1 c.c. of a
nearly saturated solution in 0°6 per cent. salt solution into
the thorax of crayfishes, and left the animals for from two

METHYLEN BLUR. 205

to four hours before killing them. 8S. Maver (Zeit. wise.
Mik., vi, 1889, p. 423) took a strength of 1 : 800 or 400 of
0°5 per cent. salt solution. The solutions of Rerzius are
of the same strength. But the tendency of more recent
practice is decidedly towards the employment of weaker
solutions. ApArTHy (cbid., ix, 1892, pp. 25, 26 e¢ seq.) finds
that it is not only superfluous, but positively disadvantageous,
to take solutions stronger than 1: 1000. Doaten (Hncycl.
Mik. Technik., 1st ed., p. 815) recommends 4 to } per cent.,
or at most $ percent. For warm-blooded animals the solution
should be warmed to 36° or 37° C., and before sending in
the injection the blood-vessels should be well washed out
with similarly warmed salt solution. The injected organs
may be removed after 20 to 30 minutes. They should be
placed on a thin layer of spun glass moistened with weak
(g to #5 per cent.) methylen blue, or simply spread out on a
slide, and the whole placed in a Petri dish with a layer of
the methylen blue on the becttom. The dish is best placed
in a stove at 86°C., and after 15 to 30 minutes (if the pieces
are thin) or 1 hour to 14 hours Gf they are thick) specimens
may be removed for examination or preservation ; or, without
using the stove, specimens may be removed 10 to 20 minutes
after injection, placed on a slide, and moistened with weak
methylen blue or salt solution, and brought under the
microscope. Then as soon as the stain is sufficiently brought
out (40 to 60 minutes) they may be fixed (§ 348).

For staining by immersion the solutions should, if anything,
be still weaker. Doan. (Arch. mik. Anat., xxxv, 1890, p.
305) places objects in a few drops of aqueous or vitreous
humour, to which are added two or three drops of a ;; to +!y
per cent. solution of methylen blue in physiological (0°75 per
cent.) salt solution, and exposes them therein to the air. In
thin pieces of tissues 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 necessary. ‘he reaction is quickened
by putting the preparations into a stove kept at 30° to 35°C.
Rover (Compt. Rend., 1893, p. 802) employed a 0°05 per
cent. solution in 0°6 per cent. solution (for muscles of Batra-
chia). ALLEN (Quart. Journ. Mier. Sct., 1894, pp. 461, 483)
takes for embryos of the lobster a solution of 0:1 per cent. in

206 CHAPTER XVI.

0°75 per cent. salt solution, and dilutes it with 15 to 20
volumes of sea-water. Sumunmann (Zeit. wiss. Mik., xvii,
1900, p. 239) takes for the choroid a solution of 0-02 per cent.
in 0°56 per cent. salt solution. Lavpowsxy (<bid., xii, 1895,
p. 177) takes 7 to } per cent. in white of egg, or serum.
Similarly Youne (abid., xv, 1898, p. 253). Micuartow (cbid.,
xxvii, 1910, p. 10) takes 4 to sz per cent. in Ringer’s salt
solution (for nerves of Mammals).

Apatuy (Zeit. wiss. Mik., ix, 1892, p. 15; see also his
Mikrotechnik, p. 172) proceeds as follows for Hirudinea and
other invertebrates. A portion of the ventral cord is ex-
posed, or dissected ont. If 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 : 10U0 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 (Lwm-
bricus 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 preparations from the 1: 1000 solution are then
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 in § 343. This is done by pouring the liquid over
them, and leaving them in it withowé moving them about in it
for at least an hour, and by preference in the dark. The
further treatment is as described in $ 343,

The object of the ammonia in these liquids is to differ-
entiate the stain—to produce an artificial “secondary diffe-
rentiation.” It acts by washing out the absorbed colour
from certain elements, others resisting longer.

See also, for Hirudinea, Sancusgz, in Trab. Lab. Invest.
Biol. Univ. Madrid, vii, 1909, fase. 1-4, or Zeit. wiss. Mik.,
xxvii, 1910, p. 393 (injection of solutions of 0:2, O-1, or 0°05
per cent., with further treatment as Apathy or Bethe).

METHYLEN BLUR. 207

343. Fixation of the Stain——The stain obtained by any of
these methods may be fixed, and more or less permanent
preparations be made by one or other of the following
methods :

ArystzIn (Anat, Anz., 1887, p. 551) puts the tissue for
half an hour into saturated aqueous solution of picrate of
ammonia.

S. Maver (Zeit. wiss. Mik., vi, 1889, p. 422) preferred a
mixture of equal parts of glycerine and saturated picrate of
ammonia solution, which served to fix the colour and mount the
preparations in, This was also in principle the method of
Rerzius (Intern. Monatschr. Anat, Phys., vii, 1890, p. 328).

Doeten (Hneycl. mik. Techn., ii, p. 105) puts for 2 to 24
hours into saturated aqueous picrate of ammonia, and then
into equal parts of glycerin and the picrate solution. (Thin
membranes, and the like, may be fixed with 1 or 2 per
cent. of 2 per cent. osmic acid solution added to the picrate
solution and stained with picro-carmine before putting into
the glycerin mixture.)

Other workers have employed saturated solution of
iodine in iodide of potassium (so ARNSTEIN) or picro-carmine
(so Fast, Arch. Anat. Hntwickel., 1890, p. 116; cf. Zeit.
wiss, Mtk., vii, 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 Lavpowsky, but this after
careful study is rejected by Doers.

ApArHy (op. cit., § 842) brings preparations either into a
concentrated aqueous solution of picrate of ammonia free
from picric acid, and containing five drops of concentrated
ammonia for every 100c.c.; or, which is generally prefer-
able, 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. 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

208 CHAPTER XVI.

prepared gum-arabic solution. When thoroughly penetrated
with this they are removed and mounted in the following
gum-syrup medium (loc. cit., p. 37) :

Picked gum-arabic . , . 50 grms.
Cane-sugar (not candied) . . 50. ,,
Distilled water . : 2 . oO,

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. Tarrants’
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.) Preparations that have
been fully differentiated (§ 342) do not keep more than
a few weeks; whilst those in which the differentiation has
not been carried to the point of thorough tinctorial isolation
of the neuro-fibrils have kept for five or six years (APATHY,
Mitth. Zool. Stat. Neapel, xii, 1897, p. 712).

PrescuKo (Anat. Anz., xii, 1897, p. 16) fixes with picrate,
and then puts into 10 per cent. formol for a few days.

The methods described next § are also available for
material not destined to be sectioned.

344. Methods for Sections——The preceding methods do not
give preparations that will resist the operations necessary
for imbedding in paraffin or mounting in balsam. A strong
solution of platimum chloride is said to do this (see Fuisv,
Arch. Anat. Entw., 1890, p. 116), but the preparations are
not very satisfactory.

For the earlier method of Parxgr (Zool. Anzeig., 1892, p.
375) with methylal see early editions. Later (Mitth. Zcol.
Stat. Neapel, xii, 1895, p. 4) he fixes the stain by dehy-
drating the objects in successive alcohols of 30, 50, 70, 95,
and 100 per cent. strength, each containing 8 per cent. of
corrosive sublimate, then brings them into a mixture of the
last with an equal volume of xylol, and lastly into pure
xylol.

For the earlier method of Brrug (Arch. mik. Anat., xliv,
1894, p. 585), see last edition.

Berun’s later method (Anat. Anz., xii, 1896, p. 488) is as
follows: After staining, pieces of tissue of 2 to 3 mm.

METHYLEN BLUE. 209

thickness are treated for ten to fifteen minutes with a con-
centrated aqueous solution of picrate of ammonia and then
brought into a solution of 1 grm. of molybdate of ammonium,
either in 20 of water, or in 10 of water and 10 of 0°5 per
cent. osmic acid or 2 per cent. chromic acid ; or into a
solution of phosphomolybdate of sodium in the same propor-
tions, each of these solutions having added to it 1 drop of
hydrochloric acid, and if desired 1 grm. of peroxide of
hydrogen. They remain in one of these solutions for three
quarters to one hour (or from four hours to twelve in the
osmic acid one), and are then passed through water, alcohol,
xylol, balsam, or paraffin. (The objects that have been
treated with one of the solutions of the sodium salt are not
thoroughly resistant to alcohol, so that for them it is well’
to cool the alcohol to under 15°C.) Sections may be after-
stained with alum carmine, or ‘‘ neutral ” tar colours.

Slight modifications of this method are given by Doarts1
(Arch. mik, Anat., xlix, 1897, p. 772; liii, 1898, p. 237;
Zeit, wiss. Zool., xvi, 1899, p. 861; and Hncycl. mik, Technik,
1903, p. 825, and 1910, p. 108). He omits the peroxide,
the hydrochloric acid, and the cooling. Bethe (Zeit. wiss.
Mik., xvii, 1900, p. 21) does not approve of these modifications,

Further modifications of the molybdenum method have
been published by Leonrowrrsca (Intern. Monatsschr. Anat.,
xviii, 1901, p. 142).

Micuaitow (Zeit. wiss. Mik., xxvii, 1910, p. 19) adds to
8 per cent. solution of molybdate 0°5 per cent. of formalin,
leaves the objects in a large quantity of it (filtered) for 24
hours at 37° C., washes with warm water, and passes through
alcohol and xylol into xylol-damar (not balsam).

See also Scumipt (Arch. Ges. Phys., citi, 1906, p. 522).

Harris (Philadelphia Medical Journ., May 14th, 1898),
after staining, rinses with water, and brings into a saturated
solution of either ferrocyanide or ferricyanide of potassium
which has been cooled to within a few degrees of zero
(a trace of osmic acid may be added to prevent macera-
tion). They remain therein for three to twenty-four
hours, and are then washed in distilled water for an hour,
and are dehydrated in absolute alcohol kept at a low
temperature, cleared in xylol or cedar oil, and imbedded in
paraffin.

14

210 CHAPTER XVI.

345, Impregaation of Epithelia, Lymph-spaces, ete. (Doaizt,
Arch, mik, Anat., xxxiii, 1889, p. 440 et seq.).—Suitable pieces
of tissue (thin membrane by preference) are brought fresh
into a 4 per cent. solution of methylen blue in physiological
salt solution (in the Hnceycl. mik. Technik, 1903, p. 827,
Dogicl gives the strength of the methylen blue as } to 1 per
cent.). 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 endo-
thelium 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-substance 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.

If it be desired to preserve the preparations permanently,
they had better be mounted in glycerin saturated with picrate
of ammonia, or (Hneycl., 1910, 11, p. 110) fixed with ammonium
molybdate and a trace of osmium.

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

S. Mayer (Zevt. wiss. Mik., vi, 1889, p. 422) stains tissues
for about ten minutes in a 1: 800 or 400 solution of methylen
blue in 0°5 per cent. salt solution, rinses in salt solution, and
puts up in the glycerin-picrate of ammonia mixture given
§ 343. The images are generally positive after injection of
the colour into the vascular system; negative after immersion
of the tissues.

Timoresew (Anat. Anz., xxxv, 1909, p. 296) impregnates
for 15 to 20 minutes in a solution of 1: 3000 or 4000 strength,
fixes with a very weak solution of ammoniuni picrate in salt
solution, and puts up in a mixture of 50 c.c. glycerin, 50 c.c.
water, and 35 c.c. saturated solution of the picrate: or fixes
with ammonium molybdate of 8 per cent. and mounts in
balsam.

346. Toluidin Blue or Thionin as succedanea of methylen
blue.—Harnis (Philadephia Med. Journ., May 14th, 1898)
has found that there is no reaction of methylen blue that

METHYLEN BLUE. QI

cannot be equally well obtained with toluidin blue or thionin.
For staining pieces of tissue he takes :
Toluidin blue, 0°1 per cent. sol. in phy-

siological salt solution . : . 2 parts
Ammonium chloride 0:25 per cent. in

water . F i : ; . 1 part
Egg albumen. Ty

For injections he uses 1 put af the aye to 1000 of physio-
logical salt solution.

Any of the methylen blue fixing methods may be employed
and the whole technique is the same.

L. Marrinorr1 (Zeit. wiss. Mik., xxvii, 1910, p. 24)
recommends a polychrome toluidin blue, made by adding 0°5
per cent. of lithium carbonate to a 1 per cent. solution of the
dye and keeping till a purple-red tone appears. Or, a stock
solution made of 1 germ. toluidin blue, 0°56 grm. lithium
carbonate, glycerin 20 grms., alcohol 5 grms., and water 75.

CHAPTER XVII.
METALLIC STAINS (IMPREGNATION METHODS).

347. The Characters of Impregnation Stains.— By impregna-
tion is understood a mode of coloration in which a colouring
matter is deposited in tissues in the form of a precipitute—
the impregnated elements becoming in consequence opaque.
By staining, on the other hand, is understood a mode of
coloration in which the colouring matter is retained by the
tissues as if in a state of solution, showing no visible solid
particles under the microscope, the stained elements remaining
in consequence transparent. But it is not right 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 the preceding chapters
give, besides a stain, a true impregnation. Methylen blue,
for instance, will give in one and the same preparation an
impregnation and a stain; and in most gold chloride prepara-
tions the coloration is in places of the nature of a finely
divided solid deposit, in others a perfectly transparent stain.

348. Negative and Positive Impregnations—In a negative
impregnation intercellular substances alone are coloured, the
cells themselves remaining colourless or very lightly tinted.
In a positive impregnation the cells are stained and the inter-
cellular spaces are unstained. (A directly contrary statement,
made in a recent Lehrbuch, is erroneous.)

Negative impregnation is generally held to be primary because brought
about by the direct reduction of a metal in the intercellular spaces; posi«
tive impregnation to be secondary (in the case of silver nitrate at least)
because it is brought about by the solution in the liquids of the tissues
of the metallic deposit formed by a primary impregnation. and the con-

METALLIC STAINS (IMPREGNATION METHODS). 213

sequent staining of the cells by the new solution of metallic salt thus
formed. These secondary impregnations takes place when the reduction
of the metal in the primary impregnation is not sufficiently energetic
(see on these points His, Schweizer Zeit. Hetlk., ii, Heft 1, p.1; GisrKe,
Zeit. wiss. Mik., i, p. 393; RANviER, Trudté, p. 107).

As to the nature of the black or brown deposit or stain formed in
the intercellular spaces in cases of primary impregnation see SCHWALBE,
Arch. mik. Anat., vi, 1870, p. 5; GIERKE’s Fiirberei 2u mikroskopischen.
Zwecken, in vols. i and ii of Zect. wiss. Mik.; Joseru, Sitzb. Akad. Wiss.
Berlin, 1888; Zeit. wiss. Mik., xi, 1, 1894, p. 42 et seg. It evidently
cannot consist of metallic silver, as it is soluble in hyposulphite of soda.
See also Macatium, Proc. Roy. Soc., lxxvi, 1905, p. 217, and ACHARD
and REYNAUD, C. R. Soc. Biol., 1xi, 1906, p. 43

349. 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 reduces them. It has been pointed ont in § 35 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 dust be absolutely denied access to them. I
have now good evidence to the effect that the same is the
case with other metallic solutions; and the point is raised
whether such solutions are not positively improved for im-
pregnation purposes by exposure to light! Dr. Linpsay
JOHNSON 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 unsatisfactory.”

ApAtuy (Milt. Zool. Stat Neapel, xii, 1897, p. 722) leaves
his gold solutions exposed to light, so long as there are no
tissues in them.

350. State cf the Tissues to be Impregnated.—The majority of
stains given by dyes are only obtained with tissues that have
been changed in their composition by the action of fixing and

214: CHAPTER XVII.

preservative reagents. With metallic impregnations the case
is different ; perfectly fresh tissues—that is, such as are either
living, or at all events have not been treated by any reagent
whatever—will also impregnate with the greatest ease and
precision, Indeed, some impregnations will not succeed at
all with tissues that are not fresh in the sense above
explained.

Silver.

351. Silver Nitrate: Generalities.—The principles of its
employment are given by Ranvizr (Traité, p. 105) as
follows :

Silver nitrate may be employed either in solution or in
the solid state. The latter method is useful for the study
of the cornea and of fibrous tissues, but is not suitable for
epithelia. For the cornea, for instance, proceed as follows :
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. It will be found that the nitrate has traversed the
epithelium and soaked into the fibrous tissue, on the surface
of which it is reduced by the light. The cells of the tissues
will be found unstained.

It is generally employed in solution, in the following
manner: In the case of a membrane, such as the epiploén,
the membrane must be stretched like a drum-head over a
porcelain dish,* and washed first with distilled water, and
then washed with a solution of silver nitrate. In order to
obtain a powerful stain it is necessary 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 begun to
turn of a blackish grey the membrane is removed, washed

* The Hoggans Histological Rings will be found much more con-
venient. They are vulcanite rings made in pairs, in which one ring just
fits into the other, so as to clip and stretch pieces of membrane hetween
them. They will be found descriked and figured in Journ. Roy. Mie,
Soc., ii, 1879, p. 857, and in Rogin’s Journ. de VAnat., 1879, p. 54.
They may be obtained of Burge & Warren, 42, Kirby Street, Hatten
Garden, London, E.C.

METALLIC STAINS (IMPREGNATION METHODS). 215

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.

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 impurities
in the specimen have been described as stomata of the tissue.

If the solution be taken too weak—for instance, | : 500 or
1 : 1000, or if the light be not brilliant—a general instead of
an interstittal stain will result; nuclei will be most stained,
then protoplasm, and the intercellular substance will contain
but very little silver. In general ina good “impregnation ”
the contents of the cells, and especially nuclei, are quite in-
visible.

The tissues should be constantly agitated in the silver-
bath in order to avoid the formation on their surfaces of
deposits of chlorides and albuminates of silver.

These impregnations only succeed with fresh tissues.

352. Silver Nitrate: the Solutions to be employed (Ranvier).
—The solutions generally employed by Ranvier vary in
strength from 1 : 300 to1: 500. Thus 1: 500 is used for the
epiploén, pulmonary endothelium, cartilage, tendon ; whilst
a strength of 1 . 5C0 is employed for the phrenic centre, and
the epithelium of the intestine. For the endothelium of
blood-vessels (by injection) solutions of 1: 500 to I : 800
are taken.

M. Douvat (Précis, p. 229) takes 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 Lymphgefisse, etc., Berlin, 1862, p. 5).

Roznsxr (Arch. de Physiol., 1869, p. 451) used solutions
varying between 0:1 and 0:2 per cent., which he allowed to
act for thirty seconds.

916 GHAPTER XVII.

Rouaer (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 Herrwias take, for marine animals, a 1 per cent.
solution (Jen. Zeit. Naturk., xvi, pp. 313 and 3824),

The Hocaans (Journ. of Anat. and Physiol., xv, 1881,
p. 477) take for lymphatics a 1 per cent. solution.

Tournrvx and Herrmann (Rostin’s Journal de V Anat., 1876,
p. 200) took for the epithelia of Invertebrates 38 : 1000, and
in some cases weaker solutions,—for one hour, washing out
with alcohol of 90 per cent.

Hover (Arch. mik. Anat., 1876, p. 619) takes a solution of
nitrate of silver, 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
ammonto-nitrate solution has the advantage of impregnating
absolutely nothing but endothelium or epithelium ; connective
tissue is not affected by it.

Ranvigr’s injection-mass for impregnating endothelium is
given under “ Injection.”

Dexsvysen (Anat. Anz, iv, 1889, No. 25, p. 789) has
applied to terrestrial animals the method of Harmer for
marine animals (§ 356). For details see previous editions.

Reeavp (Journ. Anat. et Phys., xxx, 1894, p. 719) recom-
mends for the study of lymphatics a process devised by
Rewnatvr, for the details of which see also previous editions.

3538. Other Salts of Silver—ALFEROW (Arch. Phys.,i, 1874, p. 694)
einploys the picrate, lactate, acetate, and citrate, in solution of 1. 800,
and adds a small quantity of the acid of the salt taken (10 to 15 drops of a
concentrated solution of the acid to 800 ¢.c. of the solution of the salt).
This decomposes the precipitates formed by the action of the silver salt on
the chlorides, carbonates, and other substances existing in the tissues.

ReEGAuUD and DuBREUIL (C.R. Ass. Anat., 5 Sess. 1903, p. 122) take a
fresh solution of protargol or a mixture of equal parts of 1 per cent.
protargol and 1 per cent. osmic acid, thus avoiding precipitates.

354, Silver Nitrate: Reduction—Reduction may be effected
in other media than distilled water.

v. Recxiincnausen washed his preparations in salt solution
before exposing them to the light in distilled water (Arch.

METALLIC STAINS (IMPREGNATION METHODS). 217

path, Anat., xix, p. 451). Physiological salt solution (0°75
per cent.) is commonly used for these washings.

Miter (Arch. f. path. Anat., xxxi, p. 110), after impreg-
nation 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 Grerkn,
in Zeit, wiss. Mik., i, 1884, p. 396).

Rovcet (Arch, de Physiol., 1873, p. 603) reduces in
glycerin; Sztrz (Zeit. wiss. Mik., xxix, 1912, p. 291) in
glycerin with =, of formol.

Sarrten (Arch. Mik. Anat, xxi, p. 672) exposes to the
light for a few minutes in water acidulated with acetic or
formic acid. ‘l'wannorrer (Das Mikroskop, 1880) employs a
2 per cent. solution of acetic acid.

Krauss brings his preparations, after washing, into a light
red solution of permanganate of potash. Reduction takes
place very quickly, even in the dark.

Orritz puts for two or three minutes into a 0:25 or 0°50
per cent. solution of chloride of tin.

Jaximovirce (Journ. de PAnat., xxiii, 1888, p. 142) 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, and exposes to the
light for five to seven days, the mixture being renewed
from time to time.

Dexuvrsen (op. cit., last $) reduces in oil of cloves, after

dehydration.

355. Fixation.—LeGros (Journ. de T Anat., 1868, p. 275) washes his
preparations, after reduction, in hyposulphite of soda, to prevent after-
blackening. According to Duvat (I’réczs, p. 230) they should he washed
for a few seconds only in 2 per cent. solution and then in distilled
water.

Gsrota (Arch. Anat. Phys., Phys. Abth., 1897, p. 428) reduces in a
hydroquinone developing solution, followed by fixation in hyposulphite
of soda, just as in photography.

356. Impregnation of Marine Animals—On account of the

218 CHAPTER XVII.

chlorides that bathe the tissues of marine animals, these
cannot be treated directly with nitrate of silver.

Herrwie (Jen. Zeit., xiv, 1880, p. 322) recommends fixing
them with a weak solution of osmic acid, then washing with
distilled water until the wash-water gives no more than an
insignificant 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, p. 445) washes
them for some time (half an hour) in a 5 per cent. solution
of nitrate of potash in distilled water ; they may then be
treated with silver nitrate in the usual way. For some
animals he recommends a 4°5 per cent. solution of sulphate
of soda,

357. Double-staining Silver-stained Tissues.—The nuclei of
tissues impregnated with silver may be stained with the
usual reagents, provided that solutions containing free am-
monia be avoided. ‘hese stains will only succeed, however,
with successful negative impregnations, as nuclei that have
been impregnated will not take the second stain.

Impregnation with silver may be followed by impregna-
tion 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 pro-
duced. See hereon Gurora, loc. cit., § 355.

358. Impregnation of Nerve Tissue. or this subject, which
includes the important bichromate-and-silver methcd of Goust,
and the neurofibrtl methods of Biztscuowsky and Ramon y
Cagaz, see Part ID. These give important results, not only
with Nervous tissue, but with various forms of Connective
tissue, mitochondrial formations, ete.

Gold.

359. The Characters of Gold Impregnations.—Gold chloride
differs from nitrate of silver in that it generally gives positive
(§ 348) impregnations only. It generally gives negative
images only with such tissues as have first received a
negative impregnation with silver, the gold substituting
itself for the silver. In order to obtain these images you

METALLIC STAINS (IMPREGNATION METHODS). 219

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
certain special studies on the cornea and on connective
tissue, the almost exclusive function of gold chloride is the
impregnation of nervous tissue, for which it exhibits a
remarkable selectivity.

360. Pre-impregnation and Post-impregnation.—Gold methods
may be divided into two groups: viz. pre-impregnation
methods, characterised by employing perfectly fresh tissues,
and post-impregnation methods, characterised by the employ-
ment of fixed and hardened tissues. Both are chiefly used
for nervous tissue. They give in some respects opposite
results. Pre-impregnation gives nuclei unstained, cytoplasm
rather strongly stained, axis-cylinders reddish-violet. Post-
impregnation gives nuclei sharply stained, cytoplasm pale,
axis-cylinders black, and (when successful) showing their
neurofibrils sharply distinguished from the interfibrillar
substance.

In ApdtHy’s view (Mitth. Zool. Stat. Neapel, xii, 1897,
p. 718) successful gold preparations should show a true stain,
not an impregnation (§ 347), the stain being brought about
by the formation of gold oxide (AuO) which combines with
the tissue elements. He advises in consequence that prepara-
tions should not be moved about more than can be helped in
the reducing bath, so that the colouring oxide may not be
washed away from the tissues before the stain has taken
effect.

361. As to the Commercial Salts of Gold.—Squinn’s Methods
and Formule, etc. (p. 48), says: “Commercial chloride of
gold is not the pure chloride, AuCl,, 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.”

This, however, appears not to be the case in Germany.

220 OHAPTER XVIL.

Dr. Grisier, writing to Mayur (see the Grundziige, Lex und
Mayer, p. 215), says: “ Aurwm chloratum fuscwm contains
about 53 per cent. Au, the flarwm about 48 per cent. ; in
both of them there should be only water and hydrochloric
acid besides the gold, no sodium chloride. Pure Auronatrium
chloratum contains 147 per cent. of sodium chloride, though
samples are found in commerce with much more.”

Apituy (Mitth. Zool. Stat. Neapel, xii, 1897, p. 722)
formerly employed the aurum chloratum flavum, but now
prefers the fuscum.

A. Pre-impregnation.

362. The State of the Tissues to be impregnated.—The once
classical rule, that for researches on nerve-endings the tissues
should be taken perfectly fresh, seems not to be valid for all
cases. For Drascu (Sitzb. Akad. Wiss. Wen, 1881, p. 171,
and 188!, p. 516; and Abhand. math.-phys. Cl. K. Sach.
Ges. Wiss., xiv, No. 5, 1887; Zeit. wiss. Mik., iv, 1887,
p. 492) finds that better results are obtained with tissues that
have been allowed to lie after death for twelve, twenty-four,
or even forty-eight hours in a cool place.

363. COHNHEIM’S Method (Virchow’s Arch., Bd. xxxviii, pp. 346—
349; Stricker’s Handb., p. 1100) —Fresh pieces of cornea (or other tissue)
are put into 0°5 per cent. solution of chloride of geld until 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.

Results very uncertain and anything Lut permanent.

364, Lowir’s Method (Stlzgsber. Akad. Wien, Bd. |xxi,
1875, p. 1).—The following directions are from FiscyEr’s paper
on the corpuscles of Meissner (Arch. mik. Anat., xii, 1875, p.
366).

Small pieces of fresh skin are put into dilute formic acid
(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 (14 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). Scctions are

METALLIC STAINS (IMPREGNATION METHODS). 221

then made and mounted in dammar or glycerin. Successful
preparations show the nerves alone stained.

365. Ranvier’s Formic Acid Method (Quart. Journ. Mic. Sci.
[N.S.], Ixxx, 1880, p. 456).—The tissues are placed in a
mivture of chloride of gold and formic acid (four parts of 1
per cent. gold chloride to one part of formic acid) which has
been boiled and allowed to cool (Ranvier’s Traité, p. 826).
They remain in this until thoroughly impregnated (muscle
twenty minutes, epidermis two to four hours); reduction is
affected either by daylight in acidulated water, or in the
dark in dilute formic acid (one part of the acid to four parts
of water).

366. Ranvier’s Lemonjuice Method (Traité, p. 813).—
Ranvier finds that of all acids lemon juice is the least hurt-
ful to nerve-endings. He therefore soaks pieces of tissue in
fresh lemon juice until they become transparent (five or ten
minutes in the case of muscle). They are then rapidly
washed in water, brought for about twenty minutes into 1
per cent. gold chloride solution, washed again in water, and
brought into a mixture of 50 c.c. of distilled water and two
drops of acetic acid. ‘They are exposed to the light for
twenty-four to forty-eight hours. The preparations thus
obtained are good for immediate study, but are not permanent,
the reduction of the gold being incomplete. In order to
obtain perfectly reduced, and therefore permanent, prepara-
tions, the reduction should be done in the dark in a few cubic
centimetres of dilute formic acid (1 part acid to 4 of water),
which takes about twenty-four hours.

367. Viattanz’s Osmic Acid Method (Hist. ef Dév. des
Insectes, 1888, p. 42).—The tissues are treated with osmic
acid (1 per cent. solution) until they begin to turn brown,
then with 25 per cent. 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 25 per cent. formic acid. I find this an ex-
cellent method.

Kerscuner (Arch. mik. Anat., 1xxi, 1908, p. 522) puts till
brown into a mixture of ten parts 5 per cent. formic acid

222, CHAPTER XVII.

with one part 2 per cent. osmic acid, washes, puts for two
to six hours into 1 per cent. gold chloride in the dark, washes,
puts for twelve hours into 25 per cent. formic acid in the
dark and then for twenty-four in the light, and mounts in 50
per cent. glycerin with 1 per cent. of formol.

368. 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
reductiun of the gold.

Thus Basrtan employed a solution of gold chloride of a
strength of 1 to 2000, aciduated with HCl (1 drop to 75
c.c.), and reduced in a mixture of equal parts of formic acid
and water kept warm.

Hénocqusr (Arch. de ? Anat. et de la Physiol., 1870, p. 111)
impregnates in a 0°5 per cent. solution of gold chloride,
washes in water for twelve to twenty-four hours, and reduces
in a nearly saturated solution of tartaric acid at a tempera-
ture of 40° to 50°C. Reduction is effected very rapidly,
sometimes in a quarter of an hour.

Hovur (Arch. mik. Anat., ix, 1878, p. 222) says that the
double chloride of gold and potassium has many advantages
. over the simple gold chloride. He impregnates in solutions
of 05 per cent. strength, and reduces in water containing
one or two drops of a pyrogallic acid developing solution,
such as is used in photography, or in a wari concentrated
solution of tartaric acid, at the temperature of an incubating
stove.

I have myself used the double chloride of gold and sodium
with good results.

Craccio (Journ. de Microgr., vii, 1883, p. 38) prefers the
double chloride of guld and cadmium.

Friecusie (Die Lettungsbahnen in Gehirn, 1876 ; Arch, Anat.
u. Phys., 1884, p. 453) reduces in a 10 per cent. solution of
caustic soda.

Nesterorrsky treats impregnated preparations with a drop
of ammonium sulphide, and finishes the reduction in glycerin
(quoted from GierKke’s Iirberet z. nik Zwecken).

Boum reduces in Pritchard’s solution—amyl alcohol, 1;
formic acid, 1; water, 98.

METALLIC SPAINS ([MPREGNATION MertHODS). 223

Manrrept (4rch. per le Sci. med., v, No. 15) puts fresh
tissues into gold chloride, 1 per cent., for half an hour ;
then ovalic acid, 0°5 per cent., in which they are warmed in a
water-bath to 36°. Mount in glycerin. Sunny weather is
necessary.

Boccarpr (Lavort Instit. Fisiol. Napoli, 1836, i, p. 27;
Journ. Roy. Mic, Soc., 1888, p. 155) recommends oxalic acid
of O-l per cent. or of 0:25 to 03 per cent., or a mixture of
5 ¢.c. pure formic asid, 1 c.c. of 1 per cent. oxalic acid, and
25 c.c. of water, reducing in the dark not longer than two to
four hours.

Kotossow (Zeit, wiss. Mik., v, 1888, p. 52) impregnates
for two or three hours in a 1 per cent. solution of gold
chloride acidulated with 1 per cent. of HCl, and reduces for
two or three days in the dark in a 0°01 per cent. to 0°02 per
cent. solution of chromic acid.

Gesera (Intern. Monatsschr., x, 1893, p. 205) states that
previous treatment of tissues for twenty-four hours with
lime-water (ARNsTEIN’s method) greatly helps the reduction.

Bernueim (Arch, Anat. Phys., Phys. Abth., 1892, Supp.,
p. 29) adds to Lowit’s dilute formic acid a piece of sulphite
of sodium (must be fresh and smell strongly of sulphurous
acid).

Dr. Linpsay Jounson writes to me that besides the “ sun-
ning”? of the impregnating solution recommended above
(§ 349), the gold should be carefully acidulated with a
neutral acetate or formiate, or acetic or formic acid, at
least twenty-four hours before using; and then afterwards
the tissue must be washed until no reaction occurs to test-
paper.

Apstuy (Mikrotechnik, p. 173; Mitth. Zool. Stat. Neapel,
xii, 1897, pp. 718—728) lays stress on the necessity of
having the objects thoroughly penetrated by light from all
sides during the process of reductiou. Objects, therefore,
should always be so thin that hght can readily stream
through them. He impregnates for a few hours in 1 per
cent. gold chloride (§ 361) in the dark, then brings the
objects, without washing out with water, the gold solution
being just superficially mopped up with blotting-paper, into
1 per cent. formic acid. They are to be set up in this, in a
tube or otherwise, so that the light may come through them

224, CHAPTER XVIL.

from all sides, and exposed to diffused daylight in summer,
or direct sunlight in winter, for six to eight hours without a
break, They must not be moved about more than can be
helped inthe acid. If the acid becomes brown it may be
changed for fresh. The temperature of the acid should
not be allowed to rise over 20° C., whence direct sunlight is
to be avoided during the summer. He mounts in glycerin
or his syrup (§ 348). He finds such preparations absolutely
permanent.

Post-Impregnation.

869. GertacH’s Method (Srrickur’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 HCl, and after ten to twelve
hours are washed in hydrochloric acid of 1 to 2: 3000
strength, then brought for ten minutes into a mixture of
1 part HCl to 1000 parts of 60 per cent. alcohol, then
dehydrated and mounted in balsam.

(See further, for Nerve Centres, under ‘ Nervous
System.”)

370. Goer (Mem. Accad. Torino [2], xxxii, 1880, p. 382)
puts tissues previously hardened in 2 per cent. solution of
bichromate of potash for ten to twenty minutes into 1 per
cent. solution of arsenic acid, then into } per cent. solution
of chloride of gold and potassium for half an hour, washes
in water, and reduces 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.

371. Apdtuy’s Method (Zeit. wiss. Mik., x, 1898, p. 349;
Mitth. Zool. Stat. Neapel, xii, 1897, p. 729). The material
to be used must have been fixed either in sublimate or in a
mixture of equal parts of saturated solution of sublimate in
0°5 per cent. salt solution and 1 per cent. osmic acid (this
more particularly for Vertebrates). The material should be
imbedded as quickly as possible, either in paraffin or in

METALLIC STAINS (IMPREGNATION METHODS). 225

celloidin. Sections are made and fixed on slides, and after
the usual treatment with iodine, etc., are either put into
distilled water for from two to six hours, or are rinsed in
water, treated for one minute with 1 per cent. formic acid,
and again well washed with water.

They are then put for twenty-four hours, or at least over-
night, into the gold-bath, which is preferably 1 per cent.
gold chloride (see § 361), but may be weaker, down to 0'1
per cent., after which they are just rinsed with water or
superficially dried with blotting-paper. The slides are then
set up on end in a sloping position, the sections looking
downwards, so that precipitates may not fall on them, in
glass tubes filled with 1 per cent. formic acid. The tubes are
then exposed to light until the gold is reduced, as directed
in § 368 sub. fin.

I seem to have found it advantageous to reduce in weak
solution of formaldehyde, either with or without formic acid.

Szivz (Zeit. wiss, Mik., xxix, 1912, p. 292) reduces as
APATHY for one day, then rinses and puts back for the night
into the gold, then for the next day again into the formic
acid.

872. Impregnation of Marine Animals.—For some reason
the tissues of marine animals do not readily impregnate with
gold in the fresh state. It is said by Fou that impregnation
succeeds better with spirit specimens.

373. Preservation of Impregnated Preparations.—Preparations
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 completely effected, but they are very
liable to go wrong through after-blackening. Ranvier states
that this can be avoided by putting them for a few days
into alcohol, which he says possesses the property of stopping
the reduction of the gold.

Blackened preparations may be bleached with cyanide or
ferricyanide of potassium. Rzppine employs a weak solution
of ferricyanide, CyputsKy a 0°5 per cent. solution of cyanide.

Preparations may be double-stained with the usual stains
(safranin being very much to be recommended), but nuclei

15

226 CHAPTER XVII.

will only take the second stain in the case of negative im-
pregnation,

Other Metallic Stains.

374, Osmic Acid and Pyrogallol—This method was first
published by me in 1887 (La Cellule, iv, p. 110). It consists
in putting tissues that have been treated with osmic acid
into a weak solution of pyrogallol, in which they quickly
turn greenish black, sometimes much too much so.

Hermann (Arch. mik, Anat., xxxvii, 4, 1891, p. 570)
put platino-aceto-osmic material hardened in alcohol for
twelve to eighteen hours into raw pyroligneous acid. This
acid ought (Ergebnisse der Anat., ii, 1893, p. 28) to be
as raw as possible, and to be of a dark brown colour and
evil-smelling. (The stain obtained in this way is not due to
a mere reduction of the osmic acid, but also to coloration
by the brown pyroligneous acid ; for Hermann has obtained
the same stain with sublimate material, or alcohol material
(op. cit., i, 1891 [1892], p. 7).

I find this gives much better results than the pure osmic
acid process, but not the best possible. I now proceed as
follows :

Hermann or Friemuine material is brought im lu,
directly after fixing, into a weak aqueous solution of
pyrogallol. The tissues may remain in it for twenty-four
hours, but for small objects an hour or less is sufficient. An
alcoholic solution of pyrogallol may be taken if desired.
Rawirz (Lehrbuch, p. 60) takes 20 per cent. aqueous sol. of
tannin.

There is thus obtained a black stain, which is at the same
time a plasma stain and a nuclear stain, chromatin being so
far stamed that it is not necessary to have recourse after-
wards to a special chromatin stain. With Invertebrates it
sometimes gives very elegant differentiations of nervous
tissue. It is a very easy method, and if pyrogallol be used
a very safe one (with pyroligneous acid not so safe).

If it be desired to add a chromatin stain, I greatly
recommend safranin (stain very strongly, twenty-four hours
at least, and start the extraction with acid alcohol),

METALLIC STAINS (IMPREGNATION METHODS). 227

This method has been attributed to von ManHRENTHAL. See also
under “Nervous System’ modifications of this method by AzoULAY
and HELLER and GumpPERtz; also one by Konossow (Zedt. wiss. Mik.,
ix, 1892, p. 38, and ix, 1893, p. 316).

375. Perchloride of Iron.—This reagent, introduced by PoLAILLON
(Journ. de VAnat., iii, 1866, p. 43), sometimes gives useful results,
especially in the study of peripheral nerve-ganglia, in which it stains
the nervous tissue alone, the connective tissue remaining colourless.

The Hoaeans 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; then 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.

Fou fixes in perchloride (§ 80) and treats for twenty-four hours with
alcohol containing a trace of gallic acid.

POLAILLON (loc. cit.) reduces in tannic acid.

The method is not applicable to chromic objects.

GoLoDETz and Unna (Monats. prakt. Derm. xlviii, 1909, p. 153)
put sections of skin for 5 minutes into fresh mixture of 1 per cent. per-
chloride of iron and 1 per cent. sol. of ferricyanide of potassium.
See also Unna and GOLODETZ, tbid., xlix, 1909, p. 97.

Rooseve.t (Med. Rec., ii, 1887, p. 84; Journ. Roy. Mic. Soc., 1888,
p. 157) employs a stain composed of 20 drops of saturated solution of
iron sulphate, 30 grms. water, and 15 to 20 drops pyrogallic acid.

876. Palladium Chloride (see SCHULZE, § 77). Prussian Blue (see
LEBER, Arch. Ophthalm., xiv, p. 300; RANVIER, Trazté, p. 108). Cupric
Sulphate (see LEBER, zbid.). Lead Chromate (see LEBER, 7bid.).
Sulphides (see LAanpois, Centralb. med. Wiss., 1885, No. 55; and
GIERKE, in Zevt. wiss. Mik., i, 1884, p. 497). Molybdate of Ammonia
(MERKEL; KRAUSE) (see GIERKE, tbid., i, 1884, p. 96). Oxychloride of
Ruthenium (NIcOLLE and CANTACUZENE) (see Awn. Inst. Pasteur, vii,
1893, p. 331). Ruthenium Red (Ruthenium Sesquichloride) (EIsEn,
Zertt. wiss. Mik., xiv, 1897, p. 200; in my hands totally useless). Oxide
of Manganese (GoLoDETZ and Unna, Monats. prakt. Derm., xlviii,
1909, p. 151).

CHAPTER XVIII.
OTHER STAINS AND COMBINATIONS.

377. Kernschwarz (Puatner, Zeit. wiss. Mik., iv, 1887,
p- 350).—A black liquid on sale by Griibler and Hollborn,
Mayer (Grundziige, Ler & Mayer, Ist ed., p. 202) finds
that it contains iron, combined with some gallic acid. I
use it as follows :

Sections (I have not tried material in bulk) are fixed on
slides and treated with Kernschwarz until the required depth
of stain is obtained, which will be from a few minutes to
twenty-four hours, according to the material.

There is obtained a black or neutral-tint stain, which is
either a pure chromatin stain, or at the same time a plasma
stain. If overstaining should have occurred, the stain is
easily differentiated by means of any weak acid, either in
water oralcohol. Prater took alkalies, preferably carbonate
of lithia, for differentiation.

It may be well, if a good plasma stain has been obtained,
to after-stain for twenty-four hours with safranin, followed
by differentiation in either neutral or acid alcohol, and clove
oil. The stain is perfectly permanent in balsam, and is
stated to be a good one for preparations that it is desired to
photograph. y

I greatly recommend this stain, which is safe and easy.
The combination with safranin gives a better chromatin
stain than safranin alone.

878. Brazilin, the colouring matter of Brazilian redwood or Pernam-
buco wood, has been recommended by EIsen (Zeit. wiss. Mik., xiv, 1897,
p. 198), and Hickson (Nature, Ixii, 1900, p. 589, and Quart. Journ. Mic.
Sc?., 1901, p. 469). Mayer (Grandziige, p. 203) finds that, in alum
solution, it gives a stain similar to that of hematein, but much weaker,

OTHER STAINS AND COMBINATIONS. 229

Tron-Brazilin. (Hickson, Quart. Journ. Micr. Sci., xliv,
1901, p. 470) is better. Sections are mordanted for 1 to
3 hours in 1 per cent. sol. of iron alum in alcohol of 70
per cent. (made by dissolving 1 grm. of the salt in 28 c.c.
of water, warm, and adding 77 c.c. of 90 per cent. alcohol
after cooling), rinsed with alcohol, and put for 3 to 16 hours
into 0°5 per cent. sol. of Brazilin in alcohol of 70 per cent.

379. Orchella (Orseille) see Webi (Arch. path. Anat., xxiv, p. 143) ;
and Fou (Lehrb., p. 192), and early editions of this work.

380. Orcein (IsRAEL, Virchow's Archiv, cv, 1886, p. 169; and Prakti-
cum der path. Hist., 2 Aufl., Berlin, 1893, p. 72) isa dye obtained from
the lichen, Lecanora parella, and is not to be confused with orcin,
another derivative of the same lichen. It is said to unite in itself the
staining properties of the basic and acid stains, and also the combina-
tion of two contrast colours. Israel stains sections in a solution
containing 2 grms. of orcein, 2 grms. of glacial acetic acid, and 100 c.c.
of distilled water, washes in distilled water, and passes rapidly through
absolute alcohol to thick cedar oil, in which the preparations remain
definitely mounted. Nuclei blue, protoplasm red.

See also “ Connective Tissues” in Part II, and LAURENT, Zezt. wiss.
Mik., xiii, 1896, p. 302; Ruzréxa, ibid., xiv, 1898, p. 455; und WoLrFr,
zbid., xix, 1903, p. 483.

8381. Purpurin, see Ranvigr’s Trailé technique, p. 280; Duvat’s
Précis de Technique histologique, p. 221; and GEENACHER’s formula in
Arch. Mik. Anat., xvi, 1879, p. 470. A very weak stain.

382. Indigo.—Indigo is employed in histology in the form of solu-
tions of so-called indigo carmine, or sulphindigotate of soda or potash.
The simple aqueous solution gives a diffuse stain, but is of use when
employed in conjunction with carmine, see below.

Thiersch’s Oxalic Acid Indigo-carmine (see Arch. mk. Anat., i,

1865, p. 150).

383. Other Vegetal Dyes.—See early editions. Those recom-
mended by CLaupius (Zett. wiss. Mzk., xvii, 1900,p. 52) are superfluous.

Carmine Combinations.

384. Seiler’s Carmine followed by Indigo-Carmine (Am. Quart.
Mic. Journ., i, 1879, p. 220).—Stain in borax-carmine, wash out with HCl
alcohol, wash out the acid, and after-stain in an extremely dilute alco-
holic solution of indigo-carmine (two drops of saturated aqueous solution
added to an ounce of alcohol and filtered).

I find this method gives good results with sections, but not if it be
att mpted to stain in bulk.

230 CHAPTER XVIII.

385. Merkel’s Carmine and Indigo-Carmine in One Stain
(MERKEL, Unters. anat. Anst. Rostock, 1874; Month. Mic. Jouru., 1877,
pp. 242 and 317).

Also Norris and SHAKESPEARE, Amer, Journ. Med. Set., January, 1877 ;
MERKEL, Mon. Mic. Journ., 1877, p. 242; Marsu, Section Cutting, p. 85;
BaveERL, Arch. Mik. Anat., xxiii, 1865, pp. 36, 37; MacatLtum, Trans.
Canad. Instit., ii, 1892, p. 222; Journ. Roy. Mic. Soc., v, 1892, p. 698.

386, Mayer’s Carmalum (or Hemalum) and Indigo-Carmine
in one Stain—Maver (Mitth. Zool. Stat. Neapel, xii, 1896,
p. 320) obtains very good results by taking a solution of 0-1
grm. of indigo-carmine in 50 e.c. of distilled water, or
5 per cent. alum solution, and combining it with from four
to twenty volumes of carmalum or hemalum.

387. Carmine and Picro-Indigo-Carmine (Ramon y Casa, Rev.
de Crenc. med., 1895 ; Catigsa, Rev. trim. Microgr., 11, 1897,
p. 101; Zeit. wiss. Mik., xv, 1899, p. 323).—For use after a
carmine stain, Ramén takes a solution of 0°25 grm. of
indigo-carmine in 100 grms. saturated aqueous solution
of picric acid. Stain (sectious) for five to ten minutes,
wash in weak acetic acid, then in water, then remove the
excess of picric acid with absolute alcohol, clear and
mount.

Ramon also (Elementos de Histologia, 1897 ; quoted from
La Cellule, xix, 190], p. 212) employs the picro-indigo
mixture after Magenta; stain strongly in saturated solution
of magenta, rinse in water until no more colour comes away,
and pass into the indigo mixture. See also Borner, Ann.
Inst. Pasteur, 1901, p. 57, or Let et Hennecuy, ratte,
p. 268.

388. Carmine and Anilin Blue (or Bleu Lumiere, or Bleu de
Lyon) (Duvat, Prévis de Technique Microscopique, 1878, p.
225).—Stain with carmine ; dehydrate, and stain for a few
minutes (ten minutes for a section of nerve-centres) in a
solution of ten drops of saturated solution of anilin blue in
alcohol to 10 germs. of absolute alcohol. Clear with
turpentine, without further treatment with alcohol, and
mount in balsam.

Other authors recommend, instead of anilin blue, bleu de
Lyon, dissolved in 70 per cent. alcohol acidulated with acetic
acid (Maurics and Scuutaiy), or bleu Jumiére.

OTHER STAINS AND COMBINATLONS. 231

The solutions of both these colours should be extremely
dilute for sublimate material, but strong for chrom-osmium
material. It is possible to use them for staining in bulk.

Baumearren (Arch. mik. Anat., xl, 1892, p. 512) stains
sections (of material previously stained in borax-carmine) for
twelve hours in a 0°2 per cent. solution of bleu de Lyon in
absolute alcohol, and washes out for about half that time
before mounting in balsam. He recommends the process
for cartilage and nerve-centres.

389. Carmine and Malachite Green—MaAas (Zeit. wiss. Zool., 1,
+, 1890, p. 527) recommends borax-carmine followed by weak alcoholic
solution of malachite green, with a final washing out with stronger
alcohol.

890. Carmine and Picro-nigrosin (PIANESE). See Journ. Roy.
Mic. Soc., 1892, p. 292.

891. Carmine and Picric acid. See § 298.

Hematein or Hematoxylin Combinations.

392. Hematoxylin and Picric Acid.—See § 298.

393. Hematoxylin and Eosin—This popular combination
gives results that are esthetically beautiful, but (for most
objects) is not so useful as many others, the eosin lacking
in electivity. Objects may be stained with hematoxylin
(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 recommended (Srdur, see
Zeit, wiss. Mik.,i, 1884, p. 583) to take it saturated. Hither
aqueous or alcoholic solutions of eosin may be used.

Hickson (Quart. Journ. Mic. Sci, 1898, p. 129) stains
sections for one hour in a strong solution of eosin m 90 per
cent. alcohol, washes with alcohol, and stains for twenty
minutes in a weak solution of hematoxylin.

This method is most particularly recommendable for
embryological sections, as vitellus takes the eosin stain ener-
getically, and so stands out boldly from the other germinal
layers in which the blue of the haematoxylin dominates.

See also List (Zett. wiss. Mtk., 1, 1885, p. 148) ; Busou

232, GHAPTRER XVII.

(Verh. Berl. Phys. Gses., 1887); Ginrku (Zeit. wiss, Mik., i,
1884, p. 505).

Sections should be very well washed before being passed
from eosin into hematoxylin or the reverse, as eosin very
easily precipitates haematoxylin.

For the complicated and superfluous mixtures of Runaut
and of Evrrarp, Demoor and Massarr, see Fou’s Lehrbuch,
p. 196, Ann. Inst. Pasteur, vii, 18938, p. 166, or eurly

editions.

394, Hematoxylin and Congo.See § 305.

295. Hematoxylin and Safranin.—RaBt (Morph. Jahrb., x, 1884,
p. 215) stained very lightly with very dilute DELAFIELD’s hematoxylin for
twenty-four hours, then for some hours in (PFITZNER’S) safranin and
washed out with pure alcohol. The plasma stain is here given by the
hematoxylin.

Similarly Recaup, Verh. Anat. Gres., xiv, 1900, p. 112.

Foa (Festschr. Virchow, 1891, p. 481) stains in a miature
of 25 c.c. of Bohmer’s hematoxylin, 20 of 1 per cent. solution
of safranin, and 100 of water for one to three minutes.

396. Hematoxylin and Saurefuchsin—Stain first with iron
hematoxylin or hemalum, then stain (sections) in 0°5 per

cent. aqueous solution of Saurefuchsin, dehydrate and
mount.

397. Hematoxylin and Saurefuchsin and Orange.—Proceed
as above, using for the second stain the following mixture :
Saurefuchsin, 1 grm.: orange, 6 grms.; rectified spirit,
60 c.c.; water, 240 c.c. (from Squrre’s Methods and Formule,
p. 42). Using orange G. (not mentioned by Squire), I have
had very good results.

The method of Cavazzani (Riforma Med., Napoli, 1893,
p. 604; Zeit. wiss. Mik., xi, 3, 1894, p. 344) is far too com-
plicated.

398. Hematoxylin and Picro-Saurefuchsin (van Greson, New
York Med. Jowrn., 1889, p. 57; quoted from Mogtirr, Zeit.
wiss. Mik., xv, 2, 1898, p. 172, which see for further details).
Proceed as above, using for the second stain the picro-

OTHER STAINS AND COMBINATIONS. 233

Saurefuchsin mixture, § 299. The second stain must not
be too prolonged.

Weicerr (Zeit. wiss. Mik., xxi, 1904, p. 1) stains first in
his iron-hematoxylin mixture (§ 244), rinses in water, and
stains for a short time in his picro-Siurefuchsin (§ 299),
rinses, dehydrates with 90 per cent. alcohol, and clears with
carbolic acid-xylol mixture ( $167).

CHAPTER XIX,
BXAMINATION AND PRESERVATION MEDIA,

399. Introductory —I comprehend under this heading all
the media in which an object may be examined to advantage.

All preservative media may be used for mounting, though
the only media that will afford an absolutely swre preservation
of soft tissues are the resinous ones.

400. Refractive Indices of Examination Media.—An examina-
tion medium should be of such a refractive indew as to afford a
due degree of visibility of colourless (unstained) elements.
The visibility of these is inversely as their transparency
when penetrated by the medium. It is directly proportional
to the difference between the refractive indices of the object
and of the medium in which it is mounted. The greatest
transparency is obtained when the refraction of the medium
is the same as that of the tissue elements. Media having a
lower index than that of the tissues give diminished trans-
parency, but greater visibility. Media having a higher
index than that of the tissues give great transparency, but
diminished visibility of (unstained) details. Now the index
of refraction of most tissue elements, after fixation and
dehydration, ig somewhat higher than that of Canada
balsam: so that media of the greatest clearing power (7. ¢.
giving the greatest transparency) must be looked for
amongst reagents having an index superior to that of
balsam, whilst for enhanced visibility of detail we must
employ less refractive media, such as castor oil, glycerin, or
water.

The following short list, extracted from Benrens’ Tubellen
zum Gebrauch bei mtkroskopischen Arleiten, Braunschweig,
1892, p. 42, and other sourecs, may be useful as a guide to

EXAMINATION AND PRESERVATION MEDIA. 235

the optical effects of various media. ‘The figures give the
approximate indices of refraction. They should be accepted
with some caution, on account of the variability of samples.
The figures given for balsam refer evidently to the resin in the
solid state and not to the solutions used for mounting, which

are certainly much lower, according to the lower index of
the solvent.

Air. : 1:000 | Xylol . : 1:497
Methyl aleohel . 1523 | Cedar-wood oil, not sick
Distilled water . , . 1336 ened ‘ ‘ : . 1510
Sea water . : . 13483 | Crown glass 1518
Solution of white of ege 1350 | Cedar-wood oil, thickened. 1520
Absolute alcohol s 1367 | Gum damar ; . 1520
Acetate of potash, saturated Xylol balsam. L524
aqueous sol. . . 1370 | Oil of lemons : 1527
Glycerine with an eaeat Oil of cloves . 1533
quantity of water . 1397 | Canada balsam (galt 1535
Chloride of calcium, 90 per Creasote . : . 1538
cent. in water . . 1411 | Colophonium . 1545
Glycerine, Price's. . 1460 | Carbolic acid 1549
Oil of bergamot . ' 1-464 | Oil of anise seed. : . 1557
Paraffinum liquidum . 1471 | Oil of cinnamon (or cassia) 1°567
Olive oil. ‘ 1-473 | Anilin oil . e 1580
Oil of turpentine . 1473 | Sulphide of carbon. . 1630
Glycerine, * concentrated” 1473 ) Tolu balsam. 1640
Gilson's Baume au Camsal 1478 | Monobromide of naphtha
Gilson’s Euparel 1-483 lin . ; 1-660
Terpinol . . L48t | Solution of sulphur in sul-
Castor oil 1-490 phide of carbon . 1750

It will be seen that 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
about the same extent as Canada balsam. Clove oil has a
much higher index, and therefore clears more than balsam ;
cinnamon oil higher still. Turpentine and bergamot oil have
much lower indices, and therefore clear less.

Watery Media.

401. Isotonic and “ Indifferent” Liquids.—The old distinction
of ‘‘ Indifferent ” liquids, and those which have some ie
on tissues, appears to be misleading more than helpful ;

236 CHAPTER XIX.

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 artificial.

Water may be employed for the examination of structures
that have been well fied; but this is by no means applicable
to the examination of fresh tissues. It 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).

In order to render it inoffensive to fresh tissues it must
have dissolved in it substances of similar diffusibility to
those of the liquids of the tissue, so as to prevent the occur-
rence of osmosis, to which process the destructive action of
pure water is mainly due. Now cell contents are a mixture
of colloids and erystalloids ; consequently, in order to reduce
osmotic processes to a minimum, it is necessary that the
examination medium contain a due proportion of both
crystalloids and colloids. By adding, for instance, white of
ege to salt solution this end may be in some measure
attained ; and, as a matter of fact, the liquids recommended
as “indifferent” are generally found to contain both
crystalloids and colloids. Liquids thus composed, in which
tissue-elements are in osmotic equilibrium—that is, neither
swell nor shrink—are said to be tsotonie to the tissues ;
whilst those in which they shrink are called hypertonic, and
those in which they swell hypotonic. Solutions of common
salt, in different concentrations, form the base of the most
commonly employed isotonic liquids. For marine Inverte-
brates, sea-water is generally isotonic.

402. Salt Solution (“normal salt solution,” “ physiological
salt solution”’).—0°75 per cent. sodium chloride in water.
Carnoy recommends the addition of a trace of osmic acid.

Rincer’s solution, much used in physiology, consists of
sodium chloride 0:8 parts, calcium chloride 0°02, potassium
chloride 0°02, sodium bicarbonate 0°02 and water 100 (with
or without O-l dextrose).

EXAMINATION AND PRESERVATION MEDIA. 937

According to Locks (Boston. Med. Surg. Journ., 1896,
p. 514) there should be added to salt solution (which to be
isotonic should contain, according to Hamsurerr, 0°9 to 1
per cent. of salt)—0-01 per cent. chloride of potassium, and
0°02 per cent. chloride of calcium, in order to obtain an
“indifferent” liquid.

Matassez (C. R. Soc. Biol., iii, 1896, pp. 504 and 511)
takes for erythrocytes about 1 per cent. sodium chloride.

Dexuvysen (Onderz. Phys. Lab. Leiden., 1900, p. 149)
takes for blood of Rana 0°8 per cent.

For Selachians, Musxens (Yijd. Nederb. Dierk. Ver., 1894,
p. 314) finds 2} per cent. right; and Ropin (Comptes Rend.,
1900, p. 1009) 1:5 to 2°6 per cent., according to the species.

Encetmann (Deutsch. med. Wochenschr., xxix, 1908, p. 64),
finds that 0°9 per cent. is isotonic with human blood-serum,
and 0°64 per cent. for red blood corpuscles of the frog.

Kronecxer’s “ Artificial Serum” (from Voor et Yune
Traité. @ Anat. Comp. Prat., p. 473) consists of common salt
6 parts, caustic soda 0:06, distilled water 1000.

Boum und Opren (Taschenbuch, 3 Aufl., p. 19) take car-
bonate of soda instead of caustic soda.

403. Picrer’s Liquid (Mitth. Zool. Stat. Neapel, x, 1891,
p. 89).—5 to 10 per cent. solution of chloride of manganese.
‘These proportions are for marine animals, and for terrestrial
animals will generally be found much too high. For these
from 1 per cent. to 3 per cent. will be nearer the mark. I
find this liquid excellent.

404. Aqueous Humour, Simple White of Egg—Require no
preparation beyond filtering. They may be iodised if desired
(see next §), or mixed with salt solution.

405. Iodised Serum.—Max Scuunrze (Virchow’s Archiv,
xxx, 1864, p. 263). I take the following from Ranvier
(Traité, p. 76).

The only serum that gives really good results is the amniotic
liquid of mammals. Flakes of iodine are added to it, and the
flask frequently agitated for some days. The flask should
have a wide bottom, so that the serum may form only a
shallow layer in it.

238 CHAPTER XIX.

Another method is as follows: Serum is mixed with a
large proportion of tincture of iodine; the precipitate that
forms is removed by filtration, and there remains a strong
solution of iodine in serum. ‘his should be kept in stock,
and a little of it added every two or three days to the
serum that is intended for use. In general for maceration
purposes a serum of a pale brown colour should be em-

ployed.

406. Artificial Iodised Serum (Frey, Das Mikroscop, 6 Aufl;
1877, p. 75).—Distilled water 270 grms., white of egg 30,
sodium chloride 2°5. Mix, filter, and add tincture of iodine.

407. Micua’s Glycerized Blood-serum (see the paper in Zeit. f.
wiss. Mzk., vii, 2, 1890, p. 172).

408. Chloride of Calcium (Hartine, Das Mikrozkop, 2 Aufl, p. 297).
—The aqueous solution, either saturated or diluted with 4 to 8 parts of
water, has a low refractive index and does not dry up.

409. Acetate of Potash (Max ScHuLtzx, Arch. mk. Anat., vii, 1872,

p. 180).—A nearly saturated solution in water. The index of refraction
is lower than that of glycerin.

410. Syrup.—A good strength is equal parts of loaf sugar
and water. Dissolve by boiling. ‘To preserve it from
mould, chloral hydrate may be dissolved in it (1 to 5 per
cent.)—I have used as much as 7 per cent., and found no
disadvantage—or carbolie acid (1 per cent.).

It may be used as a mounting medium, but there is always
risk of the sugar crystallising out.

Fasre-Domercur (Bull. Soc, Philomath, ix, 1899, p. 115)
dissolves 200 parts of suger in 400 of water, and adds 1
part of formaldehyde, and camphor to saturation.

411, Chloral Hydrate.—5 per cent. in water (Lapowsky,
Arch. f. mik, Anat., 1876, p. 359).

Or, 2°5 per cent. in water (Bravy, British Copepods).

Or, 1 per cent. in water (Munson, Journ. Roy. Mic. Soc.,
1881, p. 847).

EXAMINATION AND PRESERVATION MEDIA. 239

Mercurial Inquids.

(I give these as examination media only, not as permanent mounting media.
Media containing sublimate always end by making tissues granular.)

412. Giison’s Fluid (Carnoy’s Biologie cellulaire, p. 94).

Alcohol of 60 per cent. . : . 60 ce.
Water , : : : « 180 55
Glycerin. 30 ,,
Acetic acid (15 — at the lacie

to 85 of water) : : gi LB ys
Bichloride of mercury . , . O16 grm.

413. Gace’s Albumen Fluid (Zei!. f. aiss. Mik., 1886,
p- 223).

White of egg : : . 15 ce.
Water : ‘ ; . 200 ,,
Corrosive acylates : : 3 0-5 grm.
Salt. F ; ; 4 ovms.

Mix, agitate, filter, saa preserve in a cool place. Recom-
iene) for the study of red blood-corpuscles and ciliated
cells,

414. Pacini’s Fluids (Journ. de Mtt., iv, 1880; Journ. Roy. Mic. Soc.,
[N.S.] ii, 1882, p. 702, and early editions of this work).—Antiquated and
superfluous. They consist essentially of corrosive sublimate of from one
half to one third per cent. strength, with the addition of a little salt or
acetic acid.

415. GoapBy's Fluids (Micro. Dict., art. “ Preservation,” or early
editions of this work).—Quite unsuited for histological purposes.

Other Fluids.

416. Chloride and Acetate of Copper (Rirarr et Perrr’s fluid,
see § 90).

417. Tannin (Carnoy, Biol. Cellulaire, p. 95)—Water 100 grms.,
powdered tannin 0-40 grm., as an examination medium only.

418. WICKERSHEIMER’S Fluid (Zool. Anz., 1879, p. 670).— Worthless
for histological purposes.

419. Medium of Farranys (Beats, How to Work, etc.,
p- 58).--Picked gum arabic 4 ozs., water 4, glycerin 2

=

240 CHAPTER XIX.

See also the Micrographic Dictionary, and A. F. Sraniey
Kent, in Journ. Roy. Mic. Soc., 1890, p. 820.

420. Gum and Glycerin Medium (LANGERHANS, Zool. Anzeig. ii,

1879, p. 575).
Gummi arab. , r _ F . 50
Aquee . : . : . . 50
To which after twelve hours are added—
Glycerini . F ; . : . 50
Sol. aquosa acid. corhbal. (5°100) . : . 10:0

421. Attun’s Gum and Glycerin—Prof. F. J. Atuen (in.
litt.). Solution of gum arabic of the consistency of
glycerin, strained, and one eighth volume of glycerin and
one twentieth of formol gradually incorporated. Sets hard.

422. HovrEr’s Gum with Chloral Hydrate or Acetate of Potash
(Biol. Centralb., ii, 1882, pp. 23, 24).—A high 60 c.c. glass with a wide
neck is filled two thirds full with gum arabic (in pieces), and then ezther
a solution of chloral (of several per cent.) containing 5—10 per cent. of
glycerin is added or officinal solution of acetate of potash or ammonia.
Filter after solution. The solution with chloral is for carmine or
hematoxylin objects—that with acetate for anilin objects.

423. CoLr’s Gum and Syrup Medium. See § 183.

424. ApAruy’s Gum and Syrup Medium (see § 343).—This
medium sets very hard and may also be used for ringing
glycerin mounts.

425. Fasre-Domercun’s Glucose Medium (La Nature, No.
823, 9 Mars, 1889, supp.).—Glucose syrup diluted to twenty-
five degrees of the areometer (sp. gr. 11968) 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.
It is said to preserve without change almost all animal

pigments,

426. Brun’s Glucose Medium (from Faprn-Domercun’s Pre-
miers Principes dw Microscope, 1889, p. 128).—Distilled
water 140 parts, camphorated spirit 10, glucose 40,
glycerin 10. Mix the water, glucose, and glycerin, then
add the spirit, and filter. Hrnnecuvy informs me that this

EXAMINATION AND PRESERVATION MEDIA. 241

liquid preserves the colour of preparations stained with anilin
dyes, methyl green included.

427. Levulose is recommended hy Benrens, Kosset u. SCHIEF-
FERDECKER (Das Mikroskop, etc., 1889). It is uncrystallisable, and
preserves well carmine and coal-tar stains (hematoxylin 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.

428, Amann’s Lactophenol (from Lanazron, C. R. Soc. Biol.,
Ivii, 1905, p. 750).—Carbolie acid, 20; lactic acid, 20;
glycerin, 40; water, 20. For Nematodes, Acarids, etc.
Add gradually drop by drop to the water containing the
organisms. Not for mounting. Mount in glycerin jelly.

Glycerin Media.

429, Glycerin.—Glycerin diluted with water is frequently
employed as an examination and mounting medium. Dilution
with water is sometimes advisable on account of the increased
visibility that it gives to many structures. But for efficacious
preservation undiluted glycerin, the strongest that can be
procured, should be used (see Bratz, How to work, etc.).

For closing glycerin mounts, the edges of the cover should
first (after having been cleansed as far as possible from super-
fluous 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. See next chapter.

Glycerin dissolves carbonate of lime, and is therefore to
be rejected in the preparation of calcareous structures that
it is wished to preserve.

430. Extra-refractive Glycerin.—The already high index of refrac-
tion of glycerin (Price’s glycerin, n = 146) may be raised by dissolving
suitable substances in it. Thus the refractive index of a solution of
chloride of cadmium (CdCl,) in glycerin may be 1:504; that of a
saturated solution of sulphocarbolate of zinc in glycerin may he 1:501 ;
that of a saturated solution of ScHERING’s chloral hydrate (in crusts) in
glycerin is 1510: that of iodate of zinc in glycerin may be brought up

to 156. For further details see previous editions, or Journ. Roy. Mic.
Soc., 11, 1879, p. 346; iii, 1880, p. 1051; (N.S.), i, 1881, pp. 948 and 366.

431. Glycerin and Alcohol Mixtures—These 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

16

242 CHAPTER XIX.

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. Canperna’s Liquip (Zeit. wiss. Zool., xxx, 1878, p.
442).—Glycerin 1 part, alcohol 2, water 3.

2. I strongly recommend the following for very delicate
objects :—Glycerin 1 part, alcohol 1, water 2.

3. Hanrsca’s Liquip.—Glycerin 1 part, alcohol 3, water 2.

4, Jicer’s Liquip (Voer and Yuna’s Traité d’ Anat. comp.
prat., p. 16).—Glycerin 1 part, alcohol 1, sea water 10.

Glycerin Jellies.

432. Glycerin Jellies have a higher index than pure glycerin,
and set hard enough to make luting unnecessary, though it
is well to varnish the mount. To use them, you melt a small
portion on a slide, introduce the object (previously soaked in
water or glycerin), and cover. They seem very plausible,
but for delicate work I do not recommend them, and should
advise instead either pure glycerin or euparal.

433. Lawrence’s Glycerin Jelly (Davizs, Preparation and
Mounting of Microscopic Objects, p. 84).—Soak some gelatin
for two or three hours in cold water, pour off the superfluous
water, and heat until melted. ‘To each fluid ounce of the
gelatin, whilst it is fluid but cool, he adds a fluid drachm of
the white of anegg. Boil until the albumen coagulates and
the gelatin is quite clear, and to each ounce of the solution
add 6 drachms of a mixture composed of 1 part of glycerin
to 2 parts of camphor water.

434. Branp?’s Glycerin Jelly (Zeit. wiss. Afik., ii, 1880, p.
69).—Melted gelatin 1 part, glycerin 1} parts. The gelatin
to be soaked in water and melted as above. After incor-
porating the glycerin, filter through spun glass pressed into
the lower part of a funnel. He describes a simple arrange-
ment for keeping the funnel warm during the filtering (see
early editions). Some drops of carbolic acid should be
added.

435. Karsur’s Glycerin Jelly has been given § 155,

EXAMINATION AND PRESERVATION MEDIA. 243

436, Squirnw’s Glycerin Jelly (Squinw’s Methods and
Furmulx, ete., 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.

437. HemeEnuain (Zeit. wiss. Mik., xx, 1905, p. 328) takes
of gelatin 9 parts, glycerin 7, and water 42, and to the
filtrate adds drop by drop 14 parts of absolute alcohol.

438. Fiscuer (cbid., xxix, 1912, p. 65) takes 5 orms. of
borax disolved in 240 c.c. of water and adds 25 c.c. of
glycerine. To this he adds 40 grms. of gelatin, dissolves
with heat, and continues to heat gently until the solution has
somewhat thickened. This remains fluid at ordinary
temperatures.

439. Guitson’s Chloral Hydrate Jelly (communicated by
Gitson).—1 vol. of gelatin, melted secundum artem, and
1 vol. of Price’s glycerin. Mix, and add crystals of chloral
hydrate until the volume has increased by one half; warm
till dissolved. This gives a very highly refractive medium.

Gzorrroy, Journ. de Botan., 1893, p. 55 (see Zeit wiss. Mtk.,
ix, 1893, p. 476) dissolves, by the aid of as little heat as
possible, 3 to 4 grms. of gelatin in 100 c.c. of 10 per cent.
aqueous solution of chloral hydrate.

High Refractive Liquids.

440, SrepHenson’s Biniodide of Mercury and Iodide of Potas-
sium (Journ. Roy. Mic. Soc. [N.8.], ti, 1882, p. 167) —A
solution prepared by adding the two salts to water until
each is in excess; the liquid will then be found to have a
refractive index of 1°68. (If [Amann, Zeit. wiss. Mtk., xiii,
1896, p. 21] glycerin be taken instead of water, it rises to
1:78 or 1:80. Besanuns [TLabellen, 1898, p. 71] takes binio-
dide 65 parts, iodide 50,and water 25. n= 171.) Any

B44 CHAPTER XIX,

lower index can be obtained by suitable dilution with water.
This fluid is very dense, its specific gravity being 3°02. It
is highly antiseptic.

For marine animals a weak solution is probably well
adapted, as about a 1 per cent. solution (5 minims to the
ounce) will give the specific gravity of sea water.

Covers should be sealed with white wax, and the mounts
finished with two or three coatings of gold size and one of
shellac.

I have experimented both with strong and with weak
solutions. ‘They are not adapted, I find, for the purposes of
a permanent mounting medium, for the preparations are ruined
by a precipitate which forms in the fluid. But as a tempo-
vary examination medium I have occasionally found this
solution valuable. Its optical properties are wonderful; it
allows of the examination of watery tissues, without any
dehydration, in a medium of refractive index surpassing that
of any known resinous medium,

See further details in early editions,

441. Monobromide of Naphthalin.—See Journ. Roy. Mie. Soc.,
1880, p. 1043 (ABBE and Van Hevrck), and Zool. Auz., 1882, p. 555
(Max FLEscH).

Resinous Media.

442. Resins and Balsams.—Resins 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 solidi-
fied resins that should be employed for microscopical pur-
poses; 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 the preservation of stains. All solutions
should therefore 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.

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.

EXAMINATION AND PRESERVATION MEDIA. 245
Turpentine media preserve the indear of visibility of the
preparations much longer than do media made with more
volatile menstrua. Preparations made with these often
become so transparent in course of time that much fine detail
is often lost. (Such mounts may, however, be revivified
without removing the cover by putting them for a day or two
into a tube of benzol; the benzol penetrates the balsam, and
brings it down to a lower refractive index.)
For a permanent mounting medium of somewhat low index
I unhesitatingly recommend Huparal. For cases in which a
still lower index is desired, Gilson’s camsal balsam. Turpen-
tine colophonium is a safe and excellent medium, but is
injurious to alum-hematein stains. For these, and in general
where a strongly clearing medium is desired, aylol balsa
is about the most recommendable, though it is not perfectly
safe, the mounts sometimes developing granules. Seiler’s
alcohol balsam is a tine medium, and perfectly stable. Oul of
cedar is sometimes useful, it keeps perfectly, and with time it
thickens sufficiently to hold the cover in place; or if desired,
preparations may be luted with Brtu’s cement.

443. Canada Balsam—Prepare with the solid balsam as
described last §. The usual menstrua are xylol, benzol,
chloroform, and turpentine. ‘Turpentine has the advantages
pointed out last §, but the defect that it does not always
give a homogeneous solution with Canada balsam, as it does
with colophonium. For most purposes the aylol solution is the
best. If time be an object, a benzol solution should be pre-
ferred, as it sets much quicker than the xylol solution. The
chloroform solutions become very brown with age, and are
tujurious to stains made with tar dyes. Benzolis good when
chemically pure and free fiom water.

Sanur (Zeit. wiss. Mik., ii, 1885, p. 5) dissolves in cedar
oil.

Aprktay (Fauna Flora Golf. Neapel, xxii, 1909, p. 18)
takes balsain 2 parts, cedar oil (immersion) 1, and chloro-
form |,

Samples of balsam that are acéd are frequently met with, and are
injurious to some stains. Gribler & Hollhborn now prepare a neutral

balsam, in which Mayer has found that very delicate preparations, that.
lost colour immediately in any other sort of balsam, have kept perfectly

246 CHAPTER XIX.

for many months. For a process of neutralising balsam with carbonate
of soda or potash see Conuccr (Giorn. Ass. Med. Natural Napoli, vii,
1897, p. 172).

444. Sriver’s Alcohol Balsam (Proc. Amer. Soc. Mic., 188],
pp. 60-2; Journ. Roy. Mic. Soc. [N.S.], ii, 1882, pp. 126—7).—
Dissolve solid balsam in warm absolute alcohol, and filter
through absorbent cotton. Objects may be mounted in
it direct from absolute alcohol. I find it for most purposes
admirable. It is one of the most stable solutions known to
me. Care should be taken not to breathe on it, as this may
cause cloudiness.

445. Damar (Gum Damar, or Dammar, or d’Ammar).—The
menstrua are the same as for balsam. I find xylolthe best. For direc-
tions for preparing solutions, by various authors, see early editions.
After ample experience I am convinced that not one of these solutions
can be depended on for permanent preservation. Sooner or later, some-
times after a few weeks or days, or it may be only after months or years,
granules make their appearance in the mounts.

446. Colophonium.—A solution of pale colophonium in oil
of turpentine keeps well and gives very good definitions.
The solution should not be too thick, as it thickens with
age.

This medium drys very slowly (so that ample time is
afforded for arranging objects in it). In the winter a slide
will take about a month before it will be hard enough to be
safe with o@l-immerston lenses; whereas an alcohol-balsam
mount will be dry enough in a couple of days. It injures
alum-hematein stains ; but with these exceptions I find it a
most excellent mediwm.

Renn (Zeit. wiss. Mik., ix, 1893, p. 387) dissolves 1 part colophonium
in 10 of benzin. Solutions in chloroform or xylol are also used by some,
see Nissi in Encycl. mik. Techn., ii, p. 274.

447. Venice Turpentine (VOSSELER, Zeit. wiss. Mtk., vi, 1889, p.
292, et seq.).—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. Preparations may
be mounted in this medium direct from absolute alcohol. Celloidin
sections can be mounted direct from 96 per cent. Stains keep well,
according to VOssELER, but MAYER finds hemalum stains fade in it.

SucHANNEK (ibéd., vii, 1896, p. 463) prepares it with equal parts of
Venice turpentine and neutral absolute alcohol.

EXAMINATION AND PRESERVATION MEDIA. 247

448. Thickened Oil of Turpentine has been used as a mount-
ing medium by some workers. To prepare it, pour some oil
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.

449, Ginson’s Sandarac Media (La Cellule, xxiii, 1906, p.
427: the formule have not been published, on account of
the extreme difficulty of preparation, but the products are
on sale by Gritbler & Hollborn, even if not listed). There
are three of these. They are all of them solutions of gum
Sandarac in “Camsal” and other solvents (“ Camsal” is a
liquid formed by the mutual solution of the two solids salol
and camphor).

(1) Camsal balsam (baume au camsal), propylic alcohol formula ;
a mixture of sandarac, camsal, and propylic alcohol, n =
1-478.

(2) Camsal balsam, isobutylic alcohol formula, n = 1°485.

(3) Euparal, a mixture of camsal, sandarac, eucalyptol, and
paraldehyde, n = 1483. There are two sorts of this, the
colourless and the green (‘ euparal vert’’), the latter con-
taining a salt of copper, which «intensifies hematowylin
stains.

Objects may be prepared for mounting in camsal balsam
by a bath of propylic or isobutylic alcohol ; and for euparal
by a bath of the special solvent (supplied by Griibler &
Hollborn under the name of “ essence d’euparal”’). But this
is not necessary. Objects may always be mounted direct
from absolute alcohol, and even at a pinch from alcohol of 70
per cent. I myself generally prefer alcohol of 95 per cent.
(absolute is dangerously volatile for sections). In difficult
cases you may pass through a mixture of the medium and
the solvent.

These media work very kindly, and do not dry too rapidly.
They are not «aidant, and preserve delicate stains (perfectly,
so far as I know). The mounts seem to keep perfectly,
without scaling: all of mine, the oldest being eight years
old, have kept without the slightest deterioration in any
respect,

The primary intention of these media is to spare delicate
objects the usual treatment with absolute alcohol and

248 CHAPTER XIX.

essential oils. But they have another useful property—
their low index of refraction. I find that that of euparal is
just right for most delicate cytological researches, giving
just the desired increase of visibility to unstained elements.
Thus I frequently find that unstained spindles which are
totally invisible in balsam become strongly visible in
the most minute details in euparal. The camsal balsam,
n = 1-478, I have also sometimes found valuable, but its
index is a little too low for most things, and I generally
prefer euparal, which I find I am now using almost as much
as balsam. I consider that all the media which have been
recommended on the score of a slightly lower index than
balsam, such as damar, colophonium, Venice turpentine,
castor-oil, are now superseded by these media.

450. Sandarac (Lavpowsky, from Ref. Handbook Med. Sci., Supp. p.
438).—Gum sandarac 30 grs., absolute alcohol 50.¢.c. Not trustworthy,

the mounts scale badly.

451. Photographic Negative Varnish (for mounting large sections
without cover-glasses).—See WEIGERT, Zedt. wiss. Mik., iv, 1887. p. 209.

452. Castor Oil._See GRENACHER, Abhandl. naturf. Ges. Halle-a.-S.,
Bd. xvi; Zect. wiss. Mik., 1885, p. 244. Ihave not had good results
with it. .

453. Terpinol.—_n=1'484. See § 131.

454. Parolein (a pure form of paraffinum liquidum) is recom-
mended by Conus (Lancet, 1911, p. 878) as being quite neutral and
preserving certain coal tar stains. Ring mounts with Apdthy’s gum
syrup, § 343. Its index is 1-471, which I find too low for most things.

455, Cedar Oil—See § 442, sub. fin.

456. Gum Thus, dissolved in xylol, is recommended by Eisen, Zeit.
wiss. Mtk., xiv, 1897, p. 201.

457. Styrax and Liquidambar.—See Journ. Roy. Mic. Soc., 1883, p.
741; 2b., 1884, pp. 318, 475, 655, and 827; and the places there quoted.
Also Bull. Soc. Belge de Mic., 1884, p. 178; and Fou, Lehrb., p. 141.
These are very highly refractive media, therefore seldom useful in

histology.

CHAPTER XX.
CEMENTS AND VARNISHES.

458. Introduction.—T'wo, 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
(Brtt’s). I recommend this both as a cement and varnish ;
gold size may be found useful for turning cells; and Miuer’s
caoutchouc cement may be kept for occasions on which the
ntmost solidity is required. Marine glue is only necessary
for making glass cells,

For the operations of mounting in fluids, and of making
cells and ringing, see Carpenrer’s The Microscope.

CaRrENnTtER lays great stress on the principle that the
cements or varnishes used for fluid mounts should always be
such as contain xo mixture of solid particles, for those that
do always become porous after a certain lapse of time. All
fluid mounts should have the edges of the cover carefully dried
and be ringed with glycerin jelly before applying a cement ;
hy this means all danger of running in is done away with.
See §$§ 460 and 461. But no method yet devised will make
a glycerine mount absolutely permanent.

See also Ausgrr, The Microscope, xi, 1891, 150, and Journ.
Roy. Mic. Soc., 1891, p. 692 ; Bucx, The Microscope, xi, 189],
pp. 338, 368, and Journ. Roy. Mic. Soc., 1892, p. 293;
Benrens’ Tabellen zum Gebrauch bet mikroskopischen Arbeiten
(Bruhn, Braunschweig, 1892) ; Rousszner, Journ. Quek. Mic.
Club, vii, 1898, p. 93; and as to the comparative tenacity of
divers cements, Brurens, Zeit wiss. Mik, 11, 1885, p. 54, and
Ausperr Amer, Mon. Mic. Jowrn., 1885, p. 227; Journ. Roy.
Mic. Soc., 1886, p. 173.—Beurens gives the palm to amber
varnish ; Aubert places Mrutur’s caoutchouc cement at the
head of the list, Loveri’s cement coming halfway down, and

250 CHAPTER XX.

zinc white cement at the bottom, with less than one quarter
the tenacity of the caoutchouc cement.

459. Paraffin.—Temporary mounts may be closed with
paraffin, or white wax, by applying it with a bent wire, as
described § 471, and be made more or less permanent by
varnishing.

460. Gelatin Cement.—Marsu’s Section-cutting, 2nd ed.,
p. 104).—Take half an ounce of NeEzson’s opaque gelatin,
soak well in water, melt in the usual way, stir in 3 drops of
creasote. It is used warm.

When the ring of gelatin has become quite set and dry, 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 daylight, in order to render the gelatin
insoluble. The cover may then be finished with Bet1’s
cement. This process is particularly adapted for glycerin
mounts.

461. The Paper Cell Method—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) ; and as soon as the gelatin has set turn a ring of gold-
size on it, and when that is quite dry, varnish with Bet’s
cement.

For greater safety, the gelatin may be treated with bich-
romate, according to Marsn’s plan, last §.

462. RousseLet’s Method for Aqueous Mounts (op. cit., § 458).
—Close the mount with a ring of a mixture of two parts
of a solution of damar in benzol and one part gold-size.
When dry, put on three or four thin coats of pure gold-size
at intervals of twenty-four hours, and finish with a ring of
Warp’s brown cement.

463. Warp’s Brown Cement is a shellac-aleohol solution

CEMENTS AND VARNISHES. 951

made by E. Ward, Oxford Road, Manchester. Its best sol-
vent is a mixture of wood-naphtha and alcohol. Rovusse.et
considers it the best shellac varnish he has met with, better
than BEL1’s.

464. Bru’s Cement.—Composition unknown. May be ob-
tained from the opticians, or from J. Bell & Co., chemists,
338, Oxford Street, London. This varnish sets quickly.
The cover should be ringed with glycerin jelly before apply-
ing the varnish, especially with glycerin. It is soluble in
ether or chloroform. It is not attacked by oil of cedar.

465. Mitier’s Caoutchouc Cement.—Composition unknown.
May be obtained from the opticians. A very tenacious and
quickly drying cement. It may be diluted by a mixture of
equal parts of chloroform and strong alcohol (see Rousse.er,
Journ. Quek. Club., v, ii, 1895, p. 8).

466. Cuarke’s Spirit-proof Cement—Mr. Cu. Rovsse.rer has
highly recommended this to me. It may be procured from
Mr. J. Bolton, 25, Balshall Heath Road, Birmingham.

Rovusseter finds it the best he has tried for alcoholic
liquids, but not perfectly proof against watery media.

467. Asphalt Varnish (Bitwme de Judée).—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.

468. Brunswick Black.—See early editions, or Beate, How to
Work, etc., p. 49.

469. Gold-Size—Best obtained from the opticians. It is
soluble in oil of turpentine. A good cement, when of good
quality, and very useful for turning cells.

470. Marine,Glue—Found in commerce. CARPENTER says
the best is that known as GK 4, Best obtained from the
opticians. It is soluble in ether, naphtha, or solution of
potash. Its use is for attaching glass cells to slides, and for

252 CHAPTER XX.

all cases in which it is desired to cement glass to glass.
Used warm.

471. Turpentine, Venice Turpentine (Csoxor, Arch. mik.
Anat., xxi, 1882, p. 353; Parker, Amer. Mon. Mik. Journ.,
11, 1881, pp. 229-30).—Venice turpentine, or common resi-
nous turpentine, evaporated by heat until brittle on cooling,
It is used for closing glycerin mounts 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 angeles ;
the short arm of the wire should be just the length of the
side of the cover-glass. The wire is heated ina 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 and
never runs in. It sets hard in a few seconds.

472. Colophonium and Wax (Kronia, Arch. mik. Anat., 1886,
p. 657).—Seven to 9 parts of colophonium are added piece-
meal to 2 parts of melted wax, the whole filtered and left
to cool. or use, the mass is melted by placing the contain-
ing vessel in hot water. The cement is not attacked by water,
glycerin, or caustic potash.

VosseLur (Zeit. wiss. Mik., vii, 1891, p. 462) takes 1 part
of Venice turpentine to 2 to 3 of white wax.

473. ArpAruy’s Cement for Glycerin Mounts (Ze/t. wiss. Mik.,
vi, 1889, p. 171).—Equal parts of hard (60° C. melting-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. Used by warming and apply-
ing with a glass rod or brass spatula. One application is
cnough. Does not run in, and never cracks.

474. Canada Balsam, or Damar.—Cells are sometimes made with
these. They are elegant, but in my experience are not reliable for per-
manent mounts.

475, Amber Varnish.—Beurens finds this cement to possess

CEMENTS AND VARNISILES. 255

an extreme tenacity. That uscd by lim may be obtained
from Gritbler & Hollborn.

476. Amber and Copal Varnish (HEYDENREICH, Zeit. wiss. Mihk.,
1885, p. 338) —Extremely complicated.

477. Shellac Varnish (BEALE, p. 28).—A thick solution of shellac in
alcohol. The Micro. Dictionary says that the addition of 20 drops of
castor oil to the ounce is an improvement. Untrustworthy.

478. Sealing-Wax Varnish (Micro. Dict., “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 Lecome brittle and to lose its hold upon glass
after a time.

479. Tolu Balsam Cement (Carnoy’s Biol. Cell., p. 129).—
Tolu balsam, 2 parts, Canada balsam 1, saturated solution
of shellac in chloroform, 2 parts. Add enough chloroform
to bring the mixture to a syrupy consistence. Carnoy
finds this cement superior to all others.

PART ILI.

SPECIAL METHODS AND EXAMPLES.

CHAPTER XXI.
INJECTION—GELATIN MASSES (WARM).

480. Introduction.—Injection masses are composed of a
coloured substance called the colowring mass, and of a sub-
stance with which that is combined called the vehicle.

For instructions as to the operation of injecting, and the
necessary apparatus, see the Micrographic Dictionary, RutHER-
rorp’s and Scuirsr’s Practical Histology, the treatises of
Rozin and Ranvier, Beaue’s How to Work with the Mecro-
scope, the Lehrbuch der vergleichenden Mikroscopischen Ana-
tomie of For, and (for apparatus especially) the article in the
Eneycl. d. mik. Technik. For injections for the study of the
angiology of Vertebrates the practice of Robin and Ranvier
may safely be followed. For injections of Invertebrates (and
indeed, for vertebrates if it is desired to demonstrate the
minute structure of environing tissues at the same time as
the distribution of vessels) masses not containing gelatin
are, I think, generally preferable to gelatin masses; and I
would recommend as particularly convenient the Prussian
blue glycerin masses of Bratz. Glycerin masses have the
great advantage that they are used cold.

All formule which only give opaque masses, or are only
suitable for coarse injections for naked eye study, have been
suppressed,

481. Vaso-dilators.—In order that an injection may run
freely it is necessary that the vessels of the subject be in a
relaxed state. To this end the older anatomists used to wait
until rigor mortis had passed off before injecting. But it is
evidently preferable in the interest of the proper preservation
of the tissues to inject before rigor mortis has set in. Un-
fortunately, when this is done, it is found that most injection-

i?

958 CHAPTER XXI.

masses—glycerin masses especially—stimulate the contraction
of the vessels, so that frequently it is very difficult to get the
injection in. In these cases it may be advisable to use a
vaso-dilator. The animal may be anesthetised with a mix-
ture of ether and nitrite of amyl, and finally killed with pure
nitrite. Or, after killing by nitrite, a little nitrite of amy]
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 Oviarr and Sargent, in St. Louis Med. Journ.,
1886, p. 207; and Journ. Roy. Mic. Soc., 1887, p. 341).

Or, morphia may be added to the injection mags, or one
per cent. of lactic acid. Mozrsxo (Zeit. wiss. mik., xvi, 1909,
p. 545) prefers a saturated solution of Peptonwm siccum,
which has the advantage of hindering coagulation. For
warm-blooded animals the mass should be warmed to body-
temperature ; and in all cases masses that tend to dehydrate
tissues should be avoided if possible.

Rosin’s Masses.

482, Rozrn’s Gelatin Vehicle (Traité, p. 30).—One part of
gelatin soaked and melted in 7, 8, 9, or even 10 parts of
water, on a water bath.

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 2 per cent. is said to
do so.

483. Rosin’s Glycerin-gelatin Vehicle (Trazté, p. 82).—Dis-
solve in a water-bath 50 grms. of gelatin in 300 grms. of
water, in which has been dissolved some arsenious acid ; add
of glycerine 150 grms., and of carbolic acid a few drops.
Unlike the pure gelatin vehicles, this mass does keep in-
definitely.

FRANKL (Zevt. f. wiss. Zool., xiii, 1897, p. 28) prepares a similar
vehicle, and adds to it a little solution of corrosive sublimate and a
crystal of thymol.

484, Rozin’s Carmine Colouring Mass (Trait’, p. 38).—Rub
up 8 grms. of carmine with a little water and enough
ammonia to dissolve it. Add 50 germs. of glycerin and filter.

INJECTION —GELATIN MASSES (WARM). 259

Take 50 grms. of glycerin with 5 grms. of acetic acid,
and add it by degrees to the carmine-glycerine, until a
slightly acid reaction is obtained (as tested by very sensitive
blue test-paper, moistened and held over the mixture).

One part of this mixture is to be added to 38 or 4 parts of
the vehicles given above.

435. Robin’s Ferrocyaniie of Copper Colouring Mass (‘bid., p.
34).—Take—
(1) Ferrocyanide of potassium (concentrated solution) . 20 ¢.c.

Glycerin. ‘ . 50 ,,
(2) Sulphate of compere (eoncenbnited selniton) ‘ 35 ,,
Glycerin. ‘ . 60 ,,

Mix (1) and (2) slowly, with siettnidows sit fis. moment of injecting
combine with 3 volumes of qehicle,

486. Ropin’s Prussian Blue Colouring Mass (tlid., p. 35, and
2nd ed., p. 1013).

Take—
(a) Ferrocyanide of ieee (sol. sat.) . 90 c.e.
Glycerin. ‘ . 00 4,
(B) Liquid peesilends of iron a 30° Hatune 3,
Glycerin . : . 50 4,

Mix slowly and combine the siviscive 6 3 par ts of vehicle.
It is well to add a few drops of HCl.

Carmine-gelatin Masses.

487. Ranvier’s Carmine Gelatine Mass (Trart’ technique, p.
116).—Take 5 grms. Paris gelatin, soak until quite swollen
and soft, wash, drain and melt it in the water it has
absorbed over a water bath. When melted add slowly,
and with continual agitation, 21 germs. of carmine rubbed up
with a little water, and just enough ammonia, added drop
by drop, to dissolve the carmine into a transparent solution.

The mixture is now neutralised by adding cautiously, drop
by drop, with continual agitation, a solution of 1 part of
glacial acetic acid in two 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 solu-
tion, which gradually changes from ammoniacal to sour. As

* Erratim “ Sulphocyanide” in Ist edition of Rosrn’s Trazté,

260 CHAPTER XXI.

soon as the sour smell is perceived the liquid must be exa-
mined under the microscope. If it contains a granular
precipitate of carmine, too much acid has been added, and
it must be thrown away.

The mass, having been perfectly neutralised, is strained
through new flannel.

488. How to Neutralise a Carmine Mass (ViLLE, Gaz. hebd. d.
Sct. méd. de. Montpellier, Fév., 1882; may be had separately
from Delahaye et Lecrosnier, Paris).—VitLe points out that
when carmine is treated with ammonia a certain proportion
of the ammonia combines with the carmine and the rest
remains in excess. It is this excess that it is required to
neutralise precisely, not the whole of the ammonia employed.

To neutralise the acidity of commercial gelatin, it should be
washed for an hour or so in running water.

As to the neutralisation of the colouring mass, VLE is of
opinion that the sour smell cannot be safely relied on in
practice, and prefers to employ dichroic litmus paper (litmus
paper sensitised so as to be capable of being used equally
for the demonstration of acids and bases). For directions
for preparing this see loc. cit. or previous editions.

489. Hoyerr’s Carmine-Gelatin Mass (Biol. Centralb., 1882,
p. 21).—Take a concentrated gelatin solution and add to it
the needful quantity of neutral carmine staining solution (loc.
cit., p. 17). Digest in a water-bath until the dark violet-red
colour begins to pass into a bright red tint. Then add 6-10
per cent. by volumes of glycerin, and at least 2 per cent. by
weight of chloral, in a concentrated solution, and strain.

490. Fou’s Carmine-Gelatin Mass (Lehrb., p. 13). This can
be kept in the dry state for an indefinite length of time.

Gelatin in sheets is cut into strips which are macerated
for two days in carmine solution (prepared by diluting
one volume of strong ammonia with three of water and
adding carmine to saturafion, and filtering after a day
or two). The strips are then rinsed and put for a few
hours into water acidulated with acetic acid, then washed on
a sieve for several hours in running water, dried on parch-
ment paper, or on a net, and preserved for future use. To

INJECTION—GBELATIN MASSES (WARM). 261

get the mass ready for use, the strips are soaked for an hour
in water, and melted on a water-bath in 10 to 20 parts of
water.

For another process, which is said to give somewhat better results,
but is more complicated, see loc. cit., or Zett. wiss. Zool., xxxviii, p. 492,
or previous editions.

491. Krausn’s Carmine-Gelatin Mass (Zezt. wiss. Mik., xxvi, 1909,
p. 1).—100 grms. gelatin soaked in water, put for two to three days into
a solution of 15 grms. carmine in 2 litres of water with 100 grms. of borax,
washed, treated for a short time with hydrochloric acid of 2 per cent.,
washed, melted and preserved with camphor.

492. Other Carmine Gelatin Masses.—THIERSCH’S, see Arch. mik.
Anat., 1835, p. 148. Geruacu’s, see RANVIER, Tract’, p. 118, CARTER’s,
see BEALE, p. 113. Davrss, see his Prep. and Mounting of Mic. Objects,
p. 188.

Blue Gelatin Masses.
493. Rozin’s Prussian Blue Gelatin Mass (see $ 486).

494. Ranvien’s Prussian Blue Gelatin Mass (Traité, p. 119).
—Make a concentrated solution of sulphate of peroxide of
iron in distilled water, and pour it gradually into a concen-
trated solution of yellow prussiate of potash. ‘There is
produced a precipitate of insoluble Prussian blue. Wash
this on a felt strainer, underneath which is arranged a paper
filter in a glass funnel, 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 evaporated in a stove, and
the solid residuum put away in bottle.

For injections, if a simple aqueous solution be taken, it
should be satwrated. Such a mass never transudes through
the walls of vessels. Or it may be combined with one fourth
of glycerin, or with one twenty-fifth of gelatin soaked for an
hour in water and melted over a water bath in the water it
has absorbed. The gelatin is to be poured gradually into
the Prussian blue, on the water bath, stirring continually

262 CHAPTER XXI.

until the curdy precipitate that forms at first has dis-
appeared. Filter through new flannel and keep at 40° C.
until injected.

495. BRUCKE’s Soluble Berlin Blue (Arch. mzk. Anat., 1865, p. 87).—
Make a solution of ferrocyanide of potassium containing 217 grms.
of the salt to 1 litre of water, and one 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 con-
tinually. Wash the precipitate on a filter until soluble, dry it, press
between blotting paper in a press, break the mass in pieces, and dry
in the air.

The concentrated solution of the colouring matter is to be gelatinised
with just so much gelatin that the mass forms a jelly when cold. For
another method, see previous editions.

496. Other Blue Gelatin Masses.—Hover’s, Arch. mik. Anat.,
1876, p. 649; GuianEt’s, Journ. de Microgr., 1889, p. 94; Journ. Roy.
Mic. Soc., 1889, p. 463; THIERSCH’s, Arch. mk. Anat., i, 1865, p. 148;
Fou’s, Zezt. wiss. Zool., xxxvili, 1883, p. 494; and previous editions.

Other Colours.

497. Hovern’s Silver Nitrate Yellow Gelatin Mass (Biol. Cen-
tralbl., 1, 1882, pp. 19, 22)—A concentrated solution of
gelatin is mixed with an equal volume of a 4 per cent.
solution of nitrate of silver and warmed. To thisis addeda
very small quantity of an aqueous solution of pyrogallic
acid, which reduces the silver in a few seconds; chloral and
glycerin are added as directed § 489.

This mass is yellow in the capillaries and brown in the
larger vessels.

498. Other Colours.—HoyeEr’'s Green (Biol. Centralb., ii, 1882. p.
19). Made by mixing a blue mass and a yellow mass. THIERSCH’S
Green (Arch. mzk. Anat., 1865, p. 149). Rosin’s SCHEELE’s Green
(RoBIN, Traité, p. 37). Hartine’s White (see Frey, Le Microscope, p.
190). Frry’s White (cbid.). TEICHMANN'’s White (cbid., p.191). Fou’s
Brown (Zezt. wiss. Zool., xxxviii, 1883, p. 494). MILLer’s Purple (see
Amer. Mon. Mic. Journ., 1888, p.50; Journ. Roy. Mic. Soc., 1888, p. 518).
Fou’s Lead Chromate (Lehrb., p. 15). Rozsin’s Cadmium (his Traité,
p. 36). THIBRSCH’s Lead Chromate (Arch. mik. Anat., 1865, p. 149).

Hoyer’s Lead Chromate (ilid., 1867, p. 186); or, for any of these, see
early editions.

INJECTION—GELATIN MASSES (WARM). 263

499. RANVIER’s Gelatin Mass for Impregnation (Trazté, p. 123).—
Concentrated solution of gelatin, 2, 3, or 4 parts; 1 per cent. nitrate of
silver solution, 1 part.

NEvvVILLE (Ann. Sei. Nat., xiii, 1901, p. 36) takes a solution of 10 grms.
of soaked gelatin in 100 c.c. of 1 per cent. solution of nitrate of silver.

500. FRIEDENTHAL’s Hardening Mass (Centralb. Phys., xiii, 1899,
p. 267)—A 10 per cent. solution of gelatin, combined with a colouring
mass, and with 1 vol. of 4 per cent. formol, serves for injecting vessels
and hardening the tissues at the same time.

CHAPTER XXII.
INJECLIONS—OTHER MASSUS (UULD).

501. Fou’s Metagelatin Vehicle (Lehrb., p. 17).—If a slight
proportion of ammonia be added to a solution of 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 and can be injected without
warming. Colouring masses may be added to this vehicle,
which may also be thinned by the addition of weak alcohol.
After injection the preparations are thrown into strong
alcohol or chromic acid, which sets the mass.

According to the Hneycl. mik. Technik., metagelatin is
usually prepared by warming with concentrated acetic or

oxalic acid. It may be neutralised afterwards with car-
bonate of lime.

502. ‘T'anpLen’s Gold Gelatin Mass (Zeit. awiss. mik., xviii,
1901, p. 22) —Five grms. of gelatin are soaked in 100 c.c.
of water, warmed and melted, and combined with Berlin
blue. Then 5 to 6 grms. of iodide of potassium are slowly
incorporated. The mass generally remains liquid enough for
injection down to a temperature of 17° C., but if it should
coagulate a little more iodide should be added. After
injection you may fix with 5 per cent. formol. The speci-
mens will bear decaleification with hydrochloric or sul-
phurous acid.

Pear. (Journ. Appl. Micr., v, 1902, p. 1786) takes 8 to 10
per cent. of the iodide.

Maver (Grundzige Lem and Mayer, 1910, p. 250) takes
simply 10 grins. gelatin, 10 grins. hydrate of chloral and 100 c.c.
water.

Mozzsxo (Zeit. wiss. Mak., xxvii, 1910, p. 374), finds that

INJECLIONS—OTHER MASSES (COLD). 265

10 per cent (or more) of sodium salicylate will retard the
setting of gelatin for hours at normal temperatures.

Any of these masses may be made to set in the tissues by
means of weak formol.

Glycerin Masses.

503. Buarz’s Carmine Glycerine Mass (How to Jork, etc.,
p. 95).—Five grains of carmine are dissolved in a little water
with 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, gradually,
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. I have found this useful, but not so
good as the two following.

504. Beatu’s Prussian Blue (How to Work, etc., p. 98).

Common glycerin. : 5 . 1 ounce.
Spirits of wine ; F ; Soke. Gy
Ferrocyanide of potassium . , . 12 grains.
Tincture of perchloride of iron . . 1 drachm.
Water. : ; . 4 ounces.

Dissolve the forrocyanide 4 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 tron being added to the solution
of the ferrocyanide of potassium. Next the spirit and the
rest of water are to be added very gradually, the mixture
being constantly shaken.

Injected specimens should be preserved in acidulated
glycerin (e.g. with 1 per cent. acetic acid), otherwise the
colour may fade.

505. Beaw’s Acid Prussian Blue (ibid., p. 296).

Price’s glycerin : ‘ . 2 fluid ounces.
Tinct. of sesquichloride an iron . . 10 drops.
Ferrocyanide of potassium. ; . 38 grains.
Strong hydrochloric acid . : . 38 drops.

Water . ? é : ; . 1 ounce.

266 CHAPTER XXEL.

Proceed as before, dissolving the ferrocyanide in one half
of the glycerin, the iron in the other, and addiue the latter
drop by drop to the former. Finally add the water and
HCl. Two drachms of alcohol may be added to the whole
if desired.

I find this excellent.

506. RANVIER’s Prussian Blue Glycerin Mass (Traité, p. 120)—
The Prussian blue fluid, § 494, mixed with one fourth of glycerin.

507. Tuoma’s Indigo-Carmine (4rch. Anat. Phys., Anat.
Abth., 1899, p. 270).—Dissolve 0°15 germ. sulphindigotate of
soda in 50 ¢.c. water, filter, add 40 c.c. glycerin and gradually,
with agitation, 10 c.c. of a filtered 10 per cent. solution of
sodium chloride in water. If desired, 3 c.c. of a1 per cent.
solution of morphia may be added to dilate arteries. A fine
precipitate is formed, which is injected with the mass.

508. Gamboge Glycerin (Harrinc, Das Mikroskop, 1866, 2,
Theil, p. 124). —Gamboge rubbed up with water and added
to glycerin; or a saturated alcoholic solution of gamboge
added to a mixture of equal parts of glycerin and water.
Any excess of alcohol may be got rid of by allowing the
mass to stand for twenty-four hours.

509. Other Colours.—Any of the colouring masses, §§ 485 to 498,
or other suitable colouring masses, combined with glycerin, either dilute
or pure.

Purely Aqueous Masses.
510. Ranvirr’s Prussian Blue Aqueous Mass (Truité, p. 120).
—The soluble Prussian blue, § 494, injected without any
vehicle. It does not extravasate.

511. Mitier’s Berlin Blue (Arch. Mik. Anat., 1865, p.
150).—Precipitate a concentrated solution of Berlin blue by
means of 4 to 1 volume of 90 per cent. alcohol. The
precipitate is very finely divided; and the fluid may be
injected at once.

512. Mayer’s Berlin Blue (A/itth. Zool, Stat. Neapel, 1888,
p- 307.)—A solution of 10 c.c. of tincture of perchloride of

INJECTIONS—OTHER MASSES (COLD). 267

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. It is well to add a little acetic acid and to
put up the objects in an acid liquid.

513. EmEery’s Aqueous Carmine (cbéd., 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. The
supernatant clear solution is injected cold without further preparation.
The injected organs are thrown at once into strong alcohol to fix the
carmine. For injection of fishes.

§14.—Tacucur’s Indian Ink (Arch. mik. Auat., 1888, p. 565).
—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).

Deita Rosa (Ver. Anat. Ges., 1900, p. 141) recommends
the liquid Chinese ink sold in the shops.

Partially Aqueous Masses.

515. Joseru’s White-of-Egg (Ber. nalurw. Sect. Schles. Ges.,
1879, pp. 86—40 ; Journ. Roy. Mic. Soc., 1, 1882, p. 274).—
“ Filtered white-of-egg, diluted with 1 to 5 per cent. of car-
mine solution. . . . This mass remains liquid when cold,
coagulates in dilute nitric acid, chromic or osmic acid, and
remains transparent in the vessels.” For invertebrates.

Grosser (Zeit. wiss. Mik., xvii, 1900, p, 178) rubs up
Indian ink with white-of-egg; Horrmann (Zeit. Morph.
Authrop., i, 1901, p. 240) with blood-serum; so also
Hamusurcer, Zeit. wiss. Mik., xxv, 1908, p. 1 (2 vols. of the
ink—“ Perltusche ”—to 3 of serum).

516. BseLoussow’s Gum Arabic Mass (Arch. Anat. 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

268 CHAPTER XXII.

to 20f 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 clots. It should
then 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, swellmg 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. If it be desired to remove the mass
from any part of a preparation, this is easily done with
dilute acetic acid.

517. Milk has been recently recommended by Fiscuer
(Centralb. allg. Path., xiii, 1902, p. 277; Zeit. wiss. Mik.
xx, 1903, p. 224). It runs well, does not extravasate, and
can be used for auto-injection of the living subject.

After injection ib should be coagulated by putting the
organs for at least twenty-four hours into a mixture of 75
parts of formol, 15 of acetic acid, and 1000 of water (pure
formol will not do). They are then sectioned, and the
sections stained with Sudan III or Scharlach R, which stain
the milk. They cannot be mounted in balsam.

Celloidin and other Masses.

518. SCHIEFFERDECKER’s Celloidin Masses (Arch. Anat. Phys.,
1882 [Anat. Abth.], p. 201). (For Corrosion preparations).—See previous
editi.ns ; HOCHSTETTER’S Modification of SCHIEFFERDECKER’S Mass
(Anat. Anz., 1886, p. 51); Bupen’s Asphaltum Mass (Arch. Mik. Anut.,
xiv, 1877, p. 70), or early editions; HoYER’s Shellac Mass (Arch. Mik.
Anat., 1876, p. 645). For this and that of BELLARMINOW (Anat. Anz.,
1888, p. 605), see early editions; HoyEr’s Oil-colour Masses (Internat.
Monatsschr. Anat., 1887, p. 341); SEVEREANU’S, Verh. Anat. Ges., 21 vers,
1906, p. 275; Panscu’s Starch Mass (Arch. Anat. Entw., 1877, p. 480;
1880, pp. 232,371; 1881, p. 76; 1882, p. 60; 1883, p. 265; and a modification
of the same by GaGcE, Amer. Mon. Mic. Journ., 1888, p. 195); Tr1cH-
MANN’'S Linseed-Oil Masses (S. B. Math. Kl. Krakaw Akad., vii, pp.
108, 158; Journ. Roy. Mic. Soc., 1882, pp. 125 and 716, and 1895, p. 704).

INJECTIONS—OTHER MASSES (COLD). 269

Fuint’s Celluloid (Amer. Journ. Anat., i, 1902, p. 270); HusEr’s (cbid.,
vi, 1907, p. 393); Krassuskasa’s Photoxylin (Anat., Heft. 2. xiii, 1904,
p. 521).

519. Natural Injections (Rosin, Traité, 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.

Rerrerer and Zenxer use solution of Miller, see Jown.
Anat. Phys., 1894, p. 336, and Arch. Path. Anat., 1894, p. 147.

CHAPTER XXIIT.
MACERATION, DIGESTION, AND CORROSION.

Maceration.

520. 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 sepa-
rated from their place in the tissue and separately studied
after they have been isolated both from neighbouring ele-
ments and from any interstitial cement-substances that may
be present in the tissue. Simple teasing with needles is
often insufficient, as the cement-substances are frequently
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, by which is meant prolonged
soaking (generally for days rather than hours) in 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 softening 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) 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
eradually pressed down, whilst at the same time the cells of
the tissue are segregated by the repeated shocks. When the
segregation has proceeded far enough, mounting medium may
be added and the mount closed.

A good material for making war feet is obtained (VosseLer,
Zeit. wiss. Mik., vii, 1891, p. 461) by melting white wax and
stirring into it one half to two thirds of Venice turpentine,

MACERATION, DIGESTION, AND CORROSION. 271

The most desirable macerating media are those which,
whilst dissolving intercellular substances, do not attack the
cells themselves. Those which contain colloids have been
found to give the best results in this respect. Iodised
serum is an example.

521. Iodised Serum (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
jodised 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 last §; 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 maceration may be prolonged for several
weeks.

This method is 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.

522. Iodide of Potassium (ARNOLD, Arch. mik. Anat., lii, 1898,
pp. 135 and 763).—10 c.c. of 10 per cent. aqueous sol. of potassic iodide
with 5 to 10 drops of a similar solution, containing also 5 per cent. of
jodine.

528. Alcohol.—Ranvier employs one-third alcohol (1 part
of 90 per cent. alcohol to 2 parts of water). Hpithelia will
macerate well in this in twenty-four hours. It macerates
more rapidly than iodised serum.

Other strengths of alcohol may be used, either stronger
(equal parts of alcohol and water) or weaker (4 alcohol, for
isolation of the nerve-fibres of the retina, for instance—
Turn).

524, Salt Solution 10 per cent. solution of sodium chloride
is a valuable macerating medium. Weaker strengths, down
to 0°6 per cent., are also used.

525. Motescnorr and Piso Bormu’s Sodium Chloride and
Alechol (Motescuort’s Untersuchungen zw Natwlehre, xi, pp.
99—107; Ranvigr, Traité, p. 242)—10 per cent. solution of
sodium chloride, 5 volumes ; absolute alcohol, 1 volume.

272, CHAPTER XXIII.

For vibratile epithelium Ranvizr finds the mixture in-
ferior to one-third alcohol.

526. Sodium Chloride and Formaldehyde——Gace recommends
the addition of 2 parts of formalin to 1000 parts of normal
salt solution (quoted from Fisu, Proc. Amer. Mic. Soc., xvii,
1895, p. 328).

527. Caustic Potash, Caustic Soda.—These solutions should
be employed strong, 35 to 50 per cent. (MoLescHorr) ; so em-
ployed they do not greatly alter the forms of cells, whilst
weak solutions destroy all the elements. (Weak solutions
may, however, be employed for dissociating the cells of epi-
dermis, hairs, and nails.) he strong solutions may be em-
ployed 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, which constitutes a mounting
medium (see Besrens, Kosset, and Scuierrerpecker, Das
Mikroskop, i, 1889, p. 156). See also Gaag, Proc. Amer.
Soc. of Microscopists, 1889, p. 35.

528. Baryta-water, Lime-water (Fou, Lehrb., p. 110).—Baryta-
water will macerate nerve, muscle, and connective tissue in afew hours,
lime-water in a few days.

529. Sulphocyanides of Ammonium and Potassium (STIRLING,
Journ. Anat. and Phys., xvii, 1883, p. 208)—10 per cent. solution of
either of these salts, for epithelium. Macerate small pieces for twenty-
four to forty-eight hours.

Sovunier (Travaux de VInst. Zool. de Montpellier, Nouv. Sér., 2, 1891,
p. 171) has found that STrRLING’s solution greatly deteriorates cellular
elements, but that good results are obtained by combining it with a
fiwing agent. The best results were obtained with a 2 per cent. solution
of sulphocyanide combined with liquid of Ripart and Prtit; good
ones, by combining liquid of Ripart and Petit with artificial serum of
KRONECKER instead of sulphocyanide, or with pepsin, eau de Javelle,
10 per cent. sulphate of soda, or 15 per cent. solution of caustic soda;
also by combining solutions of chloride of sodium, or solutions of
caustic potash or soda, with any of the usual fixing agents.

530. Lawpors’s Solution (Arch. mikr, Anat., 1885, p. 445).

Saturated sol. of neutral chromate of ammonia 5 parts.
Saturated sol. of phosphate of potash —. ; D4,
Saturated sol. of sulphate of soda. D4,

Distilled water. : . : : . 100

»

co

MACERATION, DIGESTION, AND CORROSION. 272

Small pieces of tissue are macerated for one to three, or
even four to five days, in the liquid, then brought for twenty-
four hours into ammonia carmine diluted with one volume of
the macerating liquid.

Gierks 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
recommended for the same purpose by Nanszn (v. Zeit. wiss.
Mtk., v, 1888, p. 242).

531. Bichromate of Potash —0'2 per cent.

Hsia (Fauna u. Flora Golf. Neapel, 16 Monog., 1887,
p. 297) macerates Capitellide in 0°5 to 1 per cent. solution
for months or years, a little thymol being added against
mould.

Miuller’s Solution, diluted to same strength, or combined
with saliva, has also been used.

Brock (for nervous system of Mollusca, Intern. Monatssch,
Anat., 1, 1884, p. 249) takes equal parts of 10 per cent.
solution of bichromate of potash and visceral fluid of the
animal,

532, Permanganate of Potash.—Is recommended, either
alone or combined with alum, as the best dissociating agent
for the fibres of the cornea (Rotierr, Stricker’s Handbuch,
p. 1108). I have found it, for some objects, very energetic.

533. 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).

534, Osmic and Acetic Acid (the Hurrwics, Das Nerven-
system wu. die Sinnesorgane der Medusen, Leipzig, 1878, and
Jen. Zeitschr., xiii, 1879, p. 457).

0-05 per cent. osmic acid . 1 part.
0-2 oi acetic acid . we. des

Meduse 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

18

27-4 CHAPTER XXII.

osmic acid are removed ; they then remain for 4 day in 0:1
per cent. acetic acid, are washed in water, stained in Bratr’s
carmine, and preserved in glycerin.

For Actinia the osmic acid is taken weaker, 0°04 per cent. ;
both the solutions are made with sea water ; and the washing
out is done with 02 per cent. acetic acid. If the maceration
is complete, stain with picro-carmine; if not, with Brazu’s

carmine.

535. Mosrus’s Media (Morph. Jahrb., xii, 1887, p. 174).
1. One part of sea water with 4 to 6 parts of 0-4 per cent. solution of

bichromate of potash.
2. 0°25 per cent. chromic acid, 01 per cent. osmic acid, 01 per cent.
acetic acid, dissolved in sea water. For Lamellibranchiata. Macerate

for several days.

586. 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 pre-
served for a long time (Hopkins, Proc. Amer. Soc. of Micro-
scopists, 1890, p. 165).

Maceration is greatly aided by heat, and at a temperature
of 40° to 50° C. may be sufficiently complete in an hour
(Gage).

A mixture of equal parts of mitric acid, glycerin, and water
is recommended by Marcacci (drch. Ital. Biol., iv, 1883,
p. 293) for smooth muscle.

587. Nitric Acid and Chlorate of Potash (Ktunu, Ueber die
peripherischen Endorgane, etc., 1862; Ranvirr, Traité, 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.

538. Nitric and Acetic Acid (APATHY, Zett. wiss. Mik., x, 1898,
p. 49).—3 vols. glacial acetic acid, 3 of nitric acid, and 20 each of water,
glycerin, and absolute alcohol. Macerate leeches for twenty-four hours,
and bring them into 70 per cent. alcohol, in which they swell; then

MACERATION, DIGESTION, AND CORROSION. 275

after twenty-four hours, 50 per cent. glycerin, changed till the acid is
removed.

539. Hydrochloric Acid.—K6niIGstEIN (Sttzb. Akad. Wien, 1xxi,
1875) takes (for gold-impregnated corne) equal parts of the concen-
trated acid, glycerin, and water; Freup (ibid., lxxviii, 1879, p. 102, for
nerve-impregnations) 10 parts of acid, 7 of water, 3 of glycerin; and
SCHUBERG and ScHRODER (Zeit. wiss. Zool., lxxvi, 1904, p. 516) take
(for fresh muscles of Hirudinea) hydrochloric acid of 5 per cent.

540. Bina Hatiur’s Mixture (Morphol, Jahrb., xi, p. 321).
—One part glacial acetic acid, 1 part glycerin, 2 parts water.
For the central nervous system of Mollusca a maceration of
thirty to forty minutes may be sufficient.

541. Sulphuric Acid (Ranvier, Tratté, p. 78).—Macerate for
twenty-four hours in 30 grms. of water, to which are added
4 to 5 drops of concentrated sulphuric acid. Agitate. For
nasal mucosa, crystalline, retina, etc.

Ovrnius 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 8 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).

542. 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. mik. 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).

544, Gaan’s Picrie Alcohol (Proc. Amer. Soc. of Microscopists,
1890, p. 120).—95 per cent. alcohol, 250 parts; water, 750;
picric acid, 1. Recommended especially for epithelia and
muscle, <A few hours suffice.

545, Chloral Hydrate.—In not too strong solution, from 2 to
5 per cent. for instance, chloral hydrate is a wild macerating

276 CHAPTER XXIL.

agent that admirably preserves delicate elements. Lavpow-
sky (Arch. mik, Anat., 1876, p. 359) recommends it greatly
for salivary glands, Hickson (Quart. Jowrn, Mic. Sct., 1885,
p. 244) for the retina of Arthropods.

546. Lysol (Ruinxe, Anat. Anz., viii, 1892, p. 582).—10
per cent. solution in distilled water or in water with alcohol
and glycerin, Spermatozoa of the rat or cortical cells of
hairs are said to be resolved into fibrils in a few minutes,
epithelial cells of salamandra to be dissociated instantaneously.

Digestion.

547. Digestion is maceration in organic juices, which by
dissolving out some of the constituents of tissues earlier than
others serves to isolate those which resist. The chief liquids
employed are gastric juice (or pepsin) and pancreatic juice
(pancreatin or trypsin).

Pepsin is best employed in acidified solution, pancreatin
in alkaline.

The most favourable temperature for digestion is about
40° C.

Pepsin digests albuminoids, collagen substance and mucin,
more or less readily, elastin more slowly. Nuclein is either
not dissolved or very slowly. Keratin, neurokeratin, chitin,
fat and carbohydrates are not attacked.

Pancreatin (trypsin) digests albuminoids, nuclein, mucin,
and elastic tissue; whilst collagen substance, reticular
tissue, chitin, horny substances, fat and carbohydrates are
not attacked.

Tissues for digestion should be fresh, or fixed with alcohol,
not with chromic acid or other salts of the heavy metals.

548. Pepsin (Bratn’s, clrchives 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. Hight tenths of a grain will dissolve
100 grains of coagulated white of egg.

To prepare the digestion fluid, the powder is dissolved in
distilled water, and the solution filtered. Or the powder may
be dissolved in glycerin, The tissues to be digested may be

MACERATION, DIGESTION, AND CORROSION. 277

kept for some hours in the liquid at a temperature of 100° F.
(37° C.)
Bricxe’s (from Carnoy’s Biologie cellulaire, p. 94).
Glycerinated extract of pig’s stomach . 1 vol.
0-2 per cent. solution of HCl. ‘ . 3 vols,
Thymol, a few crystals.

Bicxratvi’s (Centrabl. med. Wiss., 1883, p. 838).—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 for not more
than half an hour to an hour.

Kusxow’s (Arch. mik, Anat., xxx, p. 32):—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 Nuche)
remain in it at the ordinary temperature for ten to forty
minutes.

549. Pancreatin—ScuterrerDEeckEr’s (Zeit. wiss. Mik., in,
1886, p. 483).—A saturated solution of the “Pankreatinum
siccum,” prepared by Dr. Witte, Rostock, 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.

Ktune’s (Unters. a. d. Phys. Inst. Univ. Heidelberg, 1,2, 1877, p. 219).
—Very complicated.

See also GeDoELst, La Cellule, iii, 1887, p. 117, and v, 1889, p. 126;
Maas, Festschr. Kupffer, 1899, p. 211, and Hornn, Arch. Anat. Phys.,
Anat. Abth., 1897, p. 136 (2 to 2 per cent. solution of Mall’s or Merck’s
pancreatin, with 0°3 per cent. of carbonate of soda; for demonstrating
adenoid tissue in paraffin sections).

Corroston.

550. Corrosion is the operation of destroying the soft parts
that surround hard parts that it is desired to study ; in
short, a means of cleansing hard parts for microscopic study,
It has been applhed to the removal of surrounding tissue
from injected vessels or cavities. For this, see ALTMANN’s
Method (Arch. mik. Anat., 1879, p. 471, or previous editions) ;
also Ressek (Bibliogr. Anat., iv, 1897, p. 229); Brian (Anta.
Anz., xiv, 1898, p. 418) ; Dewxer (Anat. Hefte., 1900, p 300) ;
Troma and Fromasrez (Arch. Entwickelungsmech, vii, 1898.

278 CHAPTER XXIII.

p. 678) ; Peanopy (Z. Bull., Boston, 1897, p. 164). The fol-
lowing sections relate chiefly to the cleansing of native hard
parts.

551. Caustic Potash, Caustic Soda, Nitric Acid —Boiling, or
long soaking in a strong solution of either of these is an
efficient means of removing soft parts from skeletal structures
(appendages of Arthropods, spicula of sponges, etc.).

552, Eau de Javelle (Hypochlorite of Potash) (Nout, Zool.
Anzeig., 122, 1882, p. 528).—A piece of sponge, or similar
object, 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, dehydrated, and mounted in balsam.

The process is applicable to calcareous structures.

553. Eau de Labarraque (Hypochlorite of Soda) may be used
in the same way as eau de Javelle. Looss (Zool. Anzeiy.,
1885, p. 383) finds that either of these solutions will com-
pletely dissolve chitin in a short time with the aid of heat.
For this purpose the commercial solution should be taken
concentrated and boiling.

If solutions diluted with 4 to 6 volumes of water be taken,
and chitinous structures be macerated in them for twenty-
four hours or more, according to size, the chitin is not dis-
solved, but becomes transparent, soft and permeable to
staining 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, and also to the removal of the
albumen from ova of Amphibia, etc.

CHAPTER XXIV.
DECALCIFICATION, DESILIC[FICATION, AND BLEACHING.

Decalcification.

554. Decalcification In order to obtain the best results, it
is important to employ only material that has been duly fixed
and hardened, and it is well not to put too much confidence in
reagents that are said to have the property of hardening and
decalcifying fresh material at the same time.

It is generally well also to employ fluids that contain
substances having a shrinking action on tissues, so as to
neutralise the swelling frequently brought about by the
decalcifying acids, Large quantities of liquid should be
employed.

After decalcification the excess of acid should be carefully
removed by washing, not in water, which favours swelling,
but in some liquid that has rather a shrinking action, e. g.
alum solution. Lastly, the tissnes should be neutralised by
treatment with carbonate of lime, or a salt of lithium or
sodium or the like.

Rousseau (Zeit. wiss. Mik., xiv, 1897, p. 207) imbeds
fixed material in celloidin, brings it into 85 per cent. alcohol,
decalcifies in a very acid mixture (15 to 40 per cent. of nitric
acid in alcohol), washes ont the acid in alcohol containing
precipitated carbonate of lime, then cuts sections. ‘lhis for
Porifera, corals, Kchinoderms, etc. Tissues are said to be
well preserved.

This process has been applied to the study of the temporal
bone of Mammals by Stun (Anat. Anz., xvii, 1900, p. 318).

Similarly Boprcker (Zeit. wiss. Mik., xii, p. 190; xxv, p.
21; xxvi, p. 206; and xxviii, p. 158), in a complicated way,
adding the acid (6 to 10 per cent.) to the thin celloidin
solution taken for imbedding.

280 CHAPTER XXIV.

555. Decalcification of Bone—I take the following from
Buscu, Arch. mik. Anat., xiv, 1877, p. 481; see also Have,
in Zeit. wiss. Mik., viii, 1891, p. 1; and Scuarrmr, 2bid., xix,
1903, pp. 308 and 441, and his paper in the Encycl. mek.
Technik.

The most widely used, though not the best, agent for
decalcification is hydrochloric acid. Its action is rapid,
even when every dilute, but causes serious swelling of the
tissues. To remedy this, chromic acid or alcohol may be
added to it. Or a3 per cent. solution of the acid may
be taken and have dissolved in it 10 to 15 per cent. of
common salt. Or (Watpryer) to a qgyq per cent. solution
of chloride of palladium may be added +5 of its volume of
HCl.

Chromic acid is also much used, but has a very weak de-
calcifying action and a strong shrinking action on tissues.
For this reason it should 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
decalcifying power, but cause great swelling. Picric acid
has a very slow action, and is only suitable for very small
structures.

556. Nitric Acid (Buscu, loc. cit.)—To all other agents
Buscu prefers nitric acid, which causes no swelling and acts
most efficaciously.

One volume of chemically pure nitric acid of sp. gr. 1:25
is diluted with 10 vols. water. Jt 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 7s
changed daily, for eight or ten days. They must be removed
as soon as the decalcification is complete, or else they wiil
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 foctal bones may be placed in the first instance

DECALCIFICATION, DESILICIFICATION, AND BLEACHING. 281

in a mixture containing 1 per cent. bichromate of potash and
zy 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 (;5 per cent.) or bichromate of potash (1 per
cent.). By putting them afterwards into alcohol a green
stain is obtained.

557. Nitric Acid (Scuarren, Zeit. wiss. Mtk., xix, 1908,
p- 460).—Scuarrer also finds nitric acid the best reagent.
It should be taken pure; the addition of formol, alcohol, or
the like, slows the reaction. The best strength is from 3 to
5 per cent. Objects must not be washed out directly with
water, and washing in salt solution, alcohol, phloroglucin, or
formol is not sufficient to prevent swelling. Alum in 5 per
cent. solution is good, but not necessary. Material should
be well fixed and imbedded in celloidin ($ 554) ; harden im
alcohol ; remove the alcohol with water; put for 12 to 24
hours (large specimens longer) into nitric acid of 3 to 5 per
cent , then into a 5 per cent. solution of sulphate of lithium
or sodium, to be changed once in the course of 12 to 24
hours ; running water, 48 hours; alcohol.

558. Nitric Acid and Alcohol.—3 per cent. of nitric acid in
70 per cent. alcohol. Mayer has long used 5 per cent. acid
in 90 per cent. alcohol. Soak specimens for several days
or weeks. Pure nitric acid, even if weak, readily exercises
a gelatinising action on bone; whilst the addition of alcohol
(or of alum) counteracts this action (Fis, Ref. Handb. Med.
Sci., Supp., p. 425).

THoma (Zeit. wiss. Mik., vil, 2, 1891, p. 191) takes five
vols. of 95 per cent. alcohol and 1 volume pure concentrated
nitric acid. Leave bones in this mixture, 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 (¢.e. for some
days after no acid reaction is obtained with litmus paper) in
95 per cent. alcohol containing an excess of precipitated
carbonate of lime. This may take eight to fourteen days,
after which the tissues will stain well and may be treated as
desired.

282 CHAPTER XXIV.

559. Nitric Acid and Formol—Scurippn (Haematol, Techn.,
Jena, 1910, p. 21) decalcifies material fixed in formol or
formol-Miiller in a mixture of 1 part of formol, 1 of nitric
acid, and 9 of water.

560. Nitric Acid and Alum ((tace, quoted from Fisn, § 558).
—A saturated aqueous solution of alum is diluted with an
equal volume of water, and to each 100 cc. of the dilute
solution is added 5 c.c. of strong nitric acid. Change every
two or three days, until the decalcification is complete. For
teeth this is said to be, perhaps, a better decalcifier than the
alcohol mixture.

561. Sulphurous Acid (Ziaur, Festschr. f. Kupffer, 1899,
p. 51).—A saturated solution in water. Wash out for 24
hours. Acts rapidly and preserves well. Best used after
fixation with formol.

562. Hydrochloric Acid (see § 555).— 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 he added
(von EBNER), see Hava’s paper quoted §555. He takes either 100 c.c.
cold saturated solution of sodium chloride in water, 100 c.c. water, and
4 c.c. hydrochloric acid. Preparations to he placed in this, and 1 to 2 c.c.
hydrochloric acid added daily until they are soft. Or, 25 parts of
hydrochloric acid, 500 of alcohol, 100 of water, and 2°5 of sodium chloride.
Have prefers the proportions of 1-0 to 5:0 of acid, 70 of alcohol, 30 of
water, and 0°5 of salt.

563. Hydrochloric Acid and Chromic Acid (BAyERL, Arch. mik.
Anat., 1885, p. 35)—Equal parts of 3 per cent. chromic acid and 1 per
cent. hydrochloric acid. For ossifying cartilage. Have recommends
equal parts of 1 per cent. hydrochloric acid and 1 per cent. chromic acid
(loc. czt.).

564. Hydrochloric Acid and Glycerin.—Glycerin, 95 ; hydrochloric
acid, 5 (Squire's Methods and Formutl:, p. 12).

565. Trichloracetic Acid—Parrscu (Verh. Ges. D. Naturf.
Aertze, 1895, 2 Theil, 2 Walfte, p. 26) uses a 5 per cent.
aqueous solution, and Neuperaur (Centralb. Phys., xi, 1897,
p- 494) a 4 per cent. one. Action energetic, preservation
said to be excellent,

DECATLCIFICATION, DESILICIFICATION, AND RLWACHING. 283

566. Picrie Acid should be taken saturated and changed frequently.
Its action is weak, but it gives good results with small objects.
Picro-nitric or Picro-hydrochloric Acid.—Action very rapid.

567. Phosphoric Acid.—10 to 15 per cent. (HavG, loc. cit. in § 555).
Somewhat slow, staining not good. According to ScHarrmr, § 557, it
produces swelling.

568. Lactic Acid.—10 per cent. or more. Fairly rapid, preserves
well, and may be recommended (Have, loc. cit.).

569. Chromic Acid is employed in strengths of from 0:1 per cent. to
2 per cent. (but see § 555), 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. Action excessively slow.

570. Chromic and Nitric Acid.—SEILER (For, Lehrb., p. 112) takes
70 volumes of 1 per cent. chromic acid, 3 of nitric acid, and 200 of water.
The action is still excessively slow, frequently requiring months to he
complete.

571. Chromo-aceto-osmic Acid (Van ver Srricut, Arch. Biol.,
ix, 1889, p. 29; and Scuarrer, Zeit. wiss. Mik., x, 1898,
p. 179).—Objects to be left in it for months, the liquid being
changed at first every two days, afterwards less frequently.
Structure well preserved.

572. Arsenic Acid.—4 per cent. aqueous solution, used at a tempera-
ture of 30° to 40° C. (SquirE’s Methods and Formule, etc., p. 11).

573. Phloroglucin with Acids (ANDEER, Centralb. med. Wiss.,
xli, xxxiii, pp. 193, 579; Intern. Monatsschr., i, p. 350; Have, Zeit.
wiss. Mik., viii, 1891, p. 8; FERrerti, zbid., ix, 1892, p. 236; Bull. R.
Avcad. Med. di Boma, 1892, p. 67).—This is said to be the most rapid
method of any. Phloroglucin by itself is not a solvent of lime salts;
its function in the mixture given below is so to protect the organic
elements of tissues against the action of the mineral acid that this can
be used in a much more concentrated form than would be otherwise
advisable.

ANDEER takes a saturated solution in warm water, and adds to it 5 to
50 per cent. of hydrochloric acid. Wash out in running water.

Other acids than hydrochloric may, of course, be taken. See Have,
Zeit. wiss. Mik., viii, 1891, p. 8, and FERRERI, Bull. Acad. Med. Roma,
1892, p. 67, or (for both) fifth edition.

Desilicification.

574. Hydrofluoric Acid (Mayzr, Zool. Anz., 1881, p. 593).—
The objects are brought in alcohol into a glass vessel coated

284 CHAPTER XXIV.

internally with paraffin. Hydrofluorie acid is then added
drop by drop (taking great care to avoid the fumes, which
attack mucous membranes with great energy). Small pieces
of siliceous sponges will be completely desilicified in a few
hours, or at most a day. The tissues do not suffer.

For sponges I find that this dangerons method can be avoided. If
well imbedded, sections may be made from them without previous

removal of the spicula, which appear to break off sharp hefore the
knife.

Rousseau imbeds the objects in celloidin, as described § 554, then
brings the block, in a covered caoutchouc dish, for a day or two into a
mixture of 50 ¢.c. alcohol and 20 to 30 drops of hydrofiuoric acid, and

washes out the acid with alcohol containing carbonate of lithia in
powder.

Bleaching.

575. Mayer’s Chlorine Method (Mitth. Zool. Stat. Neapel,
ii, 1881, p. 8).—Put into a glass tube a few crystals of
chlorate of potash, add two or three drops of hydrochloric
acid, and as soon as the green colour of the evolving chlorine
has begun to show itself, add a few cubic centimetres of
alcoho] of 50 to 70 per cent. Now put the objects (which
must have previously been soaked in alcohol of 70 to 90 per
cent.) into the tube. They float at first, but eventually
sink. They will be found bleached in from a quarter of an
hour to one or two days, without the tissues having suffered.
Only in obstinate cases should the liquid be warmed or more
acid taken. Sections on slides may be bleached in this way.
Instead of hydrochloric acid nitric acid may be taken, in
which case the active agent evolved is oxygen instead of
chlorine.

This method serves both for removing natural pigments,
such as those of the skin or of the eyes of Arthropods, and
also for bleaching material that has been blackened by
osmic acid, and, according to renewed experiments of
Mayen’s, is to be preferred to the peroxide of hydrogen
method.

For bleaching chitin of insects, not alcohol but water
should be added to the chlorate and acid (Mayer), Arch. Anat.
Phys., 1874, p. 321).

See also MAYER in Zett. wiss. Mik., xxiv, 1907, p. 353 (paraffin sections
exposed to the vapour of chlorine water),

DECALCIFIGATION, DUSILICIFICATION, AND BLUACHING. 285

GRYNFELTT and Mestrezat (C. R. Soc. Biol., 1xi, 1906, p. 87) add
2 c.c. of 20 per cent. solution of chloric acid (HC1O,) to 15 ¢.c. of aleohol
and put sections (of retina) into it for several hours at 42°C.

576. Eau de Labarraque. Eau de Javelle (see §§ 552, 553).—
These are bleaching agents. For the manner of preparing a similar
solution see early editions, or Journ. de Microgr., 1887, p. 154, or Journ.
Roy. Mie. Soc., 1887, p. 518. Of course, the method cannot be used for
bleaching soft parts which it is desired to preserve.

577. Peroxide of Hydrogen (Oxygenated Water) (Poucney’s
method, M. Duvat, Précis, etc., p. 234).—Macerate in
glycerin, to which has been added a little oxygenated water
(§ 35), 5 to 6 drops to a watch-glass of glycerin. SoLcur
(Centralbl. med. Wiss., xxi, 1883, p. 177) takes a 3 per cent.
solution of peroxide. Ftrsr (Morph. Arb. Schwalbe, vi,
1896, p. 529) points out that after a time it macerates.

The method serves both for removing pigments and for
bleaching osmic and chromic material.

578. Peroxide of Sodium (Carazzi, Zool. Anz., 444, 1894, p. 135).—
See previous editions.

579. Peroxide of Magnesium (Mayer, Grundziige, p. 290).—Use as
chlorine, § 575. A slow but delicate method.

580. Sulphurous Acid.—Prof. Gison writes me that he finds
alcoholic solution of sulphurous anhydride (SO,) very con-
venient for the rapid decoloration of bichromate objects. A
few drops suffice. Méncxepere and Berux (Arch. mik. Anat.,
liv, 1899, p. 185) obtain the acid by adding to 10 c.c. of a
2 per cent. solution of bisulphate of sodium 2 to 4 drops of
concentrated hydrochloric acid. Objects are put into the
freshly prepared solution for six to twelve hours.

581. Permanganate of Potash.—ALFIERI (Monitore Zool. Ital., viii,
1897, p. 57) bleaches celloidin sections of the choroid, etc., for eight to
twenty-four hours in a 1: 2000 solution of permanganate of potash, then
washes them out for a few hours in a solution of oxalic acid of 1: 300
strength, or weaker.

582. GRENACHER’S Mixture for Hyes of Arthropods and other
Animals (Abh. nat. Ges. Halle-a.-S., xvi; Zeit. wiss. Mtk., 1885, p. 244).
Glycerin ‘ ‘ ‘ : . . 1 part.
80 per cent. aleohol : : . 2 parts.
Mix and add 2 to 3 per cent. iat liydrodhlarto acid.

286 CHAPTER XXIV.

Pigments (2. ¢. those in question) 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.

583. Nitric Acid—ParkeER (Bull. Mus. Comp. Zool., Cambridge,
U.S.A., 1889, p. 173) treats sections (of eyes of scorpions) 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 vers), and the mixture kept cool.

JANDER (Zett, wiss. Mik., xv, 1898, p. 163) takes for removal of pig-
ments SEILER’S chromo-nitric acid (§ 570) ; twelve to forty-eight hours
is enough for small objects.

See also under “ Arthropoda.”

584. Caustic Soda.—Rawirtz (Lettfaden, p. 29) dissolves the pigment
of the mantle of Lamellibranchia by means of 3 to 9 drops of officinal
caustic soda solution added to 15 to 20 ¢.c. of 96 per cent. alcohol.

CHAPTER XXV.
EMBRYOLOGLCAL METHODS.*

585. 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 differentia, 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
obtained from the males in the same way. (It may occa-
sionally be necessary, as in the case of the Stickleback, to
kill the male, and dissect out the testes and tease them.)
The spermatozoa of fish, especially those of the Salmonide,
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. For methods of artificial Purthenogenesis
see Harvey, Biol. Bull. Wood’s Hole, 1910, p. 269.

* The sections in this chapter treating of Mammalia, Aves, and Pisces,
closely follow the Traité des Méthodes Techniques, Lyn et HennEcuy,
and are due almost entirely to Hennecuy. The corresponding parts
of the Grundziige, Luz and Mayer, are taken from this work, and there-
fore also due to Henwnercvy, which I regret to observe has not always
been understood, though duly pointed out in the Preface to the first

edition of the Grundaziige.

288 CHAPTER XXV.

586. Superficial Examination —The development of some
animals, particularly some invertebrates, may be to a certain
extent followed by observations 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, and several pelagic forms, and with Chironomus,
Asellus aquaticus, Ascidians, Planorbis, many Ccelenterata,
ete.

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 (Bausiani).

587. Fixation —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 therefcre
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 bulk.

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.

Scurippe (Zeit. wiss, Mrk., xxvii, 1910, p. 362) finds Orth’s
“ Formol-Miiller ” in general the best fixative. Fix for not
more than 24 hours, and pass through graded alcohols (20
minutes in each) into absolute (1 to 2 hours), cedar oil,
xylol, and paraffin.

EMBRYOLOGICAL METHODS. 289

Rasy (Zeit. wiss. Mik., xi, 1894, p. 165) recommends for
embryos of Vertebrates, and also for other objects, his platinic
sublimate, § 76. This serves for a large number of blasto-
derms and young embryos (Pisces, Amphibia, Aves, Mam-
malia). Advanced embryos of Teleostea ought to be fixed
in the warmed mixture, in order to avoid rupture of the
muscles and shrinkage of the chorda.

Some of his best results were obtained by a not too pro-
longed fixation in a mixture of

Platinic chloride, 1 per cent. solution . 1 vol.
Picric acid, saturated aqueous : . 2-vols
Distilled water. . : . 7

”

Ras’s picro-sublimate mixture has been given § 70. It
is recommended especially for somewhat advanced embryos,
such as embryo chicks from the third or fourth day, and
other embryos of a similar size.

Boveri (Verh. Phys. Med. Ges. Wiirzburg, xxxix, 1895, p.4), in order to
imbed and cut together numbers of ova of Echinoderms, wraps them in
pieces of sloughed epidermis of Cryptobranchus (of course, other Urodela
will do). Sosporra (Arch. mik. Anat., 1, 1897, p. 31) takes pieces of
amnios of Mammalia.

Sanzo (Zeit. wiss. Mik., xxi, 1904, p. 449) describes an automatic
apparatus for fixing material at definite stages.

588. Prerur’s Double-stain for Yolk and Tissue, see § 224.

589. Removal of Albumen.—The thick layers of albumen
that surround many ova area serious obstacle to the penetra-
tion of reagents. Cup (Arch. Ent wickelungsmech., ix, 1900,
p. 587) gives the following as of very general applicability.
After fixation (in any way except with chromic acid) the ova
are brought through graduated alcohols up to that of 80
per cent., in which they are hardened. They are then
brought down again through successive alcohols into water
acidified lightly with any acid (except chromic acid), and the
albumen is found to become transparent and dissolve.

590. Reconstruction of Embryos from Sections.—To facilitate
the study of series of sections, recourse may be had to
graphic or plastic reconstruction of the objects.

In simple cases it may be sufficient to adopt the plan

290 CHAPTER XXV.

described by Scuarrer (Zeit. wiss. Mik., vii, 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. VosmaEr (Anat. Anz., xvi, 1899, p. 269) draws
on plates of celluloid, and sets them up in a rack for ex-
amination. Kerr (Quart. Journ. Mic. Scz., xlv, 1902, p. 1)
draws on plates of ground glass which he afterwards super-
poses and makes transparent by oil of cloves run in between
them. Prensa (Zeit. wiss. Mikr., xxvii, 1910, p. 48) takes
sheets of lithographic gelatin. Woopworrn (Zeit. wiss. Mik.,
xiv, 1897, p. 15) proceeds as follows: (1) Draw an axial
line of the length of the object multiplied by the magnifi-
cation employed. (2) Measure with a micrometer the
greatest diameter of each section. (8) Plot these diameters
down transversely on the axial line at distances correspond-
ing to the thickness of the sections multiplied by the
magnification. (4) Join the extremities of these diameters ;
this will give you an outline of the object. (5) Measure off
on each section the nearest and farthest limits (from the
margin) of the organs to be filled in, and plot them down on
the transverse lines (3), and join the points as before, 7. e.
from section to section ; this will give you the outlines of
the organs.

This process is best applicable to reconstruction from
transverse sections, but it can be applied to reconstruction
from sections in any plane if the object can be provided with
a plane of definition at right angles to the plane of section.
This may be established by cutting off one end of the object,
or the like (see also Orientation, $§ 142, 161).

To make a simple plastic reconstruction, camera drawings
(or photographs) of the sections (all made at the samé
magnification) are pasted on pieces of cardboard of a thick-
ness equal to that of the sections multiplied by the magnifi-
cation employed. Then the parts of the drawings representing
the cavities of the objects are cut out with a knife or fret-
saw, cutting through the cardboard; and the pieces of fret-
work thus obtained are pasted together.

For more elaborate processes of plastic reconstruction (very compli-

cated and seldom necessary) see Born, ‘‘ Die Plattenmodellirmethode,”
in Arch. mik. Anat,, 1883, p. 591, and Zezt, wiss. Mik., v, 1888, p. 433;

EMBRYOLOGICAL METHODS. 291

STRASSER, zbid., iii, 1886, p. 179, and iv, pp. 168 and 330; KasTscHENKO,
ibid., iv, 1887, pp. 235-6 and 353, and v, 1888, p. 173; Scraper, ‘bid,
xiii, 1897, p. 446; ALExaNnpER, ibid., p. 334, and xv, 1899, p. 446;
PETER, ¢bid., xxii, 1906, p. 530; Born and Persr, ibid., xv, 1, p. 31;
and Verh, Anat. Ges., xiii, 1899, p. 184; Jounstron, Anat. Anz., xvi,
1899, p. 261; Fou, Lehrb., p. 35 or previous editions; Broman, Anat.
Hefte, xi, 1899, p. 557; Prrer, “ Die Methoden d. Rekonstruction ”
(Fischer, Jena, 1906); ScHénemann, Anat. Hefte, xviii, 1901, p. 117;
Gags, Anat. Record, i, 1907, p.167; Neumayer, Festschr. f. Kupffer,
1899, p. 459; Marx, Proc. Amer. Acad. Sez., xlii, 1907, p. 629 (electric
wax-cutter for cutting out plates).

Hy (Bull. Johns Hopkins Hosp., xvii, 1906, p. 114) finds that embryos
of mammalia taken from 95 per cent. alcohol and put into caustic potash
of 1 per cent. become so transparent that they can be studied without
cutting and reconstructing.

Mammalia.

591. Rabbit—Dissection—For the study of the early stages
the ova must be sought for in the tube a certain number of
hours after copulation. The dehiscence of the follicles takes
place about ten hours after the first coitus. The tube 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 tubes are then (if the ova are still within them, which
is the case up to the end of the third day after coition) laid
out on along slip of glass, and shit 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 lower power of the microscope.
When the ovaare 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,

292 CHAPTER XXV.

and look for the ova under the microscope by transmitted
light.

Another method, employed by K6riiker, consists in in-
jecting solution of MULLER 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 mesometrium 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 support 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 secondary laparotomy if it be desired
to preserve it for ulterior observations.

During the fourth, fifth, and sith 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
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
Mier, or O'1 per cent. solution of osmic acid, or Kizinen-
BER@s picro-sulphuric acid, or nitric acid, or acetate of
uranium solution. With a small scalpel a longitudinal in-
cision 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.

EMBRYOLOGICAL MUTIODS. 298

From the moment the ova have become adherent to the
uterine mucosa they can no longer be extracted whole. ‘he
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. Ep. v. Brnzpsn (see
Arch, de Biol., v, fase. iti, 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.)

Rerrever (C. BR. Soc. de Biol., 1887, p. 99) advises that
for ova of the seventh day the segment of uterus containing
them be opened ow the mesometrial surface, for at that date
no adhesion has yet been contracted with that side. By
running in liquid of Kleinenberg by means of a pipette
between the ovum and the free surface of the uterus, the
ovum may be got away in the shape of a closed vesicle.

For the study of living eggs (of Rats and Mice) see Marx
and Lone, Cont. Zool. Lab. Mus. Comp. Zool. Harvard Coll.,
1912, No. 225 (description of constant temperature chamber
and circulation slide).

592. Rabpir; Microscopic Preparations.—In order to make
permanent preparations of the different stages of fecundation
and segmentation, v. Bengepen (Arch. de Bivl., i, 1, 1880,
p. 149) brings the living ovum 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 after careful washing.

In order to bring out the outlines of blastoderm cells the
living ovam may be brought into one third per cent. solution
of nitrate of silver. After remaining there for half a minute
to two minntes, according to the age of the vesicle, it is
brought into pure water and exposed to the light.

After the end of the third day the blastodermic vesicle

294 CHAPTER XXV.

can be opened with fine needles, and the blastoderm washed,
stained, or impregnated with gold, and mounted in glycerin
or balsam.

For embryonic areas and more advanced embryos K61Liker
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. Jf the ovum be adherent to the
uterine mucosa the portion of the membrane to which it is
fixed should be left, stretched out with pins, in 0:1 per cent.
solution of osmic acid for from four to six hours. he blasto-
dermic vesicle can then easily be removed, and further
treated as before. For sections K6é.iixer fixes with osmic
acid. v. Benrprn treats the ova for twenty-four hours with
1 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 zona pellucida. v. BEnrprn also recommends
the liquid of Kleinenberg. Henyxrauy writes that he fre-
quently employs it for embryonic areas and embryos of
various ages, always with excellent results. Fol’s modifica-
tion of the liquid of Flemming, and Ranvier and Vignal’s
osmic acid and alcohol mixture (§ 86) also give excellent
results. For staining, Henneauy recommends borax-carmine,
or Delafield’s hematoxylin for small embryos ; for large ones
he found that his acetic acid alum-carmine was the only
reagent that would give a good stain in the mass.

For sections imbed in paraffin.

See also Weyssz, Proc. Amer. Acad. Arts. and Sci., 1894,
p. 285 (blastodermic vesicle of Sus scrofa); Sosorra, Arch.
mik, Anat., xlv, 1895, p. 15 (ovum of the Mouse ; fixation in
Firumine’s weak mixture, sections stained with Benpa’s iron
hematoxylin), and Anut. Hefte, 1 Abth., viii, 1897, p. 476
(Rabbit ; fixation with liquid of Flemming or picro-sublimate
with 2 per cent. acetic acid) ; Bonnet, ibid., ix, 1897, p. 426
(Dog; fixation in sublimate) ; Serena, Stad. Entw. d. Thicre,
Wiesbaden, 1883, p. 5, and 1887, p. 107 (picrosulphuric acid
for the mouse, and picric acid with 55 per cent. of chromic
acid for Didelphys) ; Keine, Morph. Arb., 11, 1898, p. 11
(Sus scrofa); Neumayur, Festschr. f. Kupffer, 1899, p. 458

EMBRYOLOGICAL METHODS. 295

(embryos of the sheep best fixed in Carnoy’s acetic acid,
alcohol, and chloroform, § 85); Wintwarrer, Arch. Biol.,
xvii, 1900, p. 39 (mixture of 50 parts saturated sublimate
in salt solution, 50 parts alcohol, 20 of 1 per cent. platinum
chloride, and 5 of acetic acid); Srss, Encycl. Mik. Techn.,
1910, p. 853 (cornua of Cavia fixed for 12 to 24 hours in
sublimate, and put into 0°5 per cent. osmic acid till hight
brown, then into iodine alcohol, in which the osmium is
reduced) ; Wipaxowicu, Zeit. wiss. Zool., xciv, 1909, p. 243
(Mus rattus, fixation in Genker’s mixture, or 2 parts of
alcohol of 80 per cent. with 1 of formol; also instructions
for dissection).

Aves.

593. Superficial Examination —Instructions on this head are
given in Fosrer and Barour’s Elements of Embryology. The
following is 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
(Duvat).

594. Gerlach’s Window Method (Nature, 1886, p. 497)—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 just 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.

296 CHAPTER XXV.

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 Patron, Journ. Exper. Zool., xi, 1911, p. 469 (cultivation of the
embryo zn vitro).

595. 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 to-
gether.* In later stages, when the embryo is conspicuous,
the blastoderm 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 blasto-
derm 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 Kleinenberg, 10 per cent. nitric acid, etc.). By
keeping the upper end of the pipette closed, and the lower
end in contact with the liquid on the blastoderm, the blasto-
derm 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 blastoderm, 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.

* Anprews (Zeit. wiss. Mik., xxi, 1904, p. 177) separates the blasto-
derm at this stage by injecting picro-sulphurie acid (not any rapidly
acting fixative) firstly between the blastoderm and the vitelline mem-
brane, so as to separate the two above, and then between the blastoderm
and the yolk, so as to free the blastoderm below and float it up. This

done, the membrane may be incised and the blastoderm removed. The
injection is best done with a pipette having a fine point bent upwards.

EMBRYOLOGICAL METHODS, 297

Before putting it into the hardening fluid, the portion of
vitelline membrane that covers the blastoderm should be
removed with forceps and shaking.

Fixation in 10 per cent nitric acid has the advantage of
greatly facilitating the separation of the blastoderm. The
acid should be allowed to act for ten minutes, after which it
is well to bring the preparation into 2 per cent. solution of
alum (cf. Hormann, Zeit. wiss. mik., x, 1898, p. 485). Mirxo-
pHanow (Anat. Hefte, xii, 1899, p. 200) fixes with nitric acid
of 3 per cent., Suscuxin (Nour, Mém. Soc. Nat. Moscow, xvi,
1899, p. 34) with sublimate ; Fiscuen (Morph. Jahrb., xxiv,
1896, p. 371) with Rabl’s platino-sublimate, § 76 (embryos
of the duck); Parrerson (Biol. Bull. Wood’s Hole, xiii,
1907, p. 252) with picro-sulphuric acid containing 8 per cent.
of acetic acid, for an hour (ova of Columba) ; Hoskins
(Kansas Univ. Sci. Bull., iv, 1907, p. 176), after removing
shell, for 5 to 15 minutes in a mixture of 3 parts of 10
per cent. formol with 1 of 10 per cent. nitric acid, and then
excises the embryo.

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 Hennegoy prefers the osmic acid and alcohol
mixture of Ranvier and Vignal, or Flemming’s mixture fol-
lowed by successive alcohols.

Stain and imbed by the usual methods.

Up to about the fiftieth hour embryos may be mounted
entire in glycerin or balsam.

596. M. Duvat’s Orientation Method (Amn. Sc. Nut., 1884,
p- 3).—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 direc-
tion. Duvat, starting from the fact that during incubation
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.

298 CHAPTER XXV.

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 pos-
terior 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 per cent. solution of osmic acid. As soon as the prepara-
tion 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 surface of the yolk a black triangular area, which en-
closes the cicatricula and marks its position ; you cut out
this area with scissors and a scalpel, and complete the harden-
ing with chromic acid and alcohol.

See also the method of Hirora, Journ. Roy. Mic. Soc.,
1895, p. 118.

597. Kionka’s Orientation Method (inat. He/te, 1 Abth.,
ii, 1894, p. 414).—Open the ege under salt solution, free it
from the shell and albumen, and mark the poles by sticking
into it, at about a centimetre from the blastoderm, two hedge-
hog spines, the one at the obtuse end being marked with a
red thread. Put the whole for ten minutes into water at
90° C., then bring into 70 per cent. alcohol, and after twenty-
four hours cut out the blastoderm and a little yolk round it
in the shape of an isosceles triangle, whose base marks the
anterior end of the blastoderm. Paraffin sections stained
with borax-carmine, washed out with acid alcohol containing
one drop of concentrated solution of Orange G for each 5 c.c.,
which stains the yolk.

598. VIALLETON’S Method (Anat. Anz., vii, 1892, p. 624)—Egg
opened in salt solution, blastoderm 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. alcohol for six to twelve hours in the
dark. Borax-carmine, alcohol, damar.

599. Boum and OppeL (Taschenbuch, 1896, p. 80) fix ova with fairly
large embryos in w mixture of 20 parts 3 to 5 per cent. nitric acid and
1 to 2 parts 1 per cent. silver nitrate.

EMBRYOLOGICAL MB&THODS. 299

Reptilia,

600. General Directions.—The methods described above for
birds are applicable to reptiles. During the early stages
the blastoderm should be hardened in situ on the yolk;
later the embryo can be isolated, and treated separately.

Boum and Oprret (Laschenbuch, 1900, p. 186) remove the
shell under salt solution, fix in sublimate with 20 per cent.
acetic acid, or in Lo Branco’s chromo-sublimate (§ 72), then
remove the blastoderm and bring it into alcohol.

601. Special Cases—Mirsuxuri (Journ. Coll. Sc. Japan, vi,
1894, p. 229) fixes embryos of tortoises chiefly with picro-
sulphuric acid. ‘I'o study the blastoderm he removes the
whole of the shell and as much as possible of the albumen,
marks the place where the blastoderm lies with a hair,
brings the whole, with the blastoderm uppermost, into the
fixative, and after a few hours cuts out the blastoderm and
further hardens it by itself. Young embryos generally
adhere to the shell and can, therefore, be fixed in a piece of
it made to serve as a watch-glass, then after half-an-hour
can be removed from it and further hardened alone. If the
embryonal membranes have been formed, the shell may be
scraped away at some spot and there treated with picro-
sulphuric acid until a small hole is formed ; then by working
away from this spot, by means of scraping and dropping
acid on to it, the whole of the shell may be removed.

Witt (Zvol. Jahrb, Abth. Morph., vi, 1892, p. 8) opens
ova of Platydactylus in the fixative (chiefly chromic acid, or
chromo-aceto-osmic acid with very little osmic acid) and
hardens the embryos on the yolk; so also for Cistudo and
Lacerta (1893 and 1895). Mesnerr (Anat. Anz., xi, 1895,
p- 257) does not approve of these methods ; for his own see
Morph. Arb. Schwalbe, i, 1891, p. 370.

Geruarpt (Anat, Anz, xx, 190], p. 244) fixes ova of
Tropidonotus for 24 hours in Nowak’s mixture, § 112.

Batiowi1z (Entwickl. d. Kreuzotter, 1903, p. 19) first fixes
segments of the uterus, each containing an ovum, for 1 or 2
hours, then tears them open with forceps, isolates the ova,
and puts them into fresh fixative, and thence into alcohol of
40 per cent.

300 CHAPTER XXV.

Niconas (Arch. nat. Mie., 1900, p. 457) finds the best
fixative for ova of the slow-worm, as for other large ova, is
Bourn’s picro-formol ($ 110).

See also Purenyi, $48, and Zool. Anz., 1888, pp. 189 and
196, and other methods in early editions.

Anphibea.

602. Preliminary—In order to prepare ova 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 Warrman (Amer. Natural., xxii, 1888, p. 857), and by
Brocumann (Zool. Anz, 1889, p. 269). Wuirman puts the
fixed eggs into a 10 per cent. solution of sodium hypochlorite
diluted with 5 to 6 volumes of water, and leaves them there
till they can be shaken free, which happens (for Nectwrus)
in a few minutes. Buiocamann takes eau de Javelle (potas-
sium hypochlorite), and dilutes it with 3 to 4 volumes of
water, and agitates the eges previously fixed with solution of
Flemming, for fifteen to thirty minutes in it. See also § 589.

Lesrun (La Cellule, xix, 1902, p. 316) advises fixing ova
of Anura for not less than 1} hours in liquid of Gilson, § 69.
The outer envelopes are then hard, and may be easily incised
and the ovum extracted by pressing on the pole opposite to
the incision. The operation should not be delayed until after
hardening in alcohol. Similarly (ibid., xx, 1902, p. 12), for
Urodela.

Guyer (Amer. Nat., xli, 1907, p. 400) finds it suffice to
roll the ova (either fresh or fixed, but before bringing into
alcohol) on blotting paper.

603. Imbedding.—A great difficulty with the ova of Am-
phibia hes in their becoming extremely brittle on imbedding
in paraffin. Carnoy and Lebrun (La Cellule, xii, 1897,
p. 212) fix ovaries or ovarian ova for fifteen minutes to three
quarters of an hour (but see last §) in Gilson’s mercuro-nitric
fluid, § 69, and preserve them in 80 per cent. alcohol. ‘To
iubed, they are brought for a quarter of an hour into 95 per

EMBRYOLOGICAL METHODS. 301

cent. alcohol, five minutes in absolute alcohol, then into a
mixture of alcohol and chloroform in equal parts, and as soon
as they sink in that they are put into pure chloroform.
Paraffin is added to the chloroform, enough to about double
the volume of the whole, and the whole is put for about
three hours into a stove at 35°C. Lastly, the ova are put
for not more than five minutes into a bath of pure paraffin
at 52° C,

Later (ibid., xix, 1902, p. 317) Lzsrun explains that it is
important not to dehydrate completely with absolute alcohol ;
the ova should be left in alcohol of 96 per cent. until chloro-
form can be added without the mixture becoming turbid,
and a second bath of clean paraffin should be added.

See also Morean, Devel. of the Frog's Egg, New York, 1897, p. 171.

604. Siredon.—The ova are easier to prepare than those of
the Anura, because the yolk is separated from the albu-
minous 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.

Fick (Zeit. wiss. Zool., lvi, 1893, p. 529) uses a mixture of
250 parts of 1 per cent. chromic acid, | of acetic acid, and
750 of water.

605. Triton (Scorr and Oszory, Quart. Journ. Mic. Soc.,
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 ont the ovum,
and fix it in solution of Kleinenberg.

Herrwia (Jen. Zeit. Naturw., 1881-2, p. 291) puts the eggs
into a mixture of equal parts of 2 per cent. acetic acid and
05 per cent. chromic acid. After ten hours he incises the
membranes, opening one end of the inner chorion, and turns
out the embryos and brings them into successive alcohols.

Micnagus (Arch. mik. Anat., xlvii, 1896, p. 528) fixes ova,
with their envelopes, in a mixture of concentrated sublimate
solution and concentrated picric acid, twenty parts each,
glacial acetic acid 1, and water 40, but removes the envelopes
before bringing into alcohol.

302 CHAPTER XXV.

606. Salamandra (Ras, Morphol. Jahrb., xii, 2, 1886, p. 252).
—For his more recent methods see § 587.

Grénross (Anat. Anz., xiv, 1898, p. 461) fixes the ova with
a mixture of 50 parts each of saturated sublimate and 0°5
per cent. chromic acid with one part of acetic acid.

607. Rana (O. Herrwia, Jen. Zeit. Naturw., xvi, 1883,
p- 249).—The ova are thrown into nearly boiling water (90°
to 96° C.) for five or 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. chromic
acid for not more than twelve hours, or into alcohol of 70,
80, and 90 per cent. Chromic acid makes ova brittle and
attacks the pigment, whilst alcohol preserves it, which is
frequently important for the study of the germinal layers.

Morean (Amer. Nat., xxv, 1891, p. 759, and Devel. of
the Frog’s Egg, 1897, p. 171) 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 70 per cent. alcohol containing 2 per cent. of
sulphuric acid. Wash in several changes of alcohol of 70 per
cent. About the second day in this 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 Wairman, Meth. of Research, p.156).

Scaurtze (Arch. mik. Anat., lv, 1899, p. 174) removes
with scissors the outer layers of albumen, and puts the ova
for five minutes in 2 per cent. formol warmed to 75° or
80° C. The membrane left on the ova then rises up suffi-
ciently to allow the ova to be got out with needles.

See also Born (tbid., xliti, 1894, p. 1).

Kine (Journ. Morph., xvii, 1901, p. 295, and xix, 1908, p.
370) fixes (for a few minutes) the spawn (of Bufo) in sublimate
(saturated with 5 per cent. of acetic acid), or in Flemming,
Zenker, or Hermann, brings into alcohol, first of 50 and then
80 per cent., and removes the jelly after a few days.

Buus (Trans. Roy. Soc. Hdinburgh, xli, 1905, p. 792) takes
for ova formol of 10 per cent., but for embryos and larvee
the mixture given § 109.

EBMBRYOLOGICAL METHODS. 303

Bouin takes for larve of Rana the formol-sublimate
mixture § 112.

608. Sulphate of Copper Liquid (Fou, Lehrbuch, p. 106, after
Remax and Gortte) ; for hardening ova of Amphibia:

2 per cent. solution of sulphate of copper . 50 ce.

Alcohol of 25 per cent. : : 50 ,,

Rectified wood vinegar. : . 35 drops.
Pisces,

609. 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 Salmonide must be hardened and removed
from their envelopes for the study of the external forms of
the embryo.

To this end they 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 put for
twenty-four hours into distilled water, and then into suc-
cessive alcohols. Embryos thus prepared show no deforma-
tion, 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 into Miller’s solution. Open them therein with fine
scissors—the vitellus, which immediately coagulates on con-
tact 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 (Hennzcvy) 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

304 CHAPTER XXVv.

solution of Kleinenberg, and are then brought through
alcohol of gradually increasing strength.

Cup (quoted from Sumner, Mem. New York Acad. Set.,
ii, 1900, p. 78) fixes for about a minute in sublimate with 10
per cent. of acetic acid, and brings into formalin of 10 per
cent., which is said to give a good fixation of the embryo
without the yolk becoming hard.

610. KoLLMANN’s Fixative (KoLLMANN, Arch. Anat. Phys., 1885,

p. 296).
Bichromic of potash . ; 5 per 100.
Chromic acid. x 2 4
Concentrated nitric acid 2 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.

611. Rasu’s Method see § 587; for KowaLEewsxy’s see Zeit, wiss.
Zool., xliii, 1886, p. 434, or Third Edition.

612. Salmonide.—Hennecuy’s methods have been given,
§ 609.

Kopscu (Arch. mik, Anat., li, 1897, p. 184), on the sug-
gestion of VircHow, fixes embryos for five or ten minutes
in a mixture of 1 part of chromic acid to 50 of glacial acetic
acid and 450 of water, then removes into chromic acid of
1: 500, and as soon as may be removes the capsule and yolk
under salé solution, and completes the hardening in the
chromic acid or the saturated sublimate solution.

Similarly, Benrens (Anat. Hefte, x, 1898, p. 233).—He
opens the ova in the salt solution from the antipolar side,
and frees the embryo from the yolk that remains by blowing
the latter away with a fine-pointed glass tube.

Similarly also Sozorra (ibid., 1902, p. 579).

Guperr (Proc. U.S. Nation. Mus., xxix, 1906, p. 448)
fixes blastoderms in fresh liquid of Perényi, which does not
make the yolk too hard ; later stages in Worcustsr’s liquid
(9 parts of saturated solution of sublimate in formol of 10
per cent. and 1 part of acetic acid), for half an hour to an
hour, and brings gradually into alcohol of 70 per cent.

Bourn (C. RB. Soc. Biol., lv, 1903, p. 1691) fixes for 36 to
48 hours in picro-formol.

Rasi-RvcKHARy’s Method (Arch. Anat. Entw., 1882, p. 118).—Fix in
10 per cent. nitric acid for fifteen minutes. Remove the membranes to

EMBRYOLOGICAL MBLHODS. 305

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 Goronow1tTscuH (see Morph. Juhrb., x,
1884, p. 381).

613. Selachia.—Brarp (Anat. Anz., xviii, 1900, p. 556) has
found that the best fixatives for embryos of Raja are Rabl’s
picro-platinic mixture, § 587 and sublimate.

Living embryos can be observed by scraping the shell thin
with a knife (KastscuunKxo, Anat. slnz., ii, 1888, p. 445, and
His, Arch. Anat. Phys. Anat. Abth., 1897, p. 3). See also
Bravs, Morph. Jahrb., xxxv, 1906, p. 250.

614. Amphioxus.—Sozorra (Arch, ik. Anat., 1, 1897, p.
20) fixes for twenty-four hours in liquid of Flemming ;
Harscuek (Arb. Zool. Inst. Wien., iv, 1881) in picro-sulphuric
acid. Impregnation takes place in the evening, and seg-
mentation is completed during the night.

Lueros (Grundzige, Leu and Maysr, 1910, p. 288) fixes
ova and embryos in equal parts of formol and Flemming.
Sublimate is not good; Rabl’s mixtures are better. Larvex
and young animals ought first to be anesthetised with
cocain in sea-water. After fixation they should remain only
for as short a time as possible in alcohol.

Cerrontaine (Arch. Biol., xxii, 1906, p. 287) fixes with
Flemming or Hermann. For study of ova in toto he orients
them on a slide in clove-oil-collodion which he sets with
chloroform, and adds balsam. For sectioning, he orients in
the same way on a layer of paraffin spread on a cover glass,
and imbeds the whole in paraffin.

615. Pelagic Fish Ova.—WHITMAN (Amer. Natural, xvii, 1883, pp.
1204-5; and Methods of Research, etc., p. 152).—Fix by treatment first
for five to ten minutes with a mixture of equal parts of sea-water and
4 per cent. osmic acid solution, and then for one or two days with a
solution (due to Hisig) of equal parts of 0°25 per cent. platinum chloride
and 1 per cent. chromic acid. Prick the membrane before transferring
to alcohol. See also AGassiz and WuiTman, in Proc. Amer. Acad. Arts
and Sciences, xx, 1884; and CoLiinen, Ann. and Mag. Nat. Hist., x,
1892, p. 228.

RaFFraELE (Mitth. Zool. Stat. Neapel, xii, 1895, p. 169) fixes chiefly
with liquid of Hermann (1 to 2 days), or with a mixture of Mingazzini
(absolute alcohol 1, acetic acid 1, saturated sublimate solution in water 2).

Hence and Eurensaum (Wiss. Meeresunt. Komm. Wiss. Unt. D.

20

306 CHAPTER XXV.

Meere, iii, Heligoland, 1900, pp. 205 and 213) prefer furmol with 39 vols.
of sea-water.

Tunicata,

616. Ova—Daviporr (Mitth. Zool. Stat. Neapel, ix, 1, 1889,
p. 118) fixes the ova of Dis/aplia with a mixture of 3 parts
of saturated solution of corrosive sublimate and 1 of glacial
acetic acid for from half an hour to an hour; or with a
mixture of 3 parts of saturated solution of picric acid and 1
of glacial acetic acid for three to four hours; then 70 per
cent. alcohol.

Casrte (Bull. Mus. Harvard Coll., xxvii, 1896, p. 213)
advises for ova of Ciona liquid of Perényi for twenty
minutes, followed by 70 per cent. alcohol for twenty-four
hours, and for the larvee picro-nitric acid,

617. Test-Cells of Ascidians (Morean, Journ. of Morphol., iv, 1899,
p. 195).—Tease fresh ovaries in very weak osmic acid, wash in distilled
water, treat for half un hour with 1 per cent. silver nitrate, wash for
half an hour in 2 per cent. acetic acid and reduce in sunlight. Imbed

in paraffin. By this process the liméts of the follicle cells are demon-
strated.

618. Buds —Pizon (dan. Se. Naé., xix, 1893, p. 5) studies
the gemmation of the composite Ascidiaus either on entire
corins, which he first bleaches with peroxide of hydrogen and
then stains; or by making sections, after anesthetising the
colonies with cocain of 1: 1000, fixing in glacial acetic acid
or picro-sulphuric or liquid of Flemming, and staining in tuto
with borax carmine or alum carmine, or with a_ strong
solution of methylen blue in alcohol of 90 or 100 per cent.
(after Bernarn, ibed., 1x, 1890, p. 97).

Rirrer (Journ. of Morph., xii, 1896, p. 150) recommends
for fixing Perophora and Goodstria picro-sulphuric acid.

Bryozoa.

619. Statoblasts—Brarm (Dibl. Zool., Chun and Leuckart,
6 Heft, 1890, p. 95) fixest statoblasts of Cristatella with hot
concentrated solution of sublimate for ten minutes, brings
them into water and there incises them with a razor, and

after half an hour passes them gradually into alcohol. He
stains with picro-carmine.

EMBRYOLOGICAL MBTHODS. 307

Mollusca.

620. Cephalopoda (Ussow, -Alrch. de Biol., 11, 1881, p. 582).—
Segmeuting ova are placed in 2 per cent. solution of chromic
acid for two minutes, and then in distilled water, to which a
hittle 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.

Warast (Journ. of Morphol., iv, 1891, p. 249) kills the ova
in the macerating mixture of the Hertwigs (§ 534), and as
soon as the blastoderm turns white and opaque removes it
under dilute glycerin. Treatment with hquid of Perényi is
recommended for surface views.

ViatteTon (Ann. Sc. Nat., vi, 1857, p. 168) brings
ovarian ova of Sepia into a freshly prepared mixture of
picro-sulphuric acid and 2 per cent. solution of bichromate of
potash in equal parts, and after one or two minutes incises
them in the equator, fixes for an hour and a half in picro-
sulphuric acid the halves that contain the formative vitellas,
separates this from the nutritive vitellus with a spatula,
spreads it out, and hardens it in alcohol of 70 to 90 per
cent. He fixes entire ova in liquid of Flemming or osmic
acid.

Korscuetr (Festschrift Leuckart, Leipzig, 1892, p. 348)
fixes advanced embryos of Loligo in liquid of Flemming,
sublimate, picro-sulphuric acid, or 0-2 per cent. chromic acid.
This last is specially good for young embryos if it is washed
out with many changes of picric acid.

Faussex (Mitth. Zool. Stat. Neapel, xiv, 1900, p. 88)
recommends picro-nitric acid. Fix in this, harden in alcohol,
bring the ova, still in their albumen, into hemalum, stain for
24 hours, wash in 1 per cent. alum solution for 24 hours,
when the albumen will be found softened so that the ova
can easily be extracted.

621. Gastropoda (Hennecuy).—Ova of Heliv may be fixed
for from four to six hours in Mayer’s picro-nitric acid.
The carbonate of lime that encrusts the external membrane
is thus dissolved, and the albuminous coat of the egg is

308 CHAPTER XXV.

coagulated. ‘The egg is opened with needles, the albumen
comes away in bits, and the embryo can be removed.

Hencuman (Bull. Mus. Comp. Zool., Harvard, xx, 1890,
p. 171) fixes ova of Dimaxz with 0°33 per cent. chromic acid,
or with liquid of Perényi. 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 Perényi 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.

MEISENHEIMER (Ze?t. wiss. Zool., 1xii, 1896, p. 417) dissects
out the embryos of Limaz and fixes them with picro-sulphuric
acid or concentrated sublimate. Advanced embryos are first
got into extension by means of 2 per cent. cocaine, or are
rapidly killed with hot sublimate.

Scamipr (Hnfw. Pulmonaten, Dorpat, 1891, p. 4) fixes the
ova in toto with concentrated sublimate, and dissects them
out afterwards.

Similarly Koro (Bull. Mus. Harvard Coll., xxvii, 18965,
p. 35). Or, preferably, the ova are put into salt solution,
the shell removed, the albumen removed with a pipette full
of salt solution, which dissolves it; the ova are then fixed
for one minute in Fol’s modification of liquid of Flemming,
and brought direct into Orth’s picro-lithum-carmine. See
also Linvitue, wbid., 1900, p. 215, who adopts this method of
shelling, but prefers fixing in acetic-acid-sublimate, or hquid
of Perényi.

Huyver (Zett. wiss. Zool., xciii, 1909, p. 92), before imbed-
ding embryos of Arion that have been fixed with sublimate,
treats them for an hour or two with carbonate of soda of one
tenth to one fifteenth per cent., which makes the stomach
and intestine less brittle.

Houmgs (Journ. of Merph., 1900, p. 371) teases the egg-capsules of Plan-
orbis in nitrate of silver of # per cent.,exposes to sunlight until the cell-
limits come out, rinses with 0-2 per cent. hyposulphite of soda, puts for
a few minutes into picric acid, and then through alcohol into balsam.

See also WASHBURN, Amer. Anat., xxviii, 1894, p. 528 (liquid of Flem-
ming or 0'3 per cent. chromic acid, or 1 per cent. osmic acid, followed
by liquid of Merkel).

ConkLIN (Journ. of Morph., xiii, 1897, p. 7) fixs ova of Crepidula for
fifteen to thirty minutes in picro-sulphuric acid, and stains with dilute
acidified humatoxylin of Delafield.

EMBRYOLOGICAL METHODS. 309

Kosraneckt and Wierzessxi (Arch. mik. Anat., xlvii, 1896,
p- 313) fix the spawn of Physa fontinalis either in 14 to 2
per cent. nitric acid, or in “sublimate and 3 per cent. nitric
acid in the proportion of 2:1,” and bring through suc-
cessive alcohols. They imbed entire ova in paraffin, but
isolated embryos in celloidin.

622. Cuiton, see Mercatr, Stud. Biol. Lab. Johns Hopkins Univ., v,
1893, p. 251. (Ova with young embryos put for 20 to 45 seconds into
eau de Labarraque, then into water, in which the chorion swells and can
easily be removed.)

623. Lamellibranchiata. -Sraurracner (Jena Zeit., xxviii,
1893, p. 196) fixes embryos of Cyclas in sublimate, stains
with hemalum, and cuts in paraffin.

Littie (Journ, of Morph., x, 1895, p. 7) fixes ova of Unio
for ten to twenty minutes in liquid of Perényi, and preserves
them in 70 per cent. alcohol, or advanced embryos with liquid
of Merkel or sublimate, larvee with 0°65 to 0-1 per cent.
osmic acid, preserving them in glycerin. Glochidia may be
cut with the shell in paraffin of 58° melting-point ; they may
be anzesthetised with chloral hydrate before fixing.

Ai thropoda.

624. Fixation of Ova.—In many cases the ova of Arthropods
are best fixed by heat (§ 13). This may be followed either
by alcohol or some watery hardening agent. If it be desired
to avoid heating, picro-nitric acid may be tried.

625. Removal of Membranes.—It may often be advisable not
to attempt to remove them, but to soften them with eau de
Javelle or ean de Labarraque (see § 553).

Morcan (Amer. Natural., xxii, 1888, p. 357) recommends
(for the ova of Periplaneta) eau de Labarraque diluted with
five to eight volumes of water, and slightly warmed. This
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.

626. Henxina’s Methods (Zeit. wiss. Mik., viii, 1891, p.
156).—Henxkine generally 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.

310 CHAPTER XXV.

He thinks that eaw de Javelle for softening membranes is
best avoided. They should either be dissected away or left
in situ, and cut with the rest of the egg, according to the
nature of the case. ‘T'o avoid brittleness of the yollk proceed
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. 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, oi] of bergamot, and paraffin, and (with
some exceptions, amongst which is Bombyx mort) will be
found to cut without crumbling.

627. Diptera (Henkina, Zit. wiss. Zool., xlvi, 1888, p. 289).
—Ova still contained within the fly may be fixed by plunging
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-carmine for fifteen to thirty
hours, and cut in paraffin.

Brugt (Zool. Jahrb., Abth. Morph., x, 1897, p. 569) fixes
larvee and pup in absolute aleohol heated to 70° to 75° C.,
and containing “a little” sublimate. See also Van Rezs,
thid,, iii, 1888, p. 10.

Benorsson (Handl. Fysiogr. Sallsk Lund., viii, 1897) finds
hot alcoholic solution of sublimate (Frenzel’s, § 69) the best
fixative for larva of Phalacrocera. He could not succeed in
softening the chitin with eau de Javelle.

Piruz (Arch. Zool. erprr., (4), v, 1910, p. 11) fixes pupe
in Bouin’s picro-formol, or Marchoux’s mixture, for twenty-
four hours.

628. Lepidoptera (Borrerzxy, Zeit. wiss. Zool., 1879, p. 198).
—Ova are slightly warmed in water and put for sixteen to
twenty hours in 0°5 per cent. chromic acid. The membranes
can then be removed.

EMBRYOLOGIGAL METHODS. 311

629. Hymenoptera.—Carnrinre & Biircrr (Nova Acta Acad.
Leop. Car., lxix, 1897, p. 273) kill ova of Chalicodoma by
warming in water to 60°C., and fix in aqueous picric acid,
or alcohol of 70 per cent.

Perrunkewitsca (Zool. Jahrb, Abth. Morph., xiv, 1901,
p. 576) fixes for twenty-four hours in his sublimate mixture,
and passes into alcohol of 70 per cent. with iodine.

630. Orthoptera (Parren, Quart. Journ. Mic. Sei., 1884, p.
549).—The ova or larvee (of Blattida) are placed in cold
water, which is gradually 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.

WuHee er (Jowrn. of Morph., ii, 1889, p. 292) dissects out
ovarian ova in salt solution and fixes in liquid of Perényi
(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 eges 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 envelopes to dilate and stand off
from the surface of the egg, so that they can easily be
dissected away.

Hevmons (Zeit. wiss. Zool., li, 1892, p. 434), for young
embryos, incises the cocoon at the end by which it adheres
in the body of the mothcr, brings it for two minutes into
water heated to 90°C., and opens in Flemming, in which
the embryo is dissected out.

Morean (Amer. Natural., xxii, 1888, p. 357) puts ova of
Periplaneta for thirty minutes or an hour into eau de Javelle
diluted with 4 to 8 vols. of water and slightly warmed, which
softens the capsules.

631. Coleoptera—Hirscuier (Zeit. wiss. Zool., xcii, 1909, p.
628) fixes ova of Donacia (after incising the chorion) for two
to three hours in equal parts of sublimate of 6 per cent. and
nitric acid of 3 per cent.

312 CHAPTER XXV.

Satna (Dissert. Marburg, 1906, p. 10) fixes ova of
Tenebrio for about two minutes in a hot mixture of 40 parts
of alcohol of 96 per cent., 4 of nitric acid, and 50 of saturated
aqueous sublimate ; or for 3 minutes in a hot mixture of 1
part of formol with 3 of water.

Kanrawatew (Biol, Centralb., xix, 1899, p. 124) kills larvae
of Anobiwm in hot water, freezes them with ether spray, cuts
away a lateral strip, lets them thaw, and puts for 24 hours

into picro-sulphuric acid.

632, Phalangida—The ova of Plulungiwm opilio possess a
chorion covered with yellow corpuscles that render them
opaque. Baxsranr puts them into water with a few drops of
caustic potash, and raises to boiling point. The ova are then
laid on filter paper, and the chorion removed by rubbing with
a camel’s hair brash, the vitellne membrane remaining
intact, so that the embryo can be studied through it.

Henxine’s method (Zeit. wiss. Zool., xlv, 1886, p. 86).—
Fix with boiling water or Flemming. Preserve thé 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.

633. Araneida—KisninovyE (Journ. Coll. Sei, Inp. Univ.
Japan, iv, 1891, p. 55; Zeit. wiss, Mik., ix, 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.

See also Locy, Bull. Mus. Comp. Zool. Harvard, xii, 3,
1886. Fix by hot water. The liquid of Perényi may also
be used; it has the advantage of not making the yolk so
granular.

Mowrcomery (Journ. Morph., xx, 1909, p. 628) fixes ova
of Theridiwm for 1 or 2 hours in Carnoy & Lebrun’s mixture.

Lampert (ibid., p. 420) fixes ova of Hpetra in picro-sul-
phuric acid warmed to 70° or 80°C.

Porcent (Quart. Journ. Micr. Sci., liv, 1909, p. 7) fixes
ova of Atta in boiling saturated sol. of sublimate in alcohol
of 70 per cent.

EMBRYOLOGICAL METHODS. 313

Hameburaer (Zeit. wiss. Zool., xcvi, 1910, p. 3) fixes ova of
Argyroneta in Gilson’s mixture.

634, Limulus—KinesLuy (Jowrn. Morph., vii, 1892, p. 38)
kills ova by heating in sea-water to 70° or 75° C. and brings
into alcohol of 30 to 70 per cent. Similarly Kisainovyn,
Jonrn, Coll, Sct. Japan, v, 1893, p. 56.

635. Decapoda.— Rutcumnpacn (Abh. Senckenberg Ges. Frank-
furt, xiv, 1886, p. 2) fixes ova of Astacus in water gradually
warmed to 60° or 70°C. (if the chorion should burst, that
is no evil), hardens for twenty-four hours in 1 to 2 per cent.
bichromate of potash or 0°5 per cent. chromic acid, washes
out for the same time in running water, and brings into
alcohol. Remove the chorion, and remove the embryo from
the yolk with a sharp knife.

Herrick (Bull. UL S. Fish. Comm., xv, 1896, p. 226)
kills the ova in hot water, shells and fixes in picro-sulphuric
acid.

For Homarus, see Watre, Bull. Mus. Comp. Zool., xxxv,
1899, p. 155.

636. Amphipoda—Detta Vain (Fauna u. Flora Golf.
Neapel, xx, Monog., 1893, p. 170) puts ova of Orchestia by
means of a pipette into boiling, cold-saturated sublimate
solution, removes them instantly into sea-water, and thence
into weak alcohol. If the chorion does not burst of itself it
must be pricked with a needle.

637. Cladocera.—Haexer (Zellen. w. Be/ruchtungslehre, 1899,
p. 60) fixes females of Sida with winter eggs in a hot mix-
ture of 100 c.c. alcohol of 70 per cent. with 1 to 2 c¢.c. satu-
rated sol. of sublimate. See also Samrer, Zeit. wiss. Zool.,
Ixviii, 1900, p. 176.

638. Copepoda.—Kruzcrr (Arch. Zellforsch., vi, 1911, p.
173) fixes ovaries of Harpactida in Zenker’s mixture with
10 per cent. of formol added. No other liquids give good
results.

314° CHAPTER XXV.

Vermes.

639. Rotatoria.—Junninas (Bull. Mus. Harvard Coll., xxx,
1896, p. 101) finds the best fixative for pregnant females is
the strong liquid of Flemming, but the ova must then be
bleached with chlorate of potash ($ 575).

Lenssen (La Cellule, xiv, 1898, p. 428) fixes ova of
TTydatina with sublimate for 20 seconds.

640. Turbellaria.—Garpiner (Journ. of Morph., xi, 1895,
p. 158) finds the best fixative for ova of Polychoerus is a
mixture of equal parts of absolute alcohol and glacial acetic
acid.

Bresstau (Zeit. wiss. Zool., \xxvi, 1904, p. 219) fixes
Mesostomide with summer-eges in Tellyesniczky’s mixture
(either cold or warmed to 60° or 70°C.) for 10 to 12 hours,
aud washes out for the same time. He incises winter-ova at
one pole, fixes and brings into alcohol of 95 per cent., then
makes an incision at the other pole, and imbeds in paraffin
through cedar oil. In the paraffin, slices of the shell may be
removed with a scalpel, and the ova re-imbedded when suffi-
ciently shelled.

Van per Sraicar (Arch. Biol., xv, 1898, p. 370) finds that
ova of Thysauozoon will only cut well when they have been
not more than two minutes in absolute alcohol followed by
chloroform and paraffin as used by Carnoy and Lebrun,
§ 603.

See also, for Polyclads, Francorts, Arch. Zool. Expér.,
vi, 1898, p. 196; and, for fresh-water Planaria, Ir1ma, Zevt.
wiss. Zool., xl, 1884, p. 359.

641. Cestoda (v. Beneprn, Arch. Biol., ii, 1881, p. 187).—
Ova of Zxnia 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 withdrawing the liquid by means of blotting-paper,
the cover may be made to gradually press on them so as to
burst the membranes, and the embryo may then be treated
with the usual reagents.

Haswent (Quart. Journ, Mier, Sect., liv, 1909, p. 417) fixes

ova of Temnocephala in “ sublimate alcohol,” brings them

EMBRYOLOGICAL MBTIODS. 315

into 90 per cent. alcohol with iodine added, and thence
gradually back into water, softens the shells in weak sodium
hypochlorite, washes and imbeds.

642. Trematoda:—Coxr (Zool. Jahrb., Abth. Morph., ix, 1896,
pp. 563, 566), for the special study of the excretory system
of the Miracidia of Distomum, kills with osmic acid, rinses
with distilled water, and puts for a couple of days into } per
cent. solution of silver nitrate.

Eee-capsules may be softened with 5 per cent. caustic
potash and then burst open (Hucxerr, Bibl. Zool., iv, 1889).

643. Nematoda.—The ova ot Ascaris megalocephala, a clas-
sical object of study, are one of the most impervious things in
the animal kingdom. Years ago For related to me that he
had had ova segmenting right through absolute alcohol into
balsam. Baraitton (Arch. Entwickelungsmech., 1901, p. 149)
has had ova showing living embryos after having been for
six months in liquid of Flemming, and found them to remain
alive for months after drying for 24 hours at 35°C., and
mounting in balsam, and for weeks in acids or alkalies.

Doubtless the best fixative yet made known for ova fur-
nished with their capsules will be found to be that of
Carnoy and Lzsrun, § 86 (La Cellule, xiii, 1897, p. 68). After
fixation the ova are carefully brought into 80 per cent.
alcohol, in which they are preserved. Imbedding should be
carefully done as recommended for the ova of Amphibia
(§ 603), but they ought not to remain in the pure paraffin
for more than a minute to a minute and a half. But these
authors prefer the celloidin method. At least six weeks’
soaking in the different strengths of celloidin will be neces-
sary to ensure penetration. They stain with iron haema-
toxylin.

Zur Srrassen (Arch. Untwickelungsmech., ii, 1896, p. 29)
fixes for twenty-four hours in a mixture of 4 parts 96 per
cent. alcohol and 1 part acetic acid, brings into pure alcohol,
stains with hydrochloric acid carmine, and brings gradually
into glycerin.

Similarly Zosa (Arch. mik. Anat., xlvui, 1896, p. 218) and
Eriancer (ibid., xlix, 1897, p. 309). Zoja stained with Bis-
marck brown and examined in dilute glycerin; Erlanger
made paraffin sections and stained with iron haematoxylin.

316 CHAPTER XXV.

Kostanecxr and Sinpueckr (ibid., xlviii, 1896, p. 184)
employed concentrated sublimate solution, or 8 per cent.
nitric acid or mixtures of these two, for ovarian ova.

Van Benepen and Neyv (Bull. Acad. Belg., 1887, p. 214)
took equal parts of alcohol and acetic acid. Boverr (Jena
Zeit., xxi, 1887, p. 428) fixed in his picro-acetic acid, § 95—
a clearly inadequate method. Gutick (Arch. Zellforsch., vi
1911) has “ fixed ” ova of Heterakis for 22 hours in one third
saturated picric acid with 3 per cent. of glacial acetic acid,
and had them develop in aleohol of 70 per cent. to stages
representing a normal development of several weeks,

Borne (Arch. Zellforsch., iv, 1909, p. 121) spreads ova of
Ascaris on a layer of Mayer’s albumen on a slide, sets the
albumen with a drop of formol, fixes with 4 parts of alcohol
to 1 of acetic acid, stains in alcoholic hydrochloric acid
carmine, and mounts in glycerin.

Artom (Zeit. wiss, Mik., xxv, 1908, p. 5) freezes segments
of the uteri of Ascaris in salt water, and cuts them with the
freezing microtome into disks 30 w thick, and fixes these with
divers liquids.

Crrrontaineg (ibid., xxix, 1912, p. 305) brings fixed ova
from alcohol into absolute alcohol with 1 per cent. of clove
oil, evaporates this down to one tenth, puts into absolute
alcohol with 5 per cent. of clove oil, evaporates again down
to one tenth, then into the same with 5 per cent. of collodion
added, evaporates almost entirely away, and passes through
cedar oil into paraffin.

Echinodermata, Coelenterata, and Porifera.

See the chapter on “ Zoological Methods.”

CHAPTER XXVI.
CYTOLOGICAL METHODS.

644. Study of Living Cells. —In the young larve of Am-
phibia, both Anura and Urodela, the gills and caudal “ fin,”
and sometimes other regions, may be studied in the living
state.

The larve 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.)

In the living animal the epithelial cells and nuclei (in the
state of repose) are so transparent as to be hardly visible in
the natural state. They may, however, be brought out by
curarising the larva; or, still better, by placing the cura-
rised larva for half an hour in 1 per cent. chloride of sodium
solution. Normal larvee 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
large larve, to a watch-glassful of water. From half to one
hour of immersion is necessary for curarisation. The larve
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, wrapped in
blotting-paper. Ifthey be replaced in water they return to
the normal state in eight or ten hours, and may be re-cura-
rised several times.

Other Narcottcs.—Three per cent. alcohol or 3 per cent.
ether, or infusion of tobacco, may be used in a similar way,

318 CHAPTER NXVL.

These reagents cause no obstruction to the processes of cell-
division.

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 (PeremescuKo, Arch.
mik. Anat., xvi, 1879, p. 437).

For the processes of stainiug living cells see $ 208.

645. Study of Fresh and Lightly Fixed Cells.—-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 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 out a piece of living tissue in a drop of acidulated
solution of methyl green (0°75 per cent. of acetic acid). This
ig a delicate fixing agent, killing cells instantly without
change of form. Complete the fixation by exposing the pre-
paration for a quarter of an hour to vapour of osmium, and
add a drop of solution of Ripart 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 Ripart and Petit
and cover.

Or you may kill and fix the cells by teasing in solution of
Ripart and Petit (to which you may add a trace of osmic
acid if you like), and afterwards stain with methyl green.

J have found Pictet’s chloride of manganese ($ 403) useful as
an examination medium. A little solution of dahlia may be
added to it.

Hengine (Zeit. wiss. Mik., viii, 1891, p. 156) recommends
a liquid composed of—

Water . : . 80 ce.
Glycerin ; . 16 ,,
Formic acid. : ; . 38,
Osmic acid of 1 per cent. . : a haere
Dahlia . ; ; ‘ . 0°04 orm,

CYTOLOGICAL METHODS. 319

Other fixing agents, such as picric acid or weak sublimate
solution, may of course be used. Other stains, too, such as
Bismarck brown, and of course other examination media
than solution of Ripart may be employed. But, for general
purposes, the methyl-green-osmium -and-Ripart’s-medium
method gives such good results, and is so very convenient,

that it may be called a classical method for the study of fresh
cells.

646. Some Microchemical Reactions— Methyl green is a test
for chromatin, in so far as (with fresh cells) it colours nothing
but the chromatin iu the nucleus, see § 276. It is, however,
not a perfect test, for the intensity of the coloration it pro-
duces 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.

Chromatin is distinguished from albuminoids by not being
soluble, as these are, in water and in weak mineral acids,
such as O'l 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 carbonate 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 entirely
(Carnoy, Biol. Cell., pp. 208—9). It is only partially digestible
(when i sitw in the nucleus) in the usual laboratory digestion
fluids.

The solvents of chromatin that are the most useful in
practice 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 employed in solutions of 40 to 50 per cent. strength.
If it be desired to remove all the chromatin from a nucleus
the reaction 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.

These operations must be performed on fresh cells, for
hardening agents render chromatin almost insoluble in
ammonia, potash, or sodic phosphate, etc. Hydrochloric

320 CHAPTER XXVI.

acid, however, still swells and dissolves it, though with
difficulty.

Chromatin resists the action of digestive fluids much
longer than the albumins do; so that a moderate digestion
serves to free the chromosomes from any caryoplasmic granula-
tions that may obscure them, whilst at the same time it clears
up the cytoplasm. Unwa (Monatschr. prakt. Derm., xxxiii,
1901, p. 342) digests tissues in solutions of sodium chloride,
to remove the granoplasm.

For Glycogen see CREIGHTON, I'he Formative Property of Glycogen,
London, 1896; Gaer, Trans. Amer. Micr. Sve., xxviii, 1908, p. 208;
Kato, Arch. Ges. Phys., exxvii, 1909, p. 125; Bust, Zett. wiss. Mikr.,
xx, 1904, p. 358, and xxiii, 1909, p. 319; Busou, Arch. Intern. Phys., iii,
1905, p.51; Mayer, Zett. wiss. Mikr., xxvi, 1909, p.513; Arnon,
Sitab. Hetdelberg. Acad. Wiss., 1909, p. 1, 1910, p. 3, and 1911, 14 Abh. ;
Arch. Path. Anat., exciii, 1908, p.175; Arch. mik. Anat., xxiii, 1909, p.
265; Ixxvii, 1911, p.346; Bedtr. path. Anat., li, 1911, p. 439; FramnKet,
Virchow’s Arch., 1911, p.197; Z1EGWALLNER, Zeit. wiss. Mik., xxviii,
1911, p. 152; Neussrt, Bettr. path. Anat., xlv, 1909, p. 38; ERruHarp,
Arch. Zellforsch., viii, 1912, pp. 447 and 507; HEurziicH and Lazarus,
Die Anaemie, 1898, p. 30; PEKELHARING, Petrus Camper, Deel I, 1901,
p. 231; Drizssen, Zezt. wiss. Mck., xxii, 1905, p. 422; Fiscuer, Anat.
Anz., xxvi, 1905, p. 399; Fisssineer, C. &. Soc. Biol., Ixvi, 1909, p. 183;
Nevxircu, Arch. path. Anat., cc, 1910, p. 82; VastTarrni-Crest, Att.
Ace. Med. Chir. Napold, xli, 1907, p. 350, and xliii, 1909, p. 109;
SILBERMANN and Ozorowitz, Bull. Soc. Sci. Bucarest, xvii, 1908, p. 43.

For Phosphorus see MacatLum, Proc. Roy. Soc., lxiii, 1898, p. 467 ;
Heine, Zett. Phys. Chemie, xxii, 1896, p. 132; BrEnstEy, Biol. Bull.
Wood’s Holl, x, 1906, p. 62; Scorr, Journ. Phys. Cambridge, xxxv, 1906,
p. 119.

For Iron see Macatium, Ergeb. Phys. Wiesbaden, vii, 1908, p. 565;
TIRMANN, Goerbersdorfer Veroeffentl., 11, 1898, p. 111; Macatium, Quart.
Jouin. Micr. Scz., xxxviii, 1895, p. 175; ScHNEIDER, Mitth. Zool. Stat.
Neapel, xii, 1895, p. 208; Carnoy and Leprun, La Cellule, xii, 1897,
p. 275; Sumira, Arch. path. Anat., cc, 1910, p. 280; Zauusxr, Zeit.
Phys. Chemie, xiv, 1890; WassERMANN, Anat. Hefte, xlii, 1910, p. 283.

For Copper see Boycr and HERpman, Proc. Roy. Soc., lxii, 1897, p.
35; Macauium, Journ. Phys. Cambridge, xxii, 1897, p. 92; Manrrori,
Arch. Ital. Biol., xxx, 1898, p. 186.

For Zine see MENDEL and BRADLEY, Amer. Journ. Phys., xiv, 1905,
p. 320.

For Lime salts see GRANDIS and Mainini, Arch. Ital. Biol., xxxiv,
1900, p. 75; ScHAFFER, Zezt. wiss. Zool., Ixxxix, 1908, p. 13; LeuteErt,
Encyel. mikr. Technik, ii, p. 588; STOELTZNER, Arch. path. Anat., clxxx,
1905, p. 363 MacaLium, Ergeb. Phys. Wiesbaden, vii, 1908, p. 612.

CYTOLOGICAL MirtHops. 321

For Potassium gee Macatium, Journ. Phys. Cambridge, xxxii, 1905,
p. 95; Ergeb. Phys. Wiesbaden, vii, 1908, p. 600.

For Guanin see Giacomo, Zeit. wiss. Mth, xxvii, 1910, p. 257.

Concerning the microchemistry of the cell in general see further fourth
edition; also CaRNoy & LeBrun, La Cellule, xii, 2, 1897, p. 194; Zr1mMER-
MANN, Die Morphologie wu. Physiologie des Pflanzlichen Zellkernes, Jena,
1896 (treats also of the animal cell); HarcKer, Prawis wu. Theorte der
Zellenund Befruchtungslehre, Jena; PRenant, Jown. Anat. Phys.,
xlvi, 1910, p. 343.

647. Cytological Fixing Agents.—-A fixing agent that is
good for one element of a cellis not necessarily good for all
others.

As regards the nucleus, all fixatives should be acid; for if
not they will not satisfactorily preserve either chromatin or
nucleoli.

For instance, bichromate of potash, if not rendered acid,
fixes chromosomes and nucleoli in a distended state so that
clear images of them are not obtained. Acids contract them
somewhat, and so give them sharper outlines.

The fixatives mostly employed for nuclei are liquid of
FLemmine and liquid of Hermann. ‘here is a slight differ-
ence between them. Liquid of Hermann, owing to the
platinum chloride, causes chromatin to shrink more than
liquid of Flemming does, and for this reason is supposed to
give clearer images of chromosomes, especially of their
splitting. J find that it generally make sthem shrink too
much, and that it is not at all good for spindles.

For many, if not most objects, I prefer tothese two reagents
Bouin’s picro-formol, which gives a highly faithful preserva-
tion and a more penetrating and equable fixation.

Alongside of this I would place Carnoy & Leprun’s acetic-
acid-sublimate fluid, which gives equally fine images of
chromosomes, and is still more penetrating. It is, however,
not go good for spindles. Jor these [recommend Flemming
(picro-formol does not give quite such bold images).

Some of the finest chromosomes I have seen have been
fixed with Linpsay Jounson’s mixture (§ 44), and liquid of
‘TELLYESNICZKY has given me others nearly if not quite as
good.

As regards the cyloplasm. Cytoplasm is made up of two
elements: a fibrillar element—the spongioplasm or mitome ;

21

3229 CHAPTER XXVI.

and a more or less granular liquid that bathes it—the
hyaloplasm or enchylema. It does not follow that a
reagent that will fix one of these will also fix the other.
Nor is it always desirable that both should be equally
fixed.

If you fix both, you will have a full fixation ; but in that
case the granules of the hyaloplasm (be they vital, or be they
only “ precipitation forms,’ see § 29), and the secretions or
other enclosures that may be present in it, may so mask the
fibrils of the spongioplasm as to interfere with the observa-
tion of it. So that if the latter is the principal object of
study, a thin fixation, one in which the spongioplasm is
entirely preserved, but the hyaloplasm only partly, may be
the better.

The spongioplasm is the easier to fix of the two, and the
majority of acid fixatives will preserve it more or less. The
best images I have obtained are those given by liquid of
Flemming or Hermann in cells in which the action of the
reagent has been moderate, 7.e. insufficient to thoroughly
fix the hyaloplasm at the same time. Nearly, if not quite,
as good, is Bouin’s picro-formol, which has the great advan-
tage of being very favourable for plasma-staining. I have
also had very good results with vom Rath’s picro-osmic and
picro-platinosmic mixtures, and with acid sublimate.

Hyaloplasm is not nearly so easy to fix, and there are
only two reagents in common use that readily give a really
full fixation of it; these are osmic actdand bichromate of potash.

Osmic acid acts as a fixative of hyaloplasm in liquid of
Flemming or Hermann, but only gives a full fixation in the
outer layers of the material; and in these it easily happens
that many or most of the cells are ruined by over-fixation
(see $ 35).

This defect may be toa certain degree corrected by taking
the osmic acid weaker than is usual. Thus by successively
reducing the proportion of this ingredient in liquid of
Hermann,* I have found that it can be brought down to

* Nigssine (Arch. mck. Anat., xlvi, 1895, p. 147) has the following
two modifications of Hermann’s mixture :

(1) Platinum chloride, 10 per cent. solution . 25
Osmic acid, 2 per cent. . ; 5 20
Glacial acetic acid ‘ : 5

Distilled water . : : 50

OYTOLOGICAL METHODS. 323

one eighth of the prescribed amount without loss of the dis-
tinctive characters of the fixation.

The defect of want of penetration seems to be incurable
(see §§ 35 and 42). Substitution of more highly penetrat-
ing reagents, such as picric acid, for the chromic acid or
platinum chloride, does not help in the least; you only get.
the osmic fixation outside, no whit deeper than before, and a
picro-acetic fixation, instead of a chromo- or platino-acetic
one, in the deeper layers, that is all.

In view of these defects of osmic mixtures, it may
often be advisable, where hyaloplasm, or its enclosures, is the
chief object of study, to have recourse to bichrumate of potash.
The formula that has given me the finest fixations is that of
Linpsay Jounson, but it has the drawback that there is
risk of osmication in the outer layers.

In this respect liquid of Tellyesniczky, § 42, is to be
preferred.

Corrosive sublimate gives a fairly full fixation; but I
believe it frequently produces serious artefacts, HmmEnnain’s
“Lanthanin” being one of them. Heidenhain’s solution,
§ 64, containing as it does some 11 per cent. of sublimate,
without the addition of any acid to neutralise its shrinking
action, seems to me to be an inadmissibly coarse reagent.
I have, however, obtained with liquid of Carnoy-Lebrun, § 86,
some most excellent fixations of cytoplasm. The aqueous
solutions of sublimate are frequently used in preference to
liquid of Flemming on account of the facilities they afford
for the employment of certain stams; but to that end I
prefer -Boutn’s picro-formol.

ALTMANN’s fixtives for nuclei see fi/th edition, or Arch. Anat. Entw.,
1892, p. 223, and his Elementarorganismen, 1890. His mixture for his
granula see § 43. See also THHOHARI (Journ. de ?Anat., xxxvi, 1900,
p. 216).

(2) The same with a saturated aqueous solution of corrosive
sublimate instead of the water.

They are both of them, in my opinion, as ill-imagined as possible.
They contain some three times as much platinum chloride as Hermann’s,
and Hermann’s contains already quite as much as it can bear, and, I
think, much more than is advisable: see the proportions in the mixtures
§§ 44 and 49. Rasu (Anat. Anz., iv, 1889, p. 21) employed it of from
iy to } per cent. strength, which seems to me much nearer the mark.

324 CITAPTER XXVI.

648. Chromatin Stains —For fresh tissues see § 645.

For sections of hardened tissues, stains should be chosen
amongst those that give a very intense as well as a very
sharp coloration. Some years ago safranin and gentian
violet were the most used. At the present time their place
has been taken by the iron hematoxylin of Benpa or Hurp-
ENHAIN. An alum hematoxylin such as well-ripened Dela-
field’s, or Ehrlich’s, or haemalum, may also give very good
results.

See also Thionin, Kernschwarz, and Iron Carmine, § 220.
For Barainton and Koruter’s borax-methylen-blue see Comptes
Rendus, exvii, 1893, p. 521.

649. Nucleoli are “acidophilous” in so far as, in fixed
material, they select the acid dye or dyes from mixtures such
as the Khrlich-Biondi stain. With this they stain mostly
red, sometimes orange. With fresh material they do not stain
at all with acid methyl green (distinction from chromatin).

But in fixed material treated with basic dyes (safranin,
gentian, etc.) by the regressive method they stain more
energetically than resting chromatin, and at least as much
so as chromatin in the kinetic state. With iron-hematoxylin
they stain sometimes full black, sometimes grey with a black
shell.

They can frequently be well demonstrated in unstained
preparations examined in water, being brought out by their
superior refractivity, and are sometimes visible in the living
cell.

One of the best ways of demonstrating them is to fix with
strong liquid of Flemming, and stain with safranin, followed
by differentiation with acid alcohol (§ 286).

Oxst (Zeit. wiss. Zool., xvi, 1899) fixes in sublimate, stains
in borax carmine, and then stains the sections for three
hours in very dilute aqueous methyl green or solid green.
Nucleoli blue, chromatin red.

Bucuner (Arch. Zellforsch., iii, 1909, p. 337) has found
this useful for distinguishing the accessory chromosome in
testis cells of Orthoptera—normal chromosomes red, accessory
and chromatin nucleoli blue-violet.

ZimMERMANN (Zeit. wiss. Mik., 1896, p. 468) stains for 10
minutes in a fresh mixture of 9 parts 0-1 per cent. aqueous

CYTOLOGICAL METHODS. 325

iodine green with 11 parts concentrated aqueous solution of
fuchsin, and differentiates in absolute aleohol with 1 per cent.
of acetic acid and 0:1 per cent. of iodine. Nucleoli red,
chromatin blue.

Fiscuer (Miairung, etc., p. 140) adds 30 drops of hot 0-1
per cent. fuchsin solution to 100 ¢.c. of 0°3 to 0°5 per cent.
solution of methyl green.

Monreomery (Journ. Morph., xv, 1899) stains for an hour
in IShrlich’s hematoxylin, and then for five minutes in con-
centrated aqueous eosin, or first with concentrated aqueous
methylen blue, and then with concentrated alcoholic solution
of Brazilin.

See also Reppinervs, Virchow's Arch., clxii, 1900, p. 206. For nucleoli
of ova, List, Mitth. Zool. Stat. Neapel, xii, 1896, p. 480; of nerve-cells,
Ruzicka, Zeit. wiss. Mik., xiv, 1898, p. 453, and Lavi, Riv. Pat. Nerv.
Ment. Firenze, iii, 1898, p. 289.

650. Plasma Stains—I have been unable to discover a
single thoroughly satisfactory one. Almost all of them
colour too readily the enclylema or hyaloplasm at the same
time as the spongioplasm. And, on the other hand, there
are many important elements of cells which cannot be got
to stain sufficiently.

I consider Siurefuchsin the most generally recommendable,
especially after iron haematoxylin. See also Bordeaux R.

Flemming’s Orange Method has been much used. It is
very capricious and unreliable.

Ehrlich-Bioudi mixture is a celebrated plasma stain.

The Jron-Hematein Lakes of Benda and M. Heidenhain
give good plasma stains, according to the degree of extrac-
tion, and. would be inferior to none were it not that they
stain in the same tone as the chromatin. See also Ehrlich’s
tri-acid, and his acidophilous mixture, also gold chloride,
Apathy’s process, § 371, and Kernschwarz,

Imperfectly stained plasma structures can often be well
brought out by mounting in Huparal instead of balsam.

651. Centrosomes.—These can be stained by some “acid”
anilin dyes, better by a “neutral” dye (e.g. Flemming’s
orange method, or the Ehrlich-Biondi-Heidenhain stain). But
by fur the best stain is tron-hematoxylin.

326 CHAPTER XXVI.

It is said by Heidenhain that the stain is obtained in a sharper form
by combining the hematoxylin stain with a foregoing stain with
Bordeaux R. He directs (Arch. mik. Anat., xlii, 1894, p. 665) that the
sections (sublimate sections were used by him) are to be stained for
twenty-four hours or more in “a weak ” solution of Bordeanx, until they
have attained such an intensity of colour as that “they would just be fit
for microscopic examination with high powers” (/. ¢., p. 440, note), and
that they be then brought into the ferric alum. After mordanting and
staining, the hematoxylin is to be extracted in the iron alum until the
chromatin has become entirely or almost entirely colourless. Instead
of Bordeaux, “ anilin blue” may be used in the same way.

The images of these objects given by iron-hematoxylin
require to be interpreted with special care. For they
sometimes exhibit the phenomenon termed by FiscHEr
(Fivirung, Firbung und Bau des Protoplasmas, 1899, p. 31,
et passim) ‘“ Spiegelfaerbung,” that is—a bull’s eye effect.
Globular or even elongated objects, such as chromosomes, do
not always yield up their stain simultaneously and equally
throughout their whole depth, but lose it suddenly and
entirely in their outer layers, whilst retaining it in its full
strength in their deeper layers. The still-stained parts thus
remain separated from the decoloured parts by a sharply-
defined limit ; so that a spherical granule in this state will
show a central point deeply stained—the bull’s eye—and
around it a perfectly colourless area—the white of the target.
And when the object is in balsam it is frequently quite
impossible to distinguish the outer limit of this colourless
area, so that the whole object appears to have only the
dimensions of the stained area. It seems that certain
erroneous observations that have been published have been
due to this deception.

Hermann (Arch. mk. Anut., xxxvii, 4, 1891, p. 583) recommends a
modification of the hematoxylin impregnation method of Pat, for which
see fourth edition ; also his paper, ‘‘ Methoden zum Studium des Archo-
plasmas” in Ergebnisse der Anatomie, Band ii, 1892 (1893), p. 23.

For HerpenHain’s Vanadium hee matowylin, see § 269.

Benpa (Verh. Phys. Ges. Berlin, November, 1900, Nr. 1-2; Verh.
Anat. Ges., xv, 1901, p. 167) gives the following as succedanea of the
iron hematoxylin method: The material is to be fixed in alcohol of
about 93 per cent. for two days, then treated for twenty-four hours with
nitric acid diluted with 10 vols. of water. Then bichromate of potash
of 2 per cent., twenty-four hours; chromic acid of 1 per cent., forty-eight
hours; water, twenty-four hours; alcohol; paraffin (or sections by

CYVOLOGIGAL MISTELODS. 327

freezing). The sections are stained either by don himatoxylin (twenty-
four hours in the mordant and in the stain, with differentiation in
Weigert’s borax-ferricyanide), or by either of the two following methods :

(«) The sections are oxidised for five minutes in 0°5 per cent. solution
of permanganate of potash, reduced in Pat’s oxalic mixture till they
become white (about three minutes), dried with blotting-paper flooded
with WrI¢dERt’s methyl-violet-oxalic mixture, or with the crystal-violet
solution, § 330, dried, rinsed with solution of LuGou, rinsed, dried again
with blotting paper, differentiated with a mixture of equal parts of
xylol and anilin oil, dried, rinsed with xylol, balsam.

(b) Sections mordanted twenty-four hours in iron alum of 4 per cent.
or liquor ferri, § 241, diluted with 2 vols. of water, rinsed, stained twenty-
four hours in the sol. of sulphalizarinate of soda, § 653, rinsed, mopped
with blotting-paper, warmed in 0'1 per cent. sol. of toluidin blue till
vapour is given off, stained fifteen minutes more in the same solution
whilst cooling, dipped in acetic acid of 1 per cent., dried with blotting-
paper, dipped in alcohol, differentiated about ten minutes in heech-wood
creosote, dried with blotting-paper, rinsed many times with xylol, then
balsam.

The Nebenkern of spermatic cells may be studied by the
methods indicated for centrosomes. Kernschwarz is also
very useful here.

652. Cell Granules—For the study of the conspicuous
“granules,” undoubtedly metabolic products, occurring in
certain gland-cells and blood- and lymph-corpuscles, and in
certain elements belonging to the group of connective tissues,
see the sections on “ Connective Tissues.” The most gener-
ally employed stains are the mixtures of Euriicu.

Intra vitam staining is useful here (see § 208). See also
Arno.p, Anat. Anz., xxi, 1902, p. 417.

Bunva (Verh. phys. Ges. Berlin, 1899-1900, Nr. 1-4, and
Verh. Anat. Ges., xv, 1901, p. 172) gives the following
method for demonstrating secretion-granules and distinguish-
ing them from other granules: Harden for 24 hours in 10
per cent. formalin, then for one day in 0:25 per cent.
chromic acid, one in 0°33 per cent. and 2 to 3 in 0°5 per
cent., wash one day in water, dehydrate and make paraffin
sections. Then stain with one of Ehrlich’s mixtures, accord-
ing as the granulations are basophilous, acidophilous, or
neutrophilous. The methylen-blue and eosin process of
Michaelis is recommended.

For Prenanv’s Ergastuplasm see especially Garnugr,

328 CHAPTER XXVI.

Bibliogr. Awat., Noy. 6th, 1897, p. 278, and Journ, de P.Inat.,
xxxvi, 1900, p. 22, and under “ Mitochondria,” next §.

ALTMANN (Studien iiber die Zelle, 1886; Die Hlementarorganismen
Leipzig, 1890; Arch. f. Anat. wv. Entwiekel., 1892, p. 223; also Zeit. f.
wiss. Mik., vii, 2, 1890, p. 199; ix, 3, 1893, p. 381; and L. and R. Zosa,
in Mem. R. Ist. Lombardo di Sci. e Lettere , xvi, 3, vii, p. 237) demon-
strates his “ Bioblasts” by fixing for twenty-four hours in a mixture of
equal parts of 5 per cent. bichromate of potash and 2 per cent. osimic
acid, imbedding in paraffin, staining sections for u minute on the slide
held over a flame with a solution of 20 germs. of acid fuchsin in 100 ¢.c.
of anilin water (§ 286), and washing out with saturated alcoholic solution
of picrie acid diluted with 2 volumes of water, heat being used as before
t» aid the differentiation, and finally clearing with xylol and mounting
in balsam. See hereon the critique of FiscHER, in his F’xirung,
Faerbung, uw. Baw des Protoplusmas, pp. 108, 295 (these granules
mainly artefacts).

653. Mitochondria (Chondriosomes, Chondriokonts, Chromidia,
Ergastoplasm, etc.).—These formations are fixed, more or less
abundantly, by most of the usual fixatives. But some
kinds of them seem to be attacked by organic acids; so
that it is well to reduce the proportion of these in mixtures.
Thus Benpa for this purpose makes up Flemming’s strong
mixture with only three to six drops of acetic acid to 15 c.c.
of the chromic, and 4 c.c. of the osmic. Muves (Encyel.
mik, Techn., 1910, 1, p. 476) takes 15 c.c. of chromic acid of
0°5 to 1 per cent., containing 1 per cent. of sodium chloride,
with 8 to 4 c.c. of osmic acid of 2 per cent., and three to four
drops of acetic acid. Similarly, Durspers (Arch. Zellforsch.,
iv, 1910, p. 605). Cuampy (4rch. @ Anat. Mic., xiii, 1911,
p. 55) takes 7 parts of bichromate of potash of 3 per cent.,
7 of chromic acid of 1 per cent., and 4 of osmic acid of 2
per cent. Or iodide of sodium 15 grms. ; water 800 grms. ;
formol 200 grms.; iodide of mercury to saturation. Or
simply formol. Some workers take ALTMANN’s osmic acid and
bichromate, § 43; so Mrves, Arch. mik. Anat., Ixxvi, 1911,
p. 683.

Recaup (4drch. Anat. mic., xi, 1910, fase. 2 and 8) fixes
either in 100 parts of 3 per cent. bichromate of potash with
20 of formol and 5 of acetic acid; or 80 parts of the bi-
chromate to 20 of formol without acetic acid; or in 20 parts
saturated aqueous picric acid with 5 of formol; or in formol
of 10 per cent.; and in either case mordants for one to

CYTOLOGICAL MISTHODS. 329

four weeks in bichromate of 3 per cent. and stains in
iron hematoxylin.

Mitochondria are frequently found stained by many of
the current stains, iron hematoxylin in particular sometimes
staining them with a sharpness that is not attained by any
other method. ‘hese results are more or less accidental
and sporadic: but it is claimed for Benpa’s alizarin method
that it gives a certain and specific stain of them, enabling
them to be distinguished from other morphologically similar
formations.

Benpa’s Alizarin Method (Hryebnisse der Anat., xii, 1902
(1903), p. 752, and other places) is as follows :—Harden for
eight days in strong liquid of Flemming (the acetic acid
therein being reduced to three drops). Wash for an hour
in water and put for twenty-four hours into a mixture of equal
parts of pyroligneous acid and 1 per cent. chromic acid, then for
twenty-four hours into bichromate of potash of 2 per cent.,
wash for twenty-four hours and imbed in paraffin. Sections
on the-slide are mordanted for twenty-four hours with 4 per
cent. solution of ferric alum or diluted lig. ferrt sulfur.
oxydat., then rinsed with water and put for twenty-four
hours into an amber-yellow aqueous solution of Kahlbaum’s
sulfalizarinate of soda, prepared by dropping 1 c¢.c. of
saturated alcoholic solution thereof into 80 to 100 c.c. of
water. Rinse in water, flood the slides with the solution of
crystal violet § 330, diluted with an equal vol. of water, and
warm till vapour is given off. Rinse, differentiate one or
two minutes in 60 per cent. acetic acid (till the nuclei come
out reddish), wash in running water for five to ten minutes,
dry with blotting paper, dip into absolute alcohol, pass
through bergamot oil into xylol and balsam. Mitochondria
violet, chromatin and “archoplasm” brown-red, certain
secretion granules pale violet, centrosomes red violet.

Instead of the staining solution prescribed above (which
may be kept in stock) you may take (Encycl., 11, p. 198) a
freshly prepared mixture of equal parts of anilin water and
saturated alcoholic solution of crystal violet-—and this is to
be preferred.

PRENANT (Journ. de VAnat. et Phys., xlvi, 1910, p. 217) finds that
methylen or toluidin blue, or other basic dyes, may be used instead of
the crystal violet.

330 CHAPTER XXVI.

See also some modifications by Kousrer, Beitr. path. Anat, li, 1911,
p. 209, consisting in fixation and mordanting in certain chrome alum
and chromium fluoride mixtures.

DueEsBere (loc. cit. ante) has found that the treatment with the
chromic and pyroligneous acid and bichromate may be suppressed—
with advantage.

Sztts employs the aluminium-alizarin stain given § 335, in lieu of
the iron-alizarin for Benda’s process.

Some workers (so Mrvzs) prefer to harden as Bunpa, but
to stain with iron hematoxylin instead of by the alizarin
process; the special hardening rendering the hematoxylin
stain sufficiently specific. Thus also Dinetzr, Arch. Zell-
forsch., iv, 1910, p. 673.

Arwnotp (ibid., viii, 1912, p. 256) stains first with iron
hematoxylin, differentiates, stains for twenty to thirty min-
utes with saturated aqueous solution of thionin, passes up
to absolute alcohol, stains for two minutes with Orange G.
dissolved in clove oil, and passes through xylol into balsam.
Chromatin blue, chondriosomes black.

Pensa (cbid. p. 612) has studied the mitochondria and chloroplasts in
plant cells by Ramon y Casa’s silver method for neurofibrils, applied
to sections of fresh tissues (a few minutes to an hour in silver of 1 to
2 per cent., reduction for ten minutes to an hour in a hydroquinon
bath). :

RENAUvT (Comptes rend., clii, 1911, p. 536) demonstrates mitochondria
in fresh cartilage cells by mounting sections in a mixture of artificial
serum and saturated aqueous solution of methyl violet 5 B.

CHAPTER XXVII.
TEGUMENTARY ORGANS,

654. Epithelium.—Both for surface views and for sections
good results are obtained by the nztrate of silver method, the
methylen blwe method, the perchluride of tron and pyrogallol
method of the Hoggans, § 375, the osmic acid and pyrogallol
process, § 874, and by iron-hematoaylin.

For the purpose of separating the epidermis from the
corium, Lorwy (Arch. mik. Anat., xxxvii, 1891, p. 159)
recommends macerating for twenty-four to forty-eight hours,
at a temperature of about 40° C., in 6 per cent. pyroligneous
acid. Acetic acid of } per cent. (PHILIPpson) is also good.
Minor (Amer. Nat., xx, 1886, p. 575) macerates embryos for
several days in 0°6 per cent. salt solution, MrrropHanow (Zeit.
wiss. Mik., v, 1888, p. 573) for a quarter of an hour in 3 per
cent. nitric acid, then 1 hour in one-third alcohol, and, if
need be, 24 in stronger alcohol.

Maver (Lotus, 2, x11, 1892) exposes the cornea or membrana
nictitans of Rana, Bufv, and Mus for half a minute to the
vapour of acetic acid, and then puts it into 0°5 per cent. salt
solution.

For ciliated epithelium see the methods of Engelmann
under “ Mollusca.”

655. Intercellular Bridges (and Canals), Prickle Cells.—See
Inn, in La Cellule, iv, 1888, p. 409, and v, 1889, p. 321; also
Kotossow, Arch. mik. Anat., lu, 1898, p. 1. Kotossow used
an osmic-acid-tannin stain, § 374.

See also Fremmina, Anat. Hefte, 1 Abth, vi, 1895, p. 1.

Besides maceration, impregnation may be useful ; Mrrro-
eHanow (Arch, Anat. Phys., Phys. Abth., 1884, p. 191) has
used gold chloride.

332 CHAPTER XXVII.

Unna (Monatsschr. prakt. Derm., xxxvii, 1908, p. 1) has
described a highly complicated process with Wasserblau and
orcein, see Zeit. wiss. Mik., xxi, 1904, p. 68.

656, Plasma-fibrils of Epithelium—Knromaysnrs process
(Arch. mik, Anat., xxxix, 1892, p. 141) is as follows : Sections
are stained for five minutes in a mixture of equal volumes
of anilin water (§ 286) and concentrated aqueous solution
of methyl violet 68. 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 8:3. Gentian or Krystall-
violett will do instead of methyl violet, but not quite so well.
See also Eurmawn and Japassoun, Arch. Dermatol. u. Syphilis,
1892, 1, p. 803; Zeit. wiss. Mik., ix, 1898, p. 356; Hurx-
HEIMEn, Arch. mik, Anat., li, 1899, p.510; and Rosensrapr,
ibid., Ixxv, 1910, p. 659 (takes the differentiating mixture
much weaker in anilin).

Unna (Monatsschr. prakt. Derm., xix, 1894, p. 1 and p.
277, eb seq. ; Zeit. wiss. Mik., xii, 1, 1895, pp. 61, 63) has
given a whole series of methods, from which the following
are some extracts.

(1) Wasszrpiau-Orcein.—Stain sections for ten minutes
in a neutral aqueous 1 per cent. solution of Wasserblau, rinse
and stain for five or ten minutes in a neutral alcoholic 1 per
cent. solution of Griibler’s orcein. Dehydrate, clear, and
mount in balsam. This may be varied as follows :

(a) Ten minutes in the Wasserblau and thirty minutes or
more in the orcein.

(b) Take for the second stain an acid solution of orcein.

(c) Stain for only one minute in the Wasserblau, but for
thirty or more in the neutral orcein.

(2) Stain for half an hour or more in a strong solution of
hemalum, rinse, stain for half a minute in a saturated aqueous
solution of picric acid, and dehydrate for thirty seconds in
alcohol containing 0°5 per cent. of picric acid.

TRGUMENTARY ORGANS. 333

(3) Hemalum for two hours, neutral orcein as above for
ten to twenty minutes.

More recently Unna advocates the process mentioned
last §.

See alse Ranvier, Arch. Anat. Mokr., iu, 1899, p. 1.

657. Keratohyalin—The keratohyalin granules of the cells
of the stratum granulosum are soluble in mineral acids, and
can. be digested in pepsin. They can be stained with picro-
carmine, alum hematoxylin, van Gieson’s mixture, or Unna’s
Wasserblau-orcein, last §. Fick (Centralb. allg. Path., xiii,
1902, p. 987; Zeit. wiss. Mik., xx, 1903, p. 222) stains sections
of alcohol material for three to four minutes in concentrated
aqueous solution of Kresylechtviolett, differentiates in alcohol,
clears in xylol, and mounts in balsam.

See also Unna, Monatsschr. prakt. Derm., xx, 1895, p. 69; the article
“Haut” in the Encyel. m7k. Technik.; and UNNA and GoLopEtz,
Monatsschr. prakt. Derm., xlix, 1909, p. 95; Larront, Bibl. Anat., 1909,
p. 209.

For Trichohyalin, see GAVAZZENI, Monatsschr. prakt. Derm.,xlvii, 1908,
p. 229.

658, Eleidin.— To demonstrate the stratum granuloswm and
the eleidin granules Ranvier (Arch. Anat. Micr., ii, 1899,
p- 1) hardens with alcohol, stains with picro-carmine, and
treats with lime-water. The cells swell and show up the
granules, which do not change. See loc. cit., other methods
for the study of skin.

Buzzi (see Hueycl. mik. Technik., article “ Haut ’’) stains
sections for a few minutes in a watchglassful of water with
2 to 8 drops of 1 per cent. Congo red. Similarly Werprn-
reco, Arch. mtk. Anat., lvii, 1901, p. 583. Other authors
recommend nigrosin, or Wasserblau, or orcein.

See also Josrpu, “ Dermatohist. Technik,” Berlin, 1905,
and Dreuw, Med. Klinik, Berlin, 1907, Nos. 27 and 28.

For Cholesterin see Gotoperz and Unna, Monatsschr. prakt.
Derm., xlvii, 1908, p. 1.

659. Horn, Hair, and Nails-—The elements of hairs and nails
may be isolated by prolonged maceration in 40 per cent.
potash solution, or by heating with concentrated sulphuric
acid. See also von Naruusius, Zool. Anz., xv, 1892, p. 395.

334 CHAPTER XXVIT.

Horny tissues stain well in safranin or gentian violet
(Ruinke, Arch. f. mik. Anat., xxx, 1887, p. 183; Ernst, ibid,
xlvii, 1896, p. 669; Rast, tbid., xlviti, 1896, p. 489).

Unna (op. cit. last §, p. 598) stains the tyrosin-bearing
keratin in sections of skin for a few seconds or minutes in
a mixture of 5 parts of Millon’s reagent, 5 of water,
and 1 of glycerin, treats shortly with nitric acid of 25 per
cent., and mounts in balsam.

660. Skin-nerves and Nerve-endings—Impregnate with gold
chloride. See Chap. XVII, especially § 365.

661. Tactile Corpuscles.—Sce §§ 364~-366.—Gold methods
are indicated. See also Ranvinr, Tratté, p. 919; Lanarr-
ans, Arch, mik. Anat., 1873, p. 730; Kuxrscuizxy, 2bid.,
1881, p. 858; and Smirnow, Intern. Monatsschr. f. Anat.,
etc., x, 1898, p. 241, who recommends, besides the gold
method of Léwit, the rapid bichromate of silver method of

Golgi.

662. Corpuscles of Herbst and Corpuscles of Grandry.— Docre.
(Arch, Anat. uw. Hntiickel., 1891, p. 182) has used the methylen
blue method. 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
and moistened with aqueous or vitreous humour from the
animal and left for ten to thirty minutes exposed to the air,
then brought into picrate of ammonia, and treated as
described § 343. Gersrra (Intern. Monatsschr. Anat., x,
1893, p. 205) made use of a method of Arnstein, according
to which pieces of skin are put for twenty-four hours into
lime-water, the horny layer removed, the pieces treated for
five minutes with 0°25 per cent. gold chloride, reduced in
water, and the precipitate that forms on them removed
by putting into 0°25 per cent. cyanide of potassium and
brushing.

Now (Anat. Anz, xxxvi, 1910, p. 217) takes Unwa’s
Orcein-wasserblau mixture (Wasserblau O.D., 1 part, orcein
1, acetic acid 5, glycerin 20, alcohol 50, water 100) and
adds to it 1 part more of orcein, ‘To 10 c.c. of this he adds

TEGUMENTARY ORGANS. 335

at the moment of using 10 c.c. of 1 per cent. solution of
eosin in alcohol of 80 per cent. and 3 c.c. of 1 per cent.
solution of hydroquinon. Stain for five to ten minutes,
rinse, stain for ten minutes in | per cent. aqueous solution
of safranin, wash, treat for thirty minutes with 0°5 per cent.
solution of bichromate of potash, dehydrate and mount.

Similarly Doaist, Folia Newrobiol. iv, 1910, p. 218 (also
employing Bielschowsky’s neurofibril method),

663. Corpuscles of Meissner and of Krause (Cornea and Con.
junctiva).—Doatmn (Arch. f. mik. Anat., xxxvii, 1891, p. 602,
and xliv, 1894, p. 15) employs the methylen blue method ;
for details see previous editions.

See also Loneworrn’s methods, Arch. mek. Anat., 1875,
p. 655,

664. Similar Objects.—Papille Foliate of the Rabbit, HERMANN,
see Zeit. wiss. Mik., v, 1888, p. 524; ARNSTEIN, zbid., xiii, 1897, p. 240.
Olfactive Organs of Vertebrates, Doaren, Arch. mzk. Anat., 1887, p.
74. Organs of a “Sixth Sense” in Amphibia, MirropHanow,
Zeit. wiss. Mik., v, 1888, p. 513 (details as to staining with “‘ Wasserblau,”’
for which see also Biol. Centralb., vii, 1887, p. 175). Nerve-endings
in Tongue of Frog, Faserstatn, Arch. de Zool. expér. et gén., vii, 1889,
p. 705. Tongue of Rabbit, von LENHOSSEK, Ze?t. wiss. Mik., xi, 1894,
p. 377 (Ramén y Cajal’s double Golgi-method).

665. Cornea.—There are three chief methods—the methylen
blue, the silver, and the gold method.

For the methylen blue method see particularly § 345,

Negative images of the corneal cells are easily obtained by
the dry silver method (Kinin). The conjunctival epithelium
should be removed by brushing from a living cornea, and the
corneal surface well rubbed 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 (Ranvirr) 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.

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,

336 CHAPTER XXVIU.

or by treating a negatively impregnated cornea with weak
salt solution or weak solution of hydrochloric acid (Hrs).

But the best positive images are those furnished by gold
chloride. Ranvinr prefers hig lemon-juice method. It is
important that the cornea should not remain too long in the
gold solution, ov the nerves alone will be well impregnated.

Zawarsin (Arch. mik. Anat., Ixxiv, 1909, p. 116) removes
the membrane of Descemet for study in the following manner.
A cornea, fixed in sublimate, is dissected out and put for
some hours into a mixture of alcohol and ether. Then
collodion of 4 per cent. is poured on to the inner surface,
and after some time a layer of collodion with the membrane
attached can be pecled off, and the collodion removed from
the tissue by a mixture of alcohol and ether.

See also Rotuerr, in Stricker’s Handb., pp. 1102, 1114, or
previous editions ;. Tarrurert, Anat, dnz., v, 1890, p. 524, or
previous editions ; Craccio, Arch. ital. Biol., wi, p. 75; and

Renavt, C. R. Acad. Se., 1880, p. 137.

666. Crystalline—Gepuarpr (Zeit. wiss. Mik., xiii, 1896,
p- 306) hardens the lens for one or two days in 4 to 10 per
cent. formalin; it is then casily dissociated with needles into
its fibres.

Rasi (Zeit. wiss. Zool., |xv, 1898, p. 272) fixes the
enucleated eye for half an hour in his platinum chloride or
picro-sublimate, $$ 75 and 70, divides it at the equator, and
puts the anterior half back for twenty-four hours into the
fixative.

For Maceration you may use sulphuric acid, § 541.

See also Rosinsxr, Zur Kenntniss d. Augenlinse, Berlin,
1883,

CHAPTER XXVIII.
MUSCLE AND TENDON (NERVE-ENDINGS).

Striated Muscle.

667. Muscle-cells—F'or these and allied subjects see, inter
alia, Beurens, Kossun, und Scuierrerpecker, Das Mikroskop,
etc., vol. ii, pp. 154161; and Scuarer, Proc. Roy. Soc.,
xlix, 1891, p. 280.

Iron hematoxylin gives very fine images of striped muscle,
and so does Mallory’s phospho-tungstic,

For dissociation methods see $$ 527, 536, 537, 544.

To isolate the sarcolemma Songer (Zeit. wiss. Aik , vi, 1889,
p- 189) teases fresh muscle in saturated solution of ammonium
carbonate.

668. Nerve-endings—the Methylen Blue Method —Ior Bizper-
MANN’sS procedure for the muscles of Astacus see § 842 (see
also Zeit. wiss. Mik., vi, 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.

For Hydrophilus piceus, Brspermann proceeded by inject-
ing 0°d c.c. of methylen blue solution between the ultimate
and penultimate abdominal rings, in the ventral furrow, and
keeping the animals alive in water for three to four hours,
then opened the thorax by two lateral incisions, and removed
the muscles of the first pair of legs and exposed them to the
air for three or four hours in a moist chamber, and finally
examined in salt solution.

Geriacu (Sitzb. Akad. Wiss. Miinchen, 1889, ii, p. 125)
injected frogs, either through the abdominal vein or through
the aorta, with 4 to 5¢.c. of a 1: 400 solution in 1 per cent.

22

338 CHAPTER XXVIL.

salt solution, and examined pieces of muscle in serum of the
animal, afterwards fixing with picrate of ammonia and
mounting in glycerin jelly.

The procedure of Doaign has been given, § 342.

669. Nerve-endings—the Gold Method.—l'iscuur (Arch. mek.
Anat., 1876, p. 365) used the method of Lowi:r.

Biepermann (last section) recommends for Astacus a
similar procedure, the preliminary treatment with formic acid
being omitted, and the muscles being put for a couple of days
into glycerin after reduction in the acid.

Ranvier (Lratté, p. 813) finds that for the study of the
motor terminations of Vertebrates the best method is his
lemon-juice process (§ 366).

See also the methods of Avdruy, $$ 868, 871.

670. Nerve-endings—the Silver Method.—Ranvixx employs
it as follows (ibid., p. 810): Portions. of muscle (gastro-
cnemius of frog) having been very carefully teased out in
fresh serum, are treated for ten or 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. They are then ex-
amined in a mixture of equal parts of glycerin and water.

This process gives negative images, the muscular substance
being stained brown, and the nervous arborescence unstained.
The gold process gives positive images, the nervous structures
being stained dark violet.

671. Nerve-endings—the Bichromate of Silver Method —T'he
rapid method of Gotar has been used by Ramon y Casau
for the terminations of nerves and trachez in the muscles
of insects. See Zett. wiss. Mik., vii, 1890, p. 832, or fourth
edition. A modification is used by Wunperer, Arch. mik.
Anat., Ixxi, 1908, p. 528.

672. Muscle-spindles—See Cinimpanis, Arch. mik, Anat.,
Ixxv, 1910, p. 692. Principally entra vitam methylen blue,

MUSCLE AND TENDON (NERVE-ENDINGS). 339

by injection through the internal carotid. For elastic
fibres, Weigert’s resorcin-fuchsin, followed by 1 per cent.
orcein acidified with HCI.

Hlectric Organs.

673. Electric Organs.—Ranviur (Zrait’, Chap. xvii), finds
that osmic acid is the only reagent that will Gx properly the
terminal arborisations on the Jamelle. He injects a little
2 per cent. solution under the surface of the organ, removes
a small portion of it after a few minutes, and puts it into a
quantity of the same solution for twenty-four hours. The
electric plates may then be teased out and examined in
water, and will show the stag’s horn ramifications; and the
dissepiments between the columns will show the bouquets of
Wagner. The terminal arborescence may be impregnated
with silver. A portion of the surface of the organ is
rubbed with lunar caustic until it appears opaque, then
removed and the plates teased out in water. ‘This gives
negative images.

Or, electric plates, isolated by teasing after twenty-four
hours in osmic acid as above, and kept for some days in
one-third alcohol, are washed and placed on a slide with
their ventral surface uppermost. They are then treated with
a few drops of 0°5 per cent. solution of chloride of gold
and potassium, and those which become violet are washed
and mounted in glycerin. ‘This gives positive images.

These may also be obtained by putting material fixed by
osmic acid into 2 per cent. solution of bichromate of
ammonia for a few weeks, then teasing, staining with alum
hematoxylin, and mounting in damar.

Torpedo.—Batiowirz (Arch. mik. Anat., xl, 1893, p. 460)
gets the best results by the rapid Golgi impregnation.

An electric column, with about $ to 1 cm. of tissue
round it, is dissected out, and put for three to four days
into the osmium bichromate mixture; then for one to three
days into # per cent. silver, cut without imbedding and
mounted in xylol balsam. Impregnates all the important
elements. See further, on the whole subject, BatLowrrz,
Eneycl. mik. Techn., 1910, p. 298.

Cavan (Bibl. Anat., xiii, 1904, p. 214) takes material

340 CHAPTER XXVIII.

fixed with osmic acid of 2 per cent. and impregnates it with
gold by the method of Nasias, and mounts in glycerin.

Raja.—Iwanzorr (Bull. Soc. Nat. Moscow, ix, 1895, p. 74)
fixes the organ in the tail of Raja with liquid of Flemming,
stains with hemacalcium and eosin, and makes paraffin
sections.

Batuowirz (Awut. Hefte, 1 Abth., vii, 1897, p. 285) finds
the method of Golgi excellent for this organ. He also makes
sections after fixing in saturated solution of sublimate (in
sea-water), or in liquid of Flemming, and examines them in
water. Methylen-blue may be used, intra vitam. Gold is
little good.

Gymnotus.—BaLiowirz (Hneycl. mk. Technik., p. 303) fixes
with Flemming, and makes sections. He also commends
impregnation with gold chloride, but not the Golgi method.

Malapterurus.— BsaLiowirz (ibid., p. 202) fixes with picro-
sublimate, with Flemming, or with various mixtures of
bichromate, sublimate, and formol, and uses gold chloride
and Golgi impregnations. He macerates in liquid of Miller
or saturated aqueous solution of picric acid.

Tendon.

674. Tendons.—Rerreren (C. R. Soc. Biol., x, 1898, p. 580)
fixes in equal parts of saturated solutions of sublimate and
picric acid, puts for 1 to 3 days into saturated picric acid
with 2 to 3 per cent. of sodium chloride, to remove the
mucin, and imbeds in paraffin.

674a. Union of Muscle and Tendon.—Tor tlis see Rerrunsr
aud Luwuivrs, C. R. Soc. Biol., 1911, No. 12 (orcein for
24 hours, followed by iron heematoxylin) ; and ScHunrze
(Verh. phys. med. Ges. Wtrzburg, 1911, p. 83) (treats for a
day or two with a mixture of equal parts of 2 per cent.
bichromate of potash and alcohol, in the dark, then for
2 days with 0°5 per cent. solution of hematoxylin in alcohol
of 70 per cent., then with Van Gieson’s picro-saiirefuchsin).

675. Corpuscles of Golgi (Ranviur, Traite, p. 929).—Take
the tendon of the anterior and superior insertion of the
gemini muscles of the rabbit. Treat it by the formic acid

MUSCLE AND TENDON (NERVE-ANDINGS). 341

and gold method (§ 365), and after reduction scrape with a
scalpel, in order to remove the muscle-fibres that mask the
musculo-tendinous organs.

Marcu’s methods for the tendons of the motores bulbi
oculi (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
(Gora’s method), and by the method of Manrreptr, § 368.
Mount in glycerin. The methods only succeed completely
during fine, sunny weather,

Rurrint (Atti R. Acc. Lincei Roma Rend. [5], i, 1892,
p. 442) recommends the method of Fischer.

Craccio (Mem. R. Ace. Sci. Bologna [4], t. x, 1890, p. 801)
puts tendons of Amphibia into 0°1 per cent. hydrochloric
acid or 0-2 per cent. acetic acid until transparent ; then for
five minutes into a mixture of 0-1 per cent. gold chloride
and 0:1 per cent. potassium chloride; then back into the
acetic acid, 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 glycerin acidulated with
0°5 per cent. of acetic or formic acid.

Doaten, (Arch. mik. Anat., Ixvii, 1906, p 638) stretches
tendons of eye-muscles on cardboard with hedgehog spines,
puts for 4 or 5 days into nitrate of silver of 1 to 2 per cent.,
reduces for a day in pyrogallic acid with formol, and imbeds
in celloidin.

Smooth Muscle.

676. Tests for Smooth Muscle.—Picro-siiurefuchsin, § 299,
stains muscle yellow, connective tissue red.

Picro-nigrosin, § 325, stains muscle yellowish, connective
tissue blue.

Unna (Eneycl. mik. Technik., article “ Kollagen ”’) stains
for twenty-four hours in orcein 1 part, Wasserblau 0°25,
alcohol 60, glycerin 10, water 30, which gives muscle in a
mixed tone, collagen blue, elastin reddish. See also a com-
plicated process with methylen blue in Monatssch. prakt.

342 CHAPTER XXVIII.

Dermatol., xix, 1894, p. 583, and another with orcein,
hematein, siurefuchsin and picric acid.

Rerrerer (C0. R. Soc. Biol., 1887, p. 645) fixes in 10 vols.
of alcohol with one of formic acid, washes well and stains in
alum carmine. Muscle red, connective tissue unstained.

677. General Structure—Werner (Hist. d. glatten Muscu-
latur, Dorpat, 1894, p. 22) fixes stretched intestine or bladder
in Flemming, washes well and stains in Heidenhain’s chrome
hematoxylin, § 265. For demonstrating intercellular spaces,
fresh intestine is put for 24 hours into oil, at 37° C., then for
12 hours into Flemming, and for 4 to 6 into chromo-acetic
acid.

678. Isolation of Fibres.—Gacn’s methods, see $$ 527, 544,
and 536.

Mosivs, muscle of Cardiam, see § 535.

Battowrrz, muscle of Cephalopoda, see Arch. mik. Anat.,
xxxix, 1892, p. 291.

Scuutrz (Arch. Anat. Phys., Phys. Abth., 1895-6, p. 521)
puts muscle of Vertebrates for twenty-four hours into 10
per cent. nitric acid, rinses with water, and brings pieces
for six to eight days (in the dark at first) into a mixture of
equal parts of s4 per cent. osmic acid and £ per cent. acetic
acid, teases and mounts in glycerin.

For smooth muscle of Vermes, see Aratny, Zeit f. ariss,
Mik., x, 1898, pp. 36, 319, and § 538, ante.

679. Iris—Doaigr (Arch. mik. Anat., 1886, p. 403) puts
the anterior half of an enucleated eye 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 interior and posterior plate, and these
stained according to the usual methods.

See also Koaangr, Arch. mth. Anat., 1885, p. 1; Canriezn,
ibid., 1886, p. 121 ; and Dosrormwsry, ibid., p. 91.

680. Bladder of Frog, Innervation of (Wotrr, 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 ont on removal of the syringe,

MUSCLE AND TENDON (NERVE-ENDINGS). 343

tie the frog’s thighs together.) Now open the frog, ligature
the intestine above the bladder, and cut away the abdomen
so as to have in one piece bladder, rectum and hind legs.
Put this into gold solution of 1 : 2000 for four hours; the
bladder is then excised, slit open, and pinned (with hedge-
hog spines) on to a cork (outside downwards). Place it
under running water until all the epithelium is washed away.
Use a camel’s-hair brush 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 acidnlated
water, and finally reduce in fresh water in daylight.

Ranvier (Trart’, p. 854) recommends his two gold pro-
cesses, the liquids being injected as above.

Grinstein (Arch, mik. Anat., 1899, p. 1) injects 1 per
cent. methylen blue in normal salt solution through the vena
abdoniinalis, and after twenty to thirty minutes excises the
bladder and exposes to the air. Fix the stain with picrate
of ammonia and mount in glycerin with the same ($ 343).

CHAPTER XXIX.
CONNECTIVE TISSUES.

Connective Tissue.

681. General Stains for Connective Tissue,——Connective tissue,
elastic tissue, and smooth muscle are all normally acido-
philous. Collagen, the distinctive element of connective
tissue, absolutely requires “acid”? dyes for the production
of a permanent stain, whilst elastic tissue and muscle will
also fix “basic” dyes. Collagen has a special affinity for
Siurefuchsin and Wasserblau. Elastin has a strong affinity
for acid orcein, whilst muscle has no special affinity for
either, but stains energetically with picric acid.

Picro-siurefuchsin is much used and very convenient as a
general differentiating stain, but not to be recommended for
cytological detail. See Scwarrrr, Zeit. wiss, Zool., xxx,
1905, p. 176.

E. and T. Savin recommend Benpa’s picro-siurefuchsin,
§ 299.

Enriica-Bronp! mixture gives connective tissue red, but
smooth muscle redder still.

Unna’s Wasserblau-orcein for distinguishing connective
tissue and, muscle has been given, § 676. It works after all
fixatives. Stain long, and dehydrate preferably with acid
alcohol.

Freerorn (Amer, Mon. Mice. Jowrn., 1888, p. 231) recom-
mends (for sections) picro-nigrosin, made by mixing 5 cc. of
1 per cent. aqueons 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 blue, nuclei blackish, the rest yellowish.

Ramén y Casat’s picro-indigo-carmine gives connective-
tissue fibres dark blue, with red nuclei.

S. Maver (Sttch. kh. Ahad. Wiss., Ixxxv, 1882, p. 69)

CONNECTIVE TISSUES. BA5

recommends for staining fresh tissue Violet B, § 830. Elastic
fibres and smooth muscle also stain, but of different tints.

Dusrzuin (C. R. Ass. Anat., vi Sess., 1904, p. 62) uses a
mixture of 23 vols. 1 per cent. picric acid and 2 vols.
1 per cent. methyl blue—with a foregoing stain with
carmalum or safranin.

For Ranvier’s method of artificial cedemata for the study
of areolar tissue, see his Trait¢é, p. 329.

682. Unna’s Orcein Method.—(Hncycl. mik. Techn., 1910,
p- 250). Sections are stained for ten minutes in Griibler’s
polychrome methylen blue. They are then washed with
water, mopped up, and brought for fifteen minutes into a
neutral 1 per cent. solution of orcein in absolute alcohol,
rinsed in pure alcohol, cleared in bergamot oil, and mounted.
Collagenous ground-substance dark red, muscle bluish,
elastic fibres sometimes dark red. Material may be fixed in
almost any way except with nitric or picric acid, formol, or
liquids of Miller and Hermann.

683. Unna’s Methylen-blue + Saurefuchsin (Unwa, in E'ncycl.
mik. Technik, 1910, p. 247). Stain for 2-5 minutes in poly-
chrome methylen blue solution (Griibler). Wash and stain
for 10-15 minutes in “ (0° per cent.) Saurefuchsin + (33 per
cent.) tannin-mixture (Grubler).” Water, alcohol, essence,
balsam. Collagen, protoplasm, and muscle red, nuclei and
keratin blue. On Flemming material, elastin blue. Liquids
of Hermann and Etlicki, formol and copper fixatives
incompatible.

684. Unna’s Safranin + Wasserblau (eiid.). Ten minutes
in 1 percent. safranin. Wash. Ten to 15 minutes in (“1 per
cent.) Wasserblau + (33 percent.) tannin mixture.” Wash.
Stains in opposite colours to the last. Formol and liquid of
Hermann contra-indicated for fixing.

685. Flemming’s Orange Method is said to give a very sharp
differentiation of developing fibrils.

686. Matiory (Zeit. wiss. Mik., xviii, 1901, p. 175) stains sections of
sublimate or Zenker material for a few minutes in Saiirefuchsin of 0-1
per cent., mordants for a few minutes in 1 per cent. phosphomoly)hdic

346 CHAPIER NXIX.

acid and stains for 2 to 20 minutes in anilin blue 0°5 erms., Orange G. 2,
oxalic acid 2, and water 100. His phosphotungstic haematoxylin stains
connective tissue sharply, but does not differentiate it sufficiently from
elastic tissue and muscle.

687. For the complicated procedure of Hornowsk1 see #bid., xxvi,
1909, p. 188.

688. For DeLAMARE's mixture of orcein, hematoxylin, Séurefuchsin
and picric acid see Verh. Anat. Ges., xix, 1905, p. 227.

689. Masson (C. R. Soe. Biol., Ixx, 1911, p. 573), stains first im
hemalum, then in eosin, and then for a few minutes in 1 per cent. solu-
tion of saffron in tap water (made hy boiling). Connective tissue, bone,
and cartilaye, yellow.

690. Benecke’s stain for fibrils (Verh. Anat. Ges., vii, 1893,
p. 165) is essentially that of Kromaynr, § 656.

691. Bielschowsky’s Sitver Meruop (ost, under ‘ Neuro-
fibrils’) has been used for connective-tissue fibrils. SnessAREWw
(Anat. Anz., xxxvi, 1910, p. 401) employs it as follows :
Tissue is hardened in neutral formol and sectioned with a
freezing microtome. The sections are put for at least 4 days
into won alwm of 2°5 to LO per cent., changed daily. They
are then silvered for 86 to 48 hours in nitrate of silver of 10
per cent., then treated with the oxide bath and reduced in
formol of 20 per cent. Collagen fibres grey, but fine con-
nective networks black, nerve fibres unstained or only weakly
stained.

See also Marescy, Zeit. wiss. Mik, xxiii, 1906, p. 356;
Srupyicka, ibtd., p. 416; Zimmermann, ibid., xxv, 1908,
p. 10; Lavi, Monit. zool. Ital., 1908, p. 290; Huinnicn,
Arch, Mik. Anat., xxiv, 1909, p. 786 (dentine) : Insazaro,
dvreh. Ital. Anat. Hmb., viii, 1909, p. 875 (silvers Flemming
material) ; ArHanasiu and Dracotu, (. R. Acad. Sci., chi,
1910, p. 551 (Ramon y Cajal’s silver process, with alcohol
fixation).

Elustic Tissue.

692. Elastic Tissue, Generalities —Ilastic fibres have a great
affinity for osminm, staining with much more rapidity than
most other tissue elements. They are not changed by

CONNECTIVE TISSUES. B47

caustic soda or potash, They are normally acidophilous, but
are easily rendered artificially basophilous by means of
chromic acid or other mordants, and then stain with great
energy with basic dyes. Hence a group of stains of which
those of Lustgarten and Martinotti are types. They havea
natural affinity for orcein, whence stains of the Taenzer-
Unna type.

For a review of the older methods of Barzer, Unwna,
Lusroanren, and Herxuemer, sec the paper by G. Martinorvt,
in Zeit. wiss, Mil, iv, 1887, p. 31 3 also Mneyel. nik. Teehnik.,
art. “ Klastin.”

693. Victoria Blue (Lusraarren). See § 289.

694. Safranin (G. Marrinorri, loc. cit., § 692).—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 black.

The staining will he performed quicker if it he done at the temperature
of an incubating stove (GRIESBACH, 7h7d., iv, 1887, p. 442). See also
Frrreia (7hid., v, 1888, p. 342).

See also M1BELuLI, Mon. Zool. Italiano, 1, p. 17, or Ze7t. wiss. Mik., vii,
1890, p. 225 (the report in Journ. Roy. Mic. Soc., 1890, p. 803, is
vitiated by a misprint). Other basic dyes have heen recommended.

695. Kresofuchsin (Rornre, see $ 289).

696. Orcein—This method is due to TarnnzEr, and as
modified by Unna is known as the Tannzzr-Unna method,
see third edition, or Monatssch. prakt. Dermatol., xii, 1891,
p. 394.

Uwnna’s Modified Orcein Method (Monatssch. prakt. Dermatol.,
xix, 1894, p. 397; Zeit. wiss. Mik., xii, 1895, p. 240).—
Griibler’s orcein 1 part, hydrochloric acid 1 part, absolute
alcohol 100 parts. Stain sections for thirty to sixty
minutes, or for ten to fifteen at 30°C., rinse in alcohol, clear,
and mount. Elastin dark brown, collagen light brown.

See also Merk. Sitz. Akad. Wiss. Wien., cviii, 1899, p. 335; PRANTER, cbed.,
xix, 1903, p. 361 (he takes 2 per cent. of nitric acid instead of the hydro-
chloric, and stains six to twenty-four hours); WoLFr, zbid., p.488; the
article “Elastin” in Encycl. mik. Technik.; and K.and T. SAvint, Zevt.
wiss. Mik., xxvi, 1909, p. 34.

348 CHAPTER XXIX.

697. Weaicert’s Resorcin-Fuchsin Method (Centralb. allg.
Path., ix, 1898, p. 290).—1 per cent. of basic fuchsin and 2
per cent. of resorcin (or of carbolic acid) are dissolved in
water. 200 c.c. of the solution are raised to boiling-point
in a capsule, and 25 cc. of Liquor ferrt sesquichlorate P. G.
are added, and the whole is boiled, with stirring, for two to
five minutes more. A precipitate is formed. After cooling
the liquid is filtered, and the precipitate which remains on
the filter is brought back into the capsule, and there boiled
with 200 c.c. of 94 per cent. alcohol. Allow to cool, filter,
make up the filtrate to 200 c.c. with alcohol, and add 4 e.c.

of hydrochloric acid.

Worrrum (Zeit. wiss. Mik., xxv, 1908, p. 219) adds 10 to
15 per cent. of acetone to the mixture.

Stain sections (of material fixed in any way) for twenty
minutes to an hour, wash with alcohol, clear with xylol (not
with an essence). Elastic fibres dark blue on a light ground.

MineRvini (Zett. wiss. Mck., xviii, 1901, p. 161) gives a variant with
safranin instead of fuchsin.

See also PRAaNTER, zbid., xix, 1903, p. 361; B. FrscHer, Virchow's
Arch., clxx, 1902, p. 285, or Zett. wiss. Mik., xx, 1903, p. 40 (chemistry
of the dyes obtained by these processes, which he calls “Fuchselin,”
“Safranelin.” etc.); Harv, Centralb. allg. Path., xix, 1908, p. 1; and
CitimBaRis, Arch. mik. Anat., lxxv, 1910, p. 708.

698. Hematoxylin Methods.—Harris (Zett. wiss. Mzk., xviii, 1902,
p. 290) makes an “ Hlasthematein” as follows : Hematoxylin 0:2 grms.,
aluminium chloride 01 grms., aleohol of 50 per cent. 100 ¢.c., boil and
add mercuric oxide 0°6 grms., filter and add 1 drop of HCl. Keep for
some weeks. Stain for five or ten minutes, put into alcohol with 1 per
cent. of nitric acid for one minute, then pure alcohol.

See also Dn Wirt, Anat. Rec., i, 1897, p. 74; Duercx, Arch. Path.
Anat., clxxxix, 1907, p. 62; VERHOEFF, Journ. Amer. Med. Assoc., 1908,
No. 11.

MALtory’s phosphotungstic hematoxylin is good, but not specific.

699. Other Methods for Elastic Tissue:

For the elastic tissue of the skin see PASsARGE and Krostne, Derm.
Stud., xviii, 1894.

See also for staining and dissociation AGABABOW, Arch. mik. Anat.,1,
1897, p. 566 ef seq.

For C. Martinorri’s silver impregnation see Ze?t. awiss. Mik., v, 1888,
p- 521, or Arch. Ttal. Biol., xi, 1889, p. 257.

SCHUMACHER (Arch. mihk. Anat., lv, 1899, p. 151) has had good results
(for the spleen) with picro-nigrosin, § 681.

See also § 733.

CONNECTIVE TISSUKS. 349

Plasma Cells.

700. Plasma Cells and “ Mastzellen”’; Generalities —Plasina
cells, of which “ Mastzellen” are a sub-species, are cells
found in or along with connective tissue, and distinguished
by their hypertrophied and very granular cytoplasm and
poorly staining nucleus. ‘The granules are highly basophi-
lous, much more so than the nuclei; they stain with special
energy with basic anilins, and mostly metachromatically.
They do not, however, stain with pure methyl green. ‘The
nuclei either do not stain at all or not in the normal way,
except with pure methyl green.

According to Unna in Heyel. mik, Techn, 1910, ii,
p- 411, material should be fixed in chemically pure absolute
alcohol and sectioned in celloidin. Care should be taken to
avoid contamination of the liquids by tannin; corks, and
supports for imbedding, should be soaked for some hours
before use in 2 per cent. carbonate of soda.

701. Mastzellen (NorpMANN, Beitr. z. Kenntniss d. Mastzellen,
Inaugural diss., Helmstedt, 1884).— A concentrated solution of vesuvin
containing £ to 5 per cent. of hydrochloric acid. Stain for a few minutes,
and dehydrate with absolute alcohol.

702. Plasma Cells, Unna’s Later Methods (Unna, in Hneycl.
mik. Techn., 1910, ti, p. 411).

A.—For Large Plasina Cells.

(1) Ten minutes in Griibler’s polychrome methylen blue
solution, wash aud drain. Fifteen minutes in 1 per cent.
orcein solution (Griibler), without acid; absolute alcohol, so
long as methylen blue comes away abundantly ; bergamot
oil, balsam.

(2) Methylen blue as above, 2 minutes. Wash well.
Then two minutes in glyccrin-ether mixture* (Griibler)
diluted with 4 volumes of water. Wash thoroughly (2 to 5
minutes) ; absolute alcohol, bergamot oil, balsam.

(3) Modification of a method of Paprunuuim (Virchow’s

* Glycerin ether C,H,,O;, is a glycerin anhydride. It is a differen-
tiating agent for basic dyes. The glycerin-ether mixture in question
contains alcohol and glycerin, and can be obtained from Griibler.

350 CHAPTER XXIX.

Arch., clxiv, 1901, p. 111). Ten minutes in the warm, 20° to
40° C., in Griibler’s carbol-pyronin-methyl-green mixture.
Cool rapidly, by plunging the recipient containing the tissues
into cold water. Remove the tissues with a platinum wire
and rinse. Absolute alcohol, bergamot oil, balsam.

StTRoOPENI (Zett. wiss. Mck., xxix, 1913, p. 302) takes ucridin red instead
of the pyronin. This will work after various fixatives.

B.—for small Plasina Cells.

(4) As No. 2, supra, but only half a minute in the glycerin-
ether.

(5) After removal of the celloidin from the sections with
alcohol and ether, five minutes in polychrome methylen blue,
wash, dry with blotting-paper, dehydrate (about a minute)
in «w mixture of 2 parts alcohol to 3 of xylol, then one
minute in xylol; then 5 to 10 minutes in alum-anilin (pre-
pared by allowing anilin oil to stand over a layer of powdered
aluin a couple of fingers deep) ; xylol, balsam.

(6) As No.3, supra, after a foregoing stain of two minutes
in polychrome methylen blue.

See also Wuriicu in Virchow’s Arch., clxxv, 1904, p. 198.

703. Kuxuicn’s Original Method for Mastzellen (Arch. ah.
zlnat., xii, 1876, p. 263).—Stain, for at least twelve hours
in—

Absolute alcohol . : ; 50 ce.
Water . : ; . 100 ce.
Acid. acet. glacial : . 12h ce.

—to which has been added enough dahlia to give an almost
saturated solution. Wash out with alcohol, and mount in
resinified turpentine.

See ulso SCHIEFFERDECKER and KossEL’s Geiebelehre, p. 329.
ee

704. Mastzellen, Unna’s Latest Methods (Hucycl. mik. Vechi.,
1910, ii, p. 72).—(1) Stain three hours to overnight in poly-
chrome methylen blue with a knife-pointful of alum to a
watch-glass of the stain, rinse; alcohol, oil, balsam. (2)
Stain in polychrome methylen blue quarter of an hour, rinse,
then ten minutes in glycerin-ether, § 702, wash thoroughly,
alcohol, oil, balsam.

CONNECTIVE TISSUIS. 351

These methods give a specific metachromatic stain of Mast-
zellen ona light ground. See also lor. cié., two other methods
demonstrating plasma cells at the same time.

705. Other Methods for Plasma Cells and Mastzellen.—See,
inter alivs (in previous editions) PAPPENHEIM, Virchow’s Arch., clxvi,
1901, p. 427; Berconzint, Anat. Anz., 1891, p. 596; Scurippw, Anat.
Hefte, xxviii, 1905, p. 698; Maximow, Arch. mk. Anat., Ixvii, 1906,
p. 686; Scuarrer, Centrabl. Phys., xxi, 1907, p. 261 (fixation in wbsolute
alcohol or 2 parts alcohol to 1 of formol, staining for half an hour in
methylen blue, thionin or toluidin blue, in 70 per cent. alcohol with
1 per cent. of HCl); Ranvier, C. BR. Acad. Scz., 1890, p. 165 (his
*Clasmatocytes”’: fix with osmium, stain with aqueous methyl violet 5B).

Fat.

706. Fat and Lecithin—According to Loisun (C. R. Soc.
Biol., lv, 1908, p. 703) lecithin is much less soluble than
neutral fats in turpentine or ether, and in general much
more stainable. ‘I’o demonstrate lecithin, he advises as
follows: After fixation, mordant the tissues with alum ; leave
them as little as possible in alcohol ; dehydrate with acetone,
ether, or benzin ; stain with hematoxylin, gentian, methyl
green, toluidin blue, Saurefuchsin or orange G, which stain
lecithin strongly, whilst leaving fat unstained.

Devianpre (Journ. Anat. Phys., 1904, p. 80) fixes in formol
of 4 per cent. and brings into acetone, in which fat is dissolved,
but not lecithin, which can then be stained by osmium.

See also Craccio, Arch. Zellforsch., v, 1910, p. 285; and
Fiscuner, Zeit. wiss. Mek., xxii, 1905, p. 262.

707. Mounting Fat.—Osmuicated fat can generally be mounted
in balsam without special precaution. For delicate fats,
it may suffice to avoid absolute alcohol and essences as much
as possible, and mount direct in alcohol balsam or euparal,
or cleav with cedar oil, which has little solvent action. For
very delicate fats it may be necessary to avoid alcohol of
more than 70 per cent., or avoid it altogether, and mount in
glycerin or levulose.

708. Removal of Fat.—lT’at can generally be dissolved out
by: alcohol, ether, or chloroform and the like. Osmicated

352 CHAPTER XXLX.

fat is more resistant, but can be removed in a few hours or
days by means of oil of turpentine, ether, creosote, xylol,
clove oil, of chloroform. See Frumminc in Zeit. wiss.
Mikr., 1889, pp. 39, 178.

709. Stains for Fat.—'lhe simplest, and perhaps the best is
osmicacid. This stains certain fatty bodies black, but not all.

According to Atrmann, Srarke, and Hanpwenck, only free
oleic acid and olein are directly blackened by osmic acid ;
stearin and palmitin, and stearic and palmitic acid are only
browned by it, with an after-blackening which is produced
by subsequent treatment with alcohol. Neither reaction
occurs with the fatty bodies in the solid state, and can
only be obtained when they are either in a state of fusion or
solution (from the paper of Hanpwuxrck in Zeit. wiss, Mik.,
xv, 1898, p. 177). See also Loiszt, C. R., Soc. Biol., 1903,
p. 826. Muton, Zedt. wiss. Mik., xxii, 1905, p. 138; GoLovevz,
Chem. Rev. Fett. w Harz-Industrie, xvii, 1910, p. 70 (Zeié. wiss.
Mik., xxvii, 1911, p. 218).

For quinolein blue, see § 322.

Davpi (Arch. Jtal. Biol., xxvi, 1896, p. 143) stains fat in
tissues by treating for 5 to 10 minutes with concentrated
alcoholic solution of Sudan III, washing for the same time
with alcohol, mopping up with blotting paper, and mounting
in glycerine.

Similarly Rirper, see Zeit. wiss. Alikr., xv, 1898, p. 211.

The alcohol for making the stain should be of 70 per
cent., according to most authors, though Sara (Beitr. path.
Anat., xxviii, 1900, p. 461; Zeit, wiss. Mik., xviii, 1901, p.
67) employs 96 per cent. Rosunvuat (ibid., xix, p. 469;
Verh. path, Ges., September, 1899, p. 440) insists that the
washing-out be done with alcohol of exactly 50 per cent.

Micuanuis (Virchow’s Arch., clxiv, 1901, p. 263) reeom-
inends Scharlach R (syn. “ Fettponceau’’). Stain for fifteen
to thirty minutes in a saturated solution in 70 per cent.
alcohol, and mount in glycerin or levulose.

Other authors also commend this stain. HrrxuEimMer
(Deutsche med. Wochenschr., xxvii, 1901, p. 607; Zett. wiss.
Mik., xix, 1902, p. 66) makes a solution of 70 parts of
absolute alcohol, 10 of water, 20 of 10 per cent. caustic
soda, and Scharlach R to saturation. This makes a stronger

CONNECTIVE TISSUES. 353

solution, and stains in a couple of minutes. Wash out with
alcohol of 70 per cent.

With either solution the staining. must .be done in a
covered vessel or the stain will precipitate.

Similarly Bett, Amer. Journ, Anat., ix, 1909, p. 401, and
Anat. Rec., iv, 1910, p. 199.

Herxuemeur also (Centralb. ally. Path., xiv, 1903, p. 841;
Zeit, wiss. Mik., xxi, 1904, p. 57) recommends a saturated
solution of the dye in a mixture of equal parts of acetone
and 70 per cent. alcohol.

He also (Deutsche med. Wochenschr., xxvii, 1901, p. 607;
Zeit, wiss, Mik., xix, 1902, p. 67) has had very fine results
by staining for twenty minutes in a saturated solution of
Indophenol in 70 per cent. alcohol.

Mottison (Zeit. wiss. Zool., xxvii, 1904, p. 529) las had
good results by staining gelatin sections for a few minutes
in strong extract of Alkanna in 96 per cent. alcohol, and
mounting in glycerin or syrup.

Lorain Suira (Journ. Path. Bact., xii, 1907, p. 1) finds
that Nile blue stains fatty acids blue and neutral fats reddish.

Similarly Hisewpuna (Virchow’s Arch., cxeix, 1910, p. 502)
who recommends aqueous solution of Nilblau BB.

Benpa (ibid., clxi, 1900, p. 194) finds that free fatty acids
can be detected by Weigert’s neuroglia mordant. See also
Berner, tbid., clxxxvii, 1907, p. 360, and Fiscuuur, Zeit. wiss,
Ak., xxu, 1905, p. 263.

Oxasima (cbid., xxix, 1912, p. 67) extracts red capsicum
berries for some days with alcohol, and evaporates down to
one fifth. This stains only fatty bodies: amongst them,
myelin.

See also Krnassury, Anat. Ree., v, 1911, p. 313.

Bone.*

710. Bone, Non-decalcified—Ranvinn (Traité, p. 297) has
the following :

Bones should be plunged into water, without being allowed
to dry, a8 soon as the surrounding soft parts have been

* For a detailed review of the whole sulject, see the paper of
ScHAFFER in Zezé. wiss. Mik., x, 1893, p. 167, or the article “ Knochen
und Zahne” in Encyel. mk. Techuik.

23

35-4 CHAPTER XXIX.

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 treated as follows:

An epiphysis having been removed, together with a small
portion of the diaphysis, a piece of caoutchouc tubing is
fixed by a 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; they are then put to
macerate for several months, the liquid being changed from
time to time. As soon as all the soft parts are perfectly
destroyed, the bones may be left to dry.

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 Kocu’s copal
process, and Hxurensaum’s colophonium process).

Scuarrer (Zeit. wiss. Mik., x, 1893, p.171) grinds and polishes on
stones of graduated fineness.

For the process of Wxtu for bones and teeth see § 180.

Rose (Anat. Anz., vii, 1892, pp. 512-519) follows Koch’s process. He
penetrates first with a mixture of cedar oil and xylol, then with pure
xylol, and imbeds in solution of Damar in chloroform or xylol. The
method can be combined with Golgi’s impregnation.

Wuitr (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 fuchsin in
collodion, then into spirit to harden the collodion. After
this they are ground down to the requisite thinness between
two plates of old ground glass, with water and pumice

CONNECTIVE TISSUES. 355

powder, and mounted, surface dry, in stiff balsam or styrax,
care being taken to use as little heat as possible. Lacuna,
canaliculi, and dentinal tubuli are found infiltrated by the
coloured collodion.

Marscuinsky (Arch. mihk. Anat., xxxix, 1892, p. 151, and xlvi, 1895,
p. 290), after grinding, impregnates with nitrate of silver.

For similar method of RupREcHT, see Zeit wiss. Mik., xiii, 1896, p.
21, wherein see also quoted (p. 23) a method of ZIMMERMANN.

Csoxor (Verh. anat. Ges., 1892, p. 270) describes a saw which will cut
fresh bone to 1204; and ARNDT (Zezt. wiss. Mzk., xviii, 1901, p. 146) a
double saw which will also give very thin sections.

711. Mounting.—To show lacunz and canaliculi injected
with air, take a section, or piece of very thin flat bone, quite
dry. Place on a slide a small lump of solid balsam, and
apply just enough heat to melt it. Do the same with a
cover glass, place the bone in the balsam, cover, and cool
rapidly.

712. Sections of Bones or Teeth showing the Soft Parts,—
Neary (Amer. Mon. Mic. Juourn., 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 im situ, obtained in
half an hour.

Horewe.t-Smiru (Jowrn. Brit. Dent. Ass., xi, 1890, p. 310;
Journ. Roy. Mic. Soc., 1890, p. 529) says that for preparing
sections of teeth showing odontoblasts im sitw 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.

Wet (loc. cit., § 180) fixes pieces of fresh teeth in subli-
mate, stains with borax-carmine, brings them through alcohol
into chloroform and chloroform-balsam, and after hardening
this by heat proceeds to grind as usual (§ 177).

See also Rész, $ 710.

For the study of the vessels im teeth, Lurkowsky (Anat.
Hefte, viii, 1897, p. 568) injects with Berlin blue, hardens
the teeth with a piece of the jaw for one or two days in 50
per cent. formol, decalcifies in 10 per cent. nitric acid (eight

356 GHAPTER. XXIX.

to fourteen days, change frequently) and makes celloidin
sections.

For decalcification of teeth see also § 554 (Rousszau,
Bopucker and Frewcumann). Bédecker finds Rousseau’s
process not applicable to human teeth: the acid must be
added to the fluid celloidin.

718. Vivante (Intern. Monasschr. Anat. u. Phys., ix, 1892, p. 398)
impregnates portions of frontal bone of four to six months calves, which
are not more than 3 to 4 mm. thick, by Golgi’s rapid bicromate and
silver process. After impregnation the specimens should be decalcified
in von Ebner’s mixture (§ 562), well washed with water, and brought
into solution of carbonate of soda, and finally imbedded in paraffin.
For bis quinolein blue method see fourth edition.

For UNDERWOOD'S gold process for teeth, and for that of LEPKOWSKI,
see third edition, ov Anat. Anz., 1892, p. 204.

Law (Proc. Roy. Soe. Med., i, 1908, p. 45) studies nerve-endings in
teeth of mammals by treating paraffin sections of decalcified tissue with
Berue’s molybdenum toluidin blue (details in Jowrn, Roy. Mier. Soc.,
1908, p. 518).

714. Bone, Decalcified (Fitrmmina, Zeit. wiss. mik., 1886,
p. 47).—Sections of decalcified bone are soaked in water,
dehydrated with alcohol, under pressure, dried under pressure
and mounted in hard balsam melted on the slide. They

show the lacunar system injected with air as in non-decalcified
sections ($711).

715. Stains for Cartilage and Decalcified Bone.—See hereon
Scaarrer in Zert. wiss. Mik., v, 1888, p.1; and Hneyel. mik.
Technik., art. “ Knochen.”

Kotiicer (Zeit. wiss. Zool., xliv, 1886, p. 662) treats
sections of decalcified bone with concentrated acetic acid
until they become transparent, and then puts for one quarter
to one minute into a concentrated solution of indigo-carmine,
washes and mounts in glycerin or balsam. ‘he fibres of
Sharpey appear red, the remaining bone substance blue.

Scuarrer (Zeit. wiss. Mck., v, 1888, p. 17) employed at
one time a safranin method modified from Bouma (Centralb.
med, Wiss., 1883, p. 866), for which see previous editions.
He now (Eneycl. mik. Tech., 1910, i, p. 762) stains sections
for twenty-four hours in a bath of 20 c.c. of water with one
drop of 1 per cent. solution of safranin (or thionin) and

CONNECTIVE ‘TISSUES. 357

(apparently) mounts in balsam. The safranin stain will keep
if the material is cartilage which has been fixed in picro-
sublimate ; otherwise it must be fixed with ammonium
molybdate of 5 per cent. before dehydrating.

Scamor (Centralb. allg. Path., x, 1899, p. 745) stains in
a mixture of 2 c.c. concentrated solution of thionin in
alcohol of 50 per cent. and 10 c.c. of water for ten minutes,
rinses and puts into saturated aqueous picric acid for thirty
to sixty seconds. Rinse and pass through graded alcohols
into origanum oil or carbol-xylol and balsam. Matrix
yellow, cells red, fat-cells violet. He also describes a more
complicated method with thionin and phosphotungstic or
phosphomolybdic acid.

Moti (Centralb. Physiol., xiii, 1899, p. 225) stains embry-
onic cartilage for six to twenty-four hours in orcein 0°5 gr.,
alcohol 40, water 20, hydrochloric acid 20 drops, and mounts in
balsam. Matrix blue, nuclei red.

Kauuius (Anat. Hefte, xxx, 1905, p. 9) stains first with
boraxcarmine or alum-carmine, then (sections) for ten minutes
in saturated solution of thionin, and washes out with alcohol
of 70 per cent. Said to be specific for embryonic cartilage.

Vasrarini-Crest (Att. Accad. med.-chir. Napoli, 1907, p. 4)
stains sections of embryonic cartilage with borax carmine,
then with muchzematein (alcoholic solution without acid), and
then with orange G. in alcohol.

Bayerw’s method for ossifying cartilage (Arch. mik. Anat.,
1885, p. 35) :—Portions of ossified cartilage are decalcified
as directed § 563, cut in paraffin, stained in Merkel’s carmine
and indigo-carmine mixture, and mounted in balsam.

Mayer (Grundztige, Luz & Mayzr, 1910, p. 393) prefers to
all these resorcin fuchsin, § 697, the precipitate being freed
from iron chloride by washing before dissolving in the
alcohol.

Aqueous solution of benzoazurin has been commended as a stain for
ossifying cartilage by ZscHOKKE, see Zett. wiss. Mik., x, 1893, p. 381.

A process of BaumGARTEN’s has been given, § 388.

MoERNER (Skandinavisches Arch. Physiol., i, 1889, p. 216; Zezt. wiss.
Mik., vi, 1889, p. 508) gives several stains for tracheal cartilage, chiefly
as microchemical tests, for which see third edition.

See also a critique of these methods by WoLtTERS in Arch. mik. Anat.
xxxvii, 1891, p. 492; and on the whole subject of cartilage see SCHIEF-
FERDECKER’S Gewebelehre, p. 331.

358 CHAPTER XXIX.

Fusari (Arch. Itul. Biol., xxv, 1896, p. 200) makes sections of fresh
cartilage, puts them for twenty-four hours into 1 per cent. nitrate of
silver, washes, dehydrates, and exposes to the light in balsam.

See also Dissn, Anut. Anz., xxxv, 1909, p. 318, u stain for dentine,
(hemalum followed by a mixture of Saiirerubin and Orange G); and
Retrerer and LELruévReE, C. R. Soc. Biol., xx, 1911, p. 630.

716. Cartilaginous Skeletons of embryos (VAN WIJHE, Proc. K.
Akad. Wetensch. Amsterdam, 1902, p. 47) may be studied by staining
embryos for a week in a solution of 0°25 grm. methylen blue in 100 c.c. of
70 per cent. alcohol with 1 per cent. of hydrochloric acid. Wash out
in alcohol with 1 per cent. of hydrochloric acid until no more colour
comes away (about a week) and mount in balsam. The cartilage
remains blue, all the other tissues being colourless.

Similarly, Lunpvaty (Anat. Anz., xxv, 1904, p. 219, and xl, 1912, p.
639) using toluidin blue.

Similarly also Baxay (Verh. Anat.Ges., 1902, p. 248), with Bismarck
brown (the embryos having been previously treated with nitric acid of
3 per cent.).

CHAPTINR XXX.

BLOOD AND GLANDS.

Blood.

717. Fixing and Preserving Methods.—''he school of Ehrlich
used to fix by heat. A film of blood was spread on a cover-
glass and allowed to dry in the air, and then fixed by
passing the cover a few times, three to ten or twenty,
through a flame, or by laying it fuce downwards on a hot
plate kept for several minutes or as much as two hours at a
temperature at which water not only boils but assumes the
spheroidal state (110° to 150° C.). For details see GuLtanp,
Scottish Med. Journ., April, 1899, p. 812; Ruginsvetn, Zect.
wiss, Mik., xiv, 1898, p. 456; Zietina, tbid., p. 463. But I
believe they have now well-nigh abandoned this barbarous
practice.

In wet methods either the blood is miaed at once, on
being drawn, with some fixing and preserving medium, and
studied as a fluid mount, or films are prepared and put into
a fixing liquid before they have had time to dry; or after
drying in the air without heat for a few seconds (at most 10
to 30).

To make a film, place a very small drop of blood on a
perfectly clean slide. Bring down on to the slide the edge
of another slide held over it at a slope; move this along till
it touches the edge of the drop and the blood runs along
the angle between the two slides. Then move the second
slide away from the drop, and the blood will follow it and be
drawn out into a film without being crushed, Similarly with
two cover-glasses, to make a cover-glass film, which can be
floated face down on to fixing or staining liquids in a
watch-glass.

360 CHAPTER XNX.

Some persons make films by flattening blood betweew
two cover-glasses which are afterwards separated by sliding
the one over the other; but that produces an injurious
kneading of the cellular clements.

Most of the usual fixing agents are applicable to blood.
But it is often necessary to employ only such as are favour-
able to certain stains. Those most recommended in this
respect are alcohol, formol, sublimate (should not be too
strong), osmic acid in very light fixation, or absolute
methyl alcohol, which is an energetic fixative of dried films.

Air-dried films ought to be fixed before putting into
aqueous or glycerin stains, else they will wash off; but this
is not necessary for alcoholic stains.

718. Fixing and Preserving in Bulk.— Most morphologists are
agreed that by far the most fuithful fixing agent for blood-
corpuscles is osmic acid. A drop or two of blood (Bionp1
recommends 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. As arule the osmic acid should be strong
—1 to 2 per cent. Fixed specimens may be preserved for
use in acetate of potash solution (Max Fizscu, Zeit. wiss.
Mik., v, 1888, p. 83). ;

GriksbacH also (ibid., 1890, p. 328) combines the osmic
acid with certain stains. He mentions methyl green, methyl
violet, crystal violet, safranin, eosin, Siurefuchsin, rhodamin,
and iodine in potassic iodide.

Rossr (ibid., vi, 1889, p. 475) advises a mixture 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.

Hwatp (Zeit. Biol., xxxiv, 1897, p. 257) mixes three to
four drops of blood of amphibia or reptiles with 10 c.c. of a
solution of 0°5 per cent. osmic acid in 0°5 per cent. salt
solution (for mammals 0°6 to 0°7 per cent. salt), siphons off
the supernatant liquid after twenty-four hours with his capil-
Jary siphon (§ 3, p. 4), and substitutes water, alumn-carmine,
ete., and lastly, 50 per cent. alcohol.

Weipewreicn (Arch. mik. Anat., Ixxii, 1908, p. 218) lays
a cover with a drop of blood on it on a layer of agar-agar
(1 per cent. in salt solution of 0°8 per cent.) and after five

BLOOD AND GLANDS. 361
minutes runs in osmic acid of 1 per cent., and after five
minutes more removes the cover.

Dexuuysen (Anat. Anz., xix, 1901, p. 536) sepennineie a
mixture of cither 3 or 9 vols. of 2 per cent. osmic acid with
1 of 6 per cent. acetic acid, containing } per cent. of methylen
blue, which he calls “ Osmacet.”’

The mercurial liquids of Pacini ($ 414) used to be con-
sidered good. Hayem (Du Sang,” etc., Paris, 1889; see
also Zeit. wiss. Mtk., vi, 1889, p. 335) has the following
formula : sublimate 0°5, salt 1, sulphate of soda 5, and water
200. This should be mixed with blood in the proportion of
about 1: 100. Hosin may be added to it. Lowzy’s formula
(Sitzb. k. Akud. Wiss. Wien, xcv, 1887, p. 144) consists of
d c.c. cold saturated sublimate solution, 5 grms. sulphate of
soda, 2 grms. salt, and 300 c¢.c. water. Mosso finds that both
of these are too weak in sublimate.

Dusosce (Arch. Zool. Expér., vi, 1899, p. 481) uses (for
blood of Chilopoda) a solution of acetic acid, copper acetate,
copper chloride, osmic acid, thionin, 1 grm. each, water 400,
which, mixed with the blood, fixes and stains in about two
minutes.

Formol has lately been used. Marcano (Arch. de Med.
Expér., xi, 1899, p. 434) mixes fresh blood with a mixture of
100 parts of sodium sulphate of sp. gr. 1020 and 1 of formol ;
or with water 85 to 100 parts, sodium chloride 1, and
formol 1.

Kizer (Journ. Roy. Mic. Soc., 1900, p. 128) simply mixes
1 drop of blood with 3 of 2 per cent. formalin, and allows to
stand for an hour.

Scurippe (Hamat, Techn. Jena, 1910, p. 17) lets blood drop
into a mixture of | part of formol, 9 of liquid of Miller, and
10 of water, fixes therein for 2 to 4 hours at 40°C., filters,
washes and brings through alcohol and chloroform into
paraffin for sectioning.

719. Fixing and Preserving in films.— Muir (Journ. of Anat.
and Phys., xxvi, 1892) makes cover-glass films and drops
them into saturated sublimate solution, and after half an hour
washes, dehydrates, and passes through xylol into balsam.

Guutanp (Brit. Med. Jowin., March 13th, 1897; Scottish
Med. Jowrn., April, 1899) makes cover-glass films, and after

362 CHAPTER XXX.

a few seconds drops them face downwards into a solution
of—

Absolute alcohol saturated with eosin . 28 c.c.
Pure ether ; : . 25,4,
Sublimate in sector alcohol (2. grins.

to 10 c.c.) . : . 5 drops.

After three or four minutes firey are washed, stained, and
mounted in balsam.

For Junner’s fixing and staining and staining method, see
next §.

Many recent authors fix wet films with formol. Bunario
(Deut. med. Wochenschr., 1895, p. 572) mixes | part of 10 per
cent. formol with 9 of alcohol (the mixture must be freshly
prepared), and plunges films into it for a minute.

Similarly Guizanp, with 1 part of formol to 9 of alcohol.

Similarly WermEL (see Zeit. wiss. Mik., xvi, 1899, p. 50),
who combines various stains (methylen blue, eosin, gentian,
etc.) with the formol.

Epinatron (Brit. Med. Journ., 1900, p. 19) exposes films
for 15 to 30 minutes to vapour of formol under a bell-jar.

Scorr (Journ. of Path. and Bacter., vii, 1900, p. 181)
exposes films to the vapour for about 5 seconds and drops
into absolute alcohol, and after 15 minutes stains and mounts.

A short exposure (380 seconds) to vapour of osmiwm has
also been recommended.

720. Stains for Blood.— Fresh (unfixed) blood can be stained
on the slide.

Toison (Journ. Sct. méd, de Lille, fév., 1885; Zeit. wiss.
Mik., 1885, p. 398) recommends that it be mixed with the
following fluid :

Distilled water. . 160 cc.
Glycerin (neutral, 30° Teun 30 ,,

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.) This mixture stains
leucocytes sharply, which facilitates enumeration.

BLOOD AND GLANDS. 363

Bizzozuxo and Torrw (dreh. Sed. Mediche, 1880, p. 390)
dilute a drop with normal salt solution containing a little
methyl violet, which stains nuclei intensely, cytoplasm less
intensely.

Similarly Guiatio-Tos (Zeit. wiss. Mik., 1898, p. 166),
diluting with saturated solution of neutral red in salt
solution, which stains hemoglobigenous granules in five to
ten minutes. This is also recommended by Huriicu and
Lazarus, see § 309.

Snnilarly also Ross (Zrans. Path. Soc., 1907, p. 117),
using polychrome methylen blue.

Levapitt (Journ. Phys. path. Gén., Paris, 1901, p. 425)
allows solution of Brillantkresylblaw in alcohol to dry on
a slide, puts a drop of blood on the dried layer, and
covers. Similarly Czsaxis-DemuL (Arch. path. Anat., 1909,
p. 92), with a mixture of this dye and Sudan III; and
Naxanisut (Centralb. Bakt., 1901, p. 98), with methylen
blue BB.

Fized films may be treated with the usual tissue stains,
eosin being an important one, as it stains rose-red all parts
of blood-cells that contain hemoglobin. Huruica’s acid
hematoxylin, with 0°5 gr. of eosin dissolved in it, is a good
general stain. Or, stain with hemalum, and then with eosin
(0°5 per cent. in alcohol or water).

Earuicu’s triacid, § 296, gives good general views, and
demonstrates neutrophilous granules. His mixture for eosino-
philous cells has been given, § 311.

PaPPENHEIM’s panoptic triacid (on sale by Griibler) is
Ehrlich’s triacid with methylen blue in place of the methyl
green.

CHENZINSKI’s mixture, which is good, has been given, § 313.
Stain for six to twenty-four hours in a stove. This gives
rise to precipitates. To avoid them (WILLEBRAND, Deutsch.
med. Wochenschr., 1901, p. 57) you may make a mixture of
equal parts of 0°5 per cent. solution of eosin in 70 per cent.
alcohol and saturated solution of methylen blue in water,
and add acetic acid of 1 per cent. drop by drop till the
mixture begins to turn red, and filter before use. Or
(MicHaz.is, tbid., 1899, No. 80) make (a) a mixture of 20 parts
1 per cent. aqueous methylen blue with 20 of absolute alcohol,
and (b) a mixture of 12 parts 1 per cent. aqueous eosin with

364: CHAPTER XXX.

28 of acetone, and for staining mix equal parts of these and
stain for half a minute to ten minutes.

JnnNER (Lancet, 1899, No. 6, p. 370) mixes equal parts of
1:2 to 1:25 per cent. water-soluble eosin (Griibler’s) and
1 per cent. methylen blue, filters after twenty-four hours,
washes the precipitate on the filter, dries it, and dissolves it
in 200 parts of alsolute methyl] alcohol (the solution can be
had ready made from Gribler & Hollborn). (Or, simply
mix 125 c.c. of 0°5 per cent. solution of the eosin in methyl
alcohol with 100 ¢.c. of 0°5 per cent. solution of methylen
blue.) Cover-glass films are floated on to this, in which they
are fixed and stained in three minutes. Wash off the stain
with a little water (not under the tap), dry, and mount in
balsam. Erythrocytes red, all nuclei blue, parasites blue,
but with unstained nuclei.

The methods of May and Grinwatp are closely similar
to this.

Assmann (Minch. med. Wochenschr., 1906, No. 28; “ Das
eosinsaure Methylenblau,” Leipzig, 1908, p. 85) treats fresh
films for half a minute to three minutes in a Petri dish with
a few drops of Jenner’s solution (from Griibler & Hollborn),
then pours on 20 c.c. of distilled water with five drops of
7 per cent. solution of lithium carbonate, leaves for five
minutes, rinses in distilled water, dries with blotting paper,
and mounts in neutral balsam.

The foregoing mixtures give a stain—seemingly due to
the formation of an eosinate of methylen blwe—in which the
nuclei of blood-cells are blue and their plasma red to violet.
It was made out by Romanowsxy (St. Petersburger med.
Wochenschr., 1891) that wnder certain conditions mixtures of
these two dyes give a stain which is im some respects the
inverse of this, blood-cells being stained in divers hues,
according to their kinds, and any protozoan parasites that
may be present showing red nuclei and blwe plasma; which
greatly facilitates their detection and diagnosis. This re-
action appears to be due to the formation of an eosinate—
not of methylen blue, but—of Methylenazur, § 377. The
method, only vaguely indicated by Romanowsky, has under-
gone, at the hands of Zimmann, Zerrnow, Nocur, Reurer,
Micnagtis, Ruce, Maurer, Leisuman, Giemsa and others,
numerous modifications which have culminated in the

BLOOD AND GLANDS. 365

establishment of a process worked out by Giemsa as perhaps
the most trustworthy and efficient of “ Romanowsky ”’ stains.
This is as follows :

Giemsa’s Azur-eosin process. You start with a mixture of
eosin with methylenazur (instead of methylen blue). This
mixture is very troublesome to prepare, and is best obtained
ready made from Griibler & Hollborn (their “Giemsa’sche
Loesung fiir Romanowsky-faerbung”*), Air-dried films
(Deutsch. med. Wochenschr., 1907, No. 17) ave fixed in
alcohol or in methyl-alcohol (two to three minutes), and dried
with blotting paper. ‘hey are treated for ten to fifteen
minutes with a dilution of 1 drop of the stock mixture to
1 c.c. of water, washed under a tap, dried with blotting
paper, and again dried in the air and mounted in balsam, or
(preferably) preserved unmounted. All reagents, especially
the balsam, must be strictly free from acid,

Wet films (ibid., 1909, p. 1751) are treated as follows:
Fix them for twelve to twenty-four hours in a mixture of 2
parts saturated aqueous solution of sublimate with 1| of
absolute alcohol. Wash, and treat for five to ten minutes
with a mixture of 2 parts of iodide of potassium, 100 of
water, and 8 of Lugol’s solution. Wash, and treat for ten
minutes with 0°5 per cent. solution of sodium thiosulphate.
Wash, and stain as above (changing the stain for fresh after
half an hour), for one to twelve hours. ‘hen pass through
mixtures of acetone with first 5, then 30, then 50 parts per
cent. of xylol into pure xylol, and mount in cedar oil. This.
process is applicable to sections.

Or (ibid., 1910, p. 2476) a slide is placed in a Petri dish
and covered with a mixture of equal parts of methyl-alcohol
and stock mixture. After half a minute this is poured off
and enough distilled water poured in to cover the slide, and
the whole is rocked to mix the two. After three to five
minutes, wash in running water, dry, and mount in cedar
oil.

By any of these processes nuclei (red) arc demonstrated
not only in hematozoa, but in many bacteria, spirochaete,
coccidia, sarcosporidia, ete.

* To make this up from Gribler’s powders, dissolve 3 grms. of

Azur II-eosin and 8 decigrammes of Azur II, in 125 grms. of glycerin
and 375 of methyl-alcohol.

366 CHAPTER XXX.

See also, for paraffin sections, Scuunera, in Deutsch. med,
Wochenschr., xxxv, 1909, No. 48, or Zeit. wiss. Mtk., xxvii,
1910, p. 161, who passes through acetone and xylol into
balsam.

The older Romanowsky stains published by the authors
mentioned above, as also Laveran’s “ Bleu Borrell” seem to
be superseded by Giemsa’s.

Letsuman’s Romanowsky Stain (Brit. med. Journ., March
16th and September 21st, 1901) is as follows: To a1 per cent.
solution of Griibler’s medicinal methylen blue in water add
05 per cent. of sodium carbonate, heat to 65° C. for twelve
hours and let stand for ten days. Then add an equal
volume of 0'1 per cent. solution of Griibler’s Hosin extra B,
let stand for six to twelve hours, collect the resulting pre-
cipitate on a filter, wash it until the wash water comes off
colourless, dry and powder. For staining, dissolve 0°15 grm.
in 100 c.c. of pure methyl alcohol. Stain cover-glass films
(air-dried) for five to ten minutes ; flood the film with water
for one minute, and examine, or dry (without heat) and
mount in xylol balsam. Nuclei in shades of red, cytoplasm
bluish, parasites blue with ruby red chromatin.

Raapr (Minch. med. Wochenschr., 1911, No. 27; Zeit.
wiss. Mtk., 1912, p. 286) obtains a Romanowsky stain of
blood and parasites with Jenner’s solution. Films fixed
with alcohol and ether are first stained for five to ten
minutes in solution of one part methylenblau med. puriss.
Hoechst, 0°5 part of lithium carbonate and 100 of water,
kept for at least three weeks and diluted with 10 vols. of
water. Rinse with water, dry with blotting paper, flood
with Jenner’s solution diluted with 2 or 3 vols. of water,
and stain for five to ten minutes. Wash, dry with blotting
paper, and mount.

721. PAPPENHEIM (Anat. Anz., xlii, 1912, p. 525) recommends the
following for sections of hwmopotetic tissues, and also of kidney, liver,
hypophysis, suprarenals, lung, intestinal epithelium and central nervous
system. Fix in Orth’s Formol-Miiller, stain sections for twenty minutes
in a stove in “aqueous diluted alcoholic” solution of May-GrtnwaLp

or JENNER diluted with 8 vols. of water; after-stain for forty minutes
in the stove in “aqueous GIEMSA solution (15 ¢.c. of water with 10

drops of glacial acetic acid)”; differentiate in 100 c.c. of water with
5 to 6 drops of acetic acid; wash, dry between blotting-paper ; dehydrate

in mixture of equal parts of acetone andJabsolute alcohol, and mount in

BLOOD AND GLANDS. 367

neutral balsam. The result is not a Romanowsky stain, but a pale
methylen-blue-eosin stain.

See also WricHT, Pub. Massachusetts Gen. Hosp., iii, 1910, p. 1, or
Journ. R. Micr. Soe., 1910, p. 783.

For the special technique of eosinophilous cells see Martinorri in
Zeit. wiss. Mikr., xxvi, 1909, p. 4 (alphabetical bibliography of eight
pages.)

722. Demonstration of Blood-plates of Bizzozero (Kemp, Studies
from the Biol. Lab. Johns Hopkins Univ., May, 1886, iii, No. 6 ;
Nature, 1886, p. 132). A somewhat large drop of blood is
placed on a slide, and quickly washed with a small stream of
normal salt solution. ‘I'he blood-plates are not washed away,
because they have the property of adhering to glass. They
may be stained with solution of 0°02 per cent. of methyl
violet or 1: 8000 of gentian violet, in salt solution. ‘To
make permanent preparations of them, they should first be
fixed, by putting a drop of osmic acid solution on the finger
before pricking it.

They may also be stained in films, especially by the
Romanowsky method. According to Papvennumm (“ Farb-
chemie,” p. 107) Wasserblaw is almost specific for them.

Wricar (Journ. Morph., xxi, 1910, p. 274) studies them in
tissues, after fixation with formol or sublimate (not Zenker)
by staining with a modified Giemsa stain, and bringing
through acetone and oil of turpentine into turpentine
colophonium. Details loc, cit. or Journ. Roy. mic. Soe.,
1910, p. 783.

See also Dexuuysrn, Anat. Anz., xix, 1901. p. 533;
Kopscu, Intern. Monatschr. Anat. Phys, xxi, 1904, p. 344,
and xxiii, 1906, p. 359; Duutsmn, Zeit. phys. Chem., xiii,
1909, p. 1.

723. Wuicert’s Fibrin Stain (Fortschr. d. Med., v, 1887,
No. 8, p. 228).—Sections (alcohol material) are stained in a
saturated solution of gentian or methyl violet in anilin water
($ 286). They are brought on to a slide and mopped up
with blotting-paper, and a little Lugol’s solution is poured
on tothem. After this has been allowed to act for a sufficient
time they are mopped up with blotting-paper, and a drop of
anilin is poured on to them. ‘The anilin soon becomes dark,
and is then changed for fresh once or twice. The auilin is

368 CHAPTER XXX.

then thoroughly removed by means of xylol, and a drop of
balsam and a cover are added. ‘This stain may be applied
to celloidin sections without previous removal of the celloidin.

See also the modifications of this method by KRomayer (§ 656) ;
BEneEcKE (§ 690); Unna (Monatssch. prakt. Dermat., xx, 1895, p. 140) ;
WotrF (Zezt. wiss. Mik., xv, 1899, p. 310); and one of another sort by
Kockeu, Centralb. allg. Path., x, 1899).

Glands.

724, Mucin.—Hovur (Arch. mik. Anat., xxxvi, 1890, p. 310)
finds that the mucin of mucus cells and goblet cells stains
with baste tar colours and with alum hematoxylin, but not
with aerd tar colours. He obtained his best results by means
of thionin, and good ones with toluidin blue, both of these
giving a metachromatic stain—tissues blue, mucin reddish —
and also with methylen blue (which is particularly useful
from its power of bringing out the merest traces of mucin),
safranin, ete.

Tissues should be fixed for two to eight hours in 5 per
cent. sublimate solution, and paraftin 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).

Hyaline cartilage, the jelly of Wharton, and the Mastzellen
of Ehrlich give the same reactions with basie dyes as mucin
does.

See also Sussporr, Deutsche. Zeit. Thiermed., xiv, pp. 345,
849 (Zeit. wiss. Mik., vi, 1889, p. 205); Bizzozero, Atti. 2.
Accad. di Set. di Torino, 1889 to 1892 (reports in Zeit. wiss.
Mik., vii, 1890, p. 61; and ix, 1892, p. 219) ; also Unna, ibid.,
xii, 1896, p. 42.

The safranin reaction 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. Unwa employs chiefly
polychrome methylen blue.

As regards the thionin stain, see Hari, Arch, Mth. Anat.,
Iviti, 1901, p. 678.

Bruno (Bull. Soc. Nat. Napoli, 1905, p. 220) fixes and
stains the skin of the frog in a mixture of 100 ¢.c. of formol
of 1:25 per cent. with 8 ¢.c. of I per cent. solution of thionin.
Mucus glands red.

BLOOD AND GLANDS. 369

Kuurscuizxy (Arch. mik. Anat., xlix, 1897, p. 8) fixes in
his mixture ($ 57), and stains sections either in safranin with
2 per cent. acetic acid, or in a similar solution of neutral red
(two to three days, washing out with alcohol).

MAYER (Mitt. Zuol. Stat. Neapel., xii, 1896, p. 303, or lust edition) gives
the following two formule for mixtures that stain eawclusively mucus.

725. MayEr’s Mucicarmine (op. cit., last §).—One gramme of cay:
mine, and 05 grm. of aluminium chloride with 2 ¢.c. of distilled water
heated over a small flame for two minutes, and made up to 100 c.c. with
50 per cent. alcohol. This gives a stock solution, which is as a rule to
be diluted for use tenfold with distilled or tap water.

726. MayeEr’s Muchematein (ébid.).—Hematein 0:2 grm., alu-
minium chloride 01 grm, glycerin 40 ¢.c., water 60 cc. An alcoholic
solution may be made by dissolving in 100 c.c. of 70 per cent. alcohol,
with or without the addition of two drops of nitric acid.

727. Mucicarminic Acid (RawitTz, Anat. Anz., xv, 1899, p. 439).—
One gramme of carminic acid, 2 of aluminium chloride, and 100 ¢.c., of
50 per cent. alcohol.

728. Goblet Cells—So far as these contain mucin they give
the reactions above described, see Panuru, Arch. mek. Anat.,
xxxi, 1888, p. 118 cé seg.; Lis, dbid., xxvii, 1886, p. 481 ;
and Guywissge, C. R. Suc. Biol., 1907, p. 1212.

For intestinal epithelium, especially the cells of Paneru,
see also Martin, Unters. web. Oberfldchen wu. Driisenepithel,
Leipzig, 1910; and Kuuz, Arch. mik, Anat., Ixxvii, 1911, p.
541 (sections stained with alum hamatoxylin, treated for 20
to 30 seconds with tincture of iodine, stained a few minutes
with Victoria blue, then with eosin).

729. Salivary Glands.—Soicrr (Unters. z. Naturlehre d.
Menschen, xv, 5 and 6, pp. 2—15; Festschr. f. Geyenbaur,
u, 1896, p. 211) demonstrates the granules in serous cells
and half-moons of the submaxillary gland by hardening in
a 10 per cent. solution of formol, and then making sections
and staining with hematoxylin of Delafield or of Ehrlich,
the granules taking the stain.

Krauss (Arch. mik. Anat., xlv, 1895 p. 94) stains sections
either with Heidenhain’s iron haematoxylin or with Ehrlich-
Biondi mixture or thionin. See also Krausn, ¢bid., xlix,
1897, p. 709; and Mituer, Zett. wiss, Zool., 1898, p. 640.

24

370 CHAPTER XXX.

Granp-Mourse, and Trisonpwau (C. R. Soc. Biol., lin,
1901, p. 187) recommend for pancreas Nicot.e’s “ thionine
phéniquée,” which stains the insule of Langerhans hardiy at
all, the rest strongly.

730. Gastric Glands.—Kousrur (Zeit. wiss. Mik., xii, 1895
p- 314) differentiates the two kinds of cells in stomach
glands by over-staining with hematoxylin, washing out with
alcohol containing 1 per cent. of HCl, blueing with alcohol
containing | per cent. of ammonia, and, after washing,
staining for one to five minutes in a weak solution of
Siurefuchsin. Peptic cells blue, parietal cells red. Osmic
material caunot be employed.

Cavu (Arch. Anat. Micr., iv, 1901, p. 4) stains material
fixed with Bouin’s picroformol in Victoria blue of 1 per cent,

R. and L. Mont (Rich. Lab. Anat. Roma, ix, 1902) demon-
strate ducts and canaliculi of delomorphous cells by Golgi’s
bichromate and silver impregnation, especially with reju-
venated material (see Sacerporri), leaving it for 5 or 6 days
in half-saturated sulphate of copper, then for 24 hours in the
osmic-bichromate mixture. You can imbed in paraffin

(rapidly).

731. Intestine.—Bunsiuy (Amer. Journ. Awat., v, 1906 p.
323) stains sections of glands of Lieberkiihn in a mixture
of equal parts of saturated solutions of Orange G and
Saurerubin, and then with toluidin blue, and mounts in
balsam.

732. Liver—Bravus (Denkschr. Med. Nat. Ges. Jena, v,
1896, p. 807) demonstrates the bile capillaries by the rapid
method of Gorer, hardening in a mixture of one part formol
with three parts liquid of Miller or 3 per cent. chromic
acid.

Erpincer (Beitr. path, Anat., xxxi, 1902, p. 230) studies
them by means of a complicated modification of Wuicerr’s
neuroglia stain, and Ciucuanowskr (Anat, Anz., xxi, 1902, p.
426) by means of Weicert’s myelin stain (the 1885 method).

Oppgn (Anat. Anz., v, 1890, p. 144; vi, 1891, p. 168) puts
pieces of liver or spleen (alcohol material) for twenty-four
hours into a solution of neutral chromate of potash (4 to

BLOOD AND GLANDS. 371

10 per cent.), then for twenty-four hours into a ? per cent.
solution of silver nitrate, washes, dehydrates, and cuts
without imbedding. The lattice fibres are only stained near
the surface, so that tangential sections must be made.

Similarly Berxiny, dhid., 1893, p. 772, fixing in picric
acid, then in an osmium bichromate mixture, and then
silvering.

See also RANVIER, Journ. de Microgr., ix, x, 1885-6; IGaAcuscHI, in
Arch. path, Anat., xevii, p. 142, or Zett. wiss. Mik., 1885, p. 243 (gold
process for study of fibrous networks); Kuprrer, Sitzb. Ges. f. Morph.,
etc., Minchen, Juli, 1889, or Zezt. wiss. Mck., vi, 1889, p. 506; Krause
(Arch. mik. Anat., xlii, 1893, p. 57); and Trmornsew, Anat. Anz,, xxxv
1909, p. 296 (sections of frozen tissue stained with methylen blue).

733. Spleen —For lattice fibres, see Opret, last §.

Kuuiscuirzxy (Arch, mik, Anat., xlvi, 1895, p. 675) studies
the musculature in sections (of material from liquid of
Miller) stained for a day or more in a solution of lakmoid
in ether and mounted in balsam.

For elastic fibres he puts sections for half an hour or a
day into a mixture of 800 parts 96 per cent. alcohol, 40 parts
1 per cent. solution of carbonate of potash, 2 parts Magdala
red, and 1 part methylen blue.

For the blood-vessels he puts sections of Miller inaterial
for a few minutes into a solution of one or two parts of
Saurerubin in 400 parts of 3 per cent. acetic acid, washes
out in 2 per cent. acetic acid, and after-stains in a similar
solution of helianthin or Wasserblau until the red only
remains in the erythrocytes.

See also WHITING (Trans. Roy. Soc., Edinburgh xxxviii, 1896, p. 311) ;
ScHUMACHER (Arch mik. Anat., lv; 1899, p. 151); WEIDENREICH (cbid.,
lviii, 1901, p. 251).

734. Lymphatic Glands.—For lattice-fibres especially, see
Rogsste & Yosuipa, Beitr. path. Anat., xlv, 1909, p. 110, or
Zeit. wiss. mik., xxvi, 1909, p. 295. Sections stained with
hematoxylin and eosin, or Weigert’s iron haematoxylin, or
Bielschowsky’s neurofibril stain as applied by Maruscu, luc.
cit., § 691. The sections should not remain for more than
15 to 30 minutes in the oxide bath.

372 CHAPTER XXX.

See also for the thymus some very complicated methods of
SaLKinpD, Anaé. Anz., xli, 1912, Nos. 6 and 7.

735, Kidney.—Sauvur (Arch. mik, Anat., xlvi, 1895, p. 110)
finds that for the renal epithelium the best fixative is Carnoy’s
acetic alcohol with chloroform (three to five hours, washing
out with absolute alcohol). A mixture of nine parts alcohol
with one of nitric acid is also good, as is liquid of Perényi.
He stains with iron hematoxylin, and after-stains in a very
weak solution of Siurerubin in 90 per cent. alcohol, which
stains the ciliary plateau. He macerates with iodised
serum or one-third alcohol, staining afterwards with dahlia.

ARNOLD (Anat. Anz., xxi, 1902, p. 417) employs wtra vitam
staining methods for the study of the granules of the
epithelium cells. Sections of fresh kidney are cut with a
Valentin’s knife, and brought into a very dilute solution of
neutral red, or methylen blue, in which the granules stain in
a few minutes or hours. Or saturated solutions of the dyes,
or of indigo carmine, may be injected subcutaneously during
life, at intervals of fifteen to twenty minutes, and after two
to five injections the organ may be excised and_ sections
made and examined (see $$ 208 and 342 to 344),

CHAPTER XXXI.

NERVOUS SYSTEM—GENERAI, METHODS.

736. Introduction Microscopical research into the struc-
ture of the nervous system pursues two ends. Wither it is
desired to elucidate the infernal organisation of nerve-cells
and nerve-fibres, the processes employed to this end forming a
group of cytological methods, or it is desired to study the
forms of nerve-cells, the distribution of the divers groups
of nerve-cells in the grey matter, the connections that are
formed by means of nerve-fibres between them, and to follow
out the course of the tracts of fibres that enter into the
constitution of the white matter of the cerebro-spinal axis,
the processes employed forming a group of the anatomical
methods of neurology.

A large proportion of the methods used in the study of
nerve-tissue in peripheral organs having already been des-
cribed in the chapters on “‘ Methylen Blue,” “ Impregnation
Methods,” “Tegumentary Organs,” and “Muscle and
Tendon,’ the following chapters are chiefly devoted to
methods for the study of the central nervous system.

For minute details concerning the dissection and hardening of the
encephala of Man and the larger Vertebrates see MercrER, Les Coupes
du Systeme Nerveux Central, 1894, Paris, Rueff); D&srrine, Anatomie
des Centres Nerveur, Paris, 1895; Brvan Lewis, The Human Brain,
London, Churchill; OBERSTEINER, Anleztung biem Studium des Baues
d, nervisen Centralorgane im gesunden u. kranken Zustande, Leipzig,
Toeplitz; van Watsem, Verh. Akad. Witensck. Amsterdam, vii, 1899;
Bonvicint, Zeit. wiss. Mik., xxvi, 1909, p. 410; Bayon, Die hist. Unter-
suchungensmethoden des Nervensystems, Wiirzburg, 1905; and Spre.-
MEYER, Technik d. mikro. Untersuchung d. Nervensystems, Berlin, 1911,
and VENDEROVICS, Anat. Anz., xxxix, 1911, p. 414.

Fixation.

737. Fixation by Injection.—Fixation, in the proper sense
of the word, is, of course, ont of the question in the case of

374 CHAPTER XXXI.

the human subject. But in the case of the lower animals it
is possible to introduce fixing liqnids into the living nerve-
centres by means of injection, thus ensuring a much better
penetration of the reagents than can be obtained by simple
immersion.

Goxer (Arch. Ital. de Biologie, t. vii, 1886, p. 30) injects
2°5 per cent. solution of bichromate of potash, through the
carotid if he wishes to limit the hardening to the encephalon,
or through the aorta if he desires to fix the spinal cord.

De Quervain (Virchow’s Archiv, exxxiii, 1898, p. 489)
injects solution of Miller warmed to body heat. For dogs
300 to 400 c.c. are required, for cats one third to one half
that quantity. After injection the whole organ is put into
solution of Miller for some weeks.

(Further details in fourth edition.)

Mann (Zeit. wiss., Mik., xi, 1894, p. 482) injects through
the aorta. He first mjects for about twenty seconds physio-
logical salt solution warmed to 89° C. to wash out the capil-
laries, then saturated solution of corrosive sublimate, warmed
to 89°C. After five minutes of injection the brain is ‘re-
moved and put for twelve hours into the same sublimate
solution, after which it is either put for permanent preserva-
tion into 0-1 per cent. solution of sublimate, or is at once
passed through alcohol for imbedding in paraffin.

See also § 741 (Grrora) and Strona (New York Acad. of
Sci., January 13th, 1896; Anat. Anz., xi, 21, 1896, p. 655;
Journ. Comp, Neurol., xiii, 1908, p. 291); and MecFartanp
(Journ. App. Mier., 1, 1899, p. 541).

HArpenina.

738. Hardening by the Freezing Method.—The ether freezing
method is to be preferred. The sections should be floated
on to water, treated for a minute on the slide with 0°25 per
cent. osmic acid solution, and stained or otherwise treated as
desired. See §§ 182 and 183.

For a detailed description of these manipulations see
Bevan Lrwis’s The Iuwman Brain. Also Naarorrn, C. R.
Soc. Biol., xvii, 1909, p. 542, who finds that if the tissues
are soaked for twenty-four hours before cutting in formol of
3 per cent. the formation of ice crystals is diminished.

NERVOUS SYSTEM—GENERAL METHODS. 375

739. 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. Or,
still better, the preparations may be suspended in the liquid,
see § 34.

Another plan, which is good, is to add to the hardening
liquid enough glycerin or salt to make the preparations
just float.

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
dividing 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 progress. With material intended for the
Golgi impregnation it is well not to remove them at all.

The spinal cord, the medulla oblongata, and the pons Varolit
may be hardened in toto. The dura mater should be
removed at once, and the preparation ling up in a cylinder-
glass with a weight attached to its lower end, im order to
counteract the torsions of the tissues that may otherwise occur.

The cerebrum should have plugs of cotton-wool put into
the fissure of Sylvius, and as far as possible between the
convolutions. Unless there are special reasons to the con-
trary, the brain should be divided into two symmetrical
halves by a sagittal cut passing through the median plane
of the corpus callosum. Berz recommends that after a few
hours in the hardening liquid the pia mater should be re-
moved wherever it is accessible, and the choroid plexuses also.

The cerebellum should be treated after the same manner.
The hardening action of most solutions is greatly cnchanced
by heat. Thus Weicerr (Centralb. med. Wiss., 1882,
p- 819; Zeit. wiss. Mik., 1884, p. 388) finds that at a

376 CHAPTER XXXT.

temperature of from 80° to 40°C. preparations may be
sufficiently hardened in solution of Miller 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 seems that this rapid hardening does not always
give the best results. Sanur is of opinion that it does not
(see Zeit. wiss, Mik., 1885, p. 3), and other authors are of
the same opinion.

On the other hand, the slowness of the action of chromic
salts at the normal temperature is such that decomposition
may be set up before the hardening fluid has had time to do
its work. For this reason volumimous preparations that are
to be hardened in the slow way should be put away in a
very cool place—best of all in an ice safe. A hemisphere
will require eight or nine months for hardening in this way.

Very large quantities of liquid should be taken, and be
changed, at first, every day for fresh.

Reagents (except osmic acid) should at first be taken as
weak as is consistent with the preservation of the tissue, and
be changed by degrees for stronger,

See also PFISTER in Newrol. Centralb., xvii, 1898, p. 643 (Zeit. wiss.
Mik., xv, 1899, p. 494).

740. The Reagents to be employed.—Those most used are
formol, and the chromic salts.

Burcnarpr (La Cellule, xii, 1897, p. 337) says that “accord-
ing to the unanimous judgment of all investigators, the
bichromates of potash and ammonia should not be employed
for the cytological study of nerve-cells.” Nissi (Mneyel.
mik, Technik, 1, p. 253) holds that, for this purpose, only
alcohol, formol, sublimate, and occasionally nitrie acid, are
admissible. But this does not refer to hardening for purposes
of fibre-anatomy, nor indeed in an absolute sense to cyto-
logical studies. It means that these are the reagents best
fitted for producing a “ Nervenzcllenequivalentbild,” that
is—a standard and regularly obtatable fixation, always
amenable to certain current stains, of the whole of the various
kinds of nerve-cells found in nervous centres. For fibre-
anatomy he himself uses bichromate of potash.

741, Formol—Formol gives much I tler penetration than

NERVOUS SYSTEM—GENERAL METHODS. 377

the chromic salts, and even than alcohol, and allows of the
most various stains, including silver neuro-fibril stains and
the Golgi impregnation.

Several writers insist that for nervous tissue it should not
be acid; but some prefer it acid (see ‘“ Retina”). For
neuro-fibrils it certainly should be neutral. To neutralise,
it is generally sufficient to make up the solutions with tap-
water. It is not likely to overharden.

I use 1 part of formol to 9 of water (or 8 if the formol
has been keep long).

Wercerr (Bett. Kenntn, norm. mensch, Neuroglia, 1895,
p. 1146) puts portions of material of not more than half a
centimetre in thickness for four days into a “4 per cent.
solution of formol” (meaning, presumably, 4 per cent.
formaldehyde).

Marcus (quoted from sae see below) hardens spinal cord
for two or four weeks in a ¢ per cent. solution of “formalin,”
(meaning also probably for maldehpael, then in Miiller’s fluid
for a week in an oven at 37°C,

Van Girson (Anat. Anz, x, 1895, p. 494) has used
“ solutions of formalin of 4, 6, and 10 per cent.,” followed
by 95 per cent. alcohol. Myelin was found to be well
preserved.

Lact (cf. Zeit. wiss. Mik., xii, 1895, p. 32) has had good
results with “20 per cent. solutions of formol.”

Fisu (Proc. Amer. Mic, Soc., xvii, 1895, p.319) recommends:

Water . : : . 2000 c.c.
Commercial formalin 3 ; ; 50 ,,
Sodium chloride 3 ; ; : 100 germs.
Zine chloride . : . © 15

”
Brains should be Jeft in this mixtnre for a week or ten days

or more, then transferred to a solution of water 2000 c.c.,
formalin 50 c.c., in which they may remain indefinitely if
the jar be kept tightly covered.
Parker and Froyp (Anat. Anczeiyer, Bd. ix, 1895, p. 156)
advise (for sheep’s brains) a mixture of—
Alcohol 95 per cent. : : . 6 volumes
Formol 2 per cent. . 4 55
Brains may be kept for months in the mixture (¢id.,
1896, p. 568).
Frarau (Anat, Anz., xiii, 1897, p. 325) finds that brain

378 CHAPTRR XXXT.

angments in weight slightly in 10 per cent. formol solution
(spinal cord somewhat more) ; whilst in 1 per cent. solution
it may increase as much as 24 per cent.

Gerora (Zeit, wiss. Mtk., xiii, 1896, p. 314) puts human
brains into a 5 or 10 per cent. solution of formol, and after
twenty-four hours removes the pia and changes the liqnid ;
this is also further done every five to seven days, and in one
or two weeks the hardening is complete. In the case of
foetal brains of Canis, Felis, and Homo, he first injects the
vasenlar system with a 10 to 15 per cent. solution of formol
in 85 per cent. alcohol, and then brings the heads into the
5 to 10 per cent, watery solution; after one or two days he
removes the brains from the skull and puts them back for
fifteen to twenty days into the formol.

Kapyt (Poln. Arch, Biol, Med. Wiss., i, 1901, p. 80) takes
5 parts of formol, 100 of water, and 2 of hicarbonate of soda,
for four to ten days.

Hroricxa (Proce. U.S. Nat. Mus., xxx, 1906, p. 304) takes
3 parts of formol, 25 to 45 of water, and 72 to 52 of alcohol
of 95 per cent.

STRECKER (Ze/#. wiss, Mik., xxviii, 1911, p. 17) fixes small pieces for
twenty-four to forty-eight hours in ] part of formol of 10 or 20 per cent.
with 1 part of Ehrlich-Biondi triacid mixture, and imbeds in paraffin,

thus getting a stain at the same time as a fixation. Similarly with
toluidin blue, fixing it with ammonium molybdate.

742. Chromic Salts.—That most used is the bichromate of
potash.

The hquid of Krtickr has a more rapid action than the
other solutions of chromic salts. Santi, however (loc. c7t.,
§ 739), after having studied the action of the usual solutions,
concludes that the best hardening agent for fresh tissues is
pure bichromate of potash, in 3 or 4 per cent. solution, the
hardening being done in acold place. He rejects the liquid
of Krheki on account of the precipitates it so frequently
gives rise to (see § 54).

OBERSTEINER is of the same opinion, recommending pure
bichromate 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 (§ 42) for
twenty-four hours, followed by washing with water and
hardening in SO per cent. alcohol.

NERVOUS SYSTEM—GENERATL METHODS, 379

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., or formol, before
putting them into the hardening liquid, or to add formol
(say 3 per cent.) to it, in order to avoid maceration of the
deeper layers of tissue.

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. Oserrsrimner begins with
1 per cent., and proceeds gradually during six to cight
weeks to 2 or 3 per cent. (This is at the normal tempera-
ture; at a temperature of 35° to 45° ©. one or two weeks
will do.)

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.

Nissi (Hneyel, Mik. Technik., ii, p. 245) takes (for rapid
hardening) 100 parts of liquid of Miller, 3 of formol, and
enough glycerin to make the tissues float—for a few days,—
then pure Miiller or bichromate of potash.

Brrz’s methods (Arch. mik. Anat., 1873, p. 101)—Brain
and spinal cord are first hardened, for some days or weeks,
in 70 to 80 per cent. alcohol containing enough tincture of
dodine to give it a light brown coloration. (As fast as the
alcohol becomes colourless more iodine must be added.)
Then definitely hardened in bichromate of potash, of 3 per
cent. for spinal cord, medulla oblongata, and pons, 5 per cent.
for cerebellum, and 4 per cent. for cerebrum.

Unduly neglected nowadays.

Cerebrum (Buvan Lewis, The Human Brain, p. 102).—
Methylated spirit, twenty-four hours in a cool place. Miiller’s
solution, three days in acool place. Then change the liquid ;
and after three days more 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.

380 CHAPTER XXXI.

Brain (Haminton, Journ. of Anat. and Physiol., 1878,

p. 254).—Put into—
Miiller’s fluid : ; ‘ ; 3 parts
Methylated spirit . ; ; 1 part
in an ice-safe. Change the solution in a fortnight or three
weeks ; or if it is found that the reagent has duly penetrated,
remove to—
Bichromate of ammonia : : 1 grm.
Water . 4 : F : . 400 c.c.,
for one week. Then change the solution to one of 1 per cent.
for one week, and 2 per cent. for another week, or longer.
The pieces may be kept permanently in solution of chloral
hydrate, twelve grains to the ounce.

Entire Eneephalon (Duecke, Journ. Roy. Mic. Soe., 1883,
p. 449).—Bichromate of ammonia in $ to 1 per cent. solution,
according to the consistence of the brain. If it is soft he
adds, say, 4 to 7/5 per cent. of chromic acid to the solution,
and always 4 to 1 of the whole volume of alcohol.

Encephalon (M. Dovat, Rosin’s Journal de ? Anatomie,
1876, p.497).—Bichromate of potash 25, water 1000 ; change
after twenty-four hours, and again after three or four days.
After two or three weeks place in chromic acid of 3 per 1000,
change every day for the first week and after that every
eight days until the middle of the second month. The pre-
parations must remain at least two months in the chromic acid.

Onra (Berlin. klin. Wochenschr., 1896, No. 18) takes
Formol-Miiller ($ 113), changed every few days. Small
pieces may be sufficiently hardened in a few days in a stove.
This is now very popular.

Bonvicint (Zett. woss. Mih., xxvi, 1909 p. 412) puts
entire brains into 10 per cent. formol (first injected through
the carotids or into the ventricles) for six to cight days, then
slices of it (in the dark) into 4 parts bichromate of potash
and 2°5 parts sulphate of chromium in 100 of water, changed
weekly for two months (hemispheres), or twelve to fourteen
days (medulla and pons), or five to six days (cord).

Rawirz (thid., p. 338) puts formol material for exactly
five days into alcohol with 10 per cent. of tinctura todi
P.G., then for cight to ten into saturated solution of bi-
chromate, changed after the first day, then into 95 per cent.
alcohol in the dark for three days.

NERVOUS SYSTEM—GENERAL METHODS. 3881

743. OTHER REAGENTS.—Osmic acid is hardly useful for specimens
of more than 2 or 3 millimetres’ thickness. These, at a strength of 1
per cent., it will harden well in five to ten days.

Chromic acid is not much used alone. Its action is rapid, but uneven,
and causes shrinkage and brittleness. A very little (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.

Neutral acetate of lead in 10 per cent. solution affords an excellent
preservation of ganglion cells, according to ANNA KoTLAREWSKI (see
Zeit. wiss. Mik., iv, 1887, p. 387).

TRZEBINSKI (Virchow’s Arch., 1887, p.1; Zeit. wiss. Mik., iv, 1887;
p. 497) finds that ganglion cells (of the spinal cord of the rabbit and
dog) are best preserved by hardening for eight days in 7 per cent.
solution of corrosive sublémate, followed by alcohol containing 0°5 per
cent. of iodine. Similarly, Diomrporr (cbdd., p. 499), with brain. This
process produces artificial “ pigment spots”; they may be dissolved out
by prolonged treatment with warm water, or in five minutes by strong
solution of Lugou.

Fisy (The Wilder Quarter-Century Book, 1893, p. 335) and DonaLp-
son (Journ. of Morphol., ix, 1894, p. 123) have found that bichromate of
potash produces a slight increase both in weight and volume of brains
of sheep, whereas all the other reagents tried produce a diminution of
both these factors.

Several observers have lately been using acetic alcohol. So TIMOFEEW,
Intern. Monatsschr. Anat. u. Phys., xv, 1898, p. 259 (Carnoy’s second
formula, § 85).

Mann (Methods, etc., p. 95), for cell-studies, puts for twenty-four
hours into solution of 5 parts of iodide of potassium und 25 of iodine in
100 of water, then into 70 per cent. alcohol.

Fisu (Uhe Wilder Quurter- ae Book, 1893, p. 393) takes

Water . ‘ 400 c.c.
95 per cent. alcohol : 400 _,,
Glycerin : 3 250 ,,
Zinc chloride 20 grms.
Sodium chloride 20 germs.

for about three days, then transfers for a week or more to a mixture
of equal parts of the fluid and 70 per cent. alcohol, and finally stores in
90 per cent. alcohol.

OHLMACHER recommends his sublimate mixture, § 65, Kopis (Arch.
mik. Auat., lix, 1901, p. 212) fixes in saturated solution of cyanide of
mercury, brings into 10 per cent. formol, and sections by the freezing
method.

NE Is (Bull. Acad. Se. Belg., 1809, 1900, p. 726) fixes spznal ganyliu for
twenty-four hours in a solution of 20 grms. sulphate of copper, and sub-
limate to saturation, in a litre of 7 per cent. formol with 5 c.c. of acetic
acid.

Kine (Anat. Kee., iv, 1910, p. 218) after trying over twenty-five

382 CHAPTER XXXI.

methods on brains of fifty rats, concludes that the best is Ohlmacher’s.
The brain should be put into it for two to three hours, then for one into
alcohol of 85 per cent., then 70 per cent. with iodine for at least twenty-
four hours, then passed through ether into 2 per cent. celloidin for two
to three days, and passed through chloroform and benzol into paraffin.
Bouin’s is the best of the formol liquids; Tellyesnicky’s is the only one
of the bichromate mixtures that equals it. All sublimate mixtures fix
nuclei well, but vacuolise cytoplasm.

The reader will note that these results do not allow for subsequent
impregnation operations.

744. Nervous Centres of Reptiles, Fishes, and Amphibia.—
Mason (Central Nervous System of Certain Reptiles, etc. ; WHITMAN'S
Methods, p. 196) recommends iodised alcohol, six to twelve hows; then
3 per cent. bichromate, changed once a fortnight until the hardening is
sufficient (six to ten weeks).

BurcxuHarpr (Das Centraliervensystem von Protopterus, Berlin,
1892; Zeit. wiss. Mik., ix, 1893, p. 347) recommends a liquid composed
of 500 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 Pro-
topterus are hardened in twenty-four to forty-eight hours.

Fisu (Jowrn. of Morphol, x, 1, 1895, p. 234) employed for Desmognathus
aw mixture of 100¢.c. of 50 per cent. alcohol, 5 ¢.c. of glacial acetic acid,
5 grins. of corrosive sublimate, and 1 grm. of picric acid, fixing for
twelve to twenty-four hours, and passing through the usual alcohols.

Strona (Journ. comp. Neurol., xiii, 1903, p. 296) fixes (and decalcifies
at the same time) the heads of young Acanthias in a mixture of 9 parts
of 5 per cent. iron alumand 1 part of formol, for about two weeks, makes
paraffin sections, stains with hematoxylin, and differentiates in iron alum
of 1 to 2 per cent.

SECTIONS.

745. ImBEDDING is by no means always necessary. Sections
can be obtained from any part of the central nervous system
without it. ‘lhe material should be well hardened, and
elued on to a piece of wood or cork by means of a rather
thick solution of gum arabic. As soon as it begins to stick
to the support the whole is thrown into 80 per cent. alcohol
to harden the joint, after which it may be fixed in the object-
holder of the microtome and cut.

Or, you may simply make a clean cut at the bottom of the
specimen, dry it with blotting paper, and stick it on with
sealing wax.

‘To cut, the knife should be wetted with alcohol or water.

NERVOUS SYSTEM—GENERAL METHODS. 353

If the latter, add a little soap to prevent it from runuing into
drops on the knife.

Tf the collodion method has been taken it may be found
that notwithstanding every precaution the collodion has not
thoroughly penetrated the tissues. Good sections may, how-
ever, still be obtained by Duvat’s method of collodionising
the sections. The cut surface of the tissue is dried by
blowing on it, aud is covered with a thin layer of collodion
laid on it with a brush. As soon as this layer has somewhat
dried, which happens very rapidly, a section is cut and the
cut surface is collodionised as before, and so on for each
section. This process gives very good results, and may be
advantageously employed even with material that has been
successfully imbedded, as it gives a better consistency to the
tissue, and enables thinner sections to be obtained (van
Grsucuren, in litt.).

Strasser (Zeit. wiss. Mik., ix, 1892, p. 8) obtains pavattin
sections of 10 cm. breadth by 15 cm. length. He cuts out
from hardened material slices of from 1 to 2 cm. in thickness,
de-alcoholises them with xylol-carbolic acid mixture, § 167,
allows this to evaporate, and brings them first into melted
yellow vaselin, and lastly either into a mixture of vaselin and
paraffin of 42° melting point, or into pure paraftin.

See also Poso, th., xxvii, 1910, p. 353; Dexcke, loc. cit.,
§ 742; Disexine, Anut. Centres Nervewe, p. 29.

Strasser also imbeds the slices in cclloidin, and clears
them before cutting with a mixture of xylol-carbolic acid and
80 per cent. alcohol in equal parts.

FEIst (Zeit. wiss. Mck., viii, 1892, p. 492) marks the right and left sides
of spinal cord by imbedding with each segment of it 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.

For the freezing method see p. 117, and for further details
concerning imbedding and cutting see fourth editiwn.

LIESEGANG (Zezt. wiss. Mck., xxvii, 1910, p. 369) mounts large sections,
direct from water, in a layer of 5 per cent. solution of gelatin, lets this
dry, and varnishes it, dispensing with balsam and cover.

384: GHAPTER XXXI.

GENERAL STAINS.

746. Carmines.—Ammonia-carmine is good for general views.
Stain very slowly in extremely dilute solutions. Bichromate
material ought to be brought direct into the stain without
passing through alcohol (see § 51).

Picro-carmine has much the same action, but gives a
better demonstration of non-nervous elements.

I prefer carmalwm, with formol material, as giving a more
delicate stain. I find it better than paracarmine.

Recent authors recommend soda-carmine. The Hneyel.
mik. Technik., p. 927, advises staining Miller material for a

couple of days in a 2 per cent. solution of carminate of soda
(Griibler’s).

BoRax-CARMINE, with indigo-carmine or an anilin blue to follow,
gives elegant but not very instructive images, and I have abandoned it.

See also ScHmaAvs (Minch. med Wochenschr., 1891, No. 8; Zett. wiss,
Mik., viii, 1891, p. 230) ; Upson (Neurolog. Centralb., 1888, p. 319; Zezt.
wiss. Mik., v, 1888, p. 525); FREEBoRN (Amer. Mon. Mic. Journ., 1888,
p. 231; Journ. Roy. Mic. Soc., 1889, p. 305); Kapyr, Newrol. Centralb.,
xx, 1901, p. 687; Zeit. wiss. Mik., xviii, 1902, p. 483); CHILESorTI (cbid,
xix, 1902, p. 161, and xx, 1903, p. 87); Kappers and Krryen (cbid.
xxvill, 1911, p. 275) (Weigert material after-stained with paracarmine).

747. Anilin blue-black has been much recommended by SANKEY
(Quart. Journ. Mie. Sci., 1876, p. 69);.Bevan Lewis (Human Bruin,
p. 125); Vegas (Arch. f. Psychiatrie, xvi, p. 200); GIERKE (Zezé. wiss.
Mik., S84, p. 876); Marvinorri (ebid., p. 478); JELGERSMA (Ze?t. wise.
mik., 1886, p. 39); Scumaus (Minch. med. Wochenschr., No. 8, 1891.
p. 147; Zeit. wiss. Mék., viii, 1891, p. 280),and others. As to this colour
see § 329, and for details see previous editions.

748.—Nigrosin has given useful results in some hands. I

have not succeeded, probably because the dye is of inconstant
composition, and does not keep well.

749. Picronigrosin.—Marrinorti (luc. cif., 1884, p. 478)
stains for two or three hours or days in a saturated solution
of nigrosin 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.

Jounston (Morph. Jahrb, xxxiv, 1905, p. 150) adds a
little Siurefuchsin to the mixture.

750. Kaiser (Zett. wiss. Mck., vi, 1889, p. 471) stains sections of spinal
cord for a few hours in a solution of 1 part of naphthylamin brown, 200

NERVOUS SYSTEM—GENERAL MBTHODS. 385

of water, and 100 of alcohol, washes with alcohol, clears with origanum
oil, and mounts.

751. Alizarin—Scurorrun (Neurol. Ceutralb., xxi, 1902,
p. 338 ; Zeit. wiss. Mik., xix, 1908, p. 381) stains sections
for twenty-four hours in a 1 to 2 per cent. solution of sul-
phalizarinate of soda, differentiates for $ to 1 minute in
tap-water, dehydrates, and mounts. This is a general stain,
but demonstrates Nissl bodies and other internal details.

752. Nissu’s methylen-blue is used as a general stain by some.

Rorute (Folia Newrobiol., ii, 1909, p. 885; Zedt. wiss. Mik., xxvi, 1909,
p. 252) fixes and stains for about four weeks in saturated solution of
Methylenazur I (Gribler) in formol of 10 per cent., puts for ten to fifteen
minutes into aceton, then for twelve hours into chloroform, and imbeds
in paraffin. He also has a process with trichloracetate of lead and
methylenazur.

Rawitz (Zett. wiss. Mik., xxvi, 1910, p. 341) has some complicated
methods with Indulin, Indamin blue, and Azosiureblau, which take
twenty-eight days ; and (b¢d., xxviii, 1911, p. 1) others with fuchsin and
azofuchsin which take over thirty-six days.

Kaprers (zbid., p. 417) describes a stain of chromic material with
extract of elderberries.

ScarpaTetti (Neurol. Centralb., xvi, 1897, p. 211; Zezt. wiss. Mik.,
xiv, 1897, p. 91) stains sections of formol material for five minutes in
1 per cent. hematoxylin, treats for five minutes with concentrated
solution of neutral acetate of copper, differentiates with Weigert’s
Lorax-ferri-cyanide, treats with concentrated solution of carbonate of
lithia, washes and mounts. Myelin is not stained.

758. Maztory’s Phospho-molybdic-acid Hematoxylin has been -
given, § 271.

For the extremely complicated modification of AUERBACH, see Neurol.
Centralb., xvi, 1897, p. 439, or Zect. wiss. Mik., xiv, 1897, p. 402, and for
that of Kopis see § 271.

754. Hematoxylin and Siurefuchsin.—Finorri (Virchow’s Arch.,
exliii, 1896, p. 1383; Zect. wiss. Mck., xili, 1896, p. 236) stains in heema-
toxylin, counter-stains for three minutes with 0°5 to 1 per cent. solution
of Saurefuchsin, and differentiates in 75 per cent. alcohol containing a
very little caustic potash.

Van Grrson’s hematoxylin and picro-Siurefuchsin, § 398, gives
useful general views of nerve-cells, axis-cylinders, and neuroglia.

755. ALY (Minch. med. Wochenschv., 1892, No. 4; Zezt. wiss. Mik.,
ix, 1, 1892, p. 81) stains for a couple of hours in solution of Congo red
in absolute alcohol, and washes out with pure alcohol. For peripheral
axis-cylinders, and other elements.

25

CHAPTER XXXII.
NERVOUS SYSTEM—CYTOLOGICAL METHODS.

756. Introduction.—The ordinary methods of cytology are
of course available for nerve-cells. But there are two
characteristic elements of these cells—the tigroid substance,
and the system of neuro-fibrils, which require, for minute
study, special methods such as the following.

A, Methods for Cells, demonstrating Tigroid Substance.

757. Tigroid substance is a markedly basophilous element,
occurring in the form of granules or larger irregular blocks
known as the “ bodies of Nissu.” It takes up basic anilin
dyes, but does not hold them with such special energy, as
for example, the chromatin of nuclei. It is usually stained
by the regressive method, with very careful differentiation.
The material is usually fixed with alcohol, formol, or subli-
mate. Van Guuucuten and Neus (La Cellule, xiv, 1898,
p. 374) recommend Gutson’s mixture, § 69.

All the following stains have the defect of keeping badly ;
they generally do not last more than a few months.

758, Nissi’s Methylen-blue Method (Neurol. Centralb., 1894,
p- 508).—Fresh material is hardened in 96 per cent. alcohol,
and sectioned without imbedding. The sections are floated
on to the following stain poured into a watch-glass :

Methylen blue (Alethylenblay B. ae , 3°75 parts.
Venice soap 3 i : 175,
Distilled water . : : . 1000-0

»

(This stain should not be ne fresh, but kept for at least
three months.)

The watch-glass is warmed over a flame to about 65° to
70° C. till bubbles are given off. ‘lhe sections are then
brought for an instant (5 to 20 seconds) into a mixture of

NERVOUS SYSTEM—CYTOLOGICAL METILODS. 387

10 parts of anilin oil with 90 parts of 96 per cent. alcohol,
and as soon as no more colour is given off from them are got
on to a slide, dried with filter-paper, cleared with oil of
cajeput, dried again with filter-paper, treated with a few
drops of benzin, and mounted,

Van Geuucuten (in litt.) prefers to take paraffin sections,
mounted on slides by the water method, and stain them for
five or six hours in Nissl’s mixture in a stove kept at 35° to
40° ©,

Reum (Minch. med. Wochenschr., 1892, No. 13; Zeit. wiss.
AMik., ix, 1893, p. 387) stains for half a minute to a minute
in a hot 0°1 per cent. of methylen blue, washes in 96 per cent.
alcohol till no more colour comes away, clears with origanum
oil, and mounts in balsam.

Gornarp (C. R. Soc. Biol., v, 1898, p. 530) stains ced-
loidin sections for twenty-four hours, without heat, in Unna’s
polychromatic methylen blue and differentiates in a mixture
of 5 parts of creosote, 4 of oil of cajeput, 5 of xylol, and 16
of absolute alcohol.

Lurruten and Sorco (Neurol. Centralb., xvii, 1898, p. 640)
differentiate in Unna’s glycerin-ether mixture (} 702),
remove this with absolute alcohol, and clear in origanum
oul.

Lunnuorr (¢bed., 1910, p. 1) recommends polychrome
methylen blue for 2 minutes, followed by Griibler’s “ Karbol-
Methylgriin-Pyronin ” for 20 minutes.

Lorp (Journ. Ment. Sct., October, 1898) makes sections of
fresh tissue, frozen, treats them for a few seconds with a
mixture of equal parts of 6 per cent. formaldehyde and
saturated solution of picric acid, then rinses, and warms till
bubbles appear in 5 per cent. solution of methylen blue.

Menrz von Kroau (Centralb. Bakt., viii, 1911, p. 95) stains
paraffin sections for 5 minutes in polychrome methylen blue,
treats for 1 to 15 with 2 per cent. chromic acid, differentiates
till blue with 5 per cent. tannin, and mounts in balsam.
Shows also axis cylinders.

See also GOLDSCHEIDER & Fuatau, Normale und. path, Anut. der
Nervenzellen, etc., Berlin, Kornfeld, 1898 (Zezt. wiss. Mck., xvi, 1899,
“p. 102), and Nissz’s remarks thereon, Deutsche Zeit. Nervenhertk., xiii,
1899, p. 318 (Zect. wiss. Mik., xvi, 1899, p. 370) ; Cox, Intern, Monatsschr.
Anat. Phys., xv, 1898, Heft 8; Myrrs, Anat. Recurd, 1908, p. 434;

388 CHAPTER XXXIf.

InBerG, Neurol. Centralb., 1896, No. 18; Savini, Centralb. Bukt., 1909,
p. 697.

759. Methylen BlueandErythrosin..—Heup (Anat. Phys., Anat.
Abth., 1895, 1896, p. 399) stains sections on slides, with the
aid of a gentle heat, for one or two minutes in a solution of
1 grm. of Griibler’s erythrosin in 150 of water with two
drops of glacial acetic acid, washes out with water, and
stains in a mixture of equal parts of Nissl’s methylen blue
and 5 per cent solution of acetone, warming strongly the
while, until all odour of acetone has disappeared. After
cooling he differentiates with 0°1 per cent. solution vf alum
until the sections appear reddish, rinses in water, dehydrates
as rapidly as possible in absolute alcohol, and passes through
xylol into balsam. See further hereon HEtp, op. cit., 1897,
pp. 226—238, 273—305 (Supplementband), and Boccarpt,
Mon. Zool. Ital., x, 1899, p. 141 (stains in a mixture of
erythrosin 0-1, toluidin blue, 0°2, and water 100 parts, and
differentiates in 0°5 per cent. alum solution).

760. Thionin—Lennosskix (Fein. Bau. d. Nervensystens,
Berlin, 1894, p. 149) stains sections of formol material for
five minutes in a concentrated aqueous solution of thionin,
rinses with water, and mounts as Nissl. The stain does not
keep well.

Similarly Ramon y Casat, Man. de Anat. Path. Gen., 1896
(see Zeit. wiss Mik., xv, 1899, p. 375), and Luxenzure,
Neurol. Centralb., xviti, 1899, p. 629.

761. Toluidin Blue—Lernuossex (Neurol. Centralb., xvii,
1898, p. 577; Zeit. wiss. Mik., xv, 1899, p. 492). Sections
are stained on slides for a night in concentrated solution of
toluidin blue, rinsed in water, quickly differentiated with
alcohol, cleared with xylol or carbolic-acid xylol, and mounted
in balsam.

Similarly Potumorpwinow (Zeit. wiss. Mik., xvi, 1899,
p. 371, who stains in a very weak alkaline solution, 1 part of
1 per cent. solution to 119 of water and 1 of carbonate of soda.

762. Neutral Red—Jutiuspurcer (Neurol. Centralb., xvi,.
1897, p. 259) stains sections of formol material, for half to
three quarters of a minute in warm 1 per cent, solution of

NERVOUS SYSTHM—CYLOLOGICAL MUTIHODS, 389

neutral red, dehydrates in alcohol, and passes through
bergamot oil to balsam.

Rosin (Deutsche med. Wochenschr., 1898, No. 39, p. 615;
Zeit. wiss. Mik., xvi, 1899, p. 238) stains in concentrated
aqueous solution, washes out thoroughly with water, and
passes through alcohol (must be free from acid) into xylol
and balsam. Granules of Nissl red, nucleoli red, all the
rest yellow,

763. Alizarin—See § 751.

764. Cresyl Violet. — Bienscnowsky & Puten (Neurol.
Centralb., 1900, p. 1141) stain for 24 hours in 50 c.c. of
water, with 6 to 10 drops of concentrated aqueous solution
of Cresyl violet RI. and pass through water, alcohol, cajeput
oil and xylol into balsam. The preparations keep better than
thionin or toluidin blue ones.

765. Tigroid Substance, other Methods.—See Cox, Zezt. wiss.
Mik., xiii, 1896, p. 498; xv, p. 369; xvi, 1899, p. 101; Anat. Hefte, xxxi,
1898, p. 75; Intern. Monatsschr., xv, 1898, H.8; AUERBACH, Monatsschr.
Psychiatrie, iv, 1898, p. 31; Zett. wiss. Mtk., xv, 1899, p. 493; BUEHLER,
Verh. Phys-Med. Ges. Wiirzburg, xxxi, 1898, p. 285; Zeit. wiss. Mik., xv,
1899, p. 351; Mossg, Arch. mikr. Anat., 1902, p. 403 (his argentamin
stain, which see).

B. Methods for Cells and Fibres, demonstrating Newrofibrils.

766. Neurofibrils; General Characters.—Nerve-cells, and the
fibres into which they are prolonged, contain, in addition to
the chromatic, basophilous element demonstrated by the
method of Nissl, a characteristic “achromatic” element,
consisting chiefly of fine, fairly refractive fibrils, which can
only be seen with difficulty in the unstained state, and can
only be well brought out by means of special stains. They
may be fixed with osmic acid, and made out in thin sections
of medullated nerves studied in dilute glycerin or water,
and may be, to a certain extent, isolated by maceration.

KurrrFer (Sitzb. math. Kl. Akad. wiss. Miinchen, xiii, 1884, p. 470)
proceeded as follows: A medullated 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

390 CHAPTER XXXII.

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. It
is said that if sections are mounted by the water method the stain will
be extracted ; but why not make the water acid, § 291?

The usual histological stains either leave them colourless
or stain the surrounding plasma more strongly than the fibrils
themselves. There may thus be produced a “negative”’
image of fibrils which does not really show the true neuro-
fibrils, and being taken for them may prove a source of error.
The following methods are such as have been recommended
as giving true stains of the fibrils.

767. Neurofibrils: Silver Methods—Those most used are
Ramon y Casat’s and BirtscHowsxy’s. The essential differ-
ence between the two is that Ramén employed a_ single
impregnation bath—of nitrate of silver; whilst Bielschowsky
employs two—one of nitrate and one of silver oxide dissolved
in ammonia.

Fixing agents should in general be neutral or alkaline; for
acids or oxidants divert the impregnation from the fibrils to
other elements, chiefly tigroid and nuclear elements.

Sections should be thin—not more than 15 pz to 20 pr.

Toning with gold has the effect of intensifying the stain
in the fibrils and lightening it in surrounding elements, thus
giving enhanced contrast. It also serves to favour the pre-
servation of the stain.

The object of fixing with hyposulphite is to remove from
the tissues any unreduced silver salts which might cause a
loss of contrast by darkening the ground of the preparations.
With well reduced preparations, such as Ramén’s are in
general, it is not necessary.

Bielschowsky’s methods have the advantage of being
applicable to larger specimens than Ramén’s, for they give a
more uniform impregnation through the whole thickness of
the objects (especially with the central nervous system of
man) ; whilst Ramon’s (especially his formula la) only gives
the desired results in a—sometimes very thin—layer between
an overstained outer and an understained inner one.

Bielschowsky’s are applicable to very old formol material.
Bayon (Die histologischen Untersuchungs-Mcthoden des Nerven-
systems, p. 157) has succeeded with material four years

NERVOUS SYSTEM—CYTOLOGIGAT, METHODS. 391

old. But this must be about the limit, for I find material
seven years old entirely refractory.

The two methods do not give quite the same images of
neurofibrils. Ramén’s tends to show intra-cellular fibrils
anastomosing into networks, whilst Bielschowsky’s (like
Bethe’s) tends rather to show independent fibrils traversing
cells without anastomosing.

For the demonstration of pericellular networks, buds of
Held and Auerbach, non-medullated fibres and nerve- endings
in general Ramén’s seems the better.

Bielschowsky’s method stains elastic fibres and connective
tissue fibres, which Ramén’s do not.

768. Ramon y Casat’s Methods—This section contains all
the methods described by Ramén in Trabajos del Lab. de
Investigaciones Biologicas, viii, 1910 (t. xiii of Rev. trimes-
tral micrografica). The numbering is that of Ramon.

Formula la—For small and medium nerve-cells. Small
pieces of fresh tissue are put direct (7. e. without previous
treatment with any other reagent) into nitrate of silver of
1:5 per cent.

They are kept for about three days (2$ for very small
objects, such as spinal cord of newborn rabbit; 4 for
medium-sized, such as cord or cerebellum of adult rabbit ;
5 for specimens of the size of its cerebrum) in the silver.
They must be kept in a stove at a temperature of about
35° C. all the time. (In summer, with a temperature con-
stantly over 22° C., the stove may be dispensed with, provided
that the impregnation be prolonged for two or three days
more.) ‘The tissues are known to be ripe for reduction when
a freshly cut surface shows a brownish-yellow coloration.

They are then washed for one or two minutes in distilled
water, and put into—

Pyrogallol or hydroquinon . 1 to 2 grm.
Water. . : 100 _,,
Formol* . : : Do se

* The formol is not necessary, but is useful. You may take pyridin
instead (1 to 3 per cent.). You may also add 0'5 per cent. of sodium
sulphite. The stronger the pyrogallol the greater the contrast: it is
sometimes useful to take as much as 3 per cent., but then the over-
impregnation of the outer layers will be increased. Hydroquinon (T add)
reduces more energetically than pyrogallol.

392 CHAPTER XXXII.

They remain in this for twenty-four hours. They are then
washed, hardened in alcohol, imbedded in celloidin or paraffin,
and sections mounted in damar.

TELLYESNICKY (Verh. Anat. Ges., 1904, p. 183) advises toning the
sections for five to thirty minutes in 150 cc. of water with 4 c.c. of
1 per cent. gold chloride. This is good for weak impregnations, but
not desirable for strong ones which show good contrast.

Sections from the outer layers are too dark for study,
those from the innermost too pale (if the specimens are
large ones), whilst thoso from intermediate layers are fit for
study. ‘he over-stain of the outer layers can be diminished
by diluting the silver bath with 1 volume of water for the
last twelve hours. :

This method has the defect of giving an imperfect fixation
and impregnating almost exclusively cell-bodies and dendrites.
It is not good for the large cells of adults, but excellent for
small and medium cells of newborn or very young subjects,
and for very early embryos in general.

Formula la A—As last, but with nitrate of silver of 3 to
6 per cent.

Doaten (Anat. Anz., xxv, 1904, p. 558) finds this method
gives results not attainable by other means in the study of
the corpuscles of Granpry (stoving for four to six days).
Similarly Kormur (ibid., xxvi, 1905, p. 560) with epiderm of
Inmbricus, etc.; and other authors for the ganglionic cord
of Hirudinea.

Formula la B—As before, but nitrate bath of only 0°75
per cent., and very small pieces of tissue, preferably embryos
and newborn subjects. Poor fixation, much shrinkage, but
vigorous stain of neurofibrils, of nucleolar granules, and of
the intra-nuclear rodlet of Roncoront.

Formula la C—As before, but silvering in nitrate of 2 per
cent. with one fourth of absolute alcohol or acetone added.
Better fixation than pure nitrate. Gives results very similar
to those of la with dog, cat and rabbit, and better results
with human cerebrum and cerebellum.

Formula 2a.—Mivation for 24 hours in alcohol of 96 per
cent. Tissues not washed, but mopped with blotting-
paper, and put into nitrate of silver of 1-5 per cent. for
seven days at 35° C., or six days at 40° C. The rest as

NERVOUS SYSTEM—CYTOLOGICAT, MBTHODS. 393

Formula la.* Good impregnations of nerve-centres of adults,
of peripheral nerve-endings, of regenerating nerves, and of
early embryos and of young fishes. Impregnates medullated
(and many non-medullated) fibres (black), large and medium
neurones (fibrils brown), the basket fibres of cells of
Purkinje, the granular layer, and in the cerebrum large and
medium pyramidal cells and nerve-fibres. Results fairly
constant, but sometimes showing a granular precipitate of
unknown origin.

To hinder this precipitate, and at the same time to hasten
the impregnation, it is well to add to the alcohol certain
substances which Ramén calls “accelerators.”” Such are
chloral hydrate, veronal, pyridin, nicotin, ethylamin, anti-
pyrin, and others. A

Hy pnotics, particularly veronal and chloral, and in a less
degree pyridin and ammonia, also act as rejuvenators, reviving
the susceptibility of impregnation which has been lost by
tissues that have lain too long in alcohol. Ramén has thus
succeeded with pieces of cerebrum and cerebellum that had
been half a year in alcohol.

Formula 2a A—Fixation for 24 to 48 hours in alcohol
of 96 per cent. with 2 per cent. of hydrate of chloral added.
Silver bath of 1:5 per cent. for five days in the stove. The
rest as usual. JVeronal (same proportion) gives the same
result, as do also sulphonal, trional, hedonal, ete. The results
are very constant. Medullated fibres well shown.

Formula 2a B.—F ix (time not stated) in alcohol with 10 to
20 per cent. of pyridin, wash for some hours in pure alcohol,
and silver as usual (5 days). Results regular and constant.

Formula 2a C.—Fix for 24 hours in 50 c.c. of alcohol
with ten drops of nicotin. Mop up with blotting paper,
without washing, and silver as usual for five days (or four at
40° C.). Good results with adult tissues, especially spinal
cord. Good penetration and less shrinkage than with pure
alcohol.

* Tf the impregnation of inner layers should be too weak, the sections
may be toned with—

Water 100 c.c.
Sulphocyanide of ammonium 3 gr.
Hyposulphite of sodium 3,

1 per cent. gold chloride : . afew drops.

394, CHAPTER XXXII.

Formula 2a D.—Fix for 24 hours in allyl aleohol (the
industrial product will do). Wash for some hours in
several changes of water. Put for a day into 50 c.c. of
alcohol with 4 drops of ammonia. Silver for 4 days at
35° to 38° C., and reduce as usual. Good for human tissues,
especially fibre plexuses of cerebrum and_ cerebellum.
Instead of allyl alcohol you may take acetal or aceton. Put
for six hours into aceton with 25 per cent. of water, then for
24 into pure aceton.

Formula 8a.—Fixation in anmoniacal alcohol for 20 to 48
hours. The most generally useful formula is, 50 cc. of
alcohol of 96 per cent. with four to five drops of ammonia
(of 22° strength). But for cerebrum not more than one to
three drops: for cerebellum, ganglia, spinal cord and
regenerating tracts, four drops: for neurofibrils of the large
neurons of the bulb and cord, nine to ten drops. To avoid
shrinkage, it 1s well to begin by putting for six hours into
alcohol of 70 per cent., then 85 per cent. without ammonia,
then for the rest of the time into the ammoniacal alcohol.
Do not wash, but mop up with blotting paper before putting
into the silver. Silver for four to four and a half days
(small specimens) at 40° C., or medium to large (8 to 4 mm.
thick) for 5 days at 32° to 35°C. So long as the tissues are
only yellowish-white, they are not ripe for reduction ; light
grey indicates ripeness ; dark grey over-ripeness. Reduce as
formula la.

Specimens may be decalcified, after reducing and washing,
conveniently in alcohol of 96 per cent. with a few drops of
nitric acid.

For delicate impregnation of fibrils of the soma of large
and medium neurones, this formula is superior to all others.
It gives good results with the majority of nerve centres, and
is particularly good for non-medullated fibres, peri-cellular
baskets of cerebellum, buds of Held and Auerbach in the
oblongata, for human sympathetic, and for the study of
regenerating elements.

Formula 8a A.—F ix in 50 c.c. of alcohol with 10 grm. of
elycerin and six to ten drops of ammonia. Good for retina,
non-medullated fibres, and especially the buds of Held and
Auerbach.

Formula 8a B.—TFix in 50 ¢.c. of aleohol with 1°5 c.c. of

NERVOUS SYSTEM—CYTOLOGICAL METHODS. 395

33 per cent. alcoholic solution of efhylamine. Results the
same as with ammoniacal alcohol.

Formula 4a.—Pieces of tissue of not more than 4 mm. in
thickness are fixed for six to twelve hours in formol of 15
per cent. Wash for six or more hours in running water.*
Put for 24 hours into 50 ¢.c. of alcohol with five drops of
ammonia. Wipe with blotting paper, silver for 5 days (or
four if the stove is at 38° to 40°C.). The rest as usual.
Sharp impregnation of the finer fibres of nerve centres, and
of the terminal buds of pericellular nests. Adult tissues
give better results than young ones. Energetic stain of the
arborisations of the mossy fibres of the cerebellum.

Formula 4a A.—Fix in “a mixture of formol and alcohol.”
Wash out thoroughly with running water, and silver and
reduce as usual. Fixation more rapid and better, results
similar to those of 8a.

Formula 5a—Small pieces put first for 6 to 8 hours
into a mixture of equal parts of distilled water and pyridin,
then for 18 to 24 hours into pure pyridin. Wash for several
hours in running water, and put for a day into alcohol of 90
per cent. Wipe, and put for 4 to 5 days into the silver at
35° to 88° C., and reduce as usual. Not very good for adult
organs, but superior to all others for the earliest phases of
neurogenesis, and good for regenerative processes.

Formula 6a.—Put for 24 hours -into 50 ¢.c. of water with
5 grms. of hydrate of chloral, rinse and put into 50 c.c. of
alcohol of 96 per cent. with five drops of ammonia (time not
stated). Wipe with blotting paper and put for 4 to 5 days
at 35° to 38° C. into nitrate of silver of 1°5 per cent, and
reduce as usual. Results very constant, without shrinkage.
Stains perfectly the fine plexuses of cerebrum, bulb and
cord, the Purkinje baskets and mossy fibres; also motor
plates. Nuclei sufficiently stained to enable cells to be easily
recognised. Good for regenerating nerves.

Formula 6a A—T'ix for 24 hours in chloral of 10 per cent.,
wash for 6, and put direct into the silver. Stove four days.
Results similar to those of la. Medullated fibres well
stained.

* Tf formol (or pyridin or the like) be not thoroughly removed by

washing, the stain will he weakened, and of a light reddish tone. (But
Tam not clear that this vigorous washing out is altogether advisable.

396 CHAPTER XXXII.

Formula 7a.—Fix for 24 hours in Merck’s fibro-lysin,
wash for 6, put for 24 into 50 c.c. of alcohol with five drops
of ammonia. ‘The rest as usual.

Instead of fibrolysin, lysidin may be taken.

Application of the Foregoing Methods to Different Oljects of
Study.

(1) For the study of the evolution of neuroblasts and nerve
fibres in very early embryos, it is absolutely necessary to avoid
fixing with formol, or alcohol with an accelerator, or am-
moniacal liquids. The best formule are 2aand 5a. Applic-
able to all vertebrates, but preferably to embryos of birds
and fishes. nN

(2) For late embryos and fwti of mammals.—Besides the
above, 3a, 6a and alcohol with an accelerator. Best subjects,
embryos of chick from the fifth day, and of rabbit from the
tenth to the twelfth. Or newborn birds, with ammonia
alcohol or da.

(3) For sympathetic ganglia.—Formula 8a, or pure alcohols
or 4a and 5a. Best with man. Dog, cat, and rabbit give
mostly weak reactions. ‘The visceral ganglia are the most
difficult.

(4) Sensory ganglia— Formula 2a, or 3a. Easy.

(5) Cerebelluin.—The most favourable of all nerve centres.
For Purkinje cells, la or 8a. For the baskets, climbing
fibres, and medium and small dendrites, 2a, or its variants.
For terminal rosettes and collaterals of mossy fibres, and for
the plexuses of the granular layer, 4a, or sometimes 5a or 6a.
lor the stellate cells of the molecular layer, 2a and 38a, on
the dog.

(6) Cerebrum.—In general, the same formule as for the
cerebellum, especially la, for pyramids of young dogs and
cats (of eight to twenty days). In formula 8a, the propor-
tion of ammonia should be diminished. For fine plexuses,
4a, 5a, and 6a.

(7) Spinal cord and bulb.—All the formule are applicable.
For neurofibrils of motor cells the best subject is the dog
of four to fifteen days, with formula 3a, with a large dose of
ammonia (ten drops). Also the alcoholic fixatives with an
accclerator. For medullated fibres, large and small, 2a or

NERVOUS SYSTEM—CYTOLOGICAL METILODS, 397

6a. For buds of Held and Auerbach, and for fine plexuses,
da, 8a A, or 5a.

(8) Ganglia of Invertebrates.—For the medicinal leech
(not for other leeches), la with 3 to 6 per cent. of nitrate.
For Hamopis, Aulostomum, Pontobdella and Glossiphonia, 2a
or, better 38a, with not more than two to five drops of
ammonia, and silver of 3 per cent., three to three and a half
days.

For further details see SAncuez in Trab. Lab. Invest.
Biol. Madrid, vii, 1909, p. 31, or Zeit. wiss. Mik., xxvii,
1910, p. 392.

Lumbricus is generally refractory to Ramén’s methods.
Bouter (Le Nevraze, x, 1909, p. 15) obtains good impregna-
tions by acidifying the fixatives. He takes (A) formol of 25
per cent, with 5 per cent. of acetic acid, or (B) the same
with 0°5 per cent. of ammonia added, or (C) 100 ¢.c. alcohol,
25 c.c. formol, 5 c.c. acetic acid and 0°5 c.c. ammonia. These
results are confirmed by Kowausxt, La Cellule, xxv, 1909, p.
292, who also gets impregnations by simply starving worms
for several days, or exposing them to cold (-5° C.) fora
quarter of an hour.

(9) Regenerating nerve tissue—For nerves operated a
month or more previously, 2a or 3a with not more than three
drops of ammonia will stain equally the old and the new
fibres. For nerves operated not more than two to ten days
previously, 3a with 4 to 6 drops of ammonia, 5a with pyridin
and 4a, also sometimes 6a. For regenerations in cord,
cerebrum and cerebellum, 8a with three drops of ammonia, or
5a with pyridin, or pure alcohol.

769. Variants of Ramon’s Methods.—Busta (Riv. Path. Nerv.
Ment., Firenze, xv, 1910, p. 333) fixes for 48 hours in alcohol with 5 per
cent. nitric acid, neutralises in alcohol with ammonia, and silvers and
reduces as Ramon.

Kato (Folia Neurobiul., ii, 1908, No. 3; Zett. wiss. Mikr., xxvi, 1909,
p. 281) fixes in formol of 10 to 15 per cent., and silvers for one to five
days at 35° C. in 5 per cent. argentamin to which has been added
nitrate of silver of 3 per cent. in excess, and reduces in 10 per cent.
formol with 1 per cent. of hydroquinon.

PusatTERI (Arch. Path. Anat., 195 Bd., 1909, p. 547) fixes for three to
six days at 35° to 38° C. in a mixture of 45 c.c. of tachiol (10 per cent.
solution of fluoride of silver) with 155 of water, rinses and reduces for

24 hours in formol of 5 to 10 per cent. with 1 to 2 per cent. of hydrc-
quinon. He tones the paraffin sections in 10 c.c. of water with 2

398 CHAPTER XXXII.

drops of acetic acid und five of 1 per cent. gold chloride, and fixes with
hyposulphite of soda of 5 per cent.

LIESEGANG (Kolloidchemie, Beihefte, iii, 1911, H. 7; Zedt. wiss. Mck.,
xxviii, 1912, p. 369) makes sections of formol material by the freeaing
process, and silvers them until yellow. He then adds to the silver bath
an equal volume of 50 per cent. solution of gum arabic and the same
amount of saturated solution of hydroquinon. After one or two min-
utes the sections are brought into 10 per cent. solution of hyposulphite
of sodium, washed and mounted. Results said to be the sameas by the
usual process.

770. BretscHowsky’s Methods (BretscHowsky and Wot ry,
Biol. Centralb., xxv, 1905, p. 683).—Objects of not more
than 2 mm. in thickness are fixed in neutral formol of 6 to 10
per cent. (time not stated), washed out well with distilled
water, and put for at least two days into nitrate of silver of
2 per cent.in the dark. Wash for a few minutes, and put
for half an hour to several hours into a bath made as
follows:—To nitrate of silver of 10 per cent. add drop by
drop 40 per cent. solution of caustic soda until no further pre-
cipitate is formed (this will be about 5 drops to 10 c.c.),
dissolve the precipitate almost entirely in just enough
ammonia, filter and add 4 to 5 vols. of water. The solution
will only keep for a few hours. After this bath, wash again,
put for 1 to 6 hours into formol of 4 to 5 per cent., dehydrate
and bring through xylol into paraffin. Sections are toned
on the slide for 1 to 2 hours in chloride of gold of one tenth
to one twentieth per cent. (which it is well to neutralise with
lithium carbonate), rinsed, fixed for 5 to 15 minutes in hypo-
sulphite of soda of 5 per cent., washed for 6 to 12 hours in
running water and mounted in balsam,

If nuclei take the impregnation, the fibril stain will not
have succeeded, but there may be a useful stain of other
elements, especially tigroid matter.

Scutemmer (Zeit, wiss. Mik., xxvii, 1910, p. 22) makes the
oxide bath by adding the caustic soda in excess, and washing
the precipitate by repeated decantations until the wash water
no longer gives an alkaline reaction, takes it up with ammonia
and filters through spun glass, and thus obtains a solution
which will keep for several days. I find the filtering is not
necessary.

For older forms of this method see lasé ed., and Neur,
Centralb., xxiii, 1908, p. 977, and xxiv, 1904, p. 387 (the

NERVOUS SYSTEM—CYTOLOGICAL METHODS. 399

toning bath is here 10 c.c. of water with 2 to 3 drops of |
per cent. gold chloride, and 2 to 3 of acetic acid.

BieLtscnowsky (Journ Psych. Neurol., xii, 1909, p. 135;
Zeit. wiss. Mik., xxviii, 1911, p. 226) also has a method with
pyridin. Formol material, up to one cubic centimetre for
adult tissue, and up to 5 centimetres long for embryos, is put
for 3 to 4 days into pyridin, washed for some hours in
several changes of distilled water, and pub for 3 to 5
days into nitrate of silver of 3 per cent. at a tempera-
ture of 86°C. It is then put for 24 hours into an oxide
bath made as follows : Precipitate 5 c.c. of 20 per cent. solu-
tion of silver nitrate by 5 drops of caustic soda of 40 per
cent., dissolve in ammonia, q. s., add 100 c.c. of water (and,
to hinder formation of precipitates in the tissues, a few drops
of ammonia). After this bath, wash for a couple of hours in
many changes of distilled water, and reduce in formol of
20 per cent., dehydrate and make parattin sections. These
may be toned, but there is not much gained by it.

He also (zbid.) makes sections by the freezing method, of
formol material, and puts them for 24 to 48 hours into
pyridin, and washes them out well with water as before.
They are then put for 24 hours (or more) into 3 per cent.
solution of nitrate of silver at the normal temperature ; then
into the oxide bath prepared as before, but with only 20 c.c.
of water instead of 100, and without addition of ammouia
(the bath ought not to contain an excess of ammonia recog-
nisable by the smell). They remain in this until yellowish-
brown, but not more than half an hour, are well washed, and
reduced in formol of 20 per cent. They are then toned
with gold and fixed with hyposulphite as described ante.

By these pyridin methods intra-cellular fibrils are generally
not so well shown as by the older method, but axis cylinders
come out better, and glia remains unstained. The method
succeeds even with material that has been for years in acid
formol, and gives a uniform impregnation of entire blocks
f tissue.

Scut1z (Neurol. Zentruib., xxvii, 1908, p. 909) finds that
the times given by Bielschowsky are too short—which is not
at all my experience. For toning, he puts the sections for
10 minutes into 10 cc. of water with two drops of acetic
acid, then for 30 to 45 into 10 ¢.c. of water with three drop

400 CHAPTER XXXL.

of 1 per cent. gold chloride (until blackish-grey). He fixes
for 3 to 5 minutes in 10 ec. of sodium hyposulphite
of 5 per cent. with one drop of acid sodium sulphite, and
washes out for 24 hours in distilled water.

Paron (Mitth. Zool. Stat. Neapel, xviii, 1907, p. 576) fixes
fish embryos in 4 per cent. formol neutralised with carbonate
of magnesia, makes both silver baths only # to 1 per cent,
strong, washes with weak acetic acid, aud reduces in 10 c.c.
of 1 per cent. hydroquinon with 1 cc. of formol. After
toning, he stains with 1 per cent. solution of eosin in
absolute alcohol.

Sanp (C. RB. Ass, Anat. Bruvelles, 1910; Bibliogr. anut.
Supp., 1910, p. 128) gives the following as entirely certain
for man, dog, cat and rabbit. Specimens of not more than
5 mm, in thickness are fixed for 48 hours in a freshly
prepared mixture of 90 parts of acetone and 10 of
nitric acid (change for fresh after half an hour and once
again within 24 hours). Wash out for at least 6 hours in
pure aceton, changed two or three times. Make paraffin
sections, and silver for three days at about 37°C. in
20 per cent. solution of silver nitrate. Put for ten minutes
into a uixture (at least three days old) of 1000 parts of
water, 10 of acetate of sodium, 5 of gallic acid, and 3 of
tannin (to be changed if it becomes turbid). Mount at once
or tone until grey (five minutes) in 80 parts of water with
17 of 2 per cent. solution of sulphocyanide of ammonium and
3 of 2 per cent. solution of gold chloride, and fix for a few
seconds in 5 per cent. solution of hyposulphite of soda.
Neurofibrils grey-violet, shown in cells, dendrites and axoues.
Terminal buds of Held also clearly shown, and nothing else
stained. You may after-stain in any way, even with Weigert’s
or Benda’s neuroglia stain.

Borxe (Anut. Anz., xxxv, 1909, p. 136) says that Bielschowsky’s
method will succeed after many kinds of fixation, even that by picro-
sulphuric acid. For embryos he finds the best is 10 parts of formol to
90 of 60 per cent. alcohol, the objects being washed out with pure
formol of 10 to 12 per cent. until all the alcohol is removed, before
silvering.

Luaaro (Monit, Zool. Ital., xv, 1904, p. 353) has a highly complicated
method with collargol (colloidal silver) which has not met with favour.

NERVOUS SYSTEM—CYTOLOGICAL METHODS. 401

771. Neurofibrils: Gold Methods—ArAruy’s gold method,
§ 371, gives perhaps the sharpest stain which has yet been
obtained—with certain invertebrates, but only with these:
and even with these it is difficult and uncertain.

Joris (Bull. Akad. Med. Belg., April 80th, 1904) gives the
following as being certain. Material fixed by the usual
methods is put for 8 to 12 hours into a 5 per cent. solution
of ammonium molybdate, then imbedded in paraffin. The
sections (fixed on slides by the water method) must be
washed for many hours or days in water. They are then
treated for about ten minutes with a 1:5 per cent. solution
of colloidal gold in water, rinsed and mounted. The stain
1s permanent.

The colloidal gold used was obtained from the Chemische
Fabrik Hzypey, in Radebeul-Dresden. It will dissolve in
about a day.

772. Neurofibrils, Molybdenum-Toluidin Blue (Butus, Zeit.
wixs. Mik., xvii, 1900, p. 13).—Pieces of central nervous
system (of Vertebrates) are fixed for twenty-four hours in
nitric acid of from 8 per cent. to 7°5 per cent. strength, and
brought direct into alcohol of 96 per cent. for a day or more.
They are put for twelve to twenty-four hours into a mixture
of one part of ammonia (of sp. gr. 0°95) with three of water
and eight of 96 per cent. alcohol, then for six to twelve
hours into pure alcohol; they are then put for twenty-four
hours into a mixture of one part concentrated hydrochloric
acid with three of water and eight to twelve of alcohol, then
for ten to twelve into pure alcohol, and thence for not more
than two to six hours into water. They are now mordanted
for twenty-four hours in a 4 per cent. solution of ammonium
molybdate, brought for 24 hours into alcohol, and imbedded
in paraffin (noé celloidin). Sections are seriated on albumen
(without water), then passed through xylol and alcohol into
water, and “ differentiated’””—by which the author means
washed out—with water. About 1 to 1'5 ec. of distilled
water should be poured on to the slide so as to form over the
sections a layer 1°5 to 2 mm. deep, and the slide is put for
two to ten minutes into a stove heated to not more than 55°
to 60° C. The sections are then rinsed several times with
water, a solution of one part of toluidin blue in 38000 of

26

402, CHAPTER XXXII.

water is poured on to them, they are again stoved for ten
minutes, rinsed with water, treated with 96 per cent. alcohol
till no more colour comes away, and passed through absolute
alcohol and xylol into xylol balsam.

The method is also applicable to invertebrates, for which
other fixations than nitric acid are admissible, and the
impregnation with the molybdate may be done on the
sections instead of the uncut tissues. The results are not so
certain as for vertebrates.

Lucaro (Riv. Pat. Nerv. Ment., Firenze, x, 1905, p. 269) modifies
this by fixing in nitric acid dissolved (to 1 per cent.) in aceton.

Donaceto (Aun. Nerrol. Napoli, 1904, p- 161) fixes pieces
not more than 5 mm. thick for five to six days in pyridin,
changed at least once, washes in water, and mordants for
twenty-four hours in ammonium molybdate 4 grms., water 100,
hydrochloric acid 4 drops. Wash, get into paraffin, treat
sections on slide for one minute with water, stain for three
to thirty minutes in a 1: 10,000 solution of thionin, and
mount; or, better, first treat again for fifteen to thirty
minutes with molybdate solution,

JADERHOLM (Arch. mik, Anat., Ixvii, 1905, p. 108) finds that the
pyridin causes enormous shrinkage, and that the thionin agglutinates the
fibrils.

Paravicini (Boll. Mus. Z Anat. Comp., Torino, xx, 1905, p. 514)
fixes and mordants in the dark, and differentiates after staining with
extremely weak hydrochloric acid.

See also TomasELLi, Zeit. wiss. Mik., xxiii, 1907, p. 422, and
MontTanaRl, zbid., xxviii, 1911, p. 22, who describes observations which
seem to throw doubt on the objectivity of the network described by
Donaggio.

773. Neurofibrils, APATHY’s Heematein Method (Mitth. Zool. Stat.
Neapel., xii, 1897, p. 712).—Material may be fixed with sublimate, liquid
of Zenker, picro-sulphuric acid, or any mixture that is not inimical to
staining with alum hematoxylin, and should be preserved in 90 per
cent. alcohol. Portions are stained for at least forty-eight hours in the
hematein solution 1 A, § 259, and are then washed for up to twenty-four
hours in absolutely pure distilled water, preferably suspended therein.
Before the stain has become washed out of the neurofibrils, it is fixed
therein by putting the preparations for three to five hours into spring
water, after which they are put back for not more than two hours into
distilled water, dehydrated as rapidly as possible by hanging them up

NERVOUS SYSTEM—CYTOLOUICAL METHODS. 403

in absolute alcohol, and imbedded (they must be protected from the
light whilst in the chloroform through which they are passed into
paraffin, or whilst in celloidin). Sections are made and mounted in a
resin or in neutral glycerin.

This method has given splendid results with Hirudinea and some
other invertebrates, hut I believe has not yet been successful with
vertebrates.

774. Neurofibrils, Berlin Blue (S. Msymr, Anat. Anz., xx, 1902, p.
535).—Material is best fixed in 10 per cent. formol, then mordanted for
eight to twenty days in ferrocyanide of potassium of 24 per cent. (or this
salt may be added to the fixative), then put for two to four days into
iron alum of 10 percent. Wash for several hours, imbed in paraffin, and
mount sections in balsam. Besides neurofibrils, the sheath of Schwann
and the constrictions of Ranvier are stained. Like the Golgi chrome-
silver process, this method gives a partial impregnation, only certain
elements taking on the stain.

775. The Methylen blue intra vilam method is important,

see the processes of ApAtuy, Doarmt, and Berue in Chapter
XVI.

776. The methods of Cox for the fibrils of spinal-ganglion cells, Zeit.
wiss. Mik., xiii, 1896, p. 498, and Anat. Hefte, x, 1898, p. 98, seem to be
definitively superseded.

777. Golgi’s Inrra-cettuLAR Nerworx.—For the study of
his apparato reticolare interno, Gouct (Verh. Anat. Ges., xiv,
1900, p. 174) uses a mixture due to Vurarri, consisting of—

5 per cent. bichromate . ‘ . 30 parts

0:1 per cent. platinum chloride . . 30

1 per cent. osmic acid : . 15 to 307,
and after hardening therein puts (for a time varying from a
few hours to ten days) into a mixture of 1 part of bichro-
mate of 3 or 4 per cent. with two of saturated solution of
sulphate of copper, and thence into silver nitrate of 0°8 to 1
per cent. See further Verh. Anat. Ges., xiv Vers., 1900,
p- 174.

More recently (Arch. Ital. Biol., xlix, 1908, p. 272; Mon.
Z. Ital., xix, 1908, p. 263) he fixes for six to eight hours in
a mixture of equal parts of 20 per cent. formol, saturated
solution of arsenious acid, and alcohol of 96 per cent., and then
puts for one to three hours (or days) into nitrate of silver of
1 per cent. He then reduces in any developer, usually 20 grms.

2”

404, OHAPTER XXXIL

hydroquinon, 5 grms. sodium sulphite, 50 c.c. formol of 20 per
cent., and 1000 c.c. water. Wash and imbed, preferably in
celloidin. ‘lone the sections until grey in 1000 c.c. water
with 30 grms. each of hyposulphite of soda and sulphocyanide
of ammonium and 10 per cent. of 1 per cent. gold chloride.
It is well, though not necessary, to treat them first with a
solution of 0°5 grm. of permanganate of potash and 1 grm. of
sulphuric acid in 1000 of water, and then with 1 per cent.
solution of oxalic acid, before mounting.

LEGENDRE (Anat. Anz., xxxvi, 1910, p. 209) omits the toning and per-
manganate, and imbeds in paraffin.

Similarly, CoLtry et Lucien, Bibliogr. Anat. Supp., 1909, p. 238.

SaRaGNnone (Patologica, i, 1909, p. 536; Journ. Roy. Mier. Soc., 1910,
p. 256) silvers with a mixture of 30 c¢.c. of tachzol (10 per cent. solution
of silver fluoride) with 100 of water.

Besta (Anat. Anz., xxxvi, 1910, p. 477) fixes for two days in 20 parts
of formol with two of acetic aldehyde and 80 of water, washes for
twenty-four hours in distilled water changed 7 or 8 times. and puts for
two days into 4 per cent. solution of ammonium molybdate, makes
paraffin sections, stains in a 1: 1000 solution of thionin, differentiates in
3 parts of creosote to 1 of absolute alcohol, and passes through pure
creosote and xylol into neutral balsam. Recommended for Purkinje
cells and spinal ganglia of young subjects.

Kopscu (Sztzb. Acad. Wiss. Wien, xl, 1902, p. 929; Zedt. wiss. Mik.,
xx, 1904, p. 347) demonstrates it in spinal ganglion cells as follows; the
ganglia are put for eight days (or exceptionally, a few more), into osmic
acid of 2 per cent. and paraffin sections made. The network becomes
quite black and is sharply demonstrated, unless the impregnation has
heen too prolonged, in which case the rest of the cell body becomes
blackened also.

Ss6vau (Anat. Hefte, xxx, 1906, p. 362) fixes in formol before treat-
ing with the osmic acid.

778. Medullary Sheath (Neuroceratin, etc.).—Gotc1 (quoted
from Rezzonico, Arch. per le Set. med., iv, 1879, p. 85)
puts pieces of spinal cord into 2 per cent. solution of
bichromate of potash, for eight to fifteen days in summer, or
a month in winter. Wash, and put into 6°75 per cent.
solution of nitrate of silver, for two or three days in summer,
or eight or ten or more in winter. Pass through alcohol into
oil of, turpentine, tease therein, and mount in damar. Expose
to sunlight therein for eight to ten days; or to diffused day-
light for twenty to forty days. Demonstrates funnels and
spirals,

NERVOUS SYSTEM—CYTOLOGICAL METHODS. 405

Por peripheral nerves, Goner (ibid, p. 238) puts pieces
into the bichromate for from four hours to at most two
days, passing specimens at intervals into the silver, where
they remain for twelve to twenty-four hours. Wash in
several changes of alcohol, tease therein, and pass through
oil of turpentine into damar. Reduce therein in direct sun-
light for a few days or weeks. The preparations keep well.

He also (ilid., p. 238) puts pieces into a mixture of 10
parts 2 per cent. bichromate with 2 of 1 per cent. osmic acid,
and passes them on into the silver at intervals of three hours,
after the first four, during twenty-four hours. The silver is
of 0°5 per cent. strength, and the pieces remain in it for
any time not less than eight lours. The rest as before.
Results somewhat more precise, but the stain does not keep
in damar. It is important that the nerve should not have
been stretched.

Sata (Verh. Anat, Ges., 1900, p. 176) employs the method
of Verarit for the intracellular network, last §.

See also concerning these methods Monnino, op. ctt., viii,
p. 48, and Perrone, Intern. Monatsschr. Anat., v, 1888.

GaLut (Zeit. wiss. Mik, iii, 1886, p. 467) puts for eighteen
to twenty days into solution of Miiller, then stains for fifteen
to twenty minutes in aqueous solution of China blue, washes
out in alcohol, clears in essence of turpentine, and mounts im
damar.

Pratyer (Zeit. wiss. Mek., vi, 1889, p. 186) fixes for
several days in a mixture of 1 part of Liq. Ferri Perchlor.
(Ph. G., ed. 2) and 3 to 4 parts of water or alcohol, washes
out well in water and stains for several days or weeks in a
concentrated solution of ‘ Kchtgrim” in 75 per cent.
alcohol. See also Berr, Juhrb. Psychiatrie, i, 1893, 1 Heft.

Cox (Anat. Hefte, 1, 1898, p. 75) fixes nerves in osinic acid
of 2 per cent. (rabbit) or 1 per cent. (frog), washes, de-
hydrates, clears with bergamot oil, and mounts in balsam,
The bergamot oil dissolves out the myelin, and leaves the
neuroceratin visible. It may be necessary to leave the
nerves for forty-eight hours in the oil.

Corntna (Anat. Anz., xvii, 1900, p. 309) studies the net-
work in the ischiatic of the frog on sections of sublimate
material strongly stained with iron haematoxylin.

See also Kaptan (Arch. D’sychiatr., xxxv, 1902, p. 825;

406 GHAPTER XXXIT.

Zeit. wiss, Mik., xix, 19038, p. 508)—sections stained with
Siiurefuchsin and differentiated by the method of Pat.

Gevorist (La Cellule, v, 1889, p. 136) has the following:
(a) A nerve is treated with liquid of Purinyi, either pure or
with addition of a trace of osmic acid, and examined in
glycerin. By this treatment the myelin loses its excessive
refractivity and the network comes out clearly. (b) Silver
nitrate. Good images, but uncertain. (¢) Treatment with a
mixture of osmic acid of 1 per cent. and absolute alcohol.
The network comes out black.

CHAPTER XXXIII.
MYELIN STALNS.

779. Iron Hematoxylin.—I find the simplest way to make
a inyelin stain is to make paraffin sections of formol material
and stain them with iron hematoxylin, exactly as for central
corpuscles (say twelve to fourteen hours in the mordant, six
in the hematoxylin, and about two minutes for the differen-
tiation). Sections best not over 15. You may after-stain
the cells (which are only grey) with carmalum, but not for
more than half an hour, or the hematoxylin will be attacked.
The stain is not so esthetic as Weigert’s, but quite as sharp.
Axis cylinders are not shown.

Similarly Reaaup, C. R. Acad. Set., clxviii, 1909, p. 861,
but adding a chrome mordantage either concurrently with the
formol fixation, or subsequently.

Algo Naczorrs, C. 2. Soc. Biol, lxvii, 1909, p. 542, with
sections of formol material by the freezing method ; Houser,
Journ. Comp. Neurol. and Psych. x, 1901, p. 65, and
Brooxover, tbid., xx, 1910, No. 2; Sprretmuyer, Newrol,
Zentralb., xxix, 1910, p. 348, and his Technik. d. mikro,
Untersuch. d. Nervensystems, 1911, p. 87, with sections of
25 to 85 by the freezing method ; Loyxz, C. R. Sve., Biol.,
lxix, 1910, p. 511, who differentiates first lightly, till the
grey begins to come out, in the iron alum, then washes,
and differentiates further in Weigert’s borax ferricyanide ;
Guserr, Zeit. wiss. Mik., xxviii, 1911, p. 279, who mordants
with iron alum, stains with molybdic acid hematoxylin, and
differentiates with the borax ferricyanide ; Sroznzner, ibid.,
xxiii, 1906, p. 329, who mordants celloidin sections of formol
material for five minutes in Lig. ferri sesquichloratt, stains
in hematoxylin of 0°5 per cent., and differentiates in the
mordant or in borax ferricyanide; and Kopis, Arch. mik.
Anat., lix, 1901, p. 211, who fixes for one or two days in

4.08 CHAPTER XXXII.

saturated solution of cyanide of mercury, hardens in 10 per
cent. formol, and stains sections of frozen material by
Heidenhain’s iron hematoxylin.

780. WaicErt’s Methods.—There have been in all thrce
methods of Weicgerr—the 1884 method, the 1885 method,
and the 1891 method.

The 1884 method (Fortschr. d. Med., 1884, pp. 113, 190;
Zeit. wiss. Mck., 1884, pp. 290, 564), which depends on the
formation of a chrome lake of hematoxylin, may be con-
sidered to be superseded. Not so the two others, which
depend on the formation of a copper lake in addition to the
chrome lake.

781. Weicrri’s 1885 Method (Forlschr. d. Med., 1885,
p. 136; Zeit. wiss. Mik., 1885, pp. 899, 484; Ergebnisse der
Anutomie, vi, 1896 [1897], p. 10).—The tissues are to be
hardened in bichromate of potash. Wetcert takes (Hrgeb-
nisse, p. 10) a 5 per cent. solution, and if time is an object
hardens in a stove. (Other bichromate mixtures will do,
e.g. Miiller’s, Kultschizky’s, Zenker’s ; Erlicki’s is not to be
recommended.) ‘The tissues are “ripe” for staining when
the hardening has been carried to a certain point. They
are first (Hrgebnisse, p. 18) yellow, without differentiation of
the grey matter from the white; these are unripe. Later
they show the grey matter light brown, the white matter
dark brown ; and these are ripe.

More lately (zbid., p. 14) he added to the bichromate solution 2 per
cent. of chrome alum or of fluoride of chromium, which hastens the
hardening, so that small specimens become brown and ripe in four to
five days, without stoving.

After hardening, the preparation is imbedded in celloidin
(if desired ; imbedding is not obligatory) and hardened in
the usual way. The hardened block is put for one or two
days, in an incubating stove, into saturated solution of
neutral acetate of copper diluted with one volume of water.
By this treatment the tissues hecome green and the celloidin

bluish-green. The preparation may then be kept till wanted
for sectioning in 80 per cent. alcohol.

MYELIN STAINS. 4.09

Sections are made, well washed in water, and brought
into a stain composed of —

Hematoxylin . : . 0°75 to I part.
Alcohol : ‘ : ‘ . 10 parts.
Water ; . 90

9

Saturated solution of lithium carbonate — 1 part.
They remain there for spinal cord, two hours; medullary
layers of brain, two hours; cortical layers, twenty-four
hours.
They are then again well washed with water, and brought
into a decolorising solution composed of —

Borax ‘ : ; 2°0 parts
Ferricyanide of potassium DED 55
Water F : . 2000 ,,

They remain there until complete differentiation of the
nerves (half an hour to several hours), and are then well
washed with water (running, or changed several times),
dehydrated, and mounted in balsam. ‘They may be
previously stained, if desired, with alum-carmine for the
demonstration of nuclei.

The method is applicable to the study of peripheral nerves
as well as to nerve-centres, and also to the study of lymphatic
glands, skin (see ScurerrerpeckEr, Anat. Anz., ii, 1887, p.
680), bile capillaries, and other objects.

The process is applicable to tissues that have been hardened in alcohol
or in any other way, provided that they be put into a solution of a
chromic salt until they become brown, before mordanting them in the
copper solution.

It is not necessary that the mordanting be done in bulk.
Max Fuuscu (Zeit. wiss. Mik., iii, 1886, p. 50) prefers
(following Licutueim) to make the sections first, and mordant
them separately.

VassaLe (quoted from Bayon’s Hist. Untersuchungsmeth.
d. Nervensystems, Wiirzburg, 1905, p. 124) jirst stains the
sections in 1 per cent. hematoxylin, for three to five minutes,
then puts for three to five minutes into saturated solution of
acetate of copper, and differentiates.

782. Wuicerr’s 1891 Method (Deutsche med. Wochenschr,
42, 1891, p. 11845; Zewt. wiss. Mik., vin, 1891, p. 392).—The

410 CHAPTER XXXLIT.

material is to be hardened in bichromate and imbedded in
celloidin (see last §). It is then (according to the latest form
of the process, Bneycl. mik. Technik., 1903, p. 942; for the
earlier form see last ed.) put for 24 hours in a stove into a
solution of 24 parts of fluoride of chromium, 5 of acetate of
copper, and 5 of acetic acid in 100 of water.*

Sections are then made and 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 solution of carbonate of lithium
with 98 ¢.c. of water, and 1 vol. of (8) a solution of | erm. of
hematoxylin in 10 c.c. of alcohol (A and B may be kept in
stock, but A must not be too old). The sections should be
loose ones, and not thicker than 0°025 mm. They are then
washed in several changes of water, and treated with 90 per
cent. alcohol, followed by carbolic-acid-and-xylol iixture
(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
chloroform balsam).

It was, however, found that preparations thus made,
without differentialion, did not keep well, and Weicerr
(Ergebnisse d, Anat., iii, 1894, p. 21) reverted to the practice
of differentiating with the borax-ferricyanide mixture, last §.

Later still (Eneycl. mik. Technik., 1908, p. 942) he
employed a stain composed of equal parts of (a) a mixture
of 4 c.c. of the officinal Liquor ferrt sesquichlorati with 96 of
water, and (zB) a mixture of 10 c.c. of 10 per cent. solution
of hematoxylin in alcohol with 90 of 96 per cent. alcohol.
The two must be mixed immediately before use, and the
sections should remain in the stain over night or longer, then
be rinsed and differentiated as usual. This has the advantage
of demonstrating very fine fibres, and of giving a colourless
ground,

For difficult objects the differentiating liquid may be
diluted with water, and gives better results than dilute
acetic or hydrochloric acid or the like, which were formerly
recommended.

Formol material inay (Mrgelnisse, vi, 1897, p. 14) be

* Tnstead of the chromium fluoride, you may take chrome alum, as

Weigert did at one time, and as some still do. But then you must boil,
as directed for Weigert’s Neuroglia stain, § 838.

MYELIN STAINS, 411

employed if mordanted till brown (four or five days) in 5 per

cent. solution of bichromate with 2 per cent. of chromium
fluoride.

P. Meyer (Neuwrol. Zentralb., xxviii, 1909, p. 853; Zect. wiss. Mtk.,
xxvi, 1909, p. 488) imbeds and cuts before putting into the copper fluid.

Modifications of Weigert’s Method.

783. Paw’s Method (Wien. med. Jahrb., 1886; Zeit. wiss.
Mik., iv, 1887, p. 92; Med. Jahrb., 1887, p. 589; Zeit. wiss.
Mik., 1888, p. 88).—You proceed at first as in Wuicert’s
process, but omitting the copper bath, and you stain as in
Weicert’s process. After staining in the hematoxylin
solution the sections are washed in water (if they are not
stained of a deep blue a trace of lithium carbonate 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. ; F : 1-0
Potassium Sulphite* (Kalium Sulfaro-

sum [SO,K,]) . : , : 10
Aq. Dest. . . 200-0

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 picrocarmine or
acetic-acid-carmine.

Pai’s process gives brilliant results, the ground of the
preparations being totally colourless. Weiaurr (Ergebnisse,
vi, p. 21) considers that for very thick sections it is superior
to his own. But it is not so safe for very fine fibres.

Marcus stains by the Pal method sections of material hardened in
formalin, as described § 741.

GuDDEN (Neurol. Centralb., xvi, 1897, p. 24) makes celloidin sections
of material hardened in 5-10 per cent. formol followed by alcohol,
treats them for ten hours with 0°55 per cent. chromic acid, rinses with
water, and treats with 80 per cent. alcohol, then stains by the method
of Pal, adding to the hematoxylin a few drops of dilute nitric acid
(MINNICH).

* Not “ sulphide,” as erroneously given in MERCIER’s Les Coupes du
Systeme Nei veux Central, p. 190.

412 CHAPTER XXXL.

TSCHERNYSCHEW and Karusin (Zeit. wiss. Mik., xiii, 1896, p. 354),
stains for twenty-four hours in the hwmatoaylin of KULTSCHITZKY,
next §.

So also Pavtow, (ébid., xxi, 1904, p. 14, taking the permanganate twice
as strong as Pal.

Kozowsxy (Neurol. Zentratb., xxiii, 1904, p. 1041) stains as Weigert,
and differentiates the sections first with 1 per cent. permanganate, till
the grey comes out brown, and finishes the differentiation with Liq.
ferri sesquichlorati,

Porrer (Zeit. wiss. Mik., xxvii, 1910, p. 238) stains as Weigert, last §,
and differentiates first in permanganate of 0°25 per cent., then in borax
ferricyanide.

784. Kartser (Neurol. Centralb., xii, 1893, pp. 364, 368; Zezt. wiss.
Mik., xi, 1894, p. 249) hardens first in liquid of Miiller, then for eight
days in liquid of Marcui (§ 796), mordants sections for five minutes
with sesquichloride of iron (1 part to 1 of water and 3 of 70 per cent.
alcohol), stains, and differentiates with Pal’s liquid. For details see
early editions.

Bouton (Journ. of Anat. and Phys., xxxii, 1898, p. 245) makes sections
of formalin material, and mordants them for a few minutes in 1 per cent.
osmic acid, or for a few hours in iron-alum or ammonium molybdate,
stains in Kunrscuirzky’s hematoxylin (next §), and differentiates by
Pal’s process.

Similarly Wywy, ibid., 1900, p. 381.

Laser (Lancet, 1898, p. 321; Journ. Roy. Mie. Soc., 1898, p. 600)
mordants in liquid of Marchi (1 week), makes sections, stains by Kuit-
SCHITZKY'S method, and differentiates by Pat’s.

785. Kuurscuirzky’s method (Anat. dAnz., 1839, p. 223, and
1890, p. 519).—Specimens are hardened for one or two
months in solution of Jérlicki, imbedded in celloidin or photo-
xylin, and cut. Sections are stained for from one to three
hours, or as much as twenty-four, in a stain made by adding
1 grm. of hematoxylin dissolved in a little aleohol to 100 c.c.
of 2 per cent. acefic acid. They are washed out in saturated
solution of carbonate of lithia or soda.

Differentiation is not necessary, but by adding to the ear-
bonate of lithia solution 10 per cent. of a 1 per cent. solu-
tion of red prussiate of potash, and decolorising therein for
two or three hours or more, a sharper stain is obtained.
After this the sections are well washed in water and mounted
in balsam. Myelin dark blue.

Worrers (Zert. wiss. Mik., vii, 1891, p. 466) proceeds as
Kultschitzky, except that he stains at 45° C. for twenty-four

MYELIN STAINS. AI8

hours, after which the sections are dipped in solution of
Miller, and differentiated by the method of Pal.

Similarly Kaus (it¢d., viii, 1891, p. 888 ; Neurol. Centralh.,
1891, No. 15). Myelin dark blue, cells yellow-brown.

786. MirrRopHANOw (Zeit. wiss. Mik., xiii, 1896, p. 361) mordants
photoxylin sections for at least twenty-four hours at 40° C. in a mixture
of equal parts of saturated aqueous solution of acetate of copper and 90
per cent. alcohol, stains for ten minutes in Kultschitzky's hematoxylin,
and differentiates with Weigert’s ferricyanide.

787. Bewktey’s Rapid Method (Neurol. Centralb., xi, 9,
1892, p. 270; Zeit. wise. Mik., x, 1893, p. 370).—Slices of
tissue of not more than two and a half millimetres in thick-
ness are hardened for twenty-four to thirty hours in miature
of Flemming, at a temperature of 25° C., then in absolute
alcohol, then imbedded in celloidin and cut. After washing
in water the sections are put overnight into a saturated
solution of acetate of copper (or simply warmed therein to 35°
to 40° C. for half an hour). ‘hey are then washed, and
stained for fifteen to twenty minutes in a lithium carbonate
hematoxylin similar to Weigert’s, warmed to 40° C., allowed
to cool, and differentiated for one to three minutes in Weigert’s
ferricyanide liquid, which may be diluted if desired with one
third of water.

788. Hitt (Brain, 1896, p. 1; Phil Trans., 184, B, 1894,
p. 599) stains well-washed Miiller material in bulk in alum
carmine, cuts, mordants for twenty-four hours in_half-
saturated acetate of copper, stains and differentiates as
Weigert, taking the differentiating fluid only half as strong.

789. Benpa’s Rapid Method (Berlin. klin. Wochenschr., No. 82,
1903).—Sections of formol material by the freezing process (alcohol
being avoided) are stained (without any mordanting) for twenty-four
hours in Boehmer’s hematoxylin, differentiated with Weigert’s ferri-
cyanide, and mounted in balsam. Only recommended for peripheral
nerves, or for preliminary examination of the central nervous system.

Similarly, Nacrorre, C. RB. Soc. Biol., 1908, p. 408, staining with
hemalum.

Similarly the Encycl. mik. Technzk., 1910, ii, p. 289, with fresh material
cut by the freezing process, and the sections mounted in levulose (as
alcohol somewhat extracts the stain),

414, CHAPTER XXXUT.

790. Srrewrun (Arch. Mik, Anat., lxii, 1903, p. 734) stains
small nerve-centres in bulk (after mordanting in Weigert’s
bichromate and fluoride mixture, § 781) with Weigert’s
hematoxylin (four to six days), washes for a couple of days
in 70 per cent. alcohol, makes paraffin sections, and differ-
entiates them by the method of Weigert or Pal.

791. Besta (Zeit. wiss. Mik., xxiv, 1907, p. 185) mordants nerves for
one to three days in 100 c.c. of water with 25 of formol and 4 grms. of
Merck’s ammonio-chloride of tin, cuts in paraffin, stains in MALLory’s
hematoxylin and differentiates in solution of iodine in iodide of
potassium,

792. GalleinArRronson (Centralb. med. Wixs., 1890, p. 577) stains
sections of material hardened in liquid of Erlicki or Miiller (these must
be mordanted with acetate of copper) for twelve to twenty-four hours in
a solution of 3 to 4 cc. of Gallein (Gribler & Co.) in 100 c.c. of water
with 20 of alcohol and three drops of concentrated solution of carbonate
of soda. They are then differentiated by the method of Weigert, or Pal.
Nerve-fibres red. A second stain with Methylen blue may follow (best
after differentiating with permanganate). Similarly ScuHrorrer
(Centralb. allg. Path., xiii, 1902, p. 512).

793. SCHROTTER (Neurol. Centralb., xxi, 1902, p. 338; Zert. wiss. Mik.,
xix, 1903, p. 381) also stains sections for two to three hours in a 5 per
cent. solution of sulphalizarinate of sod, to which is added a few drops
of 5 per cent. oxalic acid (enough to give an orange tint), then differ-
entiates until no more colour comes away, in carbonate of soda solution
of +55 strength, and mounts in balsam. Myelin red, on a colourless
ground.

794, Toluidin Blue and Methylen Blue.—Harnzis (Philadelphia
Med. Journ., May 14th, 1898) stains sections (of material hardened as
for Weigert’s stain) for several hours in a 1 per cent. solution of
toluidin blue in 1 per cent. borax solution, and differentiates in saturated
aqueous solution of tannic acid. Similarly, but with methylen blue,
in a complicated way FRAENKEL, Neurol. Centralb., xxii, 1903, p. 766
(Zedt. wiss., Mik., xx, 1904, p. 345).

Bine ani ELLERMANN (Arch. Anat. Phys., Phys. Abth., 1901, p. 260)
harden in 9 parts of acetone to 1 of formol, cut without embedding,
stain for five to ten minutes in saturated Methylen blue, and put for one
or two into saturated picric acid.

795. Other Modifications or Similar Methods.—FLEcusi14, Arch.
Anat. Phys., Phys. Abth., 1889, p. 537; Brees, Zeit. wiss. Mik., vii,
1890, p. 386; Rosst, zbéd., vi, 1889, p. 182; Mernrctigr, ibid., vii, 1891,
p. 480; Have, zbid., p. 153; Watsem, zbid., xi, 184, p. 236; RoBERTSON,
ibid., xiv, 1897, p. 80 (Brit. Med, Jouru., 1897, p. 651)

MYELIN STAINS. 415

Srrone (Journ. Comp. Newr., xiii, 1903, p. 291) finds bichromate of
copper (of 2 to 3 per cent.) the best mordant; and that the mordanting
is best done before bringing into celloidin. After staining, he treats
for half a minute with osmic acid of 0:25 per cent., and differentiates
as Pau.

796. Mancurs Method (for Degenerat: Nerves) (Rirista
sperim, dt Freniutria, 1887, p. 208; Zeit. wiss. Mik., ix,
1893, p. 350).—Small pieces of tissue are hardened for a
week in solution of Miiller, and then put for a few days
into a mixture of 2 parts solution of Miiller and 1 part 1 per
cent. osmic acid solution. Sections are cut, best without
imbedding, and mounted in balsam. The sheaths in normal
nerves then acquire a yellow coloration, those of degenerated
tracts a black one.

This process therefore gives positire images of the
degenerated elements, Weigert’s process only giving negative
ones.

For a critical review of this method and its modifications
see Waicurt, in Ergebnisse der Anatomie, vii, 1897 (1898),
pp. 1—8; Maruszewsk1, Arch. path. Anat., 1905, p. 12; pz
Lanan, Le Nevrawe, x, 1909, p. 83; and Lewy, Fol. Newrobiol.,
ii, 1909, p. 471 (Zeit. wiss. Mik., xxvi, 1909, p. 290).

The method has been applied to tissues that have been hardened in
formol; but this, according to WEIGERT, does not seem recommendable.

Finorti (Virchow’s <Arch., exhii, 1896, p. 183) makes
sections of material that has been in liquid of Miiller for
not more than a few weeks or months, and puts them for
four to ten hours (in the dark) into a freshly prepared
mixture of one or two parts of 1 per cent. osmic acid and
one part of a concentrated solution of picrtce acid in one-third
alcohol. For peripheral nerves, myelin (normal), black.

Orr (Journ. Path. and Bact., vi, 1900, p. 387; Journ. Roy.
Mic. Soc., 1900, p. 899) treats with a mixture of 8 «cc. of
2 per cent. osmic acid, and 2 c.c. 1 per cent. acetic acid,
which increases the penetration.

Vassate (Arch, ital. Biol., 1895, p. 91) takes 75 ¢.c. of
solution of Miller, 25 c.c. of 1 per cent. osmic acid, and
20 drops of nitric acid.

Nissi (Hneycl. mik, Technik, ii, p. 248), holding that

416 CHAPTER XXXII.

alcohol attacks the myelin, cuts without imbedding, and
hurries through alcohol and bergamot oil into balsam.

Ramon y Casa (Trab. lab. Biol. Madrid, ii, 1903, p. 93) has an
inadmissably complicated method of treating Marchi material.

Buscu (Newrol. Centralh., xvii, 1898, p. 476; Zeit. wiss.
Mik., xv, 1899, p. 373) puts formol material for five to seven
days into a solution of 1 part osmic acid, 3 of iodate of
sodium, and 3800 of water. Same stain as Marchi’s, but
more penetrating and sharper.

See also Venprrovic, Anat. Anz., xxxix, 1911, p. 414
(cuts slices of the formol material 0°5 cm. thick, and osinicates
these, thus getting increased depth of reaction).

797. Osmic Acid (ExnER, Sitzb. Ahad. Wiss. Wien, 1881, Ixxxiii, p. 151;
Bevan Lewis, The Human Brain, p. 105).—A portion of brain, not
exceeding a cubic centimetre in size, is placed in 1 per cent. osmic acid,
and after five to ten days is cut (best without imbedding). The sections
are treated by caustic ammonia (20 drops to 50 ¢.c. of water), which
clears up the general mass of the brain substance, leaving the medullated
files black. The preparations are not permanent. To make them so
(RANVIER, Trait’, 1 ed., p. 1086) they should he fixed for a quarter of
an hour in osmic acid vapour.

798. AzouLay’s Osmic Acid Method (Anat. Anz., x, 1894,
p- 25).—(a) Sections of Miiller material are put for five to
fifteen minutes into solution of osmic acid of 1 : 500 or
1: 1000 strength. Rinse with water, and put them for two
to five minutes into a 5 or 10 per cent. solution of tannin,
warming them therein over a flame till vapours are given off,
or in a stove at 50° to 55° C. Wash for five minutes in
water, double-stain if desired with carmine or eosin, and
mount in balsam. If the sections are too thick it will be
necessary to differentiate by Pat’s process, or by eau de Javelle
diluted with 50 volumes of water. (8) Material that has been
in an osmic mixture (liquid of Flemming, of Marchi, or of
Golgi). Sections as before, then the tannin bath, warming
for three to ten minutes, and the rest as before.

799. Hutter anp Gumpertz (Zeit. wiss, Mik., xii, 1896,
p. 385) give for peripheral nerves, and Hetier (op. cit., xv,
1899, p. 495) for central nervous system, the following:
Sections of Miller material are put into 1 per cent. osmic

MYELIN STAINS. 417

acid (twenty-four hours at 37° C. for peripheral nerves ; ten
minutes, or thirty at the normal temperature, for central).
They are treated with pyrogallic acid (a photographic
developer will do) till the nerves are black, then with a
violet-coloured solution of permanganate of potash till the
sections become brown, then with 2 per cent. oxalic acid till
they become yellow-green. Wash out well between each
operation.

Similarly, Tetsatni« (Neurol. Centralb., 1897, p. 521); RoBERTSON
(Brit. Med. Journ., 1897, p. 651; Jowrn. Roy. Mic. Soc., 1897, p. 175),
the material being previously mordanted with Weigert’s chrome-alum-
copper fluid for neuroglia; and Orr, Journ. Path. and Bact., vi, 1900,
p. 387. See also Rossotrmo & Buscu, Zett. wiss. Mik., xiv, 1897, p. 55

Wirrmaack (Arch. Ohrenheilk., lxi, 1905; Encycl. mek.
Techmk, ii, p. 241) mordants till green (temporal bones) in
90 parts of Miller with 10 of formol and 3 to 5 of acetic
acid, decalcifies with nitric acid and formol, treats sections
(paraffin or celloidin) for a few minutes with osmic acid of
2 per cent., and reduces in pyrogallol of 5 per cent. Shows
the least traces of myelin.

800. Iron.— ALLERHAND (Neurol. Centrailb., xvi, 1897, p. 727; Encycl.
mtk, Technik. p. 944) puts sections of Miller material for fifteen
minutes into warm 50 per cent. solution of Liquor ferrd sesquichlorati.
then for an hour or two into 20 per cent. tannin solution (old and
brown). They are then differentiated by the method of Pat, taking,
however, the liquids twice as strong.

An iron-alum process is described by StRONG in Journ, comp. Newrol.,
xili, 1903, p. 291.

801. Silver Nitrate——VasTARINI-CRESI (Att. Accad. Med.-Chir.
Napoli, 1, 1896) hardens in formol, cuts thick sections, washes them
with 40 per cent. alcohol, puts them in the dark into 1 per cent. solution
of nitrate of silver in alcohol of 40 to 70 per cent., then washes thoroughly.

Similarly, Mosss (Arch. mik. Anat., lix, 1901, p. 401), impregnating
bichromic material with 1 per cent. solution of argentamin, and reduc-
ing in 10 per cent. pyrogallic acid, and differentiating by the method of
Pau. (Argentamin is an alkaline solution of equal parts of phosphate
of silver and ethylendiamin in 10 parts of water).

802. Polarisation.—Myelin can sometimes be detected in fresh
material by the polariscope, see AMBRONN & Hep, Ber. Math. Phys.
Ges. Wiss. Leipzig., 1895, p. 37, and Gap & Heymans, Arch. Anat.

Phys., Phys. Abth., 1890, p. 531.
27

418 CHAPTER XXXII.

Myelin-and-axis-cylinder Stains.

803. Methylen Blue.—Sauut (Zeit. wiss. Mik., 1885, p. 1) stains
sections of tissue hardened in bichromate for several hours in concen-
trated aqueous solution of methylen blue, rinses with water, and stains
for five minutes in saturated aqueous solution of Siurefuchsin. If now
the sections 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.

Or, the sections are stained for a few minutes or hours in:

Water : ‘ : , , : . 40 parts.
Saturated aqueous solution of methylen blue 24 _,,
5 per cent. solution of borax. ; ~ IC 5

—then washed either in water or alcohol until the grey matter comes
out, cleared with cedar oil and mounted in balsam.

804. Saurefuchsin.—FINoTTI (Virchow's Archiv, exliii, 1896, p. 133)
stains strongly in Delafield’s hematoxylin, then for a few seconds in
concentrated solution of picric acid, then in 0°5 per cent. Saurefuchsin,
and treats with alkaline alcohol (caustic potash).

OHLMACHER (Journ. Exper. Med., ii, 1897, p. 675) stains sections for
one minute with anilin-water gentian, then for a few seconds in a solu-
tion of 05 per cent Saiurefuchsin in saturated solution of picric acid
diluted with one volume of water, and differentiates with alcohol and
clove oil.

Kapitan (Arch. Psychiatr, xxxv, 1902, p. 825) mordants (for months)
in Miller, stains sections for a day or more in 3 per cent. aqueous
Saurefuchsin, rinses in water acidulated with HCl, and differentiates by
the method of Pat (permanganate and potassium sulphite).

805. Safranin.—ADAMKIEWICS (Sitzb. k. Akad. Wiss. Wien. Math.
Naturw. KU, 1884, p. 245; Zeit. wiss. Mik., 1884, p. 587).—Stains sections
of Miller material in concentrated solution of safrunin, differentiates in
aleohol and clove oil, brings back again into water, washes in water acidi-
fied with acetic acid, and stains in methylen blue. Myelin red, nuclei
violet.

Similarly Cracuinsxi (Zezt. wiss. Mzk., vii, 1891, p. 19) and STEoEBE
(ibéd., x, 1893, p. 336), the former employing safranin followed by anilin
blue, whilst the latter first stains with anilin blue, then differentiates
with alcohol containing a very little caustic potash, and after-stains with
safranin.

806. Congo Red.—Nissu (Zeit. wiss. Mck., 1886, p. 398) stains for 3
days in Congo red (5 parts to 400 of water) and differentiates in alcohol
with 3 per cent. of nitric acid.

807. OTHER MetHops.—For PaLapino’'s palladium chloride methods
see last ed., or Rendic R. Acead, Scienze, Napoli, iv, 1890, p. 14, and
1891 [1892], p. 227; and Boll. Accad. Med. Rema, xix, 1898, p. 256;
Arch. Ital, Biol., xx, 1894, p. 40.

For WoLterRs'’ vanad/um chloride process see next chapter.

CHAPTER XXXIV.

AXIS-CYLINDER AND DENDRITE STAINS (GOLal AND OTHERS).

808. Introduction—There are three chief methods for the
anatomical (§ 736) study of axis-cylinders and protoplasmic
nerve-cell processes, viz. the methylen-blue intra-vitam
method, the bichromate-and-silver method of Goxar, and the
bichromate-and-sublimate method of Gotat. The two latter,
with some other methods suitable for the same or similar
purposes, form the subject of this chapter.

809. The Methods of Goncr. There are two methods of
Gota, viz. the Bichromate and Nitrate of Silver Method and
the Corrosive Sublimate Method.

The bichromate and nitrate of silver method has been
worked out by Gore: in three forms—the slow process, the
rapid process, and the mized process.*

The rapid process is the one that is the most in use at the
present time, and may be taken to be the classical method
of inquiry into the finer relations of the neurons in hardened
tissue.

General characters of the unpregnation.—The preparations
have not in the least the appearance of stains, and are even
very different in aspect from the impregnations obtained by
the ordinary methods of impregnating with silver or gold.

* In arecent text-book, the Lettfaden of Rawitz, the sublimate method
is called “ the slow method of Goner,” and the bichromate and silver
nitrate method is given under the form of the slow process, and called
“the rapid method of Gouer.” Rawrrz further attributes the rapid
method to Ramon ¥ Casat, which is equally erroneous. Similar con-
fusions are made by MERCIER in his Coupes du Systime Nerveux Central,
and by Ponuack and other authors. Valuable accounts of the silver
method have been given by v. LENHOSSEK in his Fecnere Bau d. Nerven-
systems, 2nd edit., 1895, and by Katrrus in the art. “ Golgische
Methode” in the Encycl. mik. Technik., 1903.

420 CHAPTER XXXIV.

The impregnation is a partial one, by which is meant that of
all the elements, whether nervous or not, that are present
in a preparation, only a limited number are coloured.
That is one of the great advantages of the method. For if
all the elements present were coloured equally, you would
not be able to see the wood for the trees, for you would hardly
be able to follow any one element for more than a very short
distance. But Golgi’s method selects from among the ele-
ments present a small number which it stains with a great
intensity and very completely—that is to say, throughout
a great length, so that they are both very clearly separated
from those elements that have remained uncoloured, and also
can be followed out for a great distance.

Axis-cylinders are generally impregnated only so long as
they are wot medullated. In the adult the method stains
nerve-cells and their processes, so far as these are not
myelinated; but if it be wished to impregnate the axis-
cylinders of the cerebro-spinal axis the method is_ best
applied to embryos or new-born animals at a time when the
fibres have not become surrounded by their sheath of
myelin.

There is no other method which will allow cell-processes
to be followed out for such great distances. But the method
does not demonstrate at the same time the histological detail
of other tissues that may be present in the preparations, and
all cytological detail is lost. It is par excellence a special
method.

Nervous tissue is not the only thing that is impregnated
in these preparations ; neuroglia, connective tissue, fibrils,
etc., are impregnated, and the method has been applied with
success to the study of such things as bile-capillaries, gland-
ducts, and the lke. Both on account of this character, and
on account of the capriciousness with which the impregnation
takes hold of only certain elements of the preparations, care
must be exercised in the interpretation of the images obtained.
A further source of possible error is found in the fact that
the method frequently gives precipitation-forms of the silver
salt that simulate dendrites and other structures (see Frizp-
LAENDER in Zeit. wiss. Mik., xii, 1895, p. 168, and the plate
in the following number.)

The method has been applied with success to the tissues

AXIS-CYLINDER AND DENDRITE STAINS. 421

of Invertebrates—Insects, Lumbricus, Tubifea, Helix, Limaa,
Distumum, Astacus, Actinida, ete.

The method has been described at length by Gotet in the
Archives Italiennes de Biologie, t. iv, 1883, p. 32 et seg., and
vil, 1886, p. 15 et seg. The following account is from the
latter paper. The earlier form of the method should not be
followed.

810. Gorai’s Bichromate and Nitrate of Silver Method, SLOW
Process (loc. cit., p. 17)—(a) The hardening.—The tissues
must be hardened in a bichromate solution. Hither pure
bichromate of potash may be employed, or liquid of Miiller
(the reaction can be obtained with liquid of Erlicki, but it is
not to be recommended). ‘The normal practice is to take
bichromate of potash, beginning with a strength of 2 per
cent., and changing this frequently for fresh solutions of
gradually increased strength, 24, 3, 4, and 5 per cent. The
tissue should be as fresh as possible; though satisfactory
results may sometimes be obtained from material taken
twenty-four to forty-eight hours after death.* Jt should be
in pieces of not more than 1 c.cm. or L$ c.cm. in size.

The most difficult point of the method consists in hitting
off the exact degree of hardening in the bichromate that
should be allowed before passing to the next stage. In
summer good results may be obtained after fifteen to twenty
days, and the material may continue in a favourable state for
impregnation up to thirty, forty, or fifty days. In cold
weather good results can seldom be obtained under a month :
when obtained, the material may continue to give good results
up to two, three, and even four months of hardening. The
only way to make sure is to pass trial portions of the tissue
at intervals into the silver-bath, in summer frequently, in
winter every eight or ten days, and observe whether the
reaction is obtained.

Good results are obtained by injecting the organs with
the hardening fluid (2°5 per cent. bichromate). See § 737.

Stoving at a temperature of 20° to 25°C. is useful for

* Material that has been hardened in formol may also be used. See
§ 817 (GuRota and Botton), and v. LENHOossEK’s Feinere Baw d.
Nervensystems, p. 23. I have had good results with material that had
been three months in formol (I have not tried older).

422 CHAPTER XXXIV.

abridging the hardening, but there is risk of over-hardening ;
and Goutal thinks the results are never quite so delicate as
after hardening in the cold.

(b) Impregnation.—As soon as the pieces of tissue have
attained the proper degree of hardening, they are brought
into a bath of nitrate of silver. The usual strength of this
bath is 0°75 per cent., but 0°50 per cent. may be taken for
material that has not been quite enough hardened, and solu-
tions of 1 per cent. may be used for material that has been
slightly over-hardened. The solution may be acidified (see
Ramon y Casa, § 819).

A large quantity of solution should be taken for the bath.

The moment the pieces of tissue are put into the silver-
bath an abundant precipitate is formed. This of course
weakens the bath pro tanto. It is therefore well, before
putting the pieces into the final silver-bath, to first wash
them well in a weaker silver solution, until on being put
into a fresh quantity of it no further precipitate is formed.
Used solutions will do for this purpose. The final silver-
bath in general needs no further attention, unless it be that
sometimes, in the case of tissues that have taken up a great
deal of bichromate of potash, the solution may after six to
ten hours become somewhat yellow, in which case it should
be changed for fresh.

It is not necessary to keep the preparations in the dark
during the impregnation bath ; in winter it is well to keep
them in a warm place.

The time normally necessary for impregnation by the
silver is from twenty-four to forty-eight hours (forty-eight
being quite exceptional). But tissues may remain in the
bath without hurt for days, weeks, or months.

(c) Preservation.—As soon as a trial has shown that a
sufficiently satisfactory impregnation has been obtained the
pieces are brought into alcohol. The alcohol is changed
two or three times, or even more, until it remains trans-
parent even after the preparations have been two or three
days in it; for in view of good preservation it is necessary
that the excess of nitrate of silver should be washed out
from them thoroughly.

Sections are now made (see § 821). ‘They are to be
washed very thoroughly in three or four changes of absolute

AXIS-CYLINDER AND DENDRITE STAINS. 423

alcohol. They are then cleared, first in creasote, in which
they should remain only a few minutes, then in oil of turpen-
tine, in which they should remain for ten to fifteen minutes
(they may remain there for days without hurt). They are
then mounted in damar (rather than in balsam), and without
a corer, Preparations mounted under covers in the usual
way always go bad sooner or later, whilst those that are
mounted without a cover keep very well, especially if they
be kept in the dark. Gorer states that he has a large number
that have kept without change for nine years.

As a general rule thick sections (50 to 60 4” or more)
show much more than thin ones, but do not scem tu keep so
well,

The order in which the elements of tissues impregnate is
generally—tirst, axis-cylinders, then ganglion cells, and lastly
neuroglia cells.

811. Gouci’s Bichromate and Nitrate of Silver Method,
RAPID process (op. cit., p. 33). Small pieces of very fresh
tissue are thrown into—

Bichromate solution of 2 to 2°5 per cent.

strength ‘ ‘ : ; . 8 parts.

Osmic acid of 1 per cent. strength w 2 5
(Or, later, two parts of bichromate of 3 per cent. to 1 of
the osmic acid.) The tissues begin to be in a fit state for
taking the silver impregnation from the second or third
day; in the next following days they are im a still more
favourable state, but the favourable moment does not last
long; the faculty of impregnation soon declines, and is
generally quite lost by the tenth or twelfth day.

The silver impregnation is conducted exactly as in the
slow process, and sections are prepared and mounted in the
same manner. (I find that they should not be left in alcohol
for more than an hour or so before mounting.)

There is this difference, that the impregnated material
cannot be preserved for any length of time in alcohol, but
must not remain for more than two days in vt. But it may
be kept in the silver solution until wanted for sectioning.
According to van Grnucuten (La Cellule, vi, 1890, p. 405),
material may be kept for six months in the silver, with
advantage, showing abundant reductions where none were

42.4, GUAPTER XXXIV.

found after forty-eight hours. But it must be kept in the
dark.

The following notes as to the proper duration of the hardening pro-
cess in different cases are taken from the papers quoted and other sources,
most of which may be found in v. LENHOSSEK, op. cit., p. 23.

Spinal cord of chick, from the sixth to the tenth day of incubation—
twelve to forty-eight hours in the mixture (up to the fifth day the em-
bryos may be treated whole, later the vertebral column should be dis-
sected out and cut into two or three segments ; it need not be opened).
The spinal column of new-born rats and mice should be treated in the
same way, and remain in the mixture for twenty-four hours (for spinal
ganglia), or for two to six days for the cord itself. (The encephalon of
these subjects may be treated in just the same way, without being dis-
sected out.)

Von LENHOSSEK (op. cit., p. 10) recommends for human (fetal) cord
two to three days for neuroglia, three to five for nerve-cells, and five to
seven for nerve-fibres and collaterals.

Cerebellum of new-born subjects, three to five days in the mixture.

Cerebral cortex of young subjects, two to three days (Mice), or as
much as five (Rabbit, Cat); cortex of adults, eight to fifteen days. The
most favourable region of the brain is the cornu Ammonis, especially in
the Rabbit.

Retina—twenty-four to forty-eight hours in the mixture, then
* double” impregnation (§ 815).

Sympathetic —three to seven days in the mixture, and two
in the silver: then double impregnation.

Spinal cord of larvee of Amphibia. ‘he entire larve
(best 2 to 2°5 centimetres long) should be put for two to five
days into the mixture, and for one to two into the silver.

Epidermis of Lumbricus—three to six days in the mixture, and two
in the silver, or double impregnation if necessary. SmrRNow makes
the mixture of equal parts of 5 per cent. bichromate and 1 per cent.
osmic acid, and leaves in it for five to twenty-eight days, and one to two
days in the silver (0°75 per cent.).

Nervous system of Helix (glia-cells). he above mixture
for eight to ten days, then silver of 0°75 to 1 per cent.

As a general rule, the younger the subject, the shorter should the
hardening be. If it has been too short, sections will have a brownish-
red opaque aspect, with precipitates, and irregular impregnation of cells
and fibres. If it has been too long, the ground will be yellow, without
precipitates, but with no impregnated elements, or hardly any.

AXIS-CYLINDER AND DENDRITE STAINS. 425

This process has the advantage of great rapidity, and of
sureness and delicacy of result, and is the one that has found
the most favour with other workers. But for methodical
study of any given part of the nervous system Gone himself
prefers the following :

812, Gotar’s Bichromate and Nitrate of Silver Method,
MIXED Process (op. cit., p. 34). —Fresh pieces of tissue are
put for periods varying from two to twenty-five or thirty
days into the usual bichromate solution ($ 810). Every two
or three or four days some of them are passed on into the
osmio-bichromate mixture of the rapid process, hardened
therein for from three or four to eight or ten days, and
finally impregnated with silver, and subsequently treated
exactly as in the rapid process.

The reasons for which Gotar prefers this process are—the
certainty of obtaining samples of the reaction in many stages
of intensity, if a sufficient number of pieces of tissue have
been operated on; the advantage of having at one’s dis-
position a notable time—some twenty-five days—during
which the tissues are in a fit state for taking the silver, and
the possibility of greatly hastening the process whenever
desired by simply bringing the pieces over at once into the
osmic mixture; lastly, a still greater delicacy of result,
especially remarkable in the demonstration of axones.

813, Theory of the Impregnation.—It used to be held that
the reaction depends on the formation in the tissues of a
precipitate of some salt-of silver. But this seems incorrect.
I find (in accordance with Lenuossix, ‘Feimere Bau d.
Nervensystems,’ p. 19) that the coloration is not due to a
visible precipitate, but is a true stain, accompanied (in
unsuccessful impregnations) by a precipitate which does not
help the stain but is injurious to it. It has been maintained
that the stain is merely superficial, and the method has been
called an “incrustation method.” JI find that it extends
throughout the whole thickness of the impregnated elements.

The chemical nature of the stain has not been made out.

A critical review of the Golgi method by WEIGERT may be found in
Ergebnisse der Anatomie, v, 1895 (1896), p. ¥.
See also H1Lu (Brain, part 73, 1896, p. 1), AzouLAyY (Comptes Rend.

426 CUAPTER XXXIV.

Soc. Biol. [10], i, 1894, p. 839); and KaLiius (Encycl. mik. Technik.,
Art. “ Golgi’sche Methode.”’

Modifications concerning the Inpregnation of the Tissues.

814. Ramon y Casan (Zeit. aiss. Mik., vii, 1890, p. 332) gives
3 per cent. as the strength of the bichromate in the mixture
for the rapid process, and in numerous other places has
given it as 3°5 per cent. ‘This latter strength has been
adopted by most of the workers who use the rapid process,
and the mixture containing this proportion of bichromate is
generally known as Ramon y Casaw’s mixture.

815. Ramén y Casav’s Double-Impregnation Process (Tab.
Lab. Hist. Med. Barcelona, 1891; La Cellule, vii, 1891, p.
130).—Sometimes the usual rapid method fails to give good
impregnations. These, however, may frequently be obtained
by putting the tissues back for a day or two into the osmium-
bichromate mixture, or into a weaker one containing only
two parts of osmic acid solution to 20 of the bichromate.
After this they are washed quickly with distilled water, and
put for a second 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 after-
wards again with the silver solution.

This modification of the original process is the most impor-
tant that has hitherto been made.

816. Go1ai’s Process for Rejuvenation of Over-hardened
Tissues.—Tissues that have been inuch too long in the osmium-
bichromate mixture will no longer take on the silver impregna-
tion. They can, however, be rejuvenated and made to
impregnate in the following manner, due to Goxel, and
published by Sacervort1 (Iuéern. Monatsschr, xi, 1894, p.
326). They are washed in a half-saturated solution of
acetate of copper until they no longer give a precipitate,
and are then put back again for five or six days into the
osmium-bichromate mixture. Sections, it is said, will bear
mounting in thickened oil of cedar under a cover.

AXIS-CYLINDER AND DENDNITE STAINS. 427

More recently (Cinquantenaire Soc. Biol., 1899, p. 514)
Golgi puts for some hours or days into a mixture of equal
parts of bichromate of 2 to 8 per cent. and sulphate of
copper of 4 to 5 per cent., or into the cupric mixture used
for the intra-cellular network, § 777.

Gemeti (Anat. Anz., xliii, 1913, p. 414) takes a mixture
of “ acetate of copper (4 per cent.) and bichromate of potash
(5 per cent.).” Time not stated.

817. Formaldehyde for the Rapid Process—Srrone (Azat.
Anz., x, 1895, p. 494) finds that formaldehyde can with
advantage be substituted for the osmic acid in the osmio-
bichromic mixture of Gorar’s rapid process. He adds from
2°5 to 5 per cent. of formalin to the (3°5 to 5 per cent.)
bichromate solution.

The advantage is that the stage of hardening favourable
for impregnation lasts longer; in other words, the formalde-
hyde bichromate does not over-harden.

Donia (ibid., p. 659) obtained the best results by means
of 3 per cent. bichromate solutions containing 4 to 6 per
cent. of formaldehyde, hardening therein for three days, and
after silvering for two days putting back into the mixture and
proceeding as in Ramon y Cajal’s double impregnation
process.

Fisu (Proc. Amer. Mic. Soc., xvii, vee 319) takes:

Formalin ‘ : ; 2 cc.

3 per cent. iiamuemens , ; . 100 ,,
leaving the tissues three days in this hquid and three days
in the silver nitrate (? per cent.).

Or, with advantage :

Liquid of Miller. : ‘ . 100 c.c.
10 per cent. formalin 3 5 : ee
1 per cent. osmic acid ; 1 ,,

Oper (La Rachicocainisation, Gensee,; 1903, p. 27) takes
two parts of undiluted formalin, instead of the 10 per cent.

The formalin and bichromate mixtures should be kept in
the dark. It is well only to make them up at the instant of
using them. Odier finds these mixtures afford a more
abundant impregnation, with fewer precipitates.

Korscu (Anat. Anz., xi, 1896, p. 727) takes 4 parts of 3°5
per cent. bichromate solution, and 1 of commercial formalde-

428 CHAPTER XXXIV.

hyde solution, and after twenty-four hours transfers to pure
3°5 per cent. bichromate for at least 2 days (retina), or 3 to
6 (central organs). He finds that by this means precipitates
are almost entirely avoided. This I also find, but I seem to
get a too abundant impregnation of capillaries.

Gerora (Intern. Monatsschr. Anat., xiii, 1896, p. 108) first
hardens (brain) for a week or two in 5 to 10 per cent. formol
solution, then puts small pieces for three to five days into
4 per cent. bichromate, then into the silver.

Similarly Bonron (Lancet, 1898, p. 218; Journ. Roy. Mic.
Suc., 1898, p. 244).

Scurzrper (Anat. Anz., xiv, 1898, p. 275) obtained good
results (on appendages of Crustacea which were impervious
to the osmic mixture) with mixtures of five parts 2°5 per
cent. bichromate to one of 4 per cent. formaldehyde, or one
part 2°5 per cent. bichromate to two of 5 per cent. formalde-
hyde, the specimens remaining for one day in the first, for
two days in the second.

Similarly DusBoscq (Arch. z. Haper., 1899, p. 483), warm-
ing the mixture to 40° C.

vAN GEHUCHTEN (in Jiét.), and other observers, have not
obtained good results with formaldehyde.

Katuius (Hneyel., p. 564) finds these mixtures good for
brain, but not so much so for other organs.

818. Acetic Aldehyde.—VassaLzE and Donaaaio (Monttore Zool.,
Ttal., vi, 1895, p. 82) harden pieces of at most 1 cm. in thickness for
fifteen to twenty days in a mixture of five parts of aldehyde with 100 of
3 to 4 per cent. bichromate, changing the fluid after a few days, as soon
as it has become dark. The rest as Golgi.

819. Modifications of the Silver Impregation._RAaAMON Y
Casa (Rev. trim. Hist., No. 2, 1888, note) found the addition of a very
little formic acid to the silver bath facilitated reduction. According to
VAN GEHUCHTEN (La Cellule, vii, 1891, p. 83), 1 drop of the acid should
be added to 100 ¢.c. of the silver. But the practice is now generally
abandoned.

BERKELEY (Johns Hopkins Hosp. Rep., vi, 1897, p. 1; Journ. Roy.
Mic. Soc., 1898, p. 242) impregnates, after hardening in the osmio-
bichromate, in a freshly prepared solution of two drops of 10 per cent.
phosphomolybic acid to 60 c.c. of 1 per cent. silver nitrate, which in
winter should be kept at a temperature of about 26° C.

HIx1 (op. cét., § 818) takes instead of silver nitrate a # per cent. solu-
tion of silver nitrite, with 0-1 per cent. of formic acid added.

AXIS-CYLINDER AND DENDRITE STAINS. 429

GuppEn (Neurol. Centralb., xx, 1901, p. 152) takes the lactate of silver
(sold as “ actol”) and finds it much more penetrating.

Faserstagn (ibid., p. 98) uses ammonio-nitrate in a complicated way.

820. Avoidance of Precipitates.—Goxar’s process frequently
gives rise to the formation at the surface of the preparations
of voluminous precipitates that are destructive of the clear-
ness of the images. Seurwawp (Zeit. wiss. Mik., vi, 1889,
p. 456) has found that this can be avoided as follows. A
10 per cent. solution of gelatin in wateris made. The tissues
are coated with this, by dipping and cooling several times,
or are imbedded in it, in a paper imbedding box, with the
aid of a little heat, and are brought therein into the silver-
bath. After the silvering the gelatin is removed before
cutting by warm water saturated with chromate of silver.

Martinorts wraps the tissue simply in blotting-paper, but
this does not appear to be efficacious.

Arias takes wafer-papers.

Ramon y Casau covers tissues with a layer of congealed
blood, which need not be removed before cutting, or with
collodion, or peritoneal membrane. See Retina.

Modifications concerning the Preservation of the Preparations.

821. Cutting —The chief quality of Goxai’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 sections. And as sufficiently thin ones can be obtained
without imbedding, the general practice is 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 minutes to harden the gum, and cut with a microtome
without imbedding.

But imbedding is possible, if it can be got through rapidly
enough. Pieces of tissue as small as possible should be
dehydrated in from half an hour to two hours, put for the
same time into thin celloidin, then coated with thick
celloidin, gummed on a cork and cut, the sections being
collodionised if necessary. ‘Thin specimens such as retina
may be soaked for a short time in celloidin, put between two
slabs of solid celloidin lightly pressed together, and the
whole cut after a short treatment with alcohol of 70 per
cent. Similarly with paraffin. ‘The tissues should be got

430 CHAPTER XXXIV.

quickly through the lower grades of alcohol, and not remain
for more than a few hours in alcohol of 95 per cent. or
absolute. They should be cleared with cedar oil (xylol
attacks the impregnation), and put direct into paraffin of
as low a melting point as possible. The cedar oil should
be used over and over again, as it takes up a little silver
(see Brooxover, Journ. Comp. Neurol., xx, 1910, p. 49).

822. Mounting.— Without special precautions, the stain will
not keep in sections mounted under a cover in the usual way.
An elaborate discussion (for which see previous editions)
between Sreurwatp (Zeit. wiss, Mik., vi, 1890, p. 448),
Samassa (ibid., vii, 1890, p. 26), and Fick (zbid., viii, 1891,
p. 168) furnishes the net practical result that watery fluids
should be avoided as much as possible during the after-
treatment, and that sections should either be mounted with-
out a cover, or on a cover raised free of contact with the
slide by means of wax feet or the like, or, for study, inverted
over the aperture of a hollowed-out wooden slide; or that
the balsam of the mount shouldbe rendered perfectly anhydrous
by careful heating on the slide, with the section in it, until
it immediately sets hard on cooling, before the cover is
applied.

This last method is also recommended by Huszr (Anat.
Anz., vii, 1892, p. 587). I think it is safer to keep the
mount uncovered till the sections have become quite dry in
it, and the balsam (applied from time to time in thin layers)
quite hard; then cover with a warmed cover pressed down.

But if mounting under a cover at once be preferred, one
of the followmg methods may be employed.

823. GREPPIN’S Process (Arch. Anat. Entw. Anat. Abth., 1889,
Supp., p. 55).—Sections are treated for thirty to forty seconds (until
whitish) with 10 per cent. solution of hydrobromic acid, and then well
washed in several changes of water and mounted under a cover in the
usual way. They can be further reduced in sunlight if desired. Further
details in previous editions.

824. OBREGIA'’s Process (Virchow’s Archiv, exxii, 1890, p. 387).—
Sections 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

AXIS-CYLINDER AND DENDRITE STAINS. 431

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 (not more) with
10 per cent. solution of hyposulphite of soda. They are lastly washed well
with water, and may be then mounted at once in balsam under a cover,
or if desired may be previously stained with carmine or hematoxylin, or
Pal’s modification of Weigert’s process, or the like.

825. Katutius (Anat. Hefte, ii, 1892, p. 271) has worked out the follow-
ing process. Take 20 c.c. commercial hydroquinone developing solution
and 230 ¢.c. distilled water (the hydroquinone solution may be made up
with 5 grms. hydroquinone, 40 grms. sodium sulphite, 75 grms. carbonate
of potassium, and 250 grms. distilled 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 (from which the unreduced silver
has been removed as far as possible by washing in many changes of
aleohol) into it for several minutes; they become dark grey to black.
They are then put for ten to fifteen minutes into 70 per cent. alcohol,
then brought for five minutes into solution of hyposulphite of soda
(about 10 parts to 50 of water), 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.
After-staining with carmine, etc., may be employed.

CurRRERI (Anat. Anz., xxxii, 1908, p. 432) after fixing tones for a short
time in 0°7 grms. gold chloride, 3 grms. sodium acetate, and 100 c.c. water.

826. ZIMMERMANN'S Process (Arch. mk. Anat., lii, 1898, p. 554).—
Sections are brought from alcohol into a large quantity of a mixture of
1 part physiological salt solution and 2 parts 96 per cent. alcohol.
They must be kept in motion therein for ten to fifteen minutes, after
which they are brought into alcohol of 75 to 96 per cent. in a bright light,
until they have become dark (half « day). They may be after-stained

with thionin (cells blue).
Later (Arch. Mik. Anat., Ixxviii, 1911, p. 199) he reduces in 20 c.c. of

saturated solution of carbonate of soda with 0°5 grm. of adurol for
several hours, and after-stains with hemalum or alum cochineal.

The Sublimate Method.

827. Goiat’s Bichromate and Sublimate Method (Archivio per
le Scienze Mediche, 1878, p. 3; Archives Italiennes de Biologie,
-iv, 1883, p. 32; vii, 1886, p. 35).—For hardening, use either
a solution of bichromate of potash progressively raised from
1 per cent. to 25 per cent., or Miiller’s solution. It is best
to take small pieces of tissue (not more than 1 to 2 ¢.c.),
large quantities of liquid, and change the latter frequently.
But the reaction can be obtained with much larger pieces,

432 CHAPTER XXXIV.

even entire hemispheres. In this case the brain should at
first be treated by repeated injections of the liquid. Fifteen
to twenty days’ immersion will suffice, or even six to eight,
but twenty to thirty should be preferred, and an immersion
of several months is not injurious.

The tissues when hardened are passed direct from the
bichromate into 0°5 per cent.solution of bichloride of mercury.
An immersion of eight to ten days therein is necessary in
order to obtain a complete reaction (or for entire hemispheres
two months or more). The solution must at first be changed
every day, and later on as often as it becomes yellow. At
the end of the reaction the preparations will be found
decolonrised, and offering the aspect of fresh tissue. They
may be left in the bichloride for any time.

In Rendiconti R. Ist. Lombardo di Sct. Milano, 2, xxiv, 1891, pp. 594,
656 (see Zeit. wiss. Mtk., viii, 3, 1891, p. 388), GoLer 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.
sublimate for a very long time, two years being not too much in some
cases.

The reaction may be said to have begun by the time the
tissues are nearly decolourised. From that time onwards
sections may be made day by day and mounted if successful.

Before mounting, the sections must be repeatedly washed
with water, otherwise they will be spoilt by the formation of
a black precipitate. (In the last place quoted Gorer 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 (prefer-
ably) glycerin.

The elements acted on are—(1) The ganglion cells, with
all their processes and ramifications. (2) Nuclei, which is
not the case with the silver process. (3) Neuroglia cells.
But the reaction in this case is far less precise and complete
than that obtained by the silver process. (4) The blood-
vessels, and particularly their muscular fibre cells.

The method is said to give 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 cere-

AXIS-CYLINDER AND DENDRITE SiTALNs. 433

bellum. It is superior to the silver method in that the
reaction can always be obtained with perfect certainty in a
certain time; that the preparations can be perfectly pre-
served by the usual methods; and that large pieces of tissue
can be impregnated. It generally gives a more abundant
lnpregnation than the silver method.

See also Frarav, in arch. mik. clnat., xlv, 1895, p. 158.

Modifications of Golgi’s Bichromate and Sublimate Mvthod.

828. Pau [Erratim “Tal,” loc. cit.] (Gazz. degli Ospituli, 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 Pat.
Wien. med. Jahrb., 1886; Zeit. wiss. Mik., v, 1887, p. 93).

For FLEcHsIG’s modifications, see Arch. Anat. Phys., Physiol. Abth.,
1889, p. 537.

829. Cox (Arch. mik. Anat., xxxvii, 1891, p. 16) finds the
sublimate and bichromate may be used together. 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 chromate
should be diluted with the water before adding it.) The
mixture should be as little acid as possible. The pieces of
tissue should be small. The duration of the impregnation is
from two to three months. ‘There is considerable difficulty
in preserving sections, which are best made with a freezing
microtome, alcohol being avoided, treated for an hour or two
with 5 per cent. solution of sodium carbonate, and mounted
without a cover, in a medium composed of—gum sandarac,
75 gr.; camphor, 15; oil of turpentine, 30; oil of lavender,
22°5 ; alcohol, 75; castor oil, 5-10 drops. Fur examination,
add a drop of castor oil, and cover. :

Dr. A. SANDERS writes me (June, 1898) that the stain keeps very well
if the sublimate be well removed by washing in many changes of alcohol,
and the tissues passed through alcohol and ether into celloidin, and the
sections mounted in chloroform-balsam under a cover. I find the statu
keeps; but the preparations quickly develop opaque granules that are
very undesirable.

Bremer (Anat. Rec., 1910, p. 265) cuts in celloidin and stains with
alum hematein and eosin.

28

434, GHAPTUR XXXIV.

For the very complicated platinum-substitution processes of RoBERT-
son and Macponatp see Journ. Ment. Se/., xlvii, 1901, p. 327; or Journ.
Roy. Mic. Soc., 1902, p. 601.

830. ZreHun’s Gold and Sublimate Method (Newrol. Centralb.,
x, 1891, p. 65).—Small pieces of fresh material are thrown
into a large quantity of a mixture of 1 per cent. sublimate
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 Lucou’s solution diluted with
four volumes of water, or with dilute tincture of iodine, until
duly differentiated, then washed and mounted in balsam.
Both medullated and non-medullated nerve-fibres are stained,
also nerve and glia cells and their processes.

831. Krounruat’s Lead Sulphide Impregnation (Neurol. Cen-
tralb., xviii, 1899, No. 5; Zeit. wiss. Mik., xvi, 1899, p. 235)
consists in treating tissues first with formate of lead and
then with hydric sulphide. The formate is prepared by
dropping formic acid slowly into solution of acetate of lead.
White crystals of formate of lead are abundantly formed ;
the mother liquor is filtered off, and the crystals are dis-
solved to saturation in water. ‘Ihe solution is mixed with
an equal volume of 10 per cent. formol ; pieces of brain or
spinal cord are put into the mixture for five days, and are
then brought direct into a mixture of equal parts of 10 per
cent. formol and hydric sulphide solution. After five days
therein they are cut in celloidin, and the sections mounted in
xylol-balsam under a cover. ‘They seem to be quite perma-
nent. Nerve-cells as well as nerve-fibres are impregnated.
The impregnation is a very complete one.

Corning (Anat. Anz, xvii, 1900, p. 108) hardens the
tissues with 10 per cent, formol before bringing them into
the formol-formate mixture, and so obtains better results.
He obtains his formate of lead direct from Marck (Plumbum
Jornucicum). He prefers to cut without imbedding. Other
details loc. cit.

AXIS-CYLINDER AND DENDRITE STAINS. 435

832. Wo.tEr’s Chloride of Vanadium process (Zeit. wiss.
Mtk., vii, 1891, p. 471):

The material (either central or peripheral nervous tissue)
is hardened in the bichromate liquid of Kuvrscurray, § 55,
followed by alcohol, as there described. Sections are mor-
danted for twenty-four hours in a mixture of 2 parts of 10
per cent. solution of chloride of vanadium and 3 parts of
3 per cent. solution of acetate of aluminium, washed for ten
minutes in water, and stained for twenty-four hours in a
solution of 2 grms. of hamatoxylin (dissolved in a little
alcohol) in 100 e.c. of 2 per cent. acetic acid. They are
washed out until they are of a light blue-red colour 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.

Chiefly an axis-cylinder stain, myelin being coloured only
if the differentiation in the acid alcohol is insufficient, but
cells are also stained.

833. Methylen Blue.—Meyer (Arch. mik. Anat., xlvi, 1895, p. 282,
and xlvii, 1896, p. 734) has obtained good results (for the central nervous
system, uot for the peripheral) by means of subcutaneous injection.
Large quantities of solution must be injected, in several portions, at
intervals of one to several hours. After some time the organs should be
thrown direct into the bath of Betus, § 344, and remain in it till the
next day.

Ramon y Cagau (ev. Trim. Micr., Madrid, i, 1896, p. 123; Zect. wiss.
Mik., xiv, 1897, p. 92) stains by “ propagation” or “diffusion.” The
brain is exposed (rabbit) and the cortex is divided into slices of « couple
of millimetres thickness by means of a razor. The slices are then
covered on both sides either with finely powdered methylen blue, or
with a saturated solution of the same, the slices are replaced in their
natural positions, the brain case is replaced for half an hour, after
which the slices are removed and fixed for a couple of hours with Bethe’s
ammonium molybdate, washed, hardened for three or four hours in a
mixture of 5 parts 1 per cent. platinum chloride, 40 parts formol, and 60
parts water, further treated for a few minutes with platinum chloride
in alcohol (1 in 300), and if small enough imbedded in paraffin. The
sections should be dehydrated with alcohol containing 0°3 per cent. of
platinum chloride, and may be cleared with xylol or bergamot oil and
mounted. The stain is stronger and more complete than that of the
other methylen blue methods.

Catois (Comptes Rend., cxxiv, 1897, p. 124) injects con-

436 CHAPTER XXXIV.

centrated methylen blue into the body-cavity of Fisuss,
removes the brain after half an hour, puts slices of it into
the same solution for half an hour, and fixcs as usual.

Lennuorr (Neurol. Zeutralb., 1910, p. 1) has some com-
plicated methods with polychrome methylen blue and
sulpho-cyanide of potassium, or ferricyanide.

834. Lunnuorr’s Iron Method (ibid.)—Sections put for
thirty seconds into 2 ¢.c. of 15 per cent. solution of tannin
with 3 drops of 5 per cent. solution of oxalic acid, rinsed in
water, then for a few seconds in 1 per cent. solution of
chloride of iron till no further blackening occurs, then
washed, dehydrated aud mounted in balsam. Axis cylinders
black, cells grey.

835. Fasursrasn’s Hematoxylin (Polu. Arch. Biol. Med.
Wiss., i, 1901, p. 189).—Sections by the freezing method of
material fixed for two to seven days in formol of 5 to 10
per cent. mordanted for five to twenty-four hours in chromic
acid of 0°25 to 0°5 per ceut., well washed, stained for twenty-
four hours in 1 per cent. aqueous hematoxylin, and differen-
tiated by the method of Pau.

Other Methods.
836. Nazias (C. R. Soc. Biol., lvi, 1904, p. 426) treats

sections until yellow with solution of 1 grm. iodine and
2 gris. iodide of potassium in 300 of water, washes, treats for
afew minutes with 1 per cent. chloride of gold, washes and
reduces in anilin or resorcin i water (1: 100, or less for the
latter) and mounts in balsam.

Avaruy’s Gold Method has been given § 871.

GERLACH’S Bichromate and Gold Process has heen given. § 369.

For aw complicated Gold Method of Ramon y Casa, see Rev. trim.
Mier, v, 1900, p. 95; or Zeit. wives. Mik., xix, 1902, p. 187.

For Upson’s exceedingly complicated Gold and Iron and Vana-
dium Methods see Mercier, in Ze/t. wiss. Mch., vii, 1891, p. 474; or in
his Coupes du Systeme Nerveux Ceutral, p. 234; or curly editions.

For Fagerstasn’s complicated Silver Method see Neurol. Ceutralb.,
xx, 1901, p. 98; or Ze/t. wiss. Mik., xviii, 1901, p. 214.

Maaini's Zine Chloride Process (see Boll. Accad. Med. di Roma,
1886; Zeit. wiss. Mik., 1888, p. 87, or early editions).

AXIS-CYLINDER AND DENDRITE STAINS. 437

Mont1's Copper Process, see Atti. R. Accad. Lineei Roma, Rendic.,
v, 1889, p. 705; Zedt. wiss. Mik., vii, 1890, p. 72.

Anthracen Ink (Leonhardi’s, obtainable from Griibler) is used in a
complicated way by Kapian, Arch. Psychiatr., xxxv, 1902, p. 825 (Zeit.
wiss. Mik., xix, 1903, p. 510)._Srranuser, Centralb. allg. Path., xiii,
1901, p. 422 (Zeit. wiss. Mik., xviii, 1902, p. 482) (pathological).
Mattory’s phospho-molybdic hematoxylin, see § 271. SaHLI’s
methods, see § 803.

Donaaero’s Tin stain, see § 278.

And see also under Neurofibrils.

CHAPTER XXXV.

NEUROGLIA, AND SENSE ORGANS.

Neuroglia.

837, InrRopuction.—Neuroglia cells may be isolated by
teasing, and may be stained in many ways (see Ranvier,
Traité, p. 1063), by osmic acid, nigrosin, carmine, orcein.
Tron hematoxylin is said to give good results with the lower
vertebrates. (JI have not found it so.) But by far the best
method for the study of the forms and relations both of
ependyma cells and astrocytes is the Bichromate-and-silver
Impregnation of Gora, the best material being that which
has been for not more than two or three days in the osmio-
bichromic mixture.

This method, however, does not tinctorially differentiate
between neuroglia-cells and nerve-cells, and is of no use for
mapping out tracts of neuroglia as a whole. The following
methods are intended for this. They either stain neuroglia
more or less specifically, leaving other tissues unstained
(WetceErt), or stain it in a different tone to other tissues.
None of them are satisfactory. Waurcrrt’s process stains the
processes of the cells (his ‘ fibres’) intensely, whilst leaving
the cell-body unstained ; and in consequence, if exclusively
followed, may lead to erroneous conclusions.

838. Weicert’s Neuroglia Stain (WuicErT’s Beitr. zur
Kenntniss der normalen menschlichen Neuroglia, Frankfurt-
a-M., 1895; and his art. “ Newrogliafarbung” in Encycl.
Mik. Technik) —Pieces of very fresh tissue of not more than
half a centimetre in thickness are put for at Jeast four days
into 10 per cent.formol. They are then mordanted for four
or five days in an incubating stove (or for at least eight days
at the temperature of the laboratory) in a solution containing

NEUROGIIA, AND SENSE ORGANS. 439

5 per cent. of neutral acetate of copper, 5 per cent. of acetic
acid, and 2} per cent. of chrome alum, in water. Add the
alum to the water, raise to boiling point, and add the acetic
acid and the acetate, powdered (or [Encycl., 2nd ed., p. 303]
instead of the chrome alum, you may take chromium fluoride,
which obviates the necessity of boiling). If preferred, the
mordant may be dissolved in the formol solution, so that the
hardening and mordanting are done at the same time.

After the mordanting the tissues are washed with water,
dehydrated, imbedded in celloidin, and sectioned. The
sections (not too thick) are treated for ten minutes with a
1 per cent. solution of permanganate of potash, and well
washed in water. They are then treated for two to four
hours with a solution of “ Chromogen.” This is a naphthalin
compound prepared by the Hoechst dye manufactory. The
solution to be used is prepared as follows: 5 per cent. of
“Chromogen” and 5 per cent. of formic acid (of 1:20 sp. gr.,
about four times as strong as the officinal) are dissolved in
water, and the solution carefully filtered. To 90 c.c. of the
filtrate are added 10 c.c. of 10 per cent. solution of sodium
sulphite.

After this bath, the sections are put till next day into a
saturated (5 per cent.) solution of Chromogen. (Instead of
the Chromogen treatment, you may simply treat the sections
with Pat’s potassium sulphite, § 788, and the results will be
nearly as good.)

They are next carefully washed and stained. This is best
done on the slide. The stain is a warm-saturated solution
of methyl violet in alcohol of 70 to 80 per cent. (to which,
after cooling and decanting, there may be added, if desired,
5 per cent. of 5 per cent. aqueous solution of oxalic acid).
The sections are treated with this for a few seconds to one
minute, and mopped up with blotting-paper, then treated for
an instant with saturated solution of iodine in iodide of
potassium of 5 per cent. ‘They are then differentiated till
clear and light blue with a mixture of anilin and xylol in
equal parts. Wash this out thoroughly with pure xylol,
and mountin balsam, or, preferably, turpentine colophonium.

Glia fibres and nuclei blue, cytoplasm invisible.

This method only gives good results with the hwnan
subject.

44.0 CITAPTER XXXV.

Maxory (Journ. Exper. Med., 1897, p. 532) fixes tissues for four days
in 10 per cent. solution of formalin, then for four to eight in saturated
solution of picrie acid (or for the same time in a mixture of the two),
then mordants for four to six days at 37° C. in 5 per cent. solution of
bichromate of ammonia, makes sections (celloidin) and stains them in
WEIGERT’S fibrin stain.

SrorcH (Virchow'’s Arch., clvii, 1899, p. 127; Zect. wiss. Mik., xvi,
1900, p. 475), instead of mordanting the material in bulk with the copper
fluid, first makes celloidin sections.

BaRTEL (Zeit. wiss. Mik., xxi, 1904, p. 18) first makes paraffin sections
and treats them with all the reagents without removing the paraffin,
until they have passed the anilin-xylol mixture, which should consist of
1 of anilin to 10 of xylol (or more), and be allowed to act for twelve to
twenty-four hours.

Sanp takes material fixed as for his neurofibril stain, p. 400, and stains
it as Weigert.

See also AGUERRE and Krause, Arch. Mck. Anat., clii, 1900, p. 509;
and WiMMER, Zett. ar7ss. Mik., xxiv, 1907, p. 192.

RusascuKin (Arch. mik. Anat., lxiv, 1904, p. 577) injects
centres of small mammals with the fixing liquid. To make
this, take 100 parts of 2°5 per cent. solution of bichromate of
potash and 0:5 to 1 of acetate of copper, boil, and add 2°5
to 3 of glacial acetic acid. To this (which may be kept in
stock) add just before use 10 per cent. of formol. Inject
warm, and after ten minutes dissect out and harden in the
liquid for five to seven days at 35° to 40° C, Dry super-
ficially, put for six to twelve hours in alcohol of 95 per cent.,
and get into celloidin or paraffin. Stain sections on slide for
six to twelve hours in saturated aqueous solution of methyl-
violet B; treat for half a minute to a minute with Gram’s
iodine in iodide of potassium (1:200 or 300); differentiate
in anilin or clove oil and pass through xylol into balsam.
Said to give very sharp results with small mammals,

839. Benda’s Methods (Neurol. Centralb., xix, 1900, p. 796, and his art
“ Neuroglinfirbung” Eneycl. Mik. Technik., ii, p. 808) are as follows:
The material js to be fixed with alcohol, and further treated with nitric
acid, etc., as directed for centrosomes, § 651, and paraffin sections are
made and fixed to slides and the paraffin removed. They are then
mordanted and stained as directed under (bd), § 651 and differentiated
and mounted as there described.

Glia fibres and nuclei blue, the rest red.

Besides this, BENDA also recommends hardening and making paraffin
s cbionsas described, then staining with the modified WEIGERTstain given
for central corpuscles under (7), § 651 ; or, staining with HerpENHAIN'S

NEUROGLIA, AND SENSE ORGANS. 441

iron hematoxylin, and differentiating with 2 per cent. iron-alum or
WEIcERT's borax-ferricyanide mixture.
See also Meves, Arch. mik. Anat., Ixxi, 1908, p. 578.

840. Ma.tory’s Hematoxylin Stains (Journ. Erper. Med., v,
1900, p. 19).—Tissues to be fixed, mordanted, and cut as
directed under Mattory, § 838. ‘The sections are put for a
quarter of an hour into 0°5 per cent. solution of permanganate
of potash, washed and put for a quarter of an hour into 1 per
cent. solution of oxalic acid, well washed and stained for
twelve to twenty-four hours or more in Mallory’s phospho-
tungstic hematoxylin. Wash, dehydrate in 95 per cent.
alcohol, clear with organum oil, mount in xylol-balsam.
Axis-cylinders and nerve-cells pink, neuroglia blue. To get
a more isolated stain of neuroglia, the sections should be
brought for five to twenty minutes, after staining, into a 30
per cent. alcoholic solution of dry sesquichloride of iron.
Neuroglia and fibrin blue, the rest colourless.

Mattory’s phospho-molybdic hematoxylin may also be
taken for the stain, but is less elective.

Da Fano (Ricerche Lab. Anat. Roma, xii, 1906, p. 111)
fixes in a mixture of 72 vols. of pyridin with 28 of nitric acid
of 50 per cent. and stains as Mallory. Or, he fixes in a
mixture of 3 vols. of nitrate of pyridin with 1 vol. of osmic acid
of 1 per cent., and stains with Benda’s alizarin toluidin blue.

Freanpt (Arch. mik. Anat. Ixxvi, 1910. p. 15) describes a very
complicated modification of Mallory’s phosphotungstic hematoxylin

method.
ALZHEIMER (quoted from SPIELMEYER’S “ Teeknik d. mik. Untersuch.

d. Nervensystems,” p. 106) fixes in Weigert’s mordant (with formol) and
stains with Mallory’s phos, homolyhdic hematoxylin.

Eisatu (Arch. Psychiatr. wu. Nervenheilk., xviii, 1911, p. 896; Zeit.
wiss. Mik., 1913, p. 420) has a highly complicated modification of the
same stain, specially for glia granules.

841. AncLapE and Morer (Rev. Neurol., ix, 1901, p. 157)
harden in a mixture of 3 parts of liquid of For (§ 42), with
1 of 7 per cent. sublimate solution, dehydrate with alcohol
followed by aceton, make paraftin sections and stain in
saturated aqueous solution of Victuria blue, heated till it
steams, rinse with liquid of Gram ($ 287), differentiate with
xylol 1 part, anilin 2 parts, and mount in balsam. Simple,
applicable to lower animals, and gives very sharp images.

4.42 CHAPTER XXXV.

Similarly, in a very complicated way, L’HERMITTE and GUCCIONE,
Semaine Médicale, xxix, 1909, No. 18, and MrrzpacHEer, Journ. f.
Psych. u. Neurol., xii, 1909, p. 1 (Zeit. wiss. Mék., xxviii, 1911, p. 229).

See also GALESEsScU, C.R. Soc. Biol., xv, 1908, p. 429 (sections mor-
danted with resorcin and stained with methyl-violet and oxalic acid).

842. Saurerubin—Kuurscuitzky (Anat. Anz., viii, 1898, p. 357)
stains paraffin sections (of material hardened in his copper liquid, § 55),
either for five to ten seconds with a mixture of 1 grm. Siurerubin
(Rubin S.), 400 c.c. 2 per cent. acetic acid, and 400 ¢.c. saturated solution
of picric acid, or for half an hour in a mixture of 3 to 5 ¢.c. of the above
stain with 100 c.c. of 96 per cent. alcohol and washes out well with
alcohol. Glia violet, ganglion cells and axis cylinders reddish.

Insignificant modifications are described by Popow, Zeit. wiss. Mtk.,
xiii, 1896, p. 358, and BurcHarpt, La Cellule, xii, 1897, p. 364.

843. Yamacriwa (Virchow's Arch., clx, 1900, p. 358) hardens very small
pieces of tissues for a month or more in liquid of Miiller, makes celloidin
sections, stains for twelve hours in saturated alcoholic solution of eosin,
then for four to six in saturated solution of anilin blue in water, and dif-
ferentiates in dilute alcohol with a very little caustic potash. Water,
alcohol, origanium oil, balsam. Neuroglia red, axis-cylinders blue.

844, Acntcarro (Bol. Soc. Hspaii. Biol., Madrid, 1911, p.
139; Zeit. woss. Mtk., xxix, 1912, p. 238) puts sections of
frozen formo] material into cold saturated solution of tannin,
warms till vapour is given off, rinses, and puts into 10 cc.
of water with 6 to 8 drops of Bielschowsky’s oxide of silver
solution (undiluted). As soon as they turn yellow they are
put into formol of 10 per cent., and after about 10 minutes
washed and mounted.

By mordanting the material with 5 per cent. acetate of
copper, or Weigert’s mordant, and silvering by Ramon y
Cajal’s process, he gets a stain of the ameeboid cells of the
cortex.

Retina.*

845, Fixation and Hardening.—Notwithstanding the Encycl.
mik, Technik., 2nd edition, p. 75, I hold that osmie acid is by
far the best fixing agent. The retina of small eyes is best
prepared by fixing the entire unopened bulb with osmium

* Besides the sources quoted in the text, see SELIGMANN, Die mikro-
skopischen Untersuchungsmethoden des Auges, Berlin, 8. Karger (Karl-
strasse 13), 1899; GREEF Anlettung zur Mokr. Untersuch. d. Auges,

Berlin, Hirschwald, 1898; and the Art. “Retina” in Enxcyel. mike.
Technik., 2nd edition, p. 575.

NEUROGLIA, AND SENSE ORGANS. 443

vapour. According to Ranvier (Traité, p. 954) you may fix
the eye of a triton (without having previously opened the
bulb—the sclerotic being very thin) by exposing it for
ten minutes to vapour of osmium. 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 alone; but most of the best recent work has been
done with chromic mixtures following the osmium.

Dr. Lindsay Johnson tells me that he now gets the best
results by suspending the globe over the steam of a 1 per
cent. osmic acid solution raised to the temperature at which
vapour is seen to be given off (but not to boiling point), for
five minutes in the case of human adults, or for one to three
minutes in the case of human infants, all monkeys and small
mammals, as in them the sclerotics are very thin. As soon
as the sclerotic is felt to be firm to the touch, it should be
opened by a small nick with a razor just behind the ciliary
body ; or if the eye be that of an adult, the cornea and lens
may be removed. ‘The eye is then put for twelve hours into
the mixture, § 44; it is then 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 successive
alcohols, beginning with 20 per cent., and ending with
absolute, taking five days from first to last.

Similarly RocHon-DuviewEAuD (Arch. Anat. Micr., ix, 1907, p. 317).

Other hardening liquids, however, also give good results,
provided that the fixation by the osmic acid has been
properly performed: amongst them liquid of Flemming, and
that of Miller. Formaldehyde mixtures he does not recom-
mend.

LEBER (Miinch. med. Wochenschr., xli, 30, 1894; Zeit. wiss. Mik., xii,

1895, p. 256) advises a solution of formol 1, water 10. After a few days’
hardening in this, the eyes may be cut through, it is said, without de-

AA. CHAPIER XXXV.

rangement of the parts. The retina lies flat, and is at least us well pre-
served as with solution of Miller.

See also Hippen (Arch. f. Ophthalm., xlv, 1898, p. 286; Zeit. wiss.
Mik., xvi, 1899, p. 79), who finds that formol fixes the lens badly, the
retina well, so far at least as the absence of folds from shrinkage is con-
cerned; and Herzoa (Arch. mik. Anat., lx, 1902, p. 517, and Encyel-
mk. Technik., p. 75), who also approves of formol, but insists that it
should be aczd, and adds 3 to 5 per cent. of acetic acid.

Koumer (Arch. Gesammte Phys., exxix, p. 35), fixes for twelve to
twenty-four hours in a mixture of 4 parts saturated solution of bichro-
mate, 4 of formol of 10 per cent., and 1 of acetic acid.

Benpda (Verh. Ges. Naturf. Afrate, lxxi Vers., 1900, p. 459) fixes in
nitric acid of 10 per cent., and hardens in liquid of Miiller, twenty-four
hours in each.

Zurn (Arch. Anat. Phys., Anat. Abth., 1902, Supp., p. 106) advises (for
mammals) fixing in saturated solution of sublimate in salt solution of
0°6 per cent., with 1 to 13 per cent. of acetic acid after removing the
anterior pole and the vitreous. Wash out in alcohol of 35 per cent.
made 5 per cent. stronger each day up to 50 per cent.; then pass on to
stronger and cedar oil and paraffin.

846. Staining —For general views I recommend iron-
hematoxylin, followed by S&urefuchsin or Picro-Siurefuch-
sin, or preceded by Bordeaux; or Kernschwarz, followed by
safranin, or the Ehrlich-Biondi stain.

The Methylen-blue intra-citam stain has given valuable
results ; see the methods of Doatgn.

But the most important method is the bichromate-and-silver
impregnation of Goal, first applied to this object by Tarrurert
(Intern. Monatsschr., 1887). This author employed the rapid
process. Soalso Ramon y Casan (La Cellule, ix, 1893, p. 121)
with the double-impregnation process, § 815. To avoid the
formation of precipitates on the tissues, he covers the retina,
before silvering, with a piece of peritoneal membrane, or a
thin layer of collodion. Or, better, he rolls the retina
(op. cit., p. 180). After removing the vitreous, the retina
is cut away around the papilla with a punch or fine scalpel,
and separated from the choroid. It is then rolled up (after
being cut into quadrants or not), so as to form a solid block.
This is painted with 2 per cent. celloidin, which is allowed
to dry for a few seconds, and the whole is put into the
bichromate mixture, and further treated as a solid mass of
tissue.

Ramén also employs his neurofibril silver method, see
Intern. Monatsschr. Anat. Phys., xxi, 1904, p- 393.

NEUROGLIA, AND SENSE ORGANS. 445

Goxar’s sublimate impregnation (Cox’s form) has also been
successfully employed by Kraus and Ramon.

The bichromate-and-silver method serves for the study of
the fibres of Miiller and neurogha cells, as well as neurones.
Weigert’s neurogha stain does not give good results.

Lennox (Arch. f. Opthulm., xxxii, 1; Zeit. wiss. Mih., iii, 1886, p. £08)
has used Weigert’s hematoxylin method.

Kuunt (Jen. Zeit. Naturw., Bd. xxiv, H. 1, 1889, p. 177) employs
Pal’s modification. Similarly ScHaFFER (Sitzb. Akad. wiss. Wien., xcix,
1890), 3, p. 110; Zett. wiss. Mik., viii, 1891, p. 227). These methods give
a differential stain of rods and cones.

For the zonula and ciliary body see Mawas, Arch. d' Anut. mier., xii,
1910, p. 103.

847. Dissociation.—I'cr maceration preparations you may
use weak solutions (0°2 to 0°5 per cent.) of osmic acid for
fixation, and then maccrate in 0°02 per cent. chromic acid
(M. Scuuzrzx), or in iodised serum (M. Scuurrze), or in
dilute alcohol (Lanpott), or in Miiller’s solution, or (Ranvier,
Traité, 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.

ScHIEFFERDECKER macerates fresh retina for several days in
the methyl mixture, § 543.

Krause (Iitern. Monatsschr. Anat., 1884, p. 225) recom-
mends treatment for several days with 10 per cent. chloral
hydrate solution ; the rods and cones are well preserved.

Inner Har.

848. Inner Ear, Dissection.—TYor the dissection of the human ear
see POLITzER, “ Die anatomische u. histologische Zergliederung d. men-
schlichen Gehéroganes,” Stuttgart (Enke), 1889 (Zect. wiss. Mck., vii,
1890, p. 364). Amongst the lower mamunalia, the gudnea-piy is a
favourable subject, as here (as with some other rodents) the cochlea
projects freely into the cavity of the bulla, and may be casily removed
with a scalpel and brought into a fixing liquid, and opened therein.
With fishes and amphibia also the membranous labyrinth may easily

be got away.

849, Preparation—Scuwatse (Bevtr. 2. Phys., 1887; Zeit.
wiss. Mik., iv, 1887, p. 90).—Fix (cochlea of guinea-pig) for

44.6 OHAPTER XXXV.

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 parattin.

Prenanv (Intern. Monatssch. Anat., ix, 1892, p. 28).—Open
the cochlea in solution of Flemming or of Hermann, and fix
therein for four to five hours. Avoid decalcification as far
as possible, but if necessary take 1 per cent. palladium
chloride. Make paraffin sections.

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.

Kara (Zeit. wiss. Mik., xxv, 1908, p. 111) fixes the inner
ear, opened, for one or two hours in 80 c.c. of 0°5 per cent.
osmic acid with 5 drops of acetic acid, then adds 10 drops
of acetic acid and 60 c¢.c. of chromic acid (or platinum
chloride) of 0°5 per cent. and leaves it for four days therein.
He then rinses, puts for twelve to twenty-four hours into
pyroligneous acid or pyrogallol vr tannin solution, decalcifies
(not necessary for mice) in 200 parts of water with 1 of
chromic acid and 4 to 10 of nitric or hydrochloric acid, and
unbeds in celloidin or sometimes paraffin.

Similarly Wirrmaack, see § 799.

Bietscpowsk: and Bruvuu (Arch. mik., Auat., xxi, 1907,
p. 27) fix the petrous in formol of 20 per cent., decalcify it
in nitric acid of 5 per cent., wash this out, and put back for
a few days into the formol, cut by the freezing method, and
silver by the neurofibril method § 770 (24 hours in nitrate
of + per cent., but only a few minutes in the oxide bath).

Sunilarly Mutienix, Bull. Mus. Comp. Zool. Harvard Cull.,
hii, 1909, p. 215.

Sremn (Anat. nz, xvi, 1900, p. 3898) decalcifies in
celloidin by the method of Rousszau. So also Kisar (Arch.
mik, Anat., lix, 1901, p. 173).

For staiing, Ranvinr (Lraite, p. 991) employs his gold
and formic-acid inethod.

The bichromate-and-silver method of GoLe: may be em-
ployed with fetal or new-born subjects. The methylen blue
intra vitam method has given good results. For the higher
vertebrates the injection method should be employed. The
Eneycl. mik. Technik, i, p. 511, recommends injection of 1 c.c.

NEUROGLIA, AND SENSE ORGANS. 44:7

of 0°5 per cent. to 1 per cent. solution every five minutes
through the vena femoralis, until the death of the animal.
The cochlea then to be got out, exposed to the air for 15 or
30 minutes, and fixed for some hours (overnight) in 10 per
cent. ammonium molybdate with a little osmic acid. Then
decalcified in trichloracetic acid of 5 per cent. with a trace
of platinum chloride, washing for 24 hours and got into
parathn.

For fishes and amphibia the immersion method will suffice.

850. Other Methods.—WaALDEYER, Stricker’s Handb., p. 958 (de-
calcification either in 0-001 per cent. palladium chloride containing 10
per cent. of HCl, or in chromic acid of 0°25 to 1 per cent.).

Ursan PritcHarpD (Journ. Roy. Mic. Soc., 1876, p. 211).—Decalecifi-
cation in | per cent. nitric acid.

Lavpowsky (Arch. 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. osmic acid solution, and mounted in glycerin.

Max Fuescw (Arch. ak. Anat., 1878, p. 800); Tarant (Arch. Ital. de
Biol., vi, p. 207); Hicuier, Abh. math-phys. Ci. Stchs. Ges. Wiss., xviii,
1892, p. 811; Zeit. wiss. Mck., ix, 1893, p. 380 (injection of blood-vessels
of the labyrinth); SizrBpenmann, Die Blutgefiisse im Labyrinthe des
meuschlichen Ohres, Wiesbaden, Bergmann, 1894; Zezt. wiss. Mik., xi,
1894, p. 386; Gray, Journ. Anat. Phys., 1903, p. 379; Scorr, zbid., xliii,
1909, p. 3829.

851. Olfactive Nerve-endings, Tactile Corpuscles, ete.—
Besides the gold method, Chapter XVII, and the wethylen-
blue methol, Chapter XVI, the rapid bichromate-and-silver
method of Goxat should be employed, and for the olfactive
mucosa gives the best results ; see van Guuucuten, Lu Cellule,
vi, 1899, p. 405. For intra-epidermic nerve-endings, besides
the inethods given Chapter XXVII, the Goa method should
be employed. According to van GunucurTen (Lu Cellule, ix,
1893, p. 319) it gives much better results than goid methods.
He uses the rapid process. For tactile corpuscles, etc., besides
the methods given § 661 and 662, see Ramon y Casau’s neuro-
fibril methods.

CHAPTER XXXVI.
METHODS FOR INVERTEBRATES,

Tunicata.

852. Fixation of Tunicata——A method of Lo Branco* for
killing simple Ascidians in an extended state has been given,
§ 25. Some forms, such as Clavellina, Perophora, Phallusia,
Molgula, Cynthia, etc., should first be narcotised by treat-
ment for from three to twelve hours with chloral bydrate
(1: 1000 in sea water), then killed in a mixture containing
chromic acid of 1 per cent. 10 parts, acetic acid of 50 per
cent. 100 parts, and finally hardened in 1 per cent. chromic
acid.

The compound Ascidians with contractile zooids may be left
in clean sea water till the zooids have become fully extended,
then fixed by van Buneprn’s acetic acid process, § 84 (steel
instruments being avoided for manipulating them). I strongly
recommend this process.

S. Lo Branco recommends for this group the chloral
hydrate process, followed by fixation with sublimate or
chromo-acetic acid.

CauLLEeRY (Bull. Sc. France Belg., xxvii, 1895, p. 5) first stupefies the

animals with cocaine (LAHILLE, a few drops of 5 per cent. solution to
30 c.c. of sea water), then fixes in liquid of Flemming or acetic acid.

Most small pelagic Tunicates are very easily fixed with
osmic acid or acid sublimate solution.

I have found the acetic acid process very good for Pyro-
soma. Lo Branco puts them for a quarter of an hour into
50 per cent. alcohol containmg 5 perv cent. of hydrochloric
acid, then into successive alcohols, beginning with 60 per
cent. He kills the hard forms of Salpa with acetic acid of
10 per cent., the semi-hard ones with 1 per cent. chromic

* References to methods of Lo Branco in this Chapter are all to his
paper in Mitth. Zool. Stat. Neapel, ix, 1890, p. 435.

METHODS FOR INVERTEBRATES. AA

acid containing 5 per cent. acetic acid, the soft ones with 1
per cent. chromic acid containing +5 per cent. osinic acid,
or 10 parts of 1 per cent. chromic acid, with 1 of formol and
9 of sea-water, Doliolide with sublimate, or the above osmic
mixture, or a mixture of 10 parts 10 per cent. solution of
sulphate of copper with 1 part concentrated sublimate solution,
or the formol mixture.

Molluscoida.

853. Bryozoa.—For some methods of killing and fixing see
§$ 18, 20, and 21. 8S. 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 Ersic, § 18. For Cristatella
see §§ 16, 20. See also Braun, § 907.

Conser (Trans. Amer. Mic. Soc., xvii, 1896, p- 810) kills
the fresh-water forms with cocaine, puts them for an hour
into 1 per cent. chromic acid, and passes through water into
alcohol, ete.

Sunilarly Carver (Hist. Nat. Bryozvaires, Montpellier,
1900, p. 15), for marine forms.

Zscurescuz (Zool. Jahrb., xxviii, 1909, p. 6) fixes larvae of
Alcyonidiwm (settled down on a layer of celloidin or paraffin)
with 8 parts of sublimate and 2 of acetic acid to 90 of sea
water, for 25 to 30 minutes.

854. Brachiopoda—Lo Bianco kills small animals in 70 per
cent. alcohol, larger ones being first narcotised with alcohol
and sea water.

Biocumann (Untersuch. fein. Bau Brachiopoden, Jena, 1892,
p. 5) fixes principally with sublimate, macerates by the
Herrwics’ method, § 534, decalcifies with 1 per cent. chromic
acid (for thick shells add a little hydrochloric or nitric acid),
or with nitric acid in alcohol of 50 to 70 per cent., and im-
beds in paraffin or celloidin.

See also Exman, Zert. wtss. Zool., lxii, 1896, p. 172.

Mollusca.

855. Fixation—Lo Branco narcotises Lamellibranchs for
six to ten hours or more with alcohol, § 18, and then kills them.

29

450 CHAPTER XXXVI.

Lisr (Mauna Flora Golf. Neapel, xxvii, 1902, p. 292)
nareotises Mytilide with 2 per cent. of cocaine in sea water,
and (for preservation of cilia) fixes in sea water, with 10 per
cent. of formol.

Lo Bianco advises that Prosobranchiata, and, amongst
the Heteropoda, Atlantida, be narcotised with 70 per cent.
alcohol, § 18. For Opisthobranchiata I recommend sudden
killmg with liquid of Perényi, or the acetic method, § 852.
Aplysia may first be narcotised by subcutaneous injection of
about 1 c.c. of a 5 to 10 per cent. solution of hydrochlorate
of cocaine (Rozerr, Bull. Scient. de la France, etc., 1890,
p. 449; Zeit. wiss. mik., ix, 1892, p. 216), or (Scuonzeiy,
Zeit. Biol., xxx, 1898, p. 187) 1 c.c. of 4 per cent. solution of
Pelletierin. For Lo Branco’s various methods see the
original, p. 467.

For Pteropoda in general, liquid of Perényi. Creseis is a
difficult form. Lo Branco advises the alcohol method, $ 18.
For the Gymnosomata he narcotises with 0-1 per cent. chloral
hydrate.

For terrestrial Gastropods see $$ 23 and 26. Marcur
(rch, mik. Anat., 1867, p. 204) gets rid of the mucus of
the integument of Limax, which may be an obstacle to
preparation, by putting the living animal into moderately
concentrated salt solution, in which it throws off its mucus
and dies in a few hours.

Lane (Anat. Hefte, 1902, p. 84) puts Helix into water with
enough chromic acid to make it of a Rhine-wine colour,
with an air-tight cover to the vessel, and when the animals
are extruded injects into them a quarter to a half of a
Pravaz syringe of 1 per cent. cocain, and after five to fifteen
minutes dissects and fixes.

Huymans (Bull. Acad. Belg., xxxii, 1896, p. 578) infects
ethyl bromide under the skin of Cephalopoda.

Lo Bianco uses for fixing them his chromo-acetic acid,
No. 1 (§ 39), with a double quantity of acetic acid, for
twenty-four hours,

856. Liver of Mollusca—Hnriques (Mttth. Zvol. Stat.
Neapel., xv, 1901, p. 289) fixes the liver of Octopus and
Sepia with sublimate. For Aplysia (especially in summer)
alcohol, formo], and chromic mixtures are counter-indicated,

METHODS FOR INVERTEBRATES. 451

on account of the carbohydrates in the cell. Sublimate is
best.

857. Nervous System of Pulmonata.—B. de Nasras (Act. Soc.
Tinn. Bordeaux, 1894; Rech. Hist. centres nerveux des
Gastéropodes, 1894, p. 23) opens the animals and fixes the
ganglia for one hour in a mixture of 6 parts glacial acetic
acid to 100 of 90 per cent. alcohol, or for fifteen to twenty
minutes in 5 per cent. sublimate with 5 per cent. acetic acid.
He stains in bulk, with Renaut’s hematoxylic eosin, or
R. Heidenhain’s hematoxylin, or a copper hematoxylin of
Viallanes, § 874, and imbeds in paraffin. He also stains by
the rapid method of Goat, imbedding, however, the ganglia
in celloidin directly after the hardening in osmic acid and
bichromate, and treating the sections with the silver (p. 34).
He stains with methylen blue by treating the ganglia in situ
for twelve to twenty-four hours with a 1 per cent. solution.

Dreyer (Zeit. wiss. Zvol., xcvi, 1910, p. 380) narcotises
Nudibranchs with cocain, and for studying the nerves fixes
them with Mayer’s picro-formol, puts for a week into a
mixture of 1 grm. of iron alum with 2 c.c. of formol and 40
of water, makes sections and stains with iron haematoxylin.

See also, for nerve-cells, McCturz, Zool. Jahrb., 1898,
p. 17 (Mawny’s methyl blue and eosin, or Benpa’s safranin
and Lichtgriin), and Leaunprz, Arch. mic. Anat., x, 1909,
p. 312.

858. Eyes of Gastropoda (FLummine, Arch. mik. Anat., 1870,
p. 441).—To obtain the excision of an exserted eye, make a
rapid cut at the base of the peduncle, 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. osmic acid, in
alcohol, or in bichromate.

Smrra (Bull. Mus. Comp. Zool. Harvard, xlvii, 1906,
p- 238) macerates eyes for at least two days in 9 parts of
water with 1 of weak mixure of Flemming, followed by
glycerin of 10 per cent. He bleaches them (in sections)
with nitric acid and chlorate of potash.

859. Eyes of Cephalopoda and Heteropoda (Grenacuer, Abh.
naturf. Ges. Halle-a.-S., Bd. xvi, 1896, p. 213).—Depigment

4592 CHAPTER XXXVI.

with hydrochloric acid (in preference to nitric acid). The
mixture § 582 may also be used. If you stain with borax-
carmine and wash out in this mixture, the pigment will be
found to be removed quicker than the stain is washed out.

Lenuossix (Zeit. wiss. Zool., lviti, 1894, p. 636; Arch. mik.
Anat., xlvii, 1896, p. 45) applies the method of Goxer to the
eyes of Cephalopods.

Similarly Korscu (Anat. Anz., xi, 1893, p. 362), but using
formol imstead of the osmic acid.

Husse (Zeit. wiss. Zool., \xviii, 1900, p. 418) fixes eyes of
Heteropoda with one of formol to 4 of water, and (p. 456)
bleaches those of Cephalopoda by the methods of GrenacHEr
and that of Janpmr, § 583.

See also Merron, ibid., 1xxix, 1905, p. 326.

860. Eyes of Lamellibranchiata.—See Parren, Mitth. Zool.
Stat, Neapel, vi, 1886, p. 733, and Rawirz, Jena. Zeit. Naturw.,
xxi, 1888, p. 115, and xxiv, 1890, p. 579 (bleaches with
caustic soda); see § 584. Hussy (op. cit., last §, p. 380)
employs the method of Janpmr for Arca. He fixes the eye
of Pecten in 10 per cent. formol for five minutes, followed by
sublimate or picro-nitric acid,

861. 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. Kocu or Exurennaum.
Mossiey (Quart. Jowrn. Mie. Sci. [2], xxv, 1885, p. 40)
decalcifies with nitric acid of 3 to 4 per cent. and then makes
sections. ‘This method serves for the study of the eyes of
CHITONIDE.

862. Injection of Acephala (FLemmine, Arch. mek. 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 the injection-pipe may
be tied in the heart, and the entire animal filled and covered
up with plaster-of-Paris, which serves to occlude cut vessels
that it is not possible to tie. As soon as the plaster has
hardened the injection may be proceeded with. See also
Dawirz, Anleit. zur Anfert. zvotom. Prép., Berlin, 1886, p. 44
(Anodonta) and p. 52 (Helix).

Daxin (Liverpool Mar. Biol. Comm., xvii, 1909, p. 76)

METHODS FOR INVERTEBRATES. 453

narcotises by adding alcohol and glycerin for eighteen to
twenty-four hours, puts for half an hour into formol of 5 per
cent., and injects from a branchial vessel.

MozesKxo (Zeit. wiss. Mik., xxvi, 1909, p. 353, and 1910,
p. 542) puts for half an hour into water at 40° to 50°C.,
removes the shell, and injects carmine by auto-injection
through the heart. For occluding vessels he takes cotton-
wool soaked with gelatin and plaster-of-Paris. He takes for
a vaso-dilator a saturated solution of peptonum siccum.

863. Maceration Methods for Epithelium. — HnauLMann
(Pfhiger’s Arch., xxiii, 1880, p. 505) macerates the intestine
of Cyclas in osmic acid of 0-2 per cent. (after having warmed
the animal for a short time to 45° to 50° C.), or in concentrated
boracic acid solution.

Cilia.—The entire intra-cellular fibre apparatus may be
isolated by teasing fresh epithelium 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 zn situ in 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 (five parts cold
saturated aqueous solution to one part water).

Beta Ha.uer’s Mixture, see § 540; Brock’s Medium, § 531;
Monius’s Media, § 535; the second of these is much recom-
mended by Drosr (Morphol. Jahrb., xii, 1866, p. 163) for
Cardinm and Mya.

Patten (Mitth. Zool. Stat. Neapel, vi, 1886, p. 736) takes
sulphuric acid, 40 drops to 60 grm. of water. Entire molluscs,
without the shell, may be kept in it for months.

Berwarp (Ann. Seti. Nat., ix, 1890, p. 191) macerates the
mantle of Prosobranchs in a mixture of one part each of
glycerin and acetic acid, two parts each of 90 per cent.
alcohol and 0:1 per cent. chromic acid and forty parts water,
which acts in from a quarter of an hour to three hours.
IIe also (pp. 102, 306) uses a weak solution of chloride of
ruthenium, especially for nerve-tracts, mucus-cells and cilia.
Alcohol material may be macerated in a mixture of one part
glycerin, two of acetic acid and forty of water.

454, CHAPTER XXXVI.

864, Mucus Glands.—Racovirza (Arch. Zool. eapér. [3], ii,
1894, p. 8) studies these in Nudibranchs (and Annelids) by
killing with acetic acid, staining in toto with methyl green
dissolved in liquid of Riparr and Psi, and after three to
six days, when only the glands show the stain, examining in
mixture of equal parts of glycerin and the liquid.

Arthropoda,

865. General Methods for Anthropoda.—As general methods
for the study of chitinous structures, the methods worked out
by Paul Mayer (see §$ 8 and 96) are excellent. It is, at all
events, absolutely necessary, in the preparation of entire
organisms or unopened organs, 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 (or
picro-nitric) is the most generally useful fixative, and 70 per
cent. alcohol is the most useful strength for washing out.
Alcoholic picro-sulphuric acid may be indicated for fixing in
some cases.

But if the animals or organs can first be properly opened,
the usual methods may be employed.

866. Crustacea.—Some forms are very satisfactorily fixed
with sublimate. Such are the. Copepoda and the larve of
Decapoda. It is sometimes indicated to use the sublimate
in alcoholic solution. Some Copepoda, however (Copilia,
Sapplhirina), are better preserved by means of weak osmic
acid, and so are the Ostracoda. In many cases the osmic
acid will produce a sufficient differentiation of the tissues, so
that further staming may be dispensed with; so for Copilia
and Phyllosona, The pyrogallic process (§ 874) may be useful.
GirsBrecut takes for marine Copepods a concentrated solution
of picric acid in sea water, to which a little osmic and acetic
acid may be added. For fresh-water forms, Zacnartas (Zool.
Anz., xxii, 1899, p. 72) takes chromo-acetic acid.

Girsprecut fixes larve of Stomatopoda for 5 to 10 minutes
in formol of 10 per cent. warmed to 40° or 50° C., opens
them in sca water and puts for 14 to 24 hours into formol 1

METHODS FOR INVERTEBRATES. 455

part and sea water 5 parts, and brings into alcohol of 70 per
cent.

Srappmrs (La Cellule, xxv, 1909, p. 356) fixes Sympoda in
Gitson’s copper formol, § 115, or in Horneti’s mixture of 100
parts of 5 per cent. formol with 40 of alcohol; and for
softening the chitin puts for 12 to 36 hours into 3 per
cent. solution of sublimate with 5 per cent. of nitric acid.

Nerrovitca (Arb. z. Inst. Wien, xiii, 1900, p. 8) fixes Argulus
with liquid of Tellyesniczky, § 52, warmed to 50°C.

For Fiscus.’s intra-vitam stain of Cladocera with alizarin
etc., see § 208.

867. Tracheata.—Kenyon (Tufts Coll. Stud., No. 4, 1896,
p. 80) fixes Pauropoda in Carnoy’s acetic alcohol and chloro-
form, § 85, cuts them in two for staining, etc., and imbeds in
celloidin followed by paraffin.

Hennines (Zeit. wiss. Mik., xvii, 1900, p. 311) takes—
Nitric acid 16 parts, chromic acid of 0°5 per cent. 16 parts,
sublimate saturated in 60 per cent. alcohol 24 parts, picric
acid saturated in water 12, and absolute alcohol 42, fixes for
twelve to twenty-four hours, and washes out with iodine
alcohol. He says that this mixture not only fixes, but softens
chitin enough to allow of paraffin sections being made through
hard parts.

Hamann (Sitz. Naturw. Freunde Berlin, 1897, p. 2) fixes
small Tracheata in 10 per cent. formol and finds the chitin
sufficiently soft for sections to be made.

Van Levuwen (Zool. Anz.,xxxii, 1907, p. 318) takes for larvee
of Hexapoda 12 parts of 1 per cent. solution of picric acid in
absolute alcohol, 2 of chloroform, 2 of formol, and 1 of acetic
acid.

Howanpr (Arch. d’ Anat. mic., xiii, 1911, p. 171), takes
12 parts of saturated solution of picric acid in formol of 40 per
cent., 54 of absolute alcohol, 3 of benzene, and 1 of nitric
acid, and finds that this fixes quickly enough not to make
chitin too hard.

Nurratr, Cooper and Rosinson (Parasitology, 1908, i,
p. 163), fix for a few minutes in hot picrosulphuric acid.

868. Methods for Clearing and Softening Chitin. —The methods
of Looss have been described § 553, those of Henninas and
Hamann last 9.

456 CHAPTER XXXVI.

Lisr (Zeit. wiss. Mik., 1886, p. 212) treats Coccide (after
hardening) for eighteen to twenty-four hours with eau de
Javelle, diluted with four volumes of water. After washing
out they may be imbedded in paraffin, and good sections
obtained.

Satine (Dissert. Marburg. 1906, p. 11) boils larvee of
Tenebrio for some minutes in eau de Labarraque, the heat
serving to fix the soft parts, which in successful cases are
well preserved. Wash out with warm water, then alcohol.

Sazerin’s method for antenne of Chilognatha (Mém. Acad.
Imp. St. Pctersh., xxxii, 1884, pp. 11, 12) consists in steep-
ing antennee (that have been dehydrated with alcohol) for
twenty-four hours in chloroform containing a drop of *
fuming nitric acid (shake occasionally).

Betnr (Zool, Jahrb., viii, 1895, p. d44) puts telsons of
AMysis for eight to fourteen days into 40 per cent. alcohol,
to which nitric acid is gradually added, so that by the end
of that time they have been brought into alcohol containing
20 per cent. of the acid. This softens the chitin, and some-
what breaks down the structure of the otolith, so that good
sections through it are occasionally obtained.

Similarly Herssr, Arch. Mntiwickelungsmech ix, 1899,
p. 291.

See also the depigmentation processes, $$ 575—584.

869. Test for Chitin (Zanper, Pfliiger’s Arch., Ixvi, 1897, p. 545).—
Treat for a short time with a drop of freshly prepared solution of iodine
in iodide of potassium and add a drop of concentrated chloride of zine.
This is then removed with water as far as possible, and the violet
reaction is obtained.

See also WusTER, Zool. Jahrb., Abth. Syst., xxviii, 1910, p. 531.

870. Beruu’s Stain for Chitin (Joc. cit., § 868).—Sections are put
for three or four minutes into a freshly prepared 10 per cent. solution of
wnilin hydrochloride, to which has been added one drop of hydrochloric
acid for every 10 ¢.c. They are then rinsed in water, and the slide is
put with the sections downwards into 10 per cent. solution of bichro-
mate of potash. The stain is at first green, but becomes blue in tap
water or alcohol containing ammonia.

Maver simply uses a solution of pyrogallol in aleohol or glycerin ;
and Hormann (Zeit. wiss. Zool., ixxxix, 1908, p. 684) puts for a day or
more into raw pyroligneous acid.

871. Trachee may be studied hy the Golgi bichromate and

METHODS FOR INVERTEBRATES. A457

silver process. Marrin (C.R. Soc. Philomath., 1893, p. 3)
injects them with indigo white (through the body cavity),
and puts into hot water from which the air has been expelled
by boiling. Trachee blue.

872, Brain of Bees—Kunyon (Journ. Comp. Newrol., vi,
1896, p. 137; Journ. Roy. Mic. Soc., 1897, p. 80) treats by
the Go.ar process (seldom successful), or hardens in a
mixture of one part formol and two of 5 per cent. sulphate
of copper, followed by staining in Mallory’s phospho-molybdic
hematoxylin.

Jonescu (Jena. Zeit., xlv, 1909, p. 111) has employed the
silver methods of Ramon y Cajal and Bielschowsky and Wolff.

873, Ventral Cord—FLovp (Mark. Anmiv. vol. 1904, p.
355) fixes the ganglia of Periplaneta for eighty minutes with
vapour of formol, and brings into alcohol.

See also Binev, Journ. Anat. Phys., xxx, 1894, p. 469.

874. Eyes of Arthropods—For the methods of LankgstEr
and Bourne (Quart. Journ. Mic. Sct., 1883, p. 180: Limulus) ;
Hickson (ihid., 1885, p. 248: Musca); Parker (Bull. Mus.
Harvard Coll., xx, 1890, p. 1; Zert. wiss. Mek., viii, 1891,
p. 82: Homarus) see early editions.

Parker (Mitth. Zool. Stat. Neapel, xii, 1895, p. 1) also
applies the methylen blue method to the retina and optic
ganglia in Decapods, especially in Astacus. He injects 0-1 c.c.
of a 0:2 per cent. solution into the ventral sinus. After
twelve to fifteen hours the animals are killed, the ganglia
quickly dissected out, and the stain fixed as described, § 344.

For his method for eyes of Scorpwns see § 583.

For the methods of Purcett for the eyes of Phalangida see
Zeit. wiss. Zool., lviii, 1894, p. 1. He has the following
stain. ‘he cephalothorax is removed and brought for
twenty minutes into 50 per cent. alcohol warmed to 45° or
50° C., and saturated with picric acid. The pigment
dissolves in this solution and stains the nuclei and some
other parts of the rhabdoms, so that no further stain is
required.

Hennines (Zeit. wiss. Aik, xvii, 1900, p. 326) depigments
sections by putting then for ten minutes (Musca) to twelve

458 OIAPTER XNXVI.

hours (Myriopoda) into a mixture of 2 parts of 80 per cent.
alcohol with one of glycerin and 2 per cent. of nitric acid,
best kept at 35° C. The elements are well preserved.

Wiomann (Zeit. wiss. Zool., xc, 1908, p. 260) makes the
lens of Arachnida fit for sectioning by putting for a day or
so into alcohol with 10 to 15 per cent. of nitric acid; and
bleaches sections with 1 part of chlorine water to 2 of alcohol.

See also Rosensrapr, Arch. mtk. Anat., xlvii, 1896, p. 478;
Viatnanes, Aun. Sci. nat., xiii, 1892, p. 854; and Dierrics,
Zeit. wiss. Zool., xcii, 1909, p. 465 (fixes in alcoholic formol,
and bleaches with dilute agua regia).

875. Injections (Arachnida and Crustacea especially).—
Aimét Scunetper (Tablettes Zool., ii, 1892, p. 123) recom-
mends lithographic Indian ink, the animals being narcotised
with chloroform, then injected and thrown into strong alcohol.
Similary Cacsarp (Bull. Se. France Belg., xxix, 1896, p. 16).

876. Arctiscoida (DoyviRE, Arch. mik. Anat., 1865, p. 105).—Exa-
mination of living animals after partial asphyxiation in boiled water.
See early editions.

Vermes.

877. Chetopoda: Cleansing Intestine—KitxunruaL (Journ.
Roy. Mic, Sce, 1888, p. 1044) puts Lumbricus intoa glass vessel
filled with bits of moistened blotting-paper. They gradually
evacuate the earthy particles from the gut, and fill it instead
with paper.

Voer and Yone (Traité @ Anat. Comp. Prat., v) recom-
mend coffee-grounds instead of paper, as they cut better after
imbedding.

Jorst (Arch, Hutwicklungsmech., v, 1897, p. 425) simply
keeps the worms for a few days in moist linen, and finds the
gut empty.

Pear, (Journ. appl. Mic., ii, 1901, p. 680) injects alcohol
of 6 per cent. through the gut of narcotised worms.

878. Chetopoda: Fixation —Lumbricus may be anesthetised
by putting the animals mto water with a few drops of chloro-
form. Purrigr puts them into water in a shallow dish, sets
up a watch-glass with chloroform in the corner of it, and
covers the whole.

METHODS FOR INVERTEBRATES. 459

Cerrontaine (Arch. de Biol., x, 1890, p. 327) injects inter-
stitially about 2 c.c. of a1: 500 solution of curare.

Jaquet (Bib. Anat., iii, 1895, p. 82) kills Lwmbricus in
extension in 1 part of nitric acid to 125 of water.

Couiin (Zeit. wiss. Zool., xlvi, 1888, p. 474) puts Criodrilus
lacuwm into a closed vessel with a little water, and hangs up
in ita strip of blotting-paper soaked in chloroform. KiKen-
THAL (Die mik. Technik, 1885 ; Zett. wiss. Mtk., 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 one to two centimeters on to the water. Tor
Opheliadee he also employs 0°1 per cent. of chloral hydrate
in sea water.

Many marine Chetopoda may be successfully narcotised
(Lo Bianco) in sea water containing 5 per cent. of alcohol,
or by means of the mixture § 18.

The Polycheta sedentaria may sometimes be satisfactorily
fixed by bringing them rapidy into corrosive sublimate.
Cold, not hot, solutions should be taken, as heat frequently
shrivels up the branchie. Hunice and Onuphis may be treated
in the same way.

Lo Bianco advises killing Chietopteride, Sternaspide,
Spirographis, Protula, by putting them for half an hour
into 1 per cent. chromic acid. Some of the sedentaria may
be got protruded from their tubes by leaving them for some
hours in 0°1 per cent. chloral hydrate in sea water.

For E:ste’s methods for Capitellide see Fauna u. Flora
Golf. Neapel, xvi, 1887, p. 295.

See also § 14 (lemon juice), and the methods $§ 20 to 26,
39 and 49.

879. Blood-vessels of Annelids (Kixunrsat, Zeit. wiss. Mtk.,
1886, p. 61).—The animals should be laid open and put for
two or three hours into agua regia (4 parts of nitric acid to
2 of hydrochloric acid). Vessels black, on a yellow ground.

Berou (Anat. Hefte, xlv, 19C0, p. 392, and xlix, 1900, p.
599) puts small Annelids for a week or more into equal parts
of 1 per cent. nitric acid and 1 per cent. nitrate of silver, or
into 50 parts of nitrate, 25 of formic acid, and 25 of water,
dissects out the organs and exposes to light. Marine forms
may be treated by Harmen’s process.

4.60 OHAPTER XXXVI.

880. Nerves of Annelids—The methylen blue method and
the bichromate of silver method of Golgi (the rapid method).
For the latter see v. Lennossix (Arch. mik. Anat., xxxix,
p. 102).

Lanapon (Journ. Comp. Neur., x, 1900, p. 4) injects strong
solution of methylen blue into the body cavity of Nereis, and
puts the animal for some hours into sea-water in the dark,
fixes the stain by Bethe’s method, and makes paraffin
sections.

See also M. Lewis, Anat. Anz., xii, 1896, p. 292 ; ArHEson,
ibid., xvi, 1899, p. 497; and the methods of ApAray $§ 342,
368, 37], and 773.

881. Hirudinea.—For the methods of killing see those given
for Lumbricus in § 878, also §§ 20 to 26, and 49.

Wurman (Meth. im mic. Anat, p. 27) recommends that
they be killed with sublimate.

I have obtained better results myself by narcotising with
carbonic acid (§ 26), and fixing with liquid of Flemming.
I have also found that lemon juice kills them in a state of
very fair extension.

ApAray succeeds with alcohol of 40 per cent.

Grav (Jen. Zett., 1893, p. 165) has obtained good results
by narcotising with decoction of tobacco.

882. Injection —Wuirman (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.

Jacquer (Mitth. Zool, Stat. Neapel, 1885, p. 298), for
artificial injections, puts leeches into water with a very small
quantity of chloroform, and allows them to remain a day or
two in the water before injecting them.

883. Nervous System.—Impregnation with gold. Brisrot
(Journ. of Morph., xv, 1898, p. 17) kills in formic acid of 15
to 20 per cent., puts for twenty-five minutes into 1 per cent.
gold chloride, reduces in formic acid of 1 per cent. (twelve
to eighteen hours), and imbeds in paraffin. See also §§ 342,
368, 371 and 778,

884. Nephridia—Sunarer (Quart, Journ. Mier. Sci., lv,
1910, p. 288) stains Hastriobdella intra vitam with very weak

METHODS FOR INVERTEBRATES. 461

solution of Methyl blue, which allows the course of the
nephridia to be made out.

885. Gephyrea—Voer and Yura (Anat. Comp. Prat., p.
373) direct that Siphunculus nudus be kept for some days in
perfectly clean basins of sea water, changed every day, in
order that the intestine of the animals may be got free from
sand, and then anesthetised with chloroform.

Warp (Bull. Mus. Comp. Zool., Cambridge, Harvard Coll.,
xxi, 3, p. 144) puts them into a shallow dish with sea water
and pours 5 per cent. alcohol ina thin film on to the surface
of the water, and as soon as they make no contractions on
being stimulated removes to 50 per cent. alcohol.

Lo Bianco says killing with 0:5 per cent. chromic acid or
with 0-1 per cent. chloral hydrate in sea water may be tried.
Phascolosoma and Phoronis should be treated by the alcohol
method, larvee of Sipunculus with cocaine, § 21.

Apr. (Zeit. wiss. Zool., xiii, 1885, p. 461) puts Priapulus
and Halicryptus into a vessel with sea water and heats on a
water-bath to 40° C.; or they may be thrown into boiling
water, which paralyses them so that they can be quickly cut
open and thrown into 3} per cent. chromic acid or picro-
sulphuric acid.

886. Rotatoria—For quieting them for study in the living
state, Weper (Arch. de Diol., vii, 4, 1888, p. 718) finds that
2 per cent. solution of hydrochlorate of cocaine gives the
best results. Warm water gave lim good results for large
species, such as those of Hydatina and Brachionus.

Harpy (Journ. Roy. Mic. Soc., 1889, p. 475) recommends
thick syrup added drop by drop to the water. Hupson (ibid.,
p- 476) mentions weak solution of salicylic acid.

Voix (Jahrb. Hamburg. wiss. Anst., xvii, 1901, p. 164)
quiets them in quince mucilage, 40 grm. of the seeds to 1
litre of water. Cf. § 917.

HirscureLper (Zeit. wiss. Zool., xevi, 1910, p. 211) studies
them living in neutral red of 1: 50,000.

See also §§ 28, 24 and 27. Methylene blue, § 339, may
be found useful.

Permanent preparations may be made by the method of
RovusseLer (Journ. Quekett Mic. Club, v, March, 1895, p. 1) :
The animals are got together in a watch glass and are nar-

4.62 CHAPTER XXXVI.

cotised by adding to the water at intervals a few drops of the
following mixture:

Hydrochlorate of cocaine 2 per cent. solution . 3 parts,

Methylated spirit . ; . : ‘ eae ee

Water . : 4 : ; 7 : » © 4;

As soon as the cilia have ceased to beat, or are seen to be
on the point of ceasing to beat, they are fixed by adding a
drop of liquid of Flemming or of 4 per cent. osmic acid.
After half a minute or less the animals are taken out with a
pipette, and thoroughly washed by passing them through two
or three watch glasses of distilled water. They are then
definitely mounted in a mixture of formol 2} parts, distilled
water 374 parts.

Zocrar (Comptes Rend., cxxiv, 1897, p. 245) narcotises as
RousseLet, but without the spirit, fixes with osmic acid for
two to four minutes, then replaces this by raw pyroligneous
acid diluted with eight to ten volumes of water, and after
five to ten minutes washes in several changes of water, and
passes through successive alcohols into glycerin or balsam.

Lenssen (La Cellule, xiv, 1898, p. 428) for the embryology
of Hydatina, kills with hot saturated sublimate, dehydrates,
stains lightly, imbeds in paraffin and stains with hemalum.

HiescurenDEr (op. cit. swpra) narcotises with cocaine, and
fixes with Fol’s picro-chromic acid.

Bzaucuaup (Arch. Zool. Hxpér., iv, 1906, p. 29) finds 1 per
cent. stovaine better than cocaine for some forms. He (evid.,
x, 1909, p. 77) fixes for five to ten minutes in four parts of
1 per cent. osmic acid with one of 6 per cent. sublimate and
five of 5 per cent. bichromate of potash, and one drop of acetic
acid for each 2 ¢.c., and imbeds in celloidin, and then through
chloroform in paraffin (three to ten minutes).

See also Tozer (Journ. Roy. Micr. Soc., 1909, p. 24).

887. Acanthocephali.—Sarrrricgn (Morph. Jahrb., x, 1884,
p. 120) obtained the best results by killing gradually with
0-1 per cent. osmic acid ; the animals placed in this contract
during the first hours, but stretch out again and die fully
extended. Similarly with 01 per cent. chromic acid; Echino-
rhynci live for days in it, but eventually die fully extended.

Hamann (Jen. Zett., xxv, 1890, p. 118) has succeeded withsub-
limate, andalso with alcohol containing a little platinum chloride.

METHODS FOR INVERTEBRATES. 463

Kaiser (Biblioth. Zool., H. vii, 1 Halfte, 1891, p. 3) found
that a saturated aqueous solution of cyanide of mercury,
warmed to 45° to 50° C., and allowed to act for from fifteen
to sixty minutes, and then washed out with 70 per cent.
alcohol, was the best of all fixing media.

888. Nematodes.—'he impermeable cuticleisa great obstacle
to preparation. According to Looss (Zool, Anz., 1885, p.
318) this difficulty may be overcome in the manner described
in § 553,

For fixing, most recent authors recommend sublimate
solutions ; chromic solutions seem to have a tendency to
make the worms brittle.

But, according to Zur Srrassen (Zeit. wiss. Zool., liv,
p-. 655), Bradynema rigidum ought to be fixed for at least
twelve hours in mixture of Flemming.

Avaestnin (Arch. Naturg., 1x, 1894, p. 255) takes for
Strongylus filaria Mayer’s picro-nitric acid.

Vuspovsky (Zert. wiss. Zool., lvii, 1894, p. 645) advises for
Gordius 0°5 per cent. chromic acid (twenty-four hours).

Lo Branco employs for marine forms concentrated subli-
mate or picro-sulphuric acid.

Looss (Zool. Anz., xxiv, 1901, p. 309) prefers hot (80° to
90° C.) alcohol of 70 per cent.

Guaug (Zeit. wiss. Zool., xev, 1910, p. 554) kills Ascaris
in a hot mixture of 100 parts of saturated sublimate, 100 of
alcohol, and 1 of acetic acid.

Staining is frequently difficult, and sometimes alcoholic
carmine, § 234, is the only thing that will give fair results.

Braun (see Journ. Roy. Mic. Soc., 1885, p. 897) recommends that
small unstained Nematodes be mounted in a mixture of 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
said to sometimes make Nematodes opaque.

Demonstration of living Trichinze.—BaRrneEs (Amer. Mon. mik. Journ,
xiv, 1893, p. 104) digests trichinised muscle (of the size of a pea) in a
mixture of 3 gr. of pepsin, 2 dr. of water, and 2 minims of hydrochloric
acid, kept at body temperature for about three hours. The fiesh and
cysts being dissolved, the fluid is poured into a conical glass, and
allowed to settle; the trichine are drawn off from the bottom with a
pipette, got on to aslide with water and examined on a hot stage.

Grawam (Arch. mik. Anat., 1, 1897, p. 216) isolates Trichinz by

164, CHAPTER XXXVI.

macerating for one or two days in 2 per cent. acetic acid, staining with
aceto-carmine, and teasing.

889. Nemertina —My best results have always been
obtained by fixing with cold saturated sublimate solution,
acidified with acetic acid. ‘he other usual fixing agents,
such as the osmic and chromic mixtures, seem to act as irri-
tants, and provoke such violent muscular contractions that the
whole of the tissues are crushed out of shape by them.

Prof. pu Pressts has suggested to me fixing with hot
(almost boiling) water. I have tried it and found the
animals die in extension, without vomiting their proboscis.
So also Jounm, Bull. Wus. Hist. Nat., 1905, p. 326.

I have tried Forrricer’s chloral hydrate method (§ 20).
My specimens died fairly extended, but vomited their pro-
boseides. According to Lo Branco narcotisation with a
solution of 0'1 to 0°2 per cent. in sea water for six to twelve
hours is useful.

Oxsrereren (§ 18) recommends his ether water.

Denpy (see Journ, Roy. Mic. Soc., 1898, p. 116) has
succeeded with Geonemertes by exposing it for half a minute
to the vapour of chloroform.

For staining fixed specimens in toto I have found that it
is well-nigh necessary to employ alcoholic stains. Borax-
carmine or Mayer’s alcoholic carmine may be recommended ;
not so cochineal or hematoxylin stains, on account of the
energy with which they are held by the mucin in the skin.

Sections by the paraffin method, after penetration with
oil of cedar (chloroform will fail to penetrate sometimes after
a lapse of weeks).

Birerr (fauna uv. Flora Golf. Neapel, xxii, 1895, p. 443)
studies the nervous system, nephridia, skin, muscle and
intestine by the wtra vitam methylen-blue method. He
injects the animals with 0°5 per cent. solution in distilled
water, or 0°5 per cent. salt water, and allows them to lie for
aix to twelve hours or more in moist blotting paper.

See also MonreomeEry (Zool. Jahrb., Abth. Morph., x, 1897, p. 6); and
Boumie (Zeit, wiss. Zool., lxiv, 1898, p, 484).

890. Cestodes—As pointed out by Voar and Yune (Zraité
@ Anat. Comp. Prat., p. 204), the observation of the living

METHODS TOR INVERTEBRATES. 465

animal may be of service, especially in the study of. the
excretory system. And, as shown by Pintygr, Teenie may
be preserved alive for several days in common water to which
a little white of ege has been added.

Tower (Zool. Jahrb., xiii, 1899, p. 363) has kept Montezia
expansa alive for several days in a mixture of 100 c.c. of
tap water, 10 gr. of white of egg, 2 of pepsin, 2 of sugar,
and 5 of prepared beef (“ Bovox”’?). Chloride of sodium, he
says, should be avoided.

Lonygere (Centralb. Bakteriol., xi, 1892, p. 89; Journ.
Roy. Mic. Soc., 1892, p. 281) has kept Trienophorus nodu-
losus alive for a month in a slightly acid pepsin-peptone
solution containing from 8 to 4 per cent. of nutritive matter
and less than 1 per cent. of NaCl.

For the nervous system, Towmr (Zool. Anz., xix, 1896,
p. 323) fixes in a picro-platin-osmic mixture (stronger than
that of O. vom Rar, § 101) for ten hours, then treats for
several hours with crude pyroligneous acid, and lastly with
alcohol, and imbeds in paraffin.

ZERNECKE (Zool. Jahrb., Abth. Anat., ix, 1895, p. 92) kills
Ligula in the osmio-bichromic mixture of Gotar (4 : 1),
impregnates as usual, makes sections in liver, and treats them
by the hydroquinon process of Katrius. Besides the peri-
pheral and central nervous system, muscle-fibres, parenchyma
cells, and the excretory vascular system are impregnated.

He has also obtained good results by the methylen-blue
method.

Biocamann (Biol. Centralb., xv, 1895, p. 14) recommends
the bichromate and sublimate method of Gota1.

See also KOHLER, Zeit. wiss. Zool., lvii, 1894, p. 386 (stretches Tania
round a glass plate or on cork, and fixes with 5 per cent. sublimate) ;
Luue, Centralb. Bakt., xxx, 1901, p. 166, and Ransom, U. S. Nation.
Mus. Bull., lxix, 1909, p. 8.

891. Trematodes (FiscHer, Zeit. wiss. Zool., 1884, p. 1).—
Opisthotrema cochleare may be mounted entire in balsam.
For sectioning, he recommends a mass made by dissolving
15 parts of soap in 17°5 parts of 96 per cent. alcohol. The
sections should be studied in glycerin.

Lo Branco fixes Trematodes with hot saturated sublimate.

Looss (Arch. mik, Anat., 1895, p. 7) takes for Bilharzia

30

466 CHAPTER XXXVI.

warm (50° to 60°C.) 1 per cent. sublimate in 70 per cent.
alcohol.

Berrenporr (Zool. Jahrb., Abth. Morph., x, 1897, p. 308)
has had good results with the rapid Golgi method only on
Distoma hepaticwm, and prefers methylen blue.

Haver (La Cellule, xvii, 1900, p. 353) has also had results
with the Golgi method on this form, and also with thionin,
(after fixing with sublimate), which demonstrates tigroid
substance.

Cercarie.—Scawarzn (Zeit. wiss. Gool., xliii, 1886, p. 45)
found that the only fixing agent that would preserve the
histological detail of these forms was cold saturated sublimate
solution warmed to 35°—40° C.

For an “ indifferent ” liquid, Hormann (Zool. Jahrb., xii,
1899, p. 176) takes 1 part of white of egg in 9 of normal
salt solution.

892, Turbellaria.—Braun (Zeit. wiss. Mik , ii, 1886, p. 398)
gets entire animals (Rhabdoccela) on to a slide, lightly
flattens out with a cover, and kills by running in a mixture
of three parts of liquid of Lang with one of 1 per cent.
osmic acid solution. (Béumie [tbid.], 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 paraffin method.

DeEuace (Arch. de Zool. exp., iv, 2, 1886) recommends fixation (of
Rhabdocela Accela) by an osmium-carmine mixture, for which see loc.

cit., or by concentrated solution of sulphate ofiron. Liquid of Lang was
not successful.

For staining, he recommends either the osmium-carmine or impreg-
nation with gold (3 formic acid, two minutes ; 1 per cent. gold chloride,
ten minutes ; 2 per cent. formic acid, two or three days in the dark.)

Bonnie (Zeit. wiss. Mik., iii, 1886, p. 239) has obtained instructive
images with Plagiostomide fixed with sublimate and stained with the
osminm-carmine.

Grarr (Lurbellaria Acela, Leipzig, 1891 ; Zeit. wiss. Mik.,
ix, 1892, p. 76) says that chromo-aceto-osmic acid, followed
by hematoxylin, is good for the skin, but not for the
Rhabdites, which in Aceela and Alloioceela seem to be
destroyed by swelling. The same method is also good for
the parenchyma of Aimphicherus cinereus, Convoluta paradora

METHODS FOR INVERTEBRATES. 467

and C. sordida. Sublimate is good for Convoluta Roscoffensis.
The nervous system may be investigated by the methods of
Detage.

For Dendroccela sublimate solutions, sometimes hot, appear
indicated for fixing; see the mixture of Lana, § 64, also
Cuicakurr (Arch, de. Biol., xii, 1892, p. 438).

Arnoup (Arch. Zellforsch., iii, 1909, p. 433) kills Dendro-
ceelum in extension (?) with strong liquid of Flemming.

OxsvERGREN narcotises Dendroccelum with his ether-water,
§ 18.

JawntcHun (Zeit. wiss. Zool., lxii, 1896, p. 256) advises
for Planaria, eyes epecially, picro-sulphuric acid for an
hour or two; osmic acid is not good, and liquid of Miiller
macerates. He stains with borax-carmine, makes sections,
and puts them for ten minutes into osmic acid, then for five
minutes into pyroligneous acid, on the top of the stove.
He macerates the visual rods in a mixture of one part
common salt, one of acetic acid, and 100 of water. He
bleaches the pigment of the eyes with peroxide of hydrogen.

Wiiseimi (tbid., xxx, 1906, p. 548) throws Triclads into
almost boiling mixture of Zenker, and after 10 to 30 minutes
removes to water for some hours, and then passes into iodine
alcohol.

Echinodermata.

893. Holothurioidea.—Tlese are difficult to fix on account
of their contracting with such violence under the influence
of irritating reagents as to expel their viscera through the
oral or cloacal aperture.

Voer and Yune (Anat. Comp. Prat., p. 641) say that
Cucumaria Planct (CO. doliolum, Marenzeller) is free from
this vice; but they recommend that it be killed with fresh
water, or by slow intoxication, § 25.

Synapta may be allowed to die in a mixture of equal
parts of sea water and ether or chloroform (S. Lo Branco).

Oxsrereren ($ 18) puts Synapta into his ether water, but
Dendrochirota first into magnesium sulphate of 1 to 2 per
cent., for some hours.

Group (Bull. Mus. Harvard Coll., xxix, 1896, p. 125)
paralyses Caudina with sulphate of magnesia, § 24, and fixes
with liquid of Perényi (or sublimate for the ovaries).

468 CHAPTER XXXVI.

Holothurids, Dr. Weser informs me, are admirably pre-
served in formaldehyde; a weak solution is sufficient.

For the staining of muscles with methylen blue, see IwANnzorr,
Arch. mtk. Anat., xlix, 1897, p. 103; and for the study of calcareous
plates, see WooDLAND, Quart, Journ. Micr. Scz., xlix, 1906, p.534 (fixation
with osmic acid, staining with picro-carmine, followed by Lichtgriin).

894. Echinoidea.—I advise that they be killed by injection
of some fixing liquid. For preservation, formaldehyde has
proved admirable in all respects, and greatly superior to
alcohol (Wexssr).

Lo Branco kills by pouring over them (mouth upwards) a
mixture of ten parts acetic acid and one of 1 per cent. chromic
acid, and brings at once into weak alcoho]. Or he makes two
holes in the shell, lets the water run out and alcohol run in.

Sections of spines may be made by grinding, see § 177.

Spicula and the skeleton of pedicellariz may be cleaned
by eau de Javelle, see DopervEin (Miss. Ligeb. Tiefsce-Exped.,
v, 1906, p. 67).

895. Asteroidea.—Hamann (Beitr. Hist. Echinodermen, ii,
1885, p. 2) iyects the living animal with a fixing liquid
through the tip of a ray. ‘The ambulacral feet and the
branchiz are soon distended by the fluid, and the animal is
then thrown into a quantity of the same reagent.

In order to study the eyes, with the pigment preserved
in situ, they 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 sectioned in a glycerin
gum mass, or some other mass that does not necessitate treat-
ment 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.

Formaldehyde is vot to be recommended for the preservation
of Asteroidea (WuzBrER).

See also Lo Branco, cp. cit. (he kills Brisinga with absolute
alcohol), also $$ 17, 20.

896. Ophiuridea should in general be killed in fresh water
if it be desired to avoid rupture of the rays (Dp Casrernarna,
La Est. Zool, du Napoles, p, 135).

METHODS FOR INVERTEBRATES. 4.69

Lo Branco kills small forms with weak alcohol, Ophiopsila
with absolute alcohol, and Ophiomyxa with 0°5 per cent.
chromic acid.

Russo (Richerche Lab. Anat. Roma, iv, 1895, p. 157) fixes
Ophiothrix« for an hour or two in 0°5 per cent. osmic acid
and then decalcifies in solution of Miiller for six to ten
days. Or he fixes for three minutes in a mixture of two
parts concentrated sublimate solution, one part 70 per cent.
alcohol, and one part acetic acid (sp. gr. 1:06), and decalcifies
in Miller or in 70 per cent. alcohol with 10 per cent. of
acetic acid. He stains with paracarmine.

897. Crinoidea.—Lo Bianco (luc. cit., p. 458) fixes Antedon
rosacea with 70 per cent. alcohol, 4. phalangium with 90
per cent.

898. Larve of Echinodermata (from instructions written down
for me by Dr. Barrois).—For the study of the metamor-
phoses of the Mchinoidea and Ophiuridea it is necessary to
obtain preparations that show the calcarcous skeleton preserved
intact (a point of considerable importance, since this skeleton
frequently affords landmarks of the greatest value), and that
give clear views of the region of formation of the young
Kchinoderm (which is generally opaque in the living larva).
They should also possess sufficient stiffness to allow of the
larva being turned about in any desired way, and placed in
any position under the microscope.

Pluteus larvee should be fixed in a cold saturated solution
of corrosive sublimate, for not more than two or three
minutes, then washed with water, and brought into dilute
Mayer’s cochineal (§ 235). This should be so dilute as to
possess a barely perceptible tinge of colour. They should
remain in it for from twelve to twenty-four hours, being
carefully watched the while, and removed from it at the
right moment and mounted in balsam, or, which is frequently
better, in oil of cloves or cedar-wood.

Auricularia and DBipinnaria—As above, but the earlier
stages of the metamorphosis of Auricularia are better studied
by fixing with osmic acid, staining with Beale’s carmine, and
mounting in glycerin.

Larve of Comatula are best fixed with liquid of Lang,

470 CHAPTER XXXVI.

and stained with dilute borax-cramine. 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 Synapte formed from Auricularia, Without this
precaution you generally get preparations of larve either
shut up (Pentacrinus), or entirely deformed by contraction
(young Synapte). .

See also MacBripu on the development of Amphiuru squamata,
Quart. Journ. Mier. Sei., xxxiv, 1892, p. 131 (osmic acid followed by liquid
of Miller and alcohol; decalcification with nitric acid in alcohol ; stain-
ing with Mayer’s paracarmine or hemalum); and SEELIGER on the de-
velopment of Autedon, Zool. Juhrb., Abth. Anat., vi, 1892, p. 161.

MacBripeE (Quart. Journ. Mier, Sci., xxxviii, 1896, p. 340) fixes larvee
of Asterina in osmic acid, brings into liquid of Muller for twelve to four-
teen hours, imbeds in celloidin followed by paraffin (see § 171). and
stains sections with carmalum or Delafield’s hematoxylin, best after a
foregoing stain of twenty-four hours in borax carmine.

Mayer (Gruudziige, Lem and Mayrr, 1910, p. 486) arranges
a number of fixed and stained Plutei on a sheet of gelatin
foil gummed to a slide with euparal, dehydrates by adding
alcohol by drops, and adds euparal and a cover. See also

Wooptann, Quart. Journ. Mier, Set., xlix, 1905, p. 807,

Cuelenterata.

899. Thread-cells.—Iwanzorr (Bull. Soc. Nut. Mcseou, x, 1896,
p. 97) advises for the Nematocysts of Actiniw maceration by the Hrrt-
wia's method, § 534, or better, fixation for two to five minutes with

vapour of osmium followed by a short washing with sea water or distilled
water.

For Meduse he also advises the HERTWIG's method, § 534, or treat-

ment with a solution containing methyl green and gentian violet with a
little osmic acid.

900, Lirrne (Journ. App. Mic., vi, 1908, p. 2116; Journ.
Roy. Mic. Soc., 1903, p. 287) kills Mydra in hot saturated
sublimate in 70 per cent. alcohol, washes with alcohol, stains
for five minutes in strong solution of methylen blue, dehy-

METHODS FOR INVERTEBRATES. 471

drates rapidly, clears with cedar or bergamot oil, and mounts
in balsam. Nematocytes blue, the rest unstained.

901. Actinida.—For narcotisution methods see §§ 15 to 26.

902, Fivation.—In Le Abttinie, Fauna u. Flora d. Golfes v.
Neapel, Anvrus says that hot corrosive sublimate often gives
good results. In the case of the larger forms the solution
should be injected into the gastric cavity.

Freezing sometimes gives good results. A vessel contain-
ing Actiniw 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.

Doerven (Journ. Inst. Jamaica, ii, 1898, p. 449) narcotises

with magnesium sulphate, § 24, and fixes with formol of 8
to 5 per cent.

908. Maceration.—For the Hrrtwias’ method (Jen. Zeit.,
1879, p. 457) see § 534. The tissues should be left to
macerate in the acetic acid for at least a day, and may then
be teased in glycerin.

Lisr (Zeté. wiss, ALik., iv, 1887, p. 211) treats tentacles of
Anthea cereus and Sagartiu parasitica for ten minutes with
a mixture of 100 c.c. of sea water with 30 c.c. of Flemming’s
strong liquid, then washes out for two or three hours in
0-2 per cent. acetic acid, and teases in dilute glycerin.
Picro-carmine may be used for staining.

904. Nervous system.—This group is generally held to be
refractory to the Golgi impregnation. Haver, however (Le
Cellule, xviii, 1901, p. 388), has obtained good results by the
rapid method on young specimens of Metridiwm dianthus.
Besides nerve-cells, there are impregnated neuro-muscular
cells, gland-cells, and nematocytes. Leave for 5 to 8 days
in the osmic micture. He has also had good results by the
iutra vitam methylen blue method (this is also good for
nematocytes). So also has Grosuts (Arb. Zool. Inst. Univ.
Wien, xvii, 1909, p. 269), adding the dye to the water with
the animals till it gives a steel-blue tint.

472, CHAPTER XXXVI.

905. 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
boiling, sometimes gives good results.

See also Lo Branco, loc. cit., p. 446.

Sections.—See §§ 177 and 178, for undecalcified specimens.

906. The Alcyonaria have also extremely contractile polyps.
In a former edition I suggested for their fixation either hot
sublimate solution or glacial acetic acid (§ 84). 8. Lo Bianco
has since recommended essentially similar processes. GaAnrBINI
(Aanuule, p. 151) drenches them with ether, and brings into
strong alcohol.

Witson (Mitth. Zool. Stat. Neapet, 1884, p. 3) kills Aleyo-
naria with a mixture of one part of strong acetic acid and
two 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.

907. Zoantharia and Alcyonaria.—Braun (Zool, Anz., 1886,p.
458) inundates Aleyonium palmatum, Sympodium coralloides,
Gorgonia verrucosa, Caryophyllia cyathus, and Palythoa
avinelle with a mixture of 20 to 25 c.c. of concentrated
solution of sublimate in sea water with four to five drops of
I per cent. osmic acid, and after five minutes passes into
successive alcohols.

(This method also gives good results with Hydra and some
Bryozoa and Rotifers.)

See also § 14.

Busor (Arch. Zool. expcr., ix, 1901, p. 50) kills Veretillum
in sea water containing 10 per cent. each of formol and ether,
and after a minute passes into 2 per cent. solution of formol
im sea water.

908. Hydroidea, Polypoid Forms.—For suitable narcotisation
methods see $§ 15 ef seq.

For killing by heat see § 13.

Hivation—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,

METHODS FOR INVERTEBRATES. 473

The solution should be employed cold in general for Gymno-
blastea, hot for most Calyptoblastea.

Ether attentively administered gives good results with
Campanularide. Hydra is very easily killed by a drop of
osmic acid on a slide.

For the methylen-blue wtra vitam method, see Chapter
XVI; also Hanzi, Arb. Zool. Inst. Wien, xvii, 1909, p. 225.

909. Meduse : Fixation —For narcotisation see § 17.

Trachymeduse and Acalephe may be fixed in the usual
way in chromic or osmic mixtures. Osmic acid may be added
to the sea water containing the animals, which should be
removed to spring water as soon as they begin to turn brown.

910. BigELow (Mem. Boston Soc. nat. Hist., v, 1900, p. 193) fixes the
scyphistomes of Cassiopeta in Lo Bianco’s mixture of 10 parts of 10
per cent. solution of cupric sulphate with 1 of saturated sublimate, and
hardens them in 5 per cent. bichromate of potash.

911. Meduse : Sections.—Paraftin and collodion are certainly
not satisfactory as all-round methods for these watery
organisms. The Herrwies (Nervensystem der Medusen, 1878,
p. 5) imbedded in liver with the aid of glycerin gum, and
hardened the objects and the mass in alcohol.

See also Jotiur’s glycerin-gum method, and the gelatin
methods in Chapter VIII.

912, Meduse : Maceration.—Sce, especially for the study
of the nervous system, § 534. Doubtless in many cases the
pyrogallic acid reaction, § 374, would give enhanced differen-
tiation.

913. Siphonophora.—For the cupric sulphate method of
Bxpor (Arch. Set. phys. et nat., xxi, 1889, p. 556), which is
admirable for the preparation of museum specimens, but not
necessary for histological work, as well as for those of Lo
Branco (op. cit., p. 454), FriepLanver (Biol. Centralb., x, 1890,
p. 483), and Daviporr (Anat. Anz., xi, 1896, p. 505) see
previous editions. Lo Bianco fixes most forms with the
mixture given § 910.

For preserving, according to Wuburr, formaldehyde is
better than alcohol. Davivorr (loc. cit.) fixes in it.

A7 4 CIEAPTER XXXVI.

914. Ctenophora: Fixation.—The small forms are very easily
prepared by means of osmic acid. For the large forms see
Lo Bianco, luc. cit., p. 457. He uses his copper sulphate
mixture, § 910.

Samassa makes sections by the double-imbedding method,
see Arch. mik. Anat., x1, 1892, p. 157.

Porifera.

915. Spongie: Fixation.—The smaller forms can be fairly
well fixed by the usual reagents, osmic acid being one of the
best. For the larger forms absolute alcohol is apparently
the best. 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. FiepLer
(Zeit. 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.

Staining —T’o avoid maceration, I hold that alcoholic
stains should be alone employed, and I recommend Mayer’s
tincture of cochineal, § 235. Von LunprnreLrp (Zett. aiss.
Mik., xi, 1894, p. 22) uses aqueous solutions of Congo red and
anilin blue for the coloration of collar-cells.

Mincuin (Quart. Journ. Mic. Sct., x], 1898, p. 569) stains
spicula sheaths with Freeborn’s picro-nigrosin, § 6381.

Rousseau (Ann. Soc. Belg. Mic., xxiv, 1899, p. 51) stains
in nigrosin, picro-nigrosin, or indulin, or Mayen’s picro-
magnesia carmine.

For intra-vitam staining, see Lorszn, $ 208 ante, p. 138.

For silvering, see § 856.

Sectioning. — Calcareous sponges may be decalcified in
alcohol, acidified with hydrochloric or nitric acid, and then
imbedded in the usual way. Siliceous sponges may be
desilicified, § 574.

For Roussgau’s methods, see § 574. Vosmarr and Prexet-
HARING decalcify with a solution of picric acid in absolute
alcohol (see Zeit. wies. Mik., xv, 1899, p. 462).

See also Johnstone-Lavis and Vosmaer, § 179,

Preparation of Hard Paits—Siliceous spicules are easily
cleaned by treating them on a shde with hot concentrated

METHODS FOR INVERTEBRATES. A475

nitric or hydrochloric acid, or solution of potash or soda.
The acids mentioned are very efficient, but may attack the
silex of some delicate spicules. Potash solution is, therefore,
frequently to be preferred, notwithstanding that, in my
experience, it does not give such clean preparations.

According to Nott, eau de Juvelle is preferable to any of
these reagents, see § 552.

Embryos and Larve.—Maas (Zool. Juhrb., Abth. Morph., vii,
1894, p. 384) fixes larvee in liquid of Flemming or Hermann,
one to three minutes, and stains with borax-carmine, or with
gentian violet and Orange G (Flemming). He also (Zeté.
wiss. Zool., xvii, 1900, p. 218) fixes young Sycones in abso-
lute alcohol and stains with ammonia carmine (spicules tn
situ).

Detace (Arch. Zool. Hxpér., x, 1892, p. 421) fixes larvae
of Spongilla that have settled down on cover-glasses for three
minutes in absolute alcohol, stains in alcoholic carmine, § 234,
and brings through alcohol into oil of bergamot, then either
mounts direct in balsam, or detaches the larve from the cover
and imbeds in paraffin (three minutes).

Protozoa.

916. Introductory.—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 (Batsrani et Hunnecuy, Compt.
rend. Soc. de Biol., 1881, p. 131).

Weak solutions of alum, potash, and borax serve to
demonstrate the striations of the cuticle, and the insertions
of the cilia of Infusoria.

Scuupere (Arch, Protistenk., vi, 1905, p. 63) stains cilia by
the Gora impregnation (will bear a cover) ; or by Logrrier’s
stain for flagella (fix with vapour of osmium, the rest under
a cover).

For the mitochondria of Protozoa, see Fauri-Fremitr,
Arch. @ Anat. micr., xi, 1910, p. 457 (intra-vitam stains for
them, Dahlia in salt solution or Picigv’s liquid being the
best}.

476 CHAPTER NNXVI.

917. Immobilisation.—See the narcotisation methods $$ 20
to 25.

According to Scniruaver (Jen. Zeit., xxiv, 1890, p. 402),
nitrate of strychnin, of 0:01 per cent. or less, gives good
results with some forms, amongst which are Stenfor and
Carchesium. Antipyrin (0°1 per cent.), or cocaine of 0°01
per cent., seems only to have given good results as regards
the extension of the stalk in stalked forms.

Ersuonp (Zool. Anz., xiti, 1890, p. 723) slows the movements
of small organisms (small worms and Crustacea as well as
Ciliata) by means of a drop of thick aqueous solution of
cherry-tree gum added to the water containing them (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.

Cerves (Bull. Soc. Zool. France, xvi, 1891, p. 93) has
found that an intra vitam stain may be obtained by adding
methyl blue or “ violet dahlia, No. 170” to the gum solution.

Jensen (after SranL; see Biol. Centralbl., xii, 1892, p.
558) makes a solution of 3 grammes of gelatin in 100 c.c.
of ordinary water by the aid of heat. This makes a jelly at
the normal temperature. It is slightly warmed, and a drop
of it is mixed in a watch glass with a drop of water con-
taining the organisms.

See also Voix, ante, § 886; Srarkewrrscu, rch. Protestenk.,
v, 1904, p. 17; Lyon, Amer. Journ. Phys, xiv, 1905, p. 427

(neutralised gum).

918. Staining intra vitui.—See hereon Branpr (Verh.
physiol. Ges. Berlin, 1878); Cerves (Bull. Soc. Zvol., 25
janv., 1881); and Hunnucuy (Soc. Philom., 12 fév., 1881).
See also § 208.

Branpr recommends a 1 : 8000 solution of Bismarck brown;
also (Biol. Centralb., 1, 1881, p. 202) ‘a dilute solution of
hematoxylin,”

Curves (op. ctt., pp. 21, 226, 264, and Zool. Anz., iv, 183],
pp. 208, 287) found that living Infusoria stain in weak solu-
tions of cyanin, Bismarck brown, dahlia, violet 5 B, chrysoidin,
nigrosin, methylen blue, malachite green, iodine green, and
other tar colours, and hematoxylin, The solutions should be
made with the liquid that constitutes the natural habitat of

METHODS FOR INVERTEBRATES. 477

the organisms. They should be very weak, that is of
strengths varying between 1 : 10,000 and 1: 100,000. For
cyanin, 1 : 500,000 is strong enough.

As to the staining of the Nucleus, see Przesmycxi, Biol.
Centralb., vii, 1897, p. 821; and as to that of the Granula,
the same author, Zeit. wiss. Mik., xiii, 1896, p. 478. Also
Lorsez, § 208,

EHvamination in a colowred mediwm in which the organisms
do not stain, but show up on a coloured background is some-
times helpful. Crrres (Bull. Suc. Zool. de France, xiii, 1888,
p. 230) recommends solution of anilin black—Infusoria will
live in it for weeks; Fasre-Domeraue (Ann. de Microgr., ti,
1889, p. 545) concentrated solution of diphenylamin blue.

919. Demonstration of Cilia (Wappinaton, Journ. Roy. Mic.
Soc., 1883, p. 185).—A drop of solution of tannin, or a trace
of alcoholic solution of sulphurous acid, added to the water
containing the living organisms.

920. Fixing and Preserving.—For killing by heat see § 13.

Pritzner (Morph. Jahrb., xi, 1885, p. 454) used concen-
trated solution of picric acid run in wnder the cover.

Enrz (Zool. Auz., iv, 1881, p. 575) adds liquid of
Kleinenberg to the water containing the organisms in a
watch glass.

Korscuetr (ibid., v, 1882, p. 217) employs in the same
way 1 per cent. osmic acid, or, for Amoebe, 2 per cent.
chromic acid.

Lanszera (ibid., p. 336) advises the same reagents, but
recommends bringing the organisms into the fixing liquid with
a pipette.

For fixation with iodine (KENT) or iodine vapour (OvERron),
see § 83.

For sulphurous acid, § 62.

Carranso (Bollettino Scientifico, iii and iv; Journ. Roy.
Mie. Soc., 1885, p. 538) fixes for a few minutes with } per
cent. solution of chloride of palladium.

Brass (Zeit. wiss. Mik., 1884, p. 89) employs a mixture of
1 part each of chromic acid, platinum chloride, and acetic
acid with 400 to 1000 parts of water.

Certes (Comptes rend., \xxxviii, 1879, p. 488) fixes with

478 CHAPTER XXXVI.

2 per cent. osmic acid, or its vapours (10 to 30 minutes).
For details see previous editions.

Lonaut (Bull. Mus. Zool. Univ. Genova, 1892, No. 4) kills
in 10 c.c. of 1 per cent. sulphate of eserin with 1 drop of 1
per cent. sublimate.

Scata (Rev. Mus. La Pluta, xv, 1908, p. 57) fixes for 5 or
10 minutes in a mixture of 2 mg. of atropin, 10 drops of
formol, 10 grm. of glycerin and 50 c.c. of water.

See also Puscaxarew, Zeit. wiss. Mtk., xxviii, 1911, p. 145
(agar process for fixing and staining Amcche).

Fon (Lehrb., p. 102) fixes delicate marine Infusoria (Tun-
tinnodea) with the perchloride of iron solution ($ 80), added
to the water containing them, and stains with gallic acid,
§ 375.

Lo Branco (loc. cit., p. 444) fixes Gregarine with picro-sul-
phuric acid (one hour), Vorticelle with hot sublimate,
Acinetee with sublimate in sea water, or with osmic acid,
‘Thalassicola with 05° per cent. chromic acid (one hour),
Acanthometre and Aulacanthe with 50 per cent. alcohol or
with concentrated sublimate, or by adding a little osmic acid
to the water. For Spherozoa he proceeds as Branpv, § 922.

Zoerar fixes Rhizopoda and Infusoria as Rotatoria, § 886,
but without narcotisation.

See also FaBrE-DOMERGUE, Ann. de Microgr., ii, 1889, p. 545, and
1890, p. 50; ScHEWw1aKoFF, Biblioth. Zool., v, 1889, p. 5; Journ. Roy.
Mic. Soc., 1889, pp. 832, 833; Zosa, Boll. Sez. Pavia, 1892; Zect. wiss.
Mik., ix, 1893, p. 485; LAuTERBORN, Zezt. wiss. Zool., lix, 1895, p. 170;
ScHAUDINN, ibid., p. 193; BauBrant, Zool. Anz., xiii, 1890, p. 133;
KaRAWAIEW, tbid., xviii, 1895, p. 286.

921. Sections —T'he organisms should be strongly fixed,
then dehydrated and cleared, and brought into melted
paraffin in a small watch glass. After a few minutes therein
they are brought on a cataract needle on to a small block of
paraffin, and arranged there with a heated needle and
sectioned. They may be stained after fixation, or the
sections may be stained on the slide, §§ 186 or 187.

Entz (Arch, Protistenk., xv, 1909, p. 98) brings the objects
from clove oil into clove oil collodion of the consistency of
honey, then brings them in this into a funnel made of
paraffin, and when they have collected at the bottom of this

=

METHODS FOR INVERTEBRATES. A79

puts it into chloroform, which dissolves the paraffin and
hardens the collodion.
See also § 137, and Przusmycki, loc. cit. § 918.

922. Spherozoa—Branot (Fauna u. Flora Golf. Neapel,
xiii, 1885, p. 7) fixes with chromic acid of 0°5 per cent. to
1 per cent. (half an hour to an hour), or with a mixture of
equal volumes of sea water and 70 per cent. alcohol with a
little tincture of iodine for a quarter to half an hour, or with
a 5 to 15 per cent. solution of sublimate in sea water.

Karawaigw (Zool. Anz., xviii, 1895, p. 286) fixes dulacantha
for 24 hours in equal parts of strong liquid of Flemming
and acetic acid, and hardens for several days in pure liquid
of Flemming.

See also Lo Branco, § 920.

923. Sporozoa —WasirLewski (Sporozvenkunde, Jena, 1896,
p- 153) studies them living in their natural medium, or in
normal salt solution, or in a medium composed of 20 parts
white of egg, 200 of water, and 1 of common salt. He fixes
Gregarinee and Coccidia with osmic acid, sublimate, or picro-
sulphuric acid, and Myxosporidia with liquid of Flemming.

Scuaupinn (Zool. Juhrb., Abth. Anat., xiii, 1900, p. 197)
fixes Coccidia with a mixture of 2 parts of saturated aqueous
sublimate and 1 of absolute alcohol, with, if desired, a trace
of acetic acid.

Sremee.t (Arch. Protistenk., xvi, 1°09, p. 389) fixes cater-
pillars infected with Nosema in two parts of saturated
sublimate with one of alcohol and a little acetic acid, and
stains sections for as much as four days in Giemsa’s mixture,
rinses with alcohol and passes through xylol into balsam.

Litaur (thed., i, 1904, p. 311) fixes cysts for a minute in
“acetic sublimate,” puts for a minute into absolute alcohol,
and stains as a smear with hemalum or iron hematoxylin.

Brasit (Arch. Zool. Hxpér., 4, iv, 1905, p. 74) fixes them for
twenty-four hours in a mixture of 1 grmn. picric acid, 15 c.c.
acetic acid, 60 c.c. formol and 150 c.c. alcohol of 80 per cent.,
and stains paraffin sections in iron hematoxylin followed by
eosin and orange G., or Lichtgriin and picric acid.

924. Hematozoa.—Grass1 (Att. Accad. Lincei, iti, 1900,
p- 357) demonstrates the Malaria-parasites in the intestine,

480 CHAPTRR XXXVI.

body-cavity and salivary glands of Anopheles by treating
them with normal salt solution containing 2 per cent. of
formol (pure formol produces swellings), or in a mixture
of 15 grm. of salt and 250 c.c. of water with the white of
an egg. He fixes with sublimate, makes paraffin sections,
and stains with hemalum or iron hematoxylin. He stains
the Sporozoites by making cover-glass preparations which
are allowed to dry, put for twenty-five minutes into absolute
alcohol, and stained by the process of Romanowsxy, § 720.

For minute instructions for the application of this process to
sections, see Giemsa, Deutsch. med. Wochenschr., xxxvi, No. 12,
1910; and Scuusere, tlid., xxxv, No. 40, 1909 (Zeit. wiss.
Mik., xxvii, 1910, pp. 160, 161 and 513).

For clinical methods, see Coins, The Diseases of the
Bl.iod, London, J. & A. Churchill, 1905.

Braprorp and Piimuer (Quart. Jowrn. Micr. Sci., xlv, 1902,
p. 452) fix Trypanosomes in vapour of equal parts of acetic
acid and 2 per cent. osmic acid, or with GotLanp’s formol and
absolute alcohol, and stain with methylen blue and eosin,
and mount in turpentine colophonium.

Hinpiz (Univ. Calif. Pub. Zool., vi, 1909, p. 129) makes
smears on cover glasses coated with albumen, fixes for five
minutes in liquid of Flemming, passes through water up to
absolute alcohol, then for ten minutes into alcohol of 80 per
cent. with a good proportion of iodine in potassic iodide, then
into 30 per cent. alcohol, and stains with iron hematoxylin
or safranin, then with polychrome methylen blue, and lastly
with Unwa’s orange with tannin, and gets quickly through
alcohol into xylol and balsam.

Mincuin (Quart. Journ. Micr. Sct., liti, 1909, p. 762) makes
cover-glass smears, fixes them with vapours of osmic acid
(with or without acetic acid), and mounts them dry, or in
balsam after fixing in liquids and various stains, amongst
these that of Tworr. Half-saturated solutions of neutral
red and Lichtgriin are mixed, the precipitate dried and dis-
solved to about 0-1 per cent. in methyl alcohol with 5 per
cent. of glycerin. Three parts of this are diluted with 1 of
water, the smears stained for an hour, differentiated with
Unna’s glycerin-ether, and mounted in balsam. This stain
works best after fixation with sublimate.

Poricarp (C. R. Soc. Biol., Ixvii, 1910, p. 505) stains

METHODS FOR INVERTEBRATES. P 481

Trypanosomes intra vitam by adding a drop of concentrated
solution of neutral red to the edge of a drop of blood spread
between slide and cover.

925. Flagellata—LavurrrBorn (Zeit. wiss. Zool., lix, 1895,
p- 170) fixes Oeratium for about ten minutes in liquid of
Flemming, puts into alcohol for twenty-four hours, brings
back into water, bleaches if necessary with hydrogen per-
oxide, and stains with picrocarmine or Delafield’s hematoxylin.
He also imbeds in paraffin, § 921, and stains sections with
iron hematoxylin.

Zacuartas (Zool. Anz., xxii, 1899, p. 72) fixes Uroglena,
etc., with a mixture of 2 vols. saturated aqueous solution of
boracic acid and 3 of saturated sublimate.

926. Stains for Flagella—The Romanowsxy stain will give
a red stain of the flagella of some forms.

The method of Lorrier (Centralbl, Bakteriol., vi, 1889, p.
209 ; vii, 1890, p. 625; Zett. wiss. Mik., vi, 1889, p. 359 ; vii,
38, 1890, p. 368; Journ. Roy. Mic. Soc., 1889, p. 711; 1890,
p. 678) is as follows. 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 fuchsin, methyl violet, or “ Wollschwarz.” 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 (t.e. the above-des-
cribed mixture), and is heated for half a minute, until the
liquid begins to vaporise, after which itis 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 (p.177), the solution being pre-
ferably neutralised to the point of precipitation by cautious
addition of 0-1 per cent. soda solution.

See also Ligprranz, Arch, Protistenk., xix, 1910, p. 28.

Bonae (Journ. Roy. Mie. Soc., 1894, p. 640; Zeit, wiss. Mik.,
xii, 1896, p. 96) makes the mordant by mixing three parts
of the tannin solution with 1 of liquor ferri sesquichlorati
diluted twentyfold with water, and lets the mixture ripen
for some days exposed to the air, or (Journ., 1895, pp. 129,
248) adds to it a few drops of hydrogen peroxide, until it

31

482 CHAPTER XXXVI.

becomes red-brown, when it is shaken up and filtered on to
the cover-glass and allowed to act for a minute. ‘The cover-
glass is then mopped up and dried, and stained with carbol-
gentian.

Koerner and Fiscugr (quoted from Hneyel. mik, Techn,
p- 514) make the mordant with 2 parts of tannin, 20 of water,
4 of ferrous sulphate solution of 1: 2 strength, and 1 of
saturated alcoholic solution of fuchsin. Warm, let it act for
a’minute, rinse and stain with anilin-water-fuchsin, or carbol-
fuchsin.

Similarly Exiuis (Centralb. Bakt., xxi, 1908, p. 241; Journ.
Roy. Mic. Soc., 1904, p. 249), but staining with Siureviolett,
1 part to 75 of alcohol and 75 of water.

PeppLer (Centralb. Bakt., xxix, 1901, p. 376; Zeit. wiss.
Mik., xviii, 1901, p. 222) makes the mordant with 20 parts of
tannin in 80 of water, and 15 parts of 2°5 per cent. chromic
acid added gradually. ‘This mordant will keep for months.

Rossr (Arch. per le Se. med., xxiv, 1900, p. 297; Zeit. wiss.
Mik., xviii, 1901, p. 226) takes for the mordant a solution of
25 grms. of tannic acid in 100 of caustic potash of 0-1 per
cent., which will keep indefinitely. The stain is Ziehl’s
carbol-fuchsin, § 289. Cover-glasses are prepared with a
drop of culture, dried, and treated with 1 drop of the
mordant and at the same time 4 to 5 of the stain, allowed
to remain for 15 to 20 minutes, washed, and mounted. See
also Centralb. Bakt., xxxiii, 1908, p. 572 (Zeit. wiss. Mik.,
xix, 1903, p. 517).

Gumettr (Centralb., xxxiii, 1908, p. 816; Zeit. wiss. Mik.,
xix, 1903, p. 516) mordants for 10 to 20 minutes in 0-025 per
cent. permanganate of potash, rinses and stains for 15 to 80
minutes in a mixture of 20 parts 0°75 per cent. aqueous
solution of calcium chloride and 1 part of 1 per cent. neutral
red solution.

A method of Pirriztp is described by Kunpaut, Journ.
app. Mic., v, 1902, p. 1836 (Journ. Roy. Mic. Soc., 1902,p. 502),
The mordant consists of 10 parts of 10 per cent. tannin solu-
tion, 5 parts of saturated sublimate solution, 5 of saturated
solution of alum, and 5 of carbol fuchsin. Mordant for a
minute with heat, and stain with a mixture of 2 parts satu-
rated aqueous solution of gentian violet with 10 of saturated
solution of alum.

METHODS FOR INVERTEBRATES. 483

Van Ermencem (Jowrn., 1894, p. 405) fixes for a few minutes
with a mixture of 1 part 2 per cent. osmic acid, and 2 parts
10 to 25 per cent. solution of tannin, washes, treats with 0°25
to 0°5 per cent. solution of nitrate of silver, then for a few
seconds with a mixture of 5 parts gallic acid, 3 of tannin,
10 of acetate of soda, and 350 of water, then puts back again
into the silver for a short time, then washes and mounts.

See also Swepuens, ibid., 1898, p. 685, and Gordon, ibid.,
1899, p. 235, and the methods of Trenxmann (Centralb., vi,
1889, p. 483; Zeit. wiss. Mik., vii, 1890, p. 79) ; Brown
(Journ. Roy. Mic. Soc., 1893, p. 268) ; Junien (ibid., 1894, p.
403); Scravo (Zeit. wiss. Mik., xiii, 1896, p. 96) ; Hussurr
(ibid., p. 96); Murr (Journ. Roy. Mic. Soc., 1899, p. 235) ;
McCrorts (cbid., 1897, p. 251; he stains for two minutes in a
mixture of equal parts of concentrated solution of night-blue,
10 per cent. solution of alum, and 10 per cent. solution of
tannic acid) ; Zerrnow (ibid., 1899, pp. 662, 66-4); Morrow
(tbed., 1900, p. 131) ; Wenexe (tbid., p. 132); Levaprri, C. RB.
Soc. Biol., lix, 1905, p. 826 (for Spirochete pallida, Ramoy’s
neurofibril stain); Mutrowskxy, Mtinch. med. Wochenschr,
lvii, 1910, No. 27; Kaus, ibid., No. 26 (Zeit. wiss. Mih.,
xxix, 1912, pp. 123, 124; both for Spirochexte).

APPENDIX.

927. Chemicals, Stains, and Apparatus.— Addresses from which
it is recommended that these be obtained are given in § 11.

928. Cleaning Slides and Covers—New ones should first be
soaked in one of the following liquids: strong sulphuric,
hydrochloric or nitric acid, or aqua regia, or a mixture of an
ounce each of sulphuric acid and bichromate of potash with
from 8 to 12 ounces of water, then washed first with water
and lastly with alcohol, and dried with a clean cloth.

For wsed ones, if a balsam mount, warm, push the cover
into a vessel with xylol or other solvent of the mount, and
put the slide into another vessel with the same, leave for a
few days, and then put into strong alcohol. If this is not
sufficient, treat as for new ones. Some persons boil in lysol,
which I do not find efficacious.

For the final treatment, see p. 121.

929. Gum for Labels —Labels stuck on glass often strip off.
This may be avoided (Marpmann, Zeit. Angew. Mtk., ii, 1896,
p. 151; Journ. Roy. Mic. Soc., 1897, p. 84) by means of the
following adhesive: 120 grammes of gum arabic are dissolved
in a quarter of a litre of water, and 30 grammes of gum tra-
gacanth in a similar quantity. After a few hours the tra-
gacanth solution is shaken until it froths, and mixed with
the gum arabic solution. Strain through linen and add 150
grammes of glycerin previously mixed with 24 grammes of
oil of thyme.

Puirce (Journ. app. Mic., ii, 1899, p. 627; Journ, Roy. Mic.
Soc., 1900, p. 404) finds that if the end of the slide be
painted with a thin solution of balsam, it may be written on
with ink when dry, and the record preserved by a second
coat painted over it.

For other receipts see early editions.

INDEX.

The numbers refer to the pages.

A.

ABBE, mounting medium, 244:

Absolute alcohol, 59—61, 235

Acanthocephali, 462

Acetate of copper, 56

Acetate of lead, 381

Acetate of potash, for bluing hema-
tein stains, 160; for mounting,
238; refractive index, 235

Acetate of silver, 216

Acetate of uranium, 57

Acetic acid, action in fixing mixtures,
22, 23—24, 45, 54; fixing with,
53; Lo Branco’s “ concentrated,”
54; due proportions in mixtures,
54; various mixtures, 55 et seq.;
for decalcification, 280

Acetic acid carmine, 145

Acetic alcohol, 55, 381; ditto with
sublimate, 55

Acetic bichromate, 42

Aceto carmine, 145

Acetone, for celloidin imbedding,
105; for dehydration, 4; for
hardening, 63; for imbedding,
81; chloroform, 15 ; formol, 67 ;
sublimate solution, 48

AcHaRD and ReEynaup, impregna-
tion, 213

AcHUCARRO, neuroglia, 442

Acid alcohol, 62

« Acid” dyes, 130

Acid, free, test for, 191

Acid fuchsin, 183

Acid magenta, 183

Acid rubin, 183

Acidophilous elements, 132

Acidophilous mixture, 193

Acids, see Acetic, Chromic, Hydro-
chloric, Nitric, Osmic, etc.

Acids, Congo red as a test for, 191

Acridin red, 350

Actini, 13, 14, 17, 274, 4'70, 471

Actinida, 470, 471

Actol, 429

ADAMKIEWICs, myelin stain, 418

Addresses, for instruments and re-
agents, 10, 11

Adipose tissue, 351, 352

Adjective staining, 133

AGABABOW, elastic tissue, 348

Agassiz and Wuitman, pelagic ova,
305

A@uERRE and Krause, neuroglia,
440

Albumen, examination media, 237,
239; freezing method, 118; in-
jection mass, 267 ; section-fixing
process, 122, 123, 125; removal
from ova, 289, 300

Alcohol, for dehydration, 3; for
preservation, 4; for narcotisa-
tion, 14; for fixing, 59-62; for
hardening, 61; for maceration,
271; removal of, 5; absolute,
61, 235; acetic, 55; one-third,
62; hydrochloric acid, 62; tests
for water, 62; table for diluting,
60; ethylic, index of refraction
235

Alcohol balsam, 246

Alcohol, methyl, for narcotisation,
15; refractive index, 235

Alcoholic carmines, 148—150

486

INDEX.

The numbers refer to the pages.

Alcoholic cochineal, 150

Alcoholic formol, 65

Alceyonaria, 472

Alcyonella, 15

Alcyonium, 13

Aldehyde, 428

ALEXANDER, reconstruction, 291

ALFEROW, silver impregnation, 216

ALFIERI, bleaching, 285

Alizarin, artificial, 197; for nerve-
tissue, 385, 414; for neuroglia,
327; for mitochondria, 329;
intra vitam, 138

Alkanna, for staining, 353

ALLEN, gum and glycerine, 240

ALLEN, methylen blue, 205

ALLERHAND, myelin stain, 417

ALT, axis-cylinder stain, 385

Atrmawn, bleaching, 31; osmicated
fat, 352; osmie and bichromic
mixture, 37; nitric acid, 39;
corrosion, 277; fixatives for
nuclei, 3823; bioblasts, 328

Alum, ammonia, solubility, 163

Alum, ferric, 157

Alum-carmine, 142, 143; ditto with
picric acid, 145

Alum-hematoxylin stains, 159 et
seq.; general characters, 159;
bluing them, 159

Aluminium chloride carmine, 144

ALZHEIMER, neuroglia, 441

Amann, lactophenol, 241; high re-
fractive medium, 2413

Amber varnish, 252, 253

Ampronn and Hexp, polarisation,
417

Ammar, résine de, 246

Ammonia-alum, 163

Ammonia-carmine, 146;
neutralise, 259, 260.

Ammonia, chromate of, 44

Ammonio-nitrate of silver, 216, 398

Ammonium molybdate impregna-
tion, 227, 401, 402

Amphibia, embryology, 300—3803

Amphibia, larve, 317

Amphioxus, 305

how to

Amphipoda, embryology, 313

Amy] nitrite, 258

Amyloid matter, 171

AnpvzEr, phloroglucin, 283

ANvREEW, sulphorhodamin, 138

Anprgs, nicotine narcotisation, 13;
Actinie, 14, 471; imbedding
squares, 79; Actinida, 471

Anprews, imbedding apparatus, 79 ;
osmic acid, 29; blastoderm of
Aves, 296

Anethol, 118

ANGLADE and Mors1, neuroglia, 441

Aneus and Co., 10

ANILE, section cutting, 94

Anilin, for clearing, 4, 73, 111; for
imbedding, 73; as a mordant,
177; refractive index, 235

Anilin blue, 195, 230

Anilin blue-black, 196, 384

Anilin dyes, generalities, 169 et
seq.; and see Coal-tar colours.

Anilin green, 170

Anilin oil, see Anilin.

Anilin red, 181

Anilin violet, 172

Anilin water, 177

Aniseed oil, 118, 235

Anirscuxow, freezing method, 117;
serial sections, 125

Annelids, 458 ; narcotisation, 15, 18,
458, 459

Anodonta, 16

Antenne, 456

Anthozoa, 470

Anthracen ink stain, 437

ApAruy, washing sublimate material,
46; alcoholic sublimate, 48;
osmic sublimate, 224; paraffin
imbedding, 81, 83; knife posi-
tion, 90, 91; knife-holder, 93 ;
section-cutting, 96; paraffin
mass, 98; gelatin imbedding,
101; celloidin imbedding, 103,
104, 105, 106, 108, 109, 110;
serial sections, water method,
121; methods for celloidin sec-
tions, 126, 127; hematein mix-

INDEX.

487

The numbers refer to the pages.

ture I A, 164; alcoholic hemo-
toxylin stain, 166; methylen
blue, 200, 203, 205, 206, 207;
cement for glycerin mounts, 240,
252; maceration, 274; muscle of
Vermes, 342; gum syrup, 208,
gold chloride, 219, 220, 223, 224;
neuro-fibrils, 371, 402; formol
mixture, 67; picro-siurefuchsin,
189 ; Canada balsam, 245 ; killing
Hirudinea, 460

APEL, Gephyrea, 461

Apparato reticolare of Gouat, 403, 404

Aqueous humour, 237

Araneida, embryology, 312

Arctiscoida, 458

Areolar tissue, 345

Argentamin, 397, 417

Argulus, fixation of, 455

ARGUTINSEY, serial sections, 125

Argyroneta, ova, 313

Arion, embryology, 308

AxknpT, bone, 355

ARNOLD G., fixing mixture, 51;
orange method, 190; Dendro-
celum, 467

Arnotp, J., neutral red, 191;
maceration, 271; glycogen, 320 ;
cell granules, 327; mitochondria,
830; kidney, 372

Agnsrpin, methylen blue, 204, 207;
gold method, 334; papille foli-
ate, 335

ARONSON, nerve-stain, with gallein,
414 .

Arsenic acid, 283

Artefacts of fixation, 22, 43

Arthropoda, 454—458 ; embryology,
309—313

Artificial alizarin, 197, 327, 329, 385,
414

Artificial fecundation, 287

Artificial serum, 238

Arvom, embryology of Ascaris, 316

Ascaris, fixation, 463; ova, 55, 56,
194, 315

Ascidians, 17,418; gemmation, 306 ;
test-cells, 3 6

Asphalt varnish, 251; injection, 268

Asphyxiation, 17 ,

Assmann, blood, 364

Astacus, embryology, 313

Astervidea, 468; larvae, 469; eyes,
457

ArHanasiu and Dragotv, connective
tissue, 346

ArHuson, Annelids, 460

Aruras, Golgi impregnation, 429

AUBERT, cements, 249

AUBURTIN, serial sections, 126

AUERBACH, staining nerve-cells, 385,
389; axis-cylinder-stain, 385

AuGSTEIN, Strongylus, 463

Aurantia, 193

Auricularia, 469

Aves, embryology, 295—298

Axis-cylinder, stains for, 418 et seq. ;
structure, 389—403

Azofuchsin, 385

Azosiureblau, 385

Azoschwarz, 196

AzouLay, osmic acid nerve-stain,
227, 416; Golgi’s impregnation,
425

Azur I, azur JI, 200, 365

Azur-eosin, 365

B.

Bascockg, celloidin blocks, 110

Basses, safranin, 177, 178

Baxay, cartilage, 358

Baker, C., address, 10

Basiani, methyl green and eosin,
193; embryological methods,
288, 312 ; Protozoa, 478

BauBiani et Hennecuy, Protozoa,
475

BaLLowirz, muscle of Cephalopoda,
342; electric organs, 339, 340;
embryologcal methods, 299

Balsam, Canada, 235, 245, 246

Balsam, for imbedding, 116, 117; as
a cement, 252

Balsam, Tolu, 235

Balsams, 244

488

INDEX.

The numbers refer to the pages.

Bauzer and Unna, elastic tissue,
847

Barium bichromate, 41

Barngs, Trichine, 463

Barrots, larve of Echinoderms, 469

Barrte., neuroglia, 440°

Baryta water, 272

“ Basic” dyes, 130

Basophilous elements, 132

Bastian, gold method, 222

BarTaluuon, ova of Ascaris, 315

Barainuon and Kornuer, methylen
blue, 324

Baumearten, bleu de Lyon, 195;
carmine and bleu de Lyon, 231

Biveru, decalcification, 232; carti-
lage, 357 ; stain, 230

Bayon, neurofibrils, 390

Beate, shellac varnish, 253; injec-
tions, 265 ; digestion, 276

Bearp, ova of Raja, 305

Beaucuanmp, Rotifers, 462

Bescx, J., cements, 249

Brecker, microtome, 11

Brvor, Siphonophora, 473

Bee, brain of, 457

Bzzr, medullated nerve, 405

Brarens, G., embryology of Sal-
monide, 304

Beurens, W., refractive indices,
234; maceration, 272; cement,
249, 252; clove oil, 71; levulose,
241; biniodide of mercury, 243

Bria Hauuer, maceration, 275

BeLLaRmMinow, injection, 268

BELL, fat, 353

Bewu’s cement, 251

BENARIO, blood, 362

Benpa, nitric acid fixation, 39; iron
hematoxylin, 155, 156; copper
hematoxylin, 167; Lichtgriin
stain, 194; Sdureviolett stain,
194; neuroglia stains, 440;
erystal violet, 197; mitochondria
stain, 329; centrosome stains,
326; alizarin, 327, 329; secre-
tion vranules, 327; rapid myelin
stain, 413; retina, 444; picro-

siurefuchsin, 189, 344; fatty

acids, 353

BENECKE, stain for fibrils, 346

Benrpecentt, formol, 6!

BENEDEN, vAN, sublimate solution,
45; acetic acid, 54; acetic
alcohol, 55; malachite green,
194; embryology of rabbit, 293,
294; of Tenia, 314; of Ascaris,
316.

Benepixt and Kyecut, dyeing, 129

Bengal rose, 192

Benersson, larve of Diptera, 310

Bens ey, fixing mixture, 51; intes-
tine, 370; phosphorus, 320

Benzin colophonium, 246

Benzoazurin, 181, 197, 357

Benzol, for clearing, 73; for im-
bedding, 81, 83

Benzopurpurin, 191

Benzoyl green, 194:

Bera, imbedding, 86

Bergamot oil, for clearing, 71, 235 ;
for imbedding, 81

Berau, Annelids, 459

BeErGonzinI, plasma cells and Mast-
zellen, 351

BERKLEY, rapid nerve-stain, 413;
Golgi impregnation, 428; liver,
871

Berlin blue impregnation, 403

Berlin blue injections, 262, 266

BERNARD, maceration of mollusea,
453

BrERner, fat, 353

Bernueim, gold method, 223

Best, glycogen, 320

Besta, silver stain, 897; intra-
cellular network, 404; myelin
stain, 414

Brrue, treatment of osmic material,
31; methylen blue, 208 ; neuro-
fibrils, 401; stain for chitin,
456; telsons, 456

Berrenvorr, Distoma, 466

Berz, nervous centres, 375, 379

Brvawn Lewis, see Lewis.

Bia>co, 8S. Lo, sce Lu Branco,

INDEX.

489

The numbers refer to the pages.

Bichloride of mercury, see Sublimate.

Bichromate of ammonia, 44, 376,
879, 380

Bichromate of barium, 41

Bichromate of calcium, 41, 44

Bichromate of copper, 41, 415

Bichromate of potash, generalities,
41; for hardening, 41; for fix-
ing, 40, and hardening, 376,
378, 379, 380; for maceration,
273

Bichromate of silver impregnation,
see GOLGI.

Bichromate and osmic mixtures, 37,
38 ; other mixtures, 42—44
Bichromate material, action of light

on, 41; bleaching, 42, 285
Bichromates, 41
BickFaLvl, digestion, 277
BizpERMANN, methylen blue, 204;
nerve and muscle, 337, 338

BIELAszEWics, iron hematoxylin,
158
BiEtscHowsky, neurofibrils, 346,

390, 398, 399

BietscHowsky and BRuEHL, inner
ear, 446

BiELScHOWsKY and PULIEN, nerve-
cells, 389

BigeLtscHowsky and Wo.Lrr, neuro-
fibrils, 398

BiaELow, scyphistomes, 473

Binet, bleaching, 30; ganglia of
Hexapods, 457

Bina and ELLERMANN, medullated
nerve, 67

Biniodide of mercury mounting me-
dium, 243

Bioblasts of ALTMANN, 328

Bronp1, staining mixture, 184; blood,
360

Bipinnaria, 469

Bismarck brown, intra vitam, 136,
188; progressive, 172; regres-
sive, 181

Bitume de Judée, 251

Bizz0zERK0, blood-plates, 367 ; glands,
368

BizzozERo and Torre, blood, 363

BszLovussow, injection, 267

Blackley blue, 195

Bladder of frog, 342

Blattida, embryology, 311

Blauschwarz, 191

Bleaching, osmic material, 30; bi-
chromate material 42; chromic
material, 33; in general, 284—
286; gold material, 225

Buss, fixative, 65; ova of Amphibia
302

Bleu alcool, 195

Bleu Borrel, 366

Bleu carmin, 196

Bleu de Lyon, 195, 230

Bleu de nuit, 195

Bleu lumitre, 195, 230

Buiocuman, series sections, 124; ova
of Amphibia, 300; Cestodes,
465; Brachiopoda, 449

Blood, 359—368

Blood-parasites, 479—481

Blue-black, 196, 384

Buum, formol, 64, 65; celloidin im-
bedding, 108

BosreEtzky, ova of Lepidoptera, 310

Boccarp1, gold method, 228; stain
for nerve-cells, 388

BopeckKER, decalcification, 279, 356

Boeck, neurofibrils, 400

Boum gold method, 222

Boum and Opren, artificial serum ;
237; egg of fowl, 298; bichro-
mate, 41; ova of reptiles, 299

Béumer, hematoxylin, 163

Boumice, Turbellaria, 466; Nemer-
tina, 464

Bouton, myelin stain, 412; Golgi’s
impregnation, 428

Bone, 280—283, 353—357, and see
Decalecification.

Bonnet, embryology of dog, 294

Bownsy, triple stain, 190

Bonvicrnt, hardening brain, 380

Borax carmine, 148

Borax methylen blue, 324

Borpaag, double imbedding, 115

4.90

INDEX,

The numbers refer to the pages.

Bordeaux R., 190, 191

Borgert, paraffin imbedding, 79

Borina, ova of Ascaris, 316

Born, section-stretcher, 94; recon-
struction, 290, 291 ; ova of Rana,
302

Borreg1, fixing mixture, 39; picro-
indigo-carmine, 230

BorReE.’s blue, 366

Bourn, picric formol, 65, 66; subli-
mate formol, 66; larve of Runa,
303 ; ova of Salmonide, 304

Bow te, neurofibrils, 397

Bouma, cartilage, 356

Boveri, picro-acetic acid, 58; im-
bedding small objects, 289; ova
of Ascaris, 316

Boyce and Herpmann, copper, 320

Brachiopoda, 449

Braprorp and Puimmer, trypano-
somes, 480

Brapy, chloral hydrate, 238

Braegm, statoblasts, 306

Brain, see Neurological methods.

Branca, formol mixture, 66

Branvt, glycerin jelly, 242; infu-
soria, 476 ; Spherozoa, 479

Brasit, Sporozoa, 479

Brass, paraffin, 81, 99; Protozoa, 40,
477

Bravn, mounting medium for Nema-
todes, 463; Turbellaria, 466;
Zoantharia and Aleyonaria, 472

Bravs, fixation by injection, 25;
ova of Selachia, 305; liver, 370

Brazilin, 228

Brecxner, double imbedding, 114

Breautia, nerve stain, 414

Bremer, methylen blue and eosin,
193 ; Cox’s sublimate impregna-
tion, 433

Bressiavu, Mesostomide, 314

Brillantschwarz, 191

BRINKMANN, paraffin imbedding, 85

Brissy, freezing, 117

Bristou, bleaching, 30; Hirudinea,
460

Brock, maceration, 273

Bromay, reconstruction, 291

BrookovER, myelin stain,
Golgi impregnation, 430

Brown cement, 250

Brown, flagella, 483

Bricks, Berlin blue, 262; digestion,
277

Brug, embryology of Diptera, 310

BRUvuL, corrosion, 277

Bron, glucose medium, 240

Bruno, mucus glands, 368

Brunortl, gelatin imbedding, 100

Brunswick black, 251

Bryozoa, 16, 449, 472; statoblasts,
306

BucHNER,
324

Bupag, injections, 268

Buds of Ascidians, 306

BvuEuLER, staining nerve-cells, 389

Bugor, Veretillum, 472

Bumpvus, thyme oil, 72; celloidin
sections, 112

Bunag, stain for flagella, 481

Burcuarpt, E., bichromates, 41,
376; paraffin, 99; fixing mix-
tures, 41; pyroligneous - acid -
carmine, 145; pyroligneous -
acid-hematoxylin, 164; methyl
green, 170; serial sections, 121;
neuroglia, 442

BurckHarpt, brain of Protopterus,
382

Birarr, Nemertina, 464

BurzynskI, gelatin imbedding, 101

Buscu, osmic mixture, 82; nerve-
stain, 416; eosin, 231; decalcifi-
cation, 280; glycogen, 320

Bussg, celloidin, 103, 104, 106, 108

Burscuu, acid hematoxylin, 163;
iron hematoxylin, 159

Buzzi, eleidin, 333

407 ;

accessory chromosome,

Cc.

Caps, gastric glands, 370
CasaL, Ramon y, see RamOn ¥ CaJAL.
Cajeput oil, 72

INDEX.

491

The numbers refer to the pages.

CaLBERLA, methyl green, 170; ditto
and eosin, 193; indulin, 195;
glycerin mixture, 242

Calcium chloride, 235, 238; bichro-
mate, 41

Causa, picro-indigo-carmine, 230

Cauvet, Bryozoa, 449

Cambridge microtomes, 10, 11

Camsal, camsal balsam, 247

Canada balsam, index, 235; for im-
bedding, 116; for mounting, 245,
246 ; as a cement, 252

CANFIELD, iris, 342

Caoutchouc cement, 249, 251

Capitellide, 15, 459

Capsicum, extract, 353

Carazzr, peroxide of sodium, 285

Carbol pyronin, 183

Carbolic acid, index, 235; for clear-
ing, 73, 111

Carbolic fuchsin, 181

Carbon sulphide, refraction, 235 ; for
imbedding, 82

Carbon tetrachloride, 81, 82

Carbonic acid for narcotisation, 18

Carmalum, 144, 146; with indigo-
carmine, 230

Carmine, generalities, 141, 142;
analysis, 141; stains in general,
142; formule for stains, 142 et
seg.; aqueous, 142; alcoholic,
148—150; combination stains,
229—231

Carmine blue, 196

Carmine solutions, to neutralise,
259, 260

Carminic acid, 141

Carnoy, cajeput oil, 72; acetic al-
cohol, 55; Congo red, 191; salt
solution, 286; tannin solution,
239; cement, 253; micro-
chemical reactions, 319

Carnoy and Lresrun, ova of Am-
phibia, 300; ditto of Ascaris,
315; micro-chemistry, 320, 321

CarpenTsErR, freezing, 117; section
grinding, 115; cements and
varnishes, 249

CaRRIERE and Buraer, ova of Chali-
codoma, 611

CaRrtTER, injection, 261

Cartilage, 356—358

Cassia oil, 71, 235

CasTELLARNAU, DE, Ophiuridea, 468

CastLe, ova of Ciona, 306

Castor oil, 235, 248

Catots, methylen blue impregnation,
435

CatTtanszo, palladium chloride, 52;
Protozoa, 477

CauLusry, Ascidians, 448

CausarD, injection of spiders, 458

Caustic soda or potash, see Soda and
Potash.

Cavazzani, hematoxylin and Siure-
fuchsin, 232

Cavattié, electric organs, 339

Cedar-wood oil, index, 235; for
clearing, 70; imbedding, 81,
82, 83; for preserving, 5; for
mounting, 245; for dissecting
in, 8

Cell division, see Cytological me-
thods.

Celloidin imbedding, 102 et seq. ;
generalities, 102, 103; prelimi-
nary preparation, 103; celloidin
bath, 104; imbedding, 105;
orientation, 106; hardening,
106 —108; preserving blocks, 108;
fixing blocks to microtome, 109 ;
cutting, 110; clearing and
mounting, 110; the new me-
thod, 112; Gilson’s rapid, 112;
the dry cutting method, 113;
celloidin and paraffin method,
114 ; injections, 268

Celloidinum inelasticum, 103

Cells, paper for mounting, 250

Cells, study of, see Cytological
methods.

Celluloid injection mass, 269

Cement for celloidin blocks, 109

Cements and varnishes, 249 e¢ seq.

Central corpuscles, centrosomes, ete.,
158, 325

492

INDEX.

The numbers refer to the pages.

Central nervous system, see Nervous
centres and Neurological me-
thods.

Cephalopoda, 450; embryology, 307;

: eyes, 451

Cercarie, 466

Cerebrum, cerebellum, see Neuro-
logical methods.

CERFONTAINE, Amphiowus, 305; As-
caris, 316; Lumbricus, 459

CrrTzs, Infusoria, 476, 477

Crs ris-DEmMEL, blood, 363

Cestodes, 464; embryology, 314

Cheetopoda, 458 —460

Chalicodoma, ova, 611

Cuampy, mitochondria, 328

Cute, dehydration apparatus, 3

Chemicals, 11

CHENZINSEY, methylen blue
eosin, 193

CuicHKorr, Turbellaria, 467

CuILp, ova of fishes, 304; removal
of albumen, 289

CHILESOTTI, nerve-stain, 384:

Chilopoda, 455

Chinablau, 196, 405

China blue, 196, 405

Chinolinblau, chinolin blue, 195

Chitin, 278, 455, 456

Chiton, eyes, 452 ; embryology, 309

Chloral hydrate, for narcotisation,
15; preservative solutions, 238,
240, 243; for maceration, 275

Chlorate of potash for maceration,
274

Chloreton, 15

Chloride of aluminium carmine, 144

Chloride of calcium, 235, 238

Chloride of copper fixative, 56

Chloride of gold, see Gold chloride.

Chloride of iridium, 52

Chloride of magnesium, narcotisa-
tion, 17

Chloride of manganese, 237

Chloride of osmium, 52

Chloride of palladium, for fixing, 52 ;
for staining, 227, 418; for de-
calcifying, 280

and

Chloride of platinum, 51; mixtures,
38, 40, 52, 289, 322

Chloride of ruthenium, 227

Chloride of sodium, see Salt.

Chloride of vanadium stain, 435

Chloride of zine, 53, 381, 436

Chlorine for bleaching, 284

Chloroform, for narcotisation, 14; for
clearing, 73; for imbedding, 81,
82, 83, 84

Cholesterin, 333

Chondriokonts, 328

Chondriosomes, 328 é

Choroid, 285

Chromate, neutral, of ammonia, 44

Chromate of lead impregnation, 227

Chromate of silver, see Goual.

Chromates, 40

Chromatin, reactions, 319

Chromatin stains, defined, 1380; the
coal-tar, 169—181; cytological,
3824

Chromatophily, 132, 135

Chrome hematoxylin, 166

Chromic acid, generalities, 32 ; fixing
with, 82; washing out, 32;
hardening with, 34; action of
light, 33; mixtures, 38 et seq.,
40, 67; for maceration, 273; for
decalcification, 280, 283

Chromidia, 328

Chromium fluoride, 408, 410

Chromium sulphate for hardening,
380

Chromo-acetic acid, 35

Chromo-aceto-osmic acid, 35—87; for
decalcification, 288

Chromo-formie acid, 35

Chromo-formol, 67

Chromogen, 439

Chromo-nitric acid, 39; for bleaching,
286

Chromo-osmic acid, 35

Chromo-picric acid, 59

Chromo-platinic mixture, 40

Chromo-sublimate, 50

Craccio, C., sublimate mixture, 48 ;
fat, 351

INDEX.

493

The numbers refer to the pages.

Craccio, G. V., gold method, 222;
corpuscles of Golgi, 341; cornea,
336

CiaGuinsxk1, myelin stain, 418

CiecHANOWSKI, liver, 370

Cilia of Infusoria, 475, 477

Ciliated epithelium, 453

CILIMBARIS, muscle-spindles, 338 ;
elastic tissue, 348

Cinnamon oil, 71, 235

Ciona, 17, 306

Citrate of silver, 216

Cladocera, 313

CLARKE’s spirit-proof cement, 251

Clasmatocytes, 351

CuauDIvS, vegetal dyes, 229

Cleaning slides and covers, 121, 484

Clearing, generalities, 5, 68 et seq ;
clearing agents, 70 et seq.;
practice of, 69; choice of an
agent, 70; celloidin sections,
110; paraffin sections, 98

Clove oil, for minute dissections, 8,
41; refractive index, 235; for
clearing, 70; for imbedding, 81;
for differentiating stains, 175

Coal-tar colours, 169 et seq ; regres-
sive staining with, 173 ; progres-
sive ditto, 170; cho of, 169;
chromatin stains, 170—181;
plasma stains, 182

Coes, differentiator, 3

Cocaine, narcotisation, 16

Coccide, 456

Coccidia, 479

Cochineal, generalities, 141; aqueous,
Partscu’s, 143; CzoKor’s, 148;
Rasv’s, 144; alcoholic, Mayzr’s,
150; iron, 146

Cochlea, 445—447

Coz, Miracidia, 315

Ccelenterata, 470—-474

Ccerulein, 194

CouNHEIM, gold method, 220

Cog, freezing method, 118

Coleoptera, embryology, 311

Couss, parolein, 248; malaria para-
sites, 480

Collagen, 344

Collargol, 400

Coun, Criodrilus, 459

Coutin and Lucien, intracellular
network, 404

Couuiner, pelagic ova, 305

Collodion for fixing sections, 123,
124, 125—128

Collodion imbedding, 102 et seq.;
and see Celloidin

Collodionisation of sections, 95, 383

Colloidal gold, 401

Colloxylin, 103

Colophonium, for imbedding, 116;
for mounting, 246 ; index, 235 ;
cement, 252

Colour-acids, 130

Colour-bases, 130

Couuccl, balsam, 246

Comatula, larve, 469

Combination stains, 228 et seq.;
carmine combinations, 229;
hematein ditto, 231

Congelation imbedding methods, 117

Congo red, 191, 385, 418, 474;—
Corinth, 191

Conx.LIn, ova of Crepidula, 308

Connective tissues, 344 e¢ seq.

ConsER, Bryozoa, 449

Cooling paraffin, 87

Copal, section method, 115; varnish,
253

Copepoda, 454; embryology, 313

Copper, detection of, 320

Copper, sulphate, 43, 303, 473;
chloride and acetate, 56; im-
pregnation, 227, 437; nitrate, 57,
67

Copper formol, 67

Copper hematoxylin, 167

Corals, 472

Corallin, 181

Cori, narcotisation, 15; cocaine, 16;
keeping osmic acid, 28 ; chromo-
aceto-osmic acid, 86

Cornea, 278, 331, 335

Corwina, Krohnthal’s impregnation,
434; medullated nerve, 405

494

INDEX.

The numbers refer to the pages.

Corpuscles, tactile, 334; of Herbst
and Grandry, 334; of Meissner,
335; of Golgi, 340; of Meissner
and Krause, 335

Corti and Ferrara, pyronin, 183

Corrosion, 268, 277, 278

Corrosive sublimate, see Sublimate.

Cotton blue, 196

Coupier’s blue, 195

Cox, Nissl’s stain, 387, 389; medul-
lated nerve, 405; sublimate im-
pregnation, 433; neurofibrils,
403

Creasote, index, 235; for clearing,
73,111; imbedding, 81

CREIGHTON, glycogen, 320

Crepidula, 308

Cresyl violet, 197, 389

Crinoidea, 469

Cristatella, 15

Crustacea, 454; embryology, 313

Crystal violet, 197

Crystalline, 336

Csokor, cement, 252; bone, 355;
and see CzoKor.

Ctenophora, 474

Cuccati, soda carmine, 146

Cupric sulphate for fixing, 48, 473;
for staining, 227

Curarisation, 317

CurRRERI, toning bath, 431

CurscHMANN, amyloid matter, 171

Cyanin, 195

Cyanide of mercury, 381

CyButsry, gold impregnations, 225

Cyclas, ova, 309

Cytological methods, 317 e¢ seq.;
living cells, 317; fresh cells,
818; microchemical reactions,
319, 320; fixing agents, 321;
chromatin stains, 324; plasma
stains, 325; granules, 327 ; cen-
trosomes, 325; nucleoli, 324;
for nerve tissue, 386

Czoxor, cochineal, 143 ; turpentine
cement, 252

D.

Danni, fat, 352

Da Fano, neuroglia, 441

Dauuaren, sublimate, 51; double
imbedding, 114

Dahlia, 180

Daxin, injection of Acephala, 452

Damar (dammar), gum, 235, 246

DanrscHakoFF, serial sections, 125

DaviporFr, ova of Distaplia, 306
Siphonophora, 473

Davis, injection, 261

De-alcoholisation, 5, 68
choice of an agent, 70

Decalcification, 279—283

Decapoda, 454; embryology, 3138

Decker, section-stretcher, 94.

Derxcxg, encephalon, 380, 383

Derianprg, fat and lecithin, 351

DéseERINE, sections of brain, 383

Dez Groot, serial sections, 121; iron-
carmalum, 146; picro-magnesia
carmine, 148; alcoholic hema-
lum, 165

Dehydration, 2—5

DEKHUYSEN,

eé seq. ;

silver impregnation,
216, 217; bichromate mixture,
43 ; blood, 361, 367; salt solu-
tion, 237

DELAFIELD, hematoxylin, 163

Detags, Turbellaria, 466; larve of
sponges, 475

DrLamare, connective-tissue stain
346

Dexa Rosa, injection, 267

Deiia VALE, ova of Orchestia, 313

Deltapurpurin, 191

Dewnpy, Geonemertes, 464

DenkER, corrosion, 277

Denne, paraffin imbedding, 84;
orientation, 87

Depigmentation, 284 —286

Dz QUERVAIN, nervous tissue, 374

Desilicification, 283

De Veccui, celloidin, 105

Dz Wirt, elastic tissue, 348

Dewitz, injection of molluses, 452

Dextrin freezing mass, 118

INDEX,

495

The numbers refer to the pages.

Differentiation, optical, by fixation,
20, 22, 37

Differentiator, Cobb’s, 3 ; Haswell’s,
3; Cheatle’s, 3; Schultze’s, 4;
Kolster’s, 4

Diffusion apparatus, 3

Digestion, 276, 277, 320

Dimmer, serial sections, 124

Dina er, mitochondria, 330

Diomiporr, nervous tissue, 381

Diptera, embryology, 310

Dissz, stain for dentine, 358

Dissections, minute, 6, 8

Dissociation, methods of, 270

Distaplia, 306

Distoma, Miracidia, 315; staining,
466

D6vDERLEIN, Echinoidea, 468

DoaieEt, methylen blue, 200, 203,
205, 207, 209, 210 ; corpuscles of
Herbst and Grandry, 334, 392 ;
corpuscles of Krause, 335; cor-
puscles of Golgi, 341; vlfactive
organs, 335; iris, 342; retina,
444,

Dosey & Co., address, 11

D6uiKEN, formol imbedding method,
118; soap imbedding, 100

Donacia, ova, 311

Donaaaio, tin hematoxylin, 168;
neurofibrils, 402

DoNALDSsON, nervous centres, 381

DosTo1EWwsky, iris, 342

Double imbedding, 114

Double stains, see Stains, combined.

DoyéreE, Arctiscoida, 458

DrascH, gold impregnation, 220

DreEvw, eleidin, 333

DREYER, nervous system of Nudi-
branchiata, 451

DrizssEn, glycogen, 320

Drost, epithelium of mollusca, 453

DRvENeR, fixation by injection, 25;
osmic sublimate, 50; Ehrlich-
Biondi stain, 185

Dvusoscq, blood, 361; Golgi impreg-
nation, 428

DUBREUIL, connective tissue, 345

Duercx, elastic tissue, 345

Duerven, Actinie, 471

DveEsBERG, mitochondria, 328, 330

Duwnuay, celloidin sections, 111

Du Puxzssis, Nemertians, 464

Dorie, Golgi’s impregnation, 427

Duvat, collodion imbedding, 102,
103; silver impregnation, 215,
217; carmine and anilin blue,
230; embryology of birds, 295,
297; hardening brain, 380; pur-
purin, 229

Dyes, basic, acid, and neutral, 130;
vegetal, 229

E.

Ear, inner, 445

Eau de Javelle, 278, 285; Eau de
Labarraque, 278, 285

EBNER, von, decalcification, 282

Echinodermata, 18, 467—470

Echtgriin, 405

Epina@er, liquid of Erlicki, 43,
bleaching, 33

Epineton, blood, 362

Eggs, of fowl, 295 e¢ seg.; and see
Embryological methods.

EHLERS, fixative, 35

ExRENBAuUM, section grinding, 116

Exruicu, acid hematoxylin, 164;
classification of dyes, 132; tri-
acid mixture, 187 ; acidophilous
mixture, 193 ; mixture C (eosino-
philous), 193 ; indulin-aurantia-
eosin, 193; neutral red, 191;
methylen blue, 200, 203; Mast-
zellen, 350; plasma cells, 350;
quinolein blue, 195; eosino-
philous cells, 193; blood films,
359

Exruicu and Lazarus, glycoyen,
320; neutral dyes, 131

Exaruicu-BionpI-HEIDENHAIN stain,
184, 344

EurmMann and JapAssoun, plasma
fibrils, 332

Eiceuer, labyrinth, 447

Ermer and Amenp, address, 11

496

INDEX.

The numbers refer to the pages.

ErsaTH, neuroglia, 441

Eisen, iridium chloride, 52 ; osmium
chloride, 52; Ehrlich - Biondi
stain, 186; ruthenium red, 227;
Brazilin, 228; gum Thus, 248

EIsenBeERG, fat, 353

Ersia, alcohol narcotisation, 15;
Capitellide, 15, 459; chromo-
platinic mixture, 40; macera-
tion, 273

E1smonp, quieting Infusoria, 476

Exman, Brachiopoda, 449

Elasthematein, 348

Elastic tissue, 344, 846—348; of
spleen, 371

Electric organs, 339

Electrification of paraffin, 97

Eleidin, 333

Exuis, flagella, 482

Exscunia, celloidin, 104

Embryological methods, 287 e¢ seq.;
generalities, 287—291; Am-
phibia, 300; Arthropoda, 309—
313; Aves, 295-298; Bryozoa,
806 ; Echinodermata, 469 ; Mam-
malia, 291—295 ; Mollusca, 307—
309 ; Pisces, 303—306 ; Porifera,
475; Reptilia, 299; Tunicata,
306; Vermes, 314—316

Emery, injection, 267

Encephalon, see Neurological me-
thods.

ENGELMANN, isotonic liquids, 237;
epithelium, 453

Enriquss, liver of Mollusca, 450

Entire objects, preparation of, 8

Ewrz, orientation, 87; Protozoa,
477, 478

Kosin, 192; with hemotoxylin, 281 ;
with methyl green, 193

Eosin and methylen blue, 193

Eosinophilous mixture, 193

E petra, ova, 312

Epidermis, 331

Epithelium, 331 ; renal, 372 ; ciliated,
453

Eppinasr, liver, 370

Ergastoplasm, 327, 328

Eruarp, glycogen, 320

ERLANGER, VON, ova of Ascaris, 315

Exuick, liquid of, 43, 378

ERMENGEM, VAN, stain for flagella,
483

Ernst, horny tissues, 334

ERReERA, nigrosin, 181

Erythrosin, 192

Essences, sce Oil, and Clearing.

ETERNOD, paraffin blocks, 89

Ether, for narcotisation, 14, 317 ; for
preserving, 5

Eucain, 16

Euparal, 247

Everarp, Dremoor, and Massart,
hematoxylin and eosin, 232

Ewa tp, capillary siphon, 4; section-
washing apparatus, 4; blood,
360

Examination and preservation me-
dia, watery, 234238; mercurial,
239 ; various, 2389-241; glycerin,
241; jellies, 242; resinous, 244—
248; high refractive liquids,
243, 244

Exner, medullated nerve-fibres, 416

Eycuesuymer, celloidin imbedding,
106, 111, 112

Eyes, of Mollusca, 451, 452; of
Arthropods, 457; of Asteroidea,
468; of Turbellaria, 467

F.

Fasru-DomeEraug, syrup, 238; glu-
cose medium, 240; Protozoa,
477, 478

FarircuHiLp, washing cylinders, 4

Faserstasn, nerve - endings, 335;
hematoxylin nerve-stain, 436;
Golgi’s nerve-stain, 429; silver
nerve-stain, 436

Fano, neuroglia, 441

Farrants, mounting medium, 239

Fast blue, 195

Fast green, 194

Fat, 71, 351 et seq.

Fatty bodies, blackening by osmic
acid, 352

INDEX.

497

The numbers refer to the pages.

Favuret-Fremier, mitochondria, 475

Faussux, ova of Cephalopoda, 307

Fecundation, artificial, 287

Fepenicr, double imbedding, 115

Frist, methylen blue, 207, 208;
spinal cord, 383

Fevizat and Branca, thionin, 180

FrERRert, decalcification, 283

Ferri, elastic tissue, 347

Ferric alum, 157

Ferricyanide of potassium for bleach-
ing, 31

Ferrocyanide of copper, injection,
259

Fettponceau, 352

Fibrin, stains for, 367, 368

Fick, Golgi’s impregnation, 430;
kresyl violet, 197; ova of Axo-
lot], 301; keratohyalin, 333

Freanpt, veuroglia, 441

FIEDLER, Spongilla, 474

Fretp and Martin, paraffin imbed-
ding, 82, 87; double imbedding,
114

Fisssinger, glycogen, 320

Finortri, Marchi’s nerve stain, £15,
myelin, 418; axis-cylinder stain;
385

Fiscus, staining, 137,138; embryos
of duck, 297

Fiscuer, A., Fixirung, Farbung und
Baw des Protoplasmas, 21; theory
of fixation, 21—24; ditto of
staining, 129; hematein stains,
160; methyl green, 170, gly-

cogen, 320; nucleoli, 325;
Spiegelfirbung, 326; bioblasts,
328

Fiscuer, B., injection, 268; fuch-
selin, safranelin, 348

Fiscugr, E., gold method, 220;
nerve and muscle, 338

Fiscuer, H., glycerin jelly, 243

Fiscuer, P. M., soap-imbedding, for
Trematodes, 465

Fiscuuer, fat, 351, 353 :

Fisu, oil of thyme, 72; celloidin
imbedding, 105, 111, 112; decals

cification, 28L; hardening ner-
vous tissuo, 377,381,382; Golgi’s
impregnation, 427; picro-subli-
nate, 50

Fixation, 2, 19, 28 eé seqg.; by injec-
tion, 373; of marine animals,
20; embryos, 288

Fixation images, 2

Fixation precipitates, 21

Fixing agents, action of, 19, 20, 134;
characters of the usual, 22;
choice of, for beginners, 24; the
various, 283—67; cytological,
321; embryological, 288; nerve
centres, 373, 375

Flagella, 481

Flagellata, 481

Fuatau, hardening brain, 377;
Golgi’s sublimate method, 433

Flattening sections, 97

Friecustia, gold method, 222; nerve-
stain, 414; Golgi’s impregnation,
433

FiLemMina, preservation, 5; acetic
acid, 53; chromo-acctic acid, 35;
chromo-aceto osmic, 85; action
of bichromate, 40; picro osmic
acid, 59; 177, 178;
dahlia, 180; connective tissue,
345 ; Orange method, 189; epi-
theliuin, 331; bone, 356; eyes
of Gastropoda, 451, injection of
Acephala, 452; nucleoli, 194

safranin,

Fiescu, chromo-osmic acid, 35;
Weivert’s mnerve-stain, 409;
inner ear, 447; blood, 360;

mounting medium, 244

Fuint, injection, 269

Fiorman, celloidin, 108

Floscularie, 16

Fioyp, ganglia of Periplaneta, 457

Fluorides, 408, 410

FoA, fixing mixture, 51; staining
mixture, 232

Forrtinagr, narcotisation, 15

Fot, narcotisation, 18; treatment of
osmic material, 30, 31; chromo-
aceto-osmic acid, 36 ; nitric acid,

32

498 INDEX.

The numbers refer to the pages.
39; perchloride of iron, 53; | FRENZEL, mercuro-nitric mixture, 48
Freup, maceration, 275

picro-chromic acid, 59; vacuum
Frey, H., artificial serum, 238 ; white

imbedding, 86; serial sections,

125; gold impregnation, 225; injection, 262

iron stain, 227; orseille, 229; | FrimpENTHAL, injection, 263; fixa-
injections, meta gelatin, 264; tive, 57

injections, carmine, 260; injec- | FrrgEDLAENDER, Golgi’s impregna-
tions, Berlin blue, 262; injec- tion, 420; cupric sulphate fixa-
tions, brown and yellow, 262; | tion, 473

maceration, 272; reconstruction
of sections, 291; ova of Ascaris,
315; Tintinnodea, 478 ; absolute
alcohol, 61
Foor and StropeE.t, section cutting,
93
Formaldehyde, 63; as a mordant,
176; for maceration, 272; for
nervous tissue, 376—878 ; for the
Golgi impregnation, 427; and
see Formol.
Formalin, see Formaldehyde.
Formalose, see Formaldehyde.
Formic acid carmine, 145
Formol, generalities, 63; for harden-
ing, 63, 64, 376-878 ; for fixing,
63, 64; mixtures, 65--67; for
hardening celloidin, 108; as an
imbedding mass, 118; as w re-
ducing agent, 64, 225; as a
mordant, 176; for gelatin im-
bedding, 101; for nerve centres,
376; for the Golgi impregna-
tion, 427
“© Formol-Miiller,” 66, 380
Fowl, embryology of, 295—298
Francorrr, vacuum imbedding, 86;
section-stretcher, 94; hematoxy-
lin, 159; ova of Polyclads, 3143
albumen fixative, 122
FRAENKEL, myelin stain, 414; gly-
cogen, 320
Franku, imbedding box, 79; injec-
tion, 258
FrErEBorN, connective tissue, 341;
nerve tissue, 384 ; picronigrosin,
344
Freezing section method, 117, 374
Frenxe1, palladium chloride, 52

FR6uLI1cH, picraminic acid, 188

Fuchselin, 348

Fuchsin, basic, 130, 181; acid, 130,
183

Fuchsin, earbolic, 181; resorein, 348

Fuchsin 8., 183

FunrmMann,imbedding, 86; celloidin,
105

Funcr, honing knives, 91

First, bleaching, 285

Fusanri, cartilage, 358

Fuss, acetone, 63

G.

Gap and Hzymans, polarisation, 417
Gaes, picric alcohol, 58; clearing
mixture, 73; section - stretcher,
94; celloidin sections, 110, 112,
126; albumen fluid, 239; starch
injection, 268; maceration, 272,
274, 275; decalcification, 282 ;
reconstruction, 291; glycogen,
320
GaLEorrt, intra vilam staining, 137 ;
neutral red, 191
GALESESCU, neuroglia, 442
Gallein, 414
Gaur, neuroceration, 405
Gamboge injection, 266
Ganpourt, double imbedding, 115
Garpini, safranin, 178; Alcyonaria,
472
GarDInER, Ova of Polychoerus, 314
Garlic water, 123
GarnizR, ergastoplasm, 327; picro-
formol, 65
GaskuLL, gelatin imbedding, 101
Gastric glands, 370

INDEX.

499

The numbers refer to the pages.

Gastropoda, 17, 449-454; embryo-
logy, 307; eyes, 451

GauDLITz, pyronin, 183

Gauts, fixing liquid, 47; serial sec-
tions, 119

Gaultheria, oil of, 72

GavazzenlI, trichohyalin, 333

Gay-Lussac, table for diluting alco-
hol, 60

Gusere, gold method, 223; corpuscles
of Herbst, 334

GEBHARDT, crystalline, 336

GEDoELST, digestion, 277; medul-
lated nerve, 406

GEHUCHTEN, VAN, acetic alcohol, 55;
sectioning nervous system, 383 ;
Nissl’s stain, 386, 387; Golgi’s
impregnation, 423, 428; nerve-
endings, 447

Gelatin, imbedding, 100; freezing
mass, 118; section fixative, 12],
125; injection masses, 257—264 ;
injection masses, carmine, 259—
261; injection masses, blue, 261,
262; other colours, 262 ; mount-
ing media, 242, 243

Gelatin cement, 250

GEMELLI, rejuvenating mixture, 427 ;
flagella, 482

Gemmation of Ascidians, 306

GEMMILL, imbedding, 86

Gentian blue, 195

Gentian violet, 178

Gxorrroy, gelatin medium, 243

Gephyrea, 461

Guruarpt, embryological methods,
299

Geruacs, J., carmine injection, 261;
gold method, 224

Geruacu, L., glycerin jelly, 100;
embryology of birds, 295 ; nerve-
endings in muscle, 337

Gsrota, formol, 64, 378; silver im-
pregnation, 217; Golgi’s im-
pregnation, 428

GEROULD, Caudina, 467

Giacomo, guanin, 321

Gunma, stain, 365, 480

GIERKE, impregnation, 213, 227;
maceration, 273; anilin, blue-
black, 384; eosin, 232

GIEsBRECHT, Clearing, 69; imbedding
trays, 78; squares, 79; paraffin
imbedding, 83 ; section grinding,
117; Copepods, 454

GIESON,VAN, origanum oil, 72; anilin
oil, 111; picro-Siurefuchsin, 188,
232; Saurefuchsin with haema-
toxylin, 232, 385; formalin for
nerve-tissue, 377

Giauio-Tos, blood, 363

GILBERT, myelin stain, 407

GILSON, mercuro-nitric fixing fluid,
49; zine chloride fixing fluid,
53; acetic alcohol with subli-
mate, 55; nitrate of copper,
57, 67; bleaching bichromate
material, 42,285 ; rapid celloidin
method, 112; mercurial examina-
tion liquid, 239; glycerin jelly,
243; sandarac mounting media,
247

Glands, 368—372

Glass, refractive index, 235

Guaug, fixation of Ascaris, 463

Glochidia, 309

Glucose mounting media, 240

Glue, marine, 251

Glycerin and alcohol mixtures, 241

Glycerin and gum, 239, 240

Glycerin ether, 349

Glycerin injections, 265, 266

Glycerin jellies, for imbedding, 100,
101; for mounting, 242, 243

Glycerin, refractive index, 235, 241;
mounting media,241— 243 ; extra
refractive, 241; method of
mounting in, 241, 249, 250

Glychemalum, 162

Glycogen, 320

Goapsy’s fluids, 239

Goblet-cells, 369

Gorrtz, hardening ova, 303

Gold chloride, impregnations, 218;
commercial salts, 219; pre-im-
pregnation, 219; post-impregna-

500

INDEX.

The numbers refer to the pages.

tion, 219, 224; marine animals,

225; preserving preparations,
225; and see the names of
authors.

Gold size, 249, 251

Gold toning baths, see Toning baths.

Gold, colloidal, 401

GoLDMANN, intra vitam stains, 138

GOLDSCHEIDER and Fuarau, Nissl’s
stain, 387

Gouat, chromate of silver impregna-
tion . introduction, 418—421 ;
slow process, 421—423; rapid
process, 423—425; mixed pro-
cess, 425; variations, 426—431 ;
gold method, 224; fixation by
injection, 374; bichromate and
sublimate method, 431; intra-
cellular net, 408 ; neuroceratin,
404:

Goual, corpuscles of, 340

GoLumAN, inira vitam stains, 138

GoxLopErz, fat, 352

GoLopreTz and Unwna, iron stain,
227; manganese stain, 227;
cholesterin, 333

Goxovin, hematoxylin, 167

GoLoving, neutral red, 192

Gorvon, stain for flagella, 483

GoronowitTscH, embryology of Sal-
monide, 305

GorHARD, Nissl’s stain, 387

GRABERG, stain, 191

GRAEFE, solvents of paraffin, 81

GraF, chromic mixture, 35; formol,
65; Hirudinea, 460

GRAFF, Von, Turbellaria, 466

GrauHamM, Trichine, 463

GRAM, staining method, 179

GRANDRY, corpuscles, 334

GrRaND-MoursEL and TRIBONDEAU,
pancreas, 370

Granpis and MAaIniny, lime salts,
320

Granule cells, 849—351; and see Leu-
cocytes.

Granules, 327

Grape-sugar imbedding, 115

Graphic or plastic reconstruction,
289; and see Orientation.

Graser, staining methods, 172, 181

Grassi, heematozoa, 479

GRay, inner ear, 447

Greer, methods for the eye, 442

Gregarine, 478, 479

GRENACHER, alum carmine, 142;
borax: carmine, 148; hydrochloric
acid carmine, 149 ; hematoxylin,
163; purpurin, 229; castor oil
for mounting, 248; bleaching
mixture, 285; eyes of molluscs,
451

GREPPIN, Golgi’s impregnation, 430

GRIEB, alum-carmine, 143

GRiESBACH, Congo red, 191; benzo-
purpurin, 191; Bengal rose, 192
iodine green, 194; elastic tissue,
347 ; blood, 360

Grinding sections, 115

GROnross, ova of Salamandra, 302

GrosELJ, Actinida, nervous system,
471

GROSSER, injection, 267

Groot, Ps, serial sections, 119; iron
carmalum, 146

GrusLer and Honzgorn, address,
11; anilin blue-black, 196; salts
of gold, 220

Griinpulver, 170

Grinstein, bladder of frog, 343

Griinstichblau, 195

GRYNFELTT and Mzsrrezat, bleach-

ing, 285
Guanin, detection of, 321
GuDDEN, Pal’s nerve-stain, 411;

Golgi’s, 429
GUDERNATSCH, serial sections, 121
Gupe@Er, embryological methods, 304
GusGUEN, methyl salicylate, 72
Guernsey blue, 195
Guityssg, double stain, 194; goblet
cells, 369
GUIGNET, injection, 262
Gutick, ova of Heterakis, 316
Guan, serial sections, 119, 124;
blood, 359, 361, 362; formol, 65

INDEX.

501

The numbers refer to the pages.

Gum, imbedding, 115; for freezing,
117, 118; mounting media, 239,
240; injection mass, 267; muci-
lage, 118; for labels, 484

Gum damar, 235, 246

Gum sandarac for mounting, 243

Gum Thus, 248

GuRWITSsCH, hematoxylin, 158

Guyer, ova of Amphibia, 300

Gymnotus, 340 '

H. i

Hani, Hydroidea, 473

Hagcxer, micro-chemistry of the’
cell, 321; Cladocera, 313

Hemacalcium, 165

Hemalum, 161, 162, 165

Hemalum and indigo-carmine, 230

Hemastrontium, 165

Hemateate of ammonia, 154

Hematein, generalities, 152—154;
formule for stains, 155 et seq.;:
and see Hematoxylin.

Hematoxylin, generalities, 152; al-
coholic stock solution, 153;
Unna’s solution, 162, 164;
characters of alum-hematoxylin
stains, 159; formule for stains,
155 et seqg.; the iron compounds, ;
155—159 ; other compounds, 159’
et seq.

Hematoxylin and eosin, 231; and
picro - Siurefuchsin, 232; and.
safranin, 232; and Siurefuchsin,.
232, 385; combination stains,,
231 i

Hamatozoa, 479 ;

HaewseEL, liquid of, 59 i

Haun, cooling paraffin, 88

Hair, 333 '

Maute and Borwy, celloidin imbed-
ding, 106 :

Hatter, Bea, maceration, 275

Hamann, Acanthocephali, 462; As-
teroidea, 468 ; Chilopoda, 455

HamBuRGER, ova of Argyroneta, 313 ;
salt solution, 237; injection, 267

Hamittron, freezing method, 117;
hardening brain, 380

i

HANDWERCK, osmicated fat, 352

Hansen, hematoxylin, 159, 163, 166 ;
picro - siiurefuchsin, 188; iron
cochineal, 146 ; eosin, 192

HAwrscu, glycerin liquid, 242

Hardening, generalities, 26; practice
of, 27; nerve-centres, 374

Hardening agents, see Fixing agents.

Harpy, Rotatoria, 461

HAr, mucin, 368

Harmer, silver impregnation, 218

Harris, hematoxylin, 348; toluidin
blue, 163, 180, 210; methylen
blue, 209; thionin, 210; nerve-
stain, 414; eucain, 16; elasthe-
matein, 348

Hartine, calcium chloride, 238;
white injection, 262; gamboge
injection, 266

Harvey, artificial parthenogenesis,
287

HasweE Lt, dehydration apparatus, 3;
embryology of Cestodes, 314

HatscuHex, Amphioxus, 305

Haver, Distoma, 466; Metridiwm,
471

Hause, decalcification, 280, 282, 283 ;
nerve-stain, 414

Hayem, blood, 361

Heat, for killing, 12

Hecxkert, ova of Trematodes, 315

HeIpEcKE, paraffin imbedding, 85

Heipennain, M.,sublimate solution,
47; paraffin imbedding, 82, 98;
serial sections, 119, 123; iron-
hematoxylin, 157, 326; Ehrlich-
Biondi stain, 184, 185, 186;
Bordeaux R., 326; vanadium
hematoxylin, 167 ; Cerulein $.,
194; Blauschwarz, Brillant-
schwarz, 191, 196; “Subtriessig,”
56; glycerin jelly, 243; salicylic
acid, 56; neutral dyes, 132;
Congo Corinth, 191; Benzo
purpurin, 191.

Herpernuain, R., chrome hematoxy-
lin, 166; Ehrlich-Biondi stain,
184

502

INDEX.

The numbers refer to the pages.

Hutwer, paraffin sections, 96

Heinz, phosphorus, 320

Heinxe and ExRensavum, pelagic
ova, 305

Heinricu, connective tissue, 346

HELD, sublimate solution, 48; stain
for nerve-cells, 388 ; iron hema-
toxylin, 159; formol mixture,
67; section cutting, 93

Helix, 16, 307, 424, 450

Heuer and Gumpertz, medullated
nerve, 416

Hatty, fixing mixture, 51; serial
sections, 121

Hencumay, ova of Limaw, 308

Henxine, section cutting, 96; em-
bryology of Arthropoda, 309,
310, 312; examination liquid,
318

HENNEGDY, overstains, 142 ; section-
fixing, 120, 123; alum-carmine,
143; permanganate method, 176;
embryological methods, 291—-
294, 296, 297, 800, 303, 307;
Protozoa, 476; re-staining old
mounts, 7

Hewnines, Chilopoda, 455; eyes of
Arthropods, 457

Hénocgqus, gold method, 222

Herest, corpuscles, 334, 392; Crus-
tacea, 456

Herwa, stain for ova, 172

Hermann, platino-aceto-osmic acid,
38, 322; safranin and gentian
stain, 179 ; osmic acid and pyro-
ligneous acid stain, 226; cyto-
logical methods, 326; papille
foliate, 335

Herrick, ova of Astacus, 313

Herrwie, silver impregnation, 216,
218; maceration, 273; Meduse
and Actinie, 273, 473; ova of
Triton, 301; ova of Rana, 302

HERXHEIMER, plasma fibrils, 332;
Kresylviolet, 197 ; fat, 352, 353 ;
elastic tissne, 347

Herzoa, retina, 444

Hescu1, amyloid degeneration, 171

Hzssz, Heteropoda, 452 ;
Pecten, 452

Hessert, flagella, 483

Heterakis, ova, 316

Heteropoda, eyes, 451

Hunvurcx, vAN, mounting medium,
244

Heryprnreicu, amber varnish, 253

Heyper, embryos of Arion, 308

Hermans, Cephalopoda, 450

Hzyrmons, embryology of Blattida,
311

Hickson, Brazilin, 228, 229; eosin
and hematoxylin, 231; macera-
tion, 276; eyes of Musca, 457

Hitt, A., nerve-stain, 413; Golgi’s
impregnation, 425, 428

Hit, E. C., embryological method,
291

Hinpie, Hematozoa, 480

Hipret, retina, 444

Hirora, egg of fowl, 298

Hirudinea, 16, 206, 397, 460

HirscHFeELDER, Rotifers, 461, 462

HrrscHuEr, ova of Donacia, 311

His, nitric acid fixation, 39 ; impreg-
nation, 213; embryos of Sela-
chia, 305

Hocusterter, injection, 268

Hoku, bichromate and osmic acid,
38; digestion, 277

Horsr, hydroxylamin, 16

Horrmann, E. H., blastoderm of
birds, 297

Horrmann, F. W., vacuum imbed-
ding, 85

Horrmann, R. W., stain for chitin,
456; orientation, 87

Hormany, K., Cercarie, 466

Hormann, M., injection, 267

Hormann’s Grin, 194

Hoaean, histological rings, 214;
iron stain, 227; silver nitrate,

eye of

216
Hott, imbedding, 81
Houianpe, serial sections, 123

fixation of Tracheata, 455
Houmess, ova of Planorbis, 308

INDEX.

503

The numbers refer to the pages.

Houmersn, fixatives, 56

Holothurioidea, 18, 467

Homarus, embryology, 3138

Honing knives, 91

Horewe.u-Smiru, odontoblasts, 355

Hopkins, maceration, 274

Horn, 333

Hornowsk1, connective tissue, 346

Hoskins, embryological method, 297

Hovussr, myelin stain, 407

Hoyer, silver impregnation, 216;
gold impregnation, 222 ; mount-
ing medium, 240; carmine-
gelatin injection, 260; blue gela-
tin injection, 262; yellow gela-
tin injection, 262 ; green gelatin
injection, 262; shellac injection,
268; oil-colour injection, 268;
mucin, 368 ; fixing mixture, 51

Hover, jun., formol, 64; imbedding,
80

Hrouicka, hardening brain, 378

Huser, Golgi’s impregnation, 420;
injection, 269

Hupson, Rotifers, 461

Hyatt, shellac imbedding, 115

Hydra, 16, 202, 472, 473

Hydrate of chloral, see Chloral.

Hydrochloric acid, for decalcifica-
tion, 280, 282; for maceration, 275

Hydrochloric acid alcohol, 62

Hydrochloric acid carmine, 149

Hydrofluoric acid, 283, 284

Hydrogen peroxide, for narcotisation,
18; for bleaching, 30, 33, 285

Hydroidea, 18, 472

Hydroxylamin, narcotisation, 16

Hymenoptera, 311

Hypertonic liquids, 236

Hypochlorite of potash, 278, 285

Hypochlorite of soda, 278, 285

Hyposulphite fixing baths, 217, 393,
398, 400

Hypotonic liquids, 236

L.
Ipz, double imbedding, 114; epithe-
lium, 331

Iaacuscul, liver, 371

I1ima, embryology of Planaria, 314

Ikxxpa, section-fixing, 123

IuBeEre, Nissl’s stain, 388

Imbedding, defined, 6; small objects,
79, 289, 478

Imbedding methods, 75; manipula-
tions, 76 e¢ seq.; trays, thimbles,
76; boxes, 78; trough, 79; in
vacuo, 85; paraffin, 81 et seq.;
soap, 100, 465; gelatin, 100;
celloidin (collodion), 102

Immobilisation of small organisms,
461, 476

Impregnation defined, 212; positive
and negative, 212; primary and
secondary, 212

Impregnation methods, 212; silver,
214—218 ; gold, 218 — 225 ; other
metals, 226, 227

Indamin blue, 385

Index of visibility, 234, 245

Indian ink injection, 267, 458

Indices of refraction, 234, 235

Indifferent liquids, 235 et seq.

Indigen, 195

Indigo, 229

Indigo carmine, 229; for injection,
266

Indigo substitute, 195

Indophenol, 353

Indulin, 195

Indulin-aurantia-eosin, 193

Infusoria, 475 et seq.

Injections, 257; gelatin, 257—264;
other masses, 264—268 ; natural,
269, 459, 460; of Mollusca, 452 ;
of Arthropods, 458; of Hiru-
dinea, 460

Inner ear, 445

InsaBaTOo, connective tissue, 346

Insects, see Arthropoda.

Instruments, 9

Intercellular bridges and canals, 331

Intestine, 370

Intracellular network of Golgi, 403,
404:

Intravitam staining, 136, 201,202, 2038

DOA

INDEX.

The numbers refer to the pages.

Inversion stains, 135

Iodate of sodium, 32

Iodide of potassium, 46, 53, 271

Todine, for removing sublimate, 46 ;
for fixing, 53; for hardening,
379; Luaot’s solution, 53

Todine green, 194

Todised serum, 237, 238; maceration
vin, 271

Tridium chloride, 52

Tris, 342

Tron alum, fixative, 53;
157

Tron, ammonio-sulphate, 157

Iron, detection of, 320

Iron, impregnations, 227

Iron perchloride, see Perchloride.

Iron, pyrogallate, 227

Tron-Brazilin, 229

Tron-carmine, 145, 146

Tron-cochineal, 146

Tron-hematoxylin, 155 —159, 407

Tsotonic liquids, 235 et seq.

IsraeL, Ehrlich-Biondi stain, 186;

mordant,

acidophilous mixture, 193;
orcein, 229
Iwanzorr, electric organs, 340;
nematocysts, 470; Holothurids,
468
J.

Jacxson, clearing, 69

Jacoss, freezing mass, 118

Jacopy, bleu de Lyon, 195

JADERHOLM, neurofibrils, 402

JAENICHEN, Planaria, 467

JAazER, glycerin liquid, 242

JaximovircH, silver impregnation,
217

JanpeEr, bleaching, 286

JANssENS, iron hematoxylin, 159,
bleu carmine, 196

Janus green, 194

Japanese section-fixing method, 123

Jaquet, leeches, 460;
459

JAVELLE, ev de, 278, 285

JELGEKSsMA, anilin blue-black, 384

Lumbricus,

JELINSE, picric acid, 57 ; “ Stabilit,”’
110

Jellies, see Glycerin.

Jenner, blood, 364, 366

Jennines, Rotatoria, 314

Jensen, Infusoria, 476

Jorst, Annelids, 458

Jouneg, freezing, 117

Jounson, Lindsay, fixing mixture,
38, 54; cement for collodion
blocks, 109; sunning metallic
solutions, 213; gold impregna-
tion, 223; retina, 443

Jounston, reconstruction, 291;
paraffin mass, 99 ; picronigrosin,
384:

Jounstons-Lavis, section grinding,
116

Jourier, gum imbedding, 115

Jonescu, brain of bees, 457

Jorpan, clearing agents, 69, 112;
imbedding box, 79; imbedding,
114; orienting, 87; cutting, 96;
serial sections, 125

Joris, neurofibrils, 401

JosmPH, silver impregnation, 213 ; in-
jection, 267; eleidin, 333

Jousin, Nemertians, 464

JULIEN, flagella, 483

JULIUSBURGER, stain for nerve-cells,
388

Juna, R., microtomes, etc, address,
10; knife-holders, 93; freezing,
117

K.

Kapyi, soap imbedding, 100; nerve-
stain, 384; hardening brain, 378

Kass, nerve-stain, 413

Kaisur, sublimate solution, 45; gly-
cerin jelly, 100 ; Bismarck brown,
181; nerve-stains, 384, 412;
Acanthocephali, 463

Kaus, stain for Spirochexte, 483

Kautivs, Golgi’s impregnation, 426,
428, 431; cartilage, 357

Kaptan, neuroceratin, 405; axis
cylinder stain, 437 ; myelin stain,
418

INDEX. 505

The numbers refer to the pages.

Kappgrrs, cooling paraffin, 88 ; elder-
berry stain, 385

Kaprrrs and Ketsen, nerve-tissue,
384

Karawaiew, Protozoa,
Anobium, 312

KastscHEnKo, reconstruction, 2913
embryos of Raja, 305

Karo, glycogen, 320; silver stain,
397

Karz, inner ear, 446

KeiBex, embryology of Sus, 294

Kemp, blood-platelets, 367

KENDALL, flagella, 482

Kent, fixative, 53

Kenyon, phospho-molybdic hema-
toxylin, 167; Pauropoda, 455;
brain of bees, 457

Keratohyalin, 333

Kernschwarz, 228

Kergz, orientation, 86; serial sections,
128; reconstruction, 290

Kerscuner, gold method, 221

Kidney, 372

Killing, generalities, 12; various pro-
cesses, 12—18

Kina, ova of Bufo, 302; hardening
brain, 381

Kinessury, fat, 358

Kinas.ey, ova of Limulus, 313

Kiongka, ege of fowl, 298

Kusur, inner ear, 446

KINsHINOUYE, embryology of Ara-
neida, 312; of Limulus, 313

478, 479;

Kissuine, melting-points of paraffin, '

99

Kizer, blood, 361

Kuzss, glycerin jelly, 100

Kern, chromic acid, 34; cornea, 335 ;
intestine, 44

KLEINENBERG, picro-sulphuric acid,
58; hematoxylin, 164

KiIn@mMuULLER and VEIEL, sublamin,
51

Knife position, 89—93

Knife-holders, 92

Knives, microtome, 90; honing, 91

Knowen, orientation, 87

Koos, von, section method, 115

Kocxg1, fibrin, 368

Kopis, molybdic hematoxylin, 167 ;
myelin stain, 407; hardening
nerve-tissue, 381

Kogrner and Fiscuer, flagella, 482

Koroip, embryology of Gastropoda,
308

Ko@angl, iris, 342

Kouter, Teenie, 465

Koay, fixing mixture, 51

KO.uiKer, ova of rabbit, 292, 294;
bone, 356

KouimM ann, fixing ova, 304

Koumsr, epiderm of Lumbricus, 392;
fixation, 25; retina, 444

Koumer and Wotrr, imbedding, 86

Koxossow, osmic mixtures, 32; gold
method, 223; osmic acid stain,
227; epithelium, 331

Kouster, gastric glands, 370; dehy-
dration, 4; imbedding, 86; mito-
chondria, 330

K6niasTeIn, maceration, 275

Korscu, embryology of Salmonide,
304; Golgi impregnation, 427;
Golgi network, 404; eyes of Ce-
phalopods, 452 ; blood-plates, 367

Korotnerr, narcotisation, 14

KorscuEtt, embryology of Loligo,
307; Protozoa, 477

KossinskI, stain, 195

KosTaNEckI and SIEDLECKI, ova of
Ascaris, 316 ; sublimate mixture
49

Kosranecki and WiERzEJSKI, ova of
Physa, 309

KorLaREwsky, ganglion cells, 381

KowaLewsky, fishes, embryology,804

Kowa.LskI, neurofibrils, 397

Kozowsky, myelin stain, 412

KrassuskaJA, injection, 269

Krauss, R, Ehrlich-Biondi stain,
184; freezing, 117; injection,
261; thiophen green, 194; liver,
371; retina, 445 ; salivary glands,
869; corpuscles of, 335 ; section
cutting, 93

506

INDIHX.

The numbers refer to the pages.

Krauss, silver impregnation, 217

Kreasote, refractive index, 235; for
clearing, 73, 111

KRecker, chloreton, 15

Kresofuchsin, 181

Kresy] violet, 197, 333

Krizespaum, washing apparatus, 4

Kroau, Mentz von, stain for nerve-
cells, 387

KrountTuat, lead impregnation, 434

Kromayer, plasma fibrils, 332;
fibrin stain, 368

KRONECKER’S serum, 237

Kr6nia, cement, 252

Kruscer,embryology of Harpactida,
313

Krysinsxy, photoxylin, 1038

Kwtunz, H., freezing method, 118

Ktune, W., maceration, 274; diges-
tion, 277

Kuunt, retina, 445

KUKENTHAL, narcotisation, 16, 459;
blood-vessels, 459; intestine of
Lumbricus, 458

Kutt, goblet cells, 369

KuurscHiTzky, preservation, 5; fix-

ing liquids, 48, 44; double
imbedding, 114; tactile cor-
puscles, 334; hematoxylin

nerve-stain, 412; neuroglia, 442 ;
elastic tissue, 871 ; mucus cells,
369; spleen, 371

Kuprrer, axis-cylinder stain, 389;
liver, 371

Kusxkow, digestion, 277

L.
LABARRAQUE, eau de, 278, 285
Labyrinth, 445—447
Lacui, formol for nerve-tissue, 377
Lactate of silver, 216, 429
Lactic acid, 280, 288
Lactophenol, 241
Larront, keratohyalin, 333
Lakes, 134
Lamellibranchiata, 274, 286, 449—
454; embryology, 3809;
452

eyes,

Lampert, ova of Epeira, 312

Lanpo1s, impregnations, 227; ma-
ceration, 272

Lanvo.t, retina, 445

Lang, liquid of, 48; Helix, 450

Lanepon, nervous system of Nereis,
460

Langs, pz, Marchi’s stain, 415

LANGERHANS, mounting medium,
240; tactile corpuscles, 334

Lanxester and Bourne, eyes of
Limulus, 457

LAnsBERG, Protozoa, 477

Larve of Amphibia, 317; of sponges,
475

Lasuert, nerve-stain, 412

Lattice fibres, 371

Laurent, methylen blue and eosin,
198 ; orcein, 229

LAUTERBORN, Protozoa, 79, 478, 481

Laut, violet, 179

Lavpowsxy, formol mixture, 65;
chromo-platinic mixture, 40;
bichromate and sublimate, 44;
methylen blue, 206; chloral
preservative solution, 238; san-
darac for mounting, 248 ; cochlea,
447 ; maceration, 276

LaveraAn, blood, 866; bleu Borrel,
366

Law, teeth, 356

Lawrence, glycerin jelly, 242

Lead acetate, 381

Lead chromate, impregnation, 227

Lead sulphide, impregnation, 227,
434

Lezer, impregnations, 227; retina,
443

Lesrun, ova of Amphibia, 300

Lecithin, 351

Ler, A. B., preservation of material,
5 ; dissections, 8 ; lemon-juice for
fixing, 13; narcotisation, 17,18;
keeping osmic acid, 28; bleach-
ing osmic material, 31; making
up chromoaceto-osmic, 36; nitric
acid for fixing, 39; sublimate
solution, 45; picro acetic acid,

INDEX.

507

The numbers refer to the pages.

58; formol, 64; cedar oil, 5, ‘70,82;
83 ; oil of turpentine, 72,81; paraf-
fin imbedding, 82, 83; paraffin
masses, 98 ; celloidin imbedding,
104, 106, 108, 113 ; Mayer’s albu-
men,122,125 ; serial sections,119 ;
stains recommended, 140, 384;
Ehrlich - Biondi stain, 186;
gentian and orange stain, 190;
neutral red, 192; gold impreg-
nation, 222, 225; intra vitam
staining, 137; iron - carmine,
145 ; iron-hematoxylin, 155, 156;
bleu de Lyon, 195; safranin,
169,177 ; stains for nerve-tissue,
384; toluidin blue, 180; Siure-
fuchsin, 183; osmic acid and
pyrogallol, 226; Kernschwarz,
228; glycerin liquid, 242; desi-
lification, 284; cedar oil for
mounting, 245; paper cell
mounting method, 250; cyto-
logical methods, 318, 321 et seq. ;
Alcyonaria, 472; Hirudinea,
460; Nemertina, 464; hardening
nerve-centres, 377; sponges,
284, 474; neuro-fibrils, 391,
398, 399; myelin stain, 407;
Golgi impregnation, 425 ; retina,
442,444; Pyrosoma, 448; Opis-
thobranchiata, 450

LEEUWEN, VAN, fixation of Hexapods,
455

Lerevre, imbedding trough, 79

LEGAL, picro-alum carmine, 145

LEGENDRE, intracellular network,
404; nervous system of Pul-
monata, 451

Laer, Sporozoa, 479

Lzeros, silver impregnation, 217;
Amphiozus, 305

LrIsHMay, stain for blood, 366

Lemon-juice for fixing, 13

Lemons, oil of, 235

LENDENFELD, Von, section cutting,
93; sponges, 474

Lenpvat, honing knives, 91

LewnuorF stain for nerve-cells, 387 ;

methylen blue impregnation,
436; iron stain for axis cylin-
ders, 436

LENHOSSEK, VON, corulein, 194;
stains for nerve-cells, 388; Golgi
method, 424; eyes of Cephalo-
pods, 452; nerves of Annelids,
460; tongue of rabbit, 335;
picro-sublimate, 50; platino-
sublimate, 52

Lennox, retina, 445

Lens, crystalline, 336

Lenssen, Rotatoria, 314, 462

LzontowirscH, methylen blue, 209

Lepidoptera, embryology, 310

‘Lerxowsry, teeth, 355, 356

Lervucxuanrt, imbedding boxes, 78

Leucobases, 202

Leucocytes, see Blood and Granules.

Lervrert, lime salts, 320

Levapiti, blood, 363;
Spirochete, 483

Levi, nucleoli of nerve-cells, 325;
connective tissue, 346

Levulose for mounting, 241

Lewis, Bzvay, anilin blue-black,
196, 384; hardening brain, 379;
staining ditto, 384

Lewis, M., nerves of

460

Lxrwy, Marchi’s stain, 415

L’yERMITTE and Guccionsz, neuro-
glia, 442

Lichtgriin, 194

LIEBERMANN, carmine, 141

Lizseranz, stain for flagella, 481

Lizszeana, sections of brain, 383;
silver stain, 398

Light, action on alcohol with chromic
material, 33; on metallic salts,
213

Light green, 194

Lituiz, embryology of Unio, 309

Limaz, ova, 308

Lime-water, 272

Lime salts, detection of, 320

Limulus, embryology, 313

Linaloa, oil of, 112

stain for

Annelids,

508

INDEX.

The numbers refer to the pages.

Linpsay JOHNSON, see JOHNSON.

Linseed oil injection, 268

Lintwarew, acetone, 63

LINVILLE, ova of Limaz, 308

Liquid of Miller, of Erlicki, of
Merkel, etc., see the names of
the respective authors.

Liquidambar, 248

Liquor ferri sulphurici oxidati, 156

Lisr, methyl green and eosin, 193;
hematoxylin and eosin, 231;
goblet-cells, 369; Coccida, 456 ;
Actinida, 471; nucleoli, 325;
Mytilide, 450.

Lithium carmine, 147

Lirrie, nematocytes, 470

Liver, 370; of Mollusca, 450

Lo Branco, tobacco narcotisation,
13; alcohol narcotisation, 14;
chloral narcotisation, 15; poi-
soning method, 17; acetic acid,
54; chromo- acetic acid, 35;
chromo-osmic acid, 35; picro-
chromic acid, 59; osmic acid
and bichromate, 37; sublimate
solution, 45 ; chromic sublimate,
50; acid alcohol, 62; chromo-
formol, 67; methods for marine
animals, 448 et seqg.; Aleyonaria,
472; Asteroidea, 468; Brachio-
poda, 449 ; Bryozoa, 449 ; Chaeto-
poda, 459; Crinoidea, 469;
Ctenophora, 474; Echinoidea,
468; Gastropoda, 450; Gephyrea,
461; Holothurioidea, 467; La-
mellibranchs, 449; Mollusca,
449, 450; Nematoda, 463; Ne-

mertina, 464; Ophiuridea, 469 ;°

Protozoa, 478, 479; Siphono-
phora, 473; Trematodes, 465;
Tunicata, 448; Zoantharia, 472
Locks, salt solution, 237
Locy, embryology of Araneida, 312
LogweEntHAL, liquid of Erlicki, 43 ;
carmine, 149
Loewy, epidermis, 331
Lorrusr, stain for flagella, 481
LorsEL, intra vitam staining, 138;
1

neutral red, 191; fat and lecithin,
351, 352

Lonaut, Protozoa, 478

Lonawortu, corpuscles of Krause,
335

Lonneera, Trienophorus, 465

Looss, eau de Labarraque, 278;
Nematodes, 463; Bilharzia, 465,

Lorp, stain for nerve-cells, 387

Loéwir, gold method, 220; blood,
361

Loyez, myelin stain, 407

Luaaro, neurofibrils, 400, 402

Luao1, iodine solution, 53

Luxe, Cestodes, 465

LuItHLEN and Sorao, Nissl's stain,
387

Lumbricus, neurofibrils, 397; epi-
dermis, 424; killing and fixing,
458, 459

Lunpvatt, cartilaginous skeletons,
358

LusreaRrtENn, Victoria blue, 180, 347

Louxrensura, stain for nerve-cells,
388

Lymphatic glands, 371

Lyon, immobilisation of Infusoria,
476

Lysol, for maceration, 276

M.

Maas, carmine and malachite green,
231; digestion, 277; larve of
sponges, 475

Macauium, carmine and indigo-car-
mine, 230; impregnation, 213;
phosphorus, 320; copper, 320;
lime salts, 320; potassium, 321

MacBripg, Amphiura, 470

Maceration, 270 et seq.; of epithe-
lium, 331, 453; of muscle, 342;
of Actinida, 471

Macponaup, impregnation method,
A434

Magdala red, 181

Magenta, 181, 230

Magenta S, 183

INDEX.

509

The numbers refer to the pages,

Maarn1, zinc impregnation, 436

Magnesia-carmine, 147

Magnesium, chloride or sulphate,
narcotisation, 17 ; peroxide, 285

MAuRENTHAL, osmic acid stain, 227

Maier, serial sections, 126

Malachite green, 194, 231

Malapterurus, 340

Malaria-parasites, 479

Matassxz, salt solution, 237

Mattory, eosin and methylen blue,
193 ; anilin blue and orange for
connective tissue, 345 ; phospho-
molybdic hematoxylin, 167;
phospho-tungstic hematoxylin,
167, 348; iron hematoxylin,
159; neuroglia, 440, 441

Mammalia, embryology, 291

Manchester brown, 172

Manrrevi, gold method, 223

Manganese chloride, 227, 237

Many, bleaching, 31; celloidin, 104;
chromo-sublimate, 50; picro-
sublimate, 50, 66; osmio-subli-
mate, 50; fixing nerve-centres,
374, 381; formol mixtures, 66 ;
hematein stain, 164; serial sec-
tions, 123; toluidin blue, 180;
methyl blue and eosin, 196

Marcacct, maceration, 274

Marcano, blood, 361

Marcu, corpuscles of Golgi, 341;
degenerate nerves, 415 ; mucus
of Gastropoda, 460

Manrcuovx, formol mixture, 67

Marcus, formol for spinal cord, 377,
All

MareEscu, connective tissue, 346

Marine animals, precautions in pre-
paring, 26; silver impregnation,
217; gold impregnation, 225

Marine glue, 251

Marx, collodionising sections, 95;
wax cutter, 291

Marx and Long, living ova, 293

Marpmann, gum for labels, 484

Manreu, carmine and indigo-carmine,
230; gelatin cement, 250

Martin, benzo -azurin, 181, 197;
goblet-cells, 369; injection of
trachew, 457

Marrinorrt, C., elastic tissue, 347,
348; Golgi impregnation, 429

Martinorri, G., anilin blue-black,
384; picro-nigrosin, 384; elastic
tissue, 347

Martinorti, L., hematoxylin, 164;
toluidin blue, 211; eosinophilous
cells, 367

Martinorriand Resecortt, safranin,
178

Mason, nervous system of reptiles,
382

Masson, connective tissue, 346

Mastzellen, 349—351

MATSCHINSKY, bone, 355

Matuszewsgl, Marchi’s stain, 415

Mavricr and ScHuuain, bleu de
Lyon, 195, 230

Mawas, retina, 445

Maximow, sublimate and _ bichro-
mate, 51; serial sections, 125;
plasma cells, 351

May and GruEenwaLp, blood, 364

Mayer, A. G., narcotisation, 17

Mayer, P., preparing marine ani-
mals, 26; bleaching osmic ob-
jects, 31; washing out chromic
objects, 33 ; formol, 64; washing
sublimate material, 46; picro-
sulphuric acid, 58; picro-nitric,
58; picro-hydrochloric, 59; acid
alcohol, 62; paraffin imbedding,
78, 80, 88, 98; water-bath, 85,
88; serial sections, water method,
119; albumen method, 122;
section stretcher, 94; theory of
staining, 136; staining with
carmine, 141; staining with
cochineal, 141; carmalum, 144;

chloride carmine

stain, 144; alum-carmine, 143 ;

aluminium

magnesia-carmine, 147; picro-
carmine, 147; picro-magnesia-
carmine, 148 ; paracarmine, 149 ;
hydrochloric acid carmine, 149 ;

510

INDEX,

The numbers refer to the pages.

alcoholic cochineal, 150; theory
of hematoxylin staining, 152,
158; hematein, 154; hemateate
of ammonia, 154; bluing hema-
tein stains, 160; hemalum;
161, 162; glychemalum, 162;
Ehrlich’s hematoxylin, 164;
hemacaleium, 165; methyl
green, 170; iodine green, 194;
triacid mixture, 187; carmine
and indigo-carmine, 230; he-
malum and indigo-carmine, 230;
mucicarmine, 369; muchema-
tein, 369; stain for chitin, 456 ,
decalcification, 281 ; desilicifica-
tion, 283; bleaching, 284, 285;
injection, 264, 266; mucus, 360;
terpinol, 73; orientation, 87,
470; cutting large sections, 95 ;
staining through paraffin, 122;
double imbedding, 115; hema-
strontium, 165; glycogen, 320;
cartilage, 357 ; mounting Plutei,
470

Mayer, P., ANDRES, and GIESBRECHT,
section-stretcher, 94

Mayer, P., and Scuorset, knife-
holders, 93

Mayer, S., neutral red, 192; violet,
196; methylen blue, 205, 207,
210; cornea, 331;
tissue, 344.

MaysgEt, Bismarck brown, 172

McCuourgg, nerve-cells of Pulmonata,
451

McCrorig,night-blue for flagella,483

McFaruanp, fixing method, 374

Medullated nerves, structure,
—406 ; stains for, 407—418

Meduse, 17, 273, 473

MeEuNERT, embryological methods,
299

Metrrowsky, stain for Spirochete, 483

MEISENHEIMER, embryology of
Limaz, 308

MEISSNER, corpuscles of, 335 ; cooling
parafiin, 88

Munpex and Braptey, zinc, 320

connective

404,

Menrctikr, nerve-stain, 414

Mercuric mixtures, 47 ef seq., 239;
and see Sublimate.

Mercury, bichloride, see Sublimate ;
biniodide, 243; cyanide, 381

Merk, elastic tissue, 347

Merxe., chromo-platinic mixture,
40; carmine and indigo-carmine,
230

Merxer and Krauss, molybdenum
impregnation, 227

MerxeEt and ScHIEFFERDECKER, cel-
loidin imbedding, 102

Merton, eyes of Mollusca, 452

MeRzBACHER, neuroglia, 442

Metachromasy, 135

Metagelatin, 264

Metallic salts, action of light on, 213

Metallic stains, 212

Metcatr, embryology of Chiton, 309

Methyl alcohol, for narcotisation, 15 ;
refractive index, 235

Methyl] blue, 196

Methyl green, 170

Methyl green and eosin, 193

Methyl] mixture, 275

Methyl] salicylate, 72

Methyl violet, 172,196; test for, 170;
progressive stain, 172; regres-
sive, 181

Methy] violet B, 196

Methylal, for dehydration, 4, 208

Methylanilin green, 170

Methylanilin violet, 172

Methylenazur, 199, 200

Methylen blue, chemistry of, 199;
uses of, 200; for intra vitam
staining, 201; for central nervous
system, 418, 435; for impregna-
tion, 203—210 ; generalities, 199 ;
staining nervous tissue, 203—
210,386—388, 414, 418, 435; stain-
ing by injection or immersion,
204 ; diffusion processes, 435 ; the
solutions employed, 204; pre-
servation of the preparations,
207—209 ; impregnation of epi-
thelia, ete, 210

INDEX.

511

The numbers refer to the pages.

Methylen blue, polychrome, 199

Methylen blu2 and eosin, 193

Methylen blue and erythrosin, 383

Methylen red, 199

Merzner, toluidin blus, 181

Mevus, liquid of Flemming, 35, 36;
imbedding capsules, 80; mito-
chondria, 328, 330; neuroglia,
441

Meyer, E, celloidin sections, 112,
115; re-staining, 7

Meyer, P., Weigert’s myelin stain,
411

Merer, Semi, methylen blue for
nerve-centres, 435; Berlin blue
stain, 403

MIsBELLI, elastic tissue, 347

MicHaAguis, ova of Triton, 301;
Janus green, 194; methylen-
azur, 199; Scharlach R, for fat,

f
|
at
|
\

M6tueEr, picro-Siurefuchsin,

Mo6sius, maceration, 274

Mozrner, cartilage, 357

Moutscuott, maceration, 272

Moxescuorr and Piso Bormes,
maceration, 271

189;

formol mixture, 66

Mott, cartilage, 357

352; stain for blood, 363; poly- |

chrome methylen blue, 200
Micita1tow, methylen blue, 203, 206,

209

Micro-chemistry of the cell, 319 ef
seq.

Microtome knives, 89—93; micro- —
tomes, 9

Miaeuta, glycerised serum, 238

Milk, injection mass, 268

MituerR, caoutchouc cement, 249,
251; injection, 262

Mincuin, sponges, 474; hematozoa,
480

MinERVINI, elastic tissue, 348

Minaazzini, sublimate mixture, 56

Minnicu, Pal’s hematoxylin, 411

Minot, microtome, 10; celloidin sec-
tions, 111; epidermis, 331

Miracidia, 315

Mitochondria, 328, 475

MirropHanow, double imbedding,
115; Wasserblau, 196; nerve-
stain, 413; blastoderm of birds,
297 ; epidermis, 331; organs of
sixth sense, 335

Mirsuxuri, embryology of tortoise,
299

Mo.tuison, fat, 353

Mollusca, 449 et seg.; embryology,
307 — 309

Molluscoida, 449

Molybdate of ammonium, impregna-
tion, 227, 401

| Molybdenum stains for neurofibrils,

401, 402; toluidin blue, 401;
thionin, 402

Molybdic hematoxylin, 167

MoncxKesere and Berue, treatment
of osmic material, 31, 285

Monp1no, medullated nerve, 405

Monobromide of naphthalin, 235, 244

Monvranart, neurofibrils, 402

Monrgomery, Nemertina, 464;
Araneida, 312; nucleoli, 325

Monti, A., copper
437

Montr, R. and L., gastric glands,
370

Moort, V. A., freezing method, 118

Mordants, 133 et seq.

Moreavux, formol mixture, 66

Moret and Bassa, hematoxylin,
159

Moret and Doteris, triacid, 187

More@an, embryology of Amphibia,
301, 802; of Ascidians, 306; of
Periplaneta, 309, 811

Morphia, as a vaso-dilator, 258

Morton, flagella, 483

Mosg.ey, shell, 452

Mossz, myelin stain, 417; Nissl
stain, 389

Motor nerve-endings, 387 e¢ seq.

Mounting in fluids, 249, 250

Mounting media, see Examination
and Preservation.

MozEsxko, injections, 258, 264, 453

impregnation,

512

INDEX.

The numbers refer to the pages.

Muchematein, 369

Mucicarmine, 369

Mucicarminic acid, 369

Mucin, 368 —370

Mucus, removal from Gastropoda,
450

Mucus cells, 868—370, 154

Mucus glands, 454

Murr, blood, 361; flagella, 483

MULLENIX, inner ear, 446

Miter, C. F., silver impregnation,
217

Miu .ter, E., salivary glands, 369

Miter, H., solution of, 42; for
maceration, 273

Miutveenr, J., paraffin imbedding, 85

Mier, W., injection, 266

Mutoy, fat, 352

Munson, chloral hydrate, 238

Muscle, smooth, 341; striated, 337 ;
nerve-endings in, 337

Muscle cells, 337

Mosxens, salt solution, 207

Myelin, 404—406

Myelin stains, 407—418

Myers, celloidin, 105; serial sections,
126; Nissl’s stain, 387

Myxosporidia, 479

N.

Nasias, R. DE, nervous system of
Pulmonata, 451; axis cylinder
stain, 436

Naggorre, freezing section method,
374; myelin stain, 407, 413

Nails, 333

NAKANISHI, blood, 363

NANSEN, maceration, 273

Naphtha, for imbedding, 82

Naphthalin, monobromide of, 235,
244

Naphthalin red, 181

Naphthylamin brown, 38+

Narcotisation, 13 e¢ seq.

NatuHusius, von, horn, 333

Natural injections, 269, 459, 460

Nuatey, bone and teeth, 355

NEELSEN and SCHIEFFERDECKER,
clearing agents, 68; cedar oil,
70; organium oil, 71 ; sandal-
wood oil, 72

Negative impregnation, 212

Netis, hardening ganglia, 67, 381

Nematocysts, 470

Nematoda, 278, 463; embryology,
315

Nemertina, 464

Nephelis, 18

Nephridia, of Histriobdella, 460

Nerve-cells, 388 eé seq.

Nerve-endings in muscle and tendon,
337—341; in skin and others,
33 4—336; and see 447 and Methy-
len blue and Neurological
methods

Nerve-fibres, structure, 389—406

Nervenzellenequivalentbild, 376

Nervous centres of Reptiles, Fishes,
and Amphibia, 382; of Gastro-
poda, 451; of Arthropods, 457 ;
of Vermes, 460; of Actinida,

471; and see Neurological
methods

Nervous system, see Neurological
methods

NESTEROFFSEY, gold method, 222

NetrovitcnH, Argulus, 455

Nerrovitcn, fixation of Argulus,
455

NEvuBERGER, decalcification, 282

NxrvuBert, glycogen, 320

Nevrincn, glycogen, 320

Neumayer, knife-wedges, 92; em-
bryos of sheep, 294; reconstruc-
tion, 291 ; celloidin, 105

Neuroceratin, 404

Neuro-fibrils, 389—403

Neuroglia, 438—442

Neurological methods, 373 ; introdue-
tion and general methods, 373—
385; cytological methods, 386—
406; myelin stains, 407—418,
myelin and axis-cylinder ditto,
418 ; axis-cylinder and proto-
plasm ditto, 419—437; retina,

INDEX.

513

The numbers refer to the pages.

inner ear, 442—447 ; neuroglia,
438

Neutral chromate of ammonia, 44

“ Neutral” dyes, 130

Neutral red, 191; for nerve-cells,
388 ; for mucus cells, 360

Neutralisation, 260

Neutrophilous elements, 132

NEvVILLE, injection, 263

New green, 194

Niconas, gelatin imbedding, 101;
osmic mixture, 32; ova of
Anguis, 300

Nicouugz, thionin, 180, 370

Nico.ue and Canracuzins, impreg-
nation, 227

Nicotin for narcotisation, 13

Nizss1n@, fixing liquids, 322

NizTzx1, hematein, 152

Night blue, 483

Nigrosin, as a chromatin stain, 181;
as a plasma stain, 195; fornerve-
tissue, 384

Nixirorow, clearing mixture, 111

Nile blue, 353

Niusson, alizarin stain, 198

Nissi, stain for nerve-cells, 386;
hardening nerve-cells, 376, 379 ;
Aquivalentbild, 376 ; Marchi’s
nerve stain, 415; Congo red
myelin stain, 418

Nissl bodies, 386

Nitrate of copper, 57

Nitrate of silver impregnation, 214—
218; generalities, 214; solu-
tions, 215; reduction, 216; fixa-
tion, 217; marine animals, 217;
injections, 262, 263; vulcanite
rings for, 214; and see Neuro-
logical methods, Connective
tissues, etc.

Nitric acid, for fixing and hardening,
39, 67, 381; for maceration,
274; for corrosion, 278; for
decalcification, 280, 281, 282 ; for
bleaching, 286

Nitrite, of amyl], 258; of silver, 428

Noack, orientation, 89

Nocut, methylen blue and red, 199

Nout, corrosion, 278; spicules, 475

Norpmany, plasma cells, 349

Normal salt solution, 236

Norris and SHAaKESPrARE, carmine
and indigo-carmine, 230

Nowak, water-bath, 97; fixing mix-
ture, 66

Nowik, corpuscles of Herbst, 334

Nuclear stains, 130; with coal-tar
dyes, 169

Nuclei, see Cytological methods.

Nuclein, reactions, 319

Nucleoli, 194, 324

Nvuszavy, serial sections, 121

Nurratu, Coopzr and Rosrnson,

Tracheata, 455
0.
OBERSTEINER, hardening nerve-

centres, 378, 379

OsreEaia, serial sections, 124, 128;
Golgi’s impregnation, 430

Osst, nucleoli, 324

OpzENIvs, maceration, 275

OpizR, Golgi impregnation, 427

OxutmacHER, sublimate alcohol, 48,
381; section fixing, 123 ; mordant-
ing with formalin, 176; safranin
artefacts, 178; picro-Siurefuch-
sin, 188; myelin stain, 418

Oil, of aniseed, 118, 235; bergamot,
71, 81; anilin, 73, 235; cassia,
71, 235; cajeput, 72; cedar, 5, 8,
70, 81, 82, 83, 285; cinnamon, 71,
235; cloves, 8, 70, 81, 235;
lemons, 2385; origanum, 71;
sandle-wood, 72; thyme, 72;
turpentine, 72, 81, 235, 247;
olive, 235; linaloa, 112

Oxagima, fat, 353

Olfactive organs, 335, 447

Olive oil, refraction, 235

Ott, freezing method, 117

Opal blue, 195

Ophiuridea, 468

OppEL, liver and spleen, 370, 371

OpriTz, silver impregnation, 217

33

514

INDEX.

The numbers refer to the pages.

Optical differentiation, 20, 22, 37
Orange G, 184,°187, 189
Orcein, Israel’s method, 229; Unna’s,
345, 847
Orcein-Wasserblau mixture, 334
Orchella, 229
Orientation, in paraffin, 86, 87; in
celloidin, 106; of blocks, 88; of
ova, 297
Origanum oil, 71
Orr, Marchi’s nerve-stain, 415, 417
Orseille, 229
OrtH, “Formol-Miiller,” 66, 380;
lithium carmine, 147
Orthoptera, embryology, 611
Osmic acid, generalities, 28, 31 ; how
to keep, 28 ; regeneration of, 29;
fixation with, 29; after-treat-
ment, 30; characters of the fixa-
tion, 31; blackening of fat, 352 ;
mixtures, 31, 35 et seg.; sub-
limate mixtures, 50; picric
mixtures, 59; stains with pyro-
gallol, pyroligneous acid or tan-
nin, 226; for hardening, 381;
stains for medullated nerve, 416,
417 ; for maceration, 273
Osmic-bichromic mixtures, 37, 38
Osmio-sublimate mixtures, 50
Osmium hematoxylin, 168
Osmium, see Osmic acid.
Osmium-carmine, 466
Osmium chloride, 52
Osmosis, to avoid, 3
Osteraren, ether-water, 15 ; Nemer-
tians, 464; Dendrocelum, 467;
Synapta, 467
Ostracoda, 454
Otocyst of Mysis, 456
Ova, see Embryological methods.
Ovens, 85
Overton, bleaching, 30, 33; fixing
with iodine, 53; fixing Algae, 44
OviaTr and SarGENT, injecting, 258
Oxalic acid, for maceration, 275
Oxychloride of ruthenium, 227
Oxygenated water, for narcotisation,
18; for bleaching, 30, 285

P.

Pacavt, picro-sublimate, 50

Pacin1, preservative liquids, 239

Pat, nerve-stain, 411; Golgi’s subli-
mate method, 433

PALADINO, nerve-stain, 418

Palladium chloride, for fixing, 52;
staining, 227, 418; decalcifying,
280

Palladium chloride, 52, 227, 280,

418

Pancreas, 370

Pancreatin digestion fluid, 276, 277

PaneETH, goblet-cells, 369

Panoptic stain, 363

Pawscu, starch injection, 268

Paper cell mounting method, 250

Paper trays and thimbles, 77, 78

Papille foliate, 335

PapPpENHEIM, plasma cells, 349, 351;
panoptic stain, 363; blood-plates,
367 ; chemistry of staining, 129;
neutral dyes, 182; methyl green
and pyronin, 183, 350; hemo-
poietic tissues, 366; sublimate
fixative, 48

Paracarmine, 149

Paraffin, for preserving material, 5;
solvents of, 81, 82; imbedding
in, 81—87, orienting in, 86; cut-
ting, 88—96; ribbons, 96; coat-
ing blocks of, 97; electrification
of, 97 ; masses recommended, 98 ;
mounting sections, 98 ; ascertain-
ment of melting point, 99; over-
heated, 99; cement, 250, 252

Parafiinum liquidum, 235

PaRAvicinI, neurofibrils, 402

Paris violet, 172

Parker, dehydration, 4; methylen
blue, 208; turpentine cement,
252; bleaching, 286; eyes of
Arthropods, 457

Parker and Ftoyp,
brain, 377

Parma blue, 195

Parolein, 248

Parthenogenesis, 287

formol for

INDEX.

515

The numbers refer to the pages.

PartscuH, cochineal, 148 ; decalcifica-
tion, 282

PassarGe and Krosina,
tissue, 348

PaTEeLLani, hematoxylin, 167

Parton, embryological method, 296
neurofibrils, 400

Patten, orientation in paraffin, 86;
embryology of Blattida, 311;
eyes of Lamellibranchs, 452
maceration of Mullusca, 453

PatTTERSON, ova of Columba, 297

Pauropoda, 455

Paviow, celloidin, 104; Pal’s nerve-
stain, 412

Pedicellarie, 468

Praxpopy, corrosion, 278

Perxrce, labelling slides, 484

PEKELHARING, glycogen, 320

Pelagic ova, 305

Pelletierin, 450

Prensa, reconstruction, 290; mito-
chondria, 330

Pentacrinus, 470

PEpP_LeER, flagella, 482

Pepsin digestion fluids, 276

Perchloride of iron, for fixing, 53 ;
for staining, 227

PEREMESCHKO,larve of Amphibia,318

PrreEnvi, chromo-nitric acid, 39, 300

Pénrxz, pupe of Diptera, 310

Periplaneta, embryology, 309, 311

Permanganate of potash, for bleach-
ing, 80, 285; for mordanting,
176; for maceration, 273

Peroxide of hydrogen, for narcotisa-
tion, 18; for bleaching, 30, 285

Peroxide of magnesium, 285

Peroxide of sodium, 285

PeRRiER, Lumbricus, 458

Peter, orienting, 87 ; iron cochineal,
146; reconstruction, 291

Petroleum-ether, 81, 82

Petrone, medullated nerve, 405

PErRUNKEWITSCH, fixing liquid, 49 ;
ova of Hymenoptera, 611

PFEIFFER VON WELLHEIM, iron-car-
mine, 145

elastic

PFISTER, hardening nerve-centres,
376

PritzER, picro-nigrosin, 195

Prirzner, safranin, 177; Protozoa,
477

Phalangida, embryology, 312

Phénicienne, La, 172

Phenol sublimate, 48

Phenylen brown, 172

Puitippson, epidermis, 331

Phloroglucin, 283

Phloxin, 192

Phospho-molybdic hematoxylin, 167

Phosphoric acid, 280, 283

Phosphorus, detection of, 320

Phospho-tungstic hematoxylin, 167

Photoxylin for imbedding, 102, 103 ;
for injection, 269

Physa, ova, 309

Physiological salt solution, 236

Prangse, formic acid carmine, 145;
methylen blue and eosin, 193;
carmine and picro-nigrosin, 231;
Saurefuchsin, 187

Piazza, ripening hematoxylin, 153

Picrate of ammonia, 130

Picrate of silver, 216

Picric acid, 57; washing out, 57; as
a plasma stain, 187 ; for decalci-
fication, 280, 283

Picric alcohol, 58, 275

Picro-acetic acid, 58

Picro-alum-carmine, 145

Picro-carmine, 147, 148

Picro-chromic acid, 59

Picro-formol, 65

Picro-hydrochloric acid, 59, 283

Picro-indigo-carmine, 230

Picro-magnesia-carmine, 148

Picro-nigrosin, 195, 231, 344, 384

Picro-nitric acid, 58, 283

Picro-nitro-chromic acid, 59

Picro-osmic acid, 59

Picro-platinic mixtures, 59, 66, 289

Picro-Saurefuchsin, 188, 344; with
hematoxylin, 232

Picro-sublimate mixtures, 49

Picro-sulphuric acid, 58

338

516

INDEX.

The numbers refer to the pages.

PictTEtT, examination liquid, 237

Pigment, removal of, 456, 457; and
see Bleaching.

Pigment spots, artificial, 43, 381

Pintner, Teenie, 465; osmic acid,
28

Pisces, embryology, 303

Pissent1, alum-carmine, 143

Pirr1e.p, flagella, 482

Pizon, gemmation of Ascidians, 306

Planaria, 466, 467; embryology, 314

Planorbis, embryology, 308

Plasma cells, 349 —351

Plasma, fibrils, 332

Plasma stains, defined, 130; the
coal-tar, 182; cytological, 325—
330

Plastic reconstruction of sections,
290

Platino-aceto-osmic acid, 38

Platino-sublimate mixtures, 52

Platinum chloride, 51 ; mixtures, 38 ;
40, 52, 289, 322

Puiarner, medullated nerve, 53, 405 ;
Kernschwarz, 228

PiEénic, imbedding, 82

PuieEscuko, methylen blue, 208

Puzssis, Du, see Du Puussis.

Pluteus, 150, 469

Popwyssozx1, fixing mixture, 36;
safranin, 178

Poisoning, 17 e¢ seq.

PouaiLton, iron impregnation, 227

Polarisation, for myelin, 417

Poxicarp, Trypanosomes, 480

Poxicr, hematoxylin, 167

Pouitzer, inner ear, 445

PoLtumorpwinow, stain for nerve-
cells, 388

Polycherus ova, 314

Polychrome toluidin blue, 211

Polychrome mythylen blue, 199

Pouzam, soap imbedding, 100

Porow, neuroglia, 44.2

Porifera, 474, 475

Positive impregnation, 212

Poso, paraffin imbedding, 85, 383

Post-impregnation, 219, 224

Potash, for maceration, 272; for
corrosion, 278; acetate of, see
Acetate; bichromate of, see Bi-
chromate; hypochlorite of, see
Hypochlorite ; permanganate of,
see Permanganate.

Potassium, detection of, 321

Potassium bichromate, see Bichro-
mate.

Potassium ferricyanide, for bleach-
ing, 31

Potassium permanganate, for bleach-
ing, 30, 285; as a mordant, 176;
for maceration, 273

Porrer, myelin stain, 412

Poucuet, bleaching, 285

PranTer, imbedding, 82;
tissue, 347, 348

Pre-impregnation, 219, 220

PreEnant, safranin, 178, 194; cochlea,
446; ergastoplasm, 327; micro-
chemistry, 321; mitochondria,
329

Preservation of material, 4

Preservative media, see Examination
and Preservation.

PReEYER, star-fishes, 14

Prickle-cells, 331

Primerose, 192

PRINGLE, vacuum imbedding, 86

PritcHaRp, chromic acid mixture,
34; reducing liquid, 222; coch-
lea, 447

Progressive staining, 138, 169—173

Protargol, 216

Protozoa, 474—483

PRowAazEK, neutral red, 192

Pruppen, hematoxylin, 163

Prussian blue, impregnation, 227;
injections, 259, 261, 262, 265,
266 ; soluble, 261, 262 ,

Przrsmycxi, Protozoa, 477, 479

Puxcexy, eyes of Phalangida, 457 ;
ova of Atta, 312

Purpurin, 229

Pusatent, silver stain, 397

elastic

- Pyridin, 62

Pyrogallate of iron stain, 227

INDEX.

517

The numbers refer to the pages.

Pyrogallate of osmium stain, 226
Pyroligneous acid, 280]
Pyroligneous acid carmine, 145
Pyroligneous acid hematoxylin, 164
Pyronin, 183, 190, 350

Pyrosin, 192

Pyrosoma, 462

Pyroxylin, 105

Q.
QUERVAIN, DE, fixation of nervous
tissue, 374
Quieting Infusoria, etc., 461, 476
Quinolein blue, 195

R.

Raanvt, blood parasites, 366

Rabbit, embryology, 291—294

Rast, chromo-formic acid, 35 ; picro-
sublimate, 49; platinum-sub-
limate, 52; platinum chloride,
52, 289; paraffin sections, 96,
98 ; cochineal, 144; hematoxylin
and safranin, 232; embryologi-
cal methods, 289, 302, 304;
horny tissues, 338 ; crystalline,
336 ; section fixing, 123

Rasyi-RicxHarp, embryology of Sal-
monide, 304

Racovitza, mucus glands, 454

RarFFrak Le, pelagic ova, 305

Raja, 305, 340

Ramon y Casau, picro-Siurefuchsin,
188 ; picro-indigo - carmine, 230,
844; nerve-endings in muscle,
838; stain for nerve-cells, 388;
Golgi impregnation, 426, 428,
429; methylen blue, 435; retina,
444; connective tissue, 344;
neurofibrils, 390—398; myelin
stain, 416; gold impregnation,
436

Rana, embryology, 302

Ranvpo.upy, chloreton, 15

Ransom, Cestodes, 465

RanvIER, osmic acid, 31; chromic
acid, 34; one-third alcohol, 62;

absolute alcohol, 62; ammonia-
carmine, 146; picro-carmine,
147; quinolein blue, 195; pur-
purin, 229; impregnation, secon-
dary, 213; impregnation, with
silver, 214, 215; with gold and
formic acid, 221; with gold and
lemon-juice, 221; after blacken-
ing of gold, 225; iodised serum,
237; injections, carmine, 259;
injections, Prussian blue, 261,
266; impregnation injections,
263 ; maceration, 271, 278, 275,
decalcification, 282; tactile
corpuscles, 334; cornea, 335,
336; nerve and muscle, 338;
plasma fibrils, 333; corpuscles
of Golgi, 340; bladder of frog,
843; medullated nerve, 416;
electric organs, 339 ; retina, 443,
445 ; areolar tissue, 345 ; eleidin,
333; clasmatocytes, 351; bone,
358 ; cochlea, 446; glands, 371

Rawitz, picro-nitro-chromic acid, 59 ;
picro-nitro-osmic acid, 59;
carmalum, 144; inversion stains,
197; artificial alizarin, 197;
bleaching, 286; mucicarminic
acid, 369; eyes of Lamelli-
branchs, 452; phospho-tungstic
fixative, 39; cochineal, 144;
glychemalum, 163; ccerulein,
194; osmium stain, 226 ; harden-
ing brain, 380; stains for nerve
centres, 385

Reagents, 11

RECKLINGHAUSEN, silver impregna-
tion, 215, 216

Reconstruction, from sections, 289;
and see Orientation.

Reppina, gold impregnations, 225

Reppinatvus, nucleoli, 325

REDENBAUGH, narcotisation, 17

Refraction, indices of, 235; of proto-
plasm, 20, 234

ReEGavupD, silver impregnation ; 216;
hematoxylin stain, 232; mito-
chondria, 328 ; myelin stain, 407

518

INDEX.

The numbers refer to the pages.

Reaaup and Dusrezvit, protargol,
216

Regressive staining, 139; with tar
colours, general directions, 173—
176, 180

Rexum, benzin colophonium, 2463
stains for nerve-cells, 387

REICHENBACH, ova of Astacus, 313

REICHERT, microtome, 10

REINHOLD-GILTAY, microtome, 11

REInKE, gentian and orange stain,
190; lysol, 276; horny tissue,
333

RgJsEK, corrosion, 277

Rejuvenation of Golgi material, 426

Remak, hardening ova, 303

Remounting, 7

Renaut, hematoxylin and eosin,
232; silver staining, 216 ; cornea,
336; mitochondria, 330

Reptilia, embryology, 299; nervous
centres, 382

Reseeorri, staining by substitution,
175; safranin, 178

Resins and balsams, 244

Resorcin, imbedding method, 118

Resorcin fuchsin, 348

Re-staining old mounts, 7

Retina, 19, 442 —4.45

RerrTerErR, embryology of rabbit,
293; bichromate sublimate, 51;
tendon, 340 ; smooth muscle, 342

RETTERER and LEeLizvre, muscle and
tendon, 340

RETTERER and ZELLNER, natural in-
jections, 269

Retzivs, methylen blue, 205, 207

Rezzonico, medullated nerve, 404

Rhabdites, 466

Rhopalea, 17

RuavuMBLER, paraffin imbedding, 79;
methyl green and eosin, 198

Rizzpert, phospho-molybdic hema-
toxylin, 167

Ribbon, section-cutting, 92, 96

RicHarbs, narcotisation, 16

Rieper, fat, 352

RiInGER’s solution, 236

Ringing wet mounts, 249, 250, 252

Rrearr and Pzrrr’s liquid, 56, 318

Ripening of hematoxylin, 152—154

Rirrer, Ascidians, 306

Roser, Aplysia, 450

Rozerrson, imbedding method, 115;
nerve-stains, 414, 417 ; platinum,
impregnation, 434

Rosin, injections, 258, 259, 262;
natural injections, 269

RosrnssI, silver impregnation, 215;
erystalline, 336

RocHon-DuvVIGNEAUD, retina, 443

Ronin, salt solutions, 237

Rozsste and YosHipa, lymphatic
glands, 371

Rouuett, freezing method, 118; cor-
nea, 273, 336

Romanowsky, stain, 364, 480

Roosrvett, pyrogallate of iron,

227

Résgz, bone, 354

Rose B a lVeau, 192

Rose de naphthaline, 181

Rosein, 181

Rosgnstapt, plasma fibrils,
eyes of Decapods, 458

RosEnTHAL, fat, 352

Rosin, “neutral” dyes, 131, 193,
neutral red, 191, 389

Ross, blood, 363

Rossi, nerve stain, 414; blood, 360;
flagella, 482

Rossotimow and Buscu, nerve-stain,
417

Rotatoria, 461,
314

Roth aus Methylenblau, 199

Roruie, Kresofuchsin, 181; stains
for nervous tissue, 385

Roveer, methylen blue, 205; silver
impregnation, 216, 217

Rovusszav, decalcification, 279, 356;

332;

472; embryology,

desilicification, 284; sponges,
474,
Rovussexet, cements, 250, 251;

aqueous mounts, 250; Rotatoria,
461

INDEX,

519

The numbers refer to the pages.

Rusascuxin, neuroglia, 440; serial
sections, 125

Rubin, basic, 181; “ acid,” Rubin §,
183

Rusinstetn, blood, 359

Rurrini, corpuscles of Golgi, 341

Ruprricut, serial sections, 124, 125

Ruprecut, bone, 355

Russo, Ophiothrix, 469

Ruthenium, impregnation, 227; red,
227

Ruzicxa, nerve-cells, 325;
229; nucleoli, 325

Ryver, double imbedding, 114

orcein,

8.

Sazsussow, double imbedding, 114

Sacerporti, Golgi’s impregnation,
426

SaEFFTicen, Echinorhyncus, 462

Saffrosin, 192

Safranelin, 348

Safranin, 176-178 ; for elastic tissue,
347; for bone, 356; for mucus
cells, 8368; for nerves, 418; with
indigo-carmine or nigrosin, 195;
with Wasserblau, 196; with
Lichtgriin or Saure-violett, 194;
with Kernschwarz, 228; with
hematoxylin, 232

Sauul, balsam, 245; hardening nerve-
centres, 376, 378; myelin and
axis-cylinder stains, 418

Sana, neuroceratin, 405

Salamandra, embryology, 302

Salicylic acid, 56

Sauine, ova of Tenebrio, 312; larve
of Tenebrio, 456

Salivary glands, 369

SaLKInD, thymus, 372

Salmonide, embryology, 304

Salpa, 448

Salt solution, 236; for maceration,
271, 272

Salts, metallic, action of light on,
213

Samassa, Golgi’s impregnation, 430 ;
Ctenophora, 474

Samrer, paraffin imbedding, 79, 87

SAncuzz, methylen blue, 206 ; neuro-
fibrils, 397

Sanp, neuro-fibrils, 400; neuroglia,
440 ;

Sandal-wood oil, 72

Sandarac for mounting, 247, 248

Sanpers, Cox’s sublimate impregna-
tion, 433

Sankey, anilin blue-black, 384

Sanzo, apparatus for fixing, 289

Saragnone, intracellular network,
404,

SARCOLEMMA, 337

Sata, fat, 352

SaTTLeR, silver impregnation, 217

Saver, kidney, 372

Saurefuchsin, 130, 188; and orange,
184, 232; and hematoxylin, 232

Saurerubin, 183

Saureviolett, 194

SavILLE Kent, Infusoria, 53

SavINI, picro-Siurefuchsin, 189, 344 ;
elastic tissue, 347; Nissl’s stain,
388

SazErin, antenne, 456

Scaua, Protozoa, 478

ScaRPAaTETTI, axis cylinder- and cell-
stain, 385

ScHAreR, muscle-cells, 337

ScuaFrrer, decalcification, 280, 281,
283 ; reconstruction, 290; retina,
445; bone, 353, 354, 356; dehy-
dration, 4; paraffin blocks, 89;
lime salts, 320; connective
tissue, 344; plasma cells, 351

ScHALLIBAUM, Collodion, 123

ScHANZE, microtome, 9

ScHarsr, reconstruction, 291

Scharlach R., 352

Scuaupinn, Protozoa, 478, 479

ScHEnK, acetate of uranium, 57

ScHEW14kKOrF, Protozoa, 478

ScCHIEFFERDECKER, clearing agents,
68, 71; serial sections, 119; cel-
loidin imbedding, 108; injec-
tions, 268 ; methyl mixture, 275 ;
digestion, 277; retina, 445; car-

520

INDEX.

The numbers refer to the pages.

tilage, 357; muscle, 337 ; micro-
tome knives, 90; Weigert’s
myelin stain, 409

ScHLEMMER, silver bath, 398

Scumaus, anilin blue-black, 384;
nerve-stain, 384

Scumipt, F., embryology of Pul-
monata, 308

Scuamipr, G., methylen blue, 209

Scumoru, bone, 357; serial sections,
122

SCHNEIDER, AIME, injection of Ar-
thropods, 458

ScHNEIDER, ANTON, aceto-carmine, |

145
ScHNEIDER, R., iron, 320
ScHOEBEL, washing apparatus, 4
Scuouz, acetone, 63
ScHONEMANN, serial sections,

reconstruction, 291
ScHONLEIN, Aplysia, 450
ScHREIBER, Golgi’s impregnation,428
Scurippg, decalcification, 282; fixa-

124;

tion, 288; plasma-cells, 351;
blood, 361

ScHROTTER, stains for nervous tissue,
385, 414

ScuvuBere, dahlia, 180; Romanowsky
stain, 366, 480 ; cilia of Infusoria,
475

ScHUBERG and SCHROEDER, macera-
tion, 275

ScuvuLtTz, smooth muscle, 342

Scuurtzs, F. E., palladium chloride,
52, 227; section-stretcher, 94;
dehydration, 4

Scuuttzg, Max, iodised serum, 287 ;
acetate of potash, 380, 288;
retina, 445

Scuutzg, O., ova of Amphibia, 302;
hematoxylin, 166, 168; muscle
and tendon, 340

ScHumacHER, elastic tissue, 348;
spleen, 371

Scutruarer, Infusoria, 476

Scutrz, neurofibrils, 399

ScHWALBE, impregnation, 213; coch-
lea, 445

Scuwarzez, Cercarie, 466

ScHYDLOwskI, paraffin imbedding,
79

Scravo, flagella, 483

Scort, F. H., blood, 362 ; phosphorus,
320

Scort, S., inner ear, 447

Scorr and Oszorn, embryology of
Triton, 301

Scyphistomes, 473

Sealing-wax varnish, 253

Secretion granules, 327

Sections, collodionisation, 95, 383;
cutting paraffin, 93; ribbons of,
96; rolling of, 983—95; flatten-
ing, 97; mounting, 119 e¢ seq.;
reconstruction from, 289

Section-flattening, 97

Section-grinding, 115

Section-stretchers, 94

Section-stretching, 93—95

SEELIGER, Antedon, 470

SEHRWALD, Golgi’s impregnation,
429, 430

SEIDENMANN, methylen blue, 206

SEILER, carmine and indigo-carmine,
229; alcohol balsam, 246; de-
calcification, 283

Selachii, embryology, 305; blood,
237

SELENKA, imbedding apparatus, 79,
88; embryology, 294

SELIGMANN, methods for the eye,
442

Serial section mounting, 119 e¢ seg. ;
paraffin sections, 119 et seq.;
watery sections, 125; celloidin
sections, 125

Serum, iodised, 237, 238 ; maceration
in, 271

SEVEREAND, injection, 268

SHEARER, nephridia, 460

Shell, 452

Shellac, for imbedding, 115, 117;
varnish, 253; injection masses,
268

Sida, ova, 313

“ Siebdosen,” 4

INDEX,

521

The numbers refer to the pages.

Srzpenmann, labyrinth, 447

Sieve-dishes, 4

SILBERMANN and OzorowiTz, glyco-
gen, 320

Silver nitrate, see Nitrate of silver.

Silver, other salts, 216

Siphon, Ewald’s capillary, 4

Siphonophora, 473

Siredon, embryology, 301

Sixth sense, 335

SsoBRine, formol, 64

SJ6vALL, intracellular network, 404

Skin, 331 e¢ seq.

SEKROBANSEY, anilin blue, 196

Slides, cleaning, 121, 484

Sumirnow, tactile corpuscles, 334;
epidermis of Lumbricus, 424

Smiru, G., eyes of Gastropoda, 451

SmitH, HopEewstt, odontoblasts, 355

Smirn, Lorrain, fat, 353

Smitu, S., serial sections, 122

Smooth muscle, 341

SNESSAREW, connective tissue, 346

Soap imbedding, 100, 465

Sosorra, imbedding ova, 289; ova of
mouse, 294; of rabbit, 294; of
Salmonide, 304; of Amphioxus,
305

Soda, for maceration, 272; for corro-
sion, 278; for bleaching, 286;
hypochlorite of, see Hypochlorite.

Soda carmine, 146, 384

Sodium, iodate, 32; chloride, 236;
peroxide, 285

Solferino, 181

SoucER, bleaching, 285; salivary
glands, 369; sarcolemma, 337

Solid green, 194

Souuas, freezing mass, 118

SonNENBRODT, bichromate of calcium
liquid, 44; ovary of fowl, 44

SovuLizR, maceration, 272

Souza, pyridin, 62

Spex, prepared paraffin, 99

Spherozoa, 479

Spicules of sponges, 278, 474

SPIELMEYER, myelin stain, 407

“ Spiegelfarbung,” 326

Spinal cord, see
methods.

Spines of Echinoidea, 468

Spirit-proof cement, 251

Spirit blue, 195

Spleen, 371

Sponges, 278, 474, 475

Sporozoa, 478, 479

Neurological

SrunLer, sublimate, 48, 51, 66;
formol mixture, 66; iron cochi-
neal, 146

SquirE, benzol, toluol, xylol, 73;
origanum oil, 72; bluing hema-
toxylin stains, 160; Kleinen-
berg’s hematoxylin, 165 ; methyl
green, 172; Saurefuchsin, 184;
Ehrlich-Biondi stain, 185; salts
of gold, 219; hematoxylin and

Saurefuchsin, 232; glycerin
jelly, 242; decalcification, 282,
283

SsoBoLew, honing knives, 91

© Stabilit,” 110

StaHL, immobilisation of Infusoria,
476

Staining, in bulk and on the slide, 7;
tubes for, 7; reagents for, 11;
generalities on, 129—142; with
carmine, 141; with hematoxylin,
152; with tar colours, 169;
practice of, 188; old mounts, 7 ;
theories of, 129 ; progressive, 138,
169; regressive, 1389, 173; sub-
stantive and adjective, 1383; by
substitution, 175; intra vitam,
1386, 201, 203

Stains, combined, 228 ef seg.; com-
bined, with carmine, 229; com-
bined, with hematein, 231

Stains, kinds of, 129; specific, 129;

nuclear, 130, 324; plasmatic,
130; inverted, 185; choice of,
140; cytological, .324—330 ;

metallic, 212—227
Srappers, fixation of Sympoda, 455
Starch, injection, 268
STAREE, osmicated fat, 352
StaTKEwitscH, Protozoa, 476

522

INDEX.

The numbers refer to the pages.

Statoblasts, 306

STauFFACHER, embryology of Cyclas,
309

Srein, temporal bone, 279, 446

STEINACH, sieve-dishes, 4

STEMPELL, Sporozoa, 479

Stepanow, celloidin, 114; double
imbedding, 114; freezing me-
thod, 118

SrePuHeEns, stain for flagella, 483

STEPHENSON’S high refractive
medium, 243

SteRLine, double imbedding, 114

StTrRLInG, maceration, 272

STOELZNER, sublimate solution, 48;
lime salts, 320; myelin stain,
407

Sr6uR, eosin, 2381

Storax, see Styrax,

Storcu, Weigert’s neuroglia stain,
440

Stovaine, for narcotisation, 462

Stoves, 85

STRAHUBER, axis cylinder stain, 437

SrrassER, section-stretcher, 94; col-
lodion-paper method, 124, 128;
gelatin method, 125; recon-
struction, 291; imbedding
nervous system, 383

Strecker, hardening and staining,
378

STREETER, myelin stain, 414

STRICHT, VAN DER, bergamot oil and
fatty granules, 71; decalcifica-
tion, 283; ova of Turbellaria,
314

STRICKER, gum imbedding, 115

STROEBE, myelin stain, 418

Strone, fixing nerve-centres, 382 ;
Golgi impregnation, 427 ; myelin
stain, 415, 417; fixing mixture,
67

Srropent, plasma cells, 350

SrupnicxKa, connective tissue, 346

Stylaria, 18

Styrax, 248

Sublamin, 51

Sublimate, corrosive, generalities,

45; fixation with, 45 ef seq.;
acetic solutions, 45; various
mixtures, 47, 48,50, 66; alcoholic,
48; preservative liquids, 239;
hardening, 381

Substantive staining, 133

Substitution in staining, 175

Subtriessig, 56

SucHANNEK. sieve-dishes, 4; berga-
mot oil, 71; anilin oil, 73 ; serial
sections, 119; Venice turpentine,
246

Sudan III, 352

Suurma, chloreton, 15

Sulphate of copper, for fixing, 43,
308, 473; for staining, 227; for
impregnation, 437

Sulphate of chromium, 380

Sulphate of iron, 156

Sulphate of magnesia, narcotisation,
17

Sulphide of carbon, 82, 235

Sulphides for impregnation, 227

Sulphindigotate of soda, 229

Sulphocyanides, 272

Sulphorhodamin, 138

Sulphur, refractive index, 235

Sulphuric acid, for maceration, 275

Sulphurous acid, for bleaching osmie
material, 31; for bichromate
ditto, 42, 285; for fixing, 44;
for decalcification, 282

Sumira, iron, 320

Summers, ether method, 125

Sunning metallic salts, 213

Suscuxin, embryos of birds, 297

SussporF, mucin, 368

Susux1, dehydration, 4

Syrup, mounting media, 238; for
freezing, 117,118

Szt71z, alizarin stain, 198; gold
method, 225 ; mito-chondria, 330

T.
Tachiol, 397
Tactile corpuscles, 33 4
Tenia, 465 ; ova, 314

TAENzER, elastic tissue, 347

INDEX. 523

The numbers refer to the pages.

TaFrant, inner ear, 447

TaGucut, injection, 267

“Taz,” Golgi’s sublimate method,
433

Tanvisr, injection, 264

Tannin, examination medium, 239;
for demonstrating cilia, 477

Tar colours, 169 e¢ seq.

Tardigrada, 458

TARTUFERI, cornea, 336; retina, 444

Teeth, 354—356

Tegumentary organs, 331—336

Tricumann, white injection, 262;
linseed oil injection, 268

Teleostea, embryology, 303

TELJATNIE, myelin stain, 417

TELLYESNICKY, nitric acid, 39; acetic
bichromate, 42 ; formol mixture,
65; toning bath, 392

Telsons, 456

Tendon, 340 ef seq.

Tenebrio, ova, 312; larve, 456

Terpinol, 72 ; index, 235

Test-cells of Ascidians, 306

Tetrachloride of carbon, 81, 82

THANHOFFER, silver nitrate, 217

THATE, microtome, 10

THEOHARI, granules, 323

Theridium, ova, 312

THIERSCH, indigo-carmine, 229 ; car-
mine injection, 261; Prussian
blue injection, 262; green and
yellow injection, 262

Thimbles, paper, 78

Turn, retina, 271

Thionin, 179; for impregnation, 210 ;
for nerve-cells, 388; for mucus-
cells, 368

Thiophen green, 194

Tuoma, microtome, 9; decalcifica-
tion, 281; corrosion, 277; injec-
tion, 260

Tuoms, Ehrlich-Biondi stain, 185

Thread-cells, 470

Thus, gum, 248

Thyme oil, 72

Thymus, 372

Tigroid bodies, 3886—389, 398

Timorrsew, hardening nerve-tissue,
381; methylen blue, 210; liver,
871

Tin hematoxylin, 168

Tintinnodea, 478

TiRMANN, iron, 320

Tiscuatxin, dehydration, 4

Tizzon1, alum-carmine, 143

Tobacco narcotisation, 13, 17, 317

Torson, blood, 362

Tolu balsam, 235; cement, 253

Toluen; see Toluol.

Toluidin blue, chromatin stain, 180;
for impregnation, 210; for nerve-
cells, 388; for neuro-fibrils, 401
for myelin, 414

Toluol, for clearing, 73; for imbed-
ding, 81; for preserving, 5

ToMASELLI, neuro-fibrils, 402

Tongue, of frog, 335 ; of rabbit, 335

Toning baths for silver stains, 392,
398, 397, 398, 399, 431

Tonxorr, bleu de Lyon, 196

TornieR, hematoxylin and subli-
mate, 166

Torpedo, 339

Tortoise, embryology, 299

TouRNEUxX and Herrmann, silver
impregnation, 216

Tower, Cestodes, 465

TozEr, Rotifers, 462

Trachee, 456

Tracheata, 455

TramBusti, Ehrlich-Biondi stain,
186

Trays for imbedding, 77

Trematodes, 465 ; embryology, 315

TRENKMANN, stain for flagella, 483

TREZEBINSEI, ganglion cells, 381

Triacid mixture, 187

Trichine, 463

Trichloracetic acid, 56, 282

Trichlorlactic acid, 56

Trichohyalin, 333

Triton, embryology, 301

Trypanosomes, 480

Trypsin digestion, 276, 277

TsCHERNISCHEFF, colloxylin, 108, 114

524

INDEX.

The numbers refer to the pages.

TscHERNYSCHEW and K aRUSIN, nerve-
stain, 412

Tscuiscu, liquid of Erlicki, 43

Tuxssy, celloidin, 104

Tubes for staining on slide, 7

TULLBERG, narcotisation, 17

Tunicata, 448; embryology,
gemmation, 306

Turbellaria, 466; embryology, 306,
314

Turpentine, cement, 252

Turpentine, oil of, for clearing, 72;
for imbedding, 81; for mount-

306 ;

ing, 247

Turpentine, Venice, 246; cement,
252

Twort, stain for blood-parasites,
480

Uz

UxExKUuLL, asphyxiation, 18

Unverwoop, teeth, 356

Unio, ova and glochidia, 309

Unna, osmic mixture, 32; washing
out chromic objects, 33; ripen-
ing hematoxylin, 152; hema-
toxylin solution, 162, 164; kera-
tohyalin, 333; plasma fibrils,
332; smooth muscle, 341; con-
nective tissue, 344, 345; plasma
cells and Mastzellen, 349, 350;
elastic tissue, 347; fibrin, 368;
mucin, 368; celloidin, 103;
prickle cells, 332; glycerin
ether, 349; oil of Gaultheria,
72; carbol-pyronin, 183; poly-
chrome methylen blue, 199;
digestion, 320; Wasserblau and
orcein, 332, 334, 341, 344; tyro-
sin, 334; orcein for elastic tissue.
347

Unna and GOoLOoDETz, iron stain,
227; keratohyalin, 333

Upson, impregnation methods, 384,
436

Uranium, acetate, 57

Ussow, ova of Cephalopoda, 307

Vv. :

Vacuum imbedding, 85, 86

Vaute, Detia, Amphipoda, 313

Van Barwepen, see BENEDEN; and
for all names with the prefix
van see the patronymics.

Vanadium chloride stain, 435

Vanadium hematoxylin, 167

Varnish, negative, for mounting,
248

Varnishes, 249 et seq.

Vaso-dilators for injections, 257, 453

VassaLz, Weigert’s nerve-stain, 409 ;
Marchi’s nerve stain, 415

VassaLeE and Donaaaio, Golgi’s im-
pregnation, 428

VASTARINI-CRESI, medullated nerve,
417 ; section cutting,'94; glyco-
gen, 320; cartilage, 357

Vesas, nerve-tissue, 384

VEJDOVSEY, Gordius, 463

Venprrovic, Marchi’s nerve stain,
416

Venice turpentine, for mounting,
246; cement, 252

Ventral cord, of Arthropods, 457

VeERATTI, Golgi impregnation, 403

VERHOEFF, elastic tissue, 348

Veretillum, 13

Véridine, 170

Vermes, 458 et seg.; embryology,
314—316

Vert en cristaux, v. lumiére, v.
d’Euséhbe, v. d’alcali, 170

VERWORN, Cristatella, 15

Vesuvin, 172

Viattanes, collodion imbedding,
107; gold method, 221; eyes of
Palinurus, 458

VIALLETON, ova of fowl, 298 3; of
Cephalopods, 307

Victoria blue, 180

Victoria green, 194

VIGNAL, osmic acid, 31

VILLE, carmine injections, 260

Violet B, 196; of Lauth, 179

Vircnow, action of light on chromic
objects, 33

INDEX.

525

The numbers refer to the pages.

Visibility, index of, 234, 245

VIVANTE, bone, 356

Voar and Yune, Annelids, 458;
Gephyrea, 461; Cestodes, 464;
Holothurids, 467

Vou, peroxide of hydrogen, 18;
Rotifers, 461

Vom Ratu, picro-sublimate mix-
tures, 50; osmio-sublimate, 50;
picro - osmic acid, 59; picro-
platinic mixtures, 59

Von Exner, see Epner; and for all
names with the prefix von see
the respective patronymics.

Vorticellide, 16

Vosm4rr, section grinding, 116; re-
construction, 290

Vosmarr and PEKELHARING, sponges,
474

VossELER, Venice turpentine, 246;
wax feet, 270; cement, 252

Ww.

WADDINGTON, narcotisation, 14; fix-
ing Infusoria, 44; demonstrating
cilia, 477

Waite, ova of Homarus, 313

WaLpEYER, decalcification,
cochlea, 447

WatLsem, van, knife warmer, 93;
paraffin, 99; nerve-stain, 414

Warp, asphyxiation, 18; Sipunculus,
461

Warn’s brown cement, 250

Wasupury, embryology of Gastro-
poda, 308

Washing out fixing liquids, 2, 25, 32

WasIELEWSEY, Sporozoa, 479

Wasserblau, 196, 345, 341

WassERMANN, iron, 320

Warast, ova of Cephalopoda, 307

Water, fresh or warm, for killing,
18; refractive index, 235; sea-
water, do., 235; as an examina-
tion medium, 236; method for
serial sections, 119; test for, 62

Water-baths, 85

Water-blue, 196

280 ;

Wax feet, 270

Wess, dextrin freezing mass, 118

Wesker, A., serial sections, 125

Weser, E., Rotatoria, 461 ; Echino-
idea, 468; Siphonophora, 473 ;
Asteroidea, 468; Holothurids,
468

Wesster, naphtha for imbedding,
82

We pt, orchella, 229

WEIDENREICH, eleidin, 333; blood,
360; spleen, 371

WEIGERT, serial sections, 127 ; clear-
ing celloidin, 111; Bismarck
brown, 172; picro-Saurefuchsin,
189, 233 ; varnish, for mounting,
248; hardening nerve centres,
875, 877; stain for nuclear
figures, 159; myelin stains, 408
—414, 415; Golgi’s impregna-
tion, 425; neuroglia stain, 438 ;
elastic tissue, 348; fibrin stain,
367; iron hematoxylin, 159, 233

Wei, section-grinding, 117; teeth,
355

Weiss, P., address, 11

We.cxe, flagella, 483

WELLHEIM, PFEIFFER VON, iron-car-

* mine, 145

WERMEL, blood, 362

WERNER, smooth muscle, 342

WESTER, chitin, 456

WevrsseE, embryology of Sus, 294

Whartonian jelly, 368

WHEELER, embryology of Blattida,
311

Whetting knives, 91

Wuitrte, bone and teeth, 354

White of egg, freezing method, 118;
section fixatives, 122, 123; exa-
mination media, 237, 239; in-
jection mass, 267; refractive
index, 235

Wuitine, spleen, 371

Wuirman, chromo-platinic mixture,
40; ova of Amphibia, 300, 302 ;
pelagic ova, 305; Hirudinea, 460

WICKERSHEIMENR’s fluid, 239

34

526

INDEX.

The numbers refer to the pages.

Wipaxkowicu, embryology of mouse,
295

Wipmany, eyes of Arachnida, 458

WIJsHE, VAN, ammonia-carmine, 146 ;
picro-carmine, 147; cartilagi-
nous skeletons, 358

Witxermt, Triclads, 67, 467 ; double
imbedding, 114; picro-Saure-
fuchsin, 189

Witt, embryological methods, 299

WI.LesRanp, blood, 363

Wimmer, neuroglia, 440

Winrwarter, embryological
thods, 295

WINIWARTER and Sarnmont, orange
method, 189

Witsoyn, Alcyonaria, 472; orienta-
tion, 87; imbedding box, 79

WINTERSTEINER, serial sections, 128

WISTINGHAUSEN, von, hematein
stains, 160

Wirt, DE, elastic tissue, 348

Wirrmaack, myelin stain, 417; inner
ear, 446

Wotrr, Exise, staining bichromate
material, 42; thionin stains,
180; fibrin, 368; elastic tissue,
347 ; orcein, 229

Wotrr, M., freezing, 117

Wo rr, W., bladder of frog, 342

WotrrvmM, elastic tissue, 348

Wollschwartz, 481

Wotters, hematoxylin nerve-stain,
412; vanadium nerve-stain, 435 ;
cartilage and bone, 357

Wooptanp, Holothurids, 468; larve
of Echinoderms, 470

Woopworth, orienting in paraffin,
87; reconstruction, 290

Wriaeut, blood - cells, 867; blood-
plates, 367

WuNDERER, nerve-endings, 338

Wyny, nerve-stain, 412

xX.

Xylol, index of, 235; for clearing
paraffin sections, 73; celloidin
sections, 111; for imbedding, 81,
82, 83; for preserving, 5

me-

Xylol balsam, 235, 245

Ys

Yamaaiwa, neuroglia, 442
Youne, methylene blue, 206
Yvon, test for water, 62

Z.

ZACHARIAS, acetic alcohol, 55; iron-
carmine, 146; Flagellata, 481;
Copepoda; 454

ZALESKI, iron, 320

ZANDER, test for Chitin, 456

ZAWARSIN, cornea, 336

ZENKER, fixing mixture, 50

ZERNECKE, Ligula, 465

ZETTNOW, flagella, 483

ZIEGLER, decalcification, 282

ZIEGWALLNER, glycogen, 320

ZIEHEN, gold and sublimate method,
434

ZIEHL, carbolic fuchsin, 181

ZIELINA, blood, 359

ZIMMER, picro-acetic acid, 58

ZIMMERMANN, A., sieve-dishes, 4;
micro-chemistry, 321; nucleoli,
324

ZIMMERMANN, K. W., Golgi’s impreg-
nation, 431; bone, 355; con-
nective tissue, 346

Zine chloride, for fixing, 53; for
hardening, 381; for impregna-
tion, 436

Zine, detection of, 320

Zoantharia, 472

Zoerar, Rotatoria, 462; Protozoa,
478

Zosa, methylen blue, 202; ova of
Ascaris, 315; Protozoa, 478;
bioblasts, 328

ZSCHIESCHE, larve of Alcyonidium,
449

ZscHOKKE, benzo-purpurin,
cartilage, 357

Zur Srrassen, ova of Ascaris, 315;
Bradynema, 463

Zurn, retina, 444

ZWAARDEMAKER, safranin, 177

191;

ADLARD AND SON, IMPR., LONDON AND DORKING.

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