:
THE MICROTOMIST'S
VADE-MECUM
A HANDBOOK OF THE METHODS OF
MICROSCOPIC ANATOMY
BY
ARTHUR BOLLES LEE, Hon. F.R.M.S.
l\
EIGHTH EDITION
EDITED BY
J. BRONTE GATENBY,
B.A., B.Sc., D.Phil. (Oxon.), D.Sc. (Lond.), F.R.M.S.,
SOMETIME LECTURER IN HISTOLOGY, OXFORD ; LECTURER IN CYTOLOGY AND SENIOR
ASSISTANT IN ZOOLOGY, UNIVERSITY COLLEGE, LONDON, AND SENIOR DEMY,
MAGDALEN COLLEGE, OXFORD.
With the collaboratian of
W. M. BAYLISS, M.A., D.Sc. (Oxon.), F.R.S., F.R.M.S.,
PROFESSOR OF GENERAL PHYSIOLOGY IN UNIVERSITY COLLEGE, LONDON ;
G. DA FANO, M.D., F.R.M.S.,
L.D. ON MORBID ANATOMY, UNIVERSITY OF PA VIA (ITALY) \ LECTURER IN
HISTOLOGY, KING'S COLLEGE, LONDON ;
A. DREW, D.Sc. (Lond.), F.R.M.S.
IMPERIAL CANCER RESEARCH FUND, LONDON ;
W. CRAMER, Ph.D., D.Sc., M.R.G.S., L.R.C.P., F.R.M.S.,
IMPERIAL CANCER RESEAJRCH FUND, LONDON ;
J. THORNTON GARTER, F.R.M.S., F.Z.S.
HON. RESEARCH ASSISTANT, UNIVERSITY COLLEGE, LONDON.
PHILADELPHIA
P. BLAKISTON'S SON & CO,
1012 WALNUT STREET
n
•-
* 0
PREFACE TO EIGHTH EDITION.
IN the preparation of this new edition of Dr. Bolles Lee's well-
known book I have received the assistance of Professor W. M.
Bayliss, Dr. C. Da Fano, Dr. A. Drew, Dr. W.. Cramer, and Mr.
J. Thornton Carter. It has been almost entirely due to these
workers that my plans for the new edition have been able to be
carried out in the way I wished. Thanks to them, this book may
be considered a most complete and stimulating book of reference for
the research worker.
Professor W. M. Bayliss, to whom I am especially grateful, has
rewritten Chapter XI. on " Staining." Dr. C. Da Fano has revised,
and in some cases almost completely rewritten, the difficult chapters
on " Neurological Techniques," which constitute indeed a special
branch of microtomy. Dr. A. Drew has completely rewritten the
valuable section on " Protozoa " ; while from Dr. W. Cramer's pen
has come most of the article on " Fatty Substances," which will
bring to the notice of embryologists and histologists the newest
advances in the micro-chemistry of this subject. Mr. J. Thornton
Carter has revised the section on " Teeth and Bone." I have
personally been responsible for the rest of the book, besides having
written special chapters or sections on " Chromatin, Chromosomes,
Nucleoli, Glycogen, Iron, Yolk, Fat, Mitochondria, Golgi Apparatus
and Benzidine Dyes " ; I have completely rewritten the section on
"Mammalian Embryological Methods." In the sections dealing
with the cytoplasmic inclusions, most of the various structures,
known as "attraction sphere rodlets," "idiozome," " Golgi-Kopsch
apparat," " nebenkern batonettes," etc., are grouped under the
term " Golgi apparatus," as now seems justifiable in view of the
results of modern researches on the subject.
A small, yet very important, addition to the present volume has
been the inclusion of two new methods for staining bacteria in tissue
(§§ 089, 761). The histologist and cytologist are often puzzled to
know whether certain enigmatic bodies they find are or are not
bacterial in nature.
: YJ :•••., ^ A ' < • PREFACE.
An addition which is likely to be welcomed, and to make the
book more useful, is a special chapter for students (Chapter
XXXVII.).
I had especially desired to have a chapter dealing with " Tissue
Culture," and had asked my friend Mr. H. M. Carleton, of Oxford,
to undertake it ; but as illness prevented his carrying out the work, I
was myself obliged at the eleventh hour to write the article. Dr.
A. Drew was most helpful here, and Dr. Strangeways, of Cambridge,
lent me some literature on the subject.
For advice and encouragement in this arduous task I have to
thank my many friends. Dr. Bolles Lee sent me his blessing and
three hundred references ; Professor J. P. Hill placed his note-
books and experience at my disposal ; Dr. J. A. Murray, of the
Cancer Research Fund, proved a perfect mine of suggestions, and I
dug freely at this source. I am very grateful to both Dr. Murray
and his amiable staff, of whom Dr. Drew and Dr. Cramer have
helped me most. Professor Starling, of University College, Pro-
fessor E. S. Goodrich, of Oxford, Professor Sherrington, of Oxford,
Professor Boycott, of University College Medical School, Professor
E. B. Wilson, of Columbia University, and Professor D. M. S.
Watson, of University College, London, helped me in one way or
another. Professor W. M. Bayliss and Dr. Oscar Brady gave me
valuable information on certain chemical aspects. My friends at
the Rothamsted Station, especially Mr. Ward Cutler and Dr. Imrns,
were very helpful. Dr. Allen, of Plymouth, and Dr. Orton kindly
answered inquiries.
Great care has been taken in the preparation of the Index, and I
have to thank my sister for much assistance.
In writing a book of this kind, it is difficult at times to ascertain
exactly who did originate some one method, and if we have occa-
sionally overlooked the original source we will be glad to have such
lapsus calami rectified in some future edition.
Finally, it must be remarked that the arrangement and method
of exposition of the new material closely follows that of Dr. Bolles
Lee, while the bulk of the book is still largely due to him.
J. BRONTE GATENBY.
UNIVERSITY COLLEGE,
LONDON.
CONTENTS.
PART I.
PAGE
CHAPTER I.
INTRODUCTORY . . . 1
CHAPTER II.
KILLING . . . . . . . . . .11
CHAPTER III.
FIXING AND HARDENING . . . . . . .18
CHAPTER IV.
FIXING AND HARDENING AGENTS — MINERAL ACIDS AND THEIR
SALTS 29
CHAPTER V.
FIXING AND HARDENING AGENTS — CHLORIDES, ORGANIC ACIDS,
AND OTHERS ......... 44
CHAPTER VI.
DE-ALCOHOLISATION AND CLEARING AGENTS .... 65
CHAPTER VII.
IMBEDDING METHODS — INTRODUCTION . . . . .71
CHAPTER VIII.
IMBEDDING METHODS : PARAFFIN AND OTHER FUSION MASSES . 76
Paraffin, 76 ; Gelatin, 92.
CHAPTER IX.
COLLODION (CELLOIDIN) AND OTHER IMBEDDING METHODS . 95
Collodion or Celloidin, 95 ; other Cold Masses, 106 ; Grind-
ing Masses, 108 ; Free/ing, 109.
>4nir '•« *•-"* v' CONTENTS.
PAGE
CHAPTER X.
SERIAL SECTION MOUNTING . . . . . . .111
Methods for Paraffin Sections, 111 ; Methods for Watery
Sections, 116 ; Methods for Celloidin Sections, 116.
CHAPTER XT.
STAINING . . . . . . . . . 120
CHAPTER XII.
CARMINE AND COCHINEAL STAINS . . . . . .135
Theory of Carmine Staining, 135 ; Aqueous Carmines, Acid,
136 ; Neutral and Alkaline, 140 ; Alcoholic Carmines and
Cochineals, 141.
CHAPTER XIII.
H^MATEIN (HJSMATOXYLIN) STAINS . . . . .145
Theory of Staining with Hsematoxylin, 145 ; Iron-haematein
Lakes, 147 ; Aluminium -ha3matein Lakes, 151 ; other Haema-
tein Compounds, 155.
CHAPTER XIV.
NUCLEAR STAINS WITH COAL-TAR DYES . . . . .159
Progressive Stains, 159 ; Regressive Stains, 162.
CHAPTER XV.
PLASMA STAINS WITH COAL-TAR DYES . .171
CHAPTER XVI.
METHYLEN BLUE .....
CHAPTER XVII.
METALLIC STAINS (IMPREGNATION METHODS) .
Silver, 198 ; Gold, 202 ; other Metallic Stains, 208.
CHAPTER XVI1L
OTHER STAINS AND COMBINATIONS .
Carmine Combinations, 212 ; Hsematein Combinations, 213.
CHAPTER XIX.
EXAMINATION AND PRESERVATION MEDIA
Aqueous Liquids, 217 ; Mercurial Liquids, 220 ; other Fluids,
220 ; Glycerin Media, 222 ; Glycerin Jellies, 223 ; High
Refractive Liquids, 224 ; Resinous Media, 225.
CONTENTS. V:-v, -..:/.;• V.JT
PAGE
CHAPTER XX.
CEMENTS AND VARNISHES 229
PAKT II.
SPECIAL METHODS AND EXAMPLES.
CHAPTER XXI.
INJECTION — GELATIN MASSES (WARM) . . 232
Carmine, 234 ; Blue, 236 ; other Colours, 237.
CHAPTER XXII.
INJECTIONS — OTHER MASSES (COLD) . . . . . 238
CHAPTER XXIII.
MACERATION, DIGESTION, AND CORROSION .... 243
Maceration, 243 ; Digestion, 248 ; Corrosion, 249.
CHAPTER XXIV.
DECALCIFICATION, DESILICIFICATION, AND BLEACHING . . 251
Decalcincation, 251 ; Desilicification, 255 ; Bleaching, 255.
CHAPTER XXV.
EMBRYOLOGICAL METHODS ....... 258
Mammalia, 263 ; Aves, 27 1 ; Reptilia, 274 ; Amphibia, 275 ;
Pisces, 279 ; Tunicata, 281 ; Bryozoa, 282 ; Mollusca, 282 ;
Arthropoda, 284 ; Vermes, 288.
CHAPTER XXVI.
CYTOLOGICAL METHODS ........ 292
Glycogen, 294 ; Iron, 297 ; Chromosomes, 303 ; Cytoplasmic
Inclusions, 316.
CHAPTER XXVII.
TEGUMENTARY ORGANS ........ 339
CHAPTER XXVIII.
MUSCLE AND TENDON (^ERVE-ENDINGS) ..... 344
Striated Muscle, 344 ; Electric Organs, 345 ; Tendon, 347 ;
Smooth Muscle, 348.
M. b
CONTENTS.
PAGE
CHAPTER XXIX.
CONNECTIVE TISSUES . 350
Connective Tissue, 350 ; Elastic, 352 ; Plasma Cells 354 ;
Fatty Substances, 356 ; Bone and Cartilage, 369 ; Skele-
tons of Embryos, 377.
CHAPTEE XXX.
BLOOD AND GLANDS 379
Blood, 379 ; Benzidine Dyes, .388 ; Glands, 391.
CHAPTER XXXI.
NERVOUS SYSTEM — GENERAL METHODS . . . . 397
CHAPTER XXXII.
NERVOUS SYSTEM — SPECIAL METHODS, CHIEFLY CYTOLOGICAL . 410
Cells, 410 ; Cells and Fibres, 416 ; Golgi's Internal Apparatus,
435 ; Medullary Sheath, 439.
CHAPTER XXXIII.
MYELIN STAINS (WEIGERT AND OTHERS) .... 442
CHAPTER XXXIV.
AXIS-CYLINDER AND DENDRITE STAINS (GOLGI AND OTHERS) . 454
CHAPTER XXXV.
NEUROGLIA AND SENSE ORGANS ...... 479
Neuroglia, 479 ; Retina, 493 ; Inner Ear, 496.
CHAPTER XXXVI.
METHODS FOR INVERTEBRATES . ' . . . . 499
Tunicata, 499 ; Molluscoida, 500 ; Mollusca, 500 ; Arthropoda,
504 ; Vermes, 509 ; Echinodermata, 518 ; Coelenterata, 521 ;
Porifera, 525 ; Protozoa, 526.
CHAPTER XXXVII.
CULTIVATION OF TISSUE " IN VITRO " AND ITS TECHNIQUE . . 550
CHAPTER XXXVIII.
A GUIDE FOR STUDENTS OF MICROTOMY . 556
APPENDIX . . 563
INDEX 564
THE MICROTOMIST'S
VADE-MECUM.
PART I.
CHAPTEK 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 carefully fixing the
structures to be examined, staining them with a nuclear stain,
dehydrating with alcohol, and mounting series of sections of the
structures in balsam. It is by this method that the work is blocked
out and very often finished. Special points are then studied, if
necessary, by Special Methods, such as examination of the living
tissue elements, in situ or in " indifferent " media ; fixation with
special fixing agents ; staining with special stains ; 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, Preliminary 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 prelimi-
nary preparation 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 substituting for it the preservative
liquid or other reagents which it is desired to employ. Ulterior
preparation comprehends the processes sketched out in §§ 3 et seq.
2. Preliminary Preparation. — The first thing to be done with any
structure is to fix its histological elements. (This statement
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
M. 1
INTRODUCTORY.
things are implied by the word " fixing " : first, the rapid killing of
the element, so that it may not have time to change the form it had
during life, but is fixed in death in the attitude it normally had
during life ; and second, the hardening of it to such a degree as may
enable it to resist without further change of form the action of the
reagents with which it may subsequently be treated. Without good
fixation it is impossible to get good stains or good sections, or prepara-
tions good in any way.
The structure, having been duly fixed by one of the processes
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 for this difference is that the first -named
reagents (and, indeed, all the compounds of the heavy metals used for
fixing) cause certain of the elements of the tissues to become less reactive,
and partly or wholly insoluble in water. The combinations of picric
acid and the elements of the tissues, on the other hand, are generally
much more soluble in watery fluids.
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 increasing strength, final dehydration with
absolute alcohol, imbibition with an essential oil or other so-called
clearing agent 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
CHAPTER I. 3
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 satura-
tion, 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 kinds 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, COBB'S differentiator (Proc. Linn. Soc., N.S.W., v, 1890,
p. 157 ; Journ. Roy. Mic. Soc., 1890, p. 821) may be employed. Or, the
apparatus of HASWELL (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.,
1892, p. 892. See also SCHULTZE (Zeit. wiss. Mile., ii, 1885, p. 537) ; and
SUSUKI, ibid., 1909, p. 211 ; KOLSTER (ibid., xvii, 1900, p. 294).
The " Siebdosen," or sieve-dishes of STEINACH, ZIMMERMANN, and
SUCHANNEK (vide Zeit. wiss. Mik., iv, 1887, p. 433, and vii, 1890, p. 158),
are useful for many purposes. See also TISCHATKIN, ibid., xxiii, p. 45.
FAIRCHILD'S perforated porcelain cylinders for washing (ibid., 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. 394).
For EWALD'S section -washing apparatus, see Zeit. Biol., xxxiv, 1897,
p. 264.
That of SCHOEBEL (ibid., xx, 1903, p. 168) is simple and efficient ; as
also that of KREIGBAUM (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 EWALD, ibid., p. 253.
It is sometimes stated that it is necessary that the last alcohol-
bath should consist of absolute alcohol. This, however, is incorrect,
a strength of 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
1—2
4 INTRODUCTORY.
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. Acetone and methylal
have been substituted for alcohol in the dehydration of methylen-
blue preparations (PARKER, Zool. Anz., 403, 1892, p. 376), and
anilin 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 histo-
logical 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 disagreeably 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 sometimes considerably altered by it ; they become over-
hard and shrink, and become brittle, and their capacity for taking
stains well becomes seriously diminished. KULTSCHITZKY (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. FLEMMING (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 recom-
mend 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, I 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 preservation of the tissues is concerned, but of course it is
not so handy for storage.
CHAPTER I. 5
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 clearing agent be a gradual one. This is
effected by placing the clearing medium under the alcohol. A
sufficient quantity of alcohol is placed in a tube (a watch-glass will
do, but tubes are generally better), and then with a pipette a suffi-
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 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 objects 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 last case the clearing may be omitted and the tissues be
imbedded direct from the alcohol). The imbedding medium con-
taining 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
6 INTRODUCTORY.
methods described in the chapter on Serial Section Methods, the
imbedding material is removed from them (in the case of paraffin),
they are stained in situ on the slide, dehydrated with alcohol,
cleared, and mounted in balsam or damar. Or they may be stained,
washed, dehydrated, and cleared in watch-glasses, and afterwards
mounted as desired — the imbedding medium being first removed if
desirable.
Or, the material may be stained in bulk, before cutting the sections.
In this case the object, after having been fixed and washed out, is
taken from the water, or while still on its way through the lower
alcohols (it should not be allowed to proceed to the higher grades of
alcohol before staining, if that can be avoided), and passed through
a bath of stain, 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 desir-
able), 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 is 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 advantage be re-stained 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 preparations, and
have succeeded in every case with series of sections mounted on
Mayer's albumen, or by the water method. For shellac-mounted
series, see E. MEYER, Bid. 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
CHAPTER I. 7
centimetres high and 27 millimetres internal diameter. Each of these
will then take two slides, English size, placed back to back.
7. Resume 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. 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 difiusibility 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.
is done with successive alcohols, water being used only in. the case
of fixation by osmic acid, or the chromic mixtures or other fixing
solutions that render washing by water imperative. Staining is
done by preference with alcoholic staining media. The stains most
to be recommended are Grenacher's borax-carmine, or one of Mayer's
alcoholic carminic acid or haematein stains. Aqueous stains are
more rarely indicated, though there are many cases in which they
are admissible, and 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 dissection,
whilst its viscosity serves to lend support to delicate structures.
Clove oil has a tendency to make tissues that have lain in it for some
8 INTRODUCTORY.
time very brittle. 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 glycerin, it may
be weirto prepare the slide by spreading on it a thin layer of MAYER'S
albumen, and on this place a small drop of glycerin, or other dissect-
ing 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, SCHALLIBAUM'S
collodion may be taken, and the organs fixed in situ on this by
adding xylol.
10. Instruments. — For all that concerns the mechanism and
manipulation of the Microscope, see vol. i of CARPENTER'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, Condensers, pp. 289 to
316, and Tube Length, pp. 158 to 159.
For information concerning the principles of construction and
•manipulation of the Microtome, see also CARPENTER'S The Micro-
scope. Microtomes are instruments for the accurate production of
thin slices of tissues. They are used both for cutting tissues that
have acquired a certain favourable consistency through having been
imbedded 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 knife set square 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
CHAPTER I. 9
material, and vice versa. 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 object are (A) in a horizontal plane,
or (B) 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 arid 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, SCHANZE,
REICHERT, and others). The THOMA, 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 above 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 (6), a type of
sliding microtomes 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 SCIENTIFIC INSTRUMENT COMPANY'S large microtome,
the MINOT precision microtome, LEITZ'S, DE GROOT'S, JUNG'S
" Tetrander." This last seems to be near perfection ; see the
description by MAYER in Zeit. 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 (JuNG, ROY, FROMME, REICHERT, THATE, and
others). I know nothing of these personally, but doubt their
constant accuracy.
Class B contains some very fine instruments, admirably 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 New and Old
Rocking Microtome, made by T«E SCIENTIFIC INSTRUMENT Co.,
Cambridge, or by SWIFT & SON, or by JUNG, or by VAN DER STAD,
Amsterdam ; the MINOT, made by BAUSCH & LOME and the SPENCER
Model, or by BECKER (Gottingen), or by Zimmermann (21, Emilien-
strasse, Leipzig) ; the REINHOLD-GILTAY, made by J. W. GILT AY,
Delft.
For descriptions of the multitudinous models on the market see
10 INTRODUCTORY.
the reports in the Zeit. wiss. Mik. and Journ. Roy. Micr. Soc., and
the price lists of the instrument makers.
11. Staining Reagents and Chemicals. — For some years it has
been difficult to obtain good dyes, but recently several new firms
have succeeded in manufacturing materials suitable for the most
critical work. The pre-war continental dyes were generally impure
products, and since the new dyes being made are mostly purer, it is
often necessary to make some slight alteration in the quantity of
dye added to a staining mixture before the optimum staining
condition is reached. Details of such alteration necessary for
British dyes should be sent to the Journal of the Royal Microscopical
Society. I advise the reader to get his reagents and dyes from
some well-known British firm which has specialised in the matter.
Great Britain. — Flatters and Garnett, Ltd., 309, Oxford Road,
Manchester (all sorts of chemicals, dyes and apparatus ; makers of
Gilson's " Euparal ") ; the British Drug Houses, Ltd., Graham
Street, City "Road, London, N. 1 (makers of many sorts of dyes) ;
The British Dyestuffs Corporation, Ltd., 70, Spring Gardens,
Manchester (important makers of dyes ; London agents are, Baird
and Tatlock, 14, Cross Street, Hatton Garden, E.G. 1) ; Hawksley
& Sons, 83, Wigmore Street, Cavendish Square, W. 1 ; and Charles
Baker, 244, High Holborn, W.C. (both firms are agents for Griibler,
and for most microscope and accessory apparatus makers). For
incubators, etc., for embedding and tissue-culture work, Charles
Hearson & Co., Ltd., 235, Regent Street, W. 1.
United States of America. — Eimer and Amend, 205 — 211, Third
Avenue, New York ; Palo Company, 90, Maiden Lane, New York ;
Edward Pennock, 3609, Woodland Avenue, Philadelphia, Pa. ;
General Biological Supply House, 5508, Kimback Avenue, Chicago,
111. ; The Will Corporation, Rochester, N.Y. ; Paul Weiss, 1620,
Arapahoe Street, Denver, Colorado.
Germany. — G. Griibler and Hollborn, Chemiker, Leipzig, Germany.
CHAPTER II.
KILLING.
12. IN the majority of cases, the first step in the preparation of
an organ or organism consists in exposing it as rapidly and as com-
pletely 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.
It should be noted that narcotisation generally implies some
change in the cells, and most narcotics have to be applied for a long
time. Such treatment is absolutely barred in material destined for
careful cytological study. This applies especially to ether and
chloroform, which are extremely injurious to cells : in the case of
larger mammals like the cat and dog a preliminary treatment in
ether or chloroform may be necessary, but directly after anaesthesia
the animals' throats should be cut or they should be killed by a blow,
if possible. Coal gas chambers are good for killing all mammals,
and I do not believe carbon monoxide is hurtful to cells. Amphi-
bians killed by chloroform are often completely spoilt for cytological
purposes ; if the brain is not wanted, pith the animal. For birds
the time-honoured custom of wringing their necks is recommended.
In the case of small lizards, newts and such live stock it is a good
plan to cut off their heads quickly with strong scissors. If the
material is wanted for chromosome or mitochondria work look up
these sections for special directions.
But these methods are by no means applicable to all cases. There
are many animals, especially such as are of a soft consistence, 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 killing must be resorted to. Speaking
generally, there are two ways of dealing with these difficult cases.
You may kill the animal so suddenly that it has not time to con-
tract : or you may paralyse it by narcotics before killing it. See
also under " Chromosomes," § 652, and " Mitochondria," § 673.
12 KILLING.
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 be necessary to go beyond 60° C. An exposure to heat for a few
seconds will generally suffice.
Small objects (Protozoa, Hydroids, Bryozoa) may be brought into a
drop of water in a watch-glass or on a slide, and heated over the flame
of a spirit-lamp. For large objects, the water or other liquid employed
as the vehicle of the heat may be heated beforehand and the animals
thrown into it.
As soon as it is supposed that the protoplasm of the tissues is 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 Alcyonium and Veretillum, and some Tunicates, such
as Pyrosoma, are very well killed by throwing them into some very
quickly acting fixing liquid, used either hot or cold. Glacial or very
strong acetic acid (VAN BENEDEN'S method) is an excellent reagent
for this purpose ; it may be used, for example, with some Medusae.
After an immersion of a few seconds or a few minutes, according to
the size of the animals, they should be brought into alcohol of at
least 50 per cent, strength. 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 anaesthetic sub-
stance very gradually, in very small doses, to the water containing
the animals, and waiting patiently for it to take effect slowly.
Menthol. — Now used with great success for anaesthetising large marine
animals. Place latter in clean vessel, and sprinkle over surface of water,
menthol crystals. As the latter dissolve the animals expand. In from
twelve to twenty-four hours they may be transferred to a fixer. Very
good for Anemones, Holothuria, Ascidia and many Mollusca. (Personal
communication from Dr. E. J. ALLEN, Plymouth.)
16. Nicotin in solution (ANDRES, Atti R. Accad. dei Lincei, v, 1880,
p. 9). Andres employs a solution of 1 gramme of nicotin in a litre
CHAPTER II. 13
of sea water. The animal is placed in a jar containing half a litre of
sea water, and the solution of nicotin is gradually 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. ii, 1880, p. 123.
17. Chloroform may be employed either in the liquid state or in
the state of vapour. The animals being extended, a 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. (KOROTNEFF, 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 thoroughly to pulverise
the chloroform, should be employed. Small quantities only should
be projected at a time, and the dose should be repeated every five
minutes until the animals are anaesthetised.
I have seen large Medusae very completely anaesthetised in ex-
tension in an hour or two by this method. ANDRES finds that it
does not succeed with Actiniae, as with them maceration of the
tissues supervenes before anaesthesia is established.
PREYER (Mitth. Zool. Stat. Neapel, Bd. vii, 1886, p. 27) recommends
chloroform water for star -fishes.
WADDINGTON employs a mixture of equal parts of 1 per cent,
sol. of cocaine (or eucain) and saturated sol. of chloroform in water
(sea or fresh), according to the habitat.
18. Ether and Alcohol may be administered in the same way.
ANDRES has obtained good results with Actiniae by the use of a
mixture (invented by SALVATORE LO 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.
EISIG (Fauna u. Flora Golf. Neapel, xvi, 1887, p. 239) benumbs
Capitellidae by putting them into a mixture of 1 part of 70 per
cent, alcohol with 9 parts of sea water.
OESTERGREN (Zeit. wiss. Mik., xix, 1903, p. 300) makes a saturated
(7 to 8 per cent.) solution of ether in sea or soft water, and uses ifc
either concentrated or diluted to about 1 per cent., and finds it to
succeed with all classes of aquatic animals.
14 KILLING.
CORI (Zeit. wiss. Mik., vi, 1890, p. 438) recommends a mixture
composed of 10 c.c. methyl-alcohol (of 96 per cent, strength), 90 c.c.
water (fresh or sea water), and 0-6 grm. 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 Cristatella ; Zeit. wiss.
Zool., Iv, 1893, p. 626).
19. Chloreton (Aceton Chloroform) is recommended for inverte-
brates and larvae of Rana by RANDOLPH (Zool. Anz., xxiii, 1900,
p. 436). KRECKER (Zeit. wiss. Zool., xcv, 1910, p. 383) takes
solutions of J to 1 per cent, for Oligochseta. 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
Scyllium and Anguilla.
For Bryozoa, see BESSIE GREEN, Journ. Roy. Mic. Soc., 1914.
20. Hydrate of Chloral.— FOETTINGER (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 centi-
grammes 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, Actiniae,
and with Asteracanthion. He did not succeed with Hydroids.
Lo BIANCO (Mitth. Zool. Stat. Neapel, Bd. ix, 1890, p. 442) employs
for various marine animals freshly prepared solutions of chloral in
sea water, of from to to i Per cent- strength.
I have never had the slightest success with Nemertians.
VERWORN (Zeit. wiss. Zool., xlvi, 1887, p. 99) puts Cristatella for a
few minutes into 10 per cent, solution of chloral, in which the animals
sooner or later become extended.
KUKENTHAL (Jena Zeit. Naturw., Bd. xx, 1887, p. 511) has obtained
good results with some Annelids by means of a solution of 1 part of
chloral in 1,000 parts of sea water.
The chloral method gives rise to maceration with some subjects,
as I can testify, and has been said to distort nuclear figures.
21. Cocaine (RICHARDS, Zool. Anz., cxcvi, 1885, p. 332). — Richards
puts a colony of Bryozoa into a watch-glass with 5 c.c. of water, and
adds gradually 1 per cent, solution of hydrochlorate of cocaine in
water. After five minutes the animals are somewhat numbed;
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
CHAPTER II. 15
certain worms. It is the best method for Rotifers (ROUSSELET).
It has also been recommended for Aplysia.
It has been pointed out (by Com, in the paper quoted § 18) 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 (EisiG).
Cocaine solutions cannot be depended on to keep for more than a few
days.
22. Eucain. HARRIS (Journ. Roy. Mic. Soc., 1900, p. 404)
recommends a 1 per cent, solution of eucain hydrochloride, as giving
far better results, with Vorticellidse, Rotatoria, and Vermes.
ROUSSELET (ibid.) reports favourably as to its action on Flosculariae.
It is stated to be perfectly stable in aqueous media. It dissolves in
sea water to about 0-5 per cent.
23. Hydroxylamin. — HOFER (Zeit. wiss. Mik., vii, 1890, p. 318).
Either the sulphate or, preferably, the hydro chlorate may be used.
This should be dissolved in water (spring or sea water, according to the
habitat) and exactly neutralised by addition of carbonate of soda. The
organisms are placed in a solution diluted to about 0*1 per cent., for
thirty minutes or less (as for Infusoria), to 0-25 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 (Helix and Anodonta).
Hydroxylamin is a powerful reducing agent, and should therefore be
well washed out before treating with easily reducible fixing agents.
24. Chloride or Sulphate of Magnesium. — TULLBERG (Arch. Zool.
Exper. et Gen., x, 1892, p. 11). For Actiniae, 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 1 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.
REDENBAUGH (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, Bid. Bull. Wood's Hole, xvii, 1909, p. 341 (puts
direct into sol. of f strength).
25. Poisoning by small doses of some fixing agent is sometimes
good. Lo BIANCO kills Ascldia and Rhopalcea in an extended state
(Mitth. Zool. 8tat. Neapel, ix, 1890, p. 471) by pouring a little 1 per cent.
16 KILLING.
chromic acid on to the surface of the water containing them, and
allowing it to diffuse slowly into it. About twelve to twenty-four
hours is necessary. He kills Ciona in a similar way with a mixture of
1 part of 1 per cent, chromic acid and 9 parts of 49 per cent, acetic
acid.
Osmic acid, or Klein enberg's solution, is sometimes employed in the
same way.
I have seen Medusae killed in a satisfactory manner by means of
crystals of corrosive sublimate added to the water containing them.
Morphia, Curare, Strychnin, Prussia 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 closing it. 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 Holothurise
and other Echinoderms in this way. WAED (see Amer. Nat., xxv,
1891, p. 398) has succeeded with Hydroids, Actiniae, and similar
forms, and UEXKULL (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 FOL, Zool. Anz.,
cxxviii, 1885, p. 698). The water containing the animals should be
saturated with the gas. The method is stated to succeed with most
Ccelenterata 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 NepJielis
of 15 or 20 millimetres in length, will require about five minutes ;
and a large Nephelis, of from 10 to 15 centimetres, will require as
many hours.
UEXKULL (Mitth. Zool. Stat. Neapel, xii, 1896, p. 463) has paralysed
CHAPTER II. 17
Echinids very rapidly with carbonic acid, likewise a small Teleostean
fish ; whilst Scyllium and Crustaceans were affected much more
slowly, and mussels not at all.
27. Peroxide of Hydrogen.— VOLK (Zool. Anz.t xix, 1896, p. 294)
kills Eotatoria 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 unrecognisable, even if not totally
destroyed. Further, in an equally short time the nerve-fibres
become varicose, and appear to be thickly studded with spindle-
shaped knots ; and other post-mortem changes rapidly occur. If,
however, a fresh piece of retina be treated with a strong solution of
osroic 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 necessary 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 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 in producing
optical differentiation in structures. By coagulating 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
CHAPTER III. 19
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 compounds 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,
however, 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 consistency and optical differentiation,
by the reagents subsequently employed. It is also sometimes an
advantage in that certain of the compounds in question have the
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 their 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 hsemalum, or, after
sectioning, with iron hsematoxylin or tar colours.
According to FISCHER (Fixirung, Fdrbung, und Bau des Proto-
20 FIXING AND HARDENING.
plasmas, 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. The 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 precipi-
tation forms, which vary according to the precipitant. Each fixing
agent gives its own characteristic fixation image, which may be more
or less lifelike, but can never be absolutely so. Fischer gives copious
descriptions of the precipitation 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 fixation by
precipitation that the most energetic fixing agents should always be
found amongst the most energetic precipitants. But on the showing
of his experiments this is not so. For 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 power cannot be
taken as a measure of the fixing power of the reagents. And further,
the study of the fixation images of tissues afforded by osmic acid,
formaldehyde, and other reagents, seems to show that the coagula-
tion 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. Fischer, studying the effects of certain fixatives on
albumose, states that mixing 10 per cent, slightly acid deutero-
albumose with Altmann's bichromate-osmic fluid causes a precipitate
of granules of from 1 to 3 /x in diameter, while corrosive sublimate
of 7 per cent, causes granules of 04 to 1 /ot in size ; one might be led
away, as was Fischer, to consider that Altmann's fluid used on cells
therefore causes artifacts to appear. As a matter of fact corrosive
sublimate is much more dangerous than Altmann's fluid, in this
respect, while Altmann's fluid merely preserves cell granules which
are visible intra vitam. The ground protoplasm after corrosive is
more granular and coarsely reticulate than after Altmann ; this
CHAPTER III. 21
does not apply to mitochondria or Golgi elements which are often
very badly preserved with corrosive. •
Fischer (op. cit.) says, " Many kinds of cell contents, indeed the
majority, have an alkaline reaction, and are thereby quite inacces-
sible to the precipitating action of certain agents, such as osmic
acid, or bichromate ; and the action of certain other fixatives, such
as platinum chloride or chromic acid, is more or less hindered by the
presence of free alkalies. For neither the chromic acid (of the
Flemming), nor the platinum chloride (of the Hermann) would be
adequate to act as acidifiers to the osmic acid of the mixtures."
My own experience is directly contrary to these conclusions of
Fischer : it is common knowledge among modern workers that a
cell fixed in acetic acid-containing solutions has a more " raked out "
appearance than when the acetic acid is omitted : this applies not
only to cell granules, but to the appearance of the ground cytoplasm,
nucleoli, and chromatin filaments. The statement that osmic acid
must be acidified before it will fix all parts of the cell is also contrary
to general experience. Formalin neutralised gives a gentler and
more precise fixation than acid formalin. While Fischer's results
may be excellent so far as concern his theoretical conclusions on the
fixation of weak solutions of egg-white, etc., too much attention
should not be paid to one who is not thoroughly acquainted with
practical cytology and histology. But from his very suggestive
observations it certainly appears that the formation of visible
precipitates is a very widespread, 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 artifacts to which it can give rise.
It has lately been pointed out by some workers (e.g. UNNA, Arch,
f. Mikr. Anat., Ixxviii), that many of the fixing reagents come under
the category of oxidisers ; Unna places great importance on the
fact that some of the most successful fixers are oxidisers, e.g.
Os04, K2Cr207, Cr03 ; but formalin, admittedly a splendid reagent,
is a reducer, while picric acid and corrosive sublimate are feeble
oxidisers, and that only under special conditions.
Of the ten common reagents used for fixing, only four are marked
oxidisers, and Unna's generalities with reference to the significance
of oxidisers may not be the correct explanation. Generalities such
as made by Unna with reference to the role of oxidisers in fixation
resemble like claims which have been made with reference to the
supposed necessity for the constant use of an acid in fixatives (and
preferably acetic acid which is really a dangerous reagent).
22 FIXING AND HARDENING.
With regard to the relative values of oxidisers or reducers in fixing
cytoplasm or* nucleus, formalin (reducer)* and Os04 (oxidiser) are
both famous cytoplasm fixers, while acetic acid (neither oxidiser nor
reducer), or alcohol (reducer) and Cr03 (oxidiser) are well-known
nuclear fixatives.
Kelly's fluid, formol-bichromate or formol-Flemming are all
splendid fixatives, and mixtures of both oxidisers and reducers ;
it is difficult to see how Unna's theories can apply here. In the case
of Flemming fluid, without acetic acid, it is certain that the fixation
process in so far as it concerns the Os04 in this mixture, is not
solely an oxidising process, at least of the same nature as the fixation
reaction by the chromic acid (Cr03). In a word, fixation of the cell
by various kinds of chemical reagents is an extremely complicated
matter concerning a large number of organic substances whose re-
actions to the chemical used are probably different in most cases.
30. The Characters of the Usual Fixing Agents. — These agents are
as follows : —
1. Osmium tetroxide,
2. Formaldehyde gas,
3. Chromium trioxide,
4. Bichromate of potassium,
5. Platinum chloride,
6. Mercury bichloride, all in water.
7. Picric acid in water,
8. Alcohol,
B 1
9. Nitric acid and
10. Acetic acid in water.
Chloroform and urea are also used. In the group marked A are
arranged the more valuable reagents, in B the less valuable or
destructive ones. Good fixatives can be made from the substances
in group A without using any of the reagents in group B. The latter
contain most of the reagents useful for chromosome work, the
former, reagents useful for fixing the cytoplasm and " resting "
nucleus.
From group A have been made the following mixtures : Altmann,
Champy, and Flemming and Hermann- without-acetic acid ; these,
are among the best mixtures known. Then there are formol (5 per
cent, to 10 per cent.), Kegaud, Helly, formol-Muller and formol-
Flemming, which are so good for mammals. Good general micro-
' anatomical fixatives from both groups A and B, are Zenker, Bouin,
* See, however, Blum, Enzykl. d. mikr. Tech., 1910.
A. 4
CHAPTER III. 23
Gilson-Petrunkewitsch and corrosive acetic, but these all destroy ]
much of the cell-contents, and give an incorrect picture of the cell, |
excepting of chromatinic structures, for which they are indicated.
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 penetra-
tion 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 mixtures. 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 chromic acid, a better differ-
entiation is obtained ; for chromic acid, whilst enhancing, and 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 homo-
geneous and with more detail observable in them.
Descriptive embryologists .often use strange illogical mixtures
containing both reducible substances and violent reducers, both
fat-solvents and fat-preservers, mixed together witjiout regard for
the chemistry of fixation. It is only the logically planned fixative
that is found generally useful, and which stands the test of time.
Fixation falls under three broad headings : —
1. Micro-anatomical, in which correct preservation of cell aggre-
gates, without shrinkage or expanding, is the desideratum. Such is
the aim of most descriptive embryologists.
2. Cytological from the point of view of the chromosome or nucleus.
3. Cytological from the point of view of fixing the cell in a state
which most resembles its condition when alive ; also so as to identify
the cell elements, especially in the cytoplasm.
In most cases the results attained by workers belonging to sections
1 and 2 can truly be said to give a caricature of the cell intra vitam.
I give below a general classification of fixatives, those in (a) being
24 FIXING AND HARDENING.
fixatives causing the maximum disturbance and destruction in the
individual cell, those in (c) the least.
A great deal, however, depends on the accessibility of the cells to
the fixative, and as to whether vertebrate or invertebrate material
is being used.
(a) Carnoy, Petrunkewitsch, alcohol, Gilson, picro-nitric, etc.
Fat, mitochondria, Golgi apparatus, and often delicate yolk discs
do not show after these. (Using alcohols and xylol subsequently.)
(6) Bouin, Zenker, corrosive acetic, Flemming-with-acetic acid, etc.
Mitochondria and Golgi apparatus rarely show after these, except
possibly in mammals, where these cell inclusions are more resistant
than in invertebrata. Fats show with the last-mentioned fixative.
(c) Osmic acid, Flemming-without-acetic, Champy, Altmann,
formalin, Mann's mercury-osmic liquid, Sjovall's method, etc.
Preserve all formed granules (except glycogen). (Using fluids
subsequently as above.)
In section (c) the formol alone will not preserve fat ; but see
Sjovall's method (§ 696).
The fixatives have not been classed according to how they them-
selves alone affect the contents of the cell, but according to how they
preserve the cell preparatory to its treatment in the liquids necessary
for embedding and sectioning.
Injurious liquids which should never be used in cytological fixation
(3, vide supra) are acetic acid, chloroform and alcohol. Acetic acid is
nearly the most destructive to delicate lipins, and its use, except
where chromosomes are being studied, is rarely indicated ; any
worker who uses acetic acid in his fixing mixtures cannot hope to get
a correct picture of any part of his cell, possibly excepting the chromo-
somes (not the. resting nucleus). The most valuable fixatives are
osmium - tetroxide, bichromate of potassium, chromium - trioxide,
and formaldehyde, possibly in the order named ; the most valuable
mixtures are Muller-formol (or Helly), Flemming-without-acetic,
Altmann, and Champy ; the three latter approach as near perfection
as present-day technique allows. Altmann's fluid (K2Cr207 +
Os04) I find to be a splendid mixture. In no case, except in small
invertebrates, do these fixatives (excluding formol) give a true
fixation of cell aggregates ; this is due to their inferior penetrating
powers, and to an unevenness of penetration. Small invertebrates,
both marine and fresh- water, and small pieces of tissue, are usually
exquisitely preserved in chrome-osmium mixtures, but are not then
generally suitable for staining and mounting whole, especially for
staining in carmine mixtures.
CHAPTER III. 25
For routine zoological work Bouin's picro-formalin-acetic is
recommended. Gilson-Petrunkewitsch is a fixative which is easy
to work and generally better than corrosive sublimate acetic.
For routine vertebrate histological work Zenker and Kelly's
Zenker-formol are indicated.
I think the beginner should avoid such things as liquid of FLEMMING
and similar mixtures.
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, bichromates, 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, picric acid, and acetic acid, are neutral, or
even favourable, in this respect.
31. The Practice of Fixation. — See that the structures are per-
fectly living at the instant of fixation, otherwise you will only fix
pathological states or post-mortem states.
Some observers have made special observations on the effect of delay in
fixation ; J. THORNTON CARTER (Phil. Trans. Eoy.Soe., Series B, vol. ccviii,
1917) has made some interesting experiments on the finely granular
ameloblasts in the developing teeth of the pike. He noticed that the
cytoplasm gave evidence of marked changes unless fixed within three
minutes of " death " ; these changes were manifested by the behaviour
of the cytoplasmic granules to stains ; the selectivity 'of the latter was
progressively altered as the rapid post-mortem changes were set in
action.
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 struc-
tures 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 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 many times
26 FIXING AND HARDENING.
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 energetically, 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 GOLGI, DE QUERVAIN, MANN, and others, given under
Nervous System.
BRATJS and DRUENER (Jena Zeit. Naturw., Bd. xxix, 1895, p. 435)
fix fishes by injection through the bulbus aortce. The vessels are first
washed out with normal salt solution, and the fixing liquid is then
thrown in.
KOLMER (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 neces-
sary to produce the desired optical differentiation with some
reagents.
Careful washing out (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 impera-
tive. 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 alcohol causes precipitates that may
ruin the preparations. Objects fixed in alcohol, formol, acetic acid,
picric acid, or 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.
CHAPTER III. 27
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. Picric
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 terrestrial forms, and fixing
solutions should in consequence be stronger (about two to three
times).
Marine animals ought to be freed from the sea water adherent 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 penetration 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-
chloric or some other appropriate acid. Picro-nitric acid is a fixing
reagent that fulfils the conditions here spoken of. (On this subject
see MAYER, in Mitth. Zool. Stat. Neapel, ii (1881), pp. 1 et seq., and
ALLEN and BROWNE in " Science of the Sea," John Murray, 1912).
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 sections from them is, in the
majority of cases, no longer necessary. But there are some excep-
tions. 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. — Employ in general a relatively
large volume of hardening liquid, and change it very frequently. If
28 FIXING AND HARDENING.
the volume of liquid be insufficient, its composition will soon become
seriously altered by the diffusion into it of the soluble substances of
the tissues ; and the result may be a macerating instead of a harden-
ing liquid. Further, as soon as, in consequence of this diffusion, the
liquid has acquired a composition similar in respect of the propor-
tions 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. This 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 reagent, increasing the
strength gradually, as fast as the tissues acquire a consistency 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 IV.
FIXING AND HARDENING AGENTS — MINERAL ACIDS AND THEIR
SALTS.
35. Osmic Acid. — The tetroxide of osmium (Os04) is the substance
commonly known as osmic acid, though it does not possess acid
properties. It is extremely volatile, and in the form of an aqueous
solution becomes pajtially reduced with great readiness in presence
of the slightest contaminating particle of organic matter. It is
generally believed that the aqueous solutions are reduced by light
alone, but this is not the case : they may be exposed to the light
with impunity if dust be absolutely denied access to them.
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 i
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.
CORI (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 a very slight rosy tint to the
liquid. From time to time, as the solution becomes colourless,
further small quantities of the salt should be added, so as to keep up
the rosy tint.
BUSCH finds that the addition of sodium iodate hinders reduction
(Neurol. Centralb., xvii, 1898, p. 476).
PINTNER finds that a slight addition of corrosive sublimate has the
same effect, e.g. 10 drops of 5 per cent, solution of sublimate added
to 100 c.c. of 1 per cent, solution of osmic acid.
For the Kopsch, Mann-Kopsch and Sjovall methods the osmic
acid solution must be free from all traces of chrome and platinum
salts, etc.
30 FIXING AND HARDENING AGENTS.
For the so-called " regeneration " of reduced solutions, see
previous editions.
Osmic acid is met with in commerce in the solid form in sealed tubes.
The assigned weights should be checked, as they may vary greatly.
Fixation by the Vapours. — 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 treated as described in § 695. 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 in order to fix the
deeper layers of relatively thick objects, such as retina, an exposure
of several hours may be desirable). It is well. to wash the objects
with water before staining, but a very slight washing will suffice.
For staining, methyl-green may be recommended for objects destined
for study in an aqueous medium, and, for permanent preparations,
alum-carmine, picro-carmine, or hsematoxylin.
In researches on nuclei, it may be useful to employ the vapours
of a freshly prepared mixture of osmic and formic or acetic acid
(Gilson, La Cellule, i, 1885, p. 96).
The reasons for preferring 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 elminated. See also
under " Cramer's Method," § 695.
Fixation by Solutions. — Osmic 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 ^ to 2 per cent. I should say
myself that, as a rule, not more than 0-1 per cent., and never
more than 2 per cent., should be used.
On account of its feeble penetrating power the objects to be fixed
should be as small as possible.
The solutions should be kept protected from bright light during
the immersion of tissues. (This precaution is not necessary if
Flemming's or Hermann's solution be used.) If the immersion is
to be a long one the tissues must be placed with the solution in well-
closed glass-stoppered vessels. The objects may be deemed to be
fixed as soon as they have become brown throughout. But see
" Mann-Kopsch Methods," § 693.
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. Notwithstanding the greatest care in
CHAPTER IV. 31
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 bichromate
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 treatment
with bichromate solutions has the great advantage of highly facili-
tating staining with carmine or hsematoxylin. 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 prepara-
tions. See " Bleaching." This may be done by means of peroxide
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 HC1, will keep well in the dark ;
but the mixture with alcohol must be made fresh for use.) Accord-
ing to BRISTOL (Amer. Natural, xxvii, 1893, p. 176) the peroxide acts
best in the sun. BINET (Journ. de VAnat. et de la Physiol., xxx,
1894, p. 449) has successfully used permanganate of potash. 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. . . . MONCKEBERG and BETHE
(Arch. Mik. Anat., liv, 1899, p. 135) have succeeded in satisfactorily
restoring the staining susceptibility of osmium material by means
of sulphurous acid (obtained by adding hydrochloric acid to bisul-
phite of sodium, 2 to 4 drops of the acid added to 10 c.c. of a 2 per
cent, solution of the salt).
FOL (Lehrb., p. 174) recommends a weak aqueous solution of ferri-
cyanide of potassium.
I find the sulphate of iron solution used in Benda's haematoxylin
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. For sections, of course, in both cases
safranin and other anilin stains may be employed with advantage,
as may hsematoxylin.
32 FIXING AND HARDENING AGENTS.
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.
36. The Osmium Tetroxide Reaction.— MANN believed that during
the osmic reaction on fatty substances the Os04 was reduced to
osmium tetra-hydroxide Os(OH)4. Other observers have assumed
the reaction to be the reduction of the Os04 to some lower oxide.
The matter has recently been reviewed by Professor J. K.
PARTINGTON and Mr. D. B. HUNTINGFORD, who find that the
reduced substance is a hydrated form of Os02, possibly Os02, 5H20,
or Os02) 6H20. In all probability, Professor PARTINGTON informs
me, the amount of water is not definite. (See also § 768 on " Fat.")
37. Osmic Mixtures. — NICOLAS (Intern. Monatsschr., 1891, p. 3) adds
| per cent, of osmic 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.
BTJSCH (Neurol. Centralb., xvii, 1898, No. 10, p. 476 ; Zeit. iciss. 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 a more energetic action in the
deeper layers. He adds 3 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 to a 1 per cent, solution. For some mixtures of KOLOSSOW,
see 5th ed., or Zeit. wiss. Mikr., v, 1888, p. 51, and ix, 1892, p. 39. See
also under " Cytology," § 677.
38. Chromic Acid. — Chromic anhydride, Cr03, is found in com-
merce in the form of red crystals that dissolve readily in water,
forming chromic acid, H2Cr04. These crystals are very deliquescent,
and it is therefore well to keep the acid in stock in the shape of a
1 per cent, solution. Care must be taken not to allow the crystals
to be contaminated by organic matter, in the presence of which the
anhydride is readily reduced into sesquioxide.
Chromic acid is generally employed in aqueous solution. Some
CHAPTER IV. 33
• observers (KLEIN ; URBAN PRITCHARD ; PERENYI) have recom-
mended 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, -^ to J 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 from
forming again and again. It has, however, been found by HANS
VIRCHOW (Arch. Mik. Anat., xxiv, 1885, p. 117) that it may be
entirely prevented by simply keeping the preparations in the dark.
The alcohol becomes yellow as usual (and should be changed as
often as this takes place), but no precipitate is formed. If this
precaution be taken, previous washing with water may be omitted,
or at all events greatly abridged.
MAYER (Grundzuge, 1st 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, preferably 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 (4- to 6 drops of
HC1 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
M. 3
34 FIXING AND HARDENING AGENTS.
usual stains. So also EDINGER (Zeit. wiss. Mik., 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 per-
oxide of hydrogen. OVERTON (Zeit. wiss. Mik., 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 prepara-
tions, § 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 hsematoxylin, or, for
sections, the basic tar colours.
Chromic acid is not a very penetrating reagent, and for this
reason, as well as for others, is now seldom used pure tor fixing.
For prolonged hardening it is generally employed in strengths
of J to \ 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. For a human spinal cord you
should take 2 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 a time. The objects should be removed from the
solution as soon as they have acquired the desired consistency, 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.
CHAPTER IV. 35
39. Chromo-acetic Acid (FLEMMING, Zellsbz., Kern. u. Zellth.,
p. 382).
Chromic acid . . . 0-2 to 0-25 per cent.
Acetic acid . . 0*1 per cent, in water.
Flemming found this the best reagent for the study of the achromatic
elements of karyokinesis. You can stain with hsematoxylin, or the basic
anilin dyes.
The following has been recommended for Annelids by EHLERS : —
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 counter-
act any shrinkage due to the chromic acid. Fix overnight, wash out
several hours in water.
Similar to this is the k' 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 (RABL, Morph. Jahrb., x, 1884, pp. 215,
216). — Four or 5 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 karyokinesis.
41. Chromo-aceto-osmic Acid (FLEMMING, Zellsubstanz, Kern und
Zetttheilung, 1882, p. 381). FIRST or WEAK formula :
Chromic acid . . . 0-25 per cent. )
Osmic acid . . . .0-1 ,, - in water.
Glacial acetic acid . .0-1 ,, )
MEVES (Encyd. mikr. Techn., 1, p. 475) sometimes adds 1 per cent,
of sodium chloride.
FOL (Lehrb. d. vergl. mik. Anat., 1884, p. 100) recommends the
following variant :
1 per cent, chromic acid . . . .25 vols.
1 per cent, osmic acid . . . 2 ,,
2 per cent, acetic acid . . . 5 „
Water 68 „
— that is to say, a mixture weaker in osmium than Flemming's.
A mixture still weaker than this in osmium, viz. with 1 vol. osmic
acid solution, instead of 2, has been recommended by Com (Zeit. wiss.
Mik., vi, 1890, p. 441).
SECOND or STRONG formula (Zeit. wiss. Mik., 1, 1884, p. 349) :
1 per cent, chromic acid . . . .15 parts.
2 per cent, osmic acid . ... . 4 ,,
Glacial acetic acid ..... 1 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.
3—2
36 FIXING AND HARDENING AGENTS.
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 FORMULA. — More recently, FLEMMING 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 "
(MEVES, 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.
Under "Cytology" Sections, § 678, see BENDA and GATENBY
modifications.
PODWYSSOZKI recommends (for glands especially) the following
modification :
1 per cent. Cr03 dissolved in 0-5 per cent, solution
of corrosive sublimate . . . . . 15 c.c.
2 per cent, osmic acid solution . . . . 4 c.c.
Glacial acetic acid . . . . . 6 to 8 drops.
The sublimate is said to augment the penetration 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 hours or days, or
according to some workers even weeks or months ; but this exagge-
rated fixation is clearly only justifiable in very special cases, if at
all. For chromosome studies some workers fix for only one hour.
Others recommend cooling the FLEMMING on ice before using.
Wash out very thoroughly in water (running, twenty-lour hours),
or treat as directed for chromic acid, § 38. Stain with alum
hsematoxylin 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 hsema-
toxylin.
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 preser-
vation 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
CHAPTER IV. 37
only for very small objects or for very small pieces of tissue, such as
suffice for cytologies} or histological work. The strong liquid
especially has not the character~of a general reagent. As a matter
of fact it was recommended by FLEMMING 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 picroformol
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.
42. Osmic Acid and Bichromate. — ALTMANN (Die Elementar-
organismen, 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. Refer to § 680.
Lo BIANCO (Mitth. Zool. Stat. Neapel, ix, 1890, p. 443) employs for
marine animals a mixture of 100 c.c. of 5 per cent, solution of bichromate
and 2 c.c. of 1 per cent, osmic acid.
HOEHL (Arch. Anat. Phys., Anat. Abth., 1896, p. 31) recommends
a mixture of 80 c.c. of 3 per cenjb. bichromate, 20 c.c. of 1 per cent,
osmic acid, and 2 c.c. of glacial acetic acid.
43. Bichromate-chromic-osmic Acid. — CHAMPY (Arch, de Zool.
Exper., 1913). — Mixture of 7 parts of 3 per cent, bichromate of
potash, 7 parts of 1 per cent, chromic acid, 4 parts of 2 per cent,
osmium tetroxide.
This mixture keeps well. Fix for from six to twenty -four hours.
Wash out in running water about the same time.
You can stain in iron hsematoxylin, or less well in ALTMANN or
BENDA. See § 681 for a description of mordanting after Champy's
fluid. This fluid is extremely useful, and I nearly always use it in
addition to FLEMMING.
44. Osmic, Bichromate, and Platinic Mixture (LINDSAY JOHNSON'S
Mixture). — Latest formula, 1895, communicated by Dr. Lindsay
Johnson :
Bichromate of potash (2-5 per cent.) . . 70 parts.
Osmic acid (2 per cent.) . . . . 10 ,,
Platinum chloride (1 per cent.) . . . 15 „
Acetic or formic acid 5
38 FIXING AND HARDENING AGENTS.
HENNEGUY, 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 FLEMMING. I think highly of it —
for certain objects. Twelve hours is probably the optimum time
for fixation. Wash out in water.
45. Platino-aceto-osmic Acid (HERMANN'S) Solution (Arch. Mik.
Anat., xxxiv, 1889, p. 58). — One per cent, platinum chloride 15 parts,
glacial acetic acid 1 part, and 2 per cent, osmic acid either 4 parts
or only 2 parts. Hermann found that protoplasm structures are
thus better preserved than with the chromic mixture. As with
Flemming, the optimum time is from twelve to sixteen hours.
Wash out at least three hours in running water.
The after-treatment and staining should be the same as for objects
treated with Flemming's solution. RENGEL (Zeil. wiss. Zool.,
Ixiii, 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 extremely
difficult to obtain good plasma stains after its action. It causes a
notable shrinkage in chromatin. It gives a full fixation of cyto-
plasm,, to which it gives a much more fine-grained aspect than liquid
of Flemming does.
Leaving out the acetic acid, the solution may be used for mito-
chondria as in § 679.
46. Rawitz (Zeit. wiss. Mikr., xxv, 1909, p. 386) takes 4 parts of
Kahlbaum's Phospho-Tungstic acid, 5 of alcohol, and 1 of acetic acid,
added just before use, fixes for twenty-four hours, and washes out
the sections before staining with water containing a little calcium
acetate.
47. Nitric Acid (ALTMANN, Arch. Anat. Phys., 1881, p. 219).—
Altmann employs for fixing embryos dilute nitric acid, containing from
3 to 3£ per cent, pure acid. Such a solution has a sp. gr. of about 1-02.
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 (ibid., 1877, p. 115) recommended a 10 per cent, solution.
CHAPTER IV. 39
Flemming at one time employed solutions of 40 to 50 per cent, for the
ova of Invertebrates.
TELLYESNICZKY (Arch. mik. Anat.,\ii, 2, 1898, p. 222) thinks that " for
general cell-fixing " the proper strength is 2 to 2| per cent., as stronger
grades act too energetically on the superficial layers.
MAYER 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 prepara-
tions 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 thrae weeks, is said to afford very tough preparations of the
encephalon.
BENDA (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-nitric Acid (PERENYI'S formula, Zool. Anseig., v, 1882,
p. 459) :
4 parts 10 per cent, nitric acid.
3 parts alcohol.
3 parts 0-5 per cent, chromic acid.
Fix for four to five 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
specimens, and found it admirable for these purposes. But preparations
made to test its value from a cytological point of view have given me
only second-rate results. It is now little used.
49. Chromic Acid and Platinum Chloride (MERKEL'S Macula lutea
des Menschen, Leipzig, 1870, p. 19). — Equal volumes of 1400
solution of chromic acid and 1400 solution of platinum chloride.
Objects should remain in it for several hours or even days. After
washing out with alcohol of 50 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 Eisig employs an immersion of three to
five hours, and transfers to 70 per cent, alcohol ; for small leeches
40 FIXING AND HARDENING AGENTS.
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 LAV-
DO WSKY 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 Eisig), viz. —
0-25 per cent, solution of platinum chloride . . 1 vol.
1 per cent, solution of chromic acid . . . 1 „
The ova to remain in it one or two days (WHITMAN, Methods in
Micro. Anat., p. 153).
Satis.
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.
FLEMMING (Arch. mik. Anat., xviii, 1880, p. 352) pointed 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 ; whilst preserving 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 BURCKHARDT, 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 combination 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 . . . . . . 5 „
(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 . 5
CHAPTER IV. 41
51. Bichromate of Potash.— Perhaps the most important of all
known hardening agents, sensu stritto. It hardens slowly, much
more so than chromic acid, but it gives an incomparably 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, § 38. They had better be kept in the dark when in alcohol.
See § 38. (B6HM and OPPEL [Taschenbuch, 3 Auf., 1896, p. 22]
fix in the dark.) // you wish to have a good stain with carmine you
should not put the objects into alcohol at all, even for a second, until
they have been stained.
You may stain either with carmine or hsematoxylin, as well as
with tar colours.
Bichromate objects have an ugly yellow colour which cannot be
removed by mere soaking in water. It is said that it can be removed
by washing for a few minutes in a 1 per cent, solution of chloral hydrate.
Prof. GILSON writes me that alcoholic solution of sulphurous anhydride
(S0.2) is very convenient for the rapid decoloration of bichromate
objects. A few drops suffice. See also § 38, and " Bleaching."
To facilitate staining with hsematoxylin, WOLFF (Zeit. wiss. Mik., xv,
3, 1899, p. 311) first stains in Boehmer's hsematoxylin for twenty-four
hours, and then for a few minutes in the same hsematoxylin 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 recom-
mended as a fixing 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 TELLYES-
NICZKY, next §.
52. Acetic Bichromate (TELLYESNICZKY, Arch. mik. Anat.} Hi,
1889, p. 242) :
Bichromate ...... 3 grms.
Glacial acetic acid ..... 5 c.c.
Water 100 „
Smaller objects to remain in the fluid for one or two days, larger
42 FIXING AND HARDENING AGENTS.
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, §§ 42,
43 and 44.
53. MULLEE'S Solution.—
Bichromate of potash . . . 2 — 2J parts.
Sulphate of soda .... 1 part.
Water 100 parts.
The duration of the reaction is about the same as with the simple
solution of chromic salt.
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 hardening properties. Fol says
that for mammalian embryos, for which it has been recommended,
it is worthless.
54. ERLICKI'S Solution (WarscJiauer med. Zeit., xxii, Nos. 15 and
18 ; Progres Medical, 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 (FoL, 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 0-5 per cent, chromic acid before putting them into alcohol
(TscHiscH, Virchow's Arch., Bd. xcvii, p. 173 ;. EDINGER, Zeit. wiss.
Mik., ii, p. 245 ; LOEWENTHAL, Rev. med. de la Suisse romande, 6me
anne"e, i, p. 20).
55. KULTSCHITZKY'S Solution (Zeit. wiss. Mik., 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, 5 or 6 drops per 100 c.c.
To make the solution, add the finely powdered salts to the alcohol in
CHAPTER IV. 43
excess, and leave them together in total darkness, for twenty-four
hours.
Fix for twelve to twenty-four hours in the dark. Then treat with
strong alcohol for twelve to twenty-four hours.
56. DEKHUYZEN'S Liquids (G. E. Acad. Sei., cxxxvii, 1903, pp. 415
and 445). — {A) 250 c.c. of 2-5 per cent. sol. of bichromate in sea -water,
25 c.c. of 6-3 per cent, nitric acid, and 54 c.c. of 2 per cent, osmic acid.
For general use with marine animals.-
(B) 173-1 c.c. of the bichromate sol. and 26-9 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.
57. Bichromate and Sublimate (KULTSCHITZKY, Arch. f. mik. Anat.,
xlix, 1897, p. 8). — Two 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. LAVDOWSKY (Zeit. wiss.
Mik., xvii, 1900, p. 301) takes 500 c.c. of 1 per cent, acetic acid, 20 to 25
grms. bichromate, and 5 to 10 c.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
solutions, up to 5 per cent.
59. Neutral Chromate of Ammonia is preferred by some. It is used
in the same strength as the bichromate. Klein has recommended it for
intestine, which it hardens, in 5 per cent, solution, in twenty-four hours.
60. Bichromate of Calcium. — SONNENBRODT (Arch. mikr. Anat.,
Ixxii, 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
or ammonia with alcohol may be employed, and have a more rapid
action than the aqueous solution. Thus HAMILTON takes for hardening
brain a mixture of 1 part methylated spirits with 3 parts of solution of
Miiller ; see also KULTSCHITZKY'S Mixture, ante, § 55). Preparations
should be kept in the dark during the process of hardening in these
mixtures.
62. Sulphurous Acid. — WADDINGTON (Journ. Roy. Mic. Soc., 1883,
p. 185) uses a saturated solution of sulphurous acid in alcohol for fixing
infusoria. OVERTON (Zeit. wiss. Mik., vii, 1890, p. 9) uses the vapours
of an aqueous solution for fixing algae.
CHAPTER V.
FIXING AND HARDENING AGENTS — CHLORIDES, ORGANIC ACIDS,
AND OTHERS.
Chlorides.
63. Bichloride of Mercury (Corrosive Sublimate). — Corrosive
sublimate is soluble in about 16 parts of cold and 3 of
boiling distilled water. It is more soluble in alcohol (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 HgCl2 in them is partly split up by
hydrolysis into Cl, H, and (HgCl)2, or HgClOH (see Chem. Centralb.,
1904, i, p. 571 ; the statements of MANN [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. KAISER'S
solution consists of 10 grms. sublimate, 3 c.c. glacial acetic acid,
and 300 c.c. distilled water (from Zeit. wiss. Mik., xi, p. 378).
VAN BENEDEN has used a saturated solution in 25 per cent, acetic
acid, and Lo BIANCO (Mitih. 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 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.
CHAPTER V. 45
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. Alcoholis almost always prefer-
able. 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 alcoholfleaves 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.
LUGOL'S) may be taken. MAYER (Zeit. wiss. Mik., xiv, 1897, p. 28)
makes it by dissolving 5 grms. of iodide of potassium in 5 c.c. of distilled
water and mixing this with a solution of 0-5 grai. 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
employed together. The iodine may be washed out in obstinate cases
with magnesia water. Similarly APATHY (MittTi. Zool. Stat. Neapel, xii,
1897, pp. 729, 730).
It has been objected to this process that iodine in potassic iodide
precipitates 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, etc. , 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 brittle, and will not stain so well.
They will also become brittle if they are kept long in alcohol.
It may happen that if the extraction of the excess of sublimate
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. SCHAPER
46 FIXING AND HARDENING AGENTS.
(Anal. Anz., xiii, 1897, p. 463), however, has shown that neglect to
extract the sublimate from the, tissues in bulk may give birth to serious
artifacts, which appear to arise during the imbedding process. So also
LOYEZ (Arch. Anat. Micr., viii, 1905, p. 71). HEIDENHAIN (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 manipu-
late 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 un-
doubtedly 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 recommended where more precise fixing
agents are counter-indicated 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 grms. sublimate,
0-5 grm. sodium chloride, and 100 c.c. water has been quoted as " solution
of GAULE."
A |- per cent, aqueous solution of sodium chloride saturated whilst
hot with sublimate was much recommended by HEIDENHAIN (Festschrift
f. Koelliker, 1892, p. 109).
The addition of sodium chloride allows a stronger solution to bo
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, Encyl. mik.
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 44 per cent., as isotonic (for
warm-blooded animals).
Liquid of Lang (Zool. Anzeiger, 1878, i, p. 14). — For Planaria.—
Distilled water . . . . . 100 parts.
Chloride of sodium . . . . 6 to 10 „
Acetic acid . . . . 6 to 8 ,,
Bichloride of mercury . . . 3 to 12 ,,
(Alum, in some cases . . . £ part.)
65. Alcoholic Solutions. — APATHY (Mikrotechnik, p. Ill) recom-
CHAPTER V. 47
mends a solution of 3 to 4 grms. of sublimate and O5 grm.
sodium chloride in 100 c.c. of 50 per cent, alcohol for general pur-
poses.
OHLMACHER (Journ. Exper. Medicine, ii, 6, 1897, p. 671) takes —
Absolute alcohol . . . . .80 parts.
Chloroform . . . . . 15 „
Glacial acetic acid . . . . 5 ,,
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. Acetone Solution. — HELD (Arch. Anat. Phys., Anat. Abth.,
1897, p. 227) fixes nerve-tissue in a 1 per cent, solution of sublimate
in 40 per cent, acetone, and washes out through increasingly con-
centrated grades of acetone.
67. Phenol Solution. — PAPPENHEIM (Arch. Path. Anat., clvii, 1899,
p. 23) shakes up carbolic acid with aqueous sublimate solution and
filters.
68. Ciaccio (Arch. Itdl. Anat. Embr., vi, 1907, p. 486) has an irrational
mixture of sublimate, iodine, and formol.
69. Mercuro-nitric Mixtures. — FRENZEL (Arch. miL Anat., xxvi,
1885, p. 232) recommends a half-saturated solution of sublimate in
80 per cent, alcohol, to which is added nitric acid in the proportion
of 1 drop to 1 or 2 c.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 un-
necessary.
GILSON'S Mixture (GILSON, in litt. 1895).
Nitric acid of 46° strength (this
would be sp. gr. 1-456, or 80 per
cent., nearly) . . . . .15 c.c.
Glacial acetic acid . . . . 4 „
Corrosive sublimate ... 20 grms.
60 per cent, alcohol . . . 100 c.c.
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.
48 FIXING AND HARDENING AGENTS.
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 recommended to beginners, as it is very easy to work with. For
some objects, as I found, the proportion of sublimate may be in-
creased with advantage.
KOSTANECKI and SIEDLECKI (Arch. mik. Anat., xlviii, 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.
PETRUNKEWITSCH (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. — EABL'S (Zeit. wiss. Mik., xi, 1894,
p. 165). — Sublimate, saturated solution in water, 1 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.
0. vom BATH (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, J to 1 per cent. Also the same with the
addition of 10 per cent, of 2 per cent, osmic acid solution.
71. Osmio-sublimate Mixtures. — MANN'S (Zeit. iviss. Mik., xi,
1894, p. 481) consists of a freshly prepared mixture of equal parts
of 1 per cent, osmic acid solution and saturated solution of sublimate
in normal salt solution (for nerve-centres). See "Mann-Kopsch
Method," § 693.
72. Chromo-sublimate. — Lo BIANCO (Mitth. Zool. Stat. Neapel,
ix, 3, 1890, p. 443). — Concentrated sublimate solution, 100 parts ;
1 per cent, chromic acid, 50 parts.
MANN (Verh. Anat. Ges., 12, 1898, p. 39) takes for nerve-cells equal
parts of 5 per cent, sublimate and 5 per cent, chromic acid.
73. Sublimate and Bichromate. — ZENKER'S Mixture (Milnchener
med. Wochenschr., xxiv., 1894, p. 534 ; quoted from MERCIER, Zeit.
iviss. MiL, xi, 4, 1894, p. 471). — Five per cent, of sublimate and
D per cent, of glacial acetic acid dissolved in solution of MULLER.
CHAPTER V. 49
Fix for several hours or overnight, wash out with water, treat the
tissues in bulk, or the sections with alcohol containing tincture of
iodine. Refer to § 684.
See also RETTERER, 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. SPULER
(Encycl. mik. Technik., 1st ed., p. 1280) says that they may be
avoided by removing the objects as soon as penetrated, and com-
pleting the hardening in liquid of MULLER. I recommend this
method.
HELLY (Zeit. wiss. Mik., xx, 1904, p. 413) omits the acetic acid,
and adds, immediately before -use, 5 per cent, of formol. This is a
splendid fixative for vertebrate material. Fix overnight, wash out
in running water for several hours. See footnote to § 684.
MAXIMOW (ib., xxvi, 1909, p. 179) 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. Hie. Sci., 1895, p. 287) takes equal parts of
saturated solution of sublimate in normal salt solution, and of liquid of
Miiller, or 5 per cent, solution of bichromate.
HOYER (Arch. Mikr. Anat., liv, 1899, p. 97) takes 1 part 5 per cent,
sublimate and 2 of 3 per cent, bichromate.
KOHN (ib., Ixx, 1907, p. 273) takes 5 parts 5 per cent, sublimate, 15
parts 3| per cent, bichromate, and 1 part acetic acid.
74. Sublamin (Ethylendiamin Sulphate of Mercury) is recommended
in 5 per cent, solution by KLINGMULLEB and VEIEL (Zeit. 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 true
platinic chloride, or tetrachloride, PtCl4, but the compound H2PtCl6,
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 — 0-5 per cent. — may be kept in the light).
It appears that some authors have stated that they were using
platinous chloride, PtCl2, but that is not possible, as this salt is not
soluble in water.
RABL (Morph. Jahrb., x, 1884, p. 216) employed an aqueous
solution of 1 : 300. The objects remained in it for twenty-four 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 " colours is
M. 4
50 FIXING AND HARDENING AGENTS.
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
Acid" and "Formol."
76. RABL (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.
LENHOSSEK (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. Ann., 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 (EISEN, Zeit. wiss. Mik., xiv, 1897, p. 195).—
Solution of | or £ per cent., acidified with 1 per cent, of glacial acetic
acid.
With the ovotestis of the snail, I have obtained about the worst
fixation I have ever seen, but with the testis of Triton much better
results.
79. Osmium Chloride (EISEN, Journ. of Morph., xvii, 1900). — Solution
of £ to ^ per cent. From specimens I have seen I should say it is
useless.
80. Perchloride of Iron (FoL, Zeit. 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 ( Zeit. wiss.
Mik., vi, 1889, p. 187).
81. Iron Alum. — STRONG (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 Zinc is sometimes used for hardening brain (see
Part II). GILSON (La Cellule, vi, 1890, p. 122) has used it as a fixative
for the silk glands of Lepidoptera, as follows :
Glacial acetic acid . . . . . 5 c.c.
Nitric acid of 46° (or 80 per cent, nearly) . 5 „
Alcohol of 80 per cent. .... 100 „
DistiUed water , . . . . . 300 „
Dry chloride of zinc .... 20 grins.
CHAPTER V. 51
83. Iodine. — KENT (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 potassium . . . . 6 „
Iodine ....... 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. See also under Goodrich's Iodine-Bourn method.
Very small objects may be instantaneously fixed by means of vapour
of Iodine. Crystals of iodine may be heated in a test-tube till the
vapours are given off ; then on inclining the tube the heavy vapours
may be made to flow over the objects arranged on a slide. The slide
should then be warmed to about 40° C. for one to three minutes in order
to evaporate the iodine from the objects, which may then be mounted
or otherwise treated as desired (OVERTON, Zeit. wiss. MiJc., vii, 1890,
p. 14).
Organic Acids, and other Agents.
84. Acetic Acid. — A substance most injurious to the finer elements
of the cytoplasm ; in some cases it is indicated for a study of the
nuclear elements. 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 1 per cent. Strengths of 5 per cent, and more
bring out the nuclein structures clearly at first, but after a time
cause them to swell and become pale, which is not the case with the
weaker strengths (ibid., p. 103). The strong acid is, however, a
valuable fixative of certain objects, which it kills with the utmost
rapidity, and leaves fixed in a state of extension.
The modus opemndi of VAN BENEDEN 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 30 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. LINDSAY JOHNSON (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. S. Lo BIANCO adds to his " concen-
trated " (49 per cent.) acid one-tenth of a 1 per cent, solution of
chromic acid. He finds that even this small proportion of chromic
4—2
52 FIXING AND HARDENING AGENTS.
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 killing, 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 to 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, 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, iii, 1886, p. 6 ; and ibid.,
1887, p. 276; v. BENEDEN et NEYT, Bull. Ac. Sci. Belg., xiv, 1887,
p. 218 ; ZACHARIAS, Anat. Anz., iii, 1888, pp. 24 — 27 ; v. GEHTJCH-
TEN, ibid., 8, p. 227). — CARNOY has given two formulae for this
important reagent. The first is —
Glacial acetic acid . . . . 1 part.
Absolute alcohol . . . .3 parts.
The second is —
Glacial acetic aci'd .... 1 part.
Absolute alcohol .... 6 parts.
Chloroform . . . . . 3 ,,
The addition of chloroform is said to render the action of the
mixture more rapid.
V. BENEDEN and NEYT take equal volumes of glacial acid and
absolute alcohol.
ZACHARIAS takes-
Glacial acetic acid .... 1 part.
Absolute alcohol . . .4 parts.
Osmic acid . . . . .a few drops.
Acetic alcohol is one of the most penetrating and quickly acting
fixatives known. It preserves both nuclei and cytoplasm, 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.
CHAPTER V. 53
86. Acetic Alcohol with Sublimate. — CARNOY and LEBRUN (La
Cellule, xiii, 1, 1887, p. 68, due to GILSON).
Absolute alcohol . . . . . 1vol.'
Glacial acetic acid . . . . . 1 „
Chloroform . . . . . . . 1 „
Sublimate to saturation.
(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.
G. S. SANSOM'S Carnoy Modification. —
Absolute alcohol . . . . .65 c.c.
Glac. acetic acid . . • . . 5 „
Chloroform 30 ,,
Corr. subl. to saturation. Leave ten minutes to half an hour ; wash
in iodine absolute, then absolute. (Personal communication.)
Eminently suitable for study of vertebrate material. I have seen
some really brilliant results obtained by the use of this fluid (§ 590).
87. Triehlor-aeetic Acid (HOLMGREN, Anat. Hefte, xviii, 1901, H.
2). — Five per cent, solution in water. Fix (nerve-cells) for eight to
twenty-four hours, wash out with alcohol. See also HEIDENHAIN, Zeit.
wiss. Mikr., xxii, 1905, p. 321, 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 " gubtriessig."
88. Triehlor-aeetie Fluid for Batrachia (CHAMPY, Arch. d. Zool
Exper. et Gen., t. lii, 1913).—
Carbolic acid cryst. in sat. aq. sol. . . 15 parts.
Formol, 40 per cent. . . . . 4 „
Trichlor-acetic acid, 20 per cent. . . 1-5 „
Outside of tissue often bad, inner parts better.
89. Salicylic 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 . . . . . 75 „
Crystallised acetic acid .... 1 grm.
Acetate of copper .... . 0-30 „
Chloride of copper 0-30 „
54 FIXING AND HARDENING AGENTS.
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 instantaneously and perfectly with
methyl green. Osmic acid may be added to the liquid to increase
the fixing action. For cytological researches a valuable medium.
91. Nitrate of Copper (G-ILSON, 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 ; cf. GILSON, 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 (Sitzb. 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 em-
ployed in the form of a strong solution whenever it is desired to make
sections or other preparations of tissues with the elements in situ, 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 hsemalum, carmalum,
methyl green.
Washing out is facilitated by heat, the extraction being about
twice as rapid at 40° C. as at the normal temperature (FoL).
It has been found by JELINEK (Zeit. iviss. 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.
CHAPTER V. 55
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, Boraxcarmine, 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. Soe. Micr., 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 1 per cent, of acetic acid. According to my experience, the
results are miserable.
ZIMMER'S mixture (from DEEGENER, Zool. Jahrb., Abth. Morph., xxvii,
1909, p. 634). — Saturated aqueous solution of picric acid, 10 parts ;
absolute alcohol, 9 ; acetic acid, 1.
96. Picro-sulphuric Acid (KLEINENBERG, Quart. Journ. Mic. Sci.,
April, 1879, p. 208 ; MAYER, Mitt. Zool. Stat. Neapel, ii, 1880, p. 2).—
MAYER 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.
I hold 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 (MAYER, Mitth. Zool. Stat. Neapel, 1881,
p. 6)-
Water 100 vols.
Nitric acid (of 25 per cent. N205) . . 5 ,,
Picric acid, as much as will dissolve.
Properties . of this fluid similar to those of pier o -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 Palinums, it gives better results than
nitric, picric, or picro-sulphuric acid. I myself consider it distinctly
superior to picro-sulphuric for most things. See Hill's fluid, § 586.
56 FIXING AND HARDENING AGENTS.
98. Picro-hydrochloric Acid (MAYER, ibid.).—
Water 100 vols.
Hydrochloric acid (of 25 per cent. HC1) . 8 ,,
Picric acid, as much as will dissolve.
99. Picro-chromic Acid (FoL, Lehrb., p. 100).—
Picric acid, sol. sat. in water . . .10 vols.
1 per cent, chromic acid solution . . 25 ,,
Water 65 „
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.
KAWITZ (Leitfaden, 1895, p. 24) takes 1 part of picro-nitric acid,
and 4 parts 1 per cent, chromic acid. Wash out in 70 per cent,
alcohol.
100. Picro-osmic Acid. — FLEMMING (Zells. Kern u. Zelltli., 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.
0. VOM RATH ( Anat. Anz., xi, 1895, p. 289) adds to 200 c.c. of saturated
aqueous solution of picric acid, 12 c.c. of 2 per cent, solution of osmic
acid, and 2 c.c. of glacial acetic acid.
EAWITZ (Leitfaden, p. 24) takes picro-nitric acid, 6 vols. ; 2 per cent,
osmic acid, 1 vol. Fix for half to three hours. Transfer direct to 70 per
cent, alcohol.
101. Piero-platinie and Picro-platin-osmic Mixtures. — 0. VOM RATH
(loo. cit., last §, pp. 282, 285) makes a picro -platinic mixture with
200 c.c. saturated aqueous solution of picric acid, 1 grm . 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 MIXTURES. See §§ 70 and 110 to 112.
Other Fixing and Hardening Agents.
102. Alcohol. — For fixing only two grades of alcohol should be
employed — very weak, or absolute. Absolute alcohol 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
CHAPTER V.
57
enhancing their penetrating power ; 70 per cent, is a good grade for
this purpose.
Table for diluting alcohol (after GAY-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.
Weaker
grade
required.
ORIGINAL GRADE.
90
p. 100.
85 80
p. 100. p. 100.
75
p. 100.
70
p. 100.
65
p. 100.
60
p. 100.
55
p. 100.
50
p. 100.
p. 100.
85
6-56
— —
— —
80
13-79
6-83
75
21-89
14-48
7-20
70
31-05
23-14
15-35
7-64
65
41-53
33-03
24-66
16-37
8-15
60
53-65
44-48
35-44
26-47
17-58
8-76
• __
55
67-87
57-90
48-07
38-32
28-63
19-02
9-47
50
84-71
73-90
63-04
52-43
41-73
31-25
20-47
10-35
45 1 105-34
93-30
81-38
69-54
57-78
46-09
34-46
22-90
11-41
40 130-80
117-34
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
87-93
73-08
58-31
43-59
30
206-22
188-57 171-05
153-61
136-04
118-94
101-71
84-54
67-45
Alcohol is an easily oxidisable substance. Chromic acid, for
instance, easily oxidises it, first into aldehyde, and then into acetic
acid. It follows that alcohol should not be combined 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 §§ 38, 39, 48.
F 'or 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 penetration. For hardening it is an im-
58 FIXING AND HARDENING AGENTS.
portant 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, alcohol. Large quantities of alcohol
should be taken. The alcohol should be frequently changed, or the
tissue should be suspended near the top of it (§ 34). 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 proportion of
alcohol. Alum-carmine is a good stain for small specimens so fixed.
For preservation, the object should be put into a weaker alcohol,
90 per cent, or less.
As to the supposed superiority of absolute alcohol over ordinary
strong alcohol, see last § ; and amongst authors upholding its superiority,
see besides KANVIER, MAYER (Mitth. Zool. Stat. Neapel, ii, 1880, p. 7) ;
BRUEL (Zool. Jahrb., Abth. Morph., x, 1897, p. 569); and VAN EEES
(ibid., iii, 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 011 exposure to
air. Fol recommends that a little quicklime be kept in it. This absorbs
part at least of the moisture drawn by it from the air.
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 is repeated until the fresh cupric sulphate no longer becomes
conspicuously blue on contact with the alcohol ; or until, on a drop of
the alcohol being mixed with a drop of turpentine, no particles of water
can be seen in it under the microscope. The cupric sulphate is prepared
by calcining common blue vitriol in a porcelain capsule over a flame
until it becomes white, and then reducing it to powder (see Proc. Acad.
Nat. Sci. Philad., 1884, p. 27 ; Journ. Eoy. Mic. Soc., 1884, pp. 322 and
984).
Test for the presence of water (YvON, G. 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 ALCOHOL. It consivsts of two parts of water and one part
CHAPTER V. 59
of alcohol of 90 per cent, (and not of absolute alcohol). See the
Traite Technique of Ranvier, p. 241, et 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 conveniently 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. 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
brain. See Comptes Rendus hebd. de la Soc. de Biologie, 8 ser., t. iv,
1887, p. 622.
This substance is strongly alkaline, and, either pure or diluted with
water, dissolves many albumens and fats. It causes considerable
shrinkage of nuclei (not so much of cytoplasm). It is now in much use
in certain neuro-fibril stains, see BIELSCHOWSKY and KAM6N. It is
soluble in water and in alcohol. Pure, it will harden and dehydrate
small brains in a week.
106. Acetone is said to harden very rapidly. It precipitates
lipins, and may yet prove an important reagent. SCHOLZ (Zeit.
iviss. Mikr.. xxii, 1905, p. 415) fixes small objects in warm acetone
for half an hour to an hour and brings them direct, or through
alcohol and ether, into celloidin.
Similarly Fuss (Arch. path. Anat., clxxxv, 1906, p. 5), using it
cold, and LINTWAREW (ibid., ccvi, 1911, p. 36) for erythrocytes, in
which it preserves the hemoglobin.
107. Lucidol. — This is a proprietary name applied to peroxide of
benzol (C6H5 — C0)202. It is a white powder insoluble in water,
but soluble in acetone and pyridin ; heated, the white powder first
of all melts and then explodes. The introduction of " Lucidol " is
clue to EHRLICH, who suggested it to SZECSI. " Lucidol " must
share with its solvents, acetone and pyridin, their defects as fixing
agents. Acetone, especially tends to tear cells to pieces, owing to
its attraction for water. So far, there have been very few references
to the use of " Lucidol " in histological literature. LANGERON
(C. R. Soc. de Biol, Ixxvi, 1914) claims that " Lucidol " is especially
convenient for work on blood, faeces and cell smears, and two of
SZECSI'S methods are given below. It should be pointed out that
the Lucidol-acetone solution gives a very active penetration, and
unlike CARNOY'S or SCHAUDINN'S fluids, does not dissolve away lipins.
60 FIXING AND HARDENING AGENTS.
In all probability some method of mordanting the fixed material in
either K2Cr207 or Cr03 dissolved in a suitable solvent might help
to " set " the lipins, and so avoid their dissolution when the material
or smears are brought into alcohols.
SZECSI (Deutsche meet. Woch., 1913) uses two solutions : one
consists of 10 grms. of " Lucidol " to 100 c.c. of acetone, the other
12 grms. of " Lucidol " to 100 c.c. of pyridin. Both solutions are
used for smears, or for pieces of tissue. After fixation, the latter
are washed out in a mixture of acetone 3 parts, and xylol or toluol
2 parts ; and then transferred to methyl alcohol or pure xylol
according to whether one is de.aling with smears, or tissue for
embedding in wax.
For tissues proceed as follows : — Fix small pieces at room tem-
perature in the acetone solution for about four or six hours, or in
the pyridin solution for ten to twelve hours. In each case transfer
to the above-mentioned acetone xylol mixture for several hours,
but not longer than 'ten. Clear in pure xylol or toluol, embed in
paraffin wax. Sections can be stained as desirable.
For the treatment of smears, etc., see under " Blood."
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-
alcohol. " Formol," " Formalin," and " Formalose " are com-
mercial 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 formol.
Solutions of formaldehyde sometimes decompose partially or
entirely, with formation of a white deposit of paraformaldehyde.
FISH 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 saturation
CHAPTER V. 61
with magnesium or sodium carbonate ; some workers use lithium
carbonate, but this should not be used for BOUIN mixtures : 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, 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. BLUM, in Anat. Anz., xi, 1896, p. 718 ;
BENEDECENTI, in Arch. Anat. u. Phys., Abth, 1897, p. 219 ; GEROTA,
in Intern. Monatschr. Anat., xiii, 1896, p. 108 ; Zeit. wiss. Mik.,
xiii, p. 311 ; SJOBRING in Anat. Anz., xvii, 1900, p. 274 ; and BLUM,
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 mixtures.
It is a powerful reducing agent, and therefore incompatible 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.
For cytological purposes a fixation of at least two days seems indi-
cated : this applies especially to gonads which are notoriously
difficult to preserve in formol. The strengths used in CAJAL'S and
DA FANG'S formol silver nitrate GOLGI apparatus methods, generally
give fine results for tissues other than genital. For these, injection
fixation may be indicated. See also § 141. MAYER 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, I
think this exaggerated, for I have found enormous over-fixation
with solutions of 1 to 2 vols. of water. Wash out 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, consider-
able volumes of liquid should be taken, and the liquid should be
62 FIXING AND HARDENING AGENTS.
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 varies greatly
with different tissues. Mucin is not precipitated and remains trans-
parent. Fat is not dissolved (see §§ 768 et seq.). Micro-organisms
retain their specific staining reactions. Formaldehyde is said to
harden celloidin as well as gelatin, and to be useful for celloidin-
imbedding (BLUM, 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 (LAVDOWSKY, 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.
GULLAND (Zeit. wiss. -Milcr., xvii, 1900, p. 222) takes (for blood)
1 part formol and 9 parts of alcohol.
BLES (Trans. Roy. Soc. Edinburgh, xli, 1905, p. 792) takes 7 of formol,
90 of alcohol of 70 per cent., and 3 of acetic acid.
TELLYESNICZKY (Encycl. mikr. Techn., i, p. 472) takes 5 of forrnol,
100 of alcohol of 70 per cent., and 5 of acetic acid.
110. Picro-Formol. — P. BOUIN (Phenomenes cytologiques anormaux
dans L' Histogenese, etc., Nancy, 1897, p. 19) recommends —
Picric acid, saturated aqueous sol. . . 75 parts.
Formol 25 „
Acetic acid . . . . . 5 „
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 pre-
served, staining qualities admirable, especially with iron-hematoxylin
and Saiirefuchsin. See also " Cytology " sections, § 656, under
heading of " Chromosomes and Urea," and GARNIER, Bibl. Anat.,
v, 1898, p. 279.
The formulae of GRAF (State Hosp. Bull. New York, 1897 ; Journ. Roy.
Mie. 8oc., 1898, p. 492) are in my view too weak.
MOKEAUX (BiU. Anat., 1910, p. 265) takes 15 parts formol, 85 of
trichlor- acetic acid of 3 per cent., and picric acid to saturation.
CHAPTER V. 63
111. Piero-platinic Formol (M. and P. BOUIN, Bibl. Anal, 1898,
f. 2, p. 2).—
Platinum chloride, 1 per cent. sol. . . 20 parts.
Picric acid, saturated sol. . . . 20 „
Formol 10
Formic or acetic acid . . . 5 „
The mixture does not keep more than a day or two, and it is probably
inferior.
BOUIN also (Arch. Biol., xvii, 1900, p. 211) simply substitutes forinol
for the osmic acid in HERMANN'S mixture, § 45.
112. Sublimate Formol (M. and P. BOUIN, loc. cit.). — A similar
mixture, in which sublimate of 1 per cent, is substituted for the platinum
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.
SPULER (Encycl. mik. TecJmik., 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 2| grms.
sublimate, 1 grm. picric acid, 5 c.c. formol, and 100 c.c. water, or (Methods,
etc., p. 97) for all tissues 2| grms. sublimate, 20 c.c. formol, and 80 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
sublimate, 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 OBTH (Bed.
klin. Wochenschr., 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.
MOELLER (Zeit. wiss. ZooL, Ixvi, 1899, p. 85) takes 1 vol. of formol
and 4 of 3 per cent, bichromate (for the intestine of mammals).
HELD (Abk. Sachs. 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 MOREL and BASSAL, Journ.
Anat. Phys., xlv, 1909, p. 632, and HELLY and MAXIMOW formulae.
Look up section on " Mitochondria," especially paragraphs on
REGAUD and SOHEIDDE, §§ 685 to 687.
114. Chromic Acid Formol. — Lo BIANCO fixes marine animals for half
to one hour in 10 parts of 1 per cent, chromic acid with 1 of forinol and
9 of sea-water, and passes into graded alcohols.
64 FIXING AND HARDENING AGENTS.
MARCHOUX (from PEREZ, Arch. Zool. Exper., 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).
These mixtures are neither so good nor reliable as Bouin's picro-
formol.
115. Copper Formol.— NELIS (Bull. Acad. Sc. Belg., 1899 (1900),
p. 726) fixes spinal ganglia for twenty-four hours in 1 litre of 7 per cent,
formol with 5 c.c. of acetic acid, 20 grms. of cupric sulphate, and sub-
limate to saturation.
STAFFERS (La Cellule, xxv, 1909, p. 356) used (for Sympoda) a mixture
of G-ILSON'S : 100 parts of formol of 5 per cent, with 2 of nitrate of
copper.
STRONG (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. Neapel,
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. — BING and ELLERMANN (Arch. Anat. Phys.,
Phys. Abth., 1901, p. 260) fix medullated nerves in 9 parts of acetone
with 1 of formol. ,
CHAPTER VI.
DE-ALCOHOLISATION AND CLEARING AGENTS.
118. Introduction.— De-alcpholisation 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 imbedding, 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 micro-
scopic preparations transparent by penetrating amongst the highly
refracting elements of which the tissues are composed, 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-alcoholi-
sation 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.
NEELSEN and SCHIEFFERDECKER (Arch. Anat. Phys., 1882, p. 206)
examined a large series of ethereal oils (prepared by Schimmel & Co.,
Leipzig), with the object of finding a not too expensive substance
that should combine the properties 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-wood.
60 DE-ALCOHOL1SATION AND CLEARING AGENTS.
To these should be added the others recommended in the following
paragraphs.
See also the paper of JORDAN (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-alcoho-
lising 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 under 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 mixture 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 of all 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 com-
bination between the essential oil and moisture, derived, I think,
rather from the air than from the objects themselves. The cloudi-
ness can usually be removed by warming (as pointed out by HATCHETT
JACKSON, Zool. Anzeig., 1889, p. 630), but in certain moist states of
the atmosphere it may persist, notwithstanding continued warming.
It is for this reason that I advise that clearing be done, whenever
possible, in shallow corked tubes, under which conditions the pheno-
menon 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 dissections 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.
CHAPTER VI. 67
121. Cedar Oil (NEELSEN and SCHIEFFERDECKEK, loc. tit., § 118).—
Clears readily tissues in 95 per cent, alcohol -without 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. I have examined the clearing properties 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 to me. 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. Doubt-
less it is, in general, best to use a pale oil, provided it be pure ; but
it is not always easy to obtain a light-coloured oil that is pure.
Clove oil passes very readily from yellow to brown with age, so that
in choosing a colourless sample you run great risk of obtaining an
adulterated sample, for clove oil is one of the most adulterated
substances in commerce.
Clove oil does not easily spread itself over the surface of a slide,
but has a tendency to form very convex drops. This property makes
it a very convenient medium for making minute 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 dissections.
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 BEHRENS
(Tabellen, 3rd ed., 1898, p. 33), it will clear from alcohol of 74 per
cent.
It has a high index of refraction, and clears objects more than
balsam mounting media. It dissolves celloidin (or collodion), 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.
6—2
68 DE-ALCOHOLISATION AND CLEARING AGENTS.
124. Oil of Bergamot (SCHIEFFERDECKER, 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,
haying a lower index than even oil of turpentine.
SUCHANNEK (Zeit. wiss. Mik., 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 DEE STRICHT (Arch, de Biol., xii, 1892, p. 741) says that
bergamot oil will, with time, dissolve out the fatty granules of
certain ova.
125. Oil of Origanum (NEELSEN and SCHIEFFERDECKER, Arch.
Anat. Phys., 1882, p. 204). — Ninety-five per cent, alcohol prepara-
tions are cleared quickly, and so are celloidin sections, without
solution of the celloidin. Anilin colours are somewhat extracted.
For work with celloidin sections care should be taken to obtain
01. Origani Cretici (" Spanisches Hopfenol "), not 01. Orig. Gallici
(v. GIBSON ; 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 Formula, etc., p. 81, says that origa-
num oil (meaning the commercial product) is nothing but oil of
white thyme more or less adulterated (see next §), and that the
product sold as 01. Origani Cretici is probably oil of marjoram.
126. Oil of Thyme.— FISH (Proc. Amer. Mic. Soc., 1893 ; Zeit.
wiss. Mik., xi, p. 503), following BUMPUS, says that for most of the
purposes for which origanum oil has been recommended, oil of
thyme will do just as well if not better. The red oil is just as efficient
as the white for clearing.
Schimmel & 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.,
Ergdnzungsh, 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 1-53. It is, unfor-
tunately, very sensitive to water.
128. Sandal-wood Oil (NEELSEN and SCHIEFFERDECKER. loc. cit.).—
Yery 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 LEBRUN
CHAPTER VI. 69
(La Cellule, xiii, 1897, p. 71) have found it useful for clearing celloidin
sections. It dissolves 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 and benzol,
are preferable for this purpose. If used for alcohol objects, it causes
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 & Co.) is recommended by
MAYER, Zeit. wiss. Mikr., xxvi, 1910, p. 523. Clears from alcohol
of 90 per cent., or even 80 per cent. One part xylol and^4
terpinol has been much used lately.
132. Carbolic Acid. — Best used in concentrated solution in alcohol.
Clears instantaneously, even very watery preparations. 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.
GAGE'S Mixture (Proc. Amer. Soc. Micr., 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 sections, for which
it is a very good medium.
134. Anilin Oil. — Common anilin oil will readily dear sections from
70 per cent, alcohol, and with certain precautions (for which see the
paper of SUCHANNEK 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 SUCHANNEK, 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 something else
(chloroform or xylol for instance for some hours), as if not removed
it will brown both the tissues and the mounting medium.
70 DE-ALCOHOLISATION AND CLEARING AGENTS.
135. Xylol, Benzol, Toluol, Chloroform. — Too volatile to be
recommendable as clearing agents in which it is desired to examine
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
(SQUIRE, Methods and Formulce, p. 20). Chloroform is injurious to
some delicate stains, but is in other respects an excellent de-alcoholisa-
tion 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 Imbedding
Methods are employed for a twofold end. Firstly, they enable us
to surround an object, too small or too delicate to be firmly held by
the fingers or by any 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 included object may be cut into sufficiently thin
slices without distortion. Secondly, they enable us to fill out with
the imbedding mass the natural cavities of the object, so that their
lining membranes or other structures contained in them may be
duly cut in situ ; and, further, they enable us 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 in 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 Imbedding Methods ; or the
object is saturated with some substance which whilst in solution is
sufficiently fluid to penetrate the object to be imbedded, whilst, after
the evaporation 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.
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 tne 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
72 IMBEDDING METHODS.
•
sections. But this only applies 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 STRASSER (Zeit. wiss. Mik., ix, 1892, p. 7) has obtained
series of frontal sections 30 p thick through the entire human brain,
in paraffin blocks measuring 10 X 15 cm. And MAYER, with the
Tetrander microtome, has obtained series of only 7 '5 JJL with a surface
of 4J X 3 cm.
For very large objects celloidin is safer, because it does not split,
and presents advantages for the manipulation of the sections ob-
tained. For all classes of objects it has the advantages of affording
a transparent mass (which facilitates orientation 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 avoid dehydrating tissues, and to.
apply chemical 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 means of pins and the tray filled with melted
mass at one pour. The pins are removed when the mass is cold.
In either case, when the mass is cold the paper is removed from it
before cutting.
To make paper trays proceed as follows. Take a piece of stout
paper or thin cardboard, of the shape of the annexed figure (Fig. 1) ;
thin (foreign) post-cards do very well indeed. Fold it along the
lines a a' and b b', then along c c and d d', taking care to fold always
the same way. Then make the folds A A', B B', C C", D D', still
folding the same way. To do this you apply A c against A a, and
pinch out the line A A', and so on for the remaining angles. This
done, you have an imperfect tray with dogs' ears at the angles. To
CHAPTER VII.
73
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 (MAYER) is described in the
Grundzilge, LEE and MAYER, 4th ed., p. 77.
GIESBRECHT now makes trays of photographic films, which, being
transparent, facilitate orientation under the dissecting microscope.
Ar
B'
\
77
/
\
A B
/:
<•'
(
^
a
x
NSX
x
'•~
%
D
c
i
U'
FIG. 1.
FIG. 2.
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 from floating when
the whole is thrown into spirit or other liquor for hardening (Fig. 2).
Or you may use short lengths of solid lead rod instead of cork.
LEUCKF ART'S Imbedding Boxes are made of two pieces of type-
metal (Fig. 3). Each of these pieces has the form of a carpenter's
" square " with the end of the shorter arm triangularly enlarged
outwards. The box is constructed by placing the two pieces to-
gether on a plate of glass which has been wetted with glycerine and
gently warmed. The area of the box will vary according to the
74
IMBEDDING METHODS.
FIG. 3.
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 1 centimetre in height, and
one of 2 centimetres, each being 8 centi-
metres in length, and 3 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. Stat.
Neapel, iv, 1883, p. 429) first smears the glass
plate with glycerine, then arranges the metal
" squares," and then fills the box with col-
lodion, which is poured out again immedi-
ately. As the ether evaporates, a thin layer
of collodion remains behind, which suffices
to keep the paraffin from running out. Even
without the collodion, the mere cooling of
the paraffin 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 paraffin may be kept in a liquid
state by warming now and then over a spirit lamp, and small objects
be placed in any desired position under the microscope (Journ. Roy.
Mic. Soc. [N.S.], ii, p. 880).
A lighter form of " squares," made of brass, and devised by
ANDRES, GIESBRECHT, and MAYER, is described loc. cit. (See Journ.
Roy. Mic. Soc., 1883, p. 913.) A more complicated sort is described
by WILSON in Zeit. wiss. Mik., xxvii, 1910, p. 228, for use with
imbedded threads to serve as orientation guides. See " Orientation."
FRANKL (Zeit. wiss. Mik., xiii, 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
any one 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 Zeit. wiss. Mik., 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 con-
CHAPTER VII. 75
taining molten paraffin. After this has been solidified 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. SAMTEB (Zeit. 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 (ibid., 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., xiii, p. 200, a paper by SCHYDLOWSKI ;
and in Zeit. wiss. Zool., Iviii, 1897, p. 144, a process of BORGERT.
BORGERT (Zeit. wiss. Zool., Iviii, 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 LEFEVRE,
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
through chloroform into paraffin in a small glass tube, and after cooling
breaks the tube and so obtains a cylinder of paraffin with the objects
ready for cutting.
HOYER (Arch. mile. Anat., liv, 1899, p. 98) performs all the operations
in a glass cylinder (5 centimetres long and 7 millimetres wide), open at
both ends, but 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 parchment 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 (Zeit. wiss. Mikr., xxiv, 1907, p. 130) takes the gelatin capsules
used by chemists ; after cooling in water the gelatin swells and is easily
removed.
MEVES (Arch. mikr. Anat., Ixxx, Abth. ii, 1912, p. 85) employs wedge-
shaped capsules made by G. Pohl, Schonbaum, Bez, Dantzig.
CHAPTER VIII.
IMBEDDING METHODS — PARAFFIN AND OTHER FUSION MASSES.
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 GRAEFE (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
APATHY, at 20° C. benzol dissolves 8 parts per cent., chloroform 10,
toluol 10, xylol 12, oil of turpentine 8, cedar oil 4 to 6, bergamot oil
0-5 to 3, creosote and clove oil hardly any. Acetone, according to
MAYER, 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 brittle.
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 HOLL (Zool. Anz.,
1885, p. 223).
Xylol is said by M. HEIDENHAIN (Kern, und Protoplasm, 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 oil of cedar, or 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
CHAPTER VIII. 77
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 WEBSTER (Journ. Anat. and
Physiol, xxv, 1891, p. 278).
FIELD and MARTIN (Zeit. wiss. Mik., xi, 1894, p. 10) recommend a
light petroleum known as " petroleum -aether." It is highly volatile,
and thus a cause of shrinkage.
Sulphide of carbon has been recommended by HEIDENHAIN (Zeit.
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. See under "Teeth" and "Chitin."
Carbon tetrachloride has been recommended by PLECNIK (op. cit., xix,
1903, p. 328) and PRANTER (ibid., p. 329) on the ground of not dissolving
out osmium -blackened fats.
MAYER finds it no better than benzol.
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 imbedding. . It penetrates
rapidly, preserves delicate structure better than any clearing agent
known to me, does not make tissues brittle, even though they may
be kept for weeks or months in it, and has the great advantage that
if it be not entirely removed from the tissues in the paraffin bath it
will not seriously impair the cutting consistency of the mass ;
indeed, I fancy it sometimes improves it by rendering it less brittle.
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 voluminous
to have brought over with them a notable quantity of clearing
agent. If the objects have been for a very long time — months or
78 IMBEDDING METHODS.
years — in the cedar oil, so that this has become thick, I remove it
partially or entirely by soaking in xylol (thirty minutes to several
hours) before putting into the paraffin. But with fresh oil of cedar
I find no advantage in doing so.
GIESBRECHT'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 to 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.
MAYER (Grundziige, LEE 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.
APATHY (Mikrotechnik, pp. 149, 150) first clears with oil 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 com-
paratively free from paraffin while the upper part is nearly pure
CHAPTER VIII. 79
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 paraffin dry— that is, protected from
vapour of water during the bath.
It is still more important to keep it as nearly as possible at melting-
point. If it be heated for some time to a point much over its normal
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 preser-
vation 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 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 (HEIDECKE, Jena.
Zeit., xxxviii, 1904, p. 506 ; MAYER, Grundzuge, LEE and MAYER,
1910, p. 85 ; BRINKMANN, Mitth. Zool. Stat. Neapel, xvi, 1903,
p. 367, three to five days for uter.us of Selachians ; MULLER, Arch,
mikr. Anat.} Ixix, 1906, p. 3, for lungs of mammals ; Poso, Esperienze
microtechniche, Napoli, 1910, p. 29, five to twelve days for uterus
and placenta of Homo). 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 Maye
or " Naples water-bath," which will be found described at p. 146 of
Journ. Eoy. Mic. Soc., 1883, or CARPENTER'S The Microscope, p. 462.
An extremely simple stove, which any one can make for himself, if
described in Centralbl. BaU., xlv, 1907, p. 191 (see Journ. Roy. Mic
1908 p 109). For others, see the price-lists of the instrument i
80 IMBEDDING METHODS.
especially JUNG, and GRUBLER and HOLLBORN ; and the descriptions in
the technical journals.
141. Imbedding IN VACUO. — There are objects which, on account of
their consistency or their size, cannot be penetrated by paraffin in the
ordinary way, even after hours or days in the bath. For such objects
the method of imbedding 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 HOFFMANN is described and figured at p. 230 of Zool. Anz.,
1884. In this arrangement the vacuum is produced by means of a
pneumatic water aspiration pump, the vessel containing the paraffin
being placed in a desiccator heated by a water- bath and furnished with
a tube that brings it into communication with the suction apparatus.
FRANCOTTE (Bull.* Soc. Belg. Mic., 1884, p. 45) produces the requisite
vacuum by the condensation of steam.
FOL (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 paraffin.
See also PRINGLE, in Journ. Path, and Bacterial, 1892, p. 117, or
Journ. Roy. Mic. Soc., 1892, p. 893 ;* KOLSTER, in Zeit. wiss. Mik., xviii,
1901, p. 170 ; BERG, Zeit. wiss. Mik., xxvi, 1909, p. 209 ; FUHRMANN,
ibid., xxi, 1904, p. 462 ; KOLMER and WOLFF, ibid., xix, 1902, p. 148 ;
GEMMILL, Journ. Boy. Mic. Soc., 1911, p. 26.
142. Imbedding and Orientation. — As soon as the objects are
thoroughly saturated with paraffin they should be imbedded 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 paraffin, 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 advantages of the method
lie in the quickness and certainty with which it can be performed.
CHAPTER VIII. 81
In using the needle it is important to melt as little paraffin as possible
at one time, in order that that which is melted may cool again as
rapidly as possible.
KERR (Quart. Journ. Micr. Sc., xlv, 1901, p. 4) employs an
electrically heated needle.
The method of PATTEN (Zeit. 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 imbedded are cleared in
clove oil or oil of 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 WOOD WORTH, Bull. Mus. Comp. Zool, xxxviii, vol. xxv, 1893, p. 45.
A similar process has also been described by FIELD and MARTIN in
Zeit. wiss. Mik., xi, 1894, p. 11, small strips of gelatin being used instead
of paper.
MAYER also (Grundzdge, LEE and MAYER, 1910, p. 89) takes strips of
photographic gelatin, and lets the collodion set in benzol.
HOFFMANN (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 vess
The drops are caused to set in xylol. See also SAMTER, ibid., xiii, 1897,
p. 441 ; JORDAN, ibid., xvi, 1899, p. 33 ; and PETER, Verb. Anat. Ges.,
xiii Vers., 1899, p. 134.
ENTZ (Arch. Protistenk., xv, 1909, p. 98) orients in clove oil collo
on a cover-glass coated with paraffin, and puts the whole into chlorofc
in which the mixture sets into a sheet which can be detached.
DENNE (Journ. Appl Mic., iii, 1902, p. 888) imbeds on disks of papei
held at the bottom of srlass tubes containing the paraffin by ben
M. 6
82 IMBEDDING METHODS.
by means of which a cylinder of paraffin containing the object may be
lifted out as soon as cool.
WILSON (Zeit. wiss. 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, ibid., 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 crystallisation 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). MAYER cools the mass in
the paraffin-tight moulds (§ 137) 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 MEISSNER
(Zeit. wiss. Mik., xviii, 1902, p. 286). Similarly, HAHN, ibid., xxv, 1908,
p. 184, and KAPPERS, ibid., xxiv, 1907, p. 254. See also FARKAS, ibid.,
xxx, 1913, p. 168, for experiments on cooling methods.
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 homo-
geneous. The same should be done if any cavities present them-
selves 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
CHAPTER VIII. 83
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 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-edge : for it is in this position that it
will offer the least resistance to the blade,
and tend the least to make the edge bend
away or dig into it. This is specially impor-
tant with longitudinal sections of worms,
Amphioxus, embryos of vertebrates, and the
like. Most especially with a square-set knife
should the narrowest diameter of the object i<v,. 4.
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 NOACK'S simple apparatus for accurately orientating small
blocks, see Zeit. wiss. MiL, xv, 1899, p. 438, or Journ. Roy. Mic.
Soc., 132, 1899, p. 550.
For ETERNOD'S machine for trimming blocks to true cubes, sec /fit.
iviss. Mik., xv, p. 421, and for that of SCIIAFFER, ibid., 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 mak s
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 knives). The position is
84 IMBEDDING METHODS.
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.
The difference between the effect of the two positions is that the
oblique position affords a more acute-angled wedge than the transverse
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 inter-
mediate positions. (See the stereometrical constructions of these
relations by SCHIEFPERDECKER, op. cit., p. 115 ; and also with more
instructive figures, APATHY, " Ueber die Bedeutung des Messer-
halters in der Mikrotomie," in Sitzber. med.-natunc. Section d.
Siebenburgischen Museumvereins, Bd. xix, Heft 7. p. 1 (Kolozsvar,
1897, A. K. Ajtai).
For honing knives see SSOBOLEW, Zeit. wiss. Mik., xxvi, 1909, p. 65 ;
LENDVAI, ibid., p. 203 ; FUNCK, ibid., 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
* 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 SCHIEFFERDECKEB, Das MikrosTcop., 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.
CHAPTER VIII. 85
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 compression 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 tilt 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 elevation 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 APATHY, loc. cit.
supra. He concludes — (1) The knife should always be tilted some-
what more than enough to bring the back of the under cutting-facet
clear of the object. (2) It should in general be less tilted for hard
and brittle objects than for soft ones ; therefore, cceteris paribus,
less for paraffin than for celloidin. (3) The extent of useful tilt
varies between 0° and 16° or occasionally 20°. (4) Excessive tilt
causes rifts (longitudinal) in the paraffin, also furrows that in bad
cases split 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. Excessive tilt may often 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 only
enough for cutting ribbons with hard paraffin.
A knife with too little tilt will often cut a second section, or frag-
ments 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. This 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
80 IMBEDDING METHODS.
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 corning 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
recent papers in Zeit. wiss. Mik., also that of APATHY, in the paper
quoted above (very complicated), and especially the description of
the two latest of Jung, viz., his model I and model n, by MAYER and
SCHOEBEL, in Zeit. wiss. Mik., xvi, 1899, p. 29 (see figure of model /
in Journ. Roy. Mic. Soc., 132, 1899, p. 546). Also CARPENTER'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.
CHAPTER VIII. 87
becomes softer on that side, and the sections have a tendency to become
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. AUh., 1897, p. 345 ; VAN WALSEM, Zeit. wise. Mik.,
xi, 1894, p. 218 ; LENDENFELD, ibid., xviii, 1901, p. 18 ; KRAUSE, 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 sections ;
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.
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, 1883, p. 100 (SCHULTZE) ; Mitth. Zool. Stat. Neapel, iv,
1883, p. 429 (MAYER, ANDRES, and GIESBRECHT) ; Arch. m,\k. Anat.,
xxiii, 1884, p. 537 (DECKER) ; Bull. Soc. Belg. Mic., x, 1883, p. 55 (FRAN-
COTTE) ; The Microscope, February, 1884 (GAGE and SMITH) ; WHIT-
MAN'S Meth. in Mic. Anat., 1885, p. 91 ; Zeit. wiss. Mik., iv, 1887, p. 2
(STRASSER) ; ibid., x, 1893, p. 157 (BORN). The best are those
and Born.
I find that MAYER'S, beautifully made by JUNG, works admirably
and is most valuable.
Another plan is to allow the sections to roll, but to contn
rolling. To this end, the block of paraffin is pared to the shape of a
88 IMBEDDING METHODS.
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 be a
very open one — indeed, very nearly flat. Lay the coil on a slide
with this end downwards, warm gently, and the part containing the
object will unroll completely and lie quite flat.
ANILE (Glandole duodenali, Napoli, 1903, p. 51) and VASTARINI-
CRESI (Mori. 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, 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 (Grundzilge, LEE 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 cutting 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.
MARK (Amer. Natural, 1885, p. 628 ; cf. Journ. Roy. Mic. Soc.,
1885, p. 738) gives the following directions :
" Have ready a little very fluid collodion in a small bottle, through
CHAPTER VIII. 80
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 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 collodionised 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.
HEIDEB (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.
RABL (ibid., xi, 2, 1894, p. 170) employs superheated paraffin (of about
100° 0.). This has the advantage of filling up any cavities there may be
in the objects, and also of preventing the sections from rolling. A compli-
cated development of this process is described by LENDENFELD in Zeit.
wiss. Mik., 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 w:» in--
process (§ 149).
JORDAN (Zeit. wiss. Mik.) adds 5 drops of oil of cedar to 15 c.c. ol
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 si
90 IMBEDDING METHODS.
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 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 /x, 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 obtained
may be cleared and mounted at once if they are quite perfect, that
is, neither rolled nor creased nor compressed. But should they in
the least degree show any of these defects, they must first be unrolled
or smoothed, or expanded to their proper dimensions.
The most efficacious plan is combined treatment with fluid 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 below 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., xir, 1896, p. 447.
CHAPTER VIII. 91
150. Clearing and Mounting.- The sections having been duly
smoothed by one of these processes, and duly fixed to 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 4 parts of essence of
turpentine with 1 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 jut, it is //.//
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 paraffin, 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 be recommended.
Paraffin varies enormously in hardness according 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 expern-m •«•
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 J55°),
Rabl (56°), Mayer (58° to 60° in summer ; in winter about 56°, but
never less than 50°). Mayer points out that at Naples the tempera-
ture during five months of the summer and autumn is over 22C
the laboratory, sometimes over 30°. Temperatures such as these are
seldom realised in the British Isles, and, whilst I quite admit that
such hard paraffin may have its raison d'etre for Naples, I hold that i
that very reason it is in general unnecessarily hard for cooler climai
My recommendation of a relatively soft paraffin refers to wor
with the Thoma sliding microtome. Microtomes with fixed knives,
92 IMBEDDING METHODS.
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. Mik., ii, 1885, p. 300) 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 commerce.
Intermediate sorts may be made by mixing hard and soft paraffin.
I find that 2 parts of paraffin melting at 50° with 1 of paraffin
melting at 36° C. give a mass melting at 48° C., and a mixture of 1
part of that melting at 53° with 1 part of that melting at 45° gives a
mass melting at 50° C.
According to E. BUKCHARDT (Jena Zeit. Naturw., xxxiv, 1900,
p. 719) mixtures of paraffins of different melting-points give better
results than an unmixed paraffin of the same melting-point as the
mixture. He recommends 10 parts of 40° paraffin + 1 of 45° -j- 1
of 52° + 1 of 58° + 6 of 60°.
For methods for ascertaining melting-points see Kissling, Chem.
Centralb. ii, 1901, p. 507.
152. Overheated Paraffin. — SPEE (Zeit. wiss. Mik., ii, 1885, p. 8) takes
paraffin of about 50° C. melting-point arid 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 Grubler. The object of this
preparation is to make the mass stickier, in view of cutting ribbons.
VAN WALSEM (Verh. Akad. Wetensch. Amsterdam, 1899, p. 132) 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).
JOHNSTON (Journ. Appl. Micr., vi, 1903, p. 2662) adds 1 per cent, of
india-rubber in very small pieces, dissolved by heating to 100° C. for
^wenty-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 POLZAM (Morph. Jahrb.,
iii, 1877, p. 558) ; KADYI (Zool. Ane., 1879, vol. ii, p. 477) ; DOLLKEN
(Zeit. wiss. Mik., xiv, 1897, p. 32).
Gelatin Masses.
154. Gelatin Imbedding is a method that has the advantage of
being applicable to tissues that have not been in the least degree
dehydrated.
CHAPTER VIII. 03
The modus operandi is, on the whole, the same as for other fusion
masses, with the difference that the objects are prepared by satura-
tion with water instead of alcohol or a clearing agent. After the
cooling of the mass it may sometimes be cut at once, but it is
generally necessary to harden it. This may be done by treatment
for a few minutes with absolute alcohol (KAISER), or for a few days
with 90 per cent, alcohol (KLEBS) or chromic acid (KLEBS) or
formaldehyde (NICOLAS), or it may be frozen (SOLLAS).
The mass can be removed from the sections by meaiis of warm
water.
155. Glycerin Gelatin, K LEES' (Arch. mik. 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 grms. 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.
GEKLACH'S (Unters. a. d. Anat. Inst. Erlangen, 1884 ; Journ. Roy. Mic.
Soc., 1885, p. 541). — Take gelatin, 40 grms. ; saturated solution of
;irsenious acid, 200 c.c. ; glycerin, 120 c.c. Clarify with white of egg.
The objects to be prepared for imbedding by a bath of one-third glycerin.
APATHY (Mitth. Z. Stat. Neapel, xii, 1897, p. 718, and Zeit. wiss.
Mikr., xxix. 1913, p. 472) soaks small objects first in glycerin and water
(equal parts) and then for at least twenty-four 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
understand — the description is not clear). Blocks are then cut out
and hardened in absolute alcohol (suspended therein) for several days
(one day per millimetre of thickness), cleared in terpinol (one day per
millimetre), and cut with a knife wetted with the same. Said to give
sections of 3 /*, without the least shrinkage.
BRUNOTTI'S Gold Gelatin Mass (Journ. de Botan., vi, 1892, p. 194).
—Twenty grms. gelatin dissolved with heat in 200 c.c. distilled
water, and 30 to 40 c.c. of glacial acetic acid with 1 grm. corrosive
sublimate added after filtering. Objects are prepared by soaking
in some of the mass diluted with 2 to 3 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,
all is required in this process.
NICOLAS'S Method (Bibliogr. Anat., Paris, 3 annee, 1 W. P- 274)
Preparations are first soaked for one or two days lu a :
94 IMBEDDING METHODS.
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 to 25 per cent, solution containing 8 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.
BURZYNSKI (Polu. Arch. Biol. Med. Wiss., i, 1901, p. 39) finds
that alkaline formol hardens gelatin better than acid.
GASKELL (Journ. Path. Bact., July, 1912, p. 58) soaks in pure
gelatin, melted s.a., for two to five hours at 37° 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.
COLLODION (CELLOIDIN) 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 veryjong.
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 celloidin sections quite
so thin as those furnished by paraffin.
In the older celloidin method the mass is cut wet, before clearing.
I strongly recommend the more recently introduced practice of
clearing before cutting, and cutting dry as described in §§ 168—170.
157. Collodion, Celloidin, Parlodion and Photoxylin. — The collodion
method is due to DUVAL (Journ. de VAnat., 1879, p. 185).
Celloidin, recommended later on by MERKEL and SCHIEFFER-
DECKER (Arch. Anat. Phys., 1882, p. 200), is merely a patent collo-
dion. ' It may be obtained from almost all of the usual dealers in
histological reagents. It is sent out in the form of tablets. These
tablets may, if desired, be dissolved at 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 APATHY, 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
96 COLLODION AND OTHER IMBEDDING METHODS.
that is more transparent and of a better consistency for cutting (Zeit.
iviss. Mik., 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. p. Dermatol., xxx, 1900, pp. 422 and
476 ; Zeit. wiss. Mik., 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. Sphering, in
Berlin.
PhotoxyUn (KRYSINSKY, VIEOHOW'S Archiv, cviii, 1887, p. 217 ;
BUSSE, 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 be
obtained from GRUBLER. 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, e.g.)
TSCHERNISCHEFF (Zeit. wiss. Mik., xvii, 1900, p. 449) recommends
Colloxylin (10 grins, dissolved in 10 grms. of eugenol or clove oil, with
the addition of 50 c.c. of ether and 1 of absolute alcohol).
The Older Celloidin Method.
158. Preparation of Objects. — The objects must first be very
thoroughly dehydrated with absolute alcohol. They are then soaked
till thoroughly penetrated in ether, or, which is better, in a mixture
of ether and absolute alcohol. DUVAL (loc. cit.) takes for this
purpose a mixture of ten parts of ether to one of alcohol ; SCHIEFFER-
DECKER (and the majority of workers) a mixture of equal parts of
ether and alcohol ; TUBBY (in Nature, November 17th, 1892, p. 51)
advises a mixture of four parts of ether and one of alcohol. FISH
advises acetone, see next §. MANN (Methods, etc., p. 172) takes
equal parts of ether and methyl alcohol. So also PAVLOW, Zeit.
wiss. Mikr., xxi, 1904, p. 15.
This stage may be omitted if the objects are of a sufficiently
permeable nature, and they may be brought direct from alcohol into
the collodion bath.
159. The Collodion Bath. — The secret of success here is to infiltrate
the objects first with thin solutions, then with the definitive thick
CHAPTER IX. 97
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.
ELSCHNIG (Zeit. wiss. Mikr., x, 1893, p. 443) 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 proportions for
the successive baths : — No. 1, 10 parts by weight of photoxylin or
perfectly dried celloidin to 150 parts of the ether and alcohol mix-
ture ; 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.
MANN (Methods, p. 172) uses solutions of 2J and 10 per cent.
APATHY (BEHRENS, Tabellen., 1898, p. 82) takes 2 per cent, and
4 per cent, for the first baths, 8 per cent, for the last.
MYERS (Arch. Anat. Phys., Anat. Abth., 1902, p. 370) takes 1J per
cent., 6 per cent., and 16 per cent.
See also NEUMAYER, Zeit. wiss. Mik., xxv, 1908, p. 38 ; DE VECCHI,
ibid., xxiii, 1906, p. 312 ; and FUHRMANN, Zeit. wiss. Zool, Ixxviii, 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 the object is duly penetrated by the thin solution, or
solutions, if more than one have been employed, it should be brought
into the thickest one. This may be done (as first described in tl
work, 1st ed., 1885, p. 194) by allowing the thin solution to coi
centrate slowly (the stopper of the containing vessel being raised,
for instance, by means of a piece of paper placed under it), a
making up <;he loss from evaporation with thick solution.
M.
98 COLLODION AND OTHER IMBEDDING METHODS.
APATHY (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 § 137. 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 in 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 manipulations
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. MiL, viii, 1892, p. 467).
161. Orientation. — Celloidin being more or less transparent, it is
seldom necessary to resort to special aids to orientation.
APATHY (Zeit. wiss. Mik., 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.
HALLE and BORN (Zeit. wiss. Mik., xii, 1896, p. 364) use plates of
hardened white of 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 EYCLESHYMER (Amer. Nat., xxvi,
1892, p. 354) see previous editions.
See also the article " Kekonstruction " in the Encycl. 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 com-
munication, with the air to set up a slow evaporation. Or porcelain
moulds or small dishes may be covered with a lightly fitting cover.
CHAPTER IX. 99
As soon as the added thick collodion (of which only just enough to
cover the object should have been taken) has so far sunk down that
the object begins to lie dry, fresh thick solution is added, and the
whole is left as before. (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 under a
bell- jar, together with a dish containing alcohol, so that the evapora-
tion is gone through in an atmosphere of alcohol. This is especially
indicated for very large objects. 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 VIALLANES (Rech. sur VHist. et k Dev. des Insectes,
1883, 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 chloro-
form, but regains its transparency after a time.
Small objects may be hardened by chloroform without preliminary
hardening by evaporation. All that is necessary is to expose the mass
to the air for a few seconds until a membrane 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.
7—2
100 COLLODION AND OTHER IMBEDDING METHODS.
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 necessary 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 con-
sistency of trie 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 practice of
different writers differs greatly. BUSSE (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.
AP!THY (Microtechnik, p. 185) mentions " glycerin-alcohol," but
without giving details. BLUM (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.
FLORMAN (Zeit. wiss. Mik., 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 carefully
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 incurring
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 dry, by dipping
CHAPTER IX. 101
them into melted paraffin (APATHY, Zeit. iviss. Mikr., v, 1888, p. 45),
or, after rinsing with water, in glycerine-jelly, which may be removed
with warm water before cutting (APATHY, Mitth. Zool Stat Neavel
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 infil-
trated 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 chloroform, or vapour of
chloroform, for a few minutes, in order that the joint may harden.
LINDSAY JOHNSON 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 collodion, 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 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. GAGE has recommended bits of glass cylinders.
JELINEK (Zeit. wiss. Mik., xi., 1894, p. 237) recommends a sort of
102 COLLODION AND OTHER IMBEDDING METHODS.
vulcanite known as " Stabilit," which is manufactured for electrical
insulation purposes. It is supplied in suitable blocks by JUNG, and
by GKUBLEK. Wood is liable to swell in alcohol so that it no longer
fits into the object-holder. BABCOCK (Journ. R. Micr. 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 recommends 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. — The 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 rem'ove 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.
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). NIKIFOROW (Zeit. wiss. Mik., viii, 1891, p. 189) recom-
mends a mixture of equal parts of alcohol and chloroform. Clear
with a substance that does not dissolve collodion. The clearing
agents most recommended are origanum oil (01. Origan. Cretici, it
is said, should be taken, not 01. 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),
CHAPTER IX. 103
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.)
FISH (Proc. Amer. Mik. Soc., 1893) advises a mixture of 1 part of
red oil of thyme with 3 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 orien-
tating. See also § 126, and under " Euparal."
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. MINOT
(Zeit. wiss. Mik., iii, 1886, p. 175) says that Dunham's mixture " clarifies
the sections very readily, and softens the cello idin just enough to prevent
the puckering which is so annoying with thyme alone."
Carbolic acid has been recommended. WEIGERT (Zeit. wiss. Mik., iii,
1886, p. 480) finds that a mixture of 3 parts of xylol with 1 part of
carbolic acid (anhydrous) clears well. But it must not be used with the
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.
For oil of cajeput see § 129 ; and for this and other clearers see
also JORDAN, Zeit. wiss. Mik., xv, 1898, p. 51, who recommends,
amongst other things', oil of Linaloa, which remains colourless.
The Newer Celloidin Method.
168. The New Method, by Clearing before Cutting.— This process
is due, I believe, in the first instance to E. MEYER (Bid,. CentraW.,
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.
knife is wetted with the clearing oil, and the same oil is employee
for covering the exposed surface of the object after each cut.
Similar recommendations are made by EYCLESHYMER (op.
pp. 354, 563), carbolic acid, or glycerin, or the mixture given §
being suggested for clearing ; and GILSON has for a long ti
104 COLLODION AND OTHER IMBEDDING METHODS.
adopted the practice of clearing before cutting with cedar oil, as
described in the next §.
FISH (loc. cit., § 167) also advocates the practice of clearing in the
mass, recommending the clearing mixture there given. Similarly
GAGE, Trans. Amer. Mik. Soc., 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. GILSON'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, 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. Infiltrate with collodion or celloidin either
by GILSON'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 teaspoonful 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 GILSON'S
mixture. This should be at first a mixture of 1 part of chloroform
with 1 or 2 parts of tedar oil. From time to time more cedar oil
CHAPTER IX. 105
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 chloroform 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. Cut
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.
STEPANOW (Zeit. wiss. Mik., xvii, 1900, p. 185) soaks and imbeds in a
solution of celloidin in a mixture of equal 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, ibid., p. 449.
JORDAN, ibid., p. 193, imbeds in a mixture of 5 parts of 8 per cent,
celloidin 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 some-
times 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.
KULTSCHITZKY'S Method (Zeit. wiss. Mik., iv, 1887, p. 48).—
After the collodion bath, the object is soaked in oil of origanum
(Oleum Origani 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 maybe preserved in the dry state, and maybe cut dry.
RYDER (Queen's Micr. Bull, 1887, p. 43 ; Journ. Roy. Micr. Soc.,
1888, p. 512) modified the process by substituting chloroform for the
origanum oil.
IDE (La Cellule, vii, 1891, p. 347, and viii, 1, 1892, p. 114) imbeds in
collodion in a tube by GILSON'S process (§ 169) ; the collodion is boiled
for forty minutes, then brought for fifteen minutes (this is for smal
objects) into chloroform heated to 30° C. containing £ part of paraffin
dissolved in it, then for ten minutes into pure melted paraffin.
FIELD and MARTIN (Bull. Soc. Zool. de France, 1894, p. 48) make
solution of dried celloidin in a mixture of equal parts of absolute alc<
and toluene, of about the consistency of clove oil. This solution 11
106 COLLODION AND OTHER IMBEDDING METHODS.
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.
STEPANOW 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.
WILHELMI (Fauna Flora Golf. Neapel, xxxii, 1909, p. 17), following
APATHY, 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, Zeit. wiss. Mik., xxv, 1908, p. 29.
STERLING (Jena Zeit., 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 ; SABUSSOW,
Mitth. Zool Stat. Neapel, xii, 1896, p. 353 ; MEYER, ibid., xiv, 1901,
p. 295 ; MITROPHANOW, Arch. Zool. Exper. (3), 3, 1896, p. 617 ;
FEDERICI, Anat. Anz., xxi, 1907, p. 602 ; BORDAGE, Bull. Sci. France
Belg., xxxix, 1905, p. 385 ; GANDOLFI, Zeit. wiss. Mik., xxv, 1909,
p. 421 ; MAYER, ibid., xxiv, 1907, p. 132.
Other Cold Masses.
172. Lead-Gum Imbedding Method of J. SALKIND (C. R. Soc. de
Bid., t. Ixxix, 1916, No. 16). — The principle of this method is that
an aqueous solution of gum treated by acetate of lead, when exposed
to the action of ammonia, is transformed into a gel, sufficiently stiff
to allow of thin sections being cut.
(1) Dissolve a quantity of gum of cherry (white for preference) in
double its weight of aq. dest. After nitration, add to the solution
one-third its volume of the liquid subacetate of lead (extract of
Saturne), to which has been added 5 per cent, of glacial acetic acid.
This gives a kind of thin collodion-like solution, in which you place
the pieces at room temperature, to be imbedded, after a fixation,
for which see below.
(2) Leave about twelve hours for pieces about a millimetre in
thickness : larger pieces must be left longer. After the correct
period has elapsed, you let the lead gum solution evaporate in the
air till the solution reaches the consistency of a thick celloidin
solution.
(3) Arrange the pieces to- be imbedded in a paper box (or on a
piece of paper), in a large drop of the thick solution. Expose to
strong ammonia vapour for about five minutes till the block hardens
to the consistency of cartilage.
CHAPTER IX. 107
(4) Trim the block, and fasten it on to the plate of a microtome
by means of some of the thick lead-gum (hardened afterwards in
ammonia vapour). Cut sections with an oblique knife, the block
being moistened with a solution 1 per cent, sodium chloride in
aq. dest. The sections are placed in the same solution, in which they
must not stay more than one hour.
(5) Sticking the sections to the slide is done by a modification
of OLT'S method, § 182. Cover the slide with albumen, then with
gelatin, arrange the sections, press down with a cloth, and harden
in formol vapour. See also J. A. Murray, below.
(6) The lead-gum is then dissolved away in 5 per cent, acetic acid.
After washing you stain and mount in any way desired.
Neither gum arabic (acacia), plum, nor apricot give quite such
good results as cherry gum. SALKIND recommends two fixatives to
precede this method of imbedding.
A. Formol, acetic acid, sub-acetate of lead, 1 part each. Aq.
dest., 5 parts.
B. Formol 10 c.c.
Acetone . . . . . 30 „
Water 40 „
Citric acid . . . . 5 to 10 grms.
Saturated with Sudan III.
After A, it is not necessary to wash out. After B, and the
majority of such fixatives as bichromate especially, you must wash
out in running water.
J. A. MURRAY (Report of Imper. Cancer Bureau, 1919) fixes cartilage
in 10 per cent, formol-salt solution for at least twenty-four hours.
After Salkind's lead-gum imbedding, cuts sections 10 — 15 /* thick with
sliding microtome. Transfers sections for from ten minutes to one hour
in 1 per cent. NaCl solution. Special slides prepared beforehand by
coating in 1 per cent, gelatin and allowing to dry.
The prepared slide is immersed in the salt solution (NaCl), sections
arranged with a smooth-pointed glass rod, superfluous liquid drained
off, and a wetted cigarette paper carefully lowered over the sections.
Firm pressure with several layers of filter paper makes the sections
adhere to the slide. Withdraw the cigarette paper and expose to formol
vapour for a few minutes. Transfer to 10 per cent, formol five minutes,
then treat in the 5 per cent, acetic to remove the lead-gum. Stain.
173. Joliet's Gum and Glycerin Method (Arch. Zool. Exper. et Oen.,
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, a
add from 6 to 10 drops of pure glycerin. In the winter or in rai
weather less glycerin should be taken than in the summer or dry wea
The object is imbedded in the mass in the watch-glass and the wlu
108 COLLODION AND OTHER IMBEDDING METHODS.
left to dry for from one to four days. When it has assumed a cartila-
ginous consistency, a block containing the object is cut out, turned over,
and allowed to dry again until wanted for use. A stove, or the sun,
may be employed for drying, but it is best to dry slowly at the normal
temperature.
174. STRICKER'S Gum Method (Hdb. d. Gewebel., p. xxiv). — A concen-
trated 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.
175. HYATT'S Shellac Method, see Am. M. Mic. Journ., i, 1880, p. 8 ;
Journ. Eoy. Mic. Soo., iii, 1880, p. 320. For sections through hard
chitinous organs consisting of several pieces, such as stings and oviposi-
tors, retaining all the parts in their natural positions.
176. BRUNOTTI'S Cold Gelatin Mass has been given, § 155.
Masses for Grinding Sections.*
177. G. VON KOCH'S Copal Method (Zool. Am., i, 1878, p. 36).—
Small pieces of the object are stained in bulk and dehydrated with
alcohol. A thin solution of copal in chloroform is prepared by
triturating small fragments of copal in a mortar with fine sand,
pouring on chloroform to the powder thus obtained and filtering.
The objects are brought into a capsule filled with the copal solution.
The solution is now 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,
* For the manipulations of section-grinding, see CARPENTER'S The
Microscope.
CHAPTER IX. 109
removing the c< pal 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. EHRENBAUM'S Colophonium and Wax Method (Zeit. wiss.
Mik., 1884, p. 414). — Ehrenbaum recommends a mass consisting of
10 parts of colophonium to 1 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. JOHNSTONE-LAVIS and VOSMAER'S Balsam Method (Journ.
Roy. Mic. Soc., 1887, p. 200). — Alcohol material is carefully 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. WEIL'S Canada Balsam Method, see Zeit. wiss. Mik., v, 1888,
p. 200.
181. GIESBRECHT'S Shellac Method.— For hard parts only, spines
of Echinus, shell, etc., see Morph. JaM., vi, 1880, p. 95, or the abstract
in LEE und MAYER, Grundziige.
Congelation Masses.
182. The Methods of Freezing.— For the requisite manipulations
and means of producing the requisite degree of cold, see CARPENTER'S
The Microscope (ether spray) ; JOHNE, Zeit. wiss. Mik., xiv, 1897,
p. 370 (liquid carbonic acid) ; WOLFF, ibid., xxv; 1908, p. 175 (ethyl
chloride) ; KRAUSE, ibid., p. 289 (solid carbonic acid) ; JUNG, Verb.
Ges. Naturf. Aertze, Ixix, 1898, p. 129 (ethyl chloride) ; BRISSY,
C. R. Soc. Biol, Ixii, 1907, p. 1115 (liquid air).
Fresh tissues may be, and are, frequently frozen without being
included in any mass. But the formation of ice crystals frequently
causes tearing of delicate elements, and it is better to infiltrate the
tissues with a mass that does not crystallise in the freezing mixture,
but becomes simply hard and tough, such as one of those given below.
When sections have been obtained, it is difficult to manipulate
them. OLT (Zeit. wiss. Mik., xxiii, 1906, p. 327) puts them into a
1 per cent, solution of gelatin, brings them therein on to a si
110 COLLODION AND OTHER IMBEDDING METHODS.
hardens for an hour in vapour of formaldehyde, and soaks for a few
minutes in formol of 10 per cent. ANITSCHKOW (ibid., xxvii, 1910,
p. 73) puts them into alcohol of 50 per cent., gets them on to a slide
prepared with Mayer's albumen, presses down with paper, puts into
alcohol of 98 per cent., and thence through lower grades into water.
183. Gum and Syrup Masses. — HAMILTON (Journ. of Anat. and
Phys., xii, 1878, p. 254) soaked tissues in syrup made with double
refined sugar, 2 ounces ; water, 1 fluid ounce ; 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.
COLE (Methods of Microscopical Research, 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 5 of gum.) Add 5 grains of pure carbolic 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 muci-
lage ; 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.
WEBB (The Microscope, ix, 1890, p. 344 ; Journ. Roy. Mic. Soc.,
1890, p. 113) 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 (JACOBS, Amer. Natural, 1885, p. 734). White
of Egg (ROLLETT, Denskschr. math, naturw. Kl. k. Acad. Wiss. Wien,
1885 ; Zeit. wiss. Mik., 1886, p. 92). — Small portions of tissue brought
in the white of a freshly laid egg on to the freezing stage, frozen and cut.
Oil of Aniseed (KUHNE, Centralb. f. Bakteriol., xii, 1892, p. 28 ; Journ.
Eoy. Mic. Soc., 1892, p. 706 ; V. A. MOORE, Amer. Mon. Mic. Journ.,
1894, p. 373 ; Journ. Eoy. Mic. Soc., 1895, p. 247). Anethol (anise
camphor) ( STEFAN ow, Zeit. wiss. Mik., xvii, 1900, p. 181).
For details of these see previous editions.
For DOLLKEN'S method of solidifying formol by means of resoroin,
see Zeit. wiss. Mik.t 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,
GRAHAM KERR'S seems the most convenient.
Methods for Paraffin Sections.
186. The Water or Desiccation Method. — GAULE (Arch. Anat.
Phys., Phys. Abth., 1881, p. 156) ; SUCHANNEK (Zeit. wiss. Mik.,
vii, 1891, p. 464) ; GULLAND (Journ. Anat. and Phys., xxvi, 1891,
p. 56) ; SCHIEFFERDECKER (Zeit. wiss. Mik., ix, 1892, p. 202) ;
HEIDENHAIN (Kern, und Protoplasma, p. 114) ; NUSBAUM (Anat.
Anz., xii, 2, 1896, p. 52) ; MAYER in the Grundzuge, LEE und MAYER,
1898, p. 113 ; DE GROOT (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 numerous. 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 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 b
allowed to melt before the sections are perfectly dry ; the sections are
sure to become detached if it does. Perfectly dry sections have a
112 SERIAL SECTION MOUNTING.
certain brilliant transparent look that is easily recognisable. As
soon as chy 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.
(6) 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 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 (DE 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.
CHAPTER X. 113
GUDERNATSCH (Zeit. wiss. Mikr., xxiv, 1908, p. 358) washes the
slide well with potash soap, and arranges the sections on it whilst
still wet. HELLY (ibid., 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. NUSBAUM adds a trace of gum
arabic (1 or 2 drops of mucilage to a glass of water) ; APATHY (Micro-
technik, p. 126) adds 1 per cent, of Mayer's albumen (§ 187) ; and
HENNEGUY (Lemons sur la Cellule, 1896, p. 62) takes a 1 : 5,000 solution
of gelatin, with a trace of bichromate of potash, added just before
using, and dries the slides exposed to light. Similarly, BURCHARDT
(Jena Zeit., xxxiv, 1900, p. 719).
Some workers have used alcohol (50 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 con-
tact with the slide. Sections of parenchymatous organs stick well ;
sections of thin- walled tubular organs stick badly. Sections of
chitinous organs are very unsafe. The larger and thinner sections
are, the better do they stick, and vice versa. 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 MAYER, Mitth. Zool. Stat.
Neapel, xii, 1896, p. 320 ; SCHMORL, Path.-hist. Untersuchungsmethoden,
1897, p. 38 ; SMITH, Journ. Anat. Phys., xxxiv, 1899, p. 151.
187. MAYER'S Albumen (Mitth. Zool Stat. Neapel, iv, 1883 ;
Internal. Monatschr.f. Anat., iv, 1887, p. 42).— White of egg, 50 c.c. ;
glycerin, 50 c.c. ; salicylate of soda, 1 grm. Shake them well
together, and filter into a clean bottle. The filtering may take days
or a week, but the preparation does not spoil meanwhile.
FBANCOTTE 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.
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
8
M.
114 SERIAL SECTION MOUNTING.
" squeegee "). The 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 slide 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 absolutely
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.
It sometimes happens that the mixture after it has stood for some
time becomes turbid, and at last coagulates, passing into a caseous
state ; or it may undergo a hyaline coagulation, drying up like
amber. But up to the very last it does not in 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.
HEIDENHAIN (Zeit. wiss. Mikr., xxii, 1905, p. 331) makes it up
with 1 grm. of blood albumen dissolved in 25 c.c. of water, and an
equal volume of 50 per cent, alcohol.
188. The Albumen and Water Method (HENNEGUY, Journ. de
VAnat. 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 OHLMACHEB, Journ. Amer. Med. Assoc., April, 1893.
The so-called " Japanese " method, attributed to IKEDA by KEINKE
(Zeit. wiss. Mik., xii, 1895, p. 21), is merely that of HENNEGUY.
MANN (Anat. Ana., viii, 1893, p. 442) shakes up white of egg with
CHAPTER X. 115
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. — HOLLANDS (Arch. d'Anat. Micr., xiii, 1911, p.
171) gives the following as more adhesive than albumen : — 50 grms. of
crushed and chopped garlic are rubbed up with 80 c.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.
KABL, however (Zeit. wiss. Mik., 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.
191. OBREGIA'S Method for Paraffin or Celloidin Sections (Neuro-
logisches Centralb., ix, 1890, p. 295 ; GULLAND, Journ. of Path.,
February, 1893). — Slides, or glass plates of any size, are coated with
a solution made of —
Syrupy solution of powdered candy-sugar made
with boiling distilled water . . . .30 c.c.
95 per cent, alcohol . . . . 20 ,,
Transparent syrupy solution of pure dextrin made
by boiling with distilled water . 10 „
They are dried slowly for two or three days until the surface is
just sticky to the moist finger. Paraffin 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 evaporation must not be artificially
hastened.
DIMMER (Zeit. wiss. 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 BLOCHMAN'S modification of Weigert's process, by means of
which large sections can be preserved unmounted, see Zeit. u
xiv, 1897, p. 189. g_2
116 SERIAL SECTION MOUNTING.
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 STRASSER'S automatic
ribbon-microtome. See Zeit. wiss. Mik., 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 SCHOENEMANN, ibid., xix, 1903, p. 333 ;
STRASSER, ibid., p. 337 ; and RUPPRICHT, ibid., xxviii, 1912, p. 281.
Methods for Watery Sections.
193. FOL'S Gelatin (For,, Lehrb., p. 132).— Four grammes of
gelatin are dissolved in 20 c.c. of glacial acetic acid by heating on a
water-bath and agitation. To 5 c.c. of the solution add 70 c.c. of
70 per cent, alcohol and 1 to 2 c.c. of 5 per cent, aqueous solution of
chrome-alum. Pour the mixture on to the slide and allow it to dry.
In a few hours the gelatin passes into the insoluble state. It retains,
however, the property of swelling and becoming somewhat sticky in
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, KUPPRICHT, loc. cit., last §, with the needless com-
plication of a seriation on Strasser's collodionised paper.
STRASSER (loc. cit., 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.
Similarly, JORDAN (Zeit. wiss. Mik., xv, 1898, p. 54), and ARGU-
TINSKY (ibid., xvii, 1900, p. 37). See also JORDAN, ibid., 192 — 194 ;
DANTSCHAKOFF, ibid., xxv, 1908, p. 35 ; MAXIMO w, ibid., xxvi,
1909, p. 184 ; ANITSCHKOW, ibid., xxvii, 1910, p. 68 ; WEBER, ibid.,
xxix, 1912, p. 186 ; EUBASCHKIN, 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 minute or
CHAPTER X. 117
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.
GAGE (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
collodion thus formed to evaporate into a thin sheet on the slide. Then
70 per cent, alcohol and other desired reagents.
Similarly, MAIEB (Munch, med. Wochensclir., Ivii, 1910, No. 12 ; Zeit.
wiss. Mik., xxvii, 1910, p. 385), but adding a treatment for ten to fifteen
minutes with sulphide of carbon.
See also MYERS, Arch. 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. MiL, 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 (APATHY, MikrotecJmik, 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.
If it be desired to stain in a watery fluid, care must have been
taken when arranging the sections to let the celloidin of each section
overlap that of its neighbours at the edges, so that the ether vapour
may fuse them all into one continuous plate. This will become
detached from the slide in watery fluids, and may then be treated
as a single section. Terpinol may be taken instead of bergamot oil.
118 SERIAL SECTION MOUNTING.
197. APATHY'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 overlapping 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. This 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. WEIGERT'S Collodion Method (Zeit. wiss. Mikr., 1885,
p. 490). — Slides, 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. 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 dehydra-
tion. 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 BLOCHMAN'S modification see § 191.
STRASSER takes gummed paper instead of the glass plates used in this
process. See the papers quoted § 192.
CHAPTER X. 119
See also WINTERSTEINER (Zeit. wiss. Mile., x, 1893, p. 316) and KUBO
(Arch. mik. Anat., Ixx, 1907, p. 173).
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 DIMMER'S modification see also § 191.
200. Collodion Film Method.*— GRAHAM 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 application
of a drop of absolute alcohol and ether (or an atmosphere of alcohol
and ether) suffices to 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 (FoL) and 182
(OLT).
* The late Dr. S. G. Scott used mica sheets, upon which he stuck
paraffin sections. These could be distributed to a class of students by
simply cutting out pieces of mica supporting the sections.
CHAPTER XL*
STAINING.
201. Dyes. — In practice, the staining materials are usually of the
nature of neutral salts. But in the so-called " basic " dyes it is the
base or the cation that consists of the complex organic compound
possessing colour ; in the " acidic " dyes it is the acid or anion that
is the staining agent. In the former case the colour-base is com-
bined with a simple acid, generally hydrochloric or sulphuric, but
sometimes acetic. In the latter case the colour-acid is combined
with an inorganic base, usually sodium. For example, the dye
called fuchsin is the hydrochloride of the base rosaniline, and its
staining properties are clearly due to the latter. Acid-fuchsin, on
the other hand, is the sodium salt of a sulphonic acid derived from
fuchsin, and its coloured constituent is present as the acid. The
free colour-base or colour-acid is in most cases insoluble in water,
although it may exist therein in the colloidal state. These are
frequently colourless in themselves. It follows that the addition
of acids to formulae for " acidic " dyes or bases to " basic " dyes is
devoid of a rational foundation.
Although the terms " acidic " and " basic " serve to indicate an
important difference between dye-salts, their careless use may lead
to unwarranted conclusions. Thus, if a cell-constituent takes up a
basic dye, it does not follow that this body has the chemical nature of
an acid. It may have, but substances other than acids are stained
by basic dyes, as we shall see below.
A " neutral " dye-salt may clearly also be formed by combination
between a colour-base and a colour-acid. These compounds are for
the most part insoluble in w^ater, although soluble in alcohol. Being
of high molecular dimensions, they have the properties of colloids,
amongst others, that of forming permanent colloidal solutions in
the presence of excess of either component. Moreover, the compo-
sition of the dye that is precipitated varies according to the relative
proportion of the two reagents in the solution. It is, therefore, not
a simple salt, but rather a " colloidal-complex " or " adsorption-
compound." These dyes have limited use, more especially in
* W. M. Baylies.
CHAPTER XI. 121
distinguishing between various kinds of leucocytes in blood (see
EHRLICH and LAZARUS, Die Anaemic, Wien, 1898).
Since the acidic dyes are salts of fairly strong sulphonic acids
with strong bases, they are electrolytically dissociated in solution to
a large extent. Thus their solutions contain coloured anions,
colourless cations (usually Na), together with undissociated salt.
They are only hydrolytically dissociated to a negligible degree, if
at all. The basic dyes, on the other hand, are salts of weak bases
(amino- or imino-derivatives) with strong acids. Although electro-
lytically dissociated as salts, so that their solutions contain coloured
cations and undissociated salt together with colourless anions, they
also undergo hydrolytic dissociation to a notable degree. Thus
they contain free colour base and free colourless acid in addition
to their ions.
It is important to remember that commercial samples of dyes
contain mineral salts, as a rule, sodium chloride or sulphate, some-
times as much as 30 per cent, or more. This must be borne in mind
in statements as to their properties. For instance, it is often said
that the Con^o-red dyes are direct dyes for cotton fibre. This is
only the case in the presence of salts, as will be shown presently.
Details of the chemical composition of different dyes are beyond
the scope of this book. The reader is referred to CAIN and THORPE'S
Synthetic Dyestuffs, 1913.
202. The Nature of the Staining Process. — From what has been
said in the preceding paragraph it will be realised that a solution of
a dye is a complex system from a physico-chemical standpoint.
Moreover, the structures to be stained are present as separate
phases, solid or liquid, of a heterogeneous system. It is clear,
therefore, that the properties of boundary surfaces must be taken
into consideration, in addition to differences of chemical composition
and of colloidal state. Much discussion has taken place with respect
to the process of dyeing, and various theories of its nature as being
essentially chemical or essentially physical, in the sense of adsorption,
mechanical or electrical, or in the sense of solid solution, involving
partition between the solution and the tissue elements according to
relative solubility of the dye therein, have been advocated. It is
probable that all these factors play their part in varying proportion
and that no one theory alone can explain all the facts.
We shall be in a better position to appreciate the complexity of
the conditions present if we examine, to begin with, the case of a pure
substance, cellulose, in relation to pure solutions of an acidic and a
basic dye respectively.
122 STAINING.
We take, then, a piece of the purest analytical filter paper, wash
it with distilled water to remove possible traces of acid, and place it
in a dilute solution of Congo red, freed from foreign salts. It is
scarcely stained at all. Add next a very small amount of a neutral
salt, say, sodium chloride. The paper is deeply stained. How are
these facts to be explained ? In view of the chemical inertness of
cellulose, it seems unlikely that a chemical combination occurs
between the dye and the paper under the influence of a neutral salt
at ordinary temperatures. Moreover, the same behaviour is shown
by such different substances as charcoal, silk, alumina, silica, and so
on. The process must be one of adsorption or deposition of the dye
on the surface by some means. In other words, it must be associated
with the decrease of surface energy of some kind. In the absence
of foreign electrolytes, adsorption may be due to decrease of surface
energy of the ordinary kind, shown as surface tension. This is
confirmed by the fact that the dye, in the absence of electrolytes, can
be washed out again by water. But since the degree of staining is
very small, there must be some influence at work restricting the
mechanical adsorption. There is, indeed, another property of the
boundary surfaces between phases which demands attention here.
This is the electrical charge, nearly always present. If we test
paper in water, we find that it has a negative charge. Similarly, by
appropriate means, we find that the dye itself has a negative charge.
Whether this is that of the coloured anion or of complex aggregates
of these ions with undissociated salt is not certain, but, according to
Freundlich, " acidic " dyes are adsorbed as a whole. In the present
case it is immaterial, because the material to be adsorbed has a
negative charge in either case. This being so, there are repellent
forces acting between the dye and the paper. Or, if we take the
point of view of energetics, the adsorption of electro-negative dye
would increase the negative charge on the paper, with an increase of
free energy, which is contrary to the Second Law of Thermo-
dynamics. Suppose, however, that we have also present the ions
into which a neutral salt dissociates. The cations, being positively
charged, are deposited on the surface of the paper, decreasing or
annulling its negative charge and reducing the free energy. There
is now little or no obstacle to the adsorption of the dye. From the
work of PERBIN we know that an ion may be adsorbed on an
oppositely charged surface to so great an extent that the charge on
this surface may actually be reversed in sign. This occurs, as it
appears, when surface energy of some kind other than electrical is
diminished by the presence of such ions, and is found mainly with
CHAPTER XL 123
plurivalent or organic ions. But since colloids are readily precipi-
tated by such ions, owing to neutralisation of their charges, it may
happen that a dye such as J^ongojred is neutralised and precipitated
before it has attached itself to the adsorbing surface by virtue of its
charge.
Since the amount adsorbed is dependent on the magnitude of the
electric charge, we have an explanation of the fact that alcohol
diminishes the effect of electrolytes. The charge on a surface is
proportional to the dielectric constant of the liquid phase in which it
is immersed, and the dielectric constant of alcohol is less than that
of water.
It is a well-known fact that colloids of the suspensoid class, such
as gold and coagulated egg white, are much more sensitive to the
action of electrolytes than are those of the emulsoid class, in which
the colloidal particles themselves contain water and differ from the
medium in which they are suspended merely by the smaller quantity
of water which they contain. Such are gum arabic, starch, colloidal
silica, and proteins in general, gelatin, raw egg white, and so on.
Faraday showed that colloidal gold could be protected from
precipitation by salt if a trace of gelatin was added. This is
explained by the adsorption of a coating of gelatin over the gold
particles, which are thereby converted into the emulsoid variety so
far as their surfaces are concerned. We find similar phenomena in
the' staining of paper by CongCLiejL It is protected from the dye
even in the presence of salts. But the conditions are made more
complex by the possibility of using in the experiment either an
electro-positive or electro-negative protein. A trace of acid or
alkali respectively has this effect on proteins, by the production of
dissociated salts. Now the former are more powerfully adsorbed
by the negative paper than the latter are, while at the same time
they reduce, instead of increasing, the electric charge. Actually
the latter effect preponderates, so that the presence of electro-
positive protein increases the depth of staining.
Turning now to the basic dyes, we find that the paper is the more
deeply stained the lower the concentration of salt present. Accord-
ing to Freundlich, it is the colour base that is chiefly adsorbed
in this case. As was pointed out above, these dyes are hydro-
lytically dissociated, so that free base is present. This free base,
being insoluble, is in the colloidal state, and, like colloidal bases in
general, has a positive charge, due to electrolytic dissociation of the
surface of the particles. See HARDY in Van Bemmelen Gedenkboek,
p. 188. Thus, not only are the coloured ions, in this case the
124 STAINING.
cations, strongly adsorbed by the negative paper, but the free base
is also. Since foreign electrolytes dimmish the charge on the paper,
their effect on staining by basic dyes is naturally of the opposite
kind to that described in the case of the acidic dyes. The ejffiect of
alcohol is in the same direction as that of electrolytes, sincejt^also
decreases the electric charge and, therefore, the amount of dye
adsorbed. Facts of the kind referred to in the preceding statements
have given rise to an " Electrical Theory of Dyeing," which probably
accounts for a larger number of them than any other single theory
is able to do. For further particulars of theories and facts relating
to dyeing and staining, the reader is referred to ALFRED FISCHER'S
Fixirung, Fdrbung und Ban des Protoplasmas, Jena, 1899 ; PELET-
JOLIVET'S Theorie des Farbeprozesses, Dresden, 1910 ; First Report
on Colloid Chemistry, Brit. Ass., 1917 ; GEE and HARRISON, Trans.
Faraday Soc., vol. vi, 1910 ; HARRISON, Journ. Soc. Dyers and
Colourists, December, 1911 ; BAYLISS, Biochem. Journ., vol. i, 1906,
p. 175.
The reducing properties, in the chemical sense, of cell constituents
play an important part in certain special staining reactions, particu-
larly those with metallic salts. Osmic acid (osmium tetroxide) is
reduced to a lower oxide or perhaps to the metallic state by un-
saturated fats. Gold and silver salts are reduced by nervous tissue
under appropriate conditions and deposited in the colloidal state.
All the various colours of colloidal gold in different degrees" of
dispersion may be seen in tissues prepared by the gold impregna-
tion methods. Details of these methods will be found in other
chapters of this book.
Effect of Heat. — Since surface tension is decreased by rise of
temperature, it would naturally be expected that, if this factor is
concerned in the process of staining, the depth of the stain would be
lessened at a higher temperature. This is actually the case with
simple substances like cellulose up to 50° C. or thereabouts. At
first sight this would seem to be at variance with the frequent
practice of fixing stains by heating the preparation. But the
temperatures used are much higher than those referred to, and, in
point of fact, if filter paper is stained in a Congo red solution at 100° C.,
the dye is firmly fixed, and cannot readily be removed by washing.
It is difficult to say what actually happens here. Chemical combina-
tion suggests itself, but the nature of the compound formed is not
easy to conceive. The temperature at which a material possesses
the maximum electric charge, as shown by GEE and HARRISON, also
plays a part in the phenomena.
CHAPTER XL 125
Chemical combination obviously occurs in some cases when
substances stained by adsorption are heated to 100° C. Thus
BAYLISS (Proc. Roy. Soc., B., vol. Ixxxiv, 1911, p. 83) showed that
various insoluble hydroxides, such as that of aluminium, are stained
blue by the free acid of Congp__red, which is its own colour. When
heated to 100° C., combination takes place with the formation of
the usual red colour of the salts of this dye. Silk behaves in the same
way, and even crystals of leucine, doubtless to be explained by the
formation of salts with the fairly strong acid.
Conditions similar to this, however, do not arise in histological
staining, where dye salts are used. It is difficult, moreover, to see
how the conditions for reaction by double decomposition could arise
in the cell, since acids and bases sufficiently strong to displace
hydrochloric acid or sodium hydroxide are required. Reactions
with precipitation might occur, but these would give rise to the
appearance of new solid structures in the cell. There is no evidence
that such precipitates are produced in a simple staining process,
although they are undoubtedly formed by fixing agents (HARDY,
Journ. of Physiology, vol. xxiv, p. 158).
The following experiment by MARTIN HEIDENHAIN is sometimes
given as evidence of the formation of salts of dyes with proteins. A
solution of Congo red, as is well known, turns blue when inadejicid
with acetic acid, owing to the separation oOEe Tree acid. If such
a blue solution is added to an acidulated solution of serum albumin,
a red solution is obtained. Since this is the characteristic colour of
the salts of Congo_red, it is natural to interpret it as a salt of the dye
acid with the protein base. But the fact that it exists in a solution
sufficiently acid to decompose the sodium salt of the dye shows that
the colour acid is more firmly combined with protein than with
sodium, a view that it is difficult or impossible to hold. Moreover,
it appears that even 5 per cent, sulphuric acid is unable to split off
the acid from some of these protein " compounds." It is clear that
the phenomena must have a different interpretation. It may be
that the free colour acid exists in two forms, a true and a pseudo-
acid — the former of a red colour and ionised; the latter, blue, in-
soluble and non-ionised. When adsorbed by protein, for some reason
or other, the acid may be for the most part in the former condition.
See the work of Wo. OSTWALD on Congo-rubin (Roll. Chem. Beihefte,
B. x, 1919). But further evidence is required.
203. Removal of Dyes.— When a stain is fixed by adsorption of
the ordinary, non-electrical type, it can be removed by frequent
washing with distilled water. This is very difficult if the dye is held
126 STAINING.
by electrical forces. The reason is, in all probability, that the dye
can only be set free by reversing the sign of the charge on the surface.
This cannot be done by pure water alone. It can be done, however,
by acid or alkali in the appropriate case. For example, if an acidic
dye has been fixed on a negative surface by the aid of cations, which
convert the charge to a positive one, OH' ions, provided by alkali,
are powerful enough to change the sign of the charge back again to
negative and thus free the dye, whereas H' ions from an acid
only increase the positive charge and fix the dye more firmly. Hence
the statement that acidic dyes are fast to acids. A basic dye,
adsorbed by a negative surface, is removed by acids and intensified
by alkalies. A corresponding explanation holds. Thus, H' ions
from acids make the surface more positive, hence the dye is released.
OH' ions make it more negative, hence the dye is held faster. In
all cases, if the acid or alkali is strong enough, any dye-salt adsorbed
is decomposed, sometimes with change of colour.
The process of " differentiation " by alcohol or other agent, to be
referred to below, is an application of these facts. Alcohol removes
a " basic " dye because it reduces the negative charge of the tissue
elements and thus releases part of the positively charged constituent
of the dye adsorbed.
204. "Specific" Stains.— Certain tissue elements and cell-
constituents have the property of staining deeply with particular
dyes. That of nervous structures with methylen blue and of
rmtp_chondria with dyes containing di-ethyl-safranin, such as Janus-
green, may be given as examples. The property may be shown
either by their taking up the stain from a dilute solution more
rapidly than other structures present do (" progressive " staining),
or by their holding on to it more tightly when excess of general
stain is washed away by appropriate treatment. This latter process
is sometimes known as " differentiation " or as " regressive "
staining.
It is natural to interpret this behaviour as due to a chemical
combination of a special kind, as did EHRLICH in his well-known
theory of " chemo-receptors," according to which certain " side-
chains " of protoplasmic molecules have special affinities for parti-
cular groups in the dye molecules. While this may be the case in
isolated instances, there are many facts which show that it cannot
be accepted as a general law. It is difficult to see what purely
chemical relationship can exist between complex, substituted,
diazo-sulphonates, as a large number of these specific dyes are, and
the chemical components of cells. Moreover, although methylen
CHAPTER XL 127
blue and other thiazines are specific vital stains for nervous tissue,
certain safranin azo-dyes — diazin-green, for example— which have
no chemical relationship to the former, are also vital nerve stains ;
while similar compounds of the safranin series itself have no such
property. See MICHAELIS, Chemie der Farbstoffe, 1902, p. 104. We
must remember also that the conception of large protoplasmic
molecules with side-chains in the chemical sense, the so-called
" biogens," is becoming more and more discredited. See HOPKINS,
Address to Physiology Section, Brit. Assoc.} 1913. We have already
seen how complex are the physical factors that intervene in such a
simple case as the staining of paper, and to these may be added
questions of solid solution, distribution between phases, diffusibility,
' and so forth. Indeed, it would seem that each individual case of
specific staining requires investigation by itself. EVANS (Amer.
Journ. ofPhysiol., vol. xxxvii., p. 255) in an investigation of " macro-
phages " comes to the conclusion that chemo-receptors are not
responsible for vital staining with the benzidine and related series
of dyes.
ALFRED FISCHER (op. cit., pp. 107 — 150) gives interesting cases of
staining differentially particles of the same substance by different
dyes. Particles can be made to take up either dye, according to
their size, the order in which the different dyes are applied and the
degree of differentiation by removal of stain.
205. Some Applications of the Theory of Staining. — It is of interest
to see how some histological facts are explained in the theory
sketched above. Most of the structures in the living cell have
negative electric charges, probably on account of the slightly alkaline
nature of the surrounding fluids. This fact accounts for the ease
with which tissues in general are stained by basic dyes. It is remark-
able that haemoglobin is one of the few constituents that have a
positive charge. Accordingly, ib is stained by acidic dyes, such as
eosin or acid fuchsin. The effect of electrolytes is shown in the
experiments of MAYR (Hofmeister's Beitrage, vol. vii., p. 560).
He finds that the affinity of Nissl bodies for basic dyes is abolished
by previous treatment with neutral salts.
206. Objects of Staining. — Most constituents of cells are, in their
natural state, either colourless or only faintly coloured. Thus they
are only visible if their refractive indices differ from those of the
media in which they are immersed. Such, for example, are fatty
globules and the granules of many secreting cells. But, as seen
thus, it is not an easy matter to judge of their true forms. This is
128 STAINING.
greatly facilitated by staining them either more deeply than or of a
different colour from their surroundings. If colourless glass beads,
although they are easily seen by refraction, could only be observed
from the direction of a line through the hole in the centre, the
recognition of their true form would be difficult. Immersing them
in a medium of the same refractive index as themselves would render
them invisible. But if they were made of coloured glass and im-
mersed in such a medium, they would be readily detected and their
shape recognised.
. t The chief object of histological staining is then to cause certain
/ Constituents of the cells to take on a different intensity of tint from
I Bothers. This may be done in various ways, as will be seen later.
It is usual to distinguish two kinds of selective staining, histological
and cytological selection. In the former an entire tissue or group
of tissue elements is prominently stained, the elements of other
kinds present remaining colourless or being differently stained, as
in the impregnation of nerve endings by the silver and gold reduction
methods. In the latter the stain is taken up or retained by some
constituent element of the cell, such as the chromatin of the nucleus
or an element of the cytoplasm.
The nuclear stains are of importance in marking out the contours
and relations of the tissues making up regions or organs as
a whole and are thus of special value to the embryologist and
morphologist.
At one time, it was thought to be possible to distinguish between
" basophilous " and " acidophilous " tissue elements, according to
their affinity for basic or acidic dyes. EHRLICH (Du Bois Reymond's
Archiv., 1879, p. 571) thought that the basic dyes have a special
affinity for the chromatin of nuclei and the acidic dyes for the
cytoplasm and intercellular substances. But we have already seen
that the same substance may take up either kind of dye, according
to the conditions present. Most staining processes are undertaken
on cells which have been acted on by fixing reagents or by the so-
called " mordants," and these may reverse the natural behaviour
to dyes. EHRLICH'S statement only applies in fact to cover-glass
preparations fixed and dried by heat, without the action of reagents.
The acidic colours, orange and acid fuchsin, although they stain
cytoplasm, may give good chromatin differentiation when used as
regressive stains. Methylen blue is basic, but stains nerves. The
widely used carmin and haematoxylin are both acidic dyes, but in
combination with alum they give nuclear stains. Other instances
might be given, but these will suffice.
CHAPTER XL 129
207. Intra-vitam Staining.— It is clear that unless the cell-membrane
of a living cell is permeable to a dye, no constituent of the cell can
be stained by it. Most dyes appear to be more or less toxic if they
enter the cell. But, while alive, the latter is to a large extent
protected, since the dye does not obtain entrance. A living Amceba
is stained by very few dyes. Neutral red, however, passes through
the membrane and stains various structures, while having no
apparent effect on the activities of the organism. The auricle of
the frog's heart can also be stained with this dye, while continuing
its normal contractions. Used in this way, the dye is applied in very
dilute solution. Since neutral red is a very sensitive indicator just
about the neutral point, the fact of its permeability and non-toxicity
makes it a valuable test for the presence of acid or alkali within the
cell.
When a dye enters a living cell, it usually stains various granules
and structures contained therein, while at the same time it is uni-
formly diffused through the liquid phase of the protoplasm. If the
process of staining is conditioned by phenomena at boundary sur-
faces, simple undifferentiated protoplasm in the living state should be
incapable of staining, and this seems to be the general experience.
As regards the question of permeability to a given dye, unless the
cell is able to show that it is still alive by movement or by con-
tractility, it is clearly a matter of difficulty to be certain that, when
a particular dye enters, it does so during life or only after it has
destroyed the normal properties of the cell-membrane. The nucleus
itself seems to be very resistant to dyes while alive, and it has been 1
stated that the appearance of stain in it is a sure indication of death. )
BOLLES LEE made a large number of observations and came to the
conclusion that most of the " intra-vitam " stains are either due to
mere diffusion through the liquid protoplasm or that the stained
constituents were not really living, being food particles or products
of cell activity.
At the same time, many of the methods which come under this
heading are of much value. Methylen blue may be injected into
the living animal and frequently gives very successful staining of
nervous structures, owing to the fact of its being conveyed into
intimate contact with the cells by means. of the blood vessels.
The various methods of preserving the stain in the structures to
which it was localised during life obviously depend on the adequacy
of the means used to fix and maintain these structures and to retain
the properties owing to which the stain was taken up. This is by
no means a simple matter. MOTT describes in living nerve cells a
130 STAINING.
number of minute particles which stain on the outside with methy-
len blue. In fixed cells, as is well known, these particles aggregate
together to form the "• Nissl granules." MICHAELIS found similar
granules in liver cells. As the cells die, the stain leaves the granules
and passes into the nucleus.
The behaviour of the living nucleus to methyl green has given
rise to some discussion. It appears that no uni-cellular organism
in which the nucleus was stained has been observed to move,
whereas the chlorophyll grains may take up the stain while the cell
is normally motile. No convincing case of staining of the living
nucleus has in fact been described.
The question as to whether the cell elements which stain during
life are to be described as living or not is scarcely putting the problem
from the right point of view. If a dye obtains contact with the
interfaces between constituents of a cell, it will in all probability
be deposited there to a degree depending on the various properties
of the interface described previously. This may occur independently
of the fact as to whether one or both of the phases is living.
Apart, however, from these questions, it must be conceded that these
so-called " vital stains " are frequently very useful. According to
BOLLES LEE'S 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 BOLLES LEE finds 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 (LoiSEL,
Journ. de VAnat. et de la PJiys., 1898, No. 2, p. 212 — a work that contains
a good deal of information on the subject of intra-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.
FISCHEL (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.
The details of the various methods used for intra^ vital staining
CHAPTER XL 131
and the fixation of the results are described in other parts of this
book. The reader may be referred to the work of GOLDMAN (Unters.
ueber die Sekretion des Organismus im Lichte der " vitalen Farbung"
Laupp : Tubingen, 1912) for certain aspects of the problem.
208. Dead Cells. — Changes take place in cells in consequence of
which they cease to respond to external stimuli and are said to be
" dead." Their staining reactions are naturally different in this
state both from those during life and from those after their structures
have been " fixed " by treatment with some appropriate solution.
Although it seems likely that valuable information would be obtained
from the investigation of their behaviour in the dead but unfixed
state, very little work appears to have been done. DOGIEL obtained
various differentiations in nerve cells by observations at various
stages after death.
209. Fixed Tissues. — The majority of staining methods are under-
taken on tissues that have been fixed and hardened by reagents.
It is sufficient to mention here that some of these reagents merely
serve to coagulate or precipitate the constituents of cells without
marked changes in their chemical nature, although their physical
state is more or less altered. Alcohol is one of these agents. Other
fixing fluids, of which those containing chromic acid are represen-
tatives, produce what seem to be compounds of cell proteins with
the reagent. In this latter case, we have what is known as a
" mordant " present.
The distinction is often made between " substantive " and
"adjective" staining. These expressions are really based on a
particular theory of the staining process and are somewhat difficult
to justify logically. When a substance takes up a dye without the
necessity of the presence of any additional reagent, the staining is
said to be " substantive." When a mordant is required, it is
" adjective." The only true case of the former process is when an
electro-negative surface adsorbs a basic dye. Such a surface is
practically unstained by an " acidic " dye, when pure. The presence
of an electrolyte is necessary. But sodium chloride may suffice, and
the question arises whether this should be called a mordant.
Mordants are usually understood to be agents which form insoluble
compounds with dyes and in this way cause their fixation in places
from which otherwise they might be washed out by the subsequent
treatment with dehydrating agents, etc. Such a substance may
clearly be either already present in the fixed preparation when the
dye is added, or it may be added together with or subsequently to
9 — 2
132 STAINING.
the staining agent. It will readily be understood that the pro-
duction of an insoluble dye salt renders solution in water more diffi-
cult, but this alone is not enough. The compound must also be
firmly attached to the surface. Thus, the barium salts of colour
acids are insoluble, but barium salts cannot be used as mordants for
acidic dyes ; the compound formed is readily washed off mechani-
cally.
The insoluble compounds between a dye and a mordant are known
as " lakes." But the chemical nature of these substances is by no
means clear — especially when they are produced in situ in stained
tissues. In fact, they do not behave as simple compounds of the
dye and the mordant. They resist the action of strong acids and
bases in moderately strong solutions. So that the stains obtained
by this method are characterised by durability and " fastness."
On the whole, we must conclude that there are factors still un-
known, but probably related to the electrical properties of the
surface, which play an important part in these reactions with
mordants. A simple chemical theory does not account for all the
phenomena. Changes in the properties of the surface by the action
of the mordant are concerned. In this connection, we may call to
mind how frequently multivalent cations are used as mordants.
Alum is one of the commonest of these.
A further mention may here be made of the " progressive " and
" regressive " methods, between which there is a more definite
distinction than that between " substantive " and " adjective "
staining. A preparation may be placed in a very dilute solution of
a dye and the action stopped when the elements with the greatest
" affinity " for the dye have taken it up. The staining of nuclei
with dilute hsematoxylin-alum may be mentioned. If the action is
prolonged, various other constituents of the cell, besides the nucleus,
take on the colour. As a rule, no great differentiation is obtained
by this method. Better results are given by the " regressive "
method, in which a general overstaining is followed by a partial
decoloration, in which certain elements retain the stain, owing to
special chemical or physical properties, after it has been removed
from the rest. Thus safranin stains the whole section of a deep red
colour ; acting on it with alcohol removes the stain from all but the
chiomatin and the nucleoli. This action of alcohol may be explained,
as already pointed out, by its effect on the magnitude of the electric
charge, since its dielectric constant is lower than that of water.
Other differentiating agents are also used. Iron-alum, in the iron-
hsematoxylin method, serves both as preliminary mordant and as
CHAPTER XL 133
differentiating agent. This double action is not easy to explain
and confirms what was said above as to the complexity of the
process.
210. Metachromasy. — There are a few dyes, mostly of the basic
aniline series, which stain certain elements in the colour of the
ordinary solutions of the dye, other elements in that of the free
colour-base. Safranin stains nuclei red ; mucin and the ground
substance of cartilage, orange. Methyl violet stains " amyloid,"
and mucin red.
Although in a few cases this behaviour may be due to the dye
being really a mixture of two dyes, as in the case of iodine green,
there is no doubt that this is not the explanation of genuine cases.
According to MICHAELIS, the appearance of the colour of the base is
not due to the alkalinity of the elements in question. The fact
that the red stain given to mucin by thionin can be changed into blue
by alcohol and back to red by water shows that the change is not
one involving great alterations of chemical structure, and a
tautomeric one is naturally suggested. It would appear that the
change is one by which an amino-group becomes freed from its
combination with the mineral acid of the salt. In the case of
thionin, the acid is supposed to change its connection to the nitrogen
which unites the two benzene rings. What'are the conditions which
regulate the change from one form to the other is unknown. A
similar difficulty is met with in the case of iodine, which is brown in
solution in alcohol, violet in chloroform.
We must, however, not overlook the changes in colour shown by
substances in the colloidal state merely in consequence of a decrease
in their degree of dispersion or increase in size of particles. Gold is
a notable case. It. may be red, violet, blue or green. All of these
tints are met with in its use as a histological reagent. Whether
similar phenomena may occur in the adsorption of dyes is uncertain,
but cannot be dismissed without further evidence.
211. The Use of Stains in Practice. — Stains for special purposes are
described in other pages of this book. It will be obvious from the
contents of the present chapter that caution must be exercised in
making deductions as to chemical composition from behaviour to
dyes.
In general, the dyes of the anilin series, although of great value
for particular purposes, are apt to be less permanent than the old
logwood and carmine stains. The Canada balsam used must be as
pure as possible if the stain is to. last for any length of time.
134 STAINING.
The most distinctive processes, involving the use of mordants and
regressive differentiation, can only be undertaken on sections.
Staining in bulk is useful when the general anatomy is the object of
study. For cytological work it is of little value.
As remarked, the main object of staining is to obtain better information
of the appearance of the structures present in the cell. The fact,
however, that this appearance is not necessarily that of the living state
should never be allowed to escape remembrance. Without special
investigation of the case, it is not permissible to draw conclusions as
to the chemical nature of a cell constituent from its behaviour to dyes.
212. Choice of a Stain. — Mr. BOLLES LEE believes that the follow-
ing may be recommended to the beginner for general work : — For
sections, MAYER'S hcemalum ; or, for chromosmium objects more
especially, BENDA'S or HEIDENHAIN'S iron hsematoxylin.
For staining in toto Grenacher's alcoholic borax-carmine, or
Mayer's carmalum, or hsemalum, unless the object be so impermeable
as to require a very highly alcoholised stain, in which case take
Mayer's paracarmine, or for chromic acid objects Mayer's hcema-
calcium.
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).
CHAPTER XII.
CARMINE AND COCHINEAL STAINS.
213. Carmine. — Carmine is by no means merely carminic acid
with at most certain impurities. According to the analysis of
LIEBERMANN (Ber. d. Chem. Ges., Jahrg. 18, 1886, pp. 1969 1975)
it is a very peculiar alumina-lime-protein compound of carminic acid,
a true chemical compound from which at all events aluminium 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, be taken
as the basis of staining solutions instead of carmine. Staining solutions
thus prepared do not give essentially better stains than those made
with carmine ; but have the advantage of being of more constant
composition. For carmine is a product which varies greatly from
sample to sample.
Carminic acid of sufficient purity is furnished by GRUBLER and
HOLLBORN (or C. A. F. KAHLBAUM, in Berlin). It is soluble in water
and weak alcohol (that of 70 per cent, only dissolves less than 3 per
cent.). It cannot be used alone for staining, as it only gives in this
way a weak and diffuse stain.
214. Cochineal. — According to MAYER (Mitth. Zool. Stat. Neapel,
x, 1892, p. 496), the active principle of extract or tincture of cochineal
(as used in histology) is not free carminic acid, but carminic acid
chemically combined with a base which is not lime, but some alkali.
The watery extract made with alum, or cochineal-alum carmine
(§ 216), owes its staining power to the formation of carminate of
alumina (last §). The tincture made with pure alcohol, on the other
hand, contains only the above-mentioned carminate of some alkali.
This carminate alone stains weakly and diffusely (like carminic acid
alone). But if in the tissues treated with it it meets with lime salts,
136 CARMINE AND COCHINEAL STAINS.
alumina or magnesia salts, or even metallic salts capable of com-
bining 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.
215. 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 hsematei'n 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 HC1 (e.g.
0-1 per cent.). Alum-solution will 6*ften suffice, or, according to
HENNEGUY (Journ. de V'Anat. et de la Physiol., xxvii, 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 CAKMINE STAINS.
a. Acid.
216. Alum-carmine (GEENACHEK, Arch. mik. Anat., xvi, 1879,
p. 465). — An aqueous solution (of 1 to 5 per cent, strength, or any
other strength that may be preferred) of common or ammonia alum
is boiled for ten to twenty minutes with J to 1 per cent, of powdered
carmine. (It is perhaps the safer plan to take the alum solution
highly concentrated in the first instance, and after boiling the carmine
in it dilute to the desired strength.) When cool, filter.
This stain must be avoided in the case of calcareous structures
that it is wished to preserve.
TIZZONI (Bull So. Med. Bologna, 1884, p. 259), PISENTI (Gasg. degli
Ospetali, No. 24; Zeit. wiss. Mik., ii, 1885, p. 378), and GRIEB (Mem.
CHAPTER XII. 137
Soc. Ital. Sci., t. vi, No. 9, 1887 ; Zeit. wiss. Mik., vii, 1, 1890, p. 47)
have given modifications of Grenadier's formula which do not appear
to me rational.
MAYER (ibid., xiv, 1897, p. 29) makes a stronger stain by taking 2
grms. carmine, 5 grms. alum, and 100 c.c. water, and boiling for an hour.
Alum-carmine is an excellent 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 consider-
able size in bulk.
217. Acetic Acid Alum-Carmine (HENNEGUY, in Traite des Metk.
Techn., LEE et HENNEGUY, 1887, p. 88). — Excess of carmine is boiled
in saturated solution of potash alum. After cooling add 10 per cent,
of glacial acetic acid, and leave to settle for some days, 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 ntfn-acidified alum-carmine.
218. Cochineal Alum-Carmine (PARTSCH, 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 CZOKOB (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.
RABL (Zeit. wiss. MiL, 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.
KAWITZ (Zeit. wiss. Mik., xxv, 1909, p. 392) takes cochineal 4 grins.,
nitrate of aluminium (or ammonio -sulphate of cobalt) 4 grms., water
100 c.c., and glycerin 100 c.c. Only for sections.
219. MAYER'S Carmalum (Mitth. Zool. Stat. Neapel, x, 1892,
p. 489).— Carminic acid, 1 grin. ; alum, 10 grms. ; distilled water,
138 CARMINE AND COCHINEAL STAINS.
200 c.c. Dissolve with heat (if necessary). Decant or filter. Add
some antiseptic, either 1 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-carrnine 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 no£ have an
alkaline reaction.
RAWITZ (Anat. Anz., xv, 1899, p. 438) 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 keep 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.
220. MAYER'S Aqueous Aluminium-Chloride 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
carmalum.
Use as carmalum. The stain is of a blue-violet colour, very powerful,
and elective, but not so purely nuclear as carmalum. It is recom-
mended 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.
221. Alum-Carmine and Picric Acid. — Alum-carmine objects may
be double-stained with picric acid. LEGAL (Morph. Jahrb., viii,
p. 353) combines the two stains by mixing 10 vols. of alum-carmine
with 1 of saturated picric acid solution. I find this very recommend-
able.
Aceto-Carmine (Acetic Acid Carmine) SCHNEIDER (Zool. Anzeig.,
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 may 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.
CHAPTER XII. 139
A similar stain has been prepared with formic acid by PIANESE (see
Zeit. wiss. Mik., x, 4, 1894, p. 502).
For BUBCHABDT'S pyroligneous-acid carmines see Arch. mik. Anat.,
liii, 1898, p. 232 ; and Jena Zeit. Naturw., xxxiv, 1900, p. 720.
222. Iron Carmine.— I recommend trial of the following, which I
have already published in the Traite des Meth. Techniques, LEE et
HENNEGUY, 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 haematoxylin), 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 hsematoxylin, but somewhat weak.
Iron Carmine. — PFEIFFER VON WELLHEIM (Zeit. wiss. Mik., xv,
1898, p. 123) mordants for six to twelve hours in a very weak solution
of chloride of iron in 50 per cent, alcohol, washes in 50 per cent, alcohol,
and stains as above. Overstains may be corrected with 0-1 to 0-5 per
cent. HC1 alcohol. I find this good, but not so good as the last.
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). Einse 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.
Hollande's Chlorcarmin Staining Method (G. R. Soc. Biol, 1916,
Ixxix, p. 662, and Jour. Roy. Micr. 800., 1920). — Place 5 c.c. pure
hydrochloric acid in a porcelain dish; add little by little 14 grms.
powdered carmine, stirring constantly to make a homogeneous doughy
mass. Allow to digest for twenty-four hours; add 250 c.c. aq. dest.,
bring to the boil, and keep boiling for half an hour. Filter; make up
to 180 c.c. with aq. dest., and then add enough 75 per cent, alcohol to
make a total volume of 200 c.c. Stain sections or pieces of tissue for
two to twenty-four hours. Rinse in aq. dest. or 30 per cent, alcohol ;
immerse in 3 per cent, iron alum solution, in which the sections become
black, and are then slowly decolourised; when differentiation is com-
plete, rinse in a 1 per cent, pyridin solution, and wash under the tap for
ten to fifteen minutes. Counterstain and mount as desired. This is a
very intense stain suitable for mitochondria and cell granules.
t. Iron Carmalum (DE GROOT, Zeit. wss. Mik., xx, 1903, 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 stain.
140 CARMINE AND COCHINEAL STAINS.
224. Iron Cochineal (SPULER, Encyclopaedic d. mile. Technik, 1903,
p. 153, and 1910, p. 240).— Stain for forty-eight hours in a stove, in
extract of cochineal (made in a highly complicated way), wash with
water, put into solution of ferric alum of f per cent, strength for twenty-
four hours or more. If the stain is not sufficiently intense, the whole
process may be repeated.
PETER (Zeit. wiss. Mik., xxi, 1904, p. 314) stains material in bulk for
forty-eight hours (sections eighteen to twenty-four) in an incubator, in
a similar extract, acidified with HC1, treats with iron-alum of 2| 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 (ibid., xxii, 1905, p. 85) stains sections or entire objects in a
solution of 5 to 10 grms. cochineal, 8 grms. ferric alum, 250 c.c. water,
and 25 c.c. sulphuric acid of 10 per cent., boiled for fifteen to twenty
minutes.
{3. 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 crystallising dish until it is
entirely dried up. It should be allowed to putrefy if possible. Dissolve
the dry deposit in pure water, and filter.
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, Zeit. wiss. Mik., iv, 1887, p. 50). It can
be obtained from GTRUBLER & HOLLBORN (Natron- Garmin).
226a. ORTH'S Lithium-Carmine (see early editions) macerates strongly,
and is superfluous. For that of BEST, see Zeit. wiss. Mik., xxiii, 1906,
p. 322.
227. Magnesia-Carmine (MAYER, Zeit. wiss. Mik., xiv, 1897, p. 23).
— Take 1 grm. carmine, 0-1 grm. magnesia usta, and 50 c.c. distilled
water, boil for five minutes, filter, and add 3 drops of forrnol. This
is the stock solution. A weak solution may be made by boiling 0-1 grm.
carmine for half an hour in 50 c.c. of magnesia water (made by leaving
0-1 grm. of magnesia usta in contact with 100 c.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.
22S. As to Picro-Carmine. — The term " picro-carmine " is
commonly used to denote a whole tribe of solutions in which
carmine, ammonia, and picric acid exist uncombined in haphazard
proportions. These solutions do not contain a double salt of picric
and carminic acid and ammonia, or picro-oarminate of ammonia.
They are always alkaline, and frequently injurious to tissues. The
CHAPTER XII. Hi
raison d'etre 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 neutral-
ise the ammonia, which it only does imperfectly. See MAYER in
Zeit. wiss. MiL, xiv, 1897, p. 18.
229. RANVIER'S Picro-Carmine, Original Formula (Traite, p. 100).
—To a saturated solution of picric acid add carmine (dissolved in
ammonia) to saturation. Evaporate down to one-fifth the original
volume in a drying oven, and separate by nitration 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.
KANVIER'S Newer Formula does not give a more constant product
(see previous editions).
230. VAN WIJHE 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 cent,
of chloral hydrate. G-ives an almost neutral preparation.
231. MAYER'S Pier o- 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 & HOLLBORN, or the solution may be made by heating
0-25 grm. of carbonate of magnesia in 200 c.c. of 0-5 per cent, solution
of picric acid, allowing to settle, and filtering.
DE GTROOT'S picro -magnesia carmine (ibid., xxix, 1912, p. 184) contains
ammonia, which is bad, and seems to me superfluous.
232. Other Formula 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 (GRENACHER, Arch. mik. Anat.,
xvi, 1879, pp. 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
142 CARMINE AND COCHINEAL STAINS.
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 HC1 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 per cent. 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 coloration. 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.
234. MAYER'S Paracarmine (Mitth. Zool. Stat. Neapel, x, 3, 1892,
p. 491). — Carminic acid, 1 grm. ; chloride of aluminium, 0-5 grm. ;
chloride of calcium, 4 grms. ; 70 per cent, alcohol, 100 c.c. 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 intended to be mounted whole may
be washed out with a weak solution of aluminium chloride in alcohol,
or if this be not sufficient, with 5 per cent, common acetic acid (or
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 penetrating ; and has less
CHAPTER XII. 143
tendency to form precipitates in the interior of objects. But, in my
hands, it does not give quite so fine a stain.
234a. Alcoholic Hydrochloric-Acid Carmine.— GRENACHER'S receipt
(Arch. f. Mik. Anat., xvi, 1879, p. 468) is troublesome. That of MAYER
(Mitth. Zool. Stat. Neapel, iv, 1883, p. 521 ; Intern. M onatsschr. f. Anat.,
etc., 1897, p. 43) is better : Carmine 4 grms. ; water, 15 c.c. ; hydro-
chloric 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 HC1.
For a complicated receipt of LOEWENTHAL see Zeit. wiss. Mik., xix,
1902, p. 56.
235. Alcoholic Cochineal, MAYER'S Old Formula (Mitth. Zool.
Stat. Neapel, ii, 1881, p. 14).— Cochineal in coarse powder is
macerated for several days in alcohol of 70 per cent. For each
gramme of the cochineal there is required 8 to 10 c.c. of the alcohol.
Stir frequently. Filter.
The objects to be 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
certain 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.
144 CARMINE AND COCHINEAL STAINS.
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 calcareous structures which it is wished to preserve —
which the new fluid cannot. For specimens of Pluteus, for instance,
I find it excellent. But it only gives good results with such objects
as contain the necessary salts, § 212.
236. MAYER'S Alcoholic Cochineal, New Formula (Mitth. Zool. Stat.
Neapel, x, 1892, p. 498). — Cochineal, 5 grms. ; chloride of calcium,
5 grms. ; chloride of aluminium, 0-5 grm. ; nitric acid of 1-20 sp. gr.,
8 drops ; 50 per cent, alcohol, 100 c.c. Powder the cochineal and rub
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 succedaneum
of paracarmine.
Since this fluid contains in itself all the necessary salts (§ 212), it gives
good results with all classes of objects.
CHAPTER XIII.
H^MATEIN (HJEMATOXYLIN) STAINS.
237. Introduction. — Hcematoxylin is a dye extracted from log-
wood. It is a substance that oxidises very readily, thus becoming
converted into hcematein, or, as often happens, into other more
highly oxidised products. It appears to be now thoroughly well
established (see NIETZKJ, Chemie der organischen Fdrbstoffe, Berlin,
Springer, 1889, pp. 215—217, and MAYEE, Mitth. Zool Stat. Neapel,
x, 1891, p. 170) that the colouring agent in solutions of log-
wood or haematoxylin is not the haematoxylin itself, but hsematein
formed in them (or, in some cases, one of the higher oxidation
products).
Haematein is an acid body, a " colour acid " (§§ 201, 206). Sut>-
stantively employed, it is a very weak plasma stain. But combined
with appropriate mordants it becomes basic, 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 em-
ployed, the former furnishing 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 haematein in staining
solutions was formerly brought about by allowing solutions of
haematoxylin 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 solutions are not fit to use for
staining.
It was discovered by MAYER and UNNA independently (see MAYER
in Mitth. Zool. Stat. Neapel, x, 1891, pp. 170—186 ; UNNA in Zeit.
wiss. MiL, viii, 1892, p. 483) that nothing is easier than to bring
about this change artificially ; all that is necessary being, for in-
stance, to add to a solution of haematoxylin containing alum a little
neutralised solution of peroxide of hydrogen or other powerful
10
146 HMMATEIN (HMMATOXYLIN} STAINS.
oxidising agent.* The solution becomes almost instantaneously
dark blue, " ripe " and fit for staining. Other methods of " ripen-
ing," or of preparing hsematein 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 ascertaining whether the solution at any moment
contains sufficient hsematein to afford a good stain. And here a
second difficulty arises : the oxidising process continuing, the
solutions become " over-ripe " ; the hsematein, through further
oxidation, passes over into colourless compounds, and the solutions
begin to precipitate. They are therefore, in reality, a mixture in
constantly varying proportions of " unripe," " ripe," and " over-
ripe " constituents (the first and last being useless for staining
purposes), and, in consequence, their staining power is very in-
constant.
Logically, therefore, as concluded by MAYER, not hsematoxylin,
but hcematein, should be taken in the first instance for making the
staining solution.
But this is not always indicated ; for such solutions may easily
over-oxidise, either in the bottle or on contact with the tissues. 80
that it is always preferable to start from hsematoxylin. In this case,
it should not be done by dissolving the hsematoxylin straight away
in the other ingredients of the staining solution. The solutions
should be made up from a strong stock solution made by dissolving
hsematoxylin crystals in absolute alcohol : one in ten is a good
proportion. This solution should be kept for a long time — months,
at leapt, a year if possible ; it gradually becomes of a vinous red,
and should not be used till it has become quite dark. It has then
become to a great extent oxidised into hsematein, and the staining
solutions made up from it will be at once fairly ripe.
Hsematein (or hsematoxylin) 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 hsematoxylin for sections.
238. Haernatoxylin is found in commerce in the form of crystals,
either colourless or browned by oxidation, easily soluble in either
water, glycerin, or alcohol.
* Ee-in vented lately (Zeit. 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.
CHAPTER XIII. U7
239. Haematein is found in commerce as a brown powder, entirely,
though with difficulty, soluble in distilled water and in alcohol'
giving a yellowish-brown solution, which remains clear on addition
of acetic acid. Alkalies dissolve it with a blue- violet tint. (See
also last edition.)
240. Iron Haematoxylin, Generalities.— This method is due to
BENDA (Verh. Phys. Ges., 1885—1886, Nos. 12, 13, 14 ; Arch. Anat,
Phys., 1886, p. 562 ; third ed. of this work, p. 365).
The method was independently worked out about the same time
by M. HEIDENHAIN. 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 mordant in Benda's liquor 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 differentia-
tion. 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 liquor ferri 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 recommends WEIGERT'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 safe, and causes swelling of the
tissues.
VAN GIBSON'S picro-saurefuchsin has been recommended as a
differentiation fluid by Benda (Deutsch. med. Wochenschr., 1898,
No. 30). I find it gives very delicate differentiations, but acts very
slowly, requiring nearly as many hours as the iron alum solution
does minutes. The addition of the saurefuchsin to the picric acid
is, I find, not necessary, and may prove an injurious complication.
In these processes haematoxylin is generally used for the stain,
not hcematein, the iron salt oxidising it into haematein, or into a
higher oxidation product. I have obtained some good stains with
10-2
148 HMMATEIN (HMMATOXYLIN) STAINS.
hsematein, but also some very bad ones ; presumably the solutions
easily over-oxidise on contact with the iron salt.
The hsematoxylin 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.
Iron hsematoxylin 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. BENDA'S later Iron Haematoxylin (Verb. d. Anat. Ges., vii, 1,
1893, p. 161). — Sections are mordanted for twenty-four hours in
liquor ferri sulphurici 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
hsematoxylin 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 30 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 differentiation, it
should be taken extremely diluted (of a very pale straw-colour, about
1 : 30 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, sometimes
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 favourable material.
* 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.
Doabtless the ferri persulphatis liquor B. P. will do instead ; the point
is, to have a per-salt, and not a proto-salt.
CHAPTER XIII. 149
242. HEIDENHAIN'S Iron Hsematoxylin (M. HEIDENHAIN, " Uber
Kern und Protoplasma," in Festschr. fur Kolliker, 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 alum (ammonio-ferric
sulphate). By this is always meant in histology the double salt of
ammonium and sesquioxide of iron (NH4)2Fe2 (S04).i, in clear violet
crystals ; the double salt of the protoxide, or salt of MOHR 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 an
aqueous solution (of about 0-5 per cent.) of haematoxylin. They
are then rinsed with water, and again treated with the iron solution,
which slowly washes out the stain. The progress of the differentia-
tion 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 differentia-
tion 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
treatment with the iron and the stain. If the baths have been of
short duration, viz. not more than half an hour in the iron and as
much in the stain, blue preparations will be obtained. These show
a very intense and highly differentiated 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
subsequent differentiation in the second iron bath also duly pro-
longed, black preparations will mult. These show chromosomes
stained, central corpuscles stained intensely black, cytoplasm some-
times colourless, sometimes grey, in which case achromatic spindle-
fibres and cell-plates are stained, connective-tissue fibres black, red
blood-corpuscles black, micro-organisms sharply stained, 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.
* Why ! I find my iron-alum solution, as well as the liquor ferri
sulph. oxid., last §, mix clear with alcohol without the least precipitate
forming.
150 HMMATEIN (HMMATOXYLIN) STAINS.
This should be a 2J 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 " hsematoxylin
solution, i.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 energetic stain, owing to its containing a trace of iron
brought over by the sections. Differentiate in a 2|- 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," and " Chromosomes," etc.
BIELASZEWICS (Bull. Acad. Cracovie, 1909, 2 serie, p. 152) differentiates
with very weak solution of calcium chloride ; GTUARNIERI (Mon. Zool.
Ital., xvii, 1906, p. 44) with saturated solution of picric acid.
GURWITSCH (Zeit. wiss. Mik., xviii, 1902, p. 291) floods sections on
the slide with mordant, warms on a water-bath till bubbles are given off
or the mordant becomes turbid, then stains with the hsematoxylin 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).
FRANCOTTE (Arch. Zool. Exper., vi, 1898, p. 200) mordants with tartrate
of iron, MALLORY (Journ. Exper. Med., v. 1900, p. 15) with chloride.
243. Iron Haematoxylin (BUTSCHLI, Unters. uber mikroskopisehe
Schaume 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 hsematoxylm. A stain of
extraordinary intensity, used by Biitschli for sections, 1 ^ in thickness,
of Protozoa.
244. Weigert's Iron Hsematoxylin Mixture (Zeit. wiss. Mik., xxi,
1904, p. 1). — Mix 1 part of a 1 per cent, solution of haematoxylin in
alcohol of 96 per cent, with 1 of a solution containing 4 c.c. of liq. ferri
sesquichlor., 1 c.c. of officinal hydrochloric acid (sp. gr. 1-124) and 95
of water. The mixture may be 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.
MOREL and BASSAL (Journ. Anat. Phys., xlv, 1909, p. 632) stain in
bulk in Weigert's mixture with the addition of 1 c.c. of 4 per cent.
solution of acetate of copper.
CHAPTER XI I L ir,i
245. JANSSENS' Iron Haematoxylin ( ' * H6matoxyline 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
Delafield's. A progressive stain, nuclear : for yeast cells.
246. HAN SEN'S Iron Haematoxylin (Zeit. wiss. Mile., xxii, 1905, p. 55).
—A solution of 10 grms. ferric alum in 150 c.c. water is added to a
solution of 1-6 grm. hsematoxylin in 75 c.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 Hsematein (Alum Haematoxylin) 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. In order to get a blue stain in preparations that have
come out red through 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 haematein 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. See SCOTT'S tap-water substi-
tute, § 676. Of course this is a different question from that oT
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 bicarbonate 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.
0-1 to 0-2 or even 0-5 per cent, of hydrochloric 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 (0-1 per cent.).
1 52 EMMA TEIN (EMMA TOX YLIN) STAINS.
Bicarbonate of soda may be used for neutralisation with 70 per
cent, alcohol as the vehicle (VON WISTINGHAUSEN, 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 hsemalum diluted ten to twenty-fold
with water. The stain obtained 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. MAYER
says that very dilute solutions will give a pure nuclear stain if they
have been diluted with alum-solution, or have been acidified. Chrom-
osmium material will not yield a pure chromatin stain unless it is
very fresh ; it is consequently next to impossible 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 solutions : MAYER finds that his
hsematein 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.
Formulae §§ 248 to 259 give aqueous solutions, and §§ 260 to 263
alcoholic ones.
248. MAYER'S Hsemalum, Newer Formula (Zeit. wiss. MiL, xx,
1903, p. 409). — Hcematoxylin, 1 grm. ; water, 1 litre. Dissolve,
and add 0-2 grm. of iodate of sodium (NaI03) 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 : 1 grm. of Jicematein (or the ammonia salt,
§§ 238, 239) dissolved with heat in 50 c.c. of 90 per cent, alcohol, and
added to a solution of 50 grms. 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. Concentrated, it
stains sometimes almost instantaneously, or in any case very rapidly.
(Spring water or tap-water containing lime must not be used for
diluting ; perhaps weak solution of alum in distilled water is the
CHAPTER XIII. 153
best means of all.) After staining, sections may be washed out
either with distilled or common water. It is admirable for staininq
J %y
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 carefully 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 turpen-
tine 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.
Hsemalum may be mixed with alum-carmine, Saurefuchsin, or
the like, to make a double staining mixture ; but it seems preferable
to use the solutions in succession.
249. MAYER'S Acid Hsemalum (Mitth. Zool. Stat. Neapel, x, 1891,
p. 174). — This is heemalum 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 (Zeit. wiss. Mik.,
viii, 1892, p. 483).
Hsematoxylin ...... 1
Alum 10
Alcohol -.100
Water 200
Sublimed sulphur ...
If the sulphur be added to the haematoxylin solution only when the
latter has become somewhat strongly blue, i.e. after two or three days'
time, the stage of oxidation attained by the solution will be fixed for
some time by the sulphur, and according to Unna the solution will
remain "constant" in staining power. MAYER (Mitth. Zool. Stat.
Neapel, xii, 1896, p. 309) finds that the sulphur process does not
preserve the solutions for long, whilst glycerin does. See below,
" GLYCHJEMALUM."
251. MAYER'S Glychsemalum (Mitth. Zool. Stat. Neapel, xii, 1896,
p. 310). — Hsematein (or hsemateate of ammonia), 0-4 grm. (to be rubbed
up in a few drops of glycerin) ; alum, 5 grms. ; glycerin, 30 ; distilled
water, 70. The stain is not purely nuclear, but may be made so by
washing out with alum solution or a weak acid. The solution keeps
admirably.
154 HMMATEIN (HMMATOXYLIN) STAINS.
KAWITZ (Leitfaden, 2nd ed., p. 63) takes 1 grin, haematein, 6 grms.
ammonia alum, 200 grms. each of water and glycerin.
Or (Zeit. wiss. Mik., xxv, 1909, p. 391) 1 grm. hsematein, 10 grms.
of nitrate of aluminium, 250 grms. each of water and glycerin.
252. HANSEN'S Solution (Zool. Anz.t 1895, p. 158). — See fourth edition.
253. HARRIS'S Solution (Micr. Bull, xy, 1898, p. 47 ; Journ. App.
Mic., iii, p. 777). — Alum-hsematoxylin solution ripened by addition of
mercuric oxide. MAYER (Grundzuge, 1901, p. 171) finds the formula
" gives too much hsematein."
254. Bohmer's Heematoxylin (Arch. mik. Anat., iv, 1868, p. 345 ;
Aerzt. Intelligenzbl., Baiern., 1865, p. 382). — Make (A) a solution of
haematox. 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 hsematoxylin ought to be old and dark
(§ 237).
A. Gr. HORNYOLD (Trans. Manch. Micr. Soc., 1915) prepares : —
Solution A : haematoxylin, 0-7 grm. ; absolute alcohol, 20 c.c. Solution
B : alum, 0-35 grm. ; aq. dest., 60 c.c. Mix A and B, expose to light
in window for three or four days, then add 20 drops of tincture of
iodine. Stain sections five to ten minutes till red-brown. Differentiate
in 70 per cent, alcohol, to which add a few drops of acetic acid. The
sections then turn blue. Grood stain after osmic fixatives.
255. Delafield's Hsematoxylin (Zeit. wiss. Mik., ii, 1885, p. 288 ;
frequently attributed erroneously to GRENACHER or PRUDDEN). —
To 400 c.c. of saturated solution of ammonia-alum (that is about
1 to 11 of water) add 4 grms. of hsematox. cryst. dissolved in 25 c.c.
of strong alcohol. Leave it exposed to the light and air in an un-
stoppered bottle for three or four days. Filter, and add 100 c.c. of
glycerin and 100 c.c. of methylic alcohol (CH40). Allow the solution
to stand (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.
BUTSCHLI (Unters. ub. mikroscopische Schdume u. das Protoplasma,
etc., 1892) recommends, under the name of "acid haematoxylin,"
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 differentiated nuclear stain than the usual solution.
MARTINOTTI (Zeit. wiss. Mik., xxvii, 1910, p. 31) makes it up with 0*2
per cent, of hcematein, and less alum (2 per cent.).
CHAPTER XI1L 155
256. Ehrlich's Acid Hsematoxylin (Zeit. wiss. Mik., 1886, p. 150).—
Water 100 c.c., absolute alcohol 100, glycerin 100, glacial acetic acid
10, haematoxylin 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 power, for years, if kept in a well-stoppered bottle. It is
very appropriate for staining in bulk, as overstaining does not
occur. I find it excellent.
MANN (ibid., xi, 1895, p. 487) makes up this stain with an equal
quantity of hsematein instead of haematoxylin.
MAYER (Grundziige, LEE and MAYER, 1st ed., p. 154) finds
that this is too much and makes the mixture overstain ; 0-4 grm.
of hsematein is quite enough.
For proper method of using EHRLICH'S hsematoxylin, see SCOTT,
§669.
257. BURCHARDT'S Pyroligneous Acid Haematoxylin (Arch. mik.
Anat., liii, 1898, p. 232) would seem to be superfluous at least.
258. UNNA'S Oxidised Haematoxylin (from MARTINOTTI, Zeit. wiss.
Mik., xxvii, 1910, p. 31). — Haematoxylin 0-5, alum 2, water 60,
alcohol 10, glycerin 20, peroxide of hydrogen solution 10, carbonate
of soda 0-05.
MARTINOTTI, loc. cit., makes it up with hcemaiein (0-2 grm.).
259. APATHY'S Hsematein Mixture I A (Mitth. 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 percent, salicylic acid in water,
and (B) a 1 per cent, solution of hsematoxylin in 70 per cent, alcohol,
preserved for six to eight weeks in a bottle not quite full. Mix
1 part of A with 1 of B and 1 of glycerin. Stains either sections or
material in bulk. Apathy uses it for staining neuro-fibrils.
260. KLEINENBERG'S Haematoxylin (Quart. Journ. Micr. Sci., Ixxiv,
1879, p. 208). — Highly irrational and very inconstant in its composition
and its effects ; see early editions ; also the criticism of MAYER (Mitth.
Zool. Stiit. Neapel, x, 1891, p. 174), and that of SQUIRE in his Methods
and Formulae, p. 25, and the alternative formulae of SQUIRE (loc. cit.)
and of VON WISTINGHAUSEN (Mitth. Zool. Stat. Neapel, x, 1891, p. 41).
261. MAYER'S Haemacalcium (Mitth. Zool. Stat. Neapel, x, 1891,
p. 182).— Hsematein (or hsemateate of ammonia, §§ 238, 239),
1 grm. ; chloride of aluminium, 1 grm. ; chloride of calcium, 50 grins. ;
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 ingre-
dients, add the acid and alcohol, dissolve either cold or with heat ;
lastlv add the chloride of calcium.
156 HMMATEIN (HMMATOXYLIN) STAINS.
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 suffice.
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 staining in acid alcohol.
Anyway objects ought NOT to have an alkaline rea.ction. 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
haemalum, and Mayer recommends it merely as a substitute for
Kleinenberg's, in cases in which an alcoholic hsematein stain seems
indicated, as being easy to prepare, and constant in its effects.
262. MAYER'S Heemastrontium (Gnmdzuge, LEE and MAYER,
1910, p. 166). — 1 grm. hsematein, 1 grm. aluminium chloride,
50 grms. strontium chloride, 600 c.c. alcohol of 70 per cent., and (if
desired) 0-25 grm. citric acid. Prepare and use as hsemacalcium.
263. DE GROOT'S Alcoholic Haemalum (Zeit. wiss. Mik., xxix, 1912,
p. 182). — Mix 20 c.c. of glycerin with 240 of alcohol of 70 per cent.
Take 4 c.c. of the mixture, 2 c.c. of hydrogen peroxide, and 0-5 grm. of
hsematoxylin, and dissolve with heat. Add 60 c.c. of the mixture,
4 grms. of calcium chloride, and 2 grms. of sodium bromide. Dissolve,
add 3 grms. of alum, heat and add 100 c.c. of the mixture. When the
alum is dissolved add 0-2 grm. of ferri-cyanide of potassium ; dissolve
and add 3 grms. more of alum and the rest of the mixture. Said to stain
almost as well as hsemalum. Wash out with alcohol of 70 per cent.
264. Other Alumina-Haematein Solutions. — A large number of
suppressed receipts will be found given in the earlier editions.
265. K. HEIDENHAIN'S Chrome Hsematoxylin (Arch. mik. Anat.,
xxiv, 1884, p. 468, and xxvii, 1886, p. 383). — Stain for twelve to twenty-
four hours in a * per cent, solution of hsematoxylin in distilled water.
Soak for the same time in a 0-5 per cent, solution of neutral chroma te
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 hsematoxylin
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. APATHY'S Modification of Heidenhain's Process (Zeit. wiss.
Mik., v, 1888, p. 47). — This is an alcoholic method. Stain in a 1 per
CHAPTER XIII. 157
cent, solution of hsematoxylin in 70 or 80 per cent, alcohol. Differentiate
sections of 10 to 15 p., half the time of staining, sections of 25 to 40 ^
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 in the dark.
For celloidin series of sections, Apathy (ibid., 1889, p. 170) stains in
the hsematoxylin solution as above for ten minutes ; then removes the
excess of heematoxylin 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.
287. SCHULTZE'S Chrome Haematoxylin (Zeit. 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, hsematoxylin in alcohol of 70 per cent., then
washed out in alcohol of 80 per cent.
268. HANSEN'S Chrome Hsematoxylin (ibid., xxii, 1905, p. 64).—
Ten grms. of chrome alum boiled in 250 c.c. of water till green, and
1 grm. hsematoxylin (dissolved in 15 c.c. of water) added ; to the
mixture when cold add 5 c.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.
269. Vanadium Haematoxylin (HEIDENHAIN, Eneyclop. mik. Technik.,
1903, p. 518). — Add 60 c.c. of a 6 per cent, solution of haematoxylin
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. BENDA'S Copper Haematoxylin (Arch. mik. Anat., xxx, 1887,
p. 49). — See fourth edition. According to my experience, not to be
compared with iron hsematoxylin, and superfluous.
271. MALLORY'S Phospho-molybdic Acid Hsematoxylin (Anat.
Am., 189i, p. 375).— One part 10 per cent, phospho-molybdic acid
solution, 1 part h&ematoxylin, 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 haematoxylin in order to obtain
the best results ; if a good stain be not obtained at once, more
hsematoxylin must be added. Water must never be used for
diluting it.
158 HMMATEIN (HMMATOXYLIN) STAINS.
See also KIBBERT (Centralb. allg. Path., vii, 1896, p. 427 ; Zeit. wiss.
Mik., xy, 1898, p. 93), PATELLANI ( 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
KEN TON'S.
KODIS (Arch. mik. Anat., lix, 1901, p. 211) takes hsematoxylin, 1 part ;
molybdic anhydride, 1-5 ; water, 100 ; H202, 0-5, or a crystal of HgO.
POLICE (Arch. Zool. Napoli, iv, 1909, p. 300) takes 0-35 grm. hsema-
toxylin, 10 drops phospho -molybdic acid of 10 per cent., 10 grms. chloral
hydrate, and 100 grms. alcohol of 70 per cent.
272. MALLORY'S Phospho-tungstic Hsematoxylin (Journ. Exp.
Med., v, 1900, p. 19 ; Zeit. wiss. Mik., xviii, 1901, p. 178) :
Hsematoxylin . . . . . .0-1
Water 80-0
10 per cent, solution of (MERCK'S) phospho-
tungstic acid . . . . . . 20-0
Peroxide of hydrogen (U.S. Ph.) . . . 0-2
(Dissolve the hsematoxylin, add the acid, then the peroxide.) Stain
sections two to twenty-four hours, wash out with water. A poly-
chromic stain, nuclei blue, intercellular substances pink. I consider
this a fine stain.
273. DONAGGIO'S Tin Hsematoxylin (Ann. Nevrol. Napoli, xxii, 1904,
p. 192). — A 1 per cent, solution of hsematoxylin 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 Hsematoxylin. — SCHULTZE (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 haematoxylin in alcohol of 35
to 50 per cent. Wash out for a day or more with alcohol of 70 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 — rare 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.
They have been little used for that purpose since the working out of
the iron haematoxylin process, which gives a more energetic stain.
But they may still be useful as a means of controlling the iron
haematoxylin 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 sections, Saurefuchsin ; for entire objects, picric
acid.
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 Meihyl-
anilin green, Griinpulver, Vert Lumiere, Lichtgrun ; these two last
are in reality the name of another colour. When first studied by
Calberla, in 1874 (Morphol. Jahrb., iii, 1887, p. 625), it went by the
160 NUCLEAR STAINS WITH COAL-TAR DYES.
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 green (Vert d'Eusebe), nor with the phenylated
rosanilins, Paris green, and Vert dAlcali, or Veridine.
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 BENEDIKT and
KNECHT'S Chemistry of the Coal-tar Colours. For tests for purity
see MAYER, Mitth. Zool. Stat. Neapel, xii, 1896, p. 312, and FISCHER,
Fixirung, Fdrbung, u. Ban 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 to? fresh, unfixed 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 (BURCKHARDT, La Cellule, xii, 1897, p. 364).
You may wash out with water (best acidulated) and mount in some
acid aqueous medium containing a little of the methyl green in
solution. The mounting medium, if aqueous, must be acidulated.
Employed in this way, with, fresh unfixed tissues, methyl green is
a pure chromatin stain, in the sense of being a precise 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 in-
variably stained of a bright green (with the exception of the nuclein
of the head of some spermatozoa), whilst extra-nuclear structures
are in general stained in tones of blue or violet. But this meta-
chromatic reaction is probably due to the methyl-violet impurity,
and is not obtained with a chemically pure methyl green.
CHAPTER XIV. 161
Staining is instantaneous ; overstating never occurs. The
solution is very penetrating, kills cells instantly without swelling or
other change of form, and preserves their forms for at least some
hours, so that it may be considered as a delicate fixative. 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 BIPART and PETIT (this is an
excellent medium for washing out 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 satis-
factorily 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
EHRLICH-BIONDI combination), considerably increased by treating
the sections for a few minutes with tincture of iodine before staining
(M. HEIDENHAIN).
Of preparations mounted with excess of colour in the usual
aqueous media, I find the most fortunate only survive for a few
moriths. Dr. HENNEGUY, however, writes to me that it keeps well
in BRUN'S glucose medium.
It was first pointed out, I believe, by HESCHL (Wiener med.
Wochenschr., 2, 1879), that methyl green is a reagent for amyloid
degeneration. His observations were confirmed by CUKSCHMANN
(Virchow's Arch., vol. Ixxix, 1880, p. 556), who showed that it colours
amyloid substance of an intense violet ; but this, as pointed out by
SQUIRE (Methods and Formula, etc., Churchill, 1892, p. 37), is un-
doubtedly 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 (WEIGERT, in Arch. mik. Anat.,
xv, 1878, p. 258). You may add a little acetic or osmic acid to the
solution. MAYSEL (ibid., xviii, 1880, pp. 237, 250) dissolves the
colour in acetic acid (this solution does not give a permanent stain).
Alcoholic solutions may also be used, e.g. saturated aqueous solution
diluted with one-third volume of 90 per cent, alcoholic ; or CAL-
BERLA'S glycerin-and-alcohol mixture or dilute glycerin (say of
40 per cent, to 50 per cent.) may very advantageously be employed.
The watery solutions must be frequently filtered (but then much
M. J1
162 NUCLEAR STAINS WITH COAL-TAR DYES.
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 over-
stains. 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. HERLA (Arch. Bid., 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 wrashes
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 intra-vitam 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. Ghirurgie, xxvii, 1888, pp. 538 — 584 ; Zeit. wiss.
Mik., 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.
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 thionin, which need not even
be acidified ; also, for fresh tissues especially, gentian violet, dahlia, and
toluidin 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 this 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 solu-
tion. 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
CHAPTER XIV. 168
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
HEIDENHAIN, EncycL mik. Technik, i, pp. 433, 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 (or Hermann's fluid) should be employed.
During the staining the tissues become overstained, that is, charged
with colour in an excessive and diffuse manner. The stain must
now be differentiated by removal of the excess of colour.
281. Differentiation, — This is generally done with alcohol, some-
times neutral, sometimes acidulated (with HC1). The stained sections,
if loose (celloidin sections), are brought into a watch-glassful of
alcohol ; if mounted in series on a slide, they are brought into a tube
of alcohol (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
transparent, and (in the case of chrom-osmium objects) have changed
colour, owing to the coming into view of the general ground colour
of the tissues. (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 differen-
tiation. This may be well in some cases, but in general 95 per cent,
is found to answer perfectly well. HEIDENHAIN (EncycL, i, p. 434)
takes methyl alcohol.
The hydrochloric-acid-alcohol extracts the colour much more
quickly from resting nuclei than from kinetic nuclei. 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.
11 — 2
164 NUCLEAR STAINS WITH COAL-TAR DYES.
The proportion of HC1 with which the alcohol should be acidi-
fied 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. The acid
process is vastly more rapid than the neutral process, and therefore
of course more risky.
There exists also a method of differentiation known as substitution —
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 RESEGOTTI in Zeit.
wise. 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 RESEGOTTI 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 BENDA'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 colour 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 nuclei 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 anilin than clove oil.
CHAPTER XIV. 165
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 that does not affect the stain
(xylol, cedar oil, etc.). Chloroform should be avoided, either as a
clearer or as the menstruum 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. Either 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. de I'Anat. et de la
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 about half the time that would have
been ta^ken 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, February
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 grm. of fuchsin or
methyleii blue dissolved in 10 c.c. of absolute alcohol may be added to
100 c.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.
,_3. Safranin.— One of the most important of these stains, on
account of its power, brilliancy, and permanence in balsam, and the
166 NUCLEAR STAINS WITH COAL-TAR DYES.
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 a good safranin.
In ordering it, from Griibler & Hollborn or elsewhere, 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 soluble in both. The
brand I have been using for a long time, which gives good results, is
the " Safranin 0 " of Griibler & Co.
Staining. — The majority of safranins are not sufficiently soluble
in water, so that solutions in other menstrua must be employed.
PFITZNER (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.
FLEMMING (Arch. mik. Anat., xix, 1881, p. 317) used a concen-
trated solution in absolute alcohol, diluted with about one-half of
water.
BABES (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 BABES (Zeit. 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.
ZWAARDEMAKER (ibid., 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.
FLEMMING'S acid differentiation (Zeit. wiss. Mik., i, 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 HC1 in watery
CHAPTER XIV. 167
solution if you prefer it.) Or you may use a lower strength, viz.
0-1 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.
PODWYSSOZKI (Beitr. z. Path. Anat., i, 1886, p. 289) differentiates
(for from a few seconds to two minutes) in a strongly alcoholic
solution of picric acid, followed by pure alcohol. Same results
(except that the stain will be brownish instead of pure red).
BABES recommends treatment with iodine, according to the
method of GRAM (see next section). This process has also been
recommended by PRENANT (Int. Monatsschr. Anat.} etc., iv, 1887,
p. 368).
It has been shown by OHLMACHER (Journ. Amer. Med. Assoc., vol. xx,
No. 5, February 4th, 1893, p. Ill) 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
precipitate.
See also the differentiation process of MARTINOTTI 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, safranin
stains, besides nuclei, elastic fibres, the cell bodies of certain horny
epithelia, and the contents of certain gland-cells (mucin, under
certain imperfectly ascertained conditions).
The stain is perfectly permanent.
287. Gentian Violet may be used in aqueous solution, or as directed
for safranin.
In some cases it may be useful to employ the method devised by
GRAM for the differentiation of bacteria in tissues (Fortschr. d.
Medicin., ii, 1884, No. 6 ; British Med. Journ., September 6th, 1884,
p. 486 ; Journ. Roy. Mic. Soc. [N.S.], iv, 1884, p. 817). In this
the sections are treated, after staining, with a solution composed of—
Iodine l grm-
Iodide of potassium . 2 grms.
Water 300 „
168 NUCLEAR STAINS WITH COAL-TAR DYES.
for two or three minutes, until they become black. They are then
differentiated with neutral alcohol, until they turn grey, and are
then finally differentiated with clove oil.
By this process, in resting nuclei the nucleoli alone are stained, or
the chromatin if stained is pale ; in dividing nuclei the chromatin is
stained with great intensity, being nearly black in the equatorial
stage.
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,
ireats 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)
in a concentrated aqueous solution, hardly anything but the chro-
matin 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 what-
ever of over-shooting the mark ; the stain will not be more extracted
in 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, q. v. Some observers have found
the stain to fade. WOLFF (Zeit. wiss. MiJc., 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. FELIZAT and BRANCA (Journ.
Anat. Phys., xxxiv, 1898, p. 590) mount without a cover. HENNEGUY
(in litt.} clears with acetone.
KING (Anat. Record, iv, 1910, p. 236) stains with a saturated solution
in carbolic acid of 1 per cent., and finds the stain permanent.
NICOLLE'S " thionine ph^niquee " consists of 1 part of saturated
solution in alcohol of 50 per cent., and 5 parts of 2 per cent, aqueous
solution of carbolic
CHAPTER XIV. 169
289. Other Regressive Stains. — The following may be useful : —
Dahlia, according to FLEMMING (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 SCHUBERG, in Zeit. wiss. ZooL,
Ixxiv, 1903, p. 7, and Ixxxvii, 1907, p. 557.
Victoria Blue (Victoriablau) (LUSTGARTEN, Med. JaJirb. k. Ges. d.
Aerzte zu Wien, 1886, pp. 285— 291).— This dye ("Victoriablau
4 A ") has a special affinity for elastic fibres. For this object Lust-
garten recommends an alcoholic solution of the dye diluted with
2 to 4 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 (Zeit. wiss. Mile., 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,
The Philadelphia Medical Journal, May 14th, 1898. It much resembles
methylen blue.
METZNER (NagePs 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 Kosanilin salts
having very similar reactions, and found in commerce under the names
of FUCHSIN, ANILIN KED, RUBIN, ROSEIN, MAGENTA, SOLFERINO,
CORALLIN). — GRASER (Deutsche Zeit. Chirurgie, xxvii, 1888, pp. 538 —
584 ; Zeit. wiss. Mik., v, 1888, p. 378) stains for twelve to twenty-four
hours in a dilute aqueous solution, washes out 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.
ZIEHL'S Carbolic Fuchsin (Zeit. wiss. Mik,, 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.
Kresofuchsin (ROTHIG, 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.
170 NUCLEAR STAINS WITH COAL-TAR DYES.
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 HC1 alcohol.
Methylen Blue.
Nigrosin (ERRERA, Proc.-Verb. Soc. Beige 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 cells 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 (§ 201) ; but some of them
are obtained by means of " neutral " dyes (§ 201), 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 succession, there is
formed in the tissues a neutral colour (§ 201) 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 combination a suitable neutral lake (cf. § 201). 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, one or more neutral colours are formed
in the mixture and stain progressively.
Excepting Biebrich scarlet, I am not acquainted with any plasma
stain that is thoroughly satisfactory for delicate work. In addition
to Biebrich scarlet, I recommend for sections Saurefuchsin, either
alone or in the form of Ehrlich-Biondi mixture, or Ehrlich's triacid :
for material in bulk, picric acid (but only for rough work).
291. Saurefuchsin (Acid Fuchsin, Fuchsin S, Acid Rubin, Rubin
S, Saurerubin, Acid Magenta, Magenta S).— The chemical descrip-
* 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," etc. It includes some double or
triple stains that affect nuclei as well as plasma, but in different hues.
172 PLASMA STAINS WITH COAL-TAR DYES.
tion of this acid colour has been given (§ 201) : 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
0-5 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. PAPPENHEIM (Arch. Path. Anat., clxvi, 1901, p. 427)
takes 2 parts 1 per cent, solution of methyl green and 1 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 CORTI and FERRARA, Mon. 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 (Encycl.
Mik. Tech., 1910, ii, p. 412 : I am indebted for the formula to Dr.
GAUDLITZ) 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 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-disul-
phonate of soda. 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 Saurefuchsin. Almost, if not quite, as precise a stain as Saure-
fuchsin. 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 unfortunately
CHAPTER XV. 173
have not noted the proportions. SQUIRE (Methods and Formula,
p. 42) takes 1 grm. Saurefuchsin, 6 grms. Orange G in 60 c.c. of
alcohol and 240 c.c. of water. See also under " Connective tissues."
295. EHRLTCH-BIONDI Mixture (or EHRLICH-BIONDI-HEIDENHAIN
Mixture) (Pfluger's Arch., xliii, 1888, p. 40).
To 100 c.c. saturated aqueous solution of orange add with con-
tinual agitation 20 c.c. saturated aqueous solution of Saurefuchsin
(Acid Fuchsin) and 50 c.c. of a like solution of methyl green.
(According to Krause (Arch. mik. Anat., xlii, 1893, p. 59), 100 parts of
water will dissolve about 20 of Saurefuchsin (Rubin S), 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. HEIDENHAIN (" Ueber Kern u. Protoplasma," in
Festschr.f. Kolliker, 1892, p. 115) the orange to be used should be
" Qrangejjr," the Acid Fuchsin or Saurefuchsin should be " Rubin
S " (" Rubin " is a synonym of Fuchsin) and the methyl green
should be " Methylgriin 00." And it is absolutely necessary that
these ingredients be those prepared under those names by the
Actienfabrikfilr Anilin-fabrikation in Berlin. They can be obtained
from Griibler & 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 precipitate on
being mixed. To avoid this it is recommended by SQUIRE (Methods
and Formula, etc., p. 37) to dilute them before mixing.
Other proportions for the mixture have been recommended by KRAUSE
(loc. cit. supra), viz. 4 c.c. of the Saurefuchsin solution, 7 of the orange G
and 8 of the methyl green ; the mixture to be diluted 50 to 100-fold with
water. THOME (Arch. mik. Anat., Hi, 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 extrac-
174 PLASMA STAINS WITH COAL-TAR DYES.
tion of the methyl green, which easily comes away in the alcohol.
DRUNER (Jena Zeit., xxix, 1894, p. 276) stains for ten minutes in
the concentrated solution, treats for one minute with alcohol con-
taining 0-1 per cent, of hydrochloric acid, and then with neutral
alcohol.
The best results are obtained with sublimate material ; chrom-
osmium material, and the like, give a much inferior stain. Prepara-
tions made with the usual mixture, as given above, are liable to
fade ; by acidifying the mixture a stronger and more sharply
selective stain is obtained, which, does not fade. But too much acid
must not be added, as this would cause a staining of the interfilar
substances. According to the Encycl. 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. HEIDENHAIN (Ueber
Kern und Protoplasma, p. 116) ; for this see fourth edition. See also
ISRAEL (Praktikum Path. Hist., 2 Aufl., Berlin, 1893, p. 69) ; TRAMBUSTI
(Eicerche 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. HEIDENHAIN, 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 add 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 Saurefuchsin 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 p in thickness, and if they are over 5 the desired results are
almost hopeless. The stain keeps very badly. I admit that the
CHAPTER XV. 175
method has its raison d'etre for the very special objects for which it
was imagined — for the researches on cell-granulations for which
EHRLICH employed the three colours, or for the researches on the
plastin element of cytoplasm for which MARTIN HEIDENHAIN em-
ployed 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 mischievous exaggeration. For
it is far from having the qualities that should be possessed by a
normal section stain. Workers have at length found this out, and
it is now but little used except for the special purposes above
indicated.
296. EHRLICH'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 EHRLICH and LAZARUS, Die
Ancemie, 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, Saurefuchsin,
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 Saurefuchsin, 15 of distilled water, 15 of alcohol,
12^ 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 less delicate, and the methyl green in it appears to have
more resistance to alcohol, so that it is better adapted for ordinary
work.
MAYER (Grundzuge,I^m and MAYER, p. 197) has simplified the formula
thus : Take 1 g. methyl green, 2 g. orange, 3 g. Saurefuchsin, and
dissolve in a mixture of 45 c.c. water, 10 c.c. glycerin, and 20 c.c. alcohol
of 90 per cent.
MOREL and DOLERIS (C. E. Soc. 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 better fixed in the
nuclei.
297. PIANESE'S Saurefuchsin-malachite Green (from MULLER, Arch.
Zellforsch., viii, 1912, p. 4) consists of 0-5 grm. malachite green, 0-1 grm.
Saurefuchsin, and 0-01 grm. Martius yellow in 150 c.c. water and 50 c.c.
alcohol. Stain for twenty-four hours, differentiate with alcohol,
containing 1 to 2 drops of HC1 per 200 c.c.
176 PLASMA STAINS WITH COAL-TAR DYES.
298. Picric Acid. — Picric acid gives useful plasma stains after
carmine and hsematoxylin. The modus operandi consists merely in
adding picric acid to the alcohols employed for dehydrating the
objects.
Picric acid has considerable power of washing cut other anilin
stains ; and in combination with hydrochloric acid it 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 afterwards. 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, LEGAL'S alum-carmine, § 221,
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 FKOHLICH (Zeit. wiss. Mik., xxvii, 1910, p. 349)
picraminic acid (from Griibler & Hollborn) has some advantages
over picric acid.
299. VAN GIBSON'S Picro-Saurefuchsin (from Zeit. wiss. Mik.,
xiii, 1896, p. 344). — To a saturated aqueous solution of picric acid
are added a few drops of saturated aqueous solution of Saurefuchsin,
until the mixture has become garnet-red. Or (Trans. Amer. Micr.
Soc., xix, 1898, p. 105) to 100 parts of the picric acid solution add
5 parts of 1 per cent, solution of Saurefuchsin. After staining
(sections only), rinse with water, dehydrate, and clear in oil of
origanum.
OHLMACHER (Journ. Exper. Med., ii, 1897, p. 675) adds 0-5 per
cent, of Saurefuchsin to a saturated solution of picric acid which
has been diluted with an equal quantity of water. He uses this
after previous staining writh gentian violet.
RAMON Y CAJAL recommends 0-1 grm. of Saurefuchsin to 100 of
saturated solution of picric acid (SCHAFFEE, Zeit. wiss. ZooL, Ixvi,
1899, p. 236).
HANSEN (Anat. Anz., xv, 1898, p. 152) adds 5 c.c. of 2 per cent,
solution of Saurefuchsin to 100 c.c. saturated solution of picric 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
3 c.c. of water with 2 drops of the acidified stain added, dehydrates
CHAPTER XV. 177
clears with xylol, and mounts in xylol-balsam. Connective-tissue
red, elastin and all other elements yellow.
WEIGERT (Zeit. wiss. Mik., 1904, p. 3) adds 10 parts of 1 per cent.
Saurefuchsin to 100 of saturated picric acid.
See also MOLLER, op. cit., xv, 1898, p. 172.
This stain is generally used as a contrast stain to follow hsemato-
xylin. APATHY (Behrens' Tabellen, 3rd ed., p. 129) takes for this
purpose 1 grm. of Saurefuchsin in 500 c.c. of saturated solution of
picrate of ammonia.
WILHELMI (Fauna Flora Golf. Neapel, xxii, 1909, p. 18) takes
0-2 grm. Saurefuchsin, 0-8 grm. picrate of ammonia, 10 grin, absolute
alcohol, and 89 grm. water.
E. and T. SAVINI (Zeit. wiss. Mik., xxvi, 1909, p. 3t) use a formula
due to BENDA. 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 c.c. of the mixture. This neither over-
stains nor attacks the primary stain.
300. FLEMMING'S Orange Method (Arch. mik. Anat., xxxvii, 1891,
pp. 249 and 685). — Stain sections of Flemming or Hermann material in
strong alcoholic safranin solution diluted with anilin water (§ 286) ;
differentiate in absolute alcohol, containing at most (M 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 SAINMONT (Zeit. wiss. Mik., xxv, 1908, p. 157,
and Arch. Biol., xxiv, 1909, p. 15) stain for twenty-four hours in the
gentian, wash out after the orange for two to three hours in 100 c.c.
absolute alcohol with 3 to 4 drops of HC1, 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.
See also FLEMMING in Arch. Anat. Phys. Anat. Abth., 1897, p. 175.
Never popular, this clumsy and uncertain process is now little used.
301. REINKE'S Orange Method (Arch. mik. Anat., xliv, 2, 1894, p. 262),
—To a concentrated aqueous solution of gentian violet are added " a few
props " of a like solution of orange G. The solution precipitates in part.
M.
178 PLASMA STAINS WITH COAL-TAR DYES.
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 the
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 four hours in saturated solution of safranin
in alcohol of 75 per cent. : then washed and put for five to fifteen
minutes into solution of 7 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 cloves. 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, connective-
tissue yellow, keratin violet. Npt adapted for blood films.
303. Bordeaux R. — An " acid " dye, giving a general stain taking
effect both on chromatin and cytoplasm, and, I consider, a very
good plasma stain. I use for chrom-osmium material a 1 per cent,
solution, and stain for twelve to twenty-four hours. The stain is
sufficiently fast.
304. Bordeaux R, Thionin, and Methyl Green (GRABERG, Zeit. wiss.
Mik., xiii, 4, 1896, p. 460).
305. Congo Red (Congoroth) (see GRIESBACH, 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
Saurefuchsin. It is useful for staining some objects during life
(see ante, § 208). CAENOY (La Cellule, xii, 1897, p. 216) has had very
CHAPTER XV. 179
good results with it after haematoxylin of DEL AFIELD. 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 (Zeit. 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 haemotoxylin, the stain of
which it does not cause to fade.
307. Benzopurpurin. — According to GRIESBACH (loc. cit., § 305),
another " acid " colour very similar in its results to Congo red. See
also ZSCHOKKE (ibid., v, 1888, p. 466), 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. Neutral Red (Neutralroth) (EHRLICH, Allg. med. Zeit., 1894,
pp. 2, 20; Zeit. wiss. Mik., xi, 1894, p. 250; GALEOTTI, 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. MiL, xvi, 2, 1899,
p. 238). Up to the present this colour has chiefly been employed
for intra-vitam staining. Tadpoles kept for a day or two in a solu-
tion 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 (EHRLICH), and to the contents of
mucus cells (GALEOTTI).
According to EHRLICH and LAZARUS (Spec. Paihol. und Therapie,
herausgeg. von NOTHNAGEL, viii, 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 methylen blue, by injection or immersion with
contact of air. It is especially a granule stain. Similar results are
recorded by ARNOLD (Anat. Am., xvi, 1899, p. 568, and xxi, 1902,
p. 418). See also EHRLICH and LAZARUS, Ancemie, i, 1898, p. 85 ;
LOISEL (Journ. de I' Anat. et de la Phijsiol, 1898, pp. 197, 210, 217)
(intra-vitam staining of sponges) ; and PROWAZEK (Zeit. iviss. Zool.,
Ixii, 1897, p. 187) (intra-vitam staining of Protozoa). I myself have
had very good results with it as an intra-vitam stain.
According to GOLOVINE (Zeit. wiss. Mik., xix, 1902, p. 176), the
12—2
180 PLASMA STAINS WITH COAL-TAR DYES.
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 NISSL (q. v.) in nerve-cells. S. MAYER (Lotos,
Prague, 1896, No. 2) states that it also stains degenerating myelin.
The solutions that have been employed for staining fixed material
are strong aqueous ones, 1 per cent, to concentrated.
309. Biebrich Scarlet (BRITISH DYES, Ltd., Huddersfield).— A. K.
GORDON (British Medical Journ., 1917, p. 828) finds this an excellent
acid dye which never overstains and is not diffuse. Use in 1 per
cent, solution, or as recommended by SCOTT (§ 676).
I have used this stain a good deal and find it better than any other
plasm dye that I have tried.
310. The Bosins, found in commerce under the names of Eosin,
Saffrosin, Primerose Soluble, Phloxin, Bengal Rose, Erythrosin,
Pyrosin B, Rose B, a PEau, 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
(" Pritnerose a VAlcool ").
They are all diffuse stains, formerly much used as contrast stains,
less so now. HANSEN (Anal. 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 GRIESBACH, Zool. Anz., 1883, p. 172.
Eosin is a specific stain for red blood-corpuscles, and also for
certain granules of leucocytes (see under " Blood ").
The yolk of some ova takes the stain strongly, so that it is useful
in some embryological researches.
311. EHRLICH'S Indulin-Aurantia-Eosin, or Acidophilous Mixture,
or Mixture C, or Mixture for Eosinophilous Cells (from the formula
kindly sent me by Dr. GRUBLER). — Indulin, aurantia, and eosin, of
each 2 parts ; glycerin, 30 parts. This gives a very thick, syrupy
solution. To 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 EHRLICH-BIONDI mixture, and is, therefore,
much more adapted to ordinary work. The stain keeps well.
ISRAEL (Prdktik. Path. Hist., Berlin, 1893, p. 68) gives a more
complicated receipt.
CHAPTER XV. 181
312. Methyl Green and Eosin (GALBERLA, Morph. Jahrb., iii, 1877,
Heft 3, p. 625 ; LIST, Zeit. wiss. Mik., ii, 1885, p. 147 ; BALBIANI, Ann.
Microgr., Paris, vii, 1895, p. 245 ; RHUMBLER, Zeit. wiss. Zool, Ixi, 1895,
p. 38). — See early editions.
313. Methylen Blue and Eosin (CHENZINSKY, quoted from Zeit. wiss
Mik., 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 . . . .40
This solution will only keep for about eight days.
PIANESE (ibid.) xi, 1894, p. 345) adds a considerable proportion of
carbonate of litljia.
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 EOSIN, Berliner klin. Wochenschr., 1898, p. 251 ; Zeit.
wiss. Mik., xvi, 1899, p. 223, and xvii, 1900, p. 333.
See also LAURENT (Centralb. allg. Path., xi, 1900, p. 86 ; Zeit. iciss.
Mik., xvii, 1900, p. 201).
314. MALLORY'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° 0.
in 5 per cent, aqueous solution of eosin, rinsed and flooded with solution
of 1 part of methylen blue, and 1 of potassium carbonate in 100 of
water, diluted with about 7 parts of water. After forty, minutes they
are flooded (not washed) with water, and differentiated for about five
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 (Lichtgriin S. F.). — An " acid " colour, soluble
in alcohol, and a good plasma stain.
BEND A (Verh. physiol. Ges. Berlin, December 18th, 1891, Nos.
4 u. 5) stains sections for twenty-four hours in a^i^^water-safraiiin
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. This 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 PRENANT, Arch. mik. Anat., vii, 1905, p. 430, and
GUIEYSSE, C.R. Soc. Biol, Ixii, 1907, p. 1212.
317. Janus Green (MICHAELIS, Arch. mik. Anat., Iv, 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
182 PLASMA STAINS WITH COAL-TAR DYES.
plasma stain for the ova of Ascaris by VAN BENEDEN and NEYT. These
authors used it for glycerin preparations ; it can hardly be got into
balsam.
FLEMMING (Arch. mik. Anat., xix, 1881, p. 324) attributes to it a
special affinity for nucleoli.
319. Iodine Green ("HOFMANN'S Grim"), see GRIESBACII (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 (Thiophengriin), see KRAUSE, Intern. Monatsschr.
Anat., etc., iv, 1887, Heft 2.
321. Coerulein S., a green " acid " dye, is recommended for the
staining of muscle-fibrils by M. v. LENHOSSEK (Anat. Anz., xvi, 1899,
p. 339). See also HEIDENHAIN, ibid., xx, 1901, p. 37, and RAWITZ, ibid.,
xxi, 1902, p. 554.
322. Quinolein Blue (Cyanin, Chinolinblau ; v. RANVIER, Traite,
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 quinol&ne " is not the preparation that usually goes under
that name. See EHRLICH, in Arch. mik. Anat., xiii, 1877, p. 266.
323. Indulin and Nigrosin. — Indulin, Nigrosin, Indigen, Coupler'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 6 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) (KOSSINSKI, Zeit.
wiss. Mik., vi, 1880, p. 61). — See early editions.
325. Picro-Nigrosin, PFITZER (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."
326. Anilin Blue. — Under this title are comprised various " basic "
derivatives of the base rosanilin. They occur under the names
CHAPTER XV. 183
Spirit Soluble Blue (Bleu Alcool), Gentian Blue 6 B, Spirit Blue 0,
Opal Blue, Bleu de Nuit, Blue Lumiere, Parma Bleu, Bleu de Lyon.
Some authors give the name Bleu de Nuit and Griindstichblau as
synonyms of Bleu de Lyon. The Encyd. 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 BAUMGARTEN and by
JACOBY). MAURICE and SCHULGUN stain in bulk with it after borax-
carmine, using a very dilute alcoholic solution. BAUMGARTEN and
JACOBY stain sections in a 0*2 per cent, alcoholic solution.
TONKOFF (Arch. mik. Anat., Ivi, 1900, p. 394) adds a little tincture
of iodine to the solution of the dye, or mordants the sections with
iodine.
SKROBANSKY (Intern. Monatsschr. Anat., xxi, 1904, p. 20) uses it
in water with picric acid.
327. Carmine Blue (Bleu Carmin Aqueux, from Mesiter, Lucius, and
Brunig, at H6chst-a-M.). — JANSSENS (La Cellule, ix, 1893, p. 9) states
that this colour possesses a special affinity for the parts of cytoplasm
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 MITROPHANOW (quoted from Zeit. wiss.
Mik., v, 1888, p. 513), used in concentrated aqueous solution it
gives a very good double stain with safranin. It is very resistant
to alcohol. Using the Wasserblau 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 35 parts 1 per cent,
solution of eosin, 45 of methyl blue 1 per cent., 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 1
The dye used by them cannot now be identified. Dr. GRUBLER writes
me that the anilin blue-black of his list is the oxyazo colour blue-bla<
or AZOSCHWARZ ; but that dye had not been discovered when Bevan
184 PLASMA STAINS WITH COAL-TAR DYES.
Lewis wrote. See also HEIDENHAIN in Zeit. wiss. Mik., xx, 1903, p. 185,
and xxv, 1909, p. 407.
330. Violet B (or Methyl Violet B) (S. MAYER, Sitzb. k. k. Akad.
wiss. Wien, in, Abth., February, 1882). — Used in solutions of 1 grm.
of the colour to 300 grms. of 0-5 per cent, salt solution, and with
fresh tissues that have not been treated with any reagent whatever,
this colour gives a stain so selective of the 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 glycerin 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 staining
sections, e.g. by KROMAYER and others. BENDA (N enrol. 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., liii, 1899, p. 519, and liv, p. 289) stains
sections of skin with Kresyl-eehtviolett Nuclei blue, plasma reddish.
Similarly TICK (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.
332. Saureviolett, see § 316.
333. Benzoazurin may be made to give either a diffuse or a nuclear
stain, according to MARTIN (see Zeit. wiss. Mik., vi, 1889, p. 193).
334. RAWITZ' «« Inversion " Plasma Stains. — It has been discovered
by RAWITZ 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
" inversion " of the usual stain. The stain, in my opinion, is a vile one.
For details see fourth edition, or RAWITZ (Sitzb. Gesnaturf. Freunde,
Berlin, 1894, p. 174 ; Zeit. wiss. Mik., xi, 1895, p. 503 ; and his Leitfaden
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.
CHAPTER XV. 185
RAWITZ (Zeit. wiss. Mik., 1909, pp. 393 and 395) also recommends
a solution of 1 grm. of Saure-Alizarinblau BB (or Sauregriin G) (both
from Hochst), 10 grms. ammonia alum, 100 c.c. glycerin, and 100 c.c.
water.
SZUTZ (ibid., xxix, 1912, p. 289) fixes n a mixture of 15 c.c. 1 per
cent, platinum chloride, 15 c.c. formol, and 30 c.c. saturated solution of
sublimate, makes paraffin sections, and stains them with Heidenhafri's
iron-hsematoxylin. 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
NILSSON, Zool Anz., xxxv, 1909, p. 196.
336. For BENDA'S Alizarin Stains, see under " Centrosomes,"
" Mitochondria," and " Neuroglia."
CHAPTER XVI.
•
METHYLEN BLUE.
337. Methylen Blue is a " basic " dye, being the chloride or the
zinc 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 impurity a
small quantity of a reddish dye, which used to be taken to be
methylen red. This impurity is present from the beginning 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. Accord-
ing to NOCHT (Central}). 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 NOCHT proposes the name
" Roth aus Methylenblau."
According to MICILELIS (Centmlb. Bakteriol., xxix, 1901, p. 763, and
xxx, 1901, p. 626 ; Zeit. wiss. Mik., xviii, 1902, p. 305, andxix, 1902,
p. 68) confirmed later by NOCHT, REUTER, and GIEMSA, this dye is
Methylenazur, an oxidation-product of methylen blue, already
described by BERNTHSEN 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 metachro-
matic).
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 metliylen blue, and is employed for staining
certain cell-granules. UNNA (Zeit. wiss. Mik., viii, 1892, p. 483)
makes this as follows : A solution of 1 part of methylen blue and
1 of carbonate of potash in 20 of alcohol and 100 of water is evapo-
rated down to 100 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 glycol, creosol, or Unna's
glycerin-ether mixture — all of which, as well as the polychrome
methylen blue, can be obtained from Griibler & Hollborn.) MICHJSLIS
CHAPTER XVI. 187
(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 T\j normal solution of
caustic soda added. Boil for a quarter of an hour ; after cooling
add 10 c.c. of -f^ 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 ( ROMAN OWSKY, 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." See also an
important paper by PROWAZEK (Zeit. wiss. Mikr. Tech., 31).
There are several sorts of methylen blue sold, the most important
being — "methylen blue, according to EHRLICH " ; "methylen
blue, according to KOCH " ; " methylen blue BX, according to
S. MAYER " ; " Methylenblau, medic, pur."
The colour to be employed for intra-vitam nerve staining should
be as pure as possible. APATHY (Zeit. wiss. Mik., ix, 1893, p. 466)
writes that the best — in fact, the only one that will give exactly the
results described by him — is that of E. MERCK, of Darmstadt,
described as " medicinisches Methylenblau." DOGIEL (Encycl.
mik. Technik., 1st edition, p. 811) has had his best results with
" Methylenblau n. Ehrlich," or " BX," obtained from Griibler &
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 granulations of " Mastzellen " and
plasma-cells, and the granules of NISSL in nerve-cells, also mucin.
It is much used — in the form of mixtures affording methylen azur—
in the study of blood, blood parasites, and similar objects. For all
of these see the respective sections in Part II. Further, it stains a
large number of tissues intra vitam, with little or no interference with
their vital functions. And last, not least, it can be made to furnish
stains of nerve tissue, intercellular cement substances, lymph spaces,
and the like, that are essentially identical with those furnished by a
successful impregnation with gold or silver, 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 in a
preparation, sometimes the totality of them ; whilst methylen blue
stains only a selection of them, so bringing them more prominently
188 METHYLEN BLUE.
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 EHRLICH (Biol Centralb., vi, 1886, p. 214 ; Abh. k. Akad.
Wiss. 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 tissue^,would be found to
be stained. This, however, is not the case. It *s 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
EHRLICH this decoloration is explained as follows : methylen blue,
on contact with reducing agents in alkaline solution, can be reduced
to a colourless 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 conditions, but that a
specific stain of one group or another of elements may be obtained
in one of two ways. If the tissue to be studied be one that stains
earlier than the others, it may be 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 § 343.
CHAPTER XVI. . 189
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. I think the tint is practically a sufficient guide,
but it may be stated that when in doubt a strength of 1 : 100,000
may be taken, and increased or diminished as occasion may seem to
require. ZOJA (Rendic. R. 1st. Lombardo, xxv, 1892 ; Zeit. wiss.
MiL, 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 epithe-
lium cells, fat cells, plasma cells, " Mastzellen," blood and lymph
corpuscles, elastic fibres, smooth muscle, striated muscle. There
are other elements that stain ih the living state, but not when the
staining is performed by simple immersion of intact animals in a
dilute staining solution in the manner we are. considering. Chief
amongst these are nerve-fibres and ganglion-cells, which remain
unstained in the intact organism. To get these stained, it is neces-
sary to isolate them sufficiently, as explained in the following
sections.
340. Staining Nervous Tissue* during Life. — It was made out by
EHRLICH (op. cit., last §) that by injecting a solution of methylen
blue into the vessels or tissues of living animals and shortly after-
wards 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. DOGIEL, however (Arch. mik. Anat., xxxv, 1890, pp. 305
et seq.), found that muscle nerves of limbs of the frog could be stained
as much as from three to eight days after the limbs had been removed
from the animal. He concludes, indeed, that the reaction shows
that the nerves were still living at that time. But it seems more
natural to conclude with AP!THY (Zeit. wiss. Mik., ix, 1892, pp. 15
et seq.) that nerve-tissue can be stained after life has ceased. APATHY
* See also p. 477.
190 . METHYLEN BLUE.
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. MICHAILOW (ibid., xxvii,
1910, p. 7) prefers tissues that have lain from one and a half to two
hours after the death of the subject in Einger'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 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 oxygen. Another consideration that
justifies the practice is that by exposure to air the preparations take
up a trace of ammonia, and APATHY has experimentally established
that this is an important factor in the sharpness of the stain. EHRLICH
also (op. Git.) holds that an alkaline reaction of the tissues is a neces-
sary condition to the stain. APATHY further holds that the stain is
a regressive one, easily washed out 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 EHRLICH) to inject methylen blue into
the vascular system or body-cavity of a living animal, wait a suffi-
cient 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 re-
moving 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 injection
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,
CHAPTER XVI. 191
p. 125) injected 1 c.c. of saturated (i.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. BIEDERMANN (Sitzb. Akad.
Wiss. 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 to four hours
before killing them. S. MAYER (Zeit. wiss. MiJc., vi, 1889, p. 423)
took a strength of 1 : 300 or 400 of 0-5 per cent, salt solution. The
solutions of EETZIUS are of the same strength. But the tendency
of more recent practice is decidedly towards the employment of
weaker solutions. APATHY (ibid., ix, 1892, pp. 25, 26 et seq.) finds
that it is not only superfluous, but positively disadvantageous, to
take solutions stronger than 1 : 1000. DOGIEL (Encycl. Mik.
Technik., 1st ed., p. 815) recommends J to J per cent., or at most
| per cent. 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 twenty to
thirty minutes. They should be placed on a thin layer of spun
glass moistened with weak (J to T^ 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 bottom. The dish is best
placed in a stove at 36° C., and after fifteen to thirty minutes (if the
pieces are thin) or one hour to one and a half hours (if they are thick)
specimens may be removed for examination or preservation ; or,
without using the stove, specimens may be removed ten to twenty
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 (forty to sixty
minutes) they may be fixed (§ 343).
For staining by immersion the solutions should, if anything, be
still weaker. DOGIEL (Arch. mik. Anat., xxxv, 1890, p. 305) places
objects in a few drops of aqueous or vitreous humour, to which are
added 2 or 3 drops of a Jg to -fg 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. The reaction is quickened
by putting the preparations into a stove kept at 30° to 35° C.
ROUGET (Compt. Rend., 1893, p. 802) employed a 0-05 per cent, solu-
tion in 0-6 per cent, salt solution (for muscles of Batrachia). ALLEN
192 METHYLEN BLUE.
(Quart. Journ. Micr. Sci., 1894, pp. 461, 483) takes for embryos
of the lobster a solution of 0-1 per cent, in 0-75 per cent, salt
solution, and dilutes it with 15 to 20 volumes of sea-water.
SEIDENMANN (Zeit. wiss. Mik., xvii, 1900, p. 239) takes for the
choroid a solution of 0-02 per cent, in 0-5 per cent, salt solution.
LAVDOWSKY (ibid., xii, 1895, p. 177) takes T3^ to J per cent, in white
of egg, or serum. Similarly YOUNG (ibid., xv, 1898, p. 253).
MICHAILOW (ibid., xxvii, 1910, p. 10) takes |- to 3^ per cent, in
Ringer's salt solution (for nerves of Mammals).
APATHY (Zeit.' wiss. Mik., ix, 1892, p. 15 ; see also his Mikro-
technik, p. 172) proceeds as follows for Hirudinea and other inverte-
brates. A portion of the ventral cord is exposed, or dissected out.
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 : 1000 solution in 0-5 to 0-75 per cent, salt
solution, allowed to act for ten minutes ; or a 1 : 10,000 solution
allowed to act for an hour to an hour and a half ; or a 1 : 100,000
solution allowed to act for three hours (Lumbricus requires twice
these times ; Astacus and Unio require three times ; medullated
nerves of vertebrates four times). For the demonstration of ganglion
cells the stain is allowed to act three or four times as long.
The 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 without
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 differentiate the
stain — to produce an artificial " secondary differentiation." It acts
by washing out the absorbed colour from certain elements, others
resisting longer.
See also, for Hirudinea, SANCHEZ, in Trab. Lab. Invest. Biol.
Univ. Madrid, vii, 1909, fasc. 1 — 4, or Zeit. wiss. Mik., xxvii, 1910,
p. 393 (injection of solutions of 0-2, 0-1, or 0-05 per cent., with
further treatment as Apathy or Bethe).
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 :
CHAPTER XVI. 193
ARNSTEIN (Anat. Anz., 1887, p. 551) puts the tissue for half an
hour into saturated aqueous solution of picrate of ammonia.
S. MAYER (Zeit. wiss. Mik., vi, 1889, p. 422) preferred a mixture
of equal parts of glycerin and saturated picrate of ammonia solu-
tion, which served to fix the colour and mount the preparations in.
This was also in principle the method of EETZIUS (Intern. Monatsschr.
Anat. Phys., vii, 1890, p. 328).
DOGIEL (Encyd. Mik. Techn., ii, p. 105) puts for two to twenty-
four hours into saturated aqueous picrate of ammonia, and then
into equal parts of glycerin and the picrate solution. (Thin mem-
branes, 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 FEIST,
Arch. Anat. Entivickel, 1890, p. 116 ; cf. Zeit. wiss. Mik., vii, 1890,
p. 231), the latter having the advantage of preserving the true blue
of the stain if it be not allowed to act too long, and the preparation
be mounted in pure glycerin.
Picric acid has been used by LAVDOWSKY, but this after careful
study is rejected by DOGIEL.
APATHY (op. cit., § 342) brings preparations either into a concen-
trated aqueous solution of picrate of ammonia free from picric add,
and containing 5 drops of concentrated ammonia for every 100 c.c. ;
or, which is generally preferable, into a 1 to 2 per cent, freshly
prepared solution of neutral carbonate of^ ammonia saturated with
picrate. They remain in either of these solutions, preferably in the
dark, for at least an hour. They are then brought into a small
quantity of saturated solution of picrate of ammonia in 50 per cent,
glycerin, where they remain until thoroughly saturated. They are
then removed into a saturated solution of the picrate in a mixture
of 2 parts 50 per cent, glycerin, 1 part cold saturated sugar solution,
and 1 part similarly prepared gum-arabic 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 50 „
Dissolve over a water-bath and add 0-05 grm. thymol. (This
mounting medium sets quickly and as hard as balsam, so that no
cementing of the mounts is necessary. Farrants' medium [with
omission of the arsenious acid] will also do. In neither case should
M. 13
194 METHYLEN BLUE.
either ammonium picrate or metliylen blue be added to the medium.)
Preparations that have been/w% 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).
PLESCHKO (Anat. Anz., xiii, 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 platinum
chloride is said to do this (see FEIST, Arch. Anat. Entw., 1890, p. 116),
but the preparations are not very satisfactory.
For the earlier method of PARKER (Zool. Anzeig., 1892, p. 375)
with methylal see early editions. Later (Mitth. Zool. Stat. Neapel,
xii, 1895, p. 4) he fixes the stain by dehydrating 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 BETHE (Arch. mik. Anat., xliv, 1894,
p. 585), see last edition.
BETHE'S later method (Anat. Anz., xii, 1896, p. 438) is as follows :
After staining, pieces of tissue of 2 to 3 mm. thickness are treated
for ten to fifteen minutes with a concentrated 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 hydro-
chloric 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 DOGIEL (Arch.
CHAPTER XVI. 195
mik. Anat,, xlix, 1897, p. 772 ; liii, 1898, p. 237 ; Zeit, iviss. Zoo?.,
Ixvi, 1899, p. 361 ; and Encycl. mik. Technik, 1903, p. 825, and
1910, p. 108.) He omits the peroxide, the hydrochloric acid, and
the cooling. Bethe (Zeit. iviss. Mik., xvii, 1900, p. 21) does not
approve of these modifications.
Further modifications of the molybdenum method have been
published by LEONTOWITSCH (Intern. Monatsschr. Anat., xviii, 1901,
p. 142).
MICHAILOW (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 twenty-four hours at 37° C.,
washes with warm water, and passes through alcohol and xylol into
xylol-damar (not balsam).
See also SCHMIDT (Arch. Ges. Phys., ciii, 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 maceration). 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 tempera-
ture, cleared in xylol or cedar oil, and imbedded in paraffin.
345. Impregnation of Epithelia, Lymph-spaces, etc. (DOGIEL,
Arch. mik. Anat., xxxiii, 1889, pp. 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 Encycl. mik. Technik, 1903, p. 827, Dogiel 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 endotheliuin
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 impregna-
tion 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 (Encycl., 1910, ii, p. 110) fixed with ammonium molybdate and
a trace of osmium.
13—2
196 METHYLEN BLUE.
The effect is practically identical (except as regards the colour)
with that of a negative impregnation with silver nitrate.
S. MAYER (Zeit. wiss. Mik., vi, 1889, p. 422) stains tissues for
about ten minutes in a 1 : 300 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.
TIMOFEJEW (Anat. Anz., xxxv, 1909, p. 296) impregnates for
fifteen to twenty minutes in a solution of 1 : 300 or 400 strength,
fixes with a very weak solution of ammonium 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. —
HARRIS (Philidelphia Med. Journ., May 14th, 1898) has found that
there is no reaction of methylen blue that 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 physio-
logical salt solution . . . 2 parts.
Ammonium chloride 0-25 per cent, in water . 1 part.
Egg albumen . . . . . . 1 ,,
For injections he uses 1 part of the dye to 1000 of physiological salt
solution.
Any of the methylen blue fixing methods may be employed and
the whole technique is the same.
L. MARTINOTTI (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 grm.
toluidin blue, 0-5 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 impregnation
is understood a mode of coloration in which a colouring matter is
deposited in tissues in the form of a precipitate — 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 impregna-
tion, 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 I
same preparation an impregnation and a stain ; and in most chloride
preparations 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 impreg-
nation intercellular substances alone are coloured, the cells them-
selves remaining colourless or very lightly tinted. In a positive
impregnation the cells are stained and the intercellular 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;
positive 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 consequent staining of the cells by the new solution of metallic salt
thus formed. These secondary impregnations take place when the
reduction of the metal in the primary impregnation is not sufficiently
energetic (see on these points His, Schweizer Zeit. Heilk., ii, Heft 1, p. 1 ;
GIERKE, Zeit. wiss. Mile., i, p. 393 ; RANVIER, Traite, 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 Fdrberei zu mikroskopischen
Zwecken, in vole, i and ii of Zeit. wiss. Mik. ; JOSEPH, Sitzb. Akad. Wiss.
198 METALLIC STAINS (IMPREGNATION METHODS).
Berlin, 1888 ; Zeit. wiss. Mik., xi, 1, 1894, p. 42 et seq. It evidently
cannot consist of metallic silver, as it is soluble in hyposulphite of soda.
See also MACALLUM, Proc. Eoy. Soc., Ixxvi, 1905, p. 217, and ACHARD
and REYNAUD, O.E. Soc. Biol, Ixi, 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 out 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 impregnation purposes by
exposure to light ! Dr. LINDSAY 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."
APATHY (Mitt. 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 of the Tissues to be Impregnated. — The majority of
stains given by dyes are only obtained with tissues that have be«n
changed in their composition by the action of fixing and 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 employ-
ment are given by RANVIEE (Traite, 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,
CHAPTER XVII. 199
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 epiploon, 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 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, 1 : 500 or 1 : 1000,
or if the light be not brilliant — a general instead of an interstitial
stain will result ; nuclei will be most stained, then protoplasm, and
the intercellular substance will contain but very little silver. In
general in a good " impregnation " the contents of the cells, and
especially nuclei, are quite invisible.
The tissues should be constantly agitated in the silver-bath in
* 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 between
them. They will be found described and figured in Journ. Eoy. Mic.
Soc., ii, 1879, p. 357, and in ROBIN'S Journ. de VAnat., 1879, p. 54.
They may be obtained of Burge & Warren, 42, Kirby Street, Hatton
Garden, London, E.G.
200 METALLIC STAINS [(IMPREGNATION METHODS).
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 to 1 : 500. Thus 1 : 300 is used for the epiploon, pulmonary
endothelium, cartilage, tendon ; whilst a strength of 1 : 500 is
employed for the phrenic centre, and the epithelium of the intestine.
For the endothelium of blood-vessels (by injection) solutions of
1 : 500 to 1 : 800 are taken.
M. DUVAL (Precis, 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 Lymphgefasse, etc., Berlin, 1862, p. 5).
ROBINSKI (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.
ROUGET (Arch, de Physiol., 1873, p. 603) employed solutions as
weak as 1 : 750, or even 1 : 1000, exposing the tissues to their action
several times over, and washing them with water after each bath.
The HERTWIGS take, for marine animals, a 1 per cent, solution
(Jen. Zeit. NaturL, xvi, pp. 313 and 324).
The HOGGANS (Journ. of Anat. and Physiol., xv, 1881, p. 477)
take for lymphatics a 1 per cent, solution.
TOURNEUX and HERRMANN (ROBIN'S Journal de I'Anat., 1876,
p. 200) took for the epithelia of Invertebrates 3 : 1000, and in some
cases weaker solutions, — for one hour, washing out with alcohol of
90 per cent.
HOYER (Arch. mik. Anat., 1876, p. 649) 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 ammonio-nitrate solution has
the advantage of impregnating absolutely nothing but endothelium
or epithelium ; connective tissue is not affected by it.
RANVIER' s injection-mass for impregnating endothelium is given
under " Injection."
DEKHUYZEN (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.
REGAUD (Journ. Anat. et Phys., xxx, 1894, p. 719) recommends
for the study of lymphatics a, process devised by RENAUT, for the
details of which see also previous editions.
CHAPTER XVII. 201
353. Other Salts of Silver. — ALFEROW (Arch. Phys., i, 1874, p. 694)
employs 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.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.
REGAUD and DUBRETJIL (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. — Keduction may be effected in
media other than distilled water.
v. RECKLINGHAUSEN washed his preparations in salt solution
before exposing them to the light in distilled water (Arch. path.
Anat., xix, p. 451). Physiological salt solution (0-75 per cent.) is
commonly used for these washings.
MULLER (Arch.f. path. Anat., xxxi, p. 110), after impregnation by
immersion for two or three minutes in a 1 per cent, solution of nitrate
of silver in the dark, adds to the solution a small quantity of 1 per
cent, solution of iodide of silver (dissolved by the aid of a little
iodide of potassium). After being agitated in this mixture the
preparations are washed with distilled water, and exposed to the
light for two days in a 1 per cent, solution of nitrate of silver (see also
GIERKE, in Zeit. wiss. Mik., i, 1884, p. 396).
ROUGET (Arch. dePhysiol., 1873, p. 603) reduces in glycerin ; SZUTZ
(Zeit. wiss. Mik., xxix, 1912, p. 291) in glycerin with -f^ of formol.
SATTLER (Arch. mik. Anat., xxi, p. 672) exposes to the light for a
few minutes in water acidulated with acetic or formic acid. THAN-
HOFFER (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.
OPPITZ puts for two or three minutes into a 0-25 or 0-50 per cent,
solution of chloride of tin.
JAKIMOVITCH (Journ. de I'Anat., 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.
DEKHUYZEN (op. cit., last §) reduces in oil of cloves, after dehydra-
tion.
355. Fixation.— LEGROS (Journ. de I' Anat., 1868, p. 275) washes his
preparations, after reduction, in hyposulphite of soda, to prevent after-
202 METALLIC STAINS (IMPREGNATION METHODS).
blackening. According to DUVAL (Precis, p. 230) they should be washed
for a few seconds only in 2 per cent, solution and then in distilled
water.
GEROTA (Arch. Ancit. Pliys., Phys. Abih., 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
chlorides that bathe the tissues of marine animals, these cannot be
treated directly with nitrate of silver.
HERTWIG (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 ammonia be avoided. These
stains will only succeed, however, with successful negative impregna-
tions, as nuclei that have been impregnated will not take the second
stain.
Impregnation with silver may be followed by impregnation with
gold. In this case the gold generally substitutes itself for the silver
in the tissues, and though the results are sharp and precise, the effect
of a double stain is not produced. See hereon GEROTA, loc. cit.,
§355.
358. Impregnation of Nerve Tissue.— For this subject, which
includes the important bichromate-and-silver method of GOLGI, and the
neurofibril methods of BIELSCHOWSKY and RAMON Y CAJAL, see
Part II. These give important results, not only with Nervous tissue,
but with various forms of Connective tissue, mitochondrial formations,
etc.
Gold.
359. The Characters of Gold Impregnations. — Gold chloride differs
from nitrate of silver in that it generally giyes positive (§ 348) im-
pregnations only. It generally gives negative images only with
such tissues as have first received a negative impregnation with
CHAPTER XVII. 203
silver, the gold substituting itself for the silver. In order to obtain
these images you first impregnate very lightly with silver ; reduce ;
treat for a few minutes with a 0-5 per cent, solution of gold chloride,
and reduce in acidulated distilled water.
This process, however, is in but little use, and except for 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.
). 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-impregna-
tion methods, characterised by the employment 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 APATHY'S view (Mitth. Zool. Stat. Neapel, xii, 1897, p. 718)
successful gold preparations should show a true stain, not an im-
pregnation (§ 347), the stain being brought about by the formation
of gold oxide (AuO) which combines with the tissue elements. He
advises in consequence that preparations 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. — SQUIRE'S Methods and
Formulas, etc. (p. 43), says : " Commercial chloride of gold is not
the pure chloride, AuCl3, but the crystallised double chloride of gold
and sodium, containing 50 per cent, of metallic gold.
" Commercial chloride of gold and sodium is the above crystallised
double chloride mixed with an equal weight of chloride of sodium,
and contains 25 per cent, of metallic gold."
This, however, appears not to be the case in Germany. Dr.
GRUBLER, writing to MAYER (see the Grundziige, LEE und MAYER,
p. 215), says : " Aurum chloratum fuscum contains about 53 per
cent. Au, the fiavum 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 14-7 per
cent, of sodium chloride, though samples are found in commerce
with much more,"
204 METALLIC STAINS (IMPREGNATION METHODS).
APATHY (Mitth. Zool Stat. Neapel, xii, 1897, p. 722) formerly
employed the aurum chlomtum flavum, but now prefers ihefuscum.
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
DRASCH (Sitzb. Akad. Wiss. Wien, 1881, p. 171, and 1884, p. 516 ;
and Abhand. maih.-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; Strieker" sHandb., p. 1100). — Fresh pieces of cornea (or other tissue)
are put into 0-5 per cent, solution of chloride of gold 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 but permanent.
364. LOWIT'S Method (Sitzgsber. Akad. Wien, Bd. bad, 1875,
p. 1). — The following directions are from FISCHER'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 (1 volume
of water to 1 of the acid of 1-12 sp. gr.), and remain there until
the epidermis peels off. They then are put for fifteen minutes into
gold chloride solution (1J 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.) Sections are 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 mixture of
chloride of gold and formic acid (4 parts of 1 per cent, gold chloride
to 1 part of formic acid) which has been boiled and allowed to cool
(RANVIER'S Traite, p. 826). They remain in this until thoroughly
impregnated (muscle twenty minutes, epidermis two to four hours) ;
reduction is affected either by daylight in acidulated water, or in the
dark in dilute formic acid (1 part of the acid to 4 parts of water).
366. RANVIER'S Lemon-juice Method (Traite, p. 813). — RANVIER
finds that of all acids lemon juice is the least hurtful to nerve-
CHAPTER XVII. 205
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
2 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 there-
fore permanent, preparations, the reduction should be done in the
dark in a few cubic centimetres of dilute formic acid (1 part acid to
4 of water), which takes about twenty-four hours.
367. VIALLANE'S Osmic Acid Method (Hist, et Dev. des Insectes,
1883, p. 42). — The tissues are treated with osmic acid (1 per cent,
solution) until they begin to turn brown, then with 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 excellent method.
KERSCHNER (Arch. mik. Anat., Ixxi, 1908, p. 522) puts till brown
into a mixture of 10 parts 5 per cent, formic acid with 1 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 reduction of the gold.
Thus BASTIAN employed a solution of gold chloride of a strength
of 1 to 2000, acidulated with HC1 (1 drop to 75 c.c.), and reduced in
a mixture of equal parts of formic acid and water kept warm.
HENOCQUE (Arch, de I'Anat. et de la Physiol, 1870, p. Ill) impreg-
nates 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 temperature of 40° to 50° C. Reduction
is effected very rapidly, sometimes in a quarter of an hour.
HOYER (Arch. mik. Anat., ix, 1873, p. 222) says that the double
chloride of gold and potassium has many advantages over the simple
gold chloride. He impregnates in solutions of 0-5 per cent, strength,
and reduces in water containing 1 or 2 drops of a pyrogallic add
206 METALLIC STAINS (IMPREGNATION METHODS).
developing solution, such as is used in photography, or in a warm
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.
CIACCIO (Journ. de Microgr., vii, 1883, p. 38) prefers the double
chloride of gold and cadmium.
FLECHSIG (Die Leitungsbahnen in Gehirn, 1876 ; Arch. Anat. u.
Phys., 1884, p. 453) reduces in a 10 per cent, solution of caustic soda.
NESTEKOFFSKY treats impregnated preparations with a drop of
ammonium sulphide, and finishes the reduction in glycerin (quoted
from GIERKE'S Fdrberei z. mik. Zivecken).
BOHM reduces in Pritchard's solution — amyl alcohol, 1 ; formic
acid, 1 ; water, 98.
MANFREDI (Arch, per le Sci. med., v, No. 15) puts fresh tissues into
gold chloride, 1 per cent., for half an hour ; then oxalic acid, 0-5 per
cent., in which they are warmed in a water-bath to 36°. Mount in
glycerin. Sunny weather is necessary.
BOCCARDI (Lavori Instit. Fisiol. Napoli, 1886, i, p. 27 ; Journ.
Roy. Mic. Soc., 1888, p. 155) recommends oxalic acid of 0-1 per cent,
or of 0-25 to 0-3 per cent., or a mixture of 5 c.c. pure formic acid,
1 c.c. of 1 per cent, oxalic acid, and 25 c.c. of water, reducing in the
dark not longer than two to four hours.
KOLOSSOW (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 HC1, 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.
GEBERG (Intern. Monatsschr., x, 1893, p. 205) states that previous
treatment of tissues for twenty-four hours with lime-water (ARN-
STEIN'S method) greatly helps the reduction.
BERNHEIM (Arch. Anat. Phys., Phys. Abth., 1892, Supp., p. 29)
adds to L 6 WIT'S dilute formic acid a piece of sulphite of sodium
(must be fresh and smell strongly of sulphurous acid).
Dr. LINDSAY JOHNSON writes to me that besides the " sunning "
of the impregnating solution recommended above (§ 349), the gold
should be carefully acidulated with a neutral acetate or formate, 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.
APATHY (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
CHAPTER XV I L 207
reduction. Objects, therefore, should always be so thin that light
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 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 in the 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 (§ 343). He finds
such preparations absolutely permanent.
Post- Impregnation .
369. GERLACH'S Method (STRICKER'S Handb., 1872, p. 678) :
Spinal cord is hardened for fifteen to twenty days in a 1 to 2 per
cent, solution of bichromate of ammonia. Thin sections are made
and thrown into a solution of 1 part of double chloride of gold and
potassium to 10,000 parts water, which is very slightly acidulated
with HC1, 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 HC1 to 1000 parts of 60 per cent, alcohol, then
dehydrated and mounted in balsam.
See further, for Nerve Centres, under " Nervous System."
370. GOLGI (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 J 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. APATHY'S Method (Zeit. wiss. Mik., x, 1893, 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 celloidin. Sections are made and fixed on slides, and after
the usual treatment with iodine, etc., are either put into distilled
208 METALLIC STAINS (IMPREGNATION METHODS).
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 overnight,
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
I seem to have found it advantageous to reduce in weak solution
of formaldehyde, either with or without formic acid.
SztiTZ (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.
372. 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 FOL 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. KANVIER 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 ferri-'
cyanide of potassium. REDDING employs a weak solution of ferri-
cyanide, CYBULSKY a 0-5 per cent, solution of cyanide.
Preparations may be double-stained with the usual stains (saf ranin
being very much to be recommended), but nuclei will only take the
second stain in the case of negative impregnation.
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
CHAPTER XVII. 209
pyrogallol, in which they quickly turn greenish black, sometimes
much too much so.
HEKMANN (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 :
HEKMANN or FLEMMING material is brought in bulk, 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. RAWITZ (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 stained that
it is not necessary to have recourse afterwards to a special chromatin
stain. With Invertebrates it sometimes gives very elegant differentia-
tions 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).
This method has been attributed to VON MAEHRENTHAL. See also
under " Nervous System " modifications of this method by AZOULAY
and HELLER and GUMPERTZ ; also one by KOLOSSOW (Zeit. wiss. Mik.,
ix, 1892, p. 38, and ix, 1893, p. 316).
375. Perehloride 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 HOGGANS proceed as follows (Journ. Quekett Club, 1876; Journ.
Boy. 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.
M. 14
210 METALLIC STAINS (IMPREGNATION METHODS).
POL 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 five minutes into fresh mixture of 1 per cent,
perchloride of iron and 1 per cent. sol. of ferricyanide of potassium.
See also UNNA and GOLODETZ, ibid., xlix, 1909, p. 97.
ROOSEVELT ( Med. Rec., ii, 1887, p. 84 ; Journ. Eoy. 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.
376. Palladium Chloride (see SCHULZE, § 77). Prussian Blue (see
LEBER, Arch. Ophthalm., xiv, p. 300 ; RANVIER, Traite, p. 108). Cuprie
Sulphate (see LEBER, ibid.). Lead Chromate (see LEBER, ibid.).
Sulphides (see LANDOIS, Centralb. med. Wiss., 1885, No. 55 ; and
GIERKE, in Zeit. wiss. Mile., i, 1884, p. 497). Molybdate of Ammonia
(MERKEL ; KRAUSE) (see GIERKE, ibid., i, 1884, p. 96). Oxychloride of
Ruthenium (NICOLLE and CANTACUZENE) (see Ann. Inst. Pasteur, vii,
1893, p. 331). Ruthenium Red (Ruthenium Sesquichloride) (EiSEN,
Zeit. 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 (PLATNER, Zeit. wiss. Mik., iv, 1887, p. 350).—
A black liquid on sale by Griibler & Hollborn. MAYER (Grundzuge,
LEE & MAYER, 1st 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 or alcohol. PLATNER
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.
I greatly recommend this stain, which is safe and easy. The
combination with safranin gives a better chromatin stain than
safranin alone.
378. 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.
Sci., 1901, p. 469). "MAYER (Grundzuge, p. 203) finds that, in alum
solution, it gives a stain similar to that of hsematein, but much weaker.
Iron- Brazilin (HICKSON, Quart. Journ. Micr. Sci., xliv, 1901,
p. 470) is better. Sections are mordanted for one to three hours in
1 per cent. sol. of iron alum in alcohol of 70 per cent, (made by
dissolving 1 grm. of the salt in 23 c.c. of water, warm, and adding
77 c.c. of 90 per cent, alcohol after cooling), rinsed with alcohol, and
put for three to sixteen hours into 0-5 per cent. sol. of Brazilin in
alcohol of 70 per cent.
379. Orehella (Orseille), see WEDL (Arch. path. Anat., Ixxiv, p. 143)
and FOL (Lehrb., p. 192), and early editions of this work.
14—2
212 OTHER STAINS AND COMBINATIONS.
380. Orcein (ISRAEL, Virchow's Archiv, cv, 1886, p. 169 ; and Prakti-
cum der path. Hist., 2 Aufl., Berlin, 1893, p. 72) is a 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 combination
of two contrast colours. Israel stains sections in a solution containing
2 grins, of orcein, 2 grins, 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, Zeit. wiss.
Mik., xiii, 1896, p. 302 ; KUZIOKA, ibid., xiv, 1898, p. 455 ; and WOLFF,
ibid., xix, 1903, p. 488. .
381. Purpurin, see RANVIER'S Traite technique, p. 280 ; DUVAL'S
Precis 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 solutions
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.
Thierseh's Oxalic Acid Indigo-Carmine (see Arvh. mik. Anat., i, 1865,
p. 150).
383. Other Vegetal Dyes. — See early editions. Those recommended
by CLAUDIUS (Zeit. wiss. Mik., xvii, 1900, p. 52) are superfluous.
Carmine Combinations.
384. Seller's Carmine followed by Indigo-Carmine (Am. Quart. Mic.
Journ., i, 1879, p. 220). — Stain in borax-carmine, wash out with HC1
alcohol, wash out the acid, and after- stain in an extremely dilute alcoholic
solution of indigo -carmine (2 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
attempted to stain in bulk.
385. Merkel's Carmine and Indigo-Carmine in One Stain (MERKEL,
Unters. anat. Anst. Rostock, 1874 ; Month. Mic. Journ., 1877, pp. 242
and 317).
Also NORRIS and SHAKESPEARE, Amer. Journ. Med. Sci., January,
1877 ; MERKEL, Mon. Mic. Journ., 1877, p. 242 ; MARSH, Section Cutting,
p. 85 ; BAYERL, Arch. Mik. Anat., xxiii, 1885, pp. 36, 37 ; MACALLUM,
Trans. Canad. Instit., ii, 1892, p. 222 ; Journ. Eoy. Mic. Soc., v, 1892,
p. 698.
386. MAYER'S Carmalum (or Hsemalum) and Indigo-Carmine in
One Stain.— MAYER (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 c.c. of distilled water, or 5 per cent, alum solution, and
combining it with from 4 to 20 volumes of carmalum or haBmalum.
387. Carmine and Picro-Indigo-Carmine (RAMON Y CAJAL, Rev.
CHAPTER XVIII. 213
de Cienc. med., 1895 ; CALLEJA, Rev. trim. Microgr., ii, 1897, p. 101 ;
Zeit. wiss. MiL, xv, 1899, p. 323). — For use after a carmine stain,
KAM6N takes a solution of 0-25 grm. of indigo-carmine in 100 grms.
saturated aqueous solution of picric acid. Stain (sections) 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, 1901, 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 BORREL, Ann. Inst. Pasteur, 1901, p. 57, or
LEE et HENNEGUY, Traite, p. 268.
388. Carmine and Anilin Blue (or Bleu Lumiere, or Bleu de Lyon)
(DuvAL, Precis 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 10 drops of saturated
solution of anilin blue in alcohol to 10 grms. 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 (MAURICE
and SCHULGIN)., or bleu lumiere.
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.
BAUMGARTEN (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. — MAAS (Zeit. wiss. Zool., 1, 4,
1890, p. 527) recommends borax-carmine followed by weak alcoholic
solution of malachite green, with a final washing out with stronger
alcohol.
390. Carmine and Picro-nigrosin (PIANESE). — See Journ. Eoy. Mic.
Soc., 1892, p. 292.
391. Carmine and Picric Acid.— See § 298.
Hcematein or Hcematoxylin Combinations.
392. Haematoxylin and Picric Acid.— See § 298.
393. Hsematoxylin and Eosin. — This popular combination gives
results that are aesthetically beautiful, but (for most objects) is not
214 OTHER STAINS AND COMBINATIONS.
so useful as many others, the eosin lacking in electivity. Objects
may be stained with hsematoxylin (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
(STOHR, see Zeit. wiss. Mik., i, 1884, p. 583) to take it saturated.
Either aqueous or alcoholic solutions of eosin may be used.
HICKSON (Quart. Journ. Mic. Sci., 1893, p. 129) stains sections
for one hour in a strong solution of eosin in 90 per cent, alcohol,
washes with alcohol, and stains for twenty minutes in a weak solution
of haematoxylin.
This method is most particularly recommendable for embryo-
logical sections, as vitellus takes the eosin stain energetically, and
so stands out boldly from the other germinal layers in which the blue
of the haematoxylin dominates.
See also LIST (Zeit. wiss. Mik., ii, 1885, p. 148) ; BUSCH (Verh.
Bed. Phys. Gses., 1887) ; GIERKE (Zeit. iviss. Mik., i, 1884, p. 505).
Sections should be very well washed before being passed from
eosin into haematoxylin or the reverse, as eosin very easily precipitates
hsematoxylin.
For the complicated and superfluous mixtures of RENAUT and of
EVERARD, DEMOOR and MASSART, see FOL'S Lehrbuch, p. 196, Ann.
Inst. Pasteur, vii, 1893, p. 166, or early editions.
See especially SCOTT'S method, § 676.
393a. Hsematoxylin and Azoeosin or Biebrich Scarlet. — See under
" Nucleoli," § 676.
394. Hsematoxylin and Congo. — See § 305.
395. Haematoxylin and Safranin. — EABL (Morph. \fahrb., x, 1884,
p. 215) stained very lightly with very dilute DELAFIELD'S haematoxylin 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
hsematoxylin.
Similarly REGAUD, Verh. Anat. Ges., xiv, 1900, p. 112.
FOA (Festschr. Virchow, 1891, p. 481) stains in a mixture of 25 c.c.
of Bohmer's haematoxylin, 20 of 1 per cent, solution of safranin,
and 100 of water for one to three minutes.
396. Hsematoxylin and Saurefuchsin. — Stain first with iron
hsematoxylin or haemalum, then stain (sections) in 0-5 per cent,
aqueous solution of Saurefuchsin, dehydrate and mount.
397. Haematoxylin and Saurefuchsin and Orange. — Proceed as
above, using for the second stain the following mixture : Saure-
fuchsin, 1 grm. ; orange, 6 grms. ; rectified spirit, 60 c.c. ; water,
CHAPTER XVIII. 215
240 c.c. (from SQUIRE'S Methods and Formula, 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 complicated.
398. Haematoxylin and Picro-Saurefuchsin (VAN GIESON, New
York Med. Journ., 1889, p. 57 ; quoted from MOELLER, Zeit. wiss.
Mik., xv, 2, 1898, p. 172, which see for further details). — Proceed
as above, using for the second stain the picro-Saurefuchsin mixture,
§ 299. The second stain must not be too prolonged.
WEIGERT (Zeit. wiss. Mik., xxi, 1904, p. 1) stains first in his iron-
haematoxylin mixture (§ 244), rinses in water, and stains for a short
time in his picro-Saurefuchsin (§ 299), rinses, dehydrates with
90 per cent, alcohol, and clears with carbolic acid-xylol mixture
(§ 167).
CHAPTER XIX.
EXAMINATION 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 sure preservation of soft
tissues are the resinous ones.
400. Refractive Indices of Examination Media. — An examination
medium should be of such a refractive index 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, is occasionally higher than
that of Canada balsam : so that media of the greatest clearing
power (i.e. 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 BEHRENS' Tabellen zum
Gebrauch bei mikroskopischen Arbeiten, Braunschweig, 1892, p. 42,
and other sources, may be useful as a guide to the optical effects of
various media. The figures give the approximate indices of refrac-
tion. 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.
CHAPTER XIX.
217
Air .... 1-000
Methyl alcohol . . 1 -323
Distilled water . . 1 -336
Sea water . . . 1-343
Solution of white of egg . 1 -350
Absolute alcohol . . 1 -367
Acetate of potash, satu-
rated aqueous sol. . 1-370
Glycerine with an equal
quantity of water . . 1-397
Chloride of calcium, 90 per
cent, in water . . -411
Glycerine, Price's . . -460
Oil of bergamot - . . -464
Paraffinum liquidum . -471
Olive oil ... -473
Oil of turpentine . . 1-473
Glycerine, " concentrated " 1 -473
Gilson's Baume au Camsal 1 -478
Gilson's Euparal . . 1-483
Terpinol . . . 1-484
Castor oil 1 -490
Xylol . . . 1-497
Cedar-wood oil, not thick-
ened .... 1-510
Crown glass . . . 1-518
Cedar- wood oil, thickened . 1 -520
Gum damar . . . 1-520
Xylol balsam . . . 1-524
Oil of lemons . . . 1-527
Oil of cloves . . *. 1-533
Canada balsam (solid) . 1-535
Creasote . . . 1-538
Colophonium . . . 1-545
Carbolic acid . 1 -549
Oil of anise seed . . 1 -557
Oil of cinnamon (or cassia) 1-567
Anilin oil . . .1 -580
Sulphide of carbon . . 1-630
Tolu balsam . . . 1-640
Monobromide of naphtha-
lin .... 1-660
Solution of sulphur in sul-
phide of carbon . . 1 -750
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 action on
tissues, appears to be misleading more than helpful ; ' for 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 fixed ; but this is by no means applicable to the
examination of fresh tissues. It is very far from being an " in-
different " 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).
218 EXAMINATION AND PRESERVATION MEDIA.
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 occurrence of osmosis, to
which process the destructive action of pure water is mainly due.
Now cell contents are a mixture of colloids and crystalloids ; conse-
quently, 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
egg to salt solution this end may be in some measure attained ; and,
as a matter of fact, the liquids recommended as " indifferent " are
generally Jiound 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 isotonic 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 Invertebrates, 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.
RINGER'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 0-1 dex-
trose).
According to LOCKE (Boston Med.-Surg. Journ., 1896, p. 514)
there should be added to salt solution (which to be isotonic should
contain, according to HAMBURGER, 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.
MALASSEZ (C. R. Soc. Biol, iii, 1896, pp. 504 and 511) takes for
erythrocytes about 1 per cent, sodium chloride.
DEKHUYZEN (Onderz. Phys. Lab. Leiden., 1900, p. 149) takes for
blood of Rana 0-8 per cent.
For Selachians, MUSKENS (Tijd. Nederb. DierL Ver., 1894, p. 314)
finds 2J per cent, right ; and EODIN (Comptes Rend., 1900, p. 1009)
1-5 to 2-6 per cent., according to the species.
ENGELMANN (Deutsch. med. Wochenschr., xxix, 1903, 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.
KRONECKER'S " Artificial Serum " (from VOGT et YUNG, Traite
CHAPTER XIX. 219
d'Anat. Comp. Prat., p. 473) consists of common salt 6 parts, caustic
soda 0-06, distilled water 1000.
BOHM und OPPEL (Taschenbuch, 3 AufL, p. 19) take carbonate of
soda instead of caustic soda.
403. PICTET'S Liquid (Mitth. Zool. Stat. Neapel, x, 1891, p. 89).—
5 to 10 per cent, solution of chloride of manganese. These propor-
tions are for marine animals, and for terrestrial animals will generally
be found much too high. For these from 1 to 3 per cent, will be
nearer the mark. I find this liquid excellent.
404. Aqueous Humour, Simple White of Egg. — Kequire no pre-
paration beyond filtering. They may be iodised if desired (see
next §), or mixed with salt solution.
405. Iodised Serum. — MAX SCHULTZE (Vir chow's Archiv., xxx,
1864, p. 263). I take the following from KANVIER (Traite, 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.
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. This should be kept in stock, and a little of it added
every two or three days to the serum that is intended for use. In
general for maceration purposes a serum of a pale brown colour
should be employed.
406. Artificial Iodised Serum (FREY, Das Mikroskop, 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. MIGULA'S Glycerised Blood-serum (see the paper in Zeit. /. wiss.
Mik., vii, 2, 1890, p. 172).
408. Chloride of Calcium (HARTING, Das Mikroskop, 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 SCHULTZE, Arch. mile. 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 carbolic acid
(1 per cent.).
220 EXAMINATION AND PRESERVATION MEDIA.
It may be used as a mounting medium, but there is always risk
of the sugar crystallising out.
FABRE-DOMERGUE (Bull. Soc. Philomath, ix, 1899, p. 115) dis-
solves 200 parts of sugar in 400 of water, and adds 1 part of formalde-
hyde, and camphor to saturation.
411. Chloral Hydrate. — 5 per cent, in water (LADOWSKY, Arch. f.
mik. Anat., 1876, p. 359).
Or, 2-5 per cent, in water (BRADY, British Copepods).
Or, 1 per cent, in water (MuNSON, Journ. Roy. Mic. Soc., 1881,
p. 847).
Mercurial Liquids.
(I give these as examination media only, not as permanent mounting media
Media containing sublimate always end by making tissues granular.)
412. GILSON'S Fluid (CARNOY'S Biologie Cellulaire, p. 94).
Alcohol of 60 per cent. . . . .60 c.c.
Water 30 „
Glycerin . . . . . . . 30 ,,
Acetic acid (15 parts of the glacial to 85 of
water) . . . . . 2 „
Bichloride of mercury . . . .0-15 grm.
413. GAGE'S Albumen Fluid (Zeit.f. wiss. Mik., 1886, p. 223).
White of egg ...... 15 c.c.
Water 200 „
Corrosive sublimate ..... 0-5 grm.
Salt ........ 4 grms.
Mix, agitate, filter, and preserve in a cool place. Recommended
for the study of red blood-corpuscles and ciliated cells.
414. P ACINI'S Fluids (Journ. de Mik., 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 |
to | per cent, strength, with the addition of a little salt or acetic acid.
415. GOADBY'S Fluids (Micro. Diet., art. "Preservation," or early
editions of this work). — Quite unsuited for histological purposes.
Other Fluids.
416. Chloride and Acetate of Copper (KIPART et PETIT'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.
CHAPTER XIX. 221
419. Medium of FARRANTS (BEALE, How to Work, etc., p. 58). —
Picked gum arable 4 ozs., water 4, glycerin 2. See also the Micro-
graphic Dictionary, and A. F. STANLEY tKENT, in Journ. Roy. Mic.
Soc., 1890, p. 820.
420. Gum and Glycerin Medium (LANGERHANS, ZooL Anzeig., ii, 1879,
p. 575).
Gummi arab. . . . . . . 5-0
Aquae ........ 5*0
to which after twelve hours are added —
Glycerin! . . . . . . . 5-0
Sol. aquosa acid, corbal. (5-100) . . . 10-0
421. ALLEN'S Gum and Glycerin. — Prof. F. J. ALLEN (in litt.).
Solution of gum arabic of the consistency of glycerin, strained, and
| volume of glycerin and -£$ of formol gradually incorporated. Sets
hard.
422. HOYER'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 either 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
hsematoxyrin objects — that with acetate for anilin objects.
423. COLE'S Gum and Syrup Medium. See § 183.
424. APATHY'S Gum and Syrup Medium (see § 343). — This medium
sets very hard and may also be used for ringing glycerin mounts.
425. FABRE-DOMERGUE'S Glucose Medium (La Nature, No. 823,
9 Mars, 1889, supp.).— Glucose syrup diluted to 25° of the areometer
(sp. gr. 1-1968) 1000 parts, methyl alcohol 200, glycerin 100, camphor
to saturation. The glucose is to be dissolved in warm water, and
the other ingredients added. The mixture, which is always acid,
must be neutralised by the addition of a little potash or soda. It
is said to preserve without change almost all animal pigments, but
the mounts do not keep indefinitely.
426. BRUN'S Glucose Medium (from FABRE-DOMERGUE'S Premiers
Principes du Microscope, 1889, p. 123).— Distilled water 140 parts,
camphorated spirit 10, glucose 40, glycerin 10. Mix the water,
glucose, and glycerin, then add the spirit, and filter. HENNEGUY
informs me that this liquid preserves the colour of preparations
stained with anilin dyes, methyl green included.
427. Levulose is recommended by BEHRENS, KOSSEL u. SCHIEF-
FERDECKEB (Das Mikroskop, etc., 1889). It is uncrystallisable, and
preserves well carmine and coal-tar stains (hsematoxylin stains fade
222 EXAMINATION AND PRESERVATION MEDIA.
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 LANGERON, C. R. Soc. BioL,
Iviii, 1905, p. 750). — Carbolic 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 em-
ployed 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 BE ALE, How to work, etc.).
For closing glycerin mounts, the edges of the cover should first
(after having been cleansed as far as possible from superfluous
glycerin) be painted with a layer of glycerin jelly ; as soon as this is
set a coat of any of the usual cements may be applied. 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 refraction
of glycerin (Price's glycerin, n — 1-46) may be raised by dissolving
suitable substances in it. Thus the refractive index of a solution of
chloride of cadmium (CdCl2) in glycerin may be 1 -504 ; that of a
saturated solution of sulphocarbolate of zinc in glycerin may be 1 -501 ;
that of a saturated solution of SCHERING'S chloral hydrate (in crusts) in
glycerin is 1-510 ; that of iodate of zinc in glycerin may be brought up
to 1 -56. For further details see previous editions, or Journ. Roy. Mic.
Soc., ii, 1879, p. 346 ; iii, 1880, p. 1051 ; (N.S.), i, 1881, pp. 943 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 being closed. By the
evaporation of the alcohol the liquid gradually increases in density,
and after some time the mount may be closed, or the object brought
into pure glycerin or glycerin jelly.
1. CALBERLA'S LIQUID (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.
CHAPTER XIX. 223
3. HANTSCH'S LIQUID. — Glycerin 1 part, alcohol 3, water 2.
4. JAGER'S LIQUID (Vo-GT and YuNG'&Traited'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 (DA VIES, Preparation and Mount-
ing 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 an egg. 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. BRANDT'S Glycerin Jelly (Zeit. wiss. Mik., ii, 1880, p. 69). —
Melted gelatin 1 part, glycerin 1J parts. The gelatin to be soaked
in water and melted as above. After incorporating the glycerin,
filter through spun glass pressed into the lower part 'of a funnel.
He describes a simple arrangement for keeping the funnel warm
during the filtering (see early editions). Some drops of carbolic acid
should be added.
435. KAISER'S Glycerin Jelly has been given § 155.
436. SQUIRE'S Glycerin Jelly (SQUIRE'S Methods and Formula,
etc., 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. HEIDENHAIN (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. FISCHER (ibid., xxix, 1912, p. 65) takes 5 grms. of borax
dissolved in 240 c.c. of water and adds 25 c.c. of glycerin. To this
224 EXAMINATION AND PRESERVATION MEDIA.
he adds 40 grins, of gelatin, dissolves with heat, and continues to
heat gently until the solution has somewhat thickened. This
remains fluid at ordinary temperatures.
439. GILSON'S Chloral Hydrate Jelly (communicated by GILSON).—
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.
GEOFFKOY, Journ. de Botan., 1893, p. 55 (see Zeit. wiss. Mik., 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. STEPHENSON'S Biniodide of Mercury and Iodide of Potassium
(Journ. Roy. Mic. Soc. [N.S.], ii, 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. Mik., xiii, 1896, p. 21] glycerin be taken instead of water,
it rises to 1-78 or 1-80. BEHRENS [Tabellen, 1898, p. 71] takes
biniodide 65 parts, iodide 50, and water 25. n = 1-71.) Any
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 mount-
ing medium, for the preparations are- ruined by a precipitate which
forms in the fluid. But as a temporary 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. Boy. Mic. Soc., 1880,
p. 1043 (ABBE and VAN HEURCK), and Zool. Anz., 1882, p. 555 (MAX
FLESOH).
CHAPTER XIX. 225
Resinous Media.
442. Resins and Balsams. — Resins and balsams consist of a
vitreous or amorphous substance held in solution by an essential
oil. By distillation or drying in the air they lose the essential oil
and pass into the solid state. It is these solidified resins that should
be employed for microscopical purposes ; for the raw resins always
contain a certain proportion of water, which makes- it difficult to
obtain a clear solution with the usual menstrua, is injurious to the
optical properties of the medium and to 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 chloro-
form, 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.
Turpentine media preserve the index of visibility of the prepara-
tions 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 Euparal. For cases in which a still
lower index is desired, Gilson's camsal balsam. Turpentine colo-
phonium is a safe and excellent medium, but is injurious to alum-
hsematein stains. For these, and in general where a strongly
clearing medium is desired, xylol balsam is about the most recom-
mendable, though it is not perfectly safe, the mounts sometimes
developing granules. Seller's alcohol balsam is a fine medium, and
perfectly stable. Oil 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 BELL'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 xylol solution is the best. If time be an object, a benzol solution
should be preferred, as it sets much quicker than the xylol solution.
M. 13
226 EXAMINATION AND PRESERVATION MEDIA.
The chloroform solutions become very brown with age, and are
injurious to stains made with tar dyes. Benzol is good when chemi-
cally pure and free from water.
SAHLI (Zeit. wiss. Mik., ii, 1885, p. 5) dissolves in cedar oil.
APATHY (Fauna Flora Golf. Neapel, xxii, 1909, p. 18) takes
balsam 2 parts, cedar oil (immersion) 1, and chloroform 1.
Samples of balsam that are acid are frequently met with, and are
injurious to some stains.
443a. How to make Neutral Balsam.— Take balsam crystals dry.
Grind up in mortar and add an equal part of sodium bicarbonate :
mix thoroughly. Transfer to a bottle and add enough xylol or
benzole to make a quite liquid solution. Allow this to stand for
three or four days, shaking occasionally. Filter, and place nitrate
on a thermostat to evaporate down to the right consistency.
S. G. SCOTT (Journ. Path., xvi), recommends keeping a piece of
marble in the balsam bottle,, and either placing the latter in a tin
box, or painting it black outside in order to protect the balsam from
the light. See COLUCCI (Giorn. Ass. Med. Natural Napoli, vii, 1897,
p. 172).
444. SEILER'S Alcohol Balsam (Proc. Amer. Soc. Mic., 1881,
pp. 60-2 ; Journ. Roy. Mic. Soc. [N.S.], ii, 1882, pp. 126-7).—
Dissolve solid balsam in warm absolute alcohol, andc 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 (TAmmar). — The menstrua
are the same as for balsam. I find xylol the best. For directions 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, sometimes 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 dries 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 oil-immersion
lenses ; whereas an alcohol-balsam mount will be dry enough in a
couple of days. It injures alum-hsematein stains ; as it sometimes
develops clouds of globules it is not to be depended upon.
CHAPTER XIX. 227
Dr. S. G. SCOTT of Oxford used both damar and colophonium instead
of balsam, and very few of his preparations left after his decease are
good. A large number have become granular.
REHM (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 NISSL in Encycl. mik. Techn., ii, p. 274.
447. Venice Turpentine (VOSSELER, Zeit. wiss. Mik., vi, 1889, pp. 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 hsemalum stains fade in it.
SUCHANNEK (ibid., vii, 1896, p. 463) prepares it with equal parts of
Venice turpentine and neutral absolute alcohol.
448. Thickened Oil of Turpentine has been used as a mounting
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. GILSON'S Sandarac Media (Lat Cellule, xxiii, 1906, p. 427 :
the formulae have not been published, on account of the extreme
difficulty of preparation, but the products are on sale by Griibler &
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 = 0-478.
(2) Camsal balsam, isobutylic alcohol formula, n = 1485.
(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 containing a salt of
copper, which intensifies hcematoxylin 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
* Now manufactured by Flatters & Garnett (§11) according to
Gilson's original formula.
15—2
228 EXAMINATION AND PRESERVATION MEDIA.
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 oxidant, 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 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 — 1478, 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 (LAVDOWSKY, form Eef. Handbook Med. Sci., Supp.,
p. 438). — Gum sandarac 30 grs., absolute alcohol 50 c.c. Not trust-
worthy, the mounts scale badly.
451. Photographic Negative Varnish (for mounting large sections
without cover-glasses). — See WEIGERT, Zeit. wiss. Mik., iv, 1887, p. 209.
452. Castor Oil.— See GBENACHER, Abhandl. naturf. Ges. Halle-a.-S.,
Bd. xvi ; Zeit. wiss. Mik., 1885, p. 244. I have not had good results
with it.
453. Terpinol. — n = 1-484. See § 131.
454. Parolein (a pure form of paramnum liquidum) is recommended
by COLES (Lancet, 1911, p. 878) as being quite neutral and preserving
certain coal tar stains. King mounts with Apathy'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. Mik., xiv, 1897, p. 201.
457. Styrax and Liquidambar.— See Journ. Roy. Mic. Soc., 1883,
p. 741 ; ibid., 1884, pp. 318, 475, 655, and 827 ; and the places there
quoted. Also Bull. Soc. Beige de Mic., 1884, p. 178 ; and FOL, Lehrb.,
p. 141. These are very highly refractive media, therefore seldom useful
in histology.
CHAPTER XX.
CEMENTS AND VARNISHES.
458. Introduction.— Two, or at most three, of the media given
below will certainly be found sufficient for all useful purposes. For
many years I have used only one cement (BELL'S). I recommend
this both as a cement and varnish ; gold size may be found useful
^for turning cells ; and MILLER'S caoutchouc cement may be kept
for occasions on which the utmost solidity is required. Marine glue
is only necessary for making glass cells.
For the operations of mounting in fluids, and of making cells and
ringing, see CARPENTER'S The Microscope.
CARPENTER lays great stress on the principle that the cements or
varnishes used for fluid mounts should always be such as contain
no mixture of solid particles, 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 ; by 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 AUBERT, The Microscope, xi, 1891, 150, and Journ. Roy.
Mic. Soc., 1891, p. 692 ; BECK, The Microscope, xi, 1891, pp. 338,
368, and Journ. Roy. Mic. Soc., 1892, p. 293 ; BEHRENS' Tabellen
zum Gebrauch bei mikroskopischen Arbeiten (Bruhn, Braunschweig,
1892) ; ROUSSELET, Journ. Quek. Mic. Club, vii, 1898, p. 93 ; and
as to the comparative tenacity of divers cements, BEHRENS, Zeit.
wiss. Mik., ii, 1885, p. 54, and AUBERT Amer. Mon. Mic. Journ.,
1885, p. 227 ; Journ. Roy. Mic. Soc., 1886, p. 173.— AUBERT places
MILLER'S caoutchouc cement at the head of the list, LOVETT'S
cement coming halfway down, and zinc white cement at the bottom,
with less than one-quarter the tenacity of the 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 (MARSH'S Section-cutting, 2nd ed., p. 104).—
Take half an ounce of NELSON'S opaque gelatin, soak well in water,
230 CEMENTS AND VARNISHES.
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 grs. 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 BELL'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 BELL'S cement.
For greater safety, the gelatin may be treated with bichromate,
according to MARSH'S plan, last §.
462. ROUSSELET'S Method for Aqueous Mounts (op. cit., § 458).—
Close the mount with a ring of a mixture of 2 parts of a solution of
damar in benzol and 1 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 WARD'S brown cement.
463. MILLER'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 ROUSSELET, Journ. Quek. Club,
v, ii, 1895, p. 8).
464. Asphalt Varnish (Bitume de Judee). — 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.
465. Brunswick Black. — See ear^y editions, or BEALE, How to
Work, etc., p. 49.
466. 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.
467. Turpentine, Venice Turpentine (CSOKOR, Arch. mik. Anat.,
xxi, 1882, p. 353 ; PARKER, Amer. Mon. MiL Journ., ii, 1881,
CHAPTER XX. 231
pp. 229 — 30). — Venice turpentine, or common resinous 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 vent at
right angles ; the short arm of the wire should be just the length of
the side of the cover-glass. The wire is heated in a spirit lamp,
plunged into the cement, some of which adheres to it, and then
brought down flat upon the slide at the margin of the cover. The
turpentine distributes itself evenly along the side of the cover, and
hardens immediately, so that the slide may be cleaned as soon as
the four sides are finished. It is claimed for this cement that it is
perfectly secure and never runs in. It sets hard in a few seconds.
468. APATHY'S Cement for Glycerin Mounts (Zeit. wiss. MiL, 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 turpen-
tine. Used by warming and applying with a glass rod or brass
spatula. One application is enough. Does not run in, and never
cracks.
469. Canada Balsam, or Damar. — Cells are sometimes made with
these. They are elegant, but in my experience are not reliable for
permanent mounts.
470. 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.
471. For the cements of WARD, BELL and CLARKE and for
KRONIG'S Colophonium and Wax, Marine Glue, Amber and Copal,
and Sealing Wax Varnish, see last edition.
PART II.
SPECIAL METHODS AND EXAMPLES.
CHAPTER XXI.
INJECTION — GELATIN MASSES (WARM).
472. Introduction. — Injection masses are composed of a coloured
substance called the colouring mass, and of a substance with which
that is combined called the vehicle.
For instructions as to the operation of injecting, and the neces-
sary apparatus, see the Micrographic Dictionary, RUTHERFORD'S
and SCHAFER'S Practical Histology, the treatises of ROBIN and
RANVIER, BEALE'S How to Work with the Microscope, the Lehrbmh
der vergleichenden Mikroscopischen Anatomic of FOL, and (for
apparatus especially) the article in the Encycl. d. mik. Technik.
For injections for the study of the angiology of Vertebrates the
practice of Robin and Ranvier may safely be followed. For injec-
tions 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 BE ALE. Glycerin masses have the great advantage that
they are used cold.
All formulse which only give opaque masses, or are only suitable
for coarse injections for naked eye study, have been suppressed.
In § 592 is a section on injection of embryos.
473. 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. Unfortunately, when this is done, it is
found that most injection masses — glycerin masses especially—
CHAPTER XXL 233
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 anaesthetised with a
mixture of ether and nitrite of amyl, and finally killed with pure
nitrite. Or, after killing by nitrite, a little nitrite of amyl in salt
solution may be injected before the injection mass is thrown in.
In any case it is advisable to add a little nitrite to the mass just
before using. The relaxing power is very great (see OVIATT and
SARGENT, in St. Louis Med. Journ., 1886, p. 207 ; and Journ. Roy.
Mic. Soc., 1887, p. 341).
BAYLISS (in personal communication) suggests for prevention of
coagulation, to wash out in citrate of soda (4 per cent.) instead of
•75 NaCl, or to add J per cent, oxalate of calcium to -75 per cent.
NaCl. To relax arterial walls, add sodium nitrite 1 in 500 to the
washing out fluid.
Or, morphia may be added to the injection mass, or 1 per cent,
of lactic acid. MOZEJKO (Zeit. wiss. MiL, xvi, 1909, p. 545) prefers
a saturated solution of neutral Peptonum 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.
ROBIN'S Masses.
474. ROBIN'S Gelatin Vehicle (Traite, 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.
475. ROBIN'S Glycerin-Gelatin Vehicle (Traite, p. 32). — Dissolve
in a water-bath 50 grms. of gelatin in 300 grms. of water, in which
has been dissolved some arsenious acid ; add of glycerin 150 grms.,
and of carbolic acid a few drops. Unlike the pure gelatin vehicles,
this mass does keep indefinitely.
FRANKL (Zeit. f. wiss. Zool, Ixiii, 1897, p. 28) prepares a similar
vehicle, and adds to it a little solution of corrosive sublimate and a
crystal of thymol.
476. ROBIN'S Carmine Colouring Mass (Traite, p. 33). — Rub up
3 grms. of carmine with a little water and enough ammonia to
dissolve it. Add 50 grms. of glycerin and filter.
234 INJECTION— GELATIN MASSES (WARM).
Take 50 grms. of glycerin with 5 grms. of acetic acid, and add it
by degrees to the carmine-glycerin, 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 3 or 4 parts of the
vehicles given above.
477. Robin's Ferrocyanide of Copper Colouring Mass (ibid., p. 34).—
Take —
(1) Ferrocyanide of potassium (concentrated
solution) . . . . . .20 c.c.
Glycerin . . . . . . 50 ,,
(2) Sulphate of copper (concentrated solution). 35 ,,
Glycerin . . . . . . 50 ,,
Mix (1) and (2) slowly, with agitation ; at the moment of injecting
combine with 3 volumes of vehicle.
478. EOBIN'S Prussian Blue Colouring Mass (ibid., p. 35, and
2nd ed., p. 1013).
Take—
(A) Ferrocyanide of potassium* (sol. sat.) . . 90 c.c.
Glycerin 50 „
(B) Liquid perchloride of iron at 30° Baume . . 3 ,,
Glycerin 50 „
Mix slowly and combine the mixture with 3 parts of vehicle. It
is well to add a few drops of HC1.
Carmine-gelatin Masses.
479. RANVIER'S Carmine-Gelatin Mass (Traite 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, 2J grms. 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 2 parts of water. (When the mass is near neutrality,
dilute the acetic acid still further.) The instant of saturation is
determined by the smell of the solution, which gradually changes
from ammoniacal to sour. As soon as the sour smell is perceived
the liquid must be examined under the microscope. If it contains
a granular precipitate of carmine, too much acid has been added,
and it must be thrown away. *
* Erratum " Sulphocyamde " in 1st ed. of ROBIN'S Traite.
CHAPTER XXL 235
The mass, having been perfectly neutralised, is strained through
new flannel.
480. How to Neutralise a Carmine Mass (VILLE, Gaz. hebd. d. Sci.
tned. de. Montpellier, Fev., 1882 ; may be had separately from
Delahaye et Lecrosnier, Paris). — VILLE 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, VILLE 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.
481. HOYER'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 5 to 10 per cent, by volume
of glycerin, and at least 2 per cent, by weight of chloral, in a con-
centrated solution, and strain.
482. FOL'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 saturation,
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 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 Zeit. wiss. Zool., xxxviii, p. 492,
or previous editions.
483. KRAUSE'S Carmine-Gelatin Mass (Zeit. wiss. Mile., 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,
236 INJECTION— GELATIN MASSES (WARM).
washed, treated for a short time with hydrochloric acid of 2 per cent.,
washed, melted and preserved with camphor.
484. Other Carmine- Gelatin Masses.— THIERSCH'S, see Arch. mik.
AnaL, 1865, p. 148. GERLACH'S, see RANVIER, Traite, p. 118, CARTER'S,
see BEALE, p. 113. DAVIES, see his Prep, and Mounting of Mic. Objects,
p. 138.
Blue Gelatin Masses.
485. KANVIER'S Prussian Blue Gelatin Mass (Traite, p. 119).—
Make a concentrated solution of sulphate of peroxide of iron in
distilled water, and pour it gradually into a concentrated 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
saturated. Such a mass never transudes though 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 until the curdy precipitate that forms at first
has disappeared. Filter through new flannel and keep at 40° C.
until injected.
486. BRUCKE'S Soluble Berlin Blue (Arch. mik. Anat., 1865, p. 87).—
Make a solution of ferrocyanide of potassium containing 217 grins, 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.
487. Other Blue Gelatin Masses.— HOYER'S, Arch. mik. Anat., 1876,
p. 649 ; GUIGNET'S, Journ. de Microgr., 1889, p. 94 ; Journ. Roy. Mic.
CHAPTER XXL 237
Soc., 1889, p. 463 ; THIERSCH'S, Arch. mik. Anat., i, 1865, p. 148 ; yFoL's,
Zeit. wiss. Zool., xxxviii, 1883, p. 494 ; and previous editions.
Other Colours.
488. HOYER'S Silver Nitrate Yellow Gelatin Mass (Biol Centralbl,
ii, 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 this is added a very small quantity of an aqueous
solution of pyrogallic acid, which reduces the silver in a few seconds ;
chloral and glycerin are added as directed § 489.
This mass is yellow in the capillaries and brown in the larger
vessels.
489. Other Colours.— HOYER'S Green (Biol. Centralb., ii, 1882, p. 19).
Made by mixing a blue mass and a yellow mass. THIERSCH'S Green
(Arch. mik. Anat., 1865, p. 149). ROBIN'S SCHEELE'S Green (ROBIN,
Traite, p. 37). HARTING'S White (see FREY, Le Microscope, p. 190).
FREY'S White (ibid.). TEICHMANN'S White (ibid., p. 191). FOL'S
Brown (Zeit. wiss. Zool, xxxviii, 1883, p. 494). MILLER'S Purple (see
Amer. Mon. Mic. Journ., 1888, p. 50 ; Journ. Roy. Mic. $oc., 1888,
p. 518). FOL'S Lead Chromate (Lehrb., p. 15). ROBIN'S Cadmium (his
Traite, p. 36). THIERSCH'S Lead Chromate (Arch. mik. Anat., 1865,
p. 149). HOYER'S Lead Chromate (ibid., 1867, p. 136) ; or, for any of
these, see early editions.
490. RANVIER'S Gelatin Mass for Impregnation (Trmte, p. 123).—
Concentrated solution of gelatin, 2, 3, or 4 parts ; 1 per cent, nitrate of
silver solution, 1 part.
NEUVILLE (Ann. Sci. 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.
4-91. 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 volume of 4 per cent, formol, serves for injecting
vessels and hardening the tissues at the same time.
CHAPTER XXII.
INJECTIONS — OTHER MASSES (COLD).
492. FOL'S Metagelatin Vehicle (Lehrb., p. 17).— If a slight pro-
portion 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 Encycl. mik. Technik., metagelatin is usually
prepared by warming with concentrated acetic or oxalic acid. It
may be neutralised afterwards with carbonate of lime.
493. TANDLER'S Gold Gelatin Mass (Zeit. wiss. 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 specimens
will bear decalcification with hydrochloric or sulphurous acid.
PEARL (Journ. Appl. Micr., v, 1902, p. 1736) takes 8 to 10 pec
cent, of the iodide.
MAYER (Grundzuge LEE and MAYER, 1910, p. 250) takes simply
10 grms. gelatin, 10 grms. hydrate of chloral and 100 c.c. water.
MOZEJKO (Zeit. wiss. Mik., xxvii, 1910, p. 374) finds that 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.
494. BEALE'S Carmine Glycerin Mass (How to Work, etc., p. 95).—
Five grains of carmine are dissolved in a little water with about
5 drops of ammonia, and added to half an ounce of glycerin. Then
add half an ounce of glycerin with 8 or 10 drops of acetic or hydro-
chloric acid, gradually, with agitation. Test with blue litmus
CHAPTER XXII. 239
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.
495. BE ALE'S Prussian Blue (How to Work, etc., p. 93).
Common glycerin .... 1 ounce.
Spirits of wine . . . 1 „
Ferrocyanide of potassium . . .12 grains.
Tincture of perchloride of iron . . 1 drachm.
Water . . . .4 ounces.
Dissolve the ferrocyanide in one ounce of the water and glycerin,
and add the tincture of iron to another ounce. These solutions
should be mixed together very gradually, and well shaken in a bottle,
the iron being added to the 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.
496. BEALE'S Acid Prussian Blue (ibid., p. 296).
Price's glycerin ..... 2 fluid ounces.
Tinct. of sesquichloride of iron . . 10 drops.
Ferrocyanide of potassium ... 3 grains.
Strong hydrochloric acid ... 3 drops.
Water ...... 1 ounce.
Proceed as before, dissolving the ferrocyanide in one half of the
glycerin, the iron in the other, and adding the latter drop by drop
to the former. Finally add the water and HC1. Two drachms of
alcohol may be added to the whole if desired.
I find this excellent.
497. RANVIER'S Prussian Blue Glycerin Mass (Traiti, p. 120).— The
Prussian blue fluid, § 494, mixed with one fourth of glycerin.
498. THOMA'S Indigo-Carmine (Arch. Anat. Phys., Anat. Abth.,
1899, p. 270).— Dissolve 0'15 grm. sulphindigotate of soda in 50 c.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 a 1 per cent, solution of morphia may be added
to dilate arteries. A fine precipitate is formed, which is injected
with the mass.
499. Gamboge Glycerin (HARTING, Das Mikroskop, 1866, 2,
Theil, p. 124). — Gamboge rubbed up with water and added to
240 INJECTIONS— OTHER MASSES (COLD).
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.
500. 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. (See § 592.)
501. RANVIER'S Prussian Blue Aqueous Mass (Traite, p. 120).—
- The soluble Prussian blue, § 494, injected without any vehicle. It
does not extravasate.
502. MULLER'S Berlin Blue (Arch. mik. Anat., 1865, p. 150).—
Precipitate a concentrated solution of Berlin blue by means of
J to 1 volume of 90 per cent, alcohol. The precipitate is very finely
divided ; and the fluid may be injected at once.
503. MAYER'S Berlin Blue (Mitth. Zool Stat. Neapel, 1888,
p. 307). — A solution of 10 c.c. of tincture of perchloride of iron in
500 c.c. of water is added to a solution of 20 gr. of yellow prussiate
of potash in 500 c.c. of water, allowed to stand for twelve hours,
decanted, the deposit washed with distilled water on a filter until
the washings come through dark blue (one to two days), and the blue
dissolved in about a litre of water. It is well to add a little acetic
acid and to put up the objects in an acid liquid.
504. EMERY'S Aqueous Carmine (ibid., 1881, p. 21). — To a 10 per cent,
ammoniacal solution of carmine is added acetic acid, with continual
stirring, until the colour of the solution changes to blood-red. 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.
505. TAGUCHI'S Indian Ink (Arch. mik. Anat., 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).
DELLA KOSA (Ver. Anat. Ges., 1900, p. 141) recommends the
liquid Chinese ink sold in the shops.
Partially Aqueous Masses.
506. JOSEPH'S White-of-Egg (Ber. natur^v. Sect. Schles. Ges.,
1879, pp. 36—40 ; Journ. Roy. Mic. Soc.t ii, 1882, p. 274).—" Fil-
CHAPTER XXII. 241
tered white-of-egg, diluted with 1 to 5 per cent, of carmine 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. MiL, xvii, 1900, p. 178) rubs up Indian ink
with white-of-egg ; HOFFMANN (Zeit. Morph. Anthrop., iii, 1901,
p. 240) with blood-serum ; so also HAMBURGER, Zeit. wiss. Mik.,
xxv, 1908, p. 1 (2 vols. of the ink — " Perltusche " — to 3 of serum).
507. BJELOUSSOW'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 to 2 of gum arabic. Rub
up the transparent, almost insoluble mass with distilled water,
added little by little, then force it through a fine-grained cloth.
Repeat these operations until there is obtained a mass that is free
from 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, swelling up as above described, and conse-
quently 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.
508. Milk has been recently recommended by FISCHER (Centralb.
allg. Path., xiii, 1902, p. 277 ; Zeit. wiss. MiL, xx, 1903, p. 224).
It runs well, does not extra vasate, and can be used for auto-injection
of the living subject.
After injection it 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 1,000 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.
509. SCHIEFFERDECKER'S Celloidin Masses (Arch. Anat. Phys., 1882
[Anat. Abth.], p. 201). (For Corrosion preparations.)— See previous
editions ; HOCHSTETTER'S Modification of SCHIEFFERDECKER'S Mass
(Anat. Anz., 1886, p. 51) ; BUDGE'S Asphaltum Mass (Arch. mil". Anat.,
16
242 INJECTIONS— OTHER MASSES (COLD).
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 ; PANSCH'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 GAGE, Amer. Mon. Mic. Journ., 1888,
p. 195) ; TEICHMANN'S Linseed-Oil Masses (8. B. Math. Kl. Krakau
Akad., vii, pp. 108, 158 ; Journ. Roy. Mic. 8oc., 1882, pp. 125 and 716,
and 1895, p. 704); FLINT'S Celluloid (Amer. Journ. Anat., i, 1902,
p. 270) ; HUBER'S (ibid., vi, 1907, p. 393) ; KRASSTJSKAJA'S Photoxylin
(Anat., Heft. 2, xiii, 1904, p. 521).
510. Natural Injections (BoBiN, Traite, p. 6). — To preserve these
throw the organs into a liquid composed of 10 parts of tincture of
perchloride of iron and 100 parts of water.
RETTERER and ZENKER use solution of Miiller, see Journ. Anat.
Phys., 1894,. p. 336, and Arch. Path. Anat., 1894, p. 147.
511. Starch Masses. See " Guides for Vertebrate Dissection,"
Kingsley, New York, 1907.
CHAPTER XXIII.
MACERATION, DIGESTION, AND CORROSION.
Maceration.
512. Methods of Dissociation. — It is sometimes necessary, in
order to obtain a complete knowledge of the forms of the elements
of a tissue, that the elements be artificially separated from their
place in the tissue and separately studied after they have been
isolated both from neighbouring elements and from any interstitial
cement-substances that may be present in the tissue. Simple
teasing with needles is often insufficient, 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 mace-
rated tissue is placed on a slide and covered with a thin glass cover
supported at the corners on four little feet made of pellets of soft
wax. By tapping the cover with a needle it is now gradually pressed
down, whilst at the same time the cells of the tissue are 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 wax feet is obtained (VOSSELER,
Zeit. wiss. MiL, vii, 1891, p. 461) by melting white wax and stirring
into it one-half to two-thirds of Venice turpentine.
The most desirable macerating media are those which, whilst
dissolving intercellular substances, do not attack the cells them-
selves. Those which contain colloids have been found to give the
best results in this respect. Iodised serum is an example.
513. Iodised Serum (Chap. XIX.). — The manner of employing it
for maceration is as follows : A piece of tissue smaller than a pea
16—2
244 MACERATION, DIGESTION, AND CORROSION.
must be taken, and placed in 4 or 5 c.c. of weakly iodised serum in
a well-closed vessel. After one day's soaking the maceration is
generally sufficient, and the preparation may be completed by
teasing or pressing out, as indicated 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.
514. Iodide of Potassium (ARNOLD, Arch. mik. Anat., Hi, 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 iodine.
515. Alcohol. — RANVIER employs one-third alcohol (1 part of
90 per cent, alcohol to 2 parts of water). Epithelia 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 (J alcohol, for isolation of the
nerve-fibres of the retina, for instance — THIN).
516. Salt Solution. — Ten per cent, solution of sodium chloride is
a valuable macerating medium. Weaker strengths, down to 0-6 per
cent., are also used.
517. MOLESCHOTT and Piso BORME'S Sodium Chloride and Alcohol
(MOLESCHOTT'S V ntersuchungen zur Naturlehre, xi, pp. 99 — 107 ;
RANVIER, Traite, p. 242). — Ten per cent, solution of sodium chloride,
5 volumes ; absolute alcohol, 1 volume.
For vibratile epithelium RANVIER finds the mixture inferior to
one-third alcohol.
518. Sodium Chloride and Formaldehyde. — GAGE recommends the
addition of 2 parts of formalin to 1,000 parts of normal salt solution
(quoted from FISH, Proc. Amer. Mic. Soc., xvii, 1895, p. 328).
519. Caustic Potash, Caustic Soda. — These solutions should be
employed strong, 35 to 50 per cent. (MOLESCHOTT) ; so employed
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 epidermis, hairs, and nails.)
The strong solutions may be employed by simply treating the
tissues with them on the slide. To make permanent preparations,
the alkali should be neutralised by adding acetic acid, which forms
CHAPTER XXIII. 245
with caustic potash acetate of potash, which constitutes a mounting
medium (see BEHRENS, KOSSEL, and SCHIEFFERDECKER, Das
Mikroskop, i, 1889, p. 156). See also GAGE, Proc. Amer. Soc. of
Microscopists, 1889, p. 35.
520. Baryta-water, Lime-water (FoL, Lehrb., p. 110).— Baryta-water
will macerate nerve, muscle, and connective tissue in a few hours, lime-
water in a few days.
521. Sulphoeyanides 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.
SOULIER (Travaux de VInst. Zool.de Montpellier, Nouv. S6r., 2, 1891,
p. 171) has found that STIRLING'S solution greatly deteriorates cellular
elements, but that good results are obtained by combining it with a
fixing agent. The best results were obtained with a 2 per cent, solution
of sulphocyanide combined with liquid of KIP ART and PETIT ; 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 1-5 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.
522. LANDOIS'S Solution (Arch. mikr. Anat., 1885, p. 445).
Saturated sol. of neutral chromate of
ammonia ..... 5 parts.
Saturated sol. of phosphate of potash . 5 „
Saturated sol. of sulphate of soda . 5 „
Distilled water 100 „
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 1 volume of the macerating
liquid.
GIERKE particularly recommends this liquid for all sorts of
macerations, but especially for the central nervous system, for
which he finds it superior to all other agents. It is also recom-
mended for the same purpose by NANSEN (v. Zeit. wiss. Milk., v,
1888, p. 242).
523. Bichromate of Potash.— 0-2 per cent.
EISIG (Fauna u. Flora Golf. Neapel, 16 Monog., 1887, p. 297)
macerates Capitellidse in 0-5 to 1 per cent, solution for months or
years, a little thymol being added against mould.
Miiller's Solution, diluted to same strength, or combined with
saliva, has also been used.
BROCK (for nervous system of Mollusca, Intern. Monatssch. Anat.,
246 MACERATION, DIGESTION, AND CORROSION.
i, 1884, p. 349) takes equal parts of 10 per cent, solution of bichro-
mate of potash and visceral fluid of the animal.
524. Permanganate of Potash is recommended, either alone or
combined with alum, as the best dissociating agent for the fibres of
the cornea (KOLLETT, Strieker's Handbuch, p. 1108). I have found
it, for some objects, very energetic.
525. 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 millimetres of
the tissue (RANVIER).
526. Osmic and Acetic Acid (the HERTWIGS, Das Nervensystem u.
die Sinnesorgane der Medusen, Leipzig, 1878, and Jen. Zeitschr.,
xiii, 1879, p. 457).
0-05 per cent, osmic acid 1 part.
0-2 „ acetic acid . . . 1 „
Medusce are to be treated with this mixture for two or three
minutes, according to size, and then washed in repeated changes
of 0-1 per cent, acetic acid until all traces of free osmic acid are
removed ; they then remain for a day in 0*1 per cent, acetic acid,
are washed in water, stained in BE ALE'S carmine, and preserved in
glycerin.
For Actinice 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 0-2 per cent, acetic acid. If the maceration is complete,
stain with picro-carmine ; if not, with BEALE'S carmine.
527. MOBIUS'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, 0-1 per cent, osmic acid, 0-1 per cent,
acetic acid, dissolved in sea water. For Lamellibranchiata. Macerate
for several days.
528. Nitric Acid. — Most useful for the maceration of muscle.
The strength used is 20 per cent. After twenty-four hours' macera-
tion in this, isolated muscle-fibres may generally be obtained by
shaking the tissue with water in a test-tube. Preparations may
afterwards be washed with water and put up in strong solution of
alum, in which they may be preserved for a long time (HOPKINS,
Proc. Amer. Soc. of Microscopists, 1890, p. 165).
Maceration is greatly aided by heat, and at a temperature of
40° to 50° C. may be sufficiently complete in an hour (GAGE).
CHAPTER XXIII. 247
A mixture of equal parts of nitric acid, glycerin, and water is
recommended by MARCACCI (Arch. Ital. Biol, iv, 1883, p. 293) for
smooth muscle.
529. Nitric Acid and Chlorate of Potash (KUHNE, Ueber die peri-
pherischen Endorgane, etc., 1862 ; RANVIEE, Traite, p. 79).—
Chlorate of potash is mixed, in a watch-glass, with four times its
volume of nitric acid. A piece of muscle is buried in the mixture
for half an hour, and then agitated with water in a test-tube, by
which means it entirely breaks up into isolated fibres.
530. Nitric and Acetic Acid (APATHY, Zeit. wiss. Mik., x, 1898, p. 49).
— 3 volumes 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
after twenty-four hours, 50 per cent, glycerin, changed till the acid is
removed.
531. Hydrochloric Acid.— KONIGSTEIN (Sitzb. Akad. Wien, Ixxi, 1875)
takes (for gold-impregnated cornese) equal parts of the concentrated
acid, glycerin, and water ; FREUD (ibid., Ixxviii, 1879, p. 102, for nerve-
impregnations), 10 parts of acid, 7 of water, 3 of glycerin ; and SCHUBERG
and SCHRODER (Zeit. wiss. Zool., Ixxvi, 1904, p. 516) take (for fresh
muscles of Hirudinea) hydrochloric acid of 5 per cent.
532. BELA HALLER'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.
533. Sulphuric Acid (RANVIER, Traite, 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.
ODENIUS found very dilute sulphuric acid to be the best reagent
for the study of nerve endings in tactile hairs. He macerated hair-
follicles for from eight to fourteen days in a solution of from 3 to
4 grs. of " English sulphuric acid " to the ounce of water.
Hot concentrated sulphuric acid serves to dissociate horny
epidermic structures (horn, hair, nails).
534. Oxalic Acid. — Maceration for many days in concentrated
solution of oxalic acid has been found useful in the study of nerve-
endings.
535. SCHIEFFERDECKER'S Methyl Mixture (for the retina) (Arch,
mik. Anat., xxviii, 1886, p. 305).— Ten parts of glycerin, 1 part of
248 MACERATION, DIGESTION, AND CORROSION.
methyl alcohol, and 20 parts of distilled water. Macerate for
several days (perfectly fresh tissue).
536. GAGE'S Picric Alcohol (Proc. Amer. Soc. of Microscopists,
1890, p. 120). — Ninety-five per cent, alcohol, 250 parts ; water,
750 ; picric acid, 1. Recommended especially for epithelia and
muscle. A few hours suffice.
537. Chloral Hydrate. — In not too strong solution, from 2 to 5 per
cent, for instance, chloral hydrate is a mild macerating agent that
admirably preserves delicate elements. LAVDOWSKY (Arch. mik.
Anat., 1876, p. 359) recommends it greatly for salivary glands,
HICKSON (Quart. Journ. Mic. Sci., 1885, p. 244) for the retina of
Arthropods.
538. Lysol (REINKE, Anat. Anz., viii, 1892, p. 582).— Ten 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.
539. 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 carbo-
hydrates 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.
540. Pepsin (BEALE'S, Archives of Medicine, i, 1858, pp. 296—
316). — The mucus expressed from the stomach glands of the pig is
rapidly dried on glass plates, powdered, and kept in stoppered
bottles. Eight- tenths of a grain will dissolve 100 grs. of coagulated
white of egg.
CHAPTER XXIII. 249
*
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 kept for some
hours in the liquid at a temperature of 100° F. (37° C.).
BRUCKE'S (from CARNOY'S Biologie cellulaire, p. 94).
Glycerinated extract of pig's stomach . 1 volume.
0-2 per cent, solution of HC1 . . 3 volumes.
Thymol, a few crystals.
BICKPALVI'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.
KUSKOW'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 Nuchse) remain in it at the ordinary tem-
perature for ten to forty minutes.
541. Pancreatin. — SCHIEFFERDECKER'S (Zeit. wiss. Mik., iii,
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.
KUHNE'S (Unters. a. d. Phys. Inst. Univ. Heidelberg, i, 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 HOEHL, Arch. Anat. Phys.,
Anat. Abth., 1897, p. 136 (£ to f 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).
Corrosion.
542. 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 applied
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 REJSEK (Bibliogr. Anat., iv, 1897, p. 229) ;
BRUHL (Anat. Anz., xiv, 1898, p. 418) ; DENKER (Anat. Hefte., 1900,
p. 300) ; THOMA and FROMHERZ (Arch. Entwickelungsmech, vii,
1898, p. 678) ; PEABODY (Z. Bull, Boston, 1897, p. 164). The
following sections relate chiefly to the cleansing of native hard
parts.
250 MACERATION, DIGESTION, AND CORROSION.
543. 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.).
544. Eau de Javelle (Hypochlorite of Potash) (NOLL, Zool. Anzeig.,
cxxii, 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.
545. Eau de Labarraque (Hypochlorite of Soda) may be used in
the same way as eau de Javelle. Looss (Zool. Anzeig., 1885,
p. 333) finds that either of these solutions will completely dissolve
chitin in a short time with the aid of heat. For this purpose the
commercial solution should be taken concentrated and boiling.
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 dissolved, 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, ett.
CHAPTER XXIV.
DECALCIFICATION, DESILICIFICATION, AND BLEACHING.
Decalcification.
546. 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 re-
moved by washing, not in water, which favours swelling, but in
some liquid that has rather a shrinking action, e.g., alum solution.
Lastly, the tissues should be neutralised by treatment with car-
bonate 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 out the acid in alcohol containing precipitated carbonate of
lime, then cuts sections. This for Porifera, corals, Echinoderms,
etc. Tissues are said to be well preserved.
This process has been applied to the study of the temporal bone
of Mammals by STEIN (Anat. Anz., xvii, 1900, p. 318).
Similarly BODECKER (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.
547. Decalcification of Bone.— I take the following from BUSCH :
Arch. mik. Anat., xiv, 1877, p. 481 ; see also HAUG, in Zeit. wiss.
Mik., viii, 1891, p. 1 ; and SCHAFFER, ibid., xix, 1903, pp. 308 and
441, and his paper in the Encycl. mik. Technik.
The most widely used, though not the best, agent for decalcifica-
tion is hydrochloric acid. Its action is rapid, even when very
252 DECALCIFICATION, DESILICIFICATION, ETC.
dilute, but causes serious swelling of the tissues. To remedy this,
chromic acid or alcohol may be added to it. Or a 3 per cent,
solution of the acid may be taken and have dissolved in it 10 to
15 per cent, of common salt. Or (WALDEYER) to a TQ\JO per cent,
solution of chloride of palladium may be added T\y of its volume of
HC1.
Chromic acid is also much used, but has a very weak decalcifying
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 decalci-
fying power, but cause great swelling. Picric acid has a very slow
action,, and is only suitable for very small structures.
548. Nitric Acid (Buscn, loc. cit.). — To all other agents BUSCH
prefers nitric acid, which causes no swelling and acts most effica-
ciously.
One volume of chemically pure nitric acid of sp. gr. 1-25 is diluted
with 10 volumes water. It may be used of this strength for very
large and tough bones ; for young bones it may be diluted down
to 1 per cent.
Fresh bones are first laid for three days in 95 per cent, alcohol ;
they are then placed in the nitric acid, which is changed daily, for
eight or ten days. They must be removed as soon as the decalcifica-
tion is complete, or else they will become stained yellow. When
removed they are washed for one or two hours in running water and
placed in 95 per cent, alcohol. This is changed after a few days for
fresh alcohol.
Young and foetal bones may be placed in the first instance in a
mixture containing 1 per cent, bichromate of potash and ^ 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
(T^ Per cen"t.) or bichromate of potash (1 per cent.). By putting them
afterwards into alcohol a green stain is obtained.
549. Nitric Acid (^CHAFFER, Zeit. wiss. Mik., xix, 1903, p. 460).—
SCHAFFER 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
CHAPTER XXIV. 253
be well fixed and imbedded in celloidin (§ 546) ; harden in alcohol ;
remove the alcohol with water ; put for twelve to twenty-four 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 twelve to twenty-four hours ; running water,
forty-eight hours ; alcohol .
550. Nitric Acid and Alcohol. — Three 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 (FiSH, Ref. Handb. Med. Sri., Supp., p. 425).
THOMA (Zeit. wiss. Mik., viii, 2, 1891, p. 191) takes 5 volumes 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 tra.ce of acid is removed (i.e., for some days after no acid
reaction is obtained with litmus paper) in 95 per cent, alcohol
containing an excess of precipitated carbonate of lime. This may
take eight to fourteen days, after which the tissues will stain well
and may be treated as desired.
551. Nitric Acid and Formol. — SCHRIDDE (Hcematol. 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.
552. Nitric Acid and Alum (GAGE, quoted from FISH, § 550}. — A
saturated aqueous solution of alum is diluted with an equal volume
of water, and to each 100 c.c. 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.
553. Sulphurous Acid (ZIEGLER, Festschr.f. Kupffer, 1899, p. 51).
— A saturated solution in water. Wash out for twenty-four hours.
Acts rapidly and preserves well. Best used after fixation with
formol.
554. Hydrochloric Acid (see § 547).— RANVIER says that it may be
taken of 50 per cent, strength, and then has a very rapid action. To
counteract the swelling action of the acid, sodium chloride may be added
(VON EBNER), see HAUG'S paper quoted § 547. He takes either 100 c.c.
254 DECALCIFICATION, DESILICIFICATION, ETC.
cold saturated solution of sodium chloride in water, 100 c.c. water, and
4 c.c. hydrochloric acid. Preparations to be placed in this, ajid 1 to 2 c.c.
hydrochloric acid added daily until they are soft. Or, 2-5 parts of
hydrochloric acid, 500 of alcohol, 100 of water, and 2-5 of sodium chloride.
HAUG prefers the proportions of 1-0 to 5-0 of acid, 70 of alcohol, 30 of
water, and 0-5 of salt.
555. 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. HAUG recommends equal
parts of 1 per cent, hydrochloric acid and 1 per cent, chromic acid
(loc. cit.).
556. Hydrochloric Acid and Glycerin.— Glycerin, 95; hydrochloric
acid, 5 (SQUIRE'S Methods and Formulas, p. 12).
557. Trichloracetic Acid. — PARTSCH (Verh. Ges. D. Naturf.
Aertze, 1895, 2 Theil, 2 Halfte, p. 26) uses a 5 per cent, aqueous
solution, and NEUBERGER (Centralb. Phys., xi, 1897, p. 494) a
4 per cent. one. Action energetic, preservation said to be excellent.
558. Picric Acid should be taken saturated arid changed frequently.
Its action is weak, but it gives good results with small objects..
Picro-nitric or Picro -hydrochloric Acid. — Action very rapid.
559. Phosphoric Acid. —10 to 15 per cent. (HAUG, loc. cit. in §547).
Somewhat slow, staining not good. According to SCIIAFFER, §549, it
produces swelling.
560. Lactic Acid. — 10 per cent, or more. Fairly rapid, preserves well,
and may be recommended (HAUG, loc. cit.).
561. Chromic Acid is employed in strengths of from 0-1 per cent, to
2 per cent, (but see § 547), 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.
562. Chromic and Nitric Acid.— SEILER (FoL, Lehrb., p. 112) takes
70 volumes of 1 per cent, chromic acid, 3 of nitric acid, arid 200 of water.
The action is still excessively slow, frequently requiring months to be
complete.
563. Chromo-aceto-osmic Acid (VAN VER STRICHT, Arch. Biol., ix,
1889, p. 29 ; and SCHAFFER, Zeit. wiss. Mik., x, 1893, p. 179).—
Objects to be left in it for months, the liquid being changed at first
every two days, afterwards less frequently. Structure well pre-
served.
564. Arsenic Acid. — 4 per cent, aqueous solution, used at a tempera-
ture of 30° to 40° C. (SQUIRE'S Methods and Formula, etc., p. 11).
565. Phlorogluein with Acids (ANDEER, Centralb. med. Wiss., xii,
xxxiii, pp. 193, 579 ; Intern. Monatsschr., i, p. 350 ; HAUG, Zeit. wiss.
CHAPTER XXIV. 255
.y viii, 1891, p. 8 ; FERRERI, ibid., ix, 1892, p. 236 ; Bull. R. Acad.
Med. di Eoma, 1892, p. 67).— This is said to be the most rapid method
ot 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 HAUG,
Zeit. wiss. Mile., viii, 1891, p. 8, and FERRERI, Bull. Acad. Med. Eoma,
1892, p. 67, or (for both) fifth edition.
Desilicification.
566. Hydrofluoric Acid (MAYER, Zool Anz., 1881, p. 593).— The
objects are brought in alcohol into a glass vessel coated internally
'with paraffin. Hydrofluoric acid is then added drop by drop
(taking great care to avoid the fumes, which attack mucous mem-
branes 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 dangerous 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 before the
knife.
KOUSSEAU imbeds the objects in celloidin, as described § 549, then
brings the block, in a covered caoutchouc dish, for a day or two into a
mixture of 50 c.c. alcohol and 20 to 30 drops of hydrofluoric acid, and
washes out the acid with alcohol containing carbonate of lithia in
powder.
Bleaching.
567. 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 2 or 3 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 alcohol 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 hydro-
chloric 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
256 DEGALOIFIGATION, DESILICIFICATION, ETC.
material that has been blackened by osmic acid, and, according to
renewed experiments of MAYER'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 Zeit. wiss. Mile., xxiv, 1907, p. 353 (paraffin sections
exposed to the vapour of chlorine water).
GRYNFELTT and MESTREZAT (C. R. Soc. Biol., Ixi, 1906, p. 87) add
2 c.c. of 20 per cent, solution of chloric acid (HC103) to 15 c.c. of alcohol
and put sections (of retina) into it for several hours at 42° C.
568. Eau de Labarraque. Eau de Javelle (see §§ 544, 545).— 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. Mic.
Soc., 1887, p. 518. Of course, the method cannot be used for bleaching
soft parts which it is desired to preserve.
569. Peroxide of Hydrogen (Oxygenated Water) (POUCHET'S
method, M. DUVAL, Precis, 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. SOLGER (Centralbl. med. Wiss., xxi,
1883, p. 177) takes a 3 per cent, solution of peroxide. FURST
(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.
570. Peroxide of Sodium (CARAZZI, Zool. Anz., 444, 1894, p. 135).—
See previous editions.
571. Peroxide of Magnesium (MAYER, Grundzuge, p. 290).— Use as
chlorine, § 567. A slow but delicate method.
572. Sulphurous Acid. — Prof. GILSON writes me that he finds
alcoholic solution of sulphurous anhydride (S02) very convenient
for the rapid decoloration of bichromate objects. A few drops suffice.
MONCKEBERG and BETHE (Arch. mik. Anat., liv, 1899, p. 135)
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.
573. 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 ot 1 ; 300
strength, or weaker.
CHAPTER XXIV. 257
574. GREN ACKER'S Mixture for Eyes of Arthropods and other Animals
(Abh. nat. Ges. Halle-a.-S., xvi ; Zeit. wiss. Mik., 1885, p. 244).
Glycerin . . . . . .1 part.
80 per cent, alcohol . . . .2 parts.
Mix and add 2 to 3 per cent, of hydrochloric acid.
Pigments (i.e. 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.
575. Nitric Acid. — PARKER (Bull. Mus. Comp. Zool., Cambridge,
U.S.A., 1889, p. 173) treats sections (of eyes of scorpions) fixed to the
slide with SCIIALLIBAUM'S medium, for about a minute with a solution of
up to 50 per cent, of nitric acid in alcohol, or, still better, with a 35 per
cent, solution of a mixture of equal parts of nitric and hydrochloric acid
in alcohol. To make the solution, the acid should be poured slowly into
the alcohol (not vice versa), and the mixture kept cool.
JANDER (Zeit. wiss. Mik., xv, 1898, p. 163) takes for removal of pig-
ments SELLER'S chromo-nitric acid (§ 562) ; twelve to forty-eight hours
is enough for small objects.
See also under " Arthropoda."
576. Caustic Soda. — RAWITZ (Leitfaden, 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.c. of 96 per cent, alcohol.
17
CHAPTER XXV.
EMBRYOLOGICAL METHODS.*
577. New Advances. — In nearly every case the newest advances
in ordinary embryological technique are constituted by the improve-
ments in fixation reported in the new sections on Cytology (§ 681).
It would be a great mistake for observers to consider that fixation
and staining methods, such as those of Champy-Kull, Kopsch, or
Flemming-without-acetic acid, and iron hsematoxylin, are of no
concern to embryologists. For instance, amphibian embryos, such
as those of Triton (Molge) prepared by Champy-Kull's method are
extremely beautiful and instructive for study, for not only does one
procure cytological perfection, but also a staining which is poly-
chromatic. For the study of invertebrate embryology, the mito-
chondrial methods open up a new field for research.
The reliability of many of the new neurological methods (see
§§ 865 to 921) has been brought to a state which should now induce
embryologists to devote even rare material for preparation, and to
use the neurological methods much more widely than at present is
the case.
In § 768 is a special treatment of the study of fats and lipoids,
which can readily be used for embryological studies. In § 646 is
a section on " Glycogen," and in § 650 one on " Iron and Copper."
In §§ 1035—1045 is a Chapter on the " Tissue Culture " methods.
In various parts of the book further notes on intra vital staining
have been inserted.
578. Artificial Fecundation. — This practice, which affords the
readiest means of obtaining the early stages of development of many
animals, may be very easily carried out in the case of the amphibia
* The sections in this chapter treating of Mammalia, Aves, and Pisces,
closely follow the Traite des Methodes Techniques, LEE et HENNEGUY,
and are due almost entirely to HENNEGUY. The corresponding parts
of the Grundzuge, LEE and MAYER, are taken from this work, and there-
fore also due to HENNEGUY, which Dr. Lee regrets to observe has not
always been understood, though duly pointed out in the Preface to the
first edition of the Grundzuge. This foot-note doss not apply to any
new material introduced into the present edition (J. B. G.).
CHAPTER XXV. 259
Anura, Teleostea, Cyclostomata, Echinodermata, 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 occasionally 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 Salmonidae, lose their vitality very rapidly in water ;
it is, therefore, advisable to add the milt immediately to the spawned
ova, then add a little water, and after a few minutes put the whole
into a suitable hatching apparatus with running water.
Artificial fecundation of Invertebrates is easily performed in a
similar way. For methods of artificial Parthenogenesis see HARVEY,
Biol Bull Wood's Hole, 1910, p. 269.
579. Superficial Examination. — The davelopment 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, etc.
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 impreg-
nated with the oil, the normal course of development is not interfered
with (BALBIANI).
580. Fixation. — Osmic acid, employed either alone or in com-
bination with other reagents, is an excellent fixing agent for small
embryos, but not at all a good one for large ones. It causes cellular
elements to shrink somewhat, and therefore brings out very clearly
the slits that separate germinal layers, and any channels or other
cavities that may be in course of formation.
In virtue of its property of blackening fatty matters, myelin
amongst them, it is of service in the study of the development of
the nervous system.
Chromic acid is indispensable for the study of the external forms
17—2
260 EMBRYOLOGICAL METHODS.
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 somewhat, 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.
SCHKIDDE (Zeit. wiss. Mik., xxvii, 1910, p. 362) finds Orth's
" Formol-Muller " in general the best fixative. Fix for not more
than twenty-four hours, and pass through graded alcohols (twenty
minutes in each) into absolute (one to two hours), cedar oil, xylol,
and paraffin.
RABL (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 blastoderms and young
embryos (Pisces, Amphibia, Aves, Mammalia). 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 prolonged
fixation in a mixture of
Platinic chloride, 1 per cent, solution . 1 volume.
Picric acid, saturated aqueous . . 2 volumes.
Distilled water . . . . . 7 „
RABL'S picro-sublimate mixture has been given § 70. 1
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. Wurzburg, 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). SOBOTTA (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.
581. PETER'S Double-stain for Yolk and Tissue, see § 224.
582. Removal of Albumen. — The thick layers of albumen that
surround many ova are a serious obstacle to the penetration of
reagents. CHILD (Arch. Entwickelungsmech., ix, 1900, p. 587)
gives the following as of very general applicability. After fixation
CHAPTER XXV. 261
(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.
583. 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 described
by SCHAFFER (Zeit. wiss. Mik., vii, 1890, p. 342). Careful outlines
of the sections to be constructed are drawn on tracing paper
with the aid of the camera lucida, superposed, 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 examination. KERR (Quart. Journ. Mic. Sci., xlv,
1902, p. 1) draws on plates of ground glass which he afterwards
superposes and makes transparent by oil of cloves run in between
them. PENSA (Zeit. wiss. Mikr., xxvii, 1910, p. 48) takes sheets
of lithographic gelatin. WOODWORTH (Zeit. wiss. Mik., xiv, 1897,
p. 15) proceeds as follows : (1) Draw an axial line of the length of
the object multiplied by the magnification employed. (2) Measure
with a micrometer the greatest diameter of each section. (3) Plot
these diameters down transversely on the axial line at distances
corresponding 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, i.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 same magnification)
are pasted on pieces of cardboard of a thickness equal to that of
the sections multiplied by the magnification employed. Then the
parts of the drawings representing the cavities of the objects are
262 EMBRYOLOGICAL METHODS.
cut out with a knife or fretsaw, cutting through the cardboard ; and
the pieces of fretwork thus obtained are pasted together.
Many useful modifications of this method have been devised.
Cardboard is rather hard to cut, and not conveniently got of the
required thickness. Professor Arthur Thompson, of Oxford, uses
numbers of sheets of blotting paper to the required thickness,
soaked in beeswax ; this makes a very tough substance, and the
models, when made, can be handled without chance of injury ;
other workers use beeswax plates alone, drawing the outline with
some sharp instrument and cutting out with a hot knife.
Mr. Pittock, of the Zoological Laboratory, University College,
London, uses a modification of K. Peter's method (vide infra).
Rather thin paper is used for drawing the outline of the object. In this
laboratory (Professor J. P. Hill), special rolls of paper are used, so
that the diagram of each of hundreds of sections may be safely
rolled up in order till wanted. A large flat stone is used for the
manufacture of the wax plates, with two brass gauges of the required
thickness placed at a distance which will accommodate in between
them the square of paper with the drawing. Instead of treating the
paper with turpentine, according to Mr. Pittock's method the
drawing is rapidly floated over the surface of a dish of water, drawing
side down, then laid upon the stone, between the metal gauge and
the superfluous moisture smoothed off with a sheet of blotting
paper. The melted wax is poured on to the paper, and a heated
metal roller passing over the metal gauge leaves just the required
amount of wax on the paper. The latter easily peels off the surface
of the stone.
For more elaborate processes of plastic reconstruction (very compli-
cated and seldom necessary) see BORN, " Die Plattenmodellirmetliode,"
in Arch. mik. Anat., 1883, p. 591, and Zeit. wiss. Mik., v, 1888, p. 433 ;
STRASSER, ibid., iii, 1886, p. 179, and iv, pp. 168 and 330 ; KASTSCHENKO,
ibid., iv, 1887, pp. 235-6 and 353, and v, 1888, p. 173 ; SCHAPER, ibid.,
xiii, 1897, p. 446 ; ALEXANDER, ibid.t p. 334, and xv, 1899, p. 446 ;
PETER, ibid., xxii, 1906, p. 530 ; BORN and PETER, ibid., xv, 1, p. 31 ;
and Verh. Anat. Ges., xiii, 1899, p. 134 ; JOHNSTON, Anat. Anz., xvi,
1899, p. 261 ; FOL, Lehrb., p. 35 or previous editions ; BROMAN, Anat.
Hefte, xi, 1899, p. 557 : PETER. "Die Methoden d. Rekonstruction "
(Fischer, Jena, 1906) ; SCHONEMANN, Anat. Hefte, xviii, 1901, p. 117 ;
GAGE, Anat. Record, i, 1907, p. 167 ; NEUMAYER, Festschr. f. Kupffer,
1899, p. 459 ; MARK, Proc. Amer. Acad. Sci., xiii, 1907, p. 629 (electric
wax-cutter for cutting out plates).
HILL (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.
CHAPTER XXV. 263
Mammalia. *
584. Times for Early Development. — The entry of the sperm into
the egg of the mouse takes place from six to ten hours after copula-
tion (SOBOTTA, Arch. mikr. Anat. Bd., 45). The pronuclei stage of
fertilisation is found from eighteen to twenty-two hours, two-cell
stage twenty-six hours, four-cell, fifty hours, eight-cell, sixty hours
after copulation : the egg remains in the tube about eighty hours.
J. A. LONG and E. L. MARK (Contrib. Zool. Lab. Museum, Harvard,
Carneg. Inst. Wash., No. 142, 1911) find in the mouse that ovarian
eggs within fifteen or sixteen hours after parturition have formed
the first maturation spindle. Fertilised eggs are obtained from
animals killed between twenty-three and thirty-one hours post
partum. The time required for the spermatozoa, after introduction
into the uterus (either artificially or by coitus) to reach the eggs in
the first part of the oviduct varies from four to seven hours in mice
inseminated about the same number of hours post partum. To
obtain free eggs for study, Mark and Long kill mice fourteen to
seventeen hours after parturition, the ova being found in a fold of
the oviduct.
In the rat the eggs are found in the oviduct about 18-7 hours
and ovulation occurs in less than eighteen hours post partum.
In the rabbit the pronuclei stage of fertilisation occurs about
fourteen hours, in the guinea-pig, twenty-two to twenty-four hours
after copulation (SOBOTTA). The rabbit's egg, like that of the
guinea-pig, remains about eighty hours, the dog's egg eight to ten
days in the tube (RoTHiG, Embryol. Technik).
Condition of Ovary as Index to Pregnancy. — On opening the body
cavity of a mammal, first of all examine the ovary. By so doing
one can estimate roughly the time that has elapsed since the dis-
charge of the ovum or ova. Prominent stigmata or areas with a
blood-shot centre indicate recent ovulation, while a smooth surface
of yellowish appearance indicates a corpus luteum, which means
that some time has elapsed since ovulation.
585. Isolation of the Eggs and Early Stages.— The tubse and uterus
or uteri are dissected out and treated in one of two ways : either
the isolated tuba after straightening is washed out from the funnel
opening with warm salt solution, or with some fixative like formalin
or weak osmic acid, or on the other hand the whole length of the
tube is laid open and spread out with a scalpel or sharp scissors and
needles, and the eggs are looked for under a dissecting microscope.
* Revised by J. B. G.
264 EMBRYOLOGICAL METHODS.
If the method of washing out is adopted, it is best to use a good
rubber bulb attached to a glass tube which has been drawn out
finely enough to pass into the oviducal opening. Kolliker used
Miiller solution or weak osmic acid for injection, collecting the
fluid in a series of watch glasses ; J. P. Hill uses solid crystal dishes,
which can easily be examined under a stereoscopic binocular
microscope. As a fluid for washing out Hill's picronitric osmic
(vide infra), weak formalin, or weak osmic acid are probably as
good as anything. The success of this injection method depends
on the amount of mucous in the tuba and on the condition of the
folds in its mucosa ; if the eggs are not found after the injection,
the walls of the tube may be opened up with scissors and the lining
scraped away with a small scalpel ; the mucus thus procured may be
diluted with a little indifferent fluid and examined on a slide under the
microscope. Both operations of injection or of opening the tuba
may succeed with comparatively large animals like the rabbit and
dog. It is practically impossible to slit open the tuba of the cat.
In cases where the subject is small, as, for instance, the mouse,
it is necessary to preserve the whole oviduct and use a fixative
sufficiently penetrative to act quickly. Even with the guinea-pig
the lumen of the tube is so small that it is difficult to remove the
ova ; we consider that attempts to press out the contents of the
tubes are dangerous. In such cases it seems better to cut the
tube into lengths with a razor and to fix whole (vide infra). BISCHOFF
in his study on the guinea-pig (Giesson, 1852), and BALLOWITZ
(Arch. Anat. Physiol, 1883) both resorted to the method of squeezing
out the contents of the tubes.
When found the ova are picked up with the point of a cataract
needle or a scalpel, on a piece of black paper cut to a point, or
with a pipette, and either examined fresh in the peritoneal fluid or
blood serum of the animal, or in Kronecker's or other artificial
serum media, or better fixed immediately.
In the case o± a large animal such as the rabbit, 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
CHAPTER XXV. 265
is desired to study these the animal may bo killed, or may be subjected
to a secondary laparotomy if it be desired to preserve it for ulterior
observations. This method, however, cannot be carried out in this
country owing to the Vivisection Acts.
This procedure was also adopted by Hartmann in his study on
Didelphys (vide infra).
586. Fixation of the Isolated Ova. — These can be fixed in a chrome-
formalin fluid of some kind : Muller-formol, Helly, Zenker- without-
acetic acid and formol are indicated. Eggs may be left in one of
these fluids overnight, then washed in distilled water and transferred
either to 1 per cent. Os04, or to some chrome-osmic fluid, this to
preserve the fat. The chrome fixation will form insoluble compounds
with lipoids, but less so with fats of the type of olein. It seems
likely that the fixation technique of Champy-Kull, of Schridde and
of Murray (see § 689) will be of great value.
For a study of the Golgi elements the methods of Cajal and
Da Fano and of Mann-Kopsch are worthy of trial, but rather more
difficult to work than chrome-osmic or chrome-formol techniques.
Where there may be a difficulty of penetration chrome-formol
fluids will be found better than chrome-osmium. A perusal of the
sections on Mitochondria and Golgi apparatus will provide sugges-
tions for the treatment of the early stages in mammalian develop-
ment. VAN BENEDEN (Arch, de Biol, 1880, p. 149) brings the living
ovum into a drop of 1 per cent. Os04 on a slide, and thence into a
solution of Miiller. 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 glycerine of gradually increasing
strength, and at last mounted in pure glycerine acidified with formic
acid. I am inclined to believe that the Champy-Kull or Regaud
fixation (the latter with a post-osmication) would be much superior
to the above method, that is, for sectioning.
Many authors have used picro-nitric, picro-sulphuric, picro-
formol with or without corrosive, chromic-acetic acid, Flemming
and Hermann, and so on, but one cannot help thinking that the
more modern and logical fixation methods will be better. This
seems borne out by the late work of LAMS (Arch, de Biol., t., xxiii),
and LEVI (Arch.f. Zellf., xiv).
J. P. HILL (Quart. Jour. Micr. Soc., 1910) gives the formula of
a " Marsupial mixture " for fixation of ova and blastocysts of Mar-
supials. This fluid is made by adding to 96 c.c. of Mayer's picro-
nitric, 2 c.c. of 1 per cent. Os04. Two c.c. of glacial acetic acid
may be added, but the picric acid is sufficiently penetrative without
the addition of acetic acid.
266 EMBRYOWGICAL METHODS.
J. A. LONG (Contrib. Zool. Lab. Museum Compar. Zool. Harvard, 1912)
describes an ingenious constant temperature box for working with fresh
egg of mammalia. A circulation slide is also described in detail. So far
J. A. Long has succeeded in keeping mice eggs alive and under observa-
tion for only twelve hours.
J. A. LONG and E. L. MARK (op. cit.) use a modified Zenker for their
study on mouse eggs. They fix for from twenty to sixty minutes.
(A) 4 per cent, bichromate of potash. (B) 4 per cent. (aq. sol.) sublimate
and 20 per cent, acetic acid. For use, mix equal portions of A and B.
Wash out in warm water for twelve to fourteen hours, 70 per cent,
alcohol and iodine twelve to fourteen hours, quickly dehydrate, clear
in xylol and embed in paraffin. Mark and Long's fixative appears to
me (on paper at least) to be far too acid. It may be indicated for
chromosome work.
587. Subsequent Treatment of Ova. — After fixation the eggs or
blastocysts should be brought into 30 per cent, alcohol and slowly
upgraded to 90 per cent, alcohol : at this stage they may be stuck
on pieces of liver or brain by MINCHIN'S albumen method ; the egg
is placed on the liver and albumen is gently pipetted over it. The
alcohol coagulates the albumen, and enables the object to be
handled more easily. Another method used by J. P. HILL (Quart.
Jour. Micr. Science, 1910) is to bring the ova into alcohol absolute
and then into equal parts of alcohol absolute and ether. Then
take a hand-cut section of liver or brain (which has been stored in
absolute) place 1 drop of 0-5 per cent, solution of photoxylin (or
celloidin) in equal parts of absolute alcohol and ether ; then transfer
the egg on a flat camel hair brush to this drop, and harden the object
in 15 per cent, chloroform in 90 per cent, alcohol. Transfer to
equal parts of absolute alcohol, xylol and chloroform. Then equal
parts of chloroform and xylol, and embed in paraffin wax.
The process of sticking the eggs to the hard cut liver or brain section
should be carried out under a dissecting microscope.
588. Uterine Eggs. — During the fourth, fifth, and sixth days after
copulation the ova of the rabbit are free in the uterine cornua ; they
are easily visible to the naked eye, and may be extracted by the
same manipulations as those of the tubes. After the sixth day
they are at rest in the uterus, but have not yet contracted adhesions
with the mucosa, so that they can still be extracted whole. At this
stage the parts of the cornua where the ova 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
CHAPTER XXV. 267
by means of two pins on the bottom of a dissecting dish, with the
mesometrial surface downwards and the ovular eminence upwards.
The dissecting-dish is then filled up with serum or liquid of MULLER,
or 0-1 per cent, solution of osmic acid, Bouin's fluid, Hill's fluid,
Helly's fluid or 10 per cent, formol. See sections on " Cytology,"
§§ 673 to 696. With a small scalpel a longitudinal incision is made
on the surface of the ovular eminence, not passing deeper than the
muscular layer ; the underlying uterine mucosa is then gently
dilacerated with two pairs of small forceps, and the ovum set free
in the liquid.
From the moment the ova have become adherent to the uterine
mucosa they can no longer be extracted whole. The embryo being
always situated on the mesometrial surface, the ovular eminence
is opened by a crucial incision, and the strip of mucosa to which the
embryo remains adherent is fixed with pins on the bottom of the
dish. ED. v. BENEDEN (see Arch, de BioL, v, fasc. iii, 1885, p. 378)
has been able by operating in this way in serum of Kronecker, and
keeping the whole at blood temperature, to observe the circulation
of the embryo for hours together. (If this be desired to be done,
the crucial incision should not be too extended, so as to leave the
terminal sinus intact.)
RETTERER (C. R. Soc. de BioL, 1887, p. 99) advises that for ova
of the seventh day the segment of uterus containing them be opened
on 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.
C. Gr. HARTMANN (Jour. Morph., 1916), in his study of the develop-
ment of the opossum, used Carney's, Bouin's, Fleming's and Hill's
fluids. He found Hill's " Marsupial mixture " a perfect fixing fluid
for marsupial eggs. J. P. Hill now recommends leaving out the
acetic acid for delicate objects.
589. Blastoderms and Later Embryos. — The routine methods of
embryology apply here in general. Great care must be exercised
to avoid rough treatment caused by upgrading the object too
quickly. The same remark applies even more particularly to clear-
ing, which to get the best result should be done very gradually.
In order to bring out the outlines of blastoderm cells the living
ovum may be brought into ^ per cent, solution of nitrate of
silver. After remaining there for half a minute to two minutes,
according to the age of the vesicle, it is brought into pure water
and exposed to the light. The preparations thus obtained are
268 EMBRYOLOGICAL METHODS.
instructive, but blacken rapidly, and cannot be permanently
preserved.
The blastodermic vesicle 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, refer to
" Cytology," §§ 673—696. KOLLIKER recommends putting the
ovum into 0-5 per cent, solution of osmic acid until it has taken
on a somewhat dark tint, which happens in about an hour, and then
treating it with successive alcohols for several hours. If the ovum
be 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. The blasto-
dermic vesicle can then easily be removed, and further treated as
before. For sections KOLLIKER fixes with osmic acid. v. BENEDEN
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. BENEDEN also recom-
mends the liquid of Kleinenberg. HENNEGUY writes that he fre-
quently employs it for embryonic areas and embryos of various
ages, always with excellent results. Fol's modification of the
liquid of Flemming, and Ranvier and Vignal's osmic acid and
alcohol mixture (§ 36) also give excellent results. For staining,
HENNEGUY recommends borax-carmine, or Delafield's hsematoxylin
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, or double imbed.
590. On the Fixation of Whole Tubes.— This may be done in
Carnoy, Bouin or Helly. For rapidity of fixation, and faithfulness
of preservation of cell aggregates Carnoy's fluid or preferably
Sansom's modification of Carnoy are to be recommended. Chrome-
formalin mixtures penetrate less readily, but often give fine results.
Bouin's fluid I have found capricious. On the whole I think that
warm Helly or Miiller-formol as a preliminary fixation are to be
recommended for small tubes. Regaud's or Schridde's methods
should give efficient fixation (§§ 684 — 689). Many workers have
used the picric mixtures like picro-sulphuric and nitric, and Kleinen-
berg's picric acid. Flemming's fluid has also been used.
In later stages of development some workers open the uterus
CHAPTER XXV. 269
under fixative, or ligature one end of the organ and inject some
fixing medium.
Corrosive formol mixtures have been much used for this purpose.
Neutral formalin of from 3 to 10 per cent, strength is often
used for preserving later stages, after the uterus has been opened
out. The advantage of this procedure from the cytological point
of view is that any methods such as those of Regaud, Bensley-
Cowdry, Sjovall, or formol-silver nitrate neurological techniques
may subsequently be used. The chrome-picric or alcoholic acetic
formol mixtures are not so suitable if one has cytological study in
view.
591. On Clearing Mammalian Material. — This is an important
matter, because delicate embryos are easily shrunken up, or even
not properly dealcoholised, by injudicious methods. J. P. Hill clears
in two stages. Dehydrated embryos are brought into cedar wood
oil in which they are left overnight. The cedar wood oil is subse-
quently washed out in benzole for several hours according to size
of object. Paraffin parings are then added to the benzole, contained
preferably in a tube, and the latter is then left overnight uncovered
on the top of the bath, and subsequently put into pure wax. This
method insures a gentle dealcoholisation, and an efficient imbedding.
Neither cedar wood oil nor benzole cause the tissue to become
brittle as happens often when one uses xylol or chloroform (see
§§ 120-135).
Imbedding. — For embryological work of a critical character,
especially with post-blastoderm stages, double-imbedding in cel-
loidin and wax is generally indispensable. It is only necessary to
contrast serial sections of chick blastoderms prepared by this
method, with those obtained by wax imbedding alone to become
convinced of the inability of the latter method to do complete
justice to the details of the structure and relations of the embryonic
tissues (WILSON and HILL, Phil. Trans. Roy. Soc., 1907).
See also J. P. HILL (Anat. Anz., Bd. xviii, 1900 ; Quart. Journ. Micr.
Science, Ivi, 1910) ; HARTMANN (Jour. Morph., xxvii, 1916). The latter
recommends punching a hole in the side of larger blastoderms to facili-
tate penetration of dehydrating and clearing fluids. WEYSSE, Proc.
Amer. Acad. Arts and Sci., 1894, p. 285 ( blast odermic vesicle of Sus
scrofa) ; SOBOTTA, Arch. mik. Anat., xlv, 1895, p. 15 (ovum of the Mouse ;
fixation in FLEMMING'S weak mixture, sections stained with BENDA'S
iron hsematoxylin), and Anat. Hefte, 1 Abth., viii, 1897, p. 476 (Kabbit ;
fixation with liquid of Flemming or picro-sublimate with 2 per cent,
acetic acid) ; BONNET, ibid., ix, 1897, p. 426 (Dog ; fixation in sub-
limate) ; SELENKA, Stud. Entw. d. Thiere, Wiesbaden, 1883, p. 5, and
270 EMBRYOLOGICAL METHODS.
1887, p. 107 (picro-sulphuric acid for the mouse, and picric acid with
i^ per cent, of chromic acid for Didelphys) ; KEIBEL, Morph. Arb., ii,
1893, p. 11 (Sus scrofa) ; NEUMAYER, Festschr. f. Kupffer, 1899, p. 458
(embryos of the sheep best fixed in Carnoy's acetic acid, alcohol, and
chloroform, § 85) ; WINIWARTER, 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) ; SPEE, Encycl. mik.
Teehn., 1910, p. 353 (cornua of Cavia fixed for twelve to twenty-four hours
in sublimate, and put into 0-5 per cent, osmic acid till light brown, then
into iodine alcohol, in which the osmium is reduced) ; WIDAKOWICH,
Zeit. wiss. Zool., xciv, 1909, p. 243 (Mus rattus, fixation in Zenker's
mixture, or 2 parts of alcohol of 80 per cent, with 1 of formol ; also
instructions for dissection).
592. Injection and Clearing of Larger Embryos. — A con-
siderable amount of useful work has lately been carried out on
embryonic blood and lymph vessels, and on the cerebro-spinal
cavities, by micro-injection apparatus. A suitable injection
medium is blown or forced into the vessels of an embryo, the latter
is fixed and then dehydrated, and cleared by the Spalteholz method
(Uber das Durchsichtigmachen von menschlichen und tierschen Prd-
paraten, und seine theoretischen Bedingungen, Leipzic, S. Herzel,
1911 ; 2 AufL, 1914).
In an early stage in the formation of embryonic vessels and
cavities the walls are thin and often ill-marked, and care must be
taken not to burst through boundaries by excessive pressure. Very
fine metal needles or, better, finely drawn out glass cannulse are
used for injecting the specimens ; the tube leading to the cannula is
filled with the injection medium, which, by means of a rubber tube
leading to the operator's mouth, is blown carefully into the per-
forated vessel or cavity. Or, one may use a rubber bulb either .
worked by hand, or placed on the floor and compressed by the foot.
See E. M. GREGORY, Anat. Record, xi, 1917.
The injection media most commonly used are india-ink, a saturated
solution of Prussian blue, an aqueous suspension of lamp black, or
silver nitrate (5 per cent.). The Prussian blue and india-ink give
about equal results, the blue clearing better, the ink being more
opaque. The ink flows the better. Silver nitrate preparations are
very beautiful and easy to analyse, but its caustic action prevents
the finer vessels from filling. Lamp black tends to precipitate in
fine flakes (CUNNINGHAM, vide infra). EVANS (vide infra), for
cerebro-spinal spaces of pig embryos, injected potassium ferro-
cyanide, 0-5 grms., iron ammonium citrate, 0-5 grm., aq. dest.,
100 c.c., and afterwards immersed the embryo for one to ten minutes
in a 10 per cent, formaldehyde solution containing 1 per cent. HC1.
CHAPTER XXV. 271
The embryo was then fixed in Bouin's fluid, but the Prussian blue
faded after about a year.
SABIN (vide infra) and CUNNINGHAM, after india-ink injection,
fix in Carnoy's fluid, place in 80 per cent, alcohol, dehydrate in
graded alcohols, clear thoroughly, first in benzine (or benzol), and
then in oil of wintergreen (Spalteholz). Embryos cleared by
Spalteholz's method may later be embedded from oil of wintergreen
by transferring to half wax, half oil of wintergreen, and then pure
wax. Tissues left in oil of wintergreen do not go brittle even after
a year or two (Sabin).
For areas of osteoblastic activity, see § 780, and cartilaginous
skeletons, § 779.
See also R. S. CUNNINGHAM (Contrib. Carng. lust. Wash., 1916,
No. 12) ; L. H. WEED (ibid., No. 14,1917) ; F. SABIN (Johns Hopkins
Hosp. Report Monographs, N.S., No. 5, Baltimore, 1913) ; Contrib.
to Embryol. Carneg. Inst. Wash., No. 7, 1915) ; P. G. SHIPLEY and
C. C. MACKLIN (Anat. Record, x, 1915-16).
Aves.
593. Superficial Examination. — Instructions on this head are
given in FOSTER and BALFOUR'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 described 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 vitelline membrane, which will be in a few
minutes, a circular incision is made in the blastoderm outside the
paper ring. The egg is put back into the salt solution, and the
paper ring removed, carrying with it the vitelline membrane and
the blastoderm, which may then be brought into a watch-glass or
on to a slide and examined under the microscope (DuvAL).
Gerlach's Window Method (Nature, 1836, 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 underneath
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
272 EMBRYOLOGICAL METHODS.
position in the incubator. The progress of the development may be
followed up to the fifth day through the window.
A description of further developments of this method, with figures of
special apparatus, will be found in Anat. Anz., ii, 1887, pp. 583, 609.
See also PATON, Journ. Exper. Zool., xi, 1911, p. 469 (cultivation of
the embryo in vitro).
594. Preparation. — During the first twenty-four hours of incuba-
tion, it is extremely difficult to separate the blastoderm from the
yolk, and they should be fixed and hardened together.* In later
stages, when the embryo is conspicuous, the 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 blastoderm
above the surface of the liquid ; the blastoderm is then treated
with some fixing solution dropped on it from a pipette (1 per cent,
solution of osmic acid, or Ranvier and Vignal's osmic acid and
alcohol mixture, iodised serum, solution of 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 blasto-
derm, the blastoderm may be kept well immersed for a few minutes,
and should then be found to be sufficiently fixed to be excised.
(Of course, if you prefer it, you can open the egg in a bath of any
fixing liquid [10 per cent, nitric acid being convenient for this
purpose] of such a depth as to cover the yolk ; and having exposed
the 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.
Before putting it into the hardening fluid, the portion of vitelline
membrane that covers the blastoderm should be removed with
forceps and shaking.
* ANDREWS (Zeit. wiss. Mik., xxi, 1904, p. 177) separates the blasto-
derm at this stage by injecting picro -sulphuric acid (not any rapidly
acting fixative) firstly, between the blastoderm arid 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 upward^
CHAPTER XXV. 273
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. HOFMANN, Zeit.
wiss. MiL, x, 1893, p. 485). MITROPHANOW (Anat. Hefte, xii, 1899,
p. 200) fixes with nitric acid of 3 per cent. ; SUSCHKIN (Nouv. Mem.
Soc. Nat. Moscow, xvi, 1899, p. 34) with sublimate ; FISCHEL
(Morph. Jahrb., xxiv, 1896, p. 371) with Rabl's platino-sublimate,
§ 76 (embryos of the duck) ; PATTERSON (Bid. 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 five to
fifteen 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 blasto-
derm 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 HENNEGUY prefers the osmic acid and alcohol
mixture of Ranvier and Vignal, or Flemming's mixture followed 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.
595. M. DUVAL'S Orientation Method (Ann. Sc. Nat., 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 cicatricula, so as to be able to
make sections in any desired direction. DUVAL, 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.
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 position that the
base of the triangle corresponds to what will be the anterior region
of the embryo, and its apex to the posterior region ; that is to say,
if the big end of the egg is to your left, the apex of the triangle will
point towards you. You now, by means of a pipette, fill the paper
triangle with 0-3 per cent, solution of osmic acid. As soon as the
preparation begins to darken you put the whole egg into weak
M. 18
274 EMBRYOLOGICAL METHODS.
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 encloses the
cicatricula and marks its position ; you cut out this area with
scissors and a scalpel, and complete the hardening with chromic
acid and alcohol.
See also the method of HIROTA, Journ. Roy. Mic. Soc., 1895,
p. 118.
596. KIONKA'S Orientation Method (Anat. Hefte, 1 Abth., iii,
1894, p. 414). — Open the egg 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 hedgehog 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 1 drop
of concentrated solution of Orange^G^for each 5 c.c., which stains
the yolk.
597. 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.
598. Chick and Reptile Blastoderms. — GERHARDT (Anat. Anz.,
xx) uses : —
Chromic acid 1 per cent. .... 150 c.c.
Sat. corr. subl. . . . . . . 150 ,,
Aq. dest. 135 „
Acetic acid . . . . . . 15 „
Formalin 150 „
Leave in twenty-four hours. Wash twenty-four hours in running
water, upgrade from 70 per cent, alcohol, 90 per cent, with iodine,
pure 90 per cent., etc. Recommended by Prof. J. P. Hill.
Reptilia.
599. 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.
BOHM and OPPEL (Taschenbuch, 1900, p. 186) remove the shell
CHAPTER XXV. 275
under salt solution, fix in sublimate with 20 per cent, acetic acid, or
in Lo BIANCO'S chromo-sublimate (§ 72), then remove the blastoderm
and bring it into alcohol.
600. Special Cases. — MITSUKURI (Journ. Coll. Sc. Japan, vi, 1894,
p. 229) fixes^mbryos of tortoises chiefly with picro-sulphuric jicid.
To study the blastoderm he removes the whole of the shell and as
much as possible of the albumen, marks the place where- the blasto-
derm lies with a hair, brings the whole, with the blastoderm upper-
most, into the fixative, and after a few hours cuts out the blasto-
derm 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^ujphuric^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.
WILL (ZooL 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 lmrj^n^Jih^embry_os,
on_the_yolk ; so also for Cistudo and Lacerta (1893 and 1895).
MEHNERT (Anat. Anz., xi, 1895, p. 257) does not approve of these
methods ; for his own see Morph. Arb. Schwalbe, i, 1891, p. 370.
GERHARDT (Anat. Anz., xx, 1901, p. 244) fixes ova of Tropido-
notus for twenty-four hours in Nowak's mixture, § 112.
BALLOWITZ (Entwickl. d. Kreuzotter, 1903, p. 19) first fixes seg-
ments of the uterus, each containing an ovum, for one or two hours,
then tears them open with forceps, isolates the ova, and puts them
into fresh fixative, and thence into -alcohol of 40 per cent.
NICOLAS (Arch. Anat. Mic., 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 PERENYI, § 48, and Zool. Anz., 1888, pp. 139 and 196,
and other methods in early editions.
Amphibia.
601. 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
18— 2
276 EMBRYOLOGICAL METHODS.
method is that described by WHITMAN (Amer. Natural., xxii, 1888,
p. 857), and by BLOCHMANN (Zool Anz., 1889, p. 269). WHITMAN
puts the fixed eggs into a 10 per cent, solution of sodium hypo-
chlorite diluted with 5 to 6 volumes of water, and leaves them there
till they can be shaken free, which happens (for Necturus) in a
few minutes. BLOCHMANN takes eau de Javelle (potassium hypo-
chlorite), and dilutes it with 3 to 4 volumes of water, and agitates
the eggs previously fixed with solution of Flemming, for fifteen to
thirty minutes in it.
LEBRUN (La Cellule, xix, 1902, p. 316) advises fixing ova of Anura
for not less than one and a half 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.
602. Imbedding. — A great difficulty with the ova of Amphibia
lies 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 imbed, they are brought for a quarter
of an hour into 95 per 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) LEBRUN explains that it is important
not to dehydrate completely with absolute alcohol ; the ova should
be left in alcohol of 96 per cent, until chloroform can be added
without the mixture becoming turbid, and a second bath of clean
paraffin should be added.
See also MORGAN, Devel. of the Frog's Egg, New York, 1897, p. 171.
603. Siredon. — The ova are easier to prepare than those of the
Anura, because the yolk is separated from the albuminous layer by
a wide space filled with a liquid that is not coagulated by reagents.
Put the eggs for a few hours into picro-sulphuric acid, then pierce
CHAPTER XXV. 277
the inner chorion with fine scissors or needles, and gently press out
the ovum. Harden in alcohol.
FICK (Zeit. wiss. ZooL, Ivi, 1893, p. 529) uses a mixture of 250 parts
of 1 per cent, chromic acid, 1 of acetic acid, and 750 of water.
604. Triton (Scorr and OSBORN, Quart. Journ. Mic. Soc., 1879,
p. 449). — The albumen is here present in the form of several con-
centric coats, which are very delicate. Incise each of them separately
with fine scissors, turn out the ovum, and fix it in solution of
Kleinenberg.
HERTWIG (Jen. Zeit. Naturw., 1881 — 2, p. 291) puts the eggs
into a mixture of equal parts of 2 per cent, acetic acid and 0«5 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.
MICHAELIS (Arch. mik. Anat., xlviii, 1896, p. 528) fixes ova,
with their envelopes, in a mixture of concentrated sublimate solution
and concentrated picric acid, 20 parts each, glacial acetic acid 1,
and water 40, but removes the envelopes before bringing into
alcohol.
605. Salamandra (RABL, Marphol. Jahrb., xii, 2, 1886, p. 252).—
For his more recent methods see § 580.
GRONROSS (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 1 part of acetic acid.
606. Rana. — I have found that the following mixture often gives
very good results for the eggs of Rana temporaria ; it dissolves
away the albumen coat, preserves yolk and mitochondria, and
leaves the eggs soft enough to cut in paraffin with a rotary micro-
tome : — •
Bichromate of potash of 2 per cent. . 100 c.c.
Chromic acid of 1 per cent. . . . 100 „
Nitric acid . . . . . 6 „
Use at least 40 c.c. to twenty or thirty eggs for fifteen to twenty-
four hours. Slightly shake, and the albumen coats fall off if not
already dissolved. Wash out for about one hour in running water
and then upgrade from 70 per cent, alcohol (one half-hour), 90 per
cent, (one hour), to absolute alcohol, two changes of one hour each.
Clear in benzol for fifteen minutes. Add chips of wax and place in
thermostat for half an hour. Transfer to pure wax for one-half
(to three-quarters) of an hour. Avoid unnecessary heat.
278 EMBRYOLOGICAL METHODS.
The eggs thus treated can often be cut 6/x on a rotary microtome
provided with a sharp knife. The method is indicated where large
numbers of stages of the early development of the frog are required
for junior class purposes. The main objection to the method is
that the chromic acid attacks pigment. Fertilisation and segmenta-
tion stages and general cytology are often extremely good ; stain in
any way (J. B. G.).
0. HERTWIG (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.
MORGAN (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 WHITMAN, Meth. of Research, p. 156.
SCHULTZE (Arch. mik. Anat., Iv, 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 sufficiently to allow the ova to be got
out with needles.
See also BORN (ibid., xliii, 1894, p. 1).
KING (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.
BLES (Trans. Roy. Soc. Edinburgh, xli, 1905, p. 792) takes for
ova formol of 10 per cent., but for embryos and larvse the mixture
given § 109.
BOUIN takes for larvse of Rana the formol-sublimate mixture
§ 112.
CHAPTER XXV. 279
607. Sulphate of Copper Liquid (FoL, Lehrbuch, p. 106, after KEMAK
and GOETTE) ; for hardening ova of Amphibia :
2 per cent, solution of sulphate of copper. 50 c.c.
Alcohol of 25 per cent. . . . . 50 ,,
Rectified wood vinegar . . . .35 drops.
Pisces.
608. 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 Salmonidee 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 con-
taining 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 successive alcohols. Embryos thus pre-
pared show no deformation, 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
Miiller's solution. Open them therein with fine scissors — the
vitellus, which immediately coagulates on contact with air, dissolves,
on the contrary, in Miiller's solution — and the germ and cortical
layer can be extracted from the capsule of the ovum. They should
be left in clean Miiller's solution for a few days, then washed with
water for twenty-four hours, and brought through successive
alcohols.
Another method (HENNEGUY) is as follows : The ova are fixed
in solution of Kleinenberg containing 10 per cent, of acetic acid.
After ten minutes they are opened in water containing 10 per
cent, of acetic acid, which dissolves the vitellus. The embryos
are put for a few hours into pure solution of Kleinenberg,
and are then brought through alcohol of gradually increasing
strength.
CHILD (quoted from SUMNER, Mem. New York Acad. Sci., 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.
280 EMBRYOLOGICAL METHODS.
609. KOLLMANN'S Fixative (KOLLMANN, Arch. Anat. Phys., 1885,
p. 296).
Bichromic of potash .... 5 per 100.
Chromic acid . . . . • . 2 ,,
Concentrated nitric acid . . 2 ,,
For ova of Teleostea. Fix for twelve hours, wash with water for
twelve hours, then remove the chorion, and put the ova into 70 per cent.
alcohol.
610. RABL'S Method, see § 587 ; for KOWALEWSKY'S see Zeit. wiss.
Zool, xliii, 1886, p. 434, or Third Edition.
611. Salmonidae. — HENNEGUY'S methods have been given, § 609.
KOPSCH (Arch. mik. Anat., li, 1897, p. 184), on the suggestion 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 salt solution, and completes
the hardening in the chromic acid or the saturated sublimate
solution.
Similarly, BEHRENS (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 SOBOTTA (ibid., 1902, p. 579).
GUDGER (Proc. U.S. Nation. Mus., xxix, 1906, p. 448) fixes
blastoderms in fresh liquid of Perenyi, which does not make the
yolk too hard ; later stages in WORCESTER'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.
BOUIN (C. R. Soc. Biol, Iv, 1903, p. 1691) fixes for thirty-six to
forty-eight hours in picro-formol.
EABL-KUCKHARD'S Method (Arch. Anat. Entw., 1882, p. 118).— Fix in
10 per cent, nitric acid for fifteen minutes. Kemove the membranes to
avoid deformation of the embryos, and put the ova back into the acid
for an hour. Wash out in 1 to 2 per cent, solution of alum for an hour
and harden in alcohol.
Modification of this method by GORONOWITSCH (see Morph. Jahrb., x,
1884, p. 381).
612. Selachia.— BEARD (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 (KASTSCHENKO, Anat. Anz., iii, 1888, p. 445, and His, Arch.
CHAPTER XXV. 281
Anat. .Phys., Anat. Abth., 1897, p. 3). See also BRAUS, Morph.
Jahrb., xxxv, 1906, p. 250.
613. Amphioxus.— SOBOTTA (Arch. mik. Anat., 1, 1897, p. 20)
fixes for twenty-four hours in liquid of Flemming ; HATSCHEK (Arb.
Zool. Inst. Wien., iv, 1881) in picro-sulphuric acid. Impregnation
takes place in the evening, and segmentation is completed during
the night.
LEGROS (Grundzuge, LEE and MAYER, 1910, p. 288) fixes ova
and embryos in equal parts of formol and Flemming. Sublimate
is not good ; Rabl's mixtures are better. Larvae and young animals
ought first to be anaesthetised with cocain in sea-water. After
fixation they should remain only for as short a time as possible in
alcohol.
CERFONTAINE (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.
614. Pelagic Fish Ova.— WHITMAN (Amvr. 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
-J- per cent, osmic acid solution, and then for one or two days with a
solution (due to Eisig) of equal parts of 0-2£ per cent, platinum chloride
and 1 per cent, chromic acid. Prick the membrane before transferring
to alcohol. See also AGASSIZ and WHITMAN, in Proc. Amer. Acad. Arts
and Sciences, xx, 1884 ; and COLLINGE, Ann. and Mag. Nat. Hist., x,
1892, p. 228.
EAFFAELE (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).
HEINKE and EHRENBAUM (<Wiss. Meeresunt. Komm. Wiss. Unt. D.
Meere, iii, Heligoland, 1900, pp. 205 and 213) prefer formol with 39
volumes of sea-water.
Tunicata.
615. Ova.— DAVIDOPF (Mitth. Zool. Stat. Neapel, ix, 1, 1889,
p. 118) fixes the ova of Distaplia 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.
CASTLE (Bull. Mus. Harvard Coll., xxvii, 1896, p. 213) advises for
ova of Ciona liquid of Perenyi for twenty minutes, followed by
282 EMBRYOLOGICAL METHODS.
70 per cent, alcohol for twenty-four hours, and for the larvae picro-
nitric acid.
616. Test-Cells of Ascidians (MORGAN, Journ. of Morphol, iv. 1890,
p. 195). — Tease fresh ovaries in very weak osmic acid, wash in distilled
water, treat for half an 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 limits of the follicle cells are demon-
strated.
617. Buds.— PIZON (Ann. Sc. Nat., xix, 1893, p. 5) studies the
gemmation of the composite Ascidians either on entire corms,
which he first bleaches with peroxide of hydrogen and then stains,
or by making sections, after anaesthetising the colonies with cocain
of 1 : 1000, fixing in glacial acetic acid or pier o -sulphuric or liquid
of Flemming, and staining in toto with borax carmine or alum
carmine, or with a strong solution of methylen blue in alcohol of
90 or 100 per cent, (after BERNARD, ibid., ix, 1890, p. 97).
RITTER (Journ. of Morph., xii, 1896, p. 150) recommends for
fixing Perophora and Goodsiria picro-sulphuric acid.
Bryozoa.
618. Statoblasts. — BRAEM (Bibl. ZooL, Chun and Leuckart,
6 Heft, 1890, p. 95) fixes statoblasts of Cristatella with hot con-
centrated 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.
Mollusca.
619. Cephalopoda (Ussow, Arch, de Biol., ii, 1881, p. 582).—
Segmenting ova are placed in 2 per cent, solution of chromic acid
for two minutes, and then in distilled w^ater, to which a little acetic
acid (1 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 clings to
it, it may be removed by pouring away the water and adding
more.
WATASE (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 liquid of Perenyi is recommended for surface views.
VIALLETON (Ann. Sc. Nat., vi, 1887, 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,
CHAPTER XXV. 283
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 vitellus, 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.
KORSCHELT (Festchrift 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.
FAUSSEK (Mitth. Zool. Stat. Neapel, xiv, 1900, p. 83) recommends
picro-nitric acid. Fix in this, harden in alcohol, bring the ova,
still in their albumen, into hsemalum, stain for twenty-four hours,
wash in 1 per cent, alum solution for twenty-four hours, when the
albumen will be found softened so that the ova can easily be
extracted.
620. Gastropoda (HENNEGUY). — Ova of Helix 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 coagulated. The egg is opened
with needles, the albumen comes away in bits, and the embryo can
be removed.
HENCHMAN (Bull. Mm. Comp. Zool., Harvard, xx, 1890, p. 171)
fixes ova of Limax with 0-33 per cent, chromic acid, or with liquid
of Perenyi. It is best to remove only the outer envelope before
putting into the chromic acid, the inner membrane being removed
after two or three minutes therein. Where Perenyi is used the
membranes must be removed first, as the albumen will else coagulate
in such a way as to prevent the removal of the embryos.
MEISENHEIMER (Zeit. wiss. Zool., Ixii, 1896, p. 417) dissects out
the embryos of Limax 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.
SCHMIDT (Entw. Pulmonaten, Dorpat, 1891, p. 4) fixes the ova
in toto with concentrated sublimate, and dissects them out afterwards.
Similarly KOFOID (Bull. Mus. Harvard Coll., xxvii, 1895, 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 LINVILLE, ibid., 1900, p. 215, who adopts this
284 EMBRYOLOGICAL METHODS.
method of shelling, but prefers fixing in acetic-acid sublimate, or
liquid of Perenyi.
HEYDER (Zeit. wiss. ZooL, xciii, 1909, p. 92), before imbedding
embryos of Arion that have been fixed with sublimate, treats them
for an hour or two with carbonate of soda of ^ to T^ per cent.,
which makes the stomach and intestine less brittle.
GATENBY (Quart. Journ. Micr. Science, 1919), for Limncea stagnalis
ova, used Flemming's strong fluid without acetic acid, Champy's
fluid for two days to a week, and Kopsch's method.
HOLMES (Journ. of Morph., 1900, p. 371) teases the egg-capsules of
Planorbis in nitrate of silver of f 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 WASHBTJRN, 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) fixes ova of Crepidula for
fifteen to thirty minutes in picro -sulphuric acid, and stains with dilute
acidified hsBmatoxylin of Delafield.
KOSTANECKI and WIERZEJSKI (Arch. mik. Anat., xlvii, 1896,
p. 313) fix the spawn of Physa fontinalis either in 1J to 2 per cent,
nitric acid, or in " sublimate and 3 per cent, nitric acid in the pro-
portion of 2:1," and bring through successive alcohols. They
imbed entire ova in paraffin, but isolated embryos in celloidin.
621. CHITON, see METCALF, Stud. Biol. Lab. Johns Hoplcins Univ., v,
1893, p. 251. (Ova with young embryos put for twenty to forty-five
seconds into eau de Labarraque, then into water, in which the chorion
swells and can easily be removed.)
622. Lamellibranchiata. — STAUFFACHER (Jena Zeit., xxviii, 1893,
p. 196) fixes embryos of Gyclas in sublimate, stains with hsemalum,
and cuts in paraffin.
LILLIE (Journ. of Morph., x, 1895, p. 7) fixes ova of Unio for
ten to twenty minutes in liquid of Perenyi, and preserves them in
70 per cent, alcohol, or advanced embryos with liquid of Merkel or
sublimate, larvae with 0-05 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 anaesthetised with chloral hydrate
before fixing.
Arthropoda.
623. 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.
CHAPTER XXV. 285
624. Removal of Membranes. — It may often be advisable not to
attempt to remove them, but to soften them with eau de Javelle or
eau de Labarraque. See § 545.
MORGAN (Amer. Natural, xxii, 1888, p. 357) recommends (for
the ova of Periplaneta) eau de Labarraque diluted with 5 to 8 volumes
of water, and slightly warmed. This will soften the chitin mem-
branes 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.
625. HENKING'S Methods (Zeit. wiss. MiL, viii, 1891, p. 156).—
HENKING 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.
He thinks that eau 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. To
avoid brittleness of the yolk 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.c. of 70 per cent, alcohol, 1 drop of concentrated
hydrochloric acid, and a knife pointful of pepsin (it is not necessary
that all the pepsin should be dissolved). The ova may then be
treated with alcohol, oil of bergamot, and paraffin, and (with some
exceptions, amongst which is Bombyx mori) will be found to cut
without crumbling.
626. Diptera (HENKING, Zeit. wiss. ZooL, xlvi; 1888, p. 289).—
Ova still contained within the fly may be fixed by plunging the
insect 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.
BRUEL (ZooL Jahrb., Abih. Morph., x, 1897, p. 569) fixes larvee
and pupae in absolute alcohol heated to 70° to 75° C., and con-
taining a " little " sublimate. See also VAN REES, ibid., iii,
1888, p. 10.
BENGTSSON (Handl. Fysiogr. Scellsk Lund., viii, 1897) finds hot
alcoholic solution of sublimate (Frenzel's, § 69) the best fixative for
286 EMBRYOLOGICAL METHODS.
larvae of Phalacrocem. He could not succeed in softening the chitin
with eau de Javelle.
PEREZ (Arch. Zool. exper., (4), v, 1910, p. 11) fixes pupse in Bouin's
picro-formol, or Marchoux's mixture, for twenty-four hours.
627. Lepidoptera (BOBRETZKY, 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.
628. Hymenoptera. — CARRIERS and BURGER (Nova Acta Acad.
Leop. Car., Ixix, 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.
PETRUNKEWITSCH (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.
629. Orthoptera (PATTEN, Quart. Journ. Mic. Sci., 1884, p. 549).—
The ova or larvae (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 succes-
sive alcohols, beginning with 20 per cent.
WHEELER (Journ. of Morph., iii, 1889, p. 292) dissects out ovarian
ova in salt solution and fixes in liquid of Perenyi (fifteen minutes),
then treats with alcohol, and stains with borax-carmine. Laid
eggs may be killed by Patten's method. After heating, the two
lips of the crista tof the capsule may be separated with fine forceps
and pieces of the walls torn away, and the eggs pushed out of the
compartments formed by their choria and hardened as desired.
Good results are also obtained by heating to 80° C. for ten minutes
in liquid of Kleinenberg, and preserving in 70 per cent, alcohol.
This causes the envelopes to dilate and stand off from the surface of
the egg, so that they can easily be dissected away.
HEYMONS (Zeit. wiss. Zool., liii, 1892, p. 434), for young embryos,
incises the cocoon at the end by which it adheres in the body of the
mother, brings it for two minutes into water heated to 90° C., and
opens in Flemming, in which the embryo is dissected out.
MORGAN (Amer. Natural, xxii, 1888, p. 357) puts ova of Peri-
planeta for thirty minutes or an hour into eau de Javelle diluted with
4 to 8 volumes of water and slightly warmed, which softens the
capsules.
630. Coleoptera.— HIRSCHLER (Zeit. wiss. Zool, xcii, 1909, p. 628)
fixes ova of Donacia (after incising the chorion) for two to three
CHAPTER XXV. 287
hours in equal parts of sublimate of 6 per cent, and nitric acid of
3 per cent.
GATENBY (Quart. Journ. Mic. Sci., 1917) for Donacia uses
Petrunkewitsch or picro-nitric. In the latter case the choriori
must be incised.
SALING (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 three minutes in a hot mixture of 1 part of formol with 3 of water.
KARAWAIEW (Biol. Centralb., xix, 1899, p. 124) kills larvae of
Anobium in hot water, freezes them with ether spray, cuts away a
lateral strip, lets them thaw, and puts for twenty-four hours into
picro-sulphuric acid.
631. Phalangida. — The ova of Phalangium opilio possess a chorion
covered with yellow corpuscles that render them opaque. BALBIANI
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 brush, the vitelline
membrane remaining intact, so that the embryo can be studied
through it.
HENKING'S method (Zeit. wiss. Zool., xlv, 1886, p. 86). — Fix with
boiling water or Flemming. Preserve the ova in 90 per cent,
alcohol. To open the chorion, bring them back into 70 per cent,
alcohol, which causes them to swell up so that the chorion can easily
be pierced with needles, and the ovum turned out.
632. Araneida. — KISHINOUYE (Journ. Coll. Sci. Imp. 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 LOGY, Bull. Mus* Comp. Zool. Harvard, xii, 3, 1886.
Fix by hot water. The liquid of Perenyi may also be used ; it has
the advantage of not making ihe yolk so granular.
MONTGOMERY (Journ. Morph., xx, 1909, p. 628) fixes ova of
Theridium for one or two hours in Carnoy & Lebrun's mixture.
LAMBERT (ibid., p. 420) fixes ova of Epeira in picro-sulphuric acid
warmed to 70° or 80° C.
PURCELL (Quart. Journ. Micr. Sci., liv, 1909, p. 7) fixes ova of
Atta in boiling saturated sol. of sublimate in alcohol of 70 per cent.
HAMBURGER (Zeit. wiss. Zool., xcvi, 1910, p. 3) fixes ova of
Argyroneta in Gilson's mixture.
288 EMBRYOLOGICAL METHODS.
633. Limulus.— KINGSLEY (Journ. 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 KISHINOUYE, Journ. Coll Sri.
Japan, v, 1893, p. 56.
634. Decapoda. — REICHENBACH (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.
HEERICK (Bull. U.S. Fish, Comm., xv, 1896, p. 226) kills the ova
in hot water, shells and fixes in picro-sulphuric acid.
For Homarus, see WAITE, Bull. Mus. Comp. Zool., xxxv, 1899,
p. 155.
635. Amphipoda.— BELLA VALLE (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.
636. Cladocera. — HAEKER (Zellen. u. Befruchtungslehre, 1899,
p. 60) fixes females of Sida with winter eggs in a hot mixture of
100 c.c. alcohol of 70 per cent, with 1 to 2 c.c. saturated sol. of
sublimate. See also SAMTER, Zeit. wiss. Zool., Ixviii, 1900, p. 176.
637. Copepoda.— KRUEGER (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.
Vermes.
638. Rotatoria.— JENNINGS (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 Hydatina with
sublimate for twenty seconds.
639. Turbellaria.— GARDINER (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.
BRESSLAU (Zeit. wiss. Zool., Ixxvi, 1904, p. 219) fixes Mesosto-
CHAPTER XXV. 289
midee with summer-eggs in Tellyesniczky's mixture (either cold or
warmed to 60° or 70° C.) for ten to twelve hours, and 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 sufficiently shelled.
VAN DER STRICHT (Arch. BioL, xv, 1898, p. 370) finds that ova of
Thysanozoon 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, § 602.
See also, for Polyclads, FRANCOTTE, Arch. Zool. Exper., vi, 1898,
p. 196 ; and, for fresh-water Planaria, IIJIMA, Zeit. wiss. ZooL,
xl, 1884, p. 359.
640. Cestoda (v. BENEDEN, Arch. BioL, ii, 1881, p. 187).— Ova
of Tcenia 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.
HASWELL (Quart. Journ. Micr. Sci., liv, 1909, p. 417) fixes ova of
Temnocephala in " sublimate alcohol," brings them into 90 per cent,
alcohol with iodine added, and thence gradually back into water,
softens the shells in weak sodium hypochlorite, washes and imbeds.
641. Trematoda. — COE (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 J per cent, solution of
silver nitrate.
Egg-capsules may be softened with 5 per cent, caustic potash
and then burst open (HECKERT, Bibl. ZooL, iv, 1889).
642. Nematoda. — The ova of Ascaris megalocephala, a classical
object of study, are one of the most impervious things in the animal
kingdom. Years ago FOL related to me that he had had ova seg-
menting right through absolute alcohol into balsam. BATAILLON
(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 twenty-
four hours at 35° C., and mounting in balsam, and for weeks in acids
or alkalies.
M. 19
290 EMBRYOLOGICAL METHODS.
Doubtless the best fixative yet made known for ova furnished
with their capsules will be found to be that of CAENOY and LEBRUN,
§ 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 (§ 602), 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 necessary to ensure penetration.
They stain with iron hsematoxylin.
ZUR STRASSEN (Arch. Entwickelungsmech., iii, 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 hydro-
chloric acid carmine, and brings gradually into glycerin.
Similarly Z,ojA(Arch. mik. Anat., xlvii, 1896, p. 218) and ERLANGER
(ibid., xlix, 1897, p. 309). Zoja stained with Bismarck brown and
examined in dilute glycerin ; Erlanger made paraffin sections and
stained with iron hsematoxylin.
KOSTANECKI and SIEDLECKI (ibid., xlviii, 1896, p. 184) employed
concentrated sublimate solution, or 3 per cent, nitric acid or mixtures
of these two, for ovarian ova.
VAN BENEDEN and NEYT (Bull. Acad. Belg., 1887, p. 214) took
equal parts of alcohol and acetic acid. BOVERI (Jena Zeit., xxi,
1887, p. 423) fixed in his picro-acetic acid, § 95 — a clearly inadequate
method. GULICK (Arch. Zellforsch., vi, 1911) has " fixed " ova of
Heterakis for twenty-two hours in one-third saturated picric acid
with 3 per cent, of glacial acetic acid, and had them develop in
alcohol of 70 per cent, to stages representing a normal development
of several weeks.
BORING (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 //, thick, and fixes these with divers
liquids.
CERFONTAINE (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
CHAPTER XXV. 291
with 5 per cent, of collodion added, evaporates almost entirely
away, and passes through cedar oil into paraffin.
For methods for the Mitochondria and Golgi apparatus it is
necessary to treat uteri as does Artom (above explained), and then
fix in the proper fluid.
Echinodermata, Ccelenterata, and Porifera.
See the chapter on " Zoological Methods."
19—2
CHAPTER XXVI.
CYTOLOGICAL METHODS.
643. Study of Living Cells. — In the young larvae of Amphibia,
both Anura and Urodela, the gills and caudal " fin," and sometimes
other regions, may be studied in the living state.
The larvae 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 curarised larva for half an hour in
1 per cent, chloride of sodium solution. Normal larvae may be used
for the study of the active state of the nucleus, but much time is
saved by using curare.
Curare. — Dissolve 1 part of curare in 100 parts water, and add
100 parts of glycerin. Of this mixture add from 5 to 10 drops
(according to the size of the larva), or even more for large larvae,
to a watch-glassful of water. From half to one hour of immersion
is necessary for curarisation. The larvae need not be left in the solu-
tion until they become quite motionless ; as soon as their move-
ments have become slow they may be taken out and placed on a
slide, wrapped in blotting-paper. If they be replaced in water they
return to the normal state in eight or ten hours, and may be
re-curarised several times.
Other Narcotics. — Three per cent, alcohol or 3 per cent, ether, or
infusion of tobacco, may be used in a similar way. 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 (PEREMESCHKO, Arch. mik. Anat., xvi, 1879,
p. 437).
For the processes of staining living cells see § 208.
CHAPTER XXVI. 293
644. Study of Fresh and Lightly Fixed Cells.— So-called " in-
different " 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 is a delicate fixing agent,
killing cells instantly without change of form. Complete the fixation
by exposing the preparation for a quarter of an hour to vapour of
osmium, and add a drop of solution of 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.
I have found Pictet's chloride of manganese (§ 403) useful as
an examination medium. A little solution of dahlia may be added
to it.
HENKING (Zeit. wiss. Mik., viii, 1891, p. 156) recommends a
liquid composed of —
Water 80 c.c.
Glycerin . . . . . 16 „
Formic acid . . . . . 3 „
Osmic acid of 1 per cent. . . . 1 „
Dahlia 0-04 grm.
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.
645. Some Microchemical Reactions. — Methyl green is a test for
chromatin, in so far as (with fresh cells) it colours nothing but the
chromatin in the nucleus, see § 276. It is, however, not a perfect
294 CYTOLOGICAL METHODS.
test, for the intensity of the coloration it produces varies greatly in
different nuclei, and may in certain nuclei be extremely weak, or
(apparently) even altogether wanting. In these cases other tests
must be applied in order to establish with certainty the presence or
absence of that element.
Chromatin is distinguished from albuminoids by not being soluble,
as these are, in water and in weak mineral acids, such as 0-1 per cent,
hydrochloric acid. It is easily soluble in concentrated mineral
acids, in alkalies, even when very dilute, and in some alkaline salts,
such as 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 in situ 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 opera-
tion 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 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 granulations that may obscure
them, whilst at the same time it clears up the cytoplasm. UNNA
(Monatschr. prakt. Derm., xxxiii, 1901, p. 342) digests tissues in
solutions of sodium chloride, to remove the granoplasm. See
also §§ 652, 664 and 668.
Glycogen.
646. Glycogen is a carbohydrate which occurs in many cells,
both glandular and genital : it is found in both inter- and intra-
cellular positions, in the form of small areas of flocculent appearance.
For its study in a tissue or organ two methods should be used : —
(1) An iodine technique ; and (2) that of Best's carmine. The
specificity of the latter method has been questioned, and both
techniques must be used for comparison (see p. 338).
CHAPTER XXVI. 295
647. Iodine Method. — Fix tissue in Carnoy, or alcohol absolute,
4 parts, acetic acid glacial, 1 part ; or in absolute alcohol ; or in
alcoholic fixatives not containing alcohol lower than a strength of
90 per cent. It is better if the tissue is cut small. Fix for one hour,
then transfer for twenty-four hours or longer, in two changes of
absolute alcohol ; then xylol and paraffin wax. Fix sections to
slide with a mixture of 50 per cent, alcohol with a few drops of
glycerin and albumen, using the alcohol as you would water ;
drain the slides dry. Remove wax in xylol, bring to 70 per cent,
alcohol. Stain sections in Ehrlich's hsematoxylin for five or ten
minutes. Blue in tap water substitute, § 669. Pass to a 2 per cent,
solution of potassium iodide saturated in iodine (a Lugol solution) ;
leave five minutes ; pour away, wipe around slide, and dehydrate
in absolute alcohol saturated in iodine. Clear in oleum origanum
cretici for about ten minutes. Mount in origanum balsam. Such
preparations should keep for years without fading much. I have
some slides of human placenta which after six years still show the •
glycogen.
648. BEST'S Carmine Stain. — Material is fixed as for the iodine
method and may be imbedded in celloidin. If paraffin sections are
used the slide must be placed in 1 per cent, celloidin overnight,
drained and allowed to dry partly, and then plunged into chloroform
and absolute alcohol (equal parts), then treated as for celloidin
sections. Transfer through alcohols 90 per cent, and 70 per cent,
to water. Stain in Ehrlich's or iron hsematoxylin as usual, but
differentiate in acid alcohol. Then proceed to BEST'S carmine
stain (Zeit. f. mikros., Bd. xxiii). Make up this stock carmine
solution : —
Carmine . . 2 grms.
Potass, carbonate 1 grm.
Potass, chloride 5 grms.
Aq. dest 60 c.c.
Boil gently for a few minutes ; cool.
Add strong liq. ammon. 20 c.c. Keep this solution in a well-
stoppered bottle in a cupboard. It may go bad in a month during
summer.
Wash sections in distilled water after staining in hsematoxylin.
Stain in following solution : —
Stock carmine solution .... 2 parts.
Liq. ammon. fort. . . . . . 3 „
Methyl alcohol (pure) . . . 3 „
for five minutes.
296 CYTOLOGICAL METHODS.
Differentiate in-
Absolute alcohol . . . . .80 parts.
Methyl alcohol 40 „
Aq. dest 100 „
till no more red comes out (three to five minutes).
Wash in 80 per cent, alcohol, absolute and clove oil, xylol and
xylol balsam ; nuclei and cytoplasm, blue, glycogen red.
It is a good plan when working on glycogen to prepare triplicate
slides, one for iodine stain, one for Best ; the other slide is brought
down to water and spat upon and set aside : the glycogen is dissolved
by the diastase of the saliva, the latter is washed off in water and the
slide stained as usual for Best's carmine. Comparison between the
first slide and this one will assist in properly identifying glycogen ;
(procedure of Dr. B. R. G. RUSSELL, Imperial Cancer Research Bureau).
One generally succeeds at first trial with such material as the liver of
a rabbit, but with invertebrate materials, especially from paraffin
sections, even though soaked in 1 per cent, celloidin, the results are often
disappointing. This can be overcome by practice and by slight modi-
fication in the time used for differentiation. For delicate material it
seems best to work with celloidin sections.
649. ZIEGLWALLNER'S Alcoholic Flemming for Glycogen and Fat.—
Neither the iodine nor Best's carmine method preserves fat as well as
glycogen. Zieglwallner has worked out the following method for
preserving both fat and glycogen. Fix small pieces of tissue in this
mixture for twenty-four to forty-eight hours : —
1 per cent, chromic acid in 80 per cent, alcohol 15-0
2 per cent. Os04 in water . . . .4-0
Acetic acid . . . . . . 1 -0
In 100 c.c. of this mixture there would be 50 per cent, alcohol.
If a corrosive sublimate fixation is necessary use this mixture in the
same way : —
Concentrated aq. sol. corrosive sublimate . 20-0
2 per cent. Os04 in water . . . .20-0
Acetic acid . . . . . .10-0
Alcohol absolute . . . . . .50-0
In washing out, a little iodine will be necessary. Transfer the pieces
of tissue to 70 per cent., then upgrade and imbed in celloidin.
In order to preserve the brownish black colour of the osmic stain of
fat, which soon disappears when the sections are brought to balsam,
one may convert the reduced osmic into its sulphide by adding a small
quantity of Na^S to the 70 per cent, alcohol which replaces the fixative.
Imbed in celloidin or wax : stain as by the iodine, or better in Best's
carmine method, from celloidin. Dr. J. A. Murray informs me that it
is generally necessary to stain sections first in warm iron alum, then
warm hsematoxylin, and then to differentiate in the cold with acid
alcohol. Afterwards proceed to Best's carmine.
PAUL BUCHNER (Praktikum der Zelknlehre /., Berlin, 1915) fixes
CHAPTER XXVL 297
overnight in a freshly-made mixture of equal parts of absolute alcohol
and strong F lemming. Wash out for two days in 50 per cent, alcohol,
imbed in celloidin, stain in Best's carmine.
See also CREIGHTON, The Formative Property of Glycogen, London,
1896 ; GAGE, Trams. Amer. Micr. Soc., xxviii, 1908, p. 203 ; KATO,
Arch. Ges. Phys., cxxvii, 1909, p. 125 ; BUSCH, Arch. Intern. Phys., iii,
1905, p. 51 ; MAYER, Zeit. wiss. Mikr., xxvi, 1909, p. 513 ; ARNOLD,
Sitzb. Heidelberg. Acad. Wiss., 1909, p. 1, 1910, p. 3, and 1911, 14 Abh. ;
Arch. path. Anat., cxciii, 1908, p. 175 ; Arch. mik. Anat., Ixxiii, 1909,
p. 2.65; Ixxvii, 1911, p. 346; Beitr. path. Anat., li, 1911, p. 439;
FRAENKEL, Virchow's Arch., 1911, p. 197 ; NEUBERT, Beitr. path. Anat.,
xlv, 1909, p. 38 ; ERHARD, Arch. Zellforsch., viii, 1912, pp. 447 and 507 ;
EHRLICH arid LAZARUS, Die Anaemie, 1898, p. 30 ; PEKELHARING,
Petrus Camper, Deel I, 1901, p. 231 ; DRIESSEN, Zeit. wiss. Mik., xxii,
1905, p. 422 ; FISCHER, Anat. Anz., xxvi, 1905, p. 399 ; FIESSINGER,
C. R. Soc. Biol., Ixvi, 1909, p. 183 ; NEUKIRCH, Arch. path. Anat., cc,
1910, p. 82.
650. Some Microchemical Tests. — IRON. — Organic compounds of
iron, which are not ionisable into ferric and ferrous ions, and in which
the iron cannot be detected by the ordinary reagents, are much
more frequently present in animal and vegetable tissues than was
previously believed to be the case. In addition to the albuminate
compounds, there exist iron compounds giving ferric and ferrous
ions, detectable with the ordinary reagents, and which, for con-
venience, may be designated Inorganic Iron Compounds.
The nature of many of the compounds of iron found in placentas,
blood-organs, the liver, etc., is obscure ; many of them appear to
be formed as degeneration or excretion products, from the breaking
down of haemoglobin. See below.
Most of our knowledge of the methods for the detection of iron
in tissues and cells is due to A. B. MACALLUM (Quart. Journ.
Micr. Science, xxxviii, 1895 ; Journ. Physiology, 1897 ; Ergebn. d.
Physiol. Wiesbaden, 1908). Macallum has shown that, to detect
organic iron, one must convert it into inorganic. This can be done
by allowing sulphuric or nitric acid alcohol to act upon sections, or
a piece of tissue, for from one to twenty-four hours at 35° C., accord-
ing to the strength of acid and the size of the object. When masked
iron is liberated in the tissues by acid alcohol, most of it is in the
form of ferric salts, particularly when the oxidising nitric acid is
used, and a small part occasionally of ferrous compounds. Inorganic
iron compounds in tissues are usually ferric, more rarely ferrous
salts.
The commonest tests for iron in tissues are the Prussian blue
reaction, and Macallum's hsematoxylin. The latter test should never
298 CYTOLOGICAL METHODS.
be used alone, because its complete specificity is somewhat
doubtful.
It is hardly necessary to point out that proper precautions should
be taken to avoid contamination of the tissue by vessels or chemicals
which may contain iron compounds. Glass needles should be used
instead of steel, and the water used should have been distilled from
a clean glass retort.
The tissues should either be fixed in redistilled formalin (10 per
cent.), chemically pure ethyl alcohol or pure 90 to 95 per cent.
alcohol, or in redistilled methylated spirit. Bouin's fluid, Flemming,
and such mixtures should not be used, as such a practice is almost
certain to introduce error. Material should be fixed or hardened
for several days in strong alcohol. Sections are made either freehand
with a bright rust-free razor wetted with absolute alcohol, or by the
paraffin method with a dry rust-free knife.
MACALLUM'S HJEMATOXYLIN METHOD. — As an indicator Macallum
uses a 0-5 per cent, solution of absolutely "pure haematoxylin "
made up in perfectly pure aqua dest. The solution should look
brownish yellow, but when alkalies or alkaline earths are added,
the colour becomes violet or red. When such a pure haematoxylin
is brought into contact with a salt of iron, the yellow colour becomes
blue-black, or bluish-black ; with organic iron compounds the
hcematoxylin is unaffected. Such compounds must be unmasked by
sulphuric or nitric acid alcohol as above mentioned.
When the compounds of iron to be investigated are found in
tissues, the latter are well hardened in alcohol (purified, vide supra),
sections prepared and washed in aq. dest., or the tissue simply
teased out, and then the haematoxylin solution is added. Those
parts which go blue-black or blue- violet contain inorganic iron ;
the remainder of the preparation may go quite dark yellowish
brown, especially nuclei, and the presence of iron may thus be
obscured. To remove this excess the preparation is treated in a
mixture of equal parts of absolute alcohol and ether, but not for
longer than one hour. The unaffected hsematoxylin is extracted,
the blue-black compound remains. Clear in oil of cloves, mount
in balsam. Such preparations are permanent.
This reaction of inorganic compounds of iron with hsematoxylin
seems to be one of oxidation (MAYER, Mitth. Zool. Stat. Neapel., x,
p. 170).
Extraordinarily small traces of inorganic iron are thus demon-
strated. The method is more sensitive than that of Prussian blue
or ammonium sulphide.
CHAPTER XXVI. 299
ORGANIC IRON COMPOUNDS. — These will not give the iron
reactions unless the complex iron compound has been broken up,
that is, the iron " unmasked " by some reagent : acid alcohol is
used for this. Sulphuric acid alcohol (4 per cent, in 95 per cent,
alcohol) and nitric acid alcohol (3 per cent, in 95 per cent, alcohol)
are better than the hydrochloric acid alcohol (Bunge's fluid).
Sulphuric acid alcohol acts very slowly especially on bulk tissues,
and even Protozoa take twenty-four hours at 35° C. before their
masked iron is revealed. Nitric acid alcohol acts more quickly
and extracts very little of the iron it liberates (which is a danger
with Bunge's fluid) ; the process is completed in about thirty-six
hours.
Sections are treated with acid alcohol, 90 per cent, alcohol,
and aq. dest., and then Macallum's hsematoxylin is added ; the
sections are washed in aq. dest., stained in safranin, as described in
next section, dehydrated and mounted in balsam.
PRUSSIAN BLUE REACTIONS ON ORGANIC COMPOUNDS. — Sections
after being treated in the acid alcohol (nitric or sulphuric) are
washed in pure 90 per cent, alcohol and then in aq. dest. They
are placed not longer than five minutes in the following solution :
aq. potassic ferrocyanide 1-5 per cent., and hydrochloric acid
0-5 per cent, in aq. solution equal parts, freshly made. Again
washed carefully in aq. dest. stained in eosin or safranin, dehy-
drated in alcohol, cleared in oil of cedar and mounted in benzole
balsam. The safranin or eosin are used in 1 per cent, strength in
30 per cent, alcohol, for three minutes for eosin, and for one half-
hour for safranin, and differentiated in 90 per cent, alcohol.
FERRIC AND FERROUS SALTS both occur in inorganic iron com-
pounds. Ferrous salts may be distinguished from ferric by the
fact that only the latter give an immediate reaction with ferro-
cyanide of potassium, while the former react with f erricyanide of
potassium. Fix material in alcohol of about 90 per cent, for several
days.
Reaction for Ferric Salts. — Wash sections in aq.. dest., transfer
to 2 per cent. aq. sol. ferrocyanide of potassium for from three to
fifteen minutes. Bring to acid alcohol (1 c.c. in 70 per cent, alcohol)
for about ten minutes. The Prussian blue reaction takes place.
Wash in pure 70 per cent, alcohol, dehydrate clear and mount in
benzole balsam.
Counter-stain if desired in eosin or safranin (op. cit.).
Ferrous Salts. — As above, substituting ferricyanide of potassium
instead of ferrocyanide.
300 CYTOLOGICAL METHODS.
Simultaneous detection of both categories of salts may be made
by using a solution of equal parts of ferricyanide and ferrocyanide.
BLOOD, AND IKON SALTS. — In § 789 is given Okajima's method
for elective staining of haematids (hemoglobin). Degeneration
products of hcemoglobin are hcemosiderin and melanin (of malaria).
Hsemosiderin is found in the liver in pernicious anaemia, and also
in large extravasations of blood. It is said that hsemosiderin will,
but melanin will not, give the Prussian blue and other iron reactions ;
both pigments survive dehydration and a clearing oil. Another
pigment derived from blood is hcematoidin or bilirubin, which
contains no iron and does not give the iron reactions.
Hcemoglobin itself is not unmasked by acid alcohol and will not give
the iron reactions, but stains bright red with eosin from Mann's
methyl-blue eosin mixture, and orange in Okajima's alizarin stain,
§ 789. Most pigments are destroyed by concentrated sulphuric
acid, which will not affect any carbon granules which may have
been fed to the cells experimentally.
See also TIRM ANN, Goerbersdorfer Veroeffentl.,ii, 1898, p. Ill ; SCHNEI-
DER, Mitth. Zool. Stat. Neapel, xii, 1895, p. 208 ; CARNOY and LEBRUN,
La Cellule, xii, 1897, p. 275 ; SUMITA, Arch. path. Anat., cc, 1910, p. 230 ;
ZALESKI, Zeit. Phys. Chemie, xiv, 1890 ; WASSERMANN, Anat. Hefte,
xlii, 1910, p. 283, JONES, Biochem. Jour. 1920.
Copper. — K. BOYCE and W. A. HERDMAN, in their paper on the
Green Leucocytosis in Oysters (Proc. Roy. Soc., Ixii, 1897 — 98), have
given directions for the application of the well-known potassic
ferrocyanide test of chemists, to sections of tissues in which copper
is to be detected. These authors fix with proper precautions (vide
supra, under " Iron ") in absolute alcohol, imbed in pure paraffin and
cut sections. Care must be taken to avoid acid solutions, such as
commercial turpentine or old xylol. Sections are brought from
absolute alcohol to distilled water, placed in a 1-5 per cent, solution
of freshly prepared potassic ferrocyanide or, preferably, in equal
parts of the same ferrocyanide solution, and a 0-5 per cent. HC1
solution, and parts where copper is present go a reddish colour.
Sections are then washed in aq. dest., dehydrated in absolute alcohol,
cleared in cedar-wood oil and mounted in Canada balsam.
See also MACALLUM, Journ. Phys. Cambridge, xxii, 1897, p. 92 ;
MARFORI, Arch. Ital. Biol, xxx, 1898, p. 186.
For Zinc see MENDEL and BRADLEY, Amer. Journ. Phys., xiv, 1905,
p. 320.
For Lime salts see GRANDIS and MAININI, Arch. Hal. Biol., xxxiv,
1900, p. 75 ; SCHAFFER, Zeit. wiss. Zool, Ixxxix, 1908, p. 13 ; LEUTERT,
Encycl. mikr. Technik, ii, p. 588 ; STOELTZNER, Arch. path. Anat.t
CHAPTER XXVI. 301
clxxx, 1905, p. 363 ; MACALLUM, Ergeb. Phys. Wiesbaden, vii, 1908,
p. 612.
For Potassium see MACALLUM, Journ. Phys. Cambridge, xxxii, 1905,
p. 95 ; Ergeb. Phys. Wiesbaden, vii, 1908, p. 600.
For Guanin see GIACOMO, Zeit. wiss. M.ik., xxvii, 1910, p. 257.
Concerning the microchemistry of the cell in general, see further fourth
edition ; also CARNOY and LEBRUN, La Cellule, xii, 2, 1897, p. 194 ;
ZIMMERMANN, Die Morphologic u. Physiologic des Pflanzlichen Zellkernes,
Jena, 1896 (treats also of the animal cell) ; HAECKER, Praxis u. Theorie
der Zellenund Befruchtungslehre, Jena ; PRENANT, Journ. Anat. Phys.,
xlvi, 1910, p. 343.
651. Cytological Fixing Agents. — A fixing agent that is good for
one element of a cell is 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 chromo-
somes 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 FLEMMING and liquid of HERMANN. There is a slight
difference 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. I find that it generally
makes them shrink too much, and that it is not at all good for spindles.
For many, if not most objects, I prefer to these two reagents
BOUIN'S picro-formol, which gives a highly faithful preservation
and a more penetrating and equable fixation.
For spindles I recommend Flemming (picro-formol does not give
quite such bold images).
Some of the finest chromosomes I have seen have been fixed with
LINDSAY JOHNSON'S mixture (§ 44), and liquid of TELLYESNICZKY
has given me others nearly if not quite as good.
As regards the cytoplasm. — Cytoplasm is made up of two elements :
a fibrillar element— the spongioplasm or mitome ; 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 " pre-
cipitation forms," see § 29), and the secretions or other enclosures
that may be present in it, may so mask the fibrils of the spongio-
302 CYTOLOGICAL METHODS.
plasm as to interfere with the observation 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,
i.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 advantage 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 acid and 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 to a 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 one-eighth of the prescribed
amount without loss of the distinctive characters of the fixation.
The defect of want of penetration seems to be incurable. See
§§ 35 and 42. Substitution of more highly penetrating 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 bichromate of potash. The formula that has
given me the finest fixations is that of LINDSAY JOHNSON, but it has
the drawback that there is risk of osmication in the outer layers.
In this respect liquid of Tellyesniczky, § 52, is to be preferred.
Corrosive sublimate gives a fairly full fixation ; but I believe it
frequently produces serious artifacts, HEIDENHAIN'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 inad-
CHAPTER XXVI. 303
missibly 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 pre-
ference to liquid of Flemming on account of the facilities they afford
for the employment of certain stains ; but to that end I prefer
BOUIN'S picro-formol.
652. Chromosomes ; Chromatin Stains.*— For fresh tissues, see
§645.
With hardly an exception modern work on chromosomes in the
germ-cell cycle is carried out by use of such fixations as strong
Flemming (§ 41), Bouin's picro-formol-acetic (§ 110), Carnoy (§ 85),
or an alcoholic nitric corrosive acetic of the Gilson or Petrunkewitsch
type. Stains now used much are iron haematoxylin of Heidenhain
or Benda, thionin, safranin, well ripened Delafield, and Mayer's
acid haemalum. Gentian violet is used by many. For the study
of the chromosomes, the desideratum seems to be some fixative
which will penetrate evenly and rapidly, which will strip out of the
cell, fats and lipoids, and which will allow the subsequent use of
some dense, preferably black or dark blue stain.
Some English workers have found Flemmiiig's strong formula, without
acetic acid to give beautiful results for chromosomes (e.g. L. HOGBEN,
Proc. Roy. Soc., B., xci, 1920). In nearly all my slides fixed for the
cytoplasmic inclusions (§ 673) good chromosome plates are found, but
I consider that fixatives which contain lipoid solvents are indicated for
chromosome work : the preparations are thereby " stripped " and
cleaner, and difficult nuclei are better interpreted when superfluous
materials are removed. SISTER MONICA TAYLOR (Quart. Jour. Micr.
Set., 1915) stains first in thionin, mounts and studies the sections ;
then, if they prove to be worth it, removes the coverslip by soaking in xylol,
and restains in iron alum hsematoxylin. Red stains are not indicated
because of the eye-strain they cause.
Iron alum hcematoxylin is especially recpmmended. Dr. Lee has
sent me the following note on the use of this stain : — " Some cyto-
logists have given up iron heematoxylin because they have found it
to clog the chromosomes ; but this will not occur if the following
precautions are observed. Mordant sections (7 to 8 /LA) for not more
than two and a half minutes in iron alum of 2 to 3 per cent. ; wash
for at least a quarter of an hour ; stain in a 0-5 per cent, solution of
ripened hsematoxylin until the sections appear dark grey, but not
black (about twenty-five minutes if the solution is fresh, and not
more than four if it has already had several slides passed through
* By J. B. G.
304 CYTOLOGICAL METHODS.
it) ; differentiate in the iron alum solution for at least two minutes
after the chromosomes, examined in water, appear to have been
sufficiently extracted, for chromosomes always appear paler in
water than- after they have been got into balsam." Personally
I have never found iron hsematoxylin to clog chromosomes, and can
only assume that something was wrong with the fixation, or a bad
specimen of stain was used.
Hot or Cold Fixation ? — Some workers advocate the use of hot
fixatives, others believe that the best results are obtained by keeping
the capsule or vial of fixative on ice while the material is being fixed.
Possibly cold, not freezing, fixatives are indicated for invertebrates
and cold-blooded animals, and fixatives at body heat for warm-
blooded. This, however, must be left to the discretion of the
worker, who may find that either very cold or hot fixatives may improve
his preparations in an unexpected manner. EZKA ALLEN recommends
using his modified chromic Bouin and urea at 38° C., while he
believes that Flemming should be used in an ice-box (Anat. Record
x, 1915 — 16). COWDRY (Contrib. Carnegie Inst. Wash., viii, 1918,
recommends the use of Regaud's formol-bichromate on ice. See
also § 31.
653. Dissection of Animals for Chromosome Work. — Some ob-
servers have claimed that dissecting out gonads of invertebrates, in
a dish of " tap- water," gives clearer chromosome figures than when
one uses Ringer or such salt solutions. If possible, avoid dissecting
out in any fluid. Fleas and lice, and such small insects, are to be
treated as follows : cut off the end of the abdomen, hold the insect
down on a glass slide by its head with the aid of a mounted needle,
and with another needle press the viscera out with a stripping
motion from the head backwards. Immediately transfer the viscera
to a fixative (DONCASTER, Quart. Jour. Micr. Sti., 1920). For
bigger insects one may qpen the body cavity after having cut off
their heads, and pipette fixative over the viscera before separating
away the gonads. Then transfer to a capsule of the fixative. Read
also directions in §§ 12 and 676.
654. Fusion of Chromosomes caused by Fixation.— It should be noted
that unsuitable or inferior fixation may cause such artifacts as fusion of
chromosomes which, intra vitam, may have merely been closely paired.
With Diptera it has been found that bodies of mosquitoes, etc., should not
be thrown whole into a fixer, but either finely teased or the gonads
should be carefully dissected out. This obtains a more rapid and there-
fore a more efficient fixation (HANCE, Jour. Morph., 1917 ; METZ,
Jour. Exp. ZooL, 1916).
CHAPTER XXVI. 305
In the same way more fluid chromosomes such as those of mammals
may be caused to run together arid so introduce error. This probably
explains the discordance in the accounts given by different workers for
such material as that of man.
655. Note on Fixatives for Chromosome Work. — For work on
vertebrate tissues there is little doubt that Bouin's picro-formol-
acetic, or one of its modifications, is the best mixture to use. For
invertebrates in general such Bouin fixatives are also very satis-
factory, but Flemming's strong formula should always be tried.
Corrosive acetic acid I hold to be a rough and unreliable fixative,
and if a corrosive fixation is desired, it is better to use Gilson or
Petrunkewitsch — these penetrate more rapidly and give a more
delicate fixation. I have seen some excellent chromosome plates
in mammals got by using Sansom's Carnoy modification (§ 86). It
should be noted carefully that many workers use chromosome
fixatives for a short time only ; for instance, Flemming may be used
on insect gonads for half to one hour only, followed by a washing
out under tap, and then upgrading from 30 per cent, alcohol (from
Professor LEONARD DONG ASTER, in literis).
656. Urea and Chromosome Fixation. — It has been claimed by
some American workers (McClung, Ezra Allen, K. T. Hance, etc.)
that the addition of from 0'5 to 2 per cent, urea crystals to fixatives
of the Flemming or Bouin type assists in penetration and gives
sharper pictures of the chromosomes. The idea of using urea is, I
understand, due to Professor McClung.
It is out of the province of this book to discuss whether a solution of
urea in such complicated fluids as Bouin or Flemming, has the same
peculiar penetrative properties as in water, or even to question whether,
after the addition of the crystals to the Bouin or Flemming, disintegra-
tion of the urea does not take place. We are prepared to accept the
statements of McClung, Allen and Hance, and to recommend a trial of
the method. WARO NAKAHARA (Jour. Morph., 1919), working on Perla,
did not find that the addition of urea crystals to his fixative made any
appreciable difference.
657. Fixation of Mammalian Chromosomes. — The material must
be absolutely fresh ; even half an hour's delay is fatal ; prepare your
fixatives and capsules, knives, etc., before you kill the animal.
Apparently chromosomes of mammals will clump together about
ten minutes after death. HANCE (Anat. Record, xii, 1917) gives the
following method : — (1) Obtain fresh specimens of tissue from as
many different animals as possible, so as to be sure of obtaining one
or more in a " cycle of cell division." (2) Place small or finely
M. 20
306 CYTOLOGICAL METHODS.
teased pieces of fresh tissue immediately into cold Flemming's
solution (on ice) plus about 0-5 per cent, urea crystals. Flemming's
solution kept on ice registers about 4° to 5° C. Leave in cold
solution for twenty-four hours or longer. (3) If this fixation fails,
try the following : Allow small pieces of fresh tissue to remain in
the air for from ten to twenty minutes after removal from the
animal before placing them in the cold Flemming.* Then fix as
before. (4) Wash in water about twenty-four hours. (5) Dehydrate
by very gradual steps. (6) Clear from 95 per cent, alcohol in cedar
oil followed by xylol. Embed in paraffin.
658. EZRA ALLEN'S Chromic Bouin and Urea. — Used for work on
the spermatogenesis of rat, etc., and generally indicated for mammals.
Picric acid, sat. sol. . . . . .75 c.c.
Formol (pure) . . . . . . 25 ,,
Glacial acetic . . . . . 5 „
To freshly-made mixture raised to temperature of 38° C. add and
dissolve, first, 1-5 grms. of chromic acid crystals, and then 2 grms.
of urea crystals. Kill animal by decapitation, remove testis imme-
diately, snip into small pieces, fix at 38° or 40° C. for from one to
two hours. Fixative replaced by " drop method " with 70 per cent,
alcohol, picric acid washed out by addition to the alcohol of a sat.
sol. of lithium carbonate, a few drops at a time ; the alcohol is
replaced by anilin oil (freshly distilled), this by synthetic oil of
wintergreen, and this by paraffin of 52° melting point. The paraffin
is slowly added till the tissue is in a bath of high paraffin concen-
tration. It is then passed through several changes of pure paraffin
to remove oil (EZRA ALLEN, Jour. Morph., 1918 ; Anat. Record,
x, 1916).
659. Amphibian Chromosomes.— CHARLES PARMENTER (Jour.
Morph., 1919) uses Ezra Allen's chromic Bouin with success for
amblystoma. Ordinary Bouin with urea crystals, Hermann, and
Flemming were also good.
660. Modified Bouin and Urea for Insects.— Miss E. ELEANOR
CAROTHERS for orthopterous chromosomes uses the following fluid
which has been developed in the Zoology Laboratory, Pennsylvania.
It is a modification of Bouin' s formula : —
Picric acid, sat. sol. aq 75 c.c.
Formalin (strong) 15
* This, of course, is directly contrary to all the rules of fixing tech-
nique, but it might work satisfactorily.
CHAPTER XXVI. 307
Glacial acetic acid ...... 10 c.c.
Urea crystals J grm.
Use for twenty-four hours. Stain in iron haematoxylin or Flemming's
tricolour mixture (Jour. Morph., xxviii., 1916 — 17).
661. Precautions in Dehydrating and Clearing. — It is well known
that too rapid dehydration or clearing will cause shrinkage and
distortion. Material to be used for chromosome study should be
dehydrated gradually, either by a syphon or some drop method
(§ 3). For clearing xylol is not good, as it causes much shrinkage.
The least shrinkage occurs with some vegetable oil, like bergamot,
origanum, cedar wood, or cassia (cinnamic aldehyde), which, if it
will not mix with paraffin, should be washed out subsequently in
some paraffin solvent, like benzole or xylol. EZRA ALLEN (Anat.
Record, x, 1915 — 16), following Suchannek (§ 134), uses distilled
aniline oil as a substitute for the higher alcohols. One gradually
brings brain and genital or such tissue to 75 per cent, alcohol, and
then adds distilled anilin oil by the drop method, shaking frequently,
or using some system for agitating the fluids, or by some diffusion
apparatus (§ 3). When nearly pure anilin oil has replaced the
alcohol, one transfers to pure oil until the tissue is cleared. From
anilin oil one embeds as follows : warm the oil and tissue slightly,
adding every ten minutes a few drops of melted paraffin, mixing
thoroughly with a pipette ; continue till the mixture has 85 to 90
per cent, paraffin. Transfer to melted paraffin. If bergamot oil
has been used for largish objects, at least four changes of pure
paraffin must be made, one half-hour to each, and the fifth bath
for at least one hour. In most cases gradual clearing in cedar- wood
oil will give satisfactory results. See also § 5.
662. Mounting Sections between Coverslips. — AGAR (Quart. Jour.
Micr. Sri., 1911) has devised a method for mounting prepara-
tions between coverslips in order that they can be observed on both
sides. Use one larger coverslip as if it were a slide. Carriers may be
made by stamping out a square in stiff cardboard, or thin metal.
See also, C. CEPEDE, C. R. Soc. Biol, civ, 1913.
663. Smear Preparations of Gonads. — In some cases smear pre-
parations of testes especially may provide useful evidence in a
research on chromosomes. One may be fortunate enough to find
nuclei at the prophase or metaphase of mitosis, with all the chromo-
somes spread out so as to be counted with ease. In many cases,
to study early stages in synapsis for which very rapid penetration
is essential, smear preparations are a sine qua non.
20—2
308 CYTOLOGICAL METHODS.
Remove the testes ; if it is large take a fragment by a pair of
forceps and quickly smear along the length of a dry slide several
times, so as to cover as much of the middle part of the slide as
possible. If the cells are likely to stick, fix immediately by pouring
on some Flemming, Bouin, or Petrunkewitsch. Set aside for a few
minutes, wash off in water, upgrade from 30 per cent, alcohol, and
leave overnight in 90 per cent, alcohol. Bring back to water,
stain in iron haematoxylin, thionin, or gentian violet, etc. See
also Groodrich's iodine-Bouin method (under "Protozoa"). Note
that smears may be fixed in steam, acetic, osmic, formalin vapour,
or " Lucidol " (§§ 107, 783), or stained and fixed simultaneously in
Leishmann, acetic Bismarck brown (§ 277), aceto-carmine (§ 221),
or such mixtures. Smears of very liquid testes, like those of
Lepidoptera, are liable to be washed away if fixative is added too
soon. It is probably best to kill the cells in some toxic vapour,
then allow them to dry a little, and then fix in a liquid.
FOOT and STROBELL (Arch. f. Zellf., Bd. xii, 1914) recommend the
following : — Place testes in drop of acidulated (acetic) " water " on end
of slide, and with fine needle (No. IX.) cut from it the area at stage
required (previously ascertained by examination oi sections). Push
this area by point of needle to middle of slide and break up the tissue by
gently tapping with the needle (never roughly spread as recommended
by some workers). This should be done under dissecting microscope.
See also Kernschwarz, and ' ' Iron Carmine," § 222. For BATAILLON and
KOEHLER'S borax-methylen-blue see Gomptes Rendm, cxvii, 1893, p. 521.
664. The So-called Microchemieal "Tests" for Chromatin.*—
Among these the " digestion tests " have met with some favour.
It is well-known that examination of the partly digested tissue from
the gut of animals which eat fresh cells (e.g. parasitic hymenoptera)
shows that the nucleus of the cell resists digestion for the longest
time. On the other hand, there are a whole series of enzymes which
are specially concerned in the hydrolysis of nucleic acid, each acting
on some particular substrate ; these enzymes are grouped together
as " nucleases." The results of tests carried out with proteolytic
enzymes on the nature of unidentified cell granules must not be
depended upon too much. Extracts of such organs as the spleen
and pancreas are known to contain more than one kind of enzyme,
* By " chromatin " I mean that material which forms the substance
of the chromosomes. The question of chromatin, nucleoli, and basophil
granules is one of the most difficult in Cytology. (See GATENBY, Quart.
Jour. Micr. Sci., vol. 64, 1920, Science Progress, January, 1921,
E. LUDFORD, Jour. Boy. Micr. Soc., 1921, and recent numbers of the
Quarterly Journal of Microscopical Science.)
CHAPTER XXVI. 309
and the zoologist especially is advised to get the help of an expert
on enzymes before attempting to interpret any experiments he may
have made with such intra-cellular enzymes. Because a certain
protease will not dissolve away a given cell granule, while a
" nuclease " may do so, does not by itself provide good evidence
for the conclusion that the granule in question is " chromatin," or
derived from the chromosomes.
VAN HERWERDEN (Arch. f. Zellf., x, 1913) for instance, using the
extract of spleen containing " nuclease," finds that the granules identi-
fied as " chromatin " emission by Schaxel are dissolved away. In all
probability, however, such granules are phospholipin and not chroma-
tinic, and were dissolved away by some lipolytic enzyme in the solution
used by Van Herwerden.
Successful experimentation on this side of cytology calls for two
desiderata : Firstly, an intimate knowledge of the nature of the
enzyme solution to be used, and of what effects the latter has on
various definitely identified categories of cell substances ; second,
a thorough study of the origin and microchemical reactions of the
body to be treated by the enzyme solution. See VERNON, Intra-
Cellular Enzymes, London, John Murray, 1908 ; BAYLISS, The
Nature of Enzyme Action, Longmans, Green & Co., 1920 ; EULEE,
General Chemistry of the Enzymes, Pope's Translation ; BAYLISS,
General Principles of Physiology, 1919.
665. Method of Using Enzyme Solutions. —These should be used on
fresh cells. It is best to begin by identifying cell or nuclear bodies or
granules in properly fixed and stained preparations ; after this one
should familiarise oneself with the appearance of such granules or
bodies in the fresh cells, with and without intra-vital staining.
The enzyme or digesting fluids may be added to fresh cells, a coverslip
provided, and the preparation observed at intervals under a high power.
Another method is to immerse pieces of fresh tissue in the digestive
fluid for suitable times, and then fix the tissue in some mixture by
previous trials indicated for the purpose ; controls should be made by
soaking other pieces of tissue for a similar time in some of the same
solution inactivated by heat, or without the added enzyme.
As indicated above, such tests are of doubtful value as regards the
identification of " chromatin " ; the solutions used should be tried
carefully on cells whose cytology is thoroughly known by the observer.
The use of proprietary "pepsins " and such-like solutions whose origin
and method of manufacture are unknown to the worker, is not likely to
lead to the best results. See also C. BECKWITH, Jour. Morph., xxv,
1914, and JORGENSEN, Arch. f. Zellf. , x, 1913, and § 645.
666. Chroinophility. — Some workers indiscriminately call all
basophil chromatic material chromatinic, which is unjustifiable :
310 CYTOLOGICAL METHODS.
the chromatic " blushes " which are sometimes found surrounding
the nuclei of eggs and other cells are sometimes believed to be
chromatinic, but the evidence for this is very slender. Chromatin,
moreover, may sometimes stain oxyphil, and it is now a well-known
fact that the nature of the fixation and the method of staining are
important factors in deciding whether the bodies in question will
stain in the basic or the acid dye. Under " Nucleoli " are given a
number of staining and fixing methods which may be tried. See
also Chapter XI, § 211.
667. "Vital" Staining of the Nucleus.— A. M. PRZESMYCKY
(C. R. Soc. Biol., Ixxviii, 1915) uses neutral red. The living nucleus
is said to have a greater affinity for neutral red than protoplasm, as
it stains more strongly and decolourises more slowly. This is not
my experience with weak neutral red. CHAMBERS (Science, 1912)
uses janus green, which shows both chromosomes and spindle
fibres. Certain physiologists doubt whether the living resting
nucleus can be stained ; some observations on this will be found in
Chapter XI., § 207. Reference may be made to the paper of P. G.
SHIPLEY (Amer. Journ. Physiol, xlix, 1919).
It has been asserted by some observers that the nucleus may also
be stained during the life of the cell by means of Bismarck brown,
Congo red, methylen blue, Nile blue, and safranin. But Bolles
Lee believes that 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. In any case there is no dye known which is a specific intra-
vital test for chromatin. It seems probable that the most " specific "
test for chromatin known to cytology is acidulated methyl green,
for which, see §§ 278 and 645.
668. Nucleoli. — The word is nowadays generally used to mean
any large rounded stainable body in the nucleus. The plasmosome
is " acidophilous " in so far as, in fixed material, it selects the acid
dye or dyes from mixtures such as the Ehrlich-Biondi, which stains
it reddish or orange. So far as known true plasmosomes of unfixed
cells do not stain green with the acid methyl green, but while this
may hold for the plastin nucleoli of somatic cells in general, there
are to be found in eggs complicated nucleoli which have not properly
CHAPTER XXVI. 311
been investigated. Even if part of these nucleoli did stain green
in acidulated methyl green, it would be unsafe to interpret such
material as chromatin, especially in view of the fact that methyl
green will stain silk and mucin, and the secretions of many gland
cells ; plasmosomes often seem to be related in some way to gland
secretion.
Most of the work on plasmosomes or true nucleoli has been
carried out on fixed material, and it cannot be too strongly emphasised
that by such means it is not possible properly to discriminate between
what is true chromatin, and what is not true chromatin. There are no
perfectly specific chromatin dyes known, the nucleoli in general
may be made to stain differently according as to whether they may
have been fixed in acid or non-acid fixatives (this applies especially
to acetic acid), or in osmicated or non-osmicated mixtures, while
true chromatin may itself stain " acidophile," as, for instance, in
the head of the sperm during spermateleosis, and in the egg during
oogenesis.
These remarks refer also to the use of so-called specific dyes for
discriminating between and identifying the various cytoplasmic
inclusions. See especially §§ 707 et seq.
The case of the so-called karyosome or chromatin nucleolus is
even less satisfactory ; one often meets with " solid " intra-nuclear
bodies which stain basophil with many so-called nuclear dyes, but
which are only doubtfully to be regarded as chromatin. In this
connection, see Bayliss (§ 204) on specificity of dyes.
Safranin and light green, and iron hsematoxylin, following 'strong
Flemming's fluid, are classic stains for nucleoli, but neither of these
methods is to be regarded as providing any useful evidence as to the
micro-chemical nature of the bodies they tinge. They simply stain
most deeply, solid bodies.
The relationship between nucleoli and the chromatin network is quite
unknown in general, but CAJAL and CARLETON (for references see
Quart. Jour. Micr. 8ci, Ixiv, 1920) have shown by formalin and silver
nitrate methods that both basophil and oxyphil nucleoli may contain
an argeiitophile core. Carleton has followed the core through mitosis,
and finds that it keeps its individuality ; the definitive nucleolus of the
" resting " nucleus is possibly derived after every mitosis from the core
or nucleolinus. - 1 have found nucleolini in the nucleoli of the gut cells
of Saccocirrus, and in foUicle -cells of the ovary of insects ; nucleolini
are known in many kinds of vertebrate cells.
For the study of nucleoli the following methods are advised :—
(1) Fixation by a corrosive sublimate, both acidified by acetic acid,
and alone. (2) Fixation by hot water or steam. (3) Fixation by a
312 CYTOLOGICAL METHODS.
variety of mixtures such as Carnoy, Bouin and Zenker with or
without acetic acid, etc. Staining in Ehrlich-Biondi, Ehrlich's
haematoxylin and azoeosin or Biebrich scarlet (by Scott's method,
described below), by Pappenheim's pyronin and methyl green, by
Auerbach's fuchsin and methyl green, Zimmermann's fuchsin iodine
green, and by Mann's methyl-blue eosin. The double and triple
simultaneous stains are valuable.
Auerbach's stain consists of equal parts of 1 per cent, methyl
green, and 1 per cent, acid fuchsin ; Pappenheim's stain (§ 292)
consists of methyl green and pyronin, a red basic stain instead of
the acid fuchsin of the Auerbach. Another stain which is very
valuable is the triple stain of Ehrlich, but it is less easy to work than
Auerbach or Pappenheim. In all work on nuclei and nucleoli,
Mann's methyl-blue eosin will be found especially helpful, because
the eosin-staining from this mixture is generally more restricted
and intense than when one stains in some basic dye followed by
eosin, or vice versa. Very beautiful results are occasionally procured
by using Unna's polychrome methylene blue (§ 337).
Recourse should be made to the methods for the mitochondria,
particularly those such as Champy-Kull and Bensley-Cowdry
(compare with Auerbach preparations), for many nucleoli are
compound bodies almost certainly containing lipoids or fats.
The formalin silver nitrate techniques of Cajal or Da Fano should
be tried, and wherever possible tests on fresh cells should be carried out
(e.g. digestion, methyl green, etc., etc.).
It seems indicated that further observations carried out on
nucleoli of live cells in tissue cultures will provide new facts,
especially with regard to the part played by these bodies during
mitosis. See sections on " Tissue Culture."
669. S. Gr. SCOTT'S Standard Hsematoxylin and Biebrich Scarlet for
Chromophility (Jour. Path, and Bact., xvi, 1912). — Fix tissue in
sublimate formalin, Zenker without acetic, Helly's Zenker-formalin,
Miiller, or formalin. All strongly acid fixatives must be avoided,
for the Ehrlich's haematoxylin will not then stain anything but
nuclei, and these only faintly. Paraffin sections are made and fixed
,to slide. After removal of paraffin with xylol, and this with alcohol,
sections of material fixed in sublimate solutions are treated with
iodine for three or four minutes (0-2 per cent, iodine in 80 per cent,
alcohol). Rinse off excess iodine with a little alcohol and remove
all iodine from tissue with a 0-25 per cent, solution of Na2S203 in
50 per cent, alcohol, not in water as recommended by HEIDENHAUN
(Arch.f. d. ges. Pkys., Bonn, 1902, Bd. xc, § 115). This is a most
CHAPTER XXVI. 313
important part of the method, as sublimate and iodine both act detri-
mentally to staining. After washing in Na2S203, wash off in a stream
of aq. dest. ; roll the water off the slide with 1 or 2 drops of alcohol
(90 per cent.), wipe the slide dry around the sections, and pour on
3 to 4 drops of Ehrlich's acid hsematoxylinVto every section. Leave
exposed on bench ten minutes. Remove the hsematoxylin by
rolling it off with drops of alcohol (90 per cent.) from a drop-bottle ;
do not wash off the stain with water as this lowers the viscosity of
the solution and allows a diffuse staining.
Wash away alcohol with a stream of aq. dest. till every trace of
stain is removed from the slide.
Blue the haematoxylin lake and remove acid from combination
with the proteins of the section by dropping on the latter 8 to 10
drops (or more for a number of sections) of the following tap water
substitute :—
KHC03 2grms.
MgS047H20 20grms.
Aq. dest 1000 c.c.
Sat. with camphor, or thymol,
After three to five minutes wash off the alkaline solution thoroughly
in aq. dest. Wipe around sections to remove superfluous water,
and add a 1 in 2000 solution of azoeosin (not eosin) or Biebrich
scarlet dissolved in this medium :—
Grlycerol 2 per cent.
Methyl or 96 per cent, ethyl alcohol .8 „
Dist. water . . . . . 90 ,,
Use azoeosin (Bayer) for bichromated material and Biebrich scarlet
for non-bichromated material. Leave in the acid dye for ten to
thirty minutes, rinse off in aq. dest., rapidly dehydrate in 90 per cent,
and absolute alcohol ; xylol, and xylol balsam.
In my hands weak watery solutions of good Eosin have not given such
satisfactory results. Scott believes that sulphonated monazo colours,
such as orange G and Bordeaux R, are useless. Eosin is much less
precise than Biebrich scarlet and azoeosin. The special points about
this method are :— (1) Getting rid of all corrosive and iodine. (2) Stain-
ing by placing hsematoxylin on slide where the solution .can evaporate,
and thus arrive at a. state of viscosity which automatically prevents
overstating. (3) Washing off stain in alcohol, not water, thus pre^
venting diffuseness. (4.) A definitely alkaline tap-water substitute.
(5) A precise counter-stain.
Histologists and cyfcologists carrying out work on chromophility are
recommended to use this method as a standard for either basophility or
oxyphility. Note, however, that granules which stain basophil by this
314 CYTOLOGICAL METHODS.
method are not necessarily chromatinic. Occasionally this method
seems to overstain genital cells, but this gives the correct degree of
chromophility of such cells in comparison with the cells of other tissues.
After having used the method critically, I have come to the conclusion
that it is more precise than the usual procedure which introduces
differentiation in an acid solution. Scott worked out this method
mainly with sections of mammalian red bone marrow.
669a. OBST (Zeit. wiss. ZooZ.,lxvi, 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.
BUCHNER (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. 463) stains for ten minutes in
a fresh mixture of 9 parts 0-1 per cent, aqueous iodine green with 1 part
concentrated aqueous solution of fuchsin, and differentiates in absolute
alcohol with 1 per cent, of acetic acid and 0-1 per cent, of iodine. Nucleoli
red, chromatin blue.
FISCHER (Fixirung, etc., p. 140) adds 30 drops of hot 0-1 per cent,
fuchsin solution to 100 c.c. of 0-3 to 0-5 per cent, solution of methyl
green.
MONTGOMERY (Journ. Morph., xv, 1899) stains for an hour in Ehrlich's
haematoxylin, and then for five minutes in concentrated aqueous eosin,
or first with concentrated aqueous methylen blue, and then with con-
centrated alcoholic solution of Brazilin.
For a series of papers on staining nucleoli, and illustrated by
profuse coloured plates, see MAX JORGENSEN'S memoirs in the Arch,
f. Zellf., x, 1913. Another paper worth consulting is CHAMPY'S
magnificent work on the male germ-cells of amphibia in the Arch,
de Zool Exper., 1913.
Sse also REDDINGIUS, Virchow's Arch., clxii, 1900, p. 206. For
nucleoli of ova, LIST, Mitth. Zool. Stat. Neapel, xii, 1896, p. 430 ; of
nerve-cells, RUZICKA, Zeit. wiss. Mile., xiv, 1898, p. 453, and LEVI, Eiv.
Pat. Nerv. Ment. Firenze, iii, 1898, p. 289.
670. Plasma Stains. — Dr. Bolles Lee states that he has been unable
to discover a single thoroughly satisfactory one. Almost all of
them colour too readily the enchylema 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.
We consider Saurefuchsin and Biebrich scarlet the most generally
recommendable, especially after iron hsematoxylin. See also
Bordeaux R.
Flemming's orange method has been much used. It is very
capricious and unreliable. Ehrlich-Biondi mixture is a celebrated
plasma stain.
CHAPTER XXVI. 315
The Iron-Hcematein Lakes of Benda and M. Heidenhain give good
plasma stains, according to the degree of extraction, 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 Euparal instead of balsam.
671. 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 far the best stain
is iron-hcematoxylin.
It is said by Heidenhain that the stain is obtained in a sharper form
by combining the hsematoxylin stain with a foregoing stain with Bor-
deaux It. He directs (Arch. mik. Anal., 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 Bordeaux, until they
have attained such an intensity of colour as that " they would just be fit
tor microscopic examination with high powers " (1. c., p. 440, note), and
that they be then brought into the ferric alum. After mordanting and
staining, the hsematoxylin 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-haematoxylin require to
be interpreted with special care. 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. It seems that certain
erroneous observations that have been published have been due to
this deception.
672. 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," " Mitochondria " and " Fat." The most generally
employed stains are the mixtures of EHRLICH.
Intra-vitam staining is useful here. See § 208. See also ARNOLD,
Anat. Anz., xxi, 1902, p. 417.
BENDA (Verh. phys. Ges. Berlin, 1899—1900, Nr. 1—4, and Verh.
Anat. Ges., xv, 1901, p. 172) gives the following method for demon-
strating secretion-granules and distinguishing them from other
granules : Harden for twenty-four hours in 10 per cent, formalin,
316 CYTOLOGICAL METHODS.
then for one day in 0-25 per cent, chromic acid, one in 0-33 per cent,
and two to three in 0-5 per cent., wash one day in water, dehydrate
and make paraffin sections. Then stain with one of Ehrlich's
mixtures, according as the granulations are basophilous, acido-
philous, or neutrophilous. The methylen-blue and eosin process of
Michaelis is recommended.
See also Mallory, §§ 271 and 272 ; Mann, § 328.
673. Mitochondria,* Golgi Apparatus,! Yolk, Fat, and other
Cytoplasmic Inclusions. I — The mitochondria and Golgi apparatus
never clearly appear in stained sections prepared by such methods
as fixation in corrosive acid, Gilson, Bouin, Carnoy or Flemming-
with-acetic acid, and staining in Ehrlich's haematoxylin and eosin,
toluidin-blue and eosin, paracarmine and borax carmine. Though
the mitochondria and Golgi apparatus are properly fixed by formalin,
Muller, Flemming- without-acetic acid, Champy, Altmann, etc., they
will rarely appear visible in stained sections which have been pre-
pared in Ehrlich's or Delafield's hsematoxylin or carmine stains, or
in fact in any of the current laboratory stains used for general
zoological purposes. The mitochondria and Golgi apparatus may
appear visible in sections fixed in formalin, Mliller, etc., and stained
in Altmann's acid, fuchsin-picric acid, iron-hsematoxylin, Benda's
alizarin and crystal- violet, etc. The Golgi apparatus rarely becomes
visible after any of the above methods, and to study it one must
use more specialised methods ; to study the Golgi apparatus and
the mitochondria by routine zoological laboratory technique is not
possible, simply because these methods will not demonstrate the
bodies in question. Nearly all of the older fixing mixtures contain
either alcohol, chloroform, or acetic acid, but the last few years of
cytological research have shown that the picture given by a fixing
mixture containing them is incorrect and inadequate, and one
cannot fail to be surprised at the improvement produced when these
reagents are omitted. Nearly all the modern research on the cyto-
plasm has to be carried out by observers using chrome- or platinum-
osmium fixatives, followed by iron-alum hsematoxylin, Benda's
crystal violet, or Altmann's acid fuchsin ; or by the important
Kopsch and Mann-Kopsch, and Sjovall osmium tetroxide methods ;
or by the useful methods of Cajal, Golgi or Da Fano's modification
* Chondriosomes, chondriokonts, plastochondria, " chromidia," bio-
blasts, chondriome, chondriomites, etc., etc.
t Nebenkern batonettes, idiozome rods, " Golgi-Kopsch apparat,"
apparato interno reticolare, dictyosomes, Binnennetz, etc.
$ By J. B. 0.
CHAPTER XXVI. 317
of Cajal, which consist of silver nitrate impregnation following
formalin fixation. Intm-vitam methods, such as janus green,
neutral red, or dahlia violet a& also used extensively. The mito-
chondria are extremely fuchsinophile, and after chrome-osmium
fixation stain strongly in iron-alum hsematoxylin. The Golgi
apparatus of somatic cells and of ovarian cells rarely stains by these
methods (Altmann or Heidenhain) unmodified, although the Golgi
apparatus of the male germ cells nearly always stains in fuchsin or
haematoxylin after chrome-osmium or formalin fixation. Besides
observing the Golgi apparatus and mitochondria, certain workers
have turned their attention to the study of fats and yolk (vitellus)
in cells.
In § 768 is a special article on fats -and lipoids, and on methods for
their study ; on the following pages are set forth various techniques
for the investigation of definite cell organs known to be partly lipoid
in nature. The application of all these methods to embryological
study opens the way to a valuable field for research. In § 768 will
be found definitions of the terms " fat," " lipoid," and " lipin."
Fats or lipoids form a special part of almost all cell-organs, as seems
to be indicated by fixing tests, and so far as we know such substances
are always intimately associated with protoplasm. Many of the
lipoids appear to be able to form with certain metallic salts
or oxides such as Cr03, K2Cr207, PtCl4, Os04, etc., compounds
insoluble or only slowly soluble in alcohol or such clearing oils as
xylol, benzole, or chloroform ; this is one of the several reactions
which take place when a cell is fixed in such a fluid as that of
Flemming (without acetic acid), Champy, or Altmann, and subse-
quently dehydrated and cleared. See §§ 29, 30 and 31.
674. Choice of Method.* — We have given below a number of
methods for lipoid granules, mitochondria, and other cell inclusions,
and not all are suitable for every piece of work. It is very rare to
find that one single method will produce the same good result in
both vertebrate and invertebrate tissues. In the same way, methods
which act satisfactorily with amphibia will often give disappointing
results with mammalia. Osmic-chrome fixation will nearly always
be found excellent for all classes of invertebrata ; Flemming- without-
acetic acid and Champy-Kull can be highly recommended. For
amphibia the addition of some K2Cr207 to the Flemming is necessary
before a correct fixation of the mitochondria is obtained ; thus
* The beginner is recommended to master such techniques as those of
Bouin's fluid and borax carmine, or Zenker and Ehrlich's hsematoxylin
before trying these methods.
318 CYTOLOGICAL METHODS.
Champy's fluid was invented for amphibia and gives very satis-
factory results (§ 43). For mammalian tissues a preliminary
fixation in osmic acid fixatives is not generally indicated ; the
tissues of mammals are far more " fatty " than those of invertebrata
or amphibia, and one finds that the Os04 becomes reduced very
rapidly and penetration is very poor. For mammalian tissues
formalin-chrome (Regaud, Bensley-Cowdry), formalin-corrosive or
formalin alone are indicated as a preliminary treatment at the least.
Formalin does not destroy lipoids, and by subsequently placing small
pieces of chrome-formalin fixed tissues in osmic acid (post-osmica-
tion), a fixation of lipoids and fats is obtained (Schridde) ; the same
result may be got by fixing tissues in chrome salts and then trans-
ferring to osmic acid. It should be noted, however, that previous
fixation in a chrome salt prevents the blackening of the Golgi
apparatus ; the formalin-osmic acid method (Sjovall) is indicated
especially for mammalian tissues, when an impregnation of the Golgi
apparatus is required by means of an osmic method, but the formalin
silver nitrate methods of Golgi, Cajal and Da Fano are always
clearly indicated for work on the Golgi apparatus of mammalian
tissues. So far as possible intra vitam and fresh smear preparations
should be used, as these nearly always give valuable results.
675. Specificity of Techniques for Cytoplasmic Inclusions, Fats,
and Lipoids. — -As a rule the lipoid granules, vacuoles, and cell organs
containing fats or lipoids are formed not of one pure substance, but
of a mixture of several. Consequently it is necessary to proceed
with caution in claiming a specificity for the techniques for various
lipoid substances : properly used, however, microchemical methods
may give valuable evidence as to the precise chemical nature of any
special body : microchemical methods, which depend for their
application on the use of complicated fixing and staining methods
are to be used cautiously. For example, Benda (§ 683) and Altmann-
Bensley methods (§§ 680 and 686), will stain granules other than
mitochondria, while the Cajal formalin uranium and silver nitrate
technique impregnates bodies apart from the Golgi apparatus. In all
these cases, however, the number of such exceptions to a specificity is
small, and suitable differentiation between two doubtful bodies can
be made by some other method. See Bayliss on " Specificity " of
staining methods, § 211.
676. On Killing Animals for Cytological Purposes. — So far as
possible avoid narcotics of any sort. Either cut off the heads of
invertebrates, or, if delicate like some worms, drop them whole in
CHAPTER XXVI. 319
the fixative ; kill vertebrates by a blow on the head, or by pithing.
If for a study of brain, bleed, or anaesthetise in coal gas, less pre-
ferably chloroform or ether. Insects can be killed with cyanide
or xylol.
Hints on removing Tissues and Cutting. — Avoid pinching the
material with forceps, as this will introduce artifacts ; it is preferable
to remove tissue without recourse to dissection under tap- water or
salt solution ; for Kopsch techniques, quickly remove blood or
lymph, etc., from surface of material with aq. dest. before placing
in fixer ; for cutting tissue the best instrument is a new safety
razor blade stuck in a special holder made for the purpose, or in a
split penholder, or held by artery forceps. When working on
arthropods, it is best to dissect the organ from the animal, instead
of preserving the whole body ; surrounding fat, etc., should be
removed. See also §§12 and 653.
677. Chrome-osmium Techniques. — Potassium bichromate or
chromium trioxide, used in watery solutions will not oxidise true fats
(see § 768) in such a way that full vacuoles of the latter will appear
in finished sections prepared by routine methods ; but combinations
of such salts with osmium tetroxide provide fixatives which will
preserve almost all cell elements in finished sections. The basis of
all chrome-osmium techniques consists in a preliminary fixation of
small pieces of tissue, small embryos or eggs, in such a fluid as Benda,
Champy, Flemming-without-acetic acid, or Altmann, for from at
least twelve hours to a week. The osmic reaction is then, in some
methods (Kull, Benda), " set " or strengthened by the reducing
effect of pyroligneous acid ; following this treatment is a further
" chroming " in 3 per cent, bichromate of potash, and, finally, a
thorough wash out under the tap. Material treated in this way is
generally perfectly preserved, and fit for selective staining. Arranged
below are chrome-osmium techniques of progressive intensity and
difficulty.
678. Modified " Flemmings " for Cell Inclusions. — BENDA : 15 c.c.
chromic acid 1 per cent., 4 c.c. osmic acid 2 per cent., 3 to 6 drops
of acetic acid. MEVES : 15 c.c. of chromic of 0-5 to 1 per cent.,
containing 1 per cent, sodium chloride, with 3 to 4 c.c. of 2 per cent,
osmic acid, and 3 to 4 drops of acetic (Encyd. mik. Techn., 1910).
G-ATENBY : Strong Flernming without acetic acid ; and same
solution diluted by one-half or one-third (Quart. Journ. Micr.
Sci., 1919). The presence of a small quantity of acetic acid is
always liable to introduce distortion, but less so among vertebrates
320 CYTOLOGICAL METHODS.
than among invertebrates. See also " Champy's Fluid " (§ 43) and
" Altmann's Fluid " (§ 42), and § 41.
679. FLEMM ING'S Strong Fluid without Acetic Acid, and Iron
Haematoxylin. — Small organs freshly dissected out in normal saline,
or parts of organs cut with safety razor blade, not more than 5 milli-
metres in diameter, are placed in about 15 c.c. of one of the above-
mentioned fixing fluids, preferably Gatenby's modification. A
glass-covered capsule or vial is the best vessel to use, and the material
is left for at least twenty hours, and not longer than one week. We
find about twenty-four hours gives a satisfactory fixation of most
tissues. After fixation the liquid is poured away, and the material
is washed for at least two hours, and not necessarily longer than
five, in running tap- water. It is then passed through up-graded
alcohols, beginning at 30 per cent., giving the material at least
three hours in the strengths 30, 50 and 70 per cent., and overnight
in 90 per cent. The pieces of tissue are dehydrated two or three
hours in two changes of absolute alcohol, and then transferred to a
mixture of half absolute alcohol and half xylol for a quarter of
an hour ; then pure xylol,. and embedded in wax. Sections are cut
from 4 to 8 //, but I generally find 6 /u, to be convenient. Leave
eight to ten hours- in iron alum, twelve to twenty hours in hsema-
toxylin. This method gives a delicate and precise stain of the
mitochondria (and Golgi apparatus or nebenkern batonettes of male
germ-cells only), fat is black, while yolk is generally greenish brown.
Especially recommended for germ-cells, and histology of Inverte-
brata, but with vertebrate tissues, and especially mammalian
material (not embryos), it often gives atrocious results ; for such
material, Helly, Zenker, or Kegaud's methods are indicated
(GATENBY, Quart. Journ. Micr. Sci., 1919).
Note that Fleming-without-acetic acid is not a suitable fixative for
after-staining in Altmann's acid fuchsin. For this the material must be
washed in distilled water for a short time after fixation, and then
transferred to 3 per cent, bichromate of potash for three days ; or the
more elaborate mordantage as for Champy-Kull may be used (§681).
680. ALTMANN'S Acid Fuchsin and Picric Acid (Die Elementaror-
ganismen, Leipzig, 1890).— Fix twenty-four hours in mixture of
equal parts of 5 per cent, bichromate of potash and 2 per cent, osmic
acid. Embed in paraffin, stain sections on slide for one minute
over flame, with a solution of 20 grms. (sic) of acid fuchsin in 100 c.c.
of aniline oil-water (§ 286). Cool, and wash out in a saturated
alcoholic solution of picric acid diluted with 2 volumes of water,
CHAPTER XXVI. 321
heat being used as before to aid differentiation ; blot, dip into
90 per cent, or absolute alcohol, xylol, balsam. This method only
stains granules which can be seen intra vitam ; properly used it
never produces artifacts, and Fischer's critique is quite wrong
(Fixirung Faerbung u. Bern des Protoplasms). Altmann's original
method has been superseded more or less by the following method
of Champy-Kull. (Both Dr. J. A. Murray and I find that the
20 grms. of acid fuchsin will not dissolve in 100 c.c. of aniline oil-
water ; only about 5 to 7 grms. will dissolve, and this quantity will
make a perfectly efficient solution.)
681. CHAMPY-KULL'S Acid Fuchsin, Toluidin Blue and Aurantia
(KuLL, Anat. Anz., Bd. xlv, 1913).— The following method, while
being generally useful, will be found very convenient for work on
Invertebrata. It gives results intermediate between those of Benda
and Altmann, but is shorter and undoubtedly better than the
method of Benda. It will be found very useful for embryological
research, and probably also for protozoology. Fix in Champy
(§ 43) (we find Flemming- without-acetic acid will do, too) for twenty-
four hours. Pieces to be fixed must be small. After fixation wash
half an hour in aq. dest., and then transfer to a mixture of 1 part
acid acet. pyrolignosum rect., and 2 parts 1 per cent, chromic acid,
for twenty hours. Wash half an hour in aq. dest., and transfer
to a 3 per cent, solution of potassium bichromate for three days.
Wash under tap for twenty-four hours ; pass through up-graded
alcohols to xylol ; embed in paraffin wax (or celloidin method, if
desired). Section 4 or 5 /a. Proceed as follows : — (1) Stain in
Altmann's acid fuchsin aniline oil mixture (5 to 10 grms. of acid
fuchsin in 100 c.c. of aniline oil-water), and heat till steaming.
(2) Set slide aside to cool for six minutes (this is important), pour
off, and wash quickly in aq. dest. (3) Counter-stain in either a
0-5 per cent, solution of toluidin blue or a saturated solution of
thionin in aq. dest. for one to two minutes. Wash in aq. dest.
In some cases the time in the blue stain must be shortened. Transfer
to a 0-5 per cent, solution of aurantia in 70 per cent, alcohol for from
twenty to forty seconds, watching extraction of fuchsin stain under
microscope. Differentiate the blue stain in 96 per cent, alcohol,
then absolute, xylol, and balsam. The chromatin is generally blue,
mitochondria (and occasionally Golgi apparatus) are red, and the
ground cytoplasm is golden-yellowish to green. This modification
of Altmann's method is a most brilliant three-colour stain which is
highly recommended. We have found that it is useful for histo-
logical as well as cytological purposes ; sections of Annelids, or of
M.
322
CYTOLOGWAL METHODS.
fiat-worms, for instance, prepared by Champy-Kull show beautiful
colour graduations in their different tissues. The preparations
begin to fade after a year.
After Champy-Kull fixation you can : (a) stain in iron haema-
toxylin (long method, § 242), (6) stain as for Benda (§ 683), (c) mount
unstained for examination of osmicated granulations, (d) stain in
safranin and light green (§ 286). For a chart illustrating Champy-
Kull technique, see below.
MAXIMOW (C. E. Soc. Biol, Paris, Ixxix, p.. 4$2) fixes in Champy,
washes slightly in water, transfers to mixture of 1 per cent, chromic acid
1 part, glacial acetic 2 parts, for twenty-four hours. Wash again for
half-an-hour, place for three days in 3 per cent. K2Cr207. Wash in
running water. Stain sections as above.
682. Champy-Kull Fixation.
Subsequent
method.
Mito-
chondria.
Fat.
Yolk.
Golgi apparatus.
Nucleus.
Mount unstained
Yellowish.
Black (extract-
able in tur-
pentine).
Yellow
to black.
Yellowish or does not
show.
Yellowish.
Ctuimpy-Kull stain .
lied or
pink.
Black.
Yellow
to black.
Generally will not
show in somatic
cells or ovaries, red
in male germ cells.
Chromatin blue to
greenish (nucleolus
red).
Iron hcematoxylin .
Black.
Black, pro-
vided it has
not been ex-
tracted in
turpentine.
Same as
above.
Same as above, but
black in male germ
cells.
Chromatin grey to
black (nucleolus
black).
Benda stain (alizarin
and crystal violet)
Violet.
Same as above.
Same as
above.
Same as above, but
violet in male germ
cells.
Chromatin brown or
yellowish (nucleolus
violet).
683. BENDA'S Alizarin Method (Ergebnisse 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 3 drops (or as for Champy-Kull or Regaud). 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 embed in paraffin. Sections on the slide
are mordanted for twenty-four hours with 4 per cent, solution of
ferric alum or diluted liq.ferri 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
CHAPTER XXVI. 323
violet (§ 330) diluted with an equal volume of water, and warm till
vapour is given off. Rinse, differentiate one or two minutes in
30 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., ii, 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.
Some workers prefer to harden as BENDA, but to stain with iron
haematoxylin instead of by the alizarin process ; the special hardening
rendering the hsematoxylin stain sufficiently specific.
ARNOLD (Arch. Zellf., viii, 1912, p. 256) stains first with iron hsema-
toxylin, differentiates, stains for twenty to thirty minutes 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.
684. Formalin-Chrome Techniques.* — The methods of Regaitd,
Bensley-Cowdry, Schridde, Murray, etc., are of importance on
account of their suitability for vertebrate, and especially mammalian
tissues. The tissues are fixed either in neutral formalin or in
formalin-chrome mixtures, washed, and then mordanted in 3 per
cent. K2Cr207. As with Champy-Kull, it is possible to stain after
such fixation by a variety of methods : — iron hsematoxylin, acid
fuchsin, alizarin and crystal violet, safranin, etc. The Regaud and
Bensley-Cowdry methods do not preserve neutral fat in the finished
sections, but by post-osmication, as for Schridde (§ 687), or Murray
(§ 689), this can be done.
685. REGAUD'S Formol- Bichromate and Iron Haematoxylin (Arch.
d'Anat. micr., t. xi, 1910). — Fix in a mixture of 3 per cent, potassium
bichromate 80 volumes, commercial formalin 20 volumes, for four
days, changing every day. Mordant in potassium bichromate for
seven days, changing every second day. Wash in running water
twenty-four hours, dehydrate (twenty-four hours each strength),
* Note that formalin-chrome mixtures consist of a reducer and an
oxidiser, and will not keep. Such solutions should always be made up
just before use. Zenker's fluid, too, keeps better without the acetic acid,
which, if b.?ing used, should be added just before the material is put in
the fixative.
21—2
324 CYTOLOGICAL METHODS.
clear and embed in paraffin. Pass sections on slide down to water ;
5 per cent, iron alum at 35° C. for twenty-four hours ; rinse in
aq. dest., not tap-water. Stain twenty-four hours in this solution :—
1 grm. of pure crystals of hsematoxylin in 10 c.c. of absolute alcohol,
added to 10 c.c. of glycerine and 80 c.c. of aq. dest. Differentiate in
5 per cent, iron alum, watching process under microscope. The
main point is to avoid washing out the mordant too much when the
slides are being transferred from the iron alum to the hsematoxylin.
Permanent stain, very good for vertebrate tissues. See also COWDRY,
Amer. Journ. Anat., xix, p. 441. I find ordinary iron hsematoxylin
is quite good after Regaud fixation.
686. BENSLEY-COWDRY Acid Fuchsin and Methyl Green Stain
(CowcRY, Contrib. Carnegie Inst. Wash., viii, 1918). — Fix as for
Regaud, either by immersion or injection ; formalin should be
neutralised in magnesium carbonate, and, if possible, the fixation
should be done in an ice-box, but this is not necessary. Pass sections
down to aq. dest. through toluol (or xylol), absolute alcohol, etc.,
thirty seconds in each ; transfer to 1 per cent, potassium per-
manganate for thirty seconds, but time must be determined experi-
mentally ; then 5 per cent, oxalic acid for thirty seconds. (Note. —
The permanganate and oxalic acid may generally be omitted.)
Then rinse in several changes of aq. dest. for about one minute
(incomplete washing prevents staining in acid fuchsin). Stain in
Altmann's fuchsin (§ 680) as follows : dry around sections with
duster, add stain, warm over spirit lamp until fumes come off ;
cool for six minutes ; wipe around sections with duster, rinse off in
aq. dest., so that the only remaining stain is in the sections (or a
precipitate forms with the methyl green) ; pipette a little 1 per
cent, methyl green over the sections for about five seconds at first,
modify time as experimentally found convenient ; drain off excess,
plunge into 95 per cent, alcohol for a second or two. Rinse in
absolute alcohol, clear in toluol, mount in balsam. Difficulties are
that the methyl green may remove the fuchsin (due to incomplete
chrome mordanting during fixation), or the fuchsin may have over-
stained (due to too much mordanting). Sometimes, if the methyl
green is too weak, it is better to omit the 95 per cent, alcohol,
dehydrating in absolute. The difficulties of this modification of
Altmann's stain are easily overcome ; I have used it for a senior
histology class, and with success. Like the Champy-Kull method,
this stain is not so permanent as Iron Hsematoxylin. See also
BENSLEY, Amer. Journ. Anat., xii, p. 308 ; DUESBERG, ibid., xxi,
p. 469.
CHAPTER XXVI. 325
687. SOHRIDDE'S Method for Mitochondria, modified (Ergeb. Anat.
u. E. Merk. Bonnet, xx, 1911). — Fix in this mixture : formol (1 part),
Miiller (9 parts), for two days ; then place in Miiller, two to four
days ; then 2 per cent. Os04, for two days. Wash overnight,
dehydrate, clear in xylol, cut paraffin sections 5 /j. Stain as follows :
iron alum hot for a quarter of an hour, then hsematoxylin hot,
a quarter of an hour. Differentiate in alum in the cold. This
has the advantage over pure formol-chrome techniques in that the
introduction of the Os()4 preserves fat ; recommended by Duesberg.
With this mordanting it should be possible to stain either as for
Altmann, Bensley-Cowdry, Champy-Kull, or Benda.
LEVI, G. (Arch. /. Zellf., Bd. xi), ovary of mammals.
10 c.c. . 2-5 per cent. K2Cr207.
10 ,, . 5 per cent, sublimate containing 2 c.c. of formol.
2 ,, . 2 per cent. Os04.
Leave for three or four days. Wash out well in running water. Stain
in Regaud, Benda. etc.
688. A. H. DREW'S Formol-Chrome-Haematoxylin Method (Journ.
R. Micr. Soc., 1920). — This method is used for demonstrating rod-
like bodies in the cytoplasm of plant cells. These rods are supposed
to be the homologue of the Golgi apparatus of animal cells. The
method will undoubtedly be useful for studying animal tissues.
Fix plant root tips, etc., for twenty-four hours in a mixture of
formol, 20 c.c. ; cobalt nitrate, 2 grms. ; sodium chloride, 0-8 grm. ;
water to 100 c.c. (preferably at temperature of 37° C.). Soak fixed
tissues in gum-syrup for at least an hour, and cut sections on freezing
microtome. Wash in water, and fix on gelatin-coated slides with
formalin. See §§ 172 and 182. Kinse in water to remove excess
formalin, mordant at 50° to 55° C. in chromic acid 4 per cent.,
osmic acid 2 per cent., equal parts, on slide for varying periods —
fifteen minutes to one hour, or longer. Rinse in water and stain
with iron alum 3 per cent, for fifteen minutes, followed by ^ per cent,
hsematoxylin for fifteen minutes, at 50° C. Differentiate in the
cold in iron alum till the nuclei show pale brown. Transfer to
2 per cent, pyridin for two minutes, dehydrate, and mount in xylol-
balsam.
In specimens chromed for short periods the mitochondria alone are
visible, while in those chromed for a longer time the mitochondria stain
less well, while gradually the long Golgi elements appear in the best
chromed cells. In animal cells, too, Drew finds short chroming shows
the mitochondria, while it requires longer treatment in the chrome to
demonstrate the Golgi apparatus. This is my own experience with the
Golgi elements or " nebenkern batonettes " of Mollusca.
326 CYTOLOGICAL METHODS.
689. -]. A. MURRAY'S Chrome-Osmic Method for Mitochondria and
Bacteria of Mammalian Tissue.— Fix tissue in formol-salt or formol-
Miiller overnight. Thin slices are then placed in Mailer's fluid for
from two to seven days, and then transferred to 2 per cent. Os04
for two days more. Wash overnight in running water, dehydrate,
embed in paraffin. Sections to be not more than 5 /n thick, fixed
on slide, and stained in 3| per cent, iron alum at 50° C. for fifteen
minutes, followed by -| per cent, aqueous hsematoxylin in same way
and for same time. Sections should now be jet black. If such
sections be decolourised in the ordinary way in iron alum, both
mitochondria and bacteria (if present) will retain the stain, and
nuclei are decolourised.
If such sections are decolourised in 0-5 per cent. HC1 in 70 per
cent, alcohol, the mitochondria give up the lake and the bacteria
remain deeply stained. At the same time the details of the nuclei
are sharply stained. Wash sections for twenty minutes in tap-water,
counters tain in Van Gieson, mount in balsam (Report Imp. Cancer
Research Bureau, 1919).
690. Double-Staining in Hsematoxylin and Acid Fuchsin.— It is well
known that different cell elements hav3 varying powers of resisting
decolourisation or differentiation after iron alum or such hsematoxylin
stains. Thus in a hermaphrodite gonad or during fertilisation it is
sometimes noticed that the mitochondria of the egg hold the hsematin
lake much faster than those of the sperm or spermatogenesis stages. It
is possible in certain cases to make use of this fact for studying differen-
tially csll granules, etc.
Fix tissue by some prolonged mordanting method, such as that of
Champy-Kull, or Eegaud. Wash out well in running water and prepare
thin paraffin sections. Stain by some intense haematoxylin method,
such as that of Benda or Heidenhain ; differentiate the csll element
which you wish to be stained subsequently a red colour, till it looks
pale greyish under the microscope : wash well in water, and counter -
stain in Altmann's acid fuchsin. Extract the fuchsin to the right stage
in 95 p3r cent, alcohol, quickly dehydrate and clear in xylol ; mount in
balsam. If necessary, aft3r staining in acid fuchsin, you may apply
the picric acid of Altmann's method (§ 680), but this necessitates under-
differentiation in the iron alum.
1 have found that after staining in the acid fuchsin you may differen-
tiate partly in aurantia as for the Kull method (§681).
A method to be tried only by experienced cytologists. The difficulty
is to differentiate the hsematoxylin just to the right stage, and to avoid
washing away the acid fuchsin (GATENBY, Journ. Eoy. Micr. Soc.,
1919 ; HANS HELD,' Arch. f. mikr. Anai., Bd. Ixxxix).
691. On Post-Chroming and Post-Osmication in General.— By
soaking tissues in K2Cr207, with or without Cr03, one produces
CHAPTER XXVI. 327
stainab'-e compounds of cell proteins and lipoids which are not easily
dissolved out by alcohol and a clearing oil. One may fix in almost any
mixture not containing alcohol; chloroform and acetic acid. Wash
the tissue in aq. dest. for a short time (say half an hour or less), cut
into small pieces, and transfer to 3 per cent, potassium bichromate
for several days, and then to 1 or 2 per cent. Os04 for a day. Wash
under tap overnight and stain in Heidenhain or an Altmann. Thus
tissues or embryos fixed in special formol-chrome, corrosive formol,
chrome-corrosive, and other mixtures which one has found most
suitable for one's purpose, may be post-chromed, or post-osmicated
as well. Schridde's and Murray's methods (above) include both
post-chroming and post-osmication.
692. KOPSCH'S Osmium Tetroxide Method (Sitzungberg. d. L
preuss. Akad. d. Wiss. zu Berlin). — Osmium tetroxide solution will
fix both fats and lipoids, and proteid substances. As has been
mentioned above, the various cell inclusions, such as mitochondria,
Golgi apparatus, yolk and fat, are nearly always mixtures of different
quantities of several definite substances, and consequently will
reduce the osmic solution in varying degrees of intensity. Kopsch
methods are somewhat capricious, but one gets results unequalled
by other methods ; for chrome-osmium, or chrome-formol, followed
by iron hsematoxylin, or Altmann, generally will not demonstrate
the Golgi apparatus (except in male germ-cells), while the Kopsch
methods preserve and demonstrate Golgi apparatus, mitochondria,
yolk, fat and chromatin structures, and occasionally neurofibrils
of embryos.
For this method dissect out organs, and cut tissue into small
pieces ; dip these quickly into aq. dest. to remove blood or cell
detritus from surface, and then transfer to a small glass-stoppered
or glass-covered capsule of 2 per cent. Os04. Leave in a darkened
cupboard for two weeks (fourteen days) at room tempera-
ture. Wash in running water for several hours, dehydrate,
embed in hard wax ; section about 3 /z. Mount unstained, or
stain chromatin in safranin or methyl-blue eosin. Unsaturated
fats black, others yellowish, Golgi apparatus, and sometimes
mitochondria, black.
This method succeeds for mollusc and many invertebrate and
vertebrate tissues, but the following Mann-Kopsch method is
generally superior. Note that a trace of chromic acid, potassium
bichromate, or platinum chloride, in the Os04 solution will inhibit
the blackening of the Golgi apparatus, but not of fat. See also
§776.
328 CYTOLOGICAL METHODS,
693. The Mann-Kopsch Method (WEIGL, Bull. Acad. Scien.
Cracovie, 1912 ; HIRSCHLER, Arch. mikr. Anat. 89 ; GATENBY,
Journ. R. Micr. Soc., 1919). For a study of cell structure, and
in general cytology, the Mann-Kopsch method gives invaluable
results. It is an alternative to the formalin-silver nitrate tech-
niques of Golgi, Cajal or Da Fano, but in addition preserves fatty
substances.
The Mann-Kopsch technique in itself is easy to work, but the
subsequent steps in staining are often extremely difficult. The
ordinary Kopsch technique may cause extreme shrinkage, and is
not generally so specific. First fix in Mann's osmo-sublimate fluid
(§ 71) for from one quarter-hour to two or three hours or more.
Pieces to be fixed must be small (not exceeding a centimetre in
diameter) and should only be left in the osmo-sublimate long
enough to complete the penetration of the fluid. For an insect
ovary, or small invertebrate, one half -hour is sufficient ; for solid
tissues like nerve, longer is' necessary. These times must be ascer-
tained experimentally. After fixation the pieces are washed in two
changes of distilled water for half an hour or less, according to the
size of the tissue and its accessibility to the water. The pieces are
transferred to a glass-stoppered bottle containing just enough
2 per cent. Os04 in aq. dest. to cover them. Then they are left in
a cupboard at room temperature, for at least ten days, and preferably
two weeks. Every few days the bottle should be examined to see
whether the Os04 is evaporating, or whether it has completely
disintegrated. Should either have happened the pieces should be
washed quickly in aq. dest., and new Os 04 solution added. It
should be noted, however, that the osmic solution nearly always
becomes slightly dark, but not until it has gone black or no longer
smells of Os04 should new liquid be added. When the right period
has elapsed the objects are taken out of the osmic, and preferably
washed for several hours in running water before transference to
50 per cent, alcohol. They are upgraded and embedded in hard
paraffin. Sections to be cut from 3 to 6 ju. They are stuck on the
slide with albumen and water in the usual way and dried over-
night. One of the slides is taken, the wax removed in xylol, and it
is mounted in xyol balsam. Examination of this slide will enable
one to ascertain to what extent the process has acted successfully.
In completely successful preparations the Golgi apparatus, yolk
and fat alone are blackened, while nuclear organs, mitochondria
and cytoplasm are stained in shades of yellow and greenish brown.
Having studied this untreated slide, and noted the extent of the
CHAPTER XXVI. 329
blackening effect of the Os04, one may then proceed to make
experiments. Several alternative methods may be tried :—
(a) The blackening may be extracted step by step in turpentine, and
the appearance of the cell granules studied at intervals.
(b) If the mitochondria are not stained black by the Os04, one may
proceed directly to Altmann's method (but preferably after
cautious treatment in -125 per cent, permanganate of potash).
(c) The nuclear structures may be stained in safranin, crystal violet,
or acid fuchsin. The sections are brought down to distilled
water and transferred to watery solutions of the dye. A few
minutes generally suffice to stain the nuclei.
We find that in successful Mann-Kopsch preparations especially of
Invertebrata, the mitochondria do not generally become black, but are
either unstained or go yellowish. In many, but not all, cases it will be
found that where the mitochondria do become black after Os04, the
colour is more readily extracted from them than from the Golgi elements,
BO that a distinction can nearly always be made by the Mann-Kopsch
method itself, without recourse to other methods which will generally
stain mitochondria and not Golgi apparatus (Regaud, Flemming, as
described in § 679). Among the most useful differentiation or extraction
methods after Mann-Kopsch, turpentine is probably the best. The wax
is removed from the sections on the slide by means of xylol, and the
slide is transferred to a jar of turpentine. After about half a minute the
section is examined under a ^th-inch objective, and the effect of the tur-
pentine is noted ; one sometimes finds that the black colour in fat
globules and yolk spheres is extracted before a quarter of an hour has
elapsed, while the Golgi apparatus retains its black condition. In most
cases it is therefore possible to distinguish between yolk and fat on the
one hand, and the Golgi apparatus on the other.
694. Mann-Kopsch-Altmann Combination (GATENBY, Journ. Roy.
Micr. Soc., 1921). — If examination of the first Mann-Kopsch section
showed that the Golgi apparatus was blackened, and the mito-
chondria were either not stained or only straw or light-brown
coloured, one may proceed directly to the Altmann stain. Should
the examination show that the mitochondria as well as the Golgi
apparatus have become blackened, the sections must be extracted
in turpentine in an endeavour to remove the blackening from the
mitochondria. If the latter treatment does not succeed properly
the only course is to make new Mann-Kopsch preparations, -allowing
less time in the Os04, say seven or eight days instead of the two
weeks.
When one has succeeded in procuring sections in which the Golgi
apparatus alone is blackened, it is possible to stain in Altmann's
aniline acid fuclisin and picric acid method, so that the mitochon-
dria (and nucleoli) become red, the Golgi apparatus is black and
the ground cytoplasm yellowish. The Mann-Kopsch sections are
330 CYTOLOGICAL METHODS.
brought down to distilled water, and cautiously treated in a '5
to -125 per cent, solution of potassium permanganate, in order
to recover the staining properties of the tissue. A very short time
suffices. Wash in water ; then stain (§ 680).
An important part of the technique is to ascertain the optimum
length of time to leave the tissue in the Mann's fluid. Examples
are as follows :—
(1) Saccocirrus (entire) overnight in Mann.
(2) Cavia testis about five hours.
(3) Chick embryos about one quarter-hour.
The Mann-Kopsch technique can be used in combination with the
Kull staining method (§ 681). We find that the cells are rather
liable to overstain in the toluidin blue, which must be left on for a
very short time.
Explanation. — Mann's osmo- sublimate fixes the cells successfully,
bscause the HgCl2 aids penetration and the Os04 is not so strong as to
cause shrinkage. Thus, before the tissue is transferred to the Kopsch
fluid (Os04 of 2 per cent.), a complete fixation has taken place and the
distorting effect of the strong Os04 is avoided. Left for two weeks, the
lipoid materials which partly form the substance oi the Golgi apparatus,
the unsaturated fats, and the special lipoids of the mitochondria, aie all
able to reduce the Os04 in varying degrees. The subsequent treatment
of the sections by turpentine (oxidiser) introduces a further differentia-
tion, and so the various inclusions can be distinguished. The acid fuchsin
stains presumably the lipoid content of the mitochondria. Sae also § 768.
695. Osmic Vapour Method (W. CRAMER, Imp. Cancer Research
Fund Report, 1919). — Choose a small glass-stoppered bottle, and
place a piece of wide glass tube open at both ends, in the bottom.
Arrange a piece of gauze over the top of the inner tube. Add
some 2 per cent. Os04 to the outer vessel. Objects to be fixed
by the osmic vapour are placed on the gauze. All the surrounding
(fatty) tissue should be removed from the organ or material to be
treated ; if too dry the outside of the material should be slightly
wetted.
The bottle, with object suspended in the Os 04 vapour, is kept at
temperature of 40° C. for one and a half hours. Removed from
bottle, the tissue is placed in 50 per cent, alcohol and upgraded and
embedded in paraffin. Cut sections, mount in balsam without
staining. Such wax sections may be treated in 10 per cent, hydrogen
peroxide in 80 per cent, alcohol for two hours, after which they may
be stained in ordinary methods (e.g. iron hsematoxylin).
GATENBY (Quart. Journ. Micr. Set., 1920) suggests two modifications.
(a) Fix as above for one and a half hours, and then transfer to 2 per
CHAPTER XXVI. 331
cent. OsO.j in water at 37° C. for several days as for Kopsch. Then wash
in water for several hours, dehydrate, embed and section. Mount
unstained, or cautiously treat in permanganate of potash or hydrogen
peroxide and then stain in acid fuchsin (Altmann) or iron hsematoxylin .
(b) Tissues may also be fixed as above, and then transferred to Alt-
mann's or Champy's fluid, and subsequently stained in Altmarm's
fuchsin and picric acid.
Cramer fixes wet films for about three minutes. We think that a
subsequent treatment of films as in above two paragraphs should be
useful. The main point to note is that substances in a tissue which
might ba dissolved out ^r altered by the water added to the Os04
crystals are fixed in situ, and without the danger of alteration. This
method should be of value to histologists and cytologists.
696. SJOVALL'S Formol Osmic Acid Method (Anat. Hrfte., Bd. xxx,
1906).
Material fixed in formalin, but without chrome salts or platinum
chloride, may be used for SjovalPs technique (Anat. Hefte., Bd. xxx).
Fix pieces of tissue or small embryos in neutral formalin (5 to 20 per cent,
neutralised with magnesium carbonate) for two days. Cut into smaller
pieces and wash in several changes of aq. dest.
Transfer to 2 per cent. Os 04 solution for from two to fourteen days at
room temperature, as for Kopsch. Wash well in water, dehydrate
clear and embed. Cut sections 3/x,it necessary decolourise in peroxide
(§ 695) and mount unstained in balsam.
This technique is capricious, much more so than Kopsch, and depends
firstly on a suitable fixation in formalin, and secondly on the right time
in Os04 : it is convenient to cut the tissue into several pieces, which are
removed from the Os04 at different intervals. At times Sjovall demon-
strates mitochondria and not Golgi elements, and in all probability it is
not of such value as Mann- Kopsch. We consider that the method is of
use to the skilled cytologist, although it may not be applicable for
general purposes.
Note. — Corrosive-formalin (§ 112) and osinicated picric (§ 100) may
also be used as preliminary fixers before a Kopsch or Sjovall osmica-
tion. We cannot recommend the latter, as the picric acid probably
introduces maceration ; the former is good.
697. CHAMPY'S Iodide of Osmium Method (Journ. de I'Anat. et Phys.,
xlix, 1913). — Champy finds that osmium iodide prepared as below has
the power of blackening fat and certain other cytoplasmic inclusions
whose identity seems doubtful, but which he calls for the time being
catalyosomes or lyosomes.
Just before use prepare the following solution : —
Osmic acid 2 per cent. . . . 1 part.
Iodide of sodium 3 per cent. . . 1 ,,
It produces a golden yellow colour. Place relatively large pieces of
tissue (5 to 6 millimetres) in the solution. If one .takes very small pieces
as for the Kopsch techniques, one gets nothing but the osmic reaction
while the iodine reaction, which takes place deeper in, is masked. Leave
tissue in for at least twenty-four hours, and use a good deal of the liquid
332 CYTOLOGICAL METHODS.
for a few pieces of tissue. Upgrade in alcohol, pass through toluol,
embed in wax. The bodies reducing the osmium iodide go black, on a
pale grey background. One may subsequently stain in Altmann (mito-
chondria red, lyosomes black) or in iron hsematoxylin. It is possible to
distinguish between lyosomes and fat, by fixing some of the same tissue
in Flemming and comparing the two preparations ; or by comparing
the central part of the osmium iodide preparation with the periphery
where the fixation is due to the Os04 exclusively.
I have tried this method, at present with disappointing results.
698. Marine or Fresh Water Organisms ^and Techniques for the
Cell Inclusions. — If you are going to use a formalin- chrome technique,
kill the animals by adding neutral formalin to the water ; if an
osmic technique is to follow, kill with Os04 ; wash slightly in aq.
dest. in both cases and then transfer to the special fixative. See
also under " Protozoa " and " Plankton."
699. The Centrifuge and Polariscope Microscope in Obgenesis Studies,
etc. — In examining large differentiated cells, such as ovarian or nerve,
most valuable help can be obtained by use of a powerful centrifuge.
Tissues or small gonads or whole invertebrates are placed in a tube and
centrifuged at high speeds (circa 3,000 revolutions) for from a quarter
of an hour to one hour. The centrifuged tissues or animals are imme-
diately divided out among several capsules and fixed by several methods
which have previously been found to show the various cytoplasm ic
inclusions : the specifically stained layers can then be examined. We
are of the opinion that no study of the inclusions during cell differentia-
tion is complete without recourse to this method. See also GATENBY,
Quart Journ. Micr. Sci., 1920 ; LILLIE, Biol. Bull., 1908—9.
FAURE-FREMIET (C. E. Soc. Biol., Paris, Ixxxiii) attaches a small
platform to the centrifuge, so that preparations on the slide, under
coverslip, can be centrifuged and examined from time to time.
The polariscope microscope has proved very useful, not only for
studying the musculature of small animals, but also for discriminating
between various fatty and lipoid materials. In working on accumu-
lations of masses of metaplastic substances in embryos, or in eggs and
other differentiated cells, polarised light is often most helpful. The
subject is dealt with in § 768 by Cramer.
700. Vital Staining of the Mitochondria. — There is probably no
specific intra vitam stain for the mitochondria alone, most of the
so-called specific stains will tinge other bodies. E. V. Cowdry has
summarised the various methods used for this purpose (Contrib. to
Embryol Carneg. Inst., Washington, viii, 1918).
Four stains (Janus green B, Janus blue, Janus black I, and
diethylsafranin) will • give an intensely positive reaction on the
mitochondria of freshly drawn human lymphocytes. All these are
Hoechst proprietary preparations; I find the Janus green of
CHAPTER XXVI. 333
Griibler is much less satisfactory. Nile blue B, Janus green G,
methylen blue med., pyronin, Bismarck brown, and methyl violet
5 B, will all tinge the mitochondria, but faintly. Cowdry claims
that in dilution 1 : 500,000 Janus green is specific, and will not
stain granules other than mitochondria : it seems certain at any
rate that exceptions to its specificity for mitochondria alone are
rare. Cowdry (loc. cit.), in his admirable discussion on the Janus
colours (pp. 86 — 93), states that the specificity of these dyes is due
to a diethylsafranin group. Janus black consists partly of Janus
green and another substance ; so also Janus blue and grey. See also
" Bayliss," § 207. For a treatment of the Benzidine dyes, see § 790.
701. Mitochondria of Blood and Cell Smears (COWDRY, Internal.
Monat. f. Anat. u. Phys., Bd. xxxi). — Janus green B, 1 : 10,000 in
0-85 per cent. NaCl. Place a drop on a series of six or more slides :
add a small amount of freshly drawn blood (etc.), and then apply
coverslip. Do not attempt to mix the cells and the Janus green.
The mitochondria stain rapidly, and the preparation, after ringing
with vaseline, will last hours.
702. Injection of Janus Green B (BENSLEY, Amer. Journ. Anat.,
xiii, 1911). — Kill the animal (we believe coal gas seems preferable
for this), inject 1 : 10,000 Janus green in salt solution, through
left ventricle or aorta by gravity pressure ; in order to obtain a
good penetration clamp up the return flow momentarily. After
ten minutes' perfusion small pieces of the gland (pancreas in this
case) may be removed and examined microscopically for mitochon-
dria. When the desired depth of staining has been reached the
entire gland is placed in salt solution pending further study.
703. Neutral Red. — Use as above for Janus green (§ 700) in
1 : 2,000 to 1 : 10,000 solution (see §§ 308, 790, and 804).
704. Toxic and other Examining Media for Mitochondria of Blood,
Protozoa, and Fresh Cells. — The mitochondria can be examined
successfully in toxic solutions such as weak Os04, with or without
an added dye.
MEVES' Victoria Green. — Add a " small " quantity of Victoria
green (malachite green) to a 4 per cent, iodic acid solution. Place
a drop on a slide with the cells to be examined (Anat. Anz., Bd.
xxvi).
Osmicated solution of Ripart and Petit, cf . § 90, recommended to
leave out the acetic acid of the original formula.
Dahlia saturated solution in 0-75 per cent. NaCl. — Stains mito-
334 CYTOLOGICAL METHODS.
chondria and Golgi apparatus (nebenkern) of male cells distinctly
violet. Both methyl violet 5 B (Griibler) and methyl blue in aqueous
solutions will stain the mitochondria. Bismarck brown gives a less
clear picture. See under these dyes in Part I.
705. Cajal, Golgi, and Da Fano Silver Nitrate Methods for the
Golgi Apparatus. — For vertebrate tissue Cajal or Golgi, and for
invertebrate tissue Cajal and Da Fano methods are a great aid to
research on the cell. Every cytologist is recommended to master
either Cajal's or Da Fano's method. These methods will be found
in the neurology section (§§ 844, 849).
706. Differentiation between Cell Inclusions. —It is frequently
somewhat difficult to distinguish between the various categories
of cell inclusions. In this section we have provided a series of tables
intended to act as a tentative guide to the interpretation of the
various images got by representative cytological techniques. These
tables are based on work carried out on animals of most orders,
but it would be injudicious for the researcher to depend upon them
implicitly, because many exceptions are met with, and the personal
factor is to be taken into consideration. The use of such tables,
if made with several methods and in conjunction with a careful
study of the origin and morphology of any doubtful cell body, will,
however, provide reliable evidence for identification. Another
warning must be given — never try to ascertain the nature of
granules in developing eggs without first studying the oogenesis
of the animal in question. Eggs after spawning or laying are difficult
objects to study by these methods, and even the most experienced
worker is unable to give a valuable interpretation until he has
worked at the oogenesis. It should also be remembered that there
are periods in the development of the cell during which the mito-
chondria are often able to resist becoming dissolved in lipoid solvents,
these periods are in the early spermatogonium in some animals,
and during the last stages of spermatogenesis (spermateleosis) in
all animals, and sometimes in large oocytes. See also the work of
REGAUD, Arch. (TAnat. micr., xi.
Nota Bene. — With regard to the oil used for clearing and im-
bedding, it should be pointed out that all these tables are based
on preparations cleared and imbedded in xylol, which occasionally
tends to extract lightly osmicated fat. Vegetable oils like cedar
wood oil seem to be less active in this way. I have not found that
chrome-osmicated fat, or " Kopsched " fat, is extracted either by
xylol or xylol-balsam. See also § 768.
CHAPTER XXVI.
335
707. Differentiation between Mitochondria and Fat (Olein, Stearin
and Palmitin Mixtures especially).
I Fresh tissue stained : Fixed in Flem-
Kopsch or Mann-
Regaud or for-
Fresh tissue
' in Herxheimer's scarlet ming with
Kopsch.
malin fixation,
stained in
Met/tod 1 red or such alcoholic ! acetic acid
iron hsema-
Janus green
employed. ] fluids.
examined in
toxylin.
1 : 10,000.
unstained sec-
! tions on slide.
Mitochon-
Will not stain bright- Do not show,
Yellow or black : if
Black.
Green.
dria.
ly, generally dis- generally dis-
black, colour often
solved away.
solved away,
difficult to extract
except in cer-
in turpentine ; if
tain cases
yellow, can be
where the
stained in acid f uch-
mitochondria
sin of Altmann.
are more re-
sistant to
acetic acid.
Fat (see §
Stains brightly. (It
Black (see §
Black : colour easily
Not stained, as
Does not
768).
should be noted here i 768).
extracted after a
it has been
stain.
that while Herx-
-
few hours in turpen-
dissolved out
heimer's scarlet red
tine.
by the clear-
will not stain mito-
ing reagent
chondria it may pos-
sibly stain lipoids
(xylol or
c h 1 oroform,
other than true fat.)
not vegetable
oils).
708. Differentiation between Golgi Apparatus and Mitochondria.*
1
2.
3.
4.
5.
Formol-
Kopsch,
Flemming-without-
Janus
_
Method ,
silver
Mann-
acetic, Regaud,
green
employed. \ nitrate
Kopsch
Champy, formalin,
1 : 10,000.
(methods of
(osmium
etc., followed by iron
Golgi,
tetroxide
alum hsematoxylin.
Cajal, Da
methods).
Fano.
Golgi appara-
tus (dictyo-
somes, ne-
b e n k e r n
Black
(even when
untoned).
Black.
Does not show except
in oogonia or very
young oocytes, and
in male germ cells ;
Rarely
stains
(e x cept
in male
In addition to these
methods a study of the
morphology and origin of
bodies found in differen-
bat onettes,
id i oz o me
rods, etc.).
rarely in other cells.
When stained gene-
rally less intense
germ
cells).
tiated cells will aid in
settling the nature of
granules or rods in ques-
than the mitochon-
tion. See COWDRY (In-
dria.
ternal. Monat., Bd. xxix);
GATBNBY (Journ. Roy.
Mitochondria.
Golden
Not stained,
Stain black, or dark
Green.
Micr. Soc., 1919, p. 93) ;
(untoned)
greyish
y e llowish,
or more
grey.
and HIRSCHIER (Arch,
f. mikr. Anat., Ixxxix).
•
(toned),
rarely
more
black.
rarely
black.
709. Mitochondria, Golgi Apparatus, Chromatin, True Chromidia,
and Nucleoli. — In § 710 is a table giving the main fixing and staining
reactions of these bodies. It will be found to hold for a discrimina-
tion between chromatin of the nucleus and mitochondria and
Golgi apparatus, especially in somatic cells ; but the chief diffi-
* See addendum, § 713.
336
CYTOLOGICAL METHODS.
culties arise in the study of oogenesis and gland secretion, where
one meets with profound changes in the chromophility of the
nucleus, and with complicated nucleoli, whose real nature is as
yet undetermined ; moreover, at certain stages in the differentiation
of a cell, true chromatin may stain oxyphil. Such exceptions must
be taken into consideration ; always ascertain carefully the origin
and behaviour of a doubtful body and remember that the safest
tests for chromatin lie not in staining, but in the behaviour of the
doubtful material during mitosis and in the sexual or reproductive
phenomena of the cell. See also GATENBY, Journ. R. Mic. Soc.,
1919 and 1920.
710. Chromatin and the Inclusions.
1.
2.
3.
4.
5.
6.
Fixation in al-
Method of
A 1 1 m a n u-
C h r o m e-o s-
Cajal's for-
Mann-Kopsch.
cohol acetic,
Carnoy (a t
C h a m p y -
Kull, i.e., fix-
B e n s 1 e y,
chrome - os-
mium fixa-
t i o n and
mol uranium
nitrate and
i.e., fixation
in corrosive-
least one
a t i o n in
mium fixa-
staining in
silver method
osmic, after-
• Technique
hour) ; Pet-
chrome - os-
tion, stain-
Ehrlich's
for Golgi ap-
treatment in
employed.
r u nkewitsch
mium, stain-
ing in acid
hsematoxylin
paratus coun-
Os04 of 2 %
or G i 1 s o n
ing in acid
fuchsin and
or toluidin
terstained in
for 1-1 days,
(over night)
fuchsin au-
methyl
blue and
safranin (or
then stain in a
and staining
rantia and
green.
cosin.
in methyl-
basic dye like
in hsematoxy-
toluidin blue.
blue eosin or
safranin.
lin of Ehr-
methyl-
lich, or tolui-
green).
din-blue and
\ eosin.
Chromatin
Blue or red,
Blue to green-
Green.
Bluish.
Blue, red or
Red in safra-
(nucleus)
etc. See
ish, rarely
green, a c -
nin, etc.
and chro-
S$ 211 and
red.
cording to
midia.
666.
colour of
basic stain
used.
Mitochon-
Will not show,
M i t ochondria
Mitochondria
Do not show,
M i tochondria
Yellowish to
dria and
because they
red, Golgi
red, Golgi
or faint ly
golden t o
black, will
Golgi ap-
paratus.
have been
nearly o r
a p para tus
rarely shows,
a p p a ratus
also as for 2.
oxyphil, gra-
nules as such
dark brown,
Golgi ap-
not stain in
safranin.
quite dis-
but when it
not identifi-
paratus
solved away,
does so it is
able.
black.
and morpho-
red.
1 ogicall y
altered b v
the fat sol-
vents of the
preparing
•
media.
Nudeolus.
Red generally.
Red.
Reddish.
Bluish.
Reddish or
Reddish.
colour of
plasma stain.
See also remarks on nucleoli in g§ 664 and 668.
711. Cytoplasmic Inclusions in Gametogenesis. — In the table
below is a summary of the fixing and staining reactions of the
inclusions during oogenesis and spermatogenesis. In the male
germ cells the Golgi apparatus (nebenkcrn batonettes) show through-
CHAPTER XXVI.
337
out ; those of the egg can generally only be demonstrated by
methods 1 and 2. The fat-methods of Daddi, Herxheimer
Martinotti, and Lorrain Smith should be tried as well, and reference
made to tables given below. If yolk granules contain olein or such
unsaturated fat they will stain in Os04 like fat, but by slow de-
colourisation as in paragraph 2 (with turpentine), their proteid
basis will be noted, and they will generally be demonstrated in
methods 1 and 4, while fat vacuoles disappear completely.
See also the special sections on " Fat," §§ 768 to 772.
712. Inclusions in Gametogenesis.
Method.
t
2.
3.
4.
5.
6.
Cajal or Da Fano
.
*
Kopsch series.
Chrome-osmium
and iron hsema-
toxylin (or Alt-
mann).
Bouin and
corrosive
acetic
and Ehr-
lich's
haem.
Champy-
Kufi:
Benda.
Mitochon-
dria.
Either do not
show or greyish
or golden
brown, accord-
i n g as to
whether sec-
tions have been
toned.
Often will not
show, or faintly
yellowish, more
rarely black or
brown, but can
often be decolour-
ised rapidly in
turps.
Black (or red).
Do not
show.
Red.
Violet.
Golgi ap-
paratus.
Black.
Black, and resists
decolourisation in
turps longer than
mitochondria, fat
or yolk.
Rarely shows,
when it does,
black (or red),
or not stained.
Does not
show.
Rarely
shows, if
so, red.
Rarely
shows, if
so, violet.
Yolk gra-
nules.
Either will not
show, greyish
or golden
brown, accord-
ing as to toning.
Yellowish, or black
easily decolour-
ised in turps.
May or may not
go black (or
unstained), very
rarely red.
Not
stained or
yellowish.
Yellowish
or black.
Yellowish
or black.
Fat vacu-
oles.
Do not show.
Black, easily de-
colourised in
turps.
Black in un-
stained pre-
paration.
Not
stained,
washed
away.
Black.
Black,
Chromatin
granules.
Do not show, but
may subse-
quently be
stained in a
basic colour,
like methyl
green or safra-
nin.
Yellowish, will sub-
sequently stain
in hsematoxylin
or safranin.
Black to grey
(or reddish
purple).
Bluish to
purple.
Blue.
Brownish
yellow.
Niicleoli .
Yellowish may
take s u b s e-
quent stain.
Yellowish.
Black or dark
grey.
Reddish,
or reddish
purple.
Red.
Violet
or brown.
713. Plan for Cytological Research.— It isjbsst to work at some animal
which is procurable in sufficient numbers, as frequently your first pre-
parations are disappointing. If a vertebrate, begin with Regaud, and
stain in iron alum haematoxylin ; if an invertebrate try Flemming
without acetic acid, and iron hsematoxylin ; the former method will
M. 22
338 CYTOLOGICAL METHODS.
give mitochondria and yolk ; zymogen but not fat ; the latter will give
all these. Charapy-Kull and Bensley-Cowdry or Altmann methods are
next worth trying. Mann-Kopsch and Sjo'vall methods may present
difficulties, but again one might succeed at the first trial : if invertebrate
tissue, the Mann-Kopsch method will be best, if vertebrate, the Cajal
or Da Fano formalin-silver nitrate methods are most indicated for a
study of the Golgi apparatus.
If your material is limited to a small amount, the Champy-Kull (or
Bensley-Cowdry) and the Mann-Kopsch methods are recommended :
successful preparations by both these methods will enable you to make
an almost complete study (sometimes of the chromosomes and) of the
cytoplasmic inclusions of every kind — excepting glycogen.* Recom-
mended in the third place is a formalin-silver nitrate method — Cajal or
Da Fano. See also GATENBY, Quart. Journ. Micr. Science, Ixiv, 1920,
p. 296, and E. V. COWPRY, Contrib. Embryology, Carneg. Inst., Washing-
ton, viii, p. 59.
Addendum: SAGUCHI (Amer. Jour. Anat., Nov. 15th, 1920) finds
that the Golgi apparatus of acinus cells of the frog pancreas is brought
into evidence by Cajal's uranic silver nitrate method, and also by the
Weigl, Kopsch, and Sjovall methods, but that in islet cells, the former
method exhibits some other sort of apparatus. See -also SAGUCHI, ibid,,
vol. 26, 1920.
* SHUN ICHI ONO (Anat., Anthrop. Ass. of China, 1920) finds that
osmicated mitochondrial fixatives preserve glycogen, which can be
stained in Best's carmine and iron hsematoxylin, the mitochondria
(grey black) and the glycogen (reddish) showing side by side.
CHAPTER XXVII.
TEGUMENT ARY ORGANS.
714. Epithelium. — Both for surface views and for sections good
results are obtained by the nitrate of silver method, the methylen
blue method, the perchloride of iron and pyrogallol method of the
Hoggans, § 375, the osmic acid and pyrogallol process, § 374, and by
iron-hcematoxylin .
For the purpose of separating the epidermis from the corium,
LOEWY (Arch. mik. Anat., xxxvii, 1891, p. 159) recommends mace-
rating 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. MINOT (Amer. Nat., xx,
1886, p. 575) macerates embryos for several days in 0-6 per cent,
salt solution, MITROPHANOW (Zeit. wiss. Mik., v, 1888, p. 573)
for a quarter of an hour in 3 per cent, nitric acid, then one hour in
one-third alcohol, and, if need be, twenty-four in stronger alcohol.
MAYER (Lotos, 2, xii, 1892) exposes the cornea or membrana
nictitans of Rana, Bufo, 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."
715. Intercellular Bridges (and Canals), Prickle Cells. — See IDE,
in La Cellule, iv, 1888, p. 409, and v, 1889, p. 321 ; also KOLOSSOW,
Arch. mik. Anat., Hi, 1898, p. 1. KOLOSSOW used an osmic-acid-
tannin stain, § 374.
See also FLEMMING, Anat. Hefte, 1 Abth, vi, 1895, p. 1.
Besides maceration, impregnation may be useful ; MITROPHANOW
(Arch. Anat. Phys., Phys. Abth., 1884, p. 191) has used gold chloride.
UNNA (Monatsschr. prakt. Derm., xxxvii, 1903, p. 1) has described
a highly complicated process with Wasserblau and orcein, see Zeit.
wiss. Mik., xxi, 1904, p. 68.
716. Plasma-fibrils of Epithelium. — KROMAYER'S 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
22—2
340 TEGUMENTARY ORGANS.
(§ 286) and concentrated aqueous solution of methyl violet 6 B.
They are well washed in water and treated with solution of iodine
in iodide of potassium until they become blue-black (one to thirty
seconds). They are again washed with water, dried with blotting-
paper, and treated with a mixture of 1 volume of anilin to 2 volumes
of xylol until sufficiently differentiated, when they are brought
into pure xylol. Very thin sections will require more xylol in pro-
portion to the anilin, viz. 1 : 3 or 1 : 4 ; thicker ones may require
more anilin, viz., 3 : 5 or 3 : 3. Gentian or Krysfcallviolett will do
instead of methyl violet, but not quite so well. See also EHRMANN
and JADASSOHN, Arch. Dermatol. u. Syphilis, 1892, 1, p. 303 ; Zeit.
iviss. Mik., ix, 1893, p. 356 ; HERXHEIMER, Arch. mik. Anat., liii,
1899, p. 510 ; and ROSENSTADT, ibid., Ixxv, 1910, p. 659 (takes the
differentiating mixture much weaker in anilin).
UNNA (Monatsschr. prakt. Derm., xix, 1894, p. 1 and pp. 277 et
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) WASSERBLAU-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 hsemalum,
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.
(3) Haemalum for two hours, neutral orcein as above for ten to
twenty minutes.
More recently UNNA advocates the process mentioned last
section.
See also KANVIER, Arch. Anat. Mikr., iii, 1899, p. 1.
717. 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
heematoxylin, 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
CHAPTER XXVII. 341
to four minutes in concentrated aqueous solution of Kresykchtviolett,
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 Encycl. mik. Technik. ; and UNNA and GOLODETZ,
Monatsschr. prakt. Derm., xlix, 1909, p. 95 ; LAFFONT, Eibl. Anat., 1909,
p. 209.
For Triehohyalin, see GAVAZZENI, Monatsschr. prakt. Derm., xlvii,
1908, p. 229.
718. Eleidin. — To demonstrate the stratum granulosum and the
eleidin granules RANVIER (Arch. Anat. Micr., iii, 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 Encycl. mik. Technik., article " Haut ") stains sections
for a few minutes in a watch-glassful of water with 2 to 3 drops of
1 per cent. Congo red. Similarly WEIDENREICH, Arch. mik. Anat.,
Ivii, 1901, p. 583. Other authors recommend nigrosin, or Wasser-
blau, or orcein.
See also JOSEPH, " Dermatohist. Technik," Berlin, 1905, and
DREUW, Med. Klinik, Berlin, 1907, Nos. 27 and 28.
For Cholesterin see GOLODETZ and UNNA, Monatsschr. prakt.
Derm., xlvii, 1908, p. 1.
719. 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
NATHUSIUS, Zool. Anz., xv, 1892, p. 395.
Horny tissues stain well in safranin or gentian violet (REINKE,
Arch. f. mik. Anat., xxx, 1887, p. 183 ; ERNST, ibid., xlvii, 1896,
p. 669 ; RABL, ibid., xlviii, 1896, p. 489).
UNNA (op. cit. last section, 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.
720. Skin-nerves and Nerve-endings. — Impregnate with gold
chloride. See Chapter XVII., especially § 365.
721. Tactile Corpuscles.-— See §§ 364— 366.— Gold methods are
indicated. See also RANVIER, Traite, p. 919 ; LANGERHANS, Arch,
mik. Anat., 1873, p. 730 ; KULTSCHIZKY, ibid., 1884, p. 358 ; and
SMIRNOW, Intern. Monatsschr. f. Anat., etc., x, 1893, p. 241, who
recommends, besides the gold method of Lowit, the rapid bichromate
of silver method of Golgi.
342 TEGUMENTARY ORGANS.
Corpuscles of Herbst and Corpuscles of Grandry. — DOGIEL
(Arch. Anat. u. EntwickeL, 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. GEBERG (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 O25 per cent, cyanide of
potassium and brushing.
NOWAK (Anat. Anz., xxxvi, 1910, p. 217) takes UNNA'S Orce'in-
wasserblau mixture (Wasserblau O.D., 1 part, orcei'n 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 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 1 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 DOGIEL, Folia NeurobioL, iv, 1910, p. 218 (also employing
Bielschowsky's neurofibril method).
723. Corpuscles of Meissner and of Krause (Cornea and Conjunc-
tiva).— DOGIEL (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 LONGWORTH'S methods, Arch. mik. Anat., 1875, p. 655.
724. Similar Objects. —Papillae Foliatae of the Rabbit, HERMANN, see
Zeit. wiss. Mik., v, 1888, p. 524 ; ARNSTEIN, ibid., xiii, 1897, p. 240.
Olf active Organs of Vertebrates, DOGIEL, Arch. mile. Anat., 1887, p. 74.
Organs of a " Sixth Sense " in Amphibia, MITROPHANOW, Zeit. wiss. Mik.,
v, 1888, p. 513 (details as to staining with " Wasserblau," for which sec
also Biol. Centralb., vii, 1887, p. 175). Nerve-endings in Tongue of Frog,
FAJERSTAIN, Arch, de Zool. exper. et gen., vii, 1889, p. 705. Tongue of
Rabbit, VON LENHOSSEK, Zeit. wiss. Mik., xi, 1894, p. 377 (Ramon y
Cajal's double Golgi methpd).
725. Cornea.— There are three chief methods-— the methylen blue,
the silver, and the gold method.
CHAPTER XXVI L 343
For the methylen blue method see particularly § 345.
Negative images of the corneal cells are easily obtained by the
dry silver method (KLEIN). The conjunct! val 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 (RANVIER) is to macerate a cornea that has been prepared as
above for two or three days in distilled water. There takes place a
secondary impregnation.
The same result may be obtained by cauterising the cornea of a
living animal as above, but allowing it to remain on the living
animal for two or three days before dissecting it out, or by treating
a negatively impregnated cornea with weak salt solution or weak
solution of hydrochloric acid (His).
But the best positive images are those furnished by gold chloride.
RANVIER prefers his lemon- juice method. It is important that
the cornea should not remain too long in the gold solution, or 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 peeled off, and the
collodion removed from the tissue by a mixture of alcohol and ether.
See also ROLLETT, in Strieker's Handb., pp. 1102, 1115, or previous
editions ; TARTUFERI, Anat. Anz., v, 1890, p. 524, or previous
editions ; CIACCIO, Arch. ital. BioL, iii, p. 75 ; and RENAULT,
C. R. Acad. Sc., 1880, p. 137.
726. Crystalline. — GERHARDT (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 easily dissociated with needles into its fibres.
RABL (Zeit. wiss. Zool, Ixv, 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, § 533.
See also ROBINSKI, Zur Kenntniss d. Augenlinse, Berlin, 1883.
CHAPTER XXVIII.
MUSCLE AND TENDON (NERVE-ENDINGS ).
Striated Muscle.
727. Muscle-cells. — For these and allied subjects see, inter alia,
BEHRENS, KOSSEL, und SCHIEFFERDECKER, Das Mikroshop, etc.,
vol. ii, pp. 154 — 161 ; and SCHAFER, Proc. Roy. Soc., xlix, 1891,
p. 280.
Iron hsematoxylin gives very fine images of striped muscle, and
so does Mallory's phospho-tungstic.
For dissociation methods see §§ 512 — 545.
To isolate the sarcolemma SOLGER (Zeit. wiss. Mik., vi, 1889,
p. 189) teases fresh muscle in saturated solution of ammonium
carbonate.
728. Nerve-endings — the Methylen Blue Method. — For BIEDER-
M ANN'S procedure for the muscles of Astacus see § 342 (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, BIEDERMANN proceeded by injecting
0-5 c.c. of methylen blue solution between the ultimate and pen-
ultimate 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.
GERLACH (Sitzb. Akad. Wiss. Munchen, 1889, ii, p. 125) injected
frogs, either through the abdominal vein or through the aorta,
with 4 to 5 c.c. of a 1 : 400 solution in 1 per cent, salt solution, and
examined pieces of muscle in serum of the animal, afterwards fixing
with picrate of ammonia and mounting in glycerin jelly.
The procedure of DOGIEL has been given, § 342.
729. Nerve-endings— the Gold Method.— FISCHER (Arch. mik.
Anat., 1876, p. 365) used the method of Lowrr.
BIEDERMANN (last section) recommends for Astacus a similar
CHAPTER XXVIII. 345
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 (Traite, 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 APATHY, §§ 368, 371.
730. Nerve-endings — the Silver Method. — RANVIER 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 examined 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.
731. Nerve-endings— the Bichromate of Silver Method.— The
rapid method of G-OLGI has been used by RAMON Y CAJAL for the
terminations of nerves and tracheae in the muscles of insects. See
Zeit. wiss. Mik., vii, 1890, p. 332, OT fourth edition. A modification
is used by WUNDERER, Arch. mik. Anat., Ixxi, 1908, p. 523.
732. Muscle-spindles. — See CILIMBARIS, Arch. mik. Anat., Ixxv,
1910, p. 692. Principally intra vitam methylen blue, by injection
through the internal carotid. For elastic fibres, Weigert's resorcin-
fuchsin, followed by 1 per cent, orcein acidified with HC1.
Electric Organs.
733. Electric Organs.— RANVIER (Traite, Chap, xviii), finds that
osmic acid is the only reagent that will fix properly the terminal
arborisations on the lamellae. 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
346 MUSCLE AND TENDON (NERVE-ENDINGS).
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 upper-
most. 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 hsematoxylin, and mounting
in damar.
Torpedo. — BALLOWITZ (Arch. mik. Anat., xlii, 1893, p. 460) gets
the best results by the rapid Golgi impregnation.
An electric column, with about \ to 1 centimetre 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 f per cent,
silver, cut without imbedding and mounted in xylol balsam. Im-
pregnates all the important elements. See further, on the whole
subject, BALLOWITZ, Encycl. mik. Techn., 1910, p. 298.
CAVALIE (Bibl. Anat., xiii, 1904, p. 214) takes material fixed
with osmic acid of 2 per cent, and impregnates it with gold by the
method of NABIAS, and mounts in glycerin.
Raja. — IWANZOFF (Bull. Soc. Nat. Moscou, ix, 1895, p. 74) fixes
the organ in the tail of Raja with liquid of Flemming, stains with
haemacalcium and eosin, and makes paraffin sections.
BALLOWITZ (Anat. 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. — BALLOWITZ (Encycl. mik. Technik., p.' 303) fixes with
Flemming, and makes sections. He also commends impregnation
with gold chloride, but not the Golgi method.
Malapterurus. — BALLOWITZ (ibid., p. 202) fixes with picro-subli-
mate, with Flemming, or with various mixtures of bichromate,
sublimate, and formol, and uses gold chloride and Golgi impregna-
tions. He macerates in liquid of Miiller or saturated aqueous
solution of picric acid.
CHAPTER XXV III. 347
Tendon.
734. Tendons.— RETTERER (C. R. Soc. BioL, x, 1898, p. 580)
fixes in equal parts of saturated solutions of sublimate and picric
acid, puts for one to three days into saturated picric acid with
2 to 3 per cent, of sodium chloride, to remove the mucin, and imbeds
in paraffin.
735. Union of Muscle and Tendon.— For this see RETTERER and
LELIEVRE, C. R. Soc. Biol., 1911, No. 12 (orcei'n for twenty-four
hours, followed by iron haematoxylin) ; and SCHULTZE (Verh. phys.
med. Ges. Wilrzburg, 1911, p. 33) (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 two days with 0-5 per cent, solution of
hsematoxylin in alcohol of 70 per cent., then with Van Gieson's
picro-sauref uchsin) .
736. Corpuscles of Golgi (RANVIER, 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 and gold method (§ 365),
and after reduction scrape with a scalpel, in order to remove the
muscle-fibres that mask the musculo-tendinous organs.
MARCHI'S methods for the tendons of the motores bulbi oculi
(Archivio per le Scienze Mediche, 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 (G-OLGi's method), and by the method of MANFREDI,
§ 368. Mount in glycerin. The methods only succeed completely
during fine, sunny weather.
RUFFINI (Atti R. Ace. Lined Roma Rend. [5], i, 1892, p. 442)
recommends the method of Fischer.
CIACCIO (Mem. R. Ace. Sci. Bologna [4], t. x, 1890, p. 301) 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.
DOGIEL (Arch. mik. Anat., Ixvii, 1906, p. 638) stretches tendons
of eye-muscles on cardboard with hedgehog spines, puts for four
348 MUSCLE AND TENDON (NERVE-ENDINGS).
or five 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.
737.- Tests for Smooth Muscle. — Picro-saurefuchsin, § 299, stains
muscle yellow, connective tissue red.
Picro-nigrosin, § 325, stains muscle yellowish, connective tissue
blue.
UNNA (Encycl. 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 complicated process with methylen
blue in Monctssch. prakt. DermatoL, xix, 1894, p. 533, and another
with orcein, hsematein, saurefuchsin and picric acid.
RETTERER (C. R. Soc. BioL, 1887, p. 645) fixes in 10 volumes of
alcohol with one of formic acid, washes well and stains in alum
carmine. Muscle red, connective tissue unstained.
738. General Structure. — WERNER (Hist. d. glatten Musculatur,
Dorpat, 1894, p. 22) fixes stretched intestine or bladder in Flemming,
washes well and stains in Heidenhain's chrome haematoxylin, § 265.
For demonstrating intercellular spaces, fresh intestine is put for
twenty-four hours into oil, at 37° C., then for twelve hours into
Flemming, and for four to six into chromo-acetic acid.
739. Isolation of Fibres. — GAGE'S methods, see §§ 518, 528, and
536.
MOBIUS, muscle of Cardiwn, see § 527.
BALLOWITZ, muscle of Cephalopoda, see Arch. mik. Anat., xxxix,
1892, p. 291.
SCHULTZ (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 -fr> per cent, osmic
acid and J per cent, acetic acid, teases and mounts in glycerin.
For smooth muscle of Vermes, see APATHY, Zeit. f. wiss. Mik., x,
1893, pp. 36, 319, and § 530, ante.
740. Iris.— DOGIEL (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.
CHAPTER XXVI I L 349
See also KOGANEI, Arch. mik. Anat., 1885, p. 1 ; CANFIELD, ibid.,
1886, p. 121 ; and DOSTOIEWSKY, ibid., p. 91.
741. Bladder of Frog, Innervation of (WOLFF, Arch./, mik. Anat.,
1881, p. 362). — A frog is killed and a solution of gold chloride of
1 : 20,000 injected into the bladder through the anus. (If the
injection flows out on removal of the syringe, tie the frog's thighs
together.) Now open the frog, 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 hedgehog 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 acidulated water, and finally
reduce in fresh water in daylight.
RANVIER (Traite, p. 854) recommends his two gold processes,
the liquids being injected as above.
GRUNSTEIN (Arch. mik. Anat., 1899, p. 1) injects, 1 per cent,
methylen blue in normal salt solution through the vena abdominalis,
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.
742. General Stains for Connective Tissue. — Connective tissue,
elastic tissue, and smooth muscle are all normally acidophilous.
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 Saurefuchsin and Wasserblau. Elastin
has a strong affinity for acid orcein, whilst muscle has no special
affinity for either, but stains energetically with picric acid.
Pier o- saurefuchsin is much used and very convenient as a general
differentiating stain, but not to be recommended for cytological
detail. See SCHAFFER, Zeit. wiss. ZooL, Ixxx, 1905, p. 176.
E. and T. SAVINT recommend BENDA'S Picro-Saurefuchsin, § 299.
EHRLICH-BIONDI mixture gives connective tissue red, but smooth
muscle redder still.
UNNA'S Wasserblau-orcein for distinguishing connective tissue
and muscle has been given, § 4%.^ It works after all fixatives.
Stain long, and dehydrate preferably with acid alcohol.
FREEBORN (Amer. Mon. Mic. Journ., 1888, p. 231) recommends
{for sections) picro-nigrosin, made by mixing 5 c.c. of 1 per cent,
aqueous solution of nigrosin, with 45 c.c. of aqueous solution of
picric acid. Stain for three to five minutes, wash with water, and
mount in balsam. Connective tissue blue, nuclei blackish, the rest
yellowish.
R.AM6N Y CAJAL'S picro-indigo-carmine gives connective-tissue
fibres dark blue, with red nuclei. /> ft z \ -v
S. MAYER (Sitzb. k. Akad. Wiss., Ixxxv, 1882, p. 69) recommends
for staining fresh tissue Violet B, § 330. Elastic fibres and smooth
muscle also stain, but of different tints.
DUBREUIL (C. R. Ass. Anat., vi Sess., 1904, p. 62) uses a mixture
of 23 volumes 1 per cent, picric acid and 2 volumes 1 per cent,
methyl blue — with a foregoing stain with carmalum or safranin.
For RANVIER'S method of artificial oedemata for the study of
areolar tissue, see his Traite, p. 329.
CHAPTER XXIX. 351
743. UNNA'S Orcein Method.— (Encyd. mik. Technik,, 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 Miiller and Hermann.
744. UNNA'S Methylen-blue + Saurefuchsin (UNNA, in Encyd.
mik. Technik, 1910, p. 247). Stain for two to five minutes in poly-
chrome methylen blue solution (Griibler). Wash and stain for ten
to fifteen minutes in " (0-5 per cent.) Saurefuchsin + (33 per
cent.) tannin-mixture (G-riibler)." Water, alcohol, essence, balsam.
Collagen, protoplasm, and muscle red, nuclei and keratin blue. On
Flemming material, etastin blue. Liquids of Hermann and Erlicki,
formol and copper fixatives incompatible.
745. UNNA'S Safranin + Wasserblau (ibid.). Ten minutes in
1 per cent, safranin. Wash. Ten to fifteen minutes in " (1 per
cent.) Wasserblau + (33 per cent.) tannin mixture." Wash.
Stains in opposite colours to the last. Formol and liquid of Hermann
contra-indicated for fixing.
746. Flemming's Orange Method is said to give a very sharp differen-
tiation of developing fibrils.
747. MALLORY (Zeit. wiss. Mik., xviii, 1901, p. 175) stains sections of
sublimate or Zenker material lor a few minutes in Saurefuchsin of 0-1
per cent, mordants for a few minutes in 1 per cent, phosphomolybdic
acid and stains for two to twenty minutes in anilin blue 0-5 grms.,
Orange G. 2, oxalic acid 2, and water 100. His phosphotungstic
hsematoxylin stains connective tissue sharply, but does not differentiate
it sufficiently .from elastic tissue and muscle.
•
748. For the complicated procedure of HORNOWSKI see ibid., xxvi,
1909, p. 138.
749. For DELAMARE'S mixture or orcein, hsematoxylin, Saurefuchsin
and picric acid see Verh. Anat. Ges., xix, 1905, p. 227.
750. MASSON (G. E. Soc. Biol, Ixx, 1911, p. 573), stains first in
hsemalum, then in eosin, and then for a, few minutes in 1 per cent, solu-
tion of saffron in tap water (made by boiling). Connective tissue, bone,
and cartilage, yellow.
751. Benecke's stain for fibrils (Verh. Anat. Ges., vii, 1893, p. 165)
is essentially that of KEOMAYER, § 716.
352 CONNECTIVE TISSUES.
.
752. Bielschowsky's SILVER METHOD (post, under " Newofibrilf')
has been used for connective-tissue fibrils. SNESSAREW (Ahat.
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 four days into iron alum of 2-5 to 10
per cent., changed daily. They are then silvered for thirty-six to
forty-eight 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 connective networks black, nerve fibres un-
stained or only weakly stained.
See also MARESCH, Zeit. wiss. Mik., xxiii, 1906, p. 356 ; STUD-
NICKA, ibid., p. 416 ; ZIMMERMANN, ibid., xxv, 1908, p. 10 ; LEVI,
Monit. zool. Ital., 1908, p. 290 ; HEINRICH, Arch. mik. Anat.,
Ixxiv, 1909, p. 786 (dentine) : INSABATO, Arch. Ital Anat. Emb.,
viii, 1909, p. 375 (silvers Flemming material) ; ATHANASTU and
DRAGOIU, C. R. Acad. Sci., cli, 1910, p. 551 (Ramon y Cajal's silver
process, with alcohol fixation).
Elastic Tissue.
753. Elastic Tissue, Generalities. — Elastic fibres have a great
affinity for osmium, staining with much more rapidity than most
other tissue elements. They are not changed by 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 have a natural affinity for orcein, whence stains of the Taenzer-
Unna type.
. For a review of the older methods of BALZER, UNNA, LUSTGARTEN,
and HERXHEIMER, see the paper by G. MARTINOTTI, in Zeit. wiss.
Mik., iv, 1887, p. 31 ; also Encycl. mik. Technik., art. " Elastin."
754. "Victoria Blue (LUSTGARTEN). See § 289.
755. Safranin (G-. MARTINOTTI, loc. cit., § 753). — 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 be performed quicker if it be done at the temperature '
of an incubating stove (GRIESBACH, ibid., iv, 1887, p. 442). See also
FERRIA (ibid,, v, 1888, p. 342).
See also MIBELLI, Mon. Zool. Italicmo, 1, p. 17, or Zeit. wiss. Mile., vii,
1890, p. 225 (the report in Journ. Roy. Mic. Soc., 1890, p. 803, is vitiated
by a misprint). Other basic dyes have been recommended.
CHAPTER XXIX. 353
756. Kresofuchsin (ROTHIG, see § 289).
757. Orcein. — This method is due to TAENZER, and as modified
by UNNA is known as the TAENZER-UNNA method, see third edition,
or Monatssch. prakt. DermatoL, xii, 1891, p. 394.
UNNA'S Modified Orcein Method (Monatssch. prakt. DermatoL ,
xix, 1894, p. 397 ; Zeit. wiss. Mik., xii, 1895, p. 240).— Grubler'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. Sits. Akad. Wiss. Wien., cviii, 1899, p. 335 ; PRANTER,
ibid., xix, 1903, p. 361 (he takes 2 per cent, of nitric acid instead of the
hydrochloric, and stains six to twenty -four hours) ; WOLFF, ibid., p. 488 ;
the article " Elastin " in Encycl. mik. Technik. ; and E. and T. SAVINI,
Zeit wiss. Mile., xxvi, 1909, p. 34.
758. WEIGERT'S Resorcin-Fuchsin Method (Centralb. allg. Path.,
ix, 1898, p. 290). — One per cent, of basic fuchsin and 2 per cent, of
resorcin (or of carbolic acid) are dissolved in water. Two hundred
cubic centimetres of the solution are raised to boiling-point in a
capsule, and 25 c.c. of Liquor ferri sesquichlorati 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 c.c. of hydrochloric acid.
WOLFRTJM (Zeit. "wiss. Mik., xxv, 1908, p. 219) adds 10 to K 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. 3*e im^u^T. b, ^l*]
MINERVINI (Zeit. wiss. Mik., xviii, 1901, p. 161) gives a variant with
safranin instead of fuchsin.
See also PRANTER, ibid., xix, 1903, p. 361 ; B. FISCHER, Virchoiv's
Arch., clxx, 1902, p. 285, or Zeit. wiss. Mik., xx, 1903, p. 40 (chemistry
of the dyes obtained by these processes, which he calls " Fuchselin,"
" Safranelin," etc.) ; HART, Centralb. allg. Path., xix, 1908, p. 1 ; and
CILIMBARIS, Arch. mik. Anat., Ixxv, 1910, p. 708.
759. Haematoxylin Methods.— HARRIS (Zeit. wiss. Mik., xviii, 1902,
p. 290) makes an " Elasthaematein " as follows : Hsematoxylin 0-2 grms.,
aluminium chloride 0-1 grms., alcohol of 50 per cent. 100 c.c., boil and
add mercuric oxide 0-6 grms., filter and add 1 drop of HC1. Keep for
M. 23
354 CONNECTIVE TISSUES.
some weeks. Stain ior five or ten minutes, put into alcohol with 1 per
cent, of nitric acid for one minute, then pure alcohol.
See also DE WITT, Anat. Eec., i, 1897, p. 74 ; DUERCK, Arch. Path.
Anat., clxxxix, 1907, p. 62 ; VERHOEFF, Journ. Amer. Ned. Assoc., 1908,
No. 11.?
MALLORY'S phosphotungstic haematoxylin is good, but not specific.
For a hsematoxylin and eosin stain for connective tissues see KRUGER
(Zeit. f. w. Mikr., xxxi, or Journ. E. Micr. Soc., 1914).
760. Other Methods for Elastic Tissue.— For the elastic tissue of the
skin see PASSARGE and KROSING, Derm. Stud., xviii, 1894.
See also for staining and dissociation AGABABOW, Arch. mik. Anat., 1,
1897, p. 566 et seq.
For C. MARTINOTTI'S silver impregnation see Zeit. wiss. Mik., v, 1888,
p. 521, or Arch. Ital. Biol., xi, 1889, p. 257.
SCHUMACHER (Arch. mik. Anat., Iv, 1899, p. 151) has had good results
(for the spleen) with picro-nigrosin, § 681.
See also § 733.
761. DREW-MURRAY van Gieson-Nile Blue Method for Connective
Tissues (and Bacteria) .—Fix in formol-salt solution. Prepare
paraffin (or frozen) sections. Stain one to three minutes in van
Gieson's picric acid-acid fuchsin solution. Wash in aq. dest. ;
treat in 2 per cent. Nile blue sulphate solution in aq. dest. for from
two to twenty-four hours. Wash in changes of aq. dest. till the
latter is tinted pale blue. Stain again in van Gieson one to five
minutes. Wash in aq. dest. till wash-water is pale yellow. Dehy-
drate rapidly with absolute alcohol from drop bottle. Clear quickly
in xylol (not more than a minute). Differentiate in clove oil from
five minutes to several hours (the longer period is for frozen sections).
Wash in xylol — Canada balsam.
In successful preparations nuclear chromatin a saturated trans-
parent blue, mast cell granules nearly black, collagen red, keratin
and erythrocytes orange yellow (if bacteria are present they stain
blue). (Report of Imper. Cancer Research Fund, 1919.)
Plasma Cells.
762. Plasma Cells and " Mastzellen " ; Generalities.— Plasma
cells, of which " Mastzellen " are a sub-species, are cells found in or
along with connective tissue, and distinguished by their hyper-
trophied and very granular cytoplasm and poorly staining nucleus.
The granules are highly basophilous, much more so than the nuclei ;
they stain with special energy with basic anilins, and mostly meta-
chromatically. 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.
CHAPTER XXIX. 355
According to UNNA in Encycl. mik. TechniL, 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
763. Mastzellen (NORDMANN, Beitr. z. Kenntniss d. Mastzellen,
Inaugural diss., Helmstedt, 1884.). — A concentrated solution of vesuvin
containing 4 to 5 per cent, of hydrochloric acid . Stain for a few minutes ,
and dehydrate with absolute alcohol.
764. Plasma Cells, UNNA'S Later Methods (UNNA, in Encycl. mik.
TechniL, 1910, ii, p. 411).
A. — For Large Plasma Cells.
(1) Ten minutes in Griibler's polychrome methylen blue solution,
wash and 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, two minutes. Wash well. Then
two minutes in glycerin- ether mixture* (Griibler) diluted with
4 volumes of water. Wash thoroughly (two to five minutes) ;
absolute alcohol, bergamot oil, balsam.
(3) Modification of a method of PAPPENHEIM (Virchow's 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.
STROPENI (Zeit. wiss. Mik., xxix, 1913, p. 302) takes acridin red
instead of the pyronin. This will work after various fixatives.
B.—For Small Plasma 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 a mixture of
2 parts alcohol to 3 of xylol, then one minute in xylol ; then five
to ten minutes in alum-anilin (prepared by allowing anilin to stand
* Glycerin ether C6H1003, 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.
23—2
356 CONNECTIVE TISSUES.
over a layer of powdered alum 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 EHRLICH in V if chow's Arch., clxxv, 1904, p. 198.
765. EHRLICH'S Original Method for Mastzellen (Arch. mik. Anat.,
xii, 1876, p. 263). — Stain for at least twelve hours in —
Absolute alcohol 50 c.c.
Water 100 „
Acid. acet. glacial . . . . 12 J „
— to which has been added enough dahlia to give an almost satu-
rated solution. Wash out with alcohol, and mount in resinified
turpentine.
See also SCHIEFFERDECKER and KOSSEL'S QewebeleJire, p. 329.
766. Mastzellen, UNNA'S Latest Methods (Encycl. mik. Techn.,
1910, ii, p. 72). — (1) Stain three hours to overnight in polychrome
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.
These methods give a specific metachromatic stain of Mastzellen
on a light ground. See also loc. cit., two other methods demon-
strating plasma cells at the same time.
767. Other Methods for Plasma Cells and Mastzellen.— See, inter olios
(in previous editions) PAPPENHEIM, Virchow's Arch., clxvi, 1901, p. 427 ;
BERGONZINI, Anat. Anz., 1891, p. 596 ; SCHRIDDE, Anat. Hefte, xxviii,
1905, p. 698 ; MAXIMOW, Arch. mik. Anat., Ixvii, 1906, p. 686 ; SCHAF-
FER, Gentralb. Phys., xxi, 1907, p. 261 (fixation in absolute 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 HC1) ;
RANVIER, C. R. Acad. Sci., 1890, p. 165 (his " Clasmatocytes " : fix
with osmium, stain with aqueous methyl violet 5B).
Fatty Substances.*
768. Fatty Substances. — The general term "fatty substances " is
used here to denote the true fats and the lipoids.
TRUE FATS are esters of the alcohol, glycerol, with the higher
fatty acids, chiefly palmitic acid, stearic acid and oleic acid. The
fatty acids may be " saturated " as, for instance, palmitic and stearic
acid, or they may be " unsaturated " as, for instance, oleic acid.
* By Dr. W. Cramer, Imper. Cancer Research Bureau, and partly by
J. B. G.
CHAPTER XXIX. 357
LIPOIDS. This term was used originally to denote substances
having solubilities similar to those of the true fats, i.e., substances
which may be present, together with the true fats in alcohol, ether
and chloroform extracts of tissues. This definition is, however,
too crude, and the term as now used is restricted to certain chemi-
cally well-defined groups of substances, which in fact constitute the
bulk of such extracts, namely, cholesterol and its esters, phospha-
tides, cerebrosides and phosphorised cerebrosides. The phos-
phorised cerebrosides are " compound lipoids." Their molecule
is very large and consists of a chemical combination of several groups
of simple lipoids, e.g., cerebrosides and a phosphatide in protagon.
The .existence of such compound lipoids is denied by some authors,
who look upon them merely as mixtures of cerebrosides and phos-
phatides. These groups of substances, although differing in their
chemical constitution from each other and from true fats, frequently
occur together in the tissues, and may, therefore, be presumed to
have a similar physiological significance. They also resemble each
other and true fats in their general staining reactions, but some of
them exhibit characteristic differences in this respect. Some
authors use the term " lipoid " as including the true fats. Others,
morphologists in particular, use the term " fat " so as to include
lipoids. It seems advisable, however, to separate the true fats
from lipoids, as is done here, since the two groups of substances
fulfil different physiological functions. The term " LIPIN " has
been proposed by some authors to denote certain groups of lipoids.
The advantage of this nomenclature is not obvious, and since
almost every author who has used this term has given it a new
definition it will not be used here.
For a detailed account of the chemistry of these substances the
reader is referred to the larger text-books and monographs, such
as HAMMARSTEN'S Text-book of Physiological Chemistry, ABDER-
HALDEN'S Biochemisches Handlexicon, Vol. iii, MACLEAN'S monograph
on Lecithin and the Allied Substances. The table on p. 358 gives
only a few elementary data concerning the chemical constitution
of these substances and their solubilities, which are of importance
in connection with their staining reactions.
In the following, the term " true fats " will always be applied to
mixtures of unsaturated and saturated fats, since in the tissues
these substances always occur together. The table shows that all
fatty substances occurring in the tissues contain somewhere in their
molecule a double linkage (marked thus ii in the table overpage),
and are therefore unsaturated compounds. This fact is of importance,
358 CONNECTIVE TISSUES.
Table of Chemical Constitution of Fatty Substances.
Group.
Examples.
Characteristic central
groups.
Additional groups.
Solubilities.
True fats .
Palmitin.
Stearin.
Olein.
Glycerine and fatty acids.
None.
Readily soluble in ace-
tone, ether, chloro-
form and benzene.
Simple
lipoids.
Sterines.
(A.) Cholesterin
(Cholesterol).
(B.) Cholesteri-
nesters.
(A.) Cholesterin free.
ii
(B.) Cholesterin and fatty
ii acids.
ii
None.
(A.) Same as true fats.
(B.) Not readily soluble
in alcohol and ace-
ton, soluble in ether,
chloroform and ben-
zene.
Phospha-
tides
(phos-
phorised
fats).
Lecithin.
Kephalin.
Glycerine, phosphoric acid,
and fattv acids.
ii
A nitrogenous base,
e.g., cholin in the
case of lecithin.
Not soluble in acetone,
readily soluble in
alcohol, ether, chloro-
form and benzene.
Kephalin, when pure,
is insoluble in alcohol.
Cerebro-
sides.
Cerebron
(Cerebrin,
Phrenosin).
Homocerebrin
(Kerasin).
Galactose and a fatty acid .
The nitrogenous base
sphingosin.
ii
Insoluble in fatty sol-
vents in the cold ex-
cept pyridine. Soluble
in hot. alcohol, ben-
zene, chloroform.
Compound
lipoids.
Phospho-
rised
cerebro-
sides.
Protagon.
A chemical combination
of the two cerebrosides,
cerebrin and homocere-
brin (vide supra), and a
substance sphingomye-
lin, allied to the phos-
phatides (see next
column).
Sphingomyelin, which
contains phosphoric
acid, a fattv acid,
sphingosin and cholin
ii ii
Same as cerebrosides.
as on it depends most of the characteristic staining reactions for
fatty substances.
The characteristic staining and other reactions for fatty sub-
stances fall into five groups : —
(1) Staining with Sudan III., or Scharlach R.
(2) Blackening with osmic acid, either with the acid itself or
mixed with bichromate solution.
(3) Staining with hsematoxylin after mordanting with bichromate.
(4) Their behaviour in polarised light.
(5) Staining with Nile blue.
The rationale of these methods will now be considered.
(1) SCHARLACH K, SUDAN III.— Mixtures of the true fats, as they
occur in the tissues, are readily stained by these dyes. Most lipoids,
when pure, do not take this stain at all, or only slightly, except
cholesterinesters, which take the stains, although not as readily as
the true fats. The staining is probably a purely physical process
and depends on the solution of the stain in the fatty material.
Such solution occurs only when the fatty material is fluid, and this
condition is fulfilled in the tissues where mixtures of the true fats
CHAPTER XXIX. 359
are always present together, as, for instance, in the cells of adipose
tissue, or in cells showing fatty degeneration. Mixtures of the true
fats and lipoids which constitute, for instance, many of the fat
globules of the adrenal cortex also take these stains. It is to be
noted, however, that Scharlach R and Sudan III. are applied in
alcoholic solution, and in the process of staining with these dyes some
of the fatty substances tend to be dissolved out. The true fats are
apparently not so readily dissolved as some substances, which show
a double refraction, presumably cholesterin — fatty acid mixtures.
The method of Herxheimer, in which Scharlach R is used in
strongly alkaline solution, has been recommended on the ground
that it is a more " energetic " fat stain for fatty substances in the
sense that it stains many cell inclusions which are not stained by
Scharlach or Sudan alone. The chemistry of the reaction has not
been worked out. It depends probably on the saponifying action
of the alkali, which liberates some fatty acids and then produces
mixtures of fatty acids and lipoids, which are more readily stained
by Scharlach. The range of staining by this method is, therefore,
probably as wide as that of osmic acid alone, and will be found to
comprise most fatty substances, but for the purpose of differentiating
between the different groups of fatty substances has lost the advantage
of restricted staining, which the ordinary staining with Sudan III. and
Scharlach R alone possesses.
(2) OSMIC ACID METHODS. — The true fats and the lipoids are all
blackened by osmic acid. This blackening indicates a reduction of
osmic acid to a lower oxide. It is due to the fact that all these
fatty substances have a double linkage (marked ii in table on p. 358)
in their molecule and are, therefore, more or less easily oxidised.
But the various groups of substances differ in the readiness with which
they are oxidised, and consequently in the rapidity with which they are
blackened by osmic acid and the depth of the blackening produced. The
true fats are blackened most rapidly and most deeply, the phospha-
tides, lecithin and kephalin, come next in order, while cholesterin, the
cerebrosides and phospho-cerebrosides are least susceptible to the
oxidising action of osmic acid. In fact, these substances when
splid are not blackened a-t all, but are so when dissolved in an appro-
priate solvent, such as chloroform. In the myelin sheath of peri-
pheral nerves they are present in the form of a colloidal solution,
since it can be shown that they contribute to the blackening of the
sheath by osmic acid.
This difference in the reducing power of the various fatty sub-
stances can be accentuated by using osmic acid together with bichro-
360 CONNECTIVE TISSUES.
mate solutions, as in the March! method. The bichromate, which is
itself an oxidising agent, acts in the various double linkages, and
prevents the osmic acid from being reduced except by the substances
having the strongest reducing power ; these are the true fats and mix-
tures of cholesterin and unsaturated fatty acids.
Since the latter show double refraction in polarised light (see
below), while the true fats do not, it is possible to differentiate these
two groups of substances.
The behaviour of mixtures of cholesterin and fatty acids is para-
doxical, since cholesterin alone and fatty acids alone do not blacken
with osmic acid after bichromate. The same paradoxical behaviour
is exhibited by these cholesterin fatty acid mixtures in their staining
reaction with hsematoxylin after mordanting with bichromate
(see next paragraph). One must assume that cholesterin is present
in these mixtures in a special physical state, in which it exhibits a
greater reducing power.
The myelin sheath of normal nerves does not contain true fats
and gives, accordingly, no blackening with bichromate-osmic acid
treatment. In the early stages of degeneration globules of true fat
are formed, which stain black with bichromate-osmic acid and then
give the positive Marchi reaction. With other tissues rich in lipoids,
such as the adrenal cortex, no such clear distinction can be obtained
because the lipoids are present therein, not only together with true
fats, but also mixed with them, so that a globule of fatty material
frequently contains both groups of substances.
(3) STAINING WITH HJEMATOXYLIN AFTER MORDANTING WITH
BICHROMATE. — This method, which was introduced originally by
Weigert for the staining of the medullary sheath of nerves, is
applicable to all fatty substances. Like the preceding method, it
also depends on the presence of a double linkage in the molecule,
as pointed out by Thorpe. Substances having a double linkage
are oxidised by the bichromate solution and, in this process of
oxidation, a chromium compound is formed which is insoluble in
fat solvents and which at one stage of the oxidation has the property
of forming a dark blue lake with haematoxylin. If oxidation be
continued, however, hsematoxylin will again cease to stain. The
rate of oxidation with bichromate varies, as might be expected,
with the concentration of the solution used and with the temperature
at which it is carried out. Working under similar conditions, it is
found again that the different fatty substances vary in the readiness
with which they are oxidised by bichromate and, consequently,
reach the stage of staining with hsematoxylin after different periods
CHAPTER XXIX. 361
of mordanting with bichromate. The different lipoids, when pure,
differ greatly in the ease with which the stainable chromium com-
pounds is formed ; thus the cerebrosides and protagon stain after
a short mordanting ; the unsaturated true fats require a slightly
longer time, while lecithin, and especially cholesterin, are very
resistant and require prolonged mordanting. But mixtures of
cholesterin and the various fatty substances show quite a different
behaviour and reach the stainable stage very rapidly. This appears
to be due to the fact that such mixtures form colloidal solutions in
a peculiar physical condition (fluid crystals). It will be clear that
a histochemical identification of the various lipoids by means of
this method is not possible. Its value lies in the possibility of
demonstrating, first, the presence of fatty substances by a method
which gives good histological details, and secondly, by comparison
with normal tissues, the occurrence of chemical changes in these
substances under pathological conditions.
As introduced by Weigert, the method stains the lipoids of the
normal myelin sheath. • By prolonging the bichromating the
degenerating nerve fibres may be made to stain in the early stage of
degeneration, or the fat droplets in fatty organs may be stained.
The same principle underlies Altmann's method for the staining
of mitochondria, which are by some believed to consist of a central
core of protein covered by an envelope of fatty material. The
original method consists in mordanting with a bichromate solution
and staining with acid fuchsin, just as Weigert originally used acid
fuchsin for the staining of the myelin sheath. In the staining of
mitochondria, the acid fuchsin can again be replaced by hsBmatoxylin
(Heidenhain's hsematoxylin).
(4) BEHAVIOUR IN POLARISED LIGHT. — The true fats and the
fatty acids are isotropic, i.e., show no double refraction in polarised
light, so that with crossed nicols the field appears dark. The lipoids
are anisotropic. In fresh teased preparations they can be seen with
crossed nicols as luminous droplets with a varying degree of brilliancy.
The double refraction disappears on gentle heating to about 60°
and reappears on cooling. In formol fixed frozen sections the aniso-
tropic lipoids appear chiefly in the form of needles and as droplets.
Heating and cooling produces the effect mentioned above.
The behaviour in polarised light is, therefore, an easy and im-
portant means of differentiating the isotropic true fats from lipoids.
(For a detailed description of the technique of the polarisation
microscope, see AMBRONN, Anleitung zur Benutzung des Polarisa-
tions Mikroskopes bei Histologischen Untersuchungen ; ADAMI,
362
CONNECTIVE TISSUES.
The Myelin and Potential Fluid Crystalline Bodies of the Organism,
1906 ; KAISERLING and OGLER, " Uber das Auftreten von Myelin
in Zellen, etc.," Vir chow's Archiv., clxvii.).
(5) NILE BLUE. — When a solution of Nile blue is boiled with
sulphuric acid the solution contains, in addition to the original basic
oxazine dye, which unites with fatty acids to form a blue compound,
a red oxazone dye, not basic in character, but soluble in liquid fat
and giving it a red colour. The stain, therefore, enables us to
distinguish neutral fat and fatty acid. If a given globule contains
neutral fat and no fatty acid, it will be coloured red ; if it contains
fatty acid only, it will be coloured blue ; but if it contains both
neutral fat and fatty acid, it will be coloured a tint between blue
and red, depending on the proportion of neutral fat and fatty acid.
The principal histochemical group reactions are summarised in the
following table : —
Table of some important Histochemical Group Reaclions.
Sudan.
Double
refrac-
tion.
Osmic
acid.
Osmic
after bi-
chromate.
Solubility in cold
Acetone.
Alcohol.
Chloro-
form.
True fats
+ + +
-
+ + +
+
+
+
+
Cholesterin
Cholesterinesters
Phosphatides .
Cerebrosides .
Phospho-cerebrosides
+
+
+
+
+
+
+
+
+ +
+
+
-
+
+
+
+
I
+
Cholesterin - fatty )
acid mixtures. j
+
+
+ +
+
+
•i-
Although different groups of fatty substances differ in their
behaviour towards different staining methods, it is, nevertheless,
necessary to proceed with caution in interpreting the results obtained
when these methods are applied to tissues. In the tissues the various
groups of fatty substances are mixed, so that one globule may contain
two or three different substances. The various substances may
then form a special kind of colloidal solution, which modifies their
physical state and alters their staining reaction completely. The
behaviour of mixtures of cholesterin and fatty acids is an example
and has been referred to above. Or one substance encloses another
substance belonging to a different group. In such a case the
staining reactions of the globule would be those of the material
which constitutes the envelope. In the adrenal cortex, for instance,
CHAPTER XXIX. 363
many globules contain a core of lipoids surrounded by true fats.
In such a case examination in polarised light is helpful. Differentia-
tion by means of different solvents is also possible within limits.
Again, the size of the globules is, for obvious reasons, an important
factor in the methods which involve mordanting with bichromate.
But even if due consideration is given to these reservations, the
application of these staining reactions has yielded important
results and has profoundly modified our conceptions of the part
taken by the fatty substances as constituents of protoplasm. In
the first place, it is important to realise that no one single method is
a specific staining method, either for all fatty substances or for any
one group of them. Thus, Sudan III. or Scharlach E. do not stain
all fatty substances, as a glance at the table of the group reactions
shows. They stain intensely the true fats, and less intensely
cholesterinesters and cholesterin-fatty acid mixtures. But many
lipoids are not stained by these dyes, and an examination in polarised
light is necessary to detect their presence. Osmic acid alone has
the widest range as a reagent for fatty substances, and stains all the
different groups. With some substances, however, such as chole-
sterin or cerebrosides, it may give negative results if they are not
present in a state of colloidal solution in the cytoplasm. But since
osmic acid stains also substances which are not fatty in nature,
e.g., adrenalin in the cells of the adrenal medulla, it cannot be looked
upon as a specific stain. In the case mentioned, a differentiation
can still be effected by immersing the section in turpentine, which
dissolves the fatty substances, even after osmication. The method
giving the highest degree of specificity is the use of osmic acid after
bichromate. This will stain only the true fats or cholesterin-fatty
acid mixtures. Or, expressed in terms of everyday histological
technique, the presence of black cell globules in material fixed either in
bichromate-osmic mixtures, such as Altmann's, Champy's or
Flemming's fixatives, or first in bichromate fixatives, such as Mutter's
or Zenker's fluids, and post-osmicated (§ 691) indicates the presence
either of true fats or of cholesterin-fatty acid mixtures. These two
can then be differentiated by examination in polarised light. It is
perhaps equally important to be able to draw the opposite con-
clusion, when other facts have indicated the presence of fatty
substances. The absence of blackening of the globules under the
conditions just mentioned definitely excludes the presence of true
fats. Thus, true fats can be proved to be absent from the myelin
sheath of normal nerves. The significance of the staining of fatty
substances after mordanting with bichromate has already been
364 CONNECTIVE TISSUES.
referred to as revealing the essential similarity of Weigert's method
for the myelin sheath of nerves and Altmann's method for mito-
chondria. The staining reactions of the Golgi apparatus — blacken-
ing after prolonged action of osmic acid, staining with hsematoxylin
after prolonged mordanting with bichromate — suggest that it is
made up of fatty substances, and, further, that these substances
are not readily oxidised. They thus correspond in their behaviour
to lipoids such as cholesterin.
From what has been said, it is clear that for the study of fatty
substances several methods must be applied to the tissues.
In so far as the selection of a fixative is concerned, it follows
that fixatives containing alcohol or chloroform must be avoided.
A fixative such as Carnoy, or such mixtures containing strong
alcohol or chloroform, are themselves fat solvents, as well as lipoid
solvents, and they dissolve away all fat-vacuoles and shrink up
cell organs which may .be formed partly of other " fatty " sub-
stances, e.g., mitochondria. Other fixatives, such as picric acid or
corrosive sublimate, while in themselves not fat or lipoid solvents,
are unable to prevent the fat from being dissolved away in any
subsequent dehydration and clearing of the tissue. Very few reagents
are known which are able to form with fat substances insoluble
or scarcely soluble in alcohol and a clearing medium, like xylol ;
osmium tetroxide and, to a less extent, potassium bichromate,
are thus valuable reagents to the histologist. Various oils act
differently towards these fat Os02 compounds ; xylol and chloro-
form do not easily disintegrate them, but an oxidising oil like tur-
pentine will quickly do so. The " compounds " of Os04 with fats
and lipoids are ill understood, but Partington and Huntingford
have recently shown (see § 772) that the reduced black substance is
a hydrated form of Os02.
Martinotti (see § 772) has recently introduced a new method
which may prove of great importance. According to this observer
the orange yellow dye chrysoidin (phenyl-azo-m-phenylene-diamide),
when applied to fatty tissue and then treated with an oxidising
agent, such as bichromate of potash, has the power of preventing the
fat globules from being dissolved away in alcohol, benzol and xylol.
In preparations the fat looks a brown orange colour.
A complete histochemical investigation of fatty cell inclusions
comprises, thus, the following methods : —
(1) In fresh preparations :
(a) Examination in polarised light.
(b) Staining with Sudan III. or Scharlach R.
CHAPTER XXIX. 365
(c) Staining with osmic acid in solution or as vapour.
(2) In frozen sections, material fixed in formol :
(a) Examination in polarised light.
(b) Staining with Sudan III. or Scharlach K.
(c) Staining with Nile blue.
(d) Repeat (b) and (c) after immersing sections in cold acetone
or cold alcohol for a few minutes.
(3) In paraffin sections :
(a) Fixation in osmic acid.
(b) Fixation in bichromate osmic acid mixtures or fixation in
bichromate solutions and post-osmication.
(c) Prolonged fixation in bichromate and staining with
Sudan III. (Ciaccio, Bell).
(d) Staining with hsematoxylin (Weigert, Lorrain, Smith,
Dietrich).
(e) Treatment with chrysoidin and subsequent fixation in
bichromate (Martinotti).
Such a plan of investigation refers especially to vertebrate
materials, but with certain small modifications is applicable to
the study of fatty substances in developing eggs, embryos, and to
the tissues of invertebrates. Paragraphs 1 and 3 are both applicable
to invertebrate tissues and embryos, while in the case of para-
graph 2 the tests can be used after the whole embryos or eggs have
been fixed in neutralised and suitably diluted formalin. It should
be remembered that in all animal cells (and possibly plant cells, too)
there exist two categories of cytoplasmic inclusions, Golgi apparatus
and mitochondria, which are partly formed of lipoid materials,
and which we now know may produce fats, or may metamorphose
into fats. In nearly all eggs where fat granules are present, exami-
nation has revealed the fact that such " yolk " is derived either from
Golgi elements or mitochondria (GATENBY and WOODGER, Journ.
Roy. Micr. Soc., 1920), and in certain cells of vertebrate tissues we
know that mitochondria may change into fat (MURRAY, Scientific
Report Cancer Research Fund, 1919).
Reference should be made to the sections on " Mitochondria,"
"Golgi Apparatus," "Fat," and "Yolk" (§§ 673—713), and
especially to the tables in §§ 702, 708, 710 and 712, where some
attempt has been made to illustrate the behaviour of the various
inclusions after the application of certain well-known techniques.
It is always necessary to ascertain exactly the condition and
behaviour of the mitochondria and Golgi apparatus in tissues or
cells being investigated for fatty and lipoid substances, particularly
366 CONNECTIVE TISSUES.
in view of any inter-relationship which may exist between the former
and the latter. See also BELL (Journ. Med. Research, xxiv,
1911, p. 539 ; Journ. of Pathol. and Bad. xix, 1914, p. 105.
CIACCIO (Centralblatt f. allg. Pathol. and Path. Anatomic, xx, 1909,
p. 771 ; Arch. f. Zellforschung, v, 1910, p. 235. CRAMER, FEISS
and BULLOCK (Proceed. Phys. Soc., 1913 ; Journ. of Physiology,
xlvi, p. 51. International Congress of Medicine, London, 1913.
Section of Pathology). DIETRICH (Erg. d. Allg. Pathologic und
Patholog. Anat., xiii, 1909, pt. 2, p. 283 ; Deutsche Patholog.
Gesellsch., xiv, 1910, p. 263). KAWAMURA (Die Cholesterinester
verfettung. Jena. Gustav Fischer. 1911). SMITH and MAIR (Journ.
Pathol. and Bact., xiii, 1909, p. 14 ; Skand. Arch. f. Physiol., xxv,
1911, p. 247).
769. Fixing and Staining. — The choice o!; the fixative depends
on the question whether the material is to be examined in frozen
sections or in paraffin sections. In any case all fixatives containing
acetone, alcohol, chloroform or other fat solvents are excluded.
For paraffin sections the material may be fixed in osmic acid alone
(1 per cent, in solution, or 2 per cent, if fixed in vapour), or in osmic
acid mixed with bichromate solution (see fixatives of Flemming,
Altmann, Champy). Or it may be fixed in formol bichromate and
treated subsequently with a bichromate-osmic mixture (see methods
of Schridde and Marchi). As stated in the general part (see
p. 363) the different methods give different results with the various
groups of fatty substances. For all these methods very small
pieces of tissue must be used. For the effects of alcohol on the
blackening of certain fatty substances by osmic acid, see HAND-
WERCK, Zeit. wiss. Mik., xv, 1898, p. 177 ; MULON, ibid., xxii, 1905,
p .138 ; GOLODETZ, ibid., xxviii, 1911, p. 213 ; and Chem. Rev. Fett
u. Harzindustrie, xvii, 1910, p. 70 ; LOISEL, C. R. Soc. Biol., 1903,
p. 826.
Another method consists in fixing and mordanting with strong
bichromate solution and subsequent staining with Sudan (see below,
Bell's method, also Ciaccio) or with hsematoxylin (methods of
Weigert for nervous system ; also Lorrain Smith, Dietrich).
For examination in frozen section the tissue may be fixed in
formol saline or formol-bichromate, or the stain may be applied
directly to the fresh tissue after teasing. For fine cytological work,
the formol should be neutralised by shaking with solid calcium
carbonate. HAYS BULLARD (Amer. Journ. Anat.. xix, 1916) recom-
mends neutralisation and distillation method of GUSTAV MANN
(Physiological Histology, Oxford, 1902) : neutralise commercial
CHAPTER XXIX. 367
solution with sodium or lithium carbonate, and freshly distil. A
20 per cent, solution is then prepared and rendered isotonic :
•75 gms. of NaCl to 100 c.c. of fluid. With short fixation (thirty-five
minutes to five hours), the quantity of fat usually does not differ
from that seen in fresh tissue (Bullard). Cut on freezing microtome,
stain by one of the methods given below, preferably Herxheimer's
alkaline scarlet red. As control use also fresh tissue.
For quinolein blue, see § 322.
DADDI (Arch. Ital. Biol., xxvi, 1896, p. 413) stains fat in tissues
by treating for five to ten minutes with concentrated alcoholic
solution of Sudan III. washing for the same time with alcohol,
mopping up with blotting paper, and mounting in glycerin.
Similarly RIEDER, see Zeit. wiss. Mik., xv, 1898, p. 211.
The alcohol for making the stain should be of 70 per cent.,
according to most authors, though SATA (Beitr. path. Anat., xxviii,
1900, p. 461 ; Zeit. wiss. Mik., xviii, 1901, p. 67) employs 96 per
cent. ROSENTHAL (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.
MICHAELIS (Virchow's Arch., clxiv, 1901, p. 263) recommends
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. HERXHEIMER (Deutsche
med. Wochenschr., xxvii, 1901, p. 607 ; Zeit. 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 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 BELL, Amer. Journ. Anat., ix, 1909, p. 401, and Anat.
Rec., iv, 1910, p. 199.
HERXHEIMER also (Centralb. allg. 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, 1909, p. 607 ; Zeit.
wiss. Mik., xix, 1902, p. 67) has had very fine results by staining
for 20 minutes in a saturated solution of Indophenol in 70 %
alcohol.
MOLLISON (Zeit. wiss. ZooL, Ixxvii, 1904, p. 529) has had good
results by staining gelatin sections for a few minutes in strong
368 CONNECTIVE TISSUES.
extract of Alkanna in 96 per cent, alcohol, and mounting in glycerin
or syrup.
LORRAIN SMITH (Journ. Path. 'Bact., xii, 1907, p. 1) finds that
Nile blue stains fatty acids blue and neutral fats reddish.
Similarly EISENBERG (Virchow's Arch., cxcix, 1910, p. 502), who
recommends aqueous solution of Nilblau BB.
BEND A (ibid., clxi, 1900, p. 194) finds that free fatty acids can be
detected by Weigert's neuroglia mordant. See also BERNER, ibid.,
clxxxvii, 1907, p. 360, and FISCHLER, Zeit. wiss. MiL, xxii, 1905,
p. 263.
OKAJIMA (ibid., 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 KINGSBURY, Anat. Rec., v, 1911, p. 313.
770. Removal of Fatty Substances. — If not treated with osmic
acid or mordanted with strong bichromate, alcohol, ether, chloroform,
pyridine, xylol, will readily dissolve fatty substances. Osmicated fats
and lipoids are more resistant, especially if osmic acid and bichromate
have been combined. It can then be removed in a few hours by
alcoholic hydrogen peroxide (10 per cent. H202 in 80 per cent, alcohol)
or in -twenty-four hours by oil of turpentine. Ether, creosote, xylol,
clove oil and chloroform will also remove osmicated fats and lipoids if
allowed to act sufficiently long.
See also FLEMMING in Zeit. iviss. Mikr., 1889, pp. 39, 178.
771. Differentiation between Fats and various Lipoids. — Fix in for-
mol and prepare frozen sections. Stain some with Sudan or Scharlach
(see above § 769), others with osmic acid. Leave some unstained.
Globules which stain with Sudan or Scharlach and osmic acid, and
which in unstained sections show no double refraction, can be identified
with certainty as true fats. This may be confirmed in paraffin sections
by fixing in bichromate and subsequent treatment with osmic acid as
in the methods of Schridde and Marchi. These globules should then
reduce osmic acid. But the presence of double refraction must not be
taken as excluding the presence of true fats since the globules may be a
mixture of true fats and double refracting lipoids. The deduction that
true fats are absent can be made when tissue containing fatty material
as indicated by blackening with osmic acid fails to give this blackening
after previous treatment with bichromate, as for instance in normal
peripheral nerve.
The histochemical differentiation between true fats and lipoids is
much more difficult when these substances are mixed in one and the
same globule than when one cell contains several globules of which
some are composed entirely of true fats while others contain lipoids.
In the latter case methods may be -applied which depend on differences
in the solubility in various solvents.
DEFLANDRE (Journ. Anat, Phys., 1904, p. 80) fixes in formol of 4 per
CHAPTER XXIX. 369
cent, and brings into acetone, in which fat is dissolved, but not lecithin,
which can then be stained by osmium.
See also CIACCIO, Arch. Zellforsch, v, 1910, p. 235 ; and FISCHLER,
Zeit. wiss. Mik., xxii, 1905, p. 262 ; LOISEL, C. R. Soc. BioL, Iv, 1903,
p. 703.
BELL with a modification of DIETRICH'S and CIACCIO' s methods
(Journ. Path, and Bact., xix) claimed to be able to distinguish between
fat drops mainly of triolein and those that principally contain " lipoids."
The former appear in annular shape, the latter are quite solid. In the
former case the centre of the droplet is not chromated, and therefore
dissolves out in xylol used for imbedding. Fix at 45° C. to 50° C. in
10 per cent, aqueous K2O207, 100 c.c., acidified by 5 c.c. of acetic.
Wash, dehydrate and imbed in paraffin. Fasten 3 to 5 ^ sections to
slide with albumen water. Treat sections in xylol, absolute alcohol,
and transfer to freshly prepared solution of Sudan III in 80 per cent,
alcohol for ten minutes. Rinse off excess stain in 50 per cent, alcohol ;
transfer to water to stop action of alcohol. Counterstain in Delafield's
hsematoxylin, wash in water, differentiate in acid alcohol, wash and
mount in glycerin gum-arabic.
772. Mounting Fat. — After treatment with osmic acid sections can
generally be mounted in balsam without special precautions. Many
lipoids, however, fade even after osmication, if kept for a long time in
balsam. In some cases it may suffice to avoid absolute' alcohol and
essences as much as possible, and mount direct in alcohol balsam or
euparal, or clear 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.
Chrysoidin (L. MARTLNOTTI, Zeit. Physiol. Chem., xci, 1914) fixes
tissue in 10 per cent, formol, sections on a freezing microtome,
washes in aq. dest., and immerses for five to ten minutes up to
several hours in a 1 per cent, aqueous solution of chrysoidin. Wash
not longer than one minute in aq. dest., and treat in 10 per cent.
K2Cr207 or Cr03 for one minute, wash, dehydrate in benzol and
xylol, and mount in neutral balsam.
Bone* |
773. Bone, Non-decalcified. — RANVIER (Traite, p. 297) has the
following : ,
Bones should be plunged into water, without being allowed to
dry, as soon as the surrounding soft parts have been removed,
and should be divided into lengths with a saw whilst wet. The
* For a detailed review of the whole subject, see the paper of SCHAF-
FER in Zeit. wiss. Mik., x, 1893, p. 167, or the article " Knochen und
Zahne " in Encycl. mik. Technik.
I This section has been revised by J. Thornton Carter, Esq.,
F.R.M.S., of the Zoological Department, University College, London.
M. 24
370 CONNECTIVE TISSUES.
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 per-
fectly 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 mois-
tened 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 litho-
graphic stone. Spongy bone should be soaked in gum and dried
before rubbing down (but see VON KOCH'S copal process and
EHRENBAUM'S colophonium process).
SCHAFFER (Zeit. wiss. Mik., x, 1893, p. 171) grinds and polishes on
stones of graduated fineness.
For the process of WEIL for bones and teeth see § 180.
KOSE (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 bs combined with Golgi's impregnation.
FANZ (Anat. Record, xiv, 1918, p. 493) employs sand or carborundum
paper of different grades of coarseness for grinding, using the back or
smooth side of a piece of sandpaper for polishing the section. He
recommends shellac in preference to balsam for attaching the section
to the glass slip.
WHITE (Journ. Roy. Mic. Soc., 1891, p. 307) recommends the
following : Sections of osseous or dental tissue should be cut or
ground down moderately thin, and soaked in ether for twenty-
four hours or more. They should then be put for two or three days
into a thin solution of 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 powder, and mounted, surface dry, in stiff balsam or styrax,
care being taken to use as little heat as possible. Lacunae, canaliculi,
and dentinal tubuli are found infiltrated by the coloured collodion.
CHAPTER XXIX. 371
HANAZAWA (Dental Cosmos, lix, 1917, pp. 125 et seq,) gives a number
of methods for staining ground and decalcified sections of dentine to
demonstrate its minute structure.
MATSCHINSKY (Arch. mile. 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. Mile., xiii, 1896, p. 21,
wherein see also quoted (p. 23) a method of ZIMMERMANN.
CSOKOR (Verh. (mat. Ges.,1892, p. 270) dascribes a saw which will cut
fresh bone to 120 p. • and ARNDT (Zeit. wiss. Mik., xviii, 1901, p. 146) a
double saw which will also give very thin sections.
774. Mounting.— To show lacunae 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.
When thin ground sections of enamel are mounted in Canada
balsam it is found often that they appear almost structureless.
To demonstrate the enamel pattern of such sections they may be
etched by immersion in -6 per cent, of hydrochloric acid in 70 per
cent, alcohol, or in a weak aqueous solution of picric acid, and
mounted in Camsal balsam or Euparal, media which, on account of
their low index of refraction, will be found to disclose the structure
of the enamel more easily.
775. Sections of Bones or Teeth showing the Soft Parts. — A
developing tooth with its epithelial enamel-organ, its mesodermal
dentinal papilla, and its layers of partially calcified enamel and den-
tine, is made up of very delicate structures of different consistency
and so peculiarly liable to unequal shrinkage, with consequent
distortion during the period of fixation and in the subsequent
processes passed through in the preparation of sections. Further,
post-mortem changes in the ameloblasts occur within a very few
minutes after death leading to a less precise behaviour to stains than
is found in the case of cells which are fixed immediately after death.
For the examination of developing teeth in situ, jaws may be
fixed in corrosive-formalin-acetic mixture, in Bouin's picro-formol,
in Zenker's mixture or Helly's modification thereof, or in Sansom's
modification of Carnoy's mixture (§ 86).
For the study of the micro-anatomy of the enamel-organ and the
dentinal papilla, a young pup or a kitten, two or three days old,
is killed, preferably by a blow on the head. The jaws are removed
and the bone of the under-surface of the mandible pared away by a
sharp scalpel until the bases of the tooth-germs are almost exposed.
24 — 2
372 CONNECTIVE TISSUES.
The muco-periosteum is grasped with a pair of forceps and stripped
from the bone, when the tooth-germs will come away attached thereto.
Sansom's modification of Carnoy's mixture, employed at blood-
heat, is particularly effective when the tooth-germs have been
exposed in the manner outlined above, fixation therein being com-
plete in from five to ten minutes. They are then passed through
successive baths of alcohol of 30 per cent, and 50 per cent., each for
fifteen minutes ; 70 per cent., to which is added tincture of iodine,
for four hours ; 90 per cent, for thirty minutes ; and into two changes
of absolute alcohol, each for fifteen minutes or longer.
The tooth-germs are then transferred to a mixture of equal parts
of absolute alcohol and carbon disulphide for one hour, two changes
of pure carbon disulphide, each of fifteen minutes, then for thirty
minutes into carbon disulphide saturated with paraffin at 30° C.,
transferred to carbon disulphide saturated with paraffin at 42° C.
for a like period, and finally into two baths of paraffin, in each
half an hour. Imbed for cutting in pure paraffin.
By the employment of this method the amount of shrinkage in
the tissues is extremely slight and the dentine does not become
hardened, so that the tooth-germs of the incisors may be cut without
decalcification. In the case of the canine and molar tooth-germs
a short period of decalcification may be necessary, and for this
purpose a rapid and delicate method lies in the employment of
ZEIGLER'S method (Festschr. f. Kupffer, 1899, p. 51), in which, by
the use of a 5 per cent, solution of sulphurous acid, the insoluble
tricalcium phosphate is changed into the readily soluble mono-
calcium phosphate.
To demonstrate cytological detail no stain equals iron-haema-
toxylin followed by a counter-stain of picric-lichtgrun or of Rubin S
in picrate of ammonia.
It cannot be too strongly emphasised that the precision of staining
methods depends on the rapidity with which fixation of the tissues is
effected after death. Refer to § 31.
For large jaws imbedding in celloidin, or, when serial sections are
required, double imbedding in celloidin, parlodion or photoxylin
and paraffin is recommended (§ 171).
MUMMERY (Phil. Trans. B., ccviii, 1917, p. 258) deprecates the
employment of paraffin for imbedding the tooth-ge%rms of fishes,
considering the heat employed to be very injurious to the delicate
enamel organs, and advocates the use of the freezing method in
obtaining sections. See carbon disulphide method above.
NBALET (Amer. Mon. Mic. Journ., 1884, p. 142 ; Journ. Eoy. Mic.
CHAPTER XXIX. 373
Soc., 1885, p. 348) says that perfectly fresh portions of bone or teeth
may be ground with emery on a dentist's lathe, and good sections, with
the soft parts in situ, obtained in half an hour.
HOPEWELL- SMITH (Journ. Brit. Dent. Ass., xi, 1890, p. 310; Journ.
Roy. Mic. Soc., 1890, p. 529) says that for preparing sections of teeth
showing odontoblasts in situ the bsst 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 microtome.
WEIL (loc. eit., § 180) fixes pieces of fresh teeth in sublimate, 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 KOSE, § 773.
775 A. — For the study of the vessels in teeth, LEPKOWSKY (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 to fourteen days, change
frequently) and makes celloidin sections.
For decalcification of teeth, see also § 546 (ROUSSEAU, BODECKER
and FLEIS.CHMANN). Bodecker finds Rousseau's process not appli-
cable to human teeth : the acid must be added to the fluid celloidin.
For the study of the lymphatics in the dental pulp, DEWEY and
NOYES (Dental Cosmos, lix, 1917, pp. 436—44) first inject the
blood vessels with carmin-gelatin. Then 2 grms. of Prussian blue
(oil colour in tubes) is stirred with 3 grms. of turpentine oil in a
glass mortar for five minutes ; 15 grms. of sulphuric ether is added,
and this fluid filtered through flannel or chamois skin. After the
injection of this fluid the head is placed for twenty-four hours or
longer in 20 per cent, formalin, and then the injected teeth are
carefully removed and the pulps examined. Later it was found
that more constant results were obtained when the injection of the
blood vessels followed that of the Prussian blue. Prussian blue
injected directly into the pulps and trypan blue or lithium
carmine injected intravenously or intraperitoneally were also
employed. See §§ 780 and 790.
WELLINGS (Proc. Sixth Internal. Dent. Cong., pp. 47 et seq.) demon-
strated intra-vitam staining of dental and adjacent tissues by means
of trypan blue (§ 780).
MUMMERY (Phil. Trans. B., ccii, 1912), for the fixation of the
nerve-tissue of the dental pulp, finds formalin to be preferable to all
other fixing agents, employing 10 parts of the 40 per cent, com-
mercial formalin to 90 parts of water.
Decalcification is effected by means of 33-3 per cent, formic acid.
After thorough washing he leaves for twenty-four hours in a strong
374 CONNECTIVE TISSUES.
solution of dextrin (which he finds preferable to gum arabic), and
sections are cut on the freezing microtome, by the employment
of which he is able usually to obtain thinner sections than when
paraffin is used for imbedding.
The sections are stained either by means of iron and tannin,
iron-hsematoxylin (Benda), Congo red, Eanvier's modification of
Lowet's gold chloride process, or by Cajal's method, where : —
(1) Small pieces of the decalcified tooth, not more than 4 milli-
metres thick, are placed in 50 c.c. of rectified spirit, to which 3 or
4 drops of ammonia may be added, and kept in this solution for
from four to six hours.
(2) Transfer to absolute alcohol for twenty-four hours.
(3) Rinse with distilled water.
(4) Place in a large quantity of 1-5 per cent, solution of silver
nitrate, and keep in warm incubator at about 35° C. for five or six
days.
(5) Rinse in distilled water for a few seconds.
(6) Place in the following solution for twenty-four hours :—
Hydrokinone . . . . . 1 to 1-5 grm.
Distilled water ..... 100 c.c.
Formol . . . . . . 5 to 10 c.c.
Rectified spirit . . . . 10 to 15 c.c.
(7) Wash in water for some minutes.
(8) Cut sections, and mount.
The presence of nerve-end cells in the dental pulp was demon-
strated by MUMMERY (Phil. Trans. B., ccix, 1920), by means of a
modification of the gold method of Beckwith.
Teeth, immediately after extraction, are placed in a solution of
formol and water or of formol and normal salt solution, preferably
4 per cent, of formol. This is, after a few days, changed to a
10 per cent, solution, and the teeth kept in this for at least a
fortnight.
Decalcification is effected by means of a 33-3 per cent, solution of
formic acid in distilled water, to which 5 per cent, of formol may be
added. (Mummery states that neither he nor Dependorf has
ever procured good nerve preparations of teeth which have been
decalcified in the mineral acids.)
Wash in running water for twenty-four hours, then for a few
minutes in distilled water.
The pieces are taken from the distilled water and suspended by
threads in a large quantity of a weak solution of gold chloride
(1 in 5,000). Each piece should be suspended in at least 100 c.c.
CHAPTER XXIX. 375
of the solution, in which it is left in the dark for from four days to
one week, according to its size. On removal from the gold solution
it is washed for a few minutes only in distilled water. Reduction
is effected by placing the pieces in a 20 per cent, solution of caustic
soda for four minutes, then rinsing in water and placing in a 10 per
cent, solution of potassium carbonate for from half an hour to an
hour. This is then drained off, and the pieces are placed in a 10
per cent, solution of potassium iodide for a short time — usually
five to ten minutes. As soon as seen to darken, the pieces are removed
from this solution to water, placed in gum for twelve hours, and
sections cut on the freezing microtome.
After dehydration the sections are mounted in camsal (propylic)
balsam.
776. VIVANTE (Intern. Monasschr. Anit. 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 millimetres thick, by Golgi's rapid bichromate
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 carbonat?- of soda, and finally imbedded in •
paraffin. For his quinolein blue method see fourth edition.
For UNDERWOOD'S gold process for teeth, and for that of LEPKOWSKI,
see third edition, or Anat. Anz., 1892, p. 294.
LAW (Proc. Roy. Soc. Med., i, 1908, p. 45) studies nerve-endings in
teeth of mammals by treating paraffin sections of decalcified tissue with
BETHE'S molybdenum toluidin blue (details in Journ. Roy. Micr. Soc.,
1908, p. 518).
VAN DER STRICHT (Carnegie Instit. Embryol. Contrib., No. 21)
fixes the isolated cochlea in a 5 per cent, aqueous solution of tri-
chloracetic acid, or in Bouin's or Zenker's fluid, and stains, before
imbedding, in borax-carmine. The sections are afterwards stained
in iron-hsematoxylin, Congo red and light green. He obtained the
best results with the membrana tectoria by making one or two
openings in the bony wall of the fresh cochlea and exposing the
piece for fifteen minutes to the vapours from an aqueous solution of
osmic acid or by submerging it in a 1 per cent, solution of the same
for one hour. Afterwards fixation was completed by immersion
in trichloracetic acid, Bouin's fluid or Zenker's fluid, and the series
of sections therefrom stained as above. By this method some of the
turns of the cochlea give very good preparations of the structure
of the membrana tectoria. The mitochondria are also visible
within osteoblasts, osteoclasts, connective-tissue cells, all epithelial
cells, and the sensorial elements.
Mitochondria in odontoblasts and osteoblasts may be demon-
strated by fixation in Flemimng- without-acetic followed by staining
376 CONNECTIVE TISSUES.
in iron-hsematoxylin (§ 679), and the Golgi apparatus in these cells is
.well shown by the employment of Golgi's method, Cajal's method,
or of Da Fano's modification thereof (§§ 844, 849), though a negative
image of this cell-element is clearly shown when the tissues are
fixed in Sansom's modification of Carnoy's mixture.
777. Bone, Decalcified (FLEMMING, 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.
778. Stains for Cartilage and Decalcified Bone. — See hereon
SCHAFFER in Zeit. wiss. MiL, v, 1888, p. 1 ; and .Encyd. mik.
Technik., art. " Knochen."
KOLLIKER (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. The fil^es_o£ Sharpey appear red, the remaining
bone substance blue.
SCHAFFER (Zeit. wiss. Mik., 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 (Encyd. mik.
Tech., 1910, i, p. 762) stains sections for twenty-four hours in a
bath of 20 c.c. of water with 1 drop of 1 per cent, solution of safranin
(or thionin) and (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.
SCHMORL (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. Kinse 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.
MOLL (Centralb. PJiysiol., xiii, 1899, p. 225) stains embryonic
cartilage for six to twenty-four hours in orcein 0-5 gr., alcohol 40,
water 20, hydrochloric acid 20 drops, and mounts iiTbalsam. Matrix
blue, nuclei red.
KALLIUS (Anat. Hefte, xxx, 1905, p. 9) stains first with borax
carmine or alum-carmine, then (sections) for ten minutes in satu-
CHAPTER XXIX. 377
rated solution of thionin, and washes out with alcohol of 70 per
cent. Said to be specific for embryonic cartilage.
VASTARINI-CRESI (Att. Accad. med.-chir. Napoli, 1907, p. 4)
stains sections of embryonic cartilage with borax carmine, then
with muchsematein (alcoholic solution without acid), and then with
Orange Gr. in alcohol.
BAYERL'S method for ossifying cartilage (Arch. mik. Anat.,. 1885,
p. 35): — Portions of ossified cartilage • are decalcified as directed,
§ 555, cut in paraffin, stained in Merkel's carmine and indigo-
carmine mixture, and mounted in balsam.
MAYER (Grundziige, LEE and MAYER, 1910, p. 393) prefers to all
these resojcin fuchsin, § 758, 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 Zeit. wiss. Mik., x, 1893, p. 381.
A process of BAUMGARTEN'S has been given, § 388.
MOERNER (Skandinavisches Arch. Physiol., i, 1889, p. 216 ; Zeit. 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 WOLTERS in Arch. mik. Anat.,
xxxvii, 1891, p. 492 ; and on the whole subject of cartilage see SCHIEF-
FERDECKER'S Gewebelehre, p. 331.
FUSARI (Arch. Ital. 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 DISSE, Anat. Anz., xxxv, 1909, p. 318, a stain for dentine
(haemalum followed by a mixture of Saurerubin and Orange G) ; and
KETTERER and LELIEVRE, C. E. Soc. Biol., Ixx, 1911, p. 630.
Skeletons of Embryos.
779. 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, LUNDVALL (Anat. Anz., xxv, 1904, p. 219, and xl, 1912,
p. 639), using toluidin blue. Thionin blue also may be used.
Similarly also BAKAY (Verh. Anat. Ges., 1902, p. 248), with Bismarck
brown (the embryos having been previously treated with nitric acid of
3 per cent.).
For fish embryos, Professor E. S. Goodrich, of Oxford, informs me that
thionin is excellent.
For the Spalteholz method of clearing such preparations see § 592.
378 CONNECTIVE TISSUES.
780. Demonstration of Centres of Osteoblastic Activity by Trypan
Blue (P. Gr. SHIPLEY and C. C. MACKLIN, Anat. Record, x, 1915 — 16).
— If an azo dye like try pan blue be administered to a very young
animal, the bones are stained quickly and very intensely with vital
colour. The dye is injected in a 1 per cent, solution into the peri-
toneal cavity (less preferably subcutaneously). The animal is
killed forty-eight hours after staining, and the tissues are fixed by
10 per cent, neutral formalin -injected through blood vessels, followed
by immersion in 10 per cent, formalin for twenty-four to forty-eight
hours. Bones are washed thoroughly, hardened in ascending
grades of alcohol, after which the soft parts are dissected away.
Clear in benzol and then in oil of wintergreen. Study with dissecting
microscope. Refer also to J. Thornton Carter, § 775A.
780A. Potash Method for Osteoblastic Centres (SCHULTZE,
Grundriss d. Entwickl. d. Menschens, 1897, and F. P. MALL, Amer.
Journ. Anat., v, No. 4, 1905-6).
Embryos of mammals after fixation in alcohol may be cleared, for
the study of the ossification centres, by means of weak potash. For
alcohol specimens Mall considers that Schultze's solution is too
strong, and uses instead a 1 per cent. KOH solution for a few hours.
With weak solutions the tissues of the smaller embryos remain firm,
and, in the end, the specimen is transparent, with the bones held in
place. After treatment with the potash, the embryo is placed in
the following solution for days, or even months :—
Water . .79
Glycerin .20
Potash . . . . . . .1
From time to time the embryo may be returned to a 3 per cent,
solution of potash for a number of hours to hasten the clearing
process ; then returned to the glycerin solution, which helps to
hold the parts together. When properly cleared, upgrade gradually
to pure glycerin, in which they may remain.
MALL (op. cit.) clears formalin embryos in 10 per cent, potash for
about a month or longer. Formalin renders the connective tissues
very tough, and this strong KOH solution is necessary. Refer also
to § 592.
CHAPTER XXX.
BLOOD AND GLANDS.
Blood.
781. Fixing and Preserving Methods. — The 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
face 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
GULLAND, Scottish Med. Journ., April, 1899, p. 312 ; RUBINSTEIN,
Zeit. wiss. Mik., xiv, 1898, p. 456 ; ZIELINA, ibid., p. 463. But I
believe they have now well-nigh abandoned this barbarous practice.
In wet methods either the blood is mixed 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 ten to thirty).
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. Simi-
larly 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.
Some persons make films by flattening blood between two cover-
glasses which are afterwards separated by sliding the one over the
other ; but that produces an injurious kneading of the cellular
elements.
Most of the usual fixing agents are applicable to blood. But it is
often necessary to employ only such as are favourable 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.
380 BLOOD AND GLANDS.
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.
782. Fixing and Preserving in Bulk. — Most morphologists are
agreed that by far the most faithful fixing agent for blood-corpuscles
is osmic acid. A drop or two of blood (BiONDi 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 a
rule the osmic acid should be strong — 1 to 2 per cent. Fixed speci-
mens may be preserved for use in acetate of potash solution (MAX
FLESCH, Zeit. wiss. Mik., v, 1888," p. 83).
GRIESBACH also (ibid., 1890, p. 328) combines the osmic acid with
certain stains. He mentions methyl green, methyl violet, crystal
violet, safranin, eosin, Saurefuchsin, rhodamin, and iodine in
potassic iodide.
Rossi (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 cau-
tiously added.
EWALD (Zeit. Biol., xxxiv, 1897, p. 257) mixes 3 to 4 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 capillary siphon (§ 3, p. 4), and substitutes water,
alum-carmine, etc., and lastly, 50 per cent, alcohol.
WEIDENBEICH (Arch. mik. Anat., Ixxii, 1908, p. 213) 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 minutes runs in osmic acid
of 1 per cent., and after five minutes more removes the cover.
DEKHUYZEN (Anat. Anz., xix, 1901, p. 536) recommends a mixture
of either 3 or 9 volumes of 2 per cent, osmic acid with 1 of 6 per cent,
acetic acid, containing J per cent, of methylen blue, which he calls
" Osmacet."
The mercurial liquids of Pacini (§ 414) used to be considered
good. HAYEM (" Du Sang" etc., Paris, 1889 ; see also Zeit. wiss:
Mik., 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. Eosin may be added
to it. LOWIT'S formula (Sitzb. k. Akad. Wiss. Wien, xcv, 1887,
p. 144) consists of 5 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.
CHAPTER XXX. 381
DUBOSCQ (Arch. Zool. Exper., 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. MAECANO (Arch, de Med. Exper.,
xi, 1899, p. 434) mixes fresh blood with a mixture of 100 parts of
sodium sulphate of sp. gr. 1-020 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.
SCHRIDDB (Hcemat. Techn. Jena, 1910, p. 17) lets blood drop into
a mixture of 1 part of formol, 9 of liquid of Miiller, and 10 of water,
fixes therein for two to four hours at 40° C., filters, washes and brings
through alcohol and chloroform into paraffin for sectioning.
783. Fixing and Preserving in Films. — Mum (Journ. of Anat. and
PJiys., xxvi, 1892) makes cover-glass films and drops them into
saturated sublimate solution, and after half an hour washes, dehy-
drates, and passes through xylol into balsam. -
GULLAND (Brit. Med. Journ., March 13th, 1897 ; Scottish Med.
Journ., April, 1899) makes cover-glass films, and after a few seconds
drops them face downwards into a solution of—
Absolute alcohol saturated with eosin . . 25 c.c.
Pure ether . . . . . . 25 „
Sublimate in absolute alcohol (2 grms. to
10 c.c.) ...... 5 drops.
After three or four minutes they are washed, stained, and mounted
in balsam.
For JENNER'S fixing and staining and staining method, see
next section.
Many recent authors fix wet films with formol. BENARIO (Deut.
med. Wochenschr., 1895, p. 572) mixes 1 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 GULLAND, 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.
EDINGTON (Brit. Med. Journ., 1900, p. 19) exposes films for
fifteen to thirty minutes to vapour of formol under a bell- jar.
SCOTT (Journ. of Path, and Bacter., vii, 1900, p. 131) exposes
382 BLOOD AND GLANDS.
films to the vapour for about five seconds and drops into absolute
alcohol, and after fifteen minutes stains and mounts.
A short exposure (thirty seconds) to vapour of osmium has also
been recommended.
SZECSI (Deutsch. med. Wochschr., 1913, p. 1584) has recommended
Lucidol for blood smears, and smears of faeces containing protozoa
and cysts. The formulae for an acetone and a pyridin solution will
be found on p. 59, § 107, and also of an acetone-xylol solution for
subsequent washing of the smears.
It is best to keep a sufficient quantity of the fixing solutions in
staining jars. Make a smear, allow it to dry, and place it in the
acetone peroxide of benzol solution for fifteen minutes ; transfer
to the acetone xylol solution for ten minutes in order to remove the
lucidol ; wash off in pure methyl alcohol ; the slide is now ready for
staining. It will be found that most of the current stains used for
such smears will act successfully after the lucidol fixation. Pappen-
heim's panoptic method (§ 784) is recommended.
For smears of fseces a fixation of twenty minutes in the pyridin-
benzol peroxide solution is used ; wash as above, in acetone-xylol,
or pyridin-xylol, and then in methyl-alcohol.
Possibly the substitution of pure acetone for the methyl alcohol
bath might prove advantageous in some ways.
784. Stains for Blood. — Fresh (unfixed) blood can be stained on
the slide. See also § 1008, et seq.
TOISON (Journ. Sci. med. de Lille, fev., 1885 ; Zeit. iviss. Mik.,
1885, p. 398) recommends that it be mixed with the following
fluid:
Distilled water .... 160 c.c.
Glycerin (neutral, 30° Baume) . 30 „
Pure sulphate of sodium . . . 8 grammes.
Pure chloride of sodium ... 1 gramme.
Methyl violet 5 B . . . . 0-5 „
(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 solutions are to be mixed and
filtered.) This mixture stains leucocytes sharply, which facilitates
enumeration.
BIZZOZERO and TORRE (Arch. Sci. Mediche, 1880, p. 390) dilute
a drop with normal salt solution containing a little methyl violet,
which stains nuclei intensely, cytoplasm less intensely.
Similarly GiGLio-Tos (Zeit. wiss. Mik., 1898, p. 166), diluting
with saturated solution of neutral red in salt solution, which stains
CHAPTER XXX. 383
hsemoglobigenous granules in five to ten minutes. This is also
recommended by EHRLICH and LAZARUS. See § 309.
Similarly also Ross (Trans. Path. Soc., 1907, p. 117), using
polychrome methylen blue.
LEVADITI (Journ. Phys. path. Gen., Paris, 1901, p. 425) allows
solution of Brillantkresylblau in alcohol to dry on a slide, puts a
drop of blood on the dried layer, and covers. Similarly CESARIS-
DEMEL (Arch. path. Anat., 1909, p. 92), with a mixture of this dye
and Sudan III ; and NAKANISHI (Centralb. Bakt., 1901, p. 98), with
methylen blue BB.
Fixed 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 haemoglobin. EHRLICH'S acid hsematoxylin, with
0-5 gr. of eosin dissolved in it, is a good general stain. Or, stain
with hsemalum, and then with eosin (0-5 per cent, in alcohol or
water).
EHRLICH'S triacid (§ 296) gives good general views, and demon-
strates neutrophilous granules. His mixture for eosinophilous
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 (MICHAELIS, ibid., 1899, No. 30) 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
28 of acetone, and for staining mix equal parts of these and stain
for half a minute to ten minutes.
JENNER (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 absolute methyl alcohol
(the solution can be had ready made from Griibler and Hollborn).
(Or, simply mix 125 c.c. of 0-5 per cent, solution of the eosin in
methyl alcohol with 100 c.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
384 BLOOD AND GLANDS.
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 GRUNWALD are closely similar to this.
ASSMANN (Munch, med. Wochenschr., 1906, No. 28 ; " Das
eosinsaure Methylenblau," Leipzig, 1908, p. 35) treats fresh films
for half a minute to three minutes in a Petri dish with a few drops
of Jenner's solution (from Griibler and Hollborn), then pours on
20 c.c. of distilled water with 5 drops of JQ 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 forma-
tion of an eosinate of methylen blue — in which the nuclei of blood-
cells are blue and their plasma red to violet. It was made out by
ROMANO WSKY (St. Petersburger med. Wochenschr., 1891) that
under certain conditions mixtures of these two dyes give a stain
which is in 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 blue plasma,
which greatly facilitates their detection and diagnosis. This
reaction 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 undergone, at the
hands of ZIEMANN, ZETTNOW, NOCHT, REUTER, MICHAELIS, RUQE,
MAURER, LEISHMAN, GIEMSA and others, numerous modifications
which have culminated in the 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 and Hollborn (their " Giemsa'sche Loesung f iir Romano wsky-
faerbung "*). Air-dried films (Deutsch. med. Wochenschr., 1907,
No. 17) are fixed in alcohol or in methyl-alcohol (two to three
minutes), and dried with blotting paper. They 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) pre-
served unmounted. All reagents, especially the balsam, must be
strictly free from acid.
* To make this up from Griibler's powders, dissolve 3 grins, of Azur
II-eosin and 8 decigrammes of Azur II in 125 grms. of glycerin and 375
of methyl-alcohol.
CHAPTER XXX. 385
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 3 of Lugol's solution. Wash, and
treat for ten minutes with 0-5 per cent, solution of sodium thio-
sulphate. Wash, and stain as above (changing the stain for fresh
after half an hour), for one to twelve hours. Then 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) are demonstrated not only
in hsematozoa, but in many bacteria, spirochsetse, coccidia, sar-
cosporidia, etc.
See also, for paraffin sections, SCHUBERG, in Deutsch. med.
Wochenschr., xxv, 1909, No. 48, or Zeit. wiss. Mik., xxvii, 1910,
p. 161, who passes through acetone and xylol into balsam.
The older Romanowsky stains published by the authors men-
tioned above, as also Laveran's " Bleu Borrel " seem to be super-
seded by Giemsa's.
IRISHMAN'S Romanowsky Stain (Brit. Med. Journ., March 16th
and September 21st, 1901) is as follows : To a 1 per cent, solution
of Grubler's medicinal methylen blue in water add 0-5 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 Eosin extra B, let stand for six to twelve hours, collect
the resulting precipitate 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.
RAADT (Munch, med. Wochenschr., 1911, No. 27 ; Zeit. wiss. Mik.,
1912, p. 236) obtains a Romanowsky stain of blood and parasites
with JENNER'S solution. Films fixed with alcohol and ether are
25
M.
386 BLOOD AND GLANDS.
first stained for five to ten minutes in solution of 1 part methy-
lenblau med. puriss. Hoechst, 0-5 part of lithium carbonate and
100 of water, kept for at least three weeks and diluted with 10
volumes of water. Rinse with water, dry with blotting paper^
flood with JENNER'S solution diluted with 2 or 3 volumes of water,
and stain for five to ten minutes. Wash, dry with blotting paper,
and mount. See also SCOTT, Folia Haem., xii, 1911.
785. PAPPENHEIM (Anat. Anz., xlii, 1912, p. 525) recommends the
following for sections of Jicemopoietic tissues, and also of kidney, liver,
hypophysis, suprarenals, lung, intestinal epithelium and central nervous
system. Fix in Orth's Formol-Muller, stain sections for twenty minutes
in a stove in " aqueous diluted alcoholic " solution of MAY-GRUNWALD
or JENNER diluted with 8 volumes of water ; after-stain for forty
minutes in the stove in " aqueous GIEMSA solution (15 c.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 and absolute alcohol, and mount in
neutral balsam. The result is not a Eomanowsky stain, but a pale
methylen-blue-eosin stain.
See also WRIGHT, Pub. Massachusetts Gen. Hosp., iii, 1910, p. 1, or
Journ. E. Micr. Soc., 1910, p. 783.
For the special technique of eosinophilous cells see MARTINOTTI in
Zeit. wiss. Mikr., xxvi, 1909, p. 4 (alphabetical bibliography of eight
pages).
786. 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. The 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 : 3,000 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 PAPPENHEIM (Farbchemie, p. 107) Wasser-
blau is almost specific for them.
WRIGHT (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. Soc., 1910, p. 783.
Bee also DEKHUYZEN, Anat. Anz.. xix, 1901, p. 533; KOPSCH,
CHAPTER XXX. 387
Intern. Monatschr. Anat. Phys., xxi, 1904, p. 344, and xxiii, 1906,
p. 359 ; DEETJEN, Zeit. phys. Chem., Ixiii, 1909, p. 1.
787. Demonstration of a New Body in Red Blood Corpuscles (GOLGI,
Boll Soc. Med. Chir. Pavia, 1919, xxxi ; Hcematologica (Napoli),
1920). This original communication of Golgi gives two methods of
interest to workers on blood : (1) Blood-films are fixed twenty-four
to forty-eight hours in equal parts of saturated solutions of mercury
chloride and potassium bichromate. They are then transferred into
equal parts of 2 per cent, mercury chloride and potassium bichro-
mate, to which 5 to 10 c.c. of 1 per cent, gold chloride and 5 to 10
drops of acetic acid are added. The films are observed in glycerol,
starting from the second or third day after the last treatment
until the fifteenth or twentieth day ; (2) drops of blood are fixed
in a watch-glass by means of a fluid composed of 2 per cent, mercury
chloride 60 c.c., saturated solution of picric acid 20 c.c., 1 per cent,
osmic acid 10 c.c., acetic acid 5 drops, with the addition either
immediately or after eight to twenty-four hours of 10 c.c. of 1 per
cent, gold chloride for every 50 c.c. of fixative. Preparations are
made from the sediment with some glycerol, from the second until
about the tenth day after fixation.
Both methods show within the red blood corpuscles a peculiar
body with a diameter of about one-third that of adult corpuscles
and of about half that of foetal ones. The body occupies the central
part of the erytrocytes, and particularly by means of the second
process it appears to have a fine, sometimes more fibrillar, sometimes
more reticular structure. Its contours, though clearly defined, are
irregular, and there is no indication whatever of a limiting membrane.
This " reticulo-fibrillar apparatus " is not a nucleus, as the latter
remains colourless by the new methods, not only in the white, but
also in the nucleated red corpuscles, in which the apparatus appears
concentrically arranged round the unstained nucleus. According
to Golgi this apparatus does not correspond to any of the structures
already described within the red corpuscles, but it reminds one a
little of the endoglobular body recently demonstrated by Petrone,
by means of a lead impregnation method, and not to be confused
with the well-known Petrone's bodies. Golgi is convinced that the
images obtainable by his two new methods are the expression of a
real structure situated within the red blood corpuscles, but he does
not feel able at present to give any opinion about their significance.
The new methods stain also the centrosome in the white corpuscles
of the blood.
25—2
388 BLOOD AND GLANDS.
788. WEIGERT'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 to them. 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
anilin is 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) ;
BENECKE (§ 690) ; UNNA (Monatssch. prakt. Dermal., xx, 1895, p. 140) ;
WOLFF (Zeit. iviss. Mile., xv, 1899, p. 310) ; and one of another sort by
KOCKEL (Centralb. allg. Path., x, 1899).
789. Elective Staining of Erythrocytes (K. OKAJIMA, Anat. Record,
xi, 1917). — This stain is based on the fact that the phosphomolybdic
acid lake of alizarin stains, shows a special affinity for haemoglobin.
Fix material in formol, sublimate, chrome, etc. Transfer sections
on slide to aq. dest. ; mordant in 10 per cent, phosphomolybdic
acid solution for thirty seconds to two minutes ; wash in water ;
stain in this mixture for twenty minutes to twenty hours : — sodium
sulfalizarinate, saturated aqueous solution, 100 c.c. ; and 10 per
cent, phosphomolybdic acid, aq. solution, 30 c.c. (10 to 50 c.c.) ;
wash in water ; alcohols, xylol, balsam. Erythrocytes go bright
yellow orange. Counter-staining may be done in Ehrlich's hsema-
toxylin.
The completeness of the " specificity " of this method is open to
doubt, but it gives interesting results.
790. Intravital Staining with Benzidine Dyes. — BOUFFARD (Ann.
de VInst. Pasteur, xx), then GOLDMANN (Beit. z. Klin. Chir., Ixiv,
have shown that animal tissues may be " stained " intra vitam by
the injection of several benzidine dyes. There are some categories
of cells in the body which seem to show a special affinity for phagocy-
tosing or, at least, ingesting in some way granules of certain of
these colloid dyes. These various cells are often called pyrrhol cells
(Goldmann), macrophages (Evans), histiocytes (Aschofi-Kiyono),
or resting wandering cells (Tschaschin).
According to HAL DOWNEY (Anat. Record, xii, 1917) the process of
" staining " is one of storage or iugestion, and not of true staining, and
attempts to classify cells according to their reactions to these colloidal
CHAPTER XXX. 389
dyes are a failure, This author believes that blood cells behave towards
these dyes just as they do to any foreign matter. Other authors do not
agree. " To dismiss these cells (pyrrhol cells) as scavengers is to do them
an injustice, for, however important this function may be, their service
to the body is a far greater one " (P. Gr. SHIPLEY, Amer. Journ. Physiol.,
xlix, 1919, p. 300).
EVANS and SCHULEMANN (Science, N.S., 1914), believe that vital
staining with azo dyes is the result of " phagocytosis " of ultra-micro-
scopic dye particles, existing in a state of fine dispersion as an hydrosol.
In using the term " phagocytosis " Evans does not quite mean an
engulfing by pseudopodia as with amoeba. P. G-. SHIPLEY (Amer.
Journ. Physiol., 1919, p. 285) points out that some cells which are
most active in phagocytosing bacteria and other coarse particles, take
no part, under ordinary conditions, in the segregation of vital dyes in
the body of the living animal.
It has been stated that the benzidine dyes are not characterised by
a propensity for staining the mitochondria, as are Janus green or
dahlia. The granules in cells which store ultramicroscopic particles of
the benzidine dyes seem to be something apart from the mitochondria.
Only occasionally the mitochondria, as such, take up a benzidine dye
like trypan blue. In tissue cultures from forty to sixty hours old
many of the cells are seen to contain large greyish granules which were
either not present in the early stages or were not very noticeable. Such
granules (" segregation granules " of Shipley, and possibly degeneration
or "neutral red" granules of the Lewises, Amer. Journ. Anat., 1915),
stain red in neutral red and deep purple in cresyl blue, and by using
a combination of trypan red and Janus green, it can be shown, according
to Shipley, that the mitochondria (green in the Janus) and the segrega-
tion granules which take up the azo red dye, are separate entities.
This opinion is not shared by TSCHASKIN (Fol. Hcern., 1914), by LEVY
(E. Accad. d. Lincei, 1916), and by MAXIMOW (Arch. Euss. d'Anat.
d. Hist, et d'Embryol., 1916).
Trypan blue is also used for demonstrating areas of osteoblastic
activity (Shipley and Macklin). • See § 780.
The Methods of using Benzidine Dyes are as follows : — Trypan blue
and pyrrol-blue of 1 per cent, strength in Ringer's solution are
injected subcutaneously, intraperitoneally or into the blood vessels.
Whereas 1 c.c. of a 1 per cent, solution per 20 grms. of the animal's
body weight injected subcutaneously has no ill-effect on the animal,
no more than 0-5 c.c. of the same solution should be used for intra-
vascular work. In the latter case coloration sets in speedily,
increases up to the second day, but rapidly fades after the fourth
day, in any case, quicker than when gradual absorption of the stain
takes place through the lymphatic channels. It is undoubtedly
safest and best for histological study to inject the staining fluids
subcutaneously. Injections of 1 c.c. of a 1 per cent, solution per
20 grms. body weight may be repeated many times once a week.
390 BLOOD AND GLANDS.
In some cases Goldmann has given fifteen consecutive injections.
These remarks refer especially to small animals like the rat ; for
bigger animals, such as the rabbit or ape, intraperitoneal injections
are preferable to subcutaneous : use the standard of 1 c.c. of a
1 per cent, solution per 20 grms. of animal's weight.
min^blue and pjrroXJ^lue anow^of^fi^ation^in
solution (injected intravenously if possible),
but it is only tissues stained in trypan blue that allow ordinary
I processes of histological technique, but even for trypan-blue, the
'fixative should contain a little formalin. Sections are cut with a
freezing microtome from tissue fixed in 10 per cent, formalin not
less than forty-eight hours. Stain as necessary in alum carmine or
hsemalum, etc. Pappenheim's pyronin and methyl green are good
for connective tissues, Ehrlich's triacid for hsemopoetic tissues
(GOLDMANN, Proc. Roy. Soc.; Ixxxv, 1912), G. B. WISLOCKI and
H. DOWNEY (Anat. Record, xii, 1917), after staining, fix in Zenker or
formpl, upgrade in alcohols, imbed in wax and section. Counter-
stain in haemalum. GOLDMANN (loc. cit.) mentions the following
" vital stains," apart from those given above : Trypan violet,
benzopurpurin, diamin blue B.B., diamin black B.H., vital " neu
rot, vital neu orange," vital " neu gelb," dianil blue E, (Griibler).
These are used in 1 per cent, strength in salt solution. Trypan blue,
trypan red, Congo red, azo blue, and beiiz^purpurin can be used on
tissue cultures by introducing some of the dye into the culture
medium (SHIPLEY, Amer. Journ. PhysioL, 1919, p. 287).
Apart from the references given above see also, HOFFMAN (Fol. Hcum.,
1914.), KENAULT (Arch. d'Anat. Micr., 1907) ; LOELE (Fol. Hcem., 1913) ;
BATCHELOR (Proc. Amer. Assoc. Anat., 1914, Anat. Record, 1914).
791. Mieroehemical Tests for the Oxygen Place in Tissues.— Recently
certain workers have claimed to be able to locate centres or regions of
oxidation in the cell by means of some substances sensitive to free oxy-
gen. Unna's method is to use a solution of rongalit white, which is a
solution of the leucobase of methylen blue kept in a state of reduction
by excess of rongalit, an absorption product of formaldehyde with
sodium sulphite. See UNNA (Die ReduMonsorte und Satierstofforte des
tierscJien Gewebes, Arch. f. Mikr. Anat., Ixxviii, 1911). A. N. DRURY
(Proc. Roy. Soc., 1914) has shown that Unna's claim is inadmissible,
and consequently his theory of staining by oxidation and reduction is
not proven. GRAHAM (Journ. Med. Research, Boston, xxxv, 1916)
claims to have demonstrated by means of H202 and naphthol, that the
granules of leucocytes and myelocytes contain a peroxidase of the
peroxide type. Schultze in his Oxydase Reaction uses a-naphthol and
dimethyl-p-phenylen-diamin (Merck). Blood and marrow smears fixed
in formalin vapour are treated firstly in the a-naphthol solution ; pre-
CHAPTER XXX. 391
pared by melting 1 grin, of naphthol on the surface of 100 c.c. of aq.
dest., and adding potassium hydrate till the naphthol dissolves. After
a few minutes in this solution (cooled) the smears are transferred to a
1 per cent, solution of the dimethyl — for the same time, when a blue
colour is seen to appear where the oxydases lie. Mount in glycerine
jelly, but blue colour fades. Myelocytes and not lymphocytes are said
to give a positive reaction.
It is doubtful how far these various colour indicators for oxygen place
in cells and tissues are reliable. It has been claimed that by means of
the last-mentioned method, it is possible to show that the staining
(oxygen place) appears especially around the nucleus of the cell. This
has not been confirmed.
Glands.
792. Mucin. — HOYER (Arch. mik. Anat., xxxvi, 1890, p. 310) finds
that the mucin of mucus cells and goblet cells stains with basic tar
colours and with alum hsematoxylin, but not with acid 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, etc.
Tissues should be fixed for two to eight hours in 5 per cent,
sublimate solution, and paraffin sections stained for five to fifteen
minutes in a very dilute aqueous solution of the dye (2 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 basic dyes as mucin does.
See also SUSSDORF, Deutsche. Zeit. Thiermed., xiv, pp. 345, 349
(Zeit. wiss. Mik., vi, 1889, p. 205) ; BIZZOZERO, Atti. R. Accad. di
Sci.'di Torino, 1889 to 1892 (reports in Zeit. wiss. Mik., vii,
1890, p. 61 ; and ix, 1892, p. 219) ; also UNNA, ibid., xiii, 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. UNNA employs chiefly polychrome methylen
blue.
As regards the thionin stain, see HARI, Arch. Mik. Anat., Iviii,
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.c. of formol of 1-25 per cent,
with 8 c.c. of 1 per cent, solution of thionin. Mucus glands red.
KULTSCHIZKY (Arch. mik. Anat., xlix, 1897, p. 8) fixes in his
mixture (§ 57), and stains sections either in safranin with 2 per
392 BLOOD AND GLANDS.
cent, acetic acid, or in a similar solution of neutral red (two to three
days, washing out with alcohol).
MAYER (Mitt. Zool. Stat. Neapel.,xu, 1896, p. 303, or last edition) gives
the following two formulae for mixtures that stain exclusively mucus.
793. MAYER'S Mucicarmine (op. cit., last §). —One gramme of carmine,
and 0-5 grm. of aluminium chloride with 2 c.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.
MAYER'S Muchaematein (ibid.). — Hsematein 0-2 grm., aluminium
chloride 0-1 grm., glycerin 40 c.c., water 60 c.c. An alcoholic solution
may be made by dissolving in 100 c.c. of 70 per cent, alcohol, with or
without the addition of 2 drops of nitric acid.
794. Mucicarminic Acid (KAWITZ, Anat. Am., xv, 1899, p. 439).—
One gramme of carminic acid, 2 of aluminium chloride, and 100 c.c. of
50 per cent, alcohol.
795. Goblet Cells. — So far as these contain mucin they give the
reactions above described. See PANETH, Arch. mik. Anat., xxxi,
1888, pp. 113 et seq. ; LIST, ibid., xxvii, 1886, p. 481 ; and GUYEISSE,
C. R. Soc. Biol, 1907, p. 1212.
For intestinal epithelium, especially the cells of PANETH, see also
MARTIN, Unters. ueb. Oberfldchen u. Drusenepithel, Leipzig, 1910 ;
and KULL, Arch. mik. Anat., Ixxvii, 1911, p. 541 (sections stained
with alum hsematoxylin, treated for twenty to thirty seconds with
tincture of iodine, stained a few minutes with Victoria blue, then
with eosin).
796. Salivary Glands. — SOLGER (Unters. z. Naturlehre d. Menschen
xv, 5 and 6, pp. 2—15; Festschr. f. Gegenbaur, ii, 1896, p. 21P
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 hsematoxylin of Delafield
or of Ehrlich, the granules taking the stain.
KRAUSE (Arch. mik. Anat., xlv, 1895, p. 94) stains sections either
with Heidenhain's iron hsematoxylin or with Ehrlich-Biondi mixture
or thionin. See also KRAUSE, ibid., xlix, 1897, p. 709 ; and MULLER,
Zeit. wiss. Zool, 1898, p. 640.
797. Gastric Glands. — KOLSTER (Zeit. wiss. Mik., xii, 1895,
p. 314) differentiates the two kinds of cells in stomach glands by
over-staining with hsematoxylin, washing out with alcohol containing
1 per cent, of HC1, blueing with alcohol containing 1 per cent, of
ammonia, and, after washing, staining for one to five minutes in a
CHAPTER XXX. 393
weak solution of Saurefuchsin. Peptic cells blue, parietal cells red.
Osmic material cannot be employed.
CADE (Arch. Anat. Micr., iv, 1901, p. 4) stains material fixed
with Bourn's picroformol in Victoria blue of 1 per cent.
B. and L. MONTI (Rich. Lab. Anat. Roma, ix, 1902) demonstrate
ducts and canaliculi of delomorphous cells by Golgi's bichromate
and silver impregnation, especially with rejuvenated material (see
SACERDOTTI), leaving it for five or six days in half -saturated sulphate
of copper, then for twenty-four hours in the osmic-bichromate
mixture. You can embed in paraffin (rapidly).
798. Intestine. — BENSLEY (Amer. Journ. Anat., 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.
799. Liver.— BRAUS (Denkschr. Med. Nat. Ges. Jena, v, 1896,
p. 307) demonstrates the bile capillaries by the rapid method of
GOLGI, hardening in a mixture of 1 part formol with 3 parts liquid
of Miiller or J per cent., chromic acid.
EPPINGER (Beitr. path. Anat., xxxi, 1902, p. 230) studies them
by means of a complicated modification of WEIGERT'S neuroglia
stain, and CIECHANOWSKI (Anat. Anz., xxi., 1902, p. 426) by means
of WEIGERT'S myelin stain (the 1885 method).
OPPEL (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 (J to 10 per cent.), then for
twenty-four hours into a f per cent, solution of silver nitrate, washes,
dehydrates and cuts without embedding. The lattice fibres are only
stained near the surface, so that tangential sections must be made.
Similarly BERKLEY, ibid., 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 ; IGACUSCHI, in
Arch. path. Anat., xcvii, p. 142, or Zeit. wiss. Mile., 1885, p. 243 (gold
process for study of fibrous networks) ; KUPFPER, Sttzb. Ges. f. Morph.,
etc., Miinchen, Juli, 1889, or Zeit. wiss. Mik., vi, 1889, p. 506 ; KRAUSE
(Arch. mik. Anat., xlii, 1893, p. 57) ; and TIMOFEJEW, Anat. Anz., xxxv,
1909, p. 296 (sections of frozen tissue stained with methylen blue).
800. Spleen. — For lattice fibres, see OPPEL, last §.
KULTSCHITZKY (Arch. mik. Anat., xlvi, 1895, p. 675) studies
the musculature in sections (of material from liquid of Miiller)
stained for a day or more in a solution of lakmoid in ether and
mounted in balsam.
394 BLOOD AND GLANDS.
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 Miiller material for a
few minutes into a solution of 1 or 2 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., Iv, 1899, p. 151) ; WEIDENREICH (ibid.,
Iviii, 1901, p. 251).
801. Lymphatic Glands. — For lattice-fibres especially, see EOESSLE
and YOSHIDA, Beitr. path. Anat., xlv, 1909, p. 110, or Zeit. wiss.
Mik., xxvi, 1909, p. 295. Sections stained with hsematoxylin and
eosin, or Weigert's iron haematoxylin, or Bielschowsky's neurofibril
stain as applied by MARESCH, loc. cit., § 752. The sections should
not remain for more than fifteen to thirty minutes in the oxide bath.
See also for the thymus some very complicated methods of SAL-
KIND, Anat. Anz., xli, 1912, Nos. 6 and 7.
802. Kidney. — SAUER (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 9 parts alcohol with 1 of nitric
acid is also good, as is liquid of Perenyi. He stains with iron
haematoxylin, and after-stains in a very weak solution of Saurerubin
in 90 per cent, alcohol, which stains the ciliary plateau. He
macerates with iodised serum or one-third alcohol, staining after-
wards with dahlia.
ARNOLD (Anat. Anz., xxi, 1902, p. 417) employs intra 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 sub-
cutaneously 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).
803. Thyroid. — BENSLEY (Amer. Journ. Anat., xxix, 1916) uses
brazilin and water blue. Fix gland in Zenker-formol. Section in
paraffin and fix sections to slide with water alone, or very little
CHAPTER XXX. 395
albumen ; pass through toluol, absolute alcohol, water, iodise, and
place in this brazilin solution for several hours : —
Phosphotungstic acid .... 1-0 grm.
Distilled water ..... 100-0 c.c.
Brazilin . . . . . . 0-05 grm.
The brazilin is first dissolved in a small quantity of distilled water
by the aid of heat and added to the phosphotungstic acid solution.
This solution goes bad after three days. After staining in the
brazilin, wash in water, and place for five minutes in this mixture : —
Phosphomolybdic acid .... 1-0 grm.
Wasserblau 0-2 „
Water . 100-0 c.c.
Wash rapidly in water, dehydrate in absolute alcohol, clear in
toluol, and mount in balsam. Cytoplasm stains blue to lilac,
nuclear chromatin deep red, contents of thyroid vacuoles sky blue,
and colloid droplets of Hiirthle deep blue to deep red.
804. Pancreas. — Most of the techniques given under the heading
of " Mitochondria " and " Golgi apparatus," etc. (§§ 673—712) give
important results with the zymogen granules of the pancreas. The
methods of Bensley-Cowdry (§ 686), Regaud (§ 685), Benda (§ 683),
and Schridde (§ 687), all apply here. For the Golgi apparatus
Cajal's formalin silver nitrate method may be used (§ 847).
BENSLEY'S Neutral Red Method (A?ner. Journ. Anat., xii, 1911 — 12).
— Animal killed by bleeding ; a cannula introduced into aorta and
a solution of neutral red in isotonic salt solution, containing 1 in
15,000 neutral red, is injected. Immediately after the pancreas has
assumed a faint rosy tint a part of the organ is removed — the islets
of Langerhans stain intense yellow red, the rest faint rosy-pink. In
a short time after mounting the islets remain the only stained
elements, owing to bleaching in the acini. Method applicable to
the counting of the islets of Langerhans.
Janus Green Method. See § 702. — Islets deep blue on a red
background.
Pyronin Method for Ducts. — Inject a 1 in 1,000 solution of pyronin,
as above, for neutral red method. The ducts stain intensely red.
Double stains may be made by injecting mixed Janus green and
pyronin (BENSLEY, op. cit.).
Methylen blue, 1 in 10,000 may also be used for this purpose.
After injection fix in 5 per cent, ammonium molybdate, for which
see also Chapter XVI.
GRAND-MOURSEL and TRIBONDEAU (C. R. Soc. BioL, liii, 1901,
396 BLOOD AND GLANDS.
p. 187) recommend for pancreas NICOLLE'S " thionine pheniquee,"
wliicli stains the insulse of Langerhans hardly at all, the rest strongly.
LANE'S Methods for Demonstration of A Cells of the Islets of
Langerhans.
(1) Fix tissue for from two to four hours in equal parts of
saturated alcoholic solution of mercury chloride, and 2J per cent,
potassium bichromate. Wash in 50 per cent, alcohol, then upgrade
and embed ; 3 jut sections are stained in neutral gentian, obtained by
precipitation of equivalent solutions of gentian violet (crystal violet)
and orange GL If the correct quantity of the latter is added to the
former, a practically complete precipitation is obtained. The
precipitate is soluble in alcohol or acetone. For staining add the
stock alcohol solution to 20 per cent, alcohol until a solution having
the colour of good haemalum is obtained. Allow to stand for
twenty-four hours. Stain for twenty-four hours, blot, dehydrate
in acetone, toluol, differentiate in absolute alcohol 1 part, oil of
cloves 3 parts, wash in toluol, and mount in balsam.
(2) Fix in 70 per cent, alcohol, then stain in neutral gentian as
above.
LANE'S Methods for Demonstration of B Cells of Islets of Langerhans.
Fix for four to twenty-four hours in :—
K2Cr207 ...... 2'5 grms.
HgCl2 ...... 5-0 „
Aq. dest . 100 -0 c.c.
Dehydrate, clear, embed, and section ; stain in neutral gentian
as above.
Formalin Bichromate Method for Fixation.— This gives a very
regular and reliable fixation, and is suitable where one is carrying
out observations which necessitate a successful routine method.
BENSLEY (op. cit.) uses 10 c.c. of neutral formalin to 90 c.c. of
Zenker's fluid without acetic acid, for twenty-four hours. Stain in
neutral gentian, acid fuchsin and toluidin blue, iron hsematoxylin
or Mallory (§ 314).
HOMANS (Journ. Med. Research, xxx, 1914) used Bensley's modified
Altmann fixative (Os04 of 4 per cent., 2 c.c. ; potassium bichromate
of 2*5 per cent., 8 c.c. ; glacial acetic acid, 1 drop), Lane's methods
(vide supra), and ordinary haematoxylin and eosin.
Very pretty results are obtainable by using Mallory's polychrome
methylen blue and eosin (§ 314).
See also BABKIN, RUBASCHKIN and SSAWITSCH, Arch. f. Mikr.
Anat., Bd. 74 ; HELLY, ibid., Bd. 67 ; LANE, Amer. Journ. Anat.
vii, 1907, SAGUCHI, ibid., Vols. 26 and 28, and § 713.
CHAPTER XXXI.*
NERVOUS SYSTEM — GENERAL METHODS.
805. Introduction. — The microscopic investigation of the nervous
system pursues two ends. Either it is desired to elucidate the
intimate structure of nerve-cells, nerve-fibres and their supporting
tissues, or to study the morphology of nerve-cells, their distribution
in the grey matter, their connections with each other, and with the
nerve fibres which chiefly constitute the white matter, and lastly to
investigate the architectural arrangement of both nerve-cells and
nerve-fibres in the various regions of the central nervous system.
The processes employed in the first case form a group of cytological
methods, whilst the processes used in the second instance are spoken
of as the anatomical methods.
The processes used in the study of nervous tissue in peripheral
organs having been described in the chapter on " Methylene Blue,"
" Impregnation Methods," " Tegumentary Organs," and " Muscle
and Tendon," the following chapters are chiefly devoted to the
description of methods for the investigation of the central nervous
system.
Fixation.
806. Fixation by Injection. — Fixation, in the proper sense of the
word, is of course out of the question for the human subject. But
in the case of the lower animals it is possible to inject fixing fluids
into their nervous centres when still in an almost living state. The
practice ensures a very rapid penetration into and even distribution
within the tissues of the fixing agents, and has, moreover, the capital
advantage of greatly helping to prevent distortion of the nerve-
tissues during their subsequent treatment. And as in most instances
the practice does not meet with special difficulties, it should be
adopted as far as possible also in the case of human subjects, but
particularly for a preliminary fixation and hardening of the very
soft cerebral mass of young individuals, which is particularly liable
to much injury and distortion in the process of removing it from
the brain case.
* Kevised and in great part rewritten by Dr. C. Da Fano, King's
College, University of London.
398 NERVOUS SYSTEM— GENERAL METHODS.
The choice of the fluid to be injected depends upon the object in
view and the subsequent treatment to which the tissues are to be
submitted. In the case of animals it is a good practice to warm the
fixing fluid to body-temperature before injecting it, and, whenever
possible, to wash out the blood by first injecting physiological
solution as suggested by Mann. The injection can be carried out
through the carotids if the fixation is to be limited to the encephalon,
and through the aorta if it is desired to fix the spinal cord too. The
above applies to higher vertebrates and particularly to mammals ;
in the case of lower vertebrates, fixation by injection has not, as a
rule, the same importance, and one must have recourse to special
methods.
See on this subject GOLGI, op. cit., in § 880 ; GEROTA, § 811 ; DE
QUERVAIN, Virchow's Arch., cxxxiii, 1893, p. 481 ; MANN, Ztschr. wiss.
MiJcr., xi, 1894, p. 482 ; STRONG, Anat. Anz., xi, 1886, p. 655 ; Journ.
Comp. Neurol., xiii, 1903, p. 291 ; McFARLAND, Journ. App. Micr,, ii,
1899, p. 541.
Hardening.
807. Hardening by the Freezing Method. — This phrase has often
given rise to confusion and should, therefore, be clearly understood.
One can harden by freezing either fresh tissues, or material already
fixed and consequently also a little hardened. In the first instance
small pieces of fresh tissue, immediately after removal and without
any previous treatment, are hardened on a freezing microtome. The
sections are generally floated on to water, and immediately after-
wards treated for a minute on the slide with a 0-25 per cent, solu-
tion of osmic acid ; or otherwise treated according to the object of
one's investigation. In this case the ether freezing method should
be preferred, bearing in mind, however, that there is considerable
difficulty in obtaining sufficiently good sections, and that the
results attainable are very limited particularly since BRODMANN
(Journ. Psychol. NeuroL, ii, 1903 — 4, p. 211) has shown that formalin
material can be used even for investigations by polarised light. (See
also p. 361.)
The hardening by freezing of already fixed material may be also
attended with some difficulty, but this will be easily overcome if
pieces are relatively small, the fixing agent properly washed away,
and one has, eventually, recourse to one or other of the processes
described in § 183. Material fixed in formalin, however, does not,
as a rule, require any soaking in gum, or syrup, or the like, and is
easily cut if the formalin has been washed away. In this case the
C03 freezing microtome is in my opinion to be preferred.
CHAPTER XXXI. 399
The hardening and section cutting by the freezing method of very
large pieces require special apparatus and special methods, for which
see NAGEOTTE, C. R. Soc. BioL, Ixvii, 1909, p. 542.
808. Hardening by Reagents. — If large pieces of nervous tissue are
to be hardened, it is necessary to take special precautions in order
to prevent them from being deformed by their weight during the
process. The spinal cord or small portions of any region of the
encephalon may be cut into thin slices, laid out on cotton wool in a
vessel into which the hardening fluid is poured. The specimens may
also be suspended in the liquid (§ 34). Another good plan consists
in adding to the hardening fluid just enough glycerine or sodium
chloride to make tissues float.
If several pieces are placed in the same vessel, they should never
be put on top of each other. Voluminous organs to be hardened
in toto should be at least incised as deeply as possible in the less
important regions. With the exception of the dura mater, the
membranes are not generally removed at first, as they serve to
protect the tissues. They can be removed partially or entirely later
on when the hardening has made some progress. In the case of
material intended for Golgi's methods it is best not to remove them
at all.
The spinal cord, medulla oblongaia and pom Varolii may be
hardened in toto, and the preparation hung up in a cylindrical
vessel with a weight attached to its lower end to prevent it from
becoming distorted.
The cerebrum should have light plugs of cotton wool in the
fissure of Sylvius, and as far as possible between the convolutions.
If it is desired not to open the lateral ventricles, the hardening fluid
may be injected into them. Unless there are special reasons to
the contrary, the brain should be divided into two portions by a
middle frontal section, or better into two symmetrical halves by
a sagittal cut passing through the median plane of the corpus
callosum.
The cerebellum should be treated in the same manner.
The action of most hardening fluids is greatly enhanced by heat.
But in the judgment of most histologists this rapid hardening is not,
as a rule, attended by good results, and one should have recourse to
it only for particular reasons and special purposes after a tentative
experiment, whenever possible, at establishing the degree of tem-
perature at which the desired results may be obtained without
otherwise injuring the delicate structure of the nervous tissues.
400 NERVOUS SYSTEM—GENERAL METHODS.
On the other hand the hardening action at room temperature of
certain reagents, such as solutions of chromic salts, proceeds so
slowly that decomposition may set in before the fluid has had time
to act effectively. For this reason voluminous preparations which
are to be hardened in toto in solutions of chromic salts, and were
not injected as described in § 806, should be put away in a very
cool place or in an ice-chest. A human cerebral hemisphere may
require eight or nine months for hardening in this way.
The volume of the fluid should always be very large in proportion
to that of the pieces of tissue and to their number. It should be
taken in solutions as weak as is consistent with the proper preserva-
tion of the tissues. It should be frequently changed and its strength
gradually increased.
MARIE'S method of fixing and hardening in situ is highly recom-
mended; for its indications and contra-indications, see SAINTON
and KATTWINKEL (Deutsche Arch. klin. Med., Ix, 1898, p. 548) and
PFISTER (Neurol Centrbl, xvii, 1898, p. 643).
809. The Reagents to be Employed. — As in the case of the fixation
by injection one should bear in mind that the preservation of tissues
for neuro-histological investigations greatly depends upon the
purpose in view. Fixing and hardening fluids which are excellent
for cytological investigations are very often unsuitable for anatomical
methods. (See § 805.) On the other hand, material collected and
prepared for cyto-architectonic or fibro-architectonic studies can
hardly be used to elucidate questions regarding the intimate structure
of nerve-cells or nerve-fibres. • Alcohol, formalin * and chromic salts
are most frequently used because they are generally ready at hand,
and because they are useful for carrying out afterwards either a
great number of methods, or certain methods, under constant
conditions of hardening and staining.
810. Alcohol. — It is generally employed in the strength of 94 to 96,
per cent., penetrates well and hardens quickly ; but as it rapidly
* Wrong as it is, I find it expedient to use the term " formalin " or
" formol " in the generally accepted sense, viz., as if it were a chemical
reagent, while it is only a commercial denomination which ought not
to have been introduced in the histological terminology. See § 108.
As is well known, the commercial formalin is only a 40 per cent, solution
of formaldehyde ; but when in this and the following chapters on the
nervous system a 5, 10 or 20 per cent, solution of formalin is mentioned,
it is intended to mean 5, 10 or 20 parts of commercial formol, and 95,
90 or 80 parts of water, respectively, while, e.g., a 20 per cent, solution
of formaldehyde is the commercial formalin diluted with half its volume
of water.— C. d. F.
CHAPTER XXXI. 401
absorbs water from the tissues the latter shrink considerably, whilst
the alcohol loses its fixing and hardening properties through hydra-
tion. It has consequently to be changed soon for a fresh supply and
used in quantities exceptionally large in proportion to the size of the
pieces, which ought to be neither too small nor too large. For this
reason one seldom hardens in alcohol voluminous organs, and its use
has become on the whole very restricted. Alcohol, however, remains
the principal fixing and hardening reagent for cytological investiga-
tions by Nissl's method (see § 826), and for carrying out some of
Ramon y Cajal's reduced silver processes (§ 827), its shrinking
influence being counteracted by having recourse for the first
fixation to weaker dilutions of alcohol (60 to 70 per cent.) to be
raised gradually up to 95 or 96 per cent, within the first nine
to twelve hours, and to be changed once or twice or more often
in the next few days.
811. Formalin. — Since the time when it was introduced into
histological technique by F. BLUM (Ztsohr. wiss Mikr., x, 1893,
p. 314) ; J. BLUM (Zool Anz., xvi, 1893, p. 434) ; HERMANN (Anat.
Anz., ix, 1893, p. 112) ; HOYER, jun. (Anat. Anz., ix, 1894, p. 236) ;
LACHI (Monit. Zool. Ital,.v, 1895, p. 15) and many others, its use
has been steadily increasing because of the many advantages it
offers. As a matter of fact it penetrates more quickly than solutions
of chromic salts, and even than alcohol ; it is not likely to over-
harden ; it allows of the most various after-treatments and methods
of staining, including neurofibril stains and Golgi's impregnation
method.
Several writers have insisted that for nervous tissue it should not
be acid, but some prefer it acid. See " Retina." For neurofibrils it
should be preferably neutral. To neutralise it, it is generally suffi-
cient to prepare its solutions with spring water, but one may shake
it with magnesium or sodium or calcium carbonate. Some authors
prefer to neutralise with ammonia. (See also § 108.)
The strength of the formalin solutions generally used for fixing
and hardening nervous tissues varies considerably with the quality
of the material in hand, but particularly with the age of the subjects.
As a rule the more delicate the material and the younger the subject,
the weaker should be the formalin solutions to be employed at first.
Generally, however, one starts with a 3 or 5 per cent, solution in the
case of very soft tissues, gradually increasing the strength up to
10 or 12 per cent. An adult human encephalon can be very well
preserved in a 10 or 15 or 20 per cent, solution with two changes of
M. 26
402 NERVOUS SYSTEM— GENERAL METHODS.
the fluid during the first days of fixation and hardening. See further
on this subject, § 108.
Formalin can be associated with, or followed by. alcohol (§ 109)
or other reagents. Thus FISH (Proc. Am. Micr. Soc., xvii, 1895,
p. 319) recommends : —
Water 2,000 c.c.
Formalin . . . . . 50 ,,
Sodium chloride .... 100 grms.
Zinc chloride . . . . . 15 „
Brains are left in the mixture eight to ten days or longer, and then
transferred into a mixture of water 2,000 c.c. and formalin 50 c.c.,
in which they may remain indefinitely if the jar is kept tightly
stoppered.
PARKER and FLOYD (Anat. Anz., ix, 1896, p. 156) advise for
sheep's brains a mixture of 6 volumes of 95 per cent, alcohol and
4 volumes of 2 per cent, formol. Brains may be kept in the mixture
for months.
FLATAU (Anat. Anz., xiii, 1897, p. 323) finds that brain increases
in weight slightly in 10 per cent, formol (spinal cord somewhat
more) ; whilst in 1 per cent, solution it may increase as much as
24 per cent.
GEROTA (Int. Monatschr. Anat., xiii, 1896, p. 108) puts human
brains into a 5 or 10 per cent, solution of formol, and after twenty-
four hours removes the pia mater, and changes the liquid ; 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 vascular 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.
KADYI (Poln. Arch. Biol. Med. Wiss., i, 1901, p. 80) takes 5 parts
of formol, 100 of water, and 2 of bicarbonate of soda, for four to ten
days.
HERDLICKA (Proc. U. S. Nat. Mus., xxx, 1906, p. 304) takes 3 parts
of formol, 25 to 45 of water, and 72 to 52 of 95 per cent, alcohol.
STRECKER fixes small pieces for twenty-four to forty-eight hours in
equal parts of 10 or 20 per cent, formol and Ehrlich-Biondi triacid
mixtures, and imbeds in paraffin, thus getting a stain at the same
time as a fixation. Similarly with toluidine blue fixing it with
ammonium molybdate. See Ztschr. wiss. Mikr., xxviii, 1911,
p. 17, and the literature discussed therein.
CHAPTER XXXI. 403
812. Chromic Salts. — The most commonly used is potassium
bichromate. The liquid of Erlicki has a more rapid action than
other solutions of chromic salts, but it has been generally abandoned
because of the alterations it very often produces.
SAHLI (Ztschr. wiss. Mikr., ii, 1885, p. 1), after investigating the
action of the usual solutions, concludes that the best hardening
agent for fresh tissues is pure potassium bichromate, in 3 or 4 per cent,
solution, the hardening being done in a cold place. He rejects the
liquid of Erlicki on account of the precipitates to which it so fre-
quently gives rise.
OBERSTEINER is of the same opinion, and recommends pure
bichromate for general hardening purposes ; whilst for the study of
delicate structural details he recommends fixing in Fol's modifica-
tion of Flemming's fluid (§47) for twenty-four hours, followed by
washing with water and hardening in 80 per cent, alcohol.
In view of the slowness of penetration of chromic salts, it is often
advisable to treat the preparations for twenty-four hours or more with
80 to 90 per cent, alcohol, or better, for a few days with a formol
solution before putting them into the hardening fluid, or to add
formol (say 3 per cent.) to it, in order to avoid maceration of the
deeper layers of the tissues. In this case, however, the fluid must
be changed after twenty-four to forty-eight hours.
Potassium bichromate should be employed 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.
Ammonium bichromate should be employed of half the strength
recommended for potassium bichromate at first ; it may be raised
to as much as 5 per cent, for cerebellum towards the end of the
hardening.
NISSL (Enzycl. Mile. Technik., ii, 1910, p. 245) uses, for rapid
hardening, large quantities of Miiller's fluid 100 parts, formol
3 parts, and enough glycerine to make the tissues float. If the
solution is often changed, even entire brains are in a few days
sufficiently hardened to be safely transferred into pure Miiller's
fluid, or potassium bichromate of about the same strength,
BETZ'S method (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 iodine tincture to give it a light
brown coloration (as soon as the alcohol becomes colourless, more
iodine must be added.) They should then be definitely hardened
in 3 per cent, potassium bichromate for spinal cord, medulla
26-2
404 NERVOUS SYSTEM- GENERAL METHODS.
oblongata, and pons, 5 per cent, for cerebellum, and 4 per cent, for
cerebrum.
The methods of BEVAN LEWIS (op. cit.) and HAMILTON (Journ.
Anat. and Physiol., 1878, p. 254) can be considered as superseded,
chiefly because they are based on a fixation with methylated spirit
(Bevan Lewis) or mixtures of Miiller's fluid and methylated spirit
(Hamilton), which cannot be used any longer for histological pur-
poses, owing to the excess of impurities it now contains. Also the
methods for encephala of DUVAL (Robin's Journ. de I' 'Anat., 1876,
p. 497) and DEECKE (Journ. R. Micr. Soc., 1883, p. 449) can be
considered as obsolete.
ORTH uses formol-Miiller changed every few days. See § 113.
BONVICINI (Ztschr. wiss. MiJcr., xxvi, 1909, p. 412) puts entire
human brains into 10 per cent, formol (first injected through the
carotids or into the ventricles) for six to eight days, cuts them into
1 cm. thick slices by means of a special macrotome, and transfers
them into a mixture of 4 parts of potassium bichromate and 2-5 parts
of chromium sulphate in 100 of water, and keeps them therein, in the
dark, for two months for cerebral hemispheres, twelve to fourteen
days for medulla and pons, five or six days for spinal cord. In the
case of slices of brain, the fluid must be changed every week.
RAWITZ (ibid., p. 338) puts formol material for exactly five days
into alcohol with 10 per cent, tinctura iodi P. G., then for eight to
ten into saturated solutions of potassium bichromate changed after
the first day, and lastly into 95 per cent, alcohol for three days in
the dark.
813. Other Reagents. — Osmic acid is hardly useful for specimens
of more than 2 or 3 mm. thickness.
Chromic acid is rarely used alone. Its action is rapid, but uneven,
and causes shrinkage and brittleness. A very little (e.g. 3 to 5 drops of
a 1 per cent, solution to every 100 c.c. of fluid), added to bichromate
solutions will do no harm and quicken the hardening.
Nitric acid has been and still is employed in strengths of 10 to 12 per
cent.
Neutral acetate of lead affords, according to KOTLAREWSKI (Ztschr.
wiss. Mikr.,iv, 1887, p. 287), an excellent preservation of ganglion cells.
Corrosive sublimate solutions either alone or mixed with other re-
agents (see Chapter V.), have been very often used for cytological
studies.
Similarly acetic alcohol.
MANN (op. cit.) for cell studies, puts small pieces for twenty-four hours
into a solution of 5 parts of potassium iodide and 25 of iodine in 100
parts of water, and then into 70 per cent, alcohol.
OLMACHER (§ 65) recommends his mixture. KODIS (Arch. mikr.
Anat., lix, 1901, p. 212) fixes tissues in a saturated solution of cyanide
CHAPTER XXXI. 405
of mercury, brings them into 10 per cent, formol, and makes sections by
the freezing method.
NELIS (Bull. Ac. 8c. Belg., 1899-1900) fixes spinal ganglia for twenty-
four hours in a solution of 20 grms. of copper sulphate and corrosive
sublimate to saturation in a litre of 7 per cent, formol with 5 c.c. of
acetic acid.
KING (Anat. Rec., iv, 1910, p. 213) after trying over twenty-five
methods on brains of rats, concludes that the best is Ohlmacher's.
The brain should be put into it for two to three hours, then for one into
85 per cent, alcohol, then into 70 per cent, with iodine for at least twenty-
four hours, then passed through alcohols of ascending strength and
alcohol-ether into 2 per cent, celloidiu for two to three days, and
through chloroform and benzol into paraffin. In her opinion, Bourn'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 the
nuclei well, but vacuolise the cytoplasm.
See further particulars on this subject in the original papers of
TRZEBINSKI, Virchow's Arch., cvii, 1887, p. 1 ; DIOMIDOFF, ibid., p. 499 ;
FISH, The Wilder Quarter-Century Book, 1893, p. 335 ; DONALDSON,
Journ. Morphol., ix, 1894, p. 123 ; MARINA, Neural. Centrbl., xvi, 1897,
p. 166 ; TIMOFEEW, Intern. Monatschr. Anat., xv, 1898, p. 259.
814. 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 hours ; then 3 per
cent, bichromate, changed once a fortnight until the hardening is
sufficient (six to ten weeks).
BURCKHARDT (Das Gentralnervensystem von Protopterus, Berlin, 1892 ;
Ztschr. wiss. MiTcr., ix, 1893, p. 347) recommends a liquid composed of
300 parts of 1 per cent, chromic acid, 10 parts of 2 per cent, osrnic
acid, and 10 parts of concentrated nitric acid, in which brains of
Protopterus are hardened in twenty-four to forty-eight hours.
FISH (Journ. of Morphol., x, 1895, p. 234) employed for Desmog-
nathus a mixture of 100 c.c. of 50 per cent, alcohol, 5 c.c. of glacial
acetic acid, 5 grms. of corrosive sublimate, and 1 grm. of picric acid, fixing
for twelve to twenty-four hours, and passing through the usual alcohols.
STRONG (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 alum and 1 part of formol, for about two weeks,
makes paraffin sections, stains with hsematoxylin, and differentiates in
1 or 2 per cent, iron alum.
JOHNSTON (Morphol. Jahrb., xxxiv, 1905, p. 150) recommends for
nerves of Petromyzon to make paraffin section from Zenker material,
and stain them with a mixture of saturated solution of nigrosin,
saturated solution of picric acid, and 1 per cent, acid fuchsin, in water
mixed in proportions arrived at by trial.
Sections.
815. Imbedding is by no means always necessary, and is objected
to in some cases. Indeed sections can be obtained from any part
406 NERVOUS SYSTEM—GENERAL METHODS.
of the central nervous system without it, if the tissues are well
hardened. Material hardened in alcohol, or in chromic solutions,
or treated according to Golgi's methods may be glued on to a piece
of wood or hard cork (or still better to a glass cube) by means of a
rather thick solution of gum arabic. As soon as it begins to stick
to the support the whole is put into 70 to 80 per cent, alcohol to
harden the gum, and then fixed in the object-holder of the microtome
and cut. Or one can simply make a clean cut at the bottom of the
specimen, dry it with blotting paper and stick it on the support with
sealing wax or paraffin of high melting point. For section cutting
the knife should be wetted with alcohol or water ; if the latter is
used some soap may be added to it to prevent it from running into
drops on the knife.
Formalin material is preferably cut by the freezing method, this
being very largely used since the introduction of C02 microtomes,
by means of which many and relatively very thin sections can be
rapidly obtained with great economy of time and imbedding reagents.
Imbedding in paraffin is not advised for the nervous system
in general, particularly after fixation in alcohol, and bichromate
solutions. One should have recourse to it only for special
cytological methods, taking care not to use paraffin of too high
a melting point.
Imbedding in celloidin is very largely used, and to great advantage,
for many purposes.
If, notwithstanding every precaution, the celloidin has not
thoroughly penetrated the tissues, good sections may still be obtained
by DUVAL'S method of collodionising the sections. The cut surface
of the block is dried by blowing on it, and 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 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 greater consistency to brittle or otherwise delicate
tissues.
The above applies to section cutting of small, medium-sized and
even relatively large pieces. Also unusually large pieces, entire
human hemispheres, and brains of high vertebrates can be cut into
thin, and, if necessary, serial sections both without, and after im-
bedding either in celloidin or paraffin or by mixed methods. The
processes used for the purpose do not differ essentially from those
above-mentioned and fully described in Chapters VII., VIII. and
CHAPTER XXXI. 407
IX., but (particularly for cy to-architectonic and fibre-architectonic
studies) special apparatus and installations are needed, the description
of which is outside the province of this book.
See FEIST, Ztschr. wiss. Mikr., viii, 1891, p. 492 ; DEECKE, op. cit. ;
DEJERINE, Anat. Centres Nerveux ; STRASSER, Ztschr. wiss. Mikr., ix,
1892, p. 8 ; BRODMANN, Journ. Psychol. u. N enrol., ii, 1903-4, p. 206 ;
WARNKE, ibid., p. 221 ; LIESEGANG, Ztschr. wiss. Mikr., xxvii, 1910
p. 369 ; VENDEROVI£, Anat. Anz., xxxix, 1911, p. 414.
General Stains.
816. Carmines. — Ammonia- carmine is good for general views.
Stain very slowly in extremely dilute solutions. Bichromate
material should 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.
Bolles Lee (see 1913 ed.) prefers carmalum with formol material
as giving a more delicate stain. He finds it better then paracarmine.
The best way of staining formol material with ammonia carmine,
carmalum, picro- carmine and the like, consists in my opinion in
cutting sections by one or the other of the freezing methods, and
transferring them for a few hours either in Miiller's fluid, or 0-5 per
cent, chromic acid as suggested by SCHWALBE (Centrbl. allg. PathoL,
xii, 1901, p. 881). Sections are then washed for a longer or shorter
time according to the amount of mordant one wishes to extract,
proceeding afterwards to stain with one of the above-mentioned
carmine solutions.
On the other hand sections of non-imbedded material fixed and
hardened in one or the other of the fluids mentioned in §§ 810 to 812
may be stained not only with carmines, but also with a great variety
of dyes if one so desires (see Chapter XL). The same applies to
sections of imbedded material, though the after-treatment to which
it has been submitted may render more or less difficult the carrying
out of certain general stains. But one should remember that in
any case the results thus obtained are not very instructive, and by
no means comparable with those attainable by the rational use of
the special methods described in the following chapters.
For other carmine processes of staining, see SCHMAUS (Munch, med.
Wochenschr., 1891, p. 147) ; UPSON (Neural. Centralb., vii, 1888, p. 319) ;
FREEBORN (Journ. Eoy. Mic. Soc., 1889, p. 305) ; KADYI (N enrol.
Centralb., xx, 1901, p. 687) ; CHILESOTTI (ibid., xix, 1902, p. 161, and
Centralb. ally. Pathol, xiii, 1892, p. 19)).
408 NERVOUS SYSTEM— GENERAL METHODS.
817. Nigrosin and Anilin-Blue-Black.— Nigrosin has given useful
results in some hands. Aniliii-blue-black has been much recommended
by SANKEY (Lancet, 2, 1875, p. 82) ; BEYAN LEWIS (Human Brain,
p. 125, and Quart. Journ. Micr. Sc., 1876, pp. 73-75) ; VEJAS (Arch. f.
Psych, xvi, 1885, p. 200) ; MARTINOTTI (ii, 1885, p. 478) ; JELGERSMA
(Ztschr. wiss. Mikr., 1886, p. 39) : SCHMAUS (Munch, med. Wochenschr.,
1891. p. 147), and others. And see also previous editions.
818. Picronigrosin. — MAKTINOTTI (loc. cit., 1885, 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.
819. KAISER (Ztschr. wiss. Mikr., vi, 1889, p. 471) stains sections of
spinal cord for a few hours in a solution of 1 part of naphthylamin brown,
200 of water, and 100 of alcohol, washes with alcohol, clears with
origanum oil and mounts.
820. Alizarine.— SCHE OTTER (Neurol. Centrbl, xxi, 1902, p. 338)
stains sections for twenty-four hours in a 1 to 2 per cent, solution
of sulphalizarinate of soda, differentiates for half to one minute in
tap-water, dehydrates, and mounts. This is a general stain, but
demonstrates Nissl bodies and other internal details.
821 MALLORY'S Phosphomolybdic Acid Hsematoxylin and KODIS'
modification, see § 271. For AUERBACH'S modification, see Neurol.
Centrbl., xvi, 1897, p. 439.
822. Hsematoxylin and Acid Fuchsin. — FINOTTI (Virchow's Arch.,
cxliii, 1896, p. 133) stains in hsematoxylin, counterstains for three
minutes with 0\5 to 1 per cent, solution of acid fiichsin, and differen-
tiates in 75 per cent, alcohol containing a very little caustic potash.
VAN GIBSON'S hsematoxylin and picro-fuchsin (§ 398) may give useful
general views of nerve-cells, axis-cylinders, and ncuroglia.
823. Other General Stains.— ALT (Munch, med. Wochenschr., 1892,
No. 4) stains for a couple of hours in a solution of Congo red in absolute
alcohol, and washes out with pure alcohol. This is useful for peripheral
axis-cylinders and other elements.
SCARPATETTI (Neurol Centrbl, xvi, 1897, p. 211) stains sections of
formol material for five minutes in 1 per cent, hsematoxylin, treats for
five minutes with concentrated solution of neutral copper acetate,
differentiates with Weigert's borax-ferricyanide, treats with con-
centrated solution of lithium carbonate, washes and mounts. Myelin
is not stained.
ROTIIIG (Folia Neurobiol, ii, 1909, p. 385) fixes and stains for about
four weeks in saturated solution of methylenazur I., in 10 per cent,
formol, puts for ten to fifteen minutes into acetone, then for twelve
hours into chloroform, and imbeds in paraffin. He also has a process
with trichloracetate of lead and methylenazur.
CHAPTER XXXI. 409
RAWITZ (Ztschr. wiss. Mikr., xxvi, 1909, p. 341) lias some compli-
cated methods with Indulin, Indaminblau, and Azosdureblau, which take
twenty-eight days: and (ibid., xxviii, 1911, p. 1) others with fuchsin
and azofuchsin which take over thirty-six days.
AKIKNS KAPPERS (ibid., xxviii, 1911, p. 417) describes a staining
method with extract of elderberries for material fixed and hardened in
Miiller's fluid or similar solutions. It is very simple and particularly
recommended for photographic purposes ; it should be carried out as
follows : Stain celloidin or paraffin sections overnight in neutralised
elderberries extract (obtained by fermentation at 20° to 25° C. ), to which
1 per cent, carbolic acid has been added. Wash in water. Differentiate
in 3 per cent. Liquor ferri sesquichlorati P.G., wash, dehydrate, and
mount.
CHAPTER XXXII.*
NERVOUS SYSTEM — SPECIAL METHODS, CHIEFLY CYTOLOGICAL.
824. Introduction. — The ordinary methods of cytology are, of
course, available for nerve cells ; but there are certain constituents
of these cells, as well as of nerve fibres, which require, for minute
study, special methods, such as the following :—
A. Methods for Cells, demonstrating Tigroid Substance and
other Granular Materials.
825. Tigroid substance or bodies, chromophilic or chromatophilic
substance or material or granules, Nissl's bodies or granules, etc., are
all denominations for a markedly basophil substance which appears
as blocks, granules or irregular patches within the cytoplasm of
nerve cells under certain conditions of fixing and staining.
It is now almost universally admitted that this substance exists
in the living cells as a fluid or semi-fluid " plasm rich in nutritive
value," and that the blocks, granules or patches are appearances
chiefly due to the coagulation of this plasm, as brought about by
the fixing agents employed for their demonstration. As, however,
these bodies or granules appear always the same under constant
optical conditions in healthy cells fixed and stained in a constant
manner, they are said to be the equivalent of such healthy cells during
life. " It follows that if the cells, prepared by the same method
and examined under the same conditions, show a difference from
the equivalent or symbol of healthy cells, the difference is the
measure of some change that has occurred during life." See
HALLIBURTON, Handbook of Physiology, London, 1920, p. 194.
This is pointed out here to make it quite clear why NISSL has
always insisted that his method should be carried out according
to his suggestions, and in a constant manner. At first (Neurol.
Centrbl, iv, 1885, p. 500) he used to stain sections of material
fixed in alcohol with a warmed watery solution of magenta red or
dahlia violet or vesuvine, and to differentiate them with alcohol.
Later (Allg. Ztschr. Psych., xlviii, 1892, p. 197) he suggested floating
sections on a warmed solution of methylene blue (B patent), with
* Re-written by Dr. C. Da Fano, King's College, University of London.
CHAPTER XXXII. 411
subsequent differentiation with a 10 per cent, solution of anilin
oil in 96 per cent, alcohol. The present form of the method was
published in 1894 (N enrol. CentrbL, xiii, p. 507) ; but NISSL con-
tinued to introduce into it slight modifications, as one understands
from many of his papers, to which due attention was paid when
preparing the following account. It must be added here that
Nissl's method has been, and still is, extremely useful for the study
of nervous tissue under various physiological and pathological
conditions, and that it stains, when properly carried out, not only
thetigroid substance and the basophil parts of nuclei of nerve cells,
but also the nuclei and certain parts of the cytoplasm of neuroglia
cells and connective tissue elements normally or abnormally present
in the nervous tissue.
826. NISSL'S Methylene-blue Method. — Not too small pieces of
fresh tissue are fixed in 96 per cent, alcohol and hardened therein
for a few days. They should not be allowed to fall to the bottom of
the bottle, but kept floating by means of some filter paper or cotton
wool. The alcohol must be in large quantities in proportion to
the number of pieces, and repeatedly changed. The pieces are cut
without embedding and the sections collected in 96 per cent, alcohol,
from which they are directly floated on some stain filtered into a
watch glass at the moment of using it. The stain should be at least
three to four months old, and shaken at the moment of filtering the
quantity needed. It is prepared by carefully dissolving 1 -75 grms.
of Venetian soap in 1 litre of distilled water and adding to it 3 -75 grms.
of methylene blue (B patent). It is a good practice to vigorously
shake the bottle from time to time, and to re-filter into the same
bottle the amount of stain left in the watch-glass after staining one
or more sections.
The watch-glass containing the stain with the section floating
on it is warmed carefully over a flame until small bubbles rise to
the surface. The section, which should not have fallen to the
bottom of the watch-glass, is immediately transferred into a mixture
of 10 parts of anilin oil and 90 parts of 96 per cent, alcohol, and as
soon as no more colour is given off (it often takes only some seconds),
it is lifted on to a slide, pressed with smooth filter paper, and cleared
with a few drops of pure anhydrous cajeput oil. Care should be
taken not to dry the section excessively with the filter paper and to
pour the cajeput oil on to the section very quickly.
The cajeput oil not only clears the section, but stops the
differentiation ; it is, therefore, advisable to renew it after a little
412 NERVOUS SYSTEM— SPECIAL METHODS.
while on the section. As soon as this has become quite transparent,
the cajeput oil is dried off with filter paper, the section thoroughly
washed with benzol and covered with a drop of thick xylol-colo-
phonium, rendered more fluid by passing the slide carefully over a
flame, and quickly covering the section with a thin cover-glass before
the colophonium sets again by cooling.
827. Suggestions regarding the Carrying-out of Nissl's Method
(as deduced from NISSL'S papers ; see chiefly Enz. d. mikr. TecJin., ii,
1910, pp. 252 — 280, and the references therein quoted at p. 287).—
For the fixation of tissues alcohol should be almost exclusively
used. Formalin, mixture of formalin and alcohol, sublimate and
mixture of the same with alcohol or picric acid and the like, nitric
acid may be occasionally employed, but no particularly good results
can be expected from them. These are, however, somewhat better if
tissues are placed for some time in alcohol after fixation with one
or the other of the above reagents. This applies particularly to
formalin material, which can be kept with advantage for many
weeks, and even months, in repeatedly changed 96 per cent, alcohol.
The bichromates of potassium and ammonium and mixtures con-
taining chromic' salts, though useful for other purposes, should be
entirely avoided for cytological investigations in Nissl's sense. See
on this subject also BURCHARDT, La Cellule, xii, 1897, p. 337.
If tissues are too brittle to be sectioned without embedding, or
if embedding is for any other reason desirable, one should have
recourse to celloidin, paraffin being used only when unavoidably
necessary or for special purposes. Pieces to be embedded in celloidin
are not to be passed through alcohol-ether, but directly from absolute
alcohol into thin celloidin. Embedding should, in any case, be carried
out as quickly as possible.
Sections of material which was not fixed in alcohol and of em-
bedded tissues, however fixed, stain, as a rule, very poorly by
Nissl's soap-methylene-blue method ; but good and even excellent
results can be obtained by staining such sections with watery
solutions (generally 0-5 to 1 per cent.) of toluidine blue, thionine,
Unna's polychrome methylene blue, dahlia violet, vesuvine, neutral
red, magenta red, Azur I, Azur II, and the like.
If one of such stains is used, it need not be warmed until
bubbles come to the surface, but only until vapour arises. For
the differentiation pure 96 per cent, alcohol, viz., without any
addition of anilin oil, should be used. In this connection I find
that very good results can be obtained from material embedded in
CHAPTER XXXII. 413
celloidin if the sections are, whenever possible, freed from the
celloidin before staining them, and if they are re-stained a second and
.a third time after having been each time completely differentiated.
See DA FANO, Proc. Physiol. Soc., Journ. Physiol., liv, 1920-1.
All preparations stained by Nissl's method keep badly, but they
keep a little better and may last almost unchanged even for
years : (1) if the anilin-alcohol or alcohol used for the differentiation
is properly washed away with pure benzol ; (2) if the xylol-colo-
phonium for the mount is prepared with pure xylol, and of the
thickness needed ; (3) if sections of material embedded in celloidin
are submitted to the above-mentioned succession of staining and
differentiation ; (4) if preparations are carefully protected from
light.
828. Modifications of Nissl's Method. — REHM (Munch, med.
Wochenschr., xxxix, 1892, p. 217) floats sections for half a minute
to a minute on a hot 0-1 per cent, solution of methylene blue,
differentiates them in 96 per cent, alcohol and clears them with
origanum oil.
LENHOSSEK (Fein. Ban. d. Nervens., 1895) stains sections of
formol material in a concentrated aqueous solution of thionine, rinses
them with water and mounts them like Nissl.
LUXENBURG (N enrol. Centrbl, xviii, 1899, p. 629) stains paraffin
serial sections either with Nissl's methylene blue or with thionine as
Lenhossek.
JULIUSBURGER (Neurol. Centrbl., xvi, 1897, p. 259) stains sections
of material fixed in Orth's fluid and embedded in celloidin, either
with Nissl's methylene blue or with warmed neutral red.
ROSIN (Deutsche med. Wochenschr., xxiv, 1898, p. 615) treats
sections of formol material similarly.
LENHOSSEK (Neurol. Centrbl., xvii, 1898, p. 577) stains paraffin or
celloidin sections of spinal ganglia fixed in Carney's fluid with a
concentrated watery solution of toluidine blue overnight, rinses with
water, differentiates quickly with alcohol and clears with xylol or
carbol-xylol.
POLUMORDWINOW (Ztschr. wiss. Mikr., xvi, 1899, p. 371), uses
1 part of 1 per cent, solution of toluidine blue to 119 of distilled
water and 1 of sodium carbonate.
VAN GEHUCHTEN and NELIS (La Cellule, xiv, 1898, p. 374) recom-
mend fixing spinal ganglia in Gilson's mixture.
VAN GEHUCHTEN (see 1913 ed.) uses paraffin sections mounted
on slides by the water method and stains them for five to six
414 NERVOUS SYSTEM— SPECIAL METHODS.
hours in Nissl's methylene blue solution in the incubator at 35°
to 40° C.
GOTHARD (C. R. Soc. BioL, v, 1898, p. 330) stains celloidin sections
for twenty-four hours in Unna's polychrome methylene blue, and
differentiates them with a mixture of 5 parts of creosote, 4 of cajeput
oil, 5 to 8 of xylol, and 16 of absolute alcohol. The mixture is
removed with absolute alcohol and sections mounted in xylol dammar
after clearing with cajeput oil.
LUITHLEN and SORGO (N enrol. Centrbl., xvii, 1898, p. 640) diffe-
rentiate in Unna's glycerin-ether mixture, remove this with absolute
alcohol, and clear with origanum oil.
Similarly LENNHOFF (ibid., 1910, p. 20) ; or, polychrome methylene
blue two minutes, distilled water quickly, carbol-pyronin-methyl
green twenty minutes ; distilled water quickly, absolute alcohol,
oil, balsam.
LORD (Journ. Ment. Sc., xliv, 1898, p. 693) makes sections from
frozen fresh tissues, 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 them with distilled water and stains them in
5 per cent, solution of methylene blue B pat.
829. BIELSCHOWSKY and PLIEN'S Cresyl Violet Method (Neural.
Centrbl., xix, 1900, p. 1141). — Celloidin or paraffin sections of material
fixed either in alcohol or formalin, or sections made by the freezing
method from formalin material, are stained for twenty-four hours
in a very diluted solution of cresyl violet R.K., prepared by adding
6 to 8 drops of a concentrated aqueous solution to every 50 c.c. of
distilled water. After a quick wash in distilled water sections are
brought through the ascending series of alcohols, cajeput oil, and
xylol, into balsam. The preparations are said to keep better than
those stained with thionine or toluidine blue.
830. Picrocarmine has been successfully used by MESSNER (Journ.
Psychol. Neurol., xviii, 1912, p. 204, and xx, 1913, p. 256). Sections
of alcohol material, embedded in celloidin or not, are washed in water
and then stained for five minutes in a warmed diluted solution of
Ranvier's picrocarmine. After a quick wash, they are differen-
tiated in 3 per cent, hydrochloric acid, dehydrated and mounted
as usual. In the case of the spinal cord, medulla oblongata and
pons the method succeeds also if material was fixed in formalin.
831. Other Methods for Tigroid Substance and Basophil Granules
in General. — See GOLDSCHEIDER u. FLATAU, Norm. u. path. Anat. d.
Nervenz, Berlin, 1898, or Ztschr. wiss. Mikr., xvi, 1899, p. 102, and
CHAPTER XXXII. 415
NISSL'S remarks thereon, Deutsche Ztschr. Nervenheilk, xiii, 1899, p. 348 ;
ILBERG, Neural Centrbl, xv, 1896, p. 831 ; Cox, Anat. Hefte, x, 1898,
p. 75 ; Int. Monatschr. Anal,x.v, 1898, p. 241 ; AUERBACH, Monatschr.
Psych. Neurol, iv, 1898, p. 31 ; MYERS, Anat. Rec., ii, 1908, p. 434 ;
SAVINI, E. u. TH., Centrbl Bakl, I Abth., xlviii, 1909, p. 697 ; MOSSE
(argentamin stain), Arch. mikr. Anal, lix, 1902, p. 403 ; MENTZ v.
KROGH, Centrbl Bakl, I Abth., Iviii, 1911, p. 95 ; JOHNSTON, Anal Bee.,
xi, 1916, p. 287.
832. HELD'S Methylene Blue and Erythrosin Method (Arch. Anat.
Phys., Anat, Abth., 1895, p. 399 ; 1897, pp. 226—233, 273—385,
Supplementband). — Material may be fixed in alcohol, but preferably
either in picro-sulphuric acid, or in van Gehuchten's mixture of
alcohol, chloroform and acetic acid, or 1 per cent, corrosive sublimate
in 40 per cent, acetone. Tissues should be carefully embedded in
paraffin and sections stuck to slides by the water method. They are
stained with the aid of gentle heat for one to two minutes in a
solution of 1 grm. erythrosin in 150 of distilled water acidulated with
2 drops of glacial acetic acid. After washing with water the slides
are transferred into a mixture of equal parts of Nissl's methylene
blue solution and 5 per cent, acetone, warming until all odour of the
latter has disappeared. Differentiation is carried out after cooling
by means of a 0 -1 per cent, solution of alum until sections are reddish.
Rinse in distilled water, dehydrate as rapidly as possible in absolute
alcohol, wash in xylol and mount in balsam.
BOCCARDI (Monit. Zool. Ital, x, 1899, p. 141) uses a mixture of
erythrosin 0-1 grm., toluidine blue 0-2 grm., and water 100 c.c., and
differentiates in 0 -5 per cent, alum solution.
By means of Held's method, besides the tigroid substance, other
granules — viz., Held's neurosomes — become stained. It may, there-
fore, be considered as a typical example of double staining of nerve-
cells. Other double stains demonstrating basophil, acidophil and
other granules have been repeatedly proposed and may be easily
obtained by the combination of an acid and a basophil dye. One
generally uses watery solutions, e.g., of acid fuchsin, and methylene
or toluidine blue, and one stains first with the acid dye and then with
the basophil one, differentiation being carried out with alcohol.
One may also have recourse to EHRLICH'S triacid (§ 296) as originally
proposed by ROSIN (Neurol. Centrbl., xii, 1893, p. 803), or to one or
other of the methods used for staining blood films (§ 784), such as
PAPPENHEIM'S panoptic triacid stain, Jenner's mixture, Leishman's
Romanowsky stain, Pappenheim's method as described in § 785,
and so on.
See on this subject COWDRY, Int. Monatschr. Anat., xxix, 1913,
416 NERVOUS SYSTEM—SPECIAL METHODS.
p. 673, and for pathological specimens ALZHEIMER'S methods 5, 6 and 9
in Histol. u. Histopath. Arb. uber d. GrossMrnr, iii, 1910, pp. 406 — 412,
which may be useful for the study of nerve-cells though originally pro-
posed for the investigation of neuroglia.
B. Methods for Cells and Fibres, demonstrating Neurofibrils.
833. Neurofibrils ; General Characters. — Nerve cells and the fibres
into which they are prolonged contain, in addition to the chromatic
constituents shown by the methods already dealt with, a character-
istic so-called achromatic element, consisting chiefly of very fine and
fairly refractive fibrils which can only be seen with great difficulty
in the unstained state, but may be fixed with osmic acid and made
out in thin sections of medullated nerve fibres observed in diluted
glycerin or water, and may be to a certain extent isolated by macera-
tion. For their demonstration, however, one or the other of the
methods chronologically described in the following paragraphs must
be employed. They are all regarded as giving true stains of neuro-
fibrils.
For the method of KUPFER (Sitzb. math. Kl. Akad. Wiss. Munchen, xiii,
1884) see former edition.
834. APATHY'S Methods. — The gold method (" Nachvergoldung ")
has been given in § 371. The stain is very sharp, but good results
are obtained only in certain invertebrates, and even in these with
considerable difficulty.
The hcemateine method (Mitth. Zool. Stat. Neapel, xii, 1897, p. 712)
has the same advantages and disadvantages, and has been little used
since the discovery of the Cajal and Bielschowsky processes. Material
may be fixed with corrosive sublimate, Zenker's fluid, picro-sulphuric
acid, or any other mixture which is not inimical to staining with
alum hsematoxylin, and should be preserved in 90 per cent, alcohol.
Portions, no more than J cm. thick, are stained for at least forty-
eight hours in hsemateine I. A. (§ 259), and then washed for up to
twenty-four hours in absolutely pure distilled water, or preferably
suspended therein. Before the stain has become washed out of the
neurofibrils entirely, it is fixed 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 in absolute alcohol, and
embedded in paraffin or celloidin, after clearing with chloroform,
and carefully protecting them from light whilst in chloroform or
celloidin. The sections are mounted either in a resin or in neutral
glycerin.
CHAPTER XXXII. 417
835. BETHE'S Molybdenum-Toluidine Blue Method (Ztschr. wiss.
Mikr., xvii, 1900, p. 13). — Pieces of the central nervous system of
vertebrates are fixed for twenty-four hours in 3 to 7'5 per cent, nitric
acid, and then brought directly into 96 per cent, alcohol for a day or
longer. They are afterwards put for twelve to twenty-four hours
in a mixture of 1 part of ammonia (of sp. gr. 0 -95) with 3 of distilled
water and 8 of 96 per cent, alcohol ; for six to twelve hours into pure
alcohol ; for twenty-four hours into a mixture of 1 part of concen-
trated hydrochloric acid, 3 of distilled water, and 8 to 12 of
alcohol ; for ten to twelve hours into pure alcohol ; for two to
six hours into water. They are now mordanted with 4 per cent,
ammonium molybdate, washed again, dehydrated and embedded in
paraffin. The sections, 8 to 10 /x thick, are seriated on slides by
means of egg albumen, but without water, then passed through xylol
and alcohol and " differentiated," viz., covered with water poured
on the sections so as to form over them a layer 1 -5 to 2 mm. deep,
and put into an incubator at 55 to 60° C. for ten minutes. They are
then rinsed with water, covered with a 1 : 3000 solution of toluidine
blue, stoved for another ten minutes, rinsed with water, and lastly
treated with 96 per cent, alcohol till no more colour comes away.
After dehydration with absolute alcohol they are mounted in the
usual way.
The method is also applicable to invertebrates for which other
fixing agents besides nitric acid are admissible, and the impregnation
with ammonium molybdate may be done on the sections instead of
previously on the uncut tissues.
For LUGARO'S modification see Eiv. pat. nerv. ment., x, 1905, p. 265.
836. DONAGGIO'S Methods (Riv. Sper. Freniatr., xxx, 1904, p. 397,
and xxxii, 1906, p. 394). — There are five methods of Donaggio. By
the first two, pieces are stained in bulk before embedding, but results
are not so good as by the other three, the most important of
. which is —
Method III. — Good for spinal cord, pons, medulla oblongata,
spinal and sympathetic ganglia. Thin slices of tissues are fixed for
five to six days in pure pyridine changed at least once, and then
treated with repeatedly changed distilled water until the pyridine
has. been entirely eliminated. The surfaces of pieces are smoothed
by means of a sharp razor, and the pieces brought for twenty-four
hours into 4 per cent, ammonium molybdate to which 4 drops of
hydrochloric acid have been added. After a quick wash, they are
rapidly dehydrated in 95 per cent, and absolute alcohols, and em-
M. 27
418 NERVOUS SYSTEM- SPECIAL METHODS.
bedded in paraffin. The sections, which must be rather thin
(3 to 6 /x), are brought through xylol, absolute and 95 per cent,
alcohols into distilled water and here washed.
This is the crucial point of the method because, by washing,
ammonium molybdate becomes extracted from the sections, and the
success of the subsequent staining depends almost entirely on
carrying out the extraction up to the right point. I find that the
only way of ensuring this consists in proceeding by trials, which must
be repeated for every series of sections. Once the right amount of
washing has been decided upon, one can proceed to stain even many
slides at the same time by means of a 1 : 10,000 solution of thionine,
to be freshly prepared every time from a less diluted stock solution.
The staining is a " progressive " one, and must be controlled
under the microscope. It generally takes about twenty minutes to
obtain it, at the end of which time the grey substance has a red-
purple tone whilst the white substance appears bluish. If the
staining is right the preparations can be quickly washed, dehydrated
and mounted. But if the neurofibrils are not quite sharply stained,
the preparations can be " differentiated " for another fifteen to
twenty minutes in the ammonium molybdate solution used for
mordanting the pieces, or for ten seconds in a diluted solution
(1 : 10 to 1 : 20) of " pink salt " (C. Erba, Milano). Preparations
last only a few months, but are sometimes of great interest. See
DA FANO, Zieglers Beitrdge, xliv, 1908, p. 495.
Method IV, which is particularly useful for the demonstration of
neurofibrils in the cells of the cortex cerebri and cerebelli, differs
from Method III only in regard to a preliminary fixation of pieces
for twenty-four hours in a mixture of pyridine nitrate 10 grms., and
pyridine, 100 c.c. ; they are then transferred for another thirty-six
hours into pure pyridine, proceeding as in Method III.
Method V may be used for the demonstration of both NissPs
substance and neurofibrils. Pieces are fixed in a saturated solution
of corrosive sublimate ; after a day they are treated for twenty-four
hours with distilled water to which a few drops of iodine tincture
have been added, then for two to three hours with pure distilled
water ; and lastly passed for forty-eight hours into pure pyridine,
this being changed at least once. The rest as in Method III.
PARAVICINI (Boll. Mus. Z. Anat. Comp. Torino, xx, 1905, p, 1)
fixes and mordants in the dark, and differentiates after staining with
extremely weak hydrochloric acid.
TOMASELLI (Ztschr. wiss. Mikr., xxiii, 1906, p. 421) fixes spinal
ganglia for six to seven hours in absolute alcohol 100 c.c. with four to
five drops of ammonia, and then transfers them for two days into pure
CHAPTER XXXII. 419
pyridine to be repeatedly changed, the vessel with the pieces being kept
at 36° to 37° C. After washing for two to three hours in running tap
water, he continues as in Donaggio's method III.
For the criticism of JADERHOLM, see Arch. mikr. Anat., Ixvii, 1906,
p. 108 ; and for that of MONTANARI, Ztschr. wiss. Mikr., xxviii, 1911,
p. 22.
837. RAMON Y CAJAL'S Methods. Introductory. — It has been said
by some authors that CajaPs methods were originally only modifica-
tions of the photographic process of Simarro. The criticism is
unjust because even the first formula of Cajal differs so profoundly
from Simarro's process as to form an entirely new method. One
cannot, however, deny the existence of a certain similarity of con-
ception between the two processes in so far as both are based on the
silver-reducing power of certain photographic reagents. For this
reason it has been thought expedient to briefly describe here Simarro's
process, which though uncertain in its results, may still be of some
value to elucidate certain histological questions.
SIMARRO'S Process (Rev. Trim. Micr., v, 1900, p. 45) consisted in
poisoning animals with subcutaneous injections of solutions of
sodium or potassium bromide or iodide in order to impregnate their
living nervous tissues with one or the other of these salts. As soon
as the animals showed that the poisoning had reached its maximum
they were killed and their central nervous system removed in the
photographic dark room. Small pieces were then immersed in a
solution of silver nitrate, which, by combining with the bromine or
iodine with which the tissues were impregnated, gave rise to the
formation of silver bromide or iodide, which is easily affected by
light. Sections were then made (always in the dark room), best by
means of a freezing microtome, and exposed for a little while to light.
There remained only treating them with a photographic developer,
such as hydroquinone, pyrogallol or the like, and fixing them with
sodium hyposulphite and so on, as if they were photographic plates ;
they were lastly washed, dehydrated and mounted in the usual way.
One can easily understand the many drawbacks of such a method
and the reason for which it was abandoned as soon as Cajal published
in 1903 his first reduced silver methods. From that time onwards,
Ramon y Cajal continued improving them and adding new formulae,
which he himself summarised in a special article of his Trab. Lab.
Invest. BioL, Madrid, viii, 1910, on which the following account is
based. The numbering is that of Ramon y Cajal.
Formula la. — Small pieces of fresh tissue are directly put into
1-5 per cent, silver nitrate and kept therein for three to four and
27—2
420 NERVOUS SYSTEM— SPECIAL METHODS.
even to five days at a temperature of about 35° C. In summer, with
a temperature constantly over 22° C., the stove may be dispensed
with, provided the impregnation is prolonged for two to three days
more. The tissues are known to be ripe for reduction when a freshly
cut surface shows a brownish-yellow colour.
They are then washed for one to two minutes in distilled water
and put into —
Pyrogallol or hydroquinone . . . 1 — 2 grms.
Distilled water 100 c.c.
Formalin 5 — 10 c.c.
The formol is not necessary but useful. One may use pyridine
instead (1 to 3 per cent.). The addition of a small quantity of
sodium sulphite (0-2 to 0-5 per cent.) has been abandoned by Cajal.
The stronger the pyrogallol, the greater the contrast, so that it may
be useful to take, sometimes, as much as 3 per cent., but then the
over-impregnation of the outer layers will be increased. Hydro-
quinone reduces more energetically than pyrogallol.
The pieces remain in the reducing fluid for about twenty-four hours
and are then quickly washed, hardened in alcohol and embedded in
paraffin or celloidin. The sections (15 to 20 //, thick) are mounted
in dammar after toning with a solution of gold chloride if the reaction
is rather weak, without toning if the impregnation is a good one.
Faintly impregnated sections can be advantageously toned with —
Distilled water 100 c.c.
Ammonium sulphocyanide ... 3 grms.
Sodium hyposulphite . . . . 3 „
1 per cent, gold chloride ... a few drops.
If subsequently found to be too dark they can be bleached by
Veratti's potassium permanganate and sulphuric acid mixture
(see § 846).
The sections from the outer layer are generally too dark for
study, those from the innermost too pale, whilst those from the
intermediate layer are good. The over-staining of the outer layer
can be diminished by diluting the silver nitrate with 1 volume of
water for the last twelve hours.
The method has the defect of giving an imperfect fixation of the
nervous tissue and of impregnating, almost exclusively, cell bodies
and dendrites. It is not good for ganglia and large cells of adult
subjects, but excellent for small and medium-sized cells of very
young subjects and early embryos.
Formula la, A. — As the last, but pieces are fixed in 3 to 6 per cent.
CHAPTER XXXII. 421
silver nitrate. This formula gives better fixation, and was success-
fully used by DOGIEL (Anat. Anz., xxv, 1904, p. 558, and Arch.
mikr. Anat., Ixvii, 1906, p. 638) for the study of Grandry's corpuscles
and other sensory nerve endings, by KOLMER (Anat. Anz., xxvi, 1905,
p. 560) for the epidermis of Lumbricus, etc., and by other authors
for the ganglionic chain of Hirudinea.
Formula la, B. — As above, but taking 0-75 per cent, silver nitrate
and very small pieces, preferably from embryos and new-born sub-
j ects. Poor fixation, much shrinkage, but vigorous stain of the neuro-
fibrils, nucleolar granules and the intranuclear rodlet of Roncoroni.
Formula la, C. — As above, but tissues are fixed in 2 per cent,
silver nitrate to which one-fourth of absolute alcohol or acetone has
been added. Better fixation than with pure silver nitrate. Results
very similar to those obtainable by Formula la with dog, cat and
rabbit, and better results with human cerebrum and cerebellum.
Formula 2a. — Fixation for twenty-four hours in 96 per cent,
alcohol. Tissues not washed, but mopped with blotting paper and
put into 1 -5 per cent, silver nitrate for seven days at 35° C., or six
days at 40° C. The rest as Formula la. Good impregnations of
nerve centres of adults, of peripheral nerve endings, of regenerating
nerves, of early embryos, and of young fishes. It impregnates
medullated and many non-medullated fibres, large and medium
nerve cells, the basket fibres of Purkinje's cells, etc. 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 Cajal calls " accelerators/' Such are chloral hydrate, veronal,
pyridine, nicotine, ethylamine, antipyrine, and others.
Hypnotics, particularly veronal and chloral, and in a less degree
pyridine and ammonia, also act as " rejuvenators," reviving the
susceptibility of impregnation in tissues which have lain too long
in alcohol.
Formula 2a, A. — Fixation for twenty-four to forty-eight hours in
96 per cent, alcohol with 2 per cent, of chloral hydrate. Silver bath
of 1 -5 per cent, for five days in the stove. The rest as usual. Veronal
(same proportion) gives the same result, as do also sulphonal,
trional, hedonal, etc. The results are very constant. Medullated
fibres well shown.
Formula 2a, B. — Fix for twenty-four hours in 96 per cent, alcohol
with 10 to 20 per cent, of pyridine ; wash for some hours in pure
alcohol and transfer pieces into 1 -5 silver nitrate for five days.
422 NERVOUS SYSTEM— SPECIAL METHODS.
I understand from Cajal's pupil, Del Rio Hortega, that this
formula may be successfully employed for the study of peripheral
nerve endings. In this case material is better fixed for twenty-four
hours in pyridine to which one-third its volume of distilled water
or 96 per cent, alcohol has been added. Pieces should be washed
in running tap-water overnight and then transferred for six hours
into pure 96 per cent, alcohol. Impregnation, reduction, embedding,
etc., as .above. Eesults are good, but pieces become extremely
hard even if dehydrated very quickly, and are consequently
difficult to cut. See also Formula 5a.
Formula 2a, C. — Fix for twenty-four hours in 50 c.c. of alcohol
with 10 drops of nicotine. 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.
Formula 2a, D. — Fix for twenty-four hours in allyl alcohol (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 four days at 35° to 38° C., and reduce as usual.
Good for human tissues, especially for fibre plexuses of cerebrum
and cerebellum. Instead of allyl alcohol one may take acetal or
acetone. Put for six hours into acetone with 25 per cent, of water,
then for twenty-four into pure acetone, wash in water, etc., as above.
Formula 3a. — Fixation in ammoniacal alcohol for twenty to forty-
eight hours. The most generally useful formula is 50 c.c. of 96 per
cent, alcohol with 4 to 5 drops of ammonia (of 22° strength). But
for cerebrum not more than 1 to 3 drops ; for cerebellum, ganglia,
spinal cord and regenerating tracts, 4 drops ; for neurofibrils of the
large nerve cells of the medulla oblongata and spinal cord, 9 to 10
drops. To avoid shrinkage, it is well to begin by putting the pieces for
six hours into 70 per cent, alcohol, then in 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 (3 to 4 mm. thick) for five 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 by Formula la.
Specimens may be decalcified, after reducing and washing,
in 96 per cent, alcohol to which a few drops of nitric acid have
been added.
For the delicate impregnation of the neurofibrils of the large and
CHAPTER XXXII. 423
medium nerve cells 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 nerve fibres.
Formula 3a, A. — Fix in 50 c.c. of alcohol with 10 grms. of glycerin
and 6 to 10 drops of ammonia. Good for retina and non-medullated
fibres, but especially for the buds of Held and Auerbach.
Formula 3a, B. — Fix in 50 c.c. of alcohol with 1 -5 c.c. of a 33 per
cent, alcoholic solution of ethylamine. 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 15 per cent, formol. Wash for
six or more hours in running tap-water. Put for twenty-four hours
into 50 c.c. of alcohol with 5 drops of ammonia. Wipe with blotting
paper, silver for five 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 arborisa-
tions of the moss fibres of the cerebellum.
Formula 4a, A. — Fix in " a mixture of formol and alcohol."
Wash out thoroughly with running tap-water, silver, and reduce
as usual. Fixation more rapid and better ; results similar to those
of 3a.
Formula 5a. — This is characterised by a preliminary fixation in
pyridine as originally suggested by HELD (Arch. Anat. PhysioL,
Anat. Abth., 1905, p. 77 ; Anat. Anz., xxix., 1906, p. 186). He
used to fix tissues in pure pyridine, but Cajal finds that this is
likely to cause much shrinkage, and he recommends fixing small
pieces first for six to eight hours in a mixture of equal parts of
distilled water and pyridine, then for eighteen to twenty-four
hours into pure pyridine. Wash for several hours in running
water, and put for a day into 90 per cent, alcohol. Wipe, and
put for four to five days into 1 -5 per cent, silver nitrate at 35° to
38° 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, as well as for peripheral nerve
endings.
Formula 6a. — Put for twenty-four hours into 50 c.c. of water
with 5 grms. of chloral hydrate, rinse, and put into 50 c.c. of 96 per
cent, alcohol with 5 drops of ammonia (time not stated). Wipe
with blotting paper ; put for four to five days at 35° to 38° C. into
424 NERVOUS SYSTEM—SPECIAL METHODS.
1-5 per cent, silver nitrate, and reduce as usual. Results very
constant, without shrinkage. Good for the fine plexuses of cere-
brum, bulb and cord, the baskets of Purkinje's cells, and moss
fibres ; also for motor plates and for regenerating nerves.
Formula 6a, A. — Fix for twenty-four hours in 10 per cent, chloral
hydrate, wash for six, and put direct into the silver. Stove for four
days. Results similar to those of Formula la. Medullated fibres
well stained.
Formula 7a. — Fix for twenty-four hours in Merck's fibrolysine,
wash for six, put for twenty-four into 50 c.c. of alcohol with 5 drops
of ammonia. The rest as by other formula?.
Instead of fibrolysin, lysidine may be taken.
838. Application of CAJAL'S Methods to different Objects. — (1) For
the study of the evolution of neuroblasts and nerve fibres in very
early embryos it is necessary to avoid fixing with formol, or alcohol
with an accelerator, or ammoniacal liquids. The best formulae are
2a and 5a which are applicable to all vertebrates, but preferably
to embryos of birds and fishes.
(2) For late embryos and foetus of mammals. Besides the above
formulae, 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 day; or new-born birds, with ammoniacal alcohol,
or 5a.
(3) For sympathetic ganglia. Formula 3a, or pure alcohol, 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) Cerebellum. For Purkinje cells, la or 3a. For the baskets,
climbing fibres, and medium and small dendrites, 2a or its variants.
For terminal rosettes and collaterals of moss fibres and for the
plexuses of the granular layer, 4a or sometimes 5a or 6a. For the
stellate cells of the molecular layer, 2a and 3a. The best subject
for the latter is the dog.
(6) Cerebrum. In general, the same formulae as for the cere-
bellum, especially la for pyramids of young dogs and cats (of eight
to twenty days). In Formula 3a the proportion of ammonia should
be diminished. For fine plexuses, 4a, 5a, and 6a.
(7) Spinal cord and bulb. All the formulae 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 (10 drops) ;
also the alcoholic fixatives with an accelerator. For medullated
CHAPTER XXXII. 425
fibres, large and small, 2a or 6a. For buds of Held and Auerbach
and for fine plexuses, 4a, 3a, A, or 5a.
(8) Ganglia of invertebrates. For the medicinal leech (not for
other leeches), la, A. For Hcemopis, Aulostomum, Pontobdella and
Glossiphonia, 2a or, better, 3a, with not more than 2 to 5 drops of
ammonia and 3 per cent, silver nitrate, stoving three or three and a
half days. For further details see SANCHEZ, Trab. Lab. Invest. Biol.,
Madrid, vii, 1909, pp. 42—47.
Lumbricus is generally refractory to Cajal's methods. BOULE
(Le Nevraxe, x, 1908, p. 15) obtained good impregnations by acidi-
fying the fixatives. He takes : (a) 25 per cent, formol with 5 per cent,
of acetic acid ; or (b) the same with 0 -5 per cent, of ammonia ;
or (c) 100 c.c. of alcohol, 25 c.c. of formol, 5 c.c. of acetic acid, and
0-5 c.c. of ammonia. For the impregnation he uses 3 per cent,
silver nitrate with 15 per cent, of alcohol, and reduces in the usual
hydroquinone-formol solution, with the addition of 15 c.c. of alcohol.
These results are confirmed by KOWALSKI (La Cellule, xxv, 1909,-
p. 292, and by SCHUTZ (Anat. Anz., xlii, 1912, p. 262). Kowalski
gets impregnations also by simply starving worms for several days,
or exposing them to cold ( — 5° C.) for a quarter of an hour.
(9) Regenerating nerve tissue. For nerves operated a month
or more previously, Formula 2a or 3a, with not more than 3 drops
of ammonia, will stain equally the old and the new fibres ; for nerves
operated not more than two to ten days previously, Formulae 3a
with 4 to 6 drops of ammonia, 5a with pyridine, and 4a, also some-
times 6a ; for regeneration in cord, cerebrum, and cerebellum,
3a with 3 drops of ammonia, or 5a, or pure alcohol.
839. Modifications of KAMON Y CAJAL'S Methods. — DA FANO (Ziegler's
Beitr., xliv, 1908, p. 495) recommends using solutions of silver nitrate
and hydro quinone in 1 : 10,000 gelatin in order to obtain a deeper and
sharper impregnation.
KAT6 (Folia neurobiol. ii, 1908) fixes in 10 to 15 per cent, formol, and
silvers for one to five days at 35° C. in 5 per cent, argentamin to which
3 per cent, of silver nitrate has been added in such a way as to have an
impregnating fluid with a little argentamin in excess ; or argentamin
eight to ten parts, with 3 per cent, potassium bichromate thirty parts
and distilled water 100 parts. For the reduction he uses 10 per cent,
formol with 1 per cent, hydro quinone.
PUSATERI (see AMATO, Virchow's Arch., clxxxv, 1908, 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, silver fluoride) and 155 of distilled water.
BESTA (Biv. pat. nerv. ment. Firenze, xv, 1910, p. 333) fixes for forty-
eight hours in alcohol with 5 per cent, nitric acid, neutralised in alcohol
with ammonia.
426 NERVOUS SYSTEM—SPECIAL METHODS.
LIESEGANG (Kolloidchemie, Beihefte, iii, 1911, H. 7 ; Ztschr. wiss. Mikr.,
xxviii, 1912, p. 369) makes sections of formol material by the freezing
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 hydro quinone. After one or two
minutes the sections are brought into 10 per cent, solution of sodium
hyposulphite, washed and mounted. Results said to be the same as by
the usual process.
ASCOLI (Boll. Soc. med. chir., Pavia, 1911, p. 177) recommends for
the sympathetic nervous system of Hirudinea the following : The
animals cut open at the back are stretched on a piece of cork and fixed
in a solution prepared by dissolving over a flame 5 gr. of pulverised
crystals of silver nitrate in 100 c.c. of 95 per cent, alcohol. After a few
minutes the animals may be detached from the cork and put back in
the same fixative for twenty-four to forty-eight hours in an incubating
stove. They are then transferred for another twenty -four to forty -eight
hours into a 10 per cent, watery solution of silver nitrate, to be kept
also in the incubator. After a quick wash they are reduced for five to
eight hours in Amidol-Hauff 0-5 gr., sodium sulphite cryst. 10 grms.,
distilled water 100 c.c., and lastly passed into glycerin. Preparations
are made by teasing, the thinner ones being toned and counterstained
as usual. For mounting he prefers Apathy's syrup.
HANSON (Anat. Anz., xlvi, 1914, p. 522) has the following for the
demonstration of non-medullated nerve fibres in cranial and peripheral
nerves : Fix in absolute alcohol containing 1 per cent, of strong
ammonia for forty-eight hours ; rinse in distilled water, put in pyridine
for twenty-four hours, wash in many changes of distilled water for
twenty -four hours, place in 2 per cent, silver nitrate at 35° C. in the
dark for three days, rinse in water, and place for one day in a 4 per cent,
solution of pyrogallic acid in 5 per cent, formalin.
As suggested by HABER and GUILD (Anat. Rec., vii, 1913, p. 253) the
results can be improved by a preliminary injection of 95 per cent, alcohol,
containing 1 per cent, of ammonia, through the arteries till tissues are
thoroughly saturated, after which they are dissected out and placed -in a
similar ammoniated alcohol solution for from two to three days. Huber
and Guild have found this method of use for the study of cranial nerves
of small animals and embryos, since the entire heads can, after fixation,
be decalcified by means of 7 per cent, .nitric acid, brought through
80, 90, and 95 per cent, alcohols, each containing 1 per cent, of ammonia,
and finally treated as above.
840. BIELSCHOWSKY'S Methods. Introductory.— 1 14 is well known
that, if ammonia be poured into a solution of silver nitrate, a pre-
cipitate is formed which is redissolved by the addition of some
more ammonia. If an alkaline solution of formaldehyde be slowly
added to this easily reducible di-ammoniacal silver nitrate
(N(NH4)AgH2N03), metallic silver is immediately precipitated and
deposited on the walls of the test tube. Both FAJERSTAJN (N enrol.
Centrbl, xx, 1901, p. 98) and BIELSCHOWSKY (ibid., xxi, 1902, p. 579)
CHAPTER XXXII. 427
thought of taking advantage of this reaction for histological purposes
with the object of finding out a silver impregnation of the nervous
tissue similar to that which characterises Golgi's method. The
results of their attempts were different : Fajerstajn was able to
obtain only a difficult method for staining axis-cylinders which
is now superseded ; Bielschowsky also published, at first, a
complicated silver method for impregnating axis-cylinders very
similar to that of Fajerstajn, but, through successive modifications
of his first process, was led to the discovery of a new method, which
is as important as Cajal's reduced silver methods from an histological
point of view, but is of still greater advantage than the latter for
histopathological investigations. Moreover, Bielschowsky 's method
is applicable to any formol material, even if very old. BAYON
(Die Untersuchungsmeth, etc.) succeeded with four-year-old material,
and I with brains which had been left in formalin for more than
eleven years.
There are at present three Bielschowsky methods : one for sections,
one for peripheral nerve-fibres and axis-cylinders, and one for pieces.
It seems better to describe them separately in the following account
which is based on the original papers of Bielschowsky, as well as
on some personal experience I gained through a visit paid to him
when in Berlin.
BIELSCHOWSKY'S Method for Sections (Journ. Psychol. N enrol,
iii, 1904, p. 169 ; and xii, 1909, p. 135). — Pieces from central nervous
organs, fixed in 15 to 20 per cent, formalin, are washed for some
hours in running tap-water and then cut by means of a C02 freezing
microtome. The sections are collected in distilled water, thoroughly
washed therein and passed in a 2 or 3 per cent, solution of silver
nitrate where they are left for twenty-four hours in a dark place,
and at room temperature. The sections can also be passed first
into pure pyridine for twenty -four to forty -eight hours, washed in
many changes of distilled water until the pyridine has been completely
eliminated and then transferred into 2 to 3 per cent, silver nitrate
as above.
The pyridine bath is optional and has the advantage of ensuring a
sharper stain of axis-cylinders whilst neuroglia, which is more or
less coloured when the pyridine bath is dispensed with, remains
unstained. Also connective tissue and nuclei are generally very
faintly stained after the pyridine treatment. Intracellular neuro-
fibrils, however, are not always so well shown as by the direct passage
of sections into the silver nitrate solution.
Before proceeding further, one should prepare the Bielschowsky
428 NERVOUS SYSTEM— SPECIAL METHODS.
ammoniacal silver nitrate-and-oxide bath as follows : — Pour 5 c.c.
of a 20 per cent, solution of silver nitrate into a measuring cylinder
and add to it first 5 drops of a 40 per cent, solution of NaOH, and
then ammonia, drop by drop, until the brown precipitate formed
disappears ; dilute to 25 c.c. with distilled water, and filter through
paper washed with the same water.
For staining take sections one by one from the silver nitrate bath,
quickly wash them in distilled water and transfer them into the
ammoniacal silver bath. Here they remain for about ten minutes
when they become yellowish-brown and should be, once more,
quickly washed in distilled water and placed in 20 per cent, formalin
prepared with spring water. The reduction takes place immediately,
and if one works with a number of sections it is advisable to re-
transfer them into a fresh bath of 20 per cent, formalin.
At the end of half an hour and even less, the reduction can be
considered as accomplished and sections can be washed in distilled
water and toned with a diluted (0-2 per cent.) solution of gold
chloride. This may be slightly acidified with acetic acid if one
wishes to obtain a faintly purple background, or neutralised with a
few drops of a diluted solution of sodium or lithium carbonate if one
prefers greyish- white backgrounds. Instead of gold chloride one
can use a slightly acid solution of chloroplatinic acid. After toning
there remains only the washing once more of sections in distilled
water, and the passing of them for a few minutes in a 5 per cent,
solution of sodium hyposulphite, or any diluted fixing bath for
photographic plates. Wash again, dehydrate in alcohols of increasing
strength up to 95 per cent., clear in carbol-xylol, and mount in balsam.
For other details about the toning and fixing of sections see the
original papers of BIELSCHOWSKT (op. cit. and Journ. Psychol. Neurol.,
iv, 1904—5, p. 227), as well as WOLFF (Biol. CentrU., xxv, 1905, p. 683),
and DA FANO (Proc. Physiol. Soc. Journ. Physiol., liii, 1920).
Bielschowsky states that this method is also suitable for sections
of celloidin or paraffin blocks of formol material, but he does not
recommend the practice, and I have no experience of it.
BIELSCHOWSKY'S Method for Peripheral Nerve-fibres (Journ.
Psychol. Neurol., iv, 1904—5, p. 227). — This method can be applied
to the study of spinal and sympathetic ganglia, peripheral nerve-
endings, and end-organs in normal conditions, but its chief applica-
tions belong to the domain of histopathology. According to my
experience good results are rarely obtained, and the method requires
important modifications to become as useful as the above and
following ones.
CHAPTER XXXII. 429
The staining is carried out on sections of formol material in the
same way as described above. There is only this difference that the
staining in the ammoniacal silver bath is carried on a few minutes
longer, viz., until the sections have taken a decidedly brown colour,
after which they are washed in 10 c.c. of distilled water acidified
with 5 drops of acetic acid, when they acquire (sometimes in a few
seconds) a yellowish tinge. They should then be immediately
transferred into the usual 20 per cent, solution of formalin. For the
toning a neutral gold bath is necessary : sections should be»left
therein until red- violet. In the finished preparations axis-cylinders
are black, myelin red- violet, connective tissue violet or blue-violet.
The washing in acidified water and the prolonged toning both answer
for the purpose of creating a sharp contrast between nerve fibres and
connective tissue fibres, which might otherwise become stained
almost as black as the axis-cylinders.
Bielschowsky has also a method for central nerve-fibres. Sections
made by freezing from formol material are placed for twenty-four
hours or longer in a 4 per cent, solution of copper sulphate or
Weigert's mordant for neuroglia stain (§ 910). After washing they
are placed for a few seconds in the usual ammoniacal silver bath and
then washed, reduced, toned and fixed as above. The preparations
are similar to those obtainable by the methods of Fajerstajn,
Strahiiber and Kaplan.
BIELSCHOWSKY'S Method for Pieces (op. cit.). — Good for peripheral
nerve-endings and embryonic material, and also for small specimens
of adult subjects. This method has been described by Bielschowsky
in various ways, probably because of the difficulty of giving fixed
rules in a case in which the greatest freedom had to be left to histo-
legists to adapt the method to the quality of their material and the
purpose of their investigations. In what follows two forms of the
method are described : one without and one with pyridine treatment
of pieces.
A. Method for Pieces without Pyridine Treatment. — Thin slices or
small pieces of formol material are washed for some hours, first in
running tap-water and afterwards in distilled water. They are then
placed in a 2 per cent, solution of silver nitrate for from one to eight
days in the dark. The use of an incubator at 35° to 37° C. is optional.
After a wash in several changes of distilled water (to be prolonged
for some minutes up to some hours according to the length of time
during which pieces have been kept in the silver bath, and if in an
incubator or not) they are transferred into an ammoniacal solution
of silver nitrate prepared as in the method for sections, but diluted
430 NERVOUS SYSTEM— SPECIAL METHODS.
up to 100 c.c. They are kept therein for from an hour up to six,
washed once more in distilled water, passed for twelve to twenty-four
hours into the usual 20 per cent, solution of formalin. Wash,
dehydrate quickly, embed, preferably in paraffin, tone sections as
described above, counterstain, if necessary, mount in balsam.
B. Method for Pieces with Pyridine Treatment. — Pieces of formol
material, up to 1 cm. thick for adult tissue, and up to 5 cm. long for
embryos, is put for two, three or four days into pure pyridine,
washed for some hours in several changes of distilled water and put
for three to five days into 3 per cent, silver nitrate at 36° C. Wash
in distilled water and transfer into the diluted ammoniacal silver
bath as above, but leaving pieces therein for twenty-four hours.
Wash for about two hours in several changes of distilled water,
reduce in 20 per cent, formalin. The rest as above.
841. Modifications of BIELSCHOWSKY'S Methods. — FAVORSKY
(Journ. Psychol. Neural., vi, 1906, p. 260) uses 10 per cent, silver
nitrate for the first silver bath instead of 2 or 3 per cent.
PATON (Mitth. Zool Stot. Neapel, xviii, 1907, p. 576) fixes fish
embryos in 4 per cent, formaldehyde neutralised with carbonate of
magnesia. For the first silver bath he uses 0-75 to 1 per cent, silver
nitrate and keeps material therein four days in summer, five to seven
in cooler weather. To make the ammoniacal silver nitrate-and-
oxide bath he takes 20 c.c. of 0 -75 to 1 per cent, silver nitrate, adds
to it 4 drops of 40 per cent, caustic soda and then ammonia drop by
drop in the usual way. The embryos are first washed in distilled water,
then kept for five to fifteen minutes in 10 c.c. of water acidified with
5 drops of acetic acid, washed once more in pure water, and trans-
ferred for twelve hours into a reducing fluid consisting of 1 per cent,
hydroquinone 20 c.c., neutralised formalin, 2 c.c. After embedding
in paraffin, the sections are toned as usual and counterstained with
1 per cent, eosin in absolute alcohol.
SCHUTZ (N enrol. Centrbl, xxvii, 1908, p. 909) finds that the times
given by Bielschowsky are too short and washes sections for twenty-
four hours after the 2 per cent, silver nitrate bath, leaves them thirty
to forty minutes in the ammoniacal silver bath, and twenty-four
hours in the 20 per cent, formalin. For toning he puts them for ten
minutes into 10 c.c. of water with 2 drops of acetic acid, then for
thirty to forty-five minutes into 10 c.c. of water with 3 drops of a
1 per cent, gold chloride solution (until blackish-grey).
BOEKE (Anat. Am., xxxv, 1910, p. 193) has obtained excellent
results by the use of Bielschowsky's method for pieces when applied
CHAPTER XXXII. 431
to the study of peripheral nerve endings. He fixes in 10 per cent,
formalin prepared with 60 per cent, alcohol, changes the fluid two
or three times, and then either leaves material therein until wanted
or keeps it in 70 to 80 per cent, alcohol. For staining, pieces are
brought into 10 to 12 per cent, formalin, and left in it until they are
quite free from alcohol. The rest as by Bielschowsky's method
for pieces, with or without pyridin treatment. Boeke finds that the
method succeeds also after other kinds of fixation.
SCHLEMMER (Ztschf. wiss Mikr., xxvii, 1910, p. 22) makes the
ammoniacal silver nitrate-and-oxide bath by adding to any silver
nitrate solution, 40 per cent, caustic soda, drop by drop, until no more
precipitate is formed. He then washes the precipitate by repeated
decantation until the wash water no longer gives an alkaline reaction,
takes it up with the smallest possible quantity of ammonia, and filters
through glass wool. This concentrated solution keeps for many
days unaltered, and should be diluted ten times its volume before
using it.
DEL Rio-HoRTEGA (Trab. Lab. Invest. BioL, Madrid, xiv, 1916,
p. 181) has made known a similar method used in those laboratories
for preparing the ammoniacal silver nitrate bath. Forty drops of
40 per cent, caustic soda are added to 30 c.c. of 10 per cent, silver
nitrate, and the precipitate washed ten to twelve times by means of
about a litre of distilled water. Fifty cubic centimetres of water are
then added to it, and ammonia, drop by drop, until the precipitate
is dissolved. The solution, brought finally to 150 c.c. and filtered
into a dark brown bottle, keeps well for many months. I find that
the ammoniacal silver bath thus prepared can be further diluted
with one, two, up to five times its volume of water, and usefully
employed for Bielschowsky's method for pieces, particularly for the
study of peripheral nerve endings.
AGDUHR (Ztschr. wiss. Mikr., xxxiv, 1917, pp. 1-99), who has
exhaustively investigated almost all questions relating to the
results obtainable by Bielschowsky's method for pieces, has come
to the conclusion that material is best fixed in neutral or slightly
acid 20 per cent, formaldehyde (50 per cent, formalin). Pieces should
then be washed in distilled water for many days until the wash-water
is free from substances reducible by an ammoniacal silver nitrate
solution used as test. For the first silver bath he uses 3 per cent,
silver nitrate, and for the second a solution obtained by adding to
10 c.c. of 10 per cent, silver nitrate, first 20 drops of 25 per cent.
NaOH, then from 200 up to 600 c.c. of distilled water, and lastly
ammonia enough to dissolve the precipitate. For the reduction he
432 NERVOUS SYSTEM— SPECIAL METHODS.
uses again 20 per cent, formaldehyde. To avoid an excessive im-
pregnation of the connective tissue he also finds it useful to. wash
pieces in acidified distilled water (see the Bielschowsky method for
peripheral nerve-fibres), but he uses as much as five times the
amount suggested by Bielschowsky.
842. DA FANG'S Modifications. — An important point of this
series of modifications of Bielschowsky's method for sections is
the use of distilled water, re-distilled on potassium permanganate,
with the object of ensuring elimination of any trace of organic
matter from the ordinary distilled water and of avoiding the forma-
tion of precipitates.
Da Fano's first modification (Mod. 1) (Atti. Soc. Lomb. Sc. Med.
BioL, Milano, iii, 1914) was meant for the study of recticular tissue
of spleen, lymph glands, and other organs, and is to be carried out
as follows : — (1) Fix small pieces of fresh tissue in 10 to 20 per cent,
formalin or in Kayserling's first fluid (forty-eight hours at least), or
in Orth's fluid (twenty-four to forty-eight hours). (2) Wash pieces
in running tap-water for twenty-four to thirty hours, and then in
distilled water for another twenty-four hours. (3) Wash sections
made by the freezing method in re-distilled water (twenty-four
hours), and then place them in filtered 2 per cent, silver nitrate
(prepared with redistilled water) in a Petri dish, taking care that
they do not touch each other. Here they are kept in the dark and
at room temperature from six hours to three days. (4) Treat
sections for twenty to thirty minutes with Bielschowsky's
ammoniacal silver nitrate solution prepared with only 2 drops of
40 per cent, caustic soda and diluted with redistilled water to 40
to 70 c.c. (5) Reduce, tone, counterstain, and mount as by Biel-
schowsky's method for sections.
Mod. 2 (Proc. Physiol. Soc. Journ. PhysioL, Hi, 1919) consists in
an application to nervous tissues of Mod. 1. The use of redistilled
water and the mode of preparing the ammoniacal silver bath are
the same, but Da Fano lays stress on the following points : —
(1) Nervous tissue must be fixed in 10 up to 20 per cent, formalin
for at least three weeks, better still for two months. Attempts to
obtain a rapid fixation with 10 to 20 per cent, formalin at 37° C.
gave bad results. (2) Sections of nervous tissues may be placed,
after washing in redistilled water, in anhydrous pyridine (six to
twelve hours), then repeatedly washed and left overnight in redis-
tilled water, to get rid of all pyridine. This treatment appears
to render neurofibrils a little thinner and, consequently, a little
CHAPTER XXXIL 433
sharper, but increases the length and cost of the method, and may
cause precipitates to form, especially where much myelin is present.
(3) It is possible to keep sections, which cannot be stained imme-
diately, for some days or even a fortnight, in redistilled water
to which a few drops of formalin have been added. Thorough
washing with redistilled water is then imperative before they are
transferred into the 2 per cent, silver nitrate solution. (4) Sections
of nervous tissues must not remain in the 2 per cent, silver
nitrate more than forty-eight hours, or precipitates may form.
The longer their stay there, the longer must be the washing
before staining ; this, however, must not, as a rule, exceed five
minutes. (5) The volume to which the ammoniacal silver nitrate
is diluted should be 35 to 45 c.c., and the sections remain in it fifteen
to twenty minutes. The subsequent washing before transferring
the sections into 20 per cent, formalin should not occupy more than
ten to fifteen seconds, and their stay in the final formalin solution
(especially for cerebral cortex) should not exceed two to three hours.
The other eight Da Fano modifications (Proc. Physiol. Soc.,
Journ. Physiol., liii, 1919-20) were all proposed for the study of
cortex cerebelli, and are characterised by a special treatment of
the sections (cut by the freezing method) with various reagents
before transferring them into the 2 per cent, silver nitrate solution,
nothing having been changed, however, in regard to the long fixation
of material in formalin and -the use of redistilled water. They may
be summarised as follows : —
Mod. 3. Place sections, after washing in redistilled water, in
2 to 3 per cent, silver nitrate at 36° to 37° C. for about twenty-four
hours ; wash quickly ; stain in ammoniacal silver nitrate solution
diluted to 40 c.c. for thirty minutes. Wash, reduce, tone, and
mount as usual.
Mod. 4. Place sections in 50 per cent, pyridine for six to eighteen
hours ; wash in redistilled water for twenty-four to forty-eight
hours ; 2 per cent, silver nitrate at 37° C. for twenty-four hours, etc.,
as in Mod. 3.
Mod. 5. Place sections in pure pyridine for four to twelve hours.
Wash in redistilled water overnight. Transfer sections into 20 per
cent, formalin prepared with redistilled water for about twenty-four
hours. Wash again in redistilled water overnight ; 2 per cent, silver
nitrate at 37° C., etc., as before.
Mod. 6. Sections are treated first with 20 per cent, formalin,
and then with pure pyridine, in the reverse order of Mod. 5.
Mods. 7 and 8. The same as Mods. 5 and 6, but replacing the
M. 28
434 NERVOUS SYSTEM-SPECIAL METHODS.
pyridine with a mixture of 3 parts of methyl-alcohol and 2 parts of
water.
Mod. 9. Place sections in a mixture of equal parts of 20 per
cent, formalin and methyl alcohol for twenty-four hours ; wash in
redistilled water for six to twenty-four hours; 2 per cent, silver
nitrate at 37° C. for twenty-four hours, etc., as before.
Mod. 10. Place sections into 20 per cent, formalin for twenty-
four hours, transfer them, without washing, into a mixture of equal
spart of 20 per cent, formalin and methyl alcohol, etc., as in Mod. 9.
Mod. 3 is particularly suitable for human material of young
individuals : Mod. 4 for adult subjects. Mods. 5 and 6 are useful
for the study of neurofibrils in the various elements of the cortex
cerebelli and for the staining of the granules. Mods. 7, 8 and 9 are
to be preferred for the demonstration of pericellular baskets and
nervous processes. Mod. 10 gives very complete stainings, and is
the most certain of all ; preparations are, however, fairly dark and,
therefore, more suitable for general view.
PERDRAU (Journ. Pathol. Bact., xxiv, 1921) has worked out a
modification which appears to be particularly suitable for the study
of the connective tissue in nervous organs. He washes pieces and
sections as in Da Fano's modifications, but without having recourse
to redistilled water. He then places sections for about ten minutes
in 0 -25 per cent, potassium permanganate, washes, and treats them
as by Pal's modification of Weigert's myelin stain (see § 857). After
another wash in distilled water, he transfers sections into 2 per cent,
silver nitrate, and continues as in Da Fano's Mod. 1. Nerve cells,
nerve fibres, neuroglia, etc., unstained ; connective tissue and
elastic fibres stained in various shades of purple-grey to black.
843. Neurofibrils ; Other Methods. — Cox's Method for fibrils of
spinal ganglion cells ; see Ztschr. wiss. Mikr. xiii, 1896, p. 498, and Anat.
Hefte, x, 1898, p. 98.
S. MEYER'S Berlin blue, see Anat. Anz., xx, 1902, p. 535.
LUGARO'S collargol (colloidal silver) method, see Monit. Zool. Ital.,
xv, .1904, p. 353.
JORIS' colloidal gold method has not been received with favour ; see
Bull. E. Aead. Med. Belg., xviii (S. iv), 1904, p. 293.
SAND (C.R. Ass. Anat. Bruxelles, 1910 ; Bibliogr. Anat. Supp.,
1910, p. 128, or Ztschr. wiss. Mikr., xxviii, 1911, p. 500) gives the
following as entirely certain for man, dog, cat, and rabbit. Speci-
mens of not more than 5 mm. in thickness are fixed for forty-eight
hours in a freshly prepared mixture of 90 parts of acetone and 10
of nitric acid, to be changed for fresh after half an hour, and once
CHAPTER XXXII. 435
again within twenty-four hours. Wash out for at^ least six hours in
pure acetone, changed two or three times. Make paraffin sections
and bring them through xylol and acetone into distilled water ;
silver for three days at about 37° C. in 20 per cent, solution of
silver nitrate. Put for ten minutes into a mixture (at least three
days old) of 1000 parts of water, 10 of sodium acetate, 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, ammonium sulphocyanide and 3 of 2 per cent, gold
chloride ; fix for a few seconds in 5 per cent, sodium hyposulphite.
Neurofibrils grey-violet, shown in cells, dendrites, and axons.
Terminal buds of Held also clearly shown, and nothing else
stained. One may counterstain in any way, even by Weigert's
or Benda's methods for neuroglia stain.
The methylene blue intra vitam method is important, and may be
usefully employed for the study of neurofibrils. See the processes
of Apathy, Dogiel, and Bethe in Chapter XVI.
C. Methods for the Demonstration of Golgi's Internal Apparatus.
844. Introduction. — The discovery of the " apparato reticolare
interno " was made by Golgi in 1898 by means of his rapid process
(see Chapter XXXIV). Soon afterwards he had recourse to a
mixture due to Veratti (see next paragraph), and Negri, Pensa, and
others of Golgi' s pupils found that the internal apparatus is not a
peculiarity of nerve cells. In 1902 Kopsch showed that the
apparatus can be stained by a simple immersion of nervous tissues
(spinal ganglia) into 2 per cent, osmic acid for eight to ten days.
Since then the apparatus was shown to exist in almost every kind
of cells, and new processes proposed for its demonstration in nervous
and other tissues by Sjovall, Golgi, Ramon y Cajal, Gatenby, Da
Fano.
As the methods of Kopsch and Sjovall, the Mann-Kopsch method,
and Gatenby's Mann-Kopsch-Altmann combination have already
been fully discussed in Chapter XXVI, and particularly described
in §§ 692 — 694 and 696, there remain to be described here only those
methods which are particularly suitable for the study of the internal
apparatus in nervous tissues, with exception of Golgi's rapid process,
for which see Chapter XXXIV, § 882.
845. GOLGI- VERATTI'S Method (see GOLGI, Anat. Anz. Verh. Anat,
Ges., xiv, 1900, p. 174). — Small pieces are hardened for a time
28—2
436 NERVOUS SYSTEM— SPECIAL METHODS.
varying from a few hours to ten days or longer in Veratti's mixture,
consisting of—
5 per cent, potassium bichromate . . 30 parts.
0-1 per cent, chloroplatinic acid . 30 „
1 per cent, osmic acid . . . 15 to 30 „
From time to time pieces are put in one or other of Golgi's
rejuvenating fluids (as described in § 888), and thence into 0 -8 to 1 per
cent, silver nitrate. Sections are cut and mounted as by Golgi's
bichromate end nitrate of silver method (see § 882).
846. GOLGI'S Arsenious Acid and Silver Nitrate Method (Arch.
Ital. Bid., xlix, 1908, p. 272). — Small pieces of quite fresh tissues
are fixed for three, six, eight or twelve hours in equal parts of 20 per
cent, formalin, saturated solution of arsenious acid, and 96 per cent,
alcohol. After a quick wash with distilled water, they are passed
for some hours (or days) into 1 per cent, silver nitrate, and then
treated with a reducing fluid, usually Cajal's hydroquinone
mixture (hydroquinone 20 grms., sodium sulphite 5 grms., formalin
50 c.c., water 1000 c.c.). Wash quickly, dehydrate, and embed
either in celloidin or paraffin. The sections are toned with equal
parts of 1 per cent, gold chloride and a mixture consisting of water
1000 c.c., with 30 grms. each of sodium hyposulphite and ammonium
sulphocyanide, and then rapidly bleached by the following method,
due to VERATTI : — Wash the toned sections in distilled water and
transfer them for one, two or three minutes into potassium per-
manganate 0-5 grm., distilled water 1000 c.c., sulphuric acid 1 c.c. ;
wash again ; transfer into 1 per cent, oxalic acid until the yellowish
colour imparted to the sections by the potassium permanganate
has disappeared ; wash thoroughly in repeatedly changed distilled
water ; counterstain, dehydrate, and mount as usual.
847. RAMON Y CAJAL'S Uranium Nitrate and Silver Nitrate Method
(Trab. Lab. Invest. Biol, Madrid, xii, 1914, p. 127).— (1) Small
pieces of quite fresh tissues are fixed for ten to fourteen hours in a
mixture of neutralised formalin 15 c.c., distilled water 85 c.c.,
uranium nitrate 1 grm. Instead of uranium nitrate, uranium
acetate, as suggested by Del Rio-Hortega, may be sometimes used.
Should a very fine reaction be desirable, the following formula may
be employed : — Uranium nitrate 1 grm., ethyl or methyl alcohol
30 c.c., distilled water 80 c.c., neutralised formalin 15 to 20 c.c.
(2) After a quick wash in distilled water, pieces are transferred into
1 -5 per cent, silver nitrate and kept therein for thirty-six to forty-
eight hours at room temperature. If the pieces are only a few and
CHAPTER XXXII. 437
small, 1 per cent, silver nitrate will be sufficient. (3) Wash quickly and
reduce for eight to twenty-four hours in hydroquinone 1 to 2 grms.,
formalin 15 c.c., distilled water 100 c.c., sodium sulphite 0-5 grm.
(4) Wash quickly, embed in paraffin or celloidin, or make sections
by the freezing method. (5) Tone and counterstain sections if
desirable. Dehydrate and mount as usual.
Best results are obtained from vertebrates, preferably kittens
and young rabbits. The method may be applied to human
material, if available in a sufficiently fresh condition. From
invertebrates results are not so good, and rather uncertain, so that
Cajal advises a simple fixation in formalin or formalin-acetone,
followed by impregnation with silver nitrate, as by his reduced
silver methods for neurofibrils.
848. DA FANG'S Cobalt Nitrate Modification (Proc. Physiol. Soc.,
Journ. Physiol, liii, 1920 ; Journ. R. Micr. Soc., 1920, p. 157).—
Small pieces of quite fresh tissues are fixed for six to eight hours at
room temperature in cobalt nitrate 1 grm., distilled water 100 c.c.,
formalin 15 c.c. The solution can be prepared beforehand, and
keeps unaltered for months. The formalin need not be neutralised
unless strongly acid or containing free sulphuric acid, in which case
it is necessary to neutralise it by one of the usual methods. For
the fixation of embryonic organs and in all cases in which a
shrinkage of delicate tissues is to be feared, the quantity of the
formalin may be reduced to 10, 8, or 6 c.c. for every 100 c.c. of distilled
water. The time of fixation should be shortened to three to four
hours or even less in the case of very small pieces, such as spinal
ganglia of mice and rats, the pituitary body of the same animals,
etc. Pieces of spinal cord, cerebrum, cerebellum of adult animals
give better results if fixed for about eight to ten hours. The fixation
may be prolonged in special cases to twelve to twenty hours, but
should not exceed twenty-four hours. The fixation in an incubator
at a temperature varying between 25° and 37° C. has been attempted
with success in the case of tissues of adult subjects, but it leads to a
staining of both the internal apparatus and intracellular formations,
which, according to their morphology, are to be considered as
mitochondria.
For the impregnation, Da Fano quickly washes the pieces in
distilled water, makes their surfaces smooth if necessary, and then
places them into 1 -5 per cent, silver nitrate in the dark for twenty-
four to forty-eight hours at room temperature. For very small
fragments, 1 per cent, silver nitrate may be used, whilst for pieces of
438 NERVOUS SYSTEM— SPECIAL METHODS.
spinal cord of adult subjects, 2 per cent, should be preferred. For
the reduction he uses Cajal's hydroquinone-formalin mixture, taking-
care in further recutting the pieces, before transferring them into
the reducing fluid, so that their thickness should not exceed 2 mm.
He dehydrates and embeds pieces, preferably in paraffin, or he makes
sections by the freezing method. He usually tones these by means
of 0-2 per cent, gold chloride, fixes with 5 per cent, sodium hypo-
sulphite, count erstains and mounts as usual.
The method gives good results also with material from lower
vertebrates and invertebrates.*
849. Other Methods and Modifications. — BESTA (Anat. Anz..
xxxvi, 1910, p. 477) fixes for two days in 20 parts of formol with 2
of acetic aldehyde and 80 of water, washes for twenty-four hours in
distilled water changed seven or eight times, and puts for two days
in 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 animals.
SUCHANOW (Neural. Central., xxi, 1902, p. 777) has obtained good
results by the use of Golgi-Veratti mixture, keeping pieces of spinal cord
and spinal ganglia for twenty to thirty days in the mixture and for two
to three days in the rejuvenating fluid.
LEGENDRE (Anat. Anz., xxxvi, 1910, p. 209) omits the toning and
bleaching by Golgi's arsenious acid method, and embeds in paraffin.
Similarly COLLIN ET LUCIEN, Bibllogr. Anat. Supp., 1909, p. 238.
SAVAGNONE (Pathologica, i, 1909) silvers pieces fixed in Golgi's arse-
nious acid mixture with 30 c.c. of tachiol (10 per cent, silver fluoride)
in 100 of water.
CARLETON (Journ. R. Micr. Soc., 1919, p. 321) reduces pieces treated
according to Cajal's uranium nitrate method for only two hours in the
usual hydro quinone mixture.
' PENFIELD (Brain, xliii, 1920) has successfully employed Cajal's
uranium nitrate method for his experimental investigations on the
alterations of Golgi's apparatus in nerve cells of spinal cord and
spinal ganglia of young cats. He adds 20 c.c. (instead of 15) to
Cajal's fixing fluid and as much as 1-5 grms. of sodium sulphite to
the hydroquinone-formalin solution. He finds it imperative to
dehydrate pieces very quickly before embedding them in paraffin.
In order to obtain perfect fixation of the spinal cord he sometimes
performs a laminectomy in the lower lumbar region of the aneesthe-
* Da Fano's method has been used by me with great success for the
study of gametogenesis of many invertebrata (J. B. G.).
CHAPTER XXXII. 439
tised animal, passes a needle in the subarachnoid space, and allows
the fixative to flow in "under a gravity pressure of 75 cm." The
heart stops about a minute after the beginning of the injection,
which is continued for twenty hours. At the end of this time the
cord is removed, pieces cut and dropped directly into the silver bath.
For counterstaining Penfield finds it particularly useful to immerse
untoned sections into a diluted solution of Unna's polychrome-
methylene blue for one to four hours, this being followed by passage
through alcohols of increasing strength and differentiation in absolute
alcohol. By this method, also Holmgren's, trophospongium is
sometimes stained. But for the study of the relationship between
the latter and Golgi's apparatus, Penfield (in litteris) prefers to make
drawings of the apparatus from certain selected cells, subsequently
removing the coverslip and bringing the slides through graded
alcohols into 5 per cent, iron alum for twelve to twenty-four hours.
This removes all silver from the cells as well as the counterstain, and
at the same time mordants the tissues for further staining by Heiden-
hain's iron-hsematoxylin method. If the proper amount of diffe-
rentiation has been secured of the particular cells already drawn, the
trophospongium will be found stained with great detail.
Addendum.
HOLMGREN'S Method for Trophospongium.^ Fix small pieces or
ganglia in trichlorolactic acid for twenty-four hours. Dehydrate
and embed in paraffin as usual. Stain thin sections for twenty-four
hours in Weigert's resorcin fuchsin solution for elastic fibres (see
§ 758) recently prepared and a little diluted. Dehydrate and mount
as usual.
D. Methods for the Demonstration of the Sustaining Apparatus
of Medullary Sheaths, Neurokeratin, etc.
850. Methods demonstrating Funnels and Spiral Filaments.—
GOLGI (see EEZZONICO, Arch. p. 1. Sc. Med., iv, 1880, p. 78 ; GOLGI,
Opera Omnia /, p. 163) puts small pieces of spinal cord in 2 per
cent, potassium bichromate for eight to fifteen days in summer, or
a month in winter. After a quick wash he transfers them into
0 -50 to 0 -75 per cent, silver nitrate for two or three days in summer,
or eight, ten or more in winter. The pieces are then washed in
95 per cent, alcohol, dehydrated in absolute alcohol, cleared in oil
of turpentine and teased therein. The preparations, mounted in
dammar, must be exposed to sunlight for eight to ten days ; or to
diffused daylight for twenty to forty days.
440 NERVOUS SYSTEM—SPECIAL METHODS.
For peripheral nerve fibres, GOLGI (Op. Omnia I, p. 162) has
proposed two methods. Of these the first is a modification of his
rapid process (see § 882), and should be carried out as follows : —
Tracts of peripheral nerves are cut with care not to stretch them,
and put in a mixture of 10 parts of 2 per cent, potassium bichromate
and 2 of 1 per cent, osmic acid. After about one hour the tract or
tracts of nerves are sufficiently hardened to be further recut in
pieces of about \ cm. in length, which are put back in the same
mixture. After another three hours, and successively at intervals
of three hours during twenty-four hours, pieces are transferred into
0 -5 per cent, silver nitrate where they may remain for any time, but
no less than eight hours. Preparations are made and mounted as
above.
The other method is a modification of that used for central nerve
fibres, the only difference consisting in keeping the pieces in the
bichromate for a much shorter period, i.e., for from four hours to
at most two days, and in transferring specimens into the silver bath
at intervals of about three hours. After twelve to twenty-four
hours preparations can be made as described above.
The preparations made by the first method show the spiral
filaments very clearly, but do not keep well. The preparations
made by the second method do not show the spiral filaments so
completely, but are more useful for the demonstration of the funnels
and last longer.
CATTANI (Arch. Ital. Biol, vii, 1886, p. 345) either fixes in Flem-
ming's fluid and teases and mounts in glycerine, or puts pieces into
Golgi's bichromate and osmic acid mixture, dehydrates and passes
into oil of turpentine to be changed until it remains colourless. The
turpentine dissolves the myelin and leaves funnels and spiral fila-
ments visible. Cattani also has a modified Golgi method, now
superseded.
SALA (Verh. Anat. Ges. Anat. Anz., 1900, p. 176) employs the
Golgi- Veratti method for the intracellular network (see § 845).
See also concerning these methods, MONDINO, Arch. p. I. Sc. Med.,
viii, p. 45.
GALLI (Ztschr. wiss. Mikr., iii, 1886, p. 467) hardens peripheral
nerves for eighteen to twenty days into Miiller's fluid, cuts out
pieces 5 to 6 mm. long, and keeps these in Miiller's fluid diluted with
2 parts of water for another two days. He then stains for fifteen to
twenty minutes in aqueous solution of China blue, washes out in
alcohol, clears in essence of turpentine, and mounts in damar.
KAM6N Y CAJAL has successfully employed some modifications of
CHAPTER XXXII. 441
his reduced silver and uranium nitrate methods, for which see Trab.
Lab. Invest. Biol, Madrid, x, 1912, p. 221.
851. Methods demonstrating Neurokeratin Network. — PLATNER
(Ztschr. wiss. Mikr., 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 " Echtgriin''
(dinitroresorcin) in 75 per cent, alcohol. See also BEER, Jahrb.
Psychiatric, ii, 1893.
Cox (Anat. Hefte, i, 1898, p. 102, note) fixes nerves in 2 per cent,
osmic acid (rabbit) or 1 per cent, (frog), washes, dehydrates, clears
with bergamot oil, and mounts in balsam. The bergamot oil dissolves
out the myelin, and leaves the neurokeratin visible. It may be
necessary to leave the nerves for forty-eight hours in the oil.
CORNING (Anat. Anz., xvii, 1900, p. 309) studies the neurokeratin
aetwork in the sciatic of the frog by means of sections of sublimate
material strongly stained with iron hsematoxylin.
KAPLAN (Arch. Psychiatr., xxxv, 1902, p. 825) stains sections with
acid fuchsin and differentiates them by Pal's method.
GEDOELST (La Cellule, v, 1889, p. 136) has the following : (a) A
nerve is treated with liquid of Perenyi, either pure or with addition
of a trace of osmic acid, and examined in glycerin. By this treat-
ment the myelin loses its excessive refractivity and the neurokeratin
network comes out clearly, (b) Silver nitrate. Good images, but
uncertain, (c) Treatment with a mixture of 1 per cent, osmic
acid and absolute alcohol. The network comes out black.
CHAPTER XXXIII.*
MYELIN STAINS.
852. Iron Hsematoxylin. — According to A. Bolles Lee (see 1913 Ed.)
the simplest way of staining myelin is to make paraffin sections of
formol material and stain them with iron hsematoxylin exactly as
for central corpuscles (say, twelve to fourteen hours in the mordant,
six in the hsematoxylin, and a few minutes for the differentiation).
Sections best not over 15 /x. One may counters tain the cells with
carmalum, but not for more than half an hour, or the hsematoxylin
will be attacked. The stain is not so aesthetic as Weigert's, but
quite as sharp. Axis cylinders are not shown.
Similarly REGAUD (C. R. Acad. Sc., cxlviii, 1909, p. 861), but
adding a chrome mordantage either concurrently with the formol
fixation, or subsequently. Also NAGEOTTE (C. R. Soc. BioL, Ixvii,
1909, p. 542), with sections of formol material by the freezing method ;
HOUSER (Journ. Comp. N enrol., x, 1901, p. 65), and BROOKOVER
(ibid., xx, 1910, No. 2) ; SPIELMEYER (Neurol. CentrbL, xxix, 1910,
p. 348) ; and his Technik d. mikrosl. Untersuch. d. Nervensy stems,
1911, p. 87, with sections of 25 to 35 /z by the freezing method ;
LOYEZ (C. R. Soc. BioL, Ixix, 1910, p. 511), who differentiates first
lightly, till the grey matter begins to appear, in the iron alum, then
washes, and differentiates further in Weigert's borax ferricyanide ;
GILBERT (Ztsch. wiss. Mikr., xxviii, 1911, p. 279), who mordants with
iron alum, stains with molybdic acid hcematoxylin, and differentiates
with the borax ferricyanide ; STOELTZNER (ibid., xxiii, 1906, p. 329),
who mordants celloidin sections of formol material for five minutes
in Liq. ferri sesquichlorati, stains in 0 -5 per cent, haematoxylin, and
differentiates in the mordant or in borax ferricyanide ; and KODIS
(Arch. mik. Anat., lix, 1902, p. 211), who fixes for one or two days
in saturated solution of mercury cyanide, hardens in 10 per cent,
formol, and stains sections, made by the freezing method, with
Heidenhain's iron heematoxylin.
853. WEIGERT'S Methods* — There have been in all three methods
of WEIGERT :— the 1884 method, the 1885 method, and the 1891
method.
* Revised by Dr. C. Da Fano, King's College, University of London.
CHAPTER XXXIII. 443
The 1884 method (Fortschr. d. Med., ii, 1884, pp. 120, 190 ; Ztschr .
wiss. Mikr., i, 1884, pp. 290, 564), which depends on the formation of
a chrome lake of hsematoxylin, may be considered as superseded.
Not so the two others, which depend on the formation of a copper
lake in addition to the chrome lake.
854. WEIGERT'S 1885 Method (Fortschr. d. Med., in, 1885, p. 236 ;
Ztschr. wiss. Mikr., 1885, pp. 399, 484 ; Ergebn. Anat., vi, 1896
(1897), p. 10). — The tissues are hardened in potassium bichro-
mate. WEIGERT takes (Ergebn., p. 10) a 5 per cent, solution, and
if time is an object hardens in a stove. (Other bichromate mixtures
will do, e.<7.,Miiller's, Kultschitzky'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
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 recently (Ergebn., p. 14) he added to the bichromate solution
2 per cent, of chrome alum or of chromium fluoride, which hastens
the hardening, so that small specimens become brown and ripe in
four to five days, without stoving.
After hardening, tissues are generally embedded in celloidin and
the blocks hardened in the usual way. They are then put for one or
two days, in an incubating stove, into a saturated solution of neutral
copper acetate diluted with 1 volume of water. By this treatment
the tissues become green and the celloidin bluish-green. They may
then be kept, till wanted for sectioning, in 80 per cent, alcohol.
Sections are made, well washed in water, and brought into a
stain composed of : —
Haamatoxylin . . . . . 0 -75 to 1 part.
Alcohol . . . . . .10 parts.
Water 90 „
Saturated solution of lithium carbonate . 1 part.
They remain there, for spinal cord, two hours ; for medullary
layers of brain, two hours ; for 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 . . . 2-5 „
Water . . . . . 200-0 „
They remain there until complete differentiation (half an hour to
444 MYELIN STAINS.
several hours), and are then well washed with water (running, or
changed several times), dehydrated, and mounted in balsam. They
may be previously counterstained, if desired, with alum-carmine.
The method is applicable to the study of peripheral nerves as
well as to nerve centres, and also the study of lymphatic glands,
skin (see SCHIEFFERDECKER, 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
FLESCH (Ztschr. wiss. Mikr., iii, 1886, p. 50) prefers (following LICH-
THEIM) to make the sections first, and to mordant them separately.
• VASSALE (Rw. sperim. Frematr.,xv, 1889, p. 102) first stains the
sections in 1 per cent. ha3matoxylin for three to five minutes,
then puts them for three to five minutes into saturated solution
of copper acetate, and differentiates as Weigert.
855. WEIGERT'S 1891 Method (Deutsche med. Wochenschr., xvii,
1891, p. 1184).— The material is hardened in bichromate and
embedded in celloidin (see last §). It is then (according to the latest
form of the process (Enzyd. mik. Technik., 1903, p. 942) ), put for
twenty-four hours in a stove into a solution of 2 \ parts of chromium
fluoride, 5 of copper acetate, and 5 of acetic acid in 100 of water.*
Sections are then made and stained for from four to twenty-
four hours at room temperature in a freshly prepared mixture
of 9 volumes of (A), a mixture of 7 c.c. of saturated aqueous solution
of lithium carbonate with 93 c.c. of water, and 1 volume of (B), a
solution of 1 grm. of haematoxylin 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 25 //. They are then washed in
several changes of water, and treated with 90 per cent, alcohol,
followed by carbol-xylol, 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
differentiation, did not keep well, and WEIGERT (Ergebn. Anat., iii,
1894, p. 21) reverted to the practice of differentiating with the
borax-ferricyanide mixture.
* Instead of the chromium fluoride one may use chrome alum, as
Weigert did at one time, and as some still do. But then one must boil,
as directed for Weigert's Neuroglia stain.
CHAPTER XXXIII. 445
Later still (Enzyd. mik. Technik., 1903, p. 942) he employed a
stain composed of equal parts of (A), a mixture of 4 c.c. of the
officinal Liquor ferri sesquichlorati P. G. with 96 of water, and (B), a
mixture of 10 c.c. of a 10 per cent, alcoholic solution of hsematoxylin
with 90 of 96 per cent, alcohol. The two (A and B) must be mixed
immediately before use, and the sections should remain in the stain
overnight or longer, then rinsed and differentiated as usual. This
has the advantage of demonstrating very fine fibres, and of giving a
colourless back ground.
For difficult objects the differentiating liquid may be diluted with
water, and gives better results than dilute acetic or hydrochloric
acids or the like, which were formerly recommended.
By means of Weigert's methods only the myelin sheaths of
normal nerve-fibres are stained, whilst those of degenerated tracts
are of a paler colour and, if the degeneration is sufficiently old,
they may even be stainless. See also § 870.
856. Formol Material (Ergebn. Anat., vi, 1896, p. 14) may be
employed if mordanted till brown (four or five days) in 5 per cent,
solution of potassium bichromate with 2 per cent, of chromium
fluoride.
I understand from Dr. Perdrau that this method is the most
satisfactory of all for routine work, and relatively small pieces ; but
particularly for the histopathological investigation of parts of the
human spinal cord, medulla oblongata, pons, and midbrain. He
generally cuts from material fixed in formalin, for no less than ten
days, slices i to J cm. thick, and places them direct in the mordant
(potassium bichromate 5 grms., chromium fluoride, 2-5 grms., water,
100 c.c.) for five to six days. Eelatively large pieces may be left in
the mordant four or five days longer. After a thorough wash in
running tap water, he dehydrates and embeds in celloidin. The
sections are stained overnight in Kultschitzky's hsematoxylin (§ 859)
several months old, washed in water, and placed in a bowl of distilled
water to which about 2 c.c. of a saturated solution of lithium
carbonate have been added. They are stirred about several times
and transferred into a fresh bath of the same solution if necessary,
until the celloidin is all but colourless. He lastly differentiates, as
by Pal's method (§ 857), washes, and counterstains either in alum
carmine for ordinary work or in an alcoholic solution of eosin if
the preparations are to be photographed.
P. MEYER (Neurol Centrbl, xxviii, 1909, p. 353) embeds formalin
material in celloidin and cuts before putting into Weigert's copper fluid.
446 MYELIN STAINS.
For Sheldon's modification, which is also based on a formalin fixation,
see Folia, Neurobiol, viii, 1914, p. 1.
Modifications of Weigert's Method.
857. PAL'S Method (Wien. med. Jahrb., N.F. i, 1886, p. 619;
Ztschr. wiss. Mikr., iv, 1887, p. 92 ; Med. Jahrb., N.F. ii, 1887,
p. 589). — One proceeds as in WEIGERT'S process, but omitting the
copper bath. After staining in the hsematoxylin 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
potassium permanganate, rinsed in water, and brought into a
decolorising solution composed of : —
Oxalic acid . . . . . . 1-0 grm.
Potassium sulphite (S03K2) . . . 1-0 „
Dist. water 200-0 c.c.
In a few seconds the grey substance of the sections is decolorised,
the white matter remaining blue. If the differentiation is not
complete the whole process can be repeated a second time, and so on.
The sections should now be well washed out, and may be counter-
stained with Magdala red or eosin, or (better) with picrocarmine or
acetic acid carmine.
Pal's process gives brilliant results, the ground of the preparations
being totally colourless. WEIGERT (Ergebn. Anat., vi, 1896, p. 21)
considered it superior to his own for thick sections, but not so safe
for very fine fibres.
MARCUS stains by the Pal method sections of material hardened in
formalin.
GUDDEN (N enrol. Centrbl., xvi, 1897, p. 24) makes celloidin sections of
material hardened in 5 to 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 hsematoxylin a few drops of dilute nitric acid (MINNICH).
TSCHERNYSCHEW and KARUSIN (Ztschr. wiss. Mikr., xiii, 1896, p. 354)
stain for twenty-four hours in KULTSCHITZKT'S haematoxylin.
PAVLOW (ibid., xxi, 1904, p. 14) uses the permanganate twice as
strong as Pal.
KOZOWSKY (N enrol. Centrbl., xxiii, 1904, p. 1041) stains as Weigert,
and differentiates the sections first with 1 per cent, permanganate, till
the grey matter comes out brown, and finishes the differentiation
with Liq. ferri sesquichlorati.
POTTER (Ztschr. wiss. Mikr., xxvii, 1910, p. 238) stains as Weigert,
last §, and differentiates first in 0-25 per cent, permanganate, then in
borax ferricyanide.
CHAPTER XXXIII. 447
858. KAISER (N enrol CentrU., xii, 1893, pp. 364) hardens first in
Miiller's fluid, then for eight days in Marchi's fluid (§ 870), mordants
sections for five minutes with sesquichloride of iron (1 part to one
of water and 3 of 70 per cent, alcohol), stains and differentiates with
Pal's liquid. For 'details see early editions.
BOLTON (Journ. Anat. & Phys., xxxii, 1898, p. 247) 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 KULTSCHITZKY'S hsematoxylin (next §), and differentiates by
Pal's process.
Similarly WYNN, ibid., xxxiv, 1900, p. 381.
LASLETT (Lancet, 1898, p. 321) mordants in Marchi's fluid (1 week),
makes sections, stains by KULTSCHITZKY'S method, and differentiates
by PAL'S.
859. KULTSCHITZKY'S Method (4wa£. ,4*13., iv, 1889, p. 223; andv,
1890, p. 519). — Specimens are hardened for one or two months in
ERLICKI'S fluid, imbedded in celloidin or photoxylin, and cut.
Sections are stained for from one to three hours, or as much as
twenty-four, in a stain made by adding 1 grm. of heematoxylin
dissolved in a little alcohol to 100 c.c. of 2 per cent, acetic acid.
They are washed out in saturated solution of lithium or sodium
carbonate. Differentiation is not necessary, but by adding to the
lithium carbonate 'solution 10 per cent, of a 1 per cent, solution of
potassium red prussiate, 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.
WOLTERS (Ztschr. wiss. Mikr., vii, 1890, p. 466) proceeds as
Kultschitzky, except that he stains at 45° C. for twenty-four hours,
after which the sections are dipped in Miiller's fluid, and
differentiated by Pal's method.
Similarly KAES (N enrol. CentrbL, x, 1891, p. 456). Myelin dark
blue, cells yellow-brown.
860. MITROPHANOW (Ztschr. wiss. Mikr., xiii, 1896, p. 470) mordants
photoxylin sections for at least twenty -four hours at 40° C. in a mixture
of equal parts of saturated aqueous solution of copper acetate and 90
per cent, alcohol, stains for ten minutes in KULTSCHITZKY'S hsematoxy-
lin, and differentiates with Weigert's ferricyanide fluid.
861. BERKLEY'S Rapid Method (Neurol. Centrbl.,xi, 1892, p. 270).
— Slices of tissue of not more than 2J mm. in thickness are
hardened for twenty-four to thirty hours in FLEMMING'S fluid, 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 copper acetate (or simply
warmed therein to 35° to 40° C. for half an hour). They are then
448 MYELIN STAINS.
washed, and stained for fifteen to twenty minutes in a lithium
carbonate hsematoxylin 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.
HILL (Brain, xix, 1896, p. 1 ; Phil Trans., 184, B, 1894,
p. 399) stains well-washed Miiller material in bulk in alum carmine,
cuts and mordants sections for twenty-four hours in half-saturated
solution of copper acetate, stains and differentiates as Weigert,
taking the differentiating fluid only half as strong.
863. BENDA'S Rapid Method (Berlin klin. Wochenschr., xl, 1903,
p. 748). Sections of formal material by the freezing process (alcohol being
avoided) are stained (without any mordanting) for twenty-four hours in
Boehmer's hsematoxylin, differentiated with Weigert's ferricyanide, and
mounted in balsam. Only recommended for peripheral nerves, or for
preliminary examination of the central nervous system.
Similarly, NAGEOTTE, C.R. Soc. Biol., ii, 1908, p. 408, staining with
hsemalum.
Similarly the Enzycl. mile. Technik., 1910, ii, p. 239, with fresh material
cut by the freezing process, and the sections mounted in laevulose (as
alcohol somewhat extracts the stain).
864. STREETER (Arch. mik. Anat., Ixii, 1903, p. 734) stains small
nerve-centres in bulk (after mordanting in Weigert's bichromate and
fluoride mixture, § 845) with Weigert's haematoxylin (four to six
days), washes for a couple of days in 70 per cent, alcohol, makes
paraffin sections, and differentiates them by the method of Weigert
or Pal.
865. BESTA'S Ammonio-Chloride of Tin Methods (Riv. Sperim.
Freniatr., xxxi, 1905, p. 569). — Pieces of peripheral nerves are fixed
for one to three days in 100 c.c. of water with 25 of formol, and 4 grms.
of Merk's ammonio-chloride of tin, and then dehydrated and em-
bedded as usual. The sections may be stained in different ways :
(a) For twenty-four hours in Mallory's phosphomolybdic-carbolic-
acid hsematoxylin with subsequent differentiation in Lugol's solu-
tion ; (b) for thirty to sixty minutes in a very diluted solution of
Delafield's hsematoxylin and then for a minute in Held's acetic
solution of erythrosin ; (c) for five to ten minutes in erythrosin,
and then for two hours in a mixture of equal parts of 1 per cent,
hsematoxylin and 4 per cent, ammonium molybdate with 3 drops of
acetic acid to every 50 c.c. of the mixture.
866. Gallein. — ARONSON (Centrbl. med. Wise., xxviii, 1890, p. 577) stains
sections of material, hardened in liquid of Erlicki or Miiller and mor-
CHAPTER XXXIII. 449
danted with copper acetate, for twelve to twenty-four hours in a solution
of 3 to 4 c.c. of Gallein in 100 c.c. of water with 20 of alcohol and three
drops of a concentrated solution of sodium carbonate. Sections are
differentiated by the method of Weigert, or Pal. Nerve-fibres red. A
second stain with methylene blue may follow (best after differentiating
with potassium permanganate). Similarly SCHROTTER (Centrabl. allg.
Path., xiii, 1902, p. 299).
867. SCHROTTER (Neurol. Gentrbl., xxi, 1902, p. 338) also stains
sections for two to three hours in a 5 per cent, solution of sodium
sulphalizarinate, to which a few drops of 5 per cent, oxalic acid (enough
to give an orange tint) are added, then differentiates until no more
colour comes away in sodium carbonate solution of ^^ strength, and
mounts in balsam. Myelin red, on a colourless ground.
868. Toluidine Blue and Methylene Blue. — HARRIS (Philadelphia Med.
Journ., i, 1898, p. 897) stains sections (of material hardened as for
Weigert's stain) for several hours in a 1 per cent, solution of toluidine
blue in 1 per cent, borax solution, and differentiates in saturated aqueous
solution of tannic acid. Similarly, but with methylene blue, in a com-
plicated way FRAENKEL (Neurol. Oentrbl., xxii, 1903, p. 766).
BING and ELLERMANN (Arch. Anat. Phys., Phys. Abth., 1901, p. 260)
harden in 9 parts of acetone to 1 of formol, cut without imbedding,
stain for five to ten minutes in saturated methylene blue solution, and
put for one or two into saturated solution of picric acid.
869. Other Modifications or Similar Methods. — FLECHSIG, Arch. Anat.
Phys., Phys. Abth., 1889, p. 537 ; BREGLIA, Ztschr. wiss. MiJcr., vii,
1890, p. 236; Rossi, ibid., vi, 1889, p. 182; MERCIER, ibid., vii, 1891,
p. 480 ; HAUG, ibid., p. 153 ; WALSEM, ibid., xi, 1894, p. 236 ; ROBERT-
SON, Brit. Med. Journ., 1897 (1), p. 651.
STRONG (Journ. Comp. Neur., xiii, 1903, p. 291) finds copper bichro-
mate (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 0-25 per cent, osmic acid and differentiates as Pal.
K. KOCH (Berl. Klin. Wochenschr., li, 1914, p. 422) makes sections by
the freezing method of formalin material imbedded in gelatine, and
after staining with Weigert's iron haematoxylin, differentiates by Pal's
method, and mounts in glycerin jelly.
870. MARCHI'S Method (for Degenerate Nerves) (Riv. sperim.
Fren., xii, 1886, p. 50). — Small pieces of nervous tissue are hardened
for a week in Mailer's solution, and then put for a few days into a
mixture of 2 parts of Miiller's solution and 1 part of 1 per cent,
osmic acid. Sections are cut, best without imbedding, and mounted
in balsam. The myelin sheaths of normal nerve fibres take a
yellowish-brown colour, those of degenerated fibres a black one.
This process, therefore, gives positive images of the degenerated
elements, Weigert's process only giving negative ones.
For a critical review of this method and its modifications, see
M. 29
450 MYELIN STAINS.
WEIGERT (Ergebn. Anat., vii, 1897 (1898), pp. 1 — 8) ; MATUSZEWSKI
(Arch. path. Anat., clxxix, 1905, p. 12) ; DE LANGE (Le Nevraxe,
x, 1908, p. 83) ; and LEWY (Fol Neurobiol, ii, 1909, p. 471).
FINOTTI (Virchow's Arch., cxliii, 1896, p. 133) makes sections of
material that has been in Miiller's fluid 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 1 or 2 parts of 1 per cent, osmic
acid, and 1 part of a concentrated solution of picric acid in one-third
alcohol. For peripheral nerves ; myelin (normal), black.
ORR (Journ. Path, and Bact., vi, 1900, p. 387) treats small pieces
of fresh tissue with a mixture of 8 c.c. of 2 per cent, osmic acid, and
2 c.c. of 1 per cent, acetic acid, which increases the penetration.
Should the mixture be darkened at the end of twenty-four hours,
then it ought to be renewed. After forty-eight hours, the pieces are
placed in 10 per cent, formalin for three days to complete reduction
and hardening.
VASSALE (Arch. Ital. Biol., xxvii, 1897, p. 131) takes 75 c.c. of
Miiller's solution, 25 c.c. of 1 per cent, osmic acid, and 20 drops of
nitric acid.
NISSL (Encycl. mik. TechniL, ii, p. 248), holding that alcohol
attacks the myelin, cuts without imbedding, and hurries sections
through alcohol and bergamot oil into balsam.
EAMON T CAJAL (Trab. lab. Biol. Madrid, ii, 1903, p. 93) has a com-
plicated method of treating Marchi material.
BUSCH (Neurol. Centralb., xvii, 1898, p. 476) puts formol material
for five to seven days into a solution of 1 part osmic acid, 3 of iodate
of sodium, and 300 of water. Same stain as Marchi's, but more
penetrating and sharper.
See also VENDEROVIC (Anat. Anz., xxxix, 1911, p. 414) who cuts
slices of formol material 0 -5 cm. thick, and treats these, with Marchi's
fluid, thus getting increased depth of reaction.
STEENSLAND (Anat. Rec., viii, 1914, p. 123) recommends clearing
sections of Marchi material with oleum origani cretici, and mounting
in chloroform-balsam.
Osmic Acid (EXNER, Sitzb. Akad. Wiss. Wien, Ixxxiii, 1881, Abth. 3,
p. 151 ; BEVAN LEWIS, The Human Brain, 1882, 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 with caustic ammonia (20 drops to 50 c.c. of
water), which clears up the general mass of the brain substance, leaving
the medullated fibres black. The preparations are not permanent, unless
(KANVIER, Traite, 1 ed., p. 1086) they are fixed for a quarter of an
hour in osmic acid vapour.
CHAPTEE^XXXIIl. 461
871. AZOULAY'S Osmic Acid Methods (Anal. Anz., x, 1894, p. 25). —
(A) Sections of Miiller material are put for five to fifteen minutes
into a solution of 1 : 500 or 1 to 1,000 of oxmic acid, rinsed with water,
and put for two to five minutes into a 5 or 10 per cent, solution of
tannin, warming them therein over a flame till vapour arises, or in
a stove at 50° to 55° C. Wash for five minutes in water, counter-
stain with carmine or eosin, and mount in balsam. If the sections
are too thick it will be necessary to differentiate by Pal's process,
or with eau de Javelle diluted with 50 volumes of water. (B) Material
that has been in an osmic mixture (fluids of Flemming, or Marchi,
or Grolgi). Sections as before, then tannin bath, warming for three
to ten minutes, the rest as before.
872. HELLER AND GUMPERTZ (Ztschr. wiss. Mikr., xii, 1895, p. 385)
give for peripheral nerves, and HELLER (ibid., xv, 1898, p. 495)
for central nervous system, the following method. Sections of
Miiller material are put into 1 per cent, osmic acid (twenty-four hours
at 37° C. for peripheral nerves ; ten to thirty minutes, at room
temperature, for central nerve fibres). They are treated with pyro-
gallic acid (a photographic developer will do) till the nerve "fibres
are black, then with a violet-coloured solution of potassium per-
manganate till the sections become brown, then with 2 per cent,
oxalic acid till they become yellow-green. Wash out well between
each operation.
Similarly, TELJATNIK (Neurol. CentrU., xvi, 1897, p. 521) ; KoBERf SON
(Brit. Med. Journ., 1897 (1), p. 651), the material being previously mor-
danted with Weigert's chrome alum- copper fluid for neuroglia ; and
OUR, Journ. Path, and Bact., vi, 1900, p. 387. See also ROSSOLIMO and
BUSCH, Ztschr. wiss. Mikr.,xiv, 1897, p. 55.
WITTMAACK (Arch. Ohrenheilk., Ixi, 1904, p. 18) mordants till
green (temporal bones) in 90 parts of Miiller's fluid 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
2 per cent, osmic acid, and reduces in 5 per cent, pyrogallol.
873. Iron. — ALLERHAND (Neurol. CentrU., xvi, 1897, p. 727) puts
sections of Miiller material for fifteen minutes into warm 50 per cent,
solution of Liquor ferri sesquichlorati, then for an hour or two into 20 per
cent, tannin solution (old and brown). They are then differentiated by
PAL'S method, using, however, the liquids twice as strong.
An iron-alum process is described by STRONG in Journ. Comp. Neurol. ,
xiii, 1903, p. 291.
874. Silver Nitrate.— VESTARINI-CRESI (Att. Accad. Med. Chir.
NapoU, 1, 1896) hardens in formol, cuts thick sections, washes them with
29—2
452 M YE LIN STAINS.
40 per cent, alcohol, puts them in the dark into 1 per cent, solution of
silver nitrate in 40 to 70 per cent, alcohol, then washes thoroughly.
Similarly, MOSSE (Arch. mik. Anat., lix, 1902, p. 401), impregnating
bichromic material with 1 per cent, solution of argentamin, and reducing
in 10 per cent, pyrogallic acid, and differentiating by the method of PAL.
Myelin - and - axis - cylinder Stains .
875. Methylene Blue. — SAHLI (Ztecftr. wiss. Mikr.,ii, 1885, p. 1) stains
sections of tissue hardened in bichromate for several hours, in con-
centrated aqueous solution of methylene blue, rinses with water, and
stains for five minutes in saturated aqueous solution of acid fuchsin. If
now the sections are rinsed first with water, then for a few seconds in a
1 : 1,000 alcoholic solution of caustic potash, and lastly brought into a
large quantity of water, the stain becomes differentiated, axis -cylinders
being shown coloured red and the myelin sheaths blue.
. Or (ibid., p. 49), the sections are stained for a few minutes or hours
in : —
Water 40 parts.
Saturated aqueous solution of methylene
blue 24 „
5 per cent, solution of borax . . . 16 „
then washed either in water or alcohol until the grey matter is distinctly
differentiated from the white substance, cleared with cedar wood oil,
and mounted in balsam. Preparations similar to those obtainable by
Weigert's method.
876. Acid Fuchsin.— FINOTTI (Virchow's Archiv., cxliii, 1896, p. 133)
stains strongly in Delafield's hsematoxylin, then for a few seconds in
concentrated solution of picric acid, then in 0-5 per cent, acid fuchsin,
and treats lastly with alkaline alcohol.
OHLMACHER (Journ. Exper. Med., ii, 1897, p. 675) stains sections for
one minute with gentian violet in anilin-water, then for a few seconds in
a 0-5 per cent, solution of acid fuchsin in saturated solution of picric acid
diluted with 1 volume of water, and differentiates with alcohol and
clove oil.
KAPLAN (Arch. Psychiatr., xxxv, 1902, p. 825) mordants for months
in Muller, stains sections for a day or more in £ per cent, aqueous
solution of acid fuchsin, rinses in water acidulated with HC1, and
differentiates by the method of Pal.
877. Safranine. — ADAMKIEWICZ (Sitzb. Akad. Wiss. Wien. Math.
Naturw. Kl.9 Ixxxix, 1884, Abth. 3, p. 245) stains sections of Muller
material in concentrated solution of safranine, differentiates in alcohol
and clove oil, brings back again into water, washes in water acidified with
acetic acid, and stains in methylene blue. Myelin red, nuclei violet.
Similarly, CIAGLINSKI (Ztschr. wiss. MiJcr., viii, 1891, p. 19) and
STROEBE (ibid., x, 1893, p. 384), the former employing safranine followed
by anilin blue, whilst the latter first stains with anilin blue, then
differentiates with alcohol containing a very little caustic potash, and
counterstains with safranine.
CHAPTER X XXIII . 453
878. Congo Red. — NISSL (Ztschr. wiss. Mikr.y iii, 1886, p. 398) stains for
three days in Congo red (5 parts to 400 of water) and differentiates in
alcohol with 3 per cent, of nitric acid.
Other Methods. KOTHIG'S Vital-Scharlach VIII Counterstain
(Neurol Centrbl, xxxiii, 1914, p. 219, and xxxiv, 1915, p. 265).—
Sections stained and differentiated by Weigert-Pal's method are
kept for twenty-four hours at room temperature in a counterstaining
fluid consisting of 90 c.c. of distilled water and 10 to 20 c.c. of a
solution of Vital-Scharlach VIII, saturated at room temperature.
They are then washed in distilled water for fifteen minutes and
differentiated in 70 per cent, alcohol for from one or two hours up to
twenty-four, when the celloidin will be found to be colourless.
After another wash in 96 per cent, alcohol, sections are mounted as
usual. Nerve cells and their processes, as well as axis-cylinders red,
the latter being visible within the deep blue myelin sheaths.
The method does not succeed if the sections were previously
treated with an osmic acid solution. Vital-Scharlach VIII may
also be used as a general stain, in which case the finished preparations
are similar to those obtainable by the usual carmine stains.
PALADINO'S palladium chloride methods ; see Eendic E. Accad.
Scienze, Napoli, iv, 1891, p. 14 ; Arch. Hal. Biol., xvii, 1892, p. 145,
and xix, 1893, p. 26.
For WOLTER'S vanadium chloride process, see next chapter.
ZOSIN'S magenta red method ; see Neurol. Centrlb., xxi, 1902, p. 207.
PERUSINI'S remarks and methods for the study of the white substance
of the spinal cord : see Journ. Psychol. Neurol., xix, 1912, p. 61.
456 AXIS-CYLINDER AND DENDRITE STAINS.
P.M. table even twenty-four to forty-eight hours after death.
It should be divided into pieces of not more than 1 cm. or 1-J cm.
in size.
The most difficult point of the method consists in finding out
the exact degree of hardening after which the material can be success-
fully submitted to the further treatment. In summer good results
may be obtained after fifteen to twenty days of hardening, and the
material may continue to be in a state suitable for the silver
impregnation up to thirty, forty or fifty days. In cold weather
good results can seldom be obtained under a month ; when this is
the -case, the material may continue to give good impregnations
for two, three or even four months. The only way to make sure
is to pass, at intervals, trial portions of the tissue into the silver
nitrate solution — in summer frequently, in winter every eight or
ten days — and observe whether and when the reaction has been
obtained.
It is a good practice to inject the organs (see § 806) with the
hardening fluid, generally 2-5 per cent, potassium bichromate, to
which, according to Golgi, 5 to 6 per cent, of gelatine may be added,
in which case, however, the fluid must be injected after warming
it to body temperature. Stoving at a temperature of 20° to 25° C.
is useful for abridging the hardening, but there is a risk of over-
hardening ; and Golgi thinks that the results are never quite so
delicate as after hardening at room temperature.
(b) Impregnation. — As soon as the pieces of tissue have attained
the proper degree of hardening, they are brought into a large
quantity of silver nitrate solution, the usual strength of which is
O75 per cent., but 0*50 per cent, may be used for material which
has not been quite enough hardened, and 1 per cent, for material
that has been slightly over-hardened.
The moment the pieces are put into the silver bath an abundant
precipitate is formed. This, of course, weakens the bath pro tanto.
It is, therefore, advisable first to 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 needs, generally, no further attention ; but it
should be changed for a fresh one if it becomes yellowish, as it some-
times does, particularly in the case of tissues which have taken
up a great deal of bichromate.
It is not necessary to keep the material in the dark during the
impregnation ; in winter it is well to keep it in a warmed room.
The time generally necessary for the impregnation is from twenty-
CHAPTER XXXI V. 457
four to forty-eight hours ; 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 80 to 90 per cent, alcohol. The alcohol is changed two, three
or more times, until it remains transparent, even after specimens
have been two or three days in it ; for, in view of good preservation,
it is necessary that the excess of silver nitrate should be washed out
from them thoroughly.
Sections are now made (see § 892). These are to be washed
thoroughly in three or four changes of absolute alcohol and cleared,
first in creosote, in which they should remain only a few minutes,
then in oil of turpentine, in which they are usually left for three to
fifteen minutes, though they may be kept in it even for some days
without being spoiled. They are then mounted in thick xylol-
damar (rather than in balsam), without coverslip. Preparations
mounted with coverslips in the usual way always go bad sooner or
later, whilst those mounted without a cover keep well for years,
especially if they are protected from dust and light.
Instead of creosote and oil of turpentine, fluid cedar-wood oil is
now used in Golgi's laboratory for clearing the sections, which are
then mounted, without cover, in thick cedar- wood oil. But care
must be taken to leave the sections in fluid cedar-wood oil no
longer than one hour or so, as otherwise they become brittle and
difficult to mount. To make sure of complete dehydration and that
no curling of the sections should take place in the fluid cedar- wood oil,
they are quickly passed through liquid absolute guaiacol, the whole
procedure being carried out as follows : A small quantity of absolute
guaiacol is poured in a watch-glass and some fluid cedar-wood oil
in two other small glass dishes. Two or three sections are carried
from the absolute alcohol into the guaiacol by means of a perforated
spatula, which is to be used for all the other passages, and cleaned
at every passage. After a few seconds the sections are transferred
into the first dish of fluid cedar- wood oil and there left for the time
necessary to pass another two or three s^tions from the absolute
alcohol into the guaiacol. The first batch of sections is now
transferred into the second dish of cedar- wood oil, the second batch
into the first cedar-wood oil and a fresh batch into guaiacol, and
so on until all sections are collected in the second dish of
cedar- wood oil.
For mounting the sections are lifted, one by one, by means of the
same small spatula, and arranged in the order and number one may
456 AXIS-CYLINDER AND DENDRITE STAINS.
P.M. table even twenty-four to forty-eight hours after death.
It should be divided into pieces of not more than 1 cm. or 1-| cm.
in size.
The most difficult point of the method consists in finding out
the exact degree of hardening after which the material can be success-
fully submitted to the further treatment. In summer good results
may be obtained after fifteen to twenty days of hardening, and the
material may continue to be in a state suitable for the silver
impregnation up to thirty, forty or fifty days. In cold weather
good results can seldom be obtained under a month ; when this is
the -case, the material may continue to give good impregnations
for two, three or even four months. The only way to make sure
is to pass, at intervals, trial portions of the tissue into the silver
nitrate solution — in summer frequently, in winter every eight or
ten days — and observe whether and when the reaction has been
obtained.
It is a good practice to inject the organs (see § 806) with the
hardening fluid, generally 2-5 per cent, potassium bichromate, to
which, according to Golgi, 5 to 6 per cent, of gelatine may be added,
in which case, however, the fluid must be injected after warming
it to body temperature. Stoving at a temperature of 20° to 25° C.
is useful for abridging the hardening, but there is a risk of over-
hardening ; and Golgi thinks that the results are never quite so
delicate as after hardening at room temperature.
(b) Impregnation. — As soon as the pieces of tissue have attained
the proper degree of hardening, they are brought into a large
quantity of silver nitrate solution, the usual strength of which is
0*75 per cent., but 0'50 per cent, may be used for material which
has not been quite enough hardened, and 1 per cent, for material
that has been slightly over-hardened.
The moment the pieces are put into the silver bath an abundant
precipitate is formed. This, of course, weakens the bath pro tanto.
It is, therefore, advisable first to 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 needs, generally, no further attention ; but it
should be changed for a fresh one if it becomes yellowish, as it some-
times does, particularly in the case of tissues which have taken
up a great deal of bichromate.
It is not necessary to keep the material in the dark during the
impregnation ; in winter it is well to keep it in a warmed room.
The time generally necessary for the impregnation is from twenty-
CHAPTER XXXI V. 457
four to forty-eight hours ; 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 80 to 90 per cent, alcohol. The alcohol is changed two, three
or more times, until it remains transparent, even after specimens
have been two or three days in it ; for, in view of good preservation,
it is necessary that the excess of silver nitrate should be washed out
from them thoroughly.
Sections are now made (see § 892). These are to be washed
thoroughly in three or four changes of absolute alcohol and cleared,
first in creosote, in which they should remain only a few minutes,
then in oil of turpentine, in which they are usually left for three to
fifteen minutes, though they may be kept in it even for some days
without being spoiled. They are then mounted in thick xylol-
damar (rather than in balsam), without coverslip. Preparations
mounted with cover-slips in the usual way always go bad sooner or
later, whilst those mounted without a cover keep well for years,
especially if they are protected from dust and light.
Instead of creosote and oil of turpentine, fluid cedar-wood oil is
now used in Golgi's laboratory for clearing the sections, which are
then mounted, without cover, in thick cedar- wood oil. But care
must be taken to leave the sections in fluid cedar-wood oil no
longer than one hour or so, as otherwise they become brittle and
difficult to mount. To make sure of complete dehydration and that
no curling of the sections should take place in the fluid cedar-wood oil,
they are quickly passed through liquid absolute guaiacol, the whole
procedure being carried out as follows : A small quantity of absolute
guaiacol is poured in a watch-glass and some fluid cedar-wood oil
in two other small glass dishes. Two or three sections are carried
from the absolute alcohol into the guaiacol by means of a perforated
spatula, which is to be used for all the other passages, and cleaned
at every passage. After a few seconds the sections are transferred
into the first dish of fluid cedar- wood oil and there left for the time
necessary to pass another two or three sections from the absolute
alcohol into the guaiacol. The first batch of sections is now
transferred into the second dish of cedar- wood oil, the second batch
into the first cedar-wood oil and a fresh batch into guaiacol, and
so on until all sections are collected in the second dish of
cedar- wood oil.
For mounting the sections are lifted, one by one, by means of the
same small spatula, and arranged in the order and number one may
458 AXIS-CYLINDER AND DENDRITE STAINS.
wish, either on ordinary slides, or on coverslips if the Golgi hollo wed-
out wooden slides are preferred for definite preservation. The
excess of cedar-wood oil carried with the spatula is removed by
covering the sections, after having definitely arranged them on the
slides, first with a sheath of cigarette paper and then with a folded
piece of filter paper, to be held by the left hand while the right is
passed over it so as to press down the sections and absorb the oil.
The whole manoeuvre may be repeated a second time, and then a
drop of thick cedar- wood oil put on each section. On the next
day the oil which may have run from the sections is cleaned from
the edges of the slides and a fresh drop of the thick cedar- wood oil
put on the sections, to be protected from dust and light at least
until the oil has become quite dry.
Preparations mounted in this way last for years unaltered ; in
fact, I have some which were made in Golgi's laboratory over
fifteen years ago and I find that they have kept without change.
1 have no experience of the use of creosote or of the mixture,
originally proposed by Andriezen, of equal parts of pyridine and
xylol instead of the guaiacol, but they should equally well serve
the purpose.
As a general rule one makes sections of 20 to 40 ju, ; thicker sections
of 50 to 60 jot, or more, show more than thin ones but do not seem to
keep so well.
The order in which the elements of nervous tissues impregnate
is generally — first, axis-cylinders, then nerve cells, and lastly,
neuroglia cells.
882. GOLGI'S Bichromate and Nitrate of Silver Method. KAPID
PROCESS. — Small pieces of very fresh tissues are hardened in a mixture
of 2 to 2 -5 per cent, potassium bichromate 8 parts, and 1 per cent,
osmic acid 2 parts. Or, if a very quick hardening is desirable,
2 parts of 3 per cent, bichromate to one of 1 per cent, osmic acid.
In Golgi's laboratory mixtures of 3 parts of 3 per cent, bichromate
and 1 of 1 per cent, osmic acid are now generally used. The tissues
begin to be in a state suitable for the silver impregnation from the
second or third day ; in the next following days they are in a still
more favourable state, but this soon declines, and is generally quite
lost by the tenth or twelfth day.
The silver impregnation is conducted exactly in the same way as
in the slow process, and sections are prepared and mounted in the
same manner, but they should not be left in alcohol for more than
an hour or so before mounting.
CHAPTER XXXIV. 459
There is this difference, that the impregnated material cannot
be preserved for any length of time in alcohol and must not remain
in it for more than one or two days. But it may be kept in the
silver solution until wanted for sectioning. According to
v. GrEHUCHTEN (La Cellule, vi, 1890, p. 405) pieces may be kept with
advantage for many days, weeks and months in the silver nitrate
solution. An abundant impregnation was found by him after many
days up to six months where almost none had been seen after
twenty-four to forty-eight hours only. But the material must be
kept in the dark.
As to the proper duration of the hardening process in different
cases, it must be pointed out that definite rules can hardly be given,
while investigators can easily find out the right moment for success-
fully transferring the pieces into the silver bath by means of attempts
made in accordance with the purpose in view and the quality of the
material with which they are working. However, the following
points should be borne in mind : —
Spinal cord of chick from the sixth to the tenth day of incubation —
twelve to forty-eight hours in the hardening mixture (up to the fifth day
the embryos may be treated whole, later the vertebral column should be
dissected out and cut into two or three segments ; it need not be
opened). The spinal column of newborn 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
dissected out.
v. LENHOSSEK (op. cit.) recommends for human fatal 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 hardening
mixture.
Cerebral cortex of young subjects two to three days (mice), or as long
as five (rabbit, cat) ; cortex of adults, eight to fifteen days. The most
favourable region of the brain is the Ammon's horn, especially in the
rabbit.
Retina — twenty-four to forty-eight hours in the mixture, then
" double " impregnation (§ 923).
Sympathetic. — SALA, L. (Mon. Zool. Ital., iii, 1892) found the
inferior cervical ganglion particularly suitable for staining by Golgi's
rapid process. He proceeds thus: osmium bichromate mixture,
three days ; quick wash in distilled water ; silver bath, two to three
days ; further wash in distilled water and passage into the same osmium-
bichromate mixture for about four days ; a third impregnation can be
resorted to, in which case pieces should remain in the hardening fluid
for five to seven days.
Spinal cord of larvw of Amphibia. — The entire larvae (best 2 to 2-5 cm.
460 AXIS-CYLINDER AND DENDRITE STAINS.
long) should be put for two to five days into the hardening mixture, and
for one to two into silver nitrate.
Epidermis of Lumbricus. — Three to six days in the mixture, and two
in the silver, or double impregnation if necessary.
Nervous system of Helix (glia- cells). The 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 be the
hardening. 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.
This process has the advantage of great rapidity, and of sureness
and delicacy of results, and it is the one that has found most
favour with other workers. But for the methodical study of any
given part of the nervous system GOLGI himself prefers the
following :—
883. GOLGI' s Bichromate and Nitrate of Silver Method. MIXED
PROCESS. — Fresh pieces of tissues are put for periods varying from
two to twenty-five or thirty days into the usual bichromate solution
(§ 881). Every two or three or four days some of them are passed
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 nitrate and subsequently treated exactly
as by the rapid process.
The reasons for which Golgi prefers this process are : The certainty
of obtaining samples of the reaction in many stages of intensity,
if a sufficient number of pieces of tissues have been used for the
purpose. The advantage of having at one's disposal a considerable
time — some twenty-five days — during which the tissues are in a
suitable state for taking the silver. The possibility of greatly
hastening the process whenever desired by simply bringing all the
pieces over at once into the osmic mixture. Lastly, a still greater
delicacy of results, particularly noticeable in the staining of axons
and their collaterals.
884. Theory of Impregnation. — It was once held that the reaction
depends on the formation in the tissues of a precipitate of some salt
of silver. And Kallius has put forward the suggestion that this
precipitate may consist of a protein-silver-chromate combination.
But this seems to B. Lee incorrect (see 1913 Ed.). In agreement
with v. Lenhossek, he finds that the colouration is not due to a
visible precipitate, but is a true stain accompanied, particularly in
unsuccessful impregnations, by precipitates which not only do not
CHAPTER XXXIV. 461
help the stain, but are injurious to it. It has been maintained that
the stain is merely superficial, and the method has been called an
" incrustation method." But it is easy to realise that it generally
extends throughout the whole thickness of the impregnated elements,
though in special cases or by slight modifications of the original
method, the stain may be limited to certain constituents of the
nerve-cell body, such as Golgi's pericellular investment and
intracellular network.
The chemical nature of the stain has not as yet been discovered.
A critical review of the Golgi method by WEIGERT may be found in
Ergebn. d. Anat., v, 1895, p. 7. See also HILL (Brain, xix, 1896, p. 1 ), and
KALLIUS (op. cit.}.
Modifications of Golgi's Bichromate and Silver Nitrate Method
concerning the Impregnation of Tissues.
885. Instead of potassium bichromate, ammonium bichromate has
been recommended by GOLGI and sodium bichromate by KALLIUS.
Both these salts appear to penetrate more quickly into the tissues
than potassium bichromate. According to STRONG (N. Y. Acad. Sc.
Proc. xiii, 1894) lithium bichromate hardens more rapidly than
potassium bichromate. The influence on the reaction of the bichro-
mates of ammonium, sodium, calcium, magnesium, rubidium,
lithium, zinc and copper, has been investigated by L. SALA (see
Kallius, op. cit., i, p. 564), but he came to the conclusion that they do
not offer any particular advantage, with the exception of calcium
bichromate, this last to be preferred for the staining of the tangential
fibres of the cerebral cortex.
RAMON Y CAJAL (Ztschr. wiss. Mikr., vii, 1890, p. 332) gives 3 per
cent, as the strength of the bichromate in the mixture for the rapid
process, but in numerous other places has given it as 3-5 per cent.
This latter strength has been adopted by many workers for the rapid
process, and the mixture containing this proportion of bichromate is
generally known as the RAMON Y CAJAL mixture.
886. RAMON Y CAJAL'S Double-Impregnation Process (La Cellule,
vii, 1891, p. 130). — Sometimes the usual rapid method fails to give
a good impregnation. This, however, may frequently be obtained
by putting the tissues back for a day or two into the osmium-
bichromate mixture used for the first hardening, or into a fresh but
weaker one containing 2 parts of 1 per cent, osmic acid and 20 parts
of 3 per cent, potassium bichromate. Tissues are then washed
quickly with distilled water or with a weak solution of silver nitrate,
462 AXIS-CYLINDER AND DENDRITE STAINS.
and put for a second time into the silver bath, where they should
remain from thirty-six to forty-eight hours. It is important to find
out the proper duration of the first hardening. 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 may be resorted
to, the objects being again treated with the weak osmium-bichromate
mixture and then again with the silver nitrate solution. I find that
this modification, which is the most important that has hitherto
been made, gives excellent results if one proceeds by tests, viz.,
re-transferring into the weak osmium-bichromate mixture those
pieces in which the reaction has been found to have succeeded
to some extent.
887. KOLOSSOW'S Modification (see ZUSCHTSCHENCO, Arch. Mikr. Anat.,
xlix, 1897). — Tissues are hardened for one to seven days in 3 to 5 per
cent, potassium bichromate containing 0-25 per cent, of osmic acid. They
are then washed quickly in distilled water, dried with filter paper and
transferred for two to three days into a bath of 2 to 3 per cent, silver
nitrate to which 0-25 to 0-5 per cent, of osmic acid has been added.
This is a good modification for sympathetic ganglia.
888. GOLGI'S Processes for the Rejuvenation of Over-hardened
Tissues. — Tissues which have been too long in the osmium-bichro-
mate mixture will no longer take on the silver impregnation. They
can, however, be made to impregnate by one or the other of Golgi's
so-called processes of rejuvenation. These can be carried out in
various ways given here with sufficient detail, as they may be of
great use not only for rejuvenating ordinary pieces of central nervous
system, but also, and particularly, for the staining of nerve-endings
in glandular and other tissues, internal apparatus, spiral filaments
of peripheral nerve-fibres, etc.
Golgi at first suggested washing the over-hardened pieces in a
half-saturated solution of copper acetate until they no longer give
a precipitate, afterwards putting them back again for five or six
days into the osmium-bichromate mixture, and subsequently
transferring them into the silver nitrate solution.
Later he advised leaving tissues in 3 to 4 per cent, copper sulphate
or 1 to 2 per cent, arsenic acid. After one, two and three days some
pieces are brought back into the osmium-bichromate mixture in
which they had been hardened, or into a weaker one, proceeding
further as in the rapid process, viz., as if the pieces had been
freshly fixed in the osmium-bichromate mixture.
More recently Golgi appears to have preferred mixtures of equal
parts of 2 or 3 or 4 per cent, copper sulphate or acetate and 4 to 5 per
CHAPTER XXXIV. 463
cent, potassium bichromate, filtering them if copper acetate was
used, and treating the pieces as stated above. As a rule these copper
acetate and potassium bichromate mixtures ought to be tried first
and in preference to others. As with other points of Golgi's methods,
so also in this case, one must proceed by tentative experiments,
according to the purpose of one's investigation and the quality of the
material in hand, but chiefly according to the length of time during
which the tissues have been left in the osmio-bichromate solution.
See on this subject SACEKDOTTI, Intern. Monatschr. Anat., xi, 1894,
p. 326 ; GOLGI, Cinquant. Soc. Biol., 1899, p. 514, and Opera Omnia II,
1903, p. 677 ; FUSAKI, Tratt. Elem. Istol. Teen. Istol., Torino, 1909 ;
SALA G., Anat. Anz.9 xviii, 1900, p. 176 ; GEMMELLI, Anat. Anz., 1913,
p. 444.
889. Formaldehyde Modifications of GOLGI'S Bichromate and Nitrate
of Silver Method. — Many investigators have found that formaldehyde
can take the place of the osmic acid in the osmio-bichromate
mixture of the rapid process. This has certain advantages : A
cheap reagent is employed instead of the expensive osmic acid.
Pieces much larger than by Golgi's original process may be used.
The stage of hardening favourable for a • good impregnation lasts
longer, i.e., formalin-bichromate mixtures do not over-harden.
Moreover, the formaldehyde modifications can be usefully resorted
to for impregnating nervous tissues of adult or young subjects, as
well as for material which after repeated attempts has been found
impervious to the osmic mixtures. However, it should be remem-
bered that many investigators have failed to obtain good results
by the formaldehyde methods and that they are unsuitable for
embryonic specimens.
HOYER, Jun. (Anat. Anz., ix, 1894, p. 236) was the first to point
out that material fixed in formalin could be used for carrying out
Golgi's method.
LACHI (Monit. Zool. Itdl., v, 1895, p. 15) used, at first, to harden
tissues for five to nine days in equal parts of 20 per cent, formalin
and 6 per cent, potassium bichromate. Afterwards "(Anal. Anz.,
x, 1895, p. 790) he adopted the mixture proposed by his pupil
DELL'ISOLA (Boll. Ace. Med. Genova, 1895, No. 2) of equal parts of
10 per cent, formalin and 10 per cent, potassium bichromate, with
the addition of 1 part of 1 per cent, osmic acid to every 10 of the
mixture, this last formula being particularly suitable for quick work,
as forty-eight hours afterwards pieces can be already transferred
into the silver bath.
STRONG (Anat. Anz., x, 1895, p. 494) suggested fixing pieces of
464 AXIS-CYLINDER AND DENDRITE STAINS.
brain of adult specimens in mixtures of 100 volumes of 3-5 per cent,
potassium bichromate and from 2-J to 5 volumes of formalin. One
or more pieces are, during several days, daily transferred into 1 per
cent, silver nitrate. Or the tissues are left for one to two days in
the above formalin-bichromate mixture and then passed into a fresh
one consisting of 2 volumes of 5 per cent, potassium bichromate
and 1 volume of formalin ; after another twelve to twenty-four
hours all the pieces are transferred into the silver bath.
DURIG (ibid., p. 659) obtained good results by fixing J cm. thick
pieces in 3 per cent, bichromate containing 4 to 6 per cent, of formalin,
and hardening therein for three days. After silvering for two days,
the pieces are brought back into the fixing mixture and one proceeds
as in Ramon y Cajal's double impregnation process.
FISH (Proc. Amer. Micr. Soc., xvii. 1895, p. 319) uses 2 c.c. of
formalin for every 100 c.c. of 3 per cent, potassium bichromate, and
leaves tissues three days in this fluid, and another three days in
0 -75 per cent, silver nitrate ; or, with advantage, Miiller's fluid,
100 c.c. ; 10 per cent, formalin, 2 c.c. ; 1 per cent, osmic acid, 1 c.c. ;
silvering as above.
KOPSCH (Anat. Anz., xi, 1896, p. 727) uses 4 parts of 3-5 per cent,
potassium bichromate and one of formalin ; after twenty-four hours
he transfers all pieces to pure 3-5 per cent, bichromate for at least
two days (retina) or three to six (central organs). He finds that by
this means, precipitates are almost entirely avoided. B. Lee
(1913 ed.) confirms this, but points out that the method gives a too
abundant impregnation of capillaries.
GEROTA (Intern. Monatsch. Anat., xiii, 1896, p. 108) first hardens
brains for a week or two in 5 to 10 per cent, formalin, then puts
small pieces for three to five days into 4 per cent, bichromate,
and lastly transfers these into the silver bath, where they are left
with advantage for ten to twenty days.
BOLTON (Lancet, 1898 (1), p. 218 ; Journ. R. Micr. Soc., 1898,
p. 244) has obtained good results from brains of cats and half-grown
kittens placed whole in 5 per cent, formalin and from human brains
hardened whole in formalin of the same strength for two to twelve
months. Small pieces are then cut out, and placed into 1 per cent.
ammonium bichromate and left therein for from a few hours up to
five days, some being transferred at intervals into 1 per cent, silver
nitrate.
SCHREIBER (Anat. Anz., xiv, 1898, p. 275) obtained good results
from appendages of Crustacea impervious to the osmic mixture, with
5 parts of 2-5 per cent, potassium bichromate to 1 of 4 per cent.
CHAPTER XXXIV. 465
formalin, or 1 part of 2-5 per cent, bichromate to 2 of 5 per cent,
formalin, the specimens remaining for one day in the first mixture
and for two in the second.
Similarly DUBOSQ (Arch, de Zool. exp., vi, 1898-90).
SMIRNOW (Arch. mikr. Anat., Hi, 1898, p. 201) fixes the cerebellum
of a freshly-killed animal in 5 per cent, potassium bichromate
4 parts, and formalin 1 part, for one to eight weeks. He then
divides the organ in two halves and places them into pure 3-5 per
cent, bichromate, where they are left for another two to five weeks.
Of the two halves one may be used for Weigert's myelin stain ; the
other is divided in pieces 1 to 2 cm. thick, and these put for one to
one and a half weeks into a mixture of 5 per cent, potassium
bichromate, 5 parts, and 2 per cent, osmic acid, 1 part. Pieces are
then transferred, at first into a weak silver bath and then into the
usual 1 per cent, silver nitrate solution. The method gives good
results also in human brains of adult subjects.
ODIER (La Rachicocainisation, Geneve, 1903, p. 27) takes 100 c.c.
of Miiller's fluid with 2 c.c. of undiluted formalin and 1 c.c. of 1 per
cent, osmic acid. The mixture should be kept in the dark and made
up at the instant of using it. Odier finds that formalin-bichromate
mixtures generally afford a more abundant impregnation with
fewer precipitates than the osmio-bichromic ones.
BROOKOVER (Journ. comp. neural. , xx, 1910, p. 49) finds useful
for adult specimens a preliminary fixation in 4 per cent. " formalde-
hyde," neutralised with lithium carbonate or ammonia, before
carrying out Golgi's rapid process.
890. Other Modifications. — VASSALE and DONAGGIO (Monit. Zool.
Ital., vi, 1895, p. 82) harden pieces of at most 1 cm. in thickness for
fifteen to twenty days in a mixture of 5 parts of acetic aldehyde and
100 of 3 to 4 per cent, potassium bichromate, changing the fluid as soon
as it has become dark. The rest as Golgi.
EAMON Y CAJAL (Rev. Trim. Histol, No. 2, 1888, note) found that 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 every 100 c.c. of the silver nitrate solution. But
the practice is now generally abandoned.
MARTINOTTI (Rif. med., 1887 ; Ztschr. wiss. Mikr., v, 1888, p. 88)
pointed out that Golgi's method can be successfully carried out on
relatively large pieces by using unusually large quantities of silver
nitrate solution with 5 per cent, glycerin added to it, and by keeping
this for thirty days at a temperature of 25° C. to impregnate nerve-cells,
and of 35° to 40° C. to stain the neuroglia.
ANDRIEZEN (Brit. Med. Journ., i, 1894, p. 909) found useful for
human brain to suspend thin slices of 2 to 4 mm. in diameter in 95 c.c.
M. 30
466 AXIS-CYLINDER AND DENDRITE STAINS.
of 2 per cent, potassium bichromate to which after ten to fifteen minutes
5 c.c. of 1 per cent, osmic acid are added. The mixture is kept in the
dark and after twenty -four hours changed for a fresh one made up
with 90 c.c. of 2£ per cent, bichromate and 10 c.c. of 1 per cent, osmic
acid. After another two days the mixture is changed over again for
one made according to the proportions given by Golgi (3 per cent,
potassium bichromate, 80 c.c. ; 1 per cent, osmic acid, 20 c.c.). Pieces are
transferred into the silver bath after three and a half days (for nerve
cells and neuroglia) up to six days. They are washed for five to fifteen
minutes in f per cent, silver nitrate, and then put into a solution of
silver nitrate of the same strength, but to which 1 drop of formic acid
to every 100 or 120 c.c. of solution has been added. The whole is kept
in an incubator at 25° to 27° C. for about three days, changing the
silver bath after the first twenty -four hours. The same author advised,
for the impregnation of neuroglia (Intern. Monatschr. Anat. x, 1893,
p. 533), adding 1 drop of a saturated solution of chromic acid and 1 drop
of formic acid to the first hardening bath.
BERKELEY (Johns Hopkins Hosp. Bep., vi, 1897, p. 1) hardens tissues
in Miiller's fluid until they are of sufficient consistency to admit of fairly
thin sections (about two weeks at room temperature). The portions
of the brain selected are cut into slices 3 mm. thick and immersed for
about three days in a mixture of 3 per cent, potassium bichromate, 100
parts, and 1 per cent, osmic acid 30 parts. For the impregnation,
tissues are removed from the hardening fluid, dried a little with filter
paper, washed in a weak solution of silver nitrate, and put for no less
than two to three days into a freshly prepared solution of 2 drops of
10 per cent, phosphomolybdic acid and 60 c.c. of 1 per cent, silver
nitrate, which in winter should be kept at a temperature of about 26° C.
HILL (op. cit. § 884) uses, instead of silver nitrate, a f per cent, solu-
tion of silver nitrite, with 0-1 per cent, formic acid added.
GUDDEN (Neurol CentrU., xx, 1901, p. 151) uses the lactate of silver
(sold as " actol "), and finds it more penetrating.
891. Avoidance of Precipitates.— Golgi's method frequently gives
rise to the formation at the surface of the pieces of irregular and
sometimes voluminous precipitates, which destroy the clearness of
preparations. To minimise this, SEHRWALD (Ztschr. wiss. Mikr.,
vi, 1889, p. 456) pours 10 per cent, gelatin, which is just liquid, into
a paper box, embeds the tissues in it with the aid of a little heat,
and brings them therein into the silver bath ; or the tissues are
coated with gelatin by dipping and cooling several times. After
the impregnation is completed the gelatin is removed, before cutting,
by means of warm water saturated with silver chroma te. MANN
(Physiol. Histol, 1902, p. 276) finds that the method gives good
results provided the gelatin is not rendered insoluble by the action
of light. To prevent this he proceeds thus :— Either in the photo-
graphic dark room or in the evening, by artificial light, tissues,
tied loosely to a thread, are immersed three times into liquefied
CHAPTER XXXI V. 467
10 per cent, gelatin, and, as soon as this has set, they are put into
the silver bath, keeping the latter in some dark place. It appears
that surrounding a tissue with gelatin makes the impregnation
slower, and for this reason Mann allows a day longer for the silver
bath.
MARTINOTTI (op. cit.) covers pieces with a layer of a pap of filter
paper and distilled water.
ATHIAS wraps tissues in wafer papers.
RAMON Y CAJAL covers them with a layer of congealed blood,
which need not be removed before cutting, or with celloidin or
peritoneal membrane. See " Retina."
Modifications concerning the Preservation of the Preparations.
892. Cutting. — As pointed out in § 880, one of the chief qualities
of Golgi's method consists in allowing one to follow nerve-cell
processes for a great distance. Evidently this cannot be done with
very thin sections ; and as sufficiently thin ones can be obtained
without embedding, the general practice is simply to wash the pieces
taken from the silver-bath with distilled water, fix them with gum
to a cork or wooden cube, put the whole into alcohol for a little
while to harden the gum, and cut by means of a sliding microtome
without embedding.
But quick embedding, particularly in celloidin, is quite possible,
and should be resorted to for material either brittle or otherwise
difficult to cut. Pieces of tissue as small as possible are brought
in the course of about two hours through the ascending series of
alcohols into absolute alcohol ; after having changed this a couple
of times, pieces are transferred for another one or two hours in thin
celloidin, then coated with thick celloidin, and by means of this
fixed to a wooden cube, the celloidin being a little hardened by
means of chloroform vapour, as usual. The whole is left for a little
while in 70 per cent, alcohol, and sections made in the usual way.
If these operations are started in the morning, when going into the
laboratory, pieces are ready for cutting at about 2 p.m., sufficient
time remaining for the further treatment of the sections according
to the directions given above (§ 881). Care should be taken, of
course, not to transfer the sections into absolute alcohol if it is not
considered safe to dissolve the celloidin. In this case dehydration
can be carried out as usual up to 98 per cent, alcohol, and the
sections transferred into fluid absolute guaiacol and cedar-wood oil
as already described in § 881.
30—2
468 AXIS-CYLINDER AND DENDRITE STAINS.
Embedding in paraffin is also possible, but results are usually
rather poor, and one should have recourse to it only for special
objects, such as muscles (see VEBATTI, Mem. R. Inst. Lomb. Sc., xix,
1902, p. 87). In any case tissues should be passed quickly through
the lower grades of alcohol, and remain only a few hours in 95 per
cent, and absolute alcohols. They should be cleared with cedar-
wood oil, as xylol and similar reagents may be injurious to the
silver impregnation. One should transfer pieces directly into
paraffin of as low a melting point as possible. According to
BROOKOVER (op. cit.), cedar-wood oil should be used over and over
again, as it becomes saturated with silver nitrate.
893. Mounting. — As pointed out in § 881, Golgi preparations do
not keep well if mounted under a cover-glass in the usual way.
How and why this happens it is very difficult to say. Though an
elaborate discussion between SEHRWALD (Ztschr. iviss. Mikr., vi,
1889, p. 443), SAMASSA (ibid., vii, 1890, p. 26), and FISH (ibid., viii,
1891, p. 168) has furnished the net practical result that watery
fluids should be avoided as much as possible during the after-
treatment, it is not clear why preparations should deteriorate, when
mounted under a cover-glass in thick cedar-wood oil or neutral
balsam ; while MANN (op. cit., p. 277) states, on the other hand, that
sections keep well if mounted under a cover-glass in Price's No. 1
pure neutral glycerin.
For these reasons the general practice is to mount sections
without a cover, either on ordinary slides or on cover-glasses to be
inverted for study over the aperture of a hollowed-out wooden
slide.
If mounting under a cover is desirable, this should either be raised
free of contact with the slide by means of wax feet or the like, or the
balsam of the mount should be rendered perfectly anhydrous by care-
ful heating it on the slide with the section in it, until it immediately
sets hard on cooling, when a slightly- warmed cover can be applied.
This last method is also recommended by HUBER (Anat. Anz., vii,
1892, p. 587). B. LEE (see previous editions) advises keeping the
preparations uncovered until the sections have become quite dry
and the balsam, applied from time to time in thin layers, quite
hard, and then to cover them with a warmed cover-glass, this
being slightly pressed down on the sections.
Various processes have been devised for mounting Golgi's prepara-
tions at once under a cover, but none of them give really satisfactory
results. One should have recourse to them either for special objects, or
if counterstaining with carmine or haematoxylin, or by Weigert-Pal's
CHAPTER XXXIV. 469
method, or the like, is particularly desirable. Tn this case one of the
following methods may be employed : —
GKEPPIN (Arch. Anat. u. Entwick., Anat. Abth., Supp., 1889, p. 55)
treats sections for thirty to forty seconds (until whitish) with 10 per
cent, hydrobromic acid, washes them in several changes of water,
dehydrates, clears with clove oil and exposes them for ten to fifteen
minutes to sunlight.
OBREGIA (Virchow's Arch., cxxii, 1890, p. 387) transfers sections into
a mixture of absolute alcohol, 10 c.c., and 1 per cent, gold chloride,
10 drops, to be previously exposed to diffuse daylight for half an hour.
Sections are then passed into it and put in a dark place. After fifteen
to thirty minutes they are washed successively in 50 per cent, alcohol,
distilled water, 10 per cent, sodium hyposulphite (five to ten minutes),
and repeatedly changed distilled water. They may be then counter-
stained, dehydrated and mounted in balsam under a cover.
KALLIUS (Anat. Hefte., ii, 1893, p. 271) uses 230 c.c. of distilled
water and 20 c.c. of commercial hydro quinone solution (hydroquinone
5 grms., sodium sulphite 40 grms., potassium carbonate 75 grms., dis-
tilled water 250 c.c.). The solution is further diluted before using with
one-third to one-half its volume of absolute alcohol and the sections
(freed from unreduced silver by washing them in many changes of
alcohol) left in it for several minutes. Here they become dark-grey to
black, and are then transferred for ten to fifteen minutes into .70 per
cent, alcohol, for five minutes into 20 per cent, sodium hyposulphite, and
for twenty-four hours into a large quantity of distilled water. Counter-
stain, dehydrate, clear and mount as usual.
EBERTH and RUNGE (Arch. mikr. Anat., xlvi, 1896, p. 370) have
successfully used a process similar to that of Greppin. They convert
the silver impregnation into silver chloride by keeping sections in
chlorine water for fifteen to twenty minutes, and they then reduce the
white silver chloride, either through exhibition to sunlight just before
mounting, or by means of Kallius' process.
BOLTON (op. cit.) has obtained good results with Kallius' process
applied to his f or mol- bichromate modification.
CUKRERI (Anat. Anz., xxxii, 1908, p. 432), after fixing by Kallius'
method, tones in 0-7 grm. of gold chloride, 3 grm. of sodium acetate
and 100 c.c. of water.
ZIMMERMANN'S process (Arch. mikr. Anat., Hi, 1898, p. 554). Paraffin
sections of formol-Golgi material are brought from alcohol into a large
quantity of a mixture of 1 part of physiological salt solution and 2 parts
of 96 per cent, alcohol. They are kept in motion therein for ten to fifteen
minutes, after which they are brought into 75 to 96 per cent, alcohol
in a bright light until they have become dark (about half a day) ; or
sections are left for half to one hour in 100 c.c. of absolute alcohol to
which a few drops of ammonium hydrosulphide have been added. In
the first case the silver deposit becomes converted into silver chloride,
in the second into silver sulphide. Later (Arch. mikr. Anat., Ixxviii,
1911, p. 199) he reduces for several hours in 20 c.c. of saturated
solution of sodium carbonate (made up with 50 per cent, alcohol) to
which 0-5 grm. of adurol are added. These processes are useful for
470 AXIS-CYLINDER AND DENDRITE STAINS.
studying the inter-relationship between gland -ducts and gland-cells
(stomach, liver) if the silver chloride sections are afterwards stained
with thionin or toluidine blue or safranin, the sulphide sections with
Delafield's hsematoxylin, and the adurol ones with hsemalum or alum
cochineal.
For toning, fixing and counterstaining sections of tissues treated by
the sublimate method and the like, see next paragraph.
The Sublimate Method.
894. GOLGI'S Bichromate and Sublimate Method (Arch. Sc. Med.,
iii, 1878 ; Rend. R. Inst, Lomb. Sc. (2), xii, 1879, p. 205 and (2),
xxiv, 1891 ; Arch. Ital. Biol, op. cit., § 880 ; Rif. Med., 1891 ; Opera
Omnia, I, p. 143, and II, pp. 505 and 607). — For hardening, use either
a solution of potassium bichromate progressively raised from 1 to 3
per cent., or Miiller's fluid. It is best to take small pieces of tissue,
large quantities of hardening fluid, and change the latter frequently.
But the reaction can be obtained with much larger pieces, even
entire hemispheres. In this case the brain should at first be treated
with " repeated " injections of the fixing agent, or this should be
injected from the carotid or the aorta. Pieces, particularly if small,
begin to be ready for the subsequent treatment eight to ten days
afterwards, but it is advisable to wait until the twentieth or thirtieth
day of immersion, this being not injurious if prolonged for several
months ; it is, on the contrary, to be recommended if the pieces are
uncommonly large.
When it is thought that the tissues have been hardened enough,
they are passed directly from the bichromate into 0 -5 to 1 per cent,
mercury bichloride. One generally prefers weak solutions (0-5 per
cent.) if pieces have been left in the fixing fluid for a relatively short
period, having recourse to the stronger ones (1 per cent.) for materials
which have been hardened for many weeks or months. The subli-
mate solution must be changed at first every day, and later as often
as it becomes yellowish. At the end of the reaction pieces will be
found decolourised and almost with the aspect of fresh tissue. To
obtain a good reaction, about ten days of immersion in the mercury
bichloride are necessary if pieces are small, longer periods, and even
months, being required for large pieces and entire hemispheres.
Particularly fine results were obtained by Golgi from brains which
had been kept in 1 per cent, sublimate for as long as two years.
The reaction may be said to have begun by the time tissues are
nearly decolourised. From that time onwards sections may be made
and mounted if successful.
CHAPTER XXXIV. 471
Embedding is not necessary, but in many cases desirable. It can
be easily carried out by washing pieces in many changes of alcohol
of ascending strengths and embedding them in celloidin. Sections,
however made, must be repeatedly washed with distilled water,
otherwise they will soon be spoilt by the formation of opaque
granules and needle-like crystals which very much hinder proper
observation. After dehydrating, sections can be passed through
creosote and turpentine and mounted, preferably without a cover-
glass, in dammar or balsam.
It is, however, preferable to treat sections by the following
fixing-and-toning process which was suggested by Golgi for trans-
forming the whitish mercury impregnation (to which the reaction
is due) into a full-black stain, much more suitable for observation
under high power. Moreover, the process helps in preventing the
formation of opaque precipitates, and allows of mounting in the
usual way without any danger of spoiling the specimens.
One proceeds thus : — Sections of pieces embedded in celloidin are
thoroughly washed in many changes of water, and then transferred
for a few minutes into a photographic fixing and toning bath to be
prepared at the moment of using, as follows :—
Solution A.
Distilled water 1,000 c.c.
Sodium hyposulphite . . . . 155 gr.
Potassium alum . . . . . 20 ,,
Ammonium thiocyanate . . . 10 „
Sodium chloride . . . . . 40 ,,
Allow to stand for eight days and then filter.
Solution B.
Gold chloride 1 gr.
Distilled water 100 c.c.
For use take 50 c.c. of sol. A, 7 c.c. of sol. B, and
40 c.c. of old combined bath.
From the fixing and toning bath sections are transferred into
distilled water and again thoroughly washed ; they are then slightly
counterstained with an acid solution of carmine diluted with some
alcohol, dehydrated, cleared, and mounted in the usual way.
The elements stained by the method are : — (1) Nerve 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 method. (4) Blood vessels, and particularly their muscle
fibre-cells.
472 AXIS-CYLINDER AND DENDRITE STAINS.
The method gives particularly good results with cerebral cortex
and Ammon's horn, very poor ones with the cerebellum and spinal
cord. It is superior to the silver method in so far that the reaction
can always be obtained with certainty in a certain time ; that the
preparation can be preserved by the usual methods ; that large
pieces of tissue can be impregnated. Moreover, it is cheaper and may
give a more abundant and finer impregnation than even the rapid
process.
895. Modifications of GOLGI'S Bichromate and Sublimate Method. —
MONDINO (Ztsch. wiss. Mikr., ii, 1885, p. 157) has obtained good results
from even whole human brain treated according to Golgi's original
method.
FLATAU (Arch. mikr. Anat., xlv, 1895, p. 158) fixes whole human
brain in 3 to 4 per cent, potassium bichromate. After two or three
months slices ^ cm. thick and 1 to 2 cm. wide are brought into 0-1
per cent, mercury bichloride to be changed every two to three days
for the first three weeks or so. Pieces are ripe for cutting after nine
to twelve months, at which time they are washed and embedded
in celloidin. Sections are passed through alcohols, cleared in carbol-
xylol and mounted in balsam.
PAL (Erratim " Tal ") (Ztschr. wiss. Mikr., iv, 1887, p. 497) converts
the whitish mercury impregnation into a black one by treating sections
with 1 per cent, sodium sulphide. They may then be counterstained
with Magdala red.
GOLGI'S sublimate method may be combined with Weigert's myelin
stain (see PAL, Wiener med. Jahrb., N.F. 1, 1886, p. 619, and the abstract
of this paper in Ztschr. wiss. Mikr., iv, 1887, p. 92, in which EDINGER
pointed out that the mercury impregnation can be turned black by
treating sections with diluted ammonia).
FLECHSIG (Arch. Anat. Phys., Physiol. Abih., 1889, p. 537) has pub-
lished a rather complicated combination of Brama's Guinea red-wood
process for medullated nerve-fibres and Golgi's sublimate method, as
slightly modified by Held.
896. Cox's Process (Arch. mikr. Anat., xxxvii, 1891, p. 16).— This
is the most important of all modifications of Golgi's bichromate and
sublimate method. Cox found that the sublimate and bichromate
can be used together, and that potassium chromate can be usefully
added to the mixture in order to reduce the normally acid reaction
of the bichromate, as otherwise axis-cylinders are not impregnated.
He used a fluid consisting of 20 parts of 5 per cent, potassium
bichromate, 20 parts of 5 per cent, corrosive sublimate, 16 parts of
5 per cent, potassium chromate, and 30 to 40 parts of distilled water.
To prepare it, the bichromate and sublimate are mixed together, the
chromate diluted with the water and added to the mixture.
One generally uses small pieces of tissues, but also relatively large
CHAPTER XXXIV. 473
ones can be employed, and whole brains of small animals, particu-
larly if some of the fluid has been previously injected through the
carotid or aorta. The duration of the impregnation is from two to
three months, but material can be left in the mixture for much
longer, certainly without danger and, very likely, with advantage.
MANN (op. cit.) recommends warming the mixture to the tem-
perature of the incubator and diluting it to one-half the strength
advocated by Cox, particularly for material of adult subjects.
Portions of the brain measuring 1 cm. in thickness or entire brains
of young animals are placed by him on cotton-wool in this solution
and left in the incubator for twenty-four hours, when the solution is
changed. After a second change on the third day the vessel (which
should contain the mixture in proportion of 30 : 1 of the brain) is
sealed with vaseline and left in the incubator for at least a month,
but preferably for two. I find this way of carrying out the Golgi-
Cox method very good, but, after incubating for a month or so, I
prefer keeping the vessel at room temperature, and cutting after
another two or three months or longer.
There is considerable difficulty in making and preserving sections
which ought to be made either by free hand or by means of a freezing
microtome after slight preliminary washing of the pieces with water,
and impregnating them with 20 per cent, dextrin' for one to three
days as suggested by Mann.
To convert the white mercury impregnation into a black one, Cox
suggested treating the sections for an hour or two with 5 per cent,
sodium carbonate, but 5 to 10 per cent, ammonia is now generally
used. They are then thoroughly washed in distilled water, carefully
dehydrated, cleared by one of the usual ways, and mounted, without
a cover, either in thick xylol balsam or in the original medium
suggested by Cox and composed of : — Gum sandarac 75 grms.,
camphor 15, oil of turpentine 30, oil of lavender 22-5, alcohol 75,
castor -oil 5 to 10 drops. For examination, add a drop of castor
oil, and cover.
897. Methods for rendering Golgi-Cox Preparations more per-
manent.— Various authors (see SANDERS, 1898, in lilt, to A. B. Lee,
Vade-Mecum, 1913 ed., p. 433 ; BREMER, Anat. Rec., iv, 1910,
p. 263) have proposed washing tissues treated according to Cox's
process in many changes of alcohol, and embedding them in celloidin
— this chiefly with the object of overcoming the difficulty of cutting
brittle pieces by means of the freezing microtome, and also of render-
ing preparations more permanent by removing the excess of corrosive
474 AXIS-CYLINDER AND DENDR1TE STAINS.
sublimate not utilised by the reaction, and which still permeates the
tissues. As a matter of fact sections of pieces thus treated are very
easily cut and can be transferred from one to another fluid without
danger of injuring them. Moreover, they can be counterstained,
and the impregnation keeps sufficiently well, particularly if sections
are mounted without a cover-glass. But in such preparations,
sometimes quickly, sometimes slowly, opaque granules and minute
needle-like crystals become almost always developed.
To avoid this I recently proposed (see DA FANO, Proc. Physiol.
Soc. Journ. Physiol., liv, 1921) to treat sections much in the same
way as by the so-called process of toning and fixing Bielschowsky
preparations and the like. (See DA FANO, ibid., liii, 1920.) I
proceed thus : — Pieces which, by a trial section, have been found
well impregnated, are washed for some hours in distilled water
and then brought, through many changes of alcohol of ascending
strengths, into absolute alcohol, and then embedded in celloidin in
the usual way. The celloidin blocks are hardened in 70 per cent,
alcohol, where they can be safely left for many days and weeks.
Sections of the desired thickness are collected in 60 per cent, alcohol,
transferred into distilled water and here thoroughly washed. They
are then treated for five to ten minutes with 5 per cent, ammonia
and washed over again in two or three changes of distilled water.
At this point toning is carried out by means of a slightly acidified
0-2 per cent, gold chloride solution, in which sections are left for
ten or fifteen or twenty minutes, according to their thickness. After
a quick washing in distilled water they are passed for three to five
minutes in 5 per cent, sodium hyposulphite and washed once more
in distilled water. From this they are transferred successively into
30, 50, and 70 per cent, alcohols, to each of which 1 drop of saturated
iodine tincture to every 5 c.c. of alcohol has been added. Sections
remain in each alcohol ten to fifteen minutes and are lastly transferred
into pure 70 per cent, alcohol.
At this point the process is ended, and one can proceed to mount
the sections in the usual way, or re-transfer them into distilled water,
counterstain them lightly with a carmine solution, dehydrate with
alcohols of ascending strength up to 95 per cent., pass them through
two changes of carbol-xylol and mount them under a thin cover-
glass in xylol-colophonium or balsam. If desirable and safe, the
celloidin can be removed before definite mounting by passing sections
through absolute alcohol, and alcohol-ether if necessary.
The process is simpler than the rather complicated platinum
substitutions of ROBERTSON and MACDONALD {Journ. Ment. Sc.,
CHAPTER XXXIV. 475
xlvii, 1901, p. 327) and is so quickly and easily carried out that
many sections can be manipulated at the same time.
Processes similar to Golgi's Methods or suitable for the
same Purposes.
898. ZIEHEN'S Gold and Sublimate Method (Neurol. Centrbl., x,
1891, p. 65). — Small pieces of fresh tissues are put into a large
quantity of a mixture of equal parts of 1 per cent, corrosive sublimate
and 1 per cent, gold chloride, and left 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 then gummed
to a cork or wooden cube and cut without embedding. Sections are
treated either with LUGOL'S solution diluted with 4 volumes of water,
or with diluted tincture of iodine, until duly differentiated, then
washed, dehydrated, and mounted in balsam. Both medullated
and non-medullated nerve-fibres, as well as nerve-cells and neuroglia
cells are stained.
899. KROHNTHAL'S Lead Sulphide Impregnation (Neurol. Centrbl.
xviii, 1889 ; Ztschr. wiss. Mikr., xvi, 1899, p. 235).— Pure formic
acid is slowly added to a saturated solution of lead acetate till white
crystals of lead formiate are abundantly formed. The mother liquid
is filtered off, and the crystals are dissolved to saturation in distilled
water. Equal volumes of this saturated solution of lead formiate
and 10 per cent, formalin form the fixing fluid in which pieces of
brain or spinal cord are left for five days. Tissues are then directly
brought into a mixture of equal parts of 10 per cent, formalin and
sulphuretted hydrogen. After a few minutes the first discoloured
portion of this mixture is poured off and replaced with fresh solution,
in which pieces remain for another five days. They are then
gradually dehydrated and embedded in celloidin. Sections are
cleared in carbol-xylol (1:1) and mounted in balsam under a cover.
Nerve-cells and nerve-fibres are extensively impregnated.
CORNING (Anat. Am., xvii, 1900, p. 108) hardens the tissues in
10 per cent, formalin and then brings them into the lead formiate,
which he buys from Merk. He prefers to cut without embedding.
900. WOLTER'S Chloride of Vanadium Process (Ztschr. wiss. Mikr.,
vii, 1891, p. 471). — Central or peripheral nervous tissues are fixed
in Kultschitzky's solution, followed by alcohol as described in § 55.
Celloidin sections, 5 to 10 /m thick, are mordanted for twenty-four
hours in a mixture of 2 parts of 10 per cent, vanadium chloride and
8 parts of 8 per cent, aluminium acetate. They are then washed
476 AXIS-CYLINDER AND DENDRITE STAINS.
for ten minutes in water, stained for twenty-four hours in an incu-
bator in Kultschitzky's haematoxylin, and differentiated in 80 per
cent, alcohol acidified with 0-5 per cent, of hydrochloric acid until
slightly blue-red. The acid is then removed by washing with pure
alcohol, and the sections dehydrated, cleared with origanum oil,
and mounted in balsam. Axis-cylinders, nerve-cells and glia cells
are stained, the myelin being coloured only when the differentiation
in the acid alcohol has been insufficient.
901. AZOULAY'S Ammonium Vanadate Process (Bull. Soc. Anat.,
Paris, Ixix, 1894, 5th S., p. 924). — Wash in water thin sections of
material fixed in a bichromate solution and embedded in celloidin.
Lift a section on a slide and pour on it a few drops of 0 -5 per cent,
ammonium vanadate, wait a moment, pour off the stain, wash with
a little distilled water and pour on the section a few drops of 2 -5 per
cent, tannin. After a few minutes pour off the tannin solution,
wash, and start all over again, and so on until axis-cylinder and
nerve-cells are stained dark green. Wash quickly, dehydrate and
mount. These preparations photograph well.
902. FAJERSTAJN'S Haematoxylin (Poln. Arch. Biol. Med. Wiss.,
i, 1901, p. 189). — Make sections, by means of the freezing microtome,
of material fixed for two to seven days in 5 to 10 per cent, formalin.
Transfer them into 0-25 to 0-5 per cent, chromic acid, and after
twenty-four hours wash them well, and put them to stain for another
twenty-four hours in 1 per cent, aqueous solution of hsematoxylin
Differentiate by Pal's method.
903. NABIAS' Method (C. R. Soc. Biol, Ivi, 1904, p. 426).— Sections
of material fixed in alcohol-corrosive sublimate or any other fixing
agent easily allowing the penetration of iodine are treated until
yellow with Lugol's solution (Gram's formula). They are then
quickly washed, and treated for a few minutes with 1 per cent,
gold chloride, quickly washed once more, and reduced in 1 per cent,
watery solution of anilin oil or resorcin. Dehydrate and mount in
balsam.
904. LENNHOFF'S Processes (Neurol. Centrbl, xxix, 1910, p. 20).—
(1) Polychrome-methylene blue and potassium sulphocyanide method
for axis-cylinders : Fixation not stated. Stain sections in polychrome
methylene blue for two to five minutes, wash them in distilled water and
transfer them for half to twenty-four hours into potassium sulpho-
cyanide (strength not stated). Wash, dehydrate, clear, and mount in
balsam. (2) Polychrome-methylene blue and potassium fcrricyanide
method for axis -cylinders and nerve cells. Sections of material fixed
in alcohol are treated as above, using potassium ferricyanide instead of
CHAPTER XXXIV. 477
the sulphocyanide. (3) Iron method. Sections are kept for thirty
seconds in 2 c.c. of a 15 per cent, solution of tannin to which 3 drops of
a 5 per cent, solution of oxalic acid have been added. Kinse them first in
distilled water and then for a few seconds in 1 per cent, solution of iron
chloride until no further blackening occurs. Wash, dehydrate and
mount in balsam. Axis-cylinders black, nerve-cells grey.
APATHY'S Gold Method. See § 371.
GERLACH'S Bichromate and Gold Process. See § 369.
EAMON Y CAJAL'S Gold Method. See Rev. trim. Micr., v, 1900, p. 95.
UPSON'S Gold and Iron and^Vanadium Methods. See MERCIEK,
Ztschr. wiss. Mikr., vii, 1891, p. 474.
MAGINI'S Zinc Chloride Process. See Boll. Ace. med. Roma, 1886, or
Ztschr. wiss. Mikr., v, 1888, p. 87.
MONTI'S Copper Process. See Rend. R. Ace. Lincei, Roma, v, 1889,
p. 705.
STRAHUBER'S Anilin Blue Method. See Centrbl. allg. Path., xii, 1901,
p. 422.
CHILESOTTI'S Carmin Stain. See Centrbl. allg. Path., xiii, 1902, p. 191 ;
Ztschr. wiss. Mikr., xix, 1902, p. 161, and xx, 1903, p. 87.
KAPLAN'S Anthracen Ink Method. See Arch. Psych., xxxv, 1902,
p. 825.
MALLORY'S Phosphomolybdic Haematoxylin. See § 271.
DONAGGIO'S Tin Stain. See § 273.
Methylene-blue Methods not considered in Chapter XVI.
905. S. MEYER'S Method for the Central Nervous System (Arch,
mikr. Anat., xlvi, 1895, p. 282, and xlvii, 1896, p. 734).— The method
consists essentially in injecting animals subcutaneously with large
quantities of a solution of methylene-blue B.X., and in treating the
central organs (brains) with Bethe's fixing bath. S. Meyer used, at
first, a 1 per cent, solution ; later, a solution of methylene-blue B.X.
saturated at the body temperature of the animal to be injected
(viz., about 5 to 6 per cent.). The injections are to be made at
short intervals and in such a way that the animal receives the total
quantity it can support in about one to two hours. A cat can
support even 150 c.c. ; half-grown rabbits, 30 to 50 c.c. ; fully
developed guinea-pigs, 30 to 50 c.c. ; new-born kittens, 15 to 25 c.c.
As soon as the animal used is dead, the brain is removed, divided into
two to four pieces, and these plunged in 10 per cent, ammonium
molybdate to which 1 drop of HC1 for every gram of ammonium
molybdate is added. Here they remain for about twenty-four
hours at 0° C. Pieces are then washed for two hours in running
tap-water, passed quickly through the ascending series of alcohols
into absolute alcohol, and, lastly, embedded in paraffin in the usual
way.
478 AXIS-CYLINDER AND DENDRITE STAINS.
906. RAMON Y CAJAL'S Diffusion Process (Rev. Trim. Micr., i,
1896, p. 123). — The brain is exposed, and by means of a sharp razor
the cortex is divided into slices about 2 mm. thick. The slices
are then covered on both sides, either with finely powdered methylene
blue or impregnated with a saturated solution of the same and
replaced in their natural situation. The brain is covered over again
with its case for about half an hour, after which the slices are removed
and fixed for a couple of hours in Bethe's ammonium molybdate
solution. They are then washed and hardened for three or four
hours in a mixture of 5 parts of chloroplatinic acid, 40 parts of
formalin, and 60 parts of distilled water. After another quick wash
and a brief treatment (? a few minutes) with a 1 : 300 alcoholic
solution of chloroplatinic acid, they are dehydrated and embedded
in paraffin. The sections may also be quickly treated with the
same weak alcoholic solution of chloro-platinic acid, cleared with
xylol or bergamot oil, and mounted in the usual way.
907. CATOIS' Method for Fishes (C. R. Ac. Sc., cxxiv, 1897, p. 204).
— Small quantities (2 to 3 c.c.) of a concentrated solution of
methylene blue, prepared with physiological salt solution, is injected
into the branchial vessels or intramuscularly. The brain is removed
after half an hour, divided into slices, and then left for another
half an hour in the same concentrated solution used for injecting
the animal. The slices are then fixed in the usual ammonium
molybdate solution, or in Cajal's chloroplatinic acid mixture.
908. See also the valuable account of DOGIEL Methylen-blau zur
Nervenfdrbung in the EnzyTd. mikr. Techn., 2nd ed., 1910, and the article
of GORDON in Anat. Rec., iv, 1910, p. 267 ; and that of MICHAILOW in
Ztschr. wiss. Mikr., xxvii,- 1910, p. 1, in which the literature of the
subject is critically discussed.
CHAPTER XXXV.
NEUKOGLIA AND SENSE ORGANS,
Neuroglia.*
909. Introduction. — Neuroglia cells may be isolated by teasing
after maceration in weak solutions of potassium bichromate or
33 per cent, alcohol, and then stained, preferably by means of dilute
picrocarmine or other carmine solutions. They may be studied,
also, in sections made from non-embedded material fixed in solutions
of chromic salts and stained with carmine, nigrosin, orcein and so
on. Sections made from either fresh material hardened by the ether
freezing method and treated with a weak solution of osmic acid
(§ 807), or from tissues hardened in potassium bichromate, can be
advantageously stained with watery solutions of anilin-blue-black
or nigrosin. Also, sections cut from material fixed, hardened and
embedded by the usual methods may, up to a point, be employed
for getting a general, though incomplete, view of the amount and
arrangement of the neuroglia in a given nervous organ. Iron
hsematoxylin, particularly after fixation in corrosive sublimate or
other fluids containing it, gives good results with sections of central
nervous organs of lower vertebrates, chiefly of fishes.
See GOLGI, Opera Omnia, i, pp. 1 and 3 to 70 ; ii, p. 61 ; KANVIER,
Traite, etc. ; BEVAN LEWIS, op. cit. ; E. MULLER, Arch. mikr. Anat.,
Iv, 1900, p. 17 ; STUDNICKA, Anat. Hefte, xv, 1900, p. 316, and the
literature quoted therein.
But the best method for the study of the morphology and relation-
ship of ependyma cells and astrocytes has been for many years,
and in a sense still is, Golgi's rapid process (§ 882), the best
material being that which has been placed for about two or three
days in the osmio-bichromic mixture.
This method, however, does not allow of any tinctorial differentia-
tion, either between neuroglia cells and nerve cells, or between
neuroglia cells and neuroglia fibres. One might even say that it
is unsuitable for the demonstration of the latter, the existence of
which was clearly established only after the publication of
* Rewritten by Dr. C. Da Fano, King's College, University of London.
480 NEUROGLIA AND SENSE ORGANS.
WEIGERT'S method (see next §), the first and, perhaps even now, most
important of all so-called specific processes for staining neuroglia
fibres.
But the Weigert method, whilst staining neuroglia fibres and
nuclei of neuroglia cells intensely and, up to a point, specifically,
leaves the cell-bodies of the latter entirely unstained. It conse-
quently led to the erroneous conclusion that the processes of
neuroglia cells were one and the same thing as the neuroglia fibres
shown by the new method, and that the latter, were, in the adult
state, only contiguous to — viz., independent of— the former.
Efforts were, therefore, made to discover new methods suitable
for the study of neuroglia fibres and neuroglia cells and their reci-
procal relations. Many modifications of Weigert 's neuroglia^ stain,
the methods of BENDA, MALLORY, ANGLADE and MOREL, HELD,
RUBASCHKIN, DA FANO, etc., may be considered as the direct
outcome of such efforts.
None of these methods, however, was sufficient to entirely solve
the problems resulting from Weigert's discovery, and from the
comparison between the results attainable by the new neuroglia
stain and Golgi's process. Hence the publication of the methods
of RAMON Y CAJAL, ACHUCARRO, DEL RIO-HORTEGA, and the modern
conception that the neuroglia consists essentially of cells provided
with variously ramified processes (protoplasmic neuroglia), and of
fibres which, though a product of differentiation of the former,
remain, very likely, throughout life continuously connected with
the protoplasmic bodies and processes of neuroglia cells (fibrous
neuroglia).
With all that, the very meaning of the word " neuroglia " and the
methods for its study are just at present the subject of fresh
discussions and investigations. It is, consequently, expedient to
fully describe in the following paragraphs only the principal methods
in use for the demonstration of neuroglia, taking this term to mean
the whole of the sustaining tissue of the central nervous organs, which
is plainly not connective tissue. For minute technical details and
methods almost exclusively used in histopathology, the original
papers quoted in the following paragraphs should be consulted, as
well as ALZHEIMER, Histol u. HistopatM. Arb., iii, 1910, pp. 406 to
412 ; NISSL, Enzykl. mikr. Techn., ii, 1910, pp. 280 to 283 ; Bonome,
Atti R. Inst. Veneto Sc. Ixvii, 1909.
910. WEIGERT'S Neuroglia Stain (WEIGERT'S Beitr. zur Kenntniss
d. norm, mensch. Neuroglia, Frankfurt-a-Main, 1895 ; and the
CHAPTER XXXV. 481
article " Neurogliafdrbung " in EnzyU. mik. Technik, ii, 1910).—
Pieces of very fresh tissue of not more than \ cm. in thickness are
put, for at least four days, into 10 per cent, formol. They are then
mordanted for four or five days at 36° to 37° C. (or for at least eight
days at the temperature of the laboratory) in a solution containing
5 per cent, of neutral copper acetate, 5 per cent, of acetic acid, and
2J 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, instead of chrome alum, 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 mordanting, the tissues are washed, dehydrated, embedded
in celloidin, and cut. The sections (not too thick) are treated for
ten minutes with a ^ per cent, solution of potassium permanganate
and well washed in water. They are then treated for two to four
hours with a solution of " chromogen." This is a naphthaline
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, 10 c.c. of a 10 per cent,
solution of sodium sulphite are added.
After this the sections are put till the next day into a saturated
(about 5 per cent.) solution of " chromogen." (According to
Bolles Lee, Pal's potassium sulphite may be used instead of the
" chromogen.")
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 70 to 80 per cent, alcohol (to which, after cooling and
decanting, there may be added, if desired, 5 per cent, of a 5 per cent,
aqueous solution of oxalic acid). The sections are treated with this
for from a few seconds to one minute, and mopped up with blotting-
paper, then treated for an instant with saturated solution of iodine in
5 per cent, potassium iodide. They are then differentiated till clear
and light blue with a mixture of equal parts of aniline oil and xylol,
washed thoroughly with pure xylol, and mounted in balsam or,
preferably, in turpentine-colophonium.
Glia fibres and nuclei blue, cytoplasm stainless.
This method only gives good results with the human subject.
911. Modifications of WEIGERT'S Method.— MALLORY (Journ. Exper.
.y 1897, p. 532) fixes tissues for four days in 10 per cent, solution of
31
482 NEUROGLIA AND SENSE ORGANS.
formalin, then for four to eight in saturated solution of picric 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 ammonium bichromate,
makes sections (celioidin) and stains them in Weigert's fibrin stain.
STORCH (Virchow's Archiv., clvii, 1899, p. 127), instead of mordanting
the material in bulk with the copper fluid, first makes celioidin sections
and then mordants them.
BARTEL (Ztschr. wiss. Mikr., xxi, 1904, p. 18) first makes paraffin
sections and treats them with all the reagents used by Weigert without
removing the paraffin, until they have passed the aniline -xylol mixture
which should consist of 1 part of aniline to 10 of xylol (or more), and be
allowed to act for twelve to twenty -four hours.
SAND uses material fixed as for his neurofibril stain (§ 843), and
stains it according to Weigert.
See also AGUERRE, Arch. mik. Anat., Ivi, 1900, p. 509 ; KRAUSE, Abh.
k. Akad. WissencJi. Berlin. Arihang, 1899; WIMMER , Centrbl. allg.
Pathol u. pathol. Anat., xvii, 1906, p. 566 ; GALESESCU, C. E. Soe.
Biol, Ixv, 1908, p. 429.
RUBASCHKIN (Arch. mik. Anat., Ixiv, 1904, p. 577) recommends
injecting centres of small mammals with the fixing liquid. To make
this, take 100 parts of 2 -5 per cent, solution of potassium bichromate
and 0 -5 to 1 of copper acetate, boil, and add 2 -5 to 5 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 same fluid for five to seven days at 35° to 40° C.
Dry superficially, put for six to twelve hours in 95 per cent, alcohol
and embed in celioidin or paraffin. Stain sections on the 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 ; differentiate in aniline or clove oil, and pass through
xylol into balsam. The method gives very sharp results with
small mammals.
912. BENDA'S Method (Neurol. Centrbl, xix, 1900, p. 796 ; and
his article " Neurogliafarbung" Enzykl. mik. Technik, ii, 1910,
p. 308) is as follows :— The material is to be fixed in 90 or 93 per
cent, alcohol for no less than two days. Pieces, not thicker than
J cm. are put for twenty-four hours in officinal nitric acid 1 part,
and distilled water 10 parts ; for another twenty-four hours in
2 per cent, potassium bichromate ; for forty-eight hours in 1 per
cent, chromic acid. After washing for twenty -four hours, they are
dehydrated in alcohols of ascending strength, cleared first in creosote
(twenty-four hours), then in benzol (twenty-four hours), and lastly
embedded slowly in paraffin, this being dissolved in benzol to
saturation first at room temperature, then successively at 38°, 42°
CHAPTER XXXV. 483
and 45° 0., so that pure paraffin, melting at 58° C., is used only for
the embedding proper.
The sections, stuck to slides, are mordanted for twenty-four hours
in 4 per cent, iron alum or in 50 per cent. Liquor ferri sulfurici
oxydati P.G-., thoroughly washed, put for. two hours into an amber-
yellow aqueous solution of sodium sulfalizarinate as directed in
§ 683, rinsed with tap water, and put to stain in 0*1 per cent, toluidine
blue either for fifteen minutes by warming until vapour arises, or
for twenty-four hours at room temperature. After rinsing in
1 per cent, acetic acid or in a very dilute solution of picric acid, the
sections are dried with filter paper, passed through absolute alcohol,
and differentiated for about ten minutes with creosote. They are
then dried once more with filter paper, washed with xylol and
mounted in balsam.
Besides this, Benda recommends hardening and making paraffin
sections as above, then staining by Weigert's method (§ 910), but
without passing the sections through the saturated solution of
" chromogen," and using instead of Weigert's methyl violet solution
a freshly prepared mixture of 1 volume of saturated solution of
crystal violet, 1 volume of 1 per cent, acid alcohol, and 2 volumes of
aniline water.
Benda also uses Heidenhain's iron haematoxylin to stain paraffin
sections of pieces treated as described, differentiating either with
2 per cent, iron alum or with Weigert's borax-ferricyanide mixture.
913. MALLORY'S Haematoxylin Stains (Journ. Exper. Med., v,
1900, p. 19). — Tissues to be fixed, mordanted and cut as directed
under MALLORY, § 911. The sections are put for a quarter of an
hour into 0 -5 per cent, solution of potassium permanganate, washed
and put for another 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 phosphotungstic hcematoxylin. Wash,
dehydrate in 95 per cent, alcohol, clear with origanum 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 iron sesquichloride. Neuroglia and fibrin
blue, the rest colourless.
MALLORY'S phospho-molybdic hwmatoxylin may also be used for
the stain, but it is less elective.
914. ANGLADE and MOREL'S Victoria Blue Method (Rev. N enrol.,
ix, 1901, p. 157). — Harden in a mixture of. 3 parts of liquid of Fol.
31-2
484 NEUROGLIA AND SENSE ORGANS.
(§ 47) with 1 of 7 per cent, sublimate solution, dehydrate with
alcohol followed by acetone, make paraffin sections, and stain in
saturated aqueous solution of Victoria blue heated till it steams ;
rinse with Gram's fluid, differentiate with xylol 1 part, aniline 2 parts,
and mount in balsam. Simple, applicable to lower animals, and
gives very sharp pictures.
915. DA FANG'S Methods (Ricerche Lab. Anal. Roma ed altri Lab.
BioL, xii, 1906). — Method I. is a modification of MALLORY'S phospho-
tungstic haematoxylin process (§ 913). Small pieces of fresh tissue are
fixed for twenty-four to forty-eight hours in a mixture of 72 volumes
of pyridine and 28 of 50 per cent, nitric acid. After washing for
about six hours, the pieces are dehydrated and embedded in paraffin.
The sections, stuck to slides by the albumin method, are treated as by
MALLORY'S method, and stained with an old solution of MALLORY'S
phosphotungstic haematoxylin, but prepared without the addition of
hydrogen peroxide. In order to increase the contrast between
neuroglia fibres (blue-violet) and the protoplasm of neuroglia cells
(pink) DA FANO dehydrates the stained sections in 95 per cent,
alcohol to which a small quantity of an alcoholic solution of eosin
has been added.
Method II. is a modification of BENDA'S process (§ 912). Very
small pieces are fixed for thirty-six to seventy-two hours in a mixture
of 2 volumes of the fixing fluid used for Method I. and 1 volume of
1 per cent, osmic acid. After washing for six to twelve hours, the
pieces are embedded in paraffin. The sections, stuck to slides, are
successively mordanted for twenty-four hours each with WEIGERT'S
copper acetate-chromium fluoride fluid (§ 910), 2 per cent, chromic
acid, and 2 per cent, iron alum rinsing in water before passing
them from one into the other mordant. They are lastly either
treated and stained as by BENDA'S alizarine-toluidine blue process,
or as by HEIDENHAIN'S iron haematoxylin method.
Method III. was arrived at in an endeavour to make use of
unsuccessful preparations made by CAJAL'S reduced silver method.
Pieces treated as by Cajal's formula la or one of its modifications
(§ 837), or simply fixed in 2 or 3 percent, silver nitrate at 36° to
37° C., are embedded in paraffin. The sections, stuck to slides, are
bleached by Pal's differentiation method for myelin stain, and then
mordanted and stained as by Method II.
916. HELD'S Method for Marginal Neuroglia (Monatschr. Psych.
NeuroL, xxvi, 1909 ; Erganzungsh., p. 360).— Tissues are preferably
fixed by means of a modified Zenker's fluid consisting of Muller's
CHAPTER XXXV. 485
fluid 100 c.c. and sublimate 3 grms., with the addition at the moment
of use of acetic acid 3 c.c., formalin 0-5 c.c. The fluid should be
warmed at 35° to 40° C. and injected through the blood-vessels,
the blood being first washed away by means of Ringer's solution to
which 1 : 1000 of amylnitrite was added. The tissues are treated
in the usual way and embedded in celloidin. The sections
are first treated for five minutes with a 1 per cent, solution of
caustic soda in 80 per cent, alcohol and washed in distilled water,
and then mordanted for a few minutes in 5 per cent, iron alum and
washed once more. For staining, Held adds to some distilled water
a few drops of a very old molybdic acid hsematoxylin, enough to
impart to the water a bluish- violet tone, and stains therein for twelve
to twenty-four hours at 50° C. The stain is prepared by dissolving
1 grm. of heematoxylin in 100 c.c. of 70 per cent, alcohol and adding
an excess of molybdic acid. Differentiation is carried out by means
of the same iron alum solution used for mordanting ; wash well ;
counterstain with v. Gieson picro-fuchsin solution ; wash in 96 per
cent, alcohol, dehydrate and mount as usual.
Neuroglia cells and fibres greyish-black ; marginal neuroglia
(membrana limitans marginalisand membrana limitans perivascularis)
sharply differentiated ; connective tissue pink-red.
917. Other similar Methods. — LHERMITTE and GUCCIONE (Semaine
Med., xxix, 1909, p. 205) have the following modification of ANGLADE and
MOREL'S method : Sections, made by the freezing method from formalin
material, are collected in distilled water and then kept for two hours
in a cold -saturated solution of sublimate and for two days in a mixture
consisting of 3 parts of 1 per cent, osmic acid, 35 of 1 per cent, chromic
acid, 7 of 2 per cent, acetic acid, 55 of distilled water. The rest as
Anglade and Morel.
Similarly MERZBACKER (Journ. Psychol. Neural. , xii, 1909, p. 1).
DE ALBERTIS (Pathologica, xii, 1920, p. 240) has recently proposed
the following combination of the methods of WEIGERT, MALLORY, and
ANGLADE and MOREL. Sections are made by means of a freezing
microtome from pieces fixed in 15 to 20 per cent, formalin for about
twenty -four hours, but not longer than three days. They are transferred
into a bath of 2 per cent, acetic acid in 1 per cent, chromic ac^d (time
not stated), and then washed for some hours in repeatedly changed
distilled water, oxidised for ten to fifteen minutes in 1 per cent,
potassium permanganate, washed again in distilled water, reduced for
fifteen to twenty minutes in 1 per cent, oxalic acid and lastly put to
stain for twelve to twenty -four hours in a saturated solution of Victoria
blue. For the further treatment sections are washed in distilled water
and from this lifted, one by one, by means of a thin glass or platinum
spatula, this to be used to plunge each section for an instant first into
concentrated Lugol's solution, then into absolute alcohol, and lastly into
equal parts of xylol and aniline oil, where the differentiation is accom-
486 NEUROGLIA AND SENSE ORGANS.
plished in a few seconds. Sections are finally collected and washed in
slightly warmed and repeatedly changed xylol, and mounted in xylol -
damar without a cover.
KULTSCHITZKY Rubin's Method (Anat. Anz., viii, 1893, p. 357) is no
longer used. For the slight modification of this method of POPOW, see
ZtscJir. wiss. Mikr., xiii, 1896, p. 358, and for that of BURCIIARDT, La
Cellule, xii, 1897, p. 364.
The method of YAMAGIWA (Virchow's Arch., clx, 1900, p. 358) is also
no longer used.
918. Methods for Protoplasmic Neuroglia and Neuroglia Granules.—
OPPENHEIM (N enrol. Centrbl., xxvii, 1908, p. 643) mordants sections
made from frozen formalin material with Weigert's copper acetate-
chromium fluoride mixture and then stains them with Weigert's iron
hsematoxylin prepared without hydrochloric acid. An important
point of this method is that the material and the sections should
not have been treated with alcohol before staining.
EISATH (Monatschr. Psych. Neurol, xx, 1906, p. 3 ; Arch. Psych.
Nervenkmnkh., xlviii, 1911, p. 897) fixes large pieces in a modified
Orth's formol-Miiller mixture consisting of water 1000 c.c., potas-
sium bichromate 25 grms., sodium sulphate 15 grms., and formalin
150 c.c., to be added at the moment of using the mixture. After
about four weeks the tissues are ready for being cut without em-
bedding, but can be kept for many months, and even years, in 4 per
cent, formalin. The sections are collected in 4 per cent, formalin,
in which they may be kept until wanted. For the staining the
sections are put for thirty seconds in a 0 -2 per cent, solution of sub-
limate, well washed in water, and lifted on to the slide, a dilution of
an old Mallory's phosphomolybdic-carbolic acid hsematoxylin being
poured on them. After a few minutes they are washed with water,
differentiated with a mixture of equal parts of 40 per cent, tannic
acid, 50 per cent, alcohol, and 20 per cent, pyrogallic acid in 80
per cent, alcohol. Wash in alcohol, dehydrate, clear and mount.
FIEANDT (Arch. mikr. Anat., Ixxvi, 1910—11, p. 125) fixes in
Heidenhain's sublimate-trichloracetic mixture, and treats pieces
for five^to seven days with 96 per cent, alcohol, to be changed three
times during the first twenty-four hours and daily in the following
days. After dehydration the pieces are embedded in paraffin as
directed by JPrantner. The sections, 3 to 5 ^ thick, are stuck to
slides, freed from sublimate by the usual iodine treatment, and then
stained for twelve to twenty-four hours with Mallory's phospho-
tungstic heematoxylin. Dry with filter paper, differentiate for a
few hours in 10 per cent, iron perchloride in absolute alcohol, blot
once more with filter paper, wash, dehydrate and mount.
CHAPTER XXXV. 487
Neuroglia fibres, cytoplasm of neuroglia cells, and glia granules
stained in various shades of blue and greyish-blue ; all other
elements yellowish-grey or yellowish-brown.
RANKE (Ztschr. ges. Neurol. u. Psych., vii, 1911, p. 355) uses for
similar purposes either celloidin sections of foetal tissues fixed in
picric acid-alcohol or sections made by freezing from formalin
(pathological) material. In the first case the sections are stuck to
slides by pressing with filter paper and then pouring on them methyl-
alcohol until all celloidin is dissolved. He next stains them for a
few minutes with his acid eosin-thionin solution (see further on),
washes with water, and re-stains them, with the help of gentle
heat, with 5 : 1000 Giemsa's " Methylenazur I " ; quick differen-
tiation with distilled water ; 96 per cent, alcohol, cajeput oil, xylol,
balsam. In the case of pathological material the sections are first
treated with 1 per cent, osmic acid in order to stain fatty products
of degeneration, etc., then pressed on to slides and stained as above.
To prepare the acid eosin-thionin mixture, mix and shake repeatedly
1000 c.c. of each 1 : 1000 watery solution of eosin W.G. and 1 : 1000
watery solution of thionin. Leave for forty-eight hours, pour
out the fluid part, and wash the sediment into a paper filter with
distilled water until the wash water is only a little stained. Dry
what remains in the filter, and dissolve it in methyl-alcohol in the
proportion of 0 -3 to 0 -5 per cent.
919. RAM6N Y CAJAL'S Gold Chloride and Sublimate Method
(Tmb. Lab. Invest. Biol, Madrid, xi, 1913, pp. 219 and 255 ; xiv,
1916, p. 155). — At first Cajal used to harden pieces of quite fresh
tissues in 14 per cent, formalin, but in his successive papers he
recommended fixing from two to ten days in —
Formol ....... 15 c.c.
Ammonium bromide .... 1 -5 — 2 grms.
Distilled water . . . . . .85 c.c.
Relatively thick sections (20 to 25 jut) are made by the freezing
method, and collected in distilled water to which a few drops of
formalin have been added. After a quick wash, batches of four to six
sections are each transferred into glass dishes of about 6 cms. in
diameter, and each containing 15 c.c. of a mixture of —
Distilled water 60 c.c.
Corrosive sublimate . . . . .0-5 grm.
1 per cent, gold chloride (Merk, brown variety) 10 c.c.
After~about four hours the sections will be found to have become
488 NEUROGLIA AND SENSE ORGANS.
an intense purple, and can be passed, for five to ten minutes, into
a fixing bath consisting of —
Concentrated solution of sodium hyposulphite 5 c.c.
Distilled water 70 „
Alcohol 30 „
Concentrated solution of sodium bisulphite . 5 ,,
Wash in 50 per cent, alcohol, lift sections on to slides, dry with filter
paper, wash with absolute alcohol, clear with origanum oil, wash
with xylol, and mount in balsam.
To ensure successful results the following points should be borne
in mind : — (1) The gold chloride must be of the brown variety.
Its solution remains unaltered for months if kept in the dark.
(2) The solution of mercury chloride becomes very easily altered,
and is best prepared when required, dissolving it with the help of
some heat, and filtering before adding it to the gold chloride solution.
(3) Best results are obtained by keeping the glass dishes, with the
sections and the gold chloride-sublimate mixture, at a temperature
of 18° to 20° C. If the reagent is freshly prepared, the reaction
will be complete in about four up to six hours. At temperatures
between 14° and 17° C. three or four hours more are necessary to
obtain good stains. With temperatures below 14° or 12° C. it is
very difficult to obtain any reaction at all. One may have recourse
to temperatures above 20° C., up to 27° or 30° C. in special cases, as
Del Rio-Hortega has done for the neuroglia of the pineal body.
(4) More diluted gold baths may be used for economical reasons,
but in this case one must have recourse either to higher temperatures
or to greater lengths of time. (5) To proceed quicker, one may
either double the proportion of sublimate in the formula given above
or double the proportion of gold chloride and treble that of sublimate.
(6) A good means to obtain rapid and vigorous reactions consists
in adding to the gold chloride-sublimate bath either 2 to 3 drops of
a 1 : 1000 solution of erythrosin or a minute quantity of the dry
dye, enough to impart to the bath a slightly orange tone. (7) All
other conditions being the same, results are greatly influenced by
the length of time during which the pieces have been kept in the
fixing fluid. As a rule, they begin to be ripe for cutting from the
end of the third day, and they continue to be in a state favourable
for obtaining good reactions for another five or six up to fifteen or
twenty days. Good stains may be exceptionally obtained after
two months of hardening. Generally, the capacity for taking the
gold disappears first from the protoplasmic, and then from the
fibrous, neuroglia.
CHAPTER XXXV. 489
By means of CajaPs method two categories of neuroglia elements
become stained a dark purple on a much lighter purplish back-
ground. The first category consists of neuroglia cells provided with
a changing number of variously ramified protoplasmic processes,
which inter-cross with those of other cells, and thus give origin to
CajaFs pleurigenic plexus. These neuroglia cells prevail in the grey
layers of the human cerebral cortex, and form the bulk of the
protoplasmic neuroglia (§ 909). In Cajal's preparations they appear
beset with vacuoles, situated both within their cytoplasm and along
their processes. The vacuoles or spaces are occupied by granules
(gliosomes), which may be stained either by Cajal's uranium nitrate
method (§ 847) (superficial sections) or by methods generally
used for the demonstration of mitochondrial formations as well as
by the methods of Eisath and Fieandt. The other category of
neuroglia elements shown by the gold chloride and sublimate
method consists of astrocytes, viz., of neuroglia cells, also provided
with a changing number of processes, but chiefly characterised by
the absence of gliosomes and the presence of fibres which, though a
product of differentiation of the protoplasmic portions of the
astrocytes, never become entirely independent of the latter. These
fibres appear to correspond to those stainable by the methods
described in §§ 910 to 916. The astrocytes prevail in the white
matter of the central nervous system, and form the bulk of the
fibrous neuroglia (§ 909). Neuroglia cells, in part protoplasmic and
in part fibrous, occur chiefly at the points of transition between the
grey and the white substances of central nervous organs.
The gold chloride and sublimate method leaves unstained a
third category of elements, the existence of which was at first recog-
nised by Cajal by means of this negative character, but they were
subsequently studied by him in superficial sections of pieces stained
by his uranium nitrate method and other cytological methods.
The cells belonging to the category now considered appear in
uranium nitrate preparations as roundish elements, but, as a matter
of fact, they also are provided with a changing number of variously-
ramified protoplasmic processes (see § 919). As Cajal was not able
to come to any definite conclusion in regard to their nature, he
proposed to term them the " third element," i.e., a category of cells
which, though non-nervous in character, do not plainly form part
either of the connective tissue (blood-vessels, pial septa) or of the
neuroglia, this term being, in Cajal's opinion, reserved for those
elements which are genetically derived from an evolution of the
ependymal epithelium.
490 NEUROGLIA AND SENSE ORGANS.
920. ACHUCARRO'S Tannin Method and DEL Rfo-HoRTEGA's
Modifications. — The methods described in this paragraph can be
considered as the direct outcome of various efforts at modifying
the Bielschowsky method for sections (§ 840) in such a way as to
obtain a neuroglia stain. As a matter of fact, they all stain both
neuroglia cells (astrocytes) and connective tissue elements. In
other words, they are not elective, and may be used for the study
of reticular tissue in non-nervous organs, as well as of other
histological details in nervous and non-nervous tissues.
PERUSINI'S modification of BIELSCHOWSKY'S method (Neurol.
CentrbL, xxix, 1910, p. 1256) should be first remembered. Pieces
of fresh material were fixed in Weigert's formalin-copper acetate-
chromium fluoride mixture for neuroglia stain (§ 910), cut by the
freezing method, and stained as by Bielschowsky 's method for
sections, without pyridine treatment. Achucarro did the same,
except for silvering by Kamon y Cajal's reduced silver process.
ACHUCARRO'S tannin method (Bol. Soc. Espan. BioL, Madrid,
1911, p. 139) consisted in putting sections made from frozen formol
material into a cold-saturated solution of tannin and warming this
until vapour arose. Without waiting for the tannin to become cool
again, the sections were, one by one, quickly rinsed in water and put to
stain for about ten minutes into three successive glass dishes, each
containing 10 c.c. of distilled water and 6 to 8 drops of Bielschowsky
ammoniacal silver nitrate-and-oxide bath, prepared beforehand, as
described in § 841. As soon as they turned dark yellow, they were
transferred into 10 per cent, formalin, and, after about ten minutes,
washed, dehydrated and mounted.
The results obtained by such a method were rather uncertain
and Achucarro himself felt the necessity of modifying it in the
following way, published by DEL Rio-HoRTEGA (Trab. Lab. Invest.
BioL, Madrid, xiv, 1916, p. 181) :— (1) Fix pieces, 2 to 3 mm. thick,
for two or three days in formalin neutralised with ammonia.
(2) Make sections of 10 ju, and mordant them in 10 per cent, tannin
until vapour arises. (3) Without waiting for the tannin to become
cool, wash the sections in distilled water alkalised with a few drops
of ammonia until they have again acquired their flexibility.
(4) Treat them with the diluted ammoniacal silver nitrate solution
as described above, but adding only 2 or 3 drops of it to every 10 c.c.
of distilled water. (5) Reduce in 20 per cent, formalin, either
neutralised as for fixing, or (according to Del Rio-Hortega) contain-
ing an excess of ammonia, say, 6 to 8 drops to every 10 c.c. of
20 per 'cent, formalin.
CHAPTER XXXV. 491
DEL Rj'o-HoRTEGA (op. cit.) found that the method could be
further modified, and usefully employed for the staining not only
of the neuroglia, but also of centrosomes of nerve cells and neuroglia
cells, mitochondria, secretion granules, intra-epithelial fibrils, reticular
tissue, collagenous fibres, etc. The modifications proposed by Del
Rio-Hortega for these various purposes are four in number, and
known as the variants of AcMcarro's method.
Modification I. — Suitable for the staining of fibrous neuroglia
as well as for elastic membranes and connective tissue cells. (1) Fix
tissues for no less than ten days in 10 per cent, formalin. (2) Make
sections by the freezing method, and mordant them for five minutes
in 3 per cent, tannin kept at a temperature of 50° to 55° C. (3) Wash
them in distilled water alkalised with ammonia, and transfer them
successively into three glass dishes, each containing 1 c.c. of
ammoniacal silver nitrate, prepared as described in § 841, and 10 c.c.
of distilled water. (4) As soon as they have taken a distinct
yellowish-brown colour, wash them in distilled water and reduce
them in a 1 : 500 gold chloride solution kept for twenty or thirty
minutes at a temperature of about 40° to 45° C. (5) Fix with 5 per
cent, sodium hyposulphite, wash, dehydrate and mount as usual.
Modification II. — Good chiefly for reticular tissue and its histo-
genesis. Material may be fixed either in 10 per cent, formalin or
Bouin's fluid, or alcohol ; if one or the other of these last two fluids
has been used, it is advisable to re-transfer pieces for a few days
into a formalin solution. Sections should, as a rule, be made by
the freezing method, but pieces may also be embedded in celloidin,
this being dissolved after cutting. The sections, however obtained,
are mordanted for five minutes at 50° to 55° C. or for fifteen to
thirty minutes at 40° to 45° C. in a 1 per cent, alcoholic solution of
tannin. Stain as in Modification I ; reduce for half a minute in
20 per cent, formalin, neutralised by shaking with chalk ; wash,
dehydrate and mount.
Modification III. — Particularly good for collagenous fibres, but
also for neuroglia fibres. Proceed as in Modification II until the
sections are placed in the staining bath ; keep them therein until
brown ; reduce and fix as in Modification I.
Modification IV (op. cit., xv, 1918, p. 375, note). — Suitable for the
demonstration of the protoplasmic neuroglia. Frozen sections of
formalin material are treated for some minutes at 45° to 50° C.
with a mixture of tannin, 3 grms. ; ammonium bromide, 1 grm. ;
distilled water, 100 c.c. Wash and stain as in Modification I ;
reduce in 20 per cent, formalin neutralised with chalk ; tone with
492 NEUROGLIA AND SENSE ORGANS.
0-2 per cent, gold chloride; fix, wash, dehydrate and mount as
usual.
921. DEL KIO-HORTEGA'S Carbonate of Silver Method (Trab. Lab.
Invest. Biol, Madrid, xv, 1918, and xvii, 1920 ; Bol Soc. Esp. Biol,
viii, 1918). — Pieces of quite fresh nervous tissues are fixed in Cajal's
ammonium bromide-formalin mixture, and kept therein for different
periods of time, according to the purposes in view. If it is desired
to stain the protoplasmic neuroglia, pieces are best fixed for twenty
to thirty or forty days ; after this time they are for some months
in a condition particularly suitable for the staining of the fibrous
neuroglia. But if the time of fixation is limited to one or two days
at the temperature of about 35° C., or to two up to four days at room
temperature, the tissues are in a state favourable to the impregnation
of Cajal's " third element " (§ 919), which Del Kio-Hortega proposes
to term either microglia or mesoglia, the first of these two denomina-
tions being simply used with reference to the smallness of the
elements thus named, the second implying that they do not belong
to the neuroglia as this term is understood by'Cajal and his pupils.
See § 919. For the staining one may choose one or the other of the
following three processes : —
Process I, for protoplasmic and fibrous neuroglia. — Sections
made by the freezing method are washed in two or three changes
of distilled water and transferred into a crystallising basin
containing 5 or 10 c.c. of ammoniacal silver carbonate solution,
prepared as follows : — To 50 c.c. of 10 per cent, silver nitrate an
equal or greater quantity of cold-saturated lithium carbonate
solution is added, so as to precipitate all silver in the form of
silver carbonate. The fluid part is poured off, and the precipitate
first washed with 200 to 300 c.c. of distilled water, and then taken
up with about 50 c.c. of diluted ammonia, by means of which it
is entirely dissolved. The solution is diluted with distilled water
up to a total volume of 250 c.c. and poured into a dark brown
bottle, where it keeps indefinitely, if put away in some dark
place.
The crystallising basin, with the ammoniacal silver carbonate and
the sections placed therein, is warmed, either in an incubating stove
at 45° to 50° C. or over a flame, until the sections become a greyish-
yellow colour. This requires only a few minutes if the sections are
moved about so that they may stain uniformly. Good results may
be also obtained by staining at 35° C. for twelve to fourteen hours
or at room temperature for one or two days. Without waiting for
CHAPTER XXXV. 493
the silver solution to become cool, the sections are quickly washed
in distilled water and then transferred, one by one, into 20 per cent,
formalin neutralised with chalk. After one or two minutes, the
reduction is complete, and the sections may be washed, toned, fixed
and dehydrated, cleared with a mixture of carbolic acid 5 parts,
xylol 45 parts, creosote 50 parts, and mounted in balsam.
Process II, for microglia. — Sections are made as above, and then
treated for ten or fifteen minutes at 50° or 55° C. with the bromide-
formalin solution used for fixing. After washing in two or three
changes of water, one continues as in Process I, but warming the
ammoniacal silver carbonate solution at 50° or 55° C. until the
sections are dark yellow.
Process III, also for microglia. — The pieces are warmed for ten
minutes in the fluid used for fixing, and then cut by the freezing
method. The sections are washed in distilled water and stained
for ten to thirty minutes, either at room temperature or by careful
gentle warming, with an ammoniacal silver carbonate solution,
prepared by adding to 10 c.c. of 10 per cent, silver nitrate, first,
30 c.c. of 5 per cent, sodium carbonate, then ammonia, drop by
drop, until the precipitated silver carbonate is dissolved, and, lastly,
distilled water up to a total volume of 150 c.c. The sections are
kept in the impregnating bath for from ten to thirty minutes at
room temperature, but they should nevertheless remain almost
colourless. For the reduction, 1 per cent, formalin is used, and
sections are left therein until they have taken a greyish-yellow
tinge ; wash, tone, etc., as in Process I.
The above refers to material fixed in Cajal's ammonium bromide-
formalin mixture ; if nervous tissues are fixed in 10 per cent,
formalin and sections treated as in Process I, nerve cells and axis
cylinders become stained as by Bielschowsky's method. If formol
sections of non-nervous tissues are treated in the same way, the
reticular tissue becomes stained.
Retina*
922. Fixation and Hardening. — Notwithstanding the Encycl. mik.
Technik., 2nd ed., p. 75, I hold that osmic acid is by far the best
fixing agent. The retina of small eyes is best prepared by fixing
the entire unopened bulb with osmium vapours.
Besides the sources quoted in the text, see SELIGMANN, Die mikro-
skopischen Untersuchungsmethoden des Auges, Berlin, S. Karger (Karl-
* By A. Bolles Lee.
494 NEUROGLIA AND SENSE ORGANS.
strasse 13), 1899 ; GREEF, Arileitung zur Mikr. Untersuch. d. Auges,
Berlin, Hirschwald, 3rd ed., 1910; and the Art. "Retina" in Encyd.
mik. Technik., 2nd ed., p. 575.
SZENT-GYORGI (Zeit. /. wiss. Mikr., xxxi, 1914), uses the following
fluid : —
Acetone . . . . . . .125 c.c.
Glacial acetic . . . . . . 5 „
Formalin . . . . . . 40 „
Sublimate 4 grins.
Aq. dest 100 c.c.
Leave whole small eyes in 100 c.c. of this mixture for two to three
days, larger whole eyes six to seven days, after which one adds an
additional 50 c.c. of acetone to the fixative and leaves for a further two
or three days. Transfer to pure acetone for three or four days, renewing
on the last day ; then bring the eyes into a vessel of acetone, with a
thick layer of desiccated calcium chloride at its bottom, for three or
four days, renewing the CaCl, if necessary. Transfer from the acetone
into a mixture of half ether, half absolute alcohol, then proceed as for
celloidin embedding.
According to RANVIER (Traite, 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
CHAPTER XXXV. 495
•
20 per cent., and ending with absolute, taking five days from first
to last.
Similarly KOCHON-DUVIGNEAUD (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 Miiller. Formalde-
hyde mixtures he does not recommend.
LEBEB, (Munch, med. Wochenschr., xli, 1894, p. 605 ; 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
derangement of the parts. The retina lies flat, and is at least as well
preserved as with solution of Miiller.
See also HIPPEL (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 HERZOG (Arch. mik. Anat., Ix, 1902, p. 517, and Encycl.
mik. Technik., p. 75), who also approves of formol, but insists that it
should be acid, and adds 3 to 5 per cent, of acetic acid.
KOLMER (Arch. Gesammte Phys., cxxix, 1909, 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.
BEND A (Verh. Ges. Naturf. Mrzte, Ixxi, 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 1£ 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.
923. Staining. — For general views I recommend iron-hsematoxylin,
followed by Saurefuchsin or Picro-Saurefuchsin, or preceded by
Bordeaux ; or Kernschwarz, followed by safranin, or the Ehrlich-
Biondi stain.
The Methylen-blue intra-vitam stain has given valuable results ;
see the methods of DOGIEL.
But the most important method is the bichromate and silver
impregnation of GOLGI, first applied to this object by TARTUFEEI
(Intern. Monatsschr., iv, 1887, p. 421). This author employed the
rapid process. So also RAM6N Y CAJAL (La Cellule, ix, 1893, p. 121)
with the double-impregnation process, § 886. To avoid the forma-
tion 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. tit,, p.' 130). After
496 NEUROGLIA AND SENSE ORGANS.
•
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.
RAMON also employs his neurofibril silver method, see Intern.
Monatsschr. Anat. Phys., xxi, 1905, p. 393.
GOLGI'S sublimate impregnation (Cox's form) has also been
successfully employed by KRAUSE and RAM6N.
The bichromate and silver method serves for the study of the
fibres of Miiller and neuroglia cells, as well as neurones. Weigert's
neuroglia stain does not give good results.
LENNOX (Arch. f. Ophthalm., xxxii, 1886, 1 ; Zeit. wiss. Mik.,iii, 1886,
p. 408) has used Weigert's hsematoxylin method.
KuHNT(t/ew. Zeit. Naturw., Bd. xxiv, 1890, p. 177) employs Pal's
modification. Similarly SCHAFFER (Sitzb. Akad. wiss. Wien., xcix,
1890, Abth. 3, p. 110; Zeit. wiss. Mik., viii, 1891,. p. 227). These
methods give a differential stain of rods and cones.
For the zonula and ciliary body see MA WAS, Arch, ft Anat. micr., xii,
1910, p. 103.
924. Dissociation. — For maceration preparations you may use
weak solutions (O2 to 0-5 per cent.) of osmic acid for fixation, and
then macerate in 0-02 per cent, chromic acid (M. SCHULTZE), or in
iodised serum (M. Schultze), or in dilute alcohol (LANDOLT), or in
Miiller's solution, or (RANVIER, Traite, p. 957) in pure water, for
two or three days. THIN (Journ. of Anat., xiii, 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 (Intern. Monatsschr. Anat., i, 1884, p. 225) recommends
treatment for several days with 10 per cent, chloral hydrate solution ;
the rods and cones are well preserved.
Inner Ear.
• 925. Inner Ear, Dissection. — For the dissection of the human ear see
POLITZER, " Die anatomische u. histologische Zergliederung d. men-
schlichen C4ehoroganes," Stuttgart (Enke), 1889 (Zeit. wiss. Mik., vii,
1890, p. 364). Amongst the lower mammalia, the guinea-pig is a
favourable subject, as here (as with some other rodents) the cochlea
projects freely into the cavity of the bulla, and may be easily removed
CHAPTER XXXV. 497
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.
926. Preparation. — SCHWALBE (Beitr. z. Phys. • (C. Ludwig's
Festschr), 1887, p. 200).— Fix (cochlea of guinea-pig) for eight to
teii hours in " Flemming," wash in water, decalcify (twenty-four
hours is enough) in 1 per cent, hydrochloric acid, wash the acid
out, dehydrate, and embed in paraffin.
PRENANT (Intern. Monatsschr. 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
neqessary 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 0-1 per cent, solution ; the stria may then
be got away whole.
KATZ (Zeit. wiss. Mik., xxv, 1908, p. Ill) fixes the inner ear,
opened, for one or two hours in 30 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 or 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
embeds in celloidin or sometimes paraffin.
Similarly WITTMAACK, see § 799.
BIELSCHOWSKY and BRUEHL (Arch. mik. Anat., Ixxi, 1908, 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 (§ 840 — twenty-four hours in nitrate of 4 per cent., but only a
few minutes in the oxide bath).
Similarly MULLENIX (Bull. Mus. Comp. Zool. Harvard Coll., liii,
1909, p. 215).
STEIN (Anat. Am., xvii, 1900, p. 398) decalcifies in celloidin by the
method of ROUSSEAU. So also KISHI (Arch. mik. Anat., lix, 1902,
p. 173).
For staining, RANVIER (Traite, p. 991) employs his gold and formic
acid method.
The bichromate and silver method of GOLGI may be employed
with/ce tal or new-born subj ects . The methylen blue intra vitam method
M.
32
498 NEUROGLIA AND SENSE ORGANS.
has given good results. For the higher vertebrates the injection
method should be employed. The Encyd. mik. Technik., i, p. 511,
recommends injection of 1 c.c. of 0-5 to 1 per cent, solution
every five minutes through the vena fernoralis until the death of
the animal. The cochlea then to be got out, exposed to the air
for fifteen or thirty 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 twenty-four hours and • got into
paraffin.
For fishes and amphibia the immersion method will suffice.
927. Other Methods. — WALDEYER, Strieker's Handb., p. 958 (decalci-
fication either in 0-001 per cent, palladium chloride containing 10 per
cent, of HC1, or in chromic acid of 0-25 to 1 per cent.).
URBAN PRITCHARD (Journ. Roy. Mic. Soc., xii, 1872, p. 380).— Decal-
cification in 1 per cent, nitric acid.
LAVDOWSKY (Arch. mik. Anat., xiii, 1877, 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 FLESCII (Arch. mik. Anat., xvi, 1879, p. 300) ; TAFANI (Arch.
Ital. de Biol., vi, 1884, p. 207) ; EICHLER ( Abh. math-phys. Cl. Sachs.
Ges. Wiss, xviii, 1892, p. 311 ; Zeit. wiss. Mik., ix, 1892, p. 380 (injection
of blood-vessels of the labyrinth) ) ; SIEBENMANN (Die Blutgejdsse im
Labyrinthe des menschlichen Ohres, Wiesbaden, Bergmann, 1894 ; Zeit.
wiss. Mik., xi, 1894, p. 386) ; GRAY (Journ. Anat. Phys., xxxvii, 1903,
p. 379) ; SCOTT (ibid., xliii, 1909, p. 329).
928. Olfactive Nerve-endings, Tactile Corpuscles, etc. — Besides
the gold method, Chapter XVII, and the methylen-blue method,
Chapter XVI, the rapid bichromate and silver method of GOLGI
should be employed, and for the olfactive mucosa gives the best
results. See VAN GEHUCHTEN, La Cellule, vi, 1890, p. 405. For
intra-epidermic nerve-endwigs, besides the methods given in Chap-
ter XXVII, the GOLGI method should be employed. According to
VAN GEHUCHTEN (La Cellule, ix, 1893, p. 319) it gives much better
results than gold methods. He uses the rapid process. For tactile
corpuscles, etc., besides the methods given in §§ 721 and 722, see
RAM6N Y CAJAL'S neuro-fibril methods.
CHAPTER XXXVI.
METHODS FOR INVERTEBRATES.
Tunica ta.
929. Fixation of Tunicata.— A method of Lo BIANCO * 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 treatment for from three to twelve
hours with chloral hydrate (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 BENEDEN'S acetic acid process, § 84 (steel instruments
being avoided for manipulating them). I strongly recommend this
process.
S. Lo BIANCO recommends for this group the chloral hydrate^
process, followed by fixation with sublimate or chromo-acetic
acid.
CAULLERY (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 Pyrosoma. Lo
BIANCO puts them for a quarter of an hour into 50 per cent, alcohol
containing 5 per 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 acid containing 5 per cent, acetic acid, the soft ones
with 1 per cent, chromic acid containing -xn per cent, osmic acid, or
10 parts of 1 per cent, chromic acid, with 1 of formol and 9 of sea
water, Doliolidse with sublimate, or the above osmic mixture, or a
* References to methods of Lo BIANCO in this Chapter are all to his
paper in Mitth. Zool Stat. Neapel, ix, 1890, p. 435.
32—2
500 METHODS FOR INVERTEBRATES.
mixture of 10 parts 10 per cent, solution of sulphate of copper with
1 part concentrated sublimate solution, or the formol mixture.
Molluscoida.
930. Bryozoa. — For some methods of killing and fixing see §§13,
20, and 21. S. Lo BIANCO employs for Pedicellina and Loxosomi
the chloral hydrate method, fixing with sublimate. For Flustra,
Cellepora, Bugula, Zoobothrium, he employs the alcohol method of
EISIG, § 18. For Cristatella see §§ 16, 20. See also BRAUN.
< CONSER (Trans. Amer. Mic. Soc., xvii, 1896, p. 310) kills the
fresh water forms with cocaine, puts them for an hour into 1 per-
cent, chromic acid, and passes through water into alcohol, etc.
Similarly CALVET (Hist. Nat. Bryozoaires, Montpellier, 1900, p. 15),
for marine forms.
ZSCHIESCHE (Zool. Jahrb., xxviii, 1909, p. 6) fixes larvae of
Alcyonidium (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
twenty-five to thirty minutes.
931. Brachiopoda. — Lo BIANCO kills small animals in 70 per cent,
alcohol, larger ones being first narcotised with alcohol a'nd sea
water.
BLOCHMANN (Untersuch.fein. Bau Brachiopoden, Jena, 1892, p. 5)
'fixes principally with sublimate, macerates by the HERT WIGS'
method, § 526, 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 embeds in paraffin or celloidin.
See also EKMAN, Zeit. wiss. Zool., Ixii, 1896, p. 172.
Mollusca.
932. Fixation. — To kill Mollusca extended for dissection make
up stock solution A : 90 parts absolute alcohol, 10 parts turpentine ;
10 per cent, of A to 90 per cent, water. Leave in twelve to twenty-
four hours. Lo BIANCO narcotises Lamellibranchs for six to ten
hours or more with alcohol, § 18, and then kills them.
LIST (Fauna Flora Golf. Neapel, xxvii, 1902, p. 292) narcotises
Mytilida3 with 2 per cent, of cocaine in sea water, and (for preserva-
tion of cilia) fixes in sea water, with 10 per cent, of formol.
Lo BIANCO advises that Prosobranchiata, and, amongst the
Heteropoda, Atlantidse, be narcotised with 70 per cent, alcohol, § 18.
For Opisthobranchiata I recommend sudden killing with liquid of
Perenyi, or the acetic method, § 929. Aplysia may first be narcotised
CHAPTER XXXVI. 501
by subcutaneous injection of about 1 c.c. of a 5 to 10 per cent,
solution of hydrochlorate of cocaine (ROBERT, Bull. Sclent, de la
France, etc., 1890, p. 449 ; Zeit. wiss. mik., ix, 1892, p. 216), or
(SCHONLEIN, Zeit. Biol., xxx, 1893, p. 187) 1 c.c. of 4 per cent,
solution of Pelletierin. For Lo BIANCO'S various methods see the
original, p. 467.
For Pteropoda in general, liquid of Perenyi. Creseis is a difficult
form. Lo BIANCO advises the alcohol method, § 18. For the
Gymnosomata he narcotises with 0-1 per cent, chloral hydrate.
For terrestrial Gastropods see §§ 23 and 26. MARCHI (Arch. 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.
LANG (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, cocaine,
and after five to fifteen minutes dissects and fixes.
HEYMANS (Bull. Acad. Belg., xxxii, 1896, p. 578) injects 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.
933. Liver of Mollusca. — ENRIQUES (Mitth. Zool. Stat. Neapel,
xv, 1901, p. 289) fixes the liver of Octopus and Sepia with sublimate.
For Aplysia (especially in summer) alcohol, formol, and chromic
mixtures are counter-indicated, on account of the carbohydrates in
the cell. Sublimate is best.
934. Nervous System of Pulmonata. — B. de NABIAS (Act. Soc.
Linn. Bordeaux, 1894 ; Rech. Hist, centres nerveux des Gasteropodes ,
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 cqnt.
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
hsematoxylic eosin, or R. Heidenhain's haematoxylin, or a copper
hsematoxylin of Viallanes, and embeds in paraffin. He also stains
by the rapid method of GOLGI, embedding, however, the ganglia
in celloidin directly after the hardening in osmic acid and bichromate,
and treating the sections with the silver (p. 458). He stains with
methylen blue by treating the ganglia in situ for twelve to twenty-
four hours with a 1 per cent, solution^
502 METHODS FOR INVERTEBRATES
DREYER (Zeit. wiss. Zool., xcvi, 1910, p. 380) narcotises Nudi-
branchs with cocaine, 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 hsematoxylin.
See also, for nerve-cells, McCLURE, Zool. Jahrb., 1898, p. 17
(MANN'S methyl blue and eosin, or BENDA'S safranin and Lichtgriin),
and LEGENDRE, Arch. mic. Anat., x, 1909, p. 312.
935. Eyes of Gastropoda (FLEMMING, 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.
SMITH (Bull. Mus. Comp. Zool. Harvard, xlviii, 1906, p. 238)
macerates eyes for at least two days in 9 parts of water with 1 of
weak mixture of Flemming, followed by glycerin of 10 per cent.
He bleaches them (in sections) with nitric acid and chlorate of
potash.
936. Eyes of Cephalopoda and Heteropoda (GRENACHER, Abh.
naturf. Ges. Halle-a.-S., Bd. xvi, 1896, p. 213). — Depigment with
hydrochloric acid (in preference to nitric acid). The mixture § 574
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.
LENHOSSEK (Zeit. wiss. Zool, Iviii, 1894, p. 636 ; Arch. mik.
Anat., xlvii, 1896, p. 45) applies the method of GOLGI to the eyes of
Cephalopods.
Similarly KOPSCH (Anat. Anz., xi, 1895, p. 362), but using formol
instead of the osmic acid.
HESSE (Zeit. wiss. Zool., Ixviii, 1900, p. 418) fixes eyes of Hetero-
poda with 1 of formol to 4 of water, and (p. 257) bleaches those of
Cephalopoda by the methods of GRENACHER and that of JANDER,
§575.
See also MERTON, ibid., Ixxix, 1905, p. 326.
937. Eyes of Lamellibranchiata.— See PATTEN, Mitth. Zool. Stat.
Neapel, vi, 1886, p. 733, and KAWITZ, Jena. Zeit. Naturw., xxii,
1888, p. 115, and xxiv, 1890, p. 579 (bleaches with caustic soda) ;
see § 576. HESSE (op. cit., last §, p. 380) employs the method of
JANDER for Area. He fixes the eye of Pecten in 10 per cent, formol
for five minutes, followed by sublimate or picro-nitric acid.
CHAPTER XXXV I. 503
938. Shell. — Sections of non-decalcified shell are easily obtained
by the usual methods of grinding, or, which is often a better plan,
by the methods of v. KOCH or EHRENBAUM. MOSELEY (Quart.
Journ. Mic. 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 CHITONID.E.
939. Injection of Acephala (FLEMMING, Arch. mik. Anat., 1878,
p. 252). — To kill the animals freeze them in a salt and ice mixture,
and throw them for half an hour into lukewarm water. They will
be found dead, and 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 DEWITZ, Anleit. zur Anfert. zootom. Prdp., Berlin, 1886, p. 44
(Anodonta) and p. 52 (Helix).
DAKIN (Liverpool Mar. Biol. Comm., xvii, 1909, p. 76) 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.
MOZEJKO (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.
940. Maceration Methods for Epithelium. — ENGELMANN (Pfluger'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 in 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
(5 parts cold saturated aqueous solution to 1 part water).
Dr. ORTON uses borax carmine and picro-nigrosin (in liter a).
BELA HALLER'S Mixture, see § 532; BROCK'S Medium, § 523;
MOBIUS'S Media, § 527 ; the second of these is much recom-
504 METHODS FOR INVERTEBRATES.
mended by DBOST (Morphol. Jahrb., xii, 1866, p. 163) for
Cardium and My a.
PATTEN (Mitth. Zool. Stat. Neapel, vi, 1886, p. 736) takes sulphuric
acid, 40 drops to 50 grms. of water. Entire molluscs, without the
shell, may be kept in it for months.
BERNARD (Ann. Sci. Nat., ix, 1890, p. 191) macerates the mantle
of Prosobranchs in a mixture of 1 part each of glycerin and acetic
acid, 2 parts each of 90 per cent, alcohol and 0-1 per cent, chromic
acid and 40 parts water, which acts in from a quarter of an hour to
three hours. He 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 1 part glycerin,
2 of acetic acid and 40 of water.
941. Mucus Glands. — RACOVITZA (Arch. Zool. exper. [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 RIPART and PETIT, 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.
942. General Methods for Arthropoda.— As general methods for
the study of chitinous structures, the methods worked out by Paul
Mayer (see §§ 8, 96 and 97) 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.
942A. Mounting Small Arthropods.— Dr. A. D. Imms informs me
that for mounting and clearing aphids and other small insects, etc., the
following formula as used by Professor Berlese for Acarina gives good
results : —
H20 20grs.
Chloral hydrate 160 „
Gum arabic .... . . 15 „
Glucose syrup . . ... . 10 ,
Acetic acid
CHAPTER XXXVI. 505
The living specimens may be placed direct on the medium on the
slide, or may be killed by a short immersion in 10 per cent, acetic acid,
or boiling water. If the specimens are in alcohol they should be washed
in 10 per cent, acetic acid before mounting. After the cover glass is
put on, gently warm the slide, then allow to cool, and leave for one or
two weeks to dry and set. Ring the slide with a waterproof substance
and finally ring with a layer of Canada balsam.
Personally, I often kill in 90 per cent, or absolute alcohol, leave for
a few days, and mount in Euparal (see § 449).
943. Crustacea. — Some forms are very satisfactorily fixed with
sublimate. Such are the Copepoda and the larvse of Decapoda.
It is sometimes indicated to use the sublimate in alcoholic solution.
Some Copepoda, however (Copilia, Sapphirina), are better preserved
by means of weak osmic acid, and so are the Ostracoda. In many
cases the osmic acid will produce a sufficient differentiation of the
tissues, so that further staining may be dispensed with ; so for
Copilia and PhyUosoma. The pyrogallic process (§ 374) may be
useful. GIESBRECHT 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, ZACHARIAS (Zool. Anz.,
xxii, 1899, p. 72) takes chromo-acetic acid.
GIESBRECHT fixes larvae of Stomatopoda for five to ten minutes
in formol of 10 per cent, warmed to 40° or 50° C., opens them in sea
water and puts for one and a half to two and a half hours into formol
1 part and sea water 5 parts, and brings into alcohol of 70 per cent.
STAFFERS (La Cellule, xxv, 1909, p. 356) fixes Sympoda in GILSON'S
copper formol, § 115, or in HORNELL'S mixture of 100 parts of 5 per
cent, formol with 40 of alcohol ; and for softening the chitin puts
for twelve to thirty-six hours into 3 per cent, solution of sublimate
with 5 per cent, of nitric acid.
NETTOVITCH (Arb. z. Inst. Wien, xiii, 1900, p. 3) fixes Argulus
with liquid of Telly esniczky, § 52, warmed to 50° C.
For FISCHEL'S intra-vitam stain of Cladocera with alizarin, etc.,
see § 207.
944. Tracheata.— KENYON (Tufts Coll Stud., No. 4, 1896, p. 80)
fixes Pauropoda in Carnoy's acetic alcohol and chloroform, § 85,
cuts them in two for staining, etc., and embeds in celloidin followed
by paraffin.
HENNINGS (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
506 METHODS FOR INVERTEBRATES.
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 LEEUWEN (Zool. Anz., xxxii, 1907, p. 318) takes for larvae
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.
HOLLANDE (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.
NUTTALL, COOPER and ROBINSON (Para&itology, 1908, i, p. 163)
fix for a few minutes in hot picrosulphuric acid.
945. Methods for Clearing and Softening Chitin. — The methods
of Looss have been described § 545, those of HENNINGS and HAMANN
last §.
LIST (Zeit. wiss. MiL, 1886, p. 212) treats Coccidse (after harden-
ing) for eighteen to twenty-four hours with eau de Javelle, diluted
with 4 volumes of water. After washing out they may be embedded
in paraffin, and good sections obtained.
BALING (Dissert. Marburg., 1906, p. 11) boils larvae 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.
SAZEPIN'S method for antennae of Chilognatha (Mem. Acad. Imp.
St. Petersb., xxxii, 1884, pp. 11, 12) consists in steeping antennae
(that have been dehydrated with alcohol) for twenty-four hours ih
chloroform containing a drop of fuming nitric acid (shake occasion-
ally).
BETHE (Zool. Jahrb., viii, 1895, p. 544) puts telsons of Mysis 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 somewhat breaks down the structure of the
otolith, so that good sections through it are occasionally obtained.
Similarly HERBST, Arch. Entwickelungsmech, ix, 1899, p. 291.
See also the depigmentation processes, §§ 567 to 576.
945 A. Double Embedding of Insects.— Dissolve 1 -5 grs. desiccated
celloidin chips in 50 c.c. clove oil, or, better, add the celloidin in
CHAPTER XXXVL 507
an ordinary ether-alcohol solution and evaporate off the solvent
in the oven overnight.
It takes many days to dissolve thoroughly, but the time may be
shortened by keeping it at 90° F. Fix objects in absolute alcohol ;
bring them into clove oil, and allow this to clear the preparations,
then transfer to the clove oil celloidin. The time in this must be
gauged according to the size and nature of the insect ; fleas, if a
rupture is made in the chitinous covering, are penetrated in twenty-
four hours or less. When ready to embed, dip a cover-glass in melted
paraffin wax, to get a smooth surface on which the celloidin solution
will not spread, but forms a thick drop. Place the insect on the
prepared glass slip in a drop sufficient to completely cover it, and
arrange in any desired position. Invert the cover-glass and float
on to some chloroform ; leave for half an hour or longer, according
to the size of the drop. The drop of celloidin should fall away from
the glass. Transfer to melted wax, and allow time for the wax
thoroughly to permeate (twenty minutes is long enough for fleas
and lice). The result will be a small tablet-shaped mass of spongy
celloidin impregnated with wax ; this can be at once embedded, or
may be put away for future use. (Professor Boycott informs me
that he has never left his preparations for more than two or three
weeks at a time, so that he has no data as to how long the same could
be kept, but they could probably be stored indefinitely.)
945B. Carbon Bisulphide Embedding of Insects, etc. — HEIDENHAIN
many years ago recommended carbon bisulphide as a medium for embed-
ding in wax. This fluid is both smelly and dangerous and should only be
used in electric thermostats. The wax is dissolved in carbon bisulphide,
and dehydrated insects, etc., are placed in some of the fluid which is
allowed to evaporate at a gentle warmth. Subsequently the material
is rapidly treated in pure wax in the thermostat. This method cer-
tainly curtails the length of time in the thermostat, and overheating is
a serious matter when one is working at chitinous or brittle organisms.
946. Test for Chitin (ZANDER, Pfluger'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 zinc.
This is then removed with water as far as possible, and the violet
reaction is obtained.
See also WESTER, Zool. Jahrb., Abth. Syst., xxviii, 1910, p. 531.
947. BETHELS Stain for Chitin (loc. cit., § 945). — Sections are put
for three or four minutes into a freshly prepared 10 per cent, solution of
anilin hydrochloride, to which has been added 1 drop of hydrochloric
acid for every 10 c.c. They are then rinsed in water, and the slide is
put with the sections downwards into 10 per cent, solution of bichro-
508 METHODS FOR INVERTEBRATES.
mate of potash. The stain is at first green, but becomes blue in tap
water or alcohol containing ammonia.
MAYER simply uses a solution of pyrogallol in alcohol or glycerin ;
and HOFMANN (Zeit. wiss. Zool., Ixxxix, 1908, p. 684) puts for a day or
more into raw pyroligneous acid.
Dr. ORTON writes to me that he simply uses picro-nigrosin and borax
carmine.
948. Tracheae may be studied by the Golgi bichromate and silver
process. MARTIN (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. Tracheae blue.
949. Brain of Bees. — KENYON (Journ. Comp. Neurol, vi, 1896,
p. 137 ; Journ. Roy. Mic. Soc., 1897, p. 80) treats by the GOLGI
process (seldom successful), or hardens in a mixture of 1 part formol
and 2 of 5 per cent, sulphate of copper, followed by staining in
Mallory's phospho-molybdic hsematoxylin.
JONESCU (Jena. Zeit., xlv, 1909, p. Ill) has employed the silver
methods of Kamon y Cajal and Bielschowsky and Wolff.
950. Ventral Cord.— FLOYD (Mark. Anniv. vol., 1904, p. 355) fixes
the ganglia of Periplaneta for eighty minutes with vapour of formol,
and brings into alcohol.
See also BINET, Journ. Anat, Phys., xxx, 1894, p. 469.
951. Eyes of Arthropods. — For the methods of LANKESTER and
BOURNE (Quart. Journ. Mic. Sci., 1883, p. 180 : Limulus) ; HICKSON
(ibid., 1885, p. 243 : Musca) ; PARKER (Bull Mus. Harvard Coll,
xx, 1890, p. 1 ; Zeit. wiss. Mik., 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 Deca-
pods, 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, § 343.
For his method for eyes of Scorpions see § 575.
For the methods of PURCELL for the eyes of Phalangida see Zeit.
wiss. Zool., Iviii, 1894, p. 1. He has the following stain. The
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.
CHAPTER XXXVI. 509
HENNINGS (Zeit. wins. Mik., xvii, 1900, p. 326) depigments
sections by putting them for ten minutes (Musca) to twelve hours
(Myriopoda) into a mixture of 2 parts of 80 per cent, alcohol with 1
of glycerin and 2 per cent, of nitric acid, best kept at 35° C. The
elements are well preserved.
WIDMANN (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 ROSENSTADT, Arch. mik. Anat., xlvii, 1896, p. 478 ;
VIALLANES, Ann. Sci. Nat., xiii, 1892, p. 354 ; and DIETRICH, Zeit.
wiss. Zool., xcii, 1909, p. 465 (fixes in alcoholic formol, and bleaches
with dilute aqua regia).
952. Injections (Arachnida" and Crustacea especially). — AIME
SCHNEIDER (Tablettes Zool., ii, 1892, p. 123) recommends lithographic
Indian ink, the animals being narcotised with chloroform, then
injected and thrown into strong alcohol. Similarly CAUSARD (Bull.
Sc. France Belg., xxix, 1896, p. 16).
953. Arctiscoida (DOYERE, Arch. mik. Anat., 1865, p. 105). — Examina-
tion of living animals after partial asphyxiation in boiled water. See
early editions.
Vermes.
954. Chsetopoda : Cleansing Intestine. — KUKENTHAL (Journ.
Roy. Mic. Soc., 1888, p. 1044) puts Lumbricus into a glass vessel
filled with bits of moistened blotting-paper. They gradually
evacuate the earthy particles from the gut, and fill it instead with
paper.
VOGT and YUNG (Traite d'Anat. Comp. Prat., v) recommend
coffee-grounds instead of paper, as they cut better after embedding.
JOEST (Arch. Entmcklungsmech., v, 1897, p. 425) simply keeps
the worms for a few days in moist linen, and finds the gut empty.
PEARL (Journ. appl. Mic., iii, 1901, p. 680) injects alcohol of 6 per
cent, through the gut of narcotised worms.
955. Chsetopoda : Fixation. — Lumbricus may be anaesthetised
by putting the animals into water with a few drops of chloroform.
PERRIER puts them into water in a shallow dish, sets up a
watch-glass with chloroform in the corner of it, and covers the
whole.
CERFONTAINE (Arch, de Biol., x, 1890, p. 327) injects interstitially
about 2 c.c. of a 1 : 500 solution of curare.
510 METHODS FOR INVERTEBRATES.
JAQUET (Bib. Anat., iii, 1895, p. 32) kills Lumbricus in extension in
1 part of nitric acid to 125 of water.
COLLIN (Zeit. wiss. Zool., xlvi, 1888, p. 474) puts Criodrilus
lacuum into a closed vessel.with a little water, and hangs up in it a
strip of blotting-paper soaked in chloroform. KUKENTHAL (Die
mik. Technik, 1885 ; Zeit. wiss. Mik., 1886, p. 61) puts Annelids into
a glass cylinder filled with, water to the height of 10 cm., and then
pours 70 per cent, alcohol to a depth of 1 to 2 cm. on to the water.
For Opheliadae he also employs 0-1 per cent, of chloral hydrate in
sea water.
Many -marine Chaetopoda may be successfully narcotised (Lo
BIANCO) in sea water containing 5 per cent, of alcohol, or by means
of the mixture, § 18.
The Polychceta sedenlaria may sometimes be satisfactorily fixed
by bringing them rapidly into corrosive sublimate. Cold, not hot,
solutions should be taken, as heat frequently shrivels up the branchiae.
Eunice and Onuphis may be treated in the same way.
Lo BIANCO advises killing Cheetopteridse, Sternaspidse, Spiro-
graphis, 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 EISIG'S methods for Capitellidse see Fauna u. Flora Golf.
Neapel, xvi, 1887, p. 295.
See also § 14 (lemon juice), and the methods §§ 20 to 26, 39 and 49.
956. Blood-vessels of Annelids (KUKENTHAL, Zeit. wiss. Mik.,
1886, p. 61). — The animals should be laid open and put for two or
three hours into aqua regia (4 parts of nitric acid to 2 of hydrochloric
acid). Vessels black, on a yellow ground.
BERGH (Anat. Hefie, xlv, 1900, 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 HARMER'S process.
957. Nerves of Annelids.— Note the methylen blue method and
the bichromate of silver method of Golgi (the rapid method). For
the latter see v. LENHOSSEK (Arch. mik. Anat., xxxix, p. 102).
LANGDON (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.
CHAPTER XXXVI. 511
See also M. LEWIS, Anat. Anz., xii, 1896, p. 292 ; ATHESON, ibid.,
xvi, 1899, p. 497 ; and the methods of APATHY §§ 342, 368, 371, and
834.
958. Hirudinea. — For the methods of killing see those given for
Lumbricus in § 955, also §§ 20 to 26, and 49.
WHITMAN (Meth. in 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.
APATHY succeeds With alcohol of 40 per cent.
GRAF (Jen. Zeit., 1893, p. 165) has obtained good results by nar-
cotising with a decoction of tobacco.
959. Injection. — WHITMAN (Amer. Natural, 1886, p. 318) states
that very perfect natural injections may often be obtained from
leeches that have been hardened in weak chromic acid or other
chromic liquid.
JACQUET (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.
960. Nervous System. — Impregnation with gold. BRISTOL (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 embeds
in paraffin. See also §§ 342, 368, 371 and 834.
961. Nephridia. — SHEARER (Quart. Journ. Micr. Sci., Iv, 1910,
p. 288) stains Histriobdella intra vitam with a very weak solution of
Methyl blue, which allows the course of the nephridia to be made out.
962. Gephyrea. — VOGT and YUNG (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
anaesthetised with chloroform.
WARD (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 in a 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.
512 METHODS FOE INVERTEBRATES.
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. Phascolo-
soma and Phoronis should be treated by the alcohol method, larvse
of Sipunculus with cocaine, § 21.
APEL (Zeit. wiss. Zool., xlii, 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 J per cent,
chromic acid or picro-sulphuric acid.
963. Rotatoria. — For quieting them for study in the living state,
WEBEK (Arch, de Biol, viii, 4, 1888, p. 713) finds that 2 per cent.
solution of hydrochlorate of cocaine gives the best results. Warm
water gave him good results for large species, such as those of
Hydatina and Brachionus.
HARDY (Journ. Roy. Mic. Soc., 1889, p. 475) recommends thick
syrup added drop by drop to the water. HUDSON (ibid., p. 476)
mentions weak solution of salicylic acid.
VOLK (Jahrb. Hamburg, wiss. Anst., xviii, 1901, p. 164) quiets
them in quince mucilage, 40 grm. of -the seeds to 1 litre of water.
Cf. § 1018.
HIRSCHFELDER (Zeit. wiss. Zool., xcvi, 1910, p. 211) studies them
living in neutral red of 1 : 50,000.
See also §§ 23, 24 and 27. Methylene blue, § 339, may be found
useful.
Permanent preparations may be made by the method of ROUSSELET
(Journ. Quekett Mic. Club, v, March, 1895, p. 1) : The animals are
got together in a watch glass and are narcotised by adding to the
water at intervals a few drops of the following mixture :
Hydrochlorate of cocaine 2 per cent, solu-
tion ....... 3 parts.
Methylated spirit . . . . . 1 „
Water 6 „
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 J 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 2J parts,
distilled water 37 J parts.
ZOGRAF (Comptes Rend., cxxiv, 1897, p. 245) narcotises as ROUSSE-
LET, but without the spirit, fixes with osmic acid for two to four
CHAPTER XXXVI. 513
minutes, then replaces this by raw pyroligneous acid diluted with
8 to 10 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, embeds in paraffin and stains with hsemamm.
HIRSCHFELDER (op. tit., supra) narcotises with cocaine, and fixes
with Fol's picro-chromic acid.
BEAUCHAMP (Arch. Zool. Exper., iv, 1906, p. 29) finds 1 per cent,
stovaine better than cocaine for some forms. He (ibid., x, 1909,
p. 77) fixes for five to ten minutes in 4 parts of 1 per cent, osmic acid,
with 1 of 6 per cent, sublimate, and 5 of 5 per cent, bichromate of
potash, and 1 drop of acetic acid for each 2 c.c., and embeds in
celloidin, and then through chloroform in paraffin (three to ten
minutes).
See also TOZER (Journ. Roy. Micr. Soc., 1909, p. 24).
964. Acanthocephali. — SAEFFTIGEN (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 0 -1 per cent, chromic acid ; Echinorhynci live for days in it,
but eventually die fully extended.
HAMANN (Jen. Zeit., xxv, 1890, p. 113) has succeeded with sub-
limate, and also with alcohol containing a little platinum chloride.
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.
965. Nematodes. — The impermeable cuticle is a great obstacle to
preparation. According to Looss (Zool. Am., 1885, p. 318) this
difficulty may be overcome in the manner described in § 545.
Wash in 1 per cent, saline (if necessary) and fix in boiling 70 per
cent, alcohol ; store in fresh 70 per cent, for examination. If this
method is properly applied the worms will die extended and straight.
For examination transfer to 70 per cent, alcohol made up with 5 per
cent, glycerine. Place small bottle of this fluid, plus worms, on
incubator at 60° C., and allow to evaporate slowly for about twenty-
four hours or even two days, which finally leaves the worms in
viscid, almost pure, glycerine. Examine in pure glycerine, or glycerine
M. 33
514 METHODS FOE INVERTEBRATES.
jelly. For rapid examination after killing in alcohol, transfer to
absolute alcohol for thirty minutes, and clear in " white " creosote.
(LEIPER, in Science of the Sea, London. John Murray. 1912.)
For fixing, most recent authors recommend sublimate solutions ;
chromic solutions seem to have a tendency to make the worms
brittle.
But, according to ZUR STRASSEN (Zeit. wiss. ZooL, liv, p. 655),
Bradynema rigidum ought to be fixed for at least twelve hours in
mixture of Flemming.
AUGSTEIN (Arch. Naturg., Ix, 1894, p. 255) takes for Strongylus
filaria Mayer's picro-nitric acid.
VEJDOVSKY (Zeit. wiss. ZooL, Ivii, 1894, p. 645) advises for
Gordius 0-5 per cent, chromic acid (twenty-four hours).
Lo BIANCO employs for marine forms concentrated sublimate or
picro-sulphuric acid.
Looss (ZooL Anz., xxiv, 1901, p. 309) prefers hot (80° to 90° C.)
alcohol of 70 per cent.
GLAUE (Zeit. wiss. ZooL, xcv, 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
§234A, 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 Trichinae. — BARNES (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 flesh and
cysts being dissolved, the fluid is poured into a conical glass, and
allowed to settle ; the trichinae are drawn off from the bottom with a
pipette, got on to a slide with water and examined on a hot stage.
GRAHAM (Arch. mik. AnaL, 1, 1897, p. 2.16) isolates Trichinae by
macerating for one or two days in 2 per cent, acetic acid, staining with
aceto -carmine, and teasing.
966. Nemertina. — My best results have always been obtained by
fixing with cold saturated sublimate solution, acidified with acetic
acid. The other usual fixing agents, such as the osmic and chromic
mixtures, seem to act as irritants, and provoke such violent muscular
contractions that the whole of the tissues are crushed out of shape by
them.
CHAPTER XXXVI. 515
Prof. DU PLESSIS has suggested to me fixing with hot (almost
boiling) water. I have tried it and found the animals die in extension,
without vomiting their proboscides. So also JOUBIN, Bull. Mm.
Hist. Nat., 1905, p. 326.
I have tried FOETTINGER'S chloral hydrate method (§ 20). My
specimens died fairly extended, but vomited their proboscides.
According to Lo BIANCO narcotisation with a solution of 0-1 to
0 *2 per cent, in sea water for six to twelve hours is useful.
OESTERGREN (§ 18) recommends his ether water.
DENDY (see Journ. Roy. Mic. Soc., 1893, 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
hsematoxylin stains, on account of the energy with which they are
helcf 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).
BURGER (Fauna u. Flora Golf. Neapel, xxii, 1895, p. 443) studies
the nervous system, nephridia, skin, muscle and intestine by the
intra 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 six to twelve hours or more in moist
blotting-paper.
See also MONTGOMERY (Zool. Jahrb., Abth. Morph., x, 1897, p. 6) ; and
BOHMIG (Zeit. wiss, Zool., Ixiv, 1898, p. 484).
967. Cestodes. — Wash gently in 1 per cent, saline, and then fix
in hot corrosive sublimate acetic (at circa 50° C.) and allow the tape-
worms to remain in the dish till the fluid becomes cold. Wash in
running water for twelve hours and transfer to 70 per cent, alcohol.
Stain as in general methods.
As pointed out by VOGT and YUNG (Traite d'Anat. Comp. Prat.,
p. 204), the observation of the living animal may be of service,
especially in the study of the excretory system. And, as shown by
PINTNER, Tseniae may be preserved alive for several days in common
water to which a little white of egg has been added.
TOWER (Zool. Jahrb., xiii, 1899, p. 363) has kept Moniezia expansa
alive for several days in a mixture of 100 c.c. of tap water, 10 grs. of
white of egg, 2 of pepsin, 2 of sugar, and 5 of prepared beef
(" Bo vox "). Chloride of sodium, he says, should be avoided.
33—2
516 METHODS FOE INVERTEBRATES.
LONNBERG (Centralb. Bakteriol., xi, 1892, p. 89 ; Journ. Roy.
Mic. Soc., 1892, p. 281) has kept Tricenophorus nodulosus alive for
a month in a slightly acid pepsin-peptone solution containing from
3 to 4 per cent, of nutritive matter and less than 1 per cent, of
NaCl.
For the nervous system, TOWER (Zool. Anz., xix, 1896, p. 323)
fixes in a picro-platin-osmic mixture (stronger than that of 0. VOM
RATH, § 101) for ten hours, then treats for several hours with crude
pyroligneous acid, and lastly with alcohol, and embeds in paraffin.
ZERNECKE (Zool. Jahrb., Abth. Anat., ix, 1895, p. 92) kills Ligula
in the osmio-bichromic mixture of GOLGI (4:1), impregnates as
usual, makes sections in liver, and treats them by the hydroquinon
process of KALLIUS. Besides the peripheral 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.
BLOCHMANN (Biol. Centralb., xv, 1895, p. 14) recommends the
bichromate and sublimate method of GOLGI.
See also KOHLER, Zeit. wiss. Zool., Ivii, 1894, p. 386 (stretches Tseniae
round a glass plate or on cork, and fixes with 5 per cent, sublimate) ;
LUHE, Centralb. Bakt., xxx, 1901, p. 166, and RANSOM, U. 8. Nation,
Mus. Bull., Ixix, 1909, p. 8.
968. Trematodes. — If necessary, clean by shaking up in 1 per cent,
saline (parasites). Decant off dirty liquid, one-third of the tube is
filled again with 1 per cent, saline, in which the worms are shaken
vigorously, and an equal quantity of HgCl2 solution is added
quickly, the vigorous shaking being continued for several minutes
thereafter. This treatment should kill the flukes in an extended
condition. Leave in the fixer as indicated (corrosive one or two
days, wash in water twelve hours if 10 per cent, formalin be substi-
tuted for the HgCl2 ; leave about same time and store in 3 per cent,
formalin).
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 BIANCO fixes Trematodes with hot saturated sublimate.
Looss (Arch. mik. Anat., 1895, p. 7) takes for Bilharzia warm
(50° to 60° C.) 1 per cent, sublimate in 70 per cent, alcohol.
BETTENDORP (Zool. Jahrb., Abth. Morph., x, 1897, p. 308) has had
good results with the rapid Golgi method only on Distoma hepaticum,
and prefers methylen blue.
CHAPTER XXXVI. 517
HAVET (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.
Cercarice. — SCHWARZE (Zeit. wiss. ZooL, 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° to 40° C.
For an " indifferent " liquid, HOFMANN (ZooL Jahrb., xii, 1899?
p. 176) takes 1 part of white of egg in 9 of normal salt solution.
969. Turbellaria.— BRAUN (Zeit. wiss. Mik., iii, 1886, p. 398) gets
entire animals (Rhabdocoela) on to a slide, lightly flattens out with
a cover, and kills by running in a mixture of 3 parts of liquid of
Lang with 1 of 1 per cent, osmic acid solution. (Bo'HMiG [ibid.],
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.
DELAGE (Arch, de Zool. exp., iv, 2, 1886) recommends fixation (of
Rhabdoccela Acoela) by an osmium -carmine mixture, for which see loc.
cit., or by concentrated solution of sulphate of iron. Liquid of Lang was
not successful.
For staining, he recommends either the osmium -carmine or impreg-
nation with gold (| formic acid, two minutes ; 1 per cent, gold chloride,
ten minutes ; 2 per cent, formic acid, two or three days in the dark).
BOHMIG (Zeit. wiss. Mik., iii, 1886, p. 239) has obtained instructive
images with Plagiostomidse fixed with sublimate and stained with the
osmium -carmine.
GRAFF (Turbellaria Acoela, Leipzig, 1891 ; Zeit. iviss. Mik., ix,
1892, p. 76) says that chromo-aceto-osmic acid, followed by heema-
toxylin, is good for the skin, but not for the Rhabdites, which in
Acoela and Alloioccela seem to be destroyed by swelling. The same
method is also good for the parenchyma of Amphichcerus cinereus,
Convoluta paradox^ and C. sordida. Sublimate is good for Convoluta
Roscoffensis. The nervous system may be investigated by the
methods of DELAGE.
For Dendroccela sublimate solutions, sometimes hot, appear
indicated for fixing ; see the mixture of LANG. § 64, also CHICHKOFF
(Arch, de Biol, xii, 1892, p. 438).
ARNOLD (Arch. Zellforsch., iii, 1909, p. 433) kills Dendroccelum in
extension (?) with strong liquid of Flemming.
OESTERGREN narcotises Dendrocoelum with his ether- water, § 18.
JAENICHEN (Zeit. wiss. Zool., Ixii, 1896, p. 256) advises for
Planaria, eyes especially, picro-sulphuric acid for an hour or two ;
518 METHODS FOR INVERTEBRATES.
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
1 part common salt, 1 of acetic acid, and 100 of water. He bleaches
the pigment of the eyes with peroxide of hydrogen.
WILHELMI (ibid., Ixxx, 1906, p. 548) throws Triclads into almost
boiling mixture of Zenker, and after ten to thirty minutes removes
to water for some hours, and then passes into iodine alcohol.
Echinodermata.
970. Holothurioidea. — These 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.
VOGT and YUNG (Anat. Comp. Prat., p. 641) say that Cucumaria
Planci (C. 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 BIANCO).
OESTERGREN (§ 18) puts Synapta into his ether water, but Dendro-
chirota first into magnesium sulphate of 1 to 2 per cent., for some
hours.
GEROULD (Bull. Mus. Harvard Coll., xxix, 1896, p. 125) paralyses
Caudina with sulphate of magnesia, § 24, and fixes with liquid of
Perenyi (or sublimate for the ovaries).
Holothurids, Dr. WEBER informs me, are admirably preserved
in formaldehyde ; a weak solution is sufficient.
For the staining of muscles with methylen blue, see IWANZOFF,
Arch. mik. Anat., xlix, 1897, p. 103 ; and for the study of calcareous
plates, see WOODLAND, Quart. Journ. Micr. 8ci., xlix, 1906, p. 534
(fixation with osmic acid, staining with picro -carmine, followed by
Lichtgriin).
971. 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 (WEBER).
Lo BIANCO kills by pouring over them (mouth upwards) a mixture
of 10 parts acetic acid and 1 of 1 per cent, chromic acid, and brings
at once into weak alcohol. 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.
CHAPTER XXXVI. 519
•
Spicula and the skeleton of pedicellari89 may be cleaned by eau de
Javelk, see DODERLEIN (Wiss. Ergeb. Tiefsee-Evped., v, 1906, p. 67).
972. Asteroidea.— HAMANN (Beitr. Hist. Echinodermen, ii, 1885,
p. 2) injects the living animal with a fixing liquid through the tip
of a ray. The ambulacral feet and the branchiae 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 treatment with alcohol (which dissolves
out the pigment, leaving the pigmented cells perfectly hyaline).
For maceration use one-third alcohol, the aceto-osmic mixture
failing to preserve the rods of the pigmented cells.
Specimens for externals only preserve in 70 per cent, alcohol or
formalin. They retain their shape better if they are put for two or
three minutes into fresh water before being placed in the fixer.
If the internal anatomy is to be studied, cut along the length of each
arm so as to allow fluid to enter, and preserve in 2 per cent, chromic
acid, etc. Wash in running water, transfer to 70 per cent, alcohol ;
or the specimen may be preserved in formalin spirit or 5 per cent,
formalin.
Formaldehyde is not to be recommended for the cell preservation
of Asteroidea (WEBER). See also Lo BIANCO, op. cit. (he kills
Brisinga with absolute alcohol), also §§ 17, 20.
973. Ophiuridea should in general be killed in fresh water if it
be desired to avoid rupture of the rays (DE CASTELLARNAU, La
Est. Zool du Napoks, p. 135).
Lo BIANCO 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 2 parts concentrated sublimate
solution, 1 part 70 per cent, alcohol, and 1 part acetic acid (sp. gr.
1-06), and decalcifies in Miiller or in 70 per cent, alcohol with 10
per cent, of acetic acid. He stains with paracarmine.
974. Crinoidea. — Lo BIANCO (loc. cit., p. 458} fixes Antedon
rosacea with 70 per cent, alcohol, A. phalangium with 90 per cent.
520 METHODS FOE INVERTEBRATES.
975. Larvae of Echinodermata (from instructions written down
for me by Dr. BARROIS). — For the study of the metamorphoses of
the Echinoidea and Ophiuridea it is necessary to obtain preparations
that show, the calcareous skeleton preserved intact (a point of con-
siderable importance, since this skeleton frequently affords land-
marks of the greatest value), and that give clear views of the region
of formation of the young Echinoderm (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 larvse 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 Bipinnaria. — 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.
Larvce of Comatula are best fixed with liquid of Lang, and stained
with dilute borax-carmine. It is important (for preparations that
are not destined to be sectioned) to use only dilute borax-carmine,
as the strong solution produces an over-stain that cannot easily
be reduced.
Narcotisation by chloral hydrate before fixing is useful, especially
for the study of Pentacrinus larvse and of the young Synaptce formed
from Auricularia. Without this precaution you generally get
preparations of larvse either shut up (Pentacrinus), or entirely
deformed by contraction (young Synaptce).
See also MACBRIDE on the development of Amphiura squamata,
Quart. Journ. Micr. Sci., xxxiv, 1892, p. 131 (osmic acid followed by
liquid of Miiller and alcohol ; decalcification with nitric acid in alcohol ;
staining with Mayer's paracarmine or hsemalum) ; and SEELIGER on the
development of Anledon, Zool. Jahrb., Abth. Anat., vi, 1892, p. 161.
MACBRIDE (Quart. Journ. Micr. Sci., xxxviii, 1896, p. 340) fixes larvse
of Asterina in osmic acid, brings into liquid of Miiller for twelve to four-
teen hours, imbeds in celloidin followed by paraffin (see § 171), and
stains sections with carmalum or Delafield's hsematoxylin, best after a
foregoing stain of twenty-four hours in borax carmine.
MAYER (Grundziige, LEE and MAYER, 1910, p. 486) arranges a
number of fixed and stained Plutei on a sheet of gelatin foil gummed
CHAPTER XXXVI. 521
to a slide with euparal, dehydrates by adding alcohol by drops, and
adds euparal and a cover. See also WOODLAND, Quart. Journ. Micr.
Sci., xlix, 1905, p. 307.
Ccelenterata.
976. Thread-Cells. — IWANZOFF (Bull. Soc. Nat. Moscou, x, 1896,
p. 97) advises for the Nematocysts of Actiniae maceration by HERT-
WIGS' 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 Medusae he also advises HERTWIGS' method, § 526, or treat-
ment with a solution containing methyl green and gentian violet with a
little osmic acid.
977. LITTLE (Journ. App. Mi-c., vi, 1903, p. 2116 ; Journ. Roy.
Mic. Soc., 1903, p. 237) kills Hydra in hot saturated sublimate in
70 per cent, alcohol, washes with alcohol, stains for" five minutes in
strong solution of methylen blue, dehydrates rapidly, clears with
cedar or bergamot oil, and mounts in balsam. Nematocysts blue,
the rest unstained.
978. Actinida. — Anaesthetise in menthol (§ 15), which will take
some twelve hours or more. For ordinary sea anemones, formalin
(5 to 10 per cent.) followed by formalin spirit is to be preferred.
For corals, such as Caryophyllia, Alcyonium or Grorgonia, anaesthe-
tise, and then add hot corrosive sublimate or 5 per cent, formalin,
followed by cold saturated corrosive sublimate. Ninety per cent,
spirit, not allowed to get weaker than 70 per cent., gives good results
for anatomical work (ALLEN and BROWNE, loc. cit.).
For other narcotisation methods see §§ 15 to 26.
979. Fixation. — In Le Attinie, Fauna u. Flora d. Golfes v. Neapel,
ANDRES 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 containing
Actiniae 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.
DUERDEN (Journ. Inst. Jamaica, ii, 1898, p. 449) narcotises with
magnesium sulphate, § 24, and fixes with formol of 3 to 5 per cent.
980. Maceration. — For the HERTWIGS' method (Jen. Zeit., 1879,
p. 457) see § 526. The tissues should be left to macerate in the
acetic acid for at least a day, and may then be teased in glycerin.
522 METHODS FOR INVERTEBRATES.
LIST (Zeit. wiss. Mik., iv, 1887, p. 211) treats tentacles of Anthea
cereus and Sagartia 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.
981. Nervous system. — This group is generally held to be refractory
to the Golgi impregnation. HA VET, however (La Cellule, xviii, 1901,
p. 388), has obtained good results by the rapid method on young
specimens of Metridium dianthus. Besides nerve-cells, there are
impregnated neuro-muscular cells, gland-cells, and nematocysts. He
leaves for five to eight days in the osmic mixture. He has also had
good results by the intra vitam methylen blue method (this is also
good for nematocysts). So also has GEOSELJ (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.
982. 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 BIANCO, loc. cit., p. 446.
Sections. — See §§ 177 and 178, for undecalcified specimens.
983. 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). S. Lo BIANCO has since recom-
mended essentially similar processes. GAKBINI (Manuale, p. 151)
drenches them with ether, and brings into strong alcohol.
WILSON (Mitth. Zool. Stat. Neapel, 1884, p. 3) kills Alcyonaria
with a mixture of 1 part of strong acetic acid and 2 parts of concen-
trated solution of corrosive sublimate, the animals being removed
as soon as dead and hardened for two or three hours in concentrated
sublimate solution.
984. Zoantharia and Alcyonaria. — BRAUN (Zool. Anz., 1886,
p. 458) inundates Alcyonium palmatum, Sympodium coralloides,
Gorgonia verrucosa, Caryophyllia cyathus, and Palythoa axinellce
with a mixture of 20 to 25 c.c. of concentrated solution of sublimate
in sea water with 4 to 5 drops of 1 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.
CHAPTER XXXVI. 523
BUJOR (Arch. Zool. exper., 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 in sea water.
985. Hydroidea in General. — Directly the tow-net comes on board,
the Plankton must be poured into a glass jar and jelly fishes at once
picked out by means of a lifter or pipette, and placed in another very
clean jar of sea water. Leave in this jar for half an hour to allow
organisms to recover from shock. Note that the slightest trace of
chemicals in the jar will prevent their expanding. The secret of
successful preservation depends on keeping the animals in motion
while you pour in the fixer. First stir the organisms very slowly and
gently, and when all are in motion begin to pour the formalin slowly
down the side of the vessel. About 10 c.c. of 10 per cent, formalin
should go to 100 c.c. of sea water, but better more than this quantity.
Keep stirring for at least two minutes after addition of fixer. Leave
for a few hours and then transfer to 5 per cent, formalin ; finally
store in 10 per cent. To obtain medusae in a nice state of expansion it
is necessary to use an anaesthetic (see especially § 15). Hydrochloride
of cocaine is possibly the best ; use a 1 or 2 per cent, solution. Place
the medusae in a small glass vessel with just enough sea water to
allow them to swim. After they have expanded add a little cocaine
(3 c.c. of 1 per cent, solution for every 100 c.c. of sea water). If the
medusae at the end of ten to fifteen minutes do not contract when
touched with a glass rod no more cocaine is needed ; if they are still
active add more narcotiser and stir ; an over-dose will cause prolonged
contraction. After ansesthetisation add the formalin and keep
stirring, and continue for a minute, or longer. Do not leave speci-
mens in solutions of cocaine longer than necessary. (ALLEN and
BROWNE in Science of the Sea. London. John Murray. 1912.)
For further description of narcotisation methods see §§ 15 et seq.
For killing by heat see § 13.
Fixation. — In general polyps may be very well killed in saturated
sublimate solution, in which they should be plunged for an instant
merely, and be brought into alcohol. The solution should be employed
cold in general for Gymnoblastea, hot for most Calyptoblastea.
Ether attentively administered gives good results with Campanu-
laridaa. Hydra is very easily killed by a drop of osmic acid on a slide.
For the methylen blue intra vitam method, see Chapter XVI ; also
HADZI, Arb. Zool Imt. Wien, xvii, 1909, p. 225.
986. Medusae : Fixation. — For narcotisation see § 17 and above.
Trachymedusas and Acalephae may be fixed in the usual way in
524 METHODS FOE INVERTEBRATES.
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.
987. BIGELOW (Mem. Boston Soc. not. Hist., v, 1900, p. 193) fixes the
scyphistomes of Cassiopeia 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.
988. Medusae : Sections. — Paraffin and collodion are certainly not
satisfactory as all-round methods for these watery organisms. The
HERTWIGS (Nervensystem der Medusen, 1878, p. 5) embedded in liver
with the aid of glycerin gum, and hardened the objects and the mass
in alcohol.
See also JOLIET'S glycerin gum method, and the gelatin methods
in Chapter VIII.
989. Medusae : Maceration. — See, especially for the study of the
nervous system, § 526. Doubtless in many cases the pyrogallic acid
reaction, § 374, would give enhanced differentiation.
990. Siphonophora. — For the cupric sulphate method of BEDOT
(Arch. Sci. 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 BIANCO (op. tit., p. 454), FRIED-
LANDER (Biol. CentrbL, x, 1890, p. 483), and DAVIDOFF (Anat. Anz.,
xi, 1896, p. 505) see previous editions. Lo BIANCO fixes most forms
with the mixture given in § 1021.
For preserving, according to WEBER, formaldehyde is better than
alcohol. DAVIDOFF (loc. cit.) fixes in it.
991. Ctenophora : Fixation. — Never store in formalin, always in 70
per cent, alcohol. Pleurobrachia are best killed in 5 per cent, formalin
in sea water. Fill large measuring jar with this fluid, drop in the
animals and leave till they sink to the bottom ; transfer to 5 per cent,
formalin in pure water. After a week or so (not longer) transfer to
very dilute alcohol, and up-grade to 70 per cent, strength. Beroe :
Bring into small quantity of sea water, and when expanded add large
quantity of corrosive sublimate saturated solution in sea water.
When specimens become white, decant and add fresh water ; wash
in several changes to remove corrosive, up-grade to 70 per cent,
alcohol. Bolina dissolves at once in formalin ; kill in Flemming,
selecting small specimens : leave half an hour, wash slightly,
up-grade to 70 per cent, alcohol. (ALLEN and BROWNE in Science
of the Sea. London. John Murray. 1912.)
CHAPTER XXXVI. 525
Small forms are very easily prepared by means of osmic acid. For
the large forms see Lo BIANCO, loc. cit., p. 457. He uses his copper
sulphate mixture, § 987.
SAMASSA makes sections by the double-embedding method. See
Arch. mik. Amt., xl, 1892, p. 157.
992. Plankton, Preservation of, without Sorting (E. J. ALLEN and
E. T. BROWNE in Science of the Sea. John Murray. 1912). — Pre-
servation of whole catch of a tow-net is performed by stirring
around the Plankton with a rod and adding a little 5 to 10 per cent,
formalin. Keep on stirring for about a minute, then allow the
organisms to settle to the bottom ; as soon as this occurs pour off
as much of the liquid as possible and transfer the Plankton to a
bottle ; again allow to settle and reduce the fluid to a minimum ;
then fill the bottle with 5 or 10 per cent, formalin. A bottle should
be not more than half full of Plankton. After a few days, or on the
appearance of opalescence of the fluid, change the liquid. Another
method is first to fill the Plankton by pouring some saturated solu-
tion of picric acid into the jar containing the organisms, then add
some formalin 5 or 10 per cent, and leave for an hour or two,
occasionally stirring. Finally decant and add 5 or 10 per cent,
formalin as before described ; the yellow colour of the fluid can be
neglected. Never use corrosive sublimate with formalin, as crystals
form, which adhere to the organisms.
Porifera.
993. Spongise : 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. FIEDLER (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. — To avoid maceration, I hold that alcoholic stains should
be alone employed, and I recommend Mayer's tincture of cochineal,
§ 235. VON LENDENFELD (Zeit. wiss. Mik., xi, 1894, p. 22) uses
aqueous solutions of Congo red and anilin blue for the coloration of
collar-cells.
MINCHIN (Quart. Journ. Mic. Sci., xl, 1898, p. 569) stains spicula
sheaths with Freeborn's picro-nigrosin, § 742.
526 METHODS FOR INVERTEBRATES.
KOUSSEAU (Ann. Soc. Belg. Mic., xxiv, 1899, p. 51) stains in
nigrosin, picro-nigrosin, or indulin, or MAYER'S picro-magnesia
carmine.
Prof. Bendy informs me that he uses Hickson's brazilin (§ 378) a
great deal in his work on sponges.
For intra vitam staining, see LOISEL, § 207, ante, p. 130.
For silvering, see § 356.
Sectioning. — Calcereous sponges may be decalcified in alcohol,
acidified with hydrochloric or nitric acid, and then embedded in the
usual way. Siliceous sponges may be desilicified, § 566.
For EOUSSEAU'S methods, see § 566. VOSMAER and PEKELHARING
decalcify with a solution of picric acid in absolute alcohol (see Zeit.
wiss. Mik., xv, 1899, p. 462).
See also Johnstone-Lavis and Vosmaer, § 179.
Preparation of Hard Parts. — Siliceous spicules are easily cleaned
by treating them on a slide with hot concentrated nitric or hydro-
chloric 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, notwith-
standing that, in my experience, it does not give such clean pre-
parations.
According to NOLL, eau de Javelle is preferable to any of these
reagents, see § 544.
Embryos and Larvae. — MAAS (Zool. Jahrb., Abth. Morph., vii,
1894, p. 334) fixes larvae 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 (Zeit. wiss. Zool, Ixvii, 1900,
p. 218) fixes young Sycons in absolute alcohol and stains with
ammonia carmine (spicules in situ).
DELAGE (Arch. Zool. Exper., x, 1892, p. 421) fixes larvae of Spon-
gilla that have settled down on cover-glasses for three minutes in
absolute alcohol, stains in alcoholic carmine, and brings through
alcohol into oil of bergamot, then either mounts direct in balsam, or
detaches the larvee from the cover and imbeds in paraffin (three,
minutes).
GATENBY (Journ. Linnean Soc., 1920) uses methods for mito-
chondria, especially Champy-Kull and Kopsch.
Protozoa.*
994. Introduction. — In the special study of the protozoa the
various reagents and methods of general cytology .are in great part
* By Dr. A. Drew, Imperial Cancer Research Fund.
CHAPTER XXXVL 527
applicable, but numerous modifications have been introduced to
meet special cases. Speaking generally, the main lines of study
applied to the protozoa may be summed up under the following
headings : — Culture, collection, determination of life cycles, general
morphology, physiology. It will perhaps be appropriate to say a
few words by way of introduction under each of these headings.
995. Culture. — No general method is applicable to all cases ; for
many of the free living protozoa, such as infusoria, amoebae and
flagellates, a simple 1 or 2 per cent, hay infusion is suitable, which
may be conveniently placed in small petri dishes ; these give the
additional advantage of providing a large surface. Amoebee are in
many cases best grown on a solid agar medium, to be described later.
Special media have been described for the culture of many patho-
genic forms, such as trypanosomes, and quite lately Sr. Monica
Taylor has described a useful method for cultivating Amoebae
proteus (§ 1013).
996. Pure Mixed Cultures of Amoebae. — When working with
protozoa it is extremely desirable to have pure cultures whenever
possible. In the case of amoebae, bacteria are necessary as food, so
that we must grow our amoebae with bacteria. A culture containing
one species of amoeba, together with a pure culture of any particular
bacterium, is spoken of as a pure mixed culture, and it is necessary
to obtain such pure mixed cultures in order properly to study the
biology of the amoebae. The preparation of a pure mixed culture
involves two distinct processes : (1) The separation from its fellows
of a single amoeba ; and (2) its subsequent cultivation with a pure
culture of some selected bacterium.
(1) Separation of a Single Cyst. — The easiest method of accom-
plishing this is by means of the following method. Take a glass
capillary tube about 5 to 10 cm. in length and 1 mm. in diameter.
Flame the centre, and draw out quickly to the fineness of a hair.
Break into two equal portions and reduce each of these to a length
of 5 cm., so that one obtains two short tubes consisting of a wider
portion and a very fine capillary portion. Next select a culture of
the amoebee rich in cysts, add a drop of sterile water, and rub a
portion of the growth into this with a sterile platinum wire. Allow
a minute portion of this emulsion to run into the capillary end of
the prepared tube, and then run in sterile water till about 0 -5 cm.
of the broad portion of the tube is filled. Mix the contents of the
tube by vigorous rotation. Now prepare an agar film on a micro-
scope slide, by melting one of the stock tubes of the agar and pouring
528 METHODS FOR INVERTEBRATES.
a few drops on a slide. Allow this to set. Place on the microscope
and focus the upper surface with an inch objective. Tap out on to
filter paper some of the liquid in the capillary tube, and then, whilst
looking through the microscope, gently touch the film with the fine
end of the tube. A small volume of the suspension of cysts will run
on to the jelly and will spread out in an area which is quite visible,
and which occupies only a small portion of the field. If no cyst is
present, or if there should be more than one, place another drop on'
a fresh film and repeat till a single cyst is obtained on the film. The
method is simple, and with practice one can make half a dozen such
cultures in an hour. Place the slide film surface downwards above
water in a petri dish (this is conveniently done by resting on two
corks), and cover the dish. Examine day by day, till numerous
amoebae are found, and then allow them to encyst. From this
culture, prepare cultures in test tubes containing the special agar
sloped as for bacteriological work. Allow these to grow for a week
till covered with cysts. This may be observed with a low-powered
lens through the wall of the test tube. Prepare a 3 per cent, solution
of hydrochloric acid in sterile distilled water and cover the jelly film
in the test tube with this solution and replace the plug. Allow this
to act for twenty-four hours. Pour off the acid, and fill up with
sterile water, and again pour off, using aseptic precautions through-
out. With a platunim loop scrape some of the emulsion of cysts off
the tube on to the surface of a fresh sterile tube of agar. Then
add a loopful of a very dilute emulsion of the selected bacterium
and rub gently over the tube. In the course of twenty-four to forty-
eight hours the amoebae will excyst, and multiply, and will generally
be found to be in pure culture with the bacterium added. Should
they not be so on bacteriological analysis, allow to encyst and again
treat the cysts with the acid and proceed as already described. It
will be found that the best organisms wherweith to grow amoebae are
those naturally occurring in water, one of the best being Bacillus
fluorescens non-liquefaciens. When pure mixed cultures have been
obtained, the stock cultures should always be kept in test tubes, as
the petri dish method over water, although excellent for impure
stock cultures, is very liable to allow contaminations to occur. By
means of the method described, amoebae have been obtained in pure
mixed culture with the following bacteria, and with the results
detailed below.
NAME OF BACTERIUM. EEMARKS.
B. fluorescens liquefaciens . . Excystation rapid, good growth.
B. fluorescens non-liquefaciens . Excystation rapid, good growth.
CHAPTER XXXVI. 529
B. pyoeyaneus . Excystation fairly rapid, growth
good but amoebae encyst
rapidly.
B. prodigiosus . . . )
B. megatherium . . .[ Ex°ystation fairly rapid, growth
B. subtilis . . . . ) g°0(L
B. proteus vulgar is . . . j Excystation fairly rapid, growth
B. coli . . . . , L poor, amoebae encyst very
B. typhosus . . . .1 soon.
B. phlei . . . . . \
B. Rabinowitch I Excystation extremely slow,
B.NdbarroZ . j growth very poor.
Pneumococcus . . . . > Excyst fairly rapidly, growth
Meningococcus . . . )" slow, encystment very slow.
Staphylococcus aureus . . Excyst fairly rapidly, growth
fair.
997. Collection. — The great majority of the free living protozoa
are inhabitants of either fresh or salt water, and to obtain satis-
factory specimens some method of collection is essential.
There are several pieces of apparatus which are 'more or less essential,
viz., a collecting stick with ring and net, collecting bottle and cutting
knife, a good pocket magnifier (about x 10), and small specimen
bottles. The collecting stick can be obtained from all opticians, and it
is usually fitted with the necessary appliances. The net is perhaps the
most essential part of the apparatus. It consists of a bag of soft mull
muslin fastened to a brass ring about 9 inches in diameter at one end,
whilst the other carries a rimmed glass bottle about 3 inches long by
1 inch wide. The cutting knife is a strong curved hook with a sharp
knife edge, which is screwed to the end of the stick, and is occasionally
useful for cutting pieces of water weeds, which would otherwise be out
of reach. A flat bottle, which can be obtained from most opticians, is
extremely useful for examining specimens secured by the net. In a
round bottle it is often extremely difficult to see minute organisms
clearly when examined with the magnifier, but with the flat bottle
one can usually quickly ascertain whether a sample is worth keeping
for study with the microscope. The magnifier should be a good one
and should be aplanatic. The most generally useful will be found to be
either Watson's aplanatic loops, or one of Zeiss's hand lenses. The most
generally useful powers will be either the x 6 or x 10, preferably both.
The various forms of pond life can be divided, for collecting purposes,
into the free, and attached, and these groups will require different
treatment in the methods of collection. The free swimming forms are
often designated by the term "plankton," and 'these will be best ob-
tained by means of the net. In order to collect material with the net
it is passed through the water half a dozen times or so and then with-
drawn ; the pond water runs out, but the various infusoria, rotifera, etc.,
are retained and are finally condensed in the bottle. They should
then be emptied either into the flat bottle for preliminary examination,
M. 34
530 METHODS FOR INVERTEBRATES.
or into one of the specimen tubes to carry home. The attached forms,
such as hydra, polyzoa, vorticella, are generally found on water weeds
or bits of stick or submerged roots. In order to secure such organisms
the weeds, etc., are severed with the cutting knife and are dragged on
shore,' placed in water in the flat bottle and examined. Polyzoa seem
to have a preference for submerged rootlets, whilst forms such as
vorticella are more frequently found on the roots and stems of duckweed.
998. Determination of Life Cycles. — The only really satisfactory
method of determining life cycles is to follow a single organism con-
tinuously throughout its various changes in the manner originally used
by the late Dr. Dallinger, but such observations should be controlled
by examination of suitably stained specimens in the different stages,
under critical illumination. In the case of many of the parasitic pro.
tozoa such methods are impossible, as cultures cannot be obtained. In
such cases we are only able to examine fixed and stained preparations
and endeavour to piece out a life cycle from the appearances observed.
This must be checked by observations of the living material wherever
possible. For free living protozoa some means of keeping a drop of
the culture fluid from drying is necessary, but any means adopted must
permit of the continuous examination of the organisms by high -power
lenses. One of the best is that used by Dallinger and Drysdale, and
described in The Microscope and its Revelations, edited by DALLINGER,
8th ed., Part I, pp. 341 to 344. For the majority of flagellates this is an
excellent arrangement. Its chief drawback seems to be due to the fact
that it does not permit the aeration of the culture fluid, and it is, there-
fore, found that organisms such as large ciliates, many amoebae, vorti-
cellse, etc., soon die out from lack of oxygen. The simplest method of
examining such forms is the well-known hanging drop arranged on a
hollow ground slide. This, however, does not permit of critical illumina-
tion and completely upsets the corrections of the condenser. To get
over this difficulty a slide with a small table ground out is used. The
table is surrounded by a trench, and a ring of vaseline is painted round
the outside of the trench, and a drop of the fluid containing the
organisms to be studied is placed on the table. A cover -glass is then
lowered on to the drop and adjusted for pressure by gently pressing
on to .the vaseline. It is advisable in most cases to arrange the drop
so that it does not spread entirely to the edge of the table ; this ensures
an air supply. For amoebae and many ciliates and flagellates the live
slide described by DREW and GRIFFIN (Journal of Royal Microscopical
Society, February 21st, 1917) may be used. This form consists of a glass
plate cut to fit the mechanical stage of the microscope, and with a glass
arm cemented along one side. A piece of linen has a hole slightly
larger than the cover- glasses to be used cut in it, and this is then
damped with water and laid upon the slide. A small glass vessel filled
with water is attached to the arm and is put in communication with the
cloth by means of a piece of linen or soft wick. The linen is thus kept
moist by capillarity. A drop of the culture is placed on the slide and a
cover-glass placed upon it, and adjusted so that the circular opening in
the linen touches it at the margins, pressure being regulated, if necessary,
CHAPTER XXXVI. 531
by the insertion of cigarette paper between slide and cover -glass. So
long as water remains in the vessel attached to the arm, the linen and
the fluid under the cover will remain moist. For many purposes the
live slide devised by Botterill and described in The Microscope and its
Revelations by DALLINGER, p. 340, will be found extremely useful.
999. General Morphology. — Here great attention must be paid to
modern cytologic methods. The most perfect fixing and staining
technique should be used in any detailed study of the protozoa, and yet
this is seldom found. Methods of fixation will necessarily differ
according to whether one wishes to study nucleus, cytoplasm or
such cytoplasmic inclusions as mitochondria or Golgi apparatus.
Different methods are also frequently necessary according to the
organisms studied. For purposes of convenience it is proposed to
treat the fixing and staining of protozoa under the following head-
ings : — Amoeba, Coccidia, Ciliata, Flagellata, Hcemamcebce.
1000. Amoebae. — For temporary purposes many amoebae may be
stained and fixed by running a drop of 1 per cent, chromic acid
under the cover-glass and then running in a little alum carmine
followed by water. Staining intra vitam is very conveniently
carried out by means of an agar jelly. A 2 per cent, solution of
agar in distilled water is made ; this is cleared with egg white and
filtered hot and distributed in 5 c.c. quantities in test tubes. For
use a tube is melted by steeping in boiling water in a beaker and a
few drops of the selected stain is added and well mixed. A little of
the molten agar is then poured on to a slide and allowed to set.
The amoebse are distributed in a drop of fluid on a cover-glass, which
is then inverted on the jelly. The preparation is at once examined
with the microscope. Staining takes place progressively till the
nucleus is tinted, when " death " occurs. One of the best stains
for such a method is Unna's polychrome methylene blue. For
permanent preparations one of the most satisfactory methods is
that used by the writer. Slides are coated with a very thin film of
molten agar. This is conveniently done by pouring a very small
quantity of the agar on one end of a perfectly clean slide and spread-
ing it out quickly into a thin film over the slide by means of a warmed
glass rod. As soon as the agar has set the slides are stored in a
moist chamber till required. A drop or two of the fluid culture
containing the amoebae is spread gently over the surface of the agar
with either a platinum loop or a glass rod, great care being taken
not to break the thin agar film. The slide is then placed film side
up in a moist chamber and allowed to remain for from ten minutes
to half an hour or more. In this time the amoebse have generally
34—2
532 METHODS FOR INVERTEBRATES.
spread themselves out on the agar and many are in states of division.
The slide is quickly removed and placed in a larger tube containing
wool saturated with 2 per cent, osmic acid. The vapour fixes the
organisms and the slide is removed in two to five minutes, and at
once carefully placed in 50 per cent, alcohol. It should remain in
this for fifteen minutes and is then transferred to 70 per cent.,
90 per cent, and absolute alcohol fifteen minutes in each. It is then
brought down to distilled water by passage through the alcohols,
and is stained by Heidenhain's iron hsematoxylin method. The
secret of success in this method is to prepare the agar coated slides
with as thin a film as possible, otherwise it will peel off in the
alcohol.
Amoebae may be mixed with 1 per cent, serum water, and spread in a
thin layer over slides, which are then fixed in Schaudinn's fluid, passed,
as before, through the upgraded alcohols and back again to water,
and stained with iron alum. Dobell's alcoholic iron hasmatin method
may be used for staining the free forms of amoebae, but is not so good
for the cysts. A modification of Mann's stain by D obeli is also an
excellent stain for amoebae and cysts, and also sections of intestinal
ulcers and tissues. The stain consists of Mann's methyl blue-eosin,
which is made up in the usual way. Differentiation is carried out in
dilute orange G- in 70 per cent, alcohol. A simple staining in Lugol's
iodine solution is also of use, especially in the routine examination of
faeces.
1001. Coccidia. — These parasites stain rather badly and conse-
quently may be examined by adding a drop or two of 1 per cent.
aqueous eosin to the material containing them. The coccidia stand
out as unstained bodies on a pink ground. Tissues may be fixed in
the following solution : —
10 per cent, cobalt chloride in distilled water 20 c.c.
2 per cent, chromic acid in distilled water . 5 „
Formic acid ...... 1 drop.
Schaudinn's alcoholic sublimate is also a good fixative. Bertarelli's
method consists in fixing in saturated perchloride of mercury and
staining in Grenacher's hsematoxylin and differentiating in acetic
alcohol.
Borrel's method is of considerable utility.
Tissues are fixed in the following solution for twenty -four hours : —
Osmic acid ...... 2 gms.
Platinum chloride . . . . 2 „
Chromic acid . . . . . 3 „
Acetic acid . . . . . 20 c.c.
Distilled water ... . 350 „
They are then washed in water for an hour or two, and passed through
CHAPTER XXXVL 533
the upgraded alcohols to paraffin. Sections are stained for one hour in
saturated magenta, and are placed for five to ten minutes in a saturated
solution of picric acid in water, 1 part, and saturated aqueous indigo
carmine, 2 parts. They are washed in water and are decolourised in
absolute alcohol and then in clove oil, and are mounted in balsam.
1002. Ciliates. — Osmic acid fixation is amongst those best suited
for the ciliates, generally being employed either as vapour or wet
on the slide. Many forms may be mixed with serum water (serum
1 part, water, 20 parts), and then spread on slides and fixed either
in the osmic vapour or by means of Schaudinn's solution. Bouin's
fluid is also very suitable for fixation of many forms such as Opalina
and Lophomonas. Tissues are best fixed in either 10 per cent,
formol saline or Flemming's solution.
Staining may be by means of methyl green or any of the stains
used for amoebae. In many cases Hollande's chloro-carmine gives
very satisfactory results.
1003. Flagellata. — For the majority of these forms the technique
employed for the study of the trypanosomes may be used. Slides
may be coated with either serum water or a very thin layer of
glycerin albumen. A drop of the fluid containing the flagellates
such as blood may be spread quickly over it before drying occurs,
and the slide at once placed in Schaudinn's sublimate or Flemming's
solution for varying periods. Slides are then treated with upgraded
alcohols, the weaker ones containing a little iodine. They are then
brought down to water and stained with Heidenhain's iron hsema-
toxylin. Dried blood films prepared in the usual manner may be
fixed in alcohol-ether or absolute methyl alcohol and stained with
Giemsa's method.
1004. Haemamoebae. — These forms, the principal of which are the
well-known malarial parasites, will be found in the blood, and
heematological methods must be used.
Blood is spread in thin layers on slides by placing a drop at one end
of the slide and touching it with the end of another. The blood will
spread out in a thin layer and may then be drawn across the slide when
a thin, usually one-cell, layer is obtained. Films made by streaking a
drop of the blood over a slide by means of cigarette paper are also
good.
Such films are best fixed either in alcohol -ether (equal parts of each)
for half an hour to one hour, or pure methyl alcohol free from acetone
one hour. For a single method of staining such films, probably Rees'
thionin is the best. It is prepared as follows. Thionin 1-5 grms.,
absolute alcohol 10 c.c., 5 per cent, carbolic acid solution. 100 c.c.
Dissolve the thionin in the alcohol and add the carbolic solution. It is
534 METHODS FOE INVERTEBRATES.
best diluted for use about one in five, and films are stained for frojn five
to thirty minutes, washed, dried and mounted in balsam or euparal.
The hsemamcebae are stained purple, nuclei blue, red cells faint blue or
grey. Films may be differentially stained by means of eosin-methylene
blue, Borrel's blue, or still better, by one of the Romanowsky methods.
1005. Eosin-Methylene Blue. — The film is prepared and fixed either
by alcohol-ether or methyl alcohol, and is then stained for thirty seconds
in a 0-5 per cent, solution of eosin, and is then washed and stained in a
saturated solution of methylene blue for thirty seconds. In a successful
preparation the red cells are stained pink and the nuclei of leucocytes
and parasites blue.
1006. Ronald Ross's Thick Film Method. — Frequently the parasites in
these cases are only present in very small numbers, and in the examina-
tion of the ordinary thin films their presence may be overlooked. To
meet such cases Ross recommends about 10 to 20 cubic mm. of the blood
to be spread in a thick film on a slide, which is dried by waving gently
over a flame and is then washed in water. By such treatment the
haemoglobin is dissolved out from the erythrocytes. The film may
now be stained for a minute in 0-5 per cent, to 1 per cent, aqueous
eosin, followed by a 1 per cent, aqueous solution of methylene blue,
made alkaline by the addition of 0-5 per cent, sodium carbonate and
heated. For this may, with advantage, be substituted a solution of
Unna's poly chrome methylene blue diluted 1 in 2 or 3 with water. Films
after staining are washed, dried, and mounted in balsam. Only the
leucocytes and the parasites are stained by this method.
1007. Borrel's Blue. — This method depends upon the formation
of an oxidation product of methylene blue. Silver oxide is prepared
by dissolving the nitrate in distilled water and precipitating the
oxide with a 10 per cent, solution of sodium hydroxide. The
precipitate obtained is washed thoroughly in distilled water and a
saturated solution of methylene blue is added. The mixture is well
shaken and allowed to stand for about a fortnight. The super-
natant fluid is then pipetted off and constitutes Borrel's blue.
Films are prepared and fixed for half an hour in either alcohol, ether-
alcohol or absolute alcohol, and are then stained with Laveran's
solution prepared as follows :—
Borrel's blue ...... 1 c.c.
0 4 per cent, aqueous eosin . . . 5 „
Distilled water . . . . . . 4 „
The slides are placed film side downwards in this fluid and allowed
to stain for from five to fifteen minutes, and should then be washed
in distilled water treated with a 5 per cent, aqueous solution of
tannic acid for one to two minutes, washed and dried. Red cells
are stained pink, nuclei of leucocytes purple violet ; whilst the
CHAPTER XXXVI. 535
cytoplasm of the parasites is stained faintly blue their nuclei is
reddish purple.
1008. Romanowsky Methods. — The Romanowsky methods depend
essentially upon the formation of Azur and other oxidation products of
methylene blue, either alone or in combination with eosin. The most
generally used are Leishman's stain, Wright's stain and the well-known
Glemsa's stain.
1009. Leishman's Stain. — This stain is an extremely useful one ;
it is best purchased, but may be prepared as described in § 784,
p. 385. Films are prepared in the ordinary manner, but are merely
dried, not fixed. Five to 10 drops of the stain are poured on from a
pipette and allowed to act for thirty seconds. An equal quantity of
distilled water is then added and the diluted stain allowed to act for
a further period of from five to ten minutes. The preparation is then
well washed in distilled water, and is dried and mounted in balsam.
Red cells are stained pale pinkish, the nuclei of the leucocytes red,
parasites blue and their nuclei reddish purple. See §§ 784 et seq.
1010. Wright's Stain. — This stain is best prepared by dissolving
Grubler's methylene blue, 1 grm. in 0-5 per cent, sodium bicarbonate
100 c.c. The mixture is then heated at 70° to 80° C. for an hour. Cool
and add 500 c.c. of a 0-1 per cent, solution of eosin (the yellowish shade
water soluble variety). The solution of eosin should be added rather
slowly, with constant stirring, till the blue colour disappears and the
mixture is purple ; at this stage the fluid should have a metallic appear-
ance on the surface, when a finely granular blackish precipitate is
formed. This is collected and dried at 37° C. and a 0-3 per cent, solution
of this dye is then made in pure absolute methyl alcohol. When required
for use it is diluted with methyl alcohol (4 stain, 1 methyl alcohol).
Stain as described for Leishman's method.
1011. Wilson's Stain. — This stain also depends upon the production
of polychrome derivatives of methylene blue. It gives very satisfactory
results when carefully used, closely resembling those obtained in good
Giemsa preparations. Two grms. of silver nitrate are dissolved in
15 c.c. of distilled water, and to the solution so obtained is added
250 c.c. of a freshly -prepared lime water. The mixture should be shaken
well and the precipitate of silver oxide collected on a filter and well
washed with distilled water. The precipitate is then dried in an oven
at a temperature not above 70° to 80° C. The moist silver oxide pre-
pared from AgNCXj and NaOH solution may be used in place of the dry
product. To the oxide so obtained 2 grms. of methylene blue dissolved
in 200 c.c. of 0-5 per cent, sodium bicarbonate solution is added. The
mixture is then gently boiled in a porcelain dish for twenty to thirty
minutes, stirring from time to time. Pour off one-third of the contents
into a cylinder and then add to the remaining solution in the dish an
amount of distilled water equal to that poured off. Boil the mixture in
the dish again for twenty to thirty minutes. Again pour one-third of
the contents of the dish into the cylinder, and boil the remainder for a
536 METHODS FOR INVERTEBRATES.
further twenty to thirty minutes. Pour the contents of the dish into
the mixture in the cylinder and make up the total volume to 200 c.c.
Filter into a cylinder and add at once solution No. II., which is prepared
as follows : 1 grm. water soluble eosin (yellow shade) in 200 c.c. dis-
tilled water. Mix and allow to stand for half an hour, and collect the
resulting precipitate on a filter. Dry the precipitate at 60° C. In order
to prepare the stain dissolve 0-2 grm. of the dried precipitate in 50 c.c.
pure acetone-free methyl alcohol. The method of staining is identical
with that of Leishman and Wright.
1012. GIEMSA'S Method. — In many ways this is the easiest and best
of the Komanowsky stains ; it is best bought ready prepared, either
as a solution or the solid products of Burroughs, Wellcome & Co. For
use, films are fixed in absolute methyl alcohol, alcohol-ether or absolute
alcohol. They are then placed film side down in the staining fluid,
which is prepared as follows : —
Giemsa's stain ... . . .1 drop.
Neutral distilled water . . .1 c.c.
They should be stained for about one hour in this stain, washed, dried
and examined.
It is highly important that the water used should be neutral. This
is best secured by boiling fresh distilled water for fifteen minutes, and
then rapidly cooling and storing in a syphon vessel over soda-lime.
1013. M. TAYLOR'S Method for Amoeba Cultures (Nature, April,
1920). — Water from such places as the drainage cuttings in birch,
alder, and willow woods, or from the margins of ordinary pools and
ponds, together with the filamentous algss and the brown scum, and
included diatoms overlying the dead leaves and the other decaying
organic matter forming the floor of such places, is gathered in
autumn or in early spring. This is allowed to stand in tap- water
for some time, until a rich brown scum appears on the top. The
surface water, with the scum, is poured off into another glass vessel,
and wheat is added (1 gram to a litre of water). In February,
minute amoebae begin to make their appearance ; these become
fully grown in May and June, and will then divide rapidly, forming
a luxuriant culture until the late autumn, when encystment of most
individuals again takes place.
Once started, amoeba cultures require no further attention than
a supply of water to compensate for evaporation, and the addition
of wheat from time to time.
Dr. J. Bronte G-atenby informs me that using Sr. Monica Taylor's
method he has made successful cultures in boiled and unboiled London
tap-water, in London rain-water and in spring-water. Sub-culturing
is easily managed. Simply take about a pint of fresh rain-water or
boiled tap -water ; about eight wheat seeds are just brought to the
boil in a test tube of water, the latter poured away and the seeds shaken
into a flat dish containing the rain-water. The dish is then inoculated
CHAPTER XXXVL 537
from an old culture and securely covered. In from two to three weeks
the cultures may be going strongly. It is best to make half a dozen
different cultures, placing them at different distances from an oven or
thermostat. Not all of them " take." Small flagellates always seem
to accompany successful cultures, but the appearance of rotifers and
small annelids usually heralds the end of the amoebae.
Sr. Monica, in a paper in press (Journ. Roy. Mic. Soc. 1921), makes
a further contribution to this subject : there are periods of depression
in the cultures, new cultures may not " take " immediately, the cultures
are best kept in the light near a window, and to avoid extinction of a
strain by means of rotifers or small worms, one can only subculture.
1014. The Growth of Paramcecium in Sterile Culture Medium (by
R. A. PETERS, Phys. Proceed., 1920). — Culture of a race of paramoecia
about 50 JJL in length, isolated from a single individual, have been
obtained upon the following medium, the cultures being considered
sterile for the reasons given below.
Sodium chloride . . . .0-06 per cent.
Potassium chloride .... 0-0014
Calcium chloride . . . .0-0012 „
Basic sodium phosphate (Na2HP04) . 0-0001 „
Acid potassium phosphate (KH2P04) . 0-0001 „
Magnesium sulphate . . . .0-001 „
Sodium bicarbonate ... . . 0-002 „
Glucose 0-03
Histidine . . . . . . 0-01
Arginin 0-01 „
Leucin ...... 0-01 „
Ammonium lactate .... 0-003 „
Ferric chloride Trace
Potassium iodide .... Trace
Manganous chloride .... Trace
The substances are made up with glass distilled water. The con-
stituents are autoclaved separately, and the final mixture sterilised
by Keating to 80° C. on three successive days.
The organisms were cultivated first in sterile media in depression
slides, experiment showing the most suitable concentration for
division. The first divisions were apt to be slow. After a number
of individuals have been obtained in this way, they are sub-cultured
in test tubes, using all sterile precautions, and when a satisfactory
test tube culture has been obtained, it can be used for culture pur-
poses as required. A culture has now been kept going by weekly
sub-culture upon this medium for three months. (Temperature from
15° to 20° C.)
From successful cultures grown in this way with the paramrecium
in question, it was impossible to obtain any bacterial growth (or other
538 METHODS FOR INVERTEBRATES.
growth) by sowing a drop of the paramcecial culture with a platinum
loop, either upon (1) nutrient broth, (2) nutrient agar, (3) glucose agar
(anaerobic) and (4) litmus milk at room temperature or at 36° C. It
was also not possible to obtain growth upon the medium itself stiffened
with a trace of agar. Examination of a growing culture under the
^ oil immersion lens, however, showed the presence of peculiar rod-
shaped bodies. These were about 10 ^ long and 2/z broad. They were
motile, but appeared as a rule to be anchored at one end to the slide.
They were never observed to divide. After a varying time they would
cease to move in the moist drop preparation. When stained they were
found to lie in rows, varying in shape from curved to straight. The
absence of any sign of parasite or symbiotic organism in the paramce-
cium and the general resemblance of these bodies to split off cilia has led
to the belief that the cultures in question really contain no other
organism than the paramoecium.
1015. Method for Examination of Faeces for Protozoa (H. M.
WOODCOCK, B. M. J., November, 1915). — A very small quantity
of the faeces is taken up on a platinum loop and well mixed with a
drop of 0 -5 per cent, salt solution sufficient in amount to run under
a coverslip. The faeces must be well diluted, otherwise cysts are
apt to be overlooked. The faeces should be examined as freshly as
possible, as after four or five hours most of the active flagellates
become motionless and die. A convenient and rapid way of making
a permanent preparation is as follows : — A thin smear of the diluted
feeces is made on a slide in the same manner as a blood film, and the
slide is immediately placed in a stain tube containing at the bottom
a small quantity of 4 per cent, osmic acid plus 1 drop of glacial
acetic acid for fixation, and is left in for about ten seconds. Allow
the slide to dry in air and then place in absolute alcohol for fifteen
minutes. Wash with tap-water and stain in Giemsa, 1 drop to
1 c.c. neutral distilled water for twenty minutes or so. Rinse with
tap-water. .
1016. DONALDSON'S Method of Detecting Protozoal Cysts in Faeces
by Means of Wet Stained Preparations (Lancet, 1917). — Donaldson
recommends the use of two solutions, A and B.
A. (1) Five per cent, aqueous potassium iodide saturated with
iodine to which is added an equal volume of ether.
B. (1) A saturated aqueous solution of Rubin S. ; or
(2) A saturated aqueous solution of eosin ; or
(3) Stephen's scarlet writing fluid.
Equal parts of stains A and B are mixed just before use. A few
loopfuls of one of the above stain combinations are placed on a
clean slide, a loopful of faeces is taken and rubbed up with the stain
CHAPTER XXX VI. 539
to form a fairly smooth emulsion, and a clean coverslip gently
lowered on to the drop. In order to get the best possible definition,
it cannot be emphasised too strongly that the film so made should
be spread out under the cover-glass by capillarity so as to form the
thinnest layer possible, preferably a layer which is no thicker than
the diameter of an Entamoeba coli cyst.
The exact amount of stain to be used will soon be learned after one
or two attempts, and depends upon the size of the loop employed and
the size of the coverslip. It is a matter of considerable importance
the way in which the film is made, especially where Rubin S., or, to a
less extent, eosin has been used in the staining combination, for if the
layer of fluid between the slide and coverslip be too thick the super-
fluous film of fluid overlying the cysts tends to render the latter less
bright. In the case of cysts of the size and shape of Entamoeba coli
or histolytica, this makes little difference, but if one is hunting specially
for the Lamblia, or, still more, Tetramitus mesnili, the colour contrast
is not so marked, owing to the smaller size of the cysts and the conse
quent thicker red layer of fluid overlying them. Where the worker has not
acquired sufficient dexterity in making such a film, the difficulty may
be got over by using Rubin S. or eosin of only half saturation in the
stain combination, or by employing the scarlet writing fluid mentioned.
In this way the effect of the super -imposed deeper red is to some extent
obviated. In a wet preparation stained by this method there is a more
or less homogeneous red background, from which the cysts stand out
as brilliant yellow or greenish- yellow spheres which even the tyro cannot
miss seeing.
1017. Method for the Tsetse Flies (M. ROBERTSON, Trans. Eoy. Soc.,
Series B, vol. 203, p. 161 ). — The newly hatched flies are starved for twenty-
four to thirty-six hours and are then fed on the infecting monkey once,
or in some cases twice. The infecting feed is the first blood ingested
by the flies. After the infecting feed the cage is starved for one or two
days and thereafter fed on clean monkey's blood every second or third
day. Daily feeding is not essential to the welfare of glossina, and does
not appear to occur in nature. Dissections are made in a drop of physio-
logical salt solution. The trypanosomes are studied both in the live
state and in fixed and stained preparations. Preserved material is
fixed while wet by dropping the coverslip film side downwards into
Schaudinn's solution ; the preparations are subsequently stained by
Heidenhain's iron hsematoxylin.
1018. Immobilisation. — See the narcotisation methods §§ 20 to 25.
According to SCHURMAYER (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 Stentor 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.
EISMOND (Zool. Anz., xiii, 1890, p. 723) slows the movements of
540 METHODS FOR INVERTEBRATES.
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.
CERTES (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 STAHL ; see Biol. Centralbl., xii, 1892, p. 558)
makes a solution of 3 grms. 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 containing the organisms.
See also VOLK, ante, § 886 ; STATKEWITSCH, Arch. Protistenk., v,
1904, p. 17 ; LYON, Amer. Journ. Phys., xiv, 1905, p. 427 (neutralised
gum).
1019. Staining intra vitam. — See hereon BRANDT (Verh. physiol.
Ges. Berlin, 1878) ; CERTES (Bull. Soc. Zool, 25 janv., 1881) ; and
HENNEGUY (Soc. Philom., 12 fev., 1881). See also § 208.
BRANDT recommends a 1 : 3000 solution of Bismarck brown ;
also (Biol. Centralb., i, 1881, p. 202) " a dilute solution of hsemato-
xylin."
CERTES (op. cit., pp. 21, 226, 264, and Zool. Anz., iv,.1881, pp. 208,
287) found that living Infusoria stain in weak solutions of cyanin,
Bismarck brown, dahlia, violet 5 B, chrysoidin, nigrosin, methylen
blue, malachite green, iodine green, and other tar colours, and
hsematoxylin. The solutions should be made with the liquid that
constitutes the natural habitat of the organisms. They should be
very weak, that is, of strengths varying between 1 : 10,000 and
1 : 100,OCO. For cyanin, 1 : 500,000 is strong enough.
As to the staining of the Nucleus, see PRZESMYCKI, Biol. Centralb.,
vii, 1897, p. 321 ; and as to that of the Granula, the same author,
Zeit. wiss. Mik., xiii, 1896, p. 478. Also LOISEL, § 208.
Examination in a coloured medium in which the organisms do not
stain, but show up on a coloured background, is sometimes helpful.
CERTES (Bull. Soc. Zool. de France, xiii, 1888, p. 230) recommends
solution of anilin black — Infusoria will live in it for weeks ; FABRE -
DOMERGUE (Ann. de Microgr., ii, 1889, p. 545) concentrated solution
of diphenylamin blue.
For the mitochondria and other granules, see FAURE-FREMIET
(Arch. d'Anat. micr., xi, p, 457). Dahlia in salt solution, Pictet's
fluid. Janus green, or crystal violet being useful.
CHAPTER XXXVI. 541
For mitochondria and Golyi apparatus of a sporozoon, J. HIRSCHLER
(Anat. Anz., xlvii, 1914 — 15) used the Mann-Kopsch method, § 693.
1020. Demonstration of Cilia (WADDINGTON, 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 is efficacious.
1021. Fixing and Preserving. — Protozoa may be killed by heat,
by toxic vapours or by toxic liquids. Almost instantaneous fixation
can sometimes be obtained by steam or by iodine (Kent) or iodine
vapour (Overton) : see § 83.
E. S. GOODRICH (Quart. Journ. Micr. Science, Ixiv, 1919) modifies
Kent's method in a way which we have found useful for amoebae.
A strong solution of iodine in potassium iodide is diluted to about
the colour of sherry with normal saline for terrestrial and fresh
water animals, and with sea water for marine organisms. Such a
solution is run under the coverslip and followed by the definitive
fixing agent, e.g., Bouin's fluid, etc., and the preparation proceeded
with in the usual way. The iodine does not appear to destroy any
of the cell contents.
With regard to fixation, read §§ 29, 30, § 655, § 663, and especially
§§ 673 to 713. See also the important section from §§ 768 to 772,
dealing with fats.
WOODCOCK and WILSON'S Modification of Schaudinn's Fixative
(Phil. Trans. Roy. Soc. B., ccvii, 1916, p. 379 ; and Univ. Calif.
Pub., xvi, 1916, p. 244). — Woodcock gives saturated aqueous
sublimate, 2 parts ; absolute alcohol, 1 part ; and acetic acid,
5 per cent. Wilson uses alcoholic sublimate -j- 5 per cent, acetic.
See also Gilson and Petrunkewitsch fluids, § 69, and acetic alcohol,
§ 86. For Schaudinn's original fixative, refer to § 1031.
Lucidol or Peroxide of Benzol. See §§ 107 and 783.
For killing by heat, see § 13.
PFITZNER (Morph. Jahrb., xi, 1885, p. 454) used concentrated
solution of picric acid run in under the cover.
ENTZ (Zool. Anz., iv, 1881, p. 575) adds liquid of Kleinenberg to
the water containing the organisms in a watch glass.
KORSCHELT (ibid., v, 1882, p. 217) employs in the same way
1 per cent, osmic acid, or, for Amoebae, 2 per cent, chromic acid.
LANSBERG (ibid., p. 336) advises the same reagents, but recom-
mends bringing the organisms into the fixing liquid with a pipette.
For sulphurous acid, § 62.
CATTANEO (Bollettino Scientifico, iii and iv ; Journ. Roy. Mic.
542 METHODS FOR INVERTEBRATES.
Soc., 1885, p. 538) fixes for a few minutes with J per cent, solution
of chloride of palladium.
BRASS (Zeit. wiss. Mik., 1884, p. 39) employs a mixture of 1 part
each of chromic acid, platinum chloride, and acetic acid with 400 to
1,000 parts of water.
CERTES (Comptes rend., Ixxxviii, 1879, p. 433) fixes with 2 per cent,
osmic acid, or its vapours (ten to thirty minutes). For details, see
previous editions.
LONGHI (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.
SCALA (Rev. Mus. La Plata, xv, 1908, p. 57) fixes for five or ten
minutes in a mixture of 2 mg. of atropin, 10 drops of f ormol, 10 grms.
of glycerin and 50 c.c. of water.
See also PUSCHKAREW, Zeit. wiss. Mik., xxviii, 1911, p. 145 (agar
process for fixing and staining Amoebae).
FOL (Lehrb., p. 102) fixes delicate marine Infusoria (Tintmnodea)
with the perchloride of iron solution (§ 80), added to the water
containing them, and stains with gallic acid.
Lo BIANCO (loc. cit., p. 444) fixes Gregarinse with picro-sulphuric
acid (one hour), Vorticellse with hot sublimate, Acinetse with subli-
mate in sea water, or with osmic acid, Thalassicola with 0 -5 per cent,
chromic acid (one hour), Acanthometras and Aulacanthse with 50 per
cent, alcohol or with concentrated sublimate, or by adding a little
osmic acid to the water. For Sphserozoa he proceeds as BRANDT,
§ 1019.
ZOGRAF 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; SCHEWIAKOFF, Biblioth. Zool., v, 1889, p. 5; Journ. Roy.
Mic. Soc., 1889, pp. 832, 833 ; ZOJA, Boll. Sci. Pavia, 1892 ; Zeit. wiss.
Mile., ix, 1893, p. 485 ; LAUTERBORN, Zeit. wiss. Zool, lix, 1895, p. 170 ;
SCHAUDINN, ibid., p. 193; BALBIANI, Zool. Ans., xiii, 1890, p. 133;
KARAWAIEW, ibid., xviii, 1895, p. 286.
1022. Embedding of Protozoa and other Small Objects (MINCHIN,
Q. J. M. S., Ix, 1915, p. 508).— A thin slice of a block of amyloid
liver preserved in alcohol is floated into a shallow glass vessel with
a flat bottom, containing alcohol. The dish is placed on the stage
of a dissecting microscope. The objects to be embedded are taken
up in a pipette and placed on the slice of liver and orientated as
desired.
A tiny drop of glycerine and albumen solution is taken up on the
CHAPTER XXXVI. 543
point of a needle and caused to touch the surface of the alcohol
immediately above the small objects. The dense albumen solution
falls at once through the alcohol and spreads out over the objects
on the liver ; at the same time the glycerine is extracted and the
albumen coagulated by the alcohol, with the result that the objects
are stuck on to the liver. The liver is now trimmed with a scalpel
into a rectangular shape and embedded in the usual way.
Minchin used this method for the stomachs of fleas. I have found
it most successful for Echinoderm and other small eggs.
Or the following method may be used : — The 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 puts it into chloroform, which
dissolves the paraffin and hardens the collodion.
Some Current Stains for Protozoa. — Below are given a number of
special staining methods suitable for routine protozoological work.
1023. HEIDENHAIN'S Alcoholic Iron Haematoxylin. — (1) Fix in
Schaudinn's solution for fifteen minutes.
(2) Place at once in 70 per cent, alcohol for at least one hour.
(3) Mordant for five hours or more in
50 per cent, alcohol 10 parts.
4 per cent, aqueous iron alum solution . 1 part.
(4) Stain for twelve to twenty-four hours in
Heidenhain's hsematoxylin ... 1 part.
70 per cent, alcohol 10 parts.
Heidenhain's heematoxylin —
Haematoxylin ...... I grm.
Absolute alcohol . . . . . 10 c.c.
Distilled water . . . . . . 90 „
Thymol 1 crystal.
Dissolve the hsematoxylin in the absolute alcohol and then add
the distilled water.
(5) Differentiate in the same solution as was used in (3) for
mordanting.
544 METHODS FOE INVERTEBRATES.
(6) Wash thoroughly in several changes of 70 per cent, alcohol to
remove mordant.
(7) Dehydrate and mount in Canada balsam.
1024. DOBELL'S Haematein Method (see Arch. f. Protistinkunde, 1914,
p. 144.) — Films or sections are transferred from 70 per cent, alcohol
into 1 per cent, solution of iron alum in 70 per cent, alcohol. This is
most easily made in the way described by HICKSON (Quart. Journ.
Micr. Sci. 44, 1901, p. 470), 1 grm. of salt dissolved in 23 c.c. of warm
water, then add 77 c.c. of 90 per cent, alcohol after cooling. They are
mordanted in this for ten minutes, rinsed in 70 per cent, alcohol and
transferred to a 1 per cent, solution of haematein in 70 per cent, alcohol.
They are left in this for ten minutes and then differentiated, either in
the iron alum or in acidified 70 per cent, alcohol (0-6 per cent. HC1 in
70 per cent, alcohol). After differentiation wash in several changes of
70 per cent, alcohol and then pass up through the alcohols into any of
the usual mounting media. Any alcoholic counterstain can be used,
light green in 90 per cent, alcohol being very good. By this method the
organism is stained a purple grey, and hard black and white contrasts
are not obtained. The various nuclear and cytoplasmic constituents
may be stained with individual intensity.
(The haematein method is good, when it works, but often it is a
complete failure ; certain amoebae, for instance, are not stained at all
by it.)
1025. DOBELL'S Modification of Mann's Methyl Blue, Eosin Stain.—
(1) Stain with Mann's methyl blue eosin mixture (see Lancet, p. 196)
in distilled water till everything is overstained (two to eight hours).
(2) Einse in distilled water and differentiate in 70 per cent, alcohol
dried from water, containing a small quantity of Orange G — just
enough to colour it. It is best to keep Orange G in concentrated solu-
tion in 90 per cent, alcohol ; add sufficient of this with a glass rod to
clear 70 per cent, before differentiating. This weak Orange G cannot
be used often as it gets discoloured very quickly.
(3) Dehydrate in absolute alcohol (not too long) and transfer to
xylol (also not too long, as eosin comes out in time if left).
(4) Mount in balsam.
By this method permanent and pretty results are obtained, often
quite as good or better than those got with Giemsa. The method is
especially good after Bonin fixation, but it is important that all the
picric acid is removed before staining.
1026. Giemsa Stain (MINCHIN'S Method, Q. J. M. S., Ix, 1915,
p. 510). — Slides are washed in tap water and put in dilute Lugol
solution (1 c.c. Lugol to 25 c.c. distilled H20) for ten minutes.
After this, rinsed in tap water and put into a 0 -5 per cent, watery
solution of hyposulphite of soda for ten minutes. Next wash in
a current of water for five minutes and then put into the stain.
The distilled water used to dilute the Giemsa stain has to be
neutralised in the way prescribed by Giemsa.
CHAPTER XXXVI. . 545
A measured volume of the distilled water is taken and to it are
added a few drops of hsematoxylin solution (5 per cent, in dist. H20)
sufficient to tint it. Then a weak solution (1 per cent, in dist. water)
of potassium carbonate is added drop by drop until the colour of the
tinted water changes from yellow-red to reddish purple. In this
way the number of drops of the carbonate solution required to
neutralise a given volume of the distilled water is found.
The slides are now placed in the stain, 1 drop Giemsa to 1 c.c.
neutralised distilled water for one hour. Transfer to a weaker
solution (1 drop stain plus 4 or 5 c.c. water) and leave overnight.
Remove excess of stain by washing in water. Differentiate stain
by carrying slides through different strengths of acetone mixed with
xylol, beginning with 95 per cent, acetone and ending with pure
xylol. Mount in dammar or Canada balsam.
(If these instructions are carefully followed the results are excellent,
but care must be used.)
1027. Giemsa Method for Staining Gut Flagellates (J. G. THOMSON).—
( 1 ) Fix wet sat. perchloride, 2 parts ; alcohol, 1 part.
(2) Wash in weak alcohol (circa, 50 per cent.).
(3) Wash in water (aq. dist.).
(4) Wash in water, to which is added a few drops of Gram's iodin.
(5) Wash in 1 per cent, solution of hypo.
(6) Wash in running water.
(7) Stain in Giemsa (1 drop, 1 c.c.) twelve hours.
(8) Differentiate in (a) acetone, 95 per cent. ; xylol, 5 per cent., for
about five minutes ; then (ft) acetone, 70 per cent. ; xylol, 30 per cent. ;
(c) pure xylol to clear.
(9) Mount in Canada balsam.
1028. Iron Brazilin Stain (HICKSON). — (1) Fix in Schaudinn's solution
for fifteen minutes.
(2) Seventy per cent, alcohol for one hour.
(3) Mordant for four hours in a 1 per cent, solution of iron alum in
70 per cent, alcohol.
(4) Stain twelve to sixteen hours in a 1 per cent, solution of brazilin
in 70 per cent, alcohol.
(5) Wash thoroughly in several changes of 70 per cent, alcohol.
(6) Dehydrate and mount in balsam.
1029. FONTANA'S Stain. — (1) Fix in Hugo's fluid for one minute.
Acetic acid (B.P.) . . . 1 c.c.
Formalin . . . . . 20 ,,
Distilled water . .100 „
(2) Wash thoroughly in tap water.
(3) Mordant. Slide should be gently warmed.
Tannic acid .... 5 grms.
Carbolic acid ..... 1 c.c.
Distilled water . . . . .100 c.c.
M. 35
546 METHODS FOR INVERTEBRATES.
(4) Wash thoroughly in tap- water.
(5) Stain and warm gently : (a) 5 per cent, solution of silver nitrate
in water ; (6) ammonia. Add the ammonia until the precipitate redis-
solves in excess of ammonia. Then add more silver nitrate drop by drop
until the precipitate reappears and remains constant.
(6) Wash thoroughly in tap-water.
(7) Dry rapidly in air.
1030. Sphserozoa. — BRANDT (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.
KARAWAIEW (Zool Anz., xviii, 1895, p. 286) fixes Aulacantha for
twenty-four 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 BIANCO, § 1021.
1031. Sporozoa. — WASIELEWSKI (Sporozoenkunde, 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 Gregarinse and
Coccidia with osmic acid, sublimate, or picro-sulphuric acid, and
Myxosporidia with liquid of Flemming.
SCHAUDINN (Zool. Jahrb., 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.
STEMPELL (Arch. ProtistenL, xvi, 1909, p. 389) fixes caterpillars
infected with Nosema in 2 parts of saturated sublimate with 1 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.
LEGER (ibid., iii, 1904, p. 311) fixes cysts for a minute in " acetic
sublimate," puts for a minute into absolute alcohol, and stains as a
smear with heemalum or iron hsematoxylin.
BRASIL (Arch. Zool. Exper., 4, iv, 1905, p. 74) fixes them for
twenty-four hours in a mixture of 1 grm. 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 hsematoxylin followed by eosin and orange G.,
or Lichtgriin and picric acid.
1032. Hsematozoa.— GRASSI (Att. Accad. Lincei, iii, 1900, p. 357)
demonstrates the Malaria-parasites in the intestine, body-cavity
and salivary glands of Anopheles by treating them with normal salt
CHAPTER XXXVI. 547
solution containing 2 per cent, of formol (pure formol produces
swellings), or in a mixture of 1 -5 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 heemalum or iron haematoxylin. He stains
the Sporozoi'tes by. making cover-glass preparations, which are
allowed to dry, put for twenty-five minutes into absolute alcohol,
and stained by the process of KOMANOWSKY, §§ 784, 1008.
For minute instructions for the application of this process to
sections, see GIEMSA, Deutsch. med. Wochenschr., xxxvi, No. 12,
1910 ; and SCHUBERG, ibid., xxxv, No. 40, 1909 (Zeit. wiss. Mik.,
xxvii, 1910, pp. 160, 161 and 513).
For clinical methods, see COLES, The Diseases of the Blood, London,
J. and A. Churchill, 1905.
BRADFORD and PLIMMER (Quart. Journ. Micr. Sci., xlv, 1902,
p. 452) fix Trypanosomes in vapour of equal parts of acetic acid and
2 per cent, osmic acid, or with GULLAND'S formol and absolute
alcohol, and stain with methylen blue and eosin, and mount in
turpentine colophonium.
KINDLE (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 propor-
tion of iodine in potassic iodide, then into 30 per cent, alcohol, and
stains with iron haematoxylin or safranin, then with polychrome
methylen blue, and lastly with UNNA'S orange with tannin, and gets
quickly through alcohol into xylol and balsam.
MINCHIN (Quart. Journ. Micr. Sci., liii, 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 TWORT. Half-
saturated solutions of neutral red and Lichtgriin are mixed, the
precipitate dried and dissolved 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.
POLICARD (C. R. Soc. Biol, Ixviii, 1910, p. 505) stains Trypano-
somes 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.
1033. Flagellata.— LAUTERBORN (Zeit. wiss. Zool., lix, 1895
p. 170) fixes Ceratium for about ten minutes in liquid of Flemming,
puts into alcohol for twenty-four hours, brings back into water,
35—2
548 METHODS FOR INVERTEBRATES.
bleaches if necessary with hydrogen peroxide, and stains with
picrocarmine or Delafield's hsematoxylin. He also embeds in
paraffin, § 1022, and stains sections with iron hsematoxylin.
ZACHARIAS (Zool. Anz., xxii, 1899, p. 72) fixes Uroglena, etc., with
a mixture of 2 volumes saturated aqueous solution of boracic acid
and 3 of saturated sublimate.
1034. Stains for Flagella.— The ROMANOWSKY stain will give a red
stain of the flagella of some forms.
The method of LOFFLER (Centralbl. BakterioL, vi, 1889, p. 209 ;
vii, 1890, p. 625 ; Zeit. wiss. Mik., vi, 1889, p. 359 ; vii, 3, 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 (i.e. the
above- described mixture), and is heated for half a minute, until the
liquid begins to vaporise, after which it is washed in distilled water
and then in alcohol. It is then treated in a similar manner with the
stain, which consists of a saturated solution of fuchsin in anilin
water (p. 177), the solution being preferably neutralised to the point
of precipitation by cautious addition of 04 per cent, soda solution.
See also LIEBETANZ, Arch. Protistenk., xix, 1910, p. 23.
BUNGE (Journ. Roy. Mic. Soc., 1894, p. 640 ; Zeit. wiss. Mik.,
xiii, 1896, p. 96) makes the mordant by mixing 3 parts of the tannin
solution with 1 of liquor ferri sesquichlorati diluted twentyf old 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 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 FISCHER (quoted from Encycl. 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 ELLIS (Centralb. Bakt., xxi, 1903, p. 241 ; Journ. Roy.
Mic. Soc., 1904, p. 249), but staining with Saureviolett, 1 part to 75
of alcohol and 75 of water.
PEPPLER (Centralb. Bakt., xxix, 1901, p. 376 ; Zeit. wiss. Mik.,
CHAPTER XXXVI. 549
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.
Rossi (Arch, per le Sc. 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 fifteen to twenty minutes, washed, and
mounted. See also Centralb. Bakt., xxxiii, 1903, p. 572 (Zeit. wiss.
Mik., xix, 1903, p. 517).
GEMELLI (Centralb.,. xxxiu, 1903, p. 316 ; Zeit. wiss. Mik., xix,
1903, p. 516) mordants for ten to twenty minutes in 0-025 per cent,
permanganate of potash, rinses and stains for fifteen to thirty
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 PITPIELD is described by KENDALL, 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 solution, 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 saturated aqueous solution of gentian
violet with 10 of saturated solution of alum.
VAN ERMENGEM (Journ., 1894, p. 405) fixes for a few minutes with
a mixture oi; 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 STEPHENS, ibid., 1898, p. 685, and G-ORDON,i&«i, 1899, p. 235,
and the methods of TRENKMANN (Centralb., vi, 1889,'p. 433 ; Zeit. wiss.
Mik., vii, 1890, p. 79) ; BROWN (Journ. Roy. Mic. Soc., 1893, p. 268) ;
JULIEN (ibid., 1894, p. 403) ; SCLAVO (Zeit. wiss. Mik., xiii, 1896, p. 96) ;
HESSERT (ibid., p. 96) ; MUIR (Journ. Roy. Mic. Soc., 1899, p. 235) ;
MCCRORIE (ibid., 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) ; ZETTNOW
(ibid., 1899, pp. 662, 664); MORTON (ibid., 1900, p. 131); WELCKE
(ibid., p. 132) ; LEVADITT, C. R. Soc. Biol., lix, 1905, p. 326 (for Spiro-
chcete pallida, KAMON'S neuroflbril stain) ; MEIROWSKY, Munch, med.
Wochenschr., Ivii, 1910, No. 27 ; KALB, ibid., No. 26 (Zeit. wiss. Mik.,
xxix, 1912, pp. 123, 124 ; both for Spirochcete).
CHAPTER XXXVII.*
THE CULTIVATION OF TISSUE " IN VITRO " AND ITS TECHNIQUE.
1035. A culture of tissue consists of a special medium, natural or
artificial, such as lymph or plasma, inoculated with small fragments
of living tissues, and is characterised by an active growth of the cells
of the fragment into the nutrient plasmatic or lymph medium.
Cells wander out into the latter, and may live up to twenty days
without any signs of necrobiosis.
The cultivation of tissues outside the body was first accomplished
successfully by Eoss Harrison, of Johns Hopkins University in the year
1907. This brilliant observer has demonstrated by a series of experi-
ments that fragments of nervous tissue of the frog embryo, covered with
fluid from the lymph sac of an adult frog, show growth of long nerve
fibres (HARRISON, Proc. Soc. Exper. Biol. and Med., iv, 1907, p. 140).
Alexis Carrel, at about the same time, had been studying the laws of
redintegration of tissues, and adapted Harrison's technique to mam-
malian tissues. M. Burrows, a pupil of Ross Harrison, at this period
began to work on tissue -culture, and first used blood plasma instead of
lymph. Subsequently Burrows adapted the technique of Harrison to
the cultivation of tissues of the chick. In September, 1910, Carrel and
Burrows, working in conjunction at the Rockefeller Institute, succeeded
in cultivating, in vitro, the adult tissues of mammals, and thus began a
series of contributions which have taught us many valuable facts
regarding senesence and rejuvenescence and the pathology of tissues
(CARREL and BURROWS, Jour. Exper. Med., xiii, No. 3, 1911).
Two methods of tissue-culture may be distinguished : —
(1) Hanging-drop or smear cultures (Harrison).
(2) Large plate cultures (Carrel and Burrows).
The former are useful for direct observation of living growing
cells, the latter can be studied when fixed and cut into sections.
There has been a great deal of work done on tissue-culture, but
most of it has been carried out by vertebrate pathologists and
histologists. There seems little doubt that this field is a most
promising one for zoologists as well as histologists. Many problems
of gametogenesis and general cytology might be settled by recourse
to tissue-culture, especially by the application of such methods to
the cells of invertebrate animals, whose cytology had previously
been examined in detail with the aid of modern techniques.
* By J. B. G.
CHAPTER XXXVII. 551
1036. Precautions to Insure Complete Sterilisation of Apparatus.—
It cannot be too strongly emphasised that the utmost precautions
must be taken to insure complete sterility of all apparatus.
Bacterial infections of the cultures are fatal. The worker who
is not familiar with the minute precautions taken by surgeons
and bacteriologists in sterilising instruments, apparatus, etc., is
advised to become so before undertaking tissue-culture work. A
rigid asepsis is necessary for the preparation of any tissue-culture.
These words are addressed especially to the zoologist who may
undertake tissue-culture work ; dirty floors and benches, dirty walls
and garments, and casual methods will all contribute towards
failure. A clean, warm room should be set aside for making the
cultures, another for making the various plasmas, and, if possible,
another for incubators and incubator microscopes. The ordinary
zoological or botanical laboratory is generally unsuitable for such
delicate work.
Dr. Drew informs me that whether in vitro culture be carried out
in a special laboratory or not, it is advantageous to use a specially
constructed glass chamber to shield the cultures from chance con-*
tamination. Such a chamber is supplied by Hearson's (§ 11), or
can be made by any carpenter. The apparatus consists essentially
of a glass box in a wooden or metal framework measuring about
2 feet 6 inches in length, 2 feet in width, and about 1 foot in height.
The top is hinged so as to allow ready access to the interior for
cleaning, etc. The side facing the worker is made of wood, with
either two small wooden doors or a piece of thick sheet rubber
pierced so as to allow the easy entrance of the hands. The box
should have glass racks to contain the hollow ground slides and a
glass table for the cover-glasses. The slides are cleaned and stored
in absolute alcohol ; they are removed from this by means of forceps
and are flamed in a Bunsen and quickly transferred to the glass
racks ; cover-glasses are cleaned in acid bichromate, washed in
water till free from all trace of the acid, rinsed in distilled water,
then in absolute alcohol, and stored in ether. They are removed by
means of forceps and flamed and placed on the previously sterilised
glass table. Here both slides and cover-glasses are protected from
all falling dust, and can be manipulated easily with the hands
through the openings in the case. The majority of failures occur
through infection taking place, and the glass chamber reduces such
a possibility very greatly.
1037. Simple Culture Technique by means of Frog Lymph (HARRI-
SON, op. cit., 1907). — Pieces of embryonic tissues of frog embryos
552 THE CULTIVATION OF TISSUE "IN VITRO."
about 3 mm. long are dissected out with clean instruments, removed
to a coverslip, and covered by a drop of lymph freshly drawn from
one of the lymph sacs of an adult frog. The coverslip is inverted
over a hollow slide, and the rim sealed with paraffin wax. When
reasonable aseptic precautions are taken, tissues will live under these
conditions for from a week up to four weeks.
GOLDSCHMIDT (Arch. /. Zellf., 1916) has investigated the sperm
cells of Lepidoptera by tissue culture methods.
1038. Technique for Culture of Mammalian Tissues. — Preparation
of the Animal for Procuring Plasma.— The animal is anaesthetised
with ether, and must be kept just at the correct depth of anaesthesia.
A. J. WALTON (Journ. Path, and Bact., xviii, 1914), from whose
article the following paragraphs are partly culled, recommends for
this purpose a wide-necked bottle, with a closely-fitting cork pierced
with two tubes of wide bore, both of which pass down to within
|-inch of the surface of the ether placed in the bottle ; one tube
communicates with a tin funnel having a mackintosh flange ; this
tube also has a side tube, and the other tube is open to the air.
The animal's head is placed in the funnel, and, when the side tube
is clamped, breathes air and ether vapour. By clamping either the
side or the short tube the amount of air or ether can be suitably
controlled.
The hair of the throat, is either shaved off, or removed by the
application of a solution of sodium sulphide §ii ad Oi, which rapidly
dissolves it. The skin is then sterilised by painting with a 2 per cent,
solution of iodine in spirit.
1039. Preparation of Instruments, etc. — Previous to the operation
the following apparatus is sterilised : — Short test tubes, 2J inches
in length ; corks kept in stoppered bottles, to fit these tubes ; small
glass cannulse in olive oil ; three glass tubes, 3 inches by 1 inch ;
several narrow-bore pipettes which are kept corked in the last-
mentioned tubes, and which just before the operation on the animal,
are removed from the tube by means of sterile forceps, dipped in a
deep tin of molten paraffin, everted to allow the paraffin to run out,
and when cool placed in another sterile tube.
Two small sterile test tubes, as mentioned above, are similarly
treated in paraffin, and should be corked with sterile corks as soon
as cool. These two tubes are placed in two other larger tubes made
to fit the centrifuge, and ice is packed between.
Just before the operation, the instruments and some rubber teats
to fit the pipettes are boiled in water for ten minutes.
CHAPTER XXXVII. 553
1040. Removal of Plasma. — When dogs, rabbits, cats, chickens,
guinea pigs and rats are used, the carotid artery is ordinarily
selected ; an incision is made in the mid-line in the neck, and as
soon as the skin is divided the edges are clipped to sterile towels.
The carotid is exposed, its distal end ligatured and its proximal end
clamped. A little sterile oil is placed on the artery, which is opened,
and one of the cannule from the sterile oil is taken, inserted and tied
in position ; on releasing the clamp the blood flows freely. This is
collected in the paraffined test tubes for centrifuging. The tubes
should be in their ice- jackets ; they are corked at once and im-
mediately centrifuged for about five minutes ; they are then removed
and placed in an ice box at 0° C.
For human plasma one may remove blood from a vein by means
of a needle pipette sterilised in olive oil.
After the centrifugilisation the supernatant plasma may be
removed with pipettes coated in paraffin (§ 1039). It should be
used immediately for making the cultures, but can be preserved for
some time in a fluid condition if kept very cool. Chicken plasma
can be so preserved for more than a week, human and dog plasma
for a few days, while rat plasma always coagulates after a few hours
(CARREL and BURROWS, Journ. Exp. Med., 1911) ; when coagulation
takes place the plasma is no longer of use.
CARREL and BURROWS (Jour. Exper. Med., xiii, 1911) found that
dilution of the plasma had a marked influence on the rate of growth of
splenic tissue ; normal plasma is not the optimum medium for growth
of tissue ; the most favourable plasma for spleen culture contains two-
fifths distilled water, and slightly less for liver and heart, and generally
for skin, too.
1041. Preparation of Tissues. — The tissues for cultures should be
in normal condition, and are best when taken from the living animal
or immediately after death. Positive results can still be obtained,
however, when the tissues have been deprived of circulation for
more than thirty minutes.
With a cataract knife and a fine needle, a small fragment of tissue
is dissected from the animal and placed on a glass plate ; the piece
is rapidly cut into smaller pieces about the size of a millet seed and
transferred to a perfectly clean sterile coverslip. This process must
be carried out rapidly because some tissues die in even as short a
time as ten seconds when exposed to the air (e.g., thyroid). To
prevent this the tissue may be dissected in serum or Ringer.
1042. Preparation of Cultures. — For cultures of the hanging-drop
type one uses a hollow ground slide of a sufficient depth to prevent
554 THE CULTIVATION OF TISSUE "IN VITRO."
the drop of plasma from touching the bottom. The tissue is quickly
placed on a coverslip, 2 drops of plasma from the paraffined pipette
(§ 1039) are added and evenly and thinly spread around the tissue ;
this must be done before coagulation occurs. If the plasma is not
spread evenly the tissue-culture will grow in many planes, and will
be less easy to observe, manipulate, and to fix and stain. When
the plasma is spread out the cover glass is inverted over a hollow
slide, of suitable depth ; a little sterile vaseline may be placed at
each side of the cell to assist adhesion preparatory to waxing down.
The latter process is done by brushing molten paraffin around the
edge of the coverslip, and on the slide, to prevent drying. Im-
mediately this has been done the preparation is transferred to an
incubator. Carrell and Burrows use a small portable electric
incubator which is used for carrying the finished cultures to a bigger
incubator in the observation room.
For the large plate cultures the same technique is used. Tissue may
be rapidly removed, cut into very small fragments, suspended in
Kinger, and then spread on the cover of a flat glass -covered (Gabrits-
chewski) box, and covered with plasma. The G abritschewski boxes
after several days' incubation (three to five days), are opened, and the
plasmatic jelly cut out into blocks and preserved.
Or, instead of using Gabritschewski boxes, one may make the culture
on large black plates, which must then be placed in glass boxes with
cotton sponges soaked in water, in order to preserve the proper hu-
midity. The boxes are then carefully sealed with paraffin and kept in
such a position that the fluid products of the culture may drain to the
bottom.
1043. Subculturing is generally difficult, the technique of culti-
vating of tissue cells in series being far from perfect. One extirpates
a piece of the primary culture at its most active period, and transfers
it to a fresh medium, growth often, but not always, beginning anew.
Tertiary cultures are made in the same way. CARREL and BURROWS
(Journ. Exper. Meet., xiii, 1911) find that a very good way is to cut
out the middle of the old culture around the original piece of tissue,
and then fill up the space with new medium. The old cells grow
into the new plasma.
1044. Fixation and Staining of Cultures. — CARREL and BURROWS
(Journ. Exper. Med., 1911) remove the cover-glass to which the
culture is adherent, and immerse in corrosive sublimate, acetic acid,
or formalin, or the various preparations of chrome salts. After-
wards the culture is stained in heematoxylin of Benda, Heidengain
or Weigert.
Dr. A. Drew informs me that he has found that the best fixatives
CHAPTER XXXVII. 555
for in vitro cultures are, 70 per cent, alcohol and 5 per cent, acetic,
Flemming, and Bouin. In all cases the cultures on the slip should
be first detached from the slide and placed in warm Ringer's solu-
tion 37° C. for five minutes. They are then placed in the fixative.
Alcohol-acetic gives the cleanest pictures. Staining is best done
by Ehrlich's heematoxylin, Delafield's hsematoxylin, iron hsema-
toxylin, carmine or Giemsa. As counterstain either eosin or
orange Gr. may be used.
1045. Artificial Culture Media.— MARGARET R. LEWIS and W. H. LEWIS
(Anat. Record, v, 1911, p. 277) have investigated tissue cultures of chick
embryo cells made in artificial media. Eighty combinations of NaCl,
CaCl2, KC1 and NaHC03 and water, to form culture media have been
proposed. It was possible to obtain growth in such media, in which
either the CaCl2 or the KC1 or the NaHC03 was omitted, but not when
the NaCl was left out. Such growths continue only for several days,
and are never as extensive as those grown in plasma media.
More recently MARGARET R. LEWIS (Gontrib. to Embryology, ix,
1920, Nos. 27-46) for tissues of chick embryos of four to twelve days'
incubation uses " Locke-Lewis " solution (90 c.c. of NaCl 0-9 per
cent. + KC1 0-042 per cent., + CaCl2 0-025 per cent., + NaHC03 0-02
per cent., + 10 c.c. of chicken bouillon -f 0-25 per cent, dextrose).
The embryo was removed from the egg and placed in a petri dish con-
taining 20 c.c. of the warmed solution. Pieces of tissue to be explanted
were removed, washed through one or more changes of warm medium,
and cut with sharp scissors into pieces about 0-5 mm. in diameter ;
each piece was then placed in the centre of a coverslip, part of the drop
drawn off, and the coverslip sealed on to a vaseline ring around the
well of a hollow slide. Cultures thus prepared were kept in an incu-
bator at 39° C., and observations made in a warm box at 39° C.
CHAPTER XXXVIII.
A GUIDE FOR STUD.ENTS OF MICROTOMY.
1046. Three Examples for Beginners : — (1) The preparation of whole
stained mounts of some small object (Daphnids).
(2) The preparation of sections of the muscle or an organ of a
vertebrate.
(3) The preparation of an embryo (or tadpole) for the making of serial
sections.
Example I. — From a pond or ditch obtain some water-fleas (Daphnia
or Simocephalus) ; allow the jar to stand for several hours till the
suspended material has settled. Capture some of the organisms as
follows : — Take a piece of glass tubing some 8 inches in length ; place
a finger over one end, dip the other end under the water and by taking
away the finger, suck up some of the Daphnids into the tube ; put your
finger over the end of the tube, remove the latter and transfer the
organisms to a capsule or watch-glass about 2 inches in diameter. With
a clean pipette carefully suck up most of the water, hardly allowing the
animals enough to swim in^; now add a fixative to kill the organisms
(see § 2), and to coagulate their protoplasm (§ 29) as rapidly as possible
so as to leave the groups of cells forming the organs intact and
in situ.
Use corrosive acetic acid (§ 63), 2 per cent, acetic acid in saturated
aqueous corrosive. Pour the fixative into the watch-glass or capsule,
till it is full (the watch-glass or capsule contains about 15 to 20 c.c,)-
Place a glass square or plate over the capsule, and leave it for thirty
minutes. The organisms become opaque, indicating the coagulation of
the proteids of their cells.
With a pipette carefully remove as much of the fixing fluid as possible.
Now that the organisms are killed, the mercury salt must be removed ;
unless the fixative is thoroughly removed, it will form masses of pin-shaped
crystals at a later stage when the animals are being mounted in
balsam.
To remove the corrosive sublimate, it is necessary to convert it into
another substance which may be more easily washed away ; this is
effected by immersing the animals in some 70 per cent, alcohol which
has been coloured light port -wine shade with tincture of iodine (§ 63),
whereupon the mercury bichloride becomes mercury iodide, which is
very soluble in 70 per cent, alcohol. The iodine and alcohol mixture
CHAPTER . XXX VIII. 557
should be used until it no longer loses its colour, which indicates excess
of iodine. The whole process should last several hours and may be
carried on overnight.
The iodine and 70 per cent, alcohol are poured away, and the animals
washed for several hours (a minimum of two) in at least two changes of
70 per cent, alcohol to remove as much of the iodine as possible. The
objects are then transferred to 50 per cent, alcohol for one half-
hour, then into 30 per cent., for the same time. They are brought
down these grades in order that shrinkage may not occur when
they are being transferred to stains containing little alcohol, or none
at all.
Two stains may be tried, Mayer's acid hsemalum (§§ 248 and 249), and
Grenadier's alcoholic borax carmine (§§ 213 and 233). The time that
both these stains should be used depends almost entirely upon the
accessibility of the cells of the object to the stain. Daphnids
are covered by a chitinous shell, which though delicate tends
to prevent instant penetration. It is a good thing to leave the
animals in the stain for about five hours at least, and overnight
preferably.
Take two clean capsules, pour into one about 10 c.c. of borax
carmine, into the other a similar quantity of the haemalum. With
a camel-hair brush or a pipette transfer some of the organisms to the
stains and leave as directed above. See that the capsules are securely
covered.
After some hours in the stain, the latter is poured away, and the
process of differentiation (§ 203) is begun. The object of differentiation
is to wash away superfluous stain from certain organs or parts of organs,
in order that a contrast in depth of colour may be obtained in the
various other organs and tissues. Both borax carmine and Mayer's
acid hsemalum may be differentiated in acid alcohol (4 to 6 drops
of HC1 to 100 c.c. of 70 per cent, alcohol), which should generally
be allowed to act at least for as long as the stain has been used, and,
if necessary, longer. In both cases when differentiation has reached
the right stage, the objects examined under a microscope have a trans-
parent appearance, and such parts as the viscera and muscles should be
well contrasted.
The borax carmine specimens are washed out for several hours in
neutral 70 per cent, alcohol. They are then upgraded to 90 per cent,
and absolute alcohol, two hours in each, or overnight in absolute alcohol,
and cleared in cedar wood or clove oil for at least two hours, and then
mounted in xylol balsam.
The hsemalum specimens have to be brought to an alkaline solution
in order to " blue " the stain, and to get rid of all acid. Some workers
" blue " the stain in 70 per cent, alcohol made slightly alkaline with
ammonia or bicarbonate of soda, but the best results are obtained by
downgrading the objects to tap- water, which is allowed to run over
them gently till they go quite blue, which should occur for small objects
within an hour. The animals are then gradually upgraded through 30,
50, 70 and 90 per cent., to absolute alcohol, and cleared as above described
for borax carmine specimens.
558 A GUIDE FOR STUDENTS OF MICROTOMY.
In order to obviate the differentiation stage, one may dilute both the
borax carmine and the acid hsemalum till they are about one-third or
one-half as strong ; dilution of the borax carmine may be carried out
with 50 per cent, alcohol (not methylated spirit) and with distilled water
in the case of haemalum. In these solutions the animals remain till
sufficiently stained. But the best results are got by the overstaining
and differentiation method.
1047. Example II. — From a frog remove a large leg or thigh muscle,
and cut it into two pieces about as big as the nail of the little
finger. If desired, the liver, a halved testis, or a kidney may also
be used.
Transfer the material to a capsule containing at least 20 c.c. of
Zenker's or Helly's fluids (§§ 73, 684). Leave till next morning, and wash
in running water under the tap for at least three hours, preferably over-
night, then transfer to 50 per cent, alcohol for an hour ; then to 70 per
cent, alcohol containing enough tincture of iodine to give the solution
a light port -wine shade. Add more iodine as the colour disappears,
prolonging the treatment overnight for large pieces. Pour away the
alcohol, and add pure 70 per cent., in which the material is washed at
least three hours. Transfer to 90 per cent, for several hours and leave
in absolute alcohol overnight. Next morning it is safest to give the
material another hour in a fresh change of alcohol absolute. Pour away
a good deal of the alcohol and add about the same quantity of xylol or
cedar oil. Shake, leave half an hour, and then transfer the material
to pure xylol or cedar oil ; leave half an hour. Pour away some
of the xylol, either add chips of hard wax to cover the tissue,
or add some of the stock xylol and wax mixture. Leave an hour
in thermostat on the upper shelf, pour off, and add molten pure
wax ; leave one or two hours on the bottom shelf. Embed blocks
(§§ 142, 143).
1048. Example III. Preparation of an Embryo for Serial Sections.—
Fix in Bourn's fluid corrosive acetic or picro-nitric, overnight (§§ 110,
63, 97). In the case of the first and last mentioned fixatives, the
embryo is afterwards transferred to 30 per cent, alcohol (half-hour),
50 per cent, (two hours), and then washed for a day in several changes
of 70 per cent. The corrosive acetic fixed specimens are similarly
treated except that at this stage iodine solution is added to the 70 per
cent, (or this may be done in 90 per cent.) alcohol till the corrosive
sublimate is removed. Leave overnight in 90 per cent, alcohol (or
at least three or four hours), and at least six hours in two changes of
absolute alcohol (preferably overnight). De-alcoholisation and clearing
must be done carefully as directed in § 591, p. 269. It is a good
plan to bring embryos from absolute alcohol, through several
gradually strengthening mixtures of alcohol and cedarwood oil — to
pure cedar-wood oil, and then wash out in benzole. Embed in wax
as described in § 591, generaUy about one hour in benzol and wax,
and two hours in pure wax. Embed blocks (§§ 142, 143). Now read
§§ 144 to 151.
CHAPTER XXXVIII.
559
1049. General Plan of Procedure Applicable to Histological Specimens.
Anaesthetise animal, kill it, quickly take out organ, cut pieces 1 cm x 1 cm x J cm.
K2 Cr-2 07
Hg
Gl.
Fix for 24 hours in Zenker's fluid
Acetic acid.
Wash in running water 24 hours.
Freezing method. I Paraffin method.
Preserve in 5% Formalin
t
Wash in water
*
Impregnate with gum
Cut sections with freezing microtome
Stain
Pass through increasing strengths of ale.
¥
70% alcohol
¥
Remove Hg deposit with iodine in 80% alcohol
(Preserve in 80% alcohol)
¥
Float in water on to slide
¥
Stain with Picro-carmine 15 minutes
Drain off and wipe away the stain around
i
Mount in Farrant's medium.
Celloidin method.
t
Ale-ether Sa .............................. 1 day
f
Thin celloidin .......................... 1 week
(15% in ale-ether)
f
Ihick celloidin ........................... 1 week
(30% in ale-ether)
After evaporation, mount on block of vulcanised
fibre
Harden celloidin in chloroform ......... 1-2 hours
and then in 80 % ale ...................... 1-6 hours
f
Cut with razor (oblique) wetted with 80 % ale.
Stain without removing celloidin
¥
(Remove celloidin with oil of cloves)
Mount in balsam.
Pass through 90% alcohol 1 day
Dehydrate in abs. ale 1 night
f
Clear in xylol or chloroform... From 1 to 2 hours
f
Pass through xylol satd. with paraffin wax 1 hour
¥
Impregnate with paraffin at 52° C 2 hours
and
Embed and make blocks
t
Cut sections with microtome
[1° Apply fixative to slide or glycerine and
albumen water]
t
2° Float section on drop of water on slide
Warm gently to open out the section
Wipe away excess of water and dry in air
f
3° Remove paraffin with xylol
t
4° Remove xylol with abs. ale.
t
. 5° Pass through 90 and 70 % alcohol to
water
6° Stain in Hsematoxylin, etc., 5-15 minutes,
etc.
7° Wash in water, 5 minutes — 1 hour
¥
8° Counter-stain in Eosin — 1 minute, etc.
¥
9° Remove excess with 90% alcohol
¥
10° Dehydrate with abs. ale.
11° Clear in clove oil or xylol
12° Mount in balsam.
(Modified from D. T. Harris' "Practical Histology.")
560 A GUIDE FOR STUDENTS OF MICROTOMY.
1050. General Rules and Hints for Students.— (1) Keep all your
bottles and capsules as clean as possible.
(2) Try to keep your bench in order (it is difficult, J. B. G.).
(3) Keep notes of the time necessary for changing reagents.
(4) Thoroughly clean your slides and coverslips in acid alcohol before
using. See addendum.
(5) Note that corrosive sublimate tends to harden material.
(6) Corrosive sublimate is difficult to remove from tissue unless you
use iodine. If not properly removed you will find numerous pin-shaped
crystals in the finished sections. § 63.
(7) Corrosive sublimate attacks the surface of steel and other metals.
Use quills, or wooden needles for manipulating tissue in sublimate.
(8) Watery stains after picric acid fixation will cause maceration if
prolonged. § 93.
(9) Unless very well washed out, picric acid should not be used in
conjunction with thionin or toluidin blue. Precipitates form. Certain
other dyes do likewise.
(10) Osmic acid crystals should be dissolved in the purest distilled
water. Wash the tube with distilled water before you break it, removing
label. Wash out capsules and bottles for osmic acid solutions in distilled
water. Keep solutions in shade or dark. § 27.
(11) Osmic acid tends to harden yolk and certain other cell materials.
The vapour of osmic acid is injurious to the eyes and nose.
(12) Osmic acid and fixatives containing it inhibit staining, but if
necessary you can induce osmicated material to stain in delicate dyes
by bringing sections down to distilled water and treating in a -25 per cent,
solution of permanganate of potash for a short time. Permanganate
also decolorises sections. See page 31.
(13) Nitric acid tends to soften chitin and yolk, but it may inhibit
staining a little. § 97.
(14) Imbed material in paraffin in the shortest time possible, for
materials left in the thermostat longer than necessary go hard,
especially from xylol ; this refers especially to vertebrate material and
yolky embryos.
(15) Alcohol and chloroform dissolve fats and lipoids, acetic acid
dissolves away lipins. Vegetable oils dissolve fats less readily than
xylol or chloroform. Read §§ 120 et seq.
(16) Strong alcohol is bad for the finger nails and skin.
(17) When diluting stains with alcohol, use solutions made up by
breaking down pure absolute alcohol. Do not use methylated spirit,
as this generally precipitates the stain.
(18) You can soon learn to tell roughly the strength of alcohols by the
smell.
(19) Don't use the dregs of the absolute alcohol bottle for dehydrating
anything. The dregs are no longer absolute. Keep a waste alcohol
bottle for used liquid.
(20) Some workers add a little bag of fused copper sulphate to their
store bottles of absolute. This keeps the alcohol dehydrated.
(21) After fixation, when dehydrating and embedding a piece of tissue,
an egg or an embryo, it is at its softest when in weak alcohol, and its
CHAPTER XXXVIII 561
hardest when in xylol or a clearing oil. Flatten or otherwise manipu-
late a fixed object, while it is still in weak alcohol, or it will break up ;
but some objects may be dissected successfully in clove oil. § 122.
(22) Cells alter soon after death : formalin fixation is the best for
corpse material. Carefully note § 31.
(23) The organs of animals over-anaesthetised by chloroform or ether
are often spoilt (especially in the vicinity of large blood vessels) and are
sometimes useless even for general purposes. § 12.
(24) Keep balsam or colophonium jar in the dark, or paint it
black outside. Acid balsam soon removes stains from tissue ; acid
balsam is the necrologists' bete noire. § 443a.
(25) After Zenker fixation sections may overstain in eosin.
(26) If finished sections have crystals in them this is due to
improper washing out of fixative, or stain.
(27) Formaldehyde gas dissolves in water up to 40 per cent. The
commercial formalin is acid and must be neutralised with magnesium
or sodium carbonate kept in a little bag in the stock bottle. § 1,08.
(28) Formaldehyde gas is injurious to the skin and mucous membrane
of nose.
(29) If after staining in delicate dyes (e.g., methyl green), all the
colour keeps coming out of the sections during passage through
alcohols, try the following method : — Wipe superfluous water from
around the sections, and dehydrate by dropping acetone on sections :
then plunge into a jar of half acetone, half xylol, then pure xylol.
(30) For clearing embryos or pieces of tissue for whole mounts, cedar-
wood oil is better than xylol. § 120.
(31) If bubbles get under the coverslip they can often be removed by
gently warming, or by placing slide under bell jar of an exhaust pump.
(32) If after mounting an object in balsam white or black lines and
blotchy areas appear, this means that dehydration was not complete.
Bring back through xylol to absolute alcohol.
(33) When, after embedding, the block is set aside for a time and it
is found that the object is surrounded by a halo of white wax, this
means that all the clearing oil was not removed and is now exuding from
the object. Ke-imbed in pure wax.
(34) When, after embedding, the material seems soft and tends to
fall out of the wax, this indicates that dehydration was not complete,
and possibly also that the time in pure wax was not long enough.
Without efficient dehydration it is impossible to make good sections.
(35) If when cutting the sections curl up, it means that either the
knife is blunt or the material has been overhardened during imbedding.
Occasionally an incorrect slope of the knife may be the cause of
curling.
(36) When the secti'ons will not form a ribbon, this means that either
the wax is too hard or the slope of the knife is not correct. If the wax
is hard, place 1 drop of soft wax on each side of the block and flatten
it out with a warm knife. Eead carefully pp. 83 to 90.
(37) The broad side of a block should be parallel to the knife.
(38) Some people use miniature drums for rolling up the wax ribbon.
Laying them on a piece of foolscap does quite well. Avoid sticky
M. 36
562 A GUIDE FOR STUDENTS OF MICROTOMY.
paper. If sections accidentally adhere you can often release them by
cautiously wetting the paper with absolute alcohol.
(39) Before placing sections on a slide, write with a diamond pencil
the number of the slide and the material used. At a pinch, a glass
wax-pencil may be used instead-
(40) If you have not used a diamond, it is always possible to tell on
which side of. the slide the section lies, simply by slightly tilting the
slide and observing the shadow thrown on the other side of the glass.
(41) Finally, if your first attempts are failures, do not be discouraged
— even the most skilful micro tomists generally produce atrocities at
their first attempts. Try again !
APPENDIX.
1051. Chemicals, Stains, and Apparatus. — Addresses of British
firms from which it is recommended that these be obtained are
given in § 11.
1052. Cleaning Slides and Covers. — New ones should first be
soaked in one of the following liquids : strong sulphuric, hydro-
chloric 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 used 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.
1053. Gum for Labels. — Labels stuck on glass often strip off.
This may be avoided (MARPMANN, Zeit. Angew. Mik., ii, 1896,
p. 151 ; Journ. Roy. Mic. Soc., 1897, p. 84) by means of the following
adhesive : 120 grms. of gum arabic are dissolved in a quarter of a
litre of water, and 30 grms. of gum tragacanth in a similar quantity.
After a few hours the tragacanth solution is shaken until it froths,
and mixed with the gum arabic solution. Strain through linen and
add 150 grms. of glycerin previously mixed with 2J grms. of oil of
thyme.
PEIRCE (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.
36—2
INDEX.
Names like Lo Bianco are given under the latter half of the name.
Abderhalden, 357
Acalephse, 523
Acanthocephali, 513
Acephala, injection, 503
Acetate of lead, brain, 404
of potash, medium, 219
Acetic acid, fixation, 51
alcohol and sublimate, 53
and alcohol, 52
bichromate, 41
decalcification, 252
alum, carmine, 137
Aceto-carmine, 138
Acetone, 168
for dehydration, 4
fixation (Lucidol), 47, 59
and formol, 64, 494
and sublimate, 47
-chloroform, for narcotisation, 14
Achucarro, neuroglia, 490
Acid fuchsin, 171
for mitochondria, 320, 321
and malachite green (Pianese), 175
and methyl green, 324
myelin, 452
and orange G, 172
Acid hsemalum, 153
haematoxylin, 155
magenta, 171
rubin, 171
Acidic dyes, 120
Acidophilous tissue, 128
Actinians, narcotisation, 13, 521
Adamkiewicz, myelin, 452
Adenoid tissue, 249
Adsorption, 120, 122
Adurol, 469
Agar, sections between coverslips, 307
Agar-Agar, growing amrebae on, 527
Agassiz and Whitman, 281
Agduhe, Bielschowsky, 431
Aguerre, 482
De Albertis, neuroglia, 485
Albumen, method of mounting sections,
113
mercurial mounting medium, 220
removal of from eggs, 260 et seq., 283,
284
Alcohol, for dehydration, preservation,
4
narcotisation, 13
fixation, 56, 58
for maceration, 244
Alcohol — contd.
absolute, 58
table for dilutions, 57
and nitric acid for decalcifying, 253
Alcoholic, mercury bichloride, 46
hsematoxylin, Apathy and de Groot,
156
Heidenhain, 543
Dobell, 544
cochineal, 144
Alcyonaria, 522
Alcyonidium, 500
Alcyonium, 12, 521, 522
Alexander, 262
Alfieri, bleaching, 256
Alizarin, 184
and crystal violet, 322
nervous system, 408
Alkanna, 368
Allen, methylene blue, 192
Allen, Ezra,' 304
chromic Bourn's fluid with urea,
306
on clearing, 307
Allen, E. J., menthol, 12
Allen and Browne, 27, 523, 524, 525
Allerhand, iron myelin method, 451
Alt, 408
Altmann, 20 et seq., 38
acid fuchsin picric acid method, 320
corrosion, 249
fat, 366
fluid, 37 «
Alum, aniline, 355
carmine, 136
and picric acid, 138
-nitric acid for decalcifying, 253
hsematoxylin, generalities, 151
Aluminium hsematein, 151
Alzheimer, 416, 480
Amann, lactophenol, 222
Amato, 425
Ammonia, carmine, 140
chromate, 43
Ammonio-chloride of tin for myelin,
Besta, 448
Ammonium, bichromate, brains, 403
sulphocyanide for maceration, 245
vanadate, Golgi method, 476
Amoebae, cultures, 527
Amphibia, 275 et seq.
brains, 405
Champy's fluid, 37, 318
INDEX.
565
Amphioxus, 281
Amphipoda, embryology, 288
Amyl nitrite, 233
Amyloid, 133
Andeer, 254
Andres, actinida, 12, 13, 521
Andrews, avian embryology, 272
Andriezen, Golgi method, 465
Anemones, narcotisation, 12
See Actinians.
Anglade and Morel, Victoria blue neuro-
glia stain, 483, 485
Anilin dyes, 159 et seq.
blue, 182
blue-black, 183, 408
blue and carmine, 213
red, 169
oil, 69
anilin oil water, 166
Anitscnkow, 110
Annelids, 39
blood vessels, 510
Champy-Kull, 322
killing, 12
nerves, 510
Antedon, 519
Apathy, 97, 98, 511
alcoholic corrosive, 46
haematoxylin, 156
bergamot oil method for celloidin
sections, 117
embedding in oil of cedar, 78
Canada balsam, 226
cement for glycerine mounts, 231
gold, 198, 206, 207
glycerine gelatin embedding, 93
gum syrup medium, 221
haematin mixture, 155
methylen blue, 189, 192, 193
on knife tilt, 85
neuron bril methods, 416
nitric acetic for maceration, 247
picro-saurefuchsin, 177
series-on-knife method for celloidin
sections, 118
theory of gold impregnation, 203
Apel, 512
Aqueous humour, 219
Araneida, 287
Arctiscoida, 509
Argentamin, 415, 452
Argyroneta ova, 287
Arndt, bone saw, 370
Arnold, 178, 297, 517
chondriosomes, 323
staining kidney, 394
Arnstein, 190, 193, 342
method for corpuscles, 342
Aronson, myelin, 448
Arsenic acid, decalcifier, 254
Arthropoda, 504 et seq.
embryology, 284 et seq.
fixation, 44, 47, 55
mounting whole, 504
Artifacts, 20, 304
Artificial fecundation, 258 et seq.
Artificial iodised serum, 219
Artom, ascaris ova, 290
Ascaris ova, 52, 289
Aschoff-Kiyono, 388
Ascidia, narcotisation, 12
buds, 282
general, 499
Ascoli, Cajal's method, 426
Asphalt varnish, 230
Asphyxiation, by boiled water, carbonic
acid gas, 16
Assmann, blood stain, 384
Astacus, eye, 508
methylen blue, 192
nerve -en dings, 344
Asteroidea, 519
Astrocytes, 479
Athanasiu and Dragoiu, 352
Atheson, 511
Athias, 467
Atta, ova, 287
Auerbach, 415
buds, 425
stain, 312
Augstein, 514
Auricularia, 520
Aves, embryology, 271 et seq.
Gerlach's window method, 271
Axis cylinder, 45£ (methylene blue)
other stains, 454 et seq.
and dendrite, advice on special
forms, 459
processes similar to Golgi
methods, 475
rapid process, 458
Azoeosin, 313
Azoulay, 209
ammonium, vanadate process, 476
osmic acid methods (myelin), 451
Babcock, 102
Babes (safranin), 166, 167
Babkin, 396
Bacteria, in amoeba cultures, 528
in tissue, 326, 354
Ballowitz, 348
electric organ, 346
mammals, 264
reptiles, 275
Balsams, 225 et seq.
cedar wood, alcoholic, 226
method, section-grinding, 109
neutral, 226
Barnes, 514
Barrois, echinoderm larvae, 520
Bartel, 482
Baryta-water, for maceration, 245
Basic dyes, 120
Basophil granules, nerve tissue, 410, 414
" Basophilous " tissue, 128
Bastian, gold, 205
566
INDEX.
Bataillon and Koehler, 308
ascaris ova, 289
Batchelor, 390
Bath (paraffin), 77
Baumgarten, 183, 213
Bayerl, decalcifier, 254
ossifying cartilage, 377
Bayliss, 125, 309
on dyes, 120
on specificity of stains, 134
vaso -dilators, 233
Beale, 232
digestion by pepsin, 248
Beard, raja embryos, 280
Beauchamp, 513
Beckwith, 309
Bedot, 524
Bees, brain, 508
Behrens, 182, 221, 344
mounting medium, 224
salmon embryos, 280
Bell, cement, 229, 366
fat, 367, 369
Benario, blood, 381
Benda, 495
alizarin method, 322
copper hsematoxylin, 157
crystal violet, 184
fatty acids, 368
" Flemming fluid," 319
iron hsematoxylin, 1*48
neuroglia stain, 482
picro-saurefuchsin, 177
rapid myelin method, 448
safranin and light green, 181
secretion, granules, 315
Benecke, fibrils, 351, 388
Van Beneden, acetic acid, fixation, 51
method against contraction, 12
acetic alcohol, 52
live mammal embryo in serum, 267
mammals, 265
taenia eggs, 289
and Neyt, ascaris ova, 290
Bengal rose, 180
Bengtsson, eggs of diptera, 285
Bensley, brazilin and water blue,
(thyroid), 394
pancreas, 395
intestine, 393
Bensley-Cowdry, acid fuchsin methyl
green stain, 324
Benzidine dyes, 388 et seq.
teeth, 373
Benzoazurin, 170, 184
cartilage, 376
Benzol, 70
embedding, 78
peroxide, 59, 382
Benzopurpurin, 179,
Benzoyl, green, 181
Bergamot oil, 68, 323
for celloidin sections, 117
Bergh, annelids, 510
Bergonzini, 356
Berkeley, Golgi method, 466
liver, 393
rapid myelin method, 447
Berlese, Acarina, 504
Berlin blue, aqueous masses, 240
of Mayer, 240
injecting teeth, 372
mass, Briicke, 236
Bernard, mollusc, 504
Berner, 368
Bernheim, 206
Beroe, 524
Bertarellis, protozoa, 532
Best's carmine stain for glycogen, 295
Besta, ammonio-chloride of tin, 448
Cajal's method, 425
Golgi apparatus, method, 438
Bethe, 507
chitin, 506
methylen blue, 194
molybdenum-toluidin blue, neuroli-
brils, 417
Bettendorf, 516
Betz, hardening nerve, 403
Bevan Lewis, 183, 404
Lo Bianco, 14, 15, 37, 510, 514, 515,
516, 518, 519, 524
acetic fixation, 51
ascidia, 499
mollusc oida, 500
chromo-sublimate, 48
corrosive acetic, 44
gephyrea, 512
mixture for narcotisation, 13
protozoa, 542
Bichloride of mercury. See under
Mercury.
Bichoff, mammals, 264
Bichromate of ammonia, 43
of calcium, 43
of potash, 41
decoloration of, 41
maceration, 245
Muller's fluid, 42
and alcohol, 43
fixation, nervous system, 403 et seq.
-osmic, 37
-platinic, 37
chromic-osmic, 37
and mercury, 48
-sublimate, Golgi method, 470
Bickfalvi, digestion, 249
Biebrich scarlet, 180, 313
Biedermann, methylen blue, 344
Bielaszewics, 150
Bielschowsky methods, 426 et seq.
Da Fano modifications, 432 et seq.
other modifications, 430 et seq.
silver method, for connective tissue,
352
and Bruehl, ear, 497
and Plien, cresyl violet, 414
Bigelow, Medusae, 524
INDEX.
567
Bile capillaries, 393
• Bilharzia, 516
Binet, 31
Bing, 64, 449
Biniodide of mercury mounting liquid,
224
Binnennetz, 316
Biondi, blood, 380
Bipinnaria, 520
Bismarck brown, 161, 169
for cartilaginous skeletons, 377
Bizzozero, 391
blood-platelets, 386
and Torre, blood, 382
Bjeloussow, gum arabic mass, 241
Bladder, frog, nerves, 349
Blastoderms, general, 260
of mammals, 267, 268
Bleaching, Mayer's chlorine method, 255
sulphurous acid, hydrogen peroxide,
chlorine, 31
Bles, frog embryology, 278
Bleu de Lyon, 183
Blochmann, 115, 500
cestodes, 516
frog embryology, 276
Blood, 379 et seq.
cells, mitochondria, 333
elective stain for reds, 388
fixation, 379 et seq.
fixing in bulk, 380
new Golgi body, 387
and iron salts, 300
platelets, 386
-serum media, 219
stains like methyl green, etc., 380
Blue gelatine mass, 236
lumiere, 183
Blum, 401
Bobretzky, 286
Boccardi, 206
erythrosin and toluidin blue, 415
Bodecker, decalcification, 251, 373
Boeke, Bielschowsky, 430—431
Bohm, 206
and Oppel, 41, 219
reptile blastoderms, 274
Bohmer, hsematoxylin, 154
Bohmig, 515, 517
Bolina, 524
Bolton, 469, 447
Golgi method, 464
Bombyx mori eggs, 285
Bone, 369 et seq.
decalcification, 251
dry sections, 369 et seq
mounting, 370
non-decalcified, 369
soft parts, 371 et seq.
saw, 370
Bonnet, 269
Bonney, 178
Bonome, 480
Bonvicini, hardening human brain, 404
Borax carmine, 141
Bordeaux R., 178
Borgert, 75
Boring, ascaris ova, 290
Born, 262, 278
Borrel, 213
-method, 532
Borrel 's blue, 534
Bouffard, Benzidine dyes, 388
Bouin, picro-formol, 62
frog larvae, 278
salmon embryos, 280
Boule, Cajal's method for lumbricus,
425
Bouma, 376
Boveri, ascaris ova, 290
embedding of echinoderm ova, 260
picro-acetic, 55
Boyce and Herdman, copper, 300
Boycott, 507
Brachiopoda, 500
Bradford and Plimmer, 547
Braem, Bryozoa, 282
Brain, cat, dog, neurofibrils, 421.
See under Nervous System.
cat, man, preliminary fixation, 402
insect, 508
preliminary treatment, 397—399
Branca, 168
sublimate formol, 63
Brandt, 546
glycerine jelly, 223
protozoa, 540
Brasil, 546
Brass, on embedding, 76
protozoa, 542
Braun, 514, 517
Braus, 26, 281
bile capillaries, 393
Brazilin, 211
for sponges, 526
Breglia, 449
Bremer, 181, 473
Bresslau, mesostomid ova, 288
Brilliant kresylblau, blood, 383
Bristol, 511
Brittle object, cutting of, 88
Brock, maceration, 245
Brodmann, 398
Bromide of soda or potash, Simarro, 419
Brookover, 442, 468
Golgi method, 465
Brown, 549
Briicke, digestion, 249
Bruel, dipterous eggs, 285
Briihl, corrosion, 249
Bruno, mucin, 391
Brunotti, gold gelatin, 93
gelatin embedding, 108
Brunswick black, 230
Bryozoa, 282, 500
heat-killing, 12
Buchner, glycogen, 296
nucleoli, 314
568
INDEX.
Budge, injection, 241
Bugula, 500
Bujor, 523
Bulb, 424
Bullard, 367
Bumpus, 103
Bunge, fluid for iron stain, 299
tiagella stain, 548
Burchardt, 139
paraffin, 92
chrome fixation, 40
protopterus brain, 405
Burzynski, 94
Burger, nemertina, 515
Burrows, 550
Busch, 29, 32, 214, 252
decalcification, 251
Marchi, 450
Butschli, 154
Buzzi, eleidin, 341
Cade, gastric glands, 393
Cajal, 435, 465., \ 7
double-impregnation, Golgi process,
461
avoidance of silver precipitates, 467
gold chloride-sublimate method of
neuroglia, 487
Golgi apparatus method, 436
methylerie blue diffusion process, 478
myelin, 450
nucleolini, 311
retina, 495
spirals and funnels, 440
Cajeput, oil of, 68, 103, 411
Calberla, 161, 181, 182
Bismarck brown, 161
liquid, 222
Calcium chloride medium, 219
Calyptoblastea, 523
Camsal balsam, 227
Canada balsam, 225 et seq.
Caoutchouc, cement of Miller, 230
Capitellidse, narcotisation, 13, 510
Capsicum berries, 368
Carazzi, 256
Car bol-pyronin -methyl green, 172, 355
Carbolic acid (clearing), 69
fuchsin, 169
thionin (King), 168
Carbon, bisulphide for embedding, 77
Carter on, 372
insects, 507
tetrachloride, 77
Carleton, Golgi apparatus, 438
nucleolini, 311
Carmalum, 137
and indigo-carmine, 212
Carmine -generalities, 136
alcoholic stains, 141
aluminium chloride solution, 138
and anil in blue, 213
ammonia, soda, lithium, magnesia,
140
Carmine-generalities — contd.
and cochineal stains, 135
-gelatine masses, 234
Hover, 235
Fol, 235
Krause, 235
glycerine mass (cold), Beale, 238
Robin, 233
Grenacher's alum-carmine, 136
and malachite green, 213
for nervous system, 407
neutral alkaline, 140
blue, 183
Carminic acid, 135
Carnoy, 151, 178
acetic alcohol, 52
and Lebrun, frog eggs, 276
iron, 300
Carothers, Bouin and urea for insect
chromosomes, 306
Carrel, 550 et seq.
Carter, J. Thornton, bone, 369 et seq.
on post-mortem changes, 25
teeth sections, 371
Cartilage and bone, 376
silver, 200
skeletons, 377
Caryophyllia, 521, 522
Cassiopeia, 524
Castellarnau, 519
Castle, Ciona, 281
Castor oil, mounting medium, 228
Catois, methylene blue method for
fishes, 478
Cattaneo, 541
Cattani, funnels and spiral filaments, 410
Caullery, 499
Causard, 509
Caustic soda bleacher, 257
potash or soda, corrosion, 250
maceration with, 244
Cavalie, electric organs, 346
Cavazzani, 215
Cedar wood oil, 66 et seq., 77
for minute dissections, 7
Cell inclusions, 316 et seq.
granules, lymph and blood cells, 315
Celloidin embedding, 95 et seq.
See also under Collodion,
knife smeared in vaseline, 102
for injection, 241
sections, Apathy's method, 118
Bolles Lee's method, 116
Summer's method, 116
staining, 102
Celloidinum inelasticum, 96
Cements and varnishes, 229 et seq.
Centrifuge, for oogenesis studies, 332
Centrosomes, 315
Cepede, 307
Cephalopoda, 282
eyes, 502
Ceratium, 547
Cercaria, 517
INDEX.
569
Cerebellum, 424.
See under Nervous System.
preliminary treatment, 399
Cerebrum, hardening, 399
Cajal's methods, 424
Cerfontaine, amphioxus, 281
Ascaris ova, 290
worm, 509
Certes, protozoa, 540
Cesaris-Demel, 383
Cestoda, 515
ova, 289
Chaetopoda, 509
marine, 510
Chalicodoma, eggs, 286
Chambers, intra vitam stain, 310
Champy, 314
fluid, 37
iodide of osmium method, 331
trichloracetic, 53
Champy- Kull,mitochondrialmethod,321
fixation, Gatenby, 322
Chenzinsky, 181
blood stain, 383
Chick, embryology, 271 et seq.
axis cylinder and dendrite, 459
Child, fish embryos, 279
Chilesotti, 407, 477
Chilopoda, blood, 381
China blue, spirals, funnels, 440
Chitin, 506 et seq.
bleaching, 256 3.5-$
tests for, 507
Chiton, eggs, 284
Chitonidse, 503
Chloral hydrate, for narcotisation, 14
jelly, 224
maceration, 248
mounting medium, 220
Chlorcarmine, 139
Chloreton, for narcotisation, 14
Chloroform, for killing, injurious in
cytology, 11
clearing, 70
embedding, 76, 78
vapour, collodion embedding, 98
Cholesterin, 356 et seq.
Chondriokonts, 316
Chondriome, 316
Chondriosomes, 316
Chorion, removal, 284 et seq.
Chromates, 40
Chromate masses, 237
Chroinatin, microchemistry, 294, 308 et
seq.
and enzymes, 308, 309
digestive fluids, 294
methyl green test, 293
Chrome salts, Burckhardt on, 40
Chromic acid, decalcification, 252, 254
fixation, 32, 33
washing, out, 33
formol, 63
for hardening, 34
Chromic acid — contd.
and hydrochloric acid, decalcifier, 254
maceration, 246
nerve tissue, 404
and platinum chloride (Merkel), 39
Chromidia, 308, 309, 310, 316
Chromo-acetic acid, 35
Chromo-aceto-osmic decalcifier, 254
Chromo-fonnic, 35
Chromo-nitric acid (Perenyi), 39
decalcifier, 254
Chromophility tests, 309, 310
Chromosomes, techniques for, Gatenby,
303 et seq.
Chrome-sublimate, 48
Chrysoidin, 364, 369
Ciacco, 206, 343
method, 369
(fat) tendon, 347
Ciaglinski, myelin, 452
Ciechanowski, bile capillaries, 393
Cilia, mollusc, 503
protozoa, 541
Ciliated epithelium, 339
Ciliates, general staining, 533
Cilimbaris, 353
Cinnamon (cassia) oil, 67
Cladocera, Haker, 288
Clasmatocytes, 356
Cleaning slides, 112, Appendix
Clearing, 5, 65
Clove oil, for minute dissections, 7, 67
Coal gas, for killing, 1 1
tar dyes (plasma), 171
Cobb, differentiator, 3
Cocaine, for narcotisation, 14
Coccidia, staining, 532
Cochineal, 135
alum carmine, 137
Cochlea, 375, 496 et seq.
Coe, Distomum, 289
Coelenterata, 521
maceration, 524
nervous system, 522
Coerulein S., 182
Cohnheim, gold, 204
Cold-blooded animals, injection, 241
Cole, gum mass, 110
Coleoptera, eggs, 286, 287
Collargol, 434
Collin, 510
and Lucien, 438
Collinge, 281
Collodion, embedding, 95 et seq.
alcohol hardening, 100
bone sections, 372
bath, 96
for celloidin sections (Weigert), 118
blocks, clearing in cedar oil, 104
clearing and mounting, 102
cutting, 101
dry cutting, 104
hardening, 98
newer method, 103
570
INDEX.
Collodion — contd.
Obregia's syrup method of mounting
sections, 115
older method, 96
preservation of blocks, 100
rapid process, 104
section mounting with albumen, 116
method of mounting paraffin sections,
115
and paraffin, 105
solution of, 97
Collodionisation, brittle objects, 88
Colloidal gold, 133
Colloidal-complex, 120
Colloids, 123
Colloxylin, 96
Colophonium, 226
and wax method, section-grinding, 109
Colucci, 226
Comatula larva, 520
Congelation masses, 109
Congo-Corinth, 179 r>1-
Congo red, 121, 122, 124 178 et seq.
nervous system, 408
myelin, 453
Conklin, Oepidula, 284
Connective tissue, 350 et seq.
Bielschowsky, 434
Conser, 500
Contraction, prevention of, 11
acetic acid in, 12
corrosive sublimate in, 12
Cooling paraffin, 82
Copal method, 108
Copepoda, 288, 505
Copper, in tissue, 300
bichromate, 64
chloride and acetate fixation, 53
mounting fluids, 220
ferrocyanide injection mass, 234
formol, 64
nitrate*, 54
sulphate and corrosive for nerve
tissue, 405
fluid for frog eggs, 279
Corallin, 169
Corals, decalcification, 251
Cori, narcotisation mixture, 14, 15
osmic solutions, 29
Cornea, 342 et seq.
maceration, 247
fibres, maceration, -246
silver method, 198
Corning, 475
neurokeratin, 441
Corpuscles, Golgi, 347
Meissner, Krause, 342
Nissl, 180
tactile, 341
Herbst and Grandry, 342
Corrosion, 249 et seq.
Corrosive sublimate. See also under
Mercury bichloride.
preventative of contraction, 12
Corrosive sublimate- — contd
washing out, 26
Cowdry, 304, 415
acid fuchsin stain, 324
Janus green, 332
Mitochondria, 338
Cox, 415, 434
Golgi process, 472 et seq.
neurokeratin, 441
Cramer, 30
fatty substances, 356
osmic vapour method, 330
Feiss and Bullock, 366
Creases (paraffin sections), 88
Creighton, 297
Creosote (clearing), 69
Crepidula, 284
Cresyl violet, nerve, 414
Crinoidea, 519
Cristatella, 282, 500
Crustacea, 505 et seq.
Crystal violet, 184
for mitochondria, 322
Crystalline lens, 247, 343
Csokor, 230
bone saw, 371
Ctenophora, 524
Cucumaria, 518
Cultures, manipulation of amoebse, 527
Cunningham, micro -injection, 270
Curare, 292
for narcotisation, 16
Curling of sections, 86
Curreri, 469
Cutting, paraffin, 86
tissue, 319
Cyclas ova, 284
Cytological methods, 292 et seq.
Cytoplasmic inclusions. 316 et seq.
Czokor, 137
Daddi, fat, 367
Dahlia, 162, 169, 333, 340
Dakin, 503
Dallinger, 530
Damar, 226
Davidoff, 524
Tunicata, 281
Dead cells, 131
Dealcoholisation, 65 et seq.
or clearing, 5
Death, 25, 131
Decalcification, 251 et seq.
teeth, 251 et seq. and 372 et seq.
Decapod, eyes, 508
Decapoda, ova, 288
Deecke, 404, 407
Dee t jen, 387
Deflandre, fat, 369
Degenerate nerve, Marchi, 449
Dehydration, 2
by alcohol, acetone, methylal, aniline
oil, 4
Dejerine, 407
INDEX.
571
Dekhuyzen, 218, 386
liquids, 43
osmacet, 380
Delafield. hsematoxylin, 154
Delage, Turbellaria, 517
sponge, 526
Delamare, 351
DelPIsola, Golgi method, 463
Delia Rosa, Indian ink mass, 240
Delia Valle, Orchestia, 288
Deltapurpurin, 179
Demoor, 214
Dendrite stains, 454 et seq.
Dendrocoelum, 517
Dendy, Geonemertes, 515
sponges, 526
Denne, orientation of objects, 81
embedding method, 78 et seq.
Dentine, 370 et seq.
Dependorf, 374
Depigmentation, 256. See Bleaching.
Descemet's membrane, 343
Desiccation method for paraffin sections,
111
Desilification, 251 et seq., 255
Dewey, 373
Dewitz, 503
Dictyosome, 316
Dietrich, 366, 509
Digestion, 248 et seq., 308
and dissociation, 243 et seq.
Dimmer, 115, 119
Diomidoff, 405
Diptera eggs, 285
Disse, 377
Dissections-minute, cedar wood oil for,
clove oil for, 7
glycerine for, 8
Distomum ova, 289
Dobell, modification of Heidenhain
stain for protozoa, 544
Mann's stain, 544
Doderlein, 519
Dog brain, 402
Dogiel, 342
corpuscles of Herbst, 342
Meisser and Krause, 342
Grandry's corpuscles, 421
iris, 348
methylen blue, 189, 190 et seq.
for epithelia, 195
tendon organs, 347
Donacia eggs, 286
Donaggio, 158
neurofibril methods, 417 et seq.
Donaldson, 405
faeces, 538
Doncaster, chromosome fixation, 305
Double-embedding collodion paraffin,
105 $ 0 <c
Double-staining in hnematoxylin and
acid fuchsin, 326
Downey, benzidine dyes, 388
Drasch, 204
Dreuw, 341
Drew, formol-chrome method. 325
manipulation of amoeba cultures, 527
protozoa, 526 et seq.
staining amoebae, etc., 531
tissue culture, 551, 554
Drew-Griffin live-slide, 530
Drew-Murray, connective tissue stain,
354
Driessen, 297
Drost, 504
Driiner, 26
Duboscq, blood, 381, 465
Dubreuil, connective tissue, 350
Duerden, coalenterates, 521
Duerk, 354
Dunham's mixture (celloidin sections),
103
Durig, Golgi method, 464
Duval, 404
carmine and anilin blue, 213
collodion embedding, 95
orientation method for blastoderms,
273
silver, 200
Dyes, nature of, 120
electric charges, 122
Ear, inner, 496 et seq.
Eau de Javelle and Eau de Labarraque
bleachers, 256
corrosion, 250
for eggs, 284, 285
for frog embryology, 276
Eberth and Runge, 469
Echinodermata, 518 et seq.
larvae, 520
Echinoderms, decalcification, 251
Echinoidea, 518
spines, 518
Edinger, 34
Edington, blood, 381
Egg-capsules, removal, 284
orthoptera, 286
Egg, white of, injection mass, 240
Eggs, Unio, 284
Ehrenbaum, grinding, 109
Ehrlich, 155
blood, 379
haematoxylin and eosin, etc., 214
Indulin-Aurantia-Eosin, 180
mast cell method, 356
methylen blue, 188 et seq.
neutral red, 179
triacid mixture, 175
Ehrlich- Biondi, 161, 173
and Lazarus, 297
Ehrmann, 340
Eichler, 498
Eisath, neuroglia granules, 486
Eisenberg, Nile blue, 368
Eisig, 13, 510
fluid, 40
maceration, 245
572
INDEX.
Eismond, 539
Ekman, 500
ElasthSmatein, 353
Elastic tissue, 352 et seq.
fibres, of spleen, 394
Elderberries, Kappers, 409
Electric organs, 345
Eleidin, 341
Ellis, 548
Elschnig, 97
Embedding, gelatine masses, 92 et seq.
collodion^ 95
paraffin, 76 et seq.
lead-gum, 106
boxes, 73
brass squares, 74
thimbles, 73
trays, 72
in vacua, 80
Embryological methods, 258 et seq.
Embryonic cartilage, 376 et seq.
Embryos, Bielschowsky methods, 429
Cajal's methods, 424
fixation, 260, 268, 278, etc. 5. 75,
Emery, aqueous carmine mass, 240
Encephala, 401, 404
Endothelium, silver, 200
Engelmann, 218
Enriques, 501
Entamoeba, 538
Entire objects, preparation of, fixing
agents for, best stains for, 7
Entz, 81, 543
protozoa, 541
Enzymes and chromatin, 308
Eosinophilous cells, 180, 383, 386
Eosins, 180 et seq.
methyl green, 181
methylen blue, 383 et seq.
Epeira eggs, 287
Ependyma cells, 479
Epidermis, digestion, 249
neurofibrils, 421
Epiploon, silver method, 199
Epithelia, silver method for, 198
Epithelium, maceration, 244, 245
Eppinger, 393
Erhard, 297
Erlanger, ascaris ova, 290
Erlicki's fluid, 42
Van Ermengem, 549
Ernst, 341
Erytnrosin, 180
with toluidin or methylen blue for
nerve tissue, 415
Eternod, 83
Ether method, for celloidin sections, 116
for narcotisation, 13
Eucaine, for narcotisation, 15
Euler, 309
Euparal, 227,1 J 3)3 fe<l, to
Evans, benzidine dyes, 388
micro-injection, 270
Everard, 214
Ewald, blood, 380
section washing apparatus, 3
Examination media, 216 et seq.
Eycleshymer, 98, 103
Eye, 493 et seq.
arthropod, 508 et seq.
bleaching, 255, 257
mollusca, 502
Fabre-Domergue, 220
protozoa, 540
Faded sections, treatment of, 6
Faeces, examination for protozoa, 538
smears, lucidol, 382
Fairchild, 3
Fajerstain, 342
nsematoxylin. Golgi method, 476
Da Fano, 428, 434, 435
nervous system, 397 et seq.
axis cylinder stains, 454 et seq.
Bielschowsky method, generalities,
426—427
modification^, 432 et seq.
Cajal's methods, 419 et seq.
advice on Cajal's methods, 424
modification of Cajal's method, 425
cobalt nitrate, Golgi apparatus
method, 437
formaldehyde, Golgi methods, 463
Golgi preparations, on cutting, 467
on mounting, 468
Golgi-Cox method, 473 .
special treatment for Golgi-Cox
preps., 474
neurofibrils, 416, 417, 418
neuroglia methods, 479, 484
Golgi's sublimate method, 470
Fanz, grinding bone, 370
Farrant's medium, 221
Fat and glycogen stain, 296
Fatty substances, Cramer and Gatenby,
356 et seq.
Faure-Fremiet, centrifuge, 332
protozoa, 540
Faussek, cephalopoda, 283
Feist, 407
Felizat, 168
Ferreri, decalcifying, 255
Ferria, 352
Ferric and ferrous salts, 297 et seq.
Fettponceau, 367
Fibres of Sharpey, 375; 374.
Fibrils, connective tissue, 350 et seq.
Fibrin, Weigert stain, 388
Fick, 184
kerato-hyalin, 340
Siredon, 277
Fieandt, neuroglia granules, 486
Fiedler, 525
Field and Martin, 105
Fiessinger, 297
Films, blood, 379
fixation, 381 et seq.
INDEX.
573
Finotti, 408
March! method, 450
myelin, 452
Fischel, 505
chick embryos, 273
Fischer, 223, 344, 353
on coagulation, 20 et seq.
nucleoli, 314
trematodes, 516
Fischler, 368
Fish, 405
embryos, Bielschowsky (Paton), 430
eggs, 279 et seq.
methylen blue nerve method, 478
Fish, on clearing celloidin sections,
103
brain of Desmognathus, 405
decalcification, 253
Golgi method, 464
Fixation, in embryology, 259 et seq.
by immersion, 25
by injection, 26, 397
by salts, 40
Fixing agents, cytological, 301
theory of, 18, 19, 131
FlageUa". stains for, 548
Flagellata, general stains, 533
Flatau, 402
Golgi, corrosive method, 472
Flattening sections, 90
Flechsig, 206, 449, 472
Flemming, 339
chromo-acetic, 35
chromo-aceto-osmic, 35
Dahlia, 169
decalcified bone sections, 375
orange method, 171, 177
picro-osmic, 56
safranin, 166
Flesch, 444, 498
blood, 380
Floyd, 508
Flustra, 500
Foa, 214
sublimate and bichromate, 49
Foettinger, 14, 515
Fol, 16, 542
mixture, 39
mounting watery sections, 116
carmine mass, 235
picro-chromic, 56
Fontana, tannin-silver protozoon stain,
545
Foot and Strobell, smears, 308
Formaldehyde, 60 et seq.
and alcohol, 62
modifications of Golgi method, 463
mordant, 165
Formalin, blood, 381
for nervous system, 401
and picric, 62
chrome techniques, 323
and sodium chloride, for maceration,
244
Formalin — contd
Formol, chromic, 63
Formol-Miiller, 63
nitric acid for decalcifying, 253
sublimate, 63
.vapour, blood, etc., 381
Formic acid, gold method, 204
Foster and Balfour, embryology, 271
Frsenkel, 297, 449
Francotte, 150, 289
Freeborn, 407
picro-nigrosin, 350
Freezing methods, 109
Frenkel, palladium and osmic, 50
Frenzel's mercuro -nitric, 47
Fresh cells, 293
Frey, 219
Friedenthal, hardening mass, 237
Friedlander, 524
Frog embryology, 275
eggs, removal of mucin, 276 et seq.
Gatenby's fluid for eggs, 277
methylen blue, 191
skin, 391
Frohlich, 176
Frozen sections, 109
brain, 406
Fuchsin (basic), 169
See Acid Fuchsin.
carbolic, 169
Furst, bleaching, 256
Fusari, 377
Gage, 101, 297
albumen and mercury mounting
medium, 220
alum, nitric acid decalcifier, 253
celloidin sections, 117
clearing mixture, 69
maceration, 248
with formalin, 244
Galesescu, 482
Gallein, myelin, 448
Galli, China blue, 440
Garbini, 522
Gardiner, ova of polychcerus, 288
Garlic water, 115
Gaskell, 94
Gastric glands, 392 et seq.
Gastropoda, 283, 500
embryology, 283
eyes, 502
Gatenby, 308, 435
amoeba culture, 536
benzidine dyes, 388 et seq.
centrifuge, 332
Champy-Kull fixation, 322
Cramer's osmic vapour method, 330
differentiation between cytoplasmic
inclusions, 334 et seq.
Donacia egjgs, 287
double stain for mitochondria, 326
(and Cramer) fatty substances, 356
on Fischer's theory, 20, 21
574
INDEX.
Gatenby — contd.
on fixing agents, 22, 23, 24, 25
Flemming modification, 36
fluid for frog eggs, 277
oft killing, 11
Limnsea embryology, 284
Mann-Kopsch method, 328
Mann-Kopsch-Altmann combination
stain, 329
on methods of mammalian embryo-
logy, 263
mitochondria, etc., 316 et seq.
mitochondria! fluid, 319
new advances in embryological tech-
nique, 258
plan for cytological research, 337,
338
sponges, 526
technique for chromosomes, 303 et
seq.
tissue culture methods, 550 et seq.
on Unna's oxypolarity theory, 21
and Woodger, 365
Gaule, desiccation method for paraffin
sections, 111
solution, 46
Gaultheria, oil of, 68
Gavazzeni, 341
Geberg, 206
corpuscles, 342
Gedoelst, 249
neurokeratin, 441
Gee and Harrison, 124
Gehardt, reptile blastoderms, 274
lens, 343
Van Gehuchten, 413, 465
axis cylinder and dendrite, 459
fixing fluid, 415
ear, 498
Gelatin, embedding masses, 92
injection masses, 232
blue, 236
green, etc., 237
red, 234
yellow, 237
cement, 229
freezing mass, 110
and glycerine, 93
Gemelli, flagella, 549
Gentian blue, 183
violet, 162, 167 et seq.
neutral, 396
for fibrin, 388
carbol, for flagella, 548
Geoffrey, mounting medium, 224
Gephyrea, 511
Gerlach (gold), 207
Gerota, 202, 398
brains, 402
Golgi method, 464
Gerould, 518
Giemsa, blood stain, 384 et seq.
for neuroglia, 487
for protozoa, 536, 544, 545
Gierke, 197, 214
maceration, 245
Giesbrecht, 505
embedding, 78
Gieson (picro-saurefuschin), 176,
215
Giglio-Tos, blood, 382
Gilbert, 442
Gilson, bleaching, 256
chloral hydrate jelly, 224
copper formol, 64
mercurial mounting medium, 220
mercuro-nitric, 47
Sandarac mounting media, 227
rapid celloidin method, 104
Glands, 391 et seq.
Glaue, 514
Glia, 479 et seq.
Glochidia, 284
Glucose mounting medium, 221
Glycerine, gelatin and glycerine injec-
tion media, 233
gelatin, 93
and HC1. decalcifier, 254
jelly, 223
for maceration, 247
for minute dissections, 8
mounting media, 222 et seq.
Glycerised blood-serum, 219
Glychaemalum, Mayer's, 153
Glycogen, 294 et seq.
and cell inclusions, 338
Goadby, 220
Goblet cells, 392
Gold, colloidal forms, 133
gelatin embedding, 93
gelatin mass, Tandler, Pearl, Mayer,
Mozejko, 238
impregnation, generalities, 202 et seq.
preservation of specimens, 208
Gold-size, 230
Gold-sublimate, Golgi method, 475
Goldmann, 131
benzidine dyes, 388 et seq.
Goldscheider and Flatau, 414
Goldschmidt, 552
Golgi, 26, 435,i*3<t
axis cylinder and dendrite stains,
454 et seq.
sublimate method, 470 et seq.
mixed process, 460
avoidance of precipitates, 466
cutting and manipulation, 467
mounting, 468
body in red cells, 387
Golgi method, modifications, 461
formaldehyde process, 463
theory of impregnation, 460
methods for funnels and spiral fila-
ments, 439
bichromate-sublimate modifications,
472
gold, 207
osmio-bichromate mixture, 458, 516
INDEX.
575
Golgi — contd.
processes for rejuvenation of over-
hardened tissue, 462
apparatus, generalities, 316
bone and teeth, 376
Kopsch techniques, 327 et seq.
Mann-Kopsch-Altmann method,
329
other methods (silver), 438
plants, 325
silver methods of Golgi, Veratti,
Da Fano, Cajal, 435 et seq.
Sjovall method, 331
corpuscles of, 347
Golgi-Cox, 472
Da Fano's special mounting method,
474
making permanent preparations, 473
Golgi-Kopsch apparatus, 316
Golgi-Veratti, Golgi apparatus method,
435
Golodetz, 8, 210, 366
ana Unna, cholesterin, 341
Golovine, 179
Goodrich, 308, 377
modification of Kent's method, 541
Goodsiria, 282
Gordon, 180, 549
Gorgonia, 521, 522
Goronowitsch, 280
Gothard, methylen blue, 414
Graeffe, paraffin solvents, 76
Graff, Hirudinea, 511
Turbellaria, 517
Graham, 390, 514
Gram, gentian violet, 167
Grandis and Mainini, 300
Grand-Moursel and Tribondeau, pan-
creas, 395
Grandry, corpuscles, 342
Grassi, hsematozoa, 546
Gray, 498
Greef, 493
Green, B., 14
Green leucocytosis, oysters, 300
Gregory, micro-injection apparatus, 270
Grenacher, borax carmine, 136, 141
bleaching mixture, 257
eyes of mollusca, 502
Greppin, 469
Griesbach, 178, 182, 352
blood, 380
Grinding sections, 108
de Groot, alcoholic haemalum, 156
iron carmalum, 139
Groselj, 522
Grosser, partially aqueous ink mass, 241
Grunpulver, 159
Griinstein, bladder, nerves, 349
Grynieltt and Mestrezat, 256
Guanin, 301
Gudden, 465
myelin, 446
Gudger, salmon embryology, 280
Gulick, Ascaris ova, 290
Gulland, 379
blood fixing fluid, 381
Gum, and chloral hydrate mount, 221
glycerine, Allen, Langerhans, 221
mounting medium, 221
syrup masses, 110
Thus, 228
Gurwitsch, 150
Guyeisse, 392
Gymnoblastea, 522
Gymnotus, electric organ, 346
Haber and Guild, Cajal's method, 426
Hadzi, 523
Haecker, 301
Hsemacalcium, Mayer, 155
Haemalum, 152
de Groot, 156
Hsemastrontium, Mayer, 156
Heematein, 145 et seq.
neurofibrils, 416
Heematoxylin, 145 et seq.
Benda and Heidenhain, 148, 149
Bohmer, 154
Delafield, 154
chemical nature of, 145
chrome, Hansen, 157
Heidenhain, 156
Schultze, 157
combination stains, 213
copper, Benda, 157
elastin, 353
Golgi-method, 476
iron, 148
Kleinenberg, 155
for iron in tissue, 298
mounting in glycerine, 152
osmium, Schultze, 158
phospho-tungstic, Mallory, 158
and picro-Saurefuchsin, 215
ripening, 146
and safranin, 214
and Saurefuchsin, 214
stock solutions, 146
tin, Donnaggio, 158
vanadium, 157
Haematoxyline noire, 151
Haemoglobin, 300
Hsemosiderin, 300
Hair, 341
Halle and Born, orientation method, 98
Bela Haller's mixture, maceration, 247
Halliburton, 410
Hamann, 506, 513
Asteroidea, 519
Hamburger, 218
Argyroneta ova, 287
Hamilton, 404
congelation method, 110
Hammarsten, 357
Hanazawa, dentine, 371
Hance, fixation of mammalian chromo-
somes, 305
576
INDEX.
Hansen, 140, 151, 176, 180
chrome-hsematoxylin, 157
Hantsch, 223
Hardening, 27
injection mass, 237
nerve tissue, 398
Hardy, 123, 512
Hari, 391
Harmer, 202
Harris, 15, 154
haematoxylin, for elastin, 353
myelin, 449
methylen blue, 195
Harrison, Ross, tissue culture, 550 et
seq.
Hart, 353
Harting, 219
gamboge glycerine mass, 239
Hartmann, 269
mammalian embryology, 265
opossum embryology, 267
Haswell, Temnocephala ova, 289
Hatschek, Amphioxus, 281
Haug, 251, 449
decalcifier, 254
Havet, 517
ccelenterates, 522
Hayem, blood fluid, 380
Heat, for killing, 12
Heckert, eggs of Distomum, 289
Heidenhain, 163, 173
alcoholic hsematoxylin for protozoa,
543
carbon bisulphide embedding, 507
centrosomes, 315
chrome-hsematoxylin, 156
theory of dyeing, 125
Ehrlich-Biondi, 173
gelatin glycerine jelly, 223
iron hsematoxylin, 147 et seq.
vanadium hsematoxylin, 157
Heinke and Ehrenbaum, 281
Heinrich, 352
Held, 150, 326
- formol Miiller, 63
method for marginal neuroglia, 484
methylen blue and erythrosin method,
415
Helix, 501
ova, 283
Heller, 209
and Gumpertz myelin, 451
Helly, sublimate and bichromate, 49
Henchman, gastropoda, 283
Henking, examination medium, 293
methods for arthropod eggs, 285
ova of Phalangida, 287
Henneguy, 38
acetic alum carmine, 137
albumen water method for section
mounting, 114
chick, 273
fish embryology, 279
gastropod embryology, 283
Henneguy — contd .
mammal blastoderm, 268
permanganate method, 165
treatment of faded sections, 6
Hennings, 505
eyes of arthropoda, 509
Insecta, 505
Henocque, gold, 205
Herbst, 506
corpuscles, 342
Herdlicka, brain, 402
Hermann, 401
fluid, 38
pyrogallol method, 209
safranin and gentian, 168
L'Hermitte and Guccione, 485
Herrick, Astacus ova, 288
Hertwig, silver for marine animals,
202
frog eggs, 278
maceration mixture, 246
Triton eggs, 277
Van Herwerden, nuclease, 309
Herxheimer, 184, 340
Scharlach R., 367
Hesse, eyes of heteropoda, 502
Hessert, 549
Heteropoda, 500
eyes, 502
Heyder, Arion embryos, 284
Heymons, 501
eggs of Orthoptera, 286
Hickson, 508
Brazilin, 211
eosin and hsematoxylin, 214
maceration, 248
High refractive mounting liquids, 224
Hill, J. P., 274
clearing and embedding, 262, 269
manipulation of ova, 266
" marsupial mixture," 265
paper for reconstructions, 262
modification of picro-nitric, 55, 264
treatment and isolation of eggs of
mammals, 264
Hill, 461, 466
myelin method, 448
Kindle, 547
Hippel, 495
Hirota, orientation, 274
; Hirschfelder, 512, 513
! Hirschler, 328
Donacia eggs, 286
protozoa, 541
Hirudinea, 511
killing, 12
methylen blue, 192
nervous system, 511
His, 38, 197
Histiocytes, 388
; Histriobdella, 511
Hochstetter, injection, 241
Hoehl, 37, 249
Hofer, 15
INDEX.
577
Hoffmann, 81, 390, 508, 517
chick embryos, 273
green, 182
Hogben, chromosomes, 303
Hoggans, histological rings, 199
perchloride of iron, 209
Hollande's chlorcarmine, 139
Holmes, Planorbis ova, 284
Holmgren, trophospongium, 439
Holothuria narcotisation, 12
Holothurioidea, 518
Homans, pancreas, 396
Homarus, 288
Honing, 84
Hopewell-Smith, teeth, 373
Hopkins, maceration, 240
Horn, hair, nails, 341
Hornowski, 351
Horny structures, maceration, 247
Hoskins, chick, 273
Hot water, for sudden killing, 12
Houser, 442
Hoyer, 75, 401
carmine mass, 235
gold, 205
Golgi method, 463
mounting medium, 221
mucin, 391
shellac mass, 242
silver method, 200
nitrate yellow gelatin mass, 237
Huber, 468
Hudson, 512
Human brain, 402, 404
neurofibrils, 421
embryos, 42
Hyatt, 108
Hydatina, 512, 513
ova, 288
Hydra, 521
methylen blue, 189
Hydrochloric acid, carmine, 143
for decalcih'cation, 251 et seq.
for maceration, 247
Hydrogen peroxide bleacher, 256
Hydroidea, general, 523
heat killing, 12
Hydroxylamin, for narcotisation, 15
Hymenoptera, eggs, 286
Hypochlorite of potash, corrosion,
250
of soda, corrosion, 250
Ide, 105
Idiozome, 316
Igacuschi, 393
Ilberg, 415
Imbedding, 71 et seq.
See also Embedding,
for amphibia, 276
in paraffin or collodion, 5
Imms, 504
Impregnation methods, 197 et seq.
M.
Impregnations other than gold, silver,
or osmium, see page 210.
iron sulphate, 210
palladium chloride, 210
perchloride of iron, 209
mass of gelatin, 237
Indian ink, injection of insects, 509
embryos, 270
mass, 240
India-rubber and paraffin, 92
Indifferent liquids, 217
media, 292
Indigo, 212
Indigo-carmine, 182, 212
with oxalic acid, 212
Indophenol, 367
Indulin, 409
aurantia-eosin, 180
and nigrosin, 182
Injection masses, purely aqueous, 240
partially aqueous, 240 et seq.
celloidin, etc., 241
Fol, Tandler, Beale, 238
gamboge glycerine, 239
gum arabic, 241
indigo carmine, Thoma, 239
milk, 241
warm, 232
cold, 238
methods, 232 et seq.
arthropods, 508
Hirudinea, 511
mammalian and other embryos, 270
mollusca, 503
"natural," 242
Inner vation, bladder, 349
Insabato, 352
Insects, 504 et seq.
double embedding, 506
carbon bisulphide, 507
• mounting whole, 504 — 505
Instruments, microscopes, microtomes. 8
Intercellular bridges, 339
Intestine, 393
Intranuclear rodlet of Roncoroni, 421
Intra vitam, methods, 292
stains, mitochondrial, 332
staining, alizarin, 130
Bismarck brown, 130, 162
Bolles Lee on, 129, 130
Congo red, 130
Fischel, 130
kidney, 394
Loisel, 130
methylen blue, 186 et seq.
neutral red, 130, 179
protozoa, 540
sulphorhodaniin, 130
theoretical, 129 et seq.
In vacuo, embedding, 80
Inversion plasma stains, 184
Invertebrates, general methods, 499 et
seq.
dendrites and axis cylinders, 454
37
578
INDEX.
Invertebrates — contd.
ganglia, Cajal, 425
nervous system, 416, 417
Iodide of osmium, 331
of potash and biniodide, "liquid, 224
for maceration, 244
Iodine, extraction of corrosive, 45
faeces examination, 538
fixation, 51
green, 182
method for glycogen, 295
vapour fixation, 51
Iodised serum, 219
for maceration, 243
Ii'idium chloride, 50
Iris, 348
Iron, 297 et seq.
alum fixation, 50
Brazilin, 211
for protozoa, 545
carmalum, 139
carmine, 139
cochineal, 140
li<%maloxylin, 147 et seq.
Benda, 148
Biitschli, 150
rapid method, 150
Weigert, 150
perchloride fixation, 50
sesquichl orate, myelin, 451
Isamin blue, 390
Islets of Langerhans, 395, 396
Isolation of single cyst, etc., 527
Israel, 180, 212
Iwanzoff, 518
electric organ, 346
Hatchett Jackson, 66
Jacoby, 183
Jacquet, leeches, 511
Jadassohn, 340
Jaderholm, 419
Jaenichen, 517
Jager, 223
Jakimovitch, 201
Jander, 502
bleaching, 257
Janssens, 151, 183
Janus green, 181, 332, 333
pancreas, 395
" Japanese " method of section mount-
ing, 114
Jaquet, 510
Jelinek, 101
Jenner, blood stain, 383
Jensen, protozoa, 540
Joest, 509
Lindsay Johnson, 206
aceto-osmic, 51
collodion method, 101
eye, 494
fluid, 37
on metallic stains, 198
Johnston, 262, 415
nerves of Petromyzon, 405
Johnstone-Lavis and Vosmaer, section
grinding, 109
Joliet, gum glycerine embedding, 107
Jones, 300
Jonescu, 508
Jordan, 66
Jorgensen, 309
nucleoli, 314
Joris, 434
Joseph, 197, 341
white -of -egg mass, 240
Juliusburger, Nissl, 413
Kadyi, 407
brain, 402
Kaes, myelin, 447
Kaiser, 170, 223, 513
glycerine gelatin, embedding, 93
myelin, 447
solution, 44
spinal cord, 408
Kalb, 549
Kallius, 455, 469
embryonic cartilage, 376
Golgi modification, 461
Kaplan, 477
myelin, 452
neurokeratin, 441
Kappers, elderberries, 409
Karawaiew, 546
anobium eggs, 287
Karger, 493
Karyosome, 310 et seq.
Kastschenko, 262
Selachian embryos, 280
Kat6, Cajal's method, 425
Kattwinkel, 400
Katz, ear, 497
Kawamura, 366
Keibel, 270
Kent, iodine fixation, 51
Kenyon, 508
Keratohyalin, 340
Kernschwarz, 211
Graham Kerr, 119
reconstruction method, 26 L
Kerschner, 205
Kidney, 394
Killing, amphibians, pithing, 11
birds, lizards, newts, 11
by sudden heat, 12
by hot water, 12
large mammals, ether and chloro-
form for, coal gas for, 11
King, brain hardening, 405
Bufo eggs, 278
carbolic thionin, 168
Kingsbury, 368
Kingsley, Limulus ova, 288
Kionka, orientation method, 274
Kishinouye, spider eggs, 287
INDEX.
579
Kizer, blood, 381
Klein, cornea, 343
Kleinenberg, hfematoxylin, 155
picro-sulphuric, 55
Knife position, paraffin cutting, 83
slope, 85
Knowen, 81
Koch, 449
Von Koch, copal method, section grind-
ing, 108
Kockel, 388 *
Kodis, 404
myelin, 442
Koerner and Fischer, tannin fuchsin
flagella stain, 548
Kofoid, gastropod embryology, 283
Kohler, 516
Kolliker, mammal blastoderm, 268
embryology of mammals, 264
indigo-carmine for bone, 376
Kollman's fixative for fish eggs, 280
Kolmer, 26, 495
Kolossow, 32, 206, 209
modification of Cajal's axis cylinder
stain, 462
prickle cells, 339
Kolster, stomach, 392
Kopsch, 386
osmic method, 327
Golgi method, 464
mollusc eyes, 502
teleost embryology, 280
Korotneff, 13
Korschelt, 541
cephalopoda, 283
Kostanecki, mercuro-nitric, 48
and Siedlecki, Ascaris ova, 290
and Wierzejski, mollusc eggs, 284
Kotlarewski, 404
Kowalewsky, teleost eggs, 280
Kowalski, 425
Kozowsky, myelin, 446
Krause, 173, 182, 393, 482, 496
carmine mass, 235
salivary glands, 392
Krauss, 201
Krecker, 14
Kresofuchsin, 169
Kresyl blue, 383
Kresyl-echtviolett, 184
Kresyl violet, 184
Krogh, 415
Krohnthal, lead sulphide impregnation,*
475
Kromayer, 184, 388
plasma fibrils, 339
Kronecker's serum, 218, 264
Kriiger, 354
Harpactida ova, 288
Kuhne, maceration, 247
Kuhnt, 496
Kukenthal, 14, 510
Lumbricus, 509
Kull, 392
Kultschizky, 341
bichromate and sublimate, 43
double embedding, 105
mucin, 391
myelin method, 447
preservation of tissue in ether or
xylol, 4
rubin for neuroglia, 486
solution, 42
spleen, 393
Kupffer, 393 .
Kuskow, digestion, 249 .
Lachi, 401
Golgi method, 463
Lactic acid, 233, 254
Lactophenol, 222
Leevulose, for myelin preparations, 448
Laffont, 341
Lahille, 499
Lake, 132
Lakmoid, 393
Lambert, Epeira eggs, 287
Lamellibranchiata, 284, 500 et seq.
cilia, 503
eyes, 502
maceration, 247
pigment, 257
Lams, 265
Landois, maceration solution, 245
Landolt, 496
Lane, pancreas, 396
Lang, corrosive liquid, 46
mollusca, 501
Langdon, 510
De Lange, 450
Langerhans, 341
gum glycerine, 221
islets of, 395
Langeron, 59
Lanis, fixation of mammal eggs, 265
Lankester and Bourne, 508
Lansberg, 541
Lanthanin, 302
Larvae, echinoderm, 520
Laslett, 447
Lattice fibres, 393, 394
Laurent, 181
Lauterborn, 75, 547
Lavdowsky, 192, 228, 498
maceration, 248
Laveran's solution, 534
Law, nerve-endings, 374
Lawrence, 223
Lead-gum embedding, 100
Lead sulphide, Golgi method, Krohn-
thal, 475
Leber, 495
Lebrun, Anurau embryology, 276
Lecithin. See under Fatty Substances,
356 et seq.
Bolles Lee, 407
decalcification, 251
37—2
580
INDEX.
Bolles Lee — contd.
celloidin section mounting, 116
chick embryos, 272
chromosome stain, 303
on choice of stain, 134
dry cutting process, celloidin, 104
ear and eye, 493—498
fish embryos, 279
fixation of ground cytoplasm, 301, 302
gold, 206
iron carmine, 139
Kernschwarz, 211
mounting fluid, 222
mounting Golgi preparations, ;468
myelin stain, 442
osmic-pyrogallol method, 208
paper cell mounting method, 230
on staining nucleus intra vitam, 310
Tunicates, 499
sponges, 525
Leeches, 511
maceration, 247
nervous system, 511
Legal, alum carmine, picric, 138
Legendre, Golgi apparatus, 438
Leger, sporozoa, 546
Legros, 201
Amphioxus, 281
Leiper, 514
Leishman, blood stain, 385
protozoa, 535
Lemon- juice gold method, 204
Von Lendenfeld, 525
Von Lenhossek, 342, 455
Nissl bodies, 413
mollusc eyes, 502
platinum chloride, 50
Lennhoff, methylen blue, nerve, 414
processes for nerve cells, etc., 476
Lennox, 496
Lens, eye, 343
Lenssen, 513
Hydatina, 288
Leon to wit sen, 195
Lepidoptera ova, 286
Lepkowsky, 375
vessels in teeth, 373
Leuckhart, embedding boxes, 73
Leutert, 300
Levaditi, 549
blood smears, 383
Levi, fixation of mammal eggs, 265
mitochondrial method, 325
Levulose, 221
Levy, 389
Bevan Lewis, 408
Lewis, 389, 511
tissue culture, 555
Lewy, 450
Lichtgrun, 159
Liebetanz, 548
Liesegang, 407
Cajal's method, 426
Ligamentum nuchse, digestion, 249
Light green, 181
Lillie, 332
Unio eggs, 284
Limax, 283, 501
Lime salts, 300
Limnsea ova, 284
Limulus, 288
Linville, mollusc ova, 283
Lipin, 357
Lipoids, 357
List, 214, 314 «
Coccids, 506
Mytilus, 500
Sagartia, 522
Lithia, for picric fixed material, 54
Lithium carmine, 140
Little, Hydra, 521
Live slide, Drew and Griffin, 530
Liver, 393
mollusc, 501
Lizard blastoderms, 275
Locke's solution, 218
Locy, spider eggs, 287
Loele, 390
Loewy, method for integument, 339
Loffler, stain for flagella, 548
Loisel, 367
fat, 369
sponge, 526
Long, constant temperature box for
mammal eggs, 266
and Mark, fixing fluid for mammals,
266
mouse embryology, 263
Longhi, 542
Longworth, 342
Lonnberg. 516
Looss, 514, 516
corrosion of chitin, 250
Nematodes, 513
Lophomonas, 533
Lord, methylen blue, nerve; 414
Lowit, blood fluid, 380
gold, 204
Loyez, 442
Lucidol, 308
in acetone, 59
blood, 382
Ludford, 308
Lugaro, 417
collargol, 434
Lugol's solution, 45
Luhe, 516
Luithlen and Sorgo, 414
Lumbricus epidermis, nerve, 421
nerve, 425
sections, 509
Lundvall, 377
Lustgarten, 169
Luxenburg, Nissl bodies, 413
Lymphatics, in teeth, 373
glands, 394
Lymph-spaces, methylen blue, 195
Lysol, for maceration, 248
INDEX.
581
Maas, 213, 249
sponges, 526
Macallum, 300, 301
iron methods, 297
Mac Bride, larvae of Echinoderms, 520
Maceration, 243 et seq.
Bela Haller's mixture, 247
epithelium of molluscs, 503
Ranvier, 244
Moleschott, 244
Landois, 245
Macklin, 271, 389
Maclean, 357
Macrophages, 388
Von Maehrenthal, 209
Magdala red, 169
for spleen fibres, 394
Magenta, 169, 171 JL>?
Magini, 477
Magnesia carmine, 140
Magnesium chloride or sulphate, for
narcotisation, 15
Malachite green, 181 .*• 2 ; 3
Malaria, 546
pigment, 300
Malassey, 218
Mall, 378
-Mallory, 354
eosin and methyl en blue, 181
neuroglia, 483
phospho-molybdic acid haematoxylin,
157
phospho-tungstic, 158
saure-fuchsm and phospho-molybdic
acid, 351
Weigert stain, 481
Mammalia, Cajal's methods, 424
eggs 266. See also under Ova.
Mammals, chromosomes, 305 et seq.
clearing and embedding, 269
embryological methods, 263
fixation of whole tubes, 268
isolation of eggs, 263 et seq.
Manchester brown, 161
Manfredi, 206
Mann, 26, 31, 114, 155, 398, 468
chromo -sublimate, 48
fat, 366
extraction of corrosive sublimate, 45
Golgi-Cox, 473
Golgi method precipitates, 466
methyl blue eosin, 183
osmio -sublimate, 48
stain, for protozoa, 544
sublimate formol, 63
Mann-Kopsch method, 328
Marcacci, maceration, 247
Marcano, blood, 381
Marchi, degenerate nerve, 449 et seq.
mollusca, 501
tendon organs, 347
Marcus, 446
Maresch, 352, 394
Marfori, 300
Marie, 400
Marina, 405
Marine animals, fixation of, 27
cell inclusions, 332
Mark, collodionisation, 88
reconstruction, 262
Marsh, 229, 230
Martin, 392
tracheae, 508
Martinotti, 167, 196, 354, 369, 386, 467,
408
Golgi method, 465
elastic tissue, 352
Mason, brains, 405
Massart, 214
Masson, connective tissue, 351
Mast cells, 354 et seq.
Unna's method, 356
Matschinsky, bone sections, 371
Matuszewski, 450
Maurice and Schulgin, 213
Mawas, 496
Maximow, 356, 389
Altmann method, 322
May-Griinwald, 384
Mayer, 15, 33, 155, 184, 297, 350
acetate of potash, 151
albumen water method for sections,
113
alcoholic cochineal, 144
aluminium chloride carmine, 138
benzol embedding, 78
bleaching, 256
carmalum, 137, 212
cartilage, 377
chitin, 508
cochineal stain, 135
decalcification, 253
desilification, 255
eye, 339
glychaemalum, 153
haemacalcium, 155
hsemalum, 152 et seq.
hsemastrontium, 156
on hsematoxylin staining, 145
iron staining, 298
methylen blue, 193, 196
mucin stains, 392
paracarmine, 142
picro-hydrochloric, 56
picro -magnesia carmine, 141
picro-nitric, 55
Plutei, 520
section stretcher, 87
triacid, 175
Maysel, 161
Me Clung, urea in fixing, 305
McClure, 502
McCrorie, 549
Medium of Farrant, 221
Medulla, hardening, 399
Medusee, 523, 524
killing, 12
maceration, 246
582
INDEX.
Medusae — contd.
narcotisation, 13
sections, 524
Mehnert, 275
Meirowsky, 549
Meisenheimer, gastropoda, 283
Melanin, 300
Melting point of paraffin, 91
Membrana, nictitans, 339
limitans, marginal neuroglia, 485
Membranes of eggs, 284, 285
Mendel and Bradley; 300
Menthol, for narcotisation, 12
Mercier, 449
Mercury, bichloride, 44
alcoholic liquids, 46 et seq.
and acetic acid, 44
extraction, 45
sodium thiosulphate, extraction by,
46
washing out, 45
and bichromate, 48
and formol, 63
mounting media, 220
nitric mixtures, 47
sublimate and salt, 46
Merkel, indigo-carmine, 212
Merkel's fluid, 39
Merton, 502
Merzbacker, 485
Mesostomidse ova, 288
Messner, picrocarmine, 414
Metachromasy, 133
Metachromatic dyes, 133
Metagelatin vehicle, Fol, 238
Metallic stains, 197 et seq.
theory of, 197 et seq.
osmic acid, pyrogallol, 208
Metcalf, Chiton ova, 284
Methyl, aniline green, 159
blue, 183
with eosin (Mann), 183
nephridia, 511
green, 159 et seq.
for chromatin, 293
with eosin, 181
mixture for maceration, 247
salicylate oil, 68
violet, 162
for fibrin, 388
fluid for blood, 382
plasma fibrils, 340
B., 184
Methylen blue, 170, 186 et seq.
blood, 383
Cajal's diffusion process, 478
Distomum, 516
and eosin (Mallory), 181
eosin stains for blood, 383 et seq.
and erythrosin, Nissl granules,
415 *
fixation of stain, 192 et seq.
frog bladder, 349
intm vitam staining, 187, 344, 477
Methyleu — contd.
for epithelia, lymph-spaces, 195
insect eyes, 508
method, cornea, 343
methods for sections, 194
modes of staining, 190
myelin, 449, 452
nerve endings, 344
methods, 477 et seq.
Nissl bodies, 411
polychrome, 186
Saurefuchsin, 351
for skeletons, 377 *
Methylenazur, 186, 187
Meves-Flemming modification, 36
Victoria green stain, 333
Meyer, Berlin blue neurofibril method,
434
methylen blue nerve method; 477
myelin, 445
new celloidin method, 103
Mibelli, 352
Mica sheets, for mounting sections, 119
Michaelis, 133, 181
blood stain. 383
fat, 367
polychrome methylen blue, 187
Triton eggs, 277
Michailow, 190, 195
Microchemistry, 293 et seq.
Microglia, 493
Micro-injection, 270
Microscopes, 8
Microtome, 8
sliding, freezing, rotary, Jung, Minot,
Tetrander, rocking, 9
Migula, 219
Miller, cement, 229
Minchin, albumen method for mammal
eggs, 266
embedding protozoa, 542
hsematozoa, 547
sponges, 525
Minervini, 353
Mitochondria, fresh examination, 332,
333,334 ist, 3b\
Gatenby on, 316
protozoa, 540
teeth and bone, 375
vital stains, 332
Mitrophanow, 183, 342
chick embryos, 273
integument, 339
myelin, 447
Mitsukuri, reptile blastoderms, 275
Mixed process of Golgi, 460
Mobius, 348
maceration medium, 246
Moerner, 377
Moleschott and Piso Borme, salt alcohol
for maceration, 244
Molge, embryology, 277
Moll, cartilage, 376
Mollison, Alkanna, 367
INDEX.
583
Mollusca, 28:2, 5UU d seq.
embryology, 282 et seq.
to kill extended, 500
maceration of epithelium, 503
mucus glands, 504
narcotisation, 12
nervous system maceration, 247
Molluscoida, 500
Molybdenum-toluidin blue, neurofibrils,
417
Monckeberg and Bethe, 31
bleacher, 256
Mondino, 472
Montanari, 419
Montgomery, 515
nucleoli, 314
ova of Theridium, 287
Monti, 477
gastric glands, 393
Mordant, 131
Mordanting, for coal tar dyes, 165
Moreaux, formol - picric - trichloracetic,
62
Morel, 175
Morgan, Ascidia, 282
cockroach eggs, 285, 286
frog embryology, 276, 278
Morphia, for narcotisation, 16
Moseley, 503
Mosse, 415
argentamin, 452
Mosso, 380
Mott, 129
Mounting media, glycerine, 222
resinous, 225
Farrant, Allen, Brun, 221 .
sections, collodion paper method, 116
Mayer's albumen method, 113
watery, 116
Mozejko, 503
vaso -dilators, 233
Muchaematein, 392
Mucicarmine, 392
Mucilage syrup mass, 110
Mucus, 391 et seq.
glands, mollusca, 504
Muir, 549
blood, 381
Mullenix, 497
Muller, 392
Berlin blue mass, 240
fluid, 42
with formol, 63
for maceration, 245
silver method, 201
Mummery, embedding tooth germs,
372
nerve tissue of teeth, 373 .
J. A. Murray, Altmann's method, 321,
365
on glycogen stain, 296
connective tissue stain, 354
chrome-osmic method, 326
lead gum, 107
Muscle -iibres, maceration, 240
and tendon, 344, 347
spindles, 345
Myelin, ammonio-chloride of tin, Besta,
448
and axis cylinder stains, 452
Berkley rapid method, 447
bulk stain, 448
iron method, 451
Kultschitzky, 447
osmic acid, 450 et seq
stains, 442
silver nitrate, 451
Weigert stains, 442
Myers, 415
Mytilus, 500
Myxosporidia, 546
Nabias, gold chloride Golgi method, 476
mollusca, 501
Nageotte, 399, 448
Nails, 341
Nakahara, urea fixation, 305
Nakanishi, blood, 383
Nansen, 245
Naphtha, clearing, 77
Naphthalin, monobromide, 224
rose, 169
Naphthol, 390
Naphthylamin brown, 408
Naples water bath, 79
Narcotisation, anemones, Holothuria,
Ascidia, Mollusca (menthol), 12
Medusae, starfishes (chloroform),
Actinias, Capitellidae (ether alcohol),
13
Cristatella, 14
larvae (chloretone), 14
Alcyonella, Bryozoa, Annellida, Mol-
lusca, Nemertians, Actiniae (chloral),
14
hydroxylamin, 15
magnesium chloride or sulphate, 15
morphia, curare, strychnin, prussic
acid, 16
asphyxiation : tobacco, carbonic acid
gas, soda water, 16
hydrogen peroxide, 17
Hydra (cocaine), 14
Vorticellidaa, Rotatoria, Vermes (eu-
caine), 15
Ascidia, Rhopalaea, 15
Ciona, 16
Medusae, 16
snails, 16
menthol, nicotine, 12
chloroform, cocaine, eucaine, ether,
alcohol, 13
methyl alcohol, chloreton, chloral hy-
drate, 14
Nasal mucosa, maceration, 247
Nathusius, 341
Nealey, bone sections, 372
584
INDEX.
Nebenkern, 316
Nelis, brain fixation, 405
Nematodes, 513 et seq.
corrosion of chitin, 250
ova, 289
Nemertina, 514 et seq.
heat killing, 12
Nephridia, Histriobdella, 511
Nerve endings, beetle, 344
Bielschowsky, 426 et seq.
frog, 344
gold method, 344
insect, 345
methylen blue method, 344
silver method, 345
bichromate of silver, 345
of teeth, 373 et seq.
staining, methylen blue, 189, 191
Nervous system, Achucarro's tannin
neuroglia method, 490
Apathy's neurofibril method, 416
axis cylinder, 452
axis cylinder and dendrite stains
(Golgi and others), 454
Cajal's double-impregnation
process, 461
Golgi bichromate - sublimate
method, 470
processes similar to Golgi
methods, 475
Cajal's method, advice as to choice
of formula, 424
(Golgi), axis cylinder and dendrite,
treatment of larvae, 459 — 460
axis cylinder and dendrite, form-
aldehyde modifications, 463
Berkley rapid myelin method, 447
Bielschowsky methods, introduc-
tory, 426
for sections, 427
for peripheral nerve fibres, 428
for pieces, 429
modifications, 430 et seq.
Cajal's gold chloride neuroglia
method, 487
methods, special objects, 424
neurofibril melhods, 419 et seq.
collodionising sections, 406
Da Fano's Bielschowsky methods,
432
Donaggio's neurofibril methods,
417
Fish's fluid, 402
fixation, 397
alcohol, 400
chromic salts, 403 et seq.
formalin, 401
general methods, 397
stains, 407
Golgi method, avoidance of preci-
pitates, 466
cutting and mounting, 467 —
469
Golgi internal apparatus, 435 et seq.
Nervous system- — could.
Golgi-Cox modification, 472
hardening, 398 et seq.
Hold's method for marginal neuro-
glia, 484
injection fixation, 397
Kultschitzky, myelin, 447
maceration, 245, 248, 247
Marchi method, 449
nzurofibrils, 416 et seq.
other methods, 434
neuroglia, 479 et seq.
Nissl substance, 410 et seq.
Pal method, 446
phosphomolybdic acid haemato-
xylin, 157, 158
polarised light, 398
preliminary fixation for Sauro-
psida, 405
Sand's neurofibril method, 434
sections, 405 et seq.
special ci/tologic.al methods, 410 et
seq.
sustaining apparatus of medullary
sheaths, neurokeratin, 439
Simarro's process, 419
Weigert's myelin stains, 442 et seq.
Nesteroffsky, 206'
Nettovitch, 505
Neuberger, decalcifier, 254
Neubert, 297
Neukirch, 297
Neumayer, 262, 270
Neurofibril methods, 416 et seq.
advice on, 424
Neuroglia, 479
Achucarro's tannin method, 496
Cajal's gold chloride sublimate
method, 487
granules, 486
protoplasmic, 486
marginal, Held's method, 484
methods, 479 et seq.
stains, Anglade and Morel, 483
Da Fano, 484
Held, 484
Oppenheim, 486
Weigert, 480
Benda, 482
Mallory, 483
Neurokeratin, 439, 441
Neurosomes, 415
Neutral, balsam, 226
dye, 120
gentian, 396
red, 179 et seq.
blood, 382
granules, 389
kidney, 394
mucin, 392
pancreas, 395
Neutralisation of carmine mass, 235
Neuville, silver impregnation mass.
237
INDEX.
585
Nicolas, 32
embedding in gelatin, 93
reptiles, 275
Nicolle, 168
Nicotine, for narcotisation, 12
Nigrosin, 170, 182, 408
Nikiforow, 102
Nile blue, connective tissue, 354
fat, 362, 368
Nissl, 410, 411, 415, 450, 480
brain hardening, 403
bodies, 410 et seq.
stains other than Nissl's, 412
granules, nature of, 130
methylen blue and erythrosin, 415
method, precautions against fading,
413
modifications, 413
Nitric acid, and acetic, for maceration,
247
bleacher, 257
and chlorate of potash, for macera-
tion, 247
corrosion, 250
decalcification, 252, '2~>:i
for fixation, 38
fixative for neurofibrils, 417
formol, 64
maceration, 247
nerve tissue, 404
Noack, 83
Noll, corrosion, 250
Nordmann, mast cells, 355
Nosema, 546
Nowak, sublimate formol, 63
tactile corpuscles, 342
Nuclear stains, coal tar, 159 et seq.
Nuclease, 308
Nucleoli, Gatenby on, 308, 310
malachite green for, 181
Nucleolini, 311
Nucleus, " vital " stains, 310
Nudibranchs, 502
Nuttall, Cooper and Robinson, 506
Obersteiner, brain, 403
Obregia, 469
method for paraffin and celloidin
sections, 115, 119
Obst, nucleoli, 314
Octopus, 501
Odenius, maceration, 247
Odier, Golgi method, 465
Oestergren, 13, 515, 517
Ohlmacher, 167
alcoholio corrosive, 47
fiuid, brain, 405
formaldehyde process, 165
myelin, 452
picric acid, 176
Oils, 65 et seq.
See also under definitive name, e.g.,
Cedarwood.
winter green, 271
Okajima, elective stain for reds, 388
fat, 368
Olmacher, 404
Olt, manipulation of frozen sections, 109
Opalina, 533
Ophiothrix, 519
Ophiuridea, 519
Oppel, lattice fibres, 393
Oppenheim, neuroglia granules, 486
Oppitz, 201
Orange G., 172, 173, 177 w
Orcein, 212
embryonic cartilage, 376
elastin, 353
method, Unna, 351
water blue, 340
Orehella, 211
Orcin, 212
Organic acids, fixation by, 51
Orientation methods, 273 et seq.
for blastoderms, 273
in embedding, 80 et seq.
Origanum oil, 68
for celloidin sections, 102
Orr, 451
March! method, 450
Orth's fluid, 260
Orthoptera eggs, 286
Orton, 503, 508
Osmacet, 380
Osmium chloride, 50
Osmium tetroxide, reduction, 29, 32, 124
acetic acid maceration, 246
bleaching, 31
restoration of staining, 31
for blood, 380
gold method (Viallane), 205
Kopsch method, 327
nerve tissue, 404
and fat, 356 et seq.
and picric, 56
pyrogallol method, 208
regeneration of, 30
fixation by vapour, 30
after-treatment, 30
sublimate, 48
vapour method, Cramer, 330
Ossifying cartilage, 254, 377
Osteoblastic areas, 378
Ostracoda, 505
Wo. Ostwald, 125
Ova, of Echinoderms, fixation, 260
fixation of mammalian, 265
frog's, 275 et seq.
Insecta, Pisces, 259
treatment of uterine eggs of mam-
mals, 266
Ovary as index to pregnancy, 263
Ovens, paraffin, 79
Overhardened tissue, rein venation of,
462
Overton, 31, 34
iodine vapour, 51
Oviatt and Sargent, 233
5.86
INDEX.
Oxalic acid, for maceration, 247
Oxidised haematoxylin (Unna), 155
Oxidisers as fixers, 21
Oxydase reaction, 390
Oxygen, place in tissue, 390
Pacini, 220
blood, 380
Pal, 472
Paladino, 453
Palladium chloride, 50
decalcification, 252
Palythoa, 522 .
Pansch, injection, 242
Pancreas, 395, 396
inclusions, 338
Pancreatin, digestion, 248
Paneth, cells, 392
Paper trays, embedding, 73
Papillse foliatse, 342
See also under Corpuscles.
Pappenheim, 356
hsemopoietic tissue, 386
mast cells, 355
panoptic stain, 383
pyronin and methyl green, 172
Paracarmine, 142
Paraffin, pure, 91
overheated, 92
sections, flattening, 90
clearing and mounting, 91, 111 et
embedding, 76
shaping block, 83 et seq.
cooling, 82
bath, 77
Paramcecium, culture, 537
Paravicini, 418
Parietal cells, 393
Paris green, 160
Parker, 508
bleaching, 257
methylen blue, 194
and Floyd, sheep brain, 402
Parlodion, 95
Parmenter, Amblystoma, 306
Parolein, 228
Parthenogenesis, 259
Partington and Huntingford, 32, 364
Partsch, cochineal alum carmine, 137
decalcification, 254
Passarge and Krosing, 354
Paton, 272
Bielschowsky modification, 430
Patten, 286, 502
mollusca, 504
orthopterous eggs, 286
orientation during embedding, 81
Patterson, chick, 273
Pauropoda, 505
Pavlov, my el in, 446
Peabody, corrosion, 249
Pearl, worms, 509
Pedicellina, 500
Pekelharing, 297
Pelagic fish ova, 281
Penfield, Golgi apparatus, 438
Holmgren's trophospongium, 439
Pensa, reconstruction method, 261
Peppier, 549
Pepsin, digestion, 248
Peptic cells, 392
Peptonum siccum, 233
Perchloride of iron impregnation, 209
Perdrau, Bielschowsky method, 434
Perenyi's fluid, 39
Perez, fly pupae, 286
Periplaneta eggs, 285
Permanganate of potash, bleacher, 31,
256
Hemieguy's mordant, 165
for osmic solution, 29
Perophora, 282
Peroxide, of ben/ol, blood, 382
of hydrogen^ bleaching, 256
for killing, 17
of magnesium, 256
of sodium, 256
Perrier, worms, 509
Perrin, 122 .
Perusini, 453
Peter, 140
reconstruction method, 262
yolk stain, 260
Peters, R. A., culture of Paramoecium,
537
Petromyzon, nerves, 405
Petronne, 387
Petrunkewitsch, Hymenoptera, 286
mercuro-nitric, 48
Pfitzner, 182
protozoa, 541
safranin, 166
Phalacrocera, 286
Phalangida ova, 287
Phenol, solution for fixation, 47
Phenylen brown, 161
Philippson, 339
Phloroglucin decalcifying mixtures, 254
Phloxin, 180
Phoronis, 512
Phosphatides, 356 et seq.
Phospho-molybdic acid haematoxylin,
Mallory, 157
Phospho-tungstic acid fixative, 38
Phosphoric acid decalcification, 252,
254
Photographic negative varnish mount,
ing medium, 228
Photoxylin, 95
Physa eggs, 284
Pianese, 175
methylen blue eosin, 181
Picraminic acid, 176
Picric acid, 54
decalcification, 252
plasma stain, 176
INDEX.
587
Picric acid — conld.
and alcohol, 55
for maceration, 248
Picro-acetic, 55
Picro-carmine, 140
general nerve stain, 414
Picro-chromic, 56
Picro-hydrochloric, ">(>
decalcifier, 254
Picro-indigo-carmine, 212
Picro-nigrosin, 182, 213, 350, 408
Picro-nitric, 55
decalcifier, 254
Picro-osmic, 56
Picro-platinic, 56
with formol, 63
Picro-Saurefuchsin, 176, 350
Picro-sublimate, 48
Picro-sulphuric, 55
Pictet's liquid, 219
Pink salt, 418
Pintner, 29, 515
Pisces, embryology, 279 et seq.
Pitfield, flagella, 549
Pittock, reconstruction method, 262
Pizon, Ascidia, 282
Placenta, glycogen, 295
Planaria, 44, 517
Plankton, general preservative, 525
Planorbis ova, 284
Plants, Golgi apparatus, 325
Plasma cells, 354 et seq.
fibrils, 339
stains, 171 et seq.
Bolles Lee, 314
Plasmodium, 546
Plasmosome, 310 et seq.
Plastic reconstruction, 261
Plastochondria, 315
Platino-aceto-osmic, 38
Platinum chloride, 49
Plainer, neurokeratin network, 441
Kernschwarz, 211
Pleschko, 194
Du Plessis, 515
Pleurobrachia, 52 1
Pluteus, 520
Podwyssozki, 167
fluid, 36
Polaillon, perchloride of iron impregna-
tion, 209
Polariscope, 332
for fat, 361 et seq.
in nervous system, 398
Policard, 547
Politzer, 496
Polumordwinow, 413
Polychcerus ova, 288
Polychrome methylen blue, mast cells,
355
nerve cells, 476
for nerve tissue, 414
Polychrome toluidin blue, 196
Polyclads ova, 289
Porifera, 525 et sefj.
decalcification, 251
Post-chroming, 326
Post-impregnation for gold, 207 et
seq.
Post-mortem changes, 25
Post-osmicating, 326
Potash method, for skeletons, 378
Potassium, 301
See under Bichromate,
bichromate, maceration with, 245
chlorate and nitric acid, for macera-
tion, 247
iodide, brain, 404
permanganate, for maceration, 246
sulphocyanide, for maceration, 245
Potter, 446
Pouchet, bleaching, 256
Pranter, 353
Precipitates, Golgi method, 466
Prenant, 181, 301, 497
Preservation media, 216
by alcohol, ether, xylol, or toluol
glycerine mixtures, acetic acid,
cedarwood oil, 4
Preyer, 13
Priapulus, 512
Prickle cells, 339
Primerose soluble, 180
Pritchard, 498
Progressive stains, coal-tar, 159
Propylic alcohol balsam, 227
Protease, 308
Protozoa, 526 et seq.
Borrel's blue, 534
Borrel method, 532
cell inclusions, see pages 316 — 338
collection, 529
culture, 527 et seq.
of paramcecium, 537
current stains, 543 et seq.
determination of life cycle, 530
Drew-Griffin live slide, 530
embedding, 542
examination in a coloured medium,
540
of faeces, 538 et seq.
•fixing and preserving, 541 et seq.
'flagellates, 533, 547
general fixation and staining, 531 et
seq.
general morphology, 531
hsematozoa, 546
killing by heat, 12
iodine, 541
Romanowsky methods, 535
immobilisation, 539
staining Coccidia, 532
ciliates, 533
haemamoabae, 533
intra vitam, 540
stains for flagella, 548
sporozoa, 546
sphaerozoa, 546
588
INDEX.
Protozoa — contd.
Taylor's culture method for amceboe,
536
trypanosomes, 533, 539, 547
Prowazek, 179
Prussian blue, aqueous mass, Ranvier,
240
acid cold, Beale, 239
cold, Beale, 239
glycerine mass, cold, Ranvier, 239
Ranvier, 236
reaction, 297 et seq.
Prussic acid, 16
Przesmycky, nucleus staining, 310
Pulmonata, nervous system, 501
Purcell, Atta ova, 287
eyes of Phalangida, 508
Purpurin, 212
Pusateri, tachiol, 425
Puschkarew, 542
Pyridine, 59
with lucidol, 59
nitrate-pyridine fixative, 418
Pyroligneous acid decaleification, 252
Pyronin, 172
and methyl green, 172
pancreas ducts, 395
Pyrosin B., 180
Pyroxylin, 97
Pyrrhol cells, 388
Pyrrol blue, 389
De Quervain, 26, 398
Quinolein blue, 182, 367
Raadt, blood stain, 385
Eabl, 341
chromoformic, 35
cochineal, 137
embryological fixation, 260
lens, 343
picro-sublimate, 48
platinum chloride, 49, 50
platinic sublimate, 260
superheated paraffin, 89
teleost eggs, 280
Rabl-Ruckhard method for salmon eggs,
280
Racovitza, mollusca, 504
Raffaele, 281
Raja, electric organ, 346
Rana, embryology, 277
Randolph, 14
Ranke, neuroglia method, 487
Ransom, 516
Ranson, Cajal's method, 426
Ranvier, 182, 340, 356, 393
alcohol, 58
bladder, nerves, 349
chromic acid maceration, 246
carmine gelatin mass, 234
cornea, 343
Ranvier — contd.
electric organs, 345
formic acid, gold, 204
eleidin granules, 341
eye of Triton, 494
lemon juice, gold, 204
maceration with alcohol, 244
nerve endings, gold, 345
silver method, 345
picro-carmine, 141
silver impregnation mass, 237
method, 198, 200
Vom Rath, picro-sublimate, 48
Rawitz, 38, 184, 502
brain hardening, 404
carmine, 138
indulin, 409
mucin, 392
picro-nitric, 56
V. Recklinghausen, silver, 200
Reconstruction from sections, 261
Red blood cells, elective stain, 388
gelatin mass, 234
Reddingius, 314
Redenbaugh, 15
Van Rees, 285
Refractive indices of media, 216
Regaud, formol-bichromate method, 323
myelin stain, 442
Regenerating nerve tissue, 421, 425
Regressive stains, 162 et seq.
. coal tar, 159
Rehm, 227
Nissl method, 413
Reichenbach, decapoda ova, 288
Reinke, 177, 341
maceration, 248
orange method, 171
Rejsek, corrosion, 249
Rejuvenation of tissue, Golgi, 462
by veronal or chloral, 421
Remak and Goette's fluid, for frog eggs,
279
Renault, 214, 343, 390
Rengel, 38
Reptilia, embryology, 274 et -sv//.
brain, 405
Resins, 225 et seq.
Resorcin-fuchsin, 353
for embryonic cartilage, 377
Retina, 493 et seq.
of arthropods, maceration, 248
bleaching, 255
maceration, 247
Retterer, smooth muscle, 348
tendon, 347
uterine eggs of mammals, 267
and Lelievre, 377
and Zenker, injection, 242
Retzius, 191
Rhumbler, 75
Ribbons, paraffin, 86 and 89 et seq.
Richards, 14
Rieder, 367
INDEX.
589
Ringer's solution, 218
Rio-Hortega, Bielschowsky, 431
neuroglia, 490—492
Ripart and Petit's fluid (copper), 53
Ritter, 282
Robertson, 449, 451
tsetse flies, 539
and Macdonald, 474
Robin, injection masses, 233 et seq.
and Ranvier, 232
Robinski, silver, 200
Rochon-Duvigneaud, 495
Roessle and Yoshida, 394
Rollett, 343
maceration, 246
Romanowsky, stains, 384 et se>{.
Roncoroni, rodlet of, 421
Roosvelt, 210
Rosaniline, 120
Rose, bone, 370
Rose B, 180
Rose de naphthalin, 169
Rosein, 169
Rosenstadt, 340, 509
Rosenthal, fat/367
Rosin, 181, 413
Ross, blood, 383
thick film method, 534
Rossi, 449
blood, 380
tiagella, 549
Rossolimo, 451
Rotatoria, 512
Jennings, 288
Rothig, 169
axis cylinder stain, 453
mammals, 263
methylen-azur, 408
Rouget, 191, 200
Rousseau, 497
decalcification, 251
desilicification of sponges, 255
sponge, 526
Rousselet, 15, 512
preparing aqueous mounts, 230
Rubaschkin, neuroglia, 482
Rubin, 169
method for neuroglia, 486
Rubin S, 171
Ruffini, 347
Russell, glycogen staining, 296
Russo, Ophiothrix, 519
Ruzicka, 314
Ryder, 105
Sabin, micro-injection, 271
Saefftigen, 513
Saffrosin, 180
Safranin, 165 et seq.,311
elastic tissue, 352
and light green, 181
mucin, 391
myelin, 452
and wasserblau, 351
Saguchi, 396
pancreas cells, 338
Sahli, 226
brain hardening, 403
myelin, 452
Sainton, 400
Sala, 440
axis cylinder dendrite stain, 459
Salamandra, embryology, 277
Salicylic acid, fixation, 53
Saling, Tenebrio, 506
eggs, 287
Salivary glands, 392
Salkind, lead gum embedding, 106
Salmonidse, embryology, 280
Rabl-Riickharcl method, 280
Salts, for fixing, 40
solutions, 218
for maceration, 244
sublimate, 46
Samassa, 468, 525
Samter, 75, 288
Sanchez, 425
Sand, 482
neurofibril method, 434
Sandal- wood oil, 68
Sandarac, 228
media, Gilson, 227
Sanders, 473
Sankey, 408
Sansom, Carnoy modification, 53
Sanzo, fixing apparatus, 260
Sarcolemma, 344
Sata, fat, 367
Sattler, 201
Saurefuchsin. See under Acid Fuchsin.
Saurerubin and Orange G, 393
Sauer, kidney, 394
Saureviolett, 181
Savini, 177, 415
Sazepin, 506
Scala, 542
Scarlet R. See Scharlach.
Scarpatetti, 408
Schafer, 232
Schaffer, 251, 261, 300, 350, 356
bone, 370
decalcification, 252, 254
thionin for bone and cartilage sec-
tions, 376
Schaper, 262
Scharlach R, 356 et seq.
fat, 367
Herxheimer, 367, 368
Schaudinn, fluid, 546
Woodcock's modification, 541
Schaxel, 309
Schiefferdecker, 67, 68, 444
celloidin masses, 241
eye, 496
maceration mixture, 247
and Kossel, 356
Schlemmer, Bielschowsky, 431
Schmaus, 407
590
INDEX.
Schmidt, gastropod embryology, 283
Schmorl, thionin for bone and cartilage
sections, 376
Schneider, 300
aceto-carmine, 138
Schonemann, 262
Schreiber, Golgi method, 404
Schridde, 356
blood, 381
mitochondrial method, 325
Schrotter, 408, 449
Schuberg, malaria, 547
Schulemann, 389
Schultze, 219, 496
chrome haematoxylin, 157
frog embryology, 278
iodised serum, 219
muscle, 348
osmium haematoxylm, 158
oxydase reaction, 390
potash method, 378
tendon, 347
Schumacher, 354
Schurmayer, 539
Schutz, 425
Bielschowsky, 430
Schwalbe, 407
cochlea, 497
Schwarze, 517
Sclavo, 549
S. G. Scott, 119, 180, 498
blood, 381
Romanowsky stain, 386
neutral balsam, 226 .
standard hsematoxylin stain, 312
Scott and Osborn,triton embryology, 277
Scyphistoma, 524
Secretion granules, 315
Section-grinding, 108
Sections, crumpling, 88
flattening, 86, 90
mounting, Henneguy's method, 114
Seeliger, 520
Segregation granules, 389
Sehrwald, 468
Golgi methods and precipitates, 466
Seidenmann, 192
Seiler, alcoholic balsam, 226
carmine and indigo, 212
decalcifier, 254
Selachia embryology, 280 et seq.
Selenka, 269
Seligmann, 493
Sensory ganglia, 424
Serial section mounting, 111
Serum, for maceration, 243, 244
media, 219
Severeanu, injection, 242
Shearer, 511
Sheep, brain, 402
Sheldon, 446
Shell, mollusc, 503
Shellac, for brittle sections, 89
embedding, 108
Shipley, 271, 310
benzidine dyes, 389
and Macklin, trypan. blue, 378
Shun Ichi Ono, glycogen, 338
Siebenmann, 498
Silver, carbonate, neuroglia method, 492
impregnation, double-staining, 202
for elastin, 354
fixation, 201
marine animals, 202
metallic stain, 198 et seq.
for nerve. See under Golgi, Ramon
y Cajal, and Bielschowsky.
nitrate, reduction, 201
picrate, lactate, acetate, for staining,
201
Simarro, neurofibrils, 419
Siphonophora, 524
Siphunculus, 511
Siredon, embryology, 276
Sjovall, 435
formol and osmic acid, 331
Skeletons, cartilage, 377
Skin nerves, 341
Slides, cleaning of, 112
Slow-worm eggs, 275
Small objects, embedding, 75
Smears, blood, 379
of gonads, 307 et seq.
lucidol fixation, 59
Smirnow, 341
Golgi method, 465
Smith, eyes of gastropods, 502
and Mair, 366
Lorrain Smith, Nile blue, 368
Smooth muscle, 348
Snails, asphyxiation, 16
Snake blastoderms, 274
Snessarew, Bielschowsky, 352
Soap masses, 92
Sobotta, 269, 280
Amphioxus, 281
mammals, 263
Soda carmine, 140
Sodium chloride and alcohol, for macera-
tion, 244
nitrite, 233
sulphalizarmate, rnyelin, 449
Solferino, 169
Solger, bleaching, 256
muscle, 344
salivary glands, 392
Solvents, for paraffin, 76
Soulier, maceration, 245
De Souza, pyridine, 59
Spalteholz, method of clearing, 270
Spee, 92, 270
Sphaerozoa, 546
Spicules, sponge, 526
Spiel meyer, 442
Spinal cord, 424
axis cylinder and dendrite, 459
hardening, 399
See under Nervous System.
INDEX.
591
Spindles, fixation, 301
Spiral filaments, 439, 440
Spleen, 393
Sponges, 525
desilicification, 255
embryos and larvae, 526
sections, spicules, 526
Sporozoa, 546
Spuler, 140
sublimate formol, 63
Squire, 173
blueing sections, 151
glycerine jelly, 223
Staining, adjective, 131
in bulk, 6
with carmine, theory of, 135
effect of heat, 124
electrolytes in, 131
hindrances to, aids to, 25
nature of, 121
progressive, 132
regressive, 132
removal of dyes, 125
substantive, 131
two kinds distinguishable, 128
unsafe criterion of chemical consti-
tution, 133, 134
vessels, 6
Stappers, 505
Starfishes, narcotisation, 13
Statoblasts, 282
Stauffacher, cyclas eggs, 284
Steensland, 450
Stein, 497
decalcification, 251
Stempell, 546
Stephens, 549
Stephenson's high ref active mounting
medium, 224
Stirling, maceration, 245
Stoeltzner, 300, 442
Stohr, 214
Storch, 482
Strahuber, 477
Zur Strassen, 514
Ascaris ova, 290
Strasser, 262, 407
Stratum granulosum, 340, 341
Strecker, brain, 402
Streeter, myelin, 448
Van der Stricht, decalcification, 254
thysanozoon eggs, 289
Strieker, gum embedding, 108
Strong, 398, 449
brain of acanthias, 405
copper bichromate, 64
Golgi method, 463
iron alum fixation, 50
Stropeni, 355
Strychnin, for narcotisation, 16
Students, guide for, 556
Studnicka, 352
Stylaria, asphyxiation, 16
Styrax, 370
Styrax and liquidambar, 228
Sublamin, 49
! Sublimate, bichromate, 43
Golgi method, 470
See under Mercury and Corrosive.
substitution stains, 164
| Suchannek, 3
anilin oil, 69
bergamot oil, 68
mounting medium, 227
Suchanow, Golgi apparatus, 438
Sudan III., for blood, 383
Daddi, 367
fat, 356 et seq.
Sulima, 14
Sulphonic acid, 121
Sulphuric acid, maceration, 247
Sulphurous acid, 43
bleacher, 256
decalcification, 253
for teeth, 372
Sumita, 300
Summers, method for celloidin sections,
116
Sumner, 279
Suschkin, chick embryos, 273
Suspensoids, 123
Sussdorf, 391
Sustaining apparatus, of medullary
sheaths, 439
Sympathetic ganglia, 424
Synapta, 518
Syrup media, 219
Szecsi, lucidol, 59*
for blood, 382
Szent-Gyorgi, eye, 494
Szutz, 185, 208
Tachiol, 425
Tactile corpuscles, 341 et seq.
hair, nerve endings, maceration, 247
Tadpoles, intra vitam staining, 179
Taenzer-Unna, orcein method, 353
Tafani, 498
Taguchi, Indian ink mass, 240
Tannin, for mounting, 220
Tannin-fuchsin, for flagella, 548
Tap water substitute, 151, 313
Tartuferi, 343
eye, 495
Taylor, Sister Monica, amoeba culture
method, 536—537
chromosome methods, 303
Teeth, Carter, 369 et seq.
decalcification, 253
embedding through carbon bisul-
phide, 372
lymphatics, 373
soft parts, 371 et seq.
vessels, 373
Tegumentary organs, 339 et seq.
Teleost eggs, 279, 280
embryology, 259
592
INDEX.
Teljatnik, 451
Telly esniczky, acetic bichromate, 41
Temnocephala ova, 289
Tendon, 344, 347
silver, 200
Tenebrio eggs, 287
Terpinol, 69
as mounting medium, 228
Test-cells of Ascidia, 282
Theory of dyeing, electrical, 124
of fixation, 18
of staining, 120
Theridium, 287
Thiersch, 212
Thin, 496
Thionin, 162, 168
cartilage, 376
for Golgi apparatus, 438
for intra litam staining of nerve, 190
mucin, 391
pancreas, 396
for skeletons of cartilage, 377
Thionine pheniquee (Nicolle), 168
Thiophen green, 182
Thoma, decalcification, 253
indigo-carmine mass, 239
and Fromherz, corrosion, 249
Thome, 173
Thompson, Arthur, reconstruction
method, 262
Thomson, J. G., Giemsa stain, 545
Thread cells, 521
Thyme, oil of, 68
Thymus, 394
Thyroid, 394, 395
Thysanozoon ova, 289
Tigroid substance, 410 et seq.
Timofejew, 196, 393, 405
Tirmann, 300
Tischatkin, 3
Tissue culture, 550 et seq.
Toison, staining fluid for blood, 382
Tolu balsam cement, 231
Toluidin blue, 169
for cartilaginous skeletons, 377
and erythrosin, 415
for intra vitam staining of nerve, 196
myelin, 449
Nissl granules, 412 — 413
Toluol, 70
Tomaselli, neurofibrils, 418
Tonkoff, 183
Torpedo, electric organ, 346
Tortoise embryos, 275
Tower, JVtoniezia, 515, 516
Tozer, 513
Tracheae, 508
Tracheata, 505
Trachymedusse, 523
Trematodes, 516 et seq.
, ova, 289
Trenkmann, 549
Triacid mixture, 175
Trichina, 514
Trichlor-acetic, 53
decalcifier, 254
Trimming blocks, Eternod, 83
Triple stain, Bonney, 178
Triton (Molge), embryology, 277
Trophospongium, 439
True fats, 356
Try pan blue, 389, 390
cartilaginous skeletons, 377
dental pulp, 373
violet, 390
Trypanosomes, 533, 539, 547
Trypsin, digestion, 248
Trzebinski, 405
Tschaskin, 389
Tschernyschew, 446
Tsetse flies, 539
Tullberg, 15
Tunicata, 499
killing, 12
ova, 281 et seq.
Turbellaria, 517 et seq.
embryology, 288
Turpentine, clearing, 69
for mounting, 227
dissolving fat 322, 320
Uexkiill, 16
Underwood, 375
Unio, methylen blue, 192
Unmasking iron, 299
Unna, 32, 34, 350, 388, 391
carbol-pyronin-methyl green, 172
collodion mass, 96
half -ripe hsematoxylin stock, 153
on ha3matoxylin staining, 145
keratin, 341
mast cells, 355
methylen blue and Saurefuchsin, 351
orcein method, 351
oxidised haematoxylin, 155
polychrome methylen blue, 186
Rongalit white, oxygen test, 390
safranin and wasserblau, 351
smooth muscle, 348
water-blue orcein, 339, 340
Upson, 407, 477
Uranium acetate, 54
nitrate silver method, for Golgi
apparatus, 436
Urea and Bouin's fluid, 306
and chromosome fixation, 305
Urodele, embryology, 276
Ussow, cephalopoda, 282
Vanadium chloride process, 475
hsematoxylin, 157
Varnishes and cements, 229 et seq.
Vaso-dilators, 232 el xeq.
Vassale, 450
Weigert method, 444
and Donnagio, Golgi method, 465
INDEX.
593
Vastarini-Cresi, embryonic cartilage, 377
silver, myelin, 451
Vejas, 408 •
Vejdovsky, r>l I
Venderovic, 407, 450
Venetian soap, 411
Venice turpentine, 227
for cementing, 230
Ventral cord, insect, 508
Veratti, 468
Veretillum, 523
killing, 12
Verhoeff, 354
Vermes, embryology, 288 et seq.
general, 509 et seq.
Vernon, 309
Vert d'Alcali, 160
Lumiere, 159
Verworn, 14
Vesuvin, 161
mast cells, 355
Viallane, 509
osmic gold method, 205
Vialleton, cephalopoda, 282
silver impregnation of chick, 274
Victoria blue, 169
mucin, 392
neuroglia stain, 483
green, 181
Violet B, 184
of Lauth, 168
Virchow, 33, 280
Vital stains, benzidine, 390
mitochondrial, 332
See under Intra Vitam.
Scharlach VIII, myelin, 453
Vivante, bone, 375
Vivisection Acts, caution against, 265
Vogt and Tung, 511
Testodes, 515
fucumaria, 518
worms, 509
Volk, 17
Vosmaer, reconstruction method, 261
and Pekelharing, sponges, 526
Vosseler, wax feet, 243
Waddington, 13, 541
Waite, 288
Waldeyer, 498
decalcification, 252
Walsem, 449
Walton, tissue culture, 552
Ward, 16
Gephyrea, 511
Warnke, 407
Washburn, mollusc ova, 284
Washing out, after fixation in alcohol,
formol, acetic acid, picric acid,
nitric acid, corrosive, osmic,
chrome, 26
liquids for, 26
Wasielewski, sporozoa, 546
Wasserblau, 183
blood-platelets, 386
and orcein, Unna, 340
and safranin, 351
spleen, 394
Wassermann, 300
Watase, cephalopoda, 282
Watch-glass, embedding in, 74
Water-baths, paraffin, 79
Water-blue. See under Wasserblau.
Watery media, 217
Wax feet, 243 et seq.
Webb, gum mass, 110
Weber, 512
Siphonophora, 524
Wedl, 211
Weed, 271
Weidenreich, 341
blood, 380
Weigert, 177, 450, 461
— • elastin stain, 353
fibrin stain, 388
hsematoxylin, 150
method for celloidin sections, 118
myelin, formol material, 444
stains, 442 et seq.
neuroglia stain, 480
•picro-Saurefuchsin, 215
Weigert-Pal, myelin method, 446
Weigl, Mann-Kopsch method, 328
Weil, 109
bone, 370
teeth sections, 373
Wellings, intra vitam staining of teeth,
373
Wermel, 381
Werner, smooth muscle, 348
Wester, 507
Weysse, 269
Wharton's jelly, 391
Wheeler, eggs of Orthoptera, 286
White, bone sections, 370
Whitman, brains, 405
frog embryology, 276
pelagic fish ova, 281
Hirudinea, 511
Wickersheimer, 220
Widakowich, 270
Widmann, lens of Arachnida, 509
Van Wijhe, ammonia carmine, 141
cartilaginous skeletons, 377
Wilhelmi, 518
Will, reptile embryology, 275
Willebrand, blood stain, 383
Wilson, J. T., and Hill, J. P., 269
Wilson, Alcyonaria, 522
stain, 535
Wimmer, 482
Winiwarter, 177, 270
Wintergreen oil, for clearing, 271
Wislocki, 390
Von Wistinghausen, 155
De Witt, 3 4
Witte, pancreatin, 249
594
INDEX.
Wittmaack, 497
myelin, 451
Wolff, 388, 428
bladder, 349
Wolfram, elastin stain, 353
Wollschwarz, 548
Wolter, 453
chloride of vanadium process, 475
Wolters, 376
myelin, 447
Woodcock, faeces, 538
and Wilson, Schaudinn fixation, 541
Woodger, 365
Woodland, 518, 521
Woodworth, reconstruction, 261
Woolf, 168
Worcester's liquid, 280
Worms, 509
Wright, blood platelets, 386
Romanowsky stain, 535
Wynn, 447
Xylol, 70
Yellow gelatin mass, 237
Yolk stain, Peter, 260 .
See, under Fat and Cytoplasmic
Inclusions, 316 et seq.
Zacharias, 139, 505
acetic alcohol, 52
protozoa, 548
Zaleski, 300
Zander, 507
Zawarsin, cornea, 343
Zenker, fluid, 48
note on, 323
Zernecke, Ligula, 516
Zettnow, 549
Ziegler, decalcification, 253
teeth sections, 372
Zieglwallner, glycogen and fat stain, 296
Ziehen, gold sublimate, Golgi method,
475
Ziehl, carbolic fuchsin, 169
Zimmermann, 301, 352
Golgi preparations, 469
nucleoli, 314
Zinc, 300
chloride, 50
Zoantharia, skeletons, 522
Zograf, 542
Rotifers, 512
Zoja, 189
Ascaris ova, 290
Zosin, 453
Zschokke, 377
Ziirn, 495
Zwaardemaker, safranin, 166
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