RESEARCH INSTITUTE, NEW DELHI

.1
IMPERIAL
AGRI~ULTURAL
...
RESEARCH INSTITUTE, NEW DELHI
,
BIOLOGICAL LABORATORY
TECHNIQUE
L1 ving
~eutral
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[P,onli.pi~,·e.
BIOLOGICAL
LABORATORY
TECHNIQUE
AN INTRODUCTION TO RESEARCH IN
EMBRYOLOGY, CYTOLOGY AND HISTOLOGY
BY
J. BRONTE GATENBY
B.A., B.Se., D.Phil. (Oxon.), M.A., Ph.D. (DuhL),
D.Se. (Lond.)
l'k'Ojc880.' (ij Zoology aad Comparative AJ'latoU1Y, 'l'l'initJl
(follpoe, Dublin enirel'sitU; 11,Jliorury MetulJcr 0/ flu! Royul
Societll of Nr'lI..~ Zertlillul; A[pnlbm' 0/ flIP ]fnyui Irish llradpJUlI.
(UHf Fellow Of the Jloyal JUicl'o,st'opical Soc.ielJ/ oj Londsu.
SOlneiilnc De))WllstJ'utor in lIistoloflJh U;z:{ord; SeriiOl' Demy,
lUao(I(lltlt Collef/B. Ox/m'fl; Seniol' .rlssistunt in Zoology,
fhliceI'sify College, J...rmdfHt; ',P('tlll'P" in CUto1orJY, London
enil~e"l'oitll; Tllfll'el.U Seesscl Bellow, }'ale VIJirersif'h U.S.II.;
PomJation VJtiN?fsitaire de ncl9iflue I..eclur'eJ', Loucllin.
DJ'u8scl ... and Glumt.
With 8 Illustrations
J"ONDON
J. & A. CHURCHILL LTD.
104 GLOUCESTER PLACE
PORTMAN SQUARE
1937
50B6
111111~lInll
IARI
TO
,TAMES
P]~'I'ER
H.ILl"
PREFACE
Tms book has been written in a practieal mannE-r for
two classes of persons, fust the University teiLcher or
student who may desire a short and simple intl'orlncLioIl
to the most recent and established pra,ctice in microtomy,
and :secondly, the amatew' who may wish to make a t-;mall
laboratory of his own, and is unaware that a good part
of modern laboratory research is caniecl out \\ ith the
simplest apparatus. Thus, many of the most modern
methods have been adapted for smear teuhnique 'which
cloCR not require fiection cutting. Having worked through
most of the methods chosen for this book it is hopecl that
t.he &tu,leHt or ~tnmteur worker will be ready to extend
his illye~tigations t.o more specialized branches of microtomy, such as are t.reated in .. The Microtomist'iI \-"clemecum" and other Himilar works. The writer thanks
his grrLdllate c;tndcuts, lY.Ii~s O. Aykroyd, lY.lr. \V. Boyle
and .Mis~ R. Jones, for their helpful intel'eHt. The pl'Oufr-.
wore alHo mad by Professor J. VV. Bigger, M.D., who made
variOllH valuable suggeRtiolls.
HOIIOOL OF ZOOLOGY,
'l'RINI'r¥ COLLEUE,
DtTBLIN.
TABLE OF CONTENTS
('lrAl'll' U
1.
II.
Ill.
1V.
V.
VL
VIr.
V111.
IX.
r\IrI
Introduction.
Laboratory Apparatus
1
Treatment of Living Cells, Saline Media, Vital
Staining
17
Fixed and Stained Smears
'27
Microchemical Tests in Smears
Whole Mounts
fiI1
Fixation Methods
fi7
N~Butyl
Paraffin, Dioxan and
Celloidin Imbedding
Alcohol and
Hl
Stains and Staining
U\l
Notes for Histology and Embryology Students,
and Addenda
vii
117
BIOLOGICAL
LABORATORY TECHNIQUE
CHAPTER I
INTRODUCTION
1. In the first part of this book some of the modern
laboratory equipment is discussed, Views on the best
type of appfLrat.us to buy have l)een given, and some
indication of the use and management of instruments,
With reference to the vnJidity 1 of' preparations made
by the methods for the cytoplasmic inclusions of the germ
and other cells, it was felt that the student's early instruc·
tion in this book shoulcl begin with cells in which t.hese
inclmdons can be seen in the living, It is important that
the student should be able to compare the living cell with
that prepared on the one hand by the BOllin or Schaudinll
and such methods, and on the other by established
techniques for the Golgi apparCLtus_, mitochondl'h1, fat,
and other cell inclusions. One of the most favourable
cells for this purpose is the spermatocyte of Helix aspe1'sa 2
(see Frontispiece), which is common on the Continent and
throughout the British Isles, and which has been intro,
duced in various other parts of the world. Failing this
mollusc, the insect germ cells, especially those of Gryllus 3
(the cricket) and Le_[)isma 4 (,the silver fish of kitchen
1 C. \V llJker and A llAll, PrOD. Royal Society oj London, Vol. 10 J, Hl:17,
p. 512; C. Dohell, Parasitology, 1925, p, 47.
2 Gatenby, P-rac, Royal8ociety oj Lomion, Vol. 104B, 1928.
3 H, Herbert Johnson, Zeit. fur WiM. Zool., Vol. 140, 1031.
• R. N. lIfukerji, J(YIlf', Royal ~Micro8Copical Society, Vol, 49, 192[1.
I,ABOJ\ATOlW TEOlllHQl1E.
1
!
2
IN1 JRODUOTION
htl'dcl's), are very favourahle. Having eX!1millCd such
cells alive, the student may then proceed to the making
of stained smears. Some of the more useful and successful
microchemical methods are given later, and the sectioncutting technique is described. Finally, a minimum of
notes on histology is given.
2. Microscopes. Nearly all modern microscopes are
gooel. If you ttre going to be a professional biologist or
microscopist the best thing is to get one of the new
binocular microscopes. Naturally a good man will do
outstanding work with a cheaper and simpler model, but
the newer binooular microscope is excellent. There are
one or two points to note. First, it is nowadays possible
to buy a stand to which either a monocular or binocular
tube may be fitted. Purchase one of these. Secondly, do
not buy a built-in mechanical stage unless you can afford
two or three microscopes. Buy a body with a large
square stage, upon which several types of mechanical
stages lllay be clamped. You oan take off the mechanical
stage and use the plain stage for staining, petri dishes,
etc. It is better, however, to have another microscope
for staining. r.rhis should have ird inch and ith inch
lenses, and a No.2 eyepiece.
You should buy a triple nose-piece at least. Very
beautifully manufaotured, accurately aligning four nosepieces are made nowadays, giving you another fitting for
a vcry low power of objective-that is to say, if you are
going to use the arrangement of trd, ~th and l\rth lenses,
which will be found most generally convenient. Some
people like a higher dry lens than the lth, but this will
depend on personal taste.
A microscope should be used in a darkened room, with no
window at your back or sides. If there is a window at the
back you should have a dark blind on it. The 'window in
front should also have a blind, or there should be a rectangular board 2 ft. X 2 ft. 6 in., clamped so as to cut. the direct
JJ110R08COPES
light from your face as you work aii the microscope. In a
continental laboratory I have seen a black eardboard fixed
on two legs fitting into lead holders, which could he shifted
to the right position. For ordinary purposes these precautions arc unneeessary, bllt it is hetter to note that, for very
difficult precise work, a dark part of the room should 1lt'~
chosen for the microscope. It is curious that this is so rarely
understood.
It has alrcady been mentioned that the combination
of lenses which is usually chosen is the ~-rd, ·~;th and ·l2-th
inch. If you wish to work on pond life, and whole mounts
generally, it would be advisable to get another power
lower than the ~rd. 1'ho T\-th or oil-immersion lens is a
very high power suitable foI' cytology and protozoology,
and is not really necessary at first. For eyepieces a
X 8 would be suitable, though for expensive apochromatic
objectives special compensating eyepieces are made.
With low-power objectives the thing to look for is flatness
of field, apart, of course, from the general clarity of the
image. With oil-immersion and higher-power lenses
there should be no coloured edge when a very small object
is focussed, and it is much better to choose a lens with a
good focal distance. Comparatively cheap oil-immersion
lenses are now on the market, which give a really excellent
performance, good enough for most research work.
So far as the amateur microscopist is concerned, a great
deal can be done with one power, a ird inch, and there al'C
a number of cheap microscopes with ird inch anr11\-th in.ch
objectives. These are quite suitable for work with whole
mounts and sections oflarger organisms such as the earthworm. For these microscopes a substage condenser is
quite unnecessary.
3. On the Care of a Microscope. If you own a valuable
microscope it is likely that you will see that it is kept well
covered eithel' with a good duster or special American
cloth cover when not in use. You should put it back in
its box if a glass or similar cover is not available! The
l-z
INrrRODuorpION
moving parts of the mierm;cope should be oiled occasionally.
If any parts seem stiff get some sperm oil, or " 3 in I oil,"
and oil it yourself. Never use, or force, stiff mechanical
stages, stiff fine adjustments or nose-pieces. Forcing the
latter eventually brings about inaccuracy of alignment.
Lenses 1Lre ruined by being brought down sharply all
a slide, or by being consbntly cleaned with xylol and a
rough duster. If you have no lens paper, use a very soft
cambric, or better, silk, handkerchief, and do not scruh
the lens. A penetrating oil like xylol may dissolve the
cement or packing of the various lenses inside the objec"
tive. If immersion oil has been left on the immersion
lens and has dried, screw off the lens carefully, rub oil off
the metal part with a silk rag only moistened with xylol,
then with lens paper (or the silk) just sweep the front of
the lens itself clean, as gently as possible. Do not polish
it. The nLg must not be dusty or the lens may be scraped.
It is expensive to have a lens repaired.
vVhen you have finished with your microscope see that
the ~rd inch or lowest power is turned around towards the
stage. Never leave the oil immersion in this position, and
never remove a slide from the niicrm,cope without lifting up
the oil immersion or tth lens frbm the surface of the slide.
4. Immersion Oil. The classical substance is slightly
thickened cedar wood oil. It has the disadvantage that it
must be wiped off the slide and lens after use, or it goes hard.
Nowadays many people use methyl benzoate, which is
rather liquid and somewhat smelly, but evaporates, and
can be used on newly mounted slides. Liquid paraffin has
also been used as a substitute for cedar wood oil. It is
chel1p, does not harden, and can be wiped off easily at any
time, but is not suitable for the most critical work ..
Delicate stains are often mounted in immersion oil
instead of bltlsam.
5. How to Use an Oil-immersion Lens. Put a small
drop of oil on that part of the slide which you have just
AllOR0800PE LAJ.1JP
13
been eXlLlllining with the f;rd or ·hth. Lower the oit
imlllersion 011 to the drop, 'watching this ea-l'efnlly frolll the
side until yun see the oil Just meet the lens, now lower
very slightly, look into the microscope aml gmduaJly
screw down with the fine adjustment until the object is
focussed. vVllen you have finished always lift the lens
fro111 the slide, tum it around and wipe it clean with lens
paper 01' a clean .piece of silk.
6. Eye Strain. An older type of binocular microscope
with one tube Rtraight,
the other Rloping inwards,
is dangerous for the ey£'s.
Never use one. When
using a monocular microscope do not shut one
eye. Never nse an arc
or Nernst lamp; th(;y
are quite unsuitable.
Either use an ordinary·
fiO or 100 wat,t frosted
hulb 01.' an inverted gas
1amI). U;ually the more
expensive
microscope
lamps sold are saJe.
Students often use t.oo
bright a light. In a
darkened corner of the
room a 100 watt bulb
is quite enough for most
work.
In Fig. 1 is a
A
o
o
L
40.",·m.
,
Fw. 1.
MieroBcope lamp made fl'OIl"l
H tin.
A IlIHrks the original
bottom of the ean, whieh has been
bored to take the eleetrie fitting.
'1'he hood in front has been solderorl on, ollcl vent holes hltve lJcet:l
cut at the top of the ean.
lamp made from an old
tin can.
Avoid long periods of
work
on red-stained
preparations. Use blue
01' greenish screens on
these.
Choose gentian violet rather than safranin, and
hoomalunl rather than carmine, if possiblc.
7. How to Measure the Size of an Object under a
Microscope. In vertebrate preparations under an oil
immersion a rough idea of the size of a body may be got
6
INTRODUOTION
by looking for a red blood corpuscle near by and com·
paring. The size of the human H. B. C. is 7-r5fk (mu) or
micra" [L micron being Trluuth of a millimetre.
The basis of more accurate measurement is a special
stage micrometer which consists of a line a millimetre in
lengt.h divided into 100 parts-that is to say, one division equals lOp. or 10 micra. These are prepared by a
photographic process and mounted on 3 X I slides. If
you are working with a camera luciela with certain known
powers, eyepieces and t.ube length, you place the micrometer unde, each power and draw the lines on paper
with the camera lucida, and measure ott the magnification
with a millimetre ruler, and compute f()r all your powers.
The size of One or more divisions of the micrometer is
drawn on a stiff cardboa,rd with the eyepiece and object
lens used in each case written beside, and this is kept for
reference.
Another way is to put the micrometer under your
microscope, slip an eyepiece micrometer inside your
eyepiece by unscrewing the top lens, and compare the
number of divisions in the eyepicce with the size of the
known divisions of the ·stage micrometer. Make a permanent note of the value of the eyepiece divisions
under each combination of powers. If you cannot get
a stage micrometer but have an eyepiece micrometer,
make a thin coverslip preparation of your blood and
knowing that a freshly-drawn R. B. C. is 7'5/L, make the
necessary computation. This is, for most purposes, quite
good.
S. On Drawing a Preparation under the Microscope.
The best way to do this is to have a }Jl'ojecting prism
or piece of mirror fixed on the microscope tube pulled
over horizontally, a very bright light, e.g., " point"o-lite "
lamp (in a dark room), and to trace the image thrown on
paper. Another way is to use a camera lucida, which
consists of a prism and a mirror. The drawing board
MIOR01'OllfE'S
7
should be sloped slightly towards the microscope. When
using artificial light it may be necessa.ry to illuminate
t.he board to the right degree. In daylight this is usually
easier. If you cannot afford a call1era lucida, buy one
of those squared discs of glass which fit inside the eyepiece, and use paper with squares of appropriate size,
counting the number of squares across different parts
of the organism and so getting the proportions correct.
9. Drawing Paper. ]'01' line block drawing, white
paper (fashion-plate board) is good enough. Use India
ink. For wash drawing, use a Bristol hoard "wit.h a slight
blue tinge, and lamp-black water paint or Chinese ink.
10. Microphotography. The "vell-appointed laboratory has
one of those excellent microphotographic cameras madc
especially for the purpose. Elaborate ones cost over £100,
which is beyond the ordinary person's powcr of buying. It is
best to know something about ordinary photography before
beginning microphotography. For yean:! we used a cheaper
microphotographic camera which fitted iuto the aperture of
the uJ)right microscope tube, and good results were got, but
never so good as with the special heavy apparatus made for
the purpose. This applies especmlly to low pow-er work. It
is well for the beginner, as weH as the research student, to
note that the India ink line drawing is more effective, cheaper,
more likely to obtain puhlication, and to be copied in future
text books. So, excellt in controversial matters, where a
good microphoto may be a necessity, never use the microphotographic camera. Of course, if you cannot draw, or
have at your disposal a laboratory artist, or if you are a very
busy person, it may be necessary to use microphotographs.
In some cases, as with tissue culture work, where the cell is
spread fiat, a microphoto may be better than a dra"wing, but
rarely in other cases_
11. Microtomes. These are expensive precision instruments and there is really no cheap substitute for them.
It may be mentioned, however, that for some classes of
microchemical and cytological work, cutting sections is
not really necessary. In other cases hand sections may
do (§ 127). Most protozoology and some branches of
8
.lN'l'BOD U 01'! 0 N
cytology actually are done with ntre recourse to the
sectioning method. This is why it is very advantageous
for the student to develop a good smear technique (§ 38).
It is not difficult to advise on the purchase of a microtome.
The Minot rotary micro tomes are the most popular,
and some firlllR now supply freezing attachments, as well
as clamps for putting the knife at a special angle for
certain types of work. Buy one of these. Practically
all classes of work can be done on a rotary microtome,
but the sliding (sledge) types are especially useful in
neurology and for cutting very hard or large sections.
The microtome :;hollld be carefully oiled, and where fouled
with paraffin wax, properly cleaned in xylol, and oiled before
use. If this is done, the microtome will last for many years,
with harel Weal', the sale upkeep being the sharpenillg of
knives. However, nowadays razor blade holders arc sold,
which arc quite goocl for orclinary work Even the clamps
for the thin Gillette blades are excellent. Such holders can
be made from a piece of iron shaped like a microtome knife.
Another small rectangle of metal acts as a cover to the blade,
which, projecting about 2 mm., is clamped firmly by means
of two bolts threaded into or passing through the iron. The
Lcitz Company sell a good clamp for Gillette blades, which we
have used with satisfaction.
I am convinced that a good clamp could be made of hard
wood (knife shaped) with a shallow slot or indentation for
one of the stiffer types of safety razor blades, covered with a
metal clamp, and provided with thumb screws. It could be
faced with metal at the ends where thc knife clamps into the
holder on the microtome.
'The hest way to learn to use a microtome is to watch an
expert. This, of course, applies to almost anything. To the
person who has no expert to watch the first thing is to notice
the safety catch which prevents the moving bar from descending on the knife. This should always be put into operation
when you are doing anything with the block you are going to
section. Otherwise yon may get a nasty eut yourself. So
look for the safety catch. The second point is to sec that the
safety catch is put out of action when you are going to cut
sections, because if you turn the wheel suddenly it gives the
OLA8SIVARE
machine 11 nasty jolt; the lom;ening of parts in SOllle types
of Minot rotary rnicrotomes is due to this treatment.
12. Bottles, Capsules, etc, These tend to be expem;ive.
In lllany htboratories much of the glassware is provided
with ground glass covers aml stoppers, Corked tubes
and the cheaper variety of medicine bottles do for most
routine work, however. What are extremely useful, and
worth while purchasing specially, are small stelllier or
petri dishes, with ground tops. These 111a.y range in size
from Il to 5 in. aeross, and are indispensable for the
general manipulation of material. \Vatch glasses are
also very useful, and can be provided with squares of
glass for covers. They can be hrought from watchmakers and hardware stores, and do quite well for coverslip smears, etc. For imbedding, washing, dehydrating,
a number of short pieces of wide glass tube of various
sizes are useful. Also some cloth gauze or butter muslin
for tying over the ends. There should be a number of
pipettes with fine and widely open ends, and rubber
teats. For staining coverslip smears, the stender or
petri dishes may be used, but for slides you wjll need
some tubes, with or without ground stoppers. Corked
or capped tubes a little higher and broader than
a slide (3 X 1) 'will be suitable. These tubes are sold
commonly in the 3t X llrr or 3t X It size, and can be
placed in a wooden rack made by boring suitable holes
in a piece of board, on t,he lines of a test tube holder. It
is usually best to have larger vessels with ground glass
stoppers for absolute alcohol and xylol used in the last
stages of dehydrating, and for clearing preparatory to
mounting.
For' writing on slides one can either use Indian ink,
a diamond, a piece of picked carborundum fixed in a
harcl glass tube with sealing wax, or smear the end of
the slide with thin amyl acetate celluloid solution. The
slide is allowed to dry, the legend written, and ltnother
10
INTRODUO'PION
layer of the solution quickly brushed over. The writing
with the diamond is the best. Many people use labels,
which, provided the end of the slide has been rubbed
free of grease (with !1bsolute aleohol), usually stick quite
well. It is a good thing to have a few drop-bottles.
In some histological laboratories nothing else is used for
students' benches.
Another useful and ingenious aid to staining i~ Guu's
staining Illate, which essentially oonsists of a plate slightly
curved so that when a slide with sections is placed face down
on the COIlcave face of the plate there is a small space left into
which stains or other fluids can be run in with a pipette.
Such a plate might be made from a flat piece of vnleanite or
cellophane by warming and giving t~ necessary slight curve.
With Gurr's plate the evaporation of the stain or other fluid
is only from the edge, and not from the whole surfaco, as with
the ordinary drop-bottle method. The method which is sure
to find favour was suggested by Dr. A. C. Coles. Using a
transparent plate it is possible with some stains to watch the
progress of the staining.
Something similar to this-a glass plato with a groove in ithas been used for years to stain large numbers of blood films
at the same time. The slides are placed smear side down on
the groove, and stain run into the latter.
13. The Arrangement of the Bench. It is always a
good thing to have a bench for one's microsoope and lamp,
a bench for the microtome, shelves for reagent bottles,
and some place on your work table which can be cleared
sufficiently to allow a space about 1 ft. square in which
are spaced the symbols, 30%, 50%, 70%, 90%, 100%,
written in chalk, or on labels stuck on the bench. When
you are dehydrating you begin at 30%, or 50%, and put
the dish or capsule over the proper figure, and movo
• up each time you change the fluid. Some people use an
oblong piece of flat glass with the figures written on
paper underneath. The main point is to have some way
of knowing what strength of alcohol the object is in.
Alternatively, you may use the method of slipping in a
OLEA.lUNa aLASS TVARE
11
piece of p~Lpel' with the grade written 011 it when you
change the fluid.
14. To Clean Slides, CoversIips and Glassware. If t.he
glass is in fairly clean condition rub wit.h strong alcohol
(70%, or 90% with 1% Hel). If very dirty, soak overnight in 1 part commercial nitric acid, 9 parts ,vater, or
in 5 gm. of potassium bichromate in 250 C.c. of 5%
sulphmic aeid in water. Do not expose your hands too
long to these fluids.
Watch glasses, petri dishes, large dishes, Winchesters,
etc., should be washed in hot water with Hudson's soap
or Soapolio, and rinsed well in running water. Some people
take coverslips from acid alcohol, through distilled water
to 90%, into absolute alcohol. Each coverslip is held in
forceps and flamed in a spirit lamp.
For glassware to be used with living cells, use the
bichromate sulphuric acid mixture, wash out at least
eight times with distilled water. Even this may not be
enough, as Oscar Richards has pointed out; soak overnight in distilled water.
15. Coverslips and Slides. Avoid very thin slides
unless they are for some special pmpose. It is better to
have good glass, but it really does not matter if there is a
slight greenish tinge in theln. If you are interested in gum
glycerine and similar whole mounts of pond organisms,
etc., you may prefer to buy circular coverslips, as these
can be very neatly ringed with a metal tube (§ 88).
The thinner types sold are graded No. 1. They are necessary for very high power (oil immersion work), but if
you are interested only in whole mounts, No.2 variety
will do. They are cheaper and stronger. If you wish to
make your own slides, this can be done by cutting 1 mm.
thick glass into pieces 3 X 1 in" and rubbing the edges
with emery paper.
16. Alcohol for Dehydration. The alcohol used ill
laboratories is e.thyl alcohol. It is still universally used
12
TNTRO])UCTION
for extracting the water from tislmes l1S '" preparation
for mounting or sectioning. There are Rubstitutes nowadays, of which the most interesting is dioxan (di-cthylene
oxide), the vapour of which is a cumulative poison at
1 in 1,000, and which has a slight and pleasant sIllell
when concentrated, and hardly any at the toxic concentration. It will be interesting to see whether dioxan
displaces ethyl alcohol. It has been stated that it is
hardly likely that in the laboratory dioxan could reach
the toxio margin. .]udging from the smell of carbon
bis'ulphide, xylol or benzol in 1'ooms where imbedding is
going on, I doubt the statement, and the hca,lth of a
perSOll cOl1stant,ly using this fluid at from 56° to 60 0 C.
in a small room with imbedding ovens is likely to be
injured. If you have a small private hLboratory and
imbed only occasionally it may be found convenient to
use dioxan instead of alcohol. At all events, we recommend the ethyl alcohol method for the present, though
the dioxan method is given below. The advantages of
ethyl alcohol as a dehydrating agent are not evident
except from the health point of view. The disadvantages
are that it is expensive, slow, it hardens some objects too
much, and it does not mix with paraffin wax. One should
remember that all the modern aniline staining methods
depend largely on the use of alcohol for passage to
balsam, and the ·l1ew method with dioxan may mean
certain readjustments for SOllle stains. The workers
who are usually most dissatisfied with the l;Llcoholmethod
are the entomologist, the embryologist dealing with hard
eggs of both insects and Sauropsida, and the person
who wishes to cut unusual objects like skin with
developing hair or feathers. Other people are usually
quite satisfied.
It is usual to find two types of ethyl alcohol in the laboratory, one industrial methylated spirit, the other absolute
alcohol. The latter is about 99'5%, the former about 95% or
ALCOH()J~
AND OLJEARINCi
13
117% pure. The so-called methylated spirit sold by chemists
and druggists is good for spirit lamps, and llreserving small
animals if you are on holiday at the seaside-hut for nothing
much else. The industrial methylated spirit varies consider[I,hly in purity and strength, and it is better not to usc it for
making stains. It is all right for most other purposes. You
dilute as follow!:!. Take the number of c.c.'s of 95% equal
to the strength required in a graduated \w:lsel, and add di!:!tilled
water to make 95 c.c. For example, to preparc 70%: take
70 C.c. of 95% alcohol and add 25 C.c. of distilled water.
Absolute and 95% alcohol should he kept in bottles with
ground glass stoppers. If you have a private laboratory it is
well to remember that you can get a permit from the Com.
missioners of Inland Revenue, 01' Customs and Excise, to
purchase industrial alcohol at, a cheap price.
17. Other Alcohols. Another alcohol commonly used
is methyl alcohol (if possible, acetone free and neutral).
It is used for making stainR and as a narcotic. N-butyl
alcohol cannot be used for clehyclrating in the lower
strengths as it dissolves very little water and most of'
the dehydration must be done ,·dth the aiel of ethyl
alcohol. However, it does not harden tissnes. We
believe the butyl alcohol method has been superseded by
clioxall imbedding. Nevertheless, the method is given in
§ 137, and both techniques can be tried on clifficult objects.
18. On Clearing. When you place a piece of dehydrated
tissue in an oil or liquid like xylol, benzol, chloroform,
carbon bisulphide, etc., it becomes more 1 or less translucent and is said to be cleared. Except in the case of
dioxan or butyl alcohol technique, this clearing process
is always carried out before imbedding in wax or mounting
in balsam. In one case it must be done because paraffin
wax and ethyl alcohol do not mix, in the second because,
while balsam and absolute alcohol do mix (Seiler's
alcoholic balsam), it will be fonnd more satisfactory to
use a balsam dissolved in benzol or xylol. Recently I
have been mounting in dioxan balsam from 95% alcohol.
1
Refer to § 100.
14
INTRODUC'['ION
The specimen of dioxan balsam I have appears t.o he
going darker; and is still under trial.
Of course, if you are going to imbed in paraffin by
one of the newer methods explained in § 136, clearing is
unnecessary, while the same applies to mounting in
glycerine, gum glycerine, or Euparal (§ 82).
Clearing previous to imbedding is a period when considerable shrinkage takes place, and also hardening.
Caution is therefore needed. There are two good ways
of clearing. When the material has been properly
dehydrated pour away all the alcohol except what is
enough to cover the pieces. Then add a quantity of
about one-quarter the amount of alcohol left, of xylol,
oarbon bisulphide, benzol, chloroform, or whatever oil
or fluid you wish to use. Shake gently, cover, and you
have the material in four parts of alcohol and one of
clearing agent. After! hour add more clearing fluid,
and so on till it is mainly clearing fluid. Then put it
into pure clearing fluid for t hoUl'. The whole process
can take 2 hours, but small objects can safely be done in
1 hour or even less.
Another way much used is to take a tub() 10-15 mIll.
in diameter, pipette in about 10 mm. in height of xylol,
etc., then very cautiously add absolute alcohol, which will
float on top of the clearing fluid. The piece to be cleared
is lowered into the alcohol and stops just above the
clearing fluid, where it clears slowly. After the two
liquids have mixed the piece drops to the bottom of the
tube, and should then be placed in pure clearing fluid.
This method is not quite so easy as the first mentioned,
but is WOl'th practising, and is much used by the chromosome workers.
Now something should be said about clearing oils or
fluids. If you cannot get good absolute alcohol you should
pass the tissue ii'oIh 96% to methyl benzoate or less preferably carbol-xylol (add phenol crystals to xylol till no more
CARE OF MATERIAL
15
dissolve), then to the fiui(l for imhedding. At the same
time it will be just 118 convenient aml cheaper to dehydrate
some 1)6%) fdeohol yourself, as described in the next
paragraph. It will be assumed that the pieces can be
dehydrated properly. Of all the clearing fluids used in
routine work in laboratories xylol is possibly the best
known, but in recent years carbon bisulphide has become
the more popular. The reason for this is that it is more
volatile, it can therefore penetrate better, and more freely
mix with the wax passing into the piece of material being
imuedded. It penetrates things like crustacea or insects
better than xylol, and hardens them less. Use carbon
bisulphide if possible.
For very small penetrable
organisms chloroform is very good.
But for clearing before mounting in balsam it is best to
use xylol or benzol, unless you intend to use Seiler's
alcoholic lalsam, or the newer dioxan balsam (which is
still on trial).
19. Dehydration of Alcohol. This can be done by
heating blue copper sulphate in a covered porcelain vessel
till the "water of crystallization~ is driven off. A bag of this
is put in a bottle of 96% alcohol. It should be changed
from time to time.
20. On Waste in the Laboratory. In large laboratories the
old used alcohol is re·distilled. It should be collected for this
purpose. Pieces of paraffin wax can be collected, melted
• and strained. Osmic acid used for Weigl and Kolatchew can
often be !Ised for making or rejuvenating Champy's or
Flemming's fluids. Used xylol or benzol is not worth keeping
except for taking out oil and grease stains on floors, etc. Old
slides and coverslips can be scraped and treated with a mixture
of used absolute alcohol and xylol, and then washed in hot
soapy water.
21. On the Care of Laboratory Material. If you leave
stained sections in the light they fade. It is very careless
to leave valuable sections on the bench in the sunlight.
When you have finished with the laboratory slides, put
INTRODUOTION
them bn,ck in a box, or at lef1st out of RUlllight. Never
lenve your beneh with sta,in and aleohol bottles uneorked
and petri dishes dirty. When stains dry in petri dishes
they are often difficult to remove. After use, pour water
into the dishes and leave in the sink.
Never pOUl' wax into the sink. There should be a
bucket for such things. Never make up stains in the
gmduated measuring jttrs. Do not cut gritty animals with
valuable microtome knives.
Never underline or mark the Departmental Library
books, and when yon leave the laboratory see that yonI'
microscope is either put baek in a locker, if such be
supplied, or eovered with a duster.
CHAPTER II
TREATMENT OF LIVING CELLS
22. On making Solutions. If you have no knowledge
of chemistry the following may be useful.
There are measuring jars for fluids graduated in cubic
centimetres (denoted by the letters c.o., c.c.'s or mI.).
Buy one of 100 e.(',. capacity, [mel if possible one of 10 e.c.
Yon should never allow these jars to stand about with
chemicals in them. They should be washed out in distilled
wnter, or good tap water, after nse, and inverted in a
stand.
The various salts are weighed on a balance. For most
purposes in a biological laboratory, it is sufficiently
accurate first carefully to balance similar square pieces
of paper on each pan (add fragments of paper to one side
where necessary), and then to place the right weights on
one side, and the chemical to be used on the paper on the
other pan. Never remove chemicals from a bottle wifh a
spatula which has just been ql8erl h~ another bottle. For
instance, if a spatula has been used to ladle out corrosive
sublimate, and is then put into the sodium chloride bottle,
you are certainly going to have trouble with your saline
media. Never put stains or chemicals directly on the
metal pans uf a balance. When making up fluids in ,,,hich
dry sa.lts are to be dissolved, choose a bottle of the right
size and do the mixing in this and not in the measuring
jar. Use this for fluids only. In general, keep all your
reagents and fixatives on a dark shelf, espeoially away
from direct sunlight.
If you are working by yourself,. the local chemist and
17
18
TR1!JATMENT OF LIVING CELLS
druggist will explain any difficultiel:l, or will make up any
solutions for you. He ,vill also sell you empty Willch('ster
bottles, and other suitable glasRware, Wash these as1jn § 14.
If you live in a district where there is no university or college,
remember that the medic!1} man has learnt to stain sections
during his student days and could give you valuable help.
If you are interested in biological science, in addition to your
text books, get and read" The Microbe Hunters," by Paul de
Kruif, and" Martin Arrowsmith," by Sinclair Lewis,
23. Saline and Artificial Media. 'rhe degree of acidity
or alkalinity of a medium may have a marked effect on
the organism or cell, and most of the reactions of the cells
of an organism take place in a liquid environment very
nearly neutral. The degree of acidity or alkalinity bears
a definite relationship to the iOllS of hydrogen present in
the volume of liquid. All aqueous solutions have a
definite hydrogen ion concentration. The pH 1 (or
hydrogen ion potential) of pure water is 7. Very acid.
solutions have pH values approximating to zero, neutral
solutions the value of 7, 'extremely alkaline solutions
approximating to 14. The proper adjustment of the pH
values of media may be vital for the growing of Protozoa,
tissue cultures, etc., and should be carefully noticed by
the student, who must learn the colorimetric method for
determining the pH if dealing with organisms living in
water, or artificial media.
Certain salts are found in tissues which act as " buffers "
in taking up acid or alkali without a corresponding change
in the condition of the rnedi'l.('In. The two principal systems
are the bicarbonate and the phosphate, the former
NaRUO g, the latter NaH 2P0 4 and Na 2RP0 4 , and are used
in buffe,ring artificial meoia, Special buffering tablets are
now on sale at chemical warehouses, ready to use.
III making up solutions of neutral red it will be found
that in distilled water the colour is red, but becomes
yellow in Hedon-Fleig Ringer, which has a pH of 8'8, and
1
See any text book of Physiology or Biochemistry.
RINOER
HI
is suitable for molluscan cells. Neutrl11 red has a pH
range of 6·5-8'4. Congo Red, a commonly used stain,
is blue in the acid, red in alkaline range, pH range 3·5-4·7.
The commonly used Ringer made from distilled watcr
supplied in my laboratory has a pH of 7·4. Human
arterial blood has a pH of 7·3, the blood of the common
snail about 8, the hay infusion giving optimum growth of
Paramoocium 7'8-8, and so on.
Another conception of the greatest importance is that
of osmotic pre881Lre. In the Hological laboratory its'
commonest appliCf1tion is in the making of iso-tonic fluids
for the examination or growing of living cells.
For simple dissections for histologioal material, '75%
NaCl (sodium chloride) for invertebrates and ,85% NaCl
for vertebrates is sufficient. Years ago Ringer showed that
both calcium and potassium salts were necessa,ry in order
to keep the perfused frog heart beating. His solution
consists of sodium, potassium and calcium ions in the
proper amounts, generally nowadays with "buffers"
added. To make up 2,000 C.c. of Ringer (that is, nearly
a Winchester full) take that quantity of distilled water,
and add 14 gm. sodium chloride, 0·6 gIll. calcium chloride,
0·5 gm. potassium chloride. This is a Ringer for coldblooded animals. For warm-blooded use 17 or 18 gm. of
sodium chloride. Nowadays bicarbonate buffer is usually
added, and if a sterile solution is needed, must be filtersterilized before adding to the sterilized Ringer j for the
quantity (2,000 c.c.) add 3 gm. of sodium bicarbonate.
Here is another fluid which is good for living invertebrate tissue mounts-known as Redan-Fleig fluid; it
also is very useful for any work on living cells of terrestrial or fresh water invertebrates. Distilled wa.ter 1,000
c.c., NaCl 7·0 gm., KCl 0·3 gm., CaCl2 0·1 gm., NaRC0 3
.1-5 gm.; NI1 2HP0 4 0·5 gm., MgS04 0'3 gm., glucose 1·0 gIll.
af yoli want a sterile fluid (not usually necessary) you
,cannot heat this, as it will partly disintegrate and its pH
20
TREATM10N'l1 OF .LIVING CELLS
(8·8) becomes altered. Divide the constituents into four
parts: (I) Nael, KOl, CaOl z, MgS0 4 • (2) GIllCQi,;e. (3)
Na 2HP0 4 . (4) NaHCO J • (1), (2) and (3) in flasks plugged
with eotton wool :tl'e sterilized in a steamer. The sodium
hicarbomtte mllst be filter-sterilized. 'l'his is the really
ollly satisfactory method. Hedon-Fleig is faintly alkaline,
and in it neutral reel changes to a yellow colour. This
does not indicate disintegration of the dye, for when
taken up by living cells kept in HMon-Fleig, the segregated droplets are bright recl.
Some other fluids which arc intended to be used with the
living tissues or eggs of various other animals are given below.
You willllotice that their salinity varies considerably. In the
fluid which should be used for dio;secting marine urustacea for
cytological or similar work, there arc 2:3 gIll. of sodium chloride
per 1,000 c.c. of distilled water, whercas in mammalian Ringer
there are only about 9 gm. per 1,000 C.c.
24. Clarke's Fl'lticZ jar Living In,,~ect Cell8. Distilled
water 200 c.c., sodium chloride 1·3 gm., potassium chloride
(}028 gIll., calcium chloride 0·024 gm., sodium bicarbonate
0·02 gm., monobasic sodium phosphate 0·002 gm.
Holtfreter's Solution for Amphibian Cell8.
Water
1,000 c.c., sodium chloride 3·50 gm., potassium chloride
0·05 gm., calcium chloride (anhyd.) 0·10 gm., sodium
bicarbonate 0·20 gm. If desired storilize first, and add
sodium bicarbonate afterwards.
~J1al'ine Crustacea Fluid. Water 1,000 c.c., sodium
chloride 29·23 gIll., potassium chloride 0·75 gm., calcium
chloride 4·44 gIll.
lYIarine Mollu8ca Fluid. Water 1,000 c.c., sodium
chloride 23·38 gm., calcium chloride 5' 55 gm., magnesium
chloride 7·62 gm.
Nicholl's Elasmobmnch l!'luid. Water 1,000 c.c., sodium
chloride 15·38 gm., potassium chloride Q·8B gIll., calcium
chloride 1·11 gill., urea 21·u gm., dextrose 1·0 gm.,., bicarbonate of soda 0'38 gm., acid hypophosphate of soda;
(NaH 2P0 4 ) 0·06 gm.
'1'188U1£ OULTURE
~l
Artzjicial Sea WaleI' (Challenger Bepol't and Allen).
Wat.or 1,000 c.e., sodium chloride 28 gm., pot.a:,;siulll
chloride 0·77 gm., calcium chloride 1·25 gIll., magnesium
chloride 2·55 gm., magnm;iulll sulphat.e 1-136 gm., sodium
bicarbonate O·1l gm. Salinity about 35.
Sea rYater R'ingel'. 0ne part sea water, three part.s
dist.illed wat.er.
25. Examination of Living Cells. The common snail,
H eUx a8pena, is nmv practiCfLlly cosmopolitan. In
America it can he got from California. Its celb are very
hardy and resistent to bacterial attack. '1'0 make a living
culture of amcehooyt.es and epithelial cells :-wound a
snail by thrusting a darning needle through its shell into
the soft part of itH body. Leave it for 4 or [) hours.
This preliminary wounding is not absolutely necessary,
but it definitely raises the proportion of successful outgrowths. Remove most of the mouth part of the shell,
pin the animal down on a piece of weighted cork and cut
away part of its mantle wall (pulmonary cavity wall),
RPC in Fig. 2, A, about 3-5 mm. SqlU1fC. It is a flat
membrane. Place this on a clean slide, and with a sharp
knife cnt into four smaller pieces (explants). Mount each
explant separately in a drop of Ht1don-Fleig fluid (§ 23),
covering with a small 20 mm. square coverslip. Leave
in a covered petri dish, where the preparations will not
evaporate. Do not use the hanging drop method, as this
is not so demonstrative for thiR Rpeeial preparation.
Examine next morning. In some cases the amcebocytes
will have begun to emigra,te (Fig. 2, C), and in 3 days many
of the preparations will bear a close resemblance to a
Hoss Harrison vertebrate 1 tissue culture. Such preparati.ons will often remain alive for 3 weeks. 2 You should
1 For an excellent short account of vertebrate tissue culture, consult
"Tissue Culture," by E. N. Wilmer, Methuen & Co .• London, 1935.
2 Gatenb~r ltnd Hill. (Juart. Jour. lYJ-icr. Science, Vol. 70, HJ34, and
Gatenby, Hill and Macdougald, ibid, Vol. 77, 1\):~4.
22
TREATJYIENT OF LIVING CELLS
notice that the slides, covers, scissors, etc., should be frce
of chemical cOllta.millation (§ 14). With these preparaOVT
A
D
F1G. 2.
A. Outline of Hellx aspersr:t, showing at OVT position of
ovotestis, at RPC position of pulmonary or mantle cavity.
B. From a smear of ovotestis of Helix, showing sperm (SPZ),
spermatogonia (SPG), spermatocyte (middJe) and
spermatid (above). N = nucleus, M = mitochondria,
GA = Golgi apparatus, YC = yolk cell.
C. Explant (EXPL) of Helix, ~howing outgrowing cells.
(By courtesy of the Editor of the Q.J.M.s.)
D. Typical insect spermatid, showing nucleus (N), mitochondria (M), Golgi apparatus (GA), "yolk granules (Y-granules
YG).
aseptic precautions are not necessary. The slides
can be left on the bench provided you ring them with
vaseline. Just melt vaseline in a vessel, warm a pipette,
tiOllS,
Ol'O'lIES'l118 SMEAR
23
and with this place It wall of vaseline half OIl the edge of
the cover, half on the slide.
Note. Helix nemoraUs and Helix pomati{t are not so
favourable for this experiment, their tissues not being so
hardy as those of H. aspersa. Pieces of mantle cavity
mounted in the blood of the snail often grow out quite
well. Ordinary salt solution buffered to pH 8 is sometimes quite successful. Commercial buffer tablets from
6'8 to 8·4 are available.
26. Hanging-drop Preparations. If you try to examine
a liquid preparation mounted between a coverslip and
slide, you will notice that even although the oil used
for the immersion lens be thin, the cells keep streaming
about as you focus. This is caused by the lifting or
pressing down of the cover. If you must use this type
of preparation try the thinner methyl benzoate instead
of cedar wood oil. In any case the hanging drop preparation is the one to use. Take some wooden matches and
cut lengths to make supports for a 20 mm. square coverslip. Smear them with vaseline and make a neat square,
upon which the coverslip can rest and be sealed. Place a
small drop of the liquid to be examined in the middle of a
coverslip of suitable size. Invert over the prepared square
of matchsticks and press down. If the drop is too big,
it will run on to the vaseline.
Note. There are round glass rings which can be bought
for making these preparations. Small size curtain rings
or rubber washers do quite nicely.
27. Helix Aspersa. Ovotestis Smear. Dissect out the
ovotestis of the snail (OVT in Fig. 2, A) and touch the
middle of a coverslip with it. Invert the smear over a prepared cell either of matchsticks or a vaselined glass ring.
Examine under the oil immersion. Both mitochondria
and Golgi bodies (nebenkern) can be seen in the living
spermatocyteR and spermatids (Frontispiece and Fig. 3,B ).1
1
Gatenby, Proc. Royal Society oj !-vndan, Vol. 104, B, 1928.
24
TRIiJAT1I:lEN'jl OF LIVING OELLS
Since junior students usually are llot provided with an
oil immersion objective, preparations of this type should
A. Making It smear of bluotl. ctl). B. lvIet,hou of
making It coverslip smear. C. Metal imbedding box
madt) from two bent pieces of brass.
]1'10.:l.
Le demonstrated by the teacher, who will possess the best
apparatus,
28. Testis of Gryllus 1 (the Cricket) or Lepisma 2 (the
Silver Fish). Dissect in a drop of saline (§ 24) on a slide
under dissecting microscope with t,yO mounted needles
and remove testiR. Tease slightly in the middle of coverslip and invert over glass ring or matches. Note in
spermatids, Golgi bodies, mitochondria, acrosome, Y
granules (for the latter use also Nile Blue sulphate, § 66).
Given a critical light and good powers, this type of
material is very favourable for studying the cytoplasmic
inclusions throughout their entire cycle in the living cell.
29. Neutral Red. In the above preparations, subsequently lift the covers carefully a,nel add a very small
drop of pink neutral red in saline or use prepared
1
H. Herbert Johnson, Zeit.Jur Wi88. ZooZ., 1.10, Band 1, Heft, 1931.
2
R. N. Mukerji, Jour, Royal :M'icro8copical Society, Vol. 49, 1!J2n.
NEU'l'RAL RIClJ
213
coverslips (§ 31). The neutral red will become segregated
in certain parts of the cells (Frontispiece). This usually
happens in about 30 minutes. Note that the neutral
red should be a pink solution, not red. The concentration
is I in 2,000 to 1 in 10,000, but if yon add a little pink
solution to ea,ch preparation, one of them is likely to be
exactly right for observing the phenomena in about
30 minutes. It has ah'eady been mentioned that in
solutions with a pH value above 7, the colour is yellow.
Note. This is an important negative test for the Golgi
apparatus. If some body or area stains in neutral red
this is a certain test that it is not Goigi material. Hed
globules often appear inside the Golgi apparatus, and
thus mark its position.
30. Janus Green. The right conccntration of this
dye for staining mitochondria vitally is less easy to
determine, and the effect is often fugitive. The correct
concentration is 1 in 10,000 to 1 in 20,000, and in a test
tube the solution, so weak that the colour can hardly be
noticed, will stain energetically. Stronger solutions are
very toxic. As with neutral red, the addition of varying
quantities of the dye solution to a number of preparations,
to be examined at intervals up to 1 honI', will generally
be satisfactory. You should notice, however, t,hat only
certain brands of .Janus Green will work. Also SOllle
specimens of Neutral Red are better than others.
31. Preparation of Coverslips or Slides for Vital Staining.
Dissolve Neutral Red, .Tanus Green and Brilliant
Cresyl Blue in plU'e absolute alcohol to 1% strength.
These solutions, or diluted .vith absolute alcohol up to
ten times, are thinly smeared on a warm cover 01'
slide and allowed to evaporate. A drop of fluid containing the organisms is put on the slide or cover
according to whether a,n ordinary or a, hanging drop
preparation is needed. The organisms or cells should
stain in from 5 to 30 minutes. Different cells or Protozoa
26
11REAT1I1ENT OF LIVING CELLS
require different concentrations, but 1/10,000 IS about
right for Neutral Reel.
32. Examination of Fresh Water Protozoa or Pond
Animals. This should be carried out in water, not in
the saline media mentioned above. But if you dissect
the pond animals, this should be done in a saline medium.
Examination of marine organisms should be carried out
in sea water, their dissection if for cytology in appropriate salt solution (§ 24).
33. Toxic Examining Media. 'rhese kill the cells or
organisms :(a) Methyl G1'een Acetic. Dark green aqueous solution
with (H% acetic acid. This solution is suitable for
examining Protozoa for class purposes. It stains the
nuclei green after a few minutes, but is non-specific for
chromatin. (b) Iodine Solution. Use a drop of Lugol
(§ 71). A dark bl'O'wll colouration generally, but not
always, means that the body is or contains glyeogell.
This solution can be used for examining f::eces for amCB ba
cysts. (c) Osmic Acid. One per cent. solution. A little is
mixed with the organisms. 'These fine preparations last
for a long time provided the osmic does not evaporate.
A good general stain for neutral fat. Not specific, however, for this. (d) Formaldehyde. Four per cent. solution
(§ 112). Usually preserves the colour of the organisms for
some time.
Toxic Vapours often used are those of osmic acid and
formaldehyde. The wet smear is held over the uncorked
bottle for a minute. Iodine vapour produced by heating
a crystal in It small tube is sometimes used after staining
in Janus Green (§ 30).
CHAPTER III
FIXED AND ST AINED SMEARS
34. The Validity of the Fixed Preparation. The following
organic substances or inclusions are soluble in either
water, acetic acid, alcohol, or xylol, or alcohol and xylol
mixed: fats, mitochondria, Golgi material, glycogen,
lecithin, cholesterol and certain other related substances.
vVhen you fix or coagulate the tissue by certain chemicals, some of these substances are converted into materials
less, or not, soluble in alcohol, dioxan, or xylol, but
usually still in a state when their partial disintegration
occurs during imbedding, dehydrating and clearing.
Thus if you fix a smear of spermatids or Protozoa in
alcohol corrosive sublimate (Schaudinn), wash in water,
bring through alcohol and xylol and mount in balsam,
you have remaining probably all the simple and conjugated proteins but nothing else (Frontispiece). Thus
the pictures and descriptions of cells and Protozoa in
most of the text books are mere skeletons, with all fats
and carbohydrates raked out. Moreover, the ground
cytoplasm and nuclear material left are nearly always
net-like or thread-like in appearance, which is artificial
(W. B. Hardy).
This difficulty has largely been surmounted by the
development of chrome-osmium fixatives and the formalin
silver techniques (Frontispiece). Bichromate of potash
and osmium tetroxide together (e.g., Champy) cause
nearly all fats, and carbohydrates like glycogen, to
become insoluble in water, alcohol and xylol. Delicate
fats always tend to become dissolved even after long
27
2R
.FlXED AND STAINED SMEARS
t,reatmellt in mllllium tetroxide. Of all the Iixillg methods,
chrome"osmiulH is the hest, and cells so fixed and rnmmted
in (llycertne are morphologically as nearly identical with
the living cells as is at present possible. The protoplasm
of such fixed cells is smooth like that of the living ones,
while the Vttrious cell grannIes are faithfully preserved.
35. Fading and Permanence of Microscopical Preparations.
The object of making microscopical preparations is to procure
a series of objects which can be studied under the microscope
in a manner which willl)rovide as much information as ]Jossible
about the nature, structure or microchemistry of the material.
]i'urthermore, it is desirable that such preparations be as
l)Grmanent as possible. This meallS that the stain, if any,
and the mounting medium must bc suitable. No preparation,
however well made, will stand the direct Hunlight for long.
All stains fade, and some mounting media become darkened,
cracked or disintegrated by exposure to sunlight and heat.
Of the common stains, the anilinc, such as methyl blue, the
carmine, ·such as borax carmine, and the hmmatoxylin, such
as Heidenhaill's alum h::ematoxylin, fade in the order given.
Ehrlich's h::ematoxyIin, especially if mounted in acid balsam,
is particularly liable to fade. Carmine and hmmatoxylin
stains are usually quite permanent if kept in thc dark. Both
osmic and silver slides are liable to fade or disintegrate.
Stained sections should be kept in special cardboard boxes
made for the purpose, or in slotted 'Wooden boxes with
accurately fitting lids.
36. Tap Water, Tap Water Substitute, Alkaline Water.
Some tap waters are alkaline enough to H blue"
hmmatoxylin, and to wash traces of acid out of stained
sections and smears. Others are not suitable, and in that
case you keep a tap water substitute, which is merely
dist.illed, or more or less neutral t.ap water with 0·2% to
0'5% sodium bicarbonate added. This is washed off with
distilled water. S. G. Scott's tap water substitute is
KHCO a, 2 gm., MgS0-t, 20 gm., Aq. dest. 1,000 e.c., with
a crystal of thymol to prevent growth of moulds. Tap
water substitute is not intended for "lvashing out after
fixations. It would be unsuitable for this purpose.
FUII'EAR 'l'JjJ(}!i.J,.\TIQUE
2ft
37. Acid Water, Acid Alcohol. Acid solutions aee llsed
to differentiate over-stained smears and sections. The
Rtrength is (}5% HCI in water or 70% alcohol.
38. Smears. The smear preparation does not call for
the cutting of sections and may therefore he dealt with
first. At the same time the making of a really good
smear is not as easy as it may seem. '1'he smear pl'epaJ:atioll is a necessity in Hrematology, Protozoology and
Cytology. Owing to the nature of the material in the tv,ro
former branches of biology, the smear is a, necessity, and
even in the last, smear preparations are almost exclusively
used in laboratories where material like DroSOl}hila is
being studied.
The main requirements of a smear are that it should
be made on a clean coverslip (No.1 preferably) awl that
the material should be thinly spread. The reason for
making smears on coverslips rather than on slides is tha,t
the slips take up less space during preparation. On the
other hand, they are fragile and not so easily manipulated as slides, and have a Wety of floating on top of one
another in a dish. Some people prefer slides.
Smears may be (a) examined fresh, (b) fixed and
stained, (c) treated with a simultaneous fixative and
stain mixed, (d) made on coverslips previously prepared
by smearing with some stain (§ 31).
The preparation of the slips for smearing begins with
their cleaning. This may be easy, or quite difficult,
depending on the state of the coverslips when bought.
At one time coverslips during manufacture became
covered with a thick layer of material which had to be
removed by chemical means. Nowadays they clean
quite easily, in acidulated alcohol (§ 37), with a chamois,
or silk handkerchief, or ordinary duster. Careful people
clean coverslips as in § 14, and store in a glass box with a
ground top.
The thickness of the coverslip is important, mainly
30
FIXED AND S':PAINED SMEARS
because if they are too thick the oil immersion objective
will not focus the SHl.ear, as the focal distance of many of
these objectives is quite small, and if too thin the slip
usuaJly breaks at the critical moment. Here again a
good box of No.1 coverslips will be all that is necessary,
as their manufacture, or sorting, has improved in recent
years. Careful protozoologists have a special instrument
for measuring the thickness of the coverslips. These are
pushed between a screw and a plate, the thickness of eac:h
c:overslip being read off, and unsuitable ones discarded.
In general, such a precaution is not necessary and you can
do quite well without a coverslip measurer.
Yon should hit ve on your bench some bottles of graded
strengths of ethyl alcohol (§ 16), some shallow petri
dishes provided with lids or glass covers. In these the
smears arc treated. The alcohols should range from
50%; 70%, 90%, to absolute. In the case of smears the
30% strength is not really needed.
Of course, if you wish to be more modern, you may
discard the alcohol, and use dioxan, which mixes with
water, paraffin wax, balsam, alcohol, and all the oils in
general use in laboratories, as well as dissolving celluloid.
Dioxan should only be used for special work where the
alcohol benzol methods are not so suitable, as for instance,
cutting sections of Crustacea and Insecta.
Then, in addition, it will be necessary to have a dealcoholising agent, which means an oil or clearing fluid
which will mix with balsam (the mounting agent) and
alcohol, the dehydrating agent. Now most people use
xylol, but I think benzol (and benzol balsam) is better, for
it becomes less acid.
39. The Smear and the Section-Differences. It may be
noticed that· the smear may not stain as uniformly as the
section. In explanation of this it should be remembered that
the section is of uniform thickness and the material from
whioh it has been cut was left at least a. day and a night in
alcohol, whereas the smear is usually hurried and uneven.
DIFFERENTIATION
31
In pracLioally all branche8 of cytological techni(IUe where
sections are \Ised, it will he noted that not all pa.rts of the
material are equlIJly advantageous to :;tudy. TIllis the
enormous aggregation of cells of the sectioned material in a
way gets over certain faults in fixation, and thence in staining.
With the smear the fixation method and time have to be just
right, for you have only one layer of cells and these become
directly exposed to the fixing fluid. In general, the fixation
times for smears may be shorter than those given for pieces
of tissue.
40. Differentiating Stained Materials. Differentiation
(i.e., the production of contrast by ext.raction of the dye
from different parts) of nearly all st.ains is done in acid
water or acid alcohol; other-s differentiate in alcohol,
or in iron alum. The object of differentiating is to remove
the stain from those parts where it is lightly held, so that
contrast between these and the organs which ho1el the
stain fast is brought about. It is the most difficult part
of staining. Generally there are two ways you can stain
an object: either use a very weak solution of the stain and
let it be taken up gradually by those parts which have the
greatest affinity for the dye-this method needs 110
differentiation and is known as progressive staining-or
leave the object in such a strong solution that almost
everything is deeply stained. You must then differentiate. This is called regressive staining. Both regressive
and progressive staining can be done well with carmine
and hoomatoxylin stains like Ehrlich (in which the alum is
mixed ·with the dye).
Generally speaking, you should remember that a
section, smear or whole mount is going to be more
transparent in balsam than when you look at it in water
or alcohol during differentiation. It is a worse fault to
under-differentiate than to over-differentiate, because
if you bring the slides or objects up to xylol you can
examine them before mounting in balsam. If they look
all right, mount in balsam; if not, it does no harm to go
:32
FIXED AND STAINED 8lYfEARS
back and continue differentiation. Over-stained preparations which have Imen years in bahutlll can be dissolved off
by leaving some hours or longer in warm xylol if the
balsltID is thick, if not in cold xylol, and transferring
i:mbsequently to alcohol and thence to the ditIerentiating
reagent.
41. To make a Schaudinn or Bouin Smear. Smear the
ovotestis of the snail, the testis of a mouse or rat, or, if
you prefer this, use a mealworm or centipede, or other
organism containing protozoan p<1rasites. Cut off one
end of the body, and by carefully detaching a part of the
othm: end pull out the gut, placing it on a, slide. Cut in
two with (t knife, lift a piece with fine forceps and smear
on a coverslip, beginning at the top and smearing with a
zig-zag motion (Fig. 3, B). Large pieces of the gut should
be taken up and smeared where they have broken frOID
the main piece. Immediately drop face down on the
Schaudinn's or Bouin's fluid in a petri dish. Leave
at least !- hour. If the coverslips sink under the
fluid, turn them smear upwards. For the Schaudinn
smears notice that corrosive sublimate will corrode the
metal of your forceps. Use matchsticks, or quickly
wash your forceps under a tap and wipe.
After t hour, pour away the fixative gently, and
wash out the petri dish carefully with tl1P water, then add.
50% alcohol (for 5 minutes), then 70%. The Schaudinn
smears must be washed in 70% alcohol (sherry coloured
with Lugol's iodine, § 71) for an hour-the Bouin's
smears shoulclremain in 70% for at least 1 hour. The
point to notice is this-you cannot be sure to stain smears
evenly by this method unless they have been in 70% for
at least 1 hour. Where there are lumps in the smear it is
better to go up to 90% strength for an hour, and the best
resttlts are got by leaving the Stnea1"8 in ~O% overnight. In
any case do not try to rush matters-it does no harm to
leave these smears in strong alcohol for several days as
33
BOUIN SMEAR
long as t.he petri dish is covered. Rcmember that you
have akeady dest.royed everything except proteins, so the
sojourn in alcohol does not matter (§ 34). When the
smears have been in clear 70% alcohol for at lear-;t 1 hour
(note therefore that the Schauclinn smears which have
been in iodine and 70% mllst have been "\vashed for that
period in clear 70% to get riel of most of the iodine) you
may think about staining.
The stain may be Delafield's, Ehrlich's or Heielenhain's
h~-ell1atoxylin, alone, or followed hy Orange G.
Take Delafield first, as it is the better of the first two
for beginners. The stock Delafield is usually too strong,
and must be diluted (take 1 part of stain and 2 parts of
distilled water, or slightly acidulated tap water (§ 37) ).
Cover the smears with stain and leave 10 minutes, when
the staining should be complete. 'When, however,
about 5 minutes have elapsed, it is a good thing to
rcmove a coverslip, place it smear side upwards on the
middle of a slide and to look at it under the microscope,
See that the smear is kept wet with stain, or water, but
elo not run the objective into the fluid while you are
looking at it. With a ~rd objective, try to find some
parasites (or cells), and see how they are staining. Possibly
their nuclei are already a good reddish colour, and it would
be the proper thing to bring the rest of the smears into
water, while you decide what you are going to do. Water
willl10t hurt them. If you arc not sure whether they are
stained rightly, put one of the smears into tap water ill a
larger petri dish, or just run the tap gently on it, or
" blue" it, as the saying is, in alkaline water (see § 36).
Then put it on the slide again and re-examine. It may
be too dark, which lUeans the colour must now be
extracted a little, or not dark enough, in which case all the
smears go back to ..the diluted Delafield, and you wait a
little longer. Now if the slides are too dark-that is, ifthe
cytoplasm is too blue, and there is not a prop~r difference
J.ADOR.!.TOIlY l'llCILNIQUE.
2
34
FIXED AND STAINED 8MEARS
in Hhacle of colom hetween nucleus and cytoplasm, you
POIlI' off the :.;taill and add acid water (§ :37) to the petri
dish. A minute in the a,cid water will uHllally suffice.
Then pour this off tl,nd place under tap water (or subRti·
tute § 30) till blue.
Now if everything has been done properly, the nuclei
will be a sharp dark blue colour, and the cytoplasm will
be grey or grey blue, and much lighter in colour than the
nuclei. Notice, however, and this is important, that
when finaily mounted in balsam, the preparations will be
more tmnsparent, and the colouring not quite so opaque,
so even if they look a little darker t,han desirable when in.
water, all may be well.
42. On Counterstaining. The nuclei are blue, and it is
desirable, but not necessary, to stain. the cytoplasm of the
cells in It contrasting colour, say red or yellow. If you want
to stain in a red dye, eosin is the stain sometimes taken,
but I prefer Orange G. It is a more delicate stain, and
easy to work. Saturate some absolute alcohol with a
little Orange G. The depth of colour will be just right.
Now transfer the smears to 70% alcohol for 5 minutes,
pour off, and add 90% alcohol, and with two mounted
needles, or forceps, lift or push aside each coverslip so that
the 90% gets underneath each slip. Unless you do this
at each stage, you may find thai; when you begin to clear
the smears in oil, a little l)ocket of water lies under each
coverslip, and your preparations are spoilt. After
10 minutes in 90%, pour off, leaving the coverslips in the
dish, and with a duster remove the last few drops from the
tilted petri dish, then add some Orange G in absolute
alcohol, cover and leave 5 or 10 minutes. Pour this back
into the bottle, and quickly add some absolute alcohol,
move the coverslips about as before, cover and leave
5 minutes. Pour off alcohol, and add xylol or benzol.
If the fluid goes millry the alcohol contained too much
water. Take Some slides, rub clean with a duster, place a
COUNTERS:f.1AINING
S1fudl drop of benzol or xylol bn,lsam in the middle of the
slide, take up t.he smear wit.h forceps, give fL quick flip to
throw off excess xylol or benzol and rapidly place smear side
down on t.he drop of balsam. The hLtt.cr should run under
the whole coverslip-if not the drop was eit.hcr not big
enough, or the balsam was t.oo thick. Be careful, howeycr,
not to have the SlUeltr resting on a nlUch too large amount.
of balsam. Set the slide aside for a few minutes, preferably
in a ,varm place. Hit goes white, 01' is full of droplets 'when
examined, you must have breathed on it, or on the slide
prepared with the drop of balsam-or your absolute is not
right. Note that if you use dioxan balsam or Euparal
you lllay mount the stained smeal' from H5% alcohol, and
ncednot take it tlll'ough absolute and xylol. If y01~ wish
to stain in Orange G, you can mount in clioxan balsam
after connterstaining and dipping in clear absolute alcohol.
43. Evaluation of the Method. Preparations made thwI
give the appearance found in the pictures in recent
manualR on Protozoology, and the older Cytology books.
That is to say, a cell or protozoon with nearly all its
internal organs except .nucleus and kinetic apparatus
completely raked out. Such preparations are useful for
identifying stages of Protozoa, but for nothing else. So
far as the validity of such preparations is concerned, that
is to say, the resemblance between the living animal or
cell, and the end fixed and stained result, see § 34 and
Frontispiece. On the question of nucleoli and chromatin,
such preparations have been superseded by the modern
"Feulgen nuclealfarbnng" method (§ 76), and give no
precise information about chromatin.
Notice. 'When it is said to " pour off" alcohol, say, of
70% or BO%, it, may be put back into the bottle from
which it came unless it has been mixed with too much
water or is badly stained. In theRe cases keep a waste
alcohol bottle. It may be re-distilled or may be useful
for some other purpose.
3G
FIXED AND S'l'AINED SMEARS
44. Staining in Ehrlich's Hrematoxylin. Bring the
smeai:s down to water, add some stain and leave 5 or
·10 minutes. When the nuclei are a deep red, differentiate
slightly in acid wateI;' (§ 37), and afterwards" blue" in
alkaline water (§ 36). Counterstain in Orange G.
45. Iron Hrematoxylin 1 Stained Smears. The smear,
.. fixed in Bouin or Schaudinn, is brought to the stage
def:lCribed ill § 41, where it is ready for staining and
has been in 70% for at least 1 hour. Bring it down to
water and wash away the alcohol (in distilled water a
few minutes), then place in 4% iron alum for 1 honr,
wash slightly in water (a few dips) and transfer to
1 % hannatoxylin in water, 01' Regaud's hrematoxylin
(§ 147). Leave 2 or 3 hours. 'rransfer to water for a
few minutes till the excess hrematoxylin has been
washed away, then into 4% iron alum in which
differentiatioll (extraction) takes place. After a minute
or so, place a smear on a slide and examine with a -ird-in.
lens. If the smear is beginning to look clear, it is better
to pour off the alum from the others in the dish, and
place them in water while you re-examine the other
smear under the microscope. Remember that it is better
to under-differentiate, as darker smears can easily be put
back into alum and extracted to the right degree, but if
you differentiate too much you will have to begin all
over again, which does not produce good preparationsso proceed cautiously with the extraction of the stain,
remembering that you can stop or slow down differentiatio.n by transferring to water, or by diluting the iron alum
solution. When the nuclei appear blue-black, the cytoplasm grey, the differentiation has gone far enough, and the
smears must llOW be washed under the tap for at least
15 minutes. Then counterstain if you wish; the method
for Orange G, described in § 42, is good. Most people
never counterstain iron alum hrematoxylin preparations.
1
Heidenhain.
HEIDE.NHAIN AND LEISHJ.1IAN.;.Y
37
Note. The reautioll can be made more intense by
using warm [.lU111 and sbin.
46. Evaluation of Method. Of COUI'He t.he method
cannot be good as it stands, beeause the fixation is a poor
one. The stain is one of the most beautiful and useful ill
biology, easily eontrolled, the right colour for long hours
of observation under the microscope, very intense, and.
permanent except in direct sunlight. vVhen, however, combined with good .fixation, this stain is prolHlbly the best
general cytological method. It is mueh used by protozoologists, and for the metaphase counts in chromosome
studies. It is llot good for nucleoli. For these use
Feulgen, Giemsa, or Mann's methyl bll1e eosin (which
see).
47. Counterstaining in Eosin. The Delafield or other
hrematoxylin smears stain well in eosin. When you have
already stained the smear and it is washing in water, dip
it for a short time in eosin (w:ttery or alcoholic) about the
colour of red ink. 'Wash in water (or 70% alcohol for
the alcoholic eosin) until most of the eosin has been
extracted, transfer to 90%, absolute, 5 minutes each,
and xylol. Mount in balsam. 'rhe danger with this
stain is that beginners usually make it too dark and get
a horrid red llless all over the smear. It is not a good
stain, and if yon· must have a counterstain, Orange G
is better.
Counterstaining in Van Gieson. This is excellent for
sections of vertebrate histological material (§ 155).
48. To make a Blood or other Smear Stained in Leishmann (Romanowsky, § 174). Leishmann's stain is: to
a 1% solution of the best medicinal methylene blue in
water add 0'5% sodium carbonate, heat to 65° C. for
12 hours and let it stand for 10 days. Then add an
equal volume of 0·1 % solution of eosin extra B, let it '
stand for 6 to 12 hours, collect precipitate on the filter
paper, and wash; wash with water until the filtrate
.FiXED AND STAINED SMEARS
3R
comes off nearly colourless; dry nnd powder. DiRsolve
0·15 gm. in 100 C.c. of pure 'In ethyl alcohol. If you got
the correct eosin and methylene blue the stain will ''lurk,
if not, it is better to buy it made up-there are numerous
good dry and. wet Leishlllann stains on the market. In
America a modification of this is called Wright's stain,l
and is almost universally used there.
49. For Blood. Take some very clean slides (there is
no advantage in using coverslips with. this stain) and
make a blood. or other smeal.'. To make a blood smear
place a small drop at one end of the slide and bring
another I>lide against the drop as shown in Fig. 3, A.
Push this slide along in the direction of the arrow. If
the slides are dirty the smear is unsuccessful. Dry the
smear (in the case of the blood Iilm) by waving it in the
air, put it on the bcnch and add sufficient stain to cover
the smear, leave 1 to 2 minutes, flood (not wash off) with
about [) times as much distilled water, agitate the slide
and leave for 1 minute. Tip off the mixed dye and water,
blot by gently placing the film on a flat piece of smooth
filter paper or blotting paper. You may either now mount
in Euparal or balsam, or as is generally done, leave without
a coverslip. "'To use, drop a little immersion oil (cedar
wood) on the slide, and examine with an oil immersion.
For gut and other not very liquid smears, the stain can be
added immediately without drying the smear. Nuclei
should be in shades of red, cytoplasm bluish, parasites
blue with ruby-red chromatin.
50. Evaluation of the Method. A remarkably beautiful
and elegant blood stain which is not likely to be superseded. Nearly everything except protein material is
raked out of the cell. The stain fades badly in most cases,
and slides without covers become smeared with. dust.
It is recommended to mount in Eupaml, after blotting.
For Wright's stain add 10 drops, leave 1 minute, add 10 drops of
water, leave 1 to 2 minutes, wash off in distilled water, blot,
mount in balsam.
1
(li~tilled
C!HIWAIE-OSfilIU.JlI :P1iJCHNIQUB
an
This stain is useful for diagnosis of blood and blood
disease!>, not for prec:ise Protozoology.
51. Re-staining Faded (Romanowsky) Slides. Remove
coverslip, if any, 'with xylol (or warm absolutp alcohol in case
of Eupaml), place in HO% overnight, bring down to water,
place in 4% iron alum for 1 hom', wash quickly in water,
place in 1% h:l'matoxylill for 3 hours, differentiate in 0·5%
HOI ,mter. Wash under tap for 15 minutes, npgrade in
aleohols, mouut in balsam.
52. Keeping Uncovered Romanowsky Slides. These should
be kept in air-tight boxes if the lallOmtory is situated in a
smoky town, as the film of sticky dust which sometimes get:;;
on them is impossihle to remove. If the slides are to 1)0
stored for long periods they may be rolled up ill stnps of paper.
53. Carnoy Smear (§ 118). This fixfLtive is suitfLhle
for al'tlll'opods, etc., containing impermeable protozoan
cysts, and is the fluid to use for such things as smears of
PScudo-llavicellre of Monocysts. Fix smears for at least
1 hour, then transfer to 90% alcohol to wash out overnight. Bring smears to 70% alcohol, transfer to 1%
iron alum in 70% alcohol for 10 minutes, rinse in 70%,
place in 1% hromatill in 70% alcohol for 10 minutes.
Differentiate in iron alum solution, or hettel', acid alcohol
70% with 0·5% Hel. Wash out in several changes of
70% alcohol. 'l'his stain is Dobell's alcoholic luematin.
54. Evaluation of Method. Destroys all fats, lipins,
and can only be regarded as a rough miero-anatomical
met,hod. The result is not ilS good as can be done with
impermeable cysts. ]'01' research work on cysts, cut frozen
sections of unfixed material and transfer to a good fixative,
such as Champy or Weigl. This will easily enter those
cysts which have been cut open by the knife. Staining
may subsequently be done in Heidenhain or :Regaud, or
acid fuchsin.
55. Champy or Flemming without Acetic Acid Smear.
These [l1'e t·wo ',vell-known fixatives which are definitely
superior to Bouin 01' Schaudinn, but the method is more
difficult. Smeal' as described in § 41 for Bonin, clrop
4n
FIXED AND STAINED 8JJIEARS
immediately on to a thin layer of Champy or F. W. A. in
a Hat petri dish, cover and lmtVe overnight. It will nob
hurt to leave the smears in fixative for 24 hours or even
several days if well covered. Now here arises the diffic:ulty. You must wash out the fixative, but for how
long 1 Generally, if the Slllears have been fixed overnight, an hour in running water next morning will be
the right amount. If fixed for several days at least 5 or
6 hours, and for thick smears, overnight. In general, the
over\Yashed smear showns chromatin centrosome and
kinetic bodies better, the underwashed, Golgi bodies
(invertebrate) and mitochondria better. Thus, if you
are getting preparations somewhat like Bouin fixed smears,
you l1re definitely washing out too long, or not staining
long enough. If you cannot get the smears to stain in
iron hmmatoxylin properly, they are probably not washed
out enough.
The stains to use are iron alum hoomatoxylin or acid
fuchsin. In either case unless you are using chlorocarmine (§ 150) before you begin staining you must leave
the smears in alcohol after they have come from the
wash in water. They will stain, it is true, without this
treatment, but not evenly or well. Here is a good plan
for iron hoomatoxylin. Leave a day and a night in
fixative, wash next morning for I hour, transfer to 70%
till next morning. Wash in distilled water for 10 minutes,
and place in 4% iron alum all day, and before you go In
the evening wash the smears slightly in distilled water,
and leave in Regaud's or Heidenhain's hromatoxylin till
next morning. Wash off excess stain in water, and
differentiate cautiously in 4% iron alum, diluted a little
if necessary to slow up the extraction. After differentiation wash at least 15 minutes under tap or in substitute
(§ 36), upgrade and mount in balsam.
Champy and F. W. A. are often post-chromed in 3%
bichromate of potassiulll for three days, then washed out
aOLGJ APPARA'PUS S.MEARS
41
several hours, brought into 50% alcohol 30 minutes,
70% ovcrnight. They lllay be stained in Regaud'H
hrematoxyliu (§§ 147, 1,18), or acid fuchsin (§ 154). Try
the method if you find your Champy smears are not.
succeeding as you would wish.
56. Gentian Violet Ita!; he en used with succeRS on Champy
films fixed overnight or hetter for shorter periods (§ 10;1). It
is a chromosome and nucleolar stain, and can be very nicc.
The method is given ill § Ifj5.
"
57. Evaluation of Method. This method is excellent
for germ cell smears, Protozoa, etc. It usually gives fat
as well, but not always, and to study the fat you must
have recourse to special methods (§ OH). Uell granules are
definitely very good by these methods. The method is
valuable for spermatogenesis where whole spcrmatids or
spermatozoa, are to he studied. One of the difficulties of
the method is to get the time of washing out, just right.
In this and in other similar difficulties, the propel' thing
to do is to have a number of smears (or pieces) washing,
and take them out at intervals.
58. Bleaching Chrome-osmium Smears. POl' nuclear and
kinetic apparatns it will often be found better to bleach in
hydrogen peroxide 4% in water for a few minutes. Then
transfer to 70% for some hours and proceed ,vith staining.
Thc botanists prefer washing out sueh material in tepid water
for 2 hours.
Some workers bleach in aqueOllR pink permanganate of
potash solution (0,5%) followed by 4% oxalic acid.
Goigi Apparatus Smears
59. The Aoyama (Cajal) Smear. This is a silver method
and often gives very interesting results on smears. The
difficulty in the formalin silver smear is a flocculent
precipitate whieh often appears over the whole coverslip,
not in cells, and which is apparently due to impreglU1tion of
broken pieces of cells and coagulated proteid body fluids.
4:3
FiXED AND 8.'1'AINElJ S.MEARS
If you make a good many smears Home are better than
others. Fix a number of coverslip smears in Aoyam!L
(§ 170) and take them out every 5 minutes up to 30
minutes. Dip in distilled water for 1 minute and transfer
to newly made-up 1·5% silver nitrate in water for several
houn, at 22° C. vVash slightly and reduce as described in
§ 170, wash in water, upgrade, mount in balsam.
~rhis method may show spinelle bridges and mitoehondria, and in some cases is remarkably specific for the
dictyosomes of the Goigi apparatus. It is an interesting
and valuable method.
60. The Weigl (Mann-Kopsch) Smear. Take some
small-size coverslips (! in.). Make smears of insect or
vertebrate gonads. Drop into Manu's oSlllo-sublimate
(§ HI). Leave 30 minutes, wash several minutes in
distilled ·water, transfer to a tube of 1 % or 2% osmic
acid (Kopsch), cork ·well, place in incubator at 30° C.
Leave 2 days, then examine. If the impregnation has
not proceeded well, return to the tube for another day.
Pour off after 4: days, fill with distilled water and put
back in incubator for the night. Wash a few minutes
in fresh distilled water again, upgrade, mount in xylol
bals~1m. If you wish to stain nuclei, use a water solution
of neutral red (water, 1,000 c.c.; N. R., 1 gm.; glacial
acetic (Lcid, 2 c.c.). Stain from water, and transfer
immediately to absolute alcohol, xylol, balsam.
61. Evaluation of the Methods. Rarely quite so good
as the sectioning methods (§ 170). The Aoyama method
is cheap, and worth trying, though definitely difficult on
account of broken cells and coagulated body fluids.
Sometimes alteration (usually shortening) of the times of
fixation and silvering may produce better results. Reductioll (§ 170) of smears takes place quiekly and need not
be llrolouged. What is critical in the Aoyama SlUear is
the time taken for washing out after fixation and silvering.
The animal spermatocyte is the best material for prac-
lVliJIOL 8.MJiJAR
tising the mcth()(l. UfiC Helix Of Mm; teHtis filllear;,; in
·which the Golgi apparatus iH well marked.
The mallie methods are, of eoun:e, the beRt known to
the cytology of the cytoplasm and can be remarkahly
i:lpecific for the Golgi bodies. Interpretatioll of the smears
is often diffieult. Your results should be compared with
those got with seetioned material.
CHAPTER IV
MICROCHEMICAL TESTS IN SMEARS
62. Many modern microchemical tests work very well
in smears. Of those given here, the fat tests are easy
~Lnd valuable. The Feulgen "Nuclealfarbung" method
is altogether admirable, and easy to work. The glycogen
methods, usually supposed to be easy, can be difficult in
material other than mammalian liver. The lecithin and
cholesterol methods work fairly well.
The living l1totoplasm in some cells (which includes uni-
cellular organislll!; likc Protozoa) may be extraordinarily
lilllpid and clear of formed granules. In other cases the
numerous granules lllay consist of starch, glycogen, fat"
volutin or protein formations associated with certain metals
and so all. Occasionally questions do arise as to the nature
of such formations, and it is well to have learnt the more
trustworthy of these methods. They must be used carefully,
and the information they supply cannot in all cases he regarded
as trustworthy.
Note. If possible use daylight when judging colours
in microchemical preparations. A yellow light is useless.
63. Fats. Some years ago we believed that the Weigl
or Kolatchew osmic methods, followed by imbedding and
sectioning in paraffin wax, would make it possible to study
all demonstrable fat. It is certain, however, that the
newly-formed fat of a more liquid and' delicate nature
(e.g., in oogenesis), even though thoroughly osmicated, is
unable to pass through heated xylol without being partly
or wholly dissolved. Moreover, some histologists are
sceptical as to the value of certain of the microchemical
tests for various types of fats, and these test~ have had
to be jettisoned. .Two tests we still give here, which are
regarded with suspicion by some chemists, are the osmic
44
FAT
45
and the Nile Blue sulphate. The osmic method is useful
for comparison with the Sudan, lH1d the Nile Blue is
certainly still useful for m:trtl .~tU'i}lin(/, the impurity of the
dye and itfl solution or segregation in three (lifferent
colou]'s being an interesting ami llseful phenomenon in
certain cases. For example, in the Y -granules of insects,
no other method is so beautiful. In ad(lition it is known
that in tissues, fats are pmctieally never pmc. hut [we a
mixture of several kinds, so that the testR for the various
tYI)es of fats were not of lllu(Jh use except in rare cases.
Compare, however, a frozen section of ovary done hy the
cholesterol method of Schultz, § 68.
64. The Osmic Test for Fats. Make a smear on tt coverslip and plaee it on a eh'op of 2% osmic acid on a slide.
Leave in a petri dish for some minutes to honrs, examining
from time to time. The true fats, phosphatides, cholesterin-fatty acid mixtures, and cholesterin, bla.cken or go
brown in almost the order mentioned, ordinary tissue fitt
blackening quickly. After fixation of tissues in biehromate of potassium (3% in water), true fat usually blackens
easily. Myelin blackens easily in osmic alone, but not in
chrome-osmic (Altmann's :fluid), while it if! said that
cholesterol will not blacken in osmic alone, out will in
Altmann's fluid. In certain Protozoa there exist granules
of unknown nature ,vhich blacken immediately in osmic,
but whieh are doubtfully of a fatty nature.
Now the osmic test is a useful start, but must not be
regarded as anything else but that. Proceed to the
Sudan IV test.
65. Sudan IV Smears. Fix smear8 in formalin vapour
as follows. Saturate a little cotton wool in strong
formalin, place in petri dish, and warm. Put smears in
. this, cover for 5 minutes, but do not let them dry.
Transfer smears to a little saturated solution of Sudn,n IV 1
dissolved in equal parts of 70% alcohol and pnre acetone
1
Not Sudan III.
Hi
j}J[OROOHJiJJ.1IIUAL 'l'ES'l'S IN SJJiEAR8
(does not keep long). Leave 5 minutes in stain. Dip into
70% alcohol, \vash in water, counterstain if you clesire in
Delafield or Ehrlioh, mount in glyoerine or Farrants.
Fats bright red.
66. Nile Blue Sulphate Vital Staining of Fat (Lol'rain
Smith). The above methods kill the cells, bnt it is possible
to stain vitally fat granules by means of Nile Blue. Make
c1, smear on a coverslip ancllower on to a drop of 0·5% Nile
Blue in n01'mal saline; or place a small drop of Nile Blue
on a ooverslip and tease a very small part of the tissue in it.
Plaoe the coverslip smear downwards on a hollow-ground
slide, or on a glass or metal ring. Seal with vaseline if necessary. The stain may take 30 minutes or longer to operate.
This is an excellent method for insect germ cells, young
ova, etc. It is recommended to try the method with
thc testes dissected from a large caterpillar (Lepidoptera).
It may be necessary to make the solution somewhat
stronger, but for delicate work 0'5% has been found
correot. It should, of course, be made up in saline. In
recent years it has been usual to decry this method, but
it is worth trying in conjunction with other teohniques.
Nile Blue Sulphate for Fixed Tissues, etc. (Smith and Mail'
Method). Make a saturated aqueous solution of the dye and
add 0'5% sulphuric acid and boil under a condenser for 1 or
2 hours. To test whether the solution is right, pipette a
little into a test tube with some xylol and shake. A fluorescent
colour should appear. If not, continue boiling. Stain
formalin smears 01' frozen sections overnight. Differentiate
in 2% acetic acid, wash in water and mount in Farrant or
glycerine.
67. Cholesterol. The Windaus-Brunswick Test is to
smeal' or tease tissue on a slide, cover with a slip, run in a
solution of 0·5% digitonin in 85% alcohol. Needles of
cholesterol-digitonin (erystiLls) appear immediately if there
is a good quantity of cholesterol, but not usually otherwise. The following test must be llsed as weU.
68. Schultz Chole~terol Reaction. Smear on coverslip
GHOL.E8TEROL, GLYOOGEN
·17
and drop on to 4% forma,ldehydc (§ 112), Len,ve at lenNi.
30 minuteR. 'l'nll1flfer to a ~·5% solution of aqueoHR
violet imJl alulll, (NHoIhS(\, Fe:l(SO,l)a, 24H 2 0, in an
incubator at 37° C. for 2 or a dltyS. I-tinse in water for
a. few minutes, Gently blot dry on slllooth filter paper.
Place 011 a slide drops of a mixture of equal parts of glacial
acetic aeid and concentrated sulphuric acid. Place
prepared smear on this. In a few seeonds cholesteroleontainillg suhstances a})pear blue green, neutl'[LI fats
yellow brown, and all nuclei brownish. The effect is
fugitive, seetiollS hecoming dirty brown 'within ~. hour.
'rhe negative test cannot be taken as certain evidence
that choleRterol is not present. This is a very nice method
and usually works well on first trial with such material
as mammalian ovary (frozen sections).
69. Glycogen. In the study of the metabolism of
organs like the Ii vel' and placenta the various tests for
glycogen have been much used. There is still a great
deal to be done in the study of placentas by the modern
microchemical methods. Glycogen or animal starch, as
it, has been called, has usually been studiecl by the iodine
method (Bernard) and Best's C!tl'luine. There is a third
mcthod which consists in fixing material in Bouin's Huid
with chromic acid (so-called Bouin-Allen), treating subsequently vdth chromic acid, and then with Schiff's reagent.
By combining the three methods correct information on
the glycogen content should be obtainable. The methods
are not easy and need to be practised carefully.
70. The Iodine Gum Preparation. Used also by protozoologists to show paraglycogell. 'fhe basis is iodine, which is
not specific for glycogcn, but stains a variety of other materials,
as for example, protein granules in sponges, lecithin, etc. On
thc other hand, if you use as a control a slide which after be,ing
washed in u'ann u'aier has been spat 1lpon and left for 30 minutes
in a warm place, the glycogen or starch is either washed away
or digested by the pytalin, and does not aI)peal' after mounting
in iodine gum.
48
llIIOROOHltMlGAL TESTS IN B.MEAR8
To malw iodine gUill, tlisFlOlvc 50 gill. of gum ambic in
Strain through
lllllslin. DissohTe 1 gill. of iorline and :3 gll1. of potassium
iodide in a little water and add to the clear gum solution. To
lise, make the smear Oil a slide, if very wet let it cvaporate a
little, if not, put a strip of gum iodine on a coverslip and lower
OIl to the smear immediately.
The preparation is ready to
examine in a few minutes.
Two important modificatiollfl of the method should be tried.
In the first YOIl fix in abRolute or !)(l% alcohol, and then mount.
In the second YOll fix and leave for 24 hours in 1 % KOH in
H6% alcohol. Thi~ method is useful for spermatozoa and
protozoa hut gives disappointing results for conventional
mammalian material. We do not recommend it for anything
except protozoa, and the information it gives about spermatozoa is of doubtful value. The method may be tried on
Sporozoa in gut of mealworm.
lOll e.c. of water, which will take Home time.
71. C. Bernard's Iodine Test (Langhan's Method). This
method is given here as used by J. W. Jenkinson for
glycogen in placentas. Owing to the fact that iodine may
stain a numbeT of other substances, among which ILre
lecithin, amyloid, chitin, and almost any clense protein
granules, it is absolutely necessary to use the warm water
and spit tests as controls. Actually the whole section is
over-stained in Lugol, and after mounting the glycogen is
mahogany brown. But, while this method is good enough
for demonstrating glycogen in mammalian liver and
placentas, it is not so clear or beltutiful as Best's carmine,
which I recommend in preference, nor can any of the three
methods given here be used on invertebrate material
without careful controls and after careful practice on
mammalian liver. The method is as follows: fix in
absolute alcohol, or Cal'lloy, see that the pieces are
properly dehydrated, transfer to xylol, and imbed in wax.
Fix sections to slide with 50% alcohol with a few drops of
Mayer's albumen (§ 128), using the alcohol instead of
water. Drain and dry. Bring to 70% alcohol through
xylol and absolute, and stain in Ehrlich or Delafield
OLYCOGE.N
4H
diluted with the same volume of absolute alcohol, wal-;h off
ill 70% alcohol; it is not, lleeesfmry to "blue." Stain
5-10 minutes in Lugol, dehydrate in absolute alcohol
stLturated in iodine, clear in oil of origanulll, and monnt
in xylol or, better, origanum balsam. These preparations
fade badly after 10 years. In organs containing large
quantities of glycogen like the liver, you can stain in
ordinary Ehrlich's lUl3lnatoxylin and blue in tap water
substitute (§ 36), but; the method given above never allows
the sections to get into less tlmll 50 %alcohol.
Note. Lugol solution is 2 gm. potassium iodide, iodine
1 gm., water 100 C.c.
72. Best's Carmine. This techniqne has been used for
over 30 years, yet its explanation is Rtill unknown. It is
not specific and must be used with the warm water a,nd
(or) spit control (§ 70).
Fix in alcohol 96%,01' absolute alcohol, and preferably
imbed in celloidin if sections are to be used. If you have
imbedded in paraffin wax (in which case you pass the
material immediately from absolute alcohol to xylol and
wax) it will be necessary to cover the sections with a film
of celloidin, which prevents the loss of the glycogen. This
is done as follows. Remove wax in xylol, bring hito
absolute alcohol, and leave overnight in 1 % celloidin in
equal parts of ether and alcohol. Drain and allow to dry
partly, then harden the celloidin for a few minutes in
chloroform vapour (in a petri dish with a few drops of
ohloroform on one side). Bring into 90% to \vhich a few
drops of chloroform have been added (to prevent solution
of celloidin) and thence into 70% alcohol and finally
water.
For smears fix in absolute, leave in thin celloidin overnight, and proceed as above for sections. You must
permeate the smear with celloidin or the glycogen will
escape.
In both smear and section you can now stain in hallna-
50
.MIOROCHENICAL TE8'PS IN
S~~fEARS
toxylin of Elll#eh or Delafield (§ 143), l1lld thcn wash well
in rUllning water. Best's carmine lllade up H"; folluws :
carmine 2 gm., potassiulll earhonate 1 gm., potassiulll
chloride 5 gIll., distilled water llO e.c. Boil gently for a
few miuutes, cool. Add 20 C.c. of strong ammonia. Keep
in a well-stoppered bottle in a cupboard. The solution
may not remain active more than a few weeks. Stain for
5 minutes or longer in 2 parts of' stock Best's carmine, 3
parts strong ammonia, and 3 parts pure methyl alcohol.
After staining, differentiate in alcohol absolute 80 parts,
methyl alcohol 40 parts, water 100 parts, till no more
red comes out (about 3-D minutes). Wash in 80% alcohol,
lLhsolute, clove oil, and Canada balsam. Glycogen bright
red. We have found this the most satisfactory method.
73. The Picric Acid Glycogen Smear (H. Bauer). 'l'hi8 is an
interesting microchemical method which is worth trying in
any material yon may be studying. Smeal' and fix for about
3 hours in Bouin-Allen, which is ordinary BOllin's fluid to a
100 C.c. of which 1·5 gm. of chromic acid crystals have been
added. Then bring the smears directly into 4% chromic
acid for 1 hour. Wash 5 minutes in running water, then
treat for 10-15 minutes in Schiff's reagent (§ 78). Afterwards rinse in three changes of 80 2 water, prepared by adding
to 200 C.c. of distilled water 10 C.c. of a 10% solution of
anhydrous sodium bisulphitc and 10 C.c. of N.RCl. (The
S02 water prevents the reformation of fuchsin.) Wash in
running water, stain in Delafield if you wish, upgrade, mount.
GJycogen is reddish violet, but so also ml1y be starch, cellulose,
tunicine, and certain other materials. This smear should be
used in conjunction with Best's carmine, iodine gum, and the
spit control (pytalin) test.
74. Lecithin Test with Smears. Romieu's iodine test for the
lecithins has been llsedlately in cytology. Lecithin is soluble
in hot alcohol, and incompletely or hardly soluble in acetone.
According to Romieu, with iodine, slightly hydl'OZy8ed lecithin
gives a mahogany 01' reddish violet colour, which is iodocholine. Whatever chemical value the test may have, it
certainly gives interesting results on smears of mammalian
sperms, showing a definitely located region at the back of the
sperm head (post-nuclear body). As far back as 1904,
LEOITH.IN
51
Defiamlre fixml material in formol (4%), hrought it into
acrtone, which dissolved fat hilt not lecithin according to him,
and after ,Yashing, staoined tlw lecithin in osmic acid. When
neces:;;ary to section material, Romiell dehydrates in aectone,
imhed;; in wax acetone, removes the wax from the scctioIl~
in a closed jar of warm acetone (in the imbedding oven) :
proceed as below for SJlle~11'S.
The srnear also is fixed in for mol and brought into warm
water to dissolve glycogen (sections cleared of paraffin wax ill
acetone as ahove are t.rcated in exactly the same way with
warlU water). Mild hydrolysis in 10%, HeI is now C<tl'ried out.
The smears (or sections) are warmed in the acid tm fuming
just begins (or hetter the acid is warmed previollsly), and then
put ....·ithont washing into the following Lugol: potassium
iodide 8 gill. ill 150 C.c. of distilled water, to which is added
(i gIll. of iodine.
Dip quickly into distilled water and mount
in glycerine. The colonr i;; fugitive. The critieal point
appears to be the degree of hydroI~'sis. This method used
in conjunction with the acetone osmic techniquc probably
gives information of valuc.
75. Material to use for the Glycogen, Fat, Lecithin and
Cholesterol Methods. Undoubtedly the proper material
for beginning on the glycogen technique is the liver of it
well-feel rat or rabbit. Fix in alcohol anelleave the pieces
overnight, and thin hand sections can be made with n,
razor next day. The point about the liver is that the
glycogen will usually be found in the cells immediately
grouped around ~L blood vessel in it very characteristic
lIlftnner. Note that for Best's carmine the pieces must
be left overnight in thin celloidin, next day put on slides
and stuck by exposure to chloroform vapour. The simple
smearing of the sections with celloidin is not enough.
After the very characteristic flaky lumps of glycogen have
been found in the liver, both with the Best and the iodine
or Bauer method, a good thick smear of snail (Helix)
ovotestis can now be tried. Very characteristic sphere:;;
and flakes of glycogen can be stained in the nurse (yolk)
cells of the molluscan ovotestis. In these cells the yolk
spheres are yellowish in colour, and the glycogen lies
52
N10ROOHEJ.lHOAL TESTS IN SMEARS
amongst them. The smear of snail ovotestis is also
excellent for practising the Sudan IV method, the yolk
grannIes of the nurse cells taking the stain avidly. The
hest material for practising the Schultz cholesterol
reaction is frozen sections of (formal fixed) mammalian
ovary, where both neut/ral fat and cholesterol will stain
side by side in different colours. For the lecithin test,
the smear of ovotestis of Helix Illay be tried. The Golgi
apparatus dictyosomes go brownish black.
76. FeuIgen's Nuc1ealfarbung Test for Chromatin. This
is an accurate and valuable test for chromatin, a,nd has
displaced the older methyl green acetic test, which was of
restricte(l value (§ 33). It should be pointed out that
:Feulgen's method is a stain as well as a, test. The preparations arc valuable for chrom.osome and nuclear studies,
and are permanent. The method is well worth learning. It is true that there is still a certain amount of
dispute about its complete specificity and its ability
always to demonstrate chromatin. It usually works well
at first trial, and on any type of smear or section containing
nuclei.
Feulgen's Nucleal Reaction in Smears. Fix in corrosive acetic (6% sublimate in water with 2 C.c. of glacial
acetic acid per 100 c.c. solution of sublimate). Other
fixatives can, however, be used, except Carnoy sublimate
alcohol mixtures (Gilson, etc.) and Bouin. Very good
preparations can be got after Champy or Flemming •
fixation. The type of fixation used is closely related to
the time needed for hydrolysis and may be fairly critical,l
Too long a sojourn in HCI weakens the reaction. Hydrolysis is carried out at 60° C. in an acid solution prepared
as follows. Add 82·5 C.c. of HOI (Sp. G. 1'19) to 1 litre
of distilled water. Leave for optimum period (4-15
minutes), then dip into cold acid and rinse in distilled
1 For corrosive acetic material it is 4, for Zenker 5, for Fleming 16,
Bouin-Allen 22, Navashin 7, Regaud 14 minutes.
Ji'EULOEN TEOHNIQUJj}
53
water. Stain for Ii hours in fuchsin sulphurous acid
(Schiff's reagent) prepared as in § 78. Wash excess st.ain
off slide ill three washes of 80 2 water, to prevent reformittion of fuchsin. If 80 2 water is not at hand it may be
prepared by adding to 200 C.c. of distilled water 10 c,o.
of a 10% solution of anhydrous sodium hi sulphite
and 10 C.c. of N.HCl. Wash in distilled water and
counterstain if you wish for t minute in Light Green
(0·5 gm. of Light Green ill 200 C.c. of alcohol 70%).
Pass to 90%, absolute, clear in xylol, ,tud mouut in
balsam. Material containing thymonuoleic acid is reddish
violet to dark violet, plasmosomes green (in Light
Green).
77. Vitamin C. The researches which led to the elaboration of !L cytological technique for this vitamin were initiated
by A. von Szent-Gyorgyi in 1927. Adrenal gland cortex
treated ""ith AgN0 3 solution rapidly blackens. A crystalline
substance" hexuronic acid" was isolated, and finally identified
with vitamin 0, partly with the assistance of the Americans,
C. King and W. A. Waugh. In 1932, Moore and Ray showed
that in scurvy the silver nitrate reducing property of the
adrenal disappeared. Giroud and Leblond showed that this
vitamin was localized in cells in connection with the mitochondria and ({algi apparatus, and the questions thus raised
are of prime importance to cytologists.
G. Bourne has carefully investigated the cytological technique for this vitamin and recommends the following procedure: .Fix tissues in a solution of 5% AgNO s, to 100 c.c.
of which 5 C.c. of glacial acetic acid has been added (modified
solution of Giroud and Leblonrl). For Protozoa, etc. smears,
a few drops of the solution are added to the liquid on the slide
with the cells. Examine after ten to fifteen minutes. Or better
isolate organisms in centrifuge, treat en masse with solution,
wash in Ilure glass distilled aCl. dest., treat in photographic
"hypo." Wash several times in aq. dest. upgrade in ethyl
alcohol, clear, imbed and section if necessary. The best
results are only obtained by ca?-rying out impregnation in dark·
neA~8. This is important.
Finally, it should be mentioned that vitamin 0 may exist
in A reduced form, reacting readily with AgN0 3 , and B
IH
J.1flC'ROCHEMIOAL TES'P8 IN SMEARS
reversihly oxidizE,d form ]lOt, ;;0 reacting. Tlw orrlinary teehlliqllf' Hilllply denlOllstrates forlll A.
To get over this difficulty A. Boul'llo expmler; pieces of tiHrme
to vapour of glaeia,l acetic acid for some miIlutml, cuts thin
slices and Ie aveR in atmosphere of hydrogen bisulphide for
fifteen minutes. This converts form B into forlll A. All
trace of hydrogen bisulphide nUGst be rem.oved. This is dono
1:n vacuo until most of the HoS has heen suckeu ant of the tissue
(15 to 30 minutes). The se~tiom; !Lre then exposed to a strong
Htream of nitrogen gas for H5 minutes. Before concluding
that vitamin C is not present the hydrogen bisulphide method
should be used. Actually there may be litt,le difference between
the various methods given when one examines the slides.
This is recognized widely as a valid method. The vitamin
hecomes black with silver nitrate and is founel in the form of
scatterod granules.
78. Schiff's Reagent. Dissolve 1 gm. of powdered
basic fuchsin (and prcferably not acid fuchsin) in 200 c.c.
of boiling distilled water. Cool to 50 0 C., then filter.
Add 20 C.c. of N.RCl, cool to about 25° C. and then acId
1 gm. of anhydrous sodium sulphite. S02 is given off
and the liquid slowly turns yellow. Allow to stand for
24 hours at least, keep well stoppered in the dark. Should
the fluid become red or rose instead of yellowish, discard
it.
79. Volutin. Fix smeal' in Schaudinn's fluid, wash out
in 70% and iodine, bring down to water and place in a
10% solution of methylene blue for 10 minutes. Rinse in
water and treat with 1 % sulphuric acid for ! minute,
upgrade \1nd mount. The nucleus and the cytoplasm
decolonrize, but volutin remains coloured. Volutin is
found commonly in Sporozoa and Flagellata.
CHAPTER V
WHOLE MOUNTS
Unstained
80. It is mmal to mouut many types of Inscets, Polyzoa,
slllall Aruehincla, Kithont. staining. The mouuting
medium often m;ed is Pa/'J'ants' med-iu'ln or one of the
improved modifications. Here is a good one: Distmed
water 50 e.c., gum arahic 4l) gm., glycerine 20 c.c., ehloml
hydrate GO gm. Dissolve the picked gum in the water
cold, \\'hil~h may take some time, then add the chloral and
dissolve wit.h gentle heat, then add the glycerine and
filter t.hrough cambric in a hot fUllnel. Another we11known fluid is that of Berle-se: Distilled water 20 c.c.,
chloral hydrate 160 gm., gum arabic 15 gm., glucose syrup
10 gm., glacial acetic acid 2 c.e. Pnri'sjlnicl for mosquito
larvm is : 'Vater 10 c.c., gum ambic (acacia) 8 gm., chloral
hydrate 70 gm., glycerine 5 c.c., glacial acetic 3 c.c.
Dissolve in a water baHl at 50 0 C. in order named, strain
through three or four thicknesRes of muslin.
When using any of these media it is best to kill the
la.rva or other animal in hot water or 10% acetic acid.
Alcohol 01" formalin spec'imen.s shmlld be washed out well
overnight in di8h~lled water 01' 10% acetic ncicl. Place the
objeet on the slide, a.dd a drop of medium, put on coverslip, and warm a little. Set aside to harden for a few
days, then ring with asphalt, Apathy's cement, cellulose
varnish or ordinary Cnnada balsam (in xylol).
8l. Dioxan balsam appears to he excellent for mounting
insects and similar object;;. Drop them either alive or
from fixative (see §§ 114, llH, IHI) into dioxan or 95%
111cohol, and mount Ilext morning in dioxa.n balsam, made
WHOLE liOUNTS
by dii'isolvillg dry Canada balsam in dioxan to the thickIless of honey.
82. Euparal. For ~O% or absolute alcohol specimens.
Transfer the objects to a drop of euparal and cover.
Will prohably he superseded largely by dioxan balsam.
83. Glycerine Jelly. Dissolve 10 gm. of best gelatine
in liO C.c. of distilled water, mix in 70 c.c. of pure glycerine,
and add 0·25 gm. of phenol crystals. These operations
should be done on a water bath, or in a beaker in a dish
of hot water. Mount objects from glycerine (§ 86).
Of the three methods above mentioned gum glycerine
medium clears the least; dioxall balsam the most.
84. Canada Balsa~. This may be dissolved in absolute
alcohol, benzol, xylol, 01' dioxan. The so-called neutral
balsam supplied by certain commercial houses goes as
acid as xylol balsam after a time in the light. Make up
your routine balsalh in benzol, not in xylol. If you wish
to mount stains which are prone to be fugitive use thick
cedar wood oil. This hardens after a time.
Canada balsam should be thin for sections, and thick
for whole ~unts, but about the viscosity of honey does
for most purposes. It is recommended to keep balsam
in a bottle covered with black paint or paper, and never
to allow' it to remain in sunlight. There are special
balsam bottles supplied by the commercial houses. Some
people put a strip of plasticine around the neck where
the loose cover comes on the bottle-this to prevent
sticking and to make an air-tight joint, which is an
excellent idea. Cork stoppers are useless for balsam.
If the-balsam lid sticks, cautiously warm it in a flame.
1'0 use Canada balsam for unstained whole mounts,
dehydrate in absolute alcohol, clear in cedar wood oil or
beechwood creosote, wash out in xylol and mount in
balsam.
85, Other Resinous Media. 8anclarac, Camsal and other
resinous or oily media have been used by Gilson for
BALSAM AND GLYr)EIUNE
57
making monutauts. Euparal is rather difficult to make
and it iR better to buy it. Here, however, is a " neutral
monnt.ing medium" which can he made in the laborn,tory, ltnd i;:; an excellent substitute f(ll' euparal in every
case:
Denham's Sandarac Camphloral is 2 parts by weight
of crystalline chloral hydrate ground in a glass mortar
with 1 part of " flowers of camphor." If any difficult,y
is found in getting the last few crystals to dissolve the
mixture is left in a warm place for 11 few hours. 'fhe
liquid is then filtered. The salldarac solution is prepared
separately. Selected crystals of sandarac gum are
dissolved in excess of isohutyl aicoholllt (jOD C. to make a
solution thin enough to filter easily. It is then shaken up
widl animal charcoal, and filtered several times through
paper, after which it is evaporated to ~1 thick SYTUP, using
a eondenser to retain the alcohol. One part of gum is
mixed with two of camphloral, and incorporated by
warming and stirring. The product should have a
refractive index of 1·485. Objects may be lllounted from
70% alcohol. Euparal is cOlllIllercially made in much
the same way, the gum being dissolved in eucalyptol.
86. Glycerine Whole Mounts. Fix in weak (4%)
formalin (§ 112) for se'veml days. Bring into water
overnight. Very gradually bring into 50% alcohol, by
dropping frmn time to time a little alcohol of this strength
into the tube of water and leaving for hours until
mixed, shaking slightly now and again. Upgrade
gradually for days to 90% alcohoP Place a layer of
glycerine at the bottom of a tube. Pipette in a similal'
layer of 90% alcohol and ether (9; 1), and transfer the
objects to it. Leave uncorked till the alcohol evaporates
and the objects fall into the glycerine. Mount in glycerine,
or glycerine jelly, ring 'with asphalt, etc. (§ 88). To mount
1 Somo people begill by fixing tho organisms in 91)% akohol.
This
would be alright for hard insects amI such like.
TVHOLE JlfOUN1'S
in glycerine jelly, warm a little on a slide, transfer object
from glycerine into this and cover. Ring after l1 few
days with cement.
87. Mounting in Balsam or Glycerine, etc. Students
often use too much mountant. You should notice tha,t
the space between the coverslip and slide, even with a
20p, section, is very small, Use a small drop of mountant,
and if it is not enough, place another small drop at the
edge of the cover and let it run in. If you wish to cement
a glycerine mounted slide this calmot be done if it is
messy. Moreover, it may be difficult to use an oil-immersion or other high power lens with short focal distance
on such preparations; and they dry very slowly. When
mounting sections, put a thin strip of balsam on the coverslip and lower one end first on the slide gently with forceps,
a pin, or sharpened matchstick.
88. Cementing Glycerine or Similar Wet Slides. TV'ith
Canada Bal8am. Some people mount in a fiat drop of
BeI'lese or Farrants, and leave away from dust and without
coverslip till dry. Then put xylol or benzol balsam on
top and cover with a coverslip. This would not do for
glycerine slides, which must be covered and ringed or
cemented with one of the following media, or ~wjth
Canada balsam.
Peter Gmy's jJ.{ediutn. Four parts anhydrous lanoline,
8 parts resin, 1 part dry Oanada balsam. Melt together,
stir. This forms a solid mass on cooling. For circular
cover glasses, dip piece of metal tube, right size, into
molten mass. For square covers, use broad, bent needle
in holder.
Amyl Acetate Celluloid Cement. This is made by clissolv~
ing some (food hard celluloid in amyl acetate. The solution should be thin enough to spread with a soft brush.
It may be used for brushing on the end of slides instead of
labels. Allow it to dry, write legend, let it (Iry, brush
over another layer of cement. It can be used for ringing
s'rAINED IVHOLE llIOUjYTS
:jfI
glycerine and gum mounts instead of other oil remellts.
In all eases slides should be grease free.
ApathY'8 C'e-!nent for Olycerine }rlolln{8. Bqual part,s of
60 0 C. paraffin wax and Canac11t balsam. Heat together
in a porcelain vessel till the lllass becomes golden and no
longer emits vapour of turpentine. To use, warm, and
apply with glass rod or spatula. An excellent medium.
Paraffin Wax. Useless for permanent slides.
89. The Stained Whole Mount. Two methods are given
here-the Borax Carmine and Mayer's Acid Hmmalum.
It is rate to see a really good whole mount made by
students. The fault most commonly noticed is 'Undel'dilfeTentiation. Fix in corrosive sublima,te acetic (§ 108) or
Petrunkewitsch (§ I HI) only if the object is rather
impenetrable. The main point h.; to use cOlTosive sublimate as it gives bright preparations. If the animal haR
to be amesthetized this must be done first (§ 02), or it
may be held between a slide and coverslip and the
fixative run in with a pipette. When fixed, which will
depend on its size, bring into 70% alcohol. (In any
case no harm will be done by leaving it in fixative
overnight.) Add iocliue (Lugol, § 71) to the 70% till
sherry colour (light brown). Leave several hours, and
if it decolourizes add a little more iodine. Next morning
pour off and add more 70%. Leave for several hours,
or as long as you find cOllvenient. Pour some borax
carmine or acid luemalum into a petri dish just to
cover the specimen. Leave overnight at least, then
pour away the stain and begin differentiation. For the
carmine stain use 70% alcohol with 0'5% HOI, for the
hremalum use water with 0'5% HOI. Leave all next day
11lld night; when this period has elapsed the stain ghould
have been extracted from cuticular structures like skin or
membranes, and only left in the internal organs and
glands. The rest of the animal should be pink (carmine)
or reddish (hremalum), and it should be possible to see
GO
WHOLE MOUNTS
:mmething of the structure. If it is opaque, it is better
to leave it a little longer. On the other hand, if it has
got rather pale after the first day in differentiating fluid
it is best to place it in non-acidulated 70% overnight and
ra-examine next morning. If differentiated rightly,
place the luemalulll preparation in alkaline water till
hlue (§ 36). Upgrade slowly to 90%, absolute, and clear
in xylol, or if your absolute alcohol is suspect, in methyl
benzoate or beechwood creosote followed by xylol, mount
in balsam. It may be necess(try to make glass feet to
support the object under the cover. Rernembel' a8 the
balsam drie..'1 the cover will press down on the pl'epamtion.
Note that if you use dioxan balsam, you mount from
95% alcohol, or from dioxan if you have dehydrated in
this.
90. Staining Chrome Fixed Material in Carmine Stains.
This cannot be done successfully if the smears, sections
or pieces have been taken to alcohol. Always stain in
the carmine first, just after washing out in water.
91. Treatment of Formalin Specimens. Organisms got
in the field are often fixed in formalin, and with ordinary
technique it seems quite hopeless to get good stains in
either acid hffimalum or borax carmine. Such materi!11
may be rejuvenated by being left in 4% hydrogen
peroxide or pink solution of permanganate of potassium
for all hour or so. The former solution is the better of
the two. Small ll1edusre stain nicely after 1 hour in
either solution, but larger or more impenetrable objects
would have to be left longer. After rejuvenating leave
overnight or longer in 70%, or 90%, alcohol before begin.
ning the staining.
92. Anresthetizing. Narcotic substances which can
be got easUy· are menthol, methyl alcohol, chloroform,
chloreton, chloral hydrate, and chloride and sulphate of
magnesium. In addition, cocaine hydrochloride or hydrochlorate" stovaine, urethrane, hydroxylamine, eucain,
AN/ESTHETICS
iiI
morphia, and curare have been used, but are not so easy
to buy. Cocaine is rather indispensable and is the basis
of ROllsselef's solution (does not kcep well) : Cooaine 2%
aqueous solution 3 c.c., methylated spirits or ethyl
alcohol of 90% 1 c.c., distilled water 6 c.o. For Protozoa
use 10% methyl alcohol, or Rousselet's solution. Oorri'8
solution, which is important for both fresh water and
marine animals, is: Methyl alcohol of 96% strength
10 c.c., water (fresh or sea water accorcling to organisms)
90 c.c., chloroform three drops. For fresh 'vater
organisms only, add O'(l% sodium ehloride. .Menthol is
used for larger marine animals; jW'It sprinkle over surfaee
of water and le~we for from 12 to 24 hours. Chloral
hydrate is used in the same WH,y, but acts sooner. .Maynesiurn sulphate (33% aqueous), added gently in water,
acts in about l hour on actinians and sueh marine
animals.
For slllall animals in general: Place them in a little
water in a very clean watch gla,ss, allow to expand and
quieten, add the narcotie gingerly. Go slowly or the
animals contract. Try thc following in this order for
Protozoa, Rotifera, and such like: 10% methyl alcohol,
Rousselet, and Cord. For larger organisms, magnesium
sulphate and ehloral hydrate are recommended.
93. Peter Gray's Technique for Rapid Permanent
Mounts of Fresh Water Organisms. (Time 5 minutes.)
Prepare some pipette drop-bottles, as used in chemical
laboratories, and fill one each with 70% alcohol, 40%
formaldehyde, glacial acetic acid and basal fixative
solution (which is picric acid 1 gm., corrosive sublimate
I gm., 100 C.c. of 95% ethyl alcohol). Have also a
stoppered drop-bottle of ether, some Mayer's albumen
(§ 128), pipettes with rubber teats, writing diamond
or carborundum pencil, a staining trough of 70% alcohol,
strips of filter paper, clean slides and several 2-in. specimen tubes in a stand.
62
WHOLE MOUNTS
Before beginning examination of ,1> collection of pond
organisms clenn slides by rubbing with acidnlated alcohol
(!)5% with 1 % acetic acid) and drying with duster.
Plewe n small drop of Mayer's albumen on centre of eaeh
slide lwd rub down with alcohol cleaned finger a patch
1 em. in diameter. Now prepare the necessary fixatives
from the pipette drop-bottles as follows: for Protozoa,
basal fixative 10 drops, ether 3 drops, glacial acetic 2
drops, 40% formaldehyde 5 drops. For heavily c1lt'icnlal'ized forms, basal fixative 10 drops, ether 1 drop,
glacial acetic 4 drops, 40% formaldehyde 5 drops. For
deNcate lal'vm, basal fixative 10 drops, ether 2 drops,
glacil1l acetic 1 drop, 40% formaldehyde 5 drops. These
are made up in the 2 X t in. specimen tubes and placed
in the stand. They are unstable, and are all modified
They cause Mayer's
YocOln-Duboscq-Brasil fluid.
albumen to become sticky, unt,il treated with alcohol, when
it hardens.
Examine your collection under a dissecting microscope,
pick up an animal with a pipette in as little water as
possible, and drop it in the middle of the patch of albumen.
Draw off surplus fluid, leaving' sufficient for the animal to
swim naturally. Some forms are surprisingly sensitive to
either the glycerine-albumen or the sodium salicylate in
it, and fixation must be hurried. Watch until the animal
is in a normal position and place a large drop of fixative
on it. If in the subsequent streaming the animal passes
outside the albumen patch it must be gently put back
with a fine glass needle. The animal must now be watched
tlntil the d1'oplet of jl1lid has evaporated 80 as to show clearly
the outlines of the object. Now flood gently with 70%
alcohol. If this has been done properly the animal is
now stuck in a natural position in the albumen. If very
small, ring the organism with the diamond, and write a
reference number or name on the slide. Now transfer to
staining trough of 70% alcohol. Drain slide, wipe around
B},fALL OROANIS.1.1JS
63
quiekly, put Oll heneh, add I drop of Ehrlieh's luemntoxylin.
Leave 30 seconds for rrotozOtt (Paralllwcium), H minutes
for miracidinlll, run off excess stain with !JO% alcohol,
never uater, hrieHy flush with 0·25% He1 ill 70 % alcohol,
wash uff with Seott'r,: tl1P Witter substitute (do not use
ammonia. for" blueing "), stain quickly in eosin (0·5% in
50% alcohol), rinse off in 95% alcohol, deftr in terpineol,
mount in balsam. You C[tIl 1ll0l1llt in euparal or dioXlLll
balsam if you wish, in whieh cn,se do not clear in terpineol.
This method is not very successful for larger organisms
like insect larvre, which should be trented as described ill
§ flu or § 80.
94. Free Living Organisms like Protozoa and Rotifers.
For those large enough, treat as described in § fl3; for
smaH flagellates and ciliates, SUlear a drop of the medium
containing them on a coverslip, kill in formalin or osmic
V!L"{)OUl' for a few minutes, now mix a small drop oflVfayer's
albumen with the liquid on the cover, and drop smeftl'
downwards on to the fixative. If there was not too much
water on the coverslip, the organisms will stick. Some
workers use gelatine water. It is a good thing previously
to concentrate the organisms in their medium by mmms
of a hand centrifuge.
95. To make Fixed Protozoa and other Small Objects
Adhere to Slide or Coverslip. ]'01' unfixed objects, which
are to be mounted and stained in Ehrlieh's hwmatoxylin
and eosin, see § 93. For fixed objects, bring into water,
prepare slides with albumen, piace objects in very little
water in middle of albumen, remove excess water with
pipette, leave for a short time until albumen and water
mix slightly, flood gently with 70% alcohol. Alternatively,
mix ohjects \vith albumen, smear on cover, [Llld drop into
70% alcohol.
For objects in absolute alcohol place drop of thin
celloidin on slide, mix objects in it, flattening the drop,
harden in chloroform vapour. Note that unless strong
WHOLE .MOUNTS
(j·1
alcoholluls a little chloroform mixed with it, the celloidin
bccomes dissolved.
Should you not wish to use alcohol, the following method
is indicated. Cut 2 gm. of sheet gelatine into small pieces,
place in flask, cover vvith distilled water and soak several
hoUl's. Make up to 100 C.c. with distilled water, warm
to 60° C. Filter only if necessalyl Add a crystal of
thymol. Take a little on the small finger and smeal' on
a slide. If the objects are not too wet they can be placed
all it immediately. If they are wet excess water must be
removed. Now fix the gelatine in a warmed slide bottle
containing a piece of cotton wool soaked in commercial
formalin. Success depends on allowing the objects to
mingle with the gelatine, which must not be diluted too
much before fixation.
96. Whole Mounts of Crustacea such as Asellus and
Gammarus (from Fresh Water Ponds). These crustacea
are good examples of rather impermeable objeets for
making whole stained mounts. The secret of success is
not to hurry matters. Soak well in 90% alcohol after
fixation, leave 2 or 3 days in carmine, and longer in
acid alcohol if necessary. It is always a good thing to
prick impermeable objects with a very fine needle to aid
penetration of fluids.
97. Whole Mounts of Nerve Endings in Muscle, etc.
These interesting preparations show the palmate or
dendritic nerve endings in muscle. Take a strip of muscle
from a newly killed frog's leg. Try to get thin sheets.
Soak in fresh lemon juice until they become transparent,
that is, for about 5 or 10 minutes. Wash in distilled water,
bring for 20 minutes into 1% aqueous gold chloride,
wash in watcr slightly, bring into 50 C.c. of distilled water
and 2 C.c. of acetic acid. Expose to light for 24-48 hours.
These prep,al'ations can be mounted in glycerine and water
1
£01'
This is often used to CO!1t old lantern slideR for making diagrams
projection.
81 L TORR 1JljJ /(RU,YA'I'IO;..Y
(1 ; I), IlIlt. are not permanent. Here iR a method which
gives permanent preparations. Take It thin .sheet of fi'eBh
muscle, suak in 1 ~~\ gokl chloride tin' 11 !e.w hou1's in it dark
cupboard. Place on a piec'c of filter Tlllper and gently mop
up solution from outside of piece. Transfer to tube of
1 % formic acid, leave in direct SUll all day in winter, 01'
in hright light in summer. If the solution goes browll
change the acid. Wash ill distilled water for a few
minutes, place on a slide, mop np water with filter paper,
add glycerine and cover.
98. Silver Impregnations of Fresh Tissue. Kill a frog
and remove it flat piece of web from hetween its toes.
\-Vash ill distilled water, stretch on a Rlide, remove eXCel-\f;
,vater with filter paper, add some (1'5% (or weaker) aqueous
silver nitrate, leave in bright sunlight unt.illight hrownish
and the cell walls are stained. The speed of the reaction
depends largely on the brightness of the light. In summer
many preparations have impregnated in less than 30
minutes. Now wfLsh in distilled water, mop up with filter
paper, re-stretch the piece, and harden with a drop of 90%
aJeohol. Stain in Ehrlich if you wish (§ 44), upgrade and
mount in balsam. Permanent.
Try this method on mesentery, lung of frog opened out,
cornea, etc.
These preparations are remarkable for the clarity with
which they show cell walls. The method is worth trying
on fresh smears of germ cells and Protozoa (Silver Line
organs). In these cases the silver mlty " come down " on
various parts of the cell.
99. Cartilaginous Skeletons of Small Fish and Embryos.
Fix in alcohol (90%) or Carnoy and wash out well in
alcohol of 70%. Stain for a week in 0·25% methylene
blue in 70% alcohol acidified with 1 % hydrochloric acid.
Differentiate in 1 % hydrochloric acid in 70% alcohol for
about a week Cartilage remains blue, all other tissues
colourless. Upgrade, mount in balsam or leave in clearing
LAROIlO\TOl\Y TEOHNIQUE.
3
WHOUf] J.110UN'I'8
Huid, next. §. Both t.hionin and tJnluidiu blue llHty IJe
m,erl in the sallle way.
100. Oil for Clearing Embryos, etc., for Observation
Whole. 'I'he t'eeol\lmeurlcrl fo;uhstallce .is Wintergreen Oil
(clear first ill benzol). Nowadays methyl benzoate (clear
straight from ahKolute) is being used also.
CHAPTER VI
FIXATION METHODS
101. Cutting Material for Cytology and Histology. It
is generally to be arlyised to avoid_ orushing the material
by using sci!':sors or a hlunt knife. vVhile this is good
advice, expcrimentK carrieel out in crushing glamluhtr
tissue did not have the had efforts expected. In any
case buy a packet of sflJet,y mzor blades, and llse ono for
(mtting thin slices of mo,terial for fixing. The:-:o hlades
can alE;o hc used in H spot·ial holder lUI' cutting paraffin
seotions (§ 1:31).
102. Dissecting and Killing Material for Cytological
and other Similar Work. The hest way is to cnt off the
head of an insect OI" snch arthropod anel pull its viscera
out into a drop of fiXative. The necessary organs are
then found and Beparated under a dissecting microsoope
or lens, and put into a capsule of fixative. The main
point is to get the material into fixat·ive as soon after the
interruption of the blood stream ns possible. If you are
working on animals like rats, kill in coal gas, not chloroform or ether. Never use these, as they tend to alter the
cells around blood vessels.
103. Vivisection Acts. You may not cut, inject. or
otherwise experiment on a living vcrtebra.te without, H,
vivisection licencc in Great Britain and Ireland and in
certain other l'Olwtries. If you do so without a licence,
and publish your work, you are exposing youmelf to
prosecution: H, vivisection licence may therefore be
necessary; hut you can vivisect any animal without a
vertebral column.
li7
FIXATION ,METHODS
104. Fixing fluids practically all consist of mixtures of
i()l'IHaJill, acet,ie [1eid, pioric acid, chromic acid, bichromate
of potassiulll, mercuric chloride (sublimate or COTI'osive
sublimate), ()smium tetroxide and alcohol. No modern
fixative consists of one of the above substances alone, with
the possible exception of formalin (in saline), which in
some eases may give a remarkably good fixation. Acetic
acici nl1(l alcohol have a destructive effect on certain
lllateriab ill the uell, and are never used when the most
fl1ithful fixation is needed (§ 34,),
Altmann'::; fluid, which consists of bichromate of
pot,i1SSiUlll and osmium tetroxide, Wl\S invented nearly
hnlf a eentuJ'Y ago, and since that time no real advance in
cytologieal fixation has becn made.
The Goigi ap7
rmratuR methods of Cajal (formalin silver) and Kopsch
(o~miurn tetroxide) twe not to be. regarded a~ advances
in fixation technique, so ll1ueh as in post-fixation
technique.
The fixatives mentioned above have very different efJeots
when brought into contact with bioehemieally isolated
globulins, nueieoproteins, lipoids, carbohydrates, or with
egg white. Some of these fixatives do not precipitate
or otherwise change all these materials, as for instance
acetic add which only fixes llucleopl'oteins, or corrosive
sublimate and picric a;cid which htwe no apparent effect
on fats. Osmic acid does not precipitate proteins, but it
changes them in a definite manner without producing the
same effect as alcohol or corrosive sublimate. It is the .
most valuable tixative known, and it is strange that it
should have figured so little in the technique of proto. zoologists.
Alcohol anci corrosive sublimate are energetic
coagulants and produce the well-known net-like protoplasm seen in .all the older pictures of cells. It is agreed
nowadays that in metazoan tissue cells, the nets are
art.ifacts. They do not allpear ill good chrome-osmium
PRNRTRA 7'lO..V OF FIXA'J 1/TE'S
nn
fixed cells, LUlCl arc be!:it. seen in Curnoy or Schaudinn fixed
cells.
Aect,ir' :wirl is an intereHt.ing subr4ance in fixation. H
d()p~ not prel"ipitate ol'{Hnnry proteins ill t·jw weak 8t1'(~ngth
ill which it is used in fixatioll. but. it, dUCH fix nn('l~o"
proteins, and in this aml t.his alone its virtue lies. In the
case of bichromate of potasBiulU the degree of acidity of
the solution is of considerable importance, hut bichromate
should be used with or after chromic acid fixatives fbr
lipoid cell organs if one wishes to get a good stain in
hrematoxylin or acid fuchsin. In this and other cases the
mere fix~1tilln is not the whole problem. This is why
Champy is superior to Flemming without acetic in cases
where an intenEe stain of various protoplasmic granules
is desired, and why pm;t-chromillg has to be resorted to
especially after Flemming fixation. Flemming, of course,
contains only chromium tl'ioxicle and osmic acid, and while
it gives an excellent fixation, it does not always prepare
the ground properly fO!' staining. The problem of penetration rates of fixatives is, in some cases at let"st, closely
related to thc added effect of the osmotic pressure of NaOl,
which may be used with such mixtures as Flemming,
Ohampy and corrosive formol. There is at present no
explanation of this, and the beneficial effects of NaOl are
even denied by some recent writers.
While there is so much work to be done on penetration
and chemistry of fixation of comparatively simple
extracted organic compounds like globulin, nucleoprotein,
glycogen and so on, the problems presented by the fixation
of cell aggregates and their products by the mixtures like
Ohampy (chromium trioxide, bichromate of potassium
and osmic acid), Regaud (formalin and bichromate of
potassium) or Zenker (corrosive sublimate, bichromate of
potassium, acetic acid) are immensely more difficult. Do
all these constituents of a fixing fluid penetrate a piece of
tissue equally, or does one constituent enter faster than
70
F'IXAT.IO;),T JJE'l'}j(JDS
the otllers '? It seems that n, substance like acetic acid,
such as in Flemming's formula (chromium trioxide, acetin
a,cid and osmic acid), does leave the others behind, and
they follow much more slowly.
Whence the statement t,hat acetic twid helptl penetration.
This is to say, the IlIlClei deeper in the piece of tissue look
hetter than in .Flemming without lleetic. because with the
latter fluid these deeper layers of cells I'(;>main for longer
unfixed in any \ray. Not all fixatives penetrate at the same
ratl~. Some like osmic acid are very slow, others like corrosive sublimate and alcohol comparatively fast. Some swell,
oth0rs shrink tissues, and the good general fixlttive Huch as
BOllin's picro-forrnol-ar,ei;ic gives a balanced result, the
tellllency of one fluid to shrink being counterbalanced by the
eiIeet of ,ulOthel' constituent of the fixative.
It should never be forgotten that in discussing the
etleets of fixatives there are t,yO possible criteria, one, the
actual effect of the fixative, two, the finaJ appeara.nce of
the cells which, having passed through ethyll.1lc:ohol and
xylol, are now ill balsalll. Corrosive sublimate and
picric acid, for example, do not injure fat or lipins, mitochondria, Golgi bodies, etc., and such cell inclusions
appeal' in eells fixed in these substances when examined
in water, after fixation. This can be shown hy fixing
smears of insect or molluscan gonads. But neither
corrosive sublimate nor picric acid is able to prevent the
solution of fats and lipin-like bodies during the dehydration, clearing and imbedding. When it is said that
corrosive sublimate and picric aeid do not fix certain cell
inclusions, this is all that can be meant. 'When, however,
you use bichromate of potash, osmic acid and formalin,
various cell inclusions are brought into a condition which
partly 01' wholly prevents their subsequent solution in
alcohol and benzol, for example. If you fix in. corrosive
sublimate or picric acid, you can (subsequently) postosmicate or post-chrome, and bring about this
desimble change in the fatty cell inclusions. This is
BOP IN';') FLU}D
"j
l
the explanation of Sehridde's method for fntH amI
mitochoncll'in.
When you come to the f01'1na]in ~ilver methods, the
matter is even more ('ornplieatcrl. Da FallO'::; method
(('obalt nitrate formalin, followed hy Hilver llitmt,c, (mel
reduction of the latter) certainly demonstrates a part of
the sperm head claimed by some observers as being
lecithin, even though the material has heen passed
through alcohol and xyl(lL Nor is the usmic af'id method
for the Golgi apparatus llnderstood. It is snppm;ed hy
SOllle to ('c)llsist in tho reductioll of Of:;(\ to a lower oxide.
Chrome-osmium iixati\'es are said to, and npparently d.o,
convert glycogen into a suhstmlCe insoluble in w~lter. AU
fixatives should be usccl intelligently hl eonjundion with
microehemim1 tests 011 smerU'H (§ 34 et .seq.).
105. Amount of Fluid to Use in Fixing, and Size of
Pieces. In l!'ig. 4 is a, ('overed sten(ler dish (seale lllHle1'neath) containing t,vo pieces of tiSS1W.
The amount of fluid ttnd the size of
pieees would he suitable for Bonin,
--=corrosive' sublimate, :Petlrnnke\vitseh,
C~aruoy" Zenker, a.nd Susa" hnt \Vl'Ollg
for Champy, :Flemming-wit,hout-acetic
40,,,,,,,.
RllCl such fluids of low penet-m.tiug FIG. 4. StendOl'
dish eontl1ining
power. Moreover, there is another
fixl1tiY(l and tWl)
serious fault if this diagmm is suppieues of tisRue.
posed to represent Champy fixation-far too much fluid
is being used, it is wasteful. Osmic add, whioh is a part
of Champy and such chrome-osmium fluids, is very
expensive. Cut the pieces very small and just cover them
,vith chrome-osmium. :For Aoyama and similar formalin
:d11'e1' nitrate method::; it would be ll11wh hetter to cut the
pieces shown in Fig. 4 into four. In this ease the
!LlllOunt of fluid is right.
106. Bouin's Fluid. This fluid is ll1uc:h used for chromosomes and fixi1lg smail arthropodB, worms, ete" for geneml
iii
---=:
72
jI'/XATIO.;_\, J.llltTHOlJS
anatomical purposes. It is nut 80 penetrating as Camoy
or Gilson-Petnmkewitseh, which do equally well for most
purpm3Ps. It ,vill not give fat or cell inclusions generally,
hilt is nowacIays used for glycogen (§ 7:3). Unlike some
other well-known mixtures, it, is just as good for vertebrate
tissues as fiJr f1mall invertebrates. It. does not tend to
forIll preeipitates in tissues as does corrosive sublimate, it
need not he ,vashed out well, does not make tissues
brittle a11cl is the best all-round anatomical fixative.
l~xcept for ehromm,omes and kinetic apparatus it is useless in cytology. Its formula is, picric acid, saturated
aqueous solution, 75 parts, good commercial formal
25 parts, acetic acir! 5 p~1rts. ,Vash out in alcohol 50%
or 70%. Fix from 12 to 24 hours. Wash out overnight
in a good quantity of alcohol, but, aR mentioned, it is not
necessftry to get rid of all the picric acid. It dissolves out
later mainly in the sections while staining. Here is an
important modification used both for chromosomes and in
microchemistry: B01lin~Allen, which is the above formula,
to 100 c.c. ofw·hich, warmed to 38° C., you first add 1·5 gm.
chromic acid crystals; then 2 glll. of urea. For fixing,
warm the solution to 38° C. Eor glycogen, etc., do not
bother to add the urea, or to warm.
Lithium carbonate washes out picric acid, if you wish to do
this. Add just a few drops of aqueous solution to the 70%
alcohol containing the pie cos or animals. Another modification of Bouin's fluid ill Dobell's Alcoholic Bouin. Saturated
solution of picric acid in 90% alcohol 15 parts, formol com.
mercial 5 parts, glacial acetic 1 part; add one or two drops
of chloroform just before use. Wash out in 90% alcohol, to
be followed bv Doball's alcoholic iron-hrematin. It is more
destructive, b~lt more penetrating, than plain :Bouin's fluid,
"'-... is us~fnl for cysts (see, however, § 53), hut is otherwise a poor
~lve.
10~nker. 'rhis is a good fixative, definitely better
than Bou~r most purposes, anel really gives quite
promising preI;-arations when the acetic acid is left out.
ZENKRR AND CORR08IVB AURTIC
7:3
It is 2! gm. of bichromate of potash, in 100 o.c. of wn,ter,
to "which 5-8 gm. of corrosive sublimate and ,} c.e. of
acetic acid have been added. Fix 24 hour.s, "wash in
running water for mORt of the following day, transfer to
70% alcohol with iocline (sherry eoloul'), and bring after"
severnl hours iuto 90% alcohol. Tissues stain hrilliantly
after this fixative, iron alum hWllULtoxylin and U nua's
methylene blue and eosin are particularly good. This
fixative has been modifien ~lS follows. Leave out the
acetic acid when the stock Zenker is made up. Befilre
fixation pour 5 C.c. of formalin into a vessel and {ill up to
the 100 c.e. mark with Zenker. 'rhis fluid iR ('allt~d
H elly, Of Zenker-Jormol, iLIld is definitely hetter than plain
Zenker. It is mllch better for geneml histology as it
preserves a good part of the granules in ceils, giving
mitochondria, and Dlany of the details of glandular
secretion and germ cell format,ioll. In Helly, material
may be left for 12-24 hours. Afterwards wash under
the tap overnight, transfer to 70% aleohol with iodine for
several hours, and then into 90% alcohol.
Helly or Zenker are specially indicated for vertehrate
material, and could be used for experimentftl or descriptive work on such subjects as the l11ammalian <:estrus cycle
where uniform results nn a large amount of materif1l are
·wanted. Bouin is also used for such problems, and is
simpler, but not so good for cell pictmes.
Note that many routine methods in pathological
laboratories are standardized for Zenker or formol
mft terial.
108. Corrosive Sublimate Acetic. Twenty-five or thirty
years ago this was the best-known fixative, leaving aside
sllch mixtures as Flemming. Corrosive aoetic Rubsequently beettme displaced by Bonin, which is better and
less troublesome. But there is OIle dass of preparation
which iH best llHLcle from corrosive fixed l1HLterial-the
earmine or hremalum whole mount. There is a number
PIXAl' /oN METHODS
offol'l1mlre for thif; fixative. A general one is the saturated
Roll1tioll of corrosive sublimate in water with 0·5 l%-1 %
glaeinl aectie acid. Flome people use up to 5% acetic
acid. Small piecei'> of tissne, flat worms, pieces of LU111,bricus and similar objeets Jix in about 2 hours. Afterw[l.l'(lH wash the pieces for SOUle minutes ,,,,ith tap ,Yater,
and then acld 70% "'ith severnl drops of Lugol's iodine
solution (§ 71) to make a sherry colou!'. Leave some
hours. If the iodine clecolOlll'izcs you mllst add lllore.
Then bring into cIear 70% for seveml more hours. It,
never hurts to leave such ohjects in 70% or 90(% alcohol.
Then p1'OooO(l to stain as described in § 89. If you want
to make soction8 it i8 far hetter to use Bouin's fluid. One
slight modification of the ftbove eorrosive acetie iH called
Kai8er'8 .tll.dcZ.
109. Heidenhain's Susa Fixative.
'Water RQ e.c.,
corrosive sublimate 4·1) gill., sodium chloride 0·5 gm.,
triehloracetic 2·0 gill., I()rmalin 20 c.e., glacial acetio
4 c.c. 'Wash out in 90(1'0 alcohol with iodine, not in 70%.
Mnch used by histologists. Gives brightly staining
preparat,ions, and as there is little deposit of sublimate,
washing out in iodine can often be left, out. It might
more often be used iU8tead of ordinary corrosive sublimate.
110. Schaudilln's Fluid. Two parts saturated watery
corrosive sublimate, absolute alcohol one part, acetic acid
0·5%. Some people use as much as 5% acetic. The
sine qua non of protozoologists. It is a destructive fluid!
You ,,,ash ont in 70% with iodine (§ 71).
111. Mann's Fluid. Freshly prepared f-mlution of equal
parts of 1 %osmic acid, and corrosive sublimate (sat,uratecl
in water, with 0·9% sodium chloride added). Exclusively
used as fixation preparatory to post-osmicatillg for
Weigl's method (§ 172).
112. Formol, which is formaldehyde gas (40%) in
water, is often a trouhlesome subst:1lloe. The commercial fluid ('ontain8 traces of formic add. It may become
[1'0 R JIA LIS
more acid by subsequent oxidation, and some eOlUmerc·ial
specimens of formalin disintegrate so much as to he
liBeleRS after It short time. It, is recummended to buy
the be8t fiJI'malin, itlld to keep it neutral (pH about (j'4)
either with ('aleiu11l carhonate, powdered and tied up ill
muslin, and left at the bottolll of the jar. or to neutralize
the diluted strength you arc going to use with borax.
Add borax till a good red colour is shown with phenolphthalein indicator.
The best strength is 4% formaldehyde in water, to
whieh if'; arMed 0'75% NaCl. In case this ltlay not be
elear, you (uhl no parts of 1.iJatc)' to 10 IJal'i8 of the conunercial
j'01'lnalin (Jannal) liquid [LIlli rli:::>;olve in it, tJl(J 0·75 gIll. of
Nael (f(mllOI-saIine). 'l'his giVCB "10% ti)l'lllOI" or
"formalin," or .JJ% formaldehyde. When one reads
"10% formalin" in the literttture, it. almost alwftys
means 10 parts of the eommeroialliquid and 00 of water.
'rhe strengths used in Cajal, Dlt Fano and Aoyau1l1 silver
nitmte methods vary from 4% to G% formaldehyde gas.
Thin slices of liver, left three or four days in 4%
formaldehyde, washed out in 70% alcohol and stftinetl in
iron alum hromatoxylin, sometimes give very good
preparations of lllltoehondria and bile ducts. Formalin
is often combined with corrosive sublimate (sublimate
formol, 1 part formalin, 3 parts saturated corrosive
sublimate) and with bichromate of potassiulll (Formal
.:Jhlllel' or Regaud). Sometimes (}75%-O'9% salt is
added. It is vwrth trying.
113. Regaud's Fluid. Very much used nowadays for
mitochondria of vertebrates. It, will give fine preparations of invertebrate neurOlle8, provided the brain or
ventral cord has been dissected out. It il:! a mixture of
an oxidiseI' and a reducer and is theoretically irra,tional.
Nevertheless it works. Regaucl used it for his studies in
spermatogenesis of the rat and got the best results by
mixing 3% aqueous bichromate of potassium, 80 vulumEs,
,(i
F'/X.-ITI(LY MRTHOD8
with j'OI11111Cl'cinl fOl'malin, 20 volullIes. He fixed for
-I days, changing the rlisintegrated flnid every clay. This
is lmneCOHf!ariJy long. Fix overnight in Regaud, mordant;
Ii II' :1 claF: ill j,iehrnmate, dWllgil1g if the Hui(l goeR
elundy. Wash out in rUllning water fur 2{' hours.
nehy~lmte slowly. Stain sections either in acid fuchsin
(§ 154), iron alum hmrnatoxylin or Regaud's special
lu:ematoxylill (§ 147).
114. Kahle's Fluid. An alcohol formol nml- much uged.
It is 115% ethyl alcohol 15 parts, forlllol 6 parts, glacial
acetic 1 part, distilled water 30 parts. Bring into 50%
aJcohol after fixing overnight. A penetrating fluid good
fiJI' hlBlllUlulll whole mounts, etc. It is useless for cell
inclusions. A good general histological fixative.
115. Altmann's famous fluid is equal parts of 5%
aqueous potassium bichromate and 2% aqueous osmic
acid. Some workers use weaker potassium bichromate,
as for instance Bensley, who recommends 2·5%; t,he
~weakeI' biehromate is an advantage, and it may be helpful
to add 0'75% sodium chloride, as advocated by C. Z.
Young, for such fixatives as this and the two following.
116. Flemming with and without Acetic Acid. '1'he
formula used nowadays for chromosomes is 1 % chromic
acid 15 parts, 2% osmic acid 4 parts, glacial acetic acid
1 part: with 01' without 0·75% sodium chloride. Very
small objects or pieces are fixed for from 1 hour to 24
hours. The points about fixation periods are that complete penetration must take place and that the hardening
effect mnst be carried on even after penetration if you
wish to use a watery stain of long duration. For eXQ,mple,
if you intend to use gentian violet, complete penetration
is all that is necessary, whereas for iron alum hrematoxylin
it is more favourable to fix .for a longer period. You will
be using the Flemming acetic formula for chromosomes,
not for cytoplasmic bodies, and there is no object in
leaving the material too long in the fluid. After fixation
]I'LE3lMLva AS]) C'lJAMPY
77
wash out in running water for at least as long as you have
fixed if the pieces are of close tissue, e.ff., mammalian
testis. Botanists prefer to ,,,[((o;h out for 2 hours in tepid
'water, changing the water every ao miuutcH.
For Flemming-withnut-aeetic-ac:id, long fixation if>
indicated beeause tho cytoplasmic granules which you
will wish to study stain better after prolonged immersion,
follovi'ed by a careful washing out. Now if you waHh out
too much of the fixative, the stf1ining il:! not intense, heing
nuclear rather than cytoplasmic. If you wash out too
little the I:-ltaining may he a failure for hoth partH of thc
eell. The best way is to lUINe a number of small piect'H
of material, transferring one ur two pieces at intorvals
during the washing into 50% aleohol. '1'he proper stain
is iron alum hWllmtoxylill (§ 145) used for the timel:-l givell
in § 148.
117. Champy's Fluid. This is a modified Flcl1lmillgwithout-arct.ic-acid, or Altmann, whirhever way fllle
prefers to regard it. It. is a Flemming with hiehromate,
and giveR a more robust Rtain with hromatoxylin than (loes
Flemming-without-acetie. The same result ('an he got,
by fixing in F. W. A. Itud post-chroming. Pix as long as
3 days, and wash out slllall pieces overnight. The proper
stain is iron alum hromatoxylin (§ 145), and the washillgout period may be rat,her critica.l (§ 121) so far as good
staining of eell inclusions is concerned. It is admirahle
for small invertebrates like Archiannelida, and for
sponges. Its penetrative power iR increased by adding
0'75% NaCI, in the case of testis and similar material.
The formula is 7 parts of 3% bichromate of pot!tssium,
7 parts of 1% osmic acid, 4 parts of 2% osmium tetroxide.
118. Carnoy's Fluid. This is the most penetrating
fluid known, and is indieated for cysts, spores, eggs with
impervious shells (Ascaris), ha.rd insects like small beetles,
and so on. It is a destructive fiXative but has a limited
application for chromosome cytology. Glacial aeetic
78
FIXATION .JIBTHOD8
[wid 1 part, absolute HIcohol 61Jarts, ohlorofol'1ll 3 parts.
Leave till penetrated (scveral hOurR) , wash out in 90%
alcohol overnighb. Another formula (Carnoy Lebrun)
contains l'(wl'osive suhlimate, anrl does not keep long.
H is equal parts of the three ahove-mentioned iluidR,
saturated with suhlimate.
119. Mecuro-nitric Alcohol of Petrunkewitsch. Les!';
rapidly penetrating than Carnoy, but also good for small
insec.ts, cmstncea, etc.. vV~1ter 3110 c.c., [Lbsolute alcohol
200 c.c., glaci'1l acetic no C.c., nitric acicllO o.c., suhlimate
to satumtion. After saturation ill corrosive, filter.
Undergoes Home disintegration after a time, but still works
well.
120. Key to the Use of Fluids.
To make sections of
sUlall inve'rtebrates such as Polyzoa, flatworms, sponges,
the more delicate Crelenterata, small embryos and ohjects
are (lCCe88ible to the fluid, use ChamI)Y overnight;
Rtain iu iron alum hrematoxylin, acid fuchsin and methyl
green. Champ}, is quite useless for vertebrate embryos,
e,g., human ernbryos. large lumps of tiHsue, 'whole mounts,
and impervious objects like insects. No piece of tisRue
should be more than :3 llllll. thick. If possible prick or
tease the objeets with needles after they have been put
in the fluid. For 'whole mounts use cOlTosive acetic or
alcohol.
For vm"iebmte tissues generally, use Zenker-Formol or
Hegaud, or if only good anatomical fixation is needed, use
Bonin or Susa. The same a,pplies to the invertebmtes
mentioned above.
For carmine or other whole mounts of vertebrate tissues
(e,g., injected material) use corrosive acetic or Sus a if
possible. For impervious things like inseots use Petrnnkewitsch or Carnoy. Stain in Borax Carmine or Acid
Hremalum. Insects like aphids do best if pricked with a
fine needle after fixation : they wash out better and stain
hetter.
~\\'hich
WASH ISU OfT1'
i!1
,For uhrolllusolIWS begin with p.lltill HOllin Hnd imll
lucmatoxylin. You may later try ]i'lelllTlliug (with aceti,~
acid). Navashin (plants), and, (If ('OUl'SO, the Heoto-cannine
I"lmear. To :mm up: the heflin'll!"/' l'ihould U:;l' Bonin for
sections a,wl corrosive sublimate acetic for whole mounts.
121. Washing Out Fixing Fluids. 'rho beginner is
usually puzzled as to what to do "bout this. As it rule
after chrome fixatives material lllust, be washed uucler a
running tap overnight, provided they hiLVe been fixed at
leaf:;t overnight. This applies to slwh l1xatives as Regau!l,
Zenker, ReIly, Altmann and _I<'l('mming. Fixativell not
contaiuing chrome salts, hut having eorrosive sublimate,
must be washed out in 70%) alcohol, to whieh enough Lugol
solution (§ 71) has been added, to make R Hherry colour,
Fixatiyes containing picrie acid should he gut into 70%
!tnd transferred to 110% fairly SOOll. \Vith fixatives
eontaining tl'ic:hlomeetie :'LCid pie'ees of vertebrate material
:-;hould go immediately into tlO% alcohol. Carlloy
material (without corrosive :-nlbliuHtte) gOOR fitl'itight into
90% or absolute aleohol, or the formula with (;01'1'0::;iV8
sublimate into 90% with Lugol llS above.
In the case of picric acid fixation, water-soluble picmtes
form (though this has been denied). 'Whatever may be
correct, it, is true that, solut,ion of part of the ground
cytoplasm takeR place if Bouin materia.l is left too long
in ,vater or watery stains.
But this is not all, especially with the chrome-formalin
and chrome-osmium fixat.ive". Th(~ fixatives nULY contain
some substance necessary to morda.nt the stain--in plainer
terms-to make the stain bite deeply into certain part,s
of the cell. 'This apl)lies both to iron a.lum hrematoxylin
and acid fuehsin staining of mitochondria of a.Il animalH,
and to the Golgi bodies of invertebrates. Thus, if you
wash out too mueh the subsequent staining is unsuccessful.
122. Method of Washing Out. This is usually done
under the tap, and presents no problem if the pieces are
prXA11lON JlfErpHODS
large. With it rubber band attach a piece of ll1uHlill on
top of 11 pet.ri dish and plaee under the t~tp. For a, large
number of pieces which are to be kept, l:Ieparate. cut some
f;hort lengths of wide glass tube, tie muslin at. both ends
with the pieces and their label (written in pencil) inside,
place in a short, narrow j,Ll', and leave uncleI' a good stream
of water. It is well to see that the stream is adjusted
before you leave at night, and that the ,vater is not turned
off overnight, as Homctimcs happens.
For numerous small objects \,...hioh are not very valuable,
wURh (lut uuder tap, using finer muslin, or sill{, hut for
vnJuablc objects wash (Jut with distilled water in a petri
dish. Remove the water from time to time with a
pipette. Small object;; obviously wa;;h out more quickly,
and call be done properly in a few hours by this method.
If impermen.ble, wash (Jut in tepid distilled water.
rl'he bot!1llists usually prefer to wash out their Flemming
fixed material in tepid water. The water is changed
every 30 minutes, for 2 hours. This treatment is quite
good and might be tried more often by zoologists. It lllay
have to be used if t,he water supply is turned off during
the night.
CHAPTER VII
PARAFFIN DIOXAN N-BUTYL
ALCOHOL AND CELLOIDIN IMBEDDING
123. The Paraffin Wax Imbedding Method iR almm;t
universally used. The fixed and dehydrated material
is passed into an oil or other liquid which clisHolves
paraffin wax and finally into meltcd pure paraffin \vax,
which permeates the tissue. Heat mUilt he URed to melt
the wax and Qil wax mixtures, and this nee:essitates some
form of irnbedcling hath, \vhinh can he cheap and primitive
l1ud yet satisfactory (Fig. 5, A), or one of the spccin1 copper
or wooden incuhators made eSIJccially for the purpose.
Recently a vcry interesting incubator has been dc!:dgned
hy Craig-Bennet 1; it costH five or six shillingi:l, and is
shown in Fig. 5, C.
The advantage of the \vax imbedding method is that it
is cheap, easy, and best for subHequent section cutting.
On the other hand, it causes shrinkage, hardcns the
material somewhat, distorts thin embryologieal and other
membranes, and is unsuitable for the study of fatty
granules.
In l'eeent years the general method has hcen much
improved, ((t) by using instead of the higher dehydrating
strengths of ethyl alcohol .'lome other alcohol i:;Qch as
butyl alcohol, (b) by adding ceresin to the wax to l)revent
crystallization on cOO~illg and to toughen the mass
(§ 124), (c) by imbedding in dioxan (§ 136), (d) by combining the dioxan and eeresin methods. vVhile these
improvements are decided advances, they do not get over
the fact that the wax medium may cause distortion of
l In the C.B. incubator only the top layer of the wax melts, thus ensur·
ing that the pieces being imbeaded do not o,,:crheat.
81
PA R.t [1'/1'0,' j)/()XAN E'f'c.
j,hill 11ICmilrn·lIeK --whcr(m~ t Ill' celloidin method i'i1l)l]Jorh.;
t.lw memhrane!:'. r~OlUpal'p, t(ll' lm.;t-anee, l,mphihinn
__
~N
~==;~=""~"=='=~"
A
P
··-6R
--
co
i¥--:'1o/-----./ B
B
'·H
loow.
c
-MS
/MT
• T4
FIG. 5.
A. Drying plate, or pdmitive imbedding plate made from
Rh"et of {'opper (P) bolted. at N to four threade!l l'()(l~
(BR). Height ean be adjusted by loosening bolts.
B. Imbedding small objects in a silver paper COllO (CO), supp01't,eel ill it stonder dish with a blook (B).
C. The Craig-Bennet Imbe<lding Bath. ('ollsisting of H. tlll'e.,,ply woollen box, with a lift-up front hingeel at H, vent;
holes in top at V, IOU watt lump nnll flhade (MS). tlmw
tray (MT) of wax-filled tubes (T.J-), 8witdl at, S. flex F.
_Hf1at.f; up ill ] 0 minutes. (By (:Ollrtesy of the Pmsident HIHI
Coundl of the Hoyal Mif'roflcopi"al Soddy of LondoIl.)
embryo sections cut from celloidin wax imbedding, or
from plain wax. The former show all the incipient spaces
and membranes in their natural positions. But for all
IMBElJDINO
general purI)oses the \vax methorl hils come to stay, and
suffices.
The main filUltS to avoid, and pnints to attend to itl
wax imhedding are: (a) '1'0 see t.hat tIl£' material has heen
properly dehydrated and is permeated with oil lwfore
being placed in the fil'f'.t oil wax hath. (b) To Hvoid
uuneeessa,ry heat in the pl'OCef'B. (c) To infiltrate fl.lowly
hut thoroughly, judging the t.ime according to the sizes
of the pieces or animals heing imbedded.
In the case of (a) the Wf~X will not penetrate if t,Ile oil
has not permeated the material previously, and in the
end the hIllel\: or animal ma.y fall out of the 'wax when
being cut, or may have 80ft patches in it whieh will
prevent cutting. This is extremely important" and It
common ±:'lult. It is, of eOlll':o:e, traceable back t.n absolute
alcohol cOllt.amina,ted with too much water, or to hnrry in
dehydrating. In the rase of (fJ) yon will make Home
tissnes like stone by overheating them. It is had for yOUl'
knife. (c) Olwionsly a large piece of' It testif' or gland,
rather impermeable material at best, or an art,hropod
with only'the mouth, anus or other poreH through whirh
the wax mixture may penetrate easily, needs careful
infiltratiol1. A frog's eggs manifestly, or a sumll organ like
a fi:og's kidney, will be penetrated more quickly. There
are no, definit.e rules, t,rial and error will be your guides,
but this much may be SfLid : if the pieces of tissue or
embryos are no bigger than 10 mIll. square, 1 hour on the
top shelf of the imbedding bath in equal parts of carbon
bisulphide and wax, followed by 2 hours in pure WILX OIl
the bottom shelf, will imbed most, t.hings suecel:lsfi.dly.
But if you are a cytologist. you Hhould not try to fix pieee~
as large as 10 111m. square, and if you are a histologist,
cleanly-ent sliceH 3-5 mm. thiek Hhoulcl be enough for
most pl1l'poses. Using such thinner pieees 1 hoUl' in the
first mixture <tud 1 hour in wax is enough. Some Htudents,
however, lw.ye enriolls i<leaR on dehydmting, whieh Illllst
PARAFPIN DIOXAN ETC.
he dOlle properly for successful imbedding. They take
a row of graded alcohols ill petri dishes, and leave pieces
of tisfme 5 minutes in each. This is useless.
Now, assuming that you have cleared the pieces as
descrihed in § 18, you now proceed to imbed them. If
yon are working in it laboratory there are likely to be
some p(':tri dishes possihly ncar or inside the oven, whieh
have previously .been used for imbedding. You transfer
your nmtel'ial and enough oil I to eover it into one of these,
seeing that it is free of water. The next step depends on
a number of things. In some laboratories there is kept a
" IRt wax oil " mixture, which reany consists of the old
WiLX I1nd oil poured a;way from previous imbeddingR.
If this is cold, it will lJe possible to remove some from its
dish with a sCl1lpel fwd [LCW it to your own capsule. The
fLmount, you 11<1(l does not matter very much, but it
should not be too lit.de, and you want to be sure that
yonI' pieces of tif'Rue are not pushed high and dry above
their oil. "When this is done put the dish on the top
shelf. Before going away you must Ree that the next
bath of pure wax is ready; it has got to he melted.
Possibly there is ,1 metal dish of "pure wax," already
melted, in the oven. Regard it with suspicion lintil you
are sure that it is pure wax. Dip a dry knife cir match
into it and cool under tall. If it goes properly hard it is
pure wax. If not, you will have to chip some hard wax
into another dish and put it to melt in the bottom of the
oven, 01' possihly melt it ca?ltiously over a bunsen. After
! hour, pour off the melted liquid (wax. and oil) from
your clish with the objects, and pour on the pure melted
WI1X. Uthe first clish of paraffin wax and oil after i hour
contains lumps of unmelted wax, you must put the dish
in the bottom of the imbedding oven and wait longer .
.Tl,le oven certainly is not at the proper temperature, and
is not suitable for imbedding. Going hack to the first
1 l\'[(,I'C11y
it
('OIlVElllil'nt
tt1m)
for whatevor wttx Bolv(1ut you aI'C uBiug ..
IMBEDDI~YO
step, if there is no wax oil mixture ready for you beside
the oven, you simply chip some (ll'Y, hrrrcl wnx into the
(liRh with the oil and ohjeets ami ipt it llW!t, 011 the top
Hhelf. On the othel' hawl, if there iN nwllerl wax oil
mixture ready in the oven YOll :-;honld place yOUl' !lit'h
and ohjects, with oil, on the top shelf' and warm it before
pouring ill the wax oil mixture. If you are :mre the WILX
oil mixture is about half-antl.half: you can pour off yom'
own oil before adding the mixture.
·When an hour or so ha,s elapsed, and inspection has
shown that the pure wax in your imhcrlding capsule is
liquid, you may proceed to eool the wax as follows. Take
a flat piece of glass 3 or 4: in. square, rub it over ·with a
little glycerine on one l:;i(le and p!tWli two pieces of flnt
metal together to make it cell, aH shown ill Fig. a. These
pieces of metal should he about 20 liun. high amI
40-00 mm. long, with 15 111m. at the end bent around at
right angles. They should be about 2 mm. thick anel
perfectly flat. Yon can make Heveml from pieces of Hat
hrass so as to give different sizes. Now quieldy pour the
wax into the mould, and with 11 pair of forceps warmed
in J;I, flame fLrrange the piece or pieces as you wish-say in a
row or closely placed together; remembering that the
sections must be small enough to go on !t 3 X 1 slide, the
closer together the better.
It is a good thing to have a bunsen or spirit lamp burning
nearby in case you are too slow and the wax hardens
before you can an'ange llmtters as you wish. By heating
an old scalpel and dipping it in the mould you can keep
the wax melted till the operation is concluded.
Now breathe lightly on the top of the wax till a film
forms, then gently lower into a dish of cold water. If a
long string of wax shoots up to the tal) of the water the
film has not been made thick enough. Leave for at least
I hour, till hardened throughout.
124. Wax and Temperatures. In warm climates, or in
Kli
PARAFFIN nJOXAiV E'l'C'.
summer. it. will be necessary to use a harder wax than
in eolde~' climes or in \vint~l'. '1'he usual melting poillt
i:; 52° C. Due to the work of Higgs, "\Vaddington and
Kriehel, we know that t.he addition of ceresin, which ii'1 f1
vcry hant wa,xy substanee, giveR the cooled wax a fine,
hard, miero-crystalline texture. In fact the hardness can
be controlled largely by adding more or less ceresin. If
YOll are just. doing ordinary work do not bother about
ceresin, but if you wish to out such objects as feathers
in skin, very yolkS eggs, 01' insects, try ·wax ceresin
mixtures. ·Waddillgton says that even 0'5% ceresin is
advantageous: in pmctice you wnuhl use higher ratios.
EspinaHse haR used successfully equal parts of "\Voolworth
eandles, paraffin wax 52° C., and ceresin for cutting bird
skin (1ncl developing feathers. If yon are planning to
cut very hard objects try ~c()lllbilling the dioxan (§ 13G)
and ceresin wax met.hods.
125. Cutting Sections. There are two types of Rectioll
{Jutting, one ea,sy, the other difficult. The first consists
in merely getting some sections of material like a gland or
genitalorgrLll (histology). The other kind is serial section
{Jutting in which all the seotions must be cut the same
thickness, and none lost (embryology, etc.). The first
thing in this latter type of section cutting is a good
microtome-that is, a rigid instrument which will cut
consecutive sections the same thickness. Not all micl'otomes will do this.
We will take the first and easy type of section cutting,
sa,y of a piece of liver or testis. The material has been
imbedded as described in § 123. The trimmed block of
wax CH'ig. 6., A) cuntaining the piece of tissue is to be fixed
on the special table (Fig. 6, B) which clamps in some way
011 the microtome bar which moves the block towards
the knife. The table is removed from the microtome,
and if the ribbed surface is free of wax some of the latter
is melted on to it with an old scalpel. rrIils makes the
CU1"P];..Yf/ SEC'l'/()X8
K7
:-;eat for the block. You now trim the latter. The iil'l.;t
thing to notice iR that the block mtl:st not Il(, longer than
1 in. on the side \vhich strikes HlP knife. and it, shuuld be
le8s. hecause, presumably, you arc going to mUltnt thc'
sections 011 a 3 ;< 1 slide, an(l you lI1U~t not !llwe t,he
H
AEl}'"
;.
l.:~
_
D
~
L
j
~B
m
K
1:-O(OICI:I~
G
o
rc?1
xUx
sect.ions as broad as an inch or t,hey GI1111l0t then he
flattened easily, using routine methods.
Inspect the hlock and ta,ke one side whieh halO a good
depth of wax as the siele which is going to be stuck on to
the microtome table. TIlis process is done with a hot
knife and there must be enough wax for this. Having
determined what side i:-; to be used for this purpose, trim
it slightly first, of alL and ~tlbseqU!mtly lJegin to trim t1w
PARA]i'Ji'lll DIOXAN E'l'U.
other sides carefully. Do not attempt to cut too deeply,
just shave a little at a time, using a stiff safety razor blade
or a good 80al}le1. At the end you should have tL block
as shown in Fig. 6, A. Fix this on to the microtome table
with a. hot knife, and dip into cold water. Inspect it and
Hee that the two sides which come down parallel to the
edge of the knife are pa,l'allel to each other. If they are
llot parallel, the ribbon of sections will turn to the left or
right QlS :;;hown in Fig. 0, M. In :Fig. 6, E, presumably the
block is to be fixed on the table with the hot knife at X.
But there is not enough wax here for the purpose. Compare with Fig. 6, A, which is correct. In Fig. 6, H, the
block has been cut too long. It will bend or vibrate too
much as the knife strikes it, or it will break. Fig. 6, I,
shows what should be done in the case of a long object;
there is plenty of support here, ,wd the lower part can
he trimmed clown later when the upper part is cut. In
:Fig. 6, ,J is a chain of three sections. There are two faults
here, hoth causeel by the fact that the block has not been
cut properly cubical and parallel. Fig. 6, 0, is one
section with the part X wasted; this should have been
trimmed off the block. Fig. 6, K, has white lines in it,
caused by pieces of grit having turned or broken the edge
of the knife. The end result is often what is shown in
Fig. 6, F, where the sections have split completely.
Fig. 6, L, seven sections cut on a defective microtome,
or on a microtome in which the wax block was insecure
or the knife blunt.
Having st/uck the block on to the table by running a
hot knife between A and B in Fig. 6, you are now ready
to cut sections. Put the safety catch in operation on the
microtome, and fix the holder in place, hut do not. screw
it tight until it is pushed in far enough to be Btruck by the
knife when you release the safety catch and lower the
moving bar towards the knife by gently turning the handle
(in n Minot microtolne). When the moving hal' is let
SERIAL BEUTIOH8
down, hring it up until the wax block ean be moved HIl
against the edge of the knife. See that the sideR are
parallel to the knife edge. Raise the moving hal' to it"
upper position and let in the safety eateh. Nnw damp
up the table holding the wax hlOl:k.
On all modem miemtmnes there is f!ome indic:ator fur
thickness; set it to 1O/-L, and begin tll1'lling the microtome
wheel until sections are cutting. Continue turning untit
the whole face is being cut. Stop awl brush off the
sections already cut. Ul-le it pointed brush, muil':!ten it
now in your mouth, cont,inne turning the wheel, and try
to catch the first few :;e(Jtions by putting the hrush under
them, and by genMy lifting them just above the knife,
you Rhould get a chain 01' ribbon of sC0ti0l1B. RoaneI' or
later, and mmally sooner, the ribbon broaks, and the piece
adhering to the brush must he laid on a sheet of foolscap.
If the ribbon keeps breaking yuu mnst inspeet the knife;
it may be at the wrong Hlope. If the hlock clears tho .i<nife
below the edge as it descends the fault lies ''lith the wax
block. It is too hard, and must be faced with softcr wax.
Turn the ·wheel up and put in the safety (lateh. Melt, a
fragment of softer wax on a scalpel and gently face both
upper an.d lower sides of the block; see that the knife is
just hot enough. Now try again. TJsuaUy this ,..·m cure
the trouble. If the sections keep curling up, it uSUl111y
means that the knife is hlunt.
126. Note on Serial Section Cutting. The microtome
should he of a rigid. pattern and the bolts which clamp t,ho
block holder and the knife in position well screwed up.
The block will have been carefully trimmed as in Fig. (I, A.
If the microtome eutt:> altel'l1ate thick and thin sections
(Fig. 6, L), or sueh varying sedions fairly dOl:'e together,
the hlocK if' loose, or the knife is not damped, or the
mechanism is out of order, unoiled, or it may want
cleaning. which can be done in xylol. If the mechmdslll
is fouled with 'Nax it obviously will not, work properly.
PA HAPFL\' DlOXAN ETO.
On the other hallfi. you should note that if yon out a
rib hun at a qnick lJa~e and slHldenly stop or slow down,
the next He(:tion will. be thinner because the wax block
hal'; warllled and expanded ·whi.1e it was being struck by
the knife, fwd in the interval cooled. Any differences in
temperature will show in the sections. Therefore, if you
have a good ri1hon coming off, and have got to the stage
whell yon are going to Rtop, do so gradually, and when
you begin again the fi1'8t sedioll will not be so different
ii'Olll the otht,l's. In all enRes turn the handle evenly and
;;teadily. Sometimes t,l18 ribhon hecomes electrified, and
ilies to\\'anIR, and sticks on to, objects nearby. Various
elallol'ltte methods to de-eleetrify the ribhons have been
sngge::;te(1. The l)eRt method is to go home fl,nd try again
next day.
127. Cutting Sections without a Microtome. By putting a
piece of jhcrl tissue taken from water or 50% alcohol between
dderherry or such pith, fairly good section;; can he cnt "ith
a RhM'P razor or a new safety razor blade. ]'01' histology these
spctiOlHl will do quite well. Put into a l)etri dish of 50%
tdeohol or water and select the thinnCloJt for subsequent treatment. For staining, dehydrating, etc., the seetions are stuek
on !t slide with lVIayer's albumen or gelatine (§ 95). }tuh
H smaU drop on the middle of the slide, press the section down
on it with it cigarette paper or JitMe fine filter or blotting paper,
:flood gently with 90% alcohol which 'will harden the albumen
anclmake the section adhere. The slide can be htmdled quite
sllecessfully if you are gentle with it.
128. On Mounting Sections. 'Vax sections are stuck on
to a 3 X 1 slide by glycerine !1ml albumen (Mayer). If
you have no glycerine and albumen at hand, use distilled
water instead, in which caf!C the sections do not stick so
seem·ely. To make glycerine and albumen, l'emove white
of egg from yolk, add an equal quantity of glycerine, stir
up with It domestic fork so as to break the large lumps of
albumen, add I gm. of salicylate of sodium dissolved in a
little wo,ter (this is to keeJ) the material sterile-yon can
use im;tead a crystal oHhymol dropped in the store bottle).
MOUNrfllNO SIiJOl'JOX8
Bl
When thoroughly stirred up, filter through a coal'se filter
paper. This will take days., and the funnel must be
covered.
To use, allel 10-15 chopi':! to 50 C.c. of distilled water
(or good tap water), Rtir. Take the brush you have used
for the sectioning, dip it in the mixture and flood. the slide.
Rub the brush backwards and forwards and see that the
slide is properly covered with liquid.
Now, when putting a length of ribbon OIl the slide you
must keep in mind what size of coverslip you intend to
use later, and remember that the ribbon stretches when
flattened by warmt.h. 'rllUS yon will havc to cut the
ribbon just a little shorter than the coverslips you are
going to use. Suppose you are mounting a large series of
sections by this method; having determined the desired
length of ribbon, you can count the sections in each length,
·which is tedious and sometimes clifficult. I ahntys use
a matchstick impaled in the middle on a mounted needle
(Fig. 6, G). The stick is previously .cut the right length.
The tYlle of scalpel to use for cutting the ribbon is shown
hl Fig. 6, N; it should be sharp. Pick up each ribbon
length with the moist brush.
When the ribbon (or ribbons) is in position c(lliliously
warm the slide over a flame, tilting and agitating the slicle
from time to time, so as to keep the warmth evenly
distributed. The sections will begin to stretch out and
flatten. When they look quite flat, set the slide on the
bench to cool for a moment, then cautiously tip the
extra albumen-water off the slide. Place ill a warlll place
to dry.
129. Drying Plate. This can be made of an iron or
glass plate on four legs (Fig. 5, A), underneath which is [L
tube with an aperture for a row of very small gas lights.
The plate must not be so hot as to melt the wax. Some
people use an electric bulb in a flat glass-topped box for
this purpose. This may become rather expensive.
.:\Jl()tilcl' way iH to put shelves near the hot-water cisterll.
Tn summeI' it usually Hufliecs to placc the slides end up
,~gaim;j:, the h,tl',k of the h(1)(·h. On a hot, l)late they Itre'
rlry next morning.
130. To Deparaffin Slides. The dried Blicle iH yently
warmed over a flame (without melting the wax), (a) to
remove moisture, (1.nd (b) to enable the xylol to dissolve
t.he para:lfin more easily, and then dipped into xylol or
henzol for a few minutes. When taken out it is dra,ined,
and it::l back "\viped ,,,ith a dm;tel': It is then ilipped into
tH) %OJ: absolute alcohol, and is ready to pass down through
graded alcohols to the stain bath.
If at any time you are not sure on which side of the slide
the sect.ions lie, hold the slide sideways towards the light.
131. Microtome Knives. For students these ha.ve been
largely sUllcrseded by safety razor blade holders, which will
do all that is necessary except cut valuable or heavy material
such as large embryos, etc. Some people buy a heavy
straight ground razor (difficult. to find nowadays) and use as
a microtome knifc. These and the heavy microtome knives
can be sharpened in the laboratory, but the process is not
easy, and certainly cannot be done properly by an unskilled
persall. :iVIicrotome knives generally need re-grinding after
cutting anill\als containing grit-e.g., tadpoles or worms, and
such animals should be fed on meat, onion or potato, or
some such soft material, before being lalled for sectioning.
There are on the market microtome knife sharpeners consisting of ioul' surfaces mounted on an oblong body with ,11
handle. Each side has either a stone or a treated leather
surface. You begin on the stone and stroke the knife on
succeeding finer sides. Mier practice you may learn to
sharpen a knife properly.
132. Thickness of Sections. For general purposes,
lOp, is the right thickness. This for two reasons; firstly,
it is thin enough, and suits the times given for staining;
secondly, it is a good ullit for measurement, or in case
you intend to make a model in embryological work.
For cytology (chromosomes) you may have to cut sections
which will contain whole cells and be no thicker. This
IMBEDDING SMALL OBJEOTS
(13
will depend on the size of the cells. For the cytology of
the cytoplasm and such work, very thin sections (3-5IL)
are usually cut.
133. Handling Protozoa and other Small Objects.
This can be a tiresome business, especially if you have only
one small object to be cut in a special direction. For a
large number of Protozoa, embryos, or eggs, the task is
easier. If you have no hand centrifuge, you can usually
manage as follows. Kill the animals in a test tube or
centrifuge tube, using a fixing fluid like Kahle's fluid
(alcohol formol) or Cal'lloy in which they are almost
certain to sink. Of course the killing can be clone in a
stender or petri dish, and the individual organisms picked
up in a pipette, and, as a matter of faet, most things will
sink in the usual fixatives. After ihatioll is complete,
remove from the test tube as much of the fluid as yon can
with a long pipette fitted with a rubber tea,t. Now add
the washing fluid, be it 50%, 70%, alcohol (for Bonin, etc.),
or water (chrqme fluids), or 1)0% (Cal'lloy with sublimate).
When the organisms settle, remove the liquid agnin and
replace with fresh water if further ehanges are necessary
(chrome-osmium, etc. (§ 121)). Finally, earry the
organisms up to the liquid to be used for imbedding
(carbon hisulphide, xylol, etc.).
Now prepare a lead foil cone (from the" silver paper"
from cigarette llackets). Take a piece of round wood, like,
but somewhat thicker than, a lead pencil. Sharpen it
to a smooth point, so that when a sUlall circle of foil is
pressed on it, and taken off, you have a neat "watertight" cone with smooth sides. Prop this up in a stender
dish with some small stones or pieces of metal (Fig. 5, B).
Now pipette the organisms with the oil into the cone.
Remove excess oil and add cold soft W~tX and place in
oven. Leave 20 minutes, with a warmed pipette remove
as much as possible of the oil wax mixtul'e and pipette
in hard wax. Leave 30 minut,eH. Change the hard wax,
PARAPPIN DIOXAN E'1'O.
aftor ] i'l minutes if you think it necessary. Remove from
the oven, and gontrly run cold water into the stender dish
to eool the paTaffin. Hemove the foil, and you have
the organisms on the top of the wax cone .. Now take a
nullo of hard wax about the size of ,1 piece of loaf sugar.
Cut a little hole in one side just big enough to take the
tip of the eone, which you cut off and dry on a filter paper,
if wet. v\Tith a, hot wire melt around the hole you have
made in the cube and drop in the tip of thc cone. Run
the wire al'olUHI it just enough to melt the outside of the
COlle.
Allow to cool. Trim the block for sectioning.
Anotlwr method much u;.;ed, but not m~arly so good, is to
hrillg thn ol'ganisl1m into a sml111 quantity of 70% alcohol.
Take It uuhc; of fb:er11ll'11in or liver, awl in a smooth part of it
malw n hole, and pipette tho organisms into the hole. COVN
the hok with a drop of egg albumen, and gently lower the
hlock into ()O~'{J or ahsolute alcohol, whioh will coagubte the
albumen ILnd sE-al tho opening. Some people simply tangle
up the objects taken from 7()% in It drop of albuDlen and
cover tht> alhumen with 00% alcohol. Anot,her method is to
flatten it drop of albulUen on a slllooth flat piece of liver 01'
twain. Pnt the object from water on it and remove excess
water 'with a pieee of filter or blotting llaper. Now gently
flood with 70% 01' 90% alcohol.
.
Now snppose you wish t,o orient a small object so that
it can 1)6 cut in a definite direction, you may use a modification of the above method. Cut a neat slice of the liver
or prefemhly brain (taken from 50%) alcohol), and fashion
it into a thin rectangular plate. The objeet (also from
50%) if; lllaced on this moist phte at right angles or
parallel to the shorter sides, as YOll wish. A little egg
albumen is run elll gently, and when l1Jl seems to 130 well,
the capsule is cantio11sly flooded with 00% alcohol. When
you are subsequently trimming the block fOT cut.t.ing you
will know which dil'eut.ion to cut.
Fur ohjects a,bout as big as tadpoles orientation can be
done fLt the end wheu the hot wax is poured illto the
L'lELLOll)JX
.~IN'/)
()()~
nil
mOlild. UHe hot needle:,;, and keep rt Hc~dppll't'ad.v io hea j,
in ease the wax starts to (iool too fJOOll.
134. Frozen Sections. ThesE~ are IllU,ric hv freezing
fresh or fixed (usually J(H'lllalin) lllHterinl ill C()2 gill';, ar~
much used in :Pathology labomtories, and are not really
very necessary except f()l' certain work, such as microchemistry. Even here the teased preparation may do.
The advantage of frozen sections is that perfectly fresh
unfixed material may be cut immediately without imhedding, a useful and quick \yay when diagnosis of a tUlllOUl'
must be made in a short time. Suppose, however, you
wish to Htndy ovarian cells by microchemical methods, the
frozen section must be used, and simil~H problems sometimes arise. Descril1tion of the method is outside the
scope of this book; refer to the "Microtomist's Vademecum," or some such book.
Note. vVhcn buying a freezing microtome Fl.ee that it
I;; provided with a kllife-eooling attachment.
, . 135. Celloidin Technique. Celloidin (gun cotton) if!
soluble in alvohol and ether, and is hardened by expmmre
to chloroform. 'rhe object is bl'Ought into absolute
alcohol, thence into a tkin mixture of dried cel10idin in
ether and alcohol (I : 1), then after a few hours placed
into a thicker mixture. After several hours (01' better,
days) the celloidin mixture, which should be just thin
enough to pour, is ti11ped into a paper mould and the
latter placed in a properly covered vessel containing a
fe·w drops of chloroform. The vapour stifl'ens the celloidin,
which can be then removed from the paper mould,
trimmed and transferred to 70% alcohol containing a
fe·w drops of chloroform. Or, the block may be tmm;fened
to chloroform just after the hardening in chlorofor111
vapoUl',· and paraffin wax subsequently chipped into the
vessel. Imbedding is clone in the ordinary way, chloro,form being the medium instead of xylol. The eelloidin
method is not very lUuch used for ordinary WOl'k It
PARAPPfN n!OXAN E'PO.
may Imve to he learnt. for Rpecial l'efiea.rch. ~Refer
HlP "Miel'otomist'r; Vade-llIE\(·.\lm;' 01' f'ome such
hook.
136. Dioxan Imbedding. Dioxan is !l cumulative
poison in I in 1,000, musing leeions of the lungs, liver and
kidneYR. It is quite ?lnsuitable for C0118tant use in 81nall
'I'OOUt8, especially when it is heated during imbedding.
However, knowing its poisunous qualities it is possible
to me it without harm. It. is a very remarkable solvent.,
dh;solving 01' lllixing freely with water, alcohol, xylol,
p[tmtIill wax, celluloid and balsam. It will dehyclrate
material, "\',hich can 8uhf:'eqnently pas!:> straight to l)araffin
wax f(JI' imbedding. It causes 110 apparent shrinkage.
Materials dehych'u,ted ill dioxan ean be mounted in dioxan
Imlsam. It therefore does away with the necessity for
uRing xylol or similar dearing agentR.
After iixatioll in Bouin, Formol, Carnoy, bring straight
into dioXllll; after COl'l'osive sublimate fixative:; it is
better to wash out in 130% or 70% 'with iodine, hut some
people add it crystal of iodine to the dioxan. Chrome
fixativeH are washed out in water, and then transferred to
dioxan. Dioxan mixes gently with water, and after
three changes of dioxa,n during the day, leave overnight
in pure dioxan, or use it dehydrating jar of dioxan. This
is a wide-mouthed short jar with a good ground glass
stO}lper. A piece of gitUZC is cut in the form of a star,
and the points are bent over so as to make a table. This
is thrust into the bottle. A small bag of calcium chloride
is arranged underneath the gauze, and dioxan is poured
in. Pieces to be dehydrated can be put on the gauze
with It pair of long forceps. 'rhis is the cheapest way of
nsing clioxan for dehydrating. Subsequently the pieces
arc put in a 1 : 1 mixture of' dioxall and wax (30 minutes),
and pure wax (1 hour) according to sizes of pieces. Yon
should notice that all modern staining methods are
designed for alcohol dehydration, and it may be necessary,
tu
B[T'PYL ALonHOL
!l7
if you u:-:e diOXltll, to modify tJw times of clifff1l'cutin.t,icnl in
Nome eaHes.
137. N-Butyl Alcohol Imbedding. Tn l'e,tlize the
hardening effed of ethyl aleohol it is unly ncnpl'sa.I'Y to
leave a piece of liver in it for 2 or :; dttys. N-butyl alcohol
has no such hardening effect, but it absorbs very little
water and part of the dehydration must be done in ethyl
alcohol. 'The most recent method (Stiles) for this technique is as fullows: From 30% ethyl alcohol the tissue
is placed in a mixture of D parts of 45% ethyl and 1 part
of N-butyl alcohol. After about 2 hours, place in an
8 : 2 mixture of 63% ethyl and butyl alcohol (2 hours),
then in a 6·5 : :3·5 mixture of 77% ethyl and butyl alcohol
(4 hours), then in a 4'0: 5'0 mixt,ure of' 90% ethyl and
butyl alcohol (6 hours to a day) and finally in i1 2'0 : 7·5
mixture of absolute ethyl and butyl, where it should
remain overnight. Now the tissue is placed in pure
N-butyl alcohol w·hich should be changed twice during the
course of several hours. Tissue may remain in N -butyl
alcohol for days without deleterious effects.
To imbed, tissue may l)e taken from N-butyl alcohol
and placed directly in melted paraffin, but it is better,
perhaps, to use an intermediate step of 2 parts of paraffin
and 1 part of butyl alcohol, leaving the tissue in this (in
a closed vessel) for 20 hours. Then the butyl alcohol
may be allowed to evaporate, or the tissue can be placed
in pure wax. In any event a long period of infiltration
is essential, 4 01' 5 days not being too much.
N-butyl mixes with dioxan, and so far as I am aware
the possibilities of combining the two techniques have
not yet been tried. It seems likely that the N-butyl
technique has already been superseded by the dioxan,
but the results of the two methods will have to be tried
out side by side byentomologists .
• 138. Storing Paraffin Blocks. This is often done on
strips of paper 01' thin card. The bottom of the trimmed
I,ADOIlATORY'fECIINIQ.UE.
4
!IS
PARAFPJR DIOXA.N ]I}'l'C.
bloek as it haH emne off the microtome is put on the paper
and <t hot knife held a.t the haek till the ,,,ax melts slightly.
On (~oolillg the numhel' (11' description of the hlock is
written beside, ~1nd the whole eoUection pinned on the
wall, 01' kept in a drawer. '
CHAPTER
VIII
STAINS AND STAINING
139. Stains and Impregnations. Tho staining substances
which you will have to learn to use are carmine and
hrematoxylin, known as natural dyes, and a nnmber of
synthetic coal tal' dyes-neutn~l red, Janus green, methyl
blue, methyl green, nigrosin, orange G, eosin, acid fuehsin,
gentian violet, Sudan IV a,nd Nile blue. There are many
others which it will be unnecessary to mention here at
length: Methylene blue, s[~fmnin, hasic fuchsin, uurantin"
light green, toluidin blue, thionin, alizarin, tl'ypan blue,
etc., are very important stains which the beginner need
not use. The most important dyes are carmine and
hrematoxylin, and maDY of the best known formulre
contain one or the other of these substances. Carmine
is the ground-up (hied bodies of the cochineal insect,
while hrematoxylill is the ether extract of logwood, a
product of South America. The extract is dried, dissolved
in water, and the hrematoxylin is crystallized out. The
other dyes mentioned are derivatives of the hydrocarbon,
benzene (C6Ho)'
What happens when a section or smear is stained ~
There are two main theories of dyeing, the chemical
(Ehrlich, Dnna, Giemsa) and the physimtl (A. Fischer,
Bayliss). From the fact that in double and triple stains,
differentiation is simply got by extracting (and recovering)
one or other of the dyes by some solvent like alcohol, it
seems that in most cases no chemical compound is formed
by dyeing, but the phenomenon is one of adsorption.
It would seem that no staining method can be speciiic~
~I.'
100
STAINS AND STAINlNO
nor is there actually any that is. It is only the microchemic,tl met,hods like Fculgen's technique which approach
complete specificity. Yet the electivity of some staining
methods is surprising and naturally has given support to
the chemical theory. l!'or instance, acid fuchsin for
mitochondria in properly fixed and lllordanted material,
Weigert's elastin stain, or methyl green for chromatin are
all remarkably elective, but other things may be stained-but not many other things.
140. Silver and Osmic Impregnations. These, especially
the former, are of tremendous import,ance in histology
and oytology. Silver can be made regularly to impregnate
anyt.hing except fat and chromatin. The process is
nearly always akin to a photographic one. The silver salt
is absorbed and held by certain bodies or surfaces, and on
subsequent reduction shows as a brown or black impregnation. Careful examination of a series of under- and
over-impregnated silver preparations will show that a
given body may be impregnated throughout with silver,
01' the effect lllay have bec:ome an enc:rusting one.
In the same way osmium tetroxide (OS04) is reduced to
a lower substance, possibly hydrated OS02. At all events,
in some caseR, this "stain" may he made to stream out
of a sec:tion by treating with turpentine in much the same
way as alcohol 'will differentiate certain coal tal' dyes.
At the present time the exact nature of osmic staining is
unknown.
141. Stains that Need not Ripen. In this book it iR
presumed thll,t you are working in a laboratory in which
ripe stains are to he got. If you are working by yourself,
and want to start staining at once, here are some general
stains which can be used immediately: 1Jlann's methyl
blue eosin for all usual purposes, especially for sections,
but not whole mounts of invertebrates or embryos. It
tends to fade in acid balsmn, but it is a, beautiful stain.
MUiJer's Acid Ham~aZ.llm, a transparent, delicate yet
HiEJJIA'POX YLJN
101
energetic alum luematoxylin stain, very useful both for
sections and large embryos or whole mounts. Boma:
carm'ine, 1:1 good general stain for crnstacea, any ·whole
llloU11ts, anel nlfoio for staining in hulk (as also iH MILyer's
above). Bon1x carmine and Mayer get better after a
litHe keeping, sa,y 8 weeks or a month. In any case it is
recommended to make up and ripen· some Delafield and
Ehrlich, '",hich will be ready later on \"\'hen wanted.
Heidenhain's and Regwud's hromatoxylill, and Leishmanll
or Giemsa, lllay be used immediately they arc made
up. For chromosomes, gClltia.n violet can be used immediately.
142. How to Use Stain Jars. When a slide is being
passed from ja.l" to jaI' it should be quickly drained into
the jar it came from, and excess stain or alcohol ruhbed
from its back with a dean ahmrbel1t duster. Keep the
stoppers or corks on the _jars when not in 1{Se.
Hrematoxylin Formulre
143. Delafield. Solution A. Ammonia alum, saturated
aqueous solution, 400 c.c. Solution B. Hrematoxylin
4 gm. Ethyl alcohol (9(i% or absolute) 25 c.c. Mix
A and B, and let it stand exposed to ai1' and light for 3 or
4 days. A large beaker does for this. Solution C.
Glycerine 100 c.c. Methyl alcohol 100 c.c.; to be added
to the other, when it is ready. Allow the mixture to
stand until it becomes quite dark. Transfer to a well. stoppered bottle. It lasts well and improves with
keeping. It, is a strong stain, and works well ,,,hen
diluted by half or one-third with distilled or acid water
(§ 37).
144. Ehrlich. Water 100 c.c., strong alcohol (95%
01' 96%) 100 c.c., glycerine 100 c.c" glacial acetic acid
10 c.c., hrematoxylin 2 gm. Dissolve the hrematoxylin
in the alcohol, aeld the acid, then glycerine and water.
102
8'llAIN8 AND 8TAININ(-I-
Add potash alum to excess. Ripen in an open beaker or
open flask in bright light unW a dark red. Pour off into
a well-stoppered hoUle.
145. Heidenhain. A 0'13% solution of halmatoxylin
in distilled water which improves on being used. Do not
throw it away when it gets black, as it is then staining
hest.
146. Mayer's Acid H<Emalum. Hffilllatoxylin 1 gIll.,
diRtilled water 1 litre. Dissolve and add 0·2 gm. sodium
iodate (NaIOa) and 50 gIll. of alum, dissolve and filter.
Add glacial acetie up to 2%: This keeps well, it stains
fairly satisfactorily at first, but is better later.
147. Regaud. One gmm hffilllatoxylin in 10 C.o. of
absolute alcohol, added. to 10 c.c. glycerine and. 80 C.c.
distilled water.
Much used nowadays instead of
Heidenhail1.
148. Times for Regaud or Heidenhain for Cell Granules.
Leave all day in 4% or 5% iron alum, rinse in water and
place in the hrematoxylin in the late afternoon. Differentiate next morning in 4% or 5% alum. Neve~" for
example, leave overnight in alum and 2 or 3 hours next
day in halmatoxylin.
Carmine Formulre
149. Borax Carmine (Grenacher). Carmine 2 or 3 gm.
4% aqueous solution of borax 100 C.c. Boil for 30
minutes. Cool, add equal quantity of 70% alcohol.
Allow to stand for a few weeks, shake now and again,
filter.
150. Hollande's Chloro-carmine. The student used
only to borax or para-carmine does not realize that some
carmine stains can be very intense and rival even the
hrematoxylin techniques. Undoubtedly one of these
stains is chloro-carmine of Hollande, which stains mitochondria in frozen or ordinary sections, and is useful in
OARMINE
103
'smears or whole pieces. Place 5 c.c. pure hyclroohlol'ie
acid in a porcelain dish, add gradually 14 gm. of powdered
carmine, stirring constantly to lllake a doughy mass.
Allow to digest for 24 hours; add 250 c.c. distilled water,
stir, and bring to the boil for t hour. Filter and make
up to 180 C.c. with distilled water, then add enough 75%
alcohol to make up to 200 c.c.
Smears or sections are stained for from 2 to 24 hours,
rinsed in distilled water or 30% alcohol, transferred to
3% iron alum solution, in which they become black,
subsequently decolourizing slowly. 'When differentiated,
rinse in 1 % pyridin solution, wash under tall for 10-15
minutes. This stain is highly recommended. If you are
using reel stains like this, and safranin, constantly, place
a blue screen in front of Y01U' microscope lamp.
151. Aceto-carmine. 'fhis is the stain now so much
used in the genetical laboratory for the rapid study of
chromosomes by, the smear method. Though fairly
permanent preparations may be made, it is really not
good for this purpose.
Schneider's aceto-carmine:
saturate boiling 45% acetic acid with carmine. This
strength of acetic is taken because it dissolves the largest
proportion of carmine.
152. Belling's Iron Aceto-carmine. Add a few drops of
ferric hydrate dissolved in 45% acetic acid to some
Schneider's aceto-carmine nntil it becomes bluish-red,
but without visible precipitate. Then add an equal
amount of ordinary aceto-carmine.
When using both the above stains with large teste8
(e.g., locust), you first smeal', add enough carmine (and
not to excess), covel' and W~1rm gently; set aside for a
few minutes. Ring with vaseline in a warIn pipette if
necessary. For very small testis (e.g., Drosophila 01'
Asellus) dissect out, add drop of aceto-carmine, cover,
leave for short time, and gently crush by moving coverslip.
'Varm if necessary, ring 'with vaseline.
STALNS AND 8JlAININO
Coal Tar Dyes
153. Mann's Methyl Blue Eosin. Mix 36 c.c. of 1 %
aqueom; solution of (water-soluble) eosin, a,nd 45 c.c. of
1 %methyl blue, and 100 c.o. distilled water. The solution
should be neither red nor blue, but purplish yellow green.
Not all eosins are suitahle. It stains instantly, but may be
left up to 24: hours.
Mann used the stain as folImn;: Fix in IJicric corrosive
sublimate (I gill. picric acid, 2 glll. tannin in 100 c.c. saturated
corrosive). Btaill sectiollfl or smears for 24 hourfl. Wash in
water, transfer to absolnte alcohol 50 C.c. with 1% NaOH in
absolnte :3 or 4 drops; sections go red. Wash quickly in
absolute alcohol, transfer to water for 2 minutes, then bring
into water slightly acidified with acetic acid. They become
blue; dehych'ate in absolute and mount in non-acid bal~alll.
Good results will be got as follows. Stain sections or
smears 15 minutes to hour. ·Wash in water, transfer to
nO% for a few mimltes, then absolute and mount in balsam.
It will be found that one of the two dyes is more soluhle
in alcohol or in water than the other-depending 011
fixation and time of staining, and you must try to keep
bot,h stains in the smear or section by observing such
details during differentiation. Note that the stain is good
for all fiXations except chrome-osmium (unless bleached).
t
C. Dobell stains for 12 hours, then differentiates in a weak
solution of Orange Gin 70% alcohol, then passes rapidly into
absolute, xylol and balsa,m .
•J. Ford recommends to stain for 3 hours at 37° C., to wash in
water and to treat with pure formalin for 5 seconds, then to
wash in water and to continue as above according to Mann.
This method is particularly vain able for Negri hodies. Mount
in cedar wood immersion oiL
154. Altmann's Acid Fuchsin. This is definitely a
very fine method with which beginners often have trouble,
traceable to one of two faults in the cll-se of failure. Firstly,
lllOrdanting is not, right, secondly, the stain is not right.
eOA L 'PAR
j)
Y]I}S
1O;}
Altmann'8 fl1lid is l,)otassiUlll bichromate 5 % (Bensley
Ul-lerl 2t% strcngth, which is better) amI osmium tetruxide
2% equal l,)arts. Fix for at least 24 hours up to 2 or 3
rIa,ys. Alternatively you may fix overnight in Flemmingwithout-acetic lwid, Champy, formalin, or Altmaun, aUfl
then transfer to 3% bichromate for a days. Hegaud's
technique is given in § 113. All these methods consiHt of
lllordallting, either in the cold, or on the top of the
incubator in bichromate. \Vithout this the fuchsin will
llO"b "bite." After lllorclrLnting, wash out overnight under
the tap (see § 122). Now this direction is quite right for
pieces of firlll tissue like mammalian testis or liver, but
may be too lllllch for a well separated locust testis, into
which water penetrates more easily. A few hours ill the
morning does for such material.
As to the stain, there are good and llad fuohsins. The
latter are due to lack of eontrol in sulphonation, and they
do not all dissolve in water to the same degree. Altmann's
specimen dissolved up to 20%, and so does the American
sample we have used. Some British specimens dissolve
up to 10% or 12%. To make up the solution, first shake
up some drops of aniline oil in excess ,vith water. Filter,
and add 10-15 gm. of acid fuchsin. Now this stain
works hetter after a, few days, and it goes bad after a
few weeks.
Bring the Bectiolls or smears to water, drain and flood
with plenty of aeid fuchsin, hold in forceps cautiously over
bunsen or spirit flame, moving it backward and forward
till the stain steams, but does not boil. Keep it hot fiJI'
30 seconds to a minute and then set aside on bench to
cool. Wash off with distilled water; the Hections should
be dark red. Dil,) quickly into methyl green (1 % aqueous),
drain and transfer straight into absolute alcohol, then xylol
and mount. If the stain does not" bite," it may be better
to t,rea,t seetiolls for 30 seconds before st.aining with fL
1 % soilltion uf permanganate of potash, followed by 5%
hili
STAINS AND S'l'AINl.NG
solution of oxalic (wid for some time. Then wash well in
water.
Now if the Rcction or smeM' is over-stained the next, one
should be differentiated in picric acid (96% alcohol
saturate(l with picric acid 1 part, water 2 ·parts) after
being washed in distilled water. The methyl green is
left out.
This method is easy to work with vertebrate material,
but is usually difficult with the invertebrates. It fades,
being reel is trying for the eyes, and has no special advantage over iron alum hrematoxylin, which we recommend in
preference.
155. Van Gieson. This counterstain is much used in
Histology fLnd Pathology lahomtories, and may be
especially recommended for connective tissue, after
Heidenhain or Regaud (§§ 145, 147). Remember that
picric acid differentiates these stains, and so you slightly
under-differentiate before counterstaining. After differentiation in iron alum wash out well under the tap, add the
Van Gieson for 3 or 4 minutes, wash in water, transfer to
95% alcohol, then dehydrate and mount in xylol. Some
workers pass through origanum oil from the absolute
alcohol into xylol.
To make Van Gieson, add to a saturated aqueous
solution of picric acid sufficient saturated aqueous solution
of acid fuchsin to give a deep gamet reel tint.
156. Safranin and Light Green. Safranin has no advantage
over gentian violet,; it is red and therefore more trying for
the eyes. It was used greatly a quarter of a century ago.
With light green stain it makes a pretty combination. Use
Babes' fluid, which is equal l)arts of saturated watery and
saturated alcoholic safranin. Stain till you get a good deep
red. This mav he an hour 01' so for corrosive fixed material
01' overnight for ehrome-osmium.
After staining, quickly
upgrade, and counterstain in -;\-% light green in clove oil.
If the result is too red, leave the next slide longer in 90%
alcohol f111d clove oil. After the clove oil, wash off well in
xylol, mount in benzol balsam or, preferably, immersion oil.
v
T.S. EARTH W()IU~[
107
157. Piero-nigrosin is a. mixture of 1 pa.rt of saturated
aqueous solution of nigrosin w'ith 9 parts of saturated
aqueous picric acid. After staining in horax or chlorocarmine, bring sections down to distilled \vater, then into
the stain for 5-10 minutes. WI1sh in tap \Yl"1ter for a
minute, then pass rapidly through 70% and 90% alcohol,
etc.
158. Piero-indigo-carmine is 2 parts of saturated
watery solution of indigo-carmine, 1 part saturated picric
acid in w[ttel'. Use after carmine, safranin, or magenta.
Both this and the above stain give very pretty preparations.
159. Transverse Sections of Earthworm by Borax
Carmine and Piero-nigrosin. Put a clean live earth WOJ'Ill
in a flat tin box lined with slightly moist filter or white
blotting paper. When Hoiled replace with cle,:m paper
till the worm is scoured. If you wish you may put SOllle
small pieces of onion in the box. When scoured free of
grit drop the worm into corrosive sublimate acetic (§ lOB)
and leave for at least 5 hours. Take out, wash in water,
and with a safety razor blade gently cut 5 nun. lengths of
the intestinal region. Do not crush the worm. It would
be quite suitable to leave the whole worm ovel'llight in
fixative before cutting. Wash the pieces in water for
15 minutes and transfer to 50% alcohol for 1 holli'. The
sublimate must now be extracted for several hours in 70%
alcohol coloured light brown with iodine (Lugol, § 71).
If the liquid decolourizes add another drop or two of Lugol
and leave an hour or so. Leave now several holli's in
clean 70% alcohol (overnight is best). After this bring
the pieces into borax carmine and leave a day and a
night. Wash the pieces quickly in water and then
upgrade at least 1 hour in each strength, beginning at
50%; .leave overnight in absolute alcohol in a wellcovered capsule. Change for at least I hour next morning
into fresh absolute alcohol. Pour off most of the absolute
and add an equal quantity of carbon bisulphide or ·xylol.
81 JAIN8 AND S'PAINING
I,cavo 1hon1', then pour off and add pure carbon bisulphide
or xylol for the same time. Imbecl in wa,x as describecl
in § ] 23, and section.
'When yon have made sections these Rhould be dark red
if the borax carmine is "biting." If they are only pink
the Cn,l'lnillC is not, right. In this case you can extract the
borax cnrmine in acid alcohol and stain in Ehrlich and
eosin (§§ 44, 47). However, if the nuclei are a good bright
reel you can cont,i.nne with the original method. You now
bring the slides down through xylol and absolute or 95%
alcohol to 70% alcohol, and dip them in acid alcohol if the
carmine is too cIal'k for a few moments and examine under
the microscope. The nuclei should be deep red, the rest
only pink. Wash off in distilled water and stain in picronigrosin (or picro-indigo-carmine) fol' a few minutes.
They should stain deeply. Wash quickly in tap or distilled
water, upgrade a minute or so in each strength, then
llenzol or xylol and benzol balsam.
Note. Try also with taupoles (feel on meat till grit
scoured out), using the same times, bnt after fixation the
tadpoles need not be cut into pieces.
Chromosomes
160. There are three main methods used nowadays.
The aceto-carmine smear technique, the gentian violet,
and the iron alum hrematoxylin. The fhst needs no
fixation provided the undiluted stain is used. 'l'he latter
Ileed careful fixation.
161. The Aceto~carmine Smear. Carmine becomes
dark if mixed with iron salts. Thus if you tease the
preparations with steel needles, a trace of iron produces
an iron carmine. This is done purposely in Belling's iron
aceto-carmine (§ 152). If you do not wish to darken the
carmine, use nickel or glass needles for smearing. Dissect
out the gonad of an insect, remove practically all salt
CHROMOSOMES
lOll
solution or liquid with filter paper, and proceed as dir0cted
in § 151.
162. With Fixation.
For subsequent staining in
gcntitLll violet use Flemming's strong formula with acetin
acid, fixing smears for 2-4, hours, slllall pieces 2-·24 hours.
\Vash out either in running ,vater overnight m' in tepid
tap water on top of the paraffin oven. Change the water
every i hour for 2 or ,3 hours. Upgrade very Riowly
from 30% to absolute, and dear by the two-layer method
deRcribecl in § 18. Some people elmtr first in cedar wood
oil, then in benzol or xylol. Smears are upgraded to
70% or 1)0%1 umlleft overnight.
It luts been recolllmended to nse the Flemming 011 iee
and to add 2% urea before fixing uULtcrial. Large teste;;
like those of locusts should be gently spnmd so that t.he
tluid gets to all park;, and pieces of vertehrate material
lllU::;t he small.
A really good Flemming gentiltll violet prepamtion i8
superior to anything else for clarity ttnd beauty.
163. A modified Flemming with formalin im;tcad of
osmie add is Navllshin'.'1 Fl1dd, whinh hag heen used
recently by botanists. Ch1'01nic acid 1% in water 15 parts,
commercial formalin 4 parts, acetic acid I part. It is
essentiltlly a botanical method, _the ordinary Flemming
being more suitable for animals. It is really excellent
for root tops. Try those of the domestic pea, slitting
them longitudinally before fixing. Stain in Heiclenhain,
hlEmal1Ull or gentian violet.
164. Bouin, Bouin-Allen. These picl'o-formol-acetic
fluids are famous for animal chromosomes (§ lOG). Fix
. material (l,t, 38°-'1,0° C. for 1 or 2 hours. Dehydra,te
carefully from aO%, a day in each; or by the drop
method, which is to begin by putting the material in a
little water in a tube, ltncl to arrange a slow drop into it
from a. bottle of 70% alcohol, and later from a hottle of
HO% alcohol.
110
STAINS AND S'I'AININO
This material stains bmmtifully in hromatoxylin and
with care in gentian violet, as well.
165. Gentian Violet (Violet Rosaniline or methyl violet).
This is an intense violet stain much used by chromosome
workers. Fix preferably in Flemming acetic for both
animah; and plants. or Navashin for plants. Bring slides
(or smears after a sojourn in 70% alcohol) into water, and
then place in 1% gentian violet solution boiled and filtered
for 3-10 minutes, according to the age of the stain. Wash
in water and mordftut for about} minute in 80% alcohol
with 1% potassium iodide and 1% iodine. Dip quickly
into Ofi% or 06% alcohol, then quickly through absolute
to clove oil, in which differentiation takes place.
Wash in three changes of xylol and mount in xylol
balsam.
Picric acid can be used with this stain as follows.
Mordant 10-20 minutes in above iodine solution, rinse in
water, stain 15 minutes in 1 % gentian violet, rinse in
water, transfer to iodine solution for a minute, then 95%
or 96% alcohol, then flooel with saturated solution of
picric acid in absolute alcohol, followed rapidly by a wash
in pure absolute. 'Pass to clove oil in which differentiation
takes place, then wash thoroughly in xylol (at least
15 minutes). This is an excellent method.
For Bouin fixed material, after staining in gentian
violet, mordant in iodine 2 minutes, rinse 2 seconds in
absolute alcohol, 15 seconds in 1% aqueous chromic
acid, 10 seconds in absolute alcohol and differentiate in
clove oil, wash off well in xylol.
166. Heidenhain. Objected to by most chromosome
workers because it is too opaque, and is really only suitable·
for animals like moths with small or few chromosomes.
Mordant sections or smears ([tfter sojourn in 70% alcohol)
in 4% iron alum, for several hours. Wash slightly for a
few minutes in mnning water, transfer to 0'5% hromatoxylin for twice as long as in alum. Wash off exees!>
OOLOf APPAR.-JTUR
111
l'taill in wat.er, tntnsfel' to 4% iron alum j,(J rlitferentiate.
If t.his happens too quickly, dilute the alum by half.
vVhen diffel'elltitttion is finished, Wm-dl uncleI' tap for at
least 10 minutes. Upgrade, mount in balsam.
The stain becomes more transparent if the times ltre
shortened, but material left a long time in chromeosmium does not stain so deeply as docs that fixed in
Bouin or Navashin.
167. Mayer's Acid Hremalum is no\v used, as it is more
transparent. Leave for at least 1 hour in a good specimen
of the stain. Differentiate in acid water, wash well in
running tap water or in ta.p water substitute.
168. Nucleoli and Chromatin. Use Feulgen's method
counterl3tained in light green.
Golgi Apparatus
169. For the Goigi i1ppa.ratus four methods may he
learnt. These will covcr the whole ground. Theyare (Cajal)
Aoyama, (Cajal) Da Fano, Weigl and Kolatchew. 'fhe
first two are formalin silver, and are cheap; the latter,
osmic, and costly. The osmic methods give a more
faithful fixation and some at least of the fat, the formalin
silver are less trustworthy so far as morphology of the
Golgi apparatus is concerned, and give no fat unless used
as in § 171. Fat can, however, be more relilLbly studied in
Sudan IV, Farrant or gum glycerine mounts.
170. Cajal-Aoyama. This and D,t ]1'ano are modifications of Cajal's method. The three fixatives for these
methods may be given here: Aoyama\~ Fluid is cadmium
chloride 1 gm., neutral commercial formalin 15 c.c.,
distilled water 85 parts. Da Fano's Fl1lid is cobalt
nitrate 1 gm., formalin 15 c.c., water 100 c.c. GajaZ's
Fluid is uranium nitrate 1 gm., formalin 15 c.c., water
100 c.c. The fluids are essentially the same except for
tlle "mordanting" substance. I have found that
STAINS AND 8'1'AININO
A( ,yCtllJa if{ t.lw h('st, 1d' the t.hree and definitely I'cmmllwnrl
it,' F'(lJ' Illalllllwlian gOllads. Ifl)'llHdin is fL PUOI' fixM,ive.
,;(1 ill (ihis 1'11(1 hegins badly.
Befter 'I'P8uff8 'll!iII fll' got fly
add in!! fl'o/ll (I·7ri'XJ to
X(/('/ to fllMI' .I'llitlN, ((lid by
8ee.i'ilf/ tha! the,{Oi'iHali-1l ,is nelti)'al-ized with calciuln c({'I'bonctte.
0'0';:,
Excellent fixatiolls of other glands or material will be got
with these iixativeR.
'The propel' time to fix is :3 or 4 hours for small pieces.
'Wash in t.wo changes of distilled 'water for n, minute,
tra.nsfer to newly made up ] 'i)% silver nitrate in water
fill' 10-15 hOLlrs at warm room temperature in the shade
or :\,t 22° C. in an incuhator. Rinse pieueH again for
] minute in distilled water, i1ncl transfer to reducing fluid
(see helow) for at least ;) hOllrs. \Vash under running
tap for a few minutes. Upgrade from :30% alcohol, imbed
in paraffin. Mount seutions withont toning or staining.
If you wish to tone this can be done in '5% gold chloride,
after bringing the sections to water, but is really unnecessary. For a countel'st,ain, dip into MaIm's methyl blue
eosin for 1 minute, then water, then into 90% alcohol,
absolute, xylol and mount balsam. Safranill and light
green (§ 156) are sometimes used as a counterstain. The
above times are for Aoyama, but cIa well for Da Fano.
In the latter method the tendency ill recent years has
been to use the fixing fluid with 0'75% salt for only a few
hours, not overnight as DR, Fana advised. Da Fano used
the silver bath for 24-48 hours; note that Aoya1lla
advises only 10-15 hours. Reduce at least 5 hours for
Ua Fano.
These silver nitrate preparations are not absolutely
specific for the Golgi apparatus (what method is '?), but
in metazoan cells there is usually no difficulty in finding
which structure is the Golgi apparatus. The method has
been used and abused by some protozoologists, who have
not hesitated to identify any blackened granules as a
Golgi apparatus. 1'he Aoyama and Da Fano methods
a.A. A}U) 11'..11' 8,]'.41117
II a
will give very intel'eRting I'm-mIt,; wit.h f.:J1WltI'R. hut, h('.re tile
teehnique it-; definitely not flllKy (~ IlH).
RedllC'inr/./1u irl iH hydrnqu iUolle 1 ~ gill., formalin I;') (1.('.,
distilled wa.tm 100 c.e., Rm\inm :-;nlphite (Ni gm. After
some weeks this fluid umy beeOllle Ilsete~s. It is Htraw
yellow at first, and later goes hlack
Ifor an unexplained reason old silver nitrate solution
does not work sometimes. vVhen starting new work it iH
better to make new reducer, new silver solution, and to
look to your formalin. 1'he latter should definitely he
of the best grade.
1'lote. If YOll intend to hegin rE's('arch work on the Goigi
apparatus of Metazoa, you should lise the silver and osmic
methods side by Hide. For some unknown reaS011 the osmic
mpthods 011 oecasiolls entirely fail to impregnate, and it i::;
neoessary to have some control on them. In the CUKe of tIle
Goigi aI)paratns of Sporozoa, the osmic methods arc the best,
the silver methods rarely giving ii, proper impregnation,
though I have seen excellent silver preparations of HpOl'OZOitll
Golgi bodies.
171. Combined Goigi Apparatus and Fat Stain. A
successful method is the combined Aoyamu (or Da Fano)
and Sudan IV. You prepare either smears or frozen
sections by, preferably, the Aoyama method. The piece
of tissue cannot be imbedded in wax as the upgrading in
alcohol to xylol removes the fat, so frozen sections must,
he cut, and the thinner the better, or the material may be
teased. After the Aoyama smeal', or the block of tissue
has been brought into reducing fluid as described in § 5H,
it should he washed in water. The smear goes immediately on to the fat stain, whereas the blook of tissue is
sectioned, and then st.ained in Sudan IV, or a smail piece
is first stained in Sudan IV and then teased up in glycerine
and water (1 ; 1). In both cases the lllountant is glycerine,
glycerine jelly, or Farrants (§ 80).
Exactly the same can be done after the Weigl or
II t
81'.-1 INS _./ NIl 81'AININ((
l\.nll1tdJew methods. Ymt stiLin ill Sudan IV and monnt.
in Farrallts, gly"t:'l'in8, or glycerine jelly.
"In l)l1th thu silycl' nitrate Hud the oRmic method the
Golgi Ilppnmt,w,; is blaek, alld fat, iR 1'0(1, hut ill some osmic
pl'cparatiollH the (;oiour of the fat is hl'owniRh red rather
thrLll the bright 1'or1 of the formalin silver preparations.
172. The Osmic Acid Golgi Methods. Osmium tetroxide
it:! one (If the mORt expensive substances known. While it
is (1, good thing to learn these methods for Metazoa, the
oumbinecl Aoyama and Sudan methods will really be
fmfficient if skilfully used, There are two main osmic
methodR, (l) prefixation in corrosive osmic of Mann for a
few houl't:! lilltil l1elletratecl, (2) prefixation overnight in
any ohromc-osmiull1 fluid without acetic acid. Subsequently the Ill£ttel'ial \vashed out for an hour 01' so for
Mann fixed pieces, but overnight and thoroughly for
c'.hrome fixed pieces. The explanation of this is that the
presence of excess chrome salts in the pieces of tissue
inhibits the osmic reaction, whereas the corrosive sublimate
in Mann's fluid has no such effect, You must see that in
both methods the pieces of material are small, After
washing out transfer to I % or 2 % aqueous osmic for
several dttys ttt 30 0 C, or thereabouts. It does not matter
very much, only at room temperature the reaction takes
weeks, at 30° C" dttys, Many people use 2% osmic, but
I % does well enough, and this strength is being used by
most workers nowadays. After 3 days break a small
piece of the ma,terial in glycerine water (I : I) and examine
nnder the oil immersion. If the Golgi apparatus is black,
pour off the osmic acid and leave the pieces in distilled
water at 30 0 C. overnight. Upgrade and cut thin sections
in wax, Counterstain as in § 60,
is
Mitochondria
173. For vertebrate material fix in Regaucl (§ 113),
stain in Regauc!'s or Heidenhain's hrematoxylin, or acid
RO J}[A NO JVSK y
lUi
fuchsin (§ 154). For invertebrates, same stains, but fix
in Champy 2 or 3 clays, wash out overnight, or fix in
Champy or Flemming-without-acetic acid overnight,
mordant 3 days in 3% potassium bichromate.
Methylene Blue
174. Polychrome Stains, with Special Reference to
Methylene Blue. If you wish to work on glands and
blood ii'om a general histological aspect, or Protozoa and
parasites in' tissue, there are three methods of great
importance. One you have ah'eady tried is J~eishmann
or "\iVl'ight for blood smears. Two others are Giemsa,
and polychrome methylene blue, with or without emlin
or some snch eouuterst,aill.
Methylene hlue solutiolls when left standing clisilltegmte
slightly, producing lo·wer homologuefl such as Azure A
and Azure B, whieh are oxidation proclucts. Tn Unna's
polychrome methylene blue these lower homologues have
heen produced by hoiling with potassium earhonate.
Unna's stahl is methylene blue I gm., potassiulll carhonate
1 gm., water 100 C.c., but it needs some months to ripen.
I give also Goodpasture's acid stain, which can be UHeu
immediately it is made (formula from Mallory and Wright).
Methylene blue I gm., potassium carbonate I gm., water
400 c.c. Dissolve and boil for 30 minutes. When cool
add 3 C.c. of glacial acetic acid, and Hhake until the
pl'ecip~tated stain is redissolved. Boil for 5 minutes until
the solution is concentrated to 200 C.c. On cooling it is
ready for use and keeps well. Use as follows. Stain in
5% aqueous eosin for 20 minutes with heat, if found
neceESt1l'Y. Wash in water, drain, and add polychrome
methylene blue diluted I part in 3 of water. Wash in
water, differentiate in 96% alcohol.
Giemsa, Leishmann, Jenner and Wright are all
Romanowsky stains. Roma,nowsky nearly 50 years ago
lIG
S'llAI NB AND 81TAINING
first used combined eosin and methylene blue. Eosinate
of methylene t'lcpamtes out in various granules and cell
elements in a rellHuknble manner, producing a polychrome
effect exceedingly striking .
.Tenner, LeiBhmann tLnd \Vright are eosinates of ]loly"
fihrOllle methylene blue dissolved in methyl alcohol,
which must be lleutral and free from acetone (though this
has reeelitly been questioned). Giel1lsa is another stain
like the above, hut containing eosinate of Azure II. All
thciie staiml are hetter bought than made.
175. Giemsa Method for Paraffin Sections. Fix in
Zenker for 24 hours, make paraffin sections 3-5f.l.
Remove sublimate in iodine, to he followed by 0·5% hypo
(sodium hyposnlphite) to get rid of all iodine. Wash in
diRtiIled water. Stain for 12-24 hours in Giemsa's liquid
stain, 1·21) c.c., pure methyl alcohol 1·5 C!.e., 0·5% sodium
hiearhonate 2 drops, distilled water 50 c.c. Differentiate
in 05% ethyl alcohol, olear, mount in thick cedar (immer"
['don) oil, as balsam is too cwid (even the "neutral "
balsam !). If t,he cytoplasmic strnctures are too blue,
omit the sorliulll bicarbonate, or mordant before staining
in 2·5% potassiulll bichTOmate; if they are too red, treat
l'e(1tiolls before staining in 1 % potassiulll permanganatc,
washed out afterwards in 5% oxalic acid, then distilled
water.
For some tissues IT. Ford recommends washing in water
after staining, rapidly flooding with strong formalin,
washing off in water, then differentiating in strong alcohol.
Mount in immersion oil.
CHAPTER IX
NOTES FOR HISTOLOGY AND
EMBRYOLOGY STUDENTS
176. Glands. (a) For superficial histological study
use Susa, Zenker or Kahle fixed material stained in a
hrematoxylin and Van Gieson, or Mann's methyl blue
eOSIn.
(b) For cytological work, Aoyama, Weigl or Kolatchew,
Regaud, and the various microchemical methods (§ ti2).
177. Connective Tissue. Zenker, Susa, Formalin,
followed by hrematoxylin and Van Gieson for collagen
fibres. For ela8tic :fibres a good method is that of'raellzer.
Fix in Bonin, Susa or Helly. Bring sections to 70%
alcohol and transfer to Orcein 1 gm. in 100 C.c. of 70%
alcohol, with 1 c.c. of hydrochloric acid. Leave for 1 hour
or longer if necessary, wash quickly in distilled water and
pass directly to 96% alcohol, and watch under microscope
until the elastic fibres stand out a clear brownish red.
178. Cartilage. Fix in sublimate formol (§ 112), stain
in safranill or Mallll's methyl blue eosin. In the borax
carmine and picro-nigrosin method the cartilage stands
ont well.
179. Muscle. As for Glands, (a) and (b).
180. Bones and Teeth. '{'hese cannot be cut without
decalcification. Fix in sublimate formol (§ 112) or Bouin
(§ 106). Transfer to a good bulk of 5% watery solution
of trichloracetic acid at least overnight, until deca,lcified.
Pass straight to 1)0 % alcohol.
Here is a good fluid (C. E. Jenkins), ·which fixes and
decalcifies at the same time: Hydrochloric acid 4 c.c.,
117
liS
H [8'1'0 LOO r
glacial acetio a O.c., chloroform 10 c.c., water 10 c.c.,
ahsolute i11coho1 73 c.c. Immersc tissue in at least
100 times its volume ofthe fluid. Leave at least 24 hours
for I-1!lULU pieces, larger objeots will take several days.
If ill doubt change the fluid and leave longer. Wash out
in 90% alcohol. Tho sections stain well hy ordinary
methods.
Central Nervous System (Brain, eto.)
181. Nissl gran'llles, fix in alcohol, Zenker, or conosive
sublimate acetic, and stain in polychrome mothylene blue
and cosin (§ 174) or 1% watery toluidin blue for several
hou!'l';, differentiating in alcohol.
Golgi Bodies (§ 101)). Spinal ganglia or cerebellum.
Mitochondria. Hegaud aeid fuchsin (§ 154), or alum.
IW:~llIat(Jxylin (§§ 145, 148).
A:ris C!ll'ind(?t" Dendrite arltZ Ne'uroglia Sta-ins. The
methods chosen here do not need a freezing microtome,
or imbedding in celloidin.
Ranson'8 Pyridin Silt'er Stain (modified Ramon y Cajal
,J\lIethod). Fix pieces of variable size in ahRolute alcohol
cont!1iniug 1 % strong ammonia for 48 hours. Rinse in
distilled water for ahout 2 minutes. Put in pyridin for
24: hours. 'Wash in many changes of distilled water fol'
24 hours. Transfer to 2% aqueous silver nitrate at 35° C.
in a dark cupboard for 3 days. Rinse in water and reduce
for 1 day in a 4% solution ofpyrogallic acid in 5% formalin.
Upgrade, cut paraffin sections. This ,method shows
various fibrillar structures, often cell bodies.
~Myelin. Make paraffin sections of formalin fixed brain,
and stain in Regaucl's hrematoxyJin (§§ 14-7, 148).
Axis Oyl'inder and Dendrites (Zeihen's Gold and Sublimate Method). Fix small pieces of fresh tissue in equal
parts of 1% gold chloride and 1% corrosive sublimate
for at least 3 weeks, but preferably even up to 3 months,
by which time they will have become reddish brown.
CLASS MA1'E.RIAL
Il!1
Cut hand sections with a razor, pass into Lugol's solution
diluted with 4 volumes of ..vater until differentiated.
'~Tash in distilled water for Heveral hours, dehydrate and
mount in balsam.
In various specimens of tissue, both medullated and
non-medullated nerve fibres, nerve cells awl neuroglia
are stained. It is advisahle to start this met.hod some
weeks before you will need the pieces. Trial specimens
can be cut and. examincd ii'om time to time.
182. On Fixatives to Use when Collecting Material for
Cytological and Histological Study. Whore the llluuhel'
of bottles nnu~t he cut down to minimum, take AOYUUHt
and Bonin-Allen and treat snbsequently as follows :Aoywrna. (a) Oontinue with method for Golgi apparatus.
(b) Post-chrome as for Regaud.
(c) Cut hmld or frozen sections and stain in Sudan IV
f(_)1' fat.
(d) rrest for lecithin and cholesterol.
Bouin-Allen. (a) Cut sec:tiollS for ordinary histology
stained in hrematoxylin and eosin, etC'.
(b) Prepare seutions for ehromosomes by Heidenhaill,
Mayer, and gentian violet.
(c) Feulgen.
(d) Glycogen (Bauer).
As a third fixative, Mann's fluid for the Weigl method is
recommended.
The best general fixative in which materieL1 may be
kept till desired for use is Bonin without aoetic acid.
This refers to marine material espeoiaJIy, though some
things harden rather much. When back in the laboratory
the material should be washed in water for a few minutes
and tl'ansferred to 70% a1c:ohol, and parts of it picked
out later for st,aining in Mayer's hremalum, or for
sectioning.
183. Recommended Material for Class Purposes. General.
III l'pring l)olleet slllall swimming tadpoles, fee(l on I110ttt,
I :.!O
HI8TOLOGY
and fix in corrosive acetic and Bouin, store in 70% alcohol.
For any time of the year use scoured earthworms,
.1rlitocllOndl'ia. Fix Rlices of mammalian liver in (a)
formol-saline for 4 days, upgrade, section and stain in
Heidenhaill's alum hrnmatoxylin. (b) Regaud, stain as
above, and by acid fuchsin. (c) Altmann (Bensley's
modification), stain in acid fuchsin and alum hrematoxylin.
Guinea-pig testis. Fix in Regaud and salt Ohu,mpy (very
small pieces). Stain as above.
In spring and SUlllmer fix Helix ovotestes in salt
Flemmillg-without-acetic or Champy. Stain in alulll
hrematoxylill.
Golgi Apparat11s. Fix t pieces of Helix ovotestis in
Mann's fluid for 2 or 3 hours, wash in distilled water for
1 hour, transfer to 1% osmic acid for 3 days at least at
30° 0., or for 3 weeks at room temperature.
Fix dorsal root-ganglia of rat or young cat in same way.
For silver method (Aoyama) use similar material and
mammalian epididymis and slllall intestine. For good
invertebrate neurones nse ganglia of Helix.
Chromo8ome8. Fix root t,ips of the d01llestic pen
(preferably), bean or onion in Navashin's ,tiuid and stain
in alum hrematoxylin and gentian violet. '
For chromatin, fix in eorrosive sublimate l1ncl stain by
Feulgen's method.
Collect testis of Salamanda or Newt from June to
August. Fix very slllall pieces in Flemming with acetin
for 3 hours, section and stain in gentian violet or alum
h£(lmatoxylin.
For mammalia, fix cavy testis in Bouin-Allen, and
stain in Mayer or Heidcnhain. In sumlller use locusts
and grasshoppers. :Fix in ]'lemming with ac:etic and stain
in gentian violet.
Fo?' Aceto-cannine Smears. In winter practise on
testis of Drosophila or AseUus, kept ill warmth for a few
(lays. Tn RUmmel' use early instal'S of hemipterous inseets,
UE.iV'l_lRIl? [TOES
1::!1
and other insects like locusts (8tenobothru8 virid-ulIlR, the
grasshopper, is useful).
Fm' JJlic1'ochmnical TC8t8. Use smears of Helix ovotestis
and Cavia testis for trials of Lecithin, Glycogcn, and
Cholesterol methods. Use also l11alllll1ali~tll material, sudl
as liver.
Protozoology. For Golgi bodies use whole intestinal
tracts of mealworlll containing grcgarines, fixed by the
Weigl (Mann-Kopsch) method. Try also centipedes, a,nd
if possible, cephalopods for AggregM,a, in whieh there is
an excellent Golgi apparatus. Ultra-centrifuge ciliates
and flagellates (next §).
184. Centrifuges. There are two types of centrifuges
commonly found in biological la boratOl'ies- ·the lutnd
centrifuge and the electric centrifuge giving up to 5,noO
revolutions per minute. '1'he latter has been used with
some success in rcsea,l'oh wOTk on eggs, hut recently the
development of the nltra-centrifuge hy J. 'V. Beams,
A. J. Weed and E. G. Pickles has marked the heginning
of a new epoch in protozoology and cytology. This is an
air-driven centrifuge and necessitates a source of compressed air, at least 50 1bs. to the square inch, preferably
up to 80. The plants used for motor tyre inflating, found
in garages, are suitable, if the reservoir is a good size.
In Fig. 7 is a diagram ofthe Beams ultra-centrifuge. The
rotor, TI, is accurately turned on a lathe out of a rod of
duralumin, brass or steel. It is top shaped, and provided
with grooves all the sides as shown in Fig. 7, B. The solid
rod is accurately turned out hollow inside (X) and provided
with a screw (or catch) lid (L) which is screwed at 8.
The lid is not absolutely necessary as liquid does not spill
when being eentl'ifnged. It is hetter to luwe a lid.
Objects to be centrifuged <11'e put into t,he cavity X. It
does not matter whether t,he rotor is balanced, as it will
revolve quite well with a piece of lead put inside. The
stator i<3 H, piece of t~\'l'geI bore brar;s tul1ing, into which the
HISTOLOGY
122
crater and tubes, H, TT, are screwed or welded. On the
sides of the crater are up to eight staggered holes, arranged
so that the air issuing from them hits the grooves OIl the
A
B
FIG. 7.
A. Rotor and stator of Beams Ultra.centrifuge.
B. Bottom of rotor, showing grooves.
C. Looking down into erater, showing eight staggered holoH
and tube aperture in middle ef (·rater.
L = lid. S = thread of serewNl lid. TI = rotor wall.
X = cavity of l·otor. G = grooves on rotor. H = hole
in stator crater. V = cavity of stator. TT = tube
running through stator ehamber and opening below.
rotor (G), thus making the latter revolve rapidly. The
size of the tube, TT, which must open below, is, in the
case of the centrifuge we possess, 3 mm. in diameter, and
in some commercial makes there is an arrangement for
controlling the airstream ,,,,hich is sucked up by the
BEAM'R CENTRIFUOE
12:1
revolving rotor, and which seems necessary for the
proper working of the apparatus. The air source is
introduced to the tuhe on the right of the 8ta,tor.
'1'he centrifuge is opera,ted in a box with double sides,
filled with sand. Sometimes the rotors burst.
For laboratories where any sort of work is being done
on the structure of Protozoa or metazoan cells this centrifuge is a necessity. It can be made by any mechanic
used to working a lathe, and absolute accuracy in the
specifications given in Fig. 7 is not necessary. For
example, the number of staggered holes on the crater is
not critical so far as we know, nor the number of grooves
cut in the rotor. The tube, TT, is necessary, but here
again a certain latitude in size of bore is possible.
The centrifuge makes a piercing whistle when in
operation. It can be turned upside down when in
operation without the rotor falling away from the column
of air.
ADDENDUM I
185. Directions for the Use of this Book. Begin by reading
Chapters 1. and II. Then start fixing and staining smears
according to the directions in Chapter III. Next make some
whole mounts as in Chapter V. This may be done before
learuing thc staining in Gha,pter III. if desired.
After some acquaintance with staining and mounting, the
fixing and imbedding of piecoR of tissue of the earth'worm or
of tadpoles (§ 159) may be tried, reading Chapter VII. If
yon are working by yourself, directions for making fixatiVes
and stains will be fonnd in Chapters VI. and VII. The
microchcmical treatmcnt of smears and sections found in
Chapter IV. should certainly be attempted last. In connection with imbedding and sectioning, the notes on histology,
Chapter IX., may be used. Some suggestions for material to
be l1sed at different times in the year hl1ve been given in
§ 183.
.
186. Amateur Microscopists living in isoll1ted places, who
may be interested in newer developments in microscopical
science, and who wish to join a Society, are recommended to
write to "The Secretary of the Royal Microscopical Society
of London," B.M.A. House, Tavistock Square, London,
W.C.I, England. This Society issues a Journal which prints
suitable and original contributions from microscopists. The
.Fellowship is open to foreign as well as British subjects;
revicws of recent advances in microtomy as well as in general
Botany and Zoology are published periodically.
187. Writing a Paper for Publication. A few short remarks
may be useful. It should be remembered that the modern
biological journal is now approaching the stage which the
chemical journal had arrived at some years ago. That is to
say, a bulky thesis has little chance of obtaining publication.
Papers must be short, with few, preferably Indian ink, illustrations. The first tIling is to look up l)revious literature on
the subject in hand. Unless the latter is something quite
new, the previous literature is certain to be fairly large,
usually very large, and often in foreign languages. Thus you
124
125
must know German and French, unless you haye friclldR wIlO
will translate for you. But even Germans and II'rene}mwIl,
'who are not specialists, will often bc unable to uuclerstnml
technical papers in their own language, amI it is better to
learn the language necessary yourself. You must read these
papers, and make notes on the ontstrmdillg pointR of iuterest,.
Usually, if you write to the authors titenlHelves, they will
send reprints of their recent papers. These translatiom; made
and digested, you will have some knowledge of the existing
state of the subject. Some of the authors may be negligiblp
and may be left out, some will be masters of their ;mhjcet,
and you must concentrate on their work and technique. If
you can get to an international congress you Illay cven meet
the authors themselves, and see their preparations. Sometimes these are worth examilling, and always you lerun some·
thing even from the occasional poomeRS of tl1eir ,yolk Yon
see the standard, at any rate, which is an education not to he
got from books and l)amphlets.
On writing your paper there is little I call say. A carefully
balanced short review of the IJrescnt state of the Bubject, as
you know it, precedes t,he original observati.ons you have
made. These should he written up in proper sequence and
clearly, and at the end of the paper you discuss other people's
work in the light of your own results. Then follows a Hhott
summary of results. This summary is an absol7ite lwcessit!l.
It should be short hut must include all outstanding results,
for you should remember that it is very helpful to three
classes of persons, the foreigner who may understand English
badly, the harassed general teacher who is trying to store in
his brain facts which may be useful for his students, and the
specialist who wishes to see at a glance what yOll have been
doing. At the end of the paper is the bihliography, which
should be aH short as possible, but should include the recent
outstanding papers, as well as the original papers written by
the perSall who initiated the special branch you are investi.
gating.
ADDENDUM
n
188. Books recommended for further study :"The Microtomist's Vade-mecum." Tenth edition. Edited
by ,J. Bronte Gatenby and Theophilus Painter. J. & A.
Churchill Ltd., 104 Gloucester Place, Portman Square,
London, W.l.
.
" Precis de l\Hcroscopie." Sixth edition. By M. Langeron.
Masson et Cie., 120 Boulevard Saint-Germain, Paris.
" Taschenbuch del' Mikroskopischen Technik." 1:3 Auflage.
By B. Romeis. R. Oldenbourg, Munich.
" Histochimie Animale." By L. Lison. Gauthier-Villars,
[i5 Quai des Grauds-Augustins, Paris (VIe).
" 'rhe Cell." By E. B. Wilson. Columbia University Press,
New York.
"Schaefer's ESllentials of Histology." Edited by H. M.
Carleton. Longmans, Green & Co., :39 Paternoster Row,
London.
" Recent Advances in Microscopy." Edited by A. Piney .
.J. & A. Churchill Ltd., 104 Gloucester Place, Portman Square,
London, W.l.
"Recent Advances in Cytology."
Second Edition.
By C. D. Darlington. J. & A. Churchill Ltd. (Deals with
recent work on chromosomes and heredity.)
189. On Buying Laboratory Material. Many of the firms
mentioned below issue illustrated catalogues, and some also
distribute lcaflets on staining methods. You arc advised
never to buy dyes or reagents with patent nameR, as research
of any kind must be carried out with materials, the formulro
of which are known. The science of biology has been built
up hy men who have cheerfully published their discoveries
and formulm for the benefit of all.
190. Firms Supplying Laboratory Apparatus, Stains, Chemicals, etc. Great Britain. Flatters & Garnett Ltd., 309
Oxford Road, Manchester (all sorts of chemicals, dyes and
apparatus, makers of Gilson's "Eupal'al"); the British Drug
Houses Ltd., Graham Street, City Road, I.Jondon, N.1 (makers
of many sorts Ot dyes); The British Dyestuffs Corporation,
126
ADDJENDtIM I J
J 27
Ltd" 70 Spring Gardens, Manchester (important makeJ's of
flyes--Loudon agents a,re Baird amI Ta,tlock, 14 CrORs St,rcet.,
Hatton Garden, E.O,1 ); G, T, Gurr, I3H New King's Road,
S,W,1i (maker of all stain;; and dyes, etc,); G. Holt & Co.,
4H Kingsv""oo(l :Road, Brixton Hill, S.W.2 (agent for Griibler) ;
Hawksley & Sons, 83 Wigmore Street, Cavendish Square,
W.I; ahd Charles Baker, 244 High Holborn, W.C. (both
firms are agents for Grubler, and for most microscope and
accessory apparatns makers), For incubators, ete., for
imbedding and tissue-culture work, Charles Hearson & Co.
Ltd., 235 Regent Street, W.l.
United Staie8 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, 550H
Kimback Avenue, Chicago, Ill.; The Will Corporation,
Rochester, N.Y.; Paul Weiss, Hl20 Arapahoe Street, Denver,
Oolorado; Natiolml Aniline Corp., New York.
INDEX
Nfl/c.
Thp nmn]Wl'R r"t'el'
Aceto-carmine formula, l:i I
Acid alcohol, :;7
Acid water, :17
Addity ami allmlinity (pH), :23
Albumen for slides, 1:28
Aleoho[ (lothyl), l(_]
"\lc'ohol (how to dehydl'l,te). 1n
Alcohol. IllL'thvl, 17
Aleuhr,( N-IlIIh·1. 17
Alkaline water,' :!li
Altmann '5 acid fuchsin method, 154
Altmann '8 fluid, lUi
Amyl acetate CPJlleJlt, 88
"\me"tlll'til'H, H:!
Aoynma fluid, 170
Aoyrnua, Hillel\-}', 5(1
Apat.hy·s "elUent, 88
Axis t:ylimloI'R, 181
t(l
paragraphs, not to )"luges.
I
Cal'lloy Lebrun formula, IlK
Citrnny smpar, i)j
Oart,i1agp, 178
(,);n·t.ilaginnlls Hkelr·tOIl", !ll)
Capsules, J:!
l!"lloiciill tC('hniqul', Jail
Celllliohl cement, SK
CelUents,81l
Centl~al Jl('rVUlIs sy~t"lll, 181
CeIT'sm, 12:1
Champy's fluid, 117
Champy, Hmea!', 55
Chloral hydmt.e, 02
Chloro-carmine formula, 100
Cholt'sterol, tl7
Chromatin (se.e .Feulgen).
Chromosomes, ltlO et 8[1[.
Clarke's sliline fluid, 24
Class material, HI:{
Clelll1ing glass, l·t
Clearing, 18
(!Icaring emhryos, 1011
Cocaine, U2
Conneetive tissne, 177
Oord's solution, !J;!
Corrosive sublimate acetic, lOS
COllntt'r"taining in O.-angl· G, 42
CoversliJls, preparation f'H' vital
staining, :n
Urustacea- sltline flui(}. ~·l
Clltt.ing ser,tiollR, 1:25
BaIKlIm,8·l
Bauer's glyeL)gen methud, n
BOlLIng' c:ent.l'ifuge, 184
I1elling, ,weto.mli·mine, 16:2
BellRlc;y'8 modification of AHmann,
115
BerleHp's medium. SO
C. 11m'nard, iodine tp8t, 71
Best's carmine, 7i
]3Iea.t1hilll,( ohromo.osmium, 08
}lones and teeth, ISO
Borax carmine formula. 141)
110mx elll'JIlim' whol,' mount., 80
]~ottles, 12
.
Dl'call'ifientioll, ISO
Deflandre's test. for lecithin, 74
Delafield formula, 14:1
Dcla1ield, staining ill, 41
Dendr-ites, IS1
Denham's sttlldarac medium, 8il
Diamond for writing on slides, 12
Digitonin, 67
..
Dioxan balsam, 81
Dioxlln imbedding, 1:~6
Bouin's fluid formula, lOU
]~Ol1ill smear, 41
Brusil fluid, n:l
Cuuad" halslllll, 8.J,
Carbolxylol, 18
C,L1·bol'undum pencil, J:2
Carnoy formula. II Il
128
INDEX
Drawing paper, 9
Drawing unde,' microscope, 8
Duboseq fluid, 93
Dyes, 18!J et seq.
Earthworm, transverse sections, 15ft
Ehrlich's hrematoxylin formula. 144
Ehrlich's h:ematuxyJin, staining in,
44
Elasmobranch saline fluid, 24
Embedding, 123
Eosin, counterstain, 47
Euparal,82
Eye strain, 6
120
Reidenhain hrematoxyJin formula,
145, 148
Reily, 107
Rollande, chloro-carmine, 150
Holtfreter's fluid, :U
Imhcchling, by dioXllll, IH6
Imbt'dding ill wax, 12;{
Imbedding, N·]mtyl alcohol, 187
Immersion oil, 4
Impregnation, fresh tissue, 98
Iodine, for glycogen, 70, 71
Iron hrematoxylin, 45'
Kahle's fluid, 114
Farrant's medium, 80
Fat, combined Aoyama. and Sudan,
171
Fats. 68
Feulgen's test, 76
Fixation images, validity of, 34
l?ixing fluids, 104
Flemming smear, 55
Flemming, with and without acetio,
formulre, 116
Formol,ll2
Formol-saline, lJ 2
Formol sublimate, 112
Fresh tissue, silver, 98
]!'rozen sections, 134
Gelatine solution, 95
Gentian violet formula. 165
Giemsa formula. 175
Glands,176
Glauber's salts, 92
Glycerine jelly, 83
Glycerine, whole mounts, 86
Glycogen, 69 et seq.
Golgi apparatus, 16!J
Golgi apparatuH, osmic methods, 172
Golgi apparatus smear, 59
Goodpasture's methylene blue, 174
Gray, Peter, medium for cementing,
88
Gray, Peter, mounting ponu
organisms, 03
Hanging drop preparations, 26
Hedon.Fleig saline, 2:1
LABORATORY'fEClllllQIJ11.
Langhan'H glyc'ngcu test, 71
Lecithin trst, 74
Leishmann blood smear, ,18
Living calls, examination of, 25
Lugol's solution, 71
Magnesium sulpht\tc, 92
Mann's fluid, 111
Mann, methyl blue eosin formula,
158
Mayer's acid hremalum formula, 146
Mayer's albumen, 128
Measurement under microscope, 7
Menthol, 92
Methyl benzoate, for clearing, 100
Methyl green acetic, 33
Methylene hIlle, for cartilage, 90
Microchemical tests, 62 el seq.
Micrometers, 7
Mierophotogmphy, 10
Microseopes, 2
Microtomes, 11
Mitochondria, 173
Mollu~c[1
fluid. 24
Mosquito Jarvie, 80
Muscle, 179
Myelin, lRI
Navashin's formula, 163
Nerve endiugs, 117
Nervous system. 181
N eutmlmountltnt, 85
Nile blue sulphate, 66
Nissl grullllles, 181
INDEX
130
Oil of Willtcrgl'('Cn, 100
Osmic method fol' Golgi bodil's, 172
Osmic test, f[Lt~, (j'1
Osmotic pressure, 2:3
OxuJic acitI, 08
J'arailin WI1X imbedding, 12:1
1'[1raglycogen, 70
PcrrilangulU1te of pobsh, b1l'l1oh, 58
Petrunkewitsoh fluid, lUI
pH,23
Picric chromic glyoogen method,
73
Piero-inc1igo-carmine, 158
Piero-nigrosin. 1.57
Polychrome methylene blue, 174
Pond life, Pete!' Gmy's method,
Progressive staining, 4U
Plll'i'~ medium, 80
ua
Hmoll!', Oal'lloy, 5:J
Smmtr (Sehltudillll or E(min), 41
Smears (living), 27
Smears, stained. :38
Solutions, how to make, 22
Stains, lao et se'].
Stemler dishes, 12
Sudan IV, (jfi
SUjJm-vital RtfLilling, 2!J-31
Suaa fixative, lOll
Tadllclcs, sectioning, 1511
Tap water substitute, 3()
Teeth, 180
Toxic examining mcdi[t, a:3
Trichlor[lcetic aokl, 180; and see
SUSI1.
Ultra-centrifuge, 184
Unoa'a stain, 17-4
Razor bh1da holdlll'R, II
Reduoing fluid formula, 171
Regaud's bichromate-formal, ll:1
Reguud'a hrematoxyliu formula, 147
BegressivD staining, 40
ltomieu, lecithin, 74
Bousselet's Rolution, \)2
Van Giesoll formula, 155
Vital staining, :20-31
Vitamin C, 77
Volut\n,71l
Safranin !tnd light grl'on method, llitl
Salt solutions, 2:1
Nanrll1r1tc, 85
Schaudinn's fluid formula, 110
Sehaudinn SI\1O[ll', 41
Rd!iff's reagent, 78
Washing out, 121, 122
Weigl method, 172
W dgl smear, eo
Whole mounts, 80 el seq.
Windaus-Bnlllswick te3t, 117
Wintergreen oil, 100
Wright, bloou stain, 48
Schneider, aceto-carmine formula,
151
Sc:hultll, cholesterol, 68
Y oCllm fluiu, 0:3
RN~
watcr (artificial), 24
Reetioll (Jutting, 1~5
Hil vel' line orgaIls, !J8
Small ohjects, handliug uf, 1:1:J
l'HlNTEII
J~
GREAT
JllllTAl~
Zenker-formel, 107
Zenker formula 107
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