MicroscopicTechnique for Zoologists

8/17/2019 MicroscopicTechnique for Zoologists

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1 A. H. I. b.::., C. P · l l t J l 4 7 · J ~ .,." 17.J,41:l 200U


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NOTES ONM I C R O S C O P I O ~ LTEOHNIQUE

FOPu ZOOLOGISTS


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" and then my wife and I to'ith great pleasure, but1vith great diiJicttlty before we cOllld corne to find themanner oj seeing any thing by my rnicroscope. At lastdid with good contmt, though not 80 much as J crcpect'When I come to understand it better."

SA1I1UEL I"'El'YS 14th August 10M"


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NOTES ON

MICROSCOPICAL TECHNIQUE

FOR ZOOLOGISTS

BY

O. F. A. P N T I N ~So.D., F.R.S.Reader in 1 nvertelrrate Zoology in

the Univer8ity of Cambridge

CAMBRIDGEAT THE UNIVERSITY PRESS

1948

IARI


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Printed 'in Great BTitain at lite Uni1 ersity Press, Cambridge(BTOoke Orutchley, UniVCTsity Printer)

and published by the Cambridge University Press(Cambridge, and Bentley lIollse, London)

Agents for U.S.A., Canada, and Indla: Macmillan

Pirst Edition 194(;Reprinted 1948


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CONTENTS

PrefaceReference Book


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vii

PREFACET H E following notes are based upon methods which have beenfound satisfactory by the staff of the Department of Zoology,Cambridge University, during the instruction of advancedstudents and those commencing research in zoology. EXI)erienceshows that the student needs some guide through the embarrassing number of methods offered to him by current handbooksof microscopy and histology. A selection of standard methods istherefore given here, and these are accompanied by COIl11nents onthe principles which underlie their use. They are set out in a formsuitable for use on the laboratory bench. In addition to these,other information and references are given to be a guide ill avariety of problems which arise in cOllnexion with microscopicalwork.

I t is a pleasure to thank Dr S. M. Manton, M1' J. E. Smith,Dr S. Smith und Dr A. Stock and others for many valuable

suggestions made during the compilation of these notes, and tothank lVlr J. R. G. Bradfield for checking numerous calculations.

C.F.A.P.

NOTE TO SECOND IMPRESSION

T H E reprinting of this book has given me the opportunity to

make a fe,,," corrections and additions. C. P. A. P.1948


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viii

R E F E R E N C E BOOKS

POT the use and Care of the ftlicroscope see:Gage, S. H. (1936). The lUicroscope. 16th elL New York: Comstock Pubishing Co.

And for an introductory account see:Beck, C. (19,1


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Part I

GENERAL METHODS

METHODS Oli' OBSERVATION

(a) Dil'ect observation under the microscope, of living or fixedmaterial, stained or unstained, should be made under critical

optical conditions. See Gage, S. H. (1936), or Beck, C. (1042).(b) Darlo-ground illnmlnation. See Carleton (1038), chap. IX.

Useful for observation of cilia, granules, etc., in living material.(c) Phase difference microscopy. A new method for unstained

material. Tissue and cell constituents are brilliantly differentiatedby optica1 interference. See Burch, C. R. & Stock, J. P. (Ul42),J. Sci.lnstnt1n. 19,71. Martin, L. C. (1f)47, Natw'e, Land., 159,827) gives a general account.

(d) .lI-luscular anatomy by polarized light (Imms's method).Mount in balsam and observe under crossed nicols. The relationsof the striated muscles of arthropods are very clearly shown.

(e) Polarized light and birefringence. For a general account ofthe theory of polarization see the Encyclopaedia Britannica,various editions. For the usc of polarized light in the analysis ofcrystalline and other structures see:Winchell, A. N. (1933). Elements of Optical Mineralogy. Part I,

ePrineiplcs and Methods', New York: J. Wiley and Sons;London: Chapman and Hall.Ambroilll, H. and li'rey, F. (1926). Das PoZarisationsmikl'oskop.

Leipzig: .Akademische Verlag:;gesellschaft M.B.H. .

(1) lI1icellm' strtlcture by birefringence. Birefringence in histological structure may be due to the intrinsic properties of themolecules of some substance composing it, or i t may be ofstructural origin owing to the presence of orientated micelles or

aggregates of molecules. Structural birefringence disappears ina medium of the same refractive index as the micelles, the wholethen becoming optically homogeneous (Picken, L. E. R. (1940),

PUT :r


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P R E PA R AT I O N OF M AT E R I A L

Biol. Rev. 15, 133). This disappearance can be sought by mounting objects successively in a series of media of graded refractiveindex such as follows: NDistilled water 1·33CZH 50H 1'3650 %glycerol in water l ' 40Glycerol 1·47Liquid paraffin 1'47Castor oil 1'49

Cedar oilCreosoteAnilinea- Chlorona phthalenca-Bromonaphthalene

N1'511·54

1·58

1'68

1'66

See also H{(Jndbook of Chemistry and Physics, 21st ed., p. 1618(Cleveland, Ohio, U.S.A.: Chemical Rubber I1ublishing Co.).

By the use of optical wedgcs and plates further importantinformation can be gained concerning the sign of the birefringence:see the l'eferences in the preceding section (e).

(g) Electron microscope. Sec v. Ardenne, M. (1940), ElectronenObermicroscopie. Berlin: Julius Springer. (Lithoprintrepl'oductiollby Edwards Brothers, Ann Arbor, Michigan.) For a brief popularaccount see Discovery (1943). 4, Bll . At present, this instrument

is only suitable for objects of very small size which can be safelyexamined dry and in vacuo. Theil' thickness should not exceedabout 2 fl'. Differentiation of internal structure in an organicparticle is very difficult to obtain, but the correct outline of anobject can be seen uudcr enormous magnification (e.g. 1 : 40,000).

P R E PA R AT I O N OF MATEl l IAL

(a) EXA:r. I INA' r ION OF L I V I N G M AT E R I A L

Always examine material alive if possible; the information gainedis often as unexpected as i t is valuable. Mount objects on amicroscope slide under a large cover-slip.

With oil-immersion objectives mount the object with as littlewater as possible under a very large cover-glass and use very thincedar oil. Otherwise the object will move during focusing.Lcitz's 'Nelkenol' is useful. .

A hanging drop on the under-surface of a cover-glass on a cell isbest for the examination of minute organisms. I t permits use of

. immersion objectives and allows free access of air to the specimen.The drop should be as shallow as possible. A cell may be


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4 P R E P A R AT I O N O F M AT E R I A L

Minttte d'issectioHs (crustacean mouthparts, etc.). Harding, J.(1939, J. Roy. Mic)'. Soc. 69, 19) describes an elegant instrument

for minute dissection. Fine needles~ r e

mounted in holders withsimple micro-movement obtained on the principle of a pantograph. The needles themselves are best made by dipping a finetungsten wire in molten NaN0 2 • This leaves a very fine point.

See also Komp, W. H. W. (1942), Publ. Hlth Rep., Wash.,57,1327.

Fine scalpel8 of all sizcs canbe made as follows. Place ahard steel safety-razor bladeobliquely between two levelblocks of hard wood held in avice (Fig. 1). Snap off the upperpart of the razor blade bypressing it over with a flatpiece of wood. A sharply pointedblade remains held between theblocks. Mount this blade in a

cleft stick or mapping-pen holderwith the aiel of marine glue orsealing wax. Fig. 1

(d) E X A M I N AT I O N O F I S O L AT E D C E L L S

Goodrich's method (1942, Quart. J. Micr. Sci. 83, 2·15). Preparea saturated solution of boric acid in 0'75 % NaCl (sea water orCa-free sea water for marine forms). Add about 2 d r o p ~ofLugol's iodine per 25 c.c. solution, giving the laUcr a pale yellowcolour \) Immerse the organism or tissue in a small volume of thesolution in a small dish. The intercellular matrix dissolves, andafter 2-3 days the individual cells will fall apart. Gently tap thedish to separate the cells. The cells will keep in saturated boricacid for many days, or even ·weeks. The method succeeds with agreat variety of animals and thcir tissues, e.g. Hydra, Lumbricus,rabbit.

Ranvier's method. Macerate the tissue for 24 hr. in 30 %alcohol.


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N A R C O T I Z AT I O N 5

(e) P n , E S E R v AT I O N OF N AT U R A L S H A P E

The shape of an active organism may be instantly prcserved byplunging it into absolute alcohol cooled in liquid air (LissmHnll,H. W. (1945), Nature, Lond., 156,391).

N A R C O T I Z AT I O N

I f possible, avoid mncotization. Prolonged exposure to anynarcotic advcrsely affects cell structure and ends in cytolysis.

I t is, however, often neccssary to narcotizc'to prevent distortionand rupture on fixation, and for work on living organisms andtissues. Always narcotize gently by gradual addition of thenarcotic without violent mechanical stimulation. The followingspecial methods are recommended.

SPECIAL METHODS

(a) A N I M A L S L I A B L E TO D I S T O RT I O N

The following method can be used in preparing most kinds ofsmall worm for fixation preparatory to section cutting, or forarranging organisms (such as Hydra) for whole mounts.

According to the size of the animal, coat a dried cover-slip orslide with paraffin wax. Narcotize the animal ill a drop of naturalmedium on the cover-slip, or by means of the slide lift the alreadynarcotized animal out of a dish of narcotizing medium.

Drain off excess fluid. Gently adjust the animal with a paintbrush. For small animals on cover-slips, cover with cigarettepaper moistened with narcotic, folding the edges on to the hackof the cover-slip. Then drop the whole face downwards on a dishof fixative. For larger animals on slides, cover with a strip ofcigarette paper. I f necessary wind a pieee of wool lightly roundthe slide to hold aU in place. Immerse in fixative. I f animals arevery sticky use, in place of the cigarette paper, a piece of old finebolting silk lightly rubbed with wax.

Do not t ry to remove animals from cover-slip or paper till

fixation is complete. I f they adhere at all strongly take animal,cover-&lip and paper to the clearing medium. The cover-slip thencomes away through solution of the wax and the paper can beremoved with less danger.


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6 N A R C O T I Z AT I O N

Perform all operations smoothly and deftly. Avoid continualreadjustment of the animal. When flaccid under narcosis its

tissues are very easily damaged. I f the animal is slightly disarranged, carryon: contraction on fixation will pull i t straight.I f you must rearrange it, flood with narcotizing medium, take upthe animal and start again.

(b) S E S S I L E A N I M A L SI f already retracted, narcotics often fail to relax these. This issometimes due to special properties of the muscles (Actinozoa),and sometimes to expansion itself being in part an active process

, resulting from muscular contraction. Therefore, let the animalsfirst become naturally expanded by placing them in clean waterand leaving them for some hours in a quiet, cool and dimly litdish of the smallest convenient size. Then anaesthetize gently andgradually in the same dish. Do not subject them to mechanicalagitation. In particular, avoid transferring the animals fTom onedish to anothel'. To fix, gently Temove as much fluid as possiblewithout disturbing the animal and then smoothly and rapidly fill

the dish with fixative by running i t into the bottom of the dishfrom a large pipette. Repeat, if excess medium has undulydiluted the fixative.

NARCOTICS

The following are rccommended for all general purposes:(a) 10 % alcohol. Recommended for fresh-water animals.

Make up from absolute alcohol not from low-grade spirit becausethis contains deleterious substances. Add the 10 %alcohol a littleat a time waiting till excitation subsides bcfOTc adding marc.Excellent for Hydra, flatworms, etc. Narcotizes in a few minutesto an hour according to spccies.

(b) MgCI2 • Recommended faT maTine animals including manysessile kinds (e.g. Actinozoa). Sea water diluted with an equalvolume of isotonic magnesium sal t solution is an ideal anaesthetic.Use 7'5 %MgCI2 .6H 20, or 20 %MgS0 4 • 7H 20. Superficial narcosisoccurs in a few minutes. After i t has supervened general narcosismay be hastened by gently injecting isotonic magnesium solutioninternally.

MIS magnesium salts (2'5 % MgC12 .6H 20) slowly narcotizesf r e s h ~ w a t e ranimals. I t l'cquires about 2 hr. for flatworms.


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N A R C O T I Z AT I O N 1

(c) Menthol. Recommended for difficult sessile organisms,both marine and fresh water (e.g. Polyzoa). Let the animal

expand in clean water in a quiet place, scatter a few crystals ofmenthol on the surface and leave overnight.(d) Ether vapour. Recommended for insects, arachnids and

terrestrial vertebrates.

(e) Tobacco smoke is an excellent narcotic for many ciliates(e.g. Parameciwn) and flagellates. I t ean also be used to slowand stop the cilia of Mytilns, and to narcotize Hydra. Fill a ShOl·ttube with the smoke. Invcrt over its mouth a slide carrying the

specimen in a drop of fluid. VVatch under a low power and removethe slidc as soon as narcotization is complete ( i - I min.). 'Thevapour from a drop of pyroligneous acid acts similarly but moreslo·wly.

I f these methods fail t ry(f) CO2 , Squirt a little soda-water from a siphon into the

water in which the animals are living.

(g),Ethyl

urethane,0·3-1'0

% in water or the appropriatenatural medium. This acts very gently and slowly (about 12 hr.for Lumbricus and Sabella). For vertebrates inject a solution insaline at body temperature.

Chloretone (0'005-0'05 %) or chloral hydrate (2 %) may alsobe used.

(h) ROU8selet's solution (J. R. Baker's modification). Recommended for Rotifera. I t must be freshly prepared;

2 % cocaine HCI90 % alcoholDistilled water

3 c.c.1 C.c.6 c.c.

Cocaine is unstable in solution. Eucaine, which is stable, may besubstituted.

(i) Gentle heating. Slowly raising the temperature towardsthe death-point (30-35° for British marine animals) may induceparalysis of the nervous system in a relaxed organism before

adverse histological changes occur. Allow organisms to relaxovernight in a bowl of water above a radiator at a suitabletemperature.


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8 F I X AT I O N

FIXATION*

The ideal fixative varies 'with the tissue, and one of the histologist's objects is to find the best fixative for the work in hand.But Heidcnhain's 'Susa', Bouin, and ' Flemming-without-acetic'are fixatives which, each for its own purpose, give very satisfactory results with a wide range of material. As in all histologicalwork success in fixation depends on practice and familiarity withthe method. Do not therefore use elaborate methods of fixationunless you have special reason. Moreover, if the standard methodfails, be certain that this is not due to lack of practice beforepassing to another method. I f animals contract rapidly 011fixation i t may be necessary to warm the fixative, thus 'Wilson,D. P. (1932, Philos. Trans. B, 221,237), uses Bouin at 3 ~ - 6 0 ° C .for the fixation of polychaete larvae. Do not t ry to fix too largean organism or picce of tissue. I f the object is large, cut off asmall portion and contrive to open up the structures to be studiedto the fixative. All parts of the tissue should be within a few mnl.of the surface exposed to the fixative.

Most fixatives act by precipitating protein constituents in sucha way that the cell and its contents retain in some degree theshape they possessed in life. But often during their action, andstill more during subsequent dehydration, important cellularconstituents are removed, that is, the lipoids. To preserve these,special fixativcs, such as Baker's Formaldehyde-calcium, areneeded and fat solvents must be avoided in subsequent treatment.

A. MICRO-ANATOMICAL F I X AT I V E S

(1) lIeidenhain's Susa. Probably the best general fixativethere is for material to be sectioned. Almost all stains act well onSus a-fixed material. Gross cell structures" e.g. protonephridia,arc prcserved well, but fine cytological detail, e.g. mitochondria,Golgi bodies, etc., is not. Its composition is:

HgCl 2 45·0 g. Trichloracetic acid 20 g.NaCl 5'0 g. Acetic acid (glacial) 40 C.c.Distilled water 800 c.c, Formalin (40

%HCHO) 200 c.c.

The first three items may be made up as a stock laboratory* See also Addenda, p. 75.


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F I X AT I O N 9

solution. For some marine animals (though not for all) there isadvantage in raising the NaCl content to 30 g. Nuel instead of

5 g. orsubstituting

seawater

for distilled water.Fix for 3-24 hr.Since collagen swells in aqueous media after exposure to tri

chloracetic acid, transfer di?'ect to 96 %alcohol (p. 17). This shouldbe made pale brown by tincture of iodine to remove any precipitateformed by the mercury. Do not touch fixative 'with metal: usea pipette, or wooden spills, or paint brushes mounted in glass orquills as sold by pharmacists.

(2) Bouin.Picric acid, saturated aqueous solution 75 c.c.Formalin (40 % HellO) 25 c.c.Acetic acid (glacial) 5 c.c.

Solution keeps indefinitely.

Fix 12 hr. or longer. Transfer direct to 70 % alcohol (p. 15).Material may be left in Bouin indefinitely. This makes i t very

suitable for use on collecting expeditions.I t is an excellent fixative for marine Invertebrata and most

mammalian tissues; good for nuclei; poor for cytoplasmic in-. elusions; bad for vertebrate kidney and some organisms con

taining numerous mucin cells. In these there may bc great distortion owing to swelling and shrinkage. Acldition of urea 1 %(Bouin-Allen) improves kidney fixation.1.-(3) Duboscq-Brasil ( = alcoholic Bouin).

Picric acid 1 g. Formalin (40 % HeHO) 60 c.c.Acetic acid (glacial) 15 C.c. 80 % alcohol 150 c.c.

A highly penetrating fixative suitable for animals covered byan impervious cuticle (Arthropoda, etc.). Fix for 2 hr. or more.Transfer direct to 90 % alcohol (p. Hi). With lal'ge or very impermeable specimens fixation must be longer, but if greatly prolongedi t causes brittleness.

To prevent hardening in Peripatopsis, Manton (11)37, Philos.Trans. B, 227,413) found i t desirable to fix for not more than 2 hr.and to complete the whole process of dehydration and embeddingin less than 24 hr.

(Cal'lloy, see p. 11, is also a highly penetrating fixative.Sometimes good for micro-anatomy of delicate tissues.).


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10

(4) Zenlcer.HgCl 2

K ZCrZ0 7

5·0 g.

2·5 g.

F I X AT I O N

Na ZS0 4 ·H 20

Distilled water

1 g.

100 C.c.The above can be made up as stock solution.

Acetic acid (glacial) 5 c.c. added just before use.

For flatworms, substitute formic for acetic acid (JI licrotomist'sVade-Mecum, 10th ed., p. 60).

FLx 3-12 hr. Wash 12-24 hr. in running tap ,vater. Thentransfer to 50 % alcohol (p. 18).

Zenker preserves fine cytoplasmic structure (mitochondria,etc.) better than Susa. I t must, however, be freshly preparedbecause it contains both oxidizing and reducing substances whichdestroy each other after some hours. Treatment after fixation ismore elaborate than with Susa. I t should not, therefore, be usedby the beginner until Susa fixation is thoroughly mastered.

There are several good variants of Zenker useful for specialpurposes, e.g. Zenker-forniol (HeIly).

B. CYTOLOGICAL F I X AT I V E SStrangeways, T. S. P. and Canti, R. G. (1927, Quart . .1. Micr.Sci. 71, 1) have shown that while many useful fixatives such asHgC12 may preserve the cell outline, the appearance of the fixedcytoplasm differs entirely from that seen in the living state.Cells fixed in OS04 solutions, on the other hand, give a veryfaithful picture of the living cytoplasm. The penetrating power of080 4 , however, is feeble, and the inner parts of tissues are there

fore poorly fixed by it. On the other hand, the outer parts areoften overfixed; they shrink and undergo changes which preventgood staining. Osmic fixatives should therefore be used for thestudy of cytoplasmic structUl"es within easy reach of the fixative,but not for the study of micro-anatomy.

All osmic fixatives keep badly and should be made up fresh.Keep in a clear bottle in a box 01' cupboard. You a m l O ' ~perceivedeterioration through a black bottle. Osmic vapour has a badeffect on the eyes and throat. OS04 is neutral, not acid.

While osmic fixatives preserve cytoplasmic structure, they donot fix nuclei well. These are best preserved by the action ofacetic acid which, however, has an adverse effect on cytoplasmic


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F I X AT I O N 11

structure. For cytological work one must choose whether topreserve the nuclear or the cytoplasmic structures.

(a) N U C L E A R F I X AT I V E S

(1) Bonin is useful for chromosome work.(2) Carnoy.

Absolute alcoholChloroformAcetic acicl (glacial)

60 C.c.30 C.c.10 c.c.

Fix for 10 min. to 3 hr. Transfer to 06%

alcohol (sevcralchanges) (p. 16).

Both (1) and (2) preserve cytoplasmic structure poorly.(3) Aceto-cannine is recommended for chromosome studies

(lIJicfotomist's Vade-1I1ecum, lOth ed., p. 142). Tease preparationswith rusty needles: the trace of iron so obtained is important.

(4) For special methods see Darlington, C. D. and La Cour,I.. F. (1042), The Handling of Chromosomes. London: Allen andUnwin.

(b) C Y T O P L A S M I C F I X AT I V E S

(1) Gatenby s Flernming-without-acctic' (Microtomisl's Vade-1I1ecum, loth cd., pp. 33 and 304) is an excellcnt cytoplasmicfixative.

1 %,chromic acid2% OS04

15 parts4 parts

To this should be added enough NaCl to make i t roughlyisotonic:

For marine invertebrates add of 15 % NaCI 5 parts.For other organisms add of 15 % N aCI 1 part.For small and delicate objects the F.W.A.' may be diluted two

.01 ' three times. Keep dilute mixtures isotonic.F.W.A. will not lceep and it is expensive. Malee up small

amounts as required.

Fix pieces of tissues not more than 5 mm. in diameter in strongF.W.A. for 24 hr. Wash 2-5 hr. in running tap water. Transferto 30 % alcohol. Dehyclrate by gentle stages, several hours ineach stage (p. 16).


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12 F I X AT I O N

(2) (a) Pm'malin, and (b) Baket s Formaldehyde-Calcium(194..1:, Quart. J. liner. Sci. 85, 1).

(a) Commercial formalin, or formol, contains about 40 % offormaldehyde (methanal) together with a variety of impuritieswhich mayor may not play some part in its properties as afixative. If diluted to eight times its volume with water, theresulting formalin (5 % HCRO) is a good cytoplasmic fixativewhich preserves lipoids. I t is also used for certain kinds of nervefixation. To prevent distortion consequent on osmotic changesduring fixation i t should be made up in sea water for marineanimals, and in suitably diluted salinc for others (e.g. 0'7 % NaCIfor terrestrial vertebrate tissues). Fix 48 hr. I f lipoid preservation is unimportant transfer direct to 50-70 % alcohol (p. 16).

Formalin is one of the few penetrating fixatives which can beused neutral and will therefore prcscrve calcareous structures.For neutral formalin add powdered CaC0 3 and a trace of phenolred as indicator. Shake and let settle. Do not use borates: theymacerate. I f for any reason Ca must be avoided, use NaHCO a .Prepare neutral formalin as required. I t may oxidize on long

standing.(b) All these advantages are combined in Baker's Formalde

hyde-calcium, particularly recommended for preservation ofGolgi bodies and other celllipoids. I t is

Formalin (40 %HCHO) 10 C.c.10 % CaC12 (anhydrous) 10 c.c.Distillcd water 80 c.c.Powdered CaCO s in slight excess

(For marine invertebrates use 10 % CaCI2 (anhydrous) 40 c.c.and distilled water 50 c.c.)Fix for 2-3 days.This is a neutral fixative. The CaC12 improves lipoid fixation as

well as preventing psmotic distortion.To preserve lipoids, store specimens in bulk or in gelatine

blocks inFormalin (40 % HCHO) 10 C.c.10 % CaCl 2 (anhydrous) 10 C.c.

10 % CdC12Distilled water

Powdered CaCO a in slight excess

10 C.c.

70 C.c.

Avoid fat solvents in subsequent treatment. Special methods


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F I X AT I O N : D E H Y D R AT I O N 18

are required for sectioning and staining; see section on 'Preparation of frozen sections', p. 26.

(c) F I X AT I O N OF Y O L K - L A D E N C E L l , S

Yolk-laden cells and embryos need special treatment. In additionto Baker's Formaldehyde-Calcium (see above) the following isrecommended:

Smith s Fonnol-Bichromate:

J{2 Cr207Formalin (40 % HCHO)Acetic acid, glacialDistilled water

0·5 g.10 C.c.

2·5 c.C.100 C.c.

Make up immediately before use; the bichromate and theformalin gradually destroy each' other.

Fix 24 hr. "Wash 12 hr. illruuning water. Transfer to formalin(4 % HCHO). Correct dehydration is important. I t must berapid to prevent brittleness, but if it is incomplete the materialwi1l be ruined. See 'Dehydration Chart' and the note on

'Dehydration of eggs and yolk cells' (pp. 14 and 17).I f jelly has to be removed, place eggs before fixation on blottiug

paper, remove jelly by slicing and rolling egg gently. Examinematerial for defects befOl'e proceeding.

C. F I X AT I O N OF PROTOZOA

See special methods (p. 57).

DEHYDRATION

IJehydrate thoroughly and remove alcohol thoroughly. Failure todo this quite completely is the commonest cause of poor histological results. I t leads to shrinkage and brittleness in paraffin,and deterioration of histological structure, defects for which thefixative sometimes unjustly gets the blame. Never return fluidsto the bottles of absolute alcohol, xylene (xylol), etc., kept foruse in preparing material for embedding. See the bottles are wellstoppered and remain so except in actual use.

Make sure that alcohols of 90 %strength and over are correctlymade up. For critical work i t is safest to make them'up directly


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14 D E H Y ] ) R AT I O N

by volume from absolute alcohol. I f made from rectified spirit,check the density which for

90 % alcohol a t 15° C. is 0·83495 % alcohol a t 15° C. is 0·816In rough laboratory practice commerciall'ectified spirit is sometimes used in place of 90 % alcohol, but since this may containas little as 4 % water, the passage into i t from 70 % alcohol maybe a severe strain on delicate organisms.

In dehydration transfer from fixative to the appropriate stagein the following series. Use small quantities in a small corked

specimen tube containing the specimen.l 'or ordinary micro-anatomical work:

50 % alcohol, 1-12 hr.70 % alcohol, 1-12 hr.90 % alcohol, 1-12 hr.Absolute alcahol (two baths), 2-12 hr.

95 % alcohol (1-12 hr.) may with advantage be emplayed beforeabsolute alcohol.

At each stage transfer the used alcohal to a dry dish lest a smallspecimen is accidentally thro.wn aut.

For very small pieces of tissue (a few mm. acrass) these timesmay, if absalutely necessary, be cut down to a minimum of nat lessthan t hr. for each diluted alcohal and an haul' far the absolutebaths.

In general, do. not prolong immersian in absolute alcohol andconcentrated alcohol beyond] 2 hr.; they harden. Over-hardened

specimens may be softened by soaking in a mixture of equal partsof alcohol, glycerol and distilled watel·.

For dioxane and other methods of dehydration, see Carleton(1938). and also Kissel', J. (1933, Cytologia, Tokyo, 4, 288).

Dehydration of eggs and yolle cells. Exposure to alcohol makesyolk harden and crumble. Therefore dehydrate rapidly. On theother hand, incomplete dehydration will ruin the specimen. Thecorrect duration of dehydration is thus very important. I t varieswith the nature and size of the material. Very small permeableobjects may be left no more than t hr. in each grade of alcohol.Larger and less permeable ones nced more time.

I t is advisable to avoid absolute alcohol because of i ts great


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D E H Y D R AT I O N 15

hardening action. This can be done by clearing from 95 %alcoholwith methyl benzoate or methyl salicylate. Clearing may then

be completed in cedar oil or benzene. Avoid xylene; i t hardens.Clearing and impregnating by Petel'ji's Celloidin-Paraffin isrecommended (p. 29).

Pixation and dehydration of very small objects. Cut off a pieceof glass tubing about I i x ± n. Narrow one end very slightly ina llame. When cold, plug this end with pyroxylin wool. P}'l'oxylinis cellulose tetranitrate; i t may explode if pressed into a hot tube.Stand the tube in fresh or sea water according to the habit of theanimal. Let the liquid rise almost to the top of the tube. Pipettein a number of animals. Lift out the tube and drain away almostall the water through the pyroxylin. Stand tube in suitablefixative. Fixative may also be dropped down the tube if desired.After fixation, dehydrate by passing the whole tube through thealcohols. I f desired, stain in passing. After thorough dehydrationin absolute alcohol lift out the tube, drain it till about n e ~ q u a r t e rfull of alcohol. Stand the tube in a very small dish (cut-off endof a specimen tube will do) containing methyl benzoate or clove

oil. These dissolve the pyroxylin (the methyl benzoate does sorapidly). Cover to exclude damp. The cleared specimens will fallto the bottom of the dish. I f necessary they can be transferred tosulutions of celloidin in these substances.

When the objects are numerous (e.g. marine eggs, embryos,etc.), fixation and dehydration can be done in the tube of a handcentrifuge. The specimens should be gently centrifuged to thebottom at each step and the supernatant fluid poured off. The

objects can, if necessary, be carried in the centrifuge tube finallyinto hot wax ready for embedding (see special methods forProtozoa, p. 59).

Fix 1 2 - 2 4 hr.}

D E H Y D R AT I O N CHART

B o t ~ i n

70 % alcohol (several changes to wash out picric), 2·10 hr.t90 % alcohol, 12 hr.}

Absolute alcohol (two changes), 12 hr.


25/88


26/88

D E H Y D R AT I O N

Schaudinn (for Protozoa)

Fix 15 min. (as directed on p. 58).1

50 % alcohol, 10-30 min.t

70 % alcohol (light brown with iodine), 10--:W min •.

90 % alcohol, 48 hr.t

Absolute alcohol, 1 hr.

Smith s Formol-Bichromate

Fix 24 hr.

Running tap water, 12 hr.t

Formalin (4% HeRO), 12-48 hr.

80 % alcohol, } hr.t50 % alcohol, ~ hr.

~

70 % alcohol, ~ 111'.t

95 % alcohol, -k hr.t

Methyl benzoate or methyl salicylate.

17

For subsequent treatment sec section on clearing, p. 19.For impregnation, Peterfi's Celloidin-Paraffin method is recommended (p. 30).

l ' M T

Fix 8-24 hr.t

S1lsa

96 % alcohol (light brown with iodine), 12 hr.

tAbsolute alcohol (two changes), 12 hr.


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18 D E H Y D R AT I O N : WA S H I N G

Fix 4-6 hr.t

Zenlcer

Running tap water, 24 hI',t

pO % alcohol, 5-10 hr.~

70 % alcohol, 5-10 hr.t

80 % alcohol (light brown with iodine), 24 hr.t

80 % alcohol (without iodine), 24 hr.t96 % alcohol, 12 hr.

tAbsolute alcohol (two changes), 12 hr.

WA S H I N G

(1) To wash specimens in tap watercut off the bottom of a test-tube A (Fig. 2).Wire a small piece of fine bolting silkB tightly over mouth. For very smallorganisms first cover the mouth with filterpaper. Stand the tube in a small dish filledwith water, as in Fig. 2. Insert specimenS. Plug the top with I t hollow COIlC ofcotton-wool. Place under a dripping tap.At the end of washing fill a smallspecimen tube completely full of water.'fake off the bolting silk with the washedspecimens and touch the surface of waterso that they sink to the bottom.

(2) K 2Cr20 7 • See that this isthoroughly washed out in water afteruse of fixatives containing it. Otherwisei t leaves precipitates. Fig. 2

B

(8) HgCl z. This is removed by iodine in alcohol at some stagein dehydration. Usually the iodine is in turn sufficiently removedduring the rest of dehydration. I f not the sections may at somestage be passed through very dilute Na2S20s (Carleton (1938),


28/88

C L E A R I N G 19

p. 120), lest traces of iodine may inhibit staining. This procedureis rarely necessary.

(4) Picric acid should be completely washed out duringdehydration. Traces increase electrification of paraflin ribbons.

CLEARING

Discard clearing agents that have absorbed moisture. Sec thatbottles are clean and dry before adding new clearing agent. Oldstocks may have a film of water on the bottom. The e1ficicncy ofanhydrous CuS0 4 in keeping xylene dry is uncertain.

(1) Xylene. Transfer from absolute alcohol to xylene tillspecimen is cleared: if less than 5 mm., for not more than 1 hr.,for larger objects up to 0 hr. Keep for a minimum time in thexylene to prevent brittleness. Delicate objects may be traIlSferred first to a mixture of . ~ xylene + lr absolute alcohol.

Test old xylene with litmus paper before use. I f acid, shakewith a little powdered CaC0 3 and filter. A trace of alcohol in

the xylene greatly increases its power to take up a trace of waterleft in the object.

Do not use xylene to clear yolky material: usc methyl benzoute.(2) Cedar oil, With a long thin dropping pipette run a few

c,c. of cedar oil carefully to bottom of a small specimen tubecontaining the specimens in absolute alcohol. Specimens at firstfloat at the interface. Gradually they clear and sink. Suck offthe alcohol. Change the cedar oil after a few hours. Specimenscan wait in cedar oil till required.

(3) Methyl benzoate (or methyl salicylate), and benzene (or cedaroil). I f specimens are to be embedded by Peterfi's CelloidinParaffin method (see p. 29), or i f absolute alcohol must beavoided, methyl benzoate or methyl salicylate may be used asintermediate clearing agents. These are not completely solublein paraffin, and before embedding in that, one must transfer'thespecimen to benzene or cedar oil.

Using the same method as for cedar oil above, transfer thespecimen from absolute alcohol to methyl benzoate (or methylsalicylate). Where there is danger of hardening by absolutealcohol (yolk or chitinous material), transfer from 95 % alcohol.


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20 S T O R A G E : M O U N T I N G M E D I A

Change the clearing agent twice or three times. Transfer tobenzene or cedar oil; thence to paraffin wax or liquid paraffin.

S TO R A G E

(1) Specimens are usually stored in 70 % alcohol.For most purposes a solution of

70 % alcoholGlycerol

95 parts5 parts

is better. Mislaid specimens do not desiccate completely in this.

A storage mixture of equal parts of alcohol, glycerol anddistilled water (Strassburger-Flemming solution) preventshardening, and softens dried material.

(2) Cedar oil is an excellent preservative, though expensive.(3) Methyl benzoate, with or without celloidin, is a good

preserving medium.(4) Specimens to be sectioned can be preserved indefinitely

when embedded in wax. But you can no longer see the specimen.Liquid paraffin however is an ideal preservative.

MOUNTING M E D I A

Refractive index

NHyrax

Saturated aqueous solutionof HgI 2 and K1

Canada balsamApathy's gum-syrupVarious artificial resinsEuparalGlycerine jelly50 % glycerol and waterWater

Apathy's gum-syrup is

1,82

1'68

1·53

1'52

1-£11-1-541'481'471·40

1'33

Soluble inXylene ( 'xylol')Benzene ('benzol )' 'Vater

Xylene, benzene, etc.WaterXylene, benzenc, etc.Absolute alcohol, xyleneWaterWater

Pure gum arabic 50 g.Pure cane sugar 50 g.

Distilled waterThymol

50 c.c.0·05 g.


30/88

M O U N T I N G M E D I A 21

I t is good for preservation of methylene blue and many otherstains, though not haematoxylill. I t sets hard.

Glycerine jelly isGelatineDistilled ·water

10 g.60 C.c.

GlycerolPhenol oryst.

70 c.c.0'25 g.

The ideal mounting medium for stained preparations shouldhave the same refractive index as the mounted object. This isabout N = 1'5-l for fixed and cleared cell constituents. Therefractive indices of Canada balsam and of YUl'ious proprictaryresins approach this value, and these arc therefore excellentmounting media.

I f i t is an advantage to monnt direct from absolute alcohol,or to avoid xylene (see section on fading of stains, p. ,18).Euparal is recommended. The refractive index of this, however,is too low for optimal l'esolution of the finest structures with anoil-immersion objective. The defect is small, but critical resolution depends on attention to all small factors. But where thehighest resolution is unnecessary, and where improved visibility

of chitin, spicules, etc., is desired, Euparal is satisfactory.

Aqueous media

'Yhen mounting in glycerol, Apathy's syrup and other aqueousmedia, transfer from ·water not from alcohol. Glycerol media havea very high osmotic pressure which may cause collapse andshrinkage even in fixed specimens. Transfer delicate objects viaintermediate dilutions of glycerol (many hours in each).Alternatively, place in excess of very dilute glycerol and slowlyevaporate (cf. 1\.:1iCi'otomist's TTade-1I1ecum, lOth ed., p. 600).

Glycerol or ,Yater mounts must be sealed to prevent absorptionor evaporation of water. Success depends on using thoroughlyclean slides and cover-slips. After covering the object suck awayall trace of fluid beyond the edge of the cover-slip. Do this with apipette pulled ont to a very fine point which has been bentthrough 45°. Then wipe round with pointed slips of clean filter

paper, taking great care not to draw air under the edge of thecover-slip in so doing and not to move the cover-slip. llaint theedge with gold size. Give a second coat when the first is dry_ The


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22 W H O L E M O U N T S

best results are obtained if round cover-slips are used and thesize laid on by a spin of a turn-table.

A thick solution of marine glue in xylene may be put on first.Build up the seal with several layers each applied when theprevious one is dry. Marine glue never completely solidifies, andthe slight flow with thermal expansion and contraction preventscracking. Cover the marine glue seal with gold size.

Glycerine jelly is applied melted. Warm the jelly and warm theslide. Place a drop on a slide, transfer the organism on to thedrop from 50 % glycerol without too much of the fluid. Coverwith a warmed cover-glass. Try a blank slide and cover-slip firstso as to gauge the quantities.

With A p a t h y ' ~syrup transfer direct from water. Withdelicate objects transfer to diluted syrup, and then concentrateas with glycerol (Langer on, 193 11,).

Polyvinyl alcohol provides a new aqueous mounting medium.Moreover, tissues can be embedded in i t for section cutting(Lubkin, V. & Carsten, M. (1942), Science, 95,633; Downs, W. G.(1943), Science, 97, 539).

WHOLE MOUNTS: PREPARATION

DISSECTION OF SMALL SPECIMENS

(a) For some purposes i t is convenient to clear in clove oil,which makes the specimen brittle. Appendages are then easilyremoved.

(b) Mounts can be prepared to show the relations of internalstructures by staining, embedding in paraffin and then slicingtissue away to permit adequate view. Dissolve the paraffin inxylene and mount.

UNST AINED OBJECTS

(a) Setde, spicules, etc. These are rendered most visible inmedia differing widely from them in refractive index, such asdilute aqueous media (N =? 1·33), or a saturated solution of HgI2in KI (N=1·68). Permanent mounts may be made in Euparalor glycerol jelly or in Hyrax (N = 1·82; see Microtomist's VadeMecum, lOth ed., p. 229).

(b) 'Tracheae. '1'0 demonstrate tracheae make a saturated


32/88

W H O L E M O U N T S 23

solution of Sudan black in a mixture of equal parts of olive oiland kerosene. Drop the fluid on the narcotized or freshly killed

insect. The black fluid runs into the tracheal system, displacingthe air. The insect may then be lightly rinsed in xylene andexamined in air or in an aqueous medium.

(c) Small arthTopods (mites, etc.). A useful combined fixativeand mounting medium is gum-chloral:

Gum arabicChloral hydrate

50 g.100 g.

32 c.c.100 C.c.

Leave slide in oven at 50 0 C. for a few days. Seal with gold size.(d) Resemblance to liv'ing condition. Fix in formalin (5 %

HCRO) so as not to destroy fats. Mount in glycerol media. Therefractive index of these is higher than water so that the clearingeffect just about offsets the opacity due to fixation of the protoplasm. The index is, however, low cnough for unstained setae,etc., to remain clearly visible.

(e) Small and delicate objects (e.g. Rotifera, Volvom, etc.).A dilute aqueous medium cvades the necessity of SUbjecting the

object to deformation dming dehydration. The low refractiveindex permits even cilia to be seen unstained. With 01' withoutprevious narcotization fix with a mild fixative, e.g. osmic vapour,or formalin (2 % HClIO). Mount in fOl'Inalin (2 % HClIO) in1 % cupric acetate. I t is bcst to mount in a shallow hollowground slide. This alloWS a very good seal. Use a No.2 cover-slip.To avoid breakage use rather low powers (6 mm. or more). Viewdirect and also under dark-ground illumination.

R.ipart and Petit 's fluid serves as a gentle fixative and a preservative for delicate organisms. I t is

Camphor waterDistilled waterAcetic acid (glacial)

75 C.c.75 c.c.

1 C.c.

Cupric acetateCupric chloride

W H O L E MOUNTS: S TA I N I N G

(a) Bomm Carmine (Grenacher)

0'30 g.O·30g.

Make a concentrated solution of carmine by adding the powderto 4 % borax solution and boiling the mixture for i hr. The liquid


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W H O L E M O U N ' l ' S

is diluted with an equal volume of 70 % alcohol, allowed tostand for a time, and filtered.

Short way. Transfer the object to be stained from 70 % alcohol,or less, to the borax carmine. Stain until the object is justthoroughly penetrated (about 10 mill.). Thoroughly differentiatein acid alcohol (4 drops of strong HC} to 100 C.c. of 70 % alcohol),lllltil the object assumes a bright transparent appearance.Dehydrate in 90 % alcohol (10 min.); pass to absolute alcohol,two lots (5-30 min. each); cleal' in xylene and mount. Mounts arecommonly spoilt by being far too heavily stained.

Long way. Stain for 6-24 hr. Differentiate in acid alcohol tillouter layers look rather white and inner parts dirty pink. Thistakes several days to 6 weeks. On mounting, various organs showvarious shades of red and orange; outer tissues clear or yellow.An excellcnt method, pmticularly after Bouin fixation.

(b) Ehrlich's Acid Hacmatoa:JvlinHacmatoxylin 2 g.Acetic acid (glacial) 10 c.c.

Glycerol 100 C.c.

Absolute alcoholWater

Alum in excess

100 c.c.100 C.c.

Dissolve the haematoxylin in the alcohol, then add the acid,then the glycerol and water. The mixture is allowed to ripenuntil i t assumes a dark red colour. rrhe stain improves withage.

Transfer to stain from 70 % alcohol. Stain until the object isof a dark Llue-black colour. Wash in 70 % alcohol. Differentiatein ad d alcohol, watching the process carefully. The object willturn a reddish colour and should bc 'blued' by washing in tapwater which is usually s l ~ g h t l yalkaline, or may be rendered soby the addition of a drop of ammonia. Dehydrate, clear andmount.

(c) Chlorazol Black E (Cannon, H. G. (1941), J. Roy. Micr. Soc.61, 88)

An excellent stain for 'Chitin ' as well as being a good generalstain. Stain progressively in a saturated solution of the stain ill70 % alcohol (abou.t 20 min.), wash with 90 % alcohol and leavein this for some time, according to the thickness of the specimen.Clear as usual.


34/88

W H O L E M O U N T S 25

(d) Robinow's methodJor cell outlines (1986, PTOtoplasma, 27_, 86)

Wash the surface of the object in water or saline. Half aminute in 20 % dilution of Fl'og Ringer is good for fresh-watersponges.

(1) l"ix in the dade for I - I min. (till the specimen is white) inAgNO a 1'0 g.()S()4 0·1 g.Distilled water 100 c.c.

(2) Wash thoroughly twice with a large excess of distilled

water.(8) Transfer to daylight or strong artificial light and develop

in 5 % hydl'oquinone.(4) Before the specimen is too dark wash well with distilled

water. Transfer to 50 or 70 % alcohol. Counterstain if desired.Dehydrate, clear and mount.

WHOLE MOUNTS: MOUNTING

(a) Large objects. Support the cover-slip with two strips ofcelluloid, Fig. 8 (a). Use plenty of balsam so that there is an

W a\ f

b~ I

Fig. 8

\a

excess (b) at the two ends of the cover-slip. On drying this ispartly sucked in. Add more balsam at the ends during drying ifnecessary. When quite dry the excess balsam is easily removedwith a safety razor blade. A weight of about 20 g. (w) on the

cover-slip helps to flatten the object.(b) Small objects. (i) Arrange these as desired face downwal'ds in a drop of rather thick balsam on the cover-slip. Set todry, covering with a slightly raised watch-glass to protect from


35/88

26 F l t O Z E N S E C T I O N S

dust. When quite dry place a chop of thin balsam on the slideand lower gently on to the cover-slip.

(ii) Objects dealt with by the pyroxylin tube method (seeabove) call be transferred to a drop of thick celloidin in clove oil011 a cover-slip, and suitably arranged. The whole is now hardenedby exposure to CHCl avapour (see below:' Celloidin sections', p. 33),after which i t may be transferred to xylene and mounted, ortaken back to equal parts of ClIC1 3 and absolute alcohol and then,to 90 % alcohol, etc., for staining.

P R E PA R AT I O N O F F R O Z E N S E C T I O N SIn certain histological methods i t is desirable that the objectremain in an aqueous medium. This is particularly necessary wherefats must be preserved unaltered. In such cases the preparation offrozen sections of material embedded in gelatine or gum-arabicsolutions is necessary. The following techniques are recommended(see Baker, J. R. (1944), Quart. J. Micr. Sci. 85, 1, for fulldescriptions ).

E M B E D D I N G AND S E C T I O N I N G

After fixation with Baker's formaldehyde-calcium wash the flXedmaterial for 4 hr. in i'unning water. For treatment after otherfixatives see Carleton (1938).

Soak 25 g. gelatine in 100 C.c. of 0'25 % aqueous cresol (preservative) for I hr. Then warm till all is dissolved. Strain throughmuslin.

(1) Soak. the specimen in the gelatine solution a t 37° C. for

24 hr.(2) Gel (preferably in a refrigerator) and cut out the block

to shape.(3) Harden block in Baker's formaldehyde-Cd-Ca fluid for

24 hr.(4) Wash in running water for 4 hr.(5) Section a t 15 (1-. For details of the freezing microtome see

Carleton (1938).

T R A N S F E R E N C E OF SECTIONS TO S L I D E S

Dilute the above gelatine solution ten times, to 2·5 g. gelatineper 100 c.c. of 0·25 % aqueous cresol.


36/88

F R O Z E N S E C T I O N S 27

(1) Wann this solution in a paraffin oven. Immerse cleanslides in it, and let these warm up

. (2) Remove slides. Wipe the back. Prop against a wall todry, gelatine surface inwards.

(3) Immerse a dry slide in distilled water together with thesection. Withdraw it, holding section in place with a pin.

(4) Dry round the section and the back of the slide.(5) As soon as the slide is dry expose i t to the vapour of

formalin (40 % HeRO).(6) Transfer slide to filtered formaldehyde-Cd-Ca fluid.

STAINING F R O Z E N SECTIONS: L I P O I D STAINS

Frozen sections may be stained in the usual aqueous stains.Lipoid stains: Sudan black and Sudan I V (Schal'lach R). The

use of these dyes to stain fatty substances in cells is stronglyrecommended. The dye Nile blue should not be used for thispurpose. Its action is complex, and the old suggestion that withit a red colour indicated neutral fat whilst a blue colour indicated

fatty acid is certainly false (Lison, 1936). The Sudan dyes, on theother hand, specifically dissolve in lipoids. Both Sudan black andthe red Sudan IV are taken up by triglycerides, but the formerhas the greater affinity for other lipoids.

The same method applies to either dye. Make a saturatedsolution of Sudan black in 70 % alcohol. Saturation is essential.There must be excess dye, and several days must be allowed forcomplete solution. Filter the solution the day i t is used.

(I) Remove the slide from formaldehyde-Cd-Ca. Wash i t for3 min. in running water.

(2) Transfer i t fairly quickly, via 50 and 70 % alcohol, toSudan solution for 7 min. Let the slide rest obliquely with thesection downwards to avoid collecting a precipitate.

(3) Pass through three successive lots of 50 % alcohol (30 sec.or less in each).

(4) Rinse in distilled water.(5) Counterstain in Mayer'S Carmalum (or Mayer's Haemalum

for Sudan IV).(6) Wash in running water for 3 min.(7) Mount in glycerol and seal (p. 21).


37/88

28 PA R A F F I N I M P R E G N AT I O N

PREPARATION OF SERIAL SECTIONS

BY PA R A F F I NCLEANING AND SOFTENING

See animals are free from grit before fixation. I f possible, starveanimals a day or so in order to clear the gut. Avoid dust reachingreagents and paraffin bath. Minute particles of grit in the waxblock are a common cause of damage to scrial sections. Decalcifyin 3-5% HNO a in 70% alcohol if necessary. Unless newlymoulted or soft, embed Al'thropods in celloidin (p. 33). beforeimpregnation. Soften dense chitin in Diaphanol eMiCl'otomist'8Vade-Mecum loth ecL, p. 597}.

IMPREGNATION

(a) Pa1'lljfin 1\<

For small specimens (5 .mm.) transfer from xylene, benzene, orcedar oil to paraffin (m.p. 52-60° C.) for 1-3 hr. in all, changingthe paraffin once or twice. Transfer with the minimum of clearingagent. Take up the specimen on a warm section lifter. Tilt to drainslightly. Rapidly insert and withdraw lifter from wax. Delicatespecimens in xylene 01' benzene should first be transferred to thesame solvent warmed on top of the embedding oven and satul'atedwith chips of paraffin.

Do not transfcr very small objects from xylene, lest theyevaporate. Large objects in cedar oil should be rinsed in xylenebefore transference to wax. Rcmember tha t cedar oil diffuses out

of large objects in molten wax rather slowly. Any object may betransferred to wax from liquid paraffin.For ordinary work (room temperature 15-20° C.) use wax of

m.p. 52-54° C. Sections can be cut with i t down to 611-. Fo];thinner sections, down to 211- use hard wax. There is rarelyadvantage in very thin sections. Hard wax helps to support brittleobjects. For hard wax, paraffin (m.p. 60 0 C.) lllay be used. Butthe following mixture (Waterman, H. C. (1939), Stain Tech. 14, 55)is nearly as hard· and has the advantage tha t the low llleltingpoint (51.13° C.) permits use in the same oven as ordinary 52° C.

* Steedman, II. F. (1947, Quart. J. Micr. Sci. 88, 123), describes a valuablenew embedding medium, ester WaaJ.


38/88

PA R A F F I N I M P R E G N AT I O N 20

paraffin wax. This wax cuts excellently on the microtome, butcare is ·needed in flattening the paraffin ribbon because the

sections flatten only just below the melting-point.Paraffin (56 0 C.)Stearic acid

80 parts16 parls

SpermacetiCeresin

Melt, stir well and filter through cotton-wool.

3 parts1 part

Embed yolley material in soft wax (m.p. < 50 0 C.) for theminimum period needed to impregnate. Harden the block in ieewater before cutting.

(b) Peter-fi's Celloidin-Pa;mffin,

Small and delicate structures may undergo distortion orrupture with ordinary_paraffin embedding. They can be supportedby first embedding in celloidin. The celloidin block can in turnbe embedded in paraffin. A method of' doing this is given onp. 35. Most of the advantages of double embedding can beachieved more quickly and easily by Peterfi's method of prcimpregnation with

celloidin.(i) Normal method

After dehydration in absolute alcohol proceed toMethyl benzoate + 1% celloidin, 3-5 hr.Run the solution to the bottom of the alcohol and let the

specimens sink before :removing the alcohol (i.e. proceed as inclearing with cedar oil) .

.j,

Fresh methyl benzoate + 1 % celloidin, 3-5 hr.j,Further fresh methyl benzoate -I-1 % celloidin, 12-24 hr.

Specimens may be stored in this if desired.t

Benzene, 15 min.i-

Fresh benzene, 15 min.t

Benzene at 30° C. (on top of oven) saturated with paraffin wax,15-30 min.

tParaffin as usual.


39/88

BO PA R A F F I N E M B E D D I N G

(ii) For yolk-laden eggs and cells

Dehydrate via 30, 50, 70 and 95 % alcohol a hr. in each).tMethyl benzoate celloidin (as above), 1-2 hr.t .

Benzene, 10 min.t

Soft wax (m.p. 45° C.), i hr.t

Fresh soft wax, t hl'.Or embed directly.

EMBEDDING

Embed in a deep watch-glass. This should be first smeared withglycerol to prevent the paraffin sticking to the glass after cooling.Orient the specimen with a hot needle. Mark the exact positionof the main. and tran.sverse axes of the specimen by pushing ashal'p chinagraph pencil into the edges of the cooling wax plate.Make additional marks to indicate the anterior end of the specimen (Fig·. 4 a). To prevent crystallization of the wax cool the

a c

d e--

irili1«1iliJ

FJg.4

watch-glass rapidly. Hold i t on the surface of the water till afirm ' skin' forms on the wax, and then slide i t vel'tically on edgeinto the water (not horizontally).

Pal'affin wam -I- t % cel'esin is less liable to crystallize than plainwax. I t cuts as well or better.

Hard paraffin wax (60 0 C.) cooled in water at room temperatureis liable to crack. Cool i t in water at 25-30° C. (Wilson, D. P.(1932), Philos. Trans. B, 221, 231).

ORIEN' l 'ATION

Very small objects are more easily seen if stained with eosinduring dehydration. Orient them in the hot wax with the aid of


40/88

PA R A F F I N S E C T I O N S 31

a binocular microscope. I t may be advantageous to arrange themin the wax on a small rectangular plate of VIva, or of animal

tissue,stained with borax

carmine. Or, a windowmay be cut in

a small rectangle of thin ash-free paper (e.g. decalcified cigarettepaper) stained with chlorazol black. The little frame so made isthen impregnated and laid in the hot wax in the embeddingwatch-glass. The object is then accurately orientated within theframe as in Fig. 4 c. The frame is cut away when the block ismounted (Fig. 4 d).

For orientating small specimens by means of celloidin blockssee section on 'Double embedding for small objects' (p. 35).

CUTTING OF SECTIONS

Cut out a rectangular block of wax containing the object, determining first of all the plane in which the sections are to be cut.Seal the block on to the microtome block holder by melting thewax on the latter with a hot knife (Fig. 4 b). Trim the block sothat the face which is to meet the microtome knife is parallel tothe opposite face. This ensures that on sectioning the resulting

ribbon will be straight. Trim the faces with single clean cuts of asharp scalpel. Successive sections in the ribboIl may break apartif the block faces are irregular.

Cut one of the lateral faces of the block obliquely so thatsections in the ribbon will be shaped as in Fig. 4 e. This preventsconfusion of direction when mounting the ribbon. In general, cutsections 8-10 .t thick.

I f sectioIls of a hard wax block fail to adhcre as a rib bon, coatthe block with softer wax (50-52° C.). Then cut away excess softwax from the sides of the block. Avoid coating with soft waxunless necessary: i t makes the ribbon more difficult to flatten.

Cut hard wax blocks a t a room temperature a t or just above15° C. if possible.

Chill soft wax blocks (50 0 C. or less) in ice water before cutting.Directions for rectifying faults which may arise duriIlg cutting

are given in Carleton (1938).

T R A N S F E R E N C E OF SECTIONS TO S L I D E S

Clean slides are essential if sections are to adhere. The best wayto clean sUdes is by using the pastes sold for cleaning windows


41/88

32 PA R A F F I N S E C T I O N S

(see these have no abrasive in them). Failing that use acidalcohol, or cleaning fluid if necessary.

Have ready some albumen adhesive. That is, a mixture ofwhite of egg 50 c.c., glycerol 50 c.c., and sodium salicylate I g.,or other preservative. Placc a very small drop on the slide andrub on well with a clean finger. Cut the ribbon into lengths equalto about -i length of the slide, and with needles or paint brushesplace the lengths-dull side upwards- in series so tha t the firstsection is in the top left-hand corner of the slide and the lastsection on the right-hand at the bottom. With a diamond, or witha glass pencil, number the slide in the bottom right-hand corner.I f the slides be always numbered in that way i t will be easy todetermine on which side of the slide the sections are mounted.and the chance of their being rubbed off accidentally duringsubsequent phases of staining and dehydration will be greatlylessened. Gently flood the slides with cold distilled water andplace on a hot plate at a temperature slightly below that of themelting-point of the wax employed. (Remember tha t sectionsfrom a hard wax block coated with soft wax are liable to separate

if the soft wax melts.) The ribbon will extend on being warmed,and when the sections are flattened the water is run off. Curvedribbons may be gently straightened while extending on the warmplate. Dry the slides overnight in an upright position on the ovenor at a temperature near BOc C. At low temperatures the sectionsmay not stick on drying.

Sections of some materials are very liable to float off the slide,particularly if prolonged treatment in dilute acids happens to benecessary during staining. To ensure adhesion in such cases takethe slides down to xylene, then through absolute alcohol to 90 %alcohol. Then dip in a 0·25, % solution of celloidin in a mixture ofequal parts of alcohol and ether. Transfer to 70 % alcohol toharden the thin celloidin film. Proceed to stain as usual.

NUMBER OF SECTIONS P E R S L I D E

Always use normal 3 x I in. slides unless for some special reason.Even with the smallest object do not t ry to get more than three

rows of about twenty sections each. Leave an ample marginb ~ t w e e nthe sections and the edge of the cover-slip, otherwise youcannot use an oil-immersion objective properly.


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C E L L O I D I N S E C T I O N S 83

Remember, a specimen 8 mm. long will give 1000 sections of8/J-. Usually you will not have the t ime to examine each of a

1000 sections. 'l'herefol'e do not cut the whole specimen and mounti t on some twenty slides. Cut and mount just what is relevant toyour work in hand.

P R E PA R AT I O N OF C E L L O I D I N S E C T I O N S

Celloidin is a pure collodion (nitrocellulose). I t is soluble in amixture of equal parts of alcohol and ether, or in methyl benzoate,or in clove oil. Thick solutions in these solvents are 'hardened'or gelled when exposed to xylene or the vapour of chloroform.Celloidin embedding is useful for

(1) hard or brittle objects;(2) the preparation of thick sections (lOOp.);(8) the orientation of minute organisms by embedding a

celloidin hlock in paraffin.

The disadvantages of celloidin embedding are

(I) i t is a very slow process;(2) i t is hard to prepare serial sections (except in the case ofthick sections);

. (3) celloidin sections wrinkle; exposure to cthel' vapourremedies t.his;

(4) both fixation to the slide and staining are more difficultthan with paraflin sections.

The general method is fully described in Carleton (1938,pp. 58 et. seq.). Two special methods involving celloidin embeddingare given below.

T H I C K C E L L O ID I N SECTIONS

The following method is one of many variants and is based onDennell, R. (1940, Sci. J. R. Call. Sci. 10,83). Thick sections are ofgreat value in microanatomy, and they are particularly suitable fororganisms 1-2 cm. long where the anatomical relations can bemuch more clearly studied by cutting into a few slices about 100 p.

thick than with the more usual and more numerous sections of 10 /J-.rfo prepal'e solutions of celloidin, wash the celloidin chips for

several hours in running water, dry well On filter paper in thePMT 3


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CELLOIDIN SECTIONS

warm, soften in absolute alcohol 12-24 hr., add an equal volumeof ether, agitate at intel'vals for some days. Make 2%, 4% and

8% solutions.

cFig. 5

The specimen is passed tlll'ough(1) Absolute alcohol.(2) Equal parts of absolute alcohol and ether.(3) 2 % celloidin in alcohol and ether (several days).(4) 4 % celloidin in alcohol and ether (several days).(5) 8

%celloidin in alcohol and ether (a week). Then:

(6) Make a paper ring from a gummed label as in Fig. 5 A.Half fill with 8 % celloidin.

(7) Harden by placing in a covered dish together with cottonwool soaked in chloroform.

(8) When hard, thoroughly soften the surface with a fainttrace of alcohol-ether, fill with 8 %celloidin, insert the object andorientate. Bubbles may be burst by cautious touching withether (Fig. 5 B).

(9) Hardcn overnight or longer in a covered dish by means ofthe vapour from a cotton-wool pad soaked in chloroform.

(IO) Remove the block by slipping a razor blade under it.


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CELLOIDIN SECTIONS 35

( t l ) Clear in cedar-wood oil + a few drops of absolute alcoholand chloroform (several hours). Clear difficult material in

CHel s 4 parts by weight Alcohol 4 partsOriganum oil 4 parts Phenol 1 partCedar oil 4 parts Na2S04 anhydrous 1 part

(12) Trim block as in ]'ig. 5 C. Wipe off excess cedar oil, andthen mount with a few drops of 8 % celloidin on the holder ofa Jung sledge microtome. Use chloroform vapour to harden theattachment of the block.

(13) Moisten the knife and block with cedar oil. Cut sections

100 JL thick. Transfer at once to cedar oil. Mark every five sectionssuccessively by clipping (see Fig. 5 D). Put each series of five ina separate dish. Stain and mount scctions individually.

For staining, pass via xylene to a mixture of equal parts ofabsolute alcohol and chloroform (not to pure absolute alcohol),then to 90 % alcohol, etc. Stain with Mallory or chlorazol black.

Clear from 90 % alcohol via a mixture of equal parts· ofabsolute alcohol and chlOl'oform (avoid absolute alcohol alone).

l'11ence pass to pure chloroform, to cedar oil and to balsam.D O U B L E E M B E D D I N G F O R SMALL O B J E C T S

D. P. Wilson's method (1933, J. Roy. Micr. Soc. 53, 220).Prepare a treacle-thick solution of celloidin in equal parts ofalcohol and ether. Add an equal volume of clove oil; stir well.

- - - - - E o s i n absolute ftlcolibl with specimens

- - - - -C love oil

- - - - -'I 'hick celloidin syrup mixed with anequal volume of clove oil

- - - - - T h i c k celloidin syrup

- - Cork support

Fig. 6


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36 CELLOIDIN SECTIONS

Place in the warm, occasionally stirring till all smell of ether hasvanished. Thin down with clove oil till the viscous residue is alittle more fluid than treacle (at room temperature a 0·1 g. leadshot should sink 1 in. in a minute).

Stain specimens in eosin in absolute alcohol to render themvisible. Place in a small upright closed glass tube as in Fig. 6.After a few days the specimens sink into the thick syrup. Thenpipette off the solutions above it.

A _ o : ~ ~ 8 T : : : : : : : I.----1 - - - -

'-b- 2 ~ 4. . . 5B D

X · ~ 4.Y3 ~ · · ~

y

F

Fig. 7

Fix a ring (glass, cardboard or metal) about ~ i n .diam. x lu in.high to a slide flooded with molten paraffin wax. Place the slidein a small Petri dish. Fill the ring with celloidin. Transfer thespecimen to i t and orientate under the microscope (Fig. 7 A).To harden, transfer Petri dish and slide to a larger dish with levelbottom, together with a small beaker of chloroform; cover. I f eftovel'night at 16-18° C. the mass will be hardened next moming.Fill Petri dishes with xylene, and after an hour or two transferthe celloidin mass to cedar oil.

Using a razor blade cut out a rectangular block of celloidin witha vertical surface parallel to the future cutting plane (Fig. 7B, C).To indicate the position of the object in the block cut off edges


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S T A I N I N G S E C T I O N S 3'7

to a point at the opposite, bottom, right-hand end from thespecimen in Fig. '7 C-E.

Embed in hard paraffin, tr im this parallel to the embeddedblock, orientate (Fig. '7 F), and section.Mount in the usual way but complete flattening of the celloidin

by exposure of the drying slide to ethel' vapour. Dry very slowlyto avoid crinkling of celloidin, and for a long time to ensureadhesion of sections.

ST AINING PA R A F F I N SECTIONS

P R E PA R AT I O N FOR STAINING

rrransfer slides of paraffin sections to xylene for 1-3 min. and thenthrough the series:

Absolute alcohol90 % alcohol70 % alcoholDistilled water

t - l min.t - l min.t - l min.

to whateverstage is

requiredfor staining.Only for very delicate work (e.g. Protozoa) is it necessary to

pass thin sections or films through 50 or 30 % alcohols.

OBJECT Ol? STAINING

The best stain depends on the object in view. One may stain toshow cytological detail, to elucidate the anatomy of a smallorganism, to trace the distribution of particular tissue elements,such as elastic fibres, 01' to detect the presence of a specificchemical substance. The most important staining method is stillHeidenhain's Iron Haernatowylin. Befm·e. all other methods thismust be practised till i t ~ C01'1'ect use is automatic. This stain isunsurpassed for photogl'aphy and for the resolution of structUl'eunder critical illumination. I t is the best stain for cytologicaldetail. I t can also be used by itself as a micro-anatomical stainwhel'e tissues al'e clearly differentiated, as in arthropods, and inmore difficult cases by making it the basis of a polychl'Ome staill

such as Masson's Ponceau-light green (Trichrome) method.Hansen's Triomyhaematin. is a useful alternative to IronHaematoxylin. I t gives a highly selective staining of the nuclei


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38 STAINI:r, ;rG SEC ' I ' IONS

which is not more disturbed by subsequent treatment than isthe case with Iron Haematoxylin. Cytoplasmic structures stainlittle, or, when the solution is fresh, not at all.

In general, for micro-anatomical work a slight modification ofthe original Mallory's Triple .stair), method is admirable, andpractice in this is recommended. Various tissues are differentiatedby their t int in a most striking manner. The method has thevirtues of speed 'and simplicity. Provided all the operations al'estandardized, i t is the easiest method by which a begillner IIlaystain a long series of slides in precisely the same manner and tothe same extent. The method suffers from certain defects.

Mallory preparations are liable to f ade -a t best in a matter ofyears, a t worst in a few months-though for most purposes, otherthan for demonstration specimens, they survive long enough.Fading can be postponed with suitable mounting media (seep. 48). Compared with some polychrome stains, Mallory is lessprecise: that is, the differentiation of tissues into those that willand those tha t will not take each component dye is not alwayssharp. A modification of Mallory known as Heidenhain's Azanmethod is more precise than the original and survives muchbetter. I t gives an exceedingly beautiful micro-anatomical stain.Its one disadvantage is the longer time it takes, and this isserious if the time available is short or liable to interruption.

Reference may also be made to Picro-Mallory (Mc]'arlane, D.(1944), Stain Tech. 19, 29). This modification is as precise as theAzan method and is not so slow. But i t is somewhat complicatedand the use of acid fuchsin probably renders the coloration somewhat less fast than that by the Azan method. On the other hand,the picro-haematoxylin basis adds to the power of tissue differentiation. A good alternative polychromatic stain, based onIron Haematoxylin, is obtained with various modifications ofMasson's T1'ichrome method. Mann's Methyl blue-eosin is auseful polychromatic stain, particularly for sections of insects.I t sharply distinguishes basophil and oxyphil substances. Themethod can be rapid, and since all the solutions employed ini t are neutral i t is suitable for use in conjunction with histo

chemical tests.Mayer'sllaemalum, with a counterstain, is a simple, quick and. vigorous micro-anatomical stain. I t is not necessary to make


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I R O N T I A E M ATO X Y L I N 89

every section. 'al l glorious within' for i t to show what is wanted.Mayer's Carmalum is useful when a red nuclear counterstain is

needed to contrast with blue, green or black cytoplasmic stains.Chlo1'azol Black stains chitin and cellulose, and is also a goodnuclear and general tissue stain. I t frequently gives markedmetachromatic staining.

Methods for special tissue elements and for histochemistrymay be sought in Carleton (1938) and Lison (1936). But becausethey are so generally useful the osmic (p. 55) and Sudan (p. 27)methods for fats are given. The section on 'Special methods' alsoincludes methods for staining of Protozoa, which require somedifferent techniques from those used for Metazoa.

Stains of different brands sometimes differ in their properties.Where necessary, therefore, in the following schedules, a brand isspecificd which has been found satisfactory. This does not implytha t another brand cannot be used, though its use might possiblyinvolve a l'evision of the schedule.

Coloured light-jiltm's (e.g. Wratten filters) may greatly enhancevisible contrast for some stains. For filter solutions, see Petersen,H. (1924), Z. wiss. Mikr. 41, 358.

H E I D E N H A I N ' S I R O N H A E M ATO X Y L I N

Solutions(a) Iron alum, ammonium ferric sulphate, 3 % solution in

water. This acts as mordant and, when diluted, as diffcl'entiator.(b) 5 % haematoxylin in 96 % alcohol 10 parts, distilled

water 90 parts.(c) As coun"terstains one may use

1 % eosin in 90 % a1l;oh01 01" i % orange G in 90 % alcohol.Haematoxylin solution slowly ' r ipens' by oxidation from the

air. Ripening can be effected in a few hours and more uniformlyif 0'2 g. sodium iodate is added for each 1 g. haematoxy1in.

Procedure(1) Bring the sections down through the alcohols and wash

well in distilled water. They can be kept for some days in water

in a covered vessel.(2) Mordant in 3 % ron alum for 30 min.-24 hr.(3) Wash rapidly in distilled water.


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40 H A N S E N ' S T R I O X Y H A E M AT I N

(4) Stain in haematoxylin for 30 min.-24 hr. To the nakedeye the whole preparation should be the colour of Indian ink.

(5) Wash rapidly in distilled water.(6) Differentiate in 1t% iron alum. The mordant solutionshould not be used for this. Follow the progress of differentiationunder the microscope. First the cytoplasmic background clears,particularly the connective tissue; then various cytoplasmicinclusions, various kinds of muscle and red blood cells, andfinally chromatin of nuclei. Correct differentiation needs practice.

(7) Wash in distilled water; dehydrate.(8) Counterstain (if needed) 1-5 min.; differentiate in 90 %

alcohol. Clear and mount.After use the solution of haematoxylin usually becomes black

owing to admixture with the iron alum, but this can be ignoredand the same solution employed again and again.

Time.'After most fixatives mordant at room temperature for 1 hr. and

stain for 1 hr. After Zenker give longer treatment. After chrome

osmium fixativesmordant and stain

for 12 hr. each.Both mordanting and staining have a high temperaturecoefficient. 10 min. suffices instead of 1 hr. at 50-60° C.

HANSEN'S I R O N T R I O X Y H A E M AT I N

Solutions(a) Iron alum (ammonium ferric sulphate)

(NH 4)2S04Distilled water

Dissolve by gentle heating.

(b) Haematoxylin (Gurr)Distilled water

Dissolve by heating.

10 g.1'4 g.

150 C.c.

1·6g.75 C.c.

Cool the solutions. Pour solution (b) into a porcelain evaporatingdish. Add solution (a) to this (not vice versa), stirring constantly.Heat slowly, without stirring, just to boiling-point. Coo.l rapidly

by floating the dish on cold water. The originally deep violetsolution should then have become dark brown without any greensheen. Filter into a well"stoppered hard glass bottle. To preventoxidation leave but little air space above the solution, which will


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MALLORY 41

keep for 6-8 months. After use the solution may be decantedback into the bottle.

ProceduJ'e(1) Bring the sections to water.(2) Stain progressively to the desired tint (1-10 min.) with

tl·ioxyhaematin.(8) Wash in tap water, 15-80 min.( 4) Coun el'stain, etc.

A variety of counterstains may be used, e.g. aniline blue andmange G. For this one may pass from stage (3) above to stage(6) in the pl'ocedures for Heidenhain' s Azan stain, or for Mallory'Sstain.

Freshly prepared trioxyhaematin gives a sharp black nuclearstain, the cytoplasm remaining almost colourless. Older solutionsmay stain cytoplasmic structures brown. This colour can beremoved by differentiating in 2 % H 2S0 4 , Romeis recommendsthe initial addition of 2-4 C.c. of 1 %H 2S 0 4 to each 8 C.c. of stainin order to ensure a pure nuclear stain, but this does not alwaysseem necessary.

MALLORY'S T R I P L E STAINSolutions

(a) Preliminary mordant. Saturated HgCl 2 in water +5 %acetic acid.

(b) Acid fuchsin, 1 % in distilled water.(0) Phosphomolybdic acid, 1 % n distilled water.(d) Mallory'S stain:

Aniline Blue W.S. (Gurr)Orange GOxalic acidDistilled water

0·5 g.2 g.2 g.

100 C.c.

, Aniline Blue' sensu stricto is a mixture of basic dyes soluble inalcohol but insoluble in water. I t is unsuitable. 'Aniline BlueW.S.' (=water soluble) is a mixture of acid dyes obtained from

this by sulphonation.I t . i s important to use Aniline Blue W.S. of a known andreliable brand, because the proportion of the component dyes


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42 MALLORY

varies in different brands. Methyl blue, a specific constituent ofAniline Blue W.S., may be used in its stead.

Procedu1'e(1) Bring slides to water.(2) Mordant in HgC12-acetic acid, 10 min.(3) Rinse in distilled water.(4) Acid fuchsin, 15 sec.(5) Differentiate by washing in distilled water, 10 sec., or

more as requircd.(6) Phosphomolybdic acid, 60 sec. (Avoid contact with metal

forceps: protect these with collodion or paraffin.)(7) Wash with distilled water, 10 sec.(8) Mallory's stain, 75 sec. Drain and wipe the bacJe of the

slide.(9) Distilled water, 10 sec. Drain and wipe.(10) Differentiate Aniline Blue W.S. ill 90 % alcohol, lOsec.,

or morc as required. -( l l ) 1st absolute alcohol, 10 sec.(12) 2nd absolute alcohol. 10 sec.

(IS) Mount direct in E ~ p a r a l ,or via pnre xylene or benzene,in good-quality balsam.

The times given in (4 )-( 9) are suitable for nemertine and planarianmaterial fixed in Susa. They vary considerably, howev }r, withdifferent tissues and diffel'e'nt brands o f stain, and for manyorganisms are considerably longer.

Standardization of operations is important. When staining withacid fuchsin and when washing, move the slide obliquely back

wards and forwards in the jar once a second.For a single slide control differentiation under the microscope.

When staining a series of slides, the first should be examined inxylene, and if the staining is imperfect bring i t back via alcoholand tap water to wash out the stain; readjust the times, andrepeat the stain ing process. I t is easier to obtain standard resultswith a series of slides by differentiation for a constant time thanby controlling the process in each slide individually under themicroscope.

Removal of excessive staining with acid fuchsin can be hastenedby substitution of tap water for distilled water at stage (5).


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M E T H Y L B L U E - E O S I N

Procedw'e(1) Stain in Hansen's stain (about 3 min.).

(2) Wash 15 min. in mnlling tap watel·.(3) Stain in Xylidene Ponceau till rather darker than finally

required (1-5 min.).(4) Rinse in distilled water.(5) Differentiate in phosphomolybdic acid, 4 min.(6) Rinse in distilled water.(7) Stain collagen in light green (about 2 min.), examining at

intervals in distilled water.(8) Dehydrate rapidly as in Mallory. Mount in balsam.Nuclei, black; collagen fibres and mucus, green; muscle, cyto

plasm, epithelium, etc., shades of pink. The method gives a veryclear picture of the distribution of collagen.

MANN'S M E T H Y L B L U E - EO S I NSolutions

(a) I % aqueous methyl blue (not methylene blue) 35 c.c.I % aqueous eosin 45 c.c.Distilled water 100 c.c.

Add a few drops of formalin as preservative.(b) 70 % alcohol with one drop of saturated solution of

orange G per c.c. (Dobell's differentiatol').

Procedure(1) Bring slides down to distilled water.(2) Stain overnight in methyl blue-eosin.(3) Rinse well in distilled water (20-30 sec.).(4) Differentiate in the Dobell's dilute orange G.(5) Dehydrate rapidly in neutral absolute alcohol.(6) Mount in Euparal, 01' clear, and mount in balsam.For mp'id work: Stain 10-30 min., differentiate in tap water,

dehydrate quickly and mount as above.Though both are acid dyes, the methyl blue behaves as though

i t were basic, so that the method sharply distinguishes basophilfrom oxyphil elements. The method is also useful for studying the

vascular and nervous systems. I t is a particularly good micro.anatomical stain for sections of insects.

Nuclei, 'chitin', connective tissue and mucus cells, blue;


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C H L O R A Z O L B L A C K 47

P1'OCedU1'e

(1) Bring sections down to water.(2) Stain in carmalum tilllluclei bright red (about 15 min.).(3) Wash 2-3 min. in distilled water.( 4) Pass tlU'ough alcohols. Counterstain in transit if necessary.(5) Absolute alcohol; xylene; balsam.Carmalum is a progressive stain. I t stains nuclei bright red

and is therefore a good nuclear counterstain when cytoplasmicstructures are stained blue or black (e.g. after silver i m p r e g n a ~tion).

CHLO RAZOL B L A C K ESee Cannon, H. G. (1941), J. Roy. Micr. Soc. 61,88.

SolutionChlorazol black E (biological quality) saturated solution m

70 % alcohol.P1'oced1J,re

(1) Bring sections to 70 % alcohol.(2) Stain with chiora black, 15-30 min.(3) Dehydrate, clear, and mount as usual.The stain is progressive. I f overstaincd, differentiate with

pyridine or dilute 'Milton'. The stain can also be used in water.Nuclei, black; cytoplasm, etc., shades of grey; 'chitin', greenish

black; glycogen, pink or red.Chlorazol black is the chief member of an interesting series of

dyes. It, has a strong affinity for cellulose as well as for ' chitin',

using that term in the zoological rather than the chemical sense,I t is not yet clear whether it stains chitin itself or the scleroprotein associated with it. I t normally stains metachromatically.I t is a valuable stain for plant tissues (Darrow, M. D., 1940,Stain Tech. 15, 67). Its staining effects are markedly influencedby the solvent. The stain can be used in water. Conn uses a 1 %

. aqueous solution for chromosomes of root-tips (1943, Stain Tech.18, 189).


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48 F A D I N G

P R E S E RVAT I O N OJ? SECTIONS

MOUNTING

In gelleral i t suffices after staining to clear sections in xylene andto mount in balsam. To prevent fading of some stains it is anadvantage to avoid xylene and Canada balsam, unless known tobe pure. One may mount in Euparal from alcohol, or one mayproceed via benzene, to neutral Canada balsam in benzene, or toa proprietary resin in benzene or pure xylene. Benzene balsamdries rapidly and is less easy to handle than xylene balsam.

I f slides must be taken rapidly out of absolute alcohol to prevent loss of stain, they may be passed to benzene via methylbenzoate. Benzene takes up even less water than xylene, andthere is more danger from imperfect dehydmtion.

FADING

Stained and mounted sections slowly fade if left in the light.Over decades, sections near the edge of a slide fade owing to slow

oxidation by air. Fading in months or years is due chiefly to(1) imperfect removal of mordants, (2) impurity of clearing agent,(3) impurity of mounting medium and its solvent. Xylene andCanada balsam commonly become gradually acid, and in thatstate stains may fade in them, sometimes in a few months.Neutral balsam. is much more satisfactory.

The causes of fading are not perfectly understood. An acidmedium will attack basic dyes. But the worst offenders for fadingarc often acid dyes such as acid fuchsin, and such stains are infact better preserved if pure balsam is saturated with salicylicacid (Langeron, 1934, p. 541). The most usual cause of fading isprobably slow reduction of the dye. Both xylene and balsam areapt to accumulate l'educing substances. An acid medium favoursreduction. Hence come the advantages of avoiding xylene byusing benzene, which is less reactive, of passing straight fromalcohol to Euparal, and of exchanging Canada balsam for anartificial resin of more stable properties. Whatever the medium,

i t should be neutral if i t is to hold both acid and basic dyes.I t may be remembered that an alkaline medium is likely to favouroxidatioll just as an acid one favours reduction. Clearing and


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50 R E C O N S T l t U C T I O N

G R A P H I C RECONSTRUC' l ' ION

For methods of }'econstruction from serial sections, both for solid

models and for diagrams, consultNorman, J. R. (J. Roy. Micr. Soc. (1928), 87).Pusey, H. K. (1989, J. Roy. Micr. Soc. 59, 232).Lison, L. (1987, 'Les Methodes de Reconstruction Graphique

en Technique Microscopique', Act. Sci. lndustr. no. 553. Paris:Hermann et Cie).

Most methods of reconstruction are laborious, but simplegraphic methods such as those below well repay the labour. Donot embark upon reconstruction unless you have a perfect seriesof sections, mounted without distortion. One may ensure cuttingthe paraffin block exactly to shape by the use of a simple instrument devised by D. P. Wilson (1933, J. Roy. Micr. Soc. 53, 25).

Method (1), for organs which extend considerably over a section,but do not involve many sections in the series. Draw the firstsection by one of the abovc methods. Orientate the drawing tofit the irnage of the second section, and then draw the latter

superimposed in a different colour. Continue in the same waywith the succeeding sections.Method (2), for organs extending over a very large number of

sections. In this case it is an advantage to make the reconstructed diagram in a plane a t right angles to that of the individual sections. Thus a horizontal plan may be made of thenervous system of an annelid worm from transverse sections.

Observe the first section with a squared eyepiece micrometer.Turn the latter till its centre line runs through some axis, such asthe dorso-ventral, that can be clearly recognized in each section.With a bilaterally symmetrical ol'ganism, thc axis may be chosento run through some central structure such as a median bloodvessel, or i t may be determined by placing the outline of thetransverse section symmetrically about the centre line of themicrometer, as in Fig. 8 B. Measure the position of the organconcerned to right or left of the axis.

Take a piece of squared paper and mark on i t a vertical axis.

Choose lines parallel with this to correspond with those in theeyepiece. Starting with the first section mark off the distance ofthe organ from the axis along one of the transverse lines of the

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MicroscopicTechnique for Zoologists

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