A PRACTICAL MANUAL OF

MEDICAL AND BIOLOGICAL

STAINING TECHNIQUES

fcr"

A PRACTICAL MANUAL OF

MEDICAL AND BIOLOGICAL

STAINING TECHNIQUES
hy

EDWARD GURR
F.R.I.C., F.R.M.S., F.L.S., M.I. Biol.

INTERSCIENCE PUBLISHERS,
NEW YORK

INC.

7^

FIRST EDITION,

SECOND EDITION,

J.

95 3
1

956

PRINTED IN GREAT BRITAIN BY

W. ARROWSMITH LTD., BRISTOL

PREFACE TO FIRST EDITION

This book has been written because it is felt that there is an urgent
need of a practical manual dealing with all or most branches of
microscopic staining, entirely divorced from theory and general
statements. It has been the writer's experience that a great deal of
time is lost in searching through volumes of theory and general
statements in order to extract a particular staining method which,
even when found, may not be complete.
It is hoped that this book will form a useful supplement to the

standard

works

on

anatomy,

bacteriology,

biology,

botany,

cytology, embryology, entomology, histology, mycology, pathology, veterinary science, zoology, etc.
It should be pointed out that this book contains

all the subject

matter, revised, rearranged and co-ordinated, of the writer's own

publications Microscopic Staining Techniques which were put out

in the form of three booklets. No. i, 2 and 3 as a temporary meas-

ure in an endeavour to meet the more pressing demands for information on the application of microscopic stains. The booklets
attained a world-wide circulation

and were in

fact

chosen by the

British Council for inclusion in their exhibits of British medical

The writer has since received many requests to incorporate

three parts of Microscopic Staining Techniques into one and this
has been done in the present book, but with many additions.
books.
all

of the methods given here are standard, some are not so

known, while others are unknown.
In writing this book, references have been made to numerous
journals and standard works, chief of which were as follows

Many

well

Stain Technology (Biotech Publication, Geneva, N.Y., U.S.A.).

Plant Microtechnique, by D. A. Johansen (McGraw-Hill, New
York).

Handbook of Practical

McCartney

(E.

&

Bacteriology,

S. Livingstone,

by T.

J.

Mackie and

J.

E.

Edinburgh).

McClung's Handbook of Microscopical Technique, edited by Ruth

McClung Jones (Paul B. Hoebber,Inc., New York, 16, U.S.A.).

PREFACE
Laboratory Tech?iique, by E. V.

Cowdry

(Williams

&

Wilkinson,

Baltimore, U.S.A.).

by R. D. Lillie (Blakiston

Histopathologic Technic,

delphia, U.S.A.).
Schaffer's Essentials of Histology,

Leach (Longmans, Green

Pathological Technique^ by

&
F.

Co., Phila-

by H. M. Carleton and E. H.

Co., London).
B. Mallory (Saunders,

Phila-

delphia, U.S.A.).
Histological Technique,

by H. M. Carleton and E. H. Leach

(Oxford University Press, England).

The section on Fluorescence Microscopy

is

a modification of

the writer's paper which was published in The Journal of the

Royal Naval Medical Service, 1951, Vol. xxxvil, No. 3, and

thanks are due to the editor of that journal for permission to include the modified paper in this book.
I

should

on record
thanks to my wife, F. P.
for her helpful criticism of the manuscript.

my

like to place

Gurr, B.Sc,

August, 1952

VI

PREFACE TO THE SECOND EDITION

I am very happy that the first edition of this book has met with so
kindly a reception; and I hope that this enlarged and revised
edition will be of greater service to medical research workers and

biologists all over the world.

Many

additions have been

book, and

made

to the subject-matter of the

wish to thank readers of the first edition for their

helpful suggestions, most of which have been incorporated in this
second edition.
A section of histochemical methods has been added as it is felt
that this may be of service to various laboratory workers, including
those engaged in cancer research, who may be interested in the
rapidly expanding science of histochemistry, which offers a rich
I

field for investigation.

The subject-matter of the book, as before, has been divided

into various headings: some of the methods which might have
"
"
been classified as
have been placed under other
Cytological
headings because the particular methods are more frequently
required by workers other than cytologists. For similar reasons,

methods which might have been transferred to the histochemical

same headings where they ap-

section have been left under the

peared in the first edition.

In addition to the standard works mentioned in the preface to
the first edition, reference has also been made to The Microscopisfs

Beams

edited by J. Bronte Gatenby and H. W.

A. Churchill, Ltd., London), and Microscopic Histo-

Vade-Mecum

(J.

&

by George Gomori (University of Chicago Press, U.S.A.).

was stated in a review in a Balkan journal, and correctly so,
that the first edition of this book gave no information on the techniques used in eastern and south-eastern European countries.
The reason for this is the scarcity here of the medical and bio-

chemistry
It

logical literature of those countries.

am not a linguist,

but

have

painfully translated some hundreds of pages of French and German literature to find some missing link for inclusion in this

present edition and,

if

any medical or biological laboratory workers

vii

PREFACE
any country in the world have any new or improved techniques
would prove to be of help to their fellow laboratory workers in
other countries, I should be very pleased to be given the opportunity of considering such techniques for inclusion in the third
edition of this work: I am most anxious that it should be of the
greatest possible service to medical and laboratory workers
throughout the world and information in any language concerning
particular techniques will be welcomed.
It has been stated in a Netherlands journal in a review, of the
first edition of this book, that Gram's Iodine and Lugol's Iodine
are identical I must correct this misleading statement which was
evidently made in error: Gram's Iodine is not identical with
Lugol's Iodine; the two formulae are given in this book.
in

that

I should like to express my appreciation of the very energetic

co-operation afforded me by my publishers, and the efficient
manner in which their production manager, Mr. R. G. Thixton,

and the printers Messrs.

J.

W. Arrowsmith

Ltd., have handled the

complicated business of getting the second edition into print.

42,

Upper Richmond Road West

East Sheen

London.
April, 1955

vui

Edward Gurr

Ji

'

'

Ti

CONTENTS
SECTION I GENERAL METHODS
Page

FIXATION
(a)

FIXATIVES

AcetoneAlcohol AbsoluteAllen's

Fixative Bouin's Fluid Bouin-Duboscq (Duboscqor Alcoholic Bouin) Carnoy's Fluid CarnoyLeBrun Champy's Fluid Flemming's Fluids
Formalin (buffered) FormolFormalin

Alcohol Helly's Fluid Hermann's Fluid Klein

enberg's Fluid Lewitsky's Fluid Marchi's Fluid

Navashin's Fluid Orth's Fluid Petrunkevitch's

Cupric p-Nitrophenol Regaud's Fluid Schau

dinn's Fluid Susa (Heidenhain) Zenker's Fluid,

Acetic

Acid

Brasil

(neutral)

etc.

(b)

DEHYDRATION, CLEARING, EMBEDDING,

SECTIONING, ETC

17

Celloidin Method
Celloidin - Paraffin Wax (double embedding)
Celloidin - Pyridin
Frozen Sections
Gelatine Embedding
Low Viscosity Nitrocellulose (L.V.N.) Method
Mercuric Chloride Precipitates in Sections Removal of
Paraffin Wax Embedding and Sectioning
Paraffin Wax - Pyridin Method

33

Water Wax

33

17

20
22
23

24
26
28

29

Miscellaneous Dehydrating and Clearing Reagents

Solvents, etc.

Butyl Alcoholy Tertiary for dehydrating and clearing

Cajeput Oil for clearing in wet climates

IX

34
34
35

CONTENTS
Page
Miscellaneous Dehydrating and Clearing Reagents Solvents,
;

etc.

{continued)

for dehydrating

Dioxane for dehydrating

Cellosohe

35

36

Ethylene and Propylene Glycols

dehydrating
Propyl Alcohol

for

for dehydrating
.

and

for

dehydrating and clearing

Terpineol

Mounting Media

as solvents

Aqueous

Syrup Mountant Aquamount Ber

Chloral
Mountant
Fluid Doetschman's Gum
&
Glychrogel MountStain Glycerin Glycerin

Apathy

37
38
38
39
39

Gum

leze

Jelly

ant.

Non-aqueous
Canada balsams

D.P.X.
Clearmount

and
Emexel
Meedol
Kirkpatrick)
(Lendrum
Balsam Fluormount Michrome Mountant, S.Q.D.
Balsam^Venetian Turpentine

41

Cristalite

SECTION 2ANIMAL HISTOLOGY

Normal and Pathological
Alcian Blue

Chlorantine Fast Red

forj mucopolysac-

charides, connective tissue, cartilage and bone

Aldehyde Fuchsin (G. Gomori),

for elastic tissue,

beta cells of the pancreatic islets

47

mast cells,
.

48

Aldehyde Fuchsin - Haematoxylin - Light Green Orange G - Chromotrope for differentiating two types
of basophils in the Adenohypophysis of the rat and

mouse

Alizarin Red,

S, for calcium deposits in cartilagenous

embryonic bone
Alizarin Red, S, for bone in small vertebrates (Dawson's
method)
Alizarin Red, S (William's method), for mammalian
embryos mature specimens of Urodele Amphibians for
distinguishing between bone and cartilage
Alizarin Red, S, for minute bones and foetal ossification
Alizarin Red, S, for nervous tissues (Benda's method)
.

$0

and

53

53

54
56
57

CONTENTS
Page
Alizarin Red,

nervous tissue in small

S, for vital staining of

vertebrates

58

Alum Carmine - Aniline Blue - Orange G for demonstrat. .

ing the various components of the Hypophysis
for vascular reticulum,
tumour cells, and connective tissue around tumour in

59

Ammoniacal Silver Carbonate

.

60

61

abnormal brain
Aniline Blue - Acid Fuchsin for elementary bodies, in
sections

Aniline Crystal Violet

nores .

Gram's Iodine

for epithelial

..

vj^

Aniline Crystal Violet Lithium Carmine

fibrin and for Gram-positive organisms in tissues
Aniline Blue - Orange G (Mallory) for collagenous and
-

Iodine for
.

reticulum
fibroglia

fibrils

;
cartilage,
elastin fibres

and

AzAN Stain (Heidenhain)

muscle

bone,
.

nuclei,

amyloid,
.

63

64

for mucin, neuroglia, chromatin,

tissue, etc.

65

Azo Carmine - Mallory Stain for islets of Langerhans

Azocarmine - Haematoxylin - Acid Green - Orange G,

66

for differential cell analysis of rat anterior hypophysis

.
.
. .
Feulgen, for glycogen, etc.

68

Bauer

BiEBRiCH Scarlet

Ethyl Violet

70

Haematoxylin, for

pepsinogen granules of the body chief cells in the gastric

glands

Bismark Brown

for mucin, cartilage,

and intestine
trachea
cells
in
goblet
embryonic tissue,
BiONDi - Ehrlich - Heidenhain Stain for nucleoli, chromatin,

Best's

etc.

mucin,

Carmine

for glycogen

Benzidine for brain capillaries

. .
Basic Fuchsin - Methylene Blue for Negri bodies in sections

Basic Fuchsin
oeotions

..

Gentian Violet
..

..

..

xi

72
73
73
75

7^

Iodine for bacteria in

.

Carbol Aniline Fuchsin for Negri bodies

Carbol Fuchsin (Ziehl Neelsen) for Nissl bodies
Carbol Fuchsin - Borrel Blue for leprosy and
ercle bacilli

7^

Methyl Green

//

. .

79
79

for tub.

80

CONTENTS
Page

Carbol Fuchsin - Haematoxylin for tubercle bacilli in

mammalian tissues
Carbol Fuchsin - Haematoxylin - Picro Acid Fuchsin
.

for

M.

leprae in sections

Carbol Fuchsin

Iodine

.-.

..
-

8i

..

82

..

Haematoxylin

Orange

demonstrating leprosy organisms together with

neurokeratin of the myelin sheath
for

Methyl Green

hyaline substance

Carbol Thionin
i.1

w LXXX

for

Carbol Fuchsin

85

Picric Acid (Schmorl) for bone cana-

Methylene Blue (Schultz-Schmitz Stain) for

demonstrating sodium urate in animal tissues
Celestin Blue - Chromotrope 2R (Lendrum), a substitute
for haematoxylin-eosin for simple diagnostic or photographic purposes
Celestin Blue - Orcein - Light Green (Lendrum) for
breast carcinoma and skin lesions

Carmine

83

demonstrating

^^

86

88

89

Chlorazol Black,

a general-purpose stain for whole tissues

and for sections
.

Congo Red for amyloid

Congo Red - Aniline Blue
.

UDlCd

Congo Red

90

91

atohyalin

for

elastic

Orange

Ehrlich Haematoxylin
.

93

pathological tissues

Dahlia, Acetic (Ehrlich) for mast

tions

DoPA Reagent

for melanoblasts

cell

collagenic fibres
-

Haematoxylin (Maximow)

ing changes of haemopoietic tissues

Ethyl Violet

93

granules in sec.

95

95

Elastin Stain (Weigert) for elastic tissues

Elastin Stain (Sheridan) for elastic tissues
Elastin - Trichrome Stain for elastic, smooth muscle and

Eosin Azur 2

and ker-

for eleidin

Toluidine Blue - Thionin, a tribasic

nerve cells and Nissl granules in normal and

Cresylfast Violet
stain for

96

97

97

for demonstrat.

99

Biebrich Scarlet (Bowie) for pepsinogen

granules in gastric mucosa

xii

100

CONTENTS
Page

Feulgen Fuchsin for chromatin in animal cells

FoNTANA Stain for argentaffine granules
FoNTANA Stain - Silver Nitrate for reticular and
fibres

Gallocyanin

Orcein

loo

102

Acid Alizarin Blue

Viridine, a general stain for animal tissues

collagen
.

GiEMSA Stain for malaria parasites, rickettsia, etc.

GiEMSA - Wright Stain, a permanent stain for differentiat.

ing the structures, particularly Nissl bodies and cytons,

..
..
..
of the spinal cord
..
..
.

Gold Chloride

Sublimate

(Cajal) for neuroglia fibres

for astrocytes in the central nervous system
.
GoLGi Method (Rapid) for nerve cells
.

104
105

106

Gram's Iodine for bacteria in sections

- EosiN for
demonstrating collagenous tissues
Haematoxylin - AzoPHLOXiN, for muscle, connective
.

102

Alizarin

Haemalum

107
108
108

109

no
ganglion cells, etc.
- Basic Fuchsin for
haemofuscin, melanin
..
..112
..
..
and haemosiderin ..
..
112
Haematoxylin (Delafield) - Eosin for general staining
tissue,

Haematoxylin

Haematoxylin
Haematoxylin
Haematoxylin
Haematoxylin

..
(Ehlrich) for keratohyalin ..
in
tissues
for
sodium
urate
(Ehrlich)

(Ehrlich)
-

Eosin for general staining

(Kultschitzky-Pal method)
.

..

positive bacteria

and

structures

..

..

Gentian Violet

113
114

114

for nuclei
..

..

..

..

..115

Iodine for Gram-

fibrin in sections

Haematoxylin (Heidenhain)

Haematoxylin
Haematoxylin

..

Fluorchrome

for myelin sheaths, etc.

Haematoxylin

..

116

and cytoplasmic
..

for identification of lipines

..

..117

..

..

118

(Kultschitzky), Weigert's Modification

for the finer studies of corticaLarchitecture and for total

brain sections

..

Haematoxylin - Phloxine

..
-

actinomyces in sections

..

..

..

Aniline Gentian Violet

.

..119
for
,

Haematoxylin, Phosphotungstic (Mallory) for pleuropneumonia organisms in sections of lung

Haematoxylin - Picro Fuchsin, for nuclei, connective
tissue, etc.

XI 11

120

121

122

CONTENTS
Page

Haematoxylin

collagen and
-

PiCRO Ponceau

S, a selective stain for

connective tissue in place of Haematoxylin

Van Gieson

Haematoxylin (Weigert)
collagen

Ponceau Fuchsin

and connective tissue

- Ponceau Fuchsin

Haematoxylin

Ponceau S

123

Fast Green, FCF,

.

Picro Aniline Blue, a

differential stain for muscle and connective tissues
Haematoxylin (Weigert) - Scarlet R for demonstrating
fatty acid crystals, soaps and neutral fats in fat

Haematoxylin

necrosis

Haematoxylin

125

126

Gieson, a selective stain for

(Ehrlich)
collagen and connective tissue
-

127

Van

Gieson, a selective stain

connective tissue

for collagen and

Hickson's Purple, a general stain for class work
Hickson's Purple - Victoria Green, G, a general stain
.

suitable for class work

Hitchcock and Ehrlich's Mixture for lymphatic,
.

124

Van

Haematoxylin (Heidenhain)

123

(Curtis) for

a differential stain for pathological tissues

-

128

130

130

ganglion,
cells of bone

plasma and basophilic cells; immature

..
..
..
marrow, striated muscle and fibrin
131
for
Stain
Jenner
blood-forming organs
132
Jenner - GiEMSA Stain for the polychromatic staining of
..
..
..
..
blood-forming organs
..133
.

Lead Haematoxylin

Acid Fuchsin (MacConaill), a

"definitive" polychrome stain for the central nervous
system and the trunks outside it
-

134

Leishman Stain

for the general differentiation of blood

for
malarial parasites trypanosomes, etc.
corpuscles;
Leuco Patent Blue for haemoglobin

135
136

Levaditi's Stain for

138

Light Green

Treponema palHdum in sections

Acid Fuchsin (Alzheimer) for demonstrat.

ing neuroglia changes

..

..

..

..

.,138

LiGNiN Pink, for whole mounts of marine vertebrates, etc.

and for chitin
Lithium Silver (Laidlaw) for staining skin and tumours
LoRRAiN - Smith - Dietrich Stain for lipoids
Lugol's Iodine for the identification of glycogen
.

xiv

140

141

143

143

CONTENTS
Page

Cresyl Fast Violet (Kluver & Barrera)

for the combined staining of cells and fibres in the

LuxoL Fast Blue

nervous system

MacCallum's Stain

organisms in tissues

insulin cells of the pancreas

Mallory Stain
pituitary

147

differential staining of

148

Mallory Stain

for differential staining of

chromophobes in mouse

and

basophils
.

144

Haematoxylin

acidophils,

and Gram-positive

Mallory Stain - Haematoxylin, for

influenza

for

sections of brain..

for Negri bodies

(Jane E.

150

in
..

..

..

..

..

Mallory Heidenhain Stain

Haematoxylin,

152

Cason's modifi-

method for connective tissue

Phosphotungstic Acid Haematoxylin, a

153

general stain for vertebrate tissues

- Victoria
Green, a general stain for class
.

154

155

156

cation), a rapid one-step

Mallory's

Marshall Red

work
Masson's Trichrome Stain, for connective tissue
Methyl Blue - Eosin (Mann) for demonstrating the
.

ous types of the

Methyl Green
Methyl Green
ceils

cells in

..

..

Stain)
etc*

for

mice

Pyronin

chromatin,

Orange

plasma

160

(Bonney's Triple
keratin,

tissue,

..1 uu

Basic Fuchsin, rapid method of dem..

..
..
^..
onstrating Negri bodies ..
Methylene Blue - Basic Fuchsin for rickettsia

Methylene Blue

159

typhus fever

for

connective

157

..158

for

Methyl Violet 6B for amyloid

Methyl Violet - Metanil Yellow,
rickettsiae in lungs of

vari-

..

..

..

Pyronin (Pappenheim-Unna)

the anterior lobe of the pituitary

for amyloid

Methyl Violet

Methylene Blue Polychrome (Unna) for mast cells

Methylene Blue Polychrome - Glycerine Ether (Unna)
for differentiating

mast

cells

and plasma

Metanil Yellow
MucicARMiNE
mucin and connective tissue
Mucicarmine (Mayer) for mucin

XV

cells

Haematoxylin
.

161

162

163

164

for
.

165
166

CONTENTS
Page
't>'

MuciCARMiNE (Southgate) for mucin

MuciHAEMATEiN for mucus
Nadi Reaction for oxidase granules
Naphthol Blue Black - Haematoxylin - Brilliant PurPURiN - AzoFUCHSiN, for collagen, smooth muscle, etc.
Naphthol Green B - Haematoxylin (Weigert) for con.

nective tissue

Neutral Red

. .

i66

neutral fats

170

171

cedents of serous or zymogenic cells

for elastic fibres and connective tissue
.

Aniline Blue

Orange G,

172

. .

173

174

gen

and keratin
Aniline Safranin for elastic and connective

tissue fibres

Orcein
Orcein

a differential stain for

elastic fibres, collagen

Orcein

175

176

177

Giemsa Stain for syphilitic tissue

Picro Fuchsin (Van Gieson) for elastic and

fibres

OsMic Acid,

169

168

Nile Blue - Picro Fuchsin (Murray-Drew) for bacteria

and actinomyces in pathological tissues
Orange G - Crystal Violet (Bensley) for secretion ante-

Orcein
Orcein

67

Fast Green

for staining both Gram-positive

and Gram-negative organisms in sections
Nile Blue Sulphate for demonstrating fatty acids and
-

i66

colla.

179

a rapid technique for staining fat in frozen

sections

179

Pasini's Stain (Improved) for differentiation of connective

tissues

Periodic Acid

Celestin Blue, for

pituitary glands, demonstrating both muco-protein

..
..
.,
precuspors of the gonadtrophins
Periodic Acid - Feulgen Fuchsin (Hotchkiss) for Polysaccharide structures
.

Phloxin
Phloxin

Haematoxylin for Hyaline in animal tissues

Methylene BIue, rapid smear technique for

Negri bodies in brain tissue

Phloxin

Methylene Blue

modification

normal and

of

(j.
-

R. Dawson's method)

Azur

B,

original
pathological tissues

very

technique

Mallory's

xvi

180

human and animal

181

183
185

186

rapid
for
.

187

CONTENTS
Page

Tartrazine {A. C. LendrunCs technique)^ a genPhloxin

eral histological stain and for the demonstration of
-

inclusion bodies

and Keratin

NiGROSiN
Protargol - Gallocyanin (Foley)
PiCRO

for Eleidin

lymphoid tissues
Saffron for connective tissue.
Safranin - Crystal Violet

193

i93

194

Fast Green

Scarlet R for staining fat

Silver Carbonate - Aniline Blue
reticulin, elastin and collagen
Silver Nitrate - Gold Chloride
.

192

Orange 2

Quadruple Stain)
Safranin - Water Blue (Unna) for Collagen fibres
Scarlet R - Ethylene Glycol, an improved technique
.

190

. .

(S. S. Kalter's

staining fat

189

for nerve fibres, sheaths

and cells
PuRPURiN for calcium deposits in pathological tissues
Quincke Reaction for Haemosiderin
Rhodamine B - Aniline Methylene Blue for splenic and
.

i88

195

197

for
.

198

97

Fast Green, for

.

199

Paracarmine (Da

Fano) for Golgi apparatus

Silver Nitrate - Hydroquinone for the detection of gold
in fixed tissues of experimental animals

202

Sudan Black (J. R. Baker's technique) for lipids

Sudan Black, a specific stain for neutral fats
Sudan Black - Ethylene Glycol, an improved technique

204
207

for lipid staining

Sudan Blue for demonstrating degenerated myelin

Sudan Brown for acute fatty degeneration not shown by
Scarlet

Sudan 2 for degenerating and intact myelin and fat

Thionin (Ehrlich) for mucin
Thionin (Ehrlich) for the differential staining of entamoeba
Thionin (Ehrlich) for nerve cells
Thionin for demonstrating malignant cells in biopsy material
ToLuiDiNE Blue for mucus
Trichrome Stain (G. Gomori) for connective tissue, etc.
Trichrome Stain (Masson), modified for pituitary gland,
.

xvii

epithelium, thyroid nerve (normal and .tumour) etc.

B

203

208

209
209
210
211

212
213
213

214
214
215

CONTENTS
Page

Urea Silver Nitrate,

Verhoeff's Stain for

Water Blue

elial fibres

Weigert

Pal Technique

217
218

for myelin in brain

Haematoxylin

tive tissue and muscle

Wright's Stain for general

Ponceau
.

220

221

and spinal
.

S. for
.

modification)

cord and for peripheral nerves and ganglia

Wool Green

Safranin for demonstrating epith-

French Elastin Stain (Moore's

for elastic tissues

Weigert

elastic fibres,

Orcein

and nerve endings

nuclei and collagen

for nerve fibres

223

connec.

224

differentiation of blood cor-

puscles ; for malarial parasites ; trypanosomes, etc.

225

SECTION 3BOTANICAL METHODS

Normal and Infected Tissues
{a)
(h)

GENERAL TREATMENT OF TISSUES

MISCELLANEOUS MICROCHEMICAL TESTS
.

229

230

Aldehydes Aleurone Amygdalin Amylodextrine

Anthocyanin Arbutin Asparagine Calcium
Calcium Oxalate
Carotin
Cellulose
Callose
Acid
Chitin
Chlorides
Formic
Chlorophyll

Glutathione Inulin Iodine Iron Lecithin

Nitrates Pectic Substances Phosphates Phyto Potassium Proteins Saponin Sodium

Sulphates Tyrosin.

sterol

{c)

STAINING TECHNIQUES

247

Acid Fuchsin - Aurantia for differentiating between bacteria and mitochondria in infected tissues
Acid Rubin - Aurantia - Toluidine Blue (KuU's Stain),
for starch grains and mitochondria
Aniline Hydrochloride, a rapid method for demonstrating
lignified tissues

247
248

Basic Fuchsin, Ammoniacal for lignified walls and cutin

Borax Carmine (Grenacher) for bulk staining and for small

whole mounts

xviii

249
249

250

CONTENTS
Page

Chlorazol Azurine, a non-fading simple double stain suitable for class work
..251
Chlorazol Black, a general-purpose stain
252
Chlorazol Paper Brown, B, a general-purpose stain
253
Cotton Blue - Safranin, for fungal hyphae in woody
.

irXOoLX^o

Erythrosin, for cellulose and lignified tissues

Delafield Haematoxylin - Cellosolve, a general-pur-

Cyanin

pose stain

Erythrosin
Lactophenol, a general-purpose stain
Gram's Iodine, a general-purpose stain
Haematoxylin (Heidenhain) - Aniline Blue (J.
.

255

255

256
256

G.

Vaughan's technique), for differential staining of nuclei,

cytoplasm and cell walls of angiosperm shoot apices
.

Haematoxylin

woody

Bismark Brown,

plants

Heidenhain Haematoxylin

for
.

phloem
.

259

Safranin, a general-purpose
.

Acid Fuchsin,
chromosomes
-

Johansen's

and

for lignified tissues

Quadruple Stain,

261

excep-

general stain,

contigous tissues
-

Martius Yellow

for callose in pollen tubes

LiGNiN Pink, a non-fading stain, specific for lignin
Magdala Red - Fast Green, for the differential staining of

host tissues and parasites

Methyl Green

Phloxin

saccharide structures

Phloroglucinol
phytes

262

264
266
268

269

269

Glycerin Jelly, for the

simultaneous double staining and mounting of pollen
grains, differentiating functional from abortive grains
Periodic Acid - Feulgen Fuchsin (Hotchkiss) for poly-

260

for

good for sections of lichens

Johansen's Quintuple Stain, a general-purpose stain,
particularly useful for leaves, roots, stems and ovaries
Lacmoid - Tannic Acid - Ferric Chloride, for phloem and
tionally

Lacmoid

257

tissues of

stain useful for the demonstration of histological and

cytological structures, as well as for algae, fungi, etc.

Iodine Green

1^"

270
183

for lignin, particularly suitable for hydro.

xix

271

CONTENTS
Page

Polyvinyl Lactophenol,
of

wood

for

for sectioning

embedding
.

brittle

specimens

Acid Fuchsin - for spermatozoids, zoopores etc.

- Aniline
Blue, for gymnosperm ovules, archegonia, embryos, angiosperm stems and roots, etc.
Safranin - Aniline Blue for cellulose walls, cytoplasm,
and for chromosomes
Safranin - Dianil Blue G, for differential staining of

Safranin
Safranin

peronosporaceae

Safranin

pose stain

274

274

..

Clove Oil

275

Clove Oil,

..

..

..

technique for staining

fat

..

..

Scarlet R for demonstrating fats

ScHULZE Solution (Chlor Zinc Iodine) for
proteins and starch
.

,.

281

282

cell

somes,

etc.

Trypan Blue,

for detection of

a nuclear stain for plant material

280

walls,

in cells

magnesium

279

270

Tetrazolium Salt, for testing the viability of seeds

Thionin - Orange G for infected plant tissues, for chromo-

Titan Yellow^

278

a general-purpose

Safranin - Picro Aniline Blue, a rapid and simple

method of demonstrating hyphae in wood sections
Safranin - Tannic Acid - Fast Green, FCF for roots
and stems
Scarlet R or Sudan 3 - Ethylene Glycol, an improved
.

277

Cellosolve, a rapid general-

Light Green

srain

FCF - Cellosolve, a rapid, non-fad-

Light Green

purpose stain

Safranin

273

ing stain in place of Sanfranin Light Green

-

Fast Green, FCF, a non-fading, general-pur-

Safranin - Fast Green,

Safranin

272
276

282

283

283
284

285

SECTION 4CYTOLOGICAL METHODS

Acid Fuchsin
chondria

Acid Fuchsin

Toluidine Blue

Aurantia

for mito.

Picroindigo Carmine, for plant tissues

cytological stain for root tips.

. .

Alizarin Red, S (Benda's method) for mitochondria,

Aniline Fuchsin - Iodine Green for mitochondria

XX

289

as a
.

etc.
.

290
291

292

CONTENTS
Page

Aniline Fuchsin - Picric Acid (Altmann) for mitochondria

Aniline Safranin (Babe's) for mitosis in animal cells
Basic Fuchsin - Picro Indigocarmine (Alcoholic) for
plant tissues as a cytological stain for root tips

293

294

295

Fuchsin - Picro Indigocarmine (Aqueous) for

chromosomes in plant cells
Breinl's Triple Stain, for chromosomes
Copper Chrome Haematoxylin (Bensley), for mitoBasic

chondria

Cotton Red

Methyl Violet

Orange

Feulgen Stain for demonstrating mitosis in plant cells

Gentian Violet Picric Acid - Iodine for chromosomes
plant cells

G for plant cells

.

sporocytes

stain for plant cells

Methyl Green

WC^lld

Methyl Violet

Acid Fuchsin

NiGROSiN, for salivary chromosomes of Drosphila

Safranin
Safranin

Crystal Violet (Hermann),

Gentian Violet - Orange

for

304

305

in plant

Eosin Scarlet, a botanical

mitosis in root tips

300
302
303

a cytological

chromosomes

for

297
299
300

in

Haematoxylin (Regaud) for mitochondria

Haematoxylin - Safranin, a cytological stain for plant cells
Iron Aceto Carmine (Belling) for chromosomes in micro-

Methyl Green - Acid Fuchsin - Erythrosin,

295
296

stain for
.

chromosomes

307
307
309

{Flemming Tri-

colour Stain) for chromosomes, etc.

. .
for demonstrating cytoplasmic changes in
.

309

Wright's Stain,
plant cells

..

..

..

..

..

..310

SECTION 5FLUORESCENCE MICROSCOPY

{b)

GENERAL INFORMATION
EQUIPMENT REQUIRED

316

{c)

STAINING METHODS

317

{a)

315

1.

Cytoplasm, Nuclei, Nucleoli and Chromatin, Differential Staining of

318

2.

Fat Staining

318

..

..

xxi

CONTENTS
Page
3.

Intravital Staining

4.

Muscle

320
321

6.

Nerve Tissues, Differential Staining of

Soil Bacteria, Staining and Counting Differentiation OF Living from Dead Cells

7.

Trypanosomes, Living, in

8.

Trypanosomes, in Dried Blood Films

324

9.

Tubercle Bacilli

325

10.

Virus Staining

5.

319

Blood

322
323

326

SECTION 6HISTOCHEMICAL METHODS

Micro- Incineration
mineral salts
3 -Hydroxy

for

the

..

detection

..

2-Naphthoic Acid

..
-

and location of
..

..331

..

Tetrazotized o-Ani-

sidine, for demonstrating sites of carbonyl activity

Iron Haematoxylin for radioauto-

graphs

333

334

Metanil Yellow
.

Methyl Green

Pyronin

Ribonuclease (Brachet), for

ribonucleic
acid
and desoxyribonucleic acid
detecting
in the same cell
Nile Blue Sulphate - Safranin, for phospholipids
Phosphomolybdic Acid - Eosin, for localization of choline
.

containing lipids

336
340
341

Sudan Black

342

SECTION 7SMEAR PREPARATIONS

Albert's Stain (Layboum's modification) for diphtheria
bacilli

Alcian Blue, for bacterial capsules and polysaccharides

Aniline Blue - Eosin B, for spermatozoa
Aniline Gentian Violet, a rapid stain for Treponema
.

348

349

pallida
L for the detection and staining of epidermophytic
infection
.
.

345
346
347

AzuR

xxii

CONTENTS
Page
Basic Fuchsin for Treponema pallida
Breed^s Stain for staining and counting bacteria in milk
Brilliant Cresyl Blue for reticulated cells, platelets, etc.
Carbol Crystal Violet for fibrin network in blood smears
.

Carbol Fuchsin
cnaeres

Carbol Fuchsin
bacteria

bacilli

sputum

and other

Borrel Blue

Brilliant Yellow for tubercle

for leprosy

'

protozoa

GiEMSA Stain

for blood,

pallida^ trypanosomes, etc.

GiEMSA

May

parasites,

trypanosomes,

etc.

356
356
357
358
358
359
359
360
361

362

363

3^3

and stain for

.

364

Treponema
.

3^5

blood, malarial
.

Aniline Gentian Violet, for spirochaetae

of Vincent's Agina and for Trep. pallidum
Gram's Stain for bacteria
Gram's Stain (Jensen) for bacteria
Gram's Stain (Kopeloff and Beerman) for bacteria
Haematoxylin (Phosphotungstic, Mallory) for entamoeba
Haematoxylin (Weigert) - Bordeaux Red for permanent

Gram's Iodine

355

for

parasites,

Grunwald Stain

fixative

malarial

Fontana Stain for spirochaetae

Gentian Violet (Noland) a combined

354

bacilli

Crystal Violet for bacterial capsules

Crystal Violet, a simple stain for spirochaetes
Dahlia, for Heinz bodies in blood smears
Dorner's Stain for spores
Ehrlich's Triacid Stain for blood
EosiN - Gentian Violet for basal bodies
EosiN - Methylene Blue - Basic Fuchsin, a general stain
for bacteria, blood and spirochaetes
Field's Stain for thick blood smears for malarial parasites
Flagella Stain (Cesares-Gil) for colon and typhoid
.

jjo

and tubercle

Casteneda's Stain, for rickettsiae and elementary bodies

organisms

349
350
351
352

acid-fast

Carbol Fuchsin
in

for tubercle bacilli

.

Carbol Fuchsin

Treponema pallida and other spiro-

for

368

preparations of anophcline midgut

xxiii

371

368
369
370
372

372

CONTENTS
Page
Indigo Carmine for the differential staining of vaginal smears
Iodine - Eosin for intestinal amoebae and flagellates

Haematoxylin

EosiN

Janus Green, B for staining oocysts of coccidia in faeces

Janus Green - Neutral Red, for supravital staining of blood
Jenner Stain for the cytological examination of blood
Leishman Stain for blood, malarial parasites, trypanosomes,
.

wLw*

Lugol's Iodine - Eosin for differentiating between spores

and vegetative forms in bacterial smears
Macchiavello's Stain, for rickettsiae
.

373

374
375

376
377
1//

379

Malachite Green a simple method of staining spores

Malachite Green - Basic Fuchsin, for yeasts
Malachite Green - Pyronin - Crystal Violet (Sandi-

380
381

ford), a contrast stain for gonococci and meningococci.

- Grunwald Stain for the
cytological examination

381

May

of blood

Methyl Green - Pyronin, for gonococci

Methyl Violet ioB, for the direct staining
bodies

Methylene Blue

Carbol Fuchsin

sules in bacterial films

of elementary
.

Methylene Blue - Gentian Violet for gonococci

Methylene Blue - Safranin for polar bodies in bacteria
New Fuchsin - Congo Red, for bacterial cell walls, particu.

B.

larly for

Newman's

colt

and B.

cereus

milk smears for counting bacteria

for protozoa, yeasts, etc.

a differential stain for vaginal smears
.

Peroxidase Stain (Cowdrey's method) for blood

PiCRO - Methyl Blue - Eosin for urinary casts

PiNACYANOL Neutral Red, for Supravital staining of blood

Ponder's Stain (Kinyoun's modification) for the differ-

entiation

organisms
-

384
385
385

386

Stain, a single solution for defatting and staining

Nile Blue Sulphate

Papanicolaou Stain,

Pyronin

383

and cap-

for flagella

382
382

of
.

metachromatic
.

Alphanaphthol (Graham)

in blood

XXIV

of

granules

387
388
388
389
390
392

diphtheria
.

393

for oxidase granules

.

394

CONTENTS
Page

Safranin - Light Green for spirochaetes, etc.

Schorr's Stain for vaginal smears
Schorr's Stain (single solution) for vaginal smears
Sudan Black - Eosin - Methylene Blue for
.

395

395

396
lipoid

granules in leucocytes

Sudan Black - Safranin for demonstrating fat in bacteria

Sudan 3 for blood in the spinal fluid, differentiating fresh
haemorrhage from old

Sudan 3 for staining fat in faeces

Tetrachrome Stain (MacNeal) for
leucocytes

398
399
differentiating types of

397
397

Thionin (Ehrlich), a rapid stain for rickettsia in smears

Vaginal Smear Stain, M.F.4 for the rapid staining

400
400

of

vaginal smears, sharply contrasting cornified from noncornified cells

401

Vaginal Smear Stain, PX,

smears
Victoria Blue
Victoria Blue
Victoria Blue

4R
4R
-

a differential stain for vaginal

for elementary bodies

for Treponema pallida

Kernechtrot Red

Light Green,

402
402
403
for

elementary bodies

404

for general differentiation of blood corfor malarial parasites, etc.

Wright's Stain
puscles

Wright's Stain

for demonstrating Trichomonas riedmuller

APPENDIX

405
406

407

I.

Atomic Weights, Tables of

409

2.

Conversion Tables

410

3-

Formulae

411

4-

Formulae (Stains and Reagents)

412

5-

Refractive Indices

426

6.

Saturated Solutions (Reagents)

428

7.

Specific Gravities

430

8.

Stain Solubilities and Molecular Weights

430

(Fixatives)

437

XXV

SECTION I GENERAL METHODS

FIXATION
The main

objects of fixation are:

To

kill the cells suddenly and uniformly so that they retain,

(a)
as near as possible, the same appearance which they possessed in
life.

(b)

To

preserve the tissues,

putrifactive

cells, etc.,

by the inhibition of

and autolytic changes.

(c) To set and hold intra-cellular bodies, cells, etc., by precipitation in the positions which they occupied in life, thereby facilitating the closest possible study of the histology and cytology of

the

cells.

(d) To facilitate differentiation in the refractive indices of certain cell elements which would otherwise be invisible owing to the

exceedingly narrow margin betw^een the refractive index of one

type of cell and that of other types.
(e) To render the cells and tissue constituents resistant to the
subsequent processes such as dehydration, clearing, embedding,
staining, etc., prior to their examination under the microscope.

(/) To facilitate proper staining of tissues. Here it should be

mentioned that some fixatives act as mordants while others act as
inhibitors for certain stains, and it is, therefore, of considerable
importance that a suitable fixative should be employed for a
particular staining technique or a particular staining technique
should be chosen to suit material which has already been treated
with a particular fixative. Recommendations as to suitable fixatives are given for most, if
cribed in this book.

not

all,

of the staining techniques des-

It is essential, if good results are to be achieved, that tissues

should be removed from the body with the least possible delay
and fixed immediately. Bacteria, protozoa and other unicellular
organisms should be living at the instant of fixation. The following points must be observed in order to avoid failure, waste of

time, effort and materials :

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

1

Pieces of tissue should, whenever possible, be cut into slices

mm. in thickness to permit penetration of the fixative

2 to 6

throughout within a reasonable time.

The

is

to be fixed should be

of sufficient size to take the pieces of tissue

without their folding

2.

container in which the material

or bending.
are to be fixed, large incisions should
3. If large organs
to allow thorough penetration of the fixing fluid.

be made

4. A volume of the fixing fluid roughly about twenty to

times as great as that of the material to be fixed is necessary.
5.

Material must not be

left in

fifty

the fixing fluid beyond the

necessary time, but should be taken out, washed, dehydrated,

cleared and embedded or stored in a fluid, suitable for the particular material, until

it is

required for embedding.

After fixation, and before proceeding to dehydration, careful

washing out of the excess fixative is necessary, except in the case
6.

of alcohol which requires no washing out. In most cases running

water

is

employed for this purpose, but for some tissue cells and
and for some fixatives, alcohol must be used. In

cell constituents,
all cases,

however,

liquids for

washing

it is

necessary to use liberal quantities of the

out.

A fixative

suitable to the material to be examined and comstains to be employed should be chosen, as diswith
the
patible
of
this
factor
will, as previously stated, lead to failure and
regard
the
as waste of time, effort and materials
as
well
disappointment
7.

importance of this rule cannot be over-emphasized.

(a)

The number
number in

the

some

of the

FIXATIVES

of these from which to choose

everyday use

is

legion, although

comparatively small.

more commonly used of these

mulae of others are given

is

are given

in the appendix.

Acetic Acid, Glacial

Recommended for :
Rapid

fixation of strongly contracting organisms.

Details of

below

for-

SECTION ONE
Technique:

Fix in the warm acid for a maximum period of fifteen minutes;

then remove excess acid by washing in 30-50% alcohol.

Remarks:
Acetic acid glacial

is

cell constituents, etc.,

rarely used alone

and

it is

it

of most value

causes the swelling of

when mixed with such

substances as formalin, alcohol, mercuric chloride,

etc., to

counter-

act their shrinking effect.

Acetone
Recommended for :
fixation of brain tissue for rabies diagnosis. (R. D. Lillie,
Histopathologic Technique.) It is also employed for fixing tissue

Rapid

enzymes, particularly phosphatases and

lipases.

Technique:

Thin

pure acetone for twelve to

they are then dehydrated by immersion in two changes of pure acetone for two hours in each at room
temperature, and afterwards cleared by immersing for half an hour
slices of tissue are fixed in

twenty-four hours

at 0 C.

in each of two changes of benzene before infiltration with paraffin

wax.*

Alcohol Absolute
Recommended for :
Glycogen,

Amyloid, Fibrin,

Hyaline,

Haeipofuscin, Phos-

phatase.

Technique:

Fix from two hours to several days according to the nature of

the material; dehydrate; clear.

Remarks:
Unsuitable for

fats

and

lipines as these are dissolved

higher concentrations of alcohol.

*Gomori, Proc. Soc. Exp.

Biol,

and Med.,

58, 362, 1945.

by the

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

Allen's Fixative (B.15)

Recommended for :

Chromosomes; plant
Formula:

tissues, particularly

buds.

SECTION ONE
mordant enhancing many staining
extracted from the fixed tissues.

effects,

should be entirely

Remarks:

which keeps indefinitely and causes only slight

is compatible with almost every staining
of
tissues,
shrinkage
it is considered to be a valuable fixative for most purtechnique
This

fixative,

and mucin. Its peneposes, although it is unsuitable for kidney

tration power is great, and delicate material should be left in contact
with this fixative only for the

minimum time, to avoid over-fixation

work in particular. There are many

modifications of Bouin, of which Allen's Fixative, B.15, has proved
to be the most satisfactory for chromosomes in mammalian
this applies to cytological

tissues.

Bouin

Duboscq

(Duboscq-Brasil, or Alcoholic Bouin)

Recommended for :
Arthropods containing parasites and protozoan
chitinous tissue.

Formula:

cysts,

and for

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

Formula:
Absolute alcohol
Chloroform

Glacial acetic acid

60 ml.
30 ml.
10 ml.

Technique:

Animal

Fix for one and a half to two hours; then

two changes of absolute alcohol before clearing and

Tissues.

transfer to

embedding.

Plant Tissues. Fix

root tips for a quarter-of-an-hour

anthers,

for one hour.

Remarks:

rapid fixative with great penetrating power.

Carnoy

LeBrun Fluid

Recommended for :
and

Insects

ticks,

and

as a rapid

and penetrating

fixative for

plant tissues.

Formula:
Absolute alcohol

. .

Glacial acetic acid

Chloroform
Mercuric chloride
.

30 ml.
30 ml.
30 ml.

to saturation (about 10

gm.)

Technique:

Fix from half to one minute

then wash in

95%

alcohol.

Remarks:

Not reconmiended

for routine fixation of plant tissues.

Champy's Fluid
Recommended for :
Plant and animal tissues in general;

mitochondria and other

cytological detail.

Formula:
Potass, dichromate

3%

aqueous

Chromic acid 1% aqueous

Osmic acid 2%
.

35 ml.
35 ml.

20 ml.

SECTION ONE
Technique:

Fix for six to twenty-four hours; then wash in running water

for the

same length of time.

Flemming's Fluid

(Strong)

Recommended for :
Plant and animal tissues, for cytological detail;
structures;

and for

for cellular

fat.

Formula:
A. Chromic acid

..

Acetic acid, glacial

B.

Osmic

acid

2%

30 ml.

..

2 ml.

8 ml.

Mix A and B immediately

before use.

Technique:

Fix for one to twenty-four hours according to the material

chromosomes an hour is sufficient. Wash with running water
;

for
for

twenty-four hours.

Remarks:

The

penetration power of this fixative

weak solution

{see

below)

is

poorer

still;

poor and that of the

however, either solution

is

results with basic stains, particularly safranin, and iron

haematoxylin. Quite apart from their high cost, Flemming fixa-

gives

good

should definitely not be employed as general fixatives, but

in
only
special cases for very small pieces of tissue where fixation
extending through a layer of about four or five cells in thickness is

tives

sufficient, as this is

the limit of their penetrating power even in

loose-celled tissues.

Flemming's Solution
Recommended for

(weak)

All purposes for

which Flemming's strong solution

is

Formula:
A. Chromic acid
Acetic acid
B.

Osmic

acid

1%
1%
1%

Distilled water

25 ml.
10 ml.
10 ml.

50 ml.

used.

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

Technique:

Used
that the

same way as the strong solution {see above), except

volume of the fixative required is about eight or ten times

in the

that of the material to be fixed.

Remarks:
See under Flemming's Fluid (strong).

Formalin Neutral
Recommended for :
Animal

tissues in general, particularly nervous tissue.

Formula:

Formalin (formaldehyde 40%)

water

Tap
Magnesium

carbonate

100 ml.

900 ml.

to excess

Shake well and allow to stand several hours

at least;

then

decant off the volume of the clear fluid required for fixation.
Technique:

Fix at least twenty-four hours at room temperature, or six to

eight hours at 50-60 C. Washing out is unnecessary.

Remarks:
Formalin penetrates well, tissues may be kept in it for long
periods without undue hardening, although there is a gradual
decrease in basophilia of cytoplasm and nuclei, and certain cytoplasmic structures are not hardened by it sufficiently to permit
paraffin embedding. Best adapted to material which is to be em-

bedded

in celloidin rather than in paraffin

wax;

also suitable for

frozen sections.

Formalin Buffered
Formula:
Neutral formalin, as above

litre

gni.

Sodium dihydrogen phosphate, monohydrate, A.R.

Disodium phosphate anhydrous, A.R.

Technique:

As

for neutral formalin.

10

6-5

gm.

SECTION ONE
Remarks:
Neutral formalin turns acid on keeping owing to the production
of formic acid, whereas the buffered fixative remains neutral.

Recommended for :
All purposes for

neutral fixative

Formol

is

which neutral formalin

employed where a

is

required.

Alcohol

Recommended for :
Glycogen in animal

tissues.

Fibrin.

Peroxidase. Plant tissues,

particularly pollen tubes in styles.

Formula:
Formalin (40% formaldehyde)
Alcohol

100 ml.

900 ml.

70%

Technique:

Fix for three to six hours; then dehydrate, clear and embed.
Alternatively, if it is not convenient to dehydrate, clear and embed
at once, the tissues may be stored for long periods without
deleterious effects in

70%

alcohol.

Remarks:
This fixative, which penetrates quickly, while compatible with
most stains, is particularly suitable for indigo carmine.
Kelly's Fluid

Recommended for :
Animal

tissues in general,

but particularly for blood-forming

organs.

Formula:

...

Potassium dichromate
Mercuric chloride

N.B.

gm.

10 gm.

Sodium sulphate crystals

Formalin (40% formaldehyde)

2gm.

Distilled water

200 ml.

The formalin should not be added

required for

immediate use.
II

10 ml.

until the fixative is

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

Technique:

Fix for twelve to twenty-four hours. Wash in running water for

the same time. Transfer to 80% alcohol; dehydrate; clear and

embed.
Remarks:

recommended

Not

for

cytoplasm

bacteria,

degeneration,

necrosis or regeneration.

Hermann's Fluid
Recommended for :
Cytological work.

Formula:
.

6 ml.

32 ml.

Glacial acetic acid

Distilled water

38 ml.

Platinic chloride

Osmic

N.B.

acid

10%

1%

nil.

The solution should be freshly prepared.

Technique:

Fix from twelve to sixteen hours, wash in running water for

then treat as for Flemming fixed-material.

three to six hours

Remarks:

While

mordants chromatin for staining with basic

inhibits
stains,
staining with acid stains. Good plasma staining
is difficult if not imposssible after this fixative.
this fixative

it

Rleinenberg's Fluid

Recommended for :
Embryos; marine organisms, arthropods, chitinous

material.

Formula:
Sulphuric acid

1%

Picric acid, saturated

aqueous
12

100 ml.

49 ml.

SECTION ONE
Technique:

Wash

out the picric acid with

warm 70%

alcohol, followed

by

increasing strengths of alcohol.

Remarks:
This

fixative

is

a powerful penetrant of chitin.

Lewitsky's Fluid
Recommended for :
Plant cytology.

Formula:
.

100 ml.
100 ml.

100 ml.

Formalin (formaldehyde 40%)

Distilled water
.

Chromic acid

5%

aqueous

Technique:

Fix tissue for twelve to twenty-four hours; then wash for six
to sixteen hours in

running water.

Marchi's Fluid
Recommended for :
Animal and plant

tissues generally.

Formula:

Note.

Potassium dichromate 2-5% aqueous

100 ml.

Sodium sulphate crystals

Osmic acid 1% aqueous

The

last

gm.

50 ml.

item should be added immediately before use.

Technique:

Immerse thin pieces of tissues, not more than 2 mm. thick in

the fixative for four to eight days then wash in running water for
twelve to sixteen hours.
Transfer to 70% alcohol, afterwards
;

dehydrating, clearing and embedding in the usual way.

Remarks:
This

fixative is also

employed

to blacken nerve fibres.

Navashin's Fluid
Recommended for :
Cytological study of plant tissues.
13

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

Formula:

Chromic acid
Acetic acid

..

10% aqueous

Distilled water

..

..

. .

Formalin (formaldehyde 40%)

Note.
tion

1-5

gm.

100 ml.
60 ml.

40 ml.

The formaldehyde should not be added

until the solu-

required for immediate use.

is

Technique:

Fix material for one to two days; then wash twelve to sixteen
hours in running water. Dehydrate clear and embed.
;

Orth's Fluid

Recommended for :
Demonstration of acute degenerative processes to be stained
with Giemsa, Wright, or Leishman Stain, and for Intestine.

Formula:
Potassium dichromate

Sodium sulphate

. .

Distilled water

crystals
. .

Formalin (formaldehyde 40%)

The

last

gm.
gm.

2-5
i

100 ml.
10 ml.

item should not be added until the fixative

is

required

for immediate use.

Technique:

Fix pieces of tissue up to i cm. in thickness for two to four days

then wash in running water from twelve to twenty-four hours.
Transfer to 80% alcohol; dehydrate; clear and embed.

Remarks:
This

fixative

but

it

as a general fixative for animal

tissue is required,

where a firm consistency of

not recommended for sharp histological detail. The value

in the above formula is extremely doubtful
that
this
constituent may be left out without any
appears

it is

of the

and

may be employed

It is useful

tissues.

sodium sulphate

noticeable effect.

14

SECTION ONE
Petrunkevitch's Cupric Paranitrophenol Fixative*

Recommended for :
Tissues in general.

Formula:
Alcohol

60%

1-42)

Nitric acid, pure (sp. gr. i*4i

Ether

Cupric nitrate Cu(N03)

2,

3H2O

Paranitrophenol pure, cryst.

100 ml.
3 ml.

6 ml.

2 gm.

gm.

Technique:

Fix material for twelve to twenty-four hours.

alcohol; dehydrate; clear and embed.

Wash

in

70%

Remarks:
All stains conmionly in use may be employed after this fixative,
which causes less hardening than most other fixatives.

Regaud's Fluid
Recommended for :
Mitochondria and

rickettsia in

animal

tissues.

Formula:
Potassium dichromate
Distilled water
Formalin (formaldehyde
.

40%)

3 gn^-

100 ml.
25 ml.

Technique:

Fix material for three days changing the fluid every day. Immerse in 3% potassium dichromate for six to eight days; then
wash in running water for twenty-four hours. Dehydrate; clear
and embed.

Remarks:
Suitable for

Giemsa

stain

and

for

Masson's trichrome

Schaudinn's Fluid
Recommended for :
Animal

tissues in general.

Protozoa.

A. Petrunkevitch,

Science 77,

15

17-18, 1933.

stain.

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

Formula:

N.B.

Mercuric chloride saturated aqueous

Absolute alcohol
Glacial acetic acid

66 ml.

33 ml.
i ml.

The acid should be added immediately before

use.

Technique:

Wash

Fix tissues for six to sixteen hours.

70%

in several changes of

alcohol.

Remarks:

The

may be used

fixative

required for

its

at

about 65 C. when

less

time

is

penetration.

Susa Fluid (Heidenhain)

Recommended for :
Animal

tissues in general.

Formula:

Mercuric chloride, saturated aqueous

50 ml.

Trichloracetic acid

Formalin (formaldehyde 40%)

20 ml.

gm.

Distilled water

30 ml.

Glacial acetic acid

nil.

Technique:

Fix for

five to

twelve hours; then wash out with

95%

alcohol.

Remarks:
Compatible with most stains, but not with Weigert's elastin
Susa offers the advantage over most other fixatives in that
causes less shrinkage and less hardening, thereby rendering

stain.
it

tissues easier to cut.

Zenker's Fluid

Recommended for :
Perfect histological detail in animal tissues in general.

16

SECTION ONE
Formula:
100 ml.

Potassium dichromate 2-5% aqueous

Mercuric chloride
Glacial acetic acid

N.B.

5 gin5 ml.

The

required for

acid should not be added until the

immediate use.

fixative

is

Technique:

Immerse

slices of tissue in the fluid for six to

twenty-four hours

according to the nature of the material and the thickness of the

slices. Wash in running water for twelve to twenty-four hours;

then transfer to

80%

alcohol.

Remarks:
Unsuitable for Mitochondria. Suitable for Mallory's connecfor demonstration of Muscle, Fibrin, Haemo-

tive tissue stain;

fuscin, Purkinje cells, etc.

(b)

DEHYDRATION, CLEARING, EMBEDDING,

SECTIONING, ETC.

CELLOIDIN

METHOD FOR EMBEDDING

TISSUES

For preserving the relations of cell layers of different consistency, as are contained in the eye; for large objects;
and for hard tissues
for pieces of central nervous system
such as decalcified bone.
;

Solutions required:

A. Celloidin

8%

Celloidin flakes

Absolute alcohol
Ether
.

25 gm. damped
with absolute alcohol*

150 ml.
163 ml.

*Note: This may be obtained in 25-gm. bottles,

ready damped with absolute alcohol.
17

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

Pour the alcohol and ether into a clean, absolutely
wide-mouth bottle of about 32 ounce capacity,
and mix by inverting the bottle several times, redry,

leasing the stopper at intervals, to release the pressure

of ether vapour; then add the Celloidin flakes and

invert the bottle as before. Leave for about 12 hours

to dissolve

inverting or shaking the bottle gently at

intervals.

B.

Celloidin

4%

8%

Celloidin solution

Absolute alcohol
Ether

Mix as
mouth
C.

2 volumes
i

volume
volume

described above, in a large-stoppered wide-

bottle.

Celloidin

Celloidin

2%
4%

2 volumes

Absolute alcohol

Ether

D. Cedarwood oil, for clearing

Chloroform

I
I

volume
volume

volume
volume

Technique:
(a)

Wet Method

1.

Pieces of tissue not thicker than 5

mm.

are fixed in the usual

manner.
2.

Wash

in

cular fixative.

running water for the prescribed time for the partiIf a fixative containing mercury has been used,

remove mercurial

by the standard technique.

in each of the
3. Immerse the pieces of tissue for two hours
alcohol.
and
following: 50%, 70%
90%
4.

Immerse

precipitate

in absolute alcohol

from two

to sixteen hours,

according to the nature and thickness of the tissue.

5.

Immerse

6.

Impregnate with

for twenty-four hours in a mixture consisting of

of
volumes
absolute alcohol and ether, in a stoppered wideequal
mouth bottle, which must be absolutely dry.

2%

Celloidin solution from five to seven

days.

18

SECTION ONE

4%

Celloidin for five to seven days.

7.

Transfer to

8.

Impregnate with

The

9.

tissue

is

8%

Celloidin for three or four days.

then taken out of the Celloidin and put into a

mould made by folding

a piece of writing paper, and the whole is

then placed in a desiccator and left for several days, lifting the
desiccator lid for a few seconds each day to accelerate the harden-

ing of the Celloidin. If, through shrinkage of the Celloidin during

this process, the tissue becomes exposed, pour on more Celloidin
solution to cover

it.

Hardening of the block may be hastened by

placing 1-2 ml. of chloroform in the bottom of the desiccator.

The block is hard enough for sectioning when no impression
left after pressing with the ball of the thumb.

The base

10.

of the hardened Celloidin block

is

dipped into

is

8%

Celloidin then fixed to a roughened wooden or a vulcanite block by

pressing firmly, afterwards leaving for at least half an hour with a

weight on top.
11.

Expose

to chloroform

vapour for half an hour; then attach

wooden or

vulcanite block to the microtome holder, or store

the Celloidin block mounted on the wooden or vulcanite block in

the

80%

alcohol until required for sectioning.

12.

The microtome

moist with
ferred

70%

knife and the Celloidin block

alcohol and each section as

by means of

it is

must be kept

cut must be trans-

a camel-hair brush, moistened with

70%

alcohol in which the

alcohol, into a suitable vessel containing
sections can be stored indefinitely until required for staining.

70%

13.

When required for staining the sections should be removed

70% alcohol by means of a small camel-hair brush, or a

from the

piece of thin glass rod bent at one end, and transferred to a series of
watch glasses containing the reagents and stains, arranged on the
bench in the order in which they are to be used. For instance, if it
is desired to stain the sections with Haematoxylin and Eosin, the
steps are as follows :
14.

Immerse

sections in

50%

alcohol for a few minutes; then

transfer to water.
15. Stain
16.

with Ehrlich Haematoxylin by the standard technique.

Blue in tap water

then stain in Eosin (aqueous solution).

19

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

Transfer to

17.

each of two

70% alcohol;
96% alcohol.

then immerse for

five

minutes in

lots of

Note: Absolute alcohol must be avoided as Celloidin

solved by
18.

Immerse

19.

Pass into two changes of xylol.

20.

Mount

{b)

is

dis-

it.

for five minutes each in

two

lots of

Carbol-Xylol.

balsam or Dammar-Xylol.

in

Dry Method

1.

Proceed exactly as described above up to and including step

then take the tissue out of the Celloidin and put it into a

No. 8

paper mould as described in step No. 9 (above).

2. Place the block in a desiccator for a day, lifting the lid for a
few seconds every hour or so; then leave in the desiccator overnight.

Next morning place the block in a mixture consisting of equal

volumes of cedarwood oil and chloroform and add another 8 volumes of cedarwood oil, one volume at a time, every hour for small
3.

day in the case of large

be wholly transparent.

objects, or every

should
4.

now

objects.

The

Celloidin

Fix the Celloidin block to the wooden or vulcanite block as

described in step 10 (above).

5.

Sections

may now be

cut without the necessity of moistening

the knife or the block.

Note: Blocks prepared by this method are stored in a dry wide-

mouth stoppered

CELLOIDIN

bottle.

WAX

PARAFFIN

(Double Embedding)

For serial sections embedded in Celloidin

Solutions required:

Celloidin

This

is

1%

in

Methyl Benzoate

prepared by adding the appropriate weight of air-dried

Methyl Benzoate in a clean dry

Celloidin flakes to a quantity of

20

SECTION ONE
corked flask or bottle. Shake well; allow the flask or bottle to
stand upright for an hour or so ; then leave it inverted for an hour,
afterwards leave it lying horizontally for a time; then stand it
upright again, and repeat the process at intervals throughout the

day and leave the bottle lying on its side overnight next morning,
solution should be complete and it is only necessary to shake the
bottle well to ensure a homogeneous solution.
:

Technique:
1.

Tissues are fixed and washed in running water, and any merremoved in the usual manner.

curial precipitate
2.

and

Immerse

90%

for

two hours

in each of the following:

50%, 70%

alcohol.

3.

Transfer to absolute alcohol for two to sixteen hours.

4.

Immerse

in

Methyl Benzoate

Celloidin solution for twenty-

four hours, at the end of which time pour oflF the solution and
replace with a fresh lot in which the tissue should remain for a
further forty-eight hours if the tissue is not now clear, transfer it
to a fresh lot of Methyl Benzoate - Celloidin solution for a further
:

period of seventy-two hours.

5.

Immerse

the first
6.

lot,

changes of pure benzene, for four hours in

in the second, and twelve in the third.
hours
eight
in three

Transfer to a mixture consisting of equal parts of paraffin

wax and pure benzene in the embedding oven for an hour.

7. Immerse in two changes of pure paraffin wax from a quarter
of an hour to six hours in each, depending
the nature of the tissue.

upon the thickness and

Note: It is of utmost importance that tissues should be kept in

the embedding oven just long enough for the paraffin wax to
penetrate fully. Prolonged heating in the oven causes shrinking
and hardening of the tissues rendering sections difficult to cut.
If,

on the other hand, any of the Methyl Benzoate remains in the

and sufficient time has not been allowed for proper pene-

tissue

wax satisfactory sections cannot be cut. It

possible, to cut thin slices of tissue for embedding,
preferably not more than 5 mm. thick so that the total time for
impregnating in the pure paraffin wax need be no longer than

tration of the paraffin

is

best,

whenever

three hours.

Large objects such

21

as

whole embryos need a

total

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

time of twelve hours in pure paraffin wax. Pieces of brain and
spinal cord about 5 to 10 mm. thick, skin, and large objects such
as whole embryos need at least three changes of pure paraffin wax
for a total time of about 12 hours, whereas organs such as spleen,
containing a large amount of blood, muscle, fibrous tissue, require

no longer than a total of three hours in the paraffin baths. It is,

however, only by experience that the technique of embedding can
be mastered.
8.

Cast the tissue in an embedding mould and proceed as in the

case of paraffin sections {see page 32).

CELLOIDIN

PYRIDIN

A rapid method of dehydrating, clearing and embedding,

which obviates the use of alcohols and the consequent
hardening of tissues
Reagents required:
A. Pyridin, extra pure, redistilled
Celloidin

4%

in equal

volume

volume

volumes of

absolute alcohol and ether

in equal volumes of
absolute alcohol and ether.

B.

Celloidin

8%

Technique:
1.

Pieces of tissue are fixed in the usual manner.

2.

Wash

in

running water for the prescribed time for the partiIf a fixative containing mercury has been

cular fixative employed.

used, remove mercurial precipitate

3.

by the standard technique.

two changes of Pyridin, from two to
each, according to the nature and the thickness of

Immerse the

eight hours in

tissue in

the tissue.
4. Immerse for twenty-four hours each in two changes of a
mixture consisting of equal volumes of Pyridin and 4% Celloidin
(formula as above).
5.

Immerse

The

in

8%

Celloidin for twelve hours.

then removed from the Celloidin bath, blocked

and cut into sections by the standard technique described on
6.

tissue

is

pages 19-20.

22

SECTION ONE

FROZEN SECTIONS
For the identification of fat in tissues for certain impregnation methods for the central nervous system; and for the
rapid examination of pathological material, such as pieces
of tumour, which may be sectioned, stained and diagnosed
within a few minutes of their removal by the surgeon in
;

the operating theatre

Sections as thin as

method

may be

^fj,

and an advantage of

cut,

this

a lesser degree of shrinkage than in the case

of paraffin-embedded material. It is not, however, possible to cut
serial sections by this method, and sections cannot be stored before
is

that there

is

staining as in the case of paraffin-embedded material. Frozen sections should be employed only for the specific purposes mentioned

above and not as an alternative to paraffin embedding. It should

be noted that frozen sections are manipulated in the same way as
Celloidin sections, but greater care must be exercised on account
of the absence of any embedding mass.
Tissues should not be frozen too hard or the sections will curl
up and split.
A special microtome is required for cutting frozen sections.

Rapid Method for Staining Fats

Solutions required:

A. Sodium chloride A.R. grade

in distilled water
Formalin
.

B.

0*9%
n^l-

95

5 i^l-

Sudan Black

B, saturated in 70%
alcohol (this should be freshly
filtered).

NOTE:

Ethylene or Propylene Glycol may be

employed as the solvent {See page 37).

C. Apathy's mountant.
Technique:
1.

Thin

slices of tissue are fixed for ten

minutes

at

37-40 C. in

Solution A.
2.

Transfer the material directly from the fixative to the freezing

microtome and cut sections about

D

23

5/z

in thickness.

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

By means

3.

of a camel-hair brush, moistened with 50% alcohol,

from the microtome knife directly to the first of a

transfer sections

series of dishes previously

use

70%

alcohol,

arranged as follows, in order of their

Sudan Black

solution (as above),

50%

alcohol

distilled water.

After immersion in

4.

alcohol (in the

70%

first

dish) for

two

minutes, stain in the Sudan Black for ten minutes, or longer

time permits.
5.

Rinse in the

6.

Transfer to the distilled water

or, if

in

there

is

50%

alcohol.
:

mount

in Apathy's

sufficient time, counterstain for

Carmalum; then

if

medium,

about three minutes

rinse in distilled water before mounting.

Results:

Neutral

fat

and myelin

blue-black to black; nuclei: red.

GELATINE EMBEDDING
This method of embedding
friable tissues are required.

is employed when sections of loose

Dehydration is entirely eliminated

embedding takes place directly from water. The gelawhich is retained during the staining processes holds the

since the
tine

tissues together without absorbing the stain itself.

Solution required:

A. Gelatine

5%

Gelatine

Phenol

aqueous

5 gi^

95

^*

Warm in an oven to about 45 C. then stir until

the gelatine has dissolved; raise to about 60 C.
before filtering through fine calico.
B.

Gelatine

10%

Phenol

Gelatine

aqueous
.

90 ml.

10 gip.

Prepare exactly as for Solution A.

24

SECTION ONE
C. Gelatine

15%

1%

Phenol

Gelatine

aqueous

85 ml.

15

gm*

Prepare exactly as for Solution A, except that

temperature should be raised to about 75 C. before
filtering.

D. Gelatine

20%

Gelatine

Phenol

1%

aqueous

20 gm.
80 ml.

Prepare as for Solution A, but raise temperature

to 95

E.

before filtering.

1%

Gelatine

Gelatine

Phenol

1%

aqueous

gm.

99

rnl.

Dissolve by warming.
Technique:
1. Small pieces of tissue, not
fixed for twenty-four hours in

more than

5%

2.

Wash

3.

Inmierse in

in

mm.

in thickness are

formalin in physiological saline.

running water.

5%

gelatine in an incubator at 37 C. for twenty-

four hours.
4.

Immerse

in

5.

Immerse

in

at 37

10% gelatine overnight in an incubator at 37 C.

15% gelatine in an incubator for several hours

C.

6.

Embed in 20%

7.

Cut out blocks of

gelatine

and leave to

tissue

set.

and immerse them

in formalin for

twenty-four hours.

Note:
solution

The
if

blocks

may be

stored indefinitely in this formalin

desired.

8.

Rinse blocks in water and trim.

9.

Freeze blocks thoroughly until they are uniformly white.

10.

Allow the block to thaw somewhat

easily.

25

until the knife cuts

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

11.

Cut

sections

up

to 5/* in thickness

and

onto slides with

float

distilled water.
12.

Drain

off excess

or three drops of

1%

water and

float sections

on

slides

with two

gelatine.

gelatine and leave the slides in

13.
at 37 C. until the sections are dry.

Drain

off excess

1%

an oven

slide in 10% formalin for ten minutes to fix the

in the usual manner with Sudan 3, or Scarlet
stain
then
gelatine;
or
osmic
blue
Nile
acid, or store the slides in the 10% formalin
R,
14.

Immerse

until required.

LOW

VISCOSITY NITROCELLULOSE

(L.V.N.)

For embedding tissues

The

following technique, using L.V.N, in place of Celloidin,

has been developed by E. H. Leach and W. Chesterman, of The

University Laboratory of Physiology, Oxford.*

I am indebted to the authors and to the Oxford University
Press for permission to print this description of the procedure.

Chesterman and Leach's technique using

Low

Viscosity Nitro-

cellulose (L.V.N.) offers advantages over the older method of embedding in Celloidin, in that penetration is quicker, considerably
it is easier to use and considerably
L.V.N, technique large blocks, such
Celloidin.
With
than
cheaper
as half a cat's brain, can be cut at i^/u; small blocks
mm.,
can be cut at 5 to jju on a paraffin microtome without any special

thinner sections can be cut,

5x5

modification or attachment.

supplied damped with normal butyl alcohol; it is

explosive than Celloidin and it should be handled with care.
When dry it will explode if hit. Exposure to sunlight should be

L.V.N,

is

more

avoided.

Solutions required:

Note: Solutions

and B each contain

*Q.y.M.Sc. Vol.

20, pt. 4,

26

20%

of Nitrocellulose:

Dec. 1949.

SECTION ONE
A. Absolute alcohol
Ether
.

Dibutyl phthalate

Mix

210 ml.

250 ml.

5 ml.*

gm. L.V.N,

well and add 140

(as

supplied

damped with N. butyl alcohol).

*

NOTE

In the original paper (Chesterman and Leach, 1949) and in the previous
edition of this book (1953), tricresyl phosphate was stipulated: this has now been
replaced by dibutyl phthalate, and I am indebted to Professor F. Bergel of
The Chester Beatty Research Institute, Institute of Cancer Research, Royal
Cancer Hospital, London, for calling my attention to the potential danger of
handling tricresyl phosphate. Professor Bergel informs me that tricresyl-ophosphate is highly toxic, more than 7-30 mg./kg. producing severe paralysis,
and while the corresponding meta- and para-compounds show little sign of
having this toxicity, the tricresyl phosphates on the market, up to the present
time, always contain some of the ortho compound.
"
References: Aldridge, W. N. (1954), Biochem.jf., 56, no. 2, 185-9,
Tricresyl

Phosphates and Cholinesterase". Thompson, R. H. S. (1954), Chem. and Ind.,

749, Martindale's Extra Pharmac. (1941), vol. i, 200-1.

B.

Absolute alcohol
Ether
.

Mix

210 ml.

250 ml.

well and add 140 gm. L.V.N,

N. butyl

(damped with

alcohol).

From Solution B prepare also 5% and 10% solutions by diluting

with a mixture consisting of equal parts of absolute alcohol and
ether.

C. Xylol
Toluol

part

part

Beechwood creosote
Procedure for embedding

2 parts

tissues:

1. Fix and dehydrate tissues as usual; then immerse in etherabsolute alcohol (equal parts) for one day.

2.

Immerse

3.

Transfer to

4.

Transfer to

in

5%

L.V.N, for three to

10% L.V.N,
20% L.V.N.

for

one

to

(Solution

five days.

two days.

above) for one to five

days.
5.

Embed

in Solution A.

6. Allow to harden slowly in a desiccator.

In one to five days
the block should be adequately hard. At this stage it should be a

27

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

but easily deformable gel not altered in shape or size by
shrinkage; it should be considerably less hard than a Celloidin
block is usually made.

Stiff

7.

Plunge the block into

75%

alcohol.

Change the alcohol

twice over a period of one to three days.

8. Trim the block, removing the hard outer rim of the L.V.N.
Use 20% L.V.N, to mount it on the wood or fibre block. Hardening is complete in a few minutes. Dip into 75% alcohol for a few
more minutes.

Cutting sections:

"

dry ". If a Celloidin microtome is used the

tilt of knife should be the same as that used for cutting Celloidin,
but the angle the knife makes with the direction of travel should
be between 25 and 45 instead of the usual 75 used for Celloidin

Cut the

sections

sections ; this prevents the rolling of sections.

Procedure for handling sections:

1.

Collect the sections in

usual.
2.

75%

Dyes tend to stain L.V.N,

Mount

sections

on

and

stain as

96%

alcohol.

alcohol; handle
than Celloidin.

less

to a slide

from a bowl of

Flatten with tissue paper moistened with

paper with a glass rod and then remove

alcohol

press the
96%
it.
Repeat this several
;

times.
3. Treat similarly several times with equal parts of absolute
alcohol and chloroform.

4.

Treat similarly several times with the solution C.

5.

Treat similarly several times with xylol.

6.

Mount

in balsam.

MERCURIC CHLORIDE PRECIPITATES IN SECTIONS:

METHOD FOR REMOVAL
Before proceeding to the staining of sections of tissues which
have been fixed in fluids containing mercuric chloride it is necessary to carry out the following procedure, which is essential for
the removal of the deposits of mercuric chloride which would

obscure the picture.

28

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

Note: Where possible use a series of corked specimen tubes for
the above procedures. By occasionally shaking the tube containing
the specimen the process of penetration of the alcohol is accelerated. Twelve hours in each change of alcohol is sufficient for small
pieces, but larger pieces of tissue usually require eighteen or
twenty-four hours. Hard tissues may be softened by Lendrum's

technique which consists of immersing the tissues in 4% aqueous

phenol for one to three days, after washing out the fixative; dehydration

is

then carried out in the manner described above.

Rapid dehydration of small slices of tissue:

Thin slices not more than 5 mm. in thickness are immersed for
half an hour in each of 50%, 70%, 90%, 96% and absolute alcohol.
{h)

Clearing

Xylol, cedarwood

oil,

benzene, toluene or chloroform are the

reagents most frequently used for this purpose.

Xylol is the most rapid of these in displacing the absolute
alcohol, but

it

has the disadvantage of rendering tissues brittle;

therefore, if xylol is used as the clearing agent tissues must be subjected to it only for the minimum time necessary to displace the

absolute alcohol.

Cedarwood oil is slow

hardening

tissues

in its action but it has the advantage of not

even after prolonged immersion.

Benzene is the best clearing agent and may be employed for the
most delicate tissues it causes the minimum shrinkage, penetrates
tissues fairly rapidly and subsequently evaporates from them in
:

the paraffin

Toluene

is

embedding bath.
also a very satisfactory clearing agent in that tissues
it for at least twenty-four hours without risk of

can be subjected to
their

undergoing shrinkage.

Chloroform hardens tissues to a lesser degree than xylol but

requires two or three times as long to penetrate and clear the tissue.
evaporates from the paraffin embedding bath, and
particularly suitable for large pieces of pathological tissue.
It rapidly

it is

Technique:
I.

Small pieces of material not more than 5 mm. thick may be

by immersing for fifteen to thirty minutes in each of two

cleared

30

SECTION ONE
changes of xylol or cedarwood

oil or benzene or toluene. Larger

cm.
thick
require one-and-a-half to three hours in
pieces up
each of two changes of the clearing agent, while bulky specimens
such as whole embryos require up to six hours in each of the two
changes. If at the end of the times prescribed above the specimens
are not translucent or transparent they should be left in the clearing
agent until they have reached that stage.

to

(c)

Embedding

Technique:
1.

Transfer the object from the clearing agent to a mixture con-

sisting of approximately equal parts of paraffin wax and the clearing

agent in a tube and place the whole in the oven set at a temperature

from about 50 to 60 C. for one half to sixteen hours, depending upon

the size and nature of the object; half an hour is sufficient for
objects up to 3 mm. thick; an hour for 5 mm., two hours for i cm.,
and from eight to sixteen hours for bulky specimens such as whole
embryos.
2.

Transfer to pure paraffin wax in the oven from a quarter to

eight hours.
3.

Transfer to another bath of pure paraffin wax for the same

length of time.

Note: Specimens up to
in each of the

mm.

thick usually require half an hour

two baths of pure

paraffiua wax, while specimens

an
hour; and i cm. about four
5
hours; very bulky objects about eight hours in each of the two
baths of wax.

mm.

in thickness require about

Pathological material containing thrombi, emboli,

etc., striated

and non-striated muscle, organs containing a large amount of

blood (spleen, etc.), and fibrous tissue should be subjected to immersion in the embedding baths for the minimum time necessary
for the

wax

to penetrate thoroughly, as they are particularly liable

and shrinkage when exposed to heat for prolonged

to hardening

periods.

Casting the Paraffin Block

I. Smear the inside of the
embedding angles and the embedding
base-plate very thinly with liquid paraffin then adjust the angles
on the plate to form a mould of a suitable size.
;

31

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

2.

Fill the

mould with molten

object in the wax and arrange

for sectioning.

When

it

paraffin

so that

it is

wax; then place the

set in the right plane

wax block

so formed is partially set, immerse it

mould, in cold water to ensure rapid
cooling and thereby obviating crystallization of the wax and consequent crumbling of the block when it is mounted on the microtome and sections are cut.
3.

the

gently, while

still

in the

Cutting of Sections
This can only be learnt by practical experience under skilled
guidance in the laboratory, and it is not proposed to make any
attempt to deal with the subject here since space does not permit a
description of the various types of microtomes available and the
technique of manipulating them. However, readers would find the
chapter on section cutting in Histological TechniquCy by H. M.
Carleton and E. H. Leach (published by Oxford University Press),
very informative.

Mounting Sections on Slides and Hydrating

1. Wet the tip of the finger slightly with glycerine albumen
(Mayer) and make a smear over an area large enough to take the
section in the centre of the slide.
2.

Pick a section up with a needle or forceps and place

it

over

the smear of albumen.

3.

that

With
it is

the thumb, gently press the section on to the smear so

quite flat and without folds or creases, taking care not to

damage the

section in the process.

Note: If the sections are curled up or folded, first place a drop of

potassium dichromate on the slide and float the section on
this; then heat very gently until the section floats out flat. Blot
round the edges of the section to remove excess solution; then
carefully but thoroughly blot until all traces of liquid are removed.
Leave the slide on a warm surface for a few minutes to drive away
the last traces of water then proceed as follows

1%

4.

Gently warm the slide

wash away
5.

all

Remove

traces of

wax with two

wax just

melts; then
or three changes of xylol.

until the paraffin

the xylol by washing the preparation thoroughly

with absolute alcohol.

32

SECTION ONE

Wash
Wash
Wash

with two changes of 90% alcohol.

with
7.
70% alcohol.
8.
with two changes of distilled water if an aqueous stain
is to be used
but if an alcoholic stain will be used staining may
commence immediately after washing with 70% alcohol.
9. Proceed to stain in accordance with the staining technique it
6.

is

desired to employ.

Note: If the section appears opalescent when the xylol or when

90% alcohol is added the presence of water is
indicated and it is necessary to retrace each step until the preparation no longer appears opalescent when taken down to alcohol.
the absolute or the

PARAFFIN

WAX

PYRIDIN TECHNIQUE

A rapid method of dehydrating and

1.

Material

is

fixed

clearing

and washed by the standard method.

remove mer-

If a fixative containing mercury has been used,

curial precipitate by the standard technique.
2.

Immerse the

tissue in two changes of Pyridin for two to

in
hours
each, according to the nature and thickness of the
eight
3.

tissue.
4. Transfer to a mixture of equal parts of molten paraffin wax
and Pyridin in the embedding oven for one half to sixteen hours,
depending upon the size and nature of the object.
5. Transfer to pure paraffin wax in the oven for a quarter to
eight hours, depending upon the thickness and nature of the

material.
6.

Cast into block and cut sections in the usual manner.

WATER WAX
(Michrome)

very rapid and simple method of embedding tissues, obviating

the use of dehydrating and clearing agents.
Water wax is an amorphous water-soluble wax which sets at
56 C. to form translucent blocks similar in appearance to paraffin
the complete absence of any trace of crystallization.

wax but with

Fresh or fixed material

may be

used.

33

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

Reagent required:

Water wax (Michrome).

Technique:
1.

Shake

off excess

water before immersing the pieces of tissue

directly into a bath of water

55-6 C. and leave for an hour.
2.
3.

4.

wax

in the

embedding oven

at

Transfer to a second bath of water wax and leave for an hour.

Transfer to a third bath of wax for an hour, or longer.
Cast the block and leave it to cool in the atmosphere or in a

refrigerator.

Care must be taken not to allow the block to come

into contact with water.

After cutting sections, float them out on water, which dissolves

away the wax, and take them up on slides.
6. Stain, dehydrate and mount immediately in the usual way.
5

Notes:
(a) Fat, if present,

should be dissolved out with several changes

of acetone, before the tissues are immersed in the wax.

and stored in airtight containers
{h) Blocks should be kept dry
as the wax is liable to take up moisture from the atmosphere.

MISCELLANEOUS DEHYDRATING AND CLEARING

REAGENTS
BUTYL ALCOHOL TERTIARY
For dehydrating and clearing tissues for paraffin embedding in
and xylol. The reagent is miscible with
water and with paraffin wax, and causes less shrinkage and hardening of tissue than does ethyl alcohol and xylol. It is also a useful

place of ethyl alcohol

substitute for ethyl alcohol for dehydrating material stained with

methylene blue and other dyes which are easily extracted by ethyl
alcohol.
Techjiique:

After fixing and washing tissues in the usual manner pass into
1. Tertiary butyl alcohol (T.B.A.) 50% aqueous for 1-2 hours.
2. 70% Aqueous T.B.A. 2 hours to several days.
:

3.

85%

aqueous T.B.A. for 1-2 hours.

34

SECTION ONE
aqueous T.B.A. for 1-3 hours.
Pure T.B.A. for 3 changes of 4 hours in each.
Equal parts of Uquid paraffin and T.B.A. for 1-2 hours.

95%

4.
5.
6.

Infihrate in paraffin wax.

7.

CAJEPUT OIL
For clearing
Phis reagent will absorb small amounts of water without cloud-

and

therefore, particularly useful in wet climates as a

clearing agent in place of xylol. Cajeput oil is considerably more
ing,

it is,

expensive than xylol, however.

CELLOSOLVE
(Ethylene glycol monethyl ether)

For dehydrating thin slices of tissue and sections

This reagent, which
cible with water,

is

a colourless, inflammable liquid, miscedarwood oil, clove oil, and

alcohol, xylol,

various other oils and solvents, and

is

also a

good solvent for

coming into increasing use both for animal and

plant histology, in place of ethyl alcohol in fact many laboratories
in Great Britain, at least, employ cellosolve in preference to the

many

stains, is

which they now regard as obsolete.

However, although some workers believe cellosolve to be superior
to all other dehydrating agents as it obviates hardening and distortion of most tissues, it is unsuitable for bulk material as it tends
ethyl alcohol technique,

to cause distortion of protoplasmic cells

its
dehydrating action.

Technique
1.

Wash

owing

to the rapidity of

pieces of tissue, not

more than

mm.

thick

and im-

merse directly into cellosolve for half an hour.

2.

Immerse

in a fresh bath of cellosolve for half to

3.

Immerse

in a third bath of cellosolve for the

one hour.

same time.

Complete the dehydration in a fourth bath of cellosolve for

an hour-and-a-half.
4.

35

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

5.

6.

7.
8.

wax
9.

10.
1 1

12.

Clear in xylol for an hour.

molten paraffin wax for half an hour.

Transfer to a second bath of paraffin wax for an hour.

Immerse

in a bath of

Complete the infiltration by immersing in a third bath of

an hour then cast the block and cut sections.

for

Fix sections to slides

then remove paraffin wax with xylol.

Rinse in two changes of cellosolve.

Apply the stain then wash with water, or alcohol.

Immerse for one to three minutes in each of three changes
;

of cellosolve.
13.

Clear in xylol, benzol, clove

14.

Mount

oil

in balsam, cristalite or

or cedarwood

oil.

D.P.X. (Lendrum and Kirk-

patrick).

DIOXANE
For the dehydration and clearing of tissues
This reagent, which is a colourless inflammable liquid, solidifying at 10 C, miscible with water and paraffin wax, alcohol and
xylol as well as many other organic solvents of the aliphatic and of
the aromatic series, is used and preferred by
of the orthodox alcohol-xylol-cedarwood

many workers in place

oil

method

for de-

hydrating and clearing tissues, as the technique is simpler and

quicker and it has the advantage of eliminating brittleness and
shrinkage of tissues. It is, however, essential that a reliable brand
of Dioxane be employed, as some makes contain relatively large

amounts of water, and are therefore unsuitable

for histology.

Warning* Care should be taken as Dioxane vapour

it

should be used only where ventilation

is

is

toxic:

abundant.

Technique:
I. Transfer tissues directly from the fixative to a well-stoppered
specimen jar containing Dioxane with a thin layer of anhydrous
calcium chloride over which is placed a circle of surgical or zinc
gauze to separate the tissue from the dehydrating agent. Allow
the Dioxane to act from three to twenty-four hours, depending
upon the size and thickness of the tissue.

36

SECTION ONE
Note: Tissues which have been treated with a
Miiller or Zenker, containing potass, dichromate,

fixative

such as

must be washed

from two to twelve hours in running water, depending upon the

size and the nature of the tissue, before being transferred to Dioxane.
2. Transfer to a mixture of equal parts of paraffin wax and Dioxane for half to one hour in a paraffin embedding oven.

3. Transfer to pure paraffin wax, allowing a somewhat longer

time for impregnation than for tissues cleared by the orthodox
method.

Note:

The Dioxane can be used

calcium chloride

is

several times provided the

changed.

ETHYLENE GLYCOL

A solvent,

inflammable

when

almost invisible flame; which

heated, burning with an intense,

also be employed as a de-

may

hydrating and diflFerentiating agent for the Sudan colours, in place

of acetone-alcohol, 70% or 50% alcohol; giving stable solutions,
without loss of stain from the lipid particles.
Solution required

Technique :

Prepare the staining solution by heating and stirring about

0*75 gm. of the dye with 100 ml. pure anhydrous ethylene glycol
to 100 C. Filter the solution while it is still hot, and again after
1.

it

has been allowed to cool.

2.

them

sections, from formalin fixed material, and wash

in water for about five minutes to remove excess formalin.

Cut frozen

3. Dehydrate the sections by agitating them gently with a camel

hair brush for three minutes in each of two changes of pure

anhydrous ethylene

glycol.

4. Transfer the sections to the staining solution for five to

seven minutes, agitating them gently at intervals.
5.

Differentiate

by agitating the sections gently in 85% ethylene

two to five minutes, controlling by examining

glycol in water for

under the microscope

at intervals.

37

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

6.

Transfer to a large dish of distilled water for three to

five

minutes.
7.

Float sections onto slides; drain and carefully blot away-

excess water.
8.

Mount

in glycerine jelly, Farrant or

Aquamount.

Note: Either ethylene or propylene glycol may be used; however, the former usually costs less than the latter.
Reference: Chiffelle, T. L. and Putt, F. A., (195

1),

Stain Tech., 26, 51-6.

PROPYL ALCOHOL, NORMAL (OR

For dehydrating and clearing tissues prior

to

ISO)

embedding

Technique:
1

Pieces of fixed tissue are placed directly into normal propyl

left therein overnight.

alcohol and
2.

Transfer directly into a bath of paraffin wax M.P. 40 C.

After infiltration of the 40 C. wax, transfer to a bath of

52-54 C. paraffin wax for a few minutes; then cast the block.
3.

Note: This method prevents hardening and distortion of tissue

particularly recommended for scirrhous carcinoma, connec:

it is

tive tissue,

tumours,

etc.

TERPINEOL
For dehydrating sections
Technique:
1. After staining and before dehydrating,
wipe the slides and
blot sections carefully, without allowing them to dry completely.

2.

Transfer to terpineol and agitate for a few seconds.

3.

Immerse

in a second lot of terpineol for a

4.

Drain and wipe the

5.

Clear with xylol, and

few seconds.

slides carefully.

mount

as usual.

Note: The destaining action of alcohol is avoided with this

method, which is harmless to the vast majority of stains. Neutral
Red being an exception. If desired, Cajeput Oil may be used for
clearing.

38

SECTION ONE

AQUEOUS MOUNTING MEDIA

APATHY GUM SYRUP MOUNTANT

fairly

quick-drying aqueous mountant which sets very hard

in place of Farrant for fat preparations; it is,

and may be used

however, usually definitely acid in reaction. Hardening of the

mountant may be hastened if the slides are left on warm plate.

Apathy may

also

be used for ringing glycerine mounts.

AQUAMOUNT
A moderately quick-drying, transparent and colourless, aqueous
mountant which, unlike Apathy and Farrant, is neutral in reaction.
It takes somewhat longer to harden than does Apathy but it is
unlike the latter in that it does not tend to crystallize or become
excessively brittle. Aquamount
for fat preparations.

is

preferable to

Apathy or Farrant

BERLEZE'S FLUID

mountant and

killing fluid

used in Entomology.

Living

specimens of Acarina and Insecta are killed by placing them

directly into a drop of this fluid on the slide, but specimens which
have been stored in alcohol should be washed with io% aqueous
acetic acid before mounting. The fluid takes from one to two weeks
or even longer to set, after which time the slides should be ringed
with a waterproof cement and ringed with a layer of Canada
balsam in benzene.

DOETSCHMAN'S

GUM CHLORAL MOUNTANT AND

STAIN

This
fluid,

is

a modification, containing basic fuchsin, of Berleze's

kills, fixes, dehydrates, clears, stains and mounts a

which

specimen in one operation.

This valuable reagent is used in entomology for killing, fixing,
dehydrating, clearing, staining and mounting specimens in one
operation. Highly chitinized specimens should be treated with
10% potassium hydroxide from sixteen to twenty-four hours,
then washed well in water before mounting directly into a drop
E

39

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

of Doetschman's fluid on a slide.

For permanent mounts the

preparations should be placed in the oven at 37-50 C. from six

to twenty-four hours. Ring the slides with a waterproof cement.
Reference: MicroscopisV s

Vade-Mecum nth

ed., p. 207.

GLYCERINE
Used

as a

mounting medium

for frozen sections.

Only the

purest neutral glycerine should be used. Permanent preparation

may be made by painting the edges of the coverslips with melted
glycerine jelly and coating the jelly,
size or asphalt varnish.

when

has

it

set,

with gold

GLYCERINE JELLY
For fat preparations, frozen sections, gelatine sections, etc. This
mountant, which should be neutral in reaction, sets sufficiently
hard to permit direct varnishing of the edges of the coverlips, of

mounted preparations, without prior luting. Preparations mounted

in glycerine jelly will last for

many

years without deterioration.

GLYCHROGEL MOUNTANT
For Marchi-stained sections, gelatine
nematodes etc.

sections,

teased pre-

parations,

A.

Chrome alum

Distilled water

0-2 gm.
30 ml.

Dissolve by warming.
B.

Gelatine granules
Distilled water

Glycerine

3 g^i-

50 ml.
20 ml.

Warm the water to 45

at a

Add

C. then shake in the gelatine a

time until dissolved and add the glycerine.

Keep

A to

solution B; shake thoroughly; then

as a preservative.
in a well-closed bottle to prevent evaporation.

solution

add about

o-i

little

filter

and

gm. camphor

Reference: Wotton, R.
21-2.

M. and Zwemer,

40

R. L. (1935), Stain Tech., 10, no.

i,

SECTION ONE

NON-AQUEOUS MOUNTING MDEIA

CANADA BALSAM
"

"

Canada balsam is natural Canada balsam,

a pale-yellow viscous fluid obtained from the balsam fir, indigenous to Canada: this fluid is useless as a histological mounting
"
medium. "Canada balsam, dried is the natural Canada balsam
which has been dried by heat until it has become a brittle solid.
"
*'
Canada balsam in xylol
(or in benzene or other suitable
Strictly speaking

prepared by dissolving the dried natural balsam in

When an histologist sees the words ** Mount in
"
*'
Canada balsam (or simply balsam ") he takes it to mean, and
correctly so, that the preparation is to be mounted in Canada
"
"
balsam in xylol. However, the term
Canada balsam
can be
solvents)
xylol,

is

etc.

who are new to the language and customs of

and such workers have been known to mount in true
Canada balsam: i.e. natural Canada balsam, and with most un-

misleading to those
histology,

satisfactory results. It
'*

is

for this reason that this definition of

"

Canada balsam is given here.

Although Canada balsam in xylol

is still used very extensively it

has a serious disadvantage in that it is prepared from a natural
resin which is uncertain and variable in chemical composition,

usually somewhat acid in reaction, and while it may be neutralized,

will revert to an acid reaction after a time, however careful one is
in handling and storing the mountant. For this reason it is better
employ a synthetic mountant of definite and unvarying chemical
composition. Short descriptions of mountants of this kind are

to

given below: they also have the advantage of being considerably

than Canada balsam, moreover, Canada balsam is conmore
siderably
costly than the synthetic mounting media desless costly

cribed below.

CLEARMOUNT
Refractive index 1-515

colourless, neutral, synthetic

mountant, with a drying time

approximately the same as Canada balsam in xylol. This mountant, which remains neutral indefinitely and does not cause the
41

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

fading of even the most delicate stains, is miscible with xylol,
absolute alcohol, benzene, toluol, dioxane and many other solvents.

CRISTALITE
Refractive index 1-515

This synthetic mountant has

except that
alcohol.

it

dries

the properties of Clearmount

rapidly and is not miscible with

all

much more

D.P.X.
Refractive index 1*515

by Lendrum and Kirkpatrick*

and probably other proprietary synthetic
mountants are based. This excellent mountant, which is colourless and neutral and does not cause fading of stains has the added
advantage of being one of the least costly of all the synthetic
synthetic mountant, devised

on which

Cristalite

resinous mountants: however, there is a considerable degree of

shrinkage of the mountant on drying and it should therefore be

applied liberally to allow for this.

*y. Path. Bact.y (1939), 49, 592; (1941), 53, 441-

MEEDOL BALSAM
Refractive index i'5i5

xylol miscible mountant, pale amber in colour and hardly

distinguishable in appearance from Canada balsam in xylol. This

one of the least costly of all resinous synthetic mounting media.

Unlike Canada balsam, but like the other synthetic mountants
described in this paragraph, Meedol balsam has the advantage of
remaining neutral indefinitely under normal conditions of storage

is

and handling.

FLUORMOUNT
Refractive index 1*515

A synthetic,
mountant

colourless, neutral, xylol miscible, non-fluorescent

for fluorescence microscopy.

42

SECTION ONE

MICHROME MOUNTANT
Refractive index 1*515

synthetic neutral mountant miscible with alcohol and with

xylol. Sections may be mounted directly from 95% alcohol if
desired.

Does not cause the fading of

years. This mountant, however,

Clearmount.

S.Q.D.

is

stains

even

after several

largely being displaced

by

BALSAM

Refractive index

i '5

15

An

exceptionally quick-drying, synthetic,

synthetic mountant, similar to Meedol balsam.

xylol

miscible,

VENETIAN TURPENTE^E
For mounting filamentous algae and other delicate material
Solutions required:

A. Glycerin 5%.
B. Venetian turpentine

10%

in abso-

lute alcohol.

Technique:
1.

Stain the material in suitable aqueous stains.

2.

Transfer to Solution A, and leave therein for several days in

an open dish.
3.

4.

Wash
Wash

with several changes of

95%

alcohol.

with two or three changes of absolute alcohol.

alcohol to
5. Transfer the material quickly from absolute
Solution B (10% Venetian turpentine in absolute alcohol), in an

open
6.

dish.

Place the dish with contents over soda lime in a desiccator

for several days until the fluid

7.

Mount on

becomes

slides.

43

viscous.

SECTION 2ANIMAL HISTOLOGY

(Normal and Pathological)

45

ALCIAN BLUE

CHLORANTINE FAST RED

For selective staining of mucopolysaccharides and for

morphological studies of connective tissue, cartilage and

bone
Solutions required:

A. Ehrlich haematoxylin
B.

Alcian blue

i%

Acetic acid

1%, aqueous

aqueous

C. Phosphomolybdic acid

D. Chlorantine

fast

1%

50 ml.
50 ml.

aqueous

red 0-5%, aqueous

Technique:

Fix tissues in Bouin or in

1.

10%

formalin and

embed

in the

usual way.
Stain sections in Ehrlich haematoxylin for ten to fifteen

2.

minutes.
3.

Blue in tap water or in lithium carbonate solution.

4.

Wash

5.

Stain for ten minutes in the Alcian blue.

6.

Wash

7.

Immerse

in distilled water.

in distilled water.
in the

phosphomolybdic acid solution for ten

minutes.
8.

Wash

9.

Stain for ten minutes in the chlorantine fast red solution.

in distilled water.

10.

Wash

1 1

Dehydrate clear in xylol and mount.

in distilled water.
;

Results:

Nuclei are stained purplish blue. Mucin, granules of mast cells,

cartilage and some types of connective tissue

ground substance of

47

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

bluish green. Collagen fibres and ossein,
Cytoplasm and muscle, pale yellow.

fibres,

Reference: Lison, L. (1954), Stain Tech., 29, no.

ALDEHYDE FUCHSIN
For elastic

tissue,

mast

cells,

(G.

13 1-8.

Gomori)

beta cells of the pancreatic

islets, etc.

Solutions required:

A.

3,

cherry red.

SECTION TWO
Note: This treatment with iodine is recommended for all
tissues, whether they have been fixed in mercurial fixatives or not,
as it often shortens the staining time necessary and makes the
shade deeper.
6.

Wash

7.

Immerse

well with water.

in solution

for about

one half to two minutes

been restored.

until the natural colour of the section has

8.

Wash

9.

Stain for five minutes to two hours in a coplin jar filled with

well with water.

solution C.

Note: Elastic

fibres, five to

ten minutes.

Beta

cells, fifteen to

thirty minutes, or longer. Pituitary, thirty minutes to two hours.

At intervals examine the

under the microscope to

slide, after rinsing with 90%

ascertain the depth of staining,
but taking care that the preparation is not allowed to dry. If the
desired depth of staining has not been attained, the slide may be

10.

alcohol,

returned to the stain and rinsed with alcohol again before further
examination: this process may be repeated any number of times
until the desired degree of staining has
1 1

If desired a counterstain

been reached.

may now be

applied haematoxylin
but for pancreas and pituiOrange
tary, a trichrome stain of the Masson type or the MalloryHeidenhain technique can be used to bring out all types of cells.
In either case. Light Green or Fast Green, FCF should be used
in place of the aniline blue as their shades contrast better with the
.

G is best for most purposes,

purple of the aldehyde fuchsin.

12.

Dehydrate with absolute alcohol; clear in xylol and mount.

Results:

The
(I)

following are stained deep purple

Elastic fibres of all tissues, whatever fixative has

been

used.
(II)

Mast

cells, after

any

fixative.

The

chief cells of the gastric mucosa, particularly well

(Ill)
stained after fixation in formalin or Bouin.

49

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

(IV) Beta cells of the pancreatic islets of all species, after
formalin, mercuric chloride-formalin or Bouin. Particular beautiful results are obtained in the islets of
cells

man and the

sheep (the beta

of the latter are particularly difficult to stain otherwise).

(V) Functioning tumours of the

islets are also

stained select-

ively.

(VI) Certain basophils of the anterior pituitary, after the same

In the pituitary of the rat and of the pig the

fixatives as in (V).

two kinds of basophils are usually quite conspicuous.

Notes:
{a) After fixatives containing mercury the background is pale
mauve. After formalin or Bouin the background is colourless.
If the stain is prepared by adding paraldehyde to Feulgen's
ifi)

fuchsin the resultant solution will stain the beta cells very distinctly
but leave the elastic fibres unstained. Old solutions of the aldehyde
fuchsin will stain elastic fibres very selectively, but leave the beta
cells unstained.
Reference: Gomori, G. (1950),

Am. J.

Clin. Path.y 20,

No.

7,

665-6.

ALDEHYDE FUCHSIN - HAEMATOXYLIN LIGHT GREEN ORANGE G . CHROMOTROPE

For the

diflFerentiation of two types of Basophils in the

Adenohypophysis of the rat and the mouse
Solutions required:

A. Benin's fixative with the acetic acid replaced

SECTION TWO
Note:

The

solution turns

purple in 24

hours, and is ripe and ready for use after being

kept at 20 C. for three days or in two days if

The

kept at about 37 C.

stain deteriorates

after four or five days.

E.

Ehrlich Haematoxylin

F.

Ethyl alcohol

70%

..

..

Hydrochloric acid, cone.

99-5 ml.
0-5 ml.

..
.

G. Lithium carbonate, saturated aqueous

H. Orange G 2% aqueous
Light Green SF 1% aqueous
.

Distilled water

Chromotrope 2R

50 ml.
20 ml.

30 ml.

0-5

0-5

Phosphotungstic acid
Glacial acetic acid

gm.
gm.

ml.

Dissolve the phosphotungstic acid in the

water, then add and dissolve the

distilled

chromotrope 2R followed by the acetic acid.

Orange G and Light green solutions. Shake
thoroughly.

Note: This solution keeps indefinitely.

Acetic Acid

I.

0*2%

Technique:

A for 24

1.

Fix in solution

2.

Wash

3.

Dehydrate, clear and

hours.

in running tap water for six to eight hours.

embed

in paraffin

wax

in the usual

manner.
4.

Cut

5.

Remove

sections, in the horizontal plane, 3 to

paraffin

wax from

4jLt

in thickness.

sections with xylol.

Pass through the usual descending grades of alcohol to distilled water.

6.

Immerse in Lugol's iodine for thirty minutes.

Transfer to the sodium thiosulphate solution until the
sections have regained their natural colour
(about two minutes).
7.
8.

9.

Rinse thoroughly in distilled water.

51

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

10. Stain in the aldehyde-fuchsin solution from two to ten
minutes, taking care not to overstain, which can be avoided by
checking the slides (after rinsing in 95% alcohol) at intervals under

the microscope: the staining should be stopped as soon as the

beta cells stand out clearly in dark purple against a colourless or
faintly purple

background.

11.

Rinse in two changes of

12.

Immerse

95%

alcohol.

for five to ten minutes in a third

change of

95%

alcohol.
13.

Rinse in

14.

Rinse in distilled water.

alcohol.

70%

15.

Stain in Ehrlich Haematoxylin for three to four minutes.

16.

Rinse in

distilled water.

by dipping three or four times

17. Diff"erentiate

in the acid

alcohol solution F.
18. Blue in the lithium carbonate solution; or in running tap
water for five to ten minutes.
19.

tion

Counterstain in the light green-orange-chromotrope solu-

H for 45 seconds.

20. Rinse quickly with

21. Rinse in
22.

Immerse

95%
for

0-2%

acetic acid.

alcohol.

two minutes

in each of

two changes of absolute

alcohol.
23. Blot slides carefully.
24.

Immerse

in xylol for

25.

Immerse

in another lot of xylol for five minutes.

26.

Mount

two minutes.

in D.P.X. or Clearmount.

Results :

Granulation of beta cells, selectively stained dark purple with

the aldehyde-fuchsin. The cells of the pas intermedia and the
Herring bodies of the neutral lobe should have little or no affinity

The delta cells are stained green, and the

acidophilic granules varying shades of orange. Nuclear chromatin,
Nucleoli are tinged bright
purplish brown to reddish brown.

for aldehyde-fuchsin.

52

SECTION TWO
red,

The non-granular

by the chromotrope 2R.

greyish green or unstained.

plasmic vacuoles, orange.

cytoplasm,

Coagulated contents of the cyto-

Reference: Halmi, Nicholas, S. (1952), Stain Tech., 27, no.

i,

61.

ALIZARIN RED, S
For calcium deposits in cartilagenous and embryonic bone
Solutions required:

A. Alizarin Red, S, aqueous 1%

B. Polychrome Methylene Blue (Unna)
Technique:

Tissues are fixed in

80-90%

embedded

alcohol and

in paraffin

wax.
1.

Sections are brought

Solution

down to

distilled

water

then stained in

A for five to

sixty minutes, according to the material.

with distilled water, followed by 95% alcohol at 60 C.

2.

Wash

3.

Counterstain with Solution

for one to three minutes.

Results:
Cartilage: intense violet.

plasm,

etc.

Calcium: red.

Nuclei: blue. Cyto-

yellow.

The method

is

particularly suitable for pathological specimens.

For bone staining in small vertebrates (Dawson's method)

Solutions required:

A. Potass, hydroxide
B. Alizarin Red, S

1%

o-i

Potass, hydroxide
Distilled water

C.

MalVs

aqueous
.

gm.

10 gm.
i litre

solution:

Glycerin

Distilled water

Potass, hydroxide

20 ml.
79
i

inl-

gm.

Technique:
I.

Whole specimens

are fixed in

days.

53

95%

alcohol for at least three

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

2.

Transfer to acetone and leave for several days to dissolve out

the fats which would otherwise stain intensely and obscure the
view of the bony structures.
3.

Wash

well with

95%

alcohol; then

immerse

in

95%

alcohol

for twenty-four hours.

4.

Immerse

the size of the

in Solution

A from one to seven

specimen, until the

days, according to

bones are clearly visible through

the muscle.
5.

Transfer to Solution

depth of colour

this takes

bones are stained the desired

until the

from one to seven days, and the solution

should be changed on the fourth day.

6.

Clear in Solution

until

no more colour comes

out.

7. Pass into a mixture of equal parts of glycerin and water, and

continue through increasing strengths of glycerin.
8.

Store in pure glycerin.

Results:

Bones are stained red

soft tissue, transparent

and unstained.

Notes:
If the initial clearing in potass, hydroxide solution has progressed to the proper stage only the bone will be stained, but
otherwise soft tissue will also be stained.

The prolonged
less liable to

preliminary fixation in alcohol renders the tissue

maceration in the potass, hydroxide solution.

Objects fixed in liquids other than alcohol may be stained by this

are soaked in 90% alcohol for at least three

method provided they

The best preparations are made with fish, but amphibia and
mammals have also been tried with a fair degree of success, although
days.

is not the same firm

consistency about the flesh of a mammal
or amphibian, prepared by this technique, as there is with that of
a fish.

there

The technique
developing bone.

is

particularly

suitable

for

demonstrating

William's modification of Dawson's method

This technique is particularly suitable for mammalian embryos
and mature specimens of Urodele amphibians for distinguishing
;

54

TWO

SECTION
between bone and cartilage and
amount of ossification.

The removal

of the viscera

is

for demonstrating the relative

unnecessary in the case of museum

specimens.
Solutions required:

A. Toluidine Blue
Alcohol 70%

0-25 gm.
100 ml.

2 ml.
Hydrochloric acid 0-5%
Allow the solution to stand for twenty-four hours
then filter and store in a tightly corked bottle.
.

Potass, hydroxide

B.

C. Alizarin Red, S
Potass hydroxide

2% aqueous
.

2%

o-ooi gm.

100 ml.
aqueous
(This solution should be freshly prepared.)

D. Methyl

salicylate

E. Methyl salicylate
F.

Methyl

salicylate

25%
50%
75%

in cellosolve

in cellosolve
in cellosolve

Technique:
1.

Wash

taining

specimens for twenty-four hours in

0-2%

of concentrated

ammonia

2.

Stain for seven days in Solution A.

3.

Harden and

95%
4.

70%

alcohol con-

solution

destain for seventy- two hours in four changes of

alcohol.

Macerate for

five to

seven days, depending on the size of the

2% aqueous potass, hydroxide.

hastened by exposure to sunlight.)

animal, in several changes of

{Note: This process

is

5. Transfer to Solution C for about twenty-four hours when the

bones should be well stained. If the specimen has been insufficiently macerated the soft tissue will be slightly stained, in which
case the specimen may be destained rapidly in acid alcohol (1%

sulphuric acid in

95%

alcohol).

Dehydrate by leaving the specimen in three changes of

cellosolve for six hours in each. Instead of cellosolve, 50%, 80%
and 90% alcohol, followed by three changes of benzol may be
used for dehydration. Small embryos require less time in the
6.

dehydrating
^

fluids.

55

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

25%, 50% and 75%

Clear by transferring to solutions of

7.

methyl salicylate in cellosolve for twenty-four hours in each.

Store in methyl salicylate.

8.

Note: If glycerin
modified as follows

used for clearing the technique has to be

is

Omit stage 6 and transfer directly from the Alizarin Red S

solution into a series of 50%, 70% and 80% glycerin for twentyfour hours in each then store in pure glycerin.
;

Results:

Soft tissues:
lage

transparent.

Osseous tissue: deep blue.

Carti-

dark blue.

Note: The relative degree of ossification and chondrogenesis

which has taken place is indicated by the intensity of the stains.
Bone and cartilage may be stained separately by omitting stage 2,
or stage 5 for cartilage.

For foetal specimens

The

technique

is

for demonstrating

mammalian embryos,

particularly suitable for

minute bones and

foetal ossification.

Solutions:

A. Alizarin

Red,

1%

S,

(freshly prepared)
Potass, hydroxide

1%

B.

Potass, hydroxide

Water
Glycerin

aqueous
.

aqueous

i litre

Note: For small specimens

ide

gm.

ml.

10 gm.

800 ml.
200 ml.
potass, hydrox-

is sufficient.

Technique:

Fix in 95% alcohol for at least two weeks after making a midline
abdominal incision to allow penetration of the fixative.
1.

Rinse in tap water.

2.

Immerse

for at least four

weeks in
56

1%

potass, carbonate.

SECTION TWO
for at least ten days in
aqueous potass, hydrox3. Immerse
ide until the bones are clearly visible through the soft tissue.

1%

Note: Formalin-fixed specimens require four to six weeks in

1% potass, hydroxide. Should the tissue become too soft it may
be hardened by immersing for twelve to twenty-four hours in a
mixture consisting of equal volumes of glycerin, water and 95%
alcohol before returning to the clearing solution. Potass, hydroxide
0*5% may be used during the last few days of the clearing.
4.
5.

men,

Wash

twenty-four hours in running tap water.

Stain one half to six hours, according to the size of the speciin Solution A.

6.

Wash

7.

Decolorize seven to fourteen days in Solution B.

for thirty minutes in running tap water.

Mount

frame and dehydrate by passing slowly

through alcohol-glycerin-water mixtures beginning with the pro2
portions 1:2:7 and then in succession 2 2 6, 3 3 4, 4 4
and finally equal parts of alcohol and glycerin only.
8.

in a glass

9.

Seal in the usual glycerin-alcohol mixture.

For nervous tissues (Benda's method)

Solutions required:

A. Nitric acid, cone.

Distilled water

B.

D. Iron alum

10 volumes

2%

Potass, dichromate

C. Chromic acid

1%

4%

E. Alizarin Red, S, saturated in absolute alcohol

Distilled water

F.

volume

Toluidine Blue

ml.

90 ml.

o-i% aqueous

Technique:
I.

Material

is

fixed in

90-95%

alcohol for at least

57

two days.

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

2.

Pieces,

in Solution

which must not be thicker than 0-5 cm., are immersed

A for twenty-four hoiirs.

3.

Transfer to Solution

for twenty-four hours.

4.

Transfer to Solution

for forty-eight hours ;

then wash in

water for twenty-four hours.

5.

Dehydrate in the usual manner.

6.

Clear in beechwood creosote for twenty-four hours

then in

benzol for twenty-four hours.

7. Embed in a saturated solution of paraffin wax
room temperature; then successively in saturated
paraffin wax in benzol at 38 C, 42 C. and 45 C.
paraffin wax only is used for the final embedding.

in benzol at

solutions of

so that pure

8. Mount sections on slides: bring down to distilled water,

mordant sections on slides with Solution D for twenty-four hours,
then wash thoroughly in water.

9.

10.
is

E then rinse in tap water.

F and warm gently until vapour

Stain for two hours with Solution

Flood

given

off;

slides

with Solution

or stain at

room temperature

for 24 hours.

then dry by blotting carefully.

11.

Rinse in

12.

Pass through absolute alcohol; then differentiate for about

1%

acetic acid;

beechwood creosote; dry by

xylol, and mount.

ten minutes in

wash with

Vital staining of

blotting carefully,

nervous tissue in small vertebrates

Solution required:

AHzarin Red, S,

2%

aqueous

Technique:
1.

Paraffin sections are brought

down

to distilled water

by the

usual method.
2.

Stain twenty-four hours in

4.

Dehydrate

2%

aqueous Alizarin Red, S.

distilled water to
3. Differentiate thirty to sixty seconds in
acetate per 10 ml.
calcium
which has been added three drops 1%
:

clear

and mount.

Note: This is a general stain which also demonstrates Nissl

bodies as well as other details.
S8

SECTION TWO

ALUM CARMINE

ANILINE BLUE

ORANGE

G.

For demonstrating the various components of the

Hypophysis
Solutions required:

A. Cresofuchsin.
B.

Alum carmine

C. Orange

(Mayer).

Distilled water

loo ml.

D. Phosphomolybdic acid
E.

2 gm.

Phosphomolybdic acid

Aniline Blue

5%

gm.

aqueous.

0-2% aqueous.

Technique:

Fix in

1.

hols;
2.

two
3.

formalin; harden and dehydrate in graded alcochloroform: embed in paraffin wax.

10%

clear in

Sections are brought

down

to

70%

alcohol and stained for

to twenty-four hours in Solution A.

Wash

quickly with distilled water; then stain with Solution

for three hours, afterwards washing with distilled water.

4.

Differentiate

and

stain the acidophil cells for five

with Solution C; then rinse in

D for two minutes

5.

Immerse

6.

Stain ten to twenty minutes with Solution E.

7.

Rinse in

no more
mount.

in Solution

distilled water;

stain

minutes

distilled water.

differentiate

then blot dry.

with

comes out; then dehydrate;

75%

alcohol until

clear in xylol

and

Results:

Chief cells, blue to grey. Pregnancy cells, blue with small bright
yellow granules; basophiles with coarse reddish blue granules.
Epithelium of the pars intermedia and pars tuberalis, variable.
Collagen

fibre,

intense

blue.

Glia fibres,

occasionally black.

59

blue-grey;

axons,

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

AMMONIACAL

SILVER CARBONATE

For vascular reticulum, tumour cells, connective tissues

around tumour, in abnormal brain tissue
Solutions required:

A. Pyridin, pure
Glycerin, pure
B.

Ammoniacal

2 volumes

volume

Silver Carbonate.

Ammonia solution is added drop by drop to

silver nitrate

io-2%

10 ml.

formed

until the precipitate

is

almost

redissolved, leaving a slightly opalescent

solution to which is then added 10 ml. sodium car-

bonate 3-1% solution and sufficient

make the volume up to 100 ml.

distilled

water to

C. Reducing solution:

Sodium carbonate anhydrous

Formalin

Distilled water

gm.

ml.

103 ml.

D. Brown gold chloride 0-2% aqueous.

E. Intensifying solution:

Oxalic acid

Formalin
F.

2%

aqueous

100 ml.

ml.

Sodium hyposulphite 10% aqueous.

N.B.

All

the above solutions must be kept in

dark bottles.
Technique:

The material is fixed in 10%

formalin or in Bouin and

embedded

in paraffin wax.
1.

tion

Bring sections

2.

Wash

3.

Immerse

4.

five

down

to distilled water

and immerse

in Solu-

A for twenty-four hours.

with

95%

alcohol, then with distilled water.

in Solution

for

two and

a half hours at 40 C.

Wash with distilled water; then reduce in Solution

minutes, afterwards washing in tap water.
60

for

SECTION TWO
5.

Tone

for five minutes in Solution

D at 30 C;

then wash in

tap water.
6. Intensify by
rinse in tap water.

N.B.
7.

for five

minutes

then

The above stages must be carried out

in the darkroom.
Fix in Solution F. {Note: Fixation should be completed in

fifteen to
8.

immersing in Solution

twenty minutes.)

Wash

in tap water;

dehydrate; clear and mount.

Results:

Tumour

cells:

reddish to greyish violet.

Vascular reticulum:

black.

Important.

^The

into contact with

tissues must not be allowed to come

mercuric chloride, as even a trace will

ruin the preparation.

ANILINE BLUE

ACID FUCHSIN

For elementary bodies in animal sections

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

2.

Sections, not thicker than 5^, are fixed to slides,

taken through descending grades of alcohol

de-waxed and

down

to water as

usual.
3.

Stain for one half to one hour in the aniline blue (Solution B)

in a stoppered staining jar.

4.

Rinse well with

5.

Drain and blot carefully to remove excess water.

6.

Rinse in absolute alcohol.

7.

Stain for twenty minutes in the acid fuchsin (Solution C).

8.

Pour

9.

Dehydrate with absolute alcohol; clear in xylol and mount

off;

distilled water.

drain and blot carefully to remove excess liquid.

in cristalite or in

Canada balsam.

Results:
cells associated with the following viruses
borna, zoster, rabies and pseudo rabies.

Elementary bodies in
are stained scarlet

ANILINE CRYSTAL VIOLET

For epithelial

GRAM'S IODINE

fibres

Solutions required:

A. AniUne crystal
B.

Gram's

violet.

iodine.

C. Aniline xylol.
Technique:
1.

Material should be fixed in absolute alcohol and

embedded

in paraffin wax.

Sections, not more than ^fi thick, are fixed to slides

brought down to distilled water in the usual manner.
2.

3.

Stain for ten to fifteen minutes in aniline crystal violet.

4.

Wash

5.

Stain with Gram's iodine for ten to thirty seconds.

6.

Wash

and

well in running water.

in water;

drain; then blot carefully but thoroughly to

remove water.
62

SECTION TWO
7.

vals
8.

Differentiate with aniline xylol, controlling at frequent inter-

by examination under the microscope.

Wash

well with xylol

mount

in

balsam or D.P.X.

Results:

Epithelial fibres are stained blue.

ANILINE CRYSTAL VIOLET - LITHIUM CARMINE

IODINE
For

fibrin

and

for Gram-positive
tissues

organisms in animal

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

and blot

6.

Rinse in

7.

Cover with Gram's iodine solution and allow the

distilled water; drain well

carefully.

stain to act

for five to ten minutes.

Pour

8.
filter

and blot carefully with

off the excess iodine solution

paper.

Differentiate with the aniline xylol solution until

purple coloration comes out.
9.

10.

Drain, and blot carefully.

1 1

Rinse with several changes of xylol.

Mount

12.

no more

in balsam or in cristalite.

Results:

Fibrin and Gram-positive organisms are blue to blue-black

while nuclei are red.

ANILINE BLUE

ORANGE G

(Mallory)

For collagenous and reticulin fibrils

cartilage, bone,
amyloid, nuclei; fibroglia and elastin fibres
;

Solutions required:

A. Acid fuchsin
Aniline Blue

B.

J-i%
-

aqueous.

Orange G.

Technique:

Tissues are fixed in Zenker and embedded in paraffin wax,

Celloidin or L.V.N.

and bring down to 90% alcohol;

then treat with iodine in the usual way to remove mercuric deposits.
1.

Mount

2.

Bring

in Solution
3.

Blue
of

down to

on

slides

distilled

water and stain for one to ten minutes

A; then without washing:

Stain for twenty minutes to one hour or longer in Aniline

then remove excess stain with several changes
Orange

95%
4.

sections

alcohol.

Dehydrate with absolute alcohol

Cristalite.

64

clear in xylol

and mount

in

SECTION TWO
Note: If Celloidin or L.V.N, sections are used the staining time
shortened and 95% alcohol should be used for decolor-

may be
izing

and dehydration

terpineol for clearing.

Results:

Collagenous fibril, intense blue. Ground substances of cartilage,

bone, mucus, amyloid varying shades of blue. Nuclei, myoglia,
:

neuroglia fibrils, axis cylinders, fibrin, nucleoli red. Blood corpuscles and myelin: yellow. Elastic fibrils: pale pink or pale
:

yellow, or unstained; fibriloglia: red or unstained.

Note: By omitting the acid fuchsin the collagenous fibres are

more sharply

defined.

AZAN STAIN
(Heidenhain)
Solutions required:

A.

Azocarmine B

..

Distilled water

100 ml.

Glacial acetic acid

Dissolve by warming
B.
JLJ

q6%

alcohol

..

0-5

cool and
.

gm.

ml.
filter.

100 ml.

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

Technique:
1

Zenker-, Bouin- or Camoy-fixed tissues are stained from fortyminutes at 55 C. in Solution A; then at room tem-

five to sixty

perature for five to ten minutes.

2.

Wash

in distilled water

until cytoplasm is pale pink,

then differentiate in Solution

and nuclei are red and

3.

Rinse for one half to one minute in Solution C.

4.

Transfer to Solution

until the connective tissue

is

for about

clear.

one to three hours or

then wash

completely decolorized

quickly in distilled water.

5. Stain for one to two hours in the diluted Solution E, examining at ten- or fifteen-minute intervals to prevent over-staining.

6. Wash quickly in distilled water;

alcohol followed by absolute alcohol.
7.

then differentiate in

95%

Clear and mount.

Results:

Collagen stained deep blue reticulum, deep blue chromatin,

red; muscle tissue, reddish to orange; erythrocytes, red; neurmucin, blue.
oglia, reddish
;

AZO CARMINE
For

Islets

MALLORY STAIN

of Langerhans

Solution required:

A. Azocarmine, B, aqueous o-i%

100 ml.
Boil for about 5 minutes; then cool and add
2 ml. glacial acetic acid. Then warm to 60 C.
.

and
B.

filter at

90%

that temperature.

alcohol 99 ml.

C. Iron alum

5%

-f-

aqueous.

66

nil.

Aniline Oil.

SECTION TWO
Aniline Blue

Orange

D. Aniline blue, aqueous

Orange G.

G (Mallory):
. .

0-5 gm.
2 gm.

Distilled water

Solution

E.

100 ml.
I

volume

2 to 3 volumes

Distilled water

Technique:
1.

Fix thin

2.

Wash

slices of

pancreas in Bouin for eight to ten hours.

in distilled water,

dehydrate, clear and

embed

in

paraffin wax.
3.

Cut

4.

Fix section to slides; dewax and take

section 4/x in thickness.

down through

the

usual grades of alcohol to distilled water.

5.

Stain in solution

for about forty-five to sixty minutes at

56 c.
6.

Rinse quickly in distilled water and blot very carefully.

7.

Destain in solution

and B
8.

cells

show red

until acinous tissue is almost colourless

against pink background of

cells.

Rinse briefly with distilled water and treat with

for 5 minutes or more.

5%

iron

alum solution
9.

Rinse again and stain two to twenty minutes in solution E

deep blue under the microscope.

until the collagenic tissue appears

10.

Rinse and blot carefully.

and dehydrate
xylol and mount.

11. Differentiate
12. Clear in

in absolute alcohol.

Results :

Cytoplasm of
red and of
;

cells

cells: rich
:

yellow orange; of

cells:

bright

sky blue.

can be demonstrated that there is no gradation

by first staining with Neutral Gentian (Bensley)
decolorizing, then restaining by the above technique.
Note :

stated that

It is

between

A and B

Reference

it

cells

Gomori, G. (1939), Anat. Rec, 74, 439-459.

Cowdry, E. V.: Laboratory Technique, 3rd cd., p 167.

67

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

AZOCARMINE

HAEMATOXYLIN
ORANGE G

ACID GREEN

For differential cell analysis of the rat anterior hypophysis

Solutions:

Formol
Potassium dichromate
Mercuric chloride

A. Zenker

Ringer's solution

(i.e.

0-9%

saline)

25 gm.
50 gm.
i litre.

I mL neutral formaldehyde 40% solution per 10 ml. of the

above solution immediately before using.

Add
B.

SECTION TWO
6.

Cedarwood

7.

Xylol, for fifteen minutes.

8.

Infiltrate

oil,

one to sixteen hours.

in paraffin

wax 56-58 C.

(four changes before

embedding).
Fix sections, 4jLt in thickness, to slides and remove paraffin
wax by immersing in two changes of xylol for three minutes each.
finally
9.

10.

Immerse

for three minutes in each of

two changes of

absolute alcohol.
11.

In

alcohol for three minutes.

95%

12. Distilled

water for three minutes.

13. Lugol's Iodine for three minutes.

14.

Sodium

thiosulphate solution for three minutes when the

been restored to their natural colour.

sections should have

15.

Stain in Delafield Haematoxylin for thirty seconds.

16.

Wash

17.

Immerse

in distilled water for three minutes.

18.

Immerse

in

in tap water for three minutes.

80%

alcohol for three minutes.

19. Aniline alcohol (Solution E) for fifteen minutes.

20. Stain in Azocarmine for forty-five minutes.

21. Rinse in distilled water.

22. Differentiate in aniline alcohol for

two

23.

Wash in acid alcohol

24.

Immerse

25.

Dehydrate by passing through 70%,

(solution

G)

to three minutes.

for thirty to sixty seconds.

in phosphotungstic acid solution for one hour.

95% and

absolute

alcohols (two minutes in each).

26. Counterstain in acid green

orange

solution for five

minutes.
27. Clear in xylol for

one minute.

Immerse for half an hour in each of two changes of xylol,

remove completely all traces of clove oil which would otherwise

28.

to

cause further decolorisation.

29.

Mount

in D.P.X. or Clearmount.

Results:

Alpha granules, purplish red. Beta cell granules, light green.

Nuclear membranes are sharply defined and mitochondria are
69

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

orange red. Erythrocytes, brilliant orange. Golgi apparatus shows
as negative image in both alpha and beta cells. Chromophobes show
little

or no cytoplasm, which is colourless to pale green.

method of counting the cells is given in the original

Note:

paper.
Reference: Briseno-Castrejon, B. and Finerty,

C. (1949), Stain Tech.y 24,

J.

I03-'7.

BAUER

FEULGEN STAIN

For Glycogen,

etc.

Solutions required:

A. Chromic acid

4%

aqueous

B.

Feulgen's fuchsin

C.

Sodium metabisulphite 10*4%

aqueous
Tap water

5 nil'

95 ml.

Technique:
1.

Fix fresh material

immediately in alcohol and embed in

paraffin wax.
2. Dewax sections; pass through the usual descending grades
of alcohol to distilled water.
3.

Immerse

4.

Wash

5.

Immerse

in the chromic acid solution for

one hour.

in running water for five to ten minutes.

in the Feulgen fuchsin for ten to twenty minutes.

6. Agitate the slides gently for about two minutes in each of

three changes of solution C.

7.

Wash

8.

Stain nuclei,

9.

in

running water for ten minutes.

Dehydrate

if

desired, in

haemalum,

as usual; clear in xylol

for

two

to five minutes.

and mount.

Results:

Glycogen, intense reddish-violet.

blue.

70

Nuclei, pale

mauve

to

navy

SECTION

BIEBRICH SCARLET

TWO

ETHYL VIOLET - HAEMATOXYLIN

(Cambel and Sgouris modification of Bowie's Stain) for

pepsinogen granules of the body chief cells in the gastric
glands
Solutions required:

A. Delafield Haematoxylin, aqueous

Ethyl Violet Biebrich Scarlet
20
alcohol
Ethyl

B.

C. Solution B.

0.5

0-5

Absolute alcohol
Distilled water

gm.

50 ml.

gm.

20 ml.
80 ml.

This solution must be freshly preeach

time it is required for use.
pared

N.B.

D. Clove Oil
Toluol

volume
volume

Technique:

Fix in Regaud's fluid for five days in a dark phial placed in a

larger amber bottle which should be wrapped round with a thick
cloth and kept in a dark room. Change the Fixative daily.
1.

2.

Dehydrate with normal propyl alcohol

(see

page 38).

Transfer to paraffin wax which should be changed three times

before the block is finally cast.
3.

4.

Cut

5.

Remove wax with two changes

6.

sections 5 to 6/x in thickness

and

fix to slides as usual.

of xylol.

Pass through absolute alcohol and the usual ascending grades

down to distilled water.

of alcohols
7.

Stain in the Haematoxylin solution for

8.

Wash and

9.

Remove

10. Stain

minute.

blue in tap water.

excess water

with solution

by draining and

blotting very carefully.

for ten to fifteen minutes, or longer

(up to twenty-four hours).

11.

Rinse briefly in distilled water, then drain and carefully

remove excess water by

G

blotting.

yi

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

12.

Differentiate in solution

(clove-toluol), controlling

under

the microscope, for about ten to fifteen minutes.

13.

Rinse with two changes of toluol.

14.

Mount

in permount-toluene or in

Clearmount or

Cristalite.

Results:

Zymogen
pepsinogen

dark

granules,

The

nuclei, blue.

Parietal

violet.

cells,

and
from the

scarlet,

parietal cells are distinctly contrasted

cells.

Reference: Cambel, P. and Sgouris,

(1951), Stain Tech., 26, 243-6.

J.

BISMARK BROWN

METHYL GREEN

For mucin, cartilage, and goblet

cells in

embryonic

tissue,

trachea and intestine

Solutions required:

A. Bismark brown
B.

1%

aqueous.

Methyl green 0-5% aqueous.

Technique:

Tissues are fixed in Bouin or Zenker and embedded in paraffin

wax.
1.

Sections are brought down to distilled water;

minutes in Solution A.

then stained

five to ten
2.

Wash

3.

Stain with Solution

with

95%

alcohol.

until the preparation appears dark

green to the naked eye.

Dehydrate with
xylol, and mount.
4.

95%

and absolute alcohol;

then clear in

Mucin:

Nuclei of

Results:

Cartilage:
cells:

dark brown.

green.

72

light

brown.

all

SECTION TWO

BIONDI

EHRLICH

HEmENHAIN STAIN

For chromatin, nucleoli, mucin,

etc.

Solution required:

Biondi

Ehrlich - Heidenhain

Distilled water

0-9 gm.
100 ml.

Dissolve by warming and stirring in a beaker.

cool add

When

Chloroform

0-25 ml.

Technique:
1

Fix tissues in saturated aqueous mercuric chloride and embed

wax in the usual manner.

in paraffin
2.

Fix sections to slides; de-wax with xylol and pass through

absolute alcohol followed

by

90%

and

70%

alcohol.

3. Treat for the removal of mercuric precipitate by the standard

technique {see page 28).

4.

Immerse

in the staining solution

from

six to twenty-four

hours.
5.

Rinse directly with

6.

Dehydrate with absolute alcohol.

7.

Clear in xylol and mount.

95%

alcohol.

Results:

Chromatin is stained bluish green, while nucleoli are red;

mucin is stained green; erythrocytes, orange. Cytoplasm and
connective tissue elements are in varying shades of red.

BEST'S

CARMINE

For glycogen
Note: This method has the advantage over the Lugol's iodine
technique in that fading does not occur so readily, and better

The

disadvantages are that the

staining of glycogen

is

stain

than in the iodine method, and the solution

is less

specific

obtained.

73

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

deteriorates after a

few weeks.

stain should be checked

If negative results are obtained the

by staining a section known to contain

glycogen.
Solutions required:

A. Ehrlich haematoxylin.
B.

Best's carmine stock solution

Methyl

alcohol,

pure

Strong ammonia

solution

lo ml.

15 ml.

10 ml.

Note: This solution should be prepared immediately before

it is

required for use.

1% in equal volumes of
absolute alcohol and ether.

C. Celloidin

D. Absolute

(ethyl) alcohol

Absolute (methyl) alcohol

Distilled water
.

80 ml.
40 ml.
100 ml.

Technique:
1.

Tissues are fixed in Bouin Fluid and

or in paraffin wax.

from stage
is

embedded

If Celloidin sections are

in Celloidin

employed proceed as
used the procedure

If paraffin sections are

5 (below).

as follows:
2.

Float sections on the slide with

then remove excess alcohol with

filter

70%

alcohol;

flatten out;

paper and blot carefully but

thoroughly.
3.

Remove

4.

Wash

paraffin

wax with

xylol in the usual manner.

with absolute alcohol as usual.

5. Transfer the slide to a stoppered staining jar containing

Celloidin (Solution C, above), for fifteen minutes.

1%

6. Transfer to a stoppered jar containing 70% alcohol, after

rapidly wiping off the Celloidin from the back of the slides. This
operation must be carried out quickly so that the Celloidin is not

allowed to dry.

Leave

in the alcohol

from ten

to fifteen minutes.

Transfer to Ehrlich haematoxylin and allow the stain to act

to ten minutes, differentiating if necessary with acid
alcohol, controlling under the microscope.
7.

from two

74

SECTION TWO
"
"
Rinse in water, and without blueing in tap water, transfer
to Best's carmine solution (formula as above) and allow the stain
to act for five to ten minutes.
8.

Differentiate in Solution

9.

until the stain ceases to

D (above) from one to five minutes

come away from the

section.

Transfer to a mixture consisting of equal volumes of ether

and absolute alcohol to dissolve out Celloidin and to dehydrate.
10.

1 1

Clear with xylol and mount.

Results:

Glycogen

is

stained as brilliant red granules, while nuclei are

blue.

BENZIDINE
For brain capillaries
Solution required:

A. Benzidine base, pure

Acetic acid
B.

Sodium

2-5% aqueous

nitroprusside

1%

200 ml.

gm.

aqueous

C. Solution

20 ml.

Solution

10 ml.

70 ml.

Distilled water

Mix

well and

filter.

N.B. : This mixture should be prepared immediately before use.

D.

100 ml.

Distilled water

Hydrogen peroxide 20

vols.

1-5 ml.

Technique:
1. Tissue should be fixed for one to three weeks in 10% formalin and frozen sections, 200 to 300^ should be employed.

2.

Immerse

in several changes of distilled water for a total

period of two hours.

75

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

3.

at

Immerse

in Solution

for half

an hour

at 37 C.

agitating

frequent intervals.
4.

Wash

5.

Immerse

in several changes of distilled water.

in Solution

for half

an hour

at 37 C. agitating

frequently.
6.

Wash

in distilled water.

Dehydrate by passing through ascending strengths of alcohol,

beginning with 70%, in the usual manner.
7.

8.

Clear in xylol and mount.

Results:

Blood vessels are stained black against a background which

almost colourless.

BASIC FUCHSIN

METHYLENE BLUE

For demonstrating Negri

is

SECTION TWO
Fix sections to slides

2.

de-wax with

xylol.

3. Pass through descending grades of alcohol and treat for the

removal of mercuric precipitate left by the fixative by the standard

technique.

Place slides, section facing upwards, over the corner of a

4.

tripod.
5. Flood slide with the staining solution (Solution C, above)
and heat gently with a small bunsen flame for five minutes with
steam rising, taking care that the preparation does not catch fire.

Allow the

6.

slide to cool for a

few seconds

then wash quickly

in water.

Decolorize and

7.

tions

assume a

diflFerentiate in

90%

alcohol until the sec-

faint violet colour.

8.

Rinse quickly in

9.

Dehydrate rapidly with absolute alcohol.

10. Clear in xylol

95%

alcohol.

and mount.

Results:

Negri bodies are stained deep red, while the granular inclusions
Nucleoli are blue-black, while cytoplasm is bluish
and
violet,
erythrocytes appear copper coloured.
are dark blue.

BASIC FUCHSIN

GENTIAN VIOLET

IODINE

For bacteria in sections

Solutions required:

A. Basic fuchsin
Alcohol absolute

Phenol

crystals
Distilled water

B.

,.

0-75 gm.
30 ml.

gm.

. .

100 ml.

Picric acid, saturated, aqueous.

C. Aniline gentian violet.

77

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

D. Gram's iodine.
E.

Aniline

Xylol

oil.

volume
volume

Technique:
Pieces of tissue should be fixed in Zenker's fluid, washed in
running water, dehydrated, cleared and embedded in paraffin wax
1

in the usual

manner.

Fix thin sections to

2.

slides,

de-wax, and treat for the removal

of mercuric precipitate by the standard technique.

3.

Stain in the fuchsin solution for ten to thirty minutes.

4.

Differentiate in formalin until the bright red colour

is

re-

duced to pink.
5.

Wash

6.

Counterstain in the picric acid solution for a few minutes

in distilled water.

until the preparation

7.

Wash

8.

Differentiate in

assumes a purplish yellow colour.

in distilled water.

95%

alcohol until the sections are red again,

and yellow and red coloration begins

Rinse in

9.

to

come

out.

distilled water.

10.

Stain in aniline gentian violet for five to ten minutes.

1 1

Rinse in distilled water.

12.

Stain in Gram's iodine solution for one minute.

13.

Drain and blot dry.

14. Differentiate

with aniline-xylol until colour ceases to come

out of the sections.

15.

Clear with two changes of xylol and mount.

Results:

Gram-positive
negative are red.

organisms

are

stained

Tissue, red and blue

78

blue,

fibrin,

while

deep blue.

Gram-

SECTION TWO

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

Technique:
1. Small pieces of tissue are fixed in io% formalin or in
95%
alcohol or in physiological saline for at least twenty-four hours,
and afterwards washed, dehydrated, cleared and embedded in

paraffin
2.

wax

in the usual

manner.

Stain sections for three to four minutes with the carbol fuch-

sin (Solution A).

4.

Wash
Wash

5.

Clear in xylol

3.

quickly in distilled water

then de-stain in Solution B.

in distilled water; then dehydrate.

then mount.

Results:

Nissl bodies and nucleoli are stained dark red; remainder unstained.

CARBOL FUCHSIN

BORREL BLUE

For Leprosy and for T.B.

Solutions required:

A. Carbol fuchsin (Ziehl Neelsen).

B.

Sulphuric acid 5%.

C. Hydrochloric acid

Alcohol

70%

D. Borrel's Blue
Distilled water

ml.

99 ml.
5 ml.

20 ml.

Technique:
1.

Material should be fixed in saturated aqueous solution of merand embedded in paraffin wax.

curic chloride
2.

Fix sections to slides and treat them for the removal of merby the standard method [see page 28).

curic precipitate
3.

to an

Immerse

in carbol fuchsin in a staining jar for thirty minutes

hour in the incubator

at 37

4.

Decolorize in Solution C.

5.

Decolorize in

70%

C.

alcohol (neutral) for two or three min-

utes until the sections appear faintly pink to the naked eye.

80

SECTION TWO
6.

Counterstain in Borrel's Blue (diluted as above:

two minutes.

Solution

for one or

D)

7.

Rinse in

distilled

water

drain and carefully blot away excess

water.
8.

Dehydrate and

differentiate the Borrel Blue, controlling

by

examination under the microscope.

9.

Clear in xylol and mount.

'

Results:

T.B. or leprosy, bright red; other bacteria, blue; cells

debris, varying shades of blue ; cell nuclei, blue.

N.B, : For demonstrating

and

cell

leprosy, differentiation of the carbol

fuchsin (stages 4 and 5) must be very carefully carried out, as this

organism is more easily completely decolorized than T.B.

CARBOL FUCHSIN
For tubercle

bacilli in

HAEMATOXYLIN
mammalian

tissue

Solutions required:

A.

Alum Haematoxylin :
Potash alum

Haematoxylin

Thymol

Distilled water

B.

Carbol fuchsin (Ziehl Neelsen)

Distilled water

20 gm.
i

gm.
gm.
400 ml.
i

volume
volumes

Technique:

Tissues are fixed in Zenker or

Flemming and embedded

in

paraffin wax, L.V.N, or Celloidin.

1.

Paraffin sections are brought

down

to distilled water;

then

stained one to five minutes in Solution A.

necessary with acid alcohol, controlling under

nuclear detail is sharp and clearly defined;
wash thoroughly in water.
2.

Differentiate

the microscope,

if

till

81

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

Stain with Solution

3.

rises

or stain at

for five minutes, heating

room temperature

till

steam

overnight.

Decolorize for twenty seconds in acid alcohol; then wash

thoroughly in water to which two or three drops of ammonia have
4.

been added to remove the

5. Differentiate in
6.

95%

acid.

alcohol.

Dehydrate; clear and mount.

If Celloidin or

or origanum

oil

L.V.N, sections are employed, clear

blot carefully on slide and mount.

in terpineol

Results:

Tubercle

bacilli,

bright red. Nuclei, blue.

CARBOL FUCHSESr

HAEMATOXYLIN

PICRO

ACID FUCHSIN
For M. leprae in sections
Solutions required:

A. Carbol fuchsin (Ziehl Neelsen).

B.

Hydrochloric acid, cone.

Absolute alcohol

C. Potassium permanganate

D. Oxalic acid
E.
F.

2%

1%

3 ml.

97

rnl*

aqueous.

aqueous.

Haematoxylin (Ehrlich).
Picric acid, saturated,

Acid fuchsin, aqueous

Distilled water

aqueous

1%
.

. .

50 ml.
10 ml.

40 ml.

Technique:
1

Pieces of tissue are fixed for three to seven days in a mixture

consisting of equal volumes of 10% formalin and absolute alcohol,

and parafHn sections are employed.

Stain sections in the carbol fuchsin solution in a stoppered

staining jar for three or four days.
2.

82

SECTION TWO
3.

Immerse

in

10%

formalin, of a slightly acid reaction, for five

minutes.
4.

Immerse

in the acid alcohol for five minutes.

w^ith potassium permanganate and

5. Flood the preparation
allow the reagent to act until the sections turn brown (this usually

takes
6.

from two

to five minutes).

Immerse

in the oxalic acid for one minute.

7. Stain with Ehrlich haematoxylin solution for two minutes

then blue in tap water or in saturated lithium carbonate solution

aqueous.
8.

Stain in picro-acid fuchsin for two to five minutes;

then

without washing:
9.

Dehydrate, clear and mount.

Results:

M.

leprae, dark blue.

yellow.

Connective tissue

fibres, red.

Muscle,

Nuclei, brown.

CARBOL FUCHSIN

IODINE

HAEMATOXYLIN

ORANGE G
For demonstrating leprosy organisms together with neurokeratin of the myelin sheath
Solutions required:

A. Lugol's iodine.
B.

Carbol fuchsin (Ziehl Neelsen).

C. Absolute alcohol
Distilled water

Hydrochloric acid concentrated.

35 ml.
65 ml.
0-5 ml.

D. Ehrlich's haematoxylin.
E.

Strong ammonia
o-88o)

Distilled water

F.

solutioix (sp. gr.

ml.

99

Orange G. aqueous 1%.

83

nil*

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

Technique:
1.

Pieces of tissue are fixed in Zenker;

washed; then trans-

ferred to a mixture of Lugol's iodine and 80% alcohol (equal volumes of each) for six to twenty-four hours.
2.

Transfer to

80%

alcohol for twelve to twenty-four hours.

3.

Immerse

95%

alcohol for

in

two

to six hours.

4. Transfer to a mixture consisting of equal volumes of absolute

alcohol and xylol, for half an hour.
5.

Immerse

in xylol for half an hour.

6. Immerse in two changes of paraffin

block and finally sectioning.

wax

before casting the

7.

Fix sections to slides and remove wax with xylol.

8.

Pass through absolute,

9.

Stain for half an hour in carbol fuchsin (Solution B).

10.

Rinse in

90% and 70%

alcohol.

distilled water.

11. Partially differentiate

with the acid alcohol (Solution C,

above).
12.

Rinse well with

13.

Stain for one to two minutes with Ehrlich haematoxylin

distilled water.

solution.
14. Differentiate in the acid alcohol (Solution C).
15.

Rinse well with

16.

Immerse

in the

distilled water.

ammonia

solution (Solution

above) for a

few seconds.
17.

Rinse well with

18.

Stain with the Orange

19.

Dehydrate rapidly with two changes of acetone.

20. Clear in xylol

distilled water.

G solution for two to three minutes.

and mount.

Results:

Leprosy organisms and neurokeratin are stained red, while

nuclei are blue and cytoplasm

is

yellow.

84

SECTION TWO

CARBOL FUCHSIN

METHYL GREEN

For demonstrating hyaline substance

Solutions required:

A. Carbol fuchsin (Ziehl Neelsen)

Distilled water
.

Methyl Green

B.

1%

in

5%

5 ml.

45

rnl.

acetic

acid.

Technique:
1.

is

Material which has been fixed in any of the standard fixatives

embedded
2.

in paraffin wax.

Fix sections to slides

down

then bring

to distilled water as

usual.
3.

Stain in Solution

4.

Wash

with

for fifteen to forty-five minutes.

drain off excess; then blot care-

distilled water;

fully.
5.

6.

Dehydrate rapidly with absolute alcohol.

Differentiate and counterstain in Solution B for two or three

minutes.
7.

Wash

8.

Clear in xylol and mount.

quickly with absolute alcohol.

Results:

Hyaline substance

is

stained bright red while nuclei are light

green.

CARBOL THIONIN

PICRIC ACID (Schmorl)

For demonstrating bone canaliculi

Solutions required:

A. Decalcifying solution:
Formalin 10%
.

Nitric acid, cone.

B.

Carbol thionin (Nicolle).

C. Picric acid

1%

aqueous.

85

100 ml.
15 ml.

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

Technique:
1.

Formalin-fixed specimens are placed in a large volume of

is changed once or twice a day.
The time

Solution A, which

required for complete decalcification will depend, of course, on the

thickness and the nature of the specimen. The bones of young

animals usually take from twenty-four to forty-eight hours, but in

other cases as long as a week may be necessary. Decalcification is
complete when the bone has become flexible and can easily be

punctured with a needle.

2.

Make

3.

Rinse in water for ten minutes.

4.

Stain with Solution

Celloidin or frozen sections.

for 10 minutes; then rinse in distilled

water.
5.

Immerse

in Solution

C for half to

one minute; then wash in

water.
6.

Differentiate with

the stain ceases to

7.

Dehydrate with

mount

70%

come out

96%

alcohol for five to ten minutes until

of the sections.
alcohol;

clear in

origanum

oil;

then

in balsam.

Results:

Ground substance, yellow to brown; bone canaliculi, dark

brown to black; cells, red; ground substance of cartilage, brilliant
purple.

Note: Carbol thionin (NicoUe) deteriorates

and

this stain is therefore best

CARMINE

when

after a

few weeks

freshly prepared.

METHYLENE BLUE

(Schultz-Schmitz Stain)
urate
in animal tissue
For demonstrating sodium
Solutions required:

A. Distilled water

Lithium carbonate
Carmine
.

Ammonium

chloride

64 ml.

0-5

. .

gm.
gm.
2 gm.
i

few minutes allow to cool

the
to
original volume and add
up
Boil for a

Strong ammonia solution

Filter before use.

86

6 ml.

then make

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

CELESTIN BLUE

CHROMOTROPE 2R

(Lendrum)

substitute for haematoxylin-eosin, for simple diagnostic

or photographic purposes, emphasizing the staining of

collagen and reticulum
Solutions required:

A. Celestin blue
Alcohol

B.

(as solution

B, page 89)

100 ml.

70%

Hydrochloric acid, cone.

C. Phosphomolybdic acid

D. Chromo trope 2R

1%

2 ml.

1%

in absolute alcohol

Technique:
1.

Fix tissues in Zenker or Bouin exactly as described on page

89, stage

i).

2.

Stain section in the celestin blue solution for fifteen minutes.

3.

Remove any cytoplasmic

4.

Wash

with water for one minute.

Mordant with
two minutes.

5.

to

staining with solution B.

1%

phosphomolybdic acid solution for one

6.

Wash

7.

Dehydrate; then stain for two minutes in the chromotrope

2R

well with water.

solution.

8.

Dehydrate; clear in

xylol,

and mount

in

D.P.X.

Results:

Nuclei, bluish purple.

Cytoplasm, pink; collagenous elements,

bright red.

Note: For photographing this stain the best

use are

filters to

those giving a spectral transmission of 5,600 to 6,000 A.U.

Reference: Lendrum, A. C. (1935), J- Path, and Bad., 40, 415-6,
blue as a nuclear stain ".

88

"

Celestin

SECTION TWO

CELESTIN BLUE

ORCEIN

LIGHT GREEN

(Lendrum)
For breast carcinoma and skin lesions

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

6.

Rinse well with distilled water.

7.

Stain the nuclei with celestin blue (Solution B) for fifteen

minutes.
8.

9.

Wash

with running water for twenty minutes.

Stain muscle and epidermis for two minutes with eosin

(Solution C).

Decolorize the

10.

somewhat

collagen

in water or in

30%

alcohol.
11.

Immerse

12.

Wash

in

phosphomolybdic acid

for

two minutes.

well in distilled water.

with the light green solution.

13. Stain collagen

14.

Dehydrate rapidly.

15.

Clear in xylol;

mount

in

D.P.X.

Results:
Elastin, light

brown. Nuclei, bluish purple. Muscle and epider-

mis, red.

Collagen, green.
From J. Path, and Bad., 40, 415-6, 1935
"
Celestin blue as a nuclear stain."

Lendrum, A. C.

CHLORAZOL BLACK

A general-purpose stain,
well as for sections.
ation,

and

it

may be

which can be used for whole

tissues as

The

stain requires no mordant or differentiemployed in aqueous or alcoholic solution.

saturated solution in

70%

teen to thirty minutes

alcohol stains ordinary sections in

fif-

the stain does not fade.

The

stain is particularly suitable for staining embryo ; kidney ;

chromatin ; nucleoli ;
intestine for demonstrating epithelial cells
;

muscle

fibres.

Solution required:

Chlorazol Black, saturated in

70%

alcohol.

Technique:
1.

Tissues should be fixed in Zenker and embedded in paraffin

wax.
2.

Bring sections

down

precipitate in the usual

to

70%

manner.
90

alcohol and

remove mercuric

SECTION TWO
3.

Stain in a freshly prepared, unfiltered, saturated solution of

70% alcohol for five to ten minutes.

Chlorazol Black in
4.

Drain

off excess

dye

dehydrate

clear in xylol

and mount.

Results:

Embryo,

epithelial cell tissues, outlined in black.

Chromatin,

Nuclei, black. Muscle fibres, intense black. Lymphocytes, intense black. Blood cells, yellowish green.
Cytoplasm,
black.

greenish grey.

Kidney and intestine, varying shades of green, grey and black.

Blood cells, light green.
Nuclei and chromosomes are stained black; cytoplasm and
secreted products grey; chitin, green; glycogen, red.
Notes:
(a) Benzl alcohol may also be
results are somewhat different.
{h) If
<(

Milton
{c)

it

is

desired

to

used as a solvent, in which case

differentiate

chlorazal

"

black,

dilute

(a proprietary antiseptic) may be used for the purpose.

The stain may be incorporated with Lactophenol.

CONGO RED
For Amyloid in tissues
Solutions required:

A. Congo Red 1% in distilled water.

B. Lithium carbonate saturated aqueous
C. Delafield or Ehrlich haematoxylin.
Technique:
1.

Formalin or alcohol-fixed material may be embedded in

may be employed.
Sections are mounted on slides and brought down to distilled

Celloidin or in paraffin wax, or frozen sections

2.

water as usual.

Congo Red

3.

Stain in the

4.

Immerse

5.

Decolorize in

solution for ten to thirty minutes.

in the lithium carbonate solution for fifteen seconds.

80%

alcohol until stain ceases to

in clouds.

91

come away

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

6.

Wash

in

running water for

minutes

fifteen

then stain with

Ehrlich or Delafield haematoxylin for five to ten minutes.

7.

Wash

dehydrate in the usual manner;

in water;

clear in

xylol and mount.

Note:

If Celloidin sections are

used dehydration should be

carried out with Isopropyl alcohol in place of ethyl alcohol.

Results:

Amyloid, red; nuclei, blue.

CONGO RED

ANILINE BLUE

ORANGE G

For elastic fibres

Solutions required:

A. Aluminium chloride,
B.

Congo Red
Sodium citrate
Glycerin

Distilled water

2%

C. Aniline Blue, aqueous

Orange

Resorcinol

Phosphomolybdic

aqueous.

acid

1%

2 gm.

gm.

2-5

ml.

97

i^il-

1-5

gm.

2-25 gm.
3

aqueous

gm100 ml.

Technique:

Tissues should be fixed in

should be employed.
1.

Wash

sections in water;

10%

formalin, and frozen sections

then immerse them in Solution

for ten minutes.

Wash

with water and drain; then stain in the Congo Red

solution for ten minutes.
2.

then plunge the slide into a dish of tap

3. Wash with tap water
water and agitate it there for ten seconds.
;

4. Wash again with tap water

utes in the Aniline Blue-Orange
5.

then stain from

G solution

five to

(Solution

Rinse carefully in tap water; drain well and blot.

92

ten min-

above).

SECTION TWO
6.

Dehydrate in absolute alcohol

and mount.

clear in

origanum

oil ;

wash

in xylol

Results:
Elastic fibres, bright red ; fibrin, dark blue.

CONGO RED

EHRLICH HAEMATOXYLIN

For eleidin and keratohyalin

Solutions required:

A. Congo Red 0-05% aqueous.

B.

Ehrlich haematoxylin.

Technique:
1.

embedded

Material should be fixed in absolute alcohol and

in paraffin wax.
2.

Sections not

brought down

more than 5^ thick

are

mounted on

slides

and

to distilled water as usual.

3.

Stain for five to ten minutes in the

4.

Rinse in

Congo Red

then stain for

distilled water;

solution.

minutes in

five to ten

Ehrlich haematoxylin.
5.

Blue in tap water in the usual manner.

6.

Dehydrate; clear in xylol and mount.

Results:

Eleidin

is

stained red, while nuclei and keratohyalin are blue.

CRESYLFAST VIOLET

TOLUIDINE BLUE

THIONIN

(EHRLICH)

non-fading tri-basic stain for nerve cells and Nissl

granules, in normal and pathological tissues
Solutions required:

A. Cresylfast violet,
Toluidine blue

CNS
.

Thionin (Ehrlich)
Ethyl Alcohol

2 gm.

gm.
gm.
200 ml.
i

0-5

30%
93

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

B.

Distilled water

Sulphuric or nitric acid, cone.

200 ml.
0-5 ml.

Technique:
1. Formalin fixed material is embedded in paraffin wax, and
sections, 4/x in thickness are fixed to slides with glycerin albumen.

at

2.

Remove wax with

xylol.

3.

Rinse with absolute alcohol.

4.

Pass through

5.

Pass through

6.

Immerse

95%
80%

alcohol.

alcohol.

slides for five to ten

seconds in the staining solution

80-90 C.
Differentiate for

7.

8. Dip and
one second.

one second in solution B.

agitate slides in a beaker of cold distilled water for

Differentiate further in

9.

80% and 95%

alcohol for one to two

seconds in each.
10.

Immerse

in

80%

alcohol for one second.

1 1
Dip and agitate the
one to two seconds.
.

slides in the still

12.

Return to

13.

Repeat steps 11 and

14.

Rinse in distilled water.

15.

95%

80%

warm

solution

for

alcohol for one second.

12.

Dehydrate by immersing
and absolute alcohol.

for

one second in each of 80%,

16.

Immerse

in xylol for one minute.

17.

Immerse

in a fresh lot of xylol for three minutes.

18.

Mount and

examine.

Results:

Neurons stand out distinctly against a pale background, and

can be followed for a considerable distance. The cytons are stained
dark purple emphasizing the blue tint, while the dendrite and axon
processes and endings present a somewhat lighter shade, bluish to
reddish. Granules in the cell body as well as in the protoplasm

94

SECTION TWO
Nuclei and nucleoli are well

processes appear purple or reddish.

differentiated.

Reference: Spoerri, Rosette (1948), Stain Tech., 23, 133-5.

DAHLIA ACETIC

(Ehrlich)

For mast cell granules in sections

Solution required:

100 ml.

Distilled water

Absolute alcohol

12-5 ml.

10 gm.

Glacial acetic acid

Dahlia

50 ml.

Dissolve by heating in a flask, lightly plugged with cotton-wool,

on a water bath. Allow to cool then filter.
;

Technique:
1.

Fix tissues in absolute alcohol and

2.

Immerse

3.

Differentiate in

embed

in Celloidin.

sections in the staining solution for twelve hours.

alcohol.

95%

4. Clear in origanum

oil

and mount

in

balsam or in

cristalite.

Results:

Granules of mast

cells are stained

reddish violet.

DOPA REAGENT
*

For melanoblasts
Solutions required:

A.

Dopa

reagent (3

4-dihydroxy-

phenylalanin)

Cold

distilled

water

0-2 gm.
200 ml.

This solution,^ which deteriorates fairly

normal temperatures, should be kept in a
refrigerator. When the solution turns dark red it is
useless and should be discarded.
Note:

rapidly at

B.

Buffer tablet

pH 7-4

Distilled water (cold)

95

tablet

100 ml.

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

C.

Solution

Solution

25 ml.
8 ml.

Note: This solution must be freshly prepared as

required.

Technique:

Tissues should be fixed not longer than two to three hours in

5% formalin, or frozen sections of fresh material may be employed.
1.

Rinse with distilled water from four to five seconds; then

in Solution C for three to four hours, controlling under

immerse

the microscope at intervals, until melanoblasts are stained black.

Note: This solution

brown.
2.

Wash

with

is

likely to overstain if

distilled water;

it

becomes sepia

dehydrate; clear; mount.

Result:

Melanoblasts, black.

Note: After dehydration (in Stage 2) 1% crystal violet in absomay be employed as a counterstain, if desired.

lute alcohol

ELASTIN STAIN
(Weigert)

Any
bedded

fixative

except Susa, and tissues

or in Celloidin or in L.V.N.

may be used

in paraffin

wax

may be em-

Preparation of the Staining Solution:

i gm. of Weigert elastin stain and
5 gm. clean, dry
sand with 100 ml. absolute alcohol and 2 ml. pure hydrochloric acid until all the stain has gone into solution; then filter.
Note: The staining solution deteriorates after two or three

Triturate

silver

weeks.

The

nuclei

may be

stained with Orth's lithium carmine prior to

if no other counterstain is desired.

the following procedure

Technique:
I

Sections are brought

down to 90%

to twelve hours according to

alcohol and stained one half

depth of staining desired.

96

The

slides

SECTION TWO
should be stained in a jar or in a Petri dish, sections face downwards, to prevent a deposit forming on the sections.
2.

Wash

with

off excess stain

differentiate in acid alcohol for a

3.

Wash quickly with 70%

95%

alcohol,

and

if

necessary

few minutes.

alcohol

then thoroughly with water.

4 Counterstain with Van Gieson, Ehrlich haematoxylin or

Safranin for about five minutes.
5.

Differentiate, if necessary, in

6.

Dehydrate

Note:

ganum

clear in xylol

If Celloidin or

oil

95%

alcohol.

and mount.

L.V.N, sections are used

or in terpineol after

95%

clear in ori-

alcohol.

Results:

Nuclei, brilliant red (if Orth's

used) or bluish black (with haematoxylin). Collagen,
pink to red other tissue elements, yellow (if Van Gieson is used).
Elastic fibres, dark blue or black.

carmine

is

ELASTIN STAIN

(Sheridan)

This stain has an advantage over Weigert's elastin stain in that

the solution may be kept for reasonably long periods without
deterioration.

The staining procedure is the same as for Weigert's elastin stain.

Results:
Elastic fibres are stained green to greenish black.

ELASTIN

TRICHROME STAIN

For the demonstration of elastic, smooth muscle and

collagenic fibres with equal clarity, particularly in the walls
of hlood vessels
Solutions required:

A. Weigert's

elastin stain

Weigert's elastin stain powder

Hydrochloric acid, cone, pure

Absolute alcohol
.

97

2 ml.

100 ml.

gm.

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

Dissolve the stain by boiling for two minutes in a
plugged lightly with cotton-wool, on a water

flask,

Allow to cool; then filter; make the

to 100 ml. with absolute alcohol; then

bath.

up

may be prepared

Alternatively the solution

acid.

volume
add the
as

described on p. 96.

Note:

This solution deteriorates

after three or

four weeks.
B.

Ehrlich haematoxylin.

C. Ponceau-acid fuchsin (Masson):

Acid fuchsin
.

Ponceau de xylidine
Distilled water

Glacial acetic acid

D. Phosphotungstic acid
E.

Light Green

1%

3%

gm.

0-3

0-7 gm.
100 ml.
ml.

aqueous.

aqueous.

Technique:
1.

Paraffin sections are

distilled

elastin stain in a staining jar for

2.

Wash

slides and brought down to

then immersed in Weigert's

mounted on

water in the usual manner

one hour.

rapidly in acid alcohol;

then dehydrate and

differ-

entiate in absolute alcohol until the sections appear only faintly

red.
3.

Immerse

in

70%

alcohol, followed

by

distilled water.

4. Stain in Ehrlich haematoxylin for eight to ten minutes

differentiate in water for five minutes.
5.

6.

then

Stain in Ponceau-acid fuchsin for five minutes.

Wash thoroughly in 3%

phosphotungstic acid

then immerse

in the phosphotungstic acid for ten minutes.

7.

Wash

Green
8.

for

thoroughly in distilled water; then stain with Light

to five minutes; then without washing:

two

Flood the preparation with 1% acetic acid and allow it

pour off excess then without washing

for three minutes

9.

Dehydrate;

clear;

mount

in

q8

Damar

xylol.

to act

SECTION TWO
Results:
Elastic tissue stained blue-black ;

smooth muscle, red

collagen

green.

EOSIN AZUR

HAEMATOXYLIN (Maximow)

For demonstration of inflammatory changes in haemopoietic tissues

Solutions required:

A. Azur 2 eosin

..

Distilled water

Heat
B.

..

..
.

o-i

gm.

loo ml.

to boiling point, then allow to cool.

Solution

(as

above)

Distilled water

lo ml.

50 ml.

C. Ehrlich haematoxylin.
Technique:

Formalin-fixed material (sections or smears) are stained

to
ten minutes with Ehrlich haematoxylin.
five
1

2.

Pour

off excess stain

immerse the preparation in tap water

then wash thoroughly with

appears blue to the naked eye

distilled water and drain well.

until

it

from

3. Stain from eighteen to twenty-four hours in Solution B (as

above) in a staining jar. If a staining jar is not available, place the
slide, resting face downwards, on two pieces of thin glass rod, so

that any precipitate

formed

4. Differentiate in

to

95%

come away from the

is

not deposited on the preparation.

alcohol until dense blue clouds cease

preparation, and the red corpuscles

and

collagen are pink.

5. Immerse the preparation in three changes of absolute alcohol,
followed by two changes of xylol then mount.
;

Results:

Cartilage stained purple;

purple to violet;

granules,

cytoplasm, pink to blue

basophil leucocytes and mast cell

nuclei, blue;
erythrocytes, pink;

eosinophil granules and secretion gran-

ules, pink.

99

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

ETHYL VIOLET

BIEBRICH SCARLET (Bowie)

For pepsinogen granules in gastric mucosa
-

Solutions required:

A. Stock

solution.

i%

Ethyl violet-Biebrich scarlet

in

B.

20%

alcohol.

Staining solution.

Stock solution
Alcohol 20%

above)

(as
.

0-5 ml.
100 ml.

Technique:
1.

Tissues should be fixed in Regaud's

water, dehydrated, cleared and

usual manner.
2.

Fix sections to

grades of alcohol
3.

slides,

down

fluid,

embedded

washed

in paraffin

in

running

wax

in the

dewax and pass through descending

to distilled water.

Stain for twenty-four hours in Solution

in a covered

staining jar.
4.

Drain and wipe

5.

Differentiate for about half an hour with a mixture con-

sisting of equal

oflF

excess liquid.

volumes of clove

examination under the microscope

6.

Wash

oil

and

xylol, controlling

at intervals.

well with several changes of xylol

by

mount

in cristalite.

Results:

Pepsinogen of the pepsin-forming

cells is stained violet,

parietal cells are red.

FEULGEN FUCHSIN
For chromatin in animal cells
Solutions required:

A. N/i hydrochloric acid.

100

while

SECTION TWO
B.

Distilled water

Boil;

allow to cool to about 70

Basic fuchsin

When

200 ml.

C;
i

gm.

dissolved, raise to boiling point

to cool to 50 C.

then add:

then allow

and add:

2 ml. pure hydrochloric acid

cone, and
2 gm. potass, metabisulphite.

Allow

to stand twenty-four hours then

add

gm.

decolorizing carbon; shake well; filter. The solution if properly prepared will be colourless.

C. Pure hydrochloric acid, cone.

Potass, metabisulphite
Distilled water
.

D. Light green 0-25% in

90%

2 ml.
2

gm.

200 ml.

alcohol.

Technique:

Tissues are fixed in Zenker or Helly for six to twelve hours

in running water for twelve to twenty-four hours.

then

washed

They

should not be treated with iodine-alcohol.

1.

tion

Sections are transferred from distilled water to ajar of Soluthen rinsed in distilled

A at 60 C. for five to fifteen minutes;

water.

2.

Transfer for one to one and a half hours to a jar of Solution

at

room temperature.

3.

Transfer for two to three minutes in each of three successive

lots of Solution

then wash thoroughly in

distilled water.

4.

Counterstain for about two to five minutes in Solution D.

5.

Dehydrate; clear and mount.

Results:

Chromatin, purple. All other elements, transparent green.

lOI

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

FONTANA STAIN
For argentaffine granules
Solutions required:

A. Silver oxide (Fontana).

Sodium

B.

thiosulphate

5%

aqueous.

Technique:
1. Tissues are fixed in 10% neutral formalin, washed, dehydrated in alcohol, cleared in cedarwood oil, and embedded in

paraffin

wax

as usual.

2. Fix sections to slides, bring down to distilled water and wash

thoroughly in two or three changes of neutral, freshly distilled

water.
3.

Immerse

in the silver oxide (Fontana) solution for twelve

to twenty-four hours in the dark in a covered, scrupulously clean

vessel.
4.

Wash

5.

Immerse

for

6.

Immerse

in tap water for ten minutes.

7.

Counterstain,

8.

Dehydrate; clear in xylol and mount.

in neutral, freshly distilled water for

one minute.

one minute in the sodium thiosulphate.

if

desired in carmalum.

Results:

Argentaffine granules, black.

FONTANA STAIN

SILVER NITRATE

For reticular and collagen fibres

Solutions required:

A. Strong ammonia solution

o-88o)

Distilled water

B.

(sp. gr.

10 ml.

90 ml.

Potassium permanganate 0-5% aqueous.

C. Oxalic acid 1-5%.

102

SECTION TWO
D.

Silver nitrate

E.

Fontana

F.

Aniline

5%

aqueous.

stain (silver oxide solution)

oil

Xylol

volume
volume

Technique:
1. Frozen sections not thicker than lo/^ are fixed in 10% formalin and afterwards washed in three changes of water for fifteen

minutes in each.

Immerse

2.

in Solution

at 60

C. for fifteen minutes, in an

oven.
3

4.

Rinse well in three changes of

Immerse

in the potassium

distilled water.

permanganate for three or four

minutes.

water for about ten or twenty seconds.

5.

Rinse with

6.

Decolorize with the oxalic acid solution until the

distilled

colour just disappears

7.

Immerse

8.

Wash

9.

Immerse

then wash well in

brown

distilled water.

in silver nitrate solution in the dark for an hour.

well with two changes of distilled water in the dark.

in Fontana's stain for fifteen

minutes

at

60 C. in

the dark.
10.

Wash

11.

Immerse

rapidly in three changes of distilled water.

in

30%

formalin for two or three minutes at

60 C.
12.

Wash

then transfer to

thoroughly in running tap water;

slides.

13. Blot

14.

away excess water.

Dehydrate with two changes of absolute

15.

Clear in the aniline-xylol (Solution F, above).

16.

Wash

with xylol; mount in

dammar

alcohol.

xylo

Results:

Reticulum

is

stained black, while collagen

N.B. : Sections must be handled with

this technique, as contact

brown.

glass needles throughout

with metal instruments will ruin the

preparations.
I

is

103

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

GALLOCYANIN

ORCEIN - ACID ALIZARIN BLUE

ALIZARIN VmiDINE

general stain for animal tissues

Solutions required:

A. Gallocyanin

..

Chrome alum

..

..

o-i

gm.

100 ml.
aqueous
Boil for ten minutes. Allow to cool then make up
the volume to 100 ml., filter and add five or six drops

5%

of formalin.
B.

Orcein 0-5% in 70% alcohol

Hydrochloric acid cone.

C. Acid Alizarin Blue

99 ml.

5 gn^-

Aluminium sulphate 10% aqueous

Boil for ten minutes.

volume

Cool and

ml.

100 ml.

filter.

to 100 ml. with distilled water

Make up the
and add

five

or six drops of formalin.

D. Phosphomolybdic acid 5%.

E.

Alizarin viridin

Buffer solution

pH

5-8

o-2 gm.
100 ml.

Technique:
1.

Fix tissues in

10%

formalin and

embed

in paraffin

wax

in

the usual manner.

2.

Fix sections to slides and take

down

to distilled water as

usual.
3.

Stain nuclei intensely by immersing the slides in the gallo-

cyanin solution in a staining jar, examining the preparations under

the microscope at intervals over a period of twenty-four hours, to
ascertain the depth of staining.
4.

Wash

5.

Stain elastic fibres in the orcein solution for ten minutes to

with two changes of

distilled water.

half an hour in a grooved, covered staining jar.

6.

Wash

7.

Stain muscle in the acid alizarin blue solution for seven

well with distilled water.

minutes.

104

SECTION TWO
8.

Wash

9.

Differentiate in the

thirty

with distilled water.

phosphomolybdic acid solution

for about

minutes, controlling by examination under the micro-

scope at intervals.
10. Wash with two changes of distilled water.
11. Stain collagen in the alizarin viridin for seven minutes.
12.

Drain and blot thoroughly but

13.

Rinse with

14.

Wash

96%

carefully.

alcohol; followed by carbol xylol.

well with two or three changes of xylol, and mount.

Results:

Muscle and epithelium, pale violet.

Nuclei, dark brown.
Erythrocytes and elastic fibres are stained a rich brown, while
mucus, collagen are in varying shades of green myelin sheaths,
;

pink

and

axis cylinders, dark blue.

GIEMSA STAIN
For malarial parasites, rickettsia,

etc.

Solution required:

Giemsa

stain

Distilled water, buffered to

pH 7-2

ml.

20 ml.

N.B.: This mixture should be freshly prepared

immediately before use.
Technique:
1.

Fix small pieces of tissue in

2.

Dehydrate; clear and embed.

10%

3. Bring down paraffin sections to

manner.

4.

formalin,

Regaud or Zenker.

distilled

water in the usual

Stain for eighteen to twenty-four hours in the diluted

Giemsa

(as above).
5.

Wash

6.

differentiate quickly in

in distilled water;

acid until the section

is

pink

then wash with

0-5%

acetic

distilled water.

Blot and dry in air and mount.

Results:

Nuclei are stained dark red erythrocytes, pink. Malaria parabluish red with red chromatin.
;

sites,

105

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

GBEMSA

WRIGHT STAIN

A permanent

stain for
particularly Nissl bodies

the

differentiating

structures,

and cytons, of the spinal cord

Solution required:

Wright's stain

Giemsa

stain

5
i

volumes
volume

Technique:
1.

Material should be fixed in neutral formalin io%.

2. Wash, dehydrate, clear, and

usual manner.
3. Fix sections to slides;
descending grades of alcohol,

4.

embed

de-wax;

down

in paraffin

wax

in the

pass through the usual

to distilled water.

Flood the sections with a measured volume of the above

it to act for two minutes.

staining solution and allow

5.

Add

an equal volume of

by rocking the slides gently.

two minutes.
6.

tilled
7.

Pour

off excess stain

distilled

Allow

water and mix with stain

this diluted stain to act for

and immerse the

slides in fresh dis-

water for one minute.

Transfer immediately into

80%

alcohol and leave therein for

fifteen seconds.

95% and

8.

Dehydrate rapidly in

9.

Clear in xylol and mount.

absolute alcohol.

Results:

Cytons and Nissl granules are stained deep blue. Nuclei of

blood-vessel structures and neuroglia are light blue. Elastic fibres
of blood vessels, deep blue. Erythrocytes, pink. Neuroglia fibres,
light red.

Note:

The

sity of the

proportion of the Giemsa stain regulates the intencyton stain.

Reference: Hanburg, L. (1935), Science, 81, 364-5.

106

SECTION TWO

GOLD CHLORroE
For neuroglia

fibres;

SUBLIMATE

(Cajal)

for astrocytes in central

nervous

system
Solutions required:

A. Neutral formalin

Ammonium

bromide

Distilled water

Gold chloride (brown or yellow)

B.

C. Mercuric chloride

5%

D. Sodium hyposulphite

15 ml.

2 gm.
85 ml.

1%

aqueous.

aqueous.

10%

aqueous.

Technique:
1

Fresh pieces of tissue are fixed for two to twenty-one days in

Solution A.
2.
3.

Frozen sections are cut 15 to 30^ thick.

Rinse in several changes of distilled water.

Immerse

sections, flattened out and not lying on top of one

for
to four hours in a freshly prepared mixture conthree
another,
4.

sisting of:

Solution

Solution

5 nil-

30 ml.

Distilled water

{Note:

3 ml. of the

mixture

is

5 t^-

required for each

section)
until the astrocytes are stained dark against a relatively light

background; the reaction should be controlled by microscopic

examination of a section while still wet.

6.

Wash
Wash

7.

Dehydrate

5.

in distilled water; then fix^in Solution

D.

thoroughly in tap water.

;

clear

and mount.

Results:

Nerve cells, red. Nerve fibres, unstained.

brownish purple, or unstained.

Astrocytes, black.

Background,

light

107

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

GOLGI METHOD

(Rapid)

For nerve cells

Solutions required:

A. Potass, bichromate

Osmic

acid

1%

3%
.

40 ml.

10 ml.

This solution must be freshly prepared.

B.

Silver nitrate

075%.

Technique.
1. Immediately the animal is killed, tissues are cut into slices
about 2 mm. thick and fixed in Solution A for one to three days

depending upon the size of the pieces.

2. After removing excess fixative by blotting, the tissues are
rinsed in Solution B until no more precipitate is formed.
3. Transfer to a fresh lot of Solution B and leave for two days
or longer.

With

brush carefully brush the precipitate from

then wash well in distilled water.
5. Dehydrate by immersing for one to four hours, depending
upon the size of the pieces, in each of the following two lots of
95% alcohol; two lots of absolute alcohol; one lot of etheralcohol (equal vols, ether and absolute alcohol).
6. Embed in Celloidin or L.V.N.
7. Sections 6o-ioo/^ are mounted on slides and covered with
Canada balsam in benzol or cristalite, without cover glasses
{see L.V.N, technique, page 26).
4.

a camel-hair

the surface of the tissue

Results:

Background, dull yellow.

Blood vessels, black.

Nerve

cells

and

their

processes,

black.

GRAM'S IODINE
For bacteria in sections
Solutions required:

A. Carbol gentian
B.

Gram's

violet.

iodine.

108

SECTION TWO
C. Carbol fuchsin (Ziehl Neelsen)
Distilled water

D. Picric

volume
9 volumes
i

acid, saturated, aqueous.

Technique:
1.

Pieces of tissue are fixed in

cleared and
2.

embedded

io%

formalin;

dehydrated;

in paraffin wax.

Fix sections to slides

de-wax and take down

to distilled

water in the usual manner.

3.

Stain in Solution

A for

about two minutes.

4.

Pour

stain

and without washing add Gram's

off excess

iodine and allow the stain to act for one minute.

5.

Differentiate in pure acetone until colour ceases to

come out

of the sections.
6.

Counterstain in the carbol fuchsin (Solution C) for about a

minute.
7.

Pour

to dry;
8.

after
9.

off excess stain,

and

drain, without allowing the sections

then without washing:

Cover the sections with the picric acid solution, pouring

one half to one minute.
Dehydrate and clear with pure acetone for about

off

fifteen

seconds.
10.

Clear in xylol and mount.

Results:

Gram-positive
negative are red.

organisms are stained violet, while GramNuclei are stained pink, while cytoplasm is

yellow.

HAEMALUM

EOSIN

For demonstrating collagenous tissue

Solution required:

A.

Haemalum

B.

Eosin, yellowish

(Mayer).

0-2%
109

in

20%

alcohol.

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

Technique:
Paraffin sections are fixed to slides, de-waxed and taken down
through descending grades of alcohol to distilled water in the
1.

usual manner.
2. Stain for five to ten minutes with the haemalum solution,
examining under the microscope at intervals until a satisfactory
degree of staining has been achieved.
3.

Rinse for a few seconds in tap water.

4.

Stain for one or two seconds with the eosin solution.

5.

Rinse for a few minutes in running tap water.

6.

Pass through

7.

Clear in xylol and mount.

70%, 90% and

absolute, alcohol.

Results:

Smooth muscle,

Collagen, deep pink.

pink.

Cytoplasm, pale

pink.

Nuclei, blue.

HAEMATOXYLIN
For muscle, connective

AZOPHLOXINE

tissue,

cells, etc.

ganglion

Solutions required:

A. Lavdowsky's Fixative:

Formalin (40% formaldehyde)

Glacial acetic acid

Alcohol

95%

Distilled water

B.

10 ml.
2 ml.

50 ml.
40 ml.

Haematoxylin (Delafield, Harris or

Ehrlich)

C. Azophloxine
Acetic acid 0*2%

Add

about

0*2

100 ml.

gm.

ml. chloroform as a preservative.

D. Acetic acid 0-2% aqueous.

E. Orange G.
.

Phosphotungstic acid
Distilled water
Fast Green FCF

gm.

4 gm.
100 ml.

0-2

gm.

no
1

SECTION TWO
Technique:

Fix material in Lavdowsky's mixture or in

in paraffin wax.

1.

io%

formalin

and embed

Stain in Harris, Ehrlich or Delafield Haematoxylin for five

2.

to ten minutes.

Blue in tap water or

3.

1%

lithium carbonate solution.

4. Stain in the azophloxine solution for two minutes.

5. Rinse in 0-2% acetic acid.

Stain in the orange G-fast green solution for one minute.

6.

7. Differentiate for about five minutes with

8. Rinse in distilled water.

0-2%

acetic acid.

9. Remove excess distilled water by draining and blotting round

the edges of the sections carefully, but do not allow to dry com-

pletely.
10. Dehydrate directly with
alcohol, or with cellosolve.

Note: Absolute and

a tendency to

70%

two or three changes of absolute

alcohol should be avoided as they have

remove the azophloxine.

Results:

Connective tissue, green. Striated muscle, brick red. Smooth

muscle, reddish violet. Nerves, blue-grey. Ganglion cells, violet.
Erythrocytes, orange. Cardiac conductive tissue is easily distinguishable from cardiac muscle as

it

takes a lighter shade of

staining.

Note: Azophloxine is used here as a substitute for ponceau de

xylidine in Goldner's modification of Masson's technique.

The

stain is also suggested in place of Eosin as a counterstain

for use with haematoxylin. The

advantages of using azophloxine
are that it gives clear and delicate pictures and it does not overstain,

recommended procedure is followed. When azophloxine

be used merely as a counterstain for haematoxylin the procedure is as follows

if

the

is

to

Proceed as steps, i, 2, 3, 4, and 5 (above).

Rinse in distilled water.
III.
Dehydrate with three changes of absolute alcohol; then
clear and mount.
I.

II.

Reference: Halper,

M. H.

(1954, November), Stain Tech., 29, no. 6, 315-7.

Ill

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

HAEMATOXYLIN

BASIC FUCHSIN

For haemofuscin, melanin and haemosiderin in animal

tissues
Solutions required:

A. Haematoxylin (Ehrlich).
Basic fuchsin

B.

0-5%

in

50%

alcohol.

Technique:

Tissues

may be

fixed in

Zenker or

in absolute alcohol or in

Paraffin or Celloidin sections

formalin.

10%

may be employed.

If

used it will be necessary to remove mercury

usual
manner.
in
the
deposits
Zenker's fixative

Stain for five to ten minutes in Ehrlich haematoxylin.

Wash

2.

3.

tion
4.

is

well in tap water, then several times in distilled water.

Stain from five to twenty minutes in the basic fuchsin soluthen pour off excess stain and wash well in distilled water.
Differentiate in

alcohol

alcohol; then dehydrate in absolute

in balsam.

95%

clear in xylol

and mount

Results:

Nuclei, blue;
their natural

melanin and haemosiderin remain unstained in

brown

colours

haemofuscin, bright red.

HAEMATOXYLIN (DELAFIELD)

EOSIN

For general staining

Solutions required:

A. Delafield haematoxylin.
B.

Eosin, yellowish,

1%, aqueous.

Technique:
I.

Tissues should be fixed in Zenker, Bouin or

in paraffin wax.

and embedded

112

10%

formalin

SECTION TWO
Sections are brought down to distilled water;
with Delafield haematoxylin for ten minutes.
2.

3.

Wash and immerse

4.

Wash

then stained

in tap water for about five minutes until

blue
to
the naked eye.
the section appears

rapidly with distilled water then stain for one to two

1% eosin yellowish, aqueous.
;

minutes with
5.

95%
6.

Wash

quickly with distilled water;

and absolute

then dehydrate with

alcohol.

Clear in xylol and mount.

Results:

Nuclei are stained blue; cytoplasm, pink.

HAEMATOXYLIN

(Ehrlich)

For keratohyalin
Solutions required:

A. Haematoxylin (Ehrlich).
B.

Potass,

permanganate o-i%.

Technique:
1.

Material should be fixed in

10%

formalin and

embedded

in

paraffin wax.
2.

Fix sections to slides and bring

down

to distilled water as

usual.
3.

Stain in Ehrlich haematoxylin for ten minutes.

4.

Pour

off excess stain

then rinse and blue in tap water,

5. Immerse in potassium permanganate

then wash well with water.

6.

o-i%

for ten seconds;

Dehydrate; clear; mount in balsam.

Results:

Keratohyalin is stained blue-black while the other elements are

unstained or faintly stained.
113

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

HAEMATOXYLIN

(Ehrlich)

For demonstrating sodium urate in animal tissue

Solution required:

Ehrlich or Delafield haematoxylin.

Technique:
1

Fix tissues in absolute alcohol and embed in Celloidin.

2.

Stain for five to ten minutes in the haematoxylin solution.

3.

Blue in saturated lithium carbonate solution.

4.

Rinse quickly in

5.

Dehydrate with

distilled water.

95%

6.

Clear in terpineol.

7.

Mount

alcohol.

in balsam.

Results:

Sodium

urate crystals and nuclei, deep blue.

HAEMATOXYLIN (Ehrlich) - EOSIN

A general stain for animal tissues
Solutions required:

A. Haematoxylin (Ehrlich).
B.

Eosin, aqueous, yellowish

1%

in tap water.

Technique:
1. Sections are mounted on slides and brought down to distilled
water in the usual manner, any mercurial deposit from the fixative
being removed by the standard technique.

Stain from two to ten minutes in haematoxylin (Ehrlich)

"
"
of the stain (a well-ripened
ripeness
depending upon the
2.

haematoxylin will act

much more

rapidly than a recently-prepared

solution).
*'

"

Rinse in water; then blue in tap water; that is, immerse

the preparation in tap water from two to ten minutes or until the
preparation appears blue to the naked eye.
3.

4.

The

preparation should

now be examined,
114

while

still

wet,

SECTION TWO
under the microscope and if the nuclei are not stained a bright and
transparent blue and the cytoplasm (except mucin and basophile
granules of mast cells, etc.) is not colourless, rinse in water and
repeat the staining and bluing process.
If, on the other hand, overstaining has taken place, immerse the

preparation in 0-5% HCl for five to thirty seconds; then immedi"

"
blue
in tap water and again examine under
ately rinse in water,
the microscope; if the sections are still overstained repeat the
"
"
blue
in tap water again.
treatment with HCl; rinse and
5.

Wash

two

well with water; then stain from

to five minutes

in eosin solution.
6.
still

Rinse quickly in water and examine the section rapidly, while

wet, under the microscope to ensure that the depth of the

counterstain

The

cytoplasm, collagen, connecshould be stained a bright

to
overstain somewhat with
advantageous

is sufficient.

cell

tive tissue fibres, erythrocytes, etc.,

transparent pink. It is
the eosin, as subsequent dehydration in the alcohols will remove
the excess eosin.
7.

Dehydrate by passing rapidly through 70%,

90%

and abso-

lute alcohol.
8.

Clear in xylol

mount

in balsam.

Results:

Nuclei are stained dark blue; karosomes, dark blue; plasmasomes, red cytoplasm (except when basophile) is stained in varying
;

shades of pink;

(when

thick),

muscle and collagen

pink;

fibres,

pink;

erythrocytes, thyroid colloid

elastic fibres

and

keratin,

bright pink.

HAEMATOXYLIN

FLUORCHROME

(Kultschitzky-Pal)

For myelin sheaths. Particularly suitable for demonstrating very fine fibres in cerebal cortex, etc.
Solutions required:

A. Weigerfs rapid fixative :

Fluorchrome
Potassium bichromate
Distilled water
.

Add

2 gm.
5 gni-

100 ml.

the potassium bichromate to the water

add the fluorchrome; cool; then filter.

115

boil ;

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

B.

Kultschitzky's haematoxylin solution.

C. Lithium carbonate,

aqueous
Potassium

saturated,

lOO ml.

i%

ferricyanide

aqueous

lo ml.

Technique:
1

more than 5 mm. thick are

from four to seven days.

Slices of formalin-fixed tissue not

immersed

in Weigert's rapid fixative

Wash

3.

running water for four to six hours.

Dehydrate and embed in Celloidin.

4.

Stain sections in Kultschitzky's haematoxylin from twelve to

2.

in

twenty-four hours.
5.

the white matter

is

hours in Solution

for several

Differentiate

controlling

one half to one hour, until

stained blue-black, and the grey matter is

under the microscope

at intervals of

stained yellowish.

Note: For human spinal cord differentiation takes up to twelve

Human cerebral cortex requires about four hours.

hours.
6.

Wash

7.

Dehydrate and clear by the standard Celloidin method.

8.

Mount

well in running water.

in cristalite.

Results:

Myelin sheaths are stained black, while ground-substance

yellow.

HAEMATOXYLIN

GENTIAN VIOLET

IODINE

For demonstrating Gram-positive bacteria

and

fibrin in sections

Solutions required:

A. Haematoxylin (Delafield).
B. Aniline gentian violet
C.

Lugol's iodine.

D. Aniline

oil.

..

Xylol
E.

Erythrosin

5%

..

..

in absolute alcohol.

116

20 ml.
10 ml.

is

SECTION TWO
Technique:

may be fixed in io% formalin or in Zenker, if the

used then mercuric precipitates must be removed from
the sections by the standard technique.
Tissues

1.

latter is

de-wax with xylol and pass through

of
alcohol
to water in usual manner.
descending grades
2.

Fix sections to slides

3.

Stain in the haematoxylin solution for five to twenty minutes.

4.

Rinse quickly in acid alcohol.

5.

Immerse

in a large

volume of tap water

for

two

to five

min-

utes.
6.

Stain in the aniline gentian violet for two to five minutes.

7.

Pour

off excess stain,

and without washing, blot the

slide

carefully.

Flood with Lugol's iodine and allow the solution to act for

8.

two

to five minutes.

9.

10.

Pour

and without washing, blot dry carefully.

Decolorize for a few seconds with Solution D.
off excess,

11. Flood the preparation with erythrosin (Solution E, above)

and allow the stain to act from one half to one minute.
12.

Pour

13.

Rinse well with xylol; drain off excess and blot dry care-

oflF

excess stain and

wash the preparation with Solution

D.

fully.

14.

Mount

in

balsam or

Cristalite or

Clearmount.

Results:

Nuclei, blue.

Fibrin and Gram-positive organisms, purplish

blue.

HAEMATOXYLIN
(Heidenhain)
Solutions required:

A. Iron alum
Distilled water

3
.

Dissolve by shaking.

117

gm.

100 ml

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

B.

Haematoxylin io% in absolute

alcohol (which has been ripened

months or longer)

for three

Distilled water

C. Eosin yellowish

1%

5 ml.

95

r^l-

aqueous.

Technique:

Fix in Zenker.

Embed

in paraffin wax.

Sections are brought down to distilled water, then morfor one half to three hours.
danted in Solution
1.

2. Wash in tap water

then rinse in tap water.

3.

one to three hours in Solution

stain

Differentiate in Solution A, controlling by examination

under

the microscope.
4.

Wash

5.

Stain with Solution

6.

Wash

in running water for five to ten minutes.

in tap water;

for one to three minutes.

then clear in xylol and

dehydrate;

mount.
Results:

Nuclei are stained black; cytoplasmic structures, pink.

HAEMATOXYLIN
For the identification of lipines
Solutions required:

A. Potassium bichromate
B.

5%

aqueous.

Haematoxylin solution (Ehrlich).

C. Potassium ferricyanide

Borax

Distilled water

gm.

2-5

2 gm.

. .

100 ml.

Technique:
I.

Tissues are fixed from twelve to twenty-four hours in

formalin in normal saline.

118

10%

SECTION TWO

3.

Wash
Make

4.

Immerse

2.

for several hours in running water.

them

frozen sections and collect

in Solution

A for

in distilled water.

twenty-four to forty-eight hours

at 37 C.
5.

Wash

in several changes of distilled water, handling the sec-

tions with care (as they

become

brittle after

immersion

in Solution

A).

6.

Immerse

7.

Wash

8.

Differentiate in Solution C, controlling

in Solution

for four to six hours at 37 C.

in distilled water.

scope, until the

ground cytoplasm
This process takes several hours.

is

under the micro-

changed from black

or six changes of
9. Wash thoroughly in five
then mount in glycerine jelly.

to yellow.

distilled water;

Result:

Lecithin and other lipines are stained black to deep blue (light
blue coloration should not be taken as positive). Lipides and
other tissue constituents are colourless.

HAEMATOXYLIN

(Kultschitzky)

(Weigert's modification)

For finer studies of cortical architecture and for

brain sections
Solutions required:

A. Weigerfs Secondary Mordant:

Cupric acetate neutral, normal

gm.
gm.

Fluorchrome

2-5

Distilled water

Boil ; allow to cool

Glacial acetic acid

then add
.

100 ml.

5 ml.

B. Haematoxylin (Kultschitzky).

C. Lithium

carbonate,

aqueous
Potassium ferricyanide
.

119

saturated,
.

1% aqueous

100 ml.
10 ml.

total

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

Technique:
1.

After fixing material in

io%

formalin mordant for four to

hwt days in Solution A.

2. Dehydrate in ascending grades of alcohol
and embed in Celloidin.

3.

Immerse

in the usual way,

for twelve to twenty-four hours in the haematoxylin

(Solution B).

C from four to twelve hours, conunder

examination
the microscope at intervals, and
trolling by
4.

Differentiate in Solution

changing the differentiating

5.

Wash

6.

Dehydrate with

7.

Clear in terpineol.

8.

Drain well and blot

9.

Mount

thoroughly in

fluid three or four times.

distilled water.

95%

alcohol.

carefully.

in balsam.

Results:

Finest myelin sheaths are stained a deep black.

HAEMATOXYLIN

- PHLOXINE
GENTIAN VIOLET

ANILINE

For actinomyces in sections

Solutions required:

A. Ehrlich haematoxylin.
B.

Phloxine

3%

aqueous.

C. Aniline gentian violet.

D. Gram's

iodine.

Technique:
1.

Tissues are fixed in

cleared and
2.

embedded

10%

in paraffin

Fix sections to slides

formalin, washed, dehydrated,

wax in the usual manner.

de-wax and bring down

to distilled

water as usual.
3. Stain with Ehrlich haematoxylin five to ten minutes;
blue and wash in lithium carbonate saturated aqueous.

120

then

SECTION TWO
Stain for fifteen to twenty-five minutes in the
then wash with distilled water.

4.

phloxine

solution;
5.

Stain in aniline gentian violet for about ten minutes.

6.

Rinse in distilled water.

7.

Immerse

8.

Wash

9.

Decolorize with aniline

in

Gram's iodine solution

for

one minute.

in distilled water.
oil until

the stain ceases to

come out

of the sections.
10.

Rinse well in several changes of xylol and mount.

Results:

Branched forms are stained blue, while clubs appear red.

HAEMATOXYLIN, PHOSPHOTUNGSTIC
For

Pleuropneumonia organisms

in

(Mallory)

sections

of lung

Solution required:

Phosphotungstic acid haematoxylin (Mallory).

Technique:
1.

Pieces of tissue should be fixed in Bouin, Carnoy or absolute

embedded in paraffin wax in the usual manner.

alcohol and
2.

Fix sections to slides

of alcohol
3.

down

de-wax, pass through descending grades

to distilled water, as usual.

Stain in Mallory's phosphotungstic acid haematoxylin in

a stoppered staining jar for twenty-four hours, without treatment

with the usual potassium permanganate and oxalic acid solutions.
4.

Pour

off excess stain

drain well without washing

then care-

fully blot dry.

5.

6.

Dehydrate quickly in absolute alcohol.

Clear in xylol and mount.

Results:

The organisms appear as masses of mycelia which are stained

deep blue.
121

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

HAEMATOXYLIN

PICRO FUCHSIN

For nuclei, connective

tissue, etc.

Solutions required:

A. Distilled water
Ferric

chloride,

aqueous
Hamaetoxylin
.

hol
B.

hydrated
.

10%
.

C. Picric acid,

1%

4%
.

2 ml.

in absolute alco.

Picric acid, saturated,

Acid fuchsin

47-5 ml.

aqueous
aqueous
.

0*4 ml.

20 ml.
0-5 ml.

saturated in absolute

alcohol.

Technique:
1

Tissues are fixed in Bouin and

embedded

in paraffin wax.

2. Sections about 8ju, in thickness are fixed to slides, dewaxed

with xylol and taken through the usual descending grades of

alcohol to distilled water.

3.

Stain for two to three minutes in solution A.

4.

Differentiate

and counterstain

for about ten to fifteen seconds

in solution B, controlling under the microscope, until only the

nuclei are stained a greyish colour with the haematoxylin.
5.

6.

7.
8.

xylol,

Rinse immediately in distilled water.

Dehydrate by dripping solution

Clear with Terpineal.

onto the

slide.

Mount directly with Michrome mountant,

then mount with Clearmount or Cristalite.

or rinse with

Results:

Chromatin, black to grey. Muscle, yellow. Connective tissue,

Keratinized regions, bright yellow. Cytoplasm, yellow.

red.

Reference: Margolena, L. A. and Dolnick, E. H. (1951), Stain Tech., vol. 26,

1 1 9-2 1.

pp.

122

SECTION TWO

HAEMATOXYLIN

PICRO PONCEAU S

A selective stain for collagen and connective tissue in place

of Haematoxylin

Van Gieson

Solutions required:

A. Haematoxylin (Heidenhain or Ehrlich).

Picro Ponceau S {Curtis):
Ponceau S i% aqueous

B.

Picric acid

Acetic acid

i% aqueous
i% aqueous

lo ml.

86 ml.

4 ml.

Technique:

Proceed exactly as for Haematoxylin (Ehrlich) - Van Gieson or

Haematoxylin (Heidenhain) - Van Gieson (pages 127-128).
Results:
-

Identical with Haematoxylin

Van Gieson.

Note: Unlike Van Gieson, Picro Ponceau does not fade when
in Canada balsam: but Van Gieson does not fade when
in D.P.X. or Clearmount or Cristalite.

mounted
mounted

HAEMATOXYLIN
Note:

PONCEAU FUCHSIN

The chief advantages

trichrome techniques,
it

(Weigert)

works well

after

is its

any

of this
simplicity

fixative.

(Curtis)

method over numerous other

and reliability and the fact that

The disadvantage

details are not as clearly revealed as after

is

that cytoplasmic

such stains as Masson or

Mallory.
Solutions required:

A. Weigert's Haematoxylin,

B.

Weigert's Haematoxylin,

C. Curtis Stain:

Ponceau

S,

2%

aqueous

Picric acid, saturated, aqueous

Acetic acid
.
.

2%

5 ml.

95 ml.
2 ml.

Technique
1
Fix material with any desired fixative,
.

embed and

section as

usual.
2.

Fix sections to slides and take

down

3. If the fixative contains a salt of

precipitate in the usual way.

123

to

70%

alcohol as usual.

mercury, remove mercurial

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

4.

Wash

with distilled water.

Stain for five to ten minutes in a mixture consisting of one

and B, and two volumes distilled
water.
5.

volume of each of solutions

6.

Wash

7.

Stain for two to four minutes in Curtis Stain.

8.

Wash

9.

10.

for five minutes in running water.

with

95%

alcohol.

Dehydrate with absolute alcohol.

Clear in xylol and mount.

Results:

Chromatin, black. Cytoplasm, yellow. Collagen and

fibres, red.

Reference: Leach, E. H. (1946), Stain Tech., 21, 107-10.

HAEMATOXYLESr

PONCEAU FUCHSIN
GREEN FCF

FAST

micro-anatomical stain, superior to haematoxylin and

eosin for the differentiation of tissues in histo-pathological

work, superior to Van Gieson for collagen fibres. The

stain has also been found very valuable in cytological work
Solutions required:

A. Iron haematoxylin (Heidenhain) No.

i.

Iron haematoxylin (Heidenhain) No.

2.

B.

C. Picric acid

8%

D. Acid fuchsin

in

1%

96%

aqueous

Glacial acetic acid

E.

Ponceau de xylidine
Distilled water

alcohol.

Glacial acetic acid

100 ml.
i

ml.

gm.

100 ml.

ml.

1% aqueous.
Phosphomolybdic
100 ml.
G. Fast Green FCF 2% aqueous
acid

F.

Glacial acetic acid

2 ml.

Technique:
I. Bouin, formalin or Susa-fixed tissues are
sectioned in the usual manner.

124

embedded and

SECTION TWO
2. Immerse sections, which have been brought down to distilled
water as usual, for one half to one hour in iron haematoxylin

(Heidenhain) No.
3.

i.

Rinse well in distilled water.

4. Stain in iron haematoxylin (Heidenhain) No. 2 for a length of

time equal to the duration of stage 2 (above) then rinse thoroughly
;

in distilled water.
5.

wash
6.

Differentiate in solution

then wash well in water

and

until only the nuclei are stained;

for five minutes :

stain in solution

in distilled water.

Stain one to five minutes in a mixture consisting of one part

E (above) and nine parts of distilled water then rinse

of Solution

thoroughly in tap water.

7. Differentiate in 1% phosphomolybdic acid for five to fifteen
minutes until collagen fibres are almost colourless; then, without

rinsing
8.

Stain in the Fast Green

FCF

Differentiate

in water.

solution for half to

two min-

utes.
9.

10.

Dehydrate

by washing
in the usual

clear

manner;

and mount.

Results:

Nuclei are stained mauve to black; cytoplasm, varying shades

and mauve muscle, red collagen fibres and mucus, green.

of red

HAEMATOXYLIN

PONCEAU

PICRO ANILINE

BLUE
For differential staining of connective tissue and muscle
Solutions required:

A. Haematoxylin (Weigert) A.
B.

Haematoxylin (Weigert) B.

C. Ponceau, S

0-2% aqueous

Glacial acetic acid

D. Picric

acid, saturated

aqueous

Aniline blue, water soluble

E.

Acetic acid

1%

aqueous.
125

100 ml.

ml.

100 ml.
o-i

gm.

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

Technique:
1.

Tissues should be fixed in

io%

formalin and paraffin sections

employed.
2.

Stain for five minutes in a freshly prepared mixture consisting

and B.

of equal parts of Weigert's Haematoxylin

3.

Wash

4.

Stain for three to five minutes in the acetic Ponceau,

in tap water.

(Solution C).
5.

Rinse in

6.

Stain for three to five minutes in the picro aniline blue

distilled water.

(Solution D).
7.

Wash

8.

Dehydrate in ascending strengths of alcohol and clear in

for three or four minutes in

1%

acetic acid solution.

xylol in the usual manner.

9.

Mount

in acid balsam.

Results:

Connective

culum, blue.

tissue, glomerular basement membrane and retiMuscle and plasma, pink. Erythrocytes, bright red.

HAEMATOXYLIN
For demonstrating

(Weigert)

SCARLET R
soaps and neutral

fatty acids crystals,

fats in fat necrosis
Solutions required:

A. Formalin
B.

Copper

10%

acetate

saturated with calcium salicylate.

10%

aqueous.

C. Weigert haematoxylin, A.

D. Weigert haematoxylin, B.
Borax 0-2% aqueous
Potassium ferricyanide

E.

F.

Scarlet

Acetone
Alcohol,
Scarlet

litre

2-5

gm.

(Herzheimer).
.

70%

Heat on

50 ml.

50 ml.

i -5

gm.

a hot water bath; then allow to cool

before filtering.

126

SECTION TWO
Technique:
1.

Fix tissues in Solution A; wash in running water and cut

frozen sections.
2.

Mordant the

sections in Solution

for three to twenty-four

hours; then wash in water.

3.

Immerse

in mixture of equal parts of Solutions

and

D for

twenty to forty-five minutes.

4.

Differentiate in Solution E, examining

under the microscope

at intervals.
5.

Wash

6.

Stain with Solution

7.

Rinse quickly with

8.

Rinse with

9.

Mount

well with distilled water.

for about five minutes.

70%

alcohol.

distilled water.

in neutral glycerine jelly.

Results:

Neutral

deep blue
be stained.

fats are stained red, whilst fatty acids are

black and haemoglobin, calcium and iron

may

also

Calcium salicylate is added to the formalin fixative to

convert soaps, which are sodium and potassium salts of fatty
Note:

calcium soaps. If it is desired to demonstrate

any, soap is present in addition to fatty acids, compare stained sections of two pieces of the same material, fixing one
and the other in ordinary 10% formalin.
piece in Solution
acids, into insoluble

how much,

if

HAEMATOXYLIN (Ehrlich) - VAN GIESON STAIN

A selective stain, for collagen and connective tissue, w^hich
requires less time than the Haematoxylin (Heidenhain) Van Gieson technique, although the results are not as
satisfactory
Solutions required:

A. Haematoxylin (Ehrlich).

Van Gieson

B.

Technique
I

stain

(Picro

Acid

fuchsin).

Mount

sections

on

slides

dewax and pass through the usual

descending grades of alcohol to water.

127

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

2.

Stain in Ehrlich haematoxylin for ten to thirty minutes.

3.

Rinse in water.

4.

Blue in tap water or

5.

Stain for three to five minutes in

6.

Rinse for one or two seconds in water.

7.

Drain and draw

paper

1%

lithium carbonate solution.

off excess

Van Gieson

stain.

water by applying a piece of

filter

to the edges of the section.

8.

Dehydrate with absolute alcohol only.

9.

Clear in xylol and

mount

in D.P.X., or Cristalite or Clear-

mount.
Note:
(a) It is essential, at stage 2, to overstain with haematoxylin as
this stain is differentiated by the picric acid of the Van Gieson
stain.
(b) Preparations mounted in Canada balsam fade, but fading
can be obviated by use of one of the recommended mountants.

HAEMATOXYLIN
A selective stain

(Heidenhain)

VAN GIESON STAIN

for collagen and connective tissue,

superior to Haematoxylin (Ehrlich) Van Gieson
Solutions required:

A. Haematoxylin (Heidenhain) A.

Haematoxylin (Heidenhain) B.
C. Van Gieson stain.

B.

Technique:
1.

Fix pieces of tissue in Bouin, Carnoy, Susa or

2.

Fix sections to slides; dewax and take

down

usual way, after removing mercurial precipitate

used as the fixative.

128

if

10%

formalin.

to water in the

Susa has been

SECTION TWO
3.

Immerse

A for one half to one hour.

than Bouin, Carnoy, Susa or formaUn

be necessary to increase the time in solution
and in solution B up to twelve hours or longer the time varies

Note :

If a fixative other

has been used

in solution

it

will

for different fixatives.

4.

Rinse in water.

5.

Stain in solution

6.

Rinse in water.

7.

Differentiate with solution A, controlling

under the microscope,

for a time exactly equal to step 3.

after the preparation has

by examination

been rinsed

briefly

in water.
8.

Wash

remove
9.

all

gently in running water for about five minutes to

traces of solution

A (iron alum).

Stain for three to five minutes in

Van

Gieson.

10.

Rinse for a few seconds in water.

11.

Examine, while

12.

Continue the staining with Van Gieson, or continue the

whichever is necessary.

still

wet, under the microscope.

differentiation with water,

13.

Drain and draw

off excess

water by means of a

filter

paper

applied carefully to the edges of the section, but do not allow the

preparation to dry completely.

14.

Dehydrate with absolute alcohol only.

15.

Clear in xylol.

16.

Mount

in D.P.X., Cristalite or Clearmount.

Results:

Nuclei of
red.

dark brown to black.

Collagen fibres: bright

and
other
tissues: yellow.
muscle,
Erythrocytes,
epithelia
cells:

Note: Van Gieson stain fades if mounted in Canada balsam, but

fading can be avoided by the use of D.P.X., or Cristalite or Clear-

mount.
129

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

mCKSON'S PURPLE
A general

stain suitable for class

work

Solution required:

Hickson purple, saturated aqueous.

Technique:
1

Bring sections

down

to water as usual.

2.

Stain in Hickson's purple for ten to twenty minutes.

3.

Dehydrate, clear and mount.

Results:

Leucocytes, purple

erythrocytes, distinct red.

The

rest of the

tissues purple.
Reference: Cannon, H. G. (1941),^- Roy. Micr. Soc, series III, 61, parts

and

4.

HICKSON'S PURPLE

VICTORIA GREEN

A general stain, particularly suitable for class work

Solutions required:

A. Hickson's Purple saturated aqueous.

B.

Victoria green, G. saturated in

70%

alcohol.

Technique:
1

Fix sections to slides

down

dewax and take through the alcohols

to distilled water as usual.

2.

Stain for ten minutes in the Hickson's purple.

3.

Rinse in distilled water.

4.

Stain in the victoria green for half to one hour.

130

SECTION TWO
5.

Rinse in 70%, followed by

6.

Clear in xylol and mount.

90%

alcohol.

Results:

Nuclear are sharply defined, purple.

Erythrocytes sharply

defined, stained vivid green, against a general blue-purple back-

ground.

Note: This method

is

an improvement of Hickson purple used

alone.
Reference: Cannon, H. G. (1941), J. Roy. Micr. Soc, series III, 61, parts 3

and

4.

fflTCHCOCK

AND

EHRLICH'S MIXTURE

For lymphatic, ganglion, plasma and basophilic cells; immature cells of bone marrow, striated muscle, and fibrin.
Technique:
1.

Fix in Zenker-acetic acid, corrosive sublimate, but not in

Miiller or formalin.
Paraffin sections are brought down to 90% alcohol;
passed through a solution of iodine in 90% alcohol.
2.

3.

The

iodine

alcohols to water

removed by passing through the graded

is

and

then

finally

washing for

fifteen

minutes in running

water.
4.

Flood with the stain and allow it to act for fifteen to thirty
then pour oif the stain and wash rapidly in water.

seconds
5.

The

preparation

is

then passed directly into absolute alcohol,

as long as the stain continues to

where it is allowed to remain only

be washed out in clouds.
6.

Clear in xylol and mount.

Note:

Sometimes the

brilliancy of the stain

is

enhanced by

re-staining.

Results:

Plasma cells: cytoplasm,

Other cells appear

green.

brilliant

in

crimson.
131

crimson: nuclei, bluishshades of green and

lighter

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

JENNER STAIN
For blood-forming organs

SECTION TWO

JENNER STAIN

GIEMSA STAIN

For the polychromatic staining of blood-forming organs

Solutions required:

A. Formol saline.
B.

Jenner Stain.

C. Giemsa stain

D. Acetic acid

008%

Distilled water (buffered to

pH 70)

ml.

20 ml.

aqueous

Technique:
1

Fix material in Solution

A (above) from twelve to forty-eight

hours.

3.

Dehydrate in the alcohols and clear as usual; embed in

wax and cut sections not exceeding 5^ in thickness.
Fix sections to slides and remove wax with xylol.

4.

Wash

5.

Stain sections with a measured

2.

paraffin

well with two changes of pure methyl alcohol.

should be freshly

Cover the

6.

volume of Jenner

stain,

which

filtered.

slides

sheets of moistened

with a Petri dish

lid lined

with two or three

prevent the evaporation of the alcohol and the consequent formation of a precipitate
on the sections), and allow the stain to act for three minutes.
filter

paper

(this is to

7. Add a volunle of distilled water (buffered to

7-0), equal to
that of the stain, to the slides, which should now be gently rocked
to ensure thorough mixing of the stain and water.

pH

8.

Allow

9.

Pour

this diluted stain to act for

off excess stain;

one minute.

then without washing, immerse the

slides in a stoppered staining jar containing diluted Giemsa

stain (Solution
above) and leave the stain to act for forty-five

minutes.
10.

Rinse and differentiate in Solution D.

1 1

Rinse thoroughly in

12. Dehydrate quickly in

of absolute alcohol.

distilled water.

95%

alcohol, followed

133

by two changes

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

13.

mount

Clear in xylol and

in Cristalite.

Results:

Erythroc3rtes are stained orange. Cytoplasm of lymphocytes

blue. Nuclei, deep blue to violet. Mast cell

and blastocytes are

granules, violet to violet-red.

LEAD HAEMATOXYLIN

"

'*

polychrome stain for the central nervous

system and the trunks outside it.

definitive

The

ACID FUCHSIN (MacConaill)

essence of this technique, which

is

due to Professor M. A.

MacConaill, of the Department of Anatomy, University College,

Cork, Ireland, is that the lead haematoxylin reduces the ammonium molybdate to form a blue lake, which makes it possible to

employ only the minimum exposure

leaving the erythrophile

(*'

to haematoxylin, thereby
Fuchsinophile ") parts of the neurone

red.

Solutions required:

A. Lead nitrate

B.

8 ml.

Acid fuchsin
Water

0-5

4%

aqueous

Solution

volume
volume

This solution should be prepared as and

it deteriorates after one day.

when required

D. Liquor ammon.

acetat. B.P.

Ammonium molybdate,
aqueous..

Water

gm.

100 ml.

Solution

Note:

gm.

92 ml.

Haematoxylin
Acetic acid

C.

2gm.

Glacial acetic acid

saturated,

..

..

..

10 ml.

70 ml.
80 ml.

Note: All the above solutions must be made withTap water may be used
the solutions must be filtered.
out the application of heat.

134

SECTION TWO
Technique:
1.

embedded

Material should be fixed in 5 or 10% formalin and

Sections are cut 6 to 12^ in thickness.

in paraffin wax.
2.

to

Fix sections to slides; remove paraffin wax and take

alcohol

70%
3.

by the usual

Pass through

30%

down

stages.

alcohol; then stain in Solution

for five

minutes.

5.

Rinse in two changes of tap water.

Immerse in Solution D for one to two minutes.

6.

Wash

4.

two

in running water for

to five minutes to

remove the

unchanged molybdate.
7.

Dehydrate; clear in xylol and mount.

Note:
deep yellow filter is of great help in microscopic
examination, although not necessary.
Results:

Nuclei, dark blue nucleoli of neurones, red axial substances

of nerve fibres, dark to pale blue cuticular substance (including
myelotheca) of nerve fibres, red neurilemma (of Glees), purplish
;

red.

Note: To eliminate all myelin, sections should be passed through

Cellosolve after the alcohols. The same precaution should be
observed when preparing tissue for embedding.

From Proceedings of the Royal Irish Academy, Vol. 53, Section B, No. i, The
Myelothecal Apparatus of Human Nerve; and from personal communications
with Professor M. A. MacConaill, m.r.i.a.

LEISHMAN STAIN
For general differentiation of blood corpuscles; for malarial
parasites; trypanosomes, etc.

This
prefer

is extensively used by British workers who generally

to Wright's stain which is used extensively in America.

stain

it

Solutions required:

A. Formol
B.

saline, neutral, buflFered.

Leishman

stain.

C. Acetic acid
L

o-o8% aqueous.
135

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

Technique:
1

Fix pieces of tissue in Solution

for sixteen to forty-eight

hours.
2. Dehydrate in the usual ascending grades of alcohol
and embed in paraffin wax.
3.

clear

Fix sections, not exceeding 5^ in thickness to slides; remove

xylol
pass through descending grades of alcohol down

wax with

to neutral distilled water.

4. Stain for five to ten minutes in freshly prepared mixture
consisting of one volume of Wright's stain and two volumes of
neutral distilled water, in a stoppered staining jar.
5.

Rinse with neutral distilled water.

6. Differentiate with the acetic acid solution, controlling by

examination under the microscope, until the protoplasm of the
cells is pink, and only nuclei are blue.
7. Wash with neutral distilled water.
8.

Dehydrate quickly with absolute alcohol;

mount

clear in xylol;

in Cristalite.

Results:

Polymorphonuclears dark purple

Erythrocytes, yellowish red.

nuclei, reddish violet granules, pale pink cytoplasm. Eosinophiles :

blue nuclei, red to orange-red granules, blue cytoplasm. Baso-

philes

purple to dark blue nuclei, dark purple to black granules.

nuclei, sky blue cytoplasm. Platelets

Lymphocytes dark purple

Malarial parasites and Leishmanial

cytoplasm, blue. Trypanosomes chromatin, red.

violet to purple granules.

chromatin, red

Note: The timing of the staining either before or after dilution

may be altered to suit individual requirements. Staining effects
similar to Giemsa are obtained by staining for ten minutes in
Leishman stain diluted with twice its volume of distilled water
buffered to

pH

6-5.

LEUCO PATENT BLUE

For the identification of haemoglobin.
Solutiofis required:

A. Patent blue AF54 (Michrome)

Distilled water
.

136

gm.

100 ml.

SECTION TWO
Dissolve; then add:

Zinc metal powder

lo gm.

Glacial acetic acid

2 ml.

Boil until the blue colour completely disappears.

i
gm. of decolorizing

Allow to cool shake with about

;

The

carbon; then filter.

be quite colourless

liquid which should then

stored in a well stoppered

is

bottle.

Solution

B.

Glacial acetic acid

10 ml.

2 ml.

Hydrogen peroxide 3%
N.B. : This solution must

ml.

be freshly prepared

and

filtered before use.

C. Safranin

o-i% aqueous

Glacial acetic acid

99 ml.

ml.

Technique:

Fix tissue blocks, not more than 3 to 5 mm. in thickness in

10% formalin buffered to pH 7-0 for 24 to 28 hours (prolonged
fixation should be avoided).
1.

2.

Embed

in paraffin

wax

as usual

and cut section

5 to 6/x in

thickness.
3.

Take

down to water as
solution B for three to

sections

usual.

minutes.

4.

Stain in

5.

Wash

6.

Counterstain for thirty to sixty sections in the safranin solu-

five

briefly in water.

tion.
7.

Rinse briefly with water.

8.

Dehydrate as usual.

9.

Clear in xylol.

10.

Mount

in Clarite or other synthetic

D.P.X., Clearmount,

mountant such

as

etc.

Results:

Haemoglobin, dark blue-green background light pink.

Note: Blood and tissue smears fixed with methyl alcohol may
also be stained by applying the stains as prescribed above.
;

Reference: Adapted from

Dunn, R. C.

(1946), Stain. Tech., 21, 65.

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

LEVADITI'S STAIN
For Treponema pallidum in sections
Solutions required:

A. Silver nitrate
B.

2-5% aqueous

Reducing solution:
Pyrogallic acid

Formalin

Distilled water

3 grn.
5 ml.

100 ml.

Technique:

Tissues about
in

10%

mm.

formalin and

thick should be fixed for twenty-four hours

in paraffin wax after staining.

embedded

After rinsing tissues in tap water,

for twenty-four hours.
2.

Immerse

of the

immerse

95%

alcohol

in distilled water until the tissue sinks to the

bottom

1.

in

jar.

Transfer to Solution

for 3 to 6 days at 37 C. in the dark,

the
solution
every twenty-four hours.
changing
3.

Wash

in distilled water; then immerse in Solution B for

twenty-four to seventy-two hours in the darkroom at room tem4.

perature.
5.

Wash

in distilled water; then dehydrate with

80%, 95%,

and absolute, alcohol.

6.

Clear in cedarwood

7.

Sections are cut

oil

and embed

in paraffin wax.

and mounted

5/^ in thickness

after

removal

of the paraffin wax.

Results:

Treponema,

Tissue, yellow to brown.

jet black.

LIGHT GREEN

ACID FUCHSIN (Alzheimer)

For demonstrating neuroglia changes

Solutions required:

A. Osmic acid

Chromic

2% aqueous
1% aqueous

acid

Glacial acetic acid

138

20 ml.
75 ml.
0-5 ml.

SECTION TWO
Acid fuchsin 25% aqueous.
C. Picric acid, saturated, alcoholic
Distilled water
B.

D. Light green

10%

15 ml.

30 ml.

aqueous.

Technique:
1.

Fix thin

slices of

the material in

formalin for twenty-

10%

four hours to three days.

2.

Wash

3.

Immerse very thin

volume of Solution

for twenty-four hours in running water.

large
if it blackens.

slices of the material in a

comparatively

A which should be changed once or twice

4.

Wash

5.

Pass through ascending grades of alcohol.

6.

Clear in the usual manner and

for several hours in running water.

7. Sections not

more than

embed

in paraffin

wax.

2 to 4/^ in thickness are fixed to

slides.
8.

De-wax with

xylol.

Rinse thoroughly with absolute alcohol and pass through the

usual descending grades of alcohol down to distilled water.
9.

10.

Stain for an hour at 60 C. with the acid fuchsin solution.

11.

Allow the preparation

to cool to

room temperature; then

wash with water.

12.

Immerse

in Solution

(picric acid)

from one second

to

two

minutes.
13.

Rinse in two changes of water.

14.

Stain from one

half to one hour in the

Light Green

solution.
15.

Rinse quickly in absolute alcohol.

16.

Rinse in xylol.

17.

Mount

in

Canada balsam or

Cristalite.

Results:

Migrating astrocytes, of varying shades of green and sometimes

containing fuchsinophile granules of brown stained lipoid inclusions. Lipoid contents of perivascular phagocytes are brown to
139

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

Neuroglia fibres and erythrocytes, red. Medullary sheaths
Connective tissue, deep green. Nerve cells are
with
red
pale green
stippling, while nerve-cell nuclei are a darker
with
red
nuclei.
green
bright
black.

are unstained.

Notes:
(a)

The

material

must be

and only small pieces should be

fresh

employed.
{b)

Sections stained by this technique should appear

lilac in

colour to the naked eye.

advantageous to experiment in order to determine the

optimum staining time in the picric acid and the light green, as
results vary according to the material to be stained.
{c)

It is

From

Pathological Technique by F. B. Mallory, by courtesy of Messrs.

W.

B.

Saunders Co., Philadelphia, U.S.A

LIGNIN PINK
For whole mounts of marine invertebrates, particularly for
crustaceans limbs, ostracod appendages, Medusa of Obelia,
etc., as well as for demonstrating chitin
Overstaining with lignin pink
out with alcohol.

is

impossible, and

it

will not

wash

Solutions required:

A. Sea water Bouin:

Sea water saturated with picric acid

Formaldehyde

40%

Glacial acetic acid

Lignin pink saturated in

B.

distilled

75 ml.
25 ml.
5

^'

water or

in Benzyl alcohol.

Technique:
1. Specimens are fixed from eighteen to forty-eight hours,
according to the material, in Solution A.
2. Wash out the fixative with 50% alcohol, followed by 70%

alcohol until the yellow coloration, due to the picric acid,

pletely extracted.
3.

Wash

4.

Immerse

in running water to

in solution

remove the

alcohol.

for fifteen minutes or longer.

140

is

com-

SECTION TWO
Results:

With the aqueous

solution of the stain

Medusa

of Obelia and limbs of crustaceans are stained deep

carmine colour. The finest structures of ostracod appendages,

uniform pink, but a better effect can, however, be obtained by

staining the specimen for a longer period (up to sixteen hours)
with a solution of the dye in benzyl alcohol the final result in this
:

a definite purple for the exoskeleton, while the other tissues

are carmine colour.

case

is

Reference: Cannon, H. G. (1941),^. R. Mic. Soc, series III, 61, parts 3 and

LITHIUM SILVER

4.

(Laidlaw)

For staining skin and tumours

A. Iodine

1%

Solutions required:
in absolute alcohol.

B. Sodium thiosulphate 5% aqueous.

C. Potassium permanganate 0-5% aqueous.
D. Oxalic acid 5% aqueous.

E.

Lithium

silver:

Dissolve 12 gm. silver nitrate in 20 ml. distilled

water in a 500 ml. stoppered bottle then add 230 ml.

lithium carbonate, saturated, aqueous, and shake well.
;

Transfer to a 250 ml. measuring cylinder; cover with

a watch glass and allow to stand undisturbed until
the precipitate formed measures about 70 ml. Pour
off the clear liquid and transfer the precipitate to
another vessel. Wash precipitate with three or four

changes of distilled water, decanting after each

washing so that the precipitate remaining measures
70 ml. Add a diluted ammonia solution (15 ml.
strong ammonia solution, sp. gr. 0-880 diluted with
35 ml. distilled water) a little at a time until the fluid
precipitate

is

almost

clear.

Filter

No. 40 filter paper.

F. Formalin 1% in tap water.
G. Gold chloride (yellow) 0-5%
Technique:
1. Fix tissues in
2.

Dehydrate,

10%

clear,

through a Whatman

in distilled water.

formalin for three days.

embed

in paraffin

141

wax

in the usual

manner.

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

Fix sections to

3.

slides,

de-wax and take down

to water as

usual.
4.

Wash

5.

Immerse

6.

Pour

in

running water for

five

minutes.

in the iodine solution for three minutes.

off excess iodine

and immerse

in the

sodium

thio-

sulphate solution for three minutes.

Rinse in tap water

7.

then immerse in the potassium perman-

ganate solution for three minutes.

8.

Rinse in tap water.

9.

Immerse

in the oxalic acid solution for five minutes.

10.

Wash

1 1

Immerse

in

running tap water for ten minutes.

in three changes of distilled water for three or four

minutes in each.
12.

Stain in an oven for five minutes with the lithium silver

solution heated to 50 C.
13.
all

all

Rinse the slide back and front with distilled water to remove

traces of excess lithium silver.

14.

Immerse

15.

Rinse both sides of the slide with

slide in ajar of

1%

formalin.
distilled

water to remove

traces of the formalin solution.

16.

Immerse

in the yellow gold chloride solution in a coplin

staining jar for ten minutes.

17.
all

Rinse both sides of the slide with

distilled

water to remove

traces of excess gold chloride.

18.

Flood the

slides

with oxalic acid and allow this reagent to

act for ten minutes.

19.

Rinse in distilled water.

20. Flood the sections with the sodium thiosulphate solution

changing the solution every time it becomes turbid over a period

of ten minutes.
21.

Wash

well in running water; then drain.

22. Dehydrate
mount.

in ascending grades of alcohol, clear in xylol

142

and

SECTION TWO
Results:

Collagen is stained a reddish purple, while reticulum appears as

black threads.

LORRAIN

SMITH

DIETRICH STAIN

For lipoids
Solutions required:

A. Potass, dichromate

5%

aqueous.

Haematoxylin (Kultschitzky)
B.

10%

Haematoxylin

absolute

in

months

alcohol (ripened three

or longer)
.

Acetic acid

2%

2%

aqueous

C. Potass, ferricyanide

Borax

aqueous

..
.

..
.

10 ml.

90 ml.
2-5

gm.

100 ml.

Technique:

Material

fixed in

is

formalin and frozen sections are

10%

em-

ployed.

Mordant

1.

tion

A at

2.

wash
3.

sections twenty-four to forty-eight hours in Soluthen wash thoroughly in distilled water.

37 C.

Immerse

in Solution

at 37 C. for four to five

hours; then

Differentiate overnight in Solution C.

Wash

thoroughly in
Aquamount or in Farrant.
4.

in distilled water.

distilled water;

drain and

mount

Results:

Lipoid substances, blue-black.

LUGOL'S IODINE
For the identification of glycogen in tissues
Solution required:

Lugol's iodine.
143

in

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

Technique:
1

Thin

slices of tissue are fixed in absolute alcohol

hydrated;

cleared,

and embedded

in paraffin

wax

then de-

in the usual

manner.
2.

Float sections on slides with

70%

alcohol and flatten

warming gently, on a warm surface (but not with

3.

Remove

excess

70%

alcohol

by

a direct flame).

by blotting very

carefully but

thoroughly.

then with absolute alcohol.

4.

Treat with xylol

5.

Stain in Lugol's iodine solution for ten minutes; then pour

off"

excess stain and carefully blot the preparation thoroughly dry.

6. Clear and differentiate with origanum

examination under the microscope.

7.

Mount

in

oil,

controlling

by

origanum balsam.

Results:

Glycogen, reddish brown; tissue constituents, pale yellow.

LUXOL FAST BLUE

(Kliiver

CRESYL FAST VIOLET

and Barrera's

Stain)

For the combined staining of cells and fibres in the nervous

system, obviating the need for chromate treatment and
haematoxylin

SECTION TWO
Technique:

Material should be fixed in

io%

formalin.

Paraffin or frozen sections give somewhat better results than

Celloidin. Affixed Celloidin give better results than loose Celloidin sections.

(a)

Frozen sections
1.

Cut

sections

in thickness

25^11

and place them

in distilled

water.
2.

Immerse

in

70%

alcohol for ten to fifteen minutes.

3. Stain from five to twenty-four, but preferably not less than

sixteen hours, in the Luxol fast blue solution, in a stoppered jar
in an oven at 40 C.

Note: For staining four sections of the brain stem of a monkey, for
example, 20 to 25 ml. of the stain should be used and then discarded.
4.

Immerse

5.

Wash

in

95%

alcohol

and wash

off the excess stain.

in distilled water.

Immerse for two or three seconds, but no longer, in the

lithium carbonate solution, as the first stage of differentiation.
6.

7.

Continue the differentiation in several changes of

70%

alcohol until the grey and white matter can be distinguished, but
taking care not to over- differentiate.
8.

Wash

9.

Immerse

in distilled water.
in the lithium carbonate solution for three to five

seconds, but no longer.

10.

Complete the

differentiation

by immersing

in

several

changes of 70% alcohol, until the white matter is stained greenishblue in sharp contrast with the colourless grey matter.
11.
12.

Wash

thoroughly in distilled water.

Stain for one to two minutes in the cresyl

fast violet solution,

which should be warmed carefully and filtered before use.

13. Wash for two or three seconds in distilled water.
14.

Differentiate in several changes of

ceases to

95%

longer tinted.
15.

16.

alcohol until colour

come away from the preparation and the

Clear in xylol-terpineol (Solution D).

Clear in xylol and mount.

H5

alcohol

is

no

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

Paraffin sections

(b)

1.

Cut sections

2.

Remove

15 to

20jLt

in thickness

wax with

paraffin

xylol

and

fix to slides.

and pass through absolute

alcohol.
3.

Rinse with several changes of

4.

Stain with the Luxol fast blue solution for five to twenty-four

95%

alcohol.

hours, but preferably not less than sixteen hours, at 57 C. in an

oven, taking precautions to prevent the loss of alcohol through
evaporation from the staining solution.
5. Proceed exactly as at Stage 4 in the technique given above
for frozen sections, except at Stage 1 1 the cresyl violet should be
allowed to act for six minutes.

Celloidin sections (loose)

(c)

1.

Cut

sections 15 to 30/z in thickness

and place them

into

75%

alcohol.
2. Stain from five to twenty-four hours, but preferably not less
than sixteen, in the Luxol fast blue solution at 57 C. in an oven,

taking precautions to prevent the loss of alcohol by evaporation

from the staining solution.
2. Proceed exactly as at Stage 4 in the technique given for
frozen sections, except at Stage 1 1 the staining time for the cresyl
fast violet should be increased to three minutes.

(d) Celloidin sections (affixed)

1.

knife
2.

Cut
and

sections 15 to 30jLt in thickness, keeping the

tissue continually flooded with 75% alcohol.

microtome

Place sections on slides, which have previously been smeared

with glycerine albumen.

en the slides by rolling

3. If necessary flatten out the sections
with a piece of glass tubing half an inch in diameter and about two
inches in length, or a small glass phial will serve the purpose.
4.

the

Drop on

sufficient clove oil to cover the sections

oil to act for five

minutes.

5.

Remove

the clove

6.

Remove

the Celloidin with absolute alcohol.

7.

Wash

with

95%

oil

with

95%

alcohol.

146

alcohol.

and leave

SECTION TWO
8. Proceed exactly as at Stage 2 in the technique given for
frozen sections, except at Stage 2 the staining time for cresyl fast
violet should be increased to six minutes.

Results:

Myelinated fibres are sharply contrasted greenish-blue against

the reddish-coloured Nissl cells. The technique shows the Nissl
picture and differentiates between the three types of glia cells:

Myelin sheaths, neurons and

glia nuclei are well demonstrated.

obtained between the three layers of
medium-sized and larger blood cells, and capillary endothelium as

Differentiation

is

also

well as mesothelial lining of Arachnoid membrane are sharply

outlined. The finer fibres of the molecular layer of the cerebral

cortex can be most effectively demonstrated in paraffin sections.

Bacteria and pigments in nerve cells are more clearly demonstrated
with this technique than with the usual Nissl stains.
References

Kliiver, Heinrich and Barrera, Elizabeth (1953), J. of Neuropath and Exp.

"
Method for the combined staining of cells
Neurology, 12, no. 4, 400-3,
and fibres in the nervous system ".

and Barrera, Elizabeth (1954),

of Psychology, yj, 199-223,
the use of Azoporphin derivatives (Phthalocyanines) in the staining of

Kliiver, Heinrich

"

On

nervous tissue ".

Note: In the original paper it is stated that this cell-fibre stain

has been employed for peripheral nerves as well as structures of the
central nervous system in amphibians, birds and mammals (rat,
guinea-pig, rabbit, cat, monkey, chimpanzee, gorilla and man),
and that with suitable counterstains Luxol fast blue will give excellent preparations of cochlea, adrenals

and numerous extraneural

tissues.

MacCALLUM'S STAIN
For influenza bacilli and Gram-positive organisms in tissues

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

B.

Picric acid saturated aqueous.

C.

Stirling's gentian violet.

D. Gram's iodine.
E.

Equal volumes of xylol and

aniline

oil.

Technique:

Tissues should be fixed in Helly and embedded in paraffin wax.

1.

Stain for ten to thirty seconds in Solution

A; then wash

in

tap water.
2.

Differentiate for a

few seconds

colour changes to a clear deep pink

3.

formaUn

in

Counterstain one to five minutes in Solution

tion appears purplish yellow to the naked eye

water.
4.

till

Differentiate in

95%

the bright red

then wash with tap water.

until the sec-

then wash with tap

alcohol until the section appears red;

then wash in tap water.

5.

wash

Stain for about five minutes in Solution C; then

in tap

water.
6.

Stain for one minute in Solution

then, without washing,

blot dry.

no more colour comes

7.

Treat in Solution

8.

Pass through two changes of xylol; then mount.

until

out.

Results:

Gram-positive organisms, blue.

varying shades of red and purple.

MALLORY'S STAIN

Gram-negative, red.

HAEMATOXYLIN

For the differential staining of the pancreatic

islets

Solutions required:

A. Distilled water

Sulphuric acid cone.

Potassium Permanganate
148

Tissues,

100 ml.
i

ml.

gm.

SECTION TWO
Harris or Ehrlich haematoxylin.

B.

C. Lithium carbonate, saturated aqueous.

D. Acid Fuchsin (Mallory) o*5% aqueous.

Phosphomolybdic Aniline Blue

E.

Orange

(Mallory).

Technique:
1.

Mammalian

washed

in

80%

2.

Dehydrate,

3.

Cut

pancreases are fixed in Bouin and afterwards

alcohol.
clear,

and embed

in paraffin wax.

sections 4 to 5 jit in thickness.

4. Fix sections to slides: remove wax with xylol and pass

through the usual grades of alcohol down to distilled water.
5.

Immerse

in solution

A for about one half to one minute, until

the sections appear uniform reddish

6.

7.

8.

brown

in colour.

Rinse in distilled water.

Stain with Ehrlich or Harris Haematoxylin for five to ten
minutes.

Blue in lithium carbonate solution.

9. Wash well in tap water.

10. Wash with distilled water.

11.

Stain in Mallory's Acid Fuchsin, controlling under microcells are red, and the B cells are pink.

scope until the

If overstained,

wash out with

distilled Vv^ater until the

above

effects are obtained.

12.

Stain in solution

for twenty minutes to twelve hours

according to the condition of the pancreas

ferentiation in the first stain.

and degree of

dif-

Results:

Nuclei are stained dark violet; nucleoli, red. Cytoplasm in A

contains red granules. Cytoplasm of B cells contains blue

cells

Cytoplasm in acinar cells varies from red to pale violet

deep violet ergastoplasm. Canahcular cells, blue-grey.
Connective tissue blue. Erythrocytes red. Mucus, azure blue.

granules.

with

Reference: Isaac,
99-102.

J.

P.

and Aron, C. (1952),

149

Bull. Mies. Appl. ser. 2, 2,

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

MALLORY STAIN

HAEMATOXYLIN

For differential staining of acidophils,

chromophobes

in

mouse

basophils

and

pituitary

Solutiotis required:
-

A. Zenker

Formal :

Zenker's Fluid

Formaldehyde
B. Formic acid 10% aqueous.
.

C. Iodine

0-5% in 70%
D. Sodium thiosulphate

E.

Harris haematoxylin.

F.

Lithium carbonate

. .

95 ml.

5 ml.

alcohol.

075%

in

10%

alcohol.

satd. aqueous.

G. Acid fuchsin 0-5% aqueous.

H. Phosphomolybdic acid 1% aqueous.
I.

Mallory's Aniline blue-orange G.

J.

Carbol

xylol.

Technique:
1. Pituitary gland together with bone to which it is attached
for 4-8 hours, or overnight in a refrigerator.
fixed in solution

is

2.

Wash

3.

Decalcify

4.

Wash

in running tap water for 8 to 10 hours.

by immersing

in

10%

formic acid for 24 hours.

in running tap water for 2 to

4 hours.

Dehydrate, clear and embed in paraffin wax 56 to 58 C. in

the usual way.
5.

6.

Cut

sections about

4)Lt

in thickness

and mount on

slides

with

glycerine albumen.
7.

Dry

the slides thoroughly in an oven at 56 C. for one to two

room temperature.
Remove wax with xylol; then wash with two changes of

hours, or overnight at
8.

absolute alcohol.

150

SECTION TWO

Wash

9.

with

10.

Immerse

1 1

Wash

12.

90%

followed by

for half to

70%

two minutes

alcohol.

in solution

(Iodine).

well with water.

Immerse in solution

D (thiosulphate) for half to two minutes

or until the natural colour of the sections

is

restored.

13.

Wash

14.

Rinse in distilled water.

15.

Stain in Harris haematoxylin for 2-3 minutes.

16.

Rinse in tap water.

17.

Blue in the lithium carbonate solution for

18.

Rinse well in distilled water.

well with tap water.

19. Stain in the acid fuchsin solution for

minute.

1-2 minutes.

20. Rinse quickly in tap water.

21.

Immerse

in the

phosphomolybdic acid solution

for 2-5

minutes.
22.

Without washing, pass the slides directly into Mallory's

orange G and leave therein for 1-2 hours.

Aniline blue

alcohol, controlling by examination

23. Differentiate with
until the blue granules are intense, but the

95%

under the microscope

fuchsinophil granules are clearly visible.

24. Rinse with absolute alcohol.

25. Rinse with carbol xylol.

26.

Immerse

27.

Mount

in

in

two changes of xylol for 10 minutes in each.

Clearmount of D.P.X.

Results:

The

nuclei of

basophils, blue.

all cells

are stained dark blue black.

Granules of

Acidophil granules, brilliant red.

cytoplasm of chromophobes, light grey.

Bone, intense blue.

Non-granular
Erythrocytes, orange to

red.

Reference: Gude, William D. (1953), Stain Tech., 28, no.

151

3,

161-2.

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

MALLORY STAIN

HAEMATOXYLIN

(Ehrlich)

For Negri bodies in sections of brain

Solutions required:

A. Ehrlich haematoxyhn.

Orange G.

B.

Phosphotungstic

aqueous
C. Acid fuchsin

acid,

1%

0-5

gm.

100 ml.

0-5

0-5

100 ml.

Phosphotungstic acid
Acetic acid

saturated

D. Phosphotungstic acid
Phosphomolybdic acid

Picric acid, saturated, aqueous

gm.
gm.

gm.
gm.

70 ml.
30 ml.

Aniline blue, aqueous

. .

Distilled water

98 ml.

Glacial acetic acid

2 ml.

Absolute alcohol
E.

gm.

Technique:
1

tilled

Fix paraffin sections to slides

water in the usual way.

dewax and take down

to dis-

Stain in Ehrlich haematoxyhn for five minutes.

2.

Blue in tap water, or in lithium carbonate solution, for two

minutes.
3.

4.

Rinse in distilled water.

5.

Stain for one minute in the orange

6.

Wash

G solution.

in tap water until only the erythrocytes are stained

yellow.
7.

Rinse in

8.

Stain for ten minutes in the acid fuchsin solution.

9.

Rinse in distilled water.

distilled water.

10. Differentiate for five

1 1

minutes in solution D.

Rinse in distilled water.

152

SECTION TWO
12.

Rinse in

i%

acetic acid.

13. Dehydrate, clear

and mount.

Results:

Negri bodies, purplish red with blue granulations. Cytoplasm

of neurones, bluish. Nucleoli, dark purple. Erythrocytes, yellow.
Reference: Zlotnik,

I.

(1953), Nature, 172, no. 4386, 962-3.

MALLORY HEIDENHAIN STAIN

(Jane E. Cason's

modification)

rapid one-step

method

for connective tissue

Solutions required:

Phosphotungstic acid crystals A. R.

Orange G.

2 gm.

Aniline blue, water soluble

Acid fuchsin

Distilled water

gm.
gm.

3 gni.

200 ml.

Technique:
I

Fix pieces of tissue in Zenker-formol for preference, although

fluid, formalin and alcohol have been used with success.

Bouin's
a.

Embed

3.

Fix sections to slides and remove wax with xylol.

in paraffin

wax and

cut sections

6//

in thickness.

4. Pass through descending grades of alcohol and if Zenkerformol has been used as the fixative, treat with iodine and sodium

thiosulphate as usual to remove mercurial precipitate.

5.

Take down

6.

Immerse

7.

Wash

8.
9.

to tap water.

for five minutes in the staining solution.

in running tap water for three to five minutes.

Dehydrate rapidly through the usual graded alcohols.

Clear in xylol and mount.
153

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

Results:

Appear

to be the

collageneous

same

as those listed

intense blue.

fibrils,

and bone, mucus, amyloid, and

by Mallory (1938), i.e.

Ground-substance of cartilage

certain other hyaline substances

are stained in varying shades of blue.

Nuclei, fibroglia, myoglia

cylinders and fibrin are stained
red. Erythrocytes and myelin, yellow. Elastic fibrils are stained
pale pink or yellow.

and neuroglia

fibrils, nucleoli, axis

Cason, Jane E., Stain Technology (1950), 25, No.

Abstract:

4,

225-6.

MALLORY'S PHOSPHOTUNGSTIC ACID HAEMATOXYLIN

A general

stain for vertebrate tissues

Solutions required:

Haematoxylin

10%

alcohol

(ripened
months or longer)

Note:

available,

If ripened

the

three

for

Phosphotungstic acid
Distilled water
.

absolute

in

ml.

gm.

100 ml.

haematoxylin solution

following

artificially

ripened

not

is

stain

should be used:

Haematoxylin (dry) o-i gm., phosphotungstic acid

2 gm., distilled water 100 ml., potassium permanganate 1% aqueous 1-77 ml.
Technique:
1

Fix in Zenker.

Embed in paraffin wax.

down to distilled water.

2.

Bring sections

3.

Treat with iodine to remove mercuric percipitate.

4.

Remove

5.

Wash

6.

iodine with

0-5% aqueous sodium

Immerse for five to ten minutes

then wash in tap water.

ganate

hyposulphite.

thoroughly in running water.

in

0-25%

potass,

perman-

7. Immerse for ten to twenty minutes

wash thoroughly with tap water.

154

in

5%

oxalic acid;

then

SECTION TWO
Stain twelve to twenty-four hours in haematoxylin solution,
prepared as above.
8.

Wash

9.

in tap

dehydrate with

water;

95%

and absolute

alcohol.

Clear in xylol and mount.

10.

Results:

Nuclei,

centrioles,

achromatic

fibroglia, myoglia,
elements of striated muscle,
blue. Collagen, reticulum, ground substances of cartilage and bone,
yellowish to brownish red. Coarse elastic fibrils, faint purple.

neuroglia

fibrils,

MARSHALL RED
A

spindles,

fibrin, contractile

VICTORIA GREEN

general stain, particularly suitable for class work

Solutions required:

A. Marshall red, saturated aqueous.

B.

Victoria

green

saturated

in

70%

alcohol.

Technique:
1.

Fix sections to slides; dewax and take

down

to distilled

water in the usual manner.

2.

Stain in the Marshall red solution for twenty minutes.

3.

Rinse in distilled water.

4.

Stain in the Victoria green solution for half an hour,

5.

Rinse in

6.

Dehydrate with absolute alcohol.

7.

Clear in xylol and mount.

70%

alcohol followed

by

90%

alcohol.

Results:

Myofibrils, sage green. Nuclei, bright carmine. The results

vary somewhat, but the muscle fibres always appear greenish to
greenish -grey, while the nuclei are red. White matter of spinal

Retina stands out well

cord, yellowish-green. Cartilage, pink.
as the rods and cones appear
bright bluish-green. Erythrocytes
unstained.
Reference: Cannon, H. G. (1941), J. Roy. Micro. Soc.y series III, 61, parts
3

and

4.

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

MASSON'S TRICHROME STAIN

For connective tissues
Solutions required:

A. Iron alum
B.

5%

aqueous.

Regaud's haematoxylin solution.

C. Picric

acid,

alcohol

Alcohol

saturated

95%

in

95%

. .

20 ml.

10 ml.

D. Ponceau fuchsin.
E.

Phosphomolybdic acid

F.

Aniline Blue

5%

in

1%

2%

aqueous.

acetic acid.

Technique:

Fix pieces of tissue in Bouin's fluid for three days or in

Regaud's fluid for one day.
1.

Wash

2.

parafiin

in running water;
as usual.

Sections

3.

dehydrate;

clear

and embed

in

wax

5/1

in thickness are fixed to slides;

passed through descending grades of alcohol

water in the usual manner.
4.

Mordant

5.

Wash

in Solution

de-waxed and

down

A for five minutes at 45

to distilled

C. to 50 C.

well in distilled water.

Stain for five minutes in Regaud's haematoxylin at 45 C.

6.

to 50 C.

Rinse in

7.

distilled water.

C above) controlling
examination
under
while
the preparation is
the
by
microscope,
DiflFerentiate in picric alcohol (Solution

8.

still

wet.

9.

Wash

in

running tap water for a minute or

so.

10.

Stain for five minutes in the Ponceau fuchsin solution.

11.

Rinse in

12.

Diff"erentiate in the

distilled water.

phosphomolybdic acid solution

minutes.

156

for five

SECTION TWO
13. Add 0-5 ml. of the acetic aniline blue (Solution F above) to
the phosphomolybdic acid on the slide and mix by rocking the
slide gently. Allow this mixture to act for five minutes.
14.

Pour

15.

Immerse

off excess liquid

and rinse

in distilled water.

phosphomolybdic acid solution again,

in

for five

minutes.
16.

Transfer to

17.

Wash

18.

% acetic acid and leave therein for five minutes.

in distilled water.

Dehydrate in 95% alcohol, followed by absolute alcohol;

mount.

clear in xylol

Results:

Collagen, deep blue. Neuroglia

Argentaffin granules, black or red.

METHYL BLUE

fibrils,

red.

Nuclei, black.

EOSIN (Mann)

For demonstrating the various types of cells in the anterior

lobe of the pituitary and for the study of the relationship
and development of the blood vessels
Solution required:

Methyl Blue-Eosin (Mann's

stain).

Technique:
1

Paraffin sections of tissues which have

been fixed in a fluid con-

taining mercuric chloride are mounted on slides and treated by the

standard technique for the removal of mercuric precipitate (see

page 28).
Bring the sections down to distilled water; then stain for a
quarter of an hour to two hours (the longer time is required if it is
2.

desired to demonstrate anterior lobe of pituitary).

3.

4.
5.

Wash and

differentiate in tap water.

Dehydrate rapidly with two changes of absolute alcohol.

Clear in xylol mount in xylol balsam and examine under the
;

low power, as the stain

is

too diffuse for critical high power work.

157

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

Results:

Nuclei are stained blue; karyosomes, dark blue; plasmosomes,

red; basophil cytoplasm, blue; oxyphil cytoplasm and oxyphil
granules, red.

METHYL GREEN
For amyloid
Solution required:

Methyl Green

i%

aqueous.

Technique:
1.

Fix tissues in absolute alcohol or in

io%

formalin and cut

frozen sections.
2.
3.

4.

Immerse

in the methyl green solution for five to ten hours.

Wash well in distilled water.

Mount in neutral glycerine, or

in

aquamount.

Results:

Amyloid

stained reddish violet, while other tissue elements

is

are green.

METHYL GREEN

PYRONm

(Pappenheim-Unna)

For plasma cells

Solution required:
Methyl Green Pyronin (Pappenheim-Unna).

Technique:
1.

Paraffin sections of absolute alcohol-fixed tissues are

on slides and brought down

method.
2.

five to thirty
3.

4.
5.

Methyl Green

Stain in

to distilled water

mounted

by the standard

Pyronin (Pappenheim-Unna) from

minutes.

Rinse in

distilled water;

drain and blot carefully.

Dehydrate rapidly in two changes of absolute alcohol.

Clear in xylol; and mount.
158

SECTION TWO
Results:

Plasmosomes and oxyphil constituents of cytoplasm are stained

red; chromatin reticulum and karyosomes, bluish green; cytoplasm of plasma

cells, brilliant

red

ground-substance of hyaline

cartilage, yellowish red; bacteria, vivid red.

METHYL VIOLET

6B

For amyloid
Solution required:

Methyl Violet 6B aqueous i%.

Technique:
1.

Material should be fixed in alcohol and

wax (although frozen

sections

may be

embedded

in paraffin

employed).

2. Paraffin sections are fixed to slides

and brought down

to dis-

tilled water.
3.

Stain for two to five minutes in the methyl violet

4.

Rinse quickly in

5.

Differentiate with

6B

solution.

distilled water.

under the microscope,

1%

acetic acid, controlling

until

by examination

the amyloid appears reddish in

colour.
6.
7.

Wash in distilled water.

Mount in glycerine jelly.

Results:

Amyloid substance

is

stained red, while cells and nuclei are in

varying shades of blue.

Notes:
(a)

gone
{h)

The

technique

is

not suitable for material which has under-

prolonged fixation in formalin.

The

stain

is

extracted from the amyloid by alcohol and for

mountant, thereby obvi-

this reason glycerine jelly is used as the

ating the use of alcohol for dehydration.

159

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

METHYL VIOLET

METANIL YELLOW

For typhus fever rickettsiae in lungs of mice

Solutions required:

A. Methyl violet loB

Distilled water

B.

Acetic acid

C. Metanil yellow

o-oi% aqueous.

90 ml.

..

..

..

10 ml.

o-oi% aqueous.

Technique :
1.

Fix pieces of lung in

10%

neutral formalin.

2. Dehydrate, clear and embed in paraffin wax in the usual

manner.
3.

down

Fix sections to slides and take

to distilled water as

usual.
4.

Stain in the methyl violet solution for half to one hour.

in solution B, controlling by examination under

5. Differentiate
the microscope until the cytoplasm is decolorized.
6.

7.
8.

Counterstain in the metanil yellow solution for a few seconds.

Dehydrate in acetone.
Mount in D.P.X. or Clearmount.

Results:

Rickettsiae are stained violet.

Reference: Nyka,

W.

J.

METHYL VIOLET

(1945),

J^.

Path, and Bad., 52, 317-24.

PYRONIN

ORANGE G

(Bonney's

Triple Stain)

For chromatin, connective

tissue, keratin

Solutions required:

A. Methyl violet 6B (Jensen)

aqueous
Pyronin 10% aqueous
Distilled water
.

160

1%

25 ml.
10 ml.
65 ml.

SECTION TWO
B.

Acetone

G aqueous 2%
Add orange G solution

Orange

loo ml.

About lo ml.

drop by drop to the ace-

tone, with shaking, until the flocculent precipitate

formed just redissolves with further addition of

orange

solution.

Technique:
1. Small pieces of tissue are fixed in acetic-alcohol or in
curie chloride.

embed

mer-

in paraffin wax.

2.

Wash; dehydrate;

3.

If mercuric chloride has been used for fixation treat sections

clear;

for the removal of mercuric precipitate

4.

Take

5.

Immerse

(Solution

sections

for

down

by the standard method.

to distilled water.

two minutes

in the

methyl

and carefully wipe the

Pour

7.

Flood the preparation with acetone-orange

8.

Pour

off excess stain

off after a

slide dry.

G solution.

few seconds.

Flood the preparation with a fresh

solution and pour off after a few seconds.
9.

10.

Wash

1 1

Rinse with two

12.

pyronin

above).

6.

violet

lot

of acetone-orange

quickly in pure acetone.

Mount

lots of xylol.

in balsam.

Results:

Cytoplasm, red; chromatin, violet; keratin, violet; connective

tissue, yellow.

METHYLENE BLUE

BASIC FUCHSIN

Rapid method of demonstrating. Negri bodies in sections

Solutions re

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

B.

Potass, hydroxide
Distilled water

N.20

C. Solution

Solution

Note: Solution

6-25 ml.
93-75 ml.

10 ml.
0-25 ml.

should be freshly prepared as

required.

Technique:

Blocks not exceeding 3

1.

mm.

in thickness of fresh tissue

from

the hippocampus major and cerebellum should be fixed in Zenker

for twelve to twenty-four hours.

Remove

2.

usual

method

mercurial precipitate with iodine in alcohol by the

page 28) then wash in running water for three

(see

to six hours.

Dehydrate in dioxane

3.

(see

page 36) and embed in paraffin

wax.
Sections not

4.

more than

4/x

thick are

mounted on

slides

brought down to distilled water.

5. Flood slides with Solution C and steam gently for
utes then cool and wash rapidly in tap water.

five

and

min-

6. Decolorize and differentiate each section separately by

waving the slide gently in a jar of 90% alcohol until the section
assumes a faint violet colour.

Dehydrate rapidly in
xylol and mount.
7.

95%

and absolute alcohol;

clear in

Results:

granular inclusions, dark blue nuclecytoplasm, bluish violet; erythrocytes, dull

Negri bodies, deep red

bluish black;
reddish brown.

oli,

METHYLENE BLUE

BASIC FUCHSIN

For rickettsia in sections

Solutions required:

A. Methylene blue
B.

Basic fuchsin

C.

Citric acid

1%

aqueous.

0-5% aqueous.

0*5% aqueous.
162

SECTION TWO
Technique:

Tissues are fixed in Regaud's

1.

fluid,

dehydrated, cleared and embedded

in running water,
as usual.

wax

Fix sections to slides; de-wax; pass through the alcohol

to distilled water in the usual manner.

2.

down
3.

washed

in paraffin

Stain in the methylene blue solution for twelve to sixteen

hours.
4.

Decolorize with

5.

Counterstain with the basic fuchsin solution for fifteen min-

95%

alcohol.

utes.
6.

Decolorize for one to three seconds in the

citric acid solu-

tion.
7.

Pour

off excess citric acid

and

rinse in distilled water; drain

blot carefully.
8.

Decolorize and dehydrate rapidly in absolute alcohol.

9.

Clear in xylol and

mount

in

dammar

xylol.

Results:

Rickettsia are stained a deep red;

background, light blue.

surrounding

METHYLENE BLUE POLYCHROME

For mast

tissue, pink;

(Unna)

cells in sections

Solutions required:
blue,
Methylene
(Unna)
Potash alum

polychrome

100 ml.
5

gm.

Technique:
1.

Material

is

fixed

in

absolute alcohol and

embedded

in

Celloidin.
2.

Stain sections in a watch glass from three to sixteen hours in

the methylene blue solution.

3.

Rinse well in

distilled water.

163

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

4.

Dehydrate with

5.

Clear in origanum

6.

Mount

in

95%

alcohol.

oil.

Canada balsm or

in Cristalite.

Results:

Mast

cell

granules are stained red

nuclei, blue.

METHYLENE BLUE POLYCHROME

ETHER (Unna)

GLYCERINE

For differentiating mast cells and plasma cells

Solutions required:

A. Formalin

(40% formaldehyde)
Absolute alcohol
.

B.

C.

50 ml.
100 ml.

Polychrome methylene blue (Unna)

Glycerine ether (Unna)
Distilled water

5 nil-

35 ml.

Technique:

Fix tissues in Solution A;

paraffin wax.
1.

2.

Immerse

clear;

dehydrate;

embed

in

sections in the methylene blue solution for ten

minutes.
3.

Rinse in

distilled water.

4. Differentiate in the glycerine ether mixture from one half to

one minute until the sections appear to be deep sky blue to the
naked eye (care should be taken that the sections are not over-

differentiated).
5.

Wash thoroughly in

6.

Fix sections to slides and carefully blot with

7.

Dehydrate rapidly with absolute alcohol.

8.

Clear in xylol and mount.

distilled

water for a few minutes.

filter

paper.

Results:

Nuclei are stained blue, while mast

blue.

164

cells are

red and plasma are

SECTION TWO

MUCICARMINE - METANIL YELLOW HAEMATOXYLIN

For mucin and connective tissue
Solutions required:

A. Haematoxylin (Weigert) A.
B.

Haematoxylin (Weigert) B.

C. Metanil yellow

0-25% aqueous.

D. Mucicarmine (Southgate).
Technique:
1.

Fix material in

2.

Dehydrate, clear; embed in paraffin wax.

3.

Fix sections to slides; de-wax with xylol and pass through

10%

formalin.

the usual descending grades of alcohol.

Rinse with

4.

distilled water.

Stain sections for one minute in a freshly prepared mixture

and B.
consisting of equal volumes of Solution
5.

6.

Wash

7.

Immerse

8.

Rinse quickly with distilled water.

9.

Immerse

in distilled water.
in Solution

in the

for about

two minutes.

mucicarmine solution for

forty-five minutes.

10.

Rinse quickly with

distilled water.

11.

Rinse quickly with

95%

12.

Dehydrate rapidly but thoroughly with absolute alcohol.

13.

Clear in xylol and mount.

alcohol.

Results:

Mucin

stained red, while connective tissue

nuclei are black.
is

6s

is

yellow, and

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

MUCICARMINE

(Mayer)

Solutions required:

Mucicarmine, stock solution

Alcohol 70%
.

volume

10 volumes

This diluted solution should be freshly

Note:
prepared.
Technique:

Tissues should be fixed in absolute alcohol for five to eight

hours and embedded in paraffin wax, Celloidin or L.V.N.
1. Bring paraffin sections down to distilled water;
for ten to twenty-five minutes in the above solution.

2.

Wash

3.

Dehydrate with 70%,

then stain

rapidly with distilled water.

95% and

absolute alcohol.

4. Paraffin sections are cleared in xylol;

sections are cleared in terpineol or

origanum

Celloidin or L.V.N.
oil.

Results:

Mucin

is

stained red.

MUCICARMINE
This

(Southgate)

used in accordance with the Mayer technique, but i volis diluted with 9 volumes of distilled
water instead of 10 volumes 70% alcohol.
Southgate's modification gives more uniform results than Mucicarmine prepared by Mayer's original formula.

ume

is

of the stock solution

MUCIHAEMATEIN
For mucus

SECTION TWO
Technique:
1.

Material

fixed in absolute alcohol; cleared;

is

and embedded

in paraffin wax.

of

2.

Sections are stained for ten minutes in the above solution.

3.

Wash

4.

Dehydrate by plunging the

95%
5.

with

distilled water.

slide into

two or three changes

alcohol.

Pass through absolute alcohol

then clear in xylol

mount.

Results:

Mucus

is

stained blue, while the remainder

is

colourless.

NADI REACTION
For oxidase granules
Solutions required:

A. a-Naphthol
Distilled water

gm.

. .

. .

100 ml.

Place in a 250-ml. flask and boil until the a-Naphthol begins to melt; then add 40% potassium hydroxide aqueous solution drop by drop until the
solution becomes yellowish-blue in colour and the
is still

a-Naphthol
B.

not completely dissolved.

Cold tap water

*D imethy -p -pheny lenediamine
.

100 ml.

base

.
0-5 gm.
Place the water in a clean amber bottle, open the
.

groove at one end then breaking

manner. Tip the contents of the ampoule into the bottle; then replace the stopper and
allow the bottle to stand, with occasional shaking,
for twenty-four hours. Care should be taken that the

ampoule by

filing a

in the usual

Dimethyl-p-phenylenediamine base does not come

into contact with the body.
This solution deteriorates

after

three

or four

weeks.

*Note:

This should be purchased in a sealed

ampoule.

167

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

C. Gram's iodine solution.

D. Carmalum.
Technique:
1. Thin pieces of fresh tissue (not more than 3 mm. thick) are
fixed for three to five hours in formalin-saUne solution.

Make

2.

frozen sections and collect

them

in distilled water.

Immerse

in a mixture consisting of equal volumes of Soluand B (Note: This mixture must be made and filtered
immediately before use) for about five minutes until the sections
3.

tions

turn blue.
4.

Rinse sections rapidly in

5.

Immerse

in

distilled water.

Gram's iodine solution

until the sections turn

brown.
6. Transfer to distilled water to which two drops of lithium carbonate 0*5% aqueous have been added for each 100 ml. of distilled
water, for a quarter to twenty-four hours until the sections have
regained their blue colour.

7.

two
8.

Wash

in distilled water then counterstain in

carmalum

for

to five minutes.

Mount

in glycerine jelly or Apathy's

medium

or in Aqua-

mount.
Results:

Oxidase granules are stained blue while nuclei are pink. Sometimes fat

is

stained also.

NAPHTHOL BLUE BLACK - HAEMATOXYLIN - BRILLIANT

PURPURIN AZOFUCHSIN
For collagen, reticulum, smooth muscle,

etc.

Solutions required:

A. Weigert's Haematoxylin A.
Weigert's Haematoxylin B.
C. Brilliant purpurin R. (C.I. No. 454)
in 1% acetic acid

B.

Azofuchsin

1%

in

1%

acetic acid.

168

30 ml.
20 ml.

SECTION TWO
D. Naphthol blue black (C.I. No. 246) i gm.
Picric acid, satd., aqueous
100 ml.
.

Technique:
1.

Paraffin sections are fixed to slides

and taken down

to

70%

alcohol in the usual manner.

Stain for six minutes in a freshly prepared mixture consisting

and B.

2.

of equal parts of solutions of

3.

Wash

4.

Counterstain for five minutes in solution C.

5.

Wash

6.

Stain in solution

7.

Rinse in

8.

Pass through the usual ascending grades of alcohol dehydrate

in tap water.

in

1%
1%

acetic acid.

for five minutes.

acetic acid for

two minutes.

in absolute.
9.

10.

Clear in Xylol.

Mount

in

D.P.X. or Clearmount.

Results:

reticulum and basement membranes, dark green.

Collagen,

Smooth muscle, brown.

Reference:

Lillie,

Nuclei, brownish black.

R. D. (1945), J. Tech. Meth., 25, 47.

NAPHTHOL GREEN

HAEMATOXYLE^

For connective tissue

Solutions required:

A. Weigert's haematoxylin, A.
B.

Weigert's haematoxylin, B.

C. Eosin, yellowish,

D. Ferric

1%

chloride, hydrated

E. Naphthol Green B,
F.

in tap water.

1%

10%.

aqueous.

Equal volumes of acetone and

169

xylol.

(Weigert)

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

Technique:
1.

Paraffin sections are

mounted on the

2.

slide

and brought down

manner.

to distilled water in the usual

Stain for six minutes in a freshly prepared mixture consisting

and B.

of equal volumes of Weigert's haematoxylin

Wash

3.
thoroughly in tap water; then stain for three minutes
in the eosin solution.

4. Wash in tap water

tion for five minutes.
5.

Rinse well in

then immerse in the ferric chloride solu-

distilled water;

then stain for

five

minutes in

the naphthol green solution.

6.

two or three minutes

Differentiate for

in

1%

acetic acid.

7. Drain well; then dehydrate with acetone, afterwards clearing in acetone-xylol (as above); then mount.

Results:

Connective

tissue, green;

Reference: Lillie, R. D. (1945),

muscle and cytoplasm, pink.

J^.

Tech. Meth., 25, 32.

NEUTRAL RED
For

staining

both

FAST GREEN
and

Gram-positive

Gram-negative

bacteria in sections
Solutions required:

A. Aniline crystal
B.

Gram's

violet.

iodine.

C. Absolute alcohol
Glacial acetic acid

D. Twort's

stain,

98 ml.

2 ml.

modified

(neutral

red-fast

green)

Technique:
1.

Fix tissue in

paraffin wax.
2.

5%

formal-saline, dehydrate, clear;

3^^ in thickness.

embed

Cut sections

Stain in aniline crystal violet for three to five minutes.

170

in

SECTION TWO
and

3.

Pour

4.

Flood with Gram's iodine and allow the stain to act for three

off excess

blot,

without washing.

minutes.

Destain with the acetic acid alcohol (Solution

5.

above)

no more colour comes away, and the sections assume a dirty

until

straw colour.
6.

Rinse quickly in

7.

Stain in neutral red-fast green diluted one part with three

distilled water.

parts of distilled water, for five minutes.

8.

Wash

9.

Decolorize with the acetic alcohol solution until no more

quickly with distilled water.

red stain comes out.

10.

Rinse quickly in absolute alcohol.

1 1

Clear in xylol and mount.

Results:

Nuclei are stained red, while cytoplasm is light green. Grambacteria, dark blue.
Gram-negative bacteria, pink.

positive

Erythrocytes, green.
J. Path.

& Bad.,

59, 357-8, Ollett,

W.

S. (1947).

NILE BLUE SULPHATE

For demonstrating

fatty acids

and neutral

fats

Solution required:

To 100 ml. saturated aqueous Nile Blue sulphate

add 0'5 ml. cone, sulphuric acid; then boil under a
reflux condenser for two hours
allow to cool then
;

use as follows

Technique:
1.

Fix small pieces of tissue in

2.

Frozen sections are stained for about ten minutes to half an

hour

at 37 C.

or overnight at

10%

formalin.

room temperature.

171

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

3.

Differentiate in

4.

Rinse in

2%

distilled

acetic acid,

water;

mount

in Farrant.

Results:

Free fatty acids, blue.

NILE BLUE

Neutral

fats, red.

PICRO FUCHSIN (Murray-Drew)

For bacteria and actinomyces in pathological tissues

Solutions required:

A. Picro fuchsin (Van Gieson).

Nile Blue sulphate aqueous

B.

1%.

Technique:
1.

Formalin-fixed material

frozen sections

embedded

is

in paraffin wax, or

may be employed.

2. Take sections down to distilled water in the usual manner

then stain in picro fuchsin for two or three minutes.
3.

Wash

4.

Stain in Nile Blue sulphate for four to twenty-four hours.

5.

Rinse in

with

distilled water.

distilled

water until the washings are tinted pale blue.

6. Drain off excess water; then blot the preparation carefully

but thoroughly.

7.
8.

Dehydrate rapidly in absolute alcohol; then

clear in xylol.

Differentiate in clove oil for five to fifteen minutes (for paraor for several hours in the case of thick frozen sections.

ffin sections)
9.

Rinse in two or three changes of xylol to remove clove

oil;

then mount.
Results:

Bacteria and chromatin are stained blue;

mast

cell granules,

blue-black;

fibrin,

erythrocytes and keratin, yellow.

172

collagen fibres, red;

blue or reddish orange;

SECTION TWO

ORANGE G

CRYSTAL VIOLET

(Bensley)

For secretion antecedents of serous or zymogenic

cells.

This stain is particularly suitable for sections of the stomach

or pancreas
Solutions required:

A. Osmic acid 2%
Potassium dichromate
.

B.

5%

Glacial acetic acid

Distilled water

4 ml.
4 ml.
i
drop
2 ml.

Neutral gentian

Orange

G 8%

Crystal violet

aqueous
4% aqueous
.

Mix thoroughly by

25 ml.
25 ml.

shaking until almost

complete

precipitation takes place then allow the preparation

to stand for an hour, afterwards collecting the preci;

pitate

on a

Wash

filter.

with about 250 ml.

solve

it

the precipitate on the

distilled water;

filter

then dry and dis-

in 25 ml. absolute alcohol.

C. Solution

Alcohol

(as

above)

20%

10 ml.

Sufficient to impart a
rich port-wine colour.

Allow to stand for twenty-four hours; then

i volume
D. Absolute alcohol
Clove oil
3 volumes
.

. .

filter.

Technique :
1.

Tissues are fixed in Solution

hours, and afterwards

embedded

(as above) for twenty-four

in paraffin wax in the usual man-

ner.
2.

Stain sections for twenty-four hours in Solution C (as above) ;

stain, and blot the preparation carefully.

then pour off excess

3.

Dehydrate by immersing

in

two or three changes of acetone.

Clear in xylol; then differentiate in Solution

controlling under the microscope.
4.

5.

(as above),

Rinse in two changes of xylol; then mount in balsam.

173

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

Results:

Zymogen granules, violet; granules of acidophil cells are

stained orange-red, while those of basophil cells are violet back;

ground, brown.

ORCEIN
For elastic

fibres

and connective

tissue

Solutions required:

A. Orcein

70%

alcohol

100 ml.

Heat on a water bath

then add

to dissolve;

filter;

ml.

well.

Unna's polychrome methylene blue.

10 or i
This should be diluted i

B.

cool;

Hydrochloric acid, cone.

Shake

gm.

I
:

may be employed);

Fix tissues (any fixative

clear; and embed.
1.

15 for use.

dehydrate;

Paraffin sections are brought down to 70% alcohol in the

flood with freshly filtered orcein solution, preas
and
warm gently for ten to fifteen minutes, until
above,
pared
2.

usual manner;

the solution thickens.

overnight

at

Alternatively, the sections

may be

stained

room temperature.

4.

Wash
Wash

5.

Stain in diluted polychrome methylene blue until the nuclei

3.

thoroughly with

70%

thoroughly with

distilled water.

alcohol.

are bright blue to blue-black; the time necessary may be ascertained by examining under the microscope at intervals.
alcohol, followed

6.

Differentiate in

7.

Clear in xylol and mount.

95%

by

absolute.

Results:
Elastic fibres,

Connective

deep brown. Nuclei, bright blue or blue to black.

brown.

tissue, pale

174

SECTION TWO
desired only to stain the elastic fibres, omit No. 5,
and treat sections with acid alcohol for a few seconds before

Note: If

it is

washing with

distilled water.

ORCEIN

A differential

ANILINE BLUE

ORANGE G

stain for elastic fibres, collagen, keratin, etc.

Solutions required:

A. Orcein
Alcohol

Hydrochloric acid, cone.

B.

Alcohol

0-6 ml.

49 ml.

50%

Hydrochloric

acid, cone.

C. Mallory's Aniline Blue

Aniline blue, aqueous

0*5 ml.

Orange G.
0-5

Orange G
Phosphomolybdic acid 1%..
D. Orange

gm.

100 ml.

70%

G 1%

gm.

2gm.
100 ml.

in absolute alcohol.

Technique:
1

Fix material in Bouin and

embed

in paraffin wax.

2. Sections, about 8/x in thickness, are fixed to slides, dewaxed

with xylol and passed through the usual descending grades of

alcohol to distilled water.

3.

Stain for one

and a half hours in the orcein solution

in a

closed staining jar.

Differentiate with solution B, controlling under the microscope, until most of the pink is extracted from the sections.
4.

Note : The duration of the differentiation

will vary according to

the nature of the material and to the thickness of the sections.

6.

Wash
Wash

7.

Immerse

5.

thoroughly with running tap water.

with distilled water.
in solution

distilled water, for

diluted with an equal

one to two minutes.

8.

Rinse with

9.

Rinse with two

95%

alcohol.

lots

of solution D.
175

volume of

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

10.

Rinse quickly with absolute alcohol.

11

Clear in xylol and mount.

Results:

Collagen, blue. Muscle fibres, pale orange

Cytoplasm, varying shades of yellow. Erythrocytes, golden yellow. Kerantinized material, bright yellow.
Elastic fibres, red.

to dirty yellow.

Reference: Margolena, L. A. and Dolnick, E. H., Stain Tech., (195 1), 26,
1

19-21.

ORCEIN

ANILINE SAFRANIN

For elastic and connective tissue fibres

Solutions required:

A. Orcein (Unna) | to
alcohol

1%

in
.

Hydrochloric acid, cone.

B.

Safranin O, water soluble

Aniline water

Absolute alcohol

80%
.

100 ml.

ml.

gm.

48 ml.

52 ml.

Technique:
1.

Sections are

mounted on

slides

and brought down

to

90%

alcohol in the usual manner.

(If the tissues have been fixed in a

fluid containing mercury, the mercurial deposit is removed by the
standard technique the sections are then taken from 70% to 90%
;

alcohol, then direct into the orcein stain.)

2.

Stain from twenty to sixty minutes with the orcein solution

in a stoppered staining jar.

3.

Rinse in acid alcohol until stain ceases to come out of the

preparation.
4.

Rinse in

5.

Stain in aniline safranin (Solution B) for five minutes; then

70%

alcohol;

then in

rinse in water.

176

distilled water.

SECTION TWO
6.

Differentiate

into

by dipping

90%

alcohol then examining

rapidly under the microscope. Repeat this process until the cell
nuclei are well brought out, stained clear bright red.
7.

Dehydrate by rinsing quickly in absolute alcohol

and mount in balsam.

then clear

in xylol

Results:
Elastic fibres are stained dark to reddish

bright red;

ground substance of hyaline

ORCEIN
For sjrphilitic

brown;

cell nuclei,

cartilage, yellow.

GIEMSA STAIN

tissue, particularly

dermatological specimens

Solutions required:

A. Orcein (Unna-Tanzer) :
Orcein

Alcohol

B.

0-5

gm.

100 ml.

70%

HCl, concentrated

0*6 ml.

Absolute alcohol
HCl, concentrated

100 ml.

C. Giemsa stain
Distilled water

Note: Solution

0'5 ml.

should be prepared freshly, as

from Giemsa

required,

D. Absolute alcohol
Eosin

1%

0-25 ml.
100 ml.

alcoholic

stain.

100 ml.

ml.

Technique:
1.

2.

two

Paraffin sections are brought

down

to

70%

Stain for one half to one hour in Solution

to five

minutes in

alcohol.

then rinse for

distilled water.

3. Wipe off excess water; dip into 95% alcohol for a few seconds; then decolorize with absolute alcohol for five to twenty-

177

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

minutes, or until the sections assume a pale brown colour and
the elastic fibres stand out, deep purple to black, under the lowfive

power
4.

objective.

B until the background

This usually takes two to seven minutes.

Decolorize in Solution

colourless.

is

almost

Note: Decolorization must not be extended more than ten

minutes, as otherwise the thin elastic fibres will become destained.
5.

Immerse

6.

Stain for

epithelial

in tap water for five to ten minutes.

two
and other

to twelve hours with Solution

cells are

deep blue

until the

connective tissue, greyish

pink or greyish blue or blue.

7. Wipe off excess stain and dehydrate and decolorize in
Solution D, controlling under the microscope.

Note: Decolorization must be stopped when the connective

tissue has lost all trace of blue

8.

tint. The
The epidermis

and has assumed a rose

blue tinge is removed fairly rapidly in Solution D.

should remain bright blue.

Immerse

in each.

in two changes of absolute alcohol for two minutes

Clear in xylol, and mount.

Results:

Nuclei, deep blue. Cytoplasm of the epidermis, muscle cells

and connective tissue cells, light blue. Plasma cells, dark greyish
blue. Eosinophilic granules, bright red. Mast cell granules, meta-

chromatic (varying shades of) purple. Neutrophilic granules, only

faintly stained. Erythrocytes, reddish brown. Collagenous fibres,
pale rose to brownish pink. Elastic fibres, dark brown to black.
Senile degenerated connective tissue (collacin, elacin and collastin),

various shades of dark grey and blue. Cartilage, metachromatic

(varying shades of) purple. Decalcified bone, light brown. Keratin,

blue (poorly stained). Stratum lucidum, dark red; keratin layer

may be light blue or light pink or colourless depending upon the

and the degree of decolorization. Inner root sheath of the

deep blue. Melanin granules, green to black. Other pigments, unstained. Bacteria and mycelia, deep blue. Demodex
folliculorum in hair follicles, brown with blue granulations.

tissue
hair,

178

SECTION TWO

ORCEIN

PICRO FUCHSIN (Van Gieson)

For elastic and collagen fibres

Solutions required:

A. Orcein (Unna) i% in 80% alcohol

Hydrochloric acid, cone.
.

B.

100 ml.
i

ml.

Picro-fuchsin (Van Gieson).

Technique:

and brought down to 70%

have been fixed in a fluid
containing mercury, the mercurial precipitate is removed by the
1.

Sections are

mounted on

alcohol in the usual manner.

slides

If tissues

standard technique.
2.

Immerse

hour or longer

in orcein solution (formula as above) for half an

if necessary ; then rinse in acid alcohol.

3.

Rinse in

4.

Stain with picro fuchsin (Van Gieson) for three to five min-

70%

alcohol; then in water.

utes.
5.

Rinse rapidly (not more than a few seconds) in water.

6.

Dehydrate rapidly;

clear,

then mount.

Results:

Collagen fibres are stained red elastic

cytes, epithelia, muscle, etc., yellow.
;

brown

erythro-

Acm

OSMIC

fibres,

rapid technique for staining fat in frozen sections

Solutions required:

A. Osmic acid 1% aqueous.

Note: Store in an amber bottle.
B.

Eosin, yellowish,

1%

aqueous.

Technique:
I.

Tissues are fixed as follows

Place 22 5 ml. distilled water in a 50-ml. beaker and heat to

about 90 C; then add 2-5 ml. formalin; raise to boiling point;

179

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

drop in a thin piece of the tissue
60 to 65 C. for ten minutes.

then place the beaker in an oven

at

2.

Cut frozen sections

lo// thick,

and place them

in another

50-ml. beaker.
3 Boil Solution A in a large test-tube and pour onto the sections,
then transfer to an oven at 60 C. for five minutes.
.

4.

Wash

sections in a dish of cold tap water after pouring back

may be used again, into the stock bottle.

the osmic acid, which

5.

Counterstain in Solution

6.

Wash

quickly in tap water

and mount

for

one minute.

transfer sections to slides ; drain

in glycerine jelly, previously

melted in an oven or on a

water bath.
Results:

Fat globules, black or greyish black against a red background.

Caution

Osmic

acid vapour

is

injurious to the eyes.

PASINI'S STAIN (Improved)

For

dififerentiation of

connective tissue

Solutions required:

A. Iron alum 2-5% aqueous.

B. PasinVs stain:

Unna's

aniline blue-orcein

Eosin bluish

2%

50%

in

Acid fuchsin
Neutral glycerine

alcohol

10 ml.

12 ml.

0-3 gm.
5 ml-

Technique:
1.

Tissues should be fixed in Heidenhain's susa mixture and

embedded

L.V.N. Sections are cut 3/^ in thickness.

After removal of mercuric precipitate in the usual manner sections are mordanted in Solution A for twenty-four hours.
in

2.

Transfer to Solution

3.

Transfer to

95%

for three to ten minutes.

alcohol and agitate for about one minute or

come out in clouds.

until the colour ceases to

4. Immerse
and mount.

in absolute alcohol for one

180

minute

then blot, clear

SECTION TWO
Results:
fibres, deep blue. Cytoplasm, red. Epithelial cells,
basal
bodies, nuclear structure, brilliant red. Erythrocentroiles,
blue. Secretory bodies,
cytes, yellowish red. Connective tissue,
of goblet cells, azure
Slime
nature.
to
their
varying according

Collagen

blue.

Connective

wandering

tissue,

cells,

smooth and

striated

muscle, well defined.

PERIODIC ACID (HOTCHKISS)

CELESTIN BLUE

For human and animal pituitary glands, demonstrating

both muco-protein precursors of the gonadotrophins

Compared with other methods, finer differences in the cytology

of the cyanophils can be appreciated. Cell counts can readily
be carried out, and the counts are more accurate, giving more
definite and clearer results than those obtained by older methods
:

for instance cells appearing by Mallory and other histological

methods, to be chromophobes, are found to belong to cyanophil
series.

The method has been applied with good cytological results to

the hypophysis of sheep and goats.
Solutions required:

5%

A. Helly Fixative

containing
neutralized formalin.

B.

Lugol's iodine.

C.

Sodium

thiosulphate

5%

of

aqueous.

D. Hotchkiss' periodic Fuchsin.

E.

Hotchkiss'

rinse

reducing

{Solution

as

page 184).
F.

Feulgen's Fuchsin.

G.

Celestin blue

{Lendrum and Mcfarlane)

Celestin blue

Iron alum
Distilled water
.

Glycerine

0-25 gm.

. .

2*5

181

gm.

50 ml.
7 n^

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

Dissolve the alum in the water: then add the

and boil for three minutes. Cool

then add the glycerine.

celestin blue,

and

filter

H. Toluidine blue 0*5% aqueous.

I.

Orange

2 gm.

100 ml.
Phosphotungstic acid 5% aqueous.
Allow to stand for 48 hours filter before use.
.

Technique:
1. Human and larger animal glands are bisected in the horizontal plane with a sharp knife, and the two halves are fixed and
embedded separately. Rat hypophyses are fixed in situ after re-

moval of the brain and overlaying meninges, by immersing the

whole base of the skull in a beaker of the fixative.
2.

Fix in Helly for eighteen hours (man, goat, sheep), or two

to four hours (rat).

3.

Wash from

six to twelve

4.

Dehydrate, clear

5.

Cut sections

hours in running water.

and embed

in paraffin wax.

4-5/x (rat), or 5-5jLt (man).

Fix sections to slides; remove paraffin wax with xylol and

pass through the usual descending grades of alcohol to distilled
6.

water.

Immerse

7.

in solution

for three minutes.

Immerse in solution C (sodium thiosulphate) for three

minutes by which time the natural colour of the sections should
have been restored and mercurial precipitate removed from the
8.

fixative.
9.

Wash

10.

well with water.

Rinse with

11.

Immerse

12.

Wash

13.

Immerse

70%

alcohol.

in periodic acid (solution

with

70%

D)

for five minutes.

alcohol.

in Hotchkiss' reducing rinse (solution E) for one

minute.
14.

Wash

15.

Immerse

with

70%

alcohol.

in Feulgen's

Fuchsin for ten to thirty minutes.

182

SECTION TWO
1 6.

Wash

in running water for ten to thirty minutes.

17. Stain in the celestin blue solution

one half to three minutes.

18.

Rinse in water.

19.

Stain in the toluidine blue (solution

H)

for

one half to three

minutes.
20.

Wash

in running water for five minutes.

21. Stain in the

phosphomolybdic orange

(solution I) for

ten seconds.
22.
is

Wash

in water for five to thirty seconds, until a yellow tinge

just visible to the

naked

eye.

Dehydrate through the usual graded alcohols.

24. Clear in xylol and mount.
Note : In place of Toluidine blue, Iron Haematoxylin may be used
23.

at step 19 in which case

before washing

will be necessary to differentiate quickly

it

in running water {step 20).

Results :

Beta granules in the cyanophils and a

vesicles in cells

which

stain as

number

of granules and
chromophobes by other methods,

magenta to deep red: the colloid is magenta. Alpha granules

of acidophils, orange yellow, and erythrocytes are a shade more
are

yellow.

Nuclei: are blue-black.

Reference: Pearse, Anthony G. Everson (1950), Stain Tech., 25, 95-102.

PERIODIC ACID

FEULGEN FUCHSIN

(Hotchkiss)

For staining polysaccharide structures in fixed animal or

plant tissue preparations
Solutions required:

A. Periodic acid

0*4 gm.

Distilled water

Sodium
B.

acetate

45

nfil-

5 ml.

M/5

Distilled water

10 ml.

Periodic acid

0-4 gm.
183

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

Sodium

acetate

M/5

Absolute alcohol

5 ml.

35 ml.

Note: This solution, which deteriorates

few days, should be kept in an amber bottle.

after a

C. Reducing rinse:

Potassium iodide

Sodium thiosulphate

Distilled water

Dissolve

then add with stirring

Absolute alcohol

2N

Hydrochloric acid

gm.
gm.

20 ml.
:

30 ml.

0-5 ml.

Note: A precipitate of sulphur is slowly formed

and this may be allowed to settle out, or the solution
may be used immediately.
The solution loses its acid reaction on keeping for
some time, and it should be tested with litmus paper
if the reaction is no longer acid a few drops of N/2
hydrochloric acid should be added until an acid
;

reaction

is

obtained.

D. Feulgen's fuchsin.
E. Sulphite wash water:
Distilled water

Hydrochloric acid, pure, cone.

Potassium metabisulphite

45-5 ml.
0-5 ml.
0-2 gm.

Technique:

Any fixative may be used but for glycogen

polysaccharides, alcohol

is

or other easily soluble

it does not dissolve

the best fixative as

such substances, and Solution B should be used in place of Solution A.

1.

The

immersed
2.

Pour

sections or smears are brought

for five minutes.
in Solution
off solution; flood

3.

Immerse

4.

Flood with

tion
5.

down

to alcohol;

then

for five

70%

with

70%

alcohol.

minutes in Solution C.
alcohol;

pour

off;

then immerse in Solu-

for fifteen to forty-five minutes.

Wash two

or three times in Solution E.

184

SECTION TWO
6.

Wash

7.

Dehydrate

thoroughly in distilled water.

;

clear

and mount in the usual manner.

Results:

The

following are stained (red) intensely by this reaction:

muscle glycogen, liver, hyaluronic acid, gastric mucin, umbilical

cord polysaccharide, chitin, crude serum albumin, crude casein,

pneumococcus type III polysaccharide, Friedlander type B polysaccharide, algin, lemon pectin, gum arable, gum tragacanth, glydihydroxyacetone, ribose, arabinose, a-glycerophosphate, mannitol, tartaric acid, gluconic acid while the following take up the stain with moderate intensity starch, glucuronic
serine,

cerine,

acid,

pneumococcus type I polysaccharide, pneumococcus type

and the following are weakly stained: celluserum
albumin, crystalline tgg albumen, glucose,
crystalline

II polysaccharide;
lose,

glucosamine, glucose-1-phosphate, galactose, maltose, saccharose,

xanthosine, adenosine, muscle adenylic acid and phlorizin.
Tryptophane is coloured brown, and the following do not take up
ribonucleic acid, desoxyribonucleic acid, cellobiose,
malic acid and tyrocidine.

the stain:
inositol,

Notes:
{a) Plant tissues are brilliantly stained, in general revealing
cellulose or cellulose-like walls of the individual cells and stored

carbohydrate such as starch granules, particularly in the region of

the chloroblasts if these are present. The cell walls of freshly cut
potato reveal every fold, wrinkle or tear.
are
(b) Accumulations of polysaccharides
animal preparations, but mucin, because of

less
its

strongly stained.
desired to demonstrate glycogen^ Solution
(c) If
used in place of Solution A.

content,

From

in

is

it is

kiss),

common

polysaccharide

Archives of Biochemistry, Vol. 16, No.

by courtesy of Academic

PHLOXIN

Press, Inc.,

i,

New

should be

pages 131-142 (R. D. HotchYork, 10, U.S.A.

HAEMATOXYLIN

For hyaline, in animal tissues

Solutions required:

A. Ehrlich haematoxylin solution.

B. Phloxin 0.5% in 25% alcohol.
185

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

C. Lithium carbonate

o-i% aqueous.

Technique:
1.

Stain in Ehrlich haematoxylin for five minutes.

2.

Wash

in water; then stain one half to one

minute in the

phloxine solution.
3.

Wash

in tap water; then decolorize in the lithium carbon-

ate solution.
4.

Wash

mount

in tap water; then dehydrate in the usual

manner and

in balsam.

Results:

Fresh hyaline appears as red droplets and threads

hyaline is pink to colourless nuclei, blue.

while older

PHLOXIN

METHYLENE BLUE

Rapid smear technique for Negri bodies in brain tissue

(J. R. Dawson's method)
Solutions required:

A. Phloxin
B.

2%

aqueous.

Methylene Blue

(Loeffler).

Technique:

The

brain to be examined should be removed as quickly as

possible; then small segments, 3 to 4 mm. thick, are cut from
1.

Ammon's horn
dish.
2.

long axis and placed in a Petri

tissue, leaving only the horn.

perpendicular to

Cut away adjacent

its

Place a segment, cut surface downwards, on the small end

new one-inch cork; then with a matchstick, wipe peripheral

tissue downward and outward, so that the segment is more firmly
of a

attached to the cork and the grey matter containing the pyramidal
Press this gently against a scrupulously
cells bulges upwards.
clean slide, and make a smear by repeating this process along the

whole length of the

slide.

rapidly before the tissue

This operation should be carried out

commences
186

to dry.

SECTION TWO
3.

Fix immediately by immersing in pure methyl alcohol for

minutes.

five to ten
4.

Rinse in running water; then stain in the phloxin solution

to five minutes.

from two
5.

Wash

in

then stain in methylene blue

running water;

(Loeffler) for ten to thirty seconds.

6.

Decolorize in

alcohol; then dehydrate in

80%

95%

alcohol

and two changes of absolute alcohol.

7. Clear in xylol and mount in balsam.

The slides should be handled with forceps throughout to

the
preparation being spoiled by coming into contact
prevent
with the fingers.
Note:

Results:

Pyramidal
brown.

cells,

PHLOXIN

Negri bodies, bright red to reddish

blue;

METHYLENE BLUE

AZUR B

For normal and pathological animal tissues

is a rapid modification of Mallory's original method rethe
staining time from one hour or more to one to two
ducing
minutes, and permitting good staining with formalin fixed tissues

This

is not possible with Mallory's original method which was

designed for Zenker-fixed material. Colophony differentiation is
obviated, and Phloxin is not washed out as in the original

which

Mallory technique.
Solutions required:

A. Phloxin
Acetic acid

0-2% aqueous

0-5

gm.

100 ml.

Filter before use.

B.

Methylene blue

Azur B

Borax..

..

Distilled water

C. Acetic acid

0-25 gm.

0-25 gm.

..
.

0-2% aqueous.
187

.,
.

..
.

0-25 gm.
100 ml.

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

Technique:

Fix sections to slides remove paraffin and pass through the

1
usual descending grades of alcohol to distilled water.
.

A for one

2.

Immerse

3.

Rinse well in water.

4.

Stain for one half to one minute in solution B.

5.

Destain partially in solution C.

6.

Differentiate in three washes of

in solution

two minutes.

to

95%

alcohol.

8.

Dehydrate with two changes of absolute alcohol.

Clear in two changes of xylol.

9.

Mount

7.

mount,

in synthetic

medium (Permount, D.P.X.

or Clear-

etc.)

Results:

Nuclei and bacteria are stained blue with some metachromasia.

Collagen and other tissue elements are bright pink to red. Erythrocytes, bright scarlet.
Reference: Thomas, John T. (1953), Stain Tech., 28, no.

PHLOXIN

6.

311

312.

TARTRAZINE

Lendrum's technique)
general histological stain and for the demonstration of
(A. C.

inclusion bodies
Note: This technique, in which use is made of a stable phloxine
and which affords the advantage of brilliant demonstration of certain inclusion bodies, is superior to Masson*s
solution,

erythrosin-saffron, which
deteriorates fairly rapidly.

is

less

readily

prepared and which

Solutions required:

A.
B.

Haemalum

100 ml.

Calcium chloride 0-5% aqueous.

Phloxine

C.

(Mayer).

Tartrazine, saturated in cellosolve.

188

0-5

gm.

SECTION TWO
Technique:
1.

Stain for five to ten minutes with the haemalum, examining

intervals, until the desired depth of

under the microscope at

staining has been attained.
2.

Wash and

blue in tap water or in saturated aqueous lithium

carbonate.
3.

4.
5.

Stain in the phloxine solution for half an hour.

Rinse quickly in water.

Drain off excess water and replace with tartrazine solution
C, as above), using a dropping bottle to control the

(Solution

differentiation.

Note:

tartrazine

is

commended
6.

7.
8.

tartrazine replaces the phloxine from collagen. As

readily soluble in water, slight overstaining is re-

The

before dehydration.

Rinse in

60%

alcohol followed

by

95%

alcohol.

Dehydrate with absolute alcohol.

Clear in xylol and mount.

Results:

Kurloff bodies in guinea pig's lung are well shown. Inclusion

bodies of a number of virus-containing tissues show retention of
phloxine.

Note:

Fixatives containing mercuric chloride give the best

results.
Abstract

J'.

Path.

& Bad.,

PICRO

Lendrum, A. C.

59, 399-404, 1947,

NIGROSIN

For eleidin and keratin

Solutions required:

A. Picric acid, saturated, aqueous.

B.

Nigrosin

1%

C. Terpineol

Origanum

aqueous.

oil

189

volume
volume

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

Technique:
1.

Tissues should be fixed in

io%

formalin, and frozen sections

employed.
2.

Sections are stained for five minutes in the picric acid solu-

tion.
3.

Rinse in

distilled water.

4.

Stain in the nigrosin solution for one minute.

5.

Wash

6.

Rinse in

7.

Clear in Solution C.

8.

Mount

in distilled water.

95%

in

alcohol.

balsam or in

Cristalite.

Results:

Eleidin, blue-black.

Keratin, bright yellow.

PROTARGOL

GALLOCYANIN

(Foley)

fibres, sheaths and cells

For nerve

Solutions:

A. Protargol

1% aqueous.
(Prepared by sprinkling the protargol powder on
the surface of the water and leaving it to dissolve.)

B.

Protargol

Alcohol

1%

aqueous

50 ml.

50 ml.
ml.
pure
0-5
Pyridine
Note: The quantity of pyridine may be varied
between o-i ml. and 2 ml. The higher concentrations
facilitate the staining of thin fibres, whereas cell
bodies and dentrites are better demonstrated with the
lower proportions of pyridine.

95%

C. Boric acid

Sodium

..

..

sulphite anhydrous

Hydroquinone
Acetone

Distilled water

..
.

..

..

..

1-4

gm.

2 gm.
0-3

15

gm.
rnl.

85 ml.
Dissolve each reagent in the above order in the
water adding the next only after the previous one has
been dissolved entirely.
.

190

ml.

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

7.

Immerse

for three to five minutes in Solution

then wash

thoroughly in distilled water.

8. Counterstain overnight in Solution G.
9.

Wash

thoroughly in

distilled

water then immerse in Solution

for thirty minutes.

10. Without washing transfer section to diluted Solution I for

one hour; then wash with 70% alcohol and differentiate the

counterstain in
11.

95%

alcohol.

Transfer to normal butyl alcohol;

clear in

cedarwood

oil

and mount.
Results:
fibres and neurofibrils, blue-black. Nissl bodies, pale
Nuclei, blue-black with silver and gold if a higher percentage of pyridine was used in solution. Myelin sheaths, bright
yellow. Connective tissues, various shades of blue and green.

Nerve

blue.

PURPURIN
For calcium deposits in pathological tissues
Solutions required:

A. Purpurin, saturated in absolute alcohol (about 0*7%).

B.

Sodium

chloride, reagent grade,

0-75% aqueous.

Technique:
1.

2.

3.

Fix material in

90%

alcohol.

embed

Dehydrate; clear;
Fix sections to slides;

in paraffin wax.

de-wax and pass through the usual

descending grades of alcohol to

distilled water.

4. Stain in the purpurin solution for about ten minutes.

5.

Immerse

6.

Rinse with

in

sodium chloride solution

for about five minutes.

70%

alcohol until the stain ceases to

90%

alcohol

come away

in clouds.
7.

Rinse with

8.

Clear in xylol and

and dehydrate with absolute

mount

in balsam.

Results:

Calcium deposits are stained

red.

192

alcohol.

SECTION TWO

QUINCKE REACTION
For haemosiderin
Solutions required:

A.

Ammonium

solution,

sulphide

concentrated

Absolute alcohol
B.

Basic fuchsin

0-5%

in

50%

volume
volumes

alcohol.

Technique:

Tissues are fixed in neutral formalin 10%, or in absolute

alcohol, and embedded in paraffin wax or Celloidin in the usual
1.

manner.
2.

Bring sections

from two

down

to distilled water,

to forty-eight hours in the

then immerse them

ammonium sulphide solution.

3.

Rinse thoroughly in distilled water.

4.

Counterstain in the basic fuchsin solution for five to twenty

minutes.
5.

Wash

in water;

drain well; rinse in

80%

alcohol.

and dehydrate in absolute alcohol;

xylol and mount in Canada balsam or in Cristalite.
6.

Differentiate

clear

Results:

Haemosiderin, dark brown to black.

RHODAMINE B

ANILINE METHYLENE BLUE

For splenic and lymphoid tissues

Solutions required:

A. Methylene Blue,
Aniline water
Distilled water
B.

Rhodamine B

2%

alcoholic
^

10 ml.
15 ml.

30 ml.

1%

aqueous

2-5 ml.

47-5 ml.

Distilled water

C. Solution

(above)

Solution

(above)

7 volumes

193

volumes

in

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

Technique:

Tissues are fixed in Zenker-Formol and embedded in paraffin

wax.
1.

After removal of mercurial precipitate by treatment with

manner {see page 28) sections are stained two

iodine in the usual

to three hours in Solution

C; then washed rapidly with absolute

alcohol.
2.

Clear in xylol

mount.

Results:

Basophile protoplasm, blue. Chromatin, violet blue. Nucleoli,

Connective tissue, faintly stained yellowish red. Muscle,
yellowish red. Erythrocytes, bright red. Acidophile granules of
red.

leucocytes, bright red. Hyalin and granules of Russell, bright

red. Nuclei of the small lymphocytes are faintly stained violet.

SAFFRON
For connective tissue
Note: Saffron is the dried stigmata of crocus sativus, and should
not be confused with safranin, which is an aniline dye.
Solution required:

A. Saffron

Distilled water

. .

2 gm.
100 ml.

Boil gently for an hour allow to cool then filter ;

I ml. of
40% formaldehyde and i ml. of
;

add

5%

tannic acid to the

filtrate.

Note: Saffron solution deteriorates after a few

weeks, and it is best to prepare the solution in small
quantities, as required.

B.

Delafield or Ehrlich haematoxylin.

C. Erythrosin,

1%

aqueous.

Technique:

Fix small pieces of tissue in Bouin, Zenker-formaldehyde or

mercuric-formaldehyde.

1.

in

2.

Wash; dehydrate; embed.

194

SECTION TWO
3.

Sections are stained for five to ten minutes with Delafield or

Ehrlich haematoxylin
4.

rinse in water.

Blue in tap water in the usual manner or in

1%

sodium phos-

phate (Na2HP04).
5.

Stain for two to five minutes in

6.

Rinse quickly with water.

7.

Differentiate with

under the microscope,

70%

1%

aqueous erythrosin.

alcohol for a few seconds, controlling

until the collagen fibres are nearly colour-

less.

Rinse in water; stain for five minutes in saffron solution

prepared as above rinse with water.
8.

9.

Wash rapidly first with 70% alcohol then with absolute alcohol

clear in xylol

and mount.

Results:

Nuclei are stained blue.

Cytoplasm, varying shades of red.

Muscle, pink. Elastic fibres, pink. Collagen, yellow.

Improved differentiation of most cells and tissues is obtained

employing

method

this

SAFRANIN

in place of haematoxylin

CRYSTAL VIOLET

ORANGE
"

&

This technique, which

is

by

eosin.

FAST GREEN

Animal Tissues

(S. S. Kalter's
Quadruple Stain for
Clin. Med., 28, 995-7, 1943.

Lab.

and

".)

Abstract from

jf.

a development of Flemming's

triple stain, is particularly useful for histology students

Solution required:

A. Safranin,
Formalin

B.

(40% formaldehyde)

Sodium

acetate

. .

Alcohol

50%

Crystal violet

0-5% aqueous.

C. Fast green F.C.F.

saturated in clove

D. Orange 2 saturated

Orange

oil

in clove

195

oil.

0-2 gm.
4 ml.
0-5

gm.

100 ml.

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

Technique:

Any fixative may be employed, but if Bouin is chosen, remove

1.

picric acid with a

few drops of saturated lithium carbonate in the

70% alcohol of dehydration series. Should a fixative containing

mercuric chloride be chosen, then it will, of course, be necessary
to remove mercuric precipitate in the usual way.
After fixation, wash, dehydrate, clear and

2.

embed

in paraffin

wax.

Cut

3.

sections

no thicker than

7-5//

and

fix to slides,

avoiding

the use of glycerine albumin, which will cloud the stain.

De-wax with

4.

xylol

then pass through absolute,

90%

and

alcohol to water.

70%

Stain in the safranin solution for twenty-four hours.

5.

7.

Rinse with water.

Stain with the crystal violet for one to two minutes.

8.

Wash

9.

Immerse

6.

10.
11.

with water.

50% alcohol for two minutes.

Immerse in 95% alcohol for two minutes.
in

Immerse

in the fast green-orange 2 (Solution

C) for

five

minutes.
12.

Differentiate,

connective tissue

is

examining under the microscope, until the

stained to the desired depth of green.

13.

Immerse

14.

Transfer to Orange 2 in clove

in clove oil for ten minutes.

oil

for ten minutes.

15. Differentiate, examining under the microscope at intervals,

until the desired depth of staining has been achieved.
16.

Immerse

17.

Mount

for ten

minutes each in two changes of

xylol.

in balsam.

Results:

Nucleoli, purple or purpHsh red. Nuclear memdark

red.
Cellular cytoplasm, pink to red, except in Henle's
brane,
loop (light green). Connective tissue, green. Elastic fibres, yellow.
Fibroblasts, green with purple nuclei. Muscle, reddish brown.

Nuclei, red.

Erythrocytes, orange.

Polymorphonuclears show purple nuclei.

196

SECTION TWO

SAFRANIN

WATER BLUE

(Unna)

For collagen fibres

Solutions required:

A. Safranin
B.

O i%

aqueous.

lo ml.
Water Blue i% aqueous.
10 ml.
Tannic acid 33% aqueous
This solution must be freshly prepared.
.

Technique:
1.

Tissues should be fixed in

1%

aqueous picric acid or in

absolute alcohol, and Celloidin sections should be employed.

2.

Stain for ten minutes in the safranin solution;

then wash

thoroughly in water.
3.

Stain for ten to fifteen minutes in Solution B.

4.

Wash

5.

Clear in Bergamot

distilled water.

thoroughly in

oil;

then mount.

Results:

Collagen fibres are stained blue, while nuclei are red

SCARLET R

ETHYLENE GLYCOL

An

improved technique for staining fat, etc, in animal tissues,

chief advantages being: (a) Excellent differentiation without
loss of stain out of the lipid particles, {b)
stable solution which
its

does not dissolve lipid materials, {c) Sections are not shrunken
but remain pliable, {d) More intense staining of fat.
Solutions required:

A. Ethylene glycol, pure, anhydrous.

B.

Scarlet

gm.

100 ml.
Ethylene Glycol, pure, anhydrous.
Heat the ethylene glycol to 100-110 C. on
a hot plate or in an oven, or over the bunsen
flame, taking care that it does not catch fire;
then add the stain and stir until all or most of
.

it is

dissolved.

Filter

when

197

cold.

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

.

C. Ethylene glycol, pure, anhydrous

Distilled water
.

85 ml.
15 ml.

D. Ehrlich or Delafield haematoxylin.

Technique:
1.

Fix material in

2.

Wash

10%

formalin and cut frozen sections.

sections in water for

two minutes or longer

to

remove

the formalin.
3.

Dehydrate the sections by agitating gently in pure anhydrous

ethylene glycol for three to five minutes.

4.

Immerse the

sections in the stain (solution B) for

two

to

three minutes, with gentle agitation.

by agitating gently in 85% ethylene glycol

to ten minutes, controlling by examination
from
one
(solution C)
under the microscope while the preparation is still wet.
5.

Differentiate

6.

Transfer to distilled water for three to

7.

Counterstain with Ehrlich or Delafield haematoxylin.

8.
9.

five

minutes.

Wash well in tap water.

Mount in glycerine jelly.

Results:

Nuclei, blue. Fat, orange to red. Cholesterol, red.

myelin, unstained. Fatty acids, unstained.

Normal

SCARLET R
For staining

fat, etc.

in

animal tissues

Solutions required:

A. Scarlet Red, saturated in equal

volumes of acetone and 70%
alcohol.

B.

Ehrlich or Delafield haematoxylin.

Technique:
I.

Tissues are fixed in formalin and frozen sections are em-

ployed.

198

SECTION TWO
2.

Sections are immersed for a second in

stained for two to five minutes, in the Scarlet

70% alcohol;
R solution.

3.

Wash

4.

Counterstain with Ehrlich or Delafield haematoxylin.

5.

Wash

quickly in 70

% alcohol, and transfer to

well in tap water;

mount

then

distilled water.

in glycerine or glycerine jelly.

Results:

Nuclei, blue. Fat, orange to red. Cholesterol, red.

myelin, unstained. Fatty acids, unstained.

Normal

SILVER CARBONATE

ORCEIN

ANILINE BLUE

FAST

GREEN
For demonstrating reticulin, elastin and collagen in the

same

tissue sections

Solutmis required:

A. Celloidin 0-5% in equal vols, of Ether and

Absolute Alcohol.
B. Pot. Perman. 0-25% aqueous.
C. Oxalic Acid

5%

aqueous.

D. Silver carbonate^ Hortega {Foot's

Silver nitrate 10% aqueous
Lithium carbonate, saturated

modification)
.

10 ml.

10 ml.
aqueous
Shake well; then allow to stand for ten
minutes or so in a 25 ml. measuring cylinder.
Pour off the supernatent fluid, then transfer the precipitate to
a 100 ml. measuring cylinder, and add about 75 ml. distilled water,
shake well allow to settle then pour off the fluid and add a second
lot of distilled water. This process should be repeated three or
.

four times.
Finally

add 25 ml. of

distilled

water to the precipitate and add

28% ammonia solution drop by drop until the precipitate is almost

dissolved.

Make up
Filter

to 100 ml. with

and warm

90%

alcohol.

to 50 C. for 15

199

minutes before using.

'

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

Neutral Formalin

E.

40%

Distilled water

Buffer to

pH

20 ml.
80 ml.

100 ml.

100 ml.

100 ml.

7-0.

Gold chloride 0*2% aqueous.

G. Sodium Thiosulphate 5%.
F.

H. Orcein

1%

in

Hydrochloric

alcohol

70%

acid, cone.

ml.

Picro Aniline Blue

Aniline blue aqueous

Picric acid, saturated aqueous

I.

o*i

gm.

or:

Picric acid, saturated aqueous

J.

Fast Green

FCF

. .

0*2

gm.

Technique:

Material should be fixed in

1.

paraffin

wax by

formalin and

embedded

in

the standard technique.

Sections 4 to

2.

immersed

10%

5/i,

in thickness are fixed to slides,

dewaxed and

in xylol for five minutes.

3.

Wash and immerse

4.

Immerse

in absolute alcohol for

in solution

A for

five

two minutes.

minutes (Celloidin).

slides for one minute.

5.

Drain

6.

Immerse

7.

Rinse in water.

8.

Immerse

in

80%

alcohol for five minutes.

in solution

(Pot.

perman.) for

five

minutes.

9. Rinse in water.

10.

Immerse

11.

Rinse in tap water.

12.

Wash

in solution

(oxalic acid) for five minutes.

in distilled water.

Immerse

in the silver carbonate solution

13.
at 50 C. for ten to fifteen minutes. (Soln. D).
14.

Rinse in

15.

Immerse

in

an oven

distilled water.

in solution

E (20%

minutes.

200

neutral formalin) for five

SECTION TWO
1 6.

Rinse in tap water.

17.

Tone

18.

Rinse in tap water.

19.

Immerse

in solution

F (Gold

in solution

chloride) for five minutes.

G for two

minutes.

20. Rinse in tap water.

21. Stain in solution

an oven

at 37 C. or for

22. Rinse in

70%

(Orcein) for ten to fifteen minutes in

one hour

at

room temperature.

alcohol, followed

tap water.

by

23. Stain in solution I (Aniline blue) for twenty to forty seconds

or solution J (Fast green) for ten to twenty seconds.
24.

Wash

with

95%

alcohol for six to eight seconds.

25. Rinse briefly with absolute alcohol.

26. Rinse with a mixture consisting of equal
until clear.

volumes of xylol

and absolute alcohol

27. Rinse with several changes of xylol,

Results:

and mount.

>

With Picro

aniline blue

brown

Elastic fibres

reddish

Collagen

blue

Reticulum
Muscle
Nuclei

blue-green
tan to brown

black

yellowish tan
pale blue

Erythrocytes

Cytoplasm

With Picro fast green

Elastic fibres

orange to reddish brown

Collagen

blue-green
black

Reticulum

Muscle
Nuclei

green
tan to

Erythrocytes

yellow to orange

Cytoplasm
Ann

Reference: Lewis,
85-7.

light
L.,

brown

green

and Jones, Russell

201

S., (1951),

Stain Tech., 26,

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

SILVER NITRATE

GOLD CHLORIDE

PARACARMINE

(Da Fano)
For Golgi apparatus
A. Cobalt nitrate
Formalin
.

B.

Silver nitrate

(N.B.

i%

aqueous
.

lOO ml.

15 ml-

1*5% aqueous.

This should be stored in an amber or blue

glass bottle.)

C.

Cajal's Reducer.

Hydroquinone

2%

Neutral formalin

Sodium

aqueous
.

100 ml.
15 ml.

gm.
N.B.: This solution should be freshly prepared.
sulphite anhydrous

0-5

D. Gold chloride 0-2% aqueous.

E.

Sodium thiosulphate

F.

Paracarmine (Mayer).

5%

aqueous.

Technique:
Pieces of tissue no thicker than 3 mm. are fixed from two to
eighteen hours in the cobalt nitrate formalin solution, according
1.

to the size

and nature of the material.

2.

Wash

3.

Immerse

the tissue quickly in a large volume of distilled waterin the silver nitrate solution in the dark for thirty-

six to forty-eight hours.

4.

Wash

quickly in a large volume of distilled water.

5.

Trim

the tissue to a thickness not exceeding 2

6.

Immerse

in Solution

(Cajal's

mm.

Reducer) for two to twenty-

four hours in the dark.

Note:
7.
8.

For most

Wash

soft tissues

in several

changes of

about four hours will

distilled water.

Dehydrate, clear and embed in paraffin wax in the usual

way.
9.

suffice.

Cut sections up

to Sju in thickness.

202

SECTION
Fix sections to slides

10.

grades of alcohol

Tone

11.

down to

sections

on

TWO

de-wax and pass through descending

distilled water.

slides

in the gold chloride

by immersing

solution for five to ten minutes.

12.

Wash

13.

Fix in

quickly in distilled water.

5%

sodium thiosulphate (Solution E)

for ten to

fifteen minutes.
14.

Wash

15.

Counterstain with paracarmine for about ten minutes.

16.

Rinse with

17.

Clear in xylol and mount.

thoroughly in

90%

distilled water.

alcohol, followed

by absolute

alcohol.

Results:

Golgi apparatus stained black while

SILVER NITRATE

cells are

pink or red.

HYDROQUINONE

For the detection of gold in filxed tissues of experimental

animals
Notes:
(a)

The

following technique must be carried out in the dark-

room.
(b) Fixatives
{c)

The

containing metals must be avoided.

use of metal instruments in handling the sections must

be avoided.
Solutions required:

A.

Gum acacia 10% aqueous, filitered

100 ml.

Silver nitrate, A.R. grade

2 gm.

Note: This solution should be prepared immediately before use.

B.

Gum acacia 10%

Hydroquinone

aqueous, filtered
.

100 ml.
i

gm.

Note: This solution should be prepared the day

before

it is

required for use.

203

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

C.

Solution

Solution

5%

Citric acid

lo ml.
10 ml.
0-5 ml.

This solution should be prepared only

required for immediate use.

Note:

when

D. Sodium thiosulphate

5%

aqueous.

Technique:
1. Small
pieces of tissue are fixed in
sections are employed.

2.

Rinse thoroughly in

3.

Immerse

formalin, and frozen

20%

distilled water.

sections in Solution

and leave therein for

five to

ten minutes.
4.

tion,
5.

6.

Plunge sections directly into the sodium thiosulphate soluwithout prior washing, and leave therein for five minutes.

Wash thoroughly in several changes of distilled

Mount in Aquamount or in Farrant's medium.

water.

Results:

The

presence of gold

is

indicated

by a black deposit

in the cells.

SUDAN BLACK
For

lipids (especially those that are not well

Sudan
(J.

III

coloured by

or IV)

R. Baker's technique)
Solutions required:

A. Formaldehyde-saline.
Formalin (Formaldehyde 40%)
Sodium chloride 10% aqueous
Distilled water
.

10 ml.
7 ml.
83 ml.

Note: Keep a few pieces of marble chips

in

the solution to maintain neutrality.

B.

Formalin (Formaldehyde 40%) neutral.

Note : Keep a few pieces of marble chips
the bottle,

204

in

SECTION TWO
C. Potassium dichromate

aqueous

Sodium

2-5%
.

chloride

88 ml.
7 ml.

10% aqueous
Note: Keep a few pieces of marble chips
.

in

the bottle.

D. Dichromate-formaldehyde.
Solution

Solution

volume.
19 volumes.
I

E. Potassium dichromate
F.

Gelatine for embedding.

Gelatine powder
.

5%

aqueous

Water
Sodium p-hydroxybenzoate
Sprinkle the gelatine on to the water and
.

25 gm.
100 ml.

0-2

leave

it

gm.

to soak for

an

hour, afterwards warming in an incubator maintained at 37 C.

until all the gelatine has dissolved, then strain through muslin while
still

warm.

Note: If Sodium p-hydroxybenzoate, which is added to prevent

the growth of moulds and bacteria, is not available in the laboratory,
then 0-25 to 0*5 gm. of Thymol should be used instead.

G. Formalum {for hardening gelatine)

Formalin (Formaldehyde 40%)
Potassium alum 5% aqueous

20 ml.
80 ml.

Keep marble chips in the bottle.

Note: Both gelatine blocks and gelatine sections may be preserved indefinitely in Formalum, which makes the gelatine very
hard, thereby facilitating the cutting of thin sections which are
non-sticky.

Important:
test for

Formalum must not be used

phospholipids (Baker 1946) as the

in the acid haematein

alum would

react with

the haematein.

H. Sudan Black
Alcohol

...

...

0-5

gm.

100 ml.

70%

Boil for ten minutes under a reflux condenser

then cool and filter.
I.

Carmalum {Mayer).
205

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

Technique:

Fix a piece of tissue not more than 3

1.

mm.

thick in the for-

maldehyde-saline for an hour.

Transfer, without washing, to the dichromate formaldehyde
(Solution D) and leave for five hours.
2.

to 5% aqueous
3. Transfer, without washing,
dichromate and leave for about eighteen hours.

Leaving the tissue in the same solution, transfer

oven at 60 C. for twenty-four hours.

4.

fin

potassium

to the paraf-

5.

Wash

6.

Leave overnight in the melted gelatine in the oven

7.

Cool the

8.

Cut out

in running water for six hours.

at 37 C.

gelatine, preferably in a refrigerator.

a rectangular block containing the specimen.

Immerse the block overnight

(or any conveniently longer

time) in formalum, placing a marble chip in the capsule or tube.
9.

10.

Cut

11.

Transfer a section to

sections 8 to

lOju,

on the freezing microtome.

70%

alcohol.

Note: It is best to transfer sections from fluid to fluid, up

16 in a Royal Worcester Porcelain thimble No. ^.4756, size
12.

to stage
2.

Transfer to the Sudan black solution, and leave for |~4

is usually about 2 J minutes.)

minutes. (The best period

in

14.

Wash
Wash

15.

Wash

in water, sinking the section gently with a camel hair

13.

brush
16.

in

alcohol for five seconds.

alcohol for one minute.

if it floats.

Transfer to

optimum time
17.

70%
50%

is

Carmalum

for

two

to three minutes.

(The

usually three minutes.)

Rinse in distilled water.

Transfer the section to a fairly large dish, or a tongue jar

of tap water, and leave for two minutes, or any conveniently longer
18.

time.
18.

Wash

19.

Transfer to a petri dish of water.

again in another large bowl of water.

20. Float the section

on

to a slide.

206

SECTION TWO
away excess water but do not allow the section
Mount in Farrant's medium, or in Aquamount.

21. Blot
22.

to dry.

23. Attach a clip to hold the coverslip to the slide: then leave
overnight in the oven to harden the mounting media, before

examining the preparation under the oil immersion objective.

Note: The slide may be examined after a quarter of an hour, if
desired; then returned to the oven to complete the hardening.
Results* :

Lipids, dark blue or blue-black. Cytoplasm

grey-blue. Chromatin: pink or red.

colourless or pale

Note: If the results are not good, another section should be tried
with variations of the staining times.

Never attempt to judge the colouring until the section is

mounted and examined under the oil immersion objective.
It is recommended that the technique be learned on the intestine
of the mouse, as it is scarcely possible to fail with this. Cut out a
piece of empty intestine about i cm. long and immerse in formaldehyde-saline for five minutes, then open it by a longitudinal
cut from one end to the other, taking care not to do any unnecessary

damage

to the

villi.

The
and

section should be left only one minute in the

two and a half minutes in the carmalum.

Sudan black

Reference: Baker, J. R. (1949), Q.jf.M.S., 90, 293-307.

"
* In the
Golgi bodies ". Dr. J. R.
original paper the method refers to
Baker, to whom I am indebted for permission to include this abstract, informs
me (March, 1955) that he regards the method as essentially for those lipids
that are not well stained by other Sudan colours. In the original paper it is
"
stated:
Simple Golgi bodies and externa: dark blue or blue-black. Golgi
vacuoles: colourless ". The changes are Dr. J. R. Baker's.

SUDAN BLACK
A

specific stain for neutral fats

Solutions required:

A. Sudan black, saturated in

70%

alcohol.

B.

Carmalum (Mayer).

Technique:

Tissues are fixed at least three days in

rinsed thoroughly in distilled water.

207

10%

formalin;

then

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

1. Frozen sections are immersed one minute in 50% alcohol;
then one minute in 70% alcohol.
2. Stain for fifteen minutes to several hours in the Sudan black.

3.

Rinse for a few minutes in

50%

alcohol;

then in

distilled

water.
4.

with

Counterstain in carmalum for about three minutes;

water mount in glycerine jelly.

distilled

wash

Results:

Neutral

fat

and myelin, blue-black

SUDAN BLACK

to black;

nuclei, red.

ETHYLENE GLYCOL

An improved
vantage

technique for lipid staining, offering the adof a stable solution, excellent differentiation v^^ith-

out loss of stain out of the lipid particles,

unshrunken sections

and

pliable

Solutions required:

A. Ethylene glycol, pure, anhydrous.

B.

Sudan black

Ethylene glycol, pure, anhydrous.

100 ml.

gm.

Heat the ethylene glycol to 100-110 C. on a hot plate or in an

oven, or over a bunsen flame, taking care that it does not catch
fire then add the stain and stir until all or most of it is dissolved.
;

Filter

when

cold.

C. Ethylene glycol, pure, anhydrous.

Distilled water
.
.

85 ml.
15 ml.

D. Carmalum (Mayer).
Technique :
1.

Fix tissues for at least three days in

2.

Wash

3.

Dehydrate the sections by agitating gently in pure anhydrous

10%

formalin.

thoroughly in running water to remove the

fixative.

ethylene glycol for three to five minutes.

4.

Immerse the

fifteen
5.

sections

in the

Sudan black solution from

minutes to one hour, agitating gently

Differentiate

by

agitating

at intervals.

gently at intervals

with

85%

ethylene glycol (solution C) from one to ten minutes, controlling

under the microscope while the section is still wet.

208

SECTION TWO
6.

Transfer the sections to distilled water for three to five

minutes.
7.
8.

9.

Counterstain with carmalum for about three minutes.

Wash with distilled water.

Mount in glycerine jelly.

Results:

Lipid substances or particles are stained blue-black to black.

Nuclei, red.
Reference: Chiffelle, T. L., and Putt, F. A. (195 1), Stain Tech., 26, no.
pages 51-6.

i,

SUDAN BLUE
For demonstrating degenerated myelin
Solution required:

Sudan

blue,

saturated

50%

in

alcohol.

Technique:
1.

Tissues should be fixed for at least three days in

10%

neutral

formalin.
2. Frozen sections are soaked one minute in 50% alcohol; then
one minute in 70% alcohol then stained from fifteen minutes to
several hours in a saturated solution of Sudan blue in 50% alcohol.
in distilled
3. Rinse for a few minutes in 50% alcohol; then
;

water.
4.

Counterstain in carmalum for three to five minutes.

5.

Rinse in water; mount in glycerine

jelly.

Result:

Degenerated myelin, blue.

SUDAN BROWN
For

fat

and

for acute fatty degeneration not

Scarlet

Solutions required:

A. Stock solution of Sudan brown

B.

saturated in isopropyl alcohol.

Mayer's acid haemalum.

209

shown by

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

Technique:
1.

Fix tissues in

2.

Frozen sections are stained for

solution of

io%

formalin.
five

minutes in a diluted

Sudan brown prepared by mixing 6 volumes of the

stock solution with 4 volumes distilled water.

{Note: This diluted solution keeps only for one day, and
should, therefore, only be prepared immediately before use.)
3.

Float out in tap water.

Stain for two to five minutes in Mayer's acid

then rinse in tap water.
4.

5.

Immerse

(Na2HP04)

in

water

tap

or

sodium phosphate

1%

in

haemalum;

until the section turns blue.

6.

Rinse in

7.

Mount

distilled water.

gum

in Apathy's

syrup.

Results:
Fat,

brown;

nuclei, bluish grey; protoplasm, colourless.

SUDAN

For degenerating and intact myelin and

fat

Solutions required:

A. Haematoxylin

1%

absolute

in

one to

alcohol, at least

five

days

50 ml.
50 ml.
This solution should be prepared immediately

old

4% iron alum aqueous

before use.
B. Borax

Potassium ferricyanide
Distilled water
.
.

210

gm.

5 gni-

100 ml.

SECTION TWO
C. Iron alum

D. Sudan

2,

0-5% aqueous

30 ml.
20 ml.
well and allow to stand for ten minutes before

alcohol

Distilled water

Mix

saturated in isopropyl
.

use.

Note: This solution deteriorates within three to

four hours.
Technique:
1

Formalin-fixed frozen sections are stained forty minutes with

A at 56 C. in a covered dish in an oven.

Solution
2.

Rinse in water and differentiate for one hour with Solution B.

3.

Rinse in

water and immerse for ten minutes in

distilled

Solution C.

Stain ten to twenty minutes in Solution

4.

then

float

out in

water.

Mount

5.

in Apathy's

gum

syrup or in Aquamount.

Results:

Normal myelin, blue-black; nerve

grey

red corpuscles, yellow to black

THIONIN

cells,

fats,

grey;

nuclei, deeper

orange yellow.

(Ehrlich)

For mucin
Solution required:

Thionin

(Ehrlich),

aqueous
Distilled water

saturated,

0-5 ml.
10 ml.

Technique:
I.

fluid, washed in running

and
embedded
sectioned in the usual

Tissues should be fixed in Zenker's

water, dehydrated, cleared,

manner.
211

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

2.

Sections are

from the
page

fixative

mounted on slides and the mercuric precipitate

is removed by the standard technique {see

28).

3. Bring down to distilled water as usual; then stain

to fifteen minutes in the thionin solution.

4.

as otherwise the stain will

Dehydrate rapidly

from

five

be removed by

the alcohol.
5.

Clear in xylol and mount.

Results:

Mucin

is metachromatically stained purple; while the basophil

of
the mast cells, Wharton's jelly of the umbilical cord, are
granules
and
the other tissue constituents are stained in varying
purple;

shades of blue.

THIONIN

(Ehrlich)

For the differential staining of entamoeba in sections

Solutions required:

A. Thionin 0-25% aqueous.

B.

Oxalic acid

2%

aqueous.

Technique:
1.

Pieces of tissue are fixed in absolute alcohol and

in Celloidin in the usual

embedded

manner.

2.

Immerse

3.

Differentiate in the oxalic acid solution for thirty to ninety

in the thionin solution for three to seven minutes.

seconds, controlling by examination under the microscope.

4.

Rinse in water.

5.

Rinse in

6.
7.

70%

alcohol.

Dehydrate by rinsing in two changes of

Clear in terpineol and mount.

95%

alcohol.

Results:

Nuclei of amoebae are stained a rich brown colour, while the

nuclei of

all

other cells are stained blue.

212

SECTION TWO

THIONIN

(Ehrlich)

For nerve cells and

fibre tracts

Solution required:

Stain and fixative combined:

Thionin 0-5%

io%

in

formalin.

Technique:
1.

Tissues are fixed and stained simultaneously by immersing

in the above solution

from

few days to three months.

2.

Wash

3.

Dehydrate in ascending grades of alcohol in the usual man-

well in running water.

ner.

embed

wax

or in Celloidin.

4.

Clear

5.

Fix sections to slides and mount in

in paraffin

Cristalite.

Results:

Cell bodies are stained blue, while the fibre tracts are red.

THIONIN

(Ehrlich)

For demonstrating malignant cells in biopsy material

Solution required:

Thionin

1%

aqueous.

Technique:
1.

Stain frozen sections for ten to sixty seconds in the thionin

solution.
2.

Rinse in water.

3.

Mount

in tap water or in

Aquamount.

Results:

Nuclei, blue to purple.

Collagen, red. Elastic tissues, green.

213

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

TOLUIDINE BLUE
For mucus
Solution required:

Toluidine Blue 1-5% aqueous.

Technique:
1.

embedded

wax

in the

to distilled water then stain for

one or

Formalin-fixed material

is

in paraffin

usual manner.
2.

Bring sections

two minutes
3.

into
4.

Wash

down

in the toluidine blue solution.

with

drain well, then plunge the slide

distilled water;

two changes of

95%

alcohol.

Dehydrate by immersing in two changes of acetone; then

and mount.

clear in xylol

Results:

Mucus, reddish

nuclei, blue;

violet;

erythrocytes,

yellow to

greenish yellow.

TRICHROME STAIN
(G.

Gomori)

A. Delafield or Ehrlich Haematoxylin.

B.

Lithium carbonate

C. Alcohol

1%

aqueous.

97 ml.

70%

HCl, concentrated
D. Picric acid

1%

in

50%

Phosphotungstic Acid 3%.

F.

Light green, or Fast green

Aniline blue
.

Neoponceau (Michrome)
G. Solution

Acetic acid

2%

3 nil-

FCF,

or

214

alcohol.

E.

3 to

gm.
gm.
i volume
4 volumes
0-5

1-5

SECTION TWO
Technique:
1.

Fix tissues in Bouin or

2.

Stain sections in the Haematoxylin solution for ten minutes.

3.

Blue in the lithium carbonate solution.

Formalin.

io%

HCl

with the

4. Differentiate, if necessary,

alcohol for pre-

dominance of the green or blue shades, or in picric alcohol for

predominance of red shades, in the final picture.
5. Immerse the preparation in 3% phosphotungstic acid for
ten to fifteen minutes.
6.

Wash

gently under the tap for one minute.

7. Stain in solution

Note: The time

not

is

for five to twenty minutes.

critical

although results will be slightly

different.
8.

Rinse in

9.

Dehydrate, clear and mount.

2%

acetic acid.

Results:

Nuclei, blue. Cytoplasm, muscle fibres, red

of red. Connective tissue green or blue.

cells, etc.,

in shades

From

personal communications with Professor G. Gomori of the

Department of Medicine University of Chicago, U.S. A., to whom
I am indebted for permission to include this hitherto unpublished
^

technique.

Note: Professor Gomori has used Woodstain scarlet, which is

not available under that name in Britain. The British equivalent
is

Neoponceau and

this case as the

I feel that this

name Woodstain

synonym

scarlet,

is

more

general biology, might suggest a botanical stain for

TRICHROME STAIN

suitable in

indexed in literature on

woody tissues.

(Masson), Modified

For epithelium, pituitary and thyroid glands, nerve (normal

and tumour), etc.
Solutions required:

A. Regaud's haematoxylin.
B. Picric acid saturated in

Alcohol
Q

95%

215

95%
.

alcohol 20 ml.
.

10 ml.

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

Violamine-acid fuchsin :

Violamine,

0-7 gm.

0-3 gm.
i ml.

100 ml.

Acid fuchsin

Glacial acetic acid

Distilled water

D. Acetic fast green

Fast green,

F C F

Glacial acetic acid

Distilled water

gm-

2 ml.

100 ml.

Technique :
1

Tissues are fixed in Bouin, Regaud, Zenker or formalin, and

in paraffin wax.

embedded
2.

Mordant

sections

on

slides

with

5%

iron

alum previously

heated to 45 C.
3.

Wash

with tap water; stain for five minutes in Regaud's

then rinse with 95% alcohol.

haematoxylin

4. Differentiate with picric alcohol;

tap water.
5.

with
6.

then wash with running

then wash

Stain for five minutes in Ponceau-acid fuchsin;

distilled water.

Differentiate for five minutes in

1%

phosphomolybdic acid;

then without rinsing:

7.

Flood the

slide

with solution

and leave for

five to ten

minutes.
8.

Rinse with

distilled water;

then return to

1%

phospho-

molybdic acid for five minutes.

9.

10.

Leave in

1%

acetic acid for five minutes.

Dehydrate in 95% alcohol, followed by absolute alcohol;

and mount.

clear in xylol

Results:

Nuclei, black. Argentaffin granules, black or red. Cytoplasm,

vermilion. Collagen, green. NeurogUa fibrils, vermilion. Mucus,
green. Keratin, vermilion. Intercellular fibrils, vermilion. Golgi
apparatus, clear.

216

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

UREA SILVER NITRATE

For nerve

and nerve endings

fibres

Note: In this technique, nerve fibres and nerve endings of

the peripheral and central nervous system are preferentially
stained.

Applied to paraffin sections on

slides,

the technique gives rapid

and eliminates the necessity of gold toning.

The following fixatives are recommended:
Chloral hydrate
25 gm.
(I).

and constant

results,

Alcohol
(II).

50%

Formalin,

Ammonium
Formalin,

aldehyde
(IV).
(V).

undiluted

aldehyde 40%).
Alcohol 95%
(III).

(i.e.

bromide

undiluted

40%

20 ml.
80 ml.
2 gm.

Form-

(i.e.

. .

100 ml.

Form-

15 ml.

or Absolute Alcohol

95%

Bouin's fluid.

I, II, III and IV are satisfactory for Central Nervous

and
nerve
trunks.
System
Solution I has been used with satisfaction for striated muscle

Solutions,

tissue.

Solution II

Solution

is

suitable for gland

and smooth muscle

tissue.

V for gland and smooth muscle tissues and for embryos.

Tissues may also be fixed in 10% formalin with good results,

but an excessive staining of connective tissue has been observed
when this fixative has been employed.
Solutions required:
A. Picric acid, saturated aqueous

Mercuric cyanide
B.

1%

Silver nitrate

Urea

aqueous

3 drops

C. Hydroquinone

Urea

2gm.
25 gm.
100 ml.

Distilled water

Sodium

gm.

100 ml.
25 gm.

Solution

50 ml.
0-5

sulphite,

anhydrous

217

10 gm.

SECTION TWO
Technique:

Fix the material in one of the above fixatives

embed in paraffin wax.

1.

(I, II, III,

IV

or V) and
2.

Fix sections to sUdes and remove paraffin wax with xylol.

3.

Rinse with two changes of absolute alcohol.

4.

Wash

with

followed by

90%

80%

alcohol.

5. Immerse slides directly into solution

half hours at 50 to 60 C. in an oven.

for

one to one and a

6.

Rinse quickly in two changes of distilled water.

7.

Reduce by immersing

25 to 30

C,

in solution

8.

Wash

thoroughly in four or

9.

Wash

with

10.

for three minutes at

agitating the slides gently for the first

five

followed by

50%

changes of

70% and 80%

two minutes.

distilled water.

alcohols.

Examine under the microscope while the preparation

wet and

found that the staining

is still

not complete, repeat step

urea-silver
nitrate
and reducing the
the
solution
original
5 using
time to ten to fifteen minutes then repeat steps 6, 7, 8 and 9.
if it is

is

11.

Rinse with

12.

Dehydrate with two changes of absolute alcohol.

13.

Clear in xylol and mount.

95%

alcohol.

Results:

Nerve

from brown to black, while nerve endand nerve cells from yellow to brown. The

fibres are stained

ings are usually black,

background is usually yellow, but its appearance depends upon

the kind of tissue and the fixative employed.
Reference: Ungewitter, L. H. (195 1), Stain Tech., 26, p. 75.

VERHOEFF'S STAIN
For elastic

nuclei and collagen

fibres,

Solutions required:

A. Haematoxylin
alcohol

5%
.

218

absolute

in
.

20 ml.

SECTION TWO
Ferric chloride (hydrated)

aqueous

Iodine solution

(i

io%

gm. KI, 50 ml. water)

Note: Solution

8 ml.

iodine, 2

gm.

8 ml.

deteriorates after twenty-four

hours.
B.

Ferric chloride hydrated

C.

Van Gieson

2% aqueous.

stain.

Tissues should be fixed in Zenker or in

former

10%

formalin:

if

the

used mercurial precipitates are removed by the iodine in

the staining solution and it is not, therefore, necessary to treat the
sections or tissues with iodine before staining.
is

Technique:
Paraffin wax, Celloidin or
1.

L.V.N, may be used for embedding.

Sections are brought down to distilled water; then imin Solution

for one quarter to one hour until quite black.

mersed

2. Differentiate for a few minutes in Solution B,

controlling by
examination in water under the low-power objective.
3.

Wash

remove
4.

with tap water;

then immerse in

Wash

Van Gieson

in tap water for five minutes;

for three to five minutes.

alcohol to

Differentiate in

6.

Paraffin sections are cleared in xylol

terpineol (after

Mount

95%

95%

then counterstain in

alcohol; then dehydrate.

5.

7.

95%

iodine.

Celloidin or L.V.N, in

alcohol).

in Cristalite or in balsam.

Results:
Elastic fibres, intense blue-black to black.

Collagen, red.

Nuclei, blue to black.

Other tissue elements, yellow.

219

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

WATER BLUE

ORCEIN

SAFRANIN

For demonstrating epithelial fibres

Solutions required:

A. Water blue
Orcein
.

Glycerine
Absolute alcohol
.

Acetic acid

5%

gm.

0-75 gm.
20 ml.

50 ml.
100 ml.

Eosin 1-25% alcoholic.

B.

C. Hydroquinone

1%

aqueous.

D. Safranin O, aqueous 1%.

Potassium dichromate 0-5%.

E.

Technique:
1.

Specimens of skin are fixed in

either in paraffin

wax

10%

formalin and

embedded

or in Celloidin.

2. Bring sections down to distilled water; then stain for ten

minutes in a mixture consisting of:

3.

Wash

Solution

Solution

Solution

10 ml.
3

^^

3 rnl.

well in distilled water.

4. Stain for ten minutes in the safranin solution.

5.

Wash

6.

Immerse

thoroughly in
in

distilled water.

0-5% potassium dichromate solution from ten

to thirty minutes.
7.

Wash

in distilled water; dehydrate in absolute alcohol ; then

clear in oil of bergamot.

8.

Examine under the microscope; then

if

necessary differ-

entiate alternatively with absolute alcohol and oil of

until the depth of the safranin stain has been reduced.
9.

Mount

in

balsam or in

bergamot

Cristalite.

Results:
Epithelial fibres are stained red, while the nuclei are pale violet

220

SECTION TWO
cytoplasm, blue to violet; granules of the
neutrophil leucocytes, sky blue; elastic fibres, red; collagen fibres,

plasmasomes, red;

blue.

WEIGERT

FRENCH ELASTIN STAIN

(Moore's modification)
This modification, which

is

due to G.

W. Moore,

of the Central

Histological Laboratory, Archway Hospital, London, gives greater

than either Sheridan or Weigert elastin stains, and

selectivity

consistently excellent results are obtained provided the stain

is

properly prepared.
Solutions of Moore's elastin stain will keep for several years
without deterioration.

The dry stain requires time and great care for its preparation;
the majority of workers will, no doubt, wish to purchase the stain
ready for use, but for those who have the time and prefer to prepare the stain themselves, the method is given below.
Preparation of the dry stain:

A. Ferric chloride, anhydrous, A.R.

grade

Distilled water

30 gm.

65 ml.

Dissolve; then make up the volume to 100 ml. with distilled water.

Note: This solution must be freshly prepared.

.

Crystal violet

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

Technique:
1.

When

Solution

begins to

boil,

add 62 ml. of Solution

slowly, in small portions at a time over a period of five minutes,

with constant

stirring.

Continue with the boiling and stirring for a further period of

two or three minutes until a coarse precipitate is obtained.
2.

3. After cooling filter by means of a Buchner funnel and flask

attached to a suction pump.

Wash

the precipitate with distilled water until the runnings

and the filtrate a clear azure blue; this usually
8
to
10
litres distilled water.
requires
4.

are colourless

5.

after

The

preparation

removing the

is

filter

then dried overnight in an incubator,

paper.

Preparation of the staining solution :

1.

The

dried elastin stain

alcohol plus

HCl

ml.

now

is

in a

dissolved in 550 ml. absolute

flask, the neck of which is

i -litre

lightly with cotton-wool. Solution is effected by boiling

gently for about thirty minutes on a water bath or electric hot-

plugged
plate.

2. Cool; filter; add 19 ml. concentrated HCl; then shake well

and allow to stand for at least twenty-four hours.

Staining technique:
1.

with
2.

Sections are brought

0'5% aqueous

potass,

Rinse and bleach with

down

to distilled water;

permanganate

5%

then treated

for five minutes.

aqueous oxalic acid; then wash in

running water.
3.

Transfer to elastin stain for at least two hours

for one half to one

hour in an oven

at 37 C. or

at 60 C.

4.

Blot and treat with absolute alcohol for three to five minutes.

5.

Rinse with water and counterstain for three minutes with

neutral red (Jensen).

6.

Rinse and differentiate the neutral red for a few seconds in

absolute alcohol.
7. Rinse in distilled water, then pour on 0-5% picric acid
aqueous and wash off immediately with running water.

222

SECTION TWO
8.

Blot;

dehydrate in absolute alcohol;

clear in xylol

and

Erythrocytes

and

mount.
Results:
Elastic

fibres,

blue-black.

Nuclei,

red.

muscle, yellow.

Notes: Best results are obtained after the stain has been kept in
stock for several weeks,
remains so indefinitely.

The

when

it

becomes perfectly

which can be

"

selective

and

**

occatopped-up
is
to
be
recommended.
loss
make
to
evaporation,
good
by
sionally
The picric acid gives a beautiful contrast to the neutral red and

use of Coplin's

jars,

enhances the appearance of the elastic fibres by causing them to

stand out against a neutral background. Care must be taken when
using it, however, as overstaining tends to give the red nuclei an
unpleasant brownish tinge. It is perhaps advisable, until the
technique has been mastered, to use neutral red only, ensuring
that

it is

properly differentiated in absolute alcohol.

WEIGERT

PAL TECHNIQUE

For myelin sheaths in brain and spinal cord and for peripheral nerves and ganglia

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

Technique:
1.

Slices of the material 2 to 5 mm. thick are fixed in 10%

for four to seven days.
then transferred to Solution

formalin
2.

Wash

and embed
sections (in

running water for several hours; then dehydrate

L.V.N, or in paraffin wax, or cut frozen
which case 2% ammon. bromide should be added to

in

in Celloidin,

the formalin fixing solution).

Sections 10 to 20fi in thickness are stained twenty-four to
forty-eight hours in a freshly prepared mixture consisting of i vol3.

ume
4.

Solution

B and

Immerse

for one half to three minutes in Solution

9 volumes Solution C.

then

rinse in distilled water.

5.

until the white matter

is

one half to three minutes or

blue-black, and the grey matter almost

Differentiate in Solution

for

colourless.
6.

Counterstain with safranin 1% aqueous, if desired, for one

two hours according to thickness of sections.

half to
7.

Wash

8.

Dehydrate

thoroughly in water.
;

clear

and mount.

Results:

Myelin sheaths, blue-black. Myelinated fibres, black or blueGrey matter, white or slightly yellow. Other structures,
unstained (unless a counterstain has been used).

black.

WOOL GREEN

HAEMATOXYLIN

PONCEAU

For connective tissue and muscle

Solutions required:

A. Picric
B.

acid, saturated in

70%

alcohol.

Weigert's haematoxylin A.

C. Ponceau S

1%

in

1%

acetic acid aqueous.

D. Weigert's haematoxylin B.
E. Wool Green S 1% in 1% aqueous

acetic acid.

F. Acetone and xylol, equal volumes of each.

224

SECTION TWO
Technique:

two minutes.

1.

Immerse

2.

Wash

3.

Stain for five to seven minutes in Weigert's haematoxylin A.

4.

Rinse in water and stain for three to five minutes in the

sections in the picric acid solution for

thoroughly in running tap water.

Ponceau S

solution.

Wash

in water.

5.

6.

Immerse

7.

Wash

8.

Stain for three to five minutes in the wool green solution.

9.

Decolorize for two minutes with

ID.

Pour

in Weigert's haematoxylin B.

thoroughly in water.

1%

acetic acid.

off excess acid; rinse well in distilled water; drain

and

blot carefully.
1 1

Rinse well with acetone.

12.

Rinse with two washings of Solution F.

13.

Clear in xylol and mount.

Results:

Muscle and cytoplasm,

membranes, green

Connective tissue and basement

red.

to dark blue.

WRIGHT'S STAIN
For

general differentiation of blood corpuscles;

malarial parasites; trypanosomes, etc.

for

This stain is extensively used in America instead of Leishman

which is preferred by British workers.

stain

Solutions required:
Formol-saline^ neutral^ buffered:

A. Formalin (40% formaldehyde)

Sodium chloride, A.R.
Distilled water
.

100 ml.
8-5

gm.

i litre

Acid sodium phosphate, monohydrate

Anhydrous disodium phosphate

225

4 gm.
6-5 gm.

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

B.

Wright's

stain.

C. Acetic acid,

o-o8% aqueous.

Technique:
1.

Fix pieces of tissue in Solution

for sixteen to forty-eight

hours.
2. Dehydrate in the usual ascending grades of alcohol;
and embed in paraffin wax.
3.

clear

Fix sections, not exceeding 5// in thickness to slides; remove

pass through descending grades of alcohol down
xylol

wax with

to neutral distilled water.

Stain for three to five minutes in a freshly prepared mixture

consisting of one volume of Wright's stain and two volumes of
4.

neutral distilled water, in a stoppered staining jar.

5.

Rinse with neutral

distilled water.

6. Differentiate with the acetic acid solution, controlling by

examination under the microscope, until the protoplasm of the

cells is pink,

and only nuclei are blue.

7.

Wash

8.

Dehydrate quickly with absolute alcohol;

mount

with neutral

distilled water.

clear in xylol;

in Cristalite.

Results:

Erythrocytes, yellowish red. Polymorphonuclears, dark purple

nuclei, reddish violet granules, pale pink cytoplasm. Eosinophiles,
blue nuclei, red to orange-red granules, blue cytoplasm. Baso-

dark blue nuclei, dark purple to black granules.

dark
purple nuclei, sky blue cytoplasm. Platelets,
Lymphocytes,
Malarial parasites and Leishmanial
violet to purple granules.

philes, purple to

chromatin, red;

cytoplasm, blue.

Trypanosomes

chromatin,

red.

Note:

may be

The timing of the staining either before or after dilution

altered to suit individual requirements.

Staining effects similar to Giemsa are obtained by staining for

ten minutes in Wright's stain diluted with four times its volume of
distilled

water buffered to

pH

6-5.

226

SECTION 3 BOTANICAL METHODS

(Normal and Infected Tissues)

(a)

GENERAL TREATMENT OF TISSUES

Material must be killed and fixed immediately it is collected to

ensure that tissues are preserved with as life-like an appearance
as possible.

Small organisms such as unicellular algae, filamentous fungi,

etc., may be placed directly into the killing fluid.
Large objects
must be cut at once into pieces not exceeding 5 mm. in any one
direction, and care must be taken to avoid rough handling and
pressure.

There are a great number of killing and fixing fluids to choose

from and the reader should refer to the chapter on fixatives in this
book as well as to standard text-books on botany such as Johansen
or Chamberlain.

The best general killing fluid, however, is 70%

o-i% glacial acetic acid; material should be

alcohol containing

immersed

in this fluid

from ten minutes

to an hour.

After killing and fixing, material may be preserved, if it is not

required for immediate examination, in 70% alcohol containing
20% glycerine, or in 10% formalin buffered to pH 7-0.
Delicate material should be stored in 85% alcohol containing
20 ml. glycerine per 100 ml. while tough hard material is best stored
in 50% alcohol containing 20 ml. glycerine per 100 ml., but here
again the reader is referred to standard text-books on botanical

technique.
Material taken from the fixing and killing fluid or from the
preserving fluid should be washed well in running water. Tissues

may be
tissues.

dehydrated, cleared and embedded, as in the case of animal

Alternatively, sections may be cut, without dehydration,

clearing

and embedding,

either

by means of a hand microtome,

or by placing thin slices of the material in a slit cut in a piece of

"
'*
with the razor edge
elder pith and cutting sections
freehand
from
the
freehand
In
sections, the
operator.
cutting
facing away
razor should be kept wet with 10% aqueous glycerine.
Glycerine (pure or diluted with water), or glycerine jelly should

be employed for mounting temporary preparations after staining,

while the preparation of permanent slides may be carried out in
229

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

same manner

as for sections of animal tissues, except that

should
be more gradual in order to avoid violent
dehydration
diflFusion and consequent shrinkage and distortion of delicate
tissues; the alcohols used should be graded 50%, 70%, 80%,

the

90%, 95% and

(b)

absolute.

MISCELLANEOUS MICROCHEMICAL TESTS

Note: In the following tests fresh material, in general, should

be employed and thick sections, up to about 40 or 50/^ are preferable to thin ones, particularly if the substances under test occur
in small proportions.

Aldehydes
Solution required:

Diphenylamine

1%

in

concen-

trated sulphuric acid.

Technique:
1.

Sections of strictly fresh tissue are placed directly into a drop

of the reagent on a
2.
3.

slide.

Heat gently until a green coloration appears.

Continue the heating for about five minutes.

Results:
If the green colour persists, formaldehyde is indicated but if the
green turns to red, aldehydes, other than formaldehyde, are present

in the tissue.

Aleurone
This occurs in the seeds of Poaceae
Solution required:

Eosin

2%

in saturated alcoholic

picric acid.

Technique:
I.

slide

Sections are immersed in a few drops of the reagent on a

and examined under the microscope.

230

SECTION THREE
As soon as the ground substance

2.

of the aleurone grains appear

add a few drops of absolute alcohol, when the globules

should be colourless and the crystals a yellowish green.
blue,

Remove

3.

excess alcohol;

and mount

clear in clove oil

in

balsam.
Results:

Protein (aleurone) crystals, yellow.

substance, dark red.

Globules, pink.

Ground

Amygdalin
This occurs in the seeds of Amygdalus; Pyrus Crataegus and
related genera, as well as the leaves of Prunus laurocerasus
Solutions required:

A. Picric acid, saturated, aqueous.

B.

Sodium carbonate 10% aqueous.

4%

C. Potassium hydroxide

in

70%

alcohol.

D. Ferrous sulphate 2-5% aqueous (prepared without

heating).

E.
F.

Ferric chloride

20%

aqueous.

Hydrochloric acid, cone.

Distilled water

volume
4 volumes
i

Technique (a):
1

on

Immerse

sections in the picric acid solution for half an hour,

slides.
2.

Wash

with water

then add one drop of the sodium carbon-

ate to a section.

Result:

A red coloration indicates

the presence of amygdalin, and this

confirmed by the odour of hydrocyanic acid (almond odour),
which is a decomposition product upon which tests for amygdalin

is

are based.

231

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

Technique (b):
1.

Place sections in the sodium hydroxide (Solution C) for

three to five minutes.

2.

Mix

(Solutions

equal volumes of the ferric sulphate and ferric chloride

and E above) in a test-tube or small beaker and heat

to boiling.
3.

Immerse sections in the above mixture and leave them therein

for five to ten

minutes.

4. Place sections on a slide in a drop of the diluted hydrochloric

acid (Solution F above).

Results:

deep blue precipitate (Berlin blue reaction) indicates the

presence of hydrocyanic acid which is indicative of amygdalin.

Amylodextrin
This product, which

intermediate between maltose and starch,

is

present in solution in storage organs where starch is hydrolysed. Its presence is indicated by the red coloration it produces
is

with Gram's iodine solution

Anthocyanin
This occurs

in the petals of the vast majority of blue and red

owe their colour to anthocyanin

flowers; such flowers

Solutions required:

A.

Glacial acetic acid.

B.

Ammonia

solution.

Technique:
1.

Place petals in a

2.

Cover with thick coverslip (No. 2 or No.

little

neutral distilled water

3)

on

slides.

and press out the

coloured liquid.
3.

Run

in a

drop of

glacial acetic acid

232

under the

coverslip.

SECTION THREE
Take another slide and repeat the process but run
ammonia solution instead of acetic acid.

4.

in a

drop

of strong
Results:

Anthocyanin

is

red in acid solution and blue violet to green in

alkaline solution.

Arbutin
This occurs in Ericacae and Pyrolacae
Solutions required:

A. Nitric acid
B.

Ammonia

10%

solution.

C. Ferric chloride

5%

aqueous.

Technique {a):

1.

Place sections in the nitric acid solution.

2.

Cover with a coverslip and examine under the microscope

mmediately.

Results:
Cells containing arbutin assume a dark orange colour, which
rapidly changes to yellow which slowly disappears altogether.

Technique (b):

This depends upon the principle that arbutin

hydrolysis, to glucose and hydroquinone.

is

converted, on

1
The dry sections are placed in a drop of water on a slide and
heated gently for two or three minutes, when the arbutin sublimes
.

in crystals.
2. Add a drop of ammonia solution when the arbutin
assume a rich brown colour.
3.

crystals

Repeat the process adding a drop of ferric chloride solution

ammonia a pale green colour confirms the presence of

instead of

arbutin.

233

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

Asparagine
This occurs widely in the plant kingdom, but is most readily
demonstrated in etiolated seedlings of Lupins and tubers of
Dahlia
Solution required:

Cupric acetate

5%

aqueous.

Technique:
1. Place strictly fresh sections in a drop of the cupric acetate
a slide and leave therein for ten to twenty minutes.

on

2. Add absolute alcohol slowly, a drop at a time, whilst examining under the microscope, until ultramarine spaerocrystals of
copper asparagine become visible, indicating the presence of

asparagine in the tissue.

Calcium
Solution required:

Oxalic acid

2%

aqueous.

Technique:
1.

Sections are placed directly onto a slide and flooded with the
and left for half an hour exposed to the air.

oxalic acid solution

2.

Pour

off

some

of the liquid

then add a coverslip.

Pipette a drop of absolute alcohol along one edge of the

coverslip, so that the alcohol is drawn under the coverslip by
capillary attraction. Examine under the microscope.
3.

Results:

Calcium,

if

present, will be indicated

by the small but

visible crystals of calcium oxalate.

Calcium Oxalate
Solutions required:

A. Cupric acetate, saturated, aqueous.

B.

Ferric sulphate
Acetic acid 20% aqueous
.

234

. .

5 gn^

100 ml.

easily

SECTION THREE
Technique:
Sections are placed directly into a drop of the cupric acetate
on a slide and left therein for about ten minutes.

1.

solution

Examine under the microscope and

2.

if

calcium oxalate crystals

are present they will have dissolved and the oxalic acid diffused
into the intracellular spaces where cupric oxalate is formed.

To test for the

dissolved oxalate, add a few drops of the ferric

sulphate (solution B) and examine under the microscope. The
appearance of yellow ferrous sulphate crystals confirms the pres3.

ence of calcium oxalate, in the

tissue.

Callose
Solution required:

Lacmoid

alcoholic

Distilled water

..

..

. .

o-i ml.

25 ml.

Technique:

Immerse

1.

minutes,

when

sections in the lacmoid solution for about fifteen

callose, if present, is stained a brilliant blue.

Mount in a drop

2.

of glycerine on a slide and examine under the

microscope.

The
callose
{a)

following solubility tests may be employed to distinguish

from other membrane substances:

Soluble in copper oxide ammonia, while cellulose and hemi-

celluloses are insoluble.

{b)

Swells but

is

insoluble in solutions of

ammonia, sodium

carbonate, potassium carbonate, whilst pectic acid

these reagents.
{c)

is

soluble in

Soluble in glycerine at 280 C. whilst cellulose and chitin are

insoluble.

Carotin
Solution required:

Potassium hydroxide

20%

in absolute alcohol.

Technique:
I

Place sections of fresh young green leaves in the potassium

235

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

hydroxide solution in a stoppered jar and leave in the dark until
the chlorophyll

is

extracted.

the sections from the jar and wash

hours in running water.

Remove

2.

them

for ten

to a large volume of distilled water and leave therein

3. Transfer
for an hour, afterwards transferring to a fresh lot of distilled water

for a further period of one hour.

Transfer to slides

4.

mount

in glycerine.

Results:
if

Carotin,
will

appear

present, appears as orange-red crystals. Xanthophyll

two or three days as yellow crystals.

after

Cellulose
Solutions required:

A. Gram's iodine.
Sulphuric acid 75%.

B.

Technique:
1.

Place sections in a drop of Gram's iodine solution on a slide.

2.

Cover with a coverslip;

examine under the microscope,

taking careful note of the location of the blue coloration.

Place a drop of the sulphuric acid solution along the edge of

the coverslip and observe the swelling of the cellulose membranes
as the sulphuric acid seeps under the coverslip, hydrolysing the
3.

cellulose to a colloid substance

known

as hydrocellulose.

As

certain other plant substances give a blue colour

with
iodine it is important to note the locality of any blue
reaction

Note:

colour which appears prior to the hydrolysation with the sul-

phuric acid.

Chitin

This

is

said to occur principally in the higher fungi.

Solution required:

A. Potassium hydroxide
Distilled water
.

236

. .

78 gm.
68 ml.

SECTION THREE
Technique:
1

Heat the potassium hydroxide solution

in an

open beaker

to

boiling point.
2.

now be covered
and continue the boiling for half an hour.

Place the sections in the beaker which should

with a clock

glass,

3.

Remove

4.

Treat the section with Gram's iodine solution.

the sections and wash in

90%

alcohol.

Results:

Chitin,

if

present in the tissue, will be indicated by a reddish-

violet colour.

Chlorides

Occur

in roots of Dauctis carota

and Beta; Primula ohconica;

solanum
Solutions required:

A. Silver nitrate
B.

Nitric acid

5%

aqueous.

1-5% aqueous.

Technique:
1
Sections, which must be cut with a scrupulously clean razor,
are placed in a drop of the silver nitrate solution on a slide.
.

2. Examine, without a
coverslip, under the microscope,
the silver chloride precipitate will appear black.

Note: The presence of chlorides in the tissue

which is white to the naked eye.

is

when

indicated

by

a precipitate

3. By means of glass needles transfer the sections to a drop of

the diluted nitric acid solution on another sHde.

4.

Examine under the microscope when

it

will

be observed that

the acid clears the sections sufficiently to allow localization of the

reaction.
5. Repeat stage 3 (above); add a few drops of ammonia solution to the slide until the precipitate just dissolves.

237

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

Examine again

6.

appear

an hour when the precipitate will re-

after

in crystalline form.

Chlorophyll
Solutions required:

A. Potassium hydroxide

methyl

20%

in

pure

alcohol.

Technique:
1

Place sections directly onto a slide and add two or three drops

of ether.
2.

Add

few drops of the potassium hydroxide solution.

Results:

Chlorophyll immediately turns brown, afterwards changing

back to green again.

Formic Acid
Solutions required:

A. Mercuric chloride
B.

1%

aqueous.

Hydrochloric acid, cone.

C. Potassium hydroxide

1% aqueous.

Technique:
Place fresh sections on slides; flood with the mercuric
chloride solution and heat on a water bath for an hour.
1.

off excess mercuric chloride solution and wash with diswater which has been acidified by the addition of i ml. hydrochloric acid cone, per 100 ml.
2.

Pour

tilled

3.

Place sections on slides; add one drop of

1%

hydroxide, and examine.

Results:

Where formic

acid

is

present, the cells are blackened.

238

potassium

SECTION THREE
Glutathione
Solutions required:

A. Acetic acid i%.

B.

Ammonium

saturated,

sulphate,

aqueous.
C.

Sodium

nitroprusside

5% aqueous.

Technique:
1.

Place thin sections of strictly fresh tissue on slides.

Flood with 1% acetic acid and heat gently till vapour rises.
This is most conveniently done by placing the slides over a corner
of a tripod and applying the heat by means of a very small bunsen
flame which should be held some distance away from the underside
2.

of the slide.
3.

Transfer to a watch glass and rinse in the

ammonium

sul-

phate solution.
in a mixture consisting of 0-5 ml. of the sodium
and 5 ml. of saturated ammonium sulphate,
solution
nitroprusside
in a watch glass.
4.

Immerse

Agitate gently but thoroughly

a few minutes.
5.

6.

by rocking the watch

Whilst watching the sections closely, add

ml. of

glass for

ammonia

solution.

Results:
If glutathione is present the cells will assume a red colour,
lasts only a second or so, with the addition of the

which usually

ammonia

solution.

Inulin

Occurs

in

bulbs of Dahlia

variabilis^ etc.

Solutions required:

A.

Thymol 15%

B.

Chloral hydrate
Distilled water

in absolute alcohol.
.

239

10 gm.
4 ml-

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

Technique:
1.

Fresh tissues are fixed in

2.

Place sections in alcohol on slides and add a drop of the

alcohol for three or four days.

70%

chloral hydrate solution.

Examine under the microscope and observe concentric

3.

layers of inulin crystals, if present.

4.

Add one

drop of the thymol solution followed by concen-

trated sulphuric acid.

Results:

On

addition of the last two reagents inulin crystals immediately

red, dissolving after a minute or so.

become

Iodine
Solutions required:

A. Starch
B.

1%

Potassium

aqueous suspension.

20%

nitrite

aqueous.

C. Hydrochloric acid cone.

Distilled water
.

ml.

19 ml.

Technique:
I
Place fresh sections in a watch glass containing 2 or 3 ml. of
the starch suspension together with three or four drops each of
Solutions B and C.
.

Results:

Iodine

is

indicated

if

the starch

is

coloured blue.

Iron
Note:

The

use of iron or steel instruments in the following

should
be avoided. The section razor, which must,
technique
be
should
be scrupulously clean.
however,
used,
Solutions required:

A. Hydrochloric acid concentrated

Distilled water
Potassium ferrocyanide 10%
.

B.

Alum

carmine.

240

2 ml.

98 ml.
2 ml.

SECTION THREE
Technique:
1

Sections of fresh material are taken from distilled water and

immersed

4.

(above) for one half to one hour.

well with several changes of distilled water.

alum carmine solution

Stain the nuclei with

3.

utes

in Solution

Wash

2.

then wash well with

for a

Dehydrate through ascending grades of alcohol

mount in D.P.X. or Cristalite.

clear in xylol

few min-

distilled water.

as usual,

Results:

Iron

Nuclei, red.

(if

present), blue.

Lecithin
Solutions required:

A. Scarlet
B.

(Botanical).

Delafield haematoxylin.

Technique:
1.

Fat

removed from

is

sections

by immersion overnight

in

acetone, in a stoppered jar or well-corked tube.

2.

Fix sections

to

slides;

rinse

in

two changes of pure

acetone.
3.

Stain

the

scarlet

solution

for

about ten

to

fifteen

minutes.
4.
5.

6.

Wash
Wash

quickly with

70%

alcohol.

in distilled water.

Counterstain with Delafield haematoxylin for two to ten

minutes.
7.

Blue and wash in tap water.

8.

Mount

in glycerine or

Aquamount.

Results:

Lecithin

(if

present), red.

Nuclei, blue.

241

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

Nitrates
Solution required:

Diphenylamine

Sulphuric acid, cone.

Distilled water

gm.

75

"il-

25 ml.
Add the sulphuric acid cautiously to the water in
small portions (about 5 ml.) at intervals in a 250-ml.
conical flask, swirling the contents round gently to
.

ensure thorough mixing.

Do

not on any account add the water to the acid as

this will result in the acid flying back and causing
serious injury to the face

and hands

clothing etc.
,

After the diluted acid has cooled somewhat,

add

the diphenylamine.
Technique:
1.

Place sections on slides under coverslips.

2.

Place two drops of the diphenylamine solution along one

edge of the coverslip.

Results:

are present a deep blue colour develops as the

diphenylamine-sulphuric acid solution seeps under the coverslip
If nitrates

and comes into contact with the section. After a few minutes, the
and the colour changes to light brown.

section disintegrates

Pectic Substances
Solution required:

A. Ruthenium red, 0-02% aqueous

Technique:
1.

Stain with the ruthenium red for thirty minutes.

2.

Mount

in glycerine

on a

slide.

Results:

Pectic substances are stained red.

242

SECTION THREE
Phosphates
Solution required:

Ammonium

io%

molybdate

in

concentrated nitric acid.

Technique:

Place sections on a slide

under the microscope.

drop of the reagent, and examine

in a

Results:

Phosphates are indicated by small black-bordered yellow drops,

into spaerocrystals and afterwards into cubes and

which develop
octahedrons.

Phjrtosterol
Solution required:

Gram's

iodine.

Technique:
1.

Place thick sections on slides and cover with concentrated

sulphuric acid.
2.

If phytosterol is present sections will assume a red colorAdd a drop of Gram's iodine solution and mix by rocking

ation.

the slide backwards and forwards gently.

Results:

The presence of phytosterol is confirmed when the colour

changes from red, after the addition of the iodine, to violet then
blue and finally to yellowish red or brown.
;

Potassinm
Solution required:

Sodium Cobalt

Sodium

nitrite

Cobalt nitrate
Distilled water

Nitrite.
.

7 gn^

Glacial acetic acid

243

g"^-

13 ml.
2 ml.

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

Technique:
I. Sections of fresh tissue are
placed in a drop of the above
reagent on a sHde and examined under the microscope.

Results:

The appearance
nitrite indicates the

of fine yellow crystals of potassium cobalt

presence of potassium.

Proteins
Solutions required:

A. Potassium ferrocyanide
Acetic acid
.

B.

Ferric chloride aqueous

o-8 gm.
loo ml.

5%.

Technique:
1.

Immerse

2.

Rinse quickly with

3.

Add

sections of fresh material in Solution

few drops of

60%

5%

A for an hour.

alcohol.
ferric chloride.

Results:

blue coloration indicates proteins.

Saponin
Method

(a)

Place a drop of concentrated sulphuric acid on a section of fresh

material.
If saponin is present in the tissue the section immediately
assumes a yellow colour which changes to red after about half an
hour, and later to violet or bluish green.

Method

(b):

Solutions required:

A. Barium hydroxide, saturated aqueous.

Calcium chloride 5%.

C. Potassium dichromate 10% aqueous.

B.

244

SECTION THREE
Technique:

To

locate the sites of saponin immerse sections in the barium

hydroxide solution for sixteen to twenty-four hours ; then examine
1.

under the microscope and observe the insoluble colourless compound formed by the interaction of saponin and barium.
2.

Wash well with the

3.

Cover with the potassium dichromate solution and watch the

calcium chloride solution.

reaction under the microscope.

Results:

The

first formed between the barium and

broken
the
barium uniting with the chromium,
down,
saponin
barium
which
is identified by its yellow colour.
chromate,
forming
Cells containing tannin assume a rich brown colour during the

insoluble substance
is

reaction.

Sodium
Solution required:

Uranium

acetate, saturated aqueous.

Technique:
1.

Sections are placed in a few drops of the reagent on

a slide.
2.

Add one

3.

Place uncovered slides in a desiccator to facilitate the slow

drop of hydrochloric acid to the preparation.

evaporation of the reagents.

4.

Examine

at

hourly intervals over a

maximum

period of eight

hours.
Results:

Pale yellow rhomboidal or tetrahedral crystals (sodium uranium

acetate) indicate the presence of sodium in the tissue.

Note: The presence of magnesium

boidal crystals, with this technique.
245

is

indicated by large

rhom-

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

Sulphates
Solutions required:

A. Benzidine hydrochloride.

B.

gm.

Hydrochloric acid, cone.

Distilled water

3 ml.

Hydrochloric acid, cone.

10 ml.

Distilled water

90 ml.

97 ml.

C. Barium chloride 10%.

Technique {a):
Sections of fresh tissue are placed in a few drops of the benzidine solution on a slide and examined under the microscope.
Results:

Scales or small glistening needles (benzidine sulphate) indicate

the presence of sulphates.

Technique (b):

For cereal seeds and other

tissues

which contain fat.

Immerse the material overnight in acetone

or well-corked tube, to remove the fat.
1.

in a stoppered jar

2. Transfer to slides; wash in two changes of pure acetone;

then allow the sections to dry on the slides.

3. Add one drop each of Solutions B and C (above) to the slide

and examine under the microscope, under a high-power objective.

Results:

granular precipitate (barium sulphate) indicates the presence

of sulphates.

Tyrosine
Solutions required:

Sodium molybdate 1%

in

sul-

phuric acid, cone.

Technique:
1.

Place sections on slide and cover with absolute alcohol.

2.

Allow the alcohol

to evaporate completely.

246

SECTION THREE
with a few drops of the sodium molybdate
3. Cover the section
and warm gently for a few minutes.
Results:

deep blue colour turning to violet after a few minutes indicates

the presence of tyrosine.

(c)

STAINING TECHNIQUES

ACID FUCHSIN

AURANTIA

For diFerentiating between bacteria and mitochondria in

sections of infected tissue

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

5.

De-Stain for a few seconds in Solution

6.

Immerse

in Solution

for a

then wash in water.

few minutes; then rinse in

water.
7.
8.

Stain for a few minutes in Solution E, then

Dehydrate very quickly with

and mount.

wash with water.

and absolute alcohol;

95%

clear in xylol

Results:
Bacteria,

deep

ACID RUBIN

violet blue.

Mitochondria and

AURANTIA

plastids, red.

TOLUIDINE BLUE (KuU's

Stain)

For starch grains and mitochondria in plant tissues

Solutions required:

A. Acid rubin
B.

C.

1% aqueous.
5% in 80% alcohol
Tannic acid 2% aqueous.
Aurantia

D. Toluidine blue

1%

aqueous.

Technique:
1.

Fix material in Regaud's

2.

Stain for five minutes in solution

fluid.

heated to about 60 or

70 C.
3.

Differentiate with solution B, controlling

by examination

under the microscope.

4.

Wash

5.

Immerse

6.

Wash

7.

Stain for five minutes in solution D.

8.

Pour

9.

Differentiate in

in water.
in solution

for 20 minutes.

well in water.

off excess stain

90%

and

rinse with

alcohol.

248

70%

alcohol.

SECTION THREE
Dehydrate with absolute alcohol.
Clear in xylol, and mount.

10.
1 1

Results:

Mitochondria are stained red, while starch grains are blue.

Note: This

is

an adaptation of one of Volkonsky's techniques.

Reference: Milovidov (1928), Arch. Anat. Micros., 24,

9.

ANILINE HYDROCHLORIDE

simple and rapid method of demonstrating lignified

tissues
Solution required:

Aniline

10%
hydrochloride
aqueous, freshly filtered before

use.

Technique:
1.

Place sections on slides and cover with a few drops of the

and allow this reagent to act for

aniline hydrochloride solution,

about
2.

minutes.

five

Pour

off excess

place a drop of glycerine

on the section and

cover with a coverglass.

Results:

Lignified tissues are stained yellow while the other tissues remain
unstained.

BASIC FUCHSIN,

AMMONIACAL

For lignified walls and cutin

Solution required:

A. Basic fuchsin,
B.

10%

alcoholic.

Strong ammonia solution

25 ml.

The fuchsin solution is added drop by drop

ammonia until a yellowish colour is produced.

Note:
to the

249

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

The fuchsin is decolorized by the ammonia and if

much fuchsin is added, then a few more drops of
ammonia must be added so that the final product is

too

pale yellowish in colour.

Technique:
1.

Immerse

sections in the solution in a stoppered jar for five to

ten minutes.
sections to watch glasses containing
for
about
five minutes, until the alcohol takes
alcohol,
2.

Transfer

absolute

on

a pink

coloration.
3.

Transfer sections to a fresh

lot

of alcohol and leave therein

for about five to ten minutes.

4.

Rinse and dehydrate in a fresh

5.

Clear in clove

6.

Mount

lot

of absolute alcohol.

oil.

Canada balsam.

in

Results:

Lignified walls and cutin, intense red;

BORAX CARMINE
For bulk staining prior

remainder, colourless.

(Grenacher)

to sectioning,

and

for

small whole

mounts
Solutions required:

A. Borax carmine, alcoholic (Grenacher).

B.

Alcohol

70%

Hydrochloric acid, concentrated

100 ml.
0-25 ml.

Technique:
1.

Immerse material

in the carmine solution

from one hour

to

four days, according to the bulk and nature of the material, until
sufficiently stained.
2.

Rinse with and immerse in acid alcohol (Solution B, above)

until clear.
3.

Wash

4.

Dehydrate with

with

70%

alcohol.

95%

alcohol, followed

250

by

absolute.

SECTION THREE
5.

Clear in xylol.

6.

Embed

in paraffin wax.

7.

Cut sections and mount on

8.

De-wax with

9.

Mount

Note:

mount

in

slides.

xylol.

Canada balsam

For whole mounts,

in xylol.
after clearing in xylol (Stage 5),

in balsam.

Results:

Nuclei are stained deep red, while cytoplasm

is

pink.

CHLORAZOL AZURINE

A simple double stain, non-fading, and particularly suitable

for

elementary class work

Solutions required:

A. Formaldehyde 40%.
Acetic acid 50%

Absolute alcohol
Distilled water
B.

Magnesium

..

..

sulphate, crystals,

6%

5 ml.

14 ml.
63 ml.

20 ml.
aqueous.

C. Chlorazol azurine, saturated aqueous.

D. Equal volumes of Solutions B and C.

Heat the mixture to 80 C. and stir well for five or ten minutes,
taking care that this temperature is not exceeded by more than a
few degrees.
Technique:
1

Sections of the material, fresh from the field, are transferred

Alternatively material may be stored in this fixative

to solution A.

for several days before sectioning.

2.

Take

3.

Immerse

sections

down

to water.

sections in solution

(the solution should

be

shaken well immediately before use) in a jar or tube overnight, or

for at least eight hours.

251

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

4.
5.
6.

Wash well with tap water.

Wash quickly in 70%, 90% and 95% or absolute alcohol.
Mount directly in Michrome mountant or in Euparal.

Result:

Non-lignified cell walls, blue, Lignified

Bark

cells,

cells,

violet to red.

orange.

Herbarium Specimens
Solution required:

Solution D,

as above.

Technique:
1.

2.

Soak or boil the specimens in water.

Cut sections and stain directly with solution D, which has

been well shaken immediately before use.

Note: Prolonged treatment in the fixatives employed bleaches
the specimens sufficiently for the purpose of staining with chlorazol
azurine otherwise bleaching is not recommended.
;

Reference: Armitage, F. D., ^. Roy. Micr. Soc.y 535, 826,

i.

/ am indebted
tory,

to Mr. F. D. Armitage^ F.R.M.S.y of The LaboraGreen End Road, Boxmoor, Herts, England, for information he

has given

me

regarding his use of this stain.

CHLORAZOL BLACK

non-fading, general-purpose stain, which may be used

whole mounts as well as for sections. The stain requires

for

no mordanting nor

differentiation

Nuclei and chromosomes are stained black, cytoplasm and

secreted products grey, by this stain, which has also been found
useful for infected plant tissues.

Solution required:

Chlorazol black, saturated in

alcohol.

252

70%

SECTION THREE
Technique:
1.

Fix tissues in Bouin or Flemming and

embed

in paraffin wax.

2. Stain in a freshly prepared, unfiltered, alcoholic solution of

chlorazol black (as above) for five to ten minutes.
3.

Drain

off excess stain;

dehydrate; clear in xylol and mount.

Results:

Vascular plant: Cell wall, jet black; cytoplasm, greyish green;

nuclei, yellowish green; nucleoli, deep amber to dark green.

Fern

leaf: Cell wall, intense black; epidermis walls, heavy

cytoplasm, light amber; nuclei, green; nucleoli, dark
green; plastids, grey; suberized walls of midrib, dark amber;
veins, dark amber.

black;

Notes:
(a)

The

{h)

Benzyl

may be incorporated with Lactophenol.

alcohol may be used as a solvent of the stain,

stain

which case the

{c)

If

it is

results are

somewhat

desired to differentiate the stain dilute

(a proprietary antiseptic)

may be

in

different.

"

Milton

'*

used.

CHLORAZOL PAPER BROWN, B

For plant tissues
Material may be stained overnight, but in many cases, e.g. where
the delicate cell contents are not germane to investigation, the
equivalent depth of staining will be produced by boiling for one
to two minutes in the staining solution. By employing the boiling

technique finished slides can be obtained in five minutes.

Solution required:

Chlorazol Paper Brown,^ B, saturated aqueous.

Technique:
Sections are stained overnight in a saturated aqueous solution
(about 3%) of the stain, or by boiling in this staining solution for
I.

one to two minutes.

253

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

i%

2.

Differentiate in

3.

Dehydrate in acetone; mount.

nitric acid.

Results:

Epidermis, yellow. Cortex, yellow. Pericycle (lignified tissues),

blood red. Zylem, salmon to blood red. Primary phloem, orange.
Cambium, pale yellow. Secondary phloem, crimson. Sieve plates,
some very bright crimson, others orange. Pith, amber.

VoL

Abstract: (1947) Stain Tech.,

COTTON BLUE

22, 4, 155-6, B. Vercourt.

SAFRANIN

For fungal h3rphae in woody tissues

Solutions required:

A. Cotton blue
B.

0-5%

in Lactophenol.

Lactophenol.

C. Safranin

1%

aqueous.

Technique:
1

Stain thin sections with solution A, which has been

about 35 C, for

five to fifteen

warmed to

minutes.

3.

Wash
Wash

4.

Counterstain with the safranin for about ten minutes.

5.

Wash

2.

with Lactophenol to remove excess stain.

with

70%

with

70%

alcohol to

remove Lactophenol.

alcohol taking care that the safranin

entirely removed.
6.

Rinse rapidly in

7.

Dehydrate with absolute alcohol.

8.

Clear in xylol.

9.

Mount

in

90%

alcohol.

Canada balsam

in xylol or in Cristalite.

Results:

Fungal hyphae, blue. Xylem, red.

Reference: Chesters (1934), Ann. Bot., 48, 820.

is

not

SECTION THREE

CYANIN

A botanical

ERYTHROSIN

stain for cellulose

and

lignified tissues

Solutions required:

A. Cyanin,
B.

in

o*i%

Note: This

is

1%

Erythrosin

50%

alcohol.

a very costly stain.

in

70%

alcohol.

Technique:
1.

Stain sections from five to twenty minutes in the cyanin

solution, placing the slide under a petri dish lid, or an evaporating

basin lined with a piece of damped filter paper to prevent evapora-

tion of the stain.

2.

Wash

3.

Stain from half to one minute in the erjrthrosin.

4.

Rinse quickly in 50%.

5.

Rinse quickly in

6.

Dehydrate by rinsing quickly in two changes of absolute

rapidly with

50%

95%

alcohol.

alcohol.

alcohol.
7.

Clear in xylol, and mount.

Results:

Lignified tissues are stained blue, while cellulose tissues are red.

DELAFIELD HAEMATOXYLIN
For botanical

CELLOSOLVE

tissues, particularly for cell walls

Solutions required:

A. Delafield haematoxylin.
B.

Alcohol

70%

Ammonia

solution (sp. gr. o-88o)

99*5 ml.
0-5 cc.

Technique:
I.

Fix sections to slides and stain in Delafield haematoxylin for

three to five minutes.

255

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

2.

Rinse in tap water.

3.

Decolorize for about thirty seconds in

4.

Rinse in

5.

Blue in Solution

6.

Rinse well in distilled water.

7.

Immerse

8.

70%

70%

alcohol.

alcohol.

(above).

two changes of cellosolve for one minute

Drain and mount in Canada balsam or in Cristalite.
in

in each.

Results:

Nuclei and chloroplasts are stained bluish purple; cytoplasm

in a paler shade of purplish blue, or colourless, lignified and cutinized tissues are yellow. Unlignified tissues, bluish purple.

ERYTHROSIN

A general

LACTOPHENOL

stain for botanical tissues

Solution required:

Erythrosin

1%

in lactophenol.

Technique:
1.

Place sections on slides and add a drop of the staining

solution.
2.

Cover with a coverslip and examine under the microscope.

Results:

Lignified and cutinized tissues are stained yellow while cellulose

walls are unstained. Nuclei and dense protoplasmic contents are
red.

Cytoplasm of vacuolated

plasts, slime

cells

plugs in sieve, tubes,

etc.,

appear pink, while chloroare red.

GRAM'S IODINE

general stain for botanical tissues

Solutions required:

A. Gram's iodine.
B.

Glycerine

50%

aqueous.

256

SECTION THREE
Technique:
1.

Stain with

2.

Pour

3.

Mount

Gram's iodine solution

few minutes.

for a

off excess stain ; rinse in distilled water.

cover with a coverslip and

in the glycerine solution;

examine under the microscope.

Results:
Proteins, brown. Cytoplasm, light brown.
brown.
dark
Nuclei,
Chloroplasts, brown or blue. Cellulose walls,

Starch, navy blue.

faint yellow.

Lignified walls, deep yellow.

HAEMATOXYLIN

(Heidenhain)

ANILINE BLUE

For the difTerential staining of nuclei, cytoplasm and cell

walls of angiosperm shoot apices
This technique which is due to Dr. J. G. Vaughan, Department of
Biology, Chelsea Polytechnic, London, S.W.3, has been used with success
on Anagallis and certain members of the Cruciferae. No mixing of the
dyes has been observed in the apical meristem region, as occurs with
other stain combinations, and the picture is clear and well defined,
thereby facilitating study and photographing.
Solutions required:

A. Haematoxylin (Heidenhain), No.

B.

Haematoxylin (Heidenhain) No.

C. Iron alum

D. Aniline
brand)

Methyl

2%

blue
.

i.

2.

aqueous.
alcohol,

cellosolve

soluble

(Michrome
i
gm.

100 ml.

Dissolve by heating on a hot plate or a

waterbath, taking care that the cellosolve
does not catch fire. Allow to cool; then
filter.

25 ml.

E. Methyl salicylate

33

Xylol
Absolute alcohol
F.

Methyl

nal-

42 ml.

40 ml.
20 ml.
20 ml.

salicylate

Xylol
Absolute alcohol

257

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

G. Xylol

Absolute alcohol

90 ml.

10 ml.

Technique:

Pass sections through descending grades of alcohol to dis-

1.

tilled water.

A for

2.

Mordant

3.

Rinse in distilled water.

4.

Stain in solution

5.

Rinse in distilled water.

6.

Differentiate in

7.

Wash

8.

Dehydrate in 25%, 50%, 70%,

in solution

2%

thirty minutes.

for twelve hours.

iron

alum

in running water for

solution.

one hour.

90% and

two changes of

absolute alcohol.
9.

Stain in the aniline blue solution for ten minutes.

10.

Remove

11.

Rinse in solution E.

excess aniline blue with absolute alcohol.

12. Clear in solution

13.

for ten minutes.

Rinse in solution G.

14. Rinse in two changes of xylol and mount in

in xylol, Clearmount, Cristalite or D.P.X.

Canada balsam

Results:

Nuclei and cytoplasm are well stained by the haematoxylin,

while cell walls are stained very satisfactorily by the aniline blue.

Note:
(a)

The

success or failure of the

the quality of the aniline blue.

[h)

Ilford Special

method

is

said to

depend on

Rapid Panchromatic

plates

were used for the

preparation of the photomicrographs which are reproduced in the

original paper.
Reference: Vaughan,

J.

G. (1955), Stain

258

Tech., 30, no. 2, 79-82.

SECTION THREE

HAEMATOXYLIN

BISMARK BROWN

For Phloem tissues of woody plants

Solutions required:

A. Iron alum

2%

aqueous.

Haematoxylin i% aqueous.
C. Bismark Brown 1% aqueous.

B.

Technique:
1.

Immerse

2.

Drain

A for ten to

sections in solution

off excess solution;

twenty minutes.

then wash in six or seven changes

of distilled water.
solution B, placing the slide on
3. Cover the preparation with
the microscope stage.
4.

Observe the progress of the stain under the low power

and when the required depth of staining has been at-

objective
tained,

pour

off excess stain

and

rinse quickly in distilled water.

5.

Rinse again with two or three changes of distilled water.

6.

Immerse the preparation

jar for three to four hours,

sections.
7.

Remove

excess stain

in solution

alcohol, followed

11. Clear in xylol,

and mount

Canada balsam

in a staining tube or

by washing well with water.

Wash
50%
Wash
with
90%
9.
10. Wash with two
8.

with

depending on the thickness of the

by

70%

alcohol.

alcohol.

changes of absolute alcohol.

in Cristalite or

Clearmount or

in xylol.

Results:

hard woods are stained cherry red; bast fibres are

and other parenchymatous
tissues are a chestnut brown, and the middle lamellae dark blue.
The bast fibres, parenchymatous tissues, and middle lamellae of
Coniferophyta are stained as indicated, but the stone cells turn a
vivid burnt orange.
Stone

cells of

brilliant orange, whilst the ray cells

259

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

HEroENHAIN HAEMATOXYLIN

SAFRANIN

A general stain for plant tissue, algae, fungi, etc., to demonstrate histological

and cytological structures

Solutions required:

A. Haematoxylin (Heidenhain) No.

i.

Haematoxylin (Heidenhain) No.

2.

B.

C. Picric acid, saturated, aqueous.

D. Safranin

1%

aqueous.

Technique:
1

Fix and dehydrate tissues and embed in paraffin wax.

Note: Algae and fungi should be treated by the Venetian turpentine method {see page 43).

remove wax with

2.

Fix paraffin sections to slides

3.

Pass through absolute alcohol and the usual descending

grades of alcohol,

down

4.

Mordant

5.

Wash

6.

Rinse well in

xylol.

to distilled water.

Heidenhain haematoxylin No. i (Solution A)

from one half to two hours but no longer, unless the material is
algae in which case as long as twelve hours may be necessary.

7.

from

in

for five

minutes in running tap water.

distilled water.

Stain algae for at least twenty-four hours, or other material

five to seven hours, in Heidenhain haematoxylin No. 2

(Solution B).

to

8.

Wash

9.

Differentiate in Solution

in

running water for

five

minutes.

(picric acid) from twenty minutes

at intervals under the

two hours, controlling by examination

microscope.
10.

Wash

for half

an hour in running tap water.

11.

Rinse well in

12.

Counterstain in the safranin solution for five to ten minutes.

13.

Rinse in distilled water.

distilled water.

260

SECTION THREE
14.

15.

and

Dehydrate as usual.
Immerse for five minutes in equal parts of absolute alcohol

xylol.

16.

Transfer to and immerse in xylol for five minutes.

17.

Mount

in

Canada balsam

in xylol.

Results:

Chromosomes, black to purple. Centrosomes and pyrenoids,

black to purple. Lignified, suberized and cutinized structures, unstained or only faintly stained. Archesporial cells and early stages
of sporogenous tissue, grey.

IODINE GREEN

ACID FUCHSIN

A botanical stain for lignified tissues, and for chromosomes

Solutions required:

A. Iodine

1%

aqueous.

Acid fuchsin

B.

1%

aqueous.

Technique:
1

Fix material in Nevashin's

2.

Take

3.

Immerse

fluid.

sections through to distilled water in the usual manner.

for at least twelve

hours in the Iodine green

solution.
4.

Wash and

intervals

differentiate with distilled water, examining at

under the microscope while the preparation is still wet,

until the non-lignified tissues retain only a faint green tint.

5.

Counterstain for about fiYQ minutes in the acid fuchsin

by examination under the microscope, while

solution, controlling

the preparation
fuchsin solution

wet, taking care to ensure that the acid

not allowed to act long enough to extract the

is still

is

Iodine green from the lignified tissues.

6.

Rinse quickly in

7.

Dehydrate quickly in two changes of absolute alcohol.

8.

Clear in clove

9.

Wash

90%

alcohol.

oil.

with xylol.
261

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

10.

Mount

in Cristalite,

Clearmount or in Canada balsam in

xylol.

Results:

Chromosomes and
to red.

nuclei, green.

Plastin

and cytoplasm, pink

Lignified tissues, green.

JOHANSEN'S QUADRUPLE STAIN

For plant tissue

SECTION THREE
Technique:

then
1. Paraffin sections are brought down to 70% alcohol;
stained for twenty-four to forty-eight hours in Solution
(over-

staining

is

not possible).

then stain ten to fifteen minutes in Solution

2.

Rinse in water

3.

Rinse in water; then rinse for fifteen seconds in Solution C.

B.

Stain ten to twenty-five minutes, according to the material

and fixative, in Solution D.
4.

5.

Rinse briefly in Solution E.

6.

Immerse

7.

Rinse in Solution G.

8.

Rinse in two changes of xylol

for about three

minutes in Solution F.

then mount in balsam.

Results:

Dividing chromatin, red resting chromatin, purplish nucleoli,

red (occasionally violet); nucleoplasm, colourless or greenish;
lignified walls, bright red; cutinized cell walls, reddish purple;
;

suberized walls, red

cellulose cell walls, greenish orange

cytomiddle
lamellae, green starch grains, purple
plasm, bright orange
with green or orange halos (the colour of the halps soon becomes
;

replaced

by purple in some types of materials)

plastids, purplish to
green; the callose portion
;

greenish; invading fungal mycelium,

of the guard cells of the stromata bright red and the remainder
purple and Casparian strips, red the remainder of the cell wall
;

of the endodermis, yellow.

In sections of roots for the origin of the lateral roots, the
cytoplasm of the latter should be stained green, with purplish
nuclei, while the cytoplasm elsewhere should be orange with

red nuclei.

The combination is exceptionally good for sections of lichens, as

the algae are well differentiated, and also for Puccinia graminis
telia and Uredinia.
From

Plant Microtechnique, by D. A. Johansen, by courtesy of McGraw-Hill

Inc., New York.

Book Company,
T

263

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

JOHANSEN'S QUINTUPLE STAIN

For plant tissue composed of a variety of cell types, such as
leaves, roots, steins

and ovaries

Solutions required:

A. Safranin O, i% in cellosolve
Alcohol 95%

4%

Sodium

acetate

Formalin
B.

Crystal violet

1%

50 ml.
25 ml.
25 ml.
i

ml.

aqueous.

C. Absolute alcohol
Cellosolve

25 ml.
25 ml.

Tertiary butyl alcohol

25 ml.

D. Fast Green, FCF.

Malachite Green

0-5

0-5

gm.
gm.

100 ml.

Cellosolve

Dissolve; then add:

Absolute alcohol

25 ml.
25 ml.

Glacial acetic acid

1*5 ml.

Orange 2

0-5

Tertiary butyl alcohol

E.

gm.

58 ml.

Cellosolve

Dissolve by warming gently, then add

14 ml.
:

Clove oil
Absolute alcohol
.

14 ml.
14 ml.

Tertiary butyl alcohol

F.

Terpineol, extra pure

Absolute alcohol
Tertiary butyl alcohol
Cellosolve
.

Methyl salicylate
Beechwood creosote
264

equal

volumes

SECTION THREE

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

12.

Rinse in Solution G.

13.

Immerse

14.

Pass through two changes of alcohol; then mount.

in Solution

H for five to ten minutes.

Notes:
{a) Isopropyl alcohol

for

which ethyl alcohol

{h)

The

schedule

may be used, if desired,

commonly used.

at first

the actual application

for every purpose

is

is

glance appears to be complicated, but

quite simple.

{c) Slides should be agitated in

all

rinses

and washes.

(d) The reason for mixing so many reagents together in Soluis that each effects certain stains and not the others.
tions F and

The

green malachite green (Solution D) is somewhat

strong at first but gradually weakens as successive slides are passed
through it consequently, the time in this stage may be reduced to
four minutes and that of the orange 2 (Solution E) in Stage 10 leng-

{e)

fast

thened to ten minutes, then equalized, and later, the changes in

time reversed as the orange becomes stronger after its initial weakness.

three hundred slides may be passed through

( / ) Approximately
each 100 ml. of each staining solution (except the safranin (Solution A), which merely needs replenishment when required), and
rinsed before replacement becomes necessary owing to contamination.

From

personal communications with Professor D. A. Johansen, Pomona,

whom this technique and my thanks are due.

California, U.S.A., to

LACMOID

TANNIC ACID

FERRIC CHLORIDE

For phloem and contiguous tissues: the technique gives

relatively stable preparations of critically stained materials
Solution required:

A. Tannic acid

1%

aqueous.

B.

Ferric chloride, hydrated

C.

Sodium bicarbonate
Distilled water

2%

aqueous.

gm.

2-5

50 ml.

Dissolve by shaking or stirring: do not apply heat,

266

SECTION THREE
D. Solution

20 ml.

Distilled water

55 ml.
25 ml.

Absolute alcohol
E.

Lacmoid

0-25 gm.
30 ml.

Absolute alcohol
Distilled water
Solution

Solution

F.

70 ml.
3 to 5 ml.

20 ml.

30 ml.
50 ml.

Distilled water

Absolute alcohol

G. Absolute alcohol
Clove

equal volumes of each

oil

Xylene
Technique:
1.

Fix material in formalin-acetic-alcohol.

2.

Unembedded

sections, 5 to 40/x in thickness

may be em-

ployed.
Sections are taken from distilled water and

3.

immersed

in

1%

tannic acid for five to ten minutes.

4.

Transfer to

5.

Wash

2%

ferric chloride for

about

five

mins.

in distilled water (three changes).

Examine under the microscope while the preparation is still

wet the colour of the walls should be medium to dark grey, and
it may be
necessary to repeat the above staining process to attain
this result as some phloem tissues stain less readily than others.
6.

7.

Wash

8.

Immerse

in three changes of distilled water.

in solution

for thirty minutes.

9. Transfer directly to solution E and leave therein for twelve

to eighteen hours, or longer if desired, as it is impossible to over-

stain.
10.

Transfer to solution

depending upon the time

1 1

12.

Wash

in

Immerse

at

F from

ten seconds to ten minutes

which the lacmoid

80% alcohol.
in 90% alcohol

for

two

267

destains.

to three minutes.

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

Immerse

13.

two changes of absolute alcohol

in

for a total time

of two to three minutes.

14.

Wash

15.

Immerse

in solution

for

two

two changes of

in

to three minutes.

xylol for a total time of

two

to

three minutes.

Mount

16.

in

D.P.X. or Clearmount or

Cristalite

mountant.

Results:
is stained sky blue to greenish blue; lignified cellulose
the
xylem cells and in most cortical or phloem fibres and
(in
sclereids) blue. Cellulose walls, nuclei, slime and cytoplasm, light

Callose

brown

to greyish

brown.

Reference: Vernon,

J.

C. and Gifford, E.

LACMOID

M.

(1953), Stain Tech., 28, 49-53.

MARTIUS YELLOW

For callose in pollen tubes

Solution required:

Lacmoid 0-01%

..

..

..

10 ml.

lo ml.
Martins Yellow 0-01% ..
Add a few drops of diluted ammonia (o-i ml.
cone, ammonia with 10 ml. distilled water) until the
solution assumes an olive tint.
..

Technique:

Slender styles or ovaries are crushed between two slides while

still wet. Larger styles or ovaries should be cut by hand into longitudinal sections, then crushed.
1.

2.

Stain two to five minutes in the

Lacmoid - Martins yellow

solution.
3.

Mount

in the stain

and examine under the microscope, using

a strong light.
Results:

Pollen tubes, blue.

Background,
268

light yellowish green.

SECTION THREE

LIGNIN PINK

A non-fading stain which is

The
and

is

specific for lignin

stain gives constant results with reasonably thin sections

particularly suitable for routine or elementary classwork.

Overstaining with Lignin Pink is impossible, and it will not wash

out with alcohol.
In combination with chlorazol black it offers a simple double
staining technique as follows

Solutions required:

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

Technique:
1.

Embed

2.

Remove

the material in paraffin

paraffin

wax from

wax

in the usual

the sections with xylol as usual.

4.

Wash
Wash

5.

Stain for five to ten minutes with solution A.

6.

Wash

3.

manner.

well with absolute alcohol.

with

90%

alcohol.

with absolute alcohol to remove excess

7. Counterstain for

two

to five

8.

Wash

9.

Clear in xylol, and mount.

stain.

minutes in solution B.

in absolute alcohol.

Results:

Host

tissues are stained green

METHYL GREEN

parasite, red.

PHLOXIN

GLYCERINE JELLY

A rapid method for the simultaneous mounting and double

staining of pollen grains, differentiating functional
abortive grains

from

This technique can be employed for the evaluation of orchid

seeds which contain naked embryo surrounded by integuments.
Solutions required:

A. Methyl Green, saturated in

B.

Phloxin, saturated in

C.

Glycerine jelly
Solution A
Solution

50%

50%

alcohol.

alcohol.
.

50 ml.

. .

2J ml.

2 ml.

Melt glycerine jelly on a water bath then measure off in a preheated measuring cylinder the 50 ml. required and pour this
amount into a pre-heated bottle to which, solutions A and B are
then added and shaken in.
;

Note: The

jelly should

now

be a port wine colour:

vary the proportions by adding

to produce this colour.

necessary to

270

it

may

he

more of one of the dyes

SECTION THREE
Technique:

amount of pollen on the centre of a slide.

2. Wash by dropping on 70% alcohol to remove any adhering
oils and resins.
Note: The alcohol will spread out and evaporate leaving a ring
1.

Place a small

a piece of tissue paper

of sludge, which should he removed with
moistened with alcohol.

no more sludge comes out.

alcohol, and just before the last drop

3.

Repeat

this process until

4.

Wash

with

finally

70%

of alcohol evaporates completely, place 2 drops of hot stained

with a needle to ensure
glycerine jelly on the pollen and stir gently

even distribution.
a coverslip, keeping the jelly under the coverslip
5. Cover with
hot by heating gently with a bunsen flame.

Note: Excessive heating will rupture many of the pollen grains.

6. Remove the flame take off the coverslip and replace it with
;

a clean, flamed coverslip.

7.

Allow the sUde

to cool

then store in a cool place.

Results:

''Functional" pollen grains are fully expanded; exine and

intine are stained green cytoplasm red. Aborted grains are either
shrunken and stained green, or expanded in varying degrees
;

depending on the amount of non-autolysed cytoplasm present at

the time of mounting, and stained a mottled, reticulate red.
Notes: The intensity of the Phloxin stain increases on standing,
whereas the action of the methyl green is practically instantaneous.
Preparations retain their brilliancy for about a year.
Reference: Owczarzak, Alfred (1952), Stain Tech., 27, no.

5, p.

249.

PHLOROGLUCINOL
An extremely

sensitive test for lignin, particularly suitable

for hydrophytes
Solution required:

Phloroglucinol

1%

in

271

70%

alcohol.

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

Technique:
Sections are placed on slides and flooded with the phloroglucinol solution which is allowed to act for about five minutes.
1.

off excess phloroglucinol and cover sections with a few

concentrated
hydrochloric acid, cover with a coverslip
drops of
the
under
and examine
microscope.
2.

Pour

Result:

Lignified tissues are stained red.

POLYVINYL LACTOPHENOL
brittle specimens of wood for sectioning.
claimed that this technique has given successful results
with wood dating back to the Roman era

For embedding
It is

In addition to softening the wood for cutting, this technique also

specimen and allows cell walls, which may have been
blackened through carbonization, to become clear and translucent,
thereby facilitating identification under the microscope.
clears the

Solution required:

Polyvinyl Lactophenol.
Technique:
1.

Immerse blocks of wood cubed

polyvinyl alcohol, and

warm

in the usual manner, in

gently for thirty minutes.

2.

Drain, and allow to cool for twenty-four hours.

3.

Cut sections from blocks with

a very sharp razor.

Results:

The wood

has lost

its

brittleness

and has acquired

rubber-like nature.
Reference: Levy,

J.

F. L. (1953), Nature, 171, 984.

272

a soft pliable

SECTION THREE

SAFRANIN

ANILINE BLUE

For plant tissues; particularly suitable for gymnosperm

ovules, archegonia, embryos and angiosperm stems and
roots
Solutions required:

A. Safranin O, 2% in Cellosolve
Absolute alcohol
.

Sodium

acetate

Formaldehyde
B.

Picric acid

C.

Ammonia

4%

aqueous

40%

0*5%

in

95%

100 ml.
50 ml.
50 ml.
8 ml.

alcohol.

solution (sp. gr. 0.88)

Absolute alcohol

0-25 ml.
100 ml.

D. Aniline Blue, alcohol soluble, saturated in

equal volumes of cellosolve and absolute
alcohol.

Technique:
1.

Take

2.

Stain from two to forty-eight hours, according to the nature

sections through to

70%

alcohol in the usual manner.

of the material, in solution A.

Note: Gymnosperms require the minimum staining time,

3.

Wash

thoroughly with running water to remove the excess

stain.
4.

Dehydrate and

5.

Immerse the preparation

differentiate carefully with solution B.

in solution

C for half to one minute.

Dehydrate morphological material in

logical preparations in absolute alcohol.
6.

7.

95%

alcohol, or cyto-

Counterstain for about one minute in a mixture consisting

of equal parts of solution

and clove

oil.

8. Clear in methyl salicylate and mount in

balsam or Emexel mountant.

Cristalite,

Canada

Results:

Lignified

bright red.

and cutinized

nuclei and chromosomes,

and cytoplasm, blue.

cell walls,

Cellulose cell walls

273

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

Note: At certain developmental and formative stages, some

cell

walls are stained sharply with the safranin, while other portions
will appear faint blue.

SAFRANIN
For plant

ANILINE BLUE

chromosomes and

tissues, particularly for

cell

walls
Solutions required:

A. Safranin
B.

i%

aqueous.

i%

Aniline blue

in absolute alcohol.

Technique:
Paraffin sections are fixed to slides ; de- waxed with xylol, and
passed through descending grades of alcohol down to distilled
1

water as usual

alternatively freehand sections

may be employed.

2.

Stain for fifteen minutes in the safranin solution.

3.

Wash

4.

Rinse in

in distilled water.

70%

alcohol, followed

by 90%.

5.

Rinse in absolute alcohol.

6.

Stain for two minutes in the aniline blue solution.

7.

Rinse quickly but thoroughly with two changes of absolute

alcohol.
8.
9.

Wash with two changes

Mount in balsam.

of xylol.

Results:

Chromosomes,

red. Nucleoli, red.

Cytoplasm almost colourless.

Cellulose walls, blue.

SAFRANIN

DIANIL BLUE G

For the differential staining of Peronosporaceae

Solutions required:

A. Phenol crystals

Lactic acid

Glycerin
Absolute alcohol
.

274

10 gm.
10 ml.

20 ml.
20 ml.

SECTION THREE
B.

Cotton blue 4B

Safranin

. .

. .

Solution

C. Safranin

in clove

0*25%

0-02 gm.
o-io gm.

100 ml.

oil,.

Technique:
1.

Embed

2.

Fix sections to slides and remove paraffin

wax by

material in paraffin

the standard technique.

wax with

xylol as

usual.
3.

Wash

well with absolute, followed

by

90%

alcohol.

4.

Immerse

in solution

A for

ten to fifteen minutes.

5.

Transfer to solution

B and

leave therein for

6.

Differentiate in solution A.

7.

Wash

8.

Immerse

9.

Differentiate in clove

10.

two hours.

with absolute alcohol.

in solution

Clear in xylol, and

Cristalite, or in

for

twenty to thirty minutes.

oil.

mount

in

Canada balsam

in xylol or in

Emexel mountant.

Results:

Host

tissue

is

stained red

Myelium

blue.

Reference: (1928), Lepik. Phtopath., 18, 869.

SAFRANIN

FAST GREEN IN CELLOSOLVE

rapid, non-fading stain for botanical tissues in place

of Safranin -light green -clove

oil

Solution required:

Safranin

Fast Green

FCF

in cellosolve.

Technique:
1.

Stain sections for five to ten minutes.

2.

Rinse in cellosolve.

4.

Mount

in

Canada balsam or

in CristaHte.

275

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

Results:

Lignified tissues are stained red, while unlignified tissues and

cytoplasm are blue-green. Nuclei, red. Chloroplasts, pink to red.

SAFRANIN

ACID FUCHSIN

For spermatozoids, zoospores, motile gametes,

etc.

Solutions required:

A. Osmic acid
B.

C.

i% aqueous.
Safranin i% aqueous.
Acid Fuchsin i% aqueous.

Technique:
1

Place a drop of an aqueous suspension of the organism on a

slide.
2.

Fix by holding the slide for about one minute over osmic

acid solution.
3.

Allow the preparation

4.

Stain for ten minutes to one hour in solution B.

5.

Wash

6.

Remove

to dry.

in water.

the slide with

excess water

by draining and

blotting the edges of

filter

paper.

95%

alcohol until only the nuclei retain the stain.

7.

Wash

8.

Stain in the acid fuchsin solution for ten to twenty seconds.

in

Wash rapidly in 70% followed by 90% alcohol.

10. Wash rapidly with two changes of absolute alcohol.

9.

11.

Clear in clove

12.

Mount

in

oil,

followed by xylol.

Canada balsam

in xylol, Cristalite,

or Emexel mountant.
Results:

Nuclei bright red.

Cytoplasm bluish pink.

Reference:

Bot. Gaz.,

(19 18), Steil.

LVX,

276

592.

Clearmount

SECTION THREE

SAFRANIN

FAST GREEN, FCF

non-fading stain, satisfactory for nearly every type of

plant material except the algae
Solutions required:

A.

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

Results:

Nuclei, chromosomes, lignified and cutinized cell walls, brilliant

Cytoplasm and

red.

cell walls at certain

will

be stained

by the

less

sharply by safranin, and other portions weakly

fast green.

SAFRANIN

cellulose cell wall, brilliant green. In some

developmental or formative stages, portions

FAST GREEN, FCF IN CELLOSOLVE

rapid non-fading stain for botanical tissues in place of

Safranin - light green - clove oil
Solution required:

Safranin

light

green in Cellosolve.

Technique:
1.

Stain sections in the safranin

five to ten

light green

cellosolve for

minutes.

2.

Rinse in Cellosolve.

3.

Mount

in

Canada balsam or

in Cristalite.

Results:

Lignified tissues are stained red, while unlignified tissues and

cytoplasm are blue-green. Nuclei, red. Chloroplasts, pink to red.

SAFRANIN

LIGHT GREEN

A general stain for botanical

CLOVE OIL
tissues

Solutions required:

A. Safranin
B.

1%

Light Green

in
-

50%

clove

alcohol.

oil.

Technique:
1.

Stain section in the safranin solution for ten minutes.

2.

Rinse in two changes of

50%

each.

278

alcohol for thirty seconds in

SECTION THREE
Rinse in two changes of

3.

70%

alcohol for thirty seconds in

each.
4.
5.

Repeat with two changes of 90% alcohol for the same time.
Immerse in two changes of absolute alcohol for two or three

minutes in each.
-

clove oil for one minute.

6.

Stain in light green

7.

Rinse and wash in clove

oil for

about

five

minutes.

Examine under the microscope and if it is found that the

safranin has been extracted by the alcohols, take the section down
through the alcohols and restain with safranin.
8.

9.

Mount

in

balsam or in

mountant.

Cristalite

Results:

Cellulose tissues and cytoplasm are stained green.

tissues

and

nuclei, red.

SAFRANIN

rapid and simple

Lignified

Chloroplasts, pink.

PICRO ANILINE BLUE

method of demonstrating hyphae

wood

in

sections

Solutions required:

A. Safranin
B.

1%

in distilled water.

Aniline blue, water soluble

Distilled water
.

Picric acid, saturated, aqueous

2-5

gm.

25 ml.
100 ml.

Technique:

then wash in water.

1.

Stain one to three minutes in Solution

2.

Flood sections with Solution B and heat over a flame until

simmer

then pour oif excess stain

cool before washing with water.

begins to

3.

oil

Wash with 70%, followed by absolute, alcohol

mount

it

allow the slide to

clear in clove

in balsam.

Results:

is

Lignified walls, red. Mycelia, clear blue. Areas where the wood
may appear bluish, but the hyphae are always well

badly decayed

defined.

279

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

SAFRANIN

TANNIC ACID

FAST GREEN

For roots and steins

Solutions required:

A. Tannic acid

i%

B.

Ferric chloride

C.

Safranin

in

3%

50%
in

alcohol.

50%

alcohol.

1% in 50% alcohol.
green FCF in clove oil

to

7%

of

D. Fast
which has been added
absolute alcohol.

Technique:
Sections are fixed to slides, treated with
in the tannic acid solution.

1.

70%

alcohol; then

immersed

Immerse

2.

for about twenty seconds each in

two

lots of

50%

alcohol.

Treat with the ferric chloride for about thirty to sixty

3.

seconds.
4.

Wash

5.

Stain with the safranin solution for twenty-four hours, in a

with

50%

stoppered staining

alcohol.

jar.

6.

Wash

7.

Differentiate for about ten seconds in

8.

Pass through

with

50%

alcohol.

80%, 90% and

70%

alcohol.

absolute alcohol, allowing ten

to thirty seconds in each.

9.

Stain for two or three minutes in the fast green.

10.

mount

Pass through absolute alcohol, followed by xylol;

then

in Cristalite.

Results:

Nuclei, red.

Cambial

cell

Parenchyma

Cytoplasm, blue-green.

walls,

black.

Collenchyma

cell walls, black.

280

Lignified

walls,

red.

walls, very dark red.

SECTION THREE

SCARLET R

or

SUDAN

An improved technique

ETHYLENE GLYCOL

for staining fat in plant tissues, offering

(a) The use of ethyl alcohol is obviated

sections remain pliable and unshrunken; {b) excellent differentiation without loss of stain from the fat; (c) More intense

the following advantages

and

staining of fat.

Solutions required:

A. Scarlet

R or

Sudan

gm.

100 ml.
Ethylene glycol, pure, anhydrous..
Heat the ethylene glycol to 100-110 C. on a hot
plate, or in an oven for preference, but if these are not
available the bunsen flame will serve the purpose so
long as care is taken to ensure that the ethylene glycol
does not take fire. Stir in the dye until all or most of it
is

B.

dissolved; then cool

Ethylene glycol
Distilled water

and

filter.

85 ml.
15 ml.

C. Delafield or Ehrlich haematoxylin.

Technique:
1.

Fix tissues in

2.

Wash

3.

Dehydrate the sections by agitating gently in the pure

10%

formalin.

out fixative with water.

ethylene glycol, anhydrous, for three to five minutes.

4. Immerse the sections in the staining solution
Sudan 3) and agitate for two to five minutes.

(Scarlet

or

Differentiate by agitating gently, at intervals, from one to

minutes in 85% ethylene glycol (solution B) controlling by
examination under the microscope while the specimen is still wet.
5.

five

6.

Transfer the sections to distilled water and leave therein for

three to five minutes.

7.
8.

9.

Counterstain with Delafield or Ehrlich haematoxylin.

Wash well in tap water.

Mount in glycerine jelly

or glycerine.

281

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

Results:

With Scarlet R: Nuclei, blue. Fat, orange

to red.

Cholesterol,

Fatty acids, unstained.

With Sudan 3 Nuclei, blue. Fat, yellow to orange. Cholesterol,
orange. Fatty acids, unstained.
red.

.*

SCARLET R
For staining

fat in plant tissues

Solutions required:

A. Scarlet R, saturated in
B.

70%

alcohol.

Delafield or Ehrlich haematoxylin.

Technique:
1.

Fix tissues in

2.

Sections are immersed for a second in

10%

formalin.

70%

alcohol;

then

stained for two to five minutes in the Scarlet R.

3.

Wash

4.

Counterstain with Ehrlich or Delafield haematoxylin.

5.

Wash

quickly and transfer to distilled water.

well in tap water;

mount in

glycerine or glycerine jelly.

Results:

Fat, orange to red.

Fatty acids, unstained.

Cholesterol, red.

SCHULZE SOLUTION

Nuclei, greyish blue.

(Chlor Zinc Iodine)

For cell walls, proteins and starch

1.

Place sections on slides and pour on a few drops of Schulze

solution.
2.

Cover with a coverslip and examine under the microscope.

Results:

Starch, blue.

Proteins, brown.

fied walls, yellow.

Cellulose walls, violet. LigniCutinized and suberized walls, yellow to brown.

282

SECTION THREE

TETRAZOLIUM SALT
For testing the viability of seeds
Solution required:

A. Tetrazolium

salt

i%

aqueous.

Technique:
1.

Select the seeds at

random and

steep in tap water for i8

hours.
2. Section longitudinally through the
half of each in a 7 cm. petri dish.
3.

Pour the tetrazolium

salt solution

embryo and place one

over the seeds and soak in

the dark for 4 hours at 20 C.

4.

Wash with

5.

Examine seeds

tap water.
for staining.

Results:

Viable seeds are stained: others unstained.

Note:

The method

not well adapted to certain seeds.

is

Reference: Cottrell, Helen

J.

(1948),

THIONIN

Ann. Appl.

Biol., 35, 123-31.

ORANGE G

For infected plant tissues

Solutions required:

A. Carbol thionin.
B.

Orange

G 0-2% in absolute alcohol.

Technique:

Fix paraffin sections to slides; pass through xylol and

descending grades of alcohol down to distilled water in the usual
manner.
1.

2.

hand

Stain for an hour in the thionin solution. Alternatively, freesections may be employed, in which case, the sections should

be stained in the thionin for only five minutes.

3. Pour off excess stain, and rinse with 70% alcohol followed

by 90%.
4.

Rinse quickly in two changes of absolute alcohol.

283

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

Differentiate for one half to one

5.

minute with the orange

solution.

90%, followed by 70%,

6.

Pass through

7.

Rinse well with water.

8.

Rinse with

9.

Rinse quickly in two changes of absolute alcohol.

70%

alcohol, followed

10.

Pass through two changes of xylol.

1 1

Mount

in

balsam or in

alcohol.

by 90%.

Cristalite.

Results:
Parasites are conspicuously stained violet-purple, with
purple nuclei. Cell walls unstained.

Note: Alternatively, stages 6, 7

case the results will be as follows

deep

and 8 may be deleted in which

parasites not so conspicuously

stained violet-purple, with deep purple nuclei. Cellulose walls,
yellow to green. Lignified tissue, blue. Tissue nuclei, pale blue
:

with purple nucleoli, and chromosomes deep blue on a purple

spindle.

TITAN YELLOW
For the detection of magnesium in plant cells
Solutions required:

A. Titan yellow, special for magnesB.

ium test 0*2% aqueous.

Sodium hydroxide 10% aqueous.

Technique:
1

mounted on slides and brought down to

water in the usual manner, or freehand sections of fresh

Paraffin sections are

distilled

may be employed.
Add one or two drops

material

of the Titan yellow solution, followed

or
of
one
two
the
sodium hydroxide solution.
drops
by
2.

Results:
If

magnesium

is

present a red coloration

284

is

produced.

SECTION THREE

TRYPAN BLUE

nuclear stain for plant material

Solutions required:

A. Formol Acetic alcohol ( Telly esniczky)

Formaldehyde

70%

40%

Glacial acetic acid

B.

ml.

Alcohol

Trypan blue

1%

aqueous

Ethylene glycol
Absolute alcohol
Zinc sulphate
C. Trypan blue 1% aqueous
Distilled water

Absolute alcohol
Cresol

HCl, concentrated

D. Exactly the same

the HCl.
E.

F.

as

solution

but omit

Absolute alcohol

Cresol

. .

. .

95 ml.
5 ml.

Naphthol yellow, saturated in equal volumes

of alcohol and ethylene glycol.

G. Sodium thiocyanate 0-5% in equal volumes of

ethylene glycol and absolute alcohol.

H. Ponceau 3R

80%

alcohol

satd.

in
.

alcohol

80%
.

volume
volume

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

scopic examination, observing the nuclei rather than the nonnuclear material, as it may not be possible to remove all the blue
stain from the latter without completely decolorizing the nuclei.

Note: If

and

alcohol

50%

is

found inadequate, then solutions

G should be tried in turn to ascertain which is the most

E, F,
satisfactory for the particular plant material.

Some material,
no
or
needs
little
differentiation.
clover
epidermis
e.g.
as
differentiators
than E or
F
and
are
more
active
Solutions
sweet

50%
4.
5.

alcohol.

Place in

95%

alcohol for two minutes.

for one
Counterstain epidermis if desired with solution
this may not be advantageous in the case of smears.

minute but
6.

Rinse in

95%

7.

Immerse

in

alcohol.

two changes of Isopropyl alcohol

for

two minutes

in each.
8.

Immerse

in a third change of Isopropyl alcohol for five

minutes.
9.

Clear in xylol and

mount

in

Clearmount or balsam.

Results:

Nuclei, bright blue.

bright red.

If counterstain is used, the

Reference: Hoffmesster, E. R. (1953), Stain Tech., 28, no.

286

background

6,

309.

is

SECTION 4-CYTOLOGICAL METHODS

ACID FUCHSIN

TOLUIDINE BLUE

For mitochondria

AURANTIA

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

7.

Rinse with distilled water;

then stain with Solution

for

thirty to fifty seconds.

8.

Differentiate with

95%

alcohol;

dehydrate;

clear in xylol

blue.

Background,

and mount.
Results:

Mitochondria are

stained

red;

nuclei,

yellow.

ACID FUCHSIN

PICROINDIGO CARMINE

A cytological

stain for root tips

Solutions required:

A. Acid Fuchsin
B.

1%

aqueous

Saturated Picric acid, aqueous

Saturated Indigo carmine, aqueous
.

C. Acetic acid

i
i

volume
volume

0-25% aqueous

Technique:

water in the usual manner.

1.

Bring sections through to

2.

Stain from five to twenty minutes in the acid fuchsin solution.

3.

Rinse in water until stain ceases to come out of the sections.

4.
5.

6.

distilled

Stain from five to fifteen minutes in solution B.

Wash
Wash

with solution C.
rapidly in

70%

alcohol, in

which the sections

will

appear red.
7.
8.

9.

Wash

rapidly in

95%

alcohol, until sections appear greenish*

Dehydrate rapidly with two changes of absolute alcohol.

Clear in xylol and mount.

Results:

With onion

root tips as an example, chromosomes and late

prophase stages are in various shades of bright or dark red. Early
prophases, bluish red. Nucleoli, clear blue. Spindle fibres and
cell walls are stained a dark blue against a light cytoplasm.

From

Plant Microtechnique by D. A. Johansen, by courtesy of McCraw-Hill

Inc., New York.

Book Company,

290

SECTION FOUR

ALIZARIN RED, S
For mitochondria, cell inclusions,

etc.

Solutions required:

A.

(Benda's method)

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

9.

in

then differentiate for two to three minutes

Rinse in water;

30%

acetic acid until the nuclei appear reddish.

10.

Wash

11.

Dip

into xylol

in

running water for

five to ten

minutes

into absolute alcohol; then pass through

blot dry.

bergamot

oil

and mount.

Results:

Mitochondria,

Chromatin and archoplasm, brownish

violet.

red. Certain secretion granules, pale violet.

Centrosomes, reddish

violet.

ANILINE FUCHSIN

IODINE GREEN

For mitochondria
Solutions required:

A. Acid fuchsin aqueous

Aniline oil.
.

Mix

well

several hours

Note:

10%
. .

by shaking
;

then

90 ml.
10 ml.

at intervals over a period of

filter.

This solution deteriorates

after three or

four weeks.

Potassium permanganate

B.

C. Oxalic acid

5%

D. Iodine green

1%

aqueous.

aqueous.

1%

aqueous.

Technique:
1.

Small pieces of tissue are fixed in Regaud's Fluid for four

days.
2.

Transfer to

3%

aqueous potassium dichromate for eight days

changing the solution at intervals of two days.

3.

Wash

4.

Dehydrate

5.

Embed

in running water overnight.

in alcohol as usual

in paraffin

wax;

and

clear in xylol.

cut sections not

more than

5/^

in

thickness.
6.

Fix sections to slides; pass through xylol and descending

292

SECTION FOUR
grades of alcohol down to distilled water in the usual manner;
then blot carefully.
7. Place the slides, sections facing upwards, on the corner of a
tripod; flood the preparation with aniline fuchsin (Solution A)
and heat gently with a small bunsen flame until vapour rises.
8.

Remove

the flame and allow the stain to act for five to ten

minutes.
9.

Pour

the excess stain and drain for a few seconds.

oflF

10.

Rinse well in distilled water.

11.

Immerse

then pour
12.

pour

off^

in the potassium

permanganate for

five

seconds;

excess.

Immerse

in the oxalic acid solution for five seconds;

then

off excess.

13. Wash with distilled water; then stain with the iodine green
solution for five to ten seconds.

14.

Pour

off excess stain;

quickly with

95%

drain for a few seconds;

and absolute

15. Clear in xylol;

mount

dehydrate

alcohol.

in Cristalite.

Results:

Mitochondria, crimson.

Nuclei, green.

ANILINE FUCHSIN

PICRIC ACID (Altmann)

For mitochondria
Solutions required:

A. AltmanrCs Fluid:

Potassium dichromate

Osmic

acid

Note:
fi[xative

2%

aqueous

poor,

i
i

the^ penetration

Although
is

5% aqueous

is

it

volume
volume
power of

fixation.

B.

Acid fuchsin

. .

Aniline water

20 gm.

95 nalC. Picric acid saturated in absolute alcohol.

.

293

this

very satisfactory for surface

. .

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

Technique:
1
Small pieces of tissue, not more than z mm. in diameter, are
fixed for twenty-four hours in Altmann's Fluid.
.

2.

Wash

for an

and embed
3.

hour in running water then dehydrate

wax in the usual manner.
;

clear,

in paraffin

Sections, not thicker than

4//,

are brought

down

to distilled

water; then stained for six minutes in Solution B.

4.

Pour

off excess stain;

differentiate

then blot section carefully;

then

and counterstain by flooding the preparation with

Solution C.
5.

Rinse quickly in

alcohol

95%

clear in xylol,

alcohol; then dehydrate with absolute

and mount.

Results:

Mitochondria are stained crimson against a vivid yellow background.

'j

ANILINE SAFRANIN

(Babe's)

For demonstrating mitosis in animal tissues

Solutions required:

<

A. Aniline Safranin (Babe).

B. Alcohol 95%
Acetic acid

1%

99 ml.
.

Technique:
1.

ml.

Fix small pieces of tissue in Flemming's or Hermann's fluid

in paraffin wax.

and embed
2.

Stain sections for five to ten minutes in the safranin solution.

3.

Rinse in water.

4. Differentiate in the acid alcohol (Solution B).

5.

Rinse in

6.

Rinse in absolute alcohol.

7.

Clear in xylol and mount.

95%

alcohol.

Results:

Mitotic figures are stained an intense red, while resting nuclei

are deep pink to colourless.

294

SECTION FOUR

BASIC FUCHSIN
For plant

PICRO INDIGOCARMINE

tissues, as

(Alcoholic)

a cytological stain for root tips

Solutions required:

A. Basic fuchsin
B.

i%

Indigocarmine
Distilled water

in

70%

alcohol.

Picric acid, saturated

0-6 gm.
50 ml.

50 ml.

aqueous

Technique:
1.

down to distilled water; then stained

minutes in the basic fuchsin solution.

Sections are brought

from ten

to twenty

water until the stain ceases to come out.

2.

Rinse in

3.

Stain from five to fifteen minutes in Solution B.

distilled

4. Rinse quickly in
the naked eye.
5.

Rinse rapidly in

70%
95%

alcohol until the sections appear red to

alcohol, followed

until the preparation appears green to the

6.

by absolute alcohol

naked

eye.

Clear in xylol; then mount.

Results:

Chromosomes and

late

prophase stages, varying shades of red

early prophases, bluish red;

and

cell walls,

nucleoli, clear blue;

spindle fibres

dark blue; cytoplasm, light blue.

BASIC FUCHSIN

PICRO INDIGOCARMINE (Aqueous)

For chromosomes in plant tissues
-

Solutions required:

A. Basic fuchsin
B.

1%

aqueous.

Picro indigocarmine.

Technique:
1.

Material

2.

Wash

paraffin
3.

is

fixed in

Nevashin or Regaud.
dehydrate, clear and

in running water;

embed

wax in the usual manner.

Fix sections to slides and remove paraffin wax with xylol.

295

in

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

4.

Pass through absolute

90% and 70% alcohol down to distilled

water, as usual.
5.

Stain in the basic fuchsin solution for about five minutes.

6.

Rinse in distilled water.

7.

Stain in the picro indigocarmine solution for ten to twenty

minutes.
8.

Rinse in

distilled

water to which a few drops of hydrochloric

acid have been added.

9.

Differentiate in

80%

red coloration ceases to

alcohol for half to one minute, until

come away.

Dehydrate with 70%, 90% and absolute alcohol.

Clear in xylol, and mount.

10.

11.

Results:

Nucleoli and other

red.

brilliant

Chromosomes,

cell

com-

ponents, sky blue.

BREINL'S TRIPLE STAIN

For chromosomes
Solutions required:

A. Iodine
Potassium iodide
Alcohol 90%
.

B.

Safranin

1%

in

50%

gm.

2 gm.
100 ml.

alcohol

C. Methylene Blue, polychrome (Unna)

D. Orange tannin
Technique:
1.

Fix sections to slides and remove paraffin wax with xylol as

usual.
2.

Rinse with two changes of absolute alcohol.

3.

Mordant by immersing

4.

Rinse well with water.

5.

Stain for at least half an hour in the safranin solution in a

in solution

closed jar or tube.

296

for fifteen minutes.

SECTION FOUR
6.

Wash

7.

Stain with solution

well with water.

for ten minutes.

8. Wash with water; drain and remove excess water, but do

not allow the preparation to dry.

Place the slide under the microscope; then cover the preparation with orange tannin, by means of a pipette.
9.

10.

Observe the progress of the staining under the microscope

until the orange tannin has replaced the blue in the cytoplasm.
1 1

Withdraw excess

fluid

from the

by means of

slide

a piece

paper to avoid the risk of spilling over the microscope stage

and condenser.
of

filter

12.

with

Take the

95%

slide

away from the microscope and wash well

alcohol.

14.

Dehydrate in absolute alcohol.

Clear and complete differentiation in aniline

15.

Rinse with cedarwood

16.

Drain and remove excess cedarwood

13.

oil

preparation by blotting or wiping with a clean

17.

Mount

in Cristalite or

oil.

oil.

Canada balsam

from around the

fluffless duster.

in Xylol or

Emexel

mountant.
Results:

Chromosome

blue.

threads,

Cytoplasm, pale yellow.

Reference: Bolles-Lee, nth

Metaphase chromosomes,

edition, p. 664.

COPPER CHROME HAEMATOXYLIN

(Bensley)

For mitochondria
Solutions required:

A. Altmann's Fluid
B.

Copper

acetate, saturated

C. Haematoxylin

aqueous

in

10%

absolute

alcohol

5 ml.

Distilled water

90 ml.

ripened

297

red.

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

D. Potassium chromate

neutral,

5%

aqueous

Weigerfs Borax Ferricyanide Mixture

100 ml.
Borax 1% aqueous

E.

. .

Potass, ferricyanide

1-25

gm.

Technique:
1

Fix very small pieces of tissue in Altmann's fluid from

2 to

24 hours.

Wash, dehydrate,

2.

and embed

clear

in paraffin

wax

in the

usual way.

Cut

3.

sections

from 4

to

5jLt

in thickness

and

affix

them

to

slides.

Remove

4.

8.

Wash
Wash
Wash
Wash

9.

Immerse

5.

6.

7.

10.

paraffin

wax with

xylol.

well with absolute alcohol.

with

alcohol.

with

90%
70%

with

distilled water.

alcohol.

in solution

Wash with several

for 5 minutes.

changes of

distilled

water for a total time

of one minute.
1 1

12.

Stain with the haematoxylin (solution C) for

Wash

minute.

in distilled water.

Immerse

one minute, which should turn

they are only light blue in colour,
rinse in distilled water and return to the copper acetate solution
13.

in solution

the sections dark blue-black

for

if

and

if necessary repeat several times until the sections are dark

blue-black after a minute in solution D, or until no increase in

colour

is

obtained.

14.

Rinse with distilled water.

15.

Immerse

in solution

again for a few seconds; then again

rinse in distilled water.

16. Differentiate

under the microscope with a mixture consistE and two volumes of distilled water.

ing of one volume of solution

17.

Wash

18.

Dehydrate, clear and mount.

with tap water for 6-8 hours.

298

SECTION FOUR
Results:

Mitochondria are stained deep blue against a yellowish background.

COTTON RED - METHYL VIOLET - ORANGE G

A cytological stain for plant tissues
Solutions required:

A. Cotton red
B.

C.

i%

Methyl

violet

Orange

aqueous.

loB

Clove

i%

aqueous.

oil.

Technique:
1.

Stain sections of Flemming-fixed material in the cotton red

from eight to twenty-four hours.

solution
2.

Wash

v/ell in

from ten minutes

3.

Pour

4.

Wash

water then stain in the methyl-violet solution

an hour according to the material.
;

to

then wash in water.

off excess stain;

well with

95%

alcohol;

followed by thorough rinsing

with absolute alcohol.

5.

Drain

6.

Immerse

7.

Pour

well,

and blot

in orange

carefully.

G - clove oil from five to ten seconds.

off excess stain

then wash the preparation with clove

oil.

8. Differentiate in a fresh lot of clove oil for about one half to

one minute, controlling by examination under the microscope

until the violet stain is satisfactory.

9.

Pour

then wash with three changes of

off excess clove oil;

xylol.
10.

Mount

in

Canada balsam

in xylol.

Results:

Metaphase and anaphase chromatin, red. Prophase chromatin,

Chromoneata, red. Chromosome matrix, purple. Spindle
fibres and plastids, violet.
Cytoplasm, light brownish grey.
violet.

Nucleoli, red.

299

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

FEULGEN STAIN
For mitosis in plant cells
Solutions required:

A. Feulgen's fuchsin.
B.

Potass, metabisulphite

io%

N/i HCl

5 ml.

Distilled water

C.

5 ml.

Fast green

FCF 0-5%

in

100 ml.

70%

alcohol.

Technique:

Tissues should be fixed in Flemming and embedded in paraffin

wax.
Sections are brought down to distilled water.
Rinse in N/i HCl; then transfer to N/i HCl at 60 C. for
four to five minutes; afterwards rinsing in cold N/i HCl.
1.

2.

3.

Rinse in distilled water;

then stain for three to four hours

in Solution A.

Drain and immediately transfer to a stoppered jar containing

B for ten minutes; then transfer to a second jar of Solution B for ten minutes, followed by ten minutes in a third jar.
Note: The jars must be kept closed.
4.

Solution

5. Rinse in distilled water; then counterstain for one half to

one minute in Solution C.

6.

Dehydrate; clear and mount.

Results:

Chromosomes, reddish

violet.

GENTIAN VIOLET

PICRIC ACID

For chromosomes in plant cells

Solutions

IODINE

SECTION FOUR
Dissolve the potassium iodide in the water, then add
till dissolved, afterwards adding

the iodine and shake

the alcohol and mixing well.

B.

i%

Crystal violet

C. Picric acid

aqueous.
in absolute alcohol.

0*5%

Technique :

Fix in Navashin or Fleming.

1.

2. Take the preparation (smear or section) through to

alcohol in the usual way.
3.

Pass through

4.

Mordant

5.

Rinse well in water.

6.

Stain with solution

7.

Rinse well in water.

8.

Immerse again

9.

Rinse in

10.

95%

70%

alcohol.

for fifteen minutes in solution A.

95%

for ten to fifteen minutes.

in solution

A for

few minutes.

alcohol.

Immerse the preparation

for about one second in the picric

acid solution.
11.

Wash

12.

Rinse in clove

immediately with absolute alcohol for a few seconds.

oil until

the violet stain ceases to

come out of

the preparation.
13.

Wash

well in two changes of xylol.

14.

Immerse

15.

Drain

in a jar of xylol for about an hour.

off excess xylol

and mount in Canada balsam

in xylol

or in Cristalite.
Results:

Chromosomes:

rich purple, each

chromosome being sharply

defined, Cytoplasm: yellow.

Note: This technique

Violet
tion

is

is

an improvement on Newton's Gentian

Iodine in that fading does not occur and sharp differentiaobtained of Chromosomes that are close together.

Reference: Smith, F. H. (1934), Stain Tech., 9 95-6.

301

SECTION FOUR

HAEMATOXYLIN

(Regaud)

For mitochondria
Solutions required:

A. Formalin (40% formaldehyde)

Potassium dichromate
Distilled water

B.

Potassium dichromate

C.

Ammonia

- ferric

25 ml.
3

gm-

98 ml.

25% aqueous.

alum 10% aqueous.

D. Haematoxylin (Regaud).
Iron

E.

Alum 5%

aqueous.

Technique:

Small pieces of tissue are fixed for three to five days in Soluwhich should be freshly prepared and changed each day.

1.

tion A,
2.

Mordant by immersing for ten

3.

Wash

in

to fourteen days in Solution B.

running water for twenty-four hours.

Dehydrate by immersing in ascending grade of alcohol, and

manner.

4.

clear in the usual

5.

Embed

6.

Fix sections to slides

in paraffin

grades of alcohol
7.

Mordant

down

wax and
;

cut sections no thicker than

5//.

de-wax and pass through descending

to water.

sections for one to three days in a stoppered jar

in the incubator at 37 C.

minutes or so in running water.

8.

Rinse for

five

9.

Immerse

for twenty-four hours in the haematoxylin (Solu-

tion D).
10. Differentiate

in

Solution E,

controlling at intervals

examination under the microscope.

11.

Rinse in running tap water for twenty to thirty minutes.

12.

Drain

13.
14.

off excess water;

then rinse with

Dehydrate with absolute alcohol.

Clear in xylol and mount.
302

95%

alcohol.

by

SECTION FOUR
Results:

Mitochondria are conspicuously stained intense black.

HAEMATOXYLIN

SAFRANIN

For differentiating nucleoli and other nuclear constituents

in plant cells

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

12.

Clear in xylol.

13.

Mount

under the

oil

in Cristalite or

immersion

Canada balsam

in xylol

and examine

objective.

Results:

Nucleoli, brilliant red. Chromosomes during metaphase and

anaphase are stained a bright red and stand out sharply against a
dark background during prophase and telophase the chromosomes
;

are considerably darker. Trabants are usually red

to the chromosomes by a black thread.

and are attached

Note: This technique gives the best results after a killing fluid
containing picric acid, but chromic acid fixatives also give good
results.

Reference Plant Microtechnique (ist ed., p. 75) by D. A. Johansen, by courtesy

McGraw-Hill Book Co.
:

of the

IRON ACETO CARMINE

(Belling)

For chromosomes in microsporocytes

Solutions required:

Iron Aceto Carmine [Belling)

Acetic acid 50% aqueous .

Carmine, powdered

100 ml.

gm.

Boil under reflux condenser for half an hour.

Cool and filter.

a few drops of

Add

ferric

hydrate in

50%

acetic acid.

Technique:

Temporary preparations :
1

2.

Anther smears are made on

slides

Place a drop of the stain on a smear

by teasing and squashing.

;

then cover with a cover-

slip.

a corner of a tripod
3. Place slide, coverslip facing upwards over
and heat gently until steam rises from the edges of the coverslip.
4. Examine under the microscope at once.

304

SECTION FOUR
Results:

Chromatin

is

stained deep translucent red, while cytoplasm

is

unstained.

Permanent preparations :
5.

Proceeding from Stage 4 (above), immerse the preparation

in a Petri dish containing

becomes loose.

acetic acid until the coverslip

10%

6. Immerse the slide and the coverslip in a mixture consisting

of equal volumes of absolute alcohol and glacial acetic acid, contained in another Petri dish.

7. Transfer to a dish containing a mixture consisting of three

volumes of absolute alcohol and one volume of glacial acetic acid.
8.

Transfer to a mixture consisting of one volume of glacial

and nine volumes of absolute alcohol.

acetic acid
9.

10.

Transfer to absolute alcohol

then into two changes of xylol.

Refix the coverslip to the slide with a drop of Cristalite.

METHYL GREEN - ACID FUCHSIN - ERYTHROSIN

A cytological stain for plant cells
Solutions required:

A. Methyl Green
B.

1% aqueous.
1% aqueous.
1% aqueous.

Acid fuchsin

C. Erythrosin
Technique:
1.

Take

sections

down

to distilled water in the usual

manner.

2.

Stain for an hour in solution A.

3.

Remove

4.

Stain in the acid fuchsin solution for one minute.

5.

Wash

6.

Stain with the er)rthrosin solution for two or three seconds.

7.

Wash

excess stain

by washing thoroughly with water.

well with distilled water to

remove excess

well with distilled water and drain

water, but do not allow the section to dry.

305

and

stain.

blot off excess

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

8.

Rinse with

9.

Rinse quickly with

10.

70%

alcohol.

90%

alcohol.

Dehydrate quickly but thoroughly with two changes of

absolute alcohol.

Clear in xylol and

11.

mount

in

Canada balsam

in xylol or in

Emexel.
Results:

Chromatin granules and nucleoli in early stages of microsporogenesis are stained green while the linin threads are red.
Chromosomes are stained brilliant green in later stages.
Reference: Cooper, D. C. (1931) American J. Bot., i8, 337.

METHYL GREEN

For chromosomes,

etc., in

ACID FUCHSIN
plant tissue

Solutions required:

A. Methyl green 2% aqueous

Acetic acid 1%
.

B.

Acid fuchsin

1%

100 ml.

2 ml.

aqueous.

Technique:
1.

Sections are brought

to seven hours in the

2.

Wash

down

to water;

then stained from

six

methyl green solution.

in water until the stain

is

almost entirely removed from

the non-lignified elements (this process should be controlled by

examination under the microscope v/hile the preparation is still
wet).
3.

Rinse in water.

4. Stain from five to ten minutes in the acid fuchsin solution,

controlling under the microscope to ensure that the green is not
extracted from the lignified tissues.
5.

Wash

6.

Clear in clove

rapidly in
oil

alcohol, followed

by absolute alcohol.
then pass through xylol and mount.

95%

Results:

Cytoplasm and

plastin, light red;

green.

306

chromosomes and

nuclei,

SECTION FOUR

METHYL VIOLET - EOSIN SCARLET

A botanical stain for mitosis in root tips
Solutions required:

A. Methyl Violet 5B aqueous 1%.

B.

Picric acid

1%

in absolute alcohol.

C. Eosin scarlet, saturated in equal

volumes of clove oil and absolute
alcohol.

Technique:
1.

Sections are fixed to slides and taken

down

to water in the

usual manner.
2.

Stain for twenty to thirty minutes in the methyl violet soluwash with distilled water.

tion ; then
3.

Differentiate with the picric acid solution for a

few seconds.

Immerse in absolute alcohol to which has been added 0-15%

strong ammonia solution, for fifteen to twenty seconds.
4.

5.

Immerse

6.

Counterstain for five to fifteen seconds in the eosin scarlet

in absolute alcohol for ten to twenty seconds.

solution.
7.

Clear in clove

oil

rinse with xylol

mount.

Results:

Resting and dividing chromatin, purple.

Cell walls, red.

Plastin,

Cytoplasm, pink.

deep red.

NIGROSINE
For the study of salivary chromosomes of Drosphila
Solutions required:

A. Acetic acid
B.

45%

Absolute alcohol
Distilled water
Hydrochloric

aqueous
.

70 ml.

29 ml.

acid, pure, cone.

307

ml.

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

C. Nigrosine,

alcoholic

brand)

70%

(Michrome
.

alcohol

gm.

100 ml.

Technique :
1.

Dissect the specimen in

2.

Treat with solution B for one to two minutes, keeping glands

45%

acetic acid.

well covered with the reagent.

3.

Wash the specimen

water, after
five

which

a large

two or three times with distilled

drop of water is left on the specimen for

carefully

minutes.

4.

Drain thoroughly and

5.

Add

6.

7.
8.

carefully.

drop of the nigrosine solution.

Place a coverslip over the preparation but do not squash.

Allow the stain

to act for ten to fifteen minutes.

Place a piece of blotting paper over the coverslip and slide

and squash the preparation.

9.

Immediately place a large drop of glycerine on one edge of

the coverslip.
10. The preparation may now be examined at once, but after
about twenty-four hours the glycerine will have penetrated the
specimen thoroughly, when it is recommended, for the sake of

convenience, to remove the excess liquid and ring the coverslip

with wax.

Note: Preparations obtained as above will last for reasonably

long periods provided they are carefully handled.
Results:

Salivary glands specifically and intensely stained only the outer

bands being coloured, except in heterochromatin regions. When
the preparation is ageing, however, the interbands as well as the
;

cytoplasm, become faintly stained.

Reference: Pares,

Ramon

(1953), Nature, 172, no. 4390.

308

SECTION FOUR

SAFRANIN CRYSTAL VIOLET (Hermann)

For chromosomes
Solution required:

A. Safranin
Aniline water

Absolute alcohol
B.

Crystal violet

1%

in

70%

gm.

50 ml.
50 ml.

alcohol.

Technique:
1.

Stain for three to twenty-four hours in the safranin solution

in a closed jar or tube.

2.

Destain briefly with

50%

alcohol.

3. Counterstain with the crystal violet from thirty seconds to

five minutes.

4.

Rinse quickly with

5.

Clear with clove

6.

Wash

90%

followed by absolute alcohol.

oil.

with xylol, and mount.

Results:

Chromosomes and
chromatin granules

may show

violet

nucleoli are stained red; resting nuclei and

propase and early telophase nuclei

violet:

chromonema and red chromatin granules

cyto-

plasm, light violet; spindle, deeper violet.

Reference: Hermann, Arch, Mik. Anat., 34, 58.

SAFRANIN

GENTIAN VIOLET

(Flemming Tricolour
For chromosomes,

ORANGE

stain)
etc.

Solutions required:

A. Safranin 3*5% in
Aniline water
B.

Gentian violet

C. Orange

G 1%

95%
.

5%

alcohol
.

aqueous.

aqueous.

309

10 ml.

10 ml.

G.

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

Technique:
Sections of material, which have previously been fixed in
Flemming's fixative, are stained for two to twenty-four hours with
1.

the safranin solution, in a stoppered staining jar.

2.

Differentiate in acid alcohol until colour almost ceases to

come out of the sections and microscopic examination shows

chromosomes in metaphase stained clearly with the safranin.
3.

Rinse with

the

distilled water.

Immerse

in the gentian violet solution, in a staining jar for one

4.
half to three hours, washing with water at intervals and examining

under the microscope until the prophase chromatin

is

clearly

stained violet.
5.

Differentiate in the orange

seconds
6.

when

pale violet clouds

solution for thirty to sixty

should be given

off.

Rinse in absolute alcohol until scarcely any more colour

comes out of the

sections.

7. Differentiate in clove oil and while very faint clouds of colour

are still coming away, rinse with benzol and mount in balsam.

Results:

Chromosomes,

red.

Nucleoli, red.

Metachromatic chromatin,

deep purple. Cytoplasm, orange. Spindle, blue-violet.

Note: The action of this stain should be so precise that in one
strand of chromatin the diffused portion will take the violet, while
the condensed section should be stained with the safranin.

WRIGHT'S STAIN
For chloroplasts in tissue spreads and for plant cytology
Note: Valuable for showing cytoplasmic changes in various tissues
and cell inclusions. Reveals cytoplasmic differentiations and experimental change. The method is generally applicable for cytological
work wherever material may be spread (not smeared) and dried rapidly.

Produces excellent preparations of chloroplasts. Where cells separate

well and where there is neither an excess of water in tissue fluid nor
concentrated protein scum, conditions are favourable for a good spread
of uninjured cells and an excellent stain.
Solution required:

Wright's

310

stain.

SECTION FOUR
Technique:
1.

A Nitella cell, carefully isolated from the plant, is placed on a

piece of

filter

paper from which

it is

transferred to a clean dry

slide.
2. The uninjured cell is pricked so that
the chloroplasts in effective spread.

it

bursts out

its

sap and

with needles and transfer to an adjacent

3. Lift the deflated cell
dry region and tear to release more chloroplasts with a small
amount of sap. Arrange areas of the cell wall smoothly so that no
portion of the cell or its contents are lost in the preparation. By
spreading the cell fluid about in this way there is no excess in any
region.

rapidly with gentle heat.

4.

Dry

5.

Place

to act for

ml. of Wright's stain

on the dried spread, and leave it

distilled water and rock the

one minute then add 2 ml.

;

slide gently to ensure

thorough mixing.

Allow this diluted stain to act for three to five minutes then
pour off and wash with distilled water until the thin portion of the
films appears pink to the naked eye then pour off excess stain.
6.

7.

Wash

with

distilled

water and allow the preparation

to dry

before examining.
Results:

Chloroplasts of normal Nitella are basophilic and of marked

Large, homogeneous-appearing vacuole plas-

alveolar structure.

which are noted in the streaming of the living cell vacuole

are brilliantly eosinophilic. However, they prove to be complex
with a central raphe, usually colourless arrangements of both
distribasophilic and eosinophilic granules; and both bilaterally
from
conditions
buted lacunae varying according to experimental

tids

The

staining of Tunicate material is

so critical that species differences are readily noted in comparative
studies of the same cells.
colourless to brilliant blue.

Reference: Koehring, Vera (1951), Stain Tech., 26, 29.

311

SECTION 5-

FLUORESCENCE MICROSCOPY

(a)

GENERAL INFORMATION

Fluorescence

is the property possessed by many substances of

short
wavelengths of Hght into longer wavelengths. In
converting
and structures of most interest are
the
substances
microscopy

those which convert ultraviolet light into light of the visible spectrum, as only substances of this character can be observed directly.
of course, well known that most living organisms are profoundly affected by short light waves, and a great deal of information as to their structure has been obtained by the study
It

is,

of the appearance of these organisms under the influence of

invisible light rays. If individual cells or structural units are

examined before, during, and after ultraviolet treatment, enough

of this effect should be discovered to impart some understanding as to the changes which occur in the animal, or plant,
as a whole.

In fluorescence microscopy structural details are rendered

visible by: {a) innate auto-fluorescence, a property possessed by
most tissues which when excited by short light waves become

"

fluorclearly visible since they become luminous and glow or

"
with a radiance of their own, or by {h) secondary fluorescence
esce
"
"
which is known as fluorochromy and is brought about by the

process of treatment of the tissues with fluorescent dyes and certain alkaloids (e.g. Berberine sulphate) and other substances.
It is proposed to deal only with fluorochromy in the short space

of this chapter.
Fluorescent dyes and other substances used for this purpose are
known collectively as " fluorochromes " these materials are
;

absorbed by certain parts of the cell. When tissues,

bacteria or protozoa, which have undergone treatment with fluorochromes are examined under the microscope, using ultraviolet
light instead of transmitted light of the visible spectrum, they
become visible as bright luminous objects against a dark background. Cells stained with fluorochromes absorb the ultraviolet

selectively

rays of short wavelength, and emit this energy in the

315

form of

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

fluorescent light in the visible spectrum. Basic and acidic fluoroact specifically to stain certain cellular structures as do

chromes
the

more common microscopic

stains

such

as,

for

instance,

methylene blue and eosin. The colour and the intensity of

fluorescence depends on the relative basophilia and acidophilia of
the individual

cells,

and upon the nature of the particular fluoro-

chrome.

Fluorochromy may be employed with advantage

in the study of

living organisms: for instance, uranin, a non-toxic stain, may be

injected into mice and frogs and the living organs can be studied

without interfering with their functioning. Fluorochromy is also

of practical importance for the demonstration of diphtheria bacilli,
tubercle and leprobacilli, malaria, etc., as well as for virus research.

(b)

EQUIPMENT REQUIRED

Contrary to the generally held belief, the apparatus required for

fluorescence microscopy is fairly simple and inexpensive.
special

microscope
1.

is

not required.

B.T.H. Mazda Mercury Vapour

Lamp

(box type)

ME 250

w/50/5.

A simple convex lens to

project the image of the light source

to
a
suitable
blue
filter
the microscope mirror. The lens
through
and light source should be encased with a black hood to prevent
2.

scattering of the rays.

3. A yellow filter which is placed in the oculars of the microscope to prevent any harmful effect of ultraviolet light to the
microscopist's eyes. For this purpose Ilford's minus blue Micro 4
is

recommended.
It

is,

ations

of course, essential that fluorescence microscopical examincarried out in a darkroom to be successful. It has

must be

been stated that microscope slides of special glass are necessary

for fluorescence microscopy, but provided they are not more than
1-3 mm. in thickness ordinary microscopic slides have been found
quite satisfactory.

316

SECTION FIVE
(c)

STAINING METHODS

Notes on fluorochromic staining technique:

(a) Fixatives containing salts of

heavy metals (with the exception

chlorine, bromine, iodine, and nitro compounds should

be avoided as these exert a quenching effect on fluorescence. The

of zinc)

most

suitable fixatives are 5 to

10%

formalin, or Kahle's fluid.

embedded in paraffin wax, all traces of the wax,

auto-fluorescent, must be removed before sections are

(b) If tissues are

which

is

stained and examined.

(c)

special grade of

immersion

oil

known

as fluoroil,

which

non-fluorescent, should be used for high-power examination,

since cedarwood oil and most of the immersion oils available for
is

ordinary microscopy are unsuitable for fluorescence work.

The

usual mounting media, as used for ordinary microcontain

scopy,
highly fluorescent materials which render them unsuitable for fluorescence work, and should, therefore, be avoided.
(d)

For temporary mounts, glycerine may be used, and for permanent

mounts there is a satisfactory medium on the market under the

name

of Fluormount.

Fluorochromes, of which auramine O, coriphosphine, acridine

Hi 07, aesculine, acridine orange, primuline, thiazole yellow
are examples, are frequently used in very dilute solutions to produce characteristic fluorescent colours, and when preparations
which have been treated with these solutions are examined in transmitted light of the visible spectrum, they appear to be unstained
yellow

or only very faintly tinted.

Some explanation as to the colour differentiation obtained by the
use of general-purpose fluorochrome of which acridine yellow

Hi 07

an example,

explained by the fact that fluorescence

by hydrogen-ion concentration, and as fluorochromes also exhibit differential absorption by various tissues, the
colour

is

is

is

effected

production of a great variety of fluorescent colours is brought

about by the influence of these two factors. Nuclei can be differentiated from cytoplasm by the use of any one of the following
general fluorochromes: acridine yellow Hi 07, coriphosphine,

phosphine 3R, or acriflavine.

Titan yellow, rhodamine B, phosphine 3R, methylene blue are
317

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

all

excellent fluorochromes for fat, while berberine sulphate is

for protozoal parasites, particularly for malaria. Thio-

much used
flavine has

been found satisfactory for virus and for

bacteria, as

well as for general fluorochromic staining, while acriflavine has

been used for trypanosomes and as a general stain, and uranin is

one of the most suitable fluorochromes for

intravital staining.

All

these fluorochromes are used in very dilute aqueous solutions,

that is to say, something to the order of o-i to o-oi per cent.

I.

Differential Staining of C3rtoplasni, Nuclei, Nucleoli

and Chromatin
Solutions required:

A. Congo red o-i% aqueous or acid

fuchsin 0'i% aqueous.
B. Acridine

Hi 07 o-i%

yellow

aqueous or coriphosphine

o-i%

aqueous.
Technique:
1.

Sections are stained for two minutes with the

Congo red

or

acid fuchsin solution.

2.

Pour

off excess

Congo red

then stain for two minutes with

the acridine yellow or coriphosphine solution.

This technique gives a very sharp

2.

Method A.

differentiation.

Staining of Fat

Solution required:

Phosphine 3R o-i% aqueous, or

methylene blue 1% aqueous.
Technique:
1.

Stain frozen section for two minutes

2.

Examine

then rinse in water.

in water.

Results:

With phosphine 3R

fat is

observed as a

318

silver fluorescence

SECTION FIVE
against a brown background and due to the omission of lipid solvents in this technique, smaller and more numerous fat droplets
can be seen than in the case of the sudan techniques as used in

ordinary microscopy.

With methylene blue

fat

gives

blue

fluorescence.

Method B.

Solution required:

Thioflavine S

i%

aqueous.

Technique:
1.

Frozen sections are stained for two minutes; then rinsed

in water.
2.

Examine

in water.

Results:

Fats appear violet against a dark background.

With

Note:

this

method fewer

lipids are stained

than with

phosphine 3R.

3.

Intravital Staining with

Dyes used

for this purpose

Fluorochromes

must be water

soluble, non-diffus-

body, non-toxic in the workable dilutions required, and highly fluorescent even in greatly diluted solutions.
Uranin possesses all these qualifications and is one of the most

ible in the living

useful fluorescent dyes for intravital work, as stated earlier in this

chapter. Acriflavine is another useful dye for this purpose, al-

though

it

is

not so intensely fluorescent as uranin.

escence of uranin

The

fluor-

impaired in basic solution so that it appears

most readily in organs of an acid reaction. It is used in o-i%
solution in physiological saline, in which form it should be injected
into the animal's blood stream or into the organ to be studied.

The

colour of

its

and consequently

is

fluorescence varies with hydrogen-ion changes

of great value as an intravital hydrogen-ion

it is

The colour changes are easily visible in dilutions to the

order of one part in ten millions and in dilute solutions the intensity of the fluorescence has a definite relation to the concentration
indicator.

of the dye and consequently the intensity of the fluorescence

319

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

serves as an indicator of the

amount

of uranin present.

rhodamine B, berberine sulphate have

thioflavine,

also

Primulin,

been found

useful for intravital work.

Many

important discoveries in the biological

field are

due to

fluorescence microscopy and although a great many beautiful and

highly contrasting colour combinations have been obtained by the

techniques devised up to the present time, fluorescence microscopy

has scarcely yet emerged from the embryonic stage, and there is

tremendous scope for experiment and improvement both

to technique and apparatus.

in regard

Staining of Muscle

4.

Solutions required:

A. Primulin o-i% aqueous.

Sodium

B.

nitrite

2%

aqueous.

C. Hydrochloric acid, cone.

Distilled water
.

D. Resorcinol,

saturated

10 ml.

90 ml.

aqueous

solution.

Technique:
1.

Sections are stained for two to five minutes with primulin

solution.
2.

Wash

nitrite to

quickly;

then immediately transfer to the sodium

stirring, an equal

which has previously been added, with

volume of the diluted hydrochloric

acid (Solution
leave therein for fifteen to twenty minutes.

above), and

3. Wash in water; then immerse for fifteen to thirty seconds in

the resorcinol solution.

Results:

Muscle

tissue

fluoresces

with a strikingly luminous green

fluorescence.

320

SECTION FIVE
Differentiation of

5.

Method A.

Nerve Tissues

Solution required:

Acridine yellow

Hi 07 aqueous o- 1%.

Technique:
1.

Sections are stained with the acridine yellow for two minutes.

2.

Pour

off excess stain

3. Dehydrate
Fluormount.

and rinse with

in the usual

distilled water.

clear in xylol;

manner;

mount

in

Results:

Neurophile nerve tissue appears

tissue

is

light blue, while cortical

nerve

light yellow to orange.

Method B.

Solution required:

Acridine orange

o-i% aqueous.

Technique:
1.

Sections are stained with the acridine orange solution for

two minutes.
2.
3.

Pour

off excess stain, rinse

Dehydrate

with

distilled water.

as usual, clear in xylol in

Fluormount.

Results:

Neurophile

is

bluish-grey while cortical tissue

is

brownish

orange.

Method C.

Solution required:
Acriflavine

o-i% aqueous.

Technique:
1.

pour
2.

Stain with the acriflavine solution for two minutes;

off excess and rinse in water.

Dehydrate rapidly

clear in xylol

and mount

in

then

Fluormount.

Results:

Neurophile

is

blue,

while cortical tissue appears brownish

yellow.

321

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

6.

Staining

and counting of bacteria, and differentiating

living from dead cells, in soil
Solutions required:

A. Buffer solution
Solution

B.

pH

200 ml.

100 ml.

Acridine Orange,

FS

o-i

gm.

C. Solution

Solution

5 ml.
5 ml.

D. Solution

Solution

10 ml.

ml.

Solution

3 ml.

Solution

9 ml.

Solution

2 ml.

Solution

8 ml.

G. Solution

Solution

9 ml.

E.

F.

ml.

Technique :
1.

label

Place

gm. of the

them consecutively

sifted soil in each of six test tubes,

B, C,

D, E, F,

and

and G.

Add

10 ml. of the appropriate solution to the five tubes

labelled B, C, D, E, and F (i.e. 10 ml. solution B to tube B;
2.

10 ml. solution

to tube C, etc.).

Note: As soils differ in their ability to absorb the stain, the same
must be stained with different concentrations of the acridine

soil

orange to ascertain the most suitable concentration. Solution G

will be used for differentiating between living and dead bacteria.
3

Shake each of the

stand for
4.

five

five

tubes thoroughly

then leave them to

or ten minutes.

The most

suitable concentration for the particular soil

is

that

contained in the tube, which shows the least amount, compared

with the others, of excess dye, and this specimen should be used
for examination as follows:
(a)

For qualitative analysis of autochthonic bacteria :

The dyed
(liquid) layer

soil
is

suspension

poured

is

strongly centrifuged

the upper

off and discarded, leaving a small quantity

322

SECTION FIVE
of sediment which

and covered with

immersion lens.
(a)

For

then well mixed with a drop of paraffin oil

a coverslip before examination with the oil

is

bacterial counts:

loopful of the stained and shaken soil suspension is placed

on a slide, covered with a coverslip, and examined with an oil
immersion lens. A counter of 20/x depth combined with a

The particles of soil covered

humus slime show a dim red
bacteria are green. The stained

counting ruled eyepiece is used.

with humus and the particles of
fluorescence

and the

living

and may be used for culturing.

To diff"erentiate between living and dead cells, solution G
should be used: living cytoplasm and nuclei show green fluorbacteria are not killed

escence, whilst dead protoplasm develops

coloured fluorescence.

bright

copper-

Reference: Strugger, S. (1948), Canad.J. Res. Sec. C, 26, pp. 188-93.

7.

Trypanosomes in Blood
Strugger 's Method)

Vital Staining of Living

(S.

Solution required:

Acridine orange

sodium

o-oi%

in

0-85%

chloride.

Technique:
1.

drop of blood suspected of containing trypanosomes

mixed on a slide with a few drops of the acridine orange

and covered with a coverslip.

is

solution,

Examine with a blue light fluorescence microscope, which

be
constructed as follows parallel rays produced by attachmay
lens to the lamp are filtered with a curvette (2 J cm.
a
convex
ing
with
saturated solution of copper oxide ammonia so
filled
thick)
that only the blue light reaches the plane mirror of the microscope.
2.

filter containing an orange glass which absorbs blue light

quantitatively, but allows green, yellow and red to pass unchanged,
slide with powdered anthracene in
is placed over the ocular.

liquid paraffin

is

used for focusing.

323

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

Results:

Trypanosomes and leucocytes are seen as motile, bright, green

luminous bodies while the erythrocytes are scarcely visible and
are non-fluorescent.

8.

Staining of Trypanosomes in Dried Blood Films

Solution required:

Auramine

o-i

Phenol crystals

%
.

aqueous
.

lOO ml.
5

gm.

Technique:

Fix air-dried blood films, suspected of containing trypanosomes, in pure methyl alcohol for two to five minutes, then wash
with distilled water.
1.

2.

Stain for about four minutes in the auramine solution then

wash with

distilled

water for one to two minutes, keeping the slide

in the dark.
Results:

Examined with blue

light fluorescence microscope, erythrocytes

as
dark
green,
appear
slightly luminous circles, while trypanosomes shine with a bright golden fluorescence against a black back-

ground. By covering the film with a drop of liquid paraffin and a

coverglass the contrast will be even more marked.

9.

Staining of Tubercle Bacilli

Solutions required:

O aqueous 0-1%
Phenol crystals
.

A. Auramine

Dissolve by shaking.

mine decomposes
B.

at

.,

Do

100 ml.
5

gm.

not apply heat as aura-

40 C.

Methylene blue 0-1% aqueous.

C. Potassium permanganate

324

0'i% aqueous.

SECTION FIVE
Technique:
Place sandalwood

Three

oil

or fluoroil between the slide and condenser.

be enclosed with a shield to

sides of the microscope should

exclude extraneous

light.

Sections:
1.

Fix tissues in 5 to

hours;

embed

10%

in paraffin

formalin for twelve to twenty-four

cut sections 5 to lo// in thick-

wax and

ness.
2.

De-wax

pletely and

as usual, taking care that all traces of

finally removed from sections.

wax

are

com-

3. Bring sections down to distilled water as usual; then flood

with the auramine solution and warm (not over 40 C.) for five to

ten minutes, afterwards washing with distilled water.

4. Counterstain in methylene blue solution for thirty seconds;
then rinse in distilled water, dehydrate as usual clear in xylol and
mount in Fluormount.
;

Smears:
1. Stain for five minutes in the auramine solution, afterwards
washing with tap water and decolorizing in 25% sulphuric acid.

in the potassium permanganate solution for about

to overcome any interfering fluorescence.
thirty seconds
2.

Immerse

Results:

Tubercle bacilli appear as thin shining,

against a dark background.

An Improved Method

slightly

curved rods

of Staining Tubercle Bacilli

(H. Lempert's

Method)

Solutions required:

A. Phenol

3% in
O

Auramine

distilled
.

Dissolve by shaking.

mine decomposes when

325

water
.

Do

100 ml.
0-3

gm.

not apply heat, as auraheated above 40 C.

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

B.

0-5 ml.

0-5

75 ml.

25 ml.

Hydrochloride acid, pure

Sodium
Methyl

chloride
alcohol,

pure

Distilled water

C. Potassium

permanganate

gm.

o*i%

aqueous.
Technique:
1

Smears of sputum or centrifuged urine deposits are fixed by

heat in the usual manner.

2.

Immerse the smears

in the

auramine solution for eight to ten

minutes, afterwards washing them with tap water.

Decolorize by immersing for two minutes in each of two
changes of Solution B; then wash in tap water.
3.

4.

Immerse

thirty seconds

in the potassium
;

permanganate solution for about

blot and dry.

then wash with tap water

Note:
It is claimed that this method gives more positives than the
Ziehl-Neelsen technique and there is a saving in time. Using
Lempert's method, tubercle bacilli are visible under the two-thirds

or the quarter-inch objective with a X 8 eyepiece, and the onesixth-inch objective may be used for confirmation.

10.

Staining Virus with Primulin

Solution required:

Primulin o-i% aqueous.

Technique:
1.

Stain for thirty seconds.

2.

Mount

pale

gum

if

desired in a

medium

consisting of 100 gm. of best

and 50 ml. glycerine with

acacia dissolved in 100 ml. water

the addition of 10 gm. chloral hydrate.

Note: With primulin, which may also be used as a general

fluorochrome, a blue-violet fluorescence is obtained.

326

SECTION FIVE

ADDITIONAL REFERENCES
Lab. Tech. in Biol, and Med., 98, 2nd edition.
Cowdrey, E. V. (1948),
"
The site of acidification of urine in frog's and rat's kidEllinger, P. (1940),
neys ", Quart, jf. Exp. "Physiol.^ 30, 205-18.
Fluorescence microscopy in biology ", Biol. Rev., 15,
Ellinger, P. (1940),
323-50.
"
Fluorescence microscopy applied to
Evans, G. H., and Singer, E. (1941),
ocular tissues ", Arch. Ophth., 25, 1007-19.
Lempert, H. (1944), Lancet, 247, 818-22.**
Fluorescence microscopy applied
Metcalf, R. L., and Patton, R. L. (1944),
to entomology and allied fields," Stain Techn., 19, i.
"
Fluorescence Microscope Examination of TrypanoStrugger, S. (1948),
somes in Blood." Canad. J. Res., Sec. E, 26, 229-31.

SECTION 6HISTOCHEMICAL METHODS

MICRO
Micro-incineration

INCINERATION

a technique

is

employed

to ascertain the

relative positions occupied by inorganic salts in fixed tissues,

the total distribution of these mineral substances in cells

and
and

tissues.

There are two methods of fixation, neither of which is perfect,

namely; (a) Freeze- dry and (b) Chemical fixation. The first
method requires reagents and apparatus which are not normally
available in the average laboratory; also the method is exceedingly
laborious and time consuming: there is a certain element of risk
of injury to the operator who may not be accustomed to handling
such substances as liquid nitrogen at a temperature of i7oC.
Moreover,

method

it is

offers

"

*'

extremely doubtful

if

any more accurate

results than those obtained

the elaborate

Freeze-Dry

by

the relatively simple chemical fixation method.

By employing either method the total mineral distribution can-

not be determined with an error of

less than, say,

io%

as all

minerals will not survive the incineration processes, and it is,

therefore, proposed to describe here the more simple and straight-

forward chemical method of fixation which can be carried out in

any laboratory.
Solution required:

Absolute alcohol

Formaldehyde

40%

..

90 ml.

..

10 ml.

Note: It is best to use freshly distilled alcohol. On no account

must the alcohol have been dried with copper sulphate nor any
other mineral reagents. The formaldehyde must not have been
treated with calcium carbonate, buffer salts or any other mineral
reagents. It is essential that all the reagents used must be free

from, and remain free from, dust and mineral matter; this refers
also to the xylol and the paraffin wax. All aparatus used must be
scrupulously clean, washed out with several lots of distilled water,
partially dried with absolute alcohol,

and

in all cases,

possible, finally dried with a clean dust-free cloth.

331

where

at all

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

1
Fix small pieces of tissue in absolute alcohol, with a mechanical agitator, for twelve to twenty-four hours, changing the
alcohol at intervals of one hour during the day.
.

Clear in freshly filtered xylol.

2.

3. Infiltrate

plunged

with and

embed

in wax,

which must not be

into water to hasten cooling.

Clean

slides, in readiness to take the sections, by washing

several times in distilled water, then partially drying them
with absolute alcohol, and finally drying them with a dust-free
4.

them

Store the slides away from dust and mineral matter until

cloth.

they are required for use.

Cut

5.

serial sections 3 to ^fi in thickness, taking particular care

at this stage that

they are not contaminated with dust or mineral

matter.

Press serial sections onto slides, without any fixative or

6.

spreading agent, with the fingers which have been washed and
dried with absolute alcohol.

Note: It is now, at this stage, more than ever necessary to guard

against contamination by dust.
7. Mount alternate serial sections on albuminized slides for
normal histological staining and comparison later with the

incinerated specimen.

The

8.

slides bearing the sections for incineration are placed in

a quartz-tube electric furnace, but if that is not available a muffle

furnace, the inside of which is entirely free from dust and debris,
will serve the purpose.

Gradually raise the temperature so that it reaches a

first three minutes.

9.

maximum

of 60 C. at the end of the

10.

During the next three minutes, gradually

raise the

tempera-

ture to 70 C.
1 1

Raise to 80 C. during the next two minutes.

12.

Raise to 200 C. during the next five minutes.

13.

During the next twenty-five minutes

at the rate of 90 C. per five minutes,

C. will be attained.

332

when

raise the

the

temperature

of 650

maximum

SECTION SIX
Turn

14.

off the heat

and allow the

slides to cool in the

oven

for about thirty minutes.

Take the

15.

slides

out of the furnace and lay coverslips over

the sections, with a pair of forceps.

16.

Seal the coverslips with

17.

De-wax and

De

Kotchinsky's cement.

stain the control serial sections,

which have

been set aside for the purpose, with suitable stains.

18. Examine the unstained, incinerated specimens comparing
them with control serial sections. This can be most conveniently

accomplished by employing two identical microscopes connected

with a comparing ocular, the microscope with the normally stained
sections being illuminated with ordinary bright-light condenser,
and the one with the incinerated sections with a dark-field illuminator this
:

method

facilitates

the location of the same structure in

both types of sections.

Notes:

There are many methods, some of which are given in this

book {see index) of identifying the numerous minerals which
might be present, but it is not possible to give them all here
without entering the realms of Mineralogy, Fluorescence Analysis

and kindred subjects which are foreign to a book of this kind, but
which are well catered for in other text-books to which the reader
is referred as well as to the various references given in McLung^s
Handbook of Microscopical Technique^ Laboratory Technique in
Biology and Medicine, by E. V. Cowdry, and the methods given
in Microscopic Histochemistry by G. Gomori.

3.HYDROXY

2-NAPHTHOIC ACID

TETRAZOTIZED

o-ANISIDINE
For demonstrating

sites

of carbonyl activity in frozen

sections

Solutions required:
this technique must be aldehyde free.
2-naphthionic acid hydrazide o-i gm.

Important All alcohol used in

:

A. 3-hydroxy

50%

alcohol

Acetic acid glacial

5%

333

95 ml.
5 ml.

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

B.

Absolute alcohol

Phosphate buffer 1/15

Distilled water

M (pH 7-2).

30 ml.

10 ml.

20 ml.

. .

Tetrazotized o-anisidine

o-o6 gm.

Technique:
1.

Tissues are fixed in

10%

Without washing out

them on slides.
2.

fixative,

3.

Allow the sections to become

the atmosphere; then

water.

Immerse in solution B
room temperature.

5.

in

for

Wash

7.

Immerse

saline).

cut frozen sections and place

affixed to slides

excess reagent (solution B)

alcohol (aldehyde free).

50%

formol

by drying

in

several changes of

one or more hours in an incubator

Remove

6.

5%

wash out formalin with

4.

at

formalin (or

by washing for two hours

in several changes of water for several hours.

in solution

for

one to two minutes.

8. Wash in several changes of distilled water to which a few

drops of acetic acid have been added.

9.

Drain

off excess liquid;

then mount in Glycerin

Jelly.

Results:
Sites of carbonyl activity are indicated by the production of a
blue pigment.
Reference: Seligman, A.

M. and

METANIL YELLOW

Ashbel, R. (195 1), Cancer,

4,

579-96.

IRON HAEMATOXYLIN

For radioautographs prepared by mounting tissue sections

directly onto photographic emulsions for study of thyroid
carcinoma and all organs of man, as well as laboratory
animals to which radio isotopes have been administered
Solutions required:

A. Metanil Yellow 0-25% aqueous.

334

SECTION SIX
B.

Haematoxylin
Ethyl alcohol

C. Ferric

95%

chloride

100 ml.

gm.

50%

hydrated

5 "nl*

Distilled water

95 ml.

aqueous

D. Solution B
Solution

. .

volume
volume

Mix well, and allow to stand for two or three

weeks before use. Filter immediately before use.

Note: The fresh solution must not he used as it darkens the emulsion
gelatine 60 ml. of this solution is sufficient for about twenty radio;

autographs.

Acetone and Xylol, equal volumes

E.

Technique:
Surgical and post-mortem specimens from patients having
recently received radioiodine (P^^) and tissues from laboratory
1.

animals to which radioiodine, radiosulphur (S^^) or radiophosphorus (P22) has been administered, are fixed in Bouin's fluid.
2.

Embed

3.

Cut

in paraffin wax, using the

Dioxan technique.

sections 7 to lo/x in thickness.

4. Transfer the section ribbons to the darkroom, place in a

waterbath, and float them onto a photographic plate. (X-ray film,
Kodak Medium lantern slides or Kodak nuclear track plates, are

suitable for this purpose.)

5.

Allow the sections

to

become permanently attached

dry on the

plate,

when they

will

to the photographic emulsion.

Note: For each isotype the processing fluids should be examined

by a Geiger counter to ensure that there is no significant loss of the
radioactive material

from the

tissue.

6. After proper photographic exposure, removal of the paraffin

wax, development, fixation, washing and drying, the autograph is

stained as follows:
7.

Stain with metanil yellow (solution

seconds.

335

A)

for five to fifteen

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

8. Wash in tap water until the slide
take about fifteen seconds.
9.

ID.

Stain in solution

is

pale yellow: this should

D for one to three minutes.

Rinse with three changes of

70%

alcohol for five seconds

in each.
11.
12.

13.
14.
15.

Wash for five seconds in 95% alcohol

Wash with acetone for five seconds.
Wash with solution E (acetone-xylol) for five seconds.
Wash with three changes of xylol.
Mount synthetic resins; e.g. D.P.X. or Cristalite.
;

Results:
Colloid, cytoplasm and connective tissue elements are stained
light yellow to light brown. Cell nuclei are stained deepblue to black.

from

Sites of radioactivity are indicated

by blackening of the photo-

graphic emulsion.
Reference: Simmel, Eva B., Fitzgerald, P.
Stain Tech., 26, 25-8.

J.

and Godwin,

J.

T. (195 1),

METHYL - GREEN - PYRONIN - RIBONUCLEASE (Brachet)

For detecting ribonucleic acid and desoxyribonucleic acid
in the

same

cell

Solutions required:
I.

Zenker's fixative containing

or

Carnoy Fixative

5%

acetic acid

SECTION SIX
2.*

Methyl green-Pyronin {Bracket)

Methyl green, 00 chloroform washed 0-15 gm.
Pyronin G
0-25 gm.
.

Alcohol

95%

2-5 ml.

Acetate Buffer

OO

pH 47

97-5 ml.

washed repeatedly with chloroform, to extract

Methyl green,
all traces of methyl violet. The washed methyl green is then dried, and
0-15 gm. is weighed out and dissolved in the acetate buffer and the
is

alcohol with the pyronin.

Note: the stain

*

See note {d)

3.

is

at the

liable to deteriorate after

end of

two or three weeks.

this technique.

fRibonuclease o-i% in distilled water which

has been adjusted to pH 6-o

t If this is not available a suitable ribonuclease extract can be prepared in the

laboratory as follows:
I. Pass 0-5 to I kilo Ox pancreas through a meat mincer.
II.

III.

Pound

it to a smooth pulp with a mortar and pestle.

Suspend the pulp in one or two volumes of N/io acetic acid

for sixteen

to twenty-four hours.
IV. Boil for ten minutes.

V. Cool; then filter.

VI. Adjust the pH of the filtrate to pH 6-o.
VII. Filter.
VIII. Add a crystal of thymol or camphor, as a preservative and store in the
refrigerator: under these conditions the solution will keep for several

months.

Technique:

Note:

not possible to give absolute rules concerning the

following procedure, as the methods of fixation, the solubility
of the ribonucleic acid of the organ to be studied, and the activity
It is

all possible variables.

The technique
which
should
be
as
a
basis
for
below,
given
regarded
experiment,
may be varied to suit individual lines of research and investigation.

of the ribonuclease, are

I
Fresh material should be used and objects or slices of tissue,
which must not be more than 2-3 mm. in thickness, are fixed for
a maximum of one hour in one of the above fixatives.
.

wash with tap water for twenty-four hours.

After Carnoy or Serra, wash in two or three changes of
alcohol for the minimum time necessary to remove the

2a. After Zenker,

2h.

95%

fixative.
3.

Dehydrate and embed

at once,

in paraffin wax.

337

through alcohol and

toluol,

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

4.

Fix sections alternatively to slides marked A,

5.

Remove

paraffin

wax with

toluol,

descending grades of alcohol to

6.

Stain slide

B and

C.

and pass through the usual

distilled water.

immediately with the methyl green-pyronin

for twenty minutes.

7.

Meanwhile place slide B in distilled water, adjusted

and leave there for an hour at 37 C.

pH

to

6-0 in the oven

8.

Place slide

oven

in the ribonuclease solution in the

at

37 C. and leave there for an hour.

9.

Meanwhile take

Slide

10.

is

it has been in the methyl

and wash it rapidly with dis-

slide A, after

green-pyronin for twenty minutes,

tilled water.

then differentiated with

95%

alcohol for five to

ten minutes.
11.

Dehydrate
and mount

toluol

slide

in

When slide B

12.

with absolute alcohol; then wash with

balsam or D.P.X. or

Cristalite.

has been in the oven for an hour, stain

the methyl green-pyronin, differentiate, dehydrate and

exactly as in the case of slide A.

When

13.

differentiate,

slide

with

mount

has been in the oven for an hour, stain,

dehydrate and mount exactly as slides

Examine and compare specimen

14.

it

with

A and

B.

and B.

Notes :
(a)

Methyl green

is

unique in that

it

will stain

some, but not

DNA as
DNA or

basophilic substances. It will stain high-polymer

occurs in the nuclei, but it will not stain depolymerized
all

RNA

it

any form. In specimen B (above) it will be found that

although the methyl green staining may have been completely
abolished, Feulgen's reaction remains unchanged.
Interesting
comparisons can be made between sets of slides as B and C (above)
with sections from the pancreas, kidney, lung, liver, genital glands,
in

(See Professor

J. Brachet's original 1942 paper*).

be
found
may
necessary, depending upon the fixative
used and the organ to be studied, to raise the concentration of the

skin, etc.
{h) It

ribonuclease solution to as

much

results.

338

as

o-6%

to obtain

optimum

SECTION SIX
of the dyes, methyl green and pyronin, is a
(c) The quality
factor of great importance if good results are to be obtained with
this technique.
(d)

In Professor Brachet's original paper (1942) the Unna

as below, was em-

Pappenheim Methyl Green-pyronin, formula

ployed

Methyl green,
Pyronin

00

0-15 gm.

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

References
"
*
Localisation des acides
Brachet, J. (1942), Archives de Biol., 53, 207-57.
les tissues animaux et les oeufs d'Amphibiens en voie
pentosenucleiques dans
"
de developpement.
"
The use of basic dyes and
Brachet, J. (1953), Q. jf. Micr. Sc, 94, i-io.
"
ribonuclease for the cytochemical detection of ribonucleic acid.
:

NILE BLUE SULPHATE

An

SAFRANIN

histochemical technique for demonstrating phospholipids in frozen sections

Solutions required:

A. Formalin

10%

Calcium Chloride 1%
Calcium Carbonate
Shake well: filter before
.

B.

Safranin

1%

Aniline Oil
C.

100 ml.
I

ml.

2gm.

aqueous
.

. .

use.
.

Nile blue sulphate, saturated aqueous

Sulphuric acid 0*5%
.

100 ml.
2 drops

100 ml.
10 ml.

Boil for 2 hours under reflux condenser.

Filter before use.

D. Acetic acid
E.

HCl

cone.

5%

aqueous.

0-5 ml.

Distilled water

99-5 ml.

Technique:
1. Fix material in solution A: then cut frozen sections, without
embedding: or the material may be embedded, if desired, in

gelatine or carbowax.

Note: Frozen sections should not be stored in water for more

than ten to fifteen minutes.
2.

Stain in the safranin solution for five minutes.

3.

Rinse in

distilled water.

4. Stain in the nile blue sulphate for ninety minutes at 60 C.

5.

Rinse in distilled water.

6.

Immerse

7.

Remove

in acetone heated to 50 C.

on

a water bath.

the acetone from the source of heat and allow the

sections to remain in

it

for half

an hour.
340

SECTION SIX

5%

acetic acid for thirty minutes.

8.

Differentiate in

9.

Rinse thoroughly in distilled water.

10.
1 1

12.

Refine the differentiation in the

Wash in several changes of

Mount in glycerine jelly.

0-5% HCl

(Solution D).

distilled water.

Results:

Phospholipids, blue. Nuclei, red.

Reference: Menschik, Z. (1953), Stain Tech., 28, 13-18.

PHOSPHOMOLYBDIC ACID

EOSIN

For the histochemical localization of choline-containing

lipids, in frozen sections
Solutions required:

A. Acetone

* .

Ether
B. Chloroform
Absolute alcohol

, .

volume
volume
volume
volume

C. Phosphomolybdic acid.
in solution B.
I

D. Stannous chloride 1%.

in

3N

Eosin

E.

hydrochloric acid.

1%

aqueous.

Technique:
1.

2.

Dip frozen sections into acetone-ether.

Immerse in the phosphomolybdic acid

solution for fifteen

minutes.
3.

Rinse in solution B.

4.

Dip

into solution

D.

5.

Counterstain with the eosin solution for one to two minutes.

6.

Mount

in glycerine jelly.

Results:

Positive areas are stained blue, whilst negative areas are red.
Reference: Landing, B. H. (1952), Uzman, L. L. and Whipple, Ann. Lab.
Invest., I, 456-2.

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

SUDAN BLACK
The

use of Sudan Black, also

known

as

Sudan Black

B, has

been described in various works on Histochemistry. As far as I

am aware, the correct structural formula of the Sudan Black
molecule has not yet been disclosed in any literature published in
any country, although it appears that this information has frequently been sought after by medical and biological research
workers. It may, therefore, be of interest and help to research
workers to give the formula here
:

^^N

N-<;^_^N
\

N-<^_^N\ yCR
\

>~N/

\CH.

H
The molecular weight and other information regarding Sudan
Black will be found on page 435.

34^

SECTION 7-SMEAR PREPARATIONS

2A

ALBERTS STAIN
(Laybourn's modification)

For diphtheria

bacilli

Solutions required:

A.

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

ALCIAN BLUE
For bacterial polysaccharides and capsules
Note: This dye will stain bacterial capsules and insoluble polysaccharides in both pure and mixed cultures of bacteria and protozoa. It
is specific for bacterial polysaccharides, and enzymatic degradation
of these results in the loss of this stain.
Solutions required:

A. Alcian blue
Alcohol
B.

Solution

95%

Distilled water

Note:

,.

This

..

..

. .

. .

solution

gm.

100 ml.

volume
9 volumes
i

which deteriorates

within a few days, should only be prepared

as

and when required

for

immediate use.

C. Carbol fuchsin (Ziehl-Neelsen)

Technique:
1

Fix air-dried smears by flaming in the usual way.

2.

Stain with solution

3.

Pour

off excess stain

preparation to dry in the

for

one minute.

and wash with water; then allow the

air.

4.

Counterstain with carbol fuchsin for a few seconds.

5.

Wash immediately

with

distilled

water to prevent over-

staining.
6.

Allow the preparation

to dry in the air

then examine.

Results:

Capsule and other bacterial polysaccharides are stained blue:

cellular material, red.

Note: Various carbohydrates including adonitol, arabinose, cellobiose, dextrin, glucose, inositol, inulin, mannitol, mannose, rhamnose,
sorbitol, trehalose, xylose, and certain enzymes including papain,
pepsin, rennin gave a positive reaction with alcian blue.

The

fact that the internal polysaccharides of the cell remain

is attributed to the
complexity of the dye molecule

unstained

which prevents

its

penetration of the

cell wall.

Reference: McKinney, Ross E. (1953), J^. Bact., (U.S.A.), 66, 453-4,

"
ing bacterial polysaccharides.

**

Stain-

SECTION SEVEN

ANILINE BLUE

EOSIN B

simple and rapid technique for spermatozoa, which

particularly suitable for

dog and human semen

Solutions required:

A.

is

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

End knobs

Dark blue

Middle piece

Sheath dark blue, centre dark pink

Axial filament

Dark blue
Dark blue

Tail

Notes:

suggested that the technique which has been tried out

on
only
dog and human semen, might be applied to other species.
The technique gives good differentiation and preservation of
cytological structure, reliable fixation and staining and undistorted
and easily recognizable detail, upon which assays of semen for
It is

fertility

depend.

There is a tendency for abnormal forms of spermatozoa

more intensely than the normal forms.
Reference: Casarett, George

W.

to stain

(1953), Stain Tech., 28, no. 3, 125-7.

ANILINE GENTIAN VIOLET

A simple and rapid stain for

Treponema pallida

Solutions required:

A. Aniline gentian violet.

B.

Sodium hydroxide
Absolute alcohol

C. Alcohol

1%

50 ml.

aqueous
.,

..

1-5 ml.

5%.

Technique:

lesion is spread into a film on a

an enlarged syphilitic gland is to be
examined, 0-5 ml. sterile saline should be injected into the gland;
then aspirated with a i-ml. syringe with a 22-gauge needle.
Haemoglobin may be removed, if necessary, with distilled water.
1

slide

loopful of

and dried

serum from the

in air.

If

2. Stain with ten drops each of Solutions A, B and

minutes, rocking the slide to ensure thorough mixing.
3.

Wash

in running tap water for twenty seconds;

examine.

348

for

two

dry and

SECTION SEVEN
Results:

Treponema pallida, deep purple, with distinct regular spirals

from precipitate. Spirochaeta refringens and other spirochaetes, purplish black, with regular open and coarse spirals.

free

AZUR L
For the detection and staining of epidermophytic infection
Solution required:

Azur

0-5% aqueous.

Technique:

Scrapings from around the margin of non-purulent areas of

infected skin or from the tops of vesicles are transferred to a slide.
1.

2. Treat three to ten minutes, depending on the size and thickness of the specimen, with Carnoy's fluid; then pour off carefully
so that any floating debris is carried away and the fungus is left

adhering to the
3.

Dry over

slide.

a flame, taking care that the remaining Carnoy's

fluid does not take fire.

4.

Stain two or three minutes with

5.

Rinse by adding

distilled

0*5% aqueous Azur L.

water drop by drop, taking care to

avoid washing away the specimen.

6.

Drain well

then dry cautiously over a flame and mount.

Results:
light blue

Fungal filaments, dark blue against a

background.

BASIC FUCHSIN
For Treponema pallida in smears
Solutions required:

A. Potassium hydroxide
B.

Basic

fuchsin

alcohol

Distilled water

10%

1%

aqueous.

in

absolute

5 rnl-

95

349

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

Technique :
Fresh, air-dried smears are fixed for five minutes in pure

1.

methyl alcohol.
then add a few drops of i
2. Allow the smears to dry in air
solution
followed immediately by
hydroxide
aqueous
potassium
twice the number of drops of basic fuchsin solution.

Rock the

slide to ensure thorough mixing of the two soluthen leave until the liquid becomes decolorized, which
should take about three minutes.
3.

tions;

Pour

4.

off excess

and rinse the preparation thoroughly with

distilled water.
5.

Drain, and blot dry carefully but thoroughly.

6.

If

it

mounted

at

desired to preserve the preparation

is

once in

it

should be

Cristalite.

Results:

Treponema pallidum,

scarlet.

Background, pink.

BREED'S STAIN
For staining and counting bacteria in milk
Solution required:

Methylene Blue

95%

alcohol

..

..

Distilled water
Phenol
Shake well until dissolved.
.

0-3

gm.

30 ml.
100 ml.

..

2| gm.

Technique:
1

Mark off

sq.

cm. on a piece of white paper and superimpose

a slide.
2.

Place

00 1

ml. of the milk to be tested on the slide and

spread, by means of a needle, into a film exactly

cated by the marking on the paper.
3. Dry on a level surface, by heating gently.

sq. cm., as indi-

well;

Immerse in xylol for a few minutes to remove the fat drain

wash with absolute alcohol; then 95% alcohol; immerse in

90%

alcohol for a few minutes.

4.

350

SECTION SEVEN
5.

Stain for two minutes with Breed's stain, prepared as above.

6.

Wash quickly in 90-95%

alcohol until the intense blue colour

changes to a faint tinge.

7.

Dry and examine.

Results:

Bacteria are stained dark blue against a lighter blue background

BRILLIANT CRESYL BLUE

For reticulated cells and platelets
Solutions required:

A. Brilliant Cresyl Blue

0-3%

in pure

absolute ethyl alcohol.

B.

Leishman

stain or Wright's stain.

Technique:
1.

Place a drop of

0-3%

alcohol on a slide and allow

Brilliant Cresyl
it to dry.

Blue stain in absolute

drop of blood 2 to 3 mm. in diameter is placed on another

slide and brought in contact with the dried stain; the two slides
are then manipulated hinge-like until all the stain has gone into
solution and the blood appears blue-black. Allow the slides to
come into contact to spread the drop then separate the slides and
2.

allow the films to dry.

3.

Counterstain with Leishman or Wright by the standard

technique.
Results :

Reticulum of immature red

cells

is

backgroiind, pale blue (fresh), or pink.

or lilac.

Note: The counterstain

count the platelets.

may be

omitted

The number
ately in a

stained clear cut blue;

Blood

platelets, pale

if it is

blue

desired only to

of red cells per cm. should be determined separhaemocytometer, and the ratio of platelets to red cells

computed from the stained preparation.

351

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

For reticulum
Stock solution:
Brilliant Cresyl Blue
saline (0-85%

Normal

NaCl)

i\ gm.
100 ml.

Technique:
1

Mix

140 times
2.

a small quantity of the stock solution of the stain with

its volume of normal saline solution.

Mix the blood

tion of

in a white-cell counting pipette in the proporto 20 vols, of the diluted staining solution.

volume blood

Shake the mixture for

blood counting

five

minutes in the pipette, and place in a

cell.

3. The fresh preparations are sealed with Vaseline to prevent

drying, and are counted immediately. At least 1,000 should be

counted for each

test.

Result:

Reticulum only stained

(blue).

CARBOL CRYSTAL VIOLET

For

fibrin

network in blood smears

Solution required:

Carbol Crystal Violet

Technique:

Note To retain the normal arrangement of the filaments in the fibrin

network, the slides must be slowly and carefully placed in, and taken
out of the reagents.
:

1.

slides

Fresh thick blood smears are made on scrupulously clean

and placed in a moist chamber, immediately, to prevent

drying.

Note:

The moist chamber may

of which has two sheets of

filter

consist of a petri dish the lid

paper, moistened with

warm

water to accelerate clotting.

After the blood has coagulated and the fibrin framework has
formed (this takes about six to ten minutes) take the slides out of
2.

352

SECTION SEVEN
the moist chamber and transfer to distilled water to haemolyse
the red corpuscles this takes about five to thirty minutes.
:

the water

Change

3.

when

it is

tinged red.

4. When the haemolysis is complete that is when all traces of

red colour has disappeared from the slides and the fibrin network
appears as a whitish film on the slide, remove slides and carefully
:

blot

away excess water, but do not

blot

network owing to

risk of

displacement.
5.

Stain for eight minutes in carbol crystal violet.

6.

Rinse in distilled water and carefully blot off excess water.

7.

Pass through three or four changes of dioxane and

mount

quickly in D.P.X. or Cristallite or Clearmount, before the net-

work

drys.

Results:

Fibrin network

is

stained violet.

arly shaped black dots.

size of a pin head.
Reference: Badertscher,

Platelets

J.

White

appear

cells

appear as irregulabout the

as black dots

A. (1952), Stain Tech., 27, no.

4,

217-20.

CARBOL FUCHSIN
For Treponema pallida and other spirochaetes
Solutions required:

A. Potassium

permanganate

5%

aqueous.
B.

Carbol fuchsin (Ziehl Neelsen)

Distilled water

volume
9 volumes
i

Technique:
1.

Air-dried smears are fixed in rnethyl alcohol for five minutes.

2.

Immerse

for three minutes in the potassium

permanganate

solution.
3.

Wash

4.

Immerse

with

distilled water.

in the diluted carbol fuchsin solution for

rites.

353

two min-

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

5.

Rinse with

6.

Drain; dry thoroughly but carefully.

7.

If

it is

distilled water.

desired to preserve the preparation,

mount

at

once in

Cristalite.

Results:

Mouth
Treponema pallidum^ light reddish brown.
chaetes and Treponema refringens, deep reddish brown.

spiro-

CARBOL FUCHSIN
For acid-fast bacteria, in sputum, including tubercle bacilli
Solutions required:

A. Ziehl Neelsen carbol fuchsin.

B.

Acid alcohol:
Alcohol

95%

Cone. Hydrochloric acid

97

nil.

3 ml.

C. Loeffler Methylene Blue.

Technique:
1

2.

slide
3.

Sputum smears

are fixed

Stain with Solution

till

steam

Pour

on

a flat surface over boiling water.

for three to five minutes, heating the

rises.

off excess

and allow the

slide to cool

somewhat before

rinsing in water.
4.

away
5.

wash

Decolorize in Solution
;

until only a faint pink colour

comes

then wash with water.

Counterstain with Solution

in water

for ten to thirty seconds; then

dry and examine.

Results:

Acid-fast bacteria, red.

Other organisms, blue.

faint blue or colourless.

354

Background,

SECTION SEVEN

CARBOL FUCHSIN

BORREL BLUE

For leprosy and for tubercle

bacilli

Solutions required:

A. Carbol fuchsin (Ziehl Neelsen).

B.

Sulphuric acid

5%.

C. Hydrochloric acid
Alcohol 70%

D. Borrel Blue
Distilled water

ml.

99

nil-

5 ml.

20 ml.

Technique:
1.

Air-dried smears are fixed in absolute alcohol for three min-

utes.
2.

Place the preparation on the corner of a tripod; flood with

then heat gently with a small bunsen flame until

carbol fuchsin

steam

rises.

3.

Allow the

4.

Decolorize with

stain to act for five minutes.

5%

sulphuric acid until only a faint pink

colour comes away.

5.

Wash

6.

Rinse in distilled water.

7.

Counterstain in the diluted Borrel Blue for one half to one

in

60%

alcohol until

no more

stain

comes

out.

minute.
8.

Pour

off excess stain;

rinse with distilled water;

drain and

blot dry.
Results:

Bacillus leprae and tubercle bacilli bright red. Other bacteria

are stained blue, while cells, cell debris and mucin in sputum are
in varying shades of blue.

Note: In the case of leprosy, differentiation of the carbol fuchand 5) must be very carefully carried out, as this
is
more
organism
easily completely decolorized than tubercle

sin (Stages 4
bacilli.

355

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

CARBOL FUCHSIN

BRILLIANT YELLOW

For tubercle bacilla in sputum smears

Solutions required:

A. Carbol fuchsin (Ziehl Neelsen).

B.

Brilliant yellow

Sulphuric acid
Distilled water

i% aqueous
25%
.

Absolute alcohol

15 ml.

40 ml.
30 ml.
10 ml.

Technique:
1

Fix sputum smears on a horizontal surface over boiling water

Stain with carbol fuchsin solution for three to five minutes,

heating over a small bunsen flame in the usual manner, until steam
2.

rises.
3.

Pour

4.

Allow the

off excess stain.

slide to cool

somewhat before

rinsing the prepar-

ation thoroughly with distilled water.

differentiate for one half to one minute
5. Counterstain and
with the brilliant yellow solution.

6.

Wash

7.

Drain; blot thoroughly, and examine.

well with distilled water.

Results:

Tubercle

bacilli are stained

red against a clear yellow back-

ground.

CASTANEDA'S STAIN
For Rickettsiae and elementary bodies
Solutions required:

A. Phosphate buffer solution pH 7-5

Formalin (Formaldehyde 40%)

Methylene blue 1% in
methyl of ethyl alcohol
356

20 ml.
i ml.

absolute
.

0-15 ml.

SECTION SEVEN

Safranin

B.

Acetic acid

aqueous 0-2%

0-1%

..

..

volume
volumes

..

Technique:
1
Spread smears of infected tissue on scrupulously clean slides
then allow to dry.
.

2.

Stain with solution

A for three

minutes.

3. Pour off excess stain; then without washing counterstain for

one to three seconds in the safranin solution.

4.

Wash

5.

Drain and dry thoroughly, and examine.

in running water.

Results:
Rickettsiae, blue

protoplasm and nuclei of the

cells, red.

CRYSTAL VIOLET
For bacterial capsules
Solutions required:

A. Crystal violet 0-5%.

B.

Copper sulphate
Distilled water

crystals
.

. .

gm.

25 ml.

Technique:
1.

Stain air-dried smears for three to four minutes in the

crystal violet solution.

2.

Rinse well in the copper sulphate solution.

3.

Blot dry, and examine.

Results:

Capsules are stained light blue, while the bacterial

blue.

357

cells are

dark

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

CRYSTAL VIOLET

simple stain for spirochaetes

Solutions required:

A. Potass, permanganate
Crystal Violet

B.

2%

i%

aqueous.

aqueous.

Technique:
1.

Air- dried films are fixed by flaming.

2.

Immerse

tilled

in Solution

A for ten minutes

then wash with dis-

water.

3.

Stain for eight to ten minutes in Solution B.

4.

Wash

in distilled water

dry and examine.

Results:

Spirochaetes, bluish black and well defined.

Note:

For Treponema pallida^ best

A on the slide.

results are obtained

by

warming Solution

DAHLIA
For the rapid staining of Heinz bodies in erythrocytes in
blood smears of animals poisoned by certain chemicals

such as aniline, phenylhydrazine, and

its

derivatives

0-2

gm.

Solutions required:

A. Dahlia

. .

Ethyl alcohol

100 ml.

95%

Technique:
1.

Moderately thick blood smears are made on scrupulously

clean slides, then allowed to dry in

air.

2. Cover the freshly dried smears with the staining solution

and allow it to act for thirty seconds.
3. Pour off excess stain and wash well with running water
remove the excess dye.

358

to

SECTION SEVEN
4.

Drain and blot dry; examine under the

oil

immersion

objective.

Results:

Heinz bodies are visible as blue granules, the depth of colour

depending somewhat on their size and the thickness of the smear.
Reference: Webster, S. H., Liljegren, E.
Stain Tech., 23, 97.

L. and Zimmer, D.

J.

(1948),

J.

DORNER'S SPORE STAIN

Solutions required:

A. Carbol fuchsin (Ziehl Neelsen).

B.

Nigrosin, water soluble

Distilled water

Dissolve by heating, cool,

Formalin

filter

10 gm.
100 ml.

and add:

0-5 ml.

Technique:
1.

A heavy suspension of the culture is made in a test-tube with

three drops of distilled water and three drops of carbol fuchsin are
then added. Heat on a boiling water bath for ten to fifteen minutes.
2.

A loopful each of the stained preparation and

mixed on a

slide,

Solution

spread into a thin smear and dried rapidly

are

then

examined.
Results:

Spores, red. Bodies of bacteria, almost colourless against a dark

grey background.

EHRLICH'S TRIACID STAIN

For blood smears
Solution required:
Ehrlich's triacid stain.
2B

359

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

Technique:
1.

Air-dried films on slides or on coverglasses are fixed by heat.

Allow to cool for a few seconds, then flood films with the
staining solution and allow the stain to act for five to ten minutes.
2.

water until the stain ceases to come out.

3.

Rinse with

4.

Drain

5.

Blot and dry thoroughly;

distilled

off excess water.

mount

in Cristalite.

Results:

Erythrocytes, orange. Eosinophile granules, bright red. Basophile granules, unstained. Neutrophile granules, purple. Lymphocytes, unstained. Nuclei, pale green.

EOSIN

GENTIAN VIOLET

For basal bodies of cilia

Solutions required:

A. Haematoxylin (Ehrlich).
B.

Eosin 0-5% aqueous.

C. Aniline gentian violet (Stirling).

D. Lugol's iodine
E.

Aniline

solution.

oil.

10 ml.

20 ml.

Xylol
Technique:
1.

Stain for two minutes in the haematoxylin solution.

2.

Blue and wash in tap water or in lithium carbonate solution.

3.

Stain for one minute in the eosin solution.

4.

Wash

5.

Stain for two hours in the aniline gentian violet.

6.

Wash

with water.

well in tap water.

7. Stain in Lugol's iodine for ten to fifteen minutes.

360

SECTION SEVEN
8.

Wash

9.

Differentiate in Solution

drain and blot dry.

well with tap water;

then wash in several changes of

xylol and mount.

Results:

Nuclei, dark blue.

EOSIN

Basal bodies, purple.

METHYLENE BLUE

BASIC FUCHSIN

general stain for bacteria, blood and spirochaetae

Solution required:

Eosin

2%

in absolute alcohol

Methylene blue 3% alcoholic

Basic fuchsin 10% alcoholic
Absolute alcohol
Distilled water

Mix

well.

2-5 ml.

7-5 ml.

3-0 ml.
12-0 ml.

25*0 ml.

Allow to stand two or three days

closed vessel; then

in a

filter.

Technique:
1. Blood and spirochaete smears are allowed to dry in the air
before fixation in methyl alcohol. Bacterial smears are fixed by
heat in the usual manner.

2.

Stain smears for one minute in the aboVe solution.

3.

Pour

4.

Drain; blot

and wash thoroughly in distilled water.

carefully but thoroughly and examine.

off excess stain

Results:

B. typhosus and paratyphoid organisms, pink.

and B. dysenteriae^ lilac. B. diphtheriae, pink with lilac
granules and bars. B. influenza^ and Leptothrix unstained but
showing lilac bars and granules. Meningicocciy violet to pink.

Bacteria

B.

coli

Blood: Erythrocytes, pink

pink.

to orange.

Lymphocyte cytoplasm, purple;

purple to dark blue;

nuclei, dark purple to black.

granules, pink to opaque white.

Spirochaetes

Neutrophiles, cytoplasm,
nuclei, blue.

Polychromatophilic

pink to violet.
361

Basophiles,

Eosinophile
cells, red.

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

FIELD'S STAIN
For thick blood films for malaria parasites
Solutions required:
stain,

Field's stain,

A. Field's
B.

Technique:
1.

A thick film is made by spreading two or three drops of blood

into a circle of about 2 cm. diameter

2.

Allow to dry

on a

slide.

in the air until the films are

no longer obviously

moist.
3.

Immerse the

film for one second in Field's stain

mediately transfer to a dish of distilled water

gently in the water until no more stain comes
is

then im-

and wave the

away and the

slide

slide

free of excess stain.

4.

rinse

Immerse the
by waving

film for one second in Field's stain B; then

gently for two or three seconds in a dish of fresh

distilled water.
5.

The

films should be placed in a vertical position

drain and dry in

and

left to

air.

Notes:

Freshly prepared films give the best results.

Staining times may be varied from one to five seconds.
Results:

General ground: creamy yellow, sometimes uniform, sometimes

mottled with pale blue.

Nuclei,

deep blue, sharply defined. Cytoplasm

pale blue, vaguely defined. Granules, eosinophilic, large, dull
red, well defined. Neutrophilic, small, pale purple, vague.
Leucocytes:

Malaria parasites : Cytoplasm, blue. Chromatin, dark, purplish

Pigment, unstained yellow of varying shades depending on
the depth of the cytoplasm in which it lies.

red.

362

SECTION SEVEN

FLAGELLA STAIN

(Cesares-Gil)

For colon and typhoid organisms

Solutions required:

A. Flagella stain (Cesares-Gil).

B.

Carbol fuchsin (Ziehl Neelsen).

Technique:

1.

freshly prepared mixture consisting of

vol. of the stain

onto unfixed films of a

hours
to
culture
old) and allowed to
twenty-four
(eighteen
young
act for one minute.

and

vol. distilled

water

is

filtered directly

2.

Wash

3.

Stain for five minutes with carbol fuchsin (Ziehl Neelsen),

with

distilled water.

freshly filtered.
4.

Wash

with

distilled water;

blot

and dry

in air.

Results:

Bacteria and flagella, red.

precipitate.

Background, very

fine granular red

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

re-dissolves.

few drops of the reserved

silver

nitrate are then added, with shaking, until the final

solution

is

slightly opalescent.

Technique:
1.

Air-dried films are flooded with Solution

then washed with

for

Flood with Solution B and heat until steam arises then pour
and allow the slide to cool somewhat before rinsing with dis-

2.
ofi^

one minute;

distilled water.
;

tilled water.
3.

Flood with Solution C; heat

steam

till

rises;

then allow the

solution to act for one half to one minute.

4. Wash with distilled water; dry and blot in air; and mount
before examining as the stain may fade immediately in contact

with immersion

oil.

Result:

Spirochaetes are stained dark

brown

to black, against a

maroon

background.

GENTIAN VIOLET

A combined fixative and

(Noland)

stain for Protozoa

Solutions required:

Gentian violet

2%

aqueous

Phenol, saturated aqueous

40%

Formaldehyde
Glycerin

ml.

80 ml.
20 ml.

rnl-

Technique :
1

Place a drop of the culture to be examined on a scrupulously

clean slide.
2.

Add

drop of the staining solution and mix and spread with

platinum wire loop into a film.

3.

Allow to dry; then examine under the

jective.

364

oil

immersion ob-

SECTION SEVEN
Results:
Flagella are stained violet, as are undulating
cirri

membranes and

of Hyportichs.

Note: The technique has been found useful for demonstrating

the various stages of Dimastogamoeba from amoeboid to flagellating forms after some of a culture has been kept in rain water for

one to several hours.

Reference: Noland, L. E. (1928), Science, 67, 535.

GIEMSA STAIN
For blood, malaria parasites, trypanosomes,
Note: Best results are obtained by buffering the
to

etc.

distilled

water

72.

Rapid method

for films:

1.

Fix air-dried films for three minutes in pure methyl alcohol.

2.

Stain for five minutes in a mixture consisting of one part of

stain and two parts of distilled water.

Giemsa
3.

blot

Wash

with

and dry

distilled

water for one half to one minute; then

in air.

Rapid method for spirochaetes

1.

Air-dried films are fixed by heat by drawing through the

flame.
2. Allow the slide to cool; then flood the slide with a freshly
prepared mixture consisting of 10 drops of Giemsa stain to 10 ml.

distilled water.
3.

Heat the

4.

Wash

till steam rises; allow to cool for about

then pour off the stain and repeat the process five

slide gently

twenty seconds
or six times.

with

Slow method

distilled water;

and mount.

for films, for demonstrating spirochaetes,

trypanosomes,
I.

blot dry

etc.

Air-dried films are fixed for three minutes in pure methyl

alcohol.

365

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

2. A fresh mixture is prepared by diluting Giemsa stain in the
proportion of lo drops of stain to lo ml. distilled water. The slide
is then placed in a staining jar and left to stain in the diluted

Giemsa for sixteen to twenty-four hours ; if a staining jar is not

available, place a piece of thin glass rod in a Petri dish; lay the
slide with one end resting on the rod, film face downwards, in the
Petri dish,

and pour in

sufficient diluted stain to cover the film;

two sheets of moist

and
cover
the
prevent evaporation,
preparation.
with
blot
and dry.
Wash
distilled
water;
3.

then line the Petri dish lid with

Method
1.

filter

for bacterial smears, throat exudate, etc.

Thin, air-dried, unfixed smears are covered with undiluted

stain for thirty seconds then a quantity of distilled water,

Giemsa

equivalent to five to ten times the volume of stain used,

and mixed with the stain by gently rocking the slide.
2. Allow the diluted stain to act for two
wash with distilled water; blot and dry.

Thin blood film method

1.

paper to

to five

added

is

minutes

then

Air-dried films are fixed for five minutes in pure methyl

then blotted and dried in air.

alcohol

2. Stain for fifteen to forty-five minutes in a mixture consisting

of one part of Giemsa stain and twenty-five parts of distilled water.
3.

Wash

in distilled water;

blot

and dry in

air.

Results:

Nuclei of the leucocytes are stained reddish purple, while the

Leishman-stained prepar-

rest of the leucocytes appear similar to

ations. Cytoplasm of plasmodia, blue.

Chromatin, red.

Thick film method (for demonstrating malaria parasites)

.*

film is prepared by spreading 3 to 5 drops of blood in a

about 15 mm. diameter over a slide; then without fixation
it is allowed to
dry on a level surface for eighteen to twenty-four
hours at room temperature protected from dust or for two to three
1.

circle

hours in an incubator
2.

at 37 C.

Stain for forty minutes in a mixture consisting of one part of

stain and fifty parts of distilled water.

Giemsa

366

SECTION SEVEN
3.

Wash

for five to ten minutes in distilled water; blot

and dry

in air.

Results:

Malaria parasites

Treponema

chromatin, clear red; cytoplasm, clear blue.

pallida.
Solutions required:

A. Giemsa
B.

stain.

Sodium

carbonate,
distilled water.

A.R.

1%

in

Technique:
1.

A lesion is rubbed roughly until

it

bleeds, with a

swab which

has been damped with absolute alcohol.

2. While bleeding is taking place, swab the chancre at intervals.
After a few minutes a clear fluid exudate will appear from the

abraded surfaces.
3.

drop of the clear

fluid

exudate (free from blood)

is

taken

lesion with a platinum wire loop, transferred to a scrupulously clean slide, and spread into a film.

from the periphery of the

4.

Allow to dry

in the air

then

fix in

absolute alcohol for fifteen

minutes.
5.

Pour

off

evaporate away

any excess alcohol, and allow the

last traces to

in the air.

6. Stain for fifteen to thirty minutes in a mixture consisting of

10 ml. distilled water buffered to 7-2, and 0-5 ml. Giemsa stain.

Rinse in running tap water for a few seconds holding the

slide with the film facing downwards.
7.

8.

Blot dry, and examine.

Results:

The spirochaetes

are stained reddish violet. Nuclei of the leucocells

reddish
purple, while the rest of the leucocytes and pus
cytes
similar to Leishman-stained preparation. Cytoplasm of

appear

Plasmodia, blue; chromatin, red.

367

Erythrocytes, yellowish pink.

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

GIEMSA STAIN

MAY-GRUNWALD STAIN

For blood and parasites

Solutions required:

A. May-Grunwald

Giemsa

B.

stain.

stain.

Technique:
1.

Unfixed air-dried films are stained for three minutes in May-

Grunwald stain; then an equal volume of distilled water is added

and mixed with the stain by rocking the slide. The diluted stain is
then allowed to act for one minute;

then drained

off,

without

washing.
2.

Stain for ten to fifteen minutes in a mixture consisting of lo

distilled water.

drops of Giemsa stain in lo ml.

3. Differentiate for about five seconds, with distilled water,

examining under the microscope to ensure that differentiation is

complete.
4.

Blot and dry in air

mount.

Results:

As Giemsa

stain

but with more intense colouring.

GRAM'S STAIN
For bacterial smears
Solutions required:

A. Aniline gentian violet or carbol

gentian violet.
B.

Gram's

iodine.

C. Carbol fuchsin (Ziehl Neelsen)

Distilled water
, .
.

368

volume

9 volumes

SECTION SEVEN
Technique:

Air-dried bacterial films are fixed by passing through the

in the usual manner.

1.

bunsen flame

Stain with aniline gentian violet or carbol gentian violet for

2.

two

to three minutes.

Pour

3.

off stain

and flood the film with Gram's iodine for one

minute.
4.

Blot and dry in

air.

Decolorize with absolute alcohol

violet
5.
(if aniline gentian
has been used) until no more colour comes out if carbol gentian
violet has been used, decolorize with aniline xylol instead of
;

alcohol.
6.

Wash

7.

Counterstain with a mixture consisting of

with water; drain.

i

volume of Ziehl

Neelsen's carbol fuchsin and 9 volumes distilled water.

8.

Wash

with

distilled water;

blot

and dry

in air.

Results:

Gram-positive organisms, violet.

red with pink nuclei.

Gram-negative organisms,

GRAM'S STAIN
Jensen's modification
Solutions required:

A. Methyl violet
B.

6B

(Jensen).

Lugol's iodine.

C. Neutral red (Jensen).

Technique:
1.

Air-dried bacterial films are fixed as above.

2.

Stain for twenty to thirty seconds with

6B

0*5% methyl

violet

in distilled water.

3.

Pour

off;

iodine to act

on

and allow the

wash

film with Lugol's iodine;

the film for one half to one minute

369

then pour

off.

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

4.

Wash

of the film

with absolute alcohol until no more colour comes out

then wash with distilled water and drain.

5. Counterstain with Jensen's neutral red (o-i gm. neutral red,

0-2 ml. 1% acetic acid, 100 ml. distilled water) for thirty to sixty

seconds.
6. Wash with distilled water; drain, blot and dry in air.
Note: Ziehl Neelsen's carbol fuchsin diluted i
15 with distilled water may be used (staining time, twenty to thirty
seconds)
for routine work. For demonstrating gonococcus and other intra:

cellular

Gram-negative bacteria, however, neutral red should be

used.

GRAM'S STAIN
(Kopelofif

and Berman modification)

For bacterial smears

Solutions required:

A. Crystal violet
B.

1%

aqueous.

Sodium bicarbonate

C. Potass, iodide
Iodine

Distilled water

N/i

NaOH

5%

aqueous.

2 gm.

89 ml.

10 ml.

gm.

D. Equal volumes of acetone and ether.

E.

Safranin

2%

aqueous.

Technique:
1. Thin, unfixed, air-dried smears are stained for five to ten
minutes in a mixture consisting of 3 ml. Solution A, and o-8 ml.

of Solution B.
2.

Rinse with

distilled

water then stain with Solution

C for two

to five minutes.
3.

Wash

with tap water and drain and blot water from the surdo not allow the films to dry.

face of the film, but

370

SECTION SEVEN
4.

Decolorize by adding Solution

drop by drop for about
more colour comes out: this takes

ten seconds, until hardly any

about ten seconds.
5.

Dry

and counterstain

in air

in Solution

for five to ten

seconds.
6.

Wash

in tap water, dry

and examine.

Results:

Gram-positive organisms, blue.

GRAM'S IODINE

Gram-negative, red.

ANILINE GENTIAN VIOLET

For spirochaetae of Vincent's Angina and for

Trep, pallidum

Solutions required:

A. N/20HCI.
B.

Gram's

iodine.

C. Aniline gentian violet.

Technique:

Spread scrapings from ulcer on a slide; then allow to dry

air before fixing by heat in the usual manner.
the
in
1.

N/20 HCl

2.

Immerse

3.

Wash

4.

Immerse

5.

Wash

6.

Stain with aniline gentian violet for 5-10 seconds.

7.

Wash

8.

Drain and blot dry.

9.

Mount

in

for 10 seconds.

in running water for 5 seconds.

in

Gram's iodine

for

5-10 seconds.

with water.

with water.

if

desired, in D.P.X., Clearmount, or

examine under the

oil

immersion

Emexel and

objective.

Results:

Spirochaetes are stained deep violet.

eference: Bailey, H. D. (1937-38), J. Lab. and Clin. Med., 23, 960.

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

HAEMATOXYLIN

(Phosphotungstic, Mallory)

For Entamoeba
Solutions required:

A. Absolute alcohol
Mercuric
chloride,

aqueous
B.

Iodine

1%

volume

saturated,
.

2 volumes

alcoholic.

C. Phosphotungstic acid haematoxylin (Mallory).

Technique:
1

are made on scrupulously clean slides and fixed

moist in the mercuric chloride solution for fifteen min-

Thin smears

while

still

utes.
2.

Wash and immerse

3.

Flood

slides

in water for a

few seconds.

with the iodine solution and leave thereon for

three minutes.

Rinse thoroughly with distilled water until the iodine colour

completely extracted.

4.
is

5.

Stain for half an hour in phosphotungstic acid haematoxylin.

6.

Rinse in water.

7. Dehydrate with
mount.

95% and absolute alcohol

clear in xylol

Results

Nuclei, deep blue;

cytoplasm, pale blue.

HAEMATOXYLIN

(Weigert)BORDEAUX

RED

For permanent preparations of anopheline midgut

Solutions required:

A. Absolute alcohol

Formalin

..

Glacial acetic acid

372

..
.

45

n^l-

..

3-5 ml.

1-5 ml.

and

SECTION SEVEN
B.

Weigert's haematoxylin, A.

C. Weigert's haematoxylin, B.

D. Bordeaux Red 0-05% in absolute

alcohol.

Technique:

The specimen together with dissecting medium is placed on a

and covered with a coverglass it is then fixed for seven minutes by drawing Solution A under the coverglass on one side,
whilst the dissecting medium is drawn off on the opposite side
1

slide

with a piece of filter paper.

2.

Flood the

A for five
3.

slide while the coverglass is

still

on with Solution

minutes.

Very

carefully

lift

the coverglass with a needle so that the

gut adheres either to the slide or to the coverglass.

4.
it

Carefully

wash the specimen with

with the alcohol for

5.

Cover with

50%

five

70%

alcohol

then cover

minutes.

alcohol for five minutes.

6. Stain for thirty seconds with a freshly prepared mixture conalcosisting of 0*5 ml. each Solutions of B and C, and 25 ml.
hol.

50%

7.

five

Very

carefully

wash with

minutes, followed by

95%

alcohol; cover with

50%

70%

for

for fiwt minutes.

8.

Stain for ten seconds in Solution D.

9.

Flush with absolute alcohol, very carefully; clear in xylol

and mount.

HAEMATOXYLIN EOSIN

INDIGO CARMINE

For the differential staining of vaginal smears

Solutions required:

A. Haematoxylin (Harris).
B.

Absolute alcohol

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

C.

Indigocarmine

0-25 gm.

Eosin, yellowish

Distilled water

. .

0-75 gm.
i ml.

Chloroform

. .

. .

drops

Technique:
1

Make smears on

scrupulously clean slides

allow to dry.

2.

Immerse

3.

Transfer to and immerse in distilled water for one minute.

in

alcohol for three minutes.

95%

Transfer to and immerse in haematoxylin (Harris) for twenty

minutes.
4.

5.

Wash

6.

Immerse

with running tap water for two minutes.

in Solution

for a

7.

Stain in Solution

8.

Rinse quickly in tap water.

9.

Dehydrate with 70%,

10.

Clear in xylol.

1 1

Mount

few seconds.

over-night.

90%

and absolute

alcohol.

in Cristalite.

Results:

Nuclei of epithelial
violet.

Cornified

cells are stained grey,

cells, brilliant

pink.

IODINE

while the cytoplasm

is

Erythrocytes, pink.

EOSIN

For intestinal amoebae and flagellates in faeces

Solutions required:

A. Saturated solution of iodine in

5%

aqueous potassium iodide.

B.

Eosin, yellowish, aqueous

10%.

C. Equal volumes of Solutions

A and

B.

Technique:
I

Spread a loopful of faeces on a

Solution C.

374

slide

with one or two drops of

SECTION SEVEN

Mix

2.

well and cover with a coverslip before examining under

the microscope.
Results:

Cysts of amoebae and flagellates are stained yellow to greenish

yellow against a red background, while glycogen bodies within
the cysts are stained brown.

JANUS GREEN B
For staining oocysts of Coccidia
Solutions required:

A. Sodium chloride, saturated, aqueous.

B.

B aqueous o-oi%.
yellowish io% in tap water.

Janus green

C. Eosin,
Technique:

The

1.

faecal material

is

strained through a double layer of

cheese muslin.
Centrifuge in ordinary pointed tubes.

2.

3. Add more water and repeat the process

most of the debris.

Add sodium

4.

when

twice, to

remove

chloride solution, shake well and centrifuge,

the oocysts will appear on the surface.

5.

Collect oocysts and transfer to a slide.

6.

Cover with a

7.

Replace the sodium chloride with glacial acetic acid by draw-

coverslip.

ing the acid under the coverslip by means of

Gently warm

8.

the slide for

^lyq.

filter

paper.

or ten minutes, taking care

not to evaporate the acid.

Replace the acid with a freshly prepared solution of janus

green B (Solution B above) by drawing the stain under the coverslip by means of a piece of filter paper; then allow the stain to act
for ten minutes.
9.

10.
1 1

2c

Wash and stain

Wash in water.

with the eosin solution for

375

five

minutes.

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

12. Blot off excess

water and seal with petroleum jelly or with

glycerine jelly.

Note: The whole of the process of staining and washing can

be observed under the low-power objective; but studying is
recommended under the oil-immersion objective.
Results:

Oocyst jelly is stained red while the walls may appear reddish,
and the structures within the sporozites are rendered visibleAbstract Science 73, 212-13, 1931, H. B. Crough and E. R. Becker.
y

JANUS GREEN, B

NEUTRAL RED

For supravital staining of blood

Solutions required:

A. Janus green,

vital stain

in neutral

0*4%

absolute alcohol.
B.

Neutral red chloride, vital stain

neutral absolute alcohol.

C. Solution

Solution

0-25%

. .

Absolute alcohol, neutral

in

0-07 ml.
1
75 ml.
10 ml.

Note: Solutions A and B are stable, but solution C deteriorates

few hours, and it should, therefore, be prepared as and
when required, for immediate use.
after a

Technique:
1.

Scrupulously clean, dry slides are flooded with solution C.

2.

Drain and leave

3.

Place a small drop of blood on each slide.

4.

Cover the blood with scrupulously

to dry.

clean,

dry coverslips and

allow the blood to spread.

Seal the edges of the coverslips with soft paraffin
38 C), and examine under the microscope.
5.

wax (M.P.

Results:

Basophilic granules:

brilliant scarlet.

376

Eosinophilic granules:

SECTION SEVEN
yellow to light orange. Neutrophilic granules: salmon colour.
Mitochondria: small blue dots or rods. Nuclei: unstained.

Note:

The

proportion of the two stains

may be

varied to suit

the particular specimen for instance, specimens very rich in cells,

such as leucaemic blood, need more concentrated mixtures of the
;

stains.

JENNER STAIN
For cytological examination of blood
Technique :
1.

face

Air-dried, unfixed blood films are stained for three minutes,

to prevent precipitate depositing on the film and

downwards

obscuring the picture.

2.

Wash

with

distilled

water until the film appears pink to the

naked eye.
3.

Blot dry and examine.

Results:

Neutrophile granules, pink. Oxyphile granules, brownish red.

Basophile granules, purple. Nucleoli (plasmosomes), pink. Cytopartially haemoglobinated precursors of erythrocytes,
of purple. Mature erythrocytes, deep pinkish
shades
varying

plasm of
orange.

LEISHMAN STAIN
For blood, malaria parasites, trypanosomes,
This stain

offers a

etc.

simple and precise method of staining blood

for diagnostic purposes.

Best results are obtained

by buffering the

distilled

water to

pH

6-6-70.
Technique for blood films :

unnecessary unless the films are to be kept in stock

for any length of time, in which case they should be fixed for five
minutes in pure methyl alcohol at room temperature.
Fixation

is

377

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

Thin film method

Air-dried films are stained, without fixing, for one minute

five to ten drops of the stain; then double the quantity of
distilled water (i.e. ten to twenty drops) is added and mixed by
1.

with

rocking the slide gently.

2.

pour
3.

Allow

this diluted stain to act for five to ten

minutes; then

off.

Wash

then differentiate by

gently with distilled water;

flooding the slide with distilled water and allowing the water to
remain on the slide for about one half to one minute, until the
film appears pink to the naked eye.
4.

Pour

blot gently

off;

and dry

in air.

Results:

Similar to Wright's stain.

Thick film method

This method should be employed in searching for blood parawhen negative results have been obtained by the thin film
method.
sites

A film is

prepared by spreading three to five drops of blood

mm. diameter over a slide; then without
it
is
to dry at room temperature from twelve to
allowed
fixation,
twenty-four hours, protected from dust. The time required for
1

in a circle about 15

drying

may be

very considerably shortened by placing the slide

in an incubator at 37 C.
2.

Remove

the haemoglobin by placing the film face down-

wards in a dish of

distilled

3.

Fix in acid alcohol for

4.

Wash

5.

Stain in

water

room temperature.

at

five to fifteen

minutes.

and dry in air.

accordance with the method described above for

well with distilled water

blot gently

thin films.

Bacterial smears, throat exudate, etc.

Unfixed, air-dried smears are stained with

two minutes; then without pouring off:

I.

for

378

ml. of the stain

SECTION SEVEN
2.

Add

an equal volume of

rock the slide to mix;

distilled water,

then allow the diluted stain to act for one minute.

3.

Pour

off;

blot

and dry

in air.

Results:

Bacteria are stained dark blue;

capsules, light blue.

Mucus,

red.

LUGOL'S IODINE
For

dififerentiating

EOSIN

between spores and vegetative forms

bacterial smears

in

Solutions required:

A. Lugol's iodine
Eosin yellowish

Phenol
B.

cryst.

Methylene blue
Absolute alcohol
Distilled water

. .

. .

. .

1%
.

2 gm.
2 gm.

10 ml.

5 ml*

85 ml.

aqueous

40 ml.

Dissolve by shaking thoroughly in a conical flask

filter before use.

Technique:
1.

Fix smears by flaming in the usual manner.

2. Place slides, smear upwards, on the corner of a tripod; flood

with the iodine-eosin stain (Solution A) and heat the underside
of the slide for five minutes, with a small flame, until the liquid

begins to boil.
3.

Remove

the flame momentarily, applying

the liquid ceases to boil

4.

Pour

repeat this process

it

off excess stain ; allow the slide to cool

plunge quickly into distilled water.

again the instant

two or three times.

somewhat

5.

Stain for two minutes in the methylene blue solution.

6.

Rinse with

7.

Drain; blot dry; examine.

distilled water.

Results:

Spores, deep pink.

Vegetative form, blue.

379

then

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

MACCHIAVELLO'S STAIN
For Rickettsiae
Solutions required:

A. Basic fuchsin

Distilled water adjusted to

7*4 with alkali

pH

0.25

gm.

7-2-

100 ml.

This solution should he freshly prepared and

filtered.

Citric acid

B.

0-5% aqueous.

C. Methylene blue

1%

aqueous.

Technique:
1.

Spread smears of the infected tissue on scrupulously clean

2.

Fix by flaming gently.

3.

Stain for four minutes with solution A.

4.

Pour

with the

off the excess stain;

citric

then wash to preparation rapidly

acid solution.

5.

Wash

6.

Stain for a few seconds with the methylene blue.

7.

Wash; dry; examine.

Rickettsiae, red.

Tissue

cells, blue.

MALACHITE GREEN

A simple method of staining

spores

Solutions required:

A. Malachite Green
B.

with tap water.

Results:

Safranin

5%

aqueous.

0-5% aqueous.

Technique:
I.

.
'

slides.

Bacterial films are dried

by heating gently over a bunsen

flame.

380

SECTION SEVEN
2. Heat on a steam bath
bottom of the slides.

till

water condenses in droplets on the

Flood films with Solution A and heat for one minute; then
pour off excess stain and wash well with water.
4. Counterstain thirty seconds in Solution B; then wash with
water; blot dry and examine.
3.

Results:

Spores, green.

Vegetative forms, red.

MALACHITE GREEN

BASIC FUCHSIN

For staining yeasts or bacteria

Solutions required:

A. Basic fuchsin

10%

Malachite green

alcoholic

0-5% aqueous

0-5 ml.
100 ml.

Technique:
1.

Spread smears on

slides

with a platinum wire loop in the

usual manner.
2.

Place slides, smear facing upwards,

Flood

on the corner of

a tripod.

with the staining solution.

Heat
the
underside
of the slide very gently over a small
4.
bunsen flame some distance away from the slide until steam rises.
3.

slide

5.

Pour

6.

Wash

7.

Drain; blot dry, and examine.

off excess stain after a

minute or

so.

well with distilled water.

Results:

Spores are stained greenish blue, while vegetative forms appear

violet to pink.
Reference: Gray, P. H. H. (1941), Nature, 147, 329.

MALACHITE GREEN

PYRONIN

CRYSTAL VIOLET

(Sandiford)

A contrast stain for gonococci and meningococci being a

Gram stain with modified Pappenheim as counterstain
381

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

Solutions required:

A. Crystal violet

Ammonium
B.

i%

in

oxalate

98% alcohol
1% aqueous

20 ml.

aqueous

5 ml.

30 ml.

Lugol's iodine.

C. Malachite green

Pyronin,

1%

B 1% aqueous

Distilled water

15 ml.

80 ml.

Technique :
1

2.

pour

Stain for thirty seconds with Solution A.

Pour off and wash

and blot.

for thirty seconds with Solution

then

off

Decolorize for three or four seconds with acetone;

wash with water.
3.

then

4.

Stain for two minutes with Solution C.

5.

Flood with water, but do not wash; blot dry and examine.

Results:

Cells

and

plish black

nuclei, bluish green.

red.

Gram-positive organisms, pur-

and Neisserieae

MAY-GRUNWALD STAIN
This stain may be used alone for the cytological examination of
blood films, but it is not suitable for parasites. The technique is
the same as that for Jenner stain. Its chief use is in conjunction
with Giemsa stain {see page 368).

METHYL GREEN

PYRONIN (UNNA-PAPPENHEIM)

For gonococci
Solutions required:

A. Methyl green

Pyronin B

. .

Phenol crystals
Absolute alcohol

382

gm.
gm.
2 gm.
i

0-2

10 ml.

SECTION SEVEN
20 ml.
.
. .
Glycerine
Grind together in a mortar and pestle adding:
100 ml.
.
Distilled water
in portions of about 10 ml. over a period of
.

about 20 minutes until solution

then filter.

is

complete;

Technique:
1

Spread smears on scrupulously clean slides and

fix

by flaming

in the usual way.

2.

Flood

slides

move flame and

3.

Pour

4.

Wash

with the stain and heat

allow the

warm

till

steam

rises,

then re-

stain to act for about 45 seconds.

off excess stain.

with distilled water; then immerse in distilled water

for about 30 to 60 seconds.

5.

Shake

off excess distilled water;

then blot dry and examine.

Results:

Gonococci are stained deep red while other bacteria, except

those of the Neisseria group, are faint purple or almost unstained.
Cytoplasm of pus cells pink while the nuclei are green.

METHYL VIOLET

loB

For the direct staining of elementary bodies

Solutions required:

A. Methyl violet loB aqueous 1%.

B.

Sodium bicarbonate.

Distilled water (cold)

..

..

100 ml.

gm.

Dissolve by shaking.
Technique:

Scrupulously clean slides are prepared by rinsing in nitric

acid, followed by water then alcohol-ether.
1.

2.

Spread films, of a

more or

less

pure suspension, on

slides.

present rinse the films with saline and then with

distilled water before allowing to dry in the air or in the incubator.
If

much protein is

3.

Fix by flaming in the usual way.

383

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

4. Mix equal volumes of solutions
onto the slides

and B and

filter at

once

5. Heat the slides over the bunsen flame or on a hot plate until
steam rises, but do not allow the stain to boil.
6.

Allow the hot stain to act for three to five minutes then pour
and allow the slide to cool somewhat before rinsing
;

off excess

with

distilled water.

7. Dry thoroughly; then

or in liquid paraffin.

mount

in

cedarwood immersion

oil

Results:

Elementary bodies are stained sharply a

light violet colour.

Note: Instead of methyl violet the following solution may be

used, in which case the elementary bodies will stain a pinkish
colour: Basic fuchsin 10% in absolute alcohol i volume; distilled
water 9 volumes.
Reference: Gutstein,

M.

(1937);

and Bad.,

of Path,

J',

METHYLENE BLUE

45, no.

i,

313-4.

CARBOL FUCHSESf

For flagella and capsules in bacterial films

Solutions required:

A. Potash alum, saturated aqueous

Tannic acid aqueous 10%

Methylene blue, saturated in absolute alcohol

40 ml.
40 ml.
10 ml.

Note: This solution deteriorates after a week or

ten days.
B.

Carbol fuchsin (Ziehl Neelsen)

Distilled water
.

5 ml.

45

n^l-

Technique:

Smears are made on scrupulously clean slides by spreading

minute speck of the culture with distilled water.
1.

2.

Allow

to dry

and

fix

by flaming
384

in the usual

manner.

SECTION SEVEN
3.

Stain with the methylene blue solution (formula as above)

for ten minutes at about 40 C.

4.

Pour

off excess stain

and rinse the preparation in

distilled

water.
5.

Counterstain for about five minutes with the diluted carbol

fuchsin solution.
6.

Pour

off excess;

rinse in distilled water;

blot dry

and

examine.
Results:

Flagella are stained blue, while capsules are red.

METHYLENE BLUE

GENTIAN VIOLET

For gonococci
Solution required:

Methylene blue
Gentian violet
Distilled water

0-5

0-5

gm.
gm.

100 ml.

Technique:
1.

Thin smears

of the secretion to be examined are fixed

by

flaming.
2.

and

Stain for thirty seconds with the above solution

then wash

dry.

Results:
violet. Nuclei of leucocytes, dark blue. Cytoof
pale reddish violet. Nuclei of epithelial cells,
leucocytes,
plasm
blue.
deep

Gonococci, dark

METHYLENE BLUE

SAFRANIN

For polar bodies of bacteria

Solutions required:

A. Methylene blue (Neisser).

B.

Safranin

1%

aqueous.

38s

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

Technique:
1.

Air-dried smears are fixed by flaming in the usual manner.

2.

Immerse

3.

Pour

in the methylene blue solution for five minutes.

off excess stain

and

carefully

wash the preparation

in

running water.
4.

Drain

then cover the preparation with

stain to act for one to two minutes.

off excess water;

safranin solution and allow the

5.

Rinse well with

distilled water;

blot dry;

drain;

examine.

Results:

The

polar bodies appear deep blue, while the bacterial cells are

distinctly red.

NEW FUCHSIN

CONGO RED

For bacterial cell walls, particularly

E, Coli

and

B. Cereus

Solutions required:

A.

New

B.

Congo Red

fuchsin

1%
.

aqueous.
.

pH 9-5.
Warm to dissolve

Buffer solution

gm.

100 ml.

Technique:

Smears of the organisms are made on

manner, with a wire loop, i mm. diameter.
1.

2.

Allow the smears to dry in the

3.

Stain in the

4.

Pour

Shake

come

without the use of heat.

fuchsin solution for three to four minutes.

off excess stain:

colour ceases to
5.

new

air

slides in the usual

then rinse the slides gently until the

out.

off excess water

and

set the slide aside to

dry in the

air.

6.

Place the air dried slides

on

warm

plate at 50 C. for ten

to twenty seconds.

the hot plate and using a wire loop i mm. in

diameter, add a loopful of the congo red solution and spread this
7.

Remove from

386

SECTION SEVEN
Stain,

by means of the wire

to act for about

Allow the

loop, over the smear.

stain

one half to one minute.

8.

Wash

off excess stain

9.

Shake

off excess distilled water;

with

thoroughly in the air before

distilled water.

then allow the smear to dry

examining under the oil immersion

objective.

Results:

Cell walls stained red.

Note Walls in the early stages of formation are not stained. Cross
:

walls are stained in the older portion of a filament of B. cereus but not
in the younger portions. The end cell of a filament has relatively heavy
side walls but the wall becomes much thinner around the free end of
the cell. The stain does not show cross walls in chains of E. Coli.

A further differentiation of the stained material may be made by

exposing the stained specimen to the fumes of hydrochloric acid which
will change the colour of the free congo red to blue.
Reference: Chance, H. L. (1953), Stain Tech., 28, 205-7.

NEWMAN'S STAIN
A

single solution for defatting and staining milk

for the enumeration of milk bacteria

smears

Solution required:

Newman! s Stain
Methylene blue
Absolute alcohol
Tetrachlorethane
Acetic acid, glacial

gm.

54 ml.
40 ml.
6 ml.

Place the methylene blue with the alcohol in a flask; then plug

the neck of the flask lightly with cotton- wool. Heat water till it
boils in a water bath then turn off the flame and place the flask
;

in the hot water until the alcohol just begins to boil. Allow the
solution to cool after swirling the liquid round inside the flask until

the methylene blue has gone into solution. Filter into the stock
bottle; add the trichlorethane replace the stopper or cork and

all

shake well.

387

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

Technique:
1.

Spread o-oi ml. of the milk over a carefully measured square

centimetre on a scrupulously clean slide.

Note:

The

area can best be measured out

by marking

it

on

piece of white paper and placing the slide over the square.
2.

Allow the film

to dry;

then

fix in

pure methyl alcohol for one

minute.
3.

Apply the

4.

Wash

stain

and allow

it

to act for

about thirty seconds.

with water.

5. Dry and examine, using a micrometre eyepiece and an

immersion lens.

oil

Results:
Bacteria, deep blue.

Background, pale blue.

NILE BLUE SULPHATE

For protozoa and yeasts
Solution required:

Nile blue sulphate

o-i% aqueous.

Technique:
1.

Stain

from

five to ten

minutes according to the material

and depth of staining desired.

2.

Rinse in distilled water; dry, and examine.

Note: This stain

may

also

be employed for living amphibian

eggs and for Hydra.

PAPANICOLAOU STAIN
For improved dififerentiation of the cells of vaginal smears
Staining solutions:

A. Ehrlich or Harris haematoxylin.

B.

Orange

Alcohol

95%

..

Phosphotungstic acid

388

..
.

..
.

0-2

gm.

100 ml.
0-0x5 gm.

SECTION SEVEN
C. Papanicolaou stain, powder

Alcohol

95%

07

gm.

100 ml.

Place in a flask, plug the neck lightly with

cotton- wool; then heat, on a waterbath until
dissolved.

Cool, then

filter.

Technique:
1

Wet smears

are fixed in a mixture of equal

and absolute alcohol for

2.

Rinse successively in 90%,

volumes of ether

minutes.

five to fifteen

70%

and

50%

alcohols

and

dis-

tilled water.
3.

Stain in Ehrlich's haematoxylin for five to ten minutes.

4.

Rinse in

distilled

water; differentiate in

0-5% hydrochloric

acid.

Rinse in distilled water; then leave for one minute in dilute

lithium carbonate solution (three drops saturated lithium carbonate to 100 ml. distilled water).
5.

6.

Rinse thoroughly in distilled water and wash successively in

50%, 70%, 95%

7.

alcohol.

Stain for one minute in Orange

solution (prepared as

above).
8.

Rinse thoroughly in

alcohol to

95%

remove

all

excess stain.

two minutes

in Papanicolaou stain; then rinse five

9.
to ten times in each of three jars of 95% alcohol.

Stain for

10.

Rinse in absolute alcohol; clear in xylol and mount.

Results:

Nuclei, red. Erythrocytes, orange. Cornified

or orange. Basophile cells, blue or green.

PEROXroASE REACTION

cells, red,

(Cowdry's method)

For blood films

Solutions required:

A. Copper sulphate crystals

Distilled water
.

389

0*5

gm.

100 ml.

pink

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

Benzidine base, pure
Distilled water

B.

Filter

o-2 gm.
200 ml.

and add 4 drops hydrogen

peroxide (20 vols.) to the

store in a dark bottle.
C. Safranin

filtrate;

1%

aqueous

Technique:
1.

Air-dried blood films are flooded with Solution

minute. Pour off

2.

then without washing and while

Flood with Solution

B and

leave for

still

two minutes

for

wet

one

then rinse

in tap water.
3.

Counterstain in Solution

4.

Wash

for one minute.

with tap water; blot and dry.

Results:

Nuclei of leucocytes, orange-red.

Peroxidase granules, blue.

Note: Unmounted specimens keep for many months without

fading.

PICRO METHYL BLUE

EOSIN

For urinary casts

&

Abstract J. of Lab.
Clin. Med. (U.S.A.), 22, 853, 1937, Jeanette Allen
Behre, and William Muhlberg.

Solutions required:

A. Eosin yellowish
B.

Methyl blue

0-5% aqueous.

aqueous
aqueous

Picric acid saturated,

Glycerine

..
.

ml.

10 ml.
10 drops

Technique:
1.

The

urine

is

centrifuged and the supernatant liquid decanted

as usual for microscopic examination.

2.

One drop

of eosin solution

mixed by shaking from

is

added

to the sediment

side to side for one to

390

two minutes.

and

SECTION SEVEN

Two

B are added and mixed. The colour

now be distinctly blue-green if it is reddish brown more of Solution B should be added till the blue-green
colour is obtained, but too much should be avoided.
3.

drops of Solution

of the sediment should

4. Some of the stained sediment is then transferred to a

covered with a coverglass and examined.

slide,

to the

The amounts of the two stains may be varied according

amount and particular character of the sediment present.

More

eosin

Note:

may be added

if

the cells have not been stained

sufficiently red. Enough of the

to stain the casts a distinct blue,

methyl blue should be added

but too much will stain them

too dark.

More permanent
to the sediment

slides may be made by adding more glycerine

and sealing the edges of the cover glass with Vase-

line or balsam.

This technique brings out the detailed structure of casts

and castlike bodies in a remarkable way. It does not furnish
a differential stain since

all

the

mucous

material

is

also stained

blue.

Results:

Hyaline casts are stained a clear blue of varying intensity. The

irregularly shaped bodies, sometimes classed as cylindroids,

more

are similarly stained. An irregular distribution of material, a

"
"
mealy structure, or a striated appearance sometimes becomes
evident in bodies which appear perfectly homogeneous before
staining.

Mucous threads and amorphous

material are also stained

blue; they are clearly differentiated from the cast-like bodies by

their structure.
Granular material is usually stained darker.
fine granular casts present a striking picture of fine, dark
Some
granules powdered over the light blue, hyaline body.
coarsely granulated casts are stained deep blue the granules of the

Mixed,

others are yellow, orange or dark reddish brown. Renal epithelial

cells are usually red, sometimes orange or yellowish. Red blood

Pus cells are usually red, occafrom the urinary passages are either

cells are stained a brighter red.

sionally blue.
red or blue.

Epithelial cells

Fat globules are unstained. In cells which are undergoing fatty

degeneration, the fat cells are seen strikingly against reddish
2D

391

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

cellular material.

The

picric acid gives a light yellowish back-

ground.
Often the stain reveals the presence of lightly stained mucus-like
envelope, apparently covering the cast material. This material is
often clearly seen at the end of a cast which has apparently been

squarely broken

off.

PINACYANOL

NEUTRAL RED

For supra-vital staining of blood

Note: Pinacyanol

superior to janus green in that it does not

fade
very selective for mitochondria, and it does not inhibit
of
the effect
neutral red. It has the disadvantage, however, of
is

it is

being extremely costly.

Solutions required:

Stock solutions:

A. Pinacyanol o-i% in absolute alcohol.

B.

Neutral

Red o-i%

in absolute alcohol.

Staining solutions:

C.

for mitochondria only

Solution

Absolute alcohol

D.

for mitochondria^ nuclei and other

Solution

Solution

ml.

20 ml.

cell granules

ml.

2 ml.

20 ml.

Absolute alcohol
Notes:

C and D deteriorate after a few hours and should,

be prepared only as and when they are required for

(a) Solutions

therefore,

immediate use.
(b) The proportions and dye concentrations of either of these
two solutions may be varied to suit particular specimens.

Technique:
I.

Scrupulously clean dry slides are flooded with solution

or D, whichever

is

required.

392

SECTION SEVEN
2.

Drain and leave to dry, then use as follows (within a few hours
D) as the dried dye combination

at latest in the case of solution

does not keep well.

on each

3.

Place a drop of blood

4.

Cover with scrupulously clean coverslip and allow the blood

slide.

to spread.

Seal the edges of the coverslips with soft paraffin

38 C), and examine under the microscope.
5.

wax (M.P.

Results:

With Solution
Mitochondria in

(Pinacyanol only)

still

living

and motile

cells are stained

deep

blue to violet.

With Solution

Mitochondria stained as above; nuclei and other

cell

granules

are stained red.

Reference: Hetherington, D. C. (1936), Stain Tech., 211, 153-4.

PONDER'S STAIN

(Kinyoun's modification)

For differentiation of metachromatic granules of diphtheria organisms

Solution required:

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

Results:

Granules, dark red.

Body

of

Other bacteria,

pale blue.

cells,

pale.

PYRONIN

ALPHANAPHTHOL

(Graham)

For oxidase granules in blood smears

Solutions required:

A. a-Naphthol, pure
Alcohol 40%

Hydrogen peroxide 20

100 ml.

vols.

gm.

0-2 ml.

Note: This solution deteriorates in four or five

days when the hydrogen peroxide has been added,
and it is, therefore, better not to add the hydrogen
peroxide until the solution is required for immediate
use.

B.

Pyronin
Alcohol

40%

Aniline

oil

C. Methylene blue

o-i

gm.

96 ml.
4 nil.

0-5% aqueous.

Technique:
1. Freshly spread air-dried blood smears are fixed in 10%
formalin for two minutes then washed well in distilled water.
;

2.

Stain for five minutes in Solution

for fifteen minutes in

3.

(as

above)

then wash

running water.

Stain for two minutes in Solution

(as

above)

then wash in

distilled water.
4. Stain for one half to one minute in Solution C (as above)
then wash with water blot and dry thoroughly mount in balsam.
;

Results:

Neutrophile granules, which give the oxidase reaction, are

stained purple to red; while eosinophile granules are lighter red,
larger and more refractile. Basophile granules are stained a deep
cell nuclei, blue;
cytoplasm, pale blue;
appear greenish yellow to pink.

purple;

394

erythrocytes

SECTION SEVEN

SAFRANIN

LIGHT GREEN

For spirochaetes,

etc.

Solutions required:

100 gm.
100 ml.

A. Tannic acid
Alcohol 95%
B.

Acetic acid, glacial

7-5 ml.

Formalin

100 ml.

i%

C. Safranin

D. Light green

aqueous.

i%

aqueous.

Technique:

The

material suspected of containing spirochaetes is placed

acetic acid on a slide. The slide is then inverted
of
drop
over the cavity of a well slide and placed in an incubator for fifteen
1.

5%

in a

minutes.

Spread the drop with a wire loop and allow it to dry in the air.
a freshly prepared mixture consisting of
3. Flood the film with
volume Solution A and 2 volumes of Solution B and steam from
2.

two
4.

to five minutes.

Wash with warm

water

distilled

then stain with Solution

for four to eight minutes.

5.

Wash

with

counterstain with light green.

distilled water;

SCHORR'S STAIN
For vaginal smears
Staining solutions:

A. Harris's haematoxylin.
B.

Biebrich Scarlet

gm.

Distilled water

0*4 gm.
99 ml.

Glacial acetic acid

Orange

C. Phosphomolybdic acid

Phosphotungstic acid
Distilled water
395

ml.

2-5
2-5

gm.
gm.

100 ml.

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

D. Fast Green

FCF

Acetic acid,

0-3%

0-25 gm.
100 ml.

Technique:
1. From the fixing solution, carry through alcohols to water;
for two minutes
stain in Solution
then wash in running water

for five minutes.

2.

3.

4.
5.

6.

B then wash in water.

one minute; then wash in water.
Stain in Solution D for two minutes then without washing
Differentiate for one minute in 1% acetic acid.
Dehydrate in the usual manner clear in xylol and mount in
Stain for one minute in Solution

Mordant

in Solution

for

dammar-xylol.

A may

Note: Solution

routine treatment of

be omitted in certain cases, as in the

menopause with oestrogens. With this omis-

sion smears can be stained in five minutes.

Results:

Cornified

orange-red.

cells,

Non-cornified

cells,

green.

SCHORR'S STAIN
(Single solution)

For vaginal smears

Solutions required

A. Harris Haematoxylin
B. Schorr's Stain (Single)

Technique:
1.

Fix wet smears one to two minutes in equal parts of absolute

then pass into two changes of absolute alcohol,

alcohol and ether

followed by
2.

Rinse with

solution
3.

80%

and

66%

distilled

A for two

alcohol.

water for ten seconds; then stain in

minutes.

Wash in running water

B for two minutes.

for five minutes; then stain with

solution
4.

Dehydrate with 66%,

and mount.

80%

and absolute alcohol; then

in xylol

Results:

Cornified

cells,

orange-red.

Non-cornified

396

cells,

green.

clear

SECTION SEVEN

SUDAN BLACK

EOSIN

METHYLENE BLUE

For lipoid granules in leucocytes

Solutions required:

A. Sudan Black B, saturated in ethylene glycol.

Note: This should be stored in a well-stoppered

bottle.

B.

Eosin, yellowish

i% in 70% alcohol.

C. Methylene blue, saturated, aqueous.

Technique:
1.

Air-dried blood films are fixed for thirty seconds in pure

methyl alcohol.
2.

Stain for half an hour in the sudan black solution in a stop-

pered staining jar.

3.

Rinse in water.

4.

Rinse thoroughly in

70%

alcohol.

5.

Counterstain for thirty seconds with the eosin solution.

6.

Wash

7.

Stain in methylene blue solution for three minutes.

in tap water.

8.

Rinse with

9.

Drain and blot thoroughly

distilled water.
;

examine under the oil-immersion

objective.

Results:

Lipoid granules are stained an intense black, whilst nuclei are

stained blue, and erythrocytes, red.

SUDAN BLACK
For demonstrating

SAFRANIN

fat in bacteria

Solutions required:

A. Sudan black,

saturated in

alcohol.

B.

Safranin O, aqueous 1%.

397

70%

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

Technique:
1. Filter about ten drops of the sudan black solution, which
should be two or three days old, into a test-tube.

2.

Emulsify bacteria from a slant culture directly into the

fil-

tered Sudan black in the test-tube.

3. Allow the tube to stand undisturbed for
minutes until the precipitate has settled.

4.

On

fifteen to

twenty

a scrupulously clean slide place a loopful of the emulsion

circular motion to facilitate rapid

from the top and smear with a

drying.
5.

Stain with the safranin solution for about fifteen seconds.

6.

Wash

7.

Blot dry, and examine under the oil-immersion objective.

with distilled water; drain.

Results:
Bacterial cells are stained pink

fatty material

is

bluish grey or

blue-black.

Note: With reasonable

unchanged
From

care,

unmounted smears

will

remain

for at least six months.

J. Bact.y 43, 717-24, 1942, Burdon, K. L., Stokes, J. C, and Kim"

Studies of the common aerobic spore-forming bacilli ".

brough, C. E.

SUDAN
A

stain for blood in the spinal fluid, differentiating fresh

haemorrhage from old

Solution required:

Sudan

3,

saturated in

70%

alcohol.

Technique:
1. Smears of spinal fluid are made on scrupulously clean slides
and allowed to dry thoroughly in the air for about half an hour.

2. Without fixing, immerse in the sudan 3 solution for five to

fifteen minutes, in a stoppered staining jar.

then immerse for one minute

3. Rinse well with distilled water
each in four or five changes of distilled water.
;

398

SECTION SEVEN
4.

Drain

5.

Mount

off excess

water

in glycerine

blot

and

dry.

and examine under the oil-immersion

objective.

Results:

Erythrocytes from haemorrhage more than twenty-four hours

old appear with ring-like peripheries and unstained centres, while
those from fresh haemorrhage in the cerebrospinal fluid are stained
yellow.

SUDAN
For staining

fat in

faeces

Solution required:

Sudan 3 saturated in equal volumes of 70% alcohol and ether.

Technique:
1.

A loopful of faeces is mixed with a drop of 50%

2.

Add one

or two drops sudan 3 solution

alcohol.

then apply a cover-

glass before examining.

Results:

Neutral
flakes

fat

which

appears as highly refractile droplets or yellowish

stain orange to orange-red.

Note: In normal faeces there is no appreciable amount of

neutral fat present.
Fatty acids appear as flakes, which stain faintly; or as fine
needles which tend to collect in clusters and do not take the stain.

Soaps appear as yellowish flakes, rounded or gnarled bodies,

everted like the pinna of the ear, or as coarse crystals, which do not
take the stain.

On warming the preparation gently, fatty acids melt, whereas

the soaps do not.
In the faeces of a healthy subject the only fat elements recognizable under the microscope are yellow calcium or colourless
soaps.

399

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

TETRACHROME STAIN
(MacNeal)
For differentiation of types of leucocytes
Staining solution:

Dissolve 0*3 gm. of the dry stain in lOO ml. of pure methyl alcohol by heating to 50 C. on a water bath. Shake well, and leave
for four or five days. with occasional shaking; then filter.
Technique:
1.

prepared blood films are dried quickly at

stained immediately one to three minutes
ml. of the staining solution prepared as above; then add

Very

thin, freshly

room temperature and

with

2 ml. buffer solution

pH

6-8 and allow to stand for about five

minutes.
2.

Pour

off the stain

and wash with buffer solution

pH 6-8 until

the thin portions of the stained film are pink.

3.

Blot carefully and examine.

Note: If it is desired to keep the films for any length of time

before staining, they should be fixed immediately while still wet,
with pure methyl alcohol for one minute.
Results:

Erythrocytes are stained yellowish red; polymorphonuclear

neutrophiles dark blue nuclei, reddish mauve granules, pale pink
:

cytoplasm. Eosinophilic leucocytes blue nuclei, red to orange-red

granules, blue cytoplasm. Basophilic leucocytes purple or dark
blue nuclei, dark purple to black granules. Lymphocytes: dark
:

purple nuclei, sky-blue cytoplasm.

Platelets: violet

granules.

TfflONIN

A rapid

(Ehrlich)

stain for rickettsia in

smears

Solutions required:

A. Thionin, saturated, aqueous.

B.

Sodium hydroxide 10% aqueous.

400

to

purple

SECTION SEVEN
C. Phenol

2%

aqueous.
the sodium hydroxide solution a little at a
time to the thionin solution until all the dye has been

Add

precipitated; then collect the precipitate and wash

with a large volume of water until the runnings show
neutral reaction. Prepare a saturated solution of the
precipitated dye in the 2% phenol.

Technique:
1.

Air-dried smears are fixed in absolute alcohol for five min-

utes.
2.

Pour

any excess alcohol and allow the film

off

to

dry in the

'

atmosphere.
3.

to

Stain in the thionin solution (prepared as above) for one half

one minute.
drain and

4.

Pour

5.

Clear in xylol and

off excess

mount

wash rapidly

in absolute alcohol.

in Cedronol.

Results:

Rickettsia are stained deep violet. Cytoplasm

while red cells are bluish green.

is

stained a light

violet,

VAGINAL SMEAR STAIN,

M.F.4

For the rapid staining of vaginal smears, sharply contrasting

cornified

from non-cornified

cells

Technique:
1.

Fix smears, while still wet, in equal parts of ether and absoone minute.

lute alcohol for

2. Rinse with two changes of

changes of 70% alcohol.
3.

80%

alcohol, followed

Rinse by dipping the slide seven times in rapid succession in

a jar of distilled water.

4.

by two

Immerse

in the staining solution for

401

two minutes.

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

5.

Rinse with 70%, followed by 90%, alcohol.

6.

Dehydrate with absolute alcohol.

7.

Clear in xylol and mount.

Results:

Comified

cells,

Non-comified

orange to red.

cells,

blue.

VAGINAL SMEAR STAIN, PX

For vaginal smears

Technique:

wet in a mixture consisting of equal

for two minutes.
alcohol
and
absolute
ether,
parts
1.

Fix smears while

2.

Rinse in

3. Rinse in

70%
50%

still

alcohol.
alcohol.

4.

Rinse in water.

5.

Immerse

in the staining solution for three minutes.

6.

Immerse

in dioxane for a

7.

Wash

8.

Rinse well in xylol and mount.

few seconds.

with absolute alcohol rapidly.

Results:

Nuclei,

stained red.

Erythrocytes, orange.

Cornified

Basophile

red, pink or orange.

blue or green.

cells,

cells,

VICTORIA BLUE 4R
For elementary bodies
Solutions required:

A. Victoria Blue 4R (Lustgarten)

Distilled water
Absolute alcohol
.

B.

10 ml.

Potassium hydroxide 0-02% aqueous.

402

gm.

90 ml.

'

SECTION SEVEN
Technique:
1.

Smears are spread on scrupulously clean

slides

and dried in

the incubator.
2.

Rinse in physiological saline solution.

3.

Rinse in

4.

Fix by immersing in pure methyl alcohol for an hour, in a

stoppered

distilled water.

jar.

a freshly prepared and filtered mixture

5. Stain overnight with
and B.
of
Solutions
of
consisting
equal parts

6.

Blot and dry and mount.

Results:

Elementary bodies of vaccinia and other viruses are stained dark

blue.
Reference: Gutstein,

M.

(1937), 7- Path,

and Bad.,

45, no.

i,

313-4-

VICTORIA BLUE 4R
For Treponema pallida
Solution required:
Victoria

2%

Blue

4R

(Lustgarten)

aqueous.

Technique:
1.

Allow smears to dry

then

in air;

fix

with methyl alcohol for

ten minutes.
2.

Stain for five minutes in the Victoria blue solution.

3.

Pour

4.

Drain

5.

If

off excess stain;

;

it is

rinse with distilled water.

dry carefully but thoroughly.

desired to preserve the specimen,

mount

at

once in

Cristalite.

Results:

the characteristic morphology of

Treponema pallida clearly distinguishes the organism from other

Spirochaetes are stained blue

spirochaetes which

may be

present.

403

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

VICTORIA BLUE

KERNECHTROT

LIGHT GREEN

For elementary bodies of various viruses, notably those of

canary pox, ectromelia, variola, and to a lesser degree,
varicella and herpes simplex
Solutions required:

A. Kernechtrot (Herzberg)
Aluminium sulphate
Distilled water
.

Dissolve by boiling
and add:

B.

..
.

allow to cool

Acetic acid

io%

aqueous

Light green

1%

aqueous

..

gm.
gm.

loo ml.

then

o-i

filter

0*5 ml.

100 ml.

Acetic acid

10 ml.

Distilled water

. ,

90 ml.

10%
C. Tartaric acid 10% aqueous.
D. Victoria blue 4R 1% aqueous
Acetic acid

10% aqueous

0-5 ml.

ml.

Technique:
1

Make smears

allow to dry in the

of infected tissue on scrupulously clean slides

2.

Fix by flaming gently.

3.

Wash

in distilled water for ten minutes.

4. Stain in
5.

air.

the Kernechtrot solution for an hour.

Rinse for thirty seconds each in two changes of distilled

water.

7.

Drain and allow to dry for half an hour in the atmosphere.

Stain in the light green solution for two minutes.

8.

Rinse for

6.

five

seconds in each of two changes of distilled

water.
9.

10.

Immerse
Drain

five to ten

11.

in the tartaric acid solution for

two minutes.

then wash for

seconds in each of two changes of distilled water.
oflp

the excess tartaric acid solution;

Stain with the victoria blue solution for two minutes.

404

SECTION SEVEN
Drain

12.

off excess stain;

then wash for

five

seconds in each

of two changes of distilled water.

Drain and leave

13.

to dry, protected

from

dust, in the air or in

the incubator.
Results:

Elementary bodies, blue.

Cell nuclei, pink.

Cell protoplasm,

green.
*'

Reference: Herzberg, K. (1934), Zentrlh. f. Bakt.

Viktoriablau zur Farbung von filtrierbarem Virus ".

Orig.,

131,

358-66,

WRIGHT'S STAIN
For general differentiation of blood corpuscles; for malaria parasites, trypanosomes, etc.
This stain
stain, which

is

extensively used in

America instead of Leishman

generally preferred by British workers.

Best results are obtained with very thin films, and the distilled
is

water used should be buffered to

pH

6 5-7-0.

Technique:

Fixation

is

unnecessary unless the films are to be kept for any

length of time before staining, in which case they should be fixed

for five minutes with pure methyl alcohol then blotted and dried
;

at

room temperature.
1.

Place

act for

ml. of the stain on a dried blood film and leave

one minute; then add 2 ml.

distilled

it

to

water and rock the

slide gently to mix.

Allow this diluted stain to act for three to five minutes; then
pour off and wash with distilled water until the thin portion of the
films appears pink to the naked eye.
2.

3.

Pour

off excess stain;

blot

and dry

at

room temperature.

Results:

Erythrocytes, yellowish red. Polymorphonuclears dark purple

nuclei, reddish violet granules, pale pink cytoplasm. Eosinophiles
:

blue nuclei, red to orange-red granules, blue cytoplasm. Basophils purple to dark blue nuclei, dark purple to black granules.
:

Lymphocytes dark purple

:

nuclei, sky-blue cytoplasm.

405

Platelets,

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

violet to purple granules.
Malarial: parasites and Leishmania
chromatin, red, cytoplasm, blue. Trypanosomes chromatin, red.
Note: The timing of the staining either before or after dilution
:

may be

altered to suit individual requirements.

Staining effects similar to Giemsa are obtained

by staining for
its volume of

ten minutes in Wright's stain diluted v^ith four times

distilled v^ater buffered to pH 6-5.

WRIGHT'S STAIN
For demonstrating Trichomonas

riedmuller

Solutions required:

A. Osmic acid

2% in 0-5% chromic

acid.

B. Wright's blood stain.

C. Buffer solution

pH

7-0.

Technique:
1. Fresh, wet smears of the material containing Trichomonas
riedmuller are made on slides and exposed to osmic acid fumes for

ten to thirty seconds when microscopic examination should

no motility among the organisms.

show

2. Cover the smear with five drops of Wright's stain and fifteen
drops of the buffer solution, and mix the solutions by gently
rocking the slide. Allow this diluted stain to act for two to five
minutes.
3.

Pour

off excess stain

and wash thoroughly with the buffer

solution.

Allow the smears to dry thoroughly

under the oil-immersion objective.
4.

in the air

then examine

Results:

Cytoplasm, blue. Posterior portion of the axostyle, pink.

Blepharoplasts, chromatin ring around the posterior part of the
axostyle, endoaxostyle granules and nucleus, dark purple. The
cytostome appears as a clear area at the anterior extremity of the
body of the organism. The anterior flagella and those bordering
the undulating membranes are stained pink.
Adapted from J^. Parasitology, 5, 473-4, 1938, H. M. Stewart.

406

APPENDIX

2E

ATOMIC WEIGHTS

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

Strontium

APPENDIX

FORMULAE
FIXATIVES

ALTMANN*S FLUID
Osmic

acid 2

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

PICRIC NITRIC ACID
Picric acid, aqueous, sat.
Nitric acid, cone.
.
.

95 i^l
5 ml.

PICRO-SUBLIMATE-ACETIC ACID (RATH'S FLUID)

Picric acid, saturated, aqueous

Mercuric chloride, hot saturated aqueous

Glacial acetic acid

. .

50 ml.
50 ml.

40 ml.

PICRO-SUBLIMATE (RAHL'S FLUID)

Picric acid, saturated aqueous

Mercuric chloride, saturated aqueous

Distilled water

50 ml.
50 ml.
100 ml.

ZENKER-FORMOL
See Helly's

fluid.

AND REAGENTS

STAINS

ACETO CARMINE (SCHNEIDER)

Carmine

Distilled water

0*4 gm.

55

45 ml.

Heat

to boiling, then
Glacial acetic acid
.

add

nil.

Raise to boiling point again; then cool and

filter.

ACID FUCHSIN (ALTMANN)

Aniline water

Acid fuchsin

100 ml.
20 gm.

ACID FUCHSIN (MALLORY)

Acid Fuchsin

Distilled water

ALBERT'S STAIN

(for

0-5

gm.

100 ml.

Diphtheria)

A.
Toluidine blue

0*3% aqueous
Methyl green 0-4% aqueous
Glacial acetic acid

Absolute alcohol

75
75

..1*5

nil.
nil.

ml.

nil.

Shake well, heat to about 80 C. then allow to stand for twenty-four

hours before filtering.

412

APPENDIX
B.
Iodine, resublimed
Potassium Iodide
Distilled water

..
.

..
.

..
.

..

gm.
gm.

1*5

150 ml.

Dissolve the potassium Iodide in about 3 to 5 ml. of the water;

then add the iodine and shake until dissolved before adding the re-

mainder of the water.

ALUM CARMINE

(MAYER)

Distilled water

Alum
Shake well

carmine powder
then boil for a minute
.

50 ml.

3-5

gm.

gm.

and

cool

filter.

AMMONIA CARMINE
Ammonia carmine powder (Michrome
Brand)

Distilled water

0-5

100 ml.

ANILINE BLUE, ACETIC (MASSON)

Aniline blue 1% aqueous
.

Aniline blue, alcohol soluble

Absolute alcohol
Distilled water

Glacial acetic acid

100 ml.
2 ml.

ANILINE BLUE, ALCOHOLIC

i

gm.

Aniline blue, water soluble

Distilled water

100 ml.

'

70 ml.
30 ml.

ANILINE BLUE, AQUEOUS

.

gm.

Store in a well-closed bottle to which a few drops of Chloroform or a

crystal of thymol is added to inhibit the growth of air-borne organisms
and consequent decomposition of the stain.

ANILINE CHLORIDE (ANILINE HYDROCHLORIDE), ACIDIFIED

Aniline chloride

Sulphuric acid

. .

25%

gm.

100 ml.

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

ANILINE CHLORIDE (ANILINE HYDROCHLORIDE)
Aniline Hydrochloride

Distilled water

ANILINE

BLUE

ORANGE G (MALLORY)
G (Michrome Brand)

Aniline blue-Orange
Distilled water

Dissolve, and

lo gm.
loo ml.

(
2*5 gm.

55

r^il-

filter.

ANILINE GENTIAN VIOLET (STIRLING)

Gentian violet
Aniline

oil

Water
Alcohol

5 gni.

2 ml.

88 ml.
10 ml.

95%

Dissolve by shaking thoroughly; allow to stand twenty-four hours

then

filter.

ANILINE SULPHATE
Aniline Sulphate
Distilled water

. ,

5 grn.

100 ml.

ANILINE WATER
Aniline

oil

Distilled water

Shake vigorously: then allow to stand.

5 nil-

100 ml.

immediately before

Filter

use.

ANILINE XYLOL
Aniline

Xylol

BEST'S

oil

volume
volume

CARMINE (STOCK SOLUTION)

Potassium carbonate
Potassium chloride
.

gm.

5gni.

Carmine

Distilled water

60 ml.

Heat gently

gm.

for five to ten minutes until dissolved: then cool

add:

Ammonia

solution (sp. gr. 0-880).

414

20 ml.

and

APPENDIX
or:
Best's

Carmine powder (Michrome Brand)

Distilled water

Heat

to boiling point; cool

lo gm.
lOO ml.

and add:

Strong ammonia solution

(sp. gr. 0'88o).

20 ml.

BORAX CARMINE (GRENACHER)

Borax
Carmine

4 gni.

Distilled water

Alcohol

..

70%

..

..

..

gm*

100 ml.
100 ml.

Shake the borax and carmine with the water then boil gently for
allow to cool make up to the original volume with
thirty minutes
distilled water; then add the 100 ml. 70% alcohol. Mix well and allow
:

to stand several days before filtering.

BORAX CARMINE (MAYER)

Borax
Carmine

Distilled water
Absolute alcohol

gm.
gm.

50 ml.
50 ml.

Shake the borax and carmine with the water: then boil gently for
thirty minutes; cool; make the volume up to 50 ml. with distilled
water; add the alcohol; then filter.

BORREL'S BLUE
Borrel's blue

powder (Michrome Brand)

Distilled water

Boil for five minutes: then cool

and

filter

2 gm.
50 ml.

and make the volume up

to 50 ml. with distilled water.

CARBOL FUCHSIN (ZIEHL NEELSEN)

Basic fuchsin

Phenol 5

Mix

10%

in absolute alcohol

% aqueous

..

..

..

10 ml.

100 ml.

well and allow to stand overnight before filtering.

CARBOL GENTIAN VIOLET

Gentian violet 10% in absolute alcohol
Phenol 2% aqueous
..
..
Alcohol 95

Mix

well; then

filter.

415

..

10 ml.
100 ml.
20 ml.

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

CARBOL METHYLENE BLUE

Phenol 5% aqueous
Methylene blue
Absolute alcohol

loo ml.

gm.
lo gm.

CARBOL THIONIN
Thionin (Ehrlich)
Phenol crystals
Alcohol

Heat

The

10%

0*125 g^i*

100 ml.

to dissolve, or triturate in a mortar; filter

when

gm.
dissolved.

solution deteriorates after a few weeks.

CARBOL TOLUIDINE BLUE

Carbol Toluidine blue powder (Michrome
Brand)
Distilled water
.

CARMALUM

. .

2-5

gm.

50 ml.

(MAYER)

Carminic acid
Potash alum 5

i gm.
200 ml.

Dissolve by heating. Cool and filter. Add a crystal of thymol or

few drops of chloroform, to the stock bottle, as preservative.

COTTON BLUE - LACTOPHENOL

Cotton blue
Lactophenol.

COTTON BLUE

100 ml.

gm.

MAGENTA LACTOPHENOL

Cotton blue

Magenta

1%

in lactophenol

Lactophenol

8 ml.

100 ml.

100 ml.

in lactophenol

DORNER'S NEGATIVE STAIN

Nigrosin

10 gm.
100 ml.

Distilled water

Formalin

9*5 ml.

100 ml.

FAST-GREEN FCF IN CELLOSOLVE

Cellosolve

Fast-green

Heat

on a water bath
then cool and filter.

in a flask

stirring;

FCF

for about half

416

3 grfi*

an hour with occasional

APPENDIX

FLAGELLA STAIN MORDANT (LOEFFLER)

Tannic acid

20% aqueous
Ferrous sulphate, cryst.
Distilled water
.

Basic fuchsin

10%

in absolute alcohol

100 ml.
20 gm.

40 ml.
10 ml.

Dissolve the ferrous sulphate by shaking vigorously v^ith the cold

distilled water (do not heat) then add the tannic acid and basic fuchsin.

GALLOCYANIN (EINARSON)
Gallocyanin

Chrome alum

Distilled water

Boil gently for five minutes; then cool;

original volume with distilled water.

0-3

gm.

10 gm.
200 ml.

and make up

filter,

to the

GIEMSA STAIN
Giemsa

stain

..

Pure methyl alcohol

Pure glycerine
.

..

..

..

3-8

gm.

250 ml.
250 ml.

Triturate with a mortar and pestle, adding the alcohol and glycerine
in small portions at a time during a period of fifteen minutes. Transfer
to a stoppered flask and allow to stand for twenty-four hours with
occasional shaking; then

filter.

GOODPASTURE'S ACID POLYCHROME METHYLENE BLUE

Methylene Blue
Potassium carbonate 0*25% aqueous
.

0-25 gm.
100 ml.

and add 0*75 ml.

Boil for half an hour, then cool under the tap

glacial acetic acid.

Shake well then boil gently for five to ten minutes cool and
the volume to 100 ml. with distilled water.
;

make up

GRAM'S IODINE
Iodine pure

Potass, iodide

Water

gm.

2 gm.
300 ml.

Dissolve the potass, iodide in about 10 ml. of the water; then add the
add the remainder of the water.
iodine, shake till dissolved
;

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

or:

Gram's Iodine powder (Michrome Brand)

Water
.

Hydrochloric acid, cone.

HAEMALUM

Potash alum

Mercuric chloride powder.

to boiling point, then allow to cool,

HAEMALUM

gm.

88 ml.
0.5 ml.

(HARRIS)

Haematoxylin 0-5% aqueous

Heat

and

100 ml.
5 gni.

0-25 gm.

filter.

(MAYER)

Haematein

Absolute alcohol
Potash alum 5
aqueous
.

. .

o*i

5 ml*

100 ml.

gm.

HAEMATOXYLIN (ALUM), AQUEOUS

Haematoxylin 0-25% aqueous

Ammonia or potash alum

Thymol
Note: The solution deteriorates
.

. .

after

..

..

400 ml.
20 gm.
i
gm.

two or three months.

HAEMATOXYLIN (DELAFIELD)
Haematoxylin 3*5% in absolute
Ammonia alum 6*25% aqueous
Glycerine

Mix thoroughly and

filter

alcohol

100 ml.

320 ml.
80 ml.

allow to ripen for at least three months;

then

before use.

HAEMATOXYLIN (EHRLICH)
Haematoxylin 2% in absolute
Potash alum
Glycerine

2*5% aqueous
.

Glacial acetic acid

Mix

alcohol

..

..

100 ml.
100 ml.
100 ml.
10 ml.

well; allow to ripen in a 500-ml. closed bottle for three months

or:
I
gm. Ehrlich haematoxylin powder (Michrome brand) in a
mixture of 4 ml. glacial acetic acid, 80 ml. 50% alcohol and 40 ml.
glycerine then filter. The solution requires no ripening and is ready

Dissolve

for

immediate use.

418

APPENDIX

HAEMATOXYLIN

(HARRIS)

Haematoxylin io% in absolute alcohol

Mercuric oxide
Potash alum 10% aqueous
Glacial acetic acid

5 ml.

0*25 gm.
100 ml.

nil.

Mix

the haematoxylin and alum solutions: raise to boiling point

then add the mercuric oxide and when the solution turns deep purple

turn off the heat then cool and add the acetic acid.
:

HAEMATOXYLIN (HEIDENHAIN)
No.

Iron alum

Distilled water

Do

Dissolve by shaking.

. .

gni.

100 ml.

not heat.

No. 2

10%

Haematoxylin

in

absolute

(ripened three to six months)

Distilled water

HAEMATOXYLIN
No.

alcohol

5 nil.

95

5 ml.

nnJ,

(IRON) (ANDERSON)

Haematoxylin

10%

Absolute alcohol
Distilled water

in absolute alcohol

aqueous

Calcium hypochloride

2%

45 nil.
50 ml.
3 ml.

No. 2
Iron alum

4%

aqueous

Sulphuric acid cone.

100 ml.
3 nil.

mix two volumes of

solution No. i with one volume of No.

few hours only, and should therefore be
prepared only when needed for immediate use.

For

use,

2; this mixture keeps for a

HAEMATOXYLIN KULTSCHITZKY
Haematoxylin
Acetic acid

10%

2%

alcoholic

aqueous

10 ml.

100 ml.

Dissolve the haematoxylin in the alcohol then shake thoroughly with

the acetic acid solution.

4^9

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

HAEMATOXYLIN PHOSPHOTUNGSTIC MALLORY

alcoholic

io%

Haematoxylin

Phosphotungstic acid
Distilled water

of

ml.

20 ml.
80 ml.

io%

Ripen by adding 4 drops Hydrogen peroxide (20 volumes) or

1 % Potassium permanganate.

5 ml.

HAEMATOXYLIN PHOSPHOMOLYBDIC MALLORY

I

Haematoxylin

..

aqueous

Chloral hydrate

Phosphomolybdic acid

10%

..

."

100 ml.
6 gm.
i ml.

HAEMATOXYLIN REGAUD
10 ml.

in absolute alcohol

10%

Haematoxylin

10 ml.

Glycerine
Distilled water

80 ml.

HAEMATOXYLIN (WEIGERT)
A.

Haematoxylin

10%

absolute alcohol

in

(ripened three to six months)

Absolute alcohol
.
.

B. Ferric chloride (hydrated)

Hydrochloric acid
Distilled water

30%

10 ml.

90 ml.

4 ml.

aqueous

95

nil*

99

nil.

ml.

IODINE GREEN, ACETIC

Iodine Green

Glacial acetic acid

aqueous
.

ml.

JENNER STAIN
Jenner stain powder
Pure methyl alcohol

0-5

gm.

100 ml.

Dissolve by heating in a flask, lightly plugged with cotton-wool, on

a water bath. Allow to cool ; then filter.

LEISHMAN STAIN
Leishman

stain

powder

0-15 gm.
100 ml.

Pure methyl alcohol

Dissolve by heating in a flask, lightly plugged with cotton-wool, on a

water bath. Allow to cool; then filter.

420

APPENDIX

LIGHT GREEN IN CELLOSOLVE

Cellosolve

Light Green

loo ml.

gm.
on a water bath for about half an hour, with occasional
shaking or stirring; then cool and filter.
Heat

in a flask

LITfflUM CARMINE

(Orth)

Lithium carbonate, saturated aqueous

Carmine
.

and

Boil for fifteen minutes then cool

loo ml.
5 giri*

filter.

or:

Lithium Carmine (Michrome Brand)

Distilled water

Heat to boiling point allow

to cool

then

2 gm.
50 ml.

filter.

LUGOL'S IODINE
Iodine pure
Potassium iodide
.

Water

, .

. .

100 ml.

gm.
gm.

MAGENTA -COTTON BLUE - LACTOPHENOL

Cotton blue

Magenta

4 ml-

2 ml.

50 ml.

lactophenol

aqueous

Lactophenol.

MAY-GRUNWALD STAIN
May-Grunwald

stain

powder

Pure methyl alcohol

Dissolve by heating in a

flask, lightly

water bath. Allow to cool, then

METHYL GREEN

Methyl green

..

...

Pyronin (B or G)
Phenol 0*5% aqueous solution
.

Glycerine
Absolute alcohol
.

plugged with cotton-wool, on

filter.

PYRONIN (PAPPENHEIM

0*25 gm.
100 ml.

..

..

UNNA)

0-15 gm.
0-25 gm.
80 ml.
20 ml.
2-5 ml.

Dissolve the powder in the phenol solution; then shake vigorously

with the glycerine and alcohol warm to dissolve then filter.
:

421

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

METHYL VIOLET (JENSEN)
Methyl

violet

6B

Distilled water

0*5

gm.

looml.

METHYLENE BLUE (BORAX)

(MANSON)
Methylene blue
Borax 5 % aqueous

2 gm.
100 ml.

Note: Methylene blue solutions must not be heated above 60 C.

METHYLENE BLUE (BORAX)

(UNNA)
Methylene blue
Borax i
aqueous

Note:

Do

gm.

100 ml.

not heat borax methylene blue solutions over 60 C.

METHYLENE BLUE (LOEFFLER)

Methylene blue
Absolute alcohol
Potass, hydroxide

..

0'0i% aqueous

3 gni.

30 ml.
100 ml.

METHYLENE BLUE, POLYCHROME (UNNA)

Heat

Methylene blue

Potass, carbonate

Absolute alcohol

20 ml.

Water

100 ml.

Cool and

to 60 C. for fifteen minutes.

gm.
gm.

filter.

or:

Polychrome methylene blue (Michrome)

Distilled water

Absolute alcohol

gm.

200 gm.

10 ml.

Dissolve by heating to 60 C and maintain at that temperature for

minutes. Cool and filter; then add the alcohol and mix

fifteen

thoroughly.

METHYLENE BLUE SULPHURIC (GABBET)

Distilled water

Sulphuric acid, cone.

Methylene blue

75 ^
25 ml.
2 gm.

Pour the sulphuric acid into the water carefully, in a

the methylene blue and shake or stir to dissolve.

422

flask,

then add

APPENDIX

MUCICARMINE (MAYER)
Stock solution:

Carmine

Aluminium

chloride,

anhydrous

gm.
gm.

0*5

Mix thoroughly by grinding to a homogeneous powder in a mortar

then place the mixture in a flask with 100 ml. of 50% alcohol and boil
for two and a half to five minutes on a water bath, shaking the flask
at intervals. Cool under the tap at once and filter when cold.
For use, dilute one volume of this solution with nine volumes distilled
;

water

or:

Shake 5 gm. of Mucicarmine powder (Michrome Brand) with 200 ml.

of 50% Alcohol; then heat to boiling point and continue boiling for

2i minutes. Cool;

filter.

MUCIHAEMATEIN
Mucihaematein powder (Michrome)

0'3 gm.
40 ml.
60 ml.

Glycerine
Distilled water
Dissolve by warming and shaking; then cool and

filter.

NEISSER'S STAIN
A. Methylene blue
Absolute alcohol

0'2 gm.
4 ml.

Glacial acetic acid

10 ml.

Distilled water

190 ml.

B. Bismark

brown

0*2

Distilled water

NEUTRAL RED

(JENSEN)

Neutral red

gm.

0*2 ml.

Distilled water

100 ml.

METHYLENE BLUE

Venetian soap
Distilled water
Dissolve then add
Methylene Blue
;

0*1

acetic acid

NISSL'S

gm.

100 ml.

0-175 gm.
80 ml.

2%

aqueous

20 ml.

Shake vigorously then store in a well-closed bottle for at least four

months, before use, shaking once or twice a week during this period.
2F

423

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

ORCEIN (UNNA

TANZER)

Orcein
70% Alcohol

100 ml.

Hydrochloric acid, cone.

gm.
ml.

PARACARMINE (MAYER)
Carminic acid

Aluminium

chloride, hydrated

Calcium chloride, anhydrous

Alcohol

PHLOXIN

70%

gm.
gm.
4 gm.
i

0-5

100 ml.

PROPYLENE GLYCOL

For eosinophile counts

Phloxin

in distilled water

0*1%

Propylene glycol
Stated to give

more

consistent results with

50 ml.
50 ml.

no destruction of eosin-

ophiles.
Reference:

Henneman, Wexler and Westenhaver

(1949), J. Lab.

and

Clin.

Med., 34, 1017-20.

PICRO ANILINE BLUE, ALCOHOLIC

Aniline blue alcohol soluble

70%

..

..

Aniline blue water soluble

Picric acid saturated, aqueous

alcohol.

Picric acid,

..

1%

in

..

70%

alcohol

gm.

50 ml.
50 ml.

PICRO ANILINE BLUE, AQUEOUS

o*i

gm.

100 ml.

PICROCARMINE
Picrocarmine powder (Michrome Brand)
Distilled water
.

Boil gently for five minutes

allow to cool ; then

gm.

50 ml.
filter

and make up

the volume to 50 ml. with distilled water.

PICRO INDIGO CARMINE

Picric acid, saturated aqueous

Indigo carmine

424

100 ml.
0-25 gm.

APPENDIX

RANDOLPH'S BLOOD COUNTING FLUID

Solution

Methylene blue
Propylene glycol
Solution

o-i

loo ml.

gm.

Eosin, bluish

o*i

Distilled water

loo ml.

gm.

Mix equal volumes of the two solutions and use as a diluting fluid
in the white cell pipette. The mixture of the blood and the diluting
fluid should remain in the pipette for fifteen minutes before filling the
counting chamber.
Reference: Randolph, T. G. (1943) Proc. S. Exp. Biol. Med., 52, p. 20-2.

RED CELL COUNTING FLUID

Sodium citrate 3% aqueous

(O. T.

George)

Formalin (Formaldehyde 40%)

Eosin, bluish

100 ml.
i

This solution stains the erythrocytes and causes the

in a single focal plane, thereby facilitating enumeration.
Reference: (1954),

SCARLET

R,

J'.

ml.

o-6

gm.

cells to lay flat

Lab. and Clin. Med., 40, 479-83.

ALKALINE (HERZHEIMER)

Absolute alcohol
Distilled water

Sodium hydroxide 10% aqueous

Scarlet

10 ml.

35 i^il.
5 ml.

gm.

Place in a flask and plug the neck lightly with cotton wool. Heat on
a hot water bath until a considerable portion of the dye has gone into
solution; then allow to cool before filtering.

SCHORR'S STAIN
A. Harris haematoxylin.
B. Biebrich scarlet

Orange

Distilled water

Glacial acetic acid

gm.

0-4 gm.
99 ml.
i

ml.

Heat the water: add the stains; stir untill dissolved allow to cool;
then add acetic acid shake well and filter.
;

C. phosphotungstic acid

10%
10%

phosphomolybdic acid
Distilled water

425

25 ml.
25 ml.
100 ml.

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

D. Fast green

FCF

acetic acid

aqueous 5

5 ml.

32 ml.
64 ml.

Distilled water

SCHORR'S STAIN
(Single solution)
Biebrich scarlet, aqueous

Orange

0*5 gi"-

Fast green FCF

Phosphotungstic acid

Phosphomolybdic acid
Glacial acetic acid

Alcohol

50%

Dissolve by warming

then allow to cool and

filter.

or:

Schorr's Stain powder (Michrome)

..
..
Alcohol 50%
..
Glacial acetic acid

..
.

Dissolve the stain by warming with the alcohol

add the acetic acid, shake well and filter.

gm.

55 ml.
0*5 ml.
allow to cool

then

APPENDIX

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

Methyl

salicylate

1-537
660

Monobromnaphthalene-alpha

Olive

1*473

oil

Optoil

1-515

Origanum

oil

1-458

Paraffin, liquid

1-471

Pyridine

1-509

S.Q.D. balsam

1-515

Turpentine

Water

oil

1*473

1-333

(distilled)

Xylol

1-492

SATURATED SOLUTIONS OF REAGENTS

To

prepare 100 ml. dissolve:

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

SPECIFIC GRAVITIES
Acetic acid glacial

(at 4 C.)

APPENDIX
Approximate
percentage solubilities
at 15 C.

Stain

..

Mol. Wt.

In water

In alcohol

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

INDEX
Absolute Alcohol Fixative, 5
Acetic Acid, 4
Acetic Aniline Blue (Masson), 65,152,
156
Aceto Carmine (Schneider), 412
Acetone, 5, 224, 335
Acetyl Yellow, 430

Allen's Fixative, 6, 7

Achromatic Spindles, 155

Acid Alcohol, 55, 65, 354, 355
Acid Alizarin Blue, 104
Acid Brown R., 430
Acid Fast Bacteria, 355
Acid Fuchsin, 61, 82, 124, 134, 138,
247, 261, 290, 292, 293, 354
Acid Fuchsin (Mallory) 412
Acid Fuchsin-Aurantia, 247, 289
Acid Green, 68, 430
Acid Haemalum, Mayer, 209
Acid Rubin, 248
Acid Violet, 430
Acid Yellow S, 430
Acidine Yellow H, 317, 318, 321, 430
Acridine Orange, 317, 321, 323, 430

Amidonaphthol Red G, 431

Acriflavine, 317, 318, 321

Aniline Blue, 59, 61, 64, 92, 125, 156,

199, 214, 257, 273, 413
Aniline Blue-Fast Green-Orange G,

Alpha cells, 67, 69

Altmann's Acid Fuchsin, 412
Altmann's Fluid, 293, 411

Alum Carmine, 59, 413

Alum Haematoxylin, 81, 418
Alzheimer, 138

Actinomyces, 120, 172

Adenohypophysis of rat and mouse, 50

Adenosine, 185
Adenylic Acid, 185
Adonitol, 347
Aesculin, 317

Amylodextrin, 232
Amyloid, 5, 64, 91, 154, 158, 159
Anagallis, 257
Angiosperm, 257, 273
Aniline Acid Fuchsin, 293
Aniline Alcohol, 65, 68
Aniline Blue Acetic (Masson), 413

no,

Orange G,

149, 150, 152,

59, 64-8,

153,

175,

414
Aniline Blue - Orcein, 180
Aniline Blue Safranin, 273, 274
191,

Alcian Blue, 47, 346

Alcohol, substitutes for, 34-8
Aldehyde Fuchsin (Gomori), 48, 50
Aldehydes, 230
Aldridge, W. N., 27
Aleuone, 230
Algae, 43, 229, 260
Algin, 185

430

382

Amnion's Horn, 186

Amoeba, 212, 374
Amphibian eggs, 388
Amygdalin, 231
Amygdalus seeds, 231

92,

Albumen, egg, 185

Alizarin Cyanin, 431

Alizarin Red S, 53-8, 291, 431
Alizarin Viridin, 104
Alkali Blue 5B, 431
Alkali Blue 6B, 431

413

Oxalate Crystal Violet,

191
Aniline Blue

Albert's Stain, 345, 412

Alizarin,

Ammonia Carmine,

Ammonium

Aniline

Chloride

(Aniline

Hydro-

chloride) 249, 413, 414

Aniline Crystal Violet, 62, 63, 170

Aniline Fuchsin - Iodine Green, 292

Aniline Gentian Violet, 77, 116, 120,
348, 360,368,

Aniline
Aniline
Aniline
Aniline
Aniline
Aniline
Aniline

371,414

Hydrochloride, 249, 428

oil, 62, 63, 78, 79
poisoning, 358

Safranin, 176, 294

Sulphate, 414, 428
water, 414
Xylol, 62, 63, 78, 103, 116,
147, 360, 414

437

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

Anisidine (ortho) tetrazotised, 333

Basic, Fuchsin, 76, 77, 70, 112, 161,

Anopheline midgut, 372

Anther smears, 285
Anthocyanin, 232

162, 193, 221, 295, 249, 349, 361,

384
Basic Fuchsin

Picro Indigo Carmine,

295

Apathy gum, 23, 39

Aquamount, 39

Bauer - Feulgen Stain, 70

Beams, H. W., vii
Becker, E. R., 378

Arabinose, 185, 346

Arbutin, 233
Archengonia, 273
Archesporial cells, 261

Beechwood
Behre,

J.

creosote, 27, 58, 264, 265

A., 390

Archoplasm, 292

Belling's Iron Aceto Carmine, 304

Argentaffin granules, 6, 102, 157, 216

Armitage, F. D., 252

Benda's
Benda's
Benda's
Benda's

C, 149
Ashbel, R., 334
Asparagine, 234
Astrocytes, 107
Astrocytes, migrating, 139
Atomic weights, 409
Aron,

method for mitochondria, 291

method for nervous tissue, 57

Bensley, R. R., 173

Bensley's Stain, 67, 173
Benzene, 30, 426, 430
Benzidine, 75, 246, 390

Benzoazurin, 431
Benzopurpurin, 431
Benzyl Alcohol, 140, 253
Berberine Sulphate, 315, 320
Bergamot Oil, 427
27
Bergel, F.
Berlezes fluid, 39
Berlin Blue reaction, 232
Best's Carmine, 73, 414
Beta cells, 50, 52, 67, 69

Auramine, O, 317, 324, 325, 431

Aurantia, 247, 248
Auto-fluorescence, 315
Axis cylinders, 105, 154
Azo Blue, 431
Azofuchsin, 168

Axons, 59

Azan

Crystal Violet, 291

Fluid, 411

Stain, 65

Azocarmine, 65, 66, 68, 430

Azo Eosin, 431

Azo Phloxin, no, 431

Biebrich Scarlet, 71, 100, 395

Biondi Ehrlich Heidenhain, 73

Azur, A, 393
Azur, B, 187
Azur, L, 349

Birds, C.N.S. of, 147

Bismarck Brown, 72, 259

Azur 2 Eosin, 99

Blastocytes, 134

Bleu de Nuit, 430

Babe's Safranin, 294

Blood, 91, 99, 130, 132, 133, 135, 137,

178, 194, 196, 226, 352, 359, 361,
365, 368, 377, 389, 392, 394, 405,

Bacteria, 3, 77, 108, 159, 170, 172, 178,

247, 322, 361, 368

Bacteria, autochthonic, 322
Bacteria and mitochondria, differentiation of,

247

Bacteria in milk, 350

Bacteria, polar bodies, 385

425
Blood counting fluids, 425
Blood films, thick, 362
Blood-forming organs, 11, 132, 133
Blood, supra-vital staining of, 351, 376

Bacteria, soil, 322

Bacterial capsules, 346, 357, 384
Bacterial cell walls, 386

Blood-vessels, 76, 97, 106, 108

Bolles - Lee, 247

Bacterial polysaccharides, 346*

Bacterial spores, 359, 379, 398

Bone Canaliculi, 85
Bone-marrow, 131

Badertscher, J. A., 353

Bailey, H. D., 371
Baker, J. R., 204, 207
Barrera, Elizabeth, 144
Basal bodies, 181, 360

Bonney's Triple Stain, 160

Borax Carmine (Grenacher), 250, 415
Borax Carmine (Mayer), 415
Borax Ferricyanide mixtur#(Weigert),
298

Bone,

43S

17, 47, 53, 56, 151, I54,

178

INDEX
Bordeaux B and R, 431
Bordeaux Red, 372

Carbol Methylene Blue, 416

Carbol Methyl Green - Pyronin, 138,
339
Carbol Thionin, 85, 283, 416
Carbol Toluidine Blue, 416
Carbonyl activity, sites of, 333
Carcinoma, breast, 89
Cardiac Conductive Tissue, in
Carleton, H. M., 32

Borna, 62
Borrel's Blue, 80, 355, 415

Bouin-Dubosq, 7
Bouin Fixative, 6, 52
Bouin Fixative Alcoholic, 7
Bouin, sea water, 140
Bowie's Stain, 71, 100
Brachet, J., 336, 337, 339
Brain, 26, 60, 119, 145, 152, 186, 223
Brain, capillaries, 75

Brain tissue, abnormal, 60

Breast carcinoma, 89
Breed's Stain, 350

24, 168, 205, 207, 208,

416

Carmine, 59, 63, 73, 86, 96

Carmine, Alum, 413
Carmine ammonia, 413
Carmine, Lithium, 96
Carmoisin, 431
Carnoy's Fluid, 7
Carnoy - Le Brun, 8
Carotin, 235

Breinl Stain, 296

Brilliant Cresyl Blue, 351
Brilliant

Carmalum,

Green, 431

Brilliant purpurin, 168

Cartilage, 53, 72, 86, 99, 154, 159, 178

Briseno-Castrejon, B, 70
Brilliant Yellow, 355
Buffer tablets, 105, 367, 377, 405

Casarett, G.

Bulk staining, 250

Burdon, K. L., 398

Casparian Strips, 263

Castanedas Stain, 356

Butyl Alcohol, tertiary, 34, 262, 264

Cajal's

Gold Chloride Sublimate, 107

Cajal's Reducer, 202

Cajeput Oil for clearing sections in

damp

P.,

390

Cat, C.N.S.

147

of,

Cedarwood oil, 30, 427

Cedronol Immersion Oil, 427
Celestin Blue, 88, 89, 181

Celloidin Pyridin, 22
Celloidin sections, 19
Cellosolve, 35, 255, 262, 264
Cellosolve in methyl salicylate, 55

Callose, 235, 268

72

Cambel and Sgouris Bowie

Castrejon-Briseno, B., 70
Casts, urinary,

Cellobiose, 185, 346

Celloidin embedding, 17
Celloidin-Paraffin Wax, 20

climates, 35, 38

Calcium, 53, 127, 234

Calcium deposits, 53, 192
Calcium Oxalate, 234
Calcium Salicylate, 127
Calcium soaps, 399

Cambel,

W.,348

Casein, 185
Cason, Jane, E., 154

Cells, alpha, 52, 67,

Stain, 71

Cambial cell walls, 280

Canada balsam dried, 41, 427
Canada balsam natural, 41, 427
Canada balsam, in xylol etc., 41, 427
Canary pox, 404
Cannon, H. G., 130, 131, 155, 269
Cancer, 334

69

Cells, archesporial, 261

Cells, beta, 50, 52, 67,

387
Cells, delta, 52, 67
Cells, epithelial, 69, 90, 181, 374, 385
Cells, ganglion,

no

Cells, malignant, 213

Carbol Crystal Violet, 352, 355, 359,

362
Carbol Fuchsin, 79, 80, 81, 82, 83, 85,

Cells, Nitella, 311

34%

353, 368, 384, 415

Carbol Gentian Violet, 108, 415

2G

402

Cells, counting of, 70, 181, 350, 351,

Capsules, bacterial, 357

Carbol Aniline Fuchsin, 79

109,

69

Cells, cornified, 374, 389, 401,

Cells, nerve, 93, 106, 107

Cells, parietal, 100
Cells, pepsin-forming, 100

Cells, polychromatophilic, 361

Cells, pyramidal, 186

439

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

bodies of, 360

Cells, renal epithelial, 391

Cilia, basal

Cells, urinary, epithelial, 391

Citric acid, 162, 204

Class work, stains

Cellulose, 185, 235, 236, 255

Cellulose cell wall, 257, 273,

282
Cellulose tissue, 268, 279
Cell walls, 255, 274, 280, 295
Central nervous system, 17, 23, 107,
134, 145, 217

Centrioles, 155, 181

Centrosomes, 261, 292

Cereal seeds, 246

Cerebellum, 161
Cerebral cortex, 115, 147
Cerebrospinal fluid, 399
Cereus, B., 386
Cesares - Gill Flagella Stain, 363

432
Connective
147

Chlorides, 237
Chloroplasts, 185, 256, 257, 276, 278,
279, 310

Chloroform, 8, 30, 59
Chlorophosphin, 431
Chlorophyll, 238
Chlor Zinc Iodine (Schulze solution),
282
Cholesterol, 198, 282
Choline, 341

Chondrogenesis, 56
'Chromatin, 66, 73, 91, 100, 122, 124,
160, 172, 194, 207, 226
acid fixative, 411

Chromoneata, 299
Chromophobes, 70, 151, 181
261, 273, 274

276

Chromotrope 2R,

51, 88, 431

Chrysoidin 3R, 431

Chrysoidin Y, 431

59, 65, 97,

walls,

102,

172

168,

280

91,

92,

93,

318,

tissue, 47, 60, 73,

386,

10, 122,

123, 126, 127, 128, 156, 160, 165,

169, 174, 176, 180, 194, 224
Contractile elements, striated muscle,

6, 91,

157,

125,

Coniferophyta, 259
Congo Corinth, 432

Chlorazol Azurin, 25 1 43 1
Chloral hydrate, 239
Chlorantine Fast Red, 47
Chlorazol Black, 90, 252, 269, 431
Chlorazol Paper Brown, 253

Chromosomes,

fibres, 48,

123,

Collenchyma

Chitin, 13, 39, 91, 140, 185, 235, 236

Chromic

Collagen

Congo Red,

Chrome Alum, 428

155,

Colloid, 183

Chiffelle,

of,

130,

127, 128, 143, 175, 201, 218

Champy's Fluid, 8, 289

Chance, H. L., 387
Chesterman, W., 26
T. L., 209
Chimpanzee, C.N.S.
China Blue, 431
China Green, 431
China Yellow, 431

for,

251
Clearing in wet climates, 35
Clearmount, 41
Clove oil, 68, 71
Coccidia, 375
Coeruline, 431
Coli organisms, 361, 363, 386
Collagen, 59, 66, 88, 97, 105, 109, 115,

274,

154
Conversion tables, 410
Cooper, D. C, 306
Copper chrome haematoxylin (Bensley), 297
Coriphosphine, 317
Cornified cells, 374, 389, 401, 402
Corollin, aqueous, 432
CoroUin, alcoholic, 432
Cortex, 254
Cortical architecture, 119

Cotton Blue, 432

Cotton Blue - Lactophenal, 254, 416
Cotton Blue - Magenta - Lactophenol,
416
Cotton Red, 299
Cottrell, Helen, J., 283

Cowdry, E. V., 67, 327, 333

Cowdry's reaction, 389
Creso Fuchsin, 59
Cresyl Fast Violet (Cresylecht violet)
93, 144
Cristalite

Mountant, 42, 427

Crocein Scarlet, 432

Crough, H. B., 378
Cruciferae, 257
Crustaceans, 140
Crystal Violet, 63, 170, 195, 221, 262,
264, 357, 358, 381, 432

440

INDEX
Crystal Violet, Sandiford, 381
Crystal Violet - Safranin, 195
Curtis stain, 123

Ectromelia, 404
Eggs, amphibian, 388
Ehrlich's triacid stain, 359

Cutin, 249
Cutinised cell walls, 256, 273, 282
Cutinised structures, 261

Elastin,

Cyanin, 255
Cyanophils, 181
Cyanosine, 432

Elastin stain (Sheridan), 97

Elastin stain (Weigert), 96, 97
Elastin trichrome stain, 97

382
Cytology, 124, 260, 289-311, 377, 392
Cytons, 93, 106
Cytoplasmic structures, 118, 123
Cyloplasmic vacoules, 53

Eleidin, 93, 189

Elementary bodies,
402, 404
Ellinger, P., 327

Fano, 202
Dahlia, 95, 358
Dahlia tubers, 234, 239
Damar Xylol, 427
Daucus Carota, 237
Dawson, J. R., 180

Enzymes,

liams's Modif.), 54
living cells, differentiation

Decalcifying solution, 85
De Kotchinsky Cement, 333
Desoxyribonucleic acid, 185, 336
Dextrin, 346

Durazal Yellow, 432

Dysentery organisms, 361

346

Epithelium, 59, 105, 179, 215

Ericacae, 233
Erie Garnet, 432

Eriochrome Violet BA, 432

Erio Green, 432
Erythrocytes, 66, 70, 73, 77, 99, 105,
106, 126, 129, 131,

214

Erythrosin, 116, 194, 255

Erythrosin - Lactophenol, 256
Ethylene Glycol, 23, 37, 197, 281, 285
Ethylene Glycol Monoethyl Ether, 35

Ethyl Violet, 71, 100

Evans, G. H. 327

95

Dorner's Negative Stain, 359, 416

Double embedding, 20

Dubosq - Brazil, 7
Dunn, R. C, 137

5,

Eosin, 89, 109, 112, 220, 230, 341,

347, 360, 374, 375, 379, 432
Eosin, Ethyl, 432
Eosin, Methylene Blue, 361, 397
Eosin, Scarlet, 307, 432
Eosinophil counts, 424
Eosin, substitute for, 88, iii, 124
Epidermis, 90, 178, 254, 285

Epithelial cells, 90, 374, 385

Epithelial fibres, 62, 220
Epithelial oi pars intermedia, 59

Dianyl Blue G, 274

Dianyl Blue 2R, 274, 432
Dibutyl Phthalate, 27
Dietrich fixative, 411
Dihydroxyphenylalanin, 3 14-, 95
Dimethyl-p-phenylene-diamine, 167
Dioxane, 36, 427
Diphenylamine, 230, 242
Diphtheria organisms, 345
Doetschman's gum chloral, 39
Dolnick, E, H., 122, 176

307

of,

Epidermis walls, 252

Epidermophytic infection, 249

322

D.P.X. mountant, 42
Drosphila, salivary chromosomes

61, 356, 383,

212

Dawson's Method for bone, 53

Dawson's Method for bone (Wil-

Dopa reagent,

6,

Embryonic tissue, 53, 72

Embryos, 53, 55, 90, 273
Entamoeba, 372
Entamoeba, differential staining

Da

of,

104,

213

Cytological stains, 124, 289-311, 377,

Dead and

16, 49, 64, 90, 96, 97,

io6, IIS, 155, 174, 176, 178, 179,

of,

Faecal material, 374

Faeces, fat in, 391, 399
Farrant's Medium, 39, 427
Fast Acid Violet, 432
Fast Green - Clove Oil - Cellosolve,

262

441

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

Fast Green F.C.F., no, 124, 191, 195,
199, 214, 216, 262, 264, 269, 396,

Fluorochromes, 315-327
Fluorochromy, 315-327
Fluoroil, 325

432
Fast Green

FCF

in cellosolve, 262,

416
Fast Green - Malachite Green, 264
Fast light Yellow, 432
Fast Yellow extra, 432
Fat, 168, 179, 198, 209, 210, 281, 318
Fat in bacteria, 397
Fat, in animal tissues, 23
Fat, in plant tissues, 23, 281,

Fontana Stain, 102, 363

Fontana - Tribondeau Stain, 363
Formalin (ten per cent) fixative, 10
Formic Acid, 238
Formaldehyde-saline, 204
Formalum, 205
Formol - Alcohol, 1 1

Formol

Saline, 132, 225

Freeze - Drying, 331
Friedlander Type B Polysaccharides,

282

Fat preparations, 23
Fat, necrosis, 126
Fat, neutral, 24, 126, 171, 207

185

Fat, stains, 23, 126, 179, 204, 207, 208,

209, 210, 281

Fatty acids, 126, 171, 198, 281, 282
Fatty degeneration, 209, 210, 391

Fern

leaf,

Frozen sections, 10, 23, 179

Frozen sections, avoidance of shrinking of, 208
139,

134,

151

197
Fibres, elastin, 49, 50, 92, 97, i55, i74,
175, 176, 178, 179, 195, 196, 201,

218
Fibres, muscle, 90
Fibres, neuroglia, 107, 140, 216
Fibres, reticulum, 64, 102

Fibrin network, 352

Fibroblasts, 196
Fibroglia, 64, 154

Gabbet's Methylene Blue, Sulphuric,

422
Galactose, 185
Gallamine Blue, 432
Gallic acid, 89

Gametes, motile, 273

Ganglion cells, no, 131, 223
Gastric glands, 71
Gastric mucin, 77, 100, 185
Gastric mucosa, 49, 71, 100

Gatenby, Bronte J., vii

Gelatine embedding, 24, 205

Field's Stain, 362

C, 70
J.

Fungal hyphae, 254

Fungi, 236, 260

Gallocyanin, 104, 190, 417, 432

Fibres, spindle, 297, 299, 310

Fibrin, 5, 11, 17, 63, 93, 116, 131, 172

Fitzgerald, P.

Granules,

Fuchsinophile

252

Ferric Chloride, 231, 233, 266

Feulgen Fuchsin, 70, 100, 183, 300
Feulgen reaction, 70, 339
Fibres, collagen, 64, 102, 124, 125,

Finerty, J.
Fish, 54

Foley's Protargol Gallocyanin, 190

Gelatin sections, 25
Gentian Blue, 432

336

Fixatives, formulae of, 411

Flagella Stains, 363, 384, 417

Flemming's Fluids, 9

Flemming's Trichrome

Stain,

195,

Gentian Violet, 77, 116, 300, 360,

364, 385, 432
Gentian Violet - Methylene Blue, 385
Gentian Violet (Noland), 364
George, O. T., 425

Giemsa

309

Fluorchrome (Weigert), 115, 223

Fluorescein, 432
Fluorescence, auto, 315, 317
Fluorescence dyes, 315-327

Stain, 14, 15, 105, 106, 133,

177, 226, 365, 368, 382, 406,

417

Gifford, E. M., 268

Gilson's Fluid, 411

Glia fibres, 59

Fluorescence interfering, 325

Fluorescence microscopy, 42, 315-327
Fluorescence, secondary, 315
Fluormount, 43, 317

Glomerular

basement

126
Gluconic Acid, 185
Glucosamine, 185

442

membranes,

INDEX
Gutstein, M., 384, 403

Glucose, 185, 346

Glucose - 1 - Phosphate, 185
Glucuronic Acid, 185
Gluthathione, 239
Glycerine, 40, 185, 229
Glycerine Albumen, 427
Glycerine Ether (Unna), 164
Glycerine Jelly, 40, 229, 270
a-Glycerophosphate, 185
Glychrogel Mountant, 40
Glycogen, 5, 7, 7, 73, Qi, I43, 185,
375

Gymnosperm
Gymnosperm

Haemalum
Haemalum

no, 112, 122, 223, 259,

260, 282, 303, 304, 334

Haematoxylin, Anderson, 419

Haematoxylin, artificial ripening

420
Haematoxylin, Delafield,

no,

93, 98,

112, 113, 114, 118, 120,

360,418
Haematoxylin-eosin substitute, 88
Haematoxylin, Harris, no, 149, 150,
373, 419
Haematoxylin, Heidenhain, 117, 123,
124, 127, 128, 257, 260, 419
Haematoxylin, Kultschitzky, 115, 119,
419
Haematoxylin, Lead, 134
Haematoxylin,
Phosphomolybdic
(Mallory), 420
Haematoxylin, Phosphotungstic (Mallory), 121, 154,420
Haematoxylin, Regaud, 156, 215,302,
420
Haematoxylin, substitute for, 88

48,' 50, 67, 214, 333

Gonadtrophins, 181
Gonococci, 370, 381, 382, 385
Goodpasture's polychrome Methylene Blue, 417
Goodpasture's Stain, 147
Gorilla, C.N.S. of, 147
Gossypimine, 432
Grahanj's Stain for oxidase granules,
394
Gram negative organisms, 78, 109,
147, 170, 369-71

positive organisms, 63, 78, 109,

116, 147, 170, 369-71

62, 78, 108, i68, 170,

Granules, Argentaffin, 6, 102

Granules, mast cell, 95
Granules, of Russel, 194
Gray, P. H. H., 381
Guard cells, 263
Gude, W. D., 151
Guinea Green B, 432
Guinea pig, C.N.S. of, 147
Gum Arabic, 185
Gum Chloral, 39
Gum Tragacanth, 185

no,

123, 127, 149, 185, 194, 198, 214,

Gomori, G.,

Gram's Iodine (formula), 417, 418

Gram's Stain, 368-71, 381

68, 71, 91,

112, 114, 116, 194, 198, 214,

241, 255, 418

Haematoxylin, Ehrlich, 74, 83, 91,

Sublimate (Cajal),

232, 236, 256 368-71

of,

154,

Golgi bodies, 207

Golgi method, rapid, 108
Golgi vacuoles, 207

Gram's Iodine,

(Mayer), 109, 418

91, 93, 99,

107
Gold, detection of in experimental
animals, 203
Goldners modification of Masson, 1 1 1
Golgi apparatus, 70, 202, 216

Gram

ovules, 273

(Harris), 418
Haematoxylin, 68, 71, 74, 81, 82, 83,

Gobletcells, 72, 81
Godwin, J. T., 336
Gold Chloride, 60, 107, 191, 200, 202

Gold Chloride

material, 265

Haematoxylin, Weigert, 123, 125, 126,

165, 168, 169, 224, 372, 420
Haemofuscin, 5, 17, 112
Haemoglobin, 127, 132, 136, 377
Haemopoietic tissues, 99

Haemorrhage, old and fresh

entiation of, 398

Haemosiderin, 112, 193

Hair Follicles, 178

Halmi, N.
Halper,

Hanburg,

Hard

S., 53

M.

H.,

in

L., 106

tissues, 17

Harris haemalum, 48

Heidenhain Azan Stain, 65

Heidenhain - Mallory, 153
Heinz bodies, 358
Helly's Fluid, 11,412
Henle's Loop, 196

443

differ-

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

Henneman, 424
Herbarium specimens, 252
Herman's Fluid, 12
Herpes simplex, 404

Isamine Blue, 433

Islets of Langerhans, 66
Isopropyl alcohol, 92
Isotopes, radio, 334

Herring bodies, 52
Hetherington, D. C, 393
Herzheimer acetone-alcohol Scarlet R,

Isaac, J. P., 149

Janus Black, 433
Janus Green, 375
Janus Red, 433
Janus Green - Neutral Red, 376
Jenner Stain, 132, 133, 377, 382, 420
Jensen's Stain, 369
Johansen, D. A., 229, 263, 266, 290,
304
Johansen's Quadruple Stain, 262

198

Herzheimer alkaline Scarlet R, 425

Herzberg, K.,405
Hickson's Purple, 130
Histochemical methods, 48,
95,

193,

100,

102,

136,

167,

230-246, 284,

70,

181,

75,

183,

331-342, 346,

389, 394, 401

Hitchcock and Erhlich Mixture, 131

Hoffmesster, E. R., 286

Hofmann's Blue, 432

Hofmann's Violet, 433
Hotchkiss, R. D., 181, 183, 185
Humus slime (soil), 322
Hyalin, 5, 85, 154, 159, 186

Hyalin casts, 391

Hyaluronic acid, 185
Hydra, 388
Hydrazine Yellow, 433
Hydrocellulose, 236
Hydrocyanic acid, 232
Hydrophytes, 271
Hydroquinone, 190, 203, 217, 220
Hyphae, fungal, 254, 279
Hydrox - 2 - naphthoic acid, 333
3
Hyphae, in wood, 254
Hypophysis, 59, 68, 181

Inclusion Bodies, 188

Indigo Carmine, 11, 295, 373, 433

Indulin, alcoholic, 433
Indulin, aqueous, 433
Influenza bacilli, 147, 361
Inositol, 185, 346
Intestinal

Amoeba, 374

Intestine, 14, 72, 90

Intravital staining (fluorescence micr.),

319
Inulin, 239, 346

Iodine, 28, 62, 68, 73, 78, 83, 240,

335
Iodine Green, 261, 292, 420, 433
Iron, 127, 240

Iron Aceto Carmine (Belling), 304

Iron haematoxylin (Anderson), 419

Johansen's Quintuple Stain, 264

Jones, Russell S., 201

Kahle's Fluid, 411

Kalter's Quad. Stain, 195
Kalter, S. S., 195

Karyosomes, 115, 158, 159

Keratin, 115, 122, 161, 172, 175, 178,
189, 216, 388, 396, 401, 402
Keratohyalin, 93, 113
Kernechtrot, 404

Kidney,

7,

90

Kimborough, C.
Kirkpatrick,

J.,

E.,

398

42

Kleinenberg's Fluid, 12
Kluver, H., 144
Koehring, Vera, 311
Kopeloff and Beerman Stain, 370
Kull's stain, 248
Kultschitzky Haematoxylin, 116, 143
Kultschitzky-Pal, 115
Kurloff bodies, 189

Lacmoid, 235, 266

Lacmoid-Martius Yellow, 268
Lactophcnol, 91, 253, 254, 427
Lactophenol - Cotton Blue, 416
Lactophenol - Cotton Blue - Magenta,
416
Landing, B. H., 341
Langerhans, islets of, 66
Lavdowsky's fixative, no
Laybourn's Stain, 345
Leach, E. H., 26, 32, 124
Lead Haematoxylin, 134
Leaves, Prunus Laurocerasus, 231
Lecithin, 119, 241
Leishmania, 136

444

INDEX
Leishman

Stain,

14,

135, 225,

Luxol fast Blue, 144

L.V.N. 26
Lymphatic cells, 131
Lymphocytes, 91, 134, 226

351,

377, 420
Lemon Pectin, 185

Lempert, H., 325, 327

Lendrum, A. C,

42, 88, 89, 181, 188

Lymphoid

tissue, 193
Macchiavello's stain, 380
MacCallum's Stain, 147
MacConaill, M. A., 134
Magdala Red, 269, 433
Magenta Cotton Blue - Lactophenal,
421
Magnesium, identification of, 245,

Leprosy, 80, 82, 83, 355, 361

Lesions, Skin, 89
Leucocytes, 99, 324, 362, 385, 397,

400
Leucocytes, differentiation of types,
400, 405
Lenco Patent Blue, 136
Levaditi's Stain for Trep. Pallidum,
138
Levy, J. F. L,, 272
Lewis, Ann, L., 201

284

Magenta - Lactophenol, 421

McKinney, R. E., 346
Malachite Green, 264, 345, 380, 381,
433
Malachite Green - Pyronin - Crystal
Violet, 381

Lewitsky's Fluid, 13
Lichens, 263
Light Green, 89, 98, loi, 138, 214,
433
Light Green - Cellosolve, 421
Light Green - Clove Oil, 278
Light Green - Safranin - Cellosolve,
275
Lignified Tissues, 249, 252, 254, 255,

Malaria, 105, 135, 225, 362, 365, 368,

377, 405
Malic acid, 185

Malignant

cells, 213
Mallory, F. B., 123, 140
Mallory's Aniline Blue - Orange G.,
59, 64-8, 92, no, 149, 150, 152,

256, 261, 272, 284

Lignified Walls, 249, 257, 273, 282
Lignin, test for, 271
Lignin Pink, 140, 269, 433

175,414
Mallory's Connective Tissue
17, 153
Mallory - Heidenhain, 153

Lignin, stains specific for, 269

Liljegren, E. J. L., 359
Lillie, R. D., 169, 170

Mallory

Mannitol, 185, 346

Mannose, 346
Mann's Stain, 157

Lithium Carmine, 63, 96, 421

Lithium Silver (Laidlaw), 141

Marchi Fluid,

Liver, 185
cells, differentiation

Loeffler's flagella

mordant, 417
Lorrain-Smith Dietrich Stain, 143
Low Viscosity Nitrocellulose (L.V.N.)
26

Lugol Iodine,

68, 73, 83,

116,

360, 369, 379

Lugol's Iodine Formula, 421

Lung, 121, 160, 189
Lung, guinea-pig, 189
Lupins, 234

Heidenhain, Rapid Tech-

nique, 153
Mall's Fluid, 53
Maltose, 185, 232
Manchester Yellow, 433

Lipases, 5
Lipids, 37, 119, 204, 208, 341
Lipines, 5, 118
Lipoid granules in leucocytes, 397
Lipoids, 139, 143, 397
Lison, L., 48

Living and dead

of, 322

Stain,

143,

13

Margolena, L. A. 122, 176

Marine Bouin, 140
Marine invertebrates, 140
Marshall Red, 155
Martius Yellow - Lacmoid, 268
Martius Yellow, 268, 433

Masson, in, 123

Masson's Acetic Aniline Blue, 413
Masson's Ponceau - Acid Fuchsin, 98
Masson Trichrome Stain, 15, 156

Mast cells, 49, 134, 163, 164, 172, 178

Mast cell granules, 47, 95, 163
Maximow's Stain, 99
May - Grunwald Stain, 368, 382, 421

445

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

Mayer's Muci Carmine, i66

Medusa

Milk, Bacteria in, 350, 387

Milovidov, 249
Mineral substances in cells, 203, 234,

of Obelia, 140

Medullary Sheaths, 140

Meedol balsam, 42
Melanin, 112, 178

237, 240, 242, 243, 245, 246, 248,

331, 334

Mitochondria, 8, 15, 69, 247, 248, 289

Mitochondria and Bacteria, differen-

Melanoblasts, 95
Meldola Blue, 433
Membranes, basement, 225
Meningococcus, 361, 381
Menschik, Z,, 341

tiation of,

Mercuric precipitates, removal of, 28

Metachromatic granules of diphtheria,
393

Metachrome Yellow 2RD, 433

Metanil Yellow, 160, 165, 334, 433
Metcalf, R. L., 327
Methyl Benzoate, 20
Methyl Benzoate Celloidin, 20

Methyl Blue, 390, 433

Methyl Blue - Eosin (Mann), 157
Methyl Green, 72, 85, 158, 270, 337,
433

Methyl Green
Methyl Green

Acid Fuchsin, 306

Acid Fuchsin - Ery-

162, 185, 186, 193, 317, 324, 350,

379, 384, 385, 394, 433

Methylene Blue Alkaline Loeffler, 79,

Blue Borax (Mason), 422

Blue Borax (Unna), 422
Blue, Nissl, 423
Blue Polychrome, 53, 163,
164, 174, 422
Methylene Blue - Rhodamine B, 193
Methylene Blue, Sulphuric, Gabbet,
422
Methylene Green, 433

Micro-incineration, 331

147

Tissues, 87

Moore's Elastin Stain, 221

Mounting media, 39-44
Mucicarmine (Mayer), 166, 423
Mucicarmine (Southgate), 165, 166
Muchihaematin, 166, 423
Mucin, 7, 47, 66, 72, 73, 165, 166, 211
Mucopolysaccharides, 47
Mucoproteins, 181
Mucus, 105, 125, 214
Muhlberg, W., 390
Miiller's Fluid, 411
Murray - Drew Technique, 172
Muscle, 17, 66, 83, 90, 97, 105, no,
III, 115, 122, 125, 126, 129, 131,
168, 178, 179, 181, 185, 194, 195,
196, 201, 224, 320

Mycelia, 121, 178, 275, 279

Myelin, 24, 65, 154, 199, 208, 209,
210, 223

Myelin Sheaths,

83,

105,

115,

120,

192, 223

Myelotheca, 135
Myelotheca, degenerate, 209
Myelothecal apparatus, 135
Myoglia, 65, 154, 155
Nacarat, 433

Nadi Reaction, 167

354, 422

M.F.4 Vaginal Smear Stain, 401

Michrome mountant, 43
Michrome Patent Blue, A.F.54, 136

of, 145,

Mouse pituitary, 150

Monosodium Urate in

throsin, 305

Methyl Green -Pyronin (Brachet), 336

Methyl Green Pyronin (Unna Pappenheim), 158, 339, 382, 421
Methyl Salicylate, 264, 265
Methyl Violet 5B, 307, 433
Methyl Violet (Jensen), 160, 422
Methyl Violet 6B, 159, 160, 433
Methyl Violet loB, 160, 383, 433
Methyl Violet - Metanil Yellow, 160
Methylene Blue, 53, 76, 79, 86, 161,

Methylene
Methylene
Methylene
Methylene

247

Monkey, C.N.S.

a-Naphthol, 167, 394

Naphthalene Brown, 433
Naphthalene Green, 433
Naphthol Blue Black, 168
Naphthol Blue R, 433

Naphthol Green B, 169, 433

Naphthol Yellow, 285
Navashin's Fluid, 13
Negri bodies, 76, 79, 152, 161, 186
Neisser Stain, 423
Neisserieae, 382, 383

Neoponceau, 214
Nerve cells, 93,

107,

108,

190, 211, 213, 215, 217

Microsporocytes, 304

446

140,

144,

INDEX
Nerve

107, 135, 144, 190,

fibres, 13,

217

Nerve fibre tracts, 213

Nerve trunks, 134
Nervous tissue, 57, 58,

Origanum

oil,

189

Orseillin, BB.,

434

Orth's Fluid, 14
Orth's Lithium Carmine, 96
147, 190, 217,

Osmic

acid, 9, 26, 108, 138, 179, 291

Ossification, 56

223, 321

Ossification, foetal, 56

Neurones, 135
Neutral fat, 171
Neutral gentian (Bensley), 67, 173
Neutral Red, 369, 392, 433
Neutral Red Fast Green, 170

Ostracod, 140
Ovaries, 264
Ovules, 264

Neurofibrils, 192

Owczarak, A., 271

Oxidase Granules, 167, 394
Oxidase Reaction, 167

Neuroglia, 66, 106, 138, 154

Ox

Neurilemma, 135

pancreas, 337
Oxydianil Yellow, 434
Oxphil Cytoplasm, 158, 159
Oxyphil Granules, 132, 158

Neuroglia fibres, 106, 107, 140, 157

Neurokeratin, 83
Neurones, 94, 134, 153
Neutrophile granules, 132
Nevashin's Fixative, 13
New Blue R, 433

New

Pancreas, 49, 149, 172

Pancreas, ox, 337
Pancreatic islets, 50, 148

Fuchsin, 386
stain, 387

Newman's

Papain, 346
Papanicolaou stain, 388

Newton's Gentian Violet, 301

Night Blue, 433

Pappenheim

Nigrosin, 189, 307, 359, 433

Nile Blue, 26, 171, 172, 340, 388, 433
Nissl*s Bodies, 58, 93, 106, 192
Nissl's stain, 79, 147, 423
Nitella cells, 311

stain, 158, 381

Paracarmine, 202, 424

Paraffin

Paraffin

Wax

Pyridin, 33

embedding, 29
Paraldehyde, 50
Parasite and host (Botanical), 269

Nitrates, test for, 242

Parasites in plant tissues,

Nitrocellulose, 26

Paratyphoid organisms, 361

Parenchyma cells, 280

Noland, L. E., 365

Nuclear membrane, 196

Pares, Ramon, 308

Parietal cells, 100

Nucleoli, 73, 77, 79, 196

Nyka,

W.

J.,

160

Pasini stain, 180

Pas Intermedia, 52, 59
Pas Tuberalis, 59
Patent Blue, 136, 137, 434
Pathological Tissues, 53, 60, 93, 124,
172, 177, 187, 192, 247, 279, 283,

Obelia, 140

Ohlmacher's Fluid, 411

Ollett,

W.

S.,

Onion root

171

tips,

290

Oocysts, 375
Optoil Immersion Oil, 428

Orange 1,433
Orange 2, 264, 433
Orange G., 59, 65,

67, 68, 83, 92,

160, 173, 182, 191,395

335
Patton, R. L., 327
Pearse, A. G. E., 183
Pentosenucleic acids, 339

no,

Orange G - Cellosolve, 262, 283

Orange 2 - Clove Oil, 195
Orange 3, 433
Orange 4, 434
Orange G - Clove Oil - Cellosolve, 262
Orcein, 89, 104, 174, 175, 176, 177,
179, 199,

424

284

Pepsin, 346
Pepsin-forming

cells, 100
Pepsinogen granules, 71, 100
Peptic substances, 242

Periodic acid, 181, 183

Perivascular phagocytes, 139

Peronosporaceae, 274
Peroxidase granules, 390
Peroxidase reaction, 389

447

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

Petrunkevitch's Fixative, 15
Phenylene Blue, 434
Phenosafranin, 434
Phenylhydrazine poisoning, 358
Phloem, primary, 254
Phloem Tissues, 254, 259
Phlorizin, 185
Phloroglucinol, 271
Phloxin, 120, 185, 186, 270

Pollen grains, 271

Pollen grains, aborted, 270
Pollen tubes, 268

Phloxin-propylene glycol, 424

Phloxin - Tartrazine, 188

Ponceau
Ponceau
Ponceau
Ponceau
Ponceau
Ponceau

Polychrome Methylene Blue (Unna),

53, 163, 164, 247, 422
Polymorphonuclears, 136, 196, 226
Polysaccharides, 183
Polysaccharide structures in
Polyvinyl lactophenol, 272

Phosphatases, 5
Phosphates, 243
Phospholipids, 340
Phospine 3R, 317, 318, 319
Phosphomolybdic Acid, 59, 152, 156,
341
Phosphotungstic Acid, 65, 68, 152,
182

Phosphotungstic haematoxylin Mallory, 154, 372

Phthalocyanins dyes, 47, 144
Phytosterol, 243
Picric Acid, 61, 77, 85, 109
Picric Alcohol, 215
Picro Aniline Blue, 125, 200, 424
Picro carmine, 424
Picro Fuchsin (Van Gieson) 82, 122,
127, 128, 172, 179
Picro Indigocarmine, 290, 424
Picro Methyl Blue, 390
Picro Nigrosin, 189
Picro Nitric acid, 412
Picro Ponceau S (Curtis), 123
Picro Sublimate, 412
Picro Sublimate Acetic acid (Rath),

412
254

Pituitary, 49, 50, 150, 157, 181, 215

Plant tissues, infected, 247, 252, 283

Plasma, 126

Plasma

Cells, 131, 158, 164, 178

Plasmodia, 366

Plasmosomes, 115, 132, 159

Plastids, 262
Plastin, 262
Platelets, 136, 226, 351, 353, 400
Pleuropneumonia in Lung, 121

Pneumococcus

Acid Fuchsin (Masson), 98

de Xylidine, 98, 124, 434
de Xylidine, Fat Stain, 434

Fuchsin, 124. 156

R, 434
S, 123, 125, 224,

434

Ponder's Stain (Kinyounn), 393

Ponder 's Toludine Blue, 426
Ponceau - Violamine, 216
Pontacyl carmine 2B, 434
Pontacyl carmine 6B, 434

Polar bodies in Bacteria, 385

Polar Yellow 5G, 434

Pontacyl Green, 434

Pontacyl Violet, 434
Pontamine Sky Blue 5BX, 434
Potassium, Tests for, 243

Pregnancy Cells, 59
Primula Obconica, 237
Primulin, 317, 320, 326
Propyl Alcohol Normal, 38
Propylene glycol, 23, 37,

38,

424.

425
Protargol, 190
Proteins, 231, 244, 257, 282

Protozoa,

3, 15,

364, 388

Prunus Laurocerasus Leaves, 231

Pseudo Rabies, 62
Pteridophyte Stems, 265
Pugh's Toluidine Blue, 426

Putt, F. A., 209

Pyramidal cells, 186

Pyrenoids, 261
Pyridin, 22, 60, 190
Pyridin paraffin wax embedding, 33
Pyrolaceae, 233
Pyronin, 160, 337, 381, 394, 434
Pyrus Crataegus, 231

185

Poaceae Seeds, 230

183

Purkinje cells, 6, 17
Purpurin, 192, 434
Pus cells, 391

Pinacyanol, 392
Pith,

cells,

Quadruple Stain (Kalter), 195

Quadruple Stain, Johansen, 262
Quinalizarin, 434

448

INDEX
Safranin
Safranin

Quincke Reaction, 193

Quinizarin Blue, 434
Quinoline Yellow, 434

Light Green, 275, 278, 395

Water Blue, 197

Salivary glands of Drosphila, 307

Sandifords Stain, 381

Saponin, 244
Saturated Solutions, reagents, 429
Scarlet R, 26, 126, 197, 241, 281, 282,

Rabbit, C.N.S. of, 147

Rabies, 62
Rabies, Pseudo, 62
Radioautographs, 334

434

HerzR, acetone-alcohol,
heimer, 198
Scarlet R, alkaline (Herzheimer), 126,
425
Schaudinn's Fluid, 15
Schmorl's Stain, 85
Schultz - Smith Stain, 86
Schulze Solution, 282

Scarlet

Radio isotopes, 334

Rahl's Fluid, 412
Randolph's blood counting fluid, 425
Reagents, Solubilities of, 428
Refractive Indices, table of, 427
Regaud's Fluid, 15
Regaud's Haematoxylin, 156, 215, 302
Rennin, 346
Resorcin Yellow, 434
Reticular Fibres, 102

Secretion Granules, 99
Secretory Bodies, 181

Sea water Bouin, 140

Reticulated Cells, 351

Seeds, testing viability

Reticulum, 66, 88, 103, 126, 143, 168,

201, 352
Rhamnose, 346

Seligman, A. M., 334

Semen, 347

Rhodamine B, 193, 3i7. 320, 434

Rhodamine B - Methylene Blue, 193
Rhodamine 6G, 434

Serous Cells, 173

Serra fixative, 336

Rhoduline Orange, 434

Rhoduline Violet, 434
Ribonuclease, 336

Setoglaucine, 434

Serum Albumen,

400
Ringer solution, 68
Roots, 264, 273
Roots, Stains for, 280

217, 237
Silver Oxide, 102

Simmel, Eva

Root Tips, Mitosis, 290, 295, 307

Rose Bengale, 434
Rosolic Acid, 434
Rosolic Acid Sodium Salt, 434
Rubens - Duval orcein, 89
Rubin S, 434

B.,

336

Skin, 89, 141, 177

Skin Infected, 177

Ruthenium Red, 242

Saccharose, 185
Saffron, 194

Safranin, 97, 137. 176, i95. i97, 220,

254, 260, 264, 274, 280, 303, 390,

434
Aniline Blue, 273, 274, 279
Cellosolve, 262, 264, 277
Crystal Violet, 262, 309
- Fast Green
FCF, 275, 277

185

Sgouris, J., 72
Sheridan's Elastin Stain, 97, 221
Shorr's Stain, 395, 396, 425, 426
Sieve Plates, 254
Silver Carbonate, 60, 199
Silver Nitrate, 102, 108, 138, 202, 203,.

Rickettsia, 15, 105, 160, 162, 356, 380,

283

Serine, 185

Ribonucleic Acid, 185, 336

Ribose, 185

Safranin
Safranin
Safranin
Safranin

of,

Skin Lesions, 89
Slime Plugs, 256
Smith, F. H., 301
Smooth, Muscle, 97, no, 168
Soaps, 126, 399
Sodium, test for, 245
Sodium Cobalt Nitrate, 243
Sodium Uranium Acetate, 245
Sodium Urate in tissues, 86, 114
Soil bacteria, 322
Soluble Blue, 434
Solochrome Black T, 434
Solochrome Blue, 434
Solochrome Violet R, 435
Solway Purple, 435

449

MEDICAL AND BIOLOGICAL STAINING TECHNIQUES

Tetrazolium

Sorbitol, 346

Specific Gravities, Tables of,

salt, 283
Tetrazotized o-Anisidine, 333
Thiazol Yellow, 231

430

Spermatozoa, 347
Spinal Cord, 106, 116, 223
Spinal Fluid, 398
Spindle Fibres, 295

Thioflavine, 319, 320, 435

Thionin, 85, 93, 211, 212, 213, 283,

400

Spirochaetes, 353, 358, 361, 363, 365,

371, 395, 403
Splenic Tissues, 193
Spoerri., R, 95

Spores, 359, 380

Spore Stain (Domer), 359
Sporogenous tissue, 261
Sporozites, 376

Sputum, 354, 356

S. Q. D. Mountant, 43, 428
Stains extracted by alcohol, remedy
for,

34

Stains, Solubilities of. Tables,

430

Starch, 185, 240, 248, 257, 282

276
Stems, Stain

Throat Exudate, 366

Thyroid, 115, 215
Tissue, embryonic, 54, 72
Tissue, friable, 24
Tissues, nervous, 57, 58
Titan Yellow, 284
Toluidine Blue, 55, 57, 93, 182, 214,
248, 289, 345, 426, 435
Toluidine Blue (Ponder), 393
Toluidine Blue (Pugh), 426
Toluol, 27, 30,71, 265
Toluylene Blue, 435

Trachea, 72
Trehalose, 346

Steil,

for, 262, 264, 273,

Thomas, J. T., 188

Thompson, R. H. S., 27

280

Stewart, H. M., 406

Stirling's Aniline Gentian Violet, 414
Stokes, J. C, 393

Treponema

Stomach, 172
Stratum lucidum, 178
Strugger, S., 323, 327

Tri-basic stain, 93

348,

349,

Treponema Refringens, 354

97,

156, 215

Trichromonas Riedmuller, 406

Tricresyl phosphate, danger of, 27

Sudan 3, 26, 204, 281, 398, 399, 435

Sudan 4, 204, 435
Sudan Black, structural formula of,
342
Black, 23, 204, 207, 397, 435
Black, stable solution of, 208

Blue, 209

Brown, 209, 435

Sulphates, 246

Sulphuric acid, 242

Susa's Fixative, 16
Syphilitic tissue, 177

Tannic

138,

Trichrome stain (Gomori), 214

Trichrome Stain (Masson), 15,

Suberised Structures, 261

Suberised Walls, 253
Sudan i, 435
Sudan 2, 210, 435

Sudan
Sudan
Sudan
Sudan

Pallida,

353, 358, 367, 368, 371, 403

acid, 245, 280

Tartaric acid, 185
Tartrazine, 188, 435
Terpineol, 38, 122, 189, 264
Tertiary Butyl Alcohol, 34, 262, 264
Terpineol-xylol, 144
Tetrachrome Stain (MacNeal), 400

Tropeolin, o. 435
Tropeolin 00., 435
Tropeolin 000. No.
Tropeolin 000. No,

435
435
Trypan Blue, 285, 435
i.,

2.,

Trypanosomes, 135, 225, 323, 324,

365, 368, 377,405
Trypanosomes, in dried blood films,
324
Trypanosomes, vital staining of, 323
Tryptophane, 185
Tubercle Bacilli, 80, 81, 324, 325, 354,
355, 356
Tumour, 23, 141, 216
Tumours of pancreatic islets, 50

Tumour,

cells,

60

Turquoise Blue, 435

Tworts Stain, modified, 170
Typhoid Organisms, 361, 363

Typhus

fever, 160

Tyrocidine, 185
Tyrosine, 246

450

INDEX
Ultra Violet Light, 315
Umbilical Cord, 185, 212
Ungewitten, L. H., 218

Unna-Pappenheim

Stain,

Volkonsky's technique, 249

Wandering Cells, 181

Water Blue, 197, 435
Water Blue - Orcein, 220
Water wax (Michrome), 33

158

Uranin, 320, 430

Uranium

Webster, S. H., 359

Weigert borax ferricyanide, 298
Weigert, Elastin Stain, 96, 97
Weigert - French Elastin Stain, 221
Weigert Kultschitsky Haematoxylin,

Acetate, 245

Urea, 217

Urea

silver nitrate, 217

Uric Acid, 87
Urinary Casts, 390
Urinary Passage epithelial

cells,

115
Weigert-Pal Technique, 223
Weigert's Rapid Fixative, 115, 223
Weigert Secondary Mordant, 119
Westerhaver, 424
Wexler, 424
Whartman's Jelly, 212

391

Urine, 391
Urine Deposits, 391

Urodele Amphibians, 54

Uzman, L.

L., 341

Vaccina, Elementary Bodies of, 403

Vacuoles, Cytoplasmic, 53
Vaginal Smear Stain (MF4), 401
Vaginal Smears, 373, 388, 395, 396,
401, 402

Van Giesen

Whipple, Ann, 34

Whole Mounts, 250, 252

Wood, brittle, sectioning
Woodstain

Stain, 97, 123, 124, 127,

Woody plants, 259

Woody tissue, 254

Stain, substitute for, 123

Wool Green S, 224, 435

Wool Violet 4BN, 435

179, 219

Van Giesen

404
Variola, 404
Varicella,

Wright's Stain,

Vascular Plants, 252

Vascular Reticulum, 60

Xanthophyll, 236
Xanthosine, 185
Xylidine Red, 435
Xylol, 30
Xylose, 346

Yeasts, 381, 388

Vincent's Agina, 371

Violamine 3B, 435
Viruses, 62, 326, 403
Virus-containing tissues, 189

New

Zenker, Formol, 68, 150, 412

Zenker's Fixative, 16

Zimmer, D.

J.,

359

Zlotnil, J., 153

Red, 435

Vital Staining of Nervous Tissue, 58

Vital stains, fluorescence, 318, 319,

Zoospores, 273
Zoster, 62
.

322, 323
Vital Staining of Trypanosomes, 323

451

Zylem, 254

Zymogen Granules, 174

Zymogenic

272

14, 106, 135, 225, 310,

351, 378, 405, 406

Vaughan, J. G., 257

Venetian Turpentine, 43
Vercourt, B., 254
Verhoeff's Stain, 218
Vernon, J. C, 268
Victoria Blue B., 435
Victoria Blue R., 435
Victoria Blue 4R., 402, 403, 404, 435
Victoria Green, 135, 155
Violamine, R,, 216

Vital

off,

Scarlet, 215

cells,

173

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