Appendix 1

Fixatives*

Immersion Fixatives Methacarn

The cytological slide is immersed in a solution Methanol 60ml containing fixative. For optimal results, the slide Chloroform 30ml should remain immersed for at least 15 min: longer Glacial acetic acid Iml fixation times do not change the cell images.

Propanol-Glycerine 95% Alcohol Isopropanol 80ml Slides are to be immersed in the alcohol (15- 30 min Glycerine 40ml is sufficient)

10%Neutral Buffered Formalin Equivalents for95% Alcohol 37-40% Formaldehyde solution 100mi 100% Methanol Water 900ml 80% Propanol Acid sod ium phosphate, monohydrate 4.0g 80% Isopropanol (can also be used as a spray Anhydrous disodium phosphate 6.5g fixative)

4%SalineFormalin Ethanol- Ether 37-40% Formaldehyde solution 5ml Mix 95% alcohol and ether in equal quantities NaCI 0.9g Water 90ml

Carnoy's Fixative The mixture haemolyses erythrocytes. It can be used for pre-fixation but also for post-fixation of 95% Alcohol 60ml air-dried smears before applying the Papanicolaou Chloroform 30ml staining method (van der Griendt, 1984, pers. Glacial acetic acid IO ml comm.). • In all cases where 'alcohol' or 'ethanol' is mentioned, ethyl alcohol is meant. 122 Appendix 1: Fixatives

Neutral Formalin Zenker's Fixative

37-40% Formaldehyde solution 10ml K2Cr20 7 2.5g Water 90ml HgCh 5-8g Calcium carbonate about I g Distilled water 100ml

Add calcium carbonate in excess. Glacial acetic acid, 5% by volume to be added at the time of use. Dissolve salts in water, heating gently. Bonin's Fluid

1.2% (= saturated) picric acid 75ml Spray Fixatives 36-40% Formalin 25ml Glacial acetic acid 5ml Hair-spray The amount of formalin may be reduced to 10mI. Any hair-spray with a high alcohol content and some lanolin or oil will do. Formalin Vapour Fixation Polyethylene Glycol Place 1-2 ml of formalin solution of required strength in Coplin jar. Immediately after taking 95% Alcohol 500 ml sample, put slide in jar with cell-coated side upper­ Ether 500ml most and tightly cover the jar. Leave the slidein the Polyethylene glycol (Carbowax) 50 g jar for required length of time depending on the procedure which is to follow. NB Ether may be omitted and instead 100ml 95% alcohol may be used. Polyethylene glycol is available in various grades 20-25% Glutaraldehyde of viscosity, indicated by a number. We prefer number 400, which has the consistency of a syrupy For prolonged storage the solutions should be kept liquid. Polyethylene glycol 1500is like vaseline and at a low temperature (4°C or lower) and low pH. stays on the slide without drying out. For that reason it may be preferred but it also means that the cell film plus coating can be damaged more easily. 2% Formaldehyde and 2% glutaraldehyde

2% Formaldehyde 25ml Leiden Spray Fixative 2% Glutaraldehyde 25ml NaCI 4.5g 96% Alcohol 700ml Acetone 200ml Polyethylene glycol (see note Osmium tetroxide above) 70ml

Osmium tetroxide 1-2g NB Perfume bottles can be used for spraying the Water 100ml fixatives on the slides. It is very important to spray from the correct distance; 25- 30cm gives optimal Slides are to be kept in the solution overnight. results. Appendix 1: Fixatives 123

For sputum, 70% alcohol is recommended. The Preserving Fluids acetone can also be replaced by alcohol. These fixatives can also be used as immersion fixatives. If Cell suspensions can be kept for a limited period of the material is bloody, it is advisable to add 50 ml time without fixation. To preserve the cells, the glacial acetic acid to 1000 ml fixative. medium should have physiological salt concentra­ tion and some essential nutrients, preferably with an anti-mould agent added. There are a number of commercial preserving fluids on the market, of Air Drying which the Minimum Essential Medium (MEM) (Eagle) is perhaps best known. It contains Some methods (like the Romanowsky-Giemsa glutamine, salts, and sodium azide as preservative. staining method or the colouring of lipids) have best results after air drying. This is usually followed by some form of post-fixation. Delft Suspension Medium

MEM 500ml 6% Foetal calf serum 30ml Fixatives for Cell Suspensions I% Glutamine 5ml 200 U jml Penicillin I ml 0.2 mgjml Streptomycin I ml Saccomano's Fixative 0.5 J.Lgjml Fungizone 1 ml

50% Alcohol 100mi pH should be 7.18. Carbowax 1540 2g

Phosphate-buffered Saline Esposti's Fluid NaCI 87.5g Methanol 225ml Na2HP04·12H20 23.25 g Purified water 225ml KH 2P04 2.15 g Glacial acetic acid 50ml Dissolve in I litre distilled water. pH is 7.4. Alcohol Solutions

50% Alcohol for cell suspensions from all body Preservative According to Marsan et al. (1982) sites except gastric 70% Alcohol for sputum, bronchial aspirates and 0.9% NaCI 249ml washings 22% bovine albumin I ml 90% Alcohol for gastric, bronchial and other saline washings Adjust to pH 7.5.

Formalin Vapour Fixation Formol-saline (van der Griendt's Fluid)

See preceding section on immersion fixatives. The Physiological saline 1000mi fixation time can be shortened to I min when the 40% Neutral formalin 2.5ml procedure is done in the microwave oven at high energy level. This solution haemolyses erythrocytes. It can also 124 Appendix 1: Fixatives

be used for incubating air-dried smears prior to Secondly, the mounting media should not in­ staining with the Papanicolaou method (seesection fluence the colour of the cells by either fading or 10.4.2). 'bleeding' (this is the running of the dye from its original site). For this reason it is important to Clearing Agents know the acidity of the medium. Thirdly, the dye or dyes should not dissolve in the mounting medium. For that reason, only water­ As the name implies, clearing agents should clear based media can be used in fat-stained preparations the cell preparation. This is done after dehydration (see below). with increasing concentrations of alcohol. The requirements mentioned above are mostly Xylene is a well established clearing agent. It is a met by the resins. These can be either natural very unpleasant chemical to have around in the semisynthetic or synthetic. The natural resins (Can: laboratory. It does not mix with water. If there ada balsam and cedar oil) are slightly acid and tend should be some water left on the cell preparation to fade basic dyes, especially cedar oil. The semisyn­ (for instance if the last dehydration bath is not thetic resins (like Euparal and Diaphane) are also 100% alcohol) , this will show as a white film on slightly acid. Euparal can fade Haematoxylin but slide. basic aniline and the Romanowsky-Giemsa stains Tertiary butanol is less demanding. It is less are well preserved. There are a number ofsynthetic unpleasant to use and does not show a white film if resins on the market. They are all quite neutral and water is left in the preparation. Its disadvantage is have a wide range of applications. that its melting point is higher than the usual Cells which are stained for fat cannot be moun­ laboratory temperature, so that it needs either ted in any of the above-mentioned media since the heating or to be mixed with a solvent to make it dye would dissolve in them . For this kind of cell fluid. Since the former possibility is not always preparation, water-based media should be used wanted, the latter is the better choice. After melting with gelatin or gum as hardener. Slides which need down the solid tertiary butanol it can bemixed with not be kept permanently can be mounted in just 96% alcohol (15 : I) after which it remains liquid glycerine for as long as necessary or practicable. (Drijver and Boon, 1983a).

Glycerine - Gelatin Mounting Media Gelatin 40g Mounting media have a double task. They should : Water 210ml (I) smooth out the cell preparation, i.e. make the Glycerine 120ml surface of the preparation even so that there are no gaps left between cells and cover glass; Soak the gelatin in the water for 2 h. Add the (2) glue together cell preparation and cover glass glycerine, stirring all the time. The solution should so that the slide can be kept without the bekept at 0-5°C and melted as needed. To prevent preparation being damaged and air getting in. mould , 50 mg Merthiolate, 100mg thymol or 5 ml melted phenol may be added. Phenol may have a Mounting media should have a number of damaging effect on Haematoxylin. properties in order to make them suitable for the purpose for which they are intended. First, they should have a refractive index similar to that of Apathy's Gum Syrup glass so that the image does not become distorted. The RI should not be the same as that of the tissue Gum arabic (acacia) 50g components. If it were the same, unstained or Cane sugar 50g slightly stained cells would be difficult to see. Water 100ml Appendix 1: Fixatives 125

Mix the ingredients while heating gently. A Blueing Solutions preservative may be added as in glycerine-gelatin. The suger is added to raise the refractive index of (I) Scott's tap water substitute (commercially the gum, which is very low. Other sugars may be available). used. The important thing is to use a sugar that does (2) Lithium carbonate solution (three drops of a not crystallise. saturated lithium carbonate solution in 100ml water). (3) Water to which a few drops of ammonia have been added .

NB Blueingsolutions should have a pH of about 8. Appendix 2

Staining Methods*

In this appendix we have collected together a number of staining methods. The Papanicolaou and Romanowsky-Giemsa methods are represented by various modifications. In addition, certain methods in which Haematoxylin is substituted by other dyes are given. Also some methods are included for specific dyeing ofnuclear and cytoplasmic components and pigments, and bacteria.

General staining methods 1-8 Nuclear staining methods 9-21 Staining methods for glycogen 22-24 Mucin staining method 25 Methods for staining fat 26,27 Fibre staining method 28 Iron staining method 29,30 Supravital staining methods 31,32 Blood staining methods 33-36 Methods for staining bacteria 37 Melanin staining method 38 Destaining methods 39

Some general remarks must now be made. First, unless otherwise mentioned, 'bring to water' or 'hydrate' means that after fixation the slides should be brought through graded baths of alcohol (96, 70, 50%) to water. Secondly, 'dehydrate' means bringing the slides to absolute alcohol through graded baths of alcohol. Unfortunately, the pH of the dye bath is rarely mentioned in staining methods described elsewhere.We have therefore included details wherever possible. We recommend measuring the pH of every dye solution and keeping a check on it if the solution is retained for a longer period or used for the staining of a large number of slides. The recipes ofthe solutions needed for a staining procedure are either given under the heading 'Solutions' needed' or marked with a superscript number, in which case they can be found in appendix 3 under the appropriate staining solution. Not all staining procedures need a specificfixation method. In that case we have recommended spray fixative or 96% alcohol since these are the methods most commonly used. This does not necessarily mean that other routinely used fixatives cannot be used or tried.

• Where 'alcohol' is mentioned, ethyl alcohol (ethanol) is meant. Appendix 2: Staining Methods 129 1 2

Papanicolaou's StainingMethodincluding Orange G Papanicolaou's StainingMethodwithout OrangeG (Leiden Modification) (Leiden Modification)

Purpose Purpose General nuclear and cytoplasmic stain. General nuclear and cytoplasmic stain.

Solutions Needed Solutions Needed Cole's Haematoxylirr' (pH 6.5) Cole's Haematoxylin' (pH 6.5) EA-Leidenl4 (pH 4.6) EA-Leidenl4 (pH 4.6) Orange G - Leiden 19 Tertiary butanol/95% alcohol, 15: I Tertiary butanol/95% alcohol, 15: I Fixation Fixation Leiden spray fixative. Leiden spray fixative. Procedure Procedure (1) Place slidesin 50% alcohol to remove polyeth­ (I) Place slides in 50% alcohol to remove polyeth­ ylene glycol for at least an hour but longer if ylene glycol for at least an hour but longer if more convenient (overnight, for instance). more convenient (overnight, for instance). (2) Rinse in tap water, 15 dips. (2) Rinse in tap water, 15 dips . (3) Stain in Cole's Haematoxylin for 2 min. (3) Stain in Cole's Haematoxylin for 2 min. (4) Rinse in tap water till water runs clear. (4) Rinse in tap water till water runs clear. (5) Dip in 50% alcohol, 15 dips. (5) Dip in 50% alcohol, 15 dips. (6) Stain in EA- Leiden for 2 min. (6) Stain in Orange G-Leiden, 1 min. (7) Dip in 50% alcohol, 15 dips. (7) Dip in two changes of 50% alcohol, 15 dips (8) Dehydrate. each. (9) Clear in tertiary butanol/alcohol for at most 5 (8) Stain in EA- Leiden, 2 min. min. (9) Dip in 50% alcohol, 15 dips. (10) Air-dry if slides are to be coverslipped (10) Dehydrate. automatically; if covered by hand , do so (11) Clear in tertiary butanol/alcohol. straight from the clearing bath without dry­ (12) Mount. ing.

Result Result Nuclei blue to bluish purple . Nuclei blue to bluish purple. Nucleoli blue or red. Nucleoli blue or red. Cytoplasm in superficial squamous cells pink, in Cytoplasm in superficial squamous cells pink, in intermediate cells turquoise blue. intermediate cells turquoise blue. Nuclei in lymphocytes and leucocytes blue. Nuclei in lymphocytes and leucocytes blue. Erythrocytes pink - orange. Erythrocytes pink-red. Highly keratinised cells orange. 130 Appendix 2: Staining Methods 3 4

Papanicolaou's StainingMethod (following Koss, Papanicolaou's StainingMethod(Gill's 1979) Modification)

Purpose Purpose General nuclear and cytoplasmic stain. General nuclear and cytoplasmic stain. Solutions Needed Solutions Needed Harris's Haematoxylirr' 0.1% Hydrochloric acid Gill's Haematoxylin" Saturated solution oflithium carbonate Gill's Orange G_617 Orange GIS Scott's tap water substitute ifpH oflocal tap water EA-35 or EA-6515 is below 7.0 Gill's EA I6 Fixation Spray fixative or 96% alcohol. Fixation Procedure 95% Alcohol, at least 15 min . (I) Leave in 95% alcohol to remove polyethylene glycol for at least an hour but longer if Procedure preferrred. (I) Dip in two changes of water, 10 dips each. (2) Hydrate. (2) Stain in Gill's Haematoxylin, 2 min . (3) Distilled water, 15 dips. (3) Dip in two changes of water, 10 dips each. (4) Harris's Haematoxylin, 6 min. (4) Leave in Scott's tap water substitute, I min. (5) Rinse in running tap water till water runs (5) Dip in two changes of water, 10 dips each . clear. (6) Dip in two changes of 95% alcohol, 10 dips (6) Dip in distilled water, 15 dips. each . (7) Stand in distilled water, 10 min. (7) Stain in Gill's OG-6, I min. (8) Differentiate in 0.1% HCI till colour of slide (8) Dip in three changes of 95% alcohol, 10 dips turns red (check!). each. (9) Dip in distilled water, 15 dips. (9) Stain in Gill's EA, 4-10 min. (10) Lithium carbonate, 2 min. (10) Dip in three changes of95% alcohol, 10 dips (II) Dehydrate. each . (12) Stain in Orange G, 2 min. (II) Dip in 100% alcohol, 10 dips. (13) Dip in 96% alcohol, IS dips. (12) Clear in xylene and mount. (14) Stain in EA -35 or 65, 4 min. (15) Dip in two changes of 96% alcohol , 15 dips Result each. (16) Leave in 100% alcohol, 2 min. Nuclei blue. (17) Clear in xylene. Cytoplasm of superficial squamous cells pink, of (18) Mount. intermediate squamous cells turquoise blue. Nucleoli dark blue. Result Nuclei of Ieucocytes and lymphocytes dark blue. Nuclei blue. Keratinised cells orange. Nucleoli dark blue or red. Erythrocytes pink - red. Cytoplasm of superficial squamous cells pink, of intermediate cells turquoise-blue or light blue, depend ing on EA used. Erythrocytes red. Nuclei of leucocytes and lymphocytes dark blue. 5 6

Rakoff'sStaining Method (Rakoff,1960) Shorr's Staining Method

Purpose Purpose In-office hormonal evaluation of gynaecological For hormonal assessment. samples. SolutionsNeeded SolutionsNeeded Shorr's stain" Rakoff''s stain" Saline solution (0.9% NaCI) Fixation 95% Alcohol. Procedure (1) Make swab with cotton tip dipped in saline. Procedure (2) Drop swab in test tube containing 1-2 ml (1) Stain in Shorr's solution, I min. saline. (2) Dehydrate. (3) Place 3 drops of stain into test tube and stir (3) Clear in xylene. solution with swab. (4) Transfer I or 2 drops to slide and coverslip. Result Result Cytoplasm of superficial squamous cells orange­ pink, of intermediate cells green. Cytoplasm of superficial cells red, of intermediate cells green. Vesicular nuclei show sharply stained margins but are otherwise pale. 132 Appendix 2: Staining Methods 7 8

Szczepanik's QuickStainingMethod(Szczepanik, Delafield's Haematoxylin-EosinY Method 1978)forPapanicolaou Method Purpose Purpose General nuclear and cytoplasmic stain. To stain cell sample during consultation. Solutions Needed Solutions Needed Delafield's Haematoxylin' Harris's Haematoxylirr' Acid alcohol : 3 drops ofconcentrated HCI in 50 ml Isopropyl alcohol 95% alcohol 0.1-0.5% Eosin Y in 25% alcohol EA-31 (Merck No. 9271 or 9272) Water at 40-70·C Fixation Spray fixative or 96% alcohol. Fixation Spray fixative. Procedure (I) Dissolve polyethylene glycol in 50% alcohol. Procedure (2) Dip in water, 15 dips. (I) Dip in two changes of propanol, I dip each. (3) Stain in Haematoxylin, 15 min. (2) Stain in Haematoxylin, 70s. (4) Rinse in tap water till water runs clear. (3) Rinse in water (40- 70·C), 5 s. (5) Stand in tap water for 10 min. (4) Dip in propanol, Is. (6) Ifslides are still very blue, add 2 drops of acid (5) Stain in EA, c. 38s. alcohol. (6) Clear in xylene, two changes, I s each. (7) Stain in Eosin Y solution. (8) Rinse in distilled water. Result (9) Dehydrate. (10) Clear in xylene. Nuclei blue. (II) Mount. Nucleoli dark blue. Cytoplasm of superficial squamous cells pink, of Result intermediate cells light blue. Erythrocytes pink . Nuclei dark blue. Nuclei of lymphocytes and leucocytes dark blue. Cytoplasm pink -red. Appendix 2: Staining Methods 133 9 10

GaUocyanin Chrome Alum (Sandritter et al., 1966) Coelestine Blue(Gray et al., 1956; Yasumatsu, 1977) Purpose Purpose To determine the total nucleic acid content of the nucleus. To be used as a general nuclear stain as a Haematoxylin substitute. Solutions Needed Solutions Needed Gallocyanin chrome alum," pH 1.64 Coelestine Blue solution according to Gray et al? Fixation or Yasumatsu" 96% Alcohol. Fixation Procedure Any fixative can be used which is also suitable for Haematoxylin staining. (1) Stain in Gallocyanin solution for 48 h. (2) Differentiate in dehydrating alcohols. Procedure (3) Clear in xylene and mount. (1) Hydrate slides. Result (2) Stain in Coelestine Blue solution, 1 min to I h. (3) Rinse in water. Nuclei blue-black. (4) Dehydrate, clear and mount. RNA-rich sites in cytoplasm blue- black. Result Nuclei blue (the colour is an intermediate between the blue from Haematoxylin and that from Methylene Blue). 134 Appendix 2: Staining Methods 11 12

NuclearStaining withCarminicAcid(Mayer, 1891) The FeulgenReaction

Purpose Purpose To be used as pure nuclear dye. To show DNA.

Solutions Needed SolutionsNeeded Carminic Acid solution7 Schiffs reagent" O.I- I% Potassium alum solution in water I N HCl at 60·C 0.05 M Metabisulphite Fixation 0.01% Fast Green FCF in 95% alcohol if wanted Spray fixative or any other routine fixative. Fixation Procedure Any fixative may be used. (I) Bring slides to water. Procedure (2) Stain in Carminic Acid solution, 15 min. (3) Rinse in water, 2-3 min. (I) Hydrolyse in HCI for 10 min. (4) If needed, differentiate briefly in potassium (2) Leave in Schiff's reagent for 10 min. alum. (3) Wash in three successive baths of metabisul- (5) Rinse thoroughly in water. phite for 2 min each. (6) Dehydrate. (4) Wash in running water. (7) Mount. (5) Counterstain, if wanted, for a few seconds. (6) Dehydrate, clear and mount. Result Result Nuclei red, very clearly defined. DNA-rich sites red. Basic cell components green. Appendix 2: Staining Methods 135 13 14

Combined Feulgen-Naphthol Yellow S Method Methyl Green-Pyronin Y (Taft, 1951; Clark, 1973) (Gaub et al., 1975) Purpose Purpose To stain DNA and RNA differentially . To demonstrate DNA and basic proteins for quan­ titative measurement. Solutions Needed Methyl Green -Pyronin Y solution!' Solutions Needed Tertiary butanol-alcohol, 3: I Schiff's reagent" 5 N HCI at 22°C Fixation 0.05 M Metabisulphite Carnoy. 0.1% Naphthol Yellow S in 1% acetic acid, pH 2.8 1% Acetic acid Procedure Tertiary butanol/absolute alcohol 15 : 1 (I) Bring slides to water. Fixation (2) Stain in Methyl Green-Pyronin Y solution, 3-5 min. 10% Neutral formalin is preferred, but it is also (3) Rinse in distilled water. possible to use alcohol-acetone. (4) Blot dry with smooth filter paper. (5) Before completely dry, differentiate for at least Procedure 2 min in tertiary butanol-alcohol mixture. (l) Hydrolyse in HCI for 60 min at 22°C. (6) Clear in two changes of xylene, 5 min each. (2) Rinse in metabisulphite. (7) Mount in synthetic resin. (3) Rinse in distilled water. (4) Stain in Naphthol Yellow S for 30 min. Result (5) Destain in 1% acetic acid, 3 baths, 0.5 min Chromatin blue-green. each. Nucleoli rose. (6) Clear in tertiary butanol-alcohol, 3 baths. Cytoplasm granules dark rose. (7) Clear in xylene 5 min. Cytoplasm of plasma cells dark rose, occasionally (8) Mount. almost purple. Mast cell granules refractile, orange- red. Result DNA-rich sites blue . Basic proteins yellow. 136 Appendix 2: Stain ing Methods 15 16

Methyl Green-Pyronin Y (Kurnick, 1952) Pontacyl DarkGreen B (Bedrick, 1970)

Purpose Purpose To stain DNA and RNA differentially. To stain nucleoli differentially.

Solutions Needed Solut ions Needed 0.2% Methyl Green in water (chloroform 2% Pontacyl Dark Green B in water; to 100ml, 2N extracted) HCI (2 ml) is added. Saturated solution ofPyron in Y in acetone n-Butyl alcohol Fixation Cedar oil 10% Formalin. Dilute solutions of Pyronin Y may be used if a more delicate stain is wanted Procedure Fixation (1) Bring slides to water. (2) Stain in Pontacyl Dark Green B solution, 3 Carnoy. min. (3) Rinse in water. Procedure (4) Dehydrate and mount. (1) Bring slides to water. (2) Stain in Methyl Green solution, 6 min. Result (3) Blot dry. Nucleoli in malignant cells more intense green than (4) Differentiate in two changes of n-butyl alcohol, those of benign cells. 2-3 min each. Good differentiation of cellular structures. (5) Stain in Pyronin Y solution, 30-90 s. (6) Transfer directly to cedar oil for clearing. (7) Further clear in xylene. (8) Mount in Permount.

Result Nucleoli pink. Cytoplasm red. Chromatin bright green. Erythrocytes brown . Appendix 2: Staining Methods 137 17 18

Love's Toluidine BlueMolybdate Method (Loveet AmidoBlack lOB(Synonym Pontacyl BlueBlack aI.,1973) SX) (Mundkur and Greenwood,1968) Purpose Purpose To stain nucleolini. To stain nucleoli differentially in lymph nodes in Solutions Needed Hodgkin's disease but can also be used for other material. 15mg% Toluidine Blue in McIlvaine's buffer" 10% Trichloroacetic acid Solutions Needed Formol sublimate (6% aqueous mercuric chloride: formaldehyde USP, 9 : I) 2% Phosphomolybdic acid in water 12 22 Lugol's iodine • 1% Amido Black lOBin water 0.2 M Aqueous sodium thiosulphate I mg% Deoxyribonuclease in Tris-hydroxymethyl Fixation aminomethane buffer" Carnoy's ethanol, chloroform, acetic acid, 6 : 3 : I, 15% Aqueous ammonium molybdate" 4h. Tertiary butanol Fixation Procedure Do not allow smears to dry! 10% Trichloroacetic (I) Bring slides to water. acid, 10 min at room temperature followed by a (2) Mordant in phosphomolybdic acid, 10 min. rinse in a dish of tap water , 5 s. Then fix in formol (3) Rinse in running tap water, 3 min. sublimate , 5 min exactly. (4) Stain in Amido Black lOB solution, 15 min. (5) Rinse in running tap water, 3 min. Procedure (6) Rinse in 30% alcohol, 15 min. (1) Rinse slides in runn ing tap water. (7) Rinse in 50% alcohol, 15 min. (2) Remove sublimate in Lugol's iodine, 5 min. (8) Rinse in 70% alcohol, 5 min. (3) Wash off Lugol's with tap water. (9) Rinse in 80% alcohol, 5 min. (4) Immerse in sodium thiosulphate, 5 min. (10) Leave overnight in 95% alcohol (shorter times (5) Rinse in running tap water, 5 min. are possible but minimum time is not (6) Leave in DNA-ase solution, 60 min at 37°C. specified). (7) Stain in Toluidine Blue, 120 min. (II) Final dehydration in 100% alcohol, 15min. (8) Rinse in running tap water, 10s exactly. (12) Clear in toluene and mount (Permount). (9) Immerse in ammonium molybdate, 7 min exactly. Result (10) Rinse in running tap water, 10s exactly. Nucleoli blue. (11) Drain off excess water. Cytoplasm very light blue. (12) Dehydrate in tertiary butanol at a tem- perature just above 25SC ( melting point). = NB A cytoplasmic counterstain may be used if (13) Clear in xylene and mount in Permount. wanted. Result Nucleolini purple. Body of the nucleolus pale blue. 138 Appendix 2: Staining Methods 19 20

Cuprolinic Bluein the Presenceof Magnesium Selective Staining of RNA witha Basic Dye and a Chloride(Mendelson et al.; 1983) CationicSurfactant (Bennionet al., 1975)

Purpose Purpose To stain nuclear and cytoplasmic RNA . To show RNA-rich sites in cytoplasm and nuclei.

Solutions Needed Solutions Needed 0.1% Cuprolinic Blue in 25 mM acetate buffer 0.1% Azure A or Toluidine Blue and 1% Hyamine with I M MgCh, pH 5.6 2389 in MIlO phosphate buffer, pH 7.0 (Hyamine 25mM acetate buffer with I M MgCh, pH 5.6 2389 is available commercially. It contains Glycerine-gelatin, if wanted (see appendix 1). approximately 50% methyldodecylbenzyltrimethyl ammonium chloride in aqueous solution.) Fixation Synthetic resin Air drying and post-fixing in modified Carnoy Fixation (ethanol-chloroform-acetic acid, 60 : 30: 5). 10% Buffered neutral formalin preferred; absolute Procedure alcohol and acetone also possible. (1) Bring slides to water. Procedure (2) Stain in Cuprolinic Blue solution, 60 min, at room temperature. (I) Bring slides to water. (3) Rinse in several changes of the dye solvent (i.e. (2) Stain in Azure A or Toluidine Blue solutions, acetate buffer with MgCh), 15 min each. 30 min. (4) Rinse in 3 changes of distilled water, 2 min (3) Rinse in water, blot or air dry. each. (4) Mount in synthetic resin (Polymount, Searl (5a) Dehydrate and mount in Euparol, or (5b) air­ Scientific). dry and mount in glycerine jelly. Result NB The same procedure can be used if both DNA Basophilic cytoplasm, like that of exocrine pan­ and RNA are to be stained. In that case the acetate creas, plasma cells, Nissl bodies and nucleoli, buffer should not contain MgCI • 2 stain purple. Chromatin stains pale blue. Result RNA in cytoplasm blue. Nucleoli blue. Appendix 2: Staining Methods 139 21 22

Guard's Method for Sex Chromatin (Guard, 1959 Best's Method (Luna, 1968). detailed in Koss, 1979) Purpose Purpose To stain glycogen. To demonstrate Barr bodies in buccal smears. Solutions Needed Solutions Needed Best's Carmine solution" Biebrich Scarlet solution (appendix 4) Any progressive Haematoxylin solution (Cole's' or Fast Green solution (appendix 4) Mayer's') Harris's Haematoxylin? Differentiating solution: 100% alcohol 20ml, Fixation methanol 10ml, distilled water 25 ml

Immediately in 95% alcohol. Fixation

Procedure Carnoy's or formol-alcohol.

(l) Transfer slide to 70% alcohol, 2 min. Procedure (2) Stain in Biebrich Scarlet, 2 min. (See Note 1.) (3) Rinse in 50% alcohol. (1) Bring slides to distilled water. (4) Differentiate in Fast Green solution, 1 to 4 h. (2) Stain in Haematoxylin, 15 min. (See Note 2.) (3) Rinse in running water, 15 min. (5) Leave in 50% alcohol, 5 min. (4) Stain in Carmine working solution, 30 min. (6) Dehydrate. (5) Leave in differentiating solution, few seconds. (7) Clear in three changes of xylene, 2 min each. (6) Rinse quickly in 80% alcohol. (8) Mount. (7) Dehydrate. (8) Clear in xylene. Result (9) Mount in Permount or Histoclad. Sex chromatin (Barr body) red. Background green. Result Glycogen pink or red. Note 1: If Haematoxylin is to be included, this Nuclei blue. should be done before staining with Biebrich Scarlet. The slides then go immediately from the Haematoxylin bath into the Biebrich Scarlet solu­ tion. The staining results are then as follows: Sex chromatin red. Nuclear chromatin blue. Cytoplasm green. Note 2: Differentiation needs to be checked micro­ scopically at hourly intervals . When the cytoplasm of all cellsis green and nuclei are vesicular, the slide is ready. Pyknotic nuclei will not differentiate and are stained red. 140 Appendix 2: Staining Methods 23 24

Periodic Acid - SchiffReaction Periodic Acid SchiffReaction inCombination with Amylase and Dimedone Purpose Purpose Demonstration of glycogen. The selective staining of glycogen, mucoproteins Solutions Needed and glycoproteins. Schiff's reagent!' Solutions Needed I% Periodic acid (H5106) in water. 0.52% NaHS03 (0.05 M) Schiff's reagent Weigert's" or Mayer's! Haematoxylin 2% Periodic acid in water 5% Dimedonef in 96% alcohol (Dimedone (5,5­ Fixation dimethylcyclohexane-3,dione) is a commercial preparation for the blocking of aldehydes.) Any fixative may be used. Amylase: saliva in phosphate-buffered saline (I : I), pH 0.0 Procedure (I) If air-dried, post-fix smear in 70% alcohol for Fixation 10min. If spray-fixed, dissolve polyethylene in Any fixative may be used. water as usual. (2) Rinse in water. Procedure (3) Oxidise in periodic acid for 5 min. (4) Rinse in distilled water. The staining procedure is the same as in Method 23. (5) Leave in Schiff's reagent for 15 min. Amylase treatment: The cells are incubated for 30

(6) Pass directly to 3 successive baths of NaHS03 min at 37"C and then stained. for 2 min each. Dimedone treatment: The cells are incubated for (7) Rinse in running tap water for 10 min each. 3h at 60°C. (8) Counterstain with Haematoxylin if wanted (Weigert's or Mayer's). Result (9) Rinse in running tap water for 10 min. PAS only: all PAS-positive material is stained. (10) Dehydrate. PAS in combination with amylase: mucoproteins (II) Clear in xylene and mount. and glycoproteins are stained, glycogen remains unstained. Result PAS in combination with dimedone: glycogen and Glycogen-rich sites red mucin are stained. Nuclei blue or black depending on Haematoxylin PAS in combination with amylase and dimedone: used. only mucin is stained. Mucin red-purple or violet. Cytoplasm grey or pale blue. Appendix 2: Staining Methods 141 25 26

A1cian BlueMethod for Mucosubstances,pH 2.5 Sudan Black B (Lillieand Fulmer, 1976) (Luna, 1968) Purpose Purpose To demonstrate fat in cytoplasm. To stain polysaccharides in mucin . Solutions Needed Fixation Sudan Black B solution" 10% Buffered neutral formalin. Propylene glycol Glycerine jelly (see appendix I) Solutions Needed Fixation 1% Alcian Blue 8GX in 3% acetic acid, adjusted to pH2.5 Formalin. 3% Acetic acid in distilled water 0.1% Kernechtrot in 5% aluminium sulphate Procedure (1) Wash in distilled water, 2-5 min. Procedure (2) Dehydrate in propylene glycol while moving (I) Bring slides to distilled water. slides, 3-5 min. (2) Mordant in 3% acetic acid, 3 min. (3) Stain in Sudan Black B solution, agitating (3) Stain in Alcian Blue, 30 min. occasionally, 5-7 min. (4) Rinse in running tap water for 10 min. (4) Differentiate in 85% propylene glycol, 2-3 (5) Rinse in distilled water. min. (6) Counterstain in Kernechtrot, 5 min. (5) Rinse in distilled water, 3-5 min. (7) Rinse in running tap water, 1 min. (6) Counterstain with nuclear stain if wanted. (8) Dehydrate and clear in xylene. (7) Rinse in tap water, two changes. . (9) Mount. (8) Mount in glycerine jelly.

Result Result At this pH, weakly acidic sulphated mucosubstan­ Neutral fats greenish black. ces, hyaluronic acid and sialomucins stain dark Myelin greenish black. blue. Mitochondria greenish black. Nuclei red. Other lipids greenish black. Cytoplasm unstained. 142 Appendix 2: Staining Methods 27 28

Oil Red 0 (ORO) (Luna, 1968) Gomori's Silver Method (Heckner Modification) Purpose Purpose Demonstration of fibres . To demonstrate fat in cytoplasm. Solutions Needed Solutions Needed 0.5% Potassium permanganate 100% Propylene glycol 1% Potassium metabisulphite 60% Propylene glycol in distilled water 2% Ammonium sulphate 0.5% Oil Red 0 solution in 100% propylene Gomori's silver solution" glycol" 4% Formalin Haematoxylin, either Harris's or Mayer's 0.5% Gold chloride Dilute acid, e.g. 0.1% HCI in water 5% Sodium thiosulphate Blueing solution if pH of tap water is below 7.0 2% Ferrous ammonium sulphate Glycerine-gelatin (see appendix I) Giemsa stain Fixation Fixation Air drying and post-fixing in methanol. Air drying. Procedure Procedure (I) Leave slides in potassium permanganate for (I) Post fix in 10% buffered neutral formalin (see 1-2 min. appendix 1). (2) Rinse in tap water,S min. (2) Rinse in distilled water. (3) Destain in potassium metabisulphite for I (3) Leave in 100% propylene glycol, 2 min. min. (4) Stain in Oil Red 0, 10 min. (4) Rinse in tap water for 5 min. (5) Differentiate in 60% propylene glycol, I min . (5) Leave in ferrous ammonium sulphate, 30 s. (6) Rinse in distilled water. (6) Rinse in distilled water, 2 baths, 2 min each. (7) Stain in Haematoxylin, 6 min . (7) Leave slide to dry. (8) Rinse in running tap water for 10 min. (8) Stain in Gomori's solution in Coplin jar, I (9) Mount in glycerine jelly. min . (9) Quickly rinse in distilled water in Coplin jar, Result 5s. (10) Develop in 4% formalin in Coplin jar, 5 min. Fat red. (11) Rinse in tap water in Coplin jar,S min. Nuclei blue. (12) Leave in gold chloride in Coplin jar,S min. (13) Rinse in distilled water. (14) Destain in potassium metabisulphite m Coplin jar, 1 min. (IS) Fix in sodium thiosulphate, I min. (16) Rinse in tap water, 10 min . (17) Stain in Giemsa (see method 35)

Result Reticulum fibres stain black. Appendix 2: Staining Meth ods 143 29 30

Perls' Method for the Demonstration of Iron Gomori's Method for the Demonstration of Iron (Original method by Peds in 1867, this version by (Luna, 1968) AFIP: seeLuna, 1968) Purpose Purpose To demonstrate iron in histiocytes, for instance in To demonstrate iron. sputum.

Fixation Solutions Needed 10% Buffered neutral formalin, air-drying with 20% Hydrochloric acid in distilled water post-fixation in 70% alcohol. 10% Ferrocyanide in distilled water (These two solutions are to be mixed in equal Solutions Needed quantities just before use.) 0.1 % Kernechtrot in 5% aluminium sulphate 10% Potassium ferrocyanide in distilled water, 70ml Fixation 10% Hydrochloric acid, 30 ml (These two solutions together make up the 10% Buffered neutral formalin or absolute alcohol. working solution ofpotassium ferrocyanide- HCl.) Procedure 0.1% Kernechtrot in 5% solution ofaluminium (1) Bring smears to distilled water. sulphate. (2) Leave in HCl-ferrocyanide solution for 30 min. Procedure (3) Rinse thoroughly in distilled water. (1) Bring slides to distilled water. (4) Counterstain in Kernechtrot if wanted. (2) Leave in potassium ferrocyanide solution, 5 (5) Rinse in distilled water. min . (6) Dehydrate in 95% alcohol and absolute alco­ (3) Leave in working solution for 20 min. hol. (4) Rinse well in distilled water. (7) Clear in two changes of xylene and mount. (5) Counterstain in Kernechtrot, 5 min . (6) Rinse well in running water. Result (7) Dehydrate in 95% alcohol and absolute alco­ Iron pigment bright blue. hol. Nuclei and background red ifcounterstain is used . (8) Clear in xylene and mount in Permount.

Result Ferric iron bright blue. Nuclei red. Cytoplasm pale pink. 144 Appendix 2: Staining Methods 31 32

Supravital Staining of Sediments in Serous Rapid Staining for Wet Sediments using either Effusions(Koss, 1979,originaUyFoot and Toluidine Blue or Methylene Blue (Koss, 1979; Holmquist, 1958) originaUyHarris and Keebler, 1976)

Purpose Purpose To differentiate histiocytes and leucocytes from To stain wet sediments. neoplastic and mesothelial cells in effusions. Solutions Needed Solutions needed Methylene Blue" or Toluidine Blue28 Working solution of Neutral Red,27 2 ml Working solution of Janus Green," 5-10 drops Procedure Mix the two solutions. (I) Place a drop ofcentrifuged sediment on a glass slide. Procedure (2) Place a drop of dye solution on the slide and (I) Place 1 or 2 drops of the dye mixture on a clean mix with an applicator stick. slide. (3) Coverslip and leave for 2-5 min. The slide is (2) Invert another slide over it. then ready for microscopic examination. It may (3) Draw the two slides apart so that an evenly be temporarily preserved by applying Vaseline distributed film is left on one side of either slide. or hot wax around the edges. The dye films dry quickly and can be kept indefinitely. Result (4) Smear sediment on dry film and coverslip. Cytoplasm and nucleus blue. Result Granules or vacuoles ofhistiocytes brilliant orange­ red. Polymorphonuclear leucocytes brilliant orange- red. Mitochondria green. Lymphocytes diffuse sky-blue. Neoplastic or mesothelial cells are not stained. Appendix 2: Staining Methods 145 33 34

May-Griinwald-Giemsa Method (Romeis, 1968) The Standardised Romanowsky -Giemsa Staining Method (as recommended by the International Purpose Committee for Standardization in Haematology) To stain blood films, but also for general staining. Purpose Solutions Needed To stain blood or bone marrow films; can also be used as general stain. May-Grimwald's Eosin Y- Methylene Blue stain (stock solution 33) Solutions Needed Giemsa stain (working solution 30, 31 or 32) Stock solution ofstandardised R- G stain Fixation Working solution of standardised R-G stain Air-drying. Procedure Procedure (I) Post-fix in stock solution for 5 min. (2) Rinse briefly in distilled water. (I) Post-fix in May-Grunwald stock solution, by (3) Stain in working solution for 25 min in the case putting 20-30 drops of the solution on the of blood films, for 35 min in the case of bone slide. marrow films. (2) Add the same number of drops of distilled (4) Rinse briefly in distilled water. water, I min. (5) Dry in air. (3) Let fluid run off the slide. (4) Add 10 drops of Giemsa solution, 15-20 min. Result (5) Rinse well in running tap water. (6) Blot dry. Chromatin purple. Nucleoli light blue. Result Basophilic cytoplasm blue. Basophilic granules purple-black. See staining result for the standardised Romanow­ Eosinophilic granules red-orange. sky-Giemsa stain (method 34) Neutrophilic granules purple. Toxic granules black. Platelet granules purple. Haemoglobinised erythrocytes pink- orange. Auer rods purple. Doehle bodies bright blue. Howell-Jolly bodies purple.

When used as a general stain: Results provided separately. 146 Appendix 2: Stain ing Methods 35 36

Giemsa Staining Method (Lopez Cardozo, personal Wright's Staining Method, Lillie Modification, communication) 1965 (Clark, 1973)

Purpose Purpose General stain for cytological material Differentiation of blood corpuscles.

Solutions Needed Solutions Needed Giemsa stock solution, Merck No . 9204 Stock stain solution : Wright's stain powder, 1g; Giemsa working solution: dilute stock solution glycerol, 50 ml; methanol, 50 ml with phosphate buffer at a ratio of 1: 9 Working solution: stock solution, 4 ml; acetone, Sorensen's phosphate buffer at pH 6.8: mix equal 3 ml; M/15 phosphate buffer, pH 6.5, 2 ml; distilled quantities ofMI15 NaH2P0 4and M/15 Na2HP04; water, 31 ml dilute the mixture with distilled water at the ratio' 2: 1 (a low molarity is needed to prevent the buffer Fixation ions from competing with the dye ions) Immediately after taking sample, in methanol for 3 mm. Fixation Air-drying. Procedure

(1) Air-dry blood film after methanol fixation. Procedure (2) Stain in Coplin jar with working solution for 5 (1) Post-fix in methanol for 15 min. min . (2) Stain in Giemsa working solution. Staining (3) Rinse in distilled water. times: in general for 20 min; material with low (4) Air-dry. cell content, like urine, ascites and pleura, 10 min; material with very low cell content, like Result liquor and eye smears, 5 min. Same as Romanowsky-Giemsa stain (method 34). (3) Rinse in tap water and leave slides to dry while they are standing upright. Understained slides can be stained again, whereas overstained slides can be destained by leaving them in methanol for 1-3 min, followed by rinsing in tap water and air-drying.

Quick Staining with Giemsa (1) Post-fix in methanol, 8 min. (2) Stain in mixture ofstock solution and buffer at a ratio of 1 : 1. Appendix 2: Staining Methods 147 37 38

Gram Stain (Koss, 1979) Ferrous Iron Uptake for Melanin (Koss, 1979)

Purpose Purpose To distinguish between Gram-positive and Gram­ To stain melanin. negative bacteria. Solutions Needed Solutions Needed 2.5% Ferrous sulphate in water Crystal Violet33 1.0% Potassium ferricyanide in I% acetic acid Lugol's iodine" 0.1% Kernechtrot in 5% ammonium sulphate 1% Neutral Red in water (dissolvedwith heating) Acetone 1% Acetic acid

Fixation Fixation Air-drying, spray-fixation. 10% Buffered formaldehyde or any routine fix­ ative. Procedure Procedure (I) Bring slide to distilled water. (2) Stain in Crystal Violet, 1-2 min. (I) Bring slides to distilled water. (3) Dip in tap water, 10 dips. (2) Leave in ferrous sulphate, I hour. (4) Stand in Lugol's iodine, 1-2 min. (3) Rinse in 4 changes of distilled water, I min (5) Dip in tap water, 10 dips. each. (6) Dip in acetone, 10-15 dips. (4) Leave in potassium ferricyanide, 30 min. (7) Rinse in running tap water 5-10 min. (5) Dip in I% acetic acid, 15 dips. (8) Stain in Neutral Red, 1-1 min. (6) Dip in distilled water, 15 dips. (9) Dip in two changes of tap water, 15dips each. (7) Stain in Kernechtrot, 1-2 min. (10) Dip in two changes of 95% alcohol, 15 dips (8) Dip in distilled water, 15 dips. each. (9) Dip in 96% alcohol, 15 dips. (II) Dip in absolute alcohol, 15 dips each. (10) Dip in 100% alcohol, 15 dips. (12) Dip in two changes of xylene, 15 dips each. (11) Clear in xylene, 15 dips. (13) Mount. (12) Mount.

Result Result Grain-positive bacteria blue-black. Melanin dark blue to green. Gram-negative bacteria red. Background red to pink. Background pink. 148 Appendix 2: Staining Methods 39 40

Destaining ofPapanicolaou Stain (Koss, 1979) Destaining ofRomanowsky-Giemsa Stain

Purpose Purpose To remove Papanicolaou stain . To remove stain after poor staining.

Procedure Procedure If slides are already coverslipped, remove coverslip Leave slides in methanol until colour has disap­ by soaking in xylene. peared. NB. The methanol should not contain any (l) Rinse slides in 2 to 3 rinses each of absolute acid. Ifit does, subsequent staining with R-G stain alcohol, 95% alcohol and water. This removes will yield not purple but blue nuclei. counterstain. (2) Leave slides in aqueous 0.2-0.5% hydrochloric acid, 5 min to I hour to remove dyes. Check under microscope to see whether all dye is removed. (3) Remove acid by rinsing in running tap water, 10-15 min. To make sure all acid is removed, place slides in Scott's tap water substitute with pH of about 8.2 and rinse again in tap water.

Result This process leaves the slides ready either for restaining with Papanicolaou's stain in case the first stain was poor or with a particular stain to colour specific cell components.

Note: Destaining in general: Leave the slides in the dye solvent until colour has disappeared. Appendix 3

Staining Solutions

Nuclear stains 1-12 Schiff's reagent 13 Cytoplasmic stains 14-21 Glycogen stains 22,23 Fat stains 24,25 Fibre stain 26 Supravital stains 27-29 Blood stains 30-32 Stain for bacteria 33 Appendix 3: Staining Solutions 151 1 2

Delafield's HaematoxyUn (Original Recipe, taken Harris's HaematoxyUn (Romeis, 1968) from BoUes-Lee's Microtomist's Vademecum) Purpose Purpose To stain nuclei regressively. To stain nuclei regressively. Chemicals Needed Chemicals Needed Haematoxylin, 5 g Haematoxylin, 3.2 g Absolute alcohol , 50 ml 95% Alcohol, 20 ml Potassium or ammonium alum, 100g Saturated aqueous aluminium alum, 320ml Mercuric oxide, 2.5 g Glycerine, 80 ml Glacial acetic acid, 40 ml or none Methanol, 80 ml Distilled water, 950 ml

Preparation Preparation (1) Dissolve Haematoxylin in alcohol. (1) Dissolve Haematoxylin in alcohol. (2) Add drop by drop to ammonium alum. (2) Dissolve alum in water by heating. (3) Leave exposed to air and light for I week. (3) Mix Haematoxylin and alum solution and heat (4) Filter. mixture to boiling point. (5) Add glycerine and methanol. (4) Remove from heat; add mercuric oxide. (6) Leave for 6-8 weeks till solution becomesdark. (5) Cool quickly by plunging in cold water as soon (7) Before using, dilute with an equal amount of as mixture turns dark purple . distilled water. (6) Filter. The solution keeps very well. The stain solution can be used immediately and Differentiation in acid and blueing in alkali are keeps for a long time. needed. Differentiation in acid and blueing in alkali are needed. Result Result Dark-blue nuclei. Dark-blue nuclei. 152 Appendix 3: Staining Solutions 3 4

Mayer's Haematoxylin Weigert's Haematoxylin (Romeis, 1968)

Purpose Purpose To stain nuclei progressively. To stain nuclei regressively.

Chemicals Needed Chemicals Needed Haematoxylin, 1g Haematoxylin, 5 g Distilled water, 1000 ml 95% Alcohol, 500 ml Sodium iodate, 0.2 g Ferric chloride, 5.8 g Aluminium alum, 50 g Concentrated HCI, 5 ml Glacial acetic acid, 40 ml or none Distilled water, 490 ml or citric acid, I g Chloral hydrate, 50 g Preparation (1) Dissolve Haematoxylin in alcohol. Preparation (2) Dissolve ferric chloride in distilled water and (I) Combine all ingredients in order of listing. add HCI. (2) Stir on magnetic mixer for about an hour at (3) Mix the two solutions in equal quantities just room temperature. before staining. The solution can be used immediately and keeps The two solutions should be kept separate. In that well. way they can be kept for years. The mixture can Differentiation in acid should not be necessary; only be kept for 8 days. blueing is. Differentiation in acid and blueing in alkali are not needed. Result Result Blue nuclei. Black nuclei. Appendix 3: Sta ining Solut ions 153 5 6

Cole's Haematoxylin (Cole, 1943) Gill's Haematoxylin (Original Recipe)

Purpose Purpose To stain nuclei progressively. To stain nuclei progressively.

Chemicals Needed Chemicals Needed Haematoxylin, 0.5 g Haematoxylin, 2.0 g Iodine, 0.5 g Sodium iodate, 0.2 g (The amount ofsodium Warm water, 250ml iodate should be very accurately measured 96% Alcohol, 50 ml (accuracy ± 0.001 g.) Saturated ammonium alum solution in water, Aluminium sulphate, A12(S04)3.18H20, 17.6 g 700ml Ethylene glycol, 250 ml Distilled water, 730 ml Preparation Glacial acetic acid, 20 ml (1) Dissolve Haematoxylin in warm water. Preparation (2) Add the iodine solution. (3) Add ammonium alum. (1) Mix all ingredients in order of listing. (4) Bring to the boil. (2) Stir for 1h on magnetic mixer at room tem- (5) Allow to cool quickly. perature. For a stronger stain the dye quantity may be The solution keeps very well. doubled. Differentiation in weak acid and blueing in Scott's The solution keeps very well. tap water substitute needed. Check pH . It should not be higher than 3.0 for optimal results. Result Dark-blue nuclei. Result Nucleoli less darkly stained. Blue nuclei. 154 Appendix 3: Staining Solutions 7 8

Carminic Acid, Carmalum (Originally Mayer, Coelestine Blue B (yasumatsu, 1977) Taken from Romeis, 1968) Purpose Purpose Nuclear stain in all procedures where Haematox­ To stain nuclei. ylin is used.

Chemicals Needed ChemicalsNeeded Carminic acid, 1.0g Coelestine Blue B, 1.0 g Potassium alum, 10.0 g 4 N RN03, 0.4 ml Distilled water, 200.0ml 5% Ferric alum, 200.0ml at 60·C

Preparation Preparation

(1) Dissolve dye and potassium alum in water with (1) Add RN03 to Coelestine Blue in beaker, stir heat. and mix with glass rod . (2) Leave to cool. (2) Add Fe-alum and mix well till dissolved. (3) Filter. (3) Filter when mixture has cooled down to room (4) Add 1ml formol or 0.2 g salicylic acid to temperature. prevent mould . (4) Filter before use. Appendix 3: Stain ing Solut ions 155 9 10

Coelestine BlueB (Gray et al., 1956) GaUocyanin ChromeAlum(Sandritter et al., 1966; alsoBrownand Scholtz, 1979) Purpose Purpose Nuclear stain in all procedures where Haematox­ ylin is used. Nuclear stain for the quantitative assessment of nucleic acids and as a general nuclear stain to Chemicals Needed replace Haematoxylin. Coelestine Blue B, 1.0 g Chemicals Needed Concentrated H2S0 4, 0.5 ml Ferric alum, 2.5 g Chrome alum, 5 g Distilled water, 100.0ml Gallocyanin, 0.15 g Glycerol, 14.0 ml Distilled water, 100ml HClorNaOH Preparation Preparation (1) Rub together dye and sulphuric acid in beaker. (2) Mix ferric alum, distilled water and glycerol at (1) Dissolve chrome alum in water. 50·C. (2) Add dye and heat slowly until dissolved. (3) Mix the two solutions. (3) Bring the mixture to pH 1.64 with a few drops (4) Leave to cool. of concentrated HC1,checking carefully with a (5) Adjust to pH 0.8 with concentrated sulphuric pH meter. acid. (4) The dye solution will only keep for about 3 weeks.

NB The stain can be used in the same sequences as Haematoxylin. It has the advantage of being a progressive stain and the disadvantage of needing a very long staining time. 156 Appendix 3: Staining Solutions 11 12

Methyl Green-Pyronin Y (Taft, 1951; also Clark, Stains and Solutions for Love's Toluidine Blue 1973) Molybdate Method for the Staining of Nucleolini (Love et aJ., 1973) Purpose Solutions Needed To stain DNA and RNA differentially. Mcllvaine's buffer pH 3.0: MilO citric acid, 16.5 ml; Chemicals Needed M/5 disodium phosphate, 3.5 ml. These two stock solutions are made in 25% methanol. The actual Methyl Green, 0.5 g buffer is made up by adding water at a ratio of I : 25 Pyronin Y (pure dye), 0.05 g Staining solution: Toluidine Blue, I g; distilled Hot distilled water, 100ml water, 100 mt. Leave the solution overnight in a Chloroform shaking machine. Working solution: stock Toluidine Blue solution, 0.75 ml; McIlvaine's buf­ Preparation fer, 49.25 ml. (I) Add Methyl Green to hot water. Lugo/'s iodine (see Stain 22) (2) Cool. Sodium thiosulphate: Na2S203.5H20, 5.0 g; distilled (3) When cool, extract the solution in separating water, 100.0 ml. funnel with successive 20-30 ml aliquots of Deoxyribonuclease solution: Trishydroxymethyl chloroform until the latter remains colourless aminomethane buffer (0.045 M MgCI2.6H20 and or is only slightly tinged with green. 5mM CaCl2per 100 ml distilled water: the buffer is (4) Add Pyronin Y and shake to dissolve. brought to pH 7.3 by adding IN HCl); DNA-ase, (5) Store in amber, glass-stoppered bottle. 1mg. Dissolve the DNA-ase in the buffer. Use the solution at 37"C No filtering needed before use. The solution can be Ammonium molybdate: 3(NH 2 0.7 Mo0 , reused. 4) 3.4H20 15.0g; demineralised water, 100.0 ml (the water should be demineralised double glass or quartz distilled). Tertiary butanol: to be used at a temperature above its melting point ( = 22SC) or mixed with a small amount of 95% alcohol. Appendix 3: Staining Solutions 157 13 14

Schiff's Reagent Leiden - EA (Drijver and Boon, 1983b)

Purpose Purpose

To be used in the periodic acid Schiff reaction for Cytoplasmic stain in Papanicolaou sequence III the demonstration ofglycogen, and in the Feulgen which acidity is variable. reaction for the demonstration of DNA. Chemicals Needed Chemicals Needed Eosin Y, 1.8g Basic Fuchsin, 1g Light Green Y, 0.7 g

NaHS03,2g 50% Alcohol, 1000ml INHC1,20ml for pH 4.6, 3.0 g Phosphotungstic acid Charcoal, 500mg Distilled water, 80 ml The pH can be adjusted as desired. When OG is used, the pH ofthe EA has to be6.5. Preparation This can be achieved by adding lithium carbonate. (1) Dissolve the dye in the water. Preparation (2) Add NaHS03 and HCI. (3) Stopper tightly and shake at intervals for 2 h. Mix all ingredients. Solution should now be clear and light yellow. Check pH . (4) Add charcoal, shake 1 min and filter. Solution should be clear and colourless. Keep the solution at 5°C. It keeps for several months at this temperature. If the solution turns pink, it should be discarded. 158 Appendix 3: Staining Solutions 15 16

Eosin-Azure (EA) in Papanicolaou's Staining Gill's Modified EA (Gill, 1977) Method (Koss, 1979, slightly modified) Purpose Purpose General cytoplasmic stain in Papanicolaou's stain­ To stain cytoplasm differentially in the Papan­ ing sequence. icolaou sequence. Chemicals Needed Chemicals Needed Eosin Y in water, 4.0 g EA 35 EA 65 EAfor Light Green Y in water, 0.3 g Saccomano 's 95% Alcohol, 700 ml method Methanol, 250 ml Eosin Y 2.25g 2.25g 2.25g Glacial acetic acid, 20 ml Light Green Y 0.45g 0.23g 0.33g Phosphotungstic acid, 2 g Phosphotungstic 2.0 g 6.0 g acid Preparation Saturated lithium 10 drops Mix all ingredients. carbonate 95% Alcohol 1000mi

Koss recommends making aqueous stock solutions first. The quantities given here are calculated from Koss's table for the preparation ofstock solutions. It is, however, quite possible to make up the solutions from the quantities given above, and dissolve immediately in 96% alcohol. We have omitted Bismarck Brown since this does not add to the staining effect. It is also omitted in most commercial EA stains. Appendix 3: Stain ing Solutions 159 17 18

Gill'sModified OrangeG-6(Gill, 1977) Orange G (Koss, 1979)

Purpose Purpose To stain highly keratinised cells in the To stain highly keratinised cells in the Papanicolaou staining sequence . Papanicolaou staining sequence

Chemicals Needed Chemicals Needed 4.0 g Orange G in 95% alcohol, 20 g OrangeG,5g 95% Alcohol , 980 ml 96% Alcohol, 1000ml Phosphotungstic acid, 0.15 g Phosphotungstic acid, 0.15 g

Preparation Preparation Mix all ingredients. Mix all ingredients. 160 Appendix 3: Staining Solutions 19 20

Orange G- Leiden (unpublished) Shorr's Stain(Shorr, 1942)

Purpose Purpose To stain highly keratinised cells differentially in For hormonal assessment. Papanicolaou's staining method. Chemicals Needed Chemicals Needed Biebrich Scarlet, 0.5 g Orange G, 2.0 g Orange G, 0.25 g Phosphotungstic acid, sufficient to give a pH Fast Green FCF, 0.075 g between 2.0 and 2.8 for optimal staining Phosphotungstic acid, 0.5 g 50% Alcohol, 1000ml Phosphomolybdic acid, 0.5 g Glacial acetic acid, 1.0 ml Preparation Preparation Mix all ingredients. Check pH. The pH ofthe EA should be around 6.5. Mix all ingredients. Appendix 3: Staining Solutions 161 21 22

Rakoff's Stain (Rakoff, 1960) Lugol's Iodine (Luna, 1968)

Purpose Purpose To stain gynaecological smears for hormonal To stain glycogen; also for the removal of assessment. sublimate.

Chemicals Needed Chemicals Needed 5% Light Green Y in water, 83 ml Iodine, 1.0g 1% Eosin Y in water, 17ml Potassium iodide, 2.0 g Distilled water, 100.0 ml Preparation Preparation Mix the two solutions. Mix all ingredients. 162 Appendix 3: Staining Solutions 23 24

Best's Carmine (Luna, 1968) Oil Red0 (Luna, 1968)

Purpose Purpose To stain glycogen. To demonstrate fat in cytoplasm.

Chemicals Needed Chemicals Needed For stock solution: Oil Red 0, 0.5 g Carmine, 2.0 g Propylene glycol, 100% 100.0 ml Potassium carbonate, 1.0 g Potassium chloride, 5.0 g Preparation Distilled water, 60.0 ml (I) Add a small amount ofpropylene glycol to the 28% NH 20.0ml 40H, dye and mix well; crush larger pieces. For working solution: (2) Gradually add the remainder of the propylene Carmine stock solution, 10.0ml glycol stirring occasionally. 28% NH 15.0ml 40H, (3) Heat gently until solution reaches 95°C, do not Methanol, 15.0 ml allow temperature to go above 100°C.Stir while heating. Preparation (4) Filter through coarse filter paper while still Stock solution: warm. (5) Allow to stand overnight at room temperature. (I) Boil together first four ingredients in an (6) Filter through Seitz filter with the aid of a evaporating dish very gently for several min­ vacuum. (When using Seitz filter put rough utes. surface of filter uppermost.) If solution be­ (2) When cool add ammonium hydroxide. Store in comes turbid, refilter. a refrigerator. Working solution: mix all ingredients. Appendix 3: Sta ining Solut ions 163 25 26

Sudan Black B (Clark, 1973) Gomori's Silver Stain for Fibres (Heckner Modification) Purpose Purpose To demonstrate fat in cytoplasm. To stain fibres. Chemicals Needed Chemicals Needed Sudan Black B, 0.7 g Propylene glycol, 100ml 10% Silver nitrate, 40 ml Distilled water, 40 ml Preparation 10% KOH solution, 3 ml 25% Ammonia The preparation method is the same as that for Oil Use acid-cleaned glassware RedO. Preparation (I) Add 2.5 ml of the KOH to 10ml of the silver nitrate solution. (2) Add 25% ammonia drop by drop while shaking the container continuously until brown precipitate is completely dissolved. (3) Add again 4 drops of silver nitrate solution for every 10ml of silver nitrate used. (4) Double the volume ofthe solution with distilled water. 164 Appendix 3: Staining Solutions 27 28

Neutral Red and Janus Green (Koss, 1979; Toluidine Blue (Koss, 1979; OriginaUy Harris and OriginaUy Foot and Holmquist, 1958) Keebler, 1976)

Purpose Purpose To stain histiocytes and leucocytes differentially To stain quickly for wet sediment examination. from mesothelial and neoplastic cells. Chemicals Needed Chemicals Needed Toluidine Blue, 0.5 g Saturated solution of Neutral Red in 100% 95% Alcohol , 20.0 ml alcohol. Distilled Water, 80 mi Saturated solution of Janus Green in 100% alcohol. Preparation (1) Dissolve dye in alcohol and add water. Preparation ofWorking Solution (2) Filter and store in a dark bottle in a re­ Neutral Red solution: 20-50 drops in 10mIIOO% frigerator. alcohol. Janus Green solution: 15-30 drops to IOml 100% alcohol. Appendix 3: Staining Solutions 165 29 30

Methylene Blue(Koss,1979; Originally Harris and Giemsa Stain(Original Recipe) Keebler, 1976) Purpose Purpose Differentiation of blood corpuscles, general stain. To use as a quick stain for wet sediment examina­ tion. Chemicals Needed Azure II-Eosin Y, 3.0 g (Azure II is a mixture of Chemicals Needed Azure B and Methylene Blue in equal quantities) Methylene Blue, 1.5 g Azure II, 0.8 g 95% Alcohol, 30.0 ml Glycerol: for blood, 250 g or 200 ml; for tissue, 0.1 N Potassium hydroxide, 2 ml 125g or 100ml Methanol (neutral, acetone free): for blood, 250g Preparation or 312ml; for tissue, 375 g or 457 ml (l) Dissolve the dye in the alcohol and add the Preparation ofStock Solution KOH. (2) Store in a dark bottle in a refrigerator. (I) Mix glycerol and methanol. (2) Dissolve the dyes in the mixture. (3) Stand at room temperature overnight. (4) Shake well for 5-10 min. (5) Pour without filtering into dark screw-cap bottle. (6) Store at room temperature.

Preparation ofWorking Solution Mix 5 ml of stock solution with 65ml of water. 166 Appendix 3: Staining Solutions 31 32

Giemsa Stain(LillieModification, 1943b) Standardised Romanowsky -Giemsa Stain (Recommended bythe International Committee for Purpose Standardization inHaematology) To stain blood corpuscles and to use as general Purpose stain. Differentiation of blood corpuscles; can also be Chemicals Needed used as general stain. Azure A- Eosinate, 0.5 g Chemicals Needed Azure B-Eosinate, 2.5 g Methylene Blue-Eosinate, 2.0 g For stock solution Methylene Blue chloride, 1.0g Azure B-thiocyanate, 3 g Glycerol, 375.0 ml Eosin-disodium salt, 1g Methanol, 375.0 ml Methanol, 600 ml Dimethylsulphoxide, 400 ml Preparation ofStock Solut ion For working solution Stock solution (1) Mix glycerol and methanol. Hepes buffer, pH 6.8 (2) Dissolve dyes in the mixture. (3) Stand at room temperature overnight. Preparation (4) Shake well for 5-10 min. (5) Pour into dark screw-cap bottle without filter­ Stock solution: ing. (1) Dissolve Azure B: add 400 ml of DMSO to the (6) Store at room temperature. Azure B powder at 37°C. (2) Add 1g of Eosin Y to 600 ml methanol. Wait Preparation ofWorking Solution until the dye is completely dissolved . (3) Slowly mix Azure B-DMSO with Eosin- Mix 5 ml of stock solution with 65 ml of water or methanol. phosphate buffer, pH 6.8. This solution keeps for many months at room temperature if stored in a glass bottle in the dark. To improve stability the stock solution may be kept at lower pH (4.0) by adding hydrochloric acid. Working solution: Mix stock solution and buffer at a ratio of 1 : 15. For rapid staining, mixtures with a higher proportion of stock solution may be used. Appendix 3: Staining Solutions 167 33 34

May-Griinwald's Eosin Y- Methylene Blue Stain Crystal Violet in Gram Stain (Koss, 1979) (from Clark's Staining Procedures, 1981) Purpose Purpose To stain Gram-positive bacteria. To stain blood films and for general staining. Chemicals Needed Chemicals Needed Crystal Violet, 5.0 g Eosin Y, 0.5 g 95% Alcohol, 50.0 ml Methylene Blue, 0.5 g Ammonium oxalate, 2.0 g Distilled water, 100 ml Distilled water, 200.0 ml Absolute methanol, 50 ml Preparation Preparation ofStock Solution (I) Dissolve dye in alcohol. (I) Mix Eosin and Methylene Blue. (2) Dissolve ammonium oxalate in water. (2) Filter. (3) Mix the two solutions and filter before use. (3) Dry filtrate. (4) Wash residue and dry. (5) Dissolve residue in methanol. Appendix 4

Dyes

This appendix provides a list ofdyes either mentioned in the text or used in one of the staining methods. The following data are provided for each dye: structure (taken from Conn's Biological Stains (Lillie, 1977), unless otherwise stated); ionic or molecular weight; Hansch 1t value; conjugated bond number; net charge; absorption maximum; solubility; main uses; and the CI number. When uses for the dyes are mentioned in either appendix 2 or 3, the reader is referred to the appropriate method or stain. Further information may be found elsewhere in the book, or in Conn's Biological Stains (Lillie, 1977). Ionic weight, Hansch 1t value, conjugated bond number and net charge are kindly provided by Dr R.W. Horobin of the University of Sheffield. Appendix 4: Dyes 171

AcidFuchsin Uses

CI no. 42685 As a cytoplasmic stain in many polychrome mix­ (Derivative of Rosanilin or Pararosanilin) tures (Mallory, 1900;Masson, 1929; Goldner, 1938; Foot, 1938; Papanicolaou, 1941).

ionic weight 540 Hansch 1t value -17.3 conjugated bond number 24 charge 2­ absorption maximum 540-546nm solubility in water + in alcohol ± Cellosolve +

Acridine Orange Uses

CI no. 46005 As fluorochrome for nucleic acids; as selective stain for tumour cells.

ionic weight 266 Hansch 1t value - 2.7 conjugated bond number 18 charge 1+ absorption maximum 467-497nm solubility in water + in alcohol + 172 Appendix 4: Dyes

AlcianBlue

CI no. 74240 Structure uncertain Uses

As differential stain for acid polysaccharides; can be used in combination with PAS reaction.

x ionic weight 1380 Hansch 1t value - 14.8 conjugated bond number 48 charge 4+ x absorption maximum blue (can also stain blue-green metachromatically) solubility in water + in alcohol ?

Alizarin Blue Uses

CI no. 67415 As a mordant nuclear dye (mordant Fe), when it stains metachromatically (Lillie, 1977).

ionic weight 291.266 without 2NaHSO) Hansch 1t value ? 2NaHS03 conjugated bond number ? charge 1 ­ absorption maximum blue solubility o OH in water ± in alcohol Appendix 4: Dyes 173

Amido BlacklOB Uses

CI no. 20470 As protein stain in paper chromatography; as (Synonym Pontacyl Blue Black SX) nucleolar stain (see Method 18). (Mundkur and Greenwood, 1968.)

ionic weight 571 Hansch It value - 5.0 conjugated bond number 34 charge 2­ absorption maximum blue solubility in water + in alcohol + in Cellosolve +

Aniline BlueWS

CI no. 42755

The dye is usually a mixture of a number of ionic weight 692 components, differing in degree of sulphonation Hansch It value - 13.3 and phenylation, conjugated bond number 36 charge 2­ Uses absorption maximum 600nm solubility As a component of connective tissue stain in water + (Mallory, 1900; Masson, 1929); in Papanicolaou's in alcohol early cytoplasmic stain (Papanicolaou, 1933); in Heidenhain's Azan (Lillie, 1977). 174 Appendix 4: Dyes

Azure A Uses

CI no. 52005 The dye often forms part of a Methylene Blue Structure by Comings (1975). solution since it is a derivative ofthat dye. Used as a nuclear dye, and as an RNA stain in combination with a surfactant (Bennion et al., 1975; see Method 20). CH3~ N -0 S+J)N/H ionic weight 256 CH / "H 3 N Hansch 1t value - 4.2 conjugated bond number 18 charge 1+ absorption maximum 630nm as orthochromatic dye solubility in water + in alcohol +

Azure B ( = Azure I) Uses

CI no. 52010 Itwas originally one ofthe components ofGiemsa's Structure by Comings (1975). blood stain; now it is the azure dye in the standard­ ised blood stain (International Committee for Standardization in Haematology recommendation, 1984). See Method 34.

ionic weight 260 Hansch 1t value - 3.6 conjugated bond number 18 charge 1 + absorption maximum 650 nm (This is the wavelength of the orthochromatic dye) solubility in water + in alcohol ± Appendix 4: Dyes 175

AzureC Uses

CI no. 52002 No known uses as individual dye, it is usually Structure by Comings (1975). present in Methylene Blue solutions since it is one of the derivatives of that dye.

ionic weight 243 Hansch 7t value - 3.0 conjugated bond number 18 charge 1 + absorption maximum 620 nm (This is the wavelength ofthe orthochromatic dye) solubility in water + in alcohol ±

BasicFuchsin Uses

CI no. 42500 In Schiff's reagent (see Stain 13);as nuclear dye in mucin and elastic tissue staining method.

Parameters given are those for Pararosanilin since this forms the main component of the dye.

ionic weight 288 Hansch 7t value - 3.5 conjugated bond number 24 charge 1+ absorption maximum 545nm solubility in water + in alcohol 176 Appendix 4: Dyes

Biebrich Scarlet Uses

CI no. 26905 As cytoplasm stain in Shorr's polychrome stain for hormonal assessment (see Method 6). For staining basic proteins at graded pH (Spicer, 1961). For staining Barr bodies in Guard's method (see Method 21). HO ionic weight 510

Nao3s-Q-N=N-q-N=ND Hansch 1t value - 3.5 conjugated bond number 30 S03Na 0 charge 2­ absorption maximum 503nm solubility in water + in alcohol in Cellosolve

BismarckBrownY Uses

CI no. 21000 For vital staining; as component in Papanicolaou's EA stain.

ionic weight 349 Hansch 1t value - 5.7 conjugated bond number 27 charge 1+ absorption maximum ? solubility in water + in alcohol ±

BrilliantCresylBlue Uses

CI no. 51010 As vital stain for the staining ofacid mucopolysac­ charides.

molecular weight 332 HC-(yN~2 Hansch 1t value ? conjugated bond number ? (HsC):NVOVNH2 charge 1+ + absorption maximum 630 CI- solubility in water + in alcohol + Appendix 4: Dyes 177

Carmine and Carminic Acid Uses

CI no. 75470 As chromalum carmine (Fyg's: Lillie, 1977) giving The dye principle of Carmine is Carminic Acid. blue - black nuclei; in Best's carmine for glycogen There are other possibilities for the structure. (see Method 22).

HO 0 ionic weight 492 OH Hansch 1t value - 2.02 conjugated bond number 24 eOOH charge I­ absorption maximum red in Best's solubility in water ± in alcohol ±

Chlorazol Black E

CI no. 30235

Uses

As direct nuclear and cytoplasmic dye, very good ionic weight 735 for photography. Hansch 1t value -3.4 conjugated bond number 49 charge 2­ absorption maximum black solubility in water + in alcohol ± in other organic solvents in Cellosolve ± 178 Appendix 4: Dyes

Coelestine BlueB Uses

CI no. 51050 As nuclear stain, as substitute for Haematoxylin.

ionic weight 328 Hansch 1t value -5.9 conjugated bond number 21 charge 1+ absorption maximum 654nm solubility OH in water + in alcohol +

Coomassie Brilliant Blue

CI no. 42655 Structure by Tas et a/. (1980).

Uses molecular weight 818 Hansch 1t value - 1.38 As protein stain in quantitative cytochemistry (Tas conjugated bond number 43 et a/., 1980) . charge 1- absorption maximum 590 nm (This is the wavelength when the dye is bound to protein , in solution the colour is dependent on pH.) solubility in water ± in alcohol + Appendix 4: Dyes J79

Cresyl Violet Uses

No CI number. As metachromatic vital stain : it stains nuclei violet and plasma blue, amyloid bodies, mast cell granules The formula is that ofCresyl Acetate, which is one and mucin red; as protein stain at graded pH; as of the forms in which the dye is used. However the Nissl stain. structure is undecided. No data on parameters are available. molecular weight 321 Hansch 1t value ? conjugated bond number ? N charge 1+ absorption maximum 600nm solubility in water H2N-Q + in alcohol +

Crystal Violet (synonym Gentian Violet) Uses

CI no. 42555 As nuclear stain; as vital stain for amyloid bodies in fresh or fixed tissue; to stain blood platelets; in Gram stain .

ionic weight 372 Hansch 1t value + 0.1 conjugated bond number 24 charge 1+ absorption maximum 589-593 nm solubility in water ± in alcohol +

Cuprolinic Blue (Quinolinic Phthalocyanin) Uses

No CI number. As specific stain for nucleic acids; in combination Structure by Tas et aJ. (1983) . with MgCI it is a specific stain for RNA (Tas et al., 1983; see Method 19).

ionic weight 640 Hansch 1t value - 21.6 conjugated bond number 44 charge 4+ absorption maximum 635nm solubility in water + in alcohol ? 180 Appendix 4: Dyes

DinitroOuorobenzene Uses

No CI number. As protein stain in quantitative cytochemistry (Tas Structure by Tas et al. (1980). et al., 1980).

ionic weight 186 Hansch 1t value + 1.78 conjugated bond number 13 charge o absorption maximum 400 solubility in water + in alcohol +

EosinY Uses

CI no. 45380 A counterstain in several polychrome stains includ­ ing Papanicolaou's in Romanowsky-Giemsa stains (Methods 1-4 and 34).

ionic weight 646 NaohO~o Hansch 1t value 0.0 conjugated bond number 31 srVCvsr charge 2- absorption maximum ""515 nm solubility OCOONa in water + in alcohol + Appendix 4: Dyes 181

FastGreen FCF

CI no. 42053

Uses ionic weight 763 Hansch 1t value - 10.6 As cytoplasmic stain in Shorr's stain (Method 20, conjugated bond number 36 Stain 6); as protein stain for quantitative cyto­ charge 2­ chemistry (Smetana and Busch, 1966; Tas et al., absorption maximum 625nm 1980; Dhar and Shah, 1982). solubility in water + in alcohol +

Fluorone Black Uses

No CI number. As mordant nuclear stain as Haematoxylin sub­ stitute, tested by Lillie et af. (l975b); it gives black nuclei.

HO~O ionic weight 320 Hansch 1t value +3.4 HO~C?A)-OH conjugated bond number 27 charge o absorption maximum black solubility in water + o in alcohol ± 182 Appendix 4: Dyes

Gallein Uses

CI no. 45445 As nuclear stain as Haematoxylin substitute, tested by Lillie et al. (I 975a); as glycogen sta in (Mur­ gatroyd and Horobin, 1969).

ionic weight 361 HO OH Hansch 1t value - 4.6 conjugated bond number 36 charge 2- HO@O absorption maximum: stains blue with Fe as mor­ dant solubility in hot water ± O-COOH in alcohol + in alkali + in acetone + in ether in chloroform in benzene

Gallo Blue E Uses

COOCH3 CI no. 51040 As nuclear dye as Haematoxylin substitute, tested by Lillie et al. (l976b).

OH No data available. OH

Gallocyanin Uses

CI no. 51030 As chrome alum mordant dye; as a substitute for Haematoxylin; for quantitative spectro­ photometry.

ionic weight 301 Hansch 1t value -8.0 conjugated bond number 22 charge 1+ absorption maximum ± 636nm solubility in water in alcohol + in Cellosolve + in glycol +

Structure by Horobin (1982). Appendix 4: Dyes 183

Haematein Uses

CI no. 75290 Since it forms the actual dye in a Haematoxylin solution, it is used as a nuclear dye.

ionic weight 300 Hansch 7t value + 1.6 conjugated bond number 18 charge o absorption maximum 445nm solubility in water + in alcohol + in Cellosolve + in glycol +

Haematoxylin Uses

CI no. 75290 Mainly as nuclear dye after oxidation to Haematein; Lillie (1977) mentioned three more HO main uses.

HoHcH2 ionic weight 302 Hansch 7t value + 1.0 U~P-OH conjugated bond number 16 charge o absorption maximum 292nm $' solubility HO OH in water + in alcohol + in Cellosolve + in glycol + 184 Appendix 4: Dyes

Janus Green B

CI no. 11050 (H5C,l2N-OnN~N-o-N(CH3)2

6 CI

Uses ionic weight 300 Hansch 7t value + 1.6 As supravital stain (see Method 31). conjugated bond number 18 charge o absorption maximum 400 and 610-623nm solubility in water + in alcohol ±

Kernechtrot Uses

CI no. 60760 For the demonstration of calcium; also for the demonstration of iron, as in Perls' and Gomori's methods (see Methods 29 and 30). a NH molecular weight 357 ~OH Hansch 7t value ? conjugated bond number ? ~S03Na charge I­ absorption maximum red a OH solubility in water ± in alcohol ? Appendix 4: Dyes 185

Light Green Y

CI no. 42095

Uses ionic weight 747 Hansch 7t value - 9.5 As cytoplasmic stain in many polychrome stains, conjugated bond number 34 including Papanicolaou's (see Stains 14-16); as charge 2- protein stain for quantitative cytochemistry (Oud et absorption maximum 428 and al., 1984). 629-634nm solubility in water + in alcohol in Cellosolve ±

LuxolFastBlueG

No CI number.

Uses Solubility in water To stain myelin sheaths (polar lipids). in alcohol + in Cellosolve + No data available on parameters. in methanol + in isopropanol + in acetone + 186 Appendix 4: Dyes

Methyl Green Uses

CI no. 42585 As differential stain for DNA in combination with Pyronin Y which stains RNA (see Methods 14and 15).

ionic weight 387 Hansch 1t value - 5.5 conjugated bond number 23 charge 2+ absorption maximum 420 and 630-634nm solubility in water + in alcohol

Methylene Blue Uses

CI no. 52015 As direct nuclear dye; as vital stain for wet Structure by Comings (1975). sediments; in the early blood stains where it was thought to be the main nuclear dye.

ionic weight 284 Hansch 1t value - 2.7 conjugated bond number 18 charge 1+ absorption maximum 660 solubility in water + in alcohol + in Cellosolve + in glycol + Appendix 4: Dyes 187

Methylene VioletBernthsen Uses

CI no . 52041 As nuclear stain in MacNeal's blood stain (Lillie, 1977).

ionic weight 256 Hansch 1t value + 2.9 conjugated bond number 18 (H C)2 3 N((Q charge 0 absorption maximum + 580 nm (This is the wavelength ofthe orthochromatic dye.) solubility in water in alcohol + in ether + in chloroform +

Naphthol Yellow S Uses

CI no . 10316 As protein stain for quantitative cytochemistry can be used in sequence with the Feulgen reaction; Structure by Tas et al. (1980). Method 13 (Tas et al., 1980).

ONa ionic weight 312 Hansch 1t value - 5.97 conjugated bond number 18 Nao3 N0 2 s-CO- charge 2- absorption maximum ± 420nm N0 solubility 2 in water + in alcohol ±

Neutral Red Uses

CI no. 50040 As vital stain in combination with Janus Green.

There are two other possibilities for the structure. ionic weight ? This is the orthoquinoid structure given by the Hansch 1t value ? Colour Index in 1956. conjugated bond number ? charge ? absorption maximum 530nm solubility in water + in alcohol ± 188 Appendix 4: Dyes

Nile Blue Uses

CI no. 51180 As fat stain; the sulphate differentiates between neutral fats and fatty acids (see Clark, 1973).

ionic weight 318 Hansch 1t value - 2.4 conjugated bond number 23 charge 1+ absorption maximum ± 640nm solubility in hot water + in alcohol +

Oil Red 0 Uses

CI no. 26125 As fat stain (see Method 27).

ionic weight 409 Hansch 1t value + 8.99 conjugated bond number 30 charge o absorption maximum ± 550nm solubility in water in alcohol + in acetone + in isopropanol +

OrangeG Uses

CI no. 16230 As a cytoplasmic stain in many polychrome stains, especially to demonstrate keratin in Papanicolaou's sequence; as a general protein stain in quantitative cytochemistry (Oud et al., 1984). HO ionic weight 406 C)-N=N-Q Hansch 1t value - 5.2 conjugated bond number 21 Na03S-Q charge 2- absorption maximum ± 480nm S03Na solubility in water + in alcohol ± in Cellosolve + in glycol + Appendix 4: Dyes 189

Orcein Uses

CI no. (Ed. I) 1242 It is used as a nuclear dye (it colours chromatin No specific structure can be given for this since bright red). several dye fractions may be presen t. No character­ istic parameters are available.

Pararosanilin Uses

CI no. 42500 Together with Rosanilin it forms the main con­ stituent of Basic Fuchsin; hence it is used for the same purposes.

ionic weight 288 Hansch 1t value - 3.5 conjugated bond number 24 charge 1+ absorption maximum ± 545nm solubility in water ± in alcohol +

Picric acid Uses

CI no. 10305 As a fixative in Bouin's; in combination with other anionic dyes for differentiation after dyeing with Haematoxylin.

ionic weight 228 II Hansch 1t value - 2.8 OH conjugated bond number 17 charge 1- absorption maximum ± 360nm O,N«NO, " in alcohol solubility in water ± N02 in alcohol + in benzene + in chloroform ± in ether ± 190 Appendix 4: Dyes

Ponceau deXylidine

CI no. 16150 CH~N=N~3Na

S03Na

Uses ionic weight 434 Hansch 7t value -4.0 As component of Masson's trichrome stain; as conjugated bond number 21 cytoplasmic stain . charge 2- absorption maximum ± 500nm solubility in water + in alcohol in acetone

Pontacyl Dark Green B Uses

CI no. 20495 As nucleolar dye (Bedrick, 1970) (see Method 16).

ionic weight 541 Hansch 7t value - 5.63 conjugated bond number 32 charge 2­ absorption maximum bluish green solubility in water + in alcohol ± in Cellosolve + Appendix 4: Dyes 191

Pyronin Y Uses

CI no. 45005 As specific dye for RNA in combination with Methyl Green which stains DNA; Methods 14and 15 (Taft, 1951; Kurnick, 1952).

ionic weight 268 Hansch 1t value - 2.5 conjugated bond number 18 charge 1+ absorption maximum 552nm solubility in water + in alcohol ±

RhodamineB Uses

CI no. 45170 As fluorescent dye; together with Nile Blue to form Rhodanile Blue. (C,H5),N-oroN(C,H5) Data not available on all parameters.

absorption maximum bluish red, ± 556 nm solubility o-COOH in water in alcohol

Rhodanile Blue

No CI number. (H5C,),N@N(C,H5),

C-N o- o"

Uses molecular weight 780 solubility A combination of Rhodamine B and Nile Blue; in water ± used as nuclear dye, as Haematoxylin substitute in alcohol ? (Gurr, 1975).

Data on all parameters not available. 192 Appendix 4: Dyes

Solochrome Cyanin R Uses

CI no. 43820 As nuclear dye as Haematoxylin substitute (stains (synonyms Chromoxane Cyanin R, Mordant Blue blue in combination with phosphoric acid: Pearse, 3) 1957; Clark, 1979).

ionic weight 467 Hansch 1t value -10 conjugated bond number 29 charge 4­ absorption maximum red solubility in water + in alcohol ?

Sudan Black B

CI no. 26150

Uses ionic weight 456 Hansch 1t value + 7.4 As fat stain. conjugated bond number 36 charge o absorption maximum ± 600nm solubility in water in alcohol + in propylene glycol + Appendix 4: Dyes 193

Thionin Uses

CI no. 52000 As nuclear stain, especially for automation (Wit­ Structure by Comings (1975). tekind and Hilgarth, 1979); as Haematoxylin sub­ stitute.

ionic weight 225 Hansch 1t value - 5.1 conjugated bond number 18 charge 1+ absorption maximum ± 598 (This is the wavelength of the orthochromatic dye.) solubility in water + in alcohol +

Toluidine Blue Uses

CI no. 52040 As direct nuclear dye; as RNA stain in combination with a surfactant (Bennion et al., 1975-see Method 20).

ionic weight 270 Hansch 1t value - 3.9 conjugated bond number 18 charge 1+ absorption maximum ± 620 solubility in water + in alcohol ± in Cellosolve + in glycol + Recommended Reading

On theHistoryof Dyeing On Dyes, theirClassification,Application and History Baker, l .R. (1963). Cytological Technique. Meth­ uen, London. Provides an excellent introduction Lillie, R.D. (1977). Conn's Biological Stains . to the mechanisms of fixation and dyeing with a Williams & Wilkins, Baltimore. An invaluable wealth of historical background. book with information on most individual dyes, Vickerstaff, T. (1950). The Physical Chemistry of the methods in which they are used and their Dyeing. Oliver & Boyd, London. history. Venkataraman, K. (1952). The Chemistry ofSynth­ etic Dyes. Academic Press, New York . ForRecipesof Staining Solutionsandfor StainingMethods On theMechanism of Fixation, apartfrom Bolles-Lee, A. (1900). Microtomist's Vademecum . Baker's 5th edn , Churchill, London. The author, who Hopwood, D. (1973). Fixation in Histochemistry was an amateur, tells of his own experience and (P.l . Stoward, Ed.) Chapman & Hall, London. that of his contemporaries; provides very Pearse, A.G.E. (1980). Histochemistry, Theoretical stimulating reading 'and Applied. I. Preparative and optical tech­ Clark, G . (1981). Staining Procedures. Published nology. Churchill Livingstone, Edinburgh. for the Biological Staining Commission. Horobin, see below. Williams & Wilkins, Baltimore. Goes hand in hand with Conn's Biological Stains. On theMechanism of Staining Romeis, B. (1968). Mikroskopische Technik . Olden­ burg Verlag, Munich. Horobin, R.W. (1982). Histochemistry. Butter­ worths , London. The author has a very original On ClinicalCytology approach to the linking mechanisms of dye and Koss, L.G. (1979). Diagnostic Cytology. Lippin­ substrate, illustrated with many useful graphs cott, Philadelphia. and drawings. Atlases On CellBiology Lopez Cardozo, P. (1977). Atlas of Clinical DeRobertis, E.D .P. and DeRobertis, E.M.F. Cytology. Lippincott, Philadelphia. (1980). Cell and Molecular Biology . Holt-Saun­ Takahashi, M. (1981). Color Atlas of Cancer ders International editions . Cytology. Thieme Verlag, Stuttgart. References

Alfert, M. and Geschwind, J.J. (1952). Selective cer cells in body fluids. Am. J. Clin. Pathol., 36, staining for the basic proteins ofcell nuclei. Proc. 462-4. Nat . Acad. sa.. 39, 991-9. Berube, G.R., Powers , M.M ., Kerkay, J. and Arata, T., Sekiba, K. and Kato, K. (1978). Clarke, G. (1966). The gallocyanin- chrome Appraisal of self-collected cervical specimens in alum stain; influence of methods of preparation cytologic screening of uterine cancer. Acta on its activity and separation of active staining Cytologica,22, 150-2. compound. Stain Technology, 41 (2), 73. Ascher, A.W., Turner, C.J. and De Boer, G.H. Beyer-Boon, M.E. (1977). Preparatory techniques. (1956). Cornification of human vaginal epith­ In: Urinary Cytology (H.J. de Voogt, P. Rathert elium. J. Anatomy, 90, 545-52. and M.E. Boon, Eds) Springer Verlag, Berlin, Baker, J.R. (1963). Cytolog ical Techn ique. Meth­ pp.7-14. uen, London. Beyer-Boon, M.E. and Van der Voorn-Den Baker, J.R. (1970). Principles of Biological Micro­ Hollander, M.J.A. (1978). Cell yield obtained technique. Methuen, London. with various cytopreparatory techniques for Becker, S.N ., Wong, J.Y., Marchiondo, A.A. and urinary cytology. Acta Cytologica, 22, 589-93. Davis, C.P. (1981). Scanning electron micros­ Beyer-Boon, M.E., Van der Voorn-Den Hollander, copy of alcohol-fixed cytopathology specimens. M.J.A., Arentz, P.W., Cornelisse, c.i, Acta Cytologica, 25, 578-84. Schaberg, A. and Fox, c.n. (I 979a). Effect of Bedrick , A.E. (1970). Differential nucleolar stain­ various routine cytopreparatory techniques on ing of malignant and benign tissues with Pon­ normal urothelial cells and their nuclei. Acta tacyl Dark Green B. Stain Technology, 45 (6), Path. Microbiol. Scand., Sect. A, 87, 63-9. 273-6. Beyer-Boon, M.E., Arentz, P.W. and Kirk, R.S. Bennion, P.J., Horobin, R.W. and Murgatroyd, (l979b). A comparison of thiomersal and 50% L.B. (1975). The use ofa basic dye (Azure A and alcohol as preservatives in urinary cytology. J. Toluidine Blue) plus a cationic surfactant for Clin. Pathol., 32, 168. selective staining of RNA: a technical and Bibbo, M., Camargo, A.e.M., Valeri, V. (1969). mechanistic study. Stain Technology, 50 (5), Studies ofcell lipids from the human vaginal and 307-13. cervical epithelium during the menstrual cycle. Bercovici, B., Diamant, Y. and Polishuk, W.Z. Acta Cytologica, 13, 260-3. (1958). The use of intra-uterine device to obtain Bibbo, M., Fennessy, J.J. and Lu, C.T. (1973). material for cytology. In: Symposium on tech­ Bronchial brushing technique for the cytologic niques for endometrial cy tological examinations. diagnosis of peripheral lung lesions. A review of Acta Cytologica, 2, 577-8. 693 cases. Acta Cytologica, 17, 251. Bernhardt,H., Gourley, R.D., Young, J.M. , She­ Bird, C.L. (1951). The Theory and Practice ofWool pherd, M.e. and Killian, J.J. (1961). A modified Dyeing. Society of Dyers and Colourists, Brad­ membrane-filter technique for detection of can- ford. 196 References and Further Reading

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Wittekind, D.H., Kretschmer, V. and Lohr, W. Yasumatsu, H. (1977). Stain using celestine blue B (1976). Kann Azur B-Eosin die May-Griinwald­ as substitute nuclear stain in routine cytologic -Giemsa Fiirbung ersetzen? Blut, 32, 71-8. examinations. Acta Cytologica, 21, 173-4. Wittekind, D.H., Kretschmer, V. and Sohmer, I. Zajicek, J. (1974). Aspiration Biopsy Cytology. Part (1982). Azure B-eosin Y stain as the standard I: Cytology of Supradiaphragmatic Organs. Romanowsky-Giemsa stain. Brit. J. Haematol., (Monographs in Clinical Cytology, vol. 4). Kar­ 51,391-3. ger, New York . Wood, M.L. and Green, A.G. (1958). Studies on Zajicek, J. (1979). Aspiration Biopsy Cytology . Part textile dyes for biological staining. V. Pontacyl II: Cytology ofInfradiaphragmatic Organs (Mon­ blue black SX, pontacyl violet 6R and luxol fast ographs in Clinical Cytology, vol. 7). Karger, yellow TN. Stain Technology, 33, 279-81. New York. Yam, L.T. and Janickla, A.J. (1983). A simple Zanker, V. (1981). Grundlagen der Farbstoff-Sub­ method of preparing smears from bloody strat Beziehungen. Acta Histochemica, Suppl. effusions for cytodiagnosis. Acta Cytologica, 27, XXIV, SI51-68. 114-18. Subject index

acetate buffer, 138 as fixative, 130, 131, 132, 133, aspiration, 94 acetic acid, 114, 135, 147 138,139 cytology, 28, 105 as acidifier, 72, 151, 152, 153, as solvent, 157, 158, 159, 165, Auer's rods , 145 158, 160 167 autolysis, 51 as fixative, 52, 53, 55, 56 for destaining, 148 auxochrome, 34 acetone, 147 aldose, 13 Ayre spatula, 102 as fixative, 52, 122, 138 Alizarin Blue, 36,172 Azocarmine, 60 for dehydration, 65, 66 as natural dye, 41 Azure A, 37, 64, 65, 88,93,95,96, in Wright's stain, 146 Alizarin Cyanin BB, 64 138,166,174 acid dyes, 42 aluminium alum , 62, 63,151,152 Azure B (synonym ofAzure I and Acid Fuchsin, 36, 60, 69, 71, 73, aluminium sulphate, 141, 143, 153 Methylene Azure), 37, 88, 90,91,171 Amido Black lOB, 37, 64, 85, 137, 94,95,96,97,98,99,100, acidophil cytoplasm, 100 173 165,166,174 acidophil granules, 100 amino acid Azure C, 37, 96,175 Acridine Orange, 36, 84, 171 as basic units ofpeptides, 8, 9 Azure I, see Azure B Acridine Red-Malachite Green, in histones, 7 Azure II, 93, 165 65 ammonium alum, 151, 153 Azures as metachromatic dyes, 47 additional nuclear sta ining, 63 ammonium carminate, 90, 91 additive fixatives, 56 ammonium molybdate, 85,137, bacteria, staining of, 147, 167 adenine, 5, 6, 40 157 bacteriostatic agents, 103, 116 adhesives, 116 ammonium oxalate, 166 Balayette brush, 102 air-dry methods, 119 ammonium sulphate, 142, 147 balloon techniques, 107 air-drying, 53, 54, 55, 78, 80, 85, amphoteric dye, 35, 38 Barr body, 3,4, 138, 139, 176 94,98,101,117,123,1 29, amphoteric Haematein, 61 basic dyes, 42 138, 140, 142, 143, 146, 147 amphoteric Light Green , 73 Basic Fuchsin, 36,175 and fat staining, 82 amylase treatment, 80,140 in Schifrs reagent, 38, 157 effects, 55 Aniline Blue, 36, 69, 72, 173 basic proteins, 68, 135 albumin, 55 anionic dye, 35 Best's carmine, 14,41, 162 for the prevention ofcell Anthocyanins as natural dyes, 41 to stain glycogen, 14, 139, 177 explosion during air­ Apathy's gum syrup, 124 Biebrich Scarlet, 37, 68, 70, 79, drying, 55 arginine, 7, 9 139,176 Alcian Blue, 36, 81, 172 artifacts, 59 as protein dye, 50 to stain mucin , 14, 141 ascites, 146 in Shorr's stain, 79, 160 Alcian Blue method, 82, 141 aspiration fluid, 146 biopsies , 116 alcohol (see also ethyl alcohol), 22, asparagine as hydrophilic agent in Bismarck Brown , 37, 68, 69, 71, 121 proteins, 13 73,88,176 as differentiator, 139 aspartic acid, 9, 49 blebs, 81

* Numbers in italics indicate the page on which the structural formula appears . 206 Index blood chelate, 42, 45, 61 cytoplasm, 3, 8, 43,61 in smears, 79 Chlamydia, 100 as site ofprotein synthesis, 5, 6 serum, 55 chloral hydrate, 152 fixation influencing size, 53 smears of, 90, 92, 93, 94, 99,101 Chlorazol Black E, 37, 177 squamous, 55 stains, 145, 146, 166, 167, 187 chloroform, 121, 156 staining, 69, 70,129,130-45, blueing, 63,124,142 chromatin, 3, 54, 95, 97, 98, 100 157,158,171,177,188 solutions, 125 effect offixation on colouring with Methylene Green­ bone marrow of,54 Pyronin,66 smears, 95, 101 pattern,55,56,86,99,101 with Romanowsky-Giemsa staining of, 145 sex, 58 stain, 99, 100 Borax Methylene, 92 staining of, 138, 139, 145, 189 cytosine, 5, 6 Bouin's fixative, 66, 67,122,189 orthochromatic, 64 . Cytospin, 24, 26, 28, 106, 109 Bouin's fluid, 122 with Methylene bovine albumin, 116 Green-Pyronin, 66, Brazilin as natural dye, 41 135 dative bonding, 42 breast chromium alum, 61,155 dehydration, 53,124,127 carcinomas, 81 chromophore, 34,42,46, 60, 80 Delft fluid, 106 cysts, 27 loss of, 38 DNA Brilliant Cresyl Blue, 36, 176 chromosomes, 3, 101 demonstration of, 64 brush cytology, 105 banding of, 94, 95, 97, 98 denaturation of, 5 brush rinsing technique, 105 chromotrope, 46, 88 denaturation brush smearing technique, 105 cilia, 100, 101 by fixation, 52, 57 n-butyl alcohol circadian rhythm, 80 ofDNA, 5 citric acid as acidifier, 152 ofproteins, 10, 12,52 calcium, demonstration of, 184 clearing agents, 65, 66, 124 deoxyribonuclease solution, 156 Canada balsam, 124 clots, 107, 113 deoxyribose, 5, 13 Carbowax (seealsopolyethylene coagulant fixatives, 52, 56 deoxyribose nucleic acid, see DNA glycol), 56, 123 coagulating agents, 53 desiccation, 55 Carmine, 37, 64,177 cochineal, 41 destaining, 47,79,146 aluminium lake ofcochineal, 41 Coelestine Blue, 36,46,64, 133, ofPapanicolaou stain, 148 Best's, 162 154,178 of Romanowsky-Giemsa stain, Carmine Picrate, 90 coelomic fluids, 82, 106 148 Carmine Picrique, 90 Cole's Haematoxylin, 153 Diaphane,124 Carminic Acid, 90, 134, 154, 177 collagen, 13, 73, 100 diastase resistance, 80 derivative ofcochineal, 41 compound lipids, 14 differential dyeing, 48,88 Carnoy's fixative, 121 concentrational centrifuge, 109 by difference in diffusion ofdye, Carnoy's fluid, 53,118,121,135, condyloma acuminata, 78 47 138,139 conjugated bond number, 43, 44 by metachromatic dyeing, 47 cationic dye, 35 connective tissue, staining of, 69, differentiation, 45, 48, 72, 86, 139 cedar oil 70,71,72,173 in mordant dyeing, 45 as clearing agent, 65, 66, 136 Coomassie Brilliant Blue, 36, 66, digestive system, 107 as mounting medium, 124 68, 178 dimedone,80, 140 cell block, 30, 31, 104 cornification, 69 dimer,95 cell blocking technique, 104, 113 coulombic bonding, 42 dimethylamine, 97 cell division, 3 covalent bonding, 42, 64 dimethylsulphoxide (DMS), 97, cell environment, 22 coverslipping, 113 166 cell loss, 28 Cresyl Violet, 36,167,179 dinitrofluorobenzene, 37, 66, 68, cell membrane, 54, 88 cross-linking agents, 56 180 cell preservation, 52 cross-linking fixation, 52, 53, 57 direct dyeing, 44 cell, superficial, 155 Cryptococcus neoformans, 81 disaccharides, 80 cell suspension, 56 Crystal Violet, 36,147,166,179 disulphide bonds in proteins, 9, 76 centrifuge, 109 culture media, 116 DNA, 5, 99,134,135,136 cephalin, 15 Cuprolinic Blue, 36, 66, 84, 138, amount of, 5 cerebrospinal fluid, 106 179 demonstration ofwith Feulgen cervix, 102 cysteine as hydrophilic agent of reaction, 40 charcoal, 156 proteins, 13 in nucleolus, 8 Index 207

splitting off by fixatives, 54 staining of pre-, 56, 121 staining with Methyl Green-Pyronin role in unveiling binding sites, with Methyl Green, 49, 135, Y,66 48 156 with the Papanicolaou stain, times, 56 with Schiff's reagent, 66 49,129,130 fixative, 52, 117, 118 structure of, 4,5,53,54,57, with the Romanowsky- coagulant, 52, 53, 56, 57 84 Giemsa stains, 99,101 cross-linking, 52, 53, 57 DNA-ase, 85,137,156 Erythrosine B, 35 immersion, 121 Doehle bodies, 145 Esposti's fluid, 123 non-coagulant, 56, 57 drying, rate of, 55 ethanol, see ethyl alcohol requirements of, 52 dye, 34 ether, 121, 122 spray, 56, 122, 129-34, 140, 147 amphoteric, 35,49 ethyl alcohol (ethanol), effect of floaters, 30 anionic, 35, 49, 90 when used as wet fixative, floating cells, 27 cationic, 35, 49, 90 32,52-60,117,121 fluorochromes,65 cytoplasmic, 68 ethylene glycol, 153 Fluorone Black, 36, 64,181 natural, 41 euchromatin, 3, 55, 58, 59,101 formaldehyde, 118 neutral,90 Euparal, 124 as fixative, 52,55-7, 121,122 nuclear, 60 exchange diffusion, 73 influence on dyeing, 50 vital, 54 exudate, 27 formalin, 58, 80, 86,101 ,118,122, dyeing, 33 135, 136, 141 differential, 48, 72, 96 in Bouin's fixative, 66 direct, 44, 68 Fast Green FCF, 36,68, 134, 139, neutral, 121-3, 138, 143 metachromatic, 46, 65, 85, 95, 181 saline, 85,121 98,99,172 for dyeing nuclear proteins, 50, vapour, 101, 122, 123 mordant,44,68, 75, 85 66 formol ,154 orthochromatic, 46, 65 in Shorr's stain , 70, 79,160 alcohol , 139 polychrome, 68, 69, 79 fat(seealsolipids),141 ,142,162, saline, 114 progressive, 47, 48, 63,152,153, 163 sublimate, 137 155 fat stains, 36, 38,40, 82 freezer, 112 regressive, 48, 61, 86, 151, 152 ferric alum, 154, 155 frozen-section histology, 87,115 stoichiometric, 64, 65, 66 ferric chloride, 62, 152 fructose, 13 times, 47,78,100 ferrous sulphate, 147 Feulgen reaction and stain, 40, 42, 64,65,66,98,134,135,157 Gallamin Blue S, 36, 64 EA (Eosin-Azure), 69, 71-9 for staining chromosomes, 98 Gallein, 36, 64, 182 EA-31,132 positive in nucleolus, 7 Gallo Blue, 36, 46,182 EA-35, 130, 158 Feulgen reagent, 63, 98, 99 Gallocyanin, 36, 64, 65, 182 EA-65, 130, 158 fibres chrome alum , 69,133,155 Gill's, 130, 158 in tumour cells, 83 G-banding, 97 Leiden, 129, 157 reticulum, 142 gelatin, 124 effusions, 144 staining of, 163 gelatin--ehrome alum, 116 electron microscopy (EM), 19, 120 fibrillar zone of nucleolus, 7, 84 Gentian Violet, 179 staining for , 40, 41,58 filter imprint technique, 112 Giemsa stain and staining method enzyme staining techniques, 34, 52 filter techniques, 110 (see also Romanowsky­ Eosin Y, 36,44,46,53,63,68,69, fine-needle aspirates, 119 Giemsa),94, 142, 146, 165, 71,73 -8,132,180 fixation, 51-9 166 as metachromatic dye, 46 by air drying, 55 Gill's Haematoxylin, 153 in Papanicolaou's stain, 49, 70, influence on IEP, 50 glacial acetic acid, 121 , 123 72,85,157,158 influence on Papanicolaou glucose, 13 in Rakoff's stain, 79,160 staining, 78 glutamic acid, 9, 49 in Romanowsky-Giemsa stains, influence on size, 53 glutaraldehyde, 56, 57,122 90-100,165,167 influence on staining of glycerine (glycerol), 61, 62, 93, 97, similarity to Erythrosine B, 35 proteins, 50 146,155,165 ,166 epithelioid cells, 28 pH-controlled,86 glycerine-gelatin (glycerinejelly), erythrocytes, 73, 76, 77, 90-4, 100, post-, 54, 55, 80, 98, 99,121 , 124, 137, 141 , 142 101,114 139,142-6 glycine, 8 208 Index glycogen, 14 hormonal assessment , 71,131 , in Romanowsky-Giemsa stain, demonstration of, 38, 80, 139, 160,176 100 140,157,161,162 hormonal studies, 81 Kernechtrot, 36,141 ,143,147,184 structure of, 13 Howell-Jolly bodies, 145 ketoses , 13 glycoproteins, 14,80,81 ,140 hyaluronic acid, 80, 141 glycerol, see glycerine hyamine, 138 Gomori's silver method, 142 hydration, 53, 127 lake,45 Gomori's silver stain, 163 hydrochloric acid laminar outer zone , 22 gradient techniques, 110 as acidifier, 72-5,136,142-4, lead salts, 40 Gram's stain, 147, 167 152,157 lecithin, 15 Gravleejet irrigator, 102 as destainer, 148 Leiden-EA,157 Griendt van der , see van der as differentiator, 130 Leiden modification, 129 Griendt as fixative, 52 Leiden spray fixative, 53, 122 guanine, 5, 6 for hydrolysation, 40, 134, 135 Leif's method, 109 Guard's method, 139, 176 hydrogen bonding, 5, 10,42 leucobase, 88 gum arabic, 124 hydroglutamic acid, 49 leucocytes, 144 hydrophilicity, 15,42,43 staining of hydrophobic bonding, 5,13,15, in Papanicolaou stain, 129, Haematein, 35, 36, 61, 63, 183 42,98 130 Haematoxylin, 55, 60-63, 68, 69, hydrophobic forces, 98 in Romanowsky-Giemsa 71,140,183 stain, 90, 93, 100 as natural dye, 41 ligand,42 as progressive stain, 61 IEPofproteins, 9,35,50,61 Light Green Y, 36, 185 as regressive stain, 48, 61 immature benign cells, 19 as amphoteric dye, 35, 38 Cole's, 46, 129, 139, 153 immersion fixatives, 121 as quantitative protein stain, 68 Delafield's, 62, 132, 151 immunotechniques, 34 inEA,49,68-76,85, 157, 158 Ehrlich's, 46 imprint techniques, 115 in Rakoff's stain, 79,161 Gill's, 61, 63,130,153 Indigo, 41 lipid Harris's, 46, 130, 132, 139, 142, influence of pH on dyeing effect offixation on, 57 151 proteins, 49 loss of, 54 in Papanicolaou's stain, 69 intermediate cells, 76 presence in cytoplasm, 14 Mayer 's, 46, 139, 140, 142, 152 interphase nucleus, 3 staining of, 40, 141, 142, 162, sensitivity to fixation, 54, 55 iodine,59,161 185,188,192 staining ofnucleolus, 85, 86 ionic bonding, 42, 64 vacuoles in mesothelial cells, 82 Haematoxylin-Eosin, 49, 118, Iron Alizarin Blue, 64 liquor, see cerebrospinal fluid 119,132 iron alum, 61 lithium carbonate, 63, 70, 71, 125, Haematoxylon campechianum, 60 Iron Coelestine Blue, 64 130,158 haemosiderin pigment, 82, 83 iron, demonstration of, 143, 184 live cells, 87 hairspray, 122 iron stains, 83 logwood,60 half-oxidised Haematoxylin, 61 Isaacs cell sampler, 102 Love's Toluidine Blue Molybdate Hansch 1t value, 43, 44,169-93 isoelectric point, see IEP of method,137 hardening, 22 proteins Lugol's iodine, 161 heavy metals , 41 isopropanol,121 Lugol's solution, 137, 147, 156 Heidenhain's Azan, 60 IUD,102 for demostration ofglycogen, Hepes buffer, 166 79,161 heterochromatin, 3, 55, 58, 59, 101 Luxol Fast Blue B, 36, 185 Janus Green, 37,88, 144, 164,184 in fixation, 58 as fat stain, 14,36 in nucleolus, 7 lymphocytes, 100, 132, 144 histiocytes, 143, 144, 164 keratin, II , 13 staining of histochemical staining, 80 demonstration of, 188 in Papanicolaou stain, 129, histological cutting, 21 pleated-sheet structure, II 130 histone, 7 precursors, 76 in Romanowsky-Giemsa proteins (see also non-histone kera tinised cells, staining of stain, 100 proteins), 3, 9, 57, 60, 98 in Papanicolaou stain, 49, 73, lysine, 4,7 Hodgkin's disease, 137 79,129,130,159,160 Iysochrome, 40 Index 209

McIlvaine's buffer, 137, 156 Mi-Mark Helix, 102 nucleolus , 3, 7,136,137 madder, 41 mitochondria, 8, 88,141 ,144 associated chromatin, 7 magnesium chloride, 66, 138 monilia, 100 granular zone of, 7 Malachite Green, 65 monocytes, 100 staining, 66, 84-6, 94,129-38, malaria parasites, 90, 92-4 monolayer, 117 145,153,173,190 malignant cells, 86 monomer, 95 Nucleopore filter technique, 112 Mallory's tricolour stain, 61, 69, mono staining, 63 nucleoproteins, 7, 63, 64 71-2 mordant, 61 blocking of, 85 manipulating colour patterns, 79 dyeing, 42, 44-46, 48 dyeing of, 66 Marsan's preservative, 123 mounting media, 124 effect offixatives, 53, 54, 57 Masson's trichrome stain, 49,69, mRNA,6 involvement in nuclear staining, 190 mucin, 80, 81, 82,100,140,141 98 May-Griinwald-Giemsa method, demonstration of, 38 nucleoside , 4, 5 93,94,144,167 in cellmembrane, 14 nucleosome, 3, 5 and fixation, 53, 58 metachromatic dyeing of, 64 nucleotide, 4, 5, 6 Mayer's Haematoxylin, 152 mucopolysaccharides, 176 nucleus, 3,43,60- 7 Medhosa cannula, 102 mucoproteins, 80,140 staining of, 129-54, 172-94 melanin, 83,146,147 mucosubstances, 141 influence ofsize, 53 MEM (minimum essential mucus, 55, 79 medium),123 mucus-producing adenocarcin- mercuric chloride, 52, 55-9, 66, omas,81 oesophagus, 107 84,179 myelin, 141 Oil Red 0 (ORO), 37,118,142, mercuric oxide, 61, 122, 137, 151 myosin, 13 162,188 mesonephroma, 81 myxomatous background, 100 Orange G , 37, 90, 91,130,188 mesothelial cells, 82 as quantitative protein stain, 68 mesothelioma, 26, 81 effect ofpH , 74-8 metabisulphite, 134, 135, 142 Nabothian cysts, 27 Gill's OG , 130, 159 metachromasia, 46, 98 Naphthol Yellow S, 37, 68, 187 in Papanicolaou's method, 49, methacarn, 121 combined with Feulgen, 66, 135 68-79 methanol, 63, 93-101,121 , 146, natural dyes, 41 in Shorr's stain, 70, 79, 160 166 Neutral Red, 36,144, 147,164,187 Leiden OG, 129, 160 as differentiator, 48, 139 neutral stain, 90 Orcein, 189 as fixative, 52, 55 neutrophil cytoplasm , 100 as chromatin stain, 189 as post-fixative, 54, 55, 98 Nile Blue, 36, 188 as natural dye, 41 for destaining, 148 as fat stain, 40 orthochromasia, 46 in preserv atives, 56 Nile Red,40 osmium tetroxide with acetone, 56 Nissl body, 138 as fixative, 52, 55, 57, 58, 122 Methyl Fluorone Black, 64 non-dyes, 38 as stain for EM, 41 Methyl Green, 36, 85, 90,186 non-histone proteins, 6,7,9,60,98 osmolarity, 117 Methyl Green-Pyronin Y, 49,65, nuclear dye, 134 oxalic acid, 72 118,136,156 nuclear envelope (membrane), 3, oxygen, 61 Methyl Violet, 46 56,58, 101 oxyhaematein,61 Methylene Azure, see Azure B nuclear protein, 66, 98 Methylene Blue, 37, 73,91-6, 144, nuclear stain, 133 164-7,186 nucleic acid, 60, 133, 155, 179 Papanicolaou's polychrome stain , as metachromatic dye, 46 composition of, 4, 5 49 as nuclear dye, 64 content, 133 Papanicolaou staining methods in combination with Schiff's re­ effectof fixation on, 57 and fixation , 53, 58, 78, 10I agent , 66, 67 loss of, 53 modification, 180-5 in Romanowsky-Giemsa nucleolar organising region, 7 staining ofthe cytoplasm, 71-9, method, 91-10I nucleolar proteins, 85 129,130,157-60, 180, Methylene Blue-Eosinate, 97 nucleolar staining methods (see 185 Methylene Violet, 37, 93,187 also nucleolus), 84-6 staining the nucleolus, 7, 84-6 microwave oven, 123 nucleolini, 7, 85 paracoccidiomycosis, 30 Millipore techniques, III staining of, 137, 156 Pararosanilin, 38, 189 210 Index parasitic protozoa, 100 100 quick staining methods, 132 PAS reaction, 38, 40, 42,80-2, polysaccharides, 13,38,80, 141, for Giemsa, 146 140,172 172 for Papanicolaou method, 132 penetration by fixatives, rate of, 55 Ponceau de Xylid ine, 37, 69, 70, quinoid group ofdyes , 35 pentose 190 in DNA, 4 Pontacyl Black CX, see Amido in RNA, 4 Black lOB Rakoff's polychrome stain, 79, peptide linkage, 8, 9 Pontacyl Dark Green, 37, 85,136, 131,161 Percoll solution, 110 190 rapid staining, 144 periodic acid, 38, 81, 140 poorly differentiated malignant red cabbage, 41 periodic acid-Schiffreaction, see cells, 20 regressive staining, 47, 48, 61 PAS port wine, 41 reserve cells, 19 Perl's method, 143 post-fixation, 55, 117, 118 resin as mounting medium, 124, pH potassium alum, 134, 151, 154 135,138 effect on potassium chromium oxide, 122 Rhodamine B, 36,191 EA,74 potassium ferricyanide, 147 Rhodanile Blue, 36, 64,191 Eosin Y, 74 potassium ferrocyanide, 143 ribonucleoprotein, 7, 84, 85 Light Green, 74 potassium iodide, 161 ribose metachromatic dyeing of preferential sampling, 27 as ketose, 13 RNA, 64, 66, 67 pre-fixed cells, 116 in RNA, 5 Orange G, 76, 77 preliminary diagnosis, 87 ribosomes, 6 proteins, 9, 49, 68 preservative, 52, 103, 116, 117 synthesis of, 7 Romanowsky-Giemsa stain, preserving fluids, 123 ripening ofHaematoxylin, 60 100 primary fixatives , 57 RNA, 4,61 -6,84 staining the nucleolus, 8, 85 progressive staining, 47,61 amount of, in nucleolus, 7, 8 offixative, 85 propanol, 121, 132 amount of, in nucleus, 7 ofstaining solution in Roman­ propylene glycol, 141, 142, 162, messenger RNA, 6 owsky-Giemsa method, 163 presence in cytoplasm, 8 100 prostate, 27 staining Phenacyanin TC, 64 proteins, 8 in Romanowsky-Giemsa phosphate buffer, 138, 146, 166 dye polymerisation effect, 98 method,99 phospholipids, 88 effects offixation, 52-7 methods, 133, 135, 136, 138 phosphomolybdic acid (PM A), fibrous, 13 Stoichiometric, 64 68-73,79,137,160 globular, 13 with Feulgen, 69 phosphoric acid, 4 in nucleus, 7 with Methylene Blue, 66, 69 phosphotungstic acid (PTA), loss of, 53 with Pyronin Y, 49, 135, 136, 68-79,157-60 precipitation of, 56 155,191 pick-and-smear method, 104 shrinking by fixation, 52 with thiazine dye s, 69 picric acid, 37, 90, 91,118,189 staining of, 69-83, 135, 173-7, structure of, 5, 6 as component ofBouin's fix- 180, 187 transfer RNA, 6 ative,66 structure of, 8-12 Romanowski effect, 86, 94, 97, pigment, 82 synthesis, 5, 6 99 -101 plasma cells, 99, 100, 135, 138 PTA , see phosphotungstic acid Romanowski- Giemsa method plasmal reaction, 38 purines, 5, 6 and stain, 56, 68, 73, 80, plasmalogen, 15, 38 pyknotic nuclei , 63 90-101, 145, 16~ 174, 180 platelets, 145 pyrimidine, 5, 6 and fixation, 98 pleura aspiration fluid, 146 Pyronin Y, 36,191 and post-fixation, 54, 55 PMA, see phosphomolybdic , acid as RNA stain in nucleoli, 7 and wet fixation, 99 polychrome dyes, 69, 70 in combination with Thionin, 65 as metachromat ic stain, 46 polyeth ylene glycol (Carbowax), in Methyl Green-Pyronin Y, 49, influence ofthickness of smear, 55, 56, 59, 104, 118, 122, 135,136,156 101 129,130 on cell blocks, 104 polylysine L, 116 staining of cytoplasm, 99 polyrnerisat ion , 98 staining of nucleolus, 8, 84, 85 polymorphonuclear leucocytes, quantitative cytochemistry, 64 staining of nucleus, 95, 97-9 Index 211

standardisation, 199, 166 silver nitrate, 163 for dyeing ofnucleolini, 85, 156 Romanowsky-type stains, 94, 97, single-stranded RNA, 84 for dyeing ofribonucleoproteins 98 sodium iodate, 61, 152, 153 in nucleoli, 7 sodium thiosulphate, 156 for dyeing RNA, 138 Solochrome Cyanin R, 36, 46, 64, for rapid staining, 144,164 saccharides 192 transcription , 4, 6 presence in cytoplasm, 8, 15 spatula, 107, 108 triacid stain, 90 structure of, 13 spectrophotometry, 68, 99 triarylmethanes, 34, 36 Saccomano technique, 24, 56,123 spray fixative , 117, 122 trichloracetic acid, 85, 137 Saccomano's fluid, 104 spreading ofcells, 22 trichomonads, 100 salicylic acid, 154 sputum triglycerides, 14 saline fixation of, 56 trisaccharides, 80 phosphate-buffered, 123, 140 preparation of, 104 Tris-buffer, 137, 156 physiological, 123, l31 squashing technique, 115 tryptophan, 8 saline formalin, 121 staining saline formol (seealsovan der equilibrium, 47,78 Griendt's fluid), 54, 114, pattern, 80 uracil, 5, 6 123 time, 78 uranyl acetate, 40, 41 salting, 50 standardised Romanowsky- urea (2 M), 114 saponin, 114 Giemsa stain, 166 urinary cytology, 103, 146, 161 Schitrs reagent, 14,38,39,57,64, stomach, 107 134,135,140,157,175 streptolysin 0, 114 in combination with Methylene striated muscle, 100 vacuoles Blue, 66, 67 Sudan Black, 14,37,40,141,162, in nucleoli , 7 in Feulgen reaction for the de­ 163,192 lipid in, 82 monstration ofDNA, 40 Sudan III, 40 ofhistiocytes, 144 reaction with aldehydes in sulphuric acid, 155 PAS-positive material in, 81 glycogen, 14 superficial cells, 76 vaginal tampon, 102 reaction with aldehydes in plas­ supravital staining, 144 vakutage device, 102 malogen,15 surfactant, 64, 139, 174 van der Griendt's fluid, 117, 118, Schmorl stain, 83 suspension technique, 116 123 Scott's solution (tap water sub­ swelling effect, 53 van der Griendt's method, 105, stitute), 63, 125, 130, 148 Szczepanik's quick staining 110 scraping technique, 115 method for Papanicolaou van der Waals forces, 42, 43, 47 secondary structure ofproteins, stain, 132 visibility ofthe nucleolus, 86 9-10 vital dye, 54, 87 disruption by fixat ion, 52 vital staining, 87-9, 144, 176, 179, sedimentation techniques, 110 tertiary butanol 184, 187 sediments, 110 as clearing agent, 65,124,129, staining ofwet sediments, 144, 135,156 164,165 as differentiator, 48 Waals van der, see van der Waals SEM,120 tertiary structure ofproteins, 13 forces sex chromatin, see Barr body disruption by fixation, 52 Water Blue, 69-71 sex hormones, 82 thiazines, 37,44,46,64,90,95 Weigert's Haematoxylin, 152 Shorr's stain and staining method, Thionin, 37, 64,193 wet-fix methods, 119 69,71,78,79,131,160,176, in combination with Pyronin Y, wet sediment, 144, 164, 165 181 65 wooden spatulas, 108 shrinkage, 22 thiosulphate, 59 Wright's staining method, 93,146 shrinking effect, 53 thymine, 5, 6, 40 sialomucins, 141 thymol ,118 side stacking, 98 toluene, 137 xylene, 65, 66,124,130,132,140, silver as stain Toluidine Blue, 156,193 143, 147 for fibrous components, 83 as metachromatic dye, 47 for nerve cells, 41 as nuclear dye, 64,65 for ribonucleoproteins, 142 as protein dye, 66, 69 Zenker's fixative, 122 Part Two Atlas 215

1.1 1.2

•!

•

1.3 1.4

PLATE 1 Microscopy without staining . 1.1, Using polarised light. Cholesterol crystals in aspiration of tumour on head. Tissue diagnosis: atheroma. 1.2. Using polarised light, with quartz red I filter, unstained . Starch crystals . 1.3, Brown colour of melanin in aspiration of lymph node metastasis of malignant melanoma. The slide is unstained. 1.4, Same case as 1.3. The melanin is stained with the modified Schmorl method (number 38) . 216 A tlas

•

2.3 2.4

PLATE 2 Components of the nuclear and cytoplasmic stains in the Papanicolaou method. 2.1, Haematoxylin only (method 3 without acid bath). Vaginal smear with intermediate cells. Note that the cytoplasm also stains blue. 2.2, Orange G, pH 2.5. Anucleated highly keratinised squames from the foot. Note that the colour is yellow. 2.3, Leiden EA (number 14). No Haematoxylin. Note turquoise colour of cytoplasm and nucleus. Vaginal smear. 2.4, Eosin Y, 0.5% with 0.2 g PTA. Note pink colour of these intermediate cells, and compare with plate 2.3. Atlas 217

3.1 3.2

3.3 3.4

PLATE 3 Comparison of EA with different pH values (omitting Bismarck Brown). 3.1, Smear from slight dysplasia. EA has pH of 4.6. No keratinised cells present. Cytoplasm stains turquoise. 3.2, Same case as 3.1. EA has pH 6.5. Cytoplasm stains blue. 3.3, Smear from condylomatous dysplasia (no koilocytotic cells in this photographic field). Keratinised cells stain red, non-keratin ised turquoise. Staining method 2. 3.4, Same case as .3.3. The keratinised cells stain the same; the non- keratinised cells stain blue instead of turquoise . Staining method 3. 218 Atlas

4.3 4.4

PLATE 4 Order of staining dishes in Papanicolaou methods including Orange G (pH 2.5). 4.1, Staining method 1, pH of EA 4.6. Normal sequence, first Orange G, followed by EA. Superficial cells stain orange , intermediate cells stain turquoise. 4.2, Same baths as in 4.1 but in reverse order (first EA, followed by Orange G). Here the orange G 'wins' over the Eosin, and thus the colour of the superficial cells is yellower. The intermediate cells are slightly destained . 4.3, Staining method 1, with laboratory-made EA, pH 6.5. Normal sequence, first Orange G, followed by EA. Here, the Eosin has replaced the Orange G in the superficial cells, colouring them red. Note orange colour of erythrocytes. 4.4, Same staining method as in 4.3, but reversed order (first EA, followed by Orange G). Here the Orange G has replaced the Eosin, staining erythrocytes and superficial cells orange-yellow. The intermediate cells are slightly destained . Atlas 219 -

5.1 5.2

• •

• • • • c • • • • • • ..

5.3 5.4

PLATE 5 Colour of nucleoli in the Papan icolaou methods. 5.1, Method 2, using EA, pH 3.5. Colo ur of nucleoli is green, due to staining with Light Gree n. Undifferentiated carcinoma. 5.2, Method 1, using EA, pH 6.5. Colou r of nucleoli is orange due to staining with Orange G. Benign endocervical cells. 5.3, Method 3. Colou r of nucleol i is red, due to staining with Eosin. Cells from ovar ian adenocarcinoma in vaginal smear . 5.4, Method 3. Colour of nucleol i is blue , due to staining with Haematoxylin. Cells from endometrial adenocarcinoma. 220 Atlas

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6.1 6.2

6.3 6.4

PLATE 6 Staining patterns of OG in cervical smears using Papanicolaou method 1. 6.1 , Koilocytotic cells. The rim of the koilos, containing dense cytoplasm, stains orange-yellow with the OG. 6.2, Same cells as in 6.1, visualised with the Nomarski method. Note that the rim of the koilos is the thickest part of the cell. 6.3 , Case of condylomatous dysplasia. The small parakeratotic cells and some of the superficial cells are highly keratinised and stain orange-yellow. 6.4 , Group of endocervical cells. Note that the inner part of these cells (without signs of keratinisation) stains orange -yellow. Atlas 221

•

7.1 7.2

7.3 7.4

PLATE 7 Different steps of keratinisation visualised in the Orange G-Papanicolaou method (1).7.1-7.4, Smears from a verruceous carcinoma of the cervix. The most highly keratinised cells stain yellow, with decreasing keratinisation orange-red-blue. Even when the malignant keratinised cells have lost their nuclei, or contain small pyknotic nuclei, they can still be recognised as originating from the carcinoma by their abnormal shape. 222 Atlas

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8.3 8.4

PLATE 8 Using the Papanicolaou method (3) on air-dried smears incubated overnight with formol-saline (van der Griendt's post-fixation method). 8.1 and 2, Slight dysplasia, note good stainability of the nuclei with Haematoxylin. 8.3 and 4, Adenocarcinoma in situ of endocervix. Note red nucleoli. Atlas 223

•• • • 9.1 9.2

• •

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9.3 9.4

PLATE 9 Length of staining times in the Romanowsky-Giemsa method . Adenocarcinoma of the lung metastasis in lymph node. Method 35 (changing staining times), pH Giemsa 6.4. 9.1, 15 min: poorly developed Romanowsky effect, bluish -purple nuclei. Note orange-pink staining of erythrocytes. 9.2, 25 min: better developed Romanowsky effect. 9.3, 35 min: well developed Romanowsky effect. 9.4, 45 min: well developed Romanowsky effect. Nuclear pattern more 'open', and therefore nucleoli more visible. 224 Atlas

10.1 10.2

• 10.3 10.4

PLATE 10 Effect of pH in the Romanowsky-Giemsa method. Adenosquamous carcinoma of the lung, method 35 (changing pH of Giemsa). 10.1 , pH Giemsa 5.5. No Romanowsky effect, blue nuclei. 10.2, pH Giemsa 6.4 . Good Romanowsky effect. Colour of mucoid material pink, of (few) erythrocytes orange-red. 10.3, pH Giemsa 7.0. Good Romanowskyeffect. Colour of mucoid material grey-pink, of erythrocytes (not in microscopic field) grey. 10.4, pH Giemsa 8.5. Good Romanowsky effect. Colour of mucoid material blue, of erythrocytes (not in microscopic field) green-blue. Atlas 225

11.1 11.2

11.3 11.4

PLATE 11 Eosin staining in the Romanowsky-Giemsa method. 11.1, Pleural fluid; asbestos body. The protein coat stains orange with Eosin. Method 35. 11.2, Lung brush, poorly differentiated adenocarcinoma . Method 35, pH Giemsa 6.4. Erythrocytes stain orange with Eosin. 11.3, Bone marrow, stained with Difquick method (commercial). Granulae stain orange with Eosin. Note grey colour of erythrocytes. 11.4, Pleural fluid, mesothelioma . Method 34. Eosin plays a role in the pinkish staining of collagen in the centre of the mesothelioma cell grouping. 226 Atlas

12.1 12.2

•

12.3 12.4

PLATE 12 Nucleoli in the Romanowsky-Giemsa method. 12.1, Pleural fluid, metastasis of ovarian adenocarcinoma, method 34. In the well spread cells the blue nucleoli are clearly visible in the purple 'open' chromatin. In the thick cell grouping the nuclei are blue, and thus the blue nucleoli are invisible. 12.2, Lung brush, adenocarcinoma, method 35. The purple chromatin is more dense than in 12.1 and therefore the nucleoli are not clearly visible. 12.3, Bone marrow, immunocytoma, Oifquick (commercial). Fat droplets on three of the nuclei, obscuring nucleoli. Blue nucleoli in the malignant immunocyte. 12.4, Aspirate of carcinoma of the breast, staining method 33. Nucleoli difficult to distinguish in dense, purple chromatin. Atlas 227

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13.1 13.2

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PLATE 13 Romanowsky-Giemsa method on air-dried cervical smears. All smears are stained with method 36.13.1 , The spindle cells originating from squamous carcinoma of the cervix have azure-blue cytoplasm and dense purple nuclei. Note the orange colour of erythrocytes. 13.2, The cells from undifferentiated carcinoma in situ of the cervix have large purple nuclei and no cytoplasm . 13.3, Screening magnification of 13.4. 13.4, Large cell non-keratinised carcinoma in situ of the cervix. The purple chromatin of the malignant cells shows a malignant pattern; the cytoplasm is pale blue. 228 Atlas

••• ., •

14.1 14.2

14.3 14.4

PLATE 14 Squamous differentiation in the Romanowsky-Giemsa method. 14.1 , Buccal smear, staining method 36 , pH 7.0. Some cells stain azure-blue but most stain blue. 14.2, Lung brush, adenosquamous carcinoma, method 35 with pH Giemsa of 6.4. One malignant squamous cell has azure-blue cytoplasm. 14.3 , Lung brush, moderately differentiated squamous cell carcinoma of the lung, staining method 35. The cytoplasm of the malignant cells stains pale blue. 14.4, Blocked sputum, formalin fixed, staining for 60 min in 2:8 Giemsa. The cytoplasm of the malignant squamous cells is red. Atlas 229

15.1 15.2

•

15.3 15.4

PLATE 15 Romanowsky-Giemsa method on cell blocks. The sections are deparaffinised in distilled water for 60 min in 2:8 Giemsa solution, dipped in 98% acetic acid, followed by 96% ethyl alcohol and dehydrated in isopropanol. 15.1, Alcohol-fixed sputum, Giemsa pH 6.8. The nuclei of the oat cells are purple. 15.2, Alcohol-fixed sputum, Giemsa pH 6.8. The nuclei of the malignant squamous cells are purple, the cytoplasm is red. 15.3, Formalin-fixed sputum, Giemsa pH 6.8. A group of adenosquamous carcinoma cells. Due to the influence of formalin there is no Romanowsky effect, thus the nuclei stain blue. 15.4, Alcohol-fixed sputum, Giemsa pH 4.8. Due to the low pH of the Giemsa there is no Romanowsky effect, thus the nuclei stain blue. ... t..) l' • 'to•, ...• • • •

16.3 16.4

PLATE 16 Effects of protein in the air-dry ing process for the Romanowsky-Giemsa method, and in wet-fixation. 16.1 , T cell lymphoma in pleural fluid , high protein content. Smear from centrifuge sediment. Note that, due to the high protein content, the cells are not well spread , and therefore nuclear cleavage is not visible. 16.2, Same case as 16.1, Cytospin slide . The protein is absorbed by the filter paper , and thus the malignant lymphoma cells do not dry in a high protein environment as in 16.1. Now, the nuclear cleavage of the malignant cells is clearly visible. 16.3, Pleural fluid , inflammation. The Cytospin slide is wet-fixed with spray fixative. The prote in in the background forms a network and therefore cytoplasm of the histiocytes cannot be delineated. 16.4, Same case as 16.3, Cytosp in slide, air-dried . Method 35 . There is no interfering prote in network and the histiocytes are well spread . Atlas 231

17.1 17.2

17.3 17.4

PLATE 17 Pleasant and unpleasant effects of air-drying for the Romanowsky-Giemsa method. 17.1, Pleural fluid, metastasis of adenocarcinoma of the breast. The sediment in the centrifuge tube was not dry enough, and thus the cell spread contained too much fluid of a low protein content. The cells look 'watery' and are not suited for diagnostic work. 17.2, Pleural fluid, mesothelioma. The laminar part of the cytoplasm is spread over the slide, forming 'blebs'. These stain positive with the PAS method, and are characteristic for mesothelial cells. 17.3, Bone marrow, immunocytoma, Oifquick (commercial). Fat globules on top of nuclei forming 'holes'. 17.4, Bone marrow, staining method 34. The central cell has a true hole in the nucleus (note granulae in it). , . -

•

• • • •

18.3 18.4

PLATE 18 Fibrin and protein in pleural fluid. 18.1, Pleural fluid with high protein content , wet-fixed , staining method 3. 18.2, Same case, air-dried smear from centrifuge sediment. Note that the cells are not well spread , and therefore there is no Romanowskyeffect. Everything stains blue. 18.3, Pleural fluid, fibrin in background. Cells better for diagnostic work than in 18.1. 18.4, Same case as 18.3. Nomarsky. Note granular appearance of fibrin. Atlas 233

• • •

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19.1 19.2

19.3 19.4

PLATE 19 Staining characteristics of mesothelial cells. 19.1, Pleural fluid, wet-fixed . Staining method 4. Malignant mesothelioma. Note the 'two-tone' staining of the cytoplasm, indicating mesothelia l different iation. 19.2, Same case as 19.1, air-dried . Giemsa staining method 34. No two-tone staining, but the laminar part of the cytoplasm stains darker blue (see plate 17). This is not the same area as the blue staining part of the two-tone cytoplasm in 19.1. In the latter cells, there is only minimal spreading of the laminar part of the cytoplasm . 19.3 , Same case as 19.1, same staining. This is a thicker part of the smear; here the malignant mesothelial cells stain orange-red, and are not recognisable as such by their staining pattern. 19.4, Same case as 19.1. Staining Method 4. Smaller mesothelial cells with blue staining cytoplasm and no two-tone effect. 234 Atlas ..

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20.1 20.2

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PLATE 20 Staining patterns, ORO stain (Method 27). 20.1, Pleural fluid, malignant mesothelioma. Note characteristic peri­ and supranuclear distribution of small ORO-positive vacuoles. 20.2, Pleural fluid, metastasis of adenocarc inoma of the breast. Adenocarcinoma cell with non-perinuc lear fat. 20.3, Pleural fluid, malignant mesothel ioma . Many histiocytes with non-perinuclear fat. 20.4, Pleural fluid, same case as 19.1 . Note that some of the peri- and supranuclear vacuoles have lost their fat content, leaving white 'holes' in the nucleus and cytoplasm . Atlas 235

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21.1 21.2

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21.3 21.4

PLATE 21 Staining patterns, PAS stain (Method 23). 21.1 and 21.2, Pleural fluid, malignant mesothelioma. Note the positive staining of the 'blebs' (see plates 17 and 19), characteristic for mesothelial cells. In addition, in 21.1 there is some central positive staining. 21.3 , Pleural fluid, metastasis of serous papillary carcinoma of the ovary. The positive dots are evenly divided over the cytoplasm. 21.4 , Ascites, metastasis of signet-ring carcinoma of the stomach . The PAS-positive large vacuole of the malignant signet ring compresses the nucleus. which therefore cannot be studied. The positive diagnosis of this fluid was based on the presence of these cells, which could not be recognised as malignant in the Papanicolaou and the Giemsa slides. 236 AtLas

22.1 22.2

22.3 22.4

PLATE 22 Staining reticulum fibres (Method 28). 22.1, Aspirate, fibrosarcoma. The fibres (thick bundles) are black (Method 28). 22.2, Same case as 22.1, Giemsa method 35. Indication of a thick bundle of fibres. 22.3, Aspirate, fibromyxosarcoma. Network of black fibres (Method 28). 22.4, Same case as 22.3. Myxomatous mass with feathery aspect, containing malignant cells. Giemsa method 36. Atlas 237

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23.3 23.4

PLATE 23 Cell arrangements. 23.1. Lymph node aspirate method 34, sarcoidosis. The epithelioid cells are arranged in dense clusters , and the lymphocytes have a 'starry sky' distribution. 23.2, Bronchial brush, method 36. Benign bronchial cells . The nuclei are arranged on the outer border of the cell cluster which displays good cohesion. Note purplish-red brush border of the cells visible in the centre of the cell grouping. 23.3, Aspirate, method 35, fibromyxosarcoma. The nuclei of the malignant cells are scattered over the microscopic field without a definite pattern, but seem to be attached to each other by the fibres. 23.4, Aspirate, method 35, pH 6.4. Poorly differentiated adenoca rcinoma of the lung. The nuclei are scattered over the microscopic field, sometimes in very loose small clusters. This pattern is different from the 'starry sky' appearance of the lymphocytes in 23.1. 238 Atlas

24.1 24.2

24.3 24.4

PLATE 24 Three-dimensionality in the Papanicolaou and the Romanowsky-Giemsa methods. 24.1 and 24.2, Pleural fluid, metastasis of ovarian adenocarcinoma, alcohol fixed. Staining method 4. Two focusing planes of the same microscopic field. Note that different parts of the cells are in focus. 24.3, Same preparation as 24.1. Nomarsky. Compare three­ dimensional cell groupings of malignant cells (right) with flat histiocytes (left). 24.4, Same case, air-dried, Giemsa method 34. Note absence of Romanowsky effect in thick cell groupings.