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.