STAINS
Stains are dyes used to colour tissues and other specimens for microscopical examination. In an acid stain, the colour is carried by an acid radical and the stain is taken up by parts of the specimen having a basic (alkaline) reaction. In a basic stain, the colour, carried by basic a radical, is attracted to parts of the specimen having an acidic reaction. Neutral stains have neither acidic nor basic affinities.
A contrast stain is used to give colour to parts of a tissue not affected by a previously applied stain. A differential stain allows different elements in a specimen to be distinguished by staining them in different colours (Schenk and Willis, 2008).
ROMANOWSKY STAINS
Romanowsky stains depend for their staining properties upon certain derivatives produced when alkaline methylene blue is combined with eosin (ICHS, 2010). Romanowsky stains are used universally for routine staining of blood films, and satisfactory results can be obtained. The remarkable property of the romanowsky dyes of making subtle distinctions in shades of staining, and of staining granules differentially, depends on two components: azure B (trimethylthionin) and eosin Y (tetrabromo-fluorescein) (Wittekind, 2003). The original romanowsky combination was polychrome methylene blue and eosin. Several of the stains now used routinely that are based on azure B also include methylene blue, but the need for this is debatable. Its presence in the stain is thought by some to enhance the staining of nucleoli and polychromatic red cells; in its absence, normal neutrophil granules tend to stain heavily and may resemble toxic granules in conventionally stained film (Marshall, 1999).
There are a number of variation in staining. One of the main factors is the presence of contaminants in the commercial dyes and a simple combination of pure azure B and eosin Y might be considered preferable to the more complex stains because this ensures consistent results from batch to batch (Wittekind et al., 2003). However, in practice, absolutely pure dyes are expensive, and it is sufficient to ensure that the stains contain at least 80% of the appropriate dye 6. Among the romanowsky stains now in use, jenner is the simplest and giemsa is the most complex. Leishman’s stain, which occupies an intermediate position, is still widely used in the routine staining of blood films, although the results are inferior to those obtained by th combined May-Grunwald-Giemsa, Jenner-Giemsa, and azure-B-eosin Y methods (Horobin and Walter, 2008).
A pH to the alkaline side of neutrality accentuates the azure component at the expense of the eosin and vice versa. A pH of 6.8 is usually recommended for general use, but to some extent this depends on personal preference (Wittekind, 2011). Romanowsky stain may be purchased in powder, tablet or liquid form. For a routine laboratory, the stain already in solution is probably the most convenient, but each batch purchased or prepared should be tested for its optimum staining time. More so, in romanowsky staining, because the aqueos dyes solution were unstable; methanol was introduced as a solvent. Leishman ans wright advocated use of methanol as a fixative prior to staining. Giemsa improved this technique by standardizing the dye solution and adding glycerol to increase stability (Marshall, 1999). The 2 mostly used Romanowsky stains in blood film staining are Leishman and Giemsa.
LEISHMAN’S STAIN
Leishman stain is designed to differentiate leucocytes and is available commercially as the precipitated powder or as a ready-prepared solution.
Components Of Leishman Stain
Solution 1
· Methylene blue 1g
· Sodium carbon (0.5% aqueous solution) 100ml
· Eosin (0.1% aqueous solution) 100ml
· Methyl alcohol 100ml
Solution 2 (Buffer solution PH 6.8)
· Disodium hydrogen phosphate (Na2HPO4) (anhydrous) 9.47g
· Potassium di-hydrogen phosphate (anhydrous) KH2PO4-m/15 sol 9.08g
· D/W 1000ml (Ochei and Kolhatkar, 2007).
GIEMSA STAIN
Giemsa stain is designed to differentiate leucocytes and is available commercially either in ready to use liquid form or as a combined powder.
Romanowky – Giemsa effect are:
· A cationic dye: the best dye is azure B and though azure A gives the nuclear purple colour, the cytoplasmic blue is inferior. No other cationic dye such as methylene blue is suitable.
· An anionic dye: most commonly eosin Y is used.
Composition Of Giemsa Stain
Solution 1
· Azure II eosin 3g
· Azure II 0.8g
· Glycerol
· Methyl alcohol 300m
Solution 2 (Buffer solution PH 7.0)
· M/15 disodium hydrogen phosphate (9.47g per litre) 61.1ml
· M/15 potassium dishydrogen phosphate (9.08g per litre) 38.9ml (Ochei and Kolhatkar, 2007).
Other romanowsky stains used in staining blood films in haematology are:
WRIGHT STAIN
Reagents
(1) Absolute methanol.
(2) Staining solution (which can be purchased as a ready-made solution or as a powder from commercial sources). Typically, 0.3 g of Wright stain powder is dissolved in 100 mL absolute methanol and left in a closed container at room temperature for 24 hours. It must be filtered before use.
(3) Sörensen’s buffer solution at pH 6.4; KH2PO4, anhydrous 6.63 g; Na2HPO4, anhydrous 2.56 g; distilled water up to 1000 mL (Berend, 2007).
MAY-GRÜNWALD-GIEMSA STAIN
Reagents
(1) Absolute methanol.
(2) Staining solution I: 0.3 g May-Grünwald powder in 100 mL absolute methanol; leave in closed container at room temperature for 24 hours. It must be filtered before use. Staining solution II (Giemsa stain): 1 g Giemsa stain powder is dissolved in 66 mL glycerol and heated to 56°C for 90 to 120 minutes. After addition of 66 mL absolute methanol and thorough mixing, the solution is left at room temperature in a closed container. It must be filtered before use.
(3) Buffer: Sörensen’s buffer solution. The pH must be at 6.8 for the May-Grünwald-Giemsa stain instead of 6.4 as in the Wright stain (Berend, 2007).
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