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
200ml
·
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).