Re: Wright Giemsa problems
|From:||"J. A. Kiernan" <email@example.com>|
On Fri, 22 Sep 2000 AndreaH@imclone.com wrote:
> Is there a way to destain a bone marrow prep after staining with Wright
> Giemsa? I ask because the Shandon kit I bought seems to have gone off
> (expired 5/2000) and I need to reorder a new kit and stain this valuable
First: The Principles. (An answer to the question will follow.)
Wright's stain (a mixture of dyes) can be bought as a "certified"
powder that is stable for many years. Its non-aqueous stock solutions
should also be stable for a few years (at least 5) if kept securely
capped and out of direct sunlight, in bottles made of glass or
unreactive plastic. Non-reactive liners (not metal foil) are needed
for screw-cap bottles. All this information has been in the published
literature for at least 50 years.
It is unlikely that the age of your kit, which you identify as
coming from a highly respected firm, is the cause of your trouble.
Most difficulties with this type of staining are attributable to
mis-matching of the fixative (nothing, formalin, alcohol, etc) with
the pH of the working solution of the dye mixture. The simple
general rule is that for a particular preparation you must determine
the optimum pH. This is used for the pre-stain rinse (if any) the
staining solution, and the wash following staining. If the pH is
correct, times in the stain and wash are not critical.
Dehydration and clearing should be rapid, because all the dyes
used in this type of technique are easily extracted by water-alcohol
mixtures, but not by absolute alcohol. Thwrefore: blot the stained
and buffer-rinsed slide with filter paper to remove pretty well all
the water. Dehydrate in 3 changes of 100% alcohol (or air-dry for at
least 2 hours). Immerse in xylene (2 X 1 minute) and apply a coverslip,
using minimum amount of a resinous mounting medium.
Second and last, The Answer:
You can de-stain by immersing the slide in acid-alcohol
(1 ml of conc HCl in 100 ml 70% or 95% alcohol) for
one minute, rinsing in 70% alcohol and then immersing in
a weakly alkaline liquid (saturated aqueous calcium hydroxide
is pretty good) until there is no remaining colour in the
slide or smear. Wash well (hours, not minutes) in water,
rinse in buffer of the correct pH, and stain again.
John A. Kiernan,
Department of Anatomy & Cell Biology,
The University of Western Ontario,
LONDON, Canada N6A 5C1
Phone: (519) 661-2111
FAX (Department): (519) 661-3936
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