Re: Wright Giemsa problems

From:"J. A. Kiernan" <jkiernan@julian.uwo.ca>

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
> slide.

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
   E-mail: kiernan@uwo.ca

  
  





<< Previous Message | Next Message >>