Re: [Histonet] Wright-Giemsa stain for sections

From:John Kiernan

Wright's and Giemsa's are different stains, both used
principally for blood and marrow to identify the
various cell types. They are mixtures of blue
cationic dyes (notably azure B) with eosin Y, an
anionic dye. Both can be used on smears or sections.

Giemsa gives excellent results with ordinary
paraffin sections once you have found the ideal
pH for the staining solution. Fixation affects the
color balance, so you have to experiment the
first time you do the method. After adequate
formaldehyde fixation the best pH is often in 
the range 4.0 to 4.5.

Solutions: 1. Acetate buffers, 0.2M from pH 3.5 to 7.
              (Only one of these will be needed when
              the optimum pH is known.)
           2. Buffered water, made by 10-fold dilution of
              of the 0.2M buffer. The pH needs adjustment
              with drops of 5% acetic acid after dilution.
           3. Giemsa stock solution. Usually this is
              bought ready-made. It should contain
              dyes certified by the Biological Stain
              Commission. (Ask the supplier.)
1. Bring paraffin sections to water.
2. Put slides in acetate buffer for 5 minutes.
3. Pour off the buffer. (It can be reused.)   
3. Add 1 ml Giemsa stock solution to 50 ml of
   buffered water in a beaker. Stir, then pour the
   diluted stain into the staining jar containing
   the slides.
4. Staining time is about 2 hours at 20C or about
   15 minutes at 50-60C (microwave oven).
5. Rinse the slides in buffered water, blot dry
   and check with a microscope. If blue predominates,
   differentiate carefully in 0.01% acetic acid, 
   rinse in buffered water and blot dry again.
   Too much blue means the pH of the buffer was too
   high. If red predominates, use a higher pH next
6. Either thoroughly air-dry the slides or dehydrate
   the almost dry sections in two changes, each 
   3 minutes, of n-butanol.
7. Clear in xylene and coverslip.

In a correctly stained preparation nuclei and 
cytoplasmic RNA are blue to purple. Erythrocytes, 
collagen and keratin are pink. The different types 
of leukocyte are recognizable by their nuclei and 
cytoplasmic details. Bacteria are dark blue to purple. 
The preparations are more informative than sections
stained with hemalum and eosin because more
cytoplasmic detail is visible in a wider range of
colors. The late R. D. Lillie perfected methods of
this type as routine staining techniques for on a
large scale (mechanized) in histopathology (see 
Lillie & Fullmer, 1976).

Lillie RD, Fullmer H (1976) Histopathologic Technic 
and Practical Histochemistry. 4th ed. New York: 
McGraw-Hill, pp. 193-197.
John A. Kiernan
Department of Anatomy and Cell Biology
The University of Western Ontario
London,   Canada   N6A 5C1
"Etheridge, Sandra AGF:EX" wrote:
> Hello fellow Histonetters!
> I am looking for a manual staining procedure for cytology or blood smears
> using the commercial Wright-Giemsa stain.  I was also wondering if it can be
> used on regular tissue sections.  I believe the method is quite fast and our
> current method does not give adaquate nuclear staining.  Our reagent expired
> in December '03, so that could also be the problem.
> Your feedback would be appreciated.
> Sandra Etheridge
> Animal Health Center
> BC Ministry of Agriculture, Food, and Fisheries
> Abbotsford, BC
> _______________________________________________
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