Fawn:  The sterile water you are using is too acidic for the staining you 
are performing.  Bottle distilled water often has a pH around 4.0 to 4.5 
Sterile water (if it has sat for any period of time) is probably as acidic 
as standard bottle distilled water.    In any Romanowsky type staining 
procedure (of which Wright Giemsa is included) the water is actually a 
differentiator. With human peripheral blood or bone marrow cells, the 
proper pH is typically 6.8 to 7.2.  Some animals require slightly lower 
pH.  Paraffin processed tissue sections for some reason I don't understand 
appear to accept lower pH values.  With the pH of the sterile water you 
are using, the color which is present in the cells is being stripped away 
by the acidic water you are using.  Regardless of how well your Wright 
Stain solution may be working, exposing the slides to 3 changes of acidic 
water is more than likely stripping any color from the slides. 

The latest edition of Humason's Animal Tissues Techniques, 5th edition has 
the formulations for Sorensen's phosphate buffer listed on pages 473 and 
474.  Sigma-Aldrich sells a slightly lower molarity product as P3288.  Try 
an appropriate pH phosphate or TRIS buffer and I think you will find your 
staining improves.

I would also suggest you fix your slides after 30 minutes of drying in the 
methanol.  Waiting overnight before fixation allows for oxidation of the 
hemoglobin.  Best staining results occur if the blood is stained right 
after collection. 

You may also want to cut back on your staining times in Wright Stain and 
Wright Giemsa solutions.  The original Wright Giemsa procedure had the 
user first staining in Wright Stain and then following up with a Giemsa 
procedure.  I do not quite understand the concept of first staining in 
Wright Stain and then staining a second time in Wright Giemsa stain.  More 
typical staining times for blood smears would range from about 15 seconds 
to 2 minutes for the Wright Stain and typically double the amount of time 
in deionized water or buffer.  The 1 to 2 ratio is a good starting point 
for the procedure, but the times can be adjusted to suit personal color 
preference.  Wright Giemsa or Giemsa solutions typically require longer 
incubations and are more commonly performed on bone marrow preparations 
sor cultured cells.  Giemsa is used for identifying malarial parasites and 
for tissue sections.    If you want to bring out more red in your red 
blood cells, try skipping the Wright  Giemsa stain and use just the Wright 
Stain. 

Best Regards,

Bob Krug 
Technical Marketing Specialist
Sigma-Aldrich
St Louis, Missouri
e-mail:  clintech@sial.com
(800) 325-0250 
 
 
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"Fawn Jones" 
Sent by: histonet-bounces@lists.utsouthwestern.edu
10/06/2004 11:40 AM

 
        To:     
        cc: 
        Subject:        [Histonet] micronucleus staining


Hi everyone,
Our histology labs is doing a wright-giemsa stain on mice micronucleus
slides and we are having a problem with our cells not picking up the
stain.  Are there any suggestions for us.
We let them dry overnight then we put them in Methanol for 5 minutes,
let them air dry for 20 minutes then they go into Wright's stain for 6
minutes, then Wright-giemsa stain for 6 minutes, and then go through 3
changes of sterile water for injection for 30 seconds each.  Then they
sit overnight before coverslipping.  If anyone could help that would be
great.
Thanks
Fawn Jones
North American Science Associates
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