May Grunwald - Giemsa problems!

From:marina goumenou

Hi to all Histonetters

I work with May Grunwald - Giemsa for about 2 years staining bone marrow 
smears from rats (I count micronucleus in polychromatic RBC) but the results 
are not constantly well. So, I still search to find what is going whrong. 
Some of the artifacts I have noticed are:
Very bright RBC, green RBC, no diferentiation between normochromatic and 
polychromatic RBC, breaked RBC, purple in the center of normochromatic RBC 
etc. I thought many different possible reasons for all these problems and I 
try to stabilize the parameters of the protocol I use but I am not sure 
about my thoughts:
I use DI water instead of buffer, is this sufficient? (I am not sure that my 
water is constantly well)
I filter Giemsa before use but what kind of filter is appropriate (pore 
size, material etc)?
I use stock solutions (botlles of 2.5L) and some times they are not in use 
for weeks after I open them. Is this a possible problem?
How long can I store my smears after fixation (5min in methanol) without 
staining them?
Is it possible having burst RBC due to FBS osmolarity and/or water in 
methanol during fixation?

I really need any help, any thought, any technical information!
Thanking you in advance
Goumenou Marina

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