[Histonet] crysel violet stainning problem

Lee & Peggy Wenk lpwenk <@t> sbcglobal.net
Tue Aug 7 19:02:37 CDT 2007

Is this the procedure for staining Nissl in brain?

I don't know if cresyl violet is still around any more. I think it stopped
being made around WWII. Most people are using cresyl echt violet, which now
a-days is actually cresyl violet acetate. So, if you are using a bottle of
cresyl violet, it's either very old, or it may be a different dye than what
you need, being called that name. After all, any company can call any dye by
any name. So the same name may be used for different dyes. Or the same dye
many be called by different dyes. 

We found that we needed to buffer the solution to a pH of 3.5, to reduce
background staining.

Also, make certain you are using cresyl violet acetate certified by the
Biological Stain Commission, if in the US. Assures good quality of dye.
There is no Color Index number, as it is a mixture of dyes.

Below is our staining procedure, which works for our lab and our students.
If in a hurry, can stain in 60 degree C. waterbath or oven for about 20-30
minutes, or heat to 60-70 degrees C. in microwave and allow to set on
counter for 5 minutes.

0.5 g Cresyl echt violet (Cresyl Violet Acetate)
0.18 g Sodium acetate (CH3COONaC3H2O)
500 ml Distilled water
1.5 mL Acetic acid, concentrated (CH3COOH)
Dissolve cresyl echt violet and sodium acetate in distilled water. Slowly
add acetic acid, drop by drop, to solution. Should have a pH of 3.5. If
solution pH is below 3.5, add more sodium acetate. If solution pH is above
3.5, add more acetic acid. Filter. Let stand overnight before using. Store
at room temperature. Stable for months. May be reused until weak.

PROCEDURE - Cresyl Echt Violet:
1.	Deparaffinize and hydrate slides through graded alcohol to distilled

2.	Place sections in cresyl echt violet solution at room temperature
1-2 hours

3. 	Differentiate in two changes of 95% ethanol until nuclei and Nissl
granules remain violet and the background is nearly colorless. Check
differentiation with the microscope. 1 to 2 drops of acetic acid many be
added to the first alcohol to speed up differentiation.

4.	Dehydrate through absolute ethanol and clear in xylene.

5.	Coverslip with a synthetic mounting media.

Nissl granules - violet
Nuclei - violet
Bacteria, fungus - blue to purple
Cartilage, mast cell granules	- blue to purple
Background - colorless

Peggy A. Wenk, HTL(ASCP)SLS
William Beaumont Hospital
Royal Oak, MI 48073

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Marta
Sent: Tuesday, August 07, 2007 7:23 PM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] crysel violet stainning problem

I'm writing because I have problem with de-staining of my samples during the
staining with crysel violet. I read from the message from 2003 that the way
is to dry samples in the oven, and don't use the water and alcoholes for
dehydratation and differentiation. I tried to add acetic acid to alcoholes,
to avoid 70, 95%...And nothing. The sections are a little bit blue after
even 12 mins in crysel violet an than they are destained. I tried it with
different samples, from formalin, Bouin's fixative. Finnaly, I have to try
with drying in the oven but now I try to find other resolution.
Crysel violet is from Sigma-Aldrich.
I will appreciate for your help.
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