[Histonet] cresylechtviolett (cresyl fast violet) staining problems

John Kiernan jkiernan <@t> uwo.ca
Mon Dec 1 10:16:30 CST 2003

Is your dye from a batch certified by the Biological
Stain Commission? If so, it will have a little sticker
on the bottle that says so, and it will probably be
called cresyl violet acetate. For variations in
names and identities (there's no correlation!) see
Conn's Biol. Stains 10th ed. (2002), Chapter 19 (by

A certified batch of cresyl violet should give a 
good result if you use the same procedure that the
B.S.C. uses to test the dye for certification. This
is described in a paper by D.P.Penney et al. 2002
"Analysis and testing of biological stains - the
Biological Stain Commission procedures." Biotech.
Histochem. 77: 237-275. This paper describes the
testing methods for all the 61 currently certifiable
stains. If your lab doesn't get this inexpensive
journal you should at least obtain Vol 77 issue 5/6 
for Sep/Nov 2002, which contains the B.S.C. 
procedures paper.
John A. Kiernan
Department of Anatomy and Cell Biology
The University of Western Ontario
London,   Canada   N6A 5C1
   kiernan <@t> uwo.ca
Sandy Thevarkunnel wrote:
> Hi,
> We just bought new cresyl fast violet from Cell point scientific to
> replace our old cresyl fast violet from Roboz which was produced in
> 1995.  The new stain seems to wash away from the tissue when we put it
> in the 95% ethanol with acetic acid step rapidly, but prior to the step
> there does not seem to be any detectable differentiation during our 75%
> ETOH, 75% ETOH with acetic acid and the 95% steps that precede the 95
> with acid step.  Our protocol for making the cresyl fast violet is to
> add 2.5g to 500mL deionized H2O stir and then filter.  This recipe was
> great with the old stain but the new does not seem to be working.  We
> are staining formalin fixed human brainstems cut at 50um.
> Staining protocol we use
> Defatting- 3hrs in 1:1 Chloroform:Ethanol
> Hydration: 7 minutes- 2x 100% ETOH, 2X 95%ETOH, 1x70%ETOH
>                      10 minutes- dH2O
> Staining- 4-6 minutes in CV
> 30 seconds dH2O
> Differentiation from 75%, 75% with Acetic acid, 95%, to 95% with acetic
> acid, variable times from 1 minute to 4 minutes
> Dehydration- 2x 100% ETOH for 4 minutes
> Clearing- 3 changes of Xylene 2min, 1min, 30 sec.
> Coverslip with Permount
> Sorry for the long message I was just hoping if it wasn't the recipe it
> just might be our protocol so if anyone has any ideas I'd really
> appreciate them, I'm kind of new at this.  By the way our old CV was
> black but the solution was still purple and the new CV is green and
> solution is the same purple colour but it seems thinner.
> Thank you very much,
> Sandy
> Boston University School of Medicine
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