[Histonet] bouin solution

[Lee & Peggy Wenk]

If the tissue is in Bouins, the excess Bouins needs to be washed out of the 
tissue immediately after fixation.

Water could be used to rinse out the excess picric acid, but I think some 
people are worried that there could be some cell swelling if the tissue is 
left in the water too long (particularly if the tissue is underfixed/not 
stablized enough).

If I remember correctly, too high of a percent of alcohol (95%, for 
example), will cause the picric acid salts to precipitate in the tissue, and 
it would be like trying to microtome tissue with sand in it.

So 50-70% alcohol is a good compromise. Have enough water to remove the 
excess picric acid salts, but not too much water to cause tissue swelling, 
and not too much percent of alcohol to cause picric acid salts to 
precipitate in the tissue.

For your slides already microtomed and on slides - these have already been 
fixed and processed. If the tissue was fixed in a picric acid fixative like 
Bouins, there is no need for the step of putting the slide in Bouins. If the 
tissue was fixed in something else (10% neutral buffered formalin, for 
example), then the slide/tissue needs to be placed into Bouins in order for 
the trichrome stains to work correctly. This is called either post-fixing or 
post-mordanting. There are several theories as to why the Bouins is needed 
for the trichromes (change tissue charges, change tissue density), but the 
end result is that Bouins fixed or Bouins post-mordanted tissue have muscles 
that are red and collagen that are blue or green (depending upon which dye 
you use). If the NBF fixed tissue is not post-mordanted, the red is a 
red/blue or red/green, and the collagen has strands of red in it. In other 
words, the colors are not as true or as crisp.

Now, the slides that were post-mordanted in Bouins have tissue that is 
yellow. We need to get the yellow out. Not so much because of the picrate 
salts that precipitate, but more because the yellow color would interfere 
with the reds and the blues or greens colors that you are trying to make.

The tissue is only 3-5 um thick, so it won't take as long to remove the 
color. And the tissue has been well fixed, dehydrated and cleared during 
tissue processing. So there are lots of cross-links to stabilize the tissue. 
So rinsing in water is only going to take a few minutes, and the tissue is 
stabilized, so there shouldn't be any cell size changes. You could rinse in 
50-70% alcohol, but that's only 50-30% water, so it is going to take a lot 
longer to get rid of the yellow color than rinsing in water.

Rinsing in tap water is fine for most labs, as long as the quality of tap 
water is good (not too acidic or basic, not too much iron or sulfur, no 
microorganisms). I like to rinse with d. water before any stain, just in 
case there is any contaminating metal that might interfere with the stains 
(we have a lot of iron in our part of the state).

By the way, putting the slides in Bouins in a coplin jar with a lid screwed 
on, then putting in a 60 degree C. oven or water bath for 45-60 minutes 
works great. This would reduce your turn around time by 17 hours! Two words 
of caution. 1) Don't breath in the hot Bouins fumes (picric acid, acetic 
acid, formaldehyde). Open up the coplin jar in a hood. 2) Don't stick the 
hot coplin jar into cold running water. Coplin jar will break/crack. We 
usually take the slides out of the hot Bouins, and put the slides into a new 
coplin jar with water, then put this under the running water. The lid is put 
back on the Bouins coplin jar, and allowed to cool. Some places reuse the 
Bouins for a number of times.

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

--------------------------------------------------
From: "wassan alkadhumi" <w_alkadhumi <@t> yahoo.com>
Sent: Tuesday, December 28, 2010 9:49 AM
To: <histonet <@t> lists.utsouthwestern.edu>
Subject: [Histonet] bouin solution

> Dear Histonet
> I have question concerning bouin solution, we use it in masson trichrome 
> before
> starting the stain. we incubate the slides trimmed at 3 micron in bouin 
> solution
> for 18 hr in room temp which is not controlled. Then we wash the slides 
> using
> running water first and then distilled water, but according to what
> i red, when using bouin solution,the tissue should be washed with (50-70)%
> alcohol to prevent deterioration of staining in time. now i should mention 
> that
> in the our  masson trichrome procedure there is no washing with alcohol 
> but only
> with water.
>
> what should i do? In the book its mentioned that we have to wash with 
> alcohol if
> the tissue is not processed yet, is that why the alcohol step is missing 
> in
> masson tri procedure?and why will the stain deteriorate if some of the 
> bouin
> solution stayed in the tissue?
> The main fixative that is used in our lab for all grooses and biopsy is 
> 10%
> formalin (except bone marrow biopsy). bouin solution is used after 
> processing of
> the tissue and deparaffinization of the sections is finished.
> Thanks
>
> Wassan
> Histotechnician
> Shorh hospital
> North of Iraq
>
>
>
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