Re: Removal of Bouin's before processing (longish)

From:"J. A. Kiernan"

"Su, Phy-Huynh" wrote a LOT of questions about using
Bouin. I can answer some of them: 

> tendons fixed with Bouins.  According to Luna ...
> ... and Carson (Histotechnology), Bouin's should be removed before
> processing, whereas Sheehan Hrapchak ... states that it will be
> removed in the 50% batch, so removal of Bouin's prior to processing 
> is not necessary.

They are probably all right. The stuff your removing is the
picric acid, which precipitates proteins as insoluble yellow
picrates. According to Luna (but nobody else, to my knowledge)
prolonged exposure to picric acid, even in solid paraffin, causes
deterioration of the tissue. The other things picric acid does
solution) are to extract some RNA (reduction or loss of
basophilia) and to convert some DNA to Schiff-positive
(a sort of Feulgen hydrolysis, though not an optimal one).

Picric acid is soluble in water (about 1%) and much more soluble
in alcohol, which washes it out more quickly. It is traditional
to move specimens from Bouin directly into 95% alcohol, to cause
irreversible coagulation of protein picrates (some of which are
soluble in water). This is probably not necessary if the duration 
of fixation is 24-48 hours - long enough for the formaldehyde in
the mixture to cross-link most of the proteins to some extent.

>                       ... The yellow colorof picric acid is 
> still there, even after several weeks in 50% EtOH, but it
> doesn't mean that Bouin's is not completely removed, does it?

If yellow is still coming out into the solvent, you are still
extracting picric acid from the tissue. The specimen remains
yellow because in contains protein picrates that are either
coagulated by alcohol or cross-linked by formaldehyde, or both.

>  How do you make 70% EtOH saturated with Licarbonate? ...  
> ...I also tried to add some solid Li Carbonate to 70% EtOH ...
> It doesn't go into solution.

Then a saturated solution is too dilute for the dissolving
to be visible. I've not heard of people using 70% alcohol
saturated with Li2CO3. It's usual to keep a stock bottle of
saturated aqueous Li2CO3 on the lab shelf for use when a
not-too-strongly alkaline liquid is needed. It's easy to
make up because no weighing is needed.  One use is for
removing the yellow colour of picric acid from sections of
Bouin-fixed tissue before staining. It does the job in
usually less than a minute. A few drops of sat. aq. Li2CO3
added to a tank of water makes an excellent blueing
solution to follow staining with haemalum. 

Finally, saturated aqueous calcium hydroxide (limewater)
is just as good for the two purposes in the preceding
paragraph, and Ca(OH)2 is much less expensive than Li2CO3.

References: JR Baker's "Principles of Biological 
  Microtechnique" (London: Methuen, 1958) is probably
  still the best review of picric acid fixation.

John A. Kiernan
Department of Anatomy and Cell Biology
The University of Western Ontario
London,   Canada   N6A 5C1

<< Previous Message | Next Message >>